A mosquito small RNA genomics resource reveals dynamic evolution and host responses to viruses and transposons

Abstract

Although mosquitoes are major transmission vectors for pathogenic arboviruses, viral infection has little impact on mosquito health. This immunity is caused in part by mosquito RNA interference (RNAi) pathways that generate antiviral small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). RNAi also maintains genome integrity by potently repressing mosquito transposon activity in the germline and soma. However, viral and transposon small RNA regulatory pathways have not been systematically examined together in mosquitoes. Therefore, we developed an integrated mosquito small RNA genomics (MSRG) resource that analyzes the transposon and virus small RNA profiles in mosquito cell cultures and somatic and gonadal tissues across four medically important mosquito species. Our resource captures both somatic and gonadal small RNA expression profiles within mosquito cell cultures, and we report the evolutionary dynamics of a novel Mosquito-Conserved piRNA Cluster Locus (MCpiRCL) made up of satellite DNA repeats. In the larger culicine mosquito genomes we detected highly regular periodicity in piRNA biogenesis patterns coinciding with the expansion of Piwi pathway genes. Finally, our resource enables detection of cross talk between piRNA and siRNA populations in mosquito cells during a response to virus infection. The MSRG resource will aid efforts to dissect and combat the capacity of mosquitoes to tolerate and spread arboviruses.

Uncovering the Worldwide Diversity and Evolution of the Virome of the Mosquitoes Aedes aegypti and Aedes albopictus

“…vpiRNAs can be determined by a ping-pong signature (U 1 -A 10 ) and 10 nt complementary overlapping reads. For the San Gabriel virus and all segments of Aedes phasma virus [ 58 , 146 ], as well as individual segments of TMTLV and WSBLV-4, we observed a ping-pong signature (U 1 -A 10 ) and over-represented complementary 10 nt overlapping pairs of sRNA from reads of 24–29 nt from these viruses ( Supplementary File S2 ). The absence of a predominant 21 nt species of vsiRNA targeting CppDNV and WSBLV-4 in Ae.…”

“…albopictus C7-10 cells from total-RNA sequencing obtained as part of the Arthropod Cell Line RNA-Seq initiative, Broad Institute (broadinstitute.org), and three independent small RNA sequencing datasets [ 129 , 164 ]. It appears that not all C7-10 cells were infected with WSBLV-4; however, [ 58 ] suggesting heterogeneity between sources of C7-10 cell lines. Additionally, we identified a convincing RNAi response (mostly piRNAs) against WSBLV-4 from C6/36 cells (SRA: SRR11252294) from this dataset [ 58 ].…”

“…It appears that not all C7-10 cells were infected with WSBLV-4; however, [ 58 ] suggesting heterogeneity between sources of C7-10 cell lines. Additionally, we identified a convincing RNAi response (mostly piRNAs) against WSBLV-4 from C6/36 cells (SRA: SRR11252294) from this dataset [ 58 ]. The RNAi profile of WSBLV-4 infection in C7-10 and C6/36 cells is in line with the Dicer-2 deficient nature of the cell line ( Figure 9 ).…”

Mayaro Virus Infection Elicits an Innate Immune Response inAnopheles stephensi

“…piRNAs and siRNAs were isolated from the small RNA libraries by selecting all 18 – 24 nt reads (siRNA) and 24 – 35 nt (piRNA) reads from the trimmed datasets and filtering out all identified mature miRNAs, and those mapping to the MAYV NC_003417.1 genome were considered potential piRNAs or siRNAs. piRNA and siRNA alignment to the AsteI2 genome was performed using Bowtie RNA sequencing aligner within the MSRG pipeline [34]. Observation of fastQC output for Control Day 7 Replicate 1 small RNA sequencing revealed poor sequencing results, so this replicate was omitted from all analyses in the small RNA focused portions of this study [35].…”

Bioinformatic and cell-based tools for pooled CRISPR knockout screening in mosquitos

“…To generate RMCE adapter cell lines as a platform for CRISPR screens, we first chose well characterized cell lines from three mosquito species that are susceptible to infection with biomedically important viruses or parasites, and for which genomic, transcriptomic, and small RNA sequencing data exist 4 : Sua-5B from Anopheles coluzzii 3 (formerly An. gambiae M form), Hsu from Culex quinquefasciatus 5 , and C6/36 from Aedes albopictus 6 .…”