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Forced-contact mating: A technique for crossing experiments with the fruit fly parasitoid, Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae)
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Biological Control 44 (2008) 73–78 www.elsevier.com/locate/ybcon
Forced-contact mating: A technique for crossing experiments with the fruit fly parasitoid, Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae) Sangvorn Kitthawee
*
Department of Biology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand Received 26 April 2007; accepted 13 September 2007 Available online 21 September 2007
Abstract In this paper, a new technique is described for successfully manipulating the mating of a braconid parasitoid (Hymenoptera: Braconidae) of tephritid fruit flies. This forced-contact-mating technique was first developed for cross-mating experiments to determine the inheritance of winglessness in Diachasmimorpha longicaudata (Ashmead). Since female D. longicaudata only result from fertilized eggs (unfertilized eggs become males), this mating technique has advantages in the mass production of females for biocontrol releases as well as in investigations on the inheritance of the wingless trait and studies of reproductive isolation among different populations of these parasitoids. Free-mated colonies were generally all winged and predominantly male. Wingless males occurred occasionally but wingless females were rare. Virgin, winged females were immobilized by chilling and placed in close contact with wingless males. Active wingless males readily mounted and mated with immobilized female. Progeny of these mated females were all winged (83% # and 17% $). When F1 females remained unmated they produced both winged and wingless males (1:1 ratio) but when immobilized F1 females were backcrossed with wingless males, both winged and wingless females (5:1 ratio) were produced in addition to winged and wingless males. The wingless character was thus determined to be controlled by a recessive gene. Crossing experiments between two different Thai populations of D. longicaudata provided evidence that these populations were reproductively isolated. Among free-mated pairs, some sperm transfer occurred but almost no female progeny were produced. Similarly, among forced-mated pairs, more than double the numbers of females had sperm transferred to their spermatheca, but few female progeny were still produced. This suggests that these two populations are reproductively isolated and are part of a closely related species complex. 2007 Elsevier Inc. All rights reserved. Keywords: Diachasmimorpha longicaudata; Bactrocera correcta; Bactrocera dorsalis; Forced-mating technique; Wingless inheritance; Species complex; Biological control
1. Introduction Several species of hymenopterous parasitoids of tephritid fruit flies have been studied and a few have been imported for mass rearing and release against pestiferous fruit flies. However, many other candidate species collected with great difficulty from the field could not be successfully maintained for extended periods in the laboratory (Clausen et al., 1965). A major problem has been *
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that females are unable to produce female progeny due to a lack of mating in captivity (Cook and Crozier, 1995; Luck et al., 1992). Therefore, more efficient procedures for mating the fruit fly parasitoid, Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae) were explored in the laboratory. Diachasmimorpha longicaudata is a solitary endoparasitoid that attacks second and third instar larvae of tephritid fruit flies (Sime et al., 2006). In Thailand, D. longicaudata has been studied in the laboratory but never used as a biological control agent in the field. The native distribution of this parasitoid includes many countries in Southeast Asia
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S. Kitthawee / Biological Control 44 (2008) 73–78
(Bess et al., 1961; Clausen et al., 1965; Wharton and Gilstrap, 1983) and has been introduced and established in several other countries for the biological control (Clausen, 1978; Clausen et al., 1965; Ovruski et al., 2000; Sivinski et al., 1996; Vargas et al., 1993; Wong and Ramadan, 1987). Although the taxonomy of D. longicaudata has been well studied, several issues remain such as the variation in color characteristics in geographically distinct populations but all are treated as D. longicaudata (Wharton and Gilstrap, 1983). In laboratory colonies of D. longicaudata, wingless males were occasionally found in the colonies but wingless female were rare (S.K., pers. observ.). Wingless individuals have physical difficulty in locating mates (Roff, 1990). Wing vibration of males is involved in the courtship sequence (Rungrojwanich and Walter, 2000) and produces acoustic signals that are associated with female receptivity and successful copulation (Sivinski and Webb, 1989). In biological control, it is essential to characterize parasitoid complexes because the performance and effectiveness of each strain or species in controlling a specific pest can differ dramatically (DeBach and Rosen, 1991). Also, taxonomic decisions should not be based solely on morphological studies but also on cross-mating experiments that verify the reproductive isolation of populations (Mayr, 1963). Previous studies on D. longicaudata revealed genetic variation that was suspected to represent a complex of sibling species (Junesirikul, 1997), but difficulty in separating them based on morphological characters remains. Cross-mating D. longicaudata populations under laboratory conditions is generally difficult at best but forced-contact-mating techniques can help to clarify reproductive isolation questions within the D. longicaudata complex. These experiments with D. longicaudata investigated the inheritance of winglessness by employing a forced-contactmating technique that was developed to manipulate parentage. The technique was then modified to investigate the phylogenetic relationship among members of the D. longicaudata complex. 2. Materials and methods
Department of Biology at Mahidol University for >5 years (>60 generations) before these experiments started. Temperature was maintained at 27 ± 2 C, with 70 ± 10% RH and a photoperiod of 12L:12D. High humidity was maintained by placing damp cloth on the cages. Adult parasitoids were kept in transparent plastic cages (12 · 33 · 18 cm) and provided with cotton wool soaked in 10% honey in distilled water. Honey (100%) was also streaked on the net cloth at the top of cages to serve as an additional food source. 2.2. Cross-mating experiments to examine wingless inheritance Wingless D. longicaudata males were found and isolated from the DLB colony. Initial crossing experiments were conducted between these wingless males and winged virgin females that were immobilized. F1 progeny were counted and their sex and wing status recorded. The proportion of males and females obtained in F1 from the cross of normal females and wingless males was compared to the proportion of males and females obtained from crosses of normal DLB by a v2 test (Sokal and Rohlf, 1995). F1 virgin females were used to examine male progeny to determine whether male offspring were winged or wingless. Winged and wingless progeny were expected in equal proportions. Backcrosses required mating F1 females with wingless males. Winged and wingless progeny of both males and females from F2 were also expected in equal proportion from these crosses. Paired comparisons between observed and predicted wing-character were performed using the G-test of goodness of fit (Sokal and Rohlf, 1995). Laboratory observations on mating behavior among wingless males revealed that wingless male could not vibrate their wings and could not stimulate females to accept copulation. D. longicaudata males produce acoustic signals that may be important in interactions between the sexes (Sivinski and Webb, 1989). Although, wingless males attempted to copulate, females did not respond and walked away or rejected wingless males. Thus, cross-mating experiments between wingless males and winged females required a special forced-contact-mating technique to achieve sperm transfer.
