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Wetterer & Moore: Fire Ants at Gopher Tortoise Burrows
RED IMPORTED FIRE ANTS (HYMENOPTERA: FORMICIDAE)
AT GOPHER TORTOISE (TESTUDINES: TESTUDINIDAE) BURROWS
JAMES K. WETTERER AND JON A. MOORE
Wilkes Honors College, Florida Atlantic University, 5353 Parkside Drive, Jupiter, FL 33458
ABSTRACT
The gopher tortoise, Gopherus polyphemus Daudin, is endemic to the southeastern US,
where its populations are declining primarily due to habitat destruction. Tortoises are
preyed upon by many species, including the red imported fire ant, Solenopsis invicta Buren,
a destructive exotic species now common throughout the tortoises' entire range. We surveyed
ants using tuna bait at 154 G. polyphemus burrows in a greenway reserve established to pro-
tect the tortoises in a residential area of southeast Florida. We found S. invicta present, typ-
ically recruiting to the bait in very high numbers, on the aprons of 33% of the tortoise
burrows. Solenopsis invicta occurred significantly more often at burrows within 30 m of the
greenway's outer edge than at burrows in more interior parts of the greenway (57% versus
16%). Among the interior burrows, S. invicta occurred significantly more often at burrows di-
rectly on two narrow strips of disturbed habitat, along an old fence line and an old pipeline,
than at burrows not on these two strips (46% versus 12%). The greenway interior appears to
offer tortoises and other species some refuge from S. invicta. However, the long thin design
typical of greenways, the inclusion of walking paths through the greenways, and the policies
of prescribed burning and reduction mowing used to maintain open habitat for the tortoises
all may increase the tortoises' exposure to S. invicta. Solenopsis invicta is also a grave threat
to other native species in these reserves, including the many animals that obligately live in-
side gopher tortoise burrows.
Key Words: edge effect, exotic species, Florida, Gopherus, greenway, Solenopsis invicta.
RESUME
La especia tortuga gopher, Gopherus polyphemus Daudin, es end6mica al sudeste de los
EE.UU., donde sus poblaciones disminuyen principalmente debido a la destrucci6n de habi-
tat. Las tortugas son atacadas por muchas species, inclusive la hormiga roja de fuego im-
portada, la Solenopsis invicta Buren, una especie ex6tica destructive ahora comun a toda la
region geografica de las tortugas. Estudiamos las hormigas que atraemos con cebo de atun
en 154 madrigueras de la G. polyphemus en una reserve ecol6gica que se estableci6 para pro-
teger las tortugas en un area residential del sudeste de la Florida. Encontramos a la S. in-
victa present, alistanda tipicamente al cebo en numeros muy altos, en los delantales de 33%
de las madrigueras de las tortugas. La presencia de la S. invicta ocurri6 apreciablemente
mas a menudo en madrigueras dentro de un parametro de 30 m de la orilla exterior de la re-
serva que en las madrigueras en parties mas interiores de la reserve (57% contra 16%). Entre
las madrigueras interiores, la S. invicta ocurri6 apreciablemente mas a menudo en madri-
gueras directamente en dos tiras estrechas del habitat perturbado, por una linea vieja de la
cerca y una tuberia vieja, que en las madrigueras que no en estaban en estas dos tiras (46%
contra 12%). El interior de la reserve ecol6gica aparece ofrecer las tortugas y otras animals
algun refugio de S. invicta. El largo y estrecho diseio tipico de las reserves ecol6gicas, la in-
clusi6n de senderos caminantes en las reserves ecol6gicas, y por la costumbre de incendios
prescritos y la reducci6n por cortes utilizados para mantener el habitat abierto para las tor-
tugas, todo ello puede aumentar la exposici6n de las tortugas a la S. invicta. La S. invicta es
tambi6n una amenaza grave a otras species nativas en estas reserves, inclusive los muchos
animals que se ven obligados a vivir en las madrigueras de las tortugas.
Translation provided by the authors.
Gopher tortoises, Gopherus polyphemus Dau- most of the tortoise's preferred upland habitat
din, are endemic to the coastal plains of the has been converted to, for example, citrus or-
southeastern United States (Diemer 1992) and chards, housing developments, and phosphate
populations are declining through most of their mines (Diemer 1992). The gopher tortoise is now
range, primarily due to habitat destruction protected in Florida as a "species of special con-
(Diemer 1986). The tortoises' decline has been cern" (Diemer 1992; Florida Game & Fresh Water
particularly great in southeastern Florida, where Fish Commission 1994).
Florida Entomologist 88(4)
Gopher tortoises prefer habitats with sandy
soils for burrows, herbaceous plants for food, and
open sunny spots for nesting and basking (Di-
emer 1992). Fire is a normal element in gopher
tortoise habitat, and when natural fires are sup-
pressed, habitats may become overgrown. To
maintain the open spaces necessary for herba-
ceous vegetation and tortoise nesting and basking
sites, land managers often use prescribed burning
or reduction mowing (Wade & Lunsford 1989;
Main & Tanner 1999).
Female tortoises lay clutches of 3-15 eggs, usu-
ally in the sand apron in front of their burrow or in
another nearby sunny spot (typically 16-67 cm
from the burrow entrance; Butler and Hull 1996).
Many predators attack gopher tortoise nests, in-
cluding raccoons, foxes, skunks, armadillos, and
snakes (Douglass & Winegarner 1977; Diemer
1992). Nest predation is often very high (Alford
1980). Landers et al. (1980) found that mammalian
predators destroyed most gopher tortoise nests
(87% within the first few weeks after laying) and
the few surviving hatchlings were often attacked
and killed by the red imported fire ant, Solenopsis
invicta Buren. Epperson & Heise (2003) found that
S. invicta predation accounted for 27% of post-
hatching mortality of gopher tortoise hatchlings.
Solenopsis invicta is probably the most de-
structive exotic ant species in the southeastern
US, negatively impacting both invertebrates and
vertebrates (Wojcik 1994; Porter & Savignano
1990). This invasive species arrived in Alabama
by ship from South America and has spread
across the southeast US from Texas to North
Carolina, now occupying the entire range of the
gopher tortoise.
Solenopsis invicta is a well-known predator on
bird hatchlings, particularly ground-nesting spe-
cies (e.g., see Ridlehuber 1982; Sikes & Arnold
1986; Steigman 1993; Drees 1994; Lockley 1995;
Powell 1995; Dickinson 1995; Giuliano et al 1996;
Mueller et al. 1999; Kopachena et al. 2000;
H. Smith, pers. comm.). Solenopsis invicta also
attacks reptile hatchlings. Cintra (1985) found
that S. invicta commonly attacked and killed
hatchling caiman (Caiman yacare (Daudin)).
Love (1997) noted records of S. invicta attacking
captive tortoises and hatching turtles and alliga-
tors. Allen et al. (1997) found that hatchling
American alligators (Alligator mississippiensis
(Daudin)) stung by fire ants showed decreased
weight gain and increased mortality. Reagan et
al. (2000) found that S. invicta in alligator nests
had a significant negative impact on hatching
success. Solenopsis invicta also appears to be an
important threat to hatching sea turtles (Wilmers
et al. 1996; Moulis 1996; Allen et al. 1998, 2001;
Krahe et al. 2003; Wetterer & Wood 2005).
Solenopsis invicta appears to be an important
threat to some adult reptiles as well, and is con-
sidered important in the decline of numerous rep-
tile species in the southeastern US (Mount 1981).
Montgomery (1996) observed six cases of S. in-
victa attacking adult three-toed box turtles (Ter-
rapene carolina triunguis (Agassiz)) in Texas,
with only one of the turtles surviving due to hu-
man intervention. "It seems that the box turtle's
defensive reaction is to withdraw the plastron,
which cannot keep the ants out completely even
when tightly closed. This reaction also renders
the turtle immobile, which enables the ants to
swarm over it, seeking out small openings be-
tween the plastron and the carapace. The turtle
must eventually relax the plastron, which allows
more ants to gain further access to its body."
Montgomery (1996) concluded, "it would be diffi-
cult to estimate just how much effect the ants
alone are having. But my observations lead me to
believe that fire ants pose a serious threat to
three-toed box turtles."
In addition to S. invicta, two other ant species
found in the gopher tortoises' range also are
known to attack vertebrates and may pose a
threat to gopher tortoises: Solenopsis geminata
(Fab.) and Wasmannia auropunctata (Roger). Al-
though not generally as virulent as its congener
S. invicta, S. geminata is also known to attack the
hatchlings of birds and reptiles (e.g., Stoddard
1931; Travis 1941; Mrazek 1974). Where W auro-
punctata occurs at high densities, it also can have
a great impact on vertebrates. For example, in
Gabon, W auropunctata has been implicated in
blinding house cats (Felis catus L.) and native
wildlife, including elephants (Loxodonta africana
(Blumenbach)) (Wetterer et al. 1999). In the So-
lomon Islands, locals reported that W auropunc-
tata commonly stings the eyes of dogs that even-
tually become blind, and attacks hatchlings of the
ground-nesting Melanesian Scrubfowl (Megapo-
dius eremita Hartlaub) (Wetterer 1997).
The present study was motivated, in part, by
the discovery in a local greenway reserve of a
Florida box turtle (Terrapene carolina bauri Tay-
lor) being attacked by S. invicta (M. Floyd, pers.
comm.). To evaluate the potential threat of S. in-
victa to gopher tortoises in the reserve, we sur-
veyed ants at gopher tortoise burrows in the
greenway.
MATERIALS AND METHODS
Study Area
Our study site was one section ("Range VIa") of
a greenway reserve set up through the Abacoa
residential development in Jupiter, Florida
(26.900N, 80.11oW), to preserve gopher tortoise
habitat. The entire Abacoa greenway system en-
compasses roughly 10% of the development's land
area. Our study segment included a 9.16-ha
wooded range and an adjacent sunken water re-
tention basin (Fig. 1). A path encircles the range
December 2005
Wetterer & Moore: Fire Ants at Gopher Tortoise Burrows
Figure 1. Aerial photograph of "Range VIa" in the Abacoa greenway, Jupiter, Florida, a wooded range bounded
in the north by Frederick Small Road and on other sides by a sunken water catchment basin. An old fence line and
a recent pipeline cross the northern and eastern parts of the range. The black bar equals approximately 30 m.
and two linear areas of disturbance cross through
the northern and eastern portions of the range;
one is an old (>6 years-old) cleared cattle fence
line (fence removed) and the other is a recently
(<3 years-old) dug pipeline (Fig. 1). A chain-link
fence, separating the reserve from Frederick
Small Road, bounds the north side of the range.
The range is bounded on the other three sides by
the water catchment basin with channels for di-
recting and holding rainwater runoff from storms.
Except for the channels, the basin is dry through
most of the year. A chain-link fence separates the
catchment basin from Central Road to the east
and several baseball fields to the south and west.
The relatively undisturbed portions of the
range consist of typical flatwood scrub (Myers and
Ewel 1990) with a sparse canopy of mature slash
pines (Pinus elliottii Engelman), an understory of
saw palmetto (Serenoa repens (Bartram)) thickets
and scrubby oaks (Quercus spp.), and open spaces
dominated by wiregrass (Aristida beyrichiana
Trin. & Rupr.), with lesser amounts of runner oak
(Quercus minima Small) and deer moss lichens
(Cladina spp. and Cladonia spp.). The old fence
line is dominated by bunches of wiregrass and
chalky bluestem (Andropogon virginicus L.) with
small stands of young slash pine saplings and
gallberry (Ilex glabra (L.)). The pipeline area is
largely open sand with low grasses and herbs
growing in patches. The path around the outer
edge of the wooded range is primarily covered
with bahiagrass (Paspalum notatum Fluegge)
that is mowed about every two to three weeks.
We surveyed ants at gopher tortoise burrows
on 26 February, 14 March, and 16 April 2002
(sampling ~1/3 of the burrows on each day) using
a folded index card with ~1 g of canned water-
packed tuna inside placed within 0.2 m of the bur-
row entrance between 1300 and 1500 h. We sur-
veyed ants using tuna bait to assess ant species
present at the burrows that recruit heavily to rich
animal protein resources. We returned 2 h (10
min) later and collected the cards, putting each in
a separate zip-lock bag. We found that two h was
enough time to allow significant recruitment, but
not complete bait removal. After killing the col-
lected ants in a freezer, we counted them, then
preserved them in alcohol for identification. Ste-
fan Cover (Museum of Comparative Zoology) and
Mark Deyrup (Archbold Biological Station) iden-
tified the ants.
As of 16 April 2002, there were 85 marked go-
pher tortoises and 164 marked burrows within
our study site. This density of 9.3 tortoises/ha is
extremely high, e.g., it is more than three times
higher than the highest mean density (2.7/ha) at
Kennedy Space Center in east-central Florida
(Breininger et al. 1994). This high density is due,
Florida Entomologist 88(4)
in part, to people releasing gopher tortoises found
elsewhere into the greenway (Diemer 1986).
From a map of burrow locations in the study
area, we calculated the distance to the nearest
edge of the wooded range for each burrow. We
classified burrows less than 30 m from the perim-
eter of the wooded range as "edge" burrows; all
others we classified as "interior" burrows.
RESULTS
We surveyed ants at 154 of the 164 marked go-
pher tortoise burrows; we excluded ten burrows
because other animals removed the bait cards. All
surveyed burrows had at least one ant present
(range = 1-637 ants; median = 62 ants). We found
19 ant species (1-3 species per bait; Table 1); the
most common was S. invicta, which occurred at 51
burrows (33%; Table 1). The only other ant found
that may pose a threat to tortoises was the little
fire ant, Wasmannia auropunctata, which oc-
curred at four burrows (3%). Solenopsis invicta
tended to recruit more workers to baits they occu-
pied (median = 160 ants) than did other ant spe-
cies (median = 40 ants).
We found S. invicta significantly more often at
edge burrows (36/63 = 57%) than at interior bur-
rows (15/91 = 16%; x2 = 27.8; P < 0.001). In con-
trast, we found one or more species of native ant
significantly less often at edge burrows (23/63 =
37%) than at interior burrows (62/91 = 68%; X2 =
15.0; P < 0.001). We found one or more species of
exotic ant other than S. invicta equally often at
edge burrows (8/63 = 13%) as at interior burrows
(18/91 = 20%; x2 = 1.3; ns). We found only one spe-
cies significantly less often at edge burrows than
at interior burrows, the native Crematogaster at-
kinsoni Wheeler (X2 = 6.4; P < 0.05); several ant
species showed a strong trend in this direction,
but we lacked sufficient sample size to demon-
strate statistical significance.
Among the 91 interior burrows, we found S. in-
victa significantly more often at burrows directly
on the narrow disturbed strips along the old fence
line and pipeline (6/13 = 46%) than at interior
burrows not on these strips (9/78 = 12%; X2 = 9.7;
P < 0.005). Sample size for edge burrow on and off
these disturbed strips was too small for statistical
comparison.
DISCUSSION
We found S. invicta present at most gopher tor-
toise burrows around the outer perimeter of the
greenway reserve, but at few burrows in the inte-
rior portions of the greenway. Conversely, native
ants were significantly less common on the outer
perimeter than in the interior of the greenway. We
also found that S. invicta was present more often
at burrows on two disturbed strips through the
interior of the greenway than at burrows in other
parts of the greenway interior. These corridors of
TABLE 1. ANTS ON TUNA BAITS AT TORTOISE BURROWS ON THE GREENWAY EDGE (n = 63) AND THE GREENWAY INTERIOR
(n = 91). t = POTENTIAL THREAT TO GOPHER TORTOISES.
Occurrence at burrows
Exotic ant species # edge # interior
$Solenopsis invicta Buren 36 15
Pheidole moerens Wheeler 3 10
Technomyrmex albipes (Smith) 1 4
tWasmannia auropunctata (Roger) 1 3
Brachymyrmex obscurior Forel 1 1
Cardiocondyla emeryi Forel 1 1
Tapinoma melanocephalum (Fabr.) 1 1
Paratrechina longicornis (Latr.) 0 1
Native ant species
Pheidole floridana Emery 13 32
Crematogaster atkinsoni Wheeler 4 21
Crematogaster ashmeadi Mayr 0 6
Forelius pruinosus (Roger) 3 3
Pheidole dentata Mayr 3 3
Dorymyrmex bureni (Trager) 3 2
Pheidole morrisii Forel 2 2
Aphaenogaster miamiana Wheeler 1 1
Temnothorax pergandei (Emery) 1 1
Aphaenogaster flemingi Smith 1 0
Dorymyrmex bossutus (Trager) 0 1
December 2005
Wetterer & Moore: Fire Ants at Gopher Tortoise Burrows
disturbance thus allowed S. invicta more access
to the interior of the greenway. Footpaths through
greenway areas may have a similar influence. The
greenway interior appears to offer tortoises and
other species some refuge from S. invicta.
Our findings have conservation and develop-
ment policy implications. For example, the long,
thin design of many segments of the greenway
may increase the exposure of gopher tortoises and
other native species to S. invicta predation by in-
creasing the spatial extent of edges (Saunders et
al. 1991). Our study site was one of the wider seg-
ments in the Abacoa greenway. Narrower por-
tions of the greenway may not have as much in-
tact interior fauna. The division of the Abacoa
greenway into sections separated by roads fur-
ther increases the amount of edge. Because S. in-
victa prefers disturbed habitats (Tschinkel 1988;
Stiles & Jones 1998), prescribed burning and re-
duction mowing, which land managers use to
maintain open habitat for the gopher tortoises,
could lead to increased S. invicta infestation and
allow these ants to penetrate interior sites.
Stings ofS. invicta may have serious detrimen-
tal effects on both adult and hatchling gopher tor-
toises. Gopher tortoise hatchlings sometimes re-
main in their nest one or more days after hatch-
ing (Butler & Hull 1996), during which time they
may be particularly at risk to S. invicta attack.
Hatching sea turtles, which typically take from
several hours to several days after pipping before
they emerge from their nests, are similarly vul-
nerable to ant attack. During this time, ants in-
vade the nests and attack trapped hatchlings,
particularly their sensitive eyes (Krahe et al.
2003). Because even a single fire ant sting may se-
riously impair a hatchling, ants may be having a
tremendous impact on sea turtle hatchlings that
are stung while emerging, through increases in
subsequent mortality, e.g., increased vulnerabil-
ity to other predators (Krahe et al. 2003).
Solenopsis invicta also poses a threat to other
animals that use gopher tortoise burrows for shel-
ter, including some species that are known only
from gopher tortoise burrows, e.g., several species
of beetle (including Copris gopheri Hubbard, On-
thophagus polyphemi Hubbard, and Aphodius
troglodytes Hubbard), a robberfly (Machimus
polyphemi Bullington & Beck), the gopher frog
(Rana capitol LeConte), and the Florida mouse
(Podomys floridanus (Chapman)) (Lago 1991;
Lips 1991; Witz et al. 1991; Diemer 1992; Hum-
phrey 1992; Deyrup & Franz 1995). Many of these
animals are likely to be poorly defended against
attacks by this exotic ant.
ACKNOWLEDGMENTS
We thank M. Deyrup and S. Cover for ant identifica-
tion; M. Wetterer, A. Wetterer, and H. Smith for com-
ments on this manuscript; and Florida Atlantic
University for financial support.
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Florida Entomologist 88(4)
Pardo-Locarno et al.: White Grub Complex in Agroecological Systems
STRUCTURE AND COMPOSITION OF THE WHITE GRUB COMPLEX
(COLEOPTERA: SCARABAEIDAE*) IN AGROECOLOGICAL SYSTEMS
OF NORTHERN CAUCA, COLOMBIA
LUIS CARLOS PARDO-LOCARNO1, JAMES MONTOYA-LERMA2, ANTHONY C. BELLOTTI3 AND AART VAN SCHOONHOVEN3
'Vegetales Organicos C.T.A.
2Departmento de Biologia, Universidad del Valle, Apartado A6reo 25360, Cali, Colombia
3Parque Cientifico Agronatura, CIAT, Centro Internacional de Agricultura Tropical
Apartado A6reo, 6713 Cali, Colombia
ABSTRACT
The larvae of some species of Scarabaeidae, known locally as "chisas" (whitegrubs), are impor-
tant pests in agricultural areas of the Cauca, Colombia. They form a complex consisting of
many species belonging to several genera that affect the roots of commercial crops. The objec-
tive of the present study was to identify the members of the complex present in two localities
(Caldono and Buenos Aires) and collect basic information on their biology, economic impor-
tance, and larval morphology. The first of two types of sampling involved sampling adults in
light traps installed weekly throughout one year. The second method involved larval collec-
tions in plots of cassava, pasture, coffee, and woodland. Each locality was visited once per
month and 10 samples per plot were collected on each occasion, with each sample from a quad-
rants 1 m2 by 15 cm deep, during 1999-2000. Light traps collected 12,512 adults belonging to
45 species and 21 genera of Scarabaeidae within the subfamilies Dynastinae, Melolonthinae,
and Rutelinae. Members of the subfamily Dynastinae predominated with 48% of the species
(mostly Cyclocephala), followed in decreasing order by Melolonthinae (35%) and Rutelinae
(15%, principally Anomala). Melolonthinae comprised 60% of the specimens (Plectris spp.
59.5% and Phyllophaga spp. 35.9%). A total of 10,261 larvae of 32 species was collected, in-
cluding 12 species each of Melolonthinae (Phyllophaga, Plectris, Astaena, Macrodactylus,
Ceraspis, Barybas, and Isonychus), Rutelinae (Anomala, Callistethus, Stigoderma, and Leu-
cothyreus) and Dynastinae (principally Cyclocephala). At least a third of the species sampled
as larvae are rhizophagous pests. Taken together, adult and larval sampling methods permit-
ted a more precise definition of the whitegrub complex in Caldono and Buenos Aires.
Key Words: beetles, scarabs, rhizophagous pests, crop pests
RESUME
Las llamadas "chisas" son larvas de algunas species de Scarabaeidae, consideradas plagas
riz6fagas, que actuan en complejos integrados por varias species y g6neros de dificl separa-
ci6n taxon6mica. La present investigaci6n tuvo como objetivos dilucidar el complejo chisa
en Caldono y Buenos Aires, dos localidades del Departamento del Cauca. Ademas se obtuvo
informaci6n basica sobre su biologia e importancia econ6mica. Los adults fueron colectados
semanalmente con trampas de luz. Las larvas se obtuvieron a partir de parcels de yuca,
pastizal, caf6 y bosque. Cada parcela se visit una vez por mes, entire 1999 y 2000. Se reali-
zaron 10 muestras por parcela en cada ocasi6n, cada muestra consisti6 en cuadrantes de
1 m2 por 15 cm de profundidad. Las trampas de luz reunieron 12.512 adults correspondien-
tes a 45 species y 21 g6neros de Scarabaeidae, la mayor diversidad correspondi6 a la subfa-
milia Dynastinae (48%, mayoria Cyclocephala) seguida por Melolonthinae (35%) y Rutelinae
(15%, principalmente Anomala). La mayor abundancia se observe en Melolonthinae (60%),
con Plectris spp., representando el 59.48% y Phyllophaga spp. el 35.89%). Se colect6 un total
de 10.261 larvas distribuidas asi: 12 species de Melolonthinae (Phyllophaga, Plectris, As-
taena, Macrodactylus, Ceraspis, Barybas e Isonychus), 12 de Rutelinae (Anomala, Cal-
listethus, Stigoderma y Leucothyreus) y 5 Dynastinae (principalmente Cyclocephala). Se
concluye que al menos la tercera parte de las species muestreadas a nivel de larvas presen-
tan habitos riz6fagos, ademas la conjugaci6n de muestreo de larvas y adults facility el re-
conocimiento del complejo chisa local lo cual se recomienda implementar en otras regions
agricolas con similares problems fitosanitarios.
Translation provided by the authors.
*Only the subfamilies Dynastinae, Rutelinae and Melolonthinae.
Florida Entomologist 88(4)
Immature stages of some species of rhizopha-
gous beetles of the family Scarabaeidae (Coleop-
tera: Scarabaeoidea) are among the most important
economic pests in Colombia (Pardo-Locarno 1994;
Londoio 1999). These insects feed on roots of vari-
ous crops in different agro-ecological zones (Pardo-
Locarno 1994; Pardo-Locarno et al. 2003a). They
comprise a species complex. Larvae are very similar
and only distinguishable based on size and, less
precisely, body form and color of the head. Several
genera and species cannot be distinguished, even
based on these criteria. Taxonomic characters are
present on the mouthparts and raster, but these
must be associated with reared specimens.
Preliminary analyses are available on some of the
white grubs of major economic importance in Colom-
bia, with details of species compositions, localities,
and crops affected (Pardo-Locarno 1994, 2000a; Re-
strepo & L6pez Avila 2000). Composition of the com-
plexes and their respective economic impact vary ac-
cording to regions and species involved (Pardo-
Locarno 2000a; Pardo-Locarno et al. 2003a). The af-
fected agro-ecosystems represent most of the physio-
graphic areas of the country, including very humid
tropical forest (e.g., Puerto Leguizamo) (Department
of Putumayo) and possibly the Choc6 region (Pardo-
Locarno 2000a; Pardo-Locarno et al. 2003a).
The complex of rhizophagous beetles consti-
tutes an important problem in cassava, sisal,
pineapple, vegetables, flowers, and pasture in the
municipalities of Santander de Quilichao, Buenos
Aires, Toribio, and Caldono, all in the Department
of Cauca (Pardo-Locarno et al. 2003c, d; Victoria
& Pardo-Locarno 2000a).
Studies carried out in one municipality of the
region (Santander de Quilichao) have identified
about 30 species of white grubs with different de-
grees of economic importance to cassava cultiva-
tion and varying patterns of adult seasonal abun-
dance (Pardo-Locarno et al. 1993). The same au-
thors identified the larvae responsible for eco-
nomic damage with notes on their natural enemies
(Pardo-Locarno et al. 1999a, b). Further, feeding
preferences were established with living larvae,
randomly collected in cassava, pasture, and other
crops and individually reared under laboratory
conditions (Victoria & Pardo 2000a, b). Neverthe-
less, there are still many gaps in the association of
adults and immatures of Scarabaeidae present in
the area. In this paper we define and compare the
regional complex of white grubs present in four
agroecosystems of the municipalities of Caldono
and Buenos Aires, Cauca, Colombia.
MATERIAL AND METHODS
Characteristics of the Study Area
Sampling was carried out in Pescador and Cas-
cajeros, localities within the municipalities of
Caldono and Buenos Aires respectively in the De-
apartment of Cauca (Fig. 1). Pescador was selected
because of previous research (Pardo-Locarno et al.
1999a, b; Pardo-Locarno 2000a; Victoria & Pardo
2000a, b). Cascajeros was chosen as it represents
a different ecology. This permitted data on the
spatial heterogeneity in the region (Wiens 1989;
Levin 1992; Hulbert 1984).
Annual rainfall in northern Cauca fluctuates be-
tween 2000-2300 mm and shows a bimodal pattern
(April-May and October-November) (IGAC, 1988).
The area is predominantly agricultural. Caldono is
the leading producer of sisal in the department al-
though as in Buenos Aires, coffee and cassava pro-
duction has increased in recent years. In Buenos
Aires the production of fruits (oranges, lemons,
guavas, etc.) predominates, followed in importance
by cash crops such as beans and tomatoes.
Field Phase
Four plots were chosen to study composition, di-
versity, and abundance of the white grub complex
in cassava, pasture, coffee, and woodland. Sam-
pling was carried out in 1999-2000 by two meth-
ods: collection of larvae in quadrants of 1 m2 by 15
cm deep and capture of adults in light traps
(Pardo-Locarno 2000a; Pardo-Locarno et al. 2003c,
d). Information was also collected on the crops,
feeding habits of the beetles and bioecological de-
tails. Live larvae were separated according to
characters of the head, mouthparts (especially the
epipharynx), and abdomen (raster, anal opening).
Adult sampling was carried out weekly with
light traps (Pardo-Locarno, et al. 1993). On each
occasion the specimens were stored in plastic 1-
gallon containers, transported to the laboratory,
identified, and counted by species.
Laboratory Phase
Approximately 2,500 larvae were collected in
different crops and allowed to complete their life
cycle under glasshouse conditions and identified as
adults. All the specimens were reared individually
in plastic cups containing sterilized soil, rich in or-
ganic matter and supplemented with roots or
pieces of carrot. Field caught larvae were kept as
individuals in disposable cups containing vapor
sterilized soil brought from the field. Larvae were
fed with fresh chopped carrot. Cups were inspected
once per week to control moisture and change food
ration. Each larva was randomly labelled accord-
ing to phenototypic characters observed on head,
feet, raster, and anal orifice. As soon as the adults
were obtained, the identification was verified. For
each morphoo" several voucher specimens were
prepared (i.e., larvae were "cooked" in boiling wa-
ter (until they float), fixed with formalin (10%) and
preserved. In very few cases (i.e., larvae with rare
or odd characters) larvae were kept alive, and their
phenotypic characteristics were drawn. For this,
December 2005
Pardo-Locarno et al.: White Grub Complex in Agroecological Systems
I C..csee
EFpMW
Ja 'a-, T*. ae
-' .IIw
C ia iD"I L E R -
Fig. 1. Map of the study area in northern Cauca, Colombia
the larva was cooled for a few minutes with ice,
and cooling was repeated as many times as neces-
sary (Pardo-Locarno 2002).
Several papers and taxonomic keys were con-
sulted for larval identification, including Ritcher
(1966); Boving (1942); Mor6n (1993); Vallejo et al.
(1996); Mor6n & Pardo-Locarno (1994); and King
(1984). Identification of adults was based on the
genitalia with the aid of diagnostic keys from En-
dridi (1985); Saylor (1942, 1945); Frey (1962,
1964, 1973, 1975); Mor6n (1986); King (1996);
Ohaus (1934); Woodruff and Beck (1989); and
Machatschke (1957, 1965). The collection of one of
the authors (LCP), which included representa-
tives of many white grub species from Cauca, was
examined (Pardo-Locarno 1999a, b). We also con-
sulted the taxonomic specialists R.P. Dechambre
(Musee de Histoire Naturelle de Paris) and M. A.
Mor6n (Instituto de Ecologia de Mexico).
RESULTS
Capture of Adults
Altogether 12,512 adults were collected in the
light traps, with 7,562 from Caldono and 4,953
from Buenos Aires. This included 21 genera and 45
species of Scarabaeidae belonging to the subfami-
lies Dynastinae, Melolonthinae, and Rutelinae
(Table 1).
The greatest species diversity was presented
by the subfamily Dynastinae with 48% of the spe-
cies, followed in decreasing order by Melolonthi-
nae (35%) and Rutelinae (15%). Among the Dy-
nastinae the diversity of the tribe Cyclocephalini
was noteworthy with 15 species identified, most
belonging to the genus Cyclocephala.
Members of the subfamily Melolonthinae were
most abundant and comprised 60% of the total
specimens collected. Of these, Plectris, with two
species, comprised 59.5%, followed by Phyllo-
phaga, with five species. Among the Dynastinae,
the Cyclocephalini predominated, particularly
C. fulgurata Burmeister, which was especially
numerous in Caldono. Among the Oryctini, the
predominant species was Strategus aloeus L.,
whose larvae are saprophagous and adults are
not recorded as pests of crops. This species was
followed in predominance by Podichnus agenor
Oliv. whose larvae are saprophagus and adults
damage sugar cane stems in the region.
Florida Entomologist 88(4)
TABLE 1. CAPTURE OF ADULTS IN LIGHT TRAPS IN CALDONO AND BUENOS AIRES.
Species Caldono Buenos Aires Specimens
Aspidolea singularis Bates
Aspidolea fuliginea Burm
Cyclocephala sp. 1
Cyclocephala amblyopsis Bates
Cyclocephala amazona Linn.
Cyclocephala melanocephala Fabr
Cyclocephala fulgurata Burm
Cyclocephala sp. 2
Cyclocephala tutilina Burm
Cyclocephala stictica Burm
Cyclocephala lunulata Burm
Cyclocephala weidneri Endr6di
Stenocrates bicarinatus Robinson
Ancognatha uulgaris Arrow
Dyscinetus dubius Olivier
Coelosis biloba Linn.
Strategus aloeus Linn.
Podischnus agenor Olivier
Golofa porteri Hope
Ligyrus gyas Er.
Ligyrus bituberculatus Beauvois
Phileurus didymus Linn
Plectris fassli Moser.
Plectris pavida Burm
Phyllophaga menetriesi Blanch.
Phyllophaga obsoleta Blanch
Phyllophaga sericata (Blanchard)
Phyllophaga thoracica (Burmeister)
Phyllophaga schneblei Frey
Phyllophaga sp. 3
Astaena valida Burmeister
Astaena sp2.
Macrodactylus subvittatus Burm
Macrodactylus sp. 1
Macrodactylus sp. 2
Isonychus sp. 1
Isonychus sp. 2
Barybas sp.
Pelidnota prasina Burmeister
Anomala cincta Say
Anomala inconstans Burm
Anomala undulata Melsh.
Anomala sp. 2
Callisthetus sp.
Leucothyreus sp.
Abundance/locality
113
18
30
162
2
4
1607
2
10
1
420
8
1
32
14
82
39
1
1
10
3
733
152
2413
213
25
2
29
1
43
9
23
1
11
2
127
22
26
1029
121
19
7562
20
1
1
119
15
751
21
1
115
25
14
4
6
3
1
4
1
3019
607
9
29
1
5
107
22
1
4
9
9
6
22
4953
133
19
30
163
121
4
1622
753
31
2
535
25
22
1
36
20
85
40
1
1
14
4
3752
759
2422
242
25
2
30
1
48
9
23
1
11
107
2
149
23
30
1038
9
127
41
12512
Members of the Rutelinae were least abun-
dant, withAnomala inconstans Burm., an agricul-
turally important species, the most numerous.
The adults of Phyllophaga spp., Plectris pavida
Burm., P fassli Moser, and other Melolonthinae of
economic importance were attracted to light traps
mainly in October and November, although some
adults were also collected during the rainy season
(March-April) and to a lesser degree in May.
Capture of Larvae
A total of 10,265 larvae, representing 32 spe-
cies of Scarabaeidae, was collected in samples
from the four plots (pasture, cassava, coffee, and
woodland) in Caldono and Buenos Aires (Table 2).
Of the total, 52% corresponded to genera of
Melolonthinae, 25% to Dynastinae and 23% to
Rutelinae (Table 2).
December 2005
Pardo-Locarno et al.: White Grub Complex in Agroecological Systems
TABLE 2. SPECIES OF LARVAE OF SCARABAEIDAE COLLECTED AND THEIR RESPECTIVE ABUNDANCES IN THE FOUR PLOTS
IN CALDONO AND BUENOS AIRES, CAUCA.
Buenos Aires Caldono
Genus/species Pasture Cassava Coffee Woodland Pasture Cassava Coffee Woodland Total
Phyllophaga sp.
Ph. Menetriesi
Phyllophaga sp. 2
Plectris pavida
P1. fdcil
Cyclocephala stictica Burm
C. amblyopsis
Cyclocephala sp.
C. lunulata
C. amazona
Rutelinae #26
Callistethus cupricollis
Anomala incostants
An. Cincta
An. undulata
An. caucana
Anomala sp. 2
Anomala sp.
Astaena sp.
Macrodactylus subvittatus
Ceraspis innotata
Barybas sp.
Dynastinae #9
Leucothyreus sp.
Scarabaeidae #27
Scarabaeidae #28
Scarabaeidae #19
Rutelinae #30
Rutelinae #21
Rutelinae #29
Isonychus sp.
Melolonthinae #4
Total
134 25
1 3
2
63 3
467 27
1
51 27 8
1
23 64
29 60
1
57 34
338 739
1
19 15
1 9
2
22 14
1 150
43
61 20
35
36 1 9
1
1 118 151
602 6 450
19 8
28 8 4
19 4 328
22 36 172
9 0 19
11 9 208
16 5 1610
3 4
1
14 1 187
26
2
20 9 269
1 169
3 1 79
10 304
56
1
10 1 146
18 348 616
33 12 592
45 163 1692
47 4 1756
92 278 501
2
8 3 67
24
1
652 652
17
23 444 686
16 19
1010 978 421 784 2388 2293 404 1987 10265
Plectris fassli was the most abundant species,
reaching densities of up to 12 larvae per quadrant
in pasture and up to six in cassava. Phyllophaga
menetriesi Bl. was collected in all the agricultural
plots and sporadically in woodland (3-4 larvae/
m2). Densities of 6-8 pupae/m2 were observed in
July-August in cassava, which suffered 33% root
yield loss according to growers. A marked season-
ality was recorded, with large numbers of first
and second instars in November-December and fi-
nal instars in February-July. These pupated in
August-September and their emergence occurred
in October-November with the rainfall at the end
of the year (data not shown).
The appearance of adults of Plectris spp. was
October and November, with abundance greater
than that of Phyllophaga (Table 1). More than
90% of the population was observed pupating in
the subsoil at 35-40 cm, with their larvae having
dug into the ground 2-3 months before emergence.
This made these species difficult to find in sample
quadrants, which only reached down to 15 cm.
Plectris fassli was eight times more abundant
than Pl. pavida and was found in all plots, pre-
dominating in pasture and cassava (Table 2),
where it reached densities of up to 40 and 34 lar-
vae per quadrant, respectively. The larvae of Pl.
pavida, active and slender, were abundant in pas-
tures where they reached densities of up to 21 lar-
vae per quadrant.
Ceraspis innotata Blancard was collected in
pastures, cassava, coffee, and rarely in forests. In
cassava it reached an abundance of up to 45 lar-
vae per quadrant, but in pastures it presented the
greatest abundance (up to 86 larvae/m2), with
most in the third instar. Their adults, little at-
tracted by light traps, were captured during the
day on the ground and on pasture foliage. They
Florida Entomologist 88(4)
were most abundant in May, almost one month af-
ter the initiation of the rains.
Larvae of the rhizophagous species Astaena
valida Burm. were collected relatively frequently
in all plots. This species demonstrated a predilec-
tion for coffee plantations and forest, where it was
collected frequently and at densities of up to 36
larvae per quadrant. It reached densities of up to
34 examples per quadrant in cassava and on pas-
ture. The genus included other species, one of
them identified tentatively as Astaena colombi-
ana Blanchard.
Macrodactylus subvittatus Burmeister, whose
adults are considered to be a pest of maize, pre-
sented abundant rhizophagous larvae, both in
pasture and cassava (with 22 and 38 larvae per
quadrant, respectively) but particularly in forest
(up to 42 larvae).
Among the larvae of Anomalini, the genera
Callistethus and Anomala predominated, the lat-
ter distributed in the upper 5 cm of the soil. One
species, provisionally identified as Anomala sp 2,
was most numerous (32 larvae per quadrant in
cassava).
DISCUSSION
The results show that the species compositions
of Scarabaeidae in Caldono and Buenos Aires are
similar, and compare to those reported for ter-
raced agroecosystems (Pardo-Locarno et al.
2003a, b). The species complex presented marked
differences to those in hot, lowland areas with
pronounced dry periods (Caribe Seco, Llanos Ori-
entales of Colombia, etc.), which are dominated
by species of Pentodontini, such as Euetheola
(ICA NNE 1972-1994; Pardo-Locarno 1994,
2000a; Pardo-Locarno et al. 2003a). It also differs
from agricultural zones on slopes at 1000 masl
with higher humidity regimens (San Jos6 del Pal-
mar-Choc6 and other mountainous regions),
where species of Cyclocephala and Anomalini pre-
dominate (Pardo-Locarno 2000b) and hot and
moderately humid cultivated land at lower eleva-
tions (Villavicencio and foothills of the Llanos
Orientales), where species of the Cyclocephalini
(Cyclocephala, Stenocrates, Dyscinetus and oth-
ers) are abundant (Pardo-Locarno 2000a; Pardo
Locarno et al. 2003a).
In general the species composition of adults re-
sembled that previously observed for the region
(Pardo-Locarno et al. 1993; Pardo-Locarno 2000a;
Pardo Locarno et al., 2003a). Nevertheless the
capture of Scarabaeidae larvae reaches values
that significantly exceed previous data obtained
in the area with 14 edaphic species recorded:
C. fulgurata Burm., C. amazonica L., C. stictica
Burm., C. lunulata Burm., Phyllophaga mene-
triesi Blanchard, Ph. obsolete Blanchard, Ph. sp.
(near elenans Saylor), Plectris sp. 2, Pl. fassli
Moser, Anomala inconstans Say, An. undulata
Melsh., An. cincta Say, Anomala sp. 2 and Leuco-
thyreus sp. (Pardo-Locarno et al. 1999a). Cassava
crops in Caldono sampled intensively during the
first semester of 1999, revealed the presence of 14
Scarabaeidae species of the genera Plectris (2 spe-
cies) Cyclocephala (4-5), Phyllophaga (5) and
Anomala (5-6). Based on study of these larvae,
specimens previously thought to be Ph. elenans
were identified as Pl. pavida.
Not surprisingly, given the geographical prox-
imity of the two areas, the white grub complex of
Caldono and Buenos Aires is similar in species
composition to that recorded for Santander de
Quilichao (Pardo-Locarno et al. 1993) and com-
plements that observed previously in Caldono
and Buenos Aires (Pardo-Locarno et al. 2003b).
Despite the greater distance, the white grub com-
plex at Caldono and Buenos Aires presents great
similarity to that recorded for the mountainous
regions of Ibagu6 and Cajamarca, Tolima (Pardo-
Locarno et al. 2003a; Vasquez & Sanchez 1996).
Cyclocephala amazonica L., C. lunulata Burm.,
Pl. pavida Burm., and Ph. menetriesi Bl. were
abundant in samples from this latter region.
With respect to the rhizophagous habit sensu
strict, Phyllophaga species are considered of
great economic importance (Vallejo et al. 1997).
This genus was represented in three of the agro-
ecosystems, i.e., pasture, cassava, and coffee. This
study provides additional evidence to corroborate
the results of previous studies (Pardo-Locarno et
al. 1999a; Pardo-Locarno 2000a, 2000b) regard-
ing the presence and relative importance of the
genera Plectris, Astaena, Leucothyreus, and Mac-
rodactylus. Many of these genera are little known
as root-eating pests. In some instances, they
reached higher population levels than Phylloph-
aga species and their inclusion in the white grub
complex of northern Cauca is suggested. It is pos-
sible that larva of the former were previously con-
fused with Phyllophaga, given their similarities
in morphology, feeding habits, reflex reactions at
the moment of capture and that all of them ap-
pear as larvae during the rains of October-
November.
The fact that Plectris spp. (especially P fassli)
predominated in pasture and cassava possibly
implicates them in the economic damage of the
root systems of these crops. An undetermined spe-
cies of this genus was incriminated as a root pest
in Rio Negro, Department of Antioquia (Londoio
1999). It is important to determine the actual eco-
nomic importance of Plectris species, for which
Colombian records are scant.
