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Caceres et al.: Quality Management Systems for Fruit Fly SIT


QUALITY MANAGEMENT SYSTEMS FOR FRUIT FLY
(DIPTERA: TEPHRITIDAE) STERILE INSECT TECHNIQUE


CARLOS CACERES1, DONALD MCINNIS2, TODD SHELLY3, ERIC JANG4, ALAN ROBINSON1 AND JORGE HENDRICHS5
1Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture
International Atomic Energy Agency, Agency's Laboratories, A-2444 Seibersdorf, Austria

2USDA-ARS USPBARC, 2727 Woodlawn Drive, Honolulu, HI 96720, U.S.A.

3USDA/APHIS/CPHST, 41-650 Ahiki St. Waimanalo, HI 96795, U.S.A.

4USDA-ARS USPBARC, P.O. Box 4459, Hilo, HI 96720, U.S.A.

5Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture
International Atomic Energy Agency, Wagramerstrasse 5. P.O. Box 100, A-1400 Vienna, Austria


ABSTRACT

The papers presented in this issue are focused on developing and validating procedures to
improve the overall quality of sterile fruit flies for use in area-wide integrated pest manage-
ment (AW-IPM) programs with a sterile insect technique (SIT) component. The group was
coordinated and partially funded by the Joint FAO/IAEA Programme of Nuclear Techniques
in Food and Agriculture, International Atomic Energy Agency, Vienna, Austria, under a five-
year Coordinated Research Project (CRP) on "Quality Assurance in Mass-Reared and Re-
leased Fruit Flies for Use in SIT Programmes". Participants in the CRP from 16 countries
came from both basic and applied fields of expertise to ensure that appropriate and relevant
procedures were developed. A variety of studies was undertaken to develop protocols to as-
sess strain compatibility and to improve colonization procedures and strain management.
Specific studies addressed issues related to insect nutrition, irradiation protocols, field dis-
persal and survival, field cage behavior assessments, and enhancement of mating competi-
tiveness. The main objective was to increase the efficiency of operational fruit fly programs
using sterile insects and to reduce their cost. Many of the protocols developed or improved
during the CRP will be incorporated into the international quality control manual for sterile
tephritid fruit flies, standardizing key components of the production, sterilization, ship-
ment, handling, and release of sterile insects.

Key Words: quality control, Tephritidae, fruit flies, behavior, SIT, sterile insects


RESUME

Los articulos presentados en este numero se enfocan en el desarrollo y la validaci6n de pro-
cedimientos para mejorar la calidad total de moscas de las frutas est6riles para su uso en
programs de manejo integrado de plagas en donde la t6cnica del insecto est6ril (TIE) es uno
de los components clave. El grupo fue coordinado y parcialmente financiado por la Divisi6n
Conjunta de T6cnicas Nucleares para la Alimentaci6n y la Agricultura de la FAO/OIEA,
Viena, Austria, por un period de cinco aios bajo el proyecto de Investigaci6n Coordinada
(PIC) sobre "el Aseguramiento de la Calidad de Moscas de las Frutas Criadas y Liberadas
para su Uso en Programas de TIE". Los participants en el PIC representan 16 paises con
experiencia en campos de investigaci6n basica y aplicada. Para asegurar que los procedi-
mientos desarrollados fueran apropiados y pertinentes, se realizaron una variedad de estu-
dios para el desarrollo de protocolos para evaluar la compatibilidad y para mejorar los
procedimientos de colonizaci6n y manejo de cepas salvajes. Estudios especificos trataron
asuntos relacionados con la nutrici6n de insects, los protocolos de irradiaci6n, la dispersion
y supervivencia en el campo, evaluaci6n del comportamiento en jaulas de campo, y el mejo-
ramiento de la competitividad sexual. Los objetivos fundamentals fueron el aumentar la
eficiencia y reducir los costs de los programs operacionales de control de moscas de las fru-
tas donde TIE es utilizada. Muchos de los protocolos desarrollados o mejorados durante el
PIC seran incorporados en el Manual Internacional de Control de Calidad para Moscas Es-
triles de la familiar Tephritidae, para estandarizar components claves como la producci6n,
esterilizaci6n, envi6, manejo y liberaci6n de insects est6riles.







Florida Entomologist 90(1)


The sterile insect technique (SIT) is rapidly be-
coming a major component of many area-wide in-
tegrated pest management (AW-IPM) programs
for fruit fly control (Dyck et al. 2005). For use in
these programs, sterile insects are produced in
large numbers, sterilized, and then released sys-
tematically into the field. In all of these processes,
quality management systems can be used to mon-
itor or improve the overall effectiveness of sterile
insects in the field and to reduce variability in
cases where production fluctuates widely in quan-
tity and quality. The insect production facility
must be a reliable source of high quality sterile
insects either for local use or shipment to pro-
grams elsewhere.
There are generally two sets of conditions that
favor the long-distance or even trans-boundary
shipment of sterile insects (Enkerlin & Quinlan
2004). First, shipment may be necessary to areas
such as California and Florida, which are typi-
cally pest-free (thus precluding the establishment
of a local fly production facility) but have repeated
introductions of fruit flies that demand a preven-
tive area-wide control program (Dowell et al.
2000). Second, shipping sterile insects is econom-
ical for small SIT programs, when the costs of in-
sect production locally would be prohibitively
high (Dowell et al. 2005). In the past, mass rear-
ing facilities were developed with public funds;
however, the private sector has recently shown in-
terest in commercializing sterile insect produc-
tion, e.g., Israel (Bassi 2005) and South Africa
(Barnes et al. this volume). This interest has been
encouraged by the wider use of sterile insects in
suppression programs (Hendrichs et al. 1995),
rather than only in eradication programs.
The International Atomic Energy Agency
(IAEA) and the Food and Agriculture Organiza-
tion of the United Nations (FAO) through their
Joint FAO/IAEA Programme of Nuclear Tech-
niques in Food and Agriculture have played a
leading role in the development and implementa-
tion of SIT technology. One mechanism used to
further this technology is through Coordinated
Research Projects (CRPs) (www.iaea.org/pro-
grammes/ri/uc_infoi.html) under the IAEA Re-
search Contract Programme (IAEA 2006). This is-
sue of Florida Entomologist summarizes the re-
sults of the five-year CRP on "Quality Assurance
in Mass-Reared and Released Fruit Flies for Use
in SIT Programmes" together with several in-
vited papers. The papers in this volume identify
components or steps in the SIT process that can
be changed or improved, such as reducing the
negative impact of mass rearing and sterilization
on the efficiency of sterile insects in the field and
establishing new standards and quality control
protocols for updating and inclusion in the inter-
national manual on "Product Quality Control and
Shipping Procedures for Sterile Mass-Reared
Tephritid Fruit Flies" (FAO/IAEA/USDA 2003).


This introductory paper highlights the rele-
vance of the papers to a quality management sys-
tem for fruit fly production, sterilization, ship-
ment, emergence, feeding, holding, and release,
and identifies gaps and areas for future research.
The papers included in this volume do not repre-
sent the total spectrum of a quality management
system as it relates to fruit fly mass rearing, but
reflect the breadth of subjects that are related to
typical quality management systems as reported
in the CRP

STRAIN COMPATIBILITY

Assessing the mating compatibility of a strain
to be released with its potential target population
remains a critical activity. In the Mediterranean
fruit fly (medfly) Ceratitis capitata (Wiedemann),
mass-reared strains have shown no significant
mating incompatibilities with any of the medfly
populations assessed to date (Cayol et al. 2002;
Lux et al. 2002), except for 2 exceptional island
cases. The first involved the use of a 38-yr old
mass-production strain from Hawaii (McInnis et
al. 1996), and the second involved the use of a tsl
genetic sexing strain (Franz 2005) against a wild
Madeira strain. However, Pereira et al. (2007a)
show in recently conducted field cage assess-
ments a low but acceptable level of compatibility
between the tsl mass-reared genetic sexing
strain, VIENNA 7 (Franz 2005), and wild popula-
tions from different islands in Madeira. Briceio
et al. (2007a) present interesting data that may
explain some of the observed mating incompati-
bilities in Madeira. Wild Madeira males appear to
have significantly longer durations of some court-
ship components (e.g., head rocking and wing
buzzing). Therefore, Pereira et al. (2007a) stress
the need for incorporation of Madeira genetic
background into the genetic sexing strain that is
used for field releases in order to overcome the
low level of compatibility and improve efficiency
in the Madeira SIT program.
For the Mexican fruit fly Anastrepha ludens
(Loew), no mating incompatibilities were observed
between mass-reared flies and populations from 6
different locations in Mexico, where programs
with an SIT component have been established
(Orozco et al. 2007). This finding is very important
because sterile insects are produced in a single
rearing facility in southern Mexico. This facility
supplies all the SIT control programs in Mexico
plus the preventive release program in southern
California. In the case of the South American fruit
flyAnastrepha fraterculus (Wiedemann), previous
studies (Petit-Marty et al. 2004) have shown good
mating compatibility among populations across
Argentina. Allinghi et al. (2007a) report that ster-
ile insects from a candidate strain for mass pro-
duction and SIT releases are reasonably compati-
ble with wild flies from Argentina and southern


March 2007







Caceres et al.: Quality Management Systems for Fruit Fly SIT


Brazil. However, in studies of wild populations of
A. fraterculus from different regions of South
America (Argentina, Brazil, Peru, Colombia),
there was evidence of substantial isolation be-
tween certain populations (Vera et al. 2006).
In a majority of cases, strain compatibility is
not a constraint for the application of the SIT;
e.g., for the Oriental fruit fly Bactrocera dorsalis
(Hendel), no mating incompatibility between the
pupal color genetic sexing strain from Hawaii and
the wild population from Thailand was observed
(McInnis; personal communication), and for
melon fly, Bactrocera cucurbitae (Coquillett) be-
tween mass reared flies from the Okinawa strain
and wild insects from Taiwan (Matsuyama &
Kuba 2004). Compatibility tests, however, remain
an essential component of SIT feasibility studies.

COLONIZATION AND STRAIN MANAGEMENT

One of the main problems faced by producers
of sterile insects is how best to colonize and mass
rear a strain while at the same time minimizing
the impact on the intrinsic characteristics of the
strain. Filter rearing systems for genetic sexing
strains (Fisher & Caceres 2000), or mother colo-
nies for bisexual strains, are a way to solve part of
this problem (Calkins & Parker 2005). In the case
of genetic sexing strains, a filter rearing system is
used for the additional purpose of maintaining
strain genetic stability.
Liedo et al. (2007) show that the use of inserts
to increase the surface resting area within adult
cages in the mother colony, as well as the use of
low adult cage density during colonization and
mass rearing, resulted in strains with increased
mating competitiveness as demonstrated in stan-
dard field cage mating tests.

QUALITY MANAGEMENT SYSTEM

New mass rearing production indices and
quality standards developed during the CRP have
been used to update the international quality con-
trol manual (FAO/IAEA/USDA 2003), contribut-
ing to the development of a new quality manage-
ment system. Such a quality management system
increases end user confidence and facility reliabil-
ity, improves work processes, SIT efficiency, and
integration with other control methods. A quality
management system can be realized through the
adoption of ISO 9000 standards, developed and
published by the International Standardization
Organization (ISO 1996) that defines, estab-
lishes, and maintains an effective quality assur-
ance system for manufacturing and service indus-
tries. The ISO 9000 standard is the most widely
known and has perhaps had the most impact of
the standards published by ISO (ISO 1996). An
organization can receive ISO 9000 certification if
an external audit recognizes successful imple-


mentation and performance of standard produc-
tion protocols.
Leppla & Fisher (1989) suggested the adoption
of industrial quality assurance protocols by mass
rearing facilities, and the medfly mass-rearing
facility in Mendoza, Argentina, became the first
facility to receive ISO 9000 certification in 1999
(G. Taret, personal communication). Barnes et al.
(2007) stress the importance of a quality manage-
ment system in maintaining high quality and pre-
dictable insect production at the Infruitec medfly
mass rearing facility in Stellenbosch, S. Africa. A
quality management system can be extended to
all other steps of the SIT, including packing and
fly emergence and handling at release centers.

STANDARDIZATION OF MASS PRODUCTION
AND QUALITY CONTROL PROCEDURES

Routine Quality Control Procedures

Standard procedures and established thresh-
old values are available and summarized in the
international quality control manual (FAO/IAEA/
USDA 2003) used by most fruit fly production fa-
cilities, which routinely keep records of the qual-
ity of their production. Barnes et al. (2007), pro-
vide a good example of how results from routine
quality control tests can be analyzed and utilized
as a feedback to help facility managers identify
quality problems and stabilize production. The
establishment of demographic and quality control
parameters for mass rearing are required in order
to develop benefit/cost scenarios that can be used
to determine the feasibility of the SIT implemen-
tation in integrated control programs. For A.
fraterculus, significant improvements in the qual-
ity control and mass rearing protocols are re-
ported for all developmental stages (Vera et al.
2007). Similar developments are reported in the
Philippines for Bactrocera philippinensis (Drew
& Hancock) (Resilva et al. 2007).
Hendrichs et al. (2007) describe a simple qual-
ity control test that might be useful to measure
predator evasion in the medfly. They showed that
current adult colony management and mass-rear-
ing procedures significantly reduce the ability of
sterile males to escape predation in comparison to
wild males, resulting in the rapid elimination due
to predation of a large proportion of the released
sterile male population. They stress the impor-
tance of measuring this parameter and propose
that simple quality control tests be developed to
routinely measure the evasive ability of sterile
males of the different mass reared strains.

Pupal Age and Development Synchronization

Synchronization and exposure of pupae to the
irradiation treatment at an optimal age is crucial
to obtain a desirable level of sterility with mini-







Florida Entomologist 90(1)


mal effect on insect quality. Day degree models to
determine the correct physiological age for irradi-
ation of a bisexual medfly strain and its relation-
ship with pupal eye color have been calculated
(Ruhm & Calkins 1981). Based on these earlier
studies, Resilva et al. (2007) present a method to
determine the relationship of pupal eye color with
physiological development of B. philippinensis
held under different temperature regimes. This
method helped determine the optimal stage for
pupal irradiation and is now being extended to
other fruit fly species.
Nestel et al. (2007a) tried to correlate respira-
tory rate in pupae with digital recordings of eye
color. Their study describes the relationship be-
tween a digitized image of pupal eye color and the
respiratory rate of pupae. This may enable irradi-
ation to be performed with greater precision.
However, the practicality of the tests under oper-
ational conditions needs to be evaluated.

Dosimetry and Irradiation Doses

X-rays, electron beam generators, or gamma
irradiators with 60Co or 17Cs can be used to steril-
ize insects. It is important to minimize the so-
matic effects induced by radiation during the
sterilization process (Bakri et al. 2005). A stan-
dard dosimetry system (Gafchromic system) has
been developed and adopted, and a standard op-
erating procedure (SOP) for the implementation
of this system as part of the quality management
system in production facilities has been compiled
(Parker & Mehta 2007; Bakri et al. 2005).
For A. fraterculus (Allinghi et al. 2007b) and
B. philippinensis (Resilva et al. 2007), optimal
doses for sterilization have been determined.
However, in choosing an optimal dose for steril-
ization, a balance needs to be reached between
the levels of sterility and fly competitiveness (To-
ledo et al. 2004). Unfortunately it appears that
many of the current operational programs apply-
ing the SIT are not achieving an appropriate bal-
ance. Parker & Mehta (2007) describe a mathe-
matical model that can be used by these programs
to derive such an optimal dose. The authors stress
that the model still requires extensive validation.

Atmosphere During Irradiation

Insect irradiation in the presence of nitrogen
or low oxygen atmospheres can improve insect
quality (Ashraf et al. 1975; Ohinata et al. 1977;
Fisher 1997). Nestel et al. (2007b) assessed the ef-
fect of irradiation under different decreased oxy-
gen levels, and the results indicated that mating
competitiveness drastically decreases when pu-
pae are irradiated under full 02 conditions, when
the packing bags remained open before irradia-
tion. Hypoxic oxygen concentrations of 2% and
10% in sealed bags at the beginning of irradiation


did not affect the mating competitiveness of
males compared to males irradiated under maxi-
mal hypoxia anoxiaa). Current practices in mass-
rearing facilities are discussed by Nestel et al.
(2007b) in light of these results.

Shipments of Biological Material

Shipments of sterile pupae or fertile eggs from
production facilities are routinely performed (En-
kerlin & Quinlan 2004). A specific insulated pu-
pal shipping container is already in use and de-
scribed in the international quality control man-
ual (FAO/IAEA/USDA 2003). A protocol for long
distance shipment of medfly eggs has been devel-
oped (Caceres et al. 2007; Maman & Caceres
2007) and a long distance shipment protocol for
sterile B. philippinensis pupae is described by
Resilva et al. (2007).

NUTRITION

Recent laboratory studies have led to the de-
velopment of chemically defined larval and adult
diets for the medfly (Chang et al. 2001). Nutri-
tional larval diet manipulations have been dem-
onstrated to affect the physiological traits of
mass-reared medfly males. In this regard, Nestel
et al. (2004) have shown that lipid and protein
levels in pupating medfly larvae were affected
and correlated with routine quality control pa-
rameters and growth factors. Nevertheless, there
is still no clear understanding of how larval diet
affects male competitiveness.
In contrast to larval nutrition, there is now in-
creasing evidence that post-teneral diet can play
an important role in sterile male competitiveness
(Blay & Yuval 1997; Taylor & Yuval 1999; Kaspi &
Yuval 2000; Kaspi et al. 2000; Yuval & Hendrichs
2000; Maor et al. 2004; Niyazi et al. 2004). Yuval et
al. (2007) discuss the effect of post-teneral feeding
of sterile medfly males and, more specifically,
whether the addition of protein to the adult diet
enhances mating success. The main conclusion is
that the addition of protein to the adult diet in-
creases male sexual performance, but in some
cases this can be associated with increased suscep-
tibility to starvation. However, when there is suffi-
cient nutrition available in the field, protein fed
and protein deprived males have equal ability find-
ing nutrients in the field. The authors suggest that
the best strategy could be to release protein fed
sterile males, which, though relatively short lived
if nutrients are not found in the field, are highly
competitive, rather than protein-deprived insects
which may live longer but mate less successfully.

DISPERSAL AND SURVIVAL

Dispersal and survival tests have been devel-
oped and validated, but there is still need for


March 2007







Caceres et al.: Quality Management Systems for Fruit Fly SIT


standardization. The release recapture method
has been used as a quality control test and was
used to evaluate the dispersal ability and survival
of mass reared fruit flies (Shaw et al. 1967; Baker
& Chan 1991). Hernandez et al. (2007) present
valuable information on longevity and dispersal
in the field for mass-rearedA. ludens and A. obli-
qua. They show that a majority of released sterile
males do not survive the first 3 to 4 d after release
in the field, and hence never reach the sexual ma-
turity required to inseminate wild females. This
information is very important for operational pro-
grams releasing sterile insects in order to modify
the adult holding and release procedures, and to
establish the frequency of releases and distance
between release points.
Meats (2007) presented both actual field data
from Bactrocera tryoni (Froggatt) sterile fly re-
leases in Australia and data from simulated mod-
eling studies comparing patterns of fly dispersion
for sterile and wild flies in the field. The more
similar the dispersion patterns, the more effec-
tive will be the sterile fly release, and the lower
will be the expected rate of population increase
for the wild flies. The author provides quantita-
tive estimates of the rate of wild fly increase
based on specific levels of mismatch between ster-
ile and wild fly dispersal patterns. A solution for
large mismatches in dispersal is closer flight
lanes for aerial release, or the use of additional
sterile fly releases in the areas surrounding traps
containing the lowest sterile to wild fly ratios.
G6mez et al. (2007) report data on the longev-
ity of mass-reared irradiated and non-irradiated
A. fraterculus in field cages. Their study showed
that within protected field cages laboratory-
reared flies lived longer than wild flies. Therefore,
they conclude that there was no adverse effect of
irradiation on longevity.

BEHAVIOR ASSESSMENTS

Segura et al. (2007) describe a field cage study
that correlates A. fraterculus male mating suc-
cess with pheromone calling activity, perching lo-
cation within the tree canopy, presence in leks,
and morphological characters. The data showed
that some components of the sexual behavior and
some morphological traits were associated with
mating success. Mating success was higher for
males grouped in a region of the tree character-
ized by the highest light intensity during the first
2 h of the morning. Highest mating success was
for pheromone calling males inside a lek.
Sciurano et al. (2007) investigated the signifi-
cance of morphological differences between suc-
cessful and unsuccessful males ofA. fraterculus in
field cage mating competitiveness tests. Morpho-
metric analyses were used to determine the rela-
tionship between phenotype and copulatory suc-
cess. The authors conclude that no linear associa-


tion existed between expected fitness and mor-
phological traits (wing width and thorax length)
that were suggested as targets of sexual selection.
Nevertheless, they suggest that sexual selection
could be affected by other morphological charac-
ters inA. fraterculus.
Pereira et al. (2007b) compare the mating suc-
cess of mass-reared, sterile medfly males after be-
ing held for different periods of time in outdoor
conditions. However, no positive effect of the
treatment could be demonstrated. Briceno et al.
(2007a) compared the duration of certain court-
ship elements of mass-reared males courting wild
females. Courtship was filmed in the laboratory,
and videotapes were analyzed. The authors re-
port comparative data on male courtship from 4
wild populations of medfly. The results document
differences of behavior between strains based on
the interaction between males and females of the
same strain. No inter-strain mating comparisons
were carried out.

ENHANCEMENT OF COPULATORY SUCCESS

Exposure of sterile male medflies to ginger root
oil or citrus peel oils significantly improves their
mating success (Shelly 2001; Katsoyannos et al.
2004) and also for Oriental fruit flies exposed to
methyl eugenol (Shelly & Nishida 2004). Progress
has been made in large-scale application of aroma-
therapy for medfly and chemotherapy for the Ori-
ental fruit fly. Incorporating ginger root oil into
medfly sterile release programs may increase the
effectiveness of the SIT and allow a reduction in the
number of sterile flies released (Barry et al. 2003).
Briceno et al. (2007b) analyzed courtship be-
havior of medfly males exposed to ginger root oil.
The results show no clear effect on courtship be-
tween treated and untreated males, though a
small sample of wild females did accept aroma-
tized males faster than control males. Thus, fur-
ther studies are needed to identify the compo-
nents that are responsible for the enhanced male
sexual performance of males treated with ginger
root oil.

CONCLUSIONS

Increasing the efficiency of the SIT is of cardi-
nal importance, both to ensure the success of such
operations and to reduce cost. The CRP has iden-
tified components that may improve quality man-
agement systems and quality control protocols for
SIT. While some reports suggest obvious improve-
ments that could be realized from implementa-
tion of changes identified, others will require
more research before they can be fully imple-
mented into a total quality management system
for fruit fly rearing, sterilization, shipment, hold-
ing, and release. It is clear, however, that this
project has succeeded in bringing together an in-







Florida Entomologist 90(1)


ternational group of researchers to take a critical
look at what factors will contribute to improve
mass rearing and handling of sterile flies for im-
proved SIT implementation. In addition, the CRP
fostered networking and collaboration among the
community of basic and applied fruit fly investi-
gators.
The major outcome of this research network
can be summarized as follows:
promotion and implementation of quality
management systems and quality control pro-
tocols
promotion and implementation of coloniza-
tion techniques to enhance and maintain
strain quality and genetic stability
promotion and dissemination of the use of
precise standards for measuring sterile male
compatibility and competitiveness under field
conditions
identification of nutritional factors affecting
sterile male performance
formulation of protocols for evaluating lon-
gevity and dispersal of sterile fruit flies and
identification of survival problems of sexually
immature sterile males in the field that need
to be addressed
development and testing of strategies for en-
hancing sterile male performance
identification of optimal doses of irradiation
in relation to mating competitiveness
development of SIT-specific dosimetry proce-
dures to allow precise and comparative mea-
surements of the applied radiation dose
development of a model to optimize radiation
dose, maximizing male competitiveness and
minimizing somatic damage
development of packaging and long-distance
shipping procedures for fertile eggs or sterile
pupae
revision of the international quality control
manual for fruit flies (FAO/IAEA/USDA 2003)
and addition of new quality control protocols.
Furthermore, it is important that operational
programs implementing the SIT continue rou-
tinely revising and updating all protocols to ensure
increased efficiency. Two-way feedback is essential
between the mass rearing facility and field opera-
tions in order to be able to correct errors during all
aspects of the process. Continued interaction be-
tween different SIT programs is recommended to
exchange information and to further standardize
operational procedures for all SIT components.
However, defining new and better methods to mea-
sure and improve the quality of mass reared in-
sects remains an elusive but overarching goal of
any program implementing the SIT.
Finally, it is necessary to point out that this se-
ries of studies did not cover all the aspects of total
quality management for fruit fly rearing, steril-
ization, shipment, holding, and release as it re-
lates to the SIT application. Therefore, is essen-


tial to continue conducting additional R&D in pri-
ority areas (many identified in this issue) to im-
prove overall efficiency of operational SIT
programs and to facilitate the establishment of
new programs for fruit fly species for which the
technology is not available.
Specific priority areas identified for further
R&D are (1) improving strain selection and col-
ony management, especially adult holding condi-
tions and a better implementation of the filter
rearing system, (2) identifying the optimal radia-
tion dose in terms of induced sterility and compet-
itiveness, (3) identifying the optimal stage for ra-
diation related to the development of the repro-
ductive system in both sexes, (4) addressing the
low survival of released mass-reared flies to the
age of sexual maturity, including the use of hor-
mones for accelerating sexual maturation (Teal
2000), and increasing the low predator evasion
capacity, (5) expanding and validating the use of
nutritional, microbiological, and semiochemical
supplements in the general adult diet to enhance
male sexual performance, and (6) evaluating the
effect of pre-release feeding, storage, and chilling
conditions on sterile insect quality.
To support rapid progress in some of the iden-
tified R&D areas, 2 new 5-year FAO/IAEA CRPs
have been initiated, involving scientists from the
world's main fruit fly research centers and major
fruit fly SIT programs. In 2004 a CRP was initi-
ated on "Improving Sterile Male Performance in
Fruit Fly Programmes", and in 2005 a second one
was started on "Development of Mass Rearing for
New World (Anastrepha) and Asian (Bactrocera)
Fruit Fly Pests".

ACKNOWLEDGMENTS

We are thankful to all the colleagues who enthusias-
tically took part in this Coordinated Research Project
and who contributed to reviewing the papers presented
in this volume.

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Florida Entomologist 90(1)


March 2007


SEXUAL PERFORMANCE OF MASS REARED AND WILD MEDITERRANEAN
FRUIT FLIES (DIPTERA: TEPHRITIDAE) FROM VARIOUS ORIGINS
OF THE MADEIRA ISLANDS

RUI PEREIRA, NATALIA SILVA, CELIO QUINTAL, RUBEN ABREU, JORDAN ANDRADE AND LUIS DANTAS
Program Madeira-Med, Estrada Eng. Abel Vieira 262, 9135-260 Camacha, Madeira, Portugal

ABSTRACT

The success of Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) control pro-
grams integrating the sterile insect technique (SIT) is based on the capacity of released the
sterile males to compete in the field for mates. The Islands of Madeira are composed of 2 pop-
ulated islands (Madeira and Porto Santo) where the medfly is present. To evaluate the com-
patibility and sexual performance of sterile flies we conducted a series of field cage tests. At
same time, the process of laboratory domestication was evaluated. 3 wild populations, one
semi-wild strain, and 1 mass reared strain were evaluated: the wild populations of (1) Ma-
deira Island (north coast), (2) Madeira Island (south coast), and (3) Porto Santo Island; (4) the
semi-wild population after 7 to 10 generations of domestication in the laboratory (respectively,
for first and second experiment); and (5) the genetic sexing strain in use at Madeira medfly fa-
cility (VIENNA 7mix2000). Field cage experiments showed that populations of all origins are
mostly compatible. There were no significant differences among wild populations in sexual
competitiveness. Semi-wild and mass-reared males performed significantly poorer in both ex-
periments than wild males in achieving matings with wild females. The study indicates that
there is no significant isolation among strains tested, although mating performance is re-
duced in mass-reared and semi-wild flies after 7 to 10 generations in the laboratory.

Key Words: Ceratitis capitata, sexual success, medfly domestication, medfly origin, propor-
tion of mating, Madeira

RESUME

El exito de los programs de control de la mosca mediterranea de la fruta (Ceratitis capitata
(Wiedemann) que integran la t6cnica del insecto est6ril (TIE) esta basado en la capacidad de
machos esteriles para competir en el campo por sus parejas. Las Islas de Madeira consistent
de 2 islas pobladas (Madeira y Porto Santo) donde la mosca mediterranea de la fruta esta
present. Para evaluar la compatibilidad y el funcionamiento sexual de moscas est6riles no-
sotros realizamos una series de pruebas dejaula en el campo. Al mismo tiempo, el process de
la domesticaci6n en el laboratorio fue evaluado. Tres poblaciones naturales, una poblaci6n
semi-natural y una poblaci6n criada en masa fueron evaluadas: las poblaciones natural de
(1) Isla de Madeira (costa norte), (2) Isla de Madeira (costa sur) y (3) Isla de Porto Santo; (4)
una poblaci6n semi-natural despu6s de 7 a 10 generaciones de domesticaci6n en el laborato-
rio (respectivamente, para el primero y segundo experimento; y (5) la raza para separar
sexos gen6ticamente que es usada en el laboratorio de la mosca mediterranea de Madeira
(VIENNA 7mix2000). Los experiments usando jaulas en el campo mostraron que las pobla-
ciones de diferentes origins fueron en su mayor parte compatibles. No hubo diferencias sig-
nificativas en la capacidad para competir sexualmente entire las poblaciones naturales. Los
machos semi-naturales y los machos criados en masa mostraron un desempeio significati-
vamente bajo en ambos experiments que los machos naturales en el logro de copula con las
hembras naturales. Este studio indica que no hay un aislamiento significativo entire las ra-
zas probadas, aunque el desempeio en el apareamiento fue reducido en las moscas criadas
en masa y semi-naturales despu6s de 7 a 10 generaciones en el laboratorio.


The Madeira Islands are located 980 km WSW dance). The main island, Madeira (740 km2), is
from mainland Portugal (32N and 17W) and are volcanic with very little level land suitable for
composed by two populated islands. Porto Santo large agricultural production. The north coast is
is small (about 50 km2) with topographic (maxi- cooler than the south coast (air temperature at
mum altitude 571 m) and temperature conditions sea level in the north corresponds to air tempera-
favourable to the Mediterranean fruit fly (medfly) ture at 300 m elevation in the south) and the max-
Ceratitis capitata (Wiedemann). However, poor imum elevation is 1881 m. The climate of Madeira
soil and low rainfall (380 mm/year) do not permit Island, particularly below 600 m over sea level on
an abundance of host fruits (the fig Ficus carica the south coast and 400 m on the north coast, is
and Opuntia spp. are exceptions in terms of abun- favourable for the development of large medfly







Pereira et al.: Sexual Success of Medfly from Various Origins


populations. Conditions in Funchal (sea level in
south coast), the capital city, and in low altitude
areas are very favourable for medfly throughout
the year. There are eight generations per year in
the Funchal area (Vieira 1952).
Insects of the same species may behave differ-
ently in different geographical areas depending
on variations in selection pressures (Thornhill &
Alcock 1983). However, recent studies with wild
medflies from different origins around the world
(Argentina, Australia, Crete, Guatemala, Kenya,
Madeira-Portugal, Reunion-France, and South
Africa) show mating compatibility with each
other (Cayol et al. 2002). The same authors tested
wild flies against mass reared genetic sexing
strains (VIENNA 4/Tol-94, VIENNA 7-97, SEIB
6-96, and AUSTRIA 6-96 (Franz 2005)) and no
sexual isolation was found. However, the data
showed that males from some origins perform
better than others, including lower compatibility
of Madeira flies with sexing strains tested.
Due to these findings, and because the Ma-
deira-Med SIT program is currently ongoing in
the Madeira Islands (Pereira et al. 2000), we per-
formed additional studies to investigate in field
cage tests the mating compatibility of locally
mass produced sterile flies. A discrepancy in ster-
ile male sexual performance observed in previous
field cage tests conducted in Madeira (Dantas et
al. 2004) underlined the need for this new study
to confirm if there is some sexual isolation be-
tween sterile flies being released and wild flies
from different origins in the Madeira Islands.
The evident difference in host structure and
the isolation of the 2 islands (50 km apart), plus
the semi-isolation between north and south
coasts of Madeira Island provided a basis for com-
parison of the different populations. We tested the
possibility of any isolation among these 3 medfly
populations Porto Santo Island, north coast of
Madeira, and south coast of Madeira. Addition-
ally, the mating compatibility of these 3 wild pop-
ulations with sterile flies and a semi-wild popula-
tion was studied.

MATERIALS AND METHODS

Five different populations of medfly were stud-
ied: (1) wild flies from south coast of Madeira Is-
land (SC); (2) wild flies from north coast of Ma-
deira Island (NC); (3) wild flies from Porto Santo
Island (PS); (4) semi-wild flies after 7 to 10 gener-
ations in the laboratory (SW); and (5) sterile flies
from Madeira-Med factory (L), strain VIENNA
7mix2000 (Franz 2005).
Two sets of field cage tests were conducted,
each comparing males of 4 of the above 5 popula-
tions. The reason for this was due to wild fly avail-
ability (an incompatibility of fly abundance peak
between wild medfly populations from Porto
Santo Island and the north coast of Madeira Is-


land). In the first set (June and July, 2002) 17 cage
tests were conducted (7 with PS females, 6 with
SC females, and 4 with SW females). In the sec-
ond set (October, 2002), 24 cage tests were carried
out (8 with females from each of NC, SC, and SW).
Wild medflies were collected as pupae from lar-
val survey samples (mixed hosts like peach,
guava, apricot, and others). Semi-wild flies came
from a small laboratory colony reared under low
stress conditions. This colony was established
with flies obtained from the fruit sampling of the
Madeira Medfly program (Pereira 2001), i.e., from
a mixture of hosts from the entire Madeira Is-
land. Pupae of the mass-reared strain were irra-
diated with 100 Gy under hypoxia in a Gamma-
cell 60Co irradiator (Nordion, Canada) 24-48 h be-
fore emergence.
Pupae from all the strains were placed in a
standard quality control plexiglas cage (30 cm x
30 cm x 40 cm) until emergence. In the first 24 h
after emergence, the insects were sexed and fe-
males were kept in separate rooms from males to
avoid contact with the male pheromone before the
tests. In the case of the L (sterile) treatment, only
males were used. All flies were maintained at 24
+ 2C, 65 5% RH and natural light (no artificial
lighting was supplied) in 2-L plastic containers
with water and adult food (1 part hydrolyzed
yeast and 3 parts sugar) ad libitum.
At the time of release into field cages wild and
semi-wild females were 11 to 13 d old, wild and
semi-wild males 9 to 11 d old, and mass reared
sterile males were 5 d old. Healthy flies were se-
lected for the experiments and marked with a dot
of water-based paint on the thorax the day before
the field cage tests.
The field cages were cylindrical, with a flat
floor and ceiling (2.9 m diameter and 2.0 m
height) supported by a PVC frame (Calkins &
Webb 1983). A potted 1.8-m high citrus tree was
placed into each cage to serve as a substrate ter-
ritory for sexual interactions.
The testing period covered the time of maxi-
mum sexual activity of medfly (sunrise to 12:00
h), and was conducted according to FAO/IAEA/
USDA (2003). Male flies were released 30 min be-
fore the females so that they could start forming
leks (Prokopy & Hendrichs 1979). In each cage, 60
males (15 from each of 4 treatments) were re-
leased to compete for 30 wild females.
Temperature, relative humidity, and light in-
tensity in the field cages were recorded every 30
min. The mating pairs were recorded during a
continuous census, and after initiation of mating,
pairs were collected in 20-mL vials. The mating
time, location on the tree, and leaf side were re-
corded. The observers had no prior information
about which kind of males were in the cages.
In the field cage tests we measured the propor-
tion of flies mating (PM) (McInnis et al. 1996),
and a mating by origin index (MOI) that mea-







Florida Entomologist 90(1)


sures the sexual success of males from different
origins. The former index was adapted from the
relative sterility index (RSI) used in SIT opera-
tions to measure the mating success of sterile
males when competing with wild males (McInnis
et al. 1996). The PM measures the suitability of
the flies and the environment for mating. It rep-
resents the overall mating activity of the flies,
both wild and sterile, and is defined as follows:

PM Number of pairs collected
Number of females released

The MOI was defined to measure the matings
achieved by males from a certain origin in rela-
tion to the total matings. The expected index
would be 0.25, since we used cages with 4 kinds of
males evenly distributed (15 from each strain).

MOI = Number of matings of a certain origin male
Total matings

Data were analyzed by analysis of variance
(ANOVA). If differences in means were detected,
a complementary multiple comparisons of means
test (Tukey's honest significant difference test)
was performed (Ott & Longnecker 2001). The sig-
nificance value was 95% (a = 0.05). Statistical
analyses were performed with R software (ver-
sion 2.1.0, www.r-project.org).

RESULTS

Results of the first set of field cage tests show
no significant differences (F= 3.16, df = 2,14; P =
0.0737) between the PM of the 3 strains of fe-
males used (Fig. 1). The PM was always above the
minimum required (0.20) for data analysis (FAO/
IAEA/USDA 2003). The MOI index that measures
the relative male sexual success competing for a
female is presented on Table 1, independently
and pooled together. The data show significantly
lower sexual success for mass-reared sterile
males against all kinds of females in the experi-
ment. Surprisingly, PS males performed signifi-
cantly better when competing for SC females, but


oas
0.7

f 0.5
I04

02
0.1
0


PS


cs
Female ogln


SW


Fig. 1. Proportion of Mediterranean fruit fly females
mating (PM SD) from different origins (PS-Porto
Santo Island, SC-south coast of Madeira Island, and
SW-semi-wild after 7 generations in laboratory). The
data show no significant differences (P = 0.0737).


not when competing for females of their own
strain (even with slightly higher PM) and the SW
females. The overall data show no significant dif-
ferences between the two wild strains (PS and
SC). However, PS males performed significantly
better than SW and L males, while SC males were
only significantly better than L males.
The second set of field cage tests again showed
no significant differences in PM (F = 1.768; df =
2,21; P = 0.1951) among the females tested (SC,
NC, and SW) (Fig. 2). As in the previous experi-
ment, the PM is always above the minimum re-
quired (0.25) for data analysis (FAO/IAEA/USDA
2003). In terms of MOI (Table 2), the data are
slightly different from the first set of experi-
ments. Males from SW and L show a significantly
lower MOI than the other males (NC and SC)
when competing for NC and SW females. The
same is true for the overall data. However, the
MOI data show no significant differences among
the 4 male treatments tested (SC, NC, SW, and L)
when competing for SC females.

DISCUSSIoN

The data obtained in this study show the normal
lower competitiveness of sterilized mass-reared
males, but clearly no significant isolation in terms
of mating compatibility among all the strains of


TABLE 1. MATING BY ORIGIN INDICES (MOI) FOR PORTO SANTO ISLAND (PS), SOUTH COAST OF MADEIRA ISLAND (SC),
SEMI-WILD (SW) AND STERILE MASS-REARED (L) MEDITERRANEAN FRUIT FLY MALES IN THE PRESENCE OF PS,
SC, SW FEMALES, AND POOLED RESULTS. ROWS WITH THE SAME LETTER PRESENT NO SIGNIFICANT DIFFER-
ENCES AMONG MALES OF DIFFERENT ORIGIN (TUKEY'S HONESTLY SIGNIFICANT DIFFERENCE TEST, a = 0.05).

Male origin

Female origin Number of cages PS SC SW L

PS 7 0.37 a 0.29 ab 0.25 b 0.09 c
SC 6 0.42 a 0.26 b 0.23 b 0.09 c
SW 4 0.39 a 0.28 a 0.27 a 0.06 b
Totals 17 0.39 a 0.28 ab 0.24 b 0.09 c


March 2007







Pereira et al.: Sexual Success of Medfly from Various Origins


TABLE 2. MATING BY ORIGIN INDICES (MOI) FOR SOUTH COAST OF MADEIRA ISLAND (SC), NORTH COAST OF MADEIRA
ISLAND (NC), SEMI-WILD (SW) AND STERILE MASS-REARED (L) MEDITERRANEAN FRUIT FLY MALES WHEN IN
PRESENCE OF SC, NC, SW FEMALES, AND POOLED RESULTS. ROWS WITH THE SAME LETTER PRESENT NO SIG-
NIFICANT DIFFERENCES AMONG MALES OF DIFFERENT ORIGIN (TUKEY'S HONESTLY SIGNIFICANT DIFFER-
ENCE TEST, a = 0.05).

Male origin

Female origin Number of cages SC NC SW L

SC 8 0.30 a 0.28 a 0.25 a 0.17 a
NC 8 0.34 a 0.37 a 0.16 b 0.13 b
SW 8 0.34 a 0.34 a 0.19 b 0.13 b
Totals 24 0.33 a 0.32 a 0.20 b 0.15 b


flies tested. These results were expected in accor-
dance with the compatibility studies of Cayol et al.
(2002) for several medfly strains from many re-
gions of the world, including Madeira Island.
Important results were obtained when re-
cently domesticated male medflies were tested in
the field cages. These semi-wild males performed
significantly worse compared to the best wild
male treatment in each of the experiments. How-
ever, the semi-wild males performed better than
sterilized mass-reared males. The phenomenon of
rapid decrease in mating sexual performance
soon after strains of flies are adapted to mass-
rearing conditions is well documented (Econo-
mopoulos 1992; Orozco & L6pez 1993; Cayol
2000). The loss of sexual competitiveness of re-
cently domesticated flies (only 7 to 10 generations
from the wild) even under low stress conditions,
i.e., low adult fly and larval density, respectively,
in adult cages and in larval diet, is likely a result
of high selection pressure that laboratory condi-
tions impose on the insects.
The phenomenon of rapid strain deterioration
after colonization is likely more evident when un-
der the high stress of mass-rearing conditions as
is common in the medfly factories around the
world. For this reason, the development of a filter
rearing system (Fisher & Caceres 2000) to man-
age mother colonies under rearing conditions (fly


Fig 2. Proportion of Mediterranean fruit fly females
mating (PM SD) from different origins (SC-south
coast and NC-north coast of Madeira Island, and SW-
semi-wild after 7 generations in the laboratory). The
data show no significant differences (P = 0.1951).


density, sex-ratio, and physical features) more
similar to conditions found in nature, as described
by Robinson et al. (2002), is recommended.
In conclusion, our data indicate no mating in-
compatibility among strains tested and also sup-
port the need to improve sterile male competitive-
ness by instituting in medfly mass rearing facili-
ties filter rearing systems to manage adult colo-
nies under less stressful conditions.

ACKNOWLEDGMENTS

This research was financially supported by the Inter-
national Atomic Energy Agency (IAEA), through research
contract POR 10842, and the Madeira-Med SIT Program.

REFERENCES CITED

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port framework for behavioral tests of fruit flies in
the field. Florida Entomol. 66: 512-514.
CAYOL, J. P. 2000. Changes in sexual behavior and in
some life history traits of tephritid species caused by
mass-rearing process, pp. 843-860 In M. Aluja and
A. L. Norrbom [eds.], Fruit Flies (Tephritidae): Phy-
logeny and Evolution of Behavior. CRC Press, Boca
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CAYOL, J. P., P. CORONADO, AND M. TAHER 2002. Sexual
compatibility in medfly (Diptera: Tephritidae) from
different origins. Florida Entomol. 85: 51-57.
DANTAS, L., R. PEREIRA, N. SILVA, A. RODRIGUES, AND
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[ed.], Proc. 6t International Symposium on Fruit Flies
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ECONOMOPOULOS, A. P. 1992. Adaptation of the Medi-
terranean fruit fly (Diptera: Tephritidae) to artificial
rearing. J. Econ. Entomol. 85: 753-758.
FAO/IAEA/USDA. 2003. Manual for Product Quality
Control and Shipping Procedures for Sterile Mass-
Reared Tephritid Fruit Flies. Version 5.0. Interna-
tional Atomic Energy Agency, Vienna, Austria.
FISHER, K., AND C. CACERES. 2000. A Filter rearing sys-
tem for mass reared genetic sexing strains of Medi-
terranean fruit fly (Diptera: Tephritidae), pp. 543-
550 In T K. Hong [ed.], Area-wide Control of Fruit
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laysia Press. Penang, Malaysia.







