Complementary Sex Determination Research Articles

Similar but not the same: insights into the evolutionary history of paralogous sex-determining genes of the dwarf honey bee Apis florea.

Studying the fate of duplicated genes provides informative insight into the evolutionary plasticity of biological pathways to which they belong. In the paralogous sex-determining genes complementary sex determiner (csd) and feminizer (fem) of honey bee species (genus Apis), only heterozygous csd initiates female development. Here, the full-length coding sequences of the genes csd and fem of the phylogenetically basal dwarf honey bee Apis florea are characterized. Compared with other Apis species, remarkable evolutionary changes in the formation and localization of a protein-interacting (coiled-coil) motif and in the amino acids coding for the csd characteristic hypervariable region (HVR) are observed. Furthermore, functionally different csd alleles were isolated as genomic fragments from a random population sample. In the predicted potential specifying domain (PSD), a high ratio of πN/πS=1.6 indicated positive selection, whereas signs of balancing selection, commonly found in other Apis species, are missing. Low nucleotide diversity on synonymous and genome-wide, non-coding sites as well as site frequency analyses indicated a strong impact of genetic drift in A. florea, likely linked to its biology. Along the evolutionary trajectory of ~30 million years of csd evolution, episodic diversifying selection seems to have acted differently among distinct Apis branches. Consistently low amino-acid differences within the PSD among pairs of functional heterozygous csd alleles indicate that the HVR is the most important region for determining allele specificity. We propose that in the early history of the lineage-specific fem duplication giving rise to csd in Apis, A. florea csd stands as a remarkable example for the plasticity of initial sex-determining signals.

Diploid males of Scaptotrigona depilis are able to join reproductive aggregations (Apidae, Meliponini)

The sex determination system in the eusocial stingless bees (Apidae, Meliponini) is based on the combination of alleles at the complementary sex determination (CSD) locus. In this system, males are haploid and females are diploid. However, diploid males can develop from fertilized eggs when they are homozygous at single or multiple sex loci. The production of such males can negatively affect population viability, since they are usually infertile or inviable. Moreover, when they are viable but infertile, or siring sterile triploid offspring, this could cause another load on the population, leading the fertilized offspring of other females to be only haploid males or triploid sterile daughters. In this context, our aim was to verify whether diploid males of the stingless bee Scaptotrigona depilis do in fact join reproductive aggregations. We showed that of 360 marked males from two different colonies, five were participating in a reproductive aggregation ca. 20 meters from their natal colonies. Using microsatellites markers, it was confirmed that three of these five males were diploid. They were captured in the mating aggregations when they were 15 to 20 days old. Further research is necessary to determine the mating success of stingless bee diploid males under natural conditions and to determine their impact on stingless bee population extinction risks. JHR 45: 125–130 (2015) doi: 10.3897/JHR.45.4769 http://jhr.pensoft.net Copyright Ayrton Vollet-Neto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. SHORT COMMUNICATION Ayrton Vollet-Neto et al. / Journal of Hymenoptera Research 45: 125–130 (2015) 126

Population-level consequences of complementary sex determination in a solitary parasitoid.

BackgroundSex 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.ResultsWe 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.ConclusionsWe 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 materialThe online version of this article (doi:10.1186/s12862-015-0340-2) contains supplementary material, which is available to authorized users.

Sterile males in a parasitoid wasp with complementary sex determination: from fitness costs to population extinction.

BackgroundSingle-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.ResultsIn 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.ConclusionThe performance of diploid males should be estimated in competitive situations. It is a crucial determinant of the probability of extinction.Electronic supplementary materialThe online version of this article (doi:10.1186/s12898-014-0032-6) contains supplementary material, which is available to authorized users.

The evolutionary dynamics of major regulators for sexual development among Hymenoptera species.

All hymenopteran species, such as bees, wasps and ants, are characterized by the common principle of haplodiploid sex determination in which haploid males arise from unfertilized eggs and females from fertilized eggs. The underlying molecular mechanism has been studied in detail in the western honey bee Apis mellifera, in which the gene complementary sex determiner (csd) acts as primary signal of the sex determining pathway, initiating female development by csd-heterozygotes. Csd arose from gene duplication of the feminizer (fem) gene, a transformer (tra) ortholog, and mediates in conjunction with transformer2 (tra2) sex-specific splicing of fem. Comparative molecular analyses identified fem/tra and its downstream target doublesex (dsx) as conserved unit within the sex determining pathway of holometabolous insects. In this study, we aim to examine evolutionary differences among these key regulators. Our main hypothesis is that sex determining key regulators in Hymenoptera species show signs of coevolution within single phylogenetic lineages. We take advantage of several newly sequenced genomes of bee species to test this hypothesis using bioinformatic approaches. We found evidences that duplications of fem are restricted to certain bee lineages and notable amino acid differences of tra2 between Apis and non-Apis species propose structural changes in Tra2 protein affecting co-regulatory function on target genes. These findings may help to gain deeper insights into the ancestral mode of hymenopteran sex determination and support the common view of the remarkable evolutionary flexibility in this regulatory pathway.

