| A series of experiments were conducted to investigate the effects of temperature on physiology, immunity, metabolite and genetic traits in thermotolerant strain of sea cucumber Apostichopus japonicus. The primary results were listed below.1 Effect of desiccation in the shade on in vitro maturation and development of Apostichopus japonicus oocytes bathed in gonad juiceTechniques for in vitro maturation of oocytes are important for studying the maturation regulatory mechanism, embryonic development and differentiation. In the present study, after gonads of Apostichopus japonicus were artificially anatomized, oocytes extracted from the gonads were bathed in gonad juice and desiccated in the shade. The effects of three different desiccation temperatures (17℃, 21℃ and 25℃) on oocyte maturation and later development of A. japonicus were studied. The results showed that, after desiccation in the shade, the follicular membranes surrounding the majority of oocytes were successfully stripped away. Most of oocytes with germinal vesicle breakdown (GVBD) could be fertilized and then sequentially developed into the blastula stage, gastrul stage, and auricularia stage. There were no significant differences in GVBDRs and the percentages of individuals in the blastula, gastrula and auricularia stages between 17℃ and 21℃. The GVBDRs after 2 h of desiccation in the shade came close to the maximum (17℃:92.6-98.6%,21℃:91.1-97.1%). After 2.5 h of desiccation in the shade, the percentage of blastula were close to maximum (17℃:87.2-97.4%,21℃: 80.7-95.4%). The percentage of gastrula (17℃:83.5-87.8%,21℃:85.8-88.3%) and auricularia (17℃:56.2%,21℃:59.6-64.5%) both came close to maximum from 2.5h to 3.5h of desiccation. When the temperature was 25℃, the GVBDRs after 3 h of desiccation in the shade was close to maximum (66.3-78.5%). The percentages of blastula, gastrula and auricularia were 53.0-61.0%,44.4-59.9% and 31.0-38.3% after 2.5-3.5 h of desiccation, respectively. The results provided a simple methods for in vitro maturation and development of oocytes in research and aquaculture of A. japonicus.2 Effect of High Temperature Stress on the Fertility of Male and Female Gametes of the Sea Cucumber Apostichopus japonicusThe effect of high temperature on Apostichopus japonicus gametes were evaluated. Gametes were exposed to control temperature (18℃) or one of six high temperature treatments (26℃,27℃,28℃,29℃,30℃ and 31℃) and the cleavage rate (CR) of fertilized eggs was measured as an indication of fertilization ability. At control temperatures (18℃), the male and female gametes had high fertilization ability (CR≥ 94.97%) in 5 h and the time at which 50% of gametes lost the ability to successfully fertilize (LT50) was 7.03 h. All gametes were non-viable after 9 h. When male and female gametes were both exposed to high temperature, the LT50 was lower than when male or female gametes were treated individually (P< 0.05). The LT50 was lower for male gametes treated alone than for female gametes treated alone. In conclusion, exposure to high temperature results in a decline in fertilization ability of A. japonicus gametes, and the higher the temperature was, the faster the declining rate of gamete fertility was. The female gametes were more resistant to high temperatures than male gametes.3 Differences in non-specific immunity, metabolic enzymes, metabolites and Hsp70 gene expression in non-aestivating and aestivating Apostichopus japonicusActive and eating Apostichopus japonicus (wet weight> 150 g/ind.) were selected during the aestivation period (29.7-17.6℃) as the non-aestivating group, and those in aestivation status were selected as the aestivating group. Differences in enzyme activities related to non-specific immunity (superoxide dismutase, catalase, and total antioxidant capacity), metabolism (hexokinase, pyruvate kinase, malate dehydrogenase, and lactate dehydrogenase) of somatic muscle tissue, intestinal metabolites and Hsp70 gene expression in respiratory tree of the two groups were compared. The results showed that temperature had a significant effect on non-specific immuno-enzyme activities in somatic tissues (P< 0.05), and that these activities were significantly higher in the non-aestivation group than those in the aestivation group at the early stage when water temperature was relatively higher (P < 0.05). However, these differences were not significant at the later stage when water temperature was relatively lower. Temperature had a significant effect on metabolic enzyme activities in the non-aestivation group (P< 0.05), whereas the effects of temperature on pyruvate kinase and lactate dehydrogenase activities in the aestivation group were not significant. Metabolic enzyme activities were significantly higher at the early stage in the non-aestivation group than those in the aestivation group, but the difference was not significant at the later stage. Glutamate, 3-aminoisobutyrate, aspartate, choline, phosphocholine, and taurine were abundant metabolites in the aestivation group, whereas 13 types of amino acids, including branched-chain amino acids, dimethylamine, glucose, glycogen, inosine, and homarine were abundant in the non-aestivation group. Hsp70 gene expression in aestivation group was significant higher than in non-aestivation group. The results above indicate that non-aestivating A. japonicus had higher non-specific immuno-enzyme and metabolic activities, vigorous metabolism, and stronger thermostable performance during the high-temperature season than those aestivating A. japonicus. These individuals represent a potential high-quality parental source of high-temperature-resistant strains for A. japonicus breeding.4 Effects of early thermal stress on the heat tolerance of Apostichopus japonicus thermotolerant strainThe F3 generations blastula (HF3-E), early auricularia (HF3-S), middle auricularia (HF3-M), late auricularia (HF3-L) and young sea cucumber (HF3-Y) were treated with 28℃, and the survival rate of every development stage was controlled in 40-50%. The Apostichopus japonieus populations that were not treated with heat