14 Florida Entc



FRANZ, G. 2005. Genetic sexing strains amenable to
large scale rearing as required for the sterile insect
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and A. S. Robinson [eds.], The Sterile Insect Tech-
nique: Principles and Practice in Area-Wide Inte-
grated Pest Management. Springer, Dordrecht, The
Netherlands. 787 pp.
MCINNIS D. O., D R. LANCE, AND C. G. JACKSON. 1996.
Behavioral resistance to sterile insect technique by
the Mediterranean fruit fly in Hawaii. Ann. Ento-
mol. Soc. Am. 89: 739-744.
OROZCO, D., AND R. O. LOPEZ. 1993. Mating competi-
tiveness of wild and laboratory mass-reared med-
flies: effect of male size, pp. 185-188 In M. Aluja and
P. Liedo [eds.], Fruit flies: Biology and Management.
Springer-Verlag, New York, New York, USA.
OTT, R. L., AND M. LONGNECKER. 2001. An Introduction
of Statistics Methods and Data Analyses (5th edn.).
Duxbury Publishers, Pacific Grove, California, USA.
PEREIRA, R. 2001. Madeira-Med, pp. 107-114 In Proc.
"Sterile Insect Technique as an Environmentally


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Friendly and Effective Insect Control System", Fun-
chal, Madeira, Portugal.
PEREIRA, R., A. BARBOSA, N. SILVA, L. DANTAS, J. CAL-
DEIRA, AND J. PACHECO. 2000. Madeira-Med pro-
gram, sterile insect technique against Mediterranean
fruit fly in Madeira, Portugal, pp. 443-438 In T K.
Hong [ed.], Area-wide Control of Fruit Flies and Other
Insect Pests. University Sains Malaysia Press. Pen-
ang, Malaysia.
PROKOPY, R. J., AND J. HENDRICHS. 1979. Mating behav-
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ROBINSON, A., J. P. CAYOL, AND J. HENDRICHS. 2002.
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tions for SIT. Florida Entomol. 85: 171-181.
THORNHILL, R., AND J. ALCOCK. 1983. The Evolution of
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219 pp.







Bricefo et al.: Courtship in Wild Medfly Strains


COURTSHIP BEHAVIOR OF DIFFERENT WILD STRAINS
OF CERATITIS CAPITATA (DIPTERA: TEPHRITIDAE)

DANIEL BRICEO'1, WILLIAM EBERHARD'2, JUAN VILARDI3, JEAN-PIERRE CAYOL4 AND TODD SHELLY5
'Escuela de Biologia, Universidad de Costa Rica, Ciudad Universitaria, Costa Rica

2Smithsonian Tropical Research Institute, Costa Rica

3Dpto. de Ciencias Biol6gicas, Facultad de Ciencias Exactas y Naturales
Universidad de Buenos Aires, 1428 Buenos Aires, Argentina

4Technical Cooperation Division, IAEA, Wagramerstrasse 5, P.O. Box 100 A-1400, Vienna, Austria

5USDA/APHIS/CPHST, 41-650 Ahiki St. Waimanalo, HI 96795 U.S.A

ABSTRACT

This study documents differences in the courtship behavior of wild strains of Ceratitis cap-
itata (Wiedemann) from Madeira (Portugal), Hawaii (U.S.A.), Costa Rica, and Patagonia (Ar-
gentina). Some traits showed large variations and others substantial overlaps. The angle at
which the male faced toward the female at the moment of transition from continuous wing
vibration and intermittent buzzing changed very little during the course of courtship in all
strains, but males from Madeira tended to face more directly toward the female than other
males. Females tended to look more, and more directly, toward the males as courtship pro-
gressed in all strains. The distance between male and female tended to decrease as courtship
proceeded in all strains, but the distances at which males initiated continuous vibration, in-
termittent buzzing, and jumped onto the female were relatively less variable between
strains, except for the strain from Costa Rica. Flies of Madeira courted for longer and the
male moved his head and buzzed his wings longer than the other strains.

Key Words: courtship behavior, wild flies, medfly, geographic differences, Madeira, Costa
Rica, Argentina, Hawaii

RESUME

Este studio document diferencias en el comportamiento de cortejo de cepas silvestres de
Ceratitis capitata (Wied.) provenientes de Madeira (Portugal), Hawaii (Estados Unidos de
Norte Am6rica), Costa Rica y Patagonia (Argentina). Algunas caracteristicas mostraron
grandes variaciones y traslape substantial. Los angulos a los cuales los machos miraron ha-
cia las hembras cambiaron muy poco en el moment de la transici6n de la vibraci6n continue
al zumbido intermitente durante el curso del cortejo en todo las cepas, pero los machos de
Madeira tendieron a enfrentar mas directamente a la hembra que otros machos. Los angulos
de las hembras disminuyeron claramente durante el cortejo en todas las cepas. La distancia
entire el macho y la hembra tendi6 a disminuir conforme el cortejo continuaba en todas las
cepas, pero las distancias a las cuales los machos iniciaron la vibraci6n continue, el zumbido
intermitente, y el salto sobre la hembra eran relativamente menos variables entire cepas ex-
cepto la cepa de Costa Rica. Moscas de Madeira cortejaron mas tiempo y el macho movio su
cabeza y zumbaba sus alas mas prolongadamente que las otras cepas.


Translation provided by the author.


The use of sterile males for the integrated con-
trol populations of Ceratitis capitata (Wiede-
mann) makes it economically important to under-
stand which male stimuli induce females to mate,
in order to design appropriate quality control
measures for mass-reared males (FAO/IAEA/
USDA 2003;Calkins & Parker 2005). Because it is
difficult to induce wild flies to reproduce in the
laboratory (R6ssler 1975), some strains have been
maintained under mass-rearing conditions for


many years. These conditions differ from those in
the wild in a number of respects (Cayol 2000).
Briceio & Eberhard (1998) found that males from
mass-reared strains court for shorter periods be-
fore attempting to mount the female, apparently
due to the crowded conditions in mass rearing
cages which result in frequent interruptions of
courtships. There are at least five differences be-
tween the sexual behavior of mass-reared males
and wild males (Briceio & Eberhard 2002a).







Florida Entomologist 90(1)


Mass-reared males are generally less able to
induce wild females to copulate than wild males.
Although several aspects of male courtship be-
havior are known to have changed in at least
some mass-reared strains (Zapien et al. 1983; Li-
imatainen et al. 1997; Briceno & Eberhard 1998;
Calcagno et al. 1999; Briceno et al. 2001), it is not
clear whether these or other male traits are more
important in producing this inferiority (Eberhard
2000). Such differences in male behavior may re-
sult in partial reproductive isolation between
strains (Lux et al. 2002).
This paper explores the possibility that there
are differences in courtship behavior among four
wild C. capitata populations from Costa Rica, Pa-
tagonia (Argentina), Hawaii (USA) and Madeira
(Portugal). Differences between wild strains may
have important implications for development
strategies for SIT implementation (Dyck et al.
2005).

MATERIALS AND METHODS

Flies of each strain were separated by sex
within 48 h of emergence as adults, and kept in
buckets topped with screen, with ad libitum ac-
cess to water and hydrolyzed yeast and sugar
(1:3). Wild flies from Costa Rica were raised from
larvae that emerged from infested tangerines col-
lected at the Estaci6n Experimental Fabio
Baudrit near Alajuela, Costa Rica. Wild flies from
Argentina were a laboratory F2 derivative from
flies raised from fruit collected in the field in the
Alto Valle region of Patagonia. Wild flies from Ha-
waii were raised from larvae collected from coffee
fruit on Kauai. Wild flies from Madeira were col-
lected from infested mixed hosts.
Flies in Costa Rica and Hawaii were video-
taped in plastic chambers that were 13.7 cm
diam. and 1.8 cm tall. They were videotaped from
below through a transparent glass table (Briceno
& Eberhard 1998) with a Sony Hi8 camcorder
equipped with +6 close-up lenses. Pairs from Pat-
agonia and Madeira wild flies 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 (or mating cham-
ber). Five min after the male began emitting
pheromone, a female was released into the cage,
and the behavior of the 2 flies was recorded for 30
min or until they copulated. Flies in mating trials
were sexually mature, 10 days old, and each fly
was used only once.
Measurements of different aspects of courtship
behavior that led to a mounting attempt by the
male were made from frame by frame analyses of
videotapes. Only a single courtship was analyzed
for each male to avoid pseudoreplication. Dura-
tions of the following male behaviors were ana-
lyzed: (1) continuous wing vibration (wings di-


rected postero-laterally and vibrated rapidly
dorso-ventrally); (2) intermittent buzzing (wings
moved back and forth from being directed poste-
riorly over to the abdomen to anteriorly and also
vibrated rapidly); for detailed descriptions of both
these wing movements, see Briceno & Eberhard
(2000b); (3) head rocking (head was rotated from
side to side and turned and laterally just before
intermittent buzzing began); (4) the total time the
female remained immobile (no walking) before
the male launched his mounting attempt; (5) and
total courtship duration from the start of contin-
uous vibration until the mounting attempt.
The directions the 2 flies were facing with re-
spect to the midpoint of the other fly's prothorax
and the distances between them were determined
at 3 stages of the courtship (initiation of continu-
ous wing vibration; initiation of intermittent
buzzing; and launch of the male's jump onto the
female) with 0 indicated that one fly was facing
directly toward the other. The "male angle" was
the angle between the direction the male faced
and the orientation directly toward the female;
the "female angle" was the equivalent for the fe-
male.
In Madeira and Hawaii strains the time dur-
ing which the male's aristae touched those of the
female was measured because contact with the
male's sexually dimorphic aristae during head
rocking and buzzing appears to be and important
part of medfly courtship (Briceno & Eberhard
2002b). The number of bouts of wing buzzing was
counted in 2 strains. All means are followed by +
SD. Statistical tests were Mann-Whitney U Tests
unless otherwise specified.

RESULTS

Data in Table 1 show that there were differ-
ences between at least 1 pair of geographic
strains in 12 of the 14 variables measured (the fe-
male angle when the male jumped, and the
amount of time the female was quiet before the
male jumped, are exceptions). There were also
large differences in most variables (especially
males vibrating and wing buzzing), and substan-
tial overlaps between different strains in most be-
havioral traits. Madeira males rocked their heads
and buzzed their wings significantly longer, and
their total courtship was also longer.
The male angles at the moment of transition
between wing vibration and wing buzzing
changed very little during the course of courtship
in all strains, but males from Madeira tended to
face more directly toward the female than other
males. Female angles clearly decreased during
courtship in all strains. The distance between the
male and female tended to decrease as courtship
proceeded in all strains. The distances at which
males initiated continuous vibration, intermit-
tent buzzing, and jumped onto the female were


March 2007







Bricefo et al.: Courtship in Wild Medfly Strains


TABLE 1. MEANS AND STANDARD DEVIATIONS OF COURTSHIP BEHAVIOR OF CERATITIS CAPITATA FLIES FROM DIFFERENT
GEOGRAPHIC AREAS, AND SIGNIFICANCE DIFFERENCES WITH MANN-WHITNEY U TESTS. VALUES IN THE SAME
ROW FOLLOWED BY THE SAME LETTER AND NUMBER ARE SIGNIFICANTLY DIFFERENT (A = p < 0.05; B = p <
0.01; C =p < 0.001, NS = NO SIGNIFICANT DIFFERENCE).

Madeira Costa Rica Hawaii Patagonia

Angles ()
contmal 1.8 2.6 ba, 6.8 + 9.1 b,, 0.8 0.9 b2a2 5.9 4.5 a,,2
confem 22.1 29.0 c, 43.9 31.4 a,c,,, 5.4 + 3.2 ab, 36.5 39.9 cb1
internal 1.2 2.7 a,,, 3.5 4.1 a, 2.7 4.6 4.4 4.7 a,
interfem 7.8 7.9 ns 13.1 16.6 ns 6.2 5.4 c, 13.7 14.8 c,
jumpmal 1.4 2.2 c, 3.8 5.1 c, 3.5 3.7 c2 2.7 5.2 c2
jumpfem 6.1 7.7 ns 8.8 13.9 ns 10.3 8.4 ns 7.6 10.8 ns
Distances (cm)
distcont 6.7 4.3 a, 1.6 2.1 a,,,, 6.7 3.7 a, 9.1 5.7 a,
distinter 1.71 0.9 a,c, 0.3 + 0.1 a,,,3 3.0 0.9 a2b,c, 1.8 1.1 a,b,
distjump 0.09 0.03 ns 0.1 0.3 ns 0.15 0.4 ns 0.10 0.03 ns
disthead 3.3 1.8 ns 3.1 1.0 ns
Duration (seconds)
femquiet 8.0 6.3 ns 5.9 3.7 ns 5.8 4.9 ns 5.9 5.6 ns
buzz 18.1 19.6 b,,, 10.6 8.3 b, 12.3 10.6 8.2 6.2 b,
vibrate 17.2 20.7 ns 14.8 19.8 ns 5.7 8.2 c, 8.6 6.8 c,
head rocking 3.9 4.6 c, 0.77 + 0.45 c, 3.3 4.8 c,,3 1.4 1.3 c,,,
court 29.4 27.1 c, 19.8 20.3 c,2 15.7 113.5 c, 16.8 10.6
antenna touches 9.4 8.7 ns 5.6 4.3 ns
number buzzes 21.3 13.7 ns 28.7 16.2 ns

N 32 56 17 38

contmal =male angle when continuous vibration began
contfem = female angle when continuous vibration began
internal= male angle when intermittent buzzing began
interfem= female angle when intermittent buzzing began
jumpmal = male angle when male jumped onto female
jumpfem = female angle when male jumped onto female
distcont = distance in cm between flies when male began continuous vibration
distinter = distance in cm between flies when male began intermittent buzzing
distjump = distance in cm between flies when male jumped onto female
disthead = distance in cm between flies when male began head rocking
femquiet = time in s female was motionless prior to the male's jump
vibrate = duration in s of continuous wing vibration
buzz = duration in s of intermittent buzzing
court = duration in s of entire courtship
antenna touches = duration in s of the antenna touches by the male during intermittent buzzing
head rocking = duration in s for head rocking


relatively less variable among strains, except for
the strain from Costa Rica.

DISCUSSION

Our results confirm several conclusions from
previous studies regarding possible female accep-
tance variables. 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 relative immobility prior
to the male's jump are in accordance with the idea
that one result of successful male courtship be-
havior is to induce the female to approach him, to
look directly toward him, and to remain immobile
(Briceio & Eberhard 2002a).


Lux et al. (2002) measured average duration of
vibration and buzzing in 3 wild populations, and
reported that in flies from Israel and Patagonia
these activities lasted longer than in flies from
Kenya (likely to be more similar to the original
ancestor of this African species) but failed to
present data or statistical tests. The values for
wing vibration in the Kenyan populations we
studied were much lower, i.e., 8.6-17.2 compared
to 57.9 (Lux et al. 2002). One behavior (head rock-
ing) that was absent in one of the wild strains (Is-
rael) studied by Lux et al. (2002) was present in
all the strains we studied.
There are several possible reasons for geo-
graphic differences in courtship behavior, includ-
ing founder effects and divergent sexual selection











in different populations. The behavioral differ-
ences between wild flies indicate that there is ap-
preciable genetic variation for these male court-
ship traits in field populations. The question of
whether variation exists in male traits under sex-
ual selection in natural populations has been con-
troversial. Our results are in accord with the
trend for genetic variation seen in other groups
(Anderson 1994). On a practical level, the vari-
ance we found means that relatively large sam-
ples of courtships are needed to test for signifi-
cant differences among strains. The differences
between strains documented here involved males
interacting with females of the same strain.
Given the probable effects of the behavior of one
sex on that of the other (Briceio & Eberhard
2002b), it is not possible to attribute differences
to one sex or the other until cross-strain pairs are
studied.
There was an apparent tendency of the wild
Madeira males to rock their heads and buzz their
wings significantly longer, and to court for longer
before mounting. Because it appears that Ma-
deira females are the "choosiest" among popula-
tions studied (Cayol et al. 2002), this could sug-
gest that males with this suite of behaviors would
be good candidates for medfly SIT operations
world-wide.

ACKNOWLEDGMENTS

RDB and WGE thank Eddy Camacho for technical
help, Bernal Burgos and Hernan Camacho for speci-
mens of wild Costa Rican flies, Jorge Lobo and Federico
Bolanos for help with statistics, and the International
Atomic Energy Agency, and Smithsonian Tropical Re-
search and Vicerrectoria of the Universidad de Costa
Rica for financial support. A BARD grant (project No IS-
2689-SGR) awarded to B. Yuval and T. E. Shelly fi-
nanced the trip of RDB to Hawaii to film flies.

REFERENCES CITED

ANDERSON, M. 1994. Sexual Selection. Princeton Univ.
Press, Princeton, New Jersey, USA.
BRICENO, R. D., AND W. G. EBERHARD. 1998. Medfly
courtship duration: a sexually selected reaction norm
changed by crowding. Eth. Ecol. Evol. 10: 369-382.
BRICENO, R. D., W. G. EBERHARD, J. C. VILARDI, AND P.
LIEDO. 2001. Geographic variation in the buzzing
songs of wild and mass-reared male medflies
(Diptera: Tephritidae: Ceratitis capitata). Florida
Entomol. 85: 32-40.
BRICENO, R. D., AND W. G. EBERHARD. 2002a. Decisions
during courtship by male and female medflies
(Diptera; Tephritidae): correlated changes in male


March 2007


behavior and female acceptance criteria in mass-
reared flies. Florida Entomol. 85: 14-31.
BRICENO, R. D., AND W. G. EBERHARD. 2002b. Courtship
in the medfly, Ceratitis capitata, includes tactile
stimulation with the male's aristae. Ent. Exp. &
Appl. 102: 221-228.
CALCAGNO, G. E., M. T. VERA, F. MANSO, S. A. LUX, F.
M. NORRY, F. N. MUNYIRI, AND J. C. VILARDI. 1999.
Courtship behavior of wild and mass-reared Medi-
terranean fruit fly (Diptera: Tephritidae) males from
Argentina. J. Econ. Entomol.92: 373-379.
CALKINS, C. O., AND A. G. PARKER. 2005. Sterile insect
quality, pp. 269-296 In V. A. Dyck, J. Hendrichs, and
A. S. Robinson [eds.], Sterile Insect Technique: Prin-
ciples and Practice in Area-wide Integrated Pest
Management. Springer, Dordrecht, The Nether-
lands. 787 pp.
CAYOL, J. P. 2000. Changes in sexual behavior and life
history traits of tephritid species caused by mass-
rearing processes, pp. 843-860 In M. Aluja and A. L.
Norrbom [eds.], Fruit Flies (Tephritidae): Phylogeny
and Evolution of Behavior. CRC Press, Boca Raton,
Florida, USA.
CAYOL, J. P., P. CORONADO, AND M. TAHER. 2002. Sex-
ual compatibility in Medfly (Diptera: Tephritidae)
from different origins. Florida Entomol. 85: 51-57.
DYCK, V. A., J. HENDRICHS, AND A. S. ROBINSON. 2005.
Sterile Insect Technique: Principles and Practice in
Area-wide Integrated Pest Management. Springer,
Dordrecht, The Netherlands. 787 pp.
EBERHARD, W. G. 2000. Sexual behavior and sexual se-
lection in the Mediterranean fruit fly, Ceratitis cap-
itata (Dacinae: Ceratitidini). pp. 457-487 In M. Aluja
and A. L. Norrbom [eds.], Fruit Flies (Tephritidae);
Phylogeny and Evolution of Behavior. CRC Press,
Boca Raton, Florida, USA.
FAO/IAEA/USDA. 2003. Manual for Product Quality
Control and Shipping Procedures for Sterile Mass-
Reared Tephritid Fruit Flies, Version 5.0. Interna-
tional Atomic Energy Agency, Vienna, Austria. 85 pp.
LIIMATAINEN, J., A. HOIKKALA, AND T. E. SHELLY. 1997.
Courtship behavior in Ceratitis capitata (Diptera:
Tephritidae): comparison of wild and mass-reared
males. Ann. Entomol. Soc. Am. 90: 836-843.
Lux, S. A., J. C. VILARDI, P. LIEDO, K. GAGGL, G. E. CAL-
CAGNO, F. N. MUNYIRI, M. T. VERA, AND F. MANSO.
2002. Effects of irradiation on the courtship behavior
of medfly (Diptera: Tephritidae) mass reared for the
sterile insect technique. Florida Entomol. 85: 102-112.
ROSSLER, Y. 1975. Reproductive differences between
laboratory-mass reared and field-collected popula-
tions of the Mediterranean fruit fly, Ceratitis capi-
tata. Ann. Entomol. Soc. Am. 68: 987-991.
ZAPIEN, G., J. HENDRICHS, P. LIEDO, AND A. CISNEROS.
1983. Comparative mating behaviour of wild and
mass-reared sterile medfly Ceratitis capitata (Wied.)
on a field caged host tree.-II. Female mate choice,
pp. 397-409 In R. Cavalloro (ed.), Fruit Flies of Eco-
nomic Importance. A. A. Balkema, Rotterdam, The
Netherlands.


Florida Entomologist 90(1)







Orozco et al.:Anastrepha ludens Sexual Competitiveness and Compatibility 19



SEXUAL COMPETITIVENESS AND COMPATIBILITY BETWEEN MASS-REARED
STERILE FLIES AND WILD POPULATIONS OF ANASTREPHA LUDENS
(DIPTERA: TEPHRITIDAE) FROM DIFFERENT REGIONS IN MEXICO


DINA OROZCO-DAVILA, REFUGIO HERNANDEZ, SALVADOR MEZA AND JULIO DOMiNGUEZ
Program Moscamed Moscafrut-Desarrollo de Metodos
Central Poniente No. 14 altos-Esq. 2' Avenida Sur. CP 30700 Tapachula, Chiapas, Mexico

ABSTRACT
The mass-reared colony ofAnastrepha ludens (Loew) currently used in Mexico for suppres-
sion of the Mexican fruit fly has been in use for over 10 years. Sterile flies are released into
a wide range of environmental conditions as part of an integrated area-wide approach to
suppress diverse populations of this pest in the Mexican Republic. This paper assesses the
performance of the sterile flies interacting with wild populations from the different environ-
ments. We investigated the sexual compatibility and competitiveness of the sterile flies
when competing with wild populations from 6 representatives Mexican states: Nuevo Le6n,
Tamaulipas, Sinaloa, Nayarit, Michoacan, and Chiapas. Results show that the males of the
wild populations differed in the time to the onset and peak of sexual activity. Nevertheless,
the index of sexual isolation (ISI) reflected sexual compatibility between the populations and
the mass-reared strain, indicating that the sterile individuals mate satisfactorily with the
wild populations from the 6 states. The male relative performance index (MRPI) showed
that the sterile male is as effective in copulating as the wild males. The female relative per-
formance index (FRPI) reflected a general tendency for wild females to copulate in greater
proportion than the sterile females, except for the strains from Tamaulipas and Chiapas. In
general, the lower participation of the sterile females in copulation increases the possibili-
ties of sterile males to mate with wild females. The relative sterility index (RSI) showed that
the acceptance by wild females of the sterile males (25-55%) was similar to that of wild
males. Females of the Chiapas strain showed the lowest acceptance of sterile males. Finally,
the results obtained in the Fried test (which measures induced sterility in eggs) showed a
competitiveness coefficient ranging from 0.2 to 0.5. This suggests that sterile males success-
fully compete and are compatible with flies from different geographic origins.

Key Words: Anastrepha ludens, Tephritidae, SIT, sexual compatibility, competitiveness,
Mexico

RESUME
La colonia actualmente usada para controlar la mosca mexicana de la fruta, Anastrepha
ludens (Loew), en M6xico tiene mas de 10 aios en cria masiva. Los insects est6riles son li-
berados en una gran variedad de condiciones ambientales como parte de un control integrado
para suprimir diversas poblaciones de esta plaga dentro de la Republica Mexicana. El obje-
tivo de este document esta dirigido a revisar el desempeno de las moscas est6riles frente a
poblaciones silvestres procedentes de diferentes ambientes y para esto se realizaron compa-
raciones de compatibilidad y competitividad sexual de las moscas est6riles contra poblaciones
silvestres de seis estados representatives de la Republica Mexicana: Nuevo Le6n, Tamauli-
pas, Sinaloa, Nayarit, Michoacan y Chiapas. Los resultados obtenidos manifiestan diferen-
cias en el horario de inicio de llamado y mayor actividad sexual del macho entire las moscas
provenientes de cada estado. Sin embargo el indice de aislamiento (ISI) reflej6 compatibilidad
sexual entire la cepa de laboratorio y todas las poblaciones analizadas, indicando que los in-
dividuos est6riles pueden aparearse satisfactoriamente con las poblaciones silvestres de los
seis estados. El indice de efectividad de apareamiento del macho (MRPI) reflej6 de manera
global que los machos est6riles son tan efectivos para copular como los silvestres. El indice de
efectividad de apareamiento de la hembra (FRPI) reflej6 que en la mayoria de los estados las
hembras silvestres copularon en mayor proporci6n que las hembras est6riles, except para
las poblaciones de Tamaulipas y Chiapas. En general, la baja participaci6n de las hembras es-
t6riles en el campo permiti6 al macho est6ril ampliar su probabilidad de apareamiento con las
hembras silvestres. En cuanto al indice de esterilidad relative (RSI), observamos que la acep-
taci6n de las hembras silvestres al macho est6ril (25-55%) fue similar a la de los machos sil-
vestres. Las hembras de la poblaci6n de Chiapas registry la menor aceptaci6n. Finalmente,
los resultados obtenidos en la prueba de Fried, la cual determine la esterilidad inducida pre-
sentaron un coeficiente de competitividad entire 0.2 y 0.5. Esto sugiere que los machos est6ri-
les compliten exitosamente y son compatibles con moscas de diferentes origenes geograficos.

Translation provided by the authors.







Florida Entomologist 90(1)


The extraordinary capacity of Anastrepha
ludens (Loew) (Diptera: Tephritidae) to adapt to
diverse environments allows its proliferation in
semitropical, tropical, and desert climates, and it
is considered a pest throughout Mexico (Aluja
1994). The effectiveness of the sterile insect tech-
nique (SIT) applied as part of area-wide integrated
pest management (AW-IPM) programs depends on
the efficient transfer of sperm carrying dominant
lethal mutations from sterile males to wild females
(Knipling 1955). Thus, the success or failure of a
sterile insect release is critically dependent on the
quality and the ability of sterile males to search for
and copulate effectively with wild females.
Mass rearing requires a broad and deep knowl-
edge of the pest insect's biology and ecology in or-
der to produce large numbers of insects without
compromising insect quality. In most mass-rear-
ing facilities there is a tendency to maintain the
same strain for long periods of time (Roessler
1975). As a consequence, and after a certain num-
ber of generations of mass-rearing, insect quality
tends to deteriorate (Partridge 1996).
Research has been conducted with field caged
host trees and different tephritids to assess the
changes that occur in the sexual behavior of mass-
reared sterile fruit flies in comparison with wild
populations. It has been found that the high den-
sities at which adult flies are commonly kept in
mass-rearing may be selecting for traits such as
males with simpler courting sequences, changes
in sexual competitiveness, shorter copulation, and
less discriminating females (Calkins 1984; Harris
et al. 1986; Boake et al. 1996; Iwahashi 1996;
Briceio & Eberhard 1998). One of the ways to
counteract this development is to regularly re-
place the colony. Nonetheless, one of the main
problems observed during colonization of a new
strain is the production bottleneck that occurs in
the initial phase of colonization, where only a
fraction of the individuals survive and reproduce
(Leppla et al. 1983; Leppla 1989). This increases
the time required to achieve the required colony
size and reduces the initial gene pool of the new
strain. Over the medium term, this reduction may
cause deviations in the behavior of laboratory
flies, such as strain incompatibility and sexual
isolation, with respect to the wild flies.
To monitor mating compatibility and competi-
tiveness changes, quality control field cage tests
have to be conducted (FAO/IAEA/USDA 2003).
For this study, mating compatibility refers to ran-
domness of mating between sterile mass-reared
insects and their wild counterparts. The competi-
tiveness tests measure the ability of sterile males
to achieve copulations with wild females and the
degree of sterility of the eggs produced by wild fe-
males when wild and sterile males compete to
mate with them.
Our goal was to determine the mating compat-
ibility and competitiveness of sterilized mass-


reared A. ludens flies of a strain that has been in
use for over 10 years in comparison with wild flies
from different regions of Mexico, where the SIT is
currently applied as a component of area-wide
campaigns to suppress this pest.

MATERIALS AND METHODS

Origin of the Biological Material

Wild pupae from the Tamaulipas region were
obtained from the townships of Guemez, Hidalgo,
Padilla, and Ciudad Victoria, where they were
collected from yellow sapote fruits (Sargenttia
greggi). In Nuevo Le6n, pupae were obtained from
Linares, El Cercado, Monterrey, and Guadalupe,
also from yellow sapote fruits. In the Sinaloa re-
gion collections covered the townships of Badira-
guato, Mocorito, and Culiacan, where sapote fruit
(Casimiroa eudilis) were the hosts. In Nayarit pu-
pae were obtained from bitter orange (Citrus au-
rentis), white sapote (Casimiroa eduleslleve),
matasano (Pouteria campechiana), and black sa-
pote (Diospyros digynajaca), collected in
Miravalles, Compostela, Xalisco, Testarazo, Aqui-
les Serdan, Emiliano Zapata, Tepic, Libertad, Lo
de Garcia, Cuachisnes, San Blas, Acaponeta, Tix-
pan, Pantanales, and San Pedro. In Michoacan
the pupae were obtained from bitter orange (Cit-
rus aurentis) collected in Uruapan, and in Chia-
pas from this same fruit collected in the Soco-
nusco region.
Fruit was collected directly from the host plant
and from the ground and taken to the laboratory
where it was placed in containers to let the larvae
mature. Once the larvae had matured, the fruit
was dissected and the larvae and/or pupae were
transferred to a pupation substrate (slightly
damp vermiculite). The pupae obtained were kept
for approximately 20 days in a room at a temper-
ature of 25 1C and 75 5% RH.
Sterile pupae were obtained directly from the
A. ludens production line at the Moscafrut mass
rearing facility in Metapa de Dominguez, Chia-
pas, Mexico (Dominguez Gordillo 1996). The orig-
inal colony is a mixture of an old colony from Mis-
sion, Texas, and wild material collected from dif-
ferent regions in Chiapas; the Mission colony had
been mass-reared for more than 10 years.

Size and Weight of the Pupae

Due to the influence of adult size on successful
mating (Burk & Webb 1983; Churchill-Standland
et al. 1986; Orozco & L6pez 1993), 2 days prior to
emergence, and when the pupal eye color was
dark brownish-green, the pupation substrate was
withdrawn and the weight and size-distribution
of the pupae were obtained with aid of a pupal siz-
ing and separating machine (FAO/IAEA/USDA
2003). This equipment was used to distribute the


March 2007







Orozco et al.:Anastrepha ludens Sexual Competitiveness and Compatibility


sterile and wild pupae into 10 different size
groups (with #1 being the smallest and #10 the
largest class, from 1.30 to 2.90 mm, respectively).
The wild and sterile pupae obtained in the size
categories 7 (2.30-2.45 mm), 8 (2.45-2.60 mm) and
9 (2.60-2.75 mm) were placed into containers, and
these containers were placed into 30 x 30 x 30-cm
cages in a room at 25 1C temperature and 75 +
5% RH. After emergence, the flies were sorted by
sex.

Field Cages

Six field cages, measuring 3 m in diameter and
2 m in height, and supported by a metal frame
(Chambers et al. 1983; Calkins & Webb 1983)
were used. Potted host mango and citrus trees
were placed alternately around the inside circum-
ference and central section of each cage. The
cages were set up in a mango (Ataulfo cv) planta-
tion in the hills of the municipality of Tapachula,
at an altitude of 137 m above sea level. The tests
were conducted in random blocks with a mini-
mum of 6 replicates

Male Calling

The numbers of calling males were record in
30-min periods. The required characteristics for
confirmation of male calling were vigorous wing
flapping, everted prostiger, and puffed pleural
glands. Observations were carried out from 15:00
to 19:30 h (summer schedule), since this is the
time when sexual activity in A. ludens is the
greatest (Aluja et al. 1983).

Sexual Compatibility

In each cage 20 males and 20 females of the
tested wild populations and 20 sterile males and
20 sterile females of the mass reared strain were
released. Wild flies were 16-21 d old while sterile
flies were 10 d old (Orozco et al. 2001). In order to
identify the individual flies, a small piece of paper
with a number was stuck to each fly's dorsal side
by white glue. Throughout the observation period
the number and type of matings was recorded as
wild male and female (WW), sterile male and fe-
male (SS), wild male and sterile female (WS), and
sterile male and wild female (SW).

Sexual Competitiveness (Induced Sterility)

For each wild population 5 field cages were set
up as follows: (1) "wild control" cage into which 32
wild males were released along with 8 wild fe-
males; (2) "sterile control" cage into which 32 ster-
ile males were released along with 8 sterile fe-
males; and (3) three "competitiveness" cages into
each of which 24 sterile males, 8 wild males and 8
wild females were released. Each cage contained


3 feeding (sugar and hydrolyzed protein in a 3:1
ratio) and watering areas, and 8 artificial host
fruits, placed into each cage in order to collect the
eggs to measure the induced sterility. The flies
were left in the cages for 5 d; after the second d,
the host fruits were changed daily to estimate fe-
cundity and fertility of the females.

Data Analysis

To estimate sexual compatibility, the index of
sexual isolation (ISI), male and female relative
performance indices MRPI and FRPI (Cayol et al.
1999), and the relative sterility index (RSI) (McIn-
nis 1996) were calculated. We used the 0.25 value
as variance limit for equal mating propensity in
ISI, MRPI, and FRPI, and for equal competitive-
ness in the RSI. The overall competitiveness value
C of sterile males, as indicated by the reduction in
egg hatch, was estimated by the Fried formula
(Fried 1971). Indices between populations were
compared by an ANOVA and Fisher's PLSD test
with StatView software ver. 5.0.

RESULTS

All the evaluations were carried out during
summer, which corresponds to the rainy season in
Mexico. Humidity ranged between 88 and 99%
and during the tests (at 17:00 h) it was usually
cloudy and rainy. The maximal light intensity re-
corded was 1440 lux and the minimal 0 lux was at
19:30 h. The temperatures recorded ranged be-
tween 24 to 32C. The only exception was with the
Chiapas strain that was evaluated during spring,
which corresponds to the hot season without rain.
In this case the temperature range was higher
but the relative humidity was significantly lower,
fluctating between 40 and 60%.
Male calling and mating activity during the
sexual activity period are presented in Fig. 1.
Some differences in the sexual activity patterns
were detected. Males from the Nayarit area be-
gan their sexual activity at 16:00 h and reached a
mating peak at 16:30 h. Males from Sinaloa,
Tamaulipas, and Michoacan initiated their sexual
activity at 16:30 h and reached their maximum
level at 19:00 h, 18:30 h and 17:30 h, respectively.
Chiapas and Nuevo Le6n initiated sexual activity
at 17:00 h and reached a maximum at 18:30 h.
The results obtained from the mating compat-
ibility test are shown in Table 1. The propensity
for mating (PM) indicates the overall percentage
of the couples that mated. All the PM values were
larger than 0.20, indicating that the conditions
under which the tests were run were satisfactory
(FAO/IAEA/USDA 2003). The index of sexual iso-
lation (ISI) is a measure of mating compatibility
between populations. The index considers the
number of couples obtained for each possible mat-
ing combination, with values range from -1 (com-







Florida Entomologist 90(1)


Novo Ledn so Naysrit so Michoacan
60 60
iI- Kl,30 lk-

20 ,


1500 I53 1*6D 1


SlaniS a 80 Tanaulp so
70 70 Ih*



11 It _10:A
1700 130 IBo0 It sm 19 00 l 5BO I5 5 e 1 175 137 If 0l f 193 1 19 is0 ,s30 Is m 03 8Im 0 II 0 m II3 mm lo 0
Time of the day


Fig. 1. Distribution of matings (bars) and males calling (lines) during the sexual activity period (the matings are
with both wild and sterile females).


plete negative assortative mating, that is, all
mating are with members of the opposite popula-
tion) through 0 (random mating) to +1 (complete
positive assortative mating, that is, total mating
isolation of the two populations). The ISI values
(Fig. 2) show satisfactory levels of compatibility
between the sterile insects and the different wild
populations, and there was no significant differ-
ence among populations (F = 1.159; df = 4,26; P =
0.3514).
The male relative performance index (MRPI)
is a measure of the propensity of sterile males to
mate with wild females, with values ranking from
-1 to +1. A value of -1 indicates that all matings
were carried out by wild males, while a value of
+1 indicates that all matings were carried out by
sterile males. Zero indicates that males from both
populations participated equally in matings. Fig.
3 shows that the sterile males were as effective at


obtaining mates as the wild males and there was
no overall differences between the populations
(F = 3.699; df = 4,26; P = 0.1702). Nevertheless,
between individual populations there was a sig-
nificant difference with the Tamaulipas popula-
tion (F = 3.699; df = 4,26; P = 0.0164). This sug-
gests that the sterile males were more effective
when competing against wild flies of the Tamauli-
pas populations.
The female relative performance index (FRPI)
is a measure of the propensity of sterile females to
mate with wild males, with values ranking from -
1 to +1. A value of -1 indicates that all matings
were carried out by wild females, while a value of
+1 indicates that all matings were carried out by
sterile females. Zero indicates that females from
both populations participated equally in mating.
In most regions, the wild females copulated more
than the sterile females (Fig. 4), with the excep-


TABLE 1. PROPENSITY OF MATING (PM), SEXUAL COMPATIBILITY (ISI) AND COMPETITIVENESS INDICES OBTAINED IN
FIELD CAGES FROM INTERACTIONS BETWEEN 6 WILD MEXICAN FRUIT FLY POPULATIONS FROM MEXICO AND
A MASS-REARED STRAIN.

Compatibility and Competitiveness Indices

State PM ISI MRPI FRPI RSI n

Tamaulipas 0.65 0.147 0.103 ab 0.200 + 0.076 c 0.148 + 0.083 c 0.530 + 0.061 d 6
Sinaloa 0.66 0.152 0.055 ab -0.114 + 0.058 ab -0.232 + 0.062 ab 0.390 + 0.024 bc 9
Nuevo Le6n 0.40 0.368 0.092 a -0.131 + 0.064 a -0.383 + 0.105 a 0.331 + 0.042 ac 11
Nayarit 0.76 0.013 0.051 b -0.094 + 0.059 ab -0.198 + 0.045 b 0.457 + 0.039 bd 7
Michoacan 0.55 -0.049 0.295 b 0.030 + 0.041 ab -0.424 + 0.118 ab 0.532 + 0.124 bd 3
Chiapas 0.57 0.361 0.052 a -0.011 + 0.032 b 0.244 + 0.055 c 0.240 + 0.053 a 12

Propensity of mating (PM) = No. of pairs collected/No, of females released.
Isolation index (ISI) = (SS+WW)-(SW+WS) / (SS+WW+SW+WS).
Male relative performance index (MRPI) = (SS+SW)-(WS+WW) / (SS+WW+SW+WS).
Female relative performance index (FRPI) = (SS+WS)-(SW+WW) / (SS+SW+WS+WW).
Relative sterile index (RSI) = (SW) / (SW+WW).
n = Number of replicates performed for each wild population.


March 2007


S'--


s







Orozco et al.:Anastrepha ludens Sexual Competitiveness and Compatibility 23



ISI


Tamaulipas.......-..........................





M ich ao a n ..................................... ........................ .. .. ............. ..............................................................
N uevo Le 6n ---- -- --- -------- --



C hiapas .............................. .......... ................. .. .................................... .
Chiapas i t:-- -

-1 -.75 -.5 -.25 0 .25 .5 .75 1
Negative Random mating Positive assortative
assortative (Sexual compatibility) mating
mating (Sexual isolation)



ISI = (SS+WW)-(SW+WS) / (SS+WW+SW+WS)
Fig. 2. Index of sexual isolation comparing the compatibility of the sterile strain with the wild strain from each
region.


tion of the Tamaulipas and Chiapas strains (F = ues range between 0 and +1. Zero means that
8.285; df = 4,26; P = 0.0002), for which a signifi- wild females mate only with wild males; a value
cant difference was found in comparison with the of +0.5 indicate that wild females mate indiscrim-
other regions. inately with wild or sterile males; a value of +1 in-
The relative sterility index (RSI) indicates the dicate that wild females mate only with sterile
sexual competitiveness between two strains. Val- males. The RSI in most cases reflected the prefer-



MRPI







Nayarit ------------. .-------
M inaloa ............................... ...........................................................

Nayartn ........................................................................... *................................................

MichoacAin....... ..
C hiapas ..................................................................................


-I -.75 -.5 -.25 0 .25 .5 .75 1
All mating by wild Equal mating All mating by sterile
individuals propensity individuals





MRPI = (SS+SW)-(WS+WW) / (SS+WW+SW+WS)

Fig. 3. Male relative performance index between each regional wild strain with the sterile strain.







Florida Entomologist 90(1)


Tamaulipas
Sinaloa

Nuevo Le6n
Nayarit

Michoacin
Chiapas


FRPI




.... ................................. .................................................... ...................................................................
. ....................... ........................ ............................. .. ...... ..-- ......................................................................




------1-p gI --^"."-" " i"


-1 -.75 -.5 -.25
All mating by wild
individuals


0 .25 .5 .75 1


Equal mating
propensity


FRPI = (SS+WS)-(SW+WW) / (SS+SW+WS+WW)

Fig. 4. Female relative performance index between each regional wild strain with the sterile strain.


ence of wild females for wild males over sterile
males. There was, however, no significant differ-
ence for the strains from Michoacan and Tamauli-
pas (F = 2.422; df = 4,26; P = 0.0136), for which
the wild males were found to be less competitive
(Fig. 5).
Values for the Fried's competitiveness coeffi-
cient range from 1 to 0. Values of 1 indicate an
equivalent level of competitiveness between the
two types of males, while values close to zero in-
dicate superior competitiveness of the wild male
(Fried 1971). The values obtained ranged from
0.23 to 0.56 (Table 2).


RSI


Tamaulipas
Sinaloa

Nuevo Le6n
Nayarit

Michoacin
Chiapas


DISCUSSION AND CONCLUSIONS

Mating competitiveness and sexual compati-
bility are important quality control parameters
that affect the performance of released sterile in-
sects. The present study analyzed the sexual com-
petitiveness and compatibility of sterile insects
from the Moscafrut mass-rearing facility with
wild populations ofA. ludens coming from differ-
ent regions from M6xico. Unlike the wild popula-
tions, which are exposed to the natural environ-
mental conditions, strains reared under labora-
tory conditions are normally exposed to fairly sta-


All mating by wild
males


Equal competitiveness of
both males


All mating by sterile
males


RSI= (SW) / (SW+WW)

Fig. 5. Relative sterility index for the sterile strain with each of the regional wild strains.


All mating by sterile
individuals


------- -- ---- ------r- ---- --- --
**- - .t-

---- ------.2 4 L ------- -


March 2007







Orozco et al.:Anastrepha ludens Sexual Competitiveness and Compatibility 25


TABLE 2. RESULTS FROM THE COMPETITIVENESS TEST (FRIED) CARRIED OUT IN FIELD CAGES BETWEEN 6 WILD MEXI-
CAN FRUIT FLY POPULATIONS FROM MEXICO AND STERILE FLIES FROM A MASS-REARED STRAIN.

Percent egg hatch
Fried competitiveness
State Wild control cage Sterile control cage Competitiveness cage value (C)

Tamaulipas 57.73 0.00 21.50 0.36
Sinaloa 88.31 0.00 46.28 0.39
Nuevo Le6n 73.60 0.00 43.07 0.29
Nayarit 76.73 0.00 32.67 0.45
Michoacan 28.06 0.00 16.71 0.23
Chiapas 57.82 0.00 19.71 0.56

Fried competitiveness value (C) = (W/S) X (Hw Hc) / (He Hs).
W = Number of wild males.
S = Number of sterile males.
Hw = Egg hatch from wild females in the wild control cage.
He = Egg hatch from wild females in the competitiveness cage.
Hs = Egg hatch from lab females in the sterile control cage.


ble environmental conditions. This may lead to a
change in the behavior of the mass-reared adults.
In general there was no evidence of any incom-
patibility between the different wild populations
and the mass reared insects, even though the re-
sults obtained from male pheromone-calling ac-
tivity revealed differences in the time of the onset
of male calling among the wild populations. The
flies from Nayarit began calling earliest in the
day, while the flies from Nuevo Le6n began the
latest, even though both states have relatively
similar latitudes (respectively, 22 and 26 degrees
north) in north-western and north-eastern Mex-
ico, and thus benefit from approximately the
same daylight h. This independence of calling
time initiation from the latitude is also evident
from the fact that the onset of pheromone-calling
in the southern-most population from Chiapas
(16 degrees north), was similar to the Nuevo Le6n
population in the northeast.
The combined data of the different indices (ISI,
MRPI, FRPI, and RSI) provide a complete and re-
liable picture of the sexual compatibility between
the wild populations and the mass reared sterile
flies, as well as their relative competitiveness.
The ISI demonstrated good sexual compatibility
between the wild populations and the mass-
reared strain, indicating that the sterile individu-
als mate satisfactorily with the wild populations
from the 6 states.
The MRPI showed that the sterile males mass-
produced at the Moscafrut facility are as effective
in copulating with wild females as the wild males.
The FRPI reflected a general tendency for wild fe-
males to copulate in greater proportion than the
sterile females, except for the populations from
Tamaulipas and Chiapas. In general, the lower
participation of the sterile females suggests that
the sterile males have greater possibilities of mat-
ing with wild females.


