The Outbreak Mechanism Of Chlorops Oryzae Based On Genome Data

Chlorops oryzae is a pest of rice crops widely distributed in China,Japan and Korea.In recent years,there have been frequent outbreaks of this pest in many counties and cities in Hunan province,causing significant economic losses.In 2017,the loss area of rice crops in Youxian of Hunan province reached 1,000 hectares,and the damage area has been nearly533,000 hectares.Previously,C.oryzae was a secondary pest of rice,but it has now become a major pest on rice in many counties and cities of Hunan province.However,the reason of frequent C.oryzae outbreaks is not clear,and there is no attention about it at home and abroad.To elucidate the reason and mechanism,this paper analyzed the genetic diversity and genetic differentiation of C.oryzae populations based on a dataset of ISSR molecular markers and COI sequences,revealed the genome structure and characteristics by insect genomics,and clarified the evolution of environmental stress-related gene families with comparative genomics.Finally,we identified the important genes regulating the reproduction of C.oryzae.These results provide a theoretical basis for revealing the internal mechanism of C.oryzae outbreak and sustainable pest management.The main contents and conclusions are as follows:1.Genetic diversity and differentiation of populations of C.oryzae.ISSR data revealed a high level of genetic diversity among the 20 populations collected from Hunan(18),Zhejiang(1)and Guizhou province(1),and only 9.97% genetic variation is between populations.There was a high level of gene flow and low,or medium,genetic differentiation among some populations.The results of a Mantel test revealed no significant correlation between genetic and geographic distance among populations,which means there is no evidence of significant genetic isolation by distance.mt DNA COI data indicates low nucleotide(0.0007)and haplotype diversity(0.36)in all populations.Fst values suggest that the 20 populations have low,or medium,levels of genetic differentiation.And the topology of a Neighbor-Joining tree suggests that there are no independent groups among the populations examined.All results are consistent with frequent gene exchange between populations,which could keep the genetic diversity,and maintain adaptability of C.oryzae,thereby promoting frequent outbreaks of this pest.2.Genome sequencing and assembly of C.oryzae.We generated a 447.60 Mb chromosome-level genome with a contig N50 of 1.17 Mb and a scaffold N50 of 117.57 Mb.Hi-C assisted assembly showed that 93.22% of contigs located on the 4 chromosomes.In addition,256.8 Mb of repeat sequences,1378 t RNAs,161 mi RNAs,130 r RNAs and 93 sn RNAs were identified,and 14,863 coding proteins were annotated by functional databases,which have important reference value for the subsequent study of functional genes.3.Comparative genomics and gene family analysis of C.oryzae.Comparative genomics analysis showed that the C.oryzae diverged from the Ceratitis capitata of the same cyclorrhaphan Diptera approximately 186 Mya ago.Compared with the common ancestor of C.oryzae and C.capitata,the C.oryzae genome displayed 561 expanded and 416 contracted gene families.In addition,69 P450 genes,57 HSP genes(6 HSP90,14 HSP70,14 HSP60,10 HSP40,and 13 s HSP)and 37 antioxidant genes(2 CAT,8 POD,5 SOD,and 22 GST)related to environmental factor stress were identified.4.Roles of heat shock proteins and antioxidant genes of C.oryzae in response to thermal stress.High-throughput RNA sequencing(RNA-seq)results show that stress response genes,such as Heat Shock Proteins(HSPs),were up-regulated by larvae subjected to heat stress.Antioxidant genes(SOD,GST and POD)were also markedly differentially expressed in different temperature treatment groups(24 ℃,33 ℃ and 39 ℃).Furthermore,quantitative real-time PCR(q RT-PCR)confirmed that expression levels of stress response genes such as HSP83,HSP70,HSP68,HSP67B2,HSP27,HSP23 and antioxidant genes such as SOD,GST and POD,were similar to the RNA-seq results.These results indicated that both stress response and antioxidant genes play important roles in counteracting thermal stress.5.Roles of P450 genes of C.oryzae in pesticide stress.Field experiments showed that dinotefuran has a better control effect on C.oryzae,while imidacloprid has a poorer control effect on C.oryzae.Transcriptome analysis showed that the expression of 4 P450 genes(CYP6A13,CYP9B2,CYP313A4 and CYP28D1)were up-regulated in larvae under pesticide stress(20% dinotefuran SC 500 and 2000 times dilution and 70% imidacloprid WDG 500 and 2000 times dilution).Quantitative real-time PCR(q RT-PCR)confirmed that the expression of CYP6A13 and CYP9B2 in larvae were significantly up-regulated under the stress of dinotefuran,indicating that these two genes were involved in detoxification metabolism of dinotefuran.The expression of CYP317A1 in C.oryzae adults was significantly up-regulated with 20mg/L dinotefuran treatment,and the expression of CYP6D1 was significantly up-regulated with 3mg/L imidacloprid.RNAi and the bioassay test after RNAi showed that the metabolic detoxification of CYP6D1 to imidacloprid enhanced its tolerance to pesticide,thereby reducing the mortality of pest with pesticide treatment.6.Effects of genes related to reproductive on ovarian development of C.oryzae.Reproductive development of insect is crucial for maintaining insect populations.Silencing of Vg(reproductive-related gene),Met,Kr-h1(juvenile hormone signaling pathway genes),In R,PI3 K,TOR,FOXO(insulin signaling pathway genes)and USP(ecdysone signaling pathway gene)significantly reduced the relative expression levels of these genes.The synchronous observation of ovarian development in RNAi group showed that the inhibition of the expression of these genes can result decrease of yolk deposition in the oocytes,thereby inhibiting ovarian development.The study on regulation of ovarian development is helpful to analyze the influence of external factors on the fitness of C.oryzae,and then analyze its outbreak mechanism from different perspectives.These results are helpful to analyze the environmental adaptation of C.oryzae and the possible causes of its frequent outbreaks.They also have important reference value for the screening of new target genes for pest control and the development of new technology systems.