Colombian references to Astaena are almost
absent in the literature. But many species have
adults that are easily confused with other seri-
cines, and some members of the genus (particu-
larly A. valida) occurred in all plots, with abun-
dant populations in cassava, pasture and second-
ary forest.
December 2005
Pardo-Locarno et al.: White Grub Complex in Agroecological Systems
Macrodactylus subvittatus Burm., is a
rhizophagous species which is abundant in pas-
tures, cassava plantations, and forest ecosystems.
This species is considered to be a maize pest al-
though Bueno et al. (1998) in their study of its life
cycle, state that its larvae "feed on the roots of
grasses and stubble and apparently do not cause
economic damage".
Other species of Macrodactylus collected in
northern Cauca include M. flavolineatus and M.
pulcripes, both apparently forest-living and at-
tracted by Saluia palaefolia H.B.K (sensu Perez
Arbelaez 1990) undergrowth or flowering plants
in general.
The findings of the present study support the
views of Pardo-Locarno (2000a) regarding the rel-
ative importance of larvae of the genus Ceraspis
in agricultural systems of the foothills and high
mountain regions. Other Colombian agroecosys-
tem still need to be sampled (ICA-NNE 1972-
1994; Posada 1989; Pardo-Locarno 1994; Re-
strepo & Lopez Avila 2000). The larvae ofC. inno-
tata are unknown to the scientific community and
have not been reported as pests of agricultural
importance (Pardo-Locarno et al. 1999a; Pardo
Locarno 2000a; Victoria & Pardo-Locarno 2000b).
However, during the present study they were col-
lected on all the crops, especially in pasture.
Based on the results of this study, the white
grub complex of Caldono and Buenos Aires, which
currently comprises 32 confirmed species, could
be increased to more than 40 species. Additional
species have been collected in light traps but some
lack corroboration in the larval form. In the latter
category are species of Stenocrates (ICA 1972-
1994; Pardo-Locarno 2000a) such as S. clipeatus
Endrodi and S. bicarinatus Robinson; two mem-
bers of Aspidolea, A. fuliginea Burm., and A. sin-
gularis Bates, Dyscinetus dubius Oliv, a species
that was moderately abundant in Buenos Aires in
light traps (Pardo-Locarno 2000a) and Ligyrus
bituberculatus and L. gyas, species whose larvae
remain unidentified. The latter are economically
important in Central America and the southern
US (King 1984; Shannon & Carballo 1986; Gor-
don & Anderson 1981) but rarely collected in light
traps. Since adults of the genus Isonychus and the
subtribe Anomalini were collected in light traps or
on the foliage of the evaluated plots, it is plausible
to expect their presence as larvae, as well.
Three large taxonomic groups of the white
grubs predominated. The first of these is the sub-
family Melolonthinae with species of greatest eco-
nomic impact, whose larvae are unable to com-
plete their life cycles without the presence of liv-
ing roots or plant tissue. Members of the tribe
Macrodactylini Plectris, Macrodactylus, Ceraspis,
Isonychus, and the genera Phyllophaga (espe-
cially Ph. menetriesi Blanchard), have the largest
rhizophagous larva. Next in importance are the
Rutelinae of the tribe Anomalini, which includes
Anomala and Callistethus, two genera with nota-
ble species diversity. Finally, the Dynastinae of
the tribe Cyclocephalini (especially Cyclocephala)
are important, but their economic importance is
unknown. Larvae of species such as C. amazon-
ica, C. lunulata, C. stictica, and C. fulgurata, As-
pidolea fuliginea, and A singularis were found to
be facultative saprophagous and able to develop
in the absence of roots or live plant tissue. The
possible saprophagous habit was also observed by
Deloya (1998) in larvae ofC. lunulata Burmeister
in maize fields in Morelos, Mexico.
The white grub complex observed in the crops
hypothetically is part of a group of edaphicolous
species pre-adapted to both an ecological and cul-
tural oversimplification due to selected crops and
agronomic practices that dominate in the region.
This has permitted selection of a group of predom-
inantly rhizophagous and saprophagous species,
whose populations are favored by the possible ex-
clusion of their natural enemies and by soil deg-
radation.
Sampling of both adults and larvae revealed a
great overlap of subpopulations associated with
the two rainy seasons. Larvae occurred in a dis-
parity of developmental stages from first to third
instars. Given the frequent capture of adults
throughout the year, this may suggest they have a
short reproductive period. This has been observed
in other species such as C. amazonica and Ph. me-
netriesi. A similar but less marked situation was
observed in An. cincta viridicollis. Current agri-
cultural practices and prevailing environmental
conditions may have intensified the seasonality of
rhizophagous and saprophagous species of Scara-
baeidae. They show relatively brief reproductive
periods, associated with rainy seasons when
adults are abundant and oviposition cycles begin.
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Florida Entomologist 88(4)
December 2005
BEHAVIORAL AND ELECTROPHYSIOLOGICAL RESPONSES OF THE
MEXICAN FRUIT FLY (DIPTERA: TEPHRITIDAE) TO GUAVA VOLATILES
EDI A. MALO, LEOPOLDO CRUZ-LOPEZ, JORGE TOLEDO, ALEJANDRO DEL MAZO,
ARMANDO VIRGEN AND JULIO C. ROJAS
Departamento de Entomologia Tropical, El Colegio de la Frontera Sur (ECOSUR)
Apdo. Postal 36, 30700, Tapachula, Chiapas, M6xico
ABSTRACT
The behavioral and electrophysiological responses of males and females of the Mexican fruit
fly Anastrepha ludens (Loew) to guava (Psidium guajava L.) volatiles were investigated in
laboratory tests. Males and females were significantly more attracted and landed more often
on guava fruits than yellow spheres used as control in the wind tunnel. Also, both sexes were
more attracted to Porapak Q extracts of guava than to solvent controls. Gas chromatography-
electroantennographic detection (GC-EAD) analysis of the behaviorally active extracts
showed that consistently eight and seven compounds elicited antennal response from male
and female, respectively. The compounds were identified by gas chromatography-mass spec-
trometry (GC-MS) as ethyl butyrate, (E)-3-hexenol, (Z)-3-hexenol, hexanol, ethyl hexanoate,
hexyl acetate, (Z)-3-hexenyl butyrate and ethyl octanoate. The electrophysiological activity of
the identified compounds at three different doses was evaluated with electroantennography
(EAG). An analysis of covariance of the EAG amplitude revealed that synthetic chemicals,
sex, dose, and the synthetic chemical x dose interaction significantly influence the antennal
response ofA. ludens. Males and females were significantly more attracted to septa loaded
with the eight-component synthetic blend compared to solvent controls in the wind tunnel.
Key Words:Anastrepha ludens, guava volatiles, attractants, GC-EAD, GC-MS
RESUME
Las respuestas conductuales y electrofisiol6gicas de machos y hembras de la mosca Mexi-
cana de la fruta Anastrepha ludens (Loew) a volatiles de guayaba (Psidium guajava L.)
fueron investigadas en experiments de laboratorio. Machos y hembras fueron significativa-
mente mas atraidos y aterrizaron mas frecuentemente en fruta de guayaba que en la esfera
amarilla usada como control en el tunel de vuelo. Asi tambi6n, ambos sexos fueron mas
atraidos al extract de frutos de guayaba que al disolvente usado como control. El andlisis
por cromatografia de gases-electroantenografia de los extractos comportalmente activos
mostr6 que consistentemente ocho y siete compuestos fueron antenalmente activos a machos
y hembras, respectivamente. Los compuestos fueron identificados por cromatografia de ga-
ses-espectrometria de masas como butirato de etilo, (E)-3-hexenol, (Z)-3-hexenol, hexanol,
hexanoato de etilo, acetato de hexilo, butirato de (Z)-3-hexenilo y octanoato de etilo. Los re-
sultados del andlisis de la amplitud del pico EAG a estimulos de compuestos sint6ticos iden-
tificados a tres diferentes dosis estudiadas, mostraron que la respuesta antenal deA. ludens
es significativamente afectada por los compuestos quimicos, sexo, dosis y la interacci6n com-
puestos quimicos x dosis. Los machos y las hembras fueron significativamente mas atraidos
a septos de hule cargadas con la mezcla de los ocho compuestos, comparadas contra el disol-
vente en el tunel de viento.
Translation provided by the authors.
The Mexican fruit fly, Anastrepha ludens
(Loew), is one of the most important species at-
tacking more than 50 tropical fruits from eight
families, including grapefruit, orange, mango,
and common guava (Eskafi & Cunningham 1987;
Norrbom & King 1988; Norrbom et al. 2000). In
Mexico losses of citrus, mango, and guava due to
A. ludens are estimated at 25% (Enkerlin et al.
1989). This species was originally native to Mex-
ico although currently it is found in Central and
South America, and southern Texas, USA
(Hernandez-Ortiz & Aluja 1993).
McPhail traps baited with fermenting sugars,
yeast and hydrolyzed protein have been used for
many years to monitor A. ludens and other spe-
cies of fruit flies. Nevertheless, low capture effi-
ciency, catch of non-target insects, difficulties in
managing the liquid-baited McPhail traps in the
field, and high cost have led to searches for more
effective attractants and traps (Epsky et al. 1993;
Heath et al. 1996). For instance, promising at-
tractants have been found in bacteria, avian fe-
ces, and human urine (Robacker et al. 1998, 2000;
Pifiero et al. 2003). Several studies have also doc-
Malo et al.: Response ofA. ludens to Fruit Volatiles
umented that both sexes of A. ludens are at-
tracted to fruit volatiles (Robacker & Fraser 2002,
2003), although generally chemical identity of the
compounds responsible for attraction remains un-
known (but see Robacker et al. 1990, 1992). The
isolation and identification of behaviorally active
compounds may be a laborious task because a rip-
ening fruit is a complex mixture of frequently
over a hundreds) detectable volatile compounds,
possessing various functional groups and ranging
from simple to complex structures (e.g., Buttery
1981; Maarse 1991). Nevertheless, analytical
tools such as gas chromatographic-electroanten-
nographic detection (GC-EAD) (Arn et al. 1975)
may facilitate rapid identification of active com-
pounds present in complex blends of fruit vola-
tiles, eliminate from consideration compounds
without biological activity, and suggest candi-
dates for behavioral and field studies (Cosse et al.
1995; Zhang et al. 1999; Nojima et al. 2003).
In this study, firstly, we evaluated the attrac-
tiveness of guava fruits and their volatiles to male
and female A. ludens in a wind tunnel; secondly,
we located electrophysiologically-active com-
pounds from guava extracts by using GC-EAD;
thirdly we identified the EAD-active compounds
by GC-mass spectrometry (GC-MS); and finally
we evaluated the electrophysiological and behav-
ioral activity of the identified compounds with an
electroantennogram (EAG) bioassay in a wind
tunnel, respectively.
MATERIALS AND METHODS
Insects
The pupae were obtained from the Moscafrut
(SAGARPA-IICA) mass rearing facility located in
Metapa de Dominguez, Chiapas, Mexico. They
were reared on an artificial diet previously de-
scribed by Dominguez et al. (2000). The adults
were maintained at 25C + 1C, 70 + 5% RH, and
a photoperiod of 12:12 (L:D) h. Adults were fed ad
libitum with a mixture of enzymatic yeast hy-
drolystate (ICN Biomedicals, Costa Mesa, CA)
and sucrose (1:3) (unless otherwise specified). Wa-
ter was provided in test tubes covered with cotton
plugs. Adult flies of 8-20 d old were used for behav-
ioral bioassays and GC-EAD and EAG analysis.
Chemicals
Synthetic chemicals were purchased from
Sigma/Aldrich (Toluca, Mexico) and Bedoukian
Research (Danbury, CT), and the purities were
>95% based on the results with capillary gas
chromatography.
Fruits
Ripe fruits were collected from a guava or-
chard (Psidium guajava L., native type) near
Tapachula, Chiapas. The fruits were collected
when they were of a yellow color and placed in
plastic bags and immediately carried to the labo-
ratory for bioassays and volatile collection. Be-
cause the color of guava fruits is not a good indi-
cator of ripeness, we included the content of sugar
determined as Brix degree. This degree is used to
measure liquid density, especially sugar concen-
tration in fruit and vegetable juices (Hogness &
Jones 1984). We measured the Brix degree from a
random sample of 10 yellow guava fruits with a
refractometer (Iroscope, Mexico City), and the
values ranged from 9.8 to 13.5 Brix degree.
Collection of Volatiles
Ripe healthy guava fruits (1.6 kg) were placed
in a cylindrical glass aeration chamber (58 cm
long x 18.5 cm i.d.). A charcoal-filtered airstream
(1 1/min) was maintained through the glass aera-
tion chamber for 16 h. Fruit volatiles were col-
lected on 350 mg of Porapak Q (50-80 mesh, Water
Associates, Inc., Milford, MA) packed between
plugs of silanized glass wool in a Pasteur pipette.
Porapak Q was cleaned previously by heating it in
a nitrogen stream at 150C for 5 h and then
washed with diethyl ether. Fruit volatiles were
eluted from the Porapak Q with 2 ml of diethyl
ether (HPCL grade) and concentrated to 600 pl by
slow evaporation under a gentle stream of nitro-
gen. The extract was stored at a -20C until bio-
assays and analysis.
Wind Tunnel Bioassays
The observations were carried out in a flight
wind tunnel, 120 cm long and 30 cm high and
wide. A fan was used to pull air through the tun-
nel with a velocity of 0.4 m/s. Activated charcoal
was used to filter intake air. Illumination was pro-
vided by four fluorescent bulbs (39 W daylight
GE, Mexico City) mounted 60 cm above the wind
tunnel giving a light intensity of 2,380 lux. The
insects were evaluated in groups of 25 individuals
(males or females), which were placed in a plastic
container (6 cm high x 8 cm diameter, release cyl-
inder) with screen top 18 h before testing. Water,
but not food, was provided with the cylinders.
They were allowed to acclimatize in the wind tun-
nel room conditions (25 1C, 60 5% relative hu-
midity) for at least one h before being assayed. In
three different trials, the responses of flies to
guava fruits, Porapak Q volatile extracts, and the
synthetic blend were evaluated in non-choice
tests. Polystyrene spheres (5 cm diameter)
painted with vinyl acrylic water-based paints
mixed to match (as detected by the human eye)
the yellow of guava fruit were used for dispensing
either the guava extracts (2nd trial) or the syn-
thetic blend (3rd trial), and as control (in all trials).
One g equivalent of guava fruit extract or 1 mg of
Florida Entomologist 88(4)
the synthetic blend prepared according to the rel-
ative proportions of each compound in the natural
extracts was loaded on a rubber septum
(Agrisense, England). The rubber septum was in-
serted into a yellow painted sphere. A rubber sep-
tum loaded with 10 pl of diethyl ether and placed
in a yellow painted sphere was used as control.
The target stimulus (e.g., fruit, unscented sphere,
or scented sphere) was hung in the center of the
wind tunnel, 16 cm from the upwind end. Each
observation was started by placing the release
cylinder on a 12 cm high platform at the down-
wind end of the tunnel and insects were released
and observed for 10 min. The insects were re-
corded for upwind flight and for landing on odor
source or control. Upwind movement was re-
corded if insects passed a point two-thirds of the
distance from the release cylinder to the odor
source or control (Robacker and Fraser 2002).
Landing behavior was scored if the flies touched
and stayed at least two minutes on the odor
source. The flies were only used once during the
bioassays. All bioassays were conducted between
8:00 and 13:00 h.
EAG Analysis
Antennal receptivity of adult males and fe-
males of A. ludens to synthetic compounds was
determined by EAG. The insect head was cut off
carefully, and a reference electrode was inserted
into its base with a glass capillary filled with
physiological saline solution (Malo et al. 2004).
The distal end of the antenna was inserted into
the tip of the recording glass capillary electrode.
One replicate was made with one fly antenna. The
signals generated by the antenna were passed
through a high-impedance amplifier (NL 1200;
Syntech, Hilversum, The Netherlands) and dis-
played on a monitor by Synthech software for pro-
cessing EAG signals. A stimulus flow controller
(CS-05; Syntech) was used to generate a stimulus
at 1-min intervals. A current of humidified pure
air (0.7 1/min) was constantly directed onto the
antenna through a 10-mm-diameter glass tube.
At least 12 individuals of each sex were used in
these tests.
Serial dilutions of the synthetic compounds
were prepared in HPLC-grade hexane to make 1,
10, and 100 pg/pl solutions. A standard aliquot (1
pl) of each test dilution was pipetted onto a piece
of filter paper (0.5 x 3.0 cm, Whatman, No. 1) ex-
posed to air 20 s to allow the solvent to evaporate,
then inserted into a glass Pasteur pipette or sam-
ple cartridge, and left for 40 s before applying. A
new cartridge was prepared for each insect. To
present a stimulus, the pipette tip containing the
test compound was inserted through a side hole
located at the midpoint of a glass tube through
which humidified pure air flowed at 0.5 1/min. The
duration of stimulus was 1 s. The continuous flow
of clean air through the airflow tube and over the
preparation ensured that odors were removed im-
mediately from the vicinity. The synthetic com-
pounds were presented in random order and the
test doses for each compound were presented se-
quentially from the lowest to highest concentra-
tion. Control stimuli (hexane) were presented at
the beginning and end of each EAG analysis, and
in the analysis the value was included as the
mean of two measures.
GC-EAD Analysis
GC-EAD analysis (Arn et al. 1975) was car-
ried out to locate the antennally active compo-
nents in Porapak Q extracts. The system con-
sisted of a gas chromatograph (Varian 3600, Palo
Alto, CA) coupled to an electroantennogram ap-
paratus (Syntech, Hilversum, The Netherlands).
The GC was equipped with a capillary column
(DB-5MS, 30 m x 0.25 mm i.d., film thickness 0.5
pm; J & W Scientific, Folsom, CA), a flame ioniza-
tion detector (FID) and a split/splitless injector.
Temperature oven was programmed at 50C for 2
min, then 3C/min to 280C, and held for 10 min.
Injector and detector temperatures were 250C
and 300C, respectively. The injector was oper-
ated in splitless mode. Helium was used as car-
rier gas at 2 ml/min and nitrogen as make-up
gas. At the end of the capillary column a fixed
outlet splitter (VSOS, Scientific Instruments
Services, Ringoes, NJ) distributed the effluent
from the column to FID and to a transfer line to-
wards the EAD preparation. Both connection
tubings were made of deactivated fused silica of
the same length and diameter such that the col-
umn effluent was split approximately 1:1. The
EAG set up was reported above. Before injection
of a sample, the antenna was stimulated with
linalool to check sensitivity. If the antennae elic-
ited a clear response different to that of signal-
noise, then the guava fruit extract or synthetic
compounds was injected. A minimum of 9 differ-
ent antennae per sex were used, and for each test
an antenna was used only once.
Chemical Analysis
The GC-MS analysis was conducted with a
Varian Star 3400 CX chromatograph linked to a
Varian Saturn 4D mass spectrometer. The sam-
ples were analyzed in a fused silica column (DB5-
MS, 30 m x 0.25 mm, film thickness 0.5 pm) that
was programmed at 50C for 2 min, then 3C/min
until 280C, and held for 10 min. The carrier gas
was helium (1 ml/min). The injector port tempera-
ture was held at 250C. Ionization was by electron
impact at 70 eV. Identifications were based on re-
tention time, mass spectral analysis of the natu-
ral compounds, and comparison with synthetic
standards.
December 2005
Malo et al.: Response ofA. ludens to Fruit Volatiles
Statistical Analysis
Data were analyzed with the Statistica Soft-
ware Package version 6.0 (StatSoft, Inc., 2003).
Data from behavioral and EAG experiments were
analyzed for homogeneity of variances and nor-
mality. When necessary, data were transformed
with log (x + 1) to stabilize the variance and nor-
mality. Results of the wind tunnel were subjected
to t-test. The values of EAG depolarization ampli-
tude after exposure to synthetic chemicals in the
dose-response studies were analyzed by three-
way ANCOVA in a block design, where the hex-
ane response was the covariate (the EAG ampli-
tude response to hexane was a typical mechanical
response produced by the air because the solvent
was evaporated), and significant ANCOVAs were
followed by a posthoc Tukey's test for multiple
comparison of means (P < 0.05). The treatments
tested were synthetic products, sex, and dose. The
insects represent replicates.
RESULTS
Response to Guava Fruits and Extracts
Males (t = 5.2; df = 28; P < 0.001), and females
(t = 2.4; df = 28; P = 0.02) were more attracted to
guava fruits than to yellow sphere (Table 1). Both
males (t = 5.0; df = 28; P < 0.001) and females (t =
5.7; df = 28; P < 0.001) landed more often on
guava fruits than on yellow spheres (Table 1).
Also, flies of both sexes were more attracted to
guava fruit extracts (males: t = -4.5; df = 18; P <
0.001; females: t = 6.7; df 18; P < 0.001), but few
females and no males landed on yellow spheres
dispensing fruit volatiles (Table 1).
GC-EAD Analysis
GC-EAD analysis of guava extracts eluted from
Porapak Q revealed eight and seven compounds
that elicited consistent antennal responses from
male and female A. ludens, respectively (Table 2).
The corresponding EAD active compounds were
identified as ethyl butyrate, (E)-3-hexenol, (Z)-3-
hexenol, hexanol, ethyl hexanoate, hexyl acetate,
(Z)-3-hexenyl butyrate and ethyl octanoate, re-
spectively, by comparison of mass spectra and GC-
MS retention times with synthetic standards.
Ethyl butyrate, ethyl hexanoate, (Z)-3-hexenol,
(E)-3-hexenol, and ethyl octanoate elicited a great
antennal response from female antennae (Table
2). The response of male antennae was lower than
the females, but again ethyl butyrate, ethyl hex-
anoate, and ethyl octanoate evoked the strongest
antennal responses (Table 2). Antennal activity of
the eight natural compounds was confirmed by
antennal responses elicited by 1 mg of their re-
spective synthetic compounds. The ratio of the dif-
ferent compounds in headspaces samples, esti-
mated by GC-MS, were: ethyl butyrate, (E)-3-hex-
enol, (Z)-3-hexenol, hexanol, ethyl hexanoate,
hexyl acetate, (Z)-3-hexenyl butyrate, and ethyl
octanoate (14:1:28:10:18:120:6: 4), respectively.
EAG Analysis
The ANCOVA analysis of the amplitude of the
EAG revealed that synthetic chemicals, sex, dose.
and the synthetic chemicals x dose interaction
significantly influenced the antennal response of
A. ludens (Table 3). The interaction between syn-
thetic chemicals x dose is shown in Fig. 1. Multi-
ple comparisons revealed that, at the dose of 1 pg,
ethyl hexanoate and ethyl octanoate evoked sig-
nificantly larger EAG responses compared with
those elicited by hexanol and hexyl acetate. The
antennal response evoked by (Z)-3-hexenyl bu-
tyrate, (Z)-3-hexenol, (E)-3-hexenol, and ethyl bu-
tyrate were intermediate between, and not signif-
icantly different from, those elicited by ethyl hex-
anoate and ethyl octanoate, and hexanol and
hexyl acetate. At a dose of 10 pg, ethyl hexanoate
evoked significantly larger antennal response
compared with those elicited by hexanol and
hexyl acetate. The EAG response evoked by (Z)-3-
hexenyl butyrate, (Z)-3-hexenol, (E)-3-hexenol,
ethyl octanoate, and ethyl butyrate were interme-
diate between and not significantly different from
TABLE 1. MEAN PERCENTAGES ( SE) OF A. LUDENS THAT EXHIBITED ATTRACTION TO AND LANDED ON GUAVA FRUITS
AND EXTRACT IN A WIND TUNNEL.
Attraction Landing
Treatment Female Male Female Male
Guava fruit 27.7 3.2 a 23.7 2.4 a 7.2 1.1 a 8.8 1.3 a
Yellow sphere 15.5 4.1 b 9.1 + 1.9 b 0.8 0.5 b 1.1 0.5 b
Guava extract 25.6 1.9 a 12.8 0.9 a 2.4 1.1 a 0.0 a
Yellow sphere + solvent 9.6 + 1.3 b 6.8 + 0.8 b 0.0 a 0.0 a
Guava fruit was compared to a yellow sphere (n = 15 replicates per treatment per sex), and guava volatiles extract compared
with ethyl ether as control (n = 10 replicates per treatment per sex). Means within columns followed by the same letter are not sig-
nificantly different (t-test, P > 0.05).
Florida Entomologist 88(4)
TABLE 2. VOLATILES ELICITING ELECTROPHYSIOLOGICAL ACTIVITY DURING GC-EAD RECORDING FROM FEMALE AND
MALE A. LUDENS ANTENNAE EXPOSED TO HEADSPACE COLLECTIONS OF GUAVA FRUITS.
Male antennae Female antennae
EAG responses EAG intensity (mV) EAG responses EAG intensity (mV)
Compound in 9 runsb (Mean + S. E.) in 10 runs' (Mean + S. E.)
Ethyl butyrate 9 0.35 + 0.07 10 0.85 + 0.18
(E)-3-Hexenol 6 0.17 + 0.07 8 0.63 + 0.18
(Z)-3-Hexenol 7 0.18 + 0.08 10 0.60 + 0.17
Hexanol 6 0.08 + 0.05 6 0.33 + 0.07
Ethyl hexanoate 9 0.60 +0.08 10 1.16 +0.19
Hexyl acetate 7 0.15 +0.07 2
(E)-3-hexenyl butyrate 8 0.18 + 0.08 5 0.62 + 0.22
Ethyl octanoate 9 0.46 + 0.07 7 0.77 + 0.12
"Identification is based on comparison of mass spectra and retention times of the natural materials with those of authentic syn-
thetic standards.
bBased on four different headspaces samples and 9 different insects.
'Based on four different headspaces samples and 10 different insects.
those elicited by ethyl hexanoate, and hexanol
and hexyl acetate. At the higher dose tested (100
pg), the EAG response to (E)-3-hexenol was sig-
nificantly higher in comparison to those evoked
by hexanol, hexyl acetate, ethyl butyrate, ethyl
hexanoate, ethyl octanoate, and (Z)-3-hexenyl bu-
tyrate. There was no significant difference in the
EAG response by (E)-3-hexenol and (Z)-3-hex-
enol. Hexanol and ethyl octanoate evoked the
lowest EAG responses.
Behavioral Response to the Blend of the EAD-Active
Compounds
Results of the behavioral response to the blend
of the EAD-active compounds are shown in Fig. 2.
Both males (t = 3.52, df = 18, P = 0.002) and fe-
males (t = 3.27, df = 18, P = 0.004) were more at-
tracted to septa loaded with the eight-component
guava blend compared to a solvent control. How-
ever, few insects landed on spheres dispensing
the guava blend, with no differences between the
number of flies landing on the blend source and
the control spheres (male: t = 0.45, df = 18, P =
0.65; females: t = 0.64, df = 18, P = 0.52).
DISCUSSION
This study showed that both males and fe-
males of A. ludens were attracted to and landed
on guava fruits more often than on yellow spheres
in a wind tunnel. The weak responses ofA. ludens
to guava fruits and their volatiles in the wind tun-
nel are similar to those obtained with other fruit
species (e.g., Robacker et al. 1990; Robacker &
Fraser 2002). The fact that Porapak Q extracts
evoked few landings on the source compared with
those observed with fruits could indicate that the
compounds eliciting this particular behavior are
lacking in these extracts or they are not in the ap-
propriate concentrations to elicit landings in the
wind tunnel. This idea seems to be supported by
TABLE 3. TEST OF SIGNIFICANCE OF THE FACTORS INVOLVED IN THE ANCOVA ANALYSIS OF THE EAG RESPONSE OF
MALES AND FEMALES OF A. LUDENS TO SYNTHETIC VOLATILES AT DIFFERENT DOSES.
Source of Variation SS df MS F P
Covariate (Hexane) 30.60 1 30.600 1117.80 <0.0001
Sex 0.54 1 0.540 19.70 <0.0001
Synthetic chemicals 4.90 7 0.690 25.50 <0.0001
Dose 8.30 2 4.150 151.60 <0.0001
Sex-Synthetic products 0.22 7 0.030 1.20 0.31
Sex-Dose 0.02 2 0.010 0.44 0.64
Synthetic products-Dose 6.70 14 0.480 17.50 <0.0001
Sex-Synthetic products-Dose 0.13 14 0.009 0.34 0.98
Block 2.90 11 0.260 9.60 <0.0001
Error 14.10 516 0.020
Total 82.60 575
December 2005
Malo et al.: Response ofA. ludens to Fruit Volatiles
O (E)-3-Hexenol
* Hexyl acetate
E Ethyl octanoate
B (Z)-3-Hexenol
0 Ethyl butyrate
* (Z)-3-Hexenyl butyrate
0 Hexanol
E Ethyl hexanoate
a ab
10 100
Load (ug)
Fig. 1. Mean EAG amplitude response (- SEM) ofA. ludens to synthetic chemicals isolated from guava fruits.
Data from both sexes per compound were combined for this figure (n = 24 EAG recordings for each compound). Sig-
nificant differences within-dose comparisons are indicated by different letters (Tukey test, P < 0.05).
the results obtained during the evaluation of syn-
thetic compounds identified from guava extracts
in which few flies landed on yellow spheres baited
with the guava blend.
In the present study we showed that GC-EAD
technique can be a useful tool for identifying
EAG-active compounds and so avoiding the eval-
uation of compounds from host fruit without bio-
logical activity. This electrophysiology approach
has been previously used for the identification of
attractants for two fruit flies species (Cosse et al.
1995; Zhang et al. 1999; Nojima et al. 2003). For
example, Zhang et al. (1999) using solid-phase
microextraction and GC-EAD were able to iden-
tify a new blend of volatiles from apples as attrac-
tants for apple-origin Rhagoletis pomonella
(Walsh) flies. The new five-component blend iden-
tified contained three compounds in common with
the previous seven-component blend (Fein et al.
1982), plus two new components. In wind tunnel
bioassays, sticky red spheres baited with the new
five-component blend caught more flies than the
previous seven-component blend or butyl hex-
anoate, a compound used commercially to moni-
tor R. pomonella. In field experiments, red
spheres baited with the five-component blend
also captured more flies than butyl hexanoate
(Zhang et al. 1999).The behavioral evaluation of
all compounds identified in host volatiles and
their possible mixtures may be a time demanding
task. For instance, Robacker et al. (1990) identi-
fied over 70 compounds from fermented chapote.
When the identified compounds were tested indi-
vidually, only 16 of the chemicals were slightly at-
tractive to A. ludens. In bioassays, the number of
compounds in an attractive mixture was reduced
to three by elimination of unnecessary com-
pounds. The three compounds identified were 1,
8-cineole, ethyl hexanoate, and hexanol and the
three-component blend (CEH) was 1.8 times more
attractive than aqueous solutions of torula dried
yeast and borax to A. ludens. In a subsequent
study, 16 compounds previously identified in vol-
atiles from chapote fruit, but not evaluated in ear-
lier work, were tested for individual attractive-
ness and for their capacity to enhance the attrac-
tiveness of CEH when combined with it (Ro-
backer et al. 1992). Of all compounds evaluated,
ethyl octanoate was found to increase the attrac-
tiveness of CEH and this four-component blend
(CEHO) was more attractive than torula yeast in
McPhail traps during flight-chamber tests.
Some of the compounds identified in the
present study have been reported to influence the
behavior of other fruit flies. For mated female
Ceratitis capitata (Wied.), the kairomonal activity
ATTRACTION
30
25
S20
S15
Female Male
LANDING
NS
Female
NS
Fig. 2. Behavioral response of male and female A.
ludens to synthetic blend compounds in the wind tun-
nel. Double asterisks (**) mean significant difference in
the response of the synthetic blend compared to control;
NS means not significant (t-test, P > 0.05).
of the odor of nectarine, a highly-preferred host
fruit, was synergistically enhanced by a blend of
ethyl hexanoate, and methyl and ethyl octanoate
(Light & Jang 1996). Hexenyl acetate individu-
ally was attractive to Bactrocera dorsalis (Hen-
del) (Hwang et al. 2002), and blended with an-
other six esters, was attractive to R. pomonella
(Fein et al. 1982).
In conclusion, this study shows that both sexes
of A. ludens are attracted to guava fruits and
guava extracts in a wind tunnel. The compounds
responsible for this attraction were identified by
GC-EAD and GC-MS techniques. The behavioral
evaluation of the identified compounds showed
that they are attractive to male and female
A. ludens in a wind tunnel. Finally, this study
showed that an approach similar to that used
here could be useful in the searching of potential
host fruit attractants for A. ludens and other
Anastrepha fruit flies. The behavioral activity of
the compounds identified here will be evaluated
in field conditions in future studies.
ACKNOWLEDGMENTS
We are grateful for the technical assistance of
Gustavo Rodas, Azucena Oropeza, and Milton A. Ras-
gado, and the statistical advice of Javier Valle-Mora
(ECOSUR). We also thank Julio Dominguez, Moscafrut
(National Fruit Fly Campaign-SAGARPA-IICA) mass
rearing facility director, for providing the flies used in
this study. This project was supported by a grant from
CONACyT (project 36490-B).
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Florida Entomologist 88(4)
December 2005
EFFECTS OF ELEVATED ATMOSPHERIC CO, ON WATER CHEMISTRY
AND MOSQUITO (DIPTERA: CULICIDAE) GROWTH UNDER
COMPETITIVE CONDITIONS IN CONTAINER HABITATS
BARRY W. ALTO1, STEPHEN P. YANOVIAK1, L. PHILIP LOUNIBOS1 AND BERT G. DRAKE2
'University of Florida, Florida Medical Entomology Laboratory, 200 9th St. SE, Vero Beach, FL 32962
2Smithsonian Environmental Research Center, P.O. Box 28, Edgewater, MD 21037
ABSTRACT
We investigated the direct and indirect effects of elevated atmospheric CO2 on freshwater
container habitats and their larval mosquito occupants. We predicted that a doubling of at-
mospheric CO, would (1) alter the chemical properties of water in this system, (2) slow deg-
radation of leaf litter, and (3) decrease larval growth ofAedes albopictus (Skuse) mosquitoes
raised on that litter under competitive conditions. Effects of elevated CO2 on water quality
parameters were not detected, but the presence of leaf litter significantly reduced pH and
dissolved oxygen relative to water-filled containers without litter. Degradation rates of oak
leaf litter from plants grown under elevated CO2 atmospheres did not differ from breakdown
rates of litter from ambient CO2 conditions. Litter from plants grown in an elevated CO2 at-
mospheres did not influence mosquito population growth, but mosquito production decreased
significantly with increasing larval density. Differences among mosquito density treatments
influenced survivorship most strongly among male Ae. albopictus and time to emergence
most strongly among females, suggesting fundamental sex-determined differences in re-
sponse to competition. Results of this and other studies indicate that direct and indirect ef-
fects of doubled atmospheric CO2 are minimal in artificial containers with freshwater.
Key Words: global change, freshwater, Aedes albopictus, larval competition, population
growth parameters
RESUME
Nosotros investigamos los efectos director e indirectos de niveles elevados de CO2 atm6sfe-
rico en medio ambiente de recipients de agua fresca y con larvas de mosquitos. Nosotros
predecimos que al doblar la cantidad de CO2 de atm6sfera resultaria: (1) en alterar las ca-
racteristicas quimicas del agua en este sistema, (2) en retrasar la degradaci6n de la hoja-
rasca, y (3) disminuir el crecimiento de las larvas de mosquitos Aedes albopictus (Skuse)
criadas en la hojarasca bajo condiciones de competencia. Efectos de un nivel elevado de CO2
sobre los parametros de la calidad de agua no fueron detectados, pero la presencia de la ho-
jarasca redujo significativamente el pH y oxigeno disuelto en relaci6n con los recipients lle-
nados con agua sin hojarasca. La tasa de degradaci6n de la hojarasca de roble en plants
sembradas bajo niveles elevados de CO2 atm6sferico no fue diferente de la tasa de degrada-
ci6n de la hojarasca bajo condiciones de CO2 ambiental. La hojarasca de plants sembradas
en una atm6sfera elevada de CO, no tuvo una influencia sobre el crecimiento de la poblaci6n
de mosquitos, pero la producci6n de mosquitos bajo significativamente al aumentar de la
densidad de las larvas. Las diferencias entire los tratamientos de densidad de mosquitos tuvo
una influencia mas marcada sobre la sobrevivencia entire los machos deAe. albopictus y con
respect al tiempo de emergir fue mas marcado entire las hembras, esto sugiere que hay di-
ferencias fundamentals determinadas por el sexo en respuesta a la competici6n. Resultados
de este y otros studios indican que los efectos director e indirectos al doblar el CO2 atmos-
f6rico son minimos en recipients artificiales de agua fresca.
Atmospheric carbon dioxide concentrations are has been paid to potential effects of elevated at-
anticipated to double by the end of this century mospheric CO2 on aquatic systems.
(Houghton et al. 1995). Increased CO2 is expected The chemical properties of water are influ-
to have a variety of direct and indirect effects on enced in part by the composition of the overlying
ecosystem processes, mediated primarily by atmosphere. Most freshwater habitats are net
changes in plant productivity and distribution sources of CO, (Wetzel 2001) and are unlikely to
(reviewed by Bazzaz 1990; Idso & Idso 1994; be greatly affected by elevated atmospheric CO,
Drake et al. 1997). Whereas these effects are be- concentrations. However, marine environments
coming increasingly predictable for terrestrial (e.g., Hein & Sand-Jensen 1997) and carbon-lim-
communities (but see Navas 1998), less attention ited freshwater ecosystems (e.g., oligotrophic
Alto et al.: Elevated CO2 Water Chemistry, and Mosquito Growth
lakes; Schindler et al. 1972) are potential sinks
for atmospheric CO,. Changes in the concentra-
tion of atmospheric CO2 may directly affect water
quality parameters, particularly pH, in these sys-
tems. Ultimately, direct effects of increased atmo-
spheric CO, on pH and other parameters are re-
lated to physicochemical properties that vary re-
gionally and among systems.
Indirect effects of elevated atmospheric CO, on
freshwater habitats primarily stem from changes
in the quality of terrestrial leaf litter, which
serves as a resource base for aquatic detritivores
(e.g., Egglishaw 1964; Cummins & Klug 1979).
Elevated atmospheric CO, generally reduces the
nutritional quality of living leaf material for ter-
restrial herbivores through reductions in N con-
centration resulting in increased C/N ratios. This
increased relative carbon content affects leaf
structural properties and may further the produc-
tion of carbon-based secondary metabolites such
as phenolics (e.g., Lindroth et al. 1993; Drake et
al. 1997; Stiling et al. 1999, 2003). Differences in
nutritional quality of naturally abscised terres-
trial leaf litter resulting from elevated atmo-
spheric CO, are less clear. Some studies show no
(or minimal) observable effects of elevated CO2 on
leaf litter (Norby & Cotrufo 1998; Strand et al.
1999; King et al. 2001), whereas others show clear
differences in leaf litter quality (Cotrufo & Ineson
1996; Cotrufo et al. 1994; Cotrufo et al. 1999; Rier
et al. 2002; Tuchman et al. 2003), perhaps due to
differences among leaf types in nutrient resorp-
tion during senescence. Abscised leaves from
plants grown in elevated CO, environments tend
to decompose more slowly due to changes in lig-
nin/N, C/N ratios, and N and lignin concentra-
tions (Cotrufo et al. 1994; Cotrufo & Ineson 1996;
Cotrufo et al. 1999; Frederiksen et al. 2001), and
reduced bacterial activity (Frederiksen et al. 2001;
Rier et al. 2002; Tuchman et al. 2003). Detritivore
productivity is often related to the decomposition
rate of litter in freshwater (Fish & Carpenter
1982; Yanoviak 1999), so even minor changes in
litter quality may have important ecological im-
plications. Despite several avenues by which ele-
vated atmospheric CO, may effect freshwater
habitats, the topic largely has been ignored.
Water-filled artificial containers (e.g., discarded
tires, flower vases, etc.) present simple systems to
examine effects of environmental change on eco-
logical processes. Water in containers is especially
likely to be directly influenced by atmospheric
conditions because it originates as rainfall, lacks
contact with soils or groundwater, and often has a
large exposed surface relative to its total volume.
Container water is not well buffered against pH
shifts, temperature fluctuations, and other effects
of atmospheric perturbations. Here we explored
the direct effects of elevated atmospheric CO, on
basic chemical properties of water in container
habitats. Assuming that these systems have poor
buffering capabilities, we expected a reduction in
pH of the container water in the elevated CO,
environment.
Container habitats also are good models for in-
vestigating the indirect effects of elevated atmo-
spheric CO2 on discrete freshwater systems. Plant
detritus, typically in the form of fallen leaf litter, is
the base of food webs in these settings. Food webs
in containers tend to be relatively simple and fre-
quently include mosquitoes as the dominant con-
sumers of litter-derived nutrients. Mosquitoes gen-
erally feed by filtering particles (e.g., microbes)
from the water column and browsing upon surfaces
(Clements 1992). Mosquito productivity is influ-
enced by the quality of litter inputs (Fish & Car-
penter 1982; Lounibos et al. 1993; Walker et al.
1997; Strand et al. 1999; Yanoviak 1999; Daugh-
erty & Juliano 2002), and reduced litter quality
due to elevated atmospheric CO, is likely to nega-
tively affect mosquito population growth parame-
ters. Strand et al. (1999) tested this hypothesis
with the eastern treehole mosquito Ochlerotatus
triseriatus (Say), and Tuchman et al. (2003) tested
it with a suite of mosquitoes that included 0. tri-
seriatus, Aedes albopictus (Skuse), Aedes aegypti
(L.), and Armigeres subalbatus (Coquillett). Re-
sults from these studies showed that the negative
effects of elevated CO, were either nonsignificant
or limited to differences in development time for a
few mosquito species or survivorship ofAe. albopic-
tus. However, in neither of these studies were the
mosquitoes reared under nutrient-limited condi-
tions typical of such habitats. If resource quality is
an important determinant of mosquito growth and
survivorship, then this should be most clearly illus-
trated by differences in the outcome of competitive
interactions (i.e., when the resource is limiting).
Here we examined the potential indirect ef-
fects of elevated atmospheric CO, on litter decom-
position and productivity of the mosquitoAe. albo-
pictus in container habitats. We chose Ae. albopic-
tus because it naturally colonizes containers and
it is an exotic species (Lounibos 2002) that out-
competes larvae of established species (Juliano
1998). In nature, intra- and interspecific larval
competition is often strong among container mos-
quitoes and it may play an important role in shap-
ing container communities (Novak et al. 1993;
Juliano 1998; Teng & Apperson 2000; Lounibos et
al. 2001, 2003). Limited effects of elevated CO, on
mosquito population growth found by Strand et
al. (1999) and Tuchman et al. (2003) may be
partly due to the experimental set-up not being
sufficiently competitive to produce differences in
mosquito performance between ambient and ele-
vated CO2 litter treatments. Differences in mos-
quito growth in ambient versus elevated CO, lit-
ter are likely to become more pronounced as in-
traspecific competition is increased.
