| Romanomermis wuchangensis,an entomophilic nematode,can actively infect a variety of mosquito larvae including Culex quinquefasciatus and Aedes aegypti etc.The parasitism rate is equal to the mortality rate of pest,and its biological control potential is great in the process of mosquito control.The nutrition of the whole life cycle of R.wuchangensis depends on the absorption of nutrients from the host hemolymph microenvironent in the parasitic period.Another characteristic is that its sex differentiat-ion of R.wuchangensis is caused by the pressure of nutrition competition.That is to say,the less parasitic nematodes in each host,the more nutrients each nematode absorbs in the hemolymph of the host,the more likely it is to develop into a female,on the contrary,it is to develop into a male.In order to explore the molecular mechanism of sex differentiation determined by nutrition in entomophilic nematode,based on sex determination pathway of Caenorhabditis elegans,we screened 10 genes related to sex determination and gonad differentiation,including fox-1,tra-2,laf 1,fem-1,tra-1,mab-3,mab-7,mab-5,gld-1,egl-4,among which fox-1,tra-2,laf-1,fem-1,tra-1,mab-3 and mab-7 genes have been preliminarily studied.However,it is not clear whether mab-5,gld-1 and egl-4 play an important role in the process of sex differentiation.In this experiment,we observed the growth of R.wuchangensis in parasitic stage and prohibited the host from feeding to explore the key period of nutrition determining sex differentiatio-n;we preliminarily explored the functions of mab-5,gld-1,and egl-4 genes in the sex differentiation of R.wuchangensis by qRT-PCR and RNAi biological technology.The main results are as follows:1.In this study,the effects of A.aegypti and C.quinquefasciatus as hosts on the ratio of female to male of R.wuchangensis were compared.The results showed that the species of host did not affect the sex ratio of this nematode,when the infection ratio was 1:1(larvae:nematodes),all nematodes developed into females,and the infection ratio is greater than or equal to 1:10,all nematodes developed into males.When the infection intensity was 1,all the nematodes developed into females,and the infection intensity was greater than or equal to 4,all the nematodes developed into males.It was also found that with the increase of infection intensity,the body of this nematode was getting smaller.The observation of the growth and development of nematodes during the parasitic stage showed that the body width of both the female and the male increased gradually from the first day to the seventh day in the parasitic period.The body length of nematodes remained unchanged on the first and the second days in the parasitic period,and firstly changed significantly on the third day in the parasitic period(p<0.001).It could be inferred that the nematode began to rapidly develop on the third day after invading the host.The body length of this nematode on the 4th and 5th increased much rapidly during parasite stage,body length of the male on the sixth day increased slowly,and body length of the female on the seventh day increased slowly.In addition,from the third day of parasitism,the host was forbidden to eat.When the host feeding was forbidden for one day,compared with the control group,the proportion of female nematodes in the experimental group decreased by 80.0±4.2%from 100%under the infection intensity of 1,and the proportion of female nematodes with infection intensity of both 2 and 3 respectively decreased from 40%±2.2%and 22.2±1.3%to 0,which meant that all nematodes developed into males;the host feeding was forbidden for two or three days,all nematodes with infection intensity of 1 also developed into males,and the body of this nematode was getting smaller with the increase of the time when the host was forbidden to eat.2.The results of qRT-PCR showed that the expression of mab-5 on the 4th day(p<0.01),the 5th day(p<0.01)of parasitism,and on the 1st day(p<0.01),the 3rd day(p<0.01)and the 5th day(p<0.001)after prolapsing were different or significantly different between the female and the maGe;the expression of gld-1 on the 4th day(p<0.001)of parasitism,the 5th day(p<0.01)after prolapsing were significantly different or different between the female and the male;egl-4 was significantly different on the 5th day of parasitism(p<0.001),and there was a significant difference in the expression of female and male in adult stage(p<0.01).Three genes were highly expressed in females in parasitism.3.RNAi results showed that the interference of mab-5?gld-1 and egl-4 gene by RNAi did not cause the change of the sex ratio of R.wuchangensis.It is speculated that mab-5,gld-1 and egl-4 do not determine the sex differentiation of this nematodes,but it is related to the gonad development of this nematode.However,the expression of fox-1,laf-1,fem-1 and egl-4 did not change significantly after gld-1 gene was interfered;the expression of mab-3 and mab-5 increased significantly after gld-1 gene was interfered;the expression of tra-2,tra-1 and mab-7 decreased significantly,which indicated that gld-1 could be in the upstream position of tra-2,tra-1,mab-3,mab-7,and mab-5 genes in sex determination pathway.The down-regulation of mab-5 gene did not change of expression fox-1,tra-2,fem-1,mab-3,egl-4,but significantly highly regulated the expression of laf-1,significantly highly down-regulated the expression of tra-1,mab-7,gld-1.It could be inferred that mab-5 might be in the upstream position of laf-1,tra-1,mab-7,gld-1.After interfering egl-4,the expression of laf-1 and tra-2 would not significantly change,and tra-1 mab-3,mab-7,mab-5 would be significantly up-regulated.fox-1,fem-1,gld-1 would be significantly down-regulated.This suggests that egl-4 might be located before fox-1,fem-1,tra-1,mab-3,mab-7,mab-5 and gld-1 genes in the sex determination pathway.To further verify those hypothesis,we selected the fox-1 gene to promote female differentiation to prove the above results.After fox-1 gene was interfered,we detected the expression of laf-1,tra-2,fem-1,mab-5,gld-1 significantly up-regulated,and tra-1,mab-3,mab-7 significantly down-regulated,which indicated that fox-1 gene might be the initial upstream position of the sex determination pathway of R.wuchangen-sis,which further indicated that egl-4 and fox-1 were located in the upstream position of gld-1 and mab-5.In summary,by observing the growth of the female and the male nematode in the parasitic stage,it was found that the developmental speed of this nematode on 3rd-5th of parasitism was much faster,during which the host diet was forbidden for two days,all nematodes developed into males,so we speculated that the key period of nutrition determining the sex differentiation of R.Wuchangensis was on the 3rd-4th of the parasitic stage.Mab-5,gld-1 and egl-4 were highly expressed in females during parasitic stage,which might be more conducive to play an important role in the development of female gonads.Furthermore,interference with mab-5,gld-1 and egl-4 genes would cause the changes of related sex determining genes,but these genes showed irregular changes.It was suggested that mab-5,gld-1 and egl-4 might be involved in the development of the reproductive system of R.wuchangensis through different regulatory pathway.The above results provide a reference for the molecular mechanism of sex differentiation determined by nutrition. |
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