Sex-determining Gene Doublesex Research Articles

Changes in the cellular makeup of motor patterning circuits drive courtship song evolution in Drosophila

How evolutionary changes in genes and neurons encode species variation in complex motor behaviors is largely unknown. Here, we develop genetic tools that permit a neural circuit comparison between the model species Drosophila melanogaster and the closely related species D.yakuba, which has undergone a lineage-specific loss of sine song, one of the two major types of male courtship song in Drosophila. Neuroanatomical comparison of song-patterning neurons called TN1 across the phylogeny demonstrates a link between the loss of sine song and a reduction both in the number of TN1 neurons and the neurites supporting the sine circuit connectivity. Optogenetic activation confirms that TN1 neurons in D.yakuba have lost the ability to drive sine song, although they have maintained the ability to drive the singing wing posture. Single-cell transcriptomic comparison shows that D.yakuba specifically lacks a cell type corresponding to TN1A neurons, the TN1 subtype that is essential for sine song. Genetic and developmental manipulation reveals a functional divergence of the sex determination gene doublesex in D.yakuba to reduce TN1 number by promoting apoptosis. Our work illustrates the contribution of motor patterning circuits and cell type changes in behavioral evolution and uncovers the evolutionary lability of sex determination genes to reconfigure the cellular makeup of neural circuits.

Candidate target genes of the male-specific expressed Doublesex in the termite Reticulitermes speratus.

Eusocial insects such as termites, ants, bees, and wasps exhibit a reproductive division of labor. The developmental regulation of reproductive organ (ovaries and testes) is crucial for distinguishing between reproductive and sterile castes. The development of reproductive organ in insects is regulated by sex-determination pathways. The sex determination gene Doublesex (Dsx), encoding transcription factors, plays an important role in this pathway. Therefore, clarifying the function of Dsx in the developmental regulation of sexual traits is important to understand the social evolution of eusocial insects. However, no studies have reported the function of Dsx in hemimetabolous eusocial group termites. In this study, we searched for binding sites and candidate target genes of Dsx in species with available genome information as the first step in clarifying the function of Dsx in termites. First, we focused on the Reticulitermes speratus genome and identified 101 candidate target genes of Dsx. Using a similar method, we obtained 112, 39, and 76 candidate Dsx target genes in Reticulitermes lucifugus, Coptotermes formosanus, and Macrotermes natalensis, respectively. Second, we compared the candidate Dsx target genes between species and identified 37 common genes between R. speratus and R. lucifugus. These included several genes probably involved in spermatogenesis and longevity. However, only a few common target genes were identified between R. speratus and the other two species. Finally, Dsx dsRNA injection resulted in the differential expression of several target genes, including piwi-like protein and B-box type zinc finger protein ncl-1 in R. speratus. These results provide valuable resource data for future functional analyses of Dsx in termites.

Molecular and cellular origins of behavioral sex differences: a tiny little fly tells a lot.

Behavioral sex differences primarily derive from the sexually dimorphic organization of neural circuits that direct the behavior. In Drosophila melanogaster, the sex-determination genes fruitless (fru) and doublesex (dsx) play pivotal roles in producing the sexual dimorphism of neural circuits for behavior. Here we examine three neural groups expressing fru and/or dsx, i.e., the P1 cluster, aSP-f and aSP-g cluster pairs and aDN cluster, in which causal relationships between the dimorphic behavior and dimorphic neural characteristics are best illustrated. aSP-f, aSP-g and aDN clusters represent examples where fru or dsx switches cell-autonomously their neurite structures between the female-type and male-type. Processed sensory inputs impinging on these neurons may result in outputs that encode different valences, which culminate in the execution of distinct behavior according to the sex. In contrast, the P1 cluster is male-specific as its female counterpart undergoes dsx-driven cell death, which lowers the threshold for the induction of male-specific behaviors. We propose that the products of fru and dsx genes, as terminal selectors in sexually dimorphic neuronal wiring, induce and maintain the sex-typical chromatin state at postembryonic stages, orchestrating the transcription of effector genes that shape single neuron structures and govern cell survival and death.

Cell-based analysis reveals that sex-determining gene signals in Ostrinia are pivotally changed by male-killing Wolbachia.

