Function Of A Vesicle Transport Gene Rop In The Major Agricultural Spider Mites

Tetranychus spider mite(Arthropoda,Arachnid,Acariformes,Tetranychidae)contains more than 1,300 species.Spider mites are of small body size and rapid reproduction.They damage crops,flowers,fruits and vegetables,causing huge economic losses to agriculture.Spider mite distributes widely in China.At present,the most prevalent spider mites in China are Tetranychus truncatus Ehara,T.urticae Koch,T.kanzawai Kishida,T.pueraricola Ehara and Goto and T.phaselus Ehara,etc.Spider mites have become highly resistant to insecticides as a result of abusing pesticides for many years.In addition,in response to the national "double reduction" policy to pesticide and fertilizer,the environmental-friendly pest control methods,such as biological control,are widely considered.The Ras opposite protein(ROP)belongs to the Sec1/Munc-18 protein family.ROP distributes in the cytoplasm and cell membrane.It can control the assembly of the Soluble N-ethylmaleimide-sensitive Factor Attachment Protein Receptor(SNARE)protein complexs to regulate the fusion of vesicle.Rop can be expressed in different insect tissues.Rop participates in a number of insect essential activities and therefore Rop is considered to be a candidate gene that can be used for control.Previous studies have shown that RNA interference(RNAi)of Rop in T.truncatus decreased adult mite oviposition significantly.However,whether Rop is conserved among Tetranychus species is not yet known,and the function of ROP in other spider mite species needs to be further studied.In this study,the ROPs of four spider mites(T.truncatus,T.urticae,T.kanzawai,and T.pueraricola)that are seriously harmful to agricultural production in China were studied.First,the conservation and evolution of Rop were analyzed by bioinformatic analysis.Secondly,we detected the expression patterns of Rop in multiple developing stages.Then,the RNAi method was used for gene functional analysis.Finally,we explored potential molecular mechanism of Rop affecting phenotypes.The main results are as follows:(1)The Rop is highly conserved among the four mite species.The similarities of nucleotide sequences and amino acid sequences are all about 98%.The phylogenetic result based on the Rop nucleotide sequence is similar to the species phylogenetic relationship(tree of mt COI),indicating that the gene is relatively conserved among relative species.But,it can be distinguished in different groups of insects.Furthermore,we found that the ROPs of spider mite were hydrophilic protein without any signal peptides nor transmembrane domains.The protein was predicted to be subcellularly localizated in cytoplasm.(2)The expression patterns of Rop are relatively consistent from larva to adult.But,different spider mites expressed different levels of Rop at the same developing stage.The expression level of Rop in nymph is reduced by 20%～40% compared with that of larva.Then the expression started to increase at the next instar.The expression level of Rop increases with the growth of the mite and reaches a peak in the adult.(3)RNAi of Rop in four species of spider mites were conducted by soaking the spider mites in ds RNA.The RNA interference efficiencies of four species mites were 29.20%,53.08%,19.06%,and 37.93%,respectively.After knocking down Rop,the reproductive ability of female mites was significantly declined.Comparing to ds GFP control,the total number of laid eggs in 3 days after emergence in ds Rop treatment dropped by 47.74%,82.40%,84.51% and 55.72%.In addition,the spider mites became darker and smaller after being treated with ds Rop.However,the decrease of Rop expression level had no significant effect on the mortality of four Tetranychus species.(4)In T.urticae,the expressions of syntaxin-1A and syntaxin-6 were significantly upregulated while that of syntaxin-5,syntaxin-12,syntaxin-16 and syntaxin-18 did not change significantly after RNAi of Rop.We speculated that ROP is indirectly regulated by syntaxin,a gene that composes SNARE protein complex.In addition,the level of vitellogen receptor gene was significantly up-regulated by 50.02% in ds Rop treatment.Increasing the number of transportors may be a compensation for the blocking of membrane fusion in spider mites.In summary,this study showed ROP is conserved among relative Tetranychus spider mite species and can significantly affects the fecundity of female adults.Accordingly,Rop is a candidate target gene for potential spider mite control.