CN107300546A - 曼氏无针乌贼耳石的钙黄绿素标记方法及其检测手段 - Google Patents
曼氏无针乌贼耳石的钙黄绿素标记方法及其检测手段 Download PDFInfo
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Abstract
本发明公开了曼氏无针乌贼耳石的钙黄绿素标记方法及其检测手段，标记方法为：将钙黄绿素加入养殖水中，制成浓度为200‑400mg/L的溶液，暴晒，再加入米赛林，混合均匀得标记浸泡液，将待标记的曼氏无针乌贼饥饿后置于标记浸泡液中浸泡，结束后用新鲜海水浸泡，即完成对曼氏无针乌贼耳石的标记。有益效果为：本发明标记方法简单，能达到无损标记的目的，能够一次性实现大规模的放流标记活动；该标记方法能形成具有高强度荧光的混合配合物，方便观察结果及对比，且标记时间持久长；该标记方法对不同阶段曼氏无针乌贼都可行，为曼氏无针乌贼的增殖放流效果评价提供有效的手段。
Description
技术领域
本发明涉及一种水生动物的标记方法，特别是涉及曼氏无针乌贼耳石的钙黄绿素标记方法及其检测手段。
背景技术
曼氏无针乌贼(Sepiella japonica)曾是我国渔场的四大渔业之一，对温度的适应性较强，在我国沿海分布很广，是一种经济价值很高的传统渔业种类。20世纪70年代以来，由于拖网、张网等多种渔具大量捕捞幼乌贼和越冬乌贼，致使曼氏无针乌贼由生长型过度捕捞向补充型过度捕捞转变，破坏了渔业资源的生态平衡，导致浙江渔场的曼氏无针乌贼资源近乎枯竭。自2006年以来，浙江沿海开展了曼氏无针乌贼受精卵和幼体的大规模增殖放流。而了解人工放流的乌贼卵和幼体的存活及分布情况，对曼氏无针乌贼的增殖放流效果评价至关重要。水生生物的标志技术（Marking technique）是研究水生生物生活史以及对其进行资源评估的重要工具。特别是对于鱼类的增殖放流，基于标志技术的标志增殖放流可用于研究鱼类洄游和资源动态，根据回捕地点记录可推测放流鱼类游动的方向、路线、范围和速度。此外结合回捕鱼体的全长、湿重和年龄资料，推测出放流群体在野外的生长和种群变动规律，进而检验增殖放流的效果。
耳石因其极强的生物惰性比利用鳞片、鳍条、鳃盖骨等进行年龄鉴定更加有效，耳石是最先钙化的组织，其生长的持续性受环境影响极小，在鱼体停止生长条件下仍能持续生长，因此其上的年轮更加精准的表明了鱼体的实际年龄。近年来，随着鱼类耳石研究的深入，耳石因其独有的生物和化学特性逐渐成为极佳的标记载体，越发得到广泛应用。利用荧光物质能在鱼类耳石上沉积的化学标记，因其效果明显、检测便捷在多种鱼类得以应用。
现有技术如授权公告号为CN 101731161 B的中国发明专利，公开了曼氏无针乌贼皮下荧光标记方法专用试剂，该标记试剂按照重量份数含有1份的茜素络合指示剂粉末和4份至8份的酸奶。该试剂在相对集中的皮下区域形成标记，较一般的染色标记具有更高的可识别性，在乌贼复苏后，无需暂养除去浮色，可以立即放流。但是乌贼幼体阶段个体很小，注射标记很困难，对乌贼幼体有损伤，影响其成活率，且不能实现大规模的放流标记。
发明内容
本发明的目的在于提供一种标记方法简单，能达到无损标记的目的，能够一次性实现大规模的放流标记活动，发光信号较强，标记时间持久，检测方便的曼氏无针乌贼耳石的钙黄绿素标记方法及其检测手段。
本发明针对上述技术中提到的问题，采取的技术方案为：曼氏无针乌贼耳石的钙黄绿素标记方法，包括以下步骤：
养殖水预处理：将经净化的海水放入缓释标记养殖箱并微充气，作为养殖水体，然后向养殖水体内加入EDTA，每吨养殖水中EDTA的添加量为4-8g，充分溶解后备用，该步骤中的EDTA能与海水中游离的钙离子发生络合反应，形成稳定的水溶性络合物，避免而后标记步骤所使用的钙黄绿素由于干扰离子而造成的损耗，确保标记浓度稳定及标记效果的稳定；
