Hendrichs et al.: Medfly Mating Behavior Studies
MEDFLY AREAWIDE STERILE INSECT TECHNIQUE PROGRAMMES
FOR PREVENTION, SUPPRESSION OR ERADICATION:
THE IMPORTANCE OF MATING BEHAVIOR STUDIES
J. HENDRICHS1, A. S. ROBINSON2, J. P. CAYOL3 AND W. ENKERLIN1
'Insect Pest Control Section, Joint FAO/IAEA Division, Wagramerstrasse 5, P.O. Box 100, A-1400 Vienna, Austria
2Entomology Unit, FAO/IAEA Agriculture and Biotechnology Laboratory, A-2444 Seibersdorf, Austria
'West Asia Section, Technical Cooperation Division, IAEA, Wagramerstrasse 5
P.O. Box 100, A-1400 Vienna, Austria
ABSTRACT
The Sterile Insect Technique (SIT) is amongst the most non-disruptive pest control methods.
Unlike some other biologically-based methods it is species specific, does not release exotic
agents into new environments and does not even introduce new genetic material into exist-
ing populations as the released organisms are not self-replicating. However, the SIT is only
effective when integrated on an areawide basis, addressing the total population of the pest,
irrespective of its distribution. There has been considerable progress in the development and
integrated application of the SIT against the Mediterranean fruit fly (medfly), Ceratitis cap-
itata, as reflected by operational programs for prevention, suppression and eradication of
this pest. There is however, considerable scope for improving the efficiency of medfly SIT, an
indispensable requirement for increased involvement of the private sector in any future ap-
plication. One way to achieve this has been the development of genetic sexing strains, mak-
ing it possible to release only sterile males. Another is improving sterile male performance
through a better understanding of the sexual behavior of this insect. Unlike other insects for
which the SIT has been successfully applied, medfly has a complex lek-based mating system
in which the females exert the mate choice selecting among aggregated and displaying wild
and sterile males. With the objective of developing a better understanding of medfly mating
behavior, an FAO/IAEA Coordinated Research Project was carried out from 1994 to 1999.
Some of the resulting work conducted during this period with the participation of research
teams from ten countries is reported in this issue.
Key Words: Mediterranean fruit fly, medfly, Ceratitis capitata, areawide IPM, sterile insect
technique, SIT, mating behavior, lek, quality control
RESUME
La t6cnica del insecto est6ril (TIE) esta entire los m6todos de control de plagas menos perju-
diciales. A diferencia de otros m6todos con base biol6gica, la TIE es especifica a nivel de es-
pecie, no transfiere agents ex6ticos hacia nuevos ambientes y ni siquiera introduce nuevo
material gen6tico dentro de las poblaciones existentes debido a que los organismos liberados
no se pueden auto replica. Sin embargo, la TIE es solamente efectiva cuando se integra en
forma extensive, considerando el total de la poblaci6n de la plaga, sin importar su distribu-
ci6n. Ha habido considerable progress en el desarrollo y la aplicaci6n integral de la TIE con-
tra la mosca del Mediterraneo, Ceratitis capitata, tal como lo reflejan los programs
operacionales para la prevenci6n, supresi6n y erradicaci6n de esta plaga. Existe sin em-
bargo, un considerable campo para mejorar la eficiencia de la TIE de la mosca del medite-
rraneo, un requerimiento indispensable por aumentar la participaci6 del sector privado en
cualquier aplicaci6n future. Una forma de lograr esto ha sido a trav6s del desarrollo de razas
gen6ticamente sexadas, haciendo possible la liberaci6n solamente de machos est6riles. Otra
es el mejoramiento del desempeno de los machos est6riles por medio de un mejor entendi-
miento del comportamiento sexual de este insecto. A diferencia de otros insects para los
cuales la TIE ha sido aplicada exitosamente, la mosca del Mediterraneo present un com-
plejo sistema de apareamiento basado la agregaci6n de machos en un "lek", deutro del cual
la hembra ejerce la selecci6n de pareja escogiendo entire el total de los machos salvajes y es-
t6riles en cortejo. Con el objetivo de desarrollar un mejor entendimiento del comportamiento
de apareamiento de la mosca del Mediterraneo, un proyecto de investigaci6n coordinado por
FAO/IAEA se llev6 a cabo de 1994 a 1999. En esta edici6n se reportan algunos de los trabajos
conducidos durante este period con la participaci6n de equipos de investigaci6n pertene-
cientes a diez pauses.
Florida Entomologist 85(1)
The effectiveness of integrating compatible
pest control methods is significantly increased by
coordinated implementation over larger contigu-
ous areas to address whole target pest popula-
tions (Knipling 1979). This areawide IPM
approach to pest management is gaining accep-
tance for some key insect pests (Tan 2000). Im-
portant tephritid fruit fly pests, such as the
Mediterranean fruit fly (medfly), Ceratitis capi-
tata, a notorious pest of quarantine importance
because of its extremely wide host range (Liquido
et al. 1991), are such key pests that invariably
cause economic damage if left uncontrolled. The
case for an areawide IPM approach arises for
these key pests as they cannot be effectively con-
trolled at the local orchard level without the sys-
tematic use of insecticide that disrupts the
biological control of secondary fruit pests and also
interferes with the use of other biologically-based
control methods (Ehler & Endicott 1984).
Among biologically-based methods, the Sterile
Insect Technique (SIT) is the most target-specific
and non-disruptive method. Unlike some other
biologically based methods it is species specific,
does not release exotic agents into new environ-
ments and does not even introduce new genetic
material into existing populations as the released
organisms are not self-replicating. However, to be
effective the released mass-reared and sterile
males have to successfully transfer their sperm
carrying dominant lethal mutations to a large
majority of females of the target population. As
when mating disruption using pheromones is ap-
plied, already-mated females that move into an
area under treatment, are largely unaffected by
the presence of sterile males, and proceed to lay
their eggs into fruit. As a result, SIT is only effec-
tive when applied on an areawide basis address-
ing the pest simultaneously over urban,
commercial, non-commercial and wild host areas.
The areawide integration of SIT with other con-
trol methods, results in significant benefits for
growers, providing them with enough incentives
to associate and to cooperate.
STATUS OF SIT FOR MEDFLY
Over the last quarter century there has been
considerable progress in the development and in-
tegrated application of SIT against medfly, as re-
flected by ongoing operational programs for
eradication, prevention, and suppression leading
to a rapid increase in sterile fruit fly production
capacity (Fig. 1). The expanding use of medfly SIT
is followed by similar trends for other fruit flies,
particularly economically-important Anastrepha
and Bactrocera species. The benefits accruing to
the domestic and export markets for fruit and
vegetable of all these programs have been of the
order of hundreds of millions of U.S. dollars annu-
ally (Hendrichs 2000).
1.5.
0 1.
05- I
1960-1970 1970-1980 1980-1990 1990-2000 2000-2002
*Other Frui es Medly(b a strain) Me y (male only strain) |
Fig. 1. Current worldwide production capacity of
sterile fruit flies.
Medfly SIT Eradication Programs
The application of SIT against medfly focused
initially on the concept of eradication, following
the successful example of the screwworm, Co-
chliomyia hominivorax, which over the last fifty
years has been eradicated from the U.S., Mexico
and recently also from all of Central America and
most of Panama (Wyss 2000). A number of medfly
SIT eradication programs have eliminated popu-
lations of this species, succeeding in the establish-
ment of medfly-free regions or whole countries.
The first large SIT program against medfly was
initiated in southern Mexico in 1977, with the
construction of a 500 million sterile fly mass rear-
ing facility in Tapachula. The aim of the Mos-
camed program was to prevent the spread of
medfly, which had become established in Central
America, into Mexico and the U.S.A. Establish-
ment of medfly in Mexico would have threatened
a multi-million fruit and vegetable export trade
with the U.S.A. The program succeeded in 1982 in
eradicating medfly from areas it had already in-
fested in southern Mexico (Hendrichs et al. 1983)
and since then a sterile fly barrier has been main-
tained from southern Belize through Guatemala
to southern Mexico to assure the fly-free status of
Mexico, U.S.A. and a large part of Guatemala (Vil-
lasenor et al. 2000). In Chile, following many un-
successful attempts to eradicate the pest using
insecticides (Olalquiaga & Lobos 1993), eradica-
tion from the northern part of the country was
achieved in 1995 with the integration of SIT,
opening trade opportunities estimated over five
years at a benefit to the Chilean fruit industry of
ca. U.S.$ 500 million (SAG 1996). In Argentina,
also as a result of SIT programs against medfly
that started in the early 1990's, fly-free areas
have been developed in various Patagonia valleys,
and Argentina recently succeeded in negotiations
with Chile to transport fruit from Mendoza and
Patagonia provinces through medfly-free Chile for
export from Chilean ports (De Longo et al. 2000).
March 2002
Hendrichs et al.: Medfly Mating Behavior Studies
Medfly SIT Prevention Programs
There are a number of examples were SIT is
being applied as a preventive control to avoid the
establishment of exotic or invasive fruit flies.
These include Southern Australia, where sterile
males are being released near Adelaide to prevent
the establishment of medfly coming from Western
Australia (Bill Woods, personal communication),
and Okinawa, Japan where preventive releases of
sterile melon flies are in progress along the south-
ern-most islands of the archipelago to avoid re-es-
tablishment of melon fly coming from Taiwan
(Kuba et al. 1996).
Probably most visible, are the repeated medfly
introductions into high-risk areas in California
and lately also Florida, threatening the exports of
a multi-billion dollar fruit industry (Siebert & Coo-
per 1995). These have required recurrent emer-
gency eradication actions, mainly consisting of
insecticide applications, costing annually millions
of U.S. dollars (Penrose 1995). Allowing the estab-
lishment of medfly in California would cost Cali-
fornia ca. U.S.$ 1.5 billion a year and result in a
drastic increase of insecticide use (Siebert 1999).
In view of the public opposition to recurrent aerial
bait-spraying over urban areas (CDFA 1994), and
the failure to eradicate these outbreaks with insec-
ticides, authorities embarked on the area-wide use
of SIT over the whole Los Angeles basin (LAB)
starting in 1994, involving the aerial release of
over 300 million sterile flies per week (Dowell &
Penrose 1995). The SIT strategy was so successful
technically, politically and environmentally, but
also from the economic point of view (costing on av-
erage less than half that of recurrent emergency
programs), that after eradication in 1996, areaw-
ide aerial releases were continued on a permanent
basis over 5,500 km2 of high risk areas in the LAB
(Dowell et al. 1999; Dowell et al. 2000).
This Preventive Release Program (PRP) has
been in operation since 1996 without major out-
breaks of medfly occurring in the LAB. It has been
expanded to ca. 6400 km2 to include additional
high-risk areas contiguous to the LAB. From 1987
until the inception of the PRP, the State of Cali-
fornia faced repeated major medfly infestation in
the LAB, with an average of 7.5 medfly infesta-
tions detected each year. Since the inception of
the PRP this has dropped to 0.2 infestations per
year (97% reduction) in the PRP area (CDFA,
2000). The few very confined medfly detections
within the PRP boundaries prove the assump-
tions on which the PRP is based: a) that Califor-
nia, especially southern California is under
constant threat of medfly invasion and b) that the
PRP can prevent the development of medfly pop-
ulations from these invasions. There is not a more
biologically efficacious, environment-friendly and
cheaper method to exclude medfly from southern
California (CDFA 2000).
Medfly SIT Suppression Programs
Two factors have been responsible for an in-
creasingly more cost-effective application of med-
fly SIT. The first factor is the development of
strains for male-only release, made possible by
continuing research supported and co-ordinated
by the IAEA and FAO over the last two decades
(Franz et al. 1996). Improved genetic sexing
strains with higher production, increased stability
and which are molecularly marked are now in use
in almost all operational SIT programs (Robinson
et al. 1999). The use of male-only strains is now
the state of the art for medfly SIT and has resulted
in a number of benefits among which are in-
creased applicability of the SIT and also increased
effectiveness of the sterile males in the absence of
sterile females (Hendrichs et al. 1995, Rendon et
al. 2000). The economic implications are signifi-
cantly reduced costs of applying SIT per square ki-
lometer per week in comparison to the use of
bisexual strains (Enkerlin et al. in preparation).
Second, as is the case in any industrial produc-
tion, biological or non-biological, significant econ-
omies of scale can be derived from larger mass
rearing factories. Whereas the cost per million for
sterile flies in small facilities producing tens of
millions per week is relatively high, this cost de-
creases by more than half when mass rearing fa-
cilities reach a capacity of hundreds of millions
per week (Enkerlin, unpublished data). The El
Pino facility in Guatemala, which has reached a
weekly production of over one billion sterile
males, actually has a sliding scale of costs for
their sales of sterile medfly males, that is in-
versely related to the production levels (Table 1).
Some of the initial medfly pilot SIT projects in
the 1960s and early 1970s confirmed the effective-
ness of SIT (De Murtas et al. 1970, Mellado et al.
1970, Ros et al. 1981, Rhode 1970, Kamburov et
al. 1975, Cheikh et al. 1975); nevertheless, appli-
cation of the SIT against medfly did not expand
mainly because sterile fly costs were higher at
that time when compared to conventional insecti-
cide sprays. However, progress on both of the
above factors has opened the possibility of using of
SIT for routine medfly suppression, rather than
only for eradication programs that are of a limited
duration. Using SIT for suppression has the major
advantage of not requiring the establishment of
quarantines to protect free areas. In addition, in
view of the increasing sensitivity to environmen-
tal concerns, there is new interest in using the
SIT, particularly in the Mediterranean region
where tourism and commercial fruit orchards co-
exist, with the aim of producing low-insecticide or
organic fruit. Sales of organically produced food,
though still small, have been growing by 20% a
year in the U.S.A. and in some European countries
as much as 40% a year (The Economist 2001). This
has stimulated the initiation of pilot SIT suppres-
Florida Entomologist 85(1)
TABLE 1. COSTS PER MILLION STERILE MALES AT VARYING PRODUCTION LEVELS AT THE MOSCAMED MEDFLY MASS
REARING FACILITY, EL PINO, GUATEMALA.1
Level of sterile
male medfly production
300 million per week
400 million per week
500 million per week
600 million per week
700 million per week
800 million per week
900 million per week
1.0 billion per week
1.1 billion per week
1.2 billion per week
1.3 billion per week
1.4 billion per week
1.5 billion per week
1.6 billion per week
Running costs2
per million sterile males
$199.29
$149.47
$119.47
$ 99.64
$ 85.41
$ 74.73
$ 66.43
$ 59.78
$ 54.35
$ 49.82
$ 45.99
$ 42.76
$ 39.87
$ 37.50
Production costs3
per million sterile males
$178.67
$178.67
$178.67
$178.67
$178.67
$178.67
$178.67
$178.67
$178.67
$178.67
$178.67
$178.67
$178.67
$178.67
Total cost
per million sterile males
$377.96
$328.14
$298.24
$278.31
$264.08
$253.40
$245.10
$238.45
$233.02
$228.49
$224.98
$221.43
$218.54
$216.17
From Vollmershausen 2001.
Includes direct administrative costs, depreciation, maintenance utilities, security and R&D.
'Includes diet materials, supplies, personnel and transportation.
sion programs in Tunisia (Cayol & Zarai 1999), Is-
rael and Jordan (R6ssler et al. 2000), Madeira
(Pereira et al. 2000), and South Africa (Barnes et
al. 2001). These SIT pilot projects have been effec-
tive in reducing insecticide applications and fruit
losses, as well as rejections of transboundary ship-
ments due to pest presence in fresh fruit exports
(Barnes et al. 2001).
The economic feasibility of using SIT for med-
fly suppression has been confirmed by benefit-cost
analyses (Enkerlin & Mumford 1997; Mumford
2000). Even without including the environmental
benefits, costs per hectare per year of protecting
orchards is now lower for an integrated areawide
approach with SIT than for non-areawide conven-
tional cover sprays, and approximately equal to
the areawide application of bait-sprays. These
savings to the fruit industries and the general en-
vironmental benefits already indicate the poten-
tial for the establishment of commercial SIT mass
rearing facilities. The continuous demand for
sterile males in SIT suppression programs should
open the way for commercialization of the SIT.
Nevertheless, further increases in the cost-effec-
tiveness of medfly SIT are a precondition before
serious private sector investment takes place in
mass rearing facilities and sterile fly production.
There is a third area, in addition to the use of
genetic sexing strains and the implementation of
economies of scale, where there is considerable
scope for improving the efficiency of medfly SIT.
This is the relatively poor performance of the
mass produced sterile males, which on average
are approximately only one third to one half as
competitive as the wild males (FAO/IAEA/USDA
2002). To compensate for this low effectiveness,
high sterile to wild over-flooding ratios are rou-
tinely applied. A better understanding of medfly
sexual behavior and the way it is affected by the
processes of colonization, mass rearing and irradi-
ation, could lead to improvements in sterile male
performance, thus lowering current over-flooding
ratios and overall costs of SIT application.
MATING SYSTEMS AND SIT
The nature of mating systems in any given
species is determined primarily by ecological fac-
tors such as the distribution of resources (Emlen
& Oring 1977). Based on resource distribution,
male mating systems of insect pests that are the
target of the SIT can be divided into three broad
polygynous categories (Thornhill & Alcock 1983).
First, there are the resource-defense systems
where the potential for mate monopolization by
males is high due to a clumped distribution of
females and the resources that are attractive to
receptive females (Table 2). Here, male mating
success is largely determined by intra-sexual
competition at these resources required by fe-
males, both to intercept females and to prevent
other males from gaining access to females. Sec-
ond, there are non-resource-based mating sys-
tems where mating takes place away from
resources required by females. The potential for
males to economically defend resources and fe-
males is rather low in this case because the re-
sources and females are more widely dispersed,
and intra-male selection involves a prolonged
searching polygyny. Males participate in a type of
continuous scramble competition, attempting to
out-race their competitors to receptive females
March 2002
Hendrichs et al.: Medfly Mating Behavior Studies
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Florida Entomologist 85(1)
that are releasing pheromones. Third, there are
lek-mating systems, also non-resource-based,
where the potential for males to monopolize re-
sources and females is also rather low. However,
in these cases, males establish "symbolic" mating
territories, and compete by attracting females
and attempting to exclude competitors from the
mating arenas. In lek mating systems, unlike the
two previous mating systems, inter-sexual selec-
tion components are also involved, with females
exerting mate choice by visiting aggregated males
and selecting a mate from amongst them.
Tsetse flies, Glossina spp. and screwworm flies
Cochliomyia hominivorax and Ci.', ..,- ;ii., bezzi-
ana, are examples of the first type of mating sys-
tem that are mainly resource-based (Table 2).
Males seek mates on animal hosts where females
forage for food (blood in the case of tsetse) or food
and oviposition sites (animal wounds in the case
of screwworms). In these species, the interaction
between the sexes is relatively simple in view of
the absence of any courtship and males compete
at such encounter sites trying to get hold of fe-
males (Jaensson 1979). Once a male manages to
grab a female, and confirms through tarsal con-
tact species-specificity of the female based on her
cuticular hydrocarbon profile particular to each
species, copulation takes place (Pomonis et al.
1993, Carlson et al. 2000). Mating success in
these species is therefore largely determined by
the sexual aggressiveness of the males, sterile or
wild, in intercepting receptive females at these
important resources.
On the other hand, the mating system of such
pest Lepidoptera as codling moth (Cydia pomo-
nella) and pink bollworm (Pectinophora gossy-
piella) is non-resource based, however, also
generally involves no male courtship, nor direct
female mate choice (Table 2). Sterile and wild
males participate in a type of scramble competi-
tion following pheromone trails originating from
receptive females, with the winner being first in
reaching the females and transferring the sper-
matophore (Snow et al. 1976).
A majority of the tropical and subtropical teph-
ritid fruit flies, including medfly and a majority of
Anastrepha and Bactrocera spp. have a lek poly-
gyny, involving both male intra-sexual selection
and also inter-sexual selection (Prokopy 1980).
Males have to find and join leks (male aggrega-
tions in mating arenas) within which they have to
participate in aggressive encounters with other
males to defend sites from which to signal and
court females. Receptive females are attracted by
male pheromone to the leks for the sole purpose of
soliciting courtships from various males, thus
comparing their performance and eventually ac-
cepting one for mating.
While in tephritids with a lek mating system
the population as a whole has a sex ratio of close
to 1:1, females are not synchronized in their sex-
ual maturation and thus only a small proportion
of females will visit leks at any given time. There-
fore the operational sex ratio, the number of
courting males for each attracted mature and re-
ceptive female in a lek, is largely biased in favor
of males. As a result, male intra-sexual competi-
tion is intense and the differential mating success
can be large, with many males getting no mates,
and a few males obtaining many matings (Arita &
Kaneshiro 1985, Hendrichs 1986). Bisexual re-
leases of sterile flies, half of which include sterile
virgin females that all become receptive within a
short time period, decrease the operational sex
ratio of males to females at leks. On the other
hand, the use of male-only strains for sterile fly
releases results in operational sex ratios that in-
crease even further the male bias at mixed leks
compared to wild male leks and thus the need for
releasing higher quality sterile males. Increasing
sterile to wild male over-flooding ratios in species
with a lek polygyny is less effective in overcoming
reduced sterile male competitiveness than in spe-
cies with the other mating systems. As wild fe-
males actively select and discriminate in favor of
males releasing timely pheromone of the ade-
quate profile (Heath et al. 1994) and performing
properly visual, sound and tactile courtship be-
haviors (Eberhard 2000), they may still favor the
courtship of a wild male even though he may rep-
resent a minority within a mixed lek. Thus the ef-
fective application of the SIT for lek species
requires a more detailed understanding of the
mating systems. They also require a much more
sophisticated quality control system to measure
and assure the sterile male performance.
STATUS OF MEDFLY SEXUAL BEHAVIOR STUDIES
AND QUALITY CONTROL OF STERILE FLIES
Even though various workers had studied var-
ious aspects of medfly behavior in the pre-SIT era
(Feron 1962), it was the implementation of SIT
that stimulated most research into medfly sexual
behavior. Concerns about whether mass-reared,
or even any laboratory-reared, medfly strains
were exhibiting wild-like characters in terms of
sexual behavior and competitiveness motivated
research as early as the 1970s. Pilot SIT activities
against medfly in Central America, Hawaii, Israel
and elsewhere resulted in assessments of the
mating competitiveness of irradiated and non-
irradiated medflies (Causse 1970, Holbrook &
Fujimoto 1970, Fried 1971, Rdssler 1975), and the
publication of a collection of quality control tests
for fruit flies in general (Boller & Chambers
1977), which included various tests relevant to
medfly mating behavior.
The initiation of the Moscamed program in the
late 1970s, resulted in the renewed interest in
medfly mating studies. This program, required a
quality control system for a weekly production of
March 2002
Hendrichs et al.: Medfly Mating Behavior Studies
500 million sterile flies. As part of this effort, the
description of the lek mating behavior of wild
medflies, observed under semi-natural conditions
in field cages, was provided (Prokopy & Hen-
drichs 1979), and the first medfly mating compat-
ibility test on a field-caged host tree was carried
out to measure female mate choice by allowing
wild females to select among competing wild and
sterile males under natural conditions (Zapien et
al. 1983). In addition, a collection of field tests
was developed for confirming and extending a se-
ries of laboratory tests (Boller et al. 1981, Cham-
bers et al. 1983) and the first quality control
manual for medfly mass rearing and field evalua-
tion was produced (Orozco et al. 1983), which has
been used extensively to measure quality of mass
produced flies. These publications formed the ba-
sis for a USDA quality control manual, compiled
to ensure that sterile medflies for SIT programs
with USDA involvement met certain quality stan-
dards (Brazzel 1986).
Further refinements have come with behav-
ioral studies in the open field to validate the med-
fly behaviors observed on field-caged host trees
(Hendrichs & Hendrichs 1990, Hendrichs et al.
1991, Whittier et al. 1992). More recent studies
addressed many other aspects including the vari-
ous effects of mass-rearing (Calkins et al. 1994,
Calkins et al. 1996), male size (Orozco & Lopez
1993), nutritional status (Blay & Yuval 1997),
mating-induced changes in female behavior (Jang
1995, Jang et al. 1998), strain differences (Liedo et
al. 1996), and behavioral incompatibility between
wild and mass reared flies (McInnis et al. 1996).
The conclusion from all these studies has been
that although sterile mass reared medflies do join
and compete within leks, achieve a portion of
matings with wild females, and transfer sperm
and induce female refractoriness and sterility in
offspring, they are clearly less competitive than
their wild counterparts. These behavioral
changes appear not to be caused by mating incom-
patibility among different medfly populations
(Cayol 2000a), but rather by mass-rearing condi-
tions, the irradiation process and the years a
strain is held in colonization (Cayol 2000b).
FAO/IAEA SPONSORED
COORDINATED RESEARCH PROJECT
The Joint Division of Nuclear Techniques in
Food in Agriculture of the Food and Agriculture
Organization (FAO) and the International Atomic
Energy Agency (IAEA) sponsors Coordinated Re-
search Projects (CRPs) or research networks that
focus participating scientists from both developing
and developed countries on applying nuclear tech-
niques to specific problems relevant to agriculture.
A CRP entitled "Medfly Mating Behavior Studies
under Field Cage Conditions" was initiated in
1994 with three objectives: a) to develop a detailed
understanding of medfly courtship behavior and
female choice through experimentation and slow-
motion video analysis, b) to measure mating com-
patibility worldwide among medfly populations,
and c) to develop harmonized mating tests to mea-
sure competitiveness and compatibility of sterile
flies. This CRP concluded in 1999 and four Re-
search Coordination Meetings (RCMs) were held
to review results and plan future research, Vi-
enna, Austria (4-7 October, 1994), Tapachula,
Mexico, (19-23 February 1996), Tel Aviv, Israel (15-
19 September) and Antigua, Guatemala (29 June-
03 July 1999). Twelve research teams from ten
countries (Argentina, Austria, Costa Rica, France,
Greece, Guatemala, Israel, Kenya, Mexico, U.S.A.)
participated and conducted research on different
aspects of medfly sexual behavior. The research
findings resulting from this CRP, published as ref-
ereed publications in scientific journals and as a
series of papers in this issue are included in the
listing of relevant references in Table 3.
APPLICATION OF RESEARCH FINDINGS:
INTERNATIONAL FRUIT FLY
QUALITY CONTROL MANUAL
On the basis of all the above studies, as well as
information from various fruit fly quality control
manuals, a concerted effort was made to develop
an FAO/IAEA/USDA manual on "Product Quality
Control and Shipping Procedures for Sterile
Mass-Reared Tephritid Fruit Flies" (FAO/IAEA/
USDA 2002). The objective was to incorporate the
improved understanding of medfly mating behav-
ior into quality control protocols, to harmonize
procedures and thus allow comparison of sterile
fly quality over time and across rearing facilities
and field release programs.
Based on the CRP findings, the FAO/IAEA/
USDA international manual emphasizes mating
competitiveness, sexual compatibility and post-
mating factors and de-emphasizes the widely used
laboratory mating propensity test. This test is car-
ried out only with mass reared males and females,
under high densities and in small Plexiglas cages,
all conditions that favor sterile males and thus it
routinely overestimates sterile male performance.
Even worse, it measures the wrong parameter,
namely speed of pair formation between mass-
reared males and females, even though sterile
males trying to achieve fast mating, short-cutting
steps in the courtship sequence, are only success-
ful under crowded colony rearing conditions but
are unsuccessful in mating with wild females
(Briceno et al. 1996, Briceno & Eberhard 1998).
The international manual recognizes that the
most important indicator of sterile male quality
control is their successful interaction with wild fe-
males of the target population. Thus a standard
field-cage test is required as an ultimate measure of
quality, where wild females of the target population
Florida Entomologist 85(1)
March 2002
TABLE 3. LISTING OF RELEVANT STUDIES CONDUCTED ON VARIOUS ASPECTS RELATED TO MEDFLY SEXUAL BEHAVIOR
AND MATING COMPETITIVENESS.
Relevant References
Courtship behavior in relation to sexual
competitiveness
Lekking behaviour in relation to sexual
competitiveness
Field cage evaluations in relation to sexual
competitiveness
Open field studies and evaluation of field
competitiveness
Feeding behavior and nutritional status
in relation to sexual competitiveness
Predation in relation to sexual behavior
Morphometric traits in relation to sexual
competitiveness
Irradiation in relation to sexual
competitiveness
Age in relation to sexual competitiveness
Factors affecting female post-mating
and re-mating behavior
Effects of mass-rearing in relation to sexual
competitiveness
Inter-population compatibility and isolation
studies
Comparative approaches to sexual behavior
in Tephritidae
Feron 1962; Sivinski et al. 1989; Whittier et al. 1994; Whittier
& Kaneshiro 1995; Briceno et al. 1996; Liimatainen et al. 1997;
Briceno & Eberhard 1998; Lance et al. 2000; Briceno &
Eberhard 2002; Briceno et al. 2002; Lux et al. 2002b
Prokopy & Hendrichs 1979; Arita & Kaneshiro 1985; Arita
& Kaneshiro 1989; Shelly et al. 1993; Shelly & Whittier 1995;
Yuval et al. 1998; Kaspi & Yuval 2000; Field et al. 2002
Wong et al. 1983; Zapien et al. 1983; Rendon et al. 1996; Cayol et
al. 1999; Katsoyannos et al. 1999; Calcagno et al. 2002; Econo-
mopoulos & Mavrikakis 2002
Hendrichs & Hendrichs 1990; Whittier et al. 1992; McInnis
et al. 1994; Rendon et al. 2000; Shelly 2000
Hendrichs et al. 1991; Warburg & Yuval 1997; Blay & Yuval
1997; Cangussu & Zucoloto 1997; Bravo & Zucoloto 1998;
Field & Yuval 1999; Kaspi & Yuval 2000; Papadopoulos et al.
2001; Shelly et al. 2002; Yuval et al. 2002
Hendrichs et al. 1993; Hendrichs & Hendrichs 1994; Hendrichs
& Hendrichs 1998
Churchill-Stanland et al. 1986; Orozco & Lopez 1993; Hunt
et al. 1998; Menez et al. 1998; Blay & Yuval 1999; Hunt et al.
2002; Hasson & Rossler 2002; Rodriguero et al. 2002
Causse 1970; Holbrook & Fujimoto 1970; Hooper 1971; Hooper
1972; Calkins et al. 1988; Heath et al. 1994; Lux et al. 2002a
Liedo et al. 1996b; Papadopoulos et al. 1998; Taylor et al. 2001;
Liedo et al. 2002
Boller et al. 1994; Jang 1995; Hendrichs et al. 1996; Jang et al.
1998; Jang 2002; McInnis et al. 2002; Vera et al. 2002
Liedo et al. 1996a; Calkins 1991; Calkins et al. 1994; Calkins
et al. 1996; Cayol 2000b
McInnis et al. 1996; Cayol 2000a; Cayol et al. 2002;
Myburgh 1962; Prokopy 1980; Sivinski et al. 2000; Yuval &
Hendrichs, 2000; Quilici et al. 2002
are the final arbiters of sterile male quality. This
test is carried out on a routine basis under semi-
natural conditions on field-caged host trees. Fur-
ther fine-tuning of the manual is a continuous pro-
cess through which the procedures evolve as new
findings emerge. In support of this process, a new 6-
year FAO/IAEA Coordinated Research Project on
"Quality Assurance of Mass Produced and released
Fruit Flies" has been initiated involving partici-
pants from all major fruit fly SIT programs.
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Florida Entomologist 85(1)
December 2002
DECISIONS DURING COURTSHIP BY MALE AND FEMALE MEDFLIES
(DIPTERA, TEPHRITIDAE): CORRELATED CHANGES IN MALE BEHAVIOR
AND FEMALE ACCEPTANCE CRITERIA IN MASS-REARED FLIES
R. D. BRICENO1 AND W. G. EBERHARD1'2
1ESCUELA DE BIOLOGIA, UNIVERSIDAD DE COSTA RICA, CIUDAD UNIVERSITARIA, COSTA RICA
2SMITHSONIAN TROPICAL RESEARCH INSTITUTE
ABSTRACT
Analyses of more than 300 videotaped courtships of wild and mass-reared medflies from
Costa Rica showed that the tendency for male and female to align themselves facing directly
toward each other increased, and that the distance between them decreased as courtship pro-
ceeded. More direct alignments and shorter distances between the flies at the moment the
male jumped onto the female were correlated with greater female acceptance of copulation.
There were no consistent differences in durations of components of intermittent buzzing
songs or male size between successful and unsuccessful courtship in either strain. Several
possible cues may release different courtship responses: males of both strains tend to initiate
both continuous vibration and intermittent buzzing after reduction of the distance to the fe-
male; slow creeping toward the female was associated with longer courtships that had failed
to lure the female close; and females tended to turn to face more directly toward the male
soon after the male began continuous vibration, and especially after he began intermittent
buzzing. Females became progressively more immobile as courtship progressed, especially
soon after intermittent buzzing began. There were numerous differences between strains.
Mass-reared males were more likely to mount females without previous courtship than were
wild males. Wild males initiated continuous wing vibration when farther from the female
and when the female was looking less directly toward them, but the two strains did not differ
in the distances and angles at which males initiated intermittent buzzing and jumped. Wild
males were more likely to creep toward the female during intermittent buzzing. Mass-reared
females but not wild females were more likely to copulate when the proportion of time the
male had spent in intermittent buzzing was low, and if the courtship began when the flies
were nearer each other. Wild but not mass-reared females were less likely to copulate if
courtship was shorter. Possible coevolution of female responses with the five different male
courtship traits that differ between mass-reared and wild flies are discussed.
Key Words: medfly, sexual selection, courtship behavior, mass-rearing, female choice
RESUME
Analisis de mas de 300 cortejos video-grabados de moscas del Mediterraneo silvestres y cria-
das en masas de Costa Rica demostraron que la tendencia de los machos y las hembras de
alinearse cara a cara el uno frente al otro aument6, y que la distancia entire ellos ha disminu6
a media que el cortejo procedia. Alineaciones mas directs y distancias mas cortas entire las
moscas en el moment en que el macho salta sobre la hembra se correlacionaron con mayor
aceptaci6n por parte de las hembras. No hubo diferencias consistentes en la duraci6n de los
components de los zumbidos intermitentes de las canciones o el tamano del macho, entire los
cortejos efectivos y no efectivos en ninguna de las dos razas. Varios estimulos posibles po-
drian inducir diferentes respuestas en el cortejo: machos de ambas razas tienden a iniciar vi-
braciones tanto continues como intermitentes que despu6s de se reduce la distancia a la
hembra; un lento acercamiento hacia la hembra se asoci6 con cortejos mas largos que no lo-
graron inducir el acercamiento de la hembra; y hembras que presentaron la tendencia de gi-
rar y encarar mas directamente al macho pronto despu6s de que el macho inici6 las
vibraciones continues, y especialmente despu6s de que iniciaron los zumbidos intermitentes.
Las hembras se hicieron progresivamente mas inm6viles a media que el cortejo continuaba,
especialmente poco tiempo despu6s que el zumbido intermitente se inici6. Existieron nume-
rosas diferencias entire las razas. Los machos criados en masa montaron a las hembras sin
ningdn tipo de cortejo previo, con mayor frecuencia que los machos silvestres. Los machos
silvestres iniciaron vibraci6n continue cuando se encontraban a mayor distancia de la hem-
bra y cuando las hembras se orientaron menos directamente hacia ellos, pero las dos razas
no difirieron en las distancias y angulos a los cuales los machos iniciaron su zumbido inter-
mitente y saltaron. Los machos silvestres se acercaron lentamente hacia las hembras con
mayor frecuencia durante el zumbido intermitente. Las hembras criadas en masa, a diferen-
Bricefio & Eberhard: Decisions by Medflies during Courtship
cia de las hembras silvestres presentaron mayor tendencia a copular cuando la proporci6n
de tiempo que el macho utilize en el zumbido intermitente fue baja, y si el cortejo comenz6
cuando las moscas estaban mas cerca las unas a las otras. Hembras silvestres a diferencia
de las hembras criadas en masa, tendieron a copular menos si el cortejo era mas corto. La po-
sibilidad de coevoluci6n en las respuestas de la hembra hacia los cinco rasgos del cortejo que
difieren entire las moscas criadas en masas y las mosca silvestres se discuten.
The success of the massive efforts to control
pest populations of the Mediterranean fruit fly,
Ceratitis capitata Wiedemann using mass-reared
sterile males depends on the abilities of these
males to successfully induce wild females to copu-
late with them. Nevertheless, current under-
standing of why it is that some courtships result
in copulation, while the majority do not, is only
fragmentary. The commonly observed mating infe-
riority of mass-reared males as compared with
wild males when they are paired with wild fe-
males (e.g., Rossler 1975b, Calkins 1984, Shelly et
al. 1994, Hendrichs et al. 1996) is apparently due
to their inadequate courtship per se, rather than
to inferior abilities to find and attend leks, or to at-
tract females pheromonally and begin to court
them once they are at a lek (Shelly et al. 1994,
Shelly & Whittier 1996, Hendrichs et al. 1996, Li-
imatainen et al. 1997, Lance et al. 2000.). Differ-
ences in courtship behavior between wild and
mass-reared males are, however, only starting to
be studied (Briceno et al. 1996, Liimatainen et al.
1997, Briceno & Eberhard 1998, 2000 in press).
Courtship in medflies was first studied in detail
by Feron (1962) and current knowledge was re-
viewed by Eberhard (2000). Usually courtship fol-
lows a relatively standard sequence of events,
during which the male courts actively but stays
more or less in one place. The female performs little
if any overt courtship behavior, but moves toward
the male and aligns herself facing him. Active male
behavioral courtship begins when the male (usu-
ally while he is in the pheromone releasing pos-
ture-stage I of Feron) responds to the presence of
the female (apparently on the basis of visual cues-
Feron 1962, Kaneshiro 2000) by turning toward
her. He bends his abdomen ventrally and starts to
vibrate his wings (stage II of Feron, "continuous
wing vibration" of Eberhard 2000). The abdominal
pleura and the rectal sac, which are everted during
stage I and presumably release pheromone (e.g.,
Nation 1981, Headrick & Goeden 1994 on other te-
phritids with similar structures), remain everted,
and the wing vibrations, which involve rapidly
twisting the wings on their longitudinal axes, pre-
sumably causes pheromone to be wafted toward
the female (Arita & Kaneshiro 1989, Briceno &
Eberhard 2000). The abdominal pleura often pulse
during continuous wing vibration (unpublished
data). After a variable amount of continuous wing
vibration, the male switches abruptly to a second
type of wing movement. He moves his wings rhyth-
mically forward and back while continuing to vi-
brate them rapidly, and he also intermittently
rocks his head from side to side and forward and
backward (stage III of Feron, "intermittent wing
buzzing" and "head rocking" of Eberhard 2000). In
some cases the male "creeps" slowly toward the fe-
male with small steps that are taken each time he
initiates a buzz (Briceno & Eberhard in press).
