Project description:We report a global survey of viral small RNAs (vsmRNAs) from >200 Aedes aegypti samples to identify many mosquito viruses that actively infect this prominent arboviral vector. Ae. aegypti viruses in the Americas were abundant, with some displaying geographical boundaries. Viruses infecting Asian Ae. aegypti were similar to those in the Americas and revealed the first wild example of dengue vsmRNAs. African Ae. aegypti displayed vsmRNAs from viruses unique to these African strains. Academic lab colonies generally lacked viruses, yet two commercial strains were deeply infected by a tombus-like virus that is related to plant viruses. Comparing matched viral long RNAs to vsmRNAs revealed viral transcripts evading the mosquito RNA interference (RNAi) pathway. By infecting mosquito cells with Ae. aegypti homogenates, we generated stably infected cell lines which produced vsmRNAs that were comparable to native mosquito vsmRNA patterns. Lastly, we demonstrate that these stably infected mosquito cells producing vsmRNAs can exert gene silencing of reporters bearing viral sequence segments, providing a potential explanation for how Ae. aegypti can tolerate the persistence of viral infections. This vsmRNA genomics approach in Ae. aegypti can add to existing vector surveillance approaches by discovering new viruses that persist in mosquito populations.
Project description:We report a global survey of viral small RNAs (vsmRNAs) from >200 Aedes aegypti samples to identify many mosquito viruses that actively infect this prominent arboviral vector. Ae. aegypti viruses in the Americas were abundant, with some displaying geographical boundaries. Viruses infecting Asian Ae. aegypti were similar to those in the Americas and revealed the first wild example of dengue vsmRNAs. African Ae. aegypti displayed vsmRNAs from viruses unique to these African strains. Academic lab colonies generally lacked viruses, yet two commercial strains were deeply infected by a tombus-like virus that is related to plant viruses. Comparing matched viral long RNAs to vsmRNAs revealed viral transcripts evading the mosquito RNA interference (RNAi) pathway. By infecting mosquito cells with Ae. aegypti homogenates, we generated stably infected cell lines which produced vsmRNAs that were comparable to native mosquito vsmRNA patterns. Lastly, we demonstrate that these stably infected mosquito cells producing vsmRNAs can exert gene silencing of reporters bearing viral sequence segments, providing a potential explanation for how Ae. aegypti can tolerate the persistence of viral infections. This vsmRNA genomics approach in Ae. aegypti can add to existing vector surveillance approaches by discovering new viruses that persist in mosquito populations.
Project description:The Flavivirus genus contains some of the most prevalent vector-borne viruses such as dengue, Zika and yellow fever viruses that cause devastating diseases in humans. However, the insect-specific clade of flaviviruses is restricted to mosquito hosts; albeit they have retained the general features of the genus such as genome structure and replication. The interaction between insect-specific flaviviruses (ISFs) and their mosquito hosts are largely unknown. Pathogenic flaviviruses are known to modulate host-derived microRNAs (miRNAs), a class of non-coding RNAs that are important in controlling gene expression. Alteration in miRNAs may represent changes in host gene expression and provide understanding of virus-host interactions. The role of miRNAs in ISF-mosquito interactions is largely unknown. A recently discovered Australian ISF, Palm Creek virus (PCV), has the ability to suppress medically relevant flaviviruses. Here, we investigated the potential involvement of miRNAs in PCV infection using the model mosquito Aedes aegypti. By combining small RNA sequencing and bioinformatics analysis, differentially expressed miRNAs were determined. Our results indicated that PCV infection hardly affects host miRNAs. Out of 101 reported miRNAs of Ae. aegypti, only aae-miR-2940-5p had significant altered expression over the course of infection. However, further analysis of aae-miR-2940-5p revealed that this miRNA does not have any direct impact on PCV replication in vitro. Thus, the results overall suggest that PCV infection has a limited effect on the mosquito miRNA profile and therefore, they may not play a significant role the PCV- Ae. aegypti interaction.
Project description:Larval application of sodium channel homologous dsRNA restores pyrethroid insecticide susceptibility in a resistant adult mosquito population
Project description:Mosquitoes are the most notorious hematophagous insects and due to their blood feeding behavior and genetic compatibility, numerous mosquito species are highly efficient vectors for certain human pathogenic parasites and viruses. The mosquito midgut is the principal organ of blood meal digestion and nutrient absorption. It is also the initial site of infection with blood meal acquired parasites and viruses. We conducted an analysis based on single-nucleus RNA sequencing(snRNA-Seq) to assess the cellular diversity of the midgut and how individual cells respond to blood meal ingestion to facilitate its digestion.
Project description:Arthropod-borne viruses (arboviruses) such as dengue virus (DENV) and Zika virus (ZIKV) pose a significant threat to global health. Novel approaches to control arbovirus spread are focused on harnessing the antiviral immune system of their main vector, the Aedes aegypti mosquito. In arthropods, genes of the Vago family are often presented as analogs of mammalian cytokines with potential antiviral functions, but the role of Vago genes upon virus infection in Ae. aegypti is largely unknown. We performed a phylogenetic analysis of the Vago gene family in Diptera that prompted us to focus on a Vago-like gene that we named VLG-1. Using CRISPR/Cas9-mediated gene editing, we generated a VLG-1 mutant line of Ae. aegypti that revealed a proviral effect of this gene upon DENV and ZIKV infection. In the absence of VLG-1, virus dissemination throughout the mosquito’s body was impaired, albeit not altering virus transmission rates. A tissue-specific transcriptome analysis revealed that the loss of VLG-1 impacted numerous biological processes potentially linked to viral replication, such as the oxidative stress response. Our results challenge the conventional understanding of Vago-like genes as antiviral factors and underscores the need for further research to elucidate the molecular mechanisms underlying mosquito-arbovirus interactions.
Project description:Aedes aegypti mosquito ovarian follicles develop synchronously after taking a blood meal. A single layer of follicular epithelial cells surrounding the oocyte is responsible for secreting a majority of eggshell structural components between 18 and 54 hours post blood meal. We previously identified a protein called eggshell organizing factor 1 (EOF1). When we knocked down EOF1 with RNA interference in female adult mosquitoes, they laid eggs that were not properly melanized, and fragile eggs did not contain viable embryos. Here, we aimed to determine putative downstream eggshell proteins in RNAi-EOF1 female mosquitoes. Our eggshell proteomics identified 220 proteins, suggesting that mosquito extracellular eggshells are composed of a complex mixture of proteins. These proteins are involved in the formation of an intact eggshell that protects the embryonic development and larva from the environment for long periods of time.