LmCDA1 And LmCDA2 Is Essential For The Gut Development And Cuticle Barrier Construction In Locusta Migratoria

The Locusta migratoria is an important agricultural pest in China,which is large size,easy to be observed and sensitive to RNA interference system.The long-term use of chemical pesticides to control locust plagues not only caused serious environmental pollution,but also increased the resistance of L.migratoria to pesticides.Therefore,to research and develop some new biological insecticides is the current research hotspot.In the life cycle of insects,the exoskeleton cannot be extended,so insects have to undergo a molting process to grow.Chitin is a very important component of insect exoskeleton.Chitin deacetylase plays an important role in the degradation of chitin and can catalyze the conversion of chitin to chitosan.Therefore,research and development of biological pesticides based on the essential genes for chitin degradation pathways is reliable strategy.In order to test the function of the LmCDA1 and LmCDA2 during the development of locusta foregut and hindgut,we observed the microstructure of cuticle in foregut and hindgut after injecting the ds LmCDAs.In order to explore the effect of chitin deacetylase on the ecological adaptability of locust,we tested whether the distribution of eosin dye in the integument was affected after interfering with LmCDA1 and LmCDA2.In addition,to further explore whether the two genes affect lipid transport and the hydrocarbon content,we observed the distribution of lipid droplets by using the Bodipy staining,and we tested the cuticular hydrocarbon content by using GC-MS technology.Then we detected the sensitivity for different pesticide control.In this study,we mainly carried out the following two aspects of work:1.Effect of chitin deacetylase on intestinal cuticle development of locustThe RT-qPCR technique was used to determine the expression patterns of chitin deacetylase(LmCDA1 and LmCDA2)in the foregut and hindgut of the fifth instar nymph every day.The results showed that the expression levels of LmCDA1 and LmCDA2 were higher in the pre-ecdysis stage and after ecdysis stage of locust foregut,while the expression levels were higheronly in the pre-ecdysis stage of locust hindgut.The double-stranded RNA of LmCDA1 and LmCDA2 was injected on the N5D2 of locust,then the external morphology of the gut dissected under light microscope was observed.The results showed that there were no visible phenotypes compared with the control.After HE staining,it was found that there were no affection on molting of foregut and hindgut after knock down the expression of these two genes,but the old and new cuticle of locust after injected ds LmCDA2 were thicker than control.We localized the position of LmCDA1 and LmCDA2 in foregut and hindgut by immunohistochemical technology,and the results showed that LmCDA1 and LmCDA2 were differentiated in the intestinal localization: LmCDA1 was mainly distributed in the epidermal cells and procuticle of foregut and hindgut,while LmCDA2 was mainly distributed in the epidermal cells and epicuticle of foregut and hindgut.2.Effect of chitin deacetylase gene on cuticle lipid composition of locustIn order to test the change of permeability after interfering with LmCDA1 and LmCDA2,we stained the cuticle of locust by Eosin Y.The results showed that the areas stained with Eosin Y changed compared with the control group,suggesting that these two genes affected the skin permeability of locust.The second instar nymph integument of locust was stained with Bodipy dye which especially combined with neutral lipid.Compared with the control group,the results showed that the number of lipid droplets outside the integument that interfered with LmCDA1 and LmCDA2 was significantly reduced,and the lipid droplets were mainly concentrated under the epidermis cells.The composition and content of cuticle hydrocarbons in the second instar-nymphs cuticle were detected by GC-MS(gas chromatography-mass spectrometry).The results showed that the total amount of hydrocarbons on the surface of the locust integument increased significantly after LmCDA1 interference.After interfering with LmCDA2,there was no significant change of hydrocarbon content in the cuticle of locust.Finally,five kinds of pesticides were applied to the control and the second instar nymphs that interfered with LmCDA1 and LmCDA2,respectively.The sensitivity of different experimental groups to five kinds of pesticides was detected,and the sensitivity to organophosphorus pesticides was enhanced after interfering with two genes,and the degree of fungal infection is weakened.