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 MOLECULAR ENTOMOLOGY

A Novel Molecular Approach to Assess Mating Success of Sterile

Ceratitis capitata (Diptera: Tephritidae) Males in
Sterile Insect Technique Programs
V. SAN ANDRES,1 A. URBANEJA,1 B. SABATER-MUNOZ,1,2 AND P. CASTANERA3
Unidad Asociada de Entomologa, Instituto Valenciano de Investigaciones Agrarias-Centro de
Investigaciones Biologicas del Consejo Superior de Investigaciones Cientcas, Valencia, Spain

J. Econ. Entomol. 100(4): 14441449 (2007)

ABSTRACT Areawide sterile insect technique (SIT) programs against Mediterranean fruit y,
Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), are increasingly implemented worldwide. A
key issue in SIT is to assess mating success of released sterile males, which could be currently estimated
by egg hatchability and by stored sperm head measurements. We report here on a novel molecular
approach that would allow detecting the presence of Mediterranean fruit y sterile male sperm in the
female spermathecae under eld conditions, as a precise marker to assess mating performance. The
simplicity (only two polymerase chain reactions) and reliability of this method, jointly with
the capability to detect Vienna sperm in wild Mediterranean fruit y maintained in monitoring
traps for 7 d under eld conditions, suggest that it could be an efcient tool when coupled with
areawide SIT programs.

KEY WORDS Ceratitis capitata, sperm, spermathecae, mating success, polymerase chain reaction-
restriction fragment length polymorphism

The Mediterranean fruit y, Ceratitis capitata (Wiede-
mann) (Diptera: Tephritidae), is a devastating fruit
pest worldwide due to its global distribution, wide
range of hosts, rapid dispersion, and adaptation to low
temperatures. The demand for insecticide-free fresh
fruit is encouraging the use of environmentally
friendly methods, such as the sterile insect technique
(SIT), for prevention, suppression, or eradication of
Mediterranean fruit y. Areawide SIT programs
against Mediterranean fruit y are currently in use
worldwide (Hendrichs et al. 2002, Lindquist 2000).
Accordingly, an areawide suppression program was
initiated in 2003 in Spanish Mediterranean citrus areas.
Because only the males contribute to induced ste-
rility, Vienna genetic sexing strains (GSS) (e.g., all-
male strains carrying a temperature-sensitive lethal
(tsl) mutation) are used in most sterile release pro-
grams, because of their increased effectiveness in
suppressing pest populations (Hendrichs et al. 1995,
2002). Male sterility is due to unbalanced chromosome
pairing (Y:5 translocations in the Vienna strain that
produces semisterility) and by the accumulation of
dominant lethal mutations in sperm after gamma ir-
radiation (Caceres 2002, Robinson 2005).
The success of the SIT against Mediterranean fruit
y depends greatly upon mating performance be-
tween released sterilized males and wild-type (wt)
females. An indirect method to assess efcacy of ster-
ile males is based on the recapture ratio steriles: wild-
type males in pheromone-baited traps (Dyck et al.
2005). This is an imprecise estimation of the efcacy
of the massively released Mediterranean fruit ies,
because no data about females, actual sterile matings,
which are the focus of these releases, are taken into
account. Additionally, there are two direct methods to
assess mating success of sterile males, which are based
on egg hatchability (IAEA 1999, Katsoyannos et al.
1999) and spermatozoids head size measurement from
wild female spermathecae (McInnis 1993). Both
methods have been implemented in Hawaii and Gua-
temala (McInnis et al. 1994, Rendon et al. 2004), but
according to Vreysen (2005), its application to area-
wide SIT programs is limited.
We report here on a novel molecular method de-
vised for the detection of Mediterranean fruit y ster-
ile male sperm in wild-type (wt) female spermathecae
as a precise marker to assist in assessing mating per-
formance of sterile males in areawide SIT programs.

1 Centro de Proteccion Vegetal y Biotecnologa, IVIA, Ctra. Materials and Methods

Moncada-Naquera Km. 4.5, 46113 Moncada, Valencia, Spain.
2 Corresponding author, e-mail: bsabater@ivia.es. Unless otherwise indicated, all molecular tech-
3 Departamento Biologa de Plantas, CIB, CSIC, C/Ramiro de niques and solutions were performed as described by
Maeztu, 9, 28040 Madrid, Spain. Sambrook et al. (1989).

