Complementary Sex Determination Research Articles

Application of Next Generation Semiconductor-Based Sequencing for the Identification of Apis mellifera Complementary Sex Determiner (csd) Alleles from Honey DNA.

Simple SummaryHoney contains traces of the DNA of the honey bees that produced it. This environmental DNA can therefore be used to investigate the genome of the honey bees. In this study, we used a next generation sequencing technology to analyze the variability of a key gene of Apis mellifera L., the complementary sex determiner (csd) gene, using honey environmental DNA as a source of honey bee DNA. This gene determines the sex of the bees. Two different alleles at this locus are needed to produce females whereas males have only one copy of this gene as they are haploid. In case two identical alleles are present in a diploid individual, the larvae are not vital and are discarded by the workers. Therefore, there is an advantage in maintaining a large csd diversity in honey bee populations. In light of the recent decline in honey bee populations, it is important to monitor the allele variability at this gene. The applied methodology provided a new strategy to disclose the genetic diversity at the csd gene at the population-wide level and identify most, if not all, csd alleles present in the colonies in a single analysis.The complementary sex determiner (csd) gene plays an essential role in the sex determination of Apis mellifera L. Females develop only if fertilized eggs have functional heterozygous genotypes at this gene whereas males, being haploids, are hemizygous. Two identical csd alleles produce non vital males. In light of the recent decline in honey bee populations, it is therefore important to monitor the allele variability at this gene. In this study, we tested the application of next generation semiconductor-based sequencing technology (Ion Torrent) coupled with environmental honey DNA as a source of honey bee genome information to retrieve massive sequencing data for the analysis of variability at the hypervariable region (HVR) of the csd gene. DNA was extracted from 12 honey samples collected from honeycombs directly retrieved from 12 different colonies. A specifically designed bioinformatic pipeline, applied to analyze a total of about 1.5 million reads, identified a total of 160 different csd alleles, 55% of which were novel. The average number of alleles per sample was compatible with the number of expected patrilines per colony, according to the mating behavior of the queens. Allele diversity at the csd could also provide information useful to reconstruct the history of the honey.

Ploidy determination in Bombus terrestris males: cost-efficiency comparison among different techniques

Diploid males represent a cost for the viability of populations in haplodiploid species with a single-locus complementary sex determination system. In such social species as bumble bees, their presence affects colony growth and reproduction, therefore, the detection of diploid males is an important aspect to implement conservation actions and for the breeding companies of this important pollinator. In this study, we applied and compared the effectiveness of morphological analyses (sperm count and testis area) with wing morphometrics (traditional and geometric) and genetic techniques (microsatellite analysis) to discriminate between diploid and haploid males originated from laboratory-reared B. terrestris colonies. Among the five techniques, microsatellite analyses and wing geometric morphometrics showed the highest efficiency in ploidy assignment and resulted in the most valuable methods according to a cost-effectiveness analysis.

Transcriptional Profiles of Diploid Mutant Apis mellifera Embryos after Knockout of csd by CRISPR/Cas9.

