Marine Fish Intestine Research Articles

Identification and characterization of a novel chromosome-encoded aminoglycoside O-nucleotidyltransferase gene, ant(9)-Id, in Providencia sp. TYF-12 isolated from the marine fish intestine

Identification and characterization of a novel chromosome-encoded aminoglycoside O-nucleotidyltransferase gene, ant(9)-Id, in Providencia sp. TYF-12 isolated from the marine fish intestine

Cellulolytic enzymes in Microbulbifer sp. Strain GL-2, a marine fish intestinal bacterium, with emphasis on endo-1,4-β-glucanases Cel5A and Cel8.

Cellulose is an abundant biomass on the planet. Various cellulases from environmental microbes have been explored for industrial use of cellulose. Marine fish intestine is of interest as one source of new enzymes. Here, we report the discovery of genes encoding two β-glucosidases (Bgl3A and Bgl3B) and four endo-1,4-β-glucanases (Cel5A, Cel8, Cel5B, and Cel9) as part of the genome sequence of a cellulolytic marine bacterium, Microbulbifer sp. Strain GL-2. Five of these six enzymes (excepting Cel5B) are presumed to localize to the periplasm or outer membrane. Transcriptional analysis demonstrated that all six genes were highly expressed in stationary phase. The transcription was induced by cello-oligosaccharides rather than by glucose, suggesting that the cellulases are produced primarily for nutrient acquisition following initial growth, facilitating the secondary growth phase. We cloned the genes encoding two of the endo-1,4-β-glucanases, Cel5A and Cel8, and purified the corresponding recombinant enzymes following expression in Escherichia coli. The activity of Cel5A was observed across a wide range of temperatures (10-40 ˚C) and pHs (6-8). This pattern differed from those of Cel8 and the commercial cellulase Enthiron, both of which exhibit decreased activities below 30 ˚C and at alkaline pHs. These characteristics suggest that Cel5A might find use in industrial applications. Overall, our results reinforce the hypothesis that marine bacteria remain a possible source of novel cellulolytic activities.

Accumulation of Microplastics and Histological Analysis on Marine Fish from Coastal Waters of Baru and Trisik Beaches, Special Region of Yogyakarta

River flow to the sea is regarded as a pathway for the dispersion and pollution of microplastics. The hydrodynamics of the coastal water of Baru and Trisik Beaches may increase microplastic concentrations in this estuarine-marine area. This research evaluated the microplastic concentration in surface seawater and microplastic accumulation in consumed marine fish and performed histological analysis on the demersal marine fish intestine under natural exposure. Sample collection was carried out at 12 stations with three replications. The microplastics analysis was performed on marine fish (dorsal muscle, gills, and gastrointestinal tract) and seawater, and microplastic characterization was based on physical and chemical characteristics. Environmental parameters for statistical analysis included air and water temperature, pH, dissolved oxygen, and salinity of seawater. Histological analysis of the intestine was performed on fish from three stations with two demersal fish species. The latter analysis included the length of the villi, the depth of the crypt cells, epithelial denudation with hematoxylin-eosin staining, and the number of goblet cells with periodic acid Schiff alcian blue staining. The results showed that the microplastic concentration was distributed throughout the fish sample for each of the stations. Microplastic concentrations for surface seawater showed the same pattern as marine fish. Microplastic accumulation in marine fish indicated the transfer of microplastic particles to various organs in the fish’s body. The histological analysis indicated, microplastic internalization in the intestine tissue, damaging intestinal structures. Further research is needed as consuming marine fish contaminated by microplastics may present increasing health risks.

An Integrative and Conjugative Element (ICE) Found in Shewanella halifaxensis Isolated from Marine Fish Intestine May Connect Genetic Materials between Human and Marine Environments.

Integrative and conjugative elements (ICEs) play a role in the horizontal transfer of antibiotic resistance genes (ARGs). We herein report an ICE from Shewanella halifaxensis isolated from fish intestine with a similar structure to both a clinical bacterial ICE and marine bacterial plasmid. The ICE was designated ICEShaJpn1, a member of the SXT/R391 family of ICEs (SRIs). ICEShaJpn1 has a common core structure with SRIs of clinical and fish origins and an ARG cassette with the pAQU1 plasmid of Photobacterium damselae subsp. damselae, suggesting that the common core of SRIs is widely distributed and ARG cassettes are collected from regional bacteria.