The RSI results show that wild female accep-
tance of the sterile males was high (25-55%). The
results obtained from the Fried test that mea-
sures induced sterility, indicate a competitiveness
coefficient ranging from 0.2 to 0.5 and suggest
that sterile males successfully competed with
flies from different geographic origin. This out-
come supports the results found in the compati-
bility tests.
Compatibility and competitiveness are regu-
lar quality control tests that are used to deter-
mine if a particular mass-rearing strain needs to
be replaced (FAO/IAEA/USDA 2003). Previous
studies have shown that long periods under
mass-rearing conditions adversely can affect the
performance of sterile fruit flies (McInnis 1996).
Other studies have shown that the geographic or-
igin of different strain might result in sexual in-
compatibility (Vera et al. 2006). Our current
work demonstrates that the mass-reared A.
ludens strain currently being produced at the
Moscafrut and used over the last 10 years in dif-
ferent geographic regions for Mexican fruit fly
control programs is still suitable for this purpose.
Our data are very similar to those recently pub-
lished (Rull et al. 2005), although they arrived at
a somewhat different conclusion due to the fact
that a different analysis was carried out. Contin-
ued careful monitoring of the performance of this
mass-reared strain under semi-natural or natu-
ral is required.

ACKNOWLEDGMENTS

We gratefully acknowledge the field evaluation team
in the Moscafrut facility; Marco P. P6rez G6mez, Jos6 L.
Zamora Palomeque, Jesus A. Escobar Trujillo, and Jos6
L. Quintero for the technical support; and International
Atomic Energy Agency and Direcci6n General de Sani-
dad Vegetal for the financial support.











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Florida Entomologist 90(1)







Allinghi et al.: Mating Compatibility between Wild and LabAnastrepha fraterculus 27



COMPATIBILITY AND COMPETITIVENESS OF A LABORATORY STRAIN
OF ANASTREPHA FRATERCULUS (DIPTERA: TEPHRITIDAE)
AFTER IRRADIATION TREATMENT


ARMANDO ALLINGHI1, GRACIELA CALCAGNO2, NATALIA PETIT-MARTY3, PAULA GOMEZ CENDRA2, DIEGO SEGURA3,
TERESA VERA4, JORGE CLADERA3, CECILIA GRAMAJO4, EDUARDO WILLING4 AND JUAN CESAR VILARDI2
'Comisi6n Nacional de Energia At6mica, CNEA, Argentina

2Depto. Ecologia, Gen6tica y Evoluci6n, Facultad de Ciencias Exactas y Naturales
Universidad Buenos Aires. (1428) Buenos Aires, Argentina

3Instituto de Gen6tica, INTA, Castelar, Argentina

4Estaci6n Experimental Agro-industrial Obispo Colombres, Tucuman, Argentina

ABSTRACT

We evaluated under semi-natural field cage conditions sexual compatibility and competitive-
ness of a laboratory strain (LAB) compared to a wild population (TUC) ofAnastrepha frater-
culus (Wiedemann). The LAB strain is produced under semi-mass rearing conditions at the
Estaci6n Experimental Agroindustrial Obispo Colombres facility (Tucuman, Argentina).
Wild flies were obtained at Horco Molle (Tucuman, Argentina) from infested guava fruits.
LAB pupae were irradiated (60Co) 48 h before adult emergence. The tested doses were 0 (con-
trol), 40, 70, and 100 Gy. Twenty-five males and 25 females each of TUC and LAB were
released into cages and mating pairs collected. Only 1 irradiation dose was considered at a
time. Females were separated and allowed to lay eggs into artificial fruits to estimate in-
duced sterility from the corresponding hatching rate. Copulation start time did not differ sig-
nificantly between strains nor among irradiation treatments. Copulation duration showed
highly significant differences among irradiation doses, but no differences between strains.
The index of sexual isolation (ISI) and the relative sterility index (RSI) indices indicated that
LAB and TUC are fully compatible, males from TUC and LAB did not differ in mating com-
petitiveness, and irradiation within the range tested did not affect these indices. Non-irradi-
ated LAB females exhibited higher mating propensity than TUC ones. However, a significant
reduction in the female relative performance index (FRPI) index was observed with increas-
ing irradiation dose. The analysis of induced sterility indicated that treatment with 40 Gy
reduces male fertility from about 80% to 0.75%, and higher doses produce total sterility.
In females, the 40 Gy dose reduces fertility to about 2% and higher doses prevent egg laying.

Key Words: mating compatibility, Anastrepha fraterculus, Irradiation, mating indices, fruit
fly, Tephritidae

RESUME

Se evalu6 bajo condiciones semi-naturales enjaulas de campo la compatibilidad y la compe-
titividad sexual de una linea de laboratorio (LAB) con respect a una poblaci6n salvaje
(TUC) de Anastrepha fraterculus (Wiedemann). La linea de laboratorio se produce en condi-
ciones de cria semi-masiva en las instalaciones de la Estaci6n Experimental Agroindustrial
Obispo Colombres (Tucuman, Argentina). Las moscas salvajes se obtuvieron de frutas infes-
tadas de guayabos en Horco Molle (Tucuman, Argentina). Las pupas de laboratorio fueron
irradiadas (60Co) 48 horas antes de la emergencia del adulto. Las dosis utilizadas fueron 0
(control), 40, 70, y 100 Gy. Se liberaron 25 machos y 25 hembras de TUC y LAB dentro de las
jaulas y se recolectaron las parejas formadas. S61o se consider 1 dosis de irradiaci6n por vez.
Las hembras apareadas fueron separadas y se les permiti6 poner huevos en frutas artificia-
les para estimar la esterilidad inducida a trav6s del porcentaje de eclosi6n. La hora de inicio
de la c6pula no difiri6 significativamente entire poblaciones ni entire los tratamientos de irra-
diaci6n. La duraci6n de la c6pula mostr6 grandes diferencias entire dosis de irradiaci6n pero
no entire cepas. Los indices ISI (aislamiento) y el RSI (esterilidad relative) indican que LAB
y TUC son totalmente compatibles, los machos de TUC y LAB no difieren en su competitivi-
dad y la irradiaci6n dentro del rango de dosis utilizadas tampoco afect6 este indice. Las hem-
bras LAB no irradiadas muestran una mayor propensi6n para el apareamiento que las
hembras de TUC. Sin embargo se observe una reducci6n significativa del indice FRPI (ac-
tuaci6n relative de hembras) a media que se aumenta la dosis de irradiaci6n. El andlisis de
la esterilidad inducida indica que con dosis de 40 Gy la fertilidad disminuye del 80% al







Florida Entomologist 90(1)


0.75%, y con dosis mayores la esterilidad fue total. Las hembras irradiadas con dosis de 40
Gy tienen una fertilidad de aproximadamente 2% y con dosis mayores no ponen huevos.

Translation provided by the authors.


Anastrepha fraterculus (Wiedemann) the South
American fruit fly (Stone 1942) is an important
pest of fruit production in Argentina and the spe-
cies is abundant in the northwestern and north-
eastern regions (Vergani 1956). The range of
A. fraterculus overlaps at least partially with that
of the Mediterranean fruit fly Ceratitis capitata
(Wiedemann). The programs of suppression or
eradication of the latter species, integrating the
Sterile Insect Technique (SIT) in this country, have
shown a remarkable success (SENASA 1997,
http://www.senasa.gov.ar/vegetal/moscal.php), and
point to the necessity of developing and applying
similar control strategies forA. fraterculus (Guillen
& Sanchez 2007).
We analyzed under laboratory conditions the
optimal irradiation dose and pupal age at the mo-
ment of irradiation to induce sterility inA. frater-
culus (Allinghi et al. 2007). Irradiated males were
able to transfer sperm and exhibited apparently
minimal effects, if any, of the irradiation on their
performance in comparison with non-irradiated
males. However, the sine qua non condition for
the SIT is sexual compatibility between sterilized
and released laboratory reared flies and wild flies.
Therefore, it is necessary to evaluate under quasi-
natural conditions the mating performance of lab-
oratory males when competing with wild males
for wild females.
In the present work we evaluated, on field-
caged host trees the sexual compatibility and
competitiveness of a laboratory strain (LAB) in
relation to a wild population from Tucuman (Ar-
gentina) (TUC). We also analyzed the effects of
different radiation doses on mating competitive-
ness, strain compatibility, fertility, copulation du-
ration, and copulation start time.

MATERIALS AND METHODS

The LAB strain used in this study was pro-
duced under semi-mass rearing conditions (Jaldo
et al. 2001) since 1997 at the Estaci6n Experi-
mental Agroindustrial Obispo Colombres facility
(Tucuman, Argentina). Wild flies were obtained
from fruiting guava trees Psidium guajava L.
(Myrtaceae) at Horco Molle (2648'S, 6520'W)
from Tucuman, Argentina. The LAB strain and
the collected fruits were sent to the Laboratory of
Insects, Instituto Nacional de Tecnologia
Agropecuaria (INTA), in Castelar, Argentina. The
collected fruits were placed on plastic trays over a
layer of sand to allow pupation. The sand was pe-
riodically sifted to obtain pupae, which were then
placed in plastic 1-L flasks. The LAB and the TUC


pupae were maintained under controlled condi-
tions (25 + 1C, 80 5% rh and a photoperiod of
12:12 L:D) until adult emergence.
LAB pupae were irradiated 48 h before adult
emergence (Allinghi et al. 2007) at the Centro
At6mico Ezeiza facility (Comisi6n Nacional de
Energia At6mica, Argentina) in a Gammacell 220
(MDS Nordion, Canada) irradiator (60Co source)
with a dose rate of 1.4 Gy/min. Lots of 500 pupae
were held in 20-mL ventilated glass containers
during the exposures of 40, 70, and 100 Gy in nor-
mal atmosphere. After irradiation, the pupae
were placed in 3-L glass containers. Flies of the
control group were subjected to all of the same
handling procedures except irradiation. Emerg-
ing adults were removed from the flasks every 24
h. To facilitate sorting by sex, flies were anaesthe-
tized by exposure to a temperature of 0C for 10
min. Fifty individuals of each sex were placed in
separate 3-L glass containers and supplied with
water and adult food. The food consisted of a 2:1
dry mixture of brown sugar: hydrolyzed corn pro-
tein (R. M. SAIC). Manso (1998) showed that lab-
oratory strain adults fed this diet developed to
sexual maturity. Adults were kept under labora-
tory conditions (25 + 2C, 60 20% r.h.), and a
photoperiod of 12:12 (L:D) until sexually mature.
De Lima et al. (1994) reported flies under such
conditions reach sexual maturity in 16 d. In a pi-
lot field cage test, we found an increasing propor-
tion of mating with fly age; however, mortality
also increased with age. The age of 20 1 d after
emergence was found to be the best compromise
between maturity and viability.
Three d prior to each experiment, flies were la-
beled to identify their origin. This was done by
placing approximately 10 flies in a mesh bag
(1 mm mesh diameter) where, one at a time, they
were gently immobilized and painted on the tho-
rax with a dot of water-based paint (Tempera
Alba, Alba, Inc., Argentina). Colors green, red,
white, and yellow were interchanged sequentially
each day. After labeling, 25 flies were placed in 1-
L containers with food and water and held under
laboratory conditions until required. Outdoor ny-
lon screened cages (2.9 m tall x 3 m diameter)
were erected over rooted 1.5 m tall, 4-year-old
tangerine trees, Citrus reticulata Blanco (Ruta-
ceae). Field cages were identified by number, and
each day treatments and observers were ran-
domly assigned to them. In field cages, 25 males
and 25 females each of TUC and LAB strains
were released. For each radiation dose, 6 repli-
cates were made. Only 1 irradiation dose was con-
sidered at a time.


March 2007







Allinghi et al.: Mating Compatibility between Wild and LabAnastrepha fraterculus 29


Because mating occurs mainly in the morning
(Malavasi et al. 1983; Morgante et al. 1983; De
Lima et al. 1994; Petit-Marty et al. 2004), the ob-
servation period was from 08.00 h to 13.00 h.
Males were released 15 min before females to al-
low establishment in the cage. Only healthy
marked flies were released, while non-active or
dead flies were replaced. For each mating pair,
the following data were recorded: copulation start
time, copulation location (fruit, net, ground, stem,
abaxial-adaxial side of a leaf, height in the tree),
and male and female colors. The pairs were gently
induced to walk into 20-mL plastic vials and
placed in the shade until the mating couple disen-
gaged. This moment was recorded as the copula-
tion end-time. These field cage tests were per-
formed at INTA Castelar (Buenos Aires Province)
between April 4 and 16, 2002. Temperatures, rel-
ative humidity, and sunshine records during this
period were favorable for fly requirements. Copu-
lation start time and copulation duration were
compared among laboratory irradiated flies by
one-way analysis of variance.
Sexual compatibility was estimated by means
of the index of sexual isolation (ISI) (Cayol et al.
1999) and the relative sterile index (RSI) (McIn-
nis et al. 1996). Male and female competitiveness
was evaluated respectively through male (MRPI)
and female relative performance (FRPI) indices
(Cayol et al. 1999). The statistical significance of
any departure from random mating or equal per-
formance of each sex was tested, following Petit-
Marty et al. (2004), by means of an independence
chi squared test taking into account the total
number of each mating combination (ISI), the to-
tal number of mated and unmated males (MRPI)
or females (FRPI), of each population. Compati-
bility and relative performance analyses were
based only on those trials where the percentage of
mating was sufficiently high (>20% of mated fe-
males). Matings occurring on the cage screen or
on the floor were not included, following the inter-


national fruit fly quality control manual (FAO/
IAEA/USDA 2003).
For each treatment, induced sterility was eval-
uated from the percent of egg hatching. At the end
of the experiments in the field cages, females
were separated according to radiation treatment
and male origin and transferred to 3-L flasks.
They were allowed to lay eggs into artificial fruits
(Manso 1998). Eggs were collected and incubated
in Petri dishes, and the hatching rate was re-
corded.

RESULTS

For both LAB and TUC flies, most matings oc-
curred on the lower side of peripheral leaves at an
intermediate canopy height. Copulation start time
(Table 1) did not differ significantly between
strains and the irradiation treatment did not show
any effect on this variable (F = 0.23, P = 0.63 and
F = 3.16, P = 0.08 for males and females, respec-
tively). Copulation duration (Table 1) showed
highly significant differences among treatments
(F = 4.97, P < 10-3 and F = 10.08, P < 10- for males
and females, respectively). These differences are
totally attributable to the irradiation treatment.
Indeed, TUC and non-irradiated LAB flies did not
differ significantly in copulation duration (P = 0.88
and P = 0.41 for males and females, respectively),
but if these two classes are grouped and compared
with irradiated flies the differences are highly sig-
nificant (F = 18.71, P < 10- and F = 40.08, P < 10-9
for males and females, respectively).
The analysis of mating compatibility by means
of the ISI indicated that LAB and TUC are fully
compatible. The estimated values did not depart
significantly from that expected for random mat-
ing, and no effect of irradiation was observed (Ta-
ble 2). Males from TUC and LAB did not differ in
mating competitiveness, and irradiation did not
affect this index. Non-irradiated LAB females ex-
hibited higher mating propensity (FRPI signifi-


TABLE 1. COPULATION START TIMES AND MATING DURATION (HRS:MIN) OF TUC1 AND LAB2 FLIES WITH DIFFERENT IR-
RADIATION DOSES.

Males Females

Start time Duration Start time Duration

Strain/dose Avg SE Avg SE n Avg SE Avg SE n

TUC 9:11 0:42 1:14 0:37 554 9:13 0:42 1:15 0:36 523
LAB/0 9:16 0:46 1:19 0:41 136 9:14 0:46 1:21 0:43 154
LAB/40 9:11 0:41 1:07 0:35 134 9:07 0:42 1:04 0:34 144
LAB/70 9:11 0:43 1:02 0:30 135 9:12 0:40 1:03 0:33 144
LAB/100 9:19 0:45 1:05 0:33 128 9:13 0:44 1:02 0:32 122

TUC = wild flies from Tucuman obtained from guava fruits; non irradiated controls.
LAB = laboratory strain reared at the Estaci6n Experimental Agroindustrial Obispo Colombres.







Florida Entomologist 90(1)


TABLE 2. COEFFICIENTS OF SEXUAL ISOLATION (ISI), MALE (MRPI) AND FEMALE (FRPI) REPRODUCTIVE PERFORMANCE
(CAYOL ET AL. 1999), AND RELATIVE STERILITY (RSI) (MCINNIS ET AL. 1996). P: SIGNIFICANCE WITH RESPECT
TO RANDOM EXPECTED VALUES.

Dose ISI P1 MRPI P FRPI P RSI

0 -0.029 0.238 0.046 0.477 0.121 0.061 0.543
40 0.008 0.909 -0.023 0.709 0.034 0.534 0.484
70 0.067 0.585 -0.052 0.392 0 1 0.440
100 -0.004 0.751 0.004 0.949 -0.050 0.273 0.504

'P = probability of obtaining the observed results assuming random mating.


cantly higher than 0) than TUC females. How-
ever, a significant reduction in the FRPI was ob-
served as irradiation dose was increased (r =
-0.98; P = 0.014). The mating competitiveness of
irradiated LAB males with TUC males for TUC
female mates was measured by the RSI. The esti-
mated RSI values approached 0.5, showing that
at all radiation doses LAB males competed effi-
ciently with TUC males for mating with TUC fe-
males (Table 2).
The analysis of induced sterility as a function
of irradiation dose was based on the proportion of
eggs hatching for the TUC strain. Egg hatch rate
was estimated from all reciprocal crosses in all
tests with the exception of the 70 Gy TUC-LAB
test, in which the data collection was missed (Ta-
ble 3). In each case 4 egg collections were obtained
during a 12 d period. The results indicated that a
treatment with 40 Gy reduces male fertility from
about 80% to 0.75% and higher doses produce to-
tal sterility. In females, the 40 Gy dose reduces


fertility to about 2% and higher doses prevent egg
laying. No differences were observed among egg
collection dates, indicating that fertility is not re-
covered after the irradiation.

DISCUSSION

The adaptation of insects to laboratory condi-
tions, mass rearing, and sterilizing by irradiation is
known to produce genetic and physiological effects
in strains (Shelly et al. 1994; Lance et al. 2000; Al-
phey 2002; Benedict & Robinson 2003). These fac-
tors can influence the efficiency of mass reared and
sterilized flies once they are released into the field
in support of control programs integrating the ster-
ile insect technique. Males of the Mexican fruit fly
Anastrepha ludens (Loew) produced in bio-facto-
ries, for example, start their sexual activity well be-
fore wild ones. This may pose a problem in conven-
tional strains involving the release of both sterile
males and females, as these may mate among


TABLE 3. NUMBER OF EGGS SCORED, NUMBER OF EGGS HATCHED AND PERCENTAGE EGG HATCH IN ALL RECIPROCAL
CROSSES AT VARIOUS RADIATION DOSES.

Mating
Treatment (male-female) No. pairs Hatched eggs Total eggs % hatch

0 Gy LAB'-TUC2 57 351 437 80.32
TUC-LAB 66 506 547 92.50
TUC-TUC 48 336 422 79.62
LAB-LAB 68 349 459 76.03
40 Gy LAB-TUC 62 4 536 0.75
TUC-LAB 68 1 46 2.17
TUC-TUC 64 425 460 92.39
LAB-LAB 66 0 0 0.00
70 Gy LAB-TUC 59 0 464 0.00
TUC-LAB 66 *
TUC-TUC 75 291 336 86.61
LAB-LAB 68 0 0 0.00
100 Gy LAB-TUC 65 0 625 0.00
TUC-TUC 64 316 398 79.40
TUC-LAB 56 0 0 0.00
LAB-LAB 56 0 0 0.00

*Missing data.
TUC = wild flies from Tucuman obtained from guava fruits; non-irradiated controls.
LAB = laboratory strain reared at the Estaci6n Experimental Agroindustrial Obispo Colombres.


March 2007







Allinghi et al.: Mating Compatibility between Wild and LabAnastrepha fraterculus 31


themselves before having the opportunity to mate
with wild counterparts (Moreno et al. 1991;
Hernandez et al. 2003). Liedo et al. (2002) observed
that laboratory-reared females of C. capitata have
greater mating propensity than wild females, and
their age of maximum mating activity is earlier.
Furthermore, Cayol (2000) reported that the high
densities of flies in breeding cages may affect court-
ship, and matings tend to be faster.
During the strain colonization process for SIT
application, the insects are faced with artificial
conditions very different from nature and may ex-
perience genetic changes due to genetic drift and
particular selective forces. These factors some-
times affect the efficiency of the SIT (Cayol 2000).
The irradiation treatment to induce sterility was
claimed to affect courtship behavior (Lux et al.
2002). Thus, the strain of A. fraterculus that is
reared under semi-mass rearing conditions at the
Obispo Colombres facility was evaluated under
conditions that imitate those in nature as a pre-
requisite to being used in control programs with
an SIT component. Outdoor field cages are an ac-
ceptable compromise between natural conditions
and a controlled laboratory experimental system
for monitoring strains (Robinson et al. 2002; FAO/
IAEA/USDA 2003).
The present results show that the behavior of
this laboratory strain is not substantially modi-
fied with respect to the natural population for
Horco Molle. Average copulation start time was
not statistically different between LAB and TUC.
The preferred position in the tree for mating was
conserved. Copulation duration was similar in
non-irradiated LAB and TUC, but irradiation
treatment significantly reduced this time. A simi-
lar trend was observed in C. capitata (Cayol et al.
1999), but the importance of this effect on the ef-
ficiency of the SIT is not clear. This is because
there is not a direct relationship between copula-
tion duration and the ability of males to transfer
sperm. However, matings that are too short might
increase the probability of female remating (FAO/
IAEA/USDA; 2003).
The estimated ISI (-0.03 to 0.07) and RSI (0.44
to 0.54) values suggest total compatibility be-
tween the laboratory strain and the natural pop-
ulation of A. fraterculus analyzed here. MRPI
(-0.05 to 0.05) did not differ from the expected, in-
dicating similar male mating competitiveness of
LAB and TUC. An important result linked to the
possibility of applying the SIT to controlA. frater-
culus is that compatibility and mating perfor-
mance of male LAB flies are not affected by irra-
diation for all tested doses. This results contrasts
with those of Cayol et al (1999), who observed
that under similar conditions LAB flies ofC. cap-
itata had reduced competitiveness (MRPI 0.09;
RSI 0.33) and compatibility ISI 0.31).
According to our FRPI estimates, LAB females
have higher mating propensity than TUC fe-


males. A similar result was observed in other te-
phritids (Cayol 2000; Liedo et al. 2002), which
suggests that LAB females are sexually more ac-
tive and less selective. However, this higher mat-
ing propensity of LAB females was reduced as the
applied irradiation dose increased.
Some authors observed that irradiated fe-
males do not lay eggs depending on the radiation
dose and the developmental stage at the time of
the irradiation treatment (Burditt et al. 1975; Ve-
lasco & Enkerlin 1982; Calkins et al. 1988). Ac-
cording to the present analysis of egg laying and
hatching, A. fraterculus females treated with 40
Gy oviposited a reduced number of eggs compared
to control females. Higher doses prevented all egg
laying. Moreover, the treatment with 70 Gy of
gamma irradiation applied 48 h before adult
emergence ensured 100% sterility both in males
and females. Furthermore, during the evaluation
period (12 d) there was no evidence of recovery of
fertility in females or males.
Recent results by Vera et al. (2006) indicate that
some A. fraterculus populations from different re-
gions in South America might be sexually incom-
patible and reproductively isolated, while Petit-
Marty et al. (2004) observed complete compatibil-
ity between TUC and several geographically iso-
lated populations from within Argentina. Alberti
et al. (2002) also concluded that TUC and other
populations from Argentina and southern Brazil
(Pelotas) are not differentiated genetically. There-
fore, it is expected that the LAB population from
Obispo Colombres facility will behave similarly
when facing natural populations from Argentina
and southern Brazil. The high compatibility of
LAB and TUC flies and the good competitiveness
of irradiated LAB males observed in the present
work encourage the application of the SIT at least
at a sub-regional level to controlA. fraterculus pop-
ulations from Argentina and southern Brazil.

ACKNOWLEDGMENTS
The authors thank R. Russo, M. E. Utg6s, A. Massa-
cane, C. Cagnotti, C. Langellotti, R. Sciurano, C. Mestre,
P. Fermani, G. Cagnoli, S. Goenaga, R. Sellaro, and
N. Rosetti for collaboration during the field cage tests.
J. C. V. is a member of Consejo Nacional de Investiga-
ciones Cientificas y T6cnicas (CONICET, Argentina).
This work was supported by the following grants: IAEA
Research Contract 9897 to B.O. Saidman, IAEA Re-
search Contract 1083, CONICET PIP 5122/05, UBA-
CYT 2004 X241, and ANPCYT PICT 6628 to J.C.V., and
Project 17, Programa de Radiaciones y Radiois6topos,
CNEA.

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Florida Entomologist 90(1)







Liedo et al.: Improving Mating Performance of Sterile Medflies


IMPROVING MATING PERFORMANCE OF MASS-REARED
STERILE MEDITERRANEAN FRUIT FLIES (DIPTERA: TEPHRITIDAE)
THROUGH CHANGES IN ADULT HOLDING CONDITIONS:
DEMOGRAPHY AND MATING COMPETITIVENESS


PABLO LIEDO, SERGIO SALGADO, AZUCENA OROPEZA AND JORGE TOLEDO
Departamento de Entomologia Tropical, El Colegio de la Frontera Sur, Apartado Postal 36
C. P. 30700, Tapachula, Chiapas, M6xico

ABSTRACT

Mass rearing conditions affect the mating behavior of Mediterranean fruit flies (medflies)
Ceratitis capitata (Wiedemann). We evaluated the effect of slight changes in the adult hold-
ing conditions of adult flies maintained for egg production on their mating performance. Col-
onization was initiated from wild flies collected as larvae from infested coffee berries (Coffea
arabica L.). When pupae were close to adult emergence, they were randomly divided into 3
groups and the emerging adults were reared under the following conditions: (1) Metapa Sys-
tem (MS, control), consisting of 70 x 45 x 15 cm aluminum frame, mesh covered cages, with
a density of 2,200 flies per cage and a 1:1 initial sex ratio; (2) Insert System (IS), with the
same type of cage, and the same fly density and sex ratio as in the MS treatment, but con-
taining twelve Plexiglas pieces (23 x 8.5 cm) to provide additional horizontal surface areas
inside the cage; and (3) Sex-ratio System (SS), same as IS, but in this case the initial male:
female ratio was 4:1. Three d later, newly emerged females were introduced, so the ratio be-
came 3:1 and on the 6th d another group of newly emerged females was added to provide a
2:1 final sex ratio, at which the final density reached 1,675 flies per cage. The eggs collected
from each of the 3 treatments were reared independently following standard procedures and
the adults were held under the same experimental conditions. This process was repeated for
over 10 to 13 generations (1 year). The experiment was repeated 3 times in 3 consecutive
years, starting each replicate with a new collection of wild flies. Life tables were constructed
for each treatment at the parental, 3rd, 6th, and 9th generations. Standard quality control
parameters (pupation at 24 h, pupal weight, adult emergence, and flight ability), were esti-
mated for each treatment every third generation in the third year. For the last generation
each year, mating competitiveness was evaluated in field cage tests with wild flies. As colo-
nization progressed, life expectancy and fecundity rates increased in the 3 rearing systems.
There was no significant difference in standard quality control parameters among the 3
rearing systems. Wild males always achieved more matings than any of the mass reared
males. Mating competitiveness of males from the IS, although surprisingly not from the SS,
was significantly greater than that of males from the MS. Our results indicate that these
slight changes in the adult holding conditions can significantly reduce the harmful effects of
mass rearing on the mating performance of sterile flies.

Key Words: Ceratitis capitata, sterile insect technique, colonization, mating behavior, insect
demography, mother colony

RESUME

Se ha demostrado que las condiciones de cria masiva afectan el comportamiento de aparea-
miento de la mosca del Mediterraneo Ceratitis capitata (Wiedemann). Nosotros evaluamos
el efecto de ligeros cambios en las condiciones en las que los adults son mantenidos para la
producci6n de huevos, en el desempeio de apareamiento de las moscas est6riles. La coloni-
zaci6n se inici6 con moscas silvestres colectadas como larvas en cerezas de caf6 (Coffea ara-
bica L.) infestadas. Cuando las pupas estuvieron cerca de la emergencia de los adults, se
dividieron en tres grupos al azar y los adults reci6n emergidos fueron criados en las siguien-
tes condiciones: (1) Sistema Metapa (MS, testigo), consistent enjaulas con marco de alumi-
nio de 70 x 45 x 15 cm, cubiertas con malla, con una densidad de 2,200 moscas por jaula y
una relaci6n de sexos inicial de 1:1; (2); Sistema Insertos (IS), con el mismo tipo de jaula,
densidad de moscas, y relaci6n de sexos que en el MS, pero conteniendo 12 piezas de plexi-
glas (23 x 8.5 cm) para proporcionar superficie horizontal al interior de lajaula; y (3) Sistema
de Relaci6n de Sexos (SS), igual que el IS, pero en este caso la relaci6n inicial macho: hembra
fue de 4:1, tres dias despu6s se introdujeron hembras reci6n emergidas para tener una rela-
ci6n de 3:1 y en el 6 dia se aiadi6 otro grupo de hembras para tener una relaci6n final de
sexos de 2:1, que equivale a una densidad final de 1,675 moscas porjaula. Los huevos colec-
tados de cada tratamiento fueron criados independientemente siguiendo los procedimientos
estandares y los adults fueron mantenidos en las mismas condiciones experimentales. Esto







Florida Entomologist 90(1)


se repiti6 por 10 a 13 generaciones (un aio). El experiment se repiti6 en tres ocasiones en
aios consecutivos, iniciando cada repetici6n con una nueva colecta de moscas silvestres. Se
construyeron tablas de vida de cada tratamiento en las generaciones parental, 3 6 y 9 Se
estimaron los parametros estandares de calidad (pupaci6n a las 24 h, peso de pupa, emer-
gencia de adults y habilidad de vuelo) para cada tratamiento, cada tercera generaci6n en el
tercer aio. En la iltima generaci6n de cada aio, se evalu6 la competitividad sexual en prue-
bas enjaulas de campo con moscas silvestres. Conforme avanz6 la colonizaci6n, se encontr6
que la esperanza de vida y las tasas de fecundidad se incrementaron en los tres sistemas de
cria. No hubo diferencia significativa en los parametros estandar de control de calidad entire
los tres sistemas. Los machos silvestres siempre lograron mas apareamientos que los ma-
chos procedentes de cada sistema de cria masiva. La competitividad de los machos del sis-
tema IS fue significativamente mayor que la de los machos del sistema MS. Nuestros
resultados indican que estas ligeras modificaciones en las condiciones de la colonia de adul-
tos reduce los efectos adversos de la cria masiva sobre el desempeno de apareamiento de los
machos est6riles.

Translation provided by the authors.


Since the early stages of the sterile insect tech-
nique (SIT), it was recognized that the mating
competitiveness of the sterile insects was a criti-
cal factor for the successful application of the
technique (Knipling 1955). Research results
showed that the exposure to irradiation for steril-
ization affected the mating performance of the
sterile fruit flies (Holbrook & Fujimoto 1970;
Hooper 1971; Ohinata et al. 1977; Knipling 1979;
Lux et al. 2002a). Later, it was found that both ir-
radiation and the selection that occurs during col-
onization could adversely affect the mating per-
formance of sterile flies (R6ssler 1975; Wong &
Nakahara 1978; Leppla et al. 1983; Wong et al.
1983; Harris et al. 1986).
In the case of the Mediterranean fruit fly (med-
fly) Ceratitis capitata (Wiedemann) however, it
has been shown that, despite a long time under
mass rearing conditions, sterile males are still ca-
pable of locating hosts, mating arenas or leks, and
mix and interact with their wild counterparts un-
der natural conditions (Zapien et al. 1983; Whit-
tier et al. 1992; Shelly & Whittier 1996; Katsoy-
annos et al. 1999). Also, it has been documented
that the courtship patterns of flies from different
geographical areas are sexually compatible
(Cayol et al. 2002; Lux et al. 2002b). However, it
has been shown that slight quantitative changes
in the courtship displays of males might result in
female rejection and that these changes could be
attributed to the selection that occurs under mass
rearing conditions. Male courtship behavior of
mass reared flies tends to be less elaborate, and
the degree to which it is affected was found to be
associated with the time under mass rearing con-
ditions (McInnis et al. 1996; Briceio & Eberhard
2002; Gaskin et al. 2002; Lux et al. 2002b; Robin-
son et al. 2002).
Harris et al. (1986) suggested that conditions
for mass rearing select for fast mating. Since most
flies in the rearing cages are of the same age and
reach sexual maturity at nearly the same time, we
speculated that the close to 1:1 "operational" sex


ratio favored short male courtships and less
choosy females, resulting in this fast mating be-
havior. Detailed observations of the mating behav-
ior of flies in the mass rearing cages showed that
male courtship was frequently interrupted (W.
Eberhard & D. Briceno, personal communication).
Under natural conditions, this fast mating be-
havior results in less competitive sterile males in
view of wild female mate choice, and therefore,
less effective programs integrating the SIT. The
goal of this study was to evaluate whether slight
changes in the colony holding conditions, where
adult flies are maintained for egg production,
could reduce this selection for fast mating and
thus produce more competitive flies. Two changes
from the standard mass-rearing procedures
(Schwarz et al. 1985) were tested: (1) horizontal
clear inserts were introduced inside the rearing
cages to increase the overall resting surface avail-
able and to imitate the undersurfaces of leaves
where males usually perform their courtship un-
der natural conditions (Prokopy & Hendrichs
1979), possibly reducing the frequency of court-
ship interruption; and (2) variation in the opera-
tional sex ratio by introducing the females into
the cages at four different times, so the number of
sexually mature males was always greater than
the number of sexually mature females (Calkins
1989).

MATERIALS AND METHODS

Biological Material

The study was initiated with wild flies col-
lected from naturally infested coffee berries (Cof-
fea arabica L.) in southwestern Guatemala. New
collections were made in each of 3 consecutive
years, each year being considered as a replicate of
the whole experiment. The location, amount of
coffee collected, and the approximate number of
larvae and adults obtained for each collection are
shown in Table 1.


March 2007







Liedo et al.: Improving Mating Performance of Sterile Medflies


TABLE 1. AMOUNT OF MATURE COFFEE BERRIES COLLECTED, APPROXIMATE NUMBER OF LARVAE AND ADULTS OB-
TAINED, AND LOCATION IN GUATEMALA OF COLLECTIONS.

Year of collection Location Coffee berries (kg) Larvae recovered Adults emerged

2000 Colomba 1,650 18,549 10,701
2001 Colomba 2,000 15,000 12,325
2002 Antigua 2,500 21,550 13,435


Male Sexual Competitiveness


Experimental work was carried out at the
Moscamed mass rearing facility in Metapa, Chia-
pas, Mexico. Standard rearing procedures and en-
vironmental conditions were used (Schwarz et al.
1985). 3 adult rearing systems were evaluated: (1)
Metapa System (MS, control), which consisted of
an aluminum frame, mesh covered cage (70 x 45 x
15 cm) with an initial density of 1,100 males and
1,100 females per cage, and an average surface
area of 3.91 cm2 per fly; (2) Insert System (IS), as
above but with the addition of 12 pieces of clear
plexiglas (polycarbonate) (23 x 8.5 cm) inside the
cage as horizontal surface areas, resulting in a
surface area of 5.85 cm2 per fly; and (3) Sex-ratio
System (SS), same as IS, but with an initial den-
sity of 1,100 males and 275 females (4:1 male: fe-
male ratio). Three d later 92 recently emerged vir-
gin females were introduced to make a 3:1 ratio,
and at the 6th day 183 recently emerged virgin fe-
males were introduced to make a 2:1 ratio, and a
total of 1,100 males and 550 females. The surface
area was 6.94 cm2 per fly.
Adults were fed ad libitum with a mixture of
enzymatic yeast hydrolysate (ICN Biomedical,
Costa Mesa, CA) and sucrose (1:3). Water was
provided in test tubes covered with cotton plugs.
On both sides at the bottom of the cages, water
channels were placed for egg collection. These
eggs were reared following the standard proce-
dures at the Metapa facility (Schwarz et al. 1985).

Demographic Analysis

To compute life tables, the number of dead flies
and the volume of eggs collected were recorded
daily from the cages. In addition, a sample of 30
pairs from each treatment, every third genera-
tion, was taken and placed in plastic cages (8 cm
diameter by 15 cm long, one male and one female
per cage) with food, water, and a 2-cm diameter
agar sphere (3 L of water + 80 g of agar dyed with
green food coloring and wrapped in Parafilm) as
an oviposition device (Boller 1968, Freeman &
Carey 1990). These spheres were replaced every
24 h and the number of eggs laid were recorded.
This was done until the last female in the cohort
died.


Each year, after 10 to 13 generations, field cage
mating tests with host trees were conducted
(FAO/IAEA/USDA 2003). In each cage, 50 wild fe-
males, 50 wild males, and 50 males of each rear-
ing system were released. Wild flies were 9-13 d
old and mass-reared sterile flies were 7-11 d old.
These ages were selected following the results of
Liedo et al. (2002). The tests were conducted at
coffee plantations in Guatemala during 5 consec-
utive d. Each d, 3 replicates (field cages) were set
up. The males were color marked on the thorax
for treatment identification.
In the third year, in addition to these mating
tests, the "Fried" field cage test was used (Fried
1971). In each cage, 50 wild females, 50 wild
males, and 150 sterile mass-reared males were
released (one field cage for each treatment), and
25 agar oviposition devices (as described above)
were placed inside each cage. After 24 h, the agar
devices were removed, and egg hatch was deter-
mined from the eggs obtained from these devices.
One hundred wild flies (1:1 male: female ratio)
were placed in a field control cage to collect eggs
and determine egg hatch without sterile fly com-
petition. Sterility induced was estimated from the
difference between egg hatch in the control and
egg hatch in competition. There were 3 cages per
treatment, and the test was run during 2 d, mak-
ing 6 replicates per adult holding system.

Standard Quality Control Tests

In the third year, standard quality control pa-
rameters (FAO/IAEA/USDA 2003) were deter-
mined for each treatment at the parental, 3rd,
6th, and 9th generations. The parameters evalu-
ated were pupal weight, adult emergence, and
flight ability.

Statistical Analysis

Life table demographic parameters used in this
study are defined by Carey (1993). Laboratory and
field tests followed the methods described in the
international quality control manual for tephritid
flies (FAO/IAEA/USDA 2003). Data from observed
proportions were transformed as I x + 0.5, and


Rearing Systems







Florida Entomologist 90(1)


subjected to analysis of variance (ANOVA), fol-
lowed by means separations by the Tukey test (P
< 0.05) (SAS Institute 1992).

RESULTS

Demographic Analysis

Survival rapidly increased through coloniza-
tion in the 3 treatments. Mean adult life expect-
ancy increased significantly from the parental to
the 3rd generation, then gradually increased or
remained stable in the following generations,
both in males and females. This trend was ob-
served when the flies were evaluated individually
(Fig. 1), although differences among generations
were not significant (F = 0.4355, P = 0.6556 for
males; F = 2.9684, P = 0.0801 for females). There
were no significant differences among rearing
systems (F = 0.0790, P = 0.9244 for males; F =
0.1569, P = 0.8561 for females) and there was no
significant interaction between rearing systems
and generations (F = 0.2214, P = 0.9225 for males;
F = 0.4244, P = 0.7888 for females).
When survival data were taken directly from
the rearing cages, there were highly significant
differences among generations (F = 7.7843, P =
0.0010 for males; F = 8.4050, P = 0.0006). How-
ever, the differences among rearing systems were
not significant (F = 1.4461, P = 0.2570 for males;
F = 0.5255, P = 0.5985 for females) and there were


) Males
so
i l



rinrt MS


also no interactions between generations and
rearing systems (F = 0.2589;P = 0.9502 for males;
F = 0.1551, P = 0.9859 for females) (Fig. 2).
Fecundity increased in a similar pattern. The
number of eggs laid per female increased signifi-
cantly from the parental generation to the 3rd
generation in all treatments, then gradually in-
creased every third generation. This was ob-
served both in the data collected from single pairs
(Fig. 3 top), as well as in those from the rearing
cages (Fig. 3 bottom). There was wide variation in
this parameter among treatments, particularly
during the first 3 to 6 generations, but no statisti-
cal differences among treatments were found (F =
2.2959, P = 0.1328 for single pairs; F = 0.0510, P
= 0.9504 for rearing cages). The difference among
generations was highly significant in both cases,
when the flies were obtained from single pairs (F
= 11.0348, P = 0.0009), and when data were col-
lected from the rearing cages (F = 35.8365, P =
1.208 x 10-8). The interaction between rearing sys-
tems and generations was not significant (F =
0.2406, P = 0.9111 for single pairs;F = 0.4048, P =
0.8678 for rearing cages). It is important to note
the demographic implications of the significant


Rta hO3 n6 aOn9


I MS

















I is
I SS


hrentl Gn.3 0GL.5 Gn.
Fig. 1. Life expectancy (e0) (days SE) of male (top)
and female (bottom) Mediterranean fruit flies from 3
different adult colony holding systems (MS = conven-
tional Metapa System, IS = Insert System, SS = Sex-ra-
tio System), estimated from single pair cages.


Ral an3 ntG an


Fig. 2. Life expectancy (e0) (days SE) of male (top)
and female (bottom) Mediterranean fruit flies from 3
adult colony holding systems (MS = conventional
Metapa System, IS = Insert System, SS = Sex-ratio Sys-
tem), estimated from rearing cages.


March 2007







Liedo et al.: Improving Mating Performance of Sterile Medflies


D
14W0


' jio1zoo Single pairs *
ion



la



Reanng cages c c






ma

-0n.I 6rn.3 G6 6 GCn-9


S50

- 40
40
3:

20


ri-
m s
-Se


IS SS MS


Fig. 4. Average percent of matings (% SE) in field
cage tests of Mediterranean fruit fly males reared under
3 different adult colony holding systems (MS = conven-
tional Metapa System, IS = Insert System, SS = Sex-ra-
tio System) (P < 0.05).


Standard Quality Control Tests


Fig. 3. Net fecundity rate (1lmJ) (eggs/female SE)
of Mediterranean fruit fly females from 3 adult colony
holding rearing systems (MS = conventional Metapa
System, IS = Insert System, SS = Sex-ratio System) at
four different generations. Estimated from single pair
cages (top) and from rearing cages (bottom).


differences between the parental and the 9th gen-
erations in both, survival and fecundity, in the 3
rearing systems.

Male Sexual Competitiveness

Results from the field cage mating tests during
the 3 years were rather consistent. Wild males
were always the most successful in terms of the
average percent of matings achieved and the dif-
ferences were statistically significant (F = 26.92;
df = 3, 6; P < 0.001) (Fig. 4). Among the 3 rearing
systems, there was a significant difference be-
tween the IS and the MS (control). The differ-
ences between the SS and the other 2 rearing sys-
tems were not significant.
The average mating index ( SE) estimated for
each rearing system, according to the interna-
tional quality control manual (FAO/IAEA/USDA
2003), also showed a significant difference be-
tween the IS and MS, and a non significant differ-
ence between the SS and the other 2 rearing sys-
tems (F = 35.08; df = 2, 6; P = 0.042) (Fig. 5).
Results from the Fried test showed that males
from the IS were the ones that induced the great-
est level of sterility (34%). Males from the SS and
MS treatments only induced 18.3 and 16.3% ste-
rility, respectively. However, differences among
treatments were not statistically significant (F =
32.87; df = 2, 15; P = 0.101). Fig. 6 shows the
average ( SE) level of sterility induced by each
treatment. Natural sterility was 13.3%.


The results of the standard quality control
tests applied to the 3 rearing systems at the pa-
rental, 3rd, 6th, and 9th generations are shown in
Fig. 7. All these values were within acceptable in-
ternational ranges (FAO/IAEA/USDA 2003).
There was a significant increase in pupal weight,
from the parental flies to the mass reared flies. In
the other 2 parameters, there were no significant
differences among generations, although a simi-
lar pattern can be observed.
Pupal weight in the 3rd generation was
greater in the SS compared with the other 2 treat-
ments (F = 0.84; df = 2, 9; P < 0.001). There were
no significant differences at the 6th generation (F
= 1.36; df= 2, 9;P = 0.052). In the 9th generation,
the IS produced the heaviest pupae (F = 1.62; df=
2, 9;P = 0.011).
Mean adult emergence was greater in the IS
and SS than in the MS at the 3rd (F = 4.66; df =


0.0o5


o.00


Fig. 5. Average mating index ( SE) for 3 adult colony
holding systems (MS = conventional Metapa System, IS
= Insert System, SS = Sex-ratio System). This index was
estimated following the quality control manual (FAO/
IAEA/USDA 2003).







