Marco Salvemini

BACKGROUND: The phlebotomine sand fly Phlebotomus perniciosus (Diptera: Psychodidae, Phlebotominae) is a major Old World vector of the protozoan Leishmania infantum, the etiological agent of visceral and cutaneous leishmaniases in humans and dogs, a worldwide re-emerging diseases of great public health concern, affecting 101 countries. Despite the growing interest in the study of this sand fly species in the last years, the development of genomic resources has been limited so far. To increase the available sequence data for P. perniciosus and to start studying the molecular basis of the sexual differentiation in sand flies, we performed whole transcriptome Illumina RNA sequencing (RNA-seq) of adult males and females and de novo transcriptome assembly.
RESULTS: We assembled 55,393 high quality transcripts, of which 29,292 were unique, starting from adult whole body male and female pools. 11,736 transcripts had at least one functional annotation, including full-length low abundance salivary transcripts, 981 transcripts were classified as putative long non-coding RNAs and 244 transcripts encoded for putative novel proteins specific of the Phlebotominae sub-family. Differential expression analysis identified 8590 transcripts significantly biased between sexes. Among them, some show relaxation of selective constraints when compared to their orthologs of the New World sand fly species Lutzomyia longipalpis.
CONCLUSIONS: In this paper, we present a comprehensive transcriptome resource for the sand fly species P. perniciosus built from short-read RNA-seq and we provide insights into sex-specific gene expression at adult stage. Our analysis represents a first step towards the identification of sex-specific genes and pathways and a foundation for forthcoming investigations into this important vector species, including the study of the evolution of sex-biased genes and of the sexual differentiation in phlebotomine sand flies.

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.

The agricultural pest Ceratitis capitata, also known as the Mediterranean fruit fly or Medfly, is a fruit crop pest of very high economic relevance in different continents. The strategy to separate Ceratitis males from females (sexing) in mass rearing facilities is a useful step before the sterilization and release of male-only flies in Sterile Insect Technique control programs (SIT). The identification of genes having early embryonic male-specific expression, including Y-linked genes, such as the Maleness factor, could help to design novel and improved methods of sexing in combination with transgenesis, aiming to confer conditional female-specific lethality or female-to-male sexual reversal.

The agricultural pest Ceratitis capitata, also known as the Mediterranean fruit fly or Medfly, belongs to the Tephritidae family, which includes a large number of other damaging pest species. The Medfly has been the first non-drosophilid fly species which has been genetically transformed paving the way for designing genetic-based pest control strategies. Furthermore, it is an experimentally tractable model, in which transient and transgene-mediated RNAi have been successfully used. We applied Illumina sequencing to total RNA preparations of 8-10 hours old embryos of C. capitata, This developmental window corresponds to the blastoderm cellularization stage. In summary, we assembled 42,614 transcripts which cluster in 26,319 unique transcripts of which 11,045 correspond to protein coding genes; we identified several hundreds of long ncRNAs; we found an enrichment of transcripts encoding RNA binding proteins among the highly expressed transcripts, such as CcTRA-2, known to be necessary to establish and, most likely, to maintain female sex of C. capitata. Our study is the first de novo assembly performed for Ceratitis capitata based on Illumina NGS technology during embryogenesis and it adds novel data to the previously published C. capitata EST databases. We expect that it will be useful for a variety of applications such as gene cloning and phylogenetic analyses, as well as to advance genetic research and biotechnological applications in the Medfly and other related Tephritidae.

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 transformer gene in Ceratitis capitata (Cctra ep ) is the founding member of a family of related SR genes that appear to act as the master epigenetic switch in sex determination in insects. A functional protein seems to be produced only in individuals with a female XX karyotype where it is required to maintain the productive mode of expression through a positive feedback loop and to direct female development by instructing the downstream target genes accordingly. When zygotic activation of this loop is prevented, male development follows. Recently, tra ep orthologues were isolated in more distantly related dipteran species including Musca domestica, Glossina morsitans and Lucilia cuprina and in the Hymenopterans Apis mellifera and Nasonia vitripennis. All of these tra ep orthologues seem to act as binary switches that govern all aspects of sexual development. Transient silencing leads to complete masculinization of individuals with a female karyotype. Reciprocally, in some systems it has been shown that transient expression of the functional TRA product is sufficient to transactivate the endogenous gene and implement female development in individuals with a male karyotype. Hence, a mechanism based on tra ep epigenetic autoregulation seems to represent a common and presumably ancestral single principle of sex determination in Insecta. The results of these studies will not only be important for understanding divergent evolution of basic developmental processes but also for designing new strategies to improve genetic sexing in different insect species of economical or medical importance.

