The Dipteran family Tephritidae (true fruit flies) comprises more than 5000 species classified in 500 genera distributed worldwide. Tephritidae include devastating agricultural pests and highly invasive species whose spread is currently facilitated by globalization, international trade and human mobility. The ability to identify and exploit a wide range of host plants for oviposition, as well as effective and diversified reproductive strategies, are among the key features supporting tephritid biological success. Intraspecific communication involves the exchange of a complex set of sensory cues that are species- and sex-specific. Chemical signals, which are standing out in tephritid communication, comprise long-distance pheromones emitted by one or both sexes, cuticular hydrocarbons with limited volatility deposited on the surrounding substrate or on the insect body regulating medium- to short-distance communication, and host-marking compounds deposited on the fruit after oviposition. In this review, the current knowledge on tephritid chemical communication was analysed with a special emphasis on fruit fly pest species belonging to the Anastrepha, Bactrocera, Ceratitis, and Rhagoletis genera. The multidisciplinary approaches adopted for characterising tephritid semiochemicals, and the real-world applications and challenges for Integrated Pest Management (IPM) and biological control strategies are critically discussed. Future perspectives for targeted research on fruit fly chemical communication are highlighted.

The Asian tiger mosquito Aedes albopictus is an invasive mosquito and a competent vector for public-health relevant arboviruses such as Chikungunya ( Alphavirus ), Dengue and Zika ( Flavivirus ) viruses. Unexpectedly, the sequencing of the genome of this mosquito revealed an unusually high number of integrated sequences with similarities to non-retroviral RNA viruses of the Flavivirus and Rhabdovirus genera. These Non-retroviral Integrated RNA Virus Sequences (NIRVS) are enriched in piRNA clusters and coding sequences and have been proposed to constitute novel mosquito immune factors. However, given the abundance of NIRVS and their variable viral origin, their relative biological roles remain unexplored. Here we used an analytical approach that intersects computational, evolutionary and molecular methods to study the genomic landscape of mosquito NIRVS. We demonstrate that NIRVS are differentially distributed across mosquito genomes, with a core set of seemingly the oldest integrations with similarity to Rhabdoviruses . Additionally, we compare the polymorphisms of NIRVS with respect to that of fast and slow-evolving genes within the Ae. albopictus genome. Overall, NIRVS appear to be less polymorphic than slow-evolving genes, with differences depending on whether they occur in intergenic regions or in piRNA clusters. Finally, two NIRVS that map within the coding sequences of genes annotated as Rhabdovirus RNA-dependent RNA polymerase and the nucleocapsid-encoding gene, respectively, are highly polymorphic and are expressed, suggesting exaptation possibly to enhance the mosquito’s antiviral responses. These results greatly advance our understanding of the complexity of the mosquito repeatome and the biology of viral integrations in mosquito genomes.

Current knowledge of the piRNA pathway is based mainly on studies on Drosophila melanogaster where three proteins of the Piwi subclade of the Argonaute family interact with PIWI-interacting RNAs to silence transposable elements in gonadal tissues. In mosquito species that transmit epidemic arboviruses such as dengue and chikungunya viruses, Piwi clade genes underwent expansion, are also expressed in the soma and cross-talk with proteins of recognized antiviral function cannot be excluded for some Piwi proteins. These observations underscore the importance of expanding our knowledge of the piRNA pathway beyond the model organism D. melanogaster. Here we focus on the emerging arboviral vector Aedes albopictus and we couple traditional approaches of expression and adaptive evolution analyses with most current computational predictions of protein structure to study evolutionary divergence among Piwi clade proteins. Superposition of protein homology models indicate possible high structure similarity among all Piwi proteins, with high levels of amino acid conservation in the inner regions devoted to RNA binding. On the contrary, solvent-exposed surfaces showed low conservation, with several sites under positive selection. Analysis of the expression profiles of Piwi transcripts during mosquito development and following infection with dengue serotype 1 or chikungunya viruses showed a concerted elicitation of all Piwi transcripts during viral dissemination of dengue viruses while maintenance of infection relied on expression of primarily Piwi5. Opposite, establishment of persistent infection by chikungunya virus is accompanied by increased expression of all Piwi genes, particularly Piwi4 and, again, Piwi5. Overall these results are consistent with functional specialization and a general antiviral role for Piwi5. Experimental evidences of sites under positive selection in Piwi1/3, Piwi4 and Piwi6, that have complex expression profiles, provide useful knowledge to design tailored functional experiments.

