The Study On The Mechanism Of Metabolic Resistance To Organophosphate Of Silkworm, Bombyx Mori

The Bombyx mori belongs to Bombycidae of Lepidoptera and was model insect ofLeidoptera which derived from the Chinese wild silkworm (Bombyx mandarin). Afterabout5000years of domestication, B.mori is highly sensitive to adverse environments, as aresult of being constantly kept isolate. Pesticide poisoning causes a lot of reduction of rawsilk annually and has become a severe threat to healthy development of silk industry inChina. And so far, there were no pesticide resistance species of B.mori in China.Organophosphorus (OP) pesticides are most commonly used in pest control. Spraysingthe OP in mulberry fields cannot control pests completely, while make the B.mori poisoned,which has become a restriction for the development of the industryIn order to study the mechanism of metabolic resistance to OP, in this research, theactivities of three enzymes (P450s, GSTs and ESTs) were studied after phoxim-induction;using the whole-genome oligonucleotide microarray to study the transcriptional expressioncharacteristics of resisitance-related genes; the characteristics of metabolism and tissuedistribution of phoxim in B.mori were studied by HPLC. And the main results are asfollows:1. The research of detoxifying enzymes1.1The activity of detoxification enzymes in B.mori after phoxim feedingWe first determined the LC₅₀(Lethal Concentration50) of phomix in3-day old,5thinstar B. mori, then we monitored the activity of cytochrome P450monooxygenase,glutathione-s-transferase, and carboxylesterase in midgut and fatbody of B.mori afterphoxim feeding. The results showed that the LC₅₀of phoxim was7.86μg/mL. Theactivities of P450s in the midgut and fatbody was1.72and6.72folds of the control; theactivity of GSTs has no change in midgut, and in fatbody was1.11folds of control; theactivity of carboxylesterase in the midgut and fatbody was0.69and1.13folds of control. The changes of carboxylesterase's activity fit the mutant aliesterase hypothesis. Theseresults demonstrated the important roles of detoxification enzymes in phoxim metabolism.In addition, the detected activities of such enzymes were generally lower than those incotton bollworms (Helicoverpa armigera), which may contribute to the high susceptibilityof B.mori to insecticides. Our findings laid the foundation for further investigations of themechanisms of resisitance to phoxim in B.mori.1.2The variation characteristics of detoxifying enzymes in the metabolic of phoximin B.moriWe studied the activity changes characteristics of three detoxifying enzymes within24hafter feeding with phoxim in B.mori. The results showed that, after feeding phoxim, infatbody, the activity of P450rised after2h, and reached a peak at24h, that was2.85foldsof control; in midgut, the maximum value reached at12h, was1.89folds of control. Infatbody the activity of GSTs reached the maximum value at12h, was1.69folds of control;in midgut, the maximum value reached at24h, was1.21folds of control. The activity ofESTs declined after feeding0.5h, at10h reached the lowest value, and were0.15foldsand0.40folds of control in the midgut and fatbody, respectively. And rised10h later, were1.14folds and0.75folds respectively of control in the midgut and fatbody at24h,respectively.The characteristics of detoxifying enzymes in the process of phoxim metabolic in B.mori:fatbody is the major organ for P450enzymes detoxification metabolic, after2h, themetabolism increased; the detoxification of GSTs is more gently; there is 'mutantaliesterase hypothesis' phenimenon in B.mori, and the metabolism turning point is10h.This study presents a foundation to explain the role of three detoxifying enzymes in theprocess of organophosphorus pesticide metabolic and can help us study the molecularmechanism of metabolic detoxification of B.mori.2. The transcriptional characteristics of resisitance-related genes2.1The gene transcriptional characteristics of B.mori after phoxim-induction fromthe whole-genomeWe used a whole-genome oligonucleotide microarray to survey the B. mori larvaeinduced by phoxim after24h. The results showed that,247genes were differentiallytranscribed in the phoxim-induced groups compared to the control groups. Among thesedifferentially expressed genes,68genes were up-regulated and179genes were down-regulated after phoxim-induced and the highest changes were26-fold up-regulatedand29-fold down-regulated, respectively. Analysis result of the differentially expressedgenes indicates that their functions could be divided into10categories. The up-regulatedgenes are mainly involved in glycometabolism, fat metabolism, protein hydrolysis electrontransfer process and the down-regulated genes are mainly involved in cell division,transcriptional regulation, intracellular signaling cascade. In this study, we firstly reportedthe whole-genome transcribed expression profile of the B. mori larvae induced by phomixand provided the gene source for studying the metabolic detoxicification mechanisms toorganophosphorus by B. mandarina and other Lepidoptera insects.2.2Molecular cloning of insect intestinal mucin (BmIIM1) and phoxim-inductiontranscription analysis from B.moriWe