Because some changes in water quality param-
eters, particularly pH, influence litter decomposi-
Florida Entomologist 88(4)
tion rates in freshwater (Groom & Hildrew 1989;
Kok & Van der Velde 1994), we hypothesized that
any direct effects of elevated atmospheric CO2 on
water quality would alter litter decomposition
rates in containers. We also expected that oak lit-
ter originating from an elevated CO2 environment
would decompose more slowly in water-filled con-
tainers. Finally, we predicted that population
growth correlates ofAe. albopictus would be de-
pressed, and intraspecific competition more in-
tense, when individuals are reared on elevated
CO2 litter. Adult aedine mosquitoes are sexually
dimorphic, with males typically smaller than fe-
males and are the first to emerge to adulthood
(Briegel & Timmermann 2001). Given that the re-
sponse to competition may be sex-determined, we
analyzed males and females separately.
MATERIALS AND METHODS
We compared water chemistry in containers set
in scrub-oak habitats at the Kennedy Space Center
(KSC), Cape Canaveral, Florida (2829'N, 8034'W).
Sixteen octagonal open-top chambers (3.6 x 2.5 m,
width x ht.) were constructed from clear polyester
film ('Mylar,' Melinex 071) overlying PVC frames.
Eight chambers were ventilated with ambient air
and 8 with CO2 concentration artificially elevated to
approximately double ambient levels (see Dijkstra
et al. 2002 for additional details).
Container Water Chemistry in Elevated vs. Ambient CO2
Black plastic cups (max. volume = 500 ml)
served as our experimental units and were ar-
ranged in a split-plot design at KSC on March 18,
2003. Each cup received distilled water to the
level of a small drain hole located ca. 3 cm from
the top (to prevent overflow), resulting in a func-
tional volume of approximately 350 ml. We added
1.0 0.005 g oak leaves (Quercus virginina Mill.)
and 5.0 ml of filtered (180 pm sieve) oak infusion
inoculum (O'Meara et al. 1989) to half of the cups
(hereafter, "litter" treatment). The litter used in
this experiment consisted of freshly fallen leaves
collected on the grounds of the Florida Medical
Entomology Laboratory (FMEL) (27.6N, 80.5W).
The leaves were dried at 60C for 48 h before
weighing. The remaining cups received distilled
water but no leaves or inoculum ("water" treat-
ment). All cups were individually covered with a
nylon screen (0.8 mm mesh) to prevent additional
litter inputs and mosquito colonization.
We placed one water treatment and one litter
treatment cup in 8 replicates of each of three ex-
perimental environments spaced 5-10 m apart: el-
evated CO2 chamber, ambient air chamber, or am-
bient air plot without a chamber (hereafter, "ele-
vated", "ambient", and "control"), for a total n = 48
cups (2 treatments x 3 environments x 8 repli-
cates). Portable meters were used to measure dis-
solved oxygen (DO), nitrate (NO3), pH, and tem-
perature of the water in each cup weekly for 9
weeks. Measurements were initiated at 0900-
1000 h on each sample date. No data were col-
lected on week 7, and equipment problems pre-
vented pH measurements on weeks 3 and 6. All
of these instances were treated as missing values
in analyses. Initial (week 0) means were obtained
for the litter treatments by measuring water
quality parameters in 10 cups (5 litter and 5 wa-
ter) in the laboratory. Final water volume was
measured in each cup on week 9, and remaining
litter was collected, dried, and weighed as above.
Water quality parameters (DO, nitrate, and pH)
were compared between treatments and among
environments with mixed model repeated-mea-
sures analysis of variance (SAS Institute 2002).
This maximum-likelihood approach is preferred
over general linear models for repeated measures
because it permits more appropriate modeling of
variance structure (von Ende 1993; Saavedra &
Douglass 2002). All repeated-measures tests em-
ployed autoregressive order 1 variance structure
and "block" (individual chamber or plot) within lit-
ter and environment treatments as the error sub-
ject. Satterthwaite-type degrees of freedom were
obtained by the Kenward-Roger method (Kenward
& Roger 1997; SAS Institute 2002). General linear
models ANOVAs were used to analyze final water
volume and litter mass. Normality was confirmed
with Kolmogorov-Smirnov tests and normal prob-
ability plots (Sokal & Rohlf 1995; SAS Institute
2002). Nitrate values were log-transformed to re-
duce variance heterogeneity and DO values were
corrected for water temperature. pH values were
not transformed since they adequately met the as-
sumptions. All means include 1 standard error
and were calculated from untransformed data.
Effects of Leaves from Elevated CO2 on Mosquito
Growth and Competition
We planned to conduct mosquito productivity
experiments in the field, but pilot data showed
that summer water temperatures in artificial con-
tainers placed in CO2 enclosures at KSC often ex-
ceeded 36C, which was lethal to Ae. albopictus
larvae reared in the lab (0.62% survivorship to
emergence from 12 cups each with 40 larvae).
Thus, we conducted the mosquito productivity por-
tion of this study in growth chambers at FMEL.
On 31 March 2003 we collected oak leaf litter (a
combination of Quercus chapmanii Sargent, Q.
myrtifolia Willd., and Q. geminata Small in ap-
proximately equal proportions) from each of the 16
experimental chambers at KSC. We chose Quercus
spp. litter because it was readily available and it is
commonly found in water-filled containers occu-
pied by mosquitoes in peninsular Florida (per-
sonal observations, BWA). The individual litter
collections were pooled by treatment, providing
December 2005
Alto et al.: Elevated CO2 Water Chemistry, and Mosquito Growth
25.0 g elevated CO2 litter and 25.0 g ambient CO2
litter. Leaves were wiped (not washed) to remove
dirt and sand, and then dried at 60C for 24 h.
Experiments were conducted in 500-ml plastic
cups containing 350 ml distilled water and 1.0 +
0.005 g elevated or ambient CO, litter. After the
litter had soaked in the cups for 3 d,Ae. albopictus
first instars (<24 h old) were added at densities 10,
20, 30, 40, or 50 per cup. Pilot data using similar
water volumes and leaves (Q. virginiana) sug-
gested these densities would provide a range of
competitive conditions. Mosquitoes used in the ex-
periment were the progeny of wild larvae collected
from artificial containers near Vero Beach, FL.
Each leaf litter and larval density treatment was
replicated 5 times (2 x 5 x 5 = 50 cups total). Cups
were individually covered with a nylon screen (0.8
mm) and housed in an incubator at a mean temper-
ature ( SD) 25.3 + 0.4C, relative humidity of 78.3
+ 5.6%, and a photoperiod of 14:10 (L:D). When pu-
pae were first detected, cups were checked daily
and pupae were removed and housed in 40-ml vials
with tap water until emergence. Adult emergence
was recorded daily, and adults were dried at 60C
for 48 h and individually weighed on a Cahn elec-
trobalance. The litter remaining in a cup was dried
and weighed after all mosquitoes from that cup
had emerged. The experiment continued until all
Ae. albopictus had emerged or died.
Multivariate analyses of variance (MANOVA)
were used to analyze the treatment effects of leaf
litter, larval density, and litter x density interac-
tion on Ae. albopictus response variables (time to
emergence, survivorship to emergence, and adult
mass). Developmental parameters are sex-spe-
cific in many mosquito species, including Ae. al-
bopictus (Juliano 1998; Teng & Apperson 2000;
Bedhomme et al. 2003). Thus, we used a separate
MANOVA for each sex. Raw data for males were
log0,-transformed to meet assumptions of univari-
ate normality and homogeneous variances. For all
analyses, significant effects were further ana-
lyzed by contrasts of all possible pairs of main ef-
fect multivariate means with experimentwise =
0.05 (sequential Bonferroni method; Rice 1989).
Standardized canonical coefficients (SCCs) were
used to determine the relative contribution of
each of the response variables to significant mul-
tivariate effects as well as their relationship to
each other (e.g., positive or negative; SAS Insti-
tute 2002; Scheiner 1993).
Additionally, we calculated an estimated finite
rate of increase, lambda (X'), for each leaf litter by
larval density replicate. X' synthesizes multiple
population growth correlates to describe popula-
tion performance (Juliano 1998). X' was calcu-
lated as follows:
(1)
= exp(r') = exp In[(1/No)xf(wx) ]
D + [xxAxf(wx)/xAxf(wx)1]
where X' is an alternative form of r', a composite
index of population performance described by
Livdahl and Sugihara (1984) and Livdahl (1982).
r' is an estimate ofr = dN/Ndt which describes the
per capital growth rate. No is the initial number of
females in a cohort (assumed to be 50% of intro-
duced larvae);A, is the number of females emerg-
ing on day x; w, is the mean female size on day x;
f(w) is a function relating the number of eggs pro-
duced by a female to her size; and D is the time (in
days) from emergence to oviposition. ForAe. albop-
ictus, D is assumed to be 14 d (Livdahl & Willey
1991). We used the following size-fecundity rela-
tionships to calculate (f(w)) (Lounibos et al. 2002):
f(wx) = 19.5 + (152.7*X)
where r2 = 0.573.
Equation 2 was derived from two separate re-
gressions in Lounibos et al. (2002). We assume
that the regression function forAe. albopictus ap-
proximates individual reproductive capacity. We
analyzed effects of leaf litter origin (elevated or
ambient CO,) and larval density onAe. albopictus
X' by two-way ANOVA. Significant effects were
further analyzed by comparisons of pairs of main
effect means with experimentwise = 0.05 (Ryan-
Einot-Gabriel-Welsch multiple range test; SAS
Institute 2002).
Finally, we used a t-test to compare the mean
proportion (arcsine square root transformed) of
litter mass remaining at the end of the experi-
ment between litter treatments (all densities
within a litter type pooled).
RESULTS
Container Water Chemistry in Elevated vs. Ambient CO2
The nitrate data set included an extreme value
(22.5 mg/L) on week 6 in the water treatment cup
located in the ambient environment. We deter-
mined this to be a statistical outlier for both the
week 6 ambient environment data (Dixon's test,
P < 0.01) and for the complete data set (P < 0.005,
according to Grubbs 1969; Sokal & Rohlf 1995),
and excluded it from the analysis (treated as
missing data). The cause of this outlier is un-
known, but excreta from a perching or passing
bird is a likely possibility.
The presence or absence of leaf litter signifi-
cantly affected nitrate concentrations, pH, and
DO in the cups (Table 1). Mean nitrate concentra-
tions were consistently greater in water contain-
ing leaf litter during weeks 1-9, whereas pH and
DO were significantly lower in cups containing
litter over the same period (Table 1; Fig. 1). All pa-
rameters varied significantly over time, and time
x litter interactions were significant for pH and
DO (Table 1). There was a trend for lower pH vari-
ance in cups with litter relative to cups without
Florida Entomologist 88(4)
TABLE 1. REPEATED-MEASURES ANOVA FOR WATER QUALITY PARAMETERS. LITTER = LEAF LITTER PRESENT OR AB-
SENT; ENVIRONMENT = AMBIENT CO2, ELEVATED CO2, OR CONTROL ENVIRONMENTS. AR(1) = AUTOREGRES-
SIVE ORDER 1 COVARIANCE ESTIMATE WITH SUBJECT = BLOCK(LITTER x ENVIRONMENT). DECIMAL DF VALUES
RESULTED FROM SATTERTHWAITE-TYPE DF CALCULATION.
Covariance
Parameter Estimates
Factor df F P AR(1) Residual
Nitrate Litter 1, 60.6 18.80 <0.0001 0.6173 0.2755
Environment 2, 60.6 5.78 0.0051
Time 7,281 48.00 <0.0001
Litter x Environment 2, 60.6 0.54 0.5829
Litter x Time 7, 281 0.80 0.5899
Environment x Time 14, 289 1.45 0.1277
Litter x Environment x Time 14, 289 1.00 0.4555
pH Litter 1, 45.3 6.95 0.0114 0.8373 0.4592
Environment 2, 45.3 2.35 0.1073
Time 5,203 37.69 <0.0001
Litter x Environment 2, 45.3 1.76 0.1830
Litter x Time 5, 203 4.07 0.0015
Environment x Time 10, 205 1.25 0.2637
Litter x Environment x Time 10, 205 1.02 0.4251
DO Litter 1, 74.9 297.20 <0.0001 0.3740 0.0053
Environment 2, 74.9 1.65 0.1983
Time 7, 271 130.80 <0.0001
Litter x Environment 2, 74.9 0.81 0.4498
Litter x Time 7, 271 3.16 0.0032
Environment x Time 14, 285 2.35 0.0043
Litter x Environment x Time 14, 285 1.16 0.3083
litter (Fig. Ib). Initial (week 0) values for water
quality parameters did not differ between litter
and water treatments (Nitrate: t = 1.07, P = 0.32;
pH: t = 0.03, P = 0.98; DO: t = 2.20, P = 0.06; df =
8 for each test; Fig. 1).
Doubling the atmospheric CO2 concentration
had minimal or no effects on water quality param-
eters in the cups. The dissolved oxygen content
and pH of container water did not differ among the
three experimental environments, whereas mean
nitrate concentrations were significantly lower in
controls (i.e., cups located outside the environmen-
tal chambers; Table 1). For nitrate values, post-hoc
univariate tests and Tukey comparisons indicate
that the only significant difference between ambi-
ent and elevated CO2 environments occurred on
week 6. Nitrate was significantly lower in cups in
the control environment than in either ambient or
elevated (or both) on weeks 2 and 4-9 (Fig. 2). Re-
analysis excluding the control environment data
nullified the significant environment effect (F,416 =
0.76, P = 0.39), but did not change the results for
litter, time, or interaction effects.
The mean ( SE) volume of water remaining in
cups at the end of the experiment did not differ
between litter treatments, but was lower in the
control environment (284 5.2 ml) than in the el-
evated CO2 chambers (309 3.6 ml; Table 2). Av-
erage final volume in the ambient environment
(297 4.6) did not differ from the final volumes in
the other environments. Elevated atmospheric
CO2 had no effect on litter degradation rates in
the cups in the field experiment (Table 2). Mean
litter mass remaining at the end of the experi-
ment was similar among ambient (0.812 0.0054
g) elevated (0.814 0.0067 g) and control (0.811
0.0041 g) environments.
Effects of Leaves from Elevated CO2 on Mosquito
Growth and Competition
ANOVA output for X' ofAe. albopictus showed
significant larval density treatment effects, but
litter type and the litter type x density treatment
interaction were not significant. Pairwise compar-
isons showed that X' increased with decreasing
larval densities when <30 larvae were present,
and was significantly lower at higher larval den-
sities, which were similar to each other (Fig. 3).
MANOVA output for males and females showed
significant effects of larval density on response
variables (time to emergence, survivorship to
emergence, adult mass; Table 3). Leaf litter type
and the litter type x density interaction did not
December 2005
Alto et al.: Elevated CO2 Water Chemistry, and Mosquito Growth
2.0
S1.5
E
0.5
0.0
7.0
6.5
5.5
5.0
4.5
1.2
C 1.0
S0.9
go.g
0.B
S
5 0.7
-- Litter
-- No Litter
0 1 2 3 4 5 6 7 8 9
Week
Fig. 1. Mean ( SE) nitrate concentration (A), pH
(B), and DO (C) in cups containing leaf litter plus water
and cups with water only. n = 16 for each mean on
weeks 1-9. Week 0 means were based on n = 5 cups
which were added to illustrate starting values and were
not included in the repeated-measures ANOVA. One ni-
trate measurement (22.5 mg/L) in the water treatment
on week 6 was excluded after significance testing for
outliers.
significantly affect these response variables (Ta-
ble 3). For males, the magnitude of the standard-
ized canonical coefficients (SCCs) showed that
survivorship contributed the most to the density
effect, followed by time to emergence and mass.
For females, SCCs showed that time to emergence
contributed the most to the density effect followed
by survivorship and mass (Table 3). For both
sexes, opposite signs of the SCCs show that time
to emergence was negatively related to survivor-
ship and mass. Multivariate contrasts for the sig-
nificant density effect for males and females
showed consistently and significantly greater sur-
vivorship and shorter time to emergence among
lower larval densities (Table 4). Overall, females
had greater survivorship and longer time to emer-
gence compared to males (Fig. 4). Similar to the
main MANOVA for males, SCCs showed that sur-
vivorship contributed the most to the significant
multivariate contrasts followed by time to emer-
gence, except for two contrasts (Table 4). Likewise,
for females, SCCs showed that time to emergence
contributed the most to the significant multivari-
ate contrasts followed by survivorship (Table 4).
Litter derived from an elevated CO, environ-
ment did not degrade more slowly than litter from
an ambient CO, environment. The mean ( SE)
proportions of elevated CO, (0.822 g 0.0074) and
ambient CO, (0.831 g 0.0081) litter remaining
were similar (t = 0.855, df = 48, P = 0.397).
DISCUSSION
Contrary to our expectations, elevated atmo-
spheric CO, had no direct effects on water quality
in the container system. The presence or absence
of leaf litter was much more important in deter-
mining the chemical characteristics of the water
than was the overlying atmosphere. Although not
measured, these effects are probably associated
with compounds leached from the litter (leading to
greater nitrate concentrations) and the metabolic
activities of microorganisms colonizing the litter
(resulting in lower dissolved oxygen content). The
difference in pH between cups with and without
litter is likely a function of both the leaf material
and the microflora acting upon it. We attribute the
lower pH in cups having litter to tannins and other
organic acids leached from the leaf material (e.g.,
Stout 1989). The trend for lower variance of pH in
cups with litter suggests that leached compounds,
and perhaps CO, production by microorganisms,
have a stabilizing effect on pH in this system.
Other studies investigating the productivity of
mosquitoes reared on litter from elevated CO2 en-
vironments either found no effect (Strand et al.
1999) or found differences in development time
for three mosquito species and in survivorship of
Ae. albopictus (Tuchman et al. 2003). Our results
concur with those of Strand and coworkers
(1999). We observed that litter derived from an el-
evated CO, atmosphere did not alter mosquito
performance even under food-limited conditions.
Not all tree species respond similarly to ele-
vated CO, (e.g., Cotrufo et al. 1994), and leaf litter
species composition can influence mosquito pro-
Florida Entomologist 88(4)
2.0
2 1.5-
1.0-
Z
0.5-
0.0
--- Ambient
--0- Elevated
-v- Control
1 2 3 4 5 6 7 8 9
Week
Fig. 2. Mean ( SE) nitrate concentration in cups located in different experimental environments (n = 8 for each
mean). One outlier (22.5 mg/L) in the ambient environment on week 6 was excluded.
ductivity (Yanoviak 1999), thus the lack of an ele-
vated CO2 effect in this study may be related to
the type of litter we used. Higher lignin concen-
tration, associated with elevated CO2 litter, slows
decomposition and bacterial productivity, as shown
by Tuchman et al. (2003) with Populus tremu-
loides Michaux. In the current study, data per-
taining to the nutritional quality of naturally ab-
scised oak litter in the KSC chambers is limited to
Q. myrtifolia, which showed no difference in C
concentration and only slight (nonsignificant) re-
ductions in N concentration between litter of ele-
vated and ambient CO2 (Stiling et al. 2002). Also,
our expectation that litter produced in an elevated
CO2 environment would degrade more slowly in
this experiment was not supported. Thus, we
speculate that perhaps microbial populations col-
onizing Quercus spp. litter may not have been suf-
ficiently different among CO2 treatments to pro-
duce differences in Ae. albopictus performance.
Quercus spp. are naturally high in lignin and tan-
nins (Stout 1989), thus CO2 mediated differences
TABLE 2. ANOVA FOR WATER VOLUME AND DRY MASS OF LITTER REMAINING IN THE CUPS AT THE END OF THE STUDY
(WEEK 9). MS = MEAN SQUARE. = P < 0.002.
Factor df MS F
Volume Litter 1 36.75 0.11
Environment 2 2562.90 7.52*
Litter x Environment 2 92.31 0.27
Error 42 340.80
Dry Mass Environment 2 1.53 x 105 0.06
Error 21 2.43 x 10"
December 2005
Alto et al.: Elevated CO2 Water Chemistry, and Mosquito Growth
1.16
1.12
E I1. 08
1.04
1.04
c
*
0 10 20 30
Larval density
Fig. 3. Mean ( SE) estimated finite ra
tion increase (K'). Different letters indicate
differences amongAe. albopictus density tr
= 90.08, df = 4, 40, P < 0.0001), but litter
litter type x density treatment interaction
nificant (all F < 3.75, P > 0.05).
in leaf quality may not be sufficient to
quito performance.
Although other studies have shown
in growth variables due to intraspe
competition (Lord 1998), as well as
ences (Teng & Apperson 2000; Bedho
2003), few have quantified competiti
differences in fitness correlates betv
and females. Here, we observed that
in survivorship were the major contrib
density effect for males, whereas dif
time to emergence were the major con
the density effect for females. The r
among the response variables for ea
asymmetrical. In particular, for fema
emergence contributed about twice a
survivorship to the density effect. For
vivorship contributed approximately
than emergence to the density effect.
Although males were significant
than females (mean SE: 0.138 0.00
+ 0.008 mg, respectively), mass contri
to the density effect, and its contribution was
nearly equal between sexes. Thus, although com-
petition had similar global effects on both sexes
(e.g., longer time to emergence, lower survivor-
ship, lower mass), the individual life history
traits affected were sex-determined, suggesting
S c fundamental differences in male and female Ae.
albopictus physiological responses to competition.
Other mosquito studies suggest that natural se-
lection may act differently on each sex, since fe-
40 50 male fitness is related to fecundity whereas male
fitness is related to number of matings (Stein-
wascher 1982; Kleckner et al. 1995). For Ae. ae-
e ofpopula- gypti, Bedhomme et al. (2003) showed sex-deter-
e significant
eatments (F mined effects of larval competition, consistent
type and the with the hypothesis that the effects of competi-
were not sig- tion on traits most important for each sex's repro-
ductive fitness (e.g., time to emergence for males
and size for females) are minimized at the ex-
pense of other traits. However, their study did not
affect mos- investigate survivorship since larval mortality
was very low (i.e., 1%), and so, individual life his-
differences tory traits were measured only in replicates
cific larval where all Ae. aegypti survived to emergence. In
sex differ- the current study we did measure survivorship,
imme et al. and our results show the costs of competition
on-induced were equally minimal for body mass of adults of
veen males both sexes, similar to other findings where re-
differences source levels were low for Ae. albopictus and Ae.
utorstothe aegypti (Juliano 1998). For males, the larger ef-
ferences in fect of competition on survivorship compared to
tributors to time to emergence suggests that this latter trait
relationship is less sensitive to effects of competition. For
ch sex was females, effects of competition on time to emer-
les, time to gence were far greater than survivorship suggest-
is much as ing that females may minimize larval mortality
males, sur- at the expense of increased time to emergence.
20% more Results of this and other studies suggest that
indirect effects of elevated atmospheric CO2 on
ly smaller freshwater systems via terrestrial litter inputs
6 and 0.293 are minimal. Ultimately, carbon inputs into fresh-
buted little water systems may increase through increased
TABLE 3. MANOVA OUTPUT FOR EFFECTS OF LEAF LITTER TYPE (ELEVATED VS. AMBIENT), LARVAL DENSITY, AND LEAF
x DENSITY INTERACTION TREATMENTS ON MALE AND FEMALE AE. ALBOPICTUS RESPONSE VARIABLES; TIME TO
EMERGENCE, SURVIVORSHIP TO EMERGENCE, AND ADULT MASS. STANDARDIZED CANONICAL COEFFICIENTS
(SCCS) SHOW THE RELATIVE CONTRIBUTION OF EACH OF THE RESPONSE VARIABLES TO SIGNIFICANT TREAT-
MENT EFFECTS.
First SSCs
Source df Pillai's Trace P Time Survivorship Mass
Males Litter Type 3,37 0.10 0.2811
Density 12, 117 1.16 <0.0001 -1.15 1.45 0.28
Litter x Density 12, 117 0.27 0.4932
Females Litter Type 3,38 0.13 0.1421
Density 12, 120 1.22 <0.0001 -2.07 1.00 0.21
Litter x Density 12, 120 0.23 0.6086
Florida Entomologist 88(4)
TABLE 4. MULTIVARIATE PAIRWISE CONTRASTS FOR EFFECTS OF LARVAL DENSITY TREATMENTS ON MALE (DF = 3, 37) AND
FEMALE (DF = 3, 38) AE. ALBOPICTUS RESPONSE VARIABLES; TIME TO EMERGENCE, SURVIVORSHIP TO EMER-
GENCE, AND ADULT MASS. STANDARDIZED CANONICAL COEFFICIENTS (SCCS) SHOW THE RELATIVE CONTRIBU-
TION OF EACH OF THE RESPONSE VARIABLES TO SIGNIFICANT PAIRWISE CONTRASTS. SIGNIFICANT DIFFERENCES
BETWEEN PAIRWISE CONTRASTS ARE SHOWN BY ASTERISKS: = P < 0.05, ** = P < 0.001, *** = P < 0.0001.
Males Females
First SCCs First SCCs
Comparison Pillai's Trace Time Surv. Mass Pillai's Trace Time Surv. Mass
10 vs. 20 0.30* -0.57 1.43 0.88 0.39** -1.43 1.25 0.66
10 vs. 30 0.65*** -0.90 1.61 0.49 0.77*** -1.97 1.10 0.12
10 vs. 40 0.84*** -0.98 1.55 0.50 0.83*** -2.05 1.03 0.21
10 vs. 50 0.87*** -1.19 1.42 0.25 0.85*** -2.00 1.04 0.35
20 vs. 30 0.36** -1.04 1.52 0.12 0.59*** 2.00 -0.83 0.26
20 vs. 40 0.72*** -1.09 1.51 0.32 0.71*** 2.17 -0.81 0.04
20 vs. 50 0.80*** -1.26 1.27 0.03 0.75*** 2.16 -0.89 -0.18
30 vs. 40 0.46*** -1.09 1.45 0.51 0.18
30 vs. 50 0.64*** 1.38 -1.09 0.03 0.37** -1.68 0.69 0.83
40 vs. 50 0.26* 1.22 -0.26 0.66 0.11
structural carbon in litter (Tuchman et al. 2003)
and increased abscission rates (Stiling et al.
2002), but the consequences of this increased car-
bon to detritivores and other consumers are likely
to be protracted and difficult to measure (e.g.,
Hungate et al. 1997). This poses the question: Is
there any freshwater system in which indirect ef-
fects of elevated atmospheric CO2 could be ex-
pected? We suggest that future studies address-
ing this topic should focus on phytotelmata
0.8
2 0.6-
t-
:0.4
0.2
0.
(plant-held waters: Varga 1928), specifically wa-
ter held in living terrestrial plants. Bromeliads,
pitcher plants (e.g., Sarracenia purpurea L.) and
Heliconia spp. flower bracts are excellent candi-
dates for such an investigation because they sup-
port a variety of heterotrophic organisms and
tend to interact physiologically with the water
they contain (e.g., Bronstein 1986; Juniper et al.
1989; Benzing 2000). If any physiological proper-
ties or products of the plants are modified by ele-
io b
10 20
B 20
30B_
40D 50 50
50
E
10 20 30 40 50 6(
Time to Emergence (days)
Fig. 4. Mean ( SE) survivorship and time to emergence for the significant Ae. albopictus density effect. Open
and filled symbols show male and female bivariate means, respectively. Different lower case and capital letters in-
dicate significant differences among means. Mass values were omitted because they contributed little to the den-
sity effect.
December 2005
Alto et al.: Elevated CO2 Water Chemistry, and Mosquito Growth
vated CO,, they should have measurable indirect
effects on the resident fauna.
Apart from potential indirect effects on litter
quality and physiological properties of phytotel-
mata, elevated atmospheric CO, has other broad-
ranging effects, such as increased regional sur-
face temperatures. Altered species distributions
resulting from these concomitant climatic effects
may be more ecologically significant than the di-
rect or indirect effects of elevated CO, (Weltzin et
al. 2003; Thomas et al. 2004). Many aquatic or-
ganisms reproduce and disperse outside of water,
thus the potential for water to act as a physical
buffer against atmospheric change is irrelevant
for many species. These types of considerations
need to be addressed if we are to make accurate
predictions about the consequences of elevated
atmospheric CO, on freshwater systems.
ACKNOWLEDGMENTS
Comments from C. Lord and J. Rey improved the
manuscript. J. Rey loaned equipment, D. Yee assisted
with the supporting literature, and N. Nishimura, R. Es-
cher, and D. Johnson provided technical support and as-
sisted with data collection. Funds were provided by
National Institutes of Health (R01-AI-44793) to L. P.
Lounibos and a DOE grant to the Smithsonian Institution
(DE-FG02-95ER61993) to B. G. Drake. This is Florida Ag-
riculture Experiment Station Journal Series R-10383.
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Florida Entomologist 88(4)
Burkett & Butler: Colored Light as an Attractant for Mosquitoes
LABORATORY EVALUATION OF COLORED LIGHT AS AN ATTRACTANT
FOR FEMALE AEDES AEGYPTI, AEDES ALBOPICTUS,
ANOPHELES QUADRIMACULATUS, AND CULEX NIGRIPALPUS
DOUGLAS A. BURKETT1 AND JERRY F. BUTLER2
'Chief, Range Operations Environmental ACC/DOPP HQACC-Ranges, Airfields & Airspace Ops
douglas.burkett@langley.af.mil
2Department of Entomology & Nematology, University of Florida, Institute of Food and Agricultural Sciences
Building 970, Hull Road, P.O. Box 110620, Gainesville, FL 32611-0620 USA
ABSTRACT
Mosquito feeding activity was monitored in an electronic apparatus (visualometer), having
ten ports, illuminated from below with narrow bandwidths of light (700, 650, 600, 550, 500,
450, 400, or 350 nm). Responses of adult female Aedes albopictus Skuse, Ae. aegypti (L.),
Anopheles quadrimaculatus, Say and Culex nigripalpus Theobald to feeding stations (blood
containers) over each light port. No-light and broad spectrum white light were used as con-
trols. Color preferences were based on electronic detection of feeding times. Aedes aegypti
showed no significant feeding preferences over any of the colors. Conversely, Ae. albopictus,
An. quadrimaculatus, and Cx. nigripalpus showed preferences for several of the wave-
lengths of light. In decreasing order,Aedes albopictus fed significantly longer at 600 nm, 500
nm, white, 450 nm, 400 nm, and black. For An. quadrimaculatus, significantly longer feed-
ing durations were found over the black or white controls and all other individual wave-
lengths had significantly longer feeding durations than 350 nm. Finally, in decreasing order,
significantly greater feeding times were recorded for Cx. nigripalpus over 500 nm, 600 nm,
450 nm, white, 650 nm, and 550 nm compared to the other wavelengths tested.
Key Words: mosquito, attractants, visual ecology, color vision, visual spectrum
RESUME
La actividad de la alimentacion de mosquitos fue estudiado en un aparato electr6nico (me-
dici6n visual), que tiene diez puertos, iluminados por debajo con secciones estrechas de luz
de banda-ancha (700, 650, 600, 550, 500, 450,400, o 350 nm). La respuesta de hembras adul-
tas deAedes albopictus Skuse,Ae. aegypti (L.),Anopheles quadrimaculatus Say y Culex ni-
gripalpus Theobald a las estaciones de alimentaci6n recipientss de sangre) sobre cada
puerto de luz. Sin luz y luz blanca de ancho espectro fueron usados como controls. Las pre-
ferencias de color fueron basadas sobre la detection electr6nica de los tiempos de alimenta-
ci6n.Aedes aegypti no mostro preferencia significativa de alimentaci6n para ninguno de los
colors. Al contrario, Ae. albopictus, An. quadrimaculatus, and Cx. nigripalpus mostraron
preferencias para various de las ondas de luz. En orden decreciente, Aedes albopictus se ali-
mento significativamente mas tiempo A 600 nm, 500 nm, blanca, 450 nm, 400 nm, y negra.
Para An. quadrimaculatus, la duraci6n de tiempo de alimentaci6n fue significativamente
mas largo en los controls de negro y blanco y todas las otras ondas tenian una duraci6n de
alimentaci6n mas larga que 350 nm. Al fin, en orden decreciente, el tiempo de alimentaci6n
fue significativamente mayor para Cx. nigripalpus sobre 500 nm, 600 nm, 450 nm, blanca,
650 nm, y 550 nm comparado con las otras ondas probadas.
That some species of mosquitoes and other
medically important Diptera are attracted to arti-
ficial light or other visual stimuli has long been
known and exploited in a variety of trap designs.
Not all mosquito species respond equally to visual
stimuli or to different wavelengths of light. In-
deed, many mosquitoes do not respond to light
traps at all (Service 1993). Mating, dispersal, ap-
petitive flight, and location of sugars, hosts, rest-
ing, oviposition and overwintering sites all are
governed to some degree by vision. Many authors
have examined the important visual components
of host/resource finding and have divided them
into shape, color (reflected and transmitted), size,
contrast, light intensity, texture and movement
(Allan et al. 1987). These factors alone or in com-
bination appear to play an important role in a fe-
male's ability to successfully locate a suitable host
or other resource.
Much of the mosquito research on color attrac-
tion has evaluated the response of diurnal species
to reflected light colors (Brett 1938; Brown 1954;
Granger 1970; Browne & Bennett 1981). Studies
using colored transmitted light are few, and even
Florida Entomologist 88(4)
fewer provide information on individual species
or emit light of known wavelengths and/or inten-
sity (Headlee 1937; Breyev 1963; Bargren & Nib-
ley 1956; Gjullin et al. 1973; Wilton & Fay 1972;
Vavra et al. 1974; Browne & Bennett 1981). None
of these studies incorporates both reflected and
transmitted light. Lack of information about the
attractiveness of different light wavelengths for
different species of mosquito is a serious void in a
science where mosquito control/research opera-
tions are based largely on the numbers and types
of mosquitoes captured in light-baited traps.
A laboratory method for the evaluation of the
relationship between various light colors (wave-
lengths) of transmitted/reflected light and feed-
ing preference (based on duration of feeding time
in seconds) is presented herein for lab reared
Aedes albopictus Skuse, Ae. aegypti (L.), Anophe-
les quadrimaculatus, Say (Type A) and wild Culex
nigripalpus Theobald. Information obtained
about mosquito responses to different wave-
lengths of light can be used to further exploit in-
sects' attraction to artificial light and enhance our
ability to conduct studies on population dynam-
ics, species specific surveys and/or improve reduc-
tion strategies.
MATERIALS AND METHODS
Visualometer and Data Collection
A pie-shaped olfactometer (Butler & Katz
1987; Marin et al. 1991; Wilson et al. 1991; Butler
& Okine 1995; Okine 1994) electronically quanti-
fies insect feeding activity on 10 compounds si-
multaneously for a set time period (Fig. la, b).
Ten holes drilled into the bottom of the aluminum
pie-shaped arena contained the tips of fiber optic
cables that emitted light upwards and illumi-
nated the artificial hosts from below (Fig. Ib, N).
The fiber optic tips were covered with recessed in-
terference filters (described below). As an addi-
tional attractant, CO2 (0.5 1/min) was released
through Tygon tubing (Norton Performance
Plastics Corp., Akron, OH), positioned directly be-
low each artificial host (Fig. 1, G) for measured
time intervals of 4 s "on" and 6 s "off". The visua-
lometer was located in a temperature-controlled,
light-proof, Faraday-cage room (Lindgren Enclo-
sures, Model No. 18-3/5-1). The apparatus (here-
after called a "visualometer") was modified to
compare 10 different light wavelengths that illu-
minated from below identical feeding stations.
Each feeding station was illuminated with unique
wavelengths (ca. 10 nm width) produced using fil-
tered broad spectrum white light. The mosquito
feeding time on the illuminated feeding stations
was recorded, logged, and analyzed using touch
and bite contact seconds created when the mos-
quito closed a circuit (Fig. 1, K and J).
Feeding Stations
The food source contained within the feeding
stations used in the visualometer, consisted of
fresh, citrated bovine blood mixed with agar and
various feeding stimulants/attractants. The food
mixture contained 1.66 g agar (U.S. Biochemical
Corp., Cleveland, OH), 33 ml fresh citrated bovine
blood; 100 ml deionized water, 7.14 mg sodium
chloride, 0.38 mg potassium chloride, 0.154 mg
calcium chloride dihydrate, 0.2 mg magnesium
chloride hexadydrate, 0.42 mg dibasic sodium
phosphate, 2.1 mg sodium bicarbonate, 0.92 mg
dextrose, and 0.184 mg glutathione disulfide (oxi-
dized glutathione), and was made to a final volume
of 133 ml and adjusted to a final pH of 7.4. The
blood/agar/feeding mixture was placed into the
"cup" on the underside of a 35-mm plastic film can-
ister lid where it was covered with a reinforced sil-
icone membrane (Butler et al. 1984) held in place
using a 4-mm retaining ring cut from the top of the
film canister. The feeding station was then in-
serted into 1 of the 10 holes cut into the transpar-
ent plexiglass visualometer lid. Between trials,
the visualometer was disassembled and washed.
Feeding stations were replaced for each replicate
and new mosquitoes were used for each trial.
Light Source and Filters
The light source was a wide spectrum tung-
sten-halogen bulb (Sylvania, no. DNF, Danvers,
MA) transmitted through fiber optic cables (RTS
Industries, Gainesville, FL) (Fig. Ib, N). Seven
VIS-NIR broadband (+ 5 nm) interference filters
(350, 400, 450, 500, 550, 600, 650, and 700 nm)
(Fig. Ib, L) with appropriate neutral density fil-
ters (Fig. Ib, M) to equalize intensities were used
for each wavelength (Oriel Instruments, Strat-
ford, CT). The "white" light (with neutral density
filter) from the fiber optic cable and no light were
used as controls.
Mosquito Species
Each trial used 150, 5-to-8d-old nulliparous,
non blood-fed females aspirated from cages con-
taining both male and females with sugar (1.0 M)
provided ad libitum.Aedes albopictus,Ae. aegypti,
An. quadrimaculatus, and Cx. nigripalpus were
the species evaluated. Each species was tested
separately. Laboratory colonies maintained at the
Center for Medical, Agricultural and Veterinary
Entomology (USDA, ARS), in Gainesville, FL pro-
vided recently colonized (1995)Ae. albopictus, and
specimens from a long-established colony of An.
quadrimaculatus (Type A). Aedes aegypti reared
as outlined in Gerberg (1970) were obtained from
an established University of Florida departmen-
tal colony. Wild Cx. nigripalpus were reared from
larva and pupae obtained from a sewage lagoon at
December 2005
Burkett & Butler: Colored Light as an Attractant for Mosquitoes
lb. Side View
From Light Source
Fig. 1. Visualometer (a) Top view (b) Side view. (A) 430 x 5-cm dia. transparent plexiglass lid (B) Artificial host,
(35-mm film canister lid) (C) 30-cm high plastic side piece with holes for tygon tubing (D) Aluminum base with holes
for fiber optic cables (E) Support leg (F) Plexiglass filter support (G) 10 cm diameter Tygon tubing for incoming
air/CO2 (H) Tubing for exhaust (I) Assembly screw (J and K) Probe inserted into top of artificial host and bottom
sensor fitted under artificial host (feeding mosquitoes complete circuit logged by computer), one sensor per artificial
host (L) Interference (bandbass) filter (M) Neutral density filter (N) Fiber optic cable (attached to light source).
Note, drawing not to scale.
Florida Entomologist 88(4)
the University of Florida Swine Research Unit.
All mosquitoes were reared and maintained at
25C, 95% RH and a 14:10 (L:D)H photoperiod. All
trials were run from 1600 to 0800 h.
Statistical Analysis
Touch/bite contact seconds were recorded for
16 h. All species trials were analyzed using the
first 4 h of feeding activity, with the exception of
Cx. nigripalpus, in which the last 4 h of feeding
times were analyzed. A 10 x 10 Latin square de-
sign (3-way ANOVA) was used forAn. quadrimac-
ulatus. For other species, a randomized complete
block (2-way ANOVA) design with 8 to 10 replica-
tions was used. Duncan's multiple range test was
used to delineate significant differences between
the colored light treatment means. Differences
between treatment means were considered signif-
icant at = 0.05. Data were log (X+1) transformed
prior to analysis.
RESULTS
With the exception of Cx nigripalpus, all spe-
cies showed a period of "orientation/ acclimation"
lasting ca. 10-15 min, after which mosquitoes
would begin aggressively probing and feeding on
the artificial hosts. Of these, Ae. albopictus was
the least aggressive and consequently had the
lowest over all feeding times on the different feed-
ing station/color combinations. The wild Cx. nigri-
palpus presumably still under circadian control
did not begin actively feeding until about 4 h into
the trial.
Aedes aegypti. Feeding duration (Fig. 2) results
for this species were not different for feeding
times (F = 1.48 df= 9, P = 0.17) over any of the col-
ors tested. Differences (day effect) for total sec-
onds of feeding (F = 2.06, df= 9, P = 0.04) were ob-
served for different replications.
Aedes albopictus. This species showed prefer-
ences (F = 2.59, df = 9, P = 0.03) for certain wave-
lengths of light (Fig. 2). Aedes albopictus fed
longer on yellow-orange (600 nm), blue-green
(500 nm), white, blue (450 nm), violet (400 nm),
and black compared to other colors tested. Aedes
albopictus had an overall mean ( SEM) feeding
time of 244 44.2 s which was significantly lower
than the feeding times (F = 9.74, P < 0.01) of the
other mosquito species. As with all other trials,
significant differences for total feeding durations
(F = 3.27, df = 9, P < 0.01) were observed for dif-
ferent replications.
Anopheles quadrimaculatus. Feeding duration
(Fig. 2) results for this species showed slight dif-
ferences for feeding times (F = 1.74, df = 9, P =
0.05) where white and black controls were the
greatest. All other individual wavelengths had
significantly longer feeding durations than 350
nm. No differences for total seconds of feeding
were observed for different replications (F = 1.87,
df = 9, P = 0.07) or positions (F = 0.67, df = 9, P >
0.73).
Culex nigripalpus. Due to lack of activity dur-
ing the first 4 h of the feeding trials, the last 4 h
(2000-2400) were analyzed and presented. Signif-
icant color preferences (F = 1.94, df = 9, P = 0.04)
were observed for this species (Fig. 2) were in de-
creasing order, blue-green (500 nm) orange (600
nm), blue (450 nm), white, red (650 nm) and yel-
low-green (550 nm) were preferred over the other
colors tested.
