刺参养殖池塘围隔育苗的基础生态学研究
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摘要
本文调查了刺参养殖池塘的理化和生物状况，筛选出两株有益池塘水质调控的亚硝酸盐氧化细菌；查明了温度、盐度和规格对刺参能量收支的影响，研究了刺参围隔育苗的技术原理；优化出池塘投苗放养的“温度、盐度和规格”适宜组合，初步建立了“工厂化产卵与孵化——池塘围隔培育”的生态育苗模式。研究结果对于刺参健康苗种培育相关理论完善、技术提升和产业化应用具有重要的参考价值。主要研究结果如下：
1.自2010年12月，对大连金砣食品有限公司刺参养殖池塘的生境特征进行了为期一年的调查。结果发现：虽然池塘的温幅和盐幅较为宽广，但变化较为缓慢，受地理纬度影响，刺参养殖池塘的水温与盐度呈负相关；pH稳定于7.9~8.2之间；COD低于10mg/L；透明度较高，营养盐含量偏低；浮游生物小型化趋势明显，其中浮游植物以硅藻占优势，而浮游动物以桡足类占优势。
2.刺参养殖池塘异养菌与硝化细菌的数量受温度影响呈季节性变化。夏、秋季较高，为152.5~214.6×10~3cfu/g；冬季较低，为36×10~3cfu/g。通过富集、分离和纯化，从高产刺参养殖池塘的底泥沉积物中筛选出两株高效亚硝化菌株，即亚硝酸盐氧化菌OH1和OB6，革兰氏染色均呈阴性；在基于16SrDNA序列比对构建的系统发育树中，OH1与Cobetia marina DSM4741(T)相似性达99.6%；OB6与Halomonas ventosae Al12(T)相似性达99.8%；两菌株OH1和OB6的24h亚硝酸降解率分别达99.6%和99.8%。
3.于2011年6至11月，采用陆基围隔法，通过搅底、施用微生态制剂、培养底栖硅藻、换水等调控措施对刺参幼、稚参进行生态育苗实验。结果表明：在围隔中刺参幼体由小耳期发育到中耳期，变态率为50%；发育到大耳期，变态率为30%；稚参投苗2d后的存活率为1.75%，实验结束后的存活率为19%，且质量由5g/m~3增加到37.9g/m~3，增重近8倍。
4.刺参养殖池塘的水温与盐度的波动较大，全凭经验选择投苗时间，产量极不稳定。本文以正交试验法，研究了水温、盐度及规格对池塘养殖刺参能量收支的影响。结果表明：水温、盐度及规格对刺参能量收支影响显著，其中盐度与规格是刺参特定增长率的主要影响因子；并优化出不同规格刺参用于生长最适温盐条件。根据实验结果，在每年春、秋季，当池塘水温与盐度分别达16°C和30时，放养湿重为37.34±4.63g的刺参，摄食生长效果最佳。
Based on aquaculture ecology theory of sea cucumber Apostichopus japonicusSelenka, community structure and seasonal variation of phytoplankton andzooplankton were investigated. The abundance of heterotrophic bacteria and nitrifyingbacteria were also investigated, and then two strains with high degradation ofnitrite-oxidizing bacteria were isolated from sediment of A. japonicus farming ponds.Furthermore, a preliminary study of ecological breeding of A. japonicus was carriedout in pond by using of enclosures on a basis of its ecological environment. Then weresearched and analyzed for feasibility of this new method. Meanwhile, the combinedinfluence of water temperature, salinity and body size on energy budget was alsostudied in the sea cucumber A. japonicus. These results could be used to optimize theconditions of sea cucumber ranching, improving our understanding ofphysioecological regulation and improving pond aquaculture management. Mainresults are as follow:
1. In order to improve efficiency of ecological breeding of A. japonicus andunderstand the habitat of farming ponds, we investigated the characteristics ofplankton community and abiotic factors of ponds and enclosures from Dec.,2010toNov.,2011. The results showed that water temperature was negative correlation withsalinity, and both had a wide range changing gradually. In conditions of watertemperature ranging from-2.5to28.8℃, salinity changing from22to33practicalsalinity unit, pH7.9~8.2, transparency getting above70cm and COD getting below10mg/L, phytoplankton and zooplankton in ponds comprised31and24species,respectively, and they tended to be miniaturization.
