Genome Sequencing Of Mythimna Separata And Functional Analysis Of Body Colar Genes Related To Phase Change

The oriental armyworm Mythimna separata(Lepidoptera: Noctuidae),one of the most devastating cereal crop pests,is a model migratory species undertaking a long-distance multigenerational roundtrip migration between southern and northern China.Larvae can transition between solitary and gregarious phases in response to population density changes,while migratory adults can be switched into residents during a sensitive stage.Migration of the armyworm is the combination of environment conditions and internal genetic features,which is simultaneously correspond with adaptive mechanism in physiology and endocrinology.In recent years,the rapid development of DNA sequencing technology has greatly promoted the research of insect genomics and lots of pests’ genome sequences in China have been published.In this study,we try to get the whole genome sequences of M.separata through the next generation sequencing,combined with transcriptome sequencing between migrants and residents to screen migrantrelated gene groups.The mechanism of density-dependent prophylaxis in the larvae of M.separata was also investigated from the perspective of biogenic amine receptors,in order to provide theoretical basis for the mechanism of its frequent outbreak and significant agriculture losses.What’s more,genome editing technology with CRISPR/Cas9 was also conducted to build a genome editing platform for the gene function study of M.separata.The main results are as follows:1.We sequence the genome of M.separata and assembled a draft genome of 694.7 Mb,with a scaffold N50 length of 1.37 Mb.The M.separata genome is suggested to have a medium degree of quality and the percentages of repetitive elements and GC content are comparable compared to other published moth genomes.We generated an official gene set by integrating ab initio gene predictions,transcriptome data from pooled M.separata samples,and insect homology.A total of 16,968 protein-coding genes was predicted,of which 84.8% of the coding genes are expressed in at least one tissue or developmental stage of the selected transcriptome sequencing samples.Orthologous genes across other 11 lepidoptera species were identified and 553 M.separata species-specific genes were annotated,while 241 orthologous gene clusters were specifically expanded in M.separata.We investigated the evolution signatures of the migration population,comparing Tajima’s D between M.separata and Spodoptera litura and found that the migration population of M.separata was experiencing bottleneck 100,000 years ago,undergoing a successive contraction stage.The genome of M.separata has a high degree of heterozygosity with 213 gustatory receptor genes,of which the bitter receptor genes are undergoing significantly expansion.2.We performed a comparative transcriptome analysis for brain and cuticle tissues over diverged development between solitary and gregarious larvae and resident and migrant adults of M.separata by RNA-seq.Differentially expressed genes(DEGs)were identified and the results shew that DEGs in phase change began to accumulate at larval forth instar,rose to the peak at sixth instar.KO pathway analyses showed upregulated genes in the forth instar were enriched most in the melanination pathway.The temporal expression patterns of the key genes in the melanination pathway was consistent with diverged development between solitary and gregarious larvae.Genes involved in the proteasome pathway were highlighted from the analysis of KO enrichment both in the development of gregarious larvae’s sixth stadium and the migratory adults’ forth day after emergence,indicated that proteasome was widely involved in various biological processes of the larvae and adults of M.separata.The insect hormone biosynthesis pathway was also enriched on the fourth day after emergence,suggesting that hormone was involved in the development of migratory individuals.3.The octopamine receptor MsOA1 and serotonin receptor Ms5HT1 belong to the superfamily of G-protein-coupled receptors,both of which are highly expressed in hemocytes and nerve cords.Stimulation of exogenous octopamine and serotonin can enhance the immune response of larvae’s hemocytes.Larvae reared at different densities experience a consistent trend on cellular immune reactions of hemocytic spreading and phagocytosis under the stimulating of different concentrations of OA and 5-HT,while the gregarious larvae of M.separata exhibit a stronger immune response than that of the solitary ones generally.Furthermore,the relative expression level of MsOA1 and Ms5HT1 of the gregarious larvae is higher than that that of the solitary ones in response to fungus Metarrhizium anisopliae infection,of which the expression trend is consistent with the immune response gene Toll1 as a positive control,suggested that both of MsOA1 and Ms5HT1 are involved in the cellular immune responses of M.separata larvae.4.Using CRISPR/Cas9 technology,pale and ebony genes were selected as target genes with Cas9 m RNA and gene-specific sg RNA synthesized in vitro and a genome editing platform of M.separata was successfully constructed with embryo microinjection.The mutants of pale injection displayed a lighter body color than the wild types on larvae,and pupae and adults showed obvious light-colored patches.On the contrast,the mutants of pebony injection showed a visibly deepened phenotype than the wild types on larvae body,pupae shell and different parts of adults,with large amount of melanin depositing.Ebony can be used as a potential Marker gene in follow-up studies for phenotypic observation and screening to identify mutants more efficiently as for its obvious mutant phenotypes.