| In the long-term interaction between Lepidoptera insects in the agriculture and forestry and Bacillus thuringiensis,the lepidoptera develops resistance due to its adaptation to Bt,however,the regulars and mechanisms of the resistance are still in its infancy.This experiment on the successive generations Clostera anachoreta Denis&Schiffermüller to BtCry1Ac pro-toxin,by their growth and development,feeding digestion,physiological metabolism,transcriptome analysis,molecular response and cellular structure of the midgut with electron microscope etc.,comprehensively analyze the response and adaptation mechanisms of Clostera anachoreta Denis&Schiffermüller of BtCry1Ac protoxin,hoping to provide certain theoretical basis for the management and prevention of lepidopteran forest insects.The results are as follows:1.The LC50 of BtCry1Ac protoxin was determined in this experiment to be 7.5 μg/ml.And the content of Bt toxin protein in the leaves of transgenic Pb29 poplar was 6.48(±3.76)ng/cm~2.The lethal rate of Clostera anachoreta Denis&Schiffermuller larvae increased with the increase of Bt concentration,however,the survival rate increased by increasing genetations.The larvae of the 3rd generation of different treatments had a mortality reduction of 11%,29.33%,23.67%,and 33.67%compared to the 1st generation,respectively.With the increase of generations,the developmental period of larvae shortened.The different treatments in third generation were shortened by 0.44 days,7.27 days,5.27 day s,and 10.49 days compared with the first generations.The B and P treatment group were significantly different.Under Bt stress,the pupal weight of the poplar moth was greatly affected.The pupal weights of different treatments of the first generation were significantly lower than that of the control.With the increase of generations,although the developmental period shortened,the pupal weights increased significantly,especially the weight of the 3rd generation of B group was not significantly different from the control.2.During Bt stress,the relative growth rate(RGR),relative consumed rate(RCR),efficiency of conversion of ingested food(ECI),and efficiency of conversion of digested food(ECD)of Clostera anachoreta Denis&Schiffermüller larvae were significantly lower than those of the control in the first and second generations.The growth and feeding rates of different treatments increased to varying degrees with increasing generations;however,the efficiency of approximate digestion of different generations and treatments did not decrease significantly,and BtCry1Ac protoxin had no significant effect on the digestibility of larvae.During the recovery period,the growth and feeding rate,and relative metabolic rate(RMR)of the different treatment groups were significantly improved compared to the stress period.Clostera anachoreta Denis&Schiffermüller larvae could increase ECI and ECD for growth.The growth,feeding rate and digestibility of the same treatments showed a downward trend with generations.3.The changes of digestive enzymes in the midgut under Bt stress:α-amylase(α-AL)and trypsin were higher than control.Larvae would increase digestive enzyme activity in the midgut to enhance the degradation and digestion of food and Bt protoxin,however,the activity decreased significantly with increasing generations.The changes of detoxification enzyme:The detoxification enzyme(GST,CarE,AchE)activities of the 3rd generation larva treatments were lower than those of the control,but the enzyme activity increased significantly with the increase of generations,and the activity of the 2nd and 3rd generations was significantly higher than that of the 1st generation.The changes of protective enzyme:Protective enzyme changes:CAT activity was not significantly different between different treatments,and decreased slightly with increasing generation:POD activity was significantly lower in the 1st and 3rd generation treatments than those in the control,while the activities of the A,B,and C groups increased first and then decreased by the increased of generations,the activity of POD fluctuated greatly between groups;the SOD activity in the first generation of the different treatments were lower than the control,and in the 2nd and 3rd generations were the same as the control.4.The percentage of Q30 bases in the raw reads and clean reads of the transcriptome data of the midgut of Clostera anachoreta Denis&Schiffermüller to Bt were greater than 92.56%.the data quality was reliable.The comparison rate of Mapped Reads after data assembly were not less than 78.42%,and the functional annotation of transcriptome data had a high comparison rate in major databases,the results were credible.In the initial response,there were 92 shared differential expressed genes between each treatment,and 27 with clear annotations(upregulated 24,downregulated 3).The JHBP,juvenile hormone binding protein,gene was highest upregulated and the numbers of digestive enzymes were the highest.The commonly genes upregulated also include insect cuticular protein,GST enzyme,gelsolin gene,hatching enzyme-like protein gene,collagenase-like gene,and so on.In the cumulative response,there were 11 differential genes.In addition to genes consistent with the initial response,arylphorin gene,CRT1 gene.and moricin/cecropin-D-like related to immunity were also found.In the initial response,only ABCF2 and ALP genes upregulated significantly by 1.75-fold and 2.59-fold at 24 hours,respectively,and other receptor genes were not differentially expressed.Among the detoxifying enzyme genes,there are 2 CarE genes and 3 P450 genes,and their expression levels were downregulated first and then upregulated.Among the digestive enzyme genes.there were 3 chymotrypsin-like enzymes,9 serine proteases,and 7 trypsin,all of which were upregulated