Effects of seawater temperature and acute Vibrio sp. challenge on the haemolymph immune and metabolic responses of adult mussels (Perna canaliculus)

The New Zealand Greenshell™ mussel (Perna canaliculus) is an endemic bivalve species with cultural importance, that is harvested recreationally and commercially. However, production is currently hampered by increasing incidences of summer mortality in farmed and wild populations. While the causative factors for these mortality events are still unknown, it is believed that increasing seawater temperatures and pathogen loads are potentially at play. To improve our understanding of these processes, challenge experiments were conducted to investigate the combined effects of increased seawater temperature and Vibrio infection on the immune and metabolic responses of adult mussels. Biomarkers that measure the physiological response of mussels to multiple-stressors can be utilised to study resilience in a changing environment, and support efforts to strengthen biosecurity management. Mussels acclimated to two temperatures (16 °C and 24 °C) were injected with either autoclaved, filtered seawater (control) or Vibrio sp. DO1 (infected). Then, haemolymph was sampled 24 h post-injection and analysed to quantify haemocyte immune responses (via flow-cytometry), antioxidant capacity (measured electrochemically) and metabolic responses (via gas chromatography-mass spectrometry) to bacterial infection. Both seawater temperature and injection type significantly influenced the immune and metabolite status of mussels. A lack of interaction effects between temperature and injection type indicated that the effects of Vibrio sp. 24 h post-infection were similar between seawater temperatures. Infected mussels had a higher proportion of dead haemocytes and lower overall haemocyte counts than uninfected controls. The proportion of haemocytes showing evidence of apoptosis was higher in mussels held at 24 °C compared with those held at 16 °C. The proportion of haemocytes producing reactive oxygen species did not differ between temperatures or injection treatments. Mussels held at 24 °C exhibited elevated levels of metabolites linked to the glycolysis pathway to support energy production. The saccharopin-lysine pathway metabolites were also increased in these mussels, indicating the role of lysine metabolism. A decrease in metabolic activity (decreases in BCAAs, GABA, urea cycle metabolites, oxidative stress metabolites) was largely seen in mussels injected with Vibrio sp. Itaconate increased as seen in previous studies, suggesting that antimicrobial activity may have been activated in infected mussels. This study highlights the complex nature of immune and metabolic responses in mussels exposed to multiple stressors and gives an insight into Vibrio sp. infection mechanisms at different seawater temperatures

Development of a non-lethal biopsy technique for estimating total tetrodotoxin concentrations in the grey side-gilled sea slug Pleurobranchaea maculata

High concentrations of tetrodotoxin (TTX) have been detected in some New Zealand populations of Pleurobranchaea maculata (grey side-gilled sea slug). Within toxic populations there is significant variability in TTX concentrations among individuals, with up to 60-fold differences measured. This variability has led to challenges when conducting controlled laboratory experiments. The current method for assessing TTX concentrations within P. maculata is lethal, thus multiple individuals must be harvested at each sampling point to produce statistically meaningful data. In this study a method was developed for taking approximately 200 mg tissue biopsies using a TemnoEvolution® 18G × 11 cm Biopsy Needle inserted transversely into the foot. Correlation between the TTX concentrations in the biopsy sample and total TTX levels and in individual tissues were assessed. Six P. maculata were biopsied twice (nine days apart) and each individual was frozen immediately following the second sampling. Tetrodotoxin concentrations in biopsy samples and in the gonad, stomach, mantle and the remaining combined tissues and fluids were measured using liquid chromatography-mass spectrometry. Based on the proportional weight of the organs/tissues a total TTX concentration for each individual was calculated. There were strong correlations between biopsy TTX concentrations and the total (r2 = 0.88), stomach (r2 = 0.92) and gonad (r2 = 0.83) TTX concentrations. This technique will enable more robust laboratory studies to be undertaken, thereby assisting in understanding TTX kinetics, ecological function and origin within P. maculata