Effects Of Parathyroid Hormone Like Receptors(iPTHRs) And Their Ligands On Insect Development And Epidermal Formation

Neuropeptide hormones are a kind of evolutionarily ancient endogenous active substances,and their main receptors,G protein coupled receptors?GPCRs?,constitute the largest protein superfamily on cell membrane.They mediate different signaling pathways,which are not only widely involved in the normal growth and development,but also closely related to the occurrence,development and treatment of various diseases in animals.Therefore,neuropeptides and their GPCR receptors can be used as important drug targets,and the corresponding researches are always at the forefront of the new medical drugs development and the potential pesticides exploration.Parathyroid hormone family members secreted by the diffuse neuroendocrine system and their receptors not only play an important role in the regulation of calcium and phosphorus,but also involve in the development of kidney and bone in vertebrates.There is also Recombinant Human Parathyroid Hormone on the medical market,which has approved for the treatment of osteoporosis,hypoparathyroidism and other diseases.However,there are few studies on this signaling system in invertebrates.Our research group and Tanaka's research group in Japan took the lead and successively found PTHR-like receptors in the representative insects such as red flour beetle Tribolium castaneum,brown rice planthopper Nilaparvata lugens and so on,and we named them i PTHRs here.However,whether there is endogenous ligand of i PTHRs in invertebrates has not been reported yet,leaving i PTHRs as orphan receptors.In addition,preliminary analysis of the function of i PTHRs revealed that knocking down the receptors seriously affected the process of molting,emergence and the formation of epidermis in T.castaneum,but the specific mechanism was not clear.Here,we analyzed the adaptive evolution mechanism of i PTHRs in invertabrates based on the phylogenetic relationship in metazoa,screened and identified the potential endogenous ligands and investigated its function.Meanwhile,we further explored the possible regulatory mechanisms by using transcriptomics and proteomics.In addition,we also carried out the function investigation of i PTHRs in N.lugens.This study attempts to provide a new clue for the comprehensive understanding of the evolution,physiological function and mechanism of PTHRs/i PTHRs,and is expected to provide a candidate target for the development of green insecticides in biological control of pests,and even provide a reference for PTH signal system to the occurrence and treatment of human calcium and phosphorus metabolism disorders,osteoporosis,hypoparathyroidism and other diseases.Firstly,we collected more i PTHRs sequences?including arachnids and molluscs?,combined the PTHRs sequences of representative vertebrates to construct the phylogenetic tree,and the adaptive evolution of i PTHRs in invertebrates was analyzed by the codeml program in PAML?Phylogenetic Analysis by Maximum Likelihood?software package.It showed that there were independent duplication and loss events in PTHRs/i PTHRs in vertebrates and invertebrates,and the number of i PTHRs genes in invertebrates is more diverse;meanwhile,i PTHRs in invertebrates experienced selection pressure,and 21 positive selection sites were screened out,which are located in the extracellular amino-terminal extracellular domain,juxtamembrane region and transmembrane domains.Because these domains are the key regions of ligand receptor binding,it may suggest that there is a large variation in the sequence of ligands in invertebrates;Secondly,PXXXamide,a candidate ligand for insect i PTHRs,was screened through extensive collection of neuropeptide information and Blast searching.The coding sequences region of PXXXamide in T.castaneum encodes 109 amino acids,which has no significant homology with PTH of vertebrates,but is relatively conservative with the peptides in other arthropods.Tests of this peptide,PXXXamide,in functional reporter assays confirmed the interaction of the ligand-receptor pair.We named it as i PTH.The temporal and spatial expression was detected by real-time quantitative PCR?q RT-PCR?and immunohistochemistry.It is highly expressed in late pupal