Significance Factors influencing Wolbachia transfer into new species remain poorly understood. This is important as Wolbachia can influence speciation and is being developed as a novel arthropod-borne disease control approach. We show the native microbiota of Anopheles impede vertical transmission of Wolbachia . Antibiotic microbiome perturbation enables Wolbachia transmission in two Anopheles species. Mosquitoes with altered microbiomes do not exhibit blood meal-induced mortality associated with Wolbachia infection, suggesting that mosquitoes are killed by interactions between Wolbachia and other bacteria present in the mosquito. We identified Asaia as the bacterium responsible for inhibiting Wolbachia transmission, and partially responsible for blood meal-induced mortality. These results suggest that microbial interactions profoundly affect the host, and that microbiome incompatibility may influence distribution of Wolbachia in arthropods.

Cas9-mediated gene editing is a powerful tool for addressing research questions in arthropods. Current approaches rely upon delivering Cas9 ribonucleoprotein (RNP) complex by embryonic microinjection, which is challenging, is limited to a small number of species, and is inefficient even in optimized taxa. Here we develop a technology termed Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) to deliver Cas9 RNP to the arthropod germline by injection into adult female mosquitoes. We identify a peptide (P2C) that mediates transduction of Cas9 RNP from the female hemolymph to the developing mosquito oocytes, resulting in heritable gene editing of the offspring with efficiency as high as 0.3 mutants per injected mosquito. We demonstrate that P2C functions in six mosquito species. Identification of taxa-specific ovary-specific ligand–receptor pairs may further extend the use of ReMOT Control for gene editing in novel species.

Highlights d Aedes aegypti harbors EVEs with high sequence identity to a contemporary RNA virus d EVE-derived piRNAs target genomic viral RNA in infected mosquitoes d Ablation of EVE results in increased viral replication in Aedes aegypti ovaries d piRNA pathway fulfills antiviral function in presence of EVE and cognate virus

Endogenous viral elements derived from nonretroviral RNA viruses have been described in various animal genomes. Whether they have a biological function, such as host immune protection against related viruses, is a field of intense study. Here, we investigated the repertoire of endogenous flaviviral elements (EFVEs) in Aedes mosquitoes, the vectors of arboviruses such as dengue and chikungunya viruses. Previous studies identified three EFVEs from Aedes albopictus cell lines and one from Aedes aegypti cell lines. However, an in-depth characterization of EFVEs in wild-type mosquito populations and individual mosquitoes in vivo has not been performed. We detected the full-length DNA sequence of the previously described EFVEs and their respective transcripts in several A. albopictus and A. aegypti populations from geographically distinct areas. However, EFVE-derived proteins were not detected by mass spectrometry. Using deep sequencing, we detected the production of PIWI-interacting RNA-like small RNAs, in an antisense orientation, targeting the EFVEs and their flanking regions in vivo. The EFVEs were integrated in repetitive regions of the mosquito genomes, and their flanking sequences varied among mosquito populations. We bioinformatically predicted several new EFVEs from a Vietnamese A. albopictus population and observed variation in the occurrence of those elements among mosquitoes. Phylogenetic analysis of an A. aegypti EFVE suggested that it integrated prior to the global expansion of the species and subsequently diverged among and within populations. The findings of this study together reveal the substantial structural and nucleotide diversity of flaviviral integrations in Aedes genomes. Unraveling this diversity will help to elucidate the potential biological function of these EFVEs.IMPORTANCE Endogenous viral elements (EVEs) are whole or partial viral sequences integrated in host genomes. Interestingly, some EVEs have important functions for host fitness and antiviral defense. Because mosquitoes also have EVEs in their genomes, characterizing these EVEs is a prerequisite for their potential use to manipulate the mosquito antiviral response. In the study described here, we focused on EVEs related to the Flavivirus genus, to which dengue and Zika viruses belong, in individual Aedes mosquitoes from geographically distinct areas. We show the existence in vivo of flaviviral EVEs previously identified in mosquito cell lines, and we detected new ones. We show that EVEs have evolved differently in each mosquito population. They produce transcripts and small RNAs but not proteins, suggesting a function at the RNA level. Our study uncovers the diverse repertoire of flaviviral EVEs in Aedes mosquito populations and contributes to an understanding of their role in the host antiviral system.

