Genomics, transcriptomics and CRISPR/CAS9 gene editing approaches to investigate embryonic sex determination in insect pest Ceratitis capitata.

During my PhD, I have carried out studies of genomic and transcriptomic in the non-model system Ceratitis capitata. C. capitata (Diptera, Tephritidae), also called “medfly”, is an insect pest that spread over almost every continent and represents a great danger to the cultivation of fruits for human alimentation. Its vast diffusion, together with its ability to parasitize many fruit hosts and the difficulties humans encountered when trying to control and eradicate it from local areas, made C. capitata one of the most dangerous species to agriculture on a global scale, and therefore, to a significant portion of national and international economies. The reproduction and the sex determination are two crucial aspects to understand the biology of insects and represent optimal targets to limit their growth and diffusion. The project of my PhD consists into two parallel and synergic research lines: 1) transcriptomic (Illumina®) and genomic (PacBio®) approaches to the identification of novel Y-linked genes potentially involved in male sex determination, and X-linked genes in “medfly”; 2) the development and perfecting of a powerful technique of gene editing, the CRISPR/Cas9, to study these novel gene functions

Syntopic Cystoseira taxa support different molluscan assemblages in the Gulf of Naples (southern Tyrrhenian Sea)

Brown macroalgae belonging to the genus Cystoseira (Fucales: Sargassaceae) are canopy-forming organisms the recent decline of which at a basin and local scale has been widely documented, which urgently calls for research to fill knowledge gaps and support new and effective measures for protection. We, hereby, characterised the molluscan assemblages associated with three Cystoseira taxa (C. amentacea, C. compressa and C. crinita) from Ischia Island (Italy, Tyrrhenian Sea), and tested whether different congeneric taxa may syntopically support a different biota. In particular, these assemblages were compared among three Cystoseira species, between two times of sampling (June–July 2015 and June–July 2016), and among six sites in terms of multivariate structure (identity and relative abundances of constituting taxa combined, and presence–absence composition), as well as for synthetic measures of diversity, including the total richness of taxa, the exponential Shannon index, and the reciprocal Simpson index. In total, 24736 molluscan individuals were collected, overall belonging to 52 taxa. The majority of the identified species included micrograzers and filter feeders, which is in agreement with similar previous studies. The composition of associated molluscan assemblages, which was mainly represented by juvenile individuals, differed among the three Cystoseira species, suggesting that even congeneric taxa do not support an analogous benthic fauna. The present findings have shedded light on the molluscan biota associated with Cystoseira taxa in the Gulf of Naples and strengthened the importance of such habitat-forming macroalgae in structuring the local infralittoral invertebrate biodiversity and as a nursery for species-specific associated molluscs

The whole genome sequence of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), reveals insights into the biology and adaptive evolution of a highly invasive pest species

The 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

RNA-seq and Insect pests: new methods for the identification and the analysis of sex-specific genes in species of economic or sanitary interests.

Insects are among the largest taxonomic animal groups on Earth. In some cases, their interactions with humans can be harmful and the knowledge of the lifecycle of insect species, e.g. disease vectors and agricultural pests, opens new chances to develop eco-sustainable control strategies, alternative to pesticides. Reproduction and sex determination are at the same time crucial aspects to understand insect biology and optimal targets to limit their population growth and diffusion. We are applying RNA-seq and digital gene expression analysis to the study of sex determination in Insect species. In particular we are comparing de novo assembled transcriptomes of early sexed embryonic populations of the agricultural pest Ceratitis capitata (Diptera, Tephritidae) to identify the unknown primary signal of sex determination in this species. We are producing de novo transcriptomes from adult males and females of the two hematophagous dipteran non-model species Aedes albopictus (Diptera, Culicidae) and Phlebotomus perniciosus (Diptera, Psychodidae), to identify genes with sex-specific or sex-biased expression, potentially involved in sex determination pathway of these vector species. We are also developing new graphical interfaces and on-line databases for comparative genomic analyses and digital gene expression analyses in biological samples with opposite sexes

