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In insect sex determination a primary signal starts the genetic sex determination cascade that, in most insect orders, is subsequently transduced down the cascade by a transformer (tra) ortholog. Only a female-specifically spliced tra mRNA yields a functional TRA-protein that forms a complex with TRA2, encoded by a transformer-2 (tra2) ortholog, to act as a sex specific splicing regulator of the downstream transcription factors doublesex (dsx) and fruitless (fru). Here, we identify the tra2 ortholog of the haplodiploid parasitoid wasp N. vitripennis (Nv-tra2) and confirm its function in N. vitripennis sex determination. Knock down of Nv-tra2 by parental RNA interference (pRNAi) results in complete sex reversal of diploid offspring from female to male, indicating the requirement of Nv-tra2 for female sex determination. As Nv-tra2 pRNAi leads to frequent lethality in early developmental stages, maternal provision of Nv-tra2 transcripts is apparently also required for another, non-sex determining function during embryogenesis. In addition, lethality following Nv-tra2 pRNAi appears more pronounced in diploid than in haploid offspring. This diploid lethal effect was also observed following Nv-tra pRNAi, which served as a positive control in our experiments. As diploid embryos from fertilized eggs have a paternal chromosome set in addition to the maternal one, this suggests that either the presence of this paternal chromosome set or the dosage effect resulting from the diploid state is incompatible with the induced male development in N. vitripennis caused by either Nv-tra2 or Nv-tra pRNAi. The role of Nv-tra2 in activating the female sex determination pathway yields more insight into the sex determination mechanism of Nasonia. (C) 2017 The Authors. Published by Elsevier Ltd.

Bumblebee species with declining population trends tend to show lower genetic diversity levels than stable species. The observed difference might be explained by abundance differences, with declining bumblebee species having lower genetic diversity levels simply due to their lower local species abundances. However, whether this holds true is not known. Here, we investigated whether bumblebee local abundances determines population genetic diversity levels. Therefore, local species abundances were measured for bumblebee species at four locations in Belgium and two locations in Estonia during bumblebee foraging years 2013–2017. These locations and countries were chosen to ensure the greatest possible variance in both local abundances and population trends for these species. Hence, genetic diversity levels were obtained for seven species by genotyping collected specimens with 16 microsatellites. Our results showed that the observed patterns in genetic diversity did not depend on local species abundance. So, although declining bumblebee species can become locally abundant, they will still show lower genetic diversity levels than stable species. This result implies that measuring bumblebees’ local abundance cannot be used to directly determine the health status of a population. Furthermore, this result has also major impact on future conservation strategies as increasing the genetic diversity levels of declining species will be very difficult, and habitat quality should be high to maintain their populations, otherwise these species are doomed to disappear first.

In hymenopterans, males are normally haploid (1n) and females diploid (2n), but individuals with divergent ploidy levels are frequently found. In species with ‘complementary sex determination’ (CSD), increasing numbers of diploid males that are often infertile or unviable arise from inbreeding, presenting a major impediment to biocontrol breeding. Non‐CSD species, which are common in some parasitoid wasp taxa, do not produce polyploids through inbreeding. Nevertheless, polyploidy also occurs in non‐CSD Hymenoptera. As a first survey on the impacts of inbreeding and polyploidy of non‐CSD species, we investigate life‐history traits of a long‐term laboratory line of the parasitoid Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) (‘Whiting polyploid line’) in which polyploids of both sexes (diploid males, triploid females) are viable and fertile. Diploid males produce diploid sperm and virgin triploid females produce haploid and diploid eggs. We found that diploid males did not differ from haploid males with respect to body size, progeny size, mate competition, or lifespan. When diploid males were mated to many females (without accounting for mating order), the females produced a relatively high proportion of male offspring, possibly indicating that these males produce less sperm and/or have reduced sperm functionality. In triploid females, parasitization rate and fecundity were reduced and body size was slightly increased, but there was no effect on lifespan. After one generation of outbreeding, lifespan as well as parasitization rate were increased, and a body size difference was no longer apparent. This suggests that outbreeding has an effect on traits observed in an inbred polyploidy background. Overall, these results indicate some phenotypic detriments of non‐CSD polyploids that must be taken into account in breeding.

