pH Regulation and Excretion in Echinoderms

Abstract

As osmoconformers with low metabolic rates, echinoderms are generally regarded as rather weak acid–base regulators. Accordingly, little attention has been placed on whether echinoderms have evolved mechanisms to regulate ionic homeostasis. In the last century, only few studies examined the acid–base physiology of echinoderms, mostly sea urchins. These studies were conducted in an environmental context as some species inhabit rock pools and experience periodic emersion from their marine environment that can cause a metabolic acidosis. Lately, acid–base physiology in marine invertebrates, especially calcifying species, has received considerable attention as these animals were considered as particularly vulnerable in the context of CO₂-induced ocean acidification. A substantial extracellular pH regulatory ability has been hypothesized to determine the degree of sensitivity in marine taxa. The emerging field of ocean acidification research in the last decade also shed new light on the acid–base physiology in echinoderms. Therefore, most of the available literature on echinoderm acid–base physiology describes the effects of CO₂-induced seawater acidification on the extracellular acid–base homeostasis of echinoderm adults and larvae. This book chapter will summarize the most recent advances of acid–base physiology and nitrogen excretion in echinoderms in the face of ocean acidification. It will cover adult pH regulation as far as mechanistic data is available and also echinoderm larval physiology in respect to intracellular and extracellular acid–base regulation. Finally, it will make a short excursion into the ocean acidification research field, since most of the conducted research started because of this.

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10.1 Adult Echinoderms

10.1.1 Extracellular Acid–Base Regulation

Echinoderms consist of five classes: Crinoidea (sea lilies), Asteroidea (sea stars), Ophiuroidea (brittle stars), Echinoidea (sea urchins), and Holothuroidea (sea cucumbers). Most of them are key stone species in many marine ecosystems and especially sea urchins and sea cucumbers are economically important organisms in many countries.

Adult echinoderms are characterized by their pentameric symmetry and an ambulacrarian vascular system (. see Fig. 10.1 for an echinoid body plan). Some of the classes have heavily calcified tests, like sea urchins, brittle stars, and crinoids. Sea stars and sea cucumbers have calcified spines or test plates and are not as heavily calcified. All echinoderms have a so-called coelomic fluid, their blood derivate with coelomic cells that resemble higher animals’ immune cells. The composition of the coelomic fluid is similar to that of seawater and is prone to acid–base disturbances when the animals encounter hypercapnic conditions or emersion. The coelomic fluid can be relatively easily sampled from the animals, and examinations regarding the extracellular acid–base status in echinoderms were primarily conducted on this fluid.

Fig. 10.1

Body plan of an adult sea urchin (a) and an echinopluteus larvae (b). Aristotle’s lantern (AL); mouth (MO); ampullae (AMP); gut (GUT); gonads (GON); anus (AN); madrepore plate (MA); spines (SPI); tube feet (TF); extracellular matrix (ECM); spicule (SP); intestine (INT); stomach (ST); esophagus (OES); outer epithelium (OEP)

Among the five classes of echinoderms, there is data on extracellular acid–base status on four of them (no data on crinoids). Of these four classes of echinoderms, only Echinoidea and Ophiuroidea were reported to actively compensate for acid–base disturbances indicated by an accumulation of bicarbonate in response to elevated pCO₂ (Stumpp et al. 2012b; Calosi et al. 2013; Hu et al. 2014). The effects of CO₂-induced seawater acidification on echinoderms have lately received considerable attention, as calcifying species were predicted to be particularly sensitive to ocean acidification. The maintenance of calcification is directly linked to acid–base regulatory capacities making this physiological process a key candidate to address sensitivities in echinoderms (Ries et al. 2009). Furthermore, echinoderms are key stone species in many marine habitats, and vulnerability of these organisms to near-future climate change could have severe repercussions on populations and ecosystem stabilities.

