Immunolocalization of Na+, K+-ATPase-rich cells in the gill and urinary system of Persian sturgeon, Acipenser persicus, fry

Localization of Na⁺, K⁺-ATPase-rich cells in the gill and urinary system of Acipenser persicus fry was performed through immunofluorescence light microscopy using a mouse monoclonal antibody IgGα₅ raised against the α-subunit of chicken Na⁺, K⁺-ATPase. Different types of epithelia were clearly identified in the gill epithelium: epithelia of branchial arch, interbranchial septum, filament and lamellar epithelium. The Na⁺, K⁺-ATPase-rich cells were found in the epithelia of branchial arch, interbranchial septum, filament, interlamellar region and also in the lamellae. Histologically, the urinary system is divided into head kidney, trunk kidney and short caudal kidney. The head kidney is composed of the pronephric tubules and the haemopoietic tissues, while the trunk kidney is composed of a large number of glomeruli and convoluted nephrons. Each nephron consisted of a large glomerulus and tubules (neck, proximal, distal and collecting tubules) which connected to ureters. Posteriorly, ureters extended and joined together to form a small urinary bladder. In the urinary system, no specific fluorescence staining was observed in the glomerulus, neck segment and proximal tubules. The distal tubule cells and collecting tubule cells showed a strong immunostaining of Na⁺, K⁺-ATPase. Epithelia of ureters and urinary bladder also showed several isolated immunofluorescent cells. Immunofluorescent cells were rich in Na⁺, K⁺-ATPase enzyme which is very important for osmoregulation.     

Comparison of physiological smolt status in descending and nondescending wild brown trout (Salmo trutta) in a Danish stream

Abstract –  Downstream movement of a wild population of brown trout was examined in a small Danish stream in relation to morphological and physiological smolt status from March to May. Downstream movement was monitored in a Wolf-type trap covering all possible passage routes in the stream. Trout caught in the trap were classified as parr, pre-smolt or smolt based on morphological criteria and compared with trout randomly caught by electrofishing upstream of the trap. Representative gill samples from trap-caught and electrofished trout were analysed for gill Na⁺, K⁺-ATPase activity and used as a measure of physiological smolt status. Only a few parr occurred in the trap. Few pre-smolts occurred in the trap evenly in March and early April. In late April, pre-smolt movement peaked. By comparison, the main downstream movement of smolts occurred in distinct peaks through late March and April. The majority of fish caught in the trap were judged as pre-smolts or smolts based on morphological criteria's and they were characterised by relatively high gill Na⁺, K⁺-ATPase activity compared with trout judged as parr. Trout caught by electrofishing upstream the trap, were classified as parr, pre-smolts and smolts early in the season (March). During and after the main smolt-run in April the distribution of the remaining trout in the brook became skewed in favour of pre-smolt and parr. The study suggests that smolting trout initiate downstream movement once having reached a certain physiological smolt condition (judged by increased gill Na⁺, K⁺-ATPase activity).     

Effects of environmental Ca2+ levels on branchial chloride cell morphology in freshwater-adapted killifish Fundulus heteroclitus

ABSTRACT: To examine the involvement of chloride cells in the uptake of Ca²⁺ in freshwater (FW) killifish, chloride cell morphology was compared in fish acclimated to different defined FW environments with Ca²⁺ concentrations of either 0.1 mM, 0.5 mM, or 2.5 mM. Numerous chloride cells were detected in whole-mount preparations of the gill filaments, which were stained with an antiserum specific for Na⁺, K⁺-ATPase. Chloride cells, located mostly on the afferent–vascular edge of the filaments, were larger at lower Ca²⁺ concentrations. Electron microscopic observations showed that in the 0.1 mM and 0.5 mM Ca²⁺ experimental groups, the apical membrane of chloride cells were flat or slightly projecting and equipped with numerous microvilli. In the 2.5 mM Ca²⁺ group, some chloride cells formed an apical pit, whereas other cells were similar to those observed in the 0.1 mM and 0.5 mM Ca²⁺ groups. Plasma osmolality decreased with decreasing ambient Ca²⁺ concentration, suggesting that environmental Ca²⁺ affects the permeability of the body surfaces. Gill Na⁺, K⁺-ATPase activity in the 0.1 mM and 0.5 mM Ca²⁺ groups were significantly higher than that in the 2.5 mM Ca²⁺ group, implying the involvement of the Na⁺–Ca²⁺ exchanger in Ca²⁺ uptake in the gills. These findings suggest that chloride cells function as the site for Ca²⁺ uptake in killifish acclimated to low Ca²⁺ environments.     