Intestinal response to ocean acidification in the European sea bass (Dicentrarchus labrax)

The intestine of marine fishes contributes to the ocean carbon cycle producing carbonate aggregates as part of the osmoregulatory process. Therefore, this study aimed to evaluate physiological adjustments of European sea bass (Dicentrarchus labrax) intestine to a higher pCO2 environment likely in the near future (~1700 μatm). At the whole-body level, hypercapnia for 5 weeks resulted in fish having a significantly diminished specific growth rate, condition factor and hepatosomatic index. An increase in plasma osmolality and HCO3− concentration was detected, paralleled by decreased metabolites concentrations. In the intestine, high seawater pCO2 was without effect on ouabain-sensitive ATPase activities, while Bafilomycin A1-sensitive ATPase activity significantly decreased in the anterior intestine. Anterior and mid intestine were mounted in Ussing chambers in order to measure bioelectrical parameters and bicarbonate secretion by pH-Stat ex-vivo. Hypercapnia induced a 2.3 and 2.8-fold increase in bicarbonate secretion rates in the anterior and mid intestine, respectively. In the intestinal fluid, HCO3− concentration increased 2.2-fold, and carbonate precipitates showed a 4.4-fold increase in response to hypercapnia, paralleled by a >3-fold increase of drinking and a >2-fold increase of intestinal volume at any given time. At the molecular level, hypercapnia elicited higher intestinal mRNA expression levels for atp6v1b (V-ATPase B subunit), slc4a4, slc26a3, and slc26a6, both in the anterior and mid intestine. As a whole, our results show that the intestine of sea bass responds to high seawater pCO2, a response that comes at a cost at the whole-body level with an impact in the fish specific growth rate, condition factor, and hepatosomatic index.

Bile salts regulate ion transport in the intestine of Senegalese sole

The regulatory role of taurocholic acid (TC) and taurolithocholic acid (TLC) in ion transport was investigated with the short-circuit current (Isc) technique in the intestine of Senegalese sole (Solea senegalensis). Under voltage clamp conditions, Isc was stable for over 3 h and averaged −15.42 ± 2.75 μA/cm2 and −65.28 + 7.69 μA/cm2 in the anterior and posterior intestine, respectively. Only bumetanide (200 μM), a loop diuretic that inhibits Na+/K+/2Cl− co-transporters, inhibited the absorptive Isc, and apical application of NPPB (200 μM), EIPA (200 μM), glibenclamide (200 μM) and DIDS (200 μM) were without effect in the absorptive current. Apical application of TC and TLC induced rapid (<1 min) increases in the absorptive current in the anterior and the posterior intestine. The responses were dose dependent in the range of physiological levels of TC or TLC previously detected in the intestine of Senegalese sole, 0.4–4 mM, with higher responses in the posterior intestine at the same doses. The response to apical application of TC and TLC was completely abolished by bumetanide (200 μM). In addition, the effect of TC or TLC in preparations with bilateral reduction of chloride (from 146 to 6 mM) in the chamber saline was reduced by 80%. Conclusions of this study indicate that the bile salts TC and TLC alter ionic transport in the intestine of marine fish enhancing the absorptive pathway. These changes revealed by regulation of Isc are characterized by their dose-dependent action, intestinal region dependency, reversibility, chloride dependence and bumetanide sensitivity.

Regulation of Bicarbonate Secretion in Marine Fish Intestine by the Calcium-Sensing Receptor.

In marine fish, high epithelial intestinal HCO3− secretion generates luminal carbonate precipitates of divalent cations that play a key role in water and ion homeostasis. The present study was designed to expose the putative role for calcium and the calcium-sensing receptor (CaSR) in the regulation of HCO3− secretion in the intestine of the sea bream (Sparus aurata L.). Effects on the expression of the CaSR in the intestine were evaluated by qPCR and an increase was observed in the anterior intestine in fed fish compared with unfed fish and with different regions of intestine. CaSR expression reflected intestinal fluid calcium concentration. In addition, anterior intestine tissue was mounted in Ussing chambers to test the putative regulation of HCO3− secretion in vitro using the anterior intestine. HCO3− secretion was sensitive to varying calcium levels in luminal saline and to calcimimetic compounds known to activate/block the CaSR i.e., R 568 and NPS-2143. Subsequent experiments were performed in intestinal sacs to measure water absorption and the sensitivity of water absorption to varying luminal levels of calcium and calcimimetics were exposed as well. It appears, that CaSR mediates HCO3− secretion and water absorption in marine fish as shown by responsiveness to calcium levels and calcimimetic compounds.

