Aquaculture Biotechnology Research Group, Department of Applied Ecology, School of Science, Faculty of Health and Environmental Sciences, Auckland University of Technology, Private Bag 92006, Auckland, 1142, New Zealand.
Aquaculture Biotechnology Research Group, Department of Applied Ecology, School of Science, Faculty of Health and Environmental Sciences, Auckland University of Technology, Private Bag 92006, Auckland, 1142, New Zealand.
Cryobiology. 2020 Apr;93:121-132. doi: 10.1016/j.cryobiol.2020.01.021. Epub 2020 Feb 7.
Cryopreservation of genetic material from farmed aquatic species is a valuable technique to advance selective breeding programs for stock improvement. In this study, effects of cryopreservation on development of trochophore and D-stage larvae of Greenshell™ mussel (Perna canaliculus) were evaluated through histology, light microscopy, scanning electron microscopy, and confocal microscopy. Larvae of both life stages were motile immediately post-thawing, but survival declined rapidly from 4 days post-fertilisation (dpf). At 18 dpf, ~23% of non-cryopreserved control larvae had progressed to the pediveliger stage, while <1% of cryopreserved larvae had survived. Control larvae grew faster and larger, and consumed more food than larvae cryopreserved at either life stage (trochophore or D-stage). Settlement competency was achieved in the control larvae at 21 days post-fertilization, with most remaining individuals developing eye spots. Organogenesis was delayed in all cryopreserved larvae, and eyespots did not appear at all. Neurogenesis was stunted in cryopreserved trochophore larvae but seemed to progress almost normally in their cryopreserved D-stage counterparts. Developing abnormalities in shell morphology rapidly became apparent in all mussels post-thaw, with trochophore larvae being most highly afflicted. These delays in organogenesis and overall development are indicative of cryo-injuries sustained at a cellular level. Our results show that D-stage larvae are somewhat more resilient to cryopreservation than trochophore larvae. D-larvae are good life-stage candidates for cryobanking genetic resources in this species because there is generally an excess of larvae from selective breeding family crosses and these can be banked and stored for later use. Further on-going research aims to improve the long-term viability of cryopreserved D-larvae for successful rearing.
冷冻保存养殖水产物种的遗传物质是推进水产养殖选种改良计划的一项有价值的技术。在这项研究中，通过组织学、光学显微镜、扫描电子显微镜和共聚焦显微镜评估了冷冻保存对绿贻贝（Perna canaliculus）担轮幼虫和 D 期幼虫发育的影响。两个生命阶段的幼虫在解冻后立即游动，但在受精后 4 天（dpf）存活率迅速下降。在 18 dpf 时，约 23%的非冷冻保存对照组幼虫已进入足丝幼虫阶段，而只有不到 1%的冷冻保存幼虫存活。对照组幼虫生长更快、更大，并且比在任何生命阶段（担轮幼虫或 D 期）冷冻保存的幼虫消耗更多的食物。在受精后 21 天，对照组幼虫达到了附着能力，大多数幼虫仍然发育出眼点。所有冷冻保存的幼虫的器官发生都被延迟，并且根本没有出现眼点。冷冻保存的担轮幼虫的神经发生受到阻碍，但它们的 D 期对应物的神经发生似乎进展正常。解冻后所有贻贝的壳形态发育异常迅速变得明显，担轮幼虫受影响最大。这些在器官发生和整体发育方面的延迟表明在细胞水平上受到了冷冻损伤。我们的研究结果表明，D 期幼虫比担轮幼虫对冷冻保存具有一定的抵抗力。D 期幼虫是该物种遗传资源冷冻保存的良好生命阶段候选者，因为选择性育种家系杂交通常会产生过多的幼虫，这些幼虫可以被冷冻保存并储存以备后用。进一步的持续研究旨在提高冷冻保存 D 期幼虫的长期存活率，以实现成功的养殖。
