Complementary Sex Determination Research Articles

Genetic diversity of Apis melliferacomplementary sex determiner (csd) in four Australian breeding populations

Honey bee (Apis mellifera) breeding has intensified as populations experience increasing stress and pollination demand increases. Breeding programmes risk genetic diversity losses as mating is focused on a small group of individuals. Loss of diversity at the complementary sex determiner (csd) locus results in decreased viability and reduced honey production. Bees that are homozygous at csd become inviable males rather than workers. We examined csd diversity in four Australian breeding populations: a queen bee breeder from New South Wales, another from Queensland, a Western Australian breeding programme involving 11 bee breeders, and a research population at the NSW Department of Primary Industries. We found 82 unique csd alleles across the four populations, 16 of which have not been previously reported. This study provides a snapshot of csd diversity in Australia which will be useful for the national honey bee genetic improvement programme (Plan Bee).

LncRNA gene ANTSR coordinates complementary sex determination in the Argentine ant.

Animals have evolved various sex determination systems. Here, we describe a newly found mechanism. A long noncoding RNA (lncRNA) transduces complementary sex determination (CSD) signal in the invasive Argentine ant. In this haplodiploid species, we identified a 5-kilobase hyper-polymorphic region underlying CSD: Heterozygous embryos become females, while homozygous and hemizygous embryos become males. Heterozygosity at the CSD locus correlates with higher expression of ANTSR, a gene that overlaps with the CSD locus and specifies an lncRNA transcript. ANTSR knockdown in CSD heterozygotes leads to male development. Comparative analyses indicated that, in Hymenoptera, ANTSR is an ancient yet rapidly evolving gene. This study reveals an lncRNA involved in genetic sex determination, alongside a previously unknown regulatory mechanism underlying sex determination based on complementarity among noncoding alleles.

Variability and Number of Circulating Complementary Sex Determiner (Csd) Alleles in a Breeding Population of Italian Honeybees under Controlled Mating.

In Apis mellifera, csd is the primary gene involved in sex determination: haploid hemizygous eggs develop as drones, while females develop from eggs heterozygous for the csd gene. If diploid eggs are homozygous for the csd gene, diploid drones will develop, but will be eaten by worker bees before they are born. Therefore, high csd allelic diversity is a priority for colony survival and breeding. This study aims to investigate the variability of the hypervariable region (HVR) of the csd gene in bees sampled in an apiary under a selection scheme. To this end, an existing dataset of 100 whole-genome sequences was analyzed with a validated pipeline based on de novo assembly of sequences within the HVR region. In total, 102 allelic sequences were reconstructed and translated into amino acid sequences. Among these, 47 different alleles were identified, 44 of which had previously been observed, while 3 are novel alleles. The results show a high variability in the csd region in this breeding population of honeybees.

Genetic Profile of Greek Indigenous Honey Bee Populations, Local Extinctions, Geographical Distinction, and Patterns, by Using mtDNA COI and Nuclear CSD Gene Analysis Markers

Evaluation and conservation of local genetic resources of the domestic honey bee populations is important, especially in regions with high diversity levels as well as high honey bee colony density. Greece is rich in honey bee biodiversity, hosting several subspecies, with the status of them being, though, doubtful. The purpose of the present study was to investigate the genetic relationships of both stationary and movable honey bee populations, originating from many location throughout Greece. Two molecular markers were utilized, namely, the conserved mitochondrial COI gene and the highly variable complementary sex determination (CSD) gene. Samples were collected from nine distant populations: eight populations from colonies that followed the traditional stationary beekeeping model and one following the modern migratory beekeeping model type, where the hives are transferred from place to place according to the season. Regardless the beekeeping model, all populations were characterized by sufficient genetic diversity indicating no signs of inbreeding or any bottleneck effects. Nevertheless, genetic differentiation and phylogenetic analysis in comparison with haplotypes obtained from GenBank revealed a genetic admixture pattern suggesting that movement causes genetic homogeneity, occasionally in the stable reared populations as well. Interestingly, two populations, namely, Kastoria and Protokklisi, belonging to A. m. macedonica population, were significantly differentiated, supporting the maintenance of their genetic integrity. Unfortunately, on the other hand, genetic structure of the populations from Crete (Sasalos population) provided evidence that the indigenous breed from the island, A. m. adami, has probably gone extinct. Future management strategies should focus on the conservation of the local genetic resources in which distinct genetic identity has been sustained.

