The gut microbiome may influence post-prandial nitrogen handling in an elasmobranch, the Pacific spiny dogfish (Squalus suckleyi).

Nitrogen recycling through the gut microbiome is an important mechanism used throughout vertebrates to reclaim valuable nitrogen trapped in urea. Evidence suggests it may be especially important in nitrogen limited animals, yet little is known about its role in marine elasmobranchs, which are said to be severely nitrogen limited. In the present study we used antibiotics to deplete the gut microbiome of Pacific spiny dogfish and assessed the role of the microbiome in nitrogen handling in both fed and fasted states. In fed animals, antibiotic treatment eliminated the activity of the microbial enzyme urease and reduced cellulase activity by 78%. This reduction in microbial enzyme activity resulted in significantly lower plasma urea levels which then trended upward as urea excretion rates decreased. Ammonia excretion rates were also significantly lower in antibiotic treated fish compared to the control fed. Finally, antibiotic treated fed individuals lost an average of 7.4% of their body mass while the fed controls lost only 1.8% of their body mass. Nitrogen handling in fasted animals was not significantly impacted by a reduction in microbial activity. These results suggest that compromising the gut microbiome significantly influences post-prandial nitrogen handling in spiny dogfish, and that the recycling of urea­nitrogen may be vital to maintaining nitrogen balance in these fish.

The role of intestinal bacteria in the ammonia detoxification ability of teleost fish.

Protein catabolism during digestion generates appreciable levels of ammonia in the gastrointestinal tract (GIT) lumen. Amelioration by the enterocyte, via enzymes such as glutamine synthetase (GS), glutamate dehydrogenase (GDH), and alanine and aspartate aminotransferases (ALT; AST), is found in teleost fish. Conservation of these enzymes across bacterial phyla suggests that the GIT microbiome could also contribute to ammonia detoxification by providing supplemental activity. Hence, the GIT microbiome, enzyme activities and ammonia detoxification were investigated in two fish occupying dissimilar niches: the carnivorous rainbow darter and the algivorous central stoneroller. There was a strong effect of fish species on the activity levels of GS, GDH, AST and ALT, as well as GIT lumen ammonia concentration, and bacterial composition of the GIT microbiome. Furthermore, removal of the intestinal bacteria impacted intestinal activities of GS and ALT in the herbivorous fish but not in the carnivore. The repeatability and robustness of this relationship was tested across field locations and years. Within an individual waterbody, there was no impact of sampling location on any of these factors. However, different waterbodies affected enzyme activities and luminal ammonia concentrations in both fish, while only the central stoneroller intestinal bacteria populations varied. Overall, a relationship between GIT bacteria, enzyme activity and ammonia detoxification was observed in herbivorous fish while the carnivorous fish displayed a correlation between enzyme activity and ammonia detoxification alone that was independent of the GIT microbiome. This could suggest that carnivorous fish are less dependent on non-host mechanisms for ammonia regulation in the GIT.

Zonation of Ca2+ transport and enzyme activity in the caeca of rainbow trout - a simple structure with complex functions.

Trout caeca are vermiform structures projecting from the anterior intestine of the gastrointestinal tract. Despite their simple gross morphology, these appendages are physically distinct along the anterior-posterior axis, and ultrastructural evidence suggests zonation of function within the structures. Individual caeca from three sections (anterior, middle and posterior) were removed from the intestine of freshwater rainbow trout and investigated for ion transport and enzyme activity. Ca2+ absorption appeared as a combination of active and passive movement, with Michaelis-Menten kinetics observable under symmetrical conditions, and was inhibited by several pharmacological agents (ouabain, La3+ and a calmodulin antagonist). There was a decrease in ion transport function from adjacent to the intestine (proximal) to the distal tip of each caecum, along with decreasing transport from anterior to posterior for the proximal portion alone. Feeding increased the JMax and KM for Ca2+ absorption within all sections, whereas ion-poor water (IPW) exposure further increased the JMax and KM for Ca2+ transport in the anterior and middle sections. Increased Na+/K+-ATPase (NKA) and citrate synthase (CS) activity rates paralleled trends seen in Ca2+ transport. Feeding in freshwater and IPW exposure increased the glycolytic capacity of the caeca via increased pyruvate kinase (PK) and decreased lactate dehydrogenase (LDH) activity, while amino acid metabolism increased with IPW exposure through increased glutamate dehydrogenase (GDH) activity. Overall, feeding and IPW exposure each altered ionoregulation within the caeca of freshwater rainbow trout in a zone-specific pattern, with the anterior and proximal portions of the caeca being most affected. Increased carbohydrate and protein metabolism fueled the increased ATP demand of NKA through CS.