Inbreeding and inbreeding depression are processes in small populations of particular interest for a range of human activities such as animal breeding, species conservation or pest management. In particular, biological control programs should benefit from a thorough understanding of the causes and consequences of inbreeding because natural enemies experience repetitive bottlenecks during importation, laboratory rearing, and introduction. Predicting the effect of inbreeding in Hymenopteran parasitoid wasps, frequently used in biological control programs, is nonetheless a difficult endeavor. In haplodiploid parasitoids, the purge of deleterious alleles via haploid males should reduce genetic load, but if these species also have complementary sex determination (CSD) abnormal diploid males will be produced, which may jeopardize the success of biological control introductions. Mastrus ridens is such a parasitoid wasp with CSD, introduced to control the codling moth, Cydia pomonella (L.). We studied its life history traits in the laboratory under two conditions: inbred (full sib) and outbred (non-sib) crosses, across five generations, in order to examine the consequences of inbreeding in this species. We found that in inbred lines non reproducing females live less, the number of daughters produced was lower, and that sex ratio (proportion of males) and proportion of diploid males were higher. Diploid males were able to produce fertile daughters, but fewer than haploid males. Lineage survival was similar for inbred and outbred lines across the five generations. The most significant decrease in fitness was thus a consequence of the production of diploid males, but this effect was not as extreme as in most other species with CSD, due to the fertility of diploid males. This study highlights the importance of determining the type of sex determination in parasitoid wasps used for biological control, and the importance of maintaining genetic diversity in species with CSD when importation or augmentation is the goal. This article is protected by copyright. All rights reserved.

Sex determination is ruled by haplodiploidy in Hymenoptera, with haploid males arising from unfertilized eggs and diploid females from fertilized eggs. However, diploid males with null fitness are produced under Complementary Sex Determination (CSD), whenindividuals are homozygous for this locus. Diploid males are expected to be more frequent in genetically eroded populations (such as islands and captive populations), as genetic diversity at the csd locus should be low. However, only a few studies have focused on the relation between population size, genetic diversity and the proportion of diploid males in the field. Here, we developed new microsatellites markers in order to assess and compare genetic diversity and diploid male proportion in populations from three distinct habitat types (mainland, island or captive), in the parasitoid waspVenturia canescens. Eroded genetic diversity and higher diploid male proportion were found in island and captive populations, and habitat type had large effect on genetic diversity. Therefore, diploid male proportion reflects the decreasing genetic diversity in small and isolated populations. Thus, Hymenopteran populations can be at high extinction risk due to habitat destruction or fragmentation.

International audience; The parasitoid wasp Mastrus ridens (Hymenoptera: Ichneumonidae) is a particularly well-suited biological model to document the history and evolution of populations used in classical biological control, repeatedly moved from laboratory to laboratory worldwide and introduced in various environments. This specialist ectoparasitoid of the codling moth was first imported from Kazakhstan to the USA in the 1990's, and then sent to Argentina, Chile and New Zealand. More recently, it was sent to Australia and France from other laboratory colonies, and imported again from field collections in Kazakhstan to Chile. Here, we used DNA sequencing to confirm the taxonomic identity of several populations used for biological control worldwide, and developed microsatellite markers for population genetics studies. A multiplex PCR amplifying 11 polymorphic markers was designed. These markers were used to compare the genetic diversity of laboratory and field populations and evaluate genetic differentiation between them. Results showed that laboratory populations with the longest rearing history had lower genetic diversity. Moreover, the geno-typing of males with the markers revealed the occurrence of diploid males, which was further confirmed by flow cytometry, suggesting complementary sex determination (CSD) in this species. The percentage of diploid males in the populations ranged from 4% to 25% and were negatively correlated with diversity indices, which is consistent with a single-locus CSD and genetic bottlenecks in laboratory rearings. Molecular tools proved to be suitable and reliable for genetic diversity analyses in M. ridens, and should be implemented more frequently in classical biological control programs.