Wolbachia, a maternally transmitted bacterium, shows male-killing, an adaptive phenotype for cytoplasmic elements, in various arthropod species during the early developmental stages. In lepidopteran insects, lethality of males is accounted for by improper dosage compensation in sex-linked genes owing to Wolbachia-induced feminization. Herein, we established Ostrinia scapulalis cell lines that retained sex specificity per the splicing pattern of the sex-determining gene doublesex (Osdsx). We found that Wolbachia transinfection in male cell lines enhanced the female-specific splice variant of Osdsx (OsdsxF ) while suppressing the male-specific variant (OsdsxM ), indicating that Wolbachia affects sex-determining gene signals even in vitro. Comparative transcriptome analysis isolated only two genes that behave differently upon Wolbachia infection. The two genes were respectively homologous to Masculinizer (BmMasc) and zinc finger-2 (Bmznf-2), male-specifically expressed sex-determining genes of the silkworm Bombyx mori that encode CCCH-type zinc finger motif proteins. By using cultured cells and organismal samples, OsMasc and Osznf-2 were found to be sex-determining genes of O. scapulalis that are subjected to sex-specific alternative splicing depending upon the chromosomal sex, developmental stage, and infection status. Overall, our findings expound the cellular autonomy in insect sex determination and the mechanism through which sex is manipulated by intracellular selfish microbes.

New self-sexing Aedes aegypti strain eliminates barriers to scalable and sustainable vector control for governments and communities in dengue-prone environments.

For more than 60years, efforts to develop mating-based mosquito control technologies have largely failed to produce solutions that are both effective and scalable, keeping them out of reach of most governments and communities in disease-impacted regions globally. High pest suppression levels in trials have yet to fully translate into broad and effective Aedes aegypti control solutions. Two primary challenges to date-the need for complex sex-sorting to prevent female releases, and cumbersome processes for rearing and releasing male adult mosquitoes-present significant barriers for existing methods. As the host range of Aedes aegypti continues to advance into new geographies due to increasing globalisation and climate change, traditional chemical-based approaches are under mounting pressure from both more stringent regulatory processes and the ongoing development of insecticide resistance. It is no exaggeration to state that new tools, which are equal parts effective and scalable, are needed now more than ever. This paper describes the development and field evaluation of a new self-sexing strain of Aedes aegypti that has been designed to combine targeted vector suppression, operational simplicity, and cost-effectiveness for use in disease-prone regions. This conditional, self-limiting trait uses the sex-determination gene doublesex linked to the tetracycline-off genetic switch to cause complete female lethality in early larval development. With no female progeny survival, sex sorting is no longer required, eliminating the need for large-scale mosquito production facilities or physical sex-separation. In deployment operations, this translates to the ability to generate multiple generations of suppression for each mosquito released, while being entirely self-limiting. To evaluate these potential benefits, a field trial was carried out in densely-populated urban, dengue-prone neighbourhoods in Brazil, wherein the strain was able to suppress wild mosquito populations by up to 96%, demonstrating the utility of this self-sexing approach for biological vector control. In doing so, it has shown that such strains offer the critical components necessary to make these tools highly accessible, and thus they harbour the potential to transition mating-based approaches to effective and sustainable vector control tools that are within reach of governments and at-risk communities who may have only limited resources.

Nucleoporin107 mediates female sexual differentiation via Dsx.

We recently identified a missense mutation in Nucleoporin107 (Nup107; D447N) underlying XX-ovarian-dysgenesis, a rare disorder characterized by underdeveloped and dysfunctional ovaries. Modeling of the human mutation in Drosophila or specific knockdown of Nup107 in the gonadal soma resulted in ovarian-dysgenesis-like phenotypes. Transcriptomic analysis identified the somatic sex-determination gene doublesex (dsx) as a target of Nup107. Establishing Dsx as a primary relevant target of Nup107, either loss or gain of Dsx in the gonadal soma is sufficient to mimic or rescue the phenotypes induced by Nup107 loss. Importantly, the aberrant phenotypes induced by compromising either Nup107 or dsx are reminiscent of bone morphogenetic protein (BMP signaling hyperactivation). Remarkably, in this context, the metalloprotease AdamTS-A, a transcriptional target of both Dsx and Nup107, is necessary for the calibration of BMP signaling. As modulation of BMP signaling is a conserved critical determinant of soma-germline interaction, the sex- and tissue-specific deployment of Dsx-F by Nup107 seems crucial for the maintenance of the homeostatic balance between the germ cells and somatic gonadal cells.