0022-0493/07/14441449$04.00/0 2007 Entomological Society of America

August 2007 SAN ANDRES ET AL.: STERILE C. capitata MATING SUCCESS
Mediterranean Fruit Fly Strains and Rearing Con-
ditions. The wt adults were obtained from a laboratory
colony maintained at the Instituto Valenciano de In-
vestigaciones Agrarias (IVIA) (Valencia, Spain) since
2002. The all-male Vienna-8 GSS strain [also named
GS1/D53 or T(Y;530C); Franz 2002] were provided
as irradiated pupae 2 d before emergence by the mass-
rearing facility in Mendoza, Argentina. Adults were
maintained at 25 4C, 75 5% RH, and a photope-
riod of 16:8 (L:D) h in an environmental chamber.
Wild-type adults were fed with a mixture of sugar and
hydrolyzed yeast (Biokar Diagnostics Co., Pantin,
France) (4:1, wt:wt) and water, whereas Vienna-8
adults were fed with a gelatinous slab consisting of
16.4% sugar, 1.5% agar (Sumilab, S.L., Madrid, Spain),
0.1% methyl-4-hydroxybenzoate (Sigma-Aldrich, St.
Louis, MO) in distilled water (Dyck et al. 2005).
Laboratory Mating Experiments. Approximately
1,000 wt and Vienna-8 pupae were set up, and adults
were allowed to emerge. Virgin females and males
were separated as they emerged, and kept in different
rooms to prevent any pheromone effect. Three mating
scenarios were designed: 1) 50 wt females (10 d old)
and 50 wt males (7 d old); 2) 50 wt females (10 d old)
and 50 Vienna-8 males (5 d old); and 3) 50 wt females
(10 d old), 50 wt males (7 d old), and 50 Vienna-8
males (5 d old). The adults were maintained as de-
scribed above. For each scenario, males were placed
in independent Perspex cages (20 by 20 by 20 cm) and
allowed to settle for 30 min. Thereafter, females were
introduced into the mating arena. In each scenario,
mating pairs were checked continuously during 2 h
and collected into vials (50-ml volume) 5 min after
initiation of mating. In total, three replicates were
conducted. After copula completion (Taylor et al.
2000), in each pair the male type was easily deter-
mined, because sterile males were labeled with uo-
rescent dye (Dyck et al. 2005). The females were
stored in 70% ethanol at 4C. Later, female spermathe-
cae were removed and used to determine sperm ori-
gin, as described below.
Field Cage Mating Experiments. Approximately
8,000 wt pupae were separated individually and in-
troduced into 1.5-ml vials to ensure virginity. Batches
of 700 newly emerged females and males (24 h
old) were placed in separated Perspex cages (20 by 20
by 20 cm). In total, 2,100 females (10 d old) and 1,400
males (7 d old) were kept in different rooms to avoid
any pheromone effect. In total, nine plastic boxes (25
by 25 by 8 cm) of Vienna-8 pupae each with 1,340
pupae were allowed to emerge and adults (1,000)
were maintained as described previously. One day
before release, Vienna-8 males were exposed for 3 h to
100 l of ginger root oil (Ginger essential oil, Guinama,
Valencia, Spain), impregnated on a piece of lter pa-
per, to enhance mating competitiveness (Shelly et al.
2004). The mating experiment was conducted in a
citrus orchard located at IVIA, which consisted of
21-yr-old Clementine trees (Clemenules variety).
Trees were 2.53 m in height and 3 4 m in width. Each
of the three clementine trees used was covered with
anti-thrip net (12 by 12 laments per cm2) held on a
1445
metallic frame (3.75 by 3.45 by 3.65 m, length by width
by height) to conne the ies. Fruits were removed
from trees and soil to avoid female attraction to ovi-
position. Three mating scenarios were tested: 1) 700
wt females with 700 wt males; 2) 700 wt females with
700 Vienna-8 males, and 3) 700 wt females, 700 wt
males and 7,000 Vienna-8 males in the ratio recom-
mended by the IAEA (10 sterile:1 wt). Males were
introduced before to let them establish mating terri-
tories, 30 min later females were released to simulate
the natural lek-based mating system. No food was
provided inside the eld cages. After 24 h, a Teph-
ri-trap (Sorygar, S.L., Madrid, Spain), baited with