Florida Entomologist 90(1)


j 40.0
44
g 30.0

W 20.0

4 10.0


Fig. 6. Sterility levels (mean SE) induced by sterile
Mediterranean fruit fly males reared under 3 different
adult colony holding systems (MS = conventional
Metapa System, IS = Insert System, SS = Sex-ratio Sys-
tem) when competing with wild males in field cages (the
natural sterility in the control was 13.3%).


2, 9; P = 0.013) and 6th generations (F = 9.36; df
= 2, 9; P = 0.021). Differences in this parameter
were not statistically significant at the 9th gener-
ation (F = 9.08; df = 2, 9; P = 0.301).
There were no significant differences among
treatments in flight ability at the 3rd (F = 2.43; df
= 2, 9; P = 0.655) and 9th (F = 3.67; df = 2, 9; P =
0.231) generations. At the 6th generation, flight
ability was significantly greater in the IS than in
the MS (F = 5.64; df = 2, 9; P = 0.037).

DISCUSSION

Demographic data confirm that mass-reared
flies have greater reproductive rates than wild
flies (Liedo & Carey 1996) and show that coloni-
zation for mass-rearing is a selection process in
which insects adapt to the new rearing conditions


w


Parental Gen. 3 Gen. Gen.9


Parental Gen. 3 Gen. 6 Gen.


Fig. 7. Standard quality control tests: (A) pupal weight (mg), (B) adult emergence (%), (C) flight a l. II;. .. *the
3 adult colony holding systems (MS = conventional Metapa System, IS = Insert System, SS = Sex-ratio System).


Parental Gen.3 Gen. 6 Gen.9


(Leppla et al. 1983; Leppla 1989). For mass rear-
ing purposes, this is desirable and necessary in
order to produce large number of insects in an ef-
ficient manner. However, this same selection pro-
cess can result in negative effects on other biolog-
ical attributes, such as mating behavior.
The results from the single pair cages and the
rearing cages showed that the conditions in which
flies are held affect the demographic parameters
obtained, with greater values at the single pair
cages than at the more stressful adult holding
cages. However, in both cases, the general trends
were similar, with mean expectation of life and
net reproductive rates increasing with genera-
tions, as the flies gradually adapted to the
crowded mass-rearing conditions. At the same
time only very small or no differences were found
among rearing systems.
Our results from the field cage mating tests
corroborate that mass-rearing adversely affects
the mating competitiveness of the reared insects
compared to wild flies (Wong & Nakahara 1978;
Wong et al. 1983; McInnis et al. 1996). The intro-
duction of horizontal inserts in the rearing cages
contributed to a significantly better mating per-
formance of the IS mass-reared insects when
compared to the standard-produced MS males.
Although there were no significant differences in
the level of sterility induced (Fried test), the pat-
tern was similar (IS > SS > MS). This suggests
that the number of matings recorded during the
observation period in the field cage mating test is
correlated with the induction of sterility in the
wild population and that males from the IS were
more competitive than males from the other 2
rearing systems.
The manipulation of the sex ratio did not have
a significant effect on the mating performance of
mass-reared flies. This result was unexpected. We


March 2007


0.0 I.







Liedo et al.: Improving Mating Performance of Sterile Medflies


were expecting that the biased sex ratio in favor
of males would allow females to be more selective
and result in more competitive males. One expla-
nation for this could be the reduced offspring pro-
duced by the smaller number of females and as a
result of harassment of ovipositing females by the
excess of males in the cage; however, there are
other potential causes that need to be investi-
gated. The small number of offspring was partic-
ularly critical in the second year. Manipulation of
operational sex ratio in adult holding cages is now
feasible due to the current availability of genetic
sexing strains. We believe that this research line,
and the interaction with increased surface area in
cages, should be further explored.
Data from the standard quality control tests
demonstrate that the 3 colonization methods
have no detrimental effect on most of these pa-
rameters. Pupal weight was the only attribute
that significantly changed (increased) through
colonization. These findings suggests that while
the demographic and mating attributes, as well
as pupal weight, were under selection pressure
during colonization, this was not the case for at-
tributes such as adult emergence and flight abil-
ity. This raises the question of whether other bio-
logical attributes could be under selection pres-
sure during colonization (Harris 1988; Calkins
1989; Miyatake & Haraguchi 1996). Rodrigueiro
et al. (2002) reported differences between wild
and mass-reared medflies in some morphological
traits. Lux et al. (2002b) found quantitative dif-
ferences in the courtship behavior of wild and
mass-reared Mediterranean fruit fly males. The
biological attributes that show significant differ-
ences between wild and mass-reared flies deserve
further research.
In the current study, we started all 3 treat-
ments from wild collected flies. It will be interest-
ing to investigate whether the introduction of in-
serts might have a reverse effect. Will a long term
mass-reared strain increase its competitiveness if
horizontal inserts are introduced to the mass rear-
ing process, without starting a new colony from
wild flies? Based on our results, the introduction
of inserts in the rearing cages is strongly recom-
mended, because its represent a minor change in
the production process, with negligible costs, and
important benefits in the application of the SIT.

ACKNOWLEDGMENTS

We thank J. P. Cayol, T. Shelly, E. Jang, and D. McIn-
nis for critical review of earlier drafts of this paper. We
are grateful to Ezequiel de Le6n, Gustavo Rodas, Reyna
Bustamante, Arnoldo Villela, Sandra Rodriguez and Ro-
drigo Rinc6n (ECOSUR-Mexico) for technical assis-
tance. Special thanks to Antonio Villasenor, Director of
Moscamed-Moscafrut Program in Chiapas, for support-
ing this research. Our appreciation to Pedro Rend6n
and Felipe Ger6nimo (USDA/ARS, Methods Develop-
ment Section, Guatemala) for their help with collection


of wild flies and setting up the field cage tests in Guate-
mala. Thanks to Pablo Matute and Felix Acajab6n
(USDA/ARS, Methods Development Section, Guate-
mala) for help in handling of biological material. We ac-
knowledge support from the Programa Moscamed in
Guatemala. This project was funded by the Interna-
tional Atomic Energy Agency Research Contract 10,774,
and Sistema de Investigaci6n Benito Juarez (SIBEJ-
Mexico, proyecto 19990501035).

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Florida Entomologist 90(1)







Barnes et al.: Quality Assurance in Medfly Production


PRODUCTION AND QUALITY ASSURANCE IN THE SIT AFRICA
MEDITERRANEAN FRUIT FLY (DIPTERA: TEPHRITIDAE)
REARING FACILITY IN SOUTH AFRICA


BRIAN BARNES', SAADIEK ROSENBERG2, LUCIANO ARNOLDS2 AND JEROME JOHNSON2
'Plant Protection Division, ARC Infruitec-Nietvoorbij Fruit, Vine and Wine Institute
Stellenbosch, 7599 South Africa

2SIT Africa (Pty) Ltd., Stellenbosch, 7599 South Africa

ABSTRACT

A mass-rearing facility for Mediterranean fruit fly Ceratitis capitata (Wiedemann) was com-
missioned in Stellenbosch in 1999 to produce sterile male fruit flies for a sterile insect tech-
nique (SIT) project in commercial fruit orchards and vineyards in the Western Cape province
of South Africa. The mass-rearing procedure was largely based on systems developed by the
FAO/IAEA Agriculture and Biotechnology Laboratory, Seibersdorf, Austria. A number of ge-
netic sexing strains were used to produce only males for release. Initial cramped rearing and
quality management conditions were alleviated in 2001 with the construction of a new adult
rearing room and quality control laboratory. In 2002 a comprehensive Quality Management
System was implemented, and in 2003 an improved genetic sexing strain, VIENNA 8, was
supplied by the FAO/IAEA Laboratory in Seibersdorf. For most of the first 3 years the facility
was unable to supply the required number of sterile male Mediterranean fruit flies for the
SIT program without importing sterile male pupae from another facility. From mid-2002, af-
ter the quality management system was implemented, both production and quality im-
proved but remained below optimum. After the introduction of the VIENNA 8 genetic sexing
strain, and together with an improvement in the climate control equipment, production sta-
bility, and quality assurance parameters improved substantially. The critical factors influ-
encing production and quality were an inadequate rearing infrastructure, problems with the
quality of the larval diet, and the initial absence of a quality management system. The re-
sults highlight the importance of effective quality management, the value of a stable and
productive genetic sexing strain, and the necessity for a sound funding base for the mass-
rearing facility.

Key Words: genetic sexing strain, mass rearing, Mediterranean fruit fly, sterile insect tech-
nique, quality management

RESUME

La facilidad para criar en masa la mosca mediterranea de la fruta, Ceratitis capitata (Wie-
demann) fue comisionada en Stellenbosch en 1999 para producer machos est6riles de moscas
para el proyecto de la t6cnica del insecto est6ril (TIE) en huertos de frutos y vifias comercia-
les en la provincia del Cabo Occidental del Sudafrica. El procedimiento de criar en masa fue
en su mayor parte basado en los sistemas desarrollados por el Laboratorio de Agricultura y
Biotecnologia de la FAO/IAEA, Seibersdorf, Austria. Un numero de razas que separara los
sexos gen6ticamente fueron utilizadas para producer solo machos para la liberaci6n. La con-
gestionada condici6n inicial para criar las moscas y su manejo de calidad fueron aliviadas en
2001 con la construcci6n de un nuevo cuarto de cria para adults y un laboratorio de control
de calidad. En 2002, un Sistema de Manejo de Calidad comprensivo fue implementado, y en
2003 una raza mejorada que separa los sexos gen6ticamente, VIENNA 8, fue proveido por el
Laboratorio de la FAO/IAEA en Seibersdorf. En la mayor parte de los primeros 3 aios la fa-
cilidad no pudo suplir el numero requerido de machos est6riles de la mosca mediterranea de
la fruta para el program de TIE sin la necesidad para importar machos est6riles de otra fa-
cilidad. Desde medio del ano de 2002, despu6s que el sistema de manejo de calidad fue im-
plementado, la producci6n y la calidad mejoraron pero aun quedaron por debajo del nivel
6ptimo. Despu6s de la introducci6n de la raza VIENNA 8 que separa los sexos gen6tica-
mente, yjunto con el equipo mejorado de control de clima, la estabilidad y los parametros de
seguridad de calidad mejoraron substancialmente. Los factors critics que influyeron en la
producci6n y la calidad fueron la infraestructura inadecuada para criar las moscas, proble-
mas con la calidad de la dieta para las larvas y la ausencia inicial de un sistema de manejo
de calidad. Los resultados muestran claramente la importancia de un manejo efectivo de la
calidad, el valor de una raza productive que separa los sexos gen6ticamente y la necesidad
de contar con una base s6lida de financimiento para la infraestructura de una cria en masa.







Florida Entomologist 90(1)


The export deciduous fruit industry is of great
economic importance to South Africa. Nearly 90
million cartons are exported annually, with total
earnings of approximately US$1 billion per an-
num. The Western Cape is the most important re-
gion for the production of deciduous fruit, with
approximately 58,000 ha under cultivation (Opti-
mal Agricultural Business Systems 2005).
The Western Cape is host to 2 species of te-
phritid fruit flies of economic importance, the
Mediterranean fruit fly (medfly) Ceratitis capi-
tata (Wiedemann), and the Natal fruit fly Cerati-
tis rosa (Karsch). Between them, they attack a
wide variety of subtropical, tropical, and decidu-
ous fruits (Annecke & Moran 1982). Both species
are international quarantine pests with the po-
tential to restrict international fruit trade with
South Africa. Further details of their occurrence,
behavior, and management in the Western Cape
is given by Myburgh (1964) and Barnes (1994). It
has been estimated that crop losses and control
costs due to fruit flies in the Western Cape alone
exceed US$3.2 million per annum (Mumford &
Tween 1997). While the economic impact of te-
phritid fruit flies country-wide has not been de-
termined, the impact on the South African export
fruit industry of a quarantine embargo on South
African fruit due to the presence of fruit flies
would be devastating. For the South African ex-
port fruit industry to remain viable, the creation
of fruit fly-free or low prevalence areas is there-
fore an urgent necessity. The sterile insect tech-
nique (SIT), integrated with other measures, is
widely regarded as the most practical and cost-ef-
fective means of establishing such areas.
A pilot project to suppress C. capitata in an iso-
lated export table grape production area in the
Western Cape, the Hex River Valley, with an SIT
component was initiated in 1997. Sterile C. capi-
tata were produced in a mass-rearing facility lo-
cated at the Infruitec-Nietvoorbij Fruit, Vine and
Wine Research Institute of the Agricultural Re-
search Council (ARC) in Stellenbosch, with a
number of different temperature-sensitive lethal
(tsl) genetic sexing strains (Franz 2005). Produc-
tion at the facility started in Apr 1999, with aerial
releases of sterile males over 10,000 ha starting
in Oct that year. Further details of the pilot
project are described by Barnes et al. (2004).
Aerial releases over the entire 10,000 ha were
later replaced by ground releases due to the high
cost of aerial releases in view of the relative small
release area.
The SIT operations were not supported finan-
cially by the national government, although the
provincial government sporadically funded the
mass-rearing facility. As a result, the facility was
initially financed through a formal SIT Partner-
ship between the ARC, which coordinated the SIT
component of the program, and the Deciduous
Fruit Producer's Trust, a fruit-grower organiza-


tion. Continued lack of national support, together
with the inability of the SIT Partnership to con-
tinue funding the mass-rearing facility, led to the
commercialization of the production and distribu-
tion of sterile medflies via SIT Africa (Pty) Ltd. in
2003. The SIT programme has since expanded to
2 other production areas, and at the time of writ-
ing a total of 6 million sterile male medflies per
week were being ground-released, specifically
targeting backyards and host plants, over a total
fruit production area of 15,600 ha.
Production volumes and quality parameters of
sterile medflies produced by the rearing facility
were well below optimum and varied a great deal
during the course of the program. This article de-
scribes the rearing process, the genetic sexing
strains used, and the production and quality pa-
rameters achieved over a period of 1 to 5 years,
and discusses causes of the poor rearing perfor-
mance and the factors that led to improved pro-
duction and quality in the facility.

MATERIALS AND METHODS

Genetic Sexing Strains

A number of genetic sexing strains obtained
from the Entomology Unit, FAO/IAEA Agricul-
ture and Biotechnology Laboratory, Seibersdorf,
Austria, were used to produce sterile male med-
flies. In such strains, the females carry a tsl mu-
tation that results in their mortality as embryos
by heat treatment so that females can be elimi-
nated before mass rearing of the males destined
for sterilization and release (Franz et al. 1994).
This results in greater cost-effectiveness as only
the males are the active agent in the SIT (Hen-
drichs et al. 1995). In addition, the females are
homozygous for the mutation white pupae (wp).
This allows the integrity of the sexing system and
the accuracy of the temperature treatment to be
monitored and is required for a Filter Rearing
System to manage the mother colony (Fisher &
Caceres 2000).
The genetic sexing strain VIENNA 7-97 was
initially used when mass-rearing started in Apr
1999. This strain was replaced in Aug/Sep 1999
with a refreshed genetic sexing strain, VIENNA
7/Mix-99 (Fisher 1999) that was initially used for
the first sterile male releases that started in Oct
1999. Due to genetic instability of this genetic
sexing strain under local rearing conditions, espe-
cially in the absence of a filter rearing system, the
colony strain was replaced three times between
May 2000 and Dec 2001, with strains VIENNA 7/
Tol 2000 in May 2000, VIENNA 7/Mix 2000 in
Nov 2000, and VIENNA 7-D53/Mix 2001 in Dec
2001 (Robinson et al. 1999).
A filter rearing system to control the accumu-
lation of genetic recombinants (Franz 2002) in the
genetic sexing strain was set up in mid-2000. In


March 2007







Barnes et al.: Quality Assurance in Medfly Production


this system, recombinant individuals (females in
brown pupae and males in white pupae; termed
'wrong sex') are removed from a mother colony
maintained under more relaxed conditions. Pro-
duction of sterile males with a filter rearing sys-
tem comprised three reproductive 'streams'-the
filter (or mother colony), amplification 1, and am-
plification 2. A fourth non-reproductive male-only
(or release) stream produced all the males for
sterilization and release (Fisher & Caceres 2000).
In Sep 2003 a new genetic sexing strain with
improved production and quality potential, VI-
ENNA 8, was provided to the SIT Africa Facility
by the Entomology Unit, FAO/IAEA Agriculture
and Biotechnology Laboratory, Seibersdorf, Aus-
tria. This facility was the first operational C. cap-
itata facility to be provided with this strain.

Production and Release Procedure

Production. Initially, an old, disused building
at the Pest Management Division of ARC In-
fruitec-Nietvoorbij was refurbished and used as a
sterile fruit fly production facility. In Apr 2001, a
new building was erected to house the adult col-
ony and a quality control laboratory, alleviating
cramped rearing conditions in the old building
and providing for better management of quality
control. This raised the maximum production po-
tential to an estimated 10 million sterile males
per week.
The mass-rearing procedure was largely devel-
oped by the FAO/IAEA Laboratory in Seibersdorf.
Larvae were reared on the following artificial
diet: digestive bran (28.75%), torula yeast
(7.00%), white sugar (13.00%), sodium benzoate
(0.25%), 30% hydrochloric acid (1.50%), formalin
(0.08%) and water (49.43%). The pH of the diet
was buffered to between 3.2 and 3.5. Five kg of
diet were placed in each rearing tray, and 3.2 mL
of eggs (for the colony stream) and 12.5 mL of eggs
(male-only stream) were seeded per tray on an
egg raft of toilet tissue.
After seeding, the trays were moved to Larvae
Room #1 (25C, 90-100% R.H.). After 3 d the trays
were moved to Larvae Room #2 (22C, 75-80%
R.H.). After 3 d, trays were then moved to Larvae
Room #3 (20C, 65-70% R.H.), where mature lar-
vae left the medium and were collected in water-
filled gutters for either 5 d (colony production) or
2 d (male-only production). Each day's larval col-
lection was mixed with fine vermiculite and kept
at 20C and 80-85% RH for pupation.
In the adult room, egging cages measuring
0.74 m x 0.84 m in cross section and 2 m in height
were surrounded on all 4 sides with fine mesh
screen through which the females oviposited. The
cages were mounted on wheels that ran on rails in
a water bath. Each cage was loaded with 3.2 liters
of pupae at a male:female ratio of 1:3, resulting in
a total cage content of approximately 186,600


flies. Adult food consisted of a 1:3 mixture of enzy-
matic yeast hydrolysate (Separations, Johannes-
burg, South Africa) and sugar, and water was pro-
vided through soft cloths protruding through
pipes filled with water. Eggs oviposited through
the screen sides fell into the water bath that was
drained once a day and the eggs were collected in
a sieve. Each cage was kept in production for 12 d.
Conditions in the adult room were maintained at
25C and 60-65% R.H., with a photoperiod of
15.5:8.5 (L:D). All eggs were bubbled in water con-
taining 0.1% sodium benzoate for 48 h under the
same conditions. Eggs for the male-only stream
were additionally heat-treated at 34C for 16 h,
which killed the female eggs.
Irradiation. Male pupae were sterilized 1 d be-
fore adult emergence (based on eye color; FAO/
IAEA/USDA 2003) with 90 Gy from 60Co in the
ARC Infruitec-Nietvoorbij walk-in irradiator. It
was equipped with a sealed point-source of 60Co
(Mayak Production Association, Ozyorsk, Russia)
stored in a below-floor, lead-filled drum that was
raised hydraulically to the required level when
needed. The dose-rate was 5.5 Gy/min, initially
determined with Fricke dosimeters and verified
by Gafchromic dosimetry (IAEA 2004). Pupae
packed in plastic bags (sample size = 180 mm x
150 mm; volume = 5 L) were irradiated under hy-
poxia on a rotating table (diameter 1.0 m; 60 s for
1 rotation) fixed around the vertical axis of the
60Co source lifting rods. Eight rotating discs (di-
ameter 0.2 m; 20 s for 1 rotation) were built into
the table around its perimeter. The resulting dual
rotation of the pupae facilitated optimum dose
distribution throughout the sample. The dose was
verified by Sterin or RadTag indicators in each
container of pupae.
Irradiated pupae were dyed with Day-Glo
fluorescent dye (Radiant Color, Houthalen, Bel-
gium) and were placed in paper bags (110 mL per
bag) in Plastic Adult Release Containers ("PARC
boxes"). During the period of aerial releases (VI-
ENNA 7 genetic sexing strain) this yielded ap-
proximately 4,250 fliers/bag at 65% flight ability.
During later ground releases (with VIENNA 8 ge-
netic sexing strain) an improved flight ability of
75% yielded approximately 5,000 fliers/bag.
Food for the flies was provided by means of
cakes of food grade agar plus sugar placed onto
gauze vents on top of each PARC box for aerial re-
leases, or for ground releases, by brown paper
strips soaked in the agar and sugar mixture and
placed into each bag. After poor performance of
the food strips, food was later provided more effec-
tively by means of small agar and sugar cakes in
plastic containers (50 mm diameter, 20 mm deep)
placed in the bottom of the bag.
Release. Details of aerial releases over the Hex
River Valley are given by Barnes et al. (2004).
These were replaced by ground releases in Jun
2003. For ground releases, bags of sterile flies







Florida Entomologist 90(1)


were transported to the release areas from 3 to 5
d after emergence and released the following day.
All flies were released in fruit fly host plants in
gardens and backyards, and in any other ne-
glected fruit trees, at a density of 2,000 flies per
hectar (Ortiz-Moreno 2002). This release system
has resulted in effective suppression ofC. capitata
in the Hex River Valley at a reduced cost to the
growers (I. Sutherland, SIT Africa [Pty] Ltd., Stel-
lenbosch, South Africa, personal communication).
The targeted nature of ground releases re-
sulted in a decrease in demand for sterile med-
flies in the Hex River Valley to 1.4 million sterile
flies per week. In Dec 2003 releases of sterile flies
started in a second area (Elgin, Grabouw, Vye-
boom and Villiersdorp) increasing the demand to
4.2 million sterile flies per week. In Aug 2004 a
third area (Riebeek Valley) joined the SIT pro-
gram, and the total requirement for releases was
increased to 6 million sterile flies per week. The
facility's production output goal was not reduced
following the decrease in demand for sterile flies -
the facility management decided rather to have
an output safety 'cushion' to accommodate any
unexpected decrease in production.

Production Performance and Quality Assurance

All rearing procedures and quality control
measurements were carried out according to the
international fruit fly quality control manual
(FAO/IAEA/USDA 2003). Up to Dec 2001 the fa-
cility had no formal quality management system.
Following consistent problems encountered with
production volumes and sterile fly quality, a com-
prehensive Quality Management System was in-
troduced in Jan 2002.
The quality management system covered two
main aspects: production and quality assurance.
A weekly review meeting evaluated each of these
aspects according to set targets as follows: (1)
Production (all reproductive streams)-number
of adult cages set up, egg production (mL per day),
number of trays seeded, number of pupae irradi-
ated, number of sterile flies delivered; (2) Quality
control (all streams)-egg hatch (%; 0 h and 48 h),
egg to pupa efficiency (%); flight ability (%), ge-
netic recombination (males in white pupae; %), fe-
males in the male-only stream (%), sterility of
males for release (% fertility). Definitions of these
parameters and the methods of assessment are
given in FAO/IAEA/USDA (2003). Other quality
parameters, e.g., of raw diet ingredients and wa-
ter, were not at that point incorporated into the
quality management system.
For the purposes of this article only the follow-
ing production parameters are discussed: produc-
tion-daily egg production, and number of pupae
irradiated per week; quality assurance-gg
hatch (48 h), egg to pupa efficiency in the male-
only stream, flight ability of sterile males, and


percentage females in the male-only stream (as
an indication of genetic recombination of the ge-
netic sexing strain). Some records from the ear-
lier part of the programme, before the new quality
control laboratory was established, are incom-
plete and the data unreliable. Results are there-
fore given from as far back in each case as they
were considered reliable. Data are presented as
means SD.

RESULTS

Egg Production

Daily egg production with the genetic sexing
strains VIENNA 7/Mix 2000 and VIENNA 7-D53/
Mix 2001 from Jan 2001, and with genetic sexing
strain VIENNA 8 from Aug 2003 to Sep 2004, is il-
lustrated in Fig. la and b. A target of 495 mL of
eggs per day was initially set in order to achieve
production levels of five million sterile males per
week for aerial releases over the Hex River Valley.
As illustrated by Fig. la between Jan 2001 and
Aug 2002, this target was seldom achieved. From
Aug 2002 the target was exceeded virtually with-
out exception. After the introduction of VIENNA
8, the daily egg production target was reduced to
360 mL per day due to the better egg to pupa effi-
ciency of this strain. Fig. lb shows that with VI-
ENNA 8, this target also was exceeded on all but
one occasion (Feb to Mar 2004). Relatively wide
fluctuations in egg production from Sep to Dec
2003 narrowed noticeably thereafter, probably as
a result of adaptation by the new strain to local
conditions.

Egg Hatch

Egg hatch after 48 h in the male-only stream is
illustrated in Fig. 2a (VIENNA 7-D53/Mix 2001)
and Fig. 2b (VIENNA 8). Except for a sharp drop
in Sep/Oct 2002, egg hatch in the VIENNA 7
strain fluctuated (mean = 55.3 9.65%) for most
of the reported period until Mar 2003, when it
dropped an average of 5% below the target level.
In the case of VIENNA 8, mean egg hatch was
somewhat lower at 38.8 11.01%, but very close
to the target of 40% for this strain (C. Caceres,
FAO/IAEA Biotechnology Laboratory, Seibers-
dorf, Austria, personal communication).

Egg to Pupa Efficiency

Egg to pupa efficiency in the male-only stream
is illustrated in Fig. 3a (VIENNA 7D53/Mix 2001)
and Fig. 3b (VIENNA 8). The target egg to pupa
efficiency for the VIENNA 7 strain was 12%. Al-
though the FAO/IAEA (2002) target for the VI-
ENNA 8 strain is 20%, the rearing facility set an
interim target of 16%. Efficiency in the VIENNA
7 strain fluctuated between 5 and 15%, with a


March 2007






Barnes et al.: Quality Assurance in Medfly Production


1200
1200 strain s strain
Vienna 7/Mix 2000 Vienna 7-D53/Mix 2001
V 7Vienna 7
E 900 --- -- -- - - -- - Target = 495 ml
Mean = 439.5 ml
SD = 313.4

0
R 600
to

300



0





1,000
Vienna 8
Target = 360 ml
S800 Mean = 567.8 ml
SD= 156.05

O 600


o 400


5 200

b
0 ---


/ /Z,,Z.;.,, Z(0-11 ZIP 4", d/,

Fig. 1. Daily egg production (14-day moving average) by two genetic sexing strains of C. capitata; (a) by two
strains of VIENNA 7 from Jan 2001to Jul 2003, and (b) by VIENNA 8 from Aug 2003 to Sep 2004.



mean of 11.2 4.60% and was characterized by from Dec 2003 to Jan 2004 and from Mar to May
three periods of substantial decreases in effi- 2004, both as a result of equipment malfunction.
ciency. There were only 2 periods of relative sta-
bility in efficiency, between May and Sep 2002 Number of Pupae Irradiated per Week
and Mar and Jul 2003. After the introduction of
the VIENNA 8 strain, egg to pupa efficiency im- The number of pupae irradiated per week from
mediately improved to a mean of 16.8 4.18%. Oct 1999 to Sep 2004, as an illustration of produc-
Two significant decreases in efficiency occurred tion of sterile flies by the facility, is presented in


/







Florida Entomologist 90(1)


100
Vienna 7
Target = 55%
S80 Mean = 55.3%
S-SD= 9.65

60


I 40
-C
20

a
0







100
Vienna 8
Target = 40%
80 Mean= 38.8%
o SD- 11.01

S60





20

b
0




Fig. 2. Egg hatch at 48 h (7-day moving average) of two genetic sexing strains of C. capitata; (a) VIENNA 7-D53/
Mix 2001, from Apr 2002 to Jul 2003, and (b) by VIENNA 8 from Sep 2003 to Sep 2004.




Fig. 4. With few exceptions, production by the fa- pupae per week for the next 10 months, albeit with
cility was very poor and variable during the first 1 major slump in Jan and Feb 2003. In Jun 2003
two and a half years until mid-2002. On only three production was deliberately decreased to approxi-
occasions, Jun and Aug 2001 and Nov/Dec 2002, mately one million sterilized pupae per week when
did production match demand. During this period, aerial releases were changed to ground releases.
sterile C. capitata pupae were frequently imported Production was then increased to 4 million and
from the El Pino facility in Guatemala to supple- later 6 million sterilized pupae per week between
ment aerial releases. From Aug 2002, production Dec 2003 and Aug 2004 to accommodate addi-
steadily increased to more than 8 million sterilized tional fruit production areas implementing SIT.


March 2007






Barnes et al.: Quality Assurance in Medfly Production


Vienna 8
Target = 16%
Mean = 16.8%
SD = 4.18

_\A


4 1',,


Fig. 3. Percentage egg to pupa efficiency (7-day moving average) in the male-only stream for (a) VIENNA 7-D53/
Mix 2001 genetic sexing strain from Jan 2002 to Jul 2003, and (b) VIENNA 8 genetic sexing strain from Aug 2003
to Sep 2004.


Flight Ability of Sterile Males
Flight ability of sterile males is given in Fig. 5a
(VIENNA 7-D53/Mix 2001) and Fig. 5b (VIENNA
8). The target flight ability for the VIENNA 7 and
VIENNA 8 strains was 75% (C. Caceres, Seibers-
dorf, Austria, personal communication). A target


of 82% for the VIENNA 8 strain is specified by
FAO/IAEA (2002), but this is for the smaller col-
ony reared by the IAEA at Seibersdorf. Flight
ability for the VIENNA 7 strain fluctuated be-
tween 60 and 85%, with a mean of 73.0 10.92%.
Flight ability improved with the VIENNA 8
strain, increasing to a mean of 78.3 7.37%.


$


4


/2O
20

*t 15-
is
CO
10


o 5-
u-l


01


~4h"


t





- -- -- - -- -







Florida Entomologist 90(1)


March 2007


1999 2000 2001 2002 2003 2004

Fig. 4. Number in millions of C. capitata pupae irradiated from Oct 1999 to Sep 2004 (3-week moving average).
The target number of sterile flies for release during different periods is indicated by dotted lines.


Females in Male-Only Stream

The occurrence of females in the male-only
stream is summarized in Fig. 6a (VIENNA 7-D53/
Mix 2001) and Fig. 6b (VIENNA 8). As sterile
stings by females in commercial fruit can lead to
infection by pathogens and secondary pests, a
maximum level of 2% females in the male-only
stream was set. From Dec 2002 to Apr 2003, the
occurrence of females from the VIENNA 7 strain
varied from about 1 to 3%, exceeding the maxi-
mum nearly 30% of the time. From Apr 2003, a
steady increase in females of up to 11% was re-
corded until just before the introduction of the VI-
ENNA 8 strain. The mean was 2.63 2.46%.
Following the introduction of VIENNA 8 in
Aug 2004, the occurrence of females in the male-
only stream dropped dramatically to a maximum
of less than 0.4%, with females being recorded on
only three occasions in 13 months. The mean was
0.02 0.09%.

DISCUSSION

During the period 1999 to mid-2002, produc-
tion of sterile C. capitata by the facility was sel-
dom sufficient to supply the requirements for
aerial releases in the Hex River Valley, i.e., 5 mil-
lion per week from Sep to May and 1 million per
week from Jun to Aug. From mid-2002, after the
positive effect of the implementation of the qual-


ity management system took effect, production
was generally adequate with only one exception.
Production and quality further improved and sta-
bilized after the introduction of the VIENNA 8
strain in Aug 2003. Many factors contributed to
the initial sub-standard production and quality
parameters, the most important of which were
the following:
(1) Lack of adequate funding. In the absence of
sustained government funding for opera-
tional expenses, and with an inadequate bud-
get, the mass-rearing facility was constantly
under financial duress. This negatively af-
fected the integrity of the entire mass-rear-
ing infrastructure, and consequently
production and quality, and affected the over-
all success of the project.
(2) Short start-up time for rearing facility. The
rearing facility was required to supply 5 mil-
lion sterile males per week to a fully opera-
tional SIT program within 7 months of
starting up in a converted facility with new
equipment and with new rearing staff with
little experience. There was little opportunity
for analyzing and solving initial problems
common in a new mass-rearing operation.
The rearing technicians had to gain most of
their experience while the release program
was in operation.
(3) Cramped rearing conditions. Until mid-2001,
all rearing took place in a small building that
was converted into a rearing facility on a low
budget. Inadequate space in both the adult







Barnes et al.: Quality Assurance in Medfly Production


120
Vienna 7
100 Target = 75%
Mean = 73.0%
0 SD 10.92
S80


60


S40


20


0








120
Vienna 8 b
Target = 75%
Mean= 78.3
SD= 7.37
'- 80


a 60


S40
40


20


0




Fig. 5. Percentage flight ability of sterile males for (a) VIENNA 7-D53/Mix 2001 genetic sexing strain from Sep
2002 to Sep 2003, and (b) VIENNA 8 genetic sexing strain from Aug 2003 to Sep 2004.


and larval rooms led to poor egg production
(e.g., Jan and Jun 2001) and poor larval pro-
duction, which in turn resulted in failure to
meet production targets. A new, spacious and
well-equipped adult room was commissioned
in Jun 2001, with a concomitant improve-
ment in egg production thereafter.


(4) Problems with larval diet. During late 1999,
very poor larval production was ascribed to
bran contaminated at source with the insecti-
cide chlorpyriphos. This resulted in reduced
numbers of pupae being produced between
Dec 1999 and Jan 2000. During Nov 2002 to
Feb 2003, a build up of rust in the larval diet






Florida Entomologist 90(1)


a


Vienna 7
Max. limit = 2%
Mean = 2.63%
SD = 2.46%


4


- Vienna 8
Max. limit = 2%
0.8 Mean = 0.02%
SSD = 0.09%
,N
S0.6

|Z 0.4 -- - - - - -- -- - - - - -- -- - -- - - -- -- - -- -

o 0.2 -

P 0.0 \



Fig. 6. Percentage females in the male-only stream for (a) VIENNA 7-D53/Mix 2001 genetic sexing strain from
Dec 2002 to Sep 2003, and (b) VIENNA 8 genetic sexing strain from Aug 2003 to Sep 2004.


mixer resulted in the diet containing toxic
levels of rust (C. Caceres, FAO/IAEA Biotech-
nology Laboratory, Seibersdorf, Austria, per-
sonal communication), that reduced the egg
to pupa efficiency and thus the number of pu-
pae irradiated from Dec 2002 to Jan/Feb
2003. On a number of occasions, bran of vary-
ing consistency was delivered, being some-
times too fine and sometimes of mixed size
grading. This resulted in sub-standard larval


diet and egg to pupa efficiency (Mar to May
2002; Apr/May 2004) and in the decrease in
the number of pupae irradiated during Mar
to May 2002.
(5) Colony replacements. The lack of an effective
filter rearing system until mid-2001 contrib-
uted to unacceptable levels of genetic recom-
bination of the genetic sexing strain and to
consequent poor production and quality. As a
result, the rearing colony had to be replaced 4


I I 11 1111


March 2007







Barnes et al.: Quality Assurance in Medfly Production


times during which production was severely
affected (May 2000, Nov 2000, Oct/Nov, and
July 2003). Production quantity and quality,
and strain stability, started improving once a
filter rearing system was introduced.
(6) Equipment malfunction. Equipment in-
stalled in the new facility in 1999, in particu-
lar climate control equipment, broke down
repeatedly. This was due mainly to budget re-
strictions precluding the purchase of higher-
specification and higher-quality equipment,
but also because high ambient temperatures
during the hot summer months (Dec to Feb)
raised temperatures in the larval rooms and
put stress on the climate control equipment.
Power failures also occurred. Production was
negatively affected on each occasion.
(7) Lack of a quality management system. Pro-
duction and quality were generally poor in
the absence of a quality management system.
The benefit of the introduction of the quality
management system in Jun 2002 can best be
seen in daily egg production and number of
pupae irradiated with the VIENNA 7 strains.
Egg production increased from an average of
250 mL per day for 17 months pre-quality
management system to 690 mL per day for 13
months post-quality management system.
The average number of VIENNA 7 pupae ir-
radiated increased from 1.4 million per week
for 32 weeks pre-quality management sys-
tem to 5.8 million per week for 13 months
post-quality management system.

Performance of the VIENNA 8 Genetic Sexing Strain

The VIENNA 8 strain has been reported to
show an approximate 20% improvement in per-
formance relative to VIENNA 7-D53/Mix 2001
(Caceres 2002). A comparison of data in Figs. la,
Ib, 3a, 3b, 5a, 5b, 6a and 6b confirms the superi-
ority of the VIENNA 8 strain. Egg production
with VIENNA 8 was easily maintained at a high
level and, once the strain had adapted to the new
conditions, was relatively stable. Mean egg to
pupa efficiency increased by 50% from 11.2% for
VIENNA 7-D53/Mix 2001 to 16.8% for VIENNA
8. Mean flight ability increased by 7.3% from
73.0% with VIENNA 7-D53/Mix 2001 to 78.3%
with VIENNA 8. The occurrence of females in the
male-only stream decreased from a mean of
2.63% with VIENNA 7-D53/Mix 2001 to 0.02%
with VIENNA 8, an improvement of 99.2%.
VIENNA 8 proved to be more genetically sta-
ble than the VIENNA 7 strains, exhibiting less
genetic recombination (occurrence of 'wrong sex'
pupae) following handling and environmental
stress. After the introduction of VIENNA 8,
equipment failure occurred on numerous occa-
sions, yet, very low levels of recombination were
recorded. Due to the better performance of VI-
ENNA 8 it was possible to reduce the number of
adult cages set up for the male-only stream from
12 per week to 8 per week without compromising


egg production. This in turn led to an estimated
savings in production costs of 30%.
In conclusion, the experiences in South Africa
have highlighted the importance for effective
fruit fly SIT operations of the following factors: (a)
sound rearing infrastructure with high quality
equipment; (b) an adequate period of staff train-
ing and equipment testing before delivering ster-
ile flies to an operational program; (c) an effective
quality management system during the produc-
tion of sterile insects; (d) a stable and productive
genetic sexing strain; and (e) a sound funding
base for the mass-rearing facility.

ACKNOWLEDGMENTS

We gratefully acknowledge Mr. David Eyles, previ-
ously of ARC Infruitec-Nietvoorbij, currently at Liver-
pool University, England, for assistance with quality
control data from the C. capitata facility; Dr. Kobus
Slabbert, iThemba Laboratories, Faure, for assistance
with Fricke dosimetry in the irradiator; Mr. Don Byers,
Private Consultant, for valuable guidance in the imple-
mentation of the Quality Management System; the In-
ternational Atomic Energy Agency, Vienna, Austria, for
continued support in the implementation of the SIT in
South Africa; the SIT Partnership (Agricultural Re-
search Council and Deciduous Fruit Producers Trust),
for financing the production of sterile medflies before
SIT was privatized; and the Provincial Government of
the Western Cape, for financial grants.

REFERENCES CITED

ANNECKE, D. P., AND V. C. MORAN. 1982. Insects and
Mites of Cultivated Plants in South Africa. Butter-
worths & Co., Durban, South Africa, 383 pp.
BARNES, B. N. 1994. Fruit fly management in pome and
stone fruit orchards: Monitoring, bait application,
cover sprays and management practices. Deciduous
Fruit Grower 44: 244-249.
BARNES, B. N., D. K. EYLES, AND G. FRANZ. 2004. South
Africa's fruit fly SIT programme-the Hex River Val-
ley pilot project and beyond, pp. 131-141 In B. N.
Barnes [ed.], Proc. 6th Int. Symp. Fruit Flies of Eco-
nomic Importance, Isteg Scientific Publications,
Irene, South Africa. 510 pp.
CACERES, C. 2002. Mass rearing of temperature sensi-
tive genetic sexing strains in the Mediterranean
fruit fly (Ceratitis capitata). Genetica 116: 107-116.
FAO/IAEA. 2002. Annual Report. Entomology Unit,
FAO/IAEA Agriculture and Biotechnology Labora-
tory, Seibersdorf, Austria.
FAO/IAEA/USDA. 2003. Manual for Product Quality
Control and Shipping Procedures for Sterile Mass-
Reared Tephritid Fruit Flies, Version 5.0. Interna-
tional Atomic Energy Agency, Vienna, Austria. 85 pp.
FISHER, K. 1999. Medfly Mass Production in Stellen-
bosch, South Africa. Stage II; The establishment of
VIENNA 7/Mix-99, a Temperature Sensitive Lethal
Genetic Sexing Strain of Medfly. Expert Mission Re-
port, 5-30 September 1999. International Atomic
Energy Agency, Vienna, Austria. 26 pp.
FISHER, K., AND C. CACERES. 2000. A filter rearing sys-
tem for mass reared genetic sexing strains of Medi-
terranean fruit fly (Diptera: Tephritidae), pp. 543-











550 In K. H. Tan [ed.], Area-Wide Control of Fruit
Flies and Other Insect Pests. Penerbit Universiti
Sains Malaysia, Penang, Malaysia. 782 pp.
FRANZ, G., E. GENCHEVA, AND P. KERREMANS. 1994. Im-
proved stability of genetic sex-separation strains for
the Mediterranean fruit fly, Ceratitis capitata. Ge-
nome 37: 72-82.
FRANZ, G. 2002. Recombination between homologous
autosomes in medfly (Ceratitis capitata) males: type-
1 recombination and the implications for the stabil-
ity of genetic sexing strains. Genetica 116: 73-84.
FRANZ, G. 2005. Genetic sexing strains amenable to
large scale rearing as required for the sterile insect
technique, pp. 427-452 In V. A. Dyck, J. Hendrichs,
and A. S. Robinson [eds.], The Sterile Insect Tech-
nique: Principles and Practice in Area-Wide Inte-
grated Pest Management. Springer, Dordrecht, The
Netherlands. 787 pp.
HENDRICHS, J., G. FRANZ, AND P. RENDON. 1995. In-
creased effectiveness and applicability of the sterile
insect technique through male-only releases for con-
trol of Mediterranean fruit flies during fruiting sea-
sons. J. Appl. Entomol. 119: 371-377.
(IAEA) INTERNATIONAL ATOMIC ENERGY AGENCY. 2004.
Dosimetry system for SIT: Standard operating pro-


March 2007


cedure for Gafchromic film, International Atomic
Energy Agency, Vienna, Austria. (http://www.iaea.
org/programmes/nafa/d4/public/d4_pbl_5_2.html).
MUMFORD, J., AND G. TWEEN. 1997. Economic feasibil-
ity study for the control of the Mediterranean and
Natal fruit fly in the Western Cape Province. Expert
Mission Report RU-7135, International Atomic En-
ergy Agency, Vienna.
MYBURGH, A. C. 1964. Orchard populations of the fruit fly,
Ceratitis capitata (Wied.), in the Western Cape Prov-
ince. J. Entomol. Soc. Southern Africa 26: 379-389.
OPTIMAL AGRICULTURAL BUSINESS SYSTEMS 2005. Key
deciduous fruit statistics 2005. Optimal Agricultural
Business Systems, Paarl, South Africa.
ORTIZ-MORENO, G. 2002. Sterile insect technique inte-
grated management of fruit fly (Phase II) SAF/5/002.
Report on field operations review of the Medfly SIT
control program at the Hex River Valley and buffer
zones. Expert Mission Report IAEA-TCR-01550, 21
Oct-8 Nov 2002, International Atomic Energy
Agency, Vienna, Austria. pp. 45.
ROBINSON, A. S., G. FRANZ, AND K. FISHER. 1999. Ge-
netic sexing strains in the medfly, Ceratitis capitata:
Development, mass rearing and field application.
Trends in Entomol. 2: 81-104.


Florida Entomologist 90(1)







Vera et al.: Demographic and Quality Control Parameters ofA. fraterculus



DEMOGRAPHIC AND QUALITY CONTROL PARAMETERS
OF ANASTREPHA FRATERCULUS (DIPTERA: TEPHRITIDAE)
MAINTAINED UNDER ARTIFICIAL REARING

TERESA VERA1, SOLANA ABRAHAM, ANDREA OVIEDO AND EDUARDO WILLING
Secci6n Zoologia Agricola, Estaci6n Experimental Agroindustrial Obispo Colombres
EEAOC, CC 9 (4101) Las Talitas, Tucuman, Argentina

'Member of Carrera de Investigador, CONICET, Argentina

ABSTRACT

The integration of the sterile insect technique (SIT) in the management of the South Amer-
ican fruit fly Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae) is a promising al-
ternative to chemically-based control in those areas where it is sympatric with Ceratitis
capitata (Wiedemann) (Diptera: Tephritidae) or other tephritid species for which the SIT is
being used. Implementation of the SIT requires the development of a cost effective mass-
rearing protocol. In this work, we present demographic and quality control parameters for
theA. fraterculus strain reared at the Estaci6n Experimental Agroindustrial Obispo Colom-
bres, Tucuman, Argentina. Considering the rearing cage as the reproduction unit, we ob-
served that fecundity is optimal during the first 3 weeks after the onset of oviposition.
Fertility was constant during this period. During 2003 and 2004, some improvements were
made to the existing rearing protocol, which resulted in increased larval viability, pupal
weight, and adult emergence. Current weekly egg production is 1 million per week. These
eggs are used to maintain the colony and to assess quality parameters. Finally, research
needs leading to improved yields and fly quality are discussed.

