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.     

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 π_N/π_S=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.     

Sequence divergence and expression of a transformer gene in the branchiopod crustacean,Daphnia magna

Environmental decline triggers a switch in reproductive strategy of Daphnia magna from asexual to sexual reproduction; however, the molecular basis of such environmental sex determination remains largely unknown. In insects most closely related to branchiopod crustaceans, orthologs of the Drosophila transformer gene such as dipteran transformer (tra), honeybee feminizer (fem) and complementary sex determiner (csd) function as sex determining factors. Therefore, we cloned a D. magna transformer gene (dmagtra) and analyzed its expression. The predicted amino acid sequence has 380 amino acids including an arginine–serine-rich region, which is characteristic of insect orthologs of Tra. Residues 180 to 205 are highly conserved with known Tra orthologs. The domain organization of DmagTra is distinct from known Tra orthologs; moreover, dmagtra does not display any detectable sexual dimorphic differences in expression or splicing patterns. We infer from these results that dmagtra may not be responsible for sex determination in D. magna.     

Molecular characterization of the gene feminizer in the stingless bee Melipona interrupta (Hymenoptera: Apidae) reveals association to sex and caste development

In highly eusocial insects, development of reproductive traits are regulated not only by sex determination pathway, but it also depends on caste fate. The molecular basis of both mechanisms in stingless bees and possible interaction with each other is still obscure. Here, we investigate sex determination in Melipona interrupta, focusing on characterization and expression analysis of the feminizer gene (Mi-fem), and its association to a major component of caste determination, the juvenile hormone (JH). We present evidence that Mi-fem mRNA is sex-specifically spliced in which only the female splice variant encodes the full length protein, following the same principle known for other bee species. We quantified Mi-fem expression among developmental stages, sexes and castes. Mi-fem expression varies considerably throughout development, with higher expression levels in embryos. Also, fem levels in pupae and newly emerged adults were significantly higher in queens than workers and males. Finally, we ectopically applied JH in cocoon spinning larvae, which correspond to the time window where queen/worker phenotypes diverge. We observed a significantly increase in Mi-fem expression compared to control groups. Since up to 100% of females turn into queens when treated with JH (while control groups are composed mainly of workers), we propose that fem might act to regulate queens' development. Our findings provide support for the conserved regulatory function of fem in Melipona bees and demonstrate a significant correlation between key elements of sex and caste determination pathways, opening the avenue to further investigate the molecular basis of these complex traits.     

The sex determination gene shows no founder effect in the giant honey bee, Apis dorsata

Background

All honey bee species (Apis spp) share the same sex determination mechanism using the complementary sex determination (csd) gene. Only individuals heterogeneous at the csd allele develop into females, and the homozygous develop into diploid males, which do not survive. The honeybees are therefore under selection pressure to generate new csd alleles. Previous studies have shown that the csd gene is under balancing selection. We hypothesize that due to the long separation from the mainland of Hainan Island, China, that the giant honey bees (Apis dorsata) should show a founder effect for the csd gene, with many different alleles clustered together, and these would be absent on the mainland.

Methodology/Principal Findings

We sampled A. dorsata workers from both Hainan and Guangxi Provinces and then cloned and sequenced region 3 of the csd gene and constructed phylogenetic trees. We failed to find any clustering of the csd alleles according to their geographical origin, i.e. the Hainan and Guangxi samples did not form separate clades. Further analysis by including previously published csd sequences also failed to show any clade-forming in both the Philippines and Malaysia.

Conclusions/Significance

Results from this study and those from previous studies did not support the expectations of a founder effect. We conclude that because of the extremely high mating frequency of A. dorsata queens, a founder effect does not apply in this species.     

Identification and functional characterization of the sex-determining gene <Emphasis Type="Italic">doublesex</Emphasis> in the sawfly, <Emphasis Type="Italic">Athalia rosae</Emphasis> (Hymenoptera: Tenthredinidae)

