Elevated seawater Pco₂differentially affects branchial acid-base transporters over the course of development in the cephalopodSepia officinalis

Abstract

The specific transporters involved in maintenance of blood pH homeostasis in cephalopod molluscs have not been identified to date. Using in situ hybridization and immunohistochemical methods, we demonstrate that Na(+)/K(+)-ATPase (soNKA), a V-type H(+)-ATPase (soV-HA), and Na(+)/HCO(3)(-) cotransporter (soNBC) are colocalized in NKA-rich cells in the gills of Sepia officinalis. mRNA expression patterns of these transporters and selected metabolic genes were examined in response to moderately elevated seawater Pco(2) (0.16 and 0.35 kPa) over a time course of 6 wk in different ontogenetic stages. The applied CO(2) concentrations are relevant for ocean acidification scenarios projected for the coming decades. We determined strong expression changes in late-stage embryos and hatchlings, with one to three log2-fold reductions in soNKA, soNBCe, socCAII, and COX. In contrast, no hypercapnia-induced changes in mRNA expression were observed in juveniles during both short- and long-term exposure. However, a transiently increased ion regulatory demand was evident during the initial acclimation reaction to elevated seawater Pco(2). Gill Na(+)/K(+)-ATPase activity and protein concentration were increased by ~15% during short (2-11 days) but not long-term (42-days) exposure. Our findings support the hypothesis that the energy budget of adult cephalopods is not significantly compromised during long-term exposure to moderate environmental hypercapnia. However, the downregulation of ion regulatory and metabolic genes in late-stage embryos, taken together with a significant reduction in somatic growth, indicates that cephalopod early life stages are challenged by elevated seawater Pco(2).

Recent advances in understanding trans-epithelial acid-base regulation and excretion mechanisms in cephalopods

“…While some marine species have been identified as rather sensitive (e.g., less active calcifying species such as corals or echinoderms) 30,47,48 others, mostly active species such as adult fish, crustaceans and cephalopods can tolerate high CO 2 concentrations over long exposure times. 49,50 A major hypothesis that had to be tested was, if the ability to compensate for a hypercapnia -induced acidosis by actively accumulating bicarbonate and eliminating protons from their body fluids is a general, unifying feature of tolerant organisms. 45 Thus, a first step was dedicated to the identification of acid-base regulatory structures in marine species and the characterization of the underlying ion-regulatory machinery including ion transporters and channels.…”

“…On the opposite site this egg capsule constitutes a diffusion barrier for gases such as O 2 and CO 2 , with diffusion coefficients of 10-20 % to that of pure seawater, leading to increasing hypoxic and hypercapnic conditions inside the egg along embryogenesis 49,51,52 (Fig. 1A).…”

“…38 Additionally, recent studies provided compelling evidence that the NKA of cephalopods is also a major player in regulating acid-base homeostasis. 12,49 In cephalopods including squid, cuttlefish and octopus, the NKA is present in high concentrations in gill epithelia located in basolateral membranes providing an electrochemical gradient that is used by a variety of secondary active transporters and ion channels.…”

“…Cloning strategies using degenerate primers designed against highly conserved regions of the NKA, VHA, Na C /H C -exchangers (NHEs), carbonic anhydrase (CA) and Na C /HCO 3 ¡ cotransporters (NBC e ) resulted in partial DNA sequences encoding these enzymes in squid and cuttlefish. 12,49 In situ hybridization and immunohistochemical methods with antibodies specifically designed against acid-base transporters of squid, revealed the sub-cellular localization of these enzymes in cephalopod ion-regulatory epithelia. 12,49 For example Na C /H C exchanger 3 (NHE3) which is an essential player for proton secretion in vertebrates 78,[91][92][93] is also expressed in apical membranes of epidermal ionocytes and gill epithelia of squid (S. lessoniana) and cuttlefish (S. oficinalis) (Fig.…”

The dynamic nitric oxide pattern in developing cuttlefish Sepia officinalis

“…The studies on the expression patterns of NK4, engrailed, Shh, Pax6 genes during Sepia development have provided some insights into nervous system differentiation, organization of the mantle, muscle formation and development (Baratte et al, 2007;Navet et al, 2008Navet et al, , 2009Grimaldi et al, 2008). Recently, the expression of branchial acid-base transporters has been followed during development and in response to environmental hypercapnia (Hu et al, 2011). In S. officinalis characterized by several morphological peculiarities within mollusc and a direct development, we report the presence of NO in embryos by detecting NO and NOS enzyme and by monitoring NOS expression levels during the various developmental stages.…”