Martin Grosell

Intestinal anion exchange in marine fish osmoregulation

SUMMARY Despite early reports, dating back three quarters of a century, of high total CO2 concentrations in the intestinal fluids of marine teleost fishes, only the past decade has provided some insight into the functional significance of this phenomenon. It is now being recognized that intestinal anion exchange is responsible for high luminal HCO3- and CO32- concentrations while at the same time contributing substantially to intestinal Cl- and thereby water absorption, which is vital for marine fish osmoregulation. In species examined to date, the majority of HCO3- secreted by the apical anion exchange process is derived from hydration of metabolic CO2 with the resulting H+ being extruded via a Na+:H+ exchange mechanism in the basolateral membrane. The basolateral H+ extrusion is critical for the apical anion exchange and relies on the Na+ gradient established by the Na+-K+-ATPase. This enzyme thereby ultimately fuels the secondary active transport of HCO3- and Cl- by the apical anion exchanger. High cellular HCO3- concentrations (>10 mmol l-1) are required for the anion exchange process and could be the result of both a high metabolic activity of the intestinal epithelium and a close association of the anion exchange protein and the enzyme carbonic anhydrase. The anion exchange activity in vivo is likely most pronounced in the anterior segment and results in net intestinal acid absorption. In contrast to other water absorbing vertebrate epithelia, the marine teleost intestine absorbs what appears to be a hypertonic fluid to displace diffusive fluid loss to the marine environment.

Deepwater Horizon crude oil impacts the developing hearts of large predatory pelagic fish

Significance The 2010 Deepwater Horizon (MC252) disaster in the northern Gulf of Mexico released more than 4 million barrels of crude oil. Oil rose from the ocean floor to the surface where many large pelagic fish spawn. Here we describe the impacts of field-collected oil samples on the rapidly developing embryos of warm-water predators, including bluefin and yellowfin tunas and an amberjack. For each species, environmentally relevant MC252 oil exposures caused serious defects in heart development. Moreover, abnormalities in cardiac function were highly consistent, indicating a broadly conserved developmental crude oil cardiotoxicity. Losses of early life stages were therefore likely for Gulf populations of tunas, amberjack, swordfish, billfish, and other large predators that spawned in oiled surface habitats. The Deepwater Horizon disaster released more than 636 million L of crude oil into the northern Gulf of Mexico. The spill oiled upper surface water spawning habitats for many commercially and ecologically important pelagic fish species. Consequently, the developing spawn (embryos and larvae) of tunas, swordfish, and other large predators were potentially exposed to crude oil-derived polycyclic aromatic hydrocarbons (PAHs). Fish embryos are generally very sensitive to PAH-induced cardiotoxicity, and adverse changes in heart physiology and morphology can cause both acute and delayed mortality. Cardiac function is particularly important for fast-swimming pelagic predators with high aerobic demand. Offspring for these species develop rapidly at relatively high temperatures, and their vulnerability to crude oil toxicity is unknown. We assessed the impacts of field-collected Deepwater Horizon (MC252) oil samples on embryos of three pelagic fish: bluefin tuna, yellowfin tuna, and an amberjack. We show that environmentally realistic exposures (1–15 µg/L total PAH) cause specific dose-dependent defects in cardiac function in all three species, with circulatory disruption culminating in pericardial edema and other secondary malformations. Each species displayed an irregular atrial arrhythmia following oil exposure, indicating a highly conserved response to oil toxicity. A considerable portion of Gulf water samples collected during the spill had PAH concentrations exceeding toxicity thresholds observed here, indicating the potential for losses of pelagic fish larvae. Vulnerability assessments in other ocean habitats, including the Arctic, should focus on the developing heart of resident fish species as an exceptionally sensitive and consistent indicator of crude oil impacts.

Intestinal bicarbonate secretion by marine teleost fish--why and how?