Feeding in Eptatretus cirrhatus: effects on metabolism, gut structure and digestive processes, and the influence of post-prandial dissolved oxygen availability.

Hagfishes are characterised by feeding behaviours that may include long intervals between meals, and a hypoxic feeding environment inside decaying carrion. The effects of feeding on metabolism (oxygen consumption rate), gut mass and morphology (gut somatic index, gut epithelium mucosal thickness), and digestive function (maltase and peptidase activity) were examined in the New Zealand hagfish, Eptatretus cirrhatus. The influence of post-prandial hypoxia on oxygen consumption rate was also investigated to replicate the immersive feeding environment. Fed hagfish displayed a 1.9-fold increase in peak oxygen consumption relative to sham controls. This elevation in post-prandial oxygen consumption continued for 72 h, during which the energy cost of digesting the meal (specific dynamic action; SDA) was 2.1 kJ. Oxygen consumption rate increased when the post-prandial environment was hypoxic, a response suggesting a lack of hypoxia tolerance in this species. Feeding did not alter gut somatic index (percentage of digesta-free gut mass to whole body mass), but there was an increase in the mucosal thickness of the gut epithelium. Maltase activity in the gut was unchanged by feeding, but the activity of gut peptidases was increased significantly, consistent with a protein-based diet. These data indicate that some postprandial responses of New Zealand hagfish are similar in nature to those seen in other animals, but this species does not exhibit the extreme post-prandial physiological and biochemical changes that are observed in other intermittently-feeding vertebrates.

Identification of the putative goldfish (Carassius auratus) magnesium transporter SLC41a1 and functional regulation in the gill, kidney, and intestine in response to dietary and environmental manipulations.

While magnesium requirements for teleost fish highlight the physiological importance of this cation for homeostasis, little is known regarding the molecular identity of transporters responsible for magnesium absorption or secretion. The recent characterization of the vertebrate magnesium transporter solute carrier 41a1 (SLC41a1) in the kidney of a euryhaline fish has provided a glimpse of possible moieties involved in piscine magnesium regulation. The present study obtained a novel SLC41a1 coding sequence for Carassius auratus and demonstrated ubiquitous expression in all tissues examined. Transcriptional regulation of SLC41a1 in response to dietary and environmental magnesium concentrations was observed across tissues. Specifically, decreased environmental magnesium correlated with decreased expression of SLC41a1 in the intestine, whereas the gill and kidney were unaffected. Dietary magnesium restriction correlated with decreased expression of SLC41a1 in the intestine and gill, while again no effects were detected in the kidney. Finally, elevated dietary magnesium correlated with increased expression of SLC41a1 in the kidney, while expression in the intestine and gill remained stable. Plasma magnesium was maintained in all treatments, and dietary assimilation efficiency increased with decreased dietary magnesium. Consumption of a single meal failed to impact SLC41a1 expression, and transcript abundance remained stable over the course of digestion in all treatments. Transcriptional regulation occurred between 7 and 14days following dietary and environmental manipulations and short-term regulation (e.g. <24h) was not observed. Overall the data supports transcriptional regulation of SLC41a1 reflecting a possible role in magnesium loss or secretion across tissues in fish.

Immunohistochemical localization of urea and ammonia transporters in two confamilial fish species, the ureotelic gulf toadfish (Opsanus beta) and the ammoniotelic plainfin midshipman (Porichthys notatus).