2.1. Parasitoid cultures Parasitoids (DLA and DLB forms of the putative D. longicaudata complex) were primarily obtained from fruit fly larvae infested ripe fruits collected from Nakhon Pathom province in central Thailand but collected at different times of the year. Parasitoids and their host flies were identified to species morphologically using taxonomic keys (Wharton and Gilstrap, 1983; White and Elson-Harris, 1992). DLA were obtained from Bactrocera correcta larvae infested guava fruit and maintained on its host fly (B. correcta) in the laboratory. DLB were obtained from B. dorsalis larvae infested Indian almond and maintained on B. dorsalis. Both were maintained in an insectary at the
2.3. Techniques for forced-contact-mating wingless males and winged females Wingless males D. longicaudata up to 3 days old (Ramadan et al., 1991) were isolated from the colony and placed in a Petri dish just prior to mating. Each dish was set as a mating unit containing 4–5 wingless males. To obtain virgin females, parasitized pupal hosts were removed from the colony and kept individually in vials until emergence. Within 4 days after emergence, females were immobilized at 10 C for 4–5 min. Virgin females are optimally receptive to mating when 1–4 days old (Ramadan et al., 1991). From preliminary observations with cold treated females,
S. Kitthawee / Biological Control 44 (2008) 73–78
cold was not found to affect female survival or male acceptance behavior. An immobilized female was placed in each mating unit containing wingless males. The position of the immobilized female was manipulated using forceps to move it into close contact with a wingless male. Wingless males responded by walking to and mounting the immobilized female. Approximately 3–5 pairings were conducted within <5 min. Females were separated and transferred to new cages provided with honey-water. Female quickly recovered from the cold-immobilization and resumed normal behavior. Ten mated females were combined in 4–5 cages for a total of 40–50 females. Fruit fly hosts were supplied to mated females until all the parasitoids had died (15–20 days). Progeny from mated females were checked to determine their sex and thereby the success or failure of sperm transfer to parent females.
2.4. Cross-mating experiments for D. longicaudata complex identification Cross-mating experiments with two populations (DLA and DLB) of D. longicaudata were conducted, as were controls. In order to compare the progeny of parasitoids from free-mated and forced-contact-mated females, free mated trial were conducted by placing 5 pairs of virgin winged males and females in a cage. After 24 h, females were separated and transferred to a new cage provided with honeywater. Fruit fly hosts were provided every day for 10 days. Success of sperm transfer and fertilization was determined by examining the sex of progeny and the spermatheca of parent females. Results from these free-mated females were used as the standard or control for comparison with forced-contact-mated females. Forced-contact mating was modified for winged male parasitoids as follows. An immobilized, virgin female was placed in a plastic vial (one per vial) and arranged in the flight position using a soft camel-hair brush. An active, winged male was then released into the same vial. The vial was slowly moved until the immobilized female was close or next to the male. The male started vibrating his wings and responded to the immobilized female when the distance between the male and female was approximately <1 cm. [Note: occasionally the female awoke and walked
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to avoid the male, i.e. rejected him, and sometime the male used his antennae to touch or tap the female’s antennae before she walked away.] After mating, females were separated and transferred in groups of 5 to a new cage provided with honey-water. Females were allowed to lay eggs into fruit fly hosts for 10 days and progeny results were compared with free-mated females. After 10 days of oviposition, surviving females were dissected and examined for the presence of sperm in their spermatheca using methods described by Ode et al. (1995). In addition, parasitoid progeny were counted and sexed to determine successful egg fertilization. Only the presence of female progeny indicated that mating, sperm transfer, and egg fertilization were all successful. The frequency of female progeny produced from forced-contactmated and free-mated females, as well as the frequency of fertilized females, was analyzed by v2 tests. Fisher’s exact test was used when the expected values were small. Analyses were performed as described in Sokal and Rohlf (1995) and with the Statistix software package (Analytical Software, 2003).