Chemical signaling is probably altered and may be
suspended during stage III, since the rectal sac is
retracted when intermittent wing buzzing begins
(Figure 3-3 of Feron 1962, Briceno et al. 1996).
Head rocking often results in contact between the
male's aristae and those of the female (Briceno &
Eberhard in press).
After a variable amount of intermittent wing
buzzing, the male jumps onto the female if she is
appropriately positioned in front of him. If she
does not dislodge him by flying or falling, as fre-
quently occurs (Eberhard 2000, Lance et al.
2000), he aligns himself on her dorsum, and, if
she everts her aculeus from the tubular eversible
membrane, he grasps it with his genitalic surstyli
and intromits (Eberhard & Pereira 1995). Males
also perform apparent courtship movements dur-
ing copulation itself (Eberhard & Pereira 1995),
suggesting that the male also attempts to influ-
ence further female decisions (e.g., transport
sperm-Yuval et al. 1996) after his genitalia have
entered the female's body (Eberhard 1991). This
stage of the male-female interaction will not be
considered further here, and the term "courtship"
will refer only to precopulatory behavior. There is
at least one alternative male behavioral se-
quence. The male does not court, but simply
jumps onto the female and immediately attempts
to copulate (Prokopy & Hendrichs 1979).
These descriptions show that both the male
and the female make a series of behavioral "deci-
sions" or transitions during courtship. The male
makes at least five decisions: whether to begin
courtship or to jump immediately; when, if he is
going to court, to begin continuous wing vibration;
when to switch from continuous vibration to inter-
mittent wing buzzing; whether to creep toward
the female during intermittent buzzing; and when
to terminate intermittent buzzing and jump onto
the female. Females also make decisions, although
some are less easily characterized. Indirect data
(Briceno et al. 1996) indicate that female behavior
which results in her being immobile, directly in
front of the male and facing directly toward him,
increases the chances that he will jump onto her.
Such female responses may include turning or not
turning, and walking or not walking. Once the
male jumps, two further female decisions are
Florida Entomologist 85(1)
more easily categorized: whether or not to dis-
lodge the male; and whether or not to evert her
aculeus (and thus allow him to intromit).
Understanding the factors that influence the
decisions of both males and females will probably
help clarify why some courtships succeed and oth-
ers fail. One attractive possibility is that males
and females exchange signals during courtship
(Lux & Gaggl 1996, Liimatainen et al. 1997). For
instance, the male decisions to switch from con-
tinuous wing vibration to intermittent buzzing or
to jump could be triggered by some particular
female behavior indicating that she is receptive.
A review of available data showed, however, that
there is no quantitative evidence that any partic-
ular female behavior has a triggering effect on
male behavior (Eberhard 2000). Briceno & Eber-
hard (1998) found three possible female signals
that showed significant associations with even-
tual mounting attempts (strike the male with her
head; lean slowly rearward and sometimes crouch;
and tap the male's legs with her front legs); but
none had a significant effect on the likelihood that
the male would mount after the female performed
them. In other words, males appeared not to pay
attention to these possible signals from the female.
One possible cue that could be used in male-
female dialogues is the female's position with re-
spect to the male. There were differences between
positions at the moment the male jumped onto
the female as compared with positions when the
courting male desisted from courting (Briceno
et al. 1996). The present study constitutes an
attempt to use similar correlational evidence, in
this case from a much larger sample of court-
ships. Not only the male's decision to jump, but
also his decision whether to court rather than
jump immediately, when to initiate courtship,
and when to switch from continuous wing vibra-
tion to intermittent buzzing, as well as the fe-
male's decision whether or not to allow a male to
mate after he has mounted her are analyzed.
MATERIALS AND METHODS
Wild flies were raised from fallen tangerines
and oranges collected at the Estaci6n Experimen-
tal Fabio Baudrit of the Universidad de Costa
Rica, el. about 900 m near Alajuela, Alajuela
Province, Costa Rica. Mass-reared flies were from
a strain that had been founded about three years
previously with flies collected in the same area,
and kept as adults thereafter (about 51 genera-
tions) in 2.30 x 0.35 x 0.50 m breeding cages with
approximately 60,000 flies/cage. Flies were sepa-
rated by sex the day after emergence, and kept in
32 x 32 x 32 cm cages with free access to water
and a mixture of sugar and hydrolyzed protein.
Immediately before taping sessions flies were as-
pirated into mating chambers (9.5 cm diameter
plastic Petri dishes with millimeter ruled paper
on the top of the lid) that had either one pair/dish
or that were divided into four equal sectors by
cardboard walls with one pair/sector. A subset of
the males was preserved by freezing, and later
measured using an ocular micrometer at 30x
(maximum width of head, maximum length and
width of the thorax in dorsal view).
All data came from analyses of videotaped
courtships in which the male mounted the female.
To avoid pseudoreplication, only the first court-
ship of each pair of animals was analyzed, and the
only courtships included were those in which the
male performed a single episode of continuous
wing vibration followed by a single episode of in-
termittent wing buzzing and then leapt onto the
female (i.e., courtships in which, for example, the
male resumed continuous vibration after inter-
mittent buzzing and then eventually mounted
were excluded). Each animal was used only once.
Several variables were measured at each of seven
moments that were associated with three transi-
tions in male behavior: one s before and one s af-
ter the male initiated continuous wing vibration
as well as the moment when he initiated this be-
havior; one s before, one s after, and the moment
when the male initiated intermittent buzzing;
and the moment when the male jumped onto the
female. Data preceding and following transitions
were analyzed to elucidate stimuli associated
with particular decisions. For instance, a change
in the female's position from one s prior to the ini-
tiation of buzzing to the moment when buzzing
began could be the stimulus used by males to trig-
ger buzzing. In contrast, only data from the
moments that transitions occurred were used in
analyses involving female acceptance of copula-
tion.
Three variables that were measured to esti-
mate the flies' relative positions are illustrated in
Fig. 1: the distance between the centers of the two
animals' prothoraces; the orientation of the fe-
male with respect to the direction in which the
male was oriented (the angle she made with his
longitudinal axis-"male angle" in Fig. 1); and the
orientation of the male with respect to the direc-
tion in which the female was oriented (the angle
he made with her longitudinal axis-"female an-
gle" in Fig. 1). These variables were measured us-
ing the NIH Image program (public domain
software) by grabbing a frame from the video with
a Genius videocapture card (Pro II series) and im-
porting it into a computer. Walking behavior was
counted as forward movements, and did not in-
clude when the fly turned but the center of its tho-
rax was immobile.
Other variables measured included the dura-
tion of each stage of courtship, and the length of
time the female had been immobile preceding the
moment the male leapt onto her. When the male
jumped onto the female, the time elapsed until
the female began to resist, whether or not she re-
March 2002
Bricefo & Eberhard: Decisions by Medflies during Courtship
female angle
/ male angle
S/ distance
Fig. 1. Angles and distances measured (male stippled).
sisted, and the time the male took to turn and
align himself facing in the same direction as the
female, were also measured. Successful mounts
were those in which the immobile female did not
dislodge the male within 60 s of his having landed
on her.
The sounds produced during intermittent
buzzing were recorded using a small, Sennheiser
MZK 80ZU, microphone inserted through a hole
in the side of the mating chamber and connected
to the camera. Recordings of sounds were im-
ported from video recordings into a PC 486dx2
computer using a 16 bit card. Durations of buzzes
and the intervals between buzzes were measured
using the real time display in the program Avisoft
using cursors to mark the beginning and the end
of the envelope curve displayed in the main win-
dow of the program (see Briceno et al. in press for
further details). The precision of these measure-
ments was determined by remeasuring the dura-
tions of 10 buzzes and 10 intervals in each of 8
different courtships. The average differences
were 2.0 ms in buzz duration, and 2.2 ms in inter-
val duration.
Most variables were not normally distributed,
and means and standard deviations are presented
for illustrative purposes only. Except where noted
otherwise, all statistical tests of differences em-
ployed two-tailed Mann-Whitney U Tests.
We performed three types of analysis. First we
made simple, variable by variable comparisons
between strains and between unsuccessful and
successful courtships that led to successful mounts.
For instance, we compared the length of intermit-
tent buzzing preceding unsuccessful and success-
ful mounts within both strains, and between
strains. These analyses had the possible problem
that some independent variables are probably
correlated. For instance, an apparently signifi-
cant effect of variable A on the female decision to
copulate rather than reject the mounted male,
might actually result from this variable's associa-
tion with another independent variable B that
truly does affect the female's decision. This possi-
ble dependence was tested with additional analy-
ses, using the statistics program SYSTAT. These
were organized into three questions:
1. Which aspects of the female's behavior
during courtship are associated with in-
creased probability that she will allow the
male to mate when he mounts?
2. Which aspects of male courtship behavior
may have induced this female receptivity?
3. Which cues are used by males to initiate
continuous wing vibration to intermittent
buzzing?
For those questions with a discontinuous re-
sponse variable (e.g., unsuccessful, successful),
we used stepwise logistic regressions (SYSTAT
forward stepwise option). For the others we used
ordinary multiple regressions. For each analysis
we provided the program with a list of variables
with possible effects, on the basis of the stimuli
likely to be available to the fly making the re-
sponse that was being tested. The program first
selected from this list the variable that had the
largest effect on the response variable, and calcu-
lated this effect. It then repeated the process with
the remaining variables on the list while correct-
ing for the effect of the first variable, and it con-
tinued this process until none of the remaining
variables had significant effects on the response
variable (P < 0.05). In each round the effects of all
variables that had already been selected in previ-
ous rounds were held constant.
An additional complication is that flies may
have multiple threshold criteria for some deci-
sions. Thus, for instance, the female may only
allow a mounted male to copulate when the dis-
tance between them is below some critical value
and in addition the duration of his courtship is
above some other critical value. Such interactions
could impede detection of decision criteria. We
thus performed an additional set of logistic re-
gressions in which we tested for interactions be-
tween pairs of independent variables in their
effects on the response variable. Regression mod-
els were constructed for different questions as be-
fore, but in this case we checked for significant
interactions between each of the variables that
Florida Entomologist 85(1)
had been found to have a significant effect in the
first model when each was combined with all of
the other variables in the list which had not had
significant effects in the original model. These in-
teractions analyses were performed separately
from other analyses because even with our large
sample sizes it was not possible to find significant
effects for more than about 4 variables at a time
with these regression techniques.
It is important to keep in mind that a given be-
havioral variable may be influenced by both the
male and the female. Consider, for example, the
length of time the female was immobile before the
male jumped onto her. The male clearly makes the
decision to jump, and it would thus seem reason-
able to include this time in the model for question 2
(male effects on female receptivity to copulation),
but not in the model for question 1 (female indica-
tors of receptivity). But it is also obvious that the fe-
male herself determines whether or not she moves,
and female movement probably inhibits jumping
by the male. This kind of interdependence some-
times made it difficult to decide which variables
should be included which models. In some cases the
same variable was included in different lists.
RESULTS
Table 1 and Figures 2, 4, and 6-8 present vari-
able-by-variable analyses of behavioral traits
with respect to strain and male copulation suc-
cess. Figures 3 and 5 illustrate changes just be-
fore and just after male initiation of continuous
vibration and intermittent buzzing behavior. Ta-
ble 2 presents the results of regression analyses
testing for independence of the effects of different
variables. Table 3 gives the results of logistic re-
gression analyses of the interactions between
those variables with significant effects in the
models in Table 2 and the rest of the variables
that did not have significant effects in these mod-
els. In general, the regression analyses confirmed
the results of the variable-by-variable analyses,
but did not reveal many additional relationships.
We will discuss the results variable by variable.
Distances between the Male and Female
The distance between the male and female
tended to decrease as courtship proceeded in both
mass-reared and wild flies (Fig. 2) (P < 0.0001
with Kruskal-Wallis Test for each strain; a poste-
riori Duncan tests showed that differences be-
tween all three pairs of values at the moment of
transition were significant in both strains P <
0.001). Wild males initiated continuous vibration
at significantly greater distances females than
did mass-reared males (Figs. 2 and 3, Table 1).
Combined values for successful and unsuccessful
courtships differed by more than a factor of 2 be-
tween the two strains (0.64 0.40 cm vs. 1.60 +
2.12 cm, P < 0.001). Distances when the male ini-
tiated subsequent stages in courtship did not dif-
fer between the two strains (Figs. 2 and 3, Table
1), even when successful and unsuccessful court-
ships were combined (P > 0.05). The distance from
which the male jumped onto the female was rela-
tively less variable within each strain than the
other distances (e.g., error bars in Fig. 2; Barlett's
homogeneity of variances test showed significant
differences between the variances in all three
variables in both strains,P < 0.001).
In both strains the distance between male and
female decreased significantly during the s prior
to initiation of continuous vibration, and during
the s prior to initiation of intermittent buzzing
(BC vs. C and BI vs. I in Fig. 3), suggesting that
reduction in the distance may be a cue used by
males to trigger both behavior patterns. Dis-
tances did not decrease significantly during the s
after continuous vibration began, nor during the s
after intermittent buzzing began, suggesting that
these male activities did not immediately induce
the female to approach him. There was no signif-
icant difference associated with successful vs. un-
successful mounts in either strain with respect to
any of the distances measured (Table 1).
Male Angles
The male angles at the moments of transition
changed very little during the course of courtship
in both strains (Fig. 4) (P = 0.021 with Kruskal-
Wallis Test on mass-reared flies, but no pairs
were significantly different with a posteriori Dun-
can Tests; the male's angle at the start of contin-
uous wing vibration was slightly larger than
either of the other two in wild flies with similar
tests). There was, however, a tendency in both
strains for the male to turn to face more directly
toward the female in the s preceding initiation of
both continuous vibration and intermittent buzz-
ing (Fig. 5). The strains did not differ consistently
(Figs. 4 and 5, Table 1).
Mounts by both mass-reared and wild males
were more likely to be successful when the male
angle was lower at the moment the male jumped
(Table 1), although in mass-reared flies this effect
was not significant in regression analyses (Table
2, Model 2). The male angle at the moment he
jumped was clearly smaller preceding successful
as compared with unsuccessful courtships when
data from the two strains were combined (2.8
6.7 vs.4.6 5.3o; P < 0.001). Male angles at other
stages showed less consistent effects (Fig. 4, Table
1), and were not significant in regression analyses
(Table 2, Model 2). Summarizing, the male re-
mained oriented looking more or less directly to-
ward the female during the entire courtship, and
he turned to face her even more directly just be-
fore beginning both continuous vibration and in-
termittent buzzing. A male's mount was more
March 2002
Bricefo & Eberhard: Decisions by Medflies during Courtship
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Florida Entomologist 85(1)
Smoss-reared successful (N-36)
1.0 i i ssd ecessu est( N=34
S1 wild unsuccessful (N=42)
C3
s r continuous start intermittent jump onto
wing vibration wing buzzing female
Male Behavior
Fig. 2. Distances between male and female at differ-
ent stages during male courtship in mass-reared and
wild flies during successful and unsuccessful court-
ships. The flies gradually came closer together as court-
ship proceeded. Distances when courtship began were
significantly greater in wild flies.
likely to be successful if he launched his jump
while looking more directly toward the female.
Female Angles
Female angles clearly decreased during court-
ship (Figs. 5 and 6) (P < 0.0001 with Kruskal-Wal-
lis Test in each strain comparing angles at the
initiations of continuous vibration, intermittent
buzzing, and the male's jump in combined data
from successful and unsuccessful courtships). A
posteriori Duncan tests showed that the female
angle when the male began continuous wing vi-
bration was significantly larger than each of the
other angles in both strains (all P < 0.001). Fe-
males of both strains turned to face more directly
toward males during the s following initiation of
intermittent and continuous buzzing, but this
trend was only weak in wild flies (Fig. 5).
Courtship was more likely to be successful in
both strains when the female was looking more
directly toward the male at the moment he
jumped (Fig. 6, Table 1), though in wild flies this
effect was not significant in regression analyses
(Table 2, Model 1). The mean female angles for
both strains combined at the moment the male
jumped were 4.0 5.6 vs. 10.8 + 12.8 comparing
successful and unsuccessful mounts (P < 0.001).
The female angles at earlier stages of courtship
did not show significant differences between suc-
cessful and unsuccessful courtships in mass-
reared flies, and only inconsistent differences in
wild flies (Tables 1 and 2). Combining successful
and unsuccessful courtships, mass-reared males
initiated courtships when females were facing
more directly toward them (female angle 31.2
41.5 vs. 42.7 + 31.6 in wild flies, P < 0.001). Sum-
marizing, the female looked more directly toward
the male later in courtship, and mounts that oc-
curred when she was looking more directly to-
ward him were more likely to result in copulation.
Initiation of continuous and intermittent buzzing
apparently induced the female to turn toward the
male, the trend was weak in wild flies. Mass-
reared males initiated courtship when females
were looking more directly toward them.
There was a positive correlation in both strains
between male and female angles, so when the
male was looking more directly toward the female,
she tended to be looking more directly toward him
(Fig. 7). This correlation seemed stronger later in
courtship, but the changes were not significant.
Female Immobility
Females were nearly always immobile when
the male jumped. The amount of time the female
had been quiet before the male jumped was signi-
ficantly shorter in successful courtships of mass-
reared flies than in those preceding unsuccessful
mounts, but there was essentially no difference in
wild flies (Fig. 8, Table 1). This difference was
independent of the effects of other variables in
mass-reared flies (Table 2, Model 1).
Female immobility was significantly shorter
when the distance between the flies at the mo-
ment of the jump was larger, and it was larger
when the distance at the beginning of intermit-
tent buzzing was larger in mass-reared but not in
wild flies (Table 3, Model 1). Mass-reared females
that were successfully mounted had been motion-
less for a marginally shorter time than wild fe-
males that were successfully mounted (P = 0.03),
but there was no difference between strains for
the females that were unsuccessfully mounted
(P > 0.05). Combining successful and unsuccess-
ful courtships in each strain, the mean durations
of female immobility did not differ significantly
between strains (6.76 7.65 s vs. 5.84 + 3.71 s for
mass-reared and wild flies (P > 0.05).
Absolute and Relative Durations
As was found previously using different flies
and a different, older mass-reared strain from
Cost Rica (Bricefio & Eberhard 1998), several as-
pects of courtship by mass-reared males were
shorter than those by wild males (Table 1). The
March 2002
Bricefo & Eberhard: Decisions by Medflies during Courtship
Mass-reared (N=240)
Wild (N=102)
()
( 20
C
o) o -
BC C AC BI I Al BC C AC BI I Al
Moment during courtship
Fig. 3. Distances between male and female 1 s before initiation of continuous vibration (BC), at the moment con-
tinuous vibration began (C) and 1 s after it began (AC), and 1 s before (BI), 1 s after (AI), and at the moment of ini-
tiation (I) of intermittent buzzing in two strains. Dots accompanying lines between bars indicate significant
differences between the two bars (one dot P < 0.05; two dots P < 0.01; three dots P < 0.001).
mass-reared successful (N=36)
mass-reared unsuccessful (N=134)
wild successful (N=34)
wild unsuccessful (N=42)
start continuous start intermittent
wing vibration wing buzzing
Male Behavior
Fig. 4. Male angles at different stages during male courtship in mass-reared and wild flies during successful and
unsuccessful courtships. Males tended to be oriented toward the female throughout courtship. Differences between
strains, and between successful and unsuccessful courtships were not significant.
20
0
< 10
0
Florida Entomologist 85(1)
iz;---female
---male
0)
C
< 40
BC C AC BI I Al BC C AC BI I Al
Moment during courtship
Fig. 5. Male and female angles 1 s before initiation of
continuous vibration (BC), at the moment continuous
vibration began (C) and 1 s after it began (AC), and 1 s
before (BI), 1 s after (AI), and at the moment of initia-
tion (I) of intermittent buzzing in two strains (mass-
reared on left, wild on right). The dots accompanying
lines between bars indicate significant differences be-
tween the two bars (one dot P < 0.05; two dots P < 0.01;
three dots P < 0.001).
difference in total courtship duration between
mass-reared and wild flies when successful and
unsuccessful courtships were combined was also
significant (15.81 12.50 s vs. 20.18 20.70 s,P <
0.05). This difference was due to different dura-
tions of continuous vibration (6.68 + 9.77 s vs.
15.86 20.20 s, P < 0.001) rather than differences
in intermittent buzzing (P > 0.05). The time spent
in intermittent buzzing was shorter in successful
courtships than in unsuccessful courtships of
mass-reared flies, but not in wild flies (Table 1),
while longer continuous vibration and total court-
ship led to greater success in wild flies but not in
mass-reared flies (Table 1). Combining data from
mass-reared and wild flies, the mean time spent
in intermittent buzzing in successful courtships
was less than that in unsuccessful courtships
(8.80 + 7.63 and 11.08 9.14 s, (P < 0.001). The
corresponding difference in durations of continu-
ous vibration was not significant.
Mass-reared females showed a strong ten-
dency to copulate when the proportion of time
during the courtship that was spent in intermit-
tent buzzing was especially low. The mean propor-
tion of time spent buzzing prior to successful
mounts was 57.0%, while the corresponding value
prior to unsuccessful mounts was 77.6% (Stu-
dent's t comparing arcsine transformations of
these proportions was 4.38, P < 0.00002). This ef-
fect was both independent of and stronger than
the effects of the other durations (Table 2, Model
2). Although wild flies showed the same trend to
copulate when the proportion of the time spent in
intermittent buzzing was lower (66.4% vs. 74.7%),
the difference was not significant (Tables 1, 2).
The decrease in this proportion in mass-reared
flies was due in large part to the decrease in the
duration of intermittent buzzing preceding suc-
cessful mounts rather than to longer durations of
continuous vibration (Table 1). In wild flies, how-
ever, there was not even a hint of a similar differ-
ence (Table 1).
There was a weak negative correlation in both
strains between the total duration of courtship
and the percentage of courtship dedicated to in-
termittent buzzing (r = -0.29, -0.60 in mass-
reared and wild flies respectively; in both cases
0.01 < P < 0.05). There was no correlation in ei-
ther strain between the absolute duration of in-
termittent buzzing and continuous vibration.
Durations of Individual Buzzes and the Rate of Buzzing
A comparison of the mean durations of the last
10 individual buzzes during intermittent buzzing
before the male jumped onto the female in a sub-
sample of the courtships of mass-reared and wild
flies that led to copulation (N = 19 and 25 respec-
tively) with those in courtships that did not lead
to copulation (N = 121 and 109 respectively)
showed that buzz duration did not differ between
successful and unsuccessful courtships in either
strain (respective means were 156 + 71 ms vs. 152
70 ms for mass-reared flies, and 114 16 ms vs.
113 + 16 ms for wild flies; P = 0.80 and 0.76 re-
spectively) (the durations of buzzes did not
change significantly during intermittent buzzing
- unpublished data). Similarly, comparisons be-
tween the average intervals between buzzes dur-
ing successful and unsuccessful courtships in
these same pairs also failed to show consistent
significant differences (respective means were
164 + 50 ms vs. 175 + 52 ms for mass-reared flies,
and 155 + 60 ms vs. 184 + 117 ms for wild flies;
P = 0.10 and 0.03 respectively). Similar compari-
sons showed no differences in the overall rates of
intermittent buzzes (number/sec) in successful
and unsuccessful courtships (respective means
2.51 + 0.7 buzzes/s vs. 3.0 + +1.0 buzzes/s for
mass-reared flies, and 3.73 + 0.40 buzzes/s vs.
3.52 + 0.71 buzzes/s for wild flies; P = 0.25 and
0.42 respectively).
March 2002
Bricefo & Eberhard: Decisions by Medflies during Courtship
100-
80
60f
start continuous
wing vibration
M
0
L._
C
U-
start intermittent
wing buzzing
mass-reared successful (N=36)
mass-reared unsuccessful (N=134)
wild successful (N=34)
wild unsuccessful (N=42)
jump onto
female
Male Behavior
Fig. 6. Female angles at different stages during male courtship in mass-reared and wild flies during successful
and unsuccessful courtships. The female tended to turn to face more toward the male after continuous wing vibra-
tion began and before intermittent wing buzzing. Differences between strains and between successful and unsuc-
cessful courtships were not significant.
Walking Behavior
There were no consistent differences between
successful and unsuccessful courtships with re-
spect to whether the female or the male was im-
mobile (= not walking) when continuous vibration
or intermittent buzzing began, or one s before or
afterward. In both strains female immobility in-
creased as courtship progressed. The female was
more likely to be immobile when intermittent
buzzing began than she had been when continu-
ous vibration began (P < 0.0001 with Chi2 for
both), and to be immobile one s after buzzing be-
gan than she had been 1 s before it began (P <
0.0001 with Chi2 for both). There was a similar,
but inconsistent trend for females to be immobile
more often one s after continuous vibration began
than they had been one s before (P = 0.002 for
mass-reared flies; P = 0.45 for wild flies).
There were several between-strain differences.
Mass-reared females were less likely to be immo-
bile after continuous vibration began (P = 0.0003
with Chi2), and one s after intermittent buzzing
began (P = 0.006). Mass-reared males were less
likely to be immobile during the s after intermit-
tent buzzing began (P = 0.001 with Chi2), but were
less likely to creep slowly toward the female dur-
ing intermittent buzzing (P = 0.0001 with Chi2).
In general, courtships were shorter in both
strains when the female was immobile one s be-
fore or one s after continuous vibration began
(means were smaller in all eight within-strain
comparisons; differences were significant in five).
There was also a significant association between
longer courtships and male creeping behavior
during intermittent buzzing (P = 0.008 and 0.01
in mass-reared and wild flies respectively). Thus
males apparently decided to creep toward the
female when relatively long courtships failed to
lure her close enough.
Male Size
Mass-reared males were smaller than wild
males in head width 0.85 + 0.06 vs. 0.89 + 0.06
mm), thorax length (2.63 + 0.15 vs. 2.81 + 0.13
mm), and thorax width (1.73 + 0.10 vs. 1.83 + 0.09
mm) (all P < 0.001 with t tests). Although mass-
reared males performed more mounting attempts
than wild males (respective means were 8.3 and
3.7; P = 0.019), there was no consistent relation
within either strain between male body size and
FL~il
1l
l
Florida Entomologist 85(1)
March 2002
Jump
*
r=0.43
()
0)
C
0)
Wild
Male angle
Male angle Male angle
vmuIL ui l Male angle Male angle
Fig. 7. Relationships between male and female angles in mass-reared and wild flies at different stages of court-
the rate of mounting failure (number of mounts
that led to copulation/ total number of mounts).
The p values for linear regression slopes of the
rate of failure on the three size measurements
were 0.047, 0.149 and 0.479 for wild flies (N = 21),
and 0.472, 0.606 and 0.939 for mass-reared flies
(N = 48). Males that copulated were not larger
than those that did not copulate among either
mass-reared or wild flies.
Mounts without Prior Courtship
Males and females frequently encountered
each other as they walked about in the confines of
the Petri dish. Mass-reared males were more
likely to jump onto the female during such an en-
counter without previous courtship (40.8% of 142
mounts were not preceded by courtship) than
were wild males (22.0% of 109 mounts without
prior courtship) (Chi2 = 9.9, df = 1, p = 0.0016).
Mounts without a previous courtship were less
likely to result in copulation in mass-reared flies
(6.9% of 58 mounts without previous courtship vs.
19.0% of 84 with previous courtship; Chi2 = 4.24,
df = 1, P < 0.05), There was no difference in accep-
tance rates in wild flies (corresponding values
were 20.8% of 24 vs. 24.7% of 85;P > 0.05).
DISCUSSION
Cues associated with Courtship Decisions
The data presented here are probably related
at two different levels of causation to the deci-
sions made by males and females during court-
ship. The difference between levels involves
cause-and-effect relations as opposed to simple
correlations. Some measurements, such as the
distance between the flies (which is largely a
function of whether or not the female approached
the male), are probably "indicator variables" that
represent the probability that a particular deci-
sion has been or will be made. They may consti-
tute, for instance, indicators from the female's
overt behavior of the likelihood that she will even-
tually accept the male's copulation attempt when
he jumps onto her. These variables may have lit-
tle or nothing to do with why the female made the
decision to accept or reject the male, but rather be
consequences of her having made a decision. A
second set of possible "cue" variables represent
possible stimuli that trigger particular decisions
by males or females. For example, the male's size
and his song characteristics, represent possible
cues that might be used by females in making the
Bricefo & Eberhard: Decisions by Medflies during Courtship
N=172
Successful Unsuccessful
Mass-reared
Successful Unsuccessful
Wild
Fig. 8. Length of time female was immobile before
the male mounted her in successful and unsuccessful
courtships of mass-reared and wild flies. Mass-reared
females were immobile for significantly shorter periods
preceding successful mounts as compared with unsuc-
cessful mounts; and successful mounts of mass-reared
flies were preceded by significantly shorter periods of fe-
male immobility than successful mounts of wild flies.
decision whether or not to accept copulation.
These two questions are discussed separately, al-
though it is possible that some variables may play
more than a single role. For instance, female be-
havior that is associated with likely acceptance
may be used as a cue by the male to trigger par-
ticular courtship behavior of his own. These dif-
ferences have not always been clear in previous
discussions of medfly courtship.
Possible Indicator Variables. Our results con-
firm and quantify several conclusions regarding
possible female "acceptance" variables from pre-
vious studies. The gradual reduction in the dis-
tance between male and female, the increase in
the female's tendency to look more directly to-
ward the male, and her increased immobility in
the later stages of courtship are in accord with the
idea that one result of successful male courtship
behavior is to induce the female to approach him
(or allow him to approach her), to look directly to-
ward him, and to remain still. Feron (1962) de-
rived these ideas from qualitative observations of
the relative mobility of females compared to
courting males, but gave no quantitative support.
Briceno et al. (1996) came to similar conclusions
from comparing A) the positions of flies at the mo-
ment a male leapt onto the female (both success-
ful and unsuccessful leaps were included), and B)
positions when males decided to abandon court-
ship (presumably relatively extremely unfavor-
able conditions).
The present data are much more extensive and
quantitative. They are also more convincing re-
garding the biological importance of male and fe-
male angles and the distance between the two
flies at the moment that the male jumps, because
they establish correlations with the likelihood
that the female will allow copulation to occur,
rather than just whether or not the male will
jump. It must be kept in mind, however, that the
data are only correlations, and thus do not allow
confident deductions regarding cause and effect.
It is thus not yet certain whether male-female
alignment and close proximity is a cause of fe-
male acceptance of copulation, or whether it is
correlated with female receptivity that is due to
other causes.
It is entirely possible that we have documented
here only manifestations of the female's likelihood
of accepting copulation, and not the reasons why
sometimes they were receptive and sometimes
not. On the other hand, our results call into ques-
tion the usefulness of studies of male-female in-
teractions and possible interchanges of signals
that do not take the angles and distances between
the flies into account (e.g., Lux & Gaggl 1996, Lii-
matainen et al. 1997). It is now clearer than be-
fore that these factors are indeed associated with
the success and failure of male courtships.
The tendency for the female to have spent less
time moving prior to courtships that terminated
with successful mounting is also in accord with
the idea that male courtship functions to arrest
female movement. The correlation between male
and female angles may be due to the difficulty of
continuing to look directly toward the female
when the female is looking (and perhaps moving)
in a direction other than toward the male.
Possible Cue Variables. The changes in distances
and angles that occurred just before and after
males began continuous vibration and intermit-
tent buzzing suggest the following interpretation.
Males are stimulated to begin continuous vibration
when the female approaches, and to begin inter-
mittent buzzing when she approaches still closer.
The male may also be induced to initiate these be-
havior patterns when the female is oriented facing
more directly toward him. Mass-reared males be-
gan both types of behavior when the female was
facing them more directly, while similar trends in
wild flies were not significant. Female decisions
may be affected by stimuli from continuous vibra-
tion and intermittent buzzing by the male.
Florida Entomologist 85(1)
March 2002
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Bricefo & Eberhard: Decisions by Medflies during Courtship
Although neither type of male behavior was associ-
ated with reductions in the distance between the
two flies, females tended to turn to orient them-
selves more directly toward the male in the s after
intermittent buzzing began, and, less consistently,
in the s after continuous vibration began.
These interpretations must be evaluated care-
fully. They assume that reductions in the distance
between the flies are due to female rather than
male movements (nearly always true prior to and
during continuous vibration and the early stages
of intermittent buzzing, but not later in buzzing).
They also attribute cause and effect relations to
what are at present only correlations (above).
Our results shed little light on the cue or cues
that trigger female acceptance of a male, other
than ruling out several possibilities. The dura-
tions of individual buzzes and the intervals be-
tween them during intermittent buzzing, the rate
of buzzing, the duration of the intermittent buzz-
ing stage, the duration of the continuous wing vi-
bration stage, and the male's size all failed to
show significant differences between successful
and unsuccessful courtships. There was a weak
tendency in wild flies for successful courtships to
have been longer than unsuccessful courtships;
the lack of such a female criterion in mass-reared
flies may be due to selection on females in mass-
rearing cages (Briceho & Eberhard 2000), where
males with shorter courtships are favored
(Briceho & Eberhard 1998). The significance of
the very strong tendency for increased female ac-
ceptance of copulation in mass-reared flies when
the proportion of time during the courtship spent
in intermittent wing buzzing was low with re-
spect to the time spent in continuous wing vibra-
tion is not clear, especially in view of the lack of a
significant trend in wild flies
Our finding that male size does not affect fe-
male acceptance is similar to the results of sev-
eral studies in Hawaii (Arita & Kaneshiro 1988,
Whittier et al. 1992, 1994, Whittier & Kaneshiro
1995), but differs from the equally clear tendency
for larger males to be more readily accepted in Is-
rael (Blay & Yuval 1997). It appears that there
may be geographic variation in this trait. Female
choice criteria are known to vary geographically
in other species (e.g., Andersson 1994).
Differences between Mass-reared and Wild Flies
Our results constitute the second set of obser-
vations showing that courtship duration is re-
duced in a mass-reared strain compared with the
wild strain from which it was derived (Briceho &
Eberhard 1998). Males of two other mass-reared
strains also perform relatively short courtships,
but in one case the behavior was recorded under
different conditions, and in the other nothing is
known of behavior of the wild flies from which it
was derived (Briceho & Eberhard 1998). The de-
gree of reduction in the present study of a 3 year-
old strain (78% of wild flies) is somewhat less
than that seen previously with older, 4.5 year-old
mass-reared strain (69%), as would be expected if
inadvertent selection due to interruptions under
mass-rearing conditions produced a gradual shift
toward shorter courtships.
The tendency for mass-reared males to initiate
continuous wing vibration when the female was
closer and when she was facing more directly to-
ward him may represent additional adaptations
by mass-reared males to the crowded conditions
of mass-rearing cages. High thresholds for initiat-
ing courtship may well be advantageous in a cage
containing 60,000 flies. These changes may be ac-
companied on the female side by a greater ten-
dency in mass-reared but not wild females to
accept courtships that were initiated at a shorter
distance. This difference was not significant in
the variable-by-variable analyses in Table 1, but
was significant in regression analyses of mass-
reared flies when the effect of relative duration of
intermittent buzzing was held constant (Table 2,
Model 2), and also showed a significant interac-
tion with the female angle when the male jumped
(Table 3, Model 1). Thus the acceptance criteria of
mass-reared females may have coevolved in ac-
cord with a reduction in the distance at which the
male begins courtship.
The greater selectivity in mass-reared males
with respect to when to initiate courtship was not
accompanied by a possibly coevolved preference
by females favoring courtships that began with
smaller female angles. However the selectivity
shown by wild females with respect to the effects
of the female's angle at the moment when contin-
uous and when intermittent wing vibration began
was absent in mass-reared females (Table 2,
Model 1). The loss of such selectivity in mass-
reared females would thus favor males that ini-
tiate courtship at smaller female angles, which is
the trend found when mass-reared and wild
males are compared. Thus this female acceptance
criterion may also have changed in mass-reared
flies (via abandonment of a former bias) in step
with changes in male behavior that probably re-
sulted from selection in crowded mass-rearing
cages, just as greater acceptance of shorter court-
ships has also coevolved with abbreviated court-
ship in mass-reared flies (Briceho & Eberhard
2000). Males in cages may thus be under selection
to respond only to females at shorter distances to
avoid interruptions; and female criteria may have
evolved to favor males whose sons were more
likely to perform uninterrupted courtships. Fur-
ther data are needed to test these ideas.
One further point regarding differences and
similarities in distances concerns the uniformity,
both between and within strains, in the distance
between male and female when the male initiated
his jump onto the female. It might have been
Florida Entomologist 85(1)
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March 2002
Bricefio & Eberhard: Decisions by Medflies during Courtship
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Florida Entomologist 85(1)
thought that the shorter courtships of mass-
reared males (e.g., Briceno & Eberhard 1998;
above) occurred because the males fail to wait un-
til the distance to the female has decreased, and
jump onto the female from farther away. This
seems not to be the case. The probable reason is
that male stimulation by the female using his
aristae (Briceno & Eberhard in press) cannot oc-
cur until the flies are quite close together.
Summarizing the results of this and previous
studies, there are now five differences known be-
tween the sexual behavior of mass-reared males
and wild males. Mass-reared male courtship prior
to mounting is shorter, is initiated at a shorter
distance from the female and when the female is
looking more directly toward the male, the male
is less likely to creep toward the female during in-
termittent buzzing, and the male is more likely to
attempt to mount a female they have encountered
without prior courtship. Acceptance criteria in
mass-reared females appear to have changed to
favor the first two and possibly also the third of
these male changes, but to act against the fifth.
The first two changes fit a Fisherian sequence of
evolution by sexual selection (see Briceno & Eber-
hard 2000). Changes in female criteria have also
been documented in other mass-reared insects
(Liu & Haynes 1994, Zhu et al. 1997).
The data documenting differences between
strains must be interpreted cautiously, because
only pairs of flies of the same strain were ob-
served. Given the probable effects of the behavior
of one sex on that of the other, it will not be possi-
ble to attribute differences to one sex or the other
with certainty until cross-strain pairs are studied.
Limitations of These Analyses
Many male courtships do not end in a mount;
the male terminates courtship when the female
moves away or otherwise fails to respond appro-
priately (Feron 1962, Briceno et al. 1996). Court-
ships that do not lead to a mounting attempt
could obviously affect a male's success, but were
omitted in the present analyses. Also omitted
were those courtships in which the male returned
to continuous wing vibration after having begun
intermittent wing buzzing. Perhaps additional
answers to why some courtships succeed and oth-
ers fail will be revealed by analyses of these types
of interaction.