10.1.1.1 Echinoidea

Sea urchins of the species Strongylocentrotus droebachiensis exposed to 1400 and 3800 μatm CO₂ were able to fully or partially compensate extracellular pH (pH_e) disturbances within 7–10 days of exposure to elevated pCO₂ (Dupont and Thorndyke 2012; Stumpp et al. 2012b) and were able to sustain this compensation for over 45 days. The maximum bicarbonate accumulation recorded in this species was 2.5 mmol L⁻¹ above their control levels of 4.0–5.3 mmol L⁻¹ HCO₃ ⁻. Interestingly, the control levels of [HCO₃ ⁻]_e were already higher than that of seawater (around 2 mmol L⁻¹). Sea urchins of the species Arbacia lixula and Paracentrotus lividus also accumulated bicarbonate up to 3.5 mmol L⁻¹ (from 2 mmol L⁻¹ control) and 5–5.1 mmol L⁻¹ (3.6–3.8 mmol L⁻¹ control levels) in response to 1500 μatm and 2500 μatm CO₂ (Calosi et al. 2013; Collard et al. 2014b). With this increase in bicarbonate, both species were able to fully or partially compensate induced extracellular acid–base disturbances (Catarino et al. 2012). Tripneustes ventricosus accumulated up to a [HCO₃ ^-]_e of 5.0 mmol L⁻¹ (from 2.9 mmol L⁻¹ control levels) in response to 2000 μatm CO₂ (Collard et al. 2014b). Psammechinus miliaris accumulated 0.5 mmol L⁻¹ HCO₃ ^- from 1.8 mmol L⁻¹ to 2.3 within 7 days of exposure to 2400 μatm CO₂ (Miles et al. 2007). These examples highlight that at least some sea urchins have a higher potential to actively regulate their extracellular pH than previously thought.

Nevertheless, there are also examples for sea urchins that do not compensate pH_e disturbances: The cidaroid pencil urchin Eucidaris tribuloides was not able to increase extracellular bicarbonate levels within 32 days of exposure to 2000 μatm CO₂ (Collard et al. 2014b). Data on irregular and/or infaunal sea urchins such as sand dollars or sea mice are still missing and may offer more conclusive patterns regarding acid–base physiology in different groups of echinoids.

In general, it seems that noncidaroid, regular sea urchins are able to accumulate up to 2.5 mmol L⁻¹ bicarbonate in order to counteract acid–base disturbances. The mechanisms underlying bicarbonate accumulation, however, are insufficiently understood. Based on coelomic Mg²⁺ and Ca²⁺ concentration measurements in response to elevated pCO₂, an accumulation based on test dissolution was suggested (Miles et al. 2007). Contrary, bicarbonate concentrations in the coelomic fluid of control animals is already significantly higher than seawater in most studied sea urchins (e.g., 2–3 mmol L⁻¹ above seawater [HCO₃ ⁻] _SW of 2–3 mmol L⁻¹) and favors the hypothesis, that already under control conditions an active HCO₃ ⁻ accumulation mechanism is employed. While temporary test dissolution may still contribute to the initial compensation phase in sea urchins, it seems unlikely that test dissolution is solely responsible for the observed reestablishment of pH_e homeostasis.

There are two tissues that could contribute to bicarbonate accumulation: the peritoneal epithelium that separates the sea urchins test from the coelomic fluid and the intestinal tissue. The peritoneal epithelium, though being tight for molecules larger than 0.3 kDa, is no barrier for ions and small molecules, such as HCO₃ ^- (Holtmann et al. 2013). Therefore, the organ that most likely contributes to the observed bicarbonate accumulation is the digestive system. The intestine prevents loss of bicarbonate ions by establishing a tight barrier for HCO₃ ⁻ ions (Holtmann et al. 2013). However, the mechanisms and a potential function of the intestine in acid–base regulation remain unexplored.

