Josi R. Taylor

Contribution of fish to the marine inorganic carbon cycle.

Oceanic production of calcium carbonate is conventionally attributed to marine plankton (coccolithophores and foraminifera). Here we report that marine fish produce precipitated carbonates within their intestines and excrete these at high rates. When combined with estimates of global fish biomass, this suggests that marine fish contribute 3 to 15% of total oceanic carbonate production. Fish carbonates have a higher magnesium content and solubility than traditional sources, yielding faster dissolution with depth. This may explain up to a quarter of the increase in titratable alkalinity within 1000 meters of the ocean surface, a controversial phenomenon that has puzzled oceanographers for decades. We also predict that fish carbonate production may rise in response to future environmental changes in carbon dioxide, and thus become an increasingly important component of the inorganic carbon cycle.

High rates of HCO3- secretion and Cl- absorption against adverse gradients in the marine teleost intestine: the involvement of an electrogenic anion exchanger and H+-pump metabolon?

SUMMARY Anion exchange contributes significantly to intestinal Cl– absorption in marine teleost fish and is thus vital for successful osmoregulation. This anion exchange process leads to high luminal HCO3– concentrations (up to ∼100 mmol l–1) and high pH and results in the formation of CaCO3 precipitates in the intestinal lumen. Recent advances in our understanding of the transport processes involved in intestinal anion exchange in marine teleost fish include the demonstration of a role for the H+-pump (V-ATPase) in apical H+ extrusion and the presence of an electrogenic (nHCO3–/Cl–) exchange protein (SLC26a6). The H+-V-ATPase defends against cellular acidification, which might otherwise occur as a consequence of the high rates of base secretion. In addition, apical H+ extrusion probably maintains lower HCO3– concentrations in the unstirred layer at the apical surface than in the bulk luminal fluids and thus facilitates continued anion exchange. Furthermore, H+-V-ATPase activity hyperpolarizes the apical membrane potential that provides the driving force for apical electrogenic nHCO3–/Cl– exchange, which appears to occur against both Cl– and HCO3– electrochemical gradients. We propose that a similar coupling between apical H+ extrusion and nHCO3–/Cl– exchange accounts for Cl– uptake in freshwater fish and amphibians against very steep Cl– gradients.

Effects of salinity on intestinal bicarbonate secretion and compensatory regulation of acid-base balance in Opsanus beta.

SUMMARY Marine teleosts have extracellular fluids less concentrated than their environment, resulting in continual water loss, which is compensated for by drinking, with intestinal water absorption driven by NaCl uptake. Absorption of Cl– occurs in part by apical Cl–/HCO3– exchange, with HCO3– provided by transepithelial transport and/or by carbonic anhydrase-mediated hydration of endogenous epithelial CO2. Hydration of CO2 also liberates H+, which is transported across the basolateral membrane. In this study, gulf toadfish (Opsanus beta) were acclimated to 9, 35 and 50 ppt. Intestinal HCO3– secretion, water and salt absorption, and the ensuing effects on acid–base balance were examined. Rectal fluid excretion greatly increased with increasing salinity from 0.17±0.05 ml kg–1 h–1 in 9 ppt to 0.70±0.19 ml kg–1 h–1 in 35 ppt and 1.46±0.22 ml kg–1 h–1 in 50 ppt. Rectal fluid composition and excretion rates allowed for estimation of drinking rates, which increased with salinity from 1.38±0.30 to 2.60±0.92 and 3.82±0.58 ml kg–1 h–1 in 9, 35 and 50 ppt, respectively. By contrast, the fraction of imbibed water absorbed decreased from 85.9±3.8% in 9 ppt to 68.8±3.2% in 35 ppt and 61.4±1.0% in 50 ppt. Despite large changes in rectal base excretion from 9.3±2.7 to 68.2±20.4 and 193.2±64.9 μmol kg–1 h–1 in 9, 35 and 50 ppt, respectively, acute or prolonged exposure to altered salinities was associated with only modest acid–base balance disturbances. Extra-intestinal, presumably branchial, net acid excretion increased with salinity (62.0±21.0, 229.7±38.5 and 403.1±32.9 μmol kg–1 h–1 at 9, 35 and 50 ppt, respectively), demonstrating a compensatory response to altered intestinal base secretion associated with osmoregulatory demand.