Natural enemies may go through genetic bottlenecks during the process of biological control introductions. Such bottlenecks are expected to be particularly detrimental in parasitoid Hymenoptera that exhibit complementary sex determination (CSD). CSD is associated with a severe form of inbreeding depression because homozygosity at one or multiple sex loci leads to the production of diploid males that are typically unviable or sterile. We observed that diploid males occur at a relatively high rate (813% of diploid adults) in a field population of Cotesia rubecula in Minnesota, USA, where this parasitoid was introduced for biological control of the cabbage white Pieris rapae. However, our laboratory crosses suggest two-locus CSD in a native Dutch population of C. rubecula and moderately high diploid males survival (approximately 70%), a scenario expected to produce low proportions of diploid males. We also show that courtship behavior of diploid males is similar to that of haploid males, but females mated to diploid males produce only very few daughters that are triploid. We use our laboratory data to estimate sex allele diversity in the field population of C. rubecula and discuss the possibility of a sex determination meltdown from two-locus CSD to effective single-locus CSD during or after introduction.

Background Haplodiploidy, where females develop from diploid, fertilized eggs and males from haploid, unfertilized eggs, is abundant in some insect lineages. Some species in these lineages reproduce by thelytoky that is caused by infection with endosymbionts: infected females lay haploid eggs that undergo diploidization and develop into females, while males are very rare or absent. It is generally assumed that in thelytokous wasps, endosymbionts merely diploidize the unfertilized eggs, which would then trigger female development. Results We found that females in the parasitoid wasp Asobara japonica infected with thelytoky-inducing Wolbachia produce 0.7–1.2 % male offspring. Seven to 39 % of these males are diploid, indicating that diploidization and female development can be uncoupled in A. japonica. Wolbachia titer in adults was correlated with their ploidy and sex: diploids carried much higher Wolbachia titers than haploids, and diploid females carried more Wolbachia than diploid males. Data from introgression lines indicated that the development of diploid individuals into males instead of females is not caused by malfunction-mutations in the host genome but that diploid males are most likely produced when the endosymbiont fails to activate the female sex determination pathway. Our data therefore support a two-step mechanism by which endosymbionts induce thelytoky in A. japonica: diploidization of the unfertilized egg is followed by feminization, whereby each step correlates with a threshold of endosymbiont titer during wasp development. Conclusions Our new model of endosymbiont-induced thelytoky overthrows the view that certain sex determination mechanisms constrain the evolution of endosymbiont-induced thelytoky in hymenopteran insects. Endosymbionts can cause parthenogenesis through feminization, even in groups in which endosymbiont-diploidized eggs would develop into males following the hosts’ sex determination mechanism. In addition, our model broadens our understanding of the mechanisms by which endosymbionts induce thelytoky to enhance their transmission to the next generation. Importantly, it also provides a novel window to study the yet-poorly known haplodiploid sex determination mechanisms in haplodiploid insects. Electronic supplementary material The online version of this article (doi:10.1186/s12862-015-0370-9) contains supplementary material, which is available to authorized users.

International audience; AbstractPresence of diploid males in wild bees reflects inbreeding and provides information about the health status of a colony or population. Detection of diploid males, and discrimination from haploid males and workers, has, however, been limited to molecular diagnostics. Here we present a novel method based on differences in wing shape, e.g., venation patterns in wings. The method is easy to apply and results, for Bombus terrestris, in very high discrimination success. Possible applications of the method are discussed.

