Identification And Functional Analyses Of Two Novel RNA Viruses In Pteromalus Puparum

Parasitoid wasps are important natural enemies of arthropod hosts in natural and agricultural ecosystems and are often associated with viruses or virion-like particles.Members of the species Pteromalus puparurum are predominantly pupal parasitoid wasps that prey on butterflies of several species including the small white(Pieris rapae),with the highest parasitism rate over 90%reported in fields of cruciferous vegetables in China.To better understand the relationship between this parasitoid wasp and its hosts,we recently sequenced the wasp's transcriptome and discovered two novel viruses.Then,we characterized their genome organization,their phylogenetic placement,geographic distribution,transmission strategy,and biological impacts on its host.1.A novel negative-stranded RNA virus mediates sex ratio in its parasitoid hostHere,we report a novel negative-stranded RNA virus from a parasitoid wasp(Pteromalus puparum).The complete viral genome is 12,230 nucleotides in length,containing five non-overlapping,linearly arranged open reading frames.Phylogenetically,the virus clusters with and is a novel member of the mononegaviral family Nyamiviridae,here designated as Pteromalus puparum negative-strand RNA virus 1(PpNSRV-1).PpNSRV-1 is present in various tissues and life stages of the parasitoid wasp,and is transmitted vertically through infected females and males.Virus infections in field populations of P.puparum wasps ranged from 16.7 to 37.5%,without linearly correlating with temperature.PpNSRV-1 increased adult longevity and impaired several fitness parameters of the wasp,but had no influence on successful parasitism.Strikingly,PpNSRV-1 mediated the offspring sex ratio by decreasing female offspring numbers.RNA interference knockdown of virus open reading frame I eliminated these PpNSRV-1-induced effects.Thus,we infer that PpNSRV-1 has complex effects on its insect host including sex ratio distortion towards males,as well as possible mutualistic benefits through increasing wasp longevity.2.A novel dicistrovirus which only had one ORF found in parasitoid waspHere we reported on the presence of a novel picorna-like virus from P.puparum,designated as Pteromalus puparum small RNA-containing virus(PpSRV),according to RNA sequencing and electron microscopy analysis of the parasitoid wasp.Depending on the transcriptomic data assembly,RACE and RT-PCR,we got the whole genomic sequence of this single-stranded RNA virus,the sequence represented that PpSRV belonged to picorna-like virus.The full length of the PpSRV genome was 8983 nucleotides.Only one single open reading frame(ORF)has been found in the PpSRV genome.Western blot analysis indicated that the big protein coded by the PpSRV ORF was present.A phylogenetic tree was generated from the RdRp domain of viruses from the order Picornavirales,and revealed that PpSRV was mostly near to BQCV(black queen cell virus,a virus originally found in dead honey bee queen larvae and pupae,Dicistroviridae,Triatovirus)and is a novel member of the picornaviral family Dicistroviridae.But it was different that all of dicistroviruses contained two ORFs.Viral load of PpSRV was highest in the digestive tract and had a significant increasing in adult wasps.Virus infections in field populations of P.puparum wasps was 100%.By RNAi,we found that PpSRV reduced the pathogens' resistance of adult wasps.