Feeding and osmoregulation: dual function of the marine teleost intestine

SUMMARY Experiments on Gulf toadfish Opsanus beta demonstrate how feeding impacts osmoregulation in the marine teleost intestine. A high Ca2+ diet of pilchards Sardina pilchardus ([Ca2+]=404.2 mmol kg-1) was compared to a low Ca2+ diet of common squid Loligo forbesi ([Ca2+]=1.3 mmol kg-1), as high [Ca2+] has been shown to stimulate intestinal anion exchange. Gastrointestinal fluids and blood plasma were collected over a time course from pre-feeding to 216 h post feeding. Following food intake, monovalent ions were largely absorbed across the intestinal epithelium, leaving a fluid rich in divalent ions, which have a lower osmotic coefficient and effectively reduce osmotic pressure in the lumen to allow for enhanced fluid absorption. Concentrations of Cl- and HCO -3 in fluid along the gastrointestinal tract of fish fed both diets, particularly 1 and 2 days post-feeding, demonstrate that apical Cl-/HCO -3 exchange plays a vital role in postprandial Cl- and water absorption. Postprandial acid-base balance disturbance as indicated by plasma alkalinization was limited or absent, indicating compensation for gastric acid secretion in this teleost fish. Plasma osmolality peaked 12 h post-feeding in toadfish fed squid, but was not accompanied by a significant increase in inorganic ion concentrations. Transient fluid secretion by the gastrointestinal tract was evident from reduced luminal Mg2+ and SO 2-4 concentrations for 24-48 h post feeding. Discrepancy between the sum of inorganic osmolytes and measured osmotic pressure was attributed to organic osmolytes, which occurred at high concentrations in the stomach and anterior intestine for up to 24 h post feeding.

The involvement of H+-ATPase and carbonic anhydrase in intestinal HCO3- secretion in seawater-acclimated rainbow trout.

SUMMARY Pyloric caeca and anterior intestine epithelia from seawater-acclimated rainbow trout exhibit different electrophysiological parameters with lower transepithelial potential and higher epithelial conductance in the pyloric caeca than the anterior intestine. Both pyloric caeca and the anterior intestine secrete HCO3– at high rates in the absence of serosal HCO3–/CO2, demonstrating that endogenous CO2 is the principal source of HCO3– under resting control conditions. Apical, bafilomycin-sensitive, H+ extrusion occurs in the anterior intestine and probably acts to control luminal osmotic pressure while enhancing apical anion exchange; both processes with implications for water absorption. Cytosolic carbonic anhydrase (CAc) activity facilitates CO2 hydration to fuel apical anion exchange while membrane-associated, luminal CA activity probably facilitates the conversion of HCO3– to CO2. The significance of membrane-bound, luminal CA may be in part to reduce HCO3– gradients across the apical membrane to further enhance anion exchange and thus Cl– absorption and to facilitate the substantial CaCO3 precipitation occurring in the lumen of marine teleosts. In this way, membrane-bound, luminal CA thus promotes the absorption of osmolytes and reduction on luminal osmotic pressure, both of which will serve to enhance osmotic gradients to promote intestinal water absorption.

Basolateral NBCe1 plays a rate-limiting role in transepithelial intestinal HCO3- secretion, contributing to marine fish osmoregulation.