Revision of the Megaperidae Manter, 1934 n. comb. (Syn. Apocreadiidae Skrjabin, 1942) including a reorganization of the Schistorchiinae Yamaguti, 1942.

Modified and/or new keys to the four subfamilies now recognized within the Megaperidae Manter, 1934 n. comb. (Syn. Apocreadiidae Skrjabin, 1942) as well as the genera within each subfamily are presented. Two new genera, Paraschistorchis n. gen. and Plesioschistorchis n. gen., both within the Schistorchiinae Yamaguti, 1942, are erected and keys are provided to the species considered in both new genera-distinguished by possessing caeca that end either in separate ani or blindly. Plesioschistorchis callyodontis (Yamaguti, 1942) n. comb. and Plesioschistorchis haridis (Nagaty, 1957) n. comb. are re-described from new material collected from the common parrotfish, Scarus psittacus Forsskål (Perciformes: Scaridae), inhabiting the Red Sea off Egypt; S. psittacus represents a new host record for both species. The taxonomic status of Schistorchis sensu stricto Lühe, 1906 is examined and revised, a key to the four species we consider in this genus offered, and the monotypic genus Megacreadium Nagaty, 1956 declared a junior synonym of Schistorchis. Members of Schistorchis sensu stricto possess a unique "complex" (i.e. highly cellular/glandular) instead of "simple" (i.e. entirely muscular) type of oral sucker that is quite large in relation to body size; an elongate, somewhat sub-rectangular-shaped body; 5+ testes arranged in at least two rows; caeca that open via separate ani; a long post-testicular region; a median genital pore either at the anterior margin of or just anterior to the ventral sucker; and species of Schistorchis sensu stricto parasitize the intestine of marine fish within the Order Tetraodontiformes Berg. With the revision of this genus, we re-describe Schistorchis carneus Lühe, 1906 from the lower and mid-intestine of the white-spotted puffer, Arothron hispidus (Linnaeus) (Tetraodontiformes: Tetraodontidae), collected in the Red Sea off Egypt. Finally, a plea is made for further study of the Megaperidae n. comb. focusing, in particular, on the following: (1) obtaining new type/voucher materials of Plesioschistorchis manteri (Gupta & Tandon, 1984) n. comb. and Schistorchis paruchini Kurochkin, 1974; (2) elucidating the life histories (i.e. intermediate hosts) of members of the Postporinae Yamaguti, 1958 and Schistorchiinae; and (3) generating DNA sequence data for more species of megaperids to help future workers produce increasingly accurate taxonomic classifications that better reflect phylogenetic relationships within this ecologically diverse group of digeneans.

Elevated CO2 increases energetic cost and ion movement in the marine fish intestine.

Energetic costs associated with ion and acid-base regulation in response to ocean acidification have been predicted to decrease the energy available to fish for basic life processes. However, the low cost of ion regulation (6–15% of standard metabolic rate) and inherent variation associated with whole-animal metabolic rate measurements have made it difficult to consistently demonstrate such a cost. Here we aimed to gain resolution in assessing the energetic demand associated with acid-base regulation by examining ion movement and O2 consumption rates of isolated intestinal tissue from Gulf toadfish acclimated to control or 1900 μatm CO2 (projected for year 2300). The active marine fish intestine absorbs ions from ingested seawater in exchange for HCO3− to maintain water balance. We demonstrate that CO2 exposure causes a 13% increase of intestinal HCO3− secretion that the animal does not appear to regulate. Isolated tissue from CO2-exposed toadfish also exhibited an 8% higher O2 consumption rate than tissue from controls. These findings show that compensation for CO2 leads to a seemingly maladaptive persistent base (HCO3−) loss that incurs an energetic expense at the tissue level. Sustained increases to baseline metabolic rate could lead to energetic reallocations away from other life processes at the whole-animal level.

Changes to Intestinal Transport Physiology and Carbonate Production at Various CO2 Levels in a Marine Teleost, the Gulf Toadfish (Opsanus beta).

