Study On The Growth,development And Enzyme System Response Of Clostera Anachoreta Stressed By Bt And Transgenic Poplar

The insect resistance to Bt has become the main ecological risk of transgenic plants,and it is also the biggest threat to the long-term effective function of Bt plants.In this paper,three Bt Cry1Ac protoxin gradient stresses were set up in Clostera anachoreta:A=15?g/ml,B=7.5?g/ml,C=1.5?g/ml,P=transgenic'Pb29'poplar?[Populus alba×?P.davidiana+P.simonii?]×P.tomentosa?×P.tomentosa Carr.]?Bt Cry1Ac gene+Arrowhead Proteinase Inhibitor?API?gene?leaf group?The expression of Bt?0.3222?g/ml?and the control group?CK?=0.05 mol/L Na₂CO₃.The larvae containing different concentrations of Bt Cry1Ac in poplar leaves were used to systematically analyze the changes of insect growth,food utilization,detoxification enzymes,protective enzymes and digestive enzymes in the midgut of larvae.The differences in growth and development,physiological enzymes,and physiological and biochemical characteristics of insects during the formation of resistance were analyzed under Bt stress.It provides a basis for the formation of insect adaptability under long-term stress,in order to clarify the long-term insecticidal activity and control effect of Bt on forestry pests,and provide a research basis for the effective and sustainable use of Bt.The main results are as follows:?1?The mortality of the 1st generation of the larvae of Clostera anachoreta was significantly increased with the increase of Bt concentration under the treatment of Cry1Ac leaves.The corrected survival rates of A,B,C and P groups were 5.17,27.58,44.81,27.58%,and those of the 3rd generation increased to 22.22,96.30,81.48 and 70.37%;each treatment started from 2³ d,and the survival rate of each group was still within 9¹3 days after stopping Bt stress.Declining,there is basically no death after the 13th day.The lower the Bt concentration,the higher the survival rate and the increase with the generation.The leaves containing Bt Cry1Ac insecticidal protein and the transgenic Pb29 poplar had strong toxic effects on the 3rd instar Clostera anachoreta larvae.?2?The development of the Clostera anachoreta larvae was greatly inhibited after the ingestion of Bt.The 1st generation larvae of the A,B,C and P groups were significantly prolonged by 14.07,9.35,4.95 and 9.91 d,respectively,while the 2nd and 3rd generation was only 12.62,7.36,3.19,6.27 d and 16.6,1.69,4.13,2.64 d longer than CK,respectively.The developmental speed of the larvae in the middle and low treatment groups was significantly accelerated,and the larva of the 3rd instar was under stress.After stress,the age of each group was obviously prolonged with the increase of concentration,but the age of the 4th to 5th instar larvae was basically the same as that of the control,and the duration was shortened with the prolongation of the generation.Most of the older larvae were able to normalize mites;the pupa weight of the 1st generation treatment group was significantly smaller than the control group?248.06 mg?,which were 134.57,138.57,163.33,and 176.99mg,respectively.The pupa weight decreased significantly with the increase of Bt concentration,but with the continuation of generations,the pupa weight of the 3rd generation was 220.28,403.88,294.40,315.11 mg,and the control group was 400.53 mg.The emergence rate of each generation and after treatment of phlegm was not affected by Bt.?3?During Bt stress,the relative growth rate,relative feeding rate,food utilization rate and food conversion rate of the larvae of the larvae were significantly lower than those of the control in the 1st and 2nd generations.The larvae could not convert the nutrients into body weight normally.The growth and feeding rate of each group increased with varying degrees.However,the approximate digestibility did not decrease significantly during the stress period,and the effect of Bt on larval digestibility of larvae was insignificant.After stopping Bt stress,the growth,feeding rate and relative metabolic rate of the treatment group were significantly higher than those during the stress period.Moreover,it could improve food utilization and conversion rate for growth,and the same treatment growth,feeding rate and digestibility tend to decrease with generations.?4?After feeding the Bt leaves,the Clostera anachoreta larvae digestive enzymes were changed:?-amylase??-AL?and trypsin were higher than the control,and the larvae increased the digestive enzyme activity in the midgut.Strengthened the decomposition of food and the ability to degrade and digest Bt protein but decreased significantly with the prolongation of the generation;the detoxification enzyme activity of the 1st generation treatment group was significantly lower than that of the control,while the 2nd and 3rd generation activities were significantly higher than the 1st generation.generation.The detoxification enzymes showed a trend of:Carboxylesterase?CarE?activity increased with generations,and the3rd generation treatment was lower than the control,but with the increasing trend of the generation,the glutathione S-transferase?GST?activity in the three generations of treatment was lower than the control,and the treatments showed an upward trend with the whole generation.The activities of the larval acetylcholinesterase?AChE?activity in the 1st and 2nd generations groups were at a low level,which was not significantly different from the control,but for 3 successive generations.The changes of larval protective enzymes were as follows:there was no significant difference between the 1-3 generations of catalase?CAT?activity,but the A,B,C and P groups showed a slight decrease with the continuation of the generation.The peroxidase?POD?activities of the 1st and 3rd generations were significantly lower than that of the control.The activities of groups A,B and C decreased with the rise of the generation,and the P group continued to increase,which was a large fluctuation between the groups.The activity of superoxide dismutase?SOD?was lower in the 1st generation of each treatment group than in the control,and the 2nd and 3rd generations were the same as the control.There was no obvious trend of protective enzyme activity in different concentrations and successive generation.Larvae might cope with Bt toxin by adjusting the relationship among SOD,CAT and POD.The overall changes of the three enzymes indicated that in the early stage of Bt stress,the larva increased the digestive enzyme activity to enhance the absorption capacity of the food,while the lower detoxification enzyme activity indicated that the larva could not effectively decompose and metabolize the toxin.With the successive generations of Bt stress,the larvae reduced the digestive enzyme activity in response to nutritional requirements,which in turn increased the activity of detoxification enzymes,indicating the change of the resistance of Clostera anachoreta to Bt toxin from passive resistance to active decomposition,and thus enhanced tolerance.