10.1.1.2 Ophiuroidea, Asteroidea, and Holothuroidea

There is only one study reporting extracellular acid–base status for an ophiuroid. The infaunal brittle star Amphiura filiformis has extracellular bicarbonate levels of 4–6 mmol L⁻¹ under control conditions (compared to 2 mmol L⁻¹ in seawater) and increased extracellular bicarbonate levels up to 8 mmol L⁻¹ when exposed to 1500 and 3200 μatm CO₂ (Hu et al. 2014). The compensation reaction happened within 24 h after onset of exposure and resulted in a full (1500 μatm) and partial (3200 μatm) recovery of pH_e. A. filiformis was able to sustain high bicarbonate levels during 14 days along the experimental period (Hu et al. 2014). Interestingly, transcript levels for V-type H⁺-ATPase were elevated in disk tissue, and not in the arm tissue, in response to CO₂ stress indicating that proton transport might also be an important mechanism for extra- and/or intracellular pH compensation.

The sea star Asterias rubens did neither accumulate bicarbonate in response to elevated pCO₂ (1200–3500 μatm, (Appelhans et al. 2012)) nor were they demonstrated to compensate acid–base disturbances in their coelomic fluid (Collard et al. 2014b) over an experimental duration ranging from 14 days to 10 weeks. The sea star Leptasterias polaris did also not compensate pHe disturbances during 7 days of exposure to 1275 μatm CO₂ (Dupont and Thorndyke 2012).

The sea cucumbers Holothuria scabra and Holothuria parva did not significantly accumulate bicarbonate to compensate their pHe in response to hypercapnic conditions (2000–3500 μatm) (Collard et al. 2014a) in the experimental period of 6–12 days that may, however, be too short an interval to initiate a compensation reaction. [HCO₃ ⁻]_e was still slightly higher (between 2.7 and 3.0 mmol L⁻¹) than in seawater (between 2.2 and 2.7 mmol L⁻¹).

These observations indicate that the ability to compensate for acid–base disturbances via active accumulation of HCO₃ ⁻ in body fluids is not a universal feature in all echinoderms but seems to be restricted to Echinoidea and Ophiuroidea. The fact that most holothurians and asteroids are less calcified than echinoids and ophiuoroids can lead to speculations that acid–base regulatory abilities are correlated to the degree of calcification in echinoderms, with the exception of the pencil urchin E. tribuloides, which is a heavily calcified sea urchin even among sea urchins. In order to test this hypothesis, a comparative approach using a larger range of species and a standardized experimental design would be needed. Moreover, additional studies on the mechanistic basis of pHe regulation in echinoderms will represent an important future task to better understand the mechanisms of acid–base transport across gut epithelia in a so far unexplored animal group.

10.1.2 Nitrogen Excretion

It has been proposed that excretion of protons may be facilitated by the excretion of ammonia (for mechanistic details on the cellular level, see chapters on crustaceans and cephalopods). There are only a few studies examining the ammonia excretion rates in response to hypercapnia in echinoderms (Stumpp et al. 2012b; Hu et al. 2014).

It seems that in all studied echinoderm species, the compensation reaction for CO₂-induced acid–base disturbances is accompanied by a significant increase in NH₃/NH₄ ⁺ excretion rates. For example, Strongylocentrotus droebachiensis increased ammonia excretion rates by 70 % in response to 2800 μatm CO₂ (Stumpp et al. 2012b). Amphiura filiformis significantly excreted 65 % more ammonia under elevated pCO₂ conditions (3400 and 6600 μatm CO₂) (Hu et al. 2014). Species, e.g., Holothuria scabra and Holothuria parva, with uncompensated perivisceral fluids did not increase their ammonia excretion in response to hypercapnic conditions (Collard et al. 2014a).

This observation suggests that pH_e regulatory mechanisms are linked to ammonia excretion as a mechanism to facilitate bicarbonate formation/accumulation and/or proton (equivalent) excretion. A potent mechanism to excrete protons is essential in species that are capable of actively accumulating HCO₃ ⁻ as the formation of HCO₃ ^- from CO₂ and H₂O is ultimately associated with the formation of H⁺ that needs to be secreted from the animals. As the present body of knowledge regarding species-specific NH₃/NH₄ ⁺ secretion characteristics and mechanisms for echinoderms are scarce, definite conclusions cannot be drawn from the present literature data.