This study revealed several strong trends with
respect to probable male and female cues and re-
sponses during the course of courtship, and also
demonstrated several clear behavioral differ-
ences between mass-reared and wild flies. But it
was much less fruitful in uncovering clear differ-
ences between successful and unsuccessful court-
ships. The critical reader cannot help but be
struck by the large standard deviations and sub-
stantial overlaps in nearly all of our data. Indeed,
large variations are ubiquitous in nearly all quan-
titative data on medfly courtship behavior
(Briceno et al. 1996, Liimatainen et al. 1997,
Briceno & Eberhard 1998, Quilici in press; an ex-
ception is behavior involving contact between
male and female aristae-Briceno & Eberhard in
press). It is clear, for instance, that when the dis-
tance to the female is shorter and when she is fac-
ing more directly toward the male, there is a
greater likelihood that the male will jump
(Briceno et al. 1996), and that the female will ac-
cept copulation with him when he does (Figs. 2
and 6). But there were numerous rejections when
both the distance and the female angle were low,
and acceptance when they were both high. Simi-
lar variation also occurs at earlier stages of court-
ship (see large error bars in Figs. 2 and 6).
These large variations and substantial over-
laps have several consequences. On a practical
level, they mean that relatively large samples of
courtships are needed to document significant dif-
ferences between successful and unsuccessful
courtships or differences between strains. They
also signal our lack of detailed understanding
why some courtships are successful and others
are not. There are several possible explanations
of this failure. Perhaps we simply have not yet fo-
cused on the male trait or traits that have the
most powerful effects on female acceptance. Such
"mystery traits" could involve factors that cannot
be measured in videotapes (e.g., sound intensities
in male songs, male pheromones).
A second possibility is that the basic approach
of searching for triggering stimuli is not biologi-
cally appropriate. Perhaps female acceptance is
sometimes "spontaneous", and does not depend
on the presence of particular stimuli. The consis-
tent superiority of wild males over mass-reared
males (above) and of some males over others (e.g.,
Whittier & Kaneshiro 1995) argues, however,
that this cannot be the complete explanation. An-
other possibility is that each particular stimulus
only slightly increases the probability of accep-
tance, rather than guaranteeing that it will occur.
Discriminating between the possibilities of mys-
tery traits and small incremental effects may be
especially difficult in medfly courtship, where a
large variety of possible stimuli are involved. Ex-
perimental manipulation of traits may be the best
tactic for future studies.
ACKNOWLEDGMENTS
We thank Ricardo Gonzalez for technical assistance,
Jorge Lobo for extensive, patient advice on statistical
matters, and Hernan Camacho for providing us with
flies. Financial support was provided by the Interna-
tional Atomic Energy Agency, the Vicerrectoria de In-
vestigaci6n of the Universidad de Costa Rica, and the
Smithsonian Tropical Research Institute.
March 2002
Bricefo & Eberhard: Decisions by Medflies during Courtship
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Florida Entomologist 85(1)
March 2002
VARIATION IN THE INTERMITTENT BUZZING SONGS
OF MALE MEDFLIES (DIPTERA: TEPHRITIDAE) ASSOCIATED
WITH GEOGRAPHY, MASS-REARING, AND COURTSHIP SUCCESS
R. D. BRICENO1, W. G. EBERHARD1'2, J. C. VILARDI3, P. LIEDO4, AND T. E. SHELLY5
1Escuela de Biologia, Universidad de Costa Rica, Ciudad Universitaria, Costa Rica
2Smithsonian Tropical Research Institute
'Laboratorio Gen6tica de Poblaciones, Depto. Cs. Biol6gicas, Fac. Cs. Exactas y Naturales
Universidad de Buenos Aires, 1428 Buenos Aires, Argentina
4El Colegio de la Frontera Sur, Tapachula, Chiapas, Mexico
5USDA-ARS, P.O. Box 2280, Honolulu HI 96804
ABSTRACT
Many aspects of the temporal pattern of sounds produced during the intermittent buzzing
displays of pre-copulatory courtship by male medflies varied between wild flies from Costa
Rica, Argentina, and Hawaii, and between mass-reared flies from Costa Rica, Argentina,
Mexico, and Hawaii. There were no consistent differences when mass-reared strains were
compared with the wild strains from the area where they originated in Costa Rica, Argen-
tina and Hawaii. Buzzing sounds produced prior to successful mounting attempts did not
differ consistently from those preceding unsuccessful mounts in flies from Costa Rica and
Argentina. In strains from all sites, however, courtships in which buzzes were interrupted
were more likely not to result in mounting of the female. There was a weak tendency for in-
terruptions to be more common in mass-reared strains.
Key Words: Courtship sounds, medfly, geographic differences
RESUME
Varios aspects de los patrons temporales de los sonidos del zumbido intermitente produ-
cido durante el cortejo pre-copulat6rio de los machos de la mosca del Mediterraneo variaron
entire moscas silvestres de Costa Rica, Argentina y Hawaii, y entire moscas de sepas de cria
masiva de Costa Rica, Argentina, Mexico y Hawaii. No se presentaron diferencias consisten-
tes cuando se compararon las sepas de cria masiva en Costa Rica, Argentina y Hawaii con
moscas silvestres de los sitios de origen. Los sonidos producidos durante cortejos que termi-
naron en c6pulas no difieron de los sonidos producidos durante cortejos que llevaron a mon-
tas que fracasaron en moscas de Costa Rica y Argentina. Pero en sepas de todos los sitios los
zumbidos intermitentes que incluyeron pequenas pausas tuvieron una mayor probabilidad
de no terminar en un intent de monta. Los zumbidos intermitentes de las moscas de las
crias masivas tuvieron una tendencia leve a interumpirse mas frecuentemente.
Male medflies (Ceratitis capitata Wied.) pro-
duce two types of wing vibration during pre-
mount courtship (Feron 1962, Rolli 1976, Webb
et al. 1983, see review of behavior in Briceno et al.
1996, Briceno & Eberhard 2002). During an early
stage of courtship, the male produces the "calling
song" with an average fundamental frequency of
about 350 Hz (Webb et al. 1983), by vibrating his
wings continuously while he looks toward the
female and holds his abdomen bent ventrally so
that the pheromone-producing everted rectal epi-
thelium is ventral to the rest of his body. Probably
a plume of pheromone is thus wafted toward the
female. After an average of about 6 sec of contin-
uous wing vibration, the rectal epithelium is
retracted, and the male begins a series of inter-
mittent wing buzzes (Fig. 1) which continue until
he leaps onto the female or abandons courtship.
Each buzz is associated with a more intense
sound that has a lower average fundamental fre-
quency of about 165 Hz (Webb et al. 1983). A sin-
gle buzz lasts on the order of 0.1 sec and is
produced during the time wings are vibrated rap-
idly with a large amplitude, from anterior of the
male's head to back over his body (Briceno &
Eberhard 2000). The softer calling song continues
during the intervals between buzzes. During in-
termittent buzzing behavior the male often also
rocks his head rapidly from side to side, often tap-
ping the female with his aristae (Briceno & Eber-
Bricefo et al.: Medfly Courtship Songs
series of buzzes
mount female
1 \
calling song
5 10 15
Time (sec)
single buzz
0.5 1.0
Time (sec)
Fig. 1. Sounds produced by a male Ceratitis capitata
during courtship and mounting.
hard in press). Finally the male leaps onto the
female, shakes his body briefly rapidly forward
and backward while producing another strong
buzzing sound (Fig. 1). Sound production ends as
the male then attempts to turn and establish gen-
italic contact; males do not resume sound produc-
tion during copulation.
The significance of sounds produced during
courtship in medflies is as yet unclear. Possible ef-
fects of sounds produced during courtship on cop-
ulation success, and of changes in the female's
ability to perceive them have been investigated
experimentally. Keiser et al. (1973) found that the
percentage of females that were inseminated
when male wings were removed dropped by about
half, and Nakagawa et al. (1973) and Levinson et
al. (1987) found that removal of the female anten-
nae nearly completely eliminated copulation.
However, the first experiment also modified possi-
ble visual stimuli, the second also modified possi-
ble tactile stimuli from the male's aristae, and
both may have also modified chemical stimula-
tion of females. There are thus as yet no conclu-
sive demonstrations that any sounds are
functionally important in courtship; it is possible
that they are all only incidental consequences of
other activities (i.e., creation of air currents) (see
review in Eberhard 2000).
There are several reasons to expect that
present day populations of medflies may not have
uniform courtship songs. The geographic range of
the species, which is native to Africa, has in-
creased dramatically, and several population bot-
tlenecks have probably occurred in recent times
(Huettel et al. 1980, Fuerst 1988). Thus both drift
and divergence under sexual selection in geo-
graphically isolated populations may have oc-
curred. In addition, mass rearing of sterile males
has often been used in attempts to control pest
populations of medflies, and mass-reared strains
have often been conserved for many years. Repro-
duction in these strains occurs under conditions
that differ sharply from those in nature in several
respects. Old mass-reared strains thus represent
the results of inadvertent experiments in which
several environmental conditions have been
changed. It is not obvious, however, which song
traits would be more advantageous under mass-
rearing conditions. Rolli (1976) reported a lack of
differences between the songs of wild medflies
from Tunis and Morocco and mass-reared males
in Germany (the age of the mass-reared strain
was not specified). The sample sizes were small,
however, and it is not clear which song characters
were compared.
This paper tests the possibility of geographic
divergence and of changes under mass-rearing in
the temporal pattern of the intermittent buzz and
the mounting songs of males of seven strains of
flies: wild flies from Costa Rica, Argentina and
Hawaii; mass-reared strains derived from these
strains 6.5, 10, and more than 40 years previ-
ously; and a three-year old mass-reared strain
from Mexico. Songs of successful and unsuccess-
ful courtships are also compared.
MATERIALS AND METHODS
Mass-reared flies from Costa Rica were from a
strain which had been initiated in 1990 using
wild flies collected near Alajeula, Costa Rica, and
maintained subsequently at the Laboratorio de
Manejo Integrado de la Mosca de la Fruta mass-
rearing facility. Wild flies were raised from larvae
that emerged from infested tangerines collected
in Jan-April, 1997, at the Estaci6n Experimental
Faubio Baudrit near Alajuela, Costa Rica.
Argentinian mass-reared flies came from the
Mendoza strain, which had been derived from
flies collected about 10 years before our observa-
tions in Mendoza province, Argentina. Wild flies
were a laboratory G2 derived from flies raised
from fruit collected in the field in the Alto Valle re-
gion of Patagonia. Mass-reared flies in Mexico
were from a three year-old strain derived from
flies collected as larvae from coffee in Costa Cuca,
Quetzaltenengo, Guatemala. Hawaiian mass-
reared flies were from the Hilab strain derived
from wild flies more than 40 years previously,
while wild flies were reared from coffee fruit col-
lected on Kauai.
Florida Entomologist 85(1)
Adult flies of all strains were separated by
sexes when they were less than two days old, and
fed mixtures of sugar and protein hydrolysate.
Male-female pairs of mass-reared flies were
placed together for video taping when they were
five days old; male-female pairs of wild flies,
whose sexual maturation is more delayed, were
placed together only after they were 10 days old.
Pairs of flies in Costa Rica and Hawaii were
videotaped in 13.7 cm diameter and 1.8 cm deep
mating chambers (clear Petri dishes) on a glass ta-
ble using a Sony CCD Video Hi 8 camera equipped
with + 6 closeup lenses. The camera was below the
table, allowing taping from below (most court-
ships occurred on the ceiling of the mating cham-
ber). A small microphone (Sennheiser System
MZK 80ZU) was inserted through a hole in the
side of the chamber and connected to the camera.
Pairs in Argentina and Mexico were videotaped in
a clear plastic cylinder 7.3 cm high and 9.0 cm in
diameter. Each morning a fresh leaf from a citrus
tree was attached to the ceiling of the cage, and a
male was released in the cage. Five minutes after
the male began emitting pheromone, a female was
released into the cage, and the flies' behavior was
recorded for 30 min or until they copulated.
All recording environments were noisier than
that used by Webb et al. (1983), and both types of
mating chambers produced strong echoes. Other
than verifying that the apparent fundamental
frequencies of the songs of the Costa Rican flies
were similar to those observed by Webb et al.
(1983), we did not attempt to analyze the frequen-
cies or power spectra of songs. Instead we concen-
trated on the temporal patterns of the songs.
There are indications in other flies that temporal
patterning may be an especially important aspect
of songs (Bennet-Clark & Ewing 1969, Kyriacou
& Hall 1982, Tomaru & Oguma 1994, Aspi &
Hoikkala 1995, Neems et al. 1997, and Hoikkala
& Kaneshiro 1997 on Drosophila).
Recordings of sounds were imported from
video recordings into a PC 486dx2 computer us-
ing a 16 bit card. The mean durations of buzzes
and intervals between buzzes were measured us-
ing the real time display in the program Avisoft
when cursors marked the beginning and the end
of the envelope curve displayed in the main win-
dow of the program (Fig. 1). The precision of these
measurements was determined by re-measuring
the duration of 10 buzzes and 10 intervals in each
of 8 different courtships. The average differences
were 2.0 ms in buzz duration, and 2.2 ms in inter-
val duration. In addition, the number and dura-
tion of interruptions during the buzzes was
determined. An interruption was defined as any
interval between buzzes that was more than
twice the mean of the intervals immediately pre-
ceding and following it (Fig. 2).
We examined two different sets of buzzes in
Costa Rican flies: the first 10 buzzes in the court-
119,6*
rT11J ~ .... I *r'r!
1... 11 r I U4 I I ll lr
2 __ 4 6 8
Fig. 2. Three intermittent buzzing courtships in which
interruptions (*) were absent (A) and present with slower
(B) and more rapid buzzing (C) (all with same time scale).
ship of a male; and the last 10 buzzes before the
male either leapt onto the female or ceased court-
ing, to determine whether the characteristics of
buzzes varied systematically during courtship.
Courtship outcome was classified in three
classes: no mount-the male ceased courting
without attempting to mount the female (failure
to mount is often associated with failure of the fe-
male to align herself properly with the courting
male and to remain still-Briceno et al. 1996,
Briceno & Eberhard 2002); failed mount-the
male mounted but was dislodged within 10 s
when the female struggled; and successful mount
- the male mounted the female and achieved gen-
italic contact.
All statistical tests were non-parametric Mann
Whitney U Tests due to the highly skewed distri-
butions of many variables. Means are presented
followed by one standard deviation for illustrative
purposes only.
RESULTS
There was no clear, consistent tendency for
buzz duration or interbuzz duration to differ be-
tween the first and last 10 buzzes of a courtship in
Costa Rican or Argentinian flies (Fig. 3). Wild flies
March 2002
I rj| |ii
Bricefo et al.: Medfly Courtship Songs
from Costa Rica, Argentina, and Hawaii differed
significantly in all five pre-mount song traits
(Table 1). Comparing wild flies, those from Argen-
tina produced longer buzzes, those from Hawaii
produced the largest number of buzzes/courtship,
and those from Costa Rica had longer intervals
between buzzes and a lower overall rate of buzz-
ing. Wild Hawaiian flies had the longest mount
buzzes. Mass-reared flies from the three sites also
differed in many traits. When mass-reared and
wild flies from the same site were compared, no
traits showed the same trends at all three sites.
In an attempt to understand the possible selec-
tive factors which might influence song character-
istics, we compared several aspects of intermit-
tent buzzes in courtships that led to copulation as
compared with those that ended in female rejec-
tion of a mount or a failure to mount in different
strains (Tables 2 and 3). There were no consistent
directional differences for traits related to dura-
tions and frequencies of intermittent buzzing
(Table 2). The duration of the mount buzz was
marginally longer in successful mounts in the
mass-reared strain from Costa Rica, but there
were no similar trends in wild Costa Rican flies or
in strains from Argentina and Hawaii.
In contrast, there was a consistent trend in each
of the six strains with good sample sizes for inter-
ruptions in intermittent buzzing to be more fre-
quent in courtships that did not lead to a mounting
attempt (first two columns in Table 3); combining
the data from all strains, the frequency with which
buzzing was interrupted was 72.3% of 166 court-
ships which did not lead to mounting, 39.2% of 263
in which the male mounted but was then rejected,
and 28.4% of 81 in which the male mounted and
succeeded in copulating. Hawaiian flies were more
likely to interrupt buzzing. Among the other sites,
mass-reared males in Costa Rica were less likely to
interrupt their courtships than mass-reared males
from Argentina or those from Mexico, but there
was no difference in this respect between wild flies
from Costa Rica and Argentina. There were no dif-
ferences when mass-reared flies were compared
with their respective wild counterparts in Costa
Rica, Argentina, and Hawaii with respect to the
proportion of courtships in which buzzing was in-
terrupted, the number of interruptions, or their
durations. But there were similar trends for more
interruptions to occur in mass-reared strains, and
when data from different sites were combined,
mass-reared flies were slightly more likely to inter-
rupt buzzing (52.7% of 262) than were wild flies
(41.7% of 156) (P = 0.0297 with Chi Squared Test).
The apparent female bias against interrupted
buzzing did not extend to events that occurred
after mounting. There was no significant associa-
tion in any strain between the occurrence of an in-
terruption, the number of interruptions, or the
mean duration of interruptions and the likelihood
that the female would reject the male once he had
mounted. Similarly, when all courtships in which
mounts occurred were combined for all strains,
there was no significant relation between male
copulation success and whether or not buzzing
had been interrupted.
When song parameters were correlated with
male body size (estimated by the maximum width
of the head in dorsal view), there was only one sig-
nificant relationship, with the total number of
buzzes/courtship (r = 0.26 with log-transformed
numbers, P < 0.05). Other correlation coefficients
were -0.01 (P = 0.99) for the mean duration of
each buzz, -0.12 (P = 0.29) for the mean duration
of interval between buzzes, -0.06 (P = 0.62) for the
number of buzzes/s, 0.22 (P = 0.20) for the log of
total duration of buzzing, and 0.17 (P = 0.41) for
the duration of the mount buzz.
DISCUSSION
Although there were several differences be-
tween mass-reared and wild Costa Rican flies in
duration and number of buzzes, similar differ-
ences did not occur between mass-reared and wild
flies from Argentina and Hawaii. This suggests
that the differences in the Costa Rican flies may
not be due to mass-rearing per se. This result is in
accord with a less-detailed study of flies from
Tunis and Morocco (Rolli 1976), and with the lack
of obvious differences in selective pressures on
the details of intermittent buzzing behavior un-
der mass-rearing conditions.
Similarly, there were no consistent differences
between successful and unsuccessful courtships in
Costa Rican, Argentinian and Hawaiian flies in
many of the song variables that we measured.
However the possibility of stabilizing sexual selec-
tion on these traits cannot be excluded until further
analyses are performed. We also did not measure
additional aspects of the song, such as its intensity
and basic frequency, and there are other ways that
vibrations may be transferred to the female, such
as through substrate and near field medium mo-
tion (Mark1 1983). Song intensity is an important
determinant of female acceptance in the tephritid
Anastrepha suspense (Loew) (Sivinski et al. 1984).
Thus while presently available data do not support
the idea that song traits influence courtship suc-
cess, the possibility cannot be ruled out.
The strongest association we found with the
eventual outcome of courtship was that between
interruptions of intermittent buzzing behavior
and failure to mount (Table 3). The significance of
this association is not clear. On the one hand, our
criterion for distinguishing interruptions was ob-
viously arbitrary. The strength and consistency of
the association we found with failure to mount
leaves little doubt that there is an association of
some sort with the pattern of buzzes, but it is un-
certain whether or not the biologically relevant
criterion is the one which we used.
Florida Entomologist 85(1)
March 2002
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Florida Entomologist 85(1)
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TABLE 3. ASSOCIATION BETWEEN THE OCCURRENCE OF AN INTERRUPTION IN INTERMITTENT BUZZING, AND THE NUMBER
AND DURATION OF INTERRUPTIONS WITH THE OUTCOMES OF COURTSHIPS IN DIFFERENT STRAINS. MEANS WERE
CALCULATED ONLY FOR THOSE COURTSHIPS IN WHICH INTERRUPTIONS OCCURRED, AND ARE FOLLOWED BY +
ONE STANDARD DEVIATION. THE SAMPLE SIZES (IN PARENTHESES) FOR MEAN NUMBER OF INTERRUPTIONS RE-
FER TO THE NUMBER OF COURTSHIPS; FOR MEAN DURATION THEY REFER TO THE NUMBER OF INTERRUPTIONS.
MC = MOUNT AND COPULATE; MF = MOUNT BUT FAIL TO COPULATE DUE TO FEMALE RESISTANCE; NM = NO
MOUNT. ASTERISKS DENOTE NM INTERRUPTION FREQUENCIES THAT WERE SIGNIFICANTLY LOWER THAN THE
FREQUENCIES FOR MC AND MF IN THE SAME STRAIN USING A CHI2 OR A FISHER EXACT TEST (F). IN NO STRAIN
DID MC FREQUENCY DIFFER SIGNIFICANTLY FROM MF FREQUENCY, NOR WAS THE FREQUENCY FOR ALL MC
(DATA FROM ALL STRAINS COMBINED) DIFFERENT FROM THAT FOR ALL MF (CHI2 TEST). VALUES FOLLOWED BY
THE SAME LETTER ARE SIGNIFICANTLY DIFFERENT (a AND = P < 0.05, B AND ** = P < 0.01, *** = P < 0.001).
Interrupt?
Mean number Mean duration
Strain and outcome Yes No of interruptions of each interruption
Costa Rica
Mass-reared MC
MF
NM***
Total ahb cc
Wild MC
MF
NM***(F)
Total c_
Argentina
Mass-reared
MC
MF
NM*(F)
Total ablbh
MC
MF
NM**(F)
Total ,c
Mexico
Mass-reared MC
MF
NM***
Total ab~gc
Hawaii
Mass-reared MC
MF
NM
Total b,214
Hawaii
Wild MC
MF
NM
Total a b5
2.8 + 3.29 (15)
4.07 3.24 (14)
3.36 3.20 (20)
2.0 (1)
1.66 1.65 (9)
2.36 1.93 (10)
2.15 + 1.87 (20)
2.00 1.15 (7)
2.23 1.42 (13)
1.92 2.60 (11)
2.34 1.84 (31)
1.75 0.95 (4)
1.37 + 0.71 (14)
2.15 + 1.35 (8)
1.64 0.98 (26)
3.30 4.07 (23)
5.31 5.44 (20)
4.28 + 4.82 (43)
3.75 2.75 (4)
5.10 + 4.86 (19)
3.80 2.62 (46)
4.15 + 3.40 (69)
3(1)
3.25 2.86 (8)
4.00 2.58 (10)
3.63 2.60 (19)
1.46 + 2.19 (43)
1.40 + 1.35 (39)
1.44 1.75 (82)
1.80 1.64 (2)
1.98 4.33 (15)
0.81 + 0.43 (26)
1.26 2.60 (43)
1.81 + 1.75 (15)
1.93 3.30 (30)
1.47 + 1.24 (31)
1.72 + 2.33 bh (76)
1.47 0.67 (7)
1.47 1.94 (27)
1.10 + 1.09 (17)
1.34 1.56 (51)
1.08 + 1.28 a (80)
1.27 + 1.30 a (116)
1.19 1.29 bj (196)
0.375 + 0.22 (4)
1.02 1.21 (19)
1.17 + 1.50 (46)
1.08 1.40 (69)
1.13 (1)
1.20 1.23 (8)
0.77 + 0.45 (10)
1.01 + 0.94 (19)
In addition, cause and effect relations in this those males whose buzzes are interrupted, failing
association are uncertain. Further analyses will to allow the male to mount by positioning them-
be required to distinguish between two possibili- selves properly. Alternatively, females may not
ties. Interruption of buzzing may be a cue used by use interruption of the buzz as a cue, but rather
females, and they may exercise selection against the male may interrupt his buzzing when he per-
Bricefo et al.: Medfly Courtship Songs
Costa Rica
First 10 Buzzes
Wild
Nor= 200
SuC5,tisl*~ Nos, =14 5s
.-^^ -- ^^ eZ '"'"-^^ ^u"CO*ft I a rncatrilN-l4 U
^ ^^^~ Ut~vecf-slul movnt N.36
^.^^^'^ ~~~ ~ I___^M rivn -2
<
----- Mas-reared
.... Wird
Last 10 Buzzes
^< / "Y /,Suwsssful .,.nf N- 2
-~-- --Unsucca Insfulm .nIr.=34
'""'" -""Sfl cceeitI N-54fl1
Moss-re ned
------ wd
Number of Buzzes After Start
First 10 Buzzes
- - -- - ueccd
1D
Number of Buzzes After Start
Argentina Number oi Buzzes Before End
Last 10 Buzzes
rNumber of BuzzeN efore End
N00
----- Mired
10 5
Number of Buzzes Before End
Fig. 3. Durations of the first and last 10 buzzes in courtships of wild and mass-reared Costa Rican and Argen-
tinian flies that led to successful mounts (copulation), unsuccessful mounts (female rejection), and failure to mount
(n = number of courtships).
ceives from the female's behavior that she is
about to reject him. However this question is re-
solved, it does not appear that our results will
help explain the common inferiority of mass-
reared males as compared with wild counter-
parts, as we found only a weak difference in inter-
ruption frequencies between mass-reared and
wild males from the same site.
There were several geographic differences be-
tween both wild and mass-reared strains in the
details of intermittent buzzing. Possible causes of
divergence include founder effects and divergent
sexual selection in different populations. The lack
of consistent association between these details of
male songs and copulatory success argues against
the possible significance of sexual selection.
Sizes of mass-reared males from Hawaii
showed little sign of strongly influencing the differ-
ent song parameters we measured in this study.
ACKNOWLEDGMENTS
We thank Stan Rand for invaluable help with sound
analyses, Hernan Camacho for providing flies, John
Vargas for technical help, an anonymous referee for use-
ful comments, and the International Atomic Energy
Agency, the Vicerrectorfa de Investigaci6n of the Uni-
versidad de Costa Rica, and the Smithsonian Tropical
Research Institute for financial support. Work in Ha-
waii was supported by a grant from BARD project No.
IS-2684-96R awarded to B. Yuval and T. E. Shelly.
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Florida Entomologist 85(1)
Calcagno et al.: Mating Performance of a Medfly Strain
COMPARISON OF MATING PERFORMANCE OF MEDFLY
(DIPTERA: TEPHRITIDAE) GENETIC SEXING AND WILD TYPE STRAINS:
FIELD CAGE AND VIDEO RECORDING EXPERIMENTS
G. E. CALCAGNO1, F. MANSO2 AND J. C. VILARDI1
1Departamento de Ciencias Biol6gicas, Facultad de Ciencias Exactas y Naturales
Universidad de Buenos Aires, 1428 Buenos Aires, Argentina
'Instituto de Gen6tica "Ewald Favret", CICA-INTA, 1712 Castelar, Argentina
ABSTRACT
To improve the efficiency of the sterile insect technique (SIT) efforts are being devoted to ob-
tain genetic sexing strains (GSS). The present work was carried out in order to compare the
mating efficiency of flies from the GSS [(Ty34228 y /X)swx] and from a wild type strain (Men-
doza). Females of the GSS (T228) exhibit longer embryonic development, while males de-
velop in a normal time period. In a field-cage experiment, mating competitiveness was
compared between the T228 and the Mendoza, Argentina mass reared strain. The number
and duration of matings and the location of copula in the tree were recorded. The analysis
was repeated using irradiated males of T228. The results showed that mating efficiency of
the GSS is good in comparison with that of the Mendoza strain. Although copulatory success
in T228 is reduced by the radiation treatment, the high numbers of sterilized males released
would compensate this effect in the control programs. In a second experiment, under labo-
ratory conditions, videorecording techniques were applied. In this case two virgin males, one
of the GSS and one emerged from wild collected fruits, competed during 30 min for a virgin
wild female. The proportion of successful males did not differ between strains, but some dif-
ferences were observed between strains in the time spent in different stages of the courtship.
Males of the T228 were more aggressive, and they attempted to copulate with the other male
more frequently than did wild males. These differences may be due to selection for more ag-
gressive individuals under the overcrowded laboratory breeding conditions for this strain.
Key Words: mating behavior, sexual selection, sperm transfer, copulatory success
RESUME
Para aumentar la efectividad de la t6cnica del insecto est6ril (TIE) se estan dedicando gran-
des esfuerzos a la obtenci6n de lineas de sexado gen6tico (LSG). El present trabajo se realize
con el fin de evaluar la eficiencia en el apareamiento de una LSG [(Ty34228 y /X)swX], en com-
paraci6n con moscas de una lfnea de tipo salvaje (Mendoza). Las hembras de la LSG (T228)
exhiben un desarrollo embrionario mas lento, mientras que los machos tienen un tiempo de
desarrollo normal. En un experiment realizado enjaulas de campo se compare el 6xito en el
apareamiento entire las lines T228 y Mendoza. Se registry el numero y duraci6n de c6pulas
y la ubicaci6n de las parejas en el arbol. El andlisis se repiti6 utilizando machos irradiados de
la lfnea T228. Los resultados mostraron que la eficiencia de la LSG es buena en comparaci6n
con la de la lfnea Mendoza. Aunque el 6xito copulatorio de la lfnea T228 disminuye por efecto
de la radiaci6n, este efecto se podria compensar en los programs de control por el alto nu-
mero de machos esterilizados liberados. En un segundo experiment se realizaron, en condi-
ciones de laboratorio, videograbaciones del cortejo. En este caso dos machos virgenes, uno de
la LSG y otro salvaje emergido de frutas colectadas en el campo, compitieron durante 30 mi-
nutos por una hembra virgen salvaje. La proporci6n de machos exitosos no difiri6 entire las li-
neas, pero se observaron algunas diferencias entire ellas en los tiempos empleados en las
distintas etapas del cortejo. Los machos de la lifnea T228 fueron mas agresivos e intentaron
copular mas frecuentemente con el otro macho que los salvajes. Estas diferencias podrfan de-
berse a selecci6n a favor de individuos mas agresivos en la LSG como consecuencia de la alta
concentraci6n de individuos caracterfstica de la crfa en laboratorio.
Although the most widespread method of in- The sterile insect technique (SIT) for the con-
sect pest control is the use of chemical insecti- trol or eradication of the Mediterranean fruit fly,
cides, multiple disadvantages have favored the Ceratitis capitata (Wiedemann), is being applied
current tendency toward replacing them by bio- successfully in different countries (Wong et al.
insecticides or methods of biological or genetic 1986, McInnis et al. 1996, Arauni et al 1996,
control. Cayol et al. 1999). Genetic sexing strains (GSS)
Florida Entomologist 85(1)
have been isolated in Argentina in which males
and females can be differentiated by the color of
the larval posterior spiracles, and the color or size
of pupae (McInnis et al. 1994, Manso & Lifschitz
1991). Although these strains have a genetic sex-
ing system compatible with mechanical sex selec-
tion, they are not accompanied by a reduction of
rearing costs. In the IG/CICA/INTA Castelar, Ar-
gentina, a mutant carrying a different eye color,
and a slower embryonic development, was iso-
lated (Manso & Lifschitz 1991, Pizarro et al.
1997). While 90% of the wild type eggs hatch after
a 36 h incubation period at 23C, the mutants can
not complete egg development before 76 h. The
corresponding gene pair was linked to the sex
chromosome through a chromosomal transloca-
tion that yielded linkage of the wild allele with the
Y chromosome. In this strain, named (Ty34228 y/
X)swx (or simply T228), males have a normal em-
bryonic development time, while females have the
mutant phenotype, allowing an early separation
of sexes (Manso & Lifschitz 1991, Pizarro et al.
1997). This strain seems to be promising for use
for mass-rearing in SIT programs.
Although SIT is being implemented thor-
oughly, it can only benefit from a better knowl-
edge of medfly biology in relation with courtship
behavior, sexual selection, and the mating sys-
tem. Variability in male copulatory success can
result from differences in the activity of males
prior to female arrival, differences in male activ-
ity displayed in its presence, and/or female choice
(Whittier et al. 1994).
An interesting example is the case of a pilot
SIT test carried out in Hawaii to eradicate the
medfly from Kauai. The program failed after sev-
eral years of continuous releasing, partially due
to the fact that the wild females of the treated
area in Kauai altered their mating preference
and began to reject more of the laboratory males
during courtship (McInnis et al. 1996). Females
that discriminate mass-reared males could per-
petuate this ability through their descendants.
The presence of a genetic basis for the discrimina-
tion would select for females that are able to re-
ject mass reared and sterilized males (McInnis et
al. 1996).
Furthermore, preliminary analyses (Favret et
al. 1995) indicate that, depending on the dose, ir-
radiation not only causes a reduction in the abil-
ity to mate, but also to transfer sperm (Favret et
al. 1995). This is important because it is neces-
sary that released sterile males are not only able
to copulate with wild females; they should also be
able to transfer sperm. If they fail to transfer
sperm, females will continue mating until finding
a male, fertile or not, that can fill its spermathe-
cae, weakening the method.
A basic technique to conduct research on med-
fly mating behavior is an analysis in field cages
(Prokopy et al. 1987, McInnis et al. 1996, Cayol et
al. 1999). Another method is video-recording that
allows detailed analysis of the courtship stages,
the factors affecting mating success, and the oc-
currence of inherited differences in courtship be-
havior between different laboratory and wild flies
(Liimatainen et al. 1997, Calcagno et al. 1999).
In the present work, mating success and dura-
tion of copula were compared between irradiated
and non-irradiated T228 males, and non-irradi-
ated Mendoza mass-reared males under field cage
conditions. Sperm transfer was also checked in
mated females. Besides, a competition experi-
ment was conducted under laboratory conditions
through the video recording of successful and
non-successful courtships. In this case, T228 and
wild males were compared.
MATERIALS AND METHODS
Field Cage Experiment
Insects. In this experiment two medfly strains
were used: (i) the Mendoza (Argentina) mass
reared strain of wild-type phenotype; (ii) the ge-
netic sexing strain (Ty34228 y/X)swx (thereafter
T228), isolated from the Mendoza strain at the
IGEF, CICA, INTA Castelar, Argentina (Favret
et al. 1995). The methods of egg collection and pu-
pae and adult rearing were described by Teran
(1977). Adults and pupae were kept in breeding
chambers at 23-250C, under a photoperiod of
12:12 (L:D). Half of the T228 pupae were irradi-
ated 48 h before emergence with an X-ray dose of
10 Krad (100 Grays) in normal air atmosphere
with a Phillips irradiator.
The day following emergence adults of each
strain were sexed. The T228 females were dis-
carded and the rest of the individuals were trans-
ferred into 2750 ml flasks, and separated
according to sex, strain, and irradiation treat-
ment. Adults were fed with sucrose:yeast (3:1),
and water was provided in the form of 1% agar.
Flies were tested at 9 + 1 d old to make sure they
were sexually mature and to avoid differences in
copulatory success due to biological development.
The males of each strain and treatment were
identified by labels painted on their pronotum
with water-based paint.
Experiment. Mating capability was compared
among the three classes of males (irradiated and
non-irradiated T228, and non-irradiated Men-
doza) using in all cases non-irradiated Mendoza
strain females as the target. The experiment was
conducted in two field cages (2.9 m diameter x 2.0
m height) in the experimental field of the Ciudad
Universitaria campus of the Universidad de Bue-
nos Aires. Each field cage contained a young pot-
ted citrus tree inside (1.5-m height, 0.80-m
diameter). The experiment lasted from February
28 to March 20, 1998. During this period, temper-
ature ranged from 15 to 320C.
March 2002
Calcagno et al.: Mating Performance of a Medfly Strain
A total of 9 replicates were made in each cage.
In each replicate, 60 males (30 of each strain)
were released into each cage at 7.00 AM. In one
cage, T228 males were irradiated while in the
other they were non-irradiated. Males were al-
lowed to establish territories and join leks for one
hour. At 8.00 AM, 30 virgin Mendoza females were
released into each cage. From 9.00 AM until 4.00
PM, the number of mating pairs and their position
within the tree were recorded once an hour. Mat-
ing pairs were removed and carefully transferred
into 300 ml vials. The vials were kept in a shady
place in order to avoid mating disruption. Copula
duration was also recorded for each pair. At the
end of the day, mated females were kept frozen (-
20C) until they were checked for sperm transfer.
Sperm transfer. The spermathecae of mated fe-
males from the above experiment were dissected
and placed onto a slide. They were stained with
2% acetic orcein, then softly squashed with a cov-
erslip. The presence of spermatozoa could then be
observed under a light microscope (20x). A total
sample of 60 females were analyzed, involving 20
females mated with each of the three groups of
males tested (irradiated and non-irradiated T228,
and non-irradiated Mendoza).
Video Recording Experiment
Insects. T228 flies were compared with wild
flies emerged from infected guava, Psidium gua-
java, collected from Concordia, Entre Rios Prov-
ince, Argentina. Pupae from both strains were
kept under controlled conditions (23-25C; L:D
12:12) until adult emergence. Flies were main-
tained under conditions described for the previ-
ous experiment, until they were 11 + 1 day old.
Experiment. The experiment was conducted
from April 30 to June 29, 1998. Males of both ori-
gins were placed in mating cages with wild, virgin
females. The cages (70-mm height x 85-mm diam-
eter) were made of a clear acrylic tube closed on the
top by a Petri dish. The bottom of the cage was
open and placed onto a transparent 2 mm thick
glass plate. Recordings were made through this
glass from below. The experiment was conducted
in a room maintained at ca. 23C, and was acous-
tically isolated. The following recording equipment
was used: a Sony Hi 8 (Model CCD-TR805, Japan)
video camera with a Novoflex Video Macro Lens
(Germany), a Phillips (Model 14GX1510/77B, Ar-
gentina) color TV, a JVC (Model H-J401EN, Ja-
pan) videocassette recorder, and a Sennheiser
(Model K6P/MKE102, Germany) microphone.
A fresh lemon, Citrus limon, leaf was placed in-
side the cage at the top in order to simulate natu-
ral conditions (males tend to establish their
territories on the underside of leaves in the field
[Prokopy & Hendrichs 1979]). The recording tech-
nique was the same as in Calcagno et al. (1999).
Courtship behavior was recorded from 10 AM to
2 PM, the typical period of highest mating (Cal-
cagno et al. 1999). Five mating cages were pre-
pared each morning at 9.00-9.30 (ca. 30-60 min
prior to the expected time of the first mating) with
one male of each origin inside. The first cage where
both males began calling (i.e., releasing phero-
mone from the abdomen) was chosen for the first
recording. This cage was recorded for 10 min, after
which time a female was gently released into the
mating cage. Courtship behaviors were recorded
during 30 min following female release. A male
was considered successful if he copulated within
that period. After concluding a recording, the cam-
era was placed under the next cage with calling
males. Two recordings were completed each day.
Recordings were analyzed to classify courtship
behaviors, and to determine the time spent in
each activity. The frame by frame function of the
video recorder, which provided 1/30 second reso-
lution, was used when necessary.
Notation for Courtship Activities. The main
courtship activities performed by males are the
following (Calcagno et al. 1999): stationary (S),
mobile (M), calling stationary (CS), calling mobile
(CM), fanning (Fa), buzzing (B), violent attempt
(VA), peaceful attempt (PA), copulation (C), fight
(Fi), and missed jump (MJ). The presence of two
males inside the cage and the analysis of female
activities, requires the description of additional
activities listed in Table 1.