Most marine teleosts defend blood pH during high CO2 exposure by sustaining elevated levels of HCO3(-) in body fluids. In contrast to the gill, where measures are taken to achieve net base retention, elevated CO2 leads to base loss in the intestine of marine teleosts studied to date. This loss is thought to occur through transport pathways previously demonstrated to be involved with routine osmoregulation in marine teleosts. The main objective of this study was to characterize the intestinal transport physiology of the gulf toadfish (Opsanus beta) when exposed to varied levels of CO2: control, 5,000, 10,000, and 20,000 μatm CO2 (0.04, 0.5, 1, and 2 kPa, respectively). Results of this study suggest that intestinal apical anion exchange is highly responsive to hypercarbia, evidenced by a dose-dependent increase in intestinal luminal HCO3(-) (mmol L(-1)) that was mirrored by a reduction in Cl(-) (mmol L(-1)). Despite activation of HCO3(-) transport pathways typically used during osmoregulation, fractional fluid absorption was only significantly lower at the highest level of CO2. Although increased HCO3(-) excretion could provide more substrate for intestinally produced carbonates, carbonate production was not significantly increased during hypercarbia at the levels tested. This study is among the first to thoroughly characterize how compensation for elevated CO2 affects transport physiology and carbonate production in the marine fish intestine. This deeper understanding may be particularly relevant when considering the impacts of future predicted ocean acidification, where prolonged base loss may alter the energetic cost of acid-base balance or osmoregulation in marine fish.

PTHrP regulates water absorption and aquaporin expression in the intestine of the marine sea bream (Sparus aurata, L.)

Water ingestion by drinking is fundamental for ion homeostasis in marine fish. However, the fluid ingested requires processing to allow net water absorption in the intestine. The formation of luminal carbonate aggregates impacts on calcium homeostasis and requires epithelial HCO3− secretion to enable water absorption. In light of its endocrine importance in calcium handling and the indication of involvement in HCO3− secretion the present study was designed to expose the role of the parathyroid hormone-related protein (PTHrP) in HCO3− secretion, water absorption and the regulation of aqp1 gene expression in the anterior intestine of the sea bream. HCO3− secretion rapidly decreased when PTHrP(1–34) was added to anterior intestine of the sea bream mounted in Ussing chambers. The effect achieved a maximum inhibition of 60% of basal secretion rates, showing a threshold effective dose of 0.1ngml−1 compatible with reported plasma values of PTHrP. When applied in combination with the adenylate cyclase inhibitor (SQ 22.536, 100μmoll−1) or the phospholipase C inhibitor (U73122, 10μmoll−1) the effect of PTHrP(1–34) on HCO3− secretion was reduced by about 50% in both cases. In parallel, bulk water absorption measured in intestinal sacs was sensitive to inhibition by PTHrP. The inhibitory action conforms to a typical dose–response curve in the range of 0.1–1000ngml−1, achieves a maximal effect of 60–65% inhibition from basal rates and shows threshold significant effects at hormone levels of 0.1ngml−1. The action of PTHrP in water absorption was completely abolished in the presence of the adenylate cyclase inhibitor (SQ 22.536, 100μmoll−1) and was insensitive to the phospholipase C inhibitor (U73122, 10μmoll−1). In vivo injections of PTHrP(1–34) or the PTH/PTHrP receptor antagonist PTHrP(7–34) evoked respectively, a significant decrease or increase of aqp1ab, but not aqp1a. Overall the present results suggest that PTHrP acts as a key regulator of carbonate aggregate formation in the intestine of marine fish via its actions on water absorption, calcium regulation and HCO3− secretion.

Endocrine regulation of carbonate precipitate formation in marine fish intestine by Stanniocalcin and PTHrP

