The effects of cryopreservation on the Pacific oyster Crassostrea gigas larval development
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Aquaculture in New Zealand is an important growing export industry and around 66 % of its total production is exported, which is worth NZ $ 400 million every year. Based on the growth strategy of this industry, the aim is to achieve an annual production of U.S $ 1 billion by 2025. In New Zealand, the Pacific oyster Crassostrea gigas, is a leading cultivated species with significant exports, along with King salmon and the New Zealand GreenshellTM mussel. Pacific oysters are native to Japan, but now are well established in many parts of the world including New Zealand. Most of the culturing for Pacific oyster in New Zealand is conducted using wild seed, the majority of which are gathered in the Kaipara Harbour on the north-western coast of the North Island. However, this source is highly unreliable, for example recent outbreaks of Ostreid herpesvirus-1 (OsHV-1) led to mass mortality of Pacific oyster larvae in 2010 leaving huge losses within this industry. To overcome these natural disasters and seasonal constraints, use of cryopreserved larvae has been suggested by many scientists. With this method of storing, larvae can be thawed to meet hatchery demands at any time. However, the degree of success using cryopreservation technique is highly variable due to lack of optimum protocols which are highly species specific. Furthermore, little is known about the freezing and cooling effects on the viability of larvae and their subsequent development. Previous studies have been focused on high survival rates just after post-thawing but recently the need to study the effect of cryopreservation on the larval quality over a longer time frame has been recognized. Before cryopreservation techniques can be applied to oyster larvae, a good understanding of the basic larval development process is necessary under normal conditions. There is lack of literature available on the detailed larval development under normal hatchery conditions for Pacific oysters. Therefore, the present study is the first to comprehensively describe the various stages of development from D-stage through to settlement of Pacific oyster Crassostrea gigas larvae under hatchery practices. To supplement this project, details of the effect of 2 cryoprotectant solutions (CPA) on the larval development from D-stage through to settlement is provided. To achieve the above aims, we used a multi-technique approach involving light microscopy, scanning electron microscopy, immunochemistry and direct visual observations. The findings indicate that these complementary techniques provide the best approach to investigate the larval stages of Pacific oysters. Both cryopreserved and normal (controls) larvae were assessed for survivability, feeding consumption, shell length, shell morphology, organogenesis and neurogenesis at regular intervals. The methodology of this study included a larval rearing process conducted at Cawthron Aquaculture Park, Nelson. Larval samples were isolated and fixed every alternate day and later transported to Auckland University of Technology on dry ice for further analysis. Raw data for parametric analyses was also supplied by Cawthron Aquaculture Park. Normal larvae (controls) show a linear downfall in survivability with lowest percentage survivability after 19 dpf, when the pediveligers were approaching metamorphosis. Feeding consumption also varied over the total larval period under investigation and was considerably low after 20 dpf. Shell length show a linear increase before showing some constancy near metamorphosis. Shell morphological observations revealed the presence of a prodissoconch I shell at D-stage with flat hinge and a pitted punctuate region further developing prodissoconch II shell characterised by comarginal growth lines at 5 days post fertilisation (dpf). After progressing to umbo (7-15 days old) stage, larvae exhibited well developed umbo and a special feature called postero dorsal notch which is characteristic of the members of Family Ostreidae. Later the larvae developed into pediveligers at day 17-19 and finally secrete dissoconch layer during transition from larval period to spat. D-stage larvae exhibited limited organogenesis with development of alimentary canal. With progression of larval days, a protruding velum with well beating cilia and velum retractor muscles were present. Further development led to appearance to posterior and anterior adductor muscles. An important mantle rejection technique called pseudofeces was also observed. In pediveligers development of functional foot and eye spot occurred along with gill rudiment. During settlement, velum was retracted inside the shell indicating metamorphosis. The larval density during settlement period was quiet low because of accidental exposure to higher dose of UV than normal and secondly it indicates healthy competitive settlement behaviour. During cryopreservation study, larvae were exposed to 2 treatments 10% ethylene glycol + 1% polyvinylpyrrolidone (PVP- 40) + either 0.2 M or 0.4 M trehalose. Three different cooling rates of 0.5, 1, 2˚C min-1 between -10 and -35ºC post-holding were investigated. Results show that there were significant differences in survivability, shell length feeding consumption and organogenesis between controls and both the treatments. Comparison between the treatments (0.2 M and 0.4 M trehalose), revealed that larvae exposed to 0.4 M especially with the cooling rate of 1˚C min-1 performed exceptionally well. Whereas larvae exposed to 0.2 M trehalose exhibit severe abnormalities with 100% mortality by day 15. Feeding consumption was significantly lower than controls and shell size was considerably small. Shell was more or less oblong giving them oval appearance at early umbo stage rather than circular shape. Organogenesis was the worst effective with severe damage to digestive diverticulum and velum. Moreover, larvae at 5-7 days post fertilisation exhibit D-stage instead of progressing towards umbo stage. All the internal deformities were indicative of larval death near future. 0.4 M cryopreservation treatment show somewhat satisfactory results at cooling rate of 1˚C min-1, the survivability and feeding consumption were significantly lower than controls but the survived larvae (658 ± 570) show almost similar development but delayed organogenesis and smaller shell size. This delay of organogenesis and other developmental characteristics were indicative of cryoinjuries sustained at the cellular level. However, the degree of cryoinjury was worse in 0.2 M exposed larvae. These results indicates future potential to cryopreserve Crassostrea gigas larvae using 0.4 M trehalose as cryopreservative agent provided optimum cooling rate which can enhance larval survivability. Finally, documentation of larval development in normal as well as in cryopreserved larvae of Crassostrea gigas in this project increased our understanding of biology of Pacific oyster larvae and fills the existing gap bridging the cryopreservation studies on relevant species. This study is an important step to reduce the commercial hatchery cultivation cost for this species as it will be easier to distinguish healthy larvae from the abnormal ones.