Expression And Regulation Of Bxy-mab-3 Genes In Pinewood Nematodes,bursaphelenchus Xylophilus

Bursaphelenchus xylophilus is a quarantine pest in worldwide and the causal agent of pine wilt disease,which has caused huge economic loss and ecological disaster in several countries,with particular respect in China.In different culturing conditions,the sexual ratio of female to male in B.xylophilus is obviously changeable.However,very few investigations were conducted and little is known about the molecular mechanism of sex determination in B.xylophilus.In Caenorhabtidis elegans,mab-3 is an important gene regulating the sex differentiation and determination,and this gene was highly conserved among different animal species.In the present thesis,the spatiotemporal expression pattern of Bxy-mab-3 and its biological functions in B.xylophilus was investigated.The results are as follows:1.The typically conserved domain of the MAB-3 family,which includes two DM motifs,was detected in Bxy-MAB-3,wherein the first amino-terminal DM domain was localized between amino acids 58 and 101,and the second one between amino acids 124 and 171.2.Whole-mount in situ hybridization showed that Bxy-mab-3 was stained in the juvenile stages J2,J3 and J4,as well as in male adults.However,no obvious staining was detected in eggs,neither in female adults.In J2,only several discrete stained points were detected in the intestine,whereas digoxigenin-labelled specific probes stained the whole intestine in J3 and in the bulk of the bodies of males and females in J4.However,the dark hybridization regions found in male adults were concentrated mainly on the spicules.3.The results of RNA interference showed that the body length and sperm size of male adults were significantly smaller than normal males(normal: 1149.33 ± 68.01 ?m in body length,3.69 ± 0.56 ?m2 in sperm cross-sectional area;after interference: 641.25 ± 97.44 ?m in males,1.98 ± 0.44 ?m2 in sperm cross-sectional area).In addition,behavioral tests showed that male adults could not mate successfully after being interfered by bxy-mab-3 gene.The interfered male could not find the vulva and is always mounted wrongly around the body of female,and the ratio of successive mate is significantly dropped compared with the normal male.Our results show that bxy-mab-3 gene is male specific and plays an important role in the spermatogenesis and male ontogeny of B.xylophilus,and can regulate the mating behavior of male adults.These results provide a basis to design some specific control measures at the molecular level in future.