Study On Beta-cypermethrin Resistance-related Proteins And Intestinal Flora In Blattella Germanica

German cockroach (Blattella germanica) is a globally common insect associated with human habitation which increasingly serious harm to people. Chemical pesticides are still the main means that control the population of German cockroach, especially pyrethroid insecticides which is the most widely used, but the pesticide-resistance has become a serious obstacle which limits its continued use. It is urgent for further study on the mechanism of the resistance of the German cockroach to pyrethroid insecticides. The thesis reveals the mechanism of Beta-cypermethrin resistance in German cockroach bred in the laboratory from multiple aspects as follows: metabolic enzyme activity, proteomics and changes in the composition of the intestinal flora. Further more, the impacts of resistance on biological fitness and anti-fungal infections in German cockroach was also studied and the following results were obtained:1. The susceptible strains of the German cockroach (Sensitive, S) were continuously selected using a single insecticide (Beta-cypermethrin) in30%to50%of the selection pressure. After11generations of continuous selection, the moderate Beta-cypermethrin resistant strains (Resistant, R) of German cockroach were obtained which the resistance ratio reached7.4.From the process of the R strain selection, the trend of resistance ratio of Beta-cypermethrin in German cockroach showed slow first and fast succedent, suggesting the resistant genes of German cockroach were gradually homozygous; R strains showed significant cross-resistance with pyrethroid insecticides (lambda-cyhalothrin, cypermethrin, deltamethrin), the resistance ratios were5.6,4.1,3.7;but showed weak cross-resistance with organophosphorus (chlorpyrifos) and carbamate (propoxur) which the resistance ratios was only1.8and1.5respectively.2. The blood hemolymph of male S strain and R strain German cockroach was collected after1ppm Beta-cypermethrin induced and the activity of three metabolic enzymes such as superoxide dismutase (SOD), acetylcholinesterase (AChE) and glutathione-S-transferase enzymes (GSTs) were test. It showed that the three enzymes activity (SOD, AChE and GSTs) in R strain were all higher than the S strain (4.9%±0.2%,2.9%±0.4%and30.3%±1.1%, respectively) and the activity of GSTs differences reached a very significant level (P<0.01); after induced by Beta-cypermethrin, the activity of SOD and GSTs increased in different levels in both S strain and R strain. SOD activity increased6.7%±0.3%and5.4%±0.6%, GSTs activity increased23.6%±0.8%and26.9%±1.6%, respectively, suggesting that SOD and GSTs played a certain role in Beta-cypermethrin resistance of the German cockroach. The adults of German cockroach were defatted by acetone and five methods (TCA/acetone, RIPA, Tris-HCl,1%SDS and lysate) were used for extraction of protein. The protein concentration was measured with Bradford methods and the characters of protein were analyzed by SDS-PAGE. It showed that defatting was beneficial to the extraction of protein from German cockroach; in five protein extraction methods, RIPA lysate was most effective, and the protein concentrations of male and female adults were14.2mg/ml and12.3mg/ml respectively.The results of SDS-PAGE showed that the protein bands of female and male were13and16respectively, followed by SDS, Tris-HCl, and lysate, TCA/acetone was the worst.Further studies showed that the male protein and haemolymph protein concentrations of S strains were lower than the R strain, a number of differences in protein band on SDS-PAGE gels, molecular weight mainly between90KD-27KD, of which90KD,78KD and55KD protein bands were abnormal increased in the R strain, may be related to the formation of resistance; After induction with1ppm Beta-cypermethrin, S strain protein concentration decreased significantly (P<0.05), R strains did not change significantly. It concluded that insecticides may inhibit the expression of some protein in S strain; a number of difference protein bands also appeared in SDS-PAGE gels, mainly around the molecular weight of118KD and20KD, it speculated that the differences in protein were mainly related the stress response to insecticides, some may be involved in resistance.4. Two-dimensional electrophoresis (2D-PAGE) was carried out on hemolymph proteins in S strain and R strain of German cockroach and the differences were analyzed. The results show that:the haemolymph protein in S strains and R strains were significant differences, a total of111different protein spots were observed, which64spots were up-regulated and47spots were down-regulated in R strains.29significantly different protein spots (magnitude>2) were identified by mass spectrometry and22spots were identified successfully. It was included defense/immune-related proteins:heat shock protein(hsp20),nitric oxide synthase (NOS), transferrin (Tf); nutrition/energy metabolism related proteins:alpha-amylase enzyme,triosephosphate isomerase, Per a3, ATP-binding protein, arginine kinase (AK), and the function of unknown proteins. Based on the results of2D-PAGE and mass spectrometry, some protein function was cleared and the relationship between Beta-cypermethrin resistances in German cockroach was