Sex determination in the Drosophila germline is dictated by the sexual identity of the surrounding soma

It has been suggested that sexual identity in the germline depends upon the combination of a nonautonomous somatic signaling pathway and an autonomous X chromosome counting system. In the studies reported here, we have examined the role of the sexual differentiation genes transformer (tra) and doublesex (dsx) in regulating the activity of the somatic signaling pathway. We asked whether ectopic somatic expression of the female products of the tra and dsx genes could feminize the germline of XY animals. We find that Tra(F) is sufficient to feminize XY germ cells, shutting off the expression of male-specific markers and activating the expression of female-specific markers. Feminization of the germline depends upon the constitutively expressed transformer-2 (tra-2) gene, but does not seem to require a functional dsx gene. However, feminization of XY germ cells by Tra(F) can be blocked by the male form of the Dsx protein (Dsx(M)). Expression of the female form of dsx, Dsx(F), in XY animals also induced germline expression of female markers. Taken together with a previous analysis of the effects of mutations in tra, tra-2, and dsx on the feminization of XX germ cells in XX animals, our findings indicate that the somatic signaling pathway is redundant at the level tra and dsx. Finally, our studies call into question the idea that a cell-autonomous X chromosome counting system plays a central role in germline sex determination.     

The Orthologue of the Fruitfly Sex Behaviour Gene Fruitless in the Mosquito Aedes aegypti: Evolution of Genomic Organisation and Alternative Splicing

In Drosophila melanogaster the doublesex (dsx) and fruitless (fru) regulatory genes act at the bottom of the somatic sex determination pathway. Both are regulated via alternative splicing by an upstream female-specific TRA/TRA-2 complex, recognizing a common cis element. dsx controls somatic sexual differentiation of non-neural as well as of neural tissues. fru, on the other hand, expresses male-specific functions only in neural system where it is required to built the neural circuits underlying proper courtship behaviour. In the mosquito Aedes aegypti sex determination is different from Drosophila. The key male determiner M, which is located on one of a pair of homomorphic sex chromosomes, controls sex-specific splicing of the mosquito dsx orthologue. In this study we report the genomic organization and expression of the fru homologue in Ae. aegypti (Aeafru). We found that it is sex-specifically spliced suggesting that it is also under the control of the sex determination pathway. Comparative analyses between the Aeafru and Anopheles gambiae fru (Angfru) genomic loci revealed partial conservation of exon organization and extensive divergence of intron lengths. We find that Aeadsx and Aeafru share novel cis splicing regulatory elements conserved in the alternatively spliced regions. We propose that in Aedes aegypti sex-specific splicing of dsx and fru is most likely under the control of splicing regulatory factors which are different from TRA and TRA-2 found in other dipteran insects and discuss the potential use of fru and dsx for developing new genetic strategies in vector control.     

The sex-determining gene doublesex in the fly Megaselia scalaris: conserved structure and sex-specific splicing.

The well-known sex-determining cascade of Drosophila melanogaster serves as a paradigm for the pathway to sexual development in insects. But the primary sex-determining signal and the subsequent step, Sex-lethal (Sxl), have been shown not to be functionally conserved in non-Drosophila flies. We isolated doublesex (dsx), which is a downstream step in the cascade, from the phorid fly Megaselia scalaris, which is a distant relative of D. melanogaster. Conserved properties, e.g., sex-specific splicing, structure of the female-specific 3' splice site, a splicing enhancer region with binding motifs for the TRA2/RBP1/TRA complex that activates female-specific splicing in Drosophila, and conserved domains for DNA-binding and oligomerization in the putative DSX protein, indicate functional conservation of dsx in M. scalaris. Hence, the dsx step of the sex-determining pathway appears to be conserved among flies and probably in an even wider group of insects, as the analysis of a published cDNA from the silkmoth indicates.     

昆虫性别决定机制研究进展

阐述昆虫的性别决定机制是理解昆虫性别分化调控的理论基础,也为人类有效控制害虫开辟了新方向。昆虫性别决定机制存在复杂性和多样性,但主要是内因即性别决定基因级联互作调控的结果。本文对近年来基于性别决定基因级联互作的昆虫性别决定机制研究进行了综述,主要包括性别决定基因概况和重要性别决定相关基因的分子级联互作关系。目前发现昆虫重要性别决定相关基因主要集中在常染色体上,且部分基因之间存在紧密的级联互作,如Sxl,tra,dsx,csd和fem等。在这些基因中,tra/fem→dsx的调控模式在已报道的昆虫中存在共性,即tra和dsx相对较保守且tra通过性特异剪切来调控下游dsx的转录形式。目前大多数昆虫的性别决定机制还不清楚,但近年来模式昆虫性别决定机制取得了一定进展,对非模式昆虫的研究还处于起步阶段但却越来越受到重视。     

The sex-determination genes fruitless and doublesex specify a neural substrate required for courtship song

Courtship song is a critical component of male courtship behavior in Drosophila, making the female more receptive to copulation and communicating species-specific information [1-6]. Sex mosaic studies have shown that the sex of certain regions of the central nervous system (CNS) is critical to song production [7]. Our examination of one of these regions, the mesothoracic ganglion (Msg), revealed the coexpression of two sex-determination genes, fruitless (fru) and doublesex (dsx). Because both genes are involved in creating a sexually dimorphic CNS [8, 9] and are necessary for song production [10-13], we investigated the individual contributions of fru and dsx to the specification of a male CNS and song production. We show a novel requirement for dsx in specifying a sexually dimorphic population of fru-expressing neurons in the Msg. Moreover, by using females constitutively expressing the male-specific isoforms of fru (Fru(M)), we show a critical requirement for the male isoform of dsx (Dsx(M)), alongside Fru(M), in the specification of courtship song. Therefore, although Fru(M) expression is sufficient for the performance of many male-specific behaviors [14], we have shown that without Dsx(M), the determination of a male-specific CNS and thus a full complement of male behaviors are not realized.     

