|关键词||皱纹盘鲍 温度 盐度 Msap Dna甲基化|
|摘要||皱纹盘鲍（Haliotis discus hannai Ino），自然分布于我国的辽东及山东半岛，是最具经济价值的贝类养殖品种之一。自20世纪80年代以来，我国皱纹盘鲍养殖业发展迅猛，养殖区域由黄海北部逐步南扩至福建沿海、广东东部沿海等地。养殖范围的大幅扩增及“南北调养”模式的不断发展，使皱纹盘鲍面临环境变化的几率急剧增加。即使在同一海域，受暴雨、潮汐、大陆径流等影响，海水的温度、盐度等因子常处于大幅变动之中。温度和盐度是海洋中的重要环境因子，它们不仅限制海洋生物的地理分布和养殖范围，还影响其存活、生长、表观遗传修饰等重要生物学过程。本文以“97”选育群体为实验材料，系统研究了温度、盐度对皱纹盘鲍生长发育的影响，确立了各发育阶段的适宜温盐范围，以期为人工养殖提供生态学参考；同时还探索了不同温盐条件下幼鲍DNA甲基化的变化模式，旨在揭示皱纹盘鲍响应温盐胁迫的表观遗传学机制，并为其抗逆品种的培育提供表观遗传学依据。|
利用计算机辅助精子分析（computer-assisted sperm analysis，CASA），检测了不同盐度下皱纹盘鲍的精子活力；同时，以受精率为评价指标，确定了受精阶段的适宜温度、盐度范围。结果表明，盐度对皱纹盘鲍的精子活力具有极显著影响（P < 0.01）。5 ~ 50内，随着盐度的升高，皱纹盘鲍的精子活率、（A+B）级精子比例、直线运动速度、曲线运动速度和平均路径速度均呈现先升高后降低的趋势。综合上述指标，确定皱纹盘鲍精子的适宜盐度为26 ~ 40。该范围内，精子活率大于90%，（A+B）级精子比例大于85%，精子直线运动速度高于80 μm/s，曲线运动速度高于100 μm/s，平均路径速度高于95 μm/s。
温度、盐度对皱纹盘鲍受精率的影响极显著（P < 0.01）。14 ~ 28ºC内，皱纹盘鲍均可受精，20ºC时，受精率最高，为94.84%；28ºC时，受精率最低，为14.39%。20 ~ 24ºC内，受精率均在85%以上，为皱纹盘鲍受精的适宜温度。皱纹盘鲍受精的盐度上限为42，下限为22，对应的受精率分别为4.46%和1.59%。皱纹盘鲍受精阶段的适宜盐度为30 ~ 34，该范围内受精率为85.05 ~ 94.84%。
观测了不同温度下的胚胎发育进程，并记录受精卵发育至二细胞、四细胞、八细胞、纤毛环形成和孵化的时间。结果表明，温度影响皱纹盘鲍的胚胎发育速率。16 ~ 26ºC内，随着温度的升高，胚胎发育速率加快，孵化时间由18.19 h逐步缩短至8.72 h。通过拟合胚胎发育时间与温度的关系，得出研究所用皱纹盘鲍的生物学零度为6.98ºC。
以孵化率和畸形率为评价指标，研究了温度、盐度单一及组合效应对皱纹盘鲍胚胎发育的影响，并利用响应曲面法估测了胚胎发育的适宜温盐范围。单因子实验结果表明，胚胎发育的适宜温度为16 ~ 24ºC（32），适宜盐度为30 ~ 34（20ºC）。双因子实验结果表明，胚胎发育的最适温盐组合为16.5 ~ 22.5ºC和31 ~ 33，该条件下的孵化率 ≥ 95%，畸形率 ≤ 5%。双因素方差分析表明，温度、盐度及其组合效应对胚胎孵化率和畸形率均有极显著影响（P < 0.01），其中盐度效应强于温度效应。
采用室内受控方法研究了温度、盐度对皱纹盘鲍变态及变态幼体生长存活的影响。结果表明，水温对变态率、变态幼体存活率和生长率的影响极显著（P < 0.01）。14 ~ 28ºC内，随着水温的升高，幼体变态率、变态幼体存活率和生长率均呈现先升高后降低的趋势。24ºC时变态率最高，为86.05%；18ºC时，变态幼体存活率最高，为96.00%；26ºC时，投苗6d内的壳长、壳宽生长率最大，分别为48.49 μm/d和43.81 μm/d；投苗15d内的壳长、壳宽生长率最大，分别为68.06 μm/d和59.69 μm/d。
盐度对皱纹盘鲍变态率及变态幼体生长率的影响极显著（P < 0.01），对幼体存活率却无显著影响（P > 0.05）。22 ~ 40内，幼体变态率先升高后降低，于32达最大值，为79.23%；变态的盐度下限为22，该盐度下仅有2.21%的个体能够完成变态。26 ~ 40内，变态幼体存活率均高达85%以上；随着盐度的升高，变态幼体生长率先升高后降低。投苗6d内壳长、壳宽生长率均于34达最大值，分别为32.03 μm/d和25.20 μm/d；投苗15d内壳长、壳宽生长率分别于34和32达最大值，为43.03 μm/d和39.00 μm/d。
以存活率和特定生长率为评价指标，研究了温度、盐度单一及组合效应对皱纹盘鲍幼鲍的影响，确定了幼鲍阶段的最适温盐条件。单因子实验结果表明，幼鲍阶段的适宜温度为16 ~ 28ºC（32），存活的温度上限为30ºC；24ºC时生长最快，壳长、壳宽和湿重的特定生长率分别为0.83 %/d、0.79 %/d和2.47 %/d。幼鲍的适宜盐度为24 ~ 40（20ºC），盐度上、下限分别为44和18，对应的存活率分别为8.33%和11.67%。双因子实验结果显示，幼鲍的最适温盐组合为23 ~ 25ºC和30 ~ 36。双因素方差分析表明，温度、盐度及其组合效应对幼鲍的存活率和特定生长率均具有显著影响；其中，在存活率方面，盐度效应强于温度效应，而在特定生长率方面，温度效应强于盐度效应。
