Pérez Sara, Fernández Nuria, Ribeiro Pedro A
Departamento de Bioloxía Animal, Bioloxía Vexetal e Ecoloxía. Facultade de Ciencias. Universidade da Coruña, Campus de A Coruña, 15071 A Coruña, Spain.
Departamento de Bioloxía Animal, Bioloxía Vexetal e Ecoloxía. Facultade de Ciencias. Universidade da Coruña, Campus de A Coruña, 15071 A Coruña, Spain.
Ecotoxicol Environ Saf. 2016 May;127:175-86. doi: 10.1016/j.ecoenv.2016.01.025. Epub 2016 Feb 4.
The use of three limpet species, Patella vulgata Linnaeus, 1758, Patella depressa Pennant, 1777 and Patella ulyssiponensis Gmelin, 1791 as model organisms in marine ecotoxicology has been evaluated. Initial laboratory experiments were aimed to standardize a biological test with embryos and larvae of Patella spp, establishing the percentage of normal trochophore larvae as endpoint. Before conducting in vitro fertilization, oocytes must be maturated artificially by incubation in an alkaline solution; therefore, alkalinizing agent, pH and time of eggs alkalinization were evaluated. Moreover, time of sperm activation, optimum sperm and oocytes concentration during fertilization, gamete contact time, use of stirring during the fertilization, egg concentration and incubation temperature were examined. Minimum sample size per treatment was also estimated. Exposure of oocytes for 10min to FSW alkalinized with NH4OH at pH 9.0, the use of undiluted sperm pre-activated during 45min and a concentration of 200 oocytesmL(-1), a gamete-contact time of 180min and egg incubation at 18°C during 24h at a concentration of 80 eggsmL(-1) were the conditions allowing maximal embryo-larval development success. With an error of 0.05, a sampling size ≥320 allows a 95% confidence in the estimate. This Patella spp. acute bioassay fulfills a number of important a priori requirements to be used in ecotoxicological studies. Nevertheless, in vitro fertilization requires considerable handling, which may lead to failure in fecundation. Such difficulties are also addressed, in order to facilitate the routine use of this protocol by other laboratories.
已评估将三种帽贝物种，即1758年的普通帽贝（Patella vulgata Linnaeus）、1777年的扁平帽贝（Patella depressa Pennant）和1791年的乌利西波帽贝（Patella ulyssiponensis Gmelin）用作海洋生态毒理学模型生物的情况。最初的实验室实验旨在标准化针对帽贝属胚胎和幼虫的生物测试，将正常担轮幼虫的百分比确定为终点。在进行体外受精之前，卵母细胞必须通过在碱性溶液中孵育进行人工成熟；因此，对碱化剂、pH值和卵碱化时间进行了评估。此外，还研究了精子激活时间、受精期间的最佳精子和卵母细胞浓度、配子接触时间、受精期间搅拌的使用、卵浓度和孵育温度。还估计了每个处理的最小样本量。将卵母细胞在pH 9.0的用氢氧化铵碱化的过滤海水（FSW）中暴露10分钟、使用在45分钟内预激活的未稀释精子、浓度为200个卵母细胞/毫升、配子接触时间为180分钟以及在18°C下以80个卵/毫升的浓度孵育24小时，这些条件可使胚胎 - 幼虫发育成功率最高。误差为0.05时，样本量≥320可使估计具有95%的置信度。这种帽贝属急性生物测定满足了在生态毒理学研究中使用的许多重要的先验要求。然而，体外受精需要大量操作，这可能导致受精失败。还解决了此类困难，以便其他实验室能够方便地常规使用该方案。