Serial founder effects slow range expansion in an invasive social insect

Invasive populations often experience founder effects: a loss of genetic diversity relative to the source population, due to a small number of founders. Even where these founder effects do not impact colonization success, theory predicts they might affect the rate at which invasive populations expand. This is because secondary founder effects are generated at advancing population edges, further reducing local genetic diversity and elevating genetic load. We show that in an expanding invasive population of the Asian honey bee (Apis cerana), genetic diversity is indeed lowest at range edges, including at the complementary sex determiner, csd, a locus that is homozygous-lethal. Consistent with lower local csd diversity, range edge colonies had lower brood viability than colonies in the range centre. Further, simulations of a newly-founded and expanding honey bee population corroborate the spatial patterns in mean colony fitness observed in our empirical data and show that such genetic load at range edges will slow the rate of population expansion.

The Potential of Complementary Sex-Determiner Gene Allelic Diversity for Studying the Number of Patrilines within Honeybee Colonies

Polyandry, a fundamental aspect of honeybee biology, influences genetic diversity within bee colonies. The Csd gene, responsible for sex determination, exhibits a high intraspecific polymorphism, making it a promising candidate for studying patrilines. This study investigates the potential of the complementary sex-determiner gene (Csd) as a marker for genetic studies of honeybee colonies, comparing its efficacy with standard microsatellite markers. A worker bee brood from five colonies was genotyped using both Csd and microsatellite markers. The results showed that Csd alleles offer higher variability than microsatellite markers, providing a superior resolution in genotyping. The number of distinct Csd alleles in each colony surpassed microsatellite markers, demonstrating the potential of Csd for detailed genetic analyses. Despite challenges in Csd amplification efficiency, a two-step nested PCR protocol proved effective. Interestingly, Csd genotyping alone identified more patrilines than the set of five microsatellite markers, emphasizing its efficacy. Combining Csd and microsatellite genotyping enhances the resolution of genetic studies in honeybee colonies, offering valuable insights into genetic diversity, reproductive success, and social dynamics. The Csd gene emerges as a promising tool for advancing genetic studies in honeybee populations.

Recognition of polymorphic Csd proteins determines sex in the honeybee.

Sex in honeybees, Apis mellifera, is genetically determined by heterozygous versus homo/hemizygous genotypes involving numerous alleles at the single complementary sex determination locus. The molecular mechanism of sex determination is however unknown because there are more than 4950 known possible allele combinations, but only two sexes in the species. We show how protein variants expressed from complementary sex determiner (csd) gene determine sex. In females, the amino acid differences between Csd variants at the potential-specifying domain (PSD) direct the selection of a conserved coiled-coil domain for binding and protein complexation. This recognition mechanism activates Csd proteins and, thus, the female pathway. In males, the absence of polymorphisms establishes other binding elements at PSD for binding and complexation of identical Csd proteins. This second recognition mechanism inactivates Csd proteins and commits male development via default pathway. Our results demonstrate that the recognition of different versus identical variants of a single protein is a mechanism to determine sex.

