| Blattella germanica（Blattodea:Blattellidae）,commonly known as the German cockroach,belongs to the class Arthropoda,Insecta,Blattaria,is one of the most widespread indoor health pests which are widely distributed and difficult to control.As a pathogen vector insect,it can carry a variety of bacteria,fungi,viruses,and parasitic eggs,leading to the spread of human diseases such as dysentery,diarrhea,and its secretions and dead insects can also cause severe allergic reactions.With the wide application of chemical insecticides in cockroach control,the problem of insecticide resistance of B.germanica has become increasingly prominent,which makes the study of insecticide resistance mechanism particularly important.As the first line of defense against insecticides,the cuticle has a great influence on the toxicity of insecticides,and penetration resistance through the cuticle plays a very important role in the formation of insect resistance.Cuticle protein is one of the main components of the cuticle,however,the mechanism of cuticle protein and its related genes in the development of insecticide resistance of B.germanica has been few studied.The research on cuticle and the mechanism of penetration resistance of cockroaches has great theoretical and practical significance for the insecticide resistance management and the control of cockroach.In this paper,the sensitive（S）strain and theβ-cypermethrin-resistant（R）strain B.germanica were used as research objects.The role of cuticle in insecticide resistance of B.germanica was preliminarily inferred by comparing the virulence of oral feeding,haemophilic injection,and cuticlular dropping ofβ-cypermethrin to the S and R strains B.germanica.The representative cuticle positions（tarsusⅠand tibia of the second appendage,tergum and mesosternum）were selected to slice and observe the changes of cuticular thickness and tissue structure caused by insecticide resistance,so as to clarify the mechanism of cuticles playing a role in insecticide resistance.At the same time,the complete sequence of the insect cuticle protein Bgicp37 gene was obtained by gene cloning technology and the BgICP37 antibody was prepared.Further,the expression characteristics and its involvement inβ-cypermethrin resistance of Bgicp37 gene were studied by real-time quantitative PCR（qRT-PCR）,Western Blotting,immunohistochemistry（IHC）and double-stranded RNA interference（RNAi）and other technical methods.The main results of the experimental research were as follows:1.Toxicity ofβ-cypermethrin against B.germanica by different administration methodsThrough the gradient experiment ofβ-cypermethrin infected with different concentrations（the S strain:1μg/m L,2μg/m L,3μg/m L,5μg/m L;the R strain:8μg/m L,10μg/m L,12μg/m L,15μg/m L）by cuticlular dropping,the LC30values ofβ-cypermethrin against the S and the R strains B.germanica were calculated as 1.532μg/m L and 9.516μg/m L by linear regression method,respectively.The S and the R strains B.germanica were infected with 1.532μg/m L and 9.516μg/m Lβ-cypermethrin by oral feeding,haemophilic injection,and cuticlular dropping,respectively.It was found that there were significant differences in the mortality within 72 h under the three administration methods,and the trend was as follows:oral feeding>haemophilic injection>cuticlular dropping（S strain:82.771±3.5283%>40.421±1.5736%>28.889±1.1111%,P<0.05;R strain:77.160±2.1605%>56.979±0.9988%>43.103±2.98863%,P<0.05）,preliminary confirmed that cuticles indeed played a role in the insecticide resistance of B.germanica.2.Histopathologic changes of cuticle of the R strain B.germanicaThe changes of cuticular thickness and tissue structure between the S and the R strains B.germanica were compared and analyzed by histological section of the representative cuticle positions（tarsusⅠand tibia of the second appendage,tergum and mesosternum）of B.germanica.The results showed that the cuticular thickness of the R strain was greater than that of the S strain in different degrees,and the differences in tarsusⅠ,tibia and mesosternum reached a significant level（P<0.05）.The thickening of tarsalⅠwas the most obvious,and that of the R strain（15.492±0.5873）was 25.788%(F[1,48]=22.126,P<0.001)thicker than that of the S strain（12.316±0.3331）.The second was the mesosternum,the R strain（11.616±0.8286）was 20.730%(F[1,48]=6.744,P<0.05)thicker than the S strain（9.208±0.4163）.The third was tibia,the R strain（11.988±0.6101）was 19.260%(F[1,48]=7.495,P<0.01)thicker than the S strain（10.052±0.3575）.But in the tergum,the R strain（12.132±0.2634）was only 2.259%(F[1,48]=0.372,P=0.545)thicker than the S strain（11.864±0.3514）,and the difference was not significant.TEM further observation of cuticular ultrastructure revealed that the characteristics of cuticular tissue structure and the changes of the R strain were different between the appendages and the tergum.In tarsusⅠand tibia of appendage,the thickness of endocuticle was significantly greater than that of exocuticle（P<0.05）,and the cuticular thickening of the R strain was mainly caused by the thickening of endocuticle,and the thickness of exocuticle had no significant change;But in the tergum,the thickness of exocuticle was significantly greater than that of endocuticle（P<0.05）,and the overall cuticular thickening of the R strain was mainly reflected in the thickening of exocuticle,and there was no significant difference in the thickness of endocuticle.According to the above results,it