Identification Of Cuticle Proteins Of Tetranychus Cinnabarinus And Their Effects On Penetration Of Acaricides

Tetranychus cinnabarinus is an important agricultural pest mite.The pyrethroids,including fenpermethrin,have been used for many years in the control of mites.The knock down resistance(kdr)caused by fenpermethrin has been widely found in the population of mites such as T.cinnabarinus.Switching acaricides with other action mechanisms is a mainly means to control the target resistance of fenpermethrin.In the detection of the toxicity against pyridaben,bifenazate and cyflumetofen in fenpermethrin-resistant strain(YN-FeR),we found that YN-FeR developed non-specific cross-resistance to these acaricides.The phenomenon is mainly caused by the thickening of the cuticle and non-specific penetration resistance to different acaricides in YN-FeR.On this basis,the author further investigated the molecular mechanism of cuticle thickening of T.cinnabarinus.The main results of this study are as follows:1.Comparison of acaricides sensitivity and determination of physicochemical properties of cuticleThe bioassay results showed that YN-FeR strains developed 22.6-,28.7-and 8.3fold cross-resistance to pyridaben,bifenazate and cyflumetofen,respectively.However,the three acaricides were injected into the susceptible strain(YN-SS)and YN-FeR strains by microinjection,and the results showed that there was no significant difference in mortality between the two strains,suggesting that the non-specific cross-resistance may be caused by the difference in penetration of acaricides to the cuticle of different strains.The results of transmission electron microscopy(TEM)showed that the cuticle thickness of YN-FeR adult female mites was 1.9-fold that of YN-SS.Further detection by acaricides spray combined with liquid chromatography/gas chromatography showed that,compared with YN-SS,YN-FeR adult female mites could block more acaricides on the cuticle,which significantly reduces the dose of acaricides entering their bodies and thus leading to the evolution of non-specific penetration resistance.2.Screening of cuticle proteins genes in T.cinnabarinusTranscriptome sequencing was performed on egg,larva,nymph1st,nymph2nd and adult3rd of T.cinnabarinus.A total of 54 cuticle proteins(CPs)genes were identified,including 40 genes belonging to the CPR family.The phylogenetic tree analysis found that the CPs genes of T.cinnabarinus converged with arachnid species,and was closely related to the CPs genes of Tetranychus urticae.The expression trend analysis showed that 31 of the 54 CPs genes showed two significant expression trends with the development process from egg to adult.The expression levels of 20 CPs genes increased gradually from egg to larva,and continued to be high from larva to nymph2nd,and then continued to decrease.The expression of 11 CPs genes was the highest in egg,and then continued to decrease in larva to adult.3.Identification and RT-qPCR analysis of important cuticle proteins genesA total of 17 up-regulated CPs genes were detected in two fenpermethrin-resistant strains(FeR and YN-FeR)from different backgrounds,and 6 CPs genes were up-regulated in the two resistant strains.Moreover,CPR24(derived from T.urticae tetur06g1680),CPR25(derived from T.urticae tetur05g2790)and CPR27(derived from T.urticae tetur01g12830)were all highly up-regulated in the two resistant strains,so they were selected as representative genes for functional study.The results of RT-qPCR showed that compared with YN-SS,CPR24,CPR25 and CPR27 were significantly up-regulated 5.0-,17.8-and 3.5-fold in YN-FeR,respectively.The expression trend of the three genes was consistent with the results of transcriptome.In addition,the expression level of the three CPs genes was the highest in the egg of T.cinnabarinus,and the expression level was relatively stable from larva to Initial adult mite,while the expression level was lower at adult3rd to adult5th,indicating that the three CPs were highly expressed in the stage involved in the cuticle and body development of T.cinnabarinus,and decreased after the completion of development.4.Functional analysis of cuticle proteins genesThe expression levels of CPR24,CPR25 and CPR27 in YN-FeR and YN-SS were successfully reduced by feeding dsRNA,and the silencing efficiency of three genes in YN-FeR was 46%,53%and 66%,and in YN-SS was 65%,81%and 73%,respectively.The results of TEM showed that silencing the expression of CPR24 and CPR25 could significantly reduce the cuticle thickness of YN-FeR,but silencing the expression of CPR27 did not change the cuticle thickness of YN-FeR,indicating that the cuticle thickening was mainly related to the overexpression of CPR24 and CPR25.In addition,silencing the expression of CPR24 and CPR25 significantly reduced the penetration ability of YN-FeR to pyridaben,bifenazate and cyflumetofen.This was evidenced by a significant decrease in the acaricides blocked on the body surface and a significant increase in the acaricides entering the body.Finally,the susceptibility of YN-FeR to the three acaricides was significantly increased.However,silencing the expression of CPR24 and CPR25 did not significantly change the susceptibility of fenpermethrin in YN-FeR although it also increased the dose of fenpermethrin entering the body.These results indicated that point mutation of kdr was still the main factor contributing to the fenpermethrin resistance in T.cinnabarinus,but the non-specific penetration resistance caused by the cuticle thickening of the resistant mites improved the adaptability to the pesticide control system,which may bring challenges to the resistance control of mite targets.In summary,the results of this study showed that when the mite developed target resistance to fenpermethrin,its CPs genes CPR24 and CPR25 were also overexpressed,which promoted cuticle development and thickening,resulting in non-specific cross-penetrating resistance to different acaricides.