Metabolomic analysis of heat-hardening in adult green-lipped mussel (Perna canaliculus): A key role for succinic acid and the GABAergic synapse pathway

We evaluated the thermotolerance (LT50) of adult green-lipped mussels (Perna canaliculus) following an acute thermal challenge in the summer of 2012 and the winter of 2013. Mussels were grouped into two treatments, naive (N, no prior heat treatment) and heat-hardened (HH = 1 h at 29 degrees C, 12 h recovery at ambient) before being immersed for 3 h in water of varying temperature, i.e. Ambient (Control), 25, 29, 31, 33, and 35 degrees C with subsequent mortality monitored for 30 days. As expected, naive mussels were less thermotolerant than heat-hardened i.e. Summer LT50, N = 31.9, HH = 33.5 degrees C; Winter LT50, N = 31.4, HH = 33.8 degrees C. Moreover, at 33 degrees C no heat-hardened mussels died compared to 100% mortality in naive specimens. At 35 degrees C all mussels died regardless of treatment. For the 'Summer' mussels, metabolite abundances in gill tissues of both naive and heat-hardened mussels were quantified. For mussels at 33 degrees C, succinic acid was significantly higher in naive mussels than heat-hardened mussels, indicating perturbations to mitochondrial pathways in these thermally stressed mussels. Additionally, analysis of biochemical pathway activity suggested a loss of neural control i.e. significantly reduced GABAergic synapse activity, in naive vs. heat-hardened mussels at 33 degrees C. Taken together these findings suggest that heat hardening improves mussel survival at higher temperatures by delaying the onset of cellular anaerobic metabolism, and by maintaining inhibition of neural pathways. Such results offer new perspectives on the complex suite of sub-cellular stress responses operating within thermally stressed organisms.