Preliminary Study On Reproductive Related Genes Of Campoletis Chlorideae

In the present paper,the endoparasitoid Canpoletis chlorideae Uchida was chosen to study the reproductive related genes.This parasitoid is the dominant parasitic wasp of Helicoverpa armigera(Hubener),a major economic pest in the world.Our team discovered the abnormal ovarian development of this paasitoid in the fields.On the basis of this finding,we firstly performed transcriptome sequencing to screen out two reproductive related genes:vitellogenin(Vg)and vitellogenin receptor(VgR).As the focus of this study,we studied its developmental expression pattern;and through Bt{Bacillus thuringiensis)toxic protein Cry 1Ac treatment,we investigated the effects of Cry 1Ac protein on the growth and development of C chlorideae,and the gene expression of Vg and VgR.Furthermore,around Reproductive abnormalities were compared with The mRNA levels of two detoxification enzyme P450 genes were compared between abnormal and normal ovary of C.chlorideae.We hope that the detoxification mechanism of parasitoids can be used to ascertain the cause of abnormal ovarian development of C.chlorideae.The following results were obtained.1.Developmental expression pattern of Vg gene of Camporetis chlorideaeVitellogenin(Vg)is a key protein in insect ovarian development and oogenesis.Until now,the research on parasitic wasp Vg is obviously insufficient,and it is still unclear whether there is Vg in larval endoparasitoids.The Vg sequence of Campoletis chlorideae(named as CcVg)was obtained on the basis of the transcriptome sequencing data of this female wasp.Through multiple alignment analysis and phylogenetic tree construction,the following results were obtained.The full-length cDNA of CcVg is 5866 bp,the full length of the ORF is 5340 bp,encoding 1779 amino acids,the protein molecular weight is 220.63 kDa,and the N-terminus contains a 16 amino acid signal peptide.The wasp Vg sequence has a typical vitellogenin superfamily gene domain,which is closely related to Pimpla nipponica.The Vg mRNA levels was compared by real-time PCR(q-PCR)s the expression of Vg gene was significantly increased in abnormal ovarian development female wasps compared with normal female wasps.Furthermore,the spatiotemporal dynamic expression pattern of the Vg gene was studied.The results showed that the expression level in the larval stage was very lows followed by a gradual increase,especially on the 0 day of cocoon and 0 day of adult eclosion,the expression of Vg increased significantly with the increase of adult age.Unlike most reports that Vg is a female characteristic gene,the expression of Vg gene is also found in male,and there is no similar expression pattern to female,it reached a peak after eclosion 1-2 days later.However,the expression level of Vg gene expression of female was significantly higher than that of male,and the fat body is the main synthetic tissue.The expression pattern of the vitellogenin gene of Campoletis chlorideae can not only enrich the reproductive biology theory of parasitoids,but also provide the theoretical basis and practical guidance for the successful breeding of this wasp.2.Characteristics of developmental expression of VgR gene of Campoletis chlorideaeThe vitellogenin receptor(VgR)is a specific receptor for vitellogenin,which is closely related to ovarian development and vitellogenesis.The study of parasitic wasp VgR is even more lacking compared with Vg.Therefore,it is clear that the expression pattern of the parasitic wasp VgR gene can lay the foundation for its functional research.The expression of VgR gene was firstly detected in two different ovarian development femles of Campoletis chlorideae by the method of real-time PCR(q-PCR),on the basis of transcriptome sequencing data.The expression level was significantly decreased in females with abnormal ovary.The expression of VgR gene in the female of Campoletis chlorideae was expressed lowly in the larval stage.From the larval stage to the adult stage,it raised gradually.and there was a small peak after cocoon 4 days later.The highest peak reached after eclosion 12 days later,which is similar to the expression pattern of Vg,indicating the cooporativety between Vg and VgR.The VgR gene in the male has a high expression level at 2-4 days in the cocoon,but the VgR gene expression of female at the initial eclosion of Od was 11.0 times higher than that of male,indicating that the VgRis also non-female specific gene.VgR is mainly expressed in the ovary,but it is also expressed in the epidermis of the male and fat body of both sexes,which is presumed to be related to non-nutritional function.3.Effects of Cry IAc toxic protein on the growth and reproductive development of Campoletis chlorideaeNumerous studies have shown that large-scale planting of transgenic Bt crops is not compatible with natural enemy insects.The Cry 1Ac toxic protein was used as an exogenous toxicant to investigate its effects on the growth and reproductive development related genes of Campoletis chlorideae according to their abnormal ovarian development in the field,Firstly,the Cry IAc was used to feed the 2nd instar larvae of the host Helicoverpa armigera,and its body weight decreased significantly,indicating that the toxic protein inhibited the growth of H.armigera larvae.The wasps were fed with honey water mixed with Cry 1Ac protein because of the characteristics of their complementary nutrition.The longevity of wasps was significantly shortened,and there was a dose effect.The Cry 1Ac imposed a significant inhibition on the growth and development of C.chlorideae,the rate of cocoon was reduced,and the eclosion rate was only 50.2%,the body length became significantly smaller than that of control.The transcriptional levels of Vg and VgR,the key genes for the reproductive development of C.chlorideae,were detected by real-time fluorescent quantitative PCR.The results showed that the mRNA expression levels of both Vg and VgR were significantly decreased in both sexes after Cry IAc treatment.The above results indicated that the Cry 1Ac protein inhibited the ontogeny of C.chlorideae,especially the reproductive development,and showed obvious incompatibility effects.These suggested that maybe some poisons in the fields generated teratogenic effects on the ovarian development of C.chlorideae.4.Effects of Cry 1Ac toxic protein on P450 gene of Campoletis chlorideaeP450 is an important detoxifying enzyme of insects and participates in the metabolism of toxic substances.The abnormal ovary of Camporletis chlorideae in the field may be induced by some toxic substances.The study of the detoxification enzyme P450 gene can verify this hypothesis.Two P450 genes were screened,Cyp6a2 and Cyp307al according to transcriptome data.The mRNA levels of two P450 genes in Campoletis chlorideae was detected by real-time PCR.The expressions of two detoxification enzyme genes were significantly lower females with abnormal ovary than that of the normal females.At the same time,it was found that the expression of these two P450 genes was female-specific,which will be demonstrated in the further study,especially their correlation with ovarian development.In addition,the expression levels of these two P450 genes also showed significant regional differences.The expression levels of P450 from Xuchang and Yuanyang were significantly lower,which may be related to the application level of insecticides or environmental factors in different regions.Furthermore,the expression levels of Cyp6a2 and Cyp307al in C.chlorideae were investigated.After the parasitized cotton bollworm treated with Cry IAc toxic protein,the expressions of Cyp6a2 and Cyp307al were both significantly increased in wasp,indicating that the Cry 1 Ac toxic protein induced up-regulation of the P450 genes.This suggested that they are involved in the detoxification of toxic proteins in parasitoid.