Detection of Flaviviral-Like DNA Sequences in Aedes aegypti (Diptera: Culicidae) Collected From Argentina

Diseases caused by flaviviruses are a major public health burden across the world. In the past decades, South America has suffered dengue epidemics, the re-emergence of yellow fever and St. Louis encephalitis viruses, and the introduction of West Nile and Zika viruses. Many insect-specific flaviviruses (ISFs) that cannot replicate in vertebrate cells have recently been described. In this study, we analyzed field-collected mosquito samples from six different ecoregions of Argentina to detect flaviviruses. We did not find any RNA belonging to pathogenic flaviviruses or ISFs in adults or immature stages. However, flaviviral-like DNA similar to flavivirus NS5 region was detected in 83–100% of Aedes aegypti (L.). Despite being previously described as an ancient element in the Ae. aegypti genome, the flaviviral-like DNA sequence was not detected in all Ae. aegypti samples and sequences obtained did not form a monophyletic group, possibly reflecting the genetic diversity of mosquito populations in Argentina.

RNA Interference Is Enhanced by Knockdown of Double-Stranded RNases in the Yellow Fever Mosquito Aedes aegypti

RNA interference (RNAi) techniques are being developed for a range of pest insect control technologies, including the sterile insect technique (SIT) and double-stranded RNA (dsRNA)-based insecticides. In SIT applications, where >99% of the released males should be sterile to meet industry standards, the efficiency of RNAi will need to be improved for many insect species if this technology is to be adopted. Endogenous dsRNases can impede dsRNA delivery in some insects, and, here, we investigated whether dsRNases in the midgut could limit RNAi efficacy in the mosquito Aedes aegypti. Ten putative dsRNases were identified in the Ae. aegypti genome, with two highly expressed in the midguts of larvae. Using an ex vivo assay, we observed that dsRNA was rapidly degraded within the mosquito larva’s gut. Double-stranded RNA targeting these two dsRNases, when fed to the larvae, effectively reduced gut dsRNase activity. When these dsRNase-specific dsRNAs were co-delivered with dsRNA targeting a cyan fluorescent protein (CFP) reporter gene, greater knockdown of CFP fluorescence was observed. These results suggest that inhibiting dsRNase activity could enable the implementation of RNAi-based mosquito control methods.

The diversity, structure and function of heritable adaptive immunity sequences in the Aedes aegypti genome

The Aedes aegypti mosquito is a major vector for arboviruses including dengue, chikungunya and Zika virus. Combating the spread of these viruses requires a more complete understanding of the mosquito immune system. Recent studies have implicated genomic endogenous viral elements (EVEs) derived from non-retroviral RNA viruses in insect immunity. Because these elements are inserted into repetitive regions of the mosquito genome, their large-scale structure and organization with respect to other genomic elements has been difficult to resolve with short-read sequencing. To better define the origin, diversity and biological role of EVEs, we employed single-molecule, real-time sequencing technology to generate a high quality, long-read assembly of the Ae. aegypti-derived Aag2 cell line genome. We leverage the quality and contiguity of this assembly to characterize the diversity and genomic context of EVEs in the genome of this important model system. We find that EVEs in the Aag2 genome are acquired through recombination by LTR retrotransposons, and organize into larger loci (>50kbp) characterized by high LTR density. These EVE containing loci are associated with increased transcription factor binding sight density and increased production of anti-genomic piRNAs. We also detected piRNA processing corresponding to on-going viral infection. This global view of EVEs and piRNA responses demonstrates the ubiquity and diversity of these heritable elements that define small-RNA mediated antiviral immunity in mosquitoes.

Generation of expressed sequence tags and sequence-tagged sites as physical landmarks in the mosquito, Aedes aegypti, genome

Twenty-three clones were developed as expressed sequence tags (ESTs) and sequence-tagged sites (STSs) that provide broad coverage of the mosquito, Aedes aegypti, genome at an average spacing of about 7.2 cM. Each of these clones had been mapped previously as a restriction fragment length polymorphism (RFLP) marker. Nineteen of these clones represent anonymous cDNAs and 4 clones represent known genes. Forty-seven percent of the anonymous cDNAs showed significant deduced amino acid sequence similarity to previously described sequences from a wide variety of organisms. STSs developed from RFLPs will provide effective tools for rapid screening of YAC and cosmid libraries, and will therefore, provide anchor points for contig construction for any region in the A. aegypti genome. Also, they facilitate the simultaneous integration of physical and genetic mapping data for A. aegypti. Key words : STS, EST, RFLP, restriction fragment length polymorphisms, QTL, quantitative trait loci.