Simple SummaryIn honey bees, males are haploid while females are diploid, leading to a fundamental difference in genetic materials between the sexes. In order to better control the comparison of gene expression between males and females, diploid mutant males were generated by knocking out the sex-determining gene, complementary sex determiner (csd), in fertilized embryos. The diploid mutant drones had male external morphological features, as well as male gonads. RNA sequencing was performed on the diploid mutant embryos and one-day-old larvae. The transcriptome analysis showed that several female-biased genes, such as worker-enriched antennal (Wat), vitellogenin (Vg), and some venom-related genes, were down-regulated in the diploid mutant males. In contrast, some male-biased genes, like takeout and apolipophorin-III-like protein (A4), were up-regulated. Moreover, the co-expression gene networks suggested that csd might interact very closely with fruitless (fru), feminizer (fem) might have connections with hexamerin 70c (hex70c), and transformer-2 (tra2) might play roles with troponin T (TpnT). Foundational information about the differences in the gene expression caused by sex differentiation was provided in this study. It is believed that this study will pave the ground for further research on the different mechanisms between males and females in honey bees.In honey bees, complementary sex determiner (csd) is the primary signal of sex determination. Its allelic composition is heterozygous in females, and hemizygous or homozygous in males. To explore the transcriptome differences after sex differentiation between males and females, with genetic differences excluded, csd in fertilized embryos was knocked out by CRISPR/Cas9. The diploid mutant males at 24 h, 48 h, 72 h, and 96 h after egg laying (AEL) and the mock-treated females derived from the same fertilized queen were investigated through RNA-seq. Mutations were detected in the target sequence in diploid mutants. The diploid mutant drones had typical male morphological characteristics and gonads. Transcriptome analysis showed that several female-biased genes, such as worker-enriched antennal (Wat), vitellogenin (Vg), and some venom-related genes, were down-regulated in the diploid mutant males. In contrast, some male-biased genes, such as takeout and apolipophorin-III-like protein (A4), had higher expressions in the diploid mutant males. Weighted gene co-expression network analysis (WGCNA) indicated that there might be interactions between csd and fruitless (fru), feminizer (fem) and hexamerin 70c (hex70c), transformer-2 (tra2) and troponin T (TpnT). The information provided by this study will benefit further research on the sex dimorphism and development of honey bees and other insects in Hymenoptera.

A scientific note defining allelic nomenclature standards for the highly diverse complementary sex-determiner (csd) locus in honey bees

A scientific note defining allelic nomenclature standards for the highly diverse complementary sex-determiner (csd) locus in honey bees

Founder effects on sex determination systems in invasive social insects.

Invasive populations are often established from a small number of individuals, and thus have low genetic diversity relative to native-range populations. Social ants, bees and wasps (social Hymenoptera) should be vulnerable to such founder effects on genetic diversity because sex in these species is determined genetically via Complementary Sex Determination (CSD). Under CSD, individuals homozygous at one or more critical sex loci are inviable or develop as infertile diploid males. Low diversity at sex loci leads to increased homozygosity and diploid male production, increasing the chance of colony death. In this review, we identify behavioral, social and reproductive traits that preserve allele richness at sex loci, allow colonies to cope with diploid male production, and eventually restore sex allele diversity in invasive populations of social Hymenoptera that experience founding bottlenecks.

Bracon brevicornis Genome Showcases the Potential of Linked-Read Sequencing in Identifying a Putative Complementary Sex Determiner Gene.

Bracon brevicornis is an ectoparasitoid of a wide range of larval-stage Lepidopterans, including several pests of important crops, such as the corn borer, Ostrinia nubilalis. It is also one of the earliest documented cases of complementary sex determination in Hymenoptera. Here, we present the linked-read-based genome of B. brevicornis, complete with an ab initio-derived annotation and protein comparisons with fellow braconids, Fopius arisanus and Diachasma alloeum. We demonstrate the potential of linked-read assemblies in exploring regions of heterozygosity and search for structural and homology-derived evidence of the complementary sex determiner gene (csd).

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.

Influence of reproductive biology on establishment capacity in introduced Hymenoptera species

Introduced species face numerous biological barriers before they can establish in a new environment. Understanding how they overcome these obstacles is crucial for the development of effective risk assessment and regulation. Reproductive biology is known to influence establishment capacity in plants and is widely used for risk assessment. This biological field should receive more attention, and particularly in the case of insects, as they display a wide range of reproductive traits and have a great impact on the economy and environment. Among insects, the order Hymenoptera is of interest for its diversity, both in terms of reproductive traits and introduction history, as invasive species and biological control agents. We review the main reproductive strategies of Hymenoptera, spanning parthenogenesis, sex determination, reproductive parasites and mating strategies, and evaluate their effect on invasive potential. For instance, thelytoky could decrease the strength of Allee effects while Arrhenotoky could increase adaptive potential. A species with complementary sex determination could be more affected by inbreeding than other species, while paternal genome elimination could lead to high levels of homozygosity. Finally, some reproductive behaviours could decrease inbreeding, facilitate mate location or adaptation by encouraging admixture. The two invasive species Apis mellifera scutellata and Leptocybe invasa and the biocontrol agent Aphidius ervi serve as case studies to illustrate the effect of reproductive traits on species capacities to become established in a new area.