Effects of Low Salinities on Oxygen Consumption of Selected Euryhaline and Stenohaline Freshwater Fish

The amount of energy required for osmoregulation depends on the difference between internal and external concentrations of ions (Rao 1968; Farmer and Beamish 1969), changes in corticosteroid hormone levels (Morgan and Iwama 1996), glomerular filtration rates (Furspan et al. 1984), gill and kidney Na⁺, K⁺-ATPase activity (McCormick et al. 1989; Morgan and Iwama 1998), tissue permeability to water and ions, and gill ventilation, perfusion, and functional surface area (Rankin and Bolis 1984). Differences in the energetic cost of osmoregulation play a significant role in the difference in growth rate between seawater-and freshwater-adapted fish (Morgan and Iwama 1991; Ron et al. 1995; Wang et al. 1997). Oxygen consumption is an indirect indicator of metabolic rate in fish (Cech 1990) and can be used to determine effects of salinity changes on energy costs.     

Changes in Plasma Cortisol, Glucose, and Selected Blood Properties in the Summer Flounder Paralichthys dentatus Associated with Sequential Movement to Three Experimental Conditions

To determine the changes in blood chemistry associated with sequential transfer of summer flounder Paralichthys dentatus (320–480 g), 300 hatchery-reared fish were moved to three different environmental conditions during a 20-d period. Fish were transferred in progression from a recirculating seawater system (22 ppt, 22.5 C) to a flow-thru seawater system (31 ppt, 20.0 C), to three small coastal net pens (33 ppt, 15.5 C), and finally to a large open ocean net pen (33 ppt, 16.0 C). For this study, eight random fish were captured at each progressive step (environmental condition), anesthetized (MS222), and bled from the caudal vein (2 mL). Transferred flounder were bled every 12 h for 48 h to collect plasma cortisol and glucose samples. Fish were bled 24 h after transport and every 3 d thereafter for osmolarity, hematocrit, hemoglobin concentration, mean corpuscular hemoglobin content, glucose, cortisol, and the electrolytes Cl^- Na⁺, K⁺ and Ca⁺. The most significant perturbations to blood chemistry (P < 0.05) occurred within 24 h of initial transfer from the recirculating to flow-thru seawater systems, suggesting an osmoregulatory rather than handling or transfer related stress. Osmolarity, electrolyte, and hematological parameters fluctuated and then recovered to stable levels by day 8 in the flow-thru seawater system. However, unlike the initial transfer, successive movement to the coastal and then the open ocean net pens produced transient increases in both plasma cortisol and glucose levels, suggesting a high level of stress associated with extended flounder handling and transfer.     

Osmoregulatory changes in wedge sole (Dicologoglossa cuneata Moreau, 1881) after acclimation to different environmental salinities

The osmoregulatory responses of 20 days of acclimation to environmental salinities of 5‰, 15‰, 25‰, 35‰ and 55‰ were assessed in juveniles of wedge sole ( Dicologoglossa cuneata Moreau, 1881). This sole shows a good capacity to adapt to this range of environmental salinities. A direct linear relationship between environmental salinity and plasma osmolality was observed, with a calculated isosmotic point of 10.4‰ (284 mOsm kg⁻¹). Na⁺, K⁺-ATPase activity in the gills followed a 'U-shaped' relationship with environmental salinity, and a direct linear relationship in kidney tissue. Plasma cortisol levels were elevated in fish held in extreme salinities, and glucose levels were higher only in the group maintained at the highest environmental salinity. In the liver, a decrease in glycogen, lactate and amino acid contents was observed in specimens acclimated to extreme salinities (5‰ and 55‰), suggesting mobilization of liver metabolites. Metabolite levels in white muscle showed a pattern similar to the liver, with lower values in specimens acclimated to extreme salinities. We conclude that wedge sole is strongly euryhaline, but acclimation to extreme salinities comes with an energetic cost.     

