Identification And Genetic Diversity Of Viruses In The Black Cutworm Agrotis Ipsilon(Lepidoptera:Noctuidae)

The diverse relationships between viruses and their hosts are formed ranging from pathogenic to mutualistic,influencing the life history of individuals and the population dynamics.The continuous development of high-throughput sequencing technology facilitates the identification of enormous uncultured virus species,enriching the genetic diversity of viruses.This work brings us a new understanding about the role of uncultured viruses,which lays a foundation for determining the interaction between viruses and their hosts.Migratory insects by strong flight ability serve as viral sustaining reservoir in the world,which providing convenient conditions for the dispersal and transmission of viruses during their longdistance migration.The black cutworm,A.ipsilon(Lepidoptera: Noctuidae)(Hufnagel),is a major worldwide migratory pest in agriculture and forestry,infesting a variety of crops such as grains,oils,cotton,hemp,potato beans,fruits and vegetables,and has frequent contact with crops and human.At present,more than two viruses in A.ipsilon have been identified,and there is few comprehensive and systematic investigation of viruses carried by A.ipsilon.Researching the viral composition in the black cutworm for identification of virus species and understanding co-evolutionary relationship between viruses and migratory insect hosts are of great significance to improve sustainable pest management strategy and control technology.The main conclusions of our work were as follows:1.This study is based on 938 A.ipsilon adults collected between 2016 and 2018.We analyzed the diverse virus composition and identified viruses by two viral metagenomics.According to high-throughput sequencing analyses,37.48% ORFs predicted based on contigs were related to viruses from 12 viral genera in the 20 viral families except the genus of doublestranded DNA bacteriophages.These ORFs account for 86.71% insect viruses(8 genera in the 12 families),0.37% animal viruses(3 genera in the4 families),0.25% plant viruses(2 genera in the 1 family),2.33%environmental viruses(2 genera in the 3 families),10.24% phages(many genera in the 5 families),<0.08% fungal viruses and protozoan viruses(1family).Through nucleic acid sequence comparison(> 92%),the sequences of three reported viruses of Agrotis ipsilon virus isolate SHAIV,Helicoverpa armigera densovirus-1 and Agaricus bisporus virus 7 were found,suggesting that the black cutworm may act as a vector for transmitting plant viruses.2.Based on the virus-like contigs,we obtained 17 novel complete viral genomes(n = 8)and near complete novel viral genomes(n = 9).Among them,according to the type of nucleic acid,11 circular singlestranded DNA viruses(ss DNA)and 6 linear single-stranded RNA viruses(ss RNA).In the mixed sample of 208 adult moths collected in the field,four novel circular ss DNA viruses and four novel linear ss RNA viruses were identified.Meanwhile,we also amplified Dicistroviridae sp.(MN918781)of birds in the family Dicistroviridae.We cloned the whole genomes of two novel circular ss DNA viruses and two novel linear ss RNA viruses,and analyzed the genetic characteristics of the four viruses.In positive individuals,11 whole genome sequences of Ai Gk V isolates were obtained,and phylogenetic analysis indicated that isolates formed a new evolutionary branch clustered with human gemycircularvirus HV-Gc V2(NC＿030448).Genetic analysis showed that 23 nucleic acid sequences of ORF3 of Ai NV isolates clustered together in a unique evolutionary branch.This work extends the host-range and diversity of viruses carried by migratory pests black cutworms.3.The infection rates of the significant four viruses were investigated with adult moths from 11 regions in Northeast and North China,Central and East China between 2014 and 2020.The investigation results in field showed that the infection rates of Ai Gm V1 and Ai Gk V were 6.85%(15/219)and 16.8%(54/322),respectively.Ai RV1 and Ai NV infection rates were 78.9%(464/588)and 8.86%(42/474).The sampling region was estimated to be a risk factor(P<0.001),associated with infection rates of Ai Gk V and Ai RV1.The season and gender were estimated to be risk factors,associated with infection rate of Ai NV.The infection rates of the four novel viruses were: Ai RV1 > Ai Gk V > Ai NV > Ai Gm V1.In addition,the co-infection analyses of the four viruses found that the co-infection rate(>2)was 20.9%(43/206),and the rate(>3)was 1.5%(3/206).4.The cp gene sequences(483bp)of 399 Ai RV1 isolates were sequenced from A.ipsilon to analyze the genetic relationship and population dynamic.The overall nucleotide and haplotype diversities of399 Ai RV1 isolates were both low,meanwhile indicating higher genetic diversity in autumn than spring and summer with significant difference.Among them,the genetic diversity of Ai RV1 isolates of migration of A.ipsilon over the Bohai Sea was relatively high,especially in the third migratory stage.Based on phylogenetic and DAPC analyses and haplotype network,the isolates from nine sampling sites clustered together and had one shared haplotype.These results suggest that Ai RV1 isolates were no significant differentiated with geographic distance and had frequently gene communication.The results of the neutral test and mismatch distribution indicated Ai RV1 isolations in migrations of A.ipsilon over the Bohai Sea were long-term stable.Using gene flow analyses,reconstruction of the spatial diffusion patterns revealed that Ai RV1 populations were more likely to diffuse from south to north in late spring and early summer and the journey of the Ai RV1 populations spreads in multiple directions in midsummer.These directionality was supported by the phylogeographic analyses.This suggests that the dispersal pattern of Ai RV1 might be related to migratory pests black cutworms.In conclusion,this is the first report of the viral composition of black cutworms and eight novel viruses,enriching the insect virus database.The black cutworm would be likely to a virus-insect vector with cross-species transmission,providing a new direction of agricultural pest management.Relationships between infection rates and the different factors provide the base data for insect virome and early warning for the dispersal and transmission of potential uncharacterized viruses.Our work also illustrated the genetic diversity and structure,and spatial diffusion pathway of Ai RV1 populations,providing reference evidences for long-distance migration of A.ipsilon,and improving scientific basis for the establishment of sustainable control and management strategy.