was speculated that the tarsusⅠ,tibia and mesosternum with higher contact frequency of insecticides were more important parts of anti-insecticide penetration than the tergum.Under the selection of insecticides,B.germanica evolved thicker endocuticle to resist the insecticide penetration into the insect body.3.Cloning and Bioinformatics Analysis of the Bgicp37 gene complete sequenceBased on the c DNA of adult male B.germanica,an intermediate segment of Bgicp37 gene was successfully cloned by PCR amplification.According to this segment,the 3’RACE’and 5’RACE were performed,and 639 bp and 573 bp segment were amplified,respectively.After splicing with the intermediate segment,the full sequence of Bgicp37 gene was obtained with 1568 bp.Bioinformatics analysis showed that the gene contained a 999 bp ORF,which was predicted to encode a protein composed of 332 amino acids.The theoretical molecular weight of the protein was 36.87086 KD,and the isoelectric point was 4.86.The protein was hydrophilicity protein.The N-terminal of this protein sequence contains a signal peptide consisting of 20 amino acids,and the cell localization prediction results showed that this protein is highly likely to be a secreted protein.The secondary structure of the protein consisted of 65.96%random coil,23.19%extended strand,7.83%αhelix and 3.01%βturn.The prediction of the structure domain showed that the protein had six low-complexity regions,which were rich in proline and rich in valine.Multi-sequence alignment and phylogenetic analysis showed that the protein had high sequence similarity and close relationship with cuticular proteins of Cryptotermes secundus and Zootermopsis nevadensis.4.Prokaryotic expression and antibody preparation of Bgicp37 geneSince the BgICP37 protein contains a signal peptide in the N-terminal region,in order to express the protein successfully,the non-signal peptide region was selected to construct the prokaryotic expression vector,the expression vector was p ET-28b,the expression strain was E.coli Rosetta.The expression conditions and solubility of recombinant engineering bacteria protein were analyzed,and 37°C,1 m M IPTG,and induction time 4 h were determined as the best conditions for prokaryotic expression of recombinant protein.The expressed recombinant protein was successfully purified by affinity chromatography.The recombinant protein was dialysed and concentrated to obtain the recombinant protein with high purity and content.The polyclonal antibody was prepared by using the concentrated recombinant protein as antigen.The antibody was detected by ELISA and Western Blotting.It was found that the titer of the antibody was greater than 128000,and the specificity was good,which met the requirements of subsequent experiments.5.Expression analysis and tissue localization of Bgicp37 gene of B.germanicaUsing qRT-PCR and Western Blotting techniques,the expression characteristics of Bgicp37 gene of the S strain and the R strain B.germanica in different developmental stages and tissue structures were compared at the level of m RNA and protein.It was found that no matter in ootheca,nymph or adult,whether in appendage,cuticle or viscera,the expression of Bgicp37 gene of the R strain was higher than that of the S strain in m RNA and protein level.IHC analysis showed that the BgICP37protein could be expressed in the cuticles,and the expression levels of BgICP37 in endocuticle and exocuticle of the tarsusⅠ,tibia,tergum and mesosternum of the R strain was higher than that of the S strain.In addition,the expression of BgICP37 was higher in the endocuticle than in the exocuticle.Associated with the changes in the thickness of cuticles,we suggested that it is the increased expression of BgICP37 that leads to the thickening of the cuticles,especially the endocuticle of the R strain.6.Functional analysis of Bgicp37 gene of B.germanicaThe Bgicp37 gene of the last instar larvae of the S strain and the R strain B.germanica was subjected to RNAi by feeding dsRNA,so that the expression of Bgicp37 gene was in a long-term low state during this molting cycle.Then the phenotypes of wing defect,cuticular thickness and cuticular tissue structure,as well as the changes of resistance toβ-cypermethrin after the downregulation of Bgicp37gene were observed and measured.The results of qRT-PCR showed that the downregulation efficiency of Bgicp37 gene in both the S strain and the R strain could reach more than 80%.Phenotypic observation and statistical analysis showed that the S strain and the R strain B.germanica with downregulated Bgicp37 gene showed significantly increased wing defect rate（P<0.05）.After the downregulation of Bgicp37 gene,the cuticle of tarsusⅠ,tibia,tergum and mesosternum were significantly thinned（P<0.05）,the degree of thinning was tarsusⅠ>tibia>mesosternum>tergum,and the cuticles thinning was mainly due to the significant thinning of the endocuticle（P<0.05）.The above results consistently suggested that the Bgicp37 gene plays a vital role in the cuticle formation of B.germanica.The change ofβ-cypermethrin resistance of B.germanica after Bgicp37 gene downregulation was detected by cuticlular dropping ofβ-cypermethrin.It was found that the mortality rate of B.germanica with downregulated Bgicp37 gene was significantly higher than that of normal B.germanica（P<0.05）,which directly proved that the cuticle played an important role inβ-cypermethrin resistance of B.germanica. |
PDF Full Text Request