Experimental support for multiple-locus complementary sex determination in the parasitoid Cotesia vestalis

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.     

Complementary sex determination in the genus Diadegma (Hymenoptera: Ichneumonidae)

In the evolution of sexual reproduction we would expect to see a close association between mating systems and sex determination mechanisms. Such associations are especially evident in the insect order Hymenoptera which shows great diversity with respect to both of these characteristics. The ancestral sex determination mechanism in this order is thought to be single‐locus complementary sex determination (sl‐CSD), which is inbreeding sensitive, and where inbreeding results in the production of sterile diploid males rather than daughters. Presently, however, there is insufficient data to give strong support to the hypothesis that sl‐CSD is truly the ancestral condition in the Hymenoptera, principally because of the difficulty of reliably determining the degree of male ploidy. Here we show that six ichneumonid parasitoids from the polyphyletic genus Diadegma are subject to sl‐CSD, using neuronal cell DNA flow cytometry to distinguish ploidy levels. The presence of sl‐CSD in these six species, together with earlier evidence from the authors for D. chrysostictos, provides considerable support for the notion that sl‐CSD was ancestral in the Aculeata/Ichneumonoidea clade, which contains all eusocial Hymenoptera. Moreover, because flow cytometry discriminates reliably between haploid and diploid males, and is independent of the maternal sex allocation or the need for genetic markers, it has considerable potential for the determination of ploidy more generally.     

Complementary Sex Determination in the Parasitic Wasp Diachasmimorpha longicaudata

We studied the sex determination in Diachasmimorpha longicaudata, a parasitoid braconid wasp widely used as biological control agent of fruit pest tephritid flies. We tested the complementary sex determination hypothesis (CSD) known in at least 60 species of Hymenoptera. According to CSD, male or female development depends on the allelic composition of one sex locus (single-locus CSD) or multiple sex loci (multiple-locus CSD). Hemizygote individuals are normal haploid males, and heterozygotes for at least one sex locus are normal diploid females, but homozygotes for all the sex loci are diploid males. In order to force the occurrence of diploid males in D. longicaudata, we established highly inbred lines and examined their offspring using chromosome counting, flow cytometry, and sex ratio analysis. We found that when mother-son crosses were studied, this wasp produced about 20% of diploid males out of the total male progeny. Our results suggest that this parasitoid may represent the second genus with multiple-locus CSD in Hymenoptera. Knowledge about the sex determination system in D. longicaudata is relevant for the improvement of mass rearing protocols of this species. This information also provides the necessary background for further investigations on the underlying molecular mechanisms of sex determination in this species, and a better insight into the evolution of this pathway in Hymenoptera in particular and insects in general.     

COMPLEMENTARY SEX DETERMINATION IN HYMENOPTERAN PARASITOIDS AND ITS IMPLICATIONS FOR BIOLOGICAL CONTROL

Abstract In haplodiploid Hymenoptera, unfertilized eggs produce haploid males while fertilized eggs lead to diploid females under most circumstances. Diploid males can also be produced from fertilization under a system of sex determination known as complementary sex determination (CSD). Under single-locus CSD, sex is determined by multiple alleles at a single sex locus. Individuals heterozygous at the sex locus are female while hemizygous and homozygous individuals develop as haploid and diploid males, respectively. In multiple-locus CSD, two or more loci, each with two or more alleles, determine sex. Diploid individuals are female if one or more sex loci are heterozygous, while a diploid is male only if homozygous at all sex loci. Diploid males are known to occur in 43 hymenopteran species and single-locus CSD has been demonstrated in 22 of these species. Diploid males are either developmentally inviable or sterile, so their production constitutes a genetic load. Because diploid male production is more likely under inbreeding, CSD is a form of inbreeding depression. It is crucial to preserve the diversity of sex alleles and reduce the loss of genetic variation in biological control. In the parasitoid species with single-locus CSD, certain precautionary procedures can prevent negative effects of single-locus CSD on biological control.     