3. Results and discussion When an initial 50 wingless male D. longicaudata were crossed with 50 winged, immobilized females, a total of 707 offspring (all winged) were produced, with a male:female sex ratio of 4.89:1 (Table 1). As expected, unmated females with unfertilized eggs produced only male progeny and all F1 females originated from successful male–female pairings. Pairings with wingless males produced only 16.97% female offspring (Table 1) whereas normal pairings with winged male DLB in forced-contact matings produced 50.00% female offspring (Table 2). There was a significant decrease in the percentage of female offspring in the cross of normal females and wingless males (v2 = 60.57; df = 1; P < 0.05). The different sex ratios may be explained by variation in the size and age of males (Ramadan et al., 1991) or since wingless males can not vibrate their wings, this could affect sperm transfer and/or female receptivity (Sivinski and Webb, 1989). The absence of wingless offspring in F1 indicated that the wingless form is recessive (Table 1). Forty virgin (unmated) F1 females (from the parental crosses) produced
Table 1 Summary of tests of wingless (wgl) inheritance using forced-contact-mating techniques between winged (wg) female and wingless (wgl) male Test
Crosses
Parent
wg $
F1
Virgin $
Backcross
F1 $
Total progeny (no. of test pairs)
·
·
wgl #
wgl #
% Winged progeny (number)
% Wingless progeny (number)
Female
Male
Female
Male
707 (50)
16.97% (120)
83.03% (587)
None
None
923 (40)
None
52% (480)
None
48% (443)
10.25% (318)
48.49% (1504)
1.87% (58)
39.39% (1222)
3102 (40)
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S. Kitthawee / Biological Control 44 (2008) 73–78
Table 2 Results of crosses between two populations of D. longicaudata, comparing forced-contact-mating and free-mating techniques % Sperm positive (number)a
Crosses female · male
No. of pairs
Total F1 progeny
% F1 female (total F1 female)
DLA · DLA Forced contact Free mated
10 10
350 371
51.43 (180) 32.88 (122)
100 (10) 100 (10)
DLB · DLB Forced contact Free mated
20 20
112 107
50.00 (56) 43.93 (47)
100 (15) 100 (15)
DLA · DLB Force contact Free mated DLB · DLA Forced contact Free mated a b
b
b
20 20 20 20
99 104 364 228
1.01 (1) 0 (0)
60 (20) 20 (20)
b
b
10.44 (38) 0.88 (2)
94.44 (18) 40 (20)
Number of mated females examined. Significant difference between forced-contact-mating and free-mating techniques.
both wingless and winged male progeny in about equal proportions with 480 winged males and 443 wingless males (G-test; G = 1.48; df = 1; P > 0.05). In 40 pairs of backcrosses between F1 hybrid females and wingless males, the progeny were as follows: winged females (10.25%), winged males (48.49%), wingless females (1.87%), and wingless males (39.39%) (Table 1). The overall percentage of winged:wingless was 58.74%:41.26% which did not closely fit the expected 1:1 ratio (G-test; G = 95.19; df = 1; P < 0.05). The low number of wingless female offspring (homozygous recessives) suggests that their survival and/or egg laying capacity were greatly reduced compared to the winged forms. This would explain why wingless forms are rare or absent in natural populations. In the colony, the wingless gene was maintained in winged, heterozygous females. Although backcrosses between F1 hybrid females and wingless males gave a low percentage of wingless females (1.87%), virgin wingless females occasionally produced viable and fertile male offspring of the wingless form. In addition, the female sex ratio of the winged form (0.21) was four times higher than in the wingless form (0.05). Apparently, the winged and wingless genes are equally expressed in term of fertility but wingless males have greatly reduced mating competitiveness as evidenced by the preponderance of males produced by seemingly mated females. Sivinski and Webb (1989) demonstrated that acoustic signals from wing vibrations were important in D. longicaudata copulation. Therefore, the absence of calling from wingless males may result in incompletely inseminated females or females that fail to perceive a specific signal that influences complete sperm transfer to the spermatheca. Rare wingless females may be caused by homozygous recessive suppressors as occurs in Drosophila willistoni (Magalhaes et al., 1965). Fertile wingless females had a decreased capacity to lay eggs but wingless females can occasionally give rise to wingless male. Thus, only hybrid females carrying the recessive wingless condition can main-
tain the wingless gene in natural populations. Although the loss of wings greatly reduces an insect’s competitiveness and capacity to move a long distance in nature, it can be useful material for genetic and behavioral investigations in the laboratory. Cross-mating experiments between wingless and winged forms of D. longicaudata could not be conducted under normal, free-mating conditions. In early observation of D. longicaudata colony, male parasitoids were often seen to attempt to mate with moribund and mostly immobile females nearing death. Active males also attempted to copulate with females just after emerging from the pupal case. These observations led to development of the forced-contact-mating technique used in crossing experiments here, and used in systematic investigation of the D. longicaudata complex. Results of systematic investigations showed that the percentage of sperm transfer within the same populations (DLA · DLA or DLB · DLB), whether forced-contact mated or free mated, was 100% (Table 2). However, the percentage of female progeny from forced-contact matings was higher (DLA = 51.43%, DLB = 50.00%) than that produced from free matings (DLA = 32.88%, DLB = 43.93%). The frequency of female progeny did not differ significantly between forced-contact-mating and free-mating techniques with the DLB population (Fisher’s exact test; P > 0.05). These results suggest that crosses within the DLB population are uniformly of a high level of genetic compatibility. Results with the DLA population were significant different in the somewhat higher frequency of female progeny from forced-contact matings (v2 = 24.69; df = 1; P < 0.05). There are clear indications that forcedcontact mating elevates the number of female progeny. For the free mated reciprocal crosses between two different populations (i.e. DLA · DLB), the percentage of sperm positive females was reduced in both directions (Table 2). In particular, the 20 natural crosses between DLA females and DLB males yielded a relatively low percentage (20%) of females with positive spermatheca and female progeny