DISCUSSION
Considering the variation in attractiveness of
different mosquito species to light-baited traps
(Huffaker & Back 1943; Bidlingmayer 1967), it is
not unreasonable to expect that individual spe-
cies will vary in wavelength preference. Such
wavelength preferences (exhibited by behavioral
responses) may or may not correspond to spectral
sensitivities. For attraction to light-baited traps,
intensity is considered more important than color
(Barr et al. 1963). As such, many studies of color
light preferences in Diptera are criticized because
they fail to compensate for intensity (and/or hue)
and make interpretation of the results difficult
(Allan et al. 1987). These visualometer tests com-
pensated for variations in light intensity by incor-
porating neutral density filters at each wave-
length so that each treatment only varied by color
and an accurate assessment of "color" preference
could be obtained. Even so, different wavelengths
may be physiologically more stimulating and re-
sult in greater behavioral responses.
For mosquitoes, electroretinograph studies for
determining spectral sensitivities have been pub-
lished for onlyAe. aegypti (Muir et al. 1992; Snow
1971). These electroretinograph studies provide
evidence of bimodal sensitivities showing a small
peak at 350 nm and a large peak at 550 nm. This
bimodal pattern is similar to those found for ta-
banids (Smith 1986; Allan et al. 1991) and other
insects (White 1985), and is assumed, but never
tested, to be similar to the spectral sensitivities of
other mosquito species. Interestingly, spectral
sensitivity research has focused mainly on diur-
nal species that are not generally attracted to
standard light-baited traps. In our visualometer
trials Cx. nigripalpus is the only species com-
monly captured in broad spectrum light baited
traps (e.g., CDC style). Results of our trials
showed none of the mosquito species tested were
highly attracted to both 350 and 550 nm and over
the other wavelengths tested. Peak spectral sen-
sitivities of approximately 350 and 550 nm may
serve to allow discrimination in an environment
dominated by greens and blues (Lythgoe 1979),
but do not necessarily correspond to attractive
wavelengths.
December 2005
Burkett & Butler: Colored Light as an Attractant for Mosquitoes
3000
Ae. aegypti
2500
2000
1500 -- A
1000 T
A A
500 A A
0
700
300 AB AB AB
S200 AB -
5 100 A
B B B-- B
o 0
0
1600
1400, An. quadrimaculatus
1400
7 1200 -
8 00 AB A A
AAB
400 T- -
0 20
S 4000
o400- Cx. nigripalpus
3500
3000
2000 A
AB
1500 ABC
B B B
B C200'B -B -- -- BC
350 400 450 500 550 600 650 700 No Light White
Wavelength (nm)
Fig 2. Duration of feeding (seconds) during a 4 h exposure (means t SEM) for Ae. aegypti; Ae. albopictus; An.
quadrimaculatus, and Cx. nigripalpus on artificial hosts illuminated with different wavelengths of light. Means
within each species group with the same letter are not significantly different (a = 0.05, Duncan's Multiple Range Test).
wihnec pce ru ihtesm etraentsgifcnl ifrn a=00,Dna' utpeRneTs)
Florida Entomologist 88(4)
Aedes aegypti and Ae. albopictus are not cap-
tured frequently in mosquito traps baited prima-
rily with light (Service 1993). Because these spe-
cies are diurnal, reflected light appears to be
more important in resource location than trans-
mitted light. In general, most successful Ae. ae-
gypti/albopictus adult traps do not use light, but
rather rely on strategic placement and low reflec-
tive colors (Fay 1968; Freier & Francy 1991). The
relatively small numbers of Ae. aegypti / albopic-
tus captured in light traps indicates that trans-
mitted light is relatively unimportant in host/re-
source choice. Indeed, the duration of feeding
times for Ae. aegypti did not differ significantly
among wavelengths tested. Duration of feeding
times for Aedes albopictus were significantly
greater for 600 nm, 500 nm, broad spectrum
white, 450 nm and 400 nm. Field trials with light
emitting diodes or other sources of monochro-
matic light might result in similar attractive col-
ors under field conditions.
Although nocturnally active, An. quadrimacu-
latus is another species poorly collected by light-
baited traps (Bradley 1943). In our visualometer
trials, An. quadrimaculatus was attracted most
strongly by the contrasting "no light" and broad
spectrum white controls followed by 550 nm light.
Although ultraviolet lamps have long been known
to increase the numbers of host or resource seek-
ing mosquitoes captured at light traps (Headlee
1937; Weiss 1943; Williams et al. 1955; Breyev
1963), 350 nm was the least attractive wave-
length for An. quadrimaculatus and most of the
other species tested in our study.
If the duration of feeding is a measure of at-
tractiveness, then the feeding time results forAn.
quadrimaculatus differed slightly with those
found in two field experiments using colored light
emitting diodes which found no significant trap
count differences for An. quadrimaculatus (Bur-
kett et al. 1998). In either case, the color of light
does not appear to be important in the host/re-
source seeking behavior of An. quadrimaculatus
based on these studies.
With the latter being more effective, field trials
with CDC-type light traps baited with light and
those with light and CO2 are effective at collecting
Cx. nigripalpus (Nayar 1982). Field research us-
ing narrow wavelength LEDs (Burkett et al.
1998) also found this mosquito attracted to light
traps, and in one field trial, Cx. nigripalpus was
significantly attracted to green (567 nm) followed
by blue (450 nm) and white over the other colors
tested. This largely agrees with what was found
in the visualometer trials for this species where
this mosquito was more attracted to the green
when compared to the other colors. Given the
weakly significant results, and general lack of
supporting field data, light color is largely unim-
portant in host/resource acquisition for both Cx.
nigripalpus andAn. quadrimaculatus.
Future trials with the visualometer need to
concentrate on species known to be attracted to
artificial light. Information obtained about medi-
cally important mosquitoes can be used to further
improve current light-based trapping methods
and, ultimately, enhance studies on their popula-
tion dynamics, surveys of species, and to improve
reduction strategies.
ACKNOWLEDGMENTS
We thank Diana Simon, Tim Robson, Haze Brown,
and Tamara Landau for assistance with the trials and
maintaining the laboratory colonies. We also thank Dr.
Sandy Allan and Dr. Clay Smith for their comments
when reviewing this manuscript. This article is pub-
lished as Florida Agricultural Experiment Station Jour-
nal Series No. R-06284.
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WILLIAMS, C. B., R. A. FRENCH, AND M. M. HOSNI. 1955.
A second experiment on testing the relative efficiency
of insect traps. Bull. Entomol. Res. 46: 193-204.
WILTON, D. P., AND R. W. FAY. 1972. Responses of adult
Anopheles stephensi to light of various wavelengths.
J. Med. Entomol. 9: 301-304.
Florida Entomologist 88(4)
December 2005
STEMBORERS ASSOCIATED WITH SMOOTH CORDGRASS,
SPARTINA ALTERNIFLORA (POACEAE), IN A NURSERY HABITAT
W. H. WHITE1, D. ADAMSKI2, G. FINE3 AND E. P. RICHARD, JR.1
'USDA, ARS, Southern Regional Research Center, Sugarcane Research Unit, Houma, LA 70360
2USDA, ARS Systematic Entomology Laboratory, c/o National Museum of Natural History, Washington, DC
3USDA, NRCS Golden Meadow Plant Materials Center, Galliano, LA 70364
ABSTRACT
Extensive ecological studies have been conducted on insects inhabiting native stands of
smooth cordgrass, Spartina alterniflora Loisel; however, this is not the case for insects found
in smooth cordgrass in a nursery habitat. We investigated species composition and larval dis-
position among stemborers (Lepidoptera: Coleophoridae and Crambidae) infesting stems of
smooth cordgrass in nursery plots. One thousand and forty stems of smooth cordgrass were
randomly selected in 2003 and examined for presence of stemborer larvae. Height of larvae
on or within stem, height of stem from ground level to top-visible dewlap, and condition of the
leaf-whorl were documented. Stemborers representing six species of Lepidoptera were recov-
ered. These species were Blastobasis graminea Adamski (Lepidoptera: Coleophoridae); a
four-species complex comprised of Diatraea saccharalis (F.), Chilo demotellus Walker, Chilo
plejadellus Zincken, and Thaumatopsis probably actuella Barnes & McDunnough (Lepi-
doptera: Crambidae); and Donacaula probably unipunctella Robinson (Lepidoptera: Cram-
bidae). Larvae were generally on different parts of the stem and, with the possible exception
of larvae of the four-species complex, seemed unlikely to contact one another. Blastobasis
graminea was the most abundant species collected (n = 128; 52%) and was located at the base
of the stem (eq x eqx- = 6.7 5.4 cm). The four-species complex was the next most abundant
(n = 85; 35%) and was always found within the stalk about mid-way up the stem (eq x = 16.7
8.6 cm). Donacaula sp. was the least abundant species (n = 32; 13%) and was always found
in the tight leaf-whorl just above the stem meristem (eq x = 23.6 15.4 cm). A total of 544
(52%) of the stems sampled had a dead leaf-whorl, but only 140 (26%) were infested. Stem-
borer species did not significantly affect the number of plants with and without deadhearts.
Key Words: Lepidoptera, Crambidae, Coleophoridae, pest introductions, sugarcane
RESUME
Estudios ecol6gicos extensivos han sido realizados sobre los insects que habitan los campos
natives de Spartina alterniflora Loisel; sin embargo, este no es el caso para los insects en-
contrados en S. alterniflora el los viveros. Nosotros investigamos la composici6n de species
como la disposici6n de larvas de los barrenadores (Lepidoptera: Coleophoridae y Crambidae)
que infestan las parceles de S. alterniflora en los viveros. Mil y cuarenta tallos de S. alterni-
flora fueron seleccionados al azar en 2003 y examinados para la presencia de larvas de bar-
renadores. La altura de la posici6n de las larvas encima o adentro el tallo, la altura desde el
nivel de tierra hasta al nivel la parte apical de la plant donde la hoja empieza doblarse, y la
condici6n de espiral de la hoja fueron documentados. Barrenadores representando seis espe-
cies de Lepidoptera fueron recolectados. Estas species son: Blastobasis graminea Adamski
(Lepidoptera: Coleophoridae); un complejo de cuatro species compuesto de [Diatraea saccha-
ralis (F.), Chilo demotellus Walker, Chilo plejadellus Zincken, y Thaumatopsis probablemente
actuella Barnes & McDunnough (Lepidoptera: Crambidae)]; y Donacaula probablemente un-
ipunctella Robinson (Lepidoptera: Crambidae). Las larvas generalmente fueron encontradas
sobre distintos parties de los tallos y con la possible excepci6n de larvas del complejo de cuatro
species, parece poco possible el tener contact entire ellas. Blastobasis graminea fue la especie
mas abundantemente recolectada (n = 128; 52%) y fue encontrada en la base del tallo (x = 6.7
5.4 cm). El complejo de cuatro species fue la siguiente categoria mas abundante (n = 85;
35%) y siempre fue encontrada dentro del tallo aproximadamente en la mitad de la altura del
tallo (x = 16.7 8.6 cm). Donacaula sp. fue la especie menos abundante (n = 32; 13%) y fue
encontrada en la espiral apretada de hojas un poco mas arriba del meristema del tallo (x =
23.6 15.4 cm). Un total de 544 (52%) de los tallos muestreados tenian una espiral de hojas
muertas, pero solamente 140 (26%) de ellos fueron infestados. Las species de barrenadores
no afectaron significativamente el numero de las plants con y sin corazones muertos.
White et al.: Stemborers Associated with Smooth Cordgrass
Smooth Cordgrass, Spartina alterniflora
Loisel, dominates the vegetation of many Atlan-
tic, Gulf, and Pacific intertidal marshes, often oc-
curring in pure stands. In Louisiana, smooth
cordgrass vegetation increases in early spring to
a peak in September that corresponds with flow-
ering. After flowering there is rapid death of the
aboveground portions of the plants to a minimum
standing crop in December, although new
sprouts are present even in December and Janu-
ary (Kirby & Gosselink 1976). For an in depth
discussion and review of the habitats and growth
form of cordgrass in the U.S., see Denno et al.
(1996).
Smooth cordgrass is a sustainable and renew-
able restoration resource that provides shoreline
protection and a foundation for near-shore build-
ing (Materne 2000). Artificial plantings of smooth
cordgrass for restoration and stabilization
projects have become widespread throughout
coastal Louisiana and, as a result, a nursery in-
dustry is developing to meet the increased need
for smooth cordgrass plant material. To assist
this developing industry, research is underway to
establish optimum procedures for propagating
plants in nursery habitats. Research thus far has
focused on agronomic and pathological aspects of
seedling propagation (Harrison et al. 2001). We
are unaware of research conducted on the impact
of insect feeding on smooth cordgrass in nursery
situations. However, planthoppers in the genus
Prokelisia are the focus of extensive ecological
studies in smooth cordgrass in natural habitats.
In mid Atlantic coast salt marshes, Prokelisia
comprise 95-99% of all herbivores samples on
smooth cordgrass (Denno et al. 1987).
In 2002, while conducting a survey in Texas
and Louisiana for the sugarcane pest, Blasto-
basis graminea Adamski (Lepidoptera: Coleo-
phoridae) (White et al. 2005), we discovered
B. graminea in smooth cordgrass nursery plots
at the USDA, NRCS Golden Meadow Plant
Materials Center, Galliano, LA. Coincidentally,
we found a second stemborer associated with
cordgrass plants in the same plots. In a follow-
up survey in 2003, we found what appeared to
be a third species of stemborer also infesting
stems of cordgrass in these plots. After numer-
ous plant dissections, it became apparent that
these stemborers were utilizing different areas
of the plant. We therefore conducted a survey of
the nursery to validate these observations and
to survey for additional stemborers that may
have heretofore gone undetected or unreported
from smooth cordgrass. In particular, we
searched for those stemborers found in smooth
cordgrass that may be important pests of sugar-
cane, such as other species of Diatraea or the
Mexican rice borer, Eoreuma loftini (Dyar) (Lep-
idoptera: Crambidae). Presented here are the
results of that survey.
MATERIALS AND METHODS
Sixteen smooth-cordgrass plots were planted
at the USDA, NRCS Golden Meadow Plant Mate-
rials Center, Galliano, LA on 18 April 2001. The
plots were established from vegetative
propagules obtained from 120 smooth cordgrass
selections collected throughout coastal Louisiana
in 1999. Propagules were blended and planted
with a one-row mechanical transplanter set on
102-cm rows. Eight plots were established; each
plot was 6 m wide by 33 m long and with a 4-m
wide buffer between plots.
On 11 June 2003, 65 stems were randomly re-
moved from the middle three rows of a five-row
plot and bundled together (1040 stems total). All
stems were cut at the base of the stem at ground
level. Bundles were brought back to the labora-
tory and immediately placed in cold storage (9C)
to reduce the possibility of larvae redistributing
themselves on stems before examination. Bun-
dles were removed from cold storage one-by-one
and the stems were carefully examined externally
for the presence of larvae and then dissected. All
larvae found were removed from the plant and
their associated height from the base of the plant
was recorded. We also measured plant height
from ground level to top-visible dewlap and noted
if the leaf-whorl emanating from the stem mer-
istem was dead or not.
Data were analyzed by the PROC MIXED pro-
cedure (SAS 2001) as a randomized complete
block design with eight replications; stalks within
plots were considered as a subsample. Chi-square
comparisons were determined by the PROC
FREQ (SAS 2001).
All larvae collected were placed on a commer-
cial sugarcane borer diet (Southland Products
Lake Village, AR) and stored in a growth chamber
[30C, 0:24 (L:D)]. Identifications were based
upon moths sent to the USDA, ARS, Systematic
Entomology Laboratory, Washington D.C. for spe-
cies determinations. Vouchers of adult moths are
deposited at the USDA, ARS, Sugarcane Re-
search Unit, Houma, LA.
RESULTS
Of the stems sampled, 218 (20%) supported at
least one larva, whereas an additional 179 (17%)
exhibited evidence of prior insect feeding but no
larvae were recovered. A total of 245 stemborer
larvae were recovered, representing four species
and two genera (species undetermined) of Lepi-
doptera (Table 1). The sugarcane pest, B. graminea,
was the most abundant species recovered (Table
2). Collected larvae readily accepted the rearing
medium, and a large number of adults developed
from larvae placed on diet. Another 85 larvae were
recovered from within the stem and, as a group,
were the next most abundant and were considered
Florida Entomologist 88(4)
TABLE 1. STEMBORERS COLLECTED FROM SMOOTH CORDGRASS DURING SURVEYS IN 2003.
Species
Previously reported from Cordgrass
Coleophoridae: Blastobasinae
Blastobasis graminea Yes (White et al. 2005)
Stemborer complex
Crambidae: Crambinae
Diatraea saccharalis No
Chilo demotellus* Yes (Stiling & Strong 1983)
C. plejadellus* Yes (Neunzig, H. H. 1987)
Thaumatopsis sp.* No
Crambidae: Schoenobiinae
Donacaula sp. No
*Collected from cordgrass in 2003, but not as part of this study.
a complex in this study, as we were unable to ob-
tain identifications from larval specimens. A sin-
gle specimen of the sugarcane borer was the only
larva from the complex to complete development
and, consequently, the only species identified from
this particular survey. None of the other 85 speci-
mens collected completed development, even
when the diet was supplemented with 20 g (10% of
total dry mix) of freeze-dried cordgrass tissue.
However, we were able to retrieve from additional
smooth cordgrass collections made in 2003, simi-
lar larvae from the same position on the cordgrass
plant that did develop into adults. These speci-
mens were identified as: Chilo demotellus Walker,
Chilo plejadellus Zincken, and Thaumatopsis
probably actuella Barnes & McDunnough. Finally,
Donacaula probably unipunctella Robinson was
the least-abundant species and difficult to rear.
Larvae of this species fed on the diet but did not
pupate. Adding freeze-dried host tissue appeared
to elicit greater feeding by larvae of this species,
but did not increase pupation. Ultimately, smooth-
grass plants were transplanted to pots in the
greenhouse and infested with additional field-col-
lected larvae before a single adult specimen was fi-
nally obtained, and was the basis for species de-
termination for this report.
Stemborer larvae were stratified on the
cordgrass plant (Fig. 1). B. graminea was gener-
ally found on the lower portions of the plant and
more narrowly distributed in that area. The stem-
borer complex was located approximately midway
up the cordgrass stem. However, as the cordgrass
stem is hollow and therefore permits free move-
ment of the larva within the stem, these larvae
were more widely distributed in individual stems
than B. graminea. Donacaula sp. also appeared
widely distributed along the stem, but was always
found in the tight leaf-whorl above the apical
meristem. For the figure, we considered all stems
as single infestation even though two of the stems
we collected contained multiple individuals of the
four-species complex. Therefore, the number col-
lected for this group reported in the figure differs
from the number reported from the table.
A total of 544 (52%) of the stems sampled had
a dead stem meristem, as evidenced by a dead
leaf-roll, a condition referred to as a deadheart or
a whitehead. Presence of a deadheart was a poor
indicator of the presence of a stemborer. Of the
TABLE 2. SUMMARY OF STEMBORER SPECIES RECOVERED, ABUNDANCE, AND LOCATION ON SMOOTH CORDGRASS IN 2003.
Mean height on plant
Species Mean number per plot' Mean height on plant (cm)1 as % of plant height'
B. graminea 8 (128)2 A 6.7 A 16.0 A
Stemborer Complex 5 (85) B 16.7 B 42.4 B
Donacaula sp. 2 (32) C 23.6 C 66.3 C
LSD (P = 0.05) 2.8 4.9 8.5
Means in a column followed by the same letter are not significantly different (0.05) based on the LSD test, PROC MIXED (SAS
2001); df = 2; Pr > F = 0.0004, <0.001, <0.001 for mean number per plot, mean height on plant, and mean height on plant as % of
plant height, respectively.
Numbers in parentheses are the total number of larvae collected.
December 2005
White et al.: Stemborers Associated with Smooth Cordgrass
-2D -10 0 10 20 30 40 50 60 70 80 90 100
Percent of Plont Height
Fig. 1. Distribution of stemborers on smooth cordgrass plants with location on plant expressed as a percentage
of plant height measured at the top visible dewlap.
544 stems collected with a deadheart, only 140
(26%) were actually infested. Stemborer species
did not significantly affect the number of plants
with and without deadhearts as determined by
chi-square test (P > 0.05).
DISCUSSION
We were unable to obtain adult specimens of
several of the stemborers collected. Consequently
we were unable to obtain definitive identifications
for all specimens collected. This deficiency plus a
relatively small sample size, prevents us from
commenting to any great extent on the degree of
interspecific or intraspecific competition that may
have existed among the various moth taxa col-
lected in our survey. With the exception of the
four-species complex, the various species were
generally found on different parts of the stem, al-
beit with some degree of overlap.
Rarely was more than a single individual
stemborer found on the same plant. We observed
B. graminea, the four-species complex, and an un-
identified curculionid sharing the same plant, al-
though not in large numbers. This was not the
case with Donacaula sp., as only once was it found
on a plant occupied by another stemborer and
never with another Donacaula. At least one spe-
cies of the complex apparently deposited eggs in
masses as we observed two plants each contain-
ing multiple larvae. One plant contained 20 indi-
viduals and the other six. We could not determine
if the larvae of this species were cannibalistic or if
the larvae disperse to other plants as their devel-
opment progresses.
Rathcke (1976) suggests that hollow stems
may increase chances of larval encounters.
Smooth cordgrass stems, with the exception of
very small stems, are hollow. Thus, interspecific
and intraspecific competition may have occurred
among the four-species complex. Stiling & Strong
(1983) report interspecific competition between
C. demotellus and Languria taedata LeConte
(Coleoptera: Languriidae), two species that share
a similar niche on the smooth cordgrass plant. We
also found specimens of an unidentified languriid
in our survey, but only a few individuals were
found and these were not found in the presence of
another stemborer.
It is not certain how important the stemborer
species reported in this study are in reducing
plant stands or causing failure of the plant to
flower, as we took no measures of damage other
than deadhearts. In native stands, none of the
larvae encountered in our surveys were ever
found in as high numbers as what we found in our
nursery situation. It will be important to validate
the species identity of those stemborers reported
in our survey and to learn more of their biology in
order to develop effective control tactics, should
they be needed. With the discovery ofB. graminea
for the first time in U.S. in smooth cordgrass in
2002, it will also be important to closely monitor
the movement of nursery material (both inter-
and intra-state), as it may serve as the host of in-
troduction of important insect pests of sugarcane
and other gramineous crops.
ACKNOWLEDGMENTS
We thank Garris Thomisee, Dawn Bagala, and Cher-
okee Trosclair of the USDA, NRCS Plant Materials Cen-
ter, Golden Meadow, LA, and Randy Richard, Chris
Finger, and Brain Duet of the USDA, ARS, Sugarcane
Research Unit, Houma, LA, for technical assistance.
Dr. M. Alma Solis, USDA, ARS, Systematic Entomology
Laboratory, provided identification of Crambidae. We
also thank Ms. Debbie Boykin, Area Statistician,
USDA, ARS, Mid-South Area, Stoneville, MS, for assis-
tance in statistical analysis. Mention of trade names or
commercial products in this article is solely for the pur-
pose of providing specific information and does not im-
ply recommendation or endorsement by the U.S.
Department of Agriculture.
REFERENCES CITED
DENNO, R. F., M. E. SCHAUFF, S. W. WILSON, AND
KAREN OLMSTEAD. 1987. Practical diagnosis and nat-
ural history of two sibling salt marsh-inhabiting plan-
December 2005
thoppers in the genus Prokelisia (Homoptera:
Delphacidae). Proc. Entomol. Soc. Wash. 89: 687-700.
DENNO, R. F., G. K RODERICK, M. A PETERSON, A. F.
HUBERY, H. G. DOBEL, M. D. EUBANKS, J. E. LOSEY,
AND G. A. LANGELLOTTO. 1996. Habitat persistence
underlies intraspecific variation in the dispersal
strategies of planthoppers. Ecological Monographs
66: 389-408.
HARRISON, S. A., T. P. C., M. D. MATERNE, B. C.
VENUTO, G. A BREITENBECK, M. A. COHN, X. FANG,
A. RYAN, R. W. SCHNEIDER, R. A. SHADOW, P. SUBU-
DHI, AND H. UTOMO. 1987. 2001. Improving native
plants to protect and preserve Louisiana's coastal
marshes. Louisiana Agriculture. 44: 4-6.
KIRBY, C. J., AND J. G. GOSSELINK. 1976. Primary pro-
duction in a Louisiana Gulf Coast Spartina alterni-
flora marsh. Ecology 57: 1052-1059.
MATERNE, M. 2000. Plant Guide Spartina alternaflora
Smooth Cordgrass. LSU AgCenter Communications.
Baton Rouge, LA.
NEUNZIG, H. H. 1987. Pyralidae (Pyraloidea), pp. 462-
494 In F. W. Stehr [ed.], Immature Insects. V. 1. Ken-
dall/Hunt Publishing Company, Dubuque, IA. 754 pp.
RATHCKE, B. J. 1976. Competition and coexistence within
a guild of herbivorous insects. Ecology. 57: 76-88.
SAS. 2001. SAS System Release 8.2. SAS Institute, Inc.,
Cary, NC, USA.
STILING, P. D., AND D. R. STRONG. 1983. Weak competi-
tion among Spartina stem borers, by means of mur-
der. Ecology. 64: 770-778.
WHITE, W. H., D. ADAMSKI, J. BROWN, T. E. REAGAN,
J. A. VILLANUEVA-JIMENEZ, M. MENDEZ-LOPEZ, AND
M. O. WAY. 2005. Survey results for the sugarcane
pest, Blastobasis graminea (Lepidoptera: Coleo-
phoridae), in Texas and Louisiana in 2002. South-
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Florida Entomologist 88(4)
Bloem et al.: Insecticides for Control of Cactoblastis cactorum
LABORATORY EVALUATION OF INSECTICIDES FOR CONTROL OF THE
INVASIVE CACTOBLASTIS CACTORUM (LEPIDOPTERA: PYRALIDAE)
STEPHANIE BLOEM1, RUSSELL F. MIZELL III, KENNETH A. BLOEM3, STEPHEN D. HIGHT4 AND JAMES E. CARPENTER'
'Center for Biological Control, Florida A&M University, Tallahassee, FL 32308
2University of Florida, North Florida Research and Education Center, Quincy, FL 32351
3USDA-APHIS-PPQ-CPHST, at Center for Biological Control, Florida A&M University, Tallahassee, FL 32307
4USDA-ARS-CMAVE, at Center for Biological Control, Florida A&M University, Tallahassee, FL 32308
5USDA-ARS-CPMRU, Tifton, GA 31794
ABSTRACT
We conducted laboratory assays of nine products registered for use on ornamental plants in
Florida for their ovicidal and larvicidal activity against the invasive cactus moth Cactoblas-
tis cactorum. One-hundred percent mortality (or 0% survival) of 1-day-old eggs was obtained
when eggstick sections were treated with cypermethrin, spinosad, or imidacloprid. These
products were equally as effective when assayed against eggs that were fully embryonated
(28 days old), when cladodes of Opuntia stricta were exposed to neonates 24 hours after dip-
ping, or to cladodes that were dipped and stored for 30 days before exposure. When Bacillus
thuringiensis (Dipel) was used to prevent neonate penetration into treated cladodes of
0. stricta, 100% mortality (or 0% survival) was recorded in the laboratory.
Key Words: insecticides, invasives, Cactoblastis cactorum, Lepidoptera, Pyralidae, cyper-
methrin, emamectin benzoate, abamectin, spinosad, azadirachtin, fenoxycarb, imidacloprid,
acephate, Bacillus thuringiensis
RESUME
Nosotros realizamos unos ensayos del laboratorio de nueve products registrados para el uso
sobre plants ornamentales en Florida para su actividad ovicida y larvacida contra la polilla
invasora de cactus Cactoblastis cactorum. Una mortalidad de cien por ciento (o 0% sobrevi-
viencia) de huevos de 1 dia de edad fue obtenida en secciones de grupos de huevos tratados
con cypermethrin, spinosad, o imidacloprid. Estos products fueron igualmente efectivos en
ensayos contra huevos con embriones completemente desarrollados (de 28 dias de edad),
cuando los cladodios de nopal de Opuntia stricta fueron expuestas a neonatas (larvas recien
nacidas) 24 horas despu6s ser emergidos, o a los cladodios que fueron emergidos en insecti-
cida y almacenados por 30 dias antes de ser expuestos. Cuando Bacillus thuringiensis (Di-
pel) fue usado para prevenir la penetraci6n de las neonatas dentro los cladadios de 0. stricta
tratados, un mortalidad de 100% (o 0% sobrevivencia) fue registrada en el laboratorio.
Cactoblastis cactorum (Berg) successfully con-
trolled several species of invasive prickly pear
cacti (Cactaceae: OpuntioideaeOpuntia) in
Australia (Dodd 1940), South Africa (Pettey
1948), and in many other parts of the world (Mo-
ran & Zimmermann 1984). In 1989 C. cactorum
was detected in the Florida Keys (Habeck & Ben-
nett 1990; Dickel 1991). The cactus moth may
have arrived through natural dispersal from the
Caribbean Islands, where it was intentionally in-
troduced in the 1950s (Simmonds & Bennett
1990), or it may have been accidentally intro-
duced by the nursery trade (Pemberton 1995).
Nevertheless, its rapid spread along the Atlantic
and Gulf Coasts has raised concerns about its un-
avoidable impact on native Opuntia cacti in the
southern United States and in Mexico (Zimmer-
mann et al. 2000). Stiling (2002) suggested that
the geographical range of C. cactorum in Florida
was expanding at an approximate rate of 50-75
km per year. However, unpublished data collected
by our group suggests that the spread rate along
coastal locations in the Gulf of Mexico was closer
to 160 km per year during 2000-2003 (S. D. Hight,
unpublished data). Given this rapid rate of geo-
graphical expansion, C. cactorum could arrive in
Texas by the year 2007. Invasion and establish-
ment of the cactus moth in the southwestern
United States and in Mexico will have serious
detrimental effects on biodiversity and stability of
native desert ecosystems and on vegetable, fruit,
and forage Opuntia industries in these areas
Florida Entomologist 88(4)
(Sober6n et al. 2001; Zimmermann et al. 2000).
Even though C. cactorum was deliberately intro-
duced into South Africa to control invasive cacti,
spineless Opuntia is still used as fodder for cattle
and other livestock during times of drought. As
such, livestock farmers manage their Opuntia
plantations in order to minimize losses due to in-
sect damage.
The biology of C. cactorum is well documented
(Dodd 1940; Pettey 1948; Zimmermann et al.
2000). Mating occurs one hour before sunrise
(Hight et al. 2003) and eggs are laid to form spine-
like eggsticks, each with 60-100 eggs. Neonates
burrow collectively into cactus cladodes (pads or
stems) where larvae feed gregariously and move
to new pads as old ones are destroyed. Pupation
occurs in plant litter or soil. The moth completes
three full generations in Florida, with peak adult
flights taking place in April, July, and October
(Zimmermann et al. 2004).
Burger (1972) was the first to report on the use
of cover sprays of methidathion and carbaryl to
protect Opuntia plantations in South Africa
against attack by both C. cactorum and Dacty-
lopius opuntiae (Cockerell) (Homoptera: Dacty-
lopiidae). Subsequently, Pretorius et al. (1986) and
Pretorius & Van Ark (1992) assayed additional
products applied either as cover sprays or stem in-
jections to prevent cladode penetration by first in-
star C. cactorum. Pretorius et al. (1986) indicated
that cover sprays of cypermethrin gave excellent
results. However, they found that stem injections
of monocrotophos gave inadequate control and
were expensive and impractical to use against the
insect. Pretorius & Van Ark (1992) evaluated addi-
tional products (mevinphos and dimethoate) as
both stem injections and cover sprays and discov-
ered that these materials applied as sprays trans-
located effectively through the plants and provided
good protection against larval attack. According to
Nel et al. (2002), the insecticides currently regis-
tered for use against C. cactorum in South Africa
include a carbamate (carbaryl), an organophos-
phate (methidathion), and two pyrethroid insecti-
cides (deltamethrin and tralomethrin).
The current infestation of C. cactorum in Flor-
ida is affecting native Opuntia species distributed
throughout large expanses of natural areas
(0. stricta (Haworth) Haworth, 0. humifusa (Raf.)
Raffinesque, and 0. pusilla (Haworth) Nutall), as
well as ornamental cactus plants (0. ficus-indica
(L.) Miller and 0. stricta) in urban settings (Hight
et al. 2002). Even though chemical control is not a
practical or environmentally responsible tactic to
protect the millions of hectares of natural Opun-
tia vegetation (Mahr 2001), insecticide controls
should be evaluated for their potential use in ur-
ban settings. Leibee & Osborne (2001) summa-
rized information on new insecticides to be as-
sayed for use against immature stages of the cac-
tus moth. If proven effective, these products could
be employed in culturally managed plantings of
Opuntia (nurseries, backyards, landscaped public
lands) either alone or in combination with other
suppression tactics. Furthermore, insecticides
could be used to treat ornamental Opuntia in
nursery settings to ensure that no infested plants
are being sold to the public. In this paper we re-
port results of laboratory assays of several insec-
ticides that are registered for use on ornamental
plants in Florida. Ovicidal and larvicidal proper-
ties of the products were examined and results
obtained are discussed in context of the area-wide
management of this invasive insect.
MATERIALS AND METHODS
Test Insects
Eggsticks used in these experiments came
from a laboratory colony of C. cactorum main-
tained at the USDA-ARS Crop Protection and
Management Research Unit, Tifton, Tift Co., GA.
The insects are reared on cladodes ofO. stricta in-
side rectangular plastic boxes (25 by 17 by 8 cm)
that are held in environmental chambers at 26
1C, a 14:10 (L:D) photoperiod, and 70% RH dur-
ing larval and pupal development. Cocoons are
collected twice per week, de-silked in a dilute
bleach solution, and pupae are sorted by gender.
Groups of 30-50 newly emerged adults of each
gender are placed together in aluminum screen
cages (35 by 35 by 35 cm) containing 1-3 cladodes
of 0. stricta for mating and oviposition. Eggsticks
are collected from the cages once per day, placed
in small plastic cups (60 ml), and maintained at
26 1C, a 14:10 (L:D), and 70% RH until needed.
Under these conditions eggsticks take approxi-
mately 30 d to complete their development.
Products Assayed
Studies were conducted during 2004 at the UF/
IFAS North Florida Research and Education Cen-
ter (NFREC), Quincy, Gadsden Co., FL. Nine dif-
ferent commercially available products were
tested in the laboratory for their ovicidal and lar-
vicidal activity against C. cactorum. The products
were cypermethrin (Ammo 2.5 E, FMC Corpora-
tion, Philadelphia, PA), emamectin benzoate
(Proclaim 5 SG, Syngenta Crop Protection Inc.,
Greensboro, NC), abamectin (Avid 1.5 EC, Syn-
genta Crop Protection, Inc., Greensboro, NC), spi-
nosad (SpinTor 2 SC, DowAgro Sciences LLC,
Indianapolis, IN), azadirachtin (Azatin EC,
AgriDyne Technologies Inc., Salt Lake City, UT),
fenoxycarb (Distance IGR, Valent U.S.A. Corpo-
ration, Walnut Creek, CA), imidacloprid (Ad-
mire 2 F, Bayer Corporation Crop Protection,
Kansas City, MO), and acephate (Orthene 75
SP, Valent U.S.A. Corp., Walnut Creek, CA). In
addition, the bacterial insecticide Bacillus thur-
December 2005
Bloem et al.: Insecticides for Control of Cactoblastis cactorum
ingiensis Berliner (Dipel, Valent U.S.A. Corp.,
Walnut Creek, CA) was evaluated against neo-
nate larvae. Two dilution rates (1.0x and 0.5x)
were chosen for each product by averaging the
high and low recommended application rates for
each material. The average dilution rate was as-
signed 1.0x and the rate was halved for the 0.5x
rate. Only the 1.0x rate was used for B. thuring-
iensis. All products were mixed with de-ionized
water and used within 30 minutes of preparation.
Ovicidal Tests
Cactus moth eggsticks were transported to
NFREC where they were divided into sections
that contained a minimum of 10 eggs and ran-
domly assigned to treatments. Egg mortality was
assessed on both newly laid (1-d-old) as well as on
fully embryonated (28-d-old) egg sticks. For each
product and dilution rate, eggstick sections were
dipped in the treatment solution for 5 s, allowed
to air-dry and placed individually inside plastic
Petri dishes. Dishes were stored in the laboratory
at ambient conditions (25 + 2C, 13:11 (L:D), and
about 30% RH). Five replicates were completed
for each egg age (1-d-old or 28-d-old), insecticidal
product (cypermethrin, emamectin benzoate, ab-
amectin, spinosad, azadirachtin, fenoxycarb, imi-
dacloprid, acephate), and dilution rate (1.0x or
0.5x). Controls were dipped in de-ionized water
and handled as above. For each experiment, the
total number of eggs per eggstick section, the
number of eggs that failed to hatch, and the per-
cent mortality was noted per replicate.
Larvicidal Tests
Only full-size mature eggsticks (28 d old;
within 2 d of neonate emergence) were used in
these evaluations. Ninety fresh cladodes of
O. strict (13 cm in length by 10 cm width) were
collected in the field and brought back to the lab-
oratory where the basal joint was allowed to heal
before initiating the tests. Ten cladodes were
dipped for one min into each product at each dilu-
tion rate and allowed to air-dry. Five cladodes of
each group were chosen at random and used in
the first experiment. The remaining cladodes
were stored for 30 d in an outdoor shed at 23 +
2C, protected from direct sunlight and rain, and
used in the evaluation of residual effects. Decom-
position from environmental factors of assayed
products on stored cladodes was at a minimum.
For both experiments, dipped and air-dried cla-
dodes were placed in plastic containers (14 by 14
by 5.1 cm) with ventilated lids. Individual egg-
sticks were placed on sections (1 by 2 cm) of filter
paper (Whatman #2) on top of each cladode. Con-
tainers were held for 14 d in the laboratory under
ambient conditions (25 + 2C, 13:11 (L:D), and
about 30% RH) to allow neonates to emerge and
larvae to penetrate the cladode. Results of each
experiment were assessed after d 15 by counting
the total number of eggs per eggstick and number
of eggs that hatched per replicate. Using this in-
formation, each cladode was destructively sam-
pled to search for emerged larvae. Five replicates
of each product (cypermethrin, emamectin ben-
zoate, abamectin, spinosad, azadirachtin, fenoxy-
carb, imidacloprid, acephate, and B. thuringien-
sis) and dilution rate (1.0x or 0.5x) were com-
pleted for both newly dipped cladodes and cla-
dodes that were dipped and stored for 30 d.
Controls were dipped in de-ionized water and
handled as above.
Statistical Analysis
Data from each experiment (ovicidal tests on
1-d-old or 28-d-old eggs and larvicidal tests for
newly dipped cladodes and for cladodes that were
dipped and stored for 30 d) were analyzed by two-
factor analysis of variance (ANOVA) with product
and dilution rate as main effects. Interaction be-
tween product and dilution rate was included in
the model (PROC ANOVA) (SAS Institute 1989).
Dependent variables included percent mortality
and percent survival, as well as the corrected
mean percent mortality with the Schneider-Orelli
formula for mortality data from a uniform popu-
lation (Zar 1984). In addition, arcsine trans-
formed data for each dependent variable were in-
cluded in the statistical model to satisfy the as-
sumptions of ANOVA. Because no significant ef-
fect due to product dilution was detected and
because no significant interactions were revealed
during the analysis, data for both dilution rates
(1.0x or 0.5x) for each product were pooled for
each experiment and differences between means
were separated by the Waller-Duncan K-ratio t-
test (P 0.05). Likewise, all dependent variables
examined yielded similar results and all signifi-
cant differences in the multiple range tests were
the same. Consequently, only data on percent sur-
vival of C. cactorum in each of the four experi-
ments are presented.
RESULTS AND DISCUSSION
Leibee & Osborne (2001) suggested possible in-
secticides to screen against the cactus moth. These
insecticides are presently registered for use on or-
namental plants in Florida and labeled as effective
against Lepidoptera that bore into plant tissue
(Leibee & Osborne 2001). Six of the nine products
suggested by these authors were evaluated in our
experiments. The three additional products that
we tested were cypermethrin (a synthetic ester
pyrethroid) which is extremely effective against
C. cactorum in South Africa (Pretorius et al. 1986),
azadirachtin, a botanical insecticide derived from
the neem tree (Meliaceae-Azadirachta indica A.
Florida Entomologist 88(4)
Juss), and the bacterial pesticide B. thuringiensis
(tested only against neonates).
A summary of our laboratory results is shown
in Table 1. Survival of immature stages of C. cac-
torum varied between 64 to 85% when eggsticks
were treated with de-ionized water (control).
However, one hundred percent mortality (or 0%
survival) of 1-d-old eggs was obtained when egg-
stick sections were treated with cypermethrin,
spinosad, or imidacloprid. These products were
equally as effective (94 to 100% mortality) when
assayed against eggs that were fully embryonated
(28 d old), when cladodes of 0. stricta were ex-
posed to neonates 24 h after dipping, or to cla-
dodes that were dipped and stored for 30 d before
exposure. Cypermethrin has been reported to be
highly toxic to bees and aquatic insects (US EPA
1989). Pretorius et al. (1986) reported that cyper-
methrin had good activity against immature
C. cactorum in South Africa when applied as a
cover spray to spineless Opuntia. The results of
our laboratory assays agree with the data re-
ported by these authors. Spinosad is a macrocyclic
lactone insecticide reported to have wide margins
of safety for many beneficial insects and related
organisms (Schoonover & Larson 1995). Imidaclo-
prid is a nicotinoid insecticide that has minimal
environmental and safety concerns associated
with its use (Leibee & Osborne 2001). However, it
has been found to be acutely toxic to a variety of
predatory insects (Mizell & Sconyers 1992).
Emamactin benzoate is an avermectin insecti-
cide that exhibits low toxicity on beneficial insects
(Leibee & Osborne 2001). This product was effec-
tive at killing eggs and larvae of C. cactorum in
the laboratory, although some survival of neo-
nates was detected in three of four laboratory as-
says (Table 1). The second avermectin insecticide
that was assayed, abamectin, showed good activ-
ity against newly laid and fully embryonated eggs
of C. cactorum, as well as against neonates that
were challenged with newly dipped cladodes.
However, the product was ineffective after the
cladodes were stored for 30 d. When B. thurin-
giensis was used to prevent neonate penetration
into treated cladodes of 0. stricta, 100% mortality
(or 0% survival) was recorded in the laboratory.
When we evaluated the results of the assays with
B. thuringiensis, we found replicates where lar-
vae had been successful at creating an entry hole
into the cladode; however, no larvae survived to
cause damage beyond this small opening. Finally,
azadirachtin, fenoxycarb (a juvenile hormone
mimic) and acephate (an organophosphate) were
moderately to totally ineffective against imma-
ture stages of the cactus moth (Table 1). Lowered
effectiveness of some products, such as insect
growth regulators (IGRs), may partially be due to
feeding behavior of neonate larvae. Eggs hatch
synchronously and larvae enter the cladode as a
group through a single to few holes. Conse-
quently, few individuals feed on the surface of the
cladodes and ingest IGRs sprayed on the surface.