The effects of diploid male production on honey bee colony evolution and survival

The order Hymenoptera includes most of the eusocial species on the planet. Correlated is the fact that many of the social species within the order are haplodiploid and use complementary sex determination (CSD) to determine the sex of offspring. CSD is the mechanism by why single sex alleles within an organism result in male development (haploid) and mismatched sex alleles develop into females (diploids). Related to this is the production of diploid males: fertilized eggs with matched sex alleles which develop as male instead of female. Honey bees are no exception to this, and as their numbers continue to suffer globally and their genetic diversity lowers, the effects of diploid male production (DMP) may pose an increased risk to the survival of bee colonies. In the present study, we develop a model for diploid male production in a honey bee colony and show that with ample resources, this phenomena has little effect on a colony’s health, but there is a limit to the sustainability of a colony suffering from diploid male production. We use our model to show that there were likely no great evolutionary pressures against CSD and DMP in wild honey bees as its effects on colony health in the wild would have been negligible but increased environmental hazards such as pesticides and monoculture crops increase the effects of DMP on colony health.

Global allele polymorphism indicates a high rate of allele genesis at a locus under balancing selection.

When selection favours rare alleles over common ones (balancing selection in the form of negative frequency-dependent selection), a locus may maintain a large number of alleles, each at similar frequency. To better understand how allelic richness is generated and maintained at such loci, we assessed 201 sequences of the complementary sex determiner (csd) of the Asian honeybee (Apis cerana), sampled from across its range. Honeybees are haplodiploid; hemizygotes at csd develop as males and heterozygotes as females, while homozygosity is lethal. Thus, csd is under strong negative frequency-dependent selection because rare alleles are less likely to end up in the lethal homozygous form. We find that in A. cerana, as in other Apis, just a few amino acid differences between csd alleles in the hypervariable region are sufficient to trigger female development. We then show that while allelic lineages are spread across geographical regions, allelic differentiation is high between populations, with most csd alleles (86.3%) detected in only one sample location. Furthermore, nucleotide diversity in the hypervariable region indicates an excess of recently arisen alleles, possibly associated with population expansion across Asia since the last glacial maximum. Only the newly invasive populations of the Austral-Pacific share most of their csd alleles. In all, the geographic patterns of csd diversity in A. cerana indicate that high mutation rates and balancing selection act together to produce high rates of allele genesis and turnover at the honeybee sex locus, which in turn leads to its exceptionally high local and global polymorphism.

Genetic diversity of the complementary sex-determiner\xa0(csd) gene in two closed breeding stocks of Varroa -resistant\xa0honey bees

Honey bee (Apis mellifera) breeding programs that use a closed mating system are particularly vulnerable to low genetic diversity. Inadequate diversity at the complementary sex-determiner (csd) locus is problematic and potentially catastrophic in honey bee populations because it causes low brood viability. In typical commercial populations, queens are open mated and csd diversity is fostered by high rates of introgression. In this study, we examine genetic diversity within the highly variable region (HVR) of csd in two stocks bred for resistance to Varroa destructor: Pol-line and Hilo, both of which use closed mating systems. We sampled 47 Pol-line colonies and 41 Hilo colonies and found 60 protein alleles that were condensed into 35 allele groupings by sequence similarity. We found that proportionately, HVR diversity levels were comparable with those in other closed breeding populations as well as open-mated populations of A. mellifera worldwide. Distinct patterns are observed among Pol-line and Hilo csd protein alleles in both the phylogeny and allele frequency distributions, suggesting early divergence of the two stocks. When compared with an African outgroup, both stocks shared alleles with the outgroup, suggesting ancestral lineages are present and not all diversity is due to new mutations. Periodic monitoring of csd diversity is recommended for closed breeding programs. The csd diversity data reported here are currently being used to make breeding decisions in these two mite-resistant populations of honey bees.

Nest-departure behaviour of gynes and drones in the invasive yellowjacket Vespula germanica (Hymenoptera: Vespidae).