This study aims to illustrate potential transport mechanisms behind the divergent approaches to nitrogen excretion seen in the ureotelic toadfish (Opsanus beta) and the ammoniotelic plainfin midshipman (Porichthys notatus). Specifically, we wish to confirm the expression of a urea transporter (UT), which is found in the gill of the toadfish and which is responsible for the unique "pulsing" nature of urea excretion and to localize the transporter within specific gill cells and at specific cellular locations. Additionally, the localization of ammonia transporters (Rhesus glycoproteins; Rhs) within the gill of both the toadfish and midshipman was explored. Toadfish UT (tUT) was found within Na(+)-K(+)-ATPase (NKA)-enriched cells, i.e., ionocytes (probably mitochondria-rich cells), especially along the basolateral membrane and potentially on the apical membrane. In contrast, midshipman UT (pnUT) immunoreactivity did not colocalize with NKA immunoreactivity and was not found along the filaments but instead within the lamellae. The cellular location of Rh proteins was also dissimilar between the two fish species. In toadfish gills, the Rh isoform Rhcg1 was expressed in both NKA-reactive cells and non-reactive cells, whereas Rhbg and Rhcg2 were only expressed in the latter. In contrast, Rhbg, Rhcg1 and Rhcg2 were expressed in both NKA-reactive and non-reactive cells of midshipman gills. In an additional transport epithelium, namely the intestine, the expression of both UTs and Rhs was similar between the two species, with only subtle differences being observed.

Uptake, handling and excretion of Na+ and Cl- from the diet in vivo in freshwater- and seawater-acclimated killifish, Fundulus heteroclitus, an agastric teleost.

A radiotracer approach using diets labelled with (22)Na(+), (36)Cl(-) and [(14)C]polyethylene-4000 (PEG-4000) was employed to investigate the role of intestinal uptake from the food in ion homeostasis in the killifish Fundulus heteroclitus. This euryhaline teleost lacks both a stomach and the capacity for Cl(-) uptake at the gills in freshwater. PEG-4000 appearance in the water was minimal up to 10-11 h post-feeding, indicating the virtual absence of Na(+) and Cl(-) loss in the faeces up until this time. Rapid uptake of dietary Na(+) and Cl(-) occurred and more than 88% of (22)Na(+) and (36)Cl(-) were absorbed in the intestine by 3 h post-feeding; excretion rates of Na(+) and Cl(-) originating from the food were greatest during this period. Uptake and excretion of Cl(-) from the diet was fivefold to sixfold greater than that of Na(+) in freshwater, and approximately threefold greater in seawater. Excretions of dietary Na(+) and Cl(-) by seawater-acclimated killifish were far greater than by freshwater-acclimated killifish in this time frame, reflecting the much greater branchial efflux rates and turnover rates of the internal exchangeable pools. At both 3 and 9 h post-feeding, the largest fraction of dietary Na(+) was found in the carcass of freshwater-acclimated fish, followed by the external water, and finally the digestive tract. However, in seawater-acclimated fish, more was excreted to the water, and less was retained in the carcass. For Cl(-), which was taken up and excreted more rapidly than Na(+), the majority of the dietary load had moved to the external water by 9 h in both freshwater and seawater animals. After 7 days training on a low-salt natural diet (live Lumbriculus variegatus worms; 31.5 µmol Na(+) g(-1) wet mass) versus a high-salt synthetic pellet diet (911 µmol Na(+) g(-1) dry food mass), freshwater killifish exhibited a lower absolute excretion rate of Na(+) from the low-salt diet, but relative uptake from the intestine and retention in the carcass were virtually identical from the two diets. Seawater killifish excreted relatively more Na(+) from the low-salt diet. Overall, our results emphasize the importance of dietary Na(+) and Cl(-) in the electrolyte economy of the killifish, particularly in freshwater, and especially for Cl(-).

Nitrogen metabolism of the intestine during digestion in a teleost fish, the plainfin midshipman (Porichthys notatus).