双翅目害虫性别分离技术的研究进展及展望

在过去的几十年中,昆虫不育技术(sterile insect technique, SIT)已被用于防治农业害虫和人类健康相关的病媒害虫。相较于传统的农药控制策略,昆虫不育技术具有物种特异性和环境友好型等特点。通过释放不育雄虫的昆虫不育技术的主要障碍是在大规模饲养阶段将雄性与雌性分离,从而提高这些防治方法的成本效率,并防止释放携带和传播疾病的雌性群体。目前大多数针对双翅目害虫的遗传防治策略没有进行性别分离,少数害虫性别分离方法是基于蛹的大小或者雌雄蛹羽化时间差异进行人工识别和机械识别分离。双翅目昆虫性别决定及分化分子机制多种多样,其性别决定主要信号差异巨大,其多种性别决定基因已用于性别分离系统的开发。性比失衡性别分离策略通过破坏性别决定途径关键基因的表达获得雄性偏向后代,雌性条件性致死分离策略利用性别决定关键基因的雌雄选择性剪接差异实现性别分离,这两种性别分离策略目前正在害虫不育防治中接受大规模饲养应用评估,而基于双翅目昆虫雌雄性二态和基因标记发展的可视化性别分离策略也已成功实现多种害虫的性别分离。我们对性比失衡分离策略、雌性条件性致死分离策略和可视化性别分离策略在双翅目害虫中的研究进展进行了综述,重点评估了这些方法在雄虫大规模饲养和释放的应用潜力,以期在更完善的性别分离技术支持下为害虫防治研究取得更多突破性进展。     

Organ-specific patterns of gene expression in the reproductive tract of Drosophila are regulated by the sex-determination genes

The sex-determination genes of Drosophila act to repress the developmental pathway for the internal somatic reproductive organs of the opposite sex. By misregulating this pathway during preadult development, the organ-specific expression pattern of the glucose dehydrogenase gene (Gld) in the reproductive tract of adult flies has been changed without a concomitant sexual transformation of the reproductive organs. Misregulation of the tra, tra-2, and dsx genes leads to very similar patterns of ectopic expression of Gld. The induced ectopic patterns of Gld expression at the adult stage occur in a small subset of organs which all normally express the Gld gene during their morphogenesis. These ectopic patterns are irrevocably set during late larval-early pupal development. The normal pattern of Gld expression in several other Drosophila species is quite similar to the ectopic patterns which we have generated in D. melanogaster, suggesting that the interspecific variation in Gld expression may result from variation in the expression of the sex-determination genes.     

Drosophila intersex orthologue in the silkworm, Bombyx mori and related species

Intersex (ix), a gene required for female sexual development in Drosophila, acts in concert with doublesex (dsx) at the end of the sex determination pathway. In the present study a homologue of ix was identified in Bombyx mori. Expression analysis of this gene by RT–PCR and RNase protection assay revealed a diagnostic alternative splice form present only in testis, whereas the most common splice form was found to express in all other tissues from early embryonic developmental stages. The present study provides evidence for the presence of an alternative splice form of ix in three species of silkmoths examined. Taken together with the results of an earlier study on ix in piralid moth, Maruca vitrata (Cavaliere et al. 2009), the present study suggests that the testis-specific splice form may be a characteristic feature of lepidopterans. Though ix lacks a conserved splicing pattern it appears to have retained its functional conservation in terminal sexual differentiation. We speculate that the presence of an additional splice form, perhaps encoding non-functional protein only in testis, may prevent the feminizing effects exerted by the functional IX protein.     

Positive and purifying selection influence the evolution of doublesex in the Anastrepha fraterculus species group

The gene doublesex (dsx) is considered to be under strong selective constraint along its evolutionary history because of its central role in somatic sex differentiation in insects. However, previous studies of dsx used global estimates of evolutionary rates to investigate its molecular evolution, which potentially miss signals of adaptive changes in generally conserved genes. In this work, we investigated the molecular evolution of dsx in the Anastrepha fraterculus species group (Diptera, Tephritidae), and test the hypothesis that this gene evolved solely by purifying selection using divergence-based and population-based methods. In the first approach, we compared sequences from Anastrepha and other Tephritidae with other Muscomorpha species, analyzed variation in nonsynonymous to synonymous rate ratios (dN/dS) in the Tephritidae, and investigated radical and conservative changes in amino acid physicochemical properties. We show a general selective constraint on dsx, but with signs of positive selection mainly in the common region. Such changes were localized in alpha-helices previously reported to be involved in dimer formation in the OD2 domain and near the C-terminal of the OD1 domain. In the population-based approach, we amplified a region of 540 bp that spanned almost all of the region common to both sexes from 32 different sites in Brazil. We investigated patterns of selection using neutrality tests based on the frequency spectrum and locations of synonymous and nonsynonymous mutations in a haplotype network. As in the divergence-based approach, these analyses showed that dsx has evolved under an overall selective constraint, but with some events of positive selection. In contrast to previous studies, our analyses indicate that even though dsx has indeed evolved as a conserved gene, the common region of dsx has also experienced bouts of positive selection, perhaps driven by sexual selection, during its evolution.