标记浸泡液配制：将钙黄绿素加入养殖水中，制成浓度为200-400mg/L的溶液，暴晒11-13h，然后再加入米赛林，钙黄绿素与米赛林的重量比为1:0.2-0.3，混合均匀，即得标记浸泡液，该步骤中米赛林能与钙黄绿素、曼氏无针乌贼耳石的钙化组织形成具有高强度荧光的混合配合物，比单一用钙黄绿素标记效果更明显，使得耳石在后续荧光检测时能产生较强的发光信号，方便观察结果及对比，同时混合配合物的稳定性更强，使得标记时间更持久，且钙黄绿素与米赛林不会影响曼氏无针乌贼贝类的存活性，该步骤中暴晒是为了恢复海水的pH，使曼氏无针乌贼能更好的在标记浸泡液中存活；
浸泡：将待标记的曼氏无针乌贼饥饿2-3d，然后置于标记浸泡液中，浸泡密度为18-23尾/L，浸泡22-26h，浸泡结束后将曼氏无针乌贼放入新鲜海水浸泡清洗2-3h以除去残余染料，继续饲养，即完成对曼氏无针乌贼耳石的标记，通过浸泡可使标记物质与耳石的钙化组织形成稳定的混合配合物，从而在耳石上生成生长标记，这种耳石上的标记能够在荧光显微镜下被清晰地检测观察，该标记方法能够减少对鱼体的操作、降低胁迫和死亡率，能达到无损标记的目的，并且能够一次性实现大规模的放流标记活动，最大限度降低人工操作对鱼苗的影响。
曼氏无针乌贼耳石的钙黄绿素标记方法的检测手段：样品采集后，将曼氏无针乌贼放入浓度80-120mg/L的麻醉剂溶液中进行麻醉，然后用新鲜海水清洗后，再用解剖针剖取出耳石，剥离附属组织，用蒸馏水洗涤、无水乙醇清洗、干燥，封存于载玻片上，耳石重叠的部分需要被磨制观察，用荧光显微镜检测耳石中的荧光标记，记录相应的标记清晰度即可，上述麻醉剂成分美托咪啶中右旋美托咪啶与左旋美托咪啶的质量比为97/3-94/6。纯右旋美托咪啶可快速加深全身麻醉，使曼氏无针乌贼迅速地进入麻醉诱导期，但麻醉维持时间短，镇痛效果差，若加大用药量，会对机体产生一定的副作用。将右旋美托咪啶与左旋美托咪啶复配而成的麻醉剂，麻醉维持时间长，且还有一定的镇痛效果，为激素的包埋提供了良好的条件。
与现有技术相比，本发明的优点在于：1）本发明标记方法简单，能够减少对鱼体的操作、降低胁迫和死亡率，能达到无损标记的目的，并且能够一次性实现大规模的放流标记活动，最大限度降低人工操作对鱼苗的影响；2）该标记方法安全可靠、毒性极低，对曼氏无针乌贼的存活无不良影响，能形成具有高强度荧光的混合配合物，使得耳石在后续荧光检测时能产生较强的发光信号，方便观察结果及对比，同时混合配合物的稳定性更强，使得标记时间更持久；3）该标记方法具有操作快速、简便、稳定可靠、成功率高和重复性好等优点，对从鱼卵到鱼苗、幼鱼或成鱼不同阶段都是可行的，可为曼氏无针乌贼的增殖放流效果评价提供有效的手段。
具体实施例
下面通过实施例对本发明方案作进一步说明：
实施例1：
曼氏无针乌贼耳石的钙黄绿素标记方法，包括以下步骤：
1）养殖水预处理：将经净化的海水放入缓释标记养殖箱并微充气，作为养殖水体，然后向养殖水体内加入EDTA，每吨养殖水中EDTA的添加量为6g，充分溶解后备用，该步骤中的EDTA能与海水中游离的钙离子发生络合反应，形成稳定的水溶性络合物，避免而后标记步骤所使用的钙黄绿素由于干扰离子而造成的损耗，确保标记浓度稳定及标记效果的稳定；
2）标记浸泡液配制：将钙黄绿素加入养殖水中，制成浓度为300mg/L的溶液，暴晒12h，然后再加入米赛林，钙黄绿素与米赛林的重量比为1:0.25，混合均匀，即得标记浸泡液，该步骤中米赛林能与钙黄绿素、曼氏无针乌贼耳石的钙化组织形成具有高强度荧光的混合配合物，比单一用钙黄绿素标记效果更明显，使得耳石在后续荧光检测时能产生较强的发光信号，方便观察结果及对比，同时混合配合物的稳定性更强，使得标记时间更持久，且钙黄绿素与米赛林不会影响曼氏无针乌贼贝类的存活性，该步骤中暴晒是为了恢复海水的pH，使曼氏无针乌贼能更好的在标记浸泡液中存活；