cloned a cDNA of B.mori insect intestinal mucin by RT-PCR in the midgut of3-dayold,5th instar larva, named BmIIM1(GeneBank accession number: JN256080). Thesequence analysis showed that, the ORF of BmIIM1was870bp, coding289aa, and thepredicted molecular weight and isoelectric point were31.78kD and4.56, respectively. Weused quantitative PCR to study the transcriptional characteristics of BmIIM1in differenttissues and induced by phoxim. The results showed that BmIIM1was only expressed inmidgut, and after phoxim-induction for24h, the transcription level was12.3times morethan that of control, indicating that BmIIM1may play important roles in protection ofmidgut from phoxim. This study lays the foundation to further research the function of IIMin B.mori, and also procids the reference to study the relation between IIM and pesticides.2.3Transcriptional characteristics of acetylcholinesterase genes in domestic B.moriexposed to phoximIn this study, we examined the transcription level of two acetylcholinesterase genes(Bm-AChE-1and Bm-AChE-2) in brain, fatbody, midgut and silkgland at24,48,72and96h later. The results showed that, after feeding with4.0μg/mL phoxim, transcription levelsof Bm-AChE-1and Bm-AChE-2in brain, fatbody and silkgland showed a trend of firstincreasing and then decreasing. At24h, transcription of Bm-AChE-1and Bm-AChE-2increased in the brain by16.22and68.71-fold, respectively, while in the fatbody andsilkglands only increased slightly. Moreover, the increase of Bm-AChE-1was greater thanthat of Bm-AChE-2. By contrast, in the midgut, transcription of Bm-AChE-1andBm-AChE-2showed a trend of first decreasing and then increasing to around their initial levels at96h. In addition, the results showed that transcription of Bm-AChE-1wasdifferent from that of Bm-AChE-2in different tissues of B.mori during the metabolismprocess of phoxim, suggesting that during metabolic process of pesticides, Bm-AChE-1plays an important role in the fat body, while Bm-AChE-2plays an important role in thebrain.This study provides a scientific rationale for studying the functional differentiation ofthe two AChE genes.2.4The transcription characteristics of genes in midgut and fatbody fromphoxim-induced B.moriIn order to study the roles of midgut and fatbody in phoxim metabolic, in this study westudied the transcription characteristics of genes in midgut and fatbody fromphoxim-induced B.mori after24h by whole-genome oligonucleotide microarray. Theresults showed that, there were299genes siginificently differentially expressed in midgut,and209genes were up-regulated,90genes were down-regulated; there were1040genessiginificently differentially expressed in fatbody, and569genes were up-regulated,471genes were down-regulated. GO analysis of the differentially expressed genes indicatedthat, in midgut, their functions could be devided into8categories; in fatbody, theirfunctions could be devided into11categories. We verified the expression level ofCYP4G25(sw20730) and CYP9a21(sw20566) by quantitative PCR, and the results wereconsistent to the hybridization which prove the hybridization result is credible. The resultsof this study showed that, after phoxim-induction, many genes up-regulated, such asadiponectin receptor gene (sw09106), CYP4G25(sw20730), and so on; the cells weredamaged by phoxim, and this causes a variety of biological processes change. We testedthe pathological section for midgut, and find that after phoxim-induction, the cylindricalcell dead, cup cell lysated, the membrane broken and other symptoms.This study lays afoundation to clarify the mechanisms of poisoning and injurying on insects by phoxim.3. The charateristics of metabolism and tissue distribution of phoxim in B.moriIn order to study the metabolism characteristics of phoxim in different tissues inLepidoptera insects, we determined the resisdue of phoxim in blood, midgut, fatbody andsilkgland at different time by HPLC. The results showed that, in midgut, the phoximcontent peak (4.35μg/g) emerged at0.5h, and then declined; in blood, the phoxim contentpeak (1.72μg/mL) emerged at1h; in fatbody, the content of phoxim rised gradually, andthe peak (3.65μg/g) emerged at4h; in silkgland, the phoxim content was stable, and rised slightly, the peak (1.45μg/g) emerged at24h.The characteristics of phoxim metabolism in B.mori were: the pesticide passed throughthe midgut, transported by blood, then accumulation and metabolic in fatbody, and it hadchronic toxic effects to silkgland. Studying on the charateristics of metabolism anddistribution of phoxim in B.mori provided an important reference value for further study onthe detoxification function of different tissues.Through this topic, we have studied the mechanism of metabolic resistance to OP inB.mori from three levels: enzyme activity level, gene and drug metabolism level, and at thesame time, discussed the mechanism of injury on B.mori by phoxim. Studying themechanism of metabolic resistance to OP provide a theoretical basis for transgenicbreeding of resistant strain, moreover, studying on the mechanism of metabolic resistanceto OP in the mode insects of Lepidoptera has great significance for the development ofeffective pesticides on Lepidoptera insect.