Key Words: Anastrepha fraterculus, sterile insect technique, mass-rearing, larval viability,
fertility, fecundity

RESUME

La integraci6n de la T6cnica del Insecto Est6ril (TIE) en el combat integrado de la mosca
Sudamericana de la fruta, Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae), es
una alternative interesante para reemplazar al control quimico en aquellas zonas donde
esta especie es simpatrica con Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) u otros
tefritidos para los que ya se utiliza la TIE. La implementaci6n de la TIE require del desa-
rrollo de un protocolo de cria masiva que sea costo-efectivo. En este trabajo presentamos pa-
rametros demograficos y de control de calidad de la cepa criada en la Estaci6n Experimental
Agroindustrial Obispo Colombres, Tucuman, Argentina. Considerando a la jaula de cria
como unidad reproductive, se observe que la fecundidad es 6ptima durante las tres primeras
semanas de iniciada la oviposici6n y que la fertilidad se mantiene constant durante ese pe-
riodo. Durante 2003-2004 se implementaron mejoras en el protocolo de cria existente lo que
result en un incremento de la viabilidad larvaria, del peso de pupas y del porcentaje de
emergencia de adults. La producci6n actual semanal es de un mill6n de huevos. Los mismos
son utilizados para mantener la colonia y realizar distintos studios de calidad de esta cepa.
Por iltimo, se sugieren necesidades de investigaci6n para alcanzar mejores rendimientos.


Translation provided by the authors.


The South American fruit fly Anastrepha
fraterculus (Wiedemann) (Diptera: Tephritidae) is
a serious pest that occurs from the southern
United States (Texas) to Argentina (Salles 1995;
Steck 1998). It attacks over 80 species of plants,
including major fruit crops, and represents a seri-
ous threat in some fruit production areas. In addi-
tion, its presence results in quarantine restric-
tions for fresh fruit exports by importing countries
(Steck 1998). At present the only control method
available is the use of bait sprays. This presents a


problem in areas where it coexists with other fruit
fly pests against which the sterile insect technique
(SIT) is being used. Such is the case for some re-
gions in Argentina, where A. fraterculus is sympa-
tric with the Mediterranean fruit fly (medfly) Cer-
atitis capitata (Wiedemann) (Diptera: Tephriti-
dae). In such situations, the application of the SIT
againstA. fraterculus can be considered an attrac-
tive alternative (Ortiz 1999).
One paramount prerequisite for SIT imple-
mentation is the development of cost-effective







Florida Entomologist 90(1)


mass-rearing protocols. Large-scale mass rearing
has not been achieved forA. fraterculus. Efforts to
colonize this species and to develop mass rearing
methods have been reported in many countries
(Ortiz 1999). Major constraints were related to
oviposition and egg fertility. A preliminary mass-
rearing strategy was developed by Jaldo et al.
(2001). This procedure followed a simple egg col-
lection method with minimal handling and high
egg fertility. However, egg to pupae recovery was
not optimal and rearing parameters reported
were obtained from small-scale rearing in Petri
dishes with egg densities lower than those used in
the routine maintenance of the colony. Additional
demographic parameters were published by
Salles (1992) and Jaldo (2001).
Although individual females are responsible
for total fecundity and fertility of the colony, un-
der mass rearing conditions, demographic param-
eters such as egg production are more informative
when the rearing cage is considered as the pro-
duction unit.
In this work, we evaluate the fecundity and
fertility of the A. fraterculus colony maintained at
the Estaci6n Experimental Agroindustrial Obispo
Colombres in Tucuman, Argentina and provide
rearing and quality control parameters.

MATERIALS AND METHODS

Colony Maintenance

The colony established at the Estaci6n Experi-
mental Agroindustrial Obispo Colombres, Tucu-
man, Argentina, was derived from infested gua-
vas collected in 1997 from the vicinity of Tafi
Viejo, Tucuman province (northwestern Argen-
tina). Since then, no wild material has been intro-
duced into the colony. Rearing conditions were
those proposed by Jaldo et al. (2001) with minor
modifications, mainly related to humidity condi-
tions in the rearing room and egg seeding density.
Adult colony cages were set up with 10,000 pu-
pae and provided with water and adult food,
which consisted of a mixture of hydrolyzed yeast,
corn protein, and sugar (1:1:4) with a supply of vi-
tamins and amino acids (Jaldo et al. 2001). Cages
were held in a rearing room with 25 1C and a
photoperiod of 12:12 (L:D). Humidity control was
difficult during 2002-2003, and ranged from 50 to
90%, but normally it was at the lower values.
From Jan 2004, with the addition of a new humid-
ifier, the relative humidity in the rearing room
was higher and more stable (80 10% R.H.).
Egg collection began 1 week after adult emer-
gence and eggs were collected 3 times per week.
Females laid their eggs through one of the panels
of the cage that was coated with a thin layer of sil-
icon rubber (hereafter referred to as the oviposi-
tion panel). To avoid dehydration, moist foam rub-
ber was applied to the outer side of the oviposition


panel and held with clips to a transparent polycar-
bonate panel which was attached to the cage. After
24 h the foam rubber was removed and eggs were
collected from the oviposition panel with the aid of
a rubber sponge. The volume of eggs obtained for
each cage was measured. Eggs were air bubbled in
water within a plastic bottle (v/v ratio of 1:100) for
48 h and later seeded on larval diet (Salles 1992).
Seeding density decreased from 34 eggs/g of
diet in 2003, to 22-15 eggs/g of diet during Jan-
May 2004, to 11 eggs/g of diet from Jun 2004 on-
wards. Egg seeding density was reduced due to an
increase in egg-pupae recovery and hence a need
to avoid larval overcrowding. Approximately at d
7, larvae started leaving the diet and pupated in
sand placed in a pan below the trays. Two d after
pupation began; sand was sieved to remove pu-
pae. The same pupal collection procedure was re-
peated twice. Pupae were placed in trays with a
thin layer of sand and held 10-12 d for matura-
tion. Two d before emergence, the total pupal pro-
duction was weighted. Each week 2 batches of 120
g of pupae were prepared to set up 2 new adult
holding cages. From Jun 2004, 2 batches of 45,000
eggs each were seeded per week to obtain enough
pupae (around 60,000) to maintain the colony;
any remaining eggs were discarded.

Demographic Analysis

Egg production was estimated by considering
the total number of eggs collected during the
lifespan of the production cages. Egg collection
was carried out for the period of 4 weeks, for a to-
tal of 12 collections per cage. For each cage in each
collection the volume of eggs collected was mea-
sured and the number of eggs obtained in each
cage for each collection was determined by multi-
plying the volume of eggs (in mL) in each collec-
tion by 14,900, which was estimated as the num-
ber of eggs in 1 mL. In addition, from each cage in
each egg collection, 3 samples of 100 eggs were
placed in a Petri dish with moistened sponge to
assess egg hatch over the oviposition period. This
procedure was repeated in 7 cages from Oct to
Nov 2003. Differences in mean values of egg
hatch along the collections dates were tested by
means of ANOVA with InfoStat (2004).

Quality Control Parameters

Several parameters related to the process of
rearing and the quality of the pupae produced were
estimated, including egg hatch, egg to pupae recov-
ery, larval viability, number of eggs obtained per
cage in each collection, number of eggs obtained per
female in each collection, weekly egg and pupal pro-
duction, pupal weight, adult emergence, and sex ra-
tio. Egg hatch was determined 3 d after eggs were
placed in Petri dishes containing a moistened cotton
sponge. The number of unhatched eggs was counted


March 2007







Vera et al.: Demographic and Quality Control Parameters ofA. fraterculus


and the percentage of egg hatch was estimated. Egg
to pupae recovery was estimated as the number of
pupae recovered in each batch/eggs seeded x 100.
For larval viability, the formula considered the
number of viable eggs (obtained from the percent-
age of egg hatch). The number of eggs per female in
each egg collection was determined by dividing the
number of eggs obtained in each cage by the num-
ber of estimated females per cage. The number of fe-
males per cage was estimated from the amount of
pupae used to set up the cage, the percentage of
adult emergence, and the sex ratio. No mortality
was assumed during this collection period.
Weekly egg production was determined by add-
ing the number of eggs obtained for each cage in
each of the 3 collections performed each week. Pu-
pal weekly production was determined consider-
ing all the pupae obtained each week from the dif-
ferent batches. Pupal weight was assessed from 3
samples of 100 pupae weighed 2 d before adult
emergence. These pupae were kept without food
and water until all flies emerged and died. The
number of emerged flies of each sex was recorded
taking into account whether they were non-
deformed, deformed, or partially emerged. Adult
emergence and sex ratio were estimated from
these figures as explained in the international
fruit fly quality control manual (FAO/IAEA/USDA
2003). According to the changes in seeding density
implemented from 2002 to the present, rearing
and quality control parameters were estimated for
4 periods: 2002, 2003, Jan-May 2004 and Jun-Aug
2004. Egg hatch, egg to pupae recovery, and larval
survival were determined for each pupal batch.

RESULTS AND DISCUSSION

Fecundity and fertility for each egg collection
during the period Oct-Nov 2003 is presented in
Table 1. Fecundity over the 4-week period totaled


413,179 + 53,026 eggs per cage. Considering the
period in which this value was obtained, as well
as the percentage of emergence, pupal weight,
and sex ratio, the number of eggs per female
along the complete oviposition period (i.e., until
the cage was discarded) was 102 eggs. In other
studies values from 394 eggs per female (Salles
1992) to 625 eggs per female (Jaldo et al. 2001)
have been recorded. Our value is underestimated
since it assumes no mortality during the collec-
tion period; and as mentioned before, values re-
ported previously come from studies in which
eggs were collected during the complete reproduc-
tive period of females confined in small cages and
under relaxed, non-crowded, conditions. As such,
they neither provide an estimate of the number of
eggs to be collected per female or cage, nor an in-
formative figure for the rearing facilities regard-
ing the optimal time to discard the production
cage. The eggs from the first 3 weeks of collection
represented approximately 90% of the total col-
lected (Table 1). During the fourth week the pro-
duction dropped and it is expected that collecting
for longer periods, where flies are more than 35 d
old, would not increase overall egg production.
This, and the fact that old cages are sources of
fungal and mite infections in the rearing rooms,
prompts us to suggest that 3 weeks of egg collec-
tion is optimal for a rearing facility, hence produc-
tion cages should be discarded on d 28.
For Anastrepha obliqua (Macquart), Anas-
trepha ludens (Loew), and Anastrepha serpentina
(Wiedemann), 41, 64, and 36 d, respectively, have
been proposed as the life of the production cage
(Liedo & Carey 1994). These periods take into ac-
count the amount of pupae that will be harvested
in the facility to be released in the field and have
been estimated from wild flies, which probably ex-
plains the higher values obtained (Liedo & Carey
1994). Our suggestion is more in agreement with


TABLE 1. ANASTREPHA FRATERCULUS FERTILITY AND FECUNDITY FROM OCT TO NOV 2003: MEAN NUMBER OF EGGS (
SEM) COLLECTED IN EACH EGG COLLECTION AND PERCENTAGE OF EGG HATCH.

Week Collection Number of eggs/cage' Cumulative percentage Egg hatch (%)

First 1 40,443 9,437 9.8 76.0 5.6
2 46,214 8,076 21.0 76.8 6.7
3 45,551 8,321 32.0 85.8 3.6
Second 1 46,829 8,003 43.3 86.3 1.8
2 43,849 5,067 53.9 87.9 1.3
3 43,423 7,786 64.5 85.2 3.0
Third 1 40,656 11,025 74.3 87.2 2.5
2 38,101 8,963 83.5 87.5 2.6
3 25,543 5,775 89.7 88.4 1.1
Fourth 1 17,241 4,940 93.9 85.6 3.2
2 10,856 2,383 96.5 82.2 2.8
3 14,474 3,230 100.0 80.0 4.3
Mean 34,432 3,895 84.1 1.2

Adult colony cages were set up with approximately 10,000 pupae which produced approximately 4,060 females.







Florida Entomologist 90(1)


values proposed by Carey & Vargas (1985), ob-
tained from other tephritids, which were already
adapted to mass rearing conditions, as well as the
21 and 17 d used in the Moscafrut Metapa facility
in Mexico to mass rear A. ludens and A. obliqua,
respectively, (Artiaga-L6pez & Hernandez, pers.
comm.). More recent values obtained from Jun-
Aug 2004 revealed that females laid approxi-
mately 135 eggs during the first 3 weeks of ovipo-
sition before the cage was discarded (Table 2).
This value increased, compared to the one ob-
tained during the trial in 2003, as a result of an
improvement of some rearing parameters such as
pupal weight, which resulted in larger and proba-
bly more fecund females (see below).
During the 4 weeks of egg collection from Oct
to Nov 2003, egg hatch averaged 84.1 1.2% (Ta-
ble 1). Although for the first collections mean val-
ues were lower, probably due to oviposition of vir-
gin females, there were no statistical differences
among the collections (F = 1.39; df = 11,56; P >
0.05). Because the differences are not significant,
and the production of eggs during the first week is
important, it is not recommended to discard these
eggs nor to start egg collection later.
Rearing and quality control data are presented
in Table 2. The higher humidity in the rearing
room from Jan 2004 may have been responsible
for the higher egg to pupae recovery, because the
larval diet did not dry out during the first days in
which first instars require high humidity. This led
to the need to reduce the seeding density from 34
eggs/g of diet to 22-15 eggs/g of diet. By Jun 2004,
it was decided to seed eggs at an even lower den-
sity (11 eggs/g of diet). To prevent water loss from
the diet, trays were covered with a polystyrene
tray for the first 5 d of larval development. Cover-
ing rearing trays to prevent dehydration is a com-
mon practice in other insectaries and for other
Anastrepha species (Pins6n et al. 1993). Subse-
quently, trays were uncovered and sugarcane ba-


gasse was added to the diet to allow larvae to
crawl outside the diet. Because the diet uses agar
as a gelling agent (Salles 1992), we found that
some larvae become stuck in the diet when trying
to leave to pupate. The addition of sugarcane ba-
gasse, which is produced locally as a by-product of
the sugar industry, provides an adequate sub-
strate for the larvae to crawl out of the diet and
enhances larval recovery. In all, changes applied
to the rearing conditions (higher and more stable
relative humidity in the rearing room, lower seed-
ing density and handling during the larval devel-
opment), resulted in the increase of the viability,
recovery, and quality control parameters (Table 2).
Egg to pupae recovery was higher than the
44% reported by Jaldo et al. (2001), even when
those values were obtained from batches of 100
eggs seeded on Petri dishes under very relaxed
conditions. Our values under such favorable con-
ditions are approximately 83% recovery. Besides
the increase in larval viability, the reduction in
egg seeding density resulted in an increase in pu-
pal weight and adult emergence. A gain in pupal
weight may have resulted in larger and probably
more fecund females. This may also explain the
increase in the number of eggs collected per fe-
male as shown for other tephritids (Krainacker et
al. 1989; Liedo et al. 1992).
Although larval viability improved, more im-
provement is still needed. The results obtained in
the small-scale trials suggest that nutrients are
not affecting the viability, but probably the struc-
ture of the diet. Finding the optimal moisture
level and a good bulking agent to maximize in-
take of nutrients from the diet is an important
goal to achieve. The present diet, which uses agar
as a gelling agent, is too expensive for mass-rear-
ing, and presents the added drawback of being
difficult for larvae to leave and pupate. Sugarcane
bagasse can be used to help the larvae leave the
diet, it is locally available from the sugarcane in-


TABLE 2. REARING AND QUALITY CONTROL PARAMETERS OF THE ANASTREPHA FRATERCULUS COLONY MAINTAINED
DURING 2002-2004 AT THE ESTACION EXPERIMENTAL AGROINDUSTRIAL OBISPO COLOMBRES, TUCUMAN,
ARGENTINA.

Parameter 2002 2003 Jan-May 2004 Jun-Aug 2004

Egg hatch (%) 74.1 4.2 84.9 1.3 83.1 1.1 84.4 0.9
Egg-pupae recovery (%)- 29.3 1.7 37.9 2.1 56.3 2.1
Larval viability (%) 33.8 1.2 49.3 3.8 66.6 2.3
Eggs/cage/collection 28,221 3,181 43,822 3,099 63,323 3,183
Eggs/female/collection 6 1 11 1 15 1
Weekly egg production 83,531 11,077 691,740 41,580 1,074,425 43,733
Weekly pupae production' 124,963 17,754 55,894 4,559
Pupal weight (mg) 10.9 0.4 11.3 0.4 12.0 0.3 13.1 0.2
Non-deformed adult emergence (%) 73.0 2.5 77.3 1.8 75.8 3.2 85.0 2.5
Adult emergence (%) 77.1 2.3 81.6 1.7 80.0 3.1 88.6 2.6
Male:female ratio 0.85 0.03 0.95 0.03 0.97 0.04 0.93 0.05

From Jun 2004 onwards only 90,000 eggs per week were seeded; this explains the drop in the production of pupae.


March 2007







Vera et al.: Demographic and Quality Control Parameters ofA. fraterculus


dustry in the region, and it is free from insecti-
cides. However, preliminary studies (unpublished
data) have shown that yields with bagasse are not
as good as those obtained with agar, indicating
more improvement is still possible.

ACKNOWLEDGMENTS

This research was supported with IAEA Research
Contract 11894 to M. T. V. We are grateful to Compaiia
Argentina de Levaduras S.A. (CALSA) and ARCOR S.A.
for kind donation of the brewer's yeast and the corn pro-
tein, respectively.

REFERENCES CITED

CAREY, J. R., AND R. I. VARGAS. 1985. Demographic
analysis of insect mass rearing: A case study of three
tephritids. J. Econ. Entomol. 78: 523-527.
FAO/IAEA/USDA. 2003. Manual for Product Quality
Control and Shipping Procedures for Sterile Mass-
Reared Tephritid Fruit Flies, Version 5.0. Interna-
tional Atomic Energy Agency, Vienna, Austria. 84 pp.
INFOSTAT. 2004. InfoStat Version 2004, Manual del
Usuario. Grupo InfoStat, FCA, Universidad Nacio-
nal de C6rdoba, Argentina. First Edition. Editorial
Brujas, Argentina.
JALDO, H. E. 2001. Estudios biol6gicos y poblacionales
de Anastrepha fraterculus (Wiedemann) (Diptera:
Tephritidae). PhD Thesis, Universidad Nacional de
Tucuman, Argentina.
JALDO, H. E., M. C. GRAMAJO, AND E. WILLINK. 2001.
Mass rearing ofAnastrepha fraterculus (Diptera: Te-
phritidae): a preliminary strategy. Florida Entomol.
84: 716-718.


KRAINACHER, D. A., J. R. CAREY, AND R. I. VARGAS.
1989. Size-specific survival and fecundity for labora-
tory strains of two tephritid (Diptera: Tephritidae)
species: implications for mass-rearing. J. Econ. En-
tomol. 82: 104-108.
LIEDO, P., AND J. R. CAREY. 1994. Mass rearing ofAnas-
trepha (Diptera: Tephritidae) fruit flies: a demo-
graphic analysis. J. Econ. Entomol. 87: 176-180.
LIEDO, P., J. R. CAREY, H. CELEDONIO, AND J. GUILLEN.
1992. Size specific demography of three species of
Anastrepha fruit flies. Entomol. Exp. & Appl. 63:
135-142.
ORTiZ, G. 1999. Potential use of the sterile insect tech-
nique against the South American fruit fly, pp. 121-
130 In The South American Fruit Fly, Anastrepha
fraterculus (Wied.); Advances in Artificial Rearing,
Taxonomic Status and Biological Studies. TECDOC-
1064, IAEA, Vienna, Austria. 202 pp.
PINSON, E., H. CELEDONIO, AND W. ENKERLIN. 1993.
Colonization and establishment of Anastrepha ser-
pentina for mass-rearing: preliminary results, pp.
281-284 In M. Aluja and P. Liedo [eds.], Fruit Flies:
Biology and Management. Springer Verlag, New
York, U.S.A. 492 pp.
SALLES, L. A. B. 1992. Metodologia de criacao de Anas-
trepha fraterculus (Wied., 1830) (Diptera: Tephriti-
dae) em dieta artificial em laboratorio. Annais da
Sociedade Entomologica do Brasil 21: 479-486.
SALLES, L. A. B. 1995. Bioecologia e Controle da Mosca
das Frutas Sul-Americana. EMBRAPA-CPACT,
Pelotas, R. S., Brazil. 58 pp.
STECK, G. J. 1998. Taxonomic status of Anastrepha
fraterculus, pp 13-20 In The South American Fruit
Fly, Anastrepha fraterculus (Wied.); Advances in Arti-
ficial Rearing, Taxonomic Status and Biological Stud-
ies. TECDOC 1064. IAEA, Vienna, Austria. 202 pp.







Florida Entomologist 90(1)


March 2007


DEVELOPMENT OF QUALITY CONTROL PROCEDURES FOR
MASS PRODUCED AND RELEASED BACTROCERA PHILIPPINENSIS
(DIPTERA: TEPHRITIDAE) FOR STERILE INSECT TECHNIQUE PROGRAMS


SOTERO RESILVA', GLENDA OBRA1, NENITA ZAMORA2 AND ERDIE GAITAN2
1Agricultural Research Group, Atomic Research Division, Philippine Nuclear Research Institute
Department of Science and Technology, Diliman, Quezon City, Philippines

2National Mango Research and Development Center, Bureau of Plant Industry
San Miguel, Jordan, Guimaras, Philippines

ABSTRACT

Quality control procedures for Bactrocera philippinensis Drew & Hancock 1994 (Diptera: Te-
phritidae) used in sterile insect technique (SIT) programs were established in the mass rear-
ing facility at the Philippine Nuclear Research Institute. Basic studies on pupal irradiation,
holding/packaging systems, shipping procedures, longevity, sterility studies, and pupal eye
color determination in relation to physiological development at different temperature re-
gimes were investigated. These studies will provide baseline data for the development of
quality control protocols for an expansion of B. philippinensis field programs with an SIT
component in the future.

Key Words: Bactrocera philippinensis, quality control, Oriental fruit flies, pupal eye color,
longevity, sterility

RESUME
Los procedimientos de control de calidad para Bactrocera philippinensis Drew & Hancock
1994 (Diptera: Tephritidae) usados en programs de la t6cnica de insect est6ril (TIE) fueron
establecidos en la facilidad de cria en masa del Instituto Filipino de Investigaci6n Nuclear.
Estudios basicos sobre la irradiaci6n de las pupas, sistemas de almacenaje/empaque, proce-
dimientos del envio, longevidad, studios de esterilidad y la determinaci6n del color de ojo de
la pupa en relaci6n con el desarrollo fisiol6gico en regimenes diferentes de temperature fue-
ron investigados. Estos studios proveeran una linea de informaci6n basica para el desarro-
1lo de protocolos de control de calidad para una expansion de los programs de campo para
B. philippinensis con un component de TIS en el future.


Quality control is important for monitoring the
performance of mass reared insects for use in the
sterile insect technique (SIT) (Boller et al. 1981).
To meet this requirement, routine quality control
tests on egg hatchability, pupal weight and size,
percent adult emergence, longevity, flight dis-
persal, and mating ability are used. The effect of
pupal holding conditions, irradiation, and pack-
aging procedures must also be assessed and
threshold values for each quality control parame-
ter need to be established.
After eradication of Bactrocera philippinensis
Drew & Hancock 1994 (Diptera: Tephritidae) with
the SIT in Naoway Islet, Philippines (Manoto et al.
1996), a feasibility study based on an integrated
control program was initiated in Guimaras Island
(Covacha et al. 2000). The Philippine Nuclear Re-
search Institute upgraded the fruit fly mass rear-
ing facility in order to produce 25 million pupae
per week. The B. philippinensis colony has been
mass reared in the laboratory for more than 100
generations (Rejesus et al. 1975). The quality con-


trol procedures being developed for this species
were based on those developed for the Mediterra-
nean fruit fly Ceratitis capitata (Wiedemann).

MATERIALS AND METHODS

Standard Specifications

Routine quality control includes measure-
ments of pupal size, percent adult emergence,
flight ability, sex ratio, response to stress, and
mating propensity. Preparation of samples, obser-
vation, and gathering of data were done by follow-
ing or modifying the procedures in the manual for
"Product Quality Control and Shipping Proce-
dures for Sterile Mass-Reared Tephritid Fruit
Flies" (FAO/IAEA/USDA 2003). Minimum specifi-
cations required for weekly and monthly routine
quality control tests were established on pre-irra-
diation, post-irradiation, and post shipment pu-
pae to serve as guides in monitoring fly quality
and for inclusion into the manual.







Resilva et al.: Oriental Fruit Fly Quality Control


Monitoring Fruit Fly Quality

Release of sterile flies in Guimaras Island by
ground commenced in Apr 2001 and for every
batch of released sterile flies routine quality con-
trol checks in the pre-irradiation, post-irradiation,
and post-shipment were carried out. Data from
quality control tests were tabulated and analyzed
to assess variation in the quality specifications. A
database of results for routine quality control
tests was constructed for reference purposes.

Pupal Irradiation

Samples of B. philippinensis pupae obtained
from the stock colony were marked with 1.5 g fluor-
escent dye (Dayglo) and held in glass vials 2 d be-
fore emergence. The glass vials containing 25 mL
of pupae were irradiated with 60Co Gamma Cell
220 Irradiator facility with doses of 0, 25, 40, 50,
75, 100, 150, and 200 Gy. After irradiation, sam-
ples of pupae were prepared for the following tests.

Emergence and Flight Ability Tests

One hundred pupae, counted into a Petri dish,
were placed at the base of a 10-cm black PVC pipe
coated with talcum powder, inside a large cage.
Percent adult emergence was based on the num-
ber of adults emerging from the pupal samples.
Non-flying, fully emerged, partially-emerged, and
deformed flies were counted and recorded. Flight
ability (flies escaping from the black PVC pipe)
was determined based on the number of une-
merged pupae and residual flies remaining in the
Petri dish.

Fecundity and Sterility Tests

Five replicates of 100 pupae of each dose were
counted and placed for emergence in screened
cages (30 x 30 x 40 cm) and provided with food
and water. The flies were allowed to lay eggs for
10 d after emergence in a small egging device con-
taining a wet sponge. Samples of eggs were
counted, held in Petri dishes on damp cloth, and
observed for egg hatch 3 d later.

Holding/Packaging of Pupae for Irradiation
and Shipment to Guimaras Island

Trials on horizontal and vertical holding/pack-
aging of pupae for irradiation and shipment were
conducted and compared with the standard pack-
aging system currently used. Each holding/pack-
aging method was evaluated to determine the ef-
fect of length, size, and position/arrangement of
polyethylene plastic bags. Three soft ice packs
measuring 11 x 7.5 inches were placed inside the
cardboard box as coolant. A laboratory thermom-
eter was inserted inside the box to check the tem-


perature at 4 h intervals for 48 h. At the same
time, samples of pupae were taken at random
from different sausage bags to determine the ef-
fect of each treatment on adult emergence and
flight ability. The packages used with their corre-
sponding specifications are shown in Fig. 1. Per-
cent adult emergence and fliers were tabulated
and checked to see if they passed the minimum
specifications (Obra & Resilva 2003).


Fig. 1. Specifications and arrangement of 3 different
packaging systems evaluated for irradiation and pupal
shipment. a. Standard arrangement size of bags (cm) =
10.16 x 28, weight of pupae/sausage = 460 g, No. of sau-
sage/box = 52, No. pupae/box = 1.9 million. b.Vertical ar-
rangement size of bags (cm) = 10 x 40, weight. of pupae/
sausage = 600 g. No. of sausage/box = 36, No. pupae/box
= 1.7 million. c. Horizontal arrangement size of bags
(cm) = 15 x 51, weight of pupae/sausage = 2000 g, No. of
sausage/box = 12, No. pupae/box = 1.9 million.







Florida Entomologist 90(1)


Determination of Pupal Eye Color
in Relation to Physiological Development

Development of pupae based on daily eye color
changes (Ruhm & Calkins 1981) at different tem-
peratures of 22-32C (room temperature), 15, 19,
and 28C were determined. Pupal samples at 15,
19, and 28C were placed in a controlled environ-
ment with an Echo Therm Chilling Incubator.
Daily changes of eye color at each temperature
were monitored by taking close up photographs
with an Intel QX3 Computer Microscope at 60x
magnification. Duration of pupal development
and eye color changes of each pupal group were
noted and matched with the color scale of the Soil
Munsel Color Charts (Anonymous 2000).

Evaluation of Different Shipping Coolants
for Sterile Pupae

Different shipping coolants such as soft ice, ice
packs, and plastic ice trays were evaluated to de-
termine suitable cooling materials for pupal ship-
ment. Three boxes of pupal samples, arranged in
the standard packaging system, were prepared
and irradiated with the multi-purpose gamma ir-
radiation facility. After irradiation, three sets of
each coolant wrapped in newspaper were placed
on the top of each cardboard box. The lids of the
boxes were closed and secured with packaging
tape. Data collection procedures were similar to
those used for packing/holding.

Determination of Optimal Irradiation Doses Range

Samples of pupae were prepared and irradi-
ated with doses inside of the irradiation chamber
ranging from 52-56 Gy, 63-67 Gy, and 67-74 Gy
Sterility was checked by mating 50 irradiated
males or females with 50 non-irradiated males or
females in cages 30 x 30 x 40 cm. Samples of eggs
were collected weekly in moistened plastic vials
over a 3-week period to determine egg hatch for
each treatment. As many as 500 eggs were col-
lected and counted during each egging.

Statistical Analysis

All data obtained in all experiments were
tested in a randomized complete block design
with 5 replications, evaluated, and subjected to
an analysis of variance (ANOVA), with the hon-
estly significant difference value calculated as
Tukey's statistic at a = 0.05 (SAS Institute 1990).

RESULTS AND DISCUSSION

Standard Specifications for B. philippinensis

Table 1 shows the standard specifications for
the essential weekly and monthly quality control


tests of mass-reared B. philippinensis. These val-
ues were based on the minimum mean data ob-
tained in a year's production in the rearing facil-
ity. The minimum weight set for pupae was 11.13
mg with a diameter of 1.75 mm. Minimum emer-
gence rate and fliers with a 10-cm flight tube in
pre-, post-irradiation, and post-shipment were
90.3, 85.2, and 80.4% for emergence, respectively,
and 77.3, 73.2, and 70.1% for fliers, respectively.
Minimum values of 50.2, 45.3, and 40.2% survival
after 28-32 h was acceptable when newly emerged
flies were subjected to stress tests in pre- and
post-irradiation and post shipment, respectively.
Mating propensity indicates an acceptable mean
mating index of 50.2% (pre-irradiation), 45.1%
(post-irradiation), and 40.3% (post-shipment) for
10 day-old flies.
Routine quality control checks were done on
sterile flies sent to Guimaras for release. Re-
leased sterile flies passed the minimum specifica-
tions set for pupal size, adult emergence, adult fli-
ers, and other quality control parameters tested.

Irradiation Studies

Table 2 shows data on the effects of different
doses of gamma radiation on adult emergence,
flight ability, fertility, and longevity. Statistical
analysis of the adult emergence data showed no
significant difference for all doses tested, com-
pared to the control group. Flight ability data in-
dicate that a high proportion of flies, 93.3-97.7%,
escaped from the flight tube after doses of 25-100
Gy. At higher doses, the number of fliers progres-
sively decreased from 73.9 to 69.0%. Females irra-
diated as pupae with 25-40 Gy were not sterile
with egg hatch of 25.0 and 3.2%, respectively.
When pupae were irradiated at 50 Gy and above,
100% sterility was achieved in all adult females.
With regard to longevity tests, no significant dif-
ference was observed following irradiation with
25-75 Gy. However, increasing the dose beyond
100 Gy progressively affected the survival, result-
ing in a decrease in adult longevity after 5 weeks.

Holding/Packaging of Pupae for Irradiation
and Shipment to Guimaras

Mean percent emergence and adult fliers in all
packaging arrangements showed satisfactory re-
sults on pupae randomly-sampled every 4 h from
0-48 h. Similar results were observed in flight
ability tests in which a high proportion of adults
capable of flight (75-99%) escaped from a 10-cm
flight tube. These findings indicate that horizon-
tal and vertical arrangement of pupae were ac-
ceptable holding/packaging methods comparable
to the standard packing system currently used for
sterile fly shipment in Guimaras. Packing of pu-
pae in cardboard boxes with 3 ice packs elimi-
nates overheating of the pupae inside the box.


March 2007







Resilva et al.: Oriental Fruit Fly Quality Control


TABLE 1. SPECIFICATIONS FOR REQUIRED WEEKLY OR MONTHLY QUALITY CONTROL PARAMETERS OF MASS-REARED B.
PHILIPPINENSIS FOR USE IN SIT PROGRAMS IN THE PHILIPPINES.

Parameter Frequency Pre-irradiation Post-irradiation Post-Shipment

A. Established minimum specifications'
Pupal size weekly
min. pupal weight (mg) 11.00 nr nr
min. pupal diameter (mm) 1.75 nr nr
Sex ratio: min.% males weekly 50.00 nr nr
Emergence & flight ability weekly
min.% emergence 90.00 85.0 80.0
min.% fliers (10-cm tube) 77.00 73.0 70.0
Stress test: min.% survival, 28-32 h weekly 50.00 45.0 45.0
Mating propensity monthly 50.00 45.0 40.0
Boller's index (min), 10-d-old flies
B. Mean QC test results2
Pupal size weekly
min. pupal weight (mg) 12.52 nr nr
min. pupal diameter (mm) 1.75 nr nr
Sex ratio: min.% males weekly 50.30 nr nr
Emergence & flight ability weekly
min.% emergence 94.50 92.7 91.6
min.% fliers (10-cm tube) 86.10 81.5 71.7
Stress test: min.% survival, 28-32 h weekly 57.60 56.3 53.4
Mating propensity monthly 46.00 39.0 48.0
Boller's index (min), 10-d-old flies

Minimum standard specification established for one year period.
'Mean quality control test results of flies used in SIT release program. All data were collected in replicates, nr =not required.


Temperature was maintained between 15 to 28C
for 48 h. In addition, lining with plastic bubble
wrap between the layers of "sausage bags" pro-
tects the pupae from mechanical injury by serv-
ing as a cushion while in transit.

Determination of Pupal Eye Color in Relation
to Physiological Development

Daily changes in eye color during pupal devel-
opment at 22-32C (room temperature), 15, 19,


and 28C are shown in Table 3. The method for es-
timation of the pupal age is based on color
changes compared with the color scale in the
Munsel Soil Color Charts (Anonymous 2000).
At 22-32C (room temperature), pupal devel-
opment is approximately 9 d. Dissection of the an-
terior part of the puparium is possible on the sec-
ond day. On d 7 when pupae are irradiated, eye
color is dark yellowish brown (HUE 10 YR 3/6).
Adult flies start to emerge at 9 d and emergence is
complete at 10 d.


TABLE 2. EFFECTS OF DIFFERENT DOSES OF GAMMA RADIATION (X SEM) ON THE ORIENTAL FRUIT FLY BACTROCERA
PHILIPPINENSIS IRRADIATED 2 DAYS BEFORE EMERGENCE.* WITHIN A ROW, MEANS FOLLOWED BY THE SAME
LETTER ARE NOT SIGNIFICANTLY DIFFERENT (a = 0.05; ANOVA TEST).

Dose (Gy) Emergence'(%) Fliers' (%) Mortality' (%) Egg hatch' (%)

0 98.7 0.4 ab 97.9 0.6 a 14.8 3.8 a 83.8 4.6 a
25 98.4 1.0 ab 94.4 1.7 ab 15.9 2.9 ab 25.1 5.2 b
40 97.4 1.9 ab 94.9 2.1b 14.9 3.8 a 3.2 2.8 c
50 98.8 0.8 a 94.2 0.9 b 12.4 2.4 a 0
75 98.1 0.6 ab 93.2 0.8 b 13.5 3.3 a 0
100 97.9 0.6 ab 93.3 0.9 b 26.3 5.45 c 0
150 97.5 0.8 b 73.9 4.6 c 19.4 3.8 b 0
200 95.3 2.7 c 69.0 5.0 d 30.7 5.2 d 0

*Data are means of 5 replicates. 'Analysis of variance results for % emergence were (F = 2.06; df= 7,16; P > 0.11030, % fliers (F
= 49.99; df = 7,16; P < 0.0001), % mortality (F = 8.43; df = 7,16; P < 0.00002), % egg hatch (F = 371.31; df = 7,16; P < 0.0001).








Florida Entomologist 90(1)


March 2007


TABLE 3. CHANGES IN EYE-COLOR OF THE ORIENTAL FRUIT FLY, B. PHILIPPINENSIS AT DIFFERENT TEMPERATURE OF PU-
PAL DEVELOPMENT.

Days of development
Colour Temperature
identification C 1 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Not dCfined 15
19 -
28
22-32
Paic yellow 15
HUE 2-5 Y S2 19

22-32
Yellow 15
HUE 2.5 Y 7/81 19

22-32
Bronish yellow 15
HUE 10YR618* 19
28
22-32
Dark ycllowish IS
HUE 10YR3/2* 19
28
22-32
Very dark grayish 15
HUE 10YR3/2* 19
28
22-32
Dark bluish gray 15
GLEY 2 3/1 19
28
22-32
Dark grayish green 15
GLEY I25/2* 19 E
28 E
22-32
*Color codes were compared to the Munsell Soil Color Charts (Year 2000 Revised Washable Edition). E = Adult emergence


At 15C, pupal development takes about 29 d.
Dissection of the puparium is possible 4 d after
pupation, and at d 22-23 the eyes are dark yellow-
ish brown (HUE 10 YR 3/6 and 3/4, respectively)
and the pupae can be irradiated. Adult flies begin
emerging on d 30.


Pupal development is approximately 15 d at
19C. Radiation can be applied at d 12 with eye
color of dark yellowish brown (HUE 10 YR 3/8).
Adult flies start to emerge after 16 d.
At 28C, pupal development requires 9 d. Eye
color is dark yellowish brown (HUE 10 YR 3/6)


TABLE 4. STERILITY OF ORIENTAL FRUIT FLY, B. PHILIPPINENSIS IRRADIATED WITH DIFFERENT RANGE DOSES OF GAMMA
IRRADIATION.


Dose range (Gy)


Crosses' (50 Males x 50 Females) Number of eggs sampled % Egg hatched


A. 52-56 Gy U U 8,000 86.76
UxIR 1,055 8.93
IR x U 8,000 0.66
IR x IR 360 3.10

B. 63-67 Gy U U 8,000 90.40
UxIR 687 7.13
IRxU 7,900 0.33
IR x IR 176 2.50

C. 67-74 Gy UxU 8,000 87.71
UxIR -
IR x U 7,956
IR x IR -

'U = non-irradiated flies; IR = irradiated flies. Eggs were collected starting 10 d after emergence for 3 consecutive weeks.







Resilva et al.: Oriental Fruit Fly Quality Control


and is noticeable on d 7 when the pupae can be ir-
radiated. Adult flies started emerging after 9 d
and emergence was complete in 10 d.

Evaluation of Different Shipping Coolants

Mean adult emergence and flight ability data
were obtained from pupae packed in boxes with
soft ice, ice packs, and plastic ice trays in a stan-
dard arrangement/packaging system. The use of
ice packs and soft ice were equivalent and met the
minimum specifications set for emergence and
adult fliers. Similar results were also observed for
plastic ice trays; however, a decrease in adult fli-
ers was noted when pupae were stored more than
44 h. A possible explanation for low fliers in plas-
tic ice trays appeared to be due to an increase in
temperature up to 32C that begins after 44 h.

Determination of Optimal Irradiation Doses Range

Table 4 shows the effects of irradiation on the
sterility of irradiated with 4 different dose
ranges. Pupal irradiation with doses lower than
67 Gy did not prevent egg hatch. When pupae
were irradiated with doses ranging from 63-67
Gy, egg hatch was between 0.3-0.7% when irradi-
ated males were paired with non-irradiated fe-
males, or from 7.1-8.9% when non-irradiated
males were paired with irradiated females. How-
ever, when the dose range was increased to 67-74
Gy, egg hatch was completely suppressed. These
results suggested that the best irradiation range
to achieve complete sterility with a Gamma-cell
220 should be between 67 and 74 Gy

ACKNOWLEDGMENTS
The authors thank the International Atomic Energy
Agency (IAEA) for partial funding of this project under
Research Contract 10841, and for procurement of the
chilling incubator under the Human Resource Develop-
ment Program No. PHI/0012. The support of Dr. Alu-
manda M. de la Rosa, PNRI Director is gratefully
acknowledged. We also give our thanks and apprecia-
tion for technical assistance of Restituto B. Ilagan, Do-
lores Lazo, Ricky Garcia, and Flora Isip.


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D. L. CHAMBERS. 1981. Measuring, monitoring and
improving the quality of mass-reared Mediterra-
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The RAPID quality control system for early warning.
Z. Angew. Entomol. 92: 67-83.
COVACHA, S. A., H. G. BIGNAYAN, E. G. GAITAN, N. F.
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S. S. RESILVA, AND M. R. REYES. 2000. Status report
on the integrated fruit fly management based on the
sterile insect technique in Guimaras Island, Philip-
pines, pp. 401-408 In K. H. Tan [ed.], Area-Wide Con-
trol of Fruit Flies and Other Insect Pests. Penerbit
University Sains Malaysia, Penang, Malaysia. 782 pp.
FAO/IAEA/USDA. 2003. Manual for Product Quality
Control and Shipping Procedures for Sterile Mass-
Reared Tephritid Fruit Flies, Version 5.0. Interna-
tional Atomic Energy agency, Vienna, Austria. 85 pp.
MANOTO, E. C., S. S., RESILVA, G. B., OBRA, M. R.
REYES, H. GOLEZ, S. A. COVACHA, H. D. BIGNAYAN,
E. G. GAITAN, AND F. ZAMORA. 1996. Pilot applica-
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OBRA, G. B., AND S. S. RESILVA. 2003. Mass production
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REJESUS, R. S., G. B. FERNANDEZ-GARCIA, AND R. C.
BAUTISTA. 1975. Screening of rice bran-yellow sweet
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Florida Entomologist 90(1)


March 2007


QUALITY CONTROL METHOD TO MEASURE PREDATOR EVASION
IN WILD AND MASS-REARED MEDITERRANEAN FRUIT FLIES
(DIPTERA: TEPHRITIDAE)


MARTHA A. HENDRICHS1, VIWAT WORNOAYPORN2, BYRON KATSOYANNOS3 AND JORGE HENDRICHS2
'Department for Conservation Biology, Vegetation and Landscape Ecology, University of Vienna
Althanstrasse 14, A-1090 Vienna, Austria

2Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture
International Atomic Energy Agency, Wagramerstrasse 5, P.O. Box 100, A-1400 Vienna, Austria

3Laboratory of Applied Zoology and Parasitology, Department of Agriculture
Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece

ABSTRACT

Sterile male insects, mass-reared and released as part of sterile insect technique (SIT) pro-
grams, must survive long enough in the field to mature sexually and compete effectively
with wild males for wild females. An often reported problem in Mediterranean fruit fly (med-
fly) Ceratitis capitata (Wiedemann) SIT programs is that numbers of released sterile males
decrease rapidly in the field for various reasons, including losses to different types of pred-
ators. This is a serious issue in view that most operational programs release sterile flies at
an age when they are still immature. Previous field and field-cage tests have confirmed that
flies of laboratory strains are less able to evade predators than wild flies. Such tests involve,
however, considerable manipulation and observation of predators and are therefore not suit-
able for routine measurements of predator evasion. Here we describe a simple quality con-
trol method with aspirators to measure agility in medflies and show that this parameter is
related to the capacity of flies to evade predators. Although further standardization of the
test is necessary to allow more accurate inter-strain comparisons, results confirm the rele-
vance of measuring predator evasion in mass-reared medfly strains. Besides being a mea-
sure of this sterile male quality parameter, the described method could be used for the
systematic selection of strains with a higher capacity for predator evasion.

