松材线虫与拟松材线虫种间不对称性引诱及分子基础

The same allelochemical could play distinct roles for different nematode species or for different sexes from the same species. It is very common in nature that Bursaphelenehus xylophilus and Bursaphelenehus mucronatus live in xylem of the same tree. It’s therefore fascinating and valuable to uncover the interspecific functions and regulation principles of allelochemicals between B. xylophilus and B. mucronatus. Our previous findings showed that there is obvious asymmetric sexual attraction between B. xylophilus and B. mucronatus, i.e., male B. xylophilus can be attracted by female B. mucronatus leading to interspecies mating. However, male B. mucronatus is usually attracted by female B. mucronatus, not by male B. xylophilus. This asymmetric sexual attraction between B. xylophilus and B. mucronatus might play important role during the course of biological invasion of B. xylophilus. Based on the previous observations, this proposal will investigate the interspecific sexual attracting behaviours between B. xylophilus and B. mucronatus in detail, and quantitatively analyze the difference of interspecific attraction between these two species. Studies in chemical ecology will also be conducted to identify the key active compounds of interspecific sexual pheromones by HPLC, NMR and behaviour tests. In addition, sexual pheromone receptors in both species will be identified at gene and protein levels, and their potential interact networks will be constructed in terms of the concept and tools of comparative genomics, proteomics and bioinformatics. The findings of this proposal will not only shed light on inter-specific interaction between B. xylophilus and B. mucronatus, but also provide the direct evidence of unbalanced sexual attraction at molecular level, which could contribute to the better understanding of biological invasion of B. xylophilus, as well as deliver the theoretical principles to design specific agents against pine wilt disease in future.

自然条件下，松材线虫与拟松材线虫的共栖现象比较普遍。已有研究结果表明，同一信息物质对不同线虫种类以及同种线虫的不同性别个体具有不同的调节功能。前期观察发现，松材线虫与拟松材线虫存在明显的种间不对称性引诱。本项目拟在前期观察的基础上，系统研究松材线虫和拟松材线虫的种间性引诱行为，定量分析其种间性引诱效率差异，明确两种线虫种间性引诱的不对称性；从化学生态学的角度，分析两种线虫种间性引诱的关键物质组分和浓度范围，并开展行为测定，阐明种间不对称性引诱的信息物质基础；采用生物信息学和比较基因组学预测两种线虫的性信息素受体基因，并利用蛋白组学分析两种线虫的性信息素受体蛋白，双向确定其性信息素信号受体，阐明种间不对称性引诱的信号交流分子基础。本研究结果不仅可以阐明两种线虫的种间互作关系，也能提供直接的分子证据，为进一步揭示松材线虫的入侵定殖机制奠定基础，同时为探索特异性的松材线虫病防控措施提供新的思路。

本项目系统地研究了松材线虫与拟松材线虫的种间不对称性引诱行为及分子机制。研究结果表明，当1对处女松材线虫成虫在1条雌或雄处女拟松材线虫干扰下，交配成功率没有显著变化；当加入2条雌或雄处女拟松材线虫，松材线虫交配成功率受到显著影响，分别下降至63.72%和25.64%；当加入3条雌或雄处女拟松材线虫，松材线虫交配率受到极强的干扰，无法完成交配，交配成功率接近于0。反之亦然，当1对处女拟松材线虫成虫在1条雌或雄处女拟松材线虫干扰下，交配成功率就会显著下降，分别为84.58%和76.35%；当加入2条雌或雄处女松材线虫，拟松材线虫交配成功率受到极大影响，分别下降到28.26%和16.34%；当加入3条雌或雄处女松材线虫，拟松材线虫无法完成交配，即交配机会被对方剥夺。这表明，两种线虫之间存在激烈的交配干扰和种间竞争。.研究发现，松材线虫和拟松材线虫的性信息素均为水溶性物质。通过C18固相萃取柱对两种线虫的信息素水溶液进行洗脱，用50%甲醇水溶液萃取后，用双蒸水去盐，再用500mM KCl阴离子交换柱萃取，收集性到信息素纯物质，之后进行核磁共振分析，确定各组分的化学结构均为大分子蛔苷类物质，且与模式线虫秀丽隐杆线虫的性信息素物质为同类物质。.本项目对松材线虫与拟松材线虫进行了三代Pacbio sequel基因组测序，确定松材线虫基因组scaffold N50的长度为4.098 Mb，总长为76.32 Mb，基因预测的数量是11059个；拟松材线虫样品的 scaffold N50的长度为1.27 Mb，组装大小为80.39 Mb，基因预测的数量是10,758个。从两个基因组中预测到的候选性信息素受体基因共25个，并对这些基因在两种线虫个体发育和繁殖行为中的调控功能进行了研究，发现Bxy11712和Bm9839基因在两种线虫的个体发和交配行为过程中发挥重要的调控功能，另外，Bxy3407与Bm9839、Bxy2120与Bm14865、Bxy11712与Bm2872三对同源基因在松材线虫和拟松材线虫存在种间特异性或雌雄差异性。本研究结果不仅可以阐明两种线虫的种间竞争关系，提供直接的分子证据，也能够为进一步揭示松材线虫的入侵定殖分子机制奠定基础，同时为探索特异性的松材线虫病防控措施提供新的思路。