In marine fish, high epithelial bicarbonate secretion by the intestine generates luminal carbonate precipitates of divalent cations that play a key role in water and ion homeostasis. In vitro studies highlight the involvement of the calciotropic hormones PTHrP (parathyroid hormone-related protein) and stanniocalcin (STC) in the regulation of epithelial bicarbonate transport. The present study tested the hypothesis that calciotropic hormones have a regulatory role in carbonate precipitate formation in vivo. Sea bream (Sparus aurata) juveniles received single intraperitoneal injections of piscine PTHrP(1-34), the PTH/PTHrP receptor antagonist PTHrP(7-34) or purified sea bream STC, or were passively immunized with polyclonal rabbit antisera raised against sea bream STC (STC-Ab). Endocrine effects on the expression of the basolateral sodium bicarbonate co-transporter (Slc4a4.A), the apical anion exchangers Slc26a6.A and Slc26a3.B, and the V-type proton pump β-subunit (Atp6v1b) in the anterior intestine were evaluated. In keeping with their calciotropic nature, the hypocalcaemic factors PTHrP(7-34) and STC up-regulated gene expression of all transporters. In contrast, the hypercalcaemic factor PTHrP(1-34) and STC antibodies down-regulated transporters involved in the bicarbonate secretion cascade. Changes in intestine luminal precipitate contents provoked by calcaemic endocrine factors validated these results: 24 h post-injection either PTHrP(1-34) or immunization with STC-Ab reduced the carbonate precipitate content in the sea bream intestine. In contrast, the PTH/PTHrP receptor antagonist PTHrP(7-34) increased not only the precipitated fraction but also the concentration of HCO3(-) equivalents in the intestinal fluid. These results confirm the hypothesis that calciotropic hormones have a regulatory role in carbonate precipitate formation in vivo in the intestine of marine fish. Furthermore, they illustrate for the first time in fish the counteracting effect of PTHrP and STC, and reveal an unexpected contribution of calcaemic factors to acid-base balance.

AVT is involved in the regulation of ion transport in the intestine of the sea bream (Sparus aurata)

The intestine of marine fish plays a crucial role in ion homeostasis by selective processing of ingested fluid. Although arginine vasotocin (AVT) is suggested to play a role in ion regulation in fish, its action in the intestine has not been demonstrated. Thus, the present study investigated in vitro the putative role of AVT in intestinal ion transport in the sea bream (Sparus aurata). A cDNA encoding part of an AVT receptor was isolated and phylogenetic analysis revealed it clustered with the V1a2-type receptor clade. V1a2 transcripts were expressed throughout the gastrointestinal tract, from esophagus to rectum, and were most abundant in the rectum regardless of long-term exposure to external salinities of 12, 35 or 55p.p.t. Basolateral addition of AVT (10−6M) to the anterior intestine and rectum of sea bream adapted to 12, 35 or 55p.p.t. mounted in Ussing chambers produced rapid salinity and region dependent responses in short circuit current (Isc), always in the absorptive direction. In addition, AVT stimulation of absorptive Isc conformed to a dose–response curve, with significant effects achieved at 10−8M, which corresponds to physiological values of plasma AVT for this species. The effect of AVT on intestinal Isc was insensitive to the CFTR selective inhibitor NPPB (200μM) applied apically, but was completely abolished in the presence of apical bumetanide (200μM). We propose a role for AVT in the regulation of ion absorption in the intestine of the sea bream mediated by an absorptive bumetanide-sensitive mechanism, likely NKCC2.

Water absorption and bicarbonate secretion in the intestine of the sea bream are regulated by transmembrane and soluble adenylyl cyclase stimulation

In the marine fish intestine luminal, HCO₃⁻ can remove divalent ions (calcium and magnesium) by precipitation in the form of carbonate aggregates. The process of epithelial HCO₃⁻ secretion is under endocrine control, therefore, in this study we aimed to characterize the involvement of transmembrane (tmACs) and soluble (sACs) adenylyl cyclases on the regulation of bicarbonate secretion (BCS) and water absorption in the intestine of the sea bream (Sparus aurata). We observed that all sections of sea bream intestine are able to secrete bicarbonate as measured by pH-Stat in Ussing chambers. In addition, gut sac preparations reveal net water absorption in all segments of the intestine, with significantly higher absorption rates in the anterior intestine that in the rectum. BCS and water absorption are positively correlated in all regions of the sea bream intestinal tract. Furthermore, stimulation of tmACs (10 μM FK + 500 μM IBMX) causes a significant decrease in BCS, bulk water absorption and short circuit current (Isc) in a region dependent manner. In turn, stimulation of sACs with elevated HCO₃⁻ results in a significant increase in BCS, and bulk water absorption in the anterior intestine, an action completely reversed by the sAC inhibitor KH7 (200 μM). Overall, the results reveal a functional relationship between BCS and water absorption in marine fish intestine and modulation by tmACs and sAC. In light of the present observations, it is hypothesized that the endocrine effects on intestinal BCS and water absorption mediated by tmACs are locally and reciprocally modulated by the action of sACs in the fish enterocyte, thus fine-tuning the process of carbonate aggregate production in the intestinal lumen.

