Inge Holm

Mosquitoes are colonized by a large but mostly uncharacterized natural virome of RNA viruses, and the composition and distribution of the natural RNA virome may influence the biology and immunity of Anopheles malaria vector populations. Anopheles mosquitoes were sampled in malaria endemic forest village sites in Senegal and Cambodia, including Anopheles funestus, Anopheles gambiae group sp., and Anopheles coustani in Senegal, and Anopheles hyrcanus group sp., Anopheles maculatus group sp., and Anopheles dirus in Cambodia. The most frequent mosquito species sampled at both study sites are human malaria vectors. Small and long RNA sequences were depleted of mosquito host sequences, de novo assembled and clustered to yield non-redundant contigs longer than 500 nucleotides. Analysis of the assemblies by sequence similarity to known virus families yielded 115 novel virus sequences, and evidence supports a functional status for at least 86 of the novel viral contigs. Important monophyletic virus clades in the Bunyavirales and Mononegavirales orders were found in these Anopheles from Africa and Asia. The remaining non-host RNA assemblies that were unclassified by sequence similarity to known viruses were clustered by small RNA profiles, and 39 high-quality independent contigs strongly matched a pattern of classic RNAi processing of viral replication intermediates, suggesting they are entirely undescribed viruses. One thousand five hundred sixty-six additional high-quality unclassified contigs matched a pattern consistent with Piwi-interacting RNAs (piRNAs), suggesting that strand-biased piRNAs are generated from the natural virome in Anopheles. To functionally query piRNA effect, we analyzed piRNA expression in Anopheles coluzzii after infection with O’nyong nyong virus (family Togaviridae), and identified two piRNAs that appear to display specifically altered abundance upon arbovirus infection. Anopheles vectors of human malaria in Africa and Asia are ubiquitously colonized by RNA viruses, some of which are monophyletic but clearly diverged from other arthropod viruses. The interplay between small RNA pathways, immunity, and the virome may represent part of the homeostatic mechanism maintaining virome members in a commensal or nonpathogenic state, and could potentially influence vector competence.

Background: Mosquitoes are colonized by a large but mostly uncharacterized natural virome of RNA viruses. Anopheles mosquitoes are efficient vectors of human malaria, and the composition and distribution of the natural RNA virome may influence the biology and immunity of Anopheles malaria vector populations. Results: Anopheles vectors of human malaria were sampled in forest village sites in Senegal and Cambodia, including Anopheles funestus, Anopheles gambiae group sp., and Anopheles coustani in Senegal, and Anopheles hyrcanus group sp., Anopheles maculatus group sp ., and Anopheles dirus in Cambodia. Small and long RNA sequences were depleted of mosquito host and de novo assembled to yield non-redundant contigs longer than 500 nucleotides. Analysis of the assemblies by sequence similarity to known virus families yielded 125 novel virus sequences, 39 from Senegal Anopheles and 86 from Cambodia. Important monophyletic virus clades in the Bunyavirales and Mononegavirales orders are found in these Anopheles from Africa and Asia. Small RNA size and abundance profiles were used to cluster non-host RNA assemblies that were unclassified by sequence similarity. 39 unclassified non-redundant contigs >500 nucleotides strongly matched a pattern of classic RNAi processing of viral replication intermediates, and 1566 unclassified contigs strongly matched a pattern consistent with piRNAs. Analysis of piRNA expression in Anopheles coluzzii after infection with O’nyong nyong virus (family Togaviridae ) suggests that virus infection can specifically alter abundance of some piRNAs. Conclusions: RNA viruses ubiquitously colonize Anopheles vectors of human malaria worldwide. At least some members of the mosquito virome are monophyletic with other arthropod viruses. However, high levels of collinearity and similarity of Anopheles viruses at the peptide level is not necessarily matched by similarity at the nucleotide level, indicating that Anopheles from Africa and Asia are colonized by closely related but clearly diverged virome members. The interplay between small RNA pathways and the virome may represent an important part of the homeostatic mechanism maintaining virome members in a commensal or nonpathogenic state, and host-virome interactions could influence variation in malaria vector competence.

Members of the Anopheles gambiae species complex are primary vectors of human malaria in Africa. It is known that a large haplotype shared between An. gambiae and Anopheles coluzzii by introgression carries point mutations of the voltage-gated sodium channel gene para, including the L1014F kdr mutation associated with insensitivity to pyrethroid insecticides. Carriage of L1014F kdr is also correlated with higher susceptibility to infection with Plasmodium falciparum. However, the genetic mechanism and causative gene(s) underlying the parasite susceptibility phenotype are not known. Mosquitoes from the wild Burkina Faso population were challenged by feeding on natural P. falciparum gametocytes. Oocyst infection phenotypes were determined and were tested for association with SNP genotypes. Candidate genes in the detected locus were prioritized and RNAi-mediated gene silencing was used to functionally test for gene effects on P. falciparum susceptibility. A genetic locus, Pfin6, was identified that influences infection levels of P. falciparum in mosquitoes. The locus segregates as a ~3 Mb haplotype carrying 65 predicted genes including the para gene. The haplotype carrying the kdr allele of para is linked to increased parasite infection prevalence, but many single nucleotide polymorphisms on the haplotype are also equally linked to the infection phenotype. Candidate genes in the haplotype were prioritized and functionally tested. Silencing of para did not influence P. falciparum infection, while silencing of a predicted immune gene, serine protease ClipC9, allowed development of significantly increased parasite numbers. Genetic variation influencing Plasmodium infection in wild Anopheles is linked to a natural ~3 megabase haplotype on chromosome 2L that carries the kdr allele of the para gene. Evidence suggests that para gene function does not directly influence parasite susceptibility, and the association of kdr with infection may be due to tight linkage of kdr with other gene(s) on the haplotype. Further work will be required to determine if ClipC9 influences the outcome of P. falciparum infection in nature, as well as to confirm the absence of a direct influence by para.