Using Efinol®L during transportation of marbled hatchetfish, Carnegiella strigata (Günther)

The objective of this experiment was to test the efficacy of a probiotic (Efinol^®L) during transportation of marbled hatchetfish, Carnegiella strigata . Wild specimens were captured from a small stream and transported for 24 h in plastic fish boxes with a probiotic (10 mg L⁻¹) and probiotic-free water. The boxes were sampled at 3, 12 and 24 h of transport. At the end of the experiment, the survival rate was close to 100% in both treatments. Dissolved oxygen diminished with time in both treatments, but the probiotic group had significantly higher levels. Conductivity, pH and ammonia increased significantly during the transport, demonstrating higher levels in the probiotic-free group. Fish from both treatments presented very high net Na⁺ and K⁺ effluxes after 3 h of transport. At 24 h, net K⁺ effluxes in fish of the probiotic treatment reached values close to zero and a significantly lower Na⁺ efflux was observed. Cortisol levels in both treatments at 3 and 12 h were significantly higher than that in control samples. Higher body cortisol levels were observed in the probiotic-free group than that in the probiotic group at 3 and 12 h. The results demonstrate that addition of a probiotic during fish transport improves water quality and leads to fish presenting a lower stress response intensity.     

Localization of Zn-binding protein in the digestive tract tissue of common carp

ABSTRACT: The concentrations of Zn and sulfhydryl (SH) groups in the digestive tract tissue of common carp and some aquatic animals were studied. It was found that Zn and bound SH groups could be used as indicators for detecting the Zn-binding protein in the digestive tract tissue of common carp. The digestive tract tissue of the fish underwent subcellular fractionation, and it was found that the nuclei/cell debris fraction contained most of the DNA (85%), Na⁺/K⁺-ATPase (82%), organic phosphate (90%) and the Zn-binding protein (79%), but only part of the 5'-nucletidase and alkaline phosphatase (<23%). The nuclei/cell debris fraction of the digestive tract tissue of common carp was treated with either collagenase type I or type IV, and subfractionated by sucrose density centrifugation. It was found that treatment with collagenase type IV could release more than 50% of the Zn-binding protein, Na⁺/K⁺-ATPase and organic phosphate from collagen. Sections of digestive tract tissue of common carp were stained for Zn. It was observed that Zn can be found mainly on the edge of the epithelial layer, and everywhere in the 'membrane-like' portion of the submucosal and muscular layers. It is proposed that most of the Zn-binding protein in the digestive tract tissue of common carp is located on the basolateral plasma membranes of the epithelial cells and on the surrounding muscle cells that are attached to the collagen type IV of basal laminae.     

Purification and characterization of the amylase from a small abalone <Emphasis Type="Italic">Haliotis sieboldii</Emphasis>

ABSTRACT:   Amylase, with MW of 59 kDa, was purified from small abalone Haliotis sieboldii by ammonium sulfate fractionation, CM Sepharose Fast Flow and Sephacryl S-100 HR chromatographies. The optimal pH and temperature of purified amylase were 6.0 and 37°C, respectively. The purified enzyme was stable at pH 6.0–8.0 and low temperatures. It was activated by Ba²⁺, Mg²⁺, Ca²⁺, Co²⁺, Ni²⁺, Mn²⁺, K⁺, Ag⁺, Na⁺ and Li⁺, but completely or partially inhibited by Al³⁺, Cu²⁺, Cd²⁺, Hg²⁺ and Zn²⁺. EDTA could completely inhibit, while iodoacetamide, N-ethylmaleimide and urea partially inhibit the purified amylase. According to the digestion mode of various polysaccharides, the purified enzyme was considered to be an α-amylase.     