10.2 Larval Echinoderms

Echinoderms generally reproduce by free-swimming larvae – with very few exceptions of brooding species. Larvae are either lecithotrophic or in most of the cases planktotrophic. Depending on the class, you find larvae with calcified endoskeletons (sea urchins and brittle stars) or larvae that are not calcified until the adult rudiment is formed (sea stars and sea cucumbers). The calcified structures, if present, are surrounded by a cytoplasmic sheet of primary mesenchyme cells, which is again embedded in a vast extracellular space between the epidermis and digestive tract. The extracellular space of echinoderm larvae is characterized by extensive almost cell-free gelatinous material occupying the primary body cavity and surrounding the calcifying primary mesenchyme cells (PMCs) in echinopluteus larvae (Strathmann 1989; Crawford 1990). The gel-like compounds of the primary body cavity are extremely flexible and the larval body is able to rebound to its original shape following muscle contraction (e.g., during swallowing of food particles). Thus, it is a highly energy-saving system for larval movement, allowing for large larvae with relatively little cellular material, a situation not unlike that in many Scyphomedusae (Strathmann 1989; Crawford 1990). The larval calcium carbonate structures, if present, combined with the gelatinous material support the larval morphology and orientation of the larvae in the water column (Pennington and Strathmann 1990). . See Fig. 10.1b for a larval body plan of an echinopluteus.

Thus, there are three morphological structures/compartments that are of interest for acid–base regulation: intracellular pH regulation in calcifying primary mesenchyme cells, the extracellular space between the ectoderm and endoderm, and the extracellular/luminal space of the digestive system. While there is genetic data available of whole larvae exposed to hypercapnic conditions, mechanistic data on acid–base regulation in echinoderms is, again, scarce and is within the echinoderms limited to larvae from sea urchins.

10.2.1 Intracellular Acid–Base Regulation in Calcifying Primary Mesenchyme Cells

Echinoderm larvae are considered to be particularly sensitive to seawater acidification and to the connected changes in calcium carbonate saturation state of seawater (Ω_Cal) since their internal skeleton is composed of high-magnesium calcite, the most soluble form of CaCO₃ (Beniash et al. 1997; Raz et al. 2003). However, sea urchin larvae are able to maintain calcification rates (when corrected for developmental or growth delay) even during exposure to unrealistically high CO₂ partial pressures (0.35 kPa CO₂, pH 7.25, Ω_Cal 1.02) (Martin et al. 2011). This indicates that the calcification machinery has substantial capacities to compensate for CaCO₃ dissolution due to CO₂-induced reductions in seawater pH. The calcification process in sea urchin larvae has been relatively well investigated (e.g., Nakano et al. 1963; Sikes et al. 1981; Wilt 2002; Raz et al. 2003; Politi et al. 2008) and primary mesenchyme cells (PMCs) were identified as the cell type mediating calcification in sea urchin larvae. These cells are located within the extracellular matrix of the primary body cavity and form a syncytium covering the spicules of the pluteus larvae. This syncytial sheath covers the entire surface of the spicules and communicates with the extracellular environment of the primary body cavity (Decker et al. 1987; Beniash et al. 1997). Recent studies using a pharmacological approach demonstrated that several loop diuretics including azosemide, bumetanide, and furosemide could significantly inhibit spicule formation at concentrations that significantly inhibit the Na⁺/K⁺/2Cl^- symporter (NKCC) (Basse et al. 2015). Using histological analyses it has been concluded that the impaired formation of spicules was due to an inhibition of cytoplasmic cord formation and maintenance in PMCs.