膜翅目寄生蜂的互补性别决定机制及其在生防上的意义(英文)

在膜翅目中 ,未受精卵形成单倍体的雄蜂 ,而在大多数情况下受精卵将产生双倍体的雌蜂。但是 ,因互补性别决定机制 (CSD)的作用 ,受精卵有时也会产生双倍体雄蜂。这种性别决定机制包括单位点的CSD和多位点的CSD。在单位点的CSD作用下 ,唯一的一个性位点上的多个等位基因决定后代个体的性别。性位点上杂合的个体将是雌性 ,半合或同型结合的个体将分别形成单倍体或双倍体的雄性。在多位点的CSD作用下 ,两个或两个以上的性位点控制后代的性别 ,每个性位点上包含两个或两个以上的等位基因。如果一个或一个以上的性位点是杂合的 ,形成的双倍体后代都是雌性的 ,但若是所有的性位点都为同型合子 ,则将产生双倍体的雄蜂。在膜翅目中 ,目前已知 4 3种具有双倍体雄蜂 ,其中 2 2种发现存在单位点的CSD ,但是多位点的CSD还有待于确认。双倍体的雄性个体或者不能存活 ,或者不育 ,这样的个体形成将对寄生蜂种群的增长带来一定的遗传负担。在生物防治上 ,保护寄生蜂种群的性等位基因的多样性及减少其遗传多异性的损失极其重要。如果利用具有单位点CSD的种类 ,采取一定的措施将可避免由于双倍体雄性的形成所带来的负面影响。     

The effects of functional response and host abundance fluctuations on genetic rescue in parasitoids with single‐locus sex determination

Many parasitoids have single‐locus complementary sex determination (sl‐CSD), which produces sterile or inviable males when homozygous at the sex determining locus. A previous study theoretically showed that small populations have elevated risks of extinction due to the positive feedback between inbreeding and small population size, referred to as the diploid male vortex. A few modeling studies have suggested that the diploid male vortex may not be as common because balancing selection at sex determining loci tends to maintain high allelic diversity in spatially structured populations. However, the generality of the conclusion is yet uncertain, as they were drawn either from models developed for particular systems or from a general‐purpose competition model. To attest the conclusion, we study several well‐studied host–parasitoid models that incorporate functional response specifying the number of attacked hosts given a host density and derive the conditions for a diploid male vortex in a single population. Then, we develop spatially structured individual‐based versions of the models to include female behavior, diploid male fertility, and temporal fluctuations. The results show that producing a handful of successful offspring per female parasitoid could enable parasitoid persistence when a typical number of CSD alleles are present. The effect of functional response depends on the levels of fluctuations in host abundance, and inviable or partially fertile diploid males and a small increase in dispersal can alleviate the risk of a diploid male vortex. Our work supports the generality of effective genetic rescue in spatially connected parasitoid populations with sl‐CSD. However, under more variable climate, the efficacy of the CSD mechanism may substantially decline.     

Contagious parthenogenesis, automixis, and a sex determination meltdown

Because of the twofold cost of sex, genes conferring asexual reproduction are expected to spread rapidly in sexual populations. However, in reality this simple prediction is often confounded by several complications observed in natural systems. Motivated by recent findings in the Cape honey bee and in the parasitoid wasp Lysiphlebus fabarum, we explore through mathematical models the spread of a recessive, parthenogenesis inducing allele in a haplodiploid population. The focus of these models is on the intricate interactions between the mode of parthenogenesis induction through automixis and complementary sex determination (CSD) systems. These interactions may result in asexual production of diploid male offspring and the spread of the parthenogenesis-inducing allele through these males. We demonstrate that if parthenogenetic females produce a substantial proportion of male offspring, this may prevent the parthenogenesis-inducing allele from spreading. However, this effect is weakened if these diploid males are at least partially fertile. We also predict a degradation of multilocus CSD systems during the spread of parthenogenesis, following which only a single polymorphic CSD locus is maintained. Finally, based on empirical parameter estimates from L. fabarum we predict that male production in parthenogens is unlikely to prevent the eventual loss of sexual reproduction in this system.     

Workers' sons rescue genetic diversity at the sex locus in an invasive honey bee population

The hallmark of eusociality is the division of labour between reproductive (queen) and nonreproductive (worker) females. Yet in many eusocial insects, workers retain the ability to produce haploid male offspring from unfertilized eggs. The reproductive potential of workers has well‐documented consequences for the structure and function of insect colonies, but its implications at the population level are less often considered. We show that worker reproduction in honey bees can have an important role in maintaining genetic diversity at the sex locus in invasive populations. The honey bee sex locus is homozygous‐lethal, and, all else being equal, a higher allele number in the population lead to higher mean brood survival. In an invasive population of the honey bee Apis cerana in Australia, workers contribute significantly to male production: 38% of male‐producing colonies are queenless, and these contribute one‐third of all males at mating congregations. Using a model, we show that such male production by queenless workers will increase the number of sex alleles retained in nascent invasive populations following founder events, relative to a scenario in which only queens reproduce. We conclude that by rescuing sex locus diversity that would otherwise be lost, workers' sons help honey bee populations to minimize the negative effects of inbreeding after founder events and so contribute to their success as invaders.     