Intestinal fluids of most marine teleosts are alkaline (pH 8.4-9.0) and contain high levels of HCO(3)(-) equivalents (40-130 mM) which are excreted at a significant rate (>100 microEq kg(-1) h(-1)). Recent research reveals the following about this substantial HCO(3)(-) secretion: (1) It is not involved in acid-base regulation or neutralisation of stomach acid, but increases in parallel with drinking rate at elevated ambient salinities suggesting a role in osmoregulation; (2) In species examined so far, all sections of the intestine can secrete bicarbonate; (3) The secretion is dependent on mucosal Cl(-), sensitive to mucosal DIDS, and immuno-histochemistry indicates involvement of an apical Cl(-)/HCO(3)(-) exchanger. In addition, hydration of CO(2) via carbonic anhydrase in combination with proton extrusion appears to be essential for bicarbonate secretion. The mode of proton extrusion is currently unknown but potential mechanisms are discussed. One consequence of the luminal alkalinity and high bicarbonate concentrations is precipitation of calcium and magnesium as carbonate complexes. This precipitation is hypothesised to reduce the osmolality of intestinal fluids and thus play a potential role in water absorption and osmoregulation. The present studies on European flounder reveal that elevated luminal calcium (but not magnesium) concentrations stimulate intestinal bicarbonate secretion both acutely and chronically, in vitro and in vivo. At the whole animal level, the result of this elevated bicarbonate secretion was increased calcium precipitation with an associated reduction in the osmolality of rectal fluids and plasma. These observations suggest direct functional links between intestinal bicarbonate secretion, divalent cation precipitation and osmoregulation in marine teleost fish.

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.

Physiological impacts of elevated carbon dioxide and ocean acidification on fish

Most fish studied to date efficiently compensate for a hypercapnic acid-base disturbance; however, many recent studies examining the effects of ocean acidification on fish have documented impacts at CO2 levels predicted to occur before the end of this century. Notable impacts on neurosensory and behavioral endpoints, otolith growth, mitochondrial function, and metabolic rate demonstrate an unexpected sensitivity to current-day and near-future CO2 levels. Most explanations for these effects seem to center on increases in Pco2 and HCO3- that occur in the body during pH compensation for acid-base balance; however, few studies have measured these parameters at environmentally relevant CO2 levels or directly related them to reported negative endpoints. This compensatory response is well documented, but noted variation in dynamic regulation of acid-base transport pathways across species, exposure levels, and exposure duration suggests that multiple strategies may be utilized to cope with hypercapnia. Understanding this regulation and changes in ion gradients in extracellular and intracellular compartments during CO2 exposure could provide a basis for predicting sensitivity and explaining interspecies variation. Based on analysis of the existing literature, the present review presents a clear message that ocean acidification may cause significant effects on fish across multiple physiological systems, suggesting that pH compensation does not necessarily confer tolerance as downstream consequences and tradeoffs occur. It remains difficult to assess if acclimation responses during abrupt CO2 exposures will translate to fitness impacts over longer timescales. Nonetheless, identifying mechanisms and processes that may be subject to selective pressure could be one of many important components of assessing adaptive capacity.

Acute embryonic or juvenile exposure to Deepwater Horizon crude oil impairs the swimming performance of mahi-mahi (Coryphaena hippurus).

The Deepwater Horizon incident likely resulted in exposure of commercially and ecologically important fish species to crude oil during the sensitive early life stages. We show that brief exposure of a water-accommodated fraction of oil from the spill to mahi-mahi as juveniles, or as embryos/larvae that were then raised for ∼25 days to juveniles, reduces their swimming performance. These physiological deficits, likely attributable to polycyclic aromatic hydrocarbons (PAHs), occurred at environmentally realistic exposure concentrations. Specifically, a 48 h exposure of 1.2 ± 0.6 μg L(-1) ΣPAHs (geometric mean ± SEM) to embryos/larvae that were then raised to juvenile stage or a 24 h exposure of 30 ± 7 μg L(-1) ΣPAHs (geometric mean ± SEM) directly to juveniles resulted in 37% and 22% decreases in critical swimming velocities (Ucrit), respectively. Oil-exposed larvae from the 48 h exposure showed a 4.5-fold increase in the incidence of pericardial and yolk sac edema relative to controls. However, this larval cardiotoxicity did not manifest in a reduced aerobic scope in the surviving juveniles. Instead, respirometric analyses point to a reduction in swimming efficiency as a potential alternative or contributing mechanism for the observed decreases in Ucrit.

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.