The high-magnesium calcitic spicules are formed through the production of a transient amorphous calcium carbonate (ACC) phase within vesicles in PMCs which is subsequently released into the spicular cavity (Beniash et al. 1997; Raz et al. 2003; Politi et al. 2004, 2008). To fuel the calcification process, bicarbonate is absorbed from the seawater (40 %) as well as generated from metabolic CO₂ (60 %) (Sikes et al. 1981). On the other hand, Ca²⁺ is exclusively obtained directly from the seawater (Nakano et al. 1963). Although the general principle of calcification is well understood, mechanistic information regarding transporters that facilitate Ca²⁺ or HCO₃ ^- uptake in PMCs is still limited. Several pharmacological studies suggested that Ca²⁺ channels and pumps are key players in the provision of Ca²⁺ for spicule formation (Hwang and Lennarz 1993; Gunaratne and Vacquier 2007). In order to remove protons generated during CaCO₃ precipitation, PMCs must possess efficient acid−base regulatory properties (McConnaughey and Whelan 1996; Buitenhuis et al. 1999). A recent study applied the intracellular pH dye acid (2,7-bis (2 carboxyethyl)-5-(and-6)-carboxy fluorescein (BCECF)) in order to investigate pH regulatory mechanisms in sea urchin PMCs (Stumpp et al. 2012a). Using pulses of NH₃/NH₄ ⁺ solution, it has been demonstrated that PMCs have significant pH_i regulatory abilities. PMCs have a control pH_i of 6.9 and 20 mmol L⁻¹ NH₃/NH₄ ⁺ solution induced an intracellular alkalinization leading to an increase of pH_i by approximately 0.7 units. Washout of the NH₃/NH₄ ⁺ solution induced an acidosis of approximately 0.7 pH units below the control value, followed by a pH recovery to control levels within 15 min. Rates of pH_i recovery after the NH₃/NH₄ ⁺ pulse were significant but weak compared to recovery times reported for strong ion regulatory cells of vertebrates (Boron and De Weer 1976; Bleich et al. 1995; Hasselblatt et al. 2000; Boron 2004) and similar to those reported for crayfish neurons (~20 min, (Moody 1981)). These experiments indicated that PMCs that are involved in skeletogenesis in sea urchins have a significant acid–base regulatory capacity.

The rate of pH compensation following an NH₃/NH₄ ⁺-induced acidosis was further investigated in the presence of low-Na⁺ and low-HCO₃ ^- artificial seawater solutions in order to test the potential role of these ions in mediating pH_i. These experiments clearly indicated that the compensation reaction after NH₃/NH₄ ⁺-induced acidosis in PMCs is highly dependent on Na⁺ and HCO₃ ^- transport and suggest the involvement of Na⁺-dependent HCO₃ ^- import mechanisms during pH recovery. These are an indirect sink of ATP mediated through secondary active transporters (e.g., transporters of the SLC4 family, including Na⁺/HCO₃ ⁻ cotransporters and Na⁺-dependent Cl^-/HCO₃ ^- exchangers) that are driven through ATP-dependent ion pumps such as the Na⁺/K⁺-ATPase or the V-type H⁺-ATPase (Boron 2004). The existence of a pH regulatory machinery in PMCs of sea urchins is underlined by the identification of genes coding for ion transporters including Na⁺/K⁺-ATPase (NKA), Na⁺/HCO₃ ^- cotransporters (NBC), H⁺-ATPases (HA), and Na⁺/H⁺-exchangers (NHE) from the sea urchin PMC transcriptome (Zhu et al. 2001). However, since protons are generated during the intracellular calcification process, the identification and characterization of proton secretion mechanisms involving transporters such as the V-type H⁺-ATPase or Na⁺/H+ exchangers represent an important future task to better understand the mechanisms of calcification in echinoid PMCs.

10.2.2 Extracellular Acid–Base Regulation

10.2.2.1 Primary Body Cavity

Microelectrode and microfluorescence measurements of extracellular pH (pH_e) within the primary body cavity of S. droebachiensis pluteus larvae revealed that the fluid of the primary body cavity pH conforms to the surrounding seawater (Stumpp et al. 2012a). Given that the fluid composition of the primary body cavity is similar to seawater, this indicates that the internal pCO₂ within the animals is similar to that of seawater pCO₂. Usually, extracellular pCO₂ of adult, heterotrophic metazoans is at least 100 Pa higher than the seawater pCO₂ in order to establish a CO₂ diffusion gradient away from the organism (Melzner et al. 2009). The result would imply that sea urchin larvae do not have an outward directed CO₂ diffusion gradient due to metabolic CO₂ production. During acute exposure of larvae to elevated seawater pCO₂, pH_e decreased linearly with pH_SW indicating that the epidermis is extremely leaky to ions and small molecules. A transepithelial KCl^- potential of 0 mV of the ectodermal epithelium also suggests that the epithelium is not a specialized transporting epithelium (Stumpp et al. 2012a). In fact, diffusion experiments using different sizes of BCECF-substituted dextrans demonstrated that the ectodermal epithelium forms no barrier for molecules up to 10 kDa if sufficient time for diffusion is allowed (60 min). Molecules up to 4 kDa penetrate the epidermis within 15 min (Stumpp et al. 2012a). Gases and small ions can therefore be expected to be in equilibrium with seawater leading to good conditions for calcification at present conditions, but on the other hand being sensitive to environmental variability in pH or future ocean acidification.

