Serean L. Adams

Towards cryopreservation of Greenshell mussel (Perna canaliculus) oocytes.

Cryopreservation is a powerful tool for selective breeding in aquaculture as it enables genetic material from selected stock to be stored and crossed at will. The aim of this study was to develop a method for cryopreserving oocytes of the Greenshelltrade mark mussel (Perna canaliculus), New Zealands main aquaculture species. The ability of oocytes to be fertilized post-thawing was used as the criterion for success in initial experiments and then subsequently, the ability of frozen oocytes to develop further to D-stage larvae was assessed. Ethylene glycol, propylene glycol, dimethyl sulphoxide and glycerol were evaluated at a range of concentrations with and without the addition of 0.2M trehalose using post-thaw fertilization as the endpoint. Ethylene glycol was most effective, particularly when used in combination with trehalose. A more detailed investigation revealed that ethylene glycol at 9% or 10% in the presence of 0.2-0.4M trehalose afforded the best protection. In experiments varying sperm to egg ratio and egg density in post-thaw fertilization procedures, D-larval yield averaged less than 1%. Following these results, a detailed experiment was conducted to determine the damaging steps in the cryopreservation process. Fertilization losses occurred at each step whereas D-larval yield approximately halved following CPA addition and was almost zero following cooling to -10 degrees C. Cryomicroscopy studies and fertilization results suggest that the inability of oocytes to develop to D-larvae stage after cooling to -10 degrees C and beyond are most likely related to some form of chilling injury rather than extracellular ice triggering intracellular ice formation. Further research is needed to determine the causes of this injury and to reduce CPA toxicity and/or osmotic effects.

Maintenance of cyanotoxin production by cryopreserved cyanobacteria in the New Zealand culture collection

Abstract A culture collection of freshwater planktonic and benthic cyanobacteria collected from sites across New Zealand has been established at the Cawthron Institute, Nelson, New Zealand. Limited resources led to uncertainty regarding the long‐term maintenance of this collection. The present study demonstrates cryopreservation to be a viable method for long‐term storage of cyanobacteria. Seventeen of 20 strains evaluated were successfully cryopreserved using the permeating cryopreservation agent dimethyl sulfoxide (Me2SO), at a final concentration of 15% (v/v). Cyanotoxin analysis was undertaken on selected strains known to produce microcystins, nodularin, anatoxin‐a, and saxitoxins. All strains retained their ability to produce these toxins following cryopreservation.

Comparative cryopreservation study of trochophore larvae from two species of bivalves: Pacific oyster (Crassostrea gigas) and Blue mussel (Mytilus galloprovincialis).

Oysters and mussels are among the most farmed species in aquaculture industry around the world. The aim of this study was to test the toxicity of cryoprotective agents to trochophore larvae from two different species of bivalves and develop an improved cryopreservation protocol to ensure greater efficiency in the development of cryopreserved trochophores (14 h old oyster larvae and 20 h old mussel larvae) to normal D-larvae for future developments of hatchery spat production. The cryopreservation protocol producing the best results for oyster trochophores (60.0 ± 6.7% normal D-larvae) was obtained by holding at 0 °C for 5 min then cooling at 1 °C min(-1) to -10 °C and holding for 5 min before cooling at 0.5 °C to -35 °C, holding 5 min and then plunging into liquid nitrogen (LN), using 10% ethylene glycol. For mussel experiments, no significant differences were found when cooling at 0.5 °C min(-1) or at 1 °C min(-1) for CPA combinations with 10% ethylene glycol and at 0.5 °C min(-1). Using these combinations, around half of trochophores were able to develop to normal D-larvae post-thawing (48.9 ± 7.6% normal D-larvae).

Cryopreservation of Greenshell™ mussel (Perna canaliculus) trochophore larvae ☆

The Greenshell™ mussel (Perna canaliculus) is the main shellfish species farmed in New Zealand. The aim of this study was to evaluate the effects of cryoprotectant concentration, loading and unloading strategy as well as freezing and thawing method in order to develop a protocol for cryopreservation of trochophore larvae (16-20 h old). Toxicity tests showed that levels of 10-15% ethylene glycol (EG) were not toxic to larvae and could be loaded and unloaded in a single step. Through cryopreservation experiments, we designed a cryopreservation protocol that enabled 40-60% of trochophores to develop to D-larvae when normalized to controls. The protocol involved: holding at 0 °C for 5 min, then cooling at 1 °C min⁻¹ to -10 °C, holding for a further 5 min, then cooling at 0.5 °C min⁻¹ to -35 °C followed by a 5 min hold and then plunging into liquid nitrogen. A final larval rearing experiment of 18 days was conducted to assess the ability of these frozen larvae to develop further. Results showed that only 2.8% of the frozen trochophores were able to develop to competent pediveligers.

Cryopreservation of Greenshell™ Mussel ( Perna canaliculus ) Sperm

Cryopreservation is a valuable technique for aquaculture as it enables a library or bank of genetically valuable animals to be maintained in a cost-effective manner. Here, we describe a method to cryopreserve the sperm of the Greenshell™ mussel (Perna canaliculus) and how to use the sperm post-thawing to maximize larval production from thawed sperm in selective breeding.

Food and Feed Applications of Algae

Microalgae and seaweeds have a long history and increasingly important applications as both food ingredients and animal feed. The vast majority of algal species have yet to be evaluated for these applications. However, due to their extensive diversity, it is likely that they will lead to the discovery of many new algal products and processes in the future. This chapter covers algae as food, feed, nutraceuticals, functional food and food ingredients as well as production systems for food from algae.