以不同温度（20ºC、24ºC、30ºC）和盐度（22、32、42）条件下培养的幼鲍为实验材料，利用MSAP（methylation-sensitive amplified polymorphism）技术进行DNA甲基化分析，探索了不同温度、盐度下幼鲍DNA甲基化的变化模式。MSAP分析表示，皱纹盘鲍幼鲍基因组DNA的总甲基化水平为33.19%，其中全甲基化水平为22.25%，半甲基化水平为10.95%。不同温度、盐度处理组间的甲基化水平无显著差异。通过对每个甲基化敏感多态性位点甲基化频率的检测，在温度、盐度处理组中各筛选出了67和63个甲基化差异位点。其中，低温组中筛选出的差异位点数为48个，高温组为20个；低盐组为40个，高盐组为33个。利用单标记分析法，检测了甲基化差异位点与表型性状的关联性。从温度、盐度处理组中各检测到23个甲基化位点与生长性状相关联。组间特征值分析表明，温度、盐度胁迫分别了引起了8.5%和6.6%的组间表观遗传差异；从分析图上可看出，不同温度、盐度处理组的表观遗传结构发生了明显分化。
|其他摘要||In China, Pacific abalone, Haliotis discus hannai Ino, is one of the most economically important molluscs, which naturally distributed along the coast of Liaodong and Shandong Peninsulas. Since the 1980s, the aquaculture of H. discus hannai Ino has developed rapidly and extended from the northern Yellow Sea to the coast of Fujian and Guangdong Provinces. With the expansion of farming area and development of north-south nursing mode, H. discus hannai Ino has been subjected to various environmental conditions. Even in the same sea area, the ambient factors, such as water temperature and salinity, are always in a state of flux due to heavy rains, tides, and continental runoff. For marine organisms, temperature and salinity are regarded as the most potent environmental factors, which not only limit the geographical distribution and farming area, but also exert significant impact on survival, growth, epigenetic modification, and so on. In this paper, with “97” selective breeding population as experimental material, we investigated the effects of temperature and salinity on growth and development of H. discus hannai Ino, and established the optimal rearing conditions for various developmental stages, in order to provide an ecological reference for farming practice. Furthermore, we also analyzed the DNA methylation alteration in abalone reared at different temperatures and salinities, expecting to reveal the epigenetic regulatory mechanism and provide an epigenetic basis for the cultivation of stress-resistant varieties.|
1. Effects of temperature and salinity on sperm motility and fertilization rate of H. discus hannai Ino
Sperm motility of H. discus hannai Ino under different salinities was measured using computer-assisted sperm analysis (CASA), and the suitable temperature and salinity ranges for fertilization were respectively determined by estimating the fertilization rate. The results showed that the salinity had significant influence on the sperm motility (P < 0.01). At 5-50, the sperm motility rate, percentage of (A+B) grade sperm, straight line velocity, curvilinear velocity, and average path velocity all rose at first and then declined. The suitable salinity range for H. discus hannai Ino sperm was 26-40, where the motility rate was greater than 90%, percentage of (A+B) grade sperm was greater than 85%, straight line velocity exceeded 80 μm/s, curvilinear velocity exceeded 100 μm/s, and average path velocity exceeded 95 μm/s.
