Branchial NH₄⁺-dependent acid-base transport mechanisms and energy metabolism of squid (Sepioteuthis lessoniana) affected by seawater acidification

Background: Cephalopods have evolved strong acid-base regulatory abilities to cope with CO₂ induced pH fluctuations in their extracellular compartments to protect gas transport via highly pH sensitive hemocyanins. To date, the mechanistic basis of branchial acid-base regulation in cephalopods is still poorly understood, and associated energetic limitations may represent a critical factor in high power squids during prolonged exposure to seawater acidification.Results: The present work used adult squid Sepioteuthis lessoniana to investigate the effects of short-term (few hours) to medium-term (up to 168 h) seawater acidification on pelagic squids. Routine metabolic rates, NH₄ ⁺ excretion, extracellular acid-base balance were monitored during exposure to control (pH 8.1) and acidified conditions of pH 7.7 and 7.3 along a period of 168 h. Metabolic rates were significantly depressed by 40% after exposure to pH 7.3 conditions for 168 h. Animals fully restored extracellular pH accompanied by an increase in blood HCO₃ ^- levels within 20 hours. This compensation reaction was accompanied by increased transcript abundance of branchial acid-base transporters including V-type H⁺-ATPase (VHA), Rhesus protein (RhP), Na⁺/HCO₃ ^- cotransporter (NBC) and cytosolic carbonic anhydrase (CAc). Immunocytochemistry demonstrated the sub-cellular localization of Na⁺/K⁺-ATPase (NKA), VHA in basolateral and Na⁺/H⁺-exchanger 3 (NHE3) and RhP in apical membranes of the ion-transporting branchial epithelium. Branchial VHA and RhP responded with increased mRNA and protein levels in response to acidified conditions indicating the importance of active NH₄ ⁺ transport to mediate acid-base balance in cephalopods.Conclusion: The present work demonstrated that cephalopods have a well developed branchial acid-base regulatory machinery. However, pelagic squids that evolved a lifestyle at the edge of energetic limits are probably more sensitive to prolonged exposure to acidified conditions compared to their more sluggish relatives including cuttlefish and octopods.