Ancestral state reconstruction analysis of hymenopteran sex determination mechanisms

We provide the first phylogenetic evidence supporting complementary sex determination (CSD) as the ancestral mechanism for haplodiploidy in the Hymenoptera. It is currently not possible, however, to distinguish the evolutionary polarity of single locus (sl) CSD and multiple‐locus (ml) CSD given the available data. In this light, we discuss the seemingly maladaptive hypothesis of ml‐CSD ancestry, suggesting that collapse from ml‐CSD to sl‐CSD should remain a viable evolutionary hypothesis based on (i) likely weakening of frequency‐dependent selection on sex alleles under ml‐CSD and (ii) recent findings with respect to the evolutionary novelty of the complementary sex determiner gene in honeybees. Our findings help provide a phylogenetically informed blueprint for future sampling of sex determination mechanisms in the Hymenoptera, as they yield hypotheses for many unsampled or ambiguous taxa and highlight taxa whose further sampling will influence reconstruction of the evolutionary polarity of sex determination mechanisms in major clades.     

Absence of Complementary Sex Determination in the Parasitoid Wasp Genus Asobara (Hymenoptera: Braconidae)

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.     

Diploid males sire triploid daughters and sons in the parasitoid wasp Cotesia vestalis

In the Hymenoptera, males develop as haploids from unfertilized eggs and females develop as diploids from fertilized eggs. In species with complementary sex determination (CSD), however, diploid males develop from zygotes that are homozygous at a highly polymorphic sex locus or loci. We investigated mating behavior and reproduction of diploid males of the parasitoid wasp Cotesia vestalis (C. plutellae), for which we recently demonstrated CSD. We show that the behavior of diploid males of C. vestalis is similar to that of haploid males, when measured as the proportion of males that display wing fanning, and the proportion of males that mount a female. Approximately 29% of diploid males sired daughters, showing their ability to produce viable sperm that can fertilize eggs. Females mated to diploid males produced all-male offspring more frequently (71%) than females mated to haploid males (27%). Daughter-producing females that had mated to diploid males produced more male-biased sex ratios than females mated to haploid males. All daughters of diploid males were triploid and sterile. Three triploid sons were also found among the offspring of diploid males. It has been suggested that this scenario, that is, diploid males mating with females and constraining them to the production of haploid sons, has a large negative impact on population growth rate and secondary sex ratio. Selection for adaptations to reduce diploid male production in natural populations is therefore likely to be strong. We discuss different scenarios that may reduce the sex determination load in C. vestalis.     

Consequences of genetic incompatibility on fitness and mate choice: the male point of view

Hymenopterans under single‐locus complementary sex determination (sl‐CSD) face inbreeding costs due to this sex determination mode. Under sl‐CSD, homozygote eggs at the sl‐CSD locus usually develop into unviable or sterile diploid males. Production of such costly males increases when sib‐mating happens because related individuals share half of their genome. In the hymenopteran Venturia canescens (a solitary parasitoid wasp), diploid males are sterile, leading to fitness costs through genetic incompatibility between parents. Whereas the costs of producing diploid males and behavioural strategies that would reduce such costs have been studied in females, the potential fitness costs faced by males have not. Here, we aimed to investigate fitness costs that males endure after a single sib‐mating and tested whether they have the ability to avoid sib‐mating through kin recognition. Our results show that males have a reduced fitness (i.e. they produce fewer daughters) when mating with their sibs. We also show that males have the ability to distinguish between non‐sib and sib females (i.e. kin). They use chemical marks emitted by the females to discriminate kin from non‐kin. We discuss the evolution of kin recognition in males in the context of mate choice for genetic compatibility. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 114 , 279–286.     

Triploid females and diploid males: underreported phenomena in <Emphasis Type="Italic">Polistes</Emphasis> wasps?

Summary In hymenopteran species, males are usually haploid and females diploid. However, in species that have complementary sex determination (CSD), diploid males arise when a female produces offspring that are homozygous at the sex-determining locus. Although diploid males are often sterile, in some species they have been shown to produce diploid sperm, thus producing triploid daughters if they mate successfully. Diploid males have been observed in very few species of social wasps, and we know of no published reports of triploid females. In this paper, we review the existing literature on diploid males and triploid females in the Hymenoptera, and report the observation of triploid females in three species of Polistes paper wasps. Although polyploid offspring may be produced parthenogenetically, the more likely scenario is that Polistes wasps have CSD and produce diploid males via homozygosity at the sex-determining locus. Therefore, female triploidy indicates that diploid males do exist in Polistes species where they are presumed to be absent, and are likely to be even more frequent among species that have experienced a genetic bottleneck. We conclude by cautioning against the assumption of a selective advantage to the production of early males, and by discussing the implications of male diploidy and female triploidy for measurement of sex ratio investment and assumptions of reproductive skew theory.Received 5 December 2003; revised 20 March 2004; accepted 19 April 2004.     

