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Stuart et al: Lady Beetles as Predators of Diaprepes
LADY BEETLES AS POTENTIAL PREDATORS OF THE ROOT WEEVIL
DIAPREPESABBREVIATUS (COLEOPTERA: CURCULIONIDAE)
IN FLORIDA CITRUS
R. J. STUART, J. P. MICHAUD, L. OLSEN AND C. W. MCCOY
University of Florida, IFAS, Citrus Research and Education Center
700 Experiment Station Road, Lake Alfred, FL 33850
ABSTRACT
Diaprepes abbreviatus (L.) is a major pest of Florida citrus. Adult females lay eggs in masses
sealed between leaves in the citrus canopy, and recently-hatched neonate larvae drop to the
soil and feed on roots. The coccinellid species, Cycloneda sanguine (L.), Harmonia axyridis
Pallas, and Olla v-nigrum Mulsant, are generalist predators that consume a wide range of
citrus pests, although they have not been observed preying on Diaprepes. We conducted ex-
periments to determine whether these species would feed on Diaprepes egg masses and ne-
onate larvae, and how an exclusive or partial diet of Diaprepes eggs would influence their
development. The three predators responded very similarly in our tests. In laboratory as-
says, coccinellid larvae and adults readily consumed exposed Diaprepes eggs and neonates
less than 48 h old; and coccinellid larvae preyed on 40% of intact egg masses laid between
wax paper strips or citrus leaves, whereas adults preyed on 8.7%. In a greenhouse assay, coc-
cinellid larvae located and preyed on 22.7% of intact egg masses laid between leaves on pot-
ted citrus trees. Although neonates might have relatively limited exposure to predation in
the canopy before they drop to the soil, predation could be an important factor selecting for
the timing of egg hatch, neonate escape from leaf envelopes, and neonate drop. The develop-
mental assays indicate that Diaprepes eggs are less suitable prey for these coccinellid spe-
cies than eggs of the flour moth, Ephestia kuhniella Zeller, but that they could be a highly
acceptable component of a mixed diet. Our experiments indicate that these coccinellid spe-
cies are potentially important predators of Diaprepes but the extent to which they contribute
to the natural biological control of this weevil remains unknown.
Key Words: predation, development, biological control, integrated pest management, Curcu-
lionidae, Coccinellidae, Cycloneda sanguine, Harmonia axyridis, Olla v-nigrum
RESUME
El picudo de los citricos, Diaprepes abbreviatus (L.) es una plaga s6ria de citricos en Florida.
Hembras adults ponen huevecillos en masas selladas dentro hojas en la copa del arbol, y las
larvas neonatas recien-eclosionadas caen al suelo y come de los raices. Las catarinitas
Cycloneda sanquinea (L.), Harmonia axyridis Pallas, and Olla v-nigrum Mulsant son depre-
dadores generalistas que consume un rango amplio de insects dafinos en citricultura,
aunque no han sido observados consumiendo Diaprepes. Experimentos fueron llevado a cabo
para determinar si estas esp6cies consumiera huevos y neonatos de Diaprepes, y como dietas
de huevecillos y neonatos, exclusivas o parciales, influiera su desarollo larval. Los tres de-
predadores respondieron en forma parecida en nuestros experiments. In pruebas de labo-
rat6rio, larvas y adults consumieron en seguida huevecillos de Diaprepes expuestos y
neonatos menos que 48 h de edad. Larvas atacaron 40% de masas de huevecillos sellado den-
tro hojas de papel encerado, o hojas de citrus, mientras que adults atacaron 8.7%. En una
prueba de invernadero, larvas de Coccinellidae atacaron 22.7% de masas de huevecillos en
arboles de citricos en tiestos. Aunque los neonatos no son exquesto a la depredacion por largo
plazo en el arbol antes de caer al suelo, la depredacion puede ser un factor important en la
evoluci6n del tiempo de eclosi6n de huevecillos, salida de neonatos del sobre de hojas, y caida
de neonatos al suelo. Las pruebas de desarollo mostraron que los huevecillos de Diaprepes
son press menos apropiadas para larvas de estas esp6cies que huevecillos del pomilla de ha-
rina, Ephestia kuhniella Zeller, pero indican que huevecillos de Diaprepes podian ser una
presa altamente acceptable dentro una dieta mesclada. Sobre todo, nuestros experiments
indican que 6stas esp6cies de Coccinellidae son depredadores de Diaprepes potencialmente
important, pero el alcance de tal depredacion en la naturaleza queda desconocido.
Translation provided by author.
The root weevil, Diaprepes abbreviatus (L.), ap- namentals in Florida (Simpson et al. 1996). In cit-
parently originated in the Caribbean and is now a rus, larval feeding damages roots, reduces yield,
major introduced pest of citrus, sugar cane, and or- and kills trees by girdling or by facilitating infec-
Florida Entomologist 85(3)
tion by plant pathogens such as Phytophthora spp.
(Graham et al. 1996). The combination ofDiaprepes
and Phytophthora can lead to rapid tree decline
and destroy groves within a few years of a weevil
infestation. Adults are long lived and feed on foli-
age, especially new growth. Mating occurs in the
canopy, and eggs are laid in masses between leaves
that are glued together by an adhesive secreted by
the female during oviposition. The larvae hatch, es-
cape from the sealed leaf envelope, drop to the soil,
and burrow down to the roots where they begin
feeding. As they grow, the larvae move to larger
roots, and pupate in the soil after 9-11 instars
(Woodruff 1985; Quintela et al. 1998; McCoy 1999).
Sources of mortality for the various life stages
of Diaprepes include numerous predators and par-
asites. Several egg parasitoids have been discov-
ered in the Caribbean and introduced into Florida
for biological control (Hall et al. 2001). Eggs are
also subject to predation by ants, crickets, earwigs,
lacewings, and spiders. Newly-hatched neonate
larvae are especially prone to predation on the soil
surface where they are attacked by ants, earwigs,
hemipterans, and spiders whereas larvae below
ground are attacked by ants and entomopatho-
genic nematodes. Adults are preyed on by ants,
birds, lizards, snakes and spiders (Whitcomb et al.
1982; Richman et al. 1983a,b; Tryon 1986; Jaffe
et al. 1990; McCoy et al. 2000; Stuart et al. in press).
In developing an effective IPM program to con-
trol D. abbreviatus, it would be advantageous to
maximize the effectiveness of as many of the nat-
ural enemies of this insect as possible. Several
coccinellid species including Cycloneda san-
guinea (L.), Harmonia axyridis Pallas, and Olla
v-nigrum Mulsant, are common in Florida citrus
groves where they prey primarily on various
aphid species (Michaud 2000). Although these
coccinellids have not been reported preying on
Diaprepes, coccinellids are known to consume a
wide range of prey including certain weevil larvae
(Kalaskar & Evans 2001). Moreover, these coc-
cinellid species coexist with Diaprepes in the cit-
rus canopy and are likely to encounter Diaprepes
egg masses and possibly neonate larvae, before
the larvae escape from their sealed leaf envelopes
and drop to the soil. Hence, coccinellids might
contribute to the natural biological control of this
weevil, and we conducted laboratory experiments
to determine whether these species could be effec-
tive predators on Diaprepes egg masses and neo-
nates. We also conducted experiments to determine
how an exclusive or partial diet ofDiaprepes eggs
would influence the development of these species.
MATERIALS AND METHODS
Rearing
Laboratory colonies ofHarmonia axyridis, Cyclo-
neda sanguinea, and Olla v-nigrum were reared
on frozen eggs of the flour moth, Ephestia kuh-
niella Zeller, and bee pollen as described by
Michaud (in press). Coccinellid eggs were col-
lected from ovipositing females and incubated as
described by Michaud (2000) to produce larvae
used in experiments. Diaprepes abbreviatus eggs
were obtained from laboratory colonies where
they were laid between strips of wax paper or be-
tween citrus leaves. Leaves that were presented
to weevils for oviposition were held together with
paper clips, and the leaf petioles were wrapped
with wet cotton and parafilm to prevent desicca-
tion. Diaprepes neonates were obtained from egg
masses laid between wax paper strips and were
used in experiments within 48 h of their emer-
gence from sealed egg masses. Aphis gossypii
Glover were reared on cotton plants (Gossypium
hirsutum, var. "SureGrow") in growth chambers
at 20 + 1C, 16:8 L:D h, 60-65% RH. Laboratory
experiments were conducted in plastic Petri
dishes (5.5 cm dia x 1.0 cm) on open laboratory
benches at 24 + 1C under fluorescent lights un-
less otherwise noted.
Prey Acceptability
Tests were performed to determine the relative
acceptability of Diaprepes eggs and neonates as
prey for larvae and adults of the three coccinellid
species. In the first test, individual first-instar coc-
cinellid larvae (<24 h old) were transferred to plas-
tic Petri dishes (as above) containing an excess of
E. kuhniella eggs (~0.1 gm) and a small D. abbre-
viatus egg mass of 10-19 eggs, which had been ex-
posed by separating the wax paper strips between
which the mass had been laid. We conducted 24-25
replicates per species, and the number ofD. abbre-
viatus eggs consumed was recorded after 24 h. In
the second test, 4-5 wk old adult coccinellids were
similarly presented with excess E. kuhniella eggs
and an exposedDiaprepes egg mass. We conducted
20 replicates per species, and predation was as-
sessed after 24 h. In the third test, coccinellid lar-
vae reared on E. kuhniella eggs to the third instar
were presented with an excess of moth eggs and 10
D. abbreviatus neonates less than 48 h old. We con-
ducted 18-20 replicates per species, and the num-
ber of D. abbreviatus larvae consumed was
recorded after 24 h. In the fourth test, individual
coccinellid adults were starved for 16 h and then
transferred to plastic Petri dishes that contained
10 A. gossypii (a mixture of 4th instars and apter-
ous adults) and 10 D. abbreviatus neonates. We
conducted 13-16 replicates per species, and the
numbers of A. gossypii and D. abbreviatus larvae
consumed were recorded after 30 min.
Prey Accessibility
Laboratory tests were conducted to determine
whether coccinellid larvae and adults could access
September 2002
Stuart et al: Lady Beetles as Predators of Diaprepes
and prey upon intact D. abbreviatus egg masses
that had been oviposited between strips of wax
paper or between citrus leaves. The first test con-
sisted of 18-20 replicates per species in which in-
dividual first instar coccinellid larvae that had
been fed E. kuhniella eggs for 24-48 h post eclo-
sion were transferred to a plastic Petri dish con-
taining a D. abbreviatus egg mass laid between
wax paper strips. After 24 h, the wax paper strips
were pulled apart and predation assessed. The
second test consisted of 10-20 replicates per spe-
cies and was similar to the first test except that
the egg mass had been laid between citrus leaves.
Predation was similarly assessed. The third test
used the same methodology and consisted of 19-20
replicates per species in which 4-5 wk old adult
coccinellids from stock colonies were presented
with egg masses that had been laid between wax
paper strips. Predation was similarly assessed.
Egg masses laid between wax paper strips were 2-
5 days old at the time of testing whereas those be-
tween leaves were less than 24 h old.
For the experiments, egg masses oviposited by
the weevils between wax paper strips were pre-
pared by trimming the paper around the masses
so that they would fit into the assay dishes, and
by folding up the edges of the wax paper slightly
to provide entry for the coccinellids without dam-
aging the egg masses themselves or the integrity
of the adhesive applied by the egg-laying weevil.
Excess leaf material around egg masses laid by
weevils between citrus leaves was also trimmed
so that they would fit into assay dishes, again
without damaging the egg masses themselves or
the integrity of the adhesive sealing them be-
tween the leaves. Some of the egg masses laid by
Diaprepes females between citrus leaves ap-
peared to be poorly sealed, such that the leaves
readily separated, exposing the eggs. Only well-
sealed egg masses were used in our experiments.
A greenhouse experiment was conducted to in-
vestigate the accessibility of egg masses to preda-
tion by coccinellid larvae of the three species
under more natural conditions. Fifty D. abbrevia-
tus adults of unknown age were caged on each of
three 3-year old Volkamer lemon trees for 24 h.
The D. abbreviatus adults and cages were then re-
moved and 50 first instar coccinellid larvae (24-36
h old) of each species were released, one species
per tree. Larvae were restricted to trees by an ap-
plication of Tanglefoot (The Tanglefoot Com-
pany, Grand Rapids, MI 40504) around the trunk.
The larvae were removed after 24 h, D. abbrevia-
tus egg masses located, and predation assessed.
Prey Suitability
Two developmental assays were performed to
assess the suitability of D. abbreviatus eggs as
food for H. axyridis, C. sanguine, and 0. v-nigrum
larvae. In the first assay, we conducted 30-32
replicates per species in which individual coc-
cinellid larvae (<24 h old) were transferred to
plastic Petri dishes with water encapsulated in
polymer beads (Entomos, LLC 4445 SW 35th Ter-
race, Suite 310, Gainesville, FL 32608), and ap-
proximately half were fed frozen E. kuhniella
eggs (control diet) and the other half frozen
D. abbreviatus eggs daily until pupation. Addi-
tional water beads were supplied every three
days. Procedures for the second assay were iden-
tical to the first except that 36-39 larvae of each
species were reared individually on the control
diet for four days until they were late second or
early third instar, whereupon approximately half
were transferred to new dishes and fed D. abbre-
viatus eggs until pupation, whereas the other half
continued receiving the control diet. Mortality,
time to pupation, time to adult emergence, and
adult dry weight were recorded for each replicate
in each experiment.
Data Analysis
Statistical analysis used the SAS System for
Windows, release 6.12 (SAS Institute, Inc. 1990).
Count data were transformed prior to analysis
using square root transformations. Comparisons
of means used PROC ANOVA, PROC GLM, and
PROC TTEST. Comparisons of proportions were
conducted using contingency table analysis and
chi-square tests or Fisher's exact tests using
PROC FREQ. Untransformed data are reported
in tables and figures as means and standard er-
rors. Differences were considered significant at
the P = 0.05 level, but sequential Bonferroni ad-
justments of critical values were used as indi-
cated below to maintain error rates at the stated
values when multiple comparisons were made
within experiments (Rice 1989).
RESULTS
Prey Acceptability
In the first choice test in which first instar coc-
cinellid larvae were offered exposed Diaprepes
eggs and an excess of wax moth eggs, C. san-
guinea consumed Diaprepes eggs in 15 of 24 rep-
licates, H. axyridis in 15 of 24 replicates, and
0. v-nigrum in 13 of 25 replicates. There was no
significant difference among coccinellid species in
the proportion of replicates in which weevil eggs
were consumed (X2 = 0.749, df= 2,P = 0.688). Sim-
ilarly, there was no significant difference among
beetle species in the number of weevil eggs that
were consumed (ANOVA, F = 1.610, df = 2, 70,
P = 0.2074; Fig. 1).
In the second choice test in which coccinellid
adults were offered exposed Diaprepes egg
masses and an excess of moth eggs, adults of each
of the three species consumed Diaprepes eggs in
Florida Entomologist 85(3)
2.0
+ 1.5
Ccc
o c 1.0
6 E illlMli|i
Z 0.5
z o.si iiiiiiiiiiiiB
0.0
Cycloneda Harmonia Olla
sanguinea axyridis v-nigrum
Fig. 1. Comparison of the number of Diaprepes eggs
consumed by first instar coccinellid larvae when offered
10-19 D. abbreuiatus eggs and an excess of flour moth
eggs. The P-value is from an ANOVA (see text).
19 of 20 replicates. The number of eggs consumed
was not recorded. In the third choice test in which
third instar coccinellid larvae were offered 10
D. abbreviatus neonates and an excess of moth
eggs, all three species ate at least some Diaprepes
neonates in all replicates, and there was no signif-
icant difference in the number of neonates con-
sumed among coccinellid species (ANOVA, F =
0.10, df = 2, 55, P = 0.9055; Fig. 2). In the fourth
choice test in which coccinellid adults were of-
fered 10 A. gossypii and 10 D. abbreviatus larvae,
all three species ate more Diaprepes larvae than
aphids (Fig. 3). Based on ANOVA, the main effect
for coccinellid species was significant (F = 4.60, df
= 2, 82, P = 0.128) with 0. v-nigrum consuming
less than the other two species, which did not dif-
fer. The main effect for prey species was also sig-
- 9-
CO
+ 8-
67
6.
Cycloneda Harmonia
sanguinea axyridis
Olla
v-nigrum
Fig. 2. Comparison of the number of Diaprepes neo-
nates consumed by third instar larvae of the three coc-
cinellid species when offered 10 neonates and an excess
of flour moth eggs. The P-value is from an ANOVA (see
text).
A. gossypii D. abbreviatus
Fig. 3. Comparison of the number of Diaprepes neo-
nates versus the number of A. gossypii consumed by
adults of the three coccinellid species. ANOVA indicates
that more Diaprepes were consumed than aphids, that
0. v-nigrum consumed less than the other two predator
species, which did not differ, and that the interaction be-
tween predator species and prey species was not signif-
icant (see text).
nificant (F = 24.15, df = 1, 82, P = 0.0001) with
more Diaprepes being consumed than aphids; and
the interaction between predator species and
prey species was not significant (F = 1.95, df = 2,
82, P = 0.1489). However, in this experiment the
Diaprepes larvae tended to stay on the bottom of
the dishes whereas the aphids often moved up the
sides and onto the lids, and this differential dis-
tribution of prey types might have contributed to
the different consumption rates.
Prey Accessibility
When Diaprepes egg masses oviposited be-
tween wax paper strips were offered to first instar
coccinellid larvae, C. sanguinea successfully pen-
etrated between the strips and preyed on eggs in
4 of 20 replicates, H. axyridis in 10 of 20 repli-
cates, and 0. v-nigrum in 7 of 18 replicates. For
egg masses between citrus leaves, C. sanguinea
larvae successfully penetrated between the leaves
P = 0.9055
t/13R T
September 2002
Stuart et al: Lady Beetles as Predators of Diaprepes
and preyed on eggs in 3 of 10 replicates,
H. axyridis larvae in 7 of 19 replicates, and
0. v-nigrum larvae in 12 of 20 replicates. There
were no significant differences among coccinellid
larvae of different species in the proportion of egg
masses that were preyed upon for wax paper (2 x
3 contingency table, x2 = 3.978, df = 2, P = 0.137),
leaves (2 x 3 contingency table, x2 = 3.239, df= 2,
P = 0.198), or for both substrates pooled (2 x 3 con-
tingency table, x2 = 5.255, df = 2, P = 0.072). Also,
pooling the data for larvae of all three predator
species, there was no significant difference for
wax paper versus citrus leaves in the proportion
of egg masses that were preyed upon (2 x 2 contin-
gency table, x2 = 0.835, df= 1, P = 0.361). Coccinel-
lid larvae successfully preyed on 36.2% of egg
masses sealed in wax paper, 44.9% sealed be-
tween citrus leaves, and 40.2% overall.
When coccinellid adults were offered egg
masses sealed between wax paper strips, C. san-
guinea penetrated between the strips and preyed
on eggs in 0 of 20 replicates, H. axyridis in 1 of 20
replicates, and 0. v-nigrum in 4 of 19 replicates,
for an overall success rate of 8.5%. The largest dif-
ference in this experiment, that between C. san-
guinea and 0. v-nigrum, was borderline signi-
ficant (Fisher's exact test, P = 0.047), but this re-
sult was rendered nonsignificant when sequential
Bonferroni adjustments of critical values were
made with respect to the total number of compar-
isons within the experiment (n = 3; Rice 1989).
Therefore, again, no significant difference among
beetle species was evident.
In the greenhouse experiment, relatively few
egg masses were laid on the lemon trees but first
instar larvae of all three coccinellid species suc-
cessfully preyed on some of them. C. sanguine
preyed on 1 of 5 egg masses, H. axyridis on 2 of 10
egg masses, and 0. v-nigrum on 2 of 7 egg masses,
for an overall predation rate of 22.7%. None of the
differences among predator species in this exper-
iment was significant (Fisher's exact tests, P >
0.05). Thus, larvae of all three coccinellid species
were capable of locating, accessing, and preying
upon Diaprepes egg masses.
Prey Suitability
Coccinellids reared on Diaprepes eggs gener-
ally did worse than those reared on flour moth
eggs. Significantly fewer larvae of each coccinellid
species survived to adulthood when reared on an
exclusive diet of Diaprepes eggs than when fed
moth eggs, adult dry weight was significantly re-
duced, and developmental time was significantly
extended (Table 1). Coccinellid larvae of each spe-
cies reared on Diaprepes eggs from the third in-
star onward survived to become adults as well as
those reared entirely on flour moth eggs, but
adult weight was still significantly lower and de-
velopment time significantly longer (Table 1).
DISCUSSION
This study demonstrates that coccinellid lar-
vae and adults are potentially important preda-
tors of Diaprepes abbreviatus eggs and neonate
larvae. The frequent consumption of Diaprepes
eggs and neonates by coccinellid larvae and adults
in these time-limited laboratory tests when other
acceptable food was present and abundant indi-
cates that these prey items are readily accepted by
these predators. Moreover, young coccinellid lar-
vae were especially capable of accessing and prey-
ing upon Diaprepes egg masses laid between wax
paper strips or citrus leaves; and coccinellid adults
exhibited this ability to a lesser extent. Diaprepes
neonates drop to the soil soon after hatching
(Woodruff 1985; McCoy 1999) and might have lim-
ited exposure to coccinellid predation in the can-
opy but predation pressure from coccinellids and
other species could be an important factor select-
ing for the timing of egg hatch, neonate escape
from leaf envelopes, and neonate drop.
The assays in this study provide little indica-
tion of differences among the three coccinellid
species that might suggest that one is a more ef-
fective predator of Diaprepes eggs or neonates
than the others. Only one of the experiments on
prey acceptability, experiment 4, revealed any
species differences. In this experiment, coccinel-
lid adults were offered a choice between aphids
and Diaprepes neonates, and 0. v-nigrum ate less
of both prey items than the other two species,
which did not differ. No differences in prey acces-
sibility or suitability were detected. Similar ex-
periments with larger sample sizes might reveal
differences, but the present data suggest that
these would be minor. Potential differences among
these species in terms of their foraging behavior
in citrus groves or their relative abundances
might make one species more effective than the
others under field conditions.
The differential predation success exhibited by
coccinellids in this study when presented with ex-
posed eggs versus intact egg masses sealed be-
tween wax paper strips or citrus leaves indicated
that egg masses laid between leaves are protected
to some extent from predation by coccinellids.
However, the success of some coccinellid larvae
and adults in penetrating intact egg masses and
preying on eggs indicates that this protection is
far from absolute. Indeed, coccinellid larvae pen-
etrated and preyed on sealed egg masses in over
40% of replicates whereas coccinellid adults did
so in 8.5%. The penetration of intact egg masses
by coccinellids suggests that these predators are
responding to chemical cues associated with egg
masses. Similar responses have been documented
for coccinellids toward other prey (Obata 1986;
Hattingh & Samways 1995).
Jaffe et al. (1990) noted that first instar Dia-
prepes larvae were somewhat repellent to various
Florida Entomologist 85(3)
TABLE 1. COMPARISONS OF THE RESULTS OF THE DEVELOPMENTAL ASSAYS IN WHICH THE THREE COCCINELLID SPECIES
WERE REARED FOR EITHER THEIR ENTIRE DEVELOPMENT OR FROM THE THIRD INSTAR ONWARD ON A DIET OF
EITHER DIAPREPES EGGS OR FLOUR MOTH EGGS.
Complete development Flour moth eggs Diaprepes eggs
Survival n % n % Statistical comparison
Cycloneda 16 68.8 15 13.3 2 = 44.771, df = 1,P < 0.001
Harmonia 16 100.0 14 42.9 X2 = 32.604, df = 1, P < 0.001
Olla 16 100.0 16 37.5 X2 = 39.062, df = 1, P < 0.001
Adult weight (g) Mean SE Mean SE
Cycloneda 3.1 0.15 1.6 0.45 F = 16.268, df= 1, 11, P = 0.002
Harmonia 9.9 0.31 5.1 0.45 F = 71.468, df= 1, 20, P < 0.001
Olla 6.0 0.19 3.0 0.30 F = 44.771, df= 1, 20, P < 0.001
Development time (d) Mean SE Mean SE
Cycloneda 9.8 0.64 20.0 1.40 F = 58.259, df= 1, 14, P < 0.001
Harmonia 9.3 0.17 20.0 0.52 F = 672.364, df= 1, 20, P < 0.001
Olla 8.1 0.11 14.1 0.51 F = 274.067, df= 1, 21, P < 0.001
Development from 3rd instar
Survival n % n %
Cycloneda 18 100.0 18 83.3 X2 = 2.789, df = 1, ns
Harmonia 18 100.0 20 85.0 X2 = 2.250, df = 1, ns
Olla 19 94.7 20 80.0 X2 = 2.282, df = 1, ns
Adult weight (g) Mean SE Mean SE
Cycloneda 3.9 0.14 2.1 0.12 F = 91.546, df= 1, 31, P < 0.001
Harmonia 10.8 1.31 6.1 0.18 F = 185.236, df= 1, 33, P < 0.001
Olla 6.2 0.21 2.9 0.11 F = 185.184, df = 1, 32, P <0.001
Development time (d) Mean SE Mean SE
Cycloneda 9.9 0.36 11.5 0.35 F = 9.513, df = 1, 32, P = 0.04
Harmonia 10.8 0.10 14.1 0.28 F = 94.119, df= 1, 33, P < 0.001
Olla 8.2 0.09 10.1 0.31 F = 39.601, df= 1, 32, P < 0.001
ant predators and that eggs less than 48 h old
were often ignored by ants. Pavis et al. (1992) ex-
tracted and identified the chemicals apparently
responsible for the repellency of the larvae and
provided further evidence for their role in larval
defense. Repellency of Diaprepes larvae to coc-
cinellids was not observed in this study and was
not evident in the experimental results. More-
over, as indicated by our leaf and greenhouse ex-
periments, eggs less than 48 h old were not
ignored by these predators. Thus, the chemical
defenses of the larvae and apparent defenses of
the eggs might only be applicable to certain taxa
of predators. Defenses targeted toward ants
would seem especially appropriate since ants are
evidently the primary predators of various life
stages of Diaprepes including egg masses and
neonate larvae (Whitcomb et al. 1982; Richman
et al. 1983a,b; Tryon 1986; Jaffe et al. 1990;
McCoy 1999; Stuart et al. 2001). Deterrence of
coccinellids from attacking alfalfa weevil larvae
has been attributed to the defensive wriggling of
the larvae (Kalaskar & Evans 2001) but it is un-
clear whether wriggling might contribute to de-
fense for Diaprepes larvae or whether alfalfa
weevil larvae also possess chemical defenses.
Seasonal cycles of coccinellid and Diaprepes
abundance in Florida citrus indicate considerable
periods of overlap when predation is likely to oc-
cur. The three coccinellid species are present in
groves year-round but are most abundant during
periods of flowering and flushing, especially
spring and fall. Flowers are attractive as sources
of pollen and nectar for all three species, and most
of their primary homopteran prey species require
newly expanding leaves for their growth and re-
production. Hot weather in summer is typically
associated with low food availability and a major-
ity of individuals of all three species aestivate
during these periods (Michaud, unpublished). In
September 2002
Stuart et al: Lady Beetles as Predators of Diaprepes
central Florida, Diaprepes adults emerge in the
spring and generally show a sharp peak in abun-
dance sometime from April through June. How-
ever, emergence continues throughout the summer,
and adults can remain abundant through mid
November (McCoy, unpublished). Egg-laying oc-
curs throughout this period, and neonate drop
has been recorded from early July through early
December (Nigg et al. in press). Diaprepes adults
feed voraciously on newly-expanding leaves but
lay egg masses between mature leaves (Woodruff
1985; McCoy 1999). Given their seasonal activity
patterns and locations in trees, it is likely that
coccinellids have frequent opportunities to prey
on D. abbreviatus egg masses and perhaps on
neonates as well.
The timing of Diaprepes egg hatch, neonate
escape from sealed leaf envelopes, and neonate
drop to the soil surface could have an important
impact on the exposure of neonates to various
predators. Jones & Schroeder (1983) found that a
considerable period often elapsed between egg
hatch and neonate escape from sealed leaf enve-
lopes, estimated average larval age at the time of
neonate drop to be about 48 h, and found that ne-
onates dropped between 1100 and 2400 h. Accord-
ing to Richman et al. (1983), ant foraging on the
soil surface during this time period is relatively
low compared to early morning hours. Thus, the
timing of neonate drop could be an adaptation to
avoid peak ant foraging periods. The diurnal
activity patterns of coccinellids in Florida citrus
groves have not been studied but these species
appear most active during daylight hours
(Michaud, unpublished), and neonate drop late in
the day might also enable avoidance of these
predators. However, since Diaprepes neonates ap-
pear to remain within sealed leaf envelopes for
relatively lengthy periods, there is a strong possi-
bility that coccinellids will encounter neonates
rather than eggs when penetrating leaf enve-
lopes. Additional research on the activity patterns
of predators, the factors that stimulate egg hatch
and neonate drop, and the conditions that pro-
mote neonate survival in the canopy, on the soil
surface, and below ground is necessary for a more
thorough understanding of how these factors
might shape D. abbreviatus life history and sur-
vival strategies.
Overall, our predation and developmental as-
says suggest that Diaprepes eggs and neonates
could be a frequent and highly acceptable compo-
nent of a mixed diet for these coccinellid species
in nature. Diaprepes eggs were less suitable prey
for these species than flour moth eggs but the
costs in terms of reduced adult weight and ex-
tended developmental time were relatively small
for larvae reared on this diet from the third in-
star. Similar suboptimal developmental results
have been obtained for various coccinellid species
and some of the aphid species that they fre-
quently consume as prey (Michaud 2000). Thus,
the results of this study, especially for coccinellid
larvae, indicate that Diaprepes egg masses and
neonates will be preyed upon quite readily when
encountered. However, the frequency of such en-
counters and the intensity of predation in nature
remain unknown; and it is unclear to what extent
coccinellids contribute to the natural biological
control of this weevil.
ACKNOWLEDGMENTS
We thank K. Crosby and the USDA-ARS (Ft. Pierce,
FL) for providing some of the D. abbreuiatus egg masses
used in these experiments, H. Nigg for providing ovipos-
iting Diaprepes for leaf and greenhouse assays, and
R. Villanueva and A. Goldarazena for reviewing the MS.
This work was supported by the Florida Agricultural
Experiment Station and grants from USDA-APHIS-
PPQ and the Florida Citrus Producers Research Advi-
sory Council and approved for publication as Journal
Series No. R-08580.
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Florida Entomologist 85(3)
Serrano & Lapointe: Mass Rearing Maconellicoccus hirsutus
EVALUATION OF HOST PLANTS AND A MERIDIC DIET FOR REARING
MACONELLICOCCUS HIRSUTUS (HEMIPTERA: PSEUDOCOCCIDAE)
AND ITS PARASITOIDANAGYRUS KAMALI (HYMENOPTERA: ENCYRTIDAE)
MIGUEL S. SERRANO AND STEPHEN L. LAPOINTE
USDA-ARS, U. S. Horticultural Research Laboratory, 2001 South Rock Road, Ft. Pierce FL 34945
ABSTRACT
Biological control programs of Maconellicoccus hirsutus (Green), in the Caribbean have re-
lied on Japanese pumpkins and sprouted potatoes as hosts for rearing both the mealybug
and its parasitoids. However, seasonal shortages of these substrates have necessitated that
others be found with equal or better qualities for sustaining large mealybug populations. In
this paper, we report experiments comparing mass-rearing M. hirsutus on acorn squash
(Cucurbita pepo L. var. 'Turbinata'), chayote (Sechium edule [Jacques]), and prickly pear,
Opuntia ficus-indica [L.]) with Japanese pumpkin and sprouted potato. In addition, a simple
meridic diet based on canned pumpkins was developed and compared. Acorn squash pro-
duced large quantities of females (up to 1,300 per squash) with a life cycle and reproductive
potential equal to that of mealybugs reared on Japanese pumpkin. Parasitoids reared on
these mealybugs developed normally and had a female-biased sex ratio similar to those
reared on mealybugs on Japanese pumpkins or potato sprouts. Development of M. hirsutus
reared on chayote and prickly pear was delayed by 1.5-5.0 days compared to that of mealy-
bugs reared on Japanese pumpkins. Mealybugs on these substrates produced parasitoids
with prolonged developmental times and male-biased sex ratios. On diet, development and
reproduction ofM. hirsutus was possible only for 3 to 4 consecutive generations. Mealybugs
with longer developmental time, lower survival, and smaller ovisacs with lower percentage
eclosion were obtained. Parasitoids reared from these mealybugs did not possess desirable
characteristics for biological control. The developmental rate of adult parasitoids increased
linearly with that of female hosts depending on the quality of the rearing substrate for the
mealybugs.
Key Words: Pink hibiscus mealybug, artificial diet, invasive species, biological control
RESUME
Desde que invadio el area del Caribe, la cochinilla rosada del hibisco, Maconellicoccus hirsu-
tus (Green) se ha criado masivamente en calabaza japonica o en papa en germinaci6n. Sin
embargo, la calabaza japonica no se puede producer a lo largo de todo el ano en la region del
caribe por lo cual se necesitan alternatives para la producci6n del insecto con el prop6sito de
producer parasitoides para su control biol6gico. En este articulo presentamos resultados de
experiments en los que comparamos la crianza masiva de la cochinilla rosada en calabacin
(Cucurbita pepo L. cv. 'turbinata'), Chayote (Sechium edule [Jacques]) y en nopal Opuntia
ficus-indica (L.) con los substratos tradicionales, calabaza japonica y plantulas de papa.
Tambi6n probamos una dieta meridica con base en puree de calabaza y sacarosa. Se produ-
jeron mas de 1,300 hembras de cochinilla por cada calabacin, con un tiempo de desarrollo y
reproducci6n iguales a los de las cochinillas obtenidas en los substratos tradicionales. Los
parasitoides que emergieron de estas cochinillas se desarrollaron normalmente y presenta-
ron una relaci6n de sexos similar a los obtenidos de papa y calabazajap6nica. Las cochinillas
que se criaron en chayote y nopal tuvieron un desarrollo 1.5 a 5.0 d mas lento y produjeron
parasitoides con tiempo de desarrollo prolongado y relaci6n de sexos sesgada hacia los ma-
chos. A pesar que el tiempo de desarrollo de las cochinillas fue mas prolongado y la supervi-
vencia y fertilidad fueron menores, fue possible producer la cochinilla rosada durante varias
generaciones completes nuestra simple dieta artificial, aunque los parasitoides que se obtu-
vieron de ellas no presentaron buenas caracteristicas para el program de control biol6gico.
Las tasas de desarrollo de ambos sexos del parasitoide fueron mas lentas cuando las cochi-
nillas donde se criaron se obtuvieron de substratos de menor calidad.
Translation provided by author.
The pink hibiscus mealybug Maconellicoccus although successful biological control programs
hirsutus (Green) continues to invade new areas of in the region appear to have slowed its spread.
the Caribbean, Central, and North America (Sa- M. hirsutus was unknown in the Western Hemi-
garra & Peterkin 1999, Michaud & Evans 2000) sphere (with the exception of Hawaii) prior to its
Florida Entomologist 85(3)
discovery on Grenada in 1994 and on Trinidad,
St. Kitts and Nevis in 1995 (Williams 1996). After
colonizing the Eastern Caribbean (Cross & Noyes
1998), the U.S. Virgin Islands, and Puerto Rico,
M. hirsutus has spread to the Americas including
California (Anonymous 1999), Mexico, Central
America, and Guyana in South America (Pollard
1998). The economic risk of invasion to U.S. agri-
culture has been estimated at $750 million per
year (Moffitt 1999) due mainly to its wide host
range that includes over 125 plant species (Ghose
1972). The largest risk would be to ornamental
crops, followed by vegetables, citrus, grapes, and
avocados. Biological control programs for the U.S.
could be implemented for as little as $500,000 per
year during the initial 3 years of invasion (Moffitt
1999).
Biological control programs have been estab-
lished in the Eastern Caribbean region with the
introduction of the coccinellids, Cryptolaemus
montrouzieri Mulsant and Scymnus coccivora
Ramkrisna, and the encyrtid parasitoid, Anagy-
rus kamali Moursi (Cross & Noyes 1998). The
U.S. Department of Agriculture introduced the
encyrtid Gyranusoidea indica Schaffe, Alam &
Agarwal to the area, including the U.S. Virgin Is-
lands and Puerto Rico (Anonymous 1997). These
two parasitoid species have been continuously
produced since 1997 by USDA-APHIS with sup-
port from USDA-ARS and local Departments of
Agriculture in laboratories on St. Thomas and Pu-
erto Rico (Anonymous 1997, Serrano et al. 2001).
To mass-produce parasitoids, M. hirsutus is
reared on the fruits of various species of cucurbits
(Babu & Azam 1987a,b, Babu & Azam 1989, Mani
& Thontadarya 1989) or on potatoes (Solanum
tuberosum L.) (Sagarra & Vincent 1999). Of these,
the preferred host has been Japanese pumpkin
(Cucurbita moschata Duchesne cv. 'Chirimen')
due to its ribbed rinds and characteristic warted
surface, which provide large settling areas for
mealybugs (Meyerdirk and Newell 1979). How-
ever, seasonal shortages of produce, and difficul-
ties in maintaining a continuous supply of
etiolated sprouts of potato, threaten production
and increase costs. We looked at alternate rearing
hosts that are available year-round. Acorn squash
(Cucurbita pepo L. cv. 'Turbinata') and chayote
fruits (Sechium edule Jacques [Schwartz]) are rel-
atively inexpensive and available throughout the
year in supermarkets on St. Croix. These fruits
have skins that resemble that of Japanese pump-
kins. Natural infestations of pink hibiscus mealy-
bug were observed on prickly pear Opuntia ficus-
indica (L.) Miller on St. Croix. The flattened stem
segments of this cactus, known as cladodes or
pads (Mizrahi et al. 1997), have traditionally
been used to mass-produce mealybug species
such as Dactylopius coccus Costa or D. opuntiae
Cockerell for industrial production of dyes
(Mizrahi et al. 1997).
These 3 hosts were compared with pumpkins
and sprouted potatoes as well as with a simple
meridic diet that permitted growth and reproduc-
tion of the mealybug. To examine the effect of host
quality on parasitoid development, we reared A.
kamali from mealybugs produced on each rearing
substrate and the diet.
MATERIALS AND METHODS
Field Production of Japanese Pumpkins
Japanese pumpkins were grown at the USDA-
ARS Research Station on St. Croix, U.S. Virgin
Islands. From August 1997 to September 2000,
monthly plantings of 0.28 ha were made with the
intent of maintaining a continuous supply of
pumpkins. Seeds (American Takii, Salinas, CA)
were hand-sown every 1.2 m in 61 m rows with
2.0 m between rows. Pumpkins were fertilized
with 680 kg.ha-'of 15-10-10 Green Crop (Ochoa
Fertilizer, Guanica, PR) at 15 and 30 d after
planting (dap). In addition, foliar applications of
6.1 kg.ha- of Nutri-Leaf (Miller Chemical Fertil-
izer Corp., Hanover, PA) were made at 45 and 60
dap. Drip irrigation was provided when neces-
sary. Pumpkins suitable for mealybug rearing
(>500 g) were hand-harvested at 90-110 dap.
Production of M. hirsutus
Japanese pumpkins infested with female
M. hirsutus and ovisacs were incubated at 27 +
1C in a crawler collection box made with a mod-
ified oven (Isotemp incubator, model 655G, Fisher
Scientific, Pittsburgh, PA). Pumpkins were dis-
tributed around a piece of white paper on the top
shelf of the oven. A microscope illuminator was
vertically inserted through the exhaust port on
top of the oven and directed towards the piece of
paper. The lamp was adjusted to 6 V at a distance
of 18 cm above the paper, to produce a round spot
of light 4 cm in diameter. Upon emergence, crawl-
ers were attracted to and concentrated in the spot
of light. The sheet of paper with crawlers was
taken out and replaced daily. Twenty-four-h-old
crawlers were distributed over the surface of
fresh Japanese pumpkins using a camel's hair
brush to maintain age-specific colonies. Infested
pumpkins were maintained on 30 x 45 cm trays in
an incubator at 27 + 2C and 70 10% RH in the
dark.
For comparison of rearing substrates, medium
to large-size Japanese pumpkins (300-700 g) were
brought to the laboratory, washed by soaking for a
minimum of 3 h in tap water, and air-dried over-
night. Freshly harvested pumpkins were used for
all experiments. Sprouted potatoes were pro-
duced by placing 20 medium-size seed potatoes
(Ryan Potato Company, East Grand Forks, MN)
on a tray with a 2 cm layer of Pro-Mix PGX (Pre-
September 2002
Serrano & Lapointe: Mass Rearing Maconellicoccus hirsutus
mier Horticulture, Inc., Red Hill, PA) for several
days in a dark room at 25 + 2C and 80 10% RH
to induce etiolation. Once sprouts reached about 5
cm in length, potatoes were cleaned and trans-
ferred to trays for infestation with M. hirsutus.
Acorn squash (Big Chuy & Sons, Inc., Nogales,
AZ) were purchased from St. Croix supermarkets.
Green-skinned fruits that weighed about 450 to
600 g were used. Small chayote gourds, approxi-
mately 250 g each, were also obtained from local
supermarkets. Uninfested, spineless pads of
prickly pear were collected from plants found at
the USDA-ARS Research Station. All fruits and
cladodes were washed and initially infested with
crawlers as previously described. After the first
generation was completed, the cycle was repeated
using only crawlers produced on the same host to
maintain a stock of at least three generations of
M. hirsutus on each host.
Depending on their size, 6 to 8 Japanese pump-
kins, acorn squash or prickly pear pads, and 10-12
chayotes or sprouted potatoes were infested per
tray per generation. Observations were made on
the production of mature females (with ovisacs)
on each host and the 'shelf life' of infested hosts,
i.e., their ability to sustain at least one generation
of M. hirsutus before physically degrading.
A meridic diet based on canned pumpkin and
sucrose was also tested. Thirty g of canned pump-
kin (Libby's Solid Pack Pumpkin, Nestle Food
Company, Glendale, CA) were dissolved in 100 ml
of distilled water by stirring for 3 h. At the end of
the stirring period, 30 g of sucrose and 0.1 g of
methyl-parabenzoic acid were added and the pH
was adjusted to 7.5 by adding potassium hydrox-
ide. The medium was poured into the tops of 5 cm
diam plastic petri dishes, in a laminar flow hood
to avoid microbial contamination. Parafilm M
(American Can Co., Greenwich, CT) was stretched
over each dish and wrinkled to provide a rough
surface for mealybug settling. Crawlers or early
second instar nymphs were placed on the para-
film with a camel's hair brush. The petri dishes
were closed, inverted, and placed in the growth
chamber at the same environmental conditions
described previously.