Statistical Analysis
In the field cage experiment, the proportion of
mated and unmated males of each group, and the
corresponding distribution of couples in the tree,
were compared using a homogeneity Chi square
test (contingency tables). Copula duration was
compared among groups through a one way anal-
ysis of variance (ANOVA), and non-planned con-
trasts were made by Scheffe's method using the
program Statistica (Statsoft 1996). In the video
recording experiment, time spent in each activity
for each strain was compared through a non-para-
metric Mann-Whitney test, using the program
Statistica (Statsoft 1996).
RESULTS
Field Cage Experiment
Males of both strains formed leks together.
Leks were usually found in the central third of the
tree and involved 3 to 6 males. Upon female ar-
rival, male displays both acoustic and visual sig-
nals. According to previous results (Calcagno et
al. 1996) and under local conditions, the highest
mating rate occurred between 10:00 AM and 2:00
PM, the period with the highest light intensity.
The copulatory success of T228 irradiated (I)
and non-irradiated (NI) males were compared
Florida Entomologist 85(1)
TABLE 1. MALE AND FEMALE ACTIVITIES OBSERVED IN THE BISEXUAL VIDEO RECORDING TEST THAT WERE NOT DE-
SCRIBED IN PREVIOUS UNISEXUAL EXPERIMENTS.
Name Symbol Description
Male-female wing signaling WS Soft front and backward wing movement. Wings in vertical
and lateral position
Stationary SL the female remains still, close (<3 cm) to the laboratory male
S, the same as previous but refers to the wild male
male-female fight Fi--mL the female attacks the laboratory male or there is mutual
aggression
Fi-*m, the female attacks the wild male or there is mutual
aggression
Fi<-mL the laboratory male attacks (fights with) the female
Fi<-m, the wild male attacks (fights with) the female
Male-male male-male fanning Fa--m the male under observation is displaying fanning as a
response to the proximity of another male
male-male buzzing B--m the same as the former but referred to buzzing
male-male attempt A--m the male under observation attempts copulation with the
other male
A<-m the other male attempts to copulate with the male under
observation
male-male fight Fi--m the male under observation attacks (fights) the other male
or there is mutual aggression
Fi<-m the male under observation is attacked by the other male,
or there is mutual aggression
head-to-head H1--H the males confront each other, head-to-head, and remain in
this attitude immobile for several seconds
Female Mobile M the female walks or flies
Ovipositing Ov the female remains still but explores with the ovipositor as
if trying to lay eggs
with that of Mendoza (M) males (Table 2). The
proportion of successful males did not differ
among strains, but irradiated T228 males did
mate significantly less than other males.
Average copula duration (min) of NI, T228
I and M males (217, 179, and 208 respectively)
(Fig. 1) differed statistically (F = 3.7; df = 2,350;
P = 0.026). The comparisons of means by Scheffe's
method indicated that the difference between I
and NI males was significant (P = 0.029), but the
remaining contrasts were not significant (P =
TABLE 2. NUMBER AND PERCENTAGE (IN PARENTHESES)
OF MATED MALES OF EACH STRAIN IN EACH EX-
PERIMENT, NI= NON-IRRADIATED T228 MALES;
1= IRRADIATED T228 MALES.
Strain NI I
T228 145 (54) 51 (26)
Mendoza 123 (46) 145 (73)
Total number of mating 268 (82) 196 (89)
Chi Square (DF= 1) 3.26 76.2
P 0.07 <106
0.106 and 0.439 for the comparisons I-M and NI-
M respectively).
The distribution of copulas in the cage did not
differ among groups (Tables 3 and 4). For the
three groups, most couples were recorded on the
underside of leaves and in the central third of the
tree.
Sperm Transfer Analysis
A total of 60 mated females were checked for
sperm transfer. Out of the 60 pairs of spermathe-
cae, 59 contained sperm. The only empty sper-
matheca belonged to a female that had mated for
60 min with an I male.
Video Recording Experiment
According to Calcagno et al. (1999), a success-
ful courtship usually exhibits the following se-
quence of activities: calling, fanning, buzzing,
peaceful attempt, and copulation. In the present
work, the courtship pattern was analyzed for 40
trios involving one virgin wild female, one virgin
T228 male, and one virgin wild male. The number
March 2002
Calcagno et al.: Mating Performance of a Medfly Strain
14 N
7
58 114 19 225 281 336 392 I M
Duration of copula (min)
Fig. 1. Duration of copula (min) for males of the Men-
doza (M) strain and non-irradiated (NI) and irradiated
(I) males of the T228 strain. The arrows indicate the
mean value for each group.
of successful males of each origin was the same (7
out of 40).
Significant differences were observed between
successful and unsuccessful males and between
strains in the time spent in some activities.
The analysis of the activities displayed by
males during the 10 min prior to female arrival
(Table 5) showed differences between successful
and unsuccessful males of the T228 strain. The
eventually successful males spent more time do-
ing mobile calling (MC) while the others tended to
be still (S).
The comparison between strains of the activi-
ties displayed in the absence of the female indi-
cated that males of the T228 strain do more
attempts to copulate (A--m) and more buzzing (B)
than wild males. By contrast, wild males tend to
spend more time in CS (Table 5).
The comparison of male activities in the pres-
ence of a female indicated that T228 males spent
more time buzzing (B--m) and in mating at-
tempts (A--m). On the contrary, wild males are
courted (A--m) and attacked (Fi*-m) by T228
males (Table 6). These results suggest that GSS
males are more aggressive, and display activities
in front of the other male that should be displayed
during a typical courtship to females.
Some differences were also observed between
successful and unsuccessful males. Unsuccessful
males tend to spend more time in activities like S,
M, Fi->m, and Fi<-m, which are not usually con-
nected with mating, and less time in B and PA,
usually required to achieve a successful courtship
(Table 6). The comparison between successful and
unsuccessful females indicated that unsuccessful
ones spent more time in S, M, and WS, and at-
tacked wild male (Fi--m'), while females that
eventually get mated did not (Table 7).
DISCUSSION
The study of biological aspects related to sexual
selection, mating systems, and courtship behavior
are very important to solve methodological prob-
lems of the sterile insect technique (Burk 1991,
Calkins 1987, Harris et al. 1988, Whittier &
Kaneshiro 1991, 1995). Preliminary analyses
(Hooper & Katyas 1971, Favret et al. 1995, Lux et
al. 1996, Calcagno et al. 1997) indicate that the
sterilization treatment results in a reduction of
the male ability to mate and transfer sperm. It is
important to clearly determine which courtship
activities are of major importance for a successful
mating (Calkins 1989, Whittier & Kaneshiro
1995, Calcagno et al. 1996), and which of them are
altered during mass-rearing.
The process of sexual selection might be di-
vided into two components: intrasexual and inter-
sexual selection. Intrasexual selection refers to
those aspects involved in fights and competition
among individuals of the same sex (usually
males). Those mechanisms involved in the ability
to attract, and be accepted by, individuals of the
opposite sex, constitute intersexual selection
(Partridge & Halliday 1984). However, determin-
ing which of the mechanisms of sexual selection
are acting is usually a very difficult task (Whittier
et al. 1994).
In this work, the experimental design applied
allowed male-male competition, and mating suc-
cess was determined by both intrasexual and in-
tersexual selection.
Results of the field cage experiment indicate
that the T228 genetic sexing strain exhibits a bet-
TABLE 3. DISTRIBUTION (IN PERCENTAGE) OF MATING PAIRS WITHIN THE TREE IN THE FIELD CAGE EXPERIMENT.
T228
Mendoza Non irradiated Irradiated
Adaxial leaf side 9 9 2
Abaxial leaf side 71 67 72
Stem 1 2 4
Net 19 22 22
Number of matings 266 145 50
x = 5.41, df = 6, P = 0.49.
Florida Entomologist 85(1)
TABLE 4. PERCENTAGES OF MATINGS AT DIFFERENT HEIGHTS IN THE TREE IN THE FIELD CAGE EXPERIMENT.
T228
Mendoza Non irradiated Irradiated
Top 25 21 24
Middle 35 35 38
Bottom 21 21 16
Net 19 22 22
Number of matings 266 145 50
x = 1.69, df = 6, p = 0.94.
ter mating performance than the Mendoza mass-
reared strain. The radiation procedure and dose
used in this experiment reduces copulatory suc-
cess in T228. This result is not completely extrap-
olable to SIT programs because we used X instead
of gamma rays, however, it shows the relevance of
an adequate dosimetry control to improve the
method. Harmful effects of irradiation might be
also compensated by the huge numbers of sterile
males released in control programs. The lower
mating success of irradiated males may be a con-
sequence of multiple effects of radiation on differ-
ent physiological levels (Favret et al. 1995, Haish
1969, Zumreoglu et al. 1979, Burk 1991).
One important result in this experiment is that
males of both strains were able to form leks and
establish territories on the abaxial side of leaves.
The number of males per lek (3-6) is comparable
with that observed by Prokopy & Hendrichs
(1979) in field cage experiments conducted in Gua-
temala. The localization of leks in the central third
of the tree, which was observed even for irradiated
individuals, also agreed with the behavior of wild
populations. Although the reasons for this prefer-
ence are not well understood, some factors such as
light intensity, foliage density, and wind protec-
tion might be involved (Arita & Kaneshiro 1989,
Hendrichs & Hendrichs 1990, Whittier et al.
1992). The circadian rhythm of laboratory and
wild flies was similar. The conclusion is that the
main aspects of the mating behavioral patterns of
wild medflies are preserved in these strains.
The copula duration was significantly short-
ened in irradiated versus non-irradiated GSS
males. Since Seo et al. (1990) observed that very
short copulas (less than 15 min) do not result in
sperm transfer, one might expect that the differ-
ence between irradiated and non-irradiated
males might be reflected in sperm transfer differ-
ences. However, several studies have indicated
that failure in sperm transfer may occur in cases
of copulas of normal duration (more than 120
min) (Camacho 1989, Seo et al. 1990). In the cur-
rent work, the difference in mating duration be-
tween irradiated and non-irradiated males was
not reflected in sperm transfer differences, since
all but one analyzed spermathecae pairs of mated
females contained sperm. Although the number of
spermatozoids transferred could not be esti-
mated, this preliminary evidence indicates that
T228 males are able to transfer sperm, a property
of major importance for a mass-reared strain.
In the video recording experiment, the mating
rate (17.5%) was much lower than in previous
ones (37.8 to 48.7%) (Calcagno et al. 1999, Norry
et al. 1999). One important difference between the
current and former experiments is that, in Cal-
cagno et al. (1999) and Norry et al. (1999), intra-
sexual selection (competition between males) had
been avoided by releasing only one male and one
female into the cage. The relatively low mating
rate observed in the present work might reflect in-
teractions between males in the limited space in-
side the cages. Intrasexual selection may involve
aggressive interactions which reduce the time
available to interact with the female. Another
cause for the reduced mating rates might be re-
lated to the female's origin. Calcagno et al. (1999)
and Norry et al. (1999) tested originally wild fe-
males that had been reared for two generations
under laboratory conditions. In the current work,
females emerged from wild collected fruits and,
perhaps, were not adapted to the experimental
conditions for video recording, which are clearly
more similar to laboratory than to wild conditions.
The results of this experiment indicate that
the copulatory success of T228 and wild males
was similar. However, important behavioral dif-
ferences were observed between strains that
might influence the copulatory process under con-
ditions different from those of the current experi-
ment. Mainly, GSS males display courtship
activities such as A--m and B->m toward the
other (wild) male, which in normal conditions
should be displayed only in presence of females.
Moreover, the GSS males were more aggressive
(Fi-*m) than wild males.
Laboratory rearing conditions are character-
ized by a dramatic reduction of space, high popu-
lation densities, and absence of natural con-
straints (lek formation, fruits, etc.). Mass rearing
conditions probably favor fast mating and short-
ened courtship (Calcagno et al. 1999), and most
probably an increase of male aggressiveness.
March 2002
Calcagno et al.: Mating Performance of a Medfly Strain
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Calcagno et al.: Mating Performance of a Medfly Strain
TABLE 7. TIME (IN SECONDS) SPENT BY FEMALES IN EACH AC-
TIVITY. *: SIGNIFICANT; **: HIGHLY SIGNIFICANT.
Activity Mated Unmated Z'
S 642.6 1270.8 3.346**
M 104.2 423.8 3.176**
SL 11.0 14.7 -0.740
S, 5.4 20.6 0.807
Fi->mL 0.2 1.0 1.048
Fi-*m, 0.0 2.7 3.104**
Fi<-mL 0.4 0.3 -0.543
Fi<-m, 0.8 0.5 0.168
WS 2.6 13.9 2.547*
Ov. 11.3 49.2 0.517
These might be the causes for the observed mat-
ing attempts with other males. If the behavioral
differences between strains have a genetic basis
they arose as a selective response to the labora-
tory rearing conditions.
Despite the behavioral differences observed,
the results of the video recording experiment in-
dicate that the T228 strain is compatible with the
Concordia wild population. However, the conclu-
sions about sexual selection are not so conclusive.
The general conclusions from both field cage
and video recording approaches are consistent in
showing that the strain T228 performs acceptably
and is a promising strain for medfly genetic con-
trol programs.
ACKNOWLEDGMENTS
We wish to thank Natalia Petit Marty for providing
the collected fruits from which wild material was ob-
tained. We are indebted to Dr. Donald 0. McInnis for the
critical reading of the manuscript. This work was car-
ried out thanks to the financial support awarded to JCV
by The International Atomic Energy Agency (IAEA), Re-
search Contract No. 7697/R2, Universidad the Buenos
Aires (PID Tw09), Consejo Nacional de Investigaciones
Cientificas y T6cnicas (CONICET) (PIP 0722/98) and
Agencia Nacional de Promoci6n Cientifica y Tecnol6gica
(PICT 2269 and 6628).
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Florida Entomologist 85(1)
Cayol et al.: Sexual Compatibility in Medfly
SEXUAL COMPATIBILITY IN MEDFLY (DIPTERA: TEPHRITIDAE)
FROM DIFFERENT ORIGINS
J. P. CAYOL1, P. CORONADO2 AND M. TAHER
FAO/IAEA Agriculture and Biotechnology Laboratory, IAEA Laboratories, A-2444 Seibersdorf, Austria
'Present address: West Asia Section, Technical Cooperation Division, IAEA
Wagramerstrasse 5, P.O. Box 100, A-1400 Vienna, Austria
Phone: (+43) 1260021630, Fax: (+43) 126007, E-mail: j.cayol@iaea.org
2University of Agricultural Sciences, Gregor Mendel Str. 33, A-1180 Vienna, Austria
ABSTRACT
The use of the Sterile Insect Technique to control and/or eradicate insect pest populations
has been extensively applied to medfly. However, patented differences in sexual compatibil-
ity between populations or strains from different origins has been a serious concern to a
wider use of sterile flies, and in particular sterile males of genetic sexing strains (GSS). In
the present experiments, the sexual compatibility and mating performance of flies from 9
countries representing 5 continents and 4 GSS were measured. It is demonstrated that, from
a qualitative standpoint, wild medfly populations world-wide have not yet evolved specific
sexual behaviors indicative of incipient pre-mating isolation mechanisms under local natu-
ral selection. Wild medfly populations are as sexually compatible with GSS as they are with
other wild populations. On that basis, the same mass reared strain can now be used world-
wide, as long as it fulfills the standard quality control requirements.
Key Words: medfly, Ceratitis capitata, sexual compatibility, comparison, wild population, gene-
tic sexing strain
RESUME
El uso de la t6cnica del insecto est6ril para controlar y o erradicar poblaciones de plagas in-
sectiles ha sido aplicado extensamente a la mosca del Mediterraneo. Sin embargo, diferencias
en compatibilidad sexual entire poblaciones o razas de diferentes origenes ha sido una seria
preocupaci6n para un uso mas amplio de las moscas est6riles y en particular machos est6riles
de Cepas gen6ticamente sexadas (CGS). En los siguientes experiments, la compatibilidad
sexual y la capacidad de apareamiento de las moscas de 9 pauses, representando 5 continents
y 4 RGS fueron evaluadas. Se ha demostrado que, desde un punto de vista cualitativo, pobla-
ciones salvajes a nivel mundial de la mosca mediterranea aun no han evolucionado bajo se-
lecci6n natural local comportamientos sexuales especificos indicativos de incipientes
mecanismos de aislamiento anteriores al apareamiento. Poblaciones salvajes de moscas me-
diterraneas son tan sexualmente compatibles con CGS que con otras poblaciones salvajes. So-
bre esas bases, la misma cepa criada en masa se puede utilizar ahora a nivel mundial con la
condici6n de que cumpla con los requerimientos estandares de control de calidad.
The Mediterranean fruit fly (medfly), Ceratitis
capitata Wiedemann (Diptera: Tephritidae), is of-
ten referred to as the most important agricultural
pest in the world (Liquido et al. 1990) and this "ti-
tle" is widely justified. From its origin in Eastern
Africa (Silvestri 1913, Bezzi 1918), the pest effi-
ciently conquered new countries and new hosts. If
medfly was present in North African and almost
all European Mediterranean countries by the
mid-19th century, its introduction in North, Cen-
tral and Latin America occurred nearly 100 years
later (Dridi 1990). Following the development of
fruit and vegetable trade worldwide, and the in-
creasing number of international, including inter-
continental, airway connections, medfly success-
fully spread over five continents in less than 150
years, and is found developing, to date, in more
than 350 wild and cultivated host plants of vari-
ous families (Liquido et al. 1990). Such a threat
for agriculture represented by a single species
turned medfly into one of the main targets of pest
control programs, including the Sterile Insect
Technique (SIT) described by Knipling (1953).
The use of SIT requires that rearing facilities
be developed to produce large numbers of insects
for sterile fly releases. In the early stages of med-
fly control using SIT, mass reared strains were es-
tablished by colonizing wild insects collected
from, or in the vicinity of, the target area. Such
strains have been reared in Mexico, Chile, Hawaii
and Guatemala rearing facilities. More recently,
with the increasing demand for sterile medflies
Florida Entomologist 85(1)
and the limited number of mass rearing facilities
available worldwide, some of these facilities be-
gan to export sterilized medflies to other coun-
tries. Eight facilities have now reached
production levels, which allow them to export
sterile insects (Fisher & Caceres 2000) on a re-
gional or inter-regional basis. When this proce-
dure is used, the flies released have to compete
with wild flies of a different geographic origin.
The increasing use of medfly genetic sexing
strains (GSS) has also resulted in the same strain
being used in different countries. To date, five
rearing facilities in the world produce GSS
(Fisher & Caceres 2000). Since GSS are assem-
bled from specific components, it is impossible to
"colonize" them from each country where sterile
GSS flies are needed. The GSS are sometimes out-
crossed with insects from the target population to
increase the genetic variability (Franz et al.
1996), although in some cases this presents prob-
lems (G. Franz, IPCS, FAO/IAEA, Vienna, unpub-
lished data). In practice, a single wild population
is used as a basis for the synthesis of the GSS.
Consequently, the same GSS based on the same
wild genetic material may be used in various
countries/continents and the question was raised
concerning the sexual compatibility of these
strains with wild medfly populations in different
countries.
In the present work, the sexual compatibility
of wild populations originating from nine coun-
tries, representing five continents, was measured
in pairwise comparisons under semi-natural field
cage conditions. In a second series of experiments,
flies from four GSS were evaluated.
MATERIALS AND METHODS
Wild Material
Wild insects were collected as pupae from in-
fested fruits in their country of origin. Pupae were
shipped by express air mail to Seibersdorf, Aus-
tria (or hand-carried), except for field cage tests
run in Argentina where wild flies were tested on
site (Cayol et al. 1999). Wild insects originating
from Argentina (Patagonia region), Australia
(Perth), France (Reunion Island), Greece (Crete
Island), Guatemala (Antigua), Israel (near Tel
Aviv and from the Arava Valley), Kenya (near
Nairobi), Portugal (Madeira Island) and South
Africa (Western Cape Province) were tested.
Their host of origin was guava (Israel, both loca-
tions; Portugal; South Africa), coffee (Guatemala,
Kenya), orange (Australia, Greece), fig and peach
(Argentina) and milkwood (France). Upon recep-
tion of a shipment, pupae were weighed and
counted. On emergence, flies were sexed and kept
in separate ventilated Plexiglas cages (11 x 15.5 x
11 cm) until tested and provided with adult food
(sugar and yeast in 3:1 ratio) and water.
Genetic Sexing Strains
Flies of several genetic sexing strains (GSS)
were obtained as pupae from the FAO/IAEA facil-
ity at Seibersdorf for green house tests. In the
field cage tests in Argentina, GSS flies were pro-
vided by the KM8 facility in Mendoza (Cayol et al.
1999). The following four GSS were tested. SEIB
6-96 is a GSS carrying a white pupa (wp) muta-
tion (R6ssler 1979) in combination with the trans-
location T(Y;5) 2-22 (Franz et al. 1994). VIENNA
4/TOL-94 is a GSS carrying wp and temperature
sensitive lethal (tsl) mutations in combination
with the translocation T(Y;5) 1-61 (Franz et al.
1994). VIENNA 7-97 is a GSS carrying wp and tsl
mutations in combination with the translocation
T(Y;5) 3-129 (Kerremans & Franz 1995). AUS-
TRIA 6-97 is a triple mutant strain carrying wp,
tsl and yellow body (y) (R6ssler & Rosenthal 1992)
selectable markers in combination with the
translocation T(Y;5) 2-22 (G. Franz, IPCS, FAO/
IAEA, Vienna, unpublished data). The genetic
background of SEIB 6-96, VIENNA 7-97 and
AUSTRIA 6-96 GSS originates from Egypt. The
genetic background of VIENNA 4/TOL-94 origi-
nates from Guatemala highlands (Lake Atitlan),
following an outcrossing of the original strain
(Franz et al. 1996). After sexing on emergence,
GSS flies were maintained under the same condi-
tions as wild flies.
Testing Cage
Flies were tested in a greenhouse located at
the FAO/IAEA Agriculture and Biotechnology
Laboratory (Seibersdorf, Austria). The green
house was temperature monitored (temperature
ranging between 24 and 32 degrees Celsius). A
cage made of netting material was placed inside
the greenhouse. The cage contained 6 potted cit-
rus trees (up to 1.8 meter height) in a total vol-
ume of 15 m3. In Argentina, flies were tested in
outdoor field cages (Chambers et al. 1983) con-
taining a single planted citrus tree (Cayol et al.
1999). In both greenhouse and field cage tests, the
cages were covered with a shading cloth filtering
85% of sunlight to avoid any "greenhouse effect".
The strains were tested in pair-wise compari-
sons. Depending on the availability of biological
material, two types of tests could be run: (i) wild-
wild comparisons, where wild flies from two dif-
ferent geographic origins were tested and (ii)
wild-GSS comparisons, where wild flies originat-
ing from one country were tested against GSS
flies. In both types of test, the protocol described
by Cayol et al. (1999) for "bisexual" type test was
applied. Two days before being tested, active and
flying flies were selected. Males and females, from
alternatively one of the two populations were
marked with a dot of water-based paint on the no-
tum for identification during the course of the
March 2002
Cayol et al.: Sexual Compatibility in Medfly
tests. On the day of the test, 30 flies of each sex
and each strain were released into the cages at
dawn. Males were released 30 minutes before fe-
males to give them time to establish a territory
and start forming leks (Prokopy & Hendrichs
1979). The number of calling males and the envi-
ronmental conditions (temperature, relative hu-
midity, light intensity and air pressure) were
checked every half-hour. The number and type of
mating pairs were checked on a continuous basis,
and 5 minutes after initiation of mating, the pairs
were collected and placed in vials (50 ml volume)
to monitor mating duration. The mated flies were
not replaced or released back into the cage after
separation (Chambers et al. 1983). Tests lasted for
7-8 consecutive hours. Tests were performed from
March 1997 until September 1998, whenever flies
were available. A total of 19 combinations were
tested as shown in Table 1. Due to the availability
of flies from different origins, the number of repli-
cations for each combination was variable.
Statistical Analysis
Raw data were transformed following an ARC-
SIN [ASIN( *(X/100)*180/PI)] transformation
to stabilize variance.
For each of the parameters measured and
whenever it was relevant, data were first pooled ac-
cording to the type of combination tested "wild ver-
sus wild" or "wild versus GSS" (later called "wild/
wild and wild/GSS comparisons"). As a second step
TABLE 1. TYPE OF MATING COMBINATIONS TESTED.
Tested against
Wild population Wild GSS
Argentinaa Seib 6-96
Australia Crete Vienna 4/Tol-94
Israel
Crete Australia Seib 6-96
Guatemala Israel Vienna 4/Tol-94
Kenya
Madeira
Israel Australia Vienna 4/Tol-94
Guatemala Austria 6-96
Madeira Vienna 7-97
Reunion
Kenya Guatemala Vienna 4/Tol-94
Madeira
Madeira Guatemala Vienna 4/Tol-94
Israel Vienna 7-97
Kenya
Reunion Israel
South Africa Vienna 7-97
Tested in field cages in San Miguel de Tucuman (Argentina) (Cayol
et al. 1999).
of the analysis, data were pooled according to the
origin of the strain (Madeira, Argentina, Vienna 7-
97, etc.) (later called "strain comparison").
In both cases, data were analyzed using Systat
9.0 (Systat, 1999) for analysis of variance
(ANOVA), followed by Tukey's HSD test.
RESULTS
Participation of Flies in Mating
This measures the suitability of the flies and
the environmental conditions of the tests for mat-
ing. It represents the overall mating activity of
the flies (Table 2). If PM < 0.20 (proportion of mat-
ing) then the results of the test must be rejected
(IAEA 1997).
Wild/Wild and Wild/GSS Comparison. The
mean PM values obtained in comparing wild/wild
and wild/GSS combinations confirmed that the
test conditions were suitable for mating, as about
40% of the possible matings were achieved. How-
ever, there was a highly significant difference be-
tween the two mean PM values, 0.407 (wild) and
0.484 (GSS), (F = 7.530; df=1,71; P = 0.008) show-
ing that somewhat more matings took place when
GSS flies were involved in the test (Table 3).
Strain Comparison. When comparing the PM
values obtained for each strain tested, even
though the overall mating activity was satisfac-
tory in each case (PM > 0.20), some significant dif-
ferences can be found among the strains (F =
2.789; df = 12,134; P = 0.002). Significantly more
matings were achieved in tests involving wild
flies from Australia (PM = 0.554) than in tests in-
volving wild flies from Kenya, Madeira or Austria
6-96 GSS flies (PM values 0.349, 0.386 and 0.345
respectively). Those differences might reflect var-
ious adaptations to the test conditions or a gener-
ally higher mating activity of Australian flies.
Sexual Compatibility
In all of the 19 comparisons involving any of the
wild populations or GSS tested, each of the four
possible types of mating was encountered confirm-
ing that there was no absolute behavioral incom-
patibility among these populations. The sexual
compatibility among the flies from different ori-
gins was assessed using the Isolation Index (ISI)
(Cayol et al. 1999) as described in Table 2. The ISI
ranges from -1 ("negative assortative mating", i.e.
flies only mate with a "foreign" partner) to +1
("positive assortative mating" or total sexual isola-
tion, i.e. flies only mate with partner of the same
origin), through an equilibrium at 0 (uniform sex-
ual compatibility, i.e. no mating preferences).
Wild/Wild and Wild/GSS Comparison. There
was no significant difference between the overall
mean value of ISI obtained when comparing wild
versus wild populations and wild versus GSS (F =
Florida Entomologist 85(1)
TABLE 2. INDICES USED TO MEASURE SEXUAL COMPATIBILITY OF MEDFLY STRAINS FROM DIFFERENT ORIGINS.
Trait measured
Index formulab
Participation in mating PM No. of pairs collected
No. of females released
Sexual isolation (aa + bb) (ab + ba)
Total no. of matings
Male relative performance MRPI (aa + ab) (bb + ba)
Total no. of matings
Female relative performance FRPI (aa + ba) (bb + ab)
Total no. of matings
Male mating competitivenessac LW
LW+WW
After Cayol et al. (1999) and McInnis et al. 1996.
'"ab": number of matings of "a" males with "b" females.
'In RSI: "L" for mass reared males and "W" for wild flies (males or females).
0.030; df 1,71; P = 0.864). Even though the two
mean ISI values showed a tendency for homolo-
gous (male and female of the same origin) mating
(Table 3), there was certainly no evidence of sex-
ual isolation. Of utmost importance, these results
show that wild flies did not discriminate against
GSS flies more than wild flies originating from a
different area or continent. In other words, wild
populations are as behaviorally compatible with
GSS as they are with other wild populations from
various geographic origins.
Strain Comparison. The mean ISI values ob-
tained for the 9 wild populations and the 4 GSS
did not differ significantly (F = 1.499; df 12,134; P
= 0.132) (Table 4). This confirms that, even if
there are some minor differences among the vari-
ous wild populations and GSS tested, none of
them developed, to date, a significant behavioral
isolation (ISI > 0.50).
Male and female Relative Mating Performance
Two other indices which look at the relative
mating performance of males (MRPI) and females
(FRPI) of the two strains, regardless of their mat-
ing partners, were measured (Cayol et al. 1999).
These indices range between -1 (all matings
achieved by one type of male (MRPI) or female
(FRPI)) and +1 (all matings achieved by the other
type of male (MRPI) or female (FRPI)) through an
equilibrium at 0 (equal mating performance of
males or females of the two strains) (Table 2).
These indices complement the ISI value by better
describing the role played by males and females of
the two strains compared.
Wild/Wild and Wild/GSS Comparison. The
male relative mating performance is significantly
higher when comparing wild versus wild popula-
tions than it is when comparing wild populations
versus GSS (F = 4.693; df 1,71; P = 0.034) (Table
3). This demonstrates that, when two types of
wild males of different geographic origin are
present in the same cage, one of the two types of
males mates more than the other. However, when
wild and GSS males are present, the relative per-
formance is more "balanced", i.e. both types of
males mate in a similar proportion (regardless of
TABLE 3. SEXUAL COMPATIBILITY AND PERFORMANCE MEASURED WHEN TESTING MEDFLY WILD POPULATIONS AGAINST
WILD OR GSS.
Combination tested
Parameter measured Wild/wild Wild/GSS
PM 0.407 b + 0.020 0.484 a + 0.018 F = 7.530; df = 1,71; P = 0.008
ISI 0.233 a + 0.057 0.221 a + 0.034 F = 0.030; df = 1,71; P = 0.864
MRPI 0.375 a + 0.042 0.265 b + 0.030 F = 4.693; df = 1,71; P = 0.034
FRPI 0.288 a + 0.033 0.345 a + 0.032 F = 1.330; df = 1,71; P = 0.253
aBased on absolute values.
'Data are presented as mean + SEM. Data followed by the same letter on the same row do not differ significantly according to Tukey's HSD test (P >
0.05).
March 2002
Cayol et al.: Sexual Compatibility in Medfly
TABLE 4. SEXUAL COMPATIBILITY AND PERFORMANCE OF WILD POPULATIONS AND GSS.b
Parameter measured
Origin of the flies PM ISI MRPI FRPI
Argentina 0.488 ab + 0.032 0.309 a + 0.062 0.360 b + 0.023 0.332 ab + 0.051
Australia 0.554 a + 0.029 0.069 a + 0.104 0.480 abc + 0.069 0.496 a + 0.075
Crete 0.508 ab + 0.025 0.108 a+ 0.123 0.586 ab + 0.066 0.300 ab + 0.114
Guatemala 0.462 ab + 0.043 0.188 a + 0.069 0.462 abc + 0.072 0.442 a+ 0.085
Israel 0.419 ab + 0.021 0.200 a + 0.056 0.316 be + 0.043 0.491 a+ 0.047
Kenya 0.349 b + 0.039 0.319 a + 0.161 0.171 c + 0.056 0.420 ab + 0.102
Madeira 0.386 b + 0.022 0.196 a + 0.079 0.600 a + 0.053 0.156 b + 0.028
Reunion 0.389 ab + 0.053 0.377 a + 0.085 0.278 c + 0.056 0.361 ab + 0.081
South Africa 0.477 ab + 0.043 0.259 a + 0.064 0.421 abc + 0.054 0.299 ab + 0.083
Vienna 4/tol-94 0.522 ab + 0.038 0.235 a + 0.070 0.456 abc + 0.061 0.335 ab + 0.042
Vienna 7-97 0.457 ab + 0.029 0.092 a + 0.084 0.236 c + 0.058 0.563 a+ 0.061
Seib 6-96 0.494 ab + 0.028 0.300 a + 0.038 0.313 be + 0.049 0.396 ab + 0.030
Austria 6-96 0.345 b 0.029 0.104 a + 0.021 0.200 c + 0.200 0.470 ab + 0.004
F = 2.789 F = 1.499 F = 4.563 F = 3.985
df= 12,134 df= 12,134 df= 12,134 df= 12,134
P = 0.002 P = 0.132 P = 0.000 P = 0.000
SSEM. Data followed by the same letter in the same column do not differ significantly according to Tukey's HSD test
the type of female). No significant difference was
found in the female relative mating performance
(F = 1.330; df= 1,71; P = 0.253) (Table 3). In both
cases (wild/wild and wild/GSS), there is a slight
tendency for one of the two types of females to
outcompete the other. In wild/GSS comparisons,
the GSS females often mate more than their wild
counterparts (regardless of the type of males).
Strain Comparison. The MRPI value of the
Madeira wild population is significantly higher
than that of the Kenya and the Reunion wild pop-
ulations, and that of the Vienna 7-97 and the Aus-
tria 6-96 GSS (F = 4.563; df = 12,134; P < 0.000)
(Table 4). Whatever strain they were compared
to, Madeira males very often, and by far, outcom-
peted the other type of males for mates. To the
contrary, and under similar conditions, Kenya,
Reunion, Vienna 7-97 and Austria 6-96 males
were outcompeted by any other type of males they
were compared to, even with their own female
counterparts. There was a significant difference
between the higher FRPI value of the Australia,
Guatemala and Israel wild populations and the
Vienna 7-97 GSS and that of the Madeira wild
population (F = 3.985; df = 12,134; P < 0.000)
(Table 4). This shows that these 4 types of females
were more prone to mate than were wild Madeira
females. This would indicate that Madeira fe-
males were more "selective" in choosing a mate
than were the other strains of females.
Mating Competitiveness of GSS Males
The mating competitiveness of GSS males
with wild males for wild female mates was mea-
sured by the Relative Sterility Index (RSI) (McIn-
nis et al. 1996) described in Table 2. When RSI =
0.5, wild and GSS males are equally competitive.
The mean RSI value has been compared for the 4
GSS tested and results are shown in Table 5.
The analysis showed that, even though all the
GSS males did compete with wild males for wild
female mates, Vienna 4/Tol-94 males were about
twice as competitive as Vienna 7-97 males (F =
2.967; df = 3,48; P = 0.041) (Table 5). This result
confirms a poor relative mating performance for
Vienna 7-97 males, which has been previously
demonstrated by the relatively low MRPI value.
Duration of Mating
Time spent in copula (duration of mating) was
measured and compared for the homologous type
of mating (male and female of the same origin) for
each GSS and wild population tested and these
results are shown in Table 6.
TABLE 5. MATING COMPETITIVENESS OF MALES FROM
THE DIFFERENT GSS.
Strain Relative Sterility Indexb
Vienna 4/Tol-94 0.448 a + 0.059
Vienna 7-97 0.217 b + 0.045
Seib 6-96 0.302 ab + 0.049
Austria 6-96 0.250 ab + 0.087
Data are presented as mean + SEM.
Data followed by the same letter do not differ significantly according
to Tukey's HSD test (F = 2.967; df= 3,48; P = 0.041).
aBased on absolute values.
bData are presented as mean
(P > 0.05).
Florida Entomologist 85(1)
TABLE 6. DURATION OF HOMOLOGOUS MATING (MALE AND
FEMALE OF THE SAME ORIGIN) FOR THE DIFFER-
ENT WILD POPULATIONS AND GSS.
Duration of
Origin of the flies homologous pairing (min)a'b
Argentina 164.466 cd + 4.777
Australia 204.920 ab + 5.962
Crete 195.500 ad + 6.859
Guatemala 156.970 d + 4.180
Israel 157.418 d+ 4.728
Kenya 198.464 ac + 8.412
Madeira 178.095 cd + 4.559
Reunion 178.538 bcd + 9.047
South Africa 227.310 a 6.241
Vienna 4/Tol-94 164.983 cd + 3.593
Vienna 7-97 158.072 d + 4.225
Seib 6-96 124.035 e + 3.700
Austria 6-96 164.278 cde + 8.664
Data are presented as mean + SEM.
Data followed by the same letter do not differ significantly according
to Tukey's HSD test (F = 28.710; df= 12,1157; P = 0.000).
A large and significant variation in mating du-
ration has been found (F = 28.710; df = 12,1157;
P < 0.000) (Table 6). Average time spent in copula
can vary from 2 hours for the Seib 6-96 GSS up to
nearly 4 hours for the South Africa wild popula-
tion. However, this duration seems to be rela-
tively stable for each strain, as demonstrated by
the low standard error values. In addition, the
GSS flies tend to mate for a relatively shorter pe-
riod (2 to 3 hours) than do wild flies (3 to 4 hours).
DISCUSSION
These data demonstrate that, from a qualita-
tive standpoint, wild medfly populations world-
wide have not yet evolved specific sexual behav-
iors indicative of incipient pre-mating isolation
mechanisms under local natural selection. In ad-
dition, it was shown that wild populations are as
sexually compatible with GSS as they are with
other wild populations.
However, some quantitative differences have
been measured among wild populations or GSS,
such as a lower or higher male or female relative
performance. In the case of Madeira for example,
the high male performance, and the relative se-
lectiveness of the females as shown in the present
experiments could, in a long run, result in a lower
mating acceptance of relatively poorly competi-
tive mass-reared males, such as Vienna 7-97,
which could affect the effectiveness of SIT. Addi-
tional tests have been run to look at this specific
case which tends to show that Madeira females do
discriminate against Vienna 7-97 males more
than other wild or GSS males (J. P. C., unpub-
lished data).
The high importance of mating behavior stud-
ies to the SIT has encouraged the Insect Pest Con-
trol Section of the International Atomic Energy
Agency to investigate this subject. The coordi-
nated research program started in 1994 by the
IAEA examined details of male courtship behavior
in wild populations from nine countries (FAO/
IAEA 1994) from both qualitative and quantitative
standpoints, using slow motion video recording.
Some minor differences have been found among
the wild populations, as demonstrated by Briceno
et al. (2002). When comparing wild flies from Costa
Rica and Argentina, the authors showed that some
significant differences of the courtship songs could
be identified and measured. In addition, it was
shown that long term rearing could affect signifi-
cantly the duration of the mass-reared male court-
ship (Eberhard & Briceno 1996, Briceno &
Eberhard 2002) and love songs (Briceno & Eber-
hard 1997). The present findings tend to show that
copula duration is also shortened in mass rearing.