Habeck & Bennett (1990) suggested that wide-
spread use of pesticides was not recommended as a
method of control for cactus moth in the Florida
Keys because of the occurrence of rare and endan-
gered lepidoptera such as the Schaus swallowtail
Papilio aristodemus ponceanus Schaus, Florida
leaf-wing Anaea floridalis Johnson & Comstock
and Bartram's scrub-hairstreak S'r ...... acis
(Drury). We believe that similar concerns exist for
TABLE 1. EFFECT OF DIFFERENT INSECTICIDES ON PERCENT SURVIVAL OF CACTOBLASTIS CACTORUM TREATED AS EGGS
THAT WERE NEWLY LAID (1-D-OLD) OR READY TO HATCH (28-D-OLD) AND LARVICIDAL ACTIVITY OF THE PROD-
UCTS WHEN NEWLY EMERGED NEONATES WERE EXPOSED TO CLADODES OF OPUNTIA STRICTA THAT HAD BEEN
DIPPED AFTER 24 H OR DIPPED AND STORED FOR 30 D.
Mean ( SD) % Survival'
Ovicidal Tests Larvicidal Tests
Eggs Eggs 24 h post 30-d post
Product 1-d-old 28-d-old cladode treatment cladode treatment
Control (H20) 80 20.8 a 85 11.0 a 64 44.4 a 81 9.4 a
Cypermethrin Oc Oc Oc Oc
Emamectin Benzoate 5.8 7.4 c 0.6 1.9 c 0 c 7.9 25.0 c
Abamectin 4.3 9.1 c 3.3 8.4 c 0 c 85.6 8.5 a
Spinosad Oc Oc Oc Oc
Azadirachtin 52.7 + 35.1 b 85.5 11.5 a 54.6 30.2 ab 43.6 40.5 b
Fenoxycarb 8.6 27.1 c 40.3 35.4 b 64.0 35.2 a 73.9 13.4 a
Imidacloprid 0 c 0.6 1.9 c 0 c 3.6 10.2 c
Acephate 38.9 33.4 b 39.7 37.0 b 35.4 38.7 b 87.3 10.2 a
B. thuringiensis -0 c 0 c
Means within each column followed by the same letter are not significantly different, Waller-Duncan K-ratio t-test (P < 0.05).
December 2005
Bloem et al.: Insecticides for Control of Cactoblastis cactorum
all natural areas in Florida and elsewhere in the
United States where Opuntia are currently in-
fested, or are at risk of being infested, with C. cac-
torum. In these settings, the application of the
Sterile Insect Technique (Carpenter et al. 2001;
Hight et al. 2004) appears to be the only reason-
able management tactic. However, the use of insec-
ticides, together with the removal and destruction
of eggsticks, infested cladodes, or entire plants, to
protect Opuntia in nursery and backyard situa-
tions and as a tool to reduce cactus moth pest pres-
sure in urban situations is still recommended. Fur-
thermore, the protection of Opuntia plantations
destined for fruit or vegetable production in Mex-
ico cannot be overlooked as the insect steadily ex-
pands its geographical range to the West.
Our laboratory results suggest possible prod-
ucts that should undergo further evaluations in
the field, in particular, B. thuringiensis, spinosad,
and imidacloprid. However, we would anticipate a
much more rapid breakdown in the effectiveness
of B. thuringiensis in the environment due to in-
creased exposure to UV light and rain events. Be-
cause these products are already registered for
use on vegetables and ornamental plants in Flor-
ida, expanding their registration in other states is
highly recommended and could perhaps lead to
the eventual acceptance of these products for use
in fruit and vegetable plantations of Opuntia in
Mexico. Lastly, when formulations become avail-
able, field tests are recommended for isolates of
AcMNPV, a nuclear polyhedrosis virus isolated
from Autographa californica (Speyer) (Lepidop-
tera: Noctuidae). This islolate has been shown by
Vail et al. (1984) to be moderately effective against
immature stages of C. cactorum in the laboratory.
ACKNOWLEDGMENTS
We thank C. Riddle (UF/IFAS, NFREC, Quincy, FL),
S. Baez (USDA-ARS-CMAVE, Tallahassee, FL), and S.
Drawdy (USDA-ARS-CPMRU, Tifton GA) for technical
assistance, and R. Layton (University of Georgia, Tifton,
GA) for assistance with the statistical analysis of the
data. We also thank T. Jackson (USDA-ARS-CMAVE,
Tallahassee, FL), D. Mahr (University of Wisconsin),
and two anonymous reviewers for helpful comments of
this manuscript. Mention of trade names or commercial
products in this publication is solely for the purpose of
providing specific information and does not imply rec-
ommendation or endorsement by the U.S. Department
of Agriculture.
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Weathersbee & McKenzie: Neem Biopesticide on Asian Citrus Psyllid
EFFECT OF A NEEM BIOPESTICIDE ON REPELLENCY, MORTALITY,
OVIPOSITION, AND DEVELOPMENT OF DIAPHORINA CITRI
(HOMOPTERA: PSYLLIDAE)
A. A. WEATHERSBEE III AND C. L. MCKENZIE
USDA, ARS, U.S. Horticultural Research Laboratory, 2001 South Rock Road, Fort Pierce, FL 34945
ABSTRACT
The biological effects of a neem-based biopesticide, containing 4.5% azadirachtin, were as-
sessed against the Asian citrus psyllid, Diaphorina citri Kuwayama, a recently introduced
insect pest and potential disease vector of citrus in the United States. Over the concentra-
tion range 11-180 ppm azadirachtin, no mortality of adult psyllids was observed when ex-
posed to treated plants. Adult psyllids demonstrated a small but significant repellent effect
from treated plants in a choice experiment, but showed no preference to oviposit on treated
or untreated plants. Psyllid nymphs were susceptible to azadirachtin at very low concentra-
tions and activity perhaps was due to developmental inhibition. At a concentration of 22.5
ppm azadirachtin, ecdysis was not observed past 4 days after treatment and all nymphs
were dead within 7 days. The densities of psyllid nymphs on treated plants exposed to a
greenhouse population were significantly reduced by concentrations as low as 10 ppm aza-
dirachtin. Over the range of concentrations used in these experiments, the product caused
no phytotoxicity to tender foliage of either citrus or orange jasmine plants. Field trials are
warranted to determine suitability of neem-based biopesticides for inclusion in citrus inte-
grated pest management programs.
Key Words: Asian citrus psyllid, Diaphorina citri, citrus, neem, azadirachtin
RESUME
Los efectos biol6gicos de un bioplaguicida producido en base de neem, que contiene 4.5% de
azadirachtina, fueron evaluados contra el psilido asi-tico de los citricos, Diaphorina citri
Kuwayama, una plaga reci6n introducida y vector potential de enfermedades en citricos en
los Estados Unidos. Atrav6z de un rango de concentraci6n de 11-180 ppm de azadirachtina,
ninguna mortalidad en los adults de psilidos fue observada cuando fueron expuestos a plan-
tas tratadas. Los adults de psilidos demonstraron un pequeno pero significativo efecto re-
pelente en las plants tratadas en un experiment de selection, pero no mostraron ninguna
preferencia para ovipositar en plants tratadas o no tratadas. Las ninfas de los psilidos fue-
ron susceptibles a la azadirachtina en concentraciones muy bajas y la actividad posible-
mente fue debida a la inhibici6n para desarrollarse. En la concentraci6n de 22.5 ppm de
azadirachtina, no se observe la ecdisis despu6s de 4 dias del tratamiento y todas las ninfas
estuvieron muertas en un period de 7 dias. La densidad de las ninfas de psilidos sobre plan-
tas tratadas expuestas a una poblaci6n del invernadero fue reducida significativamente por
concentraciones tan bajas como 10 ppm de azadirachtina. Sobre todo el rango de concentra-
ciones usadas en estos experiments, el product no causo fitotoxicidad al follaje tierno de los
citricos o en plant Murraya paniculata. Es necesario hacer mas pruebas de campo para de-
terminar el uso de los bioplaguicidas con el base de neem para incluirlos en programs de
manejo integrado de plagas en citricos.
Interest in the use ofbiopesticides with selectiv-
ity towards phytophagous insects has increased in
recent years, particularly in cropping systems that
rely on natural enemies as a major component of
integrated pest management (Tengerdy & Szakacs
1998; Rausell et al. 2000). Use of these natural
compounds in place of conventional insecticides
can reduce environmental pollution, preserve non-
target organisms, and avert insecticide-induced
Mention of a trademark or proprietary product does not con-
stitute a guarantee or warranty of the product by the U.S. De-
partment of Agriculture and does not imply its approval to the
exclusion of other products that also may be suitable.
pest resurgence. The neem tree, Azadirachta in-
dica A. Juss., produces the biodegradable and in-
secticidal liminoid azadirachtin (Isman 1999). The
compound can be efficiently extracted from neem
seeds where its concentration is greatest (Butter-
worth & Morgan 1968; Schroeder & Nakanishi
1987). The insecticidal activity of azadirachtin has
been demonstrated against numerous insect pests
(Schmutterer & Singh 1995), and its various
modes of activity can include disruption of feeding,
reproduction, or development (Mordue (Luntz) et
al. 1998; Walter 1999). The fact that azadirachtin
is selective toward phytophagous insects with min-
imal toxicity to beneficial insects increases its
Florida Entomologist 88(4)
potential value to pest management (Lowery &
Isman 1995; Naumann & Isman 1996).
Pest management programs in citrus particu-
larly rely on natural enemies to assist in main-
taining destructive insect populations below eco-
nomic injury levels. When chemical intervention
is required to reduce pest populations, the use of
selective insecticides helps to conserve natural en-
emies. Tang et al. (2002) demonstrated that aza-
dirachtin was effective in controlling the brown
citrus aphid, Toxoptera citricida (Kirkaldy), an im-
portant vector of citrus tristeza virus. Effective
control of the aphid was achieved without harm-
ing its native parasitoid, Lysiphlebus testaceipes
(Cresson). Weathersbee & Tang (2002) indicated
that azadirachtin inhibited the development of
Diaprepes abbreviatus (L.) larvae, a root weevil
pest of citrus, disrupted reproduction in the
adults, and protected the roots of citrus seedlings
from feeding damage caused by larvae.
A more recently introduced exotic pest of citrus
is the Asian citrus psyllid, Diaphorina citri Ku-
wayama, that was detected in Florida in 1998
(Hoy & Nguyen 1998). It prefers feeding on the
phloem tissues of tender shoots and leaves where
it induces abnormal growth and shoot dieback.
Furthermore, it excretes copious honeydew that
promotes the growth of sooty mold, in turn reduc-
ing photosynthesis, plant growth, and yield
(Chien & Chu 1996; Tsai et al. 2002). The greatest
threat associated with D. citri to the citrus indus-
try is its capacity to vector Liberobacter asiaticum
(L.) Jack, the bacterium that causes citrus green-
ing disease. Citrus greening has been declared the
most devastating disease of citrus worldwide re-
sulting in unmarketable fruit, yield reductions,
tree dieback, and eventual death of infected trees
(McClean & Schwarz 1970; da Graca 1991).
Natural enemies, including the parasitoids
Tamarixia radiata (Waterston) and Diaphoren-
cyrtus aligarhensis (Shafee, Alam, and Agarwal),
have been imported and released to assist in bio-
logical control of D. citri (McFarland & Hoy 2001).
Selective biopesticides that aid in managing psyl-
lids without harming these natural enemies may
be needed. Since azadirachtin has been effective
in controlling other phloem-feeding and root-feed-
ing pests of citrus (Tang et al. 2002; Weathersbee
& Tang 2002), we investigated a commercial neem
seed extract containing 4.5% azadirachtin to de-
termine its biological activity against D. citri, spe-
cifically its potential effects on repellency, oviposi-
tion, and mortality of adult psyllids; and develop-
ment and mortality of psyllid nymphs.
MATERIALS AND METHODS
Insect Source and Rearing
Laboratory colonies ofD. citri were established
with specimens collected from dooryard grape-
fruit trees (Citrus paradisi Macf) in Fort Pierce,
FL, during March 2000. Colonies were main-
tained in screened cages (70 x 70 x 70 cm) on or-
namental orange jasmine plants, Murraya panic-
ulata (L.) Jack grown in 140-cm3 containers with
potting soil (Metromix 500, Scotts, Marysville,
OH). Plants were maintained in a greenhouse
and regularly pruned and fertilized to promote
production of new growth that is preferred for ovi-
position by adult females. The colony maintained
a sex ratio of about 1:1 which is similar to the
value of 0.5224 reported by Tsai & Liu (2000).
Biopesticide Source
Neemix 4.5 (4.5% azadirachtin) was obtained
from Certis USA, Columbia, MD. The product was
screened at concentrations ranging from 0 to 0.4%
vol:vol (i.e., 0 to 180 ppm azadirachtin, diluted
with distilled water) to evaluate a range of biolog-
ical effects on psyllid adults and nymphs includ-
ing potential repellency, toxicity, reproductive dis-
ruption, and developmental inhibition.
Adult Psyllid Choice Experiment
Pineapple sweet orange, Citrus sinensis (L.),
seedlings were grown to 1.5-2.5 cm tall in Root-
cubes Growing Media (Smithers-Oasis, Inc.,
OH, USA). The seedlings were arranged four per
container inside polycarbonate Magenta vessels
(77 x 77 x 97 mm) with vented polypropylene lids
(Sigma). Two each of the seedlings per container
were treated with azadirachtin at concentrations
of 11.3, 45, or 180 ppm and two each were un-
treated to facilitate choice in the experiment. The
aerial portion of each seedling was dipped for 5 s
in the appropriate azadirachtin suspension or
distilled water and then dried for 1 h before they
were randomly arranged in the vessels. The ves-
sels were labeled according to azadirachtin con-
centration and seedlings within each vessel were
labeled according to choice option (treated or un-
treated). Twenty adult psyllids were introduced
into each vessel and were allowed to settle on the
plants or inner surfaces of the enclosure. The ves-
sels were maintained in a growth chamber at 25 +
1C, 60-80% RH, and a photoperiod of 14:10 (L:D).
There were six replications for each azadirachtin
concentration. The numbers of psyllids that set-
tled on each of the seedlings per vessel were re-
corded after 4 and 8 h, and at 1, 2, 3, 4, and 7 d.
The total numbers of eggs deposited on each seed-
ling were recorded after 7 d.
Effect on Psyllid Nymph Development
Pineapple sweet orange seedlings were grown
and treated as described above at concentrations
of 0, 11.3, 22.5, 45, 90, or 180 ppm azadirachtin.
Eight 2nd instar psyllid nymphs were transferred
December 2005
Weathersbee & McKenzie: Neem Biopesticide on Asian Citrus Psyllid
to each seedling and the seedlings were placed
singly in Petri dishes (9 cm diameter x 3 cm deep)
and maintained in a growth chamber at 25 + 1C,
60-80% RH, and a photoperiod of 14:10 (L:D).
There were eight replications for each azadirach-
tin concentration. The numbers of live nymphs re-
maining and exuviae produced in each petri dish
were recorded after 1, 2, 3, 4, and 7 d.
Effect on Survival of Nymphs in Greenhouse
Orange jasmine seedlings were pruned to 10
cm tall and fertilized to force new growth. The
seedlings (120) were placed in a screened cage (70
x 70 x 70 cm) and maintained in a small green-
house. Approximately 400 adult psyllids were in-
troduced into the cage and psyllid populations
were allowed to develop for 7 d after which time
the seedlings had become infested with 50-150
nymphs per plant. Each seedling was randomly
assigned to one of four treatments (30 replica-
tions per treatment): water containing 0, 10, 30,
or 90 ppm azadirachtin. Plants were sprayed un-
til runoff with a hand-held sprayer, dried for 1 h,
and then returned to the cage. Half of the 30 rep-
lications (plants) for each treatment were exam-
ined after 5 d to determine the numbers of eggs,
dead nymphs, and live nymphs per plant. Data
were collected on the remaining 15 replications
after 7 d.
Data Analyses and Statistics
Data collected from the adult psyllid choice ex-
periment were subjected to PROC FREQ and dif-
ferences in the frequencies of adult settling and
oviposition were determined with Pearson's chi-
square test (SAS Institute 1999). Data from the
nymph development and greenhouse population
experiments were subjected to analysis of vari-
ance by PROC GLM and treatment differences
were determined by Tukey's studentized range
test or the least-squares means procedure (SAS
Institute 1999). Differences among means were
considered significant at a probability level of five
percent (P < 0.05).
Data from the adult psyllid choice experiment
were analyzed to determine the effects of treat-
ment concentration and choice (treated or un-
treated) on the numbers of adult psyllids and eggs
observed on each citrus seedling. Data from the
nymph development experiment were analyzed to
determine the effects of treatments on daily
nymph survival and cumulative production of ex-
uviae in each petri dish. Data from the green-
house population experiment were analyzed to
determine the effects of treatments on the num-
bers of eggs, live nymphs and dead nymphs found
on orange jasmine seedlings after 5- and 7 d. Sur-
vival rates for nymphs during the greenhouse
population experiment were calculated for each
treatment as the ratio of live nymphs to total
nymphs (live plus dead) per seedling. Reduction
rates for nymphs were calculated as 1 minus the
population survival rate. Linear regression was
used to fit a polynomial model to these data to de-
scribe the dose response (SAS Institute 1999).
RESULTS
Adult Psyllid Choice Experiment
Adult psyllids given a choice of citrus seedlings
either treated or untreated with azadirachtin
demonstrated a small but highly significant pref-
erence for settling on untreated seedlings (X2 =
31.40; df = 1; P < 0.0001), indicating there was re-
pellency due to treatments over the 7-d period of
the experiment. The cumulative percentages of
adult psyllids observed settling on treated and
untreated seedlings were 44.03 and 55.97, respec-
tively (Table 1). The frequency of settling within
each treatment did not change over time (X2 =
11.20; df = 6; P = 0.0825) indicating that the re-
pellent effect was consistent for 7 d. Adult settling
was not affected by the concentration of aza-
dirachtin (x2 = 0.59; df= 2;P = 0.7438), where the
cumulative percentages settled on the seedlings
were 32.82, 34.09, and 33.09 in the high, medium,
and low treatments, respectively (data not shown
but values can be calculated from Table 1). The
interaction of azadirachtin concentration and
TABLE 1. EFFECT OF AZADIRACHTIN ON THE FREQUENCY OF D. CITRI ADULTS SETTLING ON A CHOICE OF TREATED OR
UNTREATED CITRUS SEEDLINGS. COUNTS WERE MADE AFTER 4 AND 8 H, AND 1, 2, 3, 4, AND 7 D.
Frequency (%) of psyllids settling
Concentration
(ppm azadirachtin)a Treated Untreated X2
11.3 349(47.87) 380(52.13) 1.32
45 305(40.61) 446(59.39) 26.47***
180 316(43.71) 407(56.29) 11.45***
Cumulative 970 (44.03) 1233 (55.97) 31.40***
"Each treatment was replicated 6 times and comprised 20 adult psyllids given choices of 2 treated and 2 untreated citrus seedlings.
***P < 0.001; chi-squared test of settling frequency on treated versus untreated citrus seedlings, df = 1 [SAS Institute 1999]).
Florida Entomologist 88(4)
choice was significant (X2 = 7.96; df = 2; P =
0.0187), and further examination of the data indi-
cated that no significant repellency of adult psyl-
lids occurred at the lowest rate of azadirachtin
(Table 1).
Adult psyllids demonstrated no preference to
oviposit on treated or untreated citrus seedlings.
The cumulative percentages of eggs laid on
treated (49.50) and untreated (50.50) seedlings
were similar during the 7 d of the experiment (X2
= 0.09; df = 1; P = 0.7530) (Table 2). However, the
concentration of azadirachtin affected the num-
bers of eggs produced by adult psyllids in each
treatment (x2 = 136.83; df = 2; P < 0.0001). The
frequency of eggs produced in each treatment in-
creased from 200 to 500 as the concentration of
azadirachtin increased from 11.3 to 180 ppm, in-
dicating that treatments may have influenced
adult psyllid reproduction (Table 2).
Effect on Psyllid Nymph Development
Both the survival (F = 193.89; df = 5, 140; P <
0.0001) and molting (F = 1316.74; df = 5, 140; P <
0.0001) of psyllid nymphs maintained on citrus
seedlings were affected by azadirachtin concen-
tration. Across 7-sampling d, the mean numbers
of surviving nymphs and exuviae produced in
each treatment declined with increasing treat-
ment concentration (Fig. 1). The effect of time also
was significant for both the numbers of nymphs (F
= 863.61; df= 4, 140;P < 0.0001) and exuviae (F =
203.38; df = 4, 140; P < 0.0001) indicating that the
numbers of nymphs and exuviae in each treat-
ment changed during the 7 d of the experiment.
The interaction of azadirachtin concentration and
time also was significant for both nymphs (F =
18.26; df = 20, 140; P < 0.0001) and exuviae (F =
143.15; df = 20, 140; P < 0.0001), indicating that
the responses to concentration over time differed
among one or more levels of treatment.
Survival of nymphs was significantly (P < 0.05,
least-squares means) lower in all treatments
than in the control for counts taken beyond 1 d af-
ter treatment (Fig. 1A). By d 7 of the experiment,
no psyllid nymphs survived in any of the treat-
ments with azadirachtin concentrations >11.3
ppm while survival in the control was similar to
that reported by Tsai and Liu (2000) for psyllid
nymphs developing on citrus.
The cumulative production of exuviae was sig-
nificantly (P < 0.05, least-squares means) greater
in the control than in the other treatments for all
sample intervals (Fig. 1B). Over the 7 d of the ex-
periment, an average of 23 exuviae per treatment
were produced by nymphs in the control while
less than 5 were produced by those treated with
even the lowest concentration of azadirachtin.
The data indicate that azadirachtin may have
acted as a developmental inhibitor.
Effect on Survival of Nymphs in Greenhouse
After 5 d, the numbers of eggs found on orange
jasmine seedlings treated with azadirachtin at
concentrations of 0, 10, 30, and 90 ppm were not
significantly different (F = 2.01; df = 3, 56; P =
0.1233). The average number of eggs ranged from
9.2 to 24.9 among treatments; and although there
appeared to be a trend for reduced egg eclosion,
this could not be determined due to the variability
within treatments (Table 3). The numbers of dead
nymphs differed significantly (F = 4.63; df= 3, 56;
P = 0.0058) among treatments while the numbers
of live nymphs were only marginally different (F
= 2.74; df= 3, 56; P = 0.0521). Regression analysis
of the data indicated a significant relationship be-
tween azadirachtin concentration and population
reduction of nymphs 5 d after treatment (F =
118.27; df = 3, 57; P < 0.0001). The relationship
between nymph mortality and azadirachtin con-
centration was described by the cubic equation y
= 3.096x ( 0.514) 0.100x2 ( 0.024) + 0.001x3
( 0.000); r2 = 0.86 (Fig. 2A). The intercept of the
regression model was not significant; therefore,
the data were fitted to a model that assumed a
zero intercept. The predicted values given by the
model for percent population reductions in each
treatment corresponded well with observed val-
ues. The standard errors of the predicted means
did not exceed 3.0%. Though significant, the re-
gression model indicated that azadirachtin did
TABLE 2. EFFECT OF AZADIRACHTIN ON THE FREQUENCY OF EGGS OVIPOSITED BY D. CITRI S ON A CHOICE OF TREATED
OR UNTREATED CITRUS SEEDLINGS. COUNTS OF EGGS WERE MADE AFTER 7 D.
Frequency (%) of eggs oviposited
Concentration
(ppm azadirachtin)a Treated Untreated X2
11.3 100(50.00) 100(50.00) 0.00
45 170(54.84) 140(45.16) 2.90
180 230(46.00) 270(54.00) 3.20
Cumulative 500 (49.50) 510 (50.50) 0.09
"Each treatment was replicated 6 times and comprised 20 adult psyllids given choices of 2 treated and 2 untreated citrus seedlings.
***P < 0.001; chi-square test of oviposition frequency on treated versus untreated citrus seedlings, df = 1 [SAS Institute 1999]).
December 2005
Weathersbee & McKenzie: Neem Biopesticide on Asian Citrus Psyllid
C10
5
E
03
1 2 3 4 5
Days after treatment
Fig. 1. Survival (A) and development (B
nymphs on citrus seedlings treated with a
The number of nymphs and exuviae were c
2, 3, 4, and 7 days after treatment. 0 ppm =
= 0; 22.5 ppm = V; 45 ppm = V; 90 ppm =
ppm = D. Least-squares means for the
nymphs and cumulative exuviae in each tr
presented by day; SEs of least-squares
nymphs and cumulative exuviae were 0.24
respectively.
not effectively control psyllid populate
since the highest concentration produ
50% reduction in the treated population
After 7 d, the numbers of eggs fou
seedlings were not different (F = 0.69;
P = 0.5643) among treatments. The ave
ber of eggs per seedling ranged from
among treatments (Table 3). Both the
dead nymphs (F = 19.12; df = 3, 56; P
and live nymphs (F = 9.07; df = 3, 56;
were significantly influenced by treaty
numbers of live nymphs in all azadirac
ments were significantly (P < 0.05) le
the control 7 d after treatment. After
gression of azadirachtin concentration
tion of nymph populations was signify
149.76;df= 3, 56;P < 0.0001). Both the
and regression coefficient were greater
obtained for data collected after 5 d. Th
ship between percent reduction of ny
azadirachtin concentration was descri
cubic equation y = 6.811 (+ 3.128) + 9.609x (
0.699) 0.311 x2 ( 0.029) + 0.002x3 ( 0.000); r2 =
0.89 (Fig. 2B). The intercept was significant and
representative of the population reduction in the
control. The model effectively described reduc-
tions in nymph populations over the range of
tested concentrations. The standard errors of the
predicted means did not exceed 3.1%. Aza-
dirachtin concentrations from 10 to 90 ppm pro-
vided 74 to 92% reductions of psyllid nymph pop-
ulations after 7 d, indicating that the biopesticide
was effective at low concentrations. The delayed
mortality response may have been due to inhibi-
tion of nymph development by azadirachtin.
DISCUSSION
Our laboratory and greenhouse experiments
indicated that azadirachtin was effective in con-
trolling D. citri nymphs at concentrations as low
10 ppm. The compound apparently acted as a de-
velopmental inhibitor of psyllid nymphs, since
ecdysis was completely arrested within 4 days af-
ter treatment. A similar pattern of activity was ob-
served against brown citrus aphid nymphs (Tang
et al. 2002). While azadirachtin has been observed
6 7 to act as an antifeedent against some phloem-
feeding insects (Koul 1999; Koul et al. 1997), such
effects were not observed by Lowery & Isman
3) of D. citri (1994) against aphids, or here against D. citri. We
zadirachtin. observed that psyllid nymphs remained attached
mounted on 1, to treated plants by their mouthparts after death,
*; 11.3 ppm perhaps indicating that they attempted to con-
E; and 180 tinue feeding until death. It is possible that the
number of psyllid nymphs in our experiments ingested aza-
eatment are dirachtin while feeding on the phloem tissues of
means for
Sand +0.27, the treated plants. The systemic activity of aza-
dirachtin to other insects such as leafminers and
thrips has been demonstrated previously (Wein-
traub & Horowitz 1997; Thoeming et al. 2003). Di-
ions in 5 d aphorina citri is a serious pest to the citrus indus-
ced only a try, not only for the damage it causes citrus due to
In. feeding, but also because it is the most efficient
nd on the vector of the bacterium that causes citrus green-
df = 3, 56; ing disease (Tsai et al. 2002). If this disease is in-
erage num- produced to North America, its transmission by
3.5 to 8.3 the psyllid throughout citrus producing regions
numbers of potentially could devastate the industry. Novel in-
< 0.0001) tegrated pest management strategies might pre-
S< 0.0001) vent the psyllid from achieving this end. The use
ments. The of biopesticides that are compatible with biologi-
htin treat- cal controls currently in place are desirable in
ss than in crops such as citrus that often cover large acre-
7 d, the re- ages. Neem-based products, known for their selec-
on reduc- tivity, have been shown to effectively control other
ficant (F = phloem-feeding insects including several species
F-statistic ofAphididae (Lowery et al. 1993; Lowery & Isman
than those 1994; Schmutterer & Singh 1995; Koul et al. 1997;
ie relation- Koul 1998, 1999; Tang et al. 2002) and at least one
mphs and species of Psyllidae, the pistachio psyllid, Ago-
bed by the noscena targionii (Lisht.) (Lababidi 2002).
Florida Entomologist 88(4)
TABLE 3. EFFECT OF AZADIRACHTIN ON A GREENHOUSE POPULATION OF D. CITRI ON ORANGE JASMINE SEEDLINGS.
EGGS AND NYMPHS WERE ENUMERATED ON PLANTS AT 5 AND 7 D AFTER TREATMENT.
Mean no. per seedling (n = 15)
Concentration
(ppm azadirachtin) No. eggs No. live nymphs No. dead nymphs
5 d after treatment
0 9.2 + 5.4 a 77.1 +13.5 a 2.6 + 0.5 b
10 10.6 + 4.0 a 57.7 + 13.5 ab 16.5 + 4.8 ab
30 13.1 + 6.0 a 44.0 + 9.2 ab 13.1 + 2.7 ab
90 24.9 + 4.6 a 32.5 + 9.5 b 23.8 + 6.0 a
7 d after treatment
0 3.5 + 1.6 a 74.3 + 14.4 a 5.9 + 1.4 c
10 8.3 + 3.4 a 13.3 + 2.1 b 48.7 + 9.9 ab
30 8.1 +3.0 a 9.5 + 2.5 b 36.2 + 7.8 be
90 7.5 + 2.7 a 6.3 + 1.3 b 78.3 + 15.3 a
"For a particular sample day, means within a column sharing the same letter were not significantly different (P > 0.05, Tukey's
studentized range test [SAS Institute 1999]).
bSurvival rate = no. live nymphs/(no. live nymphs + no. dead nymphs).
Azadirachtin did not kill adult psyllids within
the range of concentrations (maximum 180 ppm)
0 10 20 30 40 50 60 70 80 90
Concentration (ppm)
Fig 2. Relationship between percent reduction of pop-
ulations ofD. citri nymphs after 5 d (A) and 7 d (B), and
concentration of azadirachtin sprayed on orange jas-
mine seedlings. A polynomial model was fitted to the
data to develop the prediction equation. Vertical lines
denote standard errors of the means and dashed lines
denote 95% confidence limits for the prediction equation.
or period of evaluation (maximum 7 d) used in
these experiments. Although some repellency of
adults was demonstrated statistically, the ob-
served amount was not particularly meaningful.
Reduced or no activity has been reported for
neem-based products against the adult stage of
other insect species (Lowery & Isman 1994; Koul
1999; Tang et al. 2002; Weathersbee & Tang
2002). Also, there was no preference for adult
psyllids to oviposit on treated or untreated plants;
interestingly however, more eggs were oviposited
as the concentration of azadirachtin increased
(Table 2). Viability of the eggs was not determined
in this experiment but reproductive effects such
as egg sterility have been reported for neem-
based products with other insect species (Ascher
1993; Weathersbee & Tang 2002).
Our results indicate that azadirachtin can be
applied at very low concentrations to effectively
manage developing D. citri populations. This bo-
tanical insecticide has been found to be relatively
nontoxic to beneficial insects (Lowery & Isman
1995; Naumann & Isman 1996; Walter 1999;
Tang et al. 2002) and should be safe to apply in
the presence of natural enemies found in citrus,
particularly at concentrations required to control
psyllid nymphs. Furthermore, no phytotoxicity
was observed to any of the plant tissues used in
these experiments. Neem biopesticides may
therefore be well-suited for inclusion in citrus in-
tegrated pest management programs. Since low
concentrations of neem-based products have been
demonstrated to be effective against D. citri and
other important citrus pests (Villanueva-Jimenez
et al. 2000; Tang et al. 2002; Weathersbee & Tang
2002), field trials are warranted to determine ap-
propriate methods of application and efficacy in
commercial settings.
December 2005
Weathersbee & McKenzie: Neem Biopesticide on Asian Citrus Psyllid
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DAVIES. 2000. Field evaluation of integrated pest man-
agement-compatible pesticides for the citrus leafminer
Phyllocnistis citrella (Lepidoptera: Gracilariidae) and
its parasitoid Ageniaspis citricola (Hymenoptera: En-
cyrtidae). J. Econ. Entomol. 93: 357-367.
WALTER, J. F. 1999. Commercial experience with neem
products, pp. 155-170 In F. R. Hall and J. J. Menn
[eds.], Biopesticides: Use and Delivery. Humana, To-
towa, NJ.
WEATHERSBEE, A. A. III, AND Y. Q. TANG. 2002. Effect of
neem seed extract on feeding, growth, survival, and
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Curculionidae). J. Econ. Entomol. 95: 661-667.
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effects of a neem insecticide on Liriomyza huido-
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Florida Entomologist 88(4)
December 2005
POTENTIAL PEST MITE SPECIES COLLECTED ON ORNAMENTAL PLANTS
FROM CENTRAL AMERICA AT PORT OF ENTRY TO THE UNITED STATES
CARL C. CHILDERS AND JOSE CARLOS V. RODRIGUES
University of Florida, Department of Entomology and Nematology, Citrus Research and Education Center
700 Experiment Station Road, Lake Alfred, FL 33850
ABSTRACT
Twenty-four plant shipments arriving via air cargo from Guatemala, Honduras, and Costa
Rica to Miami International airport in Florida, were sampled on February 6-7, 2003. Ran-
dom samples of rooted plants or cuttings were washed in 80% ethanol to collect the mite spe-
cies present. Altogether 81 mites in 11 families were identified in 12 plant shipments (i.e.,
50% of the 24 shipments sampled were found to contain mites). Plant mite pests included
Breuipalpus phoenicis (Geijskes), Fungitarsonemus sp., Polyphagotarsonemus latus (Banks),
Tarsonemus sp., Tarsonemus confusus Ewing, Tetranychus urticae Koch, Tetranychus sp.,
and Rhizoglyphus sp. (Acaridae). These mites, recovered from a variety of ornamental plant
genera, are potentially serious pests. Of special importance is B. phoenicis because it is a
known vector of citrus leprosis and several related viruses of ornamental plants that occur
in Central and South America. A dilemma exists because many of these diseases, including
citrus leprosis, do not occur in the United States, but potential vectors are already present.
Relevant needs include (a) a special sampling program for mites on live plant materials re-
ceived at ports of entry, (b) new legislation that requires imported plant propagules to be free
of pest species of mites, and (c) mandatory risk mitigation in nurseries abroad where ship-
ments originate and pre-clearance at the port of export.
Key Words: Acari, Acaridae, Tenuipalpidae, Tarsonemidae, Tetranychidae, virus-like dis-
eases, citrus leprosis
RESUME
Veinte cuatro cargamentos de plants llegandos del cargo por avion de Guatemala, Hondu-
ras, y Costa Rica al Aeropuerto Internacional de Miami en Florida, fueron muestreados en
el 6-7 de febrero de 2003. Muestras tomadas al azar de plants arraigadas o cortadas fueron
lavadas en 80% de etanol para recolectar los acaros presents. Un total de 81 acaros en 11
families fueron identificados en 12 cargamentos de plants. La mitad de los 24 cargamentos
muestreados de Guatemala, Honduras, y Costa Rica tenian acaros. Los acaros plaga de plan-
tas incluyeron: Brevipalpus phoenicis (Geijskes), Fungitarsonemus sp., Polyphagotarsone-
mus latus (Banks), Tarsonemus sp., Tarsonemus confusus Ewing, Tetranychus urticae Koch,
Tetranychus sp., y Rhizoglyphus sp. (Acaridae). Estos acaros, recuperados de una variedad
de g6neros de plants ornamentales, son plagas potencialmente series. De importancia es-
pecial es B. phoenicis por ser conocido como vector de leprosis de citricos y various virus rela-
cionados con plants ornamentales que ocurren en sudamerica y centroamerica. Muchas de
estas enfermedades, incluyendo la leprosis de los citricos, no ocurren en los Estados Unidos.
Un dilema existed para la agriculture de este pais porque el acaro vector ocurre dentro de los
Estado Unidos mientras que el leprosis de los citricos no ocurre en los Estados Unidos. Las
necesidades pertinentes incluyen (a) un program de muestreo especial para los acaros so-
bre material de plants vivas recibidas en los puertos de entrada, (b) nueva legislaci6n que
require que plants importadas para propagaci6n sean libres de species de acaros que son
plagas, y (c) mitigaci6n de riesgo mandatorio en los viveros fuera del pais donde se origen los
cargamentos y pre-aprobaci6n en el puerto de exportaci6n.
More than 205 non-indigenous species (NIS)
have been introduced or first detected within the
United States between 1980 and 1993, and 59 are
expected to cause economic or environmental
harm (Office of Technology Assessment 1993).
The establishment of exotic arthropods at least in
Florida has continued at an alarming rate (Tho-
mas 2000). The potential economic losses from
these introductions were predicted to be immense
and have been proven in Florida with the devas-
tation produced by only a few examples of exotic
introductions (e.g., the bacterium, Xanthomonas
axonopodis pv. citri, that causes citrus canker; the
citrus leafminer, Phyllocnistis citrella Stainton;
and the brown citrus aphid, Toxoptera citricida
(Kirkaldy)). These and other exotic pest introduc-
tions have resulted in significant economic losses
to the Florida citrus industry.
Since 1987, at least 14 exotic mites have been
discovered in Florida (Thomas 2000; W. C. Wel-
bourn, Florida Department of Agriculture and
Consumer Services, Division of Plant Industry,
Childers & Rodrigues: Exotic Pest Mite Introductions
unpublished). Table 1 lists the reported exotic
pest mite introductions into the United States
within the past three decades. Two of these (Var-
roa destructor Anderson & Trueman andAcarapis
woodi (Rennie)) have resulted in the near elimi-
nation of feral honey bees (Apis mellifera L.).
Also, significant economic losses have resulted in
reduced availability of managed bees that are es-
sential for pollinating many crops within the
United States (Robinson et al. 1989a, b; Samma-
taro et al. 2000).
We have entered an era of unprecedented lev-
els of travel and international commerce (Klassen
et al. 2002). With these increases has come corre-
sponding increases in the movement of pests once
considered exotic. Klassen et al. (2002) estimated
that there are 130 significant arthropod pests
present in the Caribbean. Therefore, Florida
ranks near the top of states at risk from pest in-
troductions due to its position relative to the Car-
ibbean, which is the only region of the globe with-
out an active regional safeguarding organization.
Dobbs and Brodel (2004) reported an overall in-
festation rate of 10.4% of hitchhiking insect spe-
cies in cargo aircraft arriving at the Miami Inter-
national airport between September 1998 and Au-
gust 1999. However, the rate of insect infested air-
craft arriving from Central America was 23%.
Other factors relating to exotic pest introduction
are involved, including the movement of substan-
tial amounts of live plants into Florida for use as
propagative materials in the ornamental industry.
Approximately 85% of all live plant materials
imported into the United States are ornamentals
grown in Central America and shipped into Florida
through the Miami International Airport (Frank &
McCoy 1995). During 2003, plants processed at the
Miami Inspection Station constituted 72% of the
total numbers of imported ornamentals into the
United States (USDA, APHIS, Miami Interception
Records 2004). Los Angeles processed 17% of all
U.S. ornamental shipments with the remaining
11% processed by all other U.S. ports combined.
The rate of growth for such shipments is estimated
to double every 6 years (Klassen et al. 2002). The
plants are shipped into Florida through the United
States Department of Agriculture Animal, Plant,
and Health Inspection Service, Plant Protection
and Quarantine (USDA-APHIS-PPQ), Plant In-
spection Station in Miami as cuttings or rooted
plants for propagation and distribution. Between
October 2003 and September 2004 more than 1 bil-
lion plants were shipped from Central America,
Mexico, South America, Asia, and Europe with 63%
coming from Central American countries (USDA
APHIS Miami Work Accomplishment Data System
2004). Miami received over 859 million of these
plant units. Propagative plant material is but one
TABLE 1. LIST OF NON-INDIGENOUS SPECIES OF MITES INTRODUCED INTO THE UNITED STATES SINCE 1980.
Mite superfamily
or family Mite species Scientific name Pathway or distribution
Prostigmata
Eriophyidae Aculops fuchsia Keifer on plants'
Acalitus ipomocarneae Keifer on plants3
Aceria litchii Keifer on plants3
Aceria zelkoviana Kim on plants'
Cecidophyopsis n. sp. on plants3
Tegolophus perseaflorae Keifer on plants3
Vittacus bougainvillea on plants3
Abou-Awad & El-Banhawy
Phytoptidae Acathrix trymatus Keifer on plants3
Tarsonemidae Honeybee tracheal mite Acarapis woodi (Rennie) honeybees'
Tenuipalpidae Flat scarlet mite Cenopalpus pulcher apples and pears in Benton
(Camestrini & Fanzago) and Linn Counties in Oregon2
Tetranychidae Oligonychus perseae on plants'
Tuttle, Baker & Abatiello
Oligonychus grypus sugarcane3
Baker & Prichard
Eutetranychus sp. on plants3
Mesostigmata
Laelapidae Melittiphis alveartus (Berlese) honeybees1
Varroidae Varroa mite Varroa destructor honeybees'3
Anderson & Trueman
'Office of Technology Assessment 1993
'Bajwa et al. 2001
'Thomas 2000
Florida Entomologist 88(4)
of several pathways for potential introduction of
single or multiple pest mites and the diseases they
may vector into the United States.
There are over 1,000 exotic pest mite species of
potential agricultural, sylvan, or ornamental im-
portance to the United States within the acarine
families: Acaridae, Eriophyidae, Diptilomiopidae,
Laelapidae, Nalepellidae, Penthaleidae, Siterop-
tidae, Tarsonemidae, Tenuipalpidae, Tetranychi-
dae, Tuckerellidae, and Varroidae (Jeppson et al.
1975; Lindquist 1986; Ochoa et al. 1994; C. C.
Childers et al., Citrus Research and Education
Center, University of Florida, unpublished). The
Acaridae, Eriophyidae, Tarsonemidae, Tenuipalp-
idae, Tetranychidae, and Tuckerellidae have sev-
eral exotic pests of potential importance within
the Caribbean and Central American countries.
Mite-vectored diseases such as citrus leprosis and
other related virus-like pathogens pose addi-
tional threats. These concerns prompted officials
from the USDA-APHIS-PPQ to support a prelim-
inary survey (i.e., the present study) to identify
pest mite species present on imported plants.
