| The entomopathogenic nematodes are precious insect natural enemies and used to control agricultural,forestry and medical pests.Their sex determination is environment-dependent sex determination(ESD)and influenced by nutritional competitive pressure during parasitic period.So far,there are few researches and reports about the special molecular mechanism of this particular sex differentiation about animals.Therefore,in this study,we just focus on the molecular regulation mechanism,which has important biological significance and practical value.Our group used the relevant research backgrounds of Caenorhabditis elegans to perform transcriptome sequencing analysis on postparasitic juveniles(J4)and adults of Romanomermis wuchangensis,but did not carry on the transcription analysis of the parasitic juveniles(J3)that is the key stage of sex differentiation,and function of sex differentiation genes were also lack of further research.Therefore,it is of great biological significance and practical value to further explore the function of sex differentiation genes in R.wuchangensis.In this study,we first observed the morphological changes of parasitic juveniles(J3)of R.wuchangensis that infected the host Culex quinquefasciatus,proved the influence of infective ratio of nematodes to host infections and the time of parasitic juveniles(J3)emerged influencing postparasitic juveniles sex differentiation or proportion of nematodes.Then,the parasitic juveniles(3d,4d and 5d)of female and male R.wuchangensis were collected and subjected to transcriptome sequencing.Finally,according to the sex decision pathway of C.elegans,screening genes of sex differentiation,used the technique of fluorescence quantitative PCR,RNA interference and in situ hybridization to study function of these genes,to explore the expression level of each gene at different developmental stages and the effect of sex differentiation after interference.The results are as followings.1.Morphological and biological observation of parasitic juvenilesIn order to understand the morphological changes of parasitic juveniles of R.wuchangensis and their host,the infected wigglers were observed by using stereomicroscope.It was found that on parasitic 5th day the wiggler pereion was entangled with parasitic juveniles.Dissect the wiggler body found:parasitic juveniles began to grow significantly on 3rd day,and trophosome of parasitic juveniles on 5th day began to accumulated numerous.After parasitic juveniles has completed its parasite growth and development,it emerges from the host and causes host death.The postparasitic juveniles have shown significant gender characteristics.Obviously observe that female postparasitic juveniles were bigger than males,and the frontal vaginal of female can be clearly seen under stereomicroscope.The results of parasitic time and infection intensity showed that:Feeding under 26 ? conditions,the nematodes that first emerged on 6th day were smaller male postparasitic juveniles,and emerged on 8th day juveniles were the larger female postparasitic juveniles.When the ratios of parasites to host was 1:1,all postparasitic juveniles were females,and when the proportion of infection was 40:1,all postparasitic juveniles developed into males.That is,the more nematodes in the host,the less nutrient each parasitic juveniles can obtain,and it more likely to develop into males,instead,they develop into females.2.Transcriptome sequencing of parasitic juvenilesSelected parasitic juveniles 3d,4d,5d females and males were transcriptome sequenced separately and combined with the results of previous transcriptome analysis in our research group.Refered to C.elegans sex determination pathway,selected fox-1,tra-2,laf-1,tra-3,fem-1,tra-1,mab-3,mab-7,mab-5,gld-1,egl-4 and so on eleven genes related to gender differentiation.Laf-1,mab-3 and tra-2 have been studied in preliminary work,on this basis,the following four genes fem-1,fox-1,mab-7,tra-1 have been selected for further functional analysis.3.Functional studies of candidate genes(1)Analysis of candidate genes sequenceUsing bioinformatics to construct N-J phylogenetic tree for multiple alignments of sequences,the results showed that the four genes of fem-1,fox-1,mab-7 and tra-1 were all clustered together with Ovomermis sinensis,and the relatives were closest.(2)Fluorescence quantitative PCR of candidate genesQuantitative fluorescence results demonstrated that:? Rwufem-1 was extremely difference on 3rd of parasitic juveniles,female and male adults(p<0.01).The relative expression level of Rwufem-1 at 7 days postparasitic juveniles appeared difference(p<0.05);? Relative expression level of Rwufox-1 in male and female nematodes presented significant difference on 3rd day of parasitic juveniles and 1 day of postparasitic juveniles(p<0.05);? On 3rd and 5th day of parasitic juveniles,and 1 day of postparasitic juveniles,the expression level of Rwumab-7 in both of male and female nematodes were significantly higher than other developmental stages(p<0.01).The relative expression levels of the postparasitic juveniles at 5 and 7 days were significantly different(p<0.05);? The relative expression levels of female and male juveniles of Rwutra-1 on 3rd day parasitic stage and adults nematodes were extremely different(p<0.01),and the gene expression on 5 days postparasitic juveniles were significantly different(p<0.05).(3)RNAi experiment of candidate genesRNAi results show:? After interfering with Rwufem-1 gene,the proportion of female and male postparasitic juveniles were significantly increased compared with control group and the difference was extremely significant(p<0.01),indicating that Rwufem-1 was disturbed and juveniles towards female development.? After interfering with Rwufox-1,the female and male ratios of postparasitic juveniles were reduced and very significant difference(p<0.01),which showed that Rwufox-1 gene tended to male development after disturbance.? There was no difference between control group and the experimental group after Rwumab-7 was interfered(p>0.05),we speculated that the length and gene position of dsRNA for Rwumab-7 used in RNAi assay may influence their interference effect against the nematodes.? The proportion of male and female postparasitic juveniles with Rwutra-1 gene interference were significantly higher than control groups(p<0.01),indicating that the juveniles were in the direction of female development after Rwutra-1 was disturbed.(4)In situ hybridization experiment of candidate genesIn situ hybridization experiments results show:? The results of Rwufem-1 in situ hybridization showed that the gene was expressed a little in female adult with weak hybridization signal;It was abundantly expressed in male adult and the stronger hybridization signal was,the closer to the tail.It was speculated that the gene could play an 'important role in the development of male sperm and the initiation of male sex differentiation on 3rd day of parasitic juveniles.? The results of Rwufox-1 showed that this gene was abundantly expressed in female adult and the signal was stronger,a small amount of expression were found in male adult,the hybridization signal was weaker.It was speculated that Rwufox-1 was involved in the development of female gametes of R.wuchangensis.? Rwumab-7 gene results showed that the gene was slightly expressed in female adult and the hybridization signal was weaker;It was abundantly expressed in male adult and the hybridization signal was stronger;We speculated that Rwumab-7 was involved in the development of males,the formation of male gametes and development of male gonad.? The results of Rwutra-1 showed that the gene was slightly expressed in female adult and the hybridization signal was weaker,it was abundantly expressed in male adult and strong hybridization signal,speculated that Rwutra-1 might be related to gonadal development of male nematodes.Based on the results of the morphological observation about nematode and host,the sex differentiation of R.wuchangensis was verified to occur in the parasitic period.In addition,the experiment of parasitic time and infective proportion were used to verify that the sex differentiation of nematodes were related to amount of nutrients acquired during parasitic period.Based on this,preliminary inquiry of the four genes mentioned above by the methods of fluorescence quantification PCR,RNA interference,and in situ hybridization.It was speculated that Rwufem-1,Rwumab-7 and Rwutra-1 may participated in the development of male R.wuchangensis.The Rwufox-1 was associated with the development of female nematodes.The result of this study has provided materials and scientific data for the study of sex differentiation and determination pathway of R.wuchangensis. |
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