Inbreeding costs can be high in haplodiploid hymenopterans due to their particular mechanism of sex determination (i.e., single-locus complementary sex-determination system, sl-CSD), as it can lead to the production of sterile males. Therefore, mechanisms contributing to reduced inbred matings can be beneficial. In this sense, asynchronous nest departure of sibling drones and gynes could reduce kin encounters in social hymenopterans. Using six observation colonies, we determined under field conditions the nest departure behaviour of sibling reproductives of the social wasp Vespula germanica (Hymenoptera: Vespidae). We determined that sexuals leave the nests definitively and detected asynchronous departure not fixed to a particular caste at a seasonal scale in some colonies, as gynes or drones delayed their departure as a function of the departure of the opposite sex, depending on the colony. At a higher temporal resolution (i.e., within a day), we discovered that drones consistently began to leave nests 1 h before gynes and this difference was driven by those individuals that left on the same day as did the opposite-sex kin. Even though other mechanisms such as polyandry and differential dispersal could also be important at reducing inbred matings in the species, the observed departure patterns (i.e., in some colonies actually leave together with the opposite caste, while in others temporal segregation seems to occur) from nests could be complementary to the former and be important at reducing the negative effects of inbreeding in this invasive species.

An Alternative, High Throughput Method to Identify Csd Alleles of the Honey Bee.

Applying instrumental insemination in closely related honey bee colonies often leads to frequent lethality of offspring causing colony collapse. This is due to the peculiarities of honey bee reproductive biology, where the complementary sex determination (csd) gene drives sex determination within a haplodiploid system. Diploid drones containing homozygous genotypes are lethal. Tracking of csd alleles using molecular markers prevents this unwanted event in closed breeding programs. Our approach described here is based on high throughput sequencing (HTS) that provides more data than traditional molecular techniques and is capable of analysing sources containing multiple alleles, including diploid individuals as the bee queen. The approach combines HTS technique and clipping wings as a minimally invasive method to detect the complementary sex determiner (csd) alleles directly from honey bee queens. Furthermore, it might also be suitable for screening alleles of honey harvested from hives of a closed breeding facility. Data on alleles of the csd gene from different honey bee subspecies are provided. It might contribute to future databases that could potentially be used to track the origin of honey. With the help of tracking csd alleles, more focused crossings will be possible, which could in turn accelerate honey bee breeding programmes targeting increase tolerance against varroosis as well.

Drone aggregation behavior in the social wasp Vespula germanica (Hymenoptera: Vespidae): Effect of kinship and density

Inbreeding can have negative consequences on population viability because of the reduced fitness of the progeny. In general, most species have developed mechanisms to minimize inbreeding such as dispersal and kin avoidance behavior. In the eusocial Hymenoptera, related individuals typically share a common nest and have relatively short mating periods, this could lead to inbreeding, and because of their single–locus complementary sex determination system, it may generate diploid males that could result in infertile triploid progeny representing a cost for the colony. Vespula germanica, is an eusocial wasp that has invaded many parts of the world, despite likely facing a reduced genetic pool during the arrival phases. We ask whether male wasp display specific aggregation behavior that favors genetic diversity, key to reduce inbreeding. Through a set of laboratory experiments, we investigated the effects of drone nestmateship and density on the aggregation behavior of V. germanica drones. We show that drones avoid aggregating with their nestmates at all densities while non-nestmates are avoided only at high densities. This suggests that lek genetic diversity and density could be regulated through drone behavior and in the long run minimize inbreeding favoring invasion success.

Characterization of sex-specific variants of doublesex and feminizer genes in stingless bee species

Sex determination is one of the major developmental events in higher metazoans, where complex molecular mechanisms define two physiologically and behaviorally distinct organisms still genetically compatible. In honey bees, the sex determination cascade is initiated by the allelic composition of complementary sex determiner (csd) gene: males develop from hemi or homozygous embryos, whereas females develop from heterozygous embryos. In females, different alleles of csd lead to the formation of female-specific variants of feminizer (fem) and doublesex (dsx). In males, male-specific variants of fem and dsx are formed by default. In this paper, we investigated the genes of sex determination in the stingless bees Melipona quadrifasciata, Scaptotrigona postica, and Frieseomelitta varia. Our results revealed that the architecture of fem and dsx transcripts is highly conserved among the three stingless bee species, and also with honey bees.