Key Words: predator evasion, predation, survival, sterile males, Tephritidae, Ceratitis capi-
tata, Vespula germanica

RESUME

Insectos machos est6riles criados en forma masiva para ser liberados en programs que uti-
lizan la t6cnica del insecto est6ril (TIE), tienen que tener la capacidad de sobrevivir en el
campo el tiempo necesario para poder madurar sexualmente y competir efectivamente con
los machos silvestres por hembras silvestres. Un problema frecuentemente reportado por di-
chos programs de la mosca del Mediterraneo, Ceratitis capitata (Wiedemann), es que el nu-
mero de machos est6riles de laboratorio liberados en el campo, decrecen rdpidamente por
varias razones, incluyendo p6rdidas debidas a diferentes tipos de depredadores. Estudios
anteriores conducidos en el campo, y enjaulas de campo, han confirmado que las cepas de
machos de laboratorio tienen menos capacidad de evadir depredadores que los machos sil-
vestres. Estos studios involucran, sin embargo, una considerable cantidad de manipulaci6n
y observaci6n de depredadores, por lo que no son adecuados para ser usados como medidas
rutinarias en los programs de cria masiva. Aqui describimos un m6todo sencillo de control
de calidad usando aspiradores para medir agilidad en la mosca del Mediterraneo y mostra-
mos que este parametro esta relacionado a la capacidad de la moscas a evadir a depredado-
res. Aunque aun es necesario refinar la estandarizaci6n de 6ste m6todo para permitir la
comparaci6n entire cepas, los resultados confirman la importancia de tener un m6todo ruti-
nario para medir la capacidad de evasion de depredadores en cepas de cria de laboratorio de
la mosca del Mediterraneo. Ademas de medir este parametro de control de calidad de los ma-
chos est6riles, el m6todo descrito podria tambi6n ser usado para la selecci6n sistematica de
cepas con una mayor capacidad de evasion de depredadores.

Translation provided by the authors.


Sterile male insects, mass-reared and released must survive long enough in the field to compete
as part of a sterile insect technique (SIT) program, effectively with wild males for wild females.







Hendrichs et al.: Measuring Predator Evasion in C. capitata Flies


An often-reported problem in Mediterranean
fruit fly (medfly) Ceratitis capitata (Wiedemann),
area-wide control programs integrating the SIT,
is the short period after sterile male release dur-
ing which the males can be recaptured. To date, a
number of very thorough field assessments of the
dispersal and survival of sterile medflies have
been carried out (Wong et al.1986; Baker & Chan
1991; Plant & Cunningham 1991; Baker & van
der Valk 1992; Bloem et al. 1994). Most of these
studies report rapid declines in numbers of re-
leased sterile flies. On average, the last recap-
tures of released medflies in these studies have
been on d 8. This rapid decrease of flies occurred
even where the recapture covered a 1 km2 area,
and fly emigration was shown to be insignificant
as evidenced by the few flies that reached the
outer perimeter of traps (Plant & Cunningham
1991). As a result, medfly SIT programs often
have to release twice a week to ensure a sufficient
presence of sterile males in a given area (Bloem et
al. 1994; Dowell et al. 2000).
The rapid decrease of released sterile flies is
probably the result of many abiotic and biotic fac-
tors, including predation. Various studies under
natural conditions confirm the importance of ver-
tebrate and arthropod predators in medfly biology
(Hendrichs & Hendrichs 1990; Hendrichs et al.
1991; Baker & van der Valk 1992). During field-
testing of dispersal and survival of a temperature
sensitive lethal (tsl) genetic sexing strain, VI-
ENNA 42 (Franz et al. 1994) in a citrus orchard,
the rate of decrease was again high, and recap-
tured males appeared to be locating and feeding
at the same natural food sites as wild males (Hen-
drichs et al. 1993). At the same time, there was a
ten-fold difference in survival in favor of VIENNA
42 males held in control field cages in the orchard
compared with those released into the same or-
chard. However, during the same study it was
shown that in control cages with orange trees, in
which the entrance was left slightly open, sterile
males suffered heavy losses due to predation by a
yellowjacket wasp Vespula germanica L. On each
of the 4 occasions that the study was replicated,
wasps entered the open field cage in large num-
bers and within 5-7 h captured all of the flies that
had been released onto the field-caged host tree
(Hendrichs et al. 1993). In another related field
study, it could be demonstrated that foraging V.
germanica wasps followed the odor plume to pher-
omone-calling medfly males aggregated in leks
within dense host foliage (Hendrichs et al. 1994).
Other field and field-cage studies have confirmed
the sexually-biased predation suffered by mature
medflies in nature (Hendrichs & Hendrichs 1998).
In view of the above findings, the objective of
the present study was to develop a simple quality
control test to measure predator evasion in wild
and mass-reared medfly males. The development
of such a test is the first step in assessing the low


escape ability of mass-reared flies, with the even-
tual goal of improving the effectiveness of appli-
cation of SIT against medfly.

MATERIALS AND METHODS

Tests in Chios, Greece

All tests were conducted on orange trees, Cit-
rus sinensis Ob., (approximately 2.0 m tall with
2.2 m wide crowns) within field-cages used for be-
havioral studies (2.2 m height x 3 m diameter).
The first 2 tests (Tests 1 and 2) took place in a cit-
rus orchard in Chios, Greece, with wild medfly
males, originating from sour oranges and figs,
and laboratory males of a medfly genetic sexing
strain VIENNA 42 (a temperature shock in the
egg stage kills the female eggs, Franz et al. 1994),
mass-reared and sterilized (90 Gy) at the FAO/
IAEA Agriculture and Biotechnology Laboratory
in Seibersdorf, Austria. The males were shipped
from Vienna to Chios, Greece (total transport
time 12 h), for field-testing (Hendrichs et al.
1993). Wild and sterile laboratory reared males
were marked with a different color on the thorax
and released together into the field-cage the day
before testing. The methodology followed was de-
scribed in Hendrichs & Hendrichs (1998).
In Test 1, the field cage entrance zipper was
left open (approximately 0.15 m wide) to allow ac-
cess to foraging yellowjacket wasps, V germanica
L. Whenever a wasp entered the cage, the zipper
was closed and all attacks of the wasp on wild and
sterile medfly males were followed by two observ-
ers and recorded until the wasp captured a fly and
was allowed to leave the cage.
In Test 2, no wasps were allowed into the field
cage by keeping the cage zipper closed. Two observ-
ers simulated predation attacks on flies by at-
tempting to capture flies by sucking them into aspi-
rators normally used to handle flies in laboratories.
The 2 observers alternated every 20 attempts each
to capture wild (10 attempts) and sterile males (10
attempts) within the orange tree foliage. The aspi-
rators were held at the lower end of a 20-cm glass
tube inserted into a flexible plastic hose, the end of
which was held in the mouth of the person attempt-
ing to capture flies. The opposite end of the glass
tubing, through which flies were sucked, had a 5-
mm diameter opening that was bent at an angle of
about 70-75 degrees to facilitate reaching for flies
present on or below surfaces such as host foliage.
On average there were 2 capture attempts per min.
The ability of flies, involved either in pheromone-
calling or other activities such as resting or feeding,
to escape from aspirating humans was quantified.

Tests in Seibersdorf, Austria

Tests 3 and 4, carried out at Seibersdorf, Aus-
tria, were based on the use of aspirators to cap-







Florida Entomologist 90(1)


ture flies on potted orange trees in field cages
(same dimensions as above). Unmarked flies of
different treatments were placed into separate
but adjacent field cages. In Test 3, with laboratory
flies of the pupal color dimorphism medfly genetic
sexing strain SEIB 30-C (Robinson et al. 1999),
we compared non-irradiated and irradiated flies,
which were exposed to gamma radiation (90 Gy)
as mature pupae two days before adult emer-
gence. Flies were compared, while involved in dif-
ferent activities on the host tree, in their capacity
to evade capture with aspirators. This compari-
son was carried at 2-d intervals, starting with
adult emergence, to assess changes in evasive
ability during the fly maturation period.
In Test 4, we carried out inter-strain compari-
sons to measure the capacity to evade capture in 3
different laboratory strains: genetic sexing strain
VIENNA 42, pupal color genetic sexing strain
SEIB 1-61, and a strain originating from coffee in
Guatemala and held in the laboratory for 22 gen-
erations. Flies of all strains were mature (6-8 d old)
when tested. One-half of all capture attempts in
Test 4 were made by an observer with no previous
experience with aspirators, the other by an experi-
enced user of aspirators. In addition, both observ-
ers repeated the comparison of the 3 strains under
2 environmental regimes: sunny with approxi-
mately 23-25C, or overcast and only 20-21C.
In all 4 tests, conducted in Chios as well as
Seibersdorf, care was taken that flies captured
did not exceed 40% of flies released or originally
emerged in a cage, in order to prevent the fly pop-
ulation in the cage from being constituted pre-
dominantly of individuals who had succeeded in
escaping capture. Furthermore, although not re-
corded as part of the tests, every effort was made
to remove flies that had escaped in the first cap-
ture attempt to eliminate them from the popula-
tion to be sampled. However, this was not possible
in the first test where wasps were often seen at-
tacking the same fly several times in succession


before capture or definite escape of the fly. Data of
all tests were analyzed by Kruskal-Wallis non-
parametric one-way analysis of variance. In Tests
1 and 2 each replicate consisted of the percent
capture in 10 capture attempts; in Tests 3 and 4
each replicate consisted of 5 capture attempts.

RESULTS

Tests in Chios, Greece

Results of Tests 1 and 2 conducted in Chios,
comparing wasp attacks and simulated attacks
with aspirators, are shown in Table 1. Three to 4
times more sterile VIENNA 42 males than wild
males were captured by wasps. This highly signif-
icant difference was found for both pheromone-
calling males and for males engaged in other ac-
tivities (Table 1A).
In Test 2 (Table 1B), the simulated predator at-
tacks with aspirators resulted also in signifi-
cantly higher captures of sterile VIENNA 42
males, which were 2 to 3 times more likely to be
captured than wild males. Overall, the percent
capture of medfly males, irrespective of fly type or
fly activity, was 18.2 13.6% (SD) for wasps and
55.7 25.6% (SD) for aspirators. In spite of this
difference between wasps (Test 1) and observers
with aspirators (Test 2) in capturing flies (P <
0.0001), the capture ratios between fly types and
fly activities in general were similar for both
tests. In addition (Test 2), there were no differ-
ences in percentage captures between the two ob-
servers experienced with the aspirators used to
handle flies (P = 0.4576).

Tests in Seibersdorf, Austria

Results of Test 3, again measuring evasion of
capture with aspirators, are shown in Tables 2 and
3. Overall, capacity to avoid capture increased di-
rectly with days since adult emergence in the field


TABLE 1. SUCCESS OF CAPTURE ATTEMPTS (PERCENTAGE SD) OF WILD OR LABORATORY REARED MEDFLY MALES BY
YELLOWJACKET WASPS (A) OR BY EXPERIMENTERS WITH ASPIRATORS (B) ON A FIELD-CAGED ORANGE TREE IN
CHIOS, GREECE. NUMBERS IN PARENTHESIS REPRESENT FLY CAPTURE ATTEMPTS.

Successful capture attempts (%)

Fly activity Fertile wild males Sterile lab. males Significance'

A. Wasps
Sexual activities 8.2 8.7 (100) 36.7 13.4 (90) P = 0.0003**
Other activities2 13.7 6.8 (540) 40.6 12.1 (80) P < 0.0001**
B. Aspirators
Sexual activities 27.6 6.9 (29) 80.7 8.0 (150) P = 0.0058**
Other activities2 28.4 11.7 (101) 62.3 19.9 (220) P < 0.0001**

'Kruskal-Wallis One-Way Nonparametric ANOVA.
Includes feeding and resting males, both on the cage wall and foliage.


March 2007







Hendrichs et al.: Measuring Predator Evasion in C. capitata Flies


cage (Table 2). Although this trend was observable
in non-irradiated flies, it was not statistically sig-
nificant. For irradiated flies the difference was
highly significant, with recently emerged flies
having the lowest capacity for evading predators.
The comparison of non-irradiated and irradiated
flies involved in different activities on an orange
tree is shown in Table 3. Overall, irradiated flies
were significantly more susceptible to being cap-
tured than non-irradiated flies (P = 0.0019). This
was particularly so for activities or locations for
which alertness or wariness of flies is normally
higher, such as male presence on leaf tops or fe-
male approaching male leks (females on the same
or neighboring leaves within a radius of 10-15 cm
of pheromone calling males). On the other hand,
there was no significant difference for activities in
which the attention of males is diverted in sexual
activities, for example pheromone-calling, male-
male aggressive encounters, courting or mating.
Results of Test 4 are presented in Tables 4 and
5. As shown in Table 4, no differences were found
in capture avoidance between the 3 laboratory
strains under comparison (P = 0.2544) and cap-
ture rates were only slightly, but not significantly,
higher at the cooler temperatures (Table 4). While
differences in capture rates between the respec-
tive fly activities (Table 5) were similar to the pre-
ceding aspirator tests, observer experience in
with aspirators played a role for those fly activi-
ties in which alertness is normally higher, such as
male presence on leaf tops or females on fruit. For
these activities significant differences in fly cap-
ture were obtained between the experienced and
the inexperienced observer, whereas for those ac-
tivities in which flies are less wary because of in-
volvement in sexual activities or feeding no such
differences were found (Table 5).

DISCUSSION

Our results, comparing wasp attacks and sim-
ulated attacks with aspirators under semi-natu-
ral conditions, show that sterile laboratory med-


flies are less likely to evade capture than wild
flies. This confirms the relevance of measuring
predator evasion in strains mass-produced for
many generations under standard medfly mass-
rearing conditions.
Considering the significantly higher suscepti-
bility of laboratory reared flies to capture by for-
aging predators, resulting in significant losses for
fruit fly control programs that integrate the SIT,
it appears important to develop and apply a sim-
ple quality control test for tephritid fruit flies that
addresses sterile fly capacity to avoid capture.
Presently, the FAO/IAEA/USDA (2003) manual,
which is used as a standard for quality control
procedures in fruit fly programs incorporating the
SIT, does not make reference to this aspect of ster-
ile fly behavior, or fly agility or irritability, in gen-
eral. Even the "startle" test, recommended as part
of the RAPID quality control system (Boller et al.
1981), and listed in a quality control manual used
in Latin America (Orozco et al. 1983), is not in-
cluded in the above international fruit fly quality
control manual.
Open field mating tests consistently require
much higher sterile to wild male overflooding ra-
tios than are needed within relatively protected
field cages or large field enclosures to achieve the
same levels of egg sterility (Rend6n et al. 2004;
Shelly et al. 2005). As the successful food foraging
ability of sterile males has been repeatedly con-
firmed (Maor et al. 2004), this discrepancy be-
tween open field and field enclosure results must
derive from the very high mortality that sterile
males incur in the field before maturing sexually
and encountering potential mates. Thus, ability
to avoid capture is a fundamental aspect of sterile
male quality that needs to be considered in oper-
ational programs.
The startle test measures a complex of reac-
tions involved in fly response to light. It was orig-
inally developed by Schroeder et al. (1973), later
modified by Boller et al. (1981), and further im-
proved by C. O. Calkins (personal communica-
tion), including a compact mobile startle test


TABLE 2. SUCCESS OF CAPTURE ATTEMPTS (PERCENTAGE SD) OF IRRADIATED AND NON-IRRADIATED LABORATORY
REARED MALE AND FEMALE MEDFLIES BY EXPERIMENTERS WITH ASPIRATORS ON A FIELD-CAGED ORANGE
TREE OVER SUCCESSIVE PERIODS AFTER FLY EMERGENCE. NUMBERS IN PARENTHESIS REPRESENT FLY CAP-
TURE ATTEMPTS.

Fly age (days after emergence)

1-2 3-4 5-6

Treatment Successful capture attempts (%) Significance'

Non-irradiated flies2 85.0 15.5 (80) 78.0 17.4 (200) 71.3 25.3 (160) P = 0.2113NS
Irradiated flies2 93.8 12.0 (80) 83.5 14.2 (200) 73.9 23.0 (160) P = 0.0022**

'Kruskal-Wallis One-Way Nonparametric ANOVA.
Mass reared flies of pupal color sexing strain SEIB 30-C.







Florida Entomologist 90(1)


TABLE 3. SUCCESS OF CAPTURE ATTEMPTS (PERCENTAGE SD) OF IRRADIATED AND NON-IRRADIATED LABORATORY
REARED MALE AND FEMALE MEDFLIES IN RELATION TO FLY ACTIVITIES BY EXPERIMENTERS WITH ASPIRATORS
ON A FIELD-CAGED ORANGE TREE. NUMBERS IN PARENTHESIS REPRESENT FLY CAPTURE ATTEMPTS.

Successful capture attempts (%)

Fly activity Non-irradiated flies' Irradiated flies' Significance2

Females approaching leks 62.5 21.8 (80) 81.3 20.0 (80) P = 0.0157*
Males present on leaf tops 65.8 16.1 (120) 80.0 19.6 (120) P = 0.0086**
Males resting on leaf bottoms 75.8 22.1 (120) 82.5 18.9 (120) P = 0.2979NS
Males involved in sexual activities 81.3 18.6 (80) 80.0 14.6 (80) P = 0.6872N"
Mating pairs 86.7 17.3 (40) 95.0 8.9 (40) P = 0.2139NS

Mass reared flies of pupal color sexing strain SEIB 30-C.
'Kruskal-Wallis One-Way Nonparametric ANOVA.


machine with 10 test units. It is the only test
currently available that, indirectly, may give an
indication of fly agility or predator evasion capac-
ity in mass-reared flies. However, the startle test
is actually a type of flight propensity test that dis-
tinguishes between flies that will fly when stimu-
lated from those that do so at a much slower rate.
This flight is not in response to a sudden ap-
proach by a dark object (e.g., a predator) or a sud-
den falling object (Schroeder & Chambers 1977),
but to a sudden onset of light experienced by flies
held previously in total darkness for 30 min.
In terms of measuring capacity to avoid cap-
ture, there are additional disadvantages with the
startle test. Not only is the test carried out under
unnatural conditions, but previous placement of
flies into the test units requires considerable fly
manipulation, often involving CO2. There is also
the possibility of pheromone contamination of the
test units. However, it is not possible to distin-
guish between fly activities nor to detect some of
these problems because flies are concealed within
black holding containers. Finally, the startle test
lasts for a period of 3 min, allowing flies during
this rather long period to fly in the direction of the
light source, whereas responses to a predator at-
tack occur within fractions of a second to, at most,
several seconds.


Fly response to real predation attacks is com-
plex and its measurement even more so. It re-
quires extensive observation not readily amena-
ble to standardization, as well as the availability
of predators (Hendrichs & Hendrichs 1998). Pred-
ators are not available at all locations and during
all seasons. To overcome these complications, we
have described here a simple quality control
method for measuring male medfly capacity to
avoid capture. Unlike the startle test, the test re-
quires no special equipment and does not mea-
sure flight response to onset of light and other
stimuli but rather reflects more directly fly escape
ability under more realistic conditions. Although
we found absolute losses to simulated predation
with aspirators higher than to real predation by
foraging yellowjacket wasps, the relative ability
to avoid capture is similar in mass-reared and
wild males.
Our quality control method for measuring ca-
pacity to avoid capture in fruit fly males would not
require routine testing as part of the RAPID labo-
ratory quality control tests (Boller et al. 1981).
Rather, it could be conducted at longer intervals
as part of the more valuable confirmation quality
control tests carried out under semi-natural con-
ditions (Chambers et al. 1983). Only such tests,
which must be carried out in greenhouses or on


TABLE 4. SUCCESS OF CAPTURE ATTEMPTS (PERCENTAGE SD) OF LABORATORY REARED MEDFLIES WITH ASPIRATORS
ON A FIELD-CAGED ORANGE TREE UNDER DIFFERENT ENVIRONMENTAL CONDITIONS. NUMBERS IN PARENTHE-
SIS REPRESENT FLY CAPTURE ATTEMPTS.

Successful capture attempts (%)

Laboratory strain' Sunny (23-25C) Overcast (20-21C) Significance2

Pupal color sexing strain SEIB 1-61 88.9 14.1 (90) 96.0 8.2 (100) P = 0.0845N
tsl sexing strain VIENNA 42 92.0 10.1 (200) 95.0 8.9 (200) P = 0.3173"
Bisexual strain from Guatemala 95.0 8.9 (100) 97.9 6.3 (95) P = 0.2452NS

'Only those fly activities are included in which there were no differences in capture rates between experimenters (see Table 5).
No difference between the 3 strains in percent capture (P = 0.2544Ns).
Kruskal-Wallis One-Way Nonparametric ANOVA.


March 2007







Hendrichs et al.: Measuring Predator Evasion in C. capitata Flies


TABLE 5. SUCCESS OF CAPTURE ATTEMPTS (PERCENTAGE SD) OF LABORATORY REARED MEDFLIES WHEN COMPARING
EXPERIMENTERS WITH DIFFERENT EXPERIENCE IN USING ASPIRATORS ON A FIELD-CAGED ORANGE TREE.
NUMBERS IN PARENTHESIS REPRESENT FLY CAPTURE ATTEMPTS.

Successful capture attempts (%)

Fly activity' Inexperienced2 person Experienced person Significance3

Females present on fruit 72.0 11.0 (25) 96.0 8.9 (25) P = 0.0139*
Males present on leaf tops 72.0 29.1 (75) 94.7 9.2 (75) P = 0.0057**
Males resting on leaf bottoms 88.0 14.7 (75) 93.3 9.8 (75) P = 0.3373NS
Males feeding 96.0 8.3 (75) 98.7 5.2 (75) P = 0.2909NS
Males pheromone-calling 90.7 10.3 (75) 92.0 10.1 (75) P = 0.7172NS
Mating pairs 98.5 5.5 (65) 97.8 7.3 (70) P = 0.5930NS

Combined data of comparison of 3 mass reared laboratory strains.
'No previous experience.
Kruskal-Wallis One-Way Nonparametric ANOVA.


field-caged host trees, and preferably including
wild females as a quality baseline, will provide a
more definite verification of sterile fly quality.
Results, both with wasps and aspirators, con-
firm that mass-rearing conditions favor the pro-
duction of flies with a decreased irritability or "ner-
vousness". Highly irritable flies appear to die
sooner in crowded adult colony cages of mass-rear-
ing facilities than do more sedentary flies (Calkins,
personal communication), and less irritable flies
appear to have a higher mating success under
these conditions (Bricefo & Eberhard 2002).
To counter this inadvertent selection, the de-
scribed method, besides measuring capacity to
avoid capture, could be useful for the systematic
selection of strains with a lower threshold for star-
tle activity (Ewing 1963; Schroeder & Chambers
1977). The "filter rearing system" (Fisher &
Caceres 2000; Caceres et al. 2004), involves man-
aging a mother colony that is filtered to maintain
the purity of genetic sexing strains, and from
which large colonies are regularly derived for ster-
ile male mass-production. This same approach
could be extended to establish small "pre-filter"
mother colonies under relaxed low density condi-
tions, preferably in greenhouses or field-enclosures
with trees and include the selection for males that
achieve matings with wild females, and that main-
tain a certain irritability to reduce easy capture.
As in the case of the startle test, a number of
variables have to be controlled to standardize the
aspirator method to make it more reproducible.
These include temperature and light conditions,
fly location, fly sex, fly age, fly numbers (in rela-
tion to foliage surface), and nutritional history of
the flies. For example, temperatures during test-
ing should not fall outside the 24-28C range, and
flies located on the cage wall should not be in-
cluded in the measurements, in view that flies
away from the host tree are less able to evade cap-
ture attempt than those present within the host
foliage.


A major disadvantage, as shown by our tests,
is individual variation in skill in the use of aspi-
rators. However, preliminary tests have shown
that better uniformity can be achieved by with
battery-powered mechanical aspirators or by not
applying suction until just before the tip of the as-
pirator reaches the fly. In addition, as shown in
Table 5, the observer effect can largely be reduced
by restricting measurements to males involved in
sexual activities, as they are the main targets of
foraging predators (Hendrichs et al. 1994; Hen-
drichs & Hendrichs 1998), presumably because
sexually active males are, in general, less wary of
predator attacks (Nagamine & It6 1980; Burk
1982; M.A.H., unpublished data).
In summary, we present a method that allows
measuring the capacity of medflies to evade cap-
ture by predators, essential for sterile males to
reach sexual maturation under open field condi-
tions. It is recognized, that further standardiza-
tion of the test is required to allow more accurate
inter-strain comparisons to be made. However, it
can be concluded that standard medfly mass-
rearing conditions result in the production of ster-
ile males that are much less able to evade preda-
tion than are wild males. Recognition of this prob-
lem and development of a corresponding quality
control test could significantly improve the reli-
ability and economics of sterile insect technology
for fruit flies.


ACKNOWLEDGMENTS

This work was supported in part by the EU Agroin-
dustry Grant AIR3-CT92-0300 to B. I. K. We thank C. O.
Calkins, W. Klassen, P. Rend6n, L. LaChance, and A. S.
Robinson for critical reviews of an earlier version of this
manuscript, C. O. Calkins for useful information and K.
Gaggl and Harry Baumgartner (FAO/IAEA, Seibers-
dorf, Austria) for technical assistance. This work is part
of a dissertation by M. A. H. at the University ofVienna,
Austria.











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Florida Entomologist 90(1)







Nestel et al.: Medfly Pupae Respiration Patterns


GAS-EXCHANGE PATTERNS OF MEDITERRANEAN FRUIT FLY PUPAE
(DIPTERA: TEPHRITIDAE): A TOOL TO FORECAST DEVELOPMENTAL STAGE

DAVID NESTEL1, ESTHER NEMNY-LAVY AND VICTOR ALCHANATIS2
'Department of Entomology, Institute of Plant Protection, Agricultural Research Organization
Volcani Center, P.O. Box 6, 50250 Beit-Dagan, Israel

2Department of Sensing, Information and Mechanization, Institute of Agricultural Engineering
Agricultural Research Organization, Volcani Center, P.O. Box 6, 50250 Beit-Dagan, Israel


ABSTRACT

The pattern of gas-exchange (CO2 emission) was investigated for developing Mediterranean
fruit fly (medfly) Ceratitis capitata (Wiedemann) pupae incubated at different temperatures.
This study was undertaken to explore the usefulness of gas-exchange systems in the deter-
mination of physiological age in developing pupae that are mass produced for sterile insect
technique projects. The rate of CO2 emission was measured in a closed flow-through system
connected to commercial infrared gas analysis equipment. Metabolic activity (rate of CO2
emission) was related to pupal eye-color, which is the current technique used to determine
physiological age. Eye-color was characterized digitally with 3 variables (Hue, Saturation
and Intensity), and color separated by discriminant analysis. The rate of CO, emission
throughout pupal development followed a U-shape, with high levels of emission during pu-
pariation, pupal transformation and final pharate adult stages. Temperature affected the
development time of pupae, but not the basic CO2 emission patterns during development. In
all temperatures, rates of CO2 emission 1 and 2 d before adult emergence were very similar.
After mid larval-adult transition (e.g., phanerocephalic pupa), digital eye-color was signifi-
cantly correlated with CO2 emission. Results support the suggestion that gas-exchange
should be explored further as a system to determine pupal physiological age in mass produc-
tion of fruit flies.

Key Words: carbon dioxide emission, sterile insect technique, metabolic rate, pupal respira-
tion, physiological age, irradiation time, digital eye-color

RESUME

En el present studio se investigaron los patrons de intercambio gaseoso (emisi6n de CO2) en
pupas de la mosca de las frutas del Mediterraneo (Ceratitis capitata Wiedemann) incubadas a
diferentes temperatures. El studio fue realizado con la finalidad de explorer la utilizaci6n de
sistemas de intercambio gaseoso en la determinaci6n de la edad fisiol6gica de pupas durante
su producci6n masiva en proyectos de mosca est6ril. La proporci6n de emisi6n de CO2 fue me-
dido en un sistema cerrado de "flujo a trav6s del sistema" conectado a un detector infrarrojo de
gases. La actividad metab6lica de la pupa (emisi6n de CO2) fue contrastado al color del ojo de
la pupa en desarrollo, que constitute la actual t6cnica de determinaci6n de la edad fisiol6gica.
El color de ojos en pupa fue determinado digitalmente, usando tres variables (Tendencia, Sa-
turaci6n e Intensidad). Los colors fueron separados utilizando el andlisis discriminatorio. Los
patrons de emisi6n de CO2 durante el desarrollo de la pupa sugieren una tendencia de U: una
alta actividad metab6lica durante la fase inicial de pupaci6n y transformaci6n y durante la
fase final del adulto. La temperature de incubaci6n afecto el tiempo de desarrollo pero no el pa-
tr6n basico de actividad metab6lica. La proporci6n de emisi6n de CO2 uno y dos dias antes de
la emergencia del adulto fue muy similar para pupas mantenidas en las diversas temperatu-
ras. El color digital del ojo de la pupa se correlaciono significativamente con los patrons de
emisi6n de CO2 detectados a partir de la fase media de la transformaci6n de larva a adulto. Los
resultados soportan la utilizaci6n de sistemas de intercambio gaseoso como un sistema auxi-
liar para la determinaci6n de la edad fisiol6gica en cria masiva de moscas de la fruta.

Translation provided by the authors.


The sterile insect production capacity has insects; released sterile males copulate with wild
greatly expanded during the last decades and fertile females curtailing their ability to produce
evolved into an industrial process. For fruit flies, a new generation of wild flies. The success and in-
many mass-produces sterile insects for field re- creased interest in the SIT, is based on scientific
lease to control the damage exerted by these pest achievements and technological innovations de-







Florida Entomologist 90(1)


veloped during the last decades, and on the abil-
ity to produce and deliver sterile fruit flies of good
quality and sexual competitiveness (Fisher 1997;
Hendrichs et al. 2002; Tween 2002).
This technological advance has opened the
doors for the participation of the private sector in
this enterprise, which was solely financed in the
past by the public sector. New facilities, some of
them based on private capital (such as "Bio-Fly"
in Israel and "Insecta" in Europe) are being
opened, where labor is expensive and production
is capital intensive. These new tendencies in the
industrial production process of sterile insects are
driving research and development in the direc-
tion of process automation, labor saving activi-
ties, and the reduction in production uncertain-
ties. Our aim in this study was to support this
tendency by exploring the ability of available gas-
exchange measuring technologies to forecast im-
portant biological events of the developing flies.
Specifically, we characterized the gas-exchange
patterns of developing pupae and explored ana-
lytical methods to forecast and monitor key
events in the production process.
Key biological events during mass production
of Mediterranean fruit flies (medflies) Ceratitis
capitata (Wiedemann) for SIT purposes include,
among others, egg hatching, larval jumping, pu-
pation (Quesada-Allue et al. 1996), stage of pupal
development, and adult emergence. Knowledge
on the precise pupal physiological age is of impor-
tance for the management of mass-production
and for sterilization purposes (Ruhm & Calkins
1981). Medflies are sterilized by exposing pupae
to gamma-radiation. The outcome of such expo-
sure on induced sterility and fly quality depends
on the physiological age and dose at which pupae
are irradiated (Ruhm & Calkins 1981). Pupae ir-
radiated earlier than the optimal time could be
damaged, affecting their adult performance and
ability to effectively mate (Ohinata et al. 1971).
On the other hand, late irradiation could compro-
mise sterility (FAO/IAEA/USDA 2003), leading to
the release of fertile flies. These fertile flies may
copulate with wild ones, affecting the sterile/wild
relationship and effectiveness of the program. In
addition, the ability to monitor and forecast the
physiological stage of the developing pupae could
be of use during the daily activity and working
program of mass-rearing facilities. As an example,
mass-rearing facilities manipulate the environ-
mental conditions (such as temperature) of devel-
oping pupae to synchronize, and adjust, produc-
tion timing to field logistics and release schedules
(Ruhm & Calkins 1981). In this sense, a system
able to automatically determine the physiological
stage of the developing pupae could serve to man-
age and set incubation temperatures (i.e., auto-
matically) as required by field timetables.
The current system to determine pupal physi-
ological age consists of removing the pupal case


and visually inspecting the color of the eye (FAO/
IAEA/USDA 2003). This subjective system is
based on the fact that eye-color is known to
change with pupal physiological age (Ruhm &
Calkins 1981; Resilva et al. 2007). Eye color in de-
veloping pupae becomes apparent during mid-pu-
pation with the version of the head and the initi-
ation of the "phanerocephalic" pupal stage (Que-
sada-Allue et al. 1996). At this stage, pupal eyes
are whitish. Eye coloration starts to change at the
end of the pupal stage and beginning of the
pharate-adult stage, with the increase in meta-
bolic activity: at this stage, eyes become yellowish
(Quesada-Allue 1994). Subsequently, eye color
rapidly changes from yellow to orange, to red, to
brown orange and, before adult emergence, to iri-
descent (Ruhm & Calkins 1981; Quesada-Allue
1994; Quesada-Allue et al. 1996). At 23C, the
whole larval-adult transition may be accom-
plished in 13 d (Quesada-Allue et al. 1996). At
this temperature, the onset of the phanerocepha-
lic stage, where eyes have a whitish coloration, oc-
curs 48 h after larval immobilization, while the fi-
nalization of the pupal stage and initiation of the
pharate adult stage, with the subsequent acceler-
ation of metabolism and change of eye color to yel-
low, is observed 120 h (5 d) after larval immobili-
zation (Quesada-Allue et al. 1996).
Recently, Donoso et al. (2003) suggested moni-
toring temperature in pupal incubation rooms to
determine physiological age and time of irradia-
tion. These authors proposed that since insect de-
velopment is dependent upon temperature (Ratte
1984), degree-day models could be used to deter-
mine emergence time and the precise time for irra-
diation. While this method could provide us with a
good approximation of physiological age, it is an
indirect estimation of age which is based on pa-
rameters estimated under constant conditions. As
a result, it could be insensitive to temperature os-
cillations and other environmental factors inside
incubation rooms that could affect development.
Direct measurements of metabolic activity, on
the other hand, could provide a more reliable sys-
tem for the estimation of physiological age. Oxy-
gen consumption and carbon dioxide (CO2) pro-
duction in living organisms, as an example, are
the direct outcome of metabolic activity. The rates
at which these gases are consumed or produced
are directly related to the rate at which metabo-
lism proceeds in the organism (Keister & Buck
1973; Lighton & Wehner 1993). Thus, the mea-
surement of gas exchange is expected to be a good
indication of metabolic activity. The rate of gas ex-
change during metamorphosis of some Diptera
species (e.g., Calliphora erythrocephala Mac-
quart) has already been described and was shown
to follow a U-shaped curve (Agrell & Lundquist
1973), with a high metabolic activity during early
and late developmental stages and a slow-down
during mid-pupal stage. This same U-shaped


March 2007







Nestel et al.: Medfly Pupae Respiration Patterns


curve was described for the oxygen consumption
of developing medfly pupae (Langley 1970).
The present study is based on this previously
generated knowledge. We suggest that the mea-
surement of metabolic activity in medfly pupae
through gas exchange systems could provide us
with a reliable tool to forecast physiological age
and adult emergence time in mass-rearing facili-
ties. In order to investigate this idea, we first dig-
itally characterized the eye-color of developing
medfly pupae as a reference, and to have an objec-
tive method of comparison. We then characterized
the daily patterns of CO, emission on pupae de-
veloping at different constant and variable incu-
bation temperatures (ranging from 15 to 30C).
Finally, the ability of the gas-exchange system to
determine pupal physiological age was inferred
from correlating and contrasting digital eye color
with pupal respiration patterns.

MATERIALS AND METHODS

Study Insects

Larvae were obtained from the colony of the
medfly strain'Sade'(more than 20 years old) of the
Board of Fruit and Vegetable Growers, Israel. This
is a bisexual strain, reared on artificial diet, which
is regularly refreshed with material from the wild
(2-3 times a year). Larvae were collected as they
were leaving the diet ("crawling phase"). Collec-
tions were conducted during a short period of time
to synchronize immobilization and pupation.

Digital Determination of Medfly Pupal Eye-color

Approximately 330 pupae from different ages
(and developing under different temperatures)
were sampled, dissected, and their eyes exposed.
Dissected pupae were positioned, always following
the same orientation on a mini-stage (5 cm in di-
ameter), where the background was always the
same, illumination was provided from the same
sources and from the same directions, and shade
was reduced by a series of mini-reflectors sur-
rounding the stage. Pupae were photographed at a
magnification of 20x with a stereoscopic micro-
scope equipped with a 3-CCD color digital camera
(Sony DXC 990P). A small area of the pupal eye
was focused and a picture taken. The digital im-
age consisted of 3 components: Red, Green, and
Blue (RGB space). The digital image was analyzed
with Image-Pro PLUS version 4.5 software (Media
Cybernetics, Inc.). The 3 basic color components,
RGB, were transformed to an alternative color
space, defined by Hue, Saturation, and Intensity
(HSI). In the HSI color space, the information on
the object's color is expressed mainly through
Hue, because this variable is not affected by the il-
lumination intensity. The Saturation expresses
the vividness of the color, while the Intensity is af-


fected mainly by the illumination intensity. Eye
colors included: white, yellow, orange, red-orange,
brown, dark-brown, and iridescent. For each color
category we analyzed at least 20 specimens.
Eye color data derived from all the samples
were analyzed by "Discriminant Analysis" (Stat-
graphics 5 Plus 2000, Manugistics, Inc.). This
analysis produced 3 canonical variables (Fl, F2,
and F3) that are derived from the original vari-
ables (Hue, Intensity and Saturation). These ca-
nonical variables are used to classify the data into
groups ("standardized digital eye-color"). Result-
ing groups were correlated with their respiration
patterns.

Respiratory Patterns of Medfly Pupae as Affected
by Incubation Temperature

CO2 emission was measured with a closed flow-
through system connected to commercial infrared
gas analysis equipment (Model No. S-151, Qubit
Systems, Inc., Kingston, Ontario, Canada). Pupae
were placed inside a glass Erlenmeyer flask (250
mL), which was hermetically sealed with a rubber
stopper with lure connectors. Air was pumped
(0.4 L/min) through the flask to collect the pupal-
emitted CO,, which was directed to the infrared
carbon dioxide analyzer (Qubit Systems, Model
No. S-151, with a resolution of +1 ppm CO,). Air
emerging from the CO, analyzer was pumped
back into the Erlenmeyer flask. The whole system
was kept closed with vinyl tubing. Closed circula-
tion of air provided the cumulative amount of CO2
emitted by the pupae in a period of time. In order
to measure the rate of CO2 emission, we recorded
its accumulation in a period of 10 min and ob-
tained the rate from the slope. The data-logger
connected to the CO, analyzer (Vernier Software
and Technology, Beaverton, Oregon, USA) gener-
ated 1 measurement per min.
We measured the rate of CO, production in pu-
pae incubated at several constant temperatures
(15, 20, 25, and 30C), and at variable room tem-
perature (R.T.), with temperatures oscillating be-
tween 18-25C. This design was expected to cre-
ate different pupal physiological ages indepen-
dent of chronological age. Each experiment (e.g.,
replicate) consisted of simultaneously incubating
5 g of recently immobilized larvae at the different
temperatures. Incubation in the different envi-
ronments was kept constant from the moment of
larval immobilization until adult emergence.
Gas-exchange measurements took place around
mid-day. During the last days of pupal develop-
ment, there are differences in metabolic rate
throughout the day (e.g., circadian rhythms), which
tend to increase after mid-day (Nestel, unpublished
data). Due to this we kept constant the time of the
day at which gas-exchange was measured. Batches
of pupae were taken from the incubator, weighed
(pupae lose water throughout development, reduc-







Florida Entomologist 90(1)


ing their initial weight at larval immobilization by
approximately 20%, Nestel et al. 2003), placed in-
side the Erlenmeyer flask, and connected to the
flow-through Erlenmeyer flask system to measure
CO2 production during a period of 10 min. At the
end of the measurement, pupae were returned to
their respective incubator until the following mea-
surement next day. This experiment was repeated 2
to 3 times on different dates. For room temperature
(R.T.), we only conducted 1 replicate.
CO2 emission patterns during pupal develop-
ment under the different temperatures were ob-
tained by fitting the data to quadratic functions
(Statgraphics 5 Plus 2000, Manugistics, Inc.). Av-
erage rate of CO2 production 1 and 2 d before
adult emergence were extrapolated from the cal-
culated functions. During all the experiments, we
sampled more than 10 pupae per temperature
and day. These were dissected and their eye-color
determined with the digital ImagePro System.
Canonical variables were derived from the origi-
nal variables (Hue, Intensity and Saturation) as
explained earlier, and the derived standardized
digital eye-color was later correlated with rate of
CO2 emission during the specific day and temper-
ature regime (see the following section).

Relationship between Standardized Digital Eye-color
and Respiration Patterns

Pupae collected in the above experiment were
immediately dissected and their eye-color digitally
characterized by the 3 variables with ImagePro.
Based on the derived canonical variables, a "stan-
dardized digital eye-color" was determined for
each pupa. The average "standardized digital eye-
color" for a specific temperature treatment during
a specific date was related to the rate of CO2 pro-
duction measured during that day based upon lin-
ear regression (Statgraphics 5 Plus 2000,
Manugistics, Inc.). Due to the typical U-shape pat-
tern of gas exchange, we decided to investigate this
relationship with data on CO2 emission obtained in
all temperatures and replicates from mid-pupal
development period until adult emergence. Thus,
digital eye-color was correlated with CO2 emission
from d 10 in pupae developing at 15C, from d 6 in
pupae developing at 20C, from d 4 in pupae devel-
oping at 25C, from day 2 in pupae developing at
30C, and from d 6 in pupae developing at room
temperature (R.T). These dates corresponded in all
of the treatments with pupae having white-eyes.

RESULTS

Digital Characterization of Pupal Eye-color
and Standardization

After pupariation and the version of the head
during mid-pupal stage, pupal eyes have a whitish
coloration. As pupal development proceeds, eye-


color changes from white to yellow, then orange,
red-orange, brown, dark-brown, and finally irides-
cent (slightly before adult emergence). Fig. la
shows the scatter graph derived from the digital
characterization of pupal eye-color using the Hue,
Saturation and Intensity system. As can been seen,
the colors are not well separated by these 3 vari-
ables. As a result we applied a multivariate analy-
sis to separate between groups. The canonical dis-
criminant analysis resulted in 3 significant "canon-
ical variables" able to discriminate between the 7
levels of pupal eye-color (Variable Fl, X2 = 1377, df
= 18, P < 0.01; Variable F2, x2 = 651, df = 10, P <
0.01; Variable F3, X2 = 101, df = 4, P < 0.01). Fig. lb
shows the clusters of pupal eye colors formed by us-
ing the 2 first canonical variables Fl and F2. The
ability of canonical variable Fl to predict pupal
eye-color from digital data of Hue, Saturation and
Intensity used for the calibration is provided by Ta-
ble 1. The overall classification accuracy was
74.01%. The classification accuracy decreases (to
53% of cases accurately predicted) when the pupal
eye-color obtains an iridescent coloration.

Effect of Incubation Temperature upon Pupal
Development Time and CO2 Emission

Fig. 2 shows the daily patterns of CO2 emission
at mid-day as affected by incubation temperature
and chronological pupal age. The figure shows ac-
tual measurements and the fitted quadratic func-
tions. In all the cases, the fitted quadratic function
resulted in a high coefficient of determination
(above 0.85). Rate of gas exchange patterns fol-
lowed a typical U-shape, with a high metabolic ac-
tivity during the first hours and days of metamor-
phosis, and a lower level by mid-pupal period. After
this drop in metabolic activity, CO2 emission
steadily increases up to adult emergence. Using the
resultant quadratic functions obtained for each in-
cubation temperature, we derived the level of CO2
emission 1 and 2 d before adult emergence by ex-
trapolation (Table 2). Rate of CO2 emission 2 d be-
fore adult emergence ranged from 22.5 to 30.8 nmol
CO/g of pupae/minute while 1 d before adult emer-
gence it ranged from 30.3 to 39.4 nmol. In elevated
incubation temperatures, the rate of CO2 emission
was higher than at lower temperatures. In contrast,
at lower temperatures, rate of emission was more
reduced (Table 2). At 25 and 30C, rate of CO2 emis-
sion 1 and 2 d before adult emergence was compa-
rable. At 20C rate of CO2 emission was slightly
lower than at these 2 temperatures. Incubation at
room temperature produced the lowest rates of CO2
emission for 1 and 2 d before adult emergence.

Relationship between Standardized Pupal Eye-color
and Patterns of CO2 Emission

Fig. 3 shows the relationship between stan-
dardize pupal eye-color and rate of CO2 emission.


March 2007






Nestel et al.: Medfly Pupae Respiration Patterns


40 80 120

Hue


180


Saturation


Canonical Discriminant Function 1


Fig. 1. (A) Scatter plot of medfly pupal eye-color (visually determined) separated by 3 digital parameters (Hue,
Saturation and Intensity) derived from the digital-photographic characterization. (B) Classification of pupal eye-
color (visually determined) by canonical (unstandardized) variables 1 (Fl) and 2 (F2): [Fl = (-0.0272642 x hue) + (-
0.0286977 x saturation) + (-0.078355 x intensity) + 15.7097] and [F2 = (-0.010854 x hue) + (0.0815401 x saturation)
+ (0.04218 x intensity) 13.5662]. 0 white pupal eye-color, + yellow, x orange, 0 red-orange, A brown, I dark-brown,
* iridescent.

