Expression,Function and Regulation of Chitin Synthase 2 Gene in Locusta migratoria

[Objective]Chitin synthase is one of the key enzymes responsible for chitin synthesis in insects.As this enzyme is absent in higher animals,it could be served as a potential target for developing safe and effective insecticides.In our earlier research,the cDNA of chitin synthase 2 gene(LmCHS2,GenBank accession number:GU067731) in Locusta migratoria was cloned.The objectives of this paper are to further study the expression,function and regulation of LmCHS2,and to provide a scientific basis for effective pest control using RNAi methods.[Method]Based on the nucleotide sequence of LmCHS2,a pair of specific expression primers was designed,the expression patterns of LmCHS2 were studied in eggs,nymphs and adults by RT-qPCR.The dsRNA of LmCHSl was synthesized in vitro,and then injected into the female or male adults on day 1,respectively.The midguts dissected from the injected insects on day 5 were pooled for each RNA extraction.cDNA synthesis and RT-qPCR were performed to determine the down-regulation of LmCHS2.After dissected the whole gut,the midgut changes and integrity of peritrophic matrix(PM) were observed to explore the biological functions of this gene in L.migratoria adults.Locusts were maintained with no food in different times,and feeding again,to observe the changes of guts.Then the transcript levels of LmCHS2 were detected by RT-qPCR.[Result]LmCHS2 was almost undetectable during the early and middle embryogenesis,but dramatically up-regulated in late eggs.It was consistently expressed throughout the nymphal and adult stages.After dsCHS2 was injected into the female or male adults on day 1,significantly reduced transcript of LmCHS2 was observed as compared with that of the controls,and resulted in a decreased feeding and a high mortality of insects(78%for female and 85%for male adults).After dissection,it was found that there was virtually no food contained in dsCHS2-injected insects and the average length of midguts and gastric caeca was shorter than that of the control.Furthermore,histological observation of midguts showed that the control locusts contained a fully developed PM,however,locusts injected with dsCHS2 exhibited a disrupted PM or even absence of the PM.Locusts were treated under starvation for 48 h,the midguts hardly contained food and the average length of midguts was significantly shorter than that of the control midguts.From the H E stained results,it was found that the PM was almost absent in non-fed midguts while the PM of control midguts was well-structured,which was very similar with the RNAi.But after fed again,the insects contained a fully developed PM.When locusts were maintained with no food for 24 h and 48 h,the transcript levels of LmCHS2were suppressed significantly.When locusts were fed for another 0.5 h period,the transcript levels increased to the control level rapidly,which suggested that feeding affected the expression of LmCHS2.[Conclusion]LmCHS2 is responsible for chitin biosynthesis of peritrophic matrix of the midgut and plays a key role for the development of L.migratoria.The decreased expression of this gene affected the integrity of the PM,thus hindered the food absorption and led to the mortality of the locusts.In addition,feeding regulated the expression of LmCHS2.