Background Single-locus complementary sex determination (sl-CSD), which occurs in some insects of the order Hymenoptera, imposes a heavy genetic load that can drive small populations to extinction. The core process in these species is the development of individuals homozygous at the sex-determining locus into unfit diploid males. The risk of extinction of populations with sl-CSD is theoretically much higher if diploid males are viable and capable of mating but sterile, because diploid males then decrease the reproductive output of both their parents and the females with which they mate. Results In the parasitoid wasp Venturia canescens (Hymenoptera: Ichneumonidae), diploid males resembled their haploid counterparts in most respects, but their mating success was nevertheless lower than that of haploid males, especially when the two types of males were placed in competition. Furthermore, although diploid males transferred viable sperm during copulation, they sired no daughters: the females with which they mated produced only sons, like virgin females. A simulation model combining behavior, genetics and demography demonstrated that for two alternative hypotheses concerning the fertilization success of diploid sperm, the mating success of diploid males strongly affected population dynamics. Conclusion The performance of diploid males should be estimated in competitive situations. It is a crucial determinant of the probability of extinction. Electronic supplementary material The online version of this article (doi:10.1186/s12898-014-0032-6) contains supplementary material, which is available to authorized users.

Background Sex determination mechanisms are known to be evolutionarily labile but the factors driving transitions in sex determination mechanisms are poorly understood. All insects of the Hymenoptera are haplodiploid, with males normally developing from unfertilized haploid eggs. Under complementary sex determination (CSD), diploid males can be produced from fertilized eggs that are homozygous at the sex locus. Diploid males have near-zero fitness and thus represent a genetic load, which is especially severe under inbreeding. Here, we study mating structure and sex determination in the parasitoid Cotesia vestalis to investigate what may have driven the evolution of two complementary sex determination loci in this species. Results We genotyped Cotesia vestalis females collected from eight fields in four townships in Western Taiwan. 98 SNP markers were developed by aligning Illumina sequence reads of pooled DNA of eight different females against a de novo assembled genome of C. vestalis. This proved to be an efficient method for this non-model species and provides a resource for future use in related species. We found significant genetic differentiation within the sampled population but variation could not be attributed to sampling locations by AMOVA. Non-random mating was detected, with 8.1% of matings between siblings. Diploid males, detected by flow cytometry, were produced at a rate of 1.4% among diploids. Conclusions We think that the low rate of diploid male production is best explained by a CSD system with two independent sex loci, supporting laboratory findings on the same species. Fitness costs of diploid males in C. vestalis are high because diploid males can mate with females and produce infertile triploid offspring. This severe fitness cost of diploid males combined with non-random mating may have resulted in evolution from single locus CSD to CSD with two independent loci. Electronic supplementary material The online version of this article (doi:10.1186/s12862-015-0340-2) contains supplementary material, which is available to authorized users.

Diploid males are expected to occur in populations of some Hymenoptera because of the single-locus complementary sex determination (sl-CSD) mechanism. If sl-CSD is found in a species that produces gregarious broods and sib mates at a high frequency, a fraction of these sib-matings (matched mating) produces diploid male offspring. We developed models to predict the matched mating frequency in populations with different frequencies of sib mating and egg fertilization. The predictions of these models are used to determine if we can use the distributions of brood sex ratio and brood size to determine if a species has sl-CSD. The models show that sl-CSD can be detected from these brood sex ratios if the diploid male offspring survives. We applied our models to Cotesia sesamiae and its exotic congener Cotesia flavipes (Hymenoptera: Braconidae), to determine if sl-CSD occurs in these species. Parasitoids were reared from stemborer larvae sampled from maize fields in Kenya between 1992 and 1999. We found no evidence for the presence of sl-CSD with survival of diploid males in both braconid species, but we cannot exclude the possibility that sl-CSD with diploid male mortality takes place in these species.