female-targeted three-component lure (trimethyl-
amine, ammonium acetate, and putrescine; Tri-pack,
Kenogard S.A., Barcelona, Spain), and a tablet of
the insecticide dichlorvos (Biagro, S.L., Valencia,
Spain), were introduced and held in place during 24
additional hours in each tree. Captured individuals
were removed from traps, sexed, and kept separately
in 70% ethanol and stored at 4C. Females were trans-
ferred to sterilized distilled water 24 h before remov-
ing the spermathecae.
Sperm Detection Time under Field Conditions.
The procedure to obtain virgin females and males from
the eld mating trials was the same as that described
in laboratory mating experiments, although only two
mating scenarios were considered: 1) 50 wt females
(10 d old) and 50 wt males (7 d old) and 2) 50 wt
females (10 d old) and 50 Vienna-8 males (5 d old). In
total, three replicates were conducted. After copula
completion, females of each mating scenario were
placed in groups of 50 in Tephri-trap (with the en-
trances sealed with muslin), baited as described pre-
viously, and held in a Clementine tree 1, 3, and 7 d
during July 2006 to simulate the usual eld-checking
periods for monitoring traps in Spain. Females were
stored in 70% ethanol at 4C. Female spermathecae
were removed and tested as described below.
DNA Extraction. DNA extraction of entire individ-
uals was performed according to Sunnucks and Hales
(1996). DNA of female spermathecae was performed
using the same protocol, but the extraction buffer
composition was modied as follows: 50 mM Tris-HCl,
pH 7.5, 400 mM NaCl, 20 mM EDTA, pH 8.0, 0.5% SDS,
and 1% dithiothreitol (DTT). DTT enhances sperm
DNA isolation (Gill et al. 1985). Fresh proteinase K
was added at 100 g/ml after tissue homogenization.
DNA concentration was measured with a NanoDrop
ND-1000 UV-VIS spectrophotometer (Agilent Tech-
nologies, Palo Alto, CA) and adjusted to 510 ng/l for
polymerase chain reaction (PCR) amplication.
Y-Specific Sperm Detection. Four Y-specic se-
quences, AF071418, AF115330, AF116531, and AF154063,
were retrieved from the database. These sequences
were aligned using GeneDoc software (Nicholas et al.
1997) applying an MSA algorithm implemented in the
software with blossom 62 as a scoring table, a constant
cost length of 20, a gap open cost of 8, and gap ex-
tension cost of 4. A-T element core was located in the
alignment as described in Zhou et al. (2000). PCR
primers (CcYsp-5dir 5-CGA AGC CAG ACA TAC
1446 JOURNAL OF ECONOMIC ENTOMOLOGY
ACG AGG AG-3 and CcYsp-3rev 5-ACA CTT ACC
GAC ATT GAT TCC TG-3) were designed using
OLIGO version 4 primer analysis software (Rychlik
1992), out of the basic repeat unit of the A-T element,
to obtain a single PCR product, because Y-specic
primers obtained by Zhou et al. (2000) (Y114 dir and
rev) yield several products when used in PCR. This
marker yielded more than one band, as Zhous Y114
primer pair, indicating the presence of more copies of
the A-T elements than previously reported. Primers
were tested using female and male total DNA. PCR
conditions were: 300 nM dNTPs, 1 Taq buffer, 2 mM
MgCl2, 0.75 U of Taq polymerase (Invitrogen, Carls-
bad, CA), 10 pmol of each primer, and 10 ng of total
DNA. Amplication conditions were one denatur-
ation cycle at 94C for 5 min, 30 cycles at 94C for 30 s,
55C for 30 s, 72C for 45 s, followed by a nal extension
cycle at 72C for 4 min. PCR products were run in a
2% agarose D-1 low EEO gel (Pronadisa, Sumilab S.L.,
Madrid, Spain).
Discrimination between Vienna-8 and Wild-Type
Strains. To determine the origin of the sperm, a PCR-
restriction fragment length polymorphism marker has
been developed. It is based on a mitochondrial poly-
morphism found in the Vienna-8 strain (Gasparich et
al. 1995, Spanos et al. 2000). Two primers, Ccmt5495
and Ccmt5827, were designed using OLIGO version
4 software on C. capitata mitochondrial genome
(AJ242872), at anking regions of tRNA-Gly that
present the HaeIII polymorphic site: Ccmt5495, AAA
