| Ovomermis sinensis(O.sinensis)is a parasitic nematode that can parasitize a variety of lepidopteran insects in natural state,such as Spodoptera frugiperda,Spodoptera exigua and Spodoptera litura.Once the parasitic process is completed,it escapes from the host which dies due to hemolymph lost.Therefore,O.sinensis is a valuable pest control factor and has a wide range of applications in vegetables and agroforestry.Previous studies have showed that the sex determination of O.sinensis is related to the nutrition absorbed by the nematode during the parasitic period.The fewer nutrients each nematodes absorbed,the more it is likely to develop into a male.To further understand its molecular mechanism,we selected the sex-determined homologous genes based on sex determination pathway of Caenorhabditis elegans(C.elegans).They are fox-1?laf-1?tra-3?tra-2?fem-1?tra-1?mab-3?mab-7.However,the function of these genes and the role they play in the sex determination of O.sinensis are not clear.In this study,1.To understand the development of nematode in each parasitic stage,we observed the nematodes in different parasitic stages;2.To explore the relation between gene expression level and gender and parasitic stage,We detected the expression level of these genes in male and female samples at each parasitic period by qRT-PCR;3.We used RNAi technology to explore the function of the gene in sex determination.The results are as follows:1.Observing the nematodes in different parasitic periods,the body lengths of the nematodes did not vary significantly in the first 3 days of parasitization(P>0.05).The lengths of the nematodes in the first three days of parasitic stage are 0.225±0.046,0.234±0.059,0.235±0.061(Unit:cm),The body widths are 29.8±1.3,33.6±2.6,36.2±5.84(Unit:?m).From the 4th day of parasitization,the body lengths and widths of the nematodes increase rapidly(P<0.05).The lengths of they are 1.2±0.3,2.1±0.26,10.5±1.46,16.3±2.21,23.7±3.47(Unit:cm)and the widths are 95±7.4,110±6.8,242±9.8,250±16.3,285±15.6(Unit:?m).From this period,nematodes are not under synchronous development.In the same parasitic period,nematodes present two kinds of phenotype,the longer one and the shorter one,and the gap between them is getting larger and larger until the parasitic 8th day,then they break away from the host.The trophozoites rapidly accumulated from the 6th day of parasitism,and was fully filled in body cavity of the nematode on the 7th day of parasitism.On the 8th day of parasitism,the density of trophozoites was higher.2.qRT-PCR results showed that laf-1,tra-3,tra-2,fem-1,and mab-3 were highly expressed in early period of parasitization(parasitic 3-5 days)while fox-1,tra-1,mab-7 were highly expressed in the late stage of parasitism(parasitization 7-8 days).A preliminary speculation can be made that laf-1,tra-3,tra-2,fem-1 and mab-3 have been involved in the early stage of parasitism,while fox-1,tra-1 and mab-7 in the latter stage.Laf-1,fem-1,mab-3,and mab-7 are highly expressed in males(P<0.05).On the contrary,fox-1,tra-2,and tra-1 are highly expressed in females(P<0.05).3.The results of RNAi showed that(1)knocking down fox-1 or tra-3 decreased the proportion of females(P<0.05),and decreased the ratio of male when knocking down fem-1(P<0.05).The result indicates that all of these genes are involved in sex determination of O.sinensis.However,knocking down the laf-1,there was no significant change in the sex ratio of nematodes(P>0.05),although an evident split could be observed in the nematode's trophozoites when laf-1 expression turned lower.It indicates that laf-1 takes part in the formation of trophozoites in nematodes,but does not cause a change in the sex ratio of nematode.(2)We measured the body lengths of the nematodes.The results showed that there was no significant difference in the lengths of the male and female compared with the control when knocking down fox-1(P>0.05).Nevertheless,knocking down tra-3 and laf-1 caused the increase and reduction of body length of male nematodes,respectively(P<0.05),and there was no significant difference in the female nematodes(P>0.05).Similarly the length of female was significantly reduced(P<0.05)after knocking down fem-1,but there was no significant difference between males(P>0.05).In summary,according to the time of gene expression,most of the selected genes(laf-1?tra-3?tra-2?fem-1?mab-3)are highly expressed in the early stage of parasitism.And referring to the morphological results,we can speculated that the period between 3th and 5th day during parasitism is critical for sex determination of O.sinensis,while the fox-1,tra-1 and mab-7 genes may induce the development of male and female reproductive system at the end of parasitism.The results in qRT-PCR and RNAi showed that some homologous genes(tra-3?fem-1)of C.elegans play similar roles in O.sinensis,although a few of genes are less important in sex determination,for example laf-1.Down-regulated the expression of laf-1 in C.elegans can induce individual death or feminization.This study has laid a foundation of sex determination in O.sinensis,and also provided a basis for the further study on the mechanism of sex differentiation determined by the nutrition in mermithidae. |
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