The Occurrence In Jiangsu And Comparative Studies In Response To Their Responses To Temperatures And Insecticides Stress Of Two Cryptic Species Of Bemisia Tabaci

Bemisia tabaci(Gennadius)(Hemiptera:Aleyrodidae)is an important pest of cotton,vegetables and ornamental plants worldwide,it is also a species complex containing at least 44 cryptic species.The cryptic species of the Middle East-Asia Minor 1(MEAM1)and Mediterranean(MED)are the most invasive and have been widely distributed in more than 100 countries and regions except Antarctica among them,and they have caused great economic losses to the world and our country's agricultural production.Previous studies have shown that B.tabaci MED cryptic species has stronger invasive and competitive ability than MEAM1.Among the many factors that affect the competition and substitution of different cryptic species of B.tabaci,the roles of environmental temperature and pesticides as two typical abiotic factors cannot be ignored.In this study,we firstly investigated the farmland ecosystem in Jiangsu to clarify the occurrence and damage of B.tabaci and the succession of different cryptic species of B.tabaci in Jiangsu from 2015 to 2019;and to explore the competition between the two cryptic species of B.tabaci(MED and MEAM1);Then,the transcriptomes and the sequence and expression of stress-related key genes(heat shock protein gene,hsps and cytochrome P450 gene,CYP450s)of these two cryptic species of B.tabaci under temperature and pesticide stress were compared,to explore the molecular evidence of the competitiveness of these two cryptic species B.tabaci;Finally,the functions of heat shock protein gene and cytochrome P450 gene in B.tabaci MED were analyzed,and the roles of key genes related to these two types of stress in the process of resistance to external stress were discussed.The main results are as follows:(1)From 2015 to 2019,the results of the investigation on the occurrence and hazard of B.tabaci in Jiangsu and the monitoring of cryptic succession showed that:the damage of B.tabaci in the farmland ecosystem of Jiangsu was relatively light in 2016 and 2017,and the damage of B.tabaci was more serious in 2015,2018 and 2019.And it is found that the cryptic species of B.tabaci are only MED and MEAM1,and the proportion of B.tabaci MED is between 86.5%and 95.6%in these five years,it indicated that the MED was the main cryptic species of B.tabaci in the farmland ecosystem of Jiangsu.In addition,the dynamic changes in the proportion of cryptic species indicated that the two cryptic species of B.tabaci had a competitive relationship.(2)The mortality of the two cryptic species of B.tabaci under temperature and insecticide stress showed that the mortality of cryptic species of B.tabaci MED was significantly lower than that MEAM1 cryptic species under the same stress conditions.In addition,the results of the comparative study of transcriptomes of the two cryptic species of B.tabaci under these two stress factors showed that the two cryptic species of B.tabaci have similar numbers of differentially expressed genes;At the same time,the enrichment analysis of GO,COG and KEGG-related pathways showed that the two cryptic species of B.tabaci had similar stress coping mechanisms under temperature and insecticide stress,the differentially expressed genes involving related to "transmembrane transport","catalytic activity","carbohydrate transport and metabolism","secondary metabolite biosynthesis,transport and catabolism","posttranslational modification,protein turnover,chaperones and translation","lysosome","drug metabolism-other enzymes" were significantly enriched.However,the hierarchical clustering analysis of the heatmap showed that the gene clusters of key stress-related genes(heat shock protein genes and cytochrome P450 genes)showed significantly differential expression patterns in the two cryptic species of B.tabaci.(3)The full-length of four heat shock protein genes(Bt-MEDhsp70-3,Bt-MEDhsp19.5,Bt-MEDhsp19.2 and Bt-MEDhsp21.3)and five cytochrome P450 genes(Bt-MEDCYP4c1,Bt-MEDCYP4g15,Bt-MEDCYP6dz3,Bt-MEDCYP6epl and Bt-MEDCYP302a1)were cloned in B.tabaci MED by RACE(Rapid-amplification of cDNA ends),the same full-length of four heat shock protein genes(Bt-MEAM1hsp70-3,Bt-MEAM1hsp19.5,Bt-MEAM1hsp19.2 and Bt-MEAM1hsp21.3)and four cytochrome P450 genes(Bt-MEAM1CYP4c1,Bt-MEAM1CYP4g15,Bt-MEAM1CYP6dz3 and Bt-MEAM1CYP302a1)were also obtained from B.tabaci MEAM1 by using the same primers.Sequence analysis found that the sequence similarity of these genes in two B.tabaci is as high as more than 95%,and these 8 hsps and 9 CYP450s had high similarity with the corresponding family genes in Hemiptera including B.tabaci.The obtained genes were also analyzed by qPCR using the same primers,and expression comparison studies showed that the four hsps in the two cryptic species of B.tabaci could be significantly induced to express,and had the same expression pattern,but the gene expression folds in MED were significantly higher.Under the stress of two insecticide(thiamethoxam and abamectin),the activity of CYP450 enzymes in the two cryptic species of B.tabaci increased significantly;the BtCYP4c1,BtCYP6dz3 and BtCYP6epl in MED can been up-regulated under the stress of thiamethoxam,and under the stress of abamectin,the BtCYP4cl and BtCYP6epl in MED could been up-regulated,while BtCYP4g15 and BtCYP6dz3 could been down-regulated.However,under the stress of the two insecticide,only BtCYP4c1 in ME AM1 could be induced to up-regulate the expression.(4)Functional analysis of two types of stress-related key genes in B.tabaci MED.Different duration time of high temperature stress will affect the expression pattern of the 4 hsps;insecticides can inhibit the expression of the 4 hsps and increase the mortality of B.tabaci MED under heat stress.High temperature stress can significantly enhance the activity of CYP450 enzymes and promote the up-regulated expression of five CYP450s,thereby improving the survival rate of B.tabaci MED under insecticides stress;In addition,knocking down the expression of 4 hsps and 2 CYP450s(BtCYP4cl and BtCYP6ep1)by feeding double-stranded RNA(dsRNA),and the survival rate of B.tabaci MED under high temperature and thiamethoxam stress was significantly reduced,respectively.In this study,through a systematic investigation of the occurrence and cryptic species of B.tabaci in farmland ecosystems in Jiangsu,it was revealed that there is a competitive relationship between MED and MEAM1 cryptic species,the comparison of the transcriptomes and related key genes of the two cryptic species of B.tabaci under the stress of two abiotic factors(temperature and pesticides),it was further confirmed that B.tabaci MED is more advantageous under the stress of temperature and pesticides and thus explaining the reason why the B.tabaci MED can become the dominant cryptic species of B.tabaci.The results have important theoretical significance for revealing the molecular mechanism of competition and substitution among B.tabaci cryptic species.In addition,based on the functional analysis of key genes related to B.tabaci MED,this study will help to understand the adaptive mechanism of B.tabaci under stress conditions,and help guide the adoption of targeted control strategies in practical production,and provides theoretical guidance for the comprehensive management of B.tabaci.