S. Kitthawee / Biological Control 44 (2008) 73–78
were zero (N = 104 progeny). For forced-contact matings, the percentage of positive spermatheca increased significantly from 20 to 60% (Fisher’s exact test; P < 0.05) but only one F1 female progeny (1.01%) was produced by these inseminated females. These data suggest a low degree of genetic compatibility between these two populations. However, in the 20 reciprocal crosses between DLB females · DLA males, the percentage of females with positive spermathecae increased significantly from 40 to 94.44% (Fisher’s exact test; P < 0.05) and more female progeny were produced from these forced-contact matings i.e. 10.44% compared to 0.88% in free matings (v2 = 8.86; df = 1; P < 0.05). The fact that more female progeny were produced from certain inter-population crosses suggested that there may be different degrees of genetic incompatibility supporting the isolation of these populations. Crosses between DLA and DLB populations (Table 2) indicated that there was partial reproductive isolation between these two populations due to reproductive incompatibilities (Turelli et al., 2001), suggesting that they are different strains, biotypes, or species (Kazmer et al., 1996; Pinto et al., 1991). According to Fischer (1966) and Wharton and Gilstrap (1983), D. longicaudata is widely distributed with morphological variations in different geographical populations and was considered by them to be a species complex. Moreover, Kitthawee et al. (1999) studies on polymorphism in natural population of D. longicaudata in Thailand also suggested that the populations may be composed of a number of subpopulations or cryptic species. The absence of female offspring in free mating between DLA females and DLB males may be considered reproductive isolation due to pre-mating isolation such as mating behavioral isolation. In addition, the fact that they were isolated from different host flies (see Section 2) supports this conclusion. Although various authors have considered D. longicaudata to be a generalist parasitoid attacking several species of fruit flies (Wong and Ramadan, 1987), DLA was found to be specific to B. correcta while DLB preferred B. dorsalis. These populations were collected from the same location, but during different fruit seasons. Many artificial mating techniques have been developed to maintain laboratory colonies for medically and economically important insects such as mosquitoes (Burcham, 1957; Lima et al., 2004), silkworms (Takemura et al., 2000), and honeybees (Baer and Schmid-Hempel, 2000). However, such mating techniques have never been reported for parasitoids. Forced-contact-mating techniques proved to be extremely useful for crossing fruit fly parasitoids in order to determine the inheritance of the wingless trait in D. longicaudata and in documenting the reproductive isolation of different populations. It also produced a much higher percentage of females when crossed with winged males than when freely mated. This could be quite useful in mass rearing parasitoids for release in biological control programs.
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Acknowledgments I wish to thank Dr. John D. Edman and two anonymous reviewers for their valuable comments and suggestions on the manuscript. Ms. Pakamas Tabrod maintained my fruit fly and parasitoid colonies. This work was supported by the TRF/BIOTEC Special Program for Biodiversity Research and Training Grant BRT R_250007. References Analytical Software, 2003. Statistix 8. Tallahassee, FL 32317-2185. Baer, B., Schmid-Hempel, P., 2000. The artificial insemination of bumblebee queens. Insectes Sociaux 47, 183–187. Bess, H.A., van den Bosch, R., Haramoto, F.H., 1961. Fruit fly parasites and their activities in Hawaii. Proceedings of the Hawaiian Entomological Society 17, 367–378. Burcham, E., 1957. Artificial insemination of Aedes aegypti (L.). Annals of the Entomological Society of America 49, 467–493. Clausen, C.P., 1978. Introduced parasites and predators of arthropod pests and weeds: A world review. USDA-ARS, Agriculture Handbook No. 480. USDA Pubs., Washington, DC. Clausen, C.P., Clancy, D.W., Chock, Q.C., 1965. Biological control of the Oriental fruit fly (Dacus dorsalis Hendel) and other fruit flies in Hawaii. USDA Technical Bulletin 1322, 1–102. Cook, J.M., Crozier, R.H., 1995. Sex determination and population biology in the Hymenoptera. Trends in Ecology & Evolution 10, 281– 286. DeBach, P., Rosen, D., 1991. Biological Control by Natural Enemies, second ed. Cambridge University Press, Cambridge. Fischer, M., 1966. Revision der Indo-Australischen Opiinae. Dr. W. Junk, Publ., The Hague. Junesirikul, D., 1997. Study on genetic variation of fruit fly parasitoids (Diachasmimorpha sp.) in Thailand by electrophoretic techniques. M.Sc. Thesis (Environmental Biology), Faculty of Graduate Studies, Mahidol University, Bangkok, Thailand. Kazmer, K.J., Maiden, K., Ramualde, N., Coutinot, D., Hopper, K.R., 1996. Reproductive compatibility, mating behavior, and random amplified polymorphic DNA variability in some Aphelinus asychis (Hymenoptera: Aphelinidae) derived from the old world. Annals of the Entomological Society