SUMMARY Although endogenous CO2 hydration and serosal HCO3– are both known to contribute to the high rates of intestinal HCO3– secretion important to marine fish osmoregulation, the basolateral step by which transepithelial HCO3– secretion is accomplished has received little attention. Isolated intestine HCO3– secretion rates, transepithelial potential (TEP) and conductance were found to be dependent on serosal HCO3– concentration and sensitive to serosal DIDS. Elevated mucosal Cl– concentration had the unexpected effect of reducing HCO3– secretion rates, but did not affect electrophysiology. These characteristics indicate basolateral limitation of intestinal HCO3– secretion in seawater gulf toadfish, Opsanus beta. The isolated intestine has a high affinity for serosal HCO3– in the physiological range (Km=10.2 mmol l–1), indicating a potential to efficiently fine-tune systemic acid–base balance. We have confirmed high levels of intestinal tract expression of a basolateral Na+/HCO3– cotransporter of the electrogenic NBCe1 isoform in toadfish (tfNBCe1), which shows elevated expression following salinity challenge, indicating its importance in marine fish osmoregulation. When expressed in Xenopus oocytes, isolated tfNBCe1 has transport characteristics similar to those in the isolated tissue, including a similar affinity for HCO3– (Km=8.5 mmol l–1). Reported affinity constants of NBC1 for Na+ are generally much lower than physiological Na+ concentrations, suggesting that cotransporter activity is more likely to be modulated by HCO3– rather than Na+ availability in vivo. These similar functional characteristics of isolated tfNBCe1 and the intact tissue suggest a role of this cotransporter in the high HCO3– secretion rates of the marine fish intestine.

Gastro-intestinal handling of water and solutes in three species of elasmobranch fish, the white-spotted bamboo shark, Chiloscyllium plagiosum, little skate, Leucoraja erinacea and the clear nose skate Raja eglanteria.

The present study reports aspects of GI tract physiology in the white-spotted bamboo shark, Chiloscyllium plagiosum, little skate, Leucoraja erinacea and the clear nose skate, Raja eglanteria. Plasma and stomach fluid osmolality and solute values were comparable between species, and stomach pH was low in all species (2.2 to 3.4) suggesting these elasmobranchs may maintain a consistently low stomach pH. Intestinal osmolality, pH and ion values were comparable between species, however, some differences in ion values were observed. In particular Ca(2+) (19.67+/-3.65mM) and Mg(2+) (43.99+/-5.11mM) were high in L. erinacea and Mg(2+) was high (130.0+/-39.8mM) in C. palgiosum which may be an indication of drinking. Furthermore, intestinal fluid HCO(3)(-) values were low (8.19+/-2.42 and 8.63+/-1.48mM) in both skates but very high in C. plagiosum (73.3+/-16.3mM) suggesting ingested seawater may be processed by species-specific mechanisms. Urea values from the intestine to the colon dropped precipitously in all species, with the greatest decrease seen in C. plagiosum (426.0+/-8.1 to 0mM). This led to the examination of the molecular expression of both a urea transporter and a Rhesus like ammonia transporter in the intestine, rectal gland and kidney in L. erinacea. Both these transporters were expressed in all tissues; however, expression levels of the Rhesus like ammonia transporter were orders of magnitude higher than the urea transporter in the same tissue. Intestinal flux rates of solutes in L. erinacea were, for the most part, in an inward direction with the notable exception of urea. Colon flux rates of solutes in L. erinacea were all in an outward direction, although absolute rates were considerably lower than the intestine, suggestive of a much tighter epithelia. Results are discussed in the context of the potential role of the GI tract in salt and water, and nitrogen, homeostasis in elasmobranchs.

The intestinal response to feeding in seawater gulf toadfish, Opsanus beta, includes elevated base secretion and increased epithelial oxygen consumption.

SUMMARY Intestinal HCO3− secretion is essential to marine teleost fish osmoregulation and comprises a considerable source of base efflux attributable to both serosal HCO3− and endogenous CO2 hydration. The role of intestinal HCO3− secretion in dynamic acid—base balance regulation appears negligible in studies of unfed fish, but evidence of high intestinal fluid [HCO3−] in fed marine teleosts led us to investigate the source of this HCO3− and its potential role in offsetting the postprandial ‘alkaline tide’ commonly associated with digestion. Specifically, we hypothesized that elevated metabolic rate and thus endogenous CO2 production by intestinal tissue as well as increased transepithelial intestinal HCO3− secretion occur post-feeding and offset a postprandial alkaline tide. To test these hypotheses changes in HCO3− secretion and O2 consumption by gulf toadfish (Opsanus beta) isolated intestine were quantified 0, 3, 6, 12, 24 and 48 h post-feeding. Intestinal tissue of unfed fish in general showed high rates of HCO3− secretion (15.5 μmol g−1 h−1) and O2 consumption (8.9 μmol g−1 h−1). Furthermore, postprandial increases in both intestinal HCO3− secretion and O2 consumption (1.6- and 1.9-fold peak increases, respectively) were observed. Elevated intestinal HCO3− secretion rates preceded and outlasted those of O2 consumption, and occurred at a magnitude and duration sufficient to account for the lack of alkaline tide. The dependence of these high rates of postprandial intestinal base secretion on serosal HCO3− indicates transepithelial HCO3− transport increases disproportionately more than endogenous CO2 production. The magnitude of postprandial intestinal HCO3− secretion indicates the intestine certainly is capable of postprandial acid#x02014;base balance regulation.

