The housefly, Musca domestica, is an excellent model system to study the diversification of the pathway that specifies the sexual fate. A number of different mechanisms have been described in the housefly, which reflects in part the broad diversity of sex-determining strategies used in insects. In this study we present the molecular identification and characterization of F, which acts as the master switch in the housefly pathway. We provide evidence that F corresponds to the transformer ortholog in Musca (Mdtra), which, as a result of alternative processing, expresses functional products only in individuals committed to the female fate. We demonstrate that, once activated, a self-sustaining feedback loop will maintain the female-promoting functions of Mdtra. Absence of Mdtra transcripts in eggs of Arrhenogenic (Ag) mutant females suggests that maternally deployed Mdtra activity initiates this self-sustaining loop in the zygote. When an M factor is paternally transmitted to the zygote, the establishment of the loop is prevented at an early stage before cellularization and splicing of Mdtra shifts irreversibly to the male nonproductive mode. On the basis of the analysis of two mutant alleles we can explain the different sex-determining systems in the housefly largely as deviations at the level of Mdtra regulation. This plasticity in the housefly pathway may provide a suitable framework to understand the evolution of sex-determining mechanisms in other insect species. For instance, while sex determination in a close relative, the tsetse fly Glossina morsitans, differs at the level of the instructive signal, we find that its tra ortholog, Gmtra, is regulated in a mode similar to that of Mdtra.

In Drosophila melanogaster, transformer-2 (TRA-2) which is a non-sex-specific auxiliary splicing factor, is required to promote female sexual differentiation by interaction with the female-specific TRA. The two proteins positively regulate the splicing of both doublesex (dsx) and fruitless (fru) pre-mRNAs, which in turn regulate phenotypic and behavioural sexual dimorphism. In the Mediterranean fruitfly Ceratitis capitata, the female-specific CcTRA is similarly required not only for Ccdsx splicing, but also to exert a novel autoregulatory function that consists of promoting female-specific splicing of Cctra pre-mRNA. This study reports the isolation and functional analysis of the C. capitata homologue of the Drosophila transformer-2 gene (Cctra-2). Transient RNAi against Cctra-2 during embryonic development causes the full sex reversal of XX flies in adult fertile pseudo-males, as well as changes in the splicing pattern of Cctra, Ccdsx and Ccfruitless (Ccfru). We propose that: 1) Cctra-2, as in Drosophila, is necessary for promoting Ccdsx and putative Ccfru pre-mRNA female-specific splicing and that 2) unlike in Drosophila, Cctra-2 appears to be necessary for establishing female sex determination in early XX embryos and for maintaining the positive feedback regulation of Cctra during development.

Recent studies in a representative selection of holometabolous insects suggest that, despite diversity at the instructive level, the signal-relaying part of the sex-determining pathway is remarkably well conserved. In principle, it is composed of the transformer gene (tra), which acts as a common binary switch that transduces the selected sexual fate, female when ON, male when OFF, to the downstream effector doublesex(dsx) that controls overt sexual differentiation. An interesting recurrent feature is that tra is switched ON in the early zygote by maternally provisioned tra activity. Different male-determining signals evolved, which prevent maternal activation of zygotic tra to allow for male development. In some species, where lack of maternal activation leaves tra in the OFF state, novel female-determining signals were deployed to activate zygotic tra. It appears that both the instructive end of the pathway upstream of tra as well as the executive end downstream of dsx are primary targets of evolutionary divergence, while the transduction part seems less prone to changes. We propose that this is a feature shared with many other signaling cascades that regulate developmental fates.

BackgroundThe Mediterranean fruit fly (medfly), Ceratitis capitata, is a major destructive insect pest due to its broad host range, which includes hundreds of fruits and vegetables. It exhibits a unique ability to invade and adapt to ecological niches throughout tropical and subtropical regions of the world, though medfly infestations have been prevented and controlled by the sterile insect technique (SIT) as part of integrated pest management programs (IPMs). The genetic analysis and manipulation of medfly has been subject to intensive study in an effort to improve SIT efficacy and other aspects of IPM control.ResultsThe 479 Mb medfly genome is sequenced from adult flies from lines inbred for 20 generations. A high-quality assembly is achieved having a contig N50 of 45.7 kb and scaffold N50 of 4.06 Mb. In-depth curation of more than 1800 messenger RNAs shows specific gene expansions that can be related to invasiveness and host adaptation, including gene families for chemoreception, toxin and insecticide metabolism, cuticle proteins, opsins, and aquaporins. We identify genes relevant to IPM control, including those required to improve SIT.ConclusionsThe medfly genome sequence provides critical insights into the biology of one of the most serious and widespread agricultural pests. This knowledge should significantly advance the means of controlling the size and invasive potential of medfly populations. Its close relationship to Drosophila, and other insect species important to agriculture and human health, will further comparative functional and structural studies of insect genomes that should broaden our understanding of gene family evolution.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-1049-2) contains supplementary material, which is available to authorized users.

In the tephritids Ceratitis capitata and Bactrocera oleae, the gene transformer acts as the memory device for sex determination, via an auto-regulatory function; and functional Tra protein is produced only in females. This paper investigates the evolution of the gene tra, which was characterised in twelve tephritid species belonging to the less extensively analysed genus Anastrepha. Our study provided the following major conclusions. Firstly, the memory device mechanism used by this gene in sex determination in tephritids likely existed in the common ancestor of the Ceratitis, Bactrocera and Anastrepha phylogenetic lineages. This mechanism would represent the ancestral state with respect to the extant cascade seen in the more evolved Drosophila lineage. Secondly, Transformer2-specific binding intronic splicing silencer sites were found in the splicing regulatory region of transformer but not in doublesex pre-mRNAs in these tephritids. Thus, these sites probably provide the discriminating feature for the putative dual splicing activity of the Tra-Tra2 complex in tephritids. It acts as a splicing activator in dsx pre-mRNA splicing (its binding to the female-specific exon promotes the inclusion of this exon into the mature mRNA), and as a splicing inhibitor in tra pre-mRNA splicing (its binding to the male-specific exons prevents the inclusion of these exons into the mature mRNA). Further, a highly conserved region was found in the specific amino-terminal region of the tephritid Tra protein that might be involved in Tra auto-regulatory function and hence in its repressive splicing behaviour. Finally, the Tra proteins conserved the SR dipeptides, which are essential for Tra functionality.