Alex M. Zimmer

Waterborne copper exposure inhibits ammonia excretion and branchial carbonic anhydrase activity in euryhaline guppies acclimated to both fresh water and sea water

Inhibition of ammonia excretion (J(amm)) is a common response to Cu exposure in freshwater (FW) and seawater (SW) organisms. To determine the mechanism of this response, a euryhaline species of guppy (Poecilia vivipara) was exposed to 20 μg Cu/l in FW (0 ppt) and SW (25 ppt) for 96 h. In both salinities, Cu transiently inhibited ammonia excretion (J(amm)) followed by a full recovery by the end of the 96 h exposure. The activities of Na(+)/K(+)-ATPase, H(+)-ATPase, and carbonic anhydrase (CA) were examined in the gills at 12 and 96 h of Cu exposure. In both salinity acclimations, CA activity was significantly inhibited following 12h of Cu exposure in P. vivipara, marking the first in vivo evidence of Cu-induced inhibition of CA in fish. Moreover, the inhibition and recovery of this enzyme were correlated with the inhibition and recovery of J(amm) in both salinity acclimations. The blockade of CA potentially acts as a common mechanism of J(amm) inhibition in FW and SW. There were no significant effects on Na(+)/K(+)-ATPase or H(+)-ATPase activity at either time point or salinity. However, H(+)-ATPase activity was upregulated at 96 h relative to the 12h time point, potentially involving this enzyme in re-establishing J(amm).

Branchial and extra-branchial ammonia excretion in goldfish (Carassius auratus) following thermally induced gill remodeling.

Under cold acclimated conditions, goldfish (Carassius auratus) express an interlamellar cell mass (ILCM) which limits diffusive ion loss but may also impede branchial ammonia excretion (J(amm)). In the present study, goldfish were subjected to a 2-week 5 or 25 °C acclimation in order to modulate the degree of ILCM gill coverage and determine potential effects on J(amm). 25 °C-fish displayed gill coverage which was significantly lower than the 5 °C-fish, though the ILCM was not completely absent in these fish. 5 °C-fish demonstrated J(amm) values approximately 60% lower than those of 25 °C-fish. The magnitude of anterior (branchial) J(amm) strongly correlated with gill coverage (r(2)=0.83), suggesting that the ILCM may impede branchial J(amm). Divided chamber experiments demonstrated that relative to the 25 °C-fish, 5 °C-fish relied more upon posterior routes of excretion. In response to high external ammonia (HEA; 1.5mM NH(4)HCO(3)) exposures, 25 °C-fish displayed ammonia uptake while 5 °C-fish maintained excretion against HEA, suggesting that the ILCM may act as a barrier preventing ammonia uptake. In summary, the ILCM appears to impede branchial J(amm), such that 5 °C-rely more on extra-branchial routes of excretion. We hypothesize that gill remodeling in these fish may be intimately tied to physiological adjustments on the whole-body scale.

Acute exposure to waterborne copper inhibits both the excretion and uptake of ammonia in freshwater rainbow trout (Oncorhynchus mykiss)

In freshwater fish, exposure to sub-lethal concentrations of waterborne copper (Cu) results in inhibitions of ammonia excretion (Jamm) and Na(+) uptake (J(Na)in), yet the mechanisms by which these occur are not fully understood. In the present study, rainbow trout (Oncorhynchus mykiss) fry exposed to 50μg/l Cu for 24h displayed a sustained 40% decrease in Jamm and a transient 60% decrease in J(Na)in. Previously, these effects have been attributed to inhibitions of gill Na(+)/K(+)-ATPase and/or carbonic anhydrase (CA) activities by Cu. Trout fry did not display significant reductions in the branchial activities of these enzymes or H(+)-ATPase over 24h Cu exposure. Recently, Rhesus (Rh) glycoproteins, bi-directional NH3 gas channels, have been implicated in the mechanism of Cu toxicity. Juvenile trout were exposed to nominal 0, 50, and 200μg/l Cu for 3-6h under control conditions (ammonia-free water) followed by 6h exposure to high environmental ammonia (HEA; 1.5mmol/l NH4HCO3). HEA led to significant ammonia uptake in control fish (0μg/l Cu), and exposure to 50 and 200μg/l Cu resulted in significant reductions of ammonia uptake during HEA exposure. This is the first evidence that Cu inhibits both the excretion and uptake of ammonia, implicating bi-directional Rh glycoproteins as a target for Cu toxicity. We propose a model whereby Rh blockade by Cu causes the sustained inhibition of Jamm and transient inhibition of J(Na)in, with H(+)-ATPase potentially aiding in J(Na)in recovery. More work is needed to elucidate the role of Rh proteins in sub-lethal Cu toxicity.