Citrus leprosis is a serious viral disease caused
by two different viruses (one nuclear and the
other cytoplasmic). These two viruses differ mor-
phologically yet result in similar disease symp-
toms in citrus (Rodrigues et al. 2003). Citrus spe-
cies, especially oranges (Citrus sinensis (L.) Os-
beck), are highly susceptible to infection by citrus
leprosis virus. Mandarins (C. reticulata Blanco,
C. reshni Hort. ex Tanaka, C. deliciosa Tenore)
and hybrids such as 'Murcott' are considered
much less susceptible under natural and experi-
mental conditions (Rodrigues 1995). Both viruses
can kill sweet orange trees if the Brevipalpus
mite vector is not controlled with acaricides.
Identification of citrus leprosis disease in af-
fected tissues is achieved with transmission elec-
tron microscopy (TEM), the only accurate method
confirming both viral infections at this time.
Therefore, identifications based solely on visual
symptoms should not be used. Molecular primers
are only available to detect the cytoplasmic virus
type (Locali et al. 2003).
Citrus leprosis is known to occur in South
America. The disease has been recently identified
in Panama (Dominguez et al. 2001), Costa Rica
(Araya-Gonzales 2000), Honduras (J. C. V. Rod-
rigues et al., Citrus Research and Education Cen-
ter, University of Florida, unpublished), and Co-
lombia (D. S. Achor et al., Citrus Research and Ed-
ucation Center, University of Florida, unpub-
lished). Citrus leprosis reportedly occurs in
Guatemala (Mejia et al. 2002). This places parts of
Mexican citrus production at risk and ultimately
United States citrus production. The disease is be-
lieved to have occurred in Florida and disappeared
sometime prior to 1962 (Childers et al. 2003a).
The purpose of this survey was to identify mite
species found within selected ornamental sam-
ples from Central America being shipped into the
United States. We were especially interested in
possible contamination of one or more shipments
with B. phoenicis (Geijskes) or other species
within the genus Brevipalpus because of their in-
volvement as vectors of several virus-like dis-
eases of citrus and ornamental plants. Brevipal-
pus phoenicis occurs within the United States.
However, many of the diseases this genus vectors
are not present in the U.S. and pose considerable
risk to both the citrus and ornamental industries
within the United States.
MATERIALS AND METHODS
Twenty-four plant shipments arriving by air
cargo to Miami, Florida International Airport
were selected at random from a list of 486 known
host plants of B. phoenicis on February 6-7, 2003
(Childers et al. 2003c). The samples included Hed-
era spp., Scindapsus sp., Peperomia sp., Codiaeum
spp., including C. variegatum (L.) Blume, Dra-
caena spp., including D. godseffiana Hort., Schef-
flera arboricola (Hyata) Merrill, Cordyline termi-
nalis Kuth, and various species of Orchidaceae.
The 24 shipments sampled represented a min-
imum of 1,200 to 2,400 plants from the 1.6 million
shipped from Costa Rica, 3.5 million from Guate-
mala and the 71,210 plants from Honduras that
arrived on February 6-7, 2003 to Miami (USDA
APHIS Miami Work Accomplishment Data Sys-
tem 2004). These dates were within the peak sea-
son for live plant imports that occurs during Jan-
uary and February from these Central American
countries. Selection of February 6 and 7 as sam-
pling dates was done based on our availability
during that time.
Propagated plants were usually shipped in
cardboard boxes in groups (usually 10-1,000
plants of the same species depending upon indi-
vidual plant size) and wrapped in newspapers.
Each box can vary considerably in size. Many
boxes that we sampled were 60 to 90 cm long x 45
to 90 cm wide x 45 to 90 cm deep. The number of
plants per shipment varies greatly and depends
on the grower and plant material being shipped.
Shipments are very often larger than 12 boxes
from one exporter and may include more than one
plant type. Each box that we sampled contained
variable numbers of bundled plant materials usu-
ally of the same genus.
One bundle of rooted plants or cuttings per
plant type per shipment was selected, washed and
vigorously agitated in a bucket containing about
one liter of 80% ethanol. The plant materials were
then discarded and the alcohol wash was saved
with the label information for that ornamental
plant, grower, and country of origin. Each alcohol
sample was then poured into a black Petri dish di-
vided into a multiple 1-cm2 grid and examined with
a stereomicroscope (10-50x magnification) for the
December 2005
Childers & Rodrigues: Exotic Pest Mite Introductions
presence of mites. Ornamental plant species sam-
pled that were not infested with mites were not re-
corded. All mites from each sample were slide-
mounted in Hoyer's mounting medium (Krantz
1978). The slides were oven-cured at 43-45C for 2
weeks and then the preserved specimens were
identified. Species within the mite families Tenui-
palpidae, Tarsonemidae, Histiostomatidae, and
Acaridae were identified by Dr. R. Ochoa. Species
within the families Tetranychidae and Tydeidae
were identified by Dr. W. C. Welbourn.
RESULTS
Rica, Guatemala, and Honduras. A total of
81 mites in 11 families were identified in
12 ornamental plant shipments from 9 growers in
Guatemala, Honduras, and Costa Rica even with
this modest sample size (Table 2). Of the 24 plant
shipments examined, half contained at least one
mite. One sample of Dracaena godseffiana and
one of Codiaeum sp., both from Guatemala, con-
tained one or more B. phoenicis. Other potential
plant pest mites found were in the families Tar-
sonemidae, Tetranychidae, and Acaridae.
DISCUSSIoN
A very low number of plants were sampled for Small arthropods including mites desiccate rap-
pest mites on February 6-7 compared with the 5.2 idly when they die because of their large surface
million plants shipped into Miami from Costa area to volume ratios. Generally, mite body parts
TABLE 2. LIST OF ACARI COLLECTED FROM LIVE ORNAMENTAL PLANT SHIPMENTS SAMPLED FROM COSTA RICA, GUATE-
MALA, AND HONDURAS DURING FEBRUARY 6 AND 7, 2003 AT THE USDA APHIS-PPQ MIAMI PLANT INSPEC-
TION STATION.
Country
of origin Shipper Ornamental plant infested Acarine species collected
Guatemala Grower 1 Dracaena godseffiana Hort.
Guatemala Grower 1 Dracaena godseffiana Hort.
Guatemala Grower 2 Codiaeum variegatum (L.) Blume
Guatemala Grower 3 Peperomia sp.
Guatemala Grower 3 Scindapsus sp.
(Hawaiian Pothus)
Guatemala Grower 3 Scindapsus sp.
(Marble Jade)
Guatemala Grower 4 Schefflera sp.
Grower 5
Grower 6
Grower 7
Schefflera sp.
Mastasde [?]
Codiaeum sp. (Sunny Star)
Costa Rica Grower 8 Schefflera arboricola (Hayata) Merrill
Costa Rica Grower 9 Cordyline terminalis Kuth (Calypsa)
Brevipalpus phoenicis (Geijskes)'
(Tenuipalpidae)-1 specimen; Brevipalpus
sp.'(Tenuipalpidae)-1 specimen
Eupodidae-1 specimen; Lorryia formosa Coore-
man (Tydeidae)-1 specimen
Brevipalpus phoenicis1 (Tenuipalpidae)-2 spec-
imens;Fungitarsonemus sp.2 (Tarsonemidae)-2
specimens; Lorryia formosa (Tydeidae)-1 spec-
imen; Tydeidae (larva)-1 specimen
Polyphagotarsonemus latus (Banks)2 (Tarsone-
midae)-3 specimens
Tarsonemus sp.2 (Tarsonemidae)-1 specimen;
Tarsonemus confusus Ewing (Tarsonemidae)-1
specimen; Tarsonemus n. sp.2 (Tarsonemidae)-1
specimen
Tarsonemus confusus2 (Tarsonemidae)-1 speci-
men; Parapronematus sp. (Tydeidae)-2 speci-
mens; Tydeidae (larva)-1 specimen
Camerobiidae-1 specimen (probably predacious);
Orbatidae-1 specimen; Lorryia formosa (Ty-
deidae)-1 specimen
Metapronematus sp. (Tydeidae)-1 specimen
Histiostomatidae-1 specimen
Rhizoglyphus sp.2 (Acaridae)-1 specimen; Tet-
ranychus sp.2 (Tetranychidae) -14 specimens
Phytoseiidae-1 specimen (predacious mite)
Bdellidae-1 specimen (predacious mite);
Tetranychus urticae Koch3 (Tetranychidae)-6
specimens; (Tetranychidae)-4 specimens-
probably Tetranychus urticae3; Tetranychus sp.4
(Tetranychidae)-29 specimens
Plant pests and potential vectors of citrus leprosis and related ornamental viruses.
'Plant pests.
Plant pest occurring in USA.
Plant pests-only females and immatures present and males required to verify species.
Guatemala
Honduras
Costa Rica
Florida Entomologist 88(4)
are rapidly fragmented after death or whole mites
become misshapen, dried out, and lose their body
color. This is especially true for soft-bodied phy-
tophagous mite species in the families Tetrany-
chidae and Tenuipalpidae. In contrast, live, healthy
mites retain their body shape, size, and color.
Therefore, recognition of live versus dead mites is
commonly employed by use of an ethanol wash
method to extract most mites from a plant sample.
An extensive array of related Brevipalpus-
borne diseases has been identified in numerous
ornamental plants including Dracaena marginata
Lam., Hibiscus, Hedera canariensis Willdenow,
Pittosporum tobira (Thunberg), Trachelospermum
asiaticum Nakay, Brunsfelsia uniflora D. Don,
Cestrum, Malvaviscus, Pelargonium hortorum L.
H. Bailey, Thunbergia erecta T. Anders, Salvia,
Clerodendrum, and many genera of the Orchi-
daceae (Kitajima et al. 2003; Kondo et al. 2003;
Nogueira et al. 2003, 2004; and E. W. Kitajima et
al., Departmento de Entomologia, Fitopatologia e
Zoologia Agricola, Universidade de Sao Paulo,
Brazil, unpublished). Both cytoplasmic and nu-
clear types of viruses are represented and many of
these ornamental plant diseases are not known to
occur within the United States. All are vectored by
one or more species of Brevipalpus mites and the
diseases must be considered as potential threats
to the US ornamental industry. In addition, we do
not know enough about citrus leprosis to exclude
the possibility of one or more ornamental or non-
citrus hosts being silent carriers of one or both vi-
ruses currently identified as citrus leprosis (Rod-
rigues et al. 2003). Recently, Rodrigues et al.
(2005) successfully transmitted citrus leprosis (cy-
toplasmic type) from Citrus sinensis to Solanum
violaefolium Shott (Solanaceae) using viruliferous
mites (B. phoenicis). However, they were not suc-
cessful in transmitting the virus back from S. vio-
laefolium to citrus but were able to transmit the
virus between plants of S. violaefolium.
There are several reasons for concern when we
import living plant materials into the United
States. They may include live and potentially vi-
rus-infected mites within the genus Brevipalpus.
First, three Brevipalpus species (B. phoenicis,
B. obovatus Donnadieu, and B. californicus
(Banks)) have extensive and highly diverse host
plant ranges (see Childers et al. 2003c). Second,
Brevipalpus mite vectors are infected for life once
the virus is acquired during feeding on citrus.
Third, mites in this genus are long lived relative
to many tetranychid species. Fourth, Brevipalpus
populations are primarily or exclusively female.
Fifth, mites in this family disperse aerially on
wind currents. Proximity of citrus to numerous
ornamental host plant species throughout Cen-
tral America can be expected given the large rural
populations of people, agrarian economies, and
widespread distribution of dooryard and commer-
cial citrus plantings as well as an abundant array
of other host plants (Childers et al. 2003a, b, c).
Potentially large numbers of viruliferous mites
could be moved from citrus hosts in countries hav-
ing citrus leprosis to non-citrus hosts including
propagative ornamental hosts for export. The
mites are not known to successfully transmit cit-
rus leprosis to non-citrus hosts and back again to
citrus. Rather, virus-infected mites could be phys-
ically distributed onto various plants including
ornamentals via aerial dispersal. When one com-
bines the unique characteristics of mites in this
genus as described above with the huge numbers
of ornamental plants being shipped, there may be
significant risk for citrus leprosis being intro-
duced into Florida on these non-citrus host plants
from countries where this disease occurs. This
should be of immediate concern to the citrus in-
dustries of six states (Alabama, Arizona, Califor-
nia, Florida, Louisiana, and Texas).
The importation of live ornamental plants into
Florida from countries having citrus leprosis and
numerous related diseases of ornamental plants
should raise serious concerns about the potential
risk in providing pathways for introduction of
these diseases.
Numerous species of Tarsonemidae are recog-
nized as plant pests (Jeppson et al. 1975;
Lindquist 1986), as are species of acarid mites
within the genus Rhizoglyphus (Diaz et al. 2000).
The authors stated clearly that the systematics of
the genus Rhizoglyphus remains in a state of con-
fusion. Species within this genus are also impor-
tant agricultural and stored product pests. There-
fore, the presence of a mite in this genus should
be sufficient to require the shipment to be re-
jected at the port. If USDA APHIS Plant Protec-
tion and Quarantine cannot presently reject such
shipments then appropriate legislation should be
enacted to further protect United States agricul-
ture including the ornamental industry.
Spider mites are notorious pests and species
within the genus Tetranychus cannot be accu-
rately separated to species without males (Jepp-
son et al. 1975). If there is insufficient sample ma-
terial to accurately identify the species of spider
mite, then that shipment should be treated or
destroyed.
The excessive number of recent pest mite in-
troductions into the United States (Table 1) must
serve as a mandate for future actions to restrict
movement of exotic pests into the United States.
Two recommendations are presented to USDA
APHIS PPQ based on the numbers of mite in-
fested ornamental plant shipments identified in
this preliminary survey from Central American
countries. First, a sampling program specifically
for mite pests on all live ornamental plant ship-
ments coming into the United States should be
established. Visual inspection for mite infesta-
tions on large numbers of plants is inadequate
due to time constraints of inspectors, the minute
December 2005
Childers & Rodrigues: Exotic Pest Mite Introductions
size of mites, their distribution on the plants,
cryptic coloration, and behavior.
A sampling protocol for in-coming ornamental
plant shipments would include a designated sub-
sample of plants in a shipment. Use of either an
80% ethanol wash or a specified concentration of
detergent solution would be employed in combina-
tion with a filtration process to reduce the alcohol
or detergent solution volume to a desired level for
examination with a stereomicroscope. Use of the
alcohol wash method would be preferred if the
samples were to be stored before examination. Oth-
erwise, the detergent wash method would be the
preferred procedure if examination was completed
immediately after processing. Certain detergent
materials have a flushing or irritating reaction to
various arthropods including mites. Hence, many
of the mites would remain alive longer following
this procedure versus the alcohol wash method.
This assessment should be done for a mini-
mum period of one year to identify trends and
seasonal patterns of different pest mite species
(as well as other small arthropods) and provide
assurance of compliance by foreign shippers.
We only recovered three Brevipalpus specimens
from our limited sampling on February 6-7. Sea-
sonal trends of this mite and others must be deter-
mined on foreign plant shipments entering the
United States. Seasonal abundance trends of pest
mite species are known for various agricultural
crops within the United States. Such patterns of
activity and population increase by many exotic
pest mite species remain unknown for most orna-
mental plants grown in Central America and Asia.
Second, legislation is needed that would re-
quire that any shipment of ornamental plants en-
tering the United States either as ornamental
cuttings or rooted plants for propagative purposes
be free of pest mites and other plant pests. This
requires sub-sampling for mite species within dif-
ferent ornamental plant shipments other than by
visual means alone. Legislation requiring the im-
porting companies to treat their plant materials
with an insecticide and acaricide prior to ship-
ment should be enforced to protect American ag-
riculture as well as our ornamental industries
from both exotic pest mite introductions or exotic
diseases that could be transmitted by one or more
exotic or domestic mite species. The risk of intro-
ducing pest mite infested plants could be signifi-
cantly reduced by off-shore inspection and certifi-
cation of pest-free status and pre-clearance at the
port of export as recommended by The National
Plant Board (National Plant Board 1999) and
consistent with ISPM 4, Requirements for the es-
tablishment of pest free areas (FAO, 1996).
ACKNOWLEDGMENTS
This research was supported by USDA, APHIS,
PPQ, and the Florida Agricultural Experiment Station,
and approved for publication as Journal Series No. R-
10746. The authors acknowledge the support and coop-
eration of Michael J. Shannon, Fernando E. Lenis, Tho-
mas L. Skarlinsky II, USDA-APHIS-PPQ, Ronald
Ochoa, SEL-ARS-USDA, and Warren C. Welbourn, Flor-
ida Department of Agriculture and Consumer Services,
Division of Plant Industry. Appreciation is extended to
two anonymous reviewers, to Douglass Miller, SEL-
ARS-USDA, Alma Solis, USDA-NMNH, Washington,
DC, Joseph G. Morse, University of California-River-
side, and Waldemar Klassen, University of Florida,
Tropical Research and Education Center, Homestead
for helpful reviews of this manuscript.
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Florida Entomologist 88(4)
Solomon & Mikheyev: Ants of Cocos Island
THE ANT (HYMENOPTERA: FORMICIDAE)
FAUNA OF COCOS ISLAND, COSTA RICA
SCOTT E. SOLOMON AND ALEXANDER S. MIKHEYEV
Section of Integrative Biology, The University of Texas at Austin, Austin, TX
ABSTRACT
Cocos Island, Costa Rica is a 24-square kilometer volcanic island in the tropical eastern Pa-
cific Ocean, located approximately 480 kilometers from the mainland. Despite its biogeo-
graphic significance, much of the entomofauna have not been systematically surveyed. A
detailed survey of the ant (Hymenoptera: Formicidae) fauna of this island was conducted
over a three-week period. The results suggest that, despite the relatively minor presence of
humans on Cocos, much of the ant fauna is dominated by non-native species. Furthermore,
the current ant community is substantially different from that described by previous expe-
ditions. One of the previously described endemics, Camponotus biolleyi Forel, was not found
during the survey. A species known to be invasive, Wasmannia auropunctata Roger, was
found in extremely high abundance near disturbed sites, but was not present in more pris-
tine habitats. Furthermore, this population displays intraspecific aggression, which is un-
common among invasive ants, including other invasive populations of this species.
Key Words: invasion, non-native species, species richness, Wasmannia auropunctata
RESUME
La Isla del Coco, Costa Rica, es una isla volcinica de 24 kil6metros cuadrados y esta ubicada
en el oriented del Oc6ano Pacifico tropical, 480 kil6metros de la costa del continent. A pesar
de su relevancia biogeografica, la mayor parte de la entomofauna de esta isla no ha sido exa-
minada sistematicamente. Un relevamiento detallado de las hormigas (Hymenoptera: For-
micidae) de esta isla fue realizado durante tres semanas. Nuestros resultados sugieren que,
a pesar de la escasa presencia humana en La Isla del Coco, la mayor parte de la fauna de hor-
migas esta dominada por species introducidas. Ademas, la fauna actual de hormigas es
substancialmente diferente de la descrita por las expediciones anteriores. Una de las espe-
cies endemicas previamente descritas, Camponotus biolleyi Forel, no fue encontrada du-
rante nuestro studio. Una especie conocida como invasora, Wasmannia auropunctata
Roger, fue encontrada con abundancia extremadamente alta en sitios disturbados, pero esta
ausente en habitats mas pristinos. Ademas, esta poblaci6n present signos de agresi6n in-
traespecifica, la cual es infrecuente en species invasoras, incluyendo otras poblaciones in-
vasoras de esta especie.
Translation provided by the authors.
Surveys of island faunas are a basic step for
ecological and biogeographical studies, and are
critical for conservation efforts. Surveys con-
ducted over long periods are especially useful for
the insight they can offer into the processes of
community assembly, dispersal, competition, and
extinction. Species invasion may affect all of
these processes and has received increasing at-
tention as a force threatening attempts at conser-
vation. Species are defined as invasive if (1) they
are non-native (or alien) to the ecosystem under
consideration, and (2) their introduction causes or
is likely to cause economic or environmental
harm or harm to human health (Williams 1994).
The patterns by which invasive species are
spread and the impact that they have on the en-
vironment in which they are introduced are both
of fundamental interest to ecologists and conser-
vationists. Ants (Hymenoptera, Formicidae) are
particularly common invasive species (McGlynn
1999) and their impact can be quite profound (Lu-
bin 1984; Human & Gordon 1997; Vinson 1997).
Cocos Island, Costa Rica
Cocos Island (532'57"N, 8659'17"W) is a rela-
tively small (24 km2), volcanic island in the tropi-
cal eastern Pacific Ocean. It is has been of partic-
ular interest to biogeographers (see Hertlein
1963) because it is the only island in this region
that supports a lowland tropical forest, due to the
warm climate (Montoya 1990), and heavy precipi-
tation (Montoya 1990) which it receives. It is also
relatively isolated, located approximately 480 km
from the coast of Central America. Early surveys
of the flora and fauna of Cocos Island, conducted
in the late 19th and early 20th centuries (Hogue &
Miller 1981), described a diverse community of or-
Florida Entomologist 88(4)
ganisms, with a relatively high rate of endemism
(Hogue & Miller 1981; Montoya 2002). While ants
were included in several surveys (Hogue & Miller
1981), we have reason to believe that the data
from at least one of these was erroneous (see Dis-
cussion). Nevertheless, these surveys provide
baseline data for the purpose of comparison.
Cocos Island is also unique in that it has had a
minimal history of human influence (Weston-
Knight 1990). Due mostly to the presence of fresh
water, mariners have consistently used Cocos as a
refueling point for excursions in the region. How-
ever, Cocos is an extremely rugged island, sur-
rounded by steep cliffs; only 3 bays (Chatham,
Wafer, and Yglesias) provide access to the interior
of the island and have been used for anchorage by
visitors to Cocos. Although no permanent human
settlement has ever been established, several at-
tempts were made in the late 19th century, in-
cluding an agricultural colony (1884-1912) and a
penal colony (1879-1881). These settlements were
restricted to the areas around Chatham and Wa-
fer Bays, which are in the extreme northern end
of the island and are the most accessible, and to-
day house the park guards who manage the is-
land (Fig. 1). The presence of humans on Cocos Is-
land, though minimal, warrants inquiry into
their impact on the island environment, particu-
larly that of invasive species brought by humans.
We conducted a detailed survey of the ant
fauna of Cocos Island to answer the following
questions: (1) what is the current species richness
of ants, (2) how have the diversity and community
composition changed since previous surveys, and
1 h
Fig. 1. Map of Cocos Island, Costa Rica, indicating
sampling localities for leaflitter and pitfall transects,
additional leaflitter samples, and the location of park
stations.
(3) what is the impact, if any, of invasive ant spe-
cies on Cocos Island?
MATERIALS AND METHODS
Fieldwork was conducted on Cocos Island from
3-23 July 2003. Species richness was assessed by
collection of ants and other invertebrates follow-
ing the standardized "ants of the leaf litter" (ALL)
collection protocol outlined in Agosti et al. (2000),
except that transects were shortened from 200 m
to 100 m due to the extremely rugged terrain of
the island. Five 100-m transects were run on the
island (Fig. 1), collecting moist leaflitter and
other debris from a 1-m2 quadrat every 10 m
along the transect and placing a pitfall trap ap-
proximately 1 m from the site of leaflitter collec-
tion. Invertebrates were extracted from the leaf
litter by sifting the litter to remove large parti-
cles, then use of Winkler traps. The moist leaflit-
ter was allowed to dry for approximately 48 h. Pit-
fall traps consisted of plastic 60-mm diameter
drinking cups (except for transect A, in which 30-
mm-wide plastic tubes were used) containing ap-
proximately 25 milliliters of 100% ethanol cov-
ered by a plastic plate and were collected after 48
h. In addition to transects, six additional leaf lit-
ter samples (one square-meter each) were col-
lected from various sites around the island (Fig.
1), in an attempt to sample as many regions and
habitats on the island as time allowed. Baiting
was conducted at human-inhabited Chatham and
Wafer Bays, as well as at uninhabited Yglesias
Bay with Pecan Sandies cookie crumbs which
were placed in 1.5-milliliter tubes and collected in
two stages, at 10 and 30 min. General collecting
techniques were also employed, in which foraging
ants were found by visually searching and then
collected by hand; ant nests were found by search-
ing through leaflitter, rotting logs, recent tree-
falls, and under bark on living as well as decaying
trees. Species accumulation curves were gener-
ated by EstimateS (Colwell 1997).
To assess the impact of an invasive ant species
on the island, we chose to focus on Wasmannia au-
ropunctata Roger, a species that has been shown
to have profound impacts on native invertebrates
in the nearby Galapagos Islands (Clark et al.
1982; Lubin 1984) and has been spreading rapidly
to other sites worldwide (Wetterer & Porter 2003).
A Kruskall-Wallis test was used to compare ant
species richness in areas with W auropunctata
versus those without. Additionally, the effects of
W auropunctata density on ant species richness
were analyzed by Spearman rank correlation. To
measure intraspecific aggression (thought to cor-
relate with competitive ability; (Holway et al.
1998; Tsutsui et al. 2003)) within the Cocos popu-
lation of W auropunctata, worker ants were
baited to 45-ml tubes as described above. Aggres-
sion trials were conducted in a manner analogous
December 2005
Solomon & Mikheyev: Ants of Cocos Island
to Tschinkel et al. (1995) at four sites throughout
the invaded range. After 10-30 min, when the bait
tubes swarmed with several hundred (~200-500)
W auropunctata workers, they were sealed and
transported to the lab, where the open ends of the
tubes were joined. Aggression was scored as
present or absent; when present, the number of
overtly aggressive interactions (biting, grappling,
and stinging) was noted over the next five min as
the ants from the two tubes intermixed. Each trial
was repeated no less than three times with repli-
cate bait tubes. No more than an hour elapsed be-
tween the sealing of tubes in the field and the lab-
oratory aggression trials.
RESULTS
We discovered a total of 19 species of ants in 14
genera and 4 subfamilies during the survey (Table
1). Species identification was not possible for sev-
eral taxa, because either the taxonomy is presently
inadequate or because a nest series (i.e., the full
range of worker castes) was not collected (S. Cover,
MCZ: Harvard University, M. Deyrup, Archbold
Biological Station, and J. T. Longino, Evergreen
State College, pers. comm.). The observed species
richness converges on that provided by estimators
(Fig. 2), suggesting that our inventory of the leaf
litter ants was thorough in the sites sampled.
TABLE 1. SPECIES LIST OF THE ANTS OF COCOS ISLAND,
JULY 2003.
Subfamily Species
Dolichoderinae Tapinoma melanocephalum F.2
Formicinae Brachymyrmex sp.5
Camponotus cocosensis Wheeler'
Camponotus sp. 25
Paratrechina guatemalensis Forel2
Paratrechina longicornis Latreille2
Myrmicinae Adelomyrmex sp.4
Monomorium floricola Jerdon2
Pheidole moerens Wheeler2
Pheidole sp. 25
Pheidole sp. 35
Pyramica nigrescens Wheeler3
Solenopsis sp. (Diplorhoptrum)4
Solenopsis geminata F.2
Strumigenys louisianae Roger3
Wasmannia auropunctata Roger2
Ponerinae Hypoponera opacior Forel3
Odontomachus ruginodis Smith2
Pachycondyla stigma F.2
Endemic.
2Tramp species.
'Unknown origin, likely tramp species.
Unknown origin, likely native.
5Unknown origin.
The relative abundances of selected species,
based on the number of 1-m2 plots in which a spe-
cies appears, are presented in Fig. 3. In general,
more species were found at higher elevations (Fig.
4). Furthermore, the ant communities above ap-
proximately 200 meters elevation consisted of a
higher proportion of native and potentially-native
species. This pattern is exaggerated by the ab-
sence of some invasive species in higher elevation
sites. In particular, three species (Monomorium
floricola Jerdon, Solenopsis geminata F., and Tap-
inoma melanocephalum F.) were found exclusively
in the human-modified habitats around Chatham
and Wafer Bays. As a result, these species were
never present in our leaflitter or pitfall samples.
Other species that were absent from leaflitter and
pitfall samples were found to nest in rotten logs
(Camponotus sp. 2, Pachycondyla stigma F.) or
were strictly arboreal (Brachymyrmex sp.).
The presence of W auropunctata was uncorre-
lated with the number of other ant species in a
quadrat (H = 0.29, df = 1, P = 0.59). Likewise,
there was no significant effect of W auropunctata
density on the species richness of other ants (r =
-0.19, n = 31, P = 0.30). There was no variance in
the outcomes (presence or absence) of aggression
assays among replicates, although when present,
the levels of aggression (biting, grappling, and
stinging) varied between sites on the island.
DISCUSSIoN
Our survey of the ant fauna of Cocos Island re-
vealed the presence of 19 species currently inhab-
iting the island. Several lines of evidence suggest
that the majority of these are non-native, having
likely arrived on the island via human transport.
Of the species known to be common tramps (Dey-
rup et al. 2000), three were never found far from
human habitation (Monomorium floricola, Sole-
nopsis geminata, and Tapinoma melanoceph-
alum). Likewise, the species that may be native
(including the only species known to be endemic;
see Appendix) were only collected away from hu-
man-disturbed regions of the island. Of the ten
species we collected that are not known to be com-
mon tramp species, at least three of these
(Pyramica nigrescens Wheeler, Hypoponera opa-
cior Forel, Sri ....i'... *.., louisianae Roger) may
also be regularly transferred by human commerce
(J. T. Longino, Evergreen State College, and
M. Deyrup, Archbold Biological Station, pers.
comm.). The remaining six species cannot be iden-
tified because the genus needs revision or because
of insufficient material (S. Cover, MCZ: Harvard
University, M. Deyrup, Archbold Biological Sta-
tion, and J. T. Longino, Evergreen State College,
pers. comm.). Therefore, a determination of their
status as tramps is not possible. However, a con-
servative estimate would be that six of the nine-
teen ant species currently inhabiting Cocos Is-
Florida Entomologist 88(4)
Observed
o Singletons
x Doubletons
--ICE
-Chao2
Samples
Fig. 2. Species accumulation curve for leaf litter ants collected using Winkler and pitfall traps. The accumulation
curve does not include 6 additional species acquired by general collecting.
land are native (i.e., were not brought by hu-
mans). This leads us to the conclusion that most
ants on Cocos Island are non-native species, an
assertion reinforced by the apparent domination
of a few species (e.g., Wasmannia auropunctata),
especially near disturbed regions of the island.
The ant community currently inhabiting Cocos
Island is substantially different than that re-
ported from previous surveys (Hogue & Miller
1981; Table 2). Four prior expeditions collected
ants on Cocos between 1898 and 1932, and the
specimens from each of these were subsequently
described by taxonomists who did not participate
in the collection events (Forel 1902, 1908; Emery
1919; Wheeler 1919, 1933). Of these, the 1898 col-
lections by Anastasio Alfaro (Forel 1908; Emery
1919), is closest to our survey in terms of the num-
ber of species collected (17), but the identities of
the species in both surveys are quite different. In
fact, we believe it is very likely that the reports
from the 1898 surveys are erroneous for the fol-
lowing reasons. First, several of the species listed
from this survey (e.g., Atta cephalotes L.,
Nomamyrmex crassicornis Smith) are unlikely to
occur on a small oceanic island, and another,
Pseudomyrmex flavicornis, an obligate acacia spe-
cialist is unlikely to be present on Cocos, from
which no acacias have been reported (Fosberg &
Klawe 1966). Second, the material from this expe-
dition sent by Alfaro to Forel cannot be located
(J. T. Longino, Evergreen State College, pers.
comm.). Since Alfaro also sent a large amount of
specimens from the mainland to both Emery and
Forel, and since all of the species reported from the
1898 expedition also occur on mainland Costa
Rica, it seems likely that there was some confusion
between the Cocos Island and mainland material.
Further support for this hypothesis comes
from three subsequent expeditions (1902 by Pablo
Biolley; 1905 by Francis Williams; and 1932 by
Maurice Willows, Jr.), none of which included the
suspicious species mentioned above (Table 2).
These surveys reported relatively few species (5,
7, and 1, respectively), but at least two have sub-
stantial overlap between the species reported:
four of the five species (80%) reported from 1902
were also reported from 1905 (Wheeler 1919).
Only a single species, Camponotus cocosensis
Wheeler is mentioned from the 1932 expedition,
(which also was reported from 1905) but it is not
clear how extensive was the 1932 survey
(Wheeler 1933). Thus the only surveys that seem
both fairly extensive and reliable are the 1902
and 1905 surveys, which share 40% (2/5) and 57%
(4/7) of species in common with our 2003 survey.
Our 2003 survey found fourteen species that
have not been previously reported from Cocos Is-
land. Of the remaining five species, four were re-
ported by more than one previous survey. The dif-
ferences in species composition between ours and
previous surveys can be explained by one or more
of the following non-mutually- exclusive hypothe-
ses: (1) species turnover due to the arrival of new
species and extinction of others; (2) insufficient or
incomplete sampling by some expeditions; (3)
misidentification of some species; or (4) mislabel-
ing or confusion of specimens on expeditions that
visited additional sites. As previously mentioned,
i-
-
December 2005
Solomon & Mikheyev: Ants of Cocos Island
a.
E
(0
(0
a)
0)
a-
0
0.
Fig. 3. The relative abundances of selected Cocos Island ant species in litter samples.
we believe that the report from the 1898 expedi-
tion can be discarded because hypothesis 4 (spec-
imen mislabeling/confusion) likely applies. Hy-
pothesis 2 (incomplete sampling) likely applies to
the 1932 expedition. For the remaining two expe-
ditions, we are unable to determine which hy-
potheses apply, although species turnover (hy-
pothesis 1) and/or incomplete sampling (hypothe-
sis 2) seem likely.
It is noteworthy that we were not able to locate
any workers of Camponotus biolleyi, a species
thought to have been endemic to Cocos Island and
which was apparently quite abundant at the turn
of the century, commonly seen foraging on under-
story ferns (Forel 1902, p. 178). This species was
collected by two separate expeditions to Cocos Is-
land, in 1902 and 1905. Because our intensive
survey did not detect this species, it seems proba-
ble that C. biolleyi has either gone extinct or has
greatly declined in abundance. The possibility
that non-native species are displacing native ants
on Cocos Island underscores the importance of
conservation efforts on this and other oceanic is-
lands, which may be more affected by human ac-
tivity than is immediately apparent.
By far the most abundant ant species in our
survey, based on the number of plots in which it
occurred, is the little fire ant, Wasmannia auro-
punctata. This species is known to be highly inva-
sive (Wetterer & Porter 2003) and to displace na-
tive arthropods, particularly other ant species, in
areas in which it is introduced (Clark et al. 1982;
Lubin 1984; Le Breton et al. 2003). To determine
whether W auropunctata has had a similar effect
on the Cocos Island ant community, we made de-
tailed observations on the distribution, abun-
dance, and behavior of this species. Our results in-
dicate that W auropunctata appears to be re-
stricted to the regions of the island near disturbed
sites (Chatham and Wafer Bays); the 1902 expedi-
tion by Biolley observed the same distribution,
which therefore appears not to have changed sig-
nificantly for at least 100 years (Forel 1902,
p. 177). Further support for this observation
comes from the fact that W auropunctata appears
to be completely absent from Yglesias Bay, which
was never settled by humans but possesses appar-
ently suitable habitat (e.g., groves of Hibiscus tili-
aceus L. [Malvaceae], which are densely patrolled
by W auropunctata in Wafer and Chatham Bays).
60%
50% -
40%-
30% -
20% -
10%
0%
[IIILLHtL
Florida Entomologist 88(4)
O other ant species
o tramp ant species
12 -
10
0
8-
0
I 6-
-
2 4
2
0 -
Mid-
low -
elevation elevation,
riparian
High
elevation
Cloud forest
Fig. 4. Species composition among the sampling sites. Note that lower and middle elevations (also closer to
shore) are occupied almost exclusively by tramp species.
This is surprising, considering the propensity of
this species for colonizing new areas (Wetterer &
Porter 2003) and suggests that the ants' dispersal
may be limited by ecological factors, including pos-
sibly the absence of human-modified habitat.
The W auropunctata population on Cocos Is-
land appears not to be suppressing the richness of
other ant species, as has been reported on other
islands (Clark et al. 1982; Lubin 1984; Jourdan
1997). However, this may be partially due to the
high proportion of non-native ant species com-
prising the Cocos ant fauna, because non-native
species are often good competitors (lesser compet-
itors may have already been competitively ex-
cluded). Alternatively, the Cocos population of W.
auropunctata may be less competitive than other
invasive populations of this species. The presence
ofintraspecific aggression within the Cocos popu-
lation is in marked contrast with studies from
other introduced populations of this species (Le
Breton et al. 2003), and is consistent with the hy-
pothesis that the Cocos population is not as com-
petitively superior. Lack of intraspecific aggres-
sion is thought to contribute to the increased com-
petitive abilities of many invasive ant species, by
allowing them to effectively function as one enor-
mous supercolony (Tsutsui et al. 2003). If lack of
aggression is causally associated with competi-
tive superiority, then the repeated presence of ag-
gression between some colonies of W auropunc-
tata on Cocos may explain why this species has
not spread as effectively across the island as it
has in other introduced areas, and why it has not
displaced other ants even in the areas in which it
is abundant. Further study of this community is
warranted to make generalizations about the pat-
terns and processes involved in invasions of is-
lands by non-native species.
In general, our survey revealed that Cocos Is-
land appears to be dominated by non-native ant
species, despite the minimal history of human
habitation on the island. We are unable to make
meaningful comparisons between our survey and
previous surveys, because earlier collections were
either incomplete, likely to have included misla-
beled specimens, or both. Nevertheless, we are
able to conclude that at least one non-native spe-
cies, Wasmannia auropunctata, though abundant
in some parts of the island, appears not to have a
significant impact on the species richness of other
ant species. However, the fact that such a high
proportion of the ant species on Cocos are also
non-native may indicate that some sort of compet-
itive stalemate has been reached; follow-up sur-
veys would help to test whether this equilibrium
is stable or transient.
___ _______ L A ____________
Low -
elevation,
coastal
December 2005
Solomon & Mikheyev: Ants of Cocos Island
TABLE 2. SUMMARY OF THE HISTORY OF ANT COLLECTIONS ON COCOS ISLAND.
Year of expedition
Ant species (outdated names in parentheses) 18981* 19022 19053 19324 2003
Adelomyrmex sp.
Atta cephalotes L.
Azteca constructor (emmae) Emery
Brachymyrmex longicornis Forel
Brachymyrmex sp.
Camponotus atriceps (abdominalis) Smith
Camponotus biolleyi Forel
Camponotus blandus Smith
Camponotus cocosensis Wheeler
Camponotus mocsaryi Forel
Camponotus sp.
Cyphomyrmex rimosus Spinola
Hypoponera opacior Forel
Monomorium floricola Jerdon
Nomamyrmex (Eciton) crassicornis Smith
Odontomachus haematodes L.
Odontomachus ruginodis Smith
Pachycondyla stigma F.
Paratrechina guatemalensis Forel
Paratrechina longicornis Latreille
Pheidole biconstricta Mayr
Pheidole moerens Wheeler
Pheidole punctatissima Mayr
Pheidole subarmata Mayr
Pheidole sp. 2
Pheidole sp. 3
Pseudomyrmex flavicornis (belti) Smith
Pyramica nigrescens Wheeler
Solenopsis geminata F.
Solenopsis succinea Emery
Solenopsis sp. (Diplorhoptrum)
Strumigenys louisianae Roger
Tapinoma melanocephalum F.
Tetramorium guineense Bernard
Wasmannia auropunctata Roger
Zacryptocerus (Cryptocerus) cristatus Emery
Total number of species collected
X X
X X X
X
X
X
x
X
X X
X X
X X
17 5 7 1 19
1Collections by Anastasio Alfaro described by Forel (1908) and Emery (1919).
*Species list for this expedition is unreliable; see Discussion for details.
'Collections by Pablo Biolley described by Forel (1902).
'Collections by Francis X. Williams described by Wheeler (1919).
'Collections by Maurice Willows, Jr. described by Wheeler (1933).
ACKNOWLEDGMENTS
We are grateful to the Ministerio del Ambiente y En-
ergia (MINAE) and the Area de Conservaci6n Marina
Isla del Coco (ACMIC) for granting permission to conduct
this survey. We especially thank F Campos, G. Haug, W.
Gonzalez, M. Montoya, A. Vega, and L.W. Gilbert for pro-
viding logistic support and arranging transport. We
thank the Okeanos Aggressor, and the Undersea Hunter
and their crews for transporting us to and from Cocos Is-
land. Thanks also to W. Madriz for his cheerful company
and assistance during our stay on the island. We are in-
debted to the instructors and participants of the 2003
Ant Course, especially S. P. Cover, M. Deyrup, J. T. Long-
ino, and W. P. MacKay, for help in identifying ant speci-
mens. Helpful comments and suggestions by Sanford
Porter, Lloyd R. Davis Jr., and J. T. Longino improved
various aspects of this manuscript. Funding for this
project was provided by the Section of Integrative Biology
at The University of Texas at Austin and by fellowships
from the Dolores Zohrab Liebmann Foundation and the
U.S. Environmental Protection Agency to ASM.
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Florida Entomologist 88(4)
Solomon & Mikheyev: Ants of Cocos Island
APPENDIX
Miscellaneous Natural History and Taxonomy Notes
Adelomyrmex sp. This species is known only from a single leaflitter sample taken in the cloud forest
transect (Transect E) on Cerro Pel6n and therefore appears to be extremely rare. J. T. Longino has ex-
amined specimens of this morphotype and believes that it may be a new species, suggesting that it is
likely a native and possibly an endemic species (J. T. Longino, Evergreen State College, pers. comm.).
Brachymyrmex sp. We found diffuse pockets of brood and workers scattered underneath the bark of
a newly fallen tree, suggesting that this species may be arboreal.
Camponotus cocosensis Wheeler. This endemic species occurs in abundance throughout the island,
nesting in trees. By examining new treefalls, we twice found single queens establishing nests in holes
(which appear to have been made by bark-boring beetles) approximately 10 m up the tree. We once ob-
served an entire nest underneath an epiphytic fern (10-15 m high).
Camponotus sp. 2. We found a single queen in a rotten log. The queen was different than that ofC.
cocosensis and that ofC. biolleyi (B. MacKay, The University of Texas at El Paso, pers. comm.). We never
found workers of this species, suggesting that it is either extremely rare or nocturnal.
Odontomachus ruginodis Smith. This may be the same species that previous expeditions collected,
identified as 0. haematodes L. by Forel (1908) and 0. haematoda insularis Guerin by Wheeler (1919).
0. haematodes is currently a valid species name, but 0. ruginodis was previously known as 0. haema-
todes insularis var. ruginodis (Wheeler 1905). Although Wheeler (1919) did not include the variety of
the material he described from Cocos Island, he mentions (p. 303) that the specimens "agree ... in all
respects with specimens from Georgia, Florida, West Indies, and Central America in my collection."
