Parathyroid Hormone-like Receptors And Epidermal Growth Factor Receptors Co-regulate Epidermal Development In Tribulus Terrestris

The body wall of insects,which is also considered as exoskeleton,can directly contact with the external environment and play a similar role as the skin of vertebrates.It also provides a place for numerous skeletal muscles and has a great influence on the movement,feeding,spawning and other activities of insects.These special functions are much more potent than the skin of vertebrates.Once the insect's exoskeleton has hardened,it cannot continue to expand,which limits the growth of insects.Therefore,insects will develop a series of molting during their growth and development.These facts indicate that the body wall of insects is crucial to the life activities of insects.In our previous studies,knock-down of Tcpthrl1 and Tcpthrl2 in late larvae of Tribolium castaneum caused molting failure in most pupae and elytra abnormalities in adults.It suggested that Tcpthrls may play important roles in the cuticle development of T.castaneum,and this seemed to be correlated with that the altered expression of chitin synthase(CHS),cuticular proteins(CPR)and various chitinases(CHT)after RNAi of Tcpthrls in RNA-seq data.In addition,the epidermal growth factor receptor(EGFR),which also plays an important role in regulating the cuticle development of insects,was up-regulated in ds-Tcpthrls groups in RNA-seq analyses.Thus,whether Tcegfr and Tcpthrls have potential interactions is an interesting question and needs further experimental validation.Based on these results,we will further explore whether Tcpthrls affect the cuticle development through Tcegfr,and then affect the growth and development of T.castaneum.qRT-PCR analysis showed that the expression levels of Tcpthrl1 and Tcpthrl2 were almost the same in the tissues of late adults.Besides their high expression in the central nervous system(CNS),they also had high expression levels in the gut,elytra,and epidermis.Because most parts of the gut and the elytra,epidermis all belong to the body wall of insects,this suggested that Tcpthrls may play an important role in the development of the body wall in T.castaneum.Further analyses found that both Tcpthrl1 and Tcpthrl2 had the highest expression in the foregut and midgut of the late pupae and the head&thorax cuticle of late adults.In addition,Tcpthrl1 was also highly expressed in the abdominal cuticle of late adults and Tcpthrl2 was highly expressed in the elytra of late adults.Interestingly,the expression levels of Tcegfr in the CNS,gut,elytra,and epidermis were also relatively high(although it was the highest in the accessory glands).Likewise,its expression levels in various parts of the gut and other epidermal tissue were similar with Tcpthrls,which were highly expressed in the foregut and midgut of pupae,head&thorax cuticle and elytra of adults.Meanwhile,its expression levels in the larval hindgut and adult hindgut,larval abdomen cuticle and pupal elytra were also high.This result indicated that the tissue expression pattern of Tcegfr was not exactly the same with Tcpthrls,but their expression trends were consistent in the pupal and adult stages.In addition,RNAi was carried out in the early pupal stage when Tcegfr had the highest expression level.The result showed that 53%larvae could not enter the pupal stage successfully.Even the rest pupae can be eclosed into adults,but about 10%individuals had abnormal elytra.The elytra were significantly thinner compared with the control.These adults gradually became shrivelled and eventually died.These results showed that Tcpthrls and Tcegfr may have a very close relationship in regulating the cuticle development in T.castaneum.After RNAi of Tcpthrl1 and Tcpthrl2,the expression of Tcegfr gradually increased from late larval to 6-day pupae,then suddenly decreased after 7-day pupae and increased in the 1 day adults and then gradually decreased.These results indicated that Tcpthrls indeed regulated the expression of Tcegfr,especially in the critical stage of metamorphosis and metabolism.The detection of chitin content found that the chitin content was significantly increased in the ds-Tcpthrl1 pupae and adults.The chitin content also was increased but without significance in the ds-Tcpthrl2 pupae and adults.After RNAi of Tcegfr,the chitin content was significantly reduced.It is worth mentioning that the chitin content recovered after double knockdown of Tcpthrls and Tcegfr.Further analysis of the changes in chitin content in the various tissues revealed similar recovery results in the gut of late pupae and the elytra of late adult.Afterwards,we observed the changes in the microstructure of the cuticle,using paraffin sections,SEM,and TEM.We found that after RNAi of Tcpthrl1 and Tcpthrl2,the thickness of the body wall and the gut wall of late pupae increased significantly.The elytra of the adults became wrinkled,and the horizontal chitin layer and the vertical pore fiber in the microstructure of adult elytra become irregular.After RNAi of Tcegfr,the thickness of the body wall and the gut wall of late pupae were significantly reduced.Meanwhile,the elytra of the adults became thinner and the horizontal chitin layer and the vertical pore fiber in the microstructure disappeared completely.After knockdown the Tcpthrls and Tcegfr,these phenotypes recovered to a certain degree.These results suggested that Tcpthrls indeed regulate cuticle development through Tcegfr in T.castaneum.After RNAi of Tcpthrls,the key genes in chitin biosynthesis pathway were determined by qRT-PCR.Interestingly,Tcgfat and TcCHSA were up-regulated after knocking down of Tcpthrl1 and Tcpthrl2.Correspondently,after RNAi of Tcegfr,the expression of CHSA was down-regulated significantly.Therefore,we demonstrated that Tcpthrls and Tcegfr participated in cuticle development regulation and influenced the key genes expression in the chitin biosynthesis pathway in T.castaneum.