CONSERVATION GENETICS OF NEOTROPICAL POLLINATORS REVISITED: MICROSATELLITE ANALYSIS SUGGESTS THAT DIPLOID MALES ARE RARE IN ORCHID BEES

Allozyme analyses have suggested that Neotropical orchid bee (Euglossini) pollinators are vulnerable because of putative high frequencies of diploid males, a result of loss of sex allele diversity in small hymenopteran populations with single locus complementary sex determination. Our analysis of 1010 males from 27 species of euglossine bees sampled across the Neotropics at 2–11 polymorphic microsatellite loci revealed only five diploid males at an overall frequency of 0.005 (95% CIs 0.002–0.010); errors through genetic nondetection of diploid males were likely small. In contrast to allozyme‐based studies, we detected very weak or insignificant population genetic structure, even for a pair of populations >500 km apart, possibly accounting for low diploid male frequencies. Technical flaws in previous allozyme‐based analyses have probably led to considerable overestimation of diploid male production in orchid bees. Other factors may have a more immediate impact on population persistence than the genetic load imposed by diploid males on these important Neotropical pollinators.     

Ladies last: diel rhythmicity of adult emergence in a parasitoid with complementary sex determination

Protandry, the earlier adult emergence of males, is explained as either an adaptive strategy maximizing male mating opportunities at the same time as minimizing female pre‐reproductive mortality, or as an incidental by‐product of sexual dimorphism fuelled by selection for other life‐history traits. Adult emergence sequences are monitored of broods of the gregarious larval endoparasitoid Cotesia glomerata L. (Hymenoptera: Braconidae) undergoing pupal development under different temperature regimes. As a haplodiploid species with single‐locus complementary sex determination, gender in C. glomerata is determined by the genotype at one sex locus. Haploids are always male, whereas diploids are female when heterozygous but male when homozygous at the sex locus. Sibling mating promotes homozygosity and thus the production of diploid males. Diploid males are produced at the expense of females, and impose a genetic burden on individuals and populations, despite their exceptional fertility in C. glomerata. Emergence of broods is typically completed within 2 days. Irrespective of temperature, males emerge earlier and within a shorter time interval than females, and a majority of the males in a cluster emerge before the first female. The implications of an incomplete temporal segregation of the sexes on the incidence of inbreeding in C. glomerata are discussed in the light of its sex determination mechanism and its patterns of mating, host exploitation and natal dispersal.     

Mechanisms that influence sex ratio variation in the invasive hymenopteran Sirex noctilio in South Africa

Sirex noctilio is an economically important invasive pest of commercial pine forestry in the Southern Hemisphere. Newly established invasive populations of this woodwasp are characterized by highly male‐biased sex ratios that subsequently revert to those seen in the native range. This trend was not observed in the population of S. noctilio from the summer rainfall regions in South Africa, which remained highly male‐biased for almost a decade. The aim of this study was to determine the cause of this persistent male bias. As an explanation for this pattern, we test hypotheses related to mating success, female investment in male versus female offspring, and genetic diversity affecting diploid male production due to complementary sex determination. We found that 61% of females in a newly established S. noctilio population were mated. Microsatellite data analysis showed that populations of S. noctilio from the summer rainfall regions in South Africa are far less genetically diverse than those from the winter rainfall region, with mean Nei's unbiased gene diversity indexes of 0.056 and 0.273, respectively. These data also identified diploid males at low frequencies in both the winter (5%) and summer (2%) rainfall regions. The results suggest the presence of a complementary sex determination mechanism in S. noctilio, but imply that reduced genetic diversity is not the main driver of the male bias observed in the summer rainfall region. Among all the factors considered, selective investment in sons appears to have the most significant influence on male bias in S. noctilio populations. Why this investment remains different in frontier or early invasive populations is not clear but could be influenced by females laying unfertilized eggs to avoid diploid male production in populations with a high genetic relatedness.     