TCA CCA CTT TGG ATT TGA AGC; and Ccmt5827,
TGA AAA TGG TAA ACG TGA AGA GG. This
marker yields a 330-bp PCR product. After digestion
with HaeIII endonuclease, samples from the wt strain
remain undigested, whereas samples from the Vi-
enna-8 strain yield two bands of 190 and 140 pb. Prim-
ers were tested using Vienna-8 and wt male total DNA.
PCR and amplication conditions were the same as for
CcYsp. Positive amplication samples were subjected
to restriction fragment length polymorphism analysis,
by digestion of one fourth of the PCR reaction with 5
U of HaeIII nuclease (Takara Bio Inc., Shiga, Japan),
during 2 h at 37C. PCR and PCR/HaeIII products of
each sample were run side by side on a 2% agarose gel.
Results
Developing the Method. The system is based on the
application of two PCR markers: CcYsp, which allows
detecting the presence of Y chromosomes, and Ccmt,
which allows discriminating between wt and Vienna
strain males. Both markers have been tested with total
DNA from females and males from each strain (wt and
Vienna), resulting in a clear differentiation of each
type (Fig. 1). The application of both markers to
sperm enrichment DNA extraction from female sper-
mathecae results in three patterns A, B, and C (Fig. 2).
Pattern A, consisting of CcYsp positive and Ccmt-
HaeIII negative, indicates that the wt female has been
mated by a wt male. Pattern B, consisting of CcYsp
positive and Ccmt-HaeIII positive, indicates that the
wt female has been mated by a Vienna male. Pattern
Vol. 100, no. 4
Fig. 1. Validation of the two PCR markers developed.
The gure shows the PCR and PCR-restriction fragment
length polymorphism prole, which allows differentiation
between gender and strain. Head and thorax of each speci-
men, wt (lanes 2, 6, and 7), wt (lanes 3, 8, and 9), and
Vienna-8 (lanes 4, 10, and 11) were used as source for DNA
extraction. Lanes 1, 5, and 12 correspond to the molecular
weight marker 100-bp ladder (Invitrogen). Lanes 24 cor-
responds to CcYsp marker, which discriminates between
female (lane 2) and male (lanes 3 and 4) DNA irrespectively
of strain. Lanes 611 corresponds to Ccmt marker (even-
numbered lanes correspond to the PCR, and odd-numbered
lanes correspond to the PCR-restriction fragment length
polymorphism, Ccmt-HaeIII), Ccmt primers yield a 330-bp
band, which encloses the polymorphic site HaeIII. Vienna-8
strain presents a HaeIII site within Ccmt primers, rendering
two bands of 190 and 140 pb after digestion of PCR product
(lane 10) with HaeIII (lane 11). This HaeIII site is only
present in Vienna strains and in the original strain EgII
(Kourti 1997).
C, consisting of CcYsp negative and Ccmt-HaeIII neg-
ative, indicates that the wt female has not been mated
or the sperm being stored is not sufcient to be de-
tected.
Laboratory experiments were performed to ascer-
tain the feasibility of this novel approach. All females
mated with wild-type males showed a type A pattern,
those mated with Vienna males showed a type B pat-
tern, and all unmated females showed a type C pattern
(Fig. 2).
Field Cage Mating Experiments. In any mating sit-
uation, 92% of the trapped wt females were mated.
All the wt Mediterranean fruit y females analyzed
that only mated with either wt or Vienna males pre-
sented the pattern A or B, respectively. Moreover, in
the mating scenario with the ratio recommended
(10:1) for SIT operational programs, 67% of the wt
females were mated with Vienna males (Table 1).
Sperm Detection Time under Field Conditions. At
each sampling time, 1, 3, or 7 d, one female batch was
tested with both sperm markers (Table 2). The results
showed that after 7-d sperm detection in the dead
females was possible. For the wt scenario, sperm was
detected in 88% of cases, whereas in the Vienna sce-
nario a 100% was recorded.
Discussion
The molecular method reported here is a robust and
reliable marker to assess mating success between
August 2007 SAN ANDRES ET AL.: STERILE C. capitata MATING SUCCESS 1447