2. Heterotrophic bacteria and nitrifying bacteria were152.5~214.6×103cfu/gfrom spring to autumn, while about36×103cfu/g in winter. Nitrogen accumulationis a serious concern in aquaculture. Among other forms of nitrogen, nitrite is more harmful because of its low transformation rate and high toxicity. In order to isolategood strains with high decomposition ability, stability and safety, several strains wereisolated from sediment of the sea cucumber A. japonicus (Selenka) ponds using nitriteoxidizing bacteria (NOB) enrichment culture and screening techniques. The nitriteremoval efficiency of two strains, named as OH1and OB6, were up to99.6%and99.8%respectively with in24h. Total DNAs were extracted and specific primers for16S rDNA of nitrite oxidizer were used to conduct the amplification of polymerasechain reaction (PCR). Amplified product was analyzed by agarose gel electrophoresisand Sanger sequencing. Homology analysis was made by searching in Genbank andEzTaxon server. The amplified fragment range of OH1and OB6was between1300and1450bp.99.6%of OH1nucleotides were identical with a conserved fragment ofa standard strain Cobetia marina DSM4741(T), while99.8%of OB6nucleotides wereidentical with a conserved fragment of a standard strain Halomonas ventosae Al12(T).It is therefore that the nitrite oxidizing bacteria OH1and OB6isolated from the seacucumber ponds were primarily identified to be Cobetia marina DSM4741(T) andHalomonas ventosaeAl12(T).
3. The ecological breeding of larval and juvenile sea cucumber A. japonicusSelenka was studied in experimental enclosures placed in ponds of Dalian Gold ToneSeafood Co., Ltd., Liaoning Province from Jun. to Nov.,2011, with theenclosure-experimental method, by using of pond stirring, probiotics, culture benthicdiatoms and renew the water of enclosure. The results showed that the metamorphosisrates of middle and late auricularia larvae were50%and30%, respectively. Thesurvival rate of juvenile was1.75%in2days, and the survival rate was19%in70days. Finally, the average output was increased from50g/m~3to37.9g/m~3, whichmeans almost8times of the initial weight. Our study indicated that the ecologicalbreeding of larval and juvenile sea cucumber A. japonicus Selenka by using ofenclosures placed in ponds is practicable.
4. The worldwide demand for sea cucumber is outpacing the sustainable harvestcapabilities of fisheries. Sea cucumber ranches and farms are striving to supplementwild harvest, but variable temperature and salinity in pond culture systems make seacucumber production challenging. In this study, we evaluated how water temperature,salinity and body size affected the energy budget of pond-cultured sea cucumber A.japonicus (Selenka). An orthogonal design was used to identify the most suitableconditions for energy consumption and scope for growth (SFG). After the42-day experimental period, we found that water temperature, salinity and body sizesignificantly influenced energy consumption, while salinity and body size were themain influencing factors on SFG. Based on these results, a water temperature of16°Cand a salinity of30are optimal conditions for stocking sea cucumber A. japonicuswith a body size of37.34±4.63g. As such, the optimum stocking seasons for seacucumber A. japonicus may be April in the spring and October in the autumn.
1.自2010年12月，对大连金砣食品有限公司刺参养殖池塘的生境特征进行了为期一年的调查。结果发现：虽然池塘的温幅和盐幅较为宽广，但变化较为缓慢，受地理纬度影响，刺参养殖池塘的水温与盐度呈负相关；pH稳定于7.9~8.2之间；COD低于10mg/L；透明度较高，营养盐含量偏低；浮游生物小型化趋势明显，其中浮游植物以硅藻占优势，而浮游动物以桡足类占优势。
2.刺参养殖池塘异养菌与硝化细菌的数量受温度影响呈季节性变化。夏、秋季较高，为152.5~214.6×10~3cfu/g；冬季较低，为36×10~3cfu/g。通过富集、分离和纯化，从高产刺参养殖池塘的底泥沉积物中筛选出两株高效亚硝化菌株，即亚硝酸盐氧化菌OH1和OB6，革兰氏染色均呈阴性；在基于16SrDNA序列比对构建的系统发育树中，OH1与Cobetia marina DSM4741(T)相似性达99.6%；OB6与Halomonas ventosae Al12(T)相似性达99.8%；两菌株OH1和OB6的24h亚硝酸降解率分别达99.6%和99.8%。
3.于2011年6至11月，采用陆基围隔法，通过搅底、施用微生态制剂、培养底栖硅藻、换水等调控措施对刺参幼、稚参进行生态育苗实验。结果表明：在围隔中刺参幼体由小耳期发育到中耳期，变态率为50%；发育到大耳期，变态率为30%；稚参投苗2d后的存活率为1.75%，实验结束后的存活率为19%，且质量由5g/m~3增加到37.9g/m~3，增重近8倍。
4.刺参养殖池塘的水温与盐度的波动较大，全凭经验选择投苗时间，产量极不稳定。本文以正交试验法，研究了水温、盐度及规格对池塘养殖刺参能量收支的影响。结果表明：水温、盐度及规格对刺参能量收支影响显著，其中盐度与规格是刺参特定增长率的主要影响因子；并优化出不同规格刺参用于生长最适温盐条件。根据实验结果，在每年春、秋季，当池塘水温与盐度分别达16°C和30时，放养湿重为37.34±4.63g的刺参，摄食生长效果最佳。