Digestion affects nitrogen metabolism in fish, as both exogenous and endogenous proteins and amino acids are catabolized, liberating ammonia in the process. Here we present a model of local detoxification of ammonia by the intestinal tissue of the plainfin midshipman (Porichthys notatus) during digestion, resulting in an increase in urea excretion of gastrointestinal origin. Corroborating evidence indicated whole-animal ammonia and urea excretion increased following feeding, and ammonia levels within the lumen of the midshipman intestine increased to high levels (1.8±0.4 µmol N g(-1)). We propose that this ammonia entered the enterocytes and was detoxified to urea via the ornithine-urea cycle (O-UC) enzymes, as evidenced by a 1.5- to 2.9-fold post-prandial increase in glutamine synthetase activity (0.14±0.05 and 0.28±0.02 µmol min(-1) g(-1) versus 0.41±0.03 µmol min(-1) g(-1)) and an 8.7-fold increase in carbamoyl phosphate synthetase III activity (0.3±1.2 versus 2.6±0.4 nmol min(-1) g(-1)). Furthermore, digestion increased urea production by isolated gastrointestinal tissue 1.7-fold, supporting our hypothesis that intestinal tissue synthesizes urea in response to feeding. We further propose that the intestinal urea may have been excreted into the intestinal lumen via an apical urea transporter as visualized using immunohistochemistry. A portion of the urea was then excreted to the environment along with the feces, resulting in the observed increase in urea excretion, while another portion may have been used by intestinal ureolytic bacteria. Overall, we propose that P. notatus produces urea within the enterocytes via a functional O-UC, which is then excreted into the intestinal lumen. Our model of intestinal nitrogen metabolism does not appear to be universal as we were unab le to activate the O-UC in the intestine of fed rainbow trout. However, literature values suggest that multiple fish species could follow this model.

Diet influences salinity preference of an estuarine fish, the killifish Fundulus heteroclitus.

Understanding the interplay among the external environment, physiology and adaptive behaviour is crucial for understanding how animals survive in their natural environments. The external environment can have wide ranging effects on the physiology of animals, while behaviour determines which environments are encountered. Here, we identified changes in the behavioural selection of external salinity in Fundulus heteroclitus, an estuarine teleost, as a consequence of digesting a meal. Fish that consumed high levels of dietary calcium exhibited a higher preferred salinity compared with unfed fish, an effect that was exaggerated by elevated dietary sodium chloride. The mean swimming speed (calculated as a proxy of activity level) was not affected by consuming a diet of any type. Constraining fish to water of 22 p.p.t. salinity during the digestion of a meal did not alter the amount of calcium that was absorbed across the intestine. However, when denied the capacity to increase their surrounding salinity, the compromised ability to excrete calcium to the water resulted in significantly elevated plasma calcium levels, a potentially hazardous physiological consequence. This study is the first to show that fish behaviourally exploit their surroundings to enhance their ionoregulation during digestion, and to pinpoint the novel role of dietary calcium and sodium in shaping this behaviour. We conclude that in order to resolve physiological disturbances in ion balance created by digestion, fish actively sense and select the environment they inhabit. Ultimately, this may result in transient diet-dependent alteration of the ecological niches occupied by fishes, with broad implications for both physiology and ecology.

Environmental and nutritional regulation of expression and function of two peptide transporter (PepT1) isoforms in a euryhaline teleost.

Expression and function of the oligopeptide transporter PepT1 in response to changes in environmental salinity have received little study despite the important role that dipeptides play in piscine nutrition. We cloned and sequenced two novel full-length cDNAs that encode Fundulus heteroclitus PepT1-type oligopeptide transporters, and examined their expression and functional properties in freshwater- and seawater-acclimated fish and in response to fasting and re-feeding. Phylogenetic analysis of vertebrate SLC15A1 sequences confirms the presence of two PepT1 isoforms, named SLC15A1a and SLC15A1b, in fish. Similar to other vertebrate SLC15A1s, these isoforms have 12 transmembrane domains, and amino acids essential for PepT1 function are conserved. Expression analysis revealed novel environment-specific expression of the SLC15A1 isoforms in F. heteroclitus, with only SLC15A1b expressed in seawater-acclimated fish, and both isoforms expressed in freshwater-acclimated fish. Fasting and re-feeding induced changes in the expression of SLC15A1a and SLC15A1b mRNA. Short-term fasting resulted in up-regulation of PepT1 mRNA levels, while prolonged fasting resulted in down-regulation. The resumption of feeding resulted in up-regulation of PepT1 above pre-fasted levels. Experiments using the in vitro gut sac technique suggest that the PepT1 isoforms differ in functional characteristics. An increased luminal pH resulted in decreased intestinal dipeptide transport in freshwater-acclimated fish but suggested an increased dipeptide transport in seawater-acclimated fish. Overall, this is the first evidence of multiple isoforms of PepT1 in fish whose expression is environmentally dependent and results in functional differences in intestinal dipeptide transport.

