CN105340792B - 一种筛选滩涂贝类幼苗微藻饵料的方法及其应用 - Google Patents
一种筛选滩涂贝类幼苗微藻饵料的方法及其应用 Download PDFInfo
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- A01K61/00—Culture of aquatic animals
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
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Abstract
本发明提供一种筛选滩涂贝类幼苗微藻饵料的方法，即通过定量检测滩涂贝类幼体摄食混合微藻后贝类体内脂肪酸和甾醇的组成，来准确筛选滩涂贝类幼苗优质微藻饵料的方法。本发明的方法的培养周期短，避免了水质环境条件对培养效果可能产生的影响，基于贝类幼体脂肪酸甾醇组成筛选出的微藻，其饵料效果更切合实际生长过程中贝类幼体的选择性摄食和同化，从而可作为贝类幼体优质饵料的选择。
Description
技术领域
本发明属于水产养殖饵料培养技术领域，具体涉及一种筛选滩涂贝类幼苗微藻饵料的方法。
背景技术
目前我国“蛤、蚶、蛏”等滩涂贝类产量占海水贝类养殖总产量近40％。而在滩涂贝类育苗中，微藻饵料的品种直接决定着育苗的成败。长期实践发现，育苗过程中投喂不同微藻时，滩涂贝类幼体生长速度差异很大，说明微藻种类对滩涂贝类幼苗的生长发育有着直接影响。在实际滩涂贝类幼苗繁育过程中，我们需要选择对滩涂贝类幼体具有明显饵料效果的微藻饵料进行扩繁投喂。
某种饵料微藻对滩涂贝类幼体饵料效果如何，一般都通过向滩涂贝类投喂不同种类的微藻，通过观测对应贝类幼苗的生长速度和成活率来判断对应微藻的饵料效果【朱雨瑞等，2010.5种微藻对4种滩涂稚贝生长的影响，海洋学研究，28(3):60-66】。但该手段需要比较长的培养周期，另外，水质条件、环境条件均会对幼苗的生长速度和存活率产生影响。所以往往需要通过多次的反复试验才能最后确定不同微藻对贝类的饵料效果。因此，就需要提供一种有效的检测微藻作为贝类饵料的方法。而且，目前普遍采用投喂两种或多种微藻的混合投喂方式，而这种方式需要对不同的微藻进行培养，导致饵料的成本上升。
发明内容
本发明提供一种筛选滩涂贝类幼苗微藻饵料的方法，即通过定量检测滩涂贝类幼体摄食混合微藻后贝类体内脂肪酸和甾醇的组成，来准确筛选滩涂贝类幼苗优质微藻饵料的方法，从而弥补现有技术的不足。
本发明的方法，包括如下的步骤：
1)标准组PCA得分图的获得
用混合微藻投喂贝类幼体作为标准组，投喂一定周期后测定贝类幼体的脂肪酸和甾醇组成，然后对脂肪酸和甾醇组成进行主成分分析(PCA)，获得PCA 得分图；
2)在同样的养殖条件下，对贝类幼体分别投喂不同种类的单种微藻进行培养作为实验组，在投喂相同周期后测定不同实验组贝类幼体的脂肪酸和甾醇组成，然后对脂肪酸和甾醇组成进行主成分分析，根据各实验组在PCA得分图上的分布筛选出与标准组接近的实验组的微藻作为该贝类的饵料。
所述的贝类为滩涂养殖的贝类，例如泥蚶、毛蚶、青蛤、缢蛏、彩虹明樱蛤、文蛤等。
本发明所用的微藻均于位于指数生长期，浓度达到80～120cell·μl-1时进行投喂；
本发明的贝类投喂微藻饵料前，需要在经过沙滤基本不含任何微藻的洁净天然海水中不投饵饥饿8-10h以尽量排空肠胃内残饵。
本发明使用气相色谱-质谱联用来进行脂肪酸和甾醇的定量分析。
采用本发明的方法，确定泥蚶的饵料微藻为角毛藻或金藻。
与现有技术相比，本发明的优点在于：培养周期短，避免了水质环境条件对培养效果可能产生的影响，基于贝类幼体脂肪酸甾醇组成筛选出的微藻，其饵料效果更切合实际生长过程中贝类幼体的选择性摄食和同化，从而可作为贝类幼体优质饵料的选择。
附图说明
图1:基于泥蚶幼苗脂肪酸甾醇主成分分析的第一和第三主成分得分图(A) 及第二和第三主成分得分图(B)