Based on aquaculture ecology theory of sea cucumber Apostichopus japonicusSelenka, community structure and seasonal variation of phytoplankton andzooplankton were investigated. The abundance of heterotrophic bacteria and nitrifyingbacteria were also investigated, and then two strains with high degradation ofnitrite-oxidizing bacteria were isolated from sediment of A. japonicus farming ponds.Furthermore, a preliminary study of ecological breeding of A. japonicus was carriedout in pond by using of enclosures on a basis of its ecological environment. Then weresearched and analyzed for feasibility of this new method. Meanwhile, the combinedinfluence of water temperature, salinity and body size on energy budget was alsostudied in the sea cucumber A. japonicus. These results could be used to optimize theconditions of sea cucumber ranching, improving our understanding ofphysioecological regulation and improving pond aquaculture management. Mainresults are as follow:
1. In order to improve efficiency of ecological breeding of A. japonicus andunderstand the habitat of farming ponds, we investigated the characteristics ofplankton community and abiotic factors of ponds and enclosures from Dec.,2010toNov.,2011. The results showed that water temperature was negative correlation withsalinity, and both had a wide range changing gradually. In conditions of watertemperature ranging from-2.5to28.8℃, salinity changing from22to33practicalsalinity unit, pH7.9~8.2, transparency getting above70cm and COD getting below10mg/L, phytoplankton and zooplankton in ponds comprised31and24species,respectively, and they tended to be miniaturization.
2. Heterotrophic bacteria and nitrifying bacteria were152.5~214.6×103cfu/gfrom spring to autumn, while about36×103cfu/g in winter. Nitrogen accumulationis a serious concern in aquaculture. Among other forms of nitrogen, nitrite is more harmful because of its low transformation rate and high toxicity. In order to isolategood strains with high decomposition ability, stability and safety, several strains wereisolated from sediment of the sea cucumber A. japonicus (Selenka) ponds using nitriteoxidizing bacteria (NOB) enrichment culture and screening techniques. The nitriteremoval efficiency of two strains, named as OH1and OB6, were up to99.6%and99.8%respectively with in24h. Total DNAs were extracted and specific primers for16S rDNA of nitrite oxidizer were used to conduct the amplification of polymerasechain reaction (PCR). Amplified product was analyzed by agarose gel electrophoresisand Sanger sequencing. Homology analysis was made by searching in Genbank andEzTaxon server. The amplified fragment range of OH1and OB6was between1300and1450bp.99.6%of OH1nucleotides were identical with a conserved fragment ofa standard strain Cobetia marina DSM4741(T), while99.8%of OB6nucleotides wereidentical with a conserved fragment of a standard strain Halomonas ventosae Al12(T).It is therefore that the nitrite oxidizing bacteria OH1and OB6isolated from the seacucumber ponds were primarily identified to be Cobetia marina DSM4741(T) andHalomonas ventosaeAl12(T).
3. The ecological breeding of larval and juvenile sea cucumber A. japonicusSelenka was studied in experimental enclosures placed in ponds of Dalian Gold ToneSeafood Co., Ltd., Liaoning Province from Jun. to Nov.,2011, with theenclosure-experimental method, by using of pond stirring, probiotics, culture benthicdiatoms and renew the water of enclosure. The results showed that the metamorphosisrates of middle and late auricularia larvae were50%and30%, respectively. Thesurvival rate of juvenile was1.75%in2days, and the survival rate was19%in70days. Finally, the average output was increased from50g/m~3to37.9g/m~3, whichmeans almost8times of the initial weight. Our study indicated that the ecologicalbreeding of larval and juvenile sea cucumber A. japonicus Selenka by using ofenclosures placed in ponds is practicable.
4. The worldwide demand for sea cucumber is outpacing the sustainable harvestcapabilities of fisheries. Sea cucumber ranches and farms are striving to supplementwild harvest, but variable temperature and salinity in pond culture systems make seacucumber production challenging. In this study, we evaluated how water temperature,salinity and body size affected the energy budget of pond-cultured sea cucumber A.japonicus (Selenka). An orthogonal design was used to identify the most suitableconditions for energy consumption and scope for growth (SFG). After the42-day experimental period, we found that water temperature, salinity and body sizesignificantly influenced energy consumption, while salinity and body size were themain influencing factors on SFG. Based on these results, a water temperature of16°Cand a salinity of30are optimal conditions for stocking sea cucumber A. japonicuswith a body size of37.34±4.63g. As such, the optimum stocking seasons for seacucumber A. japonicus may be April in the spring and October in the autumn.
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