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.

中文翻译：

---

冷冻保存的贻贝 (Perna canaliculus) 幼虫的发育阶段影响解冻后对壳形成、器官发生、神经发生、摄食能力和存活的影响

来自养殖水生物种的遗传材料的低温保存是推进种群改良的选择性育种计划的一项宝贵技术。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 Greenshell™ 贻贝（Perna canaliculus）的承载体和 D 期幼虫发育的影响。两个生命阶段的幼虫在解冻后立即活动，但存活率从受精后 4 天 (dpf) 迅速下降。在 18 dpf 时，约 23% 的非冷冻保存的对照幼虫已经发展到 pediveliger 阶段，而 <1% 的冷冻保存的幼虫存活下来。对照幼虫生长得更快、更大，并且比在任一生命阶段（承载体或 D 阶段）冷冻保存的幼虫消耗更多的食物。在受精后 21 天，对照幼虫达到了定居能力，大多数剩余个体出现了眼斑。在所有冷冻保存的幼虫中器官发生延迟，并且根本没有出现眼斑。冷冻保存的承载体幼虫的神经发生发育迟缓，但似乎在冷冻保存的 D 期对应物中几乎正常进展。解冻后，所有贻贝的壳形态异常迅速变得明显，其中承载体幼虫受到的影响最大。这些器官发生和整体发育的延迟表明在细胞水平上持续存在冷冻损伤。我们的结果表明，D 期幼虫比 trochophore 幼虫更能适应低温保存。D-幼虫是该物种遗传资源冷冻库的良好生命阶段候选者，因为通常有过多的来自选择性育种家庭杂交的幼虫，这些幼虫可以储存起来以备后用。正在进行的进一步研究旨在提高冷冻保存的 D-幼虫的长期生存能力，以成功饲养。