图上样品分别为单种投喂P.viridis的泥蚶幼苗(IG-PV)、单种投喂C.calcitrans(TG-CC)、I.galbana(TG-IG)和N.oculata(NO)的泥蚶幼苗；两两混合投喂I.galbana和N.oculata(TG-IG-NO)、I.galbana和P.viridis (TG-IG-NO)、C.calcitrans和I.galbana(TG-CC-IG)、C.calcitrans和P.viridis (TG-CC-PV)、C.calcitrans和N.oculata(TG-CC-NO)及所有4种微藻全混合投喂的泥蚶幼苗(TG-MIX)。
具体实施方式
本发明通过对投喂混合微藻饵料和单种微藻饵料的贝类幼体测定脂肪酸甾醇组成，并进行PCA分析，在PCA得分图上，如果投喂了某种微藻后的贝类幼体样品跟混合投喂微藻饵料的贝类幼体样品比较接近，就可以说明该微藻被该贝类优先摄食和同化了，实际贝类幼体培育过程中就可以选择该藻作为该贝类幼体的优质饵料。本发明应用该技术，大大缩短了传统技术中漫长的培养过程，并避免了水质环境条件对培养效果可能产生的影响，筛选出的微藻饵料效果更切合实际过程中贝类幼体的摄食同化选择。
本发明所使用的主成分分析方法(principal components analysis，PCA)是一种多变量数据统计方法，在二维空间上显示物质聚类分布的PCA得分图，可以直观样品间物质组成的相似或非相似性【Vliet E V et al.,2008.A novel in vitro metabolomicsapproach for neurotoxicity testing,proof of principle for methyl mercurychloride and caffeine.NeuroToxicology,29(1):1–12】。
所述的混合微藻是将已报道的该贝类的各种微藻混合后完成的。
以下通过实施例对本发明作进一步详细描述。
实施例1
2014年9月在福建省宝智水产科技有限公司，选择滩涂贝类常用4种海洋微藻，分别是：硅藻门的角毛藻(Chaetoceros calcitrans)、金藻门的球等鞭金藻 (Isochrysisgalbana)和绿色巴夫藻(Pavlova viridis)、Eustigmatophyceae藻门的微绿球藻(Nannochloropsis oculata)进行培养，微藻的培养海水(盐度为20)经 0.45μm醋酸纤维滤膜过滤后，煮沸冷却。加入NMB3营养液(100mg·L-1KNO3, 10mg·L-1KH2PO4,0.25mg·L- 1MnSO4·H2O,2.50mg·L-1FeSO4·7H2O,10mg·L-1 EDTA-Na2,6μg·L-1VB1,0.05μg·L-1VB12)，同时加入硅酸钠(20mg.L-1)作为角毛藻的硅源。微藻藻种在2500mL的锥形瓶中[自然光，培养温度为(20±2)℃] 培养1周后，把它们转入50L的塑料白桶中，在自然光、自然温度(25～29℃) 下再培养1周左右，当微藻密度达到80～120cell·μl-1时投喂给贝类幼苗。
在长、宽、高分别为25、15和6cm的白色塑料盆(实验盆)中分别加入盐度为20的沙滤海水，并加入1-2mm厚200℃烘干消毒并经网孔75μm的尼龙筛绢过滤过的新鲜海泥，将湿重为1.5g平均长度0.68±0.05mm的泥蚶幼贝样品分别均匀洒入10组实验盆中(每组平行3个)，每一实验盆中分别于08:00和 20:00左右时刻投放足量对应表1中单种微藻或混合微藻(等比例混合)，在自然温度25.2-31.7℃下培养7天后，收集样品，在经过沙滤的洁净天然海水中不投饵饥饿8-10h排空肠胃内残饵，冷冻干燥后进行脂肪酸甾醇分析，同时随机取样测量30粒幼贝的生长(表2)。
表1投喂实验中选择的饵料种类