CRISPR-Cas9 targeted disruption of the yellow ortholog in the housefly identifies the brown body locus

The classic brown body (bwb) mutation in the housefly Musca domestica impairs normal melanization of the adult cuticle. In Drosophila melanogaster, a reminiscent pigmentation defect results from mutations in the yellow gene encoding dopachrome conversion enzyme (DCE). Here, we demonstrate that the bwb locus structurally and functionally represents the yellow ortholog of Musca domestica, MdY. In bwb Musca strains, we identified two mutant MdY alleles that contain lesions predicted to result in premature truncation of the MdY open reading frame. We targeted wildtype MdY by CRISPR-Cas9 RNPs and generated new mutant alleles that fail to complement existing MdY alleles, genetically confirming that MdY is the bwb locus. We further found evidence for Cas9-mediated interchromosomal recombination between wildtype and mutant bwb alleles. Our work resolves the molecular identity of the classic bwb mutation in Musca domestica and establishes the feasibility of Cas9-mediated genome editing in the Musca model

Next-generation genetic sexing strain establishment in the agricultural pest Ceratitis capitata.

Tephritid fruit fly pests pose an increasing threat to the agricultural industry due to their global dispersion and a highly invasive nature. Here we showcase the feasibility of an early-detection SEPARATOR sex sorting approach through using the non-model Tephritid pest, Ceratitis capitata. This system relies on female-only fluorescent marker expression, accomplished through the use of a sex-specific intron of the highly-conserved transformer gene from C. capitata and Anastrepha ludens. The herein characterized strains have 100% desired phenotype outcomes, allowing accurate male-female separation during early development. Overall, we describe an antibiotic and temperature-independent sex-sorting system in C. capitata, which, moving forward, may be implemented in other non-model Tephritid pest species. This strategy can facilitate the establishment of genetic sexing systems with endogenous elements exclusively, which, on a wider scale, can improve pest population control strategies like sterile insect technique

A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters.

Synthetic sex distorters have recently been developed in the malaria mosquito, relying on endonucleases that target the X-chromosome during spermatogenesis. Although inspired by naturally-occurring traits, it has remained unclear how they function and, given their potential for genetic control, how portable this strategy is across species. We established Drosophila models for two distinct mechanisms for CRISPR/Cas9 sex-ratio distortion-"X-shredding" and "X-poisoning"-and dissected their target-site requirements and repair dynamics. X-shredding resulted in sex distortion when Cas9 endonuclease activity occurred during the meiotic stages of spermatogenesis but not when Cas9 was expressed from the stem cell stages onwards. Our results suggest that X-shredding is counteracted by the NHEJ DNA repair pathway and can operate on a single repeat cluster of non-essential sequences, although the targeting of a number of such repeats had no effect on the sex ratio. X-poisoning by contrast, i.e. targeting putative haplolethal genes on the X chromosome, induced a high bias towards males (>92%) when we directed Cas9 cleavage to the X-linked ribosomal target gene RpS6. In the case of X-poisoning sex distortion was coupled to a loss in reproductive output, although a dominant-negative effect appeared to drive the mechanism of female lethality. These model systems will guide the study and the application of sex distorters to medically or agriculturally important insect target species

Inhibition studies of Brucella suis β-carbonic anhydrases with a series of 4-substituted pyridine-3-sulphonamides

The two β-carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic bacterium Brucella suis, BsuCA1 and BsuCA2, were investigated for their inhibition profile with a series of pyridine-3-sulphonamide derivatives incorporating 4-hetaryl moieties. BsuCA1 was effectively inhibited by these sulphonamides with inhibition constants ranging between 34 and 624 nM. BsuCA2 was less sensitive to these inhibitors, with KIs in the range of 62 nM - > 10 µM. The nature of the 4-substituent present on the pyridine ring was the main factor influencing the inhibitory profile against both isoforms, with 4-halogenophenylpiperazin-1-yl and 3,4,5-trisubstituted-pyrazol-1-yl derivatives showing the most effective inhibition. Some of these sulphonamides were most effective bacterial CA than human (h) CA I and II inhibitors, making them selective for the prokaryotic enzymes. Investigation of bacterial CA inhibitors may be relevant for finding antibiotics with a new mechanism of action compared to the clinically used agents for which substantial drug resistance emerged

Gene discovery in the Old World Leishmania vector Phlebotomus perniciosus (Diptera, Psychodidae): de novo assembly and digital gene expression analysis of sexed transcriptomes from a laboratory strain and Ischia island natural populations.