of America 98, 212–220. Kitthawee, S., Julsilikul, D., Sharpe, R.G., Baimai, V., 1999. Protein polymorphism in natural populations of Diachasmimorpha longicaudata (Hymanoptera: Braconidae) in Thailand. Genetica 105, 125–131. Lima, J.B., Valle, D., Peixoto, A.A., 2004. Adaptation of a South American malaria vector to laboratory colonization suggests fastermale evolution for mating ability. BMC Evolutionary Biology 4, 12. Luck, R.F., Stouthamer, R., Lunney, L.P., 1992. Sex determination and sex ratio patterns in parasitic Hymenoptera. In: Wrensch, D.L., Ebbert, M. (Eds.), Evolution and Diversity of Sex Ratio in Haplodiploid Insects and Mites. Chapman and Hall, New York, pp. 442– 476. Magalhaes, L.E., Brito Da Cunha, A., de Toledo, J.S., Toledo, F.S.A., de Souza, H.L., Targa, H.J., Setzer, V., Pavan, C., 1965. On lethals and their suppressors in experimental populations of Drosophila willistoni. Mutation Research 2, 45–54. Mayr, E., 1963. Animal Species and Evolution. Harvard University Press, Cambridge, MA. Ode, P.J., Antolin, M.F., Strand, M.R., 1995. Brood-mate avoidance in the parasitic wasp Bracon hebetor Say. Animal Behaviour 49, 1239– 1248. Ovruski, S., Aluja, M., Sivinski, J., Wharton, R.A., 2000. Hymenopteran parasitoids on fruit-infesting Tephritidae (Diptera) in Latin America and the Southern United States: Diversity, distribution, taxonomic status and their use in fruit fly biological control. Integrated Pest Management Review 5, 81–107.
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Biological Control 44 (2008) 73–78 www.elsevier.com/locate/ybcon
Forced-contact mating: A technique for crossing experiments with the fruit fly parasitoid, Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae) Sangvorn Kitthawee
*
Department of Biology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand Received 26 April 2007; accepted 13 September 2007 Available online 21 September 2007
Abstract In this paper, a new technique is described for successfully manipulating the mating of a braconid parasitoid (Hymenoptera: Braconidae) of tephritid fruit flies. This forced-contact-mating technique was first developed for cross-mating experiments to determine the inheritance of winglessness in Diachasmimorpha longicaudata (Ashmead). Since female D. longicaudata only result from fertilized eggs (unfertilized eggs become males), this mating technique has advantages in the mass production of females for biocontrol releases as well as in investigations on the inheritance of the wingless trait and studies of reproductive isolation among different populations of these parasitoids. Free-mated colonies were generally all winged and predominantly male. Wingless males occurred occasionally but wingless females were rare. Virgin, winged females were immobilized by chilling and placed in close contact with wingless males. Active wingless males readily mounted and mated with immobilized female. Progeny of these mated females were all winged (83% # and 17% $). When F1 females remained unmated they produced both winged and wingless males (1:1 ratio) but when immobilized F1 females were backcrossed with wingless males, both winged and wingless females (5:1 ratio) were produced in addition to winged and wingless males. The wingless character was thus determined to be controlled by a recessive gene. Crossing experiments between two different Thai populations of D. longicaudata provided evidence that these populations were reproductively isolated. Among free-mated pairs, some sperm transfer occurred but almost no female progeny were produced. Similarly, among forced-mated pairs, more than double the numbers of females had sperm transferred to their spermatheca, but few female progeny were still produced. This suggests that these two populations are reproductively isolated and are part of a closely related species complex. 2007 Elsevier Inc. All rights reserved. Keywords: Diachasmimorpha longicaudata; Bactrocera correcta; Bactrocera dorsalis; Forced-mating technique; Wingless inheritance; Species complex; Biological control
1. Introduction Several species of hymenopterous parasitoids of tephritid fruit flies have been studied and a few have been imported for mass rearing and release against pestiferous fruit flies. However, many other candidate species collected with great difficulty from the field could not be successfully maintained for extended periods in the laboratory (Clausen et al., 1965). A major problem has been *
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that females are unable to produce female progeny due to a lack of mating in captivity (Cook and Crozier, 1995; Luck et al., 1992). Therefore, more efficient procedures for mating the fruit fly parasitoid, Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae) were explored in the laboratory. Diachasmimorpha longicaudata is a solitary endoparasitoid that attacks second and third instar larvae of tephritid fruit flies (Sime et al., 2006). In Thailand, D. longicaudata has been studied in the laboratory but never used as a biological control agent in the field. The native distribution of this parasitoid includes many countries in Southeast Asia
