Description: 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 Pco2 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.

Description: Fish early life stages have been shown to react sensitive to simulated ocean acidification. In particular, acid–base disturbances elicited by altered seawater carbonate chemistry have been shown to induce pathologies in larval fish. However, the mechanisms underlying these disturbances are largely unknown. We used gene expression profiling of genes involved in acid–base regulation and metabolism to investigate the effects of seawater hypercapnia on developing Japanese ricefish (medaka; Oryzias latipes). Our results demonstrate that embryos respond with delayed development during the time window of 2–5 dpf when exposed to a seawater pCO2 of 0.12 and 0.42 kPa. This developmental delay is associated with strong down-regulation of genes from major metabolic pathways including glycolysis (G6PDH), Krebs cycle (CS) and the electron transport chain (CytC). In a second step we identified acid–base relevant genes in different ontogenetic stages (embryos, hatchlings and adults) and tissues (gill and intestine) that are up regulated in response to hypercapnia, including NHE3, NBCa, NBCb, AE1a, AE1b, ATP1a1a.1, ATP1a1b, ATP1b1a, Rhag, Rhbg and Rhcg. Interestingly, NHE3 and Rhcg expressions were increased in response to environmental hypercapnia in all ontogenetic stages and tissues tested, indicating the central role of these proteins in acid–base regulation. Furthermore, the increased expression of genes from amino acid metabolism pathways (ALT1, ALT2, AST1a, AST1b, AST2 and GLUD) suggests that energetic demands of hatchlings are fueled by the breakdown of amino acids. The present study provides a first detailed gene expression analysis throughout the ontogeny of a euryhaline teleost in response to seawater hypercapnia, indicating highest sensitivity in early embryonic stages, when functional ion regulatory epithelia are not yet developed.

Description: 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 pCO2 of 0.2-0.4 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 (NHE) 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.

Description: The present study provides a first finding of the acid-base regulating machinery (ion-transporters relevant for acid-base regulation) in cephalopod, and series of studies showed that they exhibit specialized ion regulatory cells (ionocytes) on their skin and yolk epithelium. A feature that was so far only reported for fish. In addition, several ion regulatory genes were identified in cephalopod to be involved in the compensation of CO2 induced acid-base disturbances, including Na+/H+-exchanger (NHE3), ammonium transporters (Rhcg) and vacuolar H+-ATPase (VHA) by being significantly up regulated in response to elevated sea water pCO2. Here we show for the first time that cephalopod embryos exhibit epidermal ionocytes and that the skin is a mayor site for proton excretion. Similar to fish, ionocytes located on the skin and yolk of cephalopod embryos are characterized by high concentrations of mitochondria. These similar responses towards elevated water pCO2 and sensitivity thresholds during life history may be explained by convergent acid-base regulatory features of cephalopods and fish.