Those differences in mating duration warrant fur-
ther study in relation to post-mating isolation.
Post-mating isolation could affect the efficacy of
SIT due to remating of wild females, shortly after
a first mating with a sterile GSS male.
Concerns about the sexual compatibility among
medflies from different origins represented some-
what of a threat to the shipment of sterile flies
from one country to another to support SIT pro-
grams. These concerns become more pronounced
when a GSS was proposed to be used in many dif-
ferent SIT programs. The findings of the present
experiments support the potential use of the same
GSS anywhere in the world. Out of the 4 GSS
tested in the present experiments, the only one,
which was outcrossed with a wild population (Vi-
enna 4/Tol-94), did show the highest mating com-
petitiveness. This strongly supported the idea of
building-up a new GSS based on mixing wild pop-
ulations from various origins. This new and very
promising GSS has now been developed and is cur-
rently under testing (G. Franz, IPCS, FAO/IAEA,
Vienna, unpublished data).
Gasparich et al. (1997) showed that the mito-
chondrial DNA of medfly populations from 100
different origins was indeed variable and that it
probably reflected the colonization pattern of
medfly from its origin in Eastern Africa about 200
years ago. However there was no evidence that
substantial genetic differentiation had occurred.
When a medfly outbreak occurs, program manag-
ers sometimes worry that the sterile flies released
might not be from the same geographic origin and
hence would not mate. A second concern is that
the "foreign" flies might introduce new genetic
material into the country. The fear is that "for-
eign" fertile flies would establish a new popula-
tion with its own genetic and behavioral
characteristics. However, the present work based
on populations representative of five continents,
March 2002
Cayol et al.: Sexual Compatibility in Medfly
clearly demonstrates that there are no significant
population specific mating behavior traits. These
observations together with the genetic data sug-
gest that the risk of introducing a more virulent
form of medfly into a specific country is remote.
In conclusion, strains to be used in SIT pro-
grams in any country must be selected to maxi-
mize the quality of the flies produced, rather than
based on the geographic origin of the strain.
ACKNOWLEDGMENTS
The authors are grateful to the team leaders and
their staffs who collected wild pupae in the different lo-
cations: Argentina, E. Rial (Programa Moscafrut Pat-
agonia); Australia, R. Johnson (AWA); Crete, A. Econo-
mopoulos (University of Heraklion); Guatemala, P. Ren-
don (USDA); Israel, Y. R6ssler (Citrus Marketing Board
of Israel); Kenya, S. Lux (ICIPE); Madeira Island, R.
Pereira (Madeira-Med Program); Reunion Island, S.
Quilici (CIRAD-FLHOR) and South Africa, B. Barnes
(INFRUITEC). The authors would also like to thank
A.S. Robinson for comments on the manuscript and K.
Fisher for advising on statistical analysis.
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Florida Entomologist 85(1)
March 2002
MATING PERFORMANCE AND SPATIAL DISTRIBUTION OF MEDFLY
(DIPTERA: TEPHRITIDAE) WHITE PUPA GENETIC SEXING MALES
UNDER FIELD CAGE CONDITIONS
A. P. ECONOMOPOULOS1' AND P. G. MAVRIKAKIS2
Department of Biology, University of Crete, Heraklion, Greece
Institute of Molecular Biology and Biotechnology, Heraklion, Greece
ABSTRACT
In mixed populations of wild and males from T (Y;5) 1-61 white female pupa genetic sexing
strain of Ceratitis capitata (Wiedemann), sterilized males of the genetic sexing strain ex-
pressed calling, lekking and mating compatibility with their wild counterparts. Neverthe-
less, their mating performance was most of time poor to very poor. For example, in a series
of studies from June-October 1996, only 0- about 1/3 of expected matings (based on insect ra-
tios) by genetic sexing sterilized males was recorded. Similar results were observed in the
other years of this study. No substantial differences between gen. sex. male x wild female
and wild male x wild female type copulations were detected in spatial distribution of couples
in copula on the orange tree. Over 83% of both mating types were detected on the underside
leaf surface.
Key Words: Ceratitis capitata, medfly, sexing strain, quality control, competitiveness
RESUME
En poblaciones mezcladas de machos salvajes y machos provenientes de pupas blancas de
hembras T (Y;5) 1-61 de razas gen6ticamente sexadas de Ceratitis capitata (Wiedemann), los
machos esterilizados de razas gen6ticamente sexadas expresaron la capacidad de llama-
miento, la acci6n de seleccionar un lugar de apareamiento y apareamiento con su contra-
parte salvaje. No obstante, su capacidad de apareamiento fue en la mayoria de las veces de
pobre a muy pobre, por ejemplo, en una series de studios llevados a cabo entire junio y octu-
bre de 1996, solamente entire 0 y 1/3 de los apareamientos esperados (basados en proporciones
de insects) por machos est6riles gen6ticamente sexados fueron registrados. Resultados si-
milares se observaron en los otros anos de este studio. No se detectaron diferencias subs-
tanciales entire el tipo de copulaci6n entire machos gen6ticamente sexados x hembras
salvajes y machos salvajes x hembras salvajes en las distribuciones espaciales de parejas en
c6pulas sobre arboles de naranja. Mas del 83% de ambos tipos de apareamiento se detecta-
ron en el lado inferior de la superficie de las hojas.
Artificial mass-rearing and sterilization may
affect field effectiveness of the Mediterranean fruit
fly, Ceratitis capitata (Wiedemann) (Diptera: Te-
phritidae), drastically. Mass rearing conditions, se-
lection genetic changes, irradiation and sterile
insect technique (SIT) handling procedures may
affect the vigor and behavior of sterile males and
reduce strikingly their mating performance with
the wild population in the field (Economopoulos
1996). Furthermore, recent evidence suggests de-
velopment of behavioral resistance in the wild flies
against sterile flies (McInnis et al. 1996). The lim-
ited success SIT had so far on several key insect
pests was connected to a large degree with the
quality of released insects. This became evident
from the initial steps of the methodology and re-
sulted in the development of tests which monitor
the quality of sterile insects and the field effective-
ness of the methodology (Calkins et al. 1996, Cayol
et al. 1999). This effort culminated in the recent
publication of a comprehensive manual of quality
control for fruit flies (IAEA 1998).
The basic and closest to field conditions mating
performance test available so far is that of field
cage (Calkins & Webb 1983). Nevertheless, al-
though the test is applied under natural condi-
tions and involves a host tree, the fact that flies
cannot freely fly away or "escape" from the host
tree, or newcomers cannot mix with the caged tree
flies reduces the value of the test. Recently, the in-
terest on sterile insect competitiveness as de-
duced from egg hatch, first described in 1971
(Fried), has been renewed. Measurements of egg
hatch from field oviposition in mock fruits are
used for a more accurate evaluation of mating
performance under completely natural conditions
(Katsoyannos et al. 1999). Unfortunately, no prac-
tical method has been standardized so far on egg
hatch measurement of field oviposited eggs in
mock fruits.
In this study, the mating performance of a
white female puparium strain has been evalu-
ated under field cage conditions in citrus planta-
tion.
Economopoulos & Mavrikakis: Medfly Genetic Sexing Male Mating
MATERIALS AND METHODS
Cylindrical field cages 2m h x 2.9m d were used
(Synthetic Industries, Dalton, GA 30720, USA).
Each cage was installed over a Navel orange tree in
a mixed plantation with Navel and Valencia orange
trees. The cage ceiling was covered by thick white
fabric to provide shade. For the study of 1996 (Table
1), most wild flies were raised from larvae in sour
oranges and loquats (early summer) and figs (late
summer). The genetic sexing males were of the
white female pupa strain T (Y;5) 1-61(95) (Franz et
al. 1994) at generations 5-10. They were gamma-
sterilized 1-2 days before adult emergence. Eight
experiments were performed from June till October
(see also caption of Table 1). Flies for the experi-
ments in 1998 (Tables 2 and 3) were similar except
that the genetic sexing males were of generations
33 and 36, respectively, and were not gamma-ster-
ilized. In the June experiments, high mortality was
observed on the second and third experiment days
because of air-born toxicity due to near-by bait
spraying. In both 1996 and 1998 matings were re-
corded from 09:00-18:00, every half hour.
In all experiments trees were pruned to fit the
cages and make easy the census of fly activities
and copulations. Males and females were sepa-
rated soon after emergence and kept on standard
adult diet (unless indicated differently in the ta-
ble) prior to introduction into the field cage. Water
was sprayed on the caged trees on the hot hours of
the day to provide the flies with drinking water.
RESULTS AND DISCUSSION
In 1996 mating performance experiments are
presented in Table 1. All genetic sexing males in-
cluded were gamma sterilized. In June-August
(highest daily temperatures under shade between
30-36C), the genetic sexing males produced only 0-
33% of observed matings while expected values ac-
cording to insect type ratios were 50-90%, i.e. 13 ob-
served instead of 87 expected matings in total. In 3
out of the 5 experiments organized in this period,
the genetic sexing males contributed zero to near
zero of mating activity observed, while in the other
2 experiments their mating share was 1/2.7 and
1/2.5 of expected values, respectively. It is noted
that the reduced performance of genetic sexing
males in the June 18 experiment could had been in-
tensified by the young age of laboratory flies mixed
in this test (refer to Table 1 caption). On October 10
and 17 (when daily temperatures were between 16-
29C), genetic sexing males succeeded in getting 1/
3.4 to 1/3.1 of their expected mating activity, i.e. 8
observed instead of 28 expected matings in total.
That is, although the results in October were not as
bad as in July, the genetic sexing males competed
again poorly with wild males for matings with wild
females. In all experiments the difference was
highly significant (P < 0.001). Both type matings
were recorded almost exclusively on the underside
leaf surface, with sterile matings located relatively
more at lower canopy than the wild type matings.
The majority of both type matings was recorded at
the same tree canopy sectors.
The results of the 1998 experiments are shown
in Tables 2 and 3. All genetic sexing males involved
were not sterilized. The genetic sexing strain was
already at generations 33 or 36 with extensive
break down. In the June experiment (warm
weather) the genetic sexing males, at 1:1:1 ratio
(wild males: wild females: gen. sex. males) and fed
complete diet, obtained more than expected mat-
ings, while at 1:1:3 ratio they obtained significantly
fewer than expected matings. In September (cooler
weather) at both insect ratios they obtained mating
percentages lower than expected. It was observed
that their performance was higher in the second
and third experiment days as compared with the
first one. At the ratio of 1:1:3, sugar fed laboratory
flies obtained fewer than expected matings in both
June and September. The difference was not signif-
icant in June, while in September it was highly sig-
nificant. When insect ratios of 1:1:3 are compared
with 1:1:1 ratios in June, the genetic sexing males
of the high overall male: female ratio (4:1) obtained
mating values lower than expected, while in the low
overall male: female ratio (2:1) they obtained mat-
ing values higher than expected ones. In September
the genetic sexing males of the low male: female ra-
tio did not perform as well. It could be that the re-
duced male: female ratio improves the sterile male
performance under warm weather. Also, if we com-
pare the mating performance of genetic sexing
males fed complete diet with sugar fed ones at 1:1:3
ratio, we observe that in the June experiments the
sugar fed males had superior mating performance
as compared with the complete diet fed ones. The
opposite was true in September. In conclusion, the
mating performance of non-irradiated genetic sex-
ing males was considerably improved as compared
with the performance of irradiated males in the
1996 experiments. This could be the result of no ir-
radiation-damage and/or even of strain perfor-
mance improvement because of sexing break down
between 1996-98. The mating performance of ge-
netic sexing males fed complete adult diet as com-
pared with sugar only feeding, did not clearly prove
superiority of any of the treatments.
The study of copulation site (1998, Table 3) con-
cluded that the preferred site by both type matings
is the underside of the leaf. There were some differ-
ences on mating site preference between June
(higher temperatures) and September (lower tem-
peratures). In June the preferred mating site by
both type matings was the lower canopy while in
September, matings moved higher in the canopy,
the phenomenon been more striking with the ge-
netic sexing type matings. As to tree sector and al-
though differences were not significant, in June
both type matings appeared to concentrate in the
north and west of the tree canopy, the phenomenon
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Florida Entomologist 85(1)
March 2002
TABLE 3. PERCENT OF TOTAL MEDFLY COPULATIONS OBSERVED ON THE DIFFERENT ORANGE TREE CANOPY SITES IN
1998. CAGED-TREE EXPERIMENTS WITH WILD FLIES (W) FROM SOUR ORANGES OR FIGS MIXED WITH NON-IR-
RADIATED GENETIC SEXING MALES OF STRAIN T (Y;5) 1-61/95 (LN) AT GENERATIONS 33-36.
June 1998 September 1998
Tree-canopy site W x W LnxW WxW LnxW
Leaf surface
Top 0.0 a 3.3 a 5.6 a 1.6 a
Bottom 85.7 b 83.3 b 88.7 c 90.2 c
Other sites (fruit, tree trunk,
cage screen and floor) 14.3 a 13.4 a 5.6 a 8.1 a
Height
High 5.3 a 3.4 a 14.9 ab 23.7 bc
Middle 15.8 a 10.3 a 40.3 c 47.5 c
Low 78.9 bc 86.2 c 44.8 c 28.8 bc
Tree sector
West 30.0 35.7 20.9 11.5
South 10.0 3.6 35.8 36.1
East 10.0 3.6 13.4 24.6
North 40.0 50.0 7.5 8.2
Center 10.0 7.1 22.4 19.7
'The above data are based on 181 matings recorded in total. Data of different sex ratio and adult feeding treatments were pooled together because no
substantial differences were observed. In the columns and the rows means followed by different letter are significantly different: P < 0.001, F = 9.033, df
11, 24 (Leaf surface) or P < 0.05, F = 2.182, df 11,24 (Height). No significant difference was found in Tree sector: P < 0.166, F=1.433,df 19,40 (Tukey's test).
'Upon mixing, virgin wild flies or genetic sexing males were 8-12 or 4-6 days old, respectively. Experiments were organized in June and September
with 2 three-day replicates of 3 field cages each time. One hundred total flies per field cage were always used at W:W:Ln 1:1:3 or 1:1:1 sex ratios, the
first sex ratio tested with Ln flies fed either complete or sugar only diet and W flies fed always complete diet. In June environmental conditions were 25-
35C, 32-48% RH and 2000-15000 Lux, while in September the conditions were 16-29C, 36-78% RH and 600-12500 Lux, respectively.
been again more intense with the genetic sexing
male matings. In September, wild type matings
concentrated primarily in south, west and center
while genetic sexing type matings concentrated in
south, east and center of tree canopy. It is interest-
ing to note that in June about half of total both-
type matings concentrated in the cooler northern
sector of the tree, while in September only 8% of
matings preferred this part of the tree canopy.
In conclusion, genetic sexing sterilized males
were found much inferior than their wild counter-
parts in mating performance under field cage condi-
tions. Nevertheless they performed sexual activity
mostly on the same canopy sites as the wild flies.
Further research is needed, especially to elucidate
the effect of protein feeding before releasing on the
survival and mating effectiveness of sterile males.
ACKNOWLEDGMENTS
Thanks are expressed to Mr. Farsarakis who allowed
use of his citrus plantations for the present experi-
ments, Mr. M. Yassar for technical help and Miss Chi-
ladaki for computer processing this manuscript. This
research was supported by IAEA RC 7657.
REFERENCES CITED
CALKINS, C. 0., AND J. C. WEBB. 1983. A cage and sup-
port framework for behavioral tests of fruit flies in
the field. Florida Entomol. 66(4): 512-514.
CALKINS, C. 0., T. R. ASHLEY, AND D. L. CHAMBERS.
1996. Implementation of technical and managerial
systems for quality control in Mediterranean fruit
fly (Ceratitis capitata) sterile release programs. pp.
399-404. In B. A. McPheron and G. C. Steck [eds.]
Fruit fly pests: a world assessment of their biology
and management. St. Lucie Press, Delray Beach, FL
CAYOL, J. P., J. VILARDI, E. RIAL, AND M. T. VERA. 1999.
New indices and method to measure the sexual com-
patibility and mating performance of medfly (Diptera:
Tephritidae) laboratory reared strains under field
cage conditions. J. Econ. Entomol. 92(1): 140-145.
ECONOMOPOULOS, A. P. 1996. Quality control and SIT
field testing with genetic sexing Mediterranean fruit
fly males. pp. 385-389. In B. A. McPheron and G. J.
Steck [eds.] "Fruit fly pests: a world assessment of
their biology and management", St. Lucie Press.
FRANZ, G., E. GENCHEVA, AND P. KERREMANS. 1994. Im-
proved stability of genetic sexing separation strains
for the Mediterranean fruit fly, Ceratitis capitata.
Genome 37: 72-82.
FRIED, M. 1971. Determination of sterile -insect compet-
itiveness. J. Econ. Entomol. 64: 869-872.
IAEA, FAO, USDA. 1998. A manual of quality control
for fruit flies. IAEA, Vienna, 52 pp.
KATSOYANNOS, B. J., N. T. PAPADOPOULOS, N. A. KOU-
LOUSSIS, R. HEATH, AND J. HENDRICHS. 1999. A
method of assessing the fertility of wild Ceratitis
capitata (Diptera: Tephritidae) females for use in
sterile insect technique programs. J. Econ. Entomol.
92(3): 590-597.
MCINNIS, D. 0., D. R. LANCE, AND C. G. JACKSON. 1996.
Behavioral resistance to the sterile insect technique
by the Mediterranean fruit fly (Diptera: Tephritidae)
in Hawaii. Ann. Entomol. Soc. Amer. 89: 739-744
Field et al.: Medfly Lek Evolution
WHY DO CALLING MEDFLIES (DIPTERA: TEPHRITIDAE) CLUSTER?
ASSESSING THE EMPIRICAL EVIDENCE FOR MODELS
OF MEDFLY LEK EVOLUTION
S. A. FIELD', R. KASPI AND B. YUVAL
Department of Entomology, Hebrew University of Jerusalem, P.O. Box 12, Rehovot, 76100, Israel
'Present address: Department of Applied & Molecular Ecology, University of Adelaide
Waite Campus Glen Osmond, 5064, SA, Australia
Phone: +61-8-83036712; Fax: +61-8-83794095; E-mail: scott.field@adelaide.edu.au
ABSTRACT
Recent years have seen a proliferation both in theoretical approaches to understanding lek
evolution and in empirical work on the lek mating system in the Mediterranean fruit fly.
This paper aims to integrate these two lines of research and to identify practical insights of
relevance to those engaged in medfly research. We begin by considering the definition of a
medfly lek and recognize the existence of male aggregations at two distinct spatial scales:
entire trees, or groups of trees within a given habitat; and small areas (ca 30 cm diameter)
within the canopy of a given tree. After summarizing the assumptions and predictions of the
main candidate models for lek evolution (predation avoidance, hotspot, hotshot and female
preferences) we review empirical evidence from previous and ongoing research that is rele-
vant to medfly lek evolution at both spatial scales. Far from being attributable to a single
cause, we conclude that the evolution and selective maintenance of lekking behavior in the
medfly can be attributed to a complex mosaic of interacting ecological pressures. We recog-
nize that much more empirical work is needed to resolve outstanding questions on medfly
lek evolution, and highlight potential benefits from the interaction between pure and ap-
plied lines of research on medfly mating behavior.
Key Words: medfly, Ceratitis capitata, lek, model, evolution
RESUME
En anos recientes se ha visto una proliferaci6n tanto en aproximaciones te6ricas para enten-
der la evoluci6n del comportamiento en la selecci6n del lugar de apareamiento y en trabajos
empiricos sobre los sistemas de selecci6n de los lugares de apareamiento en la mosca del Me-
diterraneo. Este trabajo esta dirigido hacia la integraci6n de estas dos lines investigativas
y hacia la determinaci6n de informaci6n practice y de relevancia para las personas envuel-
tas en la investigaci6n de la mosca del Mediterraneo. Comenzamos por considerar la defini-
ci6n de el m6todo de selecci6n del lugar de apareamiento de la mosca del mediterraneo y en
reconocer la existencia de grupos de machos en dos escalas espaciales diferentes: arboles
completes, o grupos de arboles dentro de un habitat dado; y pequenas areas (30 cm de dia-
metro) dentro del area foliar de un arbol determinado. Despu6s de resumir las suposiciones
y predicciones del candidate como modelo principal para la evoluci6n del comportamiento en
la selecci6n del lugar de apareamiento (evitar depredaci6n, punto clave, personaje y prefe-
rencias de la hembra) se analiz6 la evidencia empirica proveniente de investigaciones ante-
riores y ain en process, que son relevantes para la evoluci6n del comportamiento en la
selecci6n del lugar de apareamiento de la mosca del Mediterraneo en ambas escalas espacia-
les. Lejos de poderse atribuir a una sola causa, concluimos que la evoluci6n y mantenimiento
selectivo del comportamiento en la selecci6n del lugar de apareamiento de la mosca del Me-
diterraneo puede ser atribuido a un complejo mosaico de presiones interactivamente ecolo-
gicas. Reconocemos que es necesario much mas trabajo empirico para contestar las
sobresalientes preguntas en cuanto a la evoluci6n del comportamiento en la selecci6n del lu-
gar de apareamiento de la mosca del Mediterraneo, y para destacar los beneficios potenciales
de la interacci6n entire lines puras y aplicadas de investigaci6n en el comportamiento de
apareamiento de la mosca del Mediterraneo.
WHAT IS A MEDFLY LEK? 1995). Although the term 'lek' has been used by
ornithologists for over a century, only recently
Animals from a wide range of taxa, with a di- have attempts been made to give it a rigorous eco-
verse array of life-histories, are classified as hav- logical definition (see Hoglund & Alatalo 1995).
ing a lek mating system (Hoglund & Alatalo Bradbury (1977, 1981, 1985) suggested four crite-
Florida Entomologist 85(1)
ria: 1) the absence of male parental contribution;
2) most mating occurs at a specific site(s) where
males aggregate and display; 3) the display sites
contain no significant resources required by fe-
males; 4) females have the ability to choose a
mate when visiting the display site. However, in
light of numerous exceptions and ambiguities,
Hoglund & Alatalo (1995) advocated a more re-
laxed definition based simply on the presence of
aggregations of males and the absence of pair
bonding and paternal care. This definition, "ag-
gregated male display that females attend prima-
rily for the purpose of fertilization", nearly
reverts to that previously proposed by Emlen &
Oring: "communal display area where males con-
gregate for the sole purpose of attracting and
courting females and to which females come for
mating". Under this classification, more than 240
species scattered across the animal kingdom have
so far been reported to exhibit a lek mating sys-
tem (Hoglund & Alatalo 1995).
One of these species is the Mediterranean fruit
fly, Ceratitis capitata (Diptera: Tephritidae). Al-
though the details of its mating behavior are to-
day of great economic significance, this only came
about with the development of the Sterile Insect
Technique (SIT) in the 1960s. Numerous studies
on medfly mating ensued, but it was not until the
first systematic study of sexual behavior under
semi-natural conditions was undertaken by
Prokopy & Hendrichs (1979) that the existence of
lekking as the major component of the mating sys-
tem was recognized. Since that time, there have
been two distinct operational definitions of medfly
leks used in the literature. Prokopy & Hendrichs
(1979) originally defined medfly leks as "3-6 males
grouped within ca. 30 cm of one another, each on
a separate leaf". Hendrichs & Hendrichs (1990)
more or less followed this classification, as did
Field & Yuval (unpublished data), having inde-
pendently arrived at the same definition based on
field observations. However, Whittier et al. (1992),
followed by Shelly et al. (1994) and Shelly & Whit-
tier (1995), instead defined a lek as an entire tree,
because they usually found only one aggregation
formed per tree and males moved readily between
leaves, making it difficult to delimit the bound-
aries of an aggregation.
Before proceeding with a discussion of lek evo-
lution in medflies, it is important to clarify ex-
actly what is meant by the term 'lek' as applied to
medflies. We note that both existing definitions
have merits and flaws and, in the interests of
gaining ecological insight, we advocate the reten-
tion of both pending further investigations. We
thus propose the recognition of medfly leks at two
distinct spatial scales: "small-scale leks" as de-
fined by Prokopy & Hendrichs and "large-scale
leks" as defined by Whittier et al. (1992).
With respect to small-scale leks, our own expe-
rience has independently supported the original
conclusion of Prokopy & Hendrichs (1979) that
aggregations of males in the tree canopy within a
diameter of ca 30 cm are indeed functional compo-
nents of the mating system. However, we should
note that confirming the objectivity of this work-
ing definition will require studies that quantify
the spatial distribution of calling males within
and among trees, and demonstrate that males are
more clustered at this spatial level than would be
expected by chance. Nevertheless, a crucial as-
pect of lek mating systems, and one that makes
them so interesting in terms of sexual selection, is
that behavioral interactions such as dominance
contests, competitive courtship and mate compar-
isons take place within them, and can heavily in-
fluence male reproductive success. Although in
medflies communication over longer distances,
particularly by chemical means, are likely to be of
importance in male mating interactions, much of
the behavioral detail that determines the course
of sexual selection in lekking species can occur
only when males are in close proximity. The inten-
sive study of small-scale medfly aggregations is
therefore vital to our understanding of sexual se-
lection in medflies and the continued use of this
tentative definition is therefore amply justified.
One advantage of the large-scale lek definition
is that it is easier to verify quantitatively and in-
deed is already supported by empirical evidence.
Whittier et al. (1992), for example, showed that a
small subset of available trees accounted for a
large majority of recorded incidences of male call-
ing. Unpublished observations in Israel have sug-
gested the existence of a similar phenomenon
(R. K. & B. Y., unpublished data). Such large-
scale aggregations differ considerably from, for
example, classic avian leks, in which females en-
ter a discrete male aggregation within which both
male interactions and relative display efforts are
readily observable. Male medflies within a tree, or
group of trees, are relatively much more dis-
persed, separated and obscured from one another
and from females by foliage and thus cannot in-
teract or be observed to anywhere near the same
extent as males in classic leks. However, it is con-
ceivable that behavioral dynamics could at least
resemble a classic lek. Male distribution through-
out the tree could be influenced by agonistic inter-
actions, and females could enter a tree and move
about it comparing males in an analogous fashion
to a female visiting a small group of males on ad-
jacent leaves. This is especially so if, as Whittier
et al. (1992) report, males can be somewhat clus-
tered within a single tree canopy at certain times
of day. Although the existence and generality of
large-scale leks, like their small-scale counter-
parts, require further quantitative confirmation,
present evidence suggests the existence of a dis-
tinct pattern of aggregation that must be investi-
gated if we are to achieve a fuller understanding
of medfly sexual behavior.
March 2002
Field et al.: Medfly Lek Evolution
At present, therefore, we can recognize two
distinct spatial scales of medfly aggregation. Our
aim in this paper is to investigate the evolution-
ary causes of both, using existing theoretical mod-
els and emerging empirical evidence. We first
enumerate the candidate models, then present
the relevant evidence from previous and ongoing
medfly research. In light of this, we identify criti-
cal gaps in our understanding of medfly lek evolu-
tion and suggest some practical steps that can be
undertaken to fill them. We conclude with the
prediction that pursuing the path toward such an
understanding may bring many insights of bene-
fit to programs aimed at managing medfly popu-
lations.
CANDIDATE MODELS FOR LEK EVOLUTION
The main hypotheses that have been put for-
ward to explain lek evolution (see Hoglund & Al-
atalo 1995) are: i) predation risk; ii) hotspots; iii)
hotshots; iv) female preferences. Others, such as
the "passive attraction", "information-sharing"
and "black-hole" models, are either not applicable
to the biology of medflies, or are otherwise of du-
bious importance, and will not be considered here.
Hotspots. If resources used by females are
patchily distributed, or female home-ranges over-
lap, then by displaying at certain sites (hotspots)
in the habitat through which females are con-
strained to pass when traveling, males may ob-
tain an increased probability of female visitation.
Hotshots. If males differ in quality and certain
males are highly attractive to females (hotshots),
lower quality males may increase their mating
probability by clustering around the hotshots.
Female Preferences. Females may exhibit pref-
erences for mating in male aggregations due to
benefits from: a) predator deflection; b) opportu-
nity to compare males; c) increased average qual-
ity of males. Alternatively, such a preference
could evolve as an arbitrary Fisherian trait
(Andersson 1994).
Predation Risk. If males incur a risk of preda-
tion due to sexual display, and the per capital risk
decreases with increasing group size, then selec-
tion will favor aggregations.
It should be emphasized that although each of
these models generates distinct empirical predic-
tions, they are not mutually exclusive possibilities
for the evolution of lekking, and any combination
of mechanisms could be operating in a given sys-
tem. Indeed, some recent attempts at modeling
lek evolution have recognized the value of combin-
ing elements of several of these models. Below we
summarize the main theoretical features of such
models and discuss how they can be used to under-
stand lek evolution. As there are no formal math-
ematical models of the influence of predation on
lek evolution, we will concentrate on the hotspot,
hotshot and female preference hypotheses.
Before doing so, it is necessary to introduce a
critical concept in the theory surrounding lek evo-
lution, viz., the mating skew. This parameter can
be calculated in many different ways (Kokko &
Lindstrom 1997, Kokko et al. 1998), but essen-
tially measures the degree to which matings are
non-randomly distributed among males in a lek.
The interest in mating skew stems from the com-
mon empirical observation that matings in leks
are monopolized by one or a few high-ranking
males. Given that such differences exist, theoreti-
cians have attempted to explain how leks could
evolve, in spite of the fact that low-ranking males
seemingly have so little to gain by joining.
Hotspots and Hotshots
Widemo & Owens (1995) proposed that the an-
swer to this question lies in increased levels of ag-
gression and disturbance on the lek as group size
increases, and a lesser ability of the high-ranking
males to monopolize matings. This lowers the
mating skew, and leads to the prediction that low-
ranking males will have larger optimal lek sizes
than high-ranking males. Thus leks represent the
interests of low-ranking rather than high-rank-
ing males, the latter being trapped into staying in
the lek due to constraints imposed by habitat lim-
itation or the hotspot effect. Data from the ruff,
Philomachus pugnax, in which low-ranking
males actively increase the lek size by soliciting
other males to join, support this conclusion.
However, Hernandez et al. (1999) recently
demonstrated that the situation is more compli-
cated than this model suggests. Further to the in-
teraction between lek size and mating skew, it is
essential to know how relative competitive differ-
ences (RCDs) change as lek size increases. It is
not automatic that competitive differences among
males will decrease as more males join the lek. It
is conceivable that even if the mating skew de-
creases with increasing lek size as expected,
RCDs could actually increase, so that the increas-
ing lek size favors the interests of high-ranking
males rather than low-ranking ones. The analysis
of Widemo & Owens therefore represents a spe-
cial case, which may well be applicable to the ruff,
but is unlikely to be general.
Both of these models simultaneously incorpo-
rate aspects of the hotspot and hotshot hypothe-
ses. First, they assume that males are con-
strained in the locations they can choose for dis-
play (the essence of the hotspot hypothesis), and
hence they do not abandon the lek even if it grows
in size beyond what is optimal for them as indi-
viduals. Second, they assume that large differ-
ences in male quality exist, and, at least for some
combinations of RCD, mating skew and lek size,
that the lower-ranking males are benefiting from
the attractiveness of the high-ranking males (the
central idea of the hotshot hypothesis).
Florida Entomologist 85(1)
Large-Scale Leks
The evolution of leks by female preferences
has been modeled separately, using a game-theo-
retic approach (Kokko 1997). Here females assess
males by comparison both within and between
leks, subject to two constraints: within leks, they
are not able to assess the best male perfectly; and
among leks, travel costs mean they are unable to
visit and assess all available aggregations. From
the female side, the optimal strategy can be de-
duced with a straightforward mathematical cal-
culation: when choosing among leks, they should
prefer the larger ones, because the average male
quality is higher; and within leks, they should
choose the male they assess as being top-ranking,
no matter what their probability of making an er-
ror. However, from the male side, the same prob-
lem remains, viz. why should low-ranking males
join? One obvious answer is the imperfection of fe-
male choice, which gives low-ranking males at
least a small chance of being chosen by mistake.
But female travel costs are also important; if costs
are low enough so that females can sample a large
number of leks (recall they will prefer the larger
ones), then it pays males to join large leks, which
will receive the most female visits. Conversely, if
travel costs are high, optimal aggregation sizes
will remain low and lek evolution is constrained.
Empirical Evidence Concerning Lek Evolution
in Medflies
Any narrative for lek evolution in medflies
must begin by accounting for why mating does
not occur on the host fruit, as it does in many tem-
perate fruit flies. The accepted explanation, pro-
pounded by Prokopy (1980) and Burk (1981),
invokes several ecological factors (multivolt-
inism, polyphagy and high predation risk) that in
combination make it impossible or at least un-
profitable for males to monopolize resources im-
portant to females. The enormous host range and
multivoltine life cycle of C. capitata certainly
qualify it to fit this model. Furthermore, the re-
duction of oviposition to a short time-window in
the afternoon, added to the high levels of preda-
tion on females observed on fruit (Papaj et al.
1989, Hendrichs et al. 1991, Hendrichs et al.
1994, Hendrichs & Hendrichs 1998), offer strong
support for the role of predation. The secondary
mating tactic seen in C. capitata, of males guard-
ing fruit and attempting to copulate females with-
out courtship, can be seen as an evolutionary relic
in a mating system initially resource-based but
driven away from resources over evolutionary
time by the combined effects of the above-men-
tioned ecological pressures. Alternatively, it could
be an alternative mating strategy used by low-
ranking males.
Having accounted for the evolutionary shift in
medflies away from mating on host fruit, it re-
mains to address the question of why sexually ac-
tive males might aggregate at the first spatial
level, that of the tree, or group of trees, within the
habitat. The first thing to recognize is that al-
though mating away from the vulnerability of
host fruit would have reduced predation risk, it
by no means would have eliminated it. Predators
such as yellowjacket wasps, Vespula germanica
(F.) (Hymenoptera: Vespidae), which use a combi-
nation of visual and olfactory cues to hunt
amongst tree foliage for calling males and resting
or copulating flies of both sexes (Hendrichs et al.
1994, Hendrichs & Hendrichs 1998), would con-
tinue to pose a substantial risk.
Predation. There are two evolutionary routes
by which predation could lead to male aggrega-
tion in certain trees. The first is by providing a se-
lective pressure for males to display from the most
protected sites available in the habitat, viz. trees
with a dense canopy structure that would impede
the movement of larger predators and provide a
variety of refuges and escape routes. This view
has been supported by observations made by Hen-
drichs & Hendrichs (1990) in Egypt, and an anal-
ysis by Shelly & Whittier (1995) of lek distribution
in Hawaii, showing that leks were clustered in the
trees of largest volume, which also seemed (quali-
tatively) to possess the greatest horizontal foliage
density. Nevertheless, it remains for this hypoth-
esis to be explicitly tested. The second route is by
males gaining a decreased per-capita risk of mor-
tality by displaying with other males. This can oc-
cur either because the per-capita attractiveness to
a predator of an aggregation decreases with ag-
gregation size or due to the benefits of group vigi-
lance. As yet there are no data addressing per-
capita predator attraction at the large-scale lek
level. However, we can deduce that the benefits of
vigilance are unlikely to apply on a scale as large
as an entire tree. A single wasp attack in one part
of the canopy would only alert males in the imme-
diate vicinity and not elsewhere in the tree, un-
less attacked males produce a specifically
designed signal, e.g. an alarm pheromone.
Hotspot. The hotspot hypothesis is also an ap-
pealing explanation for the formation of large-
scale leks. By choosing to display in fruiting host
trees, males could maximize their chances of en-
countering females, which must visit these sites
for feeding and oviposition. Some evidence from
the field supports the hotspot interpretation, by
showing that male sexual activity is indeed con-
centrated on fruiting host trees. Hendrichs &
Hendrichs (1990, unpublished data cited therein)
found most male sexual activity occurred on fruit-
ing citrus. Hendrichs et al. (1991) found that most
leks and matings occurred in foliage of the pri-
Female Preferences
March 2002
Field et al.: Medfly Lek Evolution
mary host (orange), where females went to ovi-
posit after visiting leks. Most recently, Kaspi &
Yuval (1999b) have produced several lines of ex-
perimental support from a field-cage study. They
showed that wild males preferred to display on
trees containing real fruit and preferred trees
containing a combination of visual and olfactory
fruit stimuli over trees with either stimulus alone
or no host stimulus. However, although these
studies are consistent with the hotspot hypothe-
sis, they are also consistent with the hypothesis
that males choose such trees because of their ac-
cessibility to food sources. As lekking is energeti-
cally costly (Warburg & Yuval 1997, Yuval et al.
1998) this may be an important consideration in
male lek site selection.
Evidence inconsistent with the hotspot hy-
pothesis has also been obtained. In a field study
in Hawaii, Shelly & Whittier (1995) found lek
sites did not correspond with female oviposition
or feeding sites, as males settled preferentially on
certain persimmon trees that did not have ripe
fruit at the time. Similar observations have been
made in Israel, where in a mixed orchard, 2 par-
ticular pitanga trees have been found to harbor a
disproportionate number of calling males. Al-
though the pitanga trees were fruiting at the
time, there were also an abundance of suitable
citrus, guava (and other pitanga) trees immedi-
ately adjacent that harbored few or no males
(R. K. & B. Y., unpublished data). These observa-
tions strongly suggest that if a hotspot effect is in-
volved, it is by no means the only factor driving
male aggregation into large-scale leks.
Many authors have noted the possibility that
in addition to offering protection from predation,
trees with a dense canopy structure could offer a
more suitable microclimate for male calling activ-
ity (Arita & Kaneshiro 1985, 1989, Hendrichs &
Hendrichs 1990, Whittier et al. 1992, Shelly &
Whittier 1995). Although there are as yet no
quantitative data on microclimate variations
among trees of different size and canopy struc-
ture, Kaspi & Yuval (1999b) have recently shown
that within trees, male positioning throughout
the day tracks changes in temperature, relative
humidity, light intensity and the azimuth of the
sun. In particular, males exhibited a marked pref-
erence for leaves with microclimate characteris-
tics closely matching those in full shade. Should
males actively choose among trees according to
such preferences (or be beholden to the decisions
of females concerning where to mate), this would
provide a complementary explanation to preda-
tion avoidance for male clustering in certain trees
that have unusually dense canopies.
Hotshot and Female Preferences. With respect
to the hotshot and female preference models, we
argue that these mechanisms are not viable ex-
planations for clustering at the whole-tree level
or at best constitute only weak evolutionary
forces. If low-quality males are to gain any benefit
from settling near high-quality males, they must
be in sufficiently close proximity that visiting fe-
males will detect them and possibly mistake them
for the high-quality male to which they were ini-
tially attracted. Obviously the best position to
achieve this is as close to the high-quality male as
possible, so the evolutionary effect of the hotshot
mechanism would be the formation of groups of
males that are small and tightly clustered at a
scale well below the level of an entire tree. Simi-
larly, if female preferences for large leks are driv-
ing male aggregation, males that settle adjacent
to other male(s) will be favored over males that
take up position in the same tree, but in a rela-
tively distant part of the canopy. For these rea-
sons we feel the hotshot and female preference
hypotheses are more applicable to small-scale
male aggregations and delay discussion of them
until the following section.