The potential of RNA viruses to adapt to new environments relies on their ability to introduce changes in their genomes, which has resulted in the recent expansion of re-emergent viruses. Chikungunya virus is an important human pathogen transmitted by mosquitoes that, after 60 years of exclusive circulation in Asia and Africa, has rapidly spread in Europe and the Americas. Here, we examined the evolution of CHIKV in different hosts and uncovered host-specific requirements of the CHIKV 3’UTR. Sequence repeats are conserved at the CHIKV 3’UTR but vary in copy number among viral lineages. We found that these blocks of repeated sequences favor RNA recombination processes through copy-choice mechanism that acts concertedly with viral selection, determining the emergence of new viral variants. Functional analyses using a panel of mutant viruses indicated that opposite selective pressures in mosquito and mammalian cells impose a fitness cost during transmission that is alleviated by recombination guided by sequence repeats. Indeed, drastic changes in the frequency of viral variants with different numbers of repeats were detected during host switch. We propose that RNA recombination accelerates CHIKV adaptability, allowing the virus to overcome genetic bottlenecks within the mosquito host. These studies highlight the role of 3’UTR plasticity on CHIKV evolution, providing a new paradigm to explain the significance of sequence repetitions.

Summary Transgenerational immune priming (TGIP) allows memory-like immune responses to be transmitted from parents to offspring in many invertebrates. Despite increasing evidence for TGIP in insects, the mechanisms involved in the transfer of information remain largely unknown. Here, we show that Drosophila melanogaster and Aedes aegypti transmit antiviral immunological memory to their progeny that lasts throughout generations. We observe that TGIP, which is virus and sequence specific but RNAi independent, is initiated by a single exposure to disparate RNA viruses and also by inoculation of a fragment of viral double-stranded RNA. The progeny, which inherit a viral DNA that is only a fragment of the viral RNA used to infect the parents, display enriched expression of genes related to chromatin and DNA binding. These findings represent a demonstration of TGIP for RNA viruses in invertebrates, broadly increasing our understanding of the immune response, host genome plasticity, and antiviral memory of the germline.

28 Endogenous viral elements (EVEs) are viral sequences integrated in host genomes. A 29 large number of non-retroviral EVEs was recently detected in Aedes mosquito 30 genomes, leading to the hypothesis that mosquito EVEs may control exogenous 31 infections by closely related viruses. Here, we experimentally investigated the role of 32 an EVE naturally found in Aedes aegypti populations and derived from the widespread 33 insect-specific virus, cell-fusing agent virus (CFAV). Using CRISPR/Cas9 genome 34 editing, we created an Ae. aegypti line lacking the CFAV EVE. Absence of the EVE 35 resulted in increased CFAV replication in ovaries, possibly modulating vertical 36 transmission of the virus. Viral replication was controlled by targeting of viral RNA by 37 EVE-derived piRNAs. Our results provide evidence that antiviral piRNAs are produced 38in the presence of a naturally occurring EVE and its cognate virus, demonstrating a 39 functional link between non-retroviral EVEs and antiviral immunity in a natural insect-40

The blacklegged tick Ixodes scapularis is widely distributed in the United States and transmits multiple pathogens to humans, wildlife and domestic animals. Recently, several novel viruses in the family Bunyaviridae (South Bay virus (SBV) and Blacklegged tick phlebovirus (BTPV)) were identified infecting female I. scapularis ticks collected in New York State. We used metagenomic sequencing to investigate the distribution of viruses infecting male and female I. scapularis ticks collected in Centre County, Pennsylvania. We identified both SBV and BTPV in both male and female ticks from all collection locations. The role of male I. scapularis in pathogen epidemiology has been overlooked because they rarely bite and are not considered important pathogen vectors. However, males may act as reservoirs for pathogens that can then be transmitted to females during mating. Our data highlight the importance of examining all potential avenues of pathogen maintenance and transmission throughout the vector-pathogen life cycle in order to understand the epidemiology of tick-borne pathogens.