| The pathogen of pine wilt disease—B.xylophilus is an invasive species with the potential to cause severe damage to forests worldwide,and the pathogenic nematode is also listed as a first-class hazardous forestry pest in China and cause a serious threat to our country’s forest ecological security.In addition to the widespread host,vector insects,and existing prevention and control measures that cannot meet the demand,the reproductive ability of pine wood nematode is an important factor affecting its pathogenicity,with the female to male ratio（sex ratio）being an important factor in determining the reproductive ability of the population.Therefore,analyzing the sex ratio of pine wood nematode and its regulation mechanism are useful to elucidate the invasion and spreading mechanism of pine wood nematode from a reproductive biology perspective.Sex ratio is a core concept of modern evolutionary ecology.In addition to elucidating relationships among the sexual selection,population dynamics,and evolutionary trajectories,it is also the focal point of adaptive allocation strategies in which individuals match current and expected fitness payoffs through the adjustments to their relative investment in males and females.This thesis focusing on the effects of different environmental factors on the sex ratio of pine wood nematode offspring and the function of sex determination-related genes.The main research results include the following aspects:1.In the samples collected from different regions,the sex ratio of pinewood nematode varied from 0.95 to 2.20,indicating that pine wood nematodes may adjust the sex ratio of their offspring to adapt to environmental changes.How does it adapt to environmental changes and what factors influence it.Firstly,simulations were carried out in the laboratory,The sex ratios at different stages were calculated and found to remain at about 1.90 constantly at the third larval stage（L3）,fourth larval stage（L4）and adult,indicating a female bias.Second larval stage（L2）were incubated for48,56,72 and 96 h:at 48 and 56 h,male larvae developed into male adults at a higher rate than female larvae into female adults;while at 72 and 96 h,male and female larvae developed into adults at the same rate.In order to facilitate statistics and to avoid the effects of different rates of adult development of male and female larvae,the following experiments were conducted using the adult sex ratio of the pine wood nematode as the subject of the study.2.Effects of temperature,population density,parental age and nutrition on sex ratio,growth and development of pine wood nematode were investigated.L2 was inoculated with Botrytis cinerea after 8 d of treatment at different low temperatures:0°C,4°C and 10°C.The results showed that there was no difference in the size,hatching rate,and sex ratio of the offspring treated with different temperatures（when the eggs were collected and cultured to adults,the same below）,and the population numbers after 7 days were also not significantly different;As the population density increased（2,000,10,000,50,000）,the parental size,the eggs size and number of offspring decreased,but the offspring sex ratios did not change and were approximately 1.90;Changes in maternal age also had no effect on offspring sex ratios,which were 1.93±0.08,1.93±0.06,1.94±0.06 and 1.84±0.04 for adult at 1st,3rd,5th and 7th d,respectively.Changes in pine wood nematode development and sex ratio were counted by inoculating L2onto B.cinerea with mycelial growth concentrations of 1*PDA,1/4*PDA and 1/16*PDA,and found that parental length,width and volume of pine wood nematodes feeding on different concentrations of PDA-grown mycelium decreased significantly,with the percentage size decrease was larger in females than in males（1*PDA cf.1/16*PDA,females decreased 53.9%,males41.0%）.However,there are significant differences in the sex ratio,the average number of offspring produced by each female and the size of eggs in the offspring of different treatments:the sex ratio was 1.85±0.05a,1.67±0.04 ab and 1.41±0.05b,respectively;the number was 44.4±3.23a,44.44±3.10a,and 15±0.44b,respectively;the size of the eggs decreased gradually to16292.6±178.2μm3,15144.2±15144.2μm3,and 14114.8±134.4μm3,respectively;In order to exclude that the change in the sex ratio was caused by differences in survival rates,further studies of survival rates from egg to adult stage of the treatments showed that the differences in survival rates were not significant,at 66.06%,66.44%and 64.09%respectively.Therefore,it was hypothesized that PDA concentration influenced the growth of B.cinerea and thus the change in sex ratio of pine wood nematode progeny,and that this change was caused by the change in the prime sex ratio.3.In order to determine whether the adult sex ratio of pine nematode is caused by the changes in the prime sex ratio,the screening of sex-specific tags was carried out.Screening for female-or male-specific tags by RAPD and re-sequencing.By RAPD amplification of the male and female genomes,a total of 5624 primary bands were amplified from the 1200 random primers,but no specific tags were screened;further analysis by re-sequencing the genomes of male and female,and 80 differential fragments of males and females were selected from the sequencing results for verification,and no specific tags were found in the genomes of male and female.4.To further investigate the mechanism of sex determination in pine wood nematodes,the genes Bx-sex-1 and Bx-tra-1 were cloned and analyzed for their biological functions.The results showed that the relative expression of Bx-sex-1 was highest in the egg stage,lowest in the L2 stage and gradually increased from L2 to adult.q RT-PCR showed that Bx-sex-1 was a single copy in both female and male genomes.After Bx-sex-1 was disturbed,the hatching rate of eggs decreased and the sex ratio was reduced.Thus,it is suggested that the Bx-sex-1 gene of the pine wood nematode may have an important role in egg development and sex determination.The results of Bx-tra-1 gene expression analysis,fluorescent quantitative PCR and western blot all showed that Bx-tra-1 gene expression was highest in the L2 period.Immunohistochemical staining showed that TRA-1 protein was highly expressed in the intestine and tail of L2.The Bx-tra-1 gene of pine wood nematode may play an important role in sex determination and differentiation in L2 stage.In summary,low temperature treatment L2,different population density and maternal age did not affect the sex ratio of the offspring.Different nutritional conditions were important factors affecting the sex ratio of the offspring.It is speculated that the change of the offspring’s female-male ratio is caused by the change of the prime sex ratio.The sex-determining system and sex-determining genes may have an impact on the prime sex ratio.RAPD amplification and resequencing of male and female genomes of pine wood nematode found no specific tags for male and female worms.In the molecular mechanism of sex determination,the sex determination-related gene Bx-sex-1 plays an important role in the development of eggs and sex determination,while Bx-tra-1 plays an important role in sex determination and differentiation in the L2 period. |
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