CYP321A9 And CYP9A58 Genes Mediate The Metabolic Mechanism Of Spodoptera Frugiperda To Rice Insect-resistant Substances

Spodoptera frugiperda is a global pest that has already caused immense economic losses worldwide,which has recently invaded China in April 2019.S.frugiperda has two sympatric strains,the “corn strain”(C strain),which prefer to feed on maize and other large grasses,and the “rice strain”(R strain)that preferentially feeds on rice and various pasture grasses.The S.frugiperda that invaded China,was the “corn strain” biotype(CS).However,our results,along with findings from previous research,show that S.frugiperda CS species can complete their entire life cycle on rice.Nevertheless,the genetics behind insect pest responses to host switches,and their ability to adapt to new host plants,remains poorly understood.In this study,we compared the population fitness of S.frugiperda larvae that feeding on rice and maize.Using a combination of transcriptome,metabolome,andRNAi technologies,we discovered that SfCYP321A9 and SfCYP9A58 are involved in metabolizing allelochemicals in rice,enabling S.frugiperda to adapt to an alternative host plant(rice).Our results contribute to a greater understanding of the molecular mechanisms of host plant adaptation and the development of effective tools for managing S.frugiperda resistance.The main research contents and conclusions are as follows:1.Identification of the invasion of S.frugiperda in HunanThe genetic marker data indicates that the corn-strain is the predominant subpopulation present in the Hunan population of S.frugiperda.2.Fitness analysis of the S.frugiperda larvae feeding on rice and maizeThrough the analysis of the growth fitness of “corn strain” biotype,S.frugiperda larvae feeding on corn and rice,it was found that the larvae fed on rice had a significantly longer development time compared to those fed on corn.The mean developmental time for rice and maize fed larvae was 20.26 ± 1.43 days and 18.74 ± 1.34 days,respectively.The generation duration of S.frugiperda feeding on rice was 38.42 ± 2.50 days,and the generation duration of S.frugiperda on corn was 34.13 ± 2.58 days.The larvae weight was also affected significantly by hosts.Pupa weight was significantly different by hosts,which showed the heaviest in maize and the lightest on rice.3.Identification and functional analysis on differential genes of S.frugiperda feeding on different host plantsThrough the transcriptome sequencing from the midgut of S.frugiperda larvae that feed with rice and corn,respectively.A total of 44 080 Unigenes were obtained.The high-quality data of all samples were higher than 97.77% in Q20,bases ratio were not lower than 93.56%in Q30.N50 is 1745 bp.And 4266 transcripts were annotated DEGs.Feeding on rice induced the up-regulation of 1722 genes and the down-regulation of 2544 genes compared to feeding on corn.The up-regulated gene transcripts of S.frugiperda that were studied in the set of DEGs were members of the cytochrome P450s(6 transcripts),including SfCYP6AN4,SfCYP321A9,SfCYP6AE44,SfCYP321A10,SfCYP9A58 and SfCYP6AB12.The results of q PCR test showed that SfCYP321A9 and SfCYP9A58 have higher expression levels in individuals feeding on rice compared to those feeding on corn.Knocking down SfCYP321A9 and SfCYP9A58 resulted in a prolonged development time of S.frugiperda larvae that fed on rice.4.Differential metabolite identification and bioassay of S.frugiperda feeding on different host plantsMetabolome analysis of the differential metabolites of S.frugiperda larvae feeding on SfCYP321A9 dsRNA and EGFP dsRNA(control)rice leaves,respectively.A total of 167 significantly differential metabolites were identified in ds CYP321A9 and ds EGFP with either up-regulation or down-regulation modes.Among these metabolites,86 and 81 were up-regulated and down-regulated,respectively,in ds CYP321A9 compared to ds EGFP.Hierarchical clustering and KEGG enrichment analysis showed that the differential metabolites were mainly enriched in the pathways of serotonergic synapse,neuroactive ligand-receptor interaction,pyruvate metabolism,tryptophan metabolism,taurine and hypotaurine metabolism,and platelet activation.Comparative metabolomic analysis showed that the metabolites(carboxylic acids and derivatives)differed between SfCYP321A9 and EGFP dsRNA treatments.Additionally,the results showed that the expression levels of SfCYP321A9 and SfCYP9A58 genes was significantly increased after feeding on artificial diet containing rice insect-resistant secondary metabolites(0.8% ferulic acid,0.8% gramine and 0.05% tricin)for 48 hours.RNAi knockdown of SfCYP321A9 resulted in significantly higher mortality of S.frugiperda larvae when exposed to rice allelochemicals ferulic acid,gramine and tricin.Furthermore,overexpression of SfCYP321A9 significantly reduced mortality in Drosophila melanogaster when exposed to gramine and tricin.Our results contribute to a greater understanding of the molecular basis of host plant adaptation and provide the means to develop effective management tools for S.frugiperda resistance.Through the identification of S.frugiperda that invaded in Hunan Province,comparation of the growth fitness of S.frugiperda larvae feeding on maize and rice,and identification of the differential genes and metabolites of S.frugiperda larvae that feeding on different host plants and verified putative genes function.It is suggested that S.frugiperda invaded in Yunnan province,China,was “corn strain” biotype and revealed SfCYP321A9 and SfCYP9A58 as key detoxification genes for S.frugiperda in response to different host plants.The results provide a foundation for assess the risk to rice caused by the “corn” strain of S.frugiperda and develop effective pest management strategies.