from 6h to 24h,and the differences were mostly significant.In the cumulative response,the ABCC13 gene in treatment B was significantly downregulated,3 ALP genes were significantly upregulated,and one CAD gene was significantly down-regulated;the detoxifying enzyme genes(except CarE)showed substantially upregulated expression;among digestive enzyme genes,most of treatment B were up-regulated,the other treatments were significantly downregulated.Most genes related to Bt resistance,such as arylphorin,collagenase-like,larval cuticle protein,juvenile hormone esterase,and peritrophin,were significantly up-regulated at 24 hours and in the A/B/C treatments.The results of the initial response enrichment analysis showed that the DEGs were mainly enriched in Neuroactive ligant-recepter interaction,Protein digestion and absorption,Pancreatic secretion,Insect hormone biosynthesis,Galactose metabolism,Glutathione metabolism,Pathogenic E.coli infections.Enrichment analysis of cumulative response showed that DEGs were mainly enriched in Neuroactive ligant-recepter interaction,Galactose metabolism,Protein digestion and absorption,Pancreatic secretion,Metabolism of xenobiotics by cytochrome P450,Drugs metabolism-Cytochrome P450,PPAR signaling pathway,etc.5.In the initial response,a series of pathological responses occurred in the midgut of the 1st-generation larvae when the Bt toxin was fed for 2 hours,such as microvilli ruptured and shedding;endoplasmic reticulum swelling and breaking;mitochondrial deformation severely;chromatin condensation in the nucleus.The pathological changes of the midgut of the 4th-generation larvae were alleviated.The microvilli could be more completely attached to the cell membrane and arranged regularly.The mitochondrial morphology deformed slightly,and the spine was clearly visible.However,the endoplasmic reticulum and nucleus did not improve compared to the 1st generation larva.Analysis of the changes in the structure of midgut cells revealed that the 1 st-generation larvae had metabolisms associated with autophagy or phagocytosis at 2 to 72 h,we observed a variety of autophagy-related or phagocytosis-related cellular structures,such as autophagosomes and Autophagy lysosomes,but in the midgut of 4th generation larvae,at 8h and 72h,a small amount of cellular structures related to autophagy/phagocytosis were found.6.Through comparative analysis of the 20 DEGs,it was found that the 4th-generation larvae had a certain tolerance to Bt protoxin.However,the potential receptor proteins,ABCA Ⅲ,ALP,APN,and CAD genes were not downregulated like the resistant innsect,and upregulated significantly at some time,especially in treatment P.receptor protein genes were upregulated significantly.The detoxification digestive enzyme genes were upregulated after Bt stress,and the 4th-generation larvae responded more quickly than the 1st-generation larvae.On the contrary,the expression levels of detoxification enzyme genes were low in treatment P.The analysis of Bt-related genes is as follows:(1)the Arylphorin gene in the midgut of the larva of aspen is related to the proliferation and regeneration of midgut cells.At 1B2H,two Arylphorin genes up-regulated and activated the proliferation of midgut stem cells.Peak at 4B8H and then down.(2)the expression of insect epidermal protein gene was up-regulated with the extension of BtCry1Ac stress time,and it reached a significant level at some time points.The up-regulated level in the 4th generation was more obvious than that in the 1st generation.(3)JEHE gene was significantly up-regulated at 8h and 72h in the first generation,and the expression of 0h in the fourth generation was up-regulated compared with that in the first generation,but not significantly.4 generation 2h,24h,72h and 6day were significantly upregaged,and were upregaged to the maximum at 4B6D.(4)the two hatching enzyme genes were related to the digestion of insects,and their expression levels were up-regulated with the prolongation of the stress time of BtCry1Ac,reaching significant levels at 72 hours and 6 days after generation 1 and generation 4.(5)the changes in the expression levels of genes involved in midgut phagocytosis in larvae of a.poplar were as follows:1)the expression levels of collagenase genes in midgut of larvae of the first generation were relatively low,and were significantly up-regulated at 6 and 2h and 72 hours in the first generation,and more significantly up-regulated in the fourth generation.2)all the Tubulin beta genes were up-regulated,and the response rate of generation 4 was faster and more significant than that of generation 1.3)the CTR1 gene at different time treatments(except Pb29)was upregulated,which was significantly upregulated at 2h after generation 1.and the larvae in generation 4 were upregulated at different time periods compared with the control in generation 1.4)except the first generation 2-8h and the second generation 4-2h.the expression of Actin gene was down-regulated compared with that of the first generation.Compared with 4B0H,the expression of all treatments in the 4 generations was significantly up-regulated.5)the expression of PIK3R4 gene in the first generation of larvae was up-regulated,down-regulated and up-regulated with the extension of stress time.The results of the above studies indicate that generations of Clostera anachoreta Denis&Schiffermüller had been adaptive and tolerant to the sublethal dose of BtCry1Ac protoxin.The Clostera anachoreta Denis&Schiffermüller adapt to the long-term stress of BtCry1Ac toxin,by paying some phenotypic fitness costs;improving the activity of detoxification enzyme and decreasing the activity of digestive enzyme;and upregulation of genes related to autophagy and digestive metabolism. |
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