In the Old World, the sand fly Phlebotomus perniciosus is the main vector of Leishmania infantum, the parasitic protozoan that causes visceral and cutaneous leishmaniases in humans and canine reservoir host, as well as, of known and emerging arboviruses considered relevant from an European public health perspective. P. perniciosus is widely distributed in the western Mediterranean Basin with the most important endemic focus of canine and human visceral leishmaniases of this area located in Italy, in the Campania region. We are applying RNA-seq and digital gene expression analysis to identify genes with sex-specific or sex-biased expression at a genome-wide level and to isolate genes involved in the sex determination pathway of the P. perniciosus. We defined a de novo reference transcriptome of about 50k transcripts which includes well conserved alternatively spliced transcripts of the doublesex (dsx) and fruitless (fru) ortholog genes, which are responsible in Drosophila of somatic sexual differentition and genetic control of courtship behaviour, respectively. Interestingly, an ortholog of the key switch gene of insect female sex determination, the transformer (tra) gene, is apparently absent in P. perniciosus transcriptome and the tra auxiliary factor transformer-2 (tra-2) is highly divergent respect to other dipteran tra-2 orthologs. These preliminary data suggest only a partial conservation of the sex determination cascade in this species. Our data could contribute to the study of the evolution of sex determination pathway in the under-studied Nematocera dipteran suborder, the comparative and genome-wide analysis of haematofagy in Nematocera species and to develop new control methods for the sand fly vector species

Targeting the autosomal Ceratitis capitata transformer gene using Cas9 or dCas9 to masculinize XX individuals without inducing mutations

Background: Females of the Mediterranean fruit fly Ceratitis capitata (Medfly) are major agricultural pests, as they lay eggs into the fruit crops of hundreds of plant species. In Medfly, female sex determination is based on the activation of Cctransformer (Cctra). A maternal contribution of Cctra is required to activate Cctra itself in the XX embryos and to start and epigenetically maintain a Cctra positive feedback loop, by female-specific alternative splicing, leading to female development. In XY embryos, the male determining Maleness-on-the-Y gene (MoY) blocks this activation and Cctra produces male-specific transcripts encoding truncated CcTRA isoforms and male differentiation occurs. Results: With the aim of inducing frameshift mutations in the first coding exon to disrupt both female-specific and shorter male-specific CcTRA open reading frames (ORF), we injected Cas9 ribonucleoproteins (Cas9 and single guide RNA, sgRNA) in embryos. As this approach leads to mostly monoallelic mutations, masculinization was expected only in G1 XX individuals carrying biallelic mutations, following crosses of G0 injected individuals. Surprisingly, these injections into XX-only embryos led to G0 adults that included not only XX females but also 50% of reverted fertile XX males. The G0 XX males expressed male-specific Cctra transcripts, suggesting full masculinization. Interestingly, out of six G0 XX males, four displayed the Cctra wild type sequence. This finding suggests that masculinization by Cas9-sgRNA injections was independent from its mutagenic activity. In line with this observation, embryonic targeting of Cctra in XX embryos by a dead Cas9 (enzymatically inactive, dCas9) also favoured a male-specific splicing of Cctra, in both embryos and adults. Conclusions: Our data suggest that the establishment of Cctra female-specific autoregulation during the early embryogenesis has been repressed in XX embryos by the transient binding of the Cas9-sgRNA on the first exon of the Cctra gene. This hypothesis is supported by the observation that the shift of Cctra splicing from female to male mode is induced also by dCas9. Collectively, the present findings corroborate the idea that a transient embryonic inactivation of Cctra is sufficient for male sex determination