Diet Conditioning of Pacific Oyster, Crassostrea gigas, Broodstock to Improve Oocyte Cryopreservation Success

ABSTRACT Female broodstock of Pacific oyster (Crassostrea gigas) were conditioned using different diets to determine the effect of diet on oocyte cryopreservation success. Oocyte biochemical composition, fatty acid profile, and fertilization success before and after cryopreservation were determined. Broodstock were conditioned in 2 stages: a cold preconditioning stage during which animals were either starved or fed live microalgae, and a warm conditioning stage during which animals were again either starved or fed live microalgae, AlgaMac-2000, or a mixture of corn and rice flour. Overall, oysters that were fed microalgae during cold preconditioning produced oocytes with significantly more lipid than oysters that were starved. Similarly, those that were fed during warm conditioning produced oocytes with more lipid than starved oysters, regardless of diet. There was no effect of conditioning regime on the level of polyunsaturated fatty acids (PUFAs) in the polar lipid fraction. However, oysters that were starved or fed AlgaMac-2000 during warm conditioning had higher PUFA levels than oysters fed microalgae or flour. Broodstock conditioning regime had no effect on the fertilization success of oocytes prior to cryopreservation, but those that were fed during cold preconditioning produced oocytes that had significantly higher fertilization success after thawing.

Optimising conditions for growth and xanthophyll production in continuous culture of Tisochrysis lutea using photobioreactor arrays and central composite design experiments

ABSTRACT The production of valuable metabolites from microalgae represents a potentially sustainable source of a range of products that can be difficult to synthesise directly. Microalgae respond to the dynamic and often subtly shifting growth environment in a complex way. The optimal conditions for growth can be quite different to those needed for optimal product generation, depending on the nature of the biosynthesis of that product. This is especially so for secondary metabolites. A combination of a multi-vessel photobioreactor array, where certain growth conditions can be monitored and controlled precisely together with an experimental design matrix has been used to determine the optimal combination of temperature, irradiance and pH for a group of xanthophylls including fucoxanthin in the algae Tisochrysis lutea in continuous culture. Continuous culture as a mode is more suited for industrial production than batch mode in which the media constituents and algal population change dramatically over time. The central composite design experiment matrix has a range of set values for each parameter being investigated that bracket the optimal conditions. The three parameters investigated in this work (temperature, irradiance and pH) are major factors influencing algal growth. The method can be applied to other parameters that might affect growth or might affect production of a metabolite of interest, such as a nutrient level. The combined approach has been used previously to indicate optimal growth conditions for biomass generation and this work is one of the first to apply it to the generation of an algal product of interest.

Multi-technique approach to characterise the effects of cryopreservation on larval development of the Pacific oyster (Crassostrea gigas)

Cryopreservation experiments were conducted on D-stage larvae of the Pacific oyster (Crassostrea gigas) to investigate the effects of two cryoprotectant solutions and three cooling rates on larval development from 1 to 22 days post-fertilisation. Cryoprotectant solutions were made up to final concentrations (after 1:1 dilution with larvae) of 10% ethylene glycol, 1% polyvinylpyrrolidone and either 0.2 or 0.4 M trehalose. Three cooling rates (0.5, 1 and 2 °C min−1 between −10 and −35 °C post-holding) were tested in an orthogonal design with the two cryoprotectants. Results indicate that control larvae out-performed all cryopreservation treatments for survival, feeding consumption and shell length parameters. However, larvae exposed to 0.4 M trehalose did considerably better than those exposed to 0.2 M trehalose, regardless of cooling rate conditions. Scanning electron and light microscopy observations were used to assess larval morphology and organogenesis, indicating that treatments with surviving larvae were morphologically and developmentally similar to control larvae.

Investigation of early mussel (Perna canaliculus) development using histology, SEM imaging, immunochemistry and confocal microscopy

ABSTRACT A comprehensive study, incorporating histology, light microscopy, scanning electron microscopy, immunochemistry and confocal microscopy, was performed to investigate embryogenesis and larval development of the New Zealand Greenshell™ mussel, Perna canaliculus. Detailed observations with this multi-technique approach revealed a gastrula stage at 18 hours post-fertilization, with the appearance of a blastopore, apical sense organ and enclosing vegetal pole. Early D-stage larvae showed limited alimentary organogenesis and clear initiation of a developing nervous system. Shell morphology of D-larvae was characterized by a flat, hinged, pitted–punctate prodissoconch I shell, followed closely by commarginal growth lines within the prodissoconch II shell. Early umbo larvae had a protruding functioning velum, and well-developed posterior adductor and velar retractor muscles. Significant progression in neuronal development occurred just before the umbo stage with noticeable paired cerebral, pedal and visceral ganglia. Shell morphology was characterized by further prodissoconch II secretion with a more rounded umbonate appearance. During the transition through the pediveliger stage, rapid development of the gill rudiment, eye spot and functioning foot was observed with ongoing neuronal development. The first appearance of the dissoconch shell layer took place during this transition, at which point the nervous system was highly distinct with innervations extending throughout muscle regions and between ganglia. This study provides the first comprehensive documentation of the developmental stages of P. canaliculus larvae from fertilization to settlement. The study highlights the advantages of using a combination of techniques to understand larval development and provides crucial information to identify larval performance during larval rearing.