Table 2. Sperm detection over time on females captured

under field conditions

wt Vienna
Ccmt-HaeIII CcYsp Ccmt-HaeIII CcYsp
negative positive positive positive
Day 1 50 50 (1.00) 47 47 (1.00)
Day 3 46 43 (0.93) 48 48 (1.00)
Day 7 43 38 (0.88) 48 48 (1.00)

of Trimedlure-baited traps to ascertain the ratio ster-

ile:wt males and female-targeted (Tripack)-baited
traps to monitor wt females. In this context, the mo-
lecular method reported here could be integrated into
this schedule, and the percentage of sterile male mat-
ing could be easily estimated.
The method is cost-effective: spermathecae extir-
pation, DNA extraction, and target amplication plus
HaeIII digestion and analysis would cost $2 per sam-
ple. Furthermore, it is quick; the entire protocol for
200 samples can be completed in two working days.
The selection of only spermathecae, which is time-
Fig. 2. Molecular approach for Mediterranean fruit y consuming, as source of DNA was due to its function
sterile sperm detection. Method developed to detect sperm as long-term sperm storage organ (Twig and Yuval
accumulation in female spermathecae and its origin. The 2005) and by the negative results when other parts of
spermathecae removed from females were used for DNA the body (e.g., complete female, abdomen; data not
extraction and CcYsp and Ccmt-HaeIII PCR-restriction frag- shown) were tested.
ment length polymorphism analysis. In each pattern, the rst Sterile mating success by direct observation of mat-
lane corresponds to the CcYsp marker, the second lane cor- ing in cage trials has been reported, although at a rate
responds to the Ccmt, and the third lane corresponds to the
Ccmt-HaeIII. Pattern A corresponds to a female mated by a
lower than 50% of matings (Kraaijeveld and Chapman
wild-type male. Pattern B corresponds to a female mated by 2004, Dyck et al. 2005) was found. Two additional
a Vienna type male (arrow indicates a mixed Ccmt-HaeIII methods have been used to determine mating success
prole, 330-pb band corresponds to female DNA and 190- of sterile males. The rst method is based on the sperm
and 140-pb bands correspond to Vienna male DNA). Pattern head length average of sterile males being signicantly
C corresponds to an unmated female. MW corresponds to shorter than that of wild males (McInnis 1993). How-
molecular weight marker 100-bp ladder (Invitrogen). ever, the head sperm lengths of irradiated males de-
pend greatly on male age, irradiation dose, and male
strain used. Thus, a studio of each wild-type region
mass-reared sterilized Mediterranean fruit y males must be performed to ascertain the signicantly bigger
and wild females, because we were able to detect sperm head length than sterile males strain. The sec-
sperm in all females mated with Vienna strains, re- ond method, egg hatchability, is determined by esti-
gardless of the sampling date. The simplicity (only two mation of hatched eggs in natural infested fruits from
PCRs) jointly with the capability to detect Vienna the study areas (McInnis et al. 1994, IAEA 1999) or laid
sperm in wild Mediterranean fruit ies maintained in eggs in special traps (Katsoyanos et al. 1999). Egg
monitoring traps for 7 d (with a range of temperatures clutch recollection could be laborious, time-consum-
from 18 to 33C and a mean of 28.9C) suggests that it ing, difcult to asses in some species of fruits and an
could be an efcient tool when coupled to areawide estimation of the sterility level in nonrelease area also
SIT programs. In SIT programs, Mediterranean fruit is required. Furthermore, this method has some error