泥蚶幼贝脂肪酸甾醇分析具体步骤如下：取冷冻干燥并碾磨粉碎后的0.2g 泥蚶幼贝样品，参考改进后的Bligh-Dyer法[Bligh E G,Dyer W J.1959.A Rapid MethodLipid Extraction and Purification,Can J Biochem Physiol,37:911-917]提取总脂；总脂提取后加入2mL 5-6％KOH甲醇水(V/V＝4:1)溶液，充氮气1min，密封，60℃水浴皂化2h，冷却，皂化液加15mL饱和氯化钠溶液1mL，再用 HCl(1:1)调pH小于1，氯仿：正己烷(V/V＝1:4)6mL分三次提取，旋转蒸发仪真空干燥后，加入0.5mL 14％BF3-CH3OH溶液，充氮气1min，密封， 60℃水浴甲酯化1h，冷却，用正己烷6mL分3次提取，合并提取液，用2mL 重蒸水洗一次，上层液移出，加入1g无水硫酸钠，吸水12h，移取上层液， N2吹干后加入100μL过量BSTFA，密封，60℃水浴2h，N2吹干，正己烷定容，进行GC-MS分析。GC条件：进样口温度250℃，载气为高纯氦，柱流速0.81 mL/min，柱前压73.0Kpa，柱起始温度150℃，保持3.5min，以20℃/min升至 200℃，保持5min，再以5℃/min升至280℃/min，保持30min。用EI源分析时分流进1μL，分流比50:1。MS条件：用电子轰击电离源(EI)分析，电子能量为70eV，离子源温度200℃，接口温度250℃，选取SCAN模式，质量扫描范围为40-600，溶剂延迟3.5min。最后根据GC-EIMS-TIC中各组分的离子碎片质量图谱，通过对NIST库和WILEY库检索并参考脂肪酸和甾醇标准谱图及参考文献进行鉴定。用面积归一法计算出脂肪酸和甾醇组分的百分含量(表2，以占脂肪酸和甾醇总量的百分比即相对百分含量表示)。
表2：投喂不同饵料后泥蚶幼贝的脂肪酸甾醇组成(％)
将脂肪酸甾醇的测定结果输入SIMCA-P-12.0分析软件(瑞典Umetrics AB 公司)进行主成分分析。在软件输出的得分图上(图1)可观察到，混合投喂金藻跟巴夫藻或微绿球藻的样品(图上TG-IG-PV和TG-IG-NO)跟单独投喂金藻 (图上TG-IG)接近，而跟单独投喂巴夫藻(图上TG-PV)或微绿球藻(图上 TG-NO)的样品相距甚远；同样，混合投喂角毛藻跟巴夫藻或微绿球藻的样品 (图上TG-CC-PV和TG-CC-NO)跟单独投喂角毛藻(图上TG-CC)接近，而跟单独投喂巴夫藻(图上TG-PV)或微绿球藻(图上TG-NO)的样品相距甚远；而混合投喂金藻和角毛藻的样品(TG-CC-IG)则跟四种藻全混合投喂的样品 (TG-MIX)相近。可见，混合投喂后的泥蚶幼贝的脂肪酸甾醇组成跟单种投喂金藻或者单种投喂角毛藻的的泥蚶幼贝脂肪酸甾醇相近，说明泥蚶幼贝优先选择性摄食同化了金藻或角毛藻，这两种藻即为本发明所筛选出的该滩涂贝类幼苗的优质微藻饵料。作为证明，从4组单种投喂微藻7天后泥蚶幼贝的生长也可看出(表3)，单种投喂角毛藻和金藻的样品生长速度明显比单种投喂巴夫藻和微绿球藻快，说明这种筛选方法得到的结果是正确有效的。
表3：单种投喂微藻饵料7天后泥蚶幼贝的大小长度(mm)
Claims (6)
1.一种筛选滩涂贝类幼苗微藻饵料的方法，其特征在于，所述的方法包括如下的步骤：
1)标准组PCA得分图的获得
用混合微藻投喂贝类幼体作为标准组，投喂一定周期后测定贝类幼体的脂肪酸和甾醇组成，然后对脂肪酸和甾醇组成进行主成分分析(PCA)，获得PCA得分图；
2)在同样的养殖条件下，对贝类幼体分别投喂不同种类的单种微藻进行培养作为实验组，在投喂相同周期后测定不同实验组贝类幼体的脂肪酸和甾醇组成，然后对脂肪酸和甾醇组成进行主成分分析，根据各实验组在PCA得分图上的分布筛选出与标准组接近的实验组的微藻作为该贝类的饵料。
2.如权利要求1所述的方法，其特征在于，所述的贝类为泥蚶、毛蚶、青蛤、缢蛏、彩虹明樱蛤或文蛤。
3.如权利要求1所述的方法，其特征在于，所述的微藻位于指数生长期，浓度达到80～120cell·μl-1。
4.如权利要求1所述的方法，其特征在于，所述的贝类在投喂微藻饵料前，先在经过沙滤的天然海水中饥饿8-10h以排空肠胃内残饵。
5.如权利要求1所述的方法，其特征在于，所述的方法是使用气相色谱-质谱联用方法来进行脂肪酸和甾醇的定量分析。
6.权利要求1所述的方法在筛选泥蚶的饵料微藻中的应用，其特征在于，权利要求1所述的方法筛选的泥蚶的饵料微藻为角毛藻或金藻。
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