10.2.2.2 Stomach Lumen

Sea urchin larvae have a tripartite gut consisting of a foregut (esophagus), midgut (stomach), and a hindgut (intestine). Food particles (e.g., microalgae) are collected and concentrated in the esophagus and then swallowed by opening of the anterior cardiac sphincter. Food is processed in the stomach and absorbed in the stomach as well as the intestine. While the pH of the primary body cavity is conformed with seawater, the luminal pH_e in the stomach of S. droebachiensis increased after feeding commenced from pH_e 8.9 at 5 days post fertilization to pH_e 9.5 at 9 days post fertilization (Stumpp et al. 2013) and up to pH 9.0 in S. purpuratus (Stumpp et al. 2015). Alkaline gastric pHs are known to occur in a range of insects and their larvae such as lepidopterans (Dow 1984) and dipteran larvae (Boudko et al. 2001). Among marine species alkaline digestive conditions were additionally found in the marine copepod Calanus helgolandicus (Pond et al. 1995) as well as in tornaria larvae of the hemichordate Ptychodera flava – reaching a pH of 10.5 in their stomach lumen (Stumpp et al. 2015). Alkaline digestive conditions may represent an evolutionary advantage for marine herbivorous larvae as alkaline conditions may facilitate breakdown of plant and algal proteins (Felton and Duffey 1991). Moreover, alkaline conditions higher than pH 9.5 in larval digestive systems can be regarded as an efficient defense mechanism against environmental pathogens because most marine protists and viruses are killed or growth inhibited by alkaline conditions exceeding pH 9.5 (Pedersen and Hansen 2003; Jońezyk et al. 2011). However, these unique digestive features were proposed to be a key process that makes these larvae sensitive to climate change and the associated reductions in seawater pH (ocean acidification) by impairing digestion and exerting additional energetic costs to the larvae in future oceans (Stumpp et al. 2013) (for details, see next paragraph).

Chronic exposure of S. droebachiensis larvae to elevated pCO₂ resulted in a lower stomach lumen pH_e at 7 and 9 days post fertilization in two CO₂ treatments (1015 and 2460 μatm pCO₂). The data suggest that the environmental CO₂ induced acidification is exceeding the larval capacity of fully alkalinize the stomach lumen to control levels. The drop in stomach pH_e of 0.25 and 0.4 pH units at the high pCO₂ level (2460 μatm at 7 and 9 dpf, respectively) is, however, much smaller than the environmental pH_SW drop of 0.67 pH units, possibly indicating an activation of compensatory mechanisms. Acute acidification experiments down to pH 7 in S. purpuratus resulted in a partial compensation reaction of the gastric pH within 3 h. This compensation reaction was likely mediated by cells of the stomach epithelium.

In contrast to the thin squamous epidermal cell layers, the echinopluteus’ stomach epithelium consists of simple columnar cells (of 10–15 μm height) with microvilli extending into the stomach lumen (Burke 1981). At least two cell types are present in the stomach epithelium. Type I cells are characterized by a variety of different vacuoles and are expected to function in absorption and storage of nutrients as well as the secretion of digestive enzymes. Type II cells are assumed to phagocytose and digest whole algal cells (Burke 1981).