Prolactin regulates luminal bicarbonate secretion in the intestine of the sea bream (Sparus auratus L.)

The pituitary hormone prolactin is a pleiotropic endocrine factor that plays a major role in the regulation of ion balance in fish, with demonstrated actions mainly in the gills and kidney. The role of prolactin in intestinal ion transport remains little studied. In marine fish, which have high drinking rates, epithelial bicarbonate secretion in the intestine produces luminal carbonate aggregates believed to play a key role in water and ion homeostasis. The present study was designed to establish the putative role of prolactin in the regulation of intestinal bicarbonate secretion in a marine fish. Basolateral addition of prolactin to the anterior intestine of sea bream mounted in Ussing chambers caused a rapid (<20 min) decrease of bicarbonate secretion measured by pH-stat. A clear inhibitory dose-response curve was obtained, with a maximal inhibition of 60-65% of basal bicarbonate secretion. The threshold concentration of prolactin for a significant effect on bicarbonate secretion was 10 ng ml(-1), which is comparable with putative plasma levels in seawater fish. The effect of prolactin on apical bicarbonate secretion was independent of the generation route for bicarbonate, as shown in a preparation devoid of basolateral HCO(3)(-)/CO(2) buffer. Specific inhibitors of JAK2 (AG-490, 50 μmol l(-1)), PI3K (LY-294002, 75 μmol l(-1)) or MEK (U-012610, 10 μmol l(-1)) caused a 50-70% reduction in the effect of prolactin on bicarbonate secretion, and demonstrated the involvement of prolactin receptors. In addition to rapid effects, prolactin has actions at the genomic level. Incubation of intestinal explants of anterior intestine of the sea bream in vitro for 3 h demonstrated a specific effect of prolactin on the expression of the Slc4a4A Na(+)-HCO(3)(-) co-transporter, but not on the Slc26a6A or Slc26a3B Cl(-)/HCO(3)(-) exchanger. We propose a new role for prolactin in the regulation of bicarbonate secretion, an essential function for ion/water homeostasis in the intestine of marine fish.

Regulation of apical H+-ATPase activity and intestinal HCO3−secretion in marine fish osmoregulation

The absorption of Cl(-) and water from ingested seawater in the marine fish intestine is accomplished partly through Cl(-)/HCO(3)(-) exchange. Recently, a H(+) pump (vacuolar-type H(+)-ATPase) was found to secrete acid into the intestinal lumen, and it may serve to titrate luminal HCO(3)(-) and facilitate further Cl(-)/HCO(3)(-) exchange, especially in the posterior intestine, where adverse concentration gradients could limit Cl(-)/HCO(3)(-) exchange. The H(+) pump is expressed in all intestinal segments and in gill tissue of gulf toadfish (Opsanus beta) maintained in natural seawater. After acute transfer of toadfish to 60 ppt salinity, H(+) pump expression increased 20-fold in the posterior intestine. In agreement with these observations was a fourfold-increased H(+)-ATPase activity in the posterior intestine of animals acclimated to 60 ppt salinity. Interestingly, Na(+)-K(+)-ATPase activity was elevated in the anterior intestine and gill, but not in the posterior intestine. Apical acid secretion by isolated intestinal tissue mounted in Ussing chambers fitted with pH-stat titration systems increased after acclimation to hypersalinity in the anterior and posterior intestine, titrating >20% of secreted bicarbonate. In addition, net base secretion increased in hypersalinity-acclimated fish and was ∼70% dependent on serosal HCO(3)(-). Protein localization by immunohistochemistry confirmed the presence of the vacuolar-type H(+)-ATPase in the apical region of intestinal enterocytes. These results show that the H(+) pump, especially in the posterior intestine, plays an important role in hypersaline osmoregulation and that it likely has significant effects on HCO(3)(-) accumulation in the intestinal lumen and, therefore, the continued absorption of Cl(-) and water.

