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.

20Current knowledge of the piRNA pathway is based mainly on studies on the model organism 21 Drosophila melanogaster, where three proteins of the Piwi subclade of the Argonaute family interact 22 with PIWI-interacting RNAs to silence transposable elements in gonadal tissues. In mosquito species 23 that transmit epidemic arboviruses such as the Dengue and Chikungunya viruses, Piwi clade genes 24 underwent expansion, are also expressed in the soma, and code for proteins that may elicit antiviral 25 functions and crosstalk with other proteins of recognized antiviral mechanisms. These observations 26 underline the importance of expanding our knowledge of the piRNA pathway beyond D.27 melanogaster. 28Here we focus on the emerging arboviral vector Aedes albopictus and we couple traditional 29 approaches of expression and adaptive evolution analyses with most current computational 30 predictions of protein structure to study evolutionary divergence among Piwi clade proteins. 31Superposition of protein homology models indicate high structure similarity among all Piwi proteins, 32 with high levels of amino acid conservation in the inner regions devoted to RNA binding. On the 33 contrary, solvent-exposed surfaces showed low conservation, with several sites under positive 34 selection. Expression profiles of Piwi transcripts during mosquito development and after infection 35 with the Dengue 1 virus showed a concerted elicitation of all Piwi transcripts during viral 36 dissemination, while the maintenance of infection primarily relied on the expression of Piwi5. In 37 contrast, establishment of persistent infection by the Chikungunya virus is accompanied by an 38 increased expression of all Piwi genes, particularly Piwi4 and, again, Piwi5. Overall these results are 39 consistent with functional specialization and a general antiviral role for Piwi5. Experimental40 evidences of sites under positive selection in Piwi1/3, Piwi4 and Piwi6, further provide useful 41 knowledge to design tailored functional experiments.42 43 3 Author summary 44 Argonautes are ancient proteins involved in many cellular processes, including innate 45 immunity. Early in eukaryote evolution, Argonautes separated into Ago and Piwi clades, which 46 maintain a dynamic evolutionary history with frequent duplications and losses. The use of Drosophila 47 melanogaster as a model organism proved fundamental to understand the function of Argonautes.48 However, recent studies showed that the patterns and observations made in D. melanogaster, 49 including the number of Argonautes, their expression profile and their function, are a rarity among 50 Dipterans. 51 In vectors of epidemic arboviruses such as Dengue and Chikungunya viruses, Piwi genes 52 underwent expansion, are expressed in the soma, and some of them appear to have antiviral 53 functions. Besides being an important basic question, the identification of which (and how) Piwi 54 genes have antiviral functions may be used for the development of novel genetic-based strategies 55 of vector control. Here we co...