New insights into ion regulation of cephalopod molluscs: a role of epidermal ionocytes in acid-base regulation during embryogenesis

Abstract

The constraints of an active life in a pelagic habitat led to numerous convergent morphological and physiological adaptations that enable cephalopod molluscs and teleost fishes to compete for similar resources. Here, we show for the first time that such convergent developments are also found in the ontogenetic progression of ion regulatory tissues; as in teleost fish, epidermal ionocytes scattered on skin and yolk sac of cephalopod embryos appear to be responsible for ionic and acid-base regulation before gill epithelia become functional. Ion and acid-base regulation is crucial in cephalopod embryos, as they are surrounded by a hypercapnic egg fluid with a Pco(2) between 0.2 and 0.4 kPa. Epidermal ionocytes were characterized via immunohistochemistry, in situ hybridization, and vital dye-staining techniques. We found one group of cells that is recognized by concavalin A and MitoTracker, which also expresses Na(+)/H(+) exchangers (NHE3) and Na(+)-K(+)-ATPase. Similar to findings obtained in teleosts, these NHE3-rich cells take up sodium in exchange for protons, illustrating the energetic superiority of NHE-based proton excretion in marine systems. In vivo electrophysiological techniques demonstrated that acid equivalents are secreted by the yolk and skin integument. Intriguingly, epidermal ionocytes of cephalopod embryos are ciliated as demonstrated by scanning electron microscopy, suggesting a dual function of epithelial cells in water convection and ion regulation. These findings add significant knowledge to our mechanistic understanding of hypercapnia tolerance in marine organisms, as it demonstrates that marine taxa, which were identified as powerful acid-base regulators during hypercapnic challenges, already exhibit strong acid-base regulatory abilities during embryogenesis.

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

“…(B) Epidermal ionocytes of cephalopod early life stages were first discovered using immunohistochemical techniques showing the presence of Na C /K C -rich cells scattered over yolk and head in squid and cuttlefish embryos adopted and modiefied from. 74 Similar to the situation in teleosts the yolk constitutes the largest, well perfused surface area, in the developing embryo, making these epithelia prime sites for gas exchange and ionic regulation before the development of gill epithelia. Figure 2.…”

“…73 During ontogeny of the cuttlefish S. officinalis the activity of the NKA measured in whole animal homogenates increases exponentially in the early embryonic phase and reaches its maximum activity toward hatch of the paralarva/juvenile animal. 74 In squid, cuttlefish and octopods high concentrations of NKA were found in different tissues including neurons, renal appendages, branchial heart appendages and gills 38,75,76 (Fig. 4).…”

“…Before gill epithelia are fully developed, epidermal ionocytes on the skin mediate ion and acid-base balance in teleosts [58][59][60][61][62] ( Fig. 1).…”

“…The VHA has been demonstrated to be highly expressed in most vertebrate and invertebrate ion regulatory epithelia including kidneys, 78 skin and gills. 74,75,79,81,82 In most epithelia contributing to extracellular pH regulation the VHA is expressed in apical membranes contributing to the secretion of protons from the organism. 78,81 However, a basolateral orientation of the VHA has been associated with base secretory processes through cooperating with apical pendrin (SLC26A4) to secrete base in type B intercalated cells of the mammalian kidney 78,83 or in branchial epithelia of some elasmobranchs.…”

“…12,74,89 Proton selective electrodes in combination with 5-ethylisopropyl amiloride (EIPA) a specific inhibitor for NHE proteins could demonstrate that at least 60 % of H C secretion across the surface of epidermal ionocytes of embryonic squids is mediated through Na C /H C antiport. 74 The involvement of NHEs instead of proton pumps like the VHA to mediate apical proton secretion in marine species is thermodynamically favored by the strong Na C gradient between cytosol (30 mM) and seawater (470 mM). 94,95 It was hypothesized that NHE based proton secretion may represent a common pathway in marine organisms which however needs further confirmation by testing more marine species including crustaceans, echinoderms, annelids, and mollusks.…”