Effects of seawater temperature and acute Vibriosp. 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 Vibriosp. 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.

Oxidative Damage and Antioxidants as Markers for the Selection of Emersion Hardening Treatments in GreenshellTM Mussel Juveniles (Perna canaliculus).

Transport out of the water is one of the most challenging events for juvenile Perna canaliculus and can be a highly inefficient process, with many juveniles subsequently being lost following extended periods of emersion. Hardening techniques offer a possible method for reducing transport-related stress. In this study, different hardening treatments (short, long and intermittent sub-lethal emersion) were used to prepare ~1.2 mm P.canaliculus for transport (20 h) and subsequent reoxygenation stress during re-immersion (i.e., recovery). The oxidative stress responses, resettlement behaviour, respiration rates and survival of the mussels after transport and during recovery were all assessed. Short emersion (1 h) as a hardening treatment prior to transport did not cause major stress to the mussels, which maintained respiration at control levels, showed significantly stimulated antioxidant defences during recovery, showed greater resettlement behaviour and remained viable after 24 h of recovery. In comparison, the long and intermittent emersion treatments negatively impacted oxidative stress responses and affected the viability of the mussels after 24 h of recovery. This study showed that exposing juvenile P.canaliculus to a mild stress prior to transport may stimulate protective mechanisms, therefore eliciting a hardening response, but care must be taken to avoid overstressing the mussels. Improving the management of stress during the transport of juvenile mussels may be key to minimising mussel losses and increasing harvest production, and biomarkers associated with oxidative stress/antioxidant metabolism could be valuable tools to ensure emersion hardening does not overstress the mussels and reduce survival.