A study of the viability of small populations of Hymenoptera is a matter of importance to gain a better zoological, ethological, genetical and ecological knowledge of these insects, and for conservation purposes, mainly because of the consequences to the survival of colonies of many species of bees, wasps, and ants. Based on the Whiting (1943) principle, Kerr & Vencovski (1982) presented a hypothesis that states that viable populations of stingless bees (Meliponini) should have at least 40 colonies to survive. This number was later extended to 44 colonies by Kerr (1985). This would be necessary to avoid any substantial amount of homozygosis in the pair of chromosomic sexual loci, by keeping at least six different sexual gene alleles in a reproductive population. In most cases this would prevent the production of useless diploid males. However, several facts weigh against considering this as a general rule. From 1990 to 2001, 287 colony divisions were made, starting with 28 foundation colonies, in the inbreeding and population experiments with the Meliponini reported here. These experiments constitute the most extensive and longest scientific research ever made with Meliponini bees. In ten different experiments presented here, seven species (one with two subspecies) of Meliponini bees were inbred in five localities inside their wide-reaching native habitats, and in two localities far away from these habitats. This was done for several years. On the whole, the number of colonies increased and the loss of colonies over the years was small. In two of these experiments, although these populations were far (1,000 km and 1,200 km) from their native habitat, their foundation colonies were multiplied successfuly. It was possible to build up seven strong and three expanding medium populations, starting with one, two, three or even five colonies. However, in six other cases examined here, the Whiting (1943) principle and the hypothesis of Kerr & Vencovski (1982) and Kerr (1985), possibly hold up. In two other cases, the results are still unclear. Outside native habitats, most inbreeding experiments failed, possibly because of conditions that cause ecological stress. Although much more data are still needed, a new working hypothesis on the molecular level was presented to explain the results of the experiments described here. In the absence of any considerable stress, and in the eventuality of a good nutritive situation, even individual bees that are homozygous in the pair of chromosomic sexual locus would produce a sufficient amount of a sex determining substance. Therefore, the female genes of all the diploid individuals of a colony, both homozygous and heterozygous, would be activated. However, situations of considerable stress would cause a poor physiological and nutritive condition. This, together with homozygosis in the pair of chromosomic sexual locus, would lead to a smaller production of the sex determining substance. When this happens in the diploid homozygous individuals of a colony, in relation to sex, only male genes are activated. As a result, all such homozygous diploid bees of the colony become useless males. However, when there is a heterozygous situation in the chromosomic sexual locus of all bees of a colony, all diploid individuals would produce a high amount of the sex determining substance. Consequently, all diploid individuals of such a colony would become females (queens and workers). Stresses, including ecological stress, as well as the nutritive condition and the genetic situation in the chromosomic sexual loci, will have a key influence in the life and behavior of the Meliponini, including sex determination. In relation to genetic factors, hybrid vigour may often cause a greater production of biological substances. This may be due to the presence of a greater number of copies of allelic genes when there is heterozygosis. This is a hypothesis requiring further research. However, in the experiments presented here, this hypothesis seems to apply well to the production of a sex determining substance in bees (Apoidea) and other Hymenoptera.

Some Hymenoptera have single locus Complementary Sex Determination (sl-CSD); heterozygous individuals are female (diploid fertilised eggs) and hemizygous individuals are male (haploid unfertilised eggs). Through inbreeding homozygous diploids can arise which develop into males that are often sterile and sometimes inviable. The phylogenetic distribution of sl-CSD is unclear. In this study, we used inbred crosses to demonstrate that the parasitoid wasp Venturia canescens (Gravenhorst) has sl-CSD. Diploid males were detected through heterozygosity at a marker locus (Virus Like Particle protein). They were fully viable at 20°, 25° and 30°C as the sex ratios of inbred crosses did not deviate from controls after adjustment for diploid male frequency. These results further confirm the existence of sl-CSD in the family Ichneumonidae.