Effects of Polyploidization on Locomotor and Flight Activity, and Body Size of Males in Japanese Bumblebee, Bombus ignitus

Polyploid animals are seen in the wild in only a few groups such as planaria, weevils, ants, wasps, and bees. The sex of bumblebees is determined by the single-locus complementary sex determination (sl-CSD) system resulting in females emerging from fertilized eggs and males (normal males, haploid) emerging from unfertilized eggs. However, diploid males (polyploids) emerged from fertilized eggs when the sex-determining locus become homozygous caused by inbreeding due to population contraction. To examine the effects of polyploidization on behavioral characteristics in bumblebee males, Bombus ignitus, haploid and diploid males were compared in locomotor activity, ease of staying inside the hive, and flight activity in a laboratory experiment. The results showed that diploid males had higher locomotor activity levels, were less likely to stay inside the hive, and had lower flight activity levels than haploid males. The reduced flight activity levels may lead to lower mating rates in the field. Furthermore, the body size of diploid males was smaller than that of haploid males. Diploid males of bumblebees have a low reproductive and produce diploid sperms to emerge sterile triploids, thus creating a considerable genetic load on the population. The behavioral and morphological characteristics of diploid males may unintentionally serve as a mechanism to prevent the emergence of sterile triploids.

Half-chromatid mutation as a possible cause of mosaic males and females in Hymenoptera and rare fertile male tortoiseshell cats.

Half-chromatid mutations occur when a single base change in a gamete is transmitted to the zygote, which, after DNA replication and cleavage, will result in a mosaic individual. These mutations will be passed on through the germ plasm and also may be expressed somatically. Half-chromatid mutation has been suggested to account for the observed lower frequency of males than expected for lethal X-linked recessive disorders in humans, such as Lesch-Nyhan syndrome, incontinentia pigmenti, and Duchene muscular dystrophy. Although attention has been paid to half-chromatid mutation in humans, it otherwise has been ignored. Here I show that half-chromatid mutation in haplodiploid organisms, such as Hymenoptera, has some interesting and important consequences: (i) since all genes follow the X-linked pattern of inheritance, half-chromatid mutations should be relatively easier to detect; (ii) recessive mutations of all viabilities may be expected; (iii) mosaics of both sexes are expected in haplodiploids with half-chromatid mutation; (iv) gynandromorphs could result from half-chromatid mutation at the sex-determination locus, in species with single-locus complementary sex-determination. Finally, half-chromatid mutation can account for the rare fertile male tortoiseshell phenotype observed in the domestic cat, Felis catus, and which still has not been fully accounted for by other mechanisms.

SIMplyBee: an R package to simulate honeybee populations and breeding programs

BackgroundThe Western honeybee is an economically important species globally, but has been experiencing colony losses that lead to economical damage and decreased genetic variability. This situation is spurring additional interest in honeybee breeding and conservation programs. Stochastic simulators are essential tools for rapid and low-cost testing of breeding programs and methods, yet no existing simulator allows for a detailed simulation of honeybee populations. Here we describe SIMplyBee, a holistic simulator of honeybee populations and breeding programs. SIMplyBee is an R package and hence freely available for installation from CRAN http://cran.r-project.org/package=SIMplyBee.ImplementationSIMplyBee builds upon the stochastic simulator AlphaSimR that simulates individuals with their corresponding genomes and quantitative genetic values. To enable honeybee-specific simulations, we extended AlphaSimR by developing classes for global simulation parameters, SimParamBee, for a honeybee colony, Colony, and multiple colonies, MultiColony. We also developed functions to address major honeybee specificities: honeybee genome, haplodiploid inheritance, social organisation, complementary sex determination, polyandry, colony events, and quantitative genetics at the individual- and colony-levels.ResultsWe describe its implementation for simulating a honeybee genome, creating a honeybee colony and its members, addressing haplodiploid inheritance and complementary sex determination, simulating colony events, creating and managing multiple colonies at the same time, and obtaining genomic data and honeybee quantitative genetics. Further documentation, available at http://www.SIMplyBee.info, provides details on these operations and describes additional operations related to genomics, quantitative genetics, and other functionalities.DiscussionSIMplyBee is a holistic simulator of honeybee populations and breeding programs. It simulates individual honeybees with their genomes, colonies with colony events, and individual- and colony-level genetic and breeding values. Regarding the latter, SIMplyBee takes a user-defined function to combine individual- into colony-level values and hence allows for modeling any type of interaction within a colony. SIMplyBee provides a research platform for testing breeding and conservation strategies and their effect on future genetic gain and genetic variability. Future developments of SIMplyBee will focus on improving the simulation of honeybee genomes, optimizing the simulator’s performance, and including spatial awareness in mating functions and phenotype simulation. We invite the honeybee genetics and breeding community to join us in the future development of SIMplyBee.