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(Bess et al., 1961; Clausen et al., 1965; Wharton and Gilstrap, 1983) and has been introduced and established in several other countries for the biological control (Clausen, 1978; Clausen et al., 1965; Ovruski et al., 2000; Sivinski et al., 1996; Vargas et al., 1993; Wong and Ramadan, 1987). Although the taxonomy of D. longicaudata has been well studied, several issues remain such as the variation in color characteristics in geographically distinct populations but all are treated as D. longicaudata (Wharton and Gilstrap, 1983). In laboratory colonies of D. longicaudata, wingless males were occasionally found in the colonies but wingless female were rare (S.K., pers. observ.). Wingless individuals have physical difficulty in locating mates (Roff, 1990). Wing vibration of males is involved in the courtship sequence (Rungrojwanich and Walter, 2000) and produces acoustic signals that are associated with female receptivity and successful copulation (Sivinski and Webb, 1989). In biological control, it is essential to characterize parasitoid complexes because the performance and effectiveness of each strain or species in controlling a specific pest can differ dramatically (DeBach and Rosen, 1991). Also, taxonomic decisions should not be based solely on morphological studies but also on cross-mating experiments that verify the reproductive isolation of populations (Mayr, 1963). Previous studies on D. longicaudata revealed genetic variation that was suspected to represent a complex of sibling species (Junesirikul, 1997), but difficulty in separating them based on morphological characters remains. Cross-mating D. longicaudata populations under laboratory conditions is generally difficult at best but forced-contact-mating techniques can help to clarify reproductive isolation questions within the D. longicaudata complex. These experiments with D. longicaudata investigated the inheritance of winglessness by employing a forced-contactmating technique that was developed to manipulate parentage. The technique was then modified to investigate the phylogenetic relationship among members of the D. longicaudata complex. 2. Materials and methods
Department of Biology at Mahidol University for >5 years (>60 generations) before these experiments started. Temperature was maintained at 27 ± 2 C, with 70 ± 10% RH and a photoperiod of 12L:12D. High humidity was maintained by placing damp cloth on the cages. Adult parasitoids were kept in transparent plastic cages (12 · 33 · 18 cm) and provided with cotton wool soaked in 10% honey in distilled water. Honey (100%) was also streaked on the net cloth at the top of cages to serve as an additional food source. 2.2. Cross-mating experiments to examine wingless inheritance Wingless D. longicaudata males were found and isolated from the DLB colony. Initial crossing experiments were conducted between these wingless males and winged virgin females that were immobilized. F1 progeny were counted and their sex and wing status recorded. The proportion of males and females obtained in F1 from the cross of normal females and wingless males was compared to the proportion of males and females obtained from crosses of normal DLB by a v2 test (Sokal and Rohlf, 1995). F1 virgin females were used to examine male progeny to determine whether male offspring were winged or wingless. Winged and wingless progeny were expected in equal proportions. Backcrosses required mating F1 females with wingless males. Winged and wingless progeny of both males and females from F2 were also expected in equal proportion from these crosses. Paired comparisons between observed and predicted wing-character were performed using the G-test of goodness of fit (Sokal and Rohlf, 1995). Laboratory observations on mating behavior among wingless males revealed that wingless male could not vibrate their wings and could not stimulate females to accept copulation. D. longicaudata males produce acoustic signals that may be important in interactions between the sexes (Sivinski and Webb, 1989). Although, wingless males attempted to copulate, females did not respond and walked away or rejected wingless males. Thus, cross-mating experiments between wingless males and winged females required a special forced-contact-mating technique to achieve sperm transfer.
2.1. Parasitoid cultures Parasitoids (DLA and DLB forms of the putative D. longicaudata complex) were primarily obtained from fruit fly larvae infested ripe fruits collected from Nakhon Pathom province in central Thailand but collected at different times of the year. Parasitoids and their host flies were identified to species morphologically using taxonomic keys (Wharton and Gilstrap, 1983; White and Elson-Harris, 1992). DLA were obtained from Bactrocera correcta larvae infested guava fruit and maintained on its host fly (B. correcta) in the laboratory. DLB were obtained from B. dorsalis larvae infested Indian almond and maintained on B. dorsalis. Both were maintained in an insectary at the
2.3. Techniques for forced-contact-mating wingless males and winged females Wingless males D. longicaudata up to 3 days old (Ramadan et al., 1991) were isolated from the colony and placed in a Petri dish just prior to mating. Each dish was set as a mating unit containing 4–5 wingless males. To obtain virgin females, parasitized pupal hosts were removed from the colony and kept individually in vials until emergence. Within 4 days after emergence, females were immobilized at 10 C for 4–5 min. Virgin females are optimally receptive to mating when 1–4 days old (Ramadan et al., 1991). From preliminary observations with cold treated females,
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cold was not found to affect female survival or male acceptance behavior. An immobilized female was placed in each mating unit containing wingless males. The position of the immobilized female was manipulated using forceps to move it into close contact with a wingless male. Wingless males responded by walking to and mounting the immobilized female. Approximately 3–5 pairings were conducted within <5 min. Females were separated and transferred to new cages provided with honey-water. Female quickly recovered from the cold-immobilization and resumed normal behavior. Ten mated females were combined in 4–5 cages for a total of 40–50 females. Fruit fly hosts were supplied to mated females until all the parasitoids had died (15–20 days). Progeny from mated females were checked to determine their sex and thereby the success or failure of sperm transfer to parent females.
