Genome-Wide Identification Of Cuticular Protein Genes And Their Transcription Responses To Novaluron Exposure In Leptinotarsa Decemlineata(Say)

The Colorado potato beetle(CPB)Leptinotarsa decemlineata(Say)is a notorious defoliator of potato.The beetles often cause extremely large potato yield losses.Insect cuticle is composed of chitin filaments embedded in heterogeneous cuticular proteins(CPs).Modifications in insect cuticle decrease cuticular penetration rates of insecticides and cause penetration resistance.To understand the underlying molecular basis,an important initial step is to identify and characterize CPs.In the present paper,we identified the complete CP gene repertoire in the genome of L decemlineata,and measured the expression profiles of CPR family genes in novaluron-treated beetles.Our results facilitate further research and development of novel pest management strategies.1.Molecular cloning of cuticular protein genes in L.decemlineataThe L.decemlineata genome and transcriptome data can be used to mine the CP genes.Firstly,we performed homologous search and gene prediction,and found 175 candidate CPs.The sequence correctness of these candidates had been verified by using reverse transcripted polymerase chain reaction(RT-PCR)and/or expressed sequence tag(ESTs).These 175 CP proteins were further divided into 7 families based on motif and phylogenetic analysis,including CPR,CPF,CPFL,CPAP1,CPAP3,Twdl,and CPCFC families.The findings provide the foundation for further study.2.Analysis of each family of cuticular protein genes in L.decemlineataThe CPR is the largest family(137 members)members in L.Decemlineata(LdCPRs),were further assigned to RR-1,RR-2 and RR-3 subfamilies containing 50,85 and 2 members respectively.LdCPR family possessed a GSYS(A)XXXXDGXXXXV(R)conserved motif.The RR-1 proteins had an additional motif with a conserved YTADENGF sequence.The RR-2 members had an additional motif with a conserved RDGDVVKG region and three G-x(3)-VV sequences.CPF,CPFL,CPAP1,CPAP3,Twdl,and CPCFC families have fewer members consisting 5,4,9,8,9 and 2 respetively.Furthermore,one LdCP was not assigned to any family.The findings provide valuable information to explore the molecular basis of cuticular resistance to insecticides,and to analyze the role of CP involved in insect development and cuticle formation during ecdysis.3.Transcription responses of RR-1 and RR-2 subfamily genes to the exposure of novaluronThe transcription responses of RR-1(33 members)and RR-2(42 members)to novaluron were analyzed.Among 33 CP genes in RR-1 family,ingestion of novaluron at the concentration of 2.5 mg/L,1.25 mg/L,0.625 mg/L,and 0.3125 mg/L significantly down-regulated the expression of 18 genes,whereas the feeding has no or positive effects on the transcription of the other genes,compared with the control.Similarly,28 genes were significantly down-regulated among 42 members in RR-2 family.It appears that novaluron may disturb the transcription of CP genes to affect the formation of insect cuticle.