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.

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.

Inbreeding depression in a parasitoid wasp with single-locus complementary sex determination.

Inbreeding and inbreeding depression are key processes in small or isolated populations and are therefore central concerns for the management of threatened or (re)introduced organisms. Haplodiploid species of the order Hymenoptera have a particular status with regard to inbreeding depression. Although recessive deleterious alleles that are expressed in males should be purged, an alternative form of inbreeding depression exists in species with single-locus complementary sex determination (sl-CSD). Under sl-CSD, genetically-related parents have a high probability of producing sterile sons instead of fertile daughters. In this article, we study inbreeding depression in Venturia canescens (Hymenoptera: Ichneumonidae), a parasitoid wasp with sl-CSD. We used a crossing design to manipulate relatedness according to three levels: within-family, between-family and between-population. For each level, several fitness components were measured on parents and female offspring. We found a 20% reduction in egg load at emergence for inbred crosses. Inbred crosses also yielded a higher proportion of males, as expected in a species with sl-CSD. Mating probability, presence of daughters among offspring, body size, symmetry and longevity were unaffected by inbreeding.

Independent evolutionary origin of fem paralogous genes and complementary sex determination in hymenopteran insects.

The primary signal of sex determination in the honeybee, the complementary sex determiner (csd) gene, evolved from a gene duplication event from an ancestral copy of the fem gene. Recently, other paralogs of the fem gene have been identified in several ant and bumblebee genomes. This discovery and the close phylogenetic relationship of the paralogous gene sequences led to the hypothesis of a single ancestry of the csd genetic system of complementary sex determination in the Hymenopteran insects, in which the fem and csd gene copies evolved as a unit in concert with the mutual transfers of sequences (concerted evolution). Here, we show that the paralogous gene copies evolved repeatedly through independent gene duplication events in the honeybee, bumblebee, and ant lineage. We detected no sequence tracts that would indicate a DNA transfer between the fem and the fem1/csd genes between different ant and bee species. Instead, we found tracts of duplication events in other genomic locations, suggesting that gene duplication was a frequent event in the evolution of these genes. These and other evidences suggest that the fem1/csd gene originated repeatedly through gene duplications in the bumblebee, honeybee, and ant lineages in the last 100 million years. Signatures of concerted evolution were not detectable, implicating that the gene tree based on neutral synonymous sites represents the phylogenetic relationships and origins of the fem and fem1/csd genes. Our results further imply that the fem1 and csd gene in bumblebees, honeybees, and ants are not orthologs, because they originated independently from the fem gene. Hence, the widely shared and conserved complementary sex determination mechanism in Hymenopteran insects is controlled by different genes and molecular processes. These findings highlight the limits of comparative genomics and emphasize the requirement to study gene functions in different species and major hymenopteran lineages.

Haploid, diploid, and triploid--discrimination ability against polyploid mating partner in the parasitic wasp, Bracon brevicornis (Hymenoptera: Braconidae).

Because the quality of mating partners varies, females of several taxa have evolved the ability to discriminate against low-quality mates. Although males in the Hymenoptera are usually haploid, diploid males may occur in species with complementary sex determination. Diploid males are almost always sterile in most of the species studied so far. They are thus of very low quality as mating partners, especially when females mate only once in life. We hypothesize that hymenopteran females might have evolved the ability to discriminate against infertile diploid males and avoid mating with them. To test this hypothesis, we studied diploid male fitness in the parasitoid wasp Bracon brevicornis Wesmael (Hymenoptera: Braconidae) by measuring survival rate and fertility and then estimated their chances of actually mating with a female. Flow cytometry was used to determine the ploidy level of wasps. The fitness costs of mating a diploid male are indeed high in this species: only 15% were able to sire daughters, of which 97% were triploid and hardly able to produce viable offspring. In contrast to the hypothesis of unsuitable mate discrimination though, no evidence was found for increased rejection of diploid males by females. Male discrimination against an unsuitable partner did also not occur: triploid females elicited the same intensity of courtship behavior in males than did diploid ones.

