| Mulberry leaves are the main source of food for the economic insect silkworm.Mulberry production is often damaged by various pests,which seriously affects the safe feeding of silkworm and restricts the development of sericulture industry.Chemical control is still the main means to control mulberry pests.However,long-term application of chemical insecticides can easily lead to increased pest resistance,environmental pollution in mulberry fields and frequent poisoning of silkworms.Therefore,green pest control methods represented by parasitoid biological control have received increasing attention.In the mulberry ecosystem,the species richness of parasitic wasps is high,which plays an important role in the population growth of mulberry pests under field stress and is an important factor in the biocontrol practice of mulberry pests.In this study,the dominant parasitic wasp in mulberry field was taken as the research object.Starting from exploring its basic biological characteristics and lifelong fecundity,the key parameters of its parasitic biology were clarified,and then the composition of its venom protein and the function of key venom protein were analyzed.The mechanism of action of venom in its successful completion of parasitism lays an important foundation for completely revealing the biological characteristics of I.kuwanae and its interaction with its host and provides an important reference for the biological control of mulberry pests based on I.kuwanae.The main research results are as follows:1.Ectoparasitism,Gregarious parasitoid and host feeding characteristics of I.kuwanae are synovigenic parasitoid.The life history of I.kuwanae includes four stages: egg,larva,pupa,and adult.It takes about 14 days to complete a generation.The reproductive mode is bisexual reproduction and parthenogenesis,and the offspring of non-mating females are all males.There was no significant difference in the number of eggs laid and the number of offspring emergence of female wasps with different reproductive methods.Male and female wasps can emerge at all times of the day,but most of them emerge during the day.The male emerged 1 d～2 d earlier than the female,and the total number of male emergences in one day was greater than that of female.The oviposition behavior of I.kuwanae can be divided into host search,host inspection,ovipositor probing,host paralysis,oviposition,and other major behavioral modules.Female wasps feeding on hosts is a necessary condition for ovarian development and maturation.Feeding 10 % honey water,water and no feeding had no significant effect on ovarian development of female wasps.2.The age of parasitoids significantly affected the number of eggs laid,the length of hind tibia and the longevity of offspring.The optimum parasitism age was 10～20 days.The host weight significantly affected the number of eggs laid,the number of offspring eclosion,the size of the offspring and the longevity of the female offspring.The optimal host weight of the parasitoid was 0.4～0.6 g.Supplementation of 10% honey water significantly reduced the number of eggs laid by females,the sex ratio of offspring and the longevity of offspring females.3.The venom reservoir and venom glands of I.kuwanae were successfully observed and collected.RNA-Seq technology was used to construct the transcriptome database of the venom gland of I.kuwanae.A total of 5613 unigenes were assembled,and 34 venom protein genes such as phospholipase A2,uridine diphosphate glucosyltransferase,laccase,calreticulin,icarapin,chitinase,peptidoglycan recognition protein and trehalase were identified.Ten common venom genes were randomly selected for expression pattern verification.The results of q RT-PCR showed that phospholipase A2 gene(Ikuw PLA2),uridine diphosphate glucose transferase gene(Ikuw UGT),superoxide dismutase gene(Ikuw Sup),melanization protease gene(Ikuw Mel),laccase gene(Ikuwlac),calreticulin gene(Ikuw Cal),Ikuw Ica and trehalase gene(Ikuw Tre)were specifically expressed in venom gland tissue.On the first day after emergence,the relative expression levels of Ikuw PLA2,Ikuw Sup,Ikuwlac and,Ikuw Chi were significantly higher than those at other time points.4.A phospholipase A2 gene(IkuwPLA2)was identified from the venom gland transcriptome database of I.kuwanae.The open reading frame sequence of Ikuw PLA2 was cloned,and the recombinant phospholipase A2(Ikuw PLA2)was expressed and purified in vitro by E.coli prokaryotic expression system.The host test of recombinant protein injection showed that the triglyceride content of the host Galleria mellonella was not significantly different from that of the natural parasitic treatment group at 6 h and 12 h after injection of the host Ikuw PLA2,but significantly higher than that of the injection of pure water and mechanical damage treatment group.The expression of lipase gene(Gmellip)in G.mellonella was significantly higher than that of natural parasitism,injection of pure water and mechanical damage treatment groups at 6 h and 12 h after injection of host IkuwPLA2.The expression level of GmelUGT was significantly higher than that of the other three treatments 12 h after injection of the host Ikuw PLA2.After injection of Ikuw PLA2,the relative expression levels of immune genes such as host phospholipase A2gene(Gmel PLA2),serine protease inhibitor gene(Gmel Ser)and calreticulin gene(Gmel Cal)changed significantly,and this change was significantly affected by different time after injection.The above results showed that Ikuw PLA2 significantly affected host lipid and glucose metabolism and host immune response.5.A total of 24 UGT genes(GpUGTs)were identified from the transcriptome database of Glyphodes pyloalis.After treatment with chlorfenapyr for 24 h and 48 h,there was no difference in the expression level of Ikuw UGT compared to the control group.However,after treatment with chlorfenapyr for 24 h,the relative expression levels of four UGT genes(Gp UGT33AQ1,Gp UGT33AS1,Gp UGT50A14 and Gp UGT40AV1)were significantly up regulated.The relative expression levels of three genes(Gp UGT33AS1,Gp UGT40AM4 and Gp UGT40Ak7)were significantly up regulated after 48 h of chlorfenapyr treatment.The expression of Gp UGT33AS1 was successfully silenced by RNAi technology.The mortality of G.pyloali larvae was significantly increased after exposure to sublethal doses of chlorfenapyr,indicating that UGTs were involved in the detoxification process of G.pyloalis in response to chlorfenapyr stress. |
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