Invasion potential of hornets (Hymenoptera: Vespidae: Vespa spp.).

Hornets are large, predatory wasps that have the potential to alter biotic communities and harm honey bee colonies once established in non-native locations. Mated, diapausing females (gynes) can easily be transported to new habitats, where their behavioral flexibility allows them to found colonies using local food and nest materials. Of the 22 species in the genus Vespa, five species are now naturalized far from their endemic populations and another four have been detected either in nature or during inspections at borders of other countries. By far the most likely pathway of long-distance dispersal is the transport of gynes in transoceanic shipments of goods. Thereafter, natural dispersal of gynes in spring and accidental local transport by humans cause shorter-range expansions and contribute to the invasion process. Propagule pressure of hornets is unquantified, although it is likely low but unrelenting. The success of introduced populations is limited by low propagule size and the consequences of genetic founder effects, including the extinction vortex linked to single-locus, complementary sex determination of most hymenopterans. Invasion success is enhanced by climatic similarity between source locality and introduction site, as well as genetic diversity conferred by polyandry in some species. These and other factors that may have influenced the successful establishment of invasive populations of V. velutina, V. tropica, V. bicolor, V. orientalis, and V. crabro are discussed. The highly publicized detections of V. mandarinia in North America and research into its status provide a real-time example of an unfolding hornet invasion.

Transformer gene regulates feminization under two complementary sex determination loci in the ant, Vollenhovia emeryi

Organisms that reproduce sexually have evolved well-organized mechanisms to determine two sexes. Some hymenopterans (such as ants, bees, and wasps) have a complementary sex-determination system in which heterozygosity at one CSD locus induces female development, whereas hemi- or homozygosity at the locus induces male development. This system can generate a high cost of inbreeding, as individuals that are homozygous at the locus become sterile, diploid males. On the other hand, some hymenopterans have evolved a multi-locus, complementary, sex-determination system in which heterozygosity in at least one CSD locus induces female development. This system effectively reduces the proportion of sterile diploid males; however, how these multiple primary signals based on CSD pass through a molecular cascade to regulate downstream genes has remained unclear. To clarify this matter, we used a backcross to investigate the molecular cascade in the ant, Vollenhovia emeryi, with two CSD loci. Here we show by gene disruption that transformer (tra) is necessary for proper feminization. Expression analysis of tra and doublesex (dsx) showed that heterozygosity in at least one of the two CSD loci is sufficient to promote female sex determination. Analysis of overexpression suggested that female-type Tra protein promotes splicing of tra pre-mRNA to female isoform by a positive-regulatory-feedback loop. Our data also showed that tra affects splicing of dsx. We conclude that two-loci sex determination system in V. emeryi evolved based on tra-dsx splicing cascade that is well conserved in other insect species. Finally, we suggest a cascade model to arrive at a binary determination of sex under multiple primary signals.

In Vitro Comparison of Sex-Specific Splicing Efficiencies of fem Pre-mRNA under Monoallelic and Heteroallelic Conditions of csd, a Master Sex-Determining Gene in the Honeybee.