Development and Reproduction of M. hirsutus
After 3 M. hirsutus generations on each rear-
ing substrate, a cohort of 100 second-instar
nymphs was removed from each and placed in
groups of 10 on fresh original substrate, including
the artificial diet. Each replicate of 10 nymphs on
a substrate was placed in a 20.4 x 20.4 cm PVC
tube covered at one end with a fine mesh organdy
cloth for ventilation. The other end of the tubes
was attached with silicone to flat trays (60 x 30
cm) and placed in a rearing room at 26 2C, 60
+ 10% R.H. and complete darkness. Each tube
cage constituted a replicate. Tube cages were
checked daily under a magnifying lens. When
male nymphs reach the fourth instar, they pos-
sess noticeable wing pads (Ghose 1971) and make
a waxy 'cocoon' while remaining above a cluster of
females. When these 'pupae' were observed, the
number of days to this stage was recorded. The
number of days to the last molt for the remaining
female nymphs (pre-ovisac females) was also re-
corded to calculate female developmental time. If
not enough males were observed inside the tube
cages, five 6-h-old males from age-specific colo-
nies on their respective host were added to each
tube cage to insure enough males for fertilization
of all available females. When ovisacs were noted,
tube cages were opened and the number of live fe-
males counted to calculate percentage survival.
After this, 30 ovisacs were taken from females on
each rearing substrate. Each ovisac was isolated
in a gelatin capsule (replicate) and incubated at
27 1C. Gelatin capsules were checked daily.
When crawlers were first noticed, the number of
days to eclosion was recorded for each ovisac. Two
weeks later, all gelatin capsules were placed in a
freezer at -5C for 24 h. Crawlers and unhatched
eggs were counted under a stereoscope for each
ovisac to estimate its individual percent eclosion.
Data for developmental time for males and fe-
males, as well as the number of eggs per ovisac,
days to eclosion, and percent eclosion were ana-
lyzed by ANOVA and Ryan-Einot-Gabriel-Welsch
Multiple F test (SAS Institute 1999). Percentage
data were transformed to arcsine and days to
eclosion were transformed to In (x) to stabilize the
variance.
Effect of Host Quality on Parasitoid Development
The parasitoid A. kamali was reared for one
generation on M. hirsutus obtained from each
host described above. One hundred third instar
female M. hirsutus nymphs from colonies reared
for at least 3 generations on each substrate or 2
generations on artificial diet were transferred
with a camel's hair brush onto a fresh substrate,
and placed in tube cages in a rearing room at 26 +
2C, 60 10% R.H. and a photoperiod of 12:12
(L:D). Five tube cages (replicates) were used for
each rearing substrate. The following morning, 20
A. kamali adults were released in each tube cage
and allowed to forage and oviposit for 24 h. Honey
streaks and a water-soaked cotton ball were pro-
vided as additional food for the parasitoids. After
this period, all adult parasitoids were removed
from the cage using a vacuum aspirator. Tube
cages were checked daily thereafter for emer-
gence of adult parasitoids. Developmental time
and gender of each adult parasitoid were recorded
and analyzed by anova and Ryan-Einot-Gabriel-
Welsch Multiple F test (SAS Institute 1999). Par-
asitoid sex ratios (F/M) were transformed to arc-
sine. Sex ratios from each substrate were tested
Florida Entomologist 85(3)
against a 1:1 (unbiased) sex ratio using an un-
paired t-test. To study the relationship between
the quality of M. hirsutus on the developmental
rate of the parasitoid, linear regression models
(SAS Institute 1999) were fitted to the develop-
mental rate (day-1) of female and male parasitoids
and the developmental rate of female hosts pro-
duced on each rearing substrate.
RESULTS
Field Production of Japanese Pumpkins
Production peaked during September of each
year, but was also high from May through August
of the three-year period (Fig. 1). A noticeable de-
crease in the number and size of pumpkins har-
vested began in October and continued until
April. Production of acceptable size fruits (>500 g)
increased again during May. This repetitive pat-
tern was probably caused by variations in envi-
ronmental factors such as photoperiod or
temperature.
Production of M. hirsutus
Japanese pumpkins produced between 700
and 1500 mature M. hirsutus females per pump-
kin per generation. Individual pumpkins regu-
larly produced up to 2 complete M. hirsutus
7,000
6,000
5,000
4,000
-c
a 3,000
2,000
1,000
0
generations (>60 d) before deteriorating. Sprouted
potatoes produced between 150 and 350 females
per generation over 40 d. Each acorn squash sus-
tained between 700 and 1,300 adult female mea-
lybugs and lasted about 50 d or one generation.
Chayote fruits lasted only for one generation of
M. hirsutus (30 d) before sprouting or decaying.
Once sprouted, infested chayotes decayed within
a few days. Some chayotes started to decay as
early as 15 to 20 d after infestation. Prickly pear
pads lasted for >60 d and sustained between 90
and 100 M. hirsutus females per pad per genera-
tion. Complete development from the crawler
stage to reproductive females was observed on the
meridic diet. Between 30 and 50 females were ob-
tained per petri dish containing 100 ml of diet.
The diet typically lasted 7-14 d, before microbial
contamination was apparent.
Development and Reproduction of M. hirsutus
M. hirsutus males developed faster to the
fourth instar (pupa) on Japanese pumpkins than
on any other host tested (Table 1). On acorn
squash and sprouted potatoes, it took males on
average 0.8 to 1.4 d longer than on Japanese
pumpkins to molt to the 'pupal' stage. On chayote
males reached the fourth instar 2.2 d later than
on Japanese pumpkins. On prickly pear and diet,
males developed 4.5 and 5.1 d later, respectively,
-1 I- 1
1997 1998 1999 2000
1 9 9 ----- ----. ---..-.----........
ASOND JFMAM J J ASOND JFMAMJ J ASOND JFMAMJ J
Month
Fig. 1. Monthly production of Japanese pumpkins suitable for mealybug rearing (<500 g) per hectare from 1997
to 2000 at the USDA, ARS, research station on St. Croix, U.S. Virgin Islands.
September 2002
Serrano & Lapointe: Mass Rearing Maconellicoccus hirsutus
TABLE 1. MEAN ( STD. DEVIATION) DEVELOPMENT TIME FROM CRAWLER TO MALE PUPAE AND ADULT FEMALE, AND
PERCENT SURVIVAL OF M. HIRSUTUS AT THE END OF THE FOURTH GENERATION ON FIVE PLANT SUBSTRATES
OR A MERIDIC DIET.
Development time (d)
Host Male Pupa' Adult Female2 Percent
Japanese Pumpkin 16.2 + 1.2 a 29.6 + 1.5 a 98.0 + 4.2 a
Sprouted Potato 17.4 + 1.5 b 30.1 + 1.0 a 97.0 + 9.5 a
Acorn Squash 17.0 + 0.8 b 29.5 + 0.9 a 95.0 + 5.3 a
Chayote 18.4 + 1.1 c 31.6 + 1.6 b 92.0 + 6.3 a
Prickly pear 20.7 + 1.6 d 35.3 + 2.2 c 82.0 + 10.3 b
Meridic diet 21.3 + 1.6 d 36.9 + 2.1 d 72.0 + 9.2 c
Means within columns followed by the same letter are not significantly different by Ryan-Einot-Gabriel-Welsch Multiple F test (a = 0.05) after a sig-
nificant ANOVA.
F = 50.8; df= 5, 52; P < 0.01.
F = 123.2; df= 5,54; P < 0.01.
'F = 17.3; df = 5, 54 P < 0.01.
than on Japanese pumpkins. The developmental
time for females was similar when reared on Jap-
anese pumpkins, sprouted potatoes, and acorn
squash, at roughly 30 d. Female development
time was significantly slowed by 1.6 d on chayote,
5.3 d on prickly pear pads, and 6.9 d on the diet
(Table 1). Survival to the adult stage (>90%) was
significantly higher on Japanese pumpkins,
sprouted potatoes, acorn squash, and chayote
than on prickly pear pads (81.3%). It dropped to
73.2% on the diet (Table 1).
Females reared on all substrates, including the
artificial diet, produced 1 ovisac. Shortly after
producing the ovisac, females become inactive
and die. The number of eggs per ovisac varied de-
pending on the substrate. Females reared on Jap-
anese pumpkin and acorn squash produced
significantly more eggs per ovisac than females
reared on the other substrates evaluated (Table
2). Females reared on sprouted potatoes and
chayote had significantly more eggs per ovisac
than those from the diet. However, the number of
eggs per ovisac from females obtained on prickly
pear pads and chayote was not significantly dif-
ferent. Females reared on the meridic diet pro-
duced the smallest ovisacs, with 36.4% fewer eggs
than ovisacs produced on Japanese pumpkins
(Table 2).
The rearing substrate affected the incubation
time ofM. hirsutus eggs (Table 2). Eggs from mea-
lybugs reared on Japanese pumpkin and acorn
squash incubated faster than those from the
other rearing substrates. Eggs from mealybugs
reared on diet developed a full day later than
those from Japanese pumpkin and acorn squash.
The highest percentage eclosion was observed for
ovisacs from females reared on Japanese pump-
kin, but it was not significantly different from the
percentage eclosion of ovisacs from females
reared on acorn squash and sprouted potato. The
lowest percentages of eclosion were obtained from
females reared on prickly pear and diet (Table 2).
TABLE 2. NUMBER OF EGGS, DAYS TO ECLOSION AND PERCENT ECLOSION FOR OVISACS OBTAINED FROM FEMALES OF
M. HIRSUTUS REARED ON FIVE SUBSTRATES OR A MERIDIC DIET. DATA ARE MEANS + STD. DEVIATION FROM
A SAMPLE OF 30 OVISACS FROM EACH REARING MATERIAL.
Host No. Eggs per Ovisac' Days to Eclosion2 % Eclosion per Ovisac3
Japanese pumpkin 162.5 + 39.2 a 4.6 + 0.6 a 93.2 + 7.7 a
Acorn squash 148.2 + 39.6 a 4.8 + 0.5 a 92.3 + 4.8 ab
Sprouted potato 124.3 + 46.9 b 5.3 + 0.7 b 91.2 + 8.0 ab
Chayote 106.8 + 27.5 bc 5.4 + 0.8 b 87.5 + 7.2b
Prickly pear 84.4 + 25.9 c 5.4 + 0.9 b 76.0 + 11.5 c
Meridic diet 59.2 + 43.1 d 5.8 + 1.1 b 69.8 + 17.0 c
Means within columns followed by the same letter are not significantly different according to Ryan-Einot-Gabriel-Welsch Multiple F test ( = 0.05)
after a significant ANOVA.
F = 31.7; df= 5,174; P < 0.01.
F = 9.2; df= 5,174; P < 0.01.
F = 30.7; df= 5,174; P < 0.01.
Florida Entomologist 85(3)
Effect of Host Quality on Parasitoid Development
There was a significant effect of both rearing
substrate (F = 130.8; df = 11, 5; P < 0.01) and gen-
der of parasitoid (F = 67.0; df = 11, 1; P < 0.01) on
development time ofA. kamali, with no interac-
tion between the two factors (F = 2.1; df = 11, 5;
P = 0.08) (Fig. 2). The developmental times of fe-
male A. kamali from mealybugs reared on Japa-
nese pumpkin, sprouted potato, and acorn squash
were similar at 20.6-20.7 d (Table 3). Develop-
mental time was significantly delayed by 4.3 and
7.8 d for female parasitoids that emerged from
mealybugs reared on chayote and prickly pear, re-
spectively. When the mealybug host was obtained
from artificial diet, the developmental time of fe-
male parasitoids was 13.4 d longer than those ob-
tained from mealybugs reared on either Japanese
pumpkin or acorn squash. The developmental
time for male parasitoids followed a similar pat-
tern. The fastest development, between 18.6 and
18.9 d, occurred on M. hirsutus obtained from
Japanese pumpkin, sprouted potato, and acorn
squash. Male parasitoid development was de-
layed by 5.0-5.7 d when reared on mealybugs
from chayote and prickly pear compared with
0.055
0.050
0.045
0.040
0.035
0.030
0.025 L
0.026
males reared on mealybugs obtained on Japanese
pumpkin. On mealybugs reared on artificial diet,
male A. kamali took almost 29 d to develop, 10 d
longer than on mealybugs from Japanese pump-
kin or acorn squash. Males developed on average
2 d earlier than females on mealybugs obtained
from Japanese pumpkin, sprouted potato, and
acorn squash.
Percentage emergence of adult A. kamali was
between 59 and 62% when the mealybug was
reared on Japanese pumpkin, sprouted potato or
acorn squash. On mealybugs reared on chayote,
prickly pear and diet, the average emergence of
adult parasitoids was significantly reduced to be-
tween one half and one-third of the emergence on
Japanese pumpkin, sprouted potato or acorn
squash (Table 3).
The sex ratio was significantly female-biased
when parasitoids emerged from M. hirsutus
reared on Japanese pumpkin (P > t = 0.02),
sprouted potato (P > t = 0.01) and acorn squash
(P > t = 0.01). It was significantly male-biased on
mealybugs from chayote (P > t = 0.01), prickly
pear (P > t = 0.02), and diet (P > t = 0.02). The sex
ratio of parasitoids obtained from mealybugs pro-
duced on the artificial diet was 4.5 times more
0.028 0.030 0.032
0.034
Developmental rate (d-') of female M. hirsutus
Fig. 2. Relationship between developmental rates of the parasitoidAnagyrus kamali and developmental rate of
females of the host Maconellicoccus hirsutus produced on six rearing substrates.
September 2002
Serrano & Lapointe: Mass Rearing Maconellicoccus hirsutus
TABLE 3. MEAN ( STD. DEVIATION, N = 30) DEVELOPMENTAL TIME, ADULT EMERGENCE AND SEX RATIO OFANAGYRUS
KAMALI DEVELOPED FROM M. HIRSUTUS OBTAINED FROM DIFFERENT REARING MATERIALS.
Developmental Time (d)
Adult
Host mealybug from Females' Males2 Emergence3 (%) Sex Ratio
Japanese Pumpkin 20.6 + 0.3 a 18.7 + 0.4 a 61.6 + 3.0 a 1.8 + 0.2 a
Sprouted Potato 20.7 + 0.2 a 18.6 + 0.2 a 60.8 + 12.1 a 1.7 + 0.0 a
Acorn Squash 20.6 + 0.1 a 18.9 + 0.3 a 58.8 + 10.4 a 1.7 + 0.0 a
Chayote 24.9 + 0.8 b 23.7 + 1.1 b 29.8 + 6.4 b 0.9 + 0.2 b
Prickly pear 28.4 + 0.6 b 24.4 + 2.0 b 32.0 + 7.9 b 0.9 + 0.2 b
Meridic Diet 34.8 + 5.2 c 28.7 + 2.1 c 18.8 + 6.3 b 0.4 + 0.1 b
Means within columns followed by the same letter are not significantly different by Ryan-Einot-Gabriel-Welsch Multiple F test (c = 0.05) after a sig-
nificant ANOVA.
'F = 35.17; df= 5, 24; P < 0.01
"F = 51.60; df = 5,24; P< 0.01.
'F = 26.46; df = 5,24; P < 0.01.
F = 13.19; df= 5, 24; P < 0.01.
male-biased than that of parasitoids from mealy-
bugs reared on Japanese pumpkin (Table 3).
There was a significant linear relationship be-
tween the developmental rate of female M. hirsu-
tus and that ofA. kamali females (F = 116.1; df =
1,4; P > 0.01) and males (F = 31.0; df = 1,4; P <
0.01) (Fig. 2). When M. hirsutus females were ob-
tained from lower quality rearing substrates, i.e.,
those that induced fewer offspring and slower de-
velopment, significantly slower developmental
rates were obtained for both sexes of the parasi-
toid. Rearing substrates that favored faster devel-
opment of M. hirsutus were more likely to produce
A. kamali with faster developmental rates.
DISCUSSION
Development of M. hirsutus was completed on
all host plants tested and the artificial diet. Opti-
mal development, survival, and reproduction oc-
curred not only on the traditionally used
substrates, Japanese pumpkin and sprouted po-
tato, but also on acorn squash. Several other spe-
cies of mealybugs, including the Comstock
mealybug Pseudococcus comstocki (Kuwana), cit-
rus mealybug Planococcus citri (Risso), and
spherical mealybug Nipaecoccus viridis (New-
stead), have been successfully reared on Japanese
pumpkin (Meyerdirk & Newel 1979, Chandler
et al. 1980, Meyerdirk et al. 1988) and sprouted
potatoes (Gothilf & Beck 1966). M. hirsutus has
been routinely produced on Japanese pumpkins
for research purposes and for mass-producing
parasitoids for biological control at the USDA,
ARS Research Station on St. Croix since 1998
(Serrano et al. 2001). Acorn squash, reported here
for the first time for mass rearing M. hirsutus,
produced large numbers of insects with the same
developmental time and survival as those pro-
duced on the two traditionally used hosts. Al-
though prickly pear is a host plant frequently
found with natural infestations of M. hirsutus on
St. Croix, it did not present adequate characteris-
tics for mass-producing mealybugs under our
rearing conditions. Chayote has not yet been re-
ported as a naturally infested host. Mealybugs
could complete their life cycle and reproduce on
these gourds. However, early decay of the fruits
soon after infestation severely reduced its suit-
ability for mass-rearing mealybugs.
It is noteworthy that reproduction and sur-
vival for several generations was obtained on the
crude meridic diet. However, mealybug survival
was lower and development prolonged on this
diet. We do not consider it suitable for mass pro-
duction of mealybugs without further refine-
ments. This medium differs from artificial diets
previously developed for mealybugs (Gothilf &
Beck 1966, Calatayud et al. 1998) in that crude
materials were used. The diet included solid par-
ticles from canned pumpkin material that may
play a role in stylet penetration and the ingestion
processes, i.e., by providing an anchor for salivary
sheaths as in normal intercellular probing (Calata-
yud et al. 1994). Particles in the diet interfered
with filter sterilization. Even with the addition of
preservatives, the medium was prone to bacterial
contamination. Nonetheless, results obtained with
this method are consistent with those obtained on
chemically defined diets. Gothilf & Beck (1966)
found longer developmental times and fewer eggs
per ovisac for female P citri on an artificial diet
versus sprouted potatoes. Calatayud et al. (1998)
found reduced survival and a longer developmen-
tal time for cassava mealybug (Phenacoccus
manihoti Matile-Ferrero) reared on a defined diet
when compared with mealybugs reared directly
on cassava plants.
Developmental time, survival, and sex ratio of
A. kamali obtained in this study are similar to
results from other authors (Sagarra & Vincent
1999) when mealybug colonies were maintained
on sprouted potato. Although not an exhaustive
evaluation of parasitoid quality, the developmen-
tal time and sex ratio of parasitoids obtained from
mealybugs reared on acorn squash were within
the ranges that have been previously published
under similar environmental conditions (Sagarra
& Vincent 1999) and were not significantly differ-
ent from those occurring on the commonly used
host materials.
The quality of the mealybug host affected the
quality of parasitoids. Prolonged developmental
time, lower survival, and a male-biased sex ratio
were obtained forA. kamali reared on M. hirsutus
from chayote and prickly pear. When reared on
mealybugs raised on the artificial diet, female
A. kamali took 14 d longer to develop compared
withA. kamali reared on mealybugs on Japanese
pumpkin. These parasitoids would not be consid-
ered for field releases for biological control. No
evaluations were made, however, of adult parasi-
toid longevity or reproductive potential.
An ideal host for mass rearing M. hirsutus for
production of parasitoids for biological control
programs should have a large surface area for
mealybug settling, long 'shelf life' after infesta-
tion, and produce adults within a short develop-
mental time (~30 d). Adult females should be able
to produce large ovisacs with high percent eclo-
sion. In addition, these hosts should provide mea-
lybugs that allow a short developmental time for
A. kamali and a female-biased sex ratio. Acorn
squash, along with Japanese pumpkin and
sprouted potato, appear to satisfy these require-
ments. During periods of low production of Japa-
nese pumpkins since 1998, parasitoids produced
from M. hirsutus reared on acorn squash at the
USDA, APHIS, PPQ insectary on St. Thomas,
U.S. Virgin Islands have been mass-released for
biological control of pink hibiscus mealybug in the
U.S. Virgin Islands and Puerto Rico (USDA 1999).
ACKNOWLEDGMENTS
We thank Dale Meyerdirk, Richard Warkentin, and
Curtis Francis of USDA-APHIS, PPQ for providing ini-
tial cultures of pink hibiscus mealybug and A. kamali.
Esther Peregrine, Jos6 Gonzalez, Reinardo Vasquez and
John Lander, USDA-ARS, St. Croix, provided field assis-
tance. We also thank Rosa Franqui and Alberto Pantoja,
University of Puerto Rico, and Randall Pingel, USDA-
ARS, Mayaguez, PR, for their suggestions. Mention of a
trademark or proprietary product does not constitute a
guarantee or warranty of the product by the U.S. Depart-
ment of Agriculture and does not imply its approval to
the exclusion of other products that may also be suitable.
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Florida Entomologist 85(3)
September 2002
PHEROMONE MASS TRAPPING OF THE WEST INDIAN
SUGARCANE WEEVIL AND THE AMERICAN PALM WEEVIL
(COLEOPTERA: CURCULIONIDAE) IN PALMITO PALM
DENNIS ALPIZAR1, MARIO FALLAS1, ALLAN C. OEHLSCHLAGER2, LILLIANA M. GONZALEZ2,
CARLOS M. CHINCHILLA3 AND JUAN BULGARELLI3
'Ministerio de Agricultura y Granaderia, Guapiles, Costa Rica
2ChemTica Internacional, S. A., Apdo. 159-2150 San Jos6, Costa Rica
3ASD de Costa Rica, Apdo. 30-1000, San Jos6, Costa Rica
ABSTRACT
Experiments in Costa Rica and Honduras determined that both Metamasius hemipterus L.
and Rhynchophorus palmarum L. could be captured in the same trap using pheromone lures
emitting a mixture of their male-produced aggregation pheromones. Mass trapping of both
species was conducted in commercial palmito palm (Bactris gasipaes, Kunth) over 16 months
using a combination lure and insecticide-laden sugarcane at a density of 4 traps/ha. Capture
rates of M. hemipterus were initially high and declined significantly while capture rates of
R. palmarum were initially low and remained relatively constant. Pupae found in and dam-
age to palmito declined significantly in plots with traps compared to pre-trapping levels and
to control plots without traps. Yields of palmito palm increased in all plots but increased
most in plots with traps.
Key Words: Metamasius hemipterus, Rhynchophorus palmarum, Bactris gasipaes, mass
trapping, pheromone-baited trap, damage reduction, yield increase
RESUME
Experimentos realizados en Costa Rica y en Honduras determinaron que ambos insects
Metamasius hemipterus L. y Rhynchophorus palmarum L. pueden capturarse en la misma
trampa utilizando senuelos de feromona emitiendo una mezcla de las feromonas de agrega-
ci6n producidas por los machos de estas species. El trampeo masivo de ambas species fue
llevado a cabo en palmas de palmito commercial (Bactris gasipaes, Kunth) por un period de
16 meses utilizando trampas con el seAuelo combinado y cana de azucar impregnada con in-
secticida a densidad de 4 trampas/ha. Las razones de capture del M. hemipterus fueron altas
inicialmente y declinaron significativamente con el paso del tiempo, mientras las razones de
capture del R. palmarum fueron bajas inicialmente y permanecieron relativamente constan-
tes. El numero de pupas encontradas en el palmito dahado decline significativamente con
respect a los niveles encontrados al inicio como tambi6n comparados a los lotes sin trampas.
Los rendimientos de palma palmito incrementaron en todos los lotes pero incrementaron
mas en los lotes con trampas.
Translation provided by author.
The heart of palmito palm (Bactris gasipaes,
Kunth) is a delicacy in many countries of the
world. Increasing demand for dietary fiber contin-
ues to fuel demand for palmito heart. Areas dedi-
cated to commercial production in Central and
South America in 1996 were about 12,000 Ha of
which around 4,000 Ha were in the Atlantic Re-
gion of Costa Rica (Anonymous, Min. Agric. &
Gran., 1998 Costa Rica). Between 1986 and 1996
the amount of palmito heart exported from Costa
Rica increased by an order of magnitude (Anony-
mous, Min. Agric. & Gran., 1998 Costa Rica).
Palmito palm propagates from offshoots that
grow to a harvestable height of one meter in about
3 months. Harvesting discards all parts of the
plant except the interior of the stem. In some plan-
stations, competing offshoots are pruned to promote
more rapid growth of the remaining offshoots to
harvestable size. Harvesting and pruning provide
excellent entry points for Metamasius hemipterus
L. (Vaurie 1966) and Rhynchophorus palmarum L.
(Couturier et al. 1996; Vasquez et al. 2000) Fe-
males of these weevils are attracted to and deposit
eggs in cut stem bases. Larvae tunnel the lower
stem and rhizome destroying maturing stems.
While M. hemipterus, West Indian sugarcane
weevil, is a primary pest of sugarcane it is re-
corded as a pest of several ornamental palms. In
sugarcane females lay eggs in replanted stalk.
Over 30-60 days larvae feed on the interior stalk
before pupating in a fibrous cocoon. Adults live 2-
3 months and are good fliers (Vaurie 1966).
Alpizar et al.: Mass Trapping M. hemipterus and R. palmarum
R. palmarum, the American palm weevil, is a
primary pest of palm in Central and South Amer-
ica. In oil and coconut palm R. palmarum vectors
Bursaphelenchus cocophilis, the red ring nema-
tode that has a major economic impact on com-
mercial oil palm in the New World (Griffith 1968).
The weevil life cycle is 70-120 days of which the
larval stage is 40-60 days (Giblin-Davis et al. 1989).
Male-produced aggregation pheromones are
known for both weevil species. The aggregation
pheromone for M. hemipterus is a mixture of
4-methyl-5-nonanol and 2-methyl-4-heptanol
(Perez et al. 1997) while the aggregation phero-
mone for R. palmarum is 2-methylhept-5-en-4-ol
(Oehlschlager et al. 1992).
Trapping M. hemipterus at 4-5 traps/ha with
pheromone-baited traps effectively lowers dam-
age due to larvae of this insect in newly replanted
sugarcane (Oehlschlager et al. 1997). Trapping
R. palmarum at 1 trap per 5 ha effectively lowers
red ring incidence in commercial oil palm by 80%
over one year (Chinchilla et al. 1996).
Initial experiments conducted in Costa Rica
and Honduras in 1995 led to development of a
blend of the two pheromones that allowed trap-
ping of both species in the same trap (Chinchilla
et al. 1996). These experiments allowed combina-
tion lure trapping of both species in palmito palm.
Pheromone and sugarcane-baited traps have
been developed for M. hemipterus (Giblin-Davis
et al. 1996, Perez et al. 1997).
The purpose of this study was to determine if
mass trapping M. hemipterus and R. palmarum
in commercial palmito palm plantations using a
combination lure decreased damage due to these
weevils and increased yields.
MATERIALS AND METHODS
Combination Lure Experiments for Metamasius hemip-
terus and Rhynchophorus palmarum
Capture ofM. hemipterus was studied in 5 liter
plastic container traps modified for insect entry
(Oehlschlager et al. 1993) containing 10 pieces of
halved 20 cm long sugarcane stalk (pre-immersed
in 1% AI Sevin 80, 1-naphthyl N-methylcarbam-
ate). The ten replicate experiment was conducted
a mature oil palm plantation in Coop-California,
Quepos, Costa Rica 18-24 February 1995.
Capture of R. palmarum was studied in 20 L
plastic bucket traps (Oehlschlager et al. 1993)
containing 15 pieces of halved 20 cm long sugar-
cane stalk (pre-immersed in 1% AI Furadan,
2,3-dihydro-2,2-dimethyl-7-benzofuranyl methyl-
carbamate). The twelve replicate experiment was
conducted in a 100 Ha oil palm plantation near
La Ceiba, Honduras, 21-27 March 1995.
For both M. hemipterus and R. palmarum ex-
periments complete randomized block designs
were used. Traps were placed at 2 meters above
ground at 100 meter intervals with no trap closer
than 100 meters from any planting border. Pher-
omone lures used in both M. hemipterus and
R. palmarum experiments were 2-methylhept-
5-en-4-ol (Rhyncolure), 4-methyl-5-nonanol:2-
methyl-4-heptanol (8:1, Metalure) and a 1:1 mix-
ture of Rhyncolure and Metalure (Combolure) all
released at total rates of 3 mg/day.
Mass Trapping Experiment
Study sites for mass trapping in palmito were
in the wet tropical (<500 M above sea level) Atlan-
tic region of Costa Rica. Sites were within the
area 82' 45"-83' 46"W and 9' 39"-10' 13"N. Exper-
imental plots (100 M x 100 M) within commercial
palmito palm plantations were selected for prun-
ing practices. A 1 Ha plot in a palmito palm plan-
tation in which pruning was practiced was
selected as a control plot for evaluation of phero-
mone trapping under pruning conditions. A sec-
ond 1 Ha pruned plot in the same area was
selected as the trapping plot under pruning con-
ditions. Within the same palmito plantation a 1
Ha plot in which pruning was not conducted was
selected as a control plot for evaluation of phero-
mone trapping under non-pruning conditions. A
second non-pruning 1 Ha plot in the same area
was selected as a trapping plot under non-prun-
ing conditions. Experimental plots were sepa-
rated from each other by at least 200 M from and
from any plantation borer by at least 100 M.
On September 2, 1996 four traps were estab-
lished in a 50 meter square centered in each trap-
ping plot.
Traps were 4L yellow plastic containers with
15 cm wide x 10 cm high windows cut in each side
for insect entry similar to the square gallon traps
reported by Giblin-Davis et al. 1996. Traps were
mounted on sticks 0.5 M above ground and con-
tained a Combolure pheromone lure (as described
above) suspended by a wire from the below the
lids. Traps contained 4-5 pieces of halved 10-12
cm long sugarcane stalk (pre-immersed in 1% AI
Sevin 80, 1-naphthyl N-methylcarbamate).
Insects were counted and removed from all
traps weekly. Pheromone lures were changed
when exhausted as determined by the absence of
liquid in the lure (3-4 months). Sugarcane in
traps was renewed weekly.
Infestation and Yield Surveys
A survey of damage in palmito was conducted
in the week preceding the placement of traps.
Damage was determined by examination of all
stalks in 60 bunches (mats) of palmito palm within
each experimental plot. This was done by cutting
all stalks in each bunch at ground level and exam-
ination of each stalk for damage. Variables as-
sessed were, total stalks in each bunch, number of
Florida Entomologist 85(3)
stalks in each bunch with larval damage due to
M. hemipterus and R. palmarum and number of
M. hemipterus or R. palmarum pupae in each
stalk. We also recorded the number of stalks har-
vested from each bunch in the week of the survey.
This survey was conducted again on March 3,
1997, August 12, 1997 and January 19, 1998.
Data Analysis
Data were tested for heteroscadiscity and if
necessary, transformed to achieve homogeneity
(Zar 1984). Data was analyzed using Systat 5.2.1,
fully factorial ANOVA analysis routine. Means
are always presented untransformed.
RESULTS AND DISCUSSION
Combination Lure Experiments for Metamasius hemip-
terus and Rhynchophorus palmarum
In agreement with preliminary reports (Chin-
chilla et al. 1996) we found that in oil palm traps
containing lures with a mixture of the aggrega-
tion pheromones of M. hemipterus and R. pal-
marum were nearly as effective in capturing
these weevils as traps containing one lure emit-
ting the pheromone of each species (Figs. 1 and 2).
For both weevil species traps containing a combi-
nation pheromone lure captured 25-30% less tar-
get weevils than traps containing a pheromone
lure for the target species. The combination lure
(Combolure) was ideal for the mass trapping of
M. hemipterus and R. palmarum in palmito palm.
An experiment revealed that this lure functioned
more effectively if sugarcane rather than palmito
was used in traps (D. Alpizar, unpublished).
Mass Trapping Experiment
At the commencement of trapping capture rates
of M. hemipterus in both pruning and non-pruning
Metalure
Rhyncolure
Combolure
- a
0 250 500 750 1000
Mean (+SEM) M. hemipterus / Trap
Fig. 1. Mean (+SEM) M. hemipterus captured in
traps baited with sugarcane and 2-methylhept-5-en-4-ol
(Rhyncolure), 4-methyl-5-nonanol:2-methyl-4-heptanol
(8:1, Metalure) or a 1:1 mixture of Rhyncolure and Met-
alure (Combolure). ANOVA (n = 10) gave F = 4.45, p,
0.566 (NS). Means topped by the same letter are equiv-
alent by Bonferonni t-test (P > 0.95).
Metalure b
Rhyncholure a
Combolure a
0 10 20 30 40
Mean (+SEM) R. palmarum / Trap
Fig. 2. Mean (+SEM) R. palmarum captured in
bucket traps baited with sugarcane and 2-methylhept-
5-en-4-ol (Rhyncolure), 4-methyl-5-nonanol:2-methyl-4-
heptanol (8:1, Metalure) or a 1:1 mixture of Rhyncolure
and Metalure (Combolure). ANOVA (n = 12) gave F =
8.50, P < 0.05. Means followed by a different letter are
statistically different by Bonferonni t-test (P > 0.95).
plots were similar (Fig. 3). M. hemipterus capture
rates declined from September through December
and increased from January through March 1997.
The highest capture rates occurred in March-April
whereas a second population build-up occurred in
September 1997 (Fig. 3). The first population peak
corresponded to the end of the dry season in the At-
lantic region of Costa Rica and might be attributed
to a higher survival rate ofM. hemipterus pupae in
the dry season due to decreased fungal and bacte-
rial action on pupal cocoons. Mass trapping M.
hemipterus in banana and plantain in this region
previously revealed an increase in capture rates
during March-April (Alpizar et al. 1998). The peak
in capture rates ofM. hemipterus observed in Sep-
tember 1997 is attributed to the progeny of weevils
that emerged in March-April.
Capture rates for R. palmarum were much
lower than those of M. hemipterus at the onset of
trapping although after one year of trapping cap-
ture rates of both species were similar. Initial cap-
ture rates ofR. palmarum were ~3x higher in the
pruned plot than in the non-pruning plot and re-
mained higher for the entire trial (Fig. 3). While
the capture rates for M. hemipterus declined over
the trial period capture rates of R. palmarum re-
mained rather constant.
Infestation and Yield Surveys
The percentage of weevil damaged stalks was
assessed in both trapping and control plots the
week before commencement of trapping and 7, 12
and 17 months afterward (Fig. 4). Because
palmito palm grows to maturity in three months
and the time between assessments was five to
seven months, each assessment after the com-
mencement of trapping was conducted on palmito
stalks grown after the commencement of trap-
September 2002
|
Alpizar et al.: Mass Trapping M. hemipterus and R. palmarum
S80
40
.u
60
04 0
k
f -i
M A M J JA SON D J
1998
Fig. 3. Mean weekly capture of M. hemipterus and R. palmarum in palmito palm. Four traps were placed in one
hectare of palmito palm in which pruning was practiced and four traps were placed in one hectare in which pruning
was not practiced.
ping. The first assessment at month seven re-
vealed weevil damage in trapping plots was
reduced by >90% compared to pre-trap levels.
This occurred even though considerable numbers
ofM. hemipterus continued to be captured in this
time period. We conclude, based upon examina-
tion of capture rates and damage data that
M. hemipterus and probably R. palmarum enter-
ing trapping plots after September 1996 chose the
traps over palmito stems. A similar phenomenon
was noted during trapping Cosmopolites sordidus
and M. hemipterus in commercial banana (Al-
Trapping No Prune
Trapping Prune
Control No Prune
pizar et al. 1998). It is interesting that in control
plots, damage also decreased during the period
September 1996 to March 1997 but increased
again between March and August 1997. The same
seasonal fluctuation in damage is present but less
pronounced in the trapping plots. This fluctuation
can be attributed to the dispersal of low numbers
of adults during November-January and higher
numbers during February-May. Oviposition and
larval development would be expected to be corre-
spondingly low in November-January and high in
February-May.
tb a Pretrapping Sept.
Y March 1997
ba [ August 1997
b
[] January 1998
PERMI NS
1996
NS
Control Prune
I
% Damaged Stalks
Fig. 4. Percent of damaged stalks in palmito palm stalk prior to and after commencement of trapping for M. hemi-
pterus and R. palmarum. Statistical analysis compares each treatment at different dates and does not compare
between treatments. Means followed by a different letter are statistically different by Bonferonni t-test (P > 0.95).
N D J F
1997
S O
1996
Iv~ I-. I.-1-.~--ts
Il~ecc*)ylr~r+m.~l
Florida Entomologist 85(3)
September 2002
Trapping No Prune
Control No Prune
Trapping Prune
Control Prune
I
a
b
ab
b
a
a
b
Fb
* Pretrap September 96
El
March 97
August 97
January 98
0 0.5 1
No. of Harvested Stems / Bunch
Fig. 5. Harvested stems per bunch prior to and after commencement of trapping for M. hemipterus and R. pal-
marum. Statistical analysis compares each treatment at different dates and does not compare between treatments.
Means followed by a different letter are statistically different by Bonferonni t-test (P > 0.95).
Yield was assessed on the same dates that
damage was assessed (Fig. 5). Yields increased
dramatically in both trapping and control plots
during the trial. After commencement of trapping
those plots receiving traps consistently yielded
higher numbers of harvestable stems per bunch
than control plots without traps. Percentage yield
increase attributable to trapping was 58% in plots
in which pruning was conducted and 70% in plots
in which pruning was not conducted.
ACKNOWLEDGMENTS
The authors thank the technical staff of DEMASA of
Costa Rica for their help in execution of this study.
LITERATURE CITED
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GONZALEZ, AND S. JAYARAMAN. 1998. Pheromone-
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CHINCHILLA, C., A. C. OEHLSCHLAGER, AND J. BULGA-
RELLI. 1996. A pheromone based trapping system for
Rhynchophorus palmarum and Metamasius hemi-
pterus, ASD Oil Palm Papers No. 12, pp. 11-17.
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ida Entomologist 72: 480-488.
GIBLIN-DAVIS, R. M., J. E. PENA, A. C. OEHLSCHLAGER,
AND A. L. PEREZ. 1996. Optimization of Semiochem-
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ceus, J. Chem. Ecol. 22: 1389-1410.
GRIFFITH, R. 1968. The mechanism of transmission of
the red ring nematode. J. of the Agric. Soc. of Trin-
idad and Tobago 67:436-457.
OEHLSCHLAGER, A. C., H. D. PIERCE, JR., B. MORGAN, P.
D. C. WIMALARATNE, K. N. SLESSOR, G. G. S. KING,
G. GRIES, R. GRIES, J. H. BORDEN, L. F. JIRON, C. M.
CHINCHILLA, AND R. G. MEXZON. 1992. Chirality and
field activity of Rhynchophorol, the Aggregation
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senschaften 79: 134-135.
OEHLSCHLAGER, A. C., C. M. CHINCHILLA, L. M. GONZA-
LEZ, L. F. JIRON, R. G. MEXZON, AND B. MORGAN. 1993.
Development of a Pheromone-based trap for the
American Palm Weevil, Rhynchophorus palmarum
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OEHLSCHLAGER, A. C., L. M. GONZALEZ, AND M. GOMEZ.
1997. Pheromone-Based Trapping of the West In-
dian Sugarcane Weevil Int. Soc. of Sugarcane Tech-
nologists, Ent. Workshop, Culiacan, Sinaloa, Mexico,
February.
PEREZ, A. L., Y. CAMPOS, C. M. CHINCHILLA, G. GRIES,
R. GRIES, H. D. PIERCE, JR., L. M. GONZALEZ, A. C.
OEHLSCHLAGER, G. CASTRILLO, R. S. MCDONALD, R.
M. GIBLIN-DAVIS, J. E. PENA, R. E. DUNCAN, AND R.
ANDRADE. 1997. Aggregation Pheromones and Host
Kairomones of the West Indian Sugarcane Weevil,
Metamasius hemipterus, J. Chem. Ecol., 23: 869-888.
VASQUEZ, J., C. M. O'BRIEN, AND G. COUTURIER 2000.
Dynamis nitidulaus Guerin 1844 (Coleoptera: Cur-
culionidae: Rhynchophorinae), nueva plaga de pe-
jibaye (Bactris gasipaes) Manejo o Integrado de
Plagas (Costa Rica) 58: 70-72.
VAURIE, P. 1966. A revision of the Neotropical genus
Metamasius (Coleoptera: Curculionidae: Rhyn-
chophorinae). Species groups I and II. Bull. Ameri-
can Mus. Nat. Hist 131: 213-337.
ZAR, J. H. 1984. Biostatistical Analysis. Prentice-Hall,
Englewood Cliffs, NJ.
Reitz: Distribution of Thrips in Tomato
SEASONAL AND WITHIN PLANT DISTRIBUTION OF FRANKLINIELLA
THRIPS (THYSANOPTERA: THRIPIDAE) IN NORTH FLORIDA TOMATOES
STUART R. REITZ
USDA-ARS, Center for Biological Control, Florida A&M University, Tallahassee, FL 32307
ABSTRACT
Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), the western flower thrips,
is the primary insect pest of tomatoes and other vegetable crops in northern Florida and the
rest of the southeastern USA. However, it is not the only flower thrips present in the region
nor is it always the most abundant species. To determine the seasonal and within plant dis-
tribution of these various Frankliniella species, experimental tomato plants, grown under
different nitrogen fertilization regimes, were sampled during the fall and spring growing
seasons. Contrary to expectations, different levels of nitrogen fertilization did not affect the
abundance of thrips species. Thrips were much more abundant in the spring than in the fall.
In the spring F. occidentalis was the most abundant species, while in the fall F. tritici (Fitch)
was the most abundant species. In both the fall and spring, significantly more adults oc-
curred in flowers in the upper part of the plant canopy than in flowers in the lower part of
the plant canopy. The sex ratio tended to be female biased, but with a greater percentage of
males occurring in the upper canopy flowers. In contrast, significantly more immature
thrips occurred in the lower part of the plant canopy than in flowers in the upper part of the
plant canopy. Differences in seasonal patterns and within plant distribution should be con-
sidered in developing sampling protocols and management plans for thrips.