Under the hymenopteran single-locus complementary sex-determination system, production of diploid males results from homozygosity at the sex-determiner locus. This arises when both parents transmit identical alleles at the locus to the offspring. In species reproducing asexually through thelytokous parthenogenesis, production of diploid males may also occur when the sex locus undergoes recombination and becomes homozygous in the offspring. Diploid males represent a substantial genetic load in hymenopteran populations because they often produce unviable sperm or sire sterile triploid female offspring. In the Mediterranean ant Cataglyphis cursor, the queen and workers can produce female offspring through automictic thelytokous parthenogenesis with central fusion, a mode of parthenogenesis that increases homozygosity. We report, for the first time, the presence of about 39 % of colonies producing adult diploid males (seven colonies out of 18). Overall, 8 % of adult males were diploid (12 diploid males out of the 146 males genotyped). Genotyping workers from the seven colonies producing diploid males showed that three diploid males were sons of queens and produced by thelytoky, six were probably sons of workers also produced by thelytoky and three were non-natal. Furthermore, the mating of a diploid male with two virgin queens in the laboratory led to the production of sterile triploid workers, which shows that diploid males in C. cursor are fertile, mate successfully and produce viable and functional but probably sterile female offspring. Because diploid males originate from thelytokous reproduction, they are only produced during sexual production and hence do not impair colony growth, which could explain why they are not removed at early brood stages. © 2013 Springer-Verlag Berlin Heidelberg., SCOPUS: ar.j, info:eu-repo/semantics/published

We describe and characterize eight polymorphic microsatellite loci for the orchid bee species Eulaema meriana, an abundant species and important pollinator in wet lowland forests in tropical America. We also tested the cross-species amplification of these microsatellite loci in seven other species of the genus Eulaema. For E. meriana, number of alleles per locus ranged from four to nine and expected heterozygosity ranged from 0.377 to 0.854. Seven out of the eight loci described amplified in all seven other Eulaema species. These microsatellite loci will be of practical use for population structure, mating system and inbreeding studies in euglossine bees.

Cardinium is a bacterial symbiont infecting many species of arthropods, and is associated with manipulation of host reproduction. Cardinium is the causal agent of asexual reproduction, or thelytoky, in the chalcidoid parasitoid wasp Encarsia hispida. Feeding antibiotics to the infected adult females results in uninfected male offspring. Here, we show that these males are diploid. Diploid males are extremely unusual in the large hymenopteran superfamily Chalcidoidea, and, to our knowledge, have never before been associated with symbiont infection in this group. These findings indicate that at least in E. hispida, diploidy restoration is a necessary condition but not sufficient to elicit female development. Cardinium is required to feminize diploid male embryos and thus must interact with elements of the host sex determination system. In addition, our data suggest that Cardinium is necessary for the fertility of E. hispida; antibiotic curing of Cardinium reduces offspring production of adult females.

In temperate climates, female paper wasps typically initiate new colonies in the spring. Several nest-founding tactics have been documented in Polistes species, including solitary nest initiation, joining a cooperative association, usurping an existing nest, or adopting an abandoned nest. Occasionally, exceptionally large groups of females have also been found reusing nests from the previous season. Here we report this phenomenon in introduced populations of the Eurasian species Polistes dominulus. We describe in detail the demographic and genetic characteristics of one such spring colony from Los Angeles, California, USA, which was collected with 84 associated adults and all stages of developing brood in its 613 cells. Genetic and morphological data indicate the presence of multiple reproductively active females of varying relatedness, as well as many nonbreeding females, including probable early-produced offspring. Despite some evidence of chaotic social conditions, the colony appeared to have been highly productive. Additional observations of similar colonies are needed to determine how control is maintained within such a large breeding aggregation.