Phylogenetic Distribution and Evolutionary Dynamics of the Sex Determination Genes doublesex and transformer in Insects

Sex determination in insects is characterized by a gene cascade that is conserved at the bottom but contains diverse primary signals at the top. The bottom master switch gene doublesex is found in all insects. Its upstream regulator transformer is present in the orders Hymenoptera, Coleoptera and Diptera, but has thus far not been found in Lepidoptera and in the basal lineages of Diptera. transformer is presumed to be ancestral to the holometabolous insects based on its shared domains and conserved features of autoregulation and sex-specific splicing. We interpret that its absence in basal lineages of Diptera and its order-specific conserved domains indicate multiple independent losses or recruitments into the sex determination cascade. Duplications of transformer are found in derived families within the Hymenoptera, characterized by their complementary sex determination mechanism. As duplications are not found in any other insect order, they appear linked to the haplodiploid reproduction of the Hymenoptera. Further phylogenetic analyses combined with functional studies are needed to understand the evolutionary history of the transformer gene among insects.

Variation in the salivary proteomes of differentially virulent greenbug (Schizaphis graminum Rondani) biotypes

Greenbug (Schizaphis graminum Rondani) biotypes are classified by their differential virulence to wheat, barley, and sorghum varieties possessing greenbug resistance genes. Virulent greenbug biotypes exert phytotoxic effects upon their hosts during feeding, directly inducing physiological and metabolic alterations and accompanying foliar damage. Comparative analyses of the salivary proteomes of four differentially virulent greenbug biotypes C, E, G, and H showed significant proteomic divergence between biotypes. Thirty-two proteins were identified by LC-MS/MS; the most prevalent of which were three glucose dehydrogenase paralogs (GDH), lipophorin, complementary sex determiner, three proteins of unknown function, carbonic anhydrase, fibroblast growth factor receptor, and abnormal oocyte (ABO). Seven nucleotide-binding proteins were identified, including ABO which is involved in mRNA splicing. Quantitative variation among greenbug biotypes was detected in six proteins; two GDH paralogs, carbonic anhydrase, ABO, and two proteins of unknown function. Our findings reveal that the greenbug salivary proteome differs according to biotype and diverges substantially from those reported for other aphids. The proteomic profiles of greenbug biotypes suggest that interactions between aphid salivary proteins and the plant host result in suppression of plant defenses and cellular transport, and may manipulate transcriptional regulation in the plant host, ultimately allowing the aphid to maintain phloem ingestion. Greenbug (Schizaphis graminum Rondani, GB) is a major phytotoxic aphid pest of wheat, sorghum, and barley. Unlike non-phytotoxic aphids, GB directly damages its host, causing uniformly characteristic symptoms leading to host death. As saliva is the primary interface between the aphid and its plant host, saliva is also the primary aphid biotypic determinant, and differences in biotypic virulence are the result of biotypic variations in salivary content. This study analyzed the exuded saliva of four distinct Greenbug biotypes with a range of virulence to crop lines containing greenbug resistance traits in order to identify differences between salivary proteins of the examined biotypes. Our analyses confirmed that the salivary proteomes of the examined greenbug biotypes differ widely, identified 32 proteins of the greenbug salivary proteome, and found significant proteomic variation between six identified salivary proteins. The proteomic variation identified herein is likely the basis of biotypic virulence, and the proteins identified can serve as the basis for functional studies into both greenbug-induced phytotoxic damage and into the molecular basis of virulence in specific GB biotypes. This article is part of a Special Issue entitled: SI: Proteomics of non-model organisms.

