Genomic and molecular evolutionary analyses of insects of economic and public health relevance

Human African Trypanosomiasis, or sleeping sickness, causes 10,000 deaths per year. Despite the extensive knowledge of trypanosome biology acquired over the last 25 years, the toolbox for disease control is still limited, with neither vaccines nor effective and affordable drugs available in the near future (Aksoy and IGGI, 2010). Eradication of trypanosomiasis via tsetse populations control has a great potential and novel genomic resources are needed to improve currently available tools and develop new effective strategies. In 2015, the release of genomic resources of five Glossina species has facilitated comparative genomics analyses with the genome of G. m. morsitans (Attardo et al., 2014) and other Diptera genomes already available, providing additional information on vector competence, haematophagy, viviparity, as well as on species-specific genes involved in host seeking and vectorial capacity. In this framework, I firstly reconstructed the molecular phylogeny of the six species (G. morsitans, G. pallidipes, G. austeni, G. fuscipes, G. palpalis, G. brevipalpis) using a genome-wide set of orthologous genes. Our genomic phylogenetic analysis suggests that Morsitans, Palpalis and Fusca groups all show a monophyletic origin, with G. brevipalpis (Fusca group) being the most differentiated species. It is noteworthy that the phylogenetic relationships established by our analysis reflects the biological diversities exhibited by each of the Glossina group, such as the geographical distribution, the eco/ethological constrains, the degrees of vectorial capacity and the mating behavior. Based on this phylogeny, I used the molecular clock approach to derive the time of splitting among the Glossina groups and the included species. Orthologs genes derived from Musca domestica allowed us to date also the divergence between Musca and the Glossina genus at 209 Mya, in the Triassic period. Within the Glossina genus, the divergence time has been estimated between the most distantly related G. brevipalpis and the Palpalis and Morsitans groups at ̴ 54 Mya, i.e. Eocene period, while the divergence between the Morsitans and Palpalis groups has been dated at ̴ 21 Mya, i.e. Miocene period. The availability of genome sequences from six Glossina species provides unprecedented opportunities to study the evolution of reproductive traits relevant for interpreting the species differentiation. Indeed, reproduction is one of the fundamental processes influencing several aspects of insect’s life and it is often the target of control methods aiming at reducing the insect population size, particularly effective in reproductive systems similar to the one displayed by tsetse flies (Vreysen et al., 2000). In this framework, a key role is played by male reproductive genes that encode for proteins involved in fertilization, such as seminal fluid proteins (SFPs). These proteins are fundamental in influencing female post-mating physiological and behavioral changes as well as other processes involved in sexual competition. I identified gene candidates for positive selection among those expressed in the male reproductive tissues, male accessory glands (MAGs) and testes. Our analysis was based on 2,563 genes selected from G. m. morsitans MAGand testes-specific transcriptomes, because of their higher expression in MAG compared to testes, and vice versa. The genes were tested for the occurrence of positive selection using codon substitution models in PAML. The analysis led to the identification of a number of genes candidate for positive selection. Interestingly, most of the identified genes remained uncharacterized. These “novel” genes putatively involved in speciesor group-specific processes are particularly interesting for further evolutionary and applicative analyses. Considering the applicative potential of fast evolving and species-specific MAG sequences, I analyzed the regulatory elements responsible for the transcription of the 24 most highly transcribed accessory glands genes in G. m. morsitans. MAG-specific transcriptomic data revealed the presence of a transcription factor known as Paired. In silico analysis confirmed the transcription binding in the promoter region of most of the genes under study. The Paired transcription factor expression profile and protein localization have been demonstrated to be MAG-specific, thus suggesting a role in regulating the expression of MAG genes and, in turn, male reproduction. Its important role in reproduction is also confirmed by its sequence conservation among the six Glossina species analyzed. From the applicative point of view, it can be an important target for the manipulation of male reproduction in tsetse flies. Under this perspective, I have started performing RNAi experiments in order to evaluate the silencing effects of Paired on ejaculate composition, fertility and female post-mating behaviors. During my PhD studies, I have also been involved in the genome projects of two insect species: the Asian tiger mosquito Ae. albopictus and the medfly C. capitata. In the framework of the Ae. albopictus genome project (Italian strain), I have been involved in the determination of the genome size of the Fellini strain and, for both genomes, part of my research was dedicated to the manual curation and annotation of Odorant Binding Proteins (OBPs) and Odorant Receptor (OR) genes. Abbreviations and acronyms AAT Animal African Trypanosomiasis AW-IPM Area Wide Integrated Pest Management BBB Blood-Brain-Barrier CDS Coding Sequence CSP Chemosensory Protein dN Nonsynonymous substitution DP Differential Product DPE Downstream core Promoter Element dS Synonymous substitution FDR False Discovery rate Gaut Glossina austeni Gbre Glossina brevipalpis Gfui Glossina fuscipes Gmor Glossina morsitans GO Gene Ontology Gpai Glossina pallidipes Gppi Glossina palpalis GR Gustatory Receptor HAT Human African Trypanosomiasis IGGI International Glossina Genome Initiative Inr Initiator motif IR Ionotropic Receptor MAGs Male Accessory Glands Mya Million Years Ago NTDs Neglected Tropical Diseases OBP Odorant-Binding Proteins ODE Odorant degrading enzyme OR Odorant Receptor OR Olfactory Receptor Orco Odorant Receptor co-receptor OSN Olfactory Receptor neuron prd Paired transcription factor RDA Representational Difference Analysis SFPs Seminal Fluid Proteins SGHV Salivary Gland Hypertrophy Virus SIT Sterile Insect Technique