Use of a Free Ocean CO2 Enrichment (FOCE) System to Evaluate the Effects of Ocean Acidification on the Foraging Behavior of a Deep-Sea Urchin

The influence of ocean acidification in deep-sea ecosystems is poorly understood but is expected to be large because of the presumed low tolerance of deep-sea taxa to environmental change. We used a newly developed deep-sea free ocean CO2 enrichment (dp-FOCE) system to evaluate the potential consequences of future ocean acidification on the feeding behavior of a deep-sea echinoid, the sea urchin, Strongylocentrotus fragilis. The dp-FOCE system simulated future ocean acidification inside an experimental enclosure where observations of feeding behavior were performed. We measured the average movement (speed) of urchins as well as the time required (foraging time) for S. fragilis to approach its preferred food (giant kelp) in the dp-FOCE chamber (-0.46 pH units) and a control chamber (ambient pH). Measurements were performed during each of 4 trials (days -2, 2, 24, 27 after CO2 injection) during the month-long period when groups of urchins were continuously exposed to low pH or control conditions. Although urchin speed did not vary significantly in relation to pH or time exposed, foraging time was significantly longer for urchins in the low-pH treatment. This first deep-sea FOCE experiment demonstrated the utility of the FOCE system approach and suggests that the chemosensory behavior of a deep-sea urchin may be impaired by ocean acidification.

Selected regulation of gastrointestinal acid-base secretion and tissue metabolism for the diamondback water snake and Burmese python.

SUMMARY Snakes exhibit an apparent dichotomy in the regulation of gastrointestinal (GI) performance with feeding and fasting; frequently feeding species modestly regulate intestinal function whereas infrequently feeding species rapidly upregulate and downregulate intestinal function with the start and completion of each meal, respectively. The downregulatory response with fasting for infrequently feeding snakes is hypothesized to be a selective attribute that reduces energy expenditure between meals. To ascertain the links between feeding habit, whole-animal metabolism, and GI function and metabolism, we measured preprandial and postprandial metabolic rates and gastric and intestinal acid–base secretion, epithelial conductance and oxygen consumption for the frequently feeding diamondback water snake (Nerodia rhombifer) and the infrequently feeding Burmese python (Python molurus). Independent of body mass, Burmese pythons possess a significantly lower standard metabolic rate and respond to feeding with a much larger metabolic response compared with water snakes. While fasting, pythons cease gastric acid and intestinal base secretion, both of which are stimulated with feeding. In contrast, fasted water snakes secreted gastric acid and intestinal base at rates similar to those of digesting snakes. We observed no difference between fasted and fed individuals for either species in gastric or intestinal transepithelial potential and conductance, with the exception of a significantly greater gastric transepithelial potential for fed pythons at the start of titration. Water snakes experienced no significant change in gastric or intestinal metabolism with feeding. Fed pythons, in contrast, experienced a near-doubling of gastric metabolism and a tripling of intestinal metabolic rate. For fasted individuals, the metabolic rate of the stomach and small intestine was significantly lower for pythons than for water snakes. The fasting downregulation of digestive function for pythons is manifested in a depressed gastric and intestinal metabolism, which selectively serves to reduce basal metabolism and hence promote survival between infrequent meals. By maintaining elevated GI performance between meals, fasted water snakes incur the additional cost of tissue activity, which is expressed in a higher standard metabolic rate.