Metabolic Regulation of Copper Toxicity during Marine Mussel Embryogenesis.

The development of new tools for assessing the health of cultured shellfish larvae is crucial for aquaculture industries to develop and refine hatchery methodologies. We established a large-volume ecotoxicology/health stressor trial, exposing mussel (Perna canaliculus) embryos to copper in the presence of ethylenediaminetetraacetic acid (EDTA). GC/MS-based metabolomics was applied to identify potential biomarkers for monitoring embryonic/larval health and to characterise mechanisms of metal toxicity. Cellular viability, developmental abnormalities, larval behaviour, mortality, and a targeted analysis of proteins involved in the regulation of reactive oxygen species were simultaneously evaluated to provide a complementary framework for interpretative purposes and authenticate the metabolomics data. Trace metal analysis and speciation modelling verified EDTA as an effective copper chelator. Toxicity thresholds for P. canaliculus were low, with 10% developmental abnormalities in D-stage larvae being recorded upon exposure to 1.10 µg·L-1 bioavailable copper for 66 h. Sublethal levels of bioavailable copper (0.04 and 1.10 µg·L-1) caused coordinated fluctuations in metabolite profiles, which were dependent on development stage, treatment level, and exposure duration. Larvae appeared to successfully employ various mechanisms involving the biosynthesis of antioxidants and a restructuring of energy-related metabolism to alleviate the toxic effects of copper on cells and developing tissues. These results suggest that regulation of trace metal-induced toxicity is tightly linked with metabolism during the early ontogenic development of marine mussels. Lethal-level bioavailable copper (50.3 µg·L-1) caused severe metabolic dysregulation after 3 h of exposure, which worsened with time, substantially delayed embryonic development, induced critical oxidative damage, initiated the apoptotic pathway, and resulted in cell/organism death shortly after 18 h of exposure. Metabolite profiling is a useful approach to (1) assess the health status of marine invertebrate embryos and larvae, (2) detect early warning biomarkers for trace metal contamination, and (3) identify novel regulatory mechanisms of copper-induced toxicity.

Can additives ameliorate oxidative stress and improve development of Greenshell mussel (Perna canaliculus) oocytes during cryopreservation?

BACKGROUND: Cryopreservation of P. canaliculus oocytes has not yet been achieved. OBJECTIVE: The present study is to investigate whether the incorporation of: DMSO (0.09%), α-tocopherol (0.1 mM) plus taurine (1 mM) and ethylenediaminetetraacetic acid (EDTA; 0.1 mM), is beneficial during cryopreservation. METHODS: These three additives were incorporated to both the cryoprotectant (CPA) and recovery media, and evaluated in terms of development and oxidative stress at three key stages of cryopreservation: 1) cryoprotectant addition [10% v/v ethylene glycol plus 0.2M trehalose; final concentration], 2) cooling to -6 degrees C, and 3) cooling to -35 degrees C and liquid nitrogen immersion. RESULTS: Over all treatments (including controls) progressive cryopreservation steps resulted in a decrease in fertilization and development to D-larvae, an increase in macromolecular oxidative damage markers (protein carbonyls, lipid hydroperoxides and oxidized DNA), and a decrease in enzymatic (superoxide dismutase, catalase, glutathione S-transferase and glutathione reductase) and non-enzymatic antioxidants. CONCLUSION: Whilst results varied, the major effects of the additives were the improved percentage fertilization and a decrease in macromolecular damage.

An investigation of oxidative stress and antioxidant biomarkers during Greenshell mussel (Perna canaliculus) oocyte cryopreservation.

Oxidative damage to proteins and lipids, the enzymatic and nonenzymatic antioxidants' response, and the fertilization and development capability of Perna canaliculus oocytes were investigated at critical treatment steps in a previously published controlled-rate cryopreservation protocol. The cryoprotectant (CPA) from this protocol comprises 10% ethylene glycol (v:v) and 0.2 M trehalose (wt/vol) final concentration. Critical treatment steps included (1) seawater control, (2) CPA addition, (3) CPA addition followed by cooling to -6 °C, (4) CPA addition and cooling to -10 °C, and (5) CPA addition and cooling to -35 °C and immersion in liquid nitrogen (LN). The percentage of fertilized oocytes was 53.8 ± 13.3% in the seawater control but was reduced to 26.0 ± 15.6% after -35 °C + LN treatment, whereas development to D-larvae was 21.0 ± 6.4% in the seawater control reduced to 4.8 ± 2.9% after cooling to -6 °C, and was zero at all the subsequent cooling steps. All oxidative damage biomarkers, protein carbonyls (PCs) and lipid hydroperoxides (LPs), and antioxidants, superoxide dismutase (SOD), catalase, glutathione peroxidase, percent reduced glutathione (%GSH), and total glutathione (defined as glutathione; reduced [GSH] plus glutathione disulphide; derived from two molecules of GSH [GSSG]) were measured over all treatments on unfertilized oocytes over a post-treatment recovery period of 0 to 240 minutes in seawater. An ANOVA showed that both treatment and post-treatment periods had significant effects on the concentrations of all biomarkers (P < 0.05). Protein carbonyls and LPs increased very little after CPA addition and cooling treatments, when compared with the seawater control, but large increases up to sixfold occurred between 0 and 240 minutes for the -35 °C + LN treatment. Concentrations of SOD, catalase, total glutathione, and %GSH at 0 minutes decreased by -31.2%, -26.9%, -21.9%, and -25.0%, respectively, between the seawater control and the -35 °C + LN treatment. In contrast, glutathione peroxidase concentrations at 0 minutes increased by 34.3% between the seawater control and the -35 °C + LN treatment. Although most biochemical biomarkers showed an increasing trend over time (0-240 minutes), total glutathione decreased in concentration over time in all treatments, with the greatest decrease after the -35 °C + LN treatment (-41.2%). Significant correlations between biomarkers and D-larvae yield were negative for LPs and positive for SOD, total glutathione, and %GSH (P < 0.05). This is the first report of an investigation using these oxidative stress biomarkers and antioxidant responses on mussel oocytes, and the results have proved useful in characterizing cellular injury during the cryopreservation process.

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 Zealand's 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.