The sequenced genome of the arboviral vector mosquito, Aedes albopictus, is replete with repetitive DNA and it harbors an unusually large number of endogenous viral sequences, collectively called Nonretroviral Integrated RNA Virus Sequences (NIRVS). NIRVS are enriched both within protein-coding gene exons and PIWI-interacting RNA (piRNA) clusters, where they encode piRNAs. Based on these features, NIRVS have been proposed to function as novel mosquito antiviral immune factors. However, the relative importance and contributions of different NIRVS as functional antiviral elements and their mechanisms of action remain open questions.We apply an analytical approach that intersects computational, evolutionary and molecular methods to identify NIRVS most likely affecting mosquito immunity. Using this strategy, we show that NIRVS are a highly dynamic component of the Ae. albopictus repeatome, which nevertheless maintains a core set of seemingly the oldest NIRVS with similarity to Rhabdoviruses. Population-level polymorphism of NIRVS varies depending on whether they occur in intergenic regions, piRNA clusters or are part of gene exons. NIRVS from piRNA clusters are differentially widespread in diverse populations but conserved at the sequence level. This is consistent with the hypothesis that they act analogously to fragments of transposable elements in piRNA clusters and contribute to piRNA-based immunity. Among NIRVS from gene exons, AlbRha52 and AlbRha12 have the hallmarks of domestication as they are fixed across populations, stably expressed, and as polymorphic at the sequence level as fast-evolving genes. Overall these results support the hypothesis that NIRVS contribute to mosquito immunity, potentially through diverse modes of action.

Several true fruit flies (Tephritidae) cause major damage to agriculture worldwide. Among them, species of the genus Bactrocera are extensively studied to understand the traits associated with their invasiveness and ecology. Comparative approaches based on a reliable phylogenetic framework are particularly effective, but several nodes of the Bactrocera phylogeny are still controversial, especially concerning the reciprocal affinities of the two major pests B. dorsalis and B. tryoni. Here, we analyzed a newly assembled genomic-scaled dataset using different models of evolution to infer a phylogenomic backbone of ten representative Bactrocera species and two outgroups. We further provide the first genome-scaled inference of their divergence by calibrating the clock using fossil records and the spontaneous mutation rate. The results reveal a closer relationship of B. dorsalis with B. latifrons than to B. tryoni, contrary to what was previously supported by mitochondrial-based phylogenies. By employing coalescent-aware and heterogeneous evolutionary models, we show that this incongruence likely derives from a hitherto undetected systematic error, exacerbated by incomplete lineage sorting and possibly hybridization. This agrees with our clock analysis, which supports a rapid and recent radiation of the clade to which B. dorsalis, B. latifrons and B. tryoni belong. These results provide a new picture of Bactrocera phylogeny that can serve as the basis for future comparative analyses.