2.4. Cross-mating experiments for D. longicaudata complex identification Cross-mating experiments with two populations (DLA and DLB) of D. longicaudata were conducted, as were controls. In order to compare the progeny of parasitoids from free-mated and forced-contact-mated females, free mated trial were conducted by placing 5 pairs of virgin winged males and females in a cage. After 24 h, females were separated and transferred to a new cage provided with honeywater. Fruit fly hosts were provided every day for 10 days. Success of sperm transfer and fertilization was determined by examining the sex of progeny and the spermatheca of parent females. Results from these free-mated females were used as the standard or control for comparison with forced-contact-mated females. Forced-contact mating was modified for winged male parasitoids as follows. An immobilized, virgin female was placed in a plastic vial (one per vial) and arranged in the flight position using a soft camel-hair brush. An active, winged male was then released into the same vial. The vial was slowly moved until the immobilized female was close or next to the male. The male started vibrating his wings and responded to the immobilized female when the distance between the male and female was approximately <1 cm. [Note: occasionally the female awoke and walked
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to avoid the male, i.e. rejected him, and sometime the male used his antennae to touch or tap the female’s antennae before she walked away.] After mating, females were separated and transferred in groups of 5 to a new cage provided with honey-water. Females were allowed to lay eggs into fruit fly hosts for 10 days and progeny results were compared with free-mated females. After 10 days of oviposition, surviving females were dissected and examined for the presence of sperm in their spermatheca using methods described by Ode et al. (1995). In addition, parasitoid progeny were counted and sexed to determine successful egg fertilization. Only the presence of female progeny indicated that mating, sperm transfer, and egg fertilization were all successful. The frequency of female progeny produced from forced-contactmated and free-mated females, as well as the frequency of fertilized females, was analyzed by v2 tests. Fisher’s exact test was used when the expected values were small. Analyses were performed as described in Sokal and Rohlf (1995) and with the Statistix software package (Analytical Software, 2003).
3. Results and discussion When an initial 50 wingless male D. longicaudata were crossed with 50 winged, immobilized females, a total of 707 offspring (all winged) were produced, with a male:female sex ratio of 4.89:1 (Table 1). As expected, unmated females with unfertilized eggs produced only male progeny and all F1 females originated from successful male–female pairings. Pairings with wingless males produced only 16.97% female offspring (Table 1) whereas normal pairings with winged male DLB in forced-contact matings produced 50.00% female offspring (Table 2). There was a significant decrease in the percentage of female offspring in the cross of normal females and wingless males (v2 = 60.57; df = 1; P < 0.05). The different sex ratios may be explained by variation in the size and age of males (Ramadan et al., 1991) or since wingless males can not vibrate their wings, this could affect sperm transfer and/or female receptivity (Sivinski and Webb, 1989). The absence of wingless offspring in F1 indicated that the wingless form is recessive (Table 1). Forty virgin (unmated) F1 females (from the parental crosses) produced
Table 1 Summary of tests of wingless (wgl) inheritance using forced-contact-mating techniques between winged (wg) female and wingless (wgl) male Test
Crosses
Parent
wg $
F1
Virgin $
Backcross
F1 $
Total progeny (no. of test pairs)
·
·
wgl #
wgl #
% Winged progeny (number)
% Wingless progeny (number)
Female
Male
Female
Male
707 (50)
16.97% (120)
83.03% (587)
None
None
923 (40)
None
52% (480)
None
48% (443)
10.25% (318)
48.49% (1504)
1.87% (58)
39.39% (1222)
3102 (40)
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Table 2 Results of crosses between two populations of D. longicaudata, comparing forced-contact-mating and free-mating techniques % Sperm positive (number)a
Crosses female · male
No. of pairs
Total F1 progeny
% F1 female (total F1 female)
DLA · DLA Forced contact Free mated
10 10
350 371
51.43 (180) 32.88 (122)
100 (10) 100 (10)
DLB · DLB Forced contact Free mated
20 20
112 107
50.00 (56) 43.93 (47)
100 (15) 100 (15)
DLA · DLB Force contact Free mated DLB · DLA Forced contact Free mated a b
b
b
20 20 20 20
99 104 364 228
1.01 (1) 0 (0)
60 (20) 20 (20)
b
b
10.44 (38) 0.88 (2)
94.44 (18) 40 (20)
Number of mated females examined. Significant difference between forced-contact-mating and free-mating techniques.