The sexual fate of honeybees is determined by the complementary sex determination (CSD) model: heterozygosity at a single locus (the CSD locus) determines femaleness, while hemizygosity or homozygosity at the CSD locus determines maleness. The csd gene encodes a splicing factor that regulates sex-specific splicing of the downstream target gene feminizer (fem), which is required for femaleness. The female mode of fem splicing occurs only when csd is present in the heteroallelic condition. To gain insights into how Csd proteins are only activated under the heterozygous allelic composition, we developed an in vitro assay system to evaluate the activity of Csd proteins. Consistent with the CSD model, the co-expression of two csd alleles, both of which lack splicing activity under the single-allele condition, restored the splicing activity that governs the female mode of fem splicing. RNA immunoprecipitation quantitative PCR analyses demonstrated that the CSD protein was specifically enriched in several exonic regions in the fem pre-mRNA, and enrichment in exons 3a and 5 was significantly greater under the heterozygous allelic composition than the single-allelic condition. However, in most cases csd expression under the monoallelic condition was capable of inducing the female mode of fem splicing contrary to the conventional CSD model. In contrast, repression of the male mode of fem splicing was predominant under heteroallelic conditions. These results were reproduced by real-time PCR of endogenous fem expression in female and male pupae. These findings strongly suggest that the heteroallelic composition of csd may be more important for the repression of the male splicing mode than for the induction of the female splicing mode of the fem gene.

Revisiting the hymenopteran diploid male vortex: a review of avoidance mechanisms and incidence

AbstractAll hymenopterans (bees, ants, wasps, and sawflies) have haplodiploid sex determination. Generally, this involves haploid males developing from unfertilized eggs and diploid females developing from fertilized eggs, but diploid male production (DMP) frequently occurs. Some species have complementary sex determination (CSD), in which heterozygotes of a csd locus or loci are diploid females, hemizygotes are haploid males, and homozygotes are typically sterile diploid males. These diploid males underlie the diploid male vortex (DMV), the phenomenon of inbreeding reducing csd allelic diversity and causing increasingly higher production of sterile diploid males until the population dies out. The DMV has been cited as an extinction risk for many species across the order and a danger to both human‐controlled and natural hymenopteran populations important in biological control, pollination, and endangered species conservation. However, it has been unclear how frequently it occurs. We review the known mechanisms for DMV avoidance. Many of these mechanisms are linked to lifestyle, and so we structure our investigation around eusocial lifestyle and non‐eusocial (solitary or gregarious) species. We also review documented DMV incidence to make an inference about its prevalence. There are many means to avoid inbreeding or diploid male production, including eusocial‐exclusive polygyny, polyandry, and diploid queen execution. For both eusocial and non‐eusocial species these include biological mechanisms such as multi‐locus CSD or non‐CSD sex determination, diploid male inviability or brood removal, partial diploid male fecundity, and mating avoidance of kin or diploid males; or population genetic mechanisms such as gene flow and dispersal, balancing selection of csd alleles (with some being more prominent in eusocial vs. non‐eusocial species). Documented cases of DMV are uncommon, and incidence is often tied to exacerbatory conditions such as habitat fragmentation and host declines. With this review we suggest that due to numerous avoidance mechanisms, DMV risk may not be as high as previously believed, and requires specific circumstances. In doing so we aim to better inform hymenopteran breeding and conservation efforts.

New insights into the criteria of functional heterozygosity of the Apis mellifera complementary sex determining gene-Discovery of a functional allele pair differing by a single amino acid.

