Epigenetic Mechanism In Thermotolerence Of Bemisia Tabaci Based On DNA Methyltransferase And MiRNA

Global warming and anthropogenic climate change are expected to exacerbate the biological invasion of exotic species.For invasive insects,their high reproductive potential and genotypic plasticity allow them to modify their phenotype in response to a wide range of environmental temperatures,leading to successful adaptation,establishment,and spread in a novel environment.The whitefly,Bemisia tabaci?Gennadius?,is a globally-distributed destructive pest of agricultural and ornamental crops,with high fecundity,overlapping generations,and a wide host range.B.tabaci is considered to be a species complex that comprises at least 36 morphologically indistinguishable species.In China,there are 15 cryptic species,including 13 local cryptic species and two invasive species.The MED?Mediterranean?,which was first detected in China in 2003,rapidly spread to most provinces except for Ningxia province,and has gradually became widespread,displacing other invasive cryptic species?Middle East-Asia Minor 1?and local cryptic species?Asia?3?.Asia?3 is mainly distributed in Guangxi,Taiwan,Hunan,Zhejiang,and Hubei provinces.The successful invasion and wide displacement of MED has been investigated with respect to its broad thermal tolerance in response to a wide range of environmental temperatures.Epigenetic changes are more determinant than genetic variability in mediating environmentally-induced phenotypic variation in invasive species during successful invasions.DNA methylation and miRNAs are the main mechanisms of epigenetic.DNA methylation,which is mediated by DNA methyltransferases,modulates the response to environmental stress by regulating gene expression.miRNAs,which can inhibit the expression of target mRNAs at the post-transcriptional level through negative regulation,are involved in growth,development,and phenotypic plasticity in organism.Previous studies have shown that DNA methylation is closely related to thermal tolerance in B.tabaci.In order to assess and interpret the epigenetic mechanisms of differential thermal tolerance in response to thermal stress between invasive and indigenous whitefly species,we chose the invasive species B.tabaci Mediterranean?MED?and the indigenous species B.tabaci Asia?3 to examine their thermal tolerance at the DNA methylation and miRNA levels.We first cloned and sequenced the Dnmt2 and Dnmt3 genes from both MED and Asia?3,and then used qRT-PCR,Western blotting,and in situ hybridization to detect the distribution,localization,and expression patterns of Dnmts genes in the two whiteflies.We also determined the function of the Dnmt genes in both MED and Asia?3 using RNAi technology.We then compared the transcriptomes?RNA-Seq?and miRNA profiles?small RNA-Seq?of MED and Asia?3 adults after heat and low temperature.To identify the key temperature-related genes and signaling pathways in the two whiteflies,and to also find the differentially-expressed miRNAs and their target genes between the two whiteflies as they relate to thermotolerance.The main results and conclusions are discussed below.In this study,the full cDNA sequences of the Dnmt2 and Dnmt3 genes were determined from the MED and Asia?3,and named MED-BtDnmt2,Asia?3-BtDnmt2,MED-BtDnmt3,and Asia?3-BtDnmt3.The sequence similarity between the MED-BtDnmt2 and Asia?3-BtDnmt2 proteins was 99.04%,and 75.79%with other insect Dnmt2 proteins.MED-BtDnmt3 and Asia?3-BtDnmt3 shared 93.02%similarity,and 68.82%with other insect Dnmt3 proteins.All of these protein sequences shared a high level of identity with six highly conserved motifs within the C-terminal catalytic region that is responsible for DNA methylase activity.Combined with the previous results,we found that the B.tabaci genome encodes a full DNA methylation "tool kit",including single genes for Dnmt1,Dnmt2,and Dnmt3.We observed significantly higher expression of Dnmt1 and Dnmt3 in the abdomen and in the adult stages in both MED and Asia?3.In situ hybridization also showed that the two genes are expressed in the posterior region,which is related to reproduction.Furthermore,results of qRT-PCR assays and western blotting showed that Dnmtl expression in Asia?3 adults was significantly higher than in MED;conversely,Dnmt3 gene expression in Asia?3 adults was significantly lower than in MED.The present data suggests tissue-and sex-specific expression of both Dnmtl and Dnmt3 in B.tabaci,indicating that a functional differentiation of Dnmtl and Dnmt3 may exist between MED and AsiaII3.We hypothesize that the increased expression of Dnmt3 in MED may be related to environmental stress-induced phenotypic plasticity and adaptation.To examine whether there were differences in Dnmt gene expression between MED and Asia?3 adults after thermal exposure,we compared the expression of Dnmtl and Dnmt3 in females and males that were exposed to high?31??and