3）浸泡：将待标记的曼氏无针乌贼饥饿2.5d，然后置于标记浸泡液中，浸泡密度为20尾/L，浸泡24h，浸泡结束后将曼氏无针乌贼放入新鲜海水浸泡清洗2.5h以除去残余染料，最后转移至正常水体中继续饲养，期间观察标记引起的幼鱼急性死亡率，即完成对曼氏无针乌贼耳石的标记，通过浸泡可使标记物质与耳石的钙化组织形成稳定的混合配合物，从而在耳石上生成生长标记，这种耳石上的标记能够在荧光显微镜下被清晰地检测观察，该标记方法能够减少对鱼体的操作、降低胁迫和死亡率，能达到无损标记的目的，并且能够一次性实现大规模的放流标记活动，最大限度降低人工操作对鱼苗的影响，此外，钙黄绿素溶液有一定的酸性，浸泡时每小时监测CAL染液pH一次，并用碳酸氢钠进行缓冲调节，使染液的pH维持在7.5–8.2。
曼氏无针乌贼耳石的钙黄绿素标记方法的检测手段：饲养60天后进行样品采集，将曼氏无针乌贼放入浓度100mg/L的麻醉剂溶液中进行麻醉，然后用新鲜海水清洗后，再用解剖针剖取出耳石，剥离附属组织，用蒸馏水洗涤、无水乙醇清洗、干燥，封存于载玻片上，耳石重叠的部分需要被磨制观察，Nikon DS-Fi1高清数码摄像头的Nikon Eclipse 50i荧光显微镜，并调至CAL的激发光发射波长（如表1所示），观察耳石，记录相应的标记清晰度即可。
上述麻醉剂成分美托咪啶中右旋美托咪啶与左旋美托咪啶的质量比为95/5。纯右旋美托咪啶可快速加深全身麻醉，使曼氏无针乌贼迅速地进入麻醉诱导期，但麻醉维持时间短，镇痛效果差，若加大用药量，会对机体产生一定的副作用。将右旋美托咪啶与左旋美托咪啶复配而成的麻醉剂，麻醉维持时间长，且还有一定的镇痛效果，为激素的包埋提供了良好的条件。
表1 用于标记检测的分色镜和滤镜光片
实施例2：
曼氏无针乌贼耳石的钙黄绿素标记方法，包括以下步骤：
1）将经净化的海水放入缓释标记养殖箱并微充气，作为养殖水体，然后向养殖水体内加入EDTA，每吨养殖水中EDTA的添加量为5g，充分溶解后备用，该步骤中的EDTA能与海水中游离的钙离子发生络合反应，形成稳定的水溶性络合物，避免而后标记步骤所使用的钙黄绿素由于干扰离子而造成的损耗，确保标记浓度稳定及标记效果的稳定；
2）将钙黄绿素加入养殖水中，制成浓度为350mg/L的溶液，暴晒13h，然后再加入米赛林，钙黄绿素与米赛林的重量比为1:0.3，混合均匀，即得标记浸泡液，该步骤中米赛林能与钙黄绿素、曼氏无针乌贼耳石的钙化组织形成具有高强度荧光的混合配合物，比单一用钙黄绿素标记效果更明显，使得耳石在后续荧光检测时能产生较强的发光信号，方便观察结果及对比，同时混合配合物的稳定性更强，使得标记时间更持久，且钙黄绿素与米赛林不会影响曼氏无针乌贼贝类的存活性，该步骤中暴晒是为了恢复海水的pH，使曼氏无针乌贼能更好的在标记浸泡液中存活；
3）将待标记的曼氏无针乌贼饥饿2d，然后置于标记浸泡液中，浸泡密度为22尾/L，浸泡25h，浸泡结束后将曼氏无针乌贼放入新鲜海水浸泡清洗3h以除去残余染料，继续饲养，即完成对曼氏无针乌贼耳石的标记，通过浸泡可使标记物质与耳石的钙化组织形成稳定的混合配合物，从而在耳石上生成生长标记，这种耳石上的标记能够在荧光显微镜下被清晰地检测观察，该标记方法能够减少对鱼体的操作、降低胁迫和死亡率，能达到无损标记的目的，并且能够一次性实现大规模的放流标记活动，最大限度降低人工操作对鱼苗的影响。