Sexual fate of the sawfly, Athalia rosae (Hymenoptera: Tenthredinidae) is determined by the complementary sex determination (CSD) mechanism as is the case in honeybees. However, to date, genes involved in sex determination have not been identified in this species. In this study, we attempted to identify orthologs of complementary sex-determiner (csd), feminizer (fem), and doublesex (dsx) from the A. rosae genome, all of which are crucial components of the sex determination cascade in the honeybee. As a result, we identified a sawfly ortholog of dsx (designated as Ardsx). Rapid amplification of cDNA ends (RACE) using total RNA extracted from male and female larvae identified three male-specific variants and three female-specific variants. Comparison between the full-length Ardsx cDNAs and the genomic sequence revealed that exon 5 was differentially spliced between the male- and female-specific variants. RT-PCR analysis demonstrated that Ardsx pre-mRNA was spliced alternatively in a sex-dependent manner at almost all the developmental stages. RNAi-mediated knockdown of Ardsx in males caused severe defects in the reproductive organs and, notably, induced development of the ovipository apparatus containing the dorsal pair of blades and the sheath. These males also showed abnormalities in testes and seminal vesicles and lacked mature sperm. The present study provides the first direct evidence that dsx is essential for sexual development in hymenopteran species.     

The autoregulatory loop: A common mechanism of regulation of key sex determining genes in insects

Sex determination in most insects is structured as a gene cascade, wherein a primary signal is passed through a series of sex-determining genes, culminating in a downstream double-switch known as doublesex that decides the sexual fate of the embryo. From the literature available on sex determination cascades, it becomes apparent that sex determination mechanisms have evolved rapidly. The primary signal that provides the cue to determine the sex of the embryo varies remarkably, not only among taxa, but also within taxa. Furthermore, the upstream key gene in the cascade also varies between species and even among closely related species. The order Insecta alone provides examples of astoundingly complex diversity of upstream key genes in sex determination mechanisms. Besides, unlike key upstream genes, the downstream double-switch gene is alternatively spliced to form functional sex-specific isoforms. This sex-specific splicing is conserved across insect taxa. The genes involved in the sex determination cascade such as Sex-lethal (Sxl) in Drosophila melanogaster, transformer (tra) in many other dipterans, coleopterans and hymenopterans, Feminizer (fem) in Apis mellifera, and IGF-II mRNA-binding protein (Bmimp) in Bombyx mori are reported to be regulated by an autoregulatory positive feedback loop. In this review, by taking examples from various insects, we propose the hypothesis that autoregulatory loop mechanisms of sex determination might be a general strategy. We also discuss the possible reasons for the evolution of autoregulatory loops in sex determination cascades and their impact on binary developmental choices.     

<Emphasis Type="Italic">csd</Emphasis> gene of honeybee: Genetic structure,functioning, and evolution

At present, the sex in the honeybee is known to be controlled by the csd (complementary sex determiner) gene, which has a large allelic diversity. Heterozygotes of the csd gene develop into females, while homozygotes develop into males, which are removed by the workers at the larval stage, resulting in the observation of a “shot brood.” This review summarizes the current understanding of the genetic structure of the csd gene, the mechanism of its effect on the sex determination, and the evolution of this gene generated from the duplication of the feminizer gene. The study of the csd gene polymorphism would make it possible to develop allele specific markers for the selection of honeybee.     

Sex determination in the silkworm, Bombyx mori: a female determinant on the W chromosome and the sex-determining gene cascade

In insects, the sex is determined completely by genetic mechanisms, which at least in somatic tissues, are cell autonomous. The sex of the silkworm, Bombyx mori, is strongly controlled by the presence of the W chromosome. Genetic studies using translocations and deletions of W suggested that a presumptive feminizing gene (Fem) is located in a limited region of the W chromosome. Recent genomic studies revealed a small number of potential candidates for the Fem gene in this region. In addition, a Bombyx homologue of the Drosophila sex determining gene doublesex has been identified on an autosome and analyzed. Whereas the Drosophila doublesex gene is regulated by activation of splicing in females, the Bombyx doublesex gene (Bmdsx) encodes female- and male-specific mRNAs regulated via male-specific repression of splicing. The vitellogenin gene (Vg) is a target of the BmDSX protein, which directly binds to the Vg promoter. Furthermore, as ectopic expression of the male-type Bmdsx induces male-like transformation of the sexual organs, BmDSX may control sex-specific morphological characteristics in Bombyx. This suggests that although upstream events in Drosophila and Bombyx sex determination differ, similarities between the two species do exist in downstream genetic control of sex determination.     