Summary. In overview, a combination of eco-
logical factors appear to have acted to drive med-
fly mating activity firstly away from host fruit
and secondly to become concentrated in certain
trees within the habitat. Evidence suggests the
action of a composite predation and hotspot effect,
modulated by microclimate preferences in habitat
selection. There are compelling reasons why hot-
shot and female preference effects would not be
potent evolutionary forces at this spatial level.
However, this may not be the case when consider-
ing multiple spatially distinct leks within the
canopies of particular trees, as we discuss in the
next section.
Small-Scale Leks
Predation. As noted above, the need to avoid
predation may have driven the mating system
away from host fruit, but it is an unlikely expla-
nation for the clustering of males into large-scale
leks. However, at the level of small-scale leks, it
becomes plausible by the following possible mech-
anisms. If the number of predator attacks does
not increase in proportion to group size, animals
in the center of an aggregation are better pro-
tected than those on the periphery (Hamilton
1971), and/or efficiency in predator detection is
increased by sharing the task of vigilance with
conspecifics (Pulliam 1973). If any of these oper-
ate, then predation pressure can act as a driving
force in lek evolution.
Hendrichs & Hendrichs (1994, 1998) have
shown that medfly males lekking at a site in
Chios, Greece are at substantial risk from yellow-
jacket wasp attacks, especially when calling, an
activity that appears to significantly reduce their
vigilance. Most recently they have found strong
support for the evolutionary impact of predation
by showing that the per-capita number of wasp at-
tacks decreases with lek size, making larger ag-
Florida Entomologist 85(1)
gregations safer for calling males (M. & J.
Hendrichs, Insect Pest Control Section, IAEA, Vi-
enna, unpublished data). As the experiment was
conducted with artificial leks of caged males,
there are no data on the number of successful at-
tacks as a function of group size. However, ac-
counting for the benefits of group vigilance, we
can infer males in large leks would be even better
protected than these data imply. It is also relevant
to consider the effect of disruption of mate attrac-
tion activities by predator attacks. Each time a
predator attacks, all males in the lek are forced to
disperse and resume searching for a suitable site,
temporarily excluding them from mate-attraction
activities. The lower per-capita level of predation
in larger leks would reduce interruptions and in-
crease the amount of time available for mate at-
traction. Provided the per-capita rate of female
visitation remained comparable with that seen in
smaller aggregations (see below), calling in larger
groups would result in fitness benefits.
Hotspot. The prospects for hotspot effects to be
operating at this spatial level seem remote. A
common criticism of the hotspot model is that ar-
eas of high female density (driven by resource
patchiness) might not be localized enough to ac-
count for the tightness of male clustering (West-
cott 1994). This would certainly seem to be the
case with small-scale medfly leks.
Hotshot. The hotshot hypothesis also deserves
attention. It assumes that variations in male
quality occur, such that high quality males are
able to monopolize matings and less attractive
males then benefit by clustering around them. Ev-
idence certainly points to the necessary variation
in male quality existing, as laboratory studies
have repeatedly found a non-random distribution
of matings among males. Although this has not
yet been expressed in terms of mating skew
(sensu Kokko & Lindstrom 1997), or been verified
in a natural setting, it is nevertheless suggestive
of variations in male quality that females are able
to detect and use as a basis for discrimination dur-
ing mate choice. However, for the hotshot mecha-
nism to work in medflies, the variations in quality
must be apparent to both females and males dur-
ing long-range mate attraction, i.e. pheromone
calling. This is because the majority of small-scale
medfly leks disperse without ever receiving a fe-
male visit (S.A.F., unpublished data), making it
impossible for males to identify hotshots on the
basis of the number of matings they achieve, as
can occur in other species (see Beehler & Foster
1988). Hotshots could conceivably be identified by
the quality of a given male's pheromone, as an in-
dicator of the likelihood that he will attract a fe-
male. This could potentially be achieved by first
using the presence of pheromone to locate leks
and then the relative proportions of certain com-
ponents in the pheromone blend to discriminate
among males of different quality.
However, current evidence on the question of
whether males are attracted to the pheromone
emissions of other males at all, let alone whether
they discriminate among males of different qual-
ity, is inconclusive. It had previously been widely
assumed that males are attracted to leks by the
male pheromone and that the powerful attraction
of males to chemicals such as trimedlure was due
to its mimicking key components of the male
pheromone (Burk & Calkins 1983, Sivinski &
Calkins 1986). However, chemical analyses have
since refuted this hypothesis (Millar 1995, cited
in Eberhard, 1999), leaving the mechanism by
which male clustering occurs an open question.
Evidence against male attraction to pheromones
has come recently from Shelly (UH, Hawaii, un-
published data), who found very low attraction of
released males to artificial leks formed in the field
using caged calling males. However, positive evi-
dence has also been obtained by Kaspi & Yuval
(1999a), who showed that when selecting a calling
site in a field cage, males were more likely to set-
tle on a tree from which caged males were emit-
ting pheromone, than on a control tree containing
only caged dead males. However, we must take
into account differences in methodology. The arti-
ficial leks formed by Kaspi & Yuval (1999a) con-
tained 30 males and they released 100 males into
a confined space, whereas Shelly (UH, Hawaii,
unpublished data) used only 12 males in the leks
and released 300 males into the wild. The former
experiment thus tipped the balance in favor of de-
tecting an effect, however weak. Nevertheless,
the result raises the possibility that males do in-
deed use the male pheromone as a cue in lek site
selection, an issue worthy of further study.
It remains to be directly studied whether males
and females are able to distinguish among calling
males of different quality using variation in phero-
mone blends. However, while two studies have in-
dicated that male mating success depends on the
quantity of pheromone produced (Whittier et al.
1994, Shelly, UH, Hawaii, unpublished data), there
is as yet no evidence favoring an effect of phero-
mone quality. Shelly found that males with high
previous mating success attracted more females
than males with low success, but apparently only
because they spent more time calling. It also seems
clear that the difference in mating success fre-
quently found between sterile and wild flies is not
due to females discriminating against them on the
basis of pheromone composition; females arrive at
leks of both types of males with equal frequency
and appear to discriminate only during close-range
courtship (Calkins et al. 1994, Shelly et al. 1994,
Shelly & Whittier 1996, Shelly 1999. Nevertheless,
this could mean simply that the sterilization and/
or mass-rearing process affects courtship rather
than pheromone quality. The hypothesis that there
exist variations in pheromone quality among
males remains worthy of investigation.
March 2002
Field et al.: Medfly Lek Evolution
A prediction from the hotshot hypothesis would
be the presence of "satellite" males, who join leks
but engage in little or no calling, but nevertheless
attempt to court females that are attracted to the
lek by the pheromone emissions of others. Obser-
vations by several authors suggest that this may
be occurring. Shelly et al. (1994) observed non-
calling males in leks in the field. In our observa-
tions in the field in Israel, a substantial proportion
of males in scan samples of leks were not calling.
Further, in a field-cage study (S. A. F. & B. Y., un-
published data), non-calling males in a lek some-
times switched immediately to directed wing-
fanning when a female flew past. This may have
been simple opportunism but could also represent
an evolved strategy. The occurrence of variation in
calling activity among males with a uniform rear-
ing history and environment (Shelly, UH, Hawaii,
unpublished data) also hints that investigating
the possibility of a genetic influence on individual
calling strategies may be worthwhile.
Female Preferences. To demonstrate that fe-
male preferences are a significant factor in lek
evolution, it must be shown that more females ar-
rive per male as lek size increases, causing an in-
crease in per capital male mating success. Three
studies have examined this question. Shelly (UH,
Hawaii, unpublished data) found the increase in
female visitation rate to artificial leks in the field
remained in constant proportion to the number of
calling males, whether the males had a successful
or unsuccessful mating record in the laboratory.
Similarly, Kaspi & Yuval (unpublished data)
found that the per-capita rate of female visits to
artificial leks in a field cage remained constant
over the range of lek sizes likely to be encoun-
tered in nature (2-8). As such a response can be
accounted for by passive attraction of females to a
larger olfactory signal, we may be tempted to con-
clude that there is no evidence for a large lek pref-
erence in female medflies. However, the third
study, by M. & J. Hendrichs (IPCS, IAEA, Vienna,
unpublished data), adds a new twist by simulta-
neously considering the effect of predation. They
found that when wasp predators were allowed to
attack leks, the females preferred the smallest of
four lek sizes offered. However, when predation
was removed, not only did females prefer larger
leks, they preferred them out of proportion to the
number of males, so that the per-capita rate of fe-
male arrival did indeed increase with lek size.
This suggests that females in fact do actively pre-
fer large leks, but are constrained in their prefer-
ence due to the risk of predation. Future studies
of female preferences must take into account the
possible interaction of this factor with predation.
SUMMARY
To summarize, the evolutionary causes of
small-scale leks within trees appear to be distinct
from those favoring aggregation into large-scale
leks. Hotspot effects, apparently one of the key
factors driving large-scale lek formation, is of lit-
tle relevance for small-scale lek evolution,
whereas protection from predation, hotshot ef-
fects and female preferences could all be impor-
tant. Although plausible, no direct empirical
evidence is yet available to support the hotspot
hypothesis. The evidence is perhaps strongest for
the effect of predation acting to increase aggrega-
tion size. Evidence also exists for female prefer-
ence for large aggregations, although this
appears to be modulated by predation risk, point-
ing to an intriguing conflict of interest between
the sexes with respect to lek size.
FUTURE EXPERIMENTAL PRIORITIES
AND PRACTICAL IMPLICATIONS
Despite the passage of two decades since the
medfly was identified as a lek-mating organism,
our understanding of the evolutionary forces driv-
ing lek formation in this species remains very ru-
dimentary. It seems this is not due to a lack of
research effort into medfly mating behavior but
rather because such research has rarely been
framed with evolutionary issues specifically in
mind or been used to explicitly test evolutionary
hypotheses. Understandably, the emphasis has
been on experiments designed to bring immediate
improvements in the quality of mass-reared
males for SIT or to understand the proximate
mechanisms determining successful courtships.
However, basic and applied research questions
are never mutually exclusive, and we believe that
attempting to place medfly mating behavior in an
explicit evolutionary setting can yield practical
benefits, just as practically-oriented research has
already begun to benefit our evolutionary under-
standing, by providing critical empirical data for
testing theoretical models. Most importantly, an
evolutionary framework can facilitate ongoing
critical evaluation of empirical studies, aiding the
resolution of experimental ambiguities and con-
tradictions, and speeding the conversion of an
otherwise haphazard accumulation of results into
an orderly, coherent body of knowledge.
Although the studies cited above have pro-
vided a useful start towards understanding med-
fly lek evolution, many questions and uncertain-
ties remain. Below we identify lines of research
that appear to hold promise for teasing apart the
influences of various ecological factors and sug-
gest some experiments critical to resolving out-
standing issues.
Firstly, most of the tentative conclusions con-
cerning lek evolution drawn above rely on evi-
dence from only one or a few studies. Inevitable
variations among studies in the origin, rearing
and handling of insects, experimental methodol-
ogy and analysis make it likely that even the most
Florida Entomologist 85(1)
carefully designed and executed studies can pro-
duce ambiguous or inconclusive results. In med-
flies, the potential for discrepancies between
studies is perhaps compounded by the fact that
this insect has relatively recently colonized a va-
riety of new habitats worldwide, and different
populations have possibly undergone (or are un-
dergoing) adaptation to local conditions. It there-
fore may be necessary to accumulate numerous
tests of the same hypothesis under differing eco-
logical conditions before robust conclusions
emerge. Ideally, consensus on the evolutionary in-
fluence of an ecological factor should be quantita-
tively assessed after taking multiple similar
studies into account (Arnqvist & Wooster 1995).
Far from being a redundant exercise, repeating
experiments performed by other researchers on
different medfly populations may highlight criti-
cal ecological factors that influence mating behav-
ior and thus prove essential to the task of
understanding its evolution.
At the large-scale lek level, it would be useful to
repeat studies like that of Shelly & Whittier (1995),
which applied a multivariate analysis to confirm
which factors determine the favored sites for male
display within the habitat. Ideally, such studies
would be longitudinal in nature and would track
the location of calling males in relation to seasonal
patterns of host availability within seasons and
fluctuations in these patterns among seasons.
Combined data from different medfly populations,
climates, and habitats would provide a rich data-
base with which to identify universal factors deter-
mining large-scale lek locations. Should the
pattern of males being clumped into large-scale
leks be borne out by such studies the next task
would be to confirm that this is due to a hotspot ef-
fect rather than males simply choosing to lek near
nutritional resources. This would require tracking
of female distributions (feeding sites, oviposition
sites and movements among them) and the demon-
stration of a correlation between female distribu-
tion and the male calling sites.
Concerning specific hypotheses for lek evolu-
tion, the effect of predation is one area that has
received intense empirical attention recently
(Hendrichs et al. 1994, Hendrichs & Hendrichs
1998) and should be pursued further. To clarify
whether the formation of large-scale leks in trees
with large volumes and dense canopies is in part
a response to predation, it would be desirable to
measure predation rates in trees of different size
and canopy structure. At the level of small-scale
leks, the information already obtained on attack
rates at different lek sizes could be supplemented
by data on the rate of successful attacks at differ-
ent lek sizes, which would indicate whether indi-
viduals displaying in larger groups benefit from
increased vigilance. As this would entail mea-
surement of predation rates on naturally display-
ing males, the data would be difficult to obtain
but would be well worthwhile as they would
clinch the argument for the role of predation in
driving male aggregation.
Further investigations of the hotshot hypothe-
sis should focus on testing whether the proximate
mechanism by which males aggregate is indeed by
cueing on the pheromone emissions of other males,
and if so, which are the active components in the
blend. A positive result would add credibility to
the hypothesis that low-quality males are at-
tracted to leks occupied by hotshot males that can
be distinguished by the quality of their phero-
mone. This hypothesis could then be investigated
in an experiment similar to that performed by
Shelly (UH, Hawaii, unpublished data), who
tested attraction of males and females to calling
males of low and high mating ability, with the dif-
ference that the calling males should be classified
with respect to their ability to attract conspecifics
on the basis of their pheromone alone. Classifying
them by their mating success leaves open the pos-
sibility that the high-mating males were success-
ful not due to quality of their pheromone, but due
to the efficacy of their courtship, which is of no evo-
lutionary consequence for lek formation. If both
females and males concurred in their choice of
males, the pheromone blends of attractive and un-
attractive males could then be compared and the
physiological basis for the hotshot effect identified.
One of the most interesting research directions
to pursue is the putative interaction between pre-
dation and female preferences for large leks in de-
termining optimal lek size in small-scale leks.
While large leks appear to increase male survival
by decreasing per-capita attack rate, this may not
be true for females, at least judging by the behav-
ior observed by J. & M. Hendrichs (IPCS, IAEA,
Vienna, unpublished data). This may be ex-
plained by the fact that female vigilance toward
predators is at its lowest when receiving court-
ship, so females may be particularly sensitive to
the risk of predation when visiting a lek. This not
only brings female interests into conflict with
that of males with respect to predation, but also
sets up a counterbalance to any preference fe-
males might have for mating in larger leks due to
the opportunity to compare males. Thus it could
be an important selective force acting to set an
upper boundary to lek sizes. Further experiments
measuring female arrival rates while manipulat-
ing lek sizes and predator attack rates would be
extremely valuable.
Although the research directions outlined
above are primarily directed towards answering a
theoretical question in behavioral ecology, there
also exist potential avenues whereby such re-
search could make a positive contribution to im-
proving the efficacy of medfly control programs.
Tests of the hotspot hypothesis will provide us
with detailed knowledge about preferred lek loca-
tions in various habitats and climates, enabling
March 2002
Field et al.: Medfly Lek Evolution
more judicious selection of sites for monitoring
traps and thus improving the ability to detect and
respond to infestations. Successful pursuit of the
hotshot hypothesis could provide the key to under-
standing variations in male attractiveness, and be
a step forward in improving the quality and mat-
ing competitiveness of mass-produced males vis a
vis their wild counterparts. If specific components
of the male pheromone could be identified as re-
sponsible for a hotshot effect, they could also be
used to manufacture more effective chemical baits
and lures. It is our hope that the future will see
more interaction between applied empirical re-
search on medfly mating behavior and theoretical
modeling of lek evolution, to mutual profit.
ACKNOWLEDGMENTS
We are grateful to Marti and Jorge Hendrichs and
Todd Shelly for sharing with us their unpublished data.
Funded by grants from California Department of food
and Agriculture and BARD.
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Hasson & Rossler: Fluctuating Asymmetry and Developmental Homeostasis 7:
CHARACTER-SPECIFIC HOMEOSTASIS DOMINATES FLUCTUATING
ASYMMETRIES IN THE MEDFLY (DIPTERA: TEPHRITIDAE)
0. HASSON1 AND Y. ROSSLER2
'P.O. Box 11598, Ramat-Gan 52015, Israel
'The "Israel Cohen" Institute for Biological Control, Citrus Marketing Board of Israel, Beit Dagan 50260, Israel
ABSTRACT
Fluctuating asymmetry (FA) indicate random variation in size of bilaterally-produced traits,
which occurs during development, and hence measures the degree of developmental insta-
bility. Whole-individual homeostasis has been assumed responsible for the negative correla-
tion that has often been found between FA of many sexually selected traits and their size.
We show that, theoretically, character-specific homeostasis can provide an equally convinc-
ing explanation for this correlation. Furthermore, we tested these two hypotheses for (1) a
sexually dimorphic character and (2) sexually monomorphic characters of the male Mediter-
ranean fruit fly, Ceratitis capitata, by manipulating density during larval development. Our
results clearly support character-specific homeostasis.
Key Words: Fluctuating asymmetry, developmental homeostasis, sexual selection medfly,
Ceratitis capitata
RESUME
La fluctuaci6n asim6trica (FA) indica variaci6n aleatoria en el tamano de los rasgos produ-
cidos bilateralmente, los cuales ocurren durante el desarrollo, y por lo tanto mide el grado de
inestabilidad del desarrollo. La home6stasis de individuos completos ha sido asumida como
la responsible de la correlaci6n negative que frecuentemente ha sido encontrada entire FA
de muchos rasgos sexualmente seleccionados y de su tamano. Se demuestra te6ricamente,
que la home6stasis caracter-especifica puede proporcionar una explicaci6n igualmente con-
vincente para esta correlaci6n. Ademas, se probaron estas dos hip6tesis con respect a 1) un
character sexualmente dim6rfico y 2) caracteres sexualmente mono-m6rficos de los machos de
la mosca del Mediterraneo, Ceratitis capitata, por medio de la manipulaci6n de la densidad
durante el desarrollo larval. Nuestros resultados claramente apoyan la home6stasis carac-
ter-especifica.
Minute variations in growth rate that occur
randomly during development accumulate and
result in small variations in the final size of or-
gans. These variations (or developmental 'noise')
can be quantified by comparing the final sizes of a
character that is produced more than once under
the influence of the same genome, at the same de-
velopmental stage, while being exposed to the
same environmental conditions. Because develop-
ment repeats itself twice during growth of bilater-
ally produced organs, bilateral asymmetry can be
used to give an estimate of developmental insta-
bility (Soule 1982, Soule & Cuzin-Roudy 1982,
Leary & Allendorf 1989, Parsons 1990). This type
of asymmetry is known as 'fluctuating asymme-
try' (FA), and is usually measured as the absolute
difference between the size values of left and
right, divided by their mean (to control for the
effect of size). A number of studies have shown
that FA increases following environmental and
genetic stress. The most common environmental
factors that are known to increase FA are extreme
temperatures, starvation and chemical stresses
(Soule 1982, Parsons 1990). New mutations, in-
creased homozygosity, recombination and direc-
tional selection are documented genetic factors
that increase FA (Parsons 1990, Moller & Pomian-
kowski 1993b).
Recently, the study of FA has gained much in-
terest because of its role in sexual selection. Fe-
males of different species, including the
scorpionfly, Panorpa japonica (Thornhill 1992),
barn swallow, Hirundo rustica (Moller 1992) and
human (Thornhill & Gangestad 1992, Thornhill
et al. 1995) have been shown to mate more readily
with symmetric males. This, and other potential
uses and implications of FA (e.g., Leary & Allen-
dorf 1989, Moller & Pomiankowski 1993b), re-
quires a good understanding of the mechanisms
that affect FA.
There is a general agreement that symmetry is
enhanced by genetic modifiers that increase the
harmony of development (Leary & Allendorf
1989). Other parallel terms that have been used
are 'developmental homeostasis', 'developmental
stability' (Soule & Cuzin-Roudy 1982), and 'in-
trinsic genetic coadaptation' (Leary & Allendorf
1989), all of which imply a state of evolutionary
Florida Entomologist 85(1)
equilibrium. More explicitly, these terms reflect
the general view that evolutionary transitions
away from symmetry are followed by an evolution
of modifiers that restore symmetry (McKenzie &
Klarke 1988, Leary & Allendorf 1989). The as-
sumed nature of this stability, however, is debat-
able. Some studies argue that developmental
homeostasis evolves separately for each of the
individuals' characters, and hence that FA mea-
sures the degree of character-specific develop-
mental stability. Others contend that homeosta-
sis is holistic, affecting the development of whole
organisms. Attempts to discriminate between
these two hypotheses by examining within-indi-
vidual correlations between FA of several charac-
teristics have provided conflicting evidence (Soule
& Cuzin-Roudy 1982, Watson & Thornhill 1994).
An important reason for the conflicting evi-
dence shown by these correlations is well ex-
plained by Whitlock (1996). He points out that for
each character in a bilaterally symmetric individ-
ual there exist only two samples of the variance of
developmental instability. Because each of the
measurements of FA is an unreliable statistical
measure of variations within the individual (as-
suming common cause for different characters),
then even if developmental instabilities of differ-
ent characters are correlated, the within-individ-
uals correlations of FA are often too coarse to
show them. Therefore, to decrease the role of sta-
tistical errors due to random sampling, other
types of correlations are required, ones that can
use repetitions across individuals, or that involve
only one bilaterally symmetric character. A deci-
sion about these other measures requires a better
understanding of the possible mechanisms that
induce fluctuating asymmetry.
The known list of causes of asymmetry sug-
gests that there might be more than one mecha-
nism involved. The distinction between character-
specific and whole-individual homeostasis is often
suggestive about these mechanisms. Whole-or-
ganism homeostasis is known to be a result of
certain external factors, such as extreme temper-
atures, which results in instability in the whole
organism. Whole-organism homeostasis is often
assumed to be, however, also a function of overall
individual 'quality' that may vary between indi-
viduals due to heritable or non-heritable differ-
ences in the ability and/or opportunity to capitalize
and use resources. Heritable differences that may
exist between individuals are expected to be di-
minished by natural selection, until a state of
equilibrium between natural selection and the
source of the heritable variations is reached (Fal-
coner 1981). This point of equilibrium, character-
ized by an improved overall adaptedness, should
be reflected by reduced FA.
In contrast, character-specific homeostasis is
frequently assumed to be a result of heritable or
non-heritable variations in size of each character.
Extreme character sizes reduce harmony of devel-
opment (and, as a result, increase FA) because
they are usually not accompanied by correspond-
ing changes in developmental organization, or in
supportive tissues such as muscle, blood vessels
or nerve cells, required for the enhancement of
developmental stability. For an evolving charac-
ter, under a regime of directional selection, this
might be just a temporary stage followed by the
evolution of these other modifications that will,
eventually, increase developmental homeostasis
of the character in its final state.
Investigations of FA, in relation to studies of
sexual selection, have consistently interpreted
results assuming that developmental homeo-
stasis is a whole-organism characteristic, rather
than character-specific. This assumption nicely
links sexual selection theory with the empirical
data that frequently (but not always) show a neg-
ative correlation between FA of sexually dimor-
phic traits and their size, a pattern that is not
found in other traits (Moller & Hdglund 1991,
Moller & Pomiankowski 1993a). This pattern is
readily explained by assuming that (i) FA is de-
termined by a whole-organism developmental ho-
meostasis, (ii) this homeostasis is correlated with
the individual's overall quality or some of its com-
ponents, and (iii) the sexual trait is a handicap.
The term 'handicap' refers to a signal, such as a
sexual ornament, which is costly to produce or
maintain such that a male's optimal trait size
(i.e., his investment in advertising) is correlated
with his physical condition. As a result, better
males produce more expensive sexual ornaments
(Zahavi 1975, 1987, Nur & Hasson 1984, Pomi-
ankowski 1988, Grafen 1990). Theoretical studies
show that at evolutionary equilibrium, the male
residual quality (after developing its sexual orna-
ment) is expected to remain higher for high
quality males, despite the fact that absolute
investment in sexual ornaments by these males
is greater (Nur & Hasson 1984, Grafen 1990,
Iwasa et al. 1991). Hence, if developmental sta-
bility is caused by whole-organism mechanisms
that depend on the male quality during develop-
ment, larger sexual ornaments should be also
more symmetric.
Despite the consistency of the negative corre-
lation between the size of a sexual trait and FA
with the whole-organism stability hypothesis, it
can only provide support for this hypothesis if
character-specific mechanisms cannot explain
this pattern. Here we show that this is not the
case. In the next section we show that size-depen-
dent character-specific homeostasis provides an
equally reasonable explanation for the negative
correlation between FA of many sexual traits and
their size. In the rest of the paper we present a
simple experiment that tests these two hypothe-
ses. Its results strongly support the character-
specific mechanism.
March 2002
Hasson & Rossler: Fluctuating Asymmetry and Developmental Homeostasis
SIZE-DEPENDENT HOMEOSTASIS
The single character size-dependent homeo-
stasis hypothesis suggests that individuals in a
population evolve modifiers that improve homeo-
stasis for a certain character size. Characters
that develop to match this size will be the least
asymmetric. Hence, we can call this size the least
asymmetrical size (LAS). Sizes that deviate from
LAS (either bigger or smaller) develop to be asym-
metric. They are expected to be more asymmetric
the more they deviate from LAS (Soule 1982,
Soule & Cuzin-Roudy 1982). Therefore, traits that
undergo stabilizing selection should normally
produce a more or less symmetric V- or U-shaped
distribution of FA around the mean size of the
trait (the form of the distribution depending on
whether the effect of deviation from LAS on FA is
linear or exponential, respectively).
If there are no other size-dependent effects,
such as directional natural or sexual selection,
modifiers are most strongly favored if they im-
prove developmental stability (hence, symmetry)
of the size that they most frequently encounter
IU
4.'
size
(the term 'modifiers' has been used vaguely in the
literature of FA; here, we use this term to simply
refer to a set of heritable traits that affect a char-
acter's developmental stability, as a function of its
size, although other size-dependent responses are
also possible). Consequently, the most common
size is also expected to be the LAS. However, the
actual shape of the relationship between FA and
size, its strength (i.e., its variance around the least
squared regression line), and the value of LAS it-
self should heavily depend on the distribution of
trait sizes in the population. For a normal distri-
bution of sizes of a trait undergoing stabilizing se-
lection, LAS is expected to correspond with the
population's mean, mode and median, all of which
fall onto a single point. A high standard deviation
creates a weak selection on modifiers that improve
homeostasis of any particular size, including the
mean, because they are less likely to encounter
that size. In contrast, selection pressure on modi-
fiers that improve developmental stability of the
LAS when standard deviation of sizes is small,
must be stronger, hence also more canalizing
(Fig. 1). Furthermore, a small standard deviation
size
Fig. 1. The expected relationships between FA (bottom) and size frequency distribution (top), assuming charac-
ter-specific developmental homeostasis. The pointers show average size (top), and the least symmetric size (bottom)
for two normal curves that differ in their standard deviation (left) and a skewed curve to the left (right).
Florida Entomologist 85(1)
of sizes should also lead to a small average FA, be-
cause the population is distributed tighter around
the LAS. Empirical data show that non-sexual
traits have either a U-shape distribution of FA
with relation to size, or no pattern at all (Moller &
Pomiankowski 1993a), which may reflect different
standard deviations of sizes around the mean.
What if distribution of sizes is not normal, but
skewed to one side? This question is of particular
interest here, because many sexual ornaments
may be at equilibrium between a strong direc-
tional sexual selection toward larger sizes, and a
weak opposing force induced by biased mutations
(Iwasa et al. 1991, Pomiankowski et al. 1991,
Pomiankowski & Moller 1995). These opposing
tendencies are likely to result in a skewed distri-
bution of sizes to the left. In such cases, LAS is ex-
pected to be on the right side of the range of the
sizes frequency distribution rather than in its
center (Fig. 1). As a result, the relationship be-
tween FA and size should not be U shaped with
symmetrical arms, but a short arm on the right,
and a long arm on the left. As before, strong devi-
ations from LAS in either direction should pro-
duce, on average, poor symmetry. Because this
distribution results in a relatively weak selection
on modifiers for developmental stability at any
particular size, the tightness of the correlation be-
tween FA and size described by Figure 1 (bottom
right) should be relatively weak. Consequently, a
distribution of sizes that is strongly skewed to the
left is likely to produce an apparent negative lin-
ear regression line between FA and size.
Furthermore, if reproduction is higher for indi-
viduals whose trait size is larger, which is the
case for many sexually selected traits, then LAS
is expected to shift even further to the right, be-
cause modifiers for large sizes are frequently as-
sociated with a higher than average reproduction
rate. This will reduce further the right arm of the
already asymmetric U shaped correlation be-
tween the trait's FA and size, and improve their
apparent negative linear correlation.
PREDICTIONS
The findings of negative correlations between
FA and size in sexual traits may, therefore, be
consistent with both the whole-organism and the
character-specific homeostasis hypotheses. Both
hypotheses suggest that greater deviations from
LAS should produce, on average, greater degrees
of asymmetry at the individual level, and greater
average values of FA, at the population level.
However, there are still some different predic-
tions that can be made to distinguish between the
effects of these two hypotheses:
A. The primary factor that affects FA under the
regime of a character-specific homeostasis, is the
shape of the frequency distribution of trait sizes
(weighted by the expected fitness benefits to the
genetic modifiers of each particular size). Also,
LAS is always expected to be intermediate be-
tween minimum and maximum sizes. If the distri-
bution of sizes is highly skewed to the left, LAS is
expected to be near the maximum, hence may be
difficult to be empirically distinguished from it.
In contrast, the primary factor that affects de-
velopmental stability and FA at equilibrium
within a whole-organism homeostasis regime, is
the individual's adaptedness and the capacity to
gain, store and use resources. For a sexually se-
lected handicap at equilibrium, LAS is expected
to correspond with the maximum trait size.
B. For character-specific homeostasis, stabiliz-
ing selection is expected to maintain LAS in the
neighborhood of the mean. In response to direc-
tional selection, where mean size is driven away
from equilibrium, the evolution of modifiers
should lag behind, leaving LAS on one side of the
mean, opposite to the direction of selection (e.g.,
for a trait that increases its size, LAS is expected
to be smaller than the mean). The correlation be-
tween FA and size is therefore expected to be pos-
itive if the character is in a process of increasing
in size (Moller & Pomiankowski 1993b, making,
here, an implicit assumption of character-specific
developmental stability).
Whole-organism homeostasis is expected to re-
spond similarly in the case of stabilizing selec-
tion, but to result in an opposite correlation in the
case of directional selection (negative for increas-
ing size, positive in the case of decreasing size),
because individuals who carry the novel extreme
size have higher overall adaptedness.
METHODS
Following the predictions in section (A) above,
this study is based on the specific prediction that
if the ability to gain and use resources (reflecting
developmental stress) can be better estimated by
a parameter other than the particular trait's size,
asymmetry of the trait should nevertheless be
best correlated with its size if homeostasis is
character-specific, but better correlated with that
other parameter of quality if homeostasis is a
whole-organism trait.
We used for our experiment a laboratory stock of
the Mediterranean fruit fly (medfly) Ceratitis capi-
tata, which has been raised by one of us (YR) under
constant conditions since 1964. The adult popula-
tion has been kept constant at about 3000 individ-
uals, which is normally large enough to avoid
frequent incidences of genetic drift (Roughgarden
1979). Temperature has been kept approximately
constant at 25C. Hence, relative to wild flies, this
population has been probably kept under relatively
narrow temporal fluctuations in selective pressure
with regard to developmental conditions.
To manipulate developmental stress we raised
larvae by collecting eggs of the same age, and put-
March 2002
Hasson & Rossler: Fluctuating Asymmetry and Developmental Homeostasis
ting them, at the same day, on 3 gm food at densi-
ties of 10 (8 repetitions), 20 (4), 40 (3) and 80 (3).
We estimate typical densities of larvae in the cul-
ture stock to vary between 50 to a 100 per 3 gm
food. The average egg hatch was 0.87 (SD = 0.08),
and the proportion of larvae that pupated (from
the hatched eggs) was 0.92 (SD = 0.05). Although
the output numbers were a little smaller than the
input numbers, for clarity we continue to refer to
the original densities. Within this range of densi-
ties, the effect of density on survivorship was in-
significant (we avoided a higher density, of 160
eggs per 3 gm, which, according to a preliminary
test, reduced survivorship considerably). We col-
lected the flies on the day of emergence, kept them
alive and unfed overnight to let them fully expand
their organs, and then put them individually in
plastic tubes and stored them in a freezer, until
we measured them. To avoid temporal biases in
measurements, we sampled male flies taken from
different densities at random until we got about
20 males of densities 10 and 20 (for which there
were fewer males). We then continued to sample
males of these two densities alone until we mea-
sured 22 males of each. Our final sample sizes
were 22 (for density 10), 22 (20), 40 (40) and 29
(80). Some measurements were not recorded in all
individuals because of physical damage to the
adult flies. To minimize unconscious bias, mea-
surements were made by a research assistant who
had no knowledge of the motivation of this re-
search or the significance of our marks on the
plastic tubes in which the frozen flies were stored.
To determine the relationship between pheno-
typic quality and fluctuating asymmetry of traits
that are not sexually dimorphic we measured
head width, thorax width and length, and both
wings width and length. For a sexually dimorphic
trait we measured maximum width and length of
the bilateral supra front orbital (SFO) bristle in
males, which is the most modified sexually dimor-
phic organ in the medfly. On females this bristle is
similar in shape and size to other bristles found
on the head. On males, this bristle has an elon-
gated stem and a modified wide spatula at the
distal end, oriented forward (Fig. 2). Two recent
studies suggest that the medfly SFO bristles are
sexually selected character: Mendez et al. (un-
published) find indications that female medflies
prefer intact males as opposed to males whose
SFO bristles were removed, and Hunt et al.
(1998) show that females prefer more symmetric
SFO bristles (although not longer ones).
To measure the flies, we transferred their mag-
nified video images from a dissecting microscope
to a Power Macintosh, and used NIH Image for
scaling and measurement. We measured both left
and right of bilateral organs (bristles and wings),
where the individual's means were computed as
the average value of left and right. We removed
the wings and bristles from the flies and put them
Fig. 2. The modified supra front orbital bristles on
the head of a male Mediterranean fruit fly.
on a glass microscope slide, using a cover glass to
flatten them before taking measurements. Be-
cause the bristles' stems usually broke during re-
moval, we used measurements of the bristle's
lengths that were made on the head. Although
the bristle is slightly curved, our technique of
keeping in focus its two distal ends proved, by
comparing these measurements with those made
for stems of bristles that remained intact (21), to
be highly consistent. Size differences between
wings and bristles had no effect on the relative
degree of the measurement error because both
were enlarged to about the same size before their
image was digitized.
We calculated standardized values of FA of bi-
lateral organs as the absolute value of left minus
right, divided by their mean size. We standard-
ized sizes by dividing each of them by the trait's
mean size. This shifted the population mean of
each trait to unity, enabling comparisons of distri-
butions and standard deviations of traits of differ-
ent sizes. For statistical analyses we used JMP,
Version 3.1.5 for the PC (by SAS Institute, Inc.).
RESULTS
Larval density had a significant effect on all
measured sizes (Fig. 3) but one (bristle width).
The strongest effect was on thorax length, which
we therefore use as our best indicator of overall
ability to gain and use resources. Head and tho-
rax widths gave lower Chi-square values (Kruskal-
Wallis tests) than that of thorax length, and we
omit them from the analyses. For most purposes
we were interested in looking, in individuals
taken from the same gene pool, at the effect of
variations in trait size on FA, regardless of the
source for these variations. This roughly repre-
sents a state where individuals of different, usu-
ally unknown developmental history, are collected
Florida Entomologist 85(1)
3 E
10 20 40 0
density
240
205 -
215
210
200- t---- ---- ------ --
10 20 40 BO
density
3.4 I1
31 -
3. -
3.0-
10 20 40 MS
density
0a -
t ,
10 20 40 80
.. I ,,
iO *
density
2 *
0 2M 4n 10
density
Fig. 3. The effect of larval density on adult size. Each
figure shows all densities' mean (vertical line across), and
the mean and standard error of each density. Distances
between the horizontal ticks represent the relative sam-
ple size of each density. Statistical results (Kruskal-Wal-
lis tests) from top down-thorax length: 02[ = 26.88, P <
0.001; wing width: 02[ = 26.74, P < 0.001; wing length: 0O
= 25.85, P < 0.001; bristle width: 0[, = 3.30, P = 0.35; bris-
tle length: O,,, = 13.63, P = 0.0035.
at random in nature. Unless stated otherwise, we
therefore used pooled data.
We found tight relationships between traits' FA
and size distributions (Fig. 4). FA of bristle width
and length both showed significant negative corre-
lations with their corresponding size (Table 1).
They also showed a highly skewed distribution to
the left, and high standard deviation. In contrast,
FA of wing length showed no correlation with size,
and FA of wing width showed a parabolic, U-shape
correlation with size (y = 2.021 3.966x + 1.955x2;
F2,7 = 7.83, P < 0.001). Wing length was also the
only measure with a distribution that was not sta-
tistically different from normality. In accordance
with their much larger standard deviations, the
average FA of the bristle's width and length was
about an order of magnitude larger than that of
the wing's width and length.