Both temperature and salinity had significant influence on the fertilization rate of H. discus hannai Ino (P < 0.01). The eggs of H. discus hannai Ino could be fertilized at temperatures from 14 to 28ºC. The fertilization rate was highest at 20ºC and lowest at 28ºC, with values being 94.84% and 14.39%, respectively. The suitable temperature range was 20-24ºC, within which the fertilization rate was above 85%. The upper and lower salinity limits for fertilization were 42 and 22, respectively, with the fertilization rates being 4.46% and 1.59%. The suitable salinity range was 30-34, within which the fertilization rate ranged from 85.05% to 94.84%.
2. Effects of temperature and salinity on embryonic incubation of H. discus hannai Ino
To study the effect of temperature on the embryonic development process of H. discus hannai Ino, the timing of 1st cleavage, 2nd cleavage, 3rd cleavage, prototrochal girdle formation and hatchout at different temperatures was recorded. The results showed that the embryonic development rate of H. discus hannai Ino was affected by temperature. At 16-26ºC, the development rate increased with the rising of temperature, and the incubation period gradually reduced from 18.19 h to 8.72 h. By fitting the relationship of development time and temperature, we calculated the biological zero point (BZP) of H. discus hannai Ino to be 6.98ºC.
By estimating the hatching rate and malformation rate, we investigated the single and combined effects of temperature and salinity on embryonic incubation of H. discus hannai Ino, and determined the optimal conditions using response surface methodology. The single-factor data showed that the suitable temperature and salinity ranges for embryonic incubation were 16-24ºC (32) and 30-34 (20ºC), respectively. In the two-factor experiment, best embryonic incubation was observed at the combination of 16.5-22.5ºC and 31-33, with the hatching rate no less than 95% and malformation rate no more than 5%. Two-way ANOVA indicated that both hatching rate and malformation rate were significantly affected by temperature, salinity and their interaction (P < 0.01), and the salinity accounted for greater variance than temperature.