Adaptations to in situ feeding: novel nutrient acquisition pathways in an ancient vertebrate.

During feeding, hagfish may immerse themselves in the body cavities of decaying carcasses, encountering high levels of dissolved organic nutrients. We hypothesized that this feeding environment might promote nutrient acquisition by the branchial and epidermal epithelia. The potential for Pacific hagfish, Eptatretus stoutii, to absorb amino acids from the environment across the skin and gill was thus investigated. l-alanine and glycine were absorbed via specific transport pathways across both gill and skin surfaces, the first such documentation of direct organic nutrient acquisition in a vertebrate animal. Uptake occurred via distinct mechanisms with respect to concentration dependence, sodium dependence and effects of putative transport inhibitors across each epithelium. Significant differences in the absorbed amino acid distribution between the skin of juveniles and adults were noted. The ability to absorb dissolved organic matter across the skin and gill may be an adaptation to a scavenging lifestyle, allowing hagfish to maximize sporadic opportunities for organic nutrient acquisition. From an evolutionary perspective, hagfish represent a transitory state between the generalized nutrient absorption pathways of aquatic invertebrates and the more specialized digestive systems of aquatic vertebrates.

The gut content microbiome of wild-caught rainbow darter is altered during laboratory acclimation.

An increasing number of laboratory studies are showing that environmental stressors and diet affect the fish gut microbiome. However, the application of these results to wild populations is uncertain as little is known about how the gut microbiome shifts when fish are transitioned from the field to the laboratory. To assess this, intestinal contents (i.e. digesta) of wild-caught rainbow darter (Etheostoma caeruleum) were sampled in the field and in the lab after 14- and 42-days acclimation. In addition, from days 15-42 some fish were exposed to waterborne triclosan, an antimicrobial found in aquatic ecosystems, or to dilutions of municipal wastewater effluents, to determine how these stressors affect the bacterial communities of gut contents. 16S rRNA gene amplicon sequencing was used to determine microbial community composition, alpha, and beta diversity present in the fish gut contents. In total, there was 8,074,658 reads and 11,853 amplicon sequence variants (ASVs) identified. The gut contents of wild fish were dominant in both Proteobacteria (35%) and Firmicutes (27%), while lab fish were dominant in Firmicutes (37-47%) and had lower alpha diversity. Wild fish had greater ASVs per sample (423-1304) compared to lab fish (19-685). Similarly, the beta-diversity of these bacterial communities differed between field and lab control fish; control fish were distinct from the 10% wastewater effluent and 100 ng/L TCS treatment groups. Results indicate that the gut microbiome of wild fish changes with the transition to laboratory environments; hence, prolonged acclimation to new settings may be required to achieve a stable gut content microbiome in wild-caught fish. Research is required to understand the length of time required to reach a stable fish gut microbiome.

Acid-base responses to feeding and intestinal Cl- uptake in freshwater- and seawater-acclimated killifish, Fundulus heteroclitus, an agastric euryhaline teleost.