Immunohistochemical and in situ hybridization using antibodies and probes against specific ion transporters could demonstrate that ion pumps and transporters including Na⁺/K⁺-ATPase (NKA) and V-type H⁺-ATPase (VHA) are highly expressed in specialized epidermal cells of the stomach and intestine. The colocalization of NKA, VHA, and Na⁺H⁺ exchangers revealed the subcellular localization of these proteins in stomach cells. While NKA and VHA are localized in the luminal membrane positive Na⁺/H⁺ exchanger (NHE), immunoreactivity was mainly found in the basolateral membrane (Stumpp et al. 2015). The mechanistic basis of gastric alkalization was further examined using H⁺-selective microelectrodes in combination with specific inhibitors for acid–base relevant ion transporters. These studies underlined the histological findings by demonstrating that gastric alkalization in sea urchin larvae is inhibited by ouabain and bafilomycin, specific inhibitors for the Na⁺/K⁺-ATPase (NKA) and the V-type H⁺-ATPase (VHA), respectively. In addition, gastric alkalization was decreased by treatments with ethyl-isopropyl amiloride (EIPA, a specific inhibitor of Na⁺-dependent H⁺ exchangers (NHEs)) as well as artificial seawater with reduced Na⁺ (5 mmol L⁻¹) and K⁺ (0 mmol L⁻¹) concentrations.

Gene expression analyses of genes encoding acid–base relevant transporters in pluteus upon challenge of the gastric pH regulatory machinery with acidified seawater identified genes coding for the transporters mediating gastric alkalinization. During acute (1–3 h) challenge of the gastric pH regulatory machinery by exposing larvae to acidified conditions (pH 7.0), ion transport genes, including NKA, VHA, NHE3, and a KHE, were significantly up-regulated when compared to larvae kept under control conditions. Accordingly a first working model for gastric alkalinization in sea urchin larvae has been proposed (Stumpp et al. 2015). Gastric alkalinization in the sea urchin stomach is mainly generated through cationic exchange processes, leading to a net export of protons from the stomach lumen (summarized in . Fig. 10.2). NKA located in luminal membranes creates an electrochemical gradient, and a low intracellular [Na⁺] that drives basolateral excretion of H⁺ via NHE in the the primary body cavity (PBC). On the luminal side, H⁺ is imported via a coupling of VHA and a putative electrogenic K⁺/H⁺ antiporter. A similar cationic-based alkalinization mechanism driven by the VHA coupled to secondary active transport processes including K⁺/H⁺ antiport has been proposed for the midgut alkalinization in lepidopteran insect larvae (Wieczorek et al. 1991). The employment of NHE localized in membranes facing the PBC that mediate excretion of H⁺ in exchange for Na⁺ represents an energetically favorable mechanism due to high external [Na⁺] (≈460 mmol L⁻¹) compared to low intracellular [Na⁺] (≈30 mmol L⁻¹) (Robertson 1949).

Fig. 10.2

First working model for gastric alkalinization in sea urchin pluteus larvae. The stomach epithelium is rich in ion transporters that are capable of regulating gastric pH. The current model suggests that gastric alkalinization is achieved by a basolateral (facing the primary body cavity, PBC) NHE that is fueled by a Na⁺/K⁺-ATPase located in the luminal membrane. Protons that are excreted via NHE derive from the hydration of metabolic CO₂ via cytosolic carbonic anhydrases (CA) as well as from a luminal K⁺/H⁺ antiport mechanism energized by luminal V-type H⁺-ATPase (VHA) (modified after Stumpp et al. 2015).

10.3 Effects of Near-Future Ocean Acidification on Echinoderms

Effects of simulated ocean acidification experiments on adult echinoderms generally included reduced growth and feeding rates, decreased fertility (measured as number of eggs per female or gonad growth), and occasionally reduced metabolic rates (Appelhans et al. 2012; Dupont and Thorndyke 2012; Stumpp et al. 2012b). This most likely leads to alterations in the energy budget that may be due to elevated costs for cellular and extracellular pH homeostasis as described in sea urchins and brittle stars. However, most studies employed experimental time frames between a few days and a few months, possibly not allowing enough time for the animals to acclimate to the future scenario. For example, Dupont and colleagues demonstrated in one of the few long-term studies that the green sea urchin S. droebachiensis was able to acclimate to elevated pCO₂ conditions resulting in comparable fertility after 16 months. In contrast, animals that were acclimated for only 4 months displayed significantly reduced fertility (Dupont et al. 2012). Thus, in order to understand how ocean acidification will affect echinoderms in general, long-term studies of several months to years would yield valuable results in regard to acclimation and possibly adaptation potential.

