| The tropical bed bug,Cimex hemipterus(Hemiptera: Cimicidae),a wingless and hematophagous insect,which feeds on human beings as well as other warmblooded animals.Due to the similar morphology with temperate bed bugs(C.lectularius),they are collectively known as bed bugs.Bed bugs are important health pest.Tropical bed bugs are serious and difficult to eliminate because of resistance,especially in the Pearl River Delta region of China.Therefore,with the problem of bed bug resistance,it is urgent to study the resistance mechanism of tropical bed bug,especially the resistance mechanism of pyrethroid insecticides commonly used.In this study,bed bugs were collected from areas where bed bugs frequently occur in China,mainly taking tropical bed bugs as the research object,the main results are as follows:1.Transcriptome sequencing was performed on the tropical bed bug population in Shenzhen,and full-length voltage-gated sodium channel genes(VGSC)related to knockdown resistance(kdr)gene locus were further screened from the tropical bed bug transcriptome.A total of 5.49 Gb data were obtained from quality-controlled raw sequencing data.A total of 21 619 unigenes with a total length of 29 322 540 bp and an average length of 1 356 bp were obtained.Unigenes sequences were alignmented and annotated of the unigenes sequences with the databases of non-redundant protein sequence(NR),Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG),Evolutionary Genealogy of Genes: Non-supervised Orthologous Groups(egg NOG),and Swiss-Prot.A total of 45 362 unigenes were obtained and their functional annotations have been analyzed.A total of 10 906 coding regions were predicted,7 754 simple repeat sequence(SSR)locis and 33 144 SNP locis were detected,and 8 VGSCα genes were obtained,4 of which had full-length sequences.2.The full-length gene sequence of voltage-gated sodium channel protein genes was obtained by molecular cloning technique,and the expression pattern of the gene was determined at different developmental stages of tropical bed bug.A 6 360 bp c DNA sequence of voltage-gated sodium channel gene(Chemi Nav)was obtained,which ORF sequence was 5 931 bp encoded 1 976 amino acids.The molecular weight of the protein was 223.2 k Da,and the isoelectric point was 5.42.Chemi Nav has the highest expression in the third instar nymph stage,the lowest expression in the male adult stage,and no expression in the egg stage.The ORF sequence of tropical bed bug sodium channel encoding gene was cloned and its typical characteristics were identified.There were four transmembrane regions(I～IV),and each transmembrane region was composed of six domains(S1～S6).Kdr mutation sites were found in ⅡS4 and ⅡS6.Evolutionary analysis showed that tropical bed bugs and temperate bed bugs were clustered into one branch,indicating that they were closely related to other bed bugs and other groups had clear clustering.3.Finally,the relationship between the occurrence of kdr mutation and bed bug sensitivity to beta-cypermethrin was investigated by using three methods: bioassay by topical assay,rapid resistance identification method by using a discriminating dose and PCR method was used to detect the kdr mutation frequency.The results of the topical assays indicated that susceptibility of C.lectularius to five insecticides was: Imidacloprid and Dinotefuran > Thiamethoxam > Chlorantraniliprole and Beta-Cypermethrin.The resistance to Beta-Cypermethrin of the two field populations of C.hemipterus was higher than that in C.lectularius.Kdr mutations were found in all tested 10 field bed bug populations and four mutation sites were identified.The four C.lectularius populations can be divided into three mutation types(A: no mutated sites;B: Only L925I;C: L925 I and V419L).The seven C.hemipterus populations all had mutated loci M918 I and L1014 F.Existence of mutation sites was closely related to the susceptibility of bed bugs to a pyrethroid insecticide.Results suggest resistance to pyrethroids was widespread among the Chinese bed bug populations.Existence of mutation sites was closely related to the susceptibility of bed bugs to a pyrethroid insecticide.Results suggest resistance to pyrethroids was widespread among the Chinese bed bug populations. |
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