Key Words: Flower thrips, within plant distribution, seasonal trends, Frankliniella occiden-
talis, Frankliniella tritici, Frankliniella bispinosa
RESUME
Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), el trips occidental de flores,
es principalmente una plaga de tomate y otras hortalizas en el norte de la Florida y en el
resto del sureste de los Estados Unidos. Sin embargo, no es el inico trips de flores present
en la region ni es siempre lo mas abundante. Para determinar la distribuci6n estacional y
entire plants de las varias species de Frankliniella, mostramos plants de tomate experi-
mentales, cultivados bajos regimes de nitr6geno diferentes, durante las estaciones del otoio
y la primavera. Al contrario de las expectaciones, los niveles diferentes de fertilizante de ni-
tr6geno no afectaron la abundancia de las species de trips. Los trips fueron mas abundante
en la primavera que en el otofo. En la primavera, F. occidentalis fu6 la especie mas abun-
dante, mientras que en el otono F. tritici (Fitch) fu6 la especie mas abundante. En ambas es-
taciones, el otono y la primavera, significativamente mas adults ocurrian en las flores en la
parte superior de la copa de la plant que en las flores del parte inferior de la copa de la
plant. La proporci6n de machos y hembras adults reci6n emergidos tendia ser prejuiciada
hacia la hembras, pero con un mayor percentage de machos ocurriendo el las flores de copa
de la plant superior. Al contrast, significamente mas trips inmaduras ocurrian en la part
inferior de la copa de la plant de la plant que en las flores en la parte superior de la copa
de la plant. Se debe considerar las diferencias en los patrons estacionales y la distribuci6n
entire la plant al desarrollar un protocolo de muestreo y plan de manejo para los trips.
Several species of Frankliniella thrips are
sympatric in the southeastern USA. Members of
this complex commonly infest tomatoes and other
vegetable crops, where they are considered pri-
mary pests (Bauske 1998). Some species, such as
F occidentalis (Pergande) and F fusca (Hinds),
are known vectors of Tomato Spotted Wilt Virus
(TSWV) (Sakimura 1962, 1963). Other species,
such as F bispinosa (Morgan), are potential vec-
tors of TSWV (Webb et al. 1997). Still other spe-
cies present in the southeast, such as F tritici
(Fitch), do not vector TSWV. In addition to differ-
ences in the capacity for virus transmission, these
species differ in their phenology, population dy-
namics, host plant use, and behavior (Cho et al.
2000, Hansen 2000, Ramachandran et al. 2001).
These species-specific characteristics also can
be mediated by variation within host plants. Al-
though little is known of the nutritional ecology of
thrips, Brodbeck et al. (2001) manipulated nitro-
gen content of plants through fertilization, and
found that the peak abundance of F occidentalis
adults in tomato flowers is positively correlated
with the concentration of the primary aromatic
amino acid, phenylalinine, in the flowers. This
correlation was most pronounced for females.
Florida Entomologist 85(3)
Higher rates of nitrogen fertilization also result
in higher populations of F occidentalis in chry-
santhemums (Schuch et al. 1998). This pheno-
menon may be critically important to the
management of thrips because southeastern to-
mato growers frequently apply unnecessarily
high rates of nitrogen fertilizer to tomato crops
(Castro et al. 1993), which may then induce
higher populations of serious pests.
In addition the microhabitat within a plant
can affect observed population dynamics. Sal-
guero-Navas et al. (1991b) sampled commercial
tomato fields and found that adults of F occiden-
talis and F tritici were more abundant in flowers
in the upper canopy of tomato than in lower flow-
ers. The opposite was true of immatures, with
more being found in the lower flowers. They at-
tribute part of this difference to rapid immigra-
tion into the crop by adults while immatures near
the top would be more susceptible to frequent in-
secticide applications than those immature thrips
located lower in the canopy. However, F occiden-
talis adults also show significant differences in
the vertical distribution within nectarine or-
chards, with more adults found at lower levels
than at higher levels (Pearsall 2000), and cotton
plants, with more being found in the middle sec-
tion of plants (Atakan et al. 1996).
The specific objectives of the present research
are to determine the seasonal dynamics and
within plant distribution of male, female and im-
mature Frankliniella thrips in tomatoes grown
under different fertilizer regimes. By understand-
ing the responses of Frankliniella thrips to fertil-
ization regimes and plant architecture, more
efficient sampling protocols for thrips in vegeta-
ble crops can be developed and management pro-
grams for tomato spotted wilt improved.
MATERIALS AND METHODS
Cultural Practices
The experiments were conducted at the Flor-
ida A&M University Research Farm in Gadsden
County, Florida, from July 1999 to June 2000. Six
week-old tomato plants (Lycopersicon esculentum
Mill. cv 'Agriset') were transplanted mechanically
into raised beds. Raised beds were 15 cm high and
91 cm wide and were covered with plastic mulch,
with drip tube irrigation underneath the plastic.
In the fall season of 1999, white plastic was used
as the mulch, and plants were transplanted on
August 12. In the spring 2000 season, black plas-
tic was used as the mulch, and plants were trans-
planted on March 17. Plant spacing was 60 cm
within beds, with a bed spacing of 180 cm. Beds
were oriented north-south. Plants were treated
with fungicides every 7-10 days, with at least a
72-hour interval between spraying and subse-
quent sampling. No insecticides were applied
during these studies.
Experimental Design and Methods
To evaluate the impact of different nitrogen
treatments on thrips populations, three different
nitrogen levels were used. Three nitrogen fertil-
izer treatments (sub optimal 101 kg N/ha, opti-
mal 202 kg N/ha, super optimal 404 kg N/ha)
were applied to whole plots (Maynard & Olson
2000). One hundred and one (101) kg N/ha were
administered in the form of 10-10-10 (N-P-K) fer-
tilizer before the plastic mulch was laid. The re-
mainder of the fertilizer was applied four weeks
after transplanting. This fertilizer treatment was
administered by hand in the form of ammonium
nitrate (34-0-0, N-P-K) by cutting an opening in
the plastic and placing a band of fertilizer around
each plant approximately 15 cm from the base of
the plant. Plots were one bed wide and 15.2 m
long, and there was a 1.5 m buffer between plots
within each bed. Plots were laid out in a random-
ized complete block design with each bed forming
a block. In the fall 1999 season, the field was laid
out with four blocks. In the spring 2000 season,
the field was laid out with eight blocks. Flower po-
sition within plants was considered as a subplot
within the fertilizer treatment whole plots.
Data Collection and Analysis
Samples were collected twice per week by tak-
ing one flower from the upper third of the plant
canopy and one from the lower third on each of
three plants per plot. Flowers were collected indi-
vidually in 70% ethanol for later analysis. Sam-
pling was conducted from the onset to conclusion
of flowering. To reduce diurnal variation in sam-
pling, all samples were collected between 1000
and 1300 hours. Thrips were extracted from flow-
ers, and adults were identified to species and sex,
using a stereomicroscope. Because it was not pos-
sible to identify thrips larvae to the species level,
these were combined into a single group for anal-
ysis. The data were transformed to "(y + 0.375)
and then subjected to analysis of variance, by spe-
cies and sex for adults. The independent variables
of interest were fertilization treatment and flower
location within a plant, and the interaction of
these. Data were analyzed for each season as a
randomized complete block-split plot over time
(Steel & Torrie 1980).
RESULTS
Populations of all thrips species were consider-
ably lower in the fall season (Fig. 1) than in the
spring season (Fig. 2). The mean number of thrips
per flower in the fall was 0.70 + 0.05 (0.44 + 0.03
adults, 0.26 + 0.03 immatures) whereas in the
September 2002
Reitz: Distribution ofThrips in Tomato
0.15
F. occidentalis
0.10
0.05 -
0.00 -
1.00
F. tritici
0.80
0.60 -*- Male Upper
0.60
-/-o- Male Lower
-v Female Upper
0.40 v- Female Lower
( 0.20
0
S0.00
L- 0.50
SF. bispinosa
E. 0.40
S0.30
0.20
0.10
0.00
1.00
Immatures
0.80
0.60 -
0.40
0.20 -
0.00
06-Sep 13-Sep 20-Sep 27-Sep 04-Oct 11-Oct 18-Oct
Sample Days
Fig. 1. Mean number of thrips per flower collected from the upper and lower canopy of plants during the fall sea-
son of 1999. Because there were no significant fertilizer treatment effects, data are pooled for all treatments. Data
points are untransformed means plus their standard errors. Note different scales on the y-axes. Symbols for imma-
tures denote means for all species and sexes combined.
Florida Entomologist 85(3)
September 2002
1-May 8-May 15-May 22-May 29-May
Sample Date
Fig. 2. Mean number of thrips per flower collected from the upper and lower canopy of plants during the spring
season of 2000. Because there were no significant fertilizer treatment effects, data are pooled for all treatments.
Data points are untransformed means plus their standard errors. Note different scales on the y-axes. Symbols for
immatures denote means for all species and sexes combined. Filled symbols are for the upper canopy, and open sym-
bols are for the lower canopy.
4
3
2
1
0
8
6
4
2
0
U.
L
0. 0
0.5
S0.4
0.3
0.2
0.1
Immatures
17-Apr 24-Apr
Reitz: Distribution ofThrips in Tomato
spring the mean number of thrips per flower was
6.76 0.16 (5.02 0.15 adults, 1.74 + 0.07 imma-
tures).
Fall
In the fall season, the proportion of adult
thrips in the population was 63.3%. Of the adults,
F tritici (75.0%) was the predominant species.
F occidentalis was uncommon in the fall, com-
prising only 5.3% of adult thrips. All of these F oc-
cidentalis were female. The remaining adults
that were collected (19.7%) were F bispinosa. No
F fusca were collected in the fall. Thrips rapidly
colonized plants soon after the onset of flowering
in early September (Fig. 1). Populations peaked
in mid September and declined until the end of
this month.
There was no significant flower location by fer-
tilization treatment interaction (P > 0.05), and
there were no significant differences among the
fertilization treatments in numbers of any of the
three Frankliniella species or immatures (Table
1, P > 0.05). Significantly more F tritici and
F bispisnosa adults were found in the upper can-
opy than in the lower canopy (F1,2, = 7.81, P < 0.01;
F1, = 7.10, P < 0.01, respectively). These differ-
ences were the result of significantly more males
of F tritici and F bispinosa being in the upper
flowers than in the lower flowers (Table 2). This
difference was most pronounced during the mid-
dle of the season and decreased as populations
declined later in the season (Fig. 1). Females of
F tritici and F bispinosa did not show any differ-
ence in vertical distribution during the fall (Table
2). In contrast to the distribution of adults, signi-
ficantly more immature thrips were collected
from flowers in the lower canopy compared with
flowers from the upper canopy (Table 2). Again
this difference was strongest in the middle of the
season than at the beginning or at the end when
immature populations were low overall (Fig. 1).
Spring
Of all thrips collected in the spring, 74.4%
were adults and 25.6% were immatures. This
overall age distribution was slightly different
from the fall. However, the species composition
was markedly different from that of the fall sea-
son. F occidentalis was much more abundant in
the spring and comprised 56.1% of the adults.
F tritici comprised 41.2% of the adults in the
spring. F bispinosa comprised 2.7%. The remain-
der (<0.1%) was F fusca.
In addition to the species composition, the sea-
sonal population patterns in the spring differed
from those of the fall. In the spring, flowering be-
gan in mid April. F occidentalis began colonizing
in large numbers within a week of the onset of
flowering. These early season F occidentalis pop-
ulations were composed primarily of females.
F occidentalis populations did not peak until the
first week of May (Fig. 2), but then their popula-
tion decreased over the next three weeks. F tritici
did not begin colonizing in large numbers until al-
most a month after the onset of flowering. Popu-
lations of F tritici peaked in the middle of May,
but rapidly declined within another week. F bis-
pinosa numbers remained low throughout the
season and never showed a significant peak and
decline. The numbers of immature thrips peaked
in early May, a time corresponding to the peak
numbers of F occidentalis but low numbers of
F tritici and F bispinosa. These results suggest
that most of these larvae were F occidentalis.
As in the fall there were no significant differ-
ences among the fertilization treatments in num-
bers of thrips (P > 0.05, Table 1). However, there
were, again, differences in the within plant distri-
bution of thrips. Significantly more adults of the
primary species, F occidentalis, F tritici and
F bispinosa, were found in the upper canopy flow-
ers than in the lower canopy flowers (Table 2, Fig.
2). In contrast to the fall season, both females and
males of each species were found in significantly
greater numbers in the upper canopy flowers
than in the lower canopy flowers. The opposite
was true for immature thrips. Over the entire
season, significantly more larvae occurred in the
lower canopy flowers (Table 2, Fig. 2).
DISCUSSION
The results of this study indicate that Frank-
liniella flower thrips commonly occurring in
north Florida have different seasonal population
patterns. These results also show the importance
of understanding individual species population
dynamics when developing sampling protocols
and management programs. Frankliniella species
overwinter on numerous uncultivated plants
(Chellemi et al. 1994, Toapanta et al. 1996), and
thrips from these hosts can then rapidly colonize
crops as they begin to flower (Groves et al. 2001).
However, populations of flower thrips in tomato
and other crops typically occur in large numbers
for a relatively short period of time (Webb et al.
1970, Salguero-Navas et al. 1991b, Brodbeck et
al. 2001), and the timing of population peaks vary
according to species. In northern Florida and
southern Georgia, these peaks occur in the spring
from April through May. F occidentalis, a primary
vector of TSWV, is the predominant species early
in the spring. Its populations peak earlier than
those of F tritici (Salguero-Navas et al. 1991b,
this study). F occidentalis is virtually absent
from late spring through the fall. In the fall,
F tritici, a nonvector of TSWV, is the predomi-
nant species. Populations of F bispinosa did not
show any substantial peaks in either season.
F bispinosa was present throughout the spring
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Florida Entomologist 85(3)
and fall, but in relatively low numbers. These sea-
sonal patterns also can change according to geog-
raphy. Cho et al. (2000) report that populations of
F tritici peak earlier than those ofF occidentalis
in western North Carolina. However, the tomato-
growing season there occurs from June through
August. Although it has been argued that season-
ality is more important than host plant phenology
in determining abundance of thrips (Salguero-
Navas et al. 1991b), the definite peak and decline
in the spring and fall populations of F tritici sug-
gest that host plant phenology also plays an im-
portant role in Frankliniella population dynamics,
with younger plants being able to support greater
densities than older plants.
Contrary to expectations, the different fertil-
izer treatments did not have a significant impact
on populations of any of the thrips species. Previ-
ous studies have found that increased nitrogen
fertilization results in higher populations ofF. oc-
cidentalis in tomato (Brodbeck et al. 2001) and
chrysanthemum (Schuch et al. 1998). Brodbeck
et al. (2001) found that F occidentalis, especially
females, were responding to higher levels of the
primary aromatic amino acid phenylalanine.
Higher overall levels of aromatic amino acids tend
to promote development of F occidentalis larvae
(Mollema & Cole 1996). The lack of a fertilizer ef-
fect in the present study may result from the
method of application. The delay in final applica-
tion of fertilizer was done to keep from burning
the plants. This procedure may not have given the
plants sufficient time to assimilate the extra ni-
trogen before the peaks in thrips populations had
passed, whereas in the study done by Brodbeck et
al. (2001) all fertilizers were applied preplant.
There was a consistent significant difference in
the within plant distribution of adult and imma-
ture thrips, and this variation should be ac-
counted for in sampling protocols. Previous
studies have shown that F occidentalis, F tritici
and F bispinosa are more likely to inhabit flowers
than other plant parts (Cho et al. 2000, Hansen
2000). Therefore, part of the variation in the
within plant distribution of Frankliniella spp.
may be related to microhabitat differences at this
scale. Salguero-Navas et al. (1991b) also found
that adults were more likely to be found in flowers
in the upper parts of tomato plants than in flow-
ers lower in the canopy. Because thrips are
thought to make flights just above the plant can-
opy (Br0dsgaard 1989, Gillespie & Vernon 1990),
it is reasonable to expect that most adults would
be found in flowers in the upper part of the plant
canopy The greater proportion of males in the up-
per canopy may be related to mating aggregations
(Terry 1997). Males tend to aggregate in certain
locations for mating, while females tend to depart
these aggregation sites after mating. It also
seems likely that females make substantial
within plant movements, because most larvae
were found in the lower canopy flowers. Salguero-
Navas et al. (1991b) attribute the greater num-
bers of larvae in the lower part of tomato plants,
in the commercial tomato fields they sampled, to
differential exposure to insecticides. However, the
fact that no insecticides were applied in the
present study suggests that there is a qualitative
difference in resources within a host plant for im-
mature thrips.
A better understanding of species-specific pop-
ulation dynamics will lead to improved sampling
protocols and management plans for flower thrips.
For example, the early spring predominance of
F occidentalis females and high proportion of
thrips larvae in tomatoes that are F occidentalis
(Salguero-Navas et al. 1991a; SRR, unpublished)
suggest that a secondary cycle of TSWV in toma-
toes in the north Florida region will be an ongoing
risk. This risk may be underestimated if sampling
protocols do not adequately account for factors
such as within plant sample location and species
and sex identification.
ACKNOWLEDGMENTS
The technical assistance and advice of Marcus Ed-
wards, Florida A&M University, is greatly appreciated.
Special thanks go to Xin HuaYan for her invaluable assis-
tance. The assistance of Ignacio Baez and Erika Yearby,
Florida A&M University and USDA-ARS is greatly
appreciated. Cassell Gardner, Florida A&M University,
kindly provided access to the field site. Joe Funderburk
and Julie Stavisky, University of Florida provided valu-
able comments on an earlier draft of this manuscript.
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CHO, K., J. F. WALGENBACH, AND G. G. KENNEDY. 2000.
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Florida Entomologist 85(3)
September 2002
EXPOSURE TO GINGER ROOT OIL ENHANCES MATING SUCCESS
OF MALE MEDITERRANEAN FRUIT FLIES (DIPTERA: TEPHRITIDAE)
FROM A GENETIC SEXING STRAIN
TODD E. SHELLY', ALAN S. ROBINSON2, CARLOS CACERES2, VIWAT WORNOAYPORN2 AND AMIRUL ISLAM2
1USDA-APHIS, P.O. Box 1040, Waimanalo, HI 96795, U.S.A.
2FAO/IAEA Agriculture and Biotechnology Laboratory, A-2444 Seibersdorf, Austria
ABSTRACT
In the Mediterranean fruit fly (medfly), Ceratitis capitata (Wiedemann), exposure to a-copaene,
a botanically derived male attractant, and ginger root oil (GRO), Zingiber officinale (Roscoe),
which contains a-copaene, increased the mating success of wild males, and GRO enhanced
mating competitiveness of mass-reared males from a bisexual, mass-reared strain. The
present study extends this research by examining the effects of GRO exposure on the mating
success of mass-reared males from a genetic sexing strain based on a temperature sensitive
lethal (tsl) mutation. Such strains are currently used for nearly all sterile insect technique
(SIT) programs for this insect. In addition, potential negative effects of GRO exposure on
male survival and female remating propensity were investigated. Following exposure to
GRO, males from the tsl mass-reared strain showed enhanced mating performance against
wild-like males from two recently established colonies. Against wild-like males from a Gua-
temala strain, the proportion of matings obtained by males from the tsl mass-reared strain
increased from 16% per replicate for non-exposed (control) individuals to 30% for GRO-ex-
posed (treated) individuals. Against wild-like males from a Madeira strain, the proportion of
matings obtained by treated, tsl mass-reared males was 39% per replicate compared to only
16% for control, tsl mass-reared males. Survivorship was similar between GRO-exposed and
non-exposed males from the tsl strain, and females mated initially to treated or control tsl
mass-reared males displayed similar remating propensity. The application of pre-release,
GRO-exposure to males in the SIT against medfly is discussed.
Key Words: Ceratitis capitata, sterile insect technique, ginger root oil, mating behavior
RESUME
Los machos silvestres de la mosca mediterranea Ceratitis capitata (Wiedemann) expuestos
a a-copaene, un atrayente del macho derivado botanicamente y el aceite de la raiz de jengi-
bre (GRO), Zingiber officinale (Roscoe), lo cual contiene a-copaene, aument6 el 6xito copula-
torio de los machos silvestres, y GRO mejor6 la habilidad de machos criados en masa de un
variedad sexual gen6tica para competir en el apareamiento. Este studio extiende los efectos
de la exposici6n a GRO sobre el 6xito copulatorio de machos criados masivamente de una va-
riedad gen6tica sexual basados sobre una mutaci6n letal sensible de la temperature (tsl). Ac-
tualmente, se usa tales variedades en casi todos los programs de t6cnica de insecto est6ril
(TIS) para este insecto. Ademas, se investigaron los efectos negatives potenciales de la expo-
sici6n a GRO sobre la sobrevivencia de los machos y la propensidad de las hembras para apa-
rearse de nuevo. Despu6s de exponerlos a GRO, los machos de la variedad tsl criados en
masa mostraron un mayor capacidad para aparearse contra los machos del tipo-silvestre de
dos colonies reci6n establecidas. Contra los machos del tipo-silvestre de la variedad de Gua-
temala, la proporci6n de los apareamientos obtenidos de los machos de la variedad tsl cria-
dos en masa aument6 de 16% por replica por individuos no expuestos (control) a 30% por los
individuos expuestos a GRO (tratado). Contra los machos del tipo-silvestre de la variedad de
Madeira, la proporci6n de los apareamientos obtenidos por los machos tsl criados en masa
tratados fu6 39% por replica comparado a solo 16% en el control del los machos tsl criados
en masa. La sobrevivencia de los machos de la variedad tsl criados fu6 similar entire los ex-
puestos a GRO y no expuestos, y las hembras que se aparearon inicialmente con los machos
tsl tratados o el control machos tsl criados en masa muestraron una propensidad similar
para aparearse de nuevo. Se discute la aplicaci6n de exponer los machos a GRO en TIS, antes
de liberarlos contra la mosca mediterranean.
The Sterile Insect Technique (SIT) is an envi- pests, particularly the Mediterranean fruit fly,
ronmentally friendly approach for suppressing or Ceratitis capitata (Wiedemann) (Hendrichs et al.
eradicating insect pests and is widely used in in- 1995). The technique involves mass production,
tegrated programs against tephritid fruit fly sterilization (using irradiation), and release of
Shelly et al.: Mating Success of Male Medflies
males of the target species into the environment.
Matings between sterile males and wild females
yield infertile eggs, which reduces the reproduc-
tive population of the wild population. Because
the success of the SIT depends on the ability of
mass-reared, sterile males to obtain copulations
with wild females, it is essential that the mass-
rearing protocol itself does not produce males
with diminished mating competitiveness
(Calkins 1984).
Unfortunately, the mass-rearing procedures
inherent to the SIT do often lead to a reduction in
the mating competitiveness and viability of re-
leased C. capitata males, particularly in long-es-
tablished strains (Shelly et al. 1994; Lance et al.
2000). The deterioration of strains results from a
combination of factors, including genetic drift
with its concomitant loss of genetic variability
and intense artificial selection imposed by the
laboratory environment (Leppla & Ozaki 1991).
Aside from changing strains frequently, there is
currently no effective way to avoid this decrease
in quality. The now widespread use of genetic sex-
ing strains to produce only males for release in
medfly SIT programs (Robinson et al. 1999) has
led to the development of filter rearing systems to
maintain stability of the strains (Caceres &
Fisher 2000). This low stress, low population size
system, as well as providing a more efficient
means for strain replacement, also provides a
more natural environment for the flies and hence
helps maintain some of the important behavioral
components of fly quality.
This filter system notwithstanding, however, a
constant and important challenge for the SIT is
the development of simple and inexpensive means
to enhance the performance of released, sterile
C. capitata males in the wild. Recent research
(Shelly 2001; Shelly & McInnis 2001) in Hawaii
reveals that exposure of males to particular at-
tractants, especially those containing the com-
pound a-copaene, provides a strong advantage in
mating competition over non-exposed males. For
example, in one series of trials, Shelly and McIn-
nis (2001) recorded the mating frequency of ster-
ile, mass-reared males (from a standard bisexual
strain) and wild males competing for wild fe-
males. In the absence of chemical exposure, mass-
reared males obtained only 26% of all matings.
However, following exposure to the odor of ginger
root oil (GRO), which contains a-copaene, the
mating frequencies were reversed, and mass-
reared males accounted for 75% of all matings.
The primary objective of the present study was
to determine whether exposure to GRO similarly
enhanced the mating competitiveness of males
from a genetic sexing strain based on a tempera-
ture sensitive lethal (tsl) mutation. This is a cru-
cial undertaking as sexing strains based on the tsl
mutation are being used in nearly all SIT pro-
grams for the medfly. As described below, males
from this strain were tested against males from
two laboratory colonies established recently with
wild flies from Guatemala and Madeira, respec-
tively. In addition, we examined potential effects
of GRO exposure on male survival and remating
propensity of females.
MATERIALS AND METHODS
Study Animals
Laboratory flies were from a tsl strain desig-
nated Vienna-7-2000 Mix that had been mass-
reared at the FAO/IAEA Agriculture and Biotech-
nology Laboratory, Seibersdorf, Austria, for ap-
proximately 1 year (12 generations) prior to this
study. The strain was originally constructed
through interpopulational crosses, involving wild
flies from 7 different populations worldwide. Like
other tsl strains, Vienna-7-2000 Mix possesses a
sex-linked mutation, such that treating eggs with
high temperature kills all female zygotes, thereby
allowing production of males exclusively (Franz
et al. 1996). The rearing and release of only males
provides economic and biological benefits, and
consequently tsl genetic sexing strains are replac-
ing bisexual strains in mass-rearing facilities
worldwide (Hendrichs et al. 1995). Males used in
the present study were irradiated as pupae 1 day
prior to eclosion at 100 Gy using a cobalt 60
gamma irradiator, and following irradiation
pupae were at dusted with a pink fluorescent dye
for identification. Larvae of the tsl mass-reared
strain were reared on a wheat bran diet, and
adults were fed a mixture of sugar and protein
(yeast hydrolysate) (3:1 by volume).
Wild-like flies were from 2 recently colonized
strains. One was started using flies reared from
coffee berries (Coffea arabica L.) collected in Gua-
temala, and the other was initiated using flies
reared from oranges (Citrus sinensis (L.)) col-
lected in Madeira. In both cases, larval develop-
ment occurred in situ, pupation occurred in
sawdust, and pupae were shipped by air to the
Seibersdorf facility. Both colonies were started
with 500-1000 adults, and papayas were supplied
for oviposition and subsequent larval develop-
ment. Adults were fed the same protein-sugar
mixture as the tsl mass-reared males. When used
in the present study, the Guatemala- and Ma-
deira-derived flies were 4 and 2 generations re-
moved from the wild, respectively. Adults of the
wild-like strains were separated by sex within 1-
2 d of emergence, well before attaining sexual ma-
turity at 7-9 days.
Mating Tests
We performed 3 experiments that measured the
influence of GRO on the mating competitiveness of
tsl mass-reared males. First, wild-like males from
Florida Entomologist 85(3)
the Guatemala colony competed against GRO-
exposed (treated) or non-exposed (control) tsl
mass-reared males for wild-like females from the
Guatemala line. Second, wild-like males from the
Madeira colony competed against treated or con-
trol tsl mass-reared males for wild-like females
from the Madeira line. Wild-like males were not
exposed to GRO in either of these experiments.
Third, treated, tsl mass-reared males competed
against control, tsl mass-reared males for wild-like
females from the Guatemala line.
To expose tsl mass-reared males, we applied 20
pl of GRO to a small piece of filter paper using a
microcapillary pipette. The paper was then placed
on the bottom of a transparent, plastic drinking
cup (400 ml volume), 25 males were placed in the
cup using an aspirator, and the cup was covered
with nylon screening. Exposure commenced at
1100 h and continued until 1400 h. As reported
previously (Shelly 2001), GRO acted as an arres-
tant, and males were generally quiescent. Males
did not aggregate near the filter paper and were
not observed touching it. Following exposure,
treated males were removed from the exposure
cups, placed in holding containers, and moved to
an adjacent room. The exposure procedure was
conducted in a room isolated from any other flies
to prevent inadvertent exposure of control males.
For all tests, treated males were used the day af-
ter exposure to GRO.
Mating tests were conducted during July-Au-
gust, 2001, in 2 field-cages (2.5 m high; 3 m diam-
eter) enclosed in a greenhouse (6 by 4.8 by 3 m
high) at the Seibersdorf facility. Sections of the
ceiling and sides of the greenhouse were open, al-
lowing free movement of air. During the tests,
temperature generally ranged from 21-29C, and
relative humidity ranged from 45-80%. Each cage
contained 3 potted orange trees whose collective
canopy reached the top of the tent. In experi-
ments 1 and 2, 100 tsl mass-reared males (control
or treated) and 100 wild-like males and females
from the same strain were used. In experiment 3,
100 treated and 100 control tsl mass-reared males
and 100 wild-like females from the Guatemala
colony were used. When tested, tsl mass-reared
males were 6-7 days old, and wild-like flies were
9-12 days old.
Males were released at 0815 h, and females
were released 20 min later. Mating pairs were col-
lected over the next 4 h. At the end of a test, all
flies were removed from the field cages; new flies
were used for all tests. For experiments 1 and 2,
males were identified as tsl mass-reared or wild-
like by squashing the head of each male and ex-
amining the everted ptilinum under ultraviolet
light for the presence/absence of pink dye. For ex-
periment 3 (where only tsl mass-reared males
were used), males were marked 2 days before
testing by cooling them for several minutes and
placing a dot of enamel paint on the thorax. This
procedure had no obvious adverse effects, and
males resumed normal activities within minutes
of handling. For a given trial, we marked either
control or treated males, alternating the marked
group between successive trials.
Female Remating
The remating frequency of wild-like females
from the Guatemala strain was compared among
individuals mated first to 1) wild-like males from
the Guatemala line, 2) tsl mass-reared males ex-
posed to GRO, and 3) non-exposed, tsl mass-
reared males. To obtain initial matings, 40-60 fe-
males (9-11 d old) were placed in transparent,
plastic cages (41 by 31 by 31 cm) with an equal
number of males from a given strain or treatment
(tsl mass-reared males: 6-9 d old; wild-like males:
9-15 d old). Flies were placed in the cages at 0900
h, and mating pairs were collected for the follow-
ing 5 h. Copulation was allowed to continue with-
out interruption, but copulation durations were
not recorded. Following break-up, females were
placed in new cages containing food and water.
Groups of mated females contained 14-36 individ-
uals (x = 22 females), and each group constituted
a replicate. Three days later, males from the Gua-
temala colony (9-14 d old) were placed with the
once-mated females (in 1:1 sex ratio), and remat-
ings were recorded over the following 5 h. Cages
were held at 21-25C and were illuminated by flu-
orescent lights on a 10:14 h light:dark cycle.
Male Survivorship
To determine whether exposure to GRO af-
fected male survivorship, we placed groups of 20
treated or control males from the tsl mass-reared
strain in small plastic containers (18 by 11 by 9
cm with one end covered by nylon screening) with
ample food and water and counted survivors 5 d
later. All males were 3 d old at the start of the ex-
periment. Treated males were exposed to GRO
(following the above protocol) from 0800-1100 h
and then transferred directly to the test contain-
ers. Control males were placed in the containers
at the same time but were simply transferred
from holding cages. The males were held under
the same temperature and light regime noted
above.
Statistical Analyses
The Mann-Whitney test (test statistic T) was
used to compare 1) the number of matings ob-
tained by competing male types in a given exper-
iment and 2) the number of surviving control and
GRO-exposed males. Variation in remating
among females mated to different male types was
assessed using the Kruskal-Wallis test (test sta-
tistic H).
September 2002
Shelly et al.: Mating Success of Male Medflies
RESULTS
Mating Tests
In experiment 1, wild-like males from the Gua-
temala line obtained significantly more matings
per replicate than the control or treated males
from the tsl mass-reared strain (Table 1). Al-
though wild-like males were superior competitors
in both instances, GRO exposure nonetheless im-
proved the performance of the tsl mass-reared
males. In a given replicate, treated, tsl mass-
reared males achieved significantly more matings
(T = 109.0; P < 0.05) and a significantly higher
proportion of matings (30% versus 16%, respec-
tively; T = 114.0; P < 0.05) than control males.
There was no difference in the number of matings
obtained by wild-like Guatemala males (T = 92.5;
P > 0.05) or in the total number of matings (T =
88.0; P > 0.05) between trials involving control
versus treated tsl mass-reared males.
GRO exposure had a more marked effect on
the mating frequency of tsl mass-reared males in
experiment 2. Wild-like males from the Madeira
colony accounted for significantly more matings
per replicate than control, tsl mass-reared males,
but no difference in mating frequency was de-
tected between wild-like Madeira males and
treated, tsl mass-reared males (Table 1). In a
given replicate, treated, tsl mass-reared males ob-
tained significantly more matings (T = 38.5; P <
0.05) and a significantly higher proportion of mat-
ings (39% versus 16%, respectively; T = 40.0; P <
0.01) than control, tsl mass-reared males. There
was no difference in the number of matings ob-
tained by wild-like Madeira males (T = 32.5; P >
0.05) or in the total number of matings (T = 33.0;
P > 0.05) between trials involving control versus
treated males.
In experiment 3, treated, tsl mass-reared males
obtained significantly more matings per replicate
than control, tsl mass-reared males (Table 1). Over
all replicates, treated males accounted for 75%
(100/134) of all matings observed.
Female Remating
The incidence of female remating varied inde-
pendently of the strain and GRO treatment of the
initial mate. The average proportion of wild-like,
Guatemala females remating per replicate was
11% (range: 4-16%) for individuals first mated to
wild-like, Guatemala males, 9% (range: 5-17%)
for individuals first mated to control, tsl mass-
reared males, and 10% (range: 0-26%) for individ-
uals mated first to treated, tsl mass-reared males
(H = 0.2; df = 2; P > 0.05; 5 replicates were run for
all 3 mating combinations).
Male Survivorship
GRO exposure had no apparent effect on survi-
vorship of males from the tsl mass-reared strain.
On average, only 0.8 control males (range: 0-2)
and 1.0 treated males (range: 0-3) died over the 5-
day test period (T = 215.5; P > 0.05).
DISCUSSIoN
The present findings show that exposure to
GRO increased the mating success of males from
a tsl mass-reared strain in competition with wild-
like males from 2 different source populations.
Following GRO exposure, the mating frequency of
tsl mass-reared males increased approximately
1.9-fold (from 16% to 30% in a given replicate)
and 2.4-fold (from 16% to 39% in a given repli-
cate) in tests involving wild-like flies from Guate-
mala and Madeira, respectively. Although sub-
stantial, these increases were actually smaller
than that observed for a mass-reared, bisexual
strain in Hawaii, where the mating frequency of
TABLE 1. RESULTS OF 3 MEDFLY MATING EXPERIMENTS. IN EXPERIMENTS 1 AND 2, WILD-LIKE MALES COMPETED
AGAINST CONTROL (A) OR TREATED (B) MASS-REARED MALES, AND IN EXPERIMENT 3 CONTROL AND TREATED
MASS-REARED MALES COMPETED AGAINST ONE ANOTHER. VALUES FOR MATINGS REPRESENT MEAN NUMBER
PER REPLICATE; VALUES IN PARENTHESES ARE RANGES. SIGNIFICANCE LEVELS OF T VALUES (MANN-WHIT-
NEY TEST) ARE: ***P < 0.001; **P < 0.01; NS NOT SIGNIFICANT).
Experiment Replicates Male strain GRO? Matings/Replicate T
1A 9 Guatemala no 39.4 (21-63) 126.0***
Vienna-7 Mix no 8.3 (2-20)
1B 9 Guatemala no 34.8 (19-53) 122.0***
Vienna-7 Mix yes 15.1 (11-27)
2A 5 Madeira no 32.0 (22-41) 40.5**
Vienna-7 Mix no 6.4 (1-11)
2B 5 Madeira no 27.6 (16-33) 35.5 NS
Vienna-7 Mix yes 19.2 (10-29)
3 5 Vienna-7 Mix no 6.8 (3-10) 40.0**
Vienna-7 Mix yes 20.0 (18-22)
mass-reared males increased approximately 3-
fold (from 26% to 75% in a given replicate) after
GRO exposure (Shelly & McInnis 2001). It ap-
pears, therefore, that although GRO exposure
may consistently elevate male mating success,
the magnitude of this increase may vary among
different combinations of mass-reared and wild
flies. The mechanism by which GRO exposure af-
fects male mating success is unknown, although
it does not appear to reflect a simple elevation of
male signaling activity (Shelly & McInnis 2001).
Existing data reveal considerable variation in
the mating success of tsl mass-reared males
(without the introduction of GRO). On the one
hand, Hendrichs et al. (1996) conducted mating
tests in field cage and reported that males of tsl
mass-reared strain Vienna-42 obtained 39% of all
matings in competition with wild males. In addi-
tion, males of tsl mass-reared strains have effec-
tively reduced wild medfly populations in Guate-
mala (Rendon et al. 1996), Tunisia (Cayol & Zarai
1999), and Israel (Rossler et al. 2000). On the
other hand, the data reported herein reveal low
mating competitiveness of males from the Vi-
enna-7 Mix 200 strain against wild-like males
from two different source populations. Similarly,
in field cage trials conducted in Guatemala,
Lance et al. (2000) reported that tsl mass-reared
males (from Vienna-42 and Toliman-tsl strains
combined) accounted for only 18% of all matings
in competition with wild males.
Importantly, the present study also demon-
strates that exposure to GRO did not adversely
affect male survival. Among mass-reared males,
treated individuals displayed the same level of
survivorship as control males under laboratory
conditions. Subsequent tests conducted in field-
cages in Hawaii have similarly detected no differ-
ence in the survival level between GRO-exposed
and non-exposed males from a mass-reared, bi-
sexual strain (T.E.S., unpublished data). Al-
though open field testing would provide more
robust findings, the existing data at least indicate
that males from the tsl mass-reared strain do not
suffer any immediate and massive die-off as a
consequence of GRO exposure.
The present study also indicated that females
mated initially to GRO-exposed, tsl mass-reared
males displayed a similar tendency to remate as
females first mated to non-exposed, tsl mass-
reared males. Thus, treated and control males do
not appear to differ in their ability to induce a fe-
male refractory period following mating. In addi-
tion, there was no difference in female remating
propensity between individuals first mated to ir-
radiated, mass-reared versus wild-like males.
This latter finding differs from earlier reports.
Working exclusively with mass-reared flies, Kati-
yar and Ramirez (1970) and Bloem et al. (1993)
found that females initially mated with non-irra-
diated males were more likely to remate than fe-
September 2002
males initially mated to irradiated males. In both
of these studies, females were given multiple op-
portunities for remating over several weeks, and
consequently the present finding may have re-
flected the single remating opportunity given only
3 d after the initial mating.
Based on the present results, it appears that
pre-release exposure of males to GRO has the po-
tential to increase the effectiveness of tsl genetic
sexing strains in the SIT. Earlier work (Shelly &
McInnis 2001) on a bisexual strain demonstrated
that a male mating advantage was evident even
when males were unable to contact the GRO
source and when GRO was presented to imma-
ture males. These findings further indicate that it
may be relatively easy, in terms of both logistics
and cost, to incorporate a treatment with GRO
into the current adult male handling procedures
in operational programs.
ACKNOWLEDGMENTS
The work carried out by T. E. S. at the Seibersdorf
laboratory was supported by funds from the Interna-
tional Atomic Energy Agency.
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1993. Female medfly refractory period: effect of male
reproductive status, pp. 189-190. In M. Aluja and P.
Liedo (eds.). Fruit Flies: Biology and Management.
Springer-Verlag, New York.
CACERES, C., AND K. FISHER. 2000. A filter rearing sys-
tem for mass-produced genetic sexing strains of
Mediterranean fruit fly (Diptera: Tephritidae), pp.
543-551. In K. H. Tan (ed.). Area-Wide Control of
Fruit Flies and Other Insect Pests. Penerbit Univer-
siti Sains Malaysia, Paulau, Penang.
CALKINS, C. 0. 1984. The importance of understanding
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grams (Diptera: Tephritidae). Folia Entomol. Mex.
61: 205-213.
CAYOL, J. P., AND M. ZARAI. 1999. Field releases of two
genetic sexing strains of the Mediterranean fruit fly
(Ceratitis capitata Wied.) in two isolated oases of
Tozeur governorate, Tunisia. J. Appl. Entomol. 123:
613-619.
FRANZ, G., P. KERREMANS, P. RENDON, AND J. HEN-
DRICHS. 1996. Development and application of ge-
netic sexing systems for the Mediterranean fruit fly
based on a temperature sensitive lethal, pp. 185-191.
In B. A. McPheron and G. J. Steck (eds.). Fruit Fly
Pests: A World Assessment of their Biology and Man-
agement. St. Lucie Press, Delray Beach, FL.
HENDRICHS, J., G. FRANZ, AND P. RENDON. 1995. In-
creased effectiveness and applicability of the sterile
insect technique through male-only releases for con-
trol of Mediterranean fruit flies during fruiting sea-
sons. J. Appl. Entomol. 119: 371-377.
HENDRICHS, J., B. KATSOYANNOS, K. GAGGL, AND V.
WORNOAYPORN. 1996. Competitive behavior of males
of Mediterranean fruit fly, Ceratitis capitata, genetic
sexing strain Vienna-42, pp. 405-414. In B. A.
McPheron and G. J. Steck (eds.). Fruit Fly Pests: A
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Shelly et al.: Mating Success of Male Medflies
World Assessment of their Biology and Manage-
ment. St. Lucie Press, Delray Beach, FL.
KATIYAR, AND K. P. RAMIREZ. 1970. Mating frequency
and fertility of Mediterranean fruit fly females alter-
nately mated with normal and irradiated males. J.
Econ. Entomol. 63: 1247-1250.
LANCE, D. R., D. O. MCINNIS, P. RENDON, AND C. G.
JACKSON. 2000. Courtship among sterile and wild
Ceratitis capitata (Diptera: Tephritidae) in field
cages in Hawaii and Guatemala. Ann. Entomol. Soc.
Am. 93: 1179-1185.
LEPPLA, N. C., AND E. OZAKI. 1991. Introduction of a wild
strain and mass rearing of medfly, pp. 148-154. In K.
Kawasaki, O. Iwashashi, and K. Y Kaneshiro (eds.).
The international symposium on the biology and con-
trol of fruit flies. Univ. Ryukyus, Okinawa, Japan.
RENDON, P., D. MCINNIS, D. LANCE, AND J. STEWART.
1996. Comparison of medfly male-only and bisexual
releases in large scale field trials, pp. 517-525. In
B.A. McPheron and G. J. Steck (eds.). Fruit Fly
Pests: A World Assessment of their Biology and Man-
agement. St. Lucie Press, Delray Beach, FL.
ROBINSON, A. S., G. FRANZ, AND K. FISHER 1999. Ge-
netic sexing strains in the medfly, Ceratitis capitata:
development, mass-rearing, and field application.
Trends Entomol. 2: 81-104.
ROSSLER, Y., E. RAVINS, AND P. J. GOMES. 2000. Sterile
insect technique (SIT) in the near east-a trans-
boundary bridge for development and peace. Crop
Protection 19: 733-738.
SHELLY, T. E. 2001. Exposure to a-copaene and a-co-
paene-containing oils enhances mating success of
male Mediterranean fruit flies (Diptera: Tephriti-
dae). Ann. Entomol. Soc. Am. 94: 497-502.
SHELLY, T. E., AND D. O. MCINNIS. 2001. Exposure to
ginger root oil enhances the mating competitiveness
of irradiated, mass-reared males of the Mediterra-
nean fruit fly (Diptera: Tephritidae). J. Econ. Ento-
mol. In press.