stress thermotolerant strains F3 (F3) and the normal group (C) were used for comparison. The defferences of the survival rate and the Hsp70mRNA expression under high temperature stress were compared after 7 months. The results showed that under the temperature range of 30-32.7℃, the highest survival rate was found for group HF3-Y (55 ±7.1%), the lowest for group C (7.5± 6.5%), and the survival rate of every group declined as the following order:HF3-Y> HF3-L> HF3-S> HF3-E> HF3-M> F3> C. The survival rates of group C was significant lower than that of other groups (P<0.05), and the survival rates in group HF3-L and HF3-Y were significant higher than in other groups (P<0.05). Under the temperature of 28℃, the expression of Hsp70mRNA among different groups declined as: HF3-Y>HF3-S>HF3-M>HF3-L>F3>HF3-E>C. The Hsp70mRNA expression among group HF3-Y, HF3-S and HF3-M were not significantly different(P> 0.05), but significantly higher than that of group F3 and HF3-E (P< 0.05), and extremely significantly higher than that of group C (P< 0.001). To sum up, in the process of implementation of high-temperature tolerance Apostichopus japonieus populations breeding, the stage of young individuals can obtain more effective heat elimination select breeding effect.5 The efffects of high temperature stress on several immune enzyme activities of Apostichopus japonicus thermotolerant strain and normal speciesIn this experiment, one-year-old juveniles (13.6±1.8g) of the directive breeding population (F3 group) and wild population (C group) were used as the experimental animals. High temperature as in ponds in summer was imitated (25,27,29,31,32 and 33℃, with 0.5℃ increasing every 12h). The activities of superoxide dismutase (SOD), catalase (CAT), total antioxidant capacity (T-AOC), acid phosphatase (ACP), alkaline phosphatase (AKP) and lysozyme (LSZ) of A. japonicus(13.6±1.8g/ind.) in directive breeding population(F3 group) and wild population(C group) were determined and compared after 12h thermal stimulus at different temperatures. The results showed that:the temperature has significant influence on SOD, CAT, T-AOC, ACP, AKP and LSZ activities. The SOD activities of group F3 and group C both showed a upward trend firstly and then declined as the temperature raised from 25℃ to 33℃, and the activity of SOD in group F3 was significantly higher than group C (P<0.05) at 31 and 32℃. The CAT and T-AOC activities both showed downtrend in two groups as temperature raised from 25℃ to 33℃. The CAT activities in group F3 was higher than group C at every temperature, and the difference was significant when the temperature were 27℃,29℃,31℃ and 33℃ (P<0.05), respectively. The T-AOC activity in group F3 was significantly higher than group C (P<0.05) at temperature range from 25℃ to 33℃. The ACP and AKP activities in two groups both declined as temperature raised, the ACP activity in group F3 was significantly higher than group C (P<0.05), except at 31℃; the AKP activity in group F3 was significantly higher than group C (P<0.05) when the temperature were 25℃,27℃ and 31℃. The LSZ activity showed a upward trend firstly and then declined as the temperature raised in two groups, and the activity in group F3 was significant higher than group C at 27℃,29℃ and 31℃. The results above indicated that the the immune capacities of A. japonicus thermotolerant strain to high temperature stress has been improved after three generations directive breeding. This study will provide useful information for further research on selective breeding of A. japonicus.6 Comparison of feeding capacity and tolerance to high temperature between Apostichopus japonicus thermotolerant strain and normal speciesThe feeding capacity of thermotolerant strain F2 (small scale:13.64±2.72g/ind., large scale:42.42±7.14 g/ind.) and F3 (small scale:13.17±1.67g/ind., large scale:27.60±3.83 g/ind.) during aestivation were monitored, which was compared with normal species to evaluate the tolerance to high temperature. The results showed that, when the rate of feeding capacity to body weight (Rfb) was between 0.003 to 0.006, the capability of small scale in F2 group to high temperature was increased for 0.69-1.19 ℃ than normal species, and large scale in F2 group was 0.77-1.00℃ higher than normal species. The capability of small scale in F3 group to high temperature was increased for 1.07-1.38℃, and large scale was 0.96-1.30℃ higher than normal species. After many generations of directional breeding of high temperature resistance, the tolerance to high temperature of A.japonicus was heritable and significantly improved in the offspring, which indicated that the effect of high temperature resistant breeding is obvious.7 Influence of genetic factors on growth and heat-tolerance traits in the sea cucumber Apostichopus japonicasFast-growing and heat-tolerance strain families of the sea cucumber Apostichopus japonicus were created through the incomplete diallel cross hybrid design; then the influence of parental genetic factors on growth and heat-tolerance traits in sea cucumber was investigated, and genetic parameters of growth and heat-tolerance traits were estimated. Results showed that paternal genetic influence on growth trait was significantly higher than that of maternal, while maternal genetic influence on heat tolerance was higher than patrilineal. The heritability of body weight, growth rate, individual difference rate, and survival rate in high temperature for A. japonicus were 45.45%,31.68%,33.61%, and 23.56%, respectively. The genetic correlation between body weight and survival rate in high temperature was significantly positive, reaching 75%. Based on the combining ability of traits and selection of family phenotype, three parent and six offspring families were selected as strain resources of sea cucumber. Also, our results indicated that A. japonicus might conduct a natural selection through the accumulation of heat-tolerant additive genetic effects in offsprings and adapted to the ambient environment changed by the global warming climate. |
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