The sex determination gene doublesex regulates expression and secretion of the basement membrane protein Collagen IV

The highly conserved doublesex (dsx) and doublesex/mab-3 related (Dmrt) genes control sexually dimorphic traits across animals. The dsx gene encodes sex-specific transcription factors, DsxM in males and DsxF in females, which function differentially and often oppositely to establish sexual dimorphism. Here, we report that mutations in dsx, or overexpression of dsx, result in abnormal distribution of the basement membrane (BM) protein Collagen IV in the fat body. We find that Dsx isoforms regulate the expression of Collagen IV in the fat body and its secretion into the BM of other tissues. We identify the procollagen lysyl hydroxylase (dPlod) gene, which is involved in the biosynthesis of Collagen IV, as a direct target of Dsx. We further show that Dsx regulates Collagen IV through dPlod-dependent and independent pathways. These findings reveal how Dsx isoforms function in the secretory fat body to regulate Collagen IV and remotely establish sexual dimorphism.

Evolutionary transition of doublesex regulation from sex-specific splicing to male-specific transcription in termites

The sex determination gene doublesex (dsx) encodes a transcription factor with two domains, oligomerization domain 1 (OD1) and OD2, and is present throughout insects. Sex-specific Dsx splicing isoforms regulate the transcription of target genes and trigger sex differentiation in all Holometabola examined to date. However, in some hemimetabolous insects, dsx is not spliced sexually and its sequence is less conserved. Here, to elucidate evolutionary changes in dsx in domain organisation and regulation in termites, we searched genome and/or transcriptome databases for the dsx OD1 and OD2 in seven termite species and their sister group (Cryptocercus woodroaches). Molecular phylogenetic and synteny analyses identified OD1 sequences of termites and C. punctulatus that clustered with dsx of Holometabola and regarded them as dsx orthologues. The Cryptocercus dsx orthologue containing OD2 was spliced sexually, as previously shown in other insects. However, OD2 was not found in all termite dsx orthologues. These orthologues were encoded by a single exon in three termites for which genome information is available; they were not alternatively spliced but transcribed in a male-specific manner in two examined species. Evolution of dsx regulation from sex-specific splicing to male-specific transcription may have occurred at an early stage of social evolution in termites.

Doublesex mediates species-, sex-, environment- and trait-specific exaggeration of size and shape.

Context-dependent trait exaggeration is a major contributor to phenotypic diversity. However, the genetic modifiers instructing development across multiple contexts remain largely unknown. We use the arthropod tibia, a hotspot for segmental differentiation, as a paradigm to assess the developmental mechanisms underlying the context-dependent structural exaggeration of size and shape through nutritional plasticity, sexual dimorphism and segmental differentiation. Using an RNAseq approach in the sexually dimorphic and male-polyphenic dung beetle Digitonthophagus gazella, we find that only a small portion (3.7%) of all transcripts covary positively in expression level with trait size across contexts. However, RNAi-mediated knockdown of the conserved sex-determination gene doublesex suggests that it functions as a context-dependent master mediator of trait exaggeration in D. gazella as well as the closely related dung beetle Onthophagus taurus. Taken together, our findings suggest (i) that the gene networks associated with trait exaggeration are highly dependent on the precise developmental context, (ii) that doublesex differentially shapes morphological exaggeration depending on developmental contexts and (iii) that this context-specificity of dsx-mediated trait exaggeration may diversify rapidly. This mechanism may contribute to the resolution of conflict arising from environment-dependent antagonistic selection among sexes and divergent developmental contexts in a wide range of animals.

A role for sex-determination genes in life history evolution? Doublesex mediates sexual size dimorphism in the gazelle dung beetle.