SHELLY, T. E., T. S. WHITTIER, AND K. Y. KANESHIRO.
1994. Sterile insect release and the natural mating
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87: 470-481.
Florida Entomologist 85(3)
September 2002
SEASONAL ABUNDANCE OF THE ASIAN CITRUS PSYLLID, DIAPHORINA
CITRI (HOMOPTERA: PSYLLIDAE) IN SOUTHERN FLORIDA
JAMES H. TSAI', JIN-JUN WANG2 AND YING-HONG LIU2
1Fort Lauderdale Research and Education Center, IFAS, University of Florida, 3205 College Avenue, Fort
Lauderdale, FL 33314, USA
2Department of Plant Protection, Southwest Agricultural University, Chongqing 400716, P. R. China
ABSTRACT
Seasonal abundance of the Asian citrus psyllid, Diaphorina citri Kuwayama, was studied
weekly in two orange jasmine [Murraya paniculata (L.) Jack] plots in southern Florida from
October 1998 to October 1999. Psyllid populations occur throughout the season on orange
jasmine in southern Florida. Population peaks were observed in October, November, and De-
cember in 1998, and May and August in 1999. Psyllid population levels were positively re-
lated to the availability of new shoot flushes which were in turn related to the weekly
minimum temperature and rainfall. Natural enemies were not key factors in regulating pop-
ulations during the study period. The populations of adult psyllids were also studied weekly
on potted orange jasmine and grapefruit (Citrus paradisi Macfadyen) plants from June 1999
to July 2000. The population levels of psyllid on both host plants were not significantly dif-
ferent and general population trends on the two hosts were similar over time. Continuous
shoot flushes produced by orange jasmine could play an important role in maintaining high
populations of this insect when new shoot flushes were not available in the commercial cit-
rus groves.
Key Words: Asian citrus psyllid, seasonal abundance, temperature, rainfall
RESUME
Se estudiaron la abundancia estacional del psilido Asidtico de citricos, Diaphorina citri
Kuwayama, semanalmente en dos parcelas de Murraya paniculata (L.) Jack] en el sur de
Florida de Octubre de 1998 a Octubre de 1999. Las poblaciones del psilido ocurren atravez
de la estaci6n sobre M. paniculata en el sur de Florida. Se observaron los niveles mas altos
de la poblaci6n en octubre, noviembre, y diciembre en 1998, y en mayo y agosto de 1999. Los
niveles de poblaci6n de psilido fueron relacionadas positivamente a la disponsibilidad de
nuevas hojas que fueron a la vez relacionadas con la temperature minima y menor nivel de
lluvia semanal. Los enemigos naturales no fueron un factor clave en regular la poblaci6n du-
rante el period del studio. Tambien, se estudiaron las poblaciones de los psilidos adults
semanalmente en plants de M. paniculata y toronja (Citrus x paradisi Macfadyen) en ma-
setas de junio de 1999 hasta julio de 2000. Los niveles de la poblaci6n de los psilidos sobre
las dos plants hospederas no fueron significantemente diferentes y las tendencies genera-
les de la poblaci6n sobre los dos hospederos fueron similares atravez del tiempo del studio.
El crecimiento continue de los brotes de nuevas hojas producido por M. paniculata podria ju-
gar un papel important en mantener altas poblaciones de este insecto cuando los brotes de
nuevas hojas no estan disponibles en los huertos comerciales de citricos.
The Asian citrus psyllid, Diaphorina citri Ku-
wayama, was first found in south Florida on June
3, 1998. Subsequent findings were widespread in
Broward, Palm Beach, Martin, Dade, St. Lucie,
Hendry, and Collier Counties in a 3 month period
(Halbert et al. 1998). Diaphorina citri damages
citrus by depleting sap from the plant and inject-
ing a salivary toxin that produces malformation
of shoots and leaves. It also affects photosynthesis
of the tree by excreting honeydew, which pro-
motes the growth of sooty mold (Chien & Chu
1996). This insect is known to be the most effi-
cient vector of citrus greening disease bacterium
(Liberobacter asiaticum) or huanglungbing.
Citrus greening is the most serious disease af-
fecting most major citrus cultivars in Vietnam, Oki-
nawa, China, Taiwan, Indonesia, The Philippines,
India, Sri Lanka, Africa, and the Arabian Penin-
sula (Martinez & Wallace 1967, Moll & Van Vuuren
1977, Bove & Gamier 1984, Aubert 1987, Tsai et
al. 1988, Su & Huang 1990, Aubert et al. 1996).
Given high reproductive potential of this vector
during the period of favorable weather conditions
and food availability (Catling 1970, Mead 1977,
Tsai & Liu 2000, Liu & Tsai 2000), this pest is ex-
pected to spread throughout citrus producing ar-
eas in Florida in 2-3 years. It poses a serious threat
to other citrus producing states in the near future.
Tsai et al.: Seasonal Abundance of Diaphorina citr
Diaphorina citri is of Far Eastern origin (Mead
1977). In the last three decades, research reports
have been focused mainly on the transmission of
citrus greening agent by D. citri (Salibe & Cortez
1966, Martinez & Wallace 1967, Capoor et al.
1974, Su & Huang 1990). Since the discovery of
D. citri in south Florida, the biology of this psyllid
and associated parasitoids including Tamarixia
radiata (Waterston) and Diaphorencyrtus aligar-
hensis (Shafee, Alam & Agarwal) have been stud-
ied in Florida laboratories (Tsai & Liu 2000, Liu
& Tsai 2000, McFarland and Hoy 2001). Field
population dynamics is very limited (Tsai et al.
2000). To date, no effective control measures are
known for D. citri. As part of an effort to develop
an integrated management program for the Asian
citrus psyllid in Florida, we surveyed the sea-
sonal abundance of D. citri on orange jasmine
i3..,..... paniculataa (L.) Jack) during October
1998 to October 1999 in south Florida. We also
compared the population dynamics on potted or-
ange jasmine and grapefruit (Citrus paradise
Macfadyen) from June 1999 to July 2000.
7 -0 -- Pampanc
-0- Davie
S- pooled
6- T
MATERIALS AND METHODS
Experiment 1
Seasonal populations of D. citri were studied
weekly in two orange jasmine plots from October
1998 to October 1999 in Pompano Beach and
Davie, Broward County, Florida. Random sam-
plings for psyllid adults were made at weekly in-
tervals. One shoot (about 6-10 cm long) was
selected from each square meter area by ran-
domly throwing a pointed object made of a pencil
with a ribbon tied to one end. The shoot was se-
lected where the pencil had landed. A total of 100
shoots were selected from each field on each sam-
pling date. Numbers of psyllid adults per shoot
were counted and recorded.
Experiment 2
This experiment was conducted from June
1999 to July 2000 to compare population levels of
citrus psyllid on orange jasmine and grapefruit
10/98 11/98 12/98 01/99 02/99 03/99 04/99 05/99 06/99 07/99 08/99 09/99 10/99
Sampling date
Fig. 1. Mean + SE density of adult D. citri per shoot on orange jasmine at Pompano Beach (solid dots and line)
and Davie (circles and dashed line) from October 1998 to October 1999. Dotted line is pooled mean adult densities
of two sites.
Florida Entomologist 85(3)
(Citrus paradisi Macfadyen), at Fort Lauderdale
Research and Education Center, University of
Florida, Fort Lauderdale. About 200 potted orange
jasmine plants and 250 potted grapefruit plants
(both at 40-60 cm high) were randomly arranged.
Numbers of psyllid adults on both host plants
were sampled as described in experiment 1.
The plants in both experiments were not
sprayed with insecticides during the course of the
study. Meteorological data were kept at this cen-
ter. The mean densities per shoot were calculated
for each plot per sampling date. Paired compari-
son t tests were used to compare the difference be-
tween two sites in experiment 1 and between two
host plants in experiment 2. Correlation analysis
was employed to assess the relationship between
population levels ofD. citri and temperature and
rainfall (SAS Institute 1988).
RESULTS AND DISCUSSION
Experiment 1
The population dynamics of D. citri on orange
jasmine at two sites are shown in Fig. 1. Psyllid
adults were found on orange jasmine on all sam-
pling dates, however, mean densities of D. citri
25 F,
20 F
[Ln
varied significantly between two sites. The high-
est number of psyllid adults recorded on a shoot
was 43. Although the distance between these two
sites was only 15 miles, adult population levels
were markedly different. At the Davie site, adult
psyllid populations were high from October to De-
cember 1998, and then declined rapidly at the end
of December 1998. The populations increased rap-
idly in August 1999, and reached the highest level
in late August 1999 with a mean of 6.51 psyllids
per shoot. Thereafter, the adult populations de-
clined. At the Pompano Beach site, very few
adults were recorded from October 1998 to April
1999. The first peak was recorded at the end of
May, 1999 with a mean of 5.7 psyllids per shoot,
and second peak was recorded in August 1999
with a mean of 5.9 psyllids per shoot. Overall
adult populations were very low from December
1998 to May 1999 at both sites. Paired compari-
sons between the two sites indicated that popula-
tion dynamics were not significantly different (t =
1.20, df = 53, P = 0.2360); therefore, we pooled the
mean densities of psyllid populations at both sites
and presented them also in Fig. 1. Psyllid popula-
tions in the field from October 1998 to October
1999 had five apparent peaks, appearing in Octo-
ber, November, December 1998, and May and Au-
- - minimum temperature
- maximum temperature
Rainfall
ni
I ,n Hn
Hnfnl J1
i N''
3
2
0
2
Ca
10/98 11/98 12/98 01/99 02/99 03/99 04/99 05/99 06/99 07/99 08/99 09/99 10/99
Sampling date
Fig. 2. Mean weekly maximum and minimum temperatures (lines) and rainfall (bars) from October 1998 to Oc-
tober 1999.
I I I IIII IIIIIIII I__I I I III I I I I IIIIIIII1II I IIIIIII
September 2002
WI\YV
Tsai et al.: Seasonal Abundance of Diaphorina citr
gust 1999. The peak populations of the psyllid
seemed to coincide with the availability of the
new shoot flushes.
Atwal et al. (1968) reported that there were
four annual population peaks ofD. citri on citrus
which occurred during March, June, July, and Au-
gust-November from 1965 to 1967 in Ludhiana,
India. The highest population was observed in
March. They suggested that both high and low
temperatures were detrimental to the psyllid
population increases. However, in the current
study there were no population increases in
March, and both sites had a very low population
density from December 1998 through early May
1999.
Many psyllid species such as Paratricza cock-
erelli (Sulc.), Psylla pyricola Foerster, and Car-
diaspina albitextura Taylor were reported to be
intolerant of extreme weather (List 1939, Madsen
et al. 1963, Clark 1964). Calting (1969) also re-
ported that all stages of citrus psylla, Trioza
erytreae (Del Guercio) were sensitive to high tem-
perature together with low relative humidity. Its
populations were consistently the highest in the
cool and moist upland region and were always low
2 -
A-C
in the hot and arid lowlands of South Africa. Most
recently, Liu & Tsai (2000) reported that D. citri
failed to complete development at 10 and 33C,
and McFarland & Hoy (2001) reported increases
in D. citri population survival with increasing rel-
ative humidity in Florida.
The mean weekly maximum and minimum
temperatures and rainfall during our study are
shown in Fig. 2. Correlation analysis indicated
that mean population densities were correlated
significantly with minimum temperature (r =
0.1045, n = 60, P = 0.0045), and rainfall (r =
0.4126, n = 60, P = 0.0021), but not significantly
correlated with maximum temperature (r =
0.1045, n = 60, P = 0.4564). The lowest weekly
minimum temperature (10.5C) occurred in last
week of February 1999 with several extreme low
temperatures during the period of March and
April. Sites experienced little rainfall from Febru-
ary to late May 1999. This could be the reason
why there was no population increase in March as
compared to the study ofAtwal et al (1968). Psyl-
lid populations always increased rapidly after 1-2
weeks of heavy rain that resulted in the growth of
new shoots of orange jasmine, and psyllid adults
06/99 07/99 08/99 09/99 10/99 11/99 12/99 01/00 02/00 03/00 04/00 05/00 06/00 07/00
Sampling date
Fig. 3. Mean SE density of adult D. citri per shoot on potted orange jasmine (solid dots and line) and grapefruit
(circles and dashed line) from June 1999 to July 2000. Dotted line is pooled mean adult densities on two hosts.
Florida Entomologist 85(3)
only laid eggs on newly formed terminal buds
(Figs. 1 and 2) (Tsai & Liu 2000).
Natural enemies always were considered to
play an important role in regulating insect popu-
lation fluctuations in the field. However, during
our study we rarely found any psyllids parasit-
ized by parasitoids (<1% parasitism of nymphs)
and found only a few cocinellid predators on the
plants; natural enemies were not an important
factor affecting the psyllid populations in this
study.
Experiment 2
The densities of D. citri adults on potted or-
ange jasmine and grapefruit from June 1999 to
July 2000 are shown in Fig. 3. Although densities
of D. citri adults varied greatly between the two
host plants on various sampling dates, the rela-
tive densities and general population trends were
similar over time (Fig. 3). Of 60 samplings, 29 ob-
servations of adult on orange jasmine were higher
than those on grapefruit. The highest population
density (2.1 psyllids per shoot) on orange jasmine
was recorded in September 1999, whereas the
highest population density (1.5 psyllids per shoot)
on grapefruit was recorded in early July 1999.
35 -
30 -
20 -
15
,nfl
From June 1999 to July 2000, mean population
densities on orange jasmine and grapefruit were
0.49 and 0.45 per shoot, respectively. Paired com-
parisons did not show significant differences in
population densities between the two host plants
(t = 1.02, df = 60, P = 0.3099). This suggests that
the Asian citrus psyllid did not prefer one of the
hosts over the other which is in agreement with
that reported by Tsai & Liu (2000). They reported
that this insect had similar intrinsic population
rates of increase when reared on these two hosts.
They concluded that the continuous shoot flushes
produced by orange jasmine could play an impor-
tant role in maintaining high populations of this
insect when new flushes were not available in the
commercial citrus groves. Population peaks of
pooled observations occurred in June-July, Sep-
tember-October, December, and February-April.
Unlike experiment 1, correlation analysis did not
show the significant differences among mean pop-
ulation densities and weekly maximum tempera-
tures (r = 0.1038, n =60, P = 0.4302), minimum
temperatures (r = 0.2210, n = 60, P = 0.0899), and
rainfall (r = 0.2236, n = 60, P = 0.0860) (Fig. 4).
However, the P values of minimum temperature
and rainfall were very close to the significant
level ofP = 0.05. In their study, Liu & Tsai (2000)
-- minimum temperature
maximum temperature
I rainfall
_f BnI1n1.nn nn
06/99 07/99 08/99 09/99 10/99 11/99 12/99 01/00 02/00 03/00 04/00 05/00 06/00 07/00
Sampling date
Fig. 4. Mean weekly maximum and minimum temperatures (lines) and rainfall (bars) from June 1999 to July
2000.
,,n,
Ill I
II[I
II
September 2002
Wl\\,
25 ,,
Tsai et al.: Seasonal Abundance of Diaphorina citr
found that at 15C D. citri had higher survival
and reproductive rates than those at higher tem-
peratures.
In general,D. citri thrived very well on both or-
ange jasmine and citrus in south Florida and it
has a good potential to become a major pest. Pop-
ulation peaks were generally observed whenever
new shoot flushes became available; minimum
temperature and rainfall may be key factors af-
fecting psyllid population increases.
ACKNOWLEDGMENTS
This research was supported by the Florida Agricul-
tural Experiment Station, and approved for publication
as Journal Series No. R-08704.
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Florida Entomologist 85(3)
September 2002
OVIPOSITION BIOLOGY OF ACANTHOCINUS NODOSUS
(COLEOPTERA: CERAMBYCIDAE) IN PINUS TAEDA
KEVIN J. DODDS1, CAELIN GRABER AND FREDERICK M. STEPHEN
Department of Entomology, University of Arkansas, A-320, Fayetteville, AR 72701, U.S.A.
1Current address: Department of Forest Science, Oregon State University, Corvallis, OR 97331
ABSTRACT
Oviposition biology ofAcanthocinus nodosus was examined on southern pine beetle (SPB),
Dendroctonus frontalis, infested loblolly pine trees in Alabama, U.S.A. Components of ovipo-
sition biology, including oviposition pit description, colonization period, average number of
eggs laid per oviposition pit, use of bark beetle entrance or ventilation holes as oviposition
sites, and pit density were described. Acanthocinus nodosus oviposition pits were easily dif-
ferentiated from Monochamus titillator, another cerambycid species that also inhabits SPB-
killed trees. Colonization of trees byA. nodosus began within 2 days of initial SPB attack and
lasted for 8 to 14 days. Females laid an average of 3.33 (SE 0.48) eggs per oviposition pit
and 99% of the pits occurred on SPB entrance and ventilation holes. All pits were on the
lower bole between 18 and 163 cm above the ground. Oviposition pit density ranged from
0.22 to 0.45 pits per cm2 of bark surface. Potential interactions with other phloem inhabiting
species were noted.
Key Words: Acanthocinus nodosus, Monochamus titillator, Dendroctonus frontalis, oviposi-
tion, Cerambycidae, Scolytidae
RESUME
Se examine la biologia oviposicional de Acanthocinus nodosus sobre arboles de pino (Pinus
taeda L.) infestados con el escarabajo de pino sureno (SPB), Dendroctonus frontalis, en Ala-
bama, U.S.A. Se described los components de la biologia oviposicional, incluyendo la des-
cripci6n del hoyo de oviposici6n, el period de colonizaci6n, el promedio de huevos puestos
por hoyo de oviposici6n, el uso de la entrada del escarabajo de la corteza o los hoyos de ven-
tilaci6n como lugares de oviposici6n, y la densidad de los hoyos. Se distinguen facilmente los
hoyos de oviposici6n deAcanthocinus nodosus de los de Monochamus titillator, otra especie
de cerambicido que habitat los arboles matados por el SPB. La colonizaci6n de los arboles por
A. nodosus empez6 dentro de 2 dias del ataque inicial de SPB y dur6 por 8 a 14 dias. Las
hembras pusieron un promedio de 3.33 (SE 0.48) huevos por hoyo de oviposici6n y 99% de
los hoyos occurian en la las hoyos de entrada de SPB y los hoyos de ventilaci6n. Todos los ho-
yos fueron sobre el parte abajo del trunco entire 18 y 163 cm de encima de la tierra. La den-
sidad de los hoyos de oviposici6n varia de 0.22 a 0.45 hoyos por cm2 de superficies de corteza.
Se notaron la interacciones potenciales con otras species que habitat la floema de la plant.
Acanthocinus nodosus (F.), (Coleoptera: Ceram-
bycidae) is a frequent associate of the southern
pine beetle (SPB), Dendroctonus frontalis Zim-
mermann (Coleoptera: Scolytidae) in southern
U.S. forests (Overgaard 1968; Moser et al. 1971;
Dixon & Payne 1979). By attacking and killing
living pine trees, SPB provides extensive phloem
resources that can be utilized for reproduction by
A. nodosus. Upon arrival at host trees, adult
A. nodosus chew pits in the bark or enlarge bark
beetle ventilation holes for use as oviposition sites
(Beal 1952; USDA 1985). While mining in the
phloem, A. nodosus larvae often compete with
and/or destroy bark beetle brood (Beal 1952).
Acanthocinus nodosus is usually found in the
thick-barked lower portion of the tree bole (Savely
1939) and is reported to complete one generation
per year (USDA 1985). Acanthocinus nodosus is
found throughout the eastern United States in
dead and dying pines (Yanega 1996).
Little research has been conducted on A. no-
dosus, however, Schroeder (1997) investigated
the reproductive biology ofA. aedilis (L.) on pine
bolts in a Swedish forest. He found that 55% of
A. aedilis oviposition on cut bolts occurred in bark
beetle holes and reported a mean egg density of
3.7 eggs per pit.
The objectives of this study were to describe
the oviposition pit ofA. nodosus and aspects of its
oviposition biology including egg density, area of
infestation on tree boles, duration of oviposition,
and some interactions with SPB.
Dodds et al.: Oviposition Biology
MATERIALS AND METHODS
Study Site
Research was conducted between July 28 and
August 16, 1998, in a mixed hardwood-pine forest
on the Oakmulgee Ranger District of the Tal-
ladega National Forest, Alabama. Several species
of pine were present including loblolly, Pinus
taeda (L.), longleaf, Pinus palustris (Mill.), and
shortleaf, Pinus echinata (Mill.). Three loblolly
pines, trees A, B, and C, were used to investigate
A. nodosus oviposition biology. Additional egg
samples were removed from two P taeda in the
same SPB infestation.
Oviposition Pit Description
Oviposition of three female A. nodosus was ob-
served. After oviposition was completed, pits were
removed with a 2.54 cm arch punch and egg
placement and egg characteristics were noted.
These oviposition pits were compared to known
Monochamus titillator (Fab.) pits to establish dif-
ferences between the two species.
Arrival Period
Trees were examined for evidence of oviposi-
tion pit construction over a 20-day period. After
detecting a new SPB attack, trees were checked to
determine presence of existing A. nodosus ovipo-
sitional pits. If no pits were detected, sampling
commenced. The first day of SPB attack was the
beginning of our first sample interval. Every
other day (one sample interval) sample trees were
checked for new oviposition pits. Pits were either
marked with a permanent felt tip pen, or removed
to determine egg densities. Trees were checked
until no new pits were located for two successive
sample intervals. Previous observations of A. no-
dosus oviposition indicate that once oviposition
ceased for more than 3 days, trees were not fur-
ther colonized.
Egg Densities and SPB Interactions
Egg density was determined by removing bark
and phloem immediately surrounding an oviposi-
tion pit. A 2.54 cm arch punch was hammered
through the bark and phloem, stopping at sap-
wood, and pits were removed. Bark samples (n =
97) were then dissected by peeling the phloem
back from the bark and locating the eggs. Total
number of eggs was recorded for each pit. Each
sample was then examined to determine if a SPB
ventilation or entrance hole had been used for ovi-
position by A. nodosus. If the ventilation or en-
trance hole was the only hole present in the pit
and ended in an SPB gallery, then it was con-
cluded that A. nodosus was using an SPB hole for
an oviposition site.
Area of Infestation
After oviposition by A. nodosus was complete,
the height of each pit above the ground was mea-
sured. Measurements were grouped to create a
histogram depicting pit frequency at heights from
0 to 200 cm above the tree base.
RESULTS
Acanthocinus nodosus oviposition pits are el-
liptical to round with steeper walls than M. titil-
lator and contain a noticeably round hole at the
bottom of the pit (Fig. 1). Eggs ofA. nodosus were
oviposited as a group at one margin of the pit, ap-
parently chosen at random, as if the ovipositor
had been inserted once and all eggs deposited.
Eggs were found directly in SPB galleries as well
as in the phloem surrounding the SPB hole.
Colonization of host trees by A. nodosus
ranged from 8 to 14 days after initial SPB attack,
with an average of 11.3 (SE 1.8). On average,
80% of oviposition pits on sample trees occurred
during the first five sampling intervals following
initial SPB attack. Average number ofA. nodosus
pits on host trees was 41.3 (SE 10.6) and ranged
from 27 to 62 (Table 1). Average pit number per
100 cm2 of bark surface was 0.30 (SE 0.08).
Egg numbers per oviposition pit varied consid-
erably. Mean number of eggs was 3.33 (SE 0.48),
with a range of 0 to 33 (Fig. 2). Among 97 pits re-
moved for sampling, over 35% contained 0 eggs,
11% contained 3 eggs, 10% contained 1 egg, and
9% contained 2 eggs. Almost all (96 of 97) A. no-
dosus oviposition pits were found at the site of
SPB holes. The one pit that did not use a SPB hole
was located within a M. titillator oviposition pit.
Infested bole length varied slightly between
sample trees (Table 1). Average infested bole
length was 135.3 cm (SE 4.8), with tree C hav-
ing the longest infested bole (145 cm) and tree B
the shortest (130 cm). On average, infestation
ranged from 23 cm (SE 2.9) to 158.3 cm (SE
2.6) from the base of the tree. Tree B had the larg-
est area of infestation (1.56 m2), followed by tree A
(1.39 m2) and tree C (1.15 m2). For tree A, the
greatest number (19%) of pits occurred between
100-120 cm, with 40% occurring between 100-160
cm (Fig. 3A). On tree B, 68% of oviposition pits oc-
curred at heights above 80 cm, with 42% of the to-
tal pits concentrated in the 100-160 cm range
(Fig. 3B). In contrast to the other sample trees,
63% of the oviposition pits on tree C were located
below 100 cm (Fig. 3C). The highest density of pits
on tree C occurred between 120-140 cm and 20-40
cm from the base of the tree.
DISCUSSION
Arrival of SPB associates, especially parasi-
toids and predators, has been investigated by sev-
eral authors (Camors & Payne 1973; Dix &
Florida Entomologist 85(3)
; I
ii
Fig. 1.Acanthocinus nodosus and Monochamus titillator oviposition pits. Acanthocinus nodosus pits tended to
be more rounded than M. titillator and contain a distinct round hole at the bottom. The hole at the bottom of the
pit was most likely the result of ovipositing on a bark beetle entrance or ventilation hole.
Franklin 1978; Dixon & Payne 1979). Unfortu-
nately, fundamental knowledge concerning the
basic biology of many associates, especially those
that do not assume a direct beneficial role (i.e.,
parasitoids or predators) is lacking. This study
explored the oviposition biology ofA. nodosus, one
of those poorly studied associated insect species.
We were able to easily differentiate the ovipo-
sition pits ofA. nodosus from M. titillator. While
M. titillator pits were elliptical with a small hori-
zontal line at the bottom (Webb 1909),A. nodosus
pits tended to be more rounded in shape and con-
tained a distinct round hole at the bottom. In our
study, the round hole at the bottom of the oviposi-
tion pit was most likely a result of the bark beetle
entrance or ventilation hole. Another difference
between A. nodosus and M. titillator oviposition
pits was placement of the eggs.Acanthocinus no-
September 2002
Dodds et al.: Oviposition Biology 455
TABLE 1. ACANTHOCINUS NODOSUS INFESTATION DATA FOR SAMPLE TREES A, B, AND C.
Height at Height at Length of Area of Total No. of pits
Sample DBH base of infest. top of infest, infest, infested bole A. nodosus per 100 cm2
tree (cm) (cm) (cm) (cm) (m2) pits of bark
A 29.5 23 154 131 1.39 62 0.45
B 32 28 158 130 1.56 35 0.22
C 24.5 18 163 145 1.15 27 0.23
Mean 28.7 23 158.3 135.3 1.4 41.3 0.30
SE 2.2 2.9 2.6 4.8 0.12 10.6 0.08
dosus eggs were placed as a single group, usually
oriented in one direction. In comparison, M. titil-
lator placed eggs in a distinct circular pattern
around the center of its oviposition pits (Webb
1909). Eggs of A. nodosus were creamy-white in
color compared to the yellowish color of M. titilla-
tor eggs.
Acanthocinus nodosus was common on the
trees we sampled and had a close association with
SPB. After successful initiation of SPB attack,
A. nodosus arrived and began oviposition. Acan-
thocinus nodosus oviposition activity lasted from
8 to 14 days and occurred during the egg-oviposi-
tion stage of SPB activity. Interestingly, A. no-
dosus pits were found that had fresh resin flowing
from them. It is reported thatA. nodosus use bark
beetle exit holes as oviposition sites (USDA 1985).
The findings of our study, however, suggest that
colonization occurs much sooner. We found A. no-
dosus ovipositing while SPB adults were making
galleries and depositing eggs. Also, SPB entrance
holes are typically filled with resin and frass and
slant in relation to the bark surface, while venti-
lation holes are horizontal and filled with frass
only (Stephen & Taha 1976). In our study, we did
not differentiate between the types of bark beetle
holes. However, it was noted on several occasions
that fresh resin was in the SPB hole that was
o0
5- m a" n
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
No. of eggs per oviposition pit
Fig. 2. Frequency distribution of egg numbers in A.
nodosus oviposition pits.
used as an oviposition site byA. nodosus. Due to
the presence of SPB adults constructing galleries
and resin in the egg samples, it can be assumed
that A. nodosus were using SPB entrance holes
181-200
161-180
141-160
121-140
101-120
81-100
61-80
41-60
21-40
0-20
181-200
161-180
141-160
121-140
101-120
81-100
61-80
41-60
21-40
0-20
181-200
161-180
141-160
121-140
101-120
81-100
61-80
41-60
21-40
0-20
A
0 2 4 6 8 10 12
B
0 2 4 6 8 10 12
C
I
am
I
0 2 4 6 8 10 12
Number of Oviposition Pits
Fig. 3. Histogram depicting the distribution of num-
ber ofA. nodosus oviposition pits at different heights be-
low 2 m on trees A, B, and C.
Florida Entomologist 85(3)
and ventilation holes for oviposition sites. Dis-
crepancies between A. nodosus usage of SPB hole
types in our study and the previously recorded ob-
servations (USDA 1985) could be a factor ofA. no-
dosus oviposition timing and SPB density. In our
study, SPB mass attack density was low and rate
of colonization appeared slow in our sample trees.
For example, during the entire time A. nodosus
were ovipositing, SPB were constructing galleries
and depositing eggs.
There are a number of possible explanations to
account for the wide range of egg numbers found
in A. nodosus pits (0-33). First, the large number
of zero egg counts could be a result of predation by
egg predators (e.g., Histeridae). Because SPB ven-
tilation or entrance holes are used for oviposition
byA. nodosus, predators have easy access to eggs.
On several occasions, histerid beetles were found
in the presence of A. nodosus eggs. Because his-
terids are known SPB egg predators (Moser et al.
1971), it seems probable that they would consume
A. nodosus eggs. Second, some cerambycid species
need a period of maturation before oviposition
commences (Linsley 1961). Walsh and Linit (1985)
found that some M. carolinensis (Olivier) females
chewed oviposition pits before reaching sexual
maturity. At the other extreme, the large numbers
of eggs found in some oviposition pits could be ex-
plained as multiple oviposition events by female
A. nodosus. Because A. nodosus displays opportu-
nistic behavior by using SPB holes, it also seems
possible that females may oviposit in existing pits
created by their own species. A similar habit has
been noted in female Eucalyptus borers, Phora-
cantha semipunctata F., where up to 100 eggs
have been found in the same area under bark or in
crevices of eucalyptus trees (L. M. Hanks, pers.
comm.). Mean number ofA. nodosus eggs was sim-
ilar to estimates in Sweden by Schroeder (1997)
who found an average of 3.7A. aedilis eggs per pit.
No oviposition pits of A. nodosus occurred
above 163 cm and only one tree contained pits be-
low 20 cm. There could be several reasons for
A. nodosus not ovipositing in areas higher on the
tree bole. First, by ovipositing lower on the tree
bole,A. nodosus avoids competing with high den-
sities of other phloem inhabiting species (e.g.,
SPB, M. titillator). In addition, A. nodosus is re-
ported to pupate in the bark of host trees (USDA
1985) and may be confined to the lower bole
where thick bark provides adequate pupation
sites. The highest density of pits per 100 cm2
occurred on tree A, which had the second highest
infested bole area. The lowest density of pits oc-
curred on tree B, which had the greatest infested
bole area. However, on trees B and C pits were
marked and removed for egg counts, while not re-
moved on tree A. Release of host volatiles, com-
bined with bark removal from tree B and C could
have negatively impacted oviposition behavior of
A. nodosus. The negative effect onA. nodosus ovi-
position behavior on trees B and C seems surpris-
ing since the removal of bark surface would likely
increase host volatiles, perhaps increasing the at-
tractiveness of the tree to cerambycids. Although
there has been no work conducted on the effect of
host volatiles on A. nodosus, Schroeder and
Weslien (1994a) found A. aedilis was attracted to
alpha-pinene and 95% ethanol. It is likely that
A. nodosus exhibits a similar behavioral response
to host volatiles.
Our study provides basic observations ofA. no-
dosus oviposition behavior and its interactions
with SPB. Because both SPB andA. nodosus coex-
ist in time and space with other bark beetle and
cerambycid species, competition or intraguild
predation among these species is probable.
Schroeder and Weslien (1994b) investigated in-
teractions between A. aedilis and Tomicus pin-
iperda (L.) (Coleoptera: Scolytidae) and found
that the cerambycid species significantly im-
pacted bark beetle reproduction. Coulson et al.
(1980) reported that M. titillator competed for
food resources with SPB, reducing SPB brood by
14% when the two species occurred concurrently
in the same tree. Further, Dodds et al. (2001) con-
cluded that Monochamus carolinensis larvae
were facultative intraguild predators ofIps calli-
graphus larvae (Germar). Because A. nodosus
feeds in the same manner as M. titillator, but in
the lower portion of the bole, competition or intra-
guild predation between SPB and A. nodosus is
probable in that area of the tree. However, the
overall impact ofA. nodosus foraging on SPB sur-
vival may be less than that caused by M. titillator
since the latter inhabits much more tree surface
area than A. nodosus.
Bark beetle species that share the same tree
are typically partitioned along the length of the
bole to minimize competition (Paine et al. 1981;
Flamm et al. 1987). Like the stratification that oc-
curs in bark beetle species, a similar type of re-
source division might occur among cerambycid
species arriving on SPB infested trees. For exam-
ple, it is known that M. titillator primarily inhab-
its the middle and upper portions of infested tree
boles (Hennier 1983), while A. nodosus occurs in
the lower portion. Arhopalus rusticus obsoletus
(Randall), another cerambycid species occurring
in SPB infestations, is limited to the base and
roots of dead and dying pines (Knull 1946; Linit
et al. 1983). Xylotrechus sagittatus sagittatus
(Germar) occurs concurrently with the above cer-
ambycids, but may avoid direct competition for
phloem resources by feeding primarily in the sap-
wood (Gardiner 1957).
Further studies into cerambycid biology, includ-
ing A. nodosus, and other SPB associates are
needed. A more developed knowledge of interspe-
cific interactions of the phloem inhabiting guild as-
sociated with SPB may lead to a more complete
understanding of bark beetle population dynamics.
September 2002
Dodds et al.: Oviposition Biology
ACKNOWLEDGMENTS
We thank Joe Fowler and Bobby Lee, USFS Talla-
dega National Forest, for assistance locating study sites.
M. J. Linit (University of Missouri), L. M. Schroeder
(Swedish University of Agricultural Sciences), and R. L.
McIntosh (Saskatchewan Environment and Resource
Management) all provided valuable comments on an
early draft of this paper. Also, D. C. Steinkraus and
M. V. Meisch (University of Arkansas) provided addi-
tional comments on this work. Funding for this research
was provided by the University of Arkansas Agricul-
tural Experiment Station and the Arkansas Forest Re-
sources Center. Published with the approval of the
Director, Arkansas Agricultural Experiment Station,
manuscript #99007.
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COULSON, R. N., D. N. POPE, J. A. GAGNE, W. S. FARGO,
P. E. PULLEY, L. J. EDSON, AND T. L. WAGNER. 1980.
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Cerambycidae) on within-tree populations of Den-
droctonus frontalis (Col: Scolytidae). Entomophaga
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DIX, M. E., AND R. T. FRANKLIN. 1978. Field biology of
three hymenopterous parasitoids of the southern
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DIXON, W. N., AND T. L. PAYNE. 1979. Sequence of ar-
rival and spatial distribution of entomophagous and
associate insects on southern pine beetle-infested
trees. Texas Agri. Exp. Sta. Misc. Publ. 1432. 27 pp.
DODDS, K. J., C. GRABER, AND F. M. STEPHEN. 2001.
Facultative intraguild predation by larval Ceramby-
cidae (Coleoptera) on bark beetle larvae (Coleoptera:
Scolytidae). Environ. Entomol. 30: 17-22.
FLAMM, R. O., T. L. WAGNER, S. P. COOK, P. E. PULLEY,
R. N. COULSON, AND T. M. MCARDLE. 1987. Host col-
onization by cohabiting Dendroctonus frontalis, Ips
avulsus, and I. calligraphus (Coleoptera: Scolytidae).
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GARDINER, L. M. 1957. Deterioration of fire-killed pine
in Ontario and the causal wood-boring beetles. Ca-
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HENNIER, P. B. 1983. Monochamus titillator (F.) (Co-
leoptera: Cerambycidae) colonization and influence on
populations of Dendroctonus frontalis Zimm., Ips avul-
sus Eichhoff and Ips calligraphus Germar (Coleoptera:
Scolytidae). M.S. Thesis. Texas A&M Univ. 126 pp.
KNULL, J. N. 1946. The long-horned beetles of Ohio (Co-
leoptera: Cerambycidae). Ohio Biol. Surv. Bull. 39:
133-354.
LINIT, M. J., E. KONDO, AND M. T. SMITH. 1983. Insects
associated with the pinewood nematode, Bursaph-
elenchus xylophilus (Nematoda: Aphelenchoididae),
in Missouri. Environ. Entomol. 12: 467-470.
LINSLEY, E. G. 1961. The Cerambycidae of North Amer-
ica. University of California Publications in Ento-
mology. 135 pp.
MOSER, J. C., R. C. THATCHER, AND L. S. PICKARD. 1971.
Relative abundance of southern pine beetle associates
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OVERGAARD, N. A. 1968. Insects associated with the
southern pine beetle in Texas, Louisiana, and Mis-
sissippi. J. Econ. Entomol. 61: 1197-1201.
PAINE, T. D., M. C. BIRCH, AND P. SVIHRA. 1981. Niche
breadth and resource partitioning by four sympatric
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Oecologia. 48: 1-6.
SAVELY, H. E. 1939. Ecological relations of certain ani-
mals in dead pine and oak logs. Ecol. Monogr. 9: 323-
385.
SCHROEDER, L. M., AND J. WESLIEN. 1994a. Reduced off-
spring production in bark beetle Tomicus piniperda
in pine bolts baited with ethanol and a-pinene,
which attract antagonistic insects. J. Chem. Ecol. 20:
1429-1444.
SCHROEDER, L. M. AND J. WESLIEN. 1994b. Interactions
between the phloem-feeding Tomicus piniperda (Col.:
Scolytidae) and Acanthocinus aedilis (Col.: Ceram-
bycidae), and the predator Thanasimus formicarius
(Col.: Cleridae) with special reference to brood pro-
duction. Entomophaga. 39: 149-157.
SCHROEDER, L. M. 1997. Oviposition behavior and re-
productive success of the cerambycid Acanthocinus
aedilis in the presence and absence of the bark bee-
tle Tomicus piniperda. Entomol. Exp. Appl. 82: 9-17.
STEPHEN, F. M., AND H. A. TAHA. 1976. Optimization of
sampling effort for within-tree populations of south-
ern pine beetle and its natural enemies. Environ.
Entomol. 5: 1001-1007.
USDA FOREST SERVICE. 1985. Insects of eastern forests.
Misc. Publ. 1426. 608pp.
WALSH, K. D., AND M. J. LINIT. 1985. Oviposition biol-
ogy of the pine sawyer, Monochamus carolinensis
(Coleoptera: Cerambycidae). Ann. Entomol. Soc. Am.
78:81-85.
WEBB, J. L. 1909. The Southern Pine Sawyer. USDA,
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Florida Entomologist 85(3)
September 2002
EVALUATING ACEPHATE FOR INSECTICIDE EXCLUSION
OF OXYOPS VITIOSA (COLEOPTERA: CURCULIONIDAE)
FROM MELALEUCA QUINQUENERVIA
PHILIP W. TIPPING AND TED D. CENTER
USDA-ARS, Invasive Plant Research Laboratory, 3205 College Ave., Ft. Lauderdale, FL 33314
ABSTRACT
One method of evaluating the impact of insect weed biological control agents is to exclude
them from their host with insecticides, thereby enabling comparisons of host fitness between
infested and non-infested plants. However, the insecticide must not positively or negatively
affect the plant being protected. The insecticide acephate was tested for its effects on Oxyops
vitiosa Pascoe and Melaleuca quinquenervia (Cav.) S. T. Blake. Saplings of M. quinquenervia
were sprayed with concentrations of 0, 0.073, 0.36, and 0.73% a.i. acephate every 7, 14, and
21 days. A bioassay using leaves from sprayed plants and third instars of 0. vitiosa found
reduced defoliation up to 21 days after treatment at the 0.36 and 0.73% concentrations of
acephate. There were minor phytotoxic effects on younger, more tender leaves at the 0.73%
concentration of acephate which reduced leaf biomass. Acephate can protect M. quinquen-
ervia foliage from 0. vitiosa larvae at the 0.36% concentration and spraying every 14 days
will not affect the plant.
Key Words: Biological Control, weeds, bioassay, phytotoxicity
RESUME
Un m6todo para evaluar el impact de las agents de control biol6gico de malezas es excluir-
los de su hospedero con insecticides, en esta manera se permit las comparaciones del la
adaptabilidad 6ptima reproductive del hospedero en plants infestadas y no infestadas. Sin
embargo, la insecticide no debe afectar positivamente o negativamente la plant que esta
siendo protegida. La insecticide acephate fue probado por sus efectos sobre Oxyops vitiosa
Pascoe y Melaleuca quinquenervia (Cav.) S. T Blake. Vastagos de M. quinquenervia fueron
rociadas con concentraciones de 0, 0.073, 0.36 y 0.73% i. a. acephate cada 7, 14, y 21 dias. Se
encontraron que un bioensayo utilizando hojas de plants rocidas y la estadia tercera de
0. vitiosa reduj6 la defoliaci6n hasta 21 dias despu6s del tratamiento a las concentraciones
de 0.36 y 0.73% de acephate. Hubian efectos menores fitot6xicos sobre las hojas mas jovenes
y tiernas a la concentraci6n de 0.73% de acephate que reducieron la masa biol6gico de las ho-
jas. Acephate puede proteger el follaje de M. quinquenervia de larvas de 0. vitiosa a la con-
centraci6n de 0.36% y aplicandolo cada 14 dias sin afectar la plant.
Evaluation of the impact of introduced biologi-
cal control agents on their weed targets is an es-
sential component of any classical biological
control project (Smith & DeBach 1942). Objective
analyses can provide insights or evidence as to
why agents succeed or fail, present justification
for future projects, and may contribute to deci-
sions affecting the future direction of the project
(Farrell & Lonsdale 1997).
There are a number of techniques for conduct-
ing field evaluations, each with their own advan-
tages and disadvantages. They include: exclusion
of the biological control agent using cages, hand
removal, or insecticides; inclusion of agents using
cages; correlation of agent numbers with host fit-
ness; pre- and post-release comparisons of host
fitness; and selective releases of the agent (Adair
& Holtkamp 1999).
Oxyops vitiosa (Coleoptera: Curculionidae)
was released in south Florida during 1997 as part
of a classical biological control project for the in-
vasive Australian tree Melaleuca quinquenervia,
which infests 200,000 ha in the Everglades and
surrounding conservation areas (Center et al.