Apart from a few exceptions with nonfeeding echinoderm larvae, i.e., Crossaster papposus, which displayed increased growth rates in response to seawater acidification (Dupont et al. 2010b), exposure of echinoderm larvae to elevated pCO₂ resulted in reduced growth rates (Dupont et al. 2010a) and in some cases high mortalities (Dupont et al. 2008). However, most studies were conducted on sea urchin larvae, since pluteus larvae with their calcified endoskeleton were readily identified as being especially sensitive to seawater acidification. Only two studies looked at noncalcifying, feeding marine larvae such as bipinnaria larvae of sea stars. Interestingly, despite the lack of calcified structures, exposure of sea star larvae to environmentally realistic increases in CO₂ levels could also demonstrate slowed larval growth (Gonzalez-Bernat et al. 2012; Byrne et al. 2013). This clearly indicates that other physiological processes than calcification may cause sensitivities to seawater acidification in marine larval stages.

Based on the findings of alkaline digestive systems in sea urchin larvae (and also hemichordate larvae (Stumpp et al. 2015)) and their sensitivity to lowered pH (Stumpp et al. 2013), the hypothesis of gastric pH homeostasis being a dominant factor determining sensitivity in echinoderm larvae arises. Apart from potential elevated costs for intracellular and/or extracellular pH regulation itself (Stumpp et al. 2012a, 2015), a lowered stomach pH might have negative consequences for larval energy acquisition. Digestive enzymes secreted into the gut to hydrolyze nutrients usually exhibit specific pH optima at which the enzymes are most efficient. Sea urchin larval total protease activity exhibited a pH optimum at pH 11 with a steady increase in activity from pH 8. A potential digestive pH drop of 0.3 pH units would thus translate into 8 % less digestive efficiency. The digestive potential to digest algal cells measured in in vivo digestion assays supports the in vitro total protease measurements by demonstrating a significantly lower digestive potential in sea urchin larvae raised under future elevated pCO₂ conditions (Stumpp et al. 2013). Decreased digestive potential together with increased acid–base regulatory costs would inevitably result in modifications of the larval energy budget and could potentially explain the observed reduced growth rates in sea urchin plutei, as well as in noncalcifying but feeding echinoderm larvae, such as sea star larvae.

10.4 Conclusions

The present body of knowledge regarding pH regulatory abilities in echinoderms demonstrated that despite their relatively basal position among deuterostomes, these animals have evolved significant pH regulatory abilities. Although echinoderms were demonstrated to exhibit extracellular pH regulatory abilities, the organs and mechanisms involved remain unexplored. Few studies indicated that the pH compensatory mechanism involves accumulation of buffers (e.g., HCO₃ ⁻) as well as the excretion of proton equivalents such as NH₄ ⁺. Although extracellular pH regulatory abilities were found in a range of adult echinoids, their larval stages lack extracellular pH regulatory abilities. However, the regulation of an alkaline gastric pH homeostasis seems to play an important role in digestive function, which could be identified as an “Achilles’ heel” in the context of CO₂-induced seawater acidification.

The fact that echinoderms are an evolutionary ancient group, that has also survived acidification events during geological timescales, suggests that this animal group has the ability to successfully cope with environmental pH/pCO₂ fluctuations. In this context special attention should be dedicated to the ability of these animals to acclimate and to genetically or epigenetically adapt to changing environmental conditions. As larval stages were identified as the weakest ontogenetic link, these pelagic life stages deserve special attention. Thus, in a first step, a better understanding regarding the physiological processes in echinoderm larval stages are needed in order to better understand animal–environment interactions in this ecologically and economically important group.