Detection and isolation of p-nitrophenol-lowering bacteria from intestine of marine fishes caught in Japanese waters

To determine the existence of p-nitrophenol (PNP)-lowering bacteria in intestine of Japanese coastal fish, the gastro-intestinal contents were incubated in Brain Heart Infusion (BHI) broth and minimal medium (MM) broths containing 1mmol/L PNP at 30°C for 7days. Among 26 samples of 19 fish species, 17 samples showed a decrease in PNP of 0.5–0.8mmol/L in BHI broth, but no decrease was shown in MM broth. Eighteen PNP-lowering bacterial strains were isolated from four fishes. All of the strains were identified as Lactococcus lactis subsp. lactis. Three L. lactis strains JS1–3 isolated from Japanese seabass Lateolabrax japonicus showed the highest PNP-lowering activity (0.44mmol/L). Optimum temperature and pH for the growth and PNP decreasing corresponded with the marine environment. These results suggested that marine fishes have PNP decreasing bacteria in their intestine. These bacteria might protect host fish from toxicities of PNP and PNP related compounds.

Parathyroid hormone-related protein-stanniocalcin antagonism in regulation of bicarbonate secretion and calcium precipitation in a marine fish intestine

Bicarbonate secretion in the intestine (duodenum) of marine fish has been suggested to play a major role in regulation of calcium availability for uptake. However, while the end process may lead to carbonate precipitation, regulation of transport of calcium and/or bicarbonate may actually result in fine-tuning of calcium availability for transport. To test this hypothesis, sea bream (Sparus auratus) duodenal preparations were mounted in Ussing-type chambers and the effect of parathyroid hormone-related protein (PTHrP) and stanniocalcin 1 (STC 1) on the control of intestinal bicarbonate secretion and calcium transport was analyzed. As expected, PTHrP increased net calcium uptake, as a result of an increase of calcium uptake without changes in calcium efflux. In contrast, purified sea bream STC 1 caused a minor decrease of calcium uptake and a two- to threefold increase in calcium efflux. As a result, STC 1 was able to invert the calcium flux from net calcium uptake to net calcium loss, which is in keeping with its known actions as a hypocalcemic factor. Furthermore, both PTHrP and STC 1 regulate intestinal bicarbonate secretion. PTHrP increased calcium uptake and simultaneously reduced the single factor that induces calcium precipitation, bicarbonate secretion. In contrast, STC 1, while reversing the calcium net flux to make it secretory, promoted intestinal bicarbonate secretion, both actions directed to decrease the calcium gradient across the epithelium and promote immobilization in the form of bicarbonate in the intestinal lumen. Together our results provide robust evidence to support an antagonistic action of PTHrP and STC 1 in the fine control of movements of both calcium and bicarbonate in the intestine of seawater fish.

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

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.

The effects of pH and the iron redox state on iron uptake in the intestine of a marine teleost fish, gulf toadfish ( Opsanus beta)

In the marine teleost intestine the secretion of bicarbonate increases pH of the lumen (pH 8.4 –9.0) and importantly reduces Ca 2+ and Mg 2+ concentrations by the formation of insoluble divalent ion carbonates. The alkaline intestinal environment could potentially also cause essential metal carbonate formation reducing bioavailability. Iron accumulation was assessed in the Gulf toadfish ( Opsanus beta) gut by mounting intestine segments in modified Ussing chambers fitted to a pH-stat titration system. This system titrates to maintain lumen pH constant and in the process prevents bicarbonate accumulation. The luminal saline pH was clamped to pH 5.5 or 7.0 to investigate the effect of proton concentrations on iron uptake. In addition, redox state was altered (gassing with N 2, addition of dithiothreitol (DTT) and ascorbate) to evaluate Fe 3+ versus Fe 2+ uptake, enabling us to compare a marine teleost intestine model for iron uptake to the mammalian system for non-haem bound iron uptake that occurs via a ferrous/proton (Fe 2+/H +) symporter called Divalent Metal Transporter 1 (DMT1). None of the redox altering strategies affected iron (Fe 3+ or Fe 2+) binding to mucus, but the addition of ascorbate resulted in a 4.6-fold increase in epithelium iron accumulation. This indicates that mucus iron binding is irrespective of valency and suggests that ferrous iron is preferentially transported across the apical surface. Altering luminal saline pH from 7.0 to 5.5 did not affect ferric or ferrous iron uptake, suggesting that if iron is entering via DMT1 in marine fish intestine this transporter works efficiently under circumneutral conditions.