Abstract
BackgroundRegulation of pH homeostasis is a central feature of all animals to cope with acid–base disturbances caused by respiratory CO2. Although a large body of knowledge is available for vertebrate and mammalian pH regulatory systems, the mechanisms of pH regulation in marine invertebrates remain largely unexplored.ResultsWe used squid (Sepioteuthis lessoniana), which are known as powerful acid–base regulators to investigate the pH regulatory machinery with a special focus on proton secretion pathways during environmental hypercapnia. We cloned a Rhesus protein (slRhP), V-type H+-ATPase (slVHA) and the Na+/H+ exchanger 3 (slNHE3) from S. lessoniana, which are hypothesized to represent key players in proton secretion pathways among different animal taxa. Specifically designed antibodies for S. lessoniana demonstrated the sub-cellular localization of NKA, VHA (basolateral) and NHE3 (apical) in epidermal ionocytes of early life stages. Gene expression analyses demonstrated that slNHE3, slVHA and slRhP are up regulated in response to environmental hypercapnia (pH 7.31; 0.46 kPa pCO2) in body and yolk tissues compared to control conditions (pH 8.1; 0.045 kPa pCO2). This observation is supported by H+ selective electrode measurements, which detected increased proton gradients in CO2 treated embryos. This compensatory proton secretion is EIPA sensitive and thus confirms the central role of NHE based proton secretion in cephalopods.ConclusionThe present work shows that in convergence to teleosts and mammalian pH regulatory systems, cephalopod early life stages have evolved a unique acid–base regulatory machinery located in epidermal ionocytes. Using cephalopod molluscs as an invertebrate model this work provides important insights regarding the unifying evolutionary principles of pH regulation in different animal taxa that enables them to cope with CO2 induced acid–base disturbances.
Highlights
Regulation of pH homeostasis is a central feature of all animals to cope with acid–base disturbances caused by respiratory CO2
The wet mass (WM) of S. lessoniana late-stage embryos at the time point of 120 h was significantly reduced in 0.46 kPa CO2 treated animals (110.0 ± 11.3 mgWM), compared to control animals (151.5 ± 4.2 mgWM)
The present study demonstrated the up regulation of genes involved in primary- (e.g. V-type-H+-ATPases) and secondary-active (e.g. Na+/H+-exchanger 3 (NHE3)) acid-secretion pathways
Summary
Regulation of pH homeostasis is a central feature of all animals to cope with acid–base disturbances caused by respiratory CO2. The embryonic development of most marine fish, crustaceans and molluscs takes place within an egg capsule that creates a barrier between the developing embryo and the surrounding environment to protect from biotic (e.g. predation) and abiotic stressors (e.g. osmo-protection) [1,2,3,4]. Embryos of oviparous organisms are exposed to high respiratory pCO2 and low pO2 within the egg capsule due to their increasing metabolic rate and the egg capsule wall acting as a diffusion barrier [5,10,11]. Elevated seawater pCO2 acts in an additive fashion on the naturally hypercapnic microenvironment within the egg, which may challenge the acid–base regulatory machinery of the developing embryo
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