Everything You Wanted to Know about Honeybee Sex

There is an exquisite genetic control behind a honeybee’s fate in the hive—from the lowly drone to the almighty queen— which literally represents the bee’s knees for evolutionary scientists exploring how multiple mutations, or alleles, of a single gene called the complementary sex determiner (csd )c an have ap rofound inf luence on honeybee society. Unlike people, there are no X and Y sex chromosomes for bees. Rather, sex is determined by csd gene and its allelic composition and whether a queen bee chooses to fertilize her eggs. Female bees (queens or workers) come from fertilized eggs, always receiving two different (heterozygote) copies of csd. Fertile males always come from unfertilized eggs, receiving only one copy of csd. Two identical (homozygote) copies ofcsd in fertilized eggs is always lethal; these individuals are being killed at the early larval stage by worker bees as they would develop into diploid males which do not contribute to colony fitness. Lechner et al. (2013) have now examined the exquisite molecular control behind the sex determination, finely identifying and tracing back a comprehensive number of csd alleles to create a richer understanding of the variability of the csd gene over evolutionary time. They looked at a data set of 244 csd sequences from queens, worker bees, and drones and showed that the total number of csd alleles found in bees ranges from at least 53 (locally) to 87 (worldwide), which is much higher than previously reported. Using an evolutionary model, they also extrapolated the presence of a total 116–145 csd alleles worldwide, a great example of the enormous sequence variability within csd. They were able to finely decipher the minimum number of mutations leading to heterozygous csd, identify faster evolving hot spots within the csd gene, and how these may contribute to variability. “Comprehensive insights into the sequence variability of the sex determining gene csd in honeybees elucidate the evolutionary processes that lead to the enormous number of csd alleles found worldwide,” said Hasselmann. Finally, they traced the data back over evolutionary time and found that a novel csd function affecting sex determination arises about every 400,000 years. The study provides one of the most comprehensive views of the enormous genetic diversity and the evolutionary forces shaping sex determination in bees, as well as how changes in csd affect honeybee colony fitness.

Nucleotide Variability at Its Limit? Insights into the Number and Evolutionary Dynamics of the Sex-Determining Specificities of the Honey Bee Apis mellifera

Deciphering the evolutionary processes driving nucleotide variation in multiallelic genes is limited by the number of genetic systems in which such genes occur. The complementary sex determiner (csd) gene in the honey bee Apis mellifera is an informative example for studying allelic diversity and the underlying evolutionary forces in a well-described model of balancing selection. Acting as the primary signal of sex determination, diploid individuals heterozygous for csd develop into females, whereas csd homozygotes are diploid males that have zero fitness. Examining 77 of the functional heterozygous csd allele pairs, we established a combinatorical criteria that provide insights into the minimum number of amino acid differences among those pairs. Given a data set of 244 csd sequences, we show that the total number of csd alleles found in A. mellifera ranges from 53 (locally) to 87 (worldwide), which is much higher than was previously reported (20). Using a coupon-collector model, we extrapolate the presence of in total 116–145 csd alleles worldwide. The hypervariable region (HVR) is of particular importance in determining csd allele specificity, and we provide for this region evidence of high evolutionary rate for length differences exceeding those of microsatellites. The proportion of amino acids driven by positive selection and the rate of nonsynonymous substitutions in the HVR-flanking regions reach values close to 1 but differ with respect to the HVR length. Using a model of csd coalescence, we identified the high originating rate of csd specificities as a major evolutionary force, leading to an origin of a novel csd allele every 400,000 years. The csd polymorphism frequencies in natural populations indicate an excess of new mutations, whereas signs of ancestral transspecies polymorphism can still be detected. This study provides a comprehensive view of the enormous diversity and the evolutionary forces shaping a multiallelic gene.

The genetic population structure of two socially parasitic ants: “the active slave-maker” Myrmoxenus ravouxi and the “degenerate slave-maker” M. kraussei

Slave-making ants exploit the societies of host ant species and are typically rare and patchily distributed. IUCN considers almost all slave-making ants as vulnerable, but solid data on their actual abundance are uncommon. Here we examine the genetic structure of populations of two species of the socially parasitic genus Myrmoxenus, which differ strongly in dispersal behavior and the occurrence of slave-raids. Microsatellite genotypes suggest strong differentiation even among neighboring populations of both species. FST-, G″ST-, and D-values were considerably higher in the “degenerate slave-maker” M. kraussei from Northern Italy than in the active slavemaker M. ravouxi from Southern Germany. This matches observations that sexuals of M. kraussei mate in their natal nest and queens disperse on foot while sexuals of M. ravouxi engage in mating and dispersal flights. Allelic richness was surprisingly high in both species and did not suggest recent bottlenecks, indicating that populations are larger and less vulnerable than expected from the difficulties of locating nests. Despite of considerable inbreeding, only very few diploid males were detected, supporting the view that at least in M. kraussei sex is not determined by single locus complementary sex determination unlike most other social Hymenoptera. The mismatch between the genetic and social structure of M. ravouxi colonies indicates occasionally fusion of slave-maker colonies in the field, mutual raids, or queen replacement. Complementary analyses of the host species of the two social parasites, Temnothorax unifasciatus and T. recedens, revealed low levels of population differentiation and confirmed the colony structure with a single, singly-mated queen.