2000). Adults prefer to feed on fresh buds and
leaves that have not fully expanded. Eggs are laid
singly or in groups on stems and leaves and the
larvae typically defoliate entire tips of new vege-
tation. Adults actively oviposit and larvae are
present year round but are most abundant Octo-
ber through March, coincident with the annual
flush of foliage produced by the trees.
Evaluating the impact of 0. vitiosa in this sys-
tem by excluding them with cages would be diffi-
cult given the rapid rate of growth of the trees,
their large size, and shading effects. Hand re-
moval of insects is similarly impractical. Popula-
tions of 0. vitiosa are now widespread and
increasingly fewer suitable control sites are now
available for comparing infested versus non-in-
fested areas. A more practical evaluation method
for this system would be insecticide exclusion be-
Tipping & Center: Insecticide Exclusion of Oxyops vitiosa
cause of the aforementioned problems and the fact
that 0. vitiosa is the only significant herbivore
feeding on the plant, thereby avoiding any inter-
pretation errors caused by generalist herbivores
(Annecke et al. 1969). Potential disadvantages of
this approach include negative effects of the insec-
ticides on the plant such as phytotoxicity and in-
terference with pollination, or positive effects such
as stimulation of growth (Jones et al. 1986).
Identifying and evaluating an insecticide to
exclude feeding by 0. vitiosa was the objective of
this study. We considered factors like cost, avail-
ability of systemic formulations for large tree
studies, environmental fate, and mammalian tox-
icity. On the basis of these criteria we selected
acephate, an organophosphate compound first in-
troduced in 1969 and used in many crops (Thom-
son 1982). It is readily available, inexpensive,
with relatively low mammalian toxicity and an
antidote (Spencer 1981). Acephate has a half life
of <3 days and 6 days in aerobic and anaerobic
soils, respectively (Thomson 1982). It is also rap-
idly absorbed into leaf tissue when applied foli-
arly. For example less than 25% of the acephate
remained on the surface of cotton leaves 24 h
after application (Bouchard and Lavy 1982).
Finally, there is a commercially available formu-
lation for protecting large trees using implant-
able cartridges of acephate (AceCapstm, Creative
Sales, Inc., Fremont, NE).
MATERIALS AND METHODS
The experimental design was a randomized
complete block with 12 treatments and four
blocks replicationss). Acephate (9.4% a.i.) was ap-
plied at four concentrations, 0, 0.073% a.i.,
0.367% a.i., and 0.73 a.i.% to the plant leaves un-
til runoff. Water was applied to controls. Applica-
tions were made with a hand-pressurized garden
sprayer with approximately 0.1 liter of finished
product applied to each plant. Application fre-
quencies were once every 7, 14, or 21 days. The
same plants were sprayed up to five times (7 day
frequency) under the same regime of acephate
concentration and application frequency.
Test M. quinquenervia trees originated from
greenhouse-grown plants with similar trunk
widths that were cut back to equalize heights,
number of branches, and root mass. Plants were
then placed in water for three weeks before repot-
ting. The following data from each randomly
numbered sapling was recorded before transplan-
tation to pots: stem diameter at mid-height,
height, number of buds, and fresh weight. Once
planted in pots, plants were fertilized with 52 g of
13-13-13 controlled release fertilizer and ran-
domly assigned to a treatment.
Twelve potted plants were placed randomly into
each of four 950 liter concrete tanks (blocks)
equipped with a continual water exchange system
that completely replaced the volume of each tank
about 4 times per day. This was designed to flush
away any pesticide residues which might contami-
nate adjacent plants. Distance between pots was
maximized. Plants were removed from the tanks,
treated with the appropriate concentration of in-
secticide, and allowed to dry. The outside of the
pots was washed with water to remove any resi-
dues, allowed to dry, then placed back into the tank.
TABLE 1. MEANS (SE) OF BIOASSAY RESPONSES OF OXYOPS VITIOSA LARVAE TO LEAVES OF MELALEUCA QUINQUE-
NERVIA SPRAYED WITH ACEPHATE.
Acephate concentration (% a.i.)
Variable DAT' 0 0.073 0.367 0.73
-------- -------- --------------------------------%---------------
Defoliation (24 h) 1 66.5 + 6.6 39.0 + 5.6 11.7 + 1.4 11.0 + 1.3
7 57.5 + 6.6 24.6 + 4.2 20.6 + 3.1 14.8 + 1.8
14 68.3 + 4.3 58.3 + 4.4 25.3 + 5.1 28.3 + 4.3
21 75.0 + 8.6 68.7 + 11.7 45.0 + 10.6 35.0 + 9.3
Defoliation (48 h) 1 76.0 + 5.9 42.2 + 5.2 13.5 + 1.7 11.0 + 1.6
7 67.7 + 6.2 32.2 + 4.9 22.5 + 3.2 14.9 + 1.8
14 78.7 + 4.4 68.7 + 4.6 47.9 + 6.1 33.7 + 5.8
21 90.0 + 0.1 85.0 + 2.0 61.2 + 9.6 42.5 + 13.6
- -- --------------------- number dead after 48 h--------------------
Larval Mortality 1 2.1 + 0.3 2.0 + 0.2 3.2 + 0.3 3.3 + 0.3
7 2.3 + 0.4 2.8 + 0.2 2.9 + 0.3 3.3 + 0.3
14 3.1 +0.2 3.5 + 0.2 3.1 +0.3 3.4 + 0.3
21 1.2 + 0.2 3.0 + 0.0 2.0 + 0.1 3.7 + 0.1
'Days after treatment.
460 Florida Ento
The first acephate application was done on
Feb. 21, 2001 when at least five fully expanded
new leaves were present on all plants. Plants
were fertilized as before on March 1, 2001. Bio-
assays for the toxicity of treatments to 3rd instar
Oxyops vitiosa were conducted weekly by remov-
ing four suitable leaves from each plant, includ-
ing those plants whose leaves were not going to be
tested that week, in order to equalize the total
Control
= OU
CO
70
0
o
.'
60
50
4 50
40
Im
1 2 3 4 5
Spray Number
0.367% Acephate
ologist 85(3) September 2002
number of leaves removed from all test plants.
Fine-bladed scissors were used to cut through the
petioles of selected leaves. Suitable leaves, pre-
ferred by larvae, were those that were fully
formed but still supple and soft. Two of the four
leaves were used to assess leaf toughness using a
penetrometer (Halda Gram Gauge, Stockholm,
Sweden). Leaves were held on a plexiglass stage
that prevented movement and provided a guide
0.073% Acephate
1 2 3 4 5
Spray Number
0.73% Acephate
1 2 3 4 5
Spray Number
1 2 3 4
Spray Number
Fig. 1. Average defoliation by Oxyops vitiosa after 48 h ofMelaleuca quinquenervia leaves treated with multiple
applications of four concentrations of acephate. Vertical lines denote standard error of the mean.
Tipping & Center: Insecticide Exclusion of Oxyops vitiosa
hole for the stylus of the penetrometer (Wheeler
and Center 1996). Readings were taken at the
mid-point along the long axis of the leaf, immedi-
ately adjacent to the midrib.
The other two leaves were used for bioassays
and placed side-by-side on moistened filter paper
in a standard 15 cm petri dish. The length and
width of each leaf was recorded to estimate leaf
Control
c
C00
3.0
- 2.5
2.0
- 1.5
0
z
1 2 3 4 5
Spray Number
0.367% Acephate
1 2 3 4 5
Spray Number
area which was calculated by the formula length
x width x 0.8. Five field collected 3rd instars of
0. vitiosa were placed on the leaves and the
dishes were sealed with parafilm and held in an
environmental chamber at 27C and 12:12 (L:D)
photoperiod. After 24 and 48 h, the number of lar-
vae on plants, the number of dead larvae, and per-
cent defoliation to the nearest 10% was recorded.
0.073% Acephate
1 2 3 4
Spray Number
0.73% Acephate
1 2 3 4 5
Spray Number
Fig. 2. Average number of dead larvae of Oxyops vitiosa 48 h after exposure to Melaleuca quinquenervia leaves
treated with multiple applications of four concentrations of acephate. Vertical lines denote standard error of the
mean.
y = 3.40+0.01x r2= 0.0001
Florida Entomologist 85(3)
Defoliation was estimated visually by two evalu-
ators and the mean estimate was recorded. Lar-
vae were considered dead if they did not move
after gentle prodding with a brush. At the end of
the test, the larvae were removed and leaves were
dried at 50C for 48 h and weighed.
Bioassays were conducted 1, 7, 14, and 21 d
after treatment (DAT). The experiment was ter-
minated on April 3, 2001 and the plants were
removed from the pots and the soil washed from
the roots. Leaves were removed and counted, and
above and below ground portions of the plants
were dried and weighed separately.
Data on plant effects were analyzed with
repeated measures analysis of covariance with
initial plant height, width, and the number of
buds as the covariates (SAS Institute 1990). Main
factors in the analysis were insecticide treatment,
application frequency, and the number of applica-
tions. Data on insect effects were analyzed with
repeated measures analysis of variance with in-
secticide treatment, application frequency, DAT,
and the number of applications as main effects.
Values were transformed using square root trans-
formation when variances were heterogeneous.
Simple linear regression was used to examine re-
lationships among plant and insect responses to
application frequency, number of treatments, and
insecticide concentrations.
RESULTS AND DISCUSSION
Acephate protected M. quinquenervia foliage
from feeding by 0. vitiosa larvae. Defoliation after
24 and 48 h was affected by both the treatment
and the number of days after treatment (F =
102.8; df = 3, 189; P < 0.0001 and F = 31.8; df = 3,
189; P < 0.0001 for treatment and DAT, respec-
tively, after 48 h) (Table 1). The number of larvae
found dead after 48 h was not a useful indicator of
protection because the larvae in the non-sprayed
control consumed most of the plant tissue after
24 h and may have suffered stress from a lack of
food after then. As a result, it's difficult to discern
whether larval mortality was caused by the insec-
ticide treatment or the lack of food. The same was
true of the number of larvae found on leaves after
24 and 48 h (data not shown). It did not appear
that larvae were repelled by the insecticide before
feeding because most of the leaves showed some
feeding scars.
In addition to the acephate concentration, in-
creasing the number of applications affected defo-
liation and larval death (F = 3.34; df = 4, 189; P =
0.01 and F = 4.01; df = 4, 189; P = 0.003 for defo-
liation and the number of dead larvae after 48 h,
respectively). Defoliation declined with increas-
ing application number (Fig. 1). Larval death in-
creased as the number of applications increased
in two of four treatments, most notably the con-
trol, indicating that factors other than insecticide
toxicity were involved (Fig. 2). These results may
be explained by the increased toughness of the
leaves as the experiment progressed (F = 7.54; df
= 2, 49; P = 0.001) (Fig. 3). As the number of sam-
ple dates increased and plants were repeatedly
sampled, a larger percentage of the leaves used in
bioassays were from more proximal positions on
the branches, with concomitant increases in leaf
toughness. This effect was magnified in the
higher concentration treatments which caused
some phytotoxic effects on the tips, resulting in
burned and mis-shapened leaves, thereby forcing
the selection of more proximal leaves for the bio-
assay. Although the leaves were more proximal,
they were still suitable for normal larval growth
and development.
Leafbiomass was the only plant factor affected
by acephate. The highest concentration of ace-
phate caused reduced leaf biomass, primarily
through the previously mentioned phytotoxic ef-
fects (F = 4.2; df = 3, 46; P = 0.01). Stem and root
biomass, leaf number, leaf toughness, and trunk
width were unaffected by the concentrations of
acephate used in this study.
The general use insecticide acephate proved to
be lethal against larger larvae of 0. vitiosa and
was persistent for several weeks in the foliage of
M. quinquenervia saplings. Applications of this
compound at the 0.36% concentration every 14 d
should protect the foliage of M. quinquenervia
from 0. vitiosa without damaging the plant. A
similar period of insect protection was found by
Azarbayjani et al. (1999) for M. linariifolia using
permethrin, a non-systemic synthetic pyrethroid.
Significant larval populations are present on
M. quinquenervia in south Florida for about 6
months. Therefore, excluding 0. vitiosa larvae
from saplings and small trees for evaluation pur-
poses using acephate may require up to twelve
applications a year.
100
N 90
E
s 80
uo
c 70
ao
o 60
s 5o
S40
1 2 3 4 5
Spray Number
Fig. 3. Mean leaf toughness of Melaleuca quinquen
ervia leaves after multiple applications of acephate.
September 2002
Tipping & Center: Insecticide Exclusion of Oxyops vitiosa
ACKNOWLEDGMENT
Eileen Pokorny, Allison Forrestal, and Anna Strong as-
sisted in the field and laboratory portions of this project.
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Florida Entomologist 85(3)
DESCRIPTION, BIOLOGY, AND MATERNAL CARE
OF PACHYCORIS KLUGII (HETEROPTERA: SCUTELLERIDAE)
LUIS CERVANTES PEREDO
Institute de Ecologia, A.C., Apartado Postal 63, CP 91000, Xalapa, Veracruz, Mexico
ABSTRACT
The life cycle of Pachycoris klugii on its native host, Cnidoscoulus multilobus (Euphor-
biaceae), is reported in detail for the first time. Egg, nymphs, and adults are described and
illustrated. Extensive variation was observed in the color pattern of adults. Maternal behav-
ior was related to egg parasitism, habitat and host plant phenology. The tachinid fly Tri-
chopoda pennipes was observed parasitizing adults and the scelionid wasp Telenomus
pachycoris parasitized eggs of P. klugii.
Key Words: Pachycoris, maternal care, Euphorbiaceae, Trichopoda pennipes, Telenomus
pachycoris
RESUME
El ciclo de vida de Pachycoris klugii en su hospedera natural Cnidoscoulus multilobus
(Euphorbiaceae) se report en detalle por primera vez. Se described e ilustran el huevo, nin-
fas y adults. Se observe gran variaci6n en los patrons de coloraci6n de los adults. El com-
portamiento materno estuvo relacionado a los parasitos de huevos, al habitat y a la fenologia
de su hospedera. El tachinido, Trichopoda pennipes se observe parasitando a los adults, y
la avispa scelionida, Telenomus pachycoris parasitando a los huevos.
Translation provided by author.
There have been several reports of maternal
care in Heteroptera (Tallamy & Schaefer 1997),
including the extensive study of Antiteuchus
tripterus limbativentris Ruckes (Eberhard 1975).
This behavior appears to be more common in Ara-
didae, Scutelleridae, Pentatomidae, Belostoma-
tidae, Tingidae, and Reduviidae than in other
families (Bequaert 1935, Parker 1965, Eberhard
1975, Tallamy & Denno 1981, Taylor 1988, Smith
1997). According to Wilson (1979) it corresponds
to a subsocial behavior in which the parent offers
shelter with its body, carries the nymphs around
on its back or venter, or simply stands close by.
Wilson (1979) mentioned that insect parental be-
havior is a polyphyletic behavioral solution to ex-
ceptional environmental challenges. Only when
physical conditions are particularly rigorous,
nutritional resources are exceptionally rich or
ephemeral, or predation is unusually intense, is
selection powerful enough to stimulate parental
care in insects. In the genus Pachycoris Burmeis-
ter (Scutelleridae), females of a few species were
reported to stand guarding the eggs and first
instar nymphs (Hussey 1934, Bequaert 1935,
Grimm & Maes 1997a,b).
Pachycoris klugii Burmeister (1835) is a com-
mon species in Mexico and Central America that
in the past has been misidentified as P torridus
(Scopoli). It is an aposematic species that is
brightly colored, with metallic green, and yellow,
orange, or red spots and has been recently studied
in Nicaragua as a pest of Jatropha curcas (L.)
(Euphorbiaceae). Description of its damage,
abundance, control, and some characteristics of
its life cycle in Nicaragua are cited in several pa-
pers (Grimm 1996, Grimm & Maes 1997a, Grimm
& Maes 1997b, Grimm & Fuhrer 1998, Grimm &
Guhray 1998, Grimm & Somarriba 1998). How-
ever, these studies were done on J curcas planta-
tions which is not a natural habitat or its native
host.
In this study, the life cycle of P klugii on its na-
tive host is presented for the first time. Eggs,
nymphal stages, adult and adult genitalia and
stridulatory organs are described and illustrated.
Adult color variation is also exemplified. General
behavior of adults and nymphs as well as oviposi-
tion are described. Host plant phenology and egg
and adult parasites are also examined in relation
with maternal behavior.
MATERIALS AND METHODS
This study is based on periodic collection of
data over a long period of time and in several lo-
calities in Mexico. Most of the biological data
were obtained during several trips to the Biologi-
cal Field Station of Los Tuxtlas in the state of
Veracruz, Mexico from 1985 to 1989, and again in
1999. This area is situated between 95004' and
95009' W longitude and between 1834' and 18'36'
N latitude, with an altitude of 150 to 530 m. The
September 2002
Cervantes Peredo: Pachycoris klugii
main type of vegetation is tropical rain forest, but
the zone has been cleared for agriculture and
principally for cattle farming. More detailed in-
formation can be found in Lot-Helgueras (1976).
Additional data were acquired during other col-
lecting trips throughout Mexico, and by reviewing
specimens in the Entomological Collections of In-
stituto de Biologia of the Universidad Nacional
Autonoma de Mexico (CNIN) and the insect col-
lection at Instituto de Ecologia, A.C. in Xalapa,
Veracruz (IEXA)
Between 1985 and 1987, monthly visits were
made to Los Tuxtlas. Visits were made at irregu-
lar intervals during 1988 and 1989, and in 1999
the area was sampled every two months. The
same area was sampled on each visit. The area
sampled included the vegetation growing along
the border of the road connecting Los Tuxtlas
with the town of Catemaco. Only the first two km
of the road from the station were sampled. All
host plants (about 30 individuals) on this section
of road were sampled and all P. klugii individuals
(eggs, nymphs, and adults) were collected alive.
The bugs were put in plastic containers (11 x 11 x
11 cm), accompanied by a small stem and part of
a mature leaf of its host plant, as well as a piece
of wet cotton. Containers were covered with mus-
lin to avoid condensation and fungal growth. On
several occasions females were observed in the
field guarding eggs or nymphs; they were col-
lected with the supporting leaf. Containers were
checked daily to register eclosion, moulting or
mortality and every three days the bugs were
placed in clean containers with fresh host plant
material. Containers were maintained in the lab-
oratory at about 20C and 70% RH. Eclosion,
molting time, behavior, parasite eclosion, and
mortality were recorded. Several individuals of
each stage were fixed in 70% alcohol for illustra-
tion and description purposes. Ten individuals of
each stage were measured (measurements are
given in mm + S.E.), and used for descriptions.
Length of femur, tibia and tarsi were measured
on the posterior legs. During the study 1785 eggs,
142, 335, 155, 119, 78 nymphs of instars 1-5, re-
spectively, and 157 male and 129 female adults
were observed.
Host Plant.-The host plant of P. klugii is Cni-
doscoulus multilobus (Pax) I.M. Johnston (Eu-
phorbiaceae), a species known locally as "mala
mujer" (bad girl) or "chichicaste". It is a shrub or
tree, two to 10 meters tall that grows as second-
ary vegetation in the border of roads and forest.
In Mexico, it can be found from Colima to Chiapas
on the Pacific side and from Tamaulipas to Quin-
tana Roo in the Gulf of Mexico. It is also present
in the central plateau in San Luis Potosi, Quere-
taro, Hidalgo, Morelos, and Puebla.
Cnidoscoulus multilobus occurs in different
habitat types, from Quercus forests, to cloud for-
ests and several kinds of tropical forests. As is
typical of many Euphorbiaceae, C. multilobus has
white sap. Leaves are 20 to 25 cm long, with 15 to
30 cm long petioles. The stems and leaves have
stinging hairs which are less abundant on the up-
per side of the leaves. Leaves have five to seven
lobules with serrated margins. Flowers are white,
and fruits are small dehiscent capsules. It pro-
duces new leaves continuously and it can also be
found flowering all year depending on the cli-
matic conditions.
RESULTS
Egg.-(Figs. 1-4) Barrel shaped, yellow when
laid, turning reddish after three or four days at
which time the eyes and egg burster are visible.
Parasitized eggs turn blackish. Eggs that do not
produce nymphs or parasitoids remain yellow.
Chorion reticulated; pseudoperculum with 23 to
26 micropylar projections. Egg masses consisted
of 56 to 95 eggs (81.4 3.7) arranged in 8 to 11
regular lines. Egg length 1.65 0.05; egg width
1.1 0.03; micropilar projection length 0.05 + 0.
First Instar.-(Fig. 5) Oval, convex dorsally,
maximum width around abdominal segment two;
margin of body setose. Legs and rostrum black;
antennal segments black, except tip of segment
II, base and tip of segment III, and base of IV,
which are red. Head, pronotum, mesonotum, met-
anotum, lateral and middle plates dark brown.
Eyes and dorsal and ventral surface of abdomen
red. Head declivent, posterior margin contiguous
with anterior margin of pronotum; tylus longer
than juga and visible dorsally; eyes sessile, and
not reaching anterior angles of pronotum; ros-
trum surpassing metacoxa. Anterior margins of
pronotum, mesonotum and metanotum straight,
lateral margins rounded; metanotum half the
length of pronotum. Lateral abdominal plates
rectangular; increasing in size up to the fifth seg-
ment and then decreasing posteriorly. Middle
plates present on all abdominal segments and
characterized as follows: those on segments I and
II very thin; middle half of III compress and with
gland openings; plates on segments IV-V and V-VI
rectangular and with gland openings larger than
those on segment III; plates VI, VII, and VIII
much smaller and sometimes divided mesially,
spiracles and a pair of trichobothria present on
sternum II-VII. Body length 2.07 + 0.08; body
width 1.42 + 0.06; head length 0.6 + 0.04; head
width 0.94 + 0.02; interocular distance 0.62 +
0.02; length of antennal segments: I 0.2 + 0, II
0.26 0.005, III 0.26 0.007, IV 0.58 + 0.01;
length of rostral segments: I 0.22 + 0.01, II 0.31
0.01, III 0.28 0.008, IV 0.27 + 0.008; mesial
pronotum length 0.25 0.01; pronotal width
across humeri 1.12 + 0.08; pronotal width at ante-
rior margin 0.93 0.02; length of femur 0.63 +
0.02; length of tibia 0.59 0.01; length of tarsi: I
0.2 + 0, II 0.32 + 0.01.
Florida Entomologist 85(3)
Second Instar.-(Fig. 6) Oval, slightly flat-
tened dorsoventrally. Dorsum of head, thorax,
and abdomen punctate. Body setose. Head, anten-
nae, rostrum, legs, and lateral and middle abdom-
inal plates black. Eyes red, anuli between
antennal, leg and rostral segments yellow with
red marks. Abdomen red with numerous dorsal,
black punctures. Spiracles and trichobothria sur-
rounded by black spots. Tylus longer than juga,
rostrum extending beyond sternum VII; eyes kid-
ney shaped and slightly separated from prono-
tum; width through the eyes broader than the
width through anterior angles of pronotum; an-
tennal segments setose. Lateral margins of pro-
meso- and metatotum flattened; gland opening
present between propleura and mesopleura. Dor-
sal middle plates present on abdominal segments
I to VII; plates III-IV, IV-V, and V-VI with gland
openings; plates I and II long and narrow. Plates
VI-VII, VII, and VIII rectangular and smaller
than others; spiracles and a pair of trichobothria
present on sternum II-VII; abdomen slightly con-
vex dorsoventrally, reaching its maximum around
plate IV-V. Body length 3.29 0.09; body width
2.2 0.1; head length 1.08 0.05; head width 1.35
+ 0.01; interocular distance 0.86 0.02; length of
antennal segments: I 0.32 0.2, II 0.57 0.02, III
0.59 0.01, IV 0.8 0.02; length of rostral seg-
ments: I 0.48 + 0.01, II 1.04 + 0.02, III 0.66 0.02,
IV 0.6 0.02; mesial pronotum length 0.4 0.01;
pronotal width across humeri 1.66 0.06; prono-
tal width at anterior margin 1.31 + 0.03; length of
femur 1.07 + 0.02; length of tibia 1.14 + 0.02;
length of tarsi: I 0.22 0.01, II 0.4 + 0.008.
Third Instar.-(Fig. 7) Body eliptical, abdomen
strongly convex dorsoventrally. Maximum width
at abdominal segment II. Similar to second instar
except red coloration of abdomen turns darker
and the black areas acquire a green shine; anuli of
first antennal segment reddish. Ventral surfaces
of legs with numerous thick hairs, those of tibia
and tarsi III most dense. Dorsal middle plates
cover most of the abdomen, and appear as a con-
tinuous black longitudinal band. Middle, ventral
abdominal plates present on sternum IV, V, VI,
VII, and VIII, those on sternum V, VI, and VII
with striated areas (stridulitrum) on either side of
midline. Body length 4.51 + 0.27; body width 2.92
+ 0.16; head length 1.37 0.04; head width 1.57
0.05; interocular distance 0.98 0.03; length of
antennal segments: I 0.35 0.2, II 0.76 0.03, III
0.78 0.04, IV 1.03 0.04; length of rostral seg-
ments: I 0.73 + 0.03, II 1.41 0.05, III 0.9 0.03,
IV 0.86 0.02; mesial pronotum length 0.6 0.03;
pronotal width across humeri 2.26 0.08; prono-
tal width at anterior margin 1.54 0.04; length of
femur 1.48 + 0.02; length of tibia 1.52 + 0.08;
length of tarsi: I 0.31 + 0.01, II 0.56 + 0.02.
Fourth Instar.-(Fig. 8) Body eliptical, convex
dorsally, maximum width at abdominal segment
II. Head, thorax, antennae, rostrum, legs and lat-
eral and middle plates dark shiny green. Dark
green punctures present on head, thorax, and ab-
domen. Head declivent; tylus longer than juga;
rostrum reaching sternum VII. Lateral margin of
pronotum flattened; wing pads evident, wider
than long, reaching abdominal segment II; scutel-
lum reaching base of metanotum. Ventral abdom-
inal plates as in third instar. Body length 6.82
0.1; body width 5.05 + 0.09; head length 1.68
0.06; head width 2.61 + 0.03; interocular distance
1.63 + 0.03; length of antennal segments: I 0.54 +
0.2, II 1.17 0.03, III 1.15 0.03, IV 1.38 0.03;
length of rostral segments: I 0.9 + 0.04, II 1.84
0.03, III 1.09 0.03, IV 1.04 0.02; mesial prono-
tum length 1.11 0.02; pronotal width across hu-
meri 4.41 0.13; pronotal width at anterior
margin 1.31 + 0.03; scutellum length 1.65 0.03;
scutellum width 3.48 0.04; length of femur 2.28
0.04; length of tibia 2.47 0.05; length of tarsi:
I 0.49 + 0.02, II 0.74 + 0.03.
Fifth Instar.-(Fig. 9) Body round, convex dor-
sally, abdomen slightly concave ventrally; maxi-
mum width at mesonotum. Head, thorax, lateral
and middle plates and punctures metallic green;
antennae, rostrum, and legs dark brown with
metallic irridescent green; ventral middle plates
brown; rest of abdomen golden dorsally and yel-
lowish ventrally. Tylus longer than juga; ocelli
present for the first time; rostrum reaching base
of sternum V. Wing pads reaching base of abdom-
inal segment IV; scutellum wider than long,
reaching base of abdominal segment III. Body
length 10.11 + 0.15; body width 8.0 + 0.21; head
length 1.91 + 0.04; head width 3.41 + 0.06; in-
terocular distance 2.21 0.05; interocelar dis-
tance 0.45 0.02; length of antennal segments: I
0.7 + 0.3, II 1.75 0.03, III 1.51 + 0.05, IV 1.53
0.07; length of rostral segments: I 1.34 0.03, II
2.24 + 0.05, III 1.36 0.02, IV 1.23 0.02; mesial
pronotum length 1.91 0.02; pronotal width
across humeri 7.07 + 0.18; pronotal width at ante-
rior margin 3.77 0.09; scutellum length 3.4
0.08; scutellum width 5.66 0.17; length of femur
3.54 + 0.08; length of tibia 3.61 + 0.08; length of
tarsi: I 0.79 0.02, II 1.03 + 0.03.
Adult.-(Fig. 10) Overall, head shiny black
with metallic green sheen; antennal segments
shiny black, anuli reddish. Eyes brown and ocelli
red, other parts of the body ranging from a metal-
lic green to black, with macules that are ex-
tremely variable in size and color which ranges
from yellow to red (Figs. 11-19). Head, pronotum,
scutellum, and abdominal venter with dense
black puntuations, punctures on the macules
slightly paler. Rostrum and legs blackish. Head
wider than long, posterior margin contiguous
with anterior margin of pronotum, width through
eyes no greater than width through anterior an-
gles of pronotum; antennal segment I not reach-
ing apex of juga; juga shorter than tylus; eyes
kidney shaped; ocelli small; distance between
September 2002
Cervantes Peredo: Pachycoris klugii
1
2 .
3
I'
4r
Figs. 1-10 Different instars of P. klugii. Fig. 1. Dorsal view of egg. Fig. 2. Egg burster. Fig. 3. Lateral view of egg.
Fig. 4. Egg mass. Fig 5. First instar nymph. Fig. 6. Second instar nymph. Fig. 7. Third instar nymph. Fig. 8. Fourth
instar nymph. Fig. 9. Fifth instar nymph. Fig. 10. Adult.
t
r
Oe~o""',
116"I~
Florida Entomologist 85(3)
11 12 13
14 15
17 18 19
Figs. 11-19. Variation in the pattern of macules of P. klugii, Fig. 15 represents the basic pattern.
16
September 2002
Cervantes Peredo: Pachycoris klugii
ocelli greater than distance between ocelli and
eyes; antennal tubercule short; bucculae well de-
veloped, covering half of rostral segment I; ros-
trum almost reaching base of sternite IV.
Pronotum wider than long; anterolateral margins
slightly concave; anterior and humeral angles
with rounded apices; anterior margin concave;
posterior margin straight; posterolateral margins
sinuated; orifice of metathoracic peritreme small,
evaporative area covering three quarters of meta-
pleura. Ventral surface of all legs with numerous
thick hairs, those on metathoracic leg more dense,
forming a plectrum (Fig. 20). Sterna V, VI, and VII
striated (stridulitrum) (Fig. 21).
Female.-(Figs. 22, 23) Slightly larger than
males. Gonocoxae I rectangular, elongated;
paratergites IX rectangular with round apices.
Spermatheca spherical with two sclerotized rings
around the oviduct. Body length 17.12 + 0.23; body
width at II abdominal segment 10.76 + 0.15; head
length 3.54 + 0.08; head width 4.34 0.03; interoc-
ular distance 2.76 0.03; interocellar distance 0.5
0; length of antennal segments: I 0.92 0.2, II
1.0 + 0.02, III 1.41 0.01, IV 2.14 0.04, V 2.35
0.04; length of rostral segments: I 1.65 0.03, II
3.08 0.05, III 1.71 0.02, IV 1.61 + 0.03; mesial
pronotum length 4.62 0.07; pronotal width
across humeri 10.71 + 0.11; pronotal width at an-
terior margin 4.32 0.05; scutellum length 10.47 +
0.20; scutellum width 10.23 + 0.1; length of femur
5.21 0.05; length of tibia 4.97 + 0.05; length of
tarsi: I 0.87 0.02, II 0.41 + 0.01, III 0.73 + 0.02.
Male.-(Figs. 24-26) Pygophore large, with
posterior margin concave; parameres "L" shaped,
bases robust and apices acute; aedeagus with four
apical sclerotized projections, two broad and two
knife shaped with a small tooth three quarters
from its base. Body length 15.01 + 0.26; body
width 9.17 + 0.19; head length 3.26 + 0.06; head
width 3.95 + 0.06; interocular distance 2.48 +
0.06; interocellar distance 0.42 0.01; length of
antennal segments: I 0.86 0.2, II 0.93 0.02, III
1.37 + 0.02, IV 1.98 0.03, V 2.2 0.03; length of
rostral segments: I 1.51 0.04, II 3.54 0.06, III
1.55 + 0.03, IV 1.44 0.03; mesial pronotum
length 3.9 0.11; pronotal width across humeri
9.38 + 0.18; pronotal width at anterior margin
3.92 0.09; scutellum length 9.13 0.16; scutel-
lum width 8.68 0.21; length of femur 4.53 0.1;
length of tibia 4.45 + 0.11; length of tarsi: I 0.8 +
0.02, II 0.39 0.01, III 0.73 + 0.02.
Variation.-Figs. 11-19) Macules on pronotum
and scutellum are variable in color (red, orange, or
yellow), and number, position and size. The basic
pattern on the pronotum consists of eight macules
(Fig. 15) as follows: one semicircular macule lo-
cated on each anterolateral margin; one semicircu-
lar macule on each posterolateral margin; two
slightly elongated macules, situated on the mid-
line, one reaching the anterior margin and the
other one near the posterior margin; two round
macules laterad of the midline and between the
other macules. Macules on the midline are almost
always present and sometimes fused with each
Figs. 20, 21. Fig. 20. Posterior leg of P. klugii. Fig. 21. Sterna VII, VI, and V, showing the striated areas (stridu-
litrum).
::::: ~:.;: :::'r~:: '
i:.~:,'~'~;:: -;~~
:.li~:ili!:"~ -::: '"'
:-
::
.i:I
1:.. .;;'
:- i:~
-:.:: :::~::~
jl
Florida Entomologist 85(3)
22 23
Figs. 22, 23. Fig. 22. Genital plates of female of P. klugii. Fig. 23. Spermatheca.
other; other macules can vary in size, be fused or
disappear completely. The basic pattern on the
scutellum consists of 14 macules as follows: one
pair of semicircular macules located on the lateral
margin, near the base of scutellum; another mac-
ule situated immediately posterior to the first pair
and separated by a distance similar to the size of
the macule; one pair of triangular macules situ-
ated laterally on the posterior half of the scutel-
lum; two triangular macules situated on the
posterior end of scutellum; one triangular macule
located basally on the midline; one pair of round
macules situated on each side of midline; one irreg-
ular macule situated on middle line between the
triangular macules of the posterior half of scutel-
lum, which may be divided in three small macules.
All macules can vary in size, shape, color, can be
fused, resulting in a range of 4 to 14 macules.
Biology.-In Mexico, this subsocial bug was ob-
served feeding exclusively on C. multilobus.
Pachycoris klugii was found in all months of the
year, although it was more abundant between
March and September. It had at least two gener-
ations each year. Females began ovipositing in
early March and continued until August. Females
laid an average of 81.4 + 3.7 eggs (n = 10) in a com-
pact and regular mass. Eggs were oviposited sin-
gly following a regular pattern. After one egg was
deposited, the second one was deposited next to
the first, the third egg was deposited on a new line
between the two eggs previously deposited. The
fourth egg was positioned next to the second egg
in line with the egg one. The fifth egg was posi-
tioned between the second and fourth, next to the
third egg. The female continued in this way until
the first two lines had between 5 and 7 eggs each.
She then began the next line, which usually had
one more egg than the previous line. This pattern
was followed to make two or three lines at a time.
When a line reached 10 eggs, the number of eggs
per line started to decrease until the last row had
three to five eggs. In this way a regular and uni-
Figs. 24-26. Fig. 24. Pygophore of P. klugii. Fig. 25. Lateral view of paramere. Fig. 26. Aedeagus.
September 2002
Cervantes Peredo: Pachycoris klugii
form mass was formed, in a rhomboid shape. The
eggs are glued to the underside of a still slightly
curled new leaf.
As soon as the female deposited the last egg,
she positioned her body over the whole mass. The
egg mass surface (77.95 + 6.03 mm2) was smaller
than the females' ventral area (161.6 4.46 mm2),
so females usually covered the entire mass. She
stayed in that position until the first instar
nymphs molted. When the female was ap-
proached by an egg parasitoid, a parasitoid fly, or
predator, she moved her body down according to
the direction from which the intruder was ap-
proaching. During a frontal approach, the female
moved her antennae towards the intruder. Occa-
sionally the female also moved her legs and tried
to repel the intruder. The mother sometimes
turned around, towards the intruder, so she may
not have maintained the same position through-
out the period when she was guarding the eggs
and nymphs. Eggs hatched around 6 days after
oviposition. First instar nymphs remained under
the mother's body without much movement. After
5 days they molted and second instar nymphs
started to move away from the mother in a group.
By this time the female appeared to be weakened,
possibly because we did not observe her feeding
while guarding the eggs and nymphs. Females
kept in the lab while guarding the eggs died as
soon as the second instars appear. Third instar
nymphs appeared after six days, and remained
together. It was common to find masses of 30 to 40
individuals of third, fourth, and fifth instars on
the host plant, which probably corresponded to
the same cohort.
As with many pentatomids and other scutel-
lerids, third, fourth, fifth instar nymphs and
adults (except guarding females) dropped from
the plant and feigned death when disturbed. It
was also common to observe P klugii nymphs and
adults stridulate and expel liquid through their
anus and metathoracic scent glands when they
were disturbed. Duration of fourth and fifth in-
stars was about seven days each depending on the
quality of food and temperature. Adults were
found in groups, but numbers were much lower
than in the groups of nymphs. Three to seven
adults were found on the same plant. Males usu-
ally lived longer than females, and were kept
alive for up to a year. Females that had oviposited
usually died after the second instar nymphs ap-
pear. Females that reached September or October
without producing eggs, survived until March or
April of the next year, at which they oviposited for
first time. A large number of second instar
nymphs died for unknown reasons, as has been
observed in many other Hemiptera (Brailovsky
et al. 1992). This high mortality may be due to a
change in the habits of the nymphs, first instar
nymphs usually do not feed. It has also been men-
tioned that in order to process their food, nymphs
need to acquire some microorganisms that live in
their gut (Buchner 1965).
Both nymphs and adults feed on new leaves
and were commonly found feeding on fruits and
flowers of the host plant. When molting, nymphs
usually perched on the under side of a fully ex-
panded old leaf. From October to February the
number of P klugii on C. multilobus was lower. In
localities with colder climates, it was common to
find adults on Bromeliaceae T'ii-j.....! ., spp.).
Adults were also found on Yucca sp. Only adults
were found in these plants and were probably in
some stage of diapause until warmer tempera-
tures return.
Egg Parasitism.-Although P. klugii females
guard their eggs, they were often parasitized by
the wasp Telenomus pachycoris (L.) (Scelionidae).
More than 25 egg masses were collected in the
field but because only 10 of these masses were
complete and collected with the mother, it was
only possible to use these 10 for analysis of para-
sitism. Of these 10 masses, two of them were not
parasitized at all. The proportion of eggs attacked
varied from zero to 88.5% with a mean of 38.98 +
8.85 (n = 10). Most of the time the eggs that were
parasitized were the ones on the edges of the
mass. Eggs in the center of the mass were the
least frequently parasitized. It was noticed that
the eggs that were covered by the rear end of the
female's body were more often parasitized than
the ones near the head; however, no statistical
analysis was performed because the females
sometimes changed position. Male parasitoids
usually emerged earlier than females and re-
mained at the egg mass waiting for the females to
emerge. Unparasitized eggs hatched at the same
time as the parasitoids or not more than two days
later. Females held under laboratory conditions
occasionally laid more eggs on the top or side of
the old ones; as soon as they were laid, female
parasitoids oviposited in them. A few eggs man-
aged to escape parasitoids and produced nymphs.
Egg masses found in the field were arranged in
regular masses and did not display different
times of eclosion or parasitism, suggesting that
females did not oviposit after egg eclosion under
natural conditions.
Adult Parasitism and Predation.-Of the
adults collected in the field and the ones depos-
ited in the collections, a small proportion had eggs
of tachinid fly, Trichopoda pennipes (Fab). Five
males and two females were parasitized. The par-
asitoid eggs were usually found on top of a spira-
cle or on the margin of the scutellum. Males had
only one egg, while one female bug had two eggs
(on spiracles V and VII) and the other female had
three eggs (on the border of the scutellum). How-
ever, only one adult parasitoid was obtained from
each individual. The fly larva usually emerged
through the genital opening when the bug was
still alive, then pupated in the soil. The adult flies
Florida Entomologist 85(3)
emerged 15 to 20 days later. The bugs died after
parasitoid larvae emerged.
The spider Peucetia viridans Hentz (Oxyopi-
dae) was commonly found nesting on C. multilo-
bus and in a few occasions it was observed feeding
on nymphs and adults of P. klugii.
Distribution.-This species has been reported
several times as being present in Mexico, but with
no exact locality. The records of the specimens
studied in the present work, are as follows: MEX-
ICO. CHIAPAS. El Chorreadero, Reserva del Ocote,
Pijijiapan, El Porvenir, March, July, and Decem-
ber. DURANGO. Los Chirimollos, Km 250 Du-
rango-Mazatlan, October GUERRERO. Venta
Viga, Km 110 Coyuca de Catalan-Zihuatanejo,
Km 6 Acapulco-Filo del Caballo, April-June Sep-
tember. HIDALGO. Tlacolula, 12 Km South of
Ixmiquilpan, Km 30 Tasquillo-Huichapa, Yahalica,
Huejutla, June, October, December. JALISCO.
San Martin de Bolanos, Biology Station UNAM,A.
Somitla, 26-27-XII-1992, HB, KB, 1 9. Sierra de
Manantlan, August, October-December. MEXICO.
Acamochitlan, El Zapote, San Mateo, November,
December. MICHOACAN. Morelia, Zirahien, Tz-
intzuntzan, May, June, October. MORELOS. El
Tepozteco, February. OAXACA. Chiltepec, Hua-
juapan de Le6n, Pluma Hidalgo, Km 16 Miahua-
tlan-Puerto Escondido, Portillo del Rayo, Puente
Nacional, Km 6 Oaxaca-Guelatao, Tlacolula, Hi-
erve El Agua, Tultepec, February-November.
PUEBLA. Necaxa, Hidroel6ctrica de Tlalchichilco,
Km 7 Pahuatlan-San Pablito, Pahuatlan, Lecho
del Rio, Xicotepec de Juarez, Planta Tepexic, Km 3
Xicotepec-Barranca de Patla, January, May-July.
QUERETARO. Rancho Aztlan, 10 Km North of
Quer6taro, Higuerillas, June, August. QUIN-
TANA ROO. Coba, Chetumal, Km 146 Chetumal-
Cancin, March, May, October, November. SAN
LUIS POTOSI. C. Bolivar, Los Sabinos, Septem-
ber. TAMAULIPAS. G6mez Farfas, Rancho El
Cielo, April. VERACRUZ. Coatzintla, Esquilon,
Biology Station UNAM, Cabanas, Jalcomulco,
Tlacolulan, Xalapa, January-December.