y monitoring is conducted weekly or biweekly, de- due to killing errors of sterile sperm, because some
pending on each region. This monitoring includes use dominant mutations and unbalanced chromosome in
sterile sperm could give rise to defective eggs that
Table 1. Sperm presence and genotyping in field cage trials
hatch but larvae not reach adulthood (Robinson
2005).
Mated Sperm origin Our method could be applied to different GSS
No.
Mating scenario females
females wt Vienna strains (tested with Vienna-7 strain from Madeira and
(CcYsp (Ccmt-HaeIII (Ccmt-HaeIII Vienna-7 mix 2000 from Mendoza, irradiated at 70, 90,
analyzed
positive) negative) positive) or 140 Gy; data not shown), and it could be applied
wt wt 12 11 (0.92) 11 0 directly to dead ies collected in monitoring traps.
wt mixturea 36 33 (0.92) 11 (0.33) 22 (0.67) Moreover, other GSS from the IAEA that are in use
wt Vienna-8 28 27 (0.96) 0 27
(Caceres et al. 2004), the new transgenic (Gong et al.
a
Male mixture consisting of 10 Vienna per 1 wt, as described in 2005), or Wolbachia-infected (Zabalou et al. 2004),
Materials and Methods. strains that are under development at the agency
1448 JOURNAL OF ECONOMIC ENTOMOLOGY
(IAEA 2001, 2004), can be detected with our method,
because the two markers, which the system is based
on, have been designed in regions not affected by any
of the modications listed above. Ccmt marker has
been developed on mitochondrial DNA, more pre-
cisely, on a specic haplotype that proceeds from the
EgII strain on which all the GSS strains are based. This
haplotype is unique with only natural presence in
Egypt and Greece (Kourti 1997, IAEA 2001). CcYsp
marker has been developed on repetitive elements
located in Y-chromosome (Zhou et al. 2000). Any
translocation affecting Y-chromosome would give dif-
ferent CcYsp pattern, but it does not invalidate the
method.
In multiple mating, it would not be possible to dif-
ferentiate them properly, because the detection of
both types of sperm (wild and sterile) in the female
spermathecae would be classied as sterile success.
Nevertheless, the remating frequency in a facultative
polyandrous species such as Mediterranean fruit y is
a complex issue, which has to be considered separately
(Bonizzoni et al. 2002, 2006; Mossinson and Yuval
2003, Vera et al. 2003; Kraaijeveld et al. 2005). Further
research will be needed in this respect.
Acknowledgments
We thank J. Garca de Oteyza, I. Pla, and R. Argiles from
TRAGSA (Valencia, Spain) for providing the Vienna-8 strain.
This work was supported by EU FOOD-CT-2003-506495 and
FEOGA COOPERACION. V.S.A. was supported by an In-
stituto Nacional de Investigacion y Tecnologia Agraria y
Alimentaria (INIA) Ph.D. fellowship.
References Cited
Bonizzoni, M., B. I. Katsoyannos, R. Marguerie, C. R.
Guglielmino, G. Gasperi, A. Malacrida, and T. Chapman.
2002. Microsatellite analysis reveals remating by wild
Mediterranean fruit y females, Ceratitis capitata. Mol.
Ecol. 11: 19151921.
Bonizzoni, M., L. M. Gomulski, S. Mossinson, C. R.
Guglielmino, A. R. Malacrida, B. Yuval, and G. Gasperi.
2006. Is polyandry a common event among wild pop-
ulations of the pest Ceratitis capitata?. J. Econ. Ento-
mol. 99: 1420 1429.
Caceres, C. 2002. Mass rearing of temperature sensitive
genetic sexing strains in the Mediterranean fruit y
(Ceratitis capitata). Genetica 116: 107116.
Caceres, C., J. P. Cayol, W. Enkerlin, G. Franz, J. Hendrich,
and A. S. Robinson. 2004. Comparison of Mediterranean
fruit y (Ceratitis capitata) (Tephritidae) bisexual and