Microsatellite markers reveal population structure in gilthead sea bream Sparus auratus from the Atlantic Ocean and Mediterranean Sea

ABSTRACT:   This study was carried out to investigate the genetic variability and the population genetics of Sparus auratus. In fact, despite its importance in Mediterranean fisheries and aquaculture, very little it is known concerning its population structure. Samples of wild gilthead sea bream were collected in seven different localities along the Mediterranean and Atlantic coasts and were genetically characterized by means of microsatellite markers. Genotyping at four microsatellite loci revealed high polymorphism (7–38 alleles/locus) and expected heterozygosities, which ranged from 0.80 to 0.85. A slight but significant population structure was found ( F _ST = 0.010). In fact, at least three populations of gilthead sea bream within the Western Mediterranean Sea were identified (Sardinian Sea, Sardinian Channel and Central Tyrrhenian Sea), which are also genetically differentiated from those of the Atlantic Ocean and the Adriatic Sea.     

The effect of dietary phosphatidylcholine and its constituent fatty acids on microdiet ingestion and fatty acid absorption rate in gilthead sea bream, Sparus auratus, larvae

This study tested the effect of the level of dietary phosphatidylcholine (PC) and its constituent medium-chain fatty acids on microdiet ingestion (μg diet larva⁻¹ h⁻¹) and the absorption rate of the free fatty acid [¹⁴C]16:0 (pmole larva⁻¹ h⁻¹) in 15, 20, 21, 25, 26, 30 and 31-day-old gilthead sea bream, Sparus auratus L., larvae. Fish were fed four microdiets (A, B, C and D): microdiet A contained no phospholipid (PL), while microdiet B included 10 g kg⁻¹ Artemia nauplii PL (3.7 g kg⁻¹ PC). Microdiets C and D contained 10 g kg⁻¹ purified saturated PC dimyristoyl (C_14:0) and polyunsaturated PC dilinoleoyl (C_{18:2[cis]−9,12}), respectively.
Larvae from one or both of the PC microdiets demonstrated significantly higher ( P < 0.05) ingestion rates (μg diet larva⁻¹ h⁻¹) than the non-PL microdiet control in 15, 21, 22, 25 and 26-day-old larvae and the Artemia PL microdiet in 15, 22 and 26-day-old larvae. However, microdiet ingestion and fatty acid absorption rate appeared to be independent of the associated medium carbon chain saturated or polyunsaturated fatty acid moiety of the PC diets. Apparent absorption, as measured by the retention of radio-labelled [¹⁴C]16:0 following 8 h of non-labelled microdiet feeding, was possibly related to feeding.     

Metabolic costs of changing the cation–anion difference in the diet of juvenile African catfish Clarias gariepinus (Burchell)

The influence of dietary cation–anion difference (CAD, Na⁺ + K⁺– Cl^–, mEq kg^–1) on energy metabolism and nitrogen losses in juvenile African catfish Clarias gariepinus (Burchell) was examined in fish exposed to different dietary CAD levels (–146, 116, 497, 713 and 828 mEq kg^–1 diet). The experiment was conducted in open circuit balance respiration chambers over a 3-week period. Five 24-h monitoring periods were carried out at 3-day intervals during the experimental period with O₂ consumption, ammonia and nitrate + nitrite (NO_x) and CO₂ production being measured at 5-min intervals for each chamber. The negative dietary CAD (–146 mEq kg^–1) resulted in the highest energy expenditures (83 kJ kg^–0.8· d^–1). With increasing dietary CAD levels, heat loss gradually decreased to minimum values of 56 kJ kg^–0.8 day^–1 at a dietary CAD level of 713 mEq kg^–1. Consequently, metabolizable energy utilization efficiency (MEU, percentage of retained energy over metabolizable energy) quadratically ( P  < 0.05) increased and reached a maximum at a dietary CAD of 713 mEq kg^–1.     

Rearing conditions to develop seawater tolerance in underyearling sockeye salmon smolt