The complementary sex determiner (csd) gene is responsible for controlling the sex-determination molecular switch in western honey bees (Apis mellifera): bees that are heterozygous for csd develop into females, whereas bees that are hemizygous or homozygous develop into males. The homozygous diploid males are destroyed at an early stage of their development. It has been proposed that the minimal number of amino acid differences between two csd alleles needed to fully determine femaleness is five and it has also been shown that smaller differences may result in forming an evolutionary intermediate that is not fully capable of female determination, but has increased fitness compared to the homozygous genotype. In this study, we have implemented a terminal restriction length polymorphism-based method of identifying and distinguishing paternal alleles in a given bee colony and assigning them to a particular maternal allele in order to gather information on large number of functional csd pairs and also to identify, to some extent, genotypes that are underrepresented or absent in bee colonies. The main finding of this study is the identification of a fully functional genotype consisting of csd alleles that differed from each other by a one amino acid position. The individuals carrying this genotype expressed only female-specific transcripts of feminizer and double-sex genes. By comparing the sequences differences between the csd pair identified in our study with those described earlier, we conclude that functional heterozygosity of the csd gene is dependent not only on the number of the amino acid differences but also on the sequence context and position of the change. The discovery of a functional allele pair differing by a single amino acid also implies that the generation of a new csd specificity may also occur during a single mutation step with no need for evolutionary intermediates accumulating further mutations.

Analysis of Complementary Sex-Determiner (csd) Allele Diversity in Different Honeybee Subspecies from Italy Based on NGS Data.

Sexual regulation in Apis mellifera is controlled by the complementary sex-determiner (csd) gene: females (queens and workers) are heterozygous at this locus and males (drones) are hemizygous. When homozygous diploid drones develop, they are eaten by worker bees. High csd allelic diversity in honeybee populations is a priority for colony survival. The focus of this study is to investigate csd variability in the genomic sequence of the hypervariable region (HVR) of the csd gene in honeybee subspecies sampled in Italy. During the summer of 2017 and 2018, worker bees belonging to 125 colonies were sampled. The honeybees belonged to seven different A. mellifera subspecies: A. m. ligustica, A. m. sicula, A. m cecropia, A. m. carnica, A. m. mellifera, Buckfast and hybrid Carnica. Illumina genomic resequencing of all samples was performed and used for the characterization of global variability among colonies. In this work, a pipeline using existing resequencing data to explore the csd gene allelic variants present in the subspecies collection, based on de novo assembly of sequences falling within the HVR region, is described. On the whole, 138 allelic sequences were successfully reconstructed. Among these, 88 different alleles were identified, 68 of which match with csd alleles present in the NCBI GenBank database.

Genetic diversity and population genetic structure analysis of Apis mellifera subspecies in Algeria and Europe based on complementary sex determiner (CSD) gene

In honeybees, the mechanism of sex determination depends on genetic variation at the complementary sex determiner (CSD) locus, which has a large allelic diversity. In this study, we examined the population genetic structure and genetic diversity within the highly variable region (HVR) of CSD in five Apis mellifera subspecies, in addition to Buckfast and unknown mixed ancestry bees. We sequenced CSD in 329 drones, 146 from Algeria (A. m. intermissa and A. m. sahariensis subspecies) and 183 from Europe (A. m. ligustica, A. m. carnica, A. m. mellifera subspecies, Buckfast samples, and individuals of unknown mixed ancestry). A total of 119 nucleotide haplotypes were detected. These corresponded to 119 protein haplotypes, of which 81 were new. The analysis of these haplotypes showed that HVR diversity levels were comparable with those in other populations of honeybee worldwide. Paradoxically, this high level of diversity at the locus did not allow for a separation of the samples according to their subspecies origin, which suggested either an evolutionary convergence or a conservation of alleles across subspecies, and an absence of genetic drift. Our results can be used to provide more information about the CSD diversity to include in breeding programs of honeybee populations.