genetic sexing strains: development, evaluation and eco-
nomics, pp. 367381. In Brian N. Barnes [ed.] Proceedings
of the 6th International Symposium on Fruit Flies of
Economic Importance, 6 10 May 2002, Stellenbosch,
South Africa. Isteg Scientic Publications, Irene, South
Africa.
Dyck, V. A., J. Hendrichs, and A. S. Robinson. 2005. Sterile
insect technique: principles and practice in area-wide
integrated pest management. Springer, The Netherlands.
Franz, G. 2002. Recombination between homologous auto-
somes in medy (Ceratitis capitata) males: type-1 recom-
bination and the implications for the stability of genetic
sexing strains. Genetica 116: 73 84.
Vol. 100, no. 4
Gasparich, G. E., W. S. Sheppard, H. Y. Han, B. A. McPheron,
and G. J. Steck. 1995. Analysis of mitochondrial DNA
and development of PCR-based diagnostic molecular
markers for Mediterranean fruit y (Ceratitis capitata)
populations. Insect Mol. Biol. 4: 61 67.
Gill, P., A. J. Jeffreys, and D. J. Werret. 1985. Forensic ap-
plication of DNA ngerprints. Nature (Lond.) 318: 577
579.
Gong, P., M. J. Epton, G. L. Fu, S. Scaife, A. Hiscox, K. C.
Condon, G. C. Condon, N. I. Morrison, D. W. Kelly, T.
Dafaalla, et al. 2005. A dominant lethal genetic system
for autocidal control of the Mediterranean fruity. Nat.
Biotechnol. 23: 453 456.
Hendrichs, J., G. Franz, and P. Rendon. 1995. Increased
effectiveness and applicability of the sterile insect tech-
nique through male-only releases for control of Mediter-
ranean fruit-ies during fruiting seasons. J. Appl. Ento-
mol. 119: 371377.
Hendrichs, J., A. S. Robinson, J. P. Cayol, and W. Enkerlin.
2002. Medy area wide sterile insect technique programs
for prevention, suppression or eradication: the impor-
tance of mating behavior studies. Fla. Entomol. 85: 113.
[IAEA] International Atomic Energy Agency. 1999. Devel-
opment of female medy attractant systems for trapping
and sterility assessment. Proceedings, Final research co-
ordination meeting, Joint FAO/IAEA Division of Nuclear
Techniques in Food and Agriculture, 28 May1 June 1998,
Penang, Malaysia. IAEA-TECDOC-1099. International
Atomic Energy Agency, Vienna, Austria.
[IAEA] International Atomic Energy Agency. 2001. An-
nual Report Entomology Unit, Agencys Laboratories.
FAO/IAEA Agriculture and Biotechnology Laboratory,
Seibersdorf, Austria.
[IAEA] International Atomic Energy Agency. 2004. An-
nual Report Entomology Unit, Agencys Laboratories.
FAO/IAEA Agriculture and Biotechnology Laboratory,
Seibersdorf, Austria.
Katsoyannos, B. I., N. T. Papadopoulos, N. A. Kouloussis, R.
Heath, and J. Hendrich. 1999. Method of assessing the
fertility of wild Ceratitis capitata (Diptera: Tephritidae)
females for use in sterile insect technique programs. J.
Econ. Entomol. 92: 590 597.
Kourti, A. 1997. Comparison of mtDNA variants among
Mediterranean and New World introductions of the Med-
iterranean fruit y Ceratitis capitata (Wied.). Biochem.
Genet. 35: 363370.
Kraaijeveld, K., and T. Chapman. 2004. Effects of male ste-
rility on female remating in the Mediterranean fruity,
Ceratitis capitata. Proc. R. Soc. Lond. B. Biol. Sci. 271
(Suppl. 4): S209 S211.
Kraaijeveld, K., B. I. Katsoyannos, M. Stavrinides, N. A.
Kouloussis, and T. Chapman. 2005. Remating in wild
females of the Mediterranean fruit y, Ceratitis capitata.
Anim. Behav. 69: 771776.
Lindquist, D. 2000. Pest management strategies: area-wide
and conventional. In K. H. Tan [ed.], Area-wide man-
agement of fruit ies and other major insect pests. Uni-
versity Sains Malaysia Press, Penang, Malaysia.
McInnis, D. O. 1993. Size differences between normal and