In contrast with the tight association between
FA and size of characters, the relationships be-
tween FA and the best quality indicator, thorax
length, were weak, and fluctuating asymmetries
of the bristle's parameters, both width and length,
were much less affected by thorax length than by
the corresponding trait size (Table 1). Similarly,
FA of the wing width, which produced a signifi-
cant quadratic (U-shape) relation to wing width
(previous paragraph), showed no such pattern
when correlated against thorax length (y = 0.653
- 1.303x + 0.662x2; F2,7 = 0.702, P = 0.5). We also
estimated least asymmetric size (LAS) of bristle's
width and length by using moving averages: we
sorted the data by size of the corresponding trait
(Fig. 5, top) and grouped data points in tenths ac-
cording to their ranked size. This resulted in
groups of ten individuals, from small to large,
ranked as 1-10, 2-11, 3-12 and so on till the end of
the list. For each group we computed average FA
and estimated LAS as the average size of the least
asymmetric group. The average FA of the esti-
mated LAS was significantly lower than the aver-
age FA of the ten points of maximum bristle
width, but not in bristle length. The moving aver-
ages technique also exposed, once again, the tight
relationships between FA and trait size. When we
repeated the same procedure by grouping sizes by
thorax length rather than by bristles' width and
length (Fig. 5, bottom), the continuous trend
found in the first two figures (Fig. 5, top), was lost.
Furthermore, if symmetry develops in response
to whole-organism homeostasis, and the latter is
affected by the individual's (residual) quality, then
symmetry should be a good indicator of quality.
Because larval density affected phenotypic qual-
ity (as indicated by its effect on adult fly size), this
enabled another test of the relationships between
FA and quality. We found no effect of larval den-
sity on FA (bristle length: 02 3 = 2.769, P = 0.42;
bristle width: 02 3 = 1.607, P = 0.66; wing width:
02 3 = 3.1247, P = 0.373; wing length: 02 [ = 1.359,
P = 0.72; Kruskal-Wallis tests).
March 2002
0.8 -
Hasson & Rossler: Fluctuating Asymmetry and Developmental Homeostasis
1.3
1.2
1.1
0.9
0.8
0.7
o4 0.6
0.5
0.4
0.3
0.2
0.1
0.0
13
1.2
0.0
L.D
0.9
0,8
0.7
0.6
0.5
04
0.3
0.2
0 1
. *
.3 .4 .5 .6 .7 .1 .9 1.0 1.1 1.2 1.3 1.4
standardized wing length
I a I -i
3 .4 .5 .6 .7 .9 .9 1.0 1.1 1.2 1 3 1 4
standardized wing width
.3 .4
U "i i . i
I I i I me i il l
.5 .6 7 .9 1.0 11 1.2 1.3 1.4
standardized bristle length
Fig. 4. The relationships between size frequency distributions and FA. Distributions are represented by quantile
plots: each box shows the median as a vertical line across the middle, and the quartiles (25th and 75th percentiles)
as its ends. The means diamond identifies the mean of the sample and the 95% confidence interval about the mean.
Ticks represent 90%, 97.5%, 99.5% and maximum size from the box to the right, and 10%, 2.5%, 0.5% and minimum
size, to the left.
Finally, only bristle's width and length measure-
ments showed a positive correlation between their
FA's (N = 104, r, = 0.2918, P = 0.0027; Spearman
rank correlation). All other pairs of measurements
showed no correlation between their FA values
(giving -0.124 < r > 0.089, and 0.31
0.77).
DISCUSSION
Our study indicates that character-specific ho-
meostasis plays a major role in determining the
degree of FA in the medfly. This, however, should
not undermine the role of whole-organism homeo-
stasis, supported by studies that show within in-
dividual correlations in FA of different characters
(Soule & Cuzin-Roudy 1992, Watson & Thornhill
1994). The current study, and the fact that some of
these previous studies support the whole-organ-
ism homeostasis, may suggests that both may be
important, perhaps under different conditions, or
for different organisms. Some sources of instabil-
ity, for example, such as the effect of certain chem-
icals, or of extreme temperatures (Soule 1982,
Parsons 1989) may directly affect instability of de-
velopment of all traits, irrespective of their sizes,
resulting in a true whole-organism effect on de-
velopmental homeostasis. However, then the
source of instability affects some characteristics
more than others, a greater tendency toward
character-specific homeostasis should be de-
tected. Alatalo et al. (1988) showed that sexually
dimorphic characters have larger size variations
than other characters. They did not look at the
shapes of the size frequency distributions, but the
-
.3 .4 .5 .6 .7 .8 .9 1.0 1.1 1.2 1.3 14
standardized bristle width
Florida Entomologist 85(1)
March 2002
TABLE 1. ASSOCIATION OF BRISTLE WIDTH AND LENGTH'S FA WITH BRISTLE CORRESPONDING SIZE AND WITH THORAX
LENGTH (USING SPEARMAN'S RANK CORRELATIONS).
N r Probability
Bristle width's FA x bristle width 104 -0.4060 <0.0001
Bristle length's FA x bristle length 104 -0.5023 <0.0001
Bristle width's FA x thorax length 103 -0.2447 0.0127
Bristle length's FA x thorax length 103 -0.1120 0.2599
large size variations of sexually dimorphic char-
acters may make their FA more vulnerable to
character-specific effects (size variations) than to
whole-organism homeostasis.
In the study that we present here, larval densi-
ties affected bristles' length, but did not produce
0.4
0.35 *
0.3 I .
0.25 Ap
0.2 *?
I .
0.15
0.1
0.05 t .\
0
0 50
ranked groups by bristle width
corresponding changes in their FA. This result cor-
responds with other studies that show low herita-
bility for FA, relative to that of quantitative
morphological traits' sizes (Parsons 1990). This
suggests that in our study other sources of varia-
tion in FA were stronger than the stress created by
0.3
0.25
0.2
0.15
"o
4 0.1
4 0.05
0
ranked groups by bristle length
0
* S
go
1*
0
- C
'0
- errSiS
F
be
0
ernie ~
Vt fle
~v7*
a
ranked groups by thorax length
ranked groups by thorax length
Fig. 5. The relationships between moving averages of FA and groups ranked by sizes. Each closed circle repre-
sents its minimal rank point (horizontal axis) and the group's average FA. For bristle width, minimum average FA
is 0.0355, representing the rank order 81-90. Its corresponding average standard size is 0.224 mm. The minimum
average FA is significantly lower than that of the top ten sizes (ranked 95-104), 0.0760 (one-tail t-test for unequal
variances, t1,, = -2.35; P = 0.018). For bristle length, minimum average FA is 0.0242, rank order, 80-89 (N = 104),
and corresponding average standardized size, 0.755 mm. This minimum average is not statistically different from
the average FA of the top ten sizes (ranked 95-104), 0.0349 (one-tail t-test for equal variances, tl8 = -0.80; P = 0.216).
0.25
0.2
0.15
0.1
4., ie
..',
0.18
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02 -
,
---
Hasson & Rossler: Fluctuating Asymmetry and Developmental Homeostasis
density alone. Here, variations in FA appear to be
the consequence of the sensitivity of character-spe-
cific developmental homeostasis to size frequency
distributions. The high variance of sizes of a trait
(e.g., the highly skewed bristle length) should
weaken selection on modifiers that form stability of
each particular size. This increases the frequency
of modifiers for sizes that are at some distance from
LAS. Consequently, phenotypic changes in average
size in response to density should have a relatively
weak effect on average FA. Size-dependent charac-
ter-specific homeostasis should, therefore, weaken
the response of FA to environmental variables, es-
pecially when these variables induce changes in
size that are well within the natural range of vari-
ations of the trait size (also considering fluctua-
tions in time). Hence, FA of a trait should generally
be less sensitive to stress than the trait size itself.
This only strengthens Whitlock's (1996) argument
for the low heritability of FA.
FA has been assumed to be a measure of over-
all quality in a variety of studies of sexual selec-
tion (Watson & Thornhill 1994). However, if it is
instead strongly influenced by character-specific
modifications and size frequency distributions,
then FA should not be regarded as a direct mea-
sure of overall quality but, at best, as its approxi-
mation. When size frequency distribution of a
sexual handicap is skewed then, despite the gen-
eral correlation between symmetry and quality,
the very best individuals (whose sexual charac-
ters are largest) should develop, on average, a
greater asymmetry than males with somewhat
smaller handicap (Figs. 1 and 5). In other words,
LAS should not correspond with trait size of best
individuals. Thus, although symmetry is gener-
ally informative and correlated with quality,
within a certain range of sizes it becomes non-in-
formative, maybe even misleading criterion for
quality (depending on the degree of female selec-
tivity, and on the nature of the trait's size fre-
quency distribution).
Our results show that we need to re-evaluate
the current use of FA. For example, the assump-
tion that FA is determined by whole-organism ho-
meostasis, underlies the suggestion that a
negative correlation between FA and male orna-
ment size indicate handicaps, and a lack of it
points at Fisherian ornaments, which are not in-
formative about any of the male "qualities" except
for attractiveness to females (Moller & Pomi-
ankowski 1993c). If, however, symmetry of sexu-
ally dimorphic characters is dominated by
character-specific homeostasis, differences in cor-
relations between traits' FA and size may only re-
flect different size frequency distributions. The
correlation between characters' FA and size
would nevertheless differentiate between handi-
caps and Fisherian sexual traits only if handicaps
consistently show highly skewed distributions
and Fisherian sexual traits do not. Hence, in or-
der to make this argument, it is essential to study
also the frequency distribution of trait sizes.
The possibility that FA is less sensitive to en-
vironmental stress than traits' size poses another
important question: why do female swallows, for
example, use both tail symmetry and tail length
as criteria of the male quality (Moller 1992)? This
should only make sense in an adaptive manner if
each provides a certain different additive compo-
nent of information regarding the male quality
(Hasson 2000). The character-specific homeosta-
sis can provide an intriguing answer: while a well
developed handicap advertises a male's superior
phenotypic quality, the bilateral symmetry of a
handicap may indicate, by presenting modifiers
for its particular size, that the male is a descen-
dant of a long line of similarly good phenotypes.
Hence, while a handicap improves information
about the male phenotypic quality, the handicap's
symmetry indicates the probability that this phe-
notype is a product of "good genes".
ACKNOWLEDGMENTS
We thank Ruti Akavia and especially Golan Abend
for their dedicated technical assistance. We also thank
Phil Taylor for his many detailed comments on the
manuscript. This study was supported by a grant from
the I.A.E.A., Vienna.
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Florida Entomologist 85(1)
Hunt et al.: SFO Bristles and Mating in the Medfly
THE EFFECT OF SUPRA-FRONTO-ORBITAL (SFO) BRISTLE REMOVAL
ON MALE MATING SUCCESS IN THE MEDITERRANEAN FRUIT FLY
(DIPTERA: TEPHRITIDAE)
M. K. HUNT, E. A. Roux, R. J. WOOD AND A. S. GILBURN
School of Biological Sciences, University of Manchester, Stopford building 2.205, Oxford Road
Manchester, M13 9PT, United Kingdom
ABSTRACT
Here we present the findings of a laboratory study in which male Mediterranean fruit flies
(medflies), Ceratitis capitata, (Wiedemann) had one or both supra-fronto-orbital (SFO) bris-
tles artificially removed, for comparison with unoperated controls. All the flies were weighed
and had their wings measured. The time at which a male began pheromone-calling was cor-
related with its weight, lighter males beginning calling earlier, but there was no effect of
weight on mating success. Mated males had significantly longer wings than unmated males
although there was no correlation with wing width. Although males missing both bristles
were rejected more by females than those with one or two bristles, the loss of a single bristle
had no effect on female response. The presence of bristles was not essential for successful
mating. This study does not support the idea of females visually assessing males on the basis
of their bristle symmetry.
Key Words: Ceratitis capitata, female choice, fluctuating asymmetry, medfly, sexual selection
RESUME
A continuaci6n se presentan los descubrimientos de un studio de laboratorio en el cual ma-
chos de la mosca del Mediterraneo, Ceratitis capitata (Wiedemann) presentaban una o am-
bas setas supra-fronto-orbitales (SFO) artificialmente removidas, para comparaci6n con los
controls no modificados. Todas las moscas fueron pesadas y sus alas fueron medidas. El
tiempo en el cual un macho inici6 su llamado a trav6s de una feromona se correlacion6 con
su peso, los machos mas livianos iniciaron su llamado mas temprano, pero no hubo ningun
efecto por parte del peso en el suceso de apareamiento. Los machos apareados presentaban
alas significativamente mas largas que los machos no apareados, sin embargo, no se pre-
sent6 ninguna correlaci6n con el ancho de las alas. Aunque los machos que carecian de am-
bas setas fueron mas rechazados por las hembras en comparaci6n con aquellos que poseian
una o las dos setas, la perdida de una sola de las setas no produjo ningun efecto en la res-
puesta de las hembras. La presencia de las setas no fue esencial para un apareamiento exi-
toso. Este studio no apoya la idea de que las hembras analizan visualmente a los machos
en base a la simetria de sus setas.
Mediterranean fruit flies (medflies), Ceratitis
capitata, are sexually dimorphic, with males pos-
sessing features that are either less exaggerated
in the female or absent. These include more
brightly colored eyes, front legs with more numer-
ous and longer hairs, a brighter white 'chin' or la-
brum (which in the female is a duller cream
color), and a pair of supra-fronto-orbital (SFO)
bristles which are elongated and modified to form
a spatula shape at the tip (F6ron 1962).
The medfly has a lek mating system (Prokopy &
Hendrichs 1979, Arita & Kaneshiro 1985) in which
males gather beneath neighboring leaves on a fruit
tree and release a pheromone to attract females.
They then perform a courtship ritual which involves
more pheromone calling, wing fanning and wing
buzzing, and head rocking (F6ron 1962) before the
male jumps onto the female and attempts to mate.
The female may reject the male at any stage up to
and including the jump, simply by dropping away
from the leaf. Male mating success varies greatly,
with a small proportion of the males gaining most of
the matings (Arita & Kaneshiro 1985, Whittier et
al. 1992, 1994). It has been estimated that in the
wild over 93% of all courtships end in rejection by
the female (Whittier et al. 1994). Given these facts,
there is considerable scope for both male competi-
tion and female choice to be taking place.
Hunt et al. (1998) carried out laboratory stud-
ies to investigate a possible role for the SFO bris-
tles in the mating success of the male. Groups of
50 males and 50 females were allowed two hours
to form pairs. Symmetrical males had higher
mating success than asymmetrical males. The ex-
periments were repeated in field cages in Guate-
mala and Crete using wild flies, and again
Florida Entomologist 85(1)
symmetrical males were found to be more suc-
cessful (Hunt et al. in prep.).
When wild males emerging from oranges in
Crete were housed in wooden (rather than plexi-
glass) cages they were sometimes observed to
have lost one or both of their bristles. It was found
that males missing both bristles were less likely
to be accepted by females than males with one or
two bristles (Hunt et al. in prep.).
Interestingly there was no significant reduc-
tion observed in the female acceptance rate of
males when only one bristle was missing. It
seemed therefore that this extreme form of asym-
metry had no effect on a male's mating success, a
conclusion which rested on the assumption that
males that had lost a single bristle formed a ran-
dom sample of all fitness types. It remained pos-
sible, however, that less fit males may have been
more prone to accidental bristle loss. Here we re-
port a repeat of the experiment under laboratory
conditions, during which bristles were removed
surgically from a random sample of flies.
MATERIALS AND METHODS
A sample of flies collected from coffee beans in
Guatemala and sent directly to Manchester as
pupae, formed the basis of the laboratory colony
used in the present investigation. This colony had
been under laboratory conditions for approxi-
mately 50 generations and was reared according
to the techniques described by Hunt et al. (1998).
Virgin adults were collected in the laboratory
within 24 hours of closing. For both rearing and
mating tests, the flies were maintained at 25 + 2C
and 68 4% rh. The sexes were kept separately un-
til the adults were seven days old. The day before
an experiment, a group of nine flies was transferred
by aspirator and held immobile in a mosquito net
bag (15 cm x 30 cm) and marked individually with
a dot of paint on the thorax. While still in the bag
the flies were placed under a binocular dissecting
microscope and both SFO bristles were removed
from three of the flies with a fine pair of curved for-
ceps. Three others had one bristle removed and the
remaining three were manipulated in a similar
manner, although both bristles were left intact. The
flies were then returned to their rearing cage.
For the mate trials we simulated the natural
conditions of field cage trials by developing a
smaller indoor version (50 cm height x 40 cm
diameter) containing a small potted orange tree
measuring approximately 45 cm in height. The
cage was made of fine black mesh so the flies and
their marks were easily visible though it. The ex-
periments were begun 30 minutes after the lights
were switched on. No food and water was pro-
vided during the test due to its short duration.
On the morning of the seventh day the nine
males were released into the cage and allowed fif-
teen minutes to acclimatize before nine marked
females were released. All interactions between
the flies were noted including the time at which
the males began pheromone calling, the time at
which each courtship attempt took place and the
outcome. Each experiment lasted for two hours
and was replicated 20 times. The small number of
flies being observed allowed accurate recordings
to be made using paper and pencil. For this pur-
pose event tables were used.
At the end of each experiment, the flies were re-
moved from the cage and immobilized in the
freezer for one minute before they were weighed,
after which they were preserved in 70% ethanol.
Both wings were dissected and fixed onto micro-
scope slides under a cover-slip using glycerol gela-
tin (Sigma Diagnostics, St. Louis, USA). The length
and width of the wings were measured under a bin-
ocular microscope using a graticule eyepiece.
Statistical Analysis
Mating success was defined as whether a male
mated or not within the two hours of the experi-
ment. The mean acceptance rate of a male by fe-
males was calculated by dividing the number of its
successful copulations by the number of its court-
ship attempts (matings plus rejections). This index
was designed to take into account the amount of'ef-
fort' a male had to expend before being accepted or
not by a female. It therefore excluded those males
that made no courtship attempt. T-tests were used
to determine if there was a difference in the lengths
and widths of wings in the mated and unmated
males, and between males that attempted phero-
mone calling or courtship and those that did not.
Tests of association between wing dimensions and
acceptance rates were calculated using Spearman's
rank correlation. The association between the num-
ber of bristles and male mating success, pheromone
calling behavior and courtship attempts was calcu-
lated using Chi-squared tests. Associations be-
tween the number of bristles possessed by a male
and its mating success, the time at which it began
pheromone calling and the time to acceptance were
determined using Kruskal-Wallis tests. The associ-
ations between time to pheromone calling and time
to acceptance with weight were determined using
Spearman's rank correlations. Logistic regression
was used to determine if there was a difference in
the weight of mated and unmated males. ANOVA
was used to test for any association between the
number of bristles, male weight and male wing di-
mensions. All statistical tests were carried out us-
ing the statistical package SPSS.
RESULTS
Out of a total of 180 male flies used in the exper-
iments, 31 died during the trial or could not be
used, leaving a final total of 149 males, out of which
86 (57.7%) mated. In the 20 replicates, the percent-
age of males mating ranged from 25 to 100%.
March 2002
Hunt et al.: SFO Bristles and Mating in the Medfly
There was no difference in weight between
males with 0, 1 or 2 bristles (F2,146 = 1.294, P =
0.277). Nor was there any difference in mean
wing length (F2,146 = 0.048, P = 0.953) or mean
wing width (F2146 = 0.234, P = 0.788). The mean
wing dimensions and weight in the different bris-
tle categories are listed in Table 1.
Male SFO Bristles
The numbers of males that mated in each bris-
tle category are shown in Table 2. There was no
association overall between mating success and
the number of bristles (X2 = 1.601, df = 2, P =
0.449). Comparisons between 0, 1 and 2 bristles
also showed no significant differences (0,1 X2 =
0.344, df = 1, P = 0.558; 0,2 2 = 1.593, df = 1, P =
0.207; 1,2 X2 = 0.334, df = 1, P = 0.563).
There was no overall association between ac-
ceptance rate and number of bristles (Kruskal-
Wallis X2 = 5.64, df = 2, P = 0.06), although this
value was close to significance. However signifi-
cant differences were observed between the ac-
ceptance rate of males with no bristles and males
with either one bristle (Mann-Whitney U = 345.5,
P = 0.039) or two bristles (Mann-Whitney U =
446.0, P = 0.045) (see Fig. 1 and Table 1). No sig-
nificant difference was found in the acceptance
rate of flies with one versus two bristles (Mann-
Whitney U = 629.0, P = 0.883).
The number of bristles possessed by a male
was not associated with whether it pheromone
called or not (X2 = 1.029, df = 2, P = 0.598) or
whether or not it initiated a courtship (x2 = 0.757,
df = 2, P = 0.685).
There was no significant difference in the time
taken to begin pheromone calling between flies
with 0, 1 or 2 bristles (Kruskal-Wallis X2 = 0.328,
df = 2, P = 0.849), nor was there a significant dif-
ference in time taken to begin copulation
(Kruskal-Wallis X2 = 0.077, df = 2, P = 0.962). See
Table 1 for mean values in each bristle category.
Male Body Weight and Wing Dimensions
There was a significant positive correlation be-
tween wing length and width (rs = 0.859, N = 149,
P < 0.001), wing length and body weight (r, =
0.651, N = 149, P < 0.001), and wing width and
body weight (r, = 0.657, N = 149, P < 0.001).
Mated males had significantly longer wings
than unmated males (X2 = 7.251, df= 1,P = 0.007)
(Fig. 2), although there was no difference in wing
width (X2 = 2.415, df = 1, P = 0.120).
No association was found between male mat-
ing success and male body weight (x2 = 1.185, df=
1, P = 0.276).
Neither mean wing length (R = 0.099, N = 149,
P = 0.321) nor mean wing width (R = 0.001, N =
149, P = 0.995) were associated with acceptance
rate.
The weight of a male was not associated with
its acceptance rate (R = -0.004, N = 102, P =
0.967).
Neither wing length nor wing width were asso-
ciated with whether or not a male engaged in
pheromone calling (length: t = 1.045, df = 147,P =
0.294, width: t = 0.306, df = 147, P = 0.760). There
was also no difference in wing width between
those males which initiated a courtship and those
that did not (t = 1.665, df = 147, P = 0.098), but
males which initiated a courtship had signifi-
cantly longer wings than those which did not (t =
2.663, df = 147, P = 0.009).
The weight of a male was not associated with
whether it pheromone called or not (t = 1.472, df
= 147, P = 0.143) or whether or not it initiated a
courtship (t = 0.262, df= 147, P = 0.793).
There was no significant association between
time to begin pheromone calling and either wing
length (R = 0.02, N = P = 0.835) or wing width (R
= 0.045, N = P = 0.637). However there was a cor-
relation with weight, the lighter males beginning
pheromone calling earlier (R = 0.261, N = 111,
P = 0.006), although the weight of a male was not
correlated with the time it took to begin copula-
tion (R = -0.079, N = 149, 86, P = 0.489).
DISCUSSION
Males with no supra-fronto-orbital bristles
were less readily accepted by females than males
with either one or two intact. This suggests that
the SFO bristles play some role in either the abil-
ity of the male to perform an adequate courtship,
or in the decision of a female to accept a male as
TABLE 1. THE NUMBERS (AND PERCENTAGES) OF MALES IN EACH BRISTLE CATEGORY THAT MATED AND THOSE THAT DID
NOT.
0 bristles 1 bristle 2 bristles
Pheromone called 35 (77.8%) 34 (69.4%) 42 (76.4%)
No pheromone calling 10 (22.2%) 15 (30.6%) 13 (23.6%)
Attempted courtship 30 (66.7%) 32 (65.3%) 40 (72.7%)
No attempt 15 (33.3%) 17 (34.7%) 15 (27.3%)
Mated 23 (51.1%) 28 (57.1%) 35 (63.6%)
Unmated 22 (48.9%) 21(42.9%) 20 (36.4%)
Florida Entomologist 85(1)
March 2002
TABLE 2. MEAN WEIGHT, WING LENGTH AND WIDTH, TIME TO PHEROMONE CALL (PC) AND ACCEPTANCE RATE OF MALES
IN EACH BRISTLE CATEGORY.
0 bristles (se) 1 bristle (se) 2 bristles (se)
Mean weight (mg) 6.956 (0.172) 6.584 (0.154) 6.764 (0.152)
Mean wing length (mm) 4.322 (0.025) 4.327 (0.023) 4.332 (0.020)
Mean wing width (mm) 2.302 (0.011) 2.291 (0.013) 2.296 (0.010)
Mean time to pc secss) 1105.086 (262.167) 1420.353 (370.162) 1088.214 (276.478)
Mean acceptance rate 0.558 (0.075) 0.763 (0.064) 0.748 (0.059)
a mate. However under the experimental condi-
tions reported here, the influence of the bristles
was not strong enough to affect a male's final mat-
ing success.
Several theories have been proposed to explain
the role of the SFO bristles in the medfly. Arita &
Kaneshiro (1985) suggest that the most impor-
tant determinant of male mating success is its
ability to successfully direct a pheromone towards
the female during courtship, and the SFO bristles
may be playing a role in this (Kaneshiro cited in
Hunt et al. 1998). However Briceno et al. (1996)
and Mendez et al. (1998) note that during the
wing buzzing and head-rocking phase, when bris-
tles are in movement, the male is no longer emit-
ting pheromones.
Briceno et al. (1996) and Mendez et al. (1998)
believe it is more likely that bristles function as
display devices due to the fact that the stalk and
spatulate regions are different colors, the stalk
being clear and the spatulate end black. However
there seems to be geographical variation in this
feature since many male bristles in Hawaii are all
black (D.O. McInnis, USDA/ARS, Honolulu, un-
published data).
It has been suggested that fluctuating asym-
metry (FA) is an important component of sexually
selected characters (Moller 1990), reflecting the
quality of a male, and that males with low levels
0.9 I
0.8
0.7
t 0.6-
0.4
0.3
0 bristles
T T
1 bristle
I
2 bristles
Fig. 1. The effect of male bristle number on accep-
tance rate.
of FA are more successful than their more asym-
metrical rivals. Hunt et al. (1998) found that lab-
oratory mated male medflies with bristles that
were symmetrical in their length had a higher
mating success than males with asymmetrical
bristles, a result later confirmed in the wild in tow
separate locations, in Guatemala and Crete
(Hunt et al. in prep.). However it was impossible
to tell from these studies whether symmetrical
males were being actively selected by females or
whether such males were simply better or more
assiduous in their courtship.
Our field studies on the effect of accidental loss
of one or both bristles (to be published later) sug-
gested that female choice on the basis of visual
symmetry could not be supported. Acceptance
rates of males with one or two bristles present
were significantly higher than those of males with
no bristles. The present laboratory study also in-
dicates that the loss of one bristle has no adverse
effect on acceptance rate. Males missing one bris-
tle are absolutely asymmetrical and yet they have
the same acceptance rate as males with two bris-
tles intact. The fact that these results could be
replicated in the laboratory discounts the possibil-
ity that in the wild flies, only the less fit males
were losing bristles. The present experiments also
show that the actual removal of the bristles in the
laboratory does not adversely affect the males
4.4 .
4.36 .
4.32
4.24
4.2 1
Mated
Unmated
Fig. 2. The mean wing lengths of mated versus un-
mated males.
Hunt et al.: SFO Bristles and Mating in the Medfly
mating behavior, and that the relatively poor ac-
ceptance rate of males with no bristles was due to
their absence rather than the process by which
they were removed or any resulting trauma.
The question still remains about how the bris-
tles are actually functioning. Accepting that their
effect is visual, a recent suggestion is that they
create a 'halo' effect above the male's head as they
move from side to side during head-rocking (0.
Hasson & P. Taylor, pers. comm.). If this is so, it
might explain why males with one bristle are as
readily accepted by females as males with two
bristles, since one bristle should still be able to
create the 'halo' effect above the males head.
At a different level it is possible that the bris-
tles could be functioning as a species recognition
signal, as discussed by Hunt et al. (1998). To in-
vestigate this theory it would be necessary to
study the other species of Ceratitis in which the
males possess SFO bristles.
In summary our work on SFO bristles presents
a complex picture. Although we have demon-
strated that symmetrical males have higher mat-
ing success, whether in the laboratory (Hunt et al.
1998) or in the field (Hunt et al. in prep.), the
present study does not support the conclusion
that this occurs as a result of female mate choice
on the basis of visual symmetry. This agrees with
the suggestion of Mendez et al. (1998) that sym-
metry in bristle length is unlikely to be acting as
an indicator of male quality to the female, be-
cause the position of the bristles on the male's
head makes it difficult for the female to assess ei-
ther their size or symmetry with any degree of ac-
curacy. Despite this, we found that the presence of
one bristle, although making the male absolutely
asymmetrical, is better than having no bristles at
all. We can therefore conclude from these experi-
ments that the SFO bristles are indeed important
in encouraging acceptance by females, although
the reason remains unclear.
We have previously demonstrated in the Gua-
temalan field cage studies that males with wider
wings had a higher mating success (Hunt et al. in
prep.). In the current study, we did not find this
effect, but instead found that males with longer
wings were more successful, and were also more
likely to initiate a courtship. These results have
since been shown to be repeatable in a second lab-
oratory study (M. K. H., unpublished data). Fur-
thermore, in the Guatemalan field study, male
mating success was close to being significantly
positively associated with wing length. It is clear
that the wings are important in male mating suc-
cess. The importance of wings in determining
mating success is not surprising as they are used
in both olfactory and auditory stimulation in the
courtship sequence. A particular shape or size of
wing may be better than others at performing
these functions although wing morphology will
obviously be constrained by natural selection on
flying ability as well as its function in courtship.
The present studies indicate that the optimum
wing design for courtship may differ between
samples or between laboratory and field cages.
The reason for this variation is unclear.
Investigations of male body size made by Arita
& Kaneshiro (1988) and Whittier et al. (1992, 1994
& 1995) led to the conclusion that size has no in-
fluence on male mating success. Arita &
Kaneshiro (1988) found that smaller males from
coffee were preferred over larger males from
cherry in some mate trials, but they concluded
that 'characters other than body size are essential
determinants of mating success of the males'. In
contrast, both Churchill-Stanland (1986) and
Orozco & Lopez (1990) found that large laboratory
males had a greater mating success than small
males, although Orozco & Lopez (1990) found that
size was less important in wild strains. In our
work in field cages (Hunt et al. in prep) we found
that male body size may be important to some de-
gree, since smaller (lighter) males begin phero-
mone calling earlier. In the current study we have
demonstrated that this also occurs in laboratory
experiments, suggesting that it may be evidence of
an alternative mating strategy by the males. Per-
haps smaller males need more time to achieve the
same degree of mating success. Dunn et al. (1999)
found that smaller males of several seaweed fly
species were more willing to mount a female than
larger males and suggested that this may be be-
cause smaller males are more active, or because
they develop faster than large males and thus
have first access to females. They also suggested
that larger males may be longer lived and there-
fore have a longer time in which to gain access to
females. Several studies have shown a positive as-
sociation between male size and longevity, for ex-
ample Butlin & Day (1985) studying seaweed flies,
and Banks & Thompson (1985) studying damself-
lies. An association between size and longevity in
medflies has not been reported, although Sivinski
(1993) found it in domestic Anastrepha ludens, a
species ofTephritid related to the medfly.
Other investigations into male body size have
focused on the nutritional status of the male. Blay
& Yuval (1997) found that protein-deprived males
are smaller and have lower mating success then
the protein-fed males which mate earlier and
have a higher probability of mating. Yuval et al.
(1998) found that males of all sizes (measured by
wing length) participate in leks but that lekking
males tend to be heavier and they explain this as
a result of these males having greater nutritional
reserves. These findings cannot explain the re-
sults from our current study in which smaller
males pheromone call earlier. Their small size
cannot be explained by any nutritional deficien-
cies. If this were the case we would expect them to
begin calling later rather than earlier. However,
Blay & Yuval (1997) conclude that male medfly
size has no effect on the overall reproductive suc-
cess of the female, which suggests that size may
not be an important criterion for female choice.
ACKNOWLEDGMENTS
The authors would like to thank Charlie Nicholls for
help on earlier drafts of this paper, and two anonymous
referees for helpful comments. This research was
funded by a BBSRC special studentship (to MKH) and
an Erasmus grant (to EAR).
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Florida Entomologist 85(1)
Jang: Physiological Control of Female Olfactory Behavior
PHYSIOLOGY OF MATING BEHAVIOR IN MEDITERRANEAN FRUIT FLY
(DIPTERA: TEPHRITIDAE): CHEMORECEPTION AND MALE ACCESSORY
GLAND FLUIDS IN FEMALE POST-MATING BEHAVIOR
E. B. JANG
USDA-ARS, Pacific Basin Agricultural Research Center, P.O. Box 4459, Hilo, Hawaii 96720, USA
ABSTRACT
Studies on behavior of tephritid fruit flies have historically focused on the interaction of ex-
ternal stimuli such as temperature, semiochemicals, seasonality, etc., or the interactions of
flies between and among species for an number of observed behaviors such as mating, pher-
omone calling and oviposition. While descriptive behaviors represent much of what we know
about these pest species, less is known about the underlying physiological mechanisms
which function in priming or modulation of the observed behaviors. In the Mediterranean
fruit fly, virgin females are preferentially attracted to the volatile male pheromone over host
fruit odors. This behavior switches as a result of mating. Factors from the male accessory
gland have been shown to facilitate the switch suggesting a male role in modulation of fe-
male olfactory-driven behaviors. Other physiological factors are likely to further influence
the degree to which female behaviors are influenced.
Key Words: medfly, Ceratitis capitata, mating behavior, pheromone, host odors, accessory
glands
RESUME
Los studios en cuanto al comportamiento de las moscas tefritidas de la fruta se han enfo-
cado hist6ricamente en la interacci6n del estimulo externo tal como temperature, semio-qui-
micos, estacionalidad, etc., o en las interacciones de las moscas, entire y dentro de las
species, para un numero de comportamientos observados tales como apareamiento, lla-
mado por feromonas y oviposicion. Mientras comportamientos descriptivos representan la
mayor parte de lo que sabemos de estas species de plagas, menos se conoce de los mecanis-
mos fisiol6gicos subyacentes cuya funci6n es de preparar o modular los comportamientos ob-
servados. En la mosca del Mediterraneo, las hembras virgenes son preferentemente atraidas
a la volatil feromona masculina por encima de los olores de fruta de su hospedero. Este com-
portamiento cambia como resultado del apareamiento. Factores provenientes de la glandula
accesoria masculina han demostrado facilitar este cambio sugiriendo el papel del macho en
la modulaci6n de los comportamientos olfato conductores femeninos. Otros factors fisiol6gi-
cos son probables que influyan mas adelante en el grado en que se influye sobre el compor-
tamiento femenino.
Much of our current knowledge on mating be-
havior of the Mediterranean fruit fly, Ceratitis
capitata (medfly) has historically focused on the
role of external stimuli such as temperature,
pheromones, time of day, seasonality, etc., or the
interactions of flies with their physical surround-
ings (host trees, canopy size, lek sites, etc). De-
scriptive behaviors represent a sizable portion of
what we know about this pest. Less is known
about the underlying physiological mechanisms
which function in priming or modulation of pre
and post mating behaviors. For us to fully under-
stand mating behavior in medfly we must have
knowledge of the multiple and often complex in-
ternal factors which are involved, and the path /
mechanisms by which external stimuli results in
observed behaviors. The physiological basis of be-
havior is a vastly understudied research area
which could provide important information on
how peripheral receptors receive environmental
cues, the transduction and coding of information
centrally (from these receptors to the brain) and
how behavior is regulated biochemically. The in-
tegration of physiology to help explain behavior is
central to the goal of understanding the mecha-
nisms of mating behavior and improving the ster-
ile insect technique (SIT) in this species. In this
report I would like to review some of the work in
our laboratory looking at the link between mating
in female medfly and chemoreception.
Chemoreception and Physiological State in
Mediterranean Fruit Fly
We have been studying the mechanisms of
chemoreception and its link to behavior in the
medfly in such areas as olfaction, feeding, mating
and oviposition. Our approach is based on the hy-
pothesis that tephritid behavior is influenced by
olfactory, gustatory, visual and tactile informa-
Florida Entomologist 85(1)
tion inputs as primers of behavior and physiology
which internally modulates behavior. Behavior-
ally, tephritids are often classified within a con-
tinuum from monophagus to polyphagus, and
from "highly chemoreceptive" to "mostly visual"
(Jang & Light 1996). However what is known
about the underlying receptor systems which ac-
company these difference in behavior? Do highly
chemoreceptive species have different systems
than the non chemoreceptive species for detecting
semiochemicals? Do differences exist peripherally
(the receptor) or more centrally (the brain)?
In earlier studies on female medfly, we focused
our research on the influence of plant volatiles on
chemoreception and male pheromone on attrac-
tion of females. Both sexes exhibited sensitivity to
a range of C6-C8 aldehydes and alcohols but did
not show sex specific selectivity (Light et al.
1988). Cosse et al. (1995) identified difference in
antennal sensitivity to mango volatiles. We also
identified over 50 compounds produced by calling
male medflies and compared electroantennogram
responses of males and females to the identified
chemicals (Jang et al. 1989). Although we found
some differences in sensitivity and selectivity be-
tween male and female antennae, female sensi-
tive compounds were not attractive when assayed
individually in the wind tunnel. A blend of the
five most prevalent identified chemicals were
found to be attractive to females (Jang et al.
1994). While conducting assays of female attrac-
tion to male pheromone and/or host fruit volatiles
we noted that virgin females behaved differently
than mated females which triggered our interest
in the effects of physiological state on behavior.
Physiological state is an important concept
which, when applied to behavior, helps to explain
some of the differences observed in response of
medfly to standard stimuli. Physiological state
can be the result of multiple effectorss" which re-
sult in generalized states which we commonly
identify as age, mating status, nutritional history,
or more specific molecules such as accessory
gland fluids, sex peptides, hormones, etc. which
may be involved either directly or indirectly in a
specific behavior. These specific molecules repre-
sent an understudied area of research due to their
secondary recognition as real modulators of be-
havior. Over the last several years we have seen
increasing recognition of the influence of physio-
logical state on behavior which has heightened
our awareness of its importance to behavior.
Male Accessory Gland Fluid and Female Post Mating
Behavior
In many adult tephritids, semiochemicals
serve important roles in life history (Jang & Light
1996; Light & Jang 1996). Specific behaviors such
as feeding, attraction to pheromone, and oviposi-
tion are all probably influenced by semiochemi-
cals such as food odors, pheromones, and host
fruit odors. Our current research in Hawaii has
focused on the physiological factors which are re-
sponsible for olfactory-driven female behavior.
Virgin female medflies are attracted to and prefer
male pheromone over host fruit odors. However,
mating triggers profound physiological (and be-
havioral) changes resulting in a switch in prefer-
ence by females to host-fruit odors (Jang 1995).
Other physiological and behavioral effects such
as inhibition of remating (Nakagawa et al. 1971,
Delrio & Cavalloro 1979) have also been reported.
When females were injected (abdominally) with
extracts of accessory gland fluids from males,
they switched their behaviors like their naturally
mated counterparts (Table 1). Namely, a switch
from preference to male produced pheromone to
host fruit (guava) odors. Females injected with
saline only did not switch their behavior and con-
tinued to be attracted to the pheromone over the
host fruit odors. In addition to changes in the
TABLE 1. RESPONSE OF FEMALE MEDITERRANEAN FRUIT FLIES TO MALE-PRODUCED PHEROMONE AND GUAVA HOST
ODOR.