An organism's fitness depends strongly on its age and size at maturation. Although the evolutionary forces acting on these critical life history traits have been heavily scrutinized, the developmental mechanisms underpinning intraspecific variation in adult size and development time remain much less well-understood. Using RNA interference, I here show that the highly conserved sex-determination gene doublesex (dsx) mediates sexual size dimorphism (SSD) in the gazelle dung beetle Digitonthophagus gazella. Because doublesex undergoes sex-specific splicing and sex-limited isoforms regulate different target genes, this suggests that dsx contributes to the resolution of intralocus sexual conflict in body size. However, these results contrast with previous studies demonstrating that dsx does not affect body size or SSD in Drosophila. This indicates that intraspecific body size variation is underlain by contrasting developmental mechanisms in different insect lineages. Furthermore, although male D.gazella have a longer development time than females, sexual bimaturism was not affected by dsx expression knockdown. In addition, and in contrast to secondary sexual morphology, dsx did not significantly affect nutritional plasticity in life history. Taken together, these findings indicate that dsx signalling contributes to intraspecific life history variation but that dsx's function in mediating sexual dimorphism in life history differs among traits and species. More generally, these findings suggest that genes ancestrally tasked with sex determination have been co-opted into the developmental regulation of life history traits and may represent an underappreciated mechanism of life history evolution.

Identification and characterization of sexually dimorphic neurons that express the sex-determining gene doublesex in the brain of silkmoth Bombyx mori

Sexual differences in behavior are generated by sexually dimorphic neural circuits in animals. In insects, a highly conserved sex-determining gene doublesex (dsx) plays essential roles in the development of sexual dimorphisms. In the present study, to elucidate the neural basis of sexual differences in behaviors of silkmoth Bombyx mori, we investigated Bombyx mori dsx (Bmdsx) expression in the brains through development. In the brain, Bmdsx was differentially expressed in sex- and developmental stage-dependent manners. BmDSX protein-expressing cells were located in the dorsomedial region of the pupal and adult brains, and constituted two and one neural clusters in males and females, respectively. The number of BmDSX-positive cells was developmentally regulated and peaked at the early to middle pupal stages, suggesting that the sexually dimorphic neural circuits are established during this period. The detection of a neural activity marker protein BmHR38 suggested that the BmDSX-positive cells are not active during sexual behavior in both male and female moths, even though the cells in the vicinity of the BmDSX-positive cell clusters are active. These results imply that Bmdsx plays roles in the development of sexually dimorphic neural circuits, but the neural circuits are not related to sexual behavior in silkmoths.

Effects of prohibitin on ageing process and reproduction ofDaphnia pulex

Daphnia pulex is considered as a great natural food for aquaculture, and Prohibitin (PHB) is a functional protein that plays a key role in eukaryotic cell activities such as proliferation and ageing. To explore the function of PHB in the ageing process of Daphnia pulex (D. pulex), we cloned and analysed the full-length cDNA of phb1 and phb2. With the growth of D. pulex, expression of two genes have a N curve trend which raised up first and dropped then raised again. Western blot analysis revealed that expression of the PHB proteins increased from 1- to 10-day age, but did not significantly change from 10- to 35-day age. RNAi knockdown of phb1, reduced expression of phb2 and PHB protein, and significantly induced the expression of sex-determining related gene Doublesex (Dsx), and ageing-related pathway gene Protein kinase B (Akt). Longevity of the individuals in RNAi group was shortened to 45 days, which was significantly shorter than the other groups. Despite the growth was not significantly different, reproduction amount was significantly impacted by RNAi. These results suggest that PHB significantly affect ageing of D. pulex and may be involved in the reproductive transformation, leading to a decline of reproductive capacity. This study develops our understanding of the ageing mechanism in D. pulex as a potential model organism, provides further insight into the effect of PHB in the processes of ageing and expands our knowledge of molecular mechanisms on ageing and reproduction.

Activity-dependent visualization and control of neural circuits for courtship behavior in the fly Drosophila melanogaster

Males of Drosophila melanogaster exhibit stereotypic courtship behavior through which they assess potential mates by processing multimodal sensory information. Although previous studies revealed important neural circuits involved in this process, the full picture of circuits that participate in male courtship remains elusive. Here, we established a genetic tool to visualize or optogenetically reactivate neural circuits activated upon specific behavior, exploiting promoter activity of a neural activity-induced gene Hr38 With this approach, we visualized neural circuits activated in the male brain and the ventral nerve cord when a male interacted with a female. The labeling of neural circuits was additively dependent on inputs from antennae and foreleg tarsi. In addition, neural circuits that express the sex-determining gene fruitless or doublesex were extensively labeled by interaction with a female. Furthermore, optogenetic reactivation of the labeled neural circuits induced courtship posture. With this mapping system, we found that a fruitless-positive neural cluster aSP2 was labeled when a male interacted with a female, in addition to previously characterized neurons. Silencing of neurons including aSP2 led to frequent interruption of courtship and significant reduction of mating success rate without affecting latency to start courtship, suggesting that these neurons are required for courtship persistency important for successful copulation. Overall, these results demonstrate that activity-dependent labeling can be used as a powerful tool not only in vertebrates, but also in invertebrates, to identify neural circuits regulating innate behavior.

