Components Of Cotesia Vestalis Venoms And Its Effects On Host Hemolymph Carbohydrates

Carbohydrates are a type of macronutrient,which can help insects to carry out life activities,such as promoting development,providing energy for tissues under starvation and flight,regulating appetite,improving insect resistannce.Taking Cotesia vestalis（Hymenoptera:Braconidae）and Plutella xylostella（Lepidoptera:Plutellidae）as the research objects,we indentified the components of C.vestalis venom by mass spectrometry,and identified the types of carbohydrates in P.xylostella hemolymph,detected the effects of C.vestalis venom on host hemolymph carbohydrates.1.Combining the data from the venom mass spectrometry,the venom gland transcriptome and the C.vestalis genome,a total of 397 venom protein genes were identified in C.vestalis venom.Expression analysis revealed that three genes are specifically expressed in the venom gland（tolloid-like protein 1,ion transport peptide-like,venom protein Ci-48a）,91 genes were highly expressed in the venom glands（log₂（VG-FPKM/Carcass-FPKM）>1）.and 174 genes low expressed in the venom glands（log₂（VG-FPKM/Carcass-FPKM）<1）,129 genes has no significant difference expression levels between in the venom gland and the carcass（-1<log₂（VG-FPKM/Carcass-FPKM）<1）.The 397 venom proteins were classified into enzymes,protease inhibitors,binding proteins,others and unknowns,“enzymes”occupied the majority,reaching 48.61%,“protease inhibitors”,“binding proteins”,the“others”and the“unknowns”accounted for 0.76%,3.53%,41.31%and 5.79%respectively.The functional categories of the“enzymes”fell into hydrolases（58）,oxidoreductases（58）,and transferases（41）,ligases（16）,isomerases（14）,lyases（6）.Moreover,12representative venom genes were selected for tissue-specific analysis,and found that ion transport peptide-like,tolloid-like protein 2,Chitooligosaccharidolytic beta-N-acetylglucosaminidase,immunoevasive protein-2B,and thioredoxin-2 genes were specifically expressed in the venom gland,while tolloid-like protein 1,glutathione-S-transferase,Serpin B4,protein disulfide isomerase A3,putative hyaluronidase HYAL,hemocytin,calreticulin genes were expressed in ovary,carcass and male body other than venom glands.2.The carbohydrates in P.xylostella hemolymph were identified by HPLC,five sugars were determined:sorbitol,trehalose,fructose,glucose and sucrose.trehalose occupied the most（up to 80%）.By measuring the effects of parasitism on these five sugars in P.xylostella hemolymph at each instar（the third middle-late instar,the third late instar,the fourth early instar,the fourth middle instar and the fourth late instar）,the results showed that parasitism can cause P.xylostella hemolymph:（1）trehalose concentration increased continuously;（2）sorbitol concentration increased firstly at the third middle-late instar and the third late instar,then decreased at the fourth early instar and the fourth middle instar,and no difference at the fourth late instar;（3）fructose concentration increased during the third middle-late instar to the fourth middle instar,and no difference at the fourth late instar;（4）no significant difference of sucrose and glucose concentrations.3.The third middle instar larvae of P.xylostella were injected with the C.vestalis venom and the CvBV respectively,and their effects on the carbohydrates concentrations of P.xylostella hemolymph were compared.It was found that venom can cause P.xylostella hemolymph:（1）sorbitol concentration increased continuously（27%～55%）;（2）fructose concentration increased at the third late instar and the fourth middle instar by 37%and 20%respectively;（3）no significant difference of trehalose,glucose and sucrose concentrations.CvBV can cause P.xylostella hemolymph:（1）trehalose concentration increased continuously（17%～179%）,（2）sorbitol concentration increased at the fourth middle instar and the fourth late instar by 47%and 45%respectively,（3）fructose concentration increased during the third middle-late instar to the fourth middle instar（18%～69%）.（4）no significant difference of sucrose and glucose concentrations.4.Based on the genome and transcriptome data of P.xylostella,the genes related to sorbitol metabolism in P.xylostella were cloned,including synthesizing enzyme（AKR1B1）and others that may be involved in sorbitol synthesis（AKRs）,and sorbitol dehydrogenase（SD）.Six sequences of AKRs（PxAKR2E4,PxAKR1B1-1,PxAKR1A1-1,PxAKR2E4-l-1,PxAKR2E4-l-X1,PxAKR2E4-l-2）and one sequence of sorbitol dehydrogenase genes（Px SD）were obtained until now.In order to clarify the relationship between the increase of sorbitol concentration in P.xylostella hemolymph caused by the venom and the genes related to sorbitol metabolism,q PCR was used to detect the effect of parasitism on the transcription level of each gene.The results suggest that parasitism can cause an overall up-regulation of the transcription level of the sorbitol synthase genes in P.xylostella except PxAKR1A1-1,among which the transcription level of PxAKR1B1-l increased the most significantly,by 2.08 times;however,the transcription level of Px SD was not significant changed.Consistent with the parasitic effect,the injection of venom also up-regulated the transcription levels of PxAKR1B1-1,PxAKR2E4,PxAKR2E4-l-X1,but has no effect on the transcription levels of PxAKR2E4-l-1 and PxAKR2E4-l-2.