Renal Cu and Na excretion and hepatic Cu metabolism in both Cu acclimated and non acclimated rainbow trout (Oncorhynchus mykiss)

Abstract 64Cu and total Cu accumulation were measured in gills, plasma, liver, kidney, bile and urine during 72 h of exposure to 64Cu at 20 μg Cu l−1, in non-acclimated and Cu-acclimated (28 days of pre-exposure) rainbow trout (Oncorhynchus mykiss) fitted with urinary bladder catheters. Renal Cu excretion gradually declined from 0.03 μg Cu kg−1 h−1 in non-exposed fish to 0.01 μg Cu kg−1 h−1 after 28 days of Cu exposure. A comparison of the 64Cu-labelled Cu and the total Cu excretion rates and the corresponding renal clearance revealed apparent differences in Cu binding to plasma protein depending on whether the Cu is derived from recent branchial uptake or is already present in the plasma prior to 64Cu exposure. The plasma Cu pool derived from recent branchial uptake and the Cu pool present in the plasma prior to 64Cu exposure is accessible to renal excretion to different extents, whereas the pools seem equally accessible to hepatic accumulation and elimination. The renal Cu excretion is of minor importance compared with the hepatic Cu excretion, which was estimated to be 0.5–0.75 μg Cu kg−1 h−1 and 1.1–1.6 μg Cu kg−1 h−1 for non-acclimated and Cu-acclimated fish, respectively. Based on the biliary Cu concentration, hepatic Cu elimination appeared to be stimulated in the Cu-acclimated relative to the non-acclimated fish. Only 17% and 12% of the hepatic Cu could be accounted for by metallothionein in the control and Cu-acclimated fish, respectively. Renal Na+ efflux decreased by 40%, which was largely due to increased tubular Na+ reabsorption. Renal compensation for the impaired branchial Na+ uptake, seen during Cu exposure, thus seems to be involved in Cu acclimation in rainbow trout.

Chronic toxicity of lead to three freshwater invertebrates-Brachionus calyciflorus, Chironomus tentans, and Lymnaea stagnalis

Chronic lead (Pb) toxicity tests with Brachionus calyciflorus, Chironomus tentans, and Lymnaea stagnalis were performed in artificial freshwaters. The no-observable-effect concentration (NOEC), lowest-observable-effect concentration (LOEC), and calculated 20% effect concentration (EC20) for the rotifer B. calyciflorus were 194, 284, and 125 microg dissolved Pb/L, respectively. The midge C. tentans was less sensitive, with NOEC and LOEC of 109 and 497 microg dissolved Pb/L, respectively, and the snail L. stagnalis exhibited extreme sensitivity, evident by NOEC, LOEC, and EC20 of 12, 16, and < 4 microg dissolved Pb/L, respectively. Our findings are presented in the context of other reports on chronic Pb toxicity in freshwater organisms. The L. stagnalis results are in agreement with a previous report on pulmonate snails and should be viewed in the context of current U.S. Environmental Protection Agency (U.S. EPA) hardness adjusted water quality criteria of 8 microg Pb/L. The present findings and earlier reports indicate that freshwater pulmonate snails may not be protected by current regulatory standards. Measurements of whole-snail Na+ and Ca2+ concentrations following chronic Pb exposure revealed that Na+ homeostasis is disturbed by Pb exposure in juvenile snails in a complicated pattern, suggesting two physiological modes of action depending on the Pb exposure concentration. Substantially reduced growth in the snails that exhibit very high Ca2+ requirements may be related to reduced Ca2+ uptake and thereby reduced shell formation.

Osmoregulation and Excretion

The article discusses advances in osmoregulation and excretion with emphasis on how multicellular animals in different osmotic environments regulate their milieu intérieur. Mechanisms of energy transformations in animal osmoregulation are dealt with in biophysical terms with respect to water and ion exchange across biological membranes and coupling of ion and water fluxes across epithelia. The discussion of functions is based on a comparative approach analyzing mechanisms that have evolved in different taxonomic groups at biochemical, cellular and tissue levels and their integration in maintaining whole body water and ion homeostasis. The focus is on recent studies of adaptations and newly discovered mechanisms of acclimatization during transitions of animals between different osmotic environments. Special attention is paid to hypotheses about the diversity of cellular organization of osmoregulatory and excretory organs such as glomerular kidneys, antennal glands, Malpighian tubules and insect gut, gills, integument and intestine, with accounts on experimental approaches and methods applied in the studies. It is demonstrated how knowledge in these areas of comparative physiology has expanded considerably during the last two decades, bridging seminal classical works with studies based on new approaches at all levels of anatomical and functional organization. A number of as yet partially unanswered questions are emphasized, some of which are about how water and solute exchange mechanisms at lower levels are integrated for regulating whole body extracellular water volume and ion homeostasis of animals in their natural habitats.