Tribolium castaneum Transformer-2 regulates sex determination and development in both males and females

Tribolium castaneum Transformer (TcTra) is essential for female sex determination and maintenance through the regulation of sex-specific splicing of doublesex (dsx) pre-mRNA. In females, TcTra also regulates the sex-specific splicing of its own pre-mRNA to ensure continuous production of functional Tra protein. Transformer protein is absent in males and hence dsx pre-mRNA is spliced in a default mode. The mechanisms by which males inhibit the production of functional Tra protein are not known. Here, we report on functional characterization of transformer-2 (tra-2) gene (an ortholog of Drosophila transformer-2) in T. castaneum. RNA interference-mediated knockdown in the expression of gene coding for tra-2 in female pupae or adults resulted in the production of male-specific isoform of dsx and both female and male isoforms of tra suggesting that Tra-2 is essential for the female-specific splicing of tra and dsx pre-mRNAs. Interestingly, knockdown of tra-2 in males did not affect the splicing of dsx but resulted in the production of both female and male isoforms of tra suggesting that Tra-2 suppresses female-specific splicing of tra pre-mRNA in males. This dual regulation of sex-specific splicing of tra pre-mRNA ensures a tight regulation of sex determination and maintenance. These data suggest a critical role for Tra-2 in suppression of female sex determination cascade in males. In addition, RNAi studies showed that Tra-2 is also required for successful embryonic and larval development in both sexes.     

Male-specific phosphorylated SR proteins in adult flies of the Mediterranean Fruitfly <Emphasis Type="Italic">Ceratitis capitata</Emphasis>

Alternative splicing is a widely used mechanism of gene regulation in sex determination pathways of Insects. In species from orders as distant as Diptera, Hymenoptera and Coleoptera, female differentiation relies on the activities of conserved splicing regulators, TRA and TRA-2, promoting female-specific expression of the global effector doublesex (dsx). Less understood is to what extent post-translational modifications of splicing regulators plays a role in this pathway. In Drosophila melanogaster phosphorylation of TRA, TRA-2 and the general RBP1 factor by the LAMMER kinase doa (darkener of apricot) is required for proper female sex determination. To explore whether this is a general feature of the pathway we examined sex-specific differences in phosphorylation levels of SR splicing factors in the dipteran species D. melanogaster, Ceratitis capitata (Medfly) and Musca domestica (Housefly). We found a distinct and reproducible pattern of male-specific phosphorylation on protein extracts enriched for SR proteins in C. capitata suggesting that differential phosphorylation may also contribute to the regulation of sex-specific splicing in the Medfly.     

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.Subject terms: Evolutionary genetics, Rare variants, Ecological genetics     

Double trouble: combined action of meiotic drive and Wolbachia feminization in Eurema butterflies

Arthropod sex ratios can be manipulated by a diverse range of selfish genetic elements, including maternally inherited Wolbachia bacteria. Feminization by Wolbachia is rare but has been described for Eurema mandarina butterflies. In this species, some phenotypic and functional females, thought to be ZZ genetic males, are infected with a feminizing Wolbachia strain, wFem. Meanwhile, heterogametic WZ females are not infected with wFem. Here, we establish a quantitative PCR assay allowing reliable sexing in three Eurema species. Against expectation, all E. mandarina females, including wFem females, had only one Z chromosome that was paternally inherited. Observation of somatic interphase nuclei confirmed that W chromatin was absent in wFem females, but present in females without wFem. We conclude that the sex bias in wFem lines is due to meiotic drive (MD) that excludes the maternal Z and thus prevents formation of ZZ males. Furthermore, wFem lines may have lost the W chromosome or harbour a dysfunctional version, yet rely on wFem for female development; removal of wFem results in all-male offspring. This is the first study that demonstrates an interaction between MD and Wolbachia feminization, and it highlights endosymbionts as potentially confounding factors in MD of sex chromosomes.     

A positive feedback loop mediated by CsERF31 initiates female cucumber flower development

Sex determination is a crucially important developmental event that is pervasive throughout nature and enhances the adaptation of species. Among plants, cucumber (Cucumis sativus L.) can generate both unisexual and bisexual flowers, and the sex type is mainly controlled by several 1-aminocyclopropane-1-carboxylic acid synthases (CsACSs). However, the regulatory mechanism of these synthases remains elusive. Here, we used gene expression analysis, protein–DNA interaction assays, and transgenic plants to study the function of a gynoecium-specific gene, ETHYLENE RESPONSE FACTOR31 (CsERF31), in female flower differentiation. We found that in a predetermined female flower, ethylene signaling activates CsERF31 by CsEIN3, and then CsERF31 stimulates CsACS2, which triggers a positive feedback loop to ensure female rather than bisexual flower development. A similar interplay is functionally conserved in melon (Cucumis melo L.). Knockdown of CsERF31 by RNAi causes defective bisexual flowers to replace female flowers. Ectopic expression of CsERF31 suppresses stamen development and promotes pistil development in male flowers, demonstrating that CsERF31 functions as a sex switch. Taken together, our data confirm that CsERF31 represents the molecular link between female–male determination and female–bisexual determination, and provide mechanistic insight into how ethylene promotes female flowers, rather than bisexual flowers, in cucumber sex determination.