speculated.5. Two-dimensional electrophoresis (2D-PAGE) was carried out on hemolymph proteins in S strain and R strain of German cockroach after1ppm Beta-cypermethrin induced and the differences were analyzed.The results showed that:sub-lethal doses of Beta-cypermethrin induction caused significant changes in hemolymph protein of two strains. After induction, a total of203protein spots were difference.148spots were up-regulated and67spots were down-regulated in S strains before and after induction;65spots were up-regulated and61spots were down-regulated in R strains before and after induction;65significantly different protein spots (magnitude>2) were identified by mass spectrometry, and24were identified successfully. It included defense/immune-related proteins:heat shock proteins (HSP20), nitric oxide synthase (NOS), transferrin (Tf); detoxification related protein:protein of glutathione-S-transferase (GSTs); energy metabolism:arginine kinase (AK), Per a3, alpha-amylase and the function of unknown proteins.6. The S strain and R strain of German cockroach was compared on oothecal production, ootheca drop-out rate, nymphal hatching rate, nymphal development period, adults longevity, the proportion of male and female adults (male/female) as well as the weight of the male and female adults, and the relative fitness of R strains was calculated. The results showed that the ootheca drop-out rate of R strain of German cockroach was lower50%±1.7%, larvae hatching rate decreased by30%±0.85%, and nymphal development period delayed7d, male/female rate increased36%±1.02%and female adults longevity decreased6.5d, male adult weight loss0.0037±0.0004g, the females weight loss0.0063±0.0006g, the differences were extremely significant. R strain relative biological fitness was only0.26compared with the S strain and showed significant defective fitness.7. The ovary of female German cockroach in two strains was dissected after molting7-8d and the proteins was extracted with TCA/acetone; two-dimensional electrophoresis was carried out for the analysis of protein spots. The results showed that:the S and R strains ovarian protein were significant different in the two-dimensional gels, a total of64protein spots were different,18protein spots were up-regulated and46protein spots were down-regulated in which24protein spots reach significant difference level (magnitude≥2);24protein spots were identified by mass spectrometry,17spots were successfully identified, including5for yolk protein (vitellogenin);1for identification as heat shock proteins (heat shock protein hsp), ribosomal protein (Stubarista sa) the insect epidermis protein (putative cuticular protein ICPG), transferrin (transferrin, Tf), myokinase (adenylate kinase, adkl) an aldehyde dehydrogenase (aldehyde dehydrogenase), other proteins were unknown function. The proteome analysis of ovary revealed that some protein may play an important role in the mechanism of defective biological fitness in German cockroach. 8. The susceptibility of the German Cockroach S strain and R strain to entomopathogenic fungus (Beauveria bassiana) was compared with spore inoculation test and the composition of culturable intestinal flora was investigated. The results show that:Obvious differences in the sensitivity of the S strain and R strain of the German cockroach to Beauveria bassiana, compared to the S strain the R strain German cockroach was more susceptible to Beauveria bassian infection (P<0.05); the composition of cultured intestinal flora was different in the S strains and R strains.29bacteria strains of S strains were identified by physiological and biochemical characters, belonging to Enterobacteriaceae (9), Pseudomonas (6), Streptococcus (5), Bacillus (4), Neisseria (2), Micrococcus (2) and Lactobacillus (1). Compared with S strain, the bacteria strains were fewer (25strains) and short of Enterobacteriaceae (1), Bacillus (1) and Pseudomonas (2).9. The intestinal bacteria were tested with anti-entomopathogenic fungus Assay. A total of three strains showed significantly inhibited activity to Beauveria bassian. The phylogenetic tree was generated based on the morphology, physiological, biochemical characters and the sequence of16S rDNA. It was showed three strains were Bacillus subtilis, Bacillus atrophaeu and Pseudomonas reactans. Three bacteria all could inhibit the growth of B. bassiana significantlyin which Bacillus subtilis showed strongest inhibition activity, inhibition zone diameter of32mm; further study showed that vesicles were observed in B. bassiana hyphae on the edge of the inhibition zone. Fermentation of BGI-1reduced the conidial germination rate by12%. Further studies demonstrated that B. bassiana infections in German cockroaches orally treated with the extracts of BGI-1fermentation were significantly weakened. Cumulative mortality rate was49.5%in the treatment group at the20d, while that of the control group was62.3%. The study intends to understand the relationship between the intestinal flora and the cockroach. Those microbes with anti-entomopathogenic fungi activity might contribute to resisting the infection of pathogenic fungi. It also hinted that the bacterial strains with potential medicinal value for control fungal disease may be found in the special environment of the insect gut.