AGF injected Mated Unmated Saline injected
a. Time on Sphere
Pheromone 4203 + 786** 2061 + 312** 8143 + 1287** 9039 + 1208**
Guava 14022 + 2220 18174 + 1520 3832 + 1520 3766 + 645
b. Landings
Pheromone 6.8 + 1.9** 4.6 + 0.7** 12.8 + 2.4** 12.6 + 2.1**
Guava 16.2 + 1.7 17.2 + 1.2 5.8 + 1 5.6 + 1.2
c. Eggs
Pheromone 0 0* 0 0** 0+0 0+0
Guava 43 13 163 18 6.2 4.6 8.1 5.6
**Significance at P < 0.01 level.
*Significance at P < 0.05 level.
AGF = Accessory gland fluid.
March 2002
Jang: Physiological Control of Female Olfactory Behavior
TABLE 2. RESPONSE OF VIRGIN FEMALE MEDITERRANEAN FRUIT FLIES TO MALE-PRODUCED PHEROMONE AND GUAVA
HOST ODOR.
LRN LRS Wild
a. Time on Sphere
Pheromone 11026 + 3275** 2779 + 574 1706 + 555*
Guava 3859 + 2116 2148 + 704 259 + 152
b. Landings
Pheromone 11.94 + 0.93** 6.83 + 1.47 3.2 + 0.66**
Guava 5.56 + 0.78 4.17 + 1.01 0.6 + 24
c. Eggs
Pheromone 0 0** 0 0 0 0
Guava 20.88 3.5 0 0 0 0
**Significance at P < 0.01 level.
*Significance at P < 0.05 level.
LRN = laboratory reared normal.
LRS = laboratory reared sterile.
olfactory-stimulated switch in attraction from fruit (guava) odor. Both laboratory-reared normal
pheromone to host fruit odors, females which and wild virgin females preferred male odor over
were naturally mated or injected with accessory guava odor (Table 2) (Jang et al. 1998). Labora-
gland fluid, laid significantly more eggs than non- tory reared normal and wild females mated to
mated or saline injected controls. Females did not normal males switched their behavior and pre-
exhibit the switch in behavior immediately after ferred the guava odor over male pheromone. Both
mating suggesting that secondary mechanisms laboratory-reared and wild females also switched
may be involved in the regulation of the behav- their behavior when mated to sterile males (Table
ioral switch. 3). In both of these tests, sterile females did not
exhibit a significant switch in behavior. These
Sterile Males and Female Postmating Behavior studies concluded that irradiated males were
equally adept at altering female behavior as non-
If factors from male accessory glands are im- irradiated males (Jang et al. 1998).
portant in control of female driven olfactory be-
havior, what, if any, is the effect of irradiation on Field Cage Studies on Female Post Mating Behavior
the ability of sterile males to switch the behavior
of wild-type females? To test this question we set Laboratory-reared and wild female medflies
up a series of laboratory flight tunnel assays in were assayed in outdoor field cages to assess the
which laboratory normal, irradiated and wild- impact of the mating-induced behavioral switch
type females were mated to conspecific males and on mating behavior and oviposition activity. Lab-
observed for their response to pheromone or host- oratory and wild type virgin females mated more
Table 3. Response of laboratory normal, laboratory sterile and wild females mated to sterile males.
LRN LRS Wild
a. Time on Sphere
Pheromone 4556 + 531** 260 + 162** 270 + 140**
Guava 11368+ 825 1856+ 445 2821+ 612
b. Landings
Pheromone 6.07 + 0.67** 0.5 + 0.27** 0.67 + 0.14**
Guava 14.2 + 0.88 3.25 + 0.59 3.83 + 0.46
c. Eggs
Pheromone 89 + 14** 0 + 0 0.17 + 0.17
Guava 189 + 23 0 + 0 8.33 + 4
**Significance at P < 0.01 level.
LRN = laboratory reared normal.
LRS = laboratory reared sterile.
Florida Entomologist 85(1)
March 2002
with males on leaves and branches than females
which had previously mated with either labora-
tory normal or irradiated males (Fig. 1) (Jang
et al. in press). More of the mated females could
be found alighting and ovipositing in artificial
spheres emitting guava odor (or authentic apples)
hung in host guava trees (Figs. 2 and 3). Females
mated with either normal or sterile males exhib-
ited the behavioral switch which we had seen in
earlier laboratory studies (increased landings,
time on sphere and oviposition/eggs laid). Some
quantitative differences where observed between
responses of laboratory reared and wild females.
Both wild and laboratory reared mated females
laid significantly more eggs in artificial spheres
emitting guava odor than virgin females (Fig. 4).
Effects of Anti-JH Compounds and Chemosterilants
on Behavior
In medfly, anti-juvenile hormone compounds
such as precocene and the chemosterilant benzyl
1,3- benzodioxole have been shown to effect syn-
thesis and release of JH from the corpora allata
(Chang et al. 1994). This interference in JH pro-
duction has been shown to affect sex attractancy
of male medflies (Chang & Hsu 1982) as well as
ovarian development in females (Hsu et al. 1989).
Preliminary studies with females treated with
these compounds suggest that JH may also be in-
volved in modulation of olfactory behavior.
CONCLUSION
Chemoreception, transduction, age, mating
status and nutritional state all play important
roles in regulating behavior. Semiochemicals
aoy
Laboratory
Tiu
Wild
normal
irradiated
Fig. 2. Cumulative fly counts on spheres of labora-
tory and wild virgin and mated female Mediterranean
fruit flies.
serve as behavioral primers while hormonal ac-
tivities and cellular homeostasis are further
"downstream" in the regulatory process but no
less important. In our example we have shown
that mating and transfer of accessory gland fluid
had a direct impact on female medfly olfactory be-
havior which probably works through biochemi-
cal intermediates and possibly hormones. The
specific mechanisms which drive female behavior
await further research and promise to be complex
but exciting to uncover. Irradiation used in steril-
izing mass-reared males for sterile insect release
control programs does not appear to affect the ac-
"m 20;
16
I10.
5-.
Laboratory
S virgin
I C1 J normal
irradiated i
Fig. 1. Mating behavior of virgin an
tory and wild Mediterranean fruit flies
35
30
S25
20
Laboratory Wild
virgin
m normal
wild
Fig. 3. Cumulative ovipositions on spheres by virgin
d mated labora- and mated laboratory and wild female Mediterranean
in a field cage. fruit flies.
Jang: Physiological Control of Female Olfactory Behavior
,I
Laboratory
Wild
virgin
i normal !
i rradiated I
Fig. 4. Number of eggs deposited/h by virgin and
mated laboratory and wild female Mediterranean fruit
flies.
cessory gland fluid which switches behavior. Im-
provements in our knowledge of these processes
and their control will be the key to development of
strategies which target behavioral processes
against these pests.
REFERENCES CITED
CHANG, F., AND C. L. HSU. 1982. Effects of precocene II
on sex attractancy in the Mediterranean fruit fly, Cer-
atitis capitata. Ann. Entomol. Soc. Am. 75: 38-42.
CHANG, F., E. B. JANG, C. L. HSU, M. MA, AND L. JURD.
1994. Benzodioxole-1,3-benzodioxole derivatives and
their effects on the reproductive physiology of in-
sects. Arch. Insect Biochem. Physiol. 27: 39-51.
COSSE, A. A., J. L. TODD, J. G. MILLER, L. A. MARTINEZ,
AND T. C. BAKER. 1995. Electroantennographic and
coupled gac chromatographic-electroantennographic
response of the Mediterranean fruit fly, Ceratitis
capitata to male produced volatiles and mango odor.
J. Chem. Ecol. 21: 1823-1835.
DELRIO, G., AND R. CAVALLORO. 1979. Influenza
dell'acoppiamento sulla la recettivita sessuale e
sull'ovideposizione in femmine de Ceratitis capitata.
Entomol. Bari 15: 127-143.
HSU, C. L., F. CHANG, H. F. MOWER, L. J. GROVES, AND
L. JURD. 1989. Effect of orally administered 5 ethoxy-
6-[-4-methoxyphenyl] methyl-1,3-benzodioxole on
reproduction of the Mediterranean fruit fly. J. Econ.
Entomol. 82: 1046-1050.
JANG, E. B., D. M. LIGHT, R. A. FLATH, J. T. NAGATA,
AND T. R. MON. 1989. Electroantennogram re-
sponses of the Mediterranean fruit fly, Ceratitis cap-
itata to identified volatile constituents from calling
males. Entomol. Exp. Appl. 50: 7-19.
JANG, E. B. 1991. Olfactory stimulated behavior of fruit
flies to the odor of ripening papaya. Proc. 26th An-
nual Papaya Industry Association Conference (1990)
pp. 21-24.
JANG, E. B., D. M. LIGHT, R. G. BINDER, R. A. FLATH,
AND L. A. CARVALHO. 1994. Attraction of female
Mediterranean fruit flies to the five major compo-
nents of male-produced pheromone in a laboratory
flight tunnel. J. Chem Ecol. 20: 9-20.
JANG, E. B. 1995. Effects of mating and accessory gland
injections on olfactory-mediated behavior in female
Mediterranean fruit fly, Ceratitis capitata. J. Insect
Physiol. 41: 705-710.
JANG, E. B, AND D. M. LIGHT. 1996. Olfactory semio-
chemicals of tephritids, pp. 73-90. In B. McPheron
and G. Steck [eds.], Economic Fruit Flies: A world
assessment of their biology and management. Pro-
ceeding of the Fourth International Symposium on
Fruit Flies of Economic Importance, Sand Key Flor-
ida. 586 pp. St. Lucie Press. Delray Beach FL.
JANG, E. B., D. 0. MCINNIS, D. R. LANCE, AND L. A. CAR-
VALHO. 1998. Mating induced changes in olfactory-
mediated behavior of laboratory-reared normal,
sterile and wild female Mediterranean fruit flies
(Diptera: Tephritidae) mated to conspecific males.
Ann. Entomol. Soc. Am. 91: 139-144.
JANG, E. B., D. 0. MCINNIS, R. KURASHIMA, AND L. A.
CARVALHO. 1999. Behavioral switch of female Medi-
terranean fruit fly, Ceratitis capitata: Mating and
oviposition activity in outdoor field cages in Hawaii.
Agric. and Forest Entomol. 1: 179-184.
LIGHT, D. M., E. B. JANG, AND J. C. DICKENS. 1988. Elec-
troantennogram responses of the Mediterranean
fruit fly, Ceratitis capitata to a spectrum of plant vol-
atiles. J. Chem. Ecol. 14: 159-180.
LIGHT, D. M., AND E. B. JANG. 1996. Plant volatiles
evoke and modulate tephritid behavior, pp. 123-133.
In B. McPheron and G. Steck [eds.], Economic Fruit
Flies: A world assessment of their biology and man-
agement. Proceeding of the Fourth International
Symposium on Fruit Flies of Economic Importance,
Sand Key, Florida. St. Lucie Press. Delray Beach, FL.
586 pp.
NAKAGAWA, S., G. J. FARIAS, D. SUDA, R. T. CUNNING-
HAM, AND D. L. CHAMBERS. 1971. Reproduction of
the Mediterranean fruit fly: frequency of mating in
the laboratory. J. Econ. Entomol. 64: 949-950.
Florida Entomologist 85(1)
March 2002
EFFECT OF AGE ON THE MATING PROPENSITY OF THE
MEDITERRANEAN FRUIT FLY (DIPTERA: TEPHRITIDAE)
P. LIEDO, E. DE LEON, M. I. BARRIOS, J. F. VALLE-MORA AND G. IBARRA
El Colegio de la Frontera Sur, Apdo. postal 36, Carrt. Antiguo Aeropuerto, 30700, Tapachula, Chiapas, Mexico
ABSTRACT
The effect of age on the mating propensity of both wild and laboratory-reared Mediterranean
fruit flies, Ceratitis capitata (Wiedemann) was investigated under laboratory and field cage
conditions. The optimal age for wild flies ranged from 7 to 13 days, whereas in laboratory-
reared flies it was between 3 and 5 days old. Virgin flies were selective and more prone to
mate than flies that were held with both sexes combined and therefore, had a chance to mate
before the test. The difference among ages in laboratory-reared flies was significant only in
virgin flies. Virgin females showed a tendency to increase their mating propensity as they
got older, whereas virgin males showed a bimodal pattern, with peaks at 4 and 11 days old.
When flies of both strains and different ages were combined, laboratory-reared females ac-
counted for 72% of the all the matings and most matings were by 4-day-old females. Wild
males accounted for 67% of all the matings and the maximum number of matings were by
10-day-old males. For quality control purpose, flies should be virgin and at their optimal age,
this will produce more robust data for statistical analysis. For control purpose, it is recom-
mended to release sterile flies at 1-2 days old, because flies in the field will be at their max-
imum mating propensity. Our results support the concept that releasing males only will
make the Sterile Insect Technique more effective, since sterile males will be virgin and there-
fore, more prone to mate.
Key Words: medfly, Ceratitis capitata, Sterile Insect Technique, quality control, mating be-
havior
RESUME
Se investig6 el efecto de la edad en la propensi6n al apareamiento de moscas del Mediterra-
neo Ceratitis capitata (Wiedemann), silvestres y de cria masiva, en condiciones de laborato-
rio y jaula de campo. La edad 6ptima para las moscas silvestres estuvo entire los 7 y los 13
dias de edad, para las moscas de laboratorio estuvo entire los 3 y los 5 dias. Las moscas vir-
genes fueron mas selectivas y mas propensas a aparearse que aquellas que se mantuvieron
mezcladas antes de la prueba. Las diferencias entire edades solamente fueron significativas
en moscas virgenes de laboratorio. Las hembras virgenes mostraron una tendencia a aumen-
tar su propensi6n al apareamiento conforme aumentaba su edad, mientras que los machos
mostraron un patron bimodal, con picos a los 4 y 11 dias de edad. Cuando se combinaron am-
bas cepas y diferentes edades, las hembras de cria masiva realizaron el 72% de todos los apa-
reamientos y la mayoria fue por hembras de 4 dias. Los machos silvestres realizaron el 67%
de los apareamientos y el maximo fue por machos de 10 dias de edad. Para fines de control
de calidad, las moscas deben ser virgenes y en su edad 6ptima, esto producird datos mas ro-
bustos para los andlisis estadisticos. Para fines de control, se recomienda liberar las moscas
est6riles cuando tienen 1-2 dias de edad, asi las moscas estaran en el campo cuando sean
mas propensas a aparearse. Nuestros resultados apoyan el concept de que liberar machos
solamente hard mas efectiva la T6cnica del Insecto Est6ril, ya que los machos seran virgenes
y por lo tanto mas propensos a aparearse.
The successful application of the Sterile Insect
Technique (SIT) to suppress the Mediterranean
fruit fly Ceratitis capitata (Wied.), as well as new
developments to improve this technique, have
motivated a wider use of SIT worldwide (Hen-
drichs et al. 1995). As the demand for more envi-
ronmental friendly control methods increases, it
is likely that the use of the SIT will expand. For
wider and more efficient applications, new devel-
opments that improve it are required.
As with any other pest control method, it is
generally accepted that a better knowledge and
understanding of the biology, behavior and ecol-
ogy of the target pest will result in improvements
and more effective applications. One factor that is
likely to improve the effectiveness and efficiency
of the SIT is the development of new and better
methods to estimate or evaluate male mating
competitiveness and to determine the factors that
are important for successful mating.
After pioneering work by Prokopy & Hendrichs
(1979), the "Field Cage Test" (Calkins & Webb
1983) has been used as a research and quality con-
trol tool to characterize and understand the mat-
ing behavior of fruit flies and to evaluate the
mating competitiveness of laboratory-reared ster-
Liedo et al.: Effect of Age on Medfly Mating Propensity
ile flies (i.e. Chambers et al. 1983, Hendrichs 1986,
Guerra et al. 1986, Robinson et al. 1986, Orozco &
Lopez 1993, McInnis et al. 1996, Lance et al. 1996,
Cayol et al. 1999, Calcagno et al. 1999, IAEA
1999). However, there is not a detailed protocol on
how the test should be run for quality control pur-
poses, and factors such as density, sex ratio, and
age of the flies vary widely. Fine tuning of these
factors is important to compare results from differ-
ent locations and strains, to standardize quality
control procedures and make them more efficient,
and to determine the conditions that make the test
more sensitive, so it can be used as an early warn-
ing for mass rearing decision making.
The general goal of this research project was to
investigate the effect of age on the mating propen-
sity of both wild and laboratory-reared Mediter-
ranean fruit flies. Our specific goals were to
determine: 1) if mating propensity changes with
age, and 2) if there is an effect of the mating sta-
tus (virgin vs. non-virgin) on the mating propen-
sity of males and females.
MATERIALS AND METHODS
Five different studies were carried out. In all
cases, laboratory-reared flies (L) were obtained as
irradiated pupae from the Moscamed facility in
Metapa, Mexico. Wild flies (W) were obtained as
larvae from infested coffee berries collected in
Southwestern Guatemala. After leaving the fruit,
mature larvae were placed in screened plastic
containers for pupation. For both strains, at eclo-
sion, adults were sorted by sex and placed in plas-
tic cages with food (sugar + yeast hydrolyzate 3:1
ratio) and water. Adult flies were kept virgin be-
fore the tests, except in those cases in which the
effect of the mating status was investigated. In
these cases, in one group males and females were
held together, so they had a chance to mate before
the test (mixed). In the other group, males were
held in one cage and females in another cage, so
they could not mate before the test (virgin).
When different age groups or strains (labora-
tory-reared or wild) were tested in the same cage,
flies were marked with a small spot of water paint
on the thorax the day before the test. A different
color was used for each age group and strain. So
far we have not detected that these color mark-
ings have any effect on the mating performance of
the flies.
Field cage studies were carried out in the stan-
dard 3 m in diameter by 2 m high cages, with a cof-
fee bush inside (Calkins & Webb 1983). In those
cases in which wild flies were used, the tests were
done in a coffee plantation in Southwestern Gua-
temala. When only laboratory-reared flies were
used, the cages were located in the "coffee garden"
of ECOSUR, in Tapachula, Chiapas, Mexico.
During the test, the behavior of the flies was
observed and mating pairs were detected. When a
mating was observed, the pair was collected in a
vial and the following information was recorded:
1) Time in copula. The time at which the mating
was formed was recorded and the vials with the
mating pairs were observed frequently to record
the time at which the copulation was finished; 2)
Site of mating, whether it was on the cage screen
or over the coffee plant. In the case of matings on
the plant, whether they were on the top or bottom
part of the leaf, on a branch or on a fruit; and 3)
Kind of mating, recording the age, and strain
(where applicable) of the male and the female in
each mating, according to the colors used.
Field and laboratory observations were made
from 07:00 to 13:00 h. Temperature conditions
ranged from 22 to 32C, relative humidity from 65
to 90%, and a photoperiod of 12:12 (L:D).
Wild Flies, Mixed Ages (W1)
This was a field cage study with only wild flies.
The ages of the flies were 7, 9, 11 and 13 days old.
This age range was selected based on our previ-
ous unpublished observations and due to the lim-
iting number of flies available. Adult flies were
released in the cages between 07:00 and 08:00 h.
In each cage, 10 males and 10 females of each age
group were released. Flies that could not fly or
died during the observation period were replaced.
The experiment was repeated 10 times (5 cages
per day, two days).
Laboratory-Reared Flies, Mixed Ages (L2)
This was a field cage study similar to the previ-
ous one but with laboratory-reared flies. The effect
of the mating status (virgin vs. mixed) was evalu-
ated by running two sets of tests, one with virgin
flies (L2v) and the other with mixed flies (L2m).
The density in the cage was greater than the
one with wild flies as was the age range tested.
This was done to increase the number of potential
matings during the test and given the greater
availability of flies. Four different age groups of
flies were released in each cage. Twenty five males
and 25 females of each age group, so the total num-
ber of flies per cage was 200 (100 males and 100 fe-
males). The ages of the flies for the first day were:
2, 5, 8, and 11 days old; the second day the ages
were: 3, 6, 9, and 12; and the third day were: 4, 7,
10, and 13. Both mated and dead flies were re-
placed with individuals of the same sex, age and
mating status, so the density, as well as the sex and
age ratios in the cages were constant. Four repli-
cates were done for both virgin and mixed flies.
Laboratory-Reared Flies, Same Age (L3)
This was a field cage study with laboratory-
reared flies in which all the flies in the cage were
the same age (cohort). One hundred pairs (100
males and 100 females) of 2-day-old flies were re-
leased initially in the field cage. Flies were ob-
Florida Entomologist 85(1)
served from 07:00 to 13:00 h every day. Mated
pairs were vial collected and records on time and
location of mating were taken. At 13:00 h all the
flies from the cage were collected and transferred
to the laboratory where they were maintained in
glass cages provided with food and water. The
sexes were sorted out to prevent matings during
the time when flies were not observed (13:00 to
07:00 h). Two different approaches were followed.
In one case, both mated and dead flies were re-
placed with virgin individuals of the same age
and sex, observations were made during 15 con-
secutive days (L3a). In the other case, both mated
and dead flies were not replaced (L3b), so the test
was finished when no more females were avail-
able. Four replicates of each test were done.
Laboratory-Reared Flies, Mixed Ages for Males
and Fixed Age for Females (L4)
This was a laboratory cage study with labora-
tory-reared flies. In a 1.0 x 0.6 x 0.9 m screened
cage with a potted coffee plant inside, 20 males
and 5 females were released. Males were of 4 dif-
ferent age groups (5 males per age group) and fe-
males were of a fixed age, older than the young
males and younger than the old males. The first
day of the test, males were: 2, 5, 8, and 11 days old
and female age was 7 days old. The experiment
was conducted over 3 consecutive days, so the age
range for the males was from 2 to 13 days, and for
the females was from 7 to 9 days. Both mated and
dead flies were replaced with individuals of the
same age and sex. This experiment was carried
out with virgin females and with females that
were exposed to males before the test. Males were
always virgin. Four replicates of each approach
were done.
Wild and Laboratory-Reared Flies, Mixed Ages (WL5)
This was a field cage study. In each cage, 60 lab-
oratory-reared sterile flies (30 males and 30 fe-
males) and 60 wild flies (30 males and 30 females)
were released. Flies were sorted in 3 age groups
and the ages of the flies were the same for both
strains. The ages of the flies for the first day were 2,
5, and 8 days old; for the second day were 3, 6, and
9 days old; the third day were 4, 7, and 10 days old,
the fourth day were 5, 8, and 11 days old, and the
fifth and last day were 6, 9, and 12 days old (range
tested was 2 to 12 days old). All flies were virgin be-
fore the test. Three replicates were done.
RESULTS
Wild Flies, Mixed Ages (W1)
A total of 260 matings were recorded in the 10
replicates. Although differences among ages were
not significant, 7-day-old flies showed the lowest
mean number of matings for both sexes. Males
showed a gradual increase in the number of mat-
ings as they aged. The maximum number of mat-
ings was achieved by 13-day-old males. Females
showed the greatest number of matings at 11
days old (Table 1). The most common combination
was between 13-day-old females with of 7-day-old
males (9.3% of all combinations), but was very
similar to other combinations, such as 11-day-old
females with 11-day-old males (8.8%) and 13-day-
old females with 13-day-old males (8.4%). The
least common combinations were between 7-day-
old males and females (2.6%) and 9-day-old fe-
males with 7-day-old males (3.5%).
Laboratory-reared Flies, Mixed Ages (L2)
There was a significant effect of the mating
status on the mating propensity of both, males
and females (P = 0.0001). Greater number of mat-
ings were recorded from virgin flies than from
mixed flies at all ages, except on 2-day-old fe-
males, where the mean number of matings was
the same (Fig. 1A).
Differences among ages in both, virgin and
mixed females, were not significant (P = 0.057 for
virgin females, and P = 0.497 for mixed females).
However, in both types of females there was a ten-
dency to increase mating propensity from 2 to 4
TABLE 1. MEAN (SE) NUMBER AND PERCENTAGE OF MATINGS, PER SEX AND AGE OF WILD MEDITERRANEAN FRUIT FLIES
UNDER FIELD CAGE CONDITIONS. AGE GROUPS WERE COMBINED AND FLIES WERE VIRGIN (N = 260).
Sex Age Mean Number of matings (SE) Percent
Females' 7 4.8 (0.70) 18.46
9 6.3 (0.77) 24.23
11 8.3 (1.22) 31.54
13 6.7 (0.42) 25.77
Males" 7 5.5(0.40) 21.15
9 6.7 (0.67) 25.77
11 6.5 (0.56) 25.00
13 7.3 (0.42) 28.08
*F = 2.38, D.F. = 3, P = 0.070.
bF = 1.88, D.F. = 3, P = 0.134.
March 2002
Liedo et al.: Effect of Age on Medfly Mating Propensity
A) FEMALES
2 3 4 5 6 7 8 9 IC ii 12 13
Laboratory Cage, Mixed Ages for Males, Fixed Age
for Females (L4)
There was a significant differences among male
ages when females were virgin, but not when
females were mixed. The greatest number of mat-
ings were achieved by 5-day-old males (Table 2).
The total number of matings with mixed females
was 34, whereas in the test with virgin females
was 66. Although this greater mating propensity,
virgin females apparently were more selective
than mixed females, regarding the age of the
males and based on the statistical analysis.
B) MALES
6s
Age
' -vrs1
A -~le
10 12
Fig. 1. Age-specific mean number (SE) of matings by
laboratory-reared virgin flies and flies in which the two
sexes were mixed before the test. A) Females, B) Males.
days. Later, in the case of mixed females, their
mating propensity decreased. In the case of virgin
females, there was a second peak when females
were 8 days old and the third and greatest peak
was recorded when females were 12 days old.
There was a significant difference (F = 2.92,
P = 0.007) among ages in virgin, but not in mixed
males (F = 0.81, P = 0.63). The maximum mean
number of matings was attained by 4-day-old vir-
gin males (Fig. IB). Virgin males showed a bimo-
dal pattern in their mating propensity, increasing
from 2 to 4 days old, then decreased to a mini-
mum at age 9 and peaking again when they were
11 days old. The mean number of matings by
mixed males was never greater than 2 and with-
out any clear pattern associated with age.
Laboratory-reared Flies, Same Age (L3)
There were significant differences among ages
for both replacement (F = 4.39, P = 0.0001) and
non-replacement (F = 10.28, P = 0.0001) tests.
The maximum number of matings was recorded
when flies were 3 days old (Fig. 2). Mating activity
gradually decreased with age. In the non-replace-
ment test, by age 9 all the flies had mated or died.
In this test, over 70% of all matings were by 3 and
4-day-old flies. When mated and dead flies were
replaced, the fraction of matings at these same
ages was only 39.9% of all matings.
Field Cage-Wild and Laboratory-Reared Flies (WL5)
There was a significant difference between labo-
ratory-reared and wild flies, both in the number of
matings achieved and the age for maximum mating
activity. Laboratory-reared females showed greater
mating propensity and the age of maximum mating
activity was earlier in life, compared to wild fe-
males (Fig. 3). These females accounted for 72.0%
of all the matings and the greatest number of mat-
ings were recorded when they were 4 days old.
In the case of males, wild males achieved more
matings (67.1%) than laboratory-reared males
and the greatest number was recorded when they
were 10 days old (Fig. 4). The maximum mean
number of matings was by 4-day-old lab females
with 10-day-old wild males.
Time in Copula
The time in copula was recorded in all field
cage tests. A summary of these data is presented
in Figure 5. There was not a clear pattern or con-
20
S15
10
---Without replacement
-*..-With replacement
2 3 4 5 6 7 8 9 10 11 12 13 14 15
Age
Fig. 2. Age-specific mean number of matings by lab-
oratory-reared flies in tests with and without replace-
ment of mated and dead flies. Same letters at each point
(mean) indicate non significant differences within each
test by the Tukey Multiple Range Test.
Florida Entomologist 85(1)
March 2002
TABLE 2. MEAN (SE) NUMBER AND PERCENTAGE OF MATINGS BY MALES OF DIFFERENT AGES UNDER LABORATORY CON-
DITIONS. FEMALES WERE OF A GIVEN MEDIAN AGE, AND THEY WERE KEPT MIXED WITH MALES OR KEPT VIRGIN
BEFORE THE TEST (N = 34 AND 66 MATINGS FOR MIXED AND VIRGIN, RESPECTIVELY).
Mean (SE) number of matings Percentage of matings
Age Mixed' Virginb Mixed Virgin
2 0 0.25 (0.29) b 0 1.51
3 0.25 (0.29) 0.75 (0.87) ab 2.94 4.54
4 0 2.00 (2.31) ab 0 13.63
5 1.25(1.44) 3.50 (4.04) a 14.70 21.21
6 0.75 (0.87) 1.75 (2.02) ab 8.82 10.61
7 0.75 (1.73) 1.25 (1.44) ab 8.82 7.58
8 1.00 (1.15) 2.00 (2.31) ab 11.76 12.12
9 1.00 (1.15) 1.25 (1.44) ab 11.76 7.58
10 0.50 (0.57) 1.00 (1.15) ab 5.88 4.54
11 1.00 (1.15) 0.75 (0.87) b 11.76 7.57
12 1.00 (1.15) 0.75 (0.87) b 11.76 1.51
13 1.00 (1.15) 1.25 (1.44) b 11.76 7.57
Differences were not significant by the Chi square test, P = 0.218, d.f. = 11). Data were transformed by X for analysis.
'Means followed by the same letter are not significantly different (F = 2.85, P = 0.0086) by the Tukey Multiple Range test. Data were transformed by
/X for analysis.
sistent effect of age on the duration of copula.
However, this parameter was consistently affected
by the strain. Generally, wild flies showed greater
mean time in copula than laboratory-reared flies.
1 .
0.9 0 Laboratory Females with Laboratory Males
0.8 Wild Females with Laboratory Male
S0.7
% 0,7.
S0.5
6 0.3
0.2
0-1
0 n t I I ia rl ,
2 3 4 5 6 7 8 9 10 11 12
Female age
2 .
g 1.6-
S1.4 -
E
o6 1.2.
E
1 0.8.
'5 0.6.
S0.4
0.2
0
[Lu
laboratory Female with Wild Male
*Wild Female with Wild Male
1I n_
2 3 4 5 6 7 8 9 10 11 12
2 3 4 5 6 7 8 9 10 11 12
Female age
Fig. 3. Age-specific mean number of matings by wild
and laboratory-reared females under field cage conditions.
Considering all the tests together, the mean time
in copula for wild and laboratory-reared females
was 125.8 and 103.5 minutes, respectively. For
males, the means were 140.6 and 104.8 minutes
1,2
1
0.8
S0.4.
0.2.
1.8
16-
S1.4.
E 1.2.
E 0.8
06 0,6,
S0.4
0.2.
_
3 Laboratory Males with Wild Females
E Wild Males with Wild Females
4 I1 I1
11 12
I I
11 12
2 3 4 5 6 7 8 9 10
Male age
n Laboratory Males with Laboratory Females
*Wild Males with Laboratory Females
JLYL I I
2 3 4 5 6 7 B 9 10
Male age
Fig. 4. Age-specific mean number of matings by wild
and laboratory-reared males under field cage conditions.
[L A I I j I
'
m
Liedo et al.: Effect of Age on Medfly Mating Propensity
A) FEMALES
2500.
250
_200.
150
| 100.
50.
owl
. L2v
LW5
*L5
Do
0 0
uu u+, -,
0 5 10 15 2(
Age
B) MALES
0.
0 ,
0
0 5 10 15 20
Age
oWl
. L2v
owsl
LS W5
*L5
Fig. 5. Mean time in copula of wild and laboratory-
reared flies as a function of age. W1 represents data
from experiment 1 in which only wild flies were tested.
L2v represents data from virgin laboratory-reared flies
tested in experiment 2. W5 represents data from wild
flies in experiment 5 in which wild and laboratory-
reared flies were tested together. L5 represents data
from laboratory-reared flies in experiment 5.
for wild and laboratory reared, respectively. Copu-
las that last less than one minute were discarded
for the analysis. The maximum time in copula was
308 minutes (13-day-old wild male with 11-day-
old wild female). The mating status of the flies
(virgin or mixed) showed no significant effect on
the duration of copula (F = 1.25,P = 0.25).
Location of Matings
When only wild flies were observed, 52.5% of
the matings took place on the bottom part of the
leaves, 3.5% occurred on the top part of the leaves,
and the rest were observed on the cage screen
(44.0%). When both, wild and laboratory reared
flies were tested, the same pattern was observed,
with 78.3 and 6.3% of the matings located at the
bottom and top part of the leaves, respectively.
The rest (15.4%) occurred on the screen cage.
Regarding orientation, most matings occurred
in the East and Southeast part of the cages
(45.9%), which was the sunny part when most
mating activities took place (08:00-11:00 h).
Mating activity was strongly associated with
temperature conditions. A multiple regression
analysis was done with the number of matings
and the temperature, for the four field cage tests
with laboratory-reared flies (Fig. 6). Mating activ-
ity occurred within a range from 23 to 31C. The
greatest number of matings occurred when the
temperature was 26C.
DISCUSSION
Considering the two tests with wild flies (ex-
periments 1 and 5), we conclude that the age
range for greater mating activity (optimal range)
in wild flies is between 7 and 13 days. The non sig-
nificant difference among ages in wild flies
(Table 1), could be attributed to the range tested
(7-13) that was too small for resolution. However,
in the experiment with wild and laboratory-
reared flies (WL5), we find no matings by 3 and 4-
day-old wild females and 2-day-old wild males
(Figs. 3 and 4). This is consistent to what was re-
ported by other authors following similar meth-
ods, regardless of the geographic origin of the
flies. In Reuni6n Island, Quilici & Franck (1996)
recorded the maximum number of matings by 9-
day-old flies (optimal range: 7-9 days old), the
range they tested was from 3 to 9-day-old flies. In
Argentina, Calcagno et al. (1996), tested a range
from 3 to 17 days old and they recorded the max-
imum number of matings from 13-day-old flies
(optimal range: 11-17 days old). In Greece, Econo-
mopoulos & Mavrikakis (1996) recorded the max-
imum number of matings by 14-day-old flies
(optimal range: 8-14 days old), the range they
tested was 2 to 14-day-old flies. There were two
common features in all these tests, 1) males ma-
ture one or two days earlier than females, and 2)
in most cases, the age with the maximum number
of matings, was the oldest age tested. The only ex-
ception was in Argentina, where also was the only
y = -11 875 i 627.1X B130.4
R, = 0.8202
y -6 4193U + 341 23x 4437.2
R2= 0 7887
y -4.6246)(' 243-1 8 3140.9
R2= 0.8563
y =-2 5638+135.16 1737 2
R' 09076
A.
22 24 26 28
Temperature (C)
* L2m
* L2v
a L3a
x L3b
- Polinrmica (L2v)
. Polinemica (L3a)
Polin6amic3 (L3b)
- -Polihn6mica (L2m)
30 32
Fig. 6. Multiple regression analysis between number
of matings and temperature. L2m and L2v represent
data from experiment 2 with mixed and virgin labora-
tory-reared flies, respectively. L3a and L3b represent
data from laboratory-reared flies used in experiment 3
with and without replacement, respectively.
case where flies older than 13 days old were
tested. We believe that this characteristic might
be due, at least in part, to the fact that flies were
kept virgin before the test (see below).
The optimal age range for laboratory-reared
flies was between 3 and 5 days old. This is consis-
tent with demographic data that shows that
mass-rearing conditions select for early matura-
tion, and flies adapted to this conditions start lay-
ing eggs much earlier than wild flies normally do
(see Liedo & Carey 1996 and references therein).
Another characteristic of laboratory reared
flies, was that females were more prone to mate
than wild females. This was particularly clear in
the test in which wild and laboratory-reared flies
were compared (WL5). Despite that the particular
strain we tested was only two years under mass-
rearing conditions, 72.0% of all matings were by
laboratory-reared females. Again, this is a com-
mon characteristic of mass-reared flies (Calkins
1984, Harris et al. 1986, Hendrichs 1986).
The mating status of the flies showed a strong
effect on their mating propensity. Virgin flies
were more prone to mate, as we were expecting.
This might explain the increase in mating pro-
pensity with age (i.e., this increase could be an ar-
tifact of the experimental design, using virgin
flies) and can be attributed to physiological
changes that happen in the females after mating.
If the females have not mated, they will still be re-
sponding to male signals. However, once mated,
females will be more interested in finding a host
to laid their eggs. The reduced number of matings
by mixed males (L2), could be attributed to the
low mating propensity of the mixed females.
The mating status could also affect female
choice. Virgin females were more selective with
respect to male age than mixed females (Table 2).
We believe there are three important applied
implications of these results. For quality control
purposes, we recommend use of virgin flies at
their optimal age for mating propensity (7 to 13
for wild and 3 to 5 for laboratory-reared flies) in
field cage tests. This will increase selectivity by
females and will increase the mating probability,
producing more robust data for statistical analy-
sis and will make quality control efforts more ef-
ficient. Also, we believe that our laboratory test
(L4) represents an alternative for quality control
programs, particularly when the availability of
wild flies is a limiting factor and/or when field
cage tests represent a risk for fruit fly free zones
or areas under eradication or suppression.
Regarding the release of sterile flies, our results
indicate that releases when sterile flies are 1 or 2
days old, is the optimal condition. This will reduce
the chance of mating before release, and therefore,
these flies will be virgin and with a high propen-
sity to mate. Keeping the flies for longer time be-
fore release would reduce their mating propensity,
could result in high mortality due to crowding con-
March 2002
editions, and will increase unnecessarily program
costs because of the space required.
Finally, our results support the concept that
releasing males only will result in more effective
sterile fly release programs, since this will avoid
matings between sterile flies, and this will result
in males more motivated to court females and to
mate.
ACKNOWLEDGMENTS
We thank A. P. Economopoulos and P. G. Mavrikakis
from the Dept. of Biology, University of Crete, Heraklion,
Crete, Greece; S. Quilici and A. Franck from CIRAD-
FLHOR Reunion, Laboratoire d'Entomologie Station de
Bassin Martin, B.P. 180, 97455-Saint-Pierre Cedex,
France; and G. Calcagno, M. T. Vera, F. M. Norry, J. L.
Cladera, F. Manso and J. C. Vilardi from Lab. Gen6tica
de Poblaciones, Dept. Cs. Biol6gicas, Fac. Ciencias Exac-
tas y Naturales, Universidad de Buenos Aires, 1428
Buenos Aires, Argentina, for sharing their unpublished
data on age specific mating propensity. G. P6rez-
Lachaud, A. B. Davila, A. Oropeza and M. L. Sosa for
technical assistance. Our appreciation to P. Rendon for
his help with the collection of wild flies and setting up
the field cage tests in Guatemala. We acknowledge sup-
port from the "Programa Moscamed" in Guatemala and
Mexico. This project was funded by the International
Atomic Energy Agency (302-D4-MEX-7698) and "El
Colegio de la Frontera Sur".
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Florida Entomologist 85(1)
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