3. Effects of temperature and salinity on metamorphosis, post-larval survival and growth of H. discus hannai Ino
The effects of temperature and salinity on metamorphosis, post-larval survival and growth of H. discus hannai Ino were explored under indoor controlled conditions. The results showed that temperature significantly influenced the metamorphosis rate, post-larval survival and growth of H. discus hannai Ino (P < 0.01). At 14-28ºC, the metamorphosis rate, post-larval survival rate and growth rate all firstly increased and then decreased. Metamorphosis rate reached the peak at 24ºC with value of 86.05%. Best post-larval survival was obtained at 18ºC with value of 96.00%. 26ºC was the optimal water temperature for post-larval growth, at which the growth rates of shell length and shell width within 6 d after casting were respectively 48.49 μm/d and 43.81 μm/d, and the two growth rates within 15 d after casting were respectively 68.06 μm/d and 59.69 μm/d.
Salinity significantly affected the metamorphosis rate and post-larval growth (P < 0.01) rather than the post-larval survival (P > 0.05). At 22-40, the metamorphosis rate rose at first and then declined, and reached the peak at 32 with value of 79.23%. The lower salinity limit for metamorphosis was 22, at which only 2.21% of the individuals could complete the metamorphosis. At 26-40, the post-larval survival rate was above 85%. Within this range, the growth rate increased at first and then decreased. Maximum growth rates of shell length and shell width within 6 d after casting were obtained at 34, with the values respectively being 32.03 μm/d and 25.20 μm/d. The growth rates of shell length and shell width within 15 d after casting reached the peak at 34 and 32, respectively, which were 43.03 μm/d and 39.00 μm/d.
4. Effects of temperature and salinity on survival and growth of juvenile H. discus hannai Ino
The single and combined effects of temperature and salinity on survival and growth of juvenlie H. discus hannai Ino were simultaneously investigated, in order to determine the optimal farming conditions. The single-factor data showed that the suitable temperature range for juveniles was 16-28ºC at a constant salinity of 32, in which the survival rate was above 80%. The upper temperature limit for survival was 30ºC. Best specific growth rates of shell length, shell width and wet weight were obtained at 24ºC, which were 0.83 %/d, 0.79 %/d and 2.47 %/d, respectively. The suitable salinity range for juveniles was 24-40 at a constant temperature of 20ºC, and the upper and lower salinity limits were 44 and 18, respectively, with the survival rates being 8.33% and 11.67%. In the two-factor experiment, best survival and growth were observed at the combination of 23-25ºC and 30-36. Two-way ANOVA indicated that the survival and growth of juveniles were significantly influenced by temperature, salinity, and their interaction. Salinity accounted for greater variance than temperature in survival, while it was reverse in growth.
5. Effects of temperature and salinity on DNA methylation of juvenile H. discus hannai Ino
To explore environment-induced changes in DNA methylation, MSAP (methylation-sensitive amplified polymorphism) analysis was performed on juveniles reared at various temperature (20, 24 and 30ºC) and salinity treatments (22, 32 and 42). Results showed that the total methylation level in juvenile H. discus hannai Ino was 33.19%, with the full methylation level being 22.25% and hemi methylation level being 10.95%. There was no significant difference in the methylation level attributable to temperature or salinity. Based on the test of difference in methylation frequency at each methylation-susceptible polymorphism locus, we screened out 67 and 63 differentially methylated loci in temperature and salinity treatments, respectively. Among these loci, 48 loci were detected in 20ºC treatment, 20 in 30ºC treatment, 40 in 22 treatment and 33 in 42 treatment. Using the single marker analysis, we tested the relevance of differentially methylated loci and phenotypic traits, and obtained that 23 loci were associated with the growth-related traits in temperature and salinity treatments, respectively. The Between-group Eigen Analysis (BPCA) based on methylation profiles suggested that 8.5% and 6.6% of epigenetic variation was structured into between-group component when stimulated by temperature and salinity, respectively. The BPCA plots also showed obvious epigenetic differentiation in juvenile H. discus hannai Ino reared at different temperatures and salinities.
|孔宁. 温度、盐度对皱纹盘鲍“97”选群生长发育的影响[D]. 北京. 中国科学院大学,2016.|