What is the primary function of the early teleost gill? Evidence for Na + /NH + 4 exchange in developing rainbow trout ( Oncorhynchus mykiss )

Post-hatch fishes lack a functional gill and use cutaneous surfaces for exchange with the surrounding environment. The ionoregulatory hypothesis posits that ionoregulation is the first physiological process to be limited by cutaneous exchange, necessitating its shift to the gills. We hypothesized that the ontogeny of branchial ammonia excretion (Jamm) is coupled to Na+ uptake () in accordance with the current model for exchange in freshwater. Using divided chambers, branchial and cutaneous Jamm, and oxygen consumption (MO2) by larval rainbow trout were assessed. Following hatch, the skin accounted for 97% and 86% of total Jamm and , respectively. Jamm and shifted to the gills simultaneously at 15 days post-hatch (dph) and were highly correlated (R2 = 0.951) at the gills, but not the skin, over development. Contrastingly, MO2 shifted significantly later at 27 dph, in agreement with the ionoregulatory hypothesis. Moreover, the mRNA expression and/or enzymatic activity of Rhesus proteins, Na+/H+-exchanger, H+-ATPase, Na+/K+-ATPase and carbonic anhydrase, all key components of the -exchange system, increased in the gills over larval development. We propose that the ontogeny of branchial occurs as exchange and provide evidence for a novel element to the ionoregulatory hypothesis, the excretion of potentially lethal metabolic ammonia.

It's all in the gills: evaluation of O2 uptake in Pacific hagfish refutes a major respiratory role for the skin.

ABSTRACT Hagfish skin has been reported as an important site for ammonia excretion and as the major site of systemic oxygen acquisition. However, whether cutaneous O2 uptake is the dominant route of uptake remains under debate; all evidence supporting this hypothesis has been derived using indirect measurements. Here, we used partitioned chambers and direct measurements of oxygen consumption and ammonia excretion to quantify cutaneous and branchial exchanges in Pacific hagfish (Eptatretus stoutii) at rest and following exhaustive exercise. Hagfish primarily relied on the gills for both O2 uptake (81.0%) and ammonia excretion (70.7%). Following exercise, both O2 uptake and ammonia excretion increased, but only across the gill; cutaneous exchange was not increased. When branchial O2 availability was reduced by exposure to anteriorly localized hypoxia (∼4.6 kPa O2), cutaneous O2 consumption was only slightly elevated on an absolute basis. These results refute a major role for cutaneous O2 acquisition in the Pacific hagfish. Summary: Despite being a highly specialized transport epithelium, the skin of Pacific hagfish does not contribute substantially to O2 uptake even when branchial O2 uptake is severely impaired.

Exposure to waterborne Cu inhibits cutaneous Na+ uptake in post-hatch larval rainbow trout (Oncorhynchus mykiss)