Fertile diploid males in the ant Cataglyphis cursor: a potential cost of thelytoky?

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.

Genetic diversity and complementary sex determination (CSD) in Dolerus aeneus (Hymenoptera, Symphyta): implications for the conservation of an ecologically-important sawfly

Graminivorous sawfly numbers have declined steadily over recent years as a consequence of agricultural intensification. In spite of these declines and the importance of sawflies (Hymenoptera, Symphyta) as a food source for threatened populations of farmland birds, sawfly conservation studies have been restricted to census-based research. This is largely due to certain aspects of the sawfly life-cycle which make adequate sampling of these insects difficult. However, genetic research provides a valuable insight into population structure that cannot be obtained by traditional ecological means, and one which is essential for the development of conservation management strategies. In this study, we investigate the population genetic structure of the graminivorous sawfly Dolerus aeneus, and conduct a preliminary study to determine whether complementary sex determination (CSD), which can produce sterile diploid males under inbreeding conditions, operates in this species. Our research suggests that fragmentation of sawfly habitat as a result of agri-intensification has not yet acted to isolate D. aeneus populations, although some genetic effects are apparent (inbreeding and low diversity compared with other solitary Hymenoptera). In addition, diploid males were detected which may have compromised fertility and could indicate that CSD is operative in D. aeneus. This study highlights the need for further genetic research in sawflies to assess population structure on a UK-wide scale and to assess the prevalence of diploid males in key species. We discuss our findings in wider context of the genetics of Hymenoptera and the conservation and management of farmland biodiversity.

A New Component of the Nasonia Sex Determining Cascade Is Maternally Silenced and Regulates Transformer Expression

Although sex determination is a universal process in sexually reproducing organisms, sex determination pathways are among the most highly variable genetic systems found in nature. Nevertheless, general principles can be identified among the diversity, like the central role of transformer (tra) in insects. When a functional TRA protein is produced in early embryogenesis, the female sex determining route is activated, while prevention of TRA production leads to male development. In dipterans, male development is achieved by prevention of female-specific splicing of tra mRNA, either mediated by X-chromosome dose or masculinizing factors. In Hymenoptera, which have haplodiploid sex determination, complementary sex determination and maternal imprinting have been identified to regulate timely TRA production. In the parasitoid Nasonia, zygotic transformer (Nvtra) expression and splicing is regulated by a combination of maternal provision of Nvtra mRNA and silencing of Nvtra expression in unfertilized eggs. It is unclear, however, if this silencing is directly on the tra locus or whether it is mediated through maternal silencing of a trans-acting factor. Here we show that in Nasonia, female sex determination is dependent on zygotic activation of Nvtra expression by an as yet unknown factor. This factor, which we propose to term womanizer (wom), is maternally silenced during oogenesis to ensure male development in unfertilized eggs. This finding implicates the upstream recruitment of a novel gene in the Nasonia sex determining cascade and supports the notion that sex determining cascades can rapidly change by adding new components on top of existing regulators.

Nucleotide diversity based on csd gene of the black giant honey bee, Apis laboriosa (Hymenoptera: Apidae)

The mechanism of sex determination is common for all honeybee species (Apis spp.) by the complementary sex determi-nation (csd) gene. The csd gene has been studied in the Western honey bee (Apis mellifera L.), the Eastern honey bee (Apis cerana F.) and the giant honey bee (Apis dorsata F.), whereas no studies had been conducted on the high altitude Himalayan or black giant honey bee, Apis laboriosa Smith. In the present study, we cloned the genomic exon 6 to exon 9 region of the A. laboriosa csd gene, and identified 13 csd haplotypes. The data was analyzed and compared with the other aforementioned three honeybee species. The results showed that, as with the other three Apis species, region 3 of the csd gene contains an RS domain at the N terminal, a proline-rich domain at the C terminal, and a hypervariable region in the middle. A phylogenetic tree showed that the csd haplotypes from A. laboriosa fell into one clade with those from A. dorsata, and were separated from those from the other two species, A. mellifera and A. cerana. The network map also showed that the csd haplotypes from A. laboriosa and A. dorsata are well mixed among each other, and do not form two separate branches. Pairwise Fst analysis revealed that the value between A. laboriosa and A. dorsata was very low (0.098), confirming a close relationship to exist between them.

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.