Effective population size in Hymenoptera with complementary sex determination

Complementary sex determination in the haplodiploid Hymenoptera leads to the production of inviable or effectively sterile diploid males when diploid progeny are homozygous at the sex-determining locus. The production of diploid males reduces the number of females in a population and biases the effective breeding sex ratio in favor of haploid males. This in turn will reduce the effective population size (Ne) of hymenopteran populations with complementary sex determination relative to the expected reductions due to haplodiploidy alone. The effects of diploid male production on Ne in hymenopterans with complementary sex determination when diploid males are either inviable or effectively sterile are assessed theoretically. In both models, low allelic diversity at the sex locus reduces the Ne of hymenopteran populations, and this effect is largest when diploid males are effectively sterile.     

Unexpectedly low frequencies of diploid males in an inbreeding parasitoid with complementary sex determination

In hymenopterans with single locus complementary sex determination, sex depends on the genotype at one polymorphic locus. Haploids are always male, while diploids are female when heterozygous and male when homozygous at the sex‐determining locus. Brothers and sisters have a 50% chance of sharing a sex allele (i.e. of being ‘matched’), and hence half of all sibling matings are expected to produce diploid males at the expense of females. Nevertheless, observed frequencies of diploid males are often lower than predicted, as diploid males may succumb to pre‐imaginal mortality, or because unmatched mates or sperm enjoy a competitive advantage. We counted diploid males in broods of the parasitoid wasp Cotesia glomerata sampled in the field, and in broods produced through controlled laboratory crosses. Consistently, the frequency of diploid males fell below expectations based upon the estimated occurrence of sibling mating. In the staged broods with diploid males, females made up a disproportionately large share of the diploids. Broods with and without diploid males were of similar size. Hence, the shortage of diploid males cannot be accounted for by differential pre‐imaginal mortality alone. Instead, we postulate the existence of a mechanism that leads to preferential fertilization of eggs by sperm bearing unmatched alleles. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ●● , ●●–●●.     

Genetic sex assignment in wild populations using genotyping‐by‐sequencing data: A statistical threshold approach

Establishing the sex of individuals in wild systems can be challenging and often requires genetic testing. Genotyping‐by‐sequencing (GBS) and other reduced‐representation DNA sequencing (RRS) protocols (e.g., RADseq, ddRAD) have enabled the analysis of genetic data on an unprecedented scale. Here, we present a novel approach for the discovery and statistical validation of sex‐specific loci in GBS data sets. We used GBS to genotype 166 New Zealand fur seals (NZFS, Arctocephalus forsteri) of known sex. We retained monomorphic loci as potential sex‐specific markers in the locus discovery phase. We then used (i) a sex‐specific locus threshold (SSLT) to identify significantly male‐specific loci within our data set; and (ii) a significant sex‐assignment threshold (SSAT) to confidently assign sex in silico the presence or absence of significantly male‐specific loci to individuals in our data set treated as unknowns (98.9% accuracy for females; 95.8% for males, estimated via cross‐validation). Furthermore, we assigned sex to 86 individuals of true unknown sex using our SSAT and assessed the effect of SSLT adjustments on these assignments. From 90 verified sex‐specific loci, we developed a panel of three sex‐specific PCR primers that we used to ascertain sex independently of our GBS data, which we show amplify reliably in at least two other pinniped species. Using monomorphic loci normally discarded from large SNP data sets is an effective way to identify robust sex‐linked markers for nonmodel species. Our novel pipeline can be used to identify and statistically validate monomorphic and polymorphic sex‐specific markers across a range of species and RRS data sets.     

Dmrt1 polymorphism and sex‐chromosome differentiation in Rana temporaria

Sex‐determination mechanisms vary both within and among populations of common frogs, opening opportunities to investigate the molecular pathways and ultimate causes shaping their evolution. We investigated the association between sex‐chromosome differentiation (as assayed from microsatellites) and polymorphism at the candidate sex‐determining gene Dmrt1 in two Alpine populations. Both populations harboured a diversity of X‐linked and Y‐linked Dmrt1 haplotypes. Some males had fixed male‐specific alleles at all markers (“differentiated” Y chromosomes), others only at Dmrt1 (“proto‐” Y chromosomes), while still others were genetically indistinguishable from females (undifferentiated X chromosomes). Besides these XX males, we also found rare XY females. The several Dmrt1 Y haplotypes differed in the probability of association with a differentiated Y chromosome, which we interpret as a result of differences in the masculinizing effects of alleles at the sex‐determining locus. From our results, the polymorphism in sex‐chromosome differentiation and its association with Dmrt1, previously inferred from Swedish populations, are not just idiosyncratic features of peripheral populations, but also characterize highly diverged populations in the central range. This implies that an apparently unstable pattern has been maintained over long evolutionary times.