曼氏无针乌贼耳石的钙黄绿素标记方法的检测手段：饲养90天后进行样品采集，将曼氏无针乌贼放入浓度110mg/L的麻醉剂溶液中进行麻醉，然后用新鲜海水清洗后，再用解剖针剖取出耳石，剥离附属组织，用蒸馏水洗涤、无水乙醇清洗、干燥，封存于载玻片上，耳石重叠的部分需要被磨制观察，用Nikon DS-Fi1高清数码摄像头的Nikon Eclipse 50i荧光显微镜，并调至CAL的激发光发射波长，观察耳石，记录相应的标记清晰度即可。
实施例3：
1.标记效果：
试验组为实施例1；空白组未进行标记，其余步骤和实施例1完全相同；对照组的标记采用浓度为300mg/L的钙黄绿素溶液，未加米赛林，其余步骤和实施例1完全相同。荧光标记清晰度的等级划分为0–5级，0级：在荧光显微镜下看不到任何标记；1级：在荧光显微镜下能看见模糊的标记；2级：在荧光显微镜下容易分辨标记；3级：在荧光显微镜下能看见鲜亮的标记；4级：在自然透射光下能看见标记；5级：在自然透射光下能看见清晰地标记。标记等级≥2级，则可认为是可以检测到的良好标记效果。每组选择8条曼氏无针乌贼的耳石进行检测，取平均值，检测结果如表2所示。
2.死亡率：从标记开始到采集样品，统计曼氏无针乌贼的死亡率，结果如表2所示。
表2 标记检测结果
由上表可知，本发明实施例2的标记方法清晰度远远高于空白组和对照组，说明本发明实施例1具有良好的标记效果，方便观察结果；死亡率均为0，说明本发明标记方法安全可靠，对曼氏无针乌贼的生长无不良影响。
本发明的操作步骤中的常规操作为本领域技术人员所熟知，在此不进行赘述。
以上所述的实施例对本发明的技术方案进行了详细说明，应理解的是以上所述仅为本发明的具体实施例，并不用于限制本发明，凡在本发明的原则范围内所做的任何修改、补充或类似方式替代等，均应包含在本发明的保护范围之内。
Claims (10)
1.曼氏无针乌贼耳石的钙黄绿素标记方法，包括养殖水预处理、标记浸泡液配制、浸泡，其特征在于：所述的标记浸泡液配制步骤为：将钙黄绿素加入养殖水中制成溶液，暴晒，再加入米赛林，混合均匀，即得标记浸泡液。
2.根据权利要求1所述的曼氏无针乌贼耳石的钙黄绿素标记方法，其特征在于：所述的标记浸泡液配制步骤中钙黄绿素浓度为200-400mg/L。
3.根据权利要求1所述的曼氏无针乌贼耳石的钙黄绿素标记方法，其特征在于：所述的标记浸泡液配制步骤中暴晒11-13h。
4.根据权利要求1所述的曼氏无针乌贼耳石的钙黄绿素标记方法，其特征在于：所述的标记浸泡液配制步骤中钙黄绿素与米赛林的重量比为1:0.2-0.3。
5.根据权利要求1所述的曼氏无针乌贼耳石的钙黄绿素标记方法，其特征在于：所述的养殖水预处理中每吨养殖水中EDTA的添加量为4-8g。
6.根据权利要求1所述的曼氏无针乌贼耳石的钙黄绿素标记方法，其特征在于：所述的浸泡步骤中：将待标记的曼氏无针乌贼饥饿2-3d。
7.根据权利要求1所述的曼氏无针乌贼耳石的钙黄绿素标记方法，其特征在于：所述的浸泡步骤中浸泡密度为18-23尾/L，浸泡时间为22-26h。
8.根据权利要求1所述的曼氏无针乌贼耳石的钙黄绿素标记方法，其特征在于：所述的浸泡步骤中新鲜海水浸泡清洗时间为2-3h。
9.曼氏无针乌贼耳石的钙黄绿素标记方法的检测手段，其特征在于：所述的检测手段为：样品采集后，将曼氏无针乌贼进行麻醉后用新鲜海水清洗，取出耳石，清洗、干燥，封存于载玻片上，用荧光显微镜检测耳石中的荧光标记，记录相应的标记清晰度即可。
10.根据权利要求9所述的曼氏无针乌贼耳石的钙黄绿素标记方法的检测手段，其特征在于：所述的麻醉剂的浓度为80-120mg/L，其成分美托咪啶中右旋美托咪啶与左旋美托咪啶的质量比为97/3-94/6。
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