Courtship behaviours are common features of animal species that reproduce sexually. Typically, males are involved in courting females. Insects display an astonishing variety of courtship strategies primarily based on innate stereotyped responses to various external stimuli. In Drosophila melanogaster, male courtship requires proteins encoded by the fruitless (fru) gene that are produced in different sex-specific isoforms via alternative splicing. Drosophila mutant flies with loss-of-function alleles of the fru gene exhibit blocked male courtship behaviour. However, various individual steps in the courtship ritual are disrupted in fly strains carrying different fru alleles. These findings suggest that fru is required for specific steps in courtship. In distantly related insect species, various fru paralogues were isolated, which shows conservation of sex-specific alternative splicing and protein expression in neural tissues and suggests an evolutionary functional conservation of fru in the control of male-specific-courtship behaviour. In this review, we report the seminal findings regarding the fru gene, its splicing regulation and evolution in insects.

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.

The Doublesex (DSX) transcription factor regulates somatic sexual differentiation in Drosophila melanogaster. Female and male isoforms (DSXF and DSXM) are produced due to sex-specific RNA splicing. Here we show that in the distantly related dipteran Ceratitis capitata, the DSXM male-specific isoform is conserved and able to induce masculinization of both somatic and germline tissues when ectopically expressed in XX Drosophila transgenic individuals.

Sex determination mechanisms, differing in their modality, are widely represented in all the various animal taxa, even at the intraspecific level. Within the highly diversified class Insecta, Drosophila has been used to unravel the molecular and genetic mechanistic interactions that are involved in sex determination. Indeed, the molecularly characterized genes of the Drosophila sex determination hierarchy X:A > Sxl > tra > dsx have been fruitful starting points in the cloning of homologous genes from other insect species. This genetic cascade seems to control sex determination in all Drosophila species. Sex determination in the tephritid Mediterranean fruit fly Ceratitis capitata (Wiedemann), which diverged from Drosophila 90-100 million years ago, contrasts to that found in Drosophila. A different primary signal, a Y-linked male-determining factor (M), still to be molecularly identified, dictates maleness whereas in Drosophila, the primary signal is the X:A (X chromosome:autosome) balance. However, the Drosophila sex-determining pathway, apart from the X:A > Sxl initial regulatory segment, is functionally conserved in C. capitata. The tra gene (Cctra) of C. capitata, as in Drosophila, is the master gene for femaleness through its regulation of the dsx gene and it is dispensable for maleness. In contrast to Drosophila however, where tra is a subordinate target of Sxl, Cctra seems to initiate an autoregulatory mechanism in XX embryos that provides continuous tra female-specific function and acts as a cellular memory maintaining the female pathway. Indeed, a transient interference with Cctra expression in XX embryos by RNA interference (RNAi) treatment can cause complete sexual transformation of both germ-line and soma in adult flies, resulting in fertile XX pseudomales. The development of new transgenic sexing strains of C. capitata able to produce male-only progeny following heat-shock treatments is now feasible and a concrete possibility. Evolutionary considerations strongly suggest that this biotechnological strategy to produce maleonly progeny could be developed for many other Tephritidae and other dipteran species where the sterile insect technique (SIT) is employed within the framework of area-wide integrated pest management programmes. KEYWORDS Mediterranean fruit fly, Ceratitis capitata, Tephritidae, SIT, sexing, sex determination, RNA interference, transgenic, biotechnology, biological control

The genetic transformation of insects by transposable elements is based on the use of selectable genetic markers required to identify transgenic individuals. Conserved regulatory sequences can be used to develop single constructs capable of adequate expression of a marker, across a range of different species. We present evidence that the Drosophila GBS regulatory element (Glass-binding site), derived from the Rh1 rhodopsin gene, is able to drive in vivo eye-specific expression of a Ccwhite+ transgene in the Mediterranean fruitfly Ceratitis capitata. The Ceratitis lineage diverged from that of Drosophila∼120 Myr ago. As the GBS regulatory sequence seems to be partially conserved in the more distantly related dipteran species Anopheles gambiae (250 Myr), we propose that the GBS may be widely useful for driving eye-specific expression in a wide range of dipteran species.

The medfly Ceratitis capitata contains a gene (Cctra) with structural and functional homology to the Drosophila melanogaster sex-determining gene transformer (tra). Similar to tra in Drosophila, Cctra is regulated by alternative splicing such that only females can encode a full-length protein. In contrast to Drosophila, however, where tra is a subordinate target of Sex-lethal (Sxl), Cctra seems to initiate an autoregulatory mechanism in XX embryos that provides continuous tra female-specific function and act as a cellular memory maintaining the female pathway. Indeed, a transient interference with Cctra expression in XX embryos by RNAi treatment can cause complete sexual transformation of both germline and soma in adult flies, resulting in a fertile male XX phenotype. The male pathway seems to result when Cctra autoregulation is prevented and instead splice variants with truncated open reading frames are produced. We propose that this repression is achieved by the Y-linked male-determining factor (M).