The splicing factor transformer2 (tra2) functions in the Drosophila fat body to regulate lipid storage

Excess nutrients are stored as triglycerides mainly in the adipose tissue of an animal and these triglycerides are located in structures called lipid droplets. Previous genome-wide RNAi screens in Drosophila cells identified splicing factors as playing a role in lipid droplet formation. Our lab has recently identified the SR protein, 9G8, as an important factor in fat storage as decreasing its levels results in augmented triglyceride storage in the fat body. Previous in vitro studies have implicated 9G8 in the regulation of splicing of the sex determination gene doublesex (dsx) by binding to transformer (tra) and transformer2 (tra2); however, any function of these sex determination proteins in regulating metabolism is unknown. In this study, we have uncovered a role of tra2 to regulate fat storage in vivo. Inducing tra2dsRNA in the adult fat body resulted in an increase in triglyceride levels but had no effect on glycogen storage. Consistent with the triglyceride phenotype, tra2 knockdown flies lived longer under starvation conditions. In addition, this increase in triglycerides is due to more fat storage per cell and not an increase in the number of fat cells. Interestingly, the splicing of CPT1, an enzyme involved in the breakdown of lipids, was altered in flies with decreased tra2. The less-catalytically active isoform of CPT1 accumulated in tra2dsRNA flies suggesting a decrease in lipid breakdown, which is consistent with the increased triglyceride levels observed in these flies. Together, these results suggest a link between mRNA splicing, sex determination and lipid metabolism and may provide insight into the mechanisms underlying tissue-specific splicing and nutrient storage.

Maternal provision of non-sex-specific transformer messenger RNA in sex determination of the wasp Asobara tabida.

In many insect species maternal provision of sex-specifically spliced messenger RNA (mRNA) of sex determination genes is an essential component of the sex determination mechanism. In haplodiploid Hymenoptera, maternal provision in combination with genomic imprinting has been shown for the parasitoid Nasonia vitripennis, known as maternal effect genomic imprinting sex determination (MEGISD). Here, we characterize the sex determination cascade of Asobara tabida, another hymenopteran parasitoid. We show the presence of the conserved sex determination genes doublesex (dsx), transformer (tra) and transformer-2 (tra2) orthologues in As. tabida. Of these, At-dsx and At-tra are sex-specifically spliced, indicating a conserved function in sex determination. At-tra and At-tra2 mRNA is maternally provided to embryos but, in contrast to most studied insects, As. tabida females transmit a non-sex-specific splice form of At-tra mRNA to the eggs. In this respect, As. tabida sex determination differs from the MEGISD mechanism. How the paternal genome can induce female development in the absence of maternal provision of sex-specifically spliced mRNA remains an open question. Our study reports a hitherto unknown variant of maternal effect sex determination and accentuates the diversity of insect sex determination mechanisms.

Identification and functional characterization of the sex-determining gene doublesex in the sawfly, Athalia rosae\xa0(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.

Asymmetric interactions between doublesex and tissue- and sex-specific target genes mediate sexual dimorphism in beetles

Sexual dimorphisms fuel significant intraspecific variation and evolutionary diversification. Yet the developmental-genetic mechanisms underlying sex-specific development remain poorly understood. Here, we focus on the conserved sex-determination gene doublesex (dsx) and the mechanisms by which it mediates sex-specific development in a horned beetle species by combining systemic dsx knockdown, high-throughput sequencing of diverse tissues and a genome-wide analysis of Dsx-binding sites. We find that Dsx regulates sex-biased expression predominantly in males, that Dsx's target repertoires are highly sex- and tissue-specific and that Dsx can exercise its regulatory role via two distinct mechanisms: as a sex-specific modulator by regulating strictly sex-specific targets, or as a switch by regulating the same genes in males and females in opposite directions. More generally, our results suggest Dsx can rapidly acquire new target gene repertoires to accommodate evolutionarily novel traits, evidenced by the large and unique repertoire identified in head horns, a recent morphological innovation.