irradiated sperm heads in mated female Mediterranean
fruit ies (Diptera: Tephritidae). Ann. Entomol. Soc. Am.
86: 305308.
McInnis, D. O., S. Tam. C. Grace, and D. Miyashita. 1994.
Population suppression and sterility rates induced by
variable sex ratio, sterile insect releases of Ceratitis
capitata (Diptera: Tephritidae) in Hawaii. Ann. Entomol.
Soc. Am. 87: 231240.
August 2007 SAN ANDRES ET AL.: STERILE C. capitata MATING SUCCESS
Mossinson, S., and B. Yuval. 2003. Regulation of sexual re-
ceptivity of female Mediterranean fruit ies: old hypoth-
eses revisited and a new synthesis proposed. J. Insect
Physiol. 49: 561567.
Nicholas, K. B., H. B. Nicholas, Jr., and D. W. Deerfield, II.
1997. GeneDoc: analysis and visualization of genetic
variation EMBNEW News 4: 14.
Rendon, P., D. McInnis, D. Lance, and J. Stewart. 2004.
Medy (Diptera: Tephritidae) genetic sexing: large-scale
eld comparison of males-only and bisexual sterile y
releases in Guatemala. J Econ. Entomol 97: 15471553.
Robinson, A. S. 2005. Genetic basis of the Sterile Insect
Technique, pp. 95114. In V. A. Dyck, J. Hendrichs, and
A. S. Robinson [eds.], Sterile insect technique: principles
and practice in area-wide integrated pest management.
Springer, Dordrecht, The Netherlands.
Rychlik, W. 1992. OLIGO 4.06. Primer analysis software.
National Biosciences Inc. Publishers, Plymouth, MN.
Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular
cloning: a laboratory manual. Cold Spring Harbor Press,
Cold Spring Harbor, NY.
Shelly, T. E., D. O. McInnis, E. Pahio, and J. Edu. 2004.
Aromatherapy in the Mediterranean fruit y (Diptera:
Tephritidae): sterile males exposed to ginger root oil in
prerelease storage boxes display increased mating com-
petitiveness in eld-cage trials. J. Econ. Entomol. 97: 846
853.
Spanos, L., G. Koutroumbas, M. Kotsyfakis, and C. Louis.
2000. The mitochondrial genome of the Mediterranean
fruit y, Ceratitis capitata. Insect Mol. Biol. 9: 139 144.
Sunnucks, P., and D. F. Hales. 1996. Numerous transposed
sequences of mitochondrial cytochrome oxidase I-II in
View publication stats
1449
aphids of the genus Sitobion (Hemiptera: Aphididae).
Mol. Biol. Evol. 13: 510 524.
Taylor, P. W., R. Kaspi, and B. Yuval. 2000. Copula duration
and sperm storage in Mediterranean fruit ies from a wild
population. Physiol. Entomol. 25: 94 99.
Twig, E., and B. Yuval. 2005. Function of multiple sperm
storage organs in female Mediterranean fruit ies
(Ceratitis capitata, Diptera: Tephritidae). J. Insect
Physiol. 51: 6774.
Vera, M. T., J. L. Cladera, G. Calcagno, J. C. Vilardi, D. O.
McInnis, E. Stolar, D. Segura, N. P. Marty, F. Krsticevic,
P. G. Cendra, et al. 2003. Remating of wild Ceratitis
capitata (Diptera: Tephritidae) females in eld cages.
Ann. Entomol. Soc. Am. 96: 563570.
Vreysen, M.J.B. 2005. Monitoring sterile and wild insets in
area-wide integrated pest management programmes, pp.
325361. In V. A. Dyck, J. Hendrichs, and A. S. Robinson
[eds.], Sterile insect technique: principles and practice
in area-wide integrated pest management. Springer,
Dordrecht, The Netherlands.
Zabalou, S., M. Riegler, M. Theodorakopoulou, C. Stauffer,
C. Savakis, and K. Bourtzis. 2004. Wolbachia-induced
cytoplasmic incompatibility as a means for insect pest
population control. Proc. Natl. Acad. Sci. U.S.A. 101:
1504215045.
Zhou, Q., P. M. Untalan, and D. S. Haymer. 2000. Repetitive
A-T rich DNA sequences from the Y chromosome of the
Mediterranean fruit y, Ceratitis capitata. Genome 43:
434 438.
Received 8 November 2006; accepted 23 March 2007.