Functional identification of C-type lectin in the diamondback moth,Plutella xylostella(L.)innate immunity

C-type lectins(CTLs)are a superfamily of Ca^(2+)-dependent carbohydrate-recognition proteins,and an important pattern recognition receptor(PRR)in insect innate immunity which can mediate humoral and cellular immunity in insects.In this study,we report a novel dual carbohydrate-recognition domain(CRD)CTL from Plutella xylostella which we designate PxIML.PxIML is a protein with a 969 bp open reading frame(ORF)encoding 322 amino acids,containing a signal peptide and a dual-CRD with EPN(Glu_(124)-Pro_(125)-Asn_(126))and QPD(Gln_(274)-Pro_(275)-Asp_(276))motifs.The expression of PxIML mRNA in the fat body was significantly higher than in hemocytes and midgut.The relative expression levels of PxIML in the whole insect and the fat body were significantly inhibited after infection with Bacillus thuringiensis 8010(Bt8010)at 18 h,while they were significantly upregulated after infection with Serratia marcescens IAE6 or Pichia pastoris.The recombinant PxIML(rPxIML)protein could bind to the tested pathogen-associated molecular patterns(PAMPs),and the bacteria of Enterobacter sp.IAE5,S.marcescens IAE6,Staphylococcus aureus,Escherichia coli BL21,and Bt8010 in a Ca^(2+)-dependent manner,however,it showed limited binding to the fungus,P.pastoris.The rPxIML exhibited strong activity in the presence of Ca^(2+) to agglutinate Bt8010,Enterobacter sp.IAE5 and S.aureus,but it only weakly agglutinated with E.coli BL21,and could not agglutinate with S.marcescens IAE6 or P.pastoris.Furthermore,the rPxIML could bind to hemocytes,promote the adsorption of hemocytes to beads,and enhance the phenoloxidase(PO)activity and melanization of P.xylostella.Our results suggest that PxIML plays an important role in pathogen recognition and in mediating subsequent humoral and cellular immunity of P.xylostella.

Role of digestive protease enzymes and related genes in host plant adaptation of a polyphagous pest,Spodoptera frugiperda

The evolutionary success of phytophagous insects depends on their ability toefficiently exploit plants as a source of energy for survival.Herbivorous insects largelydepend on the efficiency,flexibility,and diversity of their digestive physiology and sophistication of their detoxification system to use chemically diverse host plants as foodsources.The fall armyworm,Spodoptera frugiperda(J.E.Smith),is a polyphagous pest ofmany commercially important crops.To elucidate the ability of this insect pest to adaptto host plant mechanisms,we evaluated the impact of primary(corn)and alternate(rice)host plants after 1l generations on gut digestive enzymatic activity and expression profiles of related genes.Results indicated that the total protease and classspecific trypsinand chymotrypsinlike protease activity of S.frugiperda significantly differed among hostplant treatments.The classspecifiq protease profiles greatly differed in S.frugiperdamidguts upon larval exposure to different treatments with inhibitors compared with treatments without inhibitors.Similarly,the single and cumulative effects of the enzymespecific inhibitors TLCK,TPCK,and E64 significantly increased larval mortality and reduced larval growth/mass across different plant treatments.Furthermore,the quantitativereverse transcription polymerase chain reaction results revealed increased transcription oftwo trypsin(SfTry3,SfTry7)and one chymotrypsin gene(Sfchym9),which indicatedthat they have roles in host plant adaptation.Knockdown of these genes resulted in significantly reduced mRNA expression levels of the trypsin genes.This was related to theincreased mortality observed in treatments compared with the dsRED control.This resultindicates possible roles of S.frugiperda gut digestive enzymes and related genes in hostplant adaptation.