Pheidole sp. 3. Though minor workers of this species were found in three pitfall traps scattered
amongst two sites, it was never found in leaflitter samples or by general collecting. Consequently, it ap-
pears possible that this is a nocturnal species.
Wasmannia auropunctata Roger. Forel (1908) adds W auropunctata var. rugosa to the list of species
from Cocos Island based on the Alfaro collection in 1902. Subsequent authors list this variety (cur-
rently recognized as a subspecies) as part of the Cocos ant fauna, but no subsequent collections have
included it. We found no morphological differences among the material we collected and therefore have
no data to suggest that there is more than one type ofW auropunctata currently present on the island.
Although this species is native to the Neotropics, its distribution on Cocos Island strongly suggests that
it was introduced, despite its presence as early as 1902 (Forel 1902).
Florida Entomologist 88(4)
December 2005
TRAPPING MOCIS SPP. (LEPIDOPTERA: NOCTUIDAE) ADULTS
WITH DIFFERENT ATTRACTANTS
ROBERT L. MEAGHER1 AND PAUL MISLEVY2
'Center for Medical, Agricultural and Veterinary Entomology, Agricultural Research Service
U.S. Department of Agriculture, Gainesville, FL 32608
2University of Florida, Range Cattle Research and Education Center, Ona, FL
ABSTRACT
Experiments conducted in a pasture agroecosystem in central Florida showed that two-com-
ponent lures composed of acetic acid and 3-methyl-l-butanol placed in Unitraps collected
adults of three species of grass looper. Mocis disseverans (Walker), M. latipes (Guen6e), and
M. marcida (Guen6e) males and females were collected from July through November, with
peaks from late September through late October. Other noctuid moths also were captured,
but constituted less than 33% of the total moths collected. In 2001 and 2002, 67.7 and 72.4%
of the Mocis spp. moths collected were females, respectively. Phenylacetaldehyde-baited
traps collected fewer Mocis spp. moths. The trapping system suggested by our results will
aid growers in monitoring for a pest complex that lacks commercially-available sex phero-
mone lures.
Key Words: Mocis latipes, Mocis disseverans, Mocis marcida, acetic acid, 3-methyl-l-bu-
tanol, grass loopers
RESUME
Experimentos realizados en un agro-ecosistema de pastos en la parte central de Florida mos-
traron que los atrayentes de dos components compuestos de acido ac6tico y 3-metil-l-buta-
nol puestos en trampas (Unitraps) recolectaron adults de tres species de gusanos
medidores. Machos y hembras de Mocis disseverans (Walker), M. latipes (Guen6e), y M. mar-
cida (Guen6e) fueron recolectados de julio a noviembre, con picos de poblaci6n desde la l1-
tima semanal de septiembre hasta la ultima semana de octubre. Otras mariposas noctuidas
tambi6n fueron recolectadas, pero constituyeron menos de 33% del total de las palomillas re-
colectadas. En 2001 y 2002, 67.7 y 72.4% de las palomillas de Mocis spp. recolectadas fueron
hembras, respectivamente. Las trampas cebadas con fenilacetoaldehido recolectaron menos
especimenes de Mocis spp. El sistema de trampa sugerido por nuestros resultados ayudara
a los agricultores en el monitoreo para un complejo de plagas que no dispone siiuelos de fe-
romonas sexuales comercialmente disponibles.
Moths in the genus Mocis (disseverans
(Walker), latipes (Guenee), marcida (Guenee),
and texana (Morrison)) are commonly called grass
loopers or striped grass loopers. Mocis spp. larvae
are important pests in the southeastern United
States of forage and pasture grasses (Watson
1933; Ogunwolu & Habeck 1975; Koehler et al.
1977) and turf grasses (Reinert 1975), but also
may infest and damage maize (Vickery 1924),
rice, sorghum (Genung 1964, 1967, 1968), and
sugarcane (Vickery 1924; Hall 1985). They also
are important pests in Central America, South
America, and the Caribbean, damaging both pas-
tures grasses and cultivated grasses (Gibbs 1990;
Portillo et al. 1991; Cave 1992).
Life history, biology, and geographic information
for Mocis spp. have been hampered by misuse of
scientific names in the literature and misidentifi-
cation in the field. For instance, M. latipes has been
recorded as Remigia repanda (F.) (a true species
known from the Caribbean), R. latipes (Guenee),
and M. repanda (F.) (Dean 1985), and adults can be
confused with velvetbean caterpillar, Anticarsia
gemmatalis Hiibner, a species belonging to the
same subfamily (Catocalinae) that shares similar
external morphology (Gregory et al. 1988).
Several sex pheromone components have been
identified for Mocis spp. Field testing of both wire
cone and bucket-style traps baited with a 2:98 load
ratio of (Z,Z,Z)-3,6,9-eicosatriene (Z)-(3,6,9)-C20)
and (Z,Z,Z)-3,6,9-heneicosatriene (Z)-(3,6,9)-C21)
or just (Z)-(3,6,9)-C21) caught male M. disseverans
(Landolt et al. 1986; McLaughlin & Heath 1989).
Mocis latipes responded best to a 20:80 ratio of
(Z,Z)-6,9-heneicosadiene (Z)-(6,9)-C21 and (Z)-
(3,6,9)-C21 (Landolt & Heath 1989). Growers and
consultants have been unable to monitor adult
Mocis spp. with these components in baited traps
because these chemicals are expensive to synthe-
size and are not commercially available.
Mocis latipes and M. disseverans moths were
collected in traps baited with aged solutions of
Meagher & Mislevy: Trapping Mocis Species Moths
molasses and jaggery (unrefined palm sugar)
(Landolt 1995). Headspace collections of chemi-
cals identified from fermented molasses solutions
included acetic acid and isobutanol (2-methyl-l-
propanol). These compounds, along with another
short-chain alcohol (3-methyl-l-butanol), were
tested as baits for vespid wasps (Landolt 1998;
Landolt et al. 2000). During testing large num-
bers of noctuid moths were also collected. Further
testing with wet and dry traps and varying
amounts of acetic acid plus 3-methyl-l-butanol
collected both sexes of the noctuids Lacanobia
subjuncta (Grote & Robinson), Mamestra configu-
rata Walker, Xestia c-nigrum (L.), and Pseudale-
tia unipuncta (Haworth) (Landolt 2000; Landolt
& Hammond 2001; Landolt & Alfaro 2001;
Landolt & Higbee 2002). Another compound, phe-
nylacetaldehyde, has been successful in trapping
Mocis spp. adults (Meagher 2001, 2002). Since
monitoring of Mocis spp. is not possible at this
time with sex pheromones as lures, our experi-
ments were conducted to determine if acetic acid,
3-methyl-l-butanol, or phenylacetaldehyde can
be used to attract adults.
MATERIALS AND METHODS
Field Site and Moth Trapping
The experiments were conducted at the Uni-
versity of Florida, Range Cattle Research and Ed-
ucation Center, Ona (2725', 8155'; 26 m eleva-
tion). This center contains over 1150 hectares of
natural and improved grasses. Mocis spp. moths
were collected in Standard Universal Moth Traps,
'Unitraps' (Great Lakes IPM, Vestaburg, MI) that
were placed on 1.5-m metal poles along roads and
pasture edges. Traps containing a treatment were
placed at least 30 m apart, and trap order was
randomized at each sample date. All traps con-
tained insecticide strips to kill moths that were
captured (Hercon Vaportape II containing 10%
2, 2-dichlorovinyl dimethyl phosphate, Hercon
Environmental Co., Emigsville, PA). Trap con-
tents were removed weekly or every two weeks,
depending on weather conditions. Moths were
placed in plastic bags and returned to the labora-
tory for identification. Since sample intervals
were variable, moth numbers were counted and
divided by the number of nights the traps were
active between sample dates.
Traps were baited with the chemicals acetic
acid, 3-methyl-l-butanol, and phenylacetalde-
hyde (Aldrich Chemical Co., Milwaukee, WI). Ace-
tic acid and 3-methyl-l-butanol as two-compo-
nent lures were loaded on cotton balls in 8-ml
polypropylene vials (Nalgene 2006-9025, Fisher,
Pittsburgh, PA). These controlled release dispens-
ers were placed on the bottom of the bucket por-
tion of the Unitraps and were secured by small
pieces of PVC pipe glued to the bucket. Attaching
vial tops with holes of different diameters modi-
fied chemical release rate (Landolt & Alfaro
2001). Phenylacetaldehyde was dispensed either
in vials (as above) or in hollow polyethylene stop-
pers (Kimble, Vineland, NJ, purchased through
Thomas Scientific, Swedesboro, NJ, #9713-F28)
(Meagher 2001, 2002).
Experiments in 2001
Two separate experiments were conducted.
Traps for Experiment 1 were placed 19 June and
removed 29 November. The experiment was de-
signed as a randomized complete block with three
replications of four treatments: (1) acetic acid and
3-methyl-l-butanol released in separate vials
with tops of 3.2 mm-hole diameters, (2) phenylac-
etaldehyde released in vials with tops of 3.2 mm-
hole diameters, (3) phenylacetaldehyde loaded
into stoppers (0.5 ml) and placed at the top of the
traps (Meagher 2001), and (4) unbaited traps.
Lures were changed 28 June, 19 July, 15 August,
30 August, 11 September, 27 September, 11 Octo-
ber, 26 October, and 7 November. Moths were re-
moved from traps on 19 separate dates. Mocis
moths were separated by sex but were not identi-
fied to species. Analysis of variance (PROC
MIXED, Contrasts, Littell et al. 1996) was used to
examine variation among treatments.
Traps for the second experiment were placed
26 October and removed 29 November. The exper-
iment was designed as a randomized complete
block with four replications (sample date) of six
treatments: (1) acetic acid and 3-methyl-l-bu-
tanol released from separate vials with top diam-
eter holes of 1.6 mm, (2) 3.2 mm, or (3) 6.4 mm, (4)
phenylacetaldehyde released from vials with a
top diameter hole of 6.4 mm or (5) in vials without
tops, and (6) unbaited traps. Lures were changed
7 November. Moths were extracted from traps on
4 separate dates. Mocis moths were separated by
sex but were not identified to species. Analysis of
variance (PROC MIXED, Contrasts, Littell et al.
1996) was used to examine variation among
treatments.
Experiments in 2002
Three separate experiments were conducted in
2002. Traps for each experiment were placed 19
July and removed 7 November. Each experiment
was designed as a randomized complete block
with twelve replications (sample date) of the
treatments. Lures were replaced 1 August, 29 Au-
gust, 11 September, 26 September, and 24 Octo-
ber. Mocis species were determined from genitalia
descriptions in Gregory et al. (1988). Moths were
separated by sex and females were dissected to
determine mating status. Analysis of variance
(PROC MIXED, Contrasts, Littell et al. 1996) was
used to examine variation among treatments.
Florida Entomologist 88(4)
The first experiment compared capture of
moths in treatments of acetic acid and 3-methyl-
1-butanol in separate vials with top diameter
holes of 1.6 mm, 3.2 mm, or 6.4 mm. The second
and third experiments compared capture of moths
in treatments where one component was released
at a constant level while the other component was
not released (= 0) or released at varying levels.
Experiment 2 contained treatments consisting of
acetic acid released from vials with top diameter
holes of 3.2 mm, while 3-methyl-l-butanol was re-
leased from 1.6, 3.2, or 6.4 mm diameter holes.
Experiment 3 contained treatments with 3-me-
thyl-l-butanol released from vials with 3.2 mm di-
ameter holes, while acetic acid was released from
1.6, 3.2, or 6.4 mm diameter holes. In each exper-
iment, there was an additional treatment of phe-
nylacetaldehyde loaded into stoppers and a treat-
ment that contained empty vials (control).
RESULTS
2001 Captures
Over 1300 Mocis spp. male (n = 431) and fe-
male (n = 904) moths were collected from late
June through late November in traps baited ei-
ther with acetic acid + 3-methyl-l-butanol or phe-
nylacetaldehyde (Fig. 1). Significantly fewer
moths were collected in traps with no lures than
in traps with either the combination lure of acetic
acid + 3-methyl-l-butanol or traps baited with
phenylacetaldehyde (total moths = 28 vs. 780 vs.
555, respectively, P < 0.05; Table 1). More female
moths per night were captured than male moths
in traps baited with an attractant (0.74 0.12 vs.
0.36 + 0.05, respectively; P = 0.0014).
Date
Fig. 1. Mean number of Mocis spp. moths collected
per night in traps baited with acetic acid (AA) + 3-me-
thyl-l-butanol (3-ME), phenylacetaldehyde (PAA) in vi-
als, phenylacetaldehyde in stoppers, or in unbaited
traps, Ona, FL, 2001.
Almost 400 Mocis spp. moths were collected in
attractant-baited traps during November in Ex-
periment 2. Fewer male Mocis moths were col-
lected in traps baited with phenylacetaldehyde or
in traps baited with acetic acid + 3-methyl-l-bu-
tanol from vials with 1.6 mm diameter holes (Ta-
ble 2). There was no difference in trap capture
among the attractants for female Mocis moths.
No moths were collected in the control traps. As in
Experiment 1, more female moths were captured
per night than male moths in traps baited with an
attractant (0.88 0.14 vs. 0.45 + 0.08, respec-
tively; P = 0.0107).
Several other noctuid species were collected in
both experiments, including Platysenta mobilis
(Walker), Spodoptera dolichos (F.), S. eridania
(Cramer), S. exigua (Hiibner), S. frugiperda (J. E.
Smith), S. latifascia (Walker), S. ornithogalli
(Guenee) (Amphipyrinae); A. gemmatalis (Cato-
calinae); Leucania spp., Pseudaletia unipuncta
(Haworth) (Hadeninae);Agrotis subterranea (F.),
Anicla infecta (Ochsenheimer) (Noctuinae);
Agrapha oxygramma (Geyer), Argyrogramma
verruca (F), and P. includes (Plusiinae). These
species comprised 26% (469/1804) of total moths
captured in Experiment 1 and 37.4% (147/393) of
moths captured in Experiment 2.
2002 Captures
Totals of 265 M. disseverans, 192 M. latipes,
and 46 M. marcida moths were collected from
July through November in traps baited individu-
ally with acetic acid or 3-methyl-l-butanol, or a
combination lure (Fig. 2). Females comprised
72.6% of the moths collected and 89.6% of them
contained a spermatophore (moths per night: fe-
males 0.077 0.009 > males 0.033 0.004 > virgin
females 0.008 0.002; P < 0.001). Traps baited
with phenylacetaldehyde collected only 42 moths.
There was no difference within a species in
numbers of M. disseverans or M. latipes collected
in traps baited with the two-component lure re-
leased from vials with holes in their tops ranging
from 1.6 mm to 6.4 mm (Experiment 1, Table 3).
Traps baited with phenylacetaldehyde collected
significantly fewer numbers of moths. In Experi-
ment 2, where 3-methyl-l-butanol was released
with varying rates, there was a trend for traps to
contain fewer moths when only acetic acid was re-
leased (Table 4). There were no differences in trap
captures among the other 3-methyl-l-butanol re-
lease rates for either M. disseverans or M. latipes.
Higher numbers of moths were collected in the ex-
periment in which acetic acid release rates var-
ied. In Experiment 3, traps releasing only 3-me-
thyl-l-butanol collected fewer M. disseverans and
tended to collect fewer M. latipes (Table 5). Differ-
ences among the varied release rates of acetic
acid were not different for either species. In all ex-
periments during 2002, traps sampled in Septem-
December 2005
Meagher & Mislevy: Trapping Mocis Species Moths
TABLE 1. COLLECTION OF MOCIS SPP. MOTHS PER NIGHT IN UNITRAPS BAITED WITH ACETIC ACID + 3-METHYL-1-BU-
TANOL (AA + 3-ME), PHENYLACETALDEHYDE (PAA) DISPENSED FROM A VIAL OR STOPPER, OR IN UNBAITED
TRAPS, EXPERIMENT 1, ONA, FL, 2001.
Lure Mocis males Mocis females Total Mocis
AA + 3-ME 0.48 0.11 a 0.88 0.27 a 1.36 0.37 a
PAA-vial 0.38 0.08 a 0.83 0.24 a 1.22 0.30 a
PAA-stopper 0.23 0.04 a 0.49 0.09 a 0.72 0.13 ab
Unbaited 0.04 0.01 b 0.03 0.02 b 0.07 0.03 b
Means within a column with the same letter are not significantly different, P > 0.05.
ber and October collected the most moths. As in
2001, many other noctuid species were collected,
comprising 32.2, 17.0, and 16.2% of all moths for
Experiment 1, 2 and 3, respectively.
DISCUSSION
Most research conducted with Mocis spp. in
Florida has been concentrated in the north-cen-
tral area. In this region, M. marcida abundance
peaked in late July to early August, while M. lati-
pes and M. disseverans densities were highest in
late September and early October (Ogunwolu &
Habeck 1975). Our results found higher numbers
of all three species from late September through
late October in south-central Florida.
Dean (1985) showed that M. latipes was the
species that was most economically important in
forage grasses, but our traps collected higher
numbers of M. disseverans than M. latipes or M.
marcida. It is possible that M. disseverans moths
are more attracted to our lures than the other
species. It is also uncertain whether M. dissever-
ans larvae have a higher incidence or are more
damaging to cultivated forage grasses grown for
P 3
C
a.
(A
2
.
Date
Fig. 2. Mean number of three species of Mocis col-
lected per night in experiments with acetic acid and 3-
methyl-1-butanol as two-components lures in baited
traps, Ona, FL, 2002.
cattle. Larval identification among species is dif-
ficult, especially between M. latipes and M. dis-
severans, and between M. marcida and M. texana
(Ogunwolu & Habeck 1979). Future research
should document the relative abundance of these
species based on larval sampling.
The two types of lures tested compared attrac-
tiveness to two different natural sources. Acetic
acid and short-chain alcohols similar to 3-methyl-
1-butanol have been isolated from fermenting so-
lutions of molasses and other sugar-containing
materials which have been used as attractants for
tortricid and noctuid moths (Frost 1926; Ditman
& Cory 1933; Norris 1935; Landolt 1995; Landolt
& Mitchell 1997). Phenylacetaldehyde has been
isolated from flowering plants and shrubs includ-
ing Zea mays L. (Poaceae) (Cantelo & Jacobson
1978), Araujia sericofera Brothero (Asclepia-
daceae) (Cantelo & Jacobson 1979), Abelia gran-
diflora (Andre) (Caprifoliaceae) (Haynes et al.
1991), Cestrum nocturnum (L.) (Solanaceae)
(Heath et al. 1992), and Gaura spp. (Onagraceae)
(Shaver et al. 1997). It has also been identified as
one of the compounds responsible for the male
scent of a papilionid butterfly (Honda 1980).
Phenylacetaldehyde attracted moths in 2001
but was not an effective lure in 2002. The mate-
rial used in 2002 was from the same bottle used in
2001. This chemical is known to be unstable
(Brown 1975) and may have polymerized to a
form that is not as attractive to moths.
Mocis latipes and M. disseverans were previ-
ously collected in Florida in traps baited with mo-
lasses or jaggery (Landolt 1995). The use of acetic
acid and 3-methyl-l-butanol as lure combinations
in the Pacific Northwest captured many noctuid
species from several subfamilies, but Mocis spp.
are not found in Washington and they were not
collected (Landolt & Hammond 2001). Release
rate experiments showed that more moths were
collected when acetic acid was released from vials
with holes 3.0 mm in diameter and 3-methyl-l-
butanol released from vials with hole diameters
<3.0 mm (Landolt & Alfaro 2001; Landolt & Hig-
bee 2002). Our results also showed that releasing
both chemicals with vial holes of 3.2 mm was ef-
fective. The single component lures were not as
effective at collecting Mocis moths.
Florida Entomologist 88(4)
TABLE 2. COLLECTION OF MOCIS SPP. MOTHS PER NIGHT IN UNITRAPS BAITED WITH ACETIC ACID + 3-METHYL-1-BU-
TANOL (AA + 3-ME) RELEASED FROM VIALS WITH 6.4-, 3.2-, OR 1.6-MM OPENINGS, PHENYLACETALDEHYDE
(PAA) DISPENSED FROM A VIAL WITH A 6.4-MM OPENING OR FROM A VIAL WITH NO TOP, OR IN UNBAITED
TRAPS, EXPERIMENT 2, ONA, FL, 2001.
Lure Mocis males Mocis females Total Mocis
AA + 3-ME (6.4) 0.61+ 0.12 ab 0.58 0.20 ab 1.19 + 0.12 a
AA + 3-ME (3.2) 0.82 0.22 a 0.98 0.36 a 1.81+ 0.51 a
AA + 3-ME (1.6) 0.19 + 0.14 c 0.85 0.17 ab 1.04 0.30 ab
PAA (6.4) 0.32 + 0.07 bc 0.82 0.25 ab 1.14 + 0.28 a
PAA-no top 0.29 + 0.17 bc 1.14 0.53 a 1.43 0.63 a
Unbaited 0.0 0.00 c 0.0 0.0 b 0.0 0.0 b
Means within a column with the same letter are not significantly different, P > 0.05.
TABLE 3. COLLECTION OF MOCIS DISSEVERANS AND M. LATIPES MOTHS PER NIGHT IN UNITRAPS BAITED WITH ACETIC
ACID + 3-METHYL-1-BUTANOL (AA + 3-ME) WITH 1.6-, 3.2-, OR 6.4-MM HOLES, EXPERIMENT 1, ONA, FL,
2002. TRAPS ALSO WERE BAITED WITH PHENYLACETALDEHYDE (PAA) AND THERE WERE UNBAITED TRAPS.
Lure M. disseverans M. latipes
AA + 3-ME (6.4) 0.22 + 0.11 ab 0.23 0.09 a
AA + 3-ME (3.2) 0.35 + 0.15 a 0.10 0.05 ab
AA + 3-ME (1.6) 0.40 + 0.18 a 0.17 0.07 ab
PAA 0.03 0.02 b 0.03 0.02 b
Unbaited 0.0 0.0 b 0.0 + 0.0 b
Means within a column with the same letter are not significantly different, P > 0.05.
TABLE 4. COLLECTION OF MOCIS DISSEVERANS AND M. LATIPES MOTHS PER NIGHT IN UNITRAPS BAITED WITH ACETIC
ACID (AA) RELEASED WITH 3.2-MM HOLES AND 3-METHYL-1-BUTANOL (3-ME) RELEASED WITH VARYING HOLE
SIZES, EXPERIMENT 2, ONA, FL, 2002. TRAPS ALSO WERE BAITED WITH PHENYLACETALDEHYDE (PAA) AND
THERE WERE UNBAITED TRAPS.
Lure M. disseverans M. latipes
AA (3.2) + 3-ME (6.4) 0.29 + 0.17 a 0.12 0.06 ab
AA (3.2) + 3-ME (3.2) 0.30 + 0.12 a 0.09 0.06 abc
AA (3.2) + 3-ME (1.6) 0.26 + 0.13 a 0.17 0.07 a
AA (3.2) 0.08 0.06 ab 0.03 0.03 bc
PAA 0.04 0.02 b 0.07 0.04 abc
Unbaited 0.0 0.0 b 0.0 + 0.0 c
Means within a column with the same letter are not significantly different, P > 0.05.
TABLE 5. COLLECTION OF MOCIS DISSEVERANS AND M. LATIPES MOTHS PER NIGHT IN UNITRAPS BAITED WITH 3-ME-
THYL-1-BUTANOL (3-ME) RELEASED WITH 3.2-MM HOLES AND ACETIC ACID (AA) RELEASED WITH VARYING
HOLE SIZES, EXPERIMENT 3, ONA, FL, 2002. TRAPS ALSO WERE BAITED WITH PHENYLACETALDEHYDE (PAA)
AND THERE WERE UNBAITED TRAPS.
Lure M. disseverans M. latipes
AA (6.4) + 3-ME (3.2) 0.50 + 0.14 a 0.55 0.28 a
AA (3.2) + 3-ME (3.2) 0.30 0.11 ab 0.21 0.11 b
AA (1.6) + 3-ME (3.2) 0.31+ 0.10 ab 0.23 + 0.11 ab
3-ME (3.2) 0.01 0.01 c 0.02 0.02 b
PAA 0.10 + 0.05 bc 0.12 0.04 b
Unbaited 0.03 0.02 c 0.04 0.03 b
Means within a column with the same letter are not significantly different, P > 0.05.
December 2005
Meagher & Mislevy: Trapping Mocis Species Moths
The lack of a commercially-available phero-
mone hampers growers' efforts to monitor these
pests. However, sugar-product lures have an ad-
vantage over pheromone lures in that they attract
both males and females (Landolt & Alfaro 2001;
Landolt & Higbee 2002). Adult trapping has been
successful in pasture agroecosystems as an early
warning management prediction tool with fall ar-
myworm (S. frugiperda) (Silvain & Ti-A-Hing
1985; Silvain 1986). The research reported here
suggests that two-component lures composed of
acetic acid and 3-methyl-l-butanol were success-
ful in capturing three Mocis species. These moths
are comparatively large and should be identifiable
by trained growers or consultants when the moths
are captured in traps baited by floral volatile
lures. Future research should determine if there
is a relationship between collection of larvae and
collection of adults and whether adult monitoring
can be used as a predictive management tool.
ACKNOWLEDGMENTS
We thank the staff at the Range Cattle Research and
Education Center for use of their facilities. P. Landolt
and R. Nagoshi (USDA-ARS) improved an early version
of this manuscript. The use of trade, firm, or corporation
names in this publication is for the information and con-
venience of the reader. Such use does not constitute an
official endorsement or approval by the United States
Department of Agriculture or the Agricultural Research
Service of any product or service to the exclusion of oth-
ers that may be suitable.
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Conklin et al.: Helicosporidia in Heterologous Hosts
INFECTIVITY OF TWO ISOLATES OF HELICOSPORIDIUM SPP.
(CHLOROPHYTA: TREBOUXIOPHYCEAE) IN HETEROLOGOUS
HOST INSECTS
TRACY CONKLIN1, VERENA-ULRIKE BLASKE-LIETZE', JAMES J. BECNEL2 AND DRION G. BOUCIAS1
'University of Florida, Entomology and Nematology, Gainesville, FL 32611-0620
2USDA ARS, Gainesville, Florida
ABSTRACT
Members of the genus Helicosporidium are the first described algal insect pathogens. They
have a close affinity to the non-photosynthetic algae of the genus Prototheca, and have a
wide host range, infecting many species of aquatic and terrestrial insects. In this study the
infectivity of two Helicosporidium spp. isolates, originating from a black fly (SjHe) and an
aquatic weevil (CsHe), was tested against a weevil Diaprepes abbreviatus (L.) and three
mosquito species Anopheles quadrimaculatus Say, Culex quinquefasciatus Say, and Aedes
aegypti (L.). The weevil constitutes a new experimental host record for helicosporidia. The
CsHe isolate was more virulent than the SjHe isolate in D. abbreviatus. Anopheles quadri-
maculatus was the most susceptible mosquito species measured by infection rate and mor-
tality. The infectivity and virulence of SjHe and CsHe isolates did not differ in any of the
mosquito species.
Key Words: Helicosporidium spp., entomopathogenic alga, mosquitoes, citrus root weevil,
bioassays
RESUME
Los miembros del g6nero Helicosporidium son los primeros pat6genos algaceos descritos
para insects. Estos pat6genos tienen una afinidad cercana a las algas no fotosint6ticas del
g6nero Prototheca, y tienen un rango amplio de hospederos, infectando muchas species de
insects acuaticos y terrestres. En este studio la capacidad de infecci6n de dos aislamientos
de Helicosporidium spp., obtenidos de una mosca negra (SjHe) y un gorgojo acuatico (CsHe),
fue probada contra el gorgojo Diaprepes abbreviatus (L.) y tres species de mosquitosAnoph-
eles quadrimaculatus Say, Culex quinquefasciatus Say, y Aedes aegypti (L.). El gorgojo es
considerado un nuevo registro de un hospedero experimental para helicosporidia. El aisla-
miento de CsHe fue mas virulento que el aislamiento de SjHe en D. abbreviatus.Anopheles
quadrimaculatus fue la especie de mosquito mas susceptible media por la tasa de infecci6n
y de mortalidad. La infectividad y virulencia de los aislamientos de SjHe y CsHe no fue di-
ferente dentro de las species de mosquitos.
Since the first description by Keilin (1921), in-
vertebrate pathogens in the genus Helicosporid-
ium have been detected worldwide in diverse
groups of arthropods, including several orders of
insects, mites, crustaceans, and trematodes (Sayre
& Clark 1978; Purrini 1984;Avery & Undeen 1987;
Pekkarinen 1993). Until recently, their taxonomic
position remained unclear. Keilin (1921) first de-
scribed Helicosporidium parasiticum as a proto-
zoan, but Weiser (1970) proposed that the heli-
cosporidia are best placed among the lower fungi.
Most recently, Boucias et al. (2001) suggested that
the vegetative development of helicosporidia was
similar to the autosporogenic growth of unicellular
algae. Significantly, genetic analysis has defined
the genus Helicosporidium as a member of the
green algal class Trebouxiophyceae (Chlorophyta)
and, as such, it represents a novel clade of inverte-
brate pathogens (Boucias et al. 2001; Tartar et al.
2002, 2003; Tartar & Boucias 2004). The treboux-
iophyte green algae are generally photosynthetic
and free-living. However, the closest relatives to
helicosporidia, the genus Prototheca, are achloro-
phyllous algae that have evolved a heterotophic
life style, infecting vertebrates.
The infectious cyst, the stage that defines the
genus Helicosporidium, is comprised of three
ovoid cells and a coiled, elongate filamentous cell
enclosed within an outer pellicle. The cyst de-
hisces when ingested by the insect host; within
the midgut lumen the pellicle ruptures, releasing
the filamentous cell and the three ovoid cells. The
invasive filamentous cells pass through the mid-
gut epithelial layer and gain ingress to the hemo-
coel. Within the hemocoel, the filamentous cells
differentiate into vegetative cells, which undergo
autosporogenic cell divisions (Bliske-Lietze et al.,
unpublished). Vegetative cells have been observed
to replicate within the phagocytic hemocytes and
to develop extracellularly in the hemolymph. Af-
Florida Entomologist 88(4)
ter multiple 2-4 cell autosporogenic cell divisions,
a portion of the hemolymph-borne vegetative cells
differentiate into cysts.
Unlike many insect pathogens, the helicospo-
ridia lack specificity and readily infect a broad
range of insects. For example, Fukuda et al.
(1976) isolated helicosporidia from Culex nigri-
palpus Theobald and they were able to infect 14
mosquito species and one non-mosquito species of
Diptera, three species of Coleoptera, and two spe-
cies of Lepidoptera with this isolate. More re-
cently, Seif & Rifaat (2001) isolated helicosporidia
from Culex pipiens L. in Egypt and cross-infected
Aedes caspius (Pallas), Culex antennatus
(Becker), Culiseta longiareolata (Macquart), and
Culex perexiguus Theobald. Hembree (1979,
1981) isolated Helicosporidium sp. from both
Aedes aegypti (L.) and Culex quinquefasciatus
Say in Thailand, and later evaluated this Heli-
cosporidium as a microbial control agent. How-
ever, Hembree (1981) dismissed the use of heli-
cosporidia as biological control agents due to its
infectivity to the predatory larvae of Toxorhyn-
chites splendens (Wiedemann). Sayre & Clark
(1978) further demonstrated the broad host range
of this group, reporting that a Helicosporidium
sp. from the cladoceran Daphnia magna (Straus)
was infectious to Ae. aegypti larvae. Avery & Un-
deen (1987) found that helicosporidia, isolated
from pond water and amplified in Helicoverpa zea
Boddie (Lepidoptera: Noctuidae), were infectious
toAnopheles quadrimaculatus Say, Culex salinar-
ius Coquillett, andAe. aegypti. In total, helicospo-
ridia have been isolated from five species of mos-
quitoes, and these mosquito isolates have been
transmitted to 21 additional species of mosqui-
toes and seven non-mosquito hosts in the labora-
tory (Chapman et al. 1967; Fukuda et al. 1976;
Hembree 1979, 1981; Seif & Rifaat 2001).
To date, four coleopteran isolates of helicospo-
ridia have been reported in the literature. In a
broad species screen, a Helicosporidium sp. iso-
late from the nitidulid Carpophilus mutilatus
(Erichson) in Texas was shown to cross-infect six
heterologous nitidulid species, the dermestid Tro-
goderma variabile Ballion, the cucujid Oryzaephi-
lus surinamensis (L.), and the anobiid Lasio-
derma serricorne (F.), as well as five pyralid lepi-
dopterans, one culicid dipteran, and three mite
species (Kellen & Lindegren 1973; Kellen &
Lindegren 1974; Lindegren & Hoffmann 1976).
Purrini (1985) identified Helicosporidium sp.
cysts in field-collected adults of the scarabaeid
Oryctes monoceros 01. in Tanzania. A weevil iso-
late originating from the curculionid Cyrtobagus
salviniae Calder & Sands in Florida infected and
replicated in three noctuid hosts (Blaske & Bou-
cias 2004). Most recently, Helicosporidium sp. in-
fections were found in German bark beetle popu-
lations ofDendroctonus micans (Coleoptera: Sco-
lytidae) (Yaman & Radek, 2005).
In this study, a comparative analysis of two
Helicosporidium spp. isolates was conducted, the
first originating from a local population of larval
Simulium jonesi Stone & Snoddy (Diptera: Sim-
uliidae) and the second isolated from a quaran-
tined lab population of an aquatic weevil C. sal-
viniae that was imported from Australia and col-
onized for release as a insect biological control
against Salvinia molesta (Mitchell). To date, the
only morphological characteristic that separates
these two isolates is the length of the filamentous
cell (Blaske-Lietze, unpublished), a potential ad-
aptation related to pathogen ingress. It was spec-
ulated that these two isolates, originating from
insects in different orders and from geographi-
cally distinct regions, might represent pathotypes
and therefore display different host range proper-
ties. Therefore in-depth bioassays of these two
helicosporidial isolates were conducted against
four insect species: the citrus root weevil, Dia-
prepes abbreviatus (L.) (Coleoptera: Curculioni-
dae), and three species of mosquitoes,An. quadri-
maculatus, Cx. quinquefasciatus, and Ae. aegypti
(Diptera: Culicidae).
MATERIALS AND METHODS
Preparation of Helicosporidium
Two Helicosporidium spp. isolates used in this
study were the black fly isolate (SjHe) from the
black fly, S. jonesi, collected in 2002 and 2003
(Alachua County, Florida), and the weevil isolate
(CsHe) from C. salviniae collected from a colony
in Alachua County. Both isolates were amplified
in H. zea and extracted on a continuous gradient
of Ludox HS40 (Perkin Elmer Life Sciences, Bos-
ton MA) (Blaske & Boucias 2004). The cyst-con-
taining band was subjected to several cycles of
low-speed centrifugation to remove residual gra-
dient material and stored at 4C. The number of
cysts in each preparation was determined with a
hemacytometer.
Infection Assays with Weevils
Four-week-old D. abbreviatus larvae were
starved for 16 h in wells of a 96-well tissue culture
dish. Individual starved larvae (15 4 mg) were
provided with a carrot cube (1 mm3) treated with
either 1 pl of water (control) or cyst suspensions
(SjHe or CsHe) at 2.5 x 105 cysts/pl and incubated
at constant conditions (26 1C, 70 + 5% RH,
darkness). Larvae that had completely consumed
the carrot after 96 h were transferred to diet cups
containing artificial diet. Three weeks after the
treatment, control and treated larvae were
weighed and examined for helicosporidial infec-
tion. Hemolymph samples were collected by nee-
dle puncture and the presence of helicosporidial
life stages (vegetative cells, cysts, pellicles) was
December 2005
Conklin et al.: Helicosporidia in Heterologous Hosts
recorded with differential interference contrast
(DIC) optics. After initial diagnosis at 3 weeks, all
larvae were transferred to fresh diet cups, incu-
bated for an additional 3 weeks, then re-exam-
ined. Throughout the experiments, survival was
recorded every other day, and dead individuals
were weighed and diagnosed for infection as de-
scribed above. In three to five replicate assays, a
total of 67, 38, and 44 larvae were used for con-
trol, SjHe, and CsHe treatments, respectively.
In a second assay, 5-week-old D. abbreviatus
adults were starved for 16 h and then force-fed 1-
pl droplets of either a 20%-sucrose solution or a
cyst suspension (2.5 x 105 cysts/pl) in 20% sucrose.
Droplets were delivered into the adults' esopha-
gus with a 1-ml syringe fitted with a blunt-tip
30G 1/2 needle mounted onto a microapplicator.
After an additional 24-h starvation period, the
adult weevils were placed in cups containing a cit-
rus leaf and moist cotton and incubated at 26 +
1C. Insects were examined daily and leaves and
cotton exchanged twice a week. After 3 weeks,
hemolymph samples were subjected to micro-
scopic examination. Ten adults were tested per
control and isolate. The experiment was repeated
three times.
Infection Assays with Mosquitoes
Bioassays assessed the activity of SjHe and
CsHe against three mosquito species,An. quadri-
maculatus, Ae. aegypti, and Cx. quinquefasciatus.
For each bioassay, 100 first-instars were placed in
a petri dish with 98 ml of deionized water
amended with a 1-ml dose of helicosporidia (treat-
ment) or deionized water (control) and a 1-ml vol-
ume of 2% alfalfa and potbelly pig chow mixture
(2:1) as a nutritional source. Final test concentra-
tions of helicosporidia in the petri dishes ranged
from 102 to 106 cysts/ml. Larvae were incubated at
constant conditions (26 1C, 12:12 photoperiod)
for 24 h then transferred to enamel pans, and wa-
ter was added to make 500 ml final volume. After
7 d, the surviving larvae were counted in each
pan and a sub-sample of 12 randomly examined
for infection under phase-contrast optics. Larvae
containing live helicosporidial cells (vegetative or
cyst stage) and those containing melanized heli-
cosporidial cells were considered infected. The de-
velopment of control and treated An. quadrimac-
ulatus andAe. aegypti at six d post-treatment was
assessed by head capsule width.
Statistical Analyses
Statistical analyses were done with the SAS
System for Windows (SAS Institute 1999). Per-
cent infection, percent survival/mortality data,
head capsule measurements, and weight gains
were subjected to analysis of variance by the pro-
cedure for general linear models (glm) in bal-
anced designs and the procedure for mixed linear
models (mixed) in unbalanced designs (Neter et
al. 1990; Rao 1998; Younger 1998). The means
were separated by the least square means state-
ment (ls means). To investigate the relationship
between the probability of larval mortality and
the weight at the time of needle puncture in wee-
vils, a correlation analysis was conducted with
the logistic regression procedure (logistic) and the
regression procedure (reg) (Rao 1998; Younger
1998). Mean values ( SD) are presented.
RESULTS
Infection Assays with Weevils
Both Helicosporidium spp. isolates were able
to infect and reproduce in D. abbreviatus larvae
and adults. Infection rates in larvae were high
(>85%, Table 1) and did not differ between iso-
lates after 3 weeks (df= 1, P = 0.1138, F = 4.07) or
after 6 weeks (df = 1, P = 0.7627, F = 0.10). Four
SjHe-treated larvae that did not show any symp-
toms of the disease after 3 weeks were diagnosed
as infected after 6 weeks, which is indicated by
the higher cumulative infection rate after 6 weeks
in Table 1. After 3 weeks, larval weight was re-
duced in CsHe-infected D. abbreviatus (105 78
mg) compared with uninfected larvae (228 75
mg) (df= 144, P < 0.0001, t = -7.94) (Table 2). The
weight of SjHe-infected larvae (191 82 mg) was
lower than in control larvae (df= 144, P = 0.0258,
t = -2.25) but higher than that of CsHe-infected
larvae (df = 144, P < 0.0001, t = 4.68). After 6
weeks, all uninfected larvae (from control and
treated groups) had reached an average weight of
478 144 mg (n = 54), approximately twice as
much as the weight of surviving SjHe- and CsHe-
infected larvae (252 115 mg, n = 27). An appar-
ent symptom of infected larvae was the cream-col-
ored hemolymph clearly visible through the integ-
ument (Fig. 1). No mortality occurred within 3
weeks post-treatment of the larvae. The percent-
age of larvae that died between 3 and 6 weeks (af-
ter the first bleeding for diagnostics) was higher
in CsHe-infected larvae than in control and SjHe-
infected larvae (df = 2, P = 0.0062, F = 10.28) (Ta-
TABLE 1. INFECTION RATES IN DIAPREPES ABBREVIATUS
LARVAE AFTER ORAL TREATMENT WITH HELICO-
SPORIDIUM SPP. CYSTS AT 2.5 x 105 CYSTS PER
LARVA.
Cumulative infection (%) after
Isolate 3 wk 6 wk
Controls 0 0 0 0
SjHe 76 3 86 8
CsHe 88 10 88 10
Florida Entomologist 88(4)
TABLE 2. LARVAL WEIGHT GAIN IN DIAPREPES ABBREVIATUS AFTER ORAL TREATMENT WITH HELICOSPORIDIUM SPP.
CYSTS AT 2.5 x 105 CYSTS PER LARVA.
Weight (mg)
Percent mortality
Isolate Infection N Initial After 3 wk After 6 wk between 3-6 wk
Controls No 67 15 4 a 228 75 a 480 149 a 25 a
SjHe Yes 32 15 4 a 191 82 b 251 112 b 38 a
No 6 16 3 a 192 85 ab 441 21 ab N/A
CsHe Yes 39 15 4 a 105 78 c 255 130 b 80 b
No 5 13 3 a 229 55 ab 472 86 a N/A
"Means followed by a different letter are significantly different (P < 0.05; SAS mixed procedure and Is means statement).
ble 2). There was a strong negative correlation be-
tween mortality and larval weight at the time of
diagnostic needle puncture (3 weeks after treat-
ment) (df = 1, P < 0.0001, x2 = 24.9177, logistic
procedure; r = -0.7575, reg procedure) (Fig. 2). Mi-
croscopic observations revealed a slower develop-
ment of the SjHe-isolate in the host hemolymph
(Fig. 3A, B). Approximately 50% of SjHe-infected
larvae showed vegetative cell development but no
cyst differentiation within 3 weeks of the treat-
ment, whereas cysts were found in all CsHe-in-
fected larvae at this time (Fig. 3B).
Oral force treatment of D. abbreviatus adults
with either isolate resulted in 100% infection af-
ter 3 weeks of challenge. No mortality occurred
within this time frame. The CsHe isolate devel-
oped faster in the host than the SjHe isolate
(Fig. 3C, D). Within 3 weeks of oral challenge,
many cysts and vegetative cells were observed in
the hemolymph of CsHe-treated weevils (Fig. 3D).