In freshwater rainbow trout (Oncorhynchus mykiss), two common responses to acute waterborne copper (Cu) exposure are reductions in ammonia excretion and Na(+) uptake at the gills, with the latter representing the likely lethal mechanism of action for Cu in adult fish. Larval fish, however, lack a functional gill following hatch and rely predominantly on cutaneous exchange, yet represent the most Cu-sensitive life stage. It is not known if Cu toxicity in larval fish occurs via the skin or gills. The present study utilized divided chambers to assess cutaneous and branchial Cu toxicity over larval development, using disruptions in ammonia excretion (Jamm) and Na(+) uptake (Jin(Na)) as toxicological endpoints. Early in development (early; 3 days post-hatch; dph), approximately 95% of Jamm and 78% of Jin(Na) occurred cutaneously, while in the late developmental stage (late; 25 dph), the gills were the dominant site of exchange (83 and 87% of Jamm and Jin(Na), respectively). Exposure to 50 μg/l Cu led to a 49% inhibition of Jamm in the late developmental stage only, while in the early and middle developmental (mid; 17 dph) stages, Cu had no effect on Jamm. Jin(Na), however, was significantly inhibited by Cu exposure at the early (53% reduction) and late (47% reduction) stages. Inhibition at the early stage of development was mediated by a reduction in cutaneous uptake, representing the first evidence of cutaneous metal toxicity in an intact aquatic organism. The inhibitions of both Jamm and Jin(Na) in the late developmental stage occurred via a reduction in branchial exchange only. The differential responses of the skin and gills to Cu exposure suggest that the mechanisms of Jamm and Jin(Na) and/or Cu toxicity differ between these tissues. Exposure to 20μg/l Cu revealed that Jamm is the more Cu-sensitive process. The results presented here have important implications in predicting metal toxicity in larval fish. The Biotic Ligand Model (BLM) is currently used to predict metal toxicity in aquatic organisms. However, for rainbow trout this is based on gill binding constants from juvenile fish. This may not be appropriate for post-hatch larval fish where the skin is the site of toxic action of Cu. Determining Cu binding constants and lethal accumulation concentrations for both skin and gills in larval fish may aid in developing a larval fish-specific BLM. Overall, the changing site of toxic action and physiology of developing larval fish present an interesting and exciting avenue for future research.

Ammonia first? The transition from cutaneous to branchial ammonia excretion in developing rainbow trout is not altered by exposure to chronically high NaCl.

ABSTRACT Larval rainbow trout (Oncorhynchus mykiss) were reared from hatch under control ([Na+]=0.60 mmol l−1) or high NaCl ([Na+]=60 mmol l−1) conditions to elucidate the driving force for the ontogeny of branchial Na+/NH4+ exchange, one of the earliest gill functions. We hypothesized that if Na+ uptake is the driving force, then in high NaCl there would be a delay in the skin-to-gill shift in ammonia excretion (Jamm) and/or an elevation in whole-body total ammonia (Tamm). In both groups, however, the skin-to-gill shift for Jamm, determined using divided chambers, occurred at the same time (13 days post-hatch; dph) and whole-body Tamm was unchanged. Moreover, high NaCl larvae displayed elevated whole-body [Na+] relative to controls by 18 dph, suggesting that maintaining branchial Jamm occurs at the expense of Na+ balance. Overall, these results support the ‘ammonia hypothesis’, which posits that ammonia excretion, probably as Na+/NH4+ exchange, is the primary function of the early fish gill. Summary: Rearing larval rainbow trout in high NaCl reveals support for the ‘ammonia hypothesis’, which posits that ammonia excretion is the earliest gill function over development and may drive gill ontogeny.

Ammonia and urea handling by early life stages of fishes

ABSTRACT Nitrogen metabolism in fishes has been a focus of comparative physiologists for nearly a century. In this Review, we focus specifically on early life stages of fishes, which have received considerable attention in more recent work. Nitrogen metabolism and excretion in early life differs fundamentally from that of juvenile and adult fishes because of (1) the presence of a chorion capsule in embryos that imposes a limitation on effective ammonia excretion, (2) an amino acid-based metabolism that generates a substantial ammonia load, and (3) the lack of a functional gill, which is the primary site of nitrogen excretion in juvenile and adult fishes. Recent findings have shed considerable light on the mechanisms by which these constraints are overcome in early life. Perhaps most importantly, the discovery of Rhesus (Rh) glycoproteins as ammonia transporters and their expression in ion-transporting cells on the skin of larval fishes has transformed our understanding of ammonia excretion by fishes in general. The emergence of larval zebrafish as a model species, together with genetic knockdown techniques, has similarly advanced our understanding of ammonia and urea metabolism and excretion by larval fishes. It has also now been demonstrated that ammonia excretion is one of the primary functions of the developing gill in rainbow trout larvae, leading to new hypotheses regarding the physiological demands driving gill development in larval fishes. Here, we highlight and discuss the dramatic changes in nitrogen handling that occur over early life development in fishes. Summary: We review the metabolic, morphological and physiological features unique to embryonic and larval fishes that are necessary to cope with an amino acid-fuelled metabolism and lack of a functional gill.