Marine teleosts generally secrete basic equivalents (HCO(3)(-)) and take up Na(+) and Cl(-) in the intestine so as to promote absorption of H(2)O. However, neither the integration of these functions with feeding nor the potential role of the gut in ionoregulation and acid-base balance in freshwater have been well studied. The euryhaline killifish (Fundulus heteroclitus) is unusual in lacking both an acid-secreting stomach and a mechanism for Cl(-) uptake at the gills in freshwater. Responses to a satiation meal were evaluated in both freshwater- and seawater-acclimated killifish. In intact animals, there was no change in acid or base flux to the external water after the meal, in accord with the absence of any post-prandial alkaline tide in the blood. Indeed, freshwater animals exhibited a post-prandial metabolic acidosis ('acidic tide'), whereas seawater animals showed no change in blood acid-base status. In vitro gut sac experiments revealed a substantially higher rate of Cl(-) absorption by the intestine in freshwater killifish, which was greatest at 1-3 h after feeding. The Cl(-) concentration of the absorbate was higher in preparations from freshwater animals than from seawater killifish and increased with fasting. Surprisingly, net basic equivalent secretion rates were also much higher in preparations from freshwater animals, in accord with the 'acidic tide'; in seawater preparations, they were lowest after feeding and increased with fasting. Bafilomycin (1 micromol l(-1)) promoted an 80% increase in net base secretion rates, as well as in Cl(-) and fluid absorption, at 1-3 h post-feeding in seawater preparations only, explaining the difference between freshwater and seawater fish. Preparations from seawater animals at 1-3 h post-feeding also acidified the mucosal saline, and this effect was associated with a marked rise in P(CO(2)), which was attenuated by bafilomycin. Measurements of chyme pH from intact animals confirmed that intestinal fluid (chyme) pH and basic equivalent concentration were lowest after feeding in seawater killifish, whereas P(CO(2)) was greatly elevated (80-95 Torr) in chyme from both seawater and freshwater animals but declined to lower levels (13 Torr) after 1-2 weeks fasting. There were no differences in pH, P(CO(2)) or the concentrations of basic equivalents in intestinal fluid from seawater versus freshwater animals at 12-24 h or 1-2 weeks post-feeding. The results are interpreted in terms of the absence of gastric HCl secretion, the limitations of the gills for acid-base balance and Cl(-) transport, and therefore the need for intestinal Cl(-) uptake in freshwater killifish, and the potential for O(2) release from the mucosal blood flow by the high P(CO(2)) in the intestinal fluids. At least in seawater killifish, H(+)-ATPase running in parallel to HCO(3)(-):Cl(-) exchange in the apical membranes of teleost enterocytes might reduce net base secretion and explain the high P(CO(2)) in the chyme after feeding.

The role of the kidney in compensating the alkaline tide, electrolyte load, and fluid balance disturbance associated with feeding in the freshwater rainbow trout, Oncorhynchus mykiss.

The effect in freshwater rainbow trout of digesting a commercial pellet meal on the renal handling of water, ions and acid-base equivalents was investigated through urine collection over a 48 h period following meal ingestion. The glomerular filtration rate (GFR) and urine flow rate (UFR) were reduced in fed fish between 12 and 24h following the meal, likely reflecting a loss of endogenous water across the gastric epithelium as a result of ingesting dry, ion-rich food pellets. The kidney was also responsible for the excretion of some excess dietary Ca(2+), and, to a much lesser extent, Na(+) and Cl(-), while the urinary excretion of K(+) was unaffected. The most dramatic effect of feeding was the elevation of renal Mg(2+) excretion, with the kidney transitioning from net Mg(2+) reabsorption to net Mg(2+) secretion during digestion. The renal handling of dietary ions accounted for 3-27% of the total ions absorbed from the diet, indicating that a majority of the ions are excreted extra-renally or incorporated into growth. However this does highlight the underestimation of renal ion handling when using unfed fish models. The metabolic alkalosis created by digestion (the alkaline tide) resulted in an increase in urine pH as well as a transition from net acidic equivalent excretion in the urine to net basic equivalent excretion. This was due to a decrease in the titratable acidity minus bicarbonate component of urine as well as a decrease in ammonia secretion. Additionally, the experimental separation of the urinary component of acid-base excretion from that of the gills highlighted the substantially larger contribution of the latter. During the alkaline tide, renal excretion accounted for approximately 5% of the total basic equivalent excretion to the external water.

Impacts of low salinity exposure and antibiotic application on gut transport activity in the Pacific spiny dogfish, Squalus acanthias suckleyi.