Despite its fundamental role in development, sex determination is highly diverse among animals. Approximately 20% of all animals are haplodiploid, with haploid males and diploid females. Haplodiploid species exhibit diverse but poorly understood mechanisms of sex determination. Some hymenopteran insect species exhibit single-locus complementary sex determination (sl-CSD), where heterozygosity at a polymorphic sex locus initiates female development. Diploid males are homozygous at the sex locus and represent a genetic load because they are inviable or sterile. Inbreeding depression associated with CSD is therefore expected to select for other modes of sex determination resulting in fewer or no diploid males. Here, we investigate an alternative, heretofore hypothetical, mode of sex determination: multiple-locus CSD (ml-CSD). Under ml-CSD, diploid males are predicted to develop only from zygotes that are homozygous at all sex loci. We show that inbreeding for eight generations in the parasitoid wasp Cotesia vestalis leads to increasing proportions of diploid males, a pattern that is consistent with ml-CSD but not sl-CSD. The proportion of diploid males (0.27 ± 0.036) produced in the first generation of inbreeding (mother–son cross) suggests that two loci are likely involved. We also modeled diploid male production under CSD with three linked loci. Our data visually resemble CSD with linked loci because diploid male production in the second generation was lower than that in the first. To our knowledge, our data provide the first experimental support for ml-CSD.

The dominant and ancestral mode of sex determination in the Hymenoptera is arrhenotokous parthenogenesis, in which diploid females develop from fertilized eggs and haploid males develop from unfertilized eggs. We discuss recent progress in the understanding of the genetic and cytoplasmic mechanisms that make arrhenotoky possible. The best-understood mode of sex determination in the Hymenoptera is complementary sex determination (CSD), in which diploid males are produced under conditions of inbreeding. The gene mediating CSD has recently been cloned in the honey bee and has been named the complementary sex determiner. However, CSD is only known from 4 of 21 hymenopteran superfamilies, with some taxa showing clear evidence of the absence of CSD. Sex determination in the model hymenopteran Nasonia vitripennis does not involve CSD, but it is consistent with a form of genomic imprinting in which activation of the female developmental pathway requires paternally derived genes. Some other hymenopterans are not arrhenotokous but instead exhibit thelytoky or paternal genome elimination.

Sex determination in the order Hymenoptera is based on haplodiploid arrhenotoky; in which males develop from unfertilized eggs and are haploid, whereas females develop from fertilized eggs and are diploid. However, some hymenopteran species produce diploid males through a mechanism known as single-locus complementary sex determination (sl-CSD). In these species, heterozygous individuals at a single sex locus develop into females, whereas hemizygotes (haploids) and homozygotes (diploids) develop into males. Inbreeding leads to homozygosity and consequent production of diploid males. We investigated the presence of sl-CSD in the braconid Cotesia flavipes using a series of inbreeding crosses among five isofemale lines. Sex ratio (proportion of females) did not differ among the within-line crosses, between-line crosses and crosses carried out between isofemale lines and a mixed (outbred) colony. Brood size of within-line and between-line crosses did not differ. Culturing populations for 25 generations did not result in changes to more male-biased sex ratios. We conclude that CSD does not exist in C. flavipes. The implications of absence of CSD in C. flavipes are discussed in context of mass rearing for classical biological control programmes.

An attractive way to improve our understanding of sex determination evolution is to study the underlying mechanisms in closely related species and in a phylogenetic perspective. Hymenopterans are well suited owing to the diverse sex determination mechanisms, including different types of Complementary Sex Determination (CSD) and maternal control sex determination. We investigated different types of CSD in four species within the braconid wasp genus Asobara that exhibit diverse life-history traits. Nine to thirteen generations of inbreeding were monitored for diploid male production, brood size, offspring sex ratio, and pupal mortality as indicators for CSD. In addition, simulation models were developed to compare these observations to predicted patterns for multilocus CSD with up to ten loci. The inbreeding regime did not result in diploid male production, decreased brood sizes, substantially increased offspring sex ratios nor in increased pupal mortality. The simulations further allowed us to reject CSD with up to ten loci, which is a strong refutation of the multilocus CSD model. We discuss how the absence of CSD can be reconciled with the variation in life-history traits among Asobara species, and the ramifications for the phylogenetic distribution of sex determination mechanisms in the Hymenoptera.