Latrophilin Participates In The Insecticide Resistance Of C. Chinensis By Regulating CSP7, OBPC01, CYP4BN1 And CYP6BQ11

Latrophilins(LPHs)belong to a unique branch of G protein-coupled receptors(GPCRs)and named adhesion GPCR(aGPCR),and firstly identified as the potential target for a potent a-latrotoxin(a-LTX)which isolated from the black widow spider venom.As a classic GPCR,LPH was associated with the multiple mental disorders such as attention-deficit/hyperactivity disorder,bipolar disorder and schizophrenia in different organisms including human,mice and fly.Latest studies from our laboratory have shown that knockdown of Tclph by RNAi not only resulted in some of the larvae morphological deficiency,but their reproductive capacity was significantly reduced in the red flour beetles,Tribolium castaneum,which is consistent with the findings of other groups.Interestingly,we also found that RNAi of Tclph at the T.castaneum larvae stage increased the susceptibilities to two different anticholinergic insecticides including dichlorvos and carbofuran,implied that Tclph was participated in insecticide resistance.However,the downstream molecular regulatory mechanisms have not yet been fully elucidated in insects.Therefore,we further screened out the downstream resistance related genes of Tclph by RNA-sequencing(RNA-seq)to clarify its signal pathway,and functional analysis was performed on these genes in T.castaneum and recivied the following results.Firstly,the RNA-seq results revealed that RNAi of Tclph led to the difference expression of two insecticide transport-related genes(TcCSP7?TcOBPC01)and two insecticide metabolism-related genes(TcCYP4BNl?TcCYP6BQll)in T.castaneum,and this result was confirmed by qRT-PCR.It is suggested that Tclph may participate in some physiological function of the red flour beetles by regulating the expression of these four genes.Subsequently,we carried out each insecticides of dichlorvos and carbofuran treatment after knockdown of Tclph at the T.castaneum larvae stage.Evidently,Tclph has been suppressed from 12 to 72 hour(hr),simultaneously,the TcCSP7?TcCYP4BNl and TcCYP6BQ11 transcripts were significantly down-regulated throughout all the time points,whereas the TcOBPC01 gene expressions have been up-regulated from 12 to 72 hr.This further suggested that Tclph plays a pivotal role in the resistance of insecticides through directly governing TcCSP7.TcCYP4BNl and TcCYP6BQ11 expression,in contrast,the over-expression of TcOBPC01 caused by reduction of Tclph is probably compensated for the insecticides resistance when the loss of Tclph in T.castaneum.Additionally,we further investigated the functions of TcCSP7?TcOBPC01?TcCYP4BNl and TcCYP6BQ11 during the T.castaneum growth and development as well as insecticides resistance process.The spatio-temporal expression pattern showed that these four genes were expressed in different developmental stages and tissues,but displayed specific characters:TcCSP7 was highly expressed in early pupae and head and antennae;TcOBPC01 was predominately expressed in late larvae and head?fat body and antennae;TcCYP4BN1 was highly expressed in early pupae as well as head and malpighian tubule;TcCYP6BQ11 was mainly expressed in pupae(early pupae?late pupae)stages and gut and malpighian tubule tissues.Moreover,TcCSP7?TcOBPC001 TcCYP4BN1 and TcCYP6BQ11 were significantly induced by dichlorvos or carbofuran from 12 to 72 hr,indicating that these four genes likely related to insecticides resistance.The functions of these four genes in the red flour beetles were further confirmed using RNAi,in which specifically suppressed the transcript levels of the corresponding genes,especially in the fifth day,the inhibition efficiency reached the maximum.Interestingly,knockdown of these four genes expression did not affect the survival of T.castaneum,suggesting that they had no effect on their growth and development.Then,we conducted the insecticides resistance bioassay for dichlorvos or carbofuran treatment on the fifth day following injection of double strand(ds)-TcCSP7,ds-TcOBPC001?ds-TcCYP4BNl or ds-TcCYP6BQ11.Although the accumulative larval mortalities increased in all groups from 12 to 72 hr due to insecticide exposures,significantly increased mortalities were observed in larvae injected with dsRNAs when compared that control groups,further suggesting that these four genes associated with insecticides resistance.These outcomes provide the necessary evidence to elucidate that Tclph genes involved in insecticides resistance through balancing the regulation of insecticide transport-related genes as well as insecticide metabolism detoxification-related genes expression in T.castaneum.