The role of the marine elasmobranch gastrointestinal tract in nitrogen-recycling and osmotic homeostasis has become increasingly apparent, with the gut microbial community likely playing a significant role converting urea, an important osmolyte in elasmobranchs, into ammonia. The Pacific spiny dogfish can experience and tolerate reduced environmental salinities, yet how this environmental challenge may affect the microbiome, and consequently nitrogen transport across the gut, is as of yet unknown. In the present study, excised gut sac preparations were made from dogfish acclimated to the following: full-strength seawater (C), low salinity for 7 days (LS), and after acute transfer of LS-acclimated fish to full-strength SW for 6 h (AT). Significantly reduced microbial derived urease activity was observed in the mucosal saline of gut sac preparations from the LS (by 81%) and AT (by 89%) treatments relative to the C treatment. Microbial derived cellulase activity from mucosal saline samples tended to follow similar patterns. To further ensure an effective decrease in the spiral valve microbial population, an antibiotic cocktail was applied to the mucosal saline used for in vitro measurements of ion, water, and nitrogen flux in these gut sac preparations. This caused a further 57-61% decrease in the mucosal saline urease activity of the C and LS treatments. Overall, we observed relatively little flux across the stomach for all measured parameters aside from water movement, which switched from a net efflux in control fish to a net influx in acutely transferred fish, indicative of drinking. While no significant differences were observed in terms of nitrogen flux (urea or ammonia), we tended to see the accumulation of ammonia in the spiral valve lumen and a switch from efflux to influx of urea in control versus acutely transferred fish. The increased ammonia production likely occurs as a result of heightened metabolism in a challenging environment, while the retention and acquisition of urea is suggestive of nitrogen scavenging under nitrogen-limiting conditions.

Quantification of Mg2+, Ca2+ and H+ transport by the gastrointestinal tract of the goldfish, Carassius auratus, using the Scanning Ion-selective Electrode Technique (SIET).

An in vitro gut-sac technique and the scanning ion-selective electrode technique (SIET) were used to characterize Mg2+, Ca2+, and H+ transport at both the mucosal and serosal surfaces of non-everted and everted gastrointestinal tissues obtained from Carassius auratus. As part of the study, two magnesium ionophores were compared (II vs. VI). Unfed animals displayed uniform transport of all ions along the intestine. Feeding resulted in elevated Mg2+ and Ca2+ transport when the gut lumen contained chyme however, under symmetrical conditions this increased transport rate was absent. Furthermore, zonation of divalent cation transport was present for both Ca2+ and Mg2+ under non-symmetrical conditions while the zonation remained for Ca2+ alone under symmetrical conditions. High dietary Mg2+ decreased absorption and induced secretion of Mg2+ in the posterior intestine. Uptake kinetics in the esophagus suggest large diffusive and/or convective components based on a linear relationship between Mg2+ transport and concentration and lack of inhibition by ouabain, an inhibitor of Na+-K+-ATPase. In contrast, kinetics in the rectum were suggestive of a low affinity, saturable carrier-mediated pathway. A decrease in Mg2+ and Ca2+ transport was observed in the posterior intestine (both at the mucosal and serosal surfaces) in response to ouabain. This impact was greatest for Ca2+ transport and when applied to the mucosal fluid and measured in everted preparations. In contrast a putative Mg2+ transport inhibitor, cobalt(III)hexamine-chloride, did not affect Mg2+ transport. This is the first study to use SIET approaches to study ion transport in the gut of teleost fish. This is also the first study to provide characterization of Mg2+ transport in the gut of C. auratus. Due to the limited selectivity of Magnesium ionophore II, subsequent studies of tissues bathed in physiological saline should be made using Magnesium Ionophore VI.

The alkaline tide goes out and the nitrogen stays in after feeding in the dogfish shark, Squalus acanthias.

