Analysis Of The Function And Signal Transduction Of Latrophilin Toxin Receptor

Latrophilins(LPHs)are adhesion G-protein-coupled receptors comprising three paralogous forms(LPH-1,LPH-2 and LPH-3)and known receptors for a-latrotoxin,which are involved in growth,development,adaptability and schizophrenia and other diseases in vertebrates.However,the functions of LPH are poorly understood in most insects.First of all,phylogenetic analysis was performed among 21 animals.The results revealed that animal lph was originated from one ancentral gene,but this gene independently evolved among the vertebrates,Cephalochordata,Urochordata and insect.It is found that lph has duplicated in vertebrates,and generated three copies(lph-1,lph-2 and lph-3),whereas lph only contain two copied(lph-2 and lph-3)in these animals in including birds Urochordata and Cephalochordata.While,lph-1 and lph-2 were obtained from Caenorhabditis briggsae and Caenorhabditis elegans.Differing from these animals,there is only one lph in the insect.Therefore,although animal lph was derived from one ancestral gene,large differentiation has occurred among them,indicating that the function of this gene diverged among these animals.Then,latrophilin(Tclph)was cloned in Tribolium castaneum,and three alternatively spliced transcripts(Tclpha,Tclphb and Tclphc)were identified.All these three Tclphs were highest expressed at the early adult stage,and strongly expressed in central nervous system of adults.Larval RNA interference(RNAi)against Tclph caused 24%adult wing abnormal,30%insect death,and led to 100%reductions in beetle fecundity.Fecundity deficiency was rescued by reciprocal crosses with wild-type females,but not males.And dissection results revealed that 63%of dslph female ovaries were atrophied.Further,exon-specific RNAi illustrated that neither knockdown of Tclpha nor Tclphc resulted in development defects and reductions in beetle fecundity.Thus,it indicated that Tclphb was essential for development and female fecundity in T.castaneum.Moreover,RNAi against Tclph increased the expression of the foxo,plc and pka genes which most related to cAMP/PKA,PLC/IP3 and IS signaling pathways,which might affect insect wing development and lifespan in T.castaneum.Additionally,PLC/IP3 and IS signaling pathways also affected Vg synthesis,further reduced the reproduction of dsTclph insects.Interestingly,RNAi of Tclph increased the larval mortality significantly in response to carbofuran and dichlorvos in T.castaneum.Further,RNAi against Tclph up-regulated transcription of Tcace-1 but down-regulated Tcchat,the keys gene on the acetylcholine(ACh)pathway.And the activity of acetylcholinesterase(AChE)and choline acetyltransferase(ChAT)was also measured,which was consistent with the change of mRNA level.These results indicated that Tclph RNAi increasing insecticides susceptibility through up-regulated Tcace-1 gene expression to compensate for the down-regulated of Tclph and Tcchat.Furthermore,the global transcriptome profiles between RNAi-treated larvae(dslph)and control larvae of T.castaneum were investigated and compared by RNA-sequencing.Totally,274 differentially expressed genes(DEGs)were identified between the dslph and control samples.These DEGs were classified into 42 GO functional groups,including such functions as the response to stimulus,developmental process and reproduction.Interestingly,the DEGs encoding Viperin,PGRP-SA,Defensinl and Defensin2 were involved in stress responses which likely modulated the effects of Tclph on stress resistance in T.castaneum.Additionally,RNAi of Tclph altered large-scale serine protease(Sp)and lipase genes expression patterns and further amplified the SP and lipase signaling pathways to regulate the development and reproduction as well as the stress response in T.castaneum.Furthermore,CSPs,ESTs,CYPs,AOXs and Gus were significantly down-regulated after RNAi of Tclph,which illustrated that they cooperated with Tclph to reduce the activity of cellular metabolism system.While,FMOs was up-regulated in dslph insects suggested it may also involve in detoxifying alkaloid of insect metabolism system.As the activity of these detoxification enzymes changed,it suggested Tclph participated in phase 0,? and ? cellular detoxification,further affected the insecticide susceptibility of dslph insects.Further,we examined two potential downstream carboxylesterases gene(Tcest4 and Tcest6)for Tclph and characterized their interactions with Tclph in T.castaneum.Tcest4 and Tcest6 were most highly expressed at the late larvae and late adults stages,and enriched in central nervous system and hemolymph of larvae,respectively.Meanwhile,the transcription of Tcest4 and Tcest6 were induced by carbofuran and dichlorvos from 12 to 72 h.When Tcest4 or Tcest6 was silenced,the susceptibility of larvae to carbofuran and dichlorvos were increased,suggesting Tcest4 and Tcest6 play important roles in detoxification of insecticides.In addition,RNAi against Tclph up-regulated transcription of Tcest4 but down-regulated Tcest6 after treated with anticholinesterase insecticides.These results indicated that Tclph RNAi increasing insecticides susceptibility through up-regulated Tcest4 gene expression to compensate for the down-regulated of Tclph and Tcest6.Furthermore,RNAi against Tcest6 led to 43%reductions in female egg laying and completely inhibition of egg hatching,suggesting Tclph affected reproduction through down-regulated Tcest6 transcription.This study used RNAi method to clarify the functions of lph in T.castaneum,and explored its signaling pathways combining with RNA-sequencing,providing vital information for LPH signaling pathway.The study on the functions and signaling pathways of LPH likely identified novel targets of insecticide due to their important activities for growth,development and insecticides susceptibility,laying a foundation for pest biology control.