A key regulator promotes female flower development by triggering a positive feedback loop during cucumber sex determination.     

Abnormal glucose homeostasis and pancreatic islet function in mice with inactivation of the Fem1b gene

Type 2 diabetes mellitus is a disorder of glucose homeostasis involving complex gene and environmental interactions that are incompletely understood. Mammalian homologs of nematode sex determination genes have recently been implicated in glucose homeostasis and type 2 diabetes mellitus. These are the Hedgehog receptor Patched and Calpain-10, which have homology to the nematode tra-2 and tra-3 sex determination genes, respectively. Here, we have developed Fem1b knockout (Fem1b-KO) mice, with targeted inactivation of Fem1b, a homolog of the nematode fem-1 sex determination gene. We show that the Fem1b-KO mice display abnormal glucose tolerance and that this is due predominantly to defective glucose-stimulated insulin secretion. Arginine-stimulated insulin secretion is also affected. The Fem1b gene is expressed in pancreatic islets, within both beta cells and non-beta cells, and is highly expressed in INS-1E cells, a pancreatic beta-cell line. In conclusion, these data implicate Fem1b in pancreatic islet function and insulin secretion, strengthening evidence that a genetic pathway homologous to nematode sex determination may be involved in glucose homeostasis and suggesting novel genes and processes as potential candidates in the pathogenesis of diabetes mellitus.     

脊椎动物性别决定和分化的分子机制研究进展

哺乳类性别决定是多种转录因子和生长因子相继表达和相互调控的结果。SRY的表达启动雄性通路并诱导下游雄性特异基因SOX9、AMH等的表达。FOXL2在雌性未分化性腺表达,WNT-4和DAX1也在雌性性别决定或分化时期表达,表明雌性通路也是受特定基因调控的,而并非“默认通路”。鸟类的性别也是由遗传基因决定的,EFT1(雌性)和DMRT1(雄性)可能是性别决定候选基因。爬行类为温度性别决定的典型,温度可能通过调节雌激素水平和控制性别特异遗传基因表达决定性别。大部分两栖类性别受环境因素影响,但发现DMRT1和DAX1可能与其精巢发育有关。鱼类性别决定和分化方式差异很大,多种因素(遗传基因、环境因素、类固醇激素等)参与了这一过程。从青Q鳉Y染色体定位克隆的DMY,被认为是第一个非哺乳类脊椎动物雄性性别决定基因。所有这些表明脊椎动物性别决定和分化机制是多样化的。     

Sex Reversal in Zebrafish fancl Mutants Is Caused by Tp53-Mediated Germ Cell Apoptosis

The molecular genetic mechanisms of sex determination are not known for most vertebrates, including zebrafish. We identified a mutation in the zebrafish fancl gene that causes homozygous mutants to develop as fertile males due to female-to-male sex reversal. Fancl is a member of the Fanconi Anemia/BRCA DNA repair pathway. Experiments showed that zebrafish fancl was expressed in developing germ cells in bipotential gonads at the critical time of sexual fate determination. Caspase-3 immunoassays revealed increased germ cell apoptosis in fancl mutants that compromised oocyte survival. In the absence of oocytes surviving through meiosis, somatic cells of mutant gonads did not maintain expression of the ovary gene cyp19a1a and did not down-regulate expression of the early testis gene amh; consequently, gonads masculinized and became testes. Remarkably, results showed that the introduction of a tp53 (p53) mutation into fancl mutants rescued the sex-reversal phenotype by reducing germ cell apoptosis and, thus, allowed fancl mutants to become fertile females. Our results show that Fancl function is not essential for spermatogonia and oogonia to become sperm or mature oocytes, but instead suggest that Fancl function is involved in the survival of developing oocytes through meiosis. This work reveals that Tp53-mediated germ cell apoptosis induces sex reversal after the mutation of a DNA–repair pathway gene by compromising the survival of oocytes and suggests the existence of an oocyte-derived signal that biases gonad fate towards the female developmental pathway and thereby controls zebrafish sex determination.