The Physiological Functions Of Corazonin Signaling System In The Hyphantria Cunea(Drury)

Corazonin signaling system is involved in regulating important physiological functions such as heart rate,body color change,and reproductive behavior in insects.However,the function of the Corazonin signaling system in the H.cunea has not been studied.In this study,the cDNA sequences of Corazonin（Crz）and its receptor gene Corazonin receptor（CrzR）were successfully identified based on the transcript library of H.cunea,and the sequence was analyzed by bioinformatics.The main expression position of HcCrzR in HEK293 cells was determined by cell localization technology,and the spatio-temporal expression specificity of HcCrz and HcCrzR genes was analyzed by real-time PCR technology.The physiological functions of the Crz signaling system involved in regulation were preliminarily analyzed via RNAi,the effects of the Crz signaling system on the growth and development of H.cunea larvae and the reproductive behavior of adults were observed,and the regulatory mechanism of the Crz signaling system in the H.cunea was analyzed.The main conclusions are as follows:1.Successfully cloned and identified the full-length sequences of HcCrz and HcCrzR cDNA of the H.cunea,HcCrz open reading frame（ORF）is 315 bp in length,encoding 104amino acids.The HcCrzR Open Reading Frame（ORF）is 1293 bp in length and encodes 430amino acids.HcCrzR has 7 highly conserved transmembrane domains and cell localization results show that it is localized on the cell membrane and is a typical G protein-coupled receptor.Phylogenetic analysis showed that HcCrz was the closest homologous to Bm Crz,and HcCrzR was closely related to Lepidoptera insects.2.HcCrz and HcCrzR were expressed at all developmental stages of H.cunea.HcCrz was mainly expressed in the 1^st and 3^rd instar larvae and adult stages of H.cunea,while HcCrzR was mainly expressed in the egg stage,4^th instar larvae,female pupae,and female adult stage.Tissue-specific expression analysis showed that HcCrz had the highest expression in the head,followed by the relative expression of the hindgut,midgut,and ovary.In contrast,HcCrzR had the highest expression in the hindgut,and the relative expression of the midgut and testis was second.3.Microinjection and real-time PCR technology were used to screen out the targets of HcCrz and HcCrzR siRNA with the best silencing effect,knockdown the HcCrz/HcCrzR gene of H.cunea larvae,and record the body weight and food intake.At 120 h after treatment,the weight of larvae in both HcCrz siRNA and HcCrzR siRNA-treated groups increased significantly,and the food intake of HcCrz siRNA and HcCrzR siRNA-treated groups increased significantly at 96 h after treatment.After the determination of silencing HcCrz/HcCrzR,the energy metabolism substance of larvae decreased,and it was found that the content of trehalose and glycogen in larvae decreased in HcCrz/HcCrzR siRNA-treated group,glucose content decreased and ATP content increased by silencing HcCrz,and membrane-bound trehalase activity decreased and ATP content decreased by silencing HcCrzR.Lipid droplets were stained with BODIPY dye,and it was found that the lipid droplets in the HcCrz/HcCrzR siRNA treatment group became smaller and the degree of lipid droplet fusion decreased.The expression of genes related to energy metabolism was detected,and it was found that the expression of trehalose metabolism-related genes and fat synthesis genes decreased,the expression of glycogen synthesis genes increased by silencing HcCrz,and the expression of the Treh2 gene decreased by silencing HcCrzR.Silencing HcCrz can significantly shorten the pupariation,but silencing HcCrzR has no significant effect on the pupal stage,and silencing HcCrz/HcCrzR has no significant effect on pupal weight in male and female pupae.4.The HcCrz/HcCrzR gene of silent H.cunea had no significant effect on the adult lifespan.The treatment group and the control adults were mated,and the number of eggs produced by adults after mating was counted.It was found that the egg production of the HcCrz/HcCrzR siRNA-treated adults was significantly reduced.Silencing HcCrz/HcCrzR also led to a significant decrease in the number of eggs in ovarioles,a decrease in Vg expression,malformation of the ovarioles,and egg blockage at the end of the ovarioles.The number of sperm in the testis in the HcCrz/HcCrzR siRNA-treatment group decreased significantly,and the expression of genes related to spermatogenesis decreased significantly.By measuring the expression of the dsx in gonad and the expression of the PBP in males and females,it was found that the expression of dsx in female gonadal was significantly upregulated,the expression of dsx in male gonadal was significantly down-regulated,and the expression of PBP was significantly upregulated in HcCrz/HcCrzR siRNA-treated females.These results indicated that the Crz signaling system of H.cunea participates in regulating the physiological metabolism of larvae in H.cunea,affects the feeding and weight of H.cunea,participates in the regulation of pupariation,affecting the oogenesis and spermiogenesis process of adults,and then participates in the regulation process of reproductive ability.The finding contribute to elucidating the mechanism of Corazonin signaling system regulating the growth and development of H.cunea,providing a theoretical basis for the research and development of environmentally friendly insecticides targeting Crz/CrzR.