| 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 1980 s, 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 InoSperm 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 InoTo 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 twofactor 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 InoThe 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, postlarval 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 InoThe 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 InoTo 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. |
PDF Full Text Request