DISCUSSION
Pachycoris species are very similar to each
other although few differences exist among their
genitalia. These similarities have led to confusion
among some species including P klugii which has
been identified as Pachycoris torridus (Scopoli) on
several occasions. Pachycoris torridus also occurs
in Mexico, although it is quite rare, and is more
commonly found in South America. The two can be
distinguished because P. klugii has a longer
scutellum and the head of the parameres are
longer than in P torridus (Eger, pers. comm.). The
great variability in the number of spots and the
different colors on the pronotum and scutellum of
P klugii has caused confusion with this species,
resulting in the description of different varieties of
P torridus, such as P torridus var. decorate Perty,
P torridus var. aguila H.S. These findings may
suggest that a revision of the genus is needed.
In 1934, Hussey described the guarding behav-
ior of P. torridus in Paraguay. His observations
were mainly anecdotal. He did not describe the
immature stages or the adult. Although the be-
havior of P torridus described by Hussey (1934) is
very similar to the one described in the present
study for P. klugii, there are some differences. The
host plant, Sapium haematospermum Mill. Arg.,
was different but belongs to the same family (Eu-
phorbiaceae). Percent parasitism was much lower
than we observed, being 15% for P torridus and
about 38% for P klugii.
Similar guarding behavior has been recorded
for P. stallii Uhler, which is species also found on
the west coast of Mexico and feeds on a species of
Croton (Williams et al. 2001). Hussey (1934) men-
tioned that P. fabricii (L.), a species found on a
number of Caribbean Islands, also exhibits this
guarding behavior. Grimm (1996, 1999) has also
reported the maternal care of P. klugii and found
that this species feeds exclusively on J curcas.
These records, suggest that species of Pachycoris
are subsocial and restricted to Euphorbiaceae.
In this study, the life cycle of P klugii was more
or less synchronized with that of its host plant.
Adults appear on C. multilobus usually between
March and April, when the plant starts producing
new shoots. The bugs feed on stems, leaves, flower
or fruit, so food resources are always present.
Also, C. multilobus (at least in Los Tuxtlas) seems
to produce flowers and fruit at different times of
the year (pers. obs.)
Grimm & Somarriba (1998) in Nicaragua, did
not find an alternative host. They found that the
life cycle of this bug is closely related to the fruit-
ing cycle of the host plant. This probably was due
to the fact that P. klugii preferred to feed on devel-
oping and ripe fruit. The system in that study was
a plantation, so reproduction of the host plant
was synchronized, facilitating the observation of
host phenology.
In this study females collected in the field ovi-
posited only once, except in two cases, where fe-
males laid more eggs above those that were
parasitized. Grimm & Somarriba (1998) reported
that each female oviposited on average 2.4
masses. Size of the egg masses may explain this
differences in the two studies. In Los Tuxtlas, fe-
males laid masses of around 81.4 eggs, while in
Nicaragua the egg masses had only 29.7 eggs. The
size of leaf surface available may play a role in the
size of egg masses. Mexican bugs in our study
were smaller than the bugs from Nicaragua
(Grimm & Somarriba 1998) suggesting that there
may be nutritional differences between the two
plant species.
As with many pentatomids (Brailovsky et al.
1992) and other scutellerids (Walt & McPherson,
September 2002
Cervantes Peredo: Pachycoris klugii
1972), first instar nymphs of P klugii remained
near the eggs. Staying under the mother's body
may help them escape predation. All nymphal
stages remained together and on the same plant
where they were born, probably increasing the
chances of finding food or a mate. They may also
escape predators and parasitoids because of their
large numbers. While guarding its young, the
mother may be an easier target for predators or
parasitoids.
According to Wilson (1979) guarding behavior
is limited to species that live in very harsh envi-
ronments in which there is a great variability in
the abundance of resources, drastic climatic con-
ditions, or that are submitted to high predation or
parasitism. For P. klugii, its host plant in Mexico
is abundant year around, so there is no variation
in the abundance of food resources. In Nicaragua
it seems that the bug is synchronized with the
phenology of its host plant, and numerically re-
spond when fruit is available. This species is
found in the tropics, where climatic conditions do
not vary in a great scale. So if we observe this be-
havior in terms of Wilson's (1979) statements, the
only probable evolutionary explanation for the
guarding behavior of this species is the egg para-
sitism and the females guard the eggs to avoid
losing their young.
ACKNOWLEDGMENTS
I gratefully acknowledge Dr. Joe Eger (Dow Agro-
Sciences) for his comments regarding this manuscript
and for checking some specimens of P. klugii. I also
thank Dr. James O'Hara (Canadian National Collec-
tion) for identifying the tachinid parasitoid. I wish to
thank three anonymous reviewers for helpful comments
on the manuscript.
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Florida Entomologist 85(3)
September 2002
ARTIFICIALLY-REARED WHITEFLIES, BEMISIA ARGENTIFOLII,
(HOMOPTERA:ALEYRODIDAE) AS HOSTS FOR PARASITIC WASPS
ELIZABETH W. DAVIDSON1, FAYE E. FARMER1 AND WALKER A. JONES2
'Department of Biology, Arizona State University, Tempe, AZ 85287-1501
2USDA-ARS Beneficial Insects Research Unit, Weslaco, TX 78596
ABSTRACT
Bemisia argentifolii (Powell & Bellows) (Homoptera: Aleyrodidae) (= B. tabaci Gennadius B
biotype) nymphs reared on an artificial feeding system were successfully parasitized by three
species of wasps, Encarsia formosa Gahan, Eretmocerus eremicus Rose and Zolnerwich, and
Eretmocerus mundus Mercet (Hymenoptera: Aphelinidae). Encarsia formosa and Eretmo-
cerus spp. preferred to parasitize third and fourth instar nymphs including the late fourth
instar "red eye" stage. Oviposition rate and developmental time compared favorably to some
reports on plants, but successful production of adult wasps was low. This system can be used
to elucidate host-parasite interactions without confounding interactions with plants.
Key Words: Encarsia formosa, Eretmocerus eremicus, Eretmocerus mundus, Bemisia tabaci
B-biotype, parasitoid, biological control, rearing
RESUME
Las ninfas de Bemisia argentifolii (Powell & Bellows) (Homoptera: Aleyrodidae) (= B. tabaci
Gennadius B biotype) criadas del sistema artificial de alimentaci6n fueron parasitadas exi-
tosamente por tres species de avispas, Encarsia formosa Gahan, Eretmocerus eremicus
Rose and Zolnerwich, y Eretmocerus mundus Mercet (Hymenoptera: Aphelinidae). Encarsia
formosa y Eretmocerus spp. preferieron parasitar la tercera y cuatra estadia ninfal inclu-
yendo la estadia cuatra tardia de "ojo rojo". La tasa de oviposici6n y el tiempo de desarrollo
comparaban favorablemente a algunos reportajes sobre plants, pero la producci6n exitosa
de las avispas adults fu6 baja. Se puede usar este sistema para elucidar las interacciones
del hospedero-parasito sin complicar las interacciones con las plants.
Parasitic wasps are important biological con-
trol agents for Bemisia argentifolii (Powell & Bel-
lows) (Homoptera: Aleyrodidae) (= B. tabaci
Gennadius B biotype), a major pest of cotton,
melon and other crops and a vector of begomovi-
rus. We report here the first successful parasitism
of whitefly nymphs reared on a sterile artificial
feeder system. Three of the major parasitoid spe-
cies commercially sold for management of B. ar-
gentifolii and other whitefly species in field and
greenhouse situations were included in these ex-
periments. Use of the artificial rearing system
should permit further studies of parasitoid phys-
iology, behavior, and host/parasite interactions
without the confounding effects of plant biochem-
istry. Refinement of this system could potentially
lead to commercial production of parasitoids
without the requirement for plants.
MATERIALS AND METHODS
Whitefly Feeder System
B. argentifolii nymphs were reared on a sterile
artificial feeding system as described by Jancov-
ich et al. (1997) and Davidson et al. (2000). The
feeder system, which is comprised of a 2-piece
polycarbonate chamber holding a 45 mm, 1.0 pm
pore-size Teflon filter membrane (Micron Sepa-
rations-Osmonics, Inc., Westboro, MA) was auto-
claved before filling with a diet consisting of filter-
sterilized 15% sucrose plus 5% Difco (Detroit,
MI) yeast extract solution. Whitefly eggs were
harvested from cotton or melon leaves using a
WaterPik dental device and surface-sterilized
using 70% ethanol and 10% Roccal II (Sterling
Drug) solution before being placed on the mem-
brane (Davidson et al. 2000). This membrane has
a regular geometric pattern on its surface that
permitted accurate mapping of each parasitized
nymph (Fig. 1). Adult B. argentifolii have been
produced on this feeder system and diet (David-
son et al. 2000).
Sterilization and Introduction of Parasitoids
Encarsia formosa pupae were harvested from
a laboratory colony maintained on B. argentifolii
on cotton in a greenhouse at Arizona State Uni-
versity or from B. argentifolii maintained on
sweet potato at the USDA-ARS Beneficial Insects
Research Unit, Weslaco, Texas. Eretmocerus ere-
micus and E. mundus pupae were obtained from
the Weslaco, Texas facility where they were reared
Davidson et al.: Artificial Rearing of Parasitoids
. .
~- P
Fig. 1. A. E. formosa-parasitized whitefly nymph on feeder membrane. B. E. mundus-parasitized nymph on
feeder membrane.
Florida Entomologist 85(3)
on a B. argentifolii colony on sweet potato. E. ere-
micus originated from a culture established by
Koppert, Inc., California; E. mundus was origi-
nally collected near Murcia, Spain. Wasp pupae
removed individually from leaves using a fine-
pointed metal needle were surface-sterilized by a
brief rinse in 70% ethanol. They were then placed
in 10% Roccal (Sterling Drug, Montvale, NJ) so-
lution for two minutes, followed by a rinse in ster-
ile water. Surface-sterilized pupae were placed in
a sterile petri dish with a small drop of sterile
honey, and held in a growth chamber at 28C, 14/
10 L/D.
Female wasps were chilled until immobile and
moved to whitefly feeders 1-3 d after emergence.
One wasp was confined to each chamber under a
sterile slide for 24 or 48 h at 28C, 14/10 L/D. Any
chambers that became contaminated by fungi
were discarded. E. formosa were not mated as
they are uniparental parasitoids. Eretmocerus spp.
females were exposed to males that emerged from
the same group of surface-sterilized pupae but
mating status was not determined. Mating is not
necessary for egg production by E. formosa,
E. mundus or E. eremicus (Gerling 1966; Vet &
van Lenteren 1981).
Estimation of Parasitism
On this feeder system, 25-50% ofB. argentifolii
nymphs develop at least to the third instar by
14 d after egg application (Davidson et al. 2000).
E. formosa females were added to feeders 19.5 d
(+1.5 d) after whitefly egg application. Female
E. mundus and E. eremicus were applied to feed-
ers 17.5 d (1.5 d) after whitefly egg application.
Whitefly nymphs were counted by stage within
two d after wasp application. The "red eye" (pre-
adult) stage was counted as a separate category
from early fourth instar. Development of nymphs
on feeders was not synchronous; all stages were
present at the time of introduction of wasps.
Whitefly nymphs were observed every three d
for 23 d (5 d) after exposure to a wasp. Nymphs
with evidence of E. formosa host-feeding or stings
(circular, brown to black holes in the host's dorsal
surface) were counted, host instar was recorded,
and the location of the host and stage of the para-
site were mapped on a computer-scanned, magni-
fied image of the feeder membrane. Twenty
feeders exposed to E. formosa for 24 h and 7 feed-
ers exposed for 48 h remained uncontaminated by
fungus for the full experimental period.
Eretmocerus eremicus and E. mundus are
initially ectoparasitoids, leaving no distinctive
marking on the host (Gerling 1966; Powell & Bel-
lows 1992). Changes in the location of the myce-
tomes, and color and shape of the whitefly nymph
were used as indicators of parasitism. Eretmo-
cerus-parasitized nymphs were also dissected 30
d after wasp exposure to confirm parasitism.
Data Analysis
Normality could not be obtained through
transformation; therefore, nonparametric statis-
tics were used in all analyses. Kruskal-Wallis
nonparametric ANOVA (SAS version 8.0; SAS
Institute, Inc., Cary, NC) was applied to raw data
to determine significant differences among: num-
ber of available whitefly nymphs within each
stage; number of nymphs stung by stage; and
number of successful wasp eggs by host stage.
Where significant differences were found, pair-
wise comparisons were performed using the
Nemenyi test, a variation of the Tukey test (Zar
1996). Because sample sizes were not the same
for 24 and 48 h experiments, these data could not
be grouped for nonparametric analysis. Statisti-
cal analyses were performed on data from E. for-
mosa only, as the numbers exposed to Eretmocerus
spp. were too small for analysis.
Whitefly stages, E. formosa stings, and "suc-
cessful" E. formosa eggs in each stage (those that
produced recognizable larvae) were pooled within
each time period and averaged using Excel
(Microsoft 2000; Microsoft Corp., Redmond, WA).
Averages and standard deviations for successful
eggs in each stage were divided by the average to-
tal number of successful eggs on the feeders to
find the proportions displayed in Figure 4.
RESULTS
E. formosa
Significant differences were found among the
numbers of available whitefly nymphs within
each stage exposed to E. formosa for 24 h (X24 =
35.233, P = 0.0001) or 48 h (x24 = 21.688, P =
0.0002). Pairwise comparison using rank sums
showed no significant differences between the
numbers of first, second, third or fourth instar
whitefly nymphs available for parasitism by
E. formosa for either 24 or 48 h; however, signifi-
cantly fewer "red-eye" stage forms were available
at both time periods (Fig. 2).
Significant differences were found among the
numbers of whitefly nymphs stung by stage at
24 h (x24 = 48.78, P < 0.0001) and 48 h (x2, = 20.68,
P = 0.0004). Pairwise comparison using rank
sums showed no significant differences between
the numbers of first, second, and "red eye"
nymphs stung. However, significantly more 3rd
and 4th instar nymphs were stung (Fig. 3).
Fourth instars, including the "red eye" stage,
received proportionally the most stings (68%). An
average of 11.8 (8.5) total whitefly nymphs per
feeder were stung by each E. formosa female in
24 h, and 19.7 (14.8) were stung in 48 h. Only
one E. formosa out of 27 failed to sting whitefly
nymphs during confinement on a feeder (for 48 h).
First or second instar whitefly nymphs were
September 2002
Davidson et al.: Artificial Rearing of Parasitoids
) 120
S100
z 80
6 60
40
20
0
A
*
Bb
124 Hour
D 48 Hour
First Second Third Fourth Red Eye
Fig. 2. Total average whitefly nymphs per feeder unit within each stage available for parasitism by E. formosa
(24 h: X24 = 35.233, P = 0.0001; 48 h: X24 = 21.688, P = 0.0002).
rarely stung by E. formosa, representing less
than 2% of total stings.
Significant differences were found among the
number of successful wasp eggs, i.e., eggs that de-
veloped into a larva or pupa, by host instar for
24 h (x24 = 26.48, P < 0.00001) and for 48 h expo-
sure to a wasp (X24 = 10.29, P = 0.0358). Pairwise
comparison showed significantly more 4th instars
were hosts to successful eggs than any other in-
star during 24 h exposure to a wasp. Due to small
sample size for 48 h exposure, pairwise compari-
son showed no significance.
Eleven of the 20 E. formosa females that re-
mained on feeders for 24 h deposited successful
eggs. On average, these females laid 2.7 (1.9)
successful eggs per wasp in 24 h, which repre-
sented 12.5% of stings produced by these females.
Four of the seven feeders that received E. formosa
females for 48 h produced successful eggs. These
wasps produced an average of 6.3 (2.9) success-
ful eggs each, representing 18.2% of their stings.
The remaining 3 females, placed on feeders for 48
h, did not produce detectable larvae or pupae. The
majority of "successful" eggs (leading to a detect-
able wasp larva, pupa or adult) produced during
either 24 or 48 h were laid in fourth instar or "red
eye" stage (Fig. 4).
Development of offspring to eclosion was ob-
served on 4 feeders (14.5% of "successful" eggs)
within twenty-eight d after exposure to female
E. formosa. Two percent of stung whitefly
nymphs produced adult female E. formosa, 9% of
the wasps remained as pupae, 12% remained in
larval stages, and 77% of stings did not develop
apparent larvae. Metamorphosis to pupae was
first observed 16.7 d (3.2 d) and eclosion of adult
wasps was first observed 24.8 d (3.9 d) after
exposure to wasps. Eighty-nine percent of E. for-
mosa that developed to pupa or adult were pro-
duced in fourth instar hosts; the remainder were
produced in third instar hosts.
Eretmocerus spp.
Eretmocerus spp. were unable to penetrate be-
neath the host when Parafilm membranes were
initially used on the whitefly feeder system (Jan-
covich et al. 1997), but successfully deposited eggs
Florida Entomologist 85(3)
124 hour
B 48 hour
Aa
First Second Third
Fourth Red Eye
Fig. 3. Whitefly nymphs per feeder unit within each stage that were stung by E. formosa (24 h: X2, = 48.78, P >
0.0001; 48 h: X2, = 20.6848, P = 0.0004).
under nymphs when the Teflon membrane with
texture was used. All Eretmocerus spp. larvae and
pupae developed in fourth instar whitefly larvae.
Host feeding was not detected in nymphs exposed
to Eretmocerus spp. females, as this species host-
feeds by probing the vasiform orifice of the host
and does not produce melanized spots on the dor-
sal surface of the host (McAuslane & Nguyen 1996).
Eleven E. mundus females were confined indi-
vidually to whitefly feeders for 24 h and 11 for
48 h. Seven E. mundus deposited successful eggs
that produced larvae during 48 h exposure to
nymphs, but none were produced during 24 h ex-
posure. By 28 d after exposure to E. mundus,
twelve wasp pupae and 7 larvae were observed.
Seven E. eremicus were confined individually to
feeders for 24 h and 7 for 48 h. Three wasp pupae
resulted from eggs oviposited by females confined
for 24 h (two from the same female), and none by
those confined for 48 h. No adult E. mundus or
E. eremicus wasps were produced from whiteflies
reared on the artificial feeder system.
DISCUSSION
E. formosa parasitism ofB. argentifolii on arti-
ficial feeders is comparable in some respects to
that observed on plants. The "successful" eggs
that developed into larvae were generally laid in
3rd instar nymphs or higher, as is also found on
plants (e.g., Nechols & Tauber 1977; Kidd &
Jervis 1991; Hoelmer 1996; Hoddel et al. 1998;
Jones & Greenberg 1999). The preference of
E. formosa for hosts that are 3rd instar and older
is reflected in the number stung within each in-
star (Fig. 3) in comparison to the number of
nymphs available (Fig. 2). Alternatively, whitefly
nymphs that had not progressed to third or fourth
instar by ca. 20 d may have been recognized by
the wasps as less desirable hosts. If eggs were laid
in first or second instar hosts, the wasp larvae
may not have developed sufficiently to be detected
as a "successful" parasitism. E. formosa females
laid about 3 "successful" eggs per female per day
on B. argentifolii on feeders, which compares fa-
vorably with oviposition rates reported by Heinz
(1996) on B. argentifolii (1.3-7.4 eggs/d) but is less
than the 8-10 eggs/d observed at similar temper-
atures by Enkegaard (1994) on plant-fed B. tabaci
and Vet & Van Lenteren (1981) on Trialeurodes
vaporariorum. The time to emergence of adult
E. formosa on B. argentifolii reared on feeders,
19-27 d, is less than the range reported by Vet &
Van Lenteren (1981) for E. formosa reared on
Aa
Aa
September 2002
Davidson et al.: Artificial Rearing of Parasitoids
124 hour
E 48 hour
First Second Third Fourth Red Eye
Fig. 4. Proportion of successful E. formosa eggs (forming detectable larva or pupa) by host stage.
T vaporariorum on plants (28-38 d for females
and 31-35 d for males).
Wasp production on the feeder system was far
less efficient in producing live adult daughter
wasps than that recorded on nymphs on plants.
The percentage of host punctures that did not
lead to development of larvae (77%) is far higher
than on plants, where around 30-40% of punc-
tures have been attributed to host feeding (En-
kegaard 1994; reviewed by Heinz 1996). On the
feeders, wasp females were confined to a much
smaller area (2 x 3 cm) and more dense host pop-
ulation (ave. 185 nymphs/feeder) than on the leaf,
which probably contributed to a high level of host
feeding. Gerling (1966) observed host feeding by
protein-starved E. formosa females, leading to
death of the hosts. Since E. formosa females were
held without a protein supply until confined to
feeders, it is probable that they were protein-
starved. Encarsia formosa is not a highly efficient
parasitoid ofB. argentifolii, B. tabaci, or T vapo-
rariorum on the plant. Parasitism rates from 2-
3% to 30% have been reported (Bethke et al. 1991;
Henter & Van Lenteren 1996; Hoddle & Van Dri-
esche 1999).
Eretmocerus spp. were less successful on the
feeder system than E. formosa, although parasit-
ism was observed. Because the feeder system was
subject to fungal contamination and greatly re-
duced survival of whitefly hosts after more than
ca. 30 d, our experiments may have been termi-
nated before Eretmocerus spp. larvae could com-
plete development.
Our study has demonstrated that artificially-
reared B. argentifolii nymphs are acceptable
hosts for both E. formosa and Eretmocerus spp.
parasitoids. However, successful production of
adult wasps from these feeders is far below that
observed on Bemisia spp. reared on plants, and is
not currently practical for commercial use. Never-
theless, the feeder system provides a method for
investigating important factors that impact the
ability of these parasitoids to control B. argenti-
folii in greenhouse or field situations. For exam-
ple, this system enables investigations on the
influence of the host plant chemicals on parasite
efficiency. Although surface characteristics of the
leaf have been shown to affect E. formosa search-
ing behavior (De Barro et al. 2000; reviewed by
Hoddle et al. 1998), much less is known about nu-
tritional and chemical factors involved in host-
plant differences in E. formosa parasitism effi-
ciency (Van Lenteren et al. 1987; Bentz et al.
1996; reviewed in Hoelmer 1996 & Hoddle et al.
1997). In other experiments, we have used this
system to investigate the effects of feeding the an-
tibiotic tetracycline to E. formosa females, which
is reported to eliminate the Wolbachia symbionts
0.25
S0.2
0.15
Cn
C 0.1
O 0.05
U)
16
(Hunter 1999). Male E. formosa offspring were
produced, demonstrating that the artificial feeder
system may be useful for studies of the wasp sym-
bionts, as it has been for studies of the whitefly
symbionts (Davidson et al., unpubl.).
ACKNOWLEDGMENTS
This research was supported by USDA CSREES
9702182 and a Cooperative Agreement with the USDA-
ARS Beneficial Insects Research Unit, Weslaco, TX. We
thank J. Blackmer and S. Naranjo for editorial and sta-
tistical advice. We are also grateful to Michelle Faye and
William Warfield for assistance.
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Florida Entomologist 85(3)
Robacker & Fraser: Mexican Fruit Fly Attraction to Chapote
DO MEXICAN FRUIT FLIES (DIPTERA: TEPHRITIDAE)
PREFER GRAPEFRUIT TO YELLOW CHAPOTE, A NATIVE HOST?
DAVID C. ROBACKER AND IVICH FRASER
Crop Quality and Fruit Insects Research, USDA, Agricultural Research Service
Kika de la Garza Subtropical Agricultural Research Center
2413 E. Highway 83, Building 200, Weslaco, TX 78596
ABSTRACT
Wild strain, mated, female Mexican fruit flies, Anastrepha ludens (Loew), with no prior expe-
rience with fruit (naive), were not attracted to and did not attempt oviposition in yellow
chapote (Sargentia ~ .. fruit more so than grapefruit (Citrus paradisi) in wind tunnel ex-
periments. Naive, mated laboratory strain females preferred grapefruit. Prior experience with
chapote increased attraction of both laboratory and wild strains to chapote. More naive than
chapote-experienced females of both strains attempted to oviposit on the sides of the wind tun-
nel. Naive laboratory strain males were more attracted to grapefruit than chapote. Naive wild
males and chapote-experienced wild and laboratory males did not prefer either fruit.
Key Words: Anastrepha ludens, fruit fly, grapefruit, yellow chapote, attraction, oviposition,
experience
RESUME
Las hembras apareadas de raza silvestre de la mosca mexicana de las frutas,Anastrepha lu-
dens (Loew), con no experiencia anterior con frutas (ingenuas), no fueron atraidas a y no tra-
taron ovopositar mas en la fruta de chapote amarillo (Sargentia ., ..- I que en toronjas
(Citrus paradisi) en experiments de tunel de viento. Las hembras ingenuas de la raza apa-
reada en el laboratorio preferieron las toronjas. La experiencia anterior con chapote au-
ment6 la atracci6n de la raza del laboratorio y de la raza silvestre hacia el chapote. Mas
hembras ingenuas que hembras con experiencia con chapote de ambas razas trataron a ovo-
positar en los lados del tunel de viento. Los machos ingenuos de la raza del laboratorio fue-
ron atraidos mas hacia las toronjas que al chapote. Los machos ingenuos silvestres y los
machos con experiencia con chapote de la raza silvestre y del laboratorio no preferieron cual-
quiera de las frutas.
The Mexican fruit fly, Anastrepha ludens
(Loew), is a polyphagous pest of citrus, mango, and
other fruits in Mexico and Central America (Norr-
bom & Kim 1988) and a perennial inhabitant of
the citrus growing areas of south Texas. Although
the original range of the fly is not completely un-
derstood, it is widely agreed that the montane re-
gions of northeastern Mexico represent at least
part of the native range and that a large citrus tree
that grows in these mountains and valleys is
among the fly's native hosts (Baker et al. 1944).
This tree, Sargentia greggii, produces small ob-
long yellow-green fruit (typical fruit 2.5-3.0 cm
long and 1.2-1.6 cm diam.) that give it its common
name, the yellow chapote (Plummer et al. 1941).
Unlike grapefruit, Citrus paradisi, in which larvae
feed on the fleshy pulp, early instar larvae feed on
the still-soft seed in immature chapote fruit before
moving into the flesh (Plummer et al. 1941). Field
workers searching for the fly in its native habitat
know to look for stands of these trees growing
along streams in mountain canyons where adults
can be found on the wing and larvae in fallen fruit
during the fruiting season. Although not reminis-
cent of typical commercial citrus in that the fruit
contains relatively little flesh and a large stone
and has only a weak non-citrus-like aroma, it ob-
viously is among the favored hosts of this fly.
Among commercial citrus, indications are that
grapefruit is the preferred host of this fly based on
high infestations in grapefruit orchards compared
with those of other citrus (Baker et al. 1944). In
laboratory wind-tunnel experiments, naive (no
previous experience with fruit), laboratory-strain,
oviposition-ready female Mexican fruit flies were
attracted to grapefruits mechanically wounded to
enhance release of peel and pulp volatiles (Ro-
backer & Fraser 2002). Surprisingly, naive, wild-
strain, oviposition-ready female Mexican fruit
flies were not attracted to grapefruit in those ex-
periments. We interpreted these results to mean
that wild flies did not recognize grapefruit as a
host because it is not a native species, whereas lab
flies, due to selection pressures from laboratory
colonization, were more opportunistic, facilitating
response to general fruit stimuli. However, wild
females that had prior experience with grapefruit
were attracted to the fruit in that work. This sug-
gested that wild fly populations were able to
adapt by learning to search for grapefruit.
Florida Entomologist 85(3)
In retrospect, it did not seem unreasonable
that wild female Mexican fruit flies would not
respond instinctively to grapefruit, a species in-
troduced to the new world. However, we hypothe-
sized that attraction of wild flies to chapote fruit
would not require learning. This made sense be-
cause chapote trees are large and the fruit incon-
spicuous, both visually and aromatically (as
judged by human olfaction), so that finding the
fruit by random searching seemed less likely than
searching by innate visual and olfactory recogni-
tion programs.
In the present work, we wanted to test the hy-
pothesis that Mexican fruit flies instinctively re-
spond to chapote fruit. Our approach was to
compare responses of naive flies to chapote fruit
with responses to grapefruit in no-choice situa-
tions. Responses to grapefruit were studied previ-
ously (Robacker & Fraser 2002) and were re-
examined here only for comparison with chapote.
We also wanted to investigate effects of experi-
ence with chapote fruit on subsequent responses
to chapote and grapefruit. Two experiments were
conducted in a wind tunnel to evaluate responses
of laboratory and wild-strain Mexican fruit flies.
MATERIALS AND METHODS
Insects, Rearing, and Handling
Laboratory flies were obtained from a culture
at our facility in Weslaco, TX. Laboratory stock
originated from 2,000 pupae collected from yellow
chapote fruit from the Montemorelos area of
Nuevo Leon in northeastern Mexico in 1997. This
culture has been maintained on artificial diet for
approximately 50 generations. Eggs are collected
after oviposition into red gel covered with para-
film. No fruit or fruit extract is used in rearing of
the laboratory culture. Wild flies were obtained
from grapefruits and sour oranges, Citrus auran-
tium, collected in orchards from the Montemore-
los area. Adults of both strains were held in
Plexiglas cages (20.5 x 20.5 x 20.5 cm) with
screened tops containing a diet mixture of sugar
and yeast hydrolysate, with water supplied sepa-
rately. Half of the cages were supplied with
chapote fruit starting one or two days after flies
closed. Laboratory conditions where test flies
were housed were 22 + 2C and 50 20% relative
humidity with a photophase of 0630 to 1930 h
provided by fluorescent lights.
Experimental Procedure
Bioassays were conducted in a plexiglass wind
tunnel with the dimensions of 0.3 x 0.3 x 1.2 m.
Each end of the wind tunnel was screened to al-
low airflow. The downwind end contained a baffle
system to create a uniform airflow through the
chamber. Air was pulled through the chamber at
0.4 m/sec by an exhaust fan connected to the
downwind end. Air exiting the chamber was di-
rected into an exhaust hose and removed to the
outdoors. The top of the chamber had two circular
openings (12.8 cm diameter) with plexiglass cov-
ers, located at each end of the chamber, to allow
easy access to the chamber's interior. A 75 W "soft
white" light bulb (General Electric Co., Cleve-
land, OH) in a reflecting lamp was positioned 17
cm above the downwind end of the chamber. The
purpose of this light was to minimize random fly-
ing into the upwind end of the chamber by using
the flies' positive phototaxis. Bioassays were con-
ducted in the same laboratory where adult flies
were held. In addition to the direct exhaust from
the wind tunnel, this room contains inlet and out-
let vents to bring new air into the room from out-
doors and remove air from the room to the
outdoors. Complete air replacement occurs 8
times per hour.
Laboratory strain and wild strain flies were
used in experiments at ages 13-22 and 17-23 d
post eclosion, respectively. This age range was
based on observations of sexual maturation, mat-
ing, and oviposition behavior by both strains of
flies in holding cages containing grapefruit and
on previous results (Robacker & Fraser 2001,
2002). Flies to be used in bioassays were trans-
ferred into cylindrical paper cartons (473 ml), ap-
proximately 12 of each sex per carton, 24 h prior
to testing. Cups were not provided with food so
flies had been starved for 24 h when trials were
conducted. Previous research demonstrated that
24 h of food deprivation enhanced attraction of
Mexican fruit flies to grapefruits and did not
affect oviposition propensity compared with non-
starved flies (Robacker & Fraser 2001). Cups
were sprayed with water several hours before tri-
als were conducted.
Grapefruits used in bioassays were ripe, Rio
Red variety grapefruits from an orchard located
near the station in Weslaco, TX. A circular piece of
the rind and pulp measuring 2.5 cm in diameter
was removed from grapefruits so that volatiles
from both the peel and pulp were present in the
aroma. This was done because previous research
showed that grapefruits wounded in this way were
more attractive than undamaged fruits to oviposi-
tion-ready females (Robacker & Fraser 2002).
Chapote fruits used in bioassays were picked from
trees and ranged from small and green to full size
and yellow green depending on season and loca-
tion where fruit were found. Small green fruit
were used whenever available because Plummer
et al. (1941) indicated that field collections of
green, half-grown fruits were more heavily in-
fested than mature fruits with Mexican fruit fly
larvae. Because of the small size of the chapote
fruits, a group of 7 fruits was used together as the
attractant source. One chapote fruit was cut in
half to increase emission of volatiles. The exposed
September 2002
Robacker & Fraser: Mexican Fruit Fly Attraction to Chapote
wounded area of the two chapote fruit halves was
roughly equal to that of a wounded grapefruit.
Grapefruits and chapote fruits were washed with
water before each trial to remove any chemicals
left by flies in the previous trial.
To conduct a trial, a grapefruit or group of
chapote fruit was suspended in a chicken-wire
basket (with standard window screening on the
bottom when chapote fruit were tested) from the
opening in the upwind end of the chamber, and
one cup of flies was placed under the downwind
opening. Flies were allowed 5 min to leave the cup
and respond to the fruit, and then were removed
from the chamber. We recorded upwind move-
ment if flies passed a point 2/ of the distance from
the release cup to the fruit, landing if flies either
landed on or walked onto the fruit, oviposition
into grapefruits and chapotes, and attempts to
oviposit onto the plexiglass walls of the bioassay
chamber. Bioassays were limited to 5 min to re-
duce accidental upwind movements and landings
due to random movements of non-responding
flies. Experiments were conducted in series of
four treatments tested in random order: chapote-
experienced flies offered a grapefruit, chapote-
experienced flies offered chapote fruit, naive flies
offered a grapefruit and naive flies offered chapote
fruit. Experiments were conducted between 1100
and 1630 h. In previous experiments, time of the
day between 0900 and 1700 h did not affect at-
traction to host fruit and oviposition behavior
(Robacker & Fraser 2001).
Statistical Analyses
All behaviors except oviposition propensity
were tested by analysis of variance using Super-
ANOVA (Abacus Concepts, 1989). Proportions of
flies that moved upwind, landed on the fruit, or
attempted oviposition on fruit or the walls of the
wind tunnel, were transformed by arcsin of the
square root (Snedecor & Cochran 1967) before
statistical analyses. Proportions of 0 were re-
placed with 1An before transformation. Effects of
fruit type, experience, and their interactions were
calculated for each fly behavior. Additional analy-
ses were performed to determine the overall
treatment effect for the 4 fruit type by experience
treatments. Separate analyses were conducted
for males and females. Means separations were
conducted using Fisher's protected least signifi-
cant difference method (Snedecor & Cochran
1967). Oviposition propensity (percentage of
females that attempted oviposition after landing
on a fruit) was analyzed by Chi-square tests
(Snedecor & Cochran 1967).
RESULTS
Results for wild females are shown in Table 1.
Upwind movements, landings, and oviposition be-
havior by naive females in response to chapote
fruit vs. grapefruit did not differ. More chapote-
experienced females moved upwind toward
chapote (F = 7.6; df = 3,75; P < 0.001) compared
with responses of naive females to chapote, naive
females to grapefruit and chapote-experienced
females to grapefruit. More chapote-experienced
females than naive females landed on either
chapote or grapefruit (F = 3.2; df= 3,75; P < 0.05).
There were no differences in total attempted ovi-
positions on either fruit type by either naive or
chapote-experienced females. Oviposition propen-
sity also did not differ significantly for the various
treatments based on a Chi-square test of single
classifications with equal expectations. Chapote-
experienced females (summed over fruit types)
attempted to oviposit on the sides of the wind tun-
TABLE 1. PERCENTAGES OF MEXICAN FRUIT FLIES WITH OR WITHOUT PRIOR EXPERIENCE WITH CHAPOTE FRUIT AT-
TRACTED TO AND ATTEMPTING OVIPOSITION IN GRAPEFRUIT OR CHAPOTE FRUIT IN A WIND TUNNEL: WILD
STRAIN FEMALES."
Attempted Oviposition Attempted
Moved Landed to oviposit propensity to oviposit on
Test fruit: experience upwindb on fruitb on fruitb on fruit" wind tunnel"
Grapefruit:
Naive 10.4 a 3.6 a 0.7 a 18.2 2.6 a
Chapote-experienced 13.9 a 5.1 ab 0.6 a 13.3 0.6 a
Chapote:
Naive 11.6 a 3.5 a 0.3 a 11.1 2.3 a
Chapote- experienced 23.4 b 10.2 b 3.3 a 32.3 1.0 a
Means followed by different letters in the same column are significantly different at the 5% level by Fisher's protected LSD.
Values are mean percentages of females responding out of the total females in the trial, n = 26 trials each test fruit/experience group; 11.7 females/
trial.
'Values are percentages of females responding out of females that landed on the fruit. Grapefruit, naive: n = 11 females landed; Grapefruit, chapote-
experienced: 15; Chapote, naive: 9; Chapote, chapote-experienced: 31. No significant differences were found by Chi-square test of single classifications
with equal expectations.
Florida Entomologist 85(3)
nel less than naive females, as indicated by a sig-
nificant experience effect by ANOVA (F = 4.1; df =
3,75; P < 0.05).
Results for laboratory females are shown in
Table 2. More naive females landed on (F = 13.1;
df = 3,69; P < 0.0001) and attempted oviposition
in (F = 9.6; df = 3,69; P < 0.0001) grapefruit than
chapote. Also, oviposition propensity of naive fe-
males was higher on grapefruit than on chapote
(X2 = 4.0; df = 1, P < 0.05). More females experi-
enced with chapote than naive females landed on
and attempted oviposition in chapote. Conversely,
more naive females than chapote-experienced
ones (summed over fruit types) attempted ovipo-
sition on the sides of the wind tunnel (F = 11.6; df
= 1,69; P < 0.01). Oviposition propensity was not
significantly affected by experience. Also, experi-
ence with chapote had little effect on any of the
responses to grapefruit. However, interaction of
fruit type with experience was significant for total
oviposition attempts (F = 5.5; df = 1,69; P < 0.05).
This effect occurred because experience with
chapote increased oviposition in chapote but de-
creased oviposition in grapefruit.
Results for males are shown in Table 3. More
wild strain males, summed over experience treat-
ments, moved upwind toward chapote than grape-
fruit (F = 4.1; df = 1,75; P < 0.05) (Table 3). More
naive laboratory strain males landed on grape-
fruit than on chapote fruit (F = 3.1; df = 3,69; P <
0.05). Also, summed over experience treatments,
more laboratory males landed on grapefruit than
on chapote (F = 7.9; df = 1,69; P < 0.01). Experi-
ence with chapote fruit had no significant effects
on responses of either strain to either fruit; how-
ever, a general trend of higher responses by
chapote-experienced flies occurred. The experi-
ence effect was borderline significant for landings
by wild males (F = 3.8; df = 1,75; P = 0.05).
DISCUSSION
Wild strain male and female Mexican fruit
flies that had no prior experience with chapote
fruit did not exhibit more attraction to or oviposi-
tion behavior on chapote, a native host, than
grapefruit, an introduced host of this species (Ta-
bles 1 and 3). Robacker and Fraser (2002) showed
that naive wild flies were not attracted to grape-
fruit compared with a plastic yellow ball indicat-
ing that they did not respond instinctively to
grapefruit as a host. Combining previous results
with those from the current work suggests that
wild flies also do not instinctively respond to
chapote as a host.
Although it is widely accepted that fruit flies
are attracted to their host fruit for oviposition
(Fletcher & Prokopy 1991; Jang & Light 1996),
most demonstrations have used laboratory flies
or wild flies with host experience. Some studies
that demonstrated host attraction by wild, naive
female tephritids are Averill et al. (1988) with ap-
ple maggot, Rhagoletis pomonella, Landolt and
Reed (1990) with papaya fruit fly, Toxotrypana
curvicauda, Prokopy et al. (1990a) with the orien-
tal fruit fly, Bactrocera dorsalis, and Prokopy and
Vargas (1996) and Katsoyannos et al. (1997) with
the Mediterranean fruit fly, Ceratitis capitata. We
know of no published research showing that wild-
strain female fruit flies are not attracted to their
natural host material. Such studies usually are
regarded as experimental failures rather than
demonstrations of actual biological phenomena.
Attraction of naive, wild male fruit flies to host
fruit volatiles has rarely been demonstrated. Kat-
soyannos et al. (1997) showed that wild, naive
male Mediterranean fruit flies are attracted to
volatiles from oranges. Another example is at-
traction of semi-wild (reared on apples for ca. 32
TABLE 2. PERCENTAGES OF MEXICAN FRUIT FLIES WITH OR WITHOUT PRIOR EXPERIENCE WITH CHAPOTE FRUIT AT-
TRACTED TO AND ATTEMPTING OVIPOSITION IN GRAPEFRUIT OR CHAPOTE FRUIT IN A WIND TUNNEL: LABORA-
TORY FEMALES."
Attempted Oviposition Attempted
Moved Landed to oviposit propensity to oviposit on
Test fruit: experience upwindb on fruitb on fruitb on fruit" wind tunnel"
Grapefruit:
Naive 35.5 a 20.8 c 10.5 c 50.8 b 8.6 c
Chapote-experienced 32.4 a 21.3 c 7.9 bc 37.3 2.2 a
Chapote:
Naive 28.0 a 5.1 a 1.1 a 21.4 a 7.4 bc
Chapote-experienced 27.6 a 11.4 b 4.3 b 37.5 2.8 ab
Means followed by different letters in the same column are significantly different at the 5% level by Fisher's protected LSD.
Values are mean percentages of females responding out of the total females in the trial, n = 24 trials each test fruit/experience group; 11.6 females/trial.
'Values are percentages of females responding out of females that landed on the fruit. Grapefruit, naive: n = 59 females landed; Grapefruit, chapote-
experienced: 59; Chapote, naive: 14; Chapote, chapote-experienced: 32. Means for response by naive females to grapefruit vs. chapote were significantly
different by Chi-square test of proportions in 2 independent samples. No significant differences among the 4 means were found by Chi-square test of sin-
gle classifications with equal expectations.
September 2002
Robacker & Fraser: Mexican Fruit Fly Attraction to Chapote
TABLE 3. PERCENTAGES OF MEXICAN FRUIT FLIES WITH OR WITHOUT PRIOR EXPERIENCE WITH CHAPOTE FRUIT AT-
TRACTED TO GRAPEFRUIT OR CHAPOTE FRUIT IN A WIND TUNNEL: MALES.
Wild strain Laboratory strain
Test fruit: experience Moved upwind Landed on fruit Moved upwind Landed on fruit
Grapefruit:
Naive 5.5 a 1.6 a 16.2 a 8.4 b
Chapote-experienced 7.0 a 3.6 a 18.1 a 7.6 b
Chapote:
Naive 7.9 a 1.0 a 15.9 a 2.1 a
Chapote-experienced 9.4 a 2.3 a 18.1 a 4.4 ab
Values are mean percentages of males responding out of the total males in the trial. Wild strain: n = 26 trials each test fruit/experience group; 11.8
males/trial. Laboratory strain: n = 24 trials each group; 11.2 males/trial. Means followed by different letters in the same column are significantly different
at the 5% level by Fisher's protected LSD.
generations) males to apple volatiles (Fein et al.
1982). Also with apple maggot, Prokopy et al.
(1989) showed that naive, wild males spent more
time on fruit when released onto hawthorn than
on apple, suggesting preferential response to a
native host.
