Cuticular Development And Molecular Mechanism Of Chitin Metabolism In The Pro-nymphs Of Locusta Migratoria

Locusta migratoria is an important agricultural pest.It has multiple epidermal formation and degradation during embryonic period,including the formation and degradation of serosal cuticle,the first embryonic cuticle and the second embryonic cuticle,and the formation of the third embryonic cuticle.The second embryonic cuticle comprise the pro-nymph and is named as pro-nymph cuticle,which is an important protective structure in the pro-nymph stage.The inhibition of its synthesis and degradation can lead to the deficiency in hatching of the locust,which is an important stage in the early control of the locust.In this study,we will clarify the epidermal structure of the pro-nymph and its formation and degradation by dynamic observation of the ultrastructure of the pro-nymph cuticle.By studying the expression characteristics,expression location and function of the chitin synthase Lm CHS1 and chitinase Lm Cht5-2 in the pro-nymph stage,we can identify the mechanism of its involvement in the chitin metabolism of the pro-nymph cuticle,which may provide a new target molecule for the control of egg-stage pests.1.Ultrastructure and chitin development of the pro-nymph cuticleThrough the dynamic observation of the ultrastructure of 9-14 d locust embryos at 30°C,we found that pro-nymph cuticle is divided into procuticle and epicuticle.The epidermis produce the epicuticle from the 10 th day(E10H0).The complete structure of pro-nymph cuticle is formed at E11H0 with the epicuticle and laminar-procuticle.The pro-nymph cuticle began to degrade at E12.The procedure is as follows: the separation of epicuticle and procuticle at E12H0-E12H12;apolysis at E12H12-E13H0;and the degradation of pro-cuticle at E13H0-E14H0.Further,paraffin sectioning technique and Fluorescent Brightener 28 staining were used to observe the chitin development of the pro-nymph cuticle.The results showed that the chitin of pro-nymph cuticle was deposited at E11,which was consistent with ultrastructural observation.At E12,the pro-nymph cuticle degraded with the chitin became thinner,and moved away from the embryo.At E13-E14,the chitin of the pro-nymph cuticle gradually degraded.2.The characteristics of Lm CHS1 and its mechanism involved in the synthesis of chitin in the pro-nymph cuticleThe mRNA expression level of LmCHS1 was detected by RT-q PCR.The expression of Lm CHS1 gene was increased at E9,reached the highest peak at E11,and remained high at E12-14.Localization of Lm CHS1 protein by immunohistochemistry revealed that Lm CHS1 was expressed in epidermal cytoplasm;it was expressed at the highest level in E11 and secreted into the epidermal layer.We synthase ds Lm CHS1 in vitro and inject it into the E7 embryo with microinjection.The RNAi significantly reduced the expression of Lm CHS1 in the pro-nymph stage(about 88%);and caused the block of the locusts hatching,with a mortality rate of 50.6%.Preparation of pro-nymph paraffin sections and HE staining showed that the formation of pro-nymphs in the ds Lm CHS1 group was blocked.Through immunohistochemistry analysis,it was found that ds Lm CHS1 significantly inhibited the expression of Lm CHS1 protein in the pro-nymph cuticle and reduced the chitin content of the cuticle.Finally,the results of transmission electron microscopy showed that the chitin layer structure of procuticle was disappeared in ds Lm CHS1 group.Therefore,ds Lm CHS1 inhibits the synthesis of Lm CHS1 in the epidermal cells,thereby hindering the synthesis of chitin,causing the formation of the pro-nymphs to be blocked,leading to embryonic developmental malformation and difficulty in hatching.3.The expression characteristics of Lm Cht5-2 and its mechanism involved in the degradation of chitin in the pro-nymph cuticleChitinase 5 is an important enzyme involved in the degradation of insect epidermis.Previous studies showed that silencing Lm Cht5-1 caused the deficiency of the pro-nymph cuticle degradation and hatching from eggshell.Based on this,this study aims to analyze the mechanism of Lm Cht5-2involved in the degradation of pro-nymph cuticle.It was found that the expression of Lm Cht5-2 gene was lower in E7-E10 in late embryonic development and significantly increased in E11,and at E12 the expression level was maintained at a relatively high level through detection.The Lm Cht5-2 protein had a high expression level in E12-E13,and red fluorescence signals were observed in epidermal cells and procuticle layers.We synthase Lm Cht5-2 in vitro and inject it into E9 embryo with microinjection.The RNAi significantly reduced the expression of Lm Cht5-2in the pro-nymph stage(about 87.5%);and caused the block of the locusts hatching,with a mortality rate of 80%.The transmission electron microscopy showed that Lm Cht5-2 plays an important role in the degradation of chitin in the pro-nymph cuticle and the separation of the procuticle and epicuticle.In summary,we found that the pro-nymph cuticle is comprised with procuticle and epicuticle.The enzyme Lm CHS1 and Lm Cht5-2 play the important role in chitin disposition and degradation in cuticular metabolism of pro-nymph stage,respectively.The results of this study will provide foundation for the development of molecular targets for the control of pests in the egg stage and the control of pests based on the regulation of molting process during the embryonic development.