low?21??temperature stresses for one generation.The expression patterns of Dnmt-specific mRNAs and the proteins after thermal stress showed a difference between MED and Asia?3.Although the expression of Dnmtl was increased in MED females in response to high temperature,the relative protein expression was decreased.However,both the survival rate and thermal resistance were significantly decreased in MED females after feeding on dsDnmtl.It is clear that both Dnmt3-specific mRNA and protein expression high temperaturewere significantly increased in MED females following exposure to high temperature,although there was no increase in Asia?3.Furthermore,both survival rate and thermal resistance were significantly decreased in MED females and males after RNAi knock-down of Dnmt3.There were no significant differences in Asia?3.We speculate that Dnmtl may play a role in thermotolerance in B.tabaci,while our results indicate that Dnmt3 plays an important role in thermotolerance in the invasive species MED.We also speculate that the increased thermal tolerance observed in MED whiteflies is regulated by Dnmt3 and can be maternally transmitted to the offspring.These findings provide indirect support for the hypothesis that the epigenetic control system is a major driver to increasing thermotolerance in the invasive whitefly species MED,that allows it to adapt to thermal stress during the invasion process.The adult MED and Asia?3 transcriptomes at 21?,31?,and 26? were sequenced using an Illumina platform.A total of 20,748 genes were obtained after mapping to the MED genome.The genes were annotated using Blast2GO searches of the Nr,KEGG,eggNOG,UniProt-sprot,and UniProt-TrEMBL databases.There was a clear difference in gene expression between MED and Asia?3 with respect to both high temperature and low temperature treatments.For MED,there were 335 differentially expression genes?DEGs??108 up-regulated and 227 down-regulated?in the high temperature treatment and 244 DEGs?213 up-regulated and 31 down-regulated?in the low temperature treatment.For Asia?3,there were 3,394 DEGs?1,723 up-regulated and 1,671 down-regulated?in the high temperature treatment and 3,969 DEGs?2,027 up-regulated and 1,942 down-regulated?in the low temperature treatment.Among these DEGs,there were 234 up-regulated and 122 down-regulated genes unique to MED,and 1,626 up-regulated and 2,060 down-regulated genes unique to AsiaII3.KEGG enrichment analysis showed that the DEGs from both MED and Asia?3 were mainly enriched in pathways involved in Metabolism,Organism Systems,and Environmental Information Processing,such as pyrimidine metabolism,purine metabolism,protein processing in the endoplasmic reticulum,the PI3K-Akt signaling pathway,the MAPK signaling pathway,and the Lysosome.Furthermore,the results showed that the unique up-regulated DEGs in MED were mainly enriched in antigen processing and presentation,starch and sucrose metabolism,and galactose metabolism,which are related to energy metabolism and the insect immune response.These results suggest that the DEGs from these special pathways may be important genes that are involved in a high thermal tolerance in MED than Asia?3.We performed small RNA sequencing on MED and Asia?3 adults treated at 21?,31?,and 26?.After bio informatics analysis,26 known and 85 novel miRNAs were identified.Among them,we identified a total of 24 differentially expressed miRNAs in the MED high temperature treatment and four in the low temperature treatment,while there were 25 differentially expressed miRNAs in the high temperature treatment and 25 in the low temperature treatment in Asia?3.Interestingly,MED and Asia?3 shared no up-or down-regulated miRNAs.The transcriptome data was used to predict and annotate the target genes,and then to screen for potential temperature-related miRNAs and target genes.We speculate that three unique up-regulated miRNAs?miR-2796,novel₉5,and novel₁3?and three unique down-regulated miRNAs?miR-8,miR-13a,and novel₃1?and their target genes in the MED high temperature treatment could be important factors leading to differences in thermal tolerance between MED and Asia?3.This study provides directions and targets for further studies on genes and miRNAs related to thermal tolerance in B.tabaci.In summary,we focused on epigenetic mechanisms,encompassing DNA methylation,the transcriptome,and miRNA expression,that could potentially explain the differences in thermotolerance between the two B.tabaci species,MED and Asia?3.Our study confirmed the role and function of DNA methyltransferases in thermal tolerance,and we also examined the possible involvement of miRNA regulation.The data presented here increase our understanding of the role played by epigenetics in temperature adaptation in B.tabaci MED,and provide a foundation for future studies aimed at understanding the potential links between epigenetics and thermal tolerance plasticity in invasive insects.