Naive laboratory A. ludens were more at-
tracted to and females attempted oviposition
more often in grapefruit than chapote (Tables 2
and 3). Robacker and Fraser (2001, 2002) demon-
strated that grapefruits were much more attrac-
tive than yellow balls to naive laboratory females.
The differential in landings on grapefruits (with
pulp wounds like those used in the current work)
vs. yellow balls was 20 to 1 for 1-d starved females
(Robacker & Fraser 2001) compared with 4 to 1
for grapefruit vs. chapote in the current study.
This suggests that chapote should be 5x more at-
tractive than a yellow ball to naive, hungry, labo-
ratory females, but this was not tested. Attraction
of males to grapefruit could not be demonstrated
in previous work unless they were starved for 2
days prior to testing (Robacker & Fraser 2001).
However, given the greater attraction of naive
laboratory males to grapefruit than to chapote in
the current work, the indication is that grapefruit
is also attractive to males starved for 1 d.
Attempts to prove attraction of naive, labora-
tory-strain male fruit flies to native host fruit
have been few. Successful demonstrations include
attraction to volatiles of fermented chapote fruit
by Mexican fruit flies (Robacker et al. 1990), at-
traction to various fruit extracts by Caribbean
fruit flies, A. suspense (Nigg et al. 1994), and at-
traction to coffee fruit by Mediterranean fruit
flies (Prokopy & Vargas 1996). Possibly, at least
some of these cases as well as those involving wild
males may represent non-specific responses to
fruit odors by flies motivated by hunger rather
than host attraction.
Previous experiments have indicated that
naive laboratory-strain Mexican fruit flies
(starved or food satiated) are attracted to grape-
fruit but naive wild flies (food satiated) are not
(Robacker & Fraser 2001, 2002). Experiments
presented here indicate that attraction of naive
laboratory females (starved for 1 d) to chapote is
much weaker than to grapefruit. Apparently, un-
known selective pressure that resulted in attrac-
tion to grapefruit but not chapote was imposed
during laboratory rearing. As stated in the meth-
ods, no fruit or fruit extract is used at any point in
the rearing procedure. A red gel in flat circular
containers is used to collect eggs, but even if this
was perceived by flies as a supernormal visual
fruit stimulus, data from Robacker and Fraser
(2002) indicate that visual stimuli from grape-
fruit are not important compared with the fruit
volatiles. As in our earlier work, we again assert
that laboratory flies are more opportunistic than
wild flies. As such, the stronger (by human olfac-
tion) although unfamiliar aroma of grapefruit
may provide a good general fruit stimulus com-
pared with the weaker aroma of chapote.
Experience with chapote increased attraction
to and oviposition behavior on chapote by wild and
laboratory strain females compared with inexperi-
enced females (Tables 1 and 2). Previously we
showed increased responses to grapefruit by wild
and laboratory females experienced with grape-
fruit (Robacker & Fraser 2002). These results were
expected based on numerous papers that have
demonstrated increased attraction to and usage of
fruits following exposure to those fruits in Medi-
terranean fruit fly and several species ofRhagole-
tis and Bactrocera (Cooley et al. 1986, Prokopy et
al. 1990a,b, 1991, 1993, Fletcher & Prokopy 1991,
Averill et al. 1996). Experience with chapote had
little effect on responses to grapefruit. Many stud-
ies have shown that experience with one fruit de-
creases responses to novel fruit types (Cooley et al.
1986, Papaj & Prokopy 1986, Prokopy et al. 1986,
Fletcher & Prokopy 1991).
Propensity of both wild and laboratory females
to attempt oviposition on the sides of the wind tun-
nel was greatly reduced if flies had previous expe-
rience with chapote. We obtained the same result
previously for Mexican fruit flies experienced with
grapefruit (Robacker & Fraser 2002). The reason
for this effect is unknown but could be related to
higher egg load of naive flies or learning from fruit
experience. A similar effect was found by Prokopy
et al. (1990b) in which acceptance of plastic ovipo-
sition spheres by Mediterranean fruit flies de-
creased for flies experienced with host fruit.
Effects of experience with chapote on re-
sponses by males were relatively small. The trend
was that experience increased attraction to
chapote. Previously we showed that experience
with grapefruit increased attraction of laboratory
males to grapefruit, although the effects were
smaller than for experienced females (Robacker
& Fraser 2002). Also, Prokopy et al. (1989)
showed that experience with apple or hawthorn
fruit increased preference by apple maggot males
for the experienced fruit. In addition, Sivinski
(1990) and Henneman and Papaj (1999) provided
evidence that male fruit flies may learn to associ-
ate host fruit with females if they are given expe-
rience with host fruit while females are present
on the fruit. However, Prokopy et al. (1989) found
no additional effect from experience with females
on fruit beyond preference gained by experience
with the fruit by itself. Thus, experience with
fruit generally increases attraction of male fruit
flies to that fruit, but the reasons are unclear.
Grapefruit and chapote differ greatly in color,
size and odor. Our experiments were not designed
to determine how important each of these charac-
teristics was in attractiveness of the 2 fruit types
to the flies. Data from earlier work indicated that
odor was very important in attraction of both
naive and experienced flies to grapefruit, but no
assessment of visual stimuli was possible (Ro-
backer & Fraser 2002). Both visual and chemical
characteristics of host fruit are known to play
roles in innate and learned attraction of fruit flies
to the fruit (Papaj & Prokopy 1986, Prokopy et al.
1990a, Fletcher & Prokopy 1991, Prokopy et al.
1994, Henneman & Papaj 1999).
Our data indicate that chapote fruit is not pre-
ferred to grapefruit by either wild or laboratory
strain Mexican fruit flies. A potentially critical
factor that may have influenced our results is the
maturity level of the chapote fruit. Plummer et al.
(1941) presented data that showed that adult
Mexican fruit flies were present in chapote stands
only during a short period when trees were in
bloom and setting fruit, then populations de-
clined rapidly as fruits matured. Taken together
with data that showed that larvae feed in the seed
before it hardens during fruit maturation, it has
been inferred that oviposition-ready females are
most attracted to chapote fruit only during fruit
set and early stages of maturation. In our work,
we used only chapote fruit picked off of trees and
attempted to obtain fruit from each of several lo-
September 2002
cations as soon as possible after set. However, it is
possible that our fruit was already beyond its
most attractive stage by the time we used it in
bioassays. In this regard, it is interesting that no
ovipositions occurred during the first month (out
of 4 months) of testing when fruits were very
small and green. This suggests that another pos-
sible problem may be changes in the attractive
quality of fruit as soon as it is picked from trees.
Thus, it may be necessary to investigate the at-
tractiveness of early stage chapote fruit in the
field by observing ovipositing females on chapote
trees. A more practical approach would be to
study fruit fly behavior on field-caged trees from
bloom to fruit drop. Perhaps such an investigation
would show that the flies are not attracted to even
the earliest fruit so much as they are attracted to
the flowers and then remain on the trees to ovi-
posit on the small fruit, not because it is attrac-
tive but because it is the only fruit present.
ACKNOWLEDGMENTS
We thank Donald Thomas (USDA-ARS, Weslaco) for
assistance in obtaining wild flies and chapote fruit;
Celestino Cervantes, Francisco Daniel, and Ronay Riley
Rodas (USDA-APHIS-IS, Mexico City) for collection of
wild fly pupae and chapote fruit; and Bob Heath
(USDA-ARS, Miami, Florida) and Peter Landolt
(USDA-ARS, Wapato, Washington) for the wind tunnel.
Use of a product brand in this work does not constitute
an endorsement by the USDA.
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Florida Entomologist 85(3)
September 2002
COMPARATIVE COST OF CHEMICAL AND BIOLOGICAL WHITEFLY
CONTROL IN POINSETTIA: IS THERE A GAP?
R. G. VAN DRIESCHE1, S. LYON1, K. JACQUES', T. SMITH2 AND P. LOPES3
'Department of Entomology, University of Massachusetts, Amherst, MA 01003
2Massachusetts Extension, University of Massachusetts, French Hall, Amherst, MA 01003
3Massachusetts Extension, Cranberry Exp. Station, E. Wareham, MA 02538
ABSTRACT
Cost is the principal constraint on the use of biological control against whiteflies in poinset-
tia (Euphorbia pulcherrima Willd. ex Koltz.) crops in the United States. Here we show that
a new, lower release rate of the whitefly parasitoid Eretmocerus eremicus Rose and Zolner-
owich (Hymenoptera: Aphelinidae), 0.5 females per plant per week, maintains whiteflies
(Bemisia argentifolii Bellows and Perring) at harvest below the economic threshold of 2 live
nymphs + pupae per leaf, when used in combination with two mid-crop applications of the
insect growth regulator fenoxycarb (Precision). Cost of this program (for 16.5 cm dia single
stem pots, with 30,000 plants under protection) varies from 21 to 34 cents per plant for the
season, for cropping periods from 12 to 18 weeks. Shipping costs are calculated and included
in estimated costs. These values compare favorably to the real cost of whitefly chemical con-
trol incurred by Massachusetts poinsettia growers in fall of 2000, which was 14 cents for a
16.5 cm dia single stem pot, with a range of 1 to 40 cents. Programs consisting of a single ap-
plication of the systemic insecticide imidacloprid alone cost 12 cents per pot per season. This
difference between 21 cents for the biological control program and 14 cents for the chemical
control program is the smallest yet reported for biological control of whiteflies in poinsettia.
Key Words: Bemisia argentifolii, Eretmocerus eremicus, poinsettia, biological control, white-
flies, cost evaluation, augmentation
RESUME
El costo es el impedimento principal del uso de control biol6gico contra la mosca blanca en
el cultivo de poinsetia [=nochebuena] (Euphorbia pulcherrima Willd. ex Koltz) en los Esta-
dos Unidos. Aqui, mostramos que liberaciones del parasite Eretmocerus eremicus Rose and
Zolnerowich (Hymenoptera: Aphelinidae) a un nuevo, menor tasa de 0.5 hembras por plant
por semana, mantiene la mosca blanca (Bemisia argentifolii Bellows and Perring) debajo el
umbral econ6mico de 2 ninfas vivas + pupas por hoja en el tiempo de la cosecha, cuando se
usa en combinaci6n con un regulador de crecimiento de insects fenoxycarb (Precision)
aplicado dos veces en medio del tiempo de crecimiento del cultivo. El costo del program por
cada maceta de 16.5 cm de dia., para plants del tallo singular, con 30,000 plants baja pro-
tecci6n varia entire 21 a 34 centavos por plant por toda la estaci6n, para el period del cul-
tivo de 12 a 18 semanas. Los costs de envio estan calculados e incluidos en la estimaci6n del
costs. Estos valores se compararon favorablemente con el costo real de control quimico de
mosca blanca incurridos por los agricultores de poinsetia en Massachusetts en el otono de
2000, lo cual fu6 14 centavos por maceta de diametro de 16.5 cm para plants del tallo sin-
gular, con un rango de 1 a 40 centavos. Los programs que consiste de una sola aplicaci6n de
la insecticide sistemica imidacloprid cost 12 centavos por maceta por cada estaci6n. La di-
ferencia entire 21 centavos por el program de control biol6gico y 14 centavos por el program
de control quimico es el menor [cuantidad] reportado jams para el control biol6gico de
mosca blanca en poinsetia.
Whiteflies (Homoptera: Aleyrodidae) continue through the roots); (2) treatment of the foliage
to be important pests of poinsettia (Euphorbia with the same material (as Marathon II ); (3) ap-
pulcherrima Willd. ex Koltz.) crops in the north- plication of other, non-systemic, insecticides to
eastern United States, even following the devel- the foliage, or (4) use of a fumigant to kill adults.
opment of more effective pesticides in the mid- In practice many growers use two or more of these
1990s. There are four commonly used options for approaches, often in an unplanned sequence in
chemical control of whiteflies in poinsettia: (1) response to whitefly problems as they are encoun-
treatment of poinsettia plants with systemic for- tered over the course of the growing season. Imi-
mulations of imidacloprid (Marathon, absorbed dacloprid is often applied first, soon after potting
Van Driesche et al.: Cost of Whitefly Biological Control
of the cuttings. This may be followed, up to the
time of bract coloration (about mid crop), by the
use of foliar-applied pesticides if whiteflies are no-
ticed later in the crop. Finally, if whiteflies are
still apparent after bract coloration, smoke fumi-
gation may be used, to replace wet sprays, which
may leave objectionable residues on bracts. Be-
cause the number of applications and the cost of
individual materials may vary greatly, many
growers do not know how much season-long
chemical control of whiteflies in poinsettia costs
per potted plant or per unit of greenhouse space.
Nor can they say if their chemical control costs
are higher or lower than the use of whitefly para-
sitoids for biological control, an alternative for
managing whiteflies in poinsettia. Growers often
compare costs of biological control to best case
scenarios, rather than the average scenario for
chemical control.
The goal of this study was to provide this miss-
ing information. We compare the cost of biological
whitefly (Bemisia argentifolii Bellows and Per-
ring) control employing a reduced release rate of
the parasite Eretmocerus eremicus Rose and Zol-
nerowich (0.5 female parasites per plant per
week) (commercially available from Koppert Bio-
logical, Inc. and other suppliers) to the cost of
chemical control as used by 22 Massachusetts
commercial poinsettia growers for the fall 2000
cropping season. We sought to answer two ques-
tions: does this newly reduced parasite release
rate provide effective control (producing plants
with acceptable market quality) and how does the
cost of this biological control program compare to
the average cost of chemical control as currently
practiced by Massachusetts poinsettia growers.
MATERIALS AND METHODS
The Biological Control Program
The efficacy trial. In previous work (Van
Driesche et al. 2001), we demonstrated that a rate
of 1 female E. eremicus per plant per week, when
combined with two mid-season applications of an
insect growth regulator, provided acceptable con-
trol of whitefly in commercial poinsettia. In fall of
2000, we conducted a further trial to determine if
a still lower parasitoid release rate might also be
effective, as this would further lower the cost. At
each of two commercial poinsettia growers in
western Massachusetts, we examined two treat-
ments, one in which we applied 1 female
E. eremicus per plant per week and one in which
we applied only 0.5 females. In both treatments,
we made two mid-season applications of the in-
sect growth regulator fenoxycarb (Precision).
At site #1 (Westover Greenhouses, Chicopee,
MA), we used two greenhouses for the trial. One
greenhouse (used for the 0.5 female parasitoids
treatment) was a 9.2 x 29.5 m plastic hoop house,
filled with the cultivar Peterstar Red, of which
there were 585 pots (22 cm dia) with 3 plants each
and 40 pots (30.5 cm dia) with five plants each.
The other Westover greenhouse (used for the 1.0
female parasitoids treatment) was a 6.2 x 31 m
glasshouse, planted to a mixture of five cultivars
(Peterstar Red, Jinglebells, Angelica White, An-
gelica Marble and Angelica Pink), in 282 pots (22
cm dia) with 3 plants each and 107 pots (30.5 cm
dia) with five plants each. At site #2 (Grandview
Farms, Chicopee, MA), a single wooden frame,
plastic covered greenhouse was divided with plas-
tic into two compartments, one for each of the
treatments. Each compartment was 8.3 m wide
by 14.8 m long. All plants were Freedom Red and
all were potted as single plants in 16.5 cm dia
pots, with 2,340 pots in each compartment.
At site #1 (Westover Greenhouses), the poin-
settias were potted in the first week of August
and the trial continued until plants were removed
for sale between 29 November and 7 December. At
site #2 (Grandview Farms), poinsettias were pot-
ted approximately 15 August and the trial contin-
ued until plants were removed for sale between
22 and 28 November.
At both sites, poinsettias were sampled weekly
by haphazardly selecting 90 plants distributed
over the greenhouse and examining 3 leaves (one
top, one middle, and one bottom), counting the
number of live whitefly nymphs and pupae per
leaf (n = 270), to assess the efficacy of control. At
mid-crop (21 and 28 of October for site #1 and 16
and 23 of October for site #2), two applications
(0.075 g A.I. per liter, equivalent to 2 weight oz of
product [25% A.I] per 50 gallons of spray solution)
of fenoxycarb were made to all plants in the trial.
Parasitoid releases were made by placing par-
asitoid pupae (purchased as loose pupae in saw-
dust from Koppert Biological, Inc.) in styrofoam
coffee cups taped to poles. Bottoms of cups were
removed and replaced with organdy fabric to pro-
vide drainage in case of wetting during watering
of the crop. Poles bearing cups were stuck into
poinsettia pots so that cups were approximately
10 cm above the foliage at the beginning of the
season. Twelve cups were placed in each green-
house for the weekly releases.
Quality control measures of E. eremicus were
taken to ensure application of the desired rate.
Weekly, we measured the sex ratio, emergence
rate, and "fill rate." The sex ratio was measured in
the laboratory by isolating in a vial a group of ap-
proximately 200-250 pupae from each shipment.
After two weeks emerged adults were examined at
random and sexed (based on antennal shape) until
100 had been sexed. Emergence rates were as-
sessed weekly by collecting material left in emer-
gence cups in test greenhouses for two weeks after
release. To achieve this, on a weekly basis the old
material from the previous release was moved to
two additional cups that were left for a second
Florida Entomologist 85(3)
week. Each subsequent week, we collected this two
week old material and took it to the laboratory and
used a 25x stereomicroscope to examine 100 exu-
viae or dead pupae chosen in random order. Each
item was classified as having produced a parasi-
toid, whitefly adult, or having died. Percent emer-
gence of parasitoids was calculated as number of
emerged parasitoids divided by total number of
items examined. The "fill rate" for each shipment
was determined by counting the number of live
parasitoid pupae per 0.2 grams for each of 10 sam-
ples taken at random from the container the day it
was received. From these values we calculated the
number of live pupae per gram. We then weighed
the total amount of product in the containers re-
ceived and multiplied it by the number of live pu-
pae per gram to calculate the total number of
pupae received (the "fill rate"). The fill rate was
then used in conjunction with the running average
of the % female and % emergence (for all previous
weeks of the trial) to calculate the number of
grams of product that had to be released in each
greenhouse (in view of the number of plants
present) to obtain the desired release rate.
Calculation of cost of biological control. We cal-
culated the cost of our biological control program
using the price for parasites in 2000 charged by a
major supplier (Koppert Biological, Inc., $168.26
for a bottle described as containing 15,000 para-
site pupae; shipping cost not included), together
with necessary information on parasitoid quality.
For this latter factor, we used the sex ratio and
emergence rates from the 2000 trial as these were
consistent with our previous experiences. Fill
rate, however, was low in 2000 (80%) compared to
earlier trials (>>100%) and so we present costs
based on an assumed fill rate of 100%. Using this
information, we calculated the cost of biological
control to growers per plant, including the cost of
application of the insect growth regulator and the
parasitoid shipping costs.
Rather than calculate the biological control
cost explicitly for the growers participating in the
efficacy trial, we determined costs in relation to
crop size and crop length, for greater generality.
Per plant costs for shipping, for example, are
higher for smaller producers because shipping
price is spread over fewer pots. We based our cal-
culations around production of the most common
pot size (16.5 cm dia, = 6.5 inch dia.). We calcu-
lated costs for two crop sizes (10,000 and 30,000
pots) and three crop durations (12, 15, and 18
weeks).
The Chemical Control Program
To estimate the number of pesticide applica-
tions made to control whiteflies and the total cost
of this control, we obtained the fall 2000 pesticide
application records for 22 Massachusetts poinset-
tia producers, chosen at random from a list of
growers. From these records we determined the
number of applications of each pesticide, noting
its brand, the rate used and the quantity applied.
One use of one insecticide constituted an applica-
tion. If label rates of two insecticides were tank
mixed and applied together, this was counted as
two applications. We quantified the cost per m2 of
greenhouse floor space of the pesticides applied
for whitefly control (not including labor) using a
spreadsheet that incorporated information on
greenhouse size, the pesticide rate, the quantity
of spray solution applied, and the cost of particu-
lar pesticide products used. We obtained 2000
pricing information for pesticides from two large
regional distributors, from whom most of the
growers surveyed had purchased their materials.
Finally, we converted the cost per m2 to cost per
pot, using information on grower crop spacing
practices, based on average values from four
growers who provided both a pot count (of a spe-
cific size, in a filled greenhouse) and greenhouse
size (i.e, 0.23 m2 per 16.5 cm dia pot, n = 3; 0.28 m2
per 22 cm dia pot, n = 2).
RESULTS
Efficacy of the Biological Control Program
At both growers, densities of whitefly (as live
nymphs + pupae per leaf) stayed below the
threshold of 2 N+P per leaf in both the green-
houses receiving the low (0.5) and those receiving
the high (1.0) parasitoid release rates (Fig. 1).
Cost of the Biological Control Program
Shipping costs are relatively fixed (one ship-
ping event per week), but the impact on the cost of
biological control per plant depends on how many
plants a grower is producing. The impact of ship-
ping becomes smaller as more plants are pro-
duced because the cost of shipping is spread over
larger numbers of plants. For a producer of 30,000
plants growing a 12 week crop, shipping costs add
only one cent per pot ($0.01) (Table 1), but for a
producer of a small (10,000 plants), long duration
crop (18 weeks), shipping alone can have costs of
five cents per pot.
Costs of whitefly biological control are further
affected by the package size purchased. Koppert
Biological, Inc. sells E. eremicus in two package
sizes: 15,000 and 3,000 pupae. Analyses pre-
sented here assume purchase in lots of 15,000. To
utilize this package size, growers need to produce
a minimum of 10,000 plants. For smaller growers
purchasing parasites in lots of 3,000 pupae, pur-
chase costs are approximately 20% higher.
Parasitoid quality (sex ratio and percentage
emergence) for E. eremicus in our trials has been
consistently high (e.g., 50 1.3% [SE]) female and
76 1.5% [SE]) emergence in our fall 2000 trial)
September 2002
Van Driesche et al.: Cost of Whitefly Biological Control
-- 1 female wasp per plant
--0.5 female wasps per plant
1.6
0U
_ 1.4
0-
+ 1.2
C-
E1
z> 0.8
0.6
a< 0.4
a 0.2
0
7
Grower 2
0 -- --
7-Aug 21-Aug 4-Sep 18-Sep 2-Oct
16-Oct 30-Oct 13-Nov 27-Nov 11-Dec
Date
-t- 1 female wasp per plant
W--0.5 female wasps per plant
16-Oct 30-Oct 13-Nov 27-Nov 11-Dec
Date
Fig. 1. Densities per leaf of live nymphs + pupae of Bemisia argentifolii on poinsettia crops in 2000 at two Massa-
chusetts growers in greenhouses treated with either a low (1 female wasp per plant per week) or superlow (0.5 female
wasps per plant per week) rate of the parasitoid Eretmocerus eremicus, plus two mid-season applications of fenoxycarb.
and does not contribute much variation to control
cost. Fill rate of packages, however, is more vari-
able. Parasites are sold in bottles advertised as
having a fixed number of pupae, but actual num-
bers present vary. Variation in fill rate, if growers
react to under filling by ordering more product,
changes the cost of the biological control program.
However, if suppliers make good on under filled
orders with free supplemental material, there is
no cost. In our 2000 trial reported here, we expe-
rienced an 80% fill rate, but in our 1997 trials, we
received an overfill rate of 173%. Assuming a
100% fill rate, costs for parasitoids (per single
stem 16.5 cm pot) for season long control range
from 18 cents for a 12 week crop to 27 cents for an
18 week crop. For multi-stem plants, cost of con-
trol increases proportionately.
Cost of the Chemical Control Program
Growers made an average of 8.3 + 2.1 (SE)
insecticide applications for season long whitefly
control (n = 22, range = 1-36). Only 7 of 22 growers
were able to control whiteflies with a single appli-
Grower 1
-Aug 21-Aug 4-Sep 18-Sep 2-Oct
.- 1.4
+ 1.2-
-n
E- 1
Sa)
z. 0.8-
> a
I. 0.6
0
a 0.4
a 0.2-
Florida Entomologist 85(3)
TABLE 1. COST (U.S. $) PER PLANT OF A WHITEFLY BIOLOGICAL CONTROL PROGRAM FOR POINSETTIA USING ERET-
MOCERUS EREMICUS.1
12 week crop 15 week crop 18 week crop
Item 10,000 plants 30,000 plants 10,000 plants 30,000 plants 10,000 plants 30,000 plants
Shipping 0.03 0.01 0.04 0.01 0.05 0.02
Parasitoids 0.18 0.18 0.22 0.22 0.27 0.27
IGR 0.02 0.02 0.02 0.02 0.02 0.02
Total program 0.23 0.21 0.28 0.25 0.34 0.31
'Assuming a 100% fill rate, 50% female sex ratio, 76% emergence rate, purchase of parasitoids in lots of 15,000 at $168.26 per 15,000 pupae, and re-
lease on single-stemmed plants in 16.5 cm pots.
cation of imidacloprid. The remaining 15 growers
applied multiple foliar applications to suppress
whiteflies, some as tank mixtures of two or more
products. The average seasonal cost of chemical
whitefly control for these growers per single stem
16.5 cm diameter pot (not counting labor) was
$0.14 $0.02 (SE), with a range from $0.01 to
$0.40. One application of imidacloprid had an
average cost per 16.5 cm pot of $0.12 (n = 18). The
average seasonal chemical control cost per triple
stem 21.6 cm pot was $0.17 $0.02 (SE).
DISCUSSION
While biological control options have been
available for whiteflies in greenhouse poinsettia
since the early work of Helgesen and Tauber
(1974), the approach has not been adopted by
growers because such programs are more expen-
sive than the use of pesticides. Use of Encarsia
formosa Gahan, the same species tested by Helge-
sen and Tauber (1974), was found by Hoddle and
Van Driesche (1996a) to cost $1.02 per plant for
season long control, over ten times the cost of
competing pesticides. Stevens et al. (2000), re-
ported the cost of use of this parasitoid to be $1.13
per pot, when the cost of scouting was included.
The initial estimate of the cost to use E. ere-
micus was even higher ($2.70 per plant per sea-
son, Hoddle and Van Driesche, 1996b). However,
progress has been made in lowering these costs.
Van Driesche et al. (2001) found that even the
standard rate of 3 females of E. eremicus per
plant per week, the same rate as tested by Hoddle
and Van Driesche (1996b) then cost only $1.18 per
plant, due to decreases in price for the parasitoid
from commercial suppliers. The same study
showed that substantially lower costs could be
achieved by simultaneously lowering the parasi-
toid application rate and combining biological
control with limited mid-crop use of insect growth
regulators, which were found to be compatible
with E. eremicus (Hoddle et al. 2001). Using this
approach, costs of whitefly biological control fell
to $0.38, when a rate of 1 female parasitoid per
plant per week was applied. In the trial presented
here, we show that a still lower rate of 0.5 female
parasitoids per plant per week also provides con-
trol. Cost for this program is only $0.25 per plant,
which includes shipping costs and the price of the
insect growth regulator, costs not included in val-
ues cited from earlier cited studies.
For the most widely grown size of poinsettia-
the standard single stem plant in a 16.5 cm pot-
we found the cost of chemical control to be 14
cents, which compared well to 25 cents for the bio-
logical control program (assuming a 15 week crop
and a 100% fill rate), including the cost of ship-
ping (assuming 30,000 plants under protection).
Our estimate of the cost of the chemical control
program is consistent with the estimate of
Stevens et al. (2000) who reported a value of 28
cents per pot when scouting costs were included.
For Massachusetts growers, the cost of our biolog-
ical control program for smaller growers (10,000
pots) would be 28 cents, due to the effect of ship-
ping costs. For very small growers (below 10,000),
purchasing parasitoids in the smaller package
size (of 3000 rather than 15,000) increases the
cost further, by about 20% per pot, to approxi-
mately 34 cents. Costs per pot for multiple stem
plants increase in direct proportion, if current
release recommendations are followed.
Trials to date have been conducted based on
release rates calculated as parasitoids per stem
rather than per pot. Under this criterion, a triple-
stemmed plant requires the release of three times
as many parasitoids as a single-stemmed plant,
at three times the cost. No trials have yet been
run to determine if this increase in release rates
is really necessary. Because of the impact on cost,
we recommend that growers release wasps at a
rate of 0.5 parasitoids per pot (rather than per
stem), recognizing that data supporting this re-
lease rate exist only for single-stemmed plants.
Efficacy data are not available for crops with mul-
tiple stems per pot treated at this rate. Growers
using this rate should monitor whitefly numbers
and be prepared to intervene chemically if neces-
sary until practical experience establishes the ef-
ficacy of this application rate.
September 2002
Van Driesche et al.: Cost of Whitefly Biological Control
Further reduction in the cost of this biological
control program seems possible. Costs to use bio-
logical control might be reduced as much as one
third in a 15 week crop by deleting parasite re-
leases in the weeks in which insect growth regu-
lators are applied (weeks 7 and 8) and the last 2
or 3 weeks before harvest (if whitefly levels are
well suppressed in week 12, as determined by
your IPM scout). While these methods have not
been validated in controlled trials, they could be
incorporated into IPM programs and validated by
whitefly monitoring during the crop. Using these
modifications, the cost of biological control in a 15
week crop for a grower producing 30,000 would be
17 cents per plant, nearly the same as the cost of
chemical control (14 cents). Costs of chemical con-
trol do not include labor costs for mixing, applica-
tion and clean up; nor are times for safety
training or record keeping considered.
REFERENCES
HELGESEN, R. G., AND M. J. TAUBER 1974. Biological
control of greenhouse whitefly, Trialeurodes uaporari-
orum (Aleyrodidae: Homoptera), on short-term crops
by manipulating biotic and abiotic factors. Canadian
Entomol. 106: 1175-1188.
HODDLE, M. S., AND R. VAN DRIESCHE. 1996a. Evalua-
tion of Encarsia formosa (Hymenoptera: Aphelini-
dae) to control Bemisia argentifolii (Homoptera:
Aleyrodidae) on poinsettia (Euphorbia pulcherrima):
a lifetable analysis. Florida Entomol. 79: 1-12.
HODDLE, M. S., AND R. VAN DRIESCHE. 1996b. Evalua-
tion of Eretmocerus eremicus and Encarsia formosa
(Hymenoptera: Aphelinidae) Beltsville strain in
commercial greenhouses for biological control of Be-
misia argentifolii (Homoptera: Aleyrodidae) on col-
ored poinsettia plants. Florida Entomol. 82: 556-569.
HODDLE, M. S., R. G. VAN DRIESCHE, S. M. LYON, AND
J. P. SANDERSON. 2001. Compatibility of insect growth
regulators with Eretmocerus eremicus (Hymenoptera:
Aphelinidae) for whitefly control (Homoptera: Alyero-
didae) control on poinsettia: I. Laboratory Assays. Bio-
logical Control. 20: 122-131.
STEVENS, T. J., III, R. L. KILMER, AND S. J. GLENN.
2000. An economic comparison of biological and con-
ventional control strategies for whiteflies (Ho-
moptera: Aleyrodidae) in greenhouse poinsettia. J.
Econ. Entomol. 93:623-629.
VAN DRIESCHE, R. G., M. S. HODDLE, S. LYON, AND J. P.
SANDERSON. 2001. Compatibility of insect growth
regulators with Eretmocerus eremicus (Hymenop-
tera: Aphelinidae) for whitefly (Homoptera: Alyero-
didae) control on poinsettia. II. Trials in commercial
poinsettia crops. Biol. Contr. 20: 132-146.
Florida Entomologist 85(3)
September 2002
SEASONAL PHENOLOGY AND NATURAL ENEMIES OF
MACONELLICOCCUS HIRSUTUS (HEMIPTERA: PSEUDOCOCCIDAE)
IN AUSTRALIA
JOHN A. GOOLSBY1, ALAN A. KIRK2 AND DALE E. MEYERDIRK3
'USDA-ARS, Australian Biological Control Laboratory, CSIRO Long Pocket Laboratories
120 Meiers Rd., Indooroopilly, QLD, Australia 4068
E-mail: john.goolsby@csiro.au
2USDA-ARS, European Biological Control Laboratory, Campus International de Baillarguet
Montferrier sur Lez, 34980, CEDEX, France
3USDA-APHIS-PPQ, National Biological Control Institute, Riverdale, MD
ABSTRACT
Foreign exploration for natural enemies of pink hibiscus mealybug, Maconellicoccus hirsutus,
was conducted in Australia from 2000 to 2002. In Queensland, the predaceous beetle Crypto-
laemus montrouzieri, the predaceous drosophilid fly, Cacoxenus perspicax and the encrytid
parasitoid Gyranusoidea indica were recovered. In Western Australia and the Northern Ter-
ritory a predatory noctuid, Mataeomera sp., an aphelinid parasitoid Coccophagus sp., and a
probable encyrtid hyperparasitoid, Coccidoctonus sp. were reared from M. hirsutus on a na-
tive Hibiscus species. A field study was conducted from February 2000 to March 2002 in Sher-
wood, Queensland to document the seasonal phenology ofM. hirsutus in its native habitat on
its preferred host, Hibiscus rosa-sinensis. Populations of the mealybug stayed at or below
detectable levels for most of the study with minor population peaks in the summer months.
Key Words: mealybugs, biological control, Cryptolaemus, Gyranusoidea indica, Australia
RESUME
Se llevo a cabo una exploraci6n ex6tica de enemigos naturales del la cochinilla harinosa ro-
sada de hibisco, Maconellicoccus hirsutus, en Australia de 2000 a 2002. En Queensland, se
recubrieron el escarabajo depredador, Cryptolaemus montrouzieri, la mosca drosofilida de-
predador, Cacoxenus perspicax y el parasitoide encirtido Gyranusoidea indica. En Australia
Occidental y en el Territorio del Norte, un noctuido depredador, Mataeomera sp., un parasi-
toide afelinido, Coccophagus sp., un encirtido, Coccidoctonus sp. lo cual es probablemente un
hiperparasitoide, fueron criados de M. hirsutus sobre una especie native de Hibiscus. Se
llevo a cabo un studio del campo en febrero 2000 a marzo 2002 en Sherwood, Queensland
para documentary la fenologia estacional de M. hirsutus en su habitat native sobre su hospe-
dero preferido, Hibiscus rosa-sinensis. Poblaciones de la cochinilla harinosa se mantenia al
nivel de o debajo del nivel detectable para la mayor parte del studio con cumbres menores
de poblaci6n en los meses de verano.
The pink hibiscus mealybug, Maconellicoccus
hirsutus (Green) (Hemiptera: Pseudococcidae) in-
vaded Caribbean islands, during the mid 1990s,
was detected in Belize, Mexico, and the Imperial
Valley of California in 1999, and the Bahamas in
2000 (Meyerdirk 2000; Michaud & Evans 2000;
Roltsch et al. 2001). In the absence of effective
natural enemies, the highly polyphagous M. hir-
sutus can reach very high population levels
(Stibick 1997). At outbreak densities, damage to
agricultural crops and ornamental landscape
plantings can be quite extensive, actually killing
hibiscus and some trees such as soursop, Annona
muricata L. and Samanea saman (Jacq.) Merr.
(Meyerdirk 1999; Kairo et al. 2000). In the pres-
ence of natural enemies, Michaud & Evans (2000)
report that the host range of the mealybug in the
field is much reduced and that noticeable damage
was restricted to its preferred host, hibiscus, Hi-
biscus rosa-sinensis L. The parasitoids Anagyrus
kamali Moursi and Gyranusoidea indica Shafee,
Alam & Agarwal (Hymenoptera: Encyrtidae), and
predator Cyrptolaemus montrouzieri Mulsant
(Coleoptera: Coccinellidae) were introduced into
the Caribbean and are providing excellent control
of M. hirsutus (Kairo et al. 2000; Meyerdirk,
1999, 2000; Michaud & Evans 2000).
The two parasitoid speciesA. kamali and G. in-
dica were recently introduced into the Imperial
Valley of California within one month after the
initial detection of M. hirsutus. It is not known
how well these natural enemies will perform in
Goolsby et al.: Phenology of Maconellicoccus hirsutus
the hot, dry Imperial Valley climate, although
preliminary results are positive (Roltsch et al.
2001). In anticipation of the need for new natural
enemy species that are climatically adapted to
the environment in the Imperial Valley, a search
for new natural enemies was conducted in climat-
ically similar parts of Australia. Maconellicoccus
hirsutus is believed to be native to Australia and
is distributed throughout the wet and dry sub-
tropical and tropical areas of Queensland, North-
ern Territory, and Western Australia (Williams
1985).
In Australia, Maconellicoccus hirsutus is un-
der excellent natural control by predators and
parasitoids and rarely reaches pest status. It has
been collected from Annona squamosa L., Citrus
spp., Erthryina sp. Glycine max L., Gossypium
spp., Sida Acuta Burm. f., Parkinsonia sp.
Parthenium hysterophorus L., and Hibiscus spp.
(Williams 1985) Radunz & Allwood (1981) report
that it is an occasional pest of ornamentals in the
Northern Territory. Perhaps due to its minor pest
status, very little is known about the seasonal
phenology of M. hirsutus in its native range. A
field study of M. hirsutus was conducted in sub-
tropical southeast Queensland (Goolsby 2000)
where it is native, on its preferred host-hibiscus.
The study is designed to be used as a comparison
for studies of M. hirsutus where it is invasive.
MATERIALS AND METHODS
Seasonal Phenology
Garden sites with hibiscus, H. rosa-sinensis
were selected in suburban Brisbane, Queensland
to study the seasonal phenology of M. hirsutus.
Monthly collections over a two-year period from
April 2000 to March 2002 were made in Sher-
wood, Graceville, Chelmer and Indooroopilly,
Queensland, all residential suburbs in the Bris-
bane metropolitan area. Each garden site had
mature plantings of hibiscus. Corrugated card-
board bands were used to estimate the density of
M. hirsutus. This technique has been used in sev-
eral biological control programs to monitor popu-
lation densities of mealybugs and evaluate the
impact of their natural enemies (DeBach 1949,
Browning 1959, Furness 1976, Berlinger 1977).
Banding has the advantage of being the least de-
structive measure of pest density. Destructive
sampling of plant terminals on individual hibis-
cus plants could have an effect on the mealybug
population. Six bands per site (ea. 10 x 7 cm) were
wrapped around the main trunk and limbs of the
hibiscus plants and secured with wire garden tie.
Bands were left on the plants for a period of one
month. Counts of mealybugs in the bands were
conducted in the laboratory. All stages of mealy-
bugs on the surface of the bands were counted;
however, the counts overwhelmingly represent
mature ovipositing females. Bands were held in
paper cartons streaked with honey for emergence
of the natural enemies.
Arthropods collected by the Australian Biolog-
ical Control Laboratory (ABCL) were assigned a
specific site collection number. Each accession is
unique prefaced by the acronym for the labora-
tory with the year collected and a serial number
associated with the field collection (i.e., ABCL
2000809). If an organism is later exported to the
U.S. for a biological control program, the number
is used as an identifier in the ROBO (Releases of
Beneficial Organisms) database that is main-
tained by USDA-Agricultural Research Service.
Genbank accession numbers for the exported
agents are also included as an identifier in ROBO
(i.e., GENBANK-1786201). This method allows
biological control workers to track the identity of
the organism, including its DNA profile, from
field collection through quarantine evaluation
and release.
Foreign Exploration
Natural enemies were collected from bands
used in the M. hirsutus field study conducted in
Brisbane, Queensland from Jan 2000 to March
2002. After field collection, the bands were held in
paper cans streaked with honey for emergence of
the natural enemies. In addition, two distinct col-
lections were made in the Northern Territory and
Western Australia in July 2000 and May 2001.
Leaves, terminals and roots were sampled for
M. hirsutus in these collections. Mealybugs of all
stages were transferred to cotton stoppered vials
with honey and held in a humiditron (Debach &
Rose 1985) at 70% RH for emergence of parasi-
toids. Mealybugs were sent to the Queensland
Department of Primary Industries for identifica-
tion. Parasitoids and predators were sent to the
ARS-Systematic Entomology Laboratory (SEL)
in Beltsville, MD for identification. Gyranusoidea
indica collected in Queensland was compared to
material from rearing colonies in the California
Dept. of Food and Agriculture in Brawley, CA. Mo-
lecular sequencing of the D2 expansion domain of
the 28S rRNA gene was used to compare the two
populations. The method was employed by Bab-
cock & Heraty (2000) and De Barro et al. (2000) to
distinguish Encarsia species of parasitic Hy-
menoptera.
RESULTS AND DISCUSSION
Seasonal Phenology
Three of the six garden sites were originally
selected because they had visible populations of
mealybugs on the terminals of the hibiscus
plants. The terminals of the plants were impacted
and exhibiting bunchy growth, but no leaf loss.
496 Florida Ento
The population density in February 2000 at these
three sites were the highest encountered in the
study with 100, 92, & 102 M. hirsutus individuals
collected in the bands respectively. The high pop-
ulation levels at these three sites were not sus-
tained. For the remainder of the study,
populations at all the sites remained at very low
levels for most of the year with two small peaks
occurring during the summer months (November-
January) of 2001 (3.2 + 0.8 M. hirsutus per band)
and 2002 (3.3 0.9 M. hirsutus per band) (Fig. 1).
Populations were below detectable levels during
the winter months (June-August) in 2001-02.
Cryptolaemus montrouzieri was consistently re-
covered from the bands throughout the study
when mealybugs were present. The characteristic
larvae were also commonly observed on infested
terminals. The parasitoid, G. indica was rarely
recovered. It may have been underrepresented in
our study due to predation by C. montrouzieri in
the bands, or due to the tendency of the bands to
harbor later instar mealybugs that are not pre-
ferred by the parasitoid. The adult female M. hir-
sutus often uses the bands for shelter during
production of ovisacs. Our observations of the
natural enemies suggest that C. montrouzierri
Im
ologist 85(3) September 2002
plays the key role in regulating M. hirsutus in
this environment.
Retrospectively, we compared population lev-
els in our study to those in Puerto Rico post re-
lease of A. kamali and G. indica (Michaud &
Evans 2000). We concluded that for most of the
two-year study, hibiscus stands showed light or
no damage with less than 5% of the plants with
live M. hirsutus, which is far below levels detected
post release of natural enemies in Puerto Rico.
Foreign Exploration
The predators, parasitoids and hyperparasi-
toids reared from all the collections of M. hirsutus
are listed in Table 1. In Queensland (QLD), C.
montrouzieri was the most commonly encoun-
tered natural enemy. Populations of the beetle
persisted year round, responding to even low den-
sities of M. hirsutus. Cryptolaemus montrouzieri
is highly polyphagous in its native habitat. Lar-
vae and adults feed on crawlers and immature
soft scales, armored scales, wax scales, fluted wax
scales, and all stages of many mealybug species
(Smith et al. 1997). However, its preference
appears to be for mealybugs (Don Sands, CSIRO
LU
Mortlake
- Bentinck
--- -- Berry S
---. Magazine I
S 15 ...... ...... Jordan
Q .: -.. -. Honour I
c* I
S. .I
10
J *
I
"I I
0 -. I
'I T T
Fig. 1. Total number ofMaconellicoccus hirsutus caught in bands at six locations in Southeast Queensland over
a two-year period. The seasons are as follows, Winter (June-August), Spring (September-November), Summer (De-
cember-February) and Fall (March-May).