stage,central nervous system?CNS?and gut tissues,and the tissue expression pattern is consistent with the receptor i PTHRs.However,RNA interference showed that i PTH knockdown had no effect on the growth and development of T.castaneum,which was quite diffenent with the abnormal phenotype caused by i PTHRs.We found that the RNAi for suppression of i PTH still displayed residual i PTH immunoreactivity in the neuroendocrine cells of CNS and gut,indicating that the i PTH RNAi did not provide an appropriate level of transcript knockdown and the leaking level of protein production was sufficient for the required levels of bioactivity.An alternative possibility is that there may be another ligand for PTH receptors that yet needs to be discovered in T.castaneum;Thirdly,the expression levels of genes related to ecdysone synthesis and metabolism,juvenile hormone synthesis and metabolism and regulation of eclosion were detected by q RT-PCR after i PTHRs knockdown in T.castaneum.It showed that the expression levels of the key enzyme genes in ecdysone synthesis pathway or metabolism process,CYP307A1,E74,Broad complex?Br-C?,FTZ-F1,did not change after the knockdown of the i PTH signaling system;Although the expression of CYP315A1 and CYP314A1 in this pathway was slightly down-regulated,the20-Hydroxyecdysone and ecdysone titer in the 3-day and 5-day pupae of the treatment group were not changed;The expression levels of the key enzyme genes in juvenile hormone synthesis pathway,CYP15A1 and JHAMT,were significantly up-regulated after i PTHRs knockdown,while there was no effect on the expression levels of CCAPRs and Rickets,the receptors of eclosion related neuropeptides CCAP,bursicon.These results indicated that the failure of ecdysis caused by i PTHRs knockdown is not through the ecdysone synthesis and metabolism,either the eclosion related regulatory genes.In comparison,their effects on the expressions of juvenile hormone synthesis genes may be one of the reasons;In addition,the downstream regulatory network of i PTHRs was analyzed by transcriptomics and quantitative proteomics in T.castaneum.At the transcription level,1231 and 1683 differentially expressed genes?DEGs?were identified in IB vs dsi PTHR1 comparison and IB vs dsi PTHR2 comparison,respectively,while only 14DEGs were identified in dsi PTHR1 vs dsi PTHR2 comparison.It showed that DEGs after i PTHRs knockdown mainly in the process and pathway of energy and nutrition metabolism?such as isocitrate dehydrogenase,ATP binding cassette protein,fructose-1,6-bisphosphatase?,epidermal structural components?chitin synthase 1,chitin deacetylase,cuticle proteins?and the juvenile hormone metabolism?juvenile hormone esterase and juvenile hormone cyclohydrolase?.At the protein level,34 and39 differentially expressed proteins?DEPs?were identified in IB vs dsi PTHR1comparison and IB vs dsi PTHR2 comparison,respectively,while 41 DEPs were identified in dsi PTHR1 vs dsi PTHR2 comparison.Consistent with the transcriptome results,the DEPs in the treatment groups were also more concentrated in energy and nutrition metabolism,and epidermal structural components.Several genes,such as ATP synthetase subunit delta,Troponin T,CPR18,CPAP1-H,were down-regulated in both transcription and protein level.All of these data indicated that i PTHRs may affect the eclosion behavior through the energy generation metabolism and then cause the abnormal synthesis in epidermal components;Finally,sequence analysis and RNA interference analysis of i PTHRs were carried out in the incomplete metamorphosis insect,N.lugens.It showed that the two i PTHRs are from intraspecific gene duplication,and their sequence identity is as high as 53.06%.These two i PTHRs in N.lugens are more similar to the i PTHR2 gene in T.castaneum,combined with the multiple alignments of i PTHRs from other insects,indicating that i PTHR2 may be the ancestor gene in insects.RNA interference results showed that part of individuals in dsi PTHR1 and dsi PTHR2 group were arrested at the time point of molting and eclosion after 4^th instar nymphs injection,and the survival rate continued decreasing to 26.7%and 55.5%,representively,which indicated that i PTHRs also play an important role in the growth and development of the incomplete metamorphosis insects.