How to get rid of diploid bumblebee males – variability in wing size and shape does not allow within‐colony ploidy discrimination

AbstractThe bumblebee Bombus terrestris L. (Hymenoptera: Apidae) is an effective pollinator of both wild and cultivated plants and essential for the pollination of various important greenhouse crops. Because of its sex determination mechanism, homozygosity of the complementary sex determination (csd) locus due to inbreeding causes fertilized eggs to develop into diploid males, which represent a serious disadvantage for the colonies as their presence decreases colony growth and survival. The identification of diploid males is thus important from both ecological and economic points of view. In the present study, we applied the geometric morphometrics method on wing shape and size of B. terrestris diploid and haploid males at overall and intra‐colony levels to investigate wing changes between the two groups. We found that changes in wing shape concerned almost all cells investigated and male diploidy resulted in a narrower and slightly longer configuration. In addition, diploid male wings presented a very high variation both in size and shape, likely due to sub‐optimal temperature of incubation and food availability during their larval development. This strong variation of diploid male wings makes the ploidy discrimination by wing geometric morphometrics inaccurate based on small reference samples at the intra‐colony level even with allometric data correction, whereas it is accurate (precision >90%) at the overall level, in which sample size is high enough (i.e., 500 individuals). Therefore, our results indicated the need to create a large dataset to allow the analysis of few samples, as is the case with studies of wild bumblebee populations, small rearing companies, or preserved museum specimens. For this reason, we provide a large database of B. terrestris wing Procrustes coordinates (500 samples) to the bumblebee scientific community for use in the analysis of the determination of diploid male individuals.

Sexual diversification of splicing regulation during embryonic development in honeybees (Apis mellifera), A haplodiploid system.

The honeybee is a haplodiploid organism in which sexual development is determined by the complementary sex determiner (csd) gene and realized by sex-specific splicing processes involving the feminizer (fem) gene. We used high throughput transcriptome sequencing (RNA-Seq) to characterize the transcriptional differences between the sexes caused by the fertilization and sex determination processes in honeybee (Apis mellifera) embryos. We identified 758, 372 and 43 differentially expressed genes (DEGs) and 58, 176 and 233 differentially spliced genes (DSGs) in 10-15-h-old, 25-40-h-old and 55-70-h-old female and male embryos, respectively. The early difference in male and female embryos in response to the fertilization and non-fertilization processes resulted mainly in differential expression of genes (758 DEGs vs. 58 DSGs). In the latest sampled embryonic stage, the transcriptional differences between the sexes were dominated by alternative splicing of transcripts (43 DEGs vs. 233 DSGs). Interestingly, differentially spliced transcripts that encode RNA-binding properties were overrepresented in 55-70-h-old embryos, indicating a more diverse regulation via alternative splicing than previous work on the sex determination pathway suggested. These stage- and sex-specific transcriptome data from honeybee embryos provide a comprehensive resource for examining the roles of fertilization and sex determination in developmental programming in a haplodiploid system.

Absence of complementary sex determination in two Leptopilina species (Figitidae, Hymenoptera) and a reconsideration of its incompatibility with endosymbiont-induced thelytoky.

Complementary sex determination (CSD) is a widespread sex determination mechanism in haplodiploid Hymenoptera. Under CSD, sex is determined by the allelic state of one or multiple CSD loci. Heterozygosity at one or more loci leads to female development, whereas hemizygosity of haploid eggs and homozygosity of diploid eggs results in male development. Sexual (arrhenotokous) reproduction normally yields haploid male and diploid female offspring. Under asexual reproduction (thelytoky), diploidized unfertilized eggs develop into females. Thelytoky is often induced by bacterial endosymbionts that achieve egg diploidization by gamete duplication. As gamete duplication leads to complete homozygosity, endosymbiont‐induced thelytokous reproduction is presumed to be incompatible with CSD, which relies on heterozygosity for female development. Previously, we excluded CSD in four Asobara (Braconidae) species and proposed a two‐step mechanism for Wolbachia‐induced thelytoky in Asobara japonica. Here, we conclusively reject CSD in two cynipid wasp species, Leptopilina heterotoma and Leptopilina clavipes. We further show that thelytoky in L. clavipes depends on Wolbachia titer but that diploidization and feminization steps cannot be separated, unlike in A. japonica. We discuss what these results reveal about the sex determination mechanism of L. clavipes and the presumed incompatibility between CSD and endosymbiont‐induced thelytoky in the Hymenoptera.