Activation of Latent Courtship Circuitry in the Brain of Drosophila Females Induces Male-like Behaviors

SummaryCourtship in Drosophila melanogaster offers a powerful experimental paradigm for the study of innate sexually dimorphic behaviors [1, 2]. Fruit fly males exhibit an elaborate courtship display toward a potential mate [1, 2]. Females never actively court males, but their response to the male’s display determines whether mating will actually occur. Sex-specific behaviors are hardwired into the nervous system via the actions of the sex determination genes doublesex (dsx) and fruitless (fru) [1]. Activation of male-specific dsx/fru+ P1 neurons in the brain initiates the male’s courtship display [3, 4], suggesting that neurons unique to males trigger this sex-specific behavior. In females, dsx+ neurons play a pivotal role in sexual receptivity and post-mating behaviors [1, 2, 5, 6, 7, 8, 9]. Yet it is still unclear how dsx+ neurons and dimorphisms in these circuits give rise to the different behaviors displayed by males and females. Here, we manipulated the function of dsx+ neurons in the female brain to investigate higher-order neurons that drive female behaviors. Surprisingly, we found that activation of female dsx+ neurons in the brain induces females to behave like males by promoting male-typical courtship behaviors. Activated females display courtship toward conspecific males or females, as well other Drosophila species. We uncovered specific dsx+ neurons critical for driving male courtship and identified pheromones that trigger such behaviors in activated females. While male courtship behavior was thought to arise from male-specific central neurons, our study shows that the female brain is equipped with latent courtship circuitry capable of inducing this male-specific behavioral program.

Molecular cloning and functional characterization of the sex-determination gene doublesex in the sexually dimorphic broad-horned beetle Gnatocerus cornutus (Coleoptera, Tenebrionidae).

Various types of weapon traits found in insect order Coleoptera are known as outstanding examples of sexually selected exaggerated characters. It is known that the sex determination gene doublesex (dsx) plays a significant role in sex-specific expression of weapon traits in various beetles belonging to the superfamily Scarabaeoidea. Although sex-specific weapon traits have evolved independently in various Coleopteran groups, developmental mechanisms of sex-specific expression have not been studied outside of the Scarabaeoidea. In order to test the hypothesis that dsx-dependent sex-specific expression of weapon traits is a general mechanism among the Coleoptera, we have characterized the dsx in the sexually dimorphic broad-horned beetle Gnatocerus cornutus (Tenebrionidea, Tenebirionidae). By using molecular cloning, we identified five splicing variants of Gnatocerus cornutus dsx (Gcdsx), which are predicted to code four different isoforms. We found one male-specific variant (GcDsx-M), two female-specific variants (GcDsx-FL and GcDsx-FS) and two non-sex-specific variants (correspond to a single isoform, GcDsx-C). Knockdown of all Dsx isoforms resulted in intersex phenotype both in male and female. Also, knockdown of all female-specific isoforms transformed females to intersex phenotype, while did not affect male phenotype. Our results clearly illustrate the important function of Gcdsx in determining sex-specific trait expression in both sexes.

Expression profile of the sex determination gene doublesex in a gynandromorph of bumblebee, Bombus ignitus.

Gynandromorphy that has both male and female features is known in many insect orders, including Hymenoptera. In most cases, however, only external morphology and behavioral aspects have been studied. We found a gynandromorph of bumblebee, Bombus ignitus, that showed almost bilateral distribution of external sexual traits, with male characters observed on the left side and female characters on the right side. This individual never exhibited sexual behavior toward new queens. The dissection of the head part showed that it had bilaterally dimorphic labial glands, only the left of which was well developed and synthesized male-specific pheromone components. In contrast, the gynandromorph possessed an ovipositor and a pair of ovaries in the abdominal part, suggesting that it had a uniformly female reproductive system. Furthermore, we characterized several internal organs of the gynandromorph by a molecular biological approach. The expression analyses of a sex determination gene, doublesex, in the brain, the fat bodies, the hindgut, and the ovaries of the gynandromorph revealed a male-type expression pattern exclusively in the left brain hemisphere and consistent female-type expression in other tissues. These findings clearly indicate the sexual discordance between external traits and internal organs in the gynandromorph. The results of genetic analyses using microsatellite markers suggested that this individual consisted of both genetically male- and female-type tissues.