Different mechanisms of Na+ uptake and ammonia excretion by the gill and yolk sac epithelium of early life stage rainbow trout

ABSTRACT In rainbow trout, the dominant site of Na+ uptake (JNa,in) and ammonia excretion (Jamm) shifts from the skin to the gills over development. Post-hatch (PH; 7 days post-hatch) larvae utilize the yolk sac skin for physiological exchange, whereas by complete yolk sac absorption (CYA; 30 days post-hatch), the gill is the dominant site. At the gills, JNa,in and Jamm occur via loose Na+/NH4+ exchange, but this exchange has not been examined in the skin of larval trout. Based on previous work, we hypothesized that, contrary to the gill model, JNa,in by the yolk sac skin of PH trout occurs independently of Jamm. Following a 12 h exposure to high environmental ammonia (HEA; 0.5 mmol l−1 NH4HCO3; 600 µmol l−1 Na+; pH 8), Jamm by the gills of CYA trout and the yolk sac skin of PH larvae, which were isolated using divided chambers, increased significantly. However, this was coupled to an increase in JNa,in across the gills only, supporting our hypothesis. Moreover, gene expression of proteins involved in JNa,in [Na+/H+-exchanger-2 (NHE2) and H+-ATPase] increased in response to HEA only in the CYA gills. We further identified expression of the apical Rhesus (Rh) proteins Rhcg2 in putative pavement cells and Rhcg1 (co-localized with apical NHE2 and NHE3b and Na+/K+-ATPase) in putative peanut lectin agglutinin-positive (PNA+) ionocytes in gill sections. Similar Na+/K+-ATPase-positive cells expressing Rhcg1 and NHE3b, but not NHE2, were identified in the yolk sac epithelium. Overall, our findings suggest that the mechanisms of JNa,in and Jamm by the dominant exchange epithelium at two distinct stages of early development are fundamentally different. Summary: Na+ uptake occurs as Na+/NH4+ exchange by the gills, but not the yolk sac skin, of early life stage rainbow trout, possibly resulting from the lack of the key transporter NHE2 in the yolk sac epithelium.

Acute exposure to high environmental ammonia (HEA) triggers the emersion response in the green shore crab.

The physiological effects of high environmental ammonia (HEA) exposure have been well documented in many aquatic species. In particular, it has recently been demonstrated that exposure to ammonia in fish leads to a similar hyperventilatory response as observed during exposure to hypoxia. In littoral crabs, such as the green crab (Carcinus maenas), exposure to severe hypoxia triggers an emersion response whereby crabs escape hypoxia to breathe air. We hypothesized that exposure to HEA in green crabs would lead to a similar behavioural response which is specific to ammonia. Using an experimental arena containing a rock bed onto which crabs could emerse, we established that exposure to HEA (4mmol/l NH4HCO3) for 15min triggers emersion in crabs. In experiments utilizing NaHCO3 controls and NH4HCO3 injections, we further determined that emersion was triggered specifically by external ammonia and was independent of secondary acid-base or respiratory disturbances caused by HEA. We then hypothesized that emersion from HEA provides a physiological benefit, similar to emersion from hypoxia. Exposure to 15min of HEA without emersion (no rock bed present) caused significant increases in arterial haemolymph total ammonia (Tamm), pH, and [HCO3-]. When emersion was allowed, arterial haemolymph Tamm and [HCO3-] increased, but no alkalosis developed. Moreover, emersion decreased haemolymph partial pressure of NH3 relative to crabs which could not emerse. Overall, we demonstrate a novel behavioural response to HEA exposure in crabs which we propose may share similar mechanistic pathways with the emersion response triggered by hypoxia.