Goolsby et al.: Phenology of Maconellicoccus hirsutus
TABLE 1. NATURAL ENEMIES RECOVERED FROM MACONELLICOCCUS HIRSUTUS IN AUSTRALIA.
Species ABCL #* Location Date Comments
Gyranusoidea indica 2000809 Sherwood, QLD 28-II-2000 D2 sequences identical to
Hym: Encyrtidae 27 31.80 S 152 58.80 E colony in Brawley, CA
Cacoxenus perspicax 2000809 Sherwood, QLD 28-II-2000 Common predator found
Diptera: Drosophilidae 27 31.80 S 152 58.80 E associated with high den-
sities of pink hibiscus
mealybug
Ophelosia bifasciata 2000803 Sherwood, QLD 28-II-2000 Not commonly recovered
Hymenoptera: Pteromalidae 27 31.80 S 152 58.80 E may be a parasitoid of
Crytpolaemus
Crytpolaemus montrouzieri 2000809 Sherwood, QLD 28-II-2000 Very common predator
Coleoptera: Coccinellidae 27 31.80 S 152 58.80 E
Encyrtidae: Hymenoptera 2000892 Kununurra, WA 8-X-2000 Collected from native
Coccidoctonus sp. 15 40.12 S 128 39.57 E Hibiscus sp. and may be
a hyperparasite
Coccophagus sp. 2000892 Kununurra, WA 8-X-2000 Collected from native
Hymenoptera: Aphelinidae 15 40.12 S 128 39.57 E Hibiscus sp.
Mataeomera sp. 2000892 Kununurra, WA 8-X-2000 Collected from native
Lepidoptera: Noctuidae 15 40.12 S 128 39.57 E Hibiscus sp.
*ABCL # = site collection number for the USDA-ARS, Australian Biological Control Laboratory.
Entomology, pers. comm.). Cryptolaemus mon-
trouzieri is extant in parts of California; therefore
there are no plans to introduce this species. A pre-
daceous drosophilid fly, Cacoxenus perspicax
Knab was collected, but only at high M. hirsutus
densities. The host range of C. perspicax and its
impact on M. hirsutus is not known. No plans
were made to introduce this species to the United
States. The encrytid parasitoid G. indica was oc-
casionally collected from M. hirsutus in Queens-
land. There were no differences in the D2
sequences between the G. indica populations
from Queensland and those in culture in Califor-
nia; therefore we elected not to ship this popula-
tion to U.S. quarantine facilities. Representative
specimens of G. indica from Queensland were
vouchered at SEL and the colony was terminated.
Collections ofM. hirsutus in the Northern Ter-
ritory (NT) and Western Australia (WA) revealed
different species of natural enemies from those
found in Queensland. Several potential wild mal-
vaceous hosts were surveyed in addition to orna-
mental hibiscus including, Gossypium australe F.
Muell, Gossypium sturtianum J. H. Willis, Gos-
sypium robinsonii F. Muell., Hibiscus pandurifor-
mis N.L. Burman var. australis Hochr., Hibiscus
sp. C andAbutilon lepidum (F. Mueller) A. Mitch.
Maconellicoccus hirsutus was collected from or-
namental hibiscus, at several locations in the NT
and WA, but no parasitoids emerged. Collections
made of G. australe in northwestern Western
Australia near Derby and Broome revealed sub-
stantial numbers of M. hirsutus just below
ground level feeding on the roots and crown of the
plant. Populations of M. hirsutus on the roots
were heavily defended by ants. Near Kununurra,
WA, M. hirsutus was collected from the terminals
of a wild malvaceous host identified using the
Flora of the Kimberley (Wheeler et al. 1992) as
Hibiscus sp. C. A predaceous noctuid larvae,
Mataeomera sp., an aphelinid parasitoid Coc-
cophagus sp., and a probable hyperparasitoid,
Coccidoctonus sp. were reared from M. hirsutus
on this native host plant (Table 1). Mataeomera
sp. could have potential as a biological control
agent as some species in this genus are believed
to have a narrow host range and can be quite
damaging to mealybug populations (Don Sands,
CSIRO Entomology, pers. comm.). This species,
as well as the primary parasitoid Coccophagus sp.
might be considered for importation if additional
natural enemies are needed for control of M. hir-
sutus in California.
CONCLUSION
In its native range, M. hirsutus is rarely a pest
and under excellent natural control by indigenous
natural enemies. The predator C. montrouzieri
appears to be the key natural enemy in subtropi-
cal eastern Australia on ornamental stands of hi-
biscus. More intensive sampling is needed to
determine the impact of parasitism by G. indica.
In addition, the natural enemies Mataeomera sp.
(Lepidoptera: Noctuidae), and Coccophagus sp.,
(Hymenoptera: Aphelinidae) from the hot, mon-
soonal environment of northern Australia and
could be useful in biological control efforts in Cal-
ifornia if A. kamali and G. indica do not provide
adequate control.
ACKNOWLEDGMENTS
The authors would like to acknowledge Anne Bourne
for statistical advice; three anonymous reviewers for
their helpful comments and Ryan Zonneveld, CSIRO
Entomology, for conducting the field and laboratory
work. We would also like to thank the staff at the
Queensland Department of Natural Resources at the
Alan Fletcher Research Station and the homeowners in
Chelmer, Graceville, Indooroopilly, and Sherwood,
Queensland for allowing us to use their gardens as test
sites. Alan Donaldson, Queensland Department of Pri-
mary Industries identified the mealybugs, Don Sands,
CSIRO Entomology, identified the predatory Noctuidae,
Mike Schauff, Alan Norboom, Michael Gates identified
the Hymenoptera and Diptera recovered in the study.
The United States Department of Agriculture; Agricul-
tural Research Service-Office of International Re-
search Programs and the Animal and Plant Health
Inspection Service-National Biological Control Insti-
tute provided funding for the research.
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Florida Entomologist 85(3)
Wang et al.: Mites and Nematodes Associated with Termites
MITES AND NEMATODES ASSOCIATED WITH THREE
SUBTERRANEAN TERMITE SPECIES (ISOPTERA: RHINOTERMITIDAE)
CHANGLU WANG'3, JANINE E. POWELL14 AND BARRY M. O'CONNOR2
'Formosan Subterranean Termite Research Unit, USDA-ARS, Stoneville, MS 38776
2Museum of Zoology, University of Michigan, Ann Arbor, MI 48109
3Current address: Center for Urban and Industrial Pest Management, Purdue University
West Lafayette, IN 47907-1158
4Current address: USDA Forest Service, Rocky Mountain Research Station, Ft. Collins, CO 80526
ABSTRACT
Mites and nematodes associated with three subterranean termite species, Reticulitermes
flavipes (Kollar), Reticulitermes virginicus (Banks), and Coptotermes formosanus Shiraki
were studied. Mites belonging to 8 families were found associated with the three termite
species.Australhypopus sp. (Acari: Acaridae) was the most common mite on R. flavipes and
R. virginicus. Histiostoma formosana Phillipsen and Coppel (Acari: Acaridae) was the dom-
inant mite species living on C. formosanus. Nematode, Rhabditis sp. (Rhabditida: Rhabditi-
dae) was found in the three termite species examined. Nematodes did not cause termite
mortality or abnormal behavior. Percentages ofR. flavipes, R. virginicus, and C. formosanus
parasitized by nematodes were 67.9, 38.8, and 3.3%, respectively. The nematodes were found
mainly in the termite heads (85.8% in R. flavipes and R. virginicus). The abundance of mites
varied with colonies and termite species. Australhypopus sp. occurred in large numbers
when injured or dead termites are present, or when moisture of the rearing medium is low
in R. flavipes and R. virginicus colonies. Histiostoma formosana and Cosmoglyphus absoloni
SamiiAdik occurred in large numbers in C. formosanus colonies. Australhypopus sp. was
tested against R. flavipes in the laboratory. It did not cause significant termite mortality at
a rate of 10 mites/termite. From a biological point of view, mites investigated were not good
candidates for controlling termites.
Key Words: Mites, nematodes, subterranean termites, Coptotermes formosanus, Reticuliter-
mes flavipes, Reticulitermes virginicus
RESUME
Se estudiaron tres species de termitas subterraneas, Reticulitermes flavipes (Kollar), Reti-
culitermes virginicus Banks) and Coptotermes formosanus (Shiraki), las cuales se encon-
traron asociadas con nematados y acaros. Ocho families de acaros se encontraron asociados
con las tres species de termitas. El acaro mas comun fue Australhypopus sp. (Acari: Acar-
idae) el cual se present en las termites R. flavipes and R. virginicus. Histiostoma formosana
Phillipsen and Coppel (Acari: Acaridae) se lo encontr6 como especie dominant viviendo so-
bre C. formosanus. Un nematodo Rhabditis sp. (Rhabditida: Rhabditidae) se encontr6 en las
tres species examinadas. Los nematodos no causaron una mortalidad en las termites ni un
comportamiento normal sobre ellas. R. flavipes, R. virginicus, and C. formosanus se encon-
traron parasitadas por nematodos cuyos porcentajes fueron 67.9, 38.8, and 3.3%, respectiv-
amente. Los nematodos se encontraron principalmente en la cabeza de las termitas (85.8%
in R. flavipes and R. virginicus). La abundancia de los acaros variaron dependiendo de la co-
lonia y especie de termitas. En colonies de R. flavipes yR. virginicus el acaroAustralhypopus
sp. aparece6 en mayor cantidad si hay presencia de termitas heridas o muertas, o cuando el
contenido de humedad del medio de cria es bajo. Histiostoma formosana y Cosmoglyphus ab-
soloni Samsinak se present en mayor cantidad en colonies de C. formosanus.Australhypo-
pus sp. fue probado contra R. falvipes bajo condiciones de laboratorio. En una relacion de 10
acaros/termitas, la mortalidad de termitas no fue significant. Desde el punto de vista bio-
16gico, los acaros investigados no fueron buenos candidates para el control de termitas.
Translation provided by author.
Many organisms are associated with termites (e.g., protozoa), phoretic (e.g., mites), predatory
(Kistner 1969). Their relationships with termites (e.g., ants), or commensal (e.g., mites, ter-
may be parasitic (e.g., phorid flies), mutualistic mitiphile beetles).
Florida Entomologist 85(3)
Among the organisms associated with ter-
mites, the most numerous and least studied are
the mites (Acari) (Eickwort 1990). Mites are com-
monly seen in termite colonies (Becker 1969,
Samsiiiak 1964, Phillipsen & Coppel 1977a,b,
Costa-Leonardo & Soares 1993). Some mites are
only incidentally found in termite nests, while
others are obligate associates (Samgiihk 1964).
Generally, most mites associated with termites
were considered saprophagous or phoretic. These
mites do not have any significant effect on the
health of their termite hosts in nature. Few mites
feed on termites. Some, such asAcotyledon formo-
sani Phillipsen and Coppel, a species that should
be assigned to the genus Australhypopus, are
abundant in weak termite colonies and cause
death (Phillipsen & Coppel 1977a). The phoretic
instar or deutonymph ofA. formosani appeared to
negatively affect a large laboratory colony of Cop-
totermes formosanus Shiraki by fastening prima-
rily to termite heads and mouthparts, thereby
impeding normal feeding (Phillipsen & Coppel
1977a). Conversely, termite-associated mites may
benefit the termites by scavenging on other ar-
thropods or fungi (Eickwort 1990). Despite the
abundant and diverse mite fauna existing with
termites, little is known for their diversity, biol-
ogy, ecology, and the nature of their associations.
Termites have been recorded as being parasit-
ized by various species of nematodes (Table 1).
Some of these nematodes caused mortality to ter-
mite hosts in laboratory observations (Merrill &
Ford 1916, Pemberton 1928). Mermis sp. and Neo-
steinernema longicurvicauda Nguyen and Smart
may kill their termite hosts upon emergence
(Ruttledge 1925, Nguyen & Smart 1994). How-
ever, their rate of parasitism apparently is very
low and has been recorded only by the above
authors. There is little information on the abun-
dance of species of nematodes associated with
termites.
In the U.S., subterranean termites cause seri-
ous damage to homeowners each year (Su 1994).
Reticulitermes flavipes (Kollar), Reticulitermes
virginicus (Banks), and C. formosanus are among
the most widely distributed and destructive sub-
terranean termites (Snyder 1954, Su & Schef-
frahn 1988). Scientists have been searching for
environmentally compatible and sustainable con-
trol methods for these termites.
In this study, we investigated the diversity and
abundance of mites and nematodes associated
with three subterranean termite species: R. flaui-
pes, R. virginicus, and C. formosanus. We exam-
ined the nature of their association with termites
and the possibility of using these mites and nem-
atodes as biological control agents.
MATERIALS AND METHODS
Collection and Maintenance of Termites
Reticulitermes flavipes and R. virginicus were
collected in mixed forests in Washington county,
and in loblolly pine (Pinus taeda L.) forests in
Pearl River and Harrison counties in Mississippi.
They were kept in cylindrical plastic containers
(15.5 cm diameter, 4 cm deep) with 1-2 cm deep
vermiculite and sand (1:1 by volume). Corrugated
cardboard and/or pine wood blocks were added as
food. One hundred to 2,000 individuals were col-
lected from each termite colony. At least 15 colo-
nies of each species were collected from each of
the three counties during the period of October,
1998-August, 2000. Samples from ten C. formosa-
nus colonies were collected from pine wood bait
buried near trees in a university campus in the
city of Guangzhou, China, in August 1999. One
sample of C. formosanus colony was collected
from a pine stump in the city of Cenxi, Guangxi,
China. Two C. formosanus samples were collected
from the cardboard bait buried in a city park in
New Orleans, Louisiana on 15 June 1999 and 17
August 1999, respectively. Each C. formosanus
sample had over 2,000 individuals. The termite
colonies were kept for a maximum period of 120 d
from field collection date at room temperature
(21-25C) in the laboratory.
TABLE 1. NEMATODES ASSOCIATED WITH TERMITES.
Nematode species Termite host Relationship References
Mikoletzkya aerivora (Cobb) Reticulitermes lucifugus Occurs in head glands; host Merrill & Ford 1916
becomes sluggish
Rhabditisjaneti Duthier R. flauipes Feeding on decaying matter Banks & Snyder 1920
Rhabditis sp. Neotermes, Reticulitermes, Parasitic Pemberton 1928
Capritermes, Coptotermes,
Macrotermes, Microceroter-
mes, Rhinotermes, Termes
Mermis sp. Cornitermes, Thoracotermes Parasitic; kills host Ruttledge 1925
Neosteinernema R. flavipes Parasitic; kills host Nguyen & Smart 1994
longicurvicauda Nguyen and
Smart
September 2002
Wang et al.: Mites and Nematodes Associated with Termites
Biology, Abundance, and Distribution
of Mites and Nematodes Associated with Termites
Termites (number varied depending on pur-
pose of the observation) from each colony were
checked for mites and nematodes under a dissect-
ing microscope (70-400x). To examine the density
of Laelaptonyssus n.sp. (Laelaptonyssidae), 300
termites (workers and soldiers) were examined
from each of the 3 colonies sampled from Wash-
ington County (total n = 900). The Termites were
kept in the laboratory up to 30 d before examina-
tion. We assumed that the mites and nematodes
associated with termites remained on/inside the
termite body after the termites were transferred
to the laboratory. General collections of mites
were made by putting termites in 70% ethanol
and immediately examining the location and
number of mites. The mites dislodged slowly from
termite body once submerged in 70% ethanol. To
check for nematodes, termites were dissected
with two No. 3 insect pins and the specimens
washed with a drop of water so the nematodes
could be seen in suspension. White traps were
used to extract adult nematodes from termites
(Kaya & Stock 1997). Ten white traps were made.
Each trap consisted a 90 x 15 cm petri dish and a
5 cm diameter moist filter paper disk resting on a
platform. In each white trap, 10 R. flavipes (from
Washington Co.) freshly killed by fine point for-
ceps were put on the filter paper. The traps were
placed in a moist large plastic container and then
maintained at 25C in an incubator. After 10
days, tap water was added to the petri dish and
the existence of nematodes was examined.
The temporal changes in abundance of mites
and nematodes were examined from one labora-
tory reared R. flavipes colony. The colony (about
5,000 individuals) was collected in a forest in
Washington Co., Mississippi and maintained in a
plastic box (31 x 24 x 11 cm) with 5 cm deep sand
and pieces of corrugated cardboard. Twenty to
120 workers were examined weekly. We also
checked weekly for presence of adult nematodes
in the rearing medium and around dead termites
using a dissecting microscope.
To examine the relationship between the
abundance of mites and nematodes, the same in-
dividuals of R. flavipes and R. virginicus were ex-
amined for both mites and nematodes. They were
first checked using a dissecting microscope for
mite densities, then they were put on a glass slide
and dissected to check for presence of nematodes.
Nematodes and mites from C. formosanus were
examined from different individuals. Termites
had been kept in the laboratory for 60-80 d at ex-
amination date since collection.
Different parts of the termite body, e.g., head,
thorax, and abdomen, were examined separately
for nematodes. Termites from three R. flavipes
colonies (total of 39 termites) and four R. virgini-
cus colonies (total of 85 termites) were examined.
Voucher specimens have been deposited in collec-
tions of the Stoneville Research Quarantine Facil-
ity, USDA Agricultural Research Service, Stone-
ville, MS. and Museum of Zoology, Michigan State
University, Ann Arbor, Michigan.
Effect ofAustralhypopus sp. on R. flavipes
The most abundant mite in Reticulitermes col-
onies was an undescribed species in the genus
Australhypopus (Acaridae). The only species cur-
rently placed in this genus is the type-species,
A. flagellifer Fain and Friend, described from
deutonymphs collected from feces of a numbat,
Myrmecobius fasciatus Waterhouse, a termitoph-
agous marsupial, in Western Australia (Fain &
Friend 1984). Examination of type material by
B. O'Connor indicates that three other termito-
philous acarid species should also be placed in
this genus: Acotyledon formosani Phillipsen and
Coppel, A. lishimei Samsifiik, and Tyroglyphus
viduus Berlese and Leonardi. A systematic revi-
sion of this genus is in preparation by B. O'Con-
nor and A. Huang (University of Michigan).
In our laboratory colonies of both species of
Reticulitermes, when dead termites were present
there were enormous numbers ofAustralhypopus
sp. This mite was propagated by mixing dead ter-
mites (killed by freezing) with healthy termites.
The termites were then placed in cylindrical plas-
tic containers (15.5 cm diameter, 4 cm deep) with
1 cm deep vermiculite and sand as rearing me-
dium and corrugated cardboard as food. Termites
were kept at room temperature (21-25C). Large
numbers of adults and nymphs ofAustralhypopus
sp. were present 15 d later and were harvested by
brushing them from the inside surface of the con-
tainer into a small round plastic container (5.0 cm
diameter, 3.5 cm deep) filled with 0.01% Triton X-
100 (wetting agent) (Sigma-Aldrich, Inc.) fluid.
Mite density was determined. Mites (in feeding
stages) were then transferred to 100 x 15 mm
petri dishes with 40 healthy termite workers per
dish and a corrugated cardboard disk at rate of 5
and 10 mites per worker (200 and 400 mites per
dish, respectively) using a 5 ml pipet. A total of 5
ml solution was added to each dish. The mites
evenly dispersed soon after being transferred to
the dishes. The experiment was a completely ran-
dom design with each treatment rate was repli-
cated three times. The control dishes received
only 0.01% Triton X-100 solution. All of the repli-
cates were from the same termite colony. Dishes
were kept at 26C, 85% RH in a dark chamber.
Observations for termite mortality were made ev-
ery 7 d for 5 wk.
Statistical Analysis
Mortality data were arcsine of the square root
transformed and analyzed using repeated mea-
Florida Entomologist 85(3)
sures Analysis of Variance (ANOVA) (Littell et al.
1996). Analysis was performed using PROC
MIXED in SAS software (SAS Institute 1999).
RESULTS
Biology, Distribution, and Abundance of Mites
Associated with Termites
Examination and dissection of R. flavipes,
R. virginicus, and C. formosanus colonies showed
that the mite fauna associated with the three ter-
mite species was quite diverse (Table 2). The
mites were found mostly on the termite heads, al-
though some were also found on the thoraces, ab-
domens, and legs.
Australhypopus sp. (Acaridae), the most com-
mon mite species found on both R. flavipes and
R. virginicus, has a distinct phoretic stage, the
deutonymph (termed "hypopus" in older litera-
ture), in addition to the trophic stages. The
deutonymphs were most often seen attaching to
termite heads and mouthparts. They do not have
feeding mouthparts, and therefore cannot feed on
termites. Once the termites die from injury or
other causes such as disease, deutonymphs soon
(as early as 24 h after host death) transform into
tritonymphs, one of the trophic stages, and feed
on the dead termite body. Adult Australhypopus
sp. emerged as early as 1 d after emergence of
tritonymphs. Adults lay eggs near dead termites.
The eggs were laid singly or in batches of 2 to 30.
The eggs hatched after 1-3 d. High mite densities
were found in weak termite colonies, which were
characterized by individuals showing lower body
weight and flatter abdomens.
Enormous numbers of Australhypopus sp.
deutonymphs were found on the inside wall of the
containers holding R. flavipes colonies in the lab-
oratory. The head and legs of R. flavipes workers
were found covered by deutonymphs of Austral-
hypopus sp. Thousands of dead Australhypopus
TABLE 2. MITES FOUND ASSOCIATED WITH THREE SUBTERRANEAN TERMITE SPECIES.
Mite species Termite host Location Relationship with termites
Histiostomatidae
Histiostoma formosani
Histiostoma sp.
Acaridae
Australhypopus sp.
Cosmoglyphus absoloni
(Samsiifik)
Schwiebea spp.
Laelapidae
Hypoaspis miles (Berlese)
Laelaptonyssidae
Laelaptonyssus n.sp.
Digmasellidae
Dendrolaelaps sp.
Microdispidae
Unknown genus
Pygmephoridae
Near Unguidispus
Scutacaridae
Unknown genus
C. formosanus
R. flavipes, R. virginicus,
and C. formosanus
R. flavipes, R. virginicus,
and C. formosanus
C. formosanus
R. flavipes, R. virginicus,
and C. formosanus
R. flavipes, R. virginicus
R. flavipes
R. flavipes
R. flavipes
R. flavipes
New Orleans, Guangzhou,
Cenxi
Washington, Pearl River,
Harrison, New Orleans,
Cenxi, Guangzhou
Washington, Pearl River,
Harrison, New Orleans,
Guangzhou, Cenxi
Cenxi, Guangzhou
Washington, Pearl River,
Harrison, New Orleans,
Guangzhou
Washington
Washington, Pearl River
Washington
Washington
Washington
R. flavipes, C. formosanus Guangzhou
Phoretic; on termite head
and legs
Phoretic; bacteriophagous;
on termite head and legs
Phoretic; mostly on termite
head
Phoretic; mostly on termite
head
Opportunistic; on termite
body and in rearing medium
Opportunistic; in rearing
medium
Phoretic; feeding habit
unknown; on termite body
Opportunistic; omnivorous;
in rearing medium
Phoretic; in rearing medium
Phoretic; in rearing medium
Opportunistic; on termite
body and in rearing medium
September 2002
Wang et al.: Mites and Nematodes Associated with Termites 503
sp. deutonymphs also could be found immediately
outside of the termite rearing containers. Two fac-
tors were found closely related with high mite t Q
populations: the existence of injured or dead ter- + + 1
mites; and very low moisture content in the rear- X X c
ing medium. 6
Schwiebea sp. and Histiostoma sp. also were
commonly seen on R. flavipes and R. virginicus,
but did not occur as often as Australhypopus sp.
Laelaptonyssus n.sp. (Laelaptonyssidae) (Krantz ci c c
2001) was found in 5 of the R. flavipes colonies of +' 6 6 6
0 +1 +1 +1
the 30-40 colonies examined. This is a rare spe- c
cies and apparently lives only on termites. Num-
bers were always low. Among three R. flavipes
colonies that had this mite, the mite density
ranged from 1.3 to 17.3 per hundred termites
(mean = 9.2%, n = 900). Up to three Laelaptonys-
sus n.sp. were seen on one termite. They were c
mostly seen on the dorsal or lateral sides of the
termite abdomen, and moved rapidly when dis- .
turbed. This mite died soon after the termite host
died.
On C. formosanus, the dominant mite species
were Histiostoma formosana Phillipsen and Cop-
pel and Cosmoglyphus absoloni (SamBiiiak). His- cc 4
tiostoma formosana deutonymphs were phoretic +1 +1 +1
and were mostly found on termite tarsi and tibia, cc
whereas deutonymphs of C. absoloni normally at- co
tached to the termite head. When H. formosana
populations were high (>15 per termite), they
could be seen on the whole body. These two spe-
cies appeared in great numbers (320 per termite) 9 H
in weak, laboratory-reared colonies. Histiostoma C ) W
sp., Australhypopus sp., and Schwiebea sp. also +1
were common in C. formosanus colonies held inm o Z
the laboratory Laelaptonyssus chinensis Sam- q C
iiiak, a rarely observed species that lives only on
termites, was found in one colony collected from
Cenxi, Guangxi, China.
Distribution and Abundance of Nematodes in Termites
Nematodes were often seen inside the termite .2
body. The only nematode observed was Rhabditis
sp. (Rhabditida: Rhabditidae). Among the three
termite species, nematodes were most common in E
R. flavipes and least common in C. formosanus. .
The maximum number of nematodes per termite .
also was different (Table 3). We did not find the q L
adult form of the nematodes either by observation
of the termites externally or by extracting nema- H z
todes from termites using the white trap method.
The distribution of nematodes in three body
parts was similar in R. flavipes and R. virginicus
(Fig. 1). Most of the nematodes were found in ter-
mite heads with an average of 85.8% of the total m |
occurrences in R. flavipes and R. virginicus. Only
7.0% and 7.2% of the nematodes were found in the
thorax and abdomen, respectively. Due to the low 6
count, distribution of nematodes in different .
C. formosanus body parts could not be described.
Florida Entomologist 85(3)
100
80
C)
S60
40
o 40
20
0
m
R. virginicus
R. j ,;. .
Head Thorax Abdomen
Location ofNematodes in Termite Body
Fig. 1. Distribution ofRhabditis sp. in termite body.
Temporal Changes in Mite and Nematode Populations
Mite and nematode populations varied not only
between termite colonies, but also changed over
time. One R. flavipes colony (about 5,000 individ-
uals) was observed in the laboratory for three
months (Fig. 2). The density of mites was 1.1 (n =
47) per termite when termites were collected from
the field. The mite densities dropped and fluctu-
ated to 0.1-0.3 mites per termite when held in the
laboratory. The percentage of termites infested
with mites varied between 10 and 32.2% (n = 20 to
120). Average nematode numbers per termite also
fluctuated between 0.8 and 2.5. The percentages of
termites with nematodes varied between 60-100%
(n = 20) from 4 examination dates.
The C. formosanus colonies collected from
China showed a dramatic increase in mite popu-
lation and decrease in body weight and termite
numbers. Five colonies were observed for their
mite populations and body weight. The weight of
C. formosanus (4% soldiers) was measured as 3.58
+ 0.01 mg/termite (279 0.6 termites/g) at collec-
tion date. The average mite density was 1.0 mite/
termite 70 d after being kept in laboratory. After
111 d of rearing in laboratory, the mite density in-
creased to 24.4 mites/termite. Most of the mites
belonged to Histiostoma spp. The average weight
of termites decreased to 0.14 mg/termite (7,407
termites/g).
High mite density could be lowered if termites
are kept in appropriate conditions. We observed a
colony ofR. flavipes (with over 5,000 individuals)
maintained in a 10-gallon aquarium (51 x 25.5 x
30.5 cm). The container was filled with 2 cm deep
vermiculite and 8 cm deep of southern pine
wooden stakes. The inside walls of the aquarium
were covered with enormous numbers ofAustral-
hypopus sp. deutonymphs 2-3 wk after termites
were transferred from the field. This was caused
by the existence of injured termites. Mites disap-
peared from the inner walls of the container later.
This suggests that there was a cycle of reproduc-
tion of mites on the dead termites, followed by a
population crash from the lack of dead termites.
Relationship between Mites and Termites
Although a high density ofAustralhypopus sp.
was observed on R. flavipes and R. virginicus, it
was not observed to cause significant mortality to
termites in the laboratory. The adults and
tritonymphs were observed feeding only on dead
termites. Termite mortality was not significantly
higher when mites were added to the termite col-
ony (F = 1.53; df = 2, 6; P = 0.29) (Fig. 3). Termite
individuals with high numbers of mites (10 mites
per termite) were capable of living for many
weeks. Therefore, the presence of large numbers
ofAustralhypopus sp. did not appear to cause sig-
nificant health problems for termite colonies. The
mite species C. absoloni from termite colonies col-
lected in China showed a similar relationship
with C. formosanus.
50
40
S30
20
2 20
0 20 40 60 80 100 0 10 20 30 40
Days of Rearing in Laboratory
Fig. 2. Temporal changes in mite and nematode pop-
ulations in a Reticulitermes flavipes colony reared in
laboratory.
Days after Inoculation of Mites
10 mites/termite 5 mites/termite -- Control
Fig. 3. Effect ofAustralhypopus sp. on Reticulitermes
flavipes.
Mites
Nematodes
September 2002
Wang et al.: Mites and Nematodes Associated with Termites
Relationship between Rhabditis sp. and Termites
The presence of nematodes in the three ter-
mite species did not cause any abnormal morphol-
ogy or behavior of the termites. We often observed
nematodes attached to the mouthparts of healthy
R. flavipes and R. virginicus when they were im-
mersed in 70% ethyl alcohol. This demonstrates
that the nematodes were located close to the im-
mediate opening of termites' mouthparts. Dead
R. flavipes, R. virginicus, and C. formosanus
placed in rearing containers and on white traps
did not yield nematodes. No nematodes were
found in dead termites when dissected in water.
DISCUSSION
This study provided information on the biol-
ogy, abundance, and mite and nematode fauna as-
sociated with three species of subterranean
termites. Information on taxonomy and biology of
the mites associated with termites is still very
limited and needs to be further clarified. From a
biological control point of view, the mites investi-
gated were not good candidates for controlling
termites. They had little effect on termites even at
high densities. Grace (1997) also reported no dis-
cernible effect of phoretic mites on survival and
feeding of R. flavipes. The presence of Austral-
hypopus sp. may even benefit the termites. We
observed that Australhypopus sp. population in-
creased in density when R. flavipes and R. virgini-
cus died from treatment with entomopathogenic
nematodes or pathogens. The mites consumed the
dead termites that were killed by nematodes or
pathogenic fungi. As a result, the nematodes and
fungus could not initiate secondary infections in
termites. Therefore, the remaining termites can
be expected to survive after initial infection with
pathogens or nematodes. We also observed Aus-
tralhypopus sp. preying upon entomopathogenic
nematodes in the laboratory.
Australhypopus sp. and Histiostoma spp. are
similar to "Acotyledon" formosani in that they
both have feeding and non-feeding life stages.
They both have the potential to develop large
numbers and were more abundant in weak and
disturbed termite colonies.
In this study, Australhypopus sp. did not cause
any apparent harm to the large termite group ob-
served in the laboratory. The mite numbers in-
creased greatly soon after disturbance to termites
and collapsed later. Phillipsen & Coppel (1977a)
stated that "Acotyledon" formosani was harmful to
C. formosanus. We speculate that huge numbers of
deutonymphs may simply overwhelm small groups
of termites. "Acotyledon" formosani was closely re-
lated withAustralhypopus sp. We suspect that they
have similar relationships with termite hosts.
The rates of nematode parasitism among the
three termite species were quite different. Copto-
terms formosanus had the lowest percentage of
parasitization and lowest maximum number of
nematodes per termite, but more studies are
needed to clarify their biology. Pemberton (1925)
did not find any nematodes from C. formosanus
collected in Honolulu, Hawaii. However, our re-
sults suggest that careful examination of large
numbers of individuals may reveal nematodes in
C. formosanus individuals. We did not find the
parasitic nematode, Neosteinernema longicur-
vicauda, reported by Nguyen & Smart (1994).
This nematode is host-specific and kills the host
upon emergence (Nguyen & Smart 1994). It could
be a potentially useful agent for control of R. fla-
vipes. More survey is warranted to search for use-
ful nematodes to control subterranean termites.
ACKNOWLEDGMENTS
We are grateful to G. W. Krantz (Oregon State Uni-
versity) and J. W. Amrine, Jr. (West Virginia University)
for identification of some of the mite specimens and pro-
viding information on mite biology. We also thank K. B.
Nguyen (University of Florida) for identification of nem-
atodes. Junhong Zhong from Guangdong Entomological
Institute, China, assisted in collecting C. formosanus
colonies. Full financial support was provided by USDA,
Agricultural Research Service via cooperative agree-
ment with Mississippi State University.
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Oehlschlager et al.: Control of Red Ring Disease by Mass Trapping of R. palmarum 507
CONTROL OF RED RING DISEASE BY MASS TRAPPING OF
RHYNCHOPHOR US PALMAR UM (COLEOPTERA: CURCULIONIDAE)
A. C. OEHLSCHLAGER1, CARLOS CHINCHILLA2, GEOVANI CASTILLO2, AND LILLIANA GONZALEZ'
'ChemTica Internacional, Valencia Industrial Park Zeta, Sto. Domingo, Heredia, Costa Rica
info@mail.pheroshop.com
2A. S. D. de Costa Rica, Apdo. 30-1000, San Jose, Costa Rica
ABSTRACT
Rhynchophorus palmarum (L), the American palm weevil, is an important pest of several
palm species in tropical America as a vector of the red ring nematode Bursaphelenchus coco-
philus Cobb. Bimonthly inspections coupled with elimination of red ring diseased (RRD) oil
palms (Elaeis guineensis Jacq.) failed to reduce infection rates of oil palms in two commercial
plantations in Costa Rica. Addition of pheromone-based trapping ofR. palmarum using trap
densities of less than one trap per five hectares lowered RRD in both plantations by over 80%
in one year. Continued removal ofRRD infected palms and trapping maintained RRD at very
low levels over several years. No matter what the initial RRD infection level, trap density or
capture rate, areas with high and areas with low RRD infection levels declined to the same
low RRD infection level after one year of trapping. An efficient strategy for management of
RRD in oil palm is based on an integrated approach where RRD and other diseased (e.g.,
spear rot) palms are promptly eliminated or properly treated and pheromone-baited traps
are used to reduce populations of R. palmarum. These strategies are complimented by re-
moval of weevil-infested palms after wind and lightning damage and periodic removal of
palms (e.g., coconut palms) in surrounding areas that serve as hosts for R. palmarum.
Key Words: American palm weevil, Rhynchophorus palmarum, pheromone trapping, oil palm,
red ring disease
RESUME
El gorgojo de la palma americana Rhynchophorus palmarum (L), es una plaga de importan-
cia en diversas species de palmas en Am6rica tropical como vector del nematodo del anillo
rojo Bursaphelenchus cocophilus Cobb. Las inspecciones bimensuales en conjunto con la eli-
minaci6n de las palmas infestadas con la enfermedad del anillo rojo (RRD, sus siglas en ing-
16s) han fallado en reducir las tasas de infeci6n en palmas aceiteras (Elaeis guineensis Jacq.)
de dos plantaciones comerciales en Costa Rica. La adici6n del trampeo basado en feromonas
del R. palmarum usando densidades de trampas menores a una trampa por cada cinco hec-
tareas, efectivamente redujo el RRD en ambas plantaciones en mas de un 80% en un ano. La
remoci6n continue de palmas infestadas con RRD y el trampeo mantuvieron el RRD a niveles
muy bajos sobre various anos. Sin importar cual fue el nivel inicial de RRD, densidad de tram-
pas o tasa de capture, las areas con infestaciones altas y las areas con infestaciones bajas de
RRD declinaron su infestaci6n al mismo nivel bajo de infestaci6n de RRD despu6s del tram-
peo por un ano. Una estrategia eficiente de manejo del RRD en cultivo de palma aceitera esta
basada en un enfoque integral en donde tanto las palmas con RRD como aquellas con otras
enfermedades (por ejemplo pudrici6n del apice central), son eliminadas o tratadas con pron-
titud y para reducir las poblaciones de R. palmarum vector del RRD se utilizan trampas ce-
badas con feromona. Esta estrategia es complementada por la remoci6n tambi6n de palmas
infestadas de gorgojos despu6s de que ocurran dahos ocasionados con el viento o descargas
el6ctricas, como tambi6n con la remoci6n peri6dica de palmas (por ejemplo palmas cocoteros)
en las areas que bordean y que sirven como hospederas del R. palmarum.
Translation provided by author.
The American palm weevil, Rhynchophorus Chinchilla 1990). It is thought that the nematode
palmarum (L) is a significant pest of cultivated oil is transmitted during weevil feeding and oviposi-
(Elaeis guineensis Jacq.) and coconut (Cocos tion (Griffith 1968, Hagley 1963). Once in the
nucifera L) palms in the Americas through direct palm the infection may result in two different
attack and as a vector of the red ring nematode symptoms. The classical symptom is yellowing of
Bursaphelenchus cocophilus, Cobb (Wattana- fronds and rapid death of the palm. A second
pongsiri 1966, Fenwick 1968, Blair 1970a, 1970b, symptom is "little leaf syndrome" which is mani-
Griffith 1967, 1968, 1969, 1987, Hagley 1963, fested in stunted growth offronds. Palms with this
Chinchilla 1988, Chinchilla et al. 1990, Morales & manifestation of B. cocophilus infection usually
Florida Entomologist 85(3)
live for many years (Chinchilla, 1988). The ring of
darkly colored necrotic tissue observed in the cut
cross-sections of stems of palms with classical
symptoms of yellowing fronds has given the afflic-
tion its common name, red ring disease (RRD).
RRD causes a significant economic impact in oil
palm. Although RRD is not a problem in plantings
under five years, losses increase thereafter and be-
come significant in older stands. Accumulated
losses due to RRD and the associated little leaf syn-
drome can reach 15% or more in commercial plan-
tations (Chinchilla et al. 1990). R. palmarum is the
only identified insect vector of RRD in oil palm in
Central America and other mechanisms of infec-
tion such as via nematode contaminated knives
during pruning or transmission via contaminated
soil are considered insignificant in comparison to
inoculation by the weevils (Chinchilla 1988, Fen-
wick 1968, Schuiling & van Dinther 1981). Meta-
masius spp. have been suggested as vectors of RRD
in Colombia (Silva 1991; Calvache et al. 1995) but
B. cocophilus infected M. hemipterus, the principal
Metamasius species in oil palm, have not been de-
tected in Costa Rica (Bulgarelli et al. 1998). The
rate of red ring infection in Costa Rican and Hon-
duran oil palm plantations is correlated with fluc-
tuations in nematode-infected R. palmarum
populations (Chinchilla et al. 1990; Morales &
Chinchilla 1990). RRD symptoms of frond yellow-
ing are not evident until two to three months after
infection and nematocidal treatments at this point
have proven fruitless in oil palm (Chinchilla 1988).
The most effective strategy to lower the incidence
of RRD is rapid elimination of nematode infected
palms coupled with reduction of weevil populations
through elimination of breeding sites (Chinchilla
1988, Griffith 1987) and trapping of adults (Chin-
chilla 1988). Treatment of palms with insecticide
(Fenwick 1967), removal of red ring-diseased trees
and trapping using insecticide-laden palm stem
have been considered appropriate phytosanitation
practices (Griffith 1969). Trapping has been prac-
ticed in the Caribbean since the 1970s (Mariau
1968, Griffith 1969). Most commonly used traps
prior to this work utilized insecticide-treated palm
stem (Mariau 1968, Griffith 1969, Morin et al.
1986, Chinchilla et al. 1990) or tropical fruits (Del-
gado & Moreno 1986).
Several years ago we demonstrated that R.
palmarum are more captured in plastic bucket
traps from which are released the male-produced
aggregation pheromone and that contain insecti-
cide-laden sugarcane or palm stem (Oehlschlager
et al., 1992a,b, 1993b). We subsequently reported
that in commercial oil palm plantations bucket
traps baited with pheromone and insecticide-
laced sugarcane, at a density of 4 traps/hectare,
effectively lowered R. palmarum populations and
new RRD infection (Oehlschlager et al. 1995). In
the 38 ha mass trapping site, R. palmarum cap-
ture rates and RRD declined from initial values
by >80% during a one year trial. During the same
period RRD in surrounding lots of the same age
and material decreased only 10% and the inci-
dence of RRD in the plantation as a whole in-
creased by 20% (Oehlschlager et al. 1995).
This high density trapping experiment was fol-
lowed by mass trapping in four plots averaging 56
hectares using trap densities ranging from 1 trap/
ha to 1 trap/3.5 ha. In all cases initial capture
rates declined after two to three months and RRD
declined compared to the same period a year pre-
vious (Chinchilla et al. 1993).
In the present paper we present results of
mass trapping of R. palmarum in two large oil
palm plantations extending over several years.
One site is a 6,514 ha plantation, located in south-
west Costa Rica and the site of previous trials.
Data from a second 8,719 ha plantation, located
in west-central Costa Rica is also included. These
studies illustrate the operational use of trapping
R. palmarum to lower RRD in commercial oil
palm that has only been published in preliminary
form (Chinchilla et al. 1993).
MATERIALS AND METHODS
Study Sites
The study was conducted in two commercial
African oil palm (E. guineensis) plantations near
the Pacific Coast of Costa Rica. The Coto planta-
tion is a 6,514 ha commercial oil palm plantation
in southern Costa Rica ~10 km from the Pacific
coast. The plant age profile of the plantation at
the beginning of 1992 was: 1,329 ha of 17-24 year-
old palms, 1,265 ha of 12-16 year-old palms, 2,602
ha of 6-11 year-old palms and 1,317 ha of 0-5 year-
old palms. The Quepos oil palm plantation is a
8,719 ha commercial plantation in central Costa
Rica ~2 km from the Pacific coast. The plant age
profile of the plantation at the beginning of 1992
was: 4,333 ha of 17-24 year-old palms, 1,643 ha of
12-16 year-old palms and 2,743 ha of 0-4 year-old
palms. The two sites are similar in variety of
plant material, plant age profile, size and climate,
but Quepos has a more extended dry season.
Disease Surveying
In both plantations periodic visual survey for
red ring disease was initiated in 1989 and was
continued bimonthly after the beginning of 1990.
Each plantation contains palm planted on a 9-m
grid at 142 palms/ha. The plantations are divided
into approximate 10 ha plots to facilitate manage-
ment. Inspection was by visual inspection of each
8 rows with the inspector walking through the
center. Inspectors recorded the position (section
location, row location and location in row) of each
palm with early symptoms of RRD. Within 1 week
a team of two revisited the infected palm and if
they verify RRD infection the palm is poisoned
September 2002
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