Eye color was standardized based upon canonical Standardized Digital Eye-color and Respiration
variable Fl (see previous section). As explained in Patterns"), the relationship between the 2 vari-
the methodology section ("Relationship between ables was performed from mid-pupal stage until


240

180

120

60


U)

3|


X X X X

x P 6L^a ><
X X
'. C + 1'
+V -+ a
0+ +

af B;: O
1p+1 CPV E
0o0 7 0 -3 1
0 00

XM
)K -K
NE
m *







Florida Entomologist 90(1)


TABLE 1. CLASSIFICATION ACCURACY OF EYE COLOR (VISUALLY DETERMINED) BY DISCRIMINANT ANALYSIS BASED ON
CANONICAL VARIABLE F1 [Fl = (-0.220486 x HUE) + (0.470136 x SATURATION) + (-1.2807 x INTENSITY)]. THE
DIAGONAL ELEMENTS IN THE TABLE REPRESENT THE PERCENTAGE OF CASES ACCURATELY CLASSIFIED.

Predicted pupal eye-color from digital parameters
Actual eye color
visually determined White Yellow Orange Red-orange Brown Dark-brown Iridescent

White (n = 150) 73.3 24.0 0.0 0.0 0.0 0.0 0.0
Yellow (n = 33) 15.2 78.8 6.1 0.0 0.0 0.0 0.0
Orange (n = 55) 0.0 3.6 76.4 20.0 0.0 0.0 0.0
Red-Orange (n = 25) 0.0 0.0 1 2.0 76.0 12.0 0.0 0.0
Brown (n = 30) 0.0 0.0 0.0 16.7 70.0 13.3 0.0
Dark-Brown (n = 17) 0.0 0.0 0.0 0.0 17.7 70.6 11.7
Iridescent (n = 17) 0.0 0.0 0.0 0.0 0.0 41.7 52.9


adult emergence (eyes in the pupae are only dis-
tinguished after the "phanerocephalic" stage). As
can be seen from the figure, there is a good linear
relationship between these 2 variables. The coef-
ficient of determination (R2) was 0.78, suggesting
that at least 78% of the variability can be ex-
plained by the linear relationship between these
2 variables.

DISCUSSION

The typical U-shape metabolic (and CO2 emis-
sion) pattern during metamorphosis is basically
the manifestation of the early histolysis and later
histogenesis and differentiation processes of the
larval-adult transition (Agrell & Lunquist 1973).
During the pre-pupal and early pupal stage, all
the machinery involved in tissue histolysis (e.g.,
proteolytic enzymes) is highly active (Agrell &
Lunquist 1973; Rabossi et al. 2000; Tolmasky et
al. 2001). This was manifested in our study by the
high levels of CO2 production during the first
hours and days of metamorphosis. Following the
evagination of the head and thoracic appendages,
and formation of definite body proportions (i.e.,
end of the pupation process), metabolic activity
and CO2 emission drops (Agrell & Lunquist 1973;
Rabossi et al. 2000; Tolmasky et al. 2001). At this
stage, pupal eyes are whitish. Metabolic activity
rises again during the pharate adult stage, when
tissues are increasing and further differentiating
(Agrell & Lundquist 1973). During this period
eyes start to change in coloration (Quesada-Allue
1994). As expected and as previously reported for
the medfly (Langley et al. 1972), reduced incuba-
tion temperatures delayed the metamorphosis
process and the time needed for the completion of
development. However, the basic U-shape meta-
bolic patterns did not differ for any of the tested
temperatures, and metabolic activity as measured
from CO2 production followed the expected trend.
The ability of the digital system and discrimi-
nant function to predict eye-color was within


acceptable ranges (overall, more than 70% of
the cases were correctly classified). The success
of this system to correctly classify pupal eye-color
decreased when the eye color became fully irides-
cent. The main source of misclassifications was
with iridescent eyes, when 43% of the cases where
classified as dark-brown when our subjective clas-
sification was iridescent. The lack of accuracy in
this case could be related to our inability of sub-
jectively classifying eye color correctly, or to the
inability of the discriminant function to com-
pletely separate between these 2 similar classes
of eye-color. In any case, this misclassification at a
sensitive moment of production highlights the
problem of the system to rely on a subjective mea-
surement to make decisions, which is mainly
based on the perspective of the human eye.
The significant linear relationships after mid-
pupal stage between standardized pupal eye-
color and average pupal emission of CO2 strength-
ened the working hypothesis of this study that
the rate of gas exchange, and therefore of meta-
bolic activity, is an indication of mid-pupal and
pharate adult physiological age. The variability of
the estimated regression (mainly at advanced pu-
pal ages and larger rates of CO2 emission) can
probably be attributed to 2 aspects: (1) the re-
duced synchronicity of pupal age resulting from
incubations at low temperatures (e.g., 15 and
20C), and (2) the mentioned misclassification of
eye-colors close to adult emergence.
At low temperatures, adult emergence usually
extends over a longer period of time (Nestel, un-
published data). In contrast, pupae incubated si-
multaneously at higher temperatures result in
adult emergence occurring more synchronously
within a shorter period of time (Nestel, unpub-
lished data). This lack of synchronicity at lower
incubation temperatures, and the resulting mix-
ture of physiological ages within the samples may
explain the slightly lower rate of CO2 emission ob-
served 1 and 2 d before adult emergence in pupae
incubated at 15C, 20C, and room temperature.


March 2007







Nestel et al.: Medfly Pupae Respiration Patterns


15 oC


5 10 15 20


20 oC


E
C



O
2
C
0


E

0)
0



Cu


R2 = 090
2D 25


25 C


6

JA


30 oC


10 15 20 25


R.T.


15 20 25


t1 R2 = 0.89
0


0 5 110


15 20


Chronological Age After Larval Immobilization (Days)

Fig. 2. Patterns of CO2 emission (rate of emission) by medfly pupae incubated at different constant temperatures
throughout development, and at room temperature (variable temperature): 15C, 20C, 25C, 30C, and R.T. (room
temperature). Marks (rectangles, triangles, and circles) show the result of actual CO2 measurements in each of the
replicated experiments. Solid line is the average gas-exchange trend per incubation temperature, and was obtained
from quadratic functions (R2, coefficient of determination for the fitted function). The arrow marks the day before
adult emergence.


This aspect requires further studies, corrections The present study was conducted under labo-
and fine-tunings before the gas-exchange system ratory conditions and a colony of a bisexual strain
can be suggested for mass-rearing facilities, was used. Production levels in this colony are


A A



0 5 10 t5O
) I







Florida Entomologist 90(1)


March 2007


TABLE 2. AVERAGE CO2 EMISSION 1 AND 2 D BEFORE ADULT EMERGENCE FROM PUPAE INCUBATED AT SEVERAL CON-
STANT TEMPERATURES, AND AT VARIABLE ROOM TEMPERATURE. THE LEVEL OF CO2 WAS DERIVED BY EXTRAP-
OLATION FROM THE QUADRATIC FUNCTIONS FITTED TO THE DATA (SEE FIG. 2).

nmol CO/g of pupae/min

Conditions of pupal incubation 2 d before adult emergence 1 d before adult emergence*

15C (constant temperature) 27.9 32.0
20C (constant temperature) 27.1 33.6
25C (constant temperature) 30.8 39.4
30C (constant temperature) 27.1 38.1
Room Temperature (variable) 22.6 30.3


small, and sample size was tailored to the avail-
ability of pupae for experimentation. If the gas ex-
change system is going to be adopted by the SIT
industry however, modifications and adaptation
would be required. One possibility includes the
sampling of developing pupae at critical stages
and measurement of CO2 emission to determine
physiological age in a similar way to the one pre-
sented in this study. A completely different ap-
proach may include the establishment of incuba-
tion rooms in the mass-rearing facilities for ad-
vanced pupal ages, and the automatic monitoring
of CO2 accumulation in the room air. These op-
tions, and other possibilities, however, would
need to be further explored.


r *
u *


-U

-30 -300 -250 -20 -150 -11

Standardized Digital Eye

Fig. 3. Relationship between the avera
ized digital pupal eye-color" (derived fr
variable 1 based on the measured Hue, Sa
Intensity) and the rate of emitted CO2 by p
ear relation was calculated for pupae that
completed the first half of their develop
are starting to increase their metabolic rat
cending portion of the polynomial functic
Data represent measurements of CO2
from pupae developing at several const
tures, and at room temperature. R2 stands
cient of determination for the linear regre


ACKNOWLEDGMENTS

The present study was partially financed by the
IAEA Research Contract 11474. We appreciate the in-
spiring discussions with Hernn F. Donoso Riffo (Centro
de Produccion de Insectos Est6riles, Arica, Chile), who
seeded the idea of this project on the authors. We thank
Yoav Gazit and Ruth Akiva (Board of Fruit and Vegeta-
ble Growers, Israel) for providing medfly larvae and pu-
pae for this project. We appreciate the suggestions
brought by several anonymous reviewers to previous
drafts; their contribution enormously helped improve
the contents and understanding of the manuscript.

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SADA-ALLUE. 2003. Lipid, carbohydrates and protein
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AND A. RABOSSI. 1996. Metamorphosis in the Medi-
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RABOSSI, A., L. ACION, AND L. A. QUESADA-ALLUE. 2000.
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RESILVA, S. S., G. B. OBRA, N. F. ZAMORA, AND E. G.
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ual. Manugistics, Inc., Rockville, Maryland, U.S.A.
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LUE. 2001. Synthesis and mobilization of glycogen
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TWEEN, G. 2002. MOSCAMED-Guatemala An evolution
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Florida Entomologist 90(1)


March 2007


EFFECTS OF PRE-IRRADIATION CONDITIONING OF MEDFLY PUPAE
(DIPTERA: TEPHRITIDAE): HYPOXIA AND QUALITY OF STERILE MALES


DAVID NESTEL1, ESTHER NEMNY-LAVY', SHEIKH MOHAMMAD ISLAM2, VIWAT WORNOAYPORN2 AND CARLOS CACERES2
'Department of Entomology, Institute of Plant Protection, Agricultural Research Organization
Volcani Center, P.O. Box 6, 50250 Beit-Dagan, Israel

2Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture
International Atomic Energy Agency, Agency's Laboratories, A-2444 Seibersdorf, Austria

ABSTRACT

Irradiation of pupae in sterile insect technique (SIT) projects is usually undertaken in hy-
poxic atmospheres, which have been shown to lessen the deleterious effects of irradiation on
the quality of adult sterile flies. Although this is the accepted technology in most mass-rear-
ing and sterilization facilities, to date no information has been generated on the actual levels
of oxygen (02) in pupae-packing containers during irradiation. The present study utilized re-
cently-developed technology to investigate the O2 level inside bags in which pupae of Medi-
terranean fruit fly (medfly) Ceratitis capitata (Wiedemann) are packed prior to irradiation,
the ability of pupae to create hypoxic environments in these bags, and the effect of 02 atmo-
spheres on the quality of irradiated males. Pupae, 1 d before adult emergence, were shown
to deplete the 02 level in sealed bags in approximately 1 h. The rate of 02 consumption was
dependent upon pupal age and incubation temperature. Incubation temperature did not sig-
nificantly affect the quality of pupae or mating capacity of resultant adult males if pupae
were irradiated under maximal hypoxic conditions inside packing bags. In contrast, mating
competitiveness drastically decreased when pupae were irradiated under ambient 02 condi-
tions, with the packing bag open. There was no difference in the mating capacity of males
when pupae were irradiated in sealed bags under either 10% or 2% 02 levels, or under max-
imal hypoxia. Normal doses of fluorescent dye, applied to pupae to mark sterile flies, did not
affect the ability of pupae to create hypoxic conditions inside packing bags, nor the quality
control parameters of either pupae or adults. Current practices in mass-rearing facilities are
discussed in the light of these results.

Key Words: Ceratitis capitata, oxygen levels, pupal respiration, mating competitiveness, ir-
radiation, sterile insect technique

RESUME

La irradiaci6n de pupas en proyectos de mosca est6ril usualmente se hace bajo condiciones
de hipoxia. Esta condici6n ha demostrado ser menos detrimente a la calidad de las moscas
que la irradiaci6n en atm6sferas con proporci6n normal de oxigeno. Aunque esta ha sido por
much tiempo parte del protocolo de irradiaci6n en plants de producci6n de mosca est6ril,
hasta ahora no se ha medido el contenido de oxigeno dentro de los recipients de empaque
de pupa durante la irradiaci6n. El present studio investig6 los contenidos de 02 en los con-
tenedores de pupas de la mosca de las frutas del Mediterraneo (Ceratitis capitata Wiede-
man), la habilidad de pupas de crear hipoxia dentro de los contenedores, y los efectos del
contenido de 02 durante la irradiaci6n del contenedor en la calidad y capacidad de aparea-
miento de moscas est6riles. Pupas de un dia antes de merger como adults crearon atm6s-
feras de maxima hipoxia dentro del empaque en aproximadamente una hora. La proporci6n
de consume de 02 en contenedores sellados es dependiente de la edad de la pupa, y de la tem-
peratura de incubaci6n. La temperature de incubaci6n no afecto significativamente la cali-
dad ni la capacidad de apareamiento de machos derivados de pupas irradiadas bajo
condiciones de hipoxia. Sin embargo, la capacidad de apareamiento de machos irradiados
como pupas en contenedores abiertos y en condiciones oxigenadas fue drasticamente afec-
tada. En comparaci6n a los resultados anteriores, atm6sferas de 2% y 10% 02 durante la
irradiaci6n no afectaron la capacidad de apareamiento de moscas est6riles. Polvo fluores-
cente, aplicado a pupas para marcar las moscas est6riles, no tuvo efectos sobre la capacidad
de las pupas de crear hipoxia. Los resultados de este studio se discuten en base a las prac-
ticas actuales de producci6n e irradiaci6n.

Translation provided by the authors.


Routine irradiation of fruit flies in control pro- (SIT) is usually undertaken when pupae are
grams integrating the sterile insect technique packed in containers that have been closed or her-







Nestel et al.: Pupal Hypoxia and Medfly Quality


metically sealed for some period prior to their ex-
posure to the radiation source (Schwarz et al.
1985). One variation of this method consists of
the constant flushing of the pupae with nitrogen
gas during irradiation (Fisher 1997). These tech-
niques have been adopted to attain "reduced-oxy-
gen atmospheres" during irradiation. These pro-
cedures have been shown to lessen the "oxygen ef-
fects" of "air irradiation" on mating performance
and competitiveness of sterile males (Hooper
1971; Ohinata et al. 1977). While these practices
are common in many fruit fly mass-rearing and
irradiation facilities, and are recommended in the
international fruit fly quality control manual
(FAO/IAEA/USDA 2003), the actual levels of oxy-
gen (02) inside sealed bags during irradiation
have not, to our knowledge, been previously de-
termined. The present study took advantage of re-
cently developed technology for 02 measurements
in air environments to characterize 02 atmo-
spheres during packing and irradiation of pupae
of the Mediterranean fruit fly (medfly) Ceratitis
capitata (Wiedemann).
Initially, we investigated how pupae of differ-
ent ages, packed in sealed polyethylene bags com-
monly used in many mass-rearing facilities, mod-
ify the 02 environment inside the bag. A second
objective investigated the effect on fruit fly qual-
ity of several "pupae packing pre-irradiation pro-
tocols" commonly performed in different medfly
mass-rearing facilities, on the 02 environments in
packing bags, and on the effect of irradiation upon
resultant adult males. Specifically, we investi-
gated the effect of incubating pupae for a certain
period of time (usually 1 h) at low temperatures
before sending the pupae to the irradiator, as re-
ported by Schwarz et al. (1985). This type of ma-
nipulation, and the transfer of pupae from the
"cold environment" into the room where the irra-
diation chamber is located, may expose the pupae
to several changing temperature regimes (in trop-
ical facilities temperatures in irradiation rooms
may be above 25C). We also investigated the ef-
fect of irradiating pupae at different periods after
the bags were sealed, and determined 02 level in
the sealed bag environment at the start of irradi-
ation. Finally, we investigated the effect of dust-
ing pupae with fluorescent dye during packing
and before irradiation upon the ability of pupae to
modify the 02 environments inside the sealed
bags. Treating pupae with fluorescent dye prior to
irradiation is a routine method commonly used to
identify released sterile flies in traps in the field.

MATERIALS AND METHODS

Source of Insects

Male medfly pupae of the temperature sensi-
tive lethal genetic sexing strain VIENNA 8
(Franz 2005) were obtained from the colony main-


trained in the research facility of the Entomology
Unit, FAO/IAEA Agriculture and Biotechnology
Laboratory, at Seibersdorf, Austria. Pupae were
collected at the appropriate age, as specified in
each of the experiments.

Measurement of Oxygen Levels

Between 400 and 500 mL of pupae were placed
inside 4.5-L polyethylene bags (15 cm width x 45
cm height, 1.5 mm thick), commonly used to pack,
irradiate, and ship medfly pupae in some mass-
rearing facilities (e.g., El Pino, Guatemala). The
bags were perforated in two places by screwing
male and female luer connectors, which opened to
the inner and outer sides of the bag (a hole in the
plastic was punctured through the connectors).
The connectors facing the outside were then at-
tached to vinyl tubing. One of the connectors di-
rected the air emerging from the sealed bag into
an 02 sensor (a lead-O2 battery, Model No. S-102,
Qubit Systems Inc., Kingston, Ontario, Canada).
The bags were sealed, leaving an empty space of
3-5 cm above the pupae. An air pump (0.4 liters/
min) pumped air through the 02 sensor after
which it was directed into the sealed polyethylene
bag. Thus, air was circulated throughout the en-
tire experimental period in a closed circuit. The
depletion of 02 over time, as affected by respira-
tion of the pupae in the sealed system, was regis-
tered by a data logger (Vernier Software and
Technology, Beaverton, Oregon, U. S. A.) that gen-
erated 1 measurement per min. Each experiment
was discontinued when measurements showed a
stable low 02 level (close to 0%) for a period of 10
min ("maximal hypoxia"). Experiments were con-
ducted at 24C except when otherwise specified.

Irradiation and Quality Control Procedures

In experiments where pupae were irradiated,
a 60Co source in a Nordion Gamacell-220 (Nor-
dion, Canada@) was used. Dose was calculated at
150 Gy A Gafchromic dosimeter placed in the cen-
ter of the bag, however, showed an average dose
during the experiments of 157 Gy. The approxi-
mate time spent in the irradiation chamber for
this dose was 8.5 min. After irradiation, samples
of pupae were used to investigate the following
quality control parameters: pupal weight, num-
ber of pupae in 5 mL, % adult emergence, and
flight ability index (FAO/IAEA/USDA 2003).
These quality control parameters were also deter-
mined for non-irradiated pupae of the same batch
of the experiment (control).
An additional test investigated the mating
competitiveness of flies in the different treat-
ments in each of the experiments (including a
non-irradiated control). For each treatment 25
sexually mature males (each marked with a dot of
differently-colored paint on the prothorax to dif-







Florida Entomologist 90(1)


ferentiate treatments) were released into a field
cage inside a greenhouse together with 25 non-ir-
radiated and sexually mature virgin females
(with an Egyptian genetic background that had
been maintained in the laboratory since 1968).
The greenhouse was kept at 25C. A potted citrus
tree, pruned to facilitate observations of mating
pairs, was placed in the center of each cage. Mat-
ing pairs were counted once an hour for 12 contin-
uous hours, giving 12 observations during the en-
tire experiment. Each observation was considered
a replicate. Differences in mating competitive-
ness between treatments was calculated from the
average and variance of the number of mating
pairs found during each 12-h observation period
based on General Linear Models (Statgraphics 5
Plus 2000, Manugistics, Inc.).

Experiment 1: Effect of Pupal Age on 02 Depletion
in Sealed Bags

Non-irradiated pupae 3, 2, and 1 d before adult
emergence were used; 500 mL of pupae of each
age were placed in separate polyethylene bags
equipped with 02 sensors as described above. For
each pupal age, we used 2-3 replicates. A hermet-
ically sealed bag with no pupae inside was used as
a control. The real pupal ages were confirmed by
using "pupal emergence grids", which included a
random sample of 100 pupae (one pupa per grid
space), and by following the emergence from pu-
pae over time. Oxygen levels were measured as
described above at a constant temperature of
24C. Airflow in the closed system was started
when the bags were sealed (= time 0). The rate of
02 depletion was determined by measuring the
02 levels every min. Differences in 02 depletion
rates (at 10 min after sealing), and time until the
attainment of maximal hypoxia inside the bags,
were investigated with a one-way ANOVA; means
were separated by LSD (Statgraphics 5 Plus
2000, Manugistics, Inc.).

Experiment 2: Influence of Incubation Temperature
on Attainment of Maximal Hypoxia and Quality
of Irradiated Males

Polyethylene bags equipped with 02 sensors as
described above were filled with 450 mL of non-ir-
radiated pupae at an estimated age of 1 d before
adult emergence. The bags were laid flat to reduce
the accumulation of metabolic heat and the pupae
were then incubated for 1 h with the bags open
preconditioningng incubation"). Two precondition-
ing incubation temperatures were used, 16C and
24C; 16C was selected because preconditioning
incubations at 16C are performed in some facili-
ties to halt pupal development before irradiation
(FAO/IAEA/USDA 2003). An incubator was used
to attain 16C while 24C was the temperature in
the experimental room. After incubation, bags


were hermetically sealed and immediately con-
nected to the closed airflow system to measure 02
consumption as specified above. Oxygen con-
sumption was investigated under two constant
temperature regimes ("post-sealing incubation
temperatures"): 16C and 24C.
Measurements at 16C were performed with
the bag and the air line inside the incubator. For
this, the door of the incubator was closed with the
vinyl tubes emerging through the rubber door
seal to conduct the air of the closed system to the
02 sensor. We ensured a free flow of air through
the system without affecting the temperature in-
side the incubator. In the case of incubation at
24C, the entire system was in a room maintained
at this constant temperature. Thus, we had four
treatments which combined the two precondition-
ing incubation temperatures (PC) and the two
post-sealing-incubation temperature (PS): (1)
24C PC and 24C PS; (2) 24C PC and 16C PS;
(3) 16C PC and 24C PS; and (4) 16C PC and
160C PS.
After reaching a constant level of maximal hy-
poxia in the sealed bag system, 02 measurements
were discontinued, pupae were irradiated inside
the sealed bags, bags were opened, and samples of
pupae were subjected to quality control tests as
specified above. We repeated the experiment
twice with different batches of pupae.

Experiment 3: Oxygen levels in Packing Bags during
Irradiation and Effect upon Male Fly Quality

As in Experiment 2 we used pupae at an esti-
mated age of 1 d before adult emergence, placing
450 mL of non-irradiated pupae in four polyethyl-
ene bags. Three out of the 4 bags were sealed si-
multaneously but only 1 of the bags was used to
monitor the 02 level. Since bags were sealed si-
multaneously with the same type and quantity of
pupae, we made the assumption that the reading
in the monitored bag would be representative of
those in the other bags. The 4th bag was left un-
sealed. The sealed bags were then individually ir-
radiated 15, 30, and 60 min after sealing, this be-
ing when the 02 level inside the monitored bag
reached approximately 10%, 2%, and maximal hy-
poxia, respectively. In addition, we irradiated the
unsealed bag that was presumed to have an ambi-
ent 02 environment during irradiation. Total irra-
diation time lasted approximately 8.5 min, during
which interval the 02 level inside the sealed bags
are presumed to have decreased further. Thus, the
02 levels mentioned above are those expected at
the onset of irradiation. The experiment was rep-
licated twice with different batches of pupae of the
same age. After irradiation the bags were opened
and samples of pupae from each bag were sepa-
rately subjected to the quality control tests. Non-
irradiated pupae which were not exposed to hy-
poxia were used as a control.


March 2007







Nestel et al.: Pupal Hypoxia and Medfly Quality


Experiment 4: Effect of Fluorescent Dye
on 02 Consumption Patterns and Irradiation Effects

Two polyethylene bags were each loaded with
500 mL of pupae at an expected age of 1 d before
adult emergence. The pupae in 1 bag were thor-
oughly mixed with 0.75 g of Day-Glo fluorescent
powder. The second bag was untreated and used
as a control. The bags were hermetically sealed
and the consumption of 02 over time measured as
described above. After reaching maximal hypoxia
the bags were irradiated, opened, and separate
samples of pupae subjected to quality control
tests. In this experiment we did not investigate
mating competitiveness.

RESULTS

All samples of pupae placed in adult emer-
gence grids confirmed that the expected ages of
pupae used for these experiments were true ages
(results not shown).

Experiment 1: Effect of Pupal Age on 02 Depletion
in Sealed Bags

Fig. 1 shows the effect of pupal age upon the
consumption of 02 by pupae in the closed air sys-
tem. These data show that older pupae consume
02 more rapidly than younger pupae. The rate of
02 consumption 10 min after the bag was sealed
was significantly faster in pupae 1 d before adult
emergence than in pupae 2 or 3 d before emer-
gence (Fig. 2; F = 20.9; df = 2,6; P < 0.01). Simi-


250




I g




- so


0


o t 20
Time Af


Fig. 1. Oxygen consump
(VIENNA 8) sealed in bags
Sd (---- ), 2 d ( ---) a
emergence. The horizontal 1
O/mL air (21% 02) is the
bag when no pupae were pr
row on the x-axis shows the
consumption by pupae was
Fig. 2).


go 0.030
80_ b .
H "
b0.025

60 0.020i

4o 0.015 %
B 40 oa
S30 0.010 '1
200
1 0.005 V
10 1 a
0 -- 0.000
-1 -2 -3
Pupal Age (days before adult emergence)

Fig. 2. Time needed by medfly pupae (VIENNA 8) to
attain maximal hypoxia (bars and left y-axis), and aver-
age and standard deviation rate of 02 consumption (bro-
ken line and right y-axis) as affected by pupal age (data
from Fig. 1, 10 min after sealing bags). Different letters
above bars stand for significant differences (P < 0.05) in
time needed to attain maximal hypoxia (Kruskal-Wal-
lis). Rate of oxygen consumption was significantly dif-
ferent for the 3 treatments (Kruskal-Wallis).


larly, the consumption rate was significantly
faster in pupae 2 d before adult emergence than 3
d before emergence. The time needed by pupae to
reach maximal hypoxia in the bag was signifi-
cantly longer in pupae 3 d before adult emergence
than in pupae 2 or 1 d before emergence (Fig. 2;
F = 9.3; df = 2,6; P < 0.05).

Experiment 2: Influence of Incubation Temperature
on Attainment of Maximal Hypoxia and Quality
of Irradiated Males


The rate at which 02 was consumed by pupae in
hermetically sealed bags when incubated at differ-
ent preconditioning (PC) and post-sealing-bag (PS)
temperatures is shown in Fig. 3. Pupae incubated
at 24C during both PC and PS consumed 02 faster
than pupae incubated first at 24C during PC and
then transferred after 1 h to 16C for PS incuba-
tion (Fig. 3A). Similarly, pupae incubated during
PC at 16C and then transferred to 24C consumed
02 faster than pupae incubated at 16C for both PC
and PS (Fig. 3B). The average durations (2 repli-
cates) needed for pupae to reach maximal hypoxic
40 60 80 100 conditions during these different schedules were
ter Sealing Bags (min) 57.5 min for 24C PC + 24C PS; 87.0 min for 24C
PC + 16C PS; 98.0 min for 16C PC + 24C PS; and
tion curves for medfly pupae 148.5 min for 16C PC + 16C PS.
s as a function of pupal age: Table 1 shows results of the quality control
ind 3 d (---) before adult
nd t appromatey 210 L tests after pupal irradiation under the 4 temper-
me at approximately 210 uiL,
line obtained for the sealed ature combination protocols and in the control.
esent inside the bag. The ar- Average and standard deviation are for 2 repli-
time at which the rate of 02 cate experiments. Mating competitiveness test
determined (data shown in was performed only with 1 of the replicates. Pupal
weight, number of pupae in 5 mL, % adult emer-







Florida Entomologist 90(1)


0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160


0 -I I I
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160


Time After Sealing Bags (min)

Fig. 3. Oxygen consumption curves for medfly pupae (VIENNA 8) 1 d before emergence packed in sealed bags
as a function of 2 pre-irradiation temperature incubation protocols. PC = pre-conditioning incubation protocol; PS
= post-sealing incubation protocol. (A) PC of 24C; (B) PC of 16C; (- ) 0O curves for PS of 24C; (- ) 0
curves for PS of 16C.


gence and flight ability did not differ significantly
between treatments and control. Similarly, incu-
bation temperature protocols did not have any
significant effect upon the mating competitive-
ness of irradiated males (Fig. 4). However, as ex-
pected the mating competitiveness of non-irradi-
ated males (control) was significantly greater
than that of irradiated males (Fig. 4).

Experiment 3: Oxygen levels in Packing Bags during
Irradiation and Effect upon Male Fly Quality

Fig. 5 shows the approximate 02 levels: I, in an
open bag (ambient 02 environment); II, 15 min
after sealing ( 10% 02 level); III, 30 min after


sealing ( 2% 02 level); IV, maximal hypoxia (close
to 0% 02 level). The rate of 02 consumption and the
time needed to reach maximal hypoxia were very
similar in the 2 replicates: at 10 min after bag-
sealing 02 consumption was 9.6 and 9.2 pL O/mL
air/min, and maximal hypoxia was reached after
52 and 58 min, in replicates 1 and 2, respectively.
Table 2 shows the effects of irradiation under
several 02 environments in packing bags upon
pupal weight, number of pupae in 5 mL, % adult
emergence, and flight ability. Average and vari-
ance are for 2 replicate experiments. The mating
competitiveness test was performed only with one
of the replicates. None of the treatments with re-
duced 02 levels had a significant effect upon the


March 2007







Nestel et al.: Pupal Hypoxia and Medfly Quality


TABLE 1. EFFECT OF DIFFERENT PRE-IRRADIATION TEMPERATURE REGIMES ON QUALITY CONTROL PARAMETERS OF IR-
RADIATED MEDFLIES.

Treatment Avg. pupal weight Avg. no. pupae in Avg. adult emergence Flight ability index
PC-PS* (mg/pupae) SD 5 mL SD (%) SD (%) SD

240C->240C 8.65 0.07 285 1 87.7 9.1 84.8 8.6
240C-160C 8.50 0.28 270 25 83.1 16.5 85.7 10.4
160C-240C 8.70 0.14 285 3 86.7 12.3 86.2 9.7
160C-160C 8.55 0.07 291 13 84.2 13.3 86.0 7.5
Control** 8.75 0.07 298 12 92.7 2.6 91.1 7.5
H*** 4.1 3.7 1.0 1.5
P >0.05 >0.05 >0.05 >0.05

*Preconditioning Temperature -> Post-Sealing Temperature.
**Control consisted of non-irradiated pupae, and pupae that did not undergo hypoxia treatment and preconditioning and post-
sealing-bag temperature incubations. Control pupae were maintained at 24C until processing.
***H-Kruskal-Wallis Non-Parametric one way ANOVA.


quality control parameters (Table 2). In contrast,
irradiation under ambient 02 level, although not
statistically significant, reduced adult emergence
and flight ability, and significantly affected the
mating competitiveness of irradiated males (Table
2, Fig. 6). As expected, non-irradiated males (con-
trol) performed significantly better than sterile
males in the mating competitiveness test (Fig. 6).

Experiment 4: Effect of Fluorescent Dye on 02
Consumption Patterns and Irradiation Effects

Oxygen consumption curves of dyed pupae and
undyed pupae 10 min after bag sealing were very


S
A





iotnd 16-116 24-'24 24 16 16-*24
Inmbtion Protcd (PCOC. PS O


Fig. 4. Mating competitiveness (ability to form copu-
lating pairs) of irradiated and non-irradiated (control)
male medflies (VIENNA 8). Irradiated males were sub-
jected to different incubation protocols preconditioningg,
PC, and post-sealing-bag, PS, incubation temperatures
protocols) before irradiation. Irradiation was performed
after reaching maximal hypoxia in sealed bags during
PS incubation. The figure includes the resultant F and P
(General Linear Models) and step-wise separation of
means (lower case letters).


similar: 8.7 and 8.6 pL 0/mL air/min, respec-
tively. Time to reach maximal hypoxia was 58 min
for dyed pupae and 60 min for undyed pupae.
Likewise, pupal weight, number of pupae in 5 mL,
% emergence and flight ability was very similar
between the 2 treatments, and comparable to the
unirradiated control (data not shown).

DISCUSSION

With no renewal of 02, pupae sealed in bags
were expected to totally deplete the 02 levels of
the air through their metabolic activity (FAO/
IAEA/USDA 2003). This study clearly showed
that the 02 level inside sealed bags loaded with
pupae steadily declines over time. Time needed
for the attainment of maximal hypoxia was de-
pendent not only on the age of the pupae, but also


a
13
U

0"


Ii m


o 1 20 a3 a a m


70 B


lme After Sealing Bags (min)

Fig. 5. Oxygen consumption curve and level of hy-
poxia attained for medfly pupae (VIENNA 8) 1 d day be-
fore emergence packed in sealed bags: I. ambient 02
level during irradiation; II. 10% 02 level at the onset of
irradiation; III. 2% 02 level at the onset of irradiation;
IV. Maximal hypoxia at the onset of irradiation.


35

30


25
^ o
2D


10


M
C
I
?08
U
I"5
ID







Florida Entomologist 90(1)


March 2007


TABLE 2. EFFECT OF DIFFERENT PRE-IRRADIATION OXYGEN LEVELS ON QUALITY CONTROL PARAMETERS OF THE IRRA-
DIATED MEDFLY.

Avg. pupal weight Avg. no. pupae in Avg. adult emergence Flight ability index
Treatment (mg/pupae) SD 5 mL SD (%) SD (%) SD

Maximum hypoxia 8.40 0.30 298 8 89.2 0.9 82.6 1.8
2% 02 8.50 0.30 303 1 90.9 2.9 84.2 1.9
10% 02 8.45 0.20 302 4 89.6 1.8 81.6 4.7
Ambient 02 8.55 0.20 303 11 85.3 6.8 67.4 13.4
Control* 8.45 0.40 301 8 89.4 2.3 86.3 0.7
H** 1.2 0.9 1.5 7.3
P >0.05 >0.05 >0.05 >0.05

*Control consisted on non-irradiated pupae that did not undergo hypoxia treatment. Control pupae were maintained at 24C un-
til processing.
**H-Kruskal-Wallis Non-Parametric One Way ANOVA.


on the temperature at which pupae are main-
tained. These results support the findings of Lan-
gley (1970) and Seo et al. (1990), that the rate of
02 consumption is strongly dependent upon the
rate of metabolic activity. Metabolic activity and
respiration rate increased significantly in pupae
close to adult emergence, and in pupae kept at
high temperatures (Keister & Buck 1973; Seo et
al. 1990).
Pre-conditioning (PC) and post-sealing (PS) in-
cubation temperatures had a marked effect upon
the consumption of 02 by pupae. As expected for a
poikilotherm organism, incubation at 24C accel-


S45
35
n s .
30
25,
U20'
S15-
S10 -
s -
g


a
F=9.9,P<.S05

b b b




iii


Control Mauimnal 2%, 1 0% Amblent
Hypoxla


Oxygen Leveb
Fig 6. Mating competitiveness (ability to form copu-
lating pairs) of irradiated and non-irradiated (control)
male medflies (VIENNA 8) as affected by irradiation
under different 02 environments. Hypoxia levels as
shown in Fig. 5:60 min = maximal hypoxia inside sealed
packing bags; 30 min = 2% 02 level at the onset of irra-
diation; 15 min = 10% 02 level at the onset of irradia-
tion; 0 min = open bags with ambient 02 levels. Control
males were neither irradiated nor underwent hypoxia.
The figure includes the resultant F and P (General Lin-
ear Models) and step-wise separation of means (lower
case letters).


rated the consumption of 02 and the metabolism
of pupae, while 16C had a depressing effect upon
both metabolism and 02 consumption. The trans-
fer of pupae from a PC temperature of 24C to a PS
temperature of 16C reduced 02 consumption to a
lesser extent than the opposite situation, suggest-
ing that it takes more time to warm up pupae and
accelerate metabolic rate than to slow down me-
tabolism with cooler temperatures. While temper-
ature manipulation prior to attainment of hypoxia
and irradiation affected the rate of 02 consump-
tion, these incubation protocols did not have any
noticeable effect upon pupal quality and mating
performance. In contrast, ambient 02 levels inside
bags during irradiation had, as previously demon-
strated (Ohinata et al. 1977), an important nega-
tive effect upon mating activity and pupal quality.
The mechanism by which different 02 environ-
ments during irradiation affect the quality of the
fly has still not been fully investigated. However,
the most acceptable hypotheses suggest that low
02 tension in pupal tissue reduces the formation of
toxic free radicals and peroxides during irradia-
tion (Ohinata et al. 1977). Regardless of the mech-
anism by which the 02 atmosphere influences the
quality of the irradiated fly, the present study con-
firmed the fact that a hypoxic environment during
irradiation enhances the mating performance and
quality of sterile pupae (Bakri et al. 2005).
It is interesting to note that irradiation under
10% and 2% 02 environments resulted in flies
with similar mating competitiveness and quality
to those irradiated under maximal hypoxia. This
phenomenon could be the result of ongoing pupal
metabolic activity, and 02 consumption, during
the time spent inside the irradiation chamber (8.5
min), which may have reduced further the 02 lev-
els inside the sealed bags. A further possibility is
that below certain 02 level inside the bags, the
"Oxygen effect" (Hooper 1971; Ohinata et al.
1977) is not manifested. Our study was not able to
detect this "threshold" 02 level, which may be of
theoretical, but not practical, interest.







Nestel et al.: Pupal Hypoxia and Medfly Quality


In practical terms, this study demonstrated
that hermetically sealing bags containing pupae
for irradiation, and keeping these pupae for ap-
proximately 1 h at temperatures of 24C, is suffi-
cient to create an optimal hypoxic environment
inside these bags. It also demonstrated that rou-
tine treatment of pupae with fluorescent dye (1.5
g/kg of pupae) did not appear to affect the ability
of pupae to consume 02 inside the bags. The study
also provided data on the effect of different incu-
bation temperatures upon the ability of pupae to
create hypoxic atmospheres inside packing bags,
and upon the effects of some pre-irradiation incu-
bation protocols currently carried out in several
medfly rearing facilities. Specifically, our data
suggests that keeping pupae in open bags at 16C
until irradiation may affect their later ability to
create an optimal hypoxic environment inside
sealed bags before and during irradiation. These
last results, and the cost/benefit evaluations of in-
cubating procedures, need to be re-assessed be-
fore final recommendations are made.

ACKNOWLEDGMENTS

This study was partially financed by the IAEA Re-
search Contract 11474. The study was undertaken in
the facilities of the Entomology Unit, FAO/IAEA Agri-
culture and Biotechnology Laboratory at Seibersdorf,
Austria. Special thanks to Dr. Victor Gaba (Agricultural
Research Organization, Israel) for comments on a previ-
ous draft of this manuscript, and to several anonymous
reviewers, whose comments and suggestions greatly im-
proved the contents of the final manuscript.

REFERENCES CITED

BAKRI, A., K. MEHTA, AND D. R. LANCE. 2005. Sterilizing
insects with ionizing radiation, pp. 233-268 In V. A.
Dyck, J. Hendrichs and A. S. Robinson [eds.], The
Sterile Insect Technique: Principles and Practice in


Area-Wide Integrated Pest Management. Springer,
Dordrecht, The Netherlands. 787 pp.
FAO/IAEA/USDA. 2003. Manual for Product Quality
Control and Shipping Procedures for Sterile Mass-
Reared Tephritidae Fruit Flies, Version 5. Interna-
tional Atomic Energy Agency, Vienna, Austria. 85 pp.
FISHER, K. 1997. Irradiation effects in air and in nitro-
gen on Mediterranean fruit fly (Diptera: Tephriti-
dae) pupae in Western Australia. J. Econ. Entomol.
90: 1609-1614.
FRANZ, G. 2005. Genetic sexing strains amenable to
large scale rearing as required for the sterile insect
technique, pp. 427-452 In V. A. Dyck, J. Hendrichs,
and A. S. Robinson [eds.], The Sterile Insect Tech-
nique: Principles and Practice in Area-Wide Inte-
grated Pest Management. Springer, Dordrecht, The
Netherlands. 787 pp.
HOOPER, G. H. S. 1971. Competitiveness of gamma-ster-
ilized males of the Mediterranean fruit fly: effect of
irradiating pupal or adult stage and of irradiating
pupae in nitrogen. J. Econ. Entomol. 64: 1364-1368.
KEISTER, M., AND J. BUCK. 1973. Respiration: some ex-
ogenous and endogenous effects on rate of respiration,
pp. 469-509 In M. Rockstein [ed.], The Physiology of
Insecta, Academic Press, New York, USA.
LANGLEY, P. A. 1970. Physiology of the Mediterranean
fruit fly in relation to the sterile-male technique, pp.
25-31 In Sterile-Male Technique for the Control of
Fruit Flies. STI/PUB/276. IAEA, Vienna, Austria.
OHINATA, K., M. ASHRAF, AND E. J. HARRIS. 1977. Med-
iterranean fruit flies: Sterility and sexual competi-
tiveness in the laboratory after treatment with
gamma irradiation in air, carbon dioxide, helium, ni-
trogen or partial vacuum. J. Econ. Entomol. 70: 165-
168.
SCHWARZ, A. J., A. ZAMBADA, D. H. S. OROZCO, AND J. L.
ZAVALA. 1985. Mass production of the Mediterra-
nean fruit fly at Metapa, Mexico. Florida Entomol.
68: 467-477.
SEO, S. T., D. L. WILLIAMSON, AND M. S. FUJIMOTO.
1990. Heat production and thermal conductivity of
respiring Mediterranean fruit fly (Diptera: Tephriti-
dae) pupae during shipment. J. Econ. Entomol. 83:
896-900.







Florida Entomologist 90(1)


March 2007


STERILE INSECT TECHNIQUE: A MODEL FOR DOSE OPTIMIZATION
FOR IMPROVED STERILE INSECT QUALITY


ANDREW PARKER1 AND KISHOR MEHTA2
1Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture
International Atomic Energy Agency, Agency's Laboratories, A-2444 Seibersdorf, Austria

2Briinner Strasse 133-3-29, A-1210, Vienna, Austria

ABSTRACT

The sterile insect technique (SIT) is an environment-friendly pest control technique with ap-
plication in the area-wide integrated control of key pests, including the suppression or elim-
ination of introduced populations and the exclusion of new introductions. Reproductive
sterility is normally induced by ionizing radiation, a convenient and consistent method that
maintains a reasonable degree of competitiveness in the released insects. The cost and effec-
tiveness of a control program integrating the SIT depend on the balance between sterility and
competitiveness, but it appears that current operational programs with an SIT component
are not achieving an appropriate balance. In this paper we discuss optimization of the steril-
ization process and present a simple model and procedure for determining the optimum dose.

Key Words: SIT, model, competitiveness, sterility, radiation dose

RESUME

La t6cnica de insecto est6ril (TIE) es una tecnologia de control de plagas favorable para el
medio ambiente con una aplicaci6n de un control integrado de plagas claves para toda la
area, incluyendo la supresi6n o eliminaci6n de poblaciones introducidas y la exclusion de
nuevas introducciones. La esterilidad reproductive es normalmente inducida por radiaci6n
ionizada, un m6todo convenient y consistent que mantiene un grado razonable para la ca-
pacidad de competencia en insects liberados. El costo y la eficacia de un program de con-
trol que incluye TIE dependent en tener un balance entire la esterilidad y la capacidad para
competir, pero parece que los programs operacionales corrientes con TIS como un compo-
nente no estan logrando el tener un balance apropiado. En esta publicaci6n, nosotros discu-
timos la optimizaci6n del process de esterilizaci6n y presentamos un modelo y procedimiento
sencillos para determinar la dosis 6ptima.


The sterile insect technique (SIT) was con-
ceived in the 1930s (Knipling 1955), and first ap-
plied on a significant scale in the 1950s against
the New World screwworm Cochliomyia homini-
vorax (Coquerel) (Baumhover et al. 1955; Knip-
ling 1960) and subsequently to a number of other
pest species (Dyck et al. 2005). The principle of the
technique is to introduce sterility by rearing large
numbers of the target pest, reproductively steril-
ize them, and release them into the wild. When
the sterile males mate with wild females, the fe-
males produce no viable offspring. With a con-
stant rate of release of sterile insects this results
in an increasingly rapid decline in the overall pop-
ulation over several generations. This technique
has been used successfully against a number of
pest species such as Mediterranean fruit fly Cer-
atitis capitata (Wiedemann), melon fly Bactrocera
cucurbitae (Coquilett), pink bollworm Pectino-
phora gossypiella (Saunders), codling moth Cydia
pomonella (L.) and tsetse fly Glossina austeni
Newstead (Tan 2000; Wyss 2000; Hendrichs et al.
2005; Klassen & Curtis 2005).