Mapping of Multiple Complementary Sex Determination Loci in a Parasitoid Wasp

Sex determination has evolved in a variety of ways and can depend on environmental and genetic signals. A widespread form of genetic sex determination is haplodiploidy, where unfertilized, haploid eggs develop into males and fertilized diploid eggs into females. One of the molecular mechanisms underlying haplodiploidy in Hymenoptera, the large insect order comprising ants, bees, and wasps, is complementary sex determination (CSD). In species with CSD, heterozygosity at one or several loci induces female development. Here, we identify the genomic regions putatively underlying multilocus CSD in the parasitoid wasp Lysiphlebus fabarum using restriction-site associated DNA sequencing. By analyzing segregation patterns at polymorphic sites among 331 diploid males and females, we identify up to four CSD candidate regions, all on different chromosomes. None of the candidate regions feature evidence for homology with the csd gene from the honey bee, the only species in which CSD has been characterized, suggesting that CSD in L. fabarum is regulated via a novel molecular mechanism. Moreover, no homology is shared between the candidate loci, in contrast to the idea that multilocus CSD should emerge from duplications of an ancestral single-locus system. Taken together, our results suggest that the molecular mechanisms underlying CSD in Hymenoptera are not conserved between species, raising the question as to whether CSD may have evolved multiple times independently in the group.

Queen Execution, Diploid Males, and Selection For and Against Polyandry in the Brazilian Stingless Bee Scaptotrigona depilis.

Female mating frequency varies. Determining the causes of this variation is an active research area. We tested the hypothesis that in stingless bees, Meliponini, single mating is due to the execution of queens that make a matched mating at the complementary sex determination locus and have diploid male offspring. We studied the Brazilian species Scaptotrigona depilis. We made up 70 test colonies so that 50% (single matched mating), 25% (double mating), 12.5% (quadruple mating), or 0% (single nonmatched mating) of the emerging brood were diploid males. Queen execution following diploid male emergence was equal and high in colonies producing 50% (77% executed) and 25% (75%) diploid males versus equal and low in colonies producing 12.5% (7%) and 0% (0%) diploid males. These results show that queens that mate with two males with similar paternity suffer an increased chance of being executed, which selects against double mating. However, double mating with unequal paternity (e.g., 25∶75), which occasionally occurs in S. depilis, is selectively neutral. Single mating and double mating with unequal paternity form one adaptive peak. The results show a second adaptive peak at quadruple mating. However, this is inaccessible via gradual evolutionary change in a selective landscape with reduced fitness at double mating.

Life-history traits of the Whiting polyploid line of the parasitoid Nasonia vitripennis.

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

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.

Patterns and drivers of genetic diversity and structure in the biological control parasitoid Habrobracon hebetor in Niger.

When a promising natural enemy of a key pest exists locally, it is a common practice in biological control (BC) to rear and release it for supplementary control in the targeted agroecosystem even though significant knowledge gaps concerning pre/post release may still exist. Incorporating genetic information into BC research fills some of these gaps. Habrobracon hebetor, a parasitoid of many economically important moths that infest stored and field crops worldwide is commonly used, particularly against the millet head miner (MHM), a key pest of millet in Sahelian countries. To advance our knowledge on how H. hebetor that occurs naturally in open-field cropping systems and grain stores as well as being mass-produced and released for MHM control, performs in millet agroecosystems in Niger we evaluated its population genetics using two mitochondrial and 21 microsatellite markers. The field samples were genetically more diverse and displayed heterozygote excess. Very few field samples had faced significant recent demographic bottlenecks. The mating system (i.e. nonrandom mating with complementary sex determination) of this species may be the major driver of these findings rather than bottlenecks caused by the small number of individuals released and the scarcity of hosts during the longlasting dry season in Niger. H. hebetor population structure was represented by several small patches and genetically distinct individuals. Gene flow occurred at local and regional scales through human-mediated and natural short-distance dispersal. These findings highlight the importance of the mating system in the genetic diversity and structure of H. hebetor populations, and contribute to our understanding of its reported efficacy against MHM in pearl millet fields.