Functional Studies Of Melanin Synthesis Pathway Related Genes In Blattella Germanica (L.)

The exoskeleton is a vital barrier for insects to survive and adapt to harsh environments.In the long-term evolution process,terrestrial insects have formed complex epidermal structures to cope with excessive water evaporation.The formation of normal cuticular structures relates to the process of melanization and sclerotization,and insect melanin is involved in the pigment deposition and hardening of insect epidermis,which is crucial to maintaining the structural and functional integrity of insect epidermis.Therefore,insect melanization may also play a key role in insect water balance.The melanin synthesis pathway is conserved in insects and has been validated in some holometabolous model species,while it was poorly studied in hemimetabolous insects.The German cockroach(Blattella germanica)is a hemimetabolous insect of the order Blattaria,commonly known as the cockroach.B.germanica a worldwide urban sanitation pest that is widely distributed,closely related to human life and extremely difficult to control.Because of the black coloration on cockroach nymphs and the yellowish-brown coloration on the adult,it can be used as an object for functional studies of cuticle melanization in insects.The functions of important enzymes involved in insect melanin biosynthesis such as tyrosine hydroxylase(TH),dopa decarboxylase(DDC),and N-β-alanyldopamine synthase(Ebony)are often pleiotropic,However,the specific functions of the genes encoding these enzymes in the epidermis of Blattella Germanica are not clear.In particular,their function in regulating insect epidermal permeability has not been fully studied.In this study,B.germanica was selected as the object.Firstly,the open reading frame sequences of these key enzyme genes in the melanin biosynthesis pathway were cloned and bioinformatically analyzed.Secondly,the spatiotemporal expression characteristics of BgTH,BgDdc,and Bg-ebony were analyzed by quantitative PCR.Then,RNA interference(RNAi)technology was used to repress the expressions of BgTH,BgDdc and Bg-ebony genes to verify their functions in the cuticle and analyze their effects on physiological functions such as cuticular pigmentation,developmental metamorphosis,and cuticular permeability.The main findings are as follows:1.Molecular identification and expression analysis of BgTH,BgDdc and Bg-ebony of B.germanicaTo investigate the role of BgTH,BgDdc,and Bg-ebony in melanin synthesis in the German cockroach,the gene sequences were first identified and cloned.Spatiotemporal expression analysis showed that BgTH and Bg-ebony were highly expressed in the head and epidermis of B.germanica,while BgDdc was expressed in the head and epidermis and high in the fat body.The expression of BgTH and Bg-ebony peaked a few hours after molting,while the expression of BgDdc peaked in the 1-day-old adults and a relatively high expression continued throughout the development.The expression of Bg-ebony in adult B.germanica was significantly higher than that in nymph.2.BgTH,BgDdc,and Bg-ebony genes affect cuticle pigmentation in the German cockroachRNAi was used to repress the melanin synthesis genes BgTH,BgDdc and Bg-ebony in B.germanica,and their functions were investigated.The results showed that BgTH-RNAi led to molting obstacles and death in some cockroaches,while the rest of the survivors showed pale and soft bodies.BgDdc-RNAi cockroaches had abnormal light brown body color,which deepened with time.RNAi knockdown of Bg-ebony in both male and female cockroaches generated a completely different black cuticle than the control,but there was no effect on their development.3.BgTH and BgDdc genes are involved in regulating the cuticular permeability of B.germanicaUnder dry conditions,BgTH knockdown male and female adults died earlier than cockroaches in control.There was no significant difference in the survival rate of male and female adults with BgDdc knockdown compared with the control.However,under high humidity conditions,nearly no cockroaches died in either treatment or control groups,and there was no significant difference in mortality of all groups.Lastly,the eosin Y staining assay showed that BgTH-RNAi resulted in a higher cuticular permeability in female cockroaches,and BgDdc-RNAi also caused slight eosin Y penetration through the cockroach cuticle.4.German cockroach BgTH and BgDdc affect cuticular hydrocarbons depositionTo further investigate the relationship between cuticle melanization and cuticular hydrocarbons(CHCs),the CHCs of BgTH-and BgDdc-silenced male and female adults were analyzed.GC-MS analysis showed that knockdown of either BgTH or BgDdc increased the total amount of CHCs in female and male cockroaches.Further studies revealed that BgTH knockdown increased the CYP4G19 expression level,while BgDdc knockdown had no significant effect on CYP4G19 expression.In summary,this study confirmed the conserved roles of BgTH,BgDdc,and Bg-ebony in the melanin synthesis in B.germanica.BgTH not only affects cuticle pigmentation but also plays essential roles in the growth and development of cockroaches.BgDdc and Bg-ebony are also crucial to the formation of body color in cockroaches,but it had no effect on insect development.It was further found that the melanization pathway plays significant roles in cuticular water retention,in which BgTH and BgDdc play key roles in cuticular permeability in B.germanica.In addition,there was an interaction between body melanization and the accumulation of cuticular hydrocarbons.The results of this study not only provide a theoretical basis for the development of new cockroach control techniques and the management of cockroach resistance by regulating the cuticle permeability of B.germanica with melanin genes,but also lay a foundation for the study of the melanin synthesis pathway in hemimetabolous insects.