| Lepidopteron pests like Helicoverpa armigera were harmful for variety of crops,leading to serious economic losses.The mermithid nematode Ovomermis sinensis has broad application prospect for biocontrol in farming and forestry pests,as its infective second-stage juveniles larval could actively seek and infect host haemolymph,and lastly kill the host to a large extent.However,the number and the distribution of this nematode were extremely limited,which hinder the control for the pest.So it was extremely urgent for mass rearing the nematode.The male and female sex ratio of O.sinensis can be used to regulate the size of this population's reproductive number.But so far,little was known for us about the sex differentiation mechanism of O.sinensis.Thus,studying on the factors which influence the sex ratio of the worm and the genes which related to sex differentiation were of significant meaning.It can not only laid the foundation for the research on sex differentiation mechanism of entomopathogenic mermithid nematodes,but also provide parameters for mass rearing and release of the natural enemy.To understand the key period of O.sinensis sex determination and sex differentiation,in this study we firstly used the stereomicroscope observing the morphological structure characteristics in the nematode development process from egg to mature adult.And found there was no obvious male and female characteristic appear during the period which developed from egg to second-stage larval and to the parasitism stage 4 day.But after the parasitism stage fourth-stage larval appeared sex differentiation distinctions in male and female body size(female was obvious bigger than males).And when they developed to adult,the different sex characteristics of orificium vaginae in female and copulatory spicules in male could observed clearly.So based on the result above we speculate sex determination and sex differentiation of O.sinensis occur during its parasitic stage.In order to explore the main physiology parameter(nutrient supply and absorb)which affect the sex ratio of O.sinensis,we respectively analyzed the parameter about parasite load,host size,host instar,host nutrients supply,and nematode nutrients absorb.The result shows that:(1)No males emerged when the parasite load is less than or equal to 5(fourth instar cotton bollworm larval,weight/length = 114.70 ± 9.43 mg/196±6.25 mm).Whereas when the parasite load added from 10 to more than 40,the sex ratio of male O.sinensis changed from 29.2 ± 3.2%to 100%,i.e.the male sex ratio increased as the parasite load added;(2)As the host size enlarge,the proportion of male O.sinensis decreased from 52.5 ± 4.1%to 15.2± 1.6%;(3)As the host added from 3 instar to 5 instar,the proportion of male O.sinensis decreased from 52.0 ± 4.3%to 29.2 ± 3.2%;(4)On the other hands,the parasitic period of males(7.5 ± 0.2 days)was significantly shorter than that of females(10.7 ± 0.5 days);(5)However,the degree of all above factors(parasite load,emergence time,host instar and host size)which affecting the sex ratio was significantly modified by restricting the host diet;(6)Host dietary restriction increased the proportion of males,and from the activity of trypsin and amylase in different parasite load group we also can summarize that each juvenile nematode which absorbed fewer nutrients tended to develop into males.Taken together,our findings suggest that factors impacting the O.sinensis sex ratio were related to host nutrients supply and nematode nutrients absorb.Based on above result,to ensure the genes which related with its sex ratio,we designed an experiment:transcriptome sequencing analysis(the genome was not known).Took the sex determination pathway of Caenorhabditis elegans as reference and through local blast,we screened out eight O.sinensis sex differentiation genes fox-1,laf-1,tra-3,tra-2,fem-1,tra-1,mab-3 and mab-7.Then analyzed the mRNA expression of the genes in different development period of this mermithid nematode when restrict the host diet(4h/day).We found that:(1)tra-3,fem-1 and mab-3 was significantly upregulated compared with the control group,while mab-7 was downregulated after restricting the host diet in 3 day.Based on the result,we supposed that the actively expression of tra-3 promote the fem-1 play role in sex determination pathway.Then though complex regulation,mab-3 and mab-7 expressed upregulate and downregulate respectively,which contribute to generate male nematode;(2)In 5 day,mab-3 expressed upregulate,whereas laf-1 and mab-7 downregulate.It was show that laf-1 may firstly began function in this time point.The same to the condition of 3 day,ultimately mab-3 and mab-7 expressed upregulate and downregulate respectively,and as well as increased the proportion of male;(3)In 7 day,only the expression of two gene laf-1 and mab-7 show the variant.We speculated that sex determination was nearly completed.The downregulate of mab-7 maybe related to male's some characteristic masculinization;(4)In post-parasitic period,the genes of fox-1 and tra-1 have evident expression distinguish.It is suggested that fox-1 and tra-1 may located in the downstream of O.sinensis sex differentiation translation pathway,and related with the gonad development;(5)In all development stage,tra-2 gene expression has no variant between the dietary restriction group and control group.The result show tra-2 was not referring to O.sinensis sex determination.Overall,in this study we firstly through observing the nematode morphological structure during its development process,and supposed sex determination period of O.sinensis occur in the parasitic stage.Furthermore,the O.sinensis sex ratio was related to host nutrients supply and nematode nutrients absorb by changing nutrients.Secondly,with transcriptome sequencing technology we obtained 8 genes which were related with sex differentiation.Lastly,to ascertain which genes really involved in sex determination of O.sinensis we compare the mRNA expression of the mermithid nematode in different development period between control group and dietary restriction group.This study stated that some environmental factors like nutrients affected the sex differentiation genes expression and finally made the effect to sex determination.The result not only laid the foundation for the research on sex differentiation mechanism of entomopathogenic mermithid nematodes,but also provided scientific parameter for the mass rearing,successfully breeding and releasing this natural enemy. |
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