The attractive features of the SIT are that it is
absolutely specific to the targeted pest, integrates
well with other controls, reduces the use of toxic
insecticides, and its action is inverse-density de-
pendent. This latter characteristic implies that as
the field population declines, the pressure in-
creases on the population from a constant rate of
sterile insect release; this characteristic makes it
desirable for eradication, suppression, contain-
ment, or the exclusion of sporadic introductions in
a preventive release program (Hendrichs et al.
2005). The inverse-density dependence of the
technique makes it possible, as part of a systems
approach, to eliminate or reduce pests to such low
levels as to allow export of important commodity
crops to areas with quarantine restrictions
against the pest.
Sterility can be induced by chemicals or ioniz-
ing radiation. Chemical sterilization was used in
early work (Boikovec 1966; LaChance 1967; La-
brecque & Smith 1968), but because of the hazard
of handling these substances, problems with con-
trolling the dose, and the risks of environmental







Parker & Mehta: A Model for Dose Optimization for SIT


contamination, chemical sterilization has been
replaced by irradiation (Hayes 1968; Bakri et al.
2005a; Bakri et al. 2005b).
When biological material is irradiated, free rad-
icals are formed, and breaks are created in the
chromosomes. If breakage occurs in chromosomes
of the germ line, this leads to the formation of dom-
inant lethal mutations in eggs and sperm
(LaChance 1967; Curtis 1971). Radiation steriliza-
tion is a simple process with easy and reliable qual-
ity control procedures. The action of the radiation
is immediate so there is no requirement to hold the
sterile insects after treatment, and radiation can
pass through packaging material allowing the in-
sects to be treated after sealing in secure packag-
ing enhancing biosecurity and reducing handling.

DOSE OPTIMIZATION

The radiation absorbed dose (referred to here-
after as dose) that is used to induce sterility is of
prime importance to programs that include the
release of sterile insects. Insects that receive too
low a dose are not sufficiently sterile and those
that receive too high a dose may be uncompeti-
tive, reducing the effectiveness of the program by
requiring that a greater number of sterile insects
must be released (Robinson 2002).
While competitiveness has often been investi-
gated (Hooper 1970; Hooper & Katiyar 1971;
Hooper 1972; Katiyar 1973a, b; Hooper 1975;
Zumreoglu et al. 1979; Winstead et al. 1990;
Haynes & Smith 1992; Boshra 1994; Saour & Ma-
kee 1997; Bloem et al. 1998; Bloem et al. 1999;
Bloem et al. 2004; Toledo et al. 2004), the critical
balance between sterility and competitiveness
has rarely been investigated or discussed in suffi-
cient detail, and few data have been presented in
the literature in a form that permits a proper
analysis of this balance (Bakri et al. 2005a). In or-
der to perform the analysis, data are required si-
multaneously for the variation of both sterility
and competitiveness with dose. Where competi-
tiveness has been studied, frequently only one or
two doses have been investigated. Further, for the
competitiveness data to be realistic, the tests
should be performed in field cages or open plots.
The relationship between residual fertility and
log(dose) is well known and is sigmoid in form.
Not enough data are available to be certain of
the relationship between competitiveness and
log(dose), but for simplicity we assume it to be
similar to most response-to-dose relationships,
which are sigmoid (Finney 1971); however any
monotonic decreasing function will lead to similar
conclusions to those presented below. Fig. 1 illus-
trates the relationships of fertility and competi-
tiveness to log(dose) following this assumption,
where the scale on the x-axis is such that one unit
represents the change in log(dose) needed to pro-
duce one a change in the response (competitive-


Log(dose) >
Fig. 1. Schematic relationship of residual fertility
and competitiveness to log(dose). Sx is the separation of
the two response curves (based on data of Hooper, 1972).


ness or fertility). The displacement, Sx (in units of
G) of the competitiveness curve to the right (or
left) of the fertility curve is generally unknown,
but must vary with species and other factors such
as the oxygen content of the atmosphere and tem-
perature during irradiation, free radical scaven-
gers provided in the diet, quality of rearing, and
possibly other factors. Considerable research re-
lated to the SIT is to improve the competitiveness
of the insects for a given sterility level, that is to
move the competitiveness line as far to the right
as possible, and thus to increase the value of Sx.
Knipling (1955) presented a simple relation-
ship for the effect of released sterile insects on a
wild population. This may be written as:


F1 = Px(1 -S)xR


where F, is the population size in the filial genera-
tion, P is the parental generation size, R is the net
population growth rate per generation, and S is
the sterility induced by the released sterilized in-
sects (IAEA 1992, pp. 108-109). In practice R is
likely to be density dependent, but for this simple
model it is assumed to be density independent,
and S is dependent on the number of sterile in-
sects released (N) if it is assumed that the released
insects are both fully sterile and fully competitive:


N
S= N
(N+P)


This, however, does not take into account either of
incomplete sterility induced by the irradiation
(S), or of the reduced competitiveness (Q). To sim-







Florida Entomologist 90(1)


plify the equations, the reduced competitiveness
of the N released insects can be represented as
NQ fully competitive insects (and N(1-Q) non-
competitive insects that have no effect on the tar-
get population), and the reduced sterility asNQS,
sterile and NQ(1-S,) fertile insects. This simplifi-
cation does not affect the final form of the rela-
tionship. These NQ(1-S,) fertile individuals add to
the pool of breeding individuals, so that:


P' = P+NQ(1 -S)


and equation [2] becomes:

NQS NQS
[4] S' = NQS1 NQS
NQS1 + P + NQ(1 SI) NQ + P

The original equation [1] now becomes:


F = P'x (1 -S')xR


F1
[6] F1
R


(P+NQ(1-S,))x( 1 NQS1
NQ + P)


Regression analysis of both Probit transformed
competitiveness (Q) and residual fertility (1-S,)
against log radiation dose will yield a relationship
that may be used to predict both parameters for
any radiation dose. Equation [6] can then be
solved numerically by iteration to find the mini-
mum value of F/R for given values of Sx and NIP.
Using values ofR = 1, P = 1 and N= 9, Fig. 2 shows
the effect on the subsequent generation (F,) of
log(dose) at 3 values of Sx for a fixed release rate.
This clearly shows that as the value of Sx increases
the value of FM.__ decreases and this minimum
point occurs at a higher sterility. At the same time
the slope of the F, curve each side of the optimum
point gets shallower, implying that a larger dose


xi


8 x3a


variation may be tolerated This has the potential
to increase the throughput of the irradiation pro-
cess as less strict limits need to be applied.
This indicates that research is essential to es-
tablish the relationship of dose to the level of ste-
rility and competitiveness in the treated insects,
and that a standardized dosimetry system and
recognized dosimetry procedures are used (ISO/
ASTM 2005a). The dose to be used for any given
SIT program is then based on the results of such
studies. The program manager should specify the
optimum dose to achieve the best combination of
competitiveness and sterility (Table 1), and this
dose should be reviewed when changes in any
procedure alter the value of Sx.
Ideally, all the insects should be irradiated to re-
ceive this optimum dose, but as the dose rate varies
spatially within a container, it is inevitable that in-
sects within will receive a range of doses. Because
of this dose variability the program manager
should also specify the minimum and maximum ac-
ceptable dose that insects may receive. If the dose
variability within the container is too high, it may
be necessary to modify the radiation field (e.g., with
a field flattener, a shaped lead shield that improves
the dose uniformity ratio) or limit the volume used
for irradiation by blocking off areas with unaccept-
ably high and/or low dose rates. The range of ac-
ceptable doses should be approximately symmetric
about the optimum dose (in log(dose)), as shown by
the symmetry of the F1 curves (Fig. 2). We suggest
that the maximum and minimum dose should be
set to yield F1 values not more than 110% of F m_.
For many insects, the dose required in the late pu-
pal stage to stop egg production or egg hatch in fe-
males is lower than the dose required to induce ste-
rility in males (Bakri et al. 2005a). For most pur-
poses, therefore, the minimum dose will be set
higher than the dose at which egg production or
hatch stops. For legal or other justifiable program
requirements a higher minimum dose may be spec-
ified, but it must be recognized that this may affect
the program efficiency (Toledo et al. 2004).


S x-5S


-3 -2 -1 0 1 2 3 4
Log(doe)


y \
F1 aim



-3 -1 0 1 2 3 4 5
Log(doae)


-3-2-1 0 1 2 3 4 5
Log(dos)


Fig. 2. Size of next generation (F1) as a function of log(dose) for 6x = lo, 30 and 50.


March 2007







Parker & Mehta: A Model for Dose Optimization for SIT


TABLE 1. VALUES FOR OPTIMUM DOSE (IN G ABOVE THE LOG(DOSE) THAT YIELDS 0.5 RESIDUAL FERTILITY), F, nim, THE
FERTILITY AND COMPETITIVENESS CORRESPONDING TO THE OPTIMUM DOSE, AND THE RANGE OF LOG(DOSE)
FORF, Mnim + 10% FOR SELECTED VALUES OF &x (DISPLACEMENT OF THE COMPETITIVENESS CURVE RIGHT OF
THE FERTILITY CURVE). THIS ANALYSES IS FOR NIP = 9.

Maximum log(dose) range
Sx/o Optimum log(dose) FlMimmm Fertility Competitiveness for F, < 1.1 x FlM.mmm

0 0.70 0.480 0.240 0.24 0.25 1.10
0.5 1.00 0.380 0.160 0.31 0.60 1.35
1 1.25 0.300 0.110 0.40 0.85 1.60
1.5 1.50 0.240 0.067 0.50 1.15 1.85
2 1.75 0.190 0.042 0.61 1.35 2.10
2.5 1.90 0.160 0.027 0.71 1.55 2.35
3 2.15 0.140 0.016 0.80 1.70 2.60
3.5 2.30 0.120 0.010 0.88 1.85 2.85
4 2.55 0.110 0.005 0.93 2.00 3.15
4.5 2.75 0.106 0.003 0.96 2.10 3.50
5 2.95 0.103 0.002 0.98 2.20 3.85


The actual dose applied in different programs
and research projects has varied widely, by a fac-
tor of almost 3 for some species (i.e., Sitophilus
granarius L. which varies between 50 and 135
Gy) as shown by the International Database on
Insect Disinfestation and Sterilization website
(IAEA 2003; Bakri et al. 2005a). From Table 1 it
can be seen that the optimum dose only yields
95% sterility (5% residual fertility) when Sx is
about 1.8 and 99.9% when Sx is greater than 5. It
is unlikely that Sx will ever be as large as 5, but
because of the lack of appreciation for the insect
competitiveness issues involved, many programs
continue to use 99.9% sterility when lower doses
would yield better control.
The optimum dose depends furthermore on the
ratio N/P (Table 2). In the early stages of a sup-
pression or eradication campaign, while the wild
population is still relatively large and the ratio
N/P is small, the optimum dose is lower than later
in the program when the value ofN/P is larger. It
would thus appear that current operational pro-
grams releasing sterile insects are not achieving
the appropriate balance between sterility and
competitiveness at each stage in the program. Ta-
ble 2 may be used to estimate the optimum dose
in Gy for any given value of Sx at various ratios of
N/P. If the regression equation for the dose-fertil-
ity relationship, with dose in Gy transformed to
log(dose) and fertility to normal equivalent devi-
ates (NED) is:

[7] NED(fertility) = a + b x log(dose)

then the actual dose in Gy can be calculated from
the values of D in Table 2 as:

(D + a)
[8] dose(Gy) = 10 b


The value of Sx can be estimated from a simple
field cage experiment, but an adequate set of field
cage data to determine the dose-response rela-
tionship has not been published. In order to illus-
trate the concept, the extensive set of laboratory
data for the Mediterranean fruit fly Ceratitis cap-
itata (Wiedemann) (Diptera: Tephritidae) pre-
sented by Hooper (1972) for fertility and competi-
tiveness (Haisch 1970; Fried 1971) at various
treatment doses is used. The main purpose of this
illustration is to demonstrate the procedure for
determining the optimum dose from relevant
data. Using Hooper's data, we show the relation-
ships of fertility and competitiveness to the radi-
ation dose in Fig. 3, with the linear regression
lines and 95% confidence intervals for the regres-
sions. The regression fit for the fertility is very
good, but there is a larger scatter in the competi-
tiveness values. This is inherent in the method of
measuring and calculating competitiveness
(Haisch 1970; Fried 1971; Hooper & Horton 1981;
Iwahashi et al. 1983). The regression coefficients
for competitiveness and fertility do not differ sig-
nificantly from each other (competitiveness: re-
gression coefficient = -3.4032, SE = 0.3326; fertil-
ity: regression coefficient = -3.8866, SE = 0.5403)
(Sokal & Rohlf 1981). The value of Sx from these
data is 1.44, and from the fertility relationship
the increase in log(dose) that results in a lo
change in fertility is 0.26, the reciprocal of the
slope of the linear regression line.
From these values the optimum dose can be es-
timated from Table 2 and equation [8]. In the
present example, where Sx = 1.44, for N/P = 8,
from the table the value of D is 1.45. Based on
equation [8]:

(1.45 + 5.4955)
[9] dose(Gy) = 10 -38866 61







Florida Entomologist 90(1)


March 2007


TABLE 2. OPTIMUM RADIATION DOSE (D, IN UNITS OF o ABOVE THE LOG(DOSE) THAT YIELDS 0.5 RESIDUAL FERTILITY)
AND CORRESPONDING STERILITY LEVEL (IN ITALICS) FOR SELECTED VALUES OF 5x AND NIP (THE RATIO OF
STERILE TO WILD MALES).

NIP

6x 1 2 4 8 16 32 64 128

0 0.22 0.34 0.50 0.67 0.86 1.04 1.22 1.40
58.7% 63.3% 69.1% 74.9% 80.5% 85.1% 88.9% 91.9%
0.5 0.48 0.61 0.77 0.95 1.14 1.32 1.50 1.68
68.4% 72.9% 77.9% 82.9% 87.3% 90.7% 93.3% 95.4%
1 0.73 0.87 1.03 1.21 1.40 1.58 1.76 1.94
76.7% 80.8% 84.8% 88.7% 91.9% 94.3% 96.1% 97.4%
1.5 0.98 1.11 1.28 1.45 1.64 1.82 2.00 2.17
83.6% 86.7% 90.0% 92.6% 94.9% 96.6% 97.7% 98.5%
2 1.22 1.35 1.51 1.68 1.86 2.04 2.22 2.39
88.9% 91.1% 93.4% 95.4% 96.9% 97.9% 98.7% 99.2%
2.5 1.46 1.58 1.73 1.90 2.08 2.25 2.42 2.59
92.8% 94.3% 95.8% 97.1% 98.1% 98.8% 99.2% 99.5%
3 1.70 1.81 1.95 2.11 2.28 2.44 2.61 2.77
95.5% 96.5% 97.4% 98.3% 98.9% 99.3% 99.5% 99.7%
3.5 1.93 2.03 2.17 2.31 2.47 2.63 2.79 2.94
97.3% 97.9% 98.5% 99.0% 99.3% 99.6% 99.7% 99.8%
4 2.16 2.26 2.38 2.52 2.66 2.81 2.96 3.11
98.5% 98.8% 99.1% 99.4% 99.6% 99.8% 99.8% 99.9%
4.5 2.40 2.49 2.60 2.72 2.86 3.00 3.14 3.28
99.2% 99.4% 99.5% 99.7% 99.8% 99.9% 99.9% 99.9%
5 2.64 2.72 2.82 2.93 3.06 3.19 3.32 3.45
99.6% 99.7% 99.8% 99.8% 99.9% 99.9% 100.0% 100.0%


Table 3 presents the calculated optimum dose,
F,1 Minmum, the corresponding values for sterility and
competitiveness and the minimum and maximum
doses to remain within 110% of F .i for Sx =
1.44 and various values of N/P. Also shown are the
values corresponding to 99% and 99.9% sterility,
103 and 162 Gy. For this value of Sx the optimum
dose lies below 90 Gy, with a range from 52-89 Gy,
which corresponds well with Hooper's own conclu-
sion that the optimum dose is about 70 Gy. Both
99% and 99.9% sterility doses fall outside this
dose range for all values of N/P shown.
The value of Sx is overestimated by the data in
Hooper (1972), as the competition was between ir-
radiated and unirradiated colony flies under labo-
ratory conditions, not between irradiated colony
flies and wild flies in the field. This colony has
been maintained under artificial conditions for
many generations and can be expected to have
competitiveness less than 1 before irradiation. Ir-
radiated colony flies could therefore be expected to
perform worse against wild flies than against col-
ony flies. Wong et al. (1983) compared mating suc-
cess between irradiated and wild males of Cerati-
tis capitata, and found no difference over a range


y = 6.0336 3.4032 x CompetiUness
t* R= 0.5771 Forlity


4







R=93 -,
y = 5.4955 3.8866 x
F= 0.9346
U, .


1.6 1.7 1.B 1.9
Log(doselGy)


2.0 2.1


Fig. 3. Relationships of fertility (squares) and com-
petitiveness (diamonds) with dose for the Mediterra-
nean fruit fly (data from Hooper 1972). NED = normal
equivalent deviates.







Parker & Mehta: A Model for Dose Optimization for SIT


TABLE 3. OPTIMUM DOSE, MINIMUM AND MAXIMUM DOSES, AND THE CORRESPONDING STERILITY, COMPETITIVENESS
AND F, FOR MEDITERRANEAN FRUIT FLY, FOR &x = 1.44 AND VARIOUS VALUES OF NIP (DERIVED FROM DATA
IN HOOPER 1972).


Sterility


74.8%
88.0%
95.3%
99.0%
99.9%


85.9%
92.7%
96.6%
99.0%
99.9%


96.7%
98.1%
99.0%
99.0%
99.9%


Competitiveness


78.0%
60.4%
40.7%
18.8%
4.9%


64.1%
49.3%
34.8%
18.8%
4.9%


34.7%
26.0%
18.5%
18.8%
4.9%


0.476
0.433
0.476
0.643
0.871

0.267
0.243
0.267
0.378
0.692


0.060
0.055
0.060
0.060
0.169


of doses, but there is an urgent need for additional
field cage or field data on competitiveness over a
range of radiation doses to determine the dose re-
lationship and thereby the magnitude of Sx.


DOSIMETRY AND THE IRRADIATION PROCESS

Dosimetry plays a crucial role throughout the
radiation sterilization process of insects. At the
research phase, where the effect of radiation on
sterility as well as on competitiveness of the in-
sects is investigated, radiation dose is the key
quantity. At the production facility, dosimetry
also has several essential roles. First, it assists in
the characterization of the irradiator, and in the
regular monitoring of its consistent operation. It
also helps in determining the correct size and
shape of the canister and other key process pa-
rameters for irradiation of the insects. And later
during the sterilization process, it provides an im-
portant element of process control.
Considering the importance of dosimetry in
programs applying the SIT, the selection of an ap-
propriate dosimetry system is critical. Such a sys-
tem should provide a systematic and repeatable
means of estimating the dose and its associated
confidence interval (ISO/ASTM 2005b). The sys-
tem should be verifiable and traceable (refer-
enced) to national or international standards.
Considering various factors, the Gafchromic do-
simetry system (Gafchromic HD-810 film; Inter-
national Specialty Products, Wayne, NJ 07470,
U.S.A.) has been selected by the IAEA based on


the specific requirements of SIT programs, espe-
cially the useful dose range of 50-600 Gy and a
low cost (IAEA 2004). This reference also de-
scribes relevant dosimetry procedures as well as
various components of this dosimetry system.
Accidental release of insects that are signifi-
cantly under-dosed will require rapid correction
by release of additional sterile insects and other
measures, especially for programs like those in
California and Florida, USA., where SIT is used
for eradication of extremely small introductions
and/or as a prophylactic measure to prevent es-
tablishment of newly introduced flies (Dowell et
al. 2000). Besides administrative control, there
are 3 main process control elements that are in
place that would minimize the chances of such ac-
cidents (FAO/IAEA/USDA 2003). These different
elements control various steps in the process and
thus complement each other as follows: (1) Steril-
ity Testing-In any SIT program, sterility testing
through bioassays should be carried out on a reg-
ular basis to confirm that all the procedures are
being followed correctly, including the rearing,
the pre-irradiation preparation (such as age-
based selection of insects, packaging for hypoxia
or nitrogen, if used), temperature control, irradi-
ation dose control, and post irradiation handling,
(2) Routine Dosimetry-The purpose of dosimetry
in process control is to monitor that all the canis-
ters (and hence all the insects) are receiving the
dose within the specified range, and (3) Radia-
tion-Sensitive Indicators-This control element
provides an immediate visual check at irradiation
facilities and at pupal reception/fly emergence


Dose


Sx = 1.44
Minimum
Optimum
Maximum
103 Gy
162 Gy
6x = 1.44
Minimum
Optimum
Maximum
103 Gy
162 Gy
6x = 1.44
Minimum
Optimum
Maximum
103 Gy
162 Gy


N/P=3
39
52
70
103
162
N/P=9
49
61
77
103
162
N/P = 100
77
89
103
103
162











centers that a given container has gone through
the irradiation process.

CONCLUSIONS

For SIT, ionizing radiation is the method of
choice for inducing reproductive sterility. The
sterilization process is important in determining
the quality of the released insects and their abil-
ity to compete with the wild population. Thus, op-
timization of the sterilization process is critical
for the efficacy of SIT programs and should be
given due consideration. We believe that doses
lower than currently applied will result in a more
effective SIT program, with any increase in resid-
ual fertility more than compensated for by the in-
creased competitiveness of the released insects.
We have developed a quantitative procedure
for determining the optimum dose based on fertil-
ity and competitiveness data. In order to estimate
the optimum dose, it will be necessary to calculate
correlations between dose and both fertility and
competitiveness. The fertility relationship is al-
ready known for many insects, so attention should
be concentrated on collecting data on competitive-
ness over a suitable range of doses. As the opti-
mum also depends on the ratio of sterile to fertile
males, the treatment dose should be reviewed
constantly during the progress of a program. Op-
timization can lead to significant reduction in pro-
gram cost and increase in programme efficiency.
The dose of radiation can be readily measured
with a standardized dosimetry system, such as the
Gafchromic system (IAEA 2004; ISO/ASTM
2005c). A dosimetry system that is traceable to na-
tional or international standards can be reliably
used both for setting the dose for the radiation ster-
ilization process and for routine process control.

ACKNOWLEDGMENTS

We thank A. S. Robinson, L. E. LaChance, and three
anonymous referees for helpful comments on an earlier
draft of this paper. Mention of trade names or commer-
cial products in this article is solely for the purpose of
providing specific information and does not imply rec-
ommendation or endorsement by the International
Atomic Energy Agency.

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Florida Entomologist 90(1)


March 2007


INDUCTION OF STERILITY IN ANASTREPHA FRATERCULUS
(DIPTERA: TEPHRITIDAE) BY GAMMA RADIATION

ARMANDO ALLINGHI1, CELIA GRAMAJO2, EDUARDO WILLING2 AND JUAN VILARDI3
'Comisi6n Nacional de Energia At6mica, CNEA

2Estaci6n Experimental Agro-industrial Obispo Columbres, Tucuman, Argentina

3Depto. Ecologia, Gen6tica y Evoluci6n, Facultad de Ciencias Exactas y Naturales
Universidad Buenos Aires. (1428) Buenos Aires, Argentina

ABSTRACT

In relation to the application of the sterile insect technique (SIT) for the South American
fruit flyAnastrepha fraterculus (Wiedemann), we analyzed the effect on adult fertility of dif-
ferent doses of gamma irradiation and the age of pupae at the time of irradiation. In a first
experiment, we applied doses of 50, 70, and 90 Gy to pupae at 24, 48, 72, and 96 h before
adult emergence. In a second experiment we irradiated pupae 48 h before emergence with
20, 40, and 60 Gy and estimated male and female fertility and sperm transfer by irradiated
males. The results indicated pupal age at irradiation does not significantly affect male fer-
tility. If males irradiated with 60 Gy are crossed to non-irradiated females the fertility is
about 1%. Females irradiated with 40 Gy did not lay eggs independently of the male to which
they mated. No significant effects of radiation were observed with respect to the ability of
males to transfer sperm. A dose of 70 Gy applied 48 h before adult emergence induces 100%
sterility in both males and females.

Key Words: SIT, South American fruit fly, fertility, sperm transfer, sterility, pupal age

RESUME

Para la aplicaci6n de la t6cnica del insecto est6ril (TIE) en Anastrepha fraterculus (Wiede-
mann), en este trabajo analizamos el efecto de diferentes dosis de irradiaci6n gamma y la
edad 6ptima de la pupa al moment de la irradiaci6n. En el primer experiment se evaluaron
las dosis de 50, 70, y 90 Gy en pupas de 24, 48, 72, y 96 h antes de la emergencia del adulto.
En el segundo experiment se irradiaron pupas 48 h antes de la emergencia con dosis de 20,
40, 60 Gy y se estim6 la fertilidad de los machos y las hembras, y la transferencia de esper-
mas por los machos irradiados. Los resultados indicaron que la irradiaci6n no modifico sig-
nificativamente la fertilidad de los machos. En las cruzas de machos irradiados a 60 Gy con
hembras no irradiadas se observe 1% de eclosi6n larvaria, mientras que las hembras irradia-
das a 40 Gy no pusieron huevos. La irradiaci6n no afect6 significativamente la transferencia
de espermas de los machos tratados. Por lo tanto, una dosis de 70 Gy aplicada 48 h antes de
la emergencia del adulto induce 100% de esterilidad tanto en machos como en hembras.


Translation provided by the authors.


The South American fruit fly Anastrepha
fraterculus (Wiedemann) (Diptera: Tephritidae)
is an important pest for fruit production in Argen-
tina (Stone 1942). This species is native to the
Americas, most probably South America, and is
widely distributed throughout the tropical and
subtropical regions (between the latitudes 27N
and 35S). Its range includes southern USA
(South Florida and Rio Grande Valley, Texas),
Central America, Caribbean Islands, and South
America, from Trinidad and Guyana to Central
Argentina (Steck 1999; Aluja 1994; Hernandez-
Ortiz 1992).
There are at least 80 host species ofA. frater-
culus, including many economically important
fruit species (Norrbom & Kim 1988). Tropical


fruit flies not only cause great losses in fruit and
vegetable production, but they also seriously im-
pede international trade because of quarantine
regulations (Klassen & Curtis 2005). In particu-
lar, the presence ofA. fraterculus in the orchards
reduces the possibility of exporting fruits and
other horticultural products to the northern
hemisphere (SENASA 1997). The export of fruits
and vegetables to pest free areas or those that
have implemented control programs against this
pest requires the application of a quarantine
treatment. Another common problem is that the
intensive use of chemical insecticides is associ-
ated with environmental contamination. Fur-
thermore, insects have been found to develop re-
sistance to almost every chemical class of insecti-







Allinghi et al.: Induction of Sterility in Anastrepha fraterculus


cide (Brown & Payne 1988). This includes some
tephritids, such as Bactrocera oleae (Gmelin)
(Vontas et al. 2002) and Bactrocera dorsalis Hen-
del (Hsu et al. 2004).
Recent studies indicate that populations of
A. fraterculus from Argentina and Southern Bra-
zil are not differentiated genetically (Alberti et al.
2002) and that 4 populations from different re-
gions of Argentina do not show reproductive isola-
tion (Petit-Marty et al. 2004). These findings sug-
gest that the sterile insect technique (SIT) might
be applied successfully against A. fraterculus at
least at a regional scale.
In other tephritids, such as Ceratitis capitata
(Wiedemann), the irradiation process may reduce
the mating performance of the sterilized males
(Calcagno et al. 2002; Lux et al. 2002). An essen-
tial requirement for a successful SIT is the appli-
cation of a sterilization protocol to mass reared
insects that ensures sterility with a minimal det-
riment of the mating competitiveness and viabil-
ity of the released insect. Germ cells (oocytes and
spermatids) are highly radiosensitive and when
exposed to ionizing radiation, dominant lethal
mutations are induced (Muller 1927). The domi-
nant lethal mutations produced by radiation in
insects depend mainly on the dose, insect type,
size, and sex (Hooper 1989). Radiosensitivity also
depends on other factors such as irradiation tem-
perature, humidity, ploidy level, mitotic cycle
phase, and metabolic condition (Enkerlin et al.
1997).
In species of the genus Anastrepha, studies on
the effect of pupal age and radiation dose on the
induced sterility are not completely consistent.
Rhode et al. (1961) reported that Anastrepha
ludens (Loew) pupae irradiated 96 h before emer-
gence with 40 Gy showed 100% male sterility. By
contrast, according to Velasco & Enkerlin (1982),
the dose needed to induce sterility in the same
species should be much higher. They reported
that 40 Gy and 100 Gy induced 90% and 99% ste-
rility, respectively, when pupae were irradiated
72 h before emergence. In the case ofAnastrepha
suspense (Loew), Burditt et al. (1975) irradiated
pupae with 40 Gy at 48 h before emergence and
observed complete adult sterility whereas
Calkins et al. (1988) reported that lower irradia-
tion doses (30 Gy) applied 24-48 h before emer-
gence induced high levels of sterility.
The efficiency of sterilized insect release pro-
grams depends to a great extent on the ability of
laboratory reared sterile males to mate with, and
transfer sperm to, wild females in the field (McIn-
nis 1993). Usually, immediately after copulation
90% of sperm is found in the spermathecae of the
female (Yuval et al. 1996). Mossinson & Yuval
(2003) have shown that females with fewer sperm
in their spermathecae show a higher tendency to
remate. Remating may reduce the efficiency of
the SIT if the second mating occurs with a wild


male. However, this is very unlikely as sterile
males far outnumber wild males in an SIT pro-
gramme.
We analyzed under laboratory conditions the
effect of different doses of gamma irradiation and
the age of pupae at the time of irradiation on the
induced sterility and the ability to transfer sperm
in A. fraterculus.

MATERIALS AND METHODS

The A. fraterculus individuals studied were
from a strain reared since 1997 at Estaci6n Ex-
perimental Provincial Obispo Colombres, Tu-
cuman, Argentina. Pupae were sent to Buenos
Aires (Centro At6mico Ezeiza, Grupo Agron6mico,
CNEA) by surface and there were kept under con-
trolled conditions (25 1C, 75 5% RH, and a
photoperiod of 12:12 (L:D). Adult diet was com-
posed of white sugar: yeast (Calsa, S:A:, Tucu-
man, Argentina) (3:1). Water was provided as 1%
agar in 12-mL vials. Food and water were
changed each once a week.
Pupae were irradiated at the Centro At6mico
Ezeiza facility (Comisi6n Nacional de Energia
At6mica, Argentina) in a Gammacell 220 (MDS
Nordion, Canada) irradiator, with 60Co source
(dose rate for the first and second experiment:
1.67 Gy min-1 and 1.60 Gy min-1).

Experiment 1: Optimal Pupal Age for the Irradiation
Treatment

Pupae were irradiated 24, 48, 72, or 96 h be-
fore adult emergence. In each case four different
radiation doses were applied: 0 (control), 50, 70,
and 90 Gy. Upon emergence adults were sepa-
rated by sex and kept for 15 d under controlled
conditions at 25C, 80% RH, and a photoperiod of
13:11 (L:D), and light intensity of 3500 lux. At
this age all individuals are sexually mature (De
Lima et al. 1994). For each treatment, male fertil-
ity was evaluated by exposing ten fertile (non-ir-
radiated) females to a sample often treated males
for 20 d in a 3000-cm3 flask.
After exposure to males, females were trans-
ferred to egg collecting flasks that were similar to
those used for the crossing, but they had an arti-
ficial egg laying substrate hanging from the top.
It consisted of 3-cm diameter sphere made of 3.5
g agar and 0.05 g red dye (color index 14700) dis-
solved in 300 mL water. The sphere was wrapped
in Parafilm (Boller 1968; Manso 1998). The arti-
ficial substrates were removed after an exposure
period of 48 h to the inseminated females, The
Parafilm was removed, and the eggs were man-
ually extracted and transferred to a wet Petri
dish. Eggs were kept for 72 h at 25C. After this
period, the numbers of hatched and non-hatched
eggs were recorded. The experiment was repli-
cated 3 times for each treatment







Florida Entomologist 90(1)


Experiment 2: Optimal Dose for the Sterilization
of Pupae 48 h before Adult Emergence

The effect of irradiation on male or female fer-
tility was analyzed by mating flies irradiated
with different doses of gamma rays with non-irra-
diated flies of the opposite sex. Pupae were
treated 48 h before emergence with 0 (control),
20, 40, and 60 Gy. Emerged adults were kept un-
der the same conditions as those of Experiment 1.
Male fertility was evaluated in a similar way as
that described for Experiment 1, but in this case
15 males and females were used. Six replicates
were obtained for the 20, 40, and 60 Gy treat-
ments. Female fertility was tested by exposing 15
irradiated females to 15 mature fertile males. In
this case 2 replicates were obtained for each radi-
ation dose. Four replicates of the crossing of fer-
tile males and females were used as the control
treatment for both male and female fertility tests.
The method of egg collecting was similar to
that described for Experiment 1. In this case fe-
males were allowed to lay eggs for 1 month. Dur-
ing this period 7 egg collections (one every 3-5
days) were made for each treatment. A total of 28
samples were obtained for the control (7 collec-
tions x 4 replicates) and 14 for each group of irra-
diated females (7 collections x 2 replicates).

Sperm Transfer

In order to determine if sterile males are able
to transfer sperm, spermathecae of fertile females
mated to irradiated and non-irradiated males in
Experiment 2 were observed. Females were sacri-
ficed and fixed in 70% ethanol. Spermathecae
were dissected on a paraffin wax layer with the
help of entomological needles. Spermathecae
were transferred onto a slide with a drop of acetic
orcein (Guillen-Aguilar 1983), covered with a cov-
erslip, and pressure applied with the thumb to
break them and release sperm into the dyeing so-
lution. The presence or absence of sperm was
scored with a stereoscopic microscope at 100x
magnification.


Statistical Methods

To determine the best time to irradiate pupae,
the percent of egg hatch was compared among
eggs laid by females inseminated by fertile males
and males irradiated with 50, 70, and 90 Gy at 24,
48, 72, and 96 h before adult emergence by means
of a homogeneity chi square test. To evaluate the
effect of different radiation doses 48 h before
adult emergence on male fertility, the percent of
egg hatch was compared among eggs laid by fer-
tile females inseminated by males treated with
different radiation doses. The method used was
non-parametric Kruskall-Wallis analysis of vari-
ance. Pair wise comparisons were performed by
Mann-Whitney U test. The percentage of egg
hatch was compared for each treatment among
the 7 consecutive egg collections by means of
Kruskall-Wallis analysis of variance.
Irradiated females tended to lay lower num-
bers of eggs and their eggs showed a reduced egg
hatch. Because only 2 classes were able to lay
eggs, i.e., control and females irradiated with 20
Gy, they were compared by means of Mann-Whit-
ney U test. The proportion of spermathecae with
or without sperm was compared by means of
Fisher's exact test. All statistical tests were per-
formed with the program STATISTICA, ver. 5.1
(StatSoft 2000).

RESULTS

Optimal Pupal Age for Irradiation

The percent egg hatch of the control group (fe-
males inseminated with non-irradiated males)
was about 87%. This values drops dramatically
(P = 0) when the males were irradiated even with
the lowest dose (50 Gy). The doses of 70 and 90 Gy
induced total sterility independent of pupal age at
the time of irradiation (Table 1). The comparison
of percent egg hatch among ages for the treatment
with 50 Gy indicated that the differences are not
significant (X2 = 2.49, P = 0.93). These results indi-
cate that within the interval considered, pupal


TABLE 1. PERCENT OF EGG HATCH AND TOTAL NUMBER OF SCORED EGGS (IN PARENTHESES) OVIPOSITED BY FERTILE FE-
MALES INSEMINATED BY NON-IRRADIATED MALES (CONTROL) AND MALES IRRADIATED WITH DIFFERENT
DOSES OF GAMMA RAYS AND AT DIFFERENT STAGES OF PUPAL DEVELOPMENT.

Dose (Gy)

Pupal age' 0 (Control) 50 70 90

24 0.56 (533) 0 (646) 0 (387)
48 0.32 (930) 0 (382) 0 (845)
72 0.18(550) 0(625) 0(886)
96 0.51(787) 0(614) 0(777)
-- 86.98 (976)

Hours before emergence.


March 2007







Allinghi et al.: Induction of Sterility in Anastrepha fraterculus


age at the time of irradiation does not affect the
sterility induced by gamma radiation in males.

Evaluation of Optimal Dose for Pupal Irradiation

Radiation reduced male fertility as deter-
mined by egg hatch, from about 80% in the control
group (0 Gy) to about 1% in the group treated
with 60 Gy (Table 2). According to Kruskall-Wal-
lis analysis of variance, the differences among
treatments were highly significant (H = 123.08, P
_ 0). Pairwise comparisons by Mann-Whitney
tests indicated that all pairs differ significantly
(U= 0-362, Z = 4.7-7.6, P < 10-5). The percentage of
egg hatch did not differ significantly within treat-
ments among the collections throughout the one-
month period the females were allowed to oviposit
(H = 7.97-11.08, P = 0.24-0.09).
In the case of females, the radiation treatment
affected both the number of eggs laid and the per-
cent hatch (Table 3). Females irradiated with 40-
60 Gy were not able to lay eggs at all. Females ir-
radiated with 20 Gy laid only a third of the eggs
as compared with non-irradiated females. The
differences between the control (0 Gy) and the
treatment with 20 Gy were highly significant for
both percent of egg hatch (U = 16, Z = -4.8, P = 1.6
10-6) and number of eggs that were laid (U = 1.5,
Z = -5.2, P = 2 10-).

Evaluation of Effects of Radiation on Sperm Transfer

The proportion of spermathecae containing
sperm was slightly higher in females mated to
fertile males (34/45) than in females mated to ir-
radiated males (46/70) but these differences were
not significant according to Fisher's exact test (P
= 0.37).

DISCUSSION

The efficient application of the SIT requires
the precise determination of the optimal condi-
tions for pupal irradiation. This question is very
important to avoid undesirable side effects of the



TABLE 2. EFFECT OF DOSE OF GAMMA IRRADIATION ON
THE PERCENT OF HATCH OF EGGS LAID BY FER-
TILE FEMALES MATED TO MALES IRRADIATED 48
H BEFORE EMERGENCE. n = NUMBER OF EGGS
SCORED.

#Dose (Gy) Egg hatch' (% SD) n

0 80.7 + 8.06 a 3384
20 9.8 4.53 b 4760
40 3.0 2.00 c 5042
60 1.3 1.70 d 5175

Different letters mean groups that differ statistically.


TABLE 3. NUMBER OF EGGS COLLECTED FROM FEMALES
TREATED WITH DIFFERENT DOSES OF GAMMA
RAYS AND THEIR CORRESPONDING PERCENT OF
HATCH.

Mean number Total number
Dose Hatching of eggs collected of eggs
(Gy) (%) SE per flask ( SE) collected

0 80.60+ 8.06 120.90+ 39.50 3384
20 39.30 22.18 37.90 13.36 530
40 -0 0
60 0 0



radiation treatment such as physiological or be-
havioral alterations that might reduce the com-
petitiveness of irradiated males. Irradiation of
larvae and young pupae may produce adult steril-
ity but also extensive somatic damage with un-
wanted effects as aspermia or reduced adult sur-
vival. The irradiation of mature pupae has been
shown to improve the field performance of mass
reared and sterilized males (Hooper 1989). In
most insects meiosis during spermatogenesis oc-
curs before the last molt (Chapman 1998) and
mature spermatozoa have already left the testis
at the time of adult emergence from the pupa
(Wigglesworth 1965). Radiation may induce high
levels of dominant lethal mutations in spermatids
and spermatozoids as well as the death of pre-
meiotic cell stages and atrophy of germinal tis-
sues. Usually, sterility is permanent, although in
exceptional cases non-damaged spermatozoids
may be regenerated from spermatogonia.
With respect to the best time to apply radia-
tion treatment, studies on other tephritid species
such as B. dorsalis, Bactrocera cucurbitae (Co-
quillett), Bactrocera oleae (Gmelin), C. capitata,
A. ludens, Anastrepha obliqua (Macquart), and
A. suspense demonstrate that pupae irradiated
24-48 h before emergence exhibit high levels of
sterility (Velasco & Enkerlin 1982; Hooper 1989;
Walder & Calkins 1993; Toledo 1993).
Our results inA. fraterculus indicate no differ-
ences in the percentage of egg hatch among eggs
produced by individuals irradiated at different
ages within the interval 24-96 h before emer-
gence. Therefore, the question remains as to what
is optimal age within this period to produce ster-
ile males with the best competitiveness. Although
this aspect remains to be studied, we adopted the
generalized criterion applied in SIT operations
against other tephritid flies of irradiating 48 h be-
fore emergence (Velasco & Enkerlin 1982; Hooper
1989; Walder & Calkins 1993; Toledo 1993). As
stated by Hooper (1989) irradiation at early de-
velopmental stages is highly detrimental due to
the high metabolic activity and morphological
changes during the metamorphosis process. If ra-
diation is applied to mature pupae 24-48 h before







Florida Entomologist 90(1)


emergence, the metamorphosis is almost com-
plete and the detrimental effects of irradiation on
organs with low metabolic rate is minimized.
However, the spermatogenesis is still ongoing,
spermatogonia and spermatozoids are still differ-
entiating and they constitute the main target for
dominant lethal mutation induction.
Competitiveness of irradiated males is nega-
tively correlated with the absorbed radiation dose
(Calcagno 2001; Calcagno et al. 2002; Lux et al.
2002). Therefore, in order to optimize the effi-
ciency of the SIT it is necessary to reach the best
compromise between sterility and competitive-
ness (Parker & Mehta 2007). Taking into account
the results obtained in Experiment 1 and the ar-
guments discussed above, Experiment 2 was
based on pupae irradiated 48 h before emergence.
The relationship between dose and percent egg
hatch observed in the present research was simi-
lar to that observed in other tephritid species. The
results are consistent with the "one-hit" hypothe-
sis (La Chance & Graham 1984) which predicts a
linear response at low doses, but as the dose in-
creases an increasing proportion of sperm carry
multiple dominant lethal mutations. One domi-
nant lethal mutation, however, is sufficient to
cause lethality. Furthermore, high irradiation
doses produce unwanted side effects reducing the
relative efficiency of the radiation (Hooper 1989).
According to the results presented here, rela-
tively low doses of radiation, e.g., 20-40 Gy, cause
90-97% sterility. This agrees with other authors
who observed that 40 Gy can sterilize A. fratercu-
lus (Gonzalez et al. 1971), 50 Gy (48 h before
emergence) produces 100% male sterility in
A. suspense (Walder & Calkins 1993), 60 Gy (24-
48 h before emergence) produces high levels of
sterility inA. obliqua (Toledo 1993), and 40 Gy at
96 h before emergence causes 100% male sterility
inA ludens (Rhode et al. 1961).
We observed that the induced sterility re-
mained across the 7 egg collections made over a
month for all treatments. This consistency among
egg collections indicates that males do not recover
their fertility during this period for any of the
doses considered. These results are consistent
with those of Gonzalez et al. (1971) in the same
species, and those ofVelasco & Enkerlin (1982) in
A. ludens.
In tephritid females the oviposition is reduced
as the irradiation dose increases. Moreover, the
eggs laid show evidence of dominant lethal muta-
tions. The doses that cause males sterility also
completely inhibit oviposition in females (Burditt
et al. 1975; Calkins et al. 1988; Hooper 1989). Ac-
cording to Velasco & Enkerlin (1982) females ofA.
ludens are more susceptible to radiation than
males are, showing effects with extremely low
doses (5-20 Gy). The reason is that 48 h before
emergence the ovaries are in an early develop-
mental stage and the radiation, depending on the


dose, may cause complete ovarian atrophy
(Walder & Calkins 1992).
According to our results, the irradiation of fe-
males of A. fraterculus results in a reduction in
the number of eggs laid, compared with non-irra-
diated females after mating to fertile males.
Doses of 20 Gy induced a 40% reduction in egg
hatch and a 67% reduction in egg laying. The dose
of 40 Gy was sufficient to induce complete steril-
ity by preventing egg laying. The fact that irradi-
ated females are not able to lay eggs is favorable
to SIT implementation based on bisexual labora-
tory strains because the potential damage to
some fruits (stings) by released females would be
eliminated.
The analysis of sperm transfer indicated that
empty spermathecae can be found in females ex-
posed to both non-irradiated and irradiated
males. The difference in the proportions of empty
spermatecae between control and males irradi-
ated with 60 Gy was not significant. Although we
did not quantify the number of sperm trans-
ferred, our results indicate that the sterility of ir-
radiated males should be attributed to the induc-
tion of dominant lethal mutations and not to atro-
phy of testis, seminal ducts, or aspermia.
Taken as a whole the results obtained in the
present work support the use of a dose of 70 Gy ap-
plied 48 h before adult emergence to induce 100%
sterility in males and females ofA. fraterculus.

ACKNOWLEDGMENTS

J. C. V. is member of Consejo Nacional de Investiga-
ciones Cientificas y T6cnicas (CONICET, Argentina).
This work was supported by the following grants: IAEA
Research Contract 1083, CONICET PIP 5122/98, UBA-
CYT 2004 X241, and ANPCYT PICT 6628 to J.C.V., and
Project 17, Programa de Radiaciones y Radiois6topos,
CNEA.

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