Background The Oriental fruit fly, Bactrocera dorsalis, is a highly polyphagous invasive species with a high reproductive potential. In many tropical and subtropical parts of the world it ranks as one of the major pests of fruits and vegetables. Due to its economic importance, genetic, cytogenetic, genomic and biotechnological approaches have been applied to understand its biology and to implement the Sterile Insect Technique, currently a part of area-wide control programmes against this fly. Its chromosome complement includes five pairs of autosomes and the sex chromosomes. The X and Y sex chromosomes are heteromorphic and the highly heterochromatic and degenerate Y harbours the male factor BdMoY. The characterization of the Y chromosome in this fly apart from elucidating its role as primary sex determination system, it is also of crucial importance to understand its role in male biology. The repetitive nature of the Y chromosome makes it challenging to sequence and characterise. Results Using Representational Difference Analysis, fluorescent in situ hybridisation on mitotic chromosomes and in silico genome resources, we show that the B. dorsalis Y chromosome harbours transcribed sequences of gyf, (typo-gyf) a homologue of the Drosophila melanogaster Gigyf gene, and of a non-LTR retrotransposon R1. Similar sequences are also transcribed on the X chromosome. Paralogues of the Gigyf gene are also present on the Y and X chromosomes of the related species B. tryoni. Another identified Y-specific repetitive sequence linked to BdMoY appears to be specific to B. dorsalis. Conclusions Our random scan of the Y chromosome provides a broad picture of its general composition and represents a starting point for further applicative and evolutionary studies. The identified repetitive sequences can provide a useful Y-marking system for molecular karyotyping of single embryos. Having a robust diagnostic marker associated with BdMoY will facilitate studies on how BdMoY regulates the male sex determination cascade during the embryonic sex-determination window. The Y chromosome, despite its high degeneracy and heterochromatic nature, harbours transcribed sequences of typo-gyf that may maintain their important function in post-transcriptional mRNA regulation. That transcribed paralogous copies of Gigyf are present also on the X and that this genomic distribution is maintained also in B. tryoni raises questions on the evolution of sex chromosomes in Bactrocera and other tephritids.

True fruit flies (Tephritidae) include several species that cause extensive damage to agriculture worldwide. Among them, species of the genus Bactrocera are widely studied to understand the traits associated to their invasiveness and ecology. Comparative approaches based on a reliable phylogenetic framework are particularly effective, but, to date, molecular phylogenies of Bactrocera are still controversial. Here, we employed a comprehensive genomic dataset to infer a robust backbone phylogeny of eleven representative Bactrocera species and two outgroups. We further provide the first genome scaled inference of their divergence using calibrated relaxed clock. The results of our analyses support a closer relationship of B. dorsalis to B. latifrons than to B. tryoni, in contrast to all mitochondrial-based phylogenies. By comparing different evolutionary models, we show that this incongruence likely derives from the fast and recent radiation of these species that occurred around 2 million years ago, which may be associated with incomplete lineage sorting and possibly (ongoing) hybridization. These results can serve as basis for future comparative analyses and highlight the utility of using large datasets and efficient phylogenetic approaches to study the evolutionary history of species of economic importance.

Aedes albopictus is an aggressive invasive species and a competent vector for over 20 arboviruses, including Chikungunya, Dengue and Zika viruses. Understanding the molecular and cellular interactions between viruses and vectors is key to implement transmission-blocking strategies to prevent viral outbreaks. However, the mechanisms that shape vector competence are poorly understood. Recent evidence reveals that the genomes of Aedes spp. harbour fragmented viral sequences which produce PIWI-interacting RNAs (piRNAs), suggesting a role in vector competence. Current knowledge of the piRNA pathway in Ae. albopictus is limited, and its possible role in the establishment of persistent infections widely unknown. We combined cutting-edge bioinformatic analyses based on next-generation sequencing data with molecular biology and virology techniques to characterise the main genes of the piRNA pathway in this mosquito species, assess their polymorphisms and analyse their expression throughout mosquito development and following infection with the Chikungunya and Dengue-1 viruses. We identified seven piwi genes which displayed high levels of polymorphism across populations and signs of adaptive evolution. Superposition of protein homology models indicate high structure similarity among all Piwi proteins, with high levels of amino acid conservation in the inner regions devoted to RNA binding. On the contrary, solvent-exposed surfaces showed low conservation, with sites under positive selection. Infection experiments indicated specific responses depending on viral species, time of infection and mosquito tissue, highlighting distinct roles for specific Piwi proteins. In conclusion, this work helps define the role of the piRNA pathway in persistent arboviral infections and understand the evolutionary divergence among piwi proteins.