In light of previous work showing a marked metabolic alkalosis ("alkaline tide") in the bloodstream after feeding in the dogfish shark (Squalus acanthias), we evaluated whether there was a corresponding net base excretion to the water at this time. In the 48 h after a natural voluntary meal (teleost tissue, averaging 5.5% of body weight), dogfish excreted 10,470 micromol kg(-1) more base (i.e. HCO3- equivalents) than the fasted control animals (which exhibited a negative base excretion of -2160 micromol kg(-1)). This large activation of branchial base excretion after feeding thereby prevented a potentially fatal alkalinization of the body fluids by the alkaline tide. The rate peaked at 330 micromol kg(-1) h(-1) at 12.5-24 h after the meal. Despite a prolonged 1.7-fold elevation in MO2 after feeding ("specific dynamic action"), urea-N excretion decreased by 39% in the same 48 h period relative to fasted controls. In contrast, ammonia-N excretion did not change appreciably. The N/O2 ratio declined from 0.51 in fasted animals to 0.19 in fed sharks, indicating a stimulation of N-anabolic processes at this time. These results, which differ greatly from those in teleost fish, are interpreted in terms of the fundamentally different ureotelic osmoregulatory strategy of elasmobranchs, and recent discoveries on base excretion and urea-retention mechanisms in elasmobranch gills.

A broader look at ammonia production, excretion, and transport in fish: a review of impacts of feeding and the environment.

For nearly a century, researchers have studied ammonia production and excretion in teleost fish. Stemming from past investigations a body of knowledge now exists on various aspects including biochemical mechanisms of ammonia formation and specific routes and tissues used for ammonia transport, culminating in a current detailed theoretical model of branchial transport, including the molecular identities of the moieties involved. However, typical studies examining ammonia balance use routine laboratory conditions and fasted fish. While avoiding additional variables that influence nitrogen balance, these studies are arguably idealistic and do not reflect the natural variety of conditions that fish encounter. Further studies have revealed the impacts of extrinsic factors (e.g. salinity, pH, temperature) on ammonia handling in fasted fish whereas others have explored intrinsic factors, such as life history and developmental impacts. One routine challenge for ammonia balance that fish encounter is feeding and digestion. Fortunately, many new studies have revealed the impact of feeding and digestion on several aspects of ammonia balance; from production to excretion and to transport, and several have done so incorporating supplemental extrinsic and/or intrinsic factors. Together, these complex studies reveal realistic responses to multifactorial challenges encountered by animals in the wild and begin to provide a holistic view of ammonia balance in freshwater teleost fish.

The interactive effect of digesting a meal and thermal acclimation on maximal enzyme activities in the gill, kidney, and intestine of goldfish (Carassius auratus).

Surrounding environmental temperatures affect many aspects of ectotherm physiology. Generally, organisms can compensate at one or more biological levels, or allow temperature to dictate processes such as enzyme activities through kinetic effects on reaction rates. As digestion also alters physiological processes such as enzyme activities, this study determined the interacting effect of thermal acclimation (8 and 20 °C) and digesting a single meal on maximal enzyme activities in three tissues of the goldfish (Carrassius auratus). Acclimation to elevated temperatures decreased branchial Na+, K+, ATPase (NKA) activity. In contrast, acclimation to elevated temperatures had no effect on citrate synthase (CS) or pyruvate kinase (PK) activity in any tissue, nor were renal NKA or glutamine synthetase (GS) activities impacted. Warm water-acclimation exaggerated the positive impact of digestion on intestinal and branchial NKA activities and intestinal GS activity only, but digestion had no effect in the kidney. CS and PK did not display intestinal zonation; however, there was a distinct increase towards the distal intestine in NKA and GS activities. Zonation of NKA was more prominent in warm-acclimated animals, while acclimation temperature did not affect intestinal heterogeneity of GS. Finally, the impact of tissue protein content on enzyme activity was discussed. We conclude that the intestine and gill of warm-acclimated goldfish exhibited an augmented capacity for increasing several enzyme activities in response to digestion while the kidney was unaffected by thermal acclimation or digesting a single meal. However, this amplified capacity was ameliorated by alterations in tissue protein content. Amplified increases in NKA activity may ultimately have implications for ATP demand in these tissues, while increased GS activity may beneficially increase ammonia-detoxifying capacity in the intestine.