拟松材线虫个体发育研究（英文）

Ontogenesis of pine parasitic nematode Bursaphelenchus mucronatus (Nematoda: Aphelenchoididae)

【目的】探索拟松材线虫(Bursaphelenchus mucronatus)的繁殖能力,并对其胚胎发育过程中经历的重要阶段和卵的形态变化进行研究,了解胚胎发育和完成整个生活史所需时间,为进一步研究其生长发育并进行有效防控提供参考。【方法】分别挑取3组180条拟松材线虫雌成虫,观察记录雌虫在25℃条件下的产卵情况,每隔2 h统计每组线虫的累积产卵量,直至卵的数量基本不再增加。挑取尚未产卵的拟松材线虫雌虫于载玻片上,待其产卵后,将卵置于蔡司体视显微镜下观察。连续观察胚胎的发育过程并使用照片记录不同发育阶段胚胎的形态变化,记录卵发育至不同阶段所需时间。挑取约200个刚产下的拟松材线虫卵,在25℃条件下发育24 h后每隔4 h统计其总孵化率,直至孵化数不再增加,设置3组重复。将刚孵化的2龄幼虫接种于灰葡萄孢(Botrytis cinerea)上,分为3组,每组设置3个重复,分别在接种1、2、3 d后使用贝尔曼漏斗法收集线虫,计算混合龄线虫中每龄期线虫所占比例,计算拟松材线虫胚后发育及完成整个生活史所需时间。【结果】①在拟松材线虫产卵能力方面,0~10 h拟松材线虫产卵总量增长较快,16 h后产卵量逐渐趋于稳定,28 h内雌虫平均累积产卵12粒/条。②拟松材线虫的胚胎发育过程主要经历以下几个关键阶段:单胞期、双胞期、3胞期、4胞期、5胞期、8胞期、16胞期、囊胚期、利马豆期、蝌蚪期、蠕虫期、1龄幼虫(J1),至孵化为2龄幼虫(J2)时结束。③在胚胎发育前期,第1次卵裂发生的位置存在两种情况,即卵的1/2和1/3处。双胞发育至5胞时也存在两种不同的发育方式,一种是双胞不移动直接分裂成3胞并列排列,另外一种是细胞进行移动,3胞呈三角形排列。通过观察30个卵的第1次卵裂和100个卵双胞的发育过程发现,这些不同的发育方式均是普遍存在的。④在25℃条件下,拟松材线虫卵的累积孵化率随时间增加而增加,在32 h时达到最高(93.31%),随后逐渐趋于稳定。⑤在25℃条件下记录了拟松材线虫卵从单胞发育至各个阶段的时间,完成整个胚胎发育过程需要约28 h。2龄幼虫接种于灰葡萄孢3 d后即可获得新的2龄幼虫,因此拟松材线虫完成整个生活史只需要3 d。【结论】对拟松材线虫卵从单胞阶段直至孵化的整个胚胎发育过程进行观察发现,拟松材线虫完成胚胎发育大约需要28 h,完成整个生活史需要3 d。对拟松材线虫产卵能力和卵的孵化率进行统计,收集拟松材线虫卵和2龄幼虫的最佳时间分别为16 h和36 h。拟松材线虫胚胎发育前期,在第1次卵裂期以及由双胞期发育至5胞期的两个过程中均存在两种与同属线虫不同的发育方式。这种现象还有待进一步研究。

【Objective】Explore the reproductive capacity, key stages and morphological changes of eggs in the embryonic development of Bursaphelenchus mucronatus. In addition, time required for the embryonic development and completion of the entire lifecycle were studied to provide a scientific basis for further study of its growth and development.【Method】A total of 180 female adult nematodes in three groups were selected to observe their behavior of egg-laying process at 25 ℃. The cumulative production of the eggs of each group of nematodes was counted every 2 hours until the number of eggs became constant. To observe the entire embryonic development process, the pregnant female nematodes in petri dishes were placed under a Zeiss stereo microscope, and the process of embryonic development was photographed. The time required from egg laying to each key stages of the B. mucronatus embryo was recorded at 25 ℃. Approximately 200 eggs of B. mucronatus were selected and their total hatching rates were calculated every 4 h after 24 hours of development at 25 ℃ until the number of hatchings no longer increased, in 3 sets of replicates. The newly hatched J2 were inoculated onto PDA plates containing the well-grown Botrytis cinerea. Then, on the day 1, 2 and 3 after inoculation of the J2, the nematodes were collected using the Berman funnel method, and the proportions of each age line were calculated to learn the time for nematodes to complete their entire lifecycle.【Result】① B. mucronatus has a good egg-laying ability. The total number of eggs increases rapidly within the first 10 hours. After 16 hours, the numbers of eggs laid gradually reached constant, with an average of 12 eggs/nematode in 28 hours. ② The development process of B. mucronatus embryos mainly comprised of the following key stages: single-cell stage, 2-cell stage, 3-cell stage, 4-cell stage, 5-cell stage, 8-cell stage, 16-cell stage, blastocyst stage, Lima bean stage, tadpole stage, worm stage, first-stage juveniles(J1) and the stage of second-generation juveniles(J2). ③ In the early stages of embryonic development, two types of depressions, i.e., one-half and one-third, occurred. In addition, two different developmental patterns were observed during the 2-cell stage to 5-cell stage. The first one is that two cells do not move but split directly into three cells;the other one is that the cells move to a form of triangle. It was revealed by observing the development of the first cleavage of 30 eggs and the development of the 2-cell stage of 100 eggs that these development patterns are common. ④ At 25 ℃, the cumulative hatching rate of B. mucronatus eggs increased with time, reaching 93.31% after 32 h, and then gradually leveled off. ⑤ The time required for the entire embryogenesis of B. mucronatus eggs from single cell to each of the key stages at 25 ℃ took about 28 hours. The J2 of B. mucronatus were able to generate new J2 after 3 days of inoculation with B. cinerea, and the entire life cycle of B. mucronatus completed in just 3 days.【Conclusion】 The entire embryogenesis process of B. mucronatus eggs from single cell to hatching took about 28 hours at 25 ℃. B. mucronatus can complete its entire lifecycle in just 3 days. The 16 hours are the best time to collect a large number of spawning eggs, and the 36 hours are the best time to collect J2. Further studies are worthwhile on the significance of each of the development stages in the early embryonic development of B. mucronatus.