In SjHe-treated weevils, the majority of helicospo-
ridial life stages were contained within
hemocytes (Fig. 3C), and only a few vegetative
cells could be seen circulating freely in the
hemolymph.
Infection Assays with Mosquitoes
All three of the tested mosquito species were
susceptible to helicosporidial infection.Anopheles
quadrimaculatus was the most susceptible of the
three species, experiencing higher mortality (df =
4, P = 0.0229, F = 3.14) (Fig. 4) and infection rates
(df = 4, P < 0.0001, F = 48.91) (Fig. 5) at 7 d post-
treatment than the other two species tested. Mor-
tality in treated Cx. quinquefasciatus and Ae. ae-
gypti did not differ from mortality in any of the
control groups (Fig. 4). The SjHe isolate did not
cause any infection in Cx. quinquefasciatus at 105
cysts/ml (Fig. 5).
For both isolates, the hemolymph of infected
mosquito larvae was filled with vegetative stages
7 d after exposure. Cyst production was also seen
at this time. Melanized helicosporidial cells were
observed in the head and thorax region of mosqui-
toes infected with the CsHe isolate. The SjHe iso-
late did not cause this melanization response
unless vegetative stages were present in the
hemolymph.
An. quadrimaculatus larvae exhibited a clear
dosage response to both the SjHe and CsHe iso-
Fig. 1. Infected (left) and control (right) Diaprepes abbreviatus larvae 6 weeks after oral challenge with the CsHe
Helicosporidium sp. isolate. Bar: 5 mm.
December 2005
Conklin et al.: Helicosporidia in Heterologous Hosts
. 60
S40
0
20
0
50 100 150 200
Average larval weig
Fig. 2. Probability of mortality as
ofDiaprepes abbreviatus larvae at the
ture (SAS reg procedure). Data are ex
replicate control assays and three rep
says with each isolate of Helicosporidi
late of Helicosporidium sp. Moi
tion rates increased with incre
cysts (Table 3). These increases
infectivity were statistically significant for both
r=-0.7575 isolates except for the mortality due to SjHe.
Over a dosage range from 102 to 105 cysts/ml,
mortality increased from 20 26 to 59 36% (df
= 5, P = 0.134, F = 1.91) and from 10 9 to 93
+ 2% (df = 5, P = < 0.0001, F = 69.08) after expo-
sure to the SjHe and CsHe isolates, respec-
tively. Treatments with the SjHe and CsHe iso-
late over a dose range from 102 to 105 cysts/ml
resulted in increasing infection rates from 2 4
250 300 to 64 + 48% (df = 4, P = < 0.0001, F = 13.72) and
ht (mg) from 14 24 to 71 51% (df = 5, P = 0.0007, F
a function of weight = 6.24), respectively. On average, 35% of the 7-
time of needle punc- day mortality of An. quadrimaculatus was ex-
trapolated from five pressed within the first 24 h after exposure to
'licate treatment as- 105 cysts/ml (Fig. 6). This pronounced 24-h mor-
ium sp. tality was not seen in the other two species of
mosquito. When exposed to 105 cysts/ml, the
mortality of An. quadrimaculatus at 24 h was
rtality and infec- higher than the 24-h mortality of Cx. quinque-
asing dosages of fasciatus andAe. aegypti (df= 8, P = 0.0015, F =
in mortality and 3.82) (Fig. 6).
Fig. 3. Hemolymph samples from infected Diaprepes abbreviatus larvae (A, B) and adults (C, D) three weeks af-
ter oral challenge with Helicosporidium sp. A, C: SjHe isolate. B, D: CsHe isolate. Bars: 10 lim.
Florida Entomologist 88(4)
6 ___ Control
60
1 [ SjHe
50 OCsHe
o 40
30
20
10
Cx. quinquefasciatus Ae. aegypti An. quadrimaculatus
Fig. 4. Seven-day mean ( SD) percent mortality of Culex quinquefasciatus, Aedes aegypti, and Anopheles
quadrimaculatus at a dosage of 105 cysts/ml. Different letters indicate significant differences (P < 0.05; SAS glm
procedure and Is means statement).
Infection did not have an effect on the develop-
ment of An. quadrimaculatus larvae. At 6 d post-
treatment, head capsule size of An. quadrimacu-
latus exposed to SjHe or CsHe helicosporidia did
not differ from control head capsule size (df = 2,
P = 0.2999, F = 1.27) (Fig. 7). InAe. aegypti, infec-
tion with the SjHe isolate did affect larval devel-
opment. The head capsule size of larvae exposed
to SjHe was smaller than control head capsule
sizes (df = 1, P = 0.0185, F = 6.88).
DISCUSSION
Our ability to infect a new coleopteran host,
D. abbreviatus, and three mosquito species with
the same two isolates of helicosporidia supports
the wide host range of helicosporidia. In addition,
these two isolates infect three species of noctuid
Lepidoptera (Blaske & Boucias 2004; Blaske-
Lietze & Boucias 2005) and the SjHe isolate also
infects three other dipteran hosts (Boucias et al.
2001). However, in the present study, suitability
for helicosporidial development varied among dif-
ferent host and isolate systems. Of the mosqui-
toes tested, An. quadrimaculatus was the most
susceptible, whereas Cx. quinquefasciatus andAe.
aegypti were less suitable hosts. Similar results
were obtained with a Helicosporidium sp. isolated
from Cx. nigripalpus (Fukuda et al. 1976). The de-
velopmental cycle of the pathogen appeared to be
prolonged in the weevil host. In contrast to the
known susceptible lepidopteran and dipteran spe-
cies (Boucias et al. 2001; Blaske & Boucias 2004;
Blaske-Lietze & Boucias 2005), D. abbreviatus
has a long life cycle with an extended larval pe-
riod. When reared on artificial diet under con-
trolled conditions, the larval stages of the weevil
D. abbreviatus, the noctuid Spodoptera exigua
(Hiibner), and the mosquitoAn. quadrimaculatus
last 106-125 d (Lapointe 2000), 13-15 d, and 7-9 d,
respectively. After three weeks, four of the weevil
larvae exposed to SjHe appeared uninfected, but
at six weeks, these larvae were diagnosed as in-
fected, indicating that the infection progressed
slowly. Lepidopteran larvae have been found in-
fected with this isolate within 2 d post-treatment
(Blaske-Lietze & Boucias 2005), while mosquito
larvae have been found infected with this isolate
within 3-4 d post-treatment (Boucias et al. 2001).
Also, cyst differentiation in the hemolymph ap-
pears to be adjusted to the speed of development
of the host insect. Whereas the SjHe isolate
formed mature cysts within 6-7 d in S. exigua
(Blaske-Lietze & Boucias 2005), and within 7 d in
different mosquito hosts, in approximately 50% of
SjHe-infected weevil larvae and in 100% of SjHe-
infected weevil adults, no cysts had differentiated
within 3 weeks. Mortality ofD. abbreviatus larvae
was highest between 3 and 6 weeks post-treat-
ment, while the majority of infected S. exigua lar-
vae died at pupation (10 d post-treatment), and
Fukuda et al. (1976) and Hembree (1981) re-
ported that mosquito mortality occurred in the
fourth instar (5-7 d post-treatment).
The interaction between host development and
helicosporidia development is complex, and while
the host life span may influence the helicosporidia
development, the helicosporidia also influence the
December 2005
Conklin et al.: Helicosporidia in Heterologous Hosts
70
60 SjHe
SOCsHe
S40
30
20 -
10
Cx. quinquefasciatus Ae. aegypti An. quadrimaculatus
Fig. 5. Seven-day mean ( SD) percent infection of Culex quinquefasciatus,Aedes aegypti, andAnopheles quadri-
maculatus at a dosage of 105 cysts/ml. Different letters indicate significant differences (P < 0.05; SAS glm procedure
and Is means statement).
host's development, as seen in the larval weight
gain and head capsule data. It appears that heli-
cosporidia cause a variety of developmental re-
sponses in its many hosts. Recently, Blaske &
Boucias (2004) reported that helicosporidia infec-
tion did not affect larval weight gain in three noc-
tuid hosts. However, infection reduced adult lon-
gevity and produced deformities in adults, indi-
cating that development of adults was affected
though larval weight gain was not. Larval weight
gain in D. abbreviatus was severely affected by in-
fection with helicosporidia, though what effect
this infection would have on the larvae as they
pupate and emerge as adults remains to be seen.
Unlike the D. abbreviatus, An. quadrimaculatus
did not experience a delay in development due to
infection with helicosporidia. This might be ex-
plained by the short developmental time of mos-
quito larvae compared to the weevil. However, ex-
posedAe. aegypti larvae did experience a develop-
mental delay. It is difficult to say what would ac-
count for the differences in the two mosquito
species. Mosquitoes are known to exhibit a vari-
ety of developmental responses to pathogen infec-
tion. Giblin & Platzer (1985) found that mosquito
larvae parasitized by a mermithid nematode ex-
hibited a reduced feeding rate, and developed
more slowly than uninfected larvae. Alterna-
tively, Agnew et al. (1999) found that larval fe-
males infected with a microsporidian parasite de-
veloped more quickly than uninfected females
and emerged as smaller adults. The high 24-h
mortality ofAn. quadrimaculatus observed in the
present study may have had an effect on the ap-
parent development rate, as the mosquito larvae
are sensitive to density, and develop more quickly
under low-density conditions than high-density
conditions (Christophers 1960).
While both isolates infected the experimental
hosts, the two isolates did exhibit differences.
Within 3 weeks, the lower virulence of the SjHe
isolate was evident in orally challengedD. abbre-
viatus. The SjHe isolate had less of an effect on
body weights of infected larvae and developed
more slowly in the hemolymph of both larvae
and adults of the citrus root weevil. Despite high
infection rates caused by both isolates (76 and
88% by SjHe and CsHe isolate, respectively), no
larval mortality occurred within 3 weeks. Be-
tween 3 and 6 weeks, however, a high number of
CsHe-infected larvae died (80%), whereas of the
SjHe-infected and control larvae, the majority
survived (62 and 75%, respectively). Mortality
was negatively correlated to larval weight at the
time of the first needle puncture (3 weeks post-
treatment), which accounts for the similar
weights of CsHe- and SjHe-infected weevil lar-
vae recorded after 6 weeks. The CsHe isolate has
also been shown to be very virulent in suscepti-
ble noctuid hosts; following oral challenge with 2
x 105 cysts per larva, 55% of infected S. exigua
died within 13 d and 85% of infected Trichoplu-
sia ni (Hiibner) died within 15 d (Blaske & Bou-
cias 2004).
In the mosquito system, the two isolates
showed no difference in their influence on the de-
velopment of An. quadrimaculatus or infection
and mortality of the three tested species of mos-
Florida Entomologist 88(4)
TABLE 3. MEAN ( SD) MORTALITY AND INFECTION RATES IN ANOPHELES QUADRIMACULATUS 7 D AFTER EXPOSURE TO
SJHE AND CSHE AT DIFFERENT DOSAGES.
Dose (cysts/ml) Isolate N Percent Mortality" Percent Infection"
0 SjHe 6 15+ 10 a Ot0a
102 SjHe 5 20 + 26 a 2 4a
103 SjHe 6 22 + 34 a 7 6a
104 SjHe 6 28 + 31 a 44 22 b
105 SjHe 3 59 36 a 64 + 48 b
0 CsHe 6 11 9 a 0 a
102 CsHe 3 10 9 a 14 + 24 a
103 CsHe 3 11 + 3 a 27 + 25 b
104 CsHe 6 23 10 b 65 + 26 c
105 CsHe 3 93 2 c 71+ 51c
"Means followed by a different letter are significantly different (P < 0.05; SAS gim procedure and Is means statement).
quitoes. While there was no statistical difference
between mortality and infection rates with the
CsHe and SjHe isolates, the observed melaniza-
tion of the CsHe isolate indicates that mosquitoes
are less suitable hosts for the CsHe isolate as op-
posed to the SjHe isolate. Several other research-
ers have observed melanization of coleopteran
and other isolates of Helicosporidium sp. in mos-
quitoes. Fukuda et al. (1976) reported that mela-
nization occurred with mosquitoes exposed to
helicosporidia isolated from the nitidulid beetle C.
mutilatus. Avery & Undeen (1987) reported simi-
lar melanization in mosquito larvae infected with
a pond-water isolate and suggested that the mel-
anization may be an indication that mosquitoes
are abnormal hosts for helicosporidia. The phylo-
genetic affinity of mosquitoes to the original sim-
70
a a a a a
60
50
;40
30
20
0
10 -
0- T I- T
ulid host may explain the lack of melanization in
individuals infected with the SjHe isolate.
The results of this study demonstrate that
both the SjHe and the CsHe Helicosporidium sp.
isolates caused dosage-dependent infection rates
in An. quadrimaculatus that were directly pro-
portional to the applied cyst concentration. Dos-
age-dependent mortality was confined to the
CsHe isolate. A dose response to helicosporidial
challenge has been shown in other studies con-
ducted with different host insects and different
Helicosporidium sp. isolates. For example, treat-
ment ofAe. aegypti larvae with a mosquito isolate
from Thailand (isolated from Ae. aegypti) at con-
centrations ranging from 5 x 102 to 5 x 104 cysts
per milliliter (100-fold increase) resulted in infec-
tion rates increasing from 4 to 100% (Hembree,
a ab bc c
Control
SeSjHe
O- CsHe
Cx. quinquefasciatus Ae. aegypti An. quadrimaculatus
Fig. 6. Mean percent ( SD) mortality of Culex quinquefasciatus,Aedes aegypti, andAnopheles quadrimaculatus
recorded 24 h post-treatment with 105 cysts/ml. Different letters indicate significant differences (P < 0.05; SAS glm
procedure and Is means statement).
December 2005
Conklin et al.: Helicosporidia in Heterologous Hosts
80
70
60
E50
4 40
0 30
I
20
10
0
Ae. aegypti An. quadrimaculatus
Fig. 7. Mean ( SD) head capsule width ofAedes aegypti andAnopheles quadrimaculatus 7 d post-treatment. An
asterisk (*) indicates a significant difference from the control (P < 0.05); NS indicates no significant difference from
the control (P > 0.05) (SAS glm procedure and Is means statement).
1981). In contrast to these findings, no dosage re-
sponse was seen in a study conducted with the
CsHe isolate and larvae of the noctuid H. zea
(Bliske & Boucias 2004). Regardless of a 20-fold
increase in the administered dosage (ranging
from 104 to 2 x 105 cysts per insect), only 50 to 60%
of the challenged H. zea showed manifestation of
the disease.
In the present study, helicosporidia also had a
pronounced effect on the 24-h mortality of An.
quadrimaculatus. This early pulse of mortality
was likely the result of septicemia facilitated by
the ingestion of helicosporidia. Dead first-instar
larvae of An. quadrimaculatus exposed to SjHe
Helicosporidium sp. at 106 cysts/ml contained nu-
merous helicosporidial filamentous cells piercing
the midgut epithelium; the resulting damage to
the midgut barriers potentially allowed for the in-
gress of opportunistic bacteria. Factors that influ-
enced the filamentous cell's penetration of the
gut-the number of cysts ingested, dehiscement
rate of cysts in the midgut, the strength of the
midgut wall, or the resident gut microflora-thus
may have dictated the early larval mortality ob-
served with An. quadrimaculatus. Avery & Un-
deen (1987) reported a similar effect of high 72-h
mortality in An. quadrimaculatus, Cx. salinarius,
and Ae. aegypti exposed to helicosporidia isolated
from pond water.
The helicosporidia remain cryptic pathogens.
This study has demonstrated both the wide host
range and the discrete species effects of two iso-
lates. While these two isolates are capable of in-
fecting a similar range of species, there are defi-
nite isolate effects in the timing, intensity, and
symptoms of infection. Further research is
needed to understand the evolutionary relation-
ship between these two isolates and their hosts.
ACKNOWLEDGMENTS
We thank Hannah Snyder and Natalie van Hoose for
technical assistance; Suzanne Fraser (DPI, Gainesville,
FL) and Heather Furlong and Genie White (USDA,
Gainesville, FL) for generously providing insects and
diet; Janice Col (Statistical Consulting Unit, Depart-
ment of Statistics, UFL, IFAS, Gainesville, Florida) for
statistical advice; and two anonymous reviewers for crit-
ical comments on an earlier draft of the manuscript.
This work was supported by a grant from the National
Science Foundation (NSF, MCB-0131017). Florida Agri-
cultural Experiment Station Journal Series No. R-
10762.
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Florida Entomologist 88(4)
Nava et al.: Population Dynamics ofS. catenifer and Parasitoids
POPULATION DYNAMICS OF STENOMA CATENIFER
(LEPIDOPTERA: ELACHISTIDAE) AND RELATED LARVAL PARASITOIDS
IN MINAS GERAIS, BRAZIL
DORI E. NAVA1'4, JOSE R. P. PARRA1, VALMIR A. COSTA2, TANIA M. GUERRA3 AND FERNANDO L. CONSOLI1
'Depto. de Entomologia, Fitopatologia e Zoologia Agricola, ESALQ/USP, Caixa Postal 9, 13418-900, Brazil
2Instituto Biol6gico. Centro Experimental do Instituto Biol6gico. Laborat6rio de Controle Biologico
Caixa Postal 70, 13001-970 Campinas, SP, Brazil
3Laborat6rio de Entomologia, Depto. Fitotecnia, CCA/UFSC, Caixa Postal 476, 88040-900, Florian6polis, SC, Brazil
4Corresponding author; e-mail:
ABSTRACT
The avocado seed moth Stenoma catenifer Walsingham (Lepidoptera: Elachistidae) can
cause total production losses and is considered a key pest of avocado in Brazil. This research
aimed to evaluate the larval dynamics of S. catenifer and its associated parasitoids in an av-
ocado growing region throughout two consecutive agricultural seasons. The highest percent-
age of fruits attacked by S. catenifer was found in the period near harvest, starting in June.
The percentage of infested fruits in the first agricultural season was approximately 60%,
near 11% above that of the second season. Five undetermined braconids within five genera
(Dolichogenidea sp., Hypomicrogaster sp.,Apanteles sp., Chelonus sp., and Hymenochaonia
sp.) and two ichneumonids (Eudeleboea sp. and Pristomerus sp.) were found. Dolichogenidea
sp. andApanteles sp. were the most abundant, and therefore are the species with the highest
potential as S. catenifer population regulators. In both seasons, the emergence peak of par-
asitoids occurred at the end of the winter (August), when at least 30 and 40% total larval
parasitism was observed.
Key Words: Persea americana, biological control, larval parasitoids
RESUMO
A broca-do-abacate, Stenoma catenifer Walsingham, 1912, 6 considerada praga-chave do
abacateiro no Brasil, podendo acarretar perdas totals da producgo. O objetivo deste trabalho
foi quantificar, identificar os parasit6ides larvais de S. catenifer e verificar as 6pocas de oco-
rr6ncia da fase larval deste hospedeiro e de cada esp6cie de parasit6ide em duas safras agri-
colas consecutivas. A maior porcentagem de frutos atacados por S. catenifer foi verificada no
period pr6ximo a colheita, a partir do m6s de junho. A porcentagem de frutos infestados na
primeira safra agricola foi de aproximadamente 60%, sendo superior a da segunda safra,
cerca de 11%. Foram encontradas cinco parasit6ides da familiar Braconidae (Dolichogenidea
sp., Hypomicrogaster sp.,Apanteles sp., Chelonus sp. e Hymenochaonia sp.) e dois da familiar
Ichneumonidae (Eudeleboea sp. e Pristomerus sp.). Dolichogenidea sp. eApanteles sp. foram
as esp6cies que apresentaram maior potential como reguladoras populacionais de S. cateni-
fer. Nas duas safras, o pico de emerg6ncia dos parasit6ides ocorreu no m6s de agosto, quando
foi verificado um parasitismo larval entire 30 e 40%.
Translation provided by the authors.
Mealybugs, leaf caterpillars, wood borer and
the Limeira beetle are among the many species of
insects reported from avocado orchards, but re-
ports of these species as pests in avocado orchards
in Brazil occurred only early in the 90s (Teixeira
1992). By the end of the 1980s and beginning of
the 1990s, groves in the States of Parana, Sao
Paulo, Minas Gerais, and Espirito Santo were
fairly infested by the avocado borer, Stenoma
catenifer Walsingham. Currently, this insect is
considered the most important avocado pest in
the main avocado producing regions, where it can
cause complete crop failure (Hohmann & Mene-
guim 1993).
Several studies have been conducted to estab-
lish suitable management practices to control
this pest, especially through chemical control
(Fornazieri et al. 1994; Hohmann et al. 2000).
However, the use of insecticides is hindered by a
lack of larval sampling methods and the fact that
the larvae are protected from contact insecticides
since they feed inside the fruit.
Florida Entomologist 88(4)
Biological control is a suitable alternative
method for S. catenifer control. Natural rates of
parasitism as high as 40% by egg parasitoids have
been recorded in Parand State (Hohmann & Mene-
guim 1993), and the possibility of using these nat-
ural enemies has fostered further research on the
thermal requirements and parasitoid strain selec-
tion (Maceda et al. 2003; Nava & Parra 2003). Lar-
val parasitoids also seem to be an important com-
ponent in the natural control of S. catenifer, with
natural rates of parasitism ranging from 9 to 30%
(Boscan de Martinez & Godoy 1982; Hohmann &
Meneguim 1993). Despite the potential use of lar-
val parasitoids as biological control agents of the
avocado moth through augmentative or inundative
releases, studies on the efficiency and rearing of
these larval parasitoids were limited until recently
due to the lack of an artificial rearing system for
S. catenifer (Nava & Parra 2005).
Identifying larval parasitoids and determining
their temporal occurrence will advance avocado
borer biological control efforts. The objective of
this research was to identify and quantify larval
parasitoid temporal occurrence throughout the
avocado production cycle in the state of Minas
Gerais (MG), Brazil, to support the establishment
of control strategies against this pest.
MATERIALS AND METHODS
Study Site
The present study was carried out in a com-
mercial avocado grove (cultivar Margarida) lo-
cated in Sao Tomas de Aquino, MG (2052'30"S,
47007'30"W and 1,000 m elevation). The study
was conducted during the 2001-2002 and 2002-
2003 cropping seasons, from December to Sep-
tember of the following year.
The avocado grove occupied an area of approx-
imately 4 hectares, with trees spaced at 10 m
within rows and 15 m between rows, totaling
around 100 trees per hectare. The survey was con-
ducted every 15 d, totaling 15 and 21 collections of
fruits for avocado borer population dynamics and
larval parasitoid studies, respectively. In the first
cropping season, seven sprays were made by al-
ternating pyrethroids and organophosphates,
while the second cropping season required 11 ap-
plications.
Population Dynamics of S. catenifer
Eleven trees were selected in a zigzag sampling
technique. Each tree was circled and one fruit was
collected from each side (north, south, east, and
west) of the tree at an intermediate height, total-
ing four fruits/tree, to evaluate the population dy-
namics of S. catenifer. Fruits were brought to the
lab to evaluate the population dynamics of the av-
ocado moth after fruit dissection and determina-
tion of the percentage of infested fruits through-
out the production cycle (January-August).
Parasitoids Associated with S. catenifer
In the larval parasitoid survey, 50 infested
fruits were collected at random. The fruits were
taken to the laboratory for dissection and removal
of larvae. Larvae were individually placed in plas-
tic containers (10 cm height x 6 and 8 cm at the
top and base, respectively) and fed avocado seeds
until emergence of the S. catenifer adult or para-
sitoids. The stage at which the host was at the mo-
ment parasitoid emergence occurred, and the
number of parasitoids emerged per host were de-
termined. All insects were maintained under con-
trolled conditions (25 + 1C, 70 10% relative hu-
midity and 14:10 (L/D) photoperiod).
The percentage of parasitoids was determined
based on the number of parasitized larvae from
which parasitoids developed successfully in rela-
tion to the total larvae collected. We did not score
for cases of superparasitism or parasitization of
hosts at an unsuitable stage which would lead to
an unsuccessful parasitoid development and/or
host's death. The specimens obtained were pre-
served in 70% alcohol for identification, and
voucher specimens were deposited in the entomo-
logical collection of the Museu "Oscar Monte",
Centro Experimental do Instituto Biol6gico,
Campinas, SP
RESULTS AND DISCUSSION
Population Dynamics of S. catenifer
Infestation of fruits by S. catenifer was less fre-
quent during the initial stages of the survey (De-
cember, January, and February), but increased as
fruits were getting ready to harvest (Fig. 1). The
infestation in the 2001-2002 cropping season
reached 60% in June and was much higher than
the peak observed during 2002-2003 season. Al-
though the infestation in the first cropping season
was considerable, there is a report from Venezu-
ela demonstrating it can reach up to 80% of the
fruits (Boscan de Martinez & Godoy 1982).
The difference in infestation between both
cropping seasons was probably related to the
more intensive use of insecticides in the second
cropping season (11 vs. 7 applications). The diffi-
culty in establishing control strategies is also due
to the fact that cultivar Margarida is a medium-
and late-cycle plant, and the fruit may remain in
the field for approximately one year. Therefore, a
more detailed sampling of groves should be made
by initial growers, and insecticide application
should be made when the first fruits showing at-
tack symptoms (punctuations with white exudate
on the fruits due to larvae boring through the exo-
carp) are detected.
December 2005
Nava et al.: Population Dynamics ofS. catenifer and Parasitoids
--Cropping Season 2001-2002 -O-Cropping Season 2002-2003
60-
50
40
S30
20
10 -
Dec Jan Feb Mar Apr May Jun Jul Aug
Fig. 1. Seasonal abundance of the larval stage of Stenoma catenifer on cultivar Margarida avocado trees, during
the 2001-2002 and 2002-2003 cropping seasons (December-August). Sao Tomas de Aquino, MG.
Parasitoids Associated with S. catenifer
Undetermined species of the genera Dolicho-
genidea sp., Hypomicrogaster sp., Apanteles sp.,
Chelonus sp., Hymenochaonia sp. (Hymenoptera,
Braconidae); and Eudeleboea sp., and Pris-
tomerus sp. (Hymenoptera, Ichneumonidae) were
detected (Fig. 2A). The first genus is gregarious,
while the others are solitary, but all are koino-
biont endoparasitoids. The braconids emerged
from the host early during the last larval stage,
while ichneumonids emerged late in this stage as
the host approached pupation.
This is the first report of Dolichogenidea sp.,
Hypomicrogaster sp., Hymenochaonia sp., and
Pristomerus sp. as avocado borer parasitoids. Che-
lonus and Eudeleboea have already been associ-
ated with S. catenifer in Guiana (Cervantes et al.
1999), andApanteles has been recorded in Venezu-
ela (Boscan de Martinez & Godoy 1982). In Brazil,
Hohmann & Meneguim (1993) reported parasit-
ism of S. catenifer larvae by two unidentified bra-
conids and one ichneumonid species. In another
study, Hohmann et al. (2003) mentioned the collec-
tion of an ichneumonid, possibly in the genus
Eudeleboea, and a braconid in the genusApanteles.
Dolichogenidea sp. and Chelonus sp. were the
first parasitoids to emerge from S. catenifer lar-
vae, followed by Apanteles sp. and Hypomicro-
gaster sp., in the first cropping season (Fig. 3). In
the second cropping season, Dolichogenidea sp.
followed the same emergence pattern found dur-
ing the first season, but the occurrence of Apan-
teles sp. and Hypomicrogaster sp. was delayed.
Apanteles sp. was first recorded in April and Doli-
chogenidea sp. only in June (Fig. 3). The emer-
gence of Eudeleboea sp. suggested an irregular
behavior because specimens were collected in
March and April in the 2001-2002 cropping sea-
son, and in September in the following year. Pris-
tomerus sp. and Hymenochaonia sp. were re-
corded in the months of May and June, respec-
tively (Fig. 3).
Dolichogenidea sp. and Apanteles sp. were col-
lected throughout both cropping seasons, indicat-
ing they are present and are able to exploit hosts
even when insecticides are applied (Fig. 3). The gre-
gariousness of Dolichogenidea sp. and the exploita-
tion of hosts early in the season may be advanta-
geous to this species as a biological control agent of
S. catenifer. Two other species were also abundant,
Apanteles sp. and Hypomicrogaster sp., and are also
good candidates for the implementation of biologi-
cal control programs in avocado orchards.
Chelonus sp., Hymenochaonia sp. and Pris-
tomerus sp. were not found in the second cropping
season (Fig. 2B), which could be associated to a
reduced host searching capacity as indicated by
their low abundance in this season (3%, 2%, and
1%, respectively). Also, these species could have
been either (1) displaced by the most abundant
ones as the host population was dramatically re-
duced after adoption of a more intensive use of
chemicals during the second crop season, or (2)
they might have suffered more the side-effects of
the chemicals utilized for the control of the avo-
cado seed moth.
According to Elzen & King (1999), the use of
insecticides is the main factor to be taken into ac-
count when natural enemy conservation is the
goal. In the present study, we observed that the
increased use of insecticides in the second crop-
ping season could have contributed to decrease
the number of parasitoid species (Fig. 2). How-
Florida Entomologist 88(4)
Eudeleboea sp.
2%
Hymenochaonia sp.
2%
Pristomerus sp.
1%
Dolichogenidea sp.
39%
Hypomicrogaster sp.
35%
Apanteies sp.
18%
Hypomicrogaster sp.
9%
Eudeleboea sp.
1%
s sp. .
i IoIic11genidea
Oolichogenidea
77%
sp.
Fig. 2. Relative frequency of larval parasitoid species collected on cultivar Margarida avocado plants during the
2001-2002 production cycle (December-September) (A) and 2002-2003 production cycle (B). Sao Tomas de Aquino, MG.
ever, the greater number of insecticide applica-
tions did not decrease total parasitism (sum of
parasitism of all species) in the second cropping
season (2002-2003), which was near 47% in the
month ofAugust, higher than the 32% recorded in
the 2001-2002 cropping season (Fig. 4). It can also
be observed that the percentage of parasitism in-
creases as S. catenifer population grows (Figs. 1
and 4), thus revealing a reciprocal density mech-
anism, where the population density of the mor-
tality agent depends directly on the population
density of the herbivore host (Huffaker & Mes-
senger 1976).
Boscan de Martinez & Godoy (1982) related a
S. catenifer parasitism in Venezuela of up to 30%
byApanteles. In the State of Parand, Brazil, Hoh-
mann & Meneguim (1993) observed parasitism of
9%, also caused by Apanteles. The present study
verified that parasitism level was variable
throughout the cropping seasons (Fig. 4). In addi-
tion, the comparison of results is difficult, since
the authors quoted above did not mention the
time during the season when their surveys were
carried out.
The occurrence of Perilampus sp. (Hymenop-
tera: Perilampidae) as a hyperparasitoid of Doli-
chogenidea sp. was also observed. The interaction
between hyperparasitoid and primary parasitoid
may often reduce the population of the latter and
negatively interfere with the population reduc-
tion of the herbivore. In this case, even though 7%
parasitism was recorded, it is believed that this
hyperparasitoid would not interfere with the con-
trol of S. catenifer, since it was recorded only in
December 2005
Apantele
13%
Nava et al.: Population Dynamics ofS. catenifer and Parasitoids
Dolichogenidea sp.
Apanteles sp.
Hypomicrogaster sp.
Chelonus sp.
Pristonerus sp.
Hymenochaonia sp.
Eudeleboca sp,
Dolichogenidea sp.
Axpanieles sp.
Hypomicrogaster sp.
Eudeleboea sp.
I I I
Dec Jan Feb Mar Apr May Jun Jul Aug Sep
Fig. 3. Distribution of Stenoma catenifer larval parasitoid species, collected in a cultivar Margarida avocado or-
chard along a production cycle (December-September), in the 2001-2002 (A) and 2002-2003 (B) cropping seasons.
Sao Tomas de Aquino, MG.
the month of August, the moment at which fruit
harvest had begun, not resulting in a numeric re-
duction ofDolichogenidea sp.
Our study suggests that these larval parasi-
toids may play an important role in S. catenifer
population dynamics, and that these parasitoids
should be conserved as much as possible as they
may make a significant contribution toward the
integrated management of the pest. Our study
also points for the necessity of a better look on the
host-parasitoid population dynamics as parasit-
ism rates were very similar even when host pop-
ulation was reduced 6-fold, indicating augmenta-
tive biological approaches could be required for
successful control of the avocado seed moth at
higher host densities. Finally, as to efficiently de-
velop parasitoid conservation strategies or to im-
45
40 O -Cropping season 2001/2002 -0-C
35
30
E 25 -
20
a 15 -
10
5
0 -
plement an augmentative biological control pro-
gram, further studies on the structure of the par-
asitoid community associated to this moth and
the identification of selective chemicals should be
conducted.
ACKNOWLEDGMENTS
Thanks to Agronomist Jos6 Carlos Goncalves, owner
of the Caf6 Total Company, for funding the research, to
laboratory technician Neide Graciano Z6rio, to Agrono-
mist Peter Kasten Junior, M.Sc., for support in the re-
search, to Coordenacao de Aperfeicoamento de Pessoal
de Nivel Superior (Capes) for a scholarship granted to
the first author, and to Conselho Nacional de Desen-
volvimento Cientifico e Tecnol6gico (CNPq) for a newly-
graduated-Ph.D. fellowship granted to the last author.
droppingg season 2002/2003
Dec Jan Feb Mar Apr May Jun Jul Aug Sep
Fig. 4. Total parasitism of Stenoma catenifer caterpillars collected in a cultivar Margarida avocado orchard along
a production cycle (December-September), in the 2001-2002 (A) and 2002-2003 (B) cropping seasons. Sao Tomas de
Aquino, MG.
I I
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Florida Entomologist 88(4)
Childers et al.: Thrips Species Biting Man
A REVIEW OF THRIPS SPECIES BITING MAN INCLUDING RECORDS
IN FLORIDA AND GEORGIA BETWEEN 1986-1997
CARL C. CHILDERS1, RAMONA J. BESHEAR2, GALEN FRANTZ3 AND MARLON NELMS4
'University of Florida, IFAS, Department of Entomology and Nematology CREC
700 Experiment Station Road, Lake Alfred, FL 33850
2University of Georgia, Experiment Station, Griffin, GA 30223-1731
'1415 Greenwood Ave., Lehigh Acres, FL 33972-1625
4Metropolitan Dade County Public Works Dept., Mosquito Control Division, 8901 N.W. 58th St., Miami, FL 33178
ABSTRACT
Two species of thrips (Frankliniella tritici and Limothrips cerealium) were reported biting
people in Georgia during 1986 and 1990. Frankliniella bispinosa was the only thrips species
documented biting people in Florida between 1986 and 1997. This species occurs in great
abundance throughout Florida and is a recognized plant pest on numerous crops. Because of
the tremendous numbers ofF. bispinosa that are produced on dozens of different cultivated
and weed host plants during the spring months, this species can become a serious nuisance
problem. Frankliniella bispinosa will bite people and produce variable reactions from slight
irritation to formation of a rash with associated intense itching that can last for several days.
The problem usually is seasonal and associated with maximum seasonal increase in F. bis-
pinosa populations and favorable environmental conditions that can occur anytime between
February and April in central and south Florida. Colored sticky-trap tests in Florida dem-
onstrated the strong attraction of both white and blue colors to F bispinosa. People wearing
white or blue colored clothing tend to be more at risk of being bitten by this thrips pest.
Key Words: Thysanoptera, Thripidae, Phlaeothripidae, nuisance insects, biting insects
RESUME
Dos species de trips (Frankliniella tritici y Limothrips cerealium) son reportadas picando
personas en Georgia durante 1986 y 1990. Frankliniella bispinosa es la unica especie de
trips conocida que pico personas en la Florida entire 1986 y 1997. Esta especie occure en
abundancia en toda la Florida y es reconocida como plaga en numerosos cultivos. Debido al
gran numero de F bispinosa que se reproducen en docenas de plants cultivadas y malezas
diferentes durante los meses de la primavera, esta especie puede llegar a ser un problema
fastidioso y serio. Frankliniella bispinosa produce reacciones en las personas que pica, desde
una irritaci6n ligera hasta un sarpullido associado con una picaz6n intense que puede durar
various dias. El problema usualmente es estacional y en asocio con el aumento maximo por pe-
riodo en la poblaci6n de F bispinosa, y con condiciones ambientales favorables que occuren
entire febrero y abril en la parte central y sur de Florida. En pruebas utilizando trampas pe-
gajosas con color en Florida, F bispinosa domonstro una atracci6n fuerte al color blanco y
azul. Personas llevando ropa de color blanco o azul son mas propensas al riego de ser picadas
por esta plaga trips.
Insects in the Order Thysanoptera, commonly
known as "thrips" have a wide variety of niches
and include phytophagous (several are serious
economic pests), fungivorous, and predacious spe-
cies (Lewis 1997). Most adults have two pairs of
developed wings and are capable of flying. Several
out of 5500 species disperse aerially (Lewis 1973).
Because of their small size, rapid movement, and
usually light color, thrips are often over-looked by
most people except when causing damage to flow-
ers, leaves, or fruits of various agricultural or syl-
van crops. An infrequently reported problem di-
rectly affecting people occurs when thrips become
a nuisance by traveling in large swarms and land-
ing on clothing and exposed parts of their heads,
necks, and arms.
Some confusion has lingered in recent years
with the idea that thrips have rasping-sucking
mouthparts (Davidson & Peairs 1966; Pfadt
1985). All thrips have piercing-sucking mouth-
parts that consist of a single mandibular stylet
and two opposable and interlocking maxillary
stylets (Heming 1978; Chisholm & Lewis 1984;
Hunter & Ullman 1992). The mandibular stylet
lacks an opening or food channel and is used prin-
cipally to punch a hole in the substrate followed
by insertion of the paired, tongue-in-grooved
maxillary stylets (Heming 1978). The stylets are
Florida Entomologist 88(4)
housed within the mouthcone situated just ante-
rior of the first pair of legs. Thrips feeding stages
include adults of both sexes and two instars.
Adult and larval thrips can bite people (Bailey
1936) and cause welts and rashes or other dermal
reactions (Lewis 1973). When thrips bite people
the stylets are apparently used to pierce a per-
son's skin and extract fluids (Johnson 1925; Hood
1927; Bailey 1936). Irritation by thrips to people
likely results from the rapid extension and retrac-
tion of the insect's maxillary stylets singly or to-
gether. Subsequent skin penetration and release
of thrips saliva could ensue. The right maxillary
stylet tip is larger and slightly broader than that
of the left, at least in Frankliniella bispinosa
(Morgan) (Childers & Achor 1991a). Thus, rapid
extension and retraction of the paired stylets can
result in different stylet pathways, some of which
could penetrate a person's exposed skin. The max-
illary stylets provide for both excretion of saliva
and uptake of partially digested food or liquid.
Although past instances of thrips biting people
and other nuisance problems have been reported
in publications from several countries including
the United States, more recent reports from Flor-
ida and Georgia have not been published. It is
helpful to educate people to the fact that some
thrips species can bite humans. Such biting does
not result in any known disease transmission but
skin irritations are known to occur. In some cases,
it is possible that thrips biting people could be
confused with mosquitoes or biting midges such
as Culicoides species (no-see-ums). The objectives
of this paper are to review the documented re-
ports of thrips biting humans and to further doc-
ument incidences of specific thrips species biting
people in Florida and Georgia between 1986 and
1997. Symptoms and effects to humans associated
with thrips irritation and biting are presented.
Review of Documented Cases of Thrips Biting People
One of the earliest instances of thrips being a
nuisance to people was reported by Curtis (1883).
He observed a species of black thrips causing skin
irritation during hot weather but did not mention
that humans were bitten. This report was similar
to Korting (1930), who discussed swarms ofLimo-
thrips sp in Europe, and the insects subsequently
crawled around on people's skin and produced un-
pleasant itching sensations (localized inflamma-
tions) from bites. The author concluded that these
thrips were probing to obtain moisture. Limo-
thrips cerealium Haliday produced an itching
sensation and inflammation of the nose and ears
of people in grain fields near Kiel, Germany dur-
ing July and August (Bailey 1936).
Karnyothrips flavipes (Jones), a predacious
species was observed feeding on human blood on
two occasions in Trinidad (Hood 1927). The insect
became distended and reddish in color while feed-
ing. The bites caused local raised lesions about 6
mm in diameter with a surrounding blotched area
of 19 mm by 13 mm. According to Bailey (1936)
second instars of both Thrips tabaci Lindeman
and Frankliniella moultoni Hood (= F occidenta-
lis Pergande; S. Nakahara, pers. comm. 2005) are
capable of piercing human skin and sucking
blood. This results in a pinkish dotted area being
formed at the site of the bite. There was slight
itching with no associated swelling at the location
of the bites. Other predacious thrips species such
as Scolothrips sexmaculatus (Pergande), Lepto-
thrips mali (Fitch), Aeolothrips fasciatus (L.), and
A. kuwanii Moulton were less likely to bite man
and cause inflammation.
Heliothrips indicus Bagnall (= Caliothrips in-
dicus (Bagnall) (S. Nakahara, pers. comm. 2005)
was reported to bite man on several instances at
Khartoum in Sudan during October 15, 1924
(Johnston 1925). According to the author, the bite
was sharp and painful causing severe irritation
followed by localized inflammation. Blood corpus-
cles were not found in the digestive tract of the
thrips, and Johnston concluded that the thrips
were merely seeking moisture. He also suggested
that the irritation from the stylet penetration was
caused by the insect's saliva.
According to Lewis (1973), thrips occasionally
irritate people out of doors and can be a minor nui-
sance inside buildings. Attacks on people outside
usually occurred in hot, sultry weather when
large numbers of migrating thrips alighted on
their bare skin. The itching and prickling sensa-
tions produced by the thrips were believed to be
caused by their attempts to obtain water from the
moist surface of human skin, or perhaps they were
attracted to volatile compounds in perspiration. In
laboratory studies, Holtman (1963) found that
Limothrips cerealium and Haplothrips aculeatus
(Fabricius) were attracted to capronic and lactic
acids in human perspiration. People developed
rashes on their faces from bites by Thrips imagi-
nis Bagnall in Melbourne, Australia (Bailey 1936).
The author suggested that saliva was injected into
the skin punctures during probing. Denmark
(1967) reported that the bites of Gynaikothrips fi-
corum (Marchal) occasionally could be quite an-
noying to people in Florida. This thrips species is
commonly found on various Ficus species. South-
cott (1986) reported several thrips species biting
people in Australia. Frankliniella shultzei (Try-
bom) was recorded biting at dusk in the Adelaide
vicinity during the summer months of January
and February. A small stinging bite left a small
red area about one mm in diameter that lasted for
about one h. Multiple bites from a single F
schultzei were documented as it moved around
Southcott's forearm at dusk. Bites by Haplothrips
various Hood did not result in a rash. However, this
species was attracted to white clothing hung out-
side to dry in the Adelaide vicinity during early
December 2005
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