both wingless and winged male progeny in about equal proportions with 480 winged males and 443 wingless males (G-test; G = 1.48; df = 1; P > 0.05). In 40 pairs of backcrosses between F1 hybrid females and wingless males, the progeny were as follows: winged females (10.25%), winged males (48.49%), wingless females (1.87%), and wingless males (39.39%) (Table 1). The overall percentage of winged:wingless was 58.74%:41.26% which did not closely fit the expected 1:1 ratio (G-test; G = 95.19; df = 1; P < 0.05). The low number of wingless female offspring (homozygous recessives) suggests that their survival and/or egg laying capacity were greatly reduced compared to the winged forms. This would explain why wingless forms are rare or absent in natural populations. In the colony, the wingless gene was maintained in winged, heterozygous females. Although backcrosses between F1 hybrid females and wingless males gave a low percentage of wingless females (1.87%), virgin wingless females occasionally produced viable and fertile male offspring of the wingless form. In addition, the female sex ratio of the winged form (0.21) was four times higher than in the wingless form (0.05). Apparently, the winged and wingless genes are equally expressed in term of fertility but wingless males have greatly reduced mating competitiveness as evidenced by the preponderance of males produced by seemingly mated females. Sivinski and Webb (1989) demonstrated that acoustic signals from wing vibrations were important in D. longicaudata copulation. Therefore, the absence of calling from wingless males may result in incompletely inseminated females or females that fail to perceive a specific signal that influences complete sperm transfer to the spermatheca. Rare wingless females may be caused by homozygous recessive suppressors as occurs in Drosophila willistoni (Magalhaes et al., 1965). Fertile wingless females had a decreased capacity to lay eggs but wingless females can occasionally give rise to wingless male. Thus, only hybrid females carrying the recessive wingless condition can main-
tain the wingless gene in natural populations. Although the loss of wings greatly reduces an insect’s competitiveness and capacity to move a long distance in nature, it can be useful material for genetic and behavioral investigations in the laboratory. Cross-mating experiments between wingless and winged forms of D. longicaudata could not be conducted under normal, free-mating conditions. In early observation of D. longicaudata colony, male parasitoids were often seen to attempt to mate with moribund and mostly immobile females nearing death. Active males also attempted to copulate with females just after emerging from the pupal case. These observations led to development of the forced-contact-mating technique used in crossing experiments here, and used in systematic investigation of the D. longicaudata complex. Results of systematic investigations showed that the percentage of sperm transfer within the same populations (DLA · DLA or DLB · DLB), whether forced-contact mated or free mated, was 100% (Table 2). However, the percentage of female progeny from forced-contact matings was higher (DLA = 51.43%, DLB = 50.00%) than that produced from free matings (DLA = 32.88%, DLB = 43.93%). The frequency of female progeny did not differ significantly between forced-contact-mating and free-mating techniques with the DLB population (Fisher’s exact test; P > 0.05). These results suggest that crosses within the DLB population are uniformly of a high level of genetic compatibility. Results with the DLA population were significant different in the somewhat higher frequency of female progeny from forced-contact matings (v2 = 24.69; df = 1; P < 0.05). There are clear indications that forcedcontact mating elevates the number of female progeny. For the free mated reciprocal crosses between two different populations (i.e. DLA · DLB), the percentage of sperm positive females was reduced in both directions (Table 2). In particular, the 20 natural crosses between DLA females and DLB males yielded a relatively low percentage (20%) of females with positive spermatheca and female progeny
S. Kitthawee / Biological Control 44 (2008) 73–78
were zero (N = 104 progeny). For forced-contact matings, the percentage of positive spermatheca increased significantly from 20 to 60% (Fisher’s exact test; P < 0.05) but only one F1 female progeny (1.01%) was produced by these inseminated females. These data suggest a low degree of genetic compatibility between these two populations. However, in the 20 reciprocal crosses between DLB females · DLA males, the percentage of females with positive spermathecae increased significantly from 40 to 94.44% (Fisher’s exact test; P < 0.05) and more female progeny were produced from these forced-contact matings i.e. 10.44% compared to 0.88% in free matings (v2 = 8.86; df = 1; P < 0.05). The fact that more female progeny were produced from certain inter-population crosses suggested that there may be different degrees of genetic incompatibility supporting the isolation of these populations. Crosses between DLA and DLB populations (Table 2) indicated that there was partial reproductive isolation between these two populations due to reproductive incompatibilities (Turelli et al., 2001), suggesting that they are different strains, biotypes, or species (Kazmer et al., 1996; Pinto et al., 1991). According to Fischer (1966) and Wharton and Gilstrap (1983), D. longicaudata is widely distributed with morphological variations in different geographical populations and was considered by them to be a species complex. Moreover, Kitthawee et al. (1999) studies on polymorphism in natural population of D. longicaudata in Thailand also suggested that the populations may be composed of a number of subpopulations or cryptic species. The absence of female offspring in free mating between DLA females and DLB males may be considered reproductive isolation due to pre-mating isolation such as mating behavioral isolation. In addition, the fact that they were isolated from different host flies (see Section 2) supports this conclusion. Although various authors have considered D. longicaudata to be a generalist parasitoid attacking several species of fruit flies (Wong and Ramadan, 1987), DLA was found to be specific to B. correcta while DLB preferred B. dorsalis. These populations were collected from the same location, but during different fruit seasons. Many artificial mating techniques have been developed to maintain laboratory colonies for medically and economically important insects such as mosquitoes (Burcham, 1957; Lima et al., 2004), silkworms (Takemura et al., 2000), and honeybees (Baer and Schmid-Hempel, 2000). However, such mating techniques have never been reported for parasitoids. Forced-contact-mating techniques proved to be extremely useful for crossing fruit fly parasitoids in order to determine the inheritance of the wingless trait in D. longicaudata and in documenting the reproductive isolation of different populations. It also produced a much higher percentage of females when crossed with winged males than when freely mated. This could be quite useful in mass rearing parasitoids for release in biological control programs.
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