ABSTRACT:   In order to confirm the conditions required to produce underyearling smolts, juvenile sockeye salmon Oncorhynchus nerka were reared under different photoperiod and temperature conditions. The level of smoltification was assessed by plasma sodium concentration 24 h after transfer to sea water (plasma-Na), gill Na⁺,K⁺-ATPase activity (gill-ATPase), and plasma thyroxine (plasma-T4) and growth hormone (plasma-GH) concentrations. Mean fork length of all groups reached more than approximately 8.5 cm at the end of the experiment. The fish that experienced an alteration in photoperiod from short day (8:16 h light–dark cycle) to long day (16:8 h light–dark cycle) showed significant elevation in gill-ATPase from 9 to 16 µmol inorganic phosphate per milligram protein per hour (µmol Pi/mg pro/h) and reduction in plasma-Na from 194 to 154 mM. This group displayed distinct pigmentation on their fin margins. However, in groups reared under constant short day and constant long day, changes in gill-ATPase and plasma-Na occurred vaguely at each different time and pigmentation on fin margins were not observed. The observed photoperiod-induced changes were greater at 7°C than at 11°C. Further, the alteration of the photoperiod induced two–threefold increases in plasma-GH and plasma-T4. These results suggest that lengthening of photoperiod from short to long daylight at 7°C causes smoltification accompanied by active secretion of growth hormone and thyroxine in underyearling sockeye salmon when the fish are larger than 8.5 cm in fork length.     

Role of sodium bicarbonate on the initiation of sperm motility in the Japanese eel

ABSTRACT:   In order to find out the role of sodium bicarbonate (NaHCO₃) on the initiation of sperm motility in the Japanese eel Anguilla japonica , interactions were investigated between NaHCO₃ and various reagents (K⁺ channel blocker 4-aminopyridine [4-AP], ammonium chloride [NH₄Cl], sodium acetate and calcium chloride [CaCl₂]) that could regulate internal factors (intracellular K⁺, intracellular pH [[pH]_i] and intracellular Ca²⁺) in sperm motility. Contradictory effects of NaHCO₃ were observed (i.e. an inhibitory effect when 4-AP was absent and a promoting effect when 4-AP was present). Sodium bicarbonate inhibited the initiation of sperm motility in the Japanese eel. However, NaHCO₃ restored the motility of immotile sperm that 4-AP inhibited. The inhibitory effect of NaHCO₃ disappeared with the addition of NH₄Cl, which raised [pH]_i, but the promoting effect was not affected by [pH]_i. Although NaHCO₃ recovered motility in the presence of 4-AP, this recovery was also observed with the addition of CaCl₂ instead of NaHCO₃. In the initiation of sperm motility in the Japanese eel, two roles for NaHCO₃ are suggested: an inhibitory role relating to the regulation of [pH]_i and a promoting role relating to the uptake of another initiation factor, which could be Ca²⁺.     

Dietary pyridoxine requirement of juvenile Jian carp (Cyprinus carpio var. Jian)

In a 80-day feeding trial, a total of 1050 juvenile Jian carp ( Cyprinus carpio var. Jian) with an average initial weight of 10.71 ± 0.05 g were fed semi-purified diets containing seven graded levels of pyridoxine (0.20, 1.71, 3.23, 4.96, 6.32, 8.58 and 12.39 mg pyridoxine kg⁻¹ diet). Results indicated that with increasing dietary pyridoxine levels up to 4.96 mg kg⁻¹ diet, percent weight gain (PWG) and specific growth rate (SGR) were improved, and no differences were found with further increase of pyridoxine levels. Feed intake also followed the similar pattern to that observed with PWG and SGR when dietary pyridoxine levels were ≤6.32 mg kg⁻¹ diet. But feed efficiency and protein efficiency ratio were not affected by pyridoxine levels. Crude protein of carcass, productive protein value and plasma ammonia concentration were improved with increasing dietary pyridoxine levels up to 4.96 mg kg⁻¹ diet. Amylase activities in the intestine were improved with increasing dietary pyridoxine levels up to 4.96 mg kg⁻¹ diet, but protease and lipase activities in the intestine were not affected by pyridoxine levels. Na⁺, K⁺-ATPase and Gamma-glutamyl transpeptidase activities in proximal intestine, mid intestine (MI) and distal intestine (DI) were lowest when fed the diet containing 1.71 mg pyridoxine kg⁻¹ diet. The alkaline phosphatase activities in MI and DI followed the same pattern. The dietary pyridoxine requirement of juvenile Jian carp based on PWG estimated by broken line model was 6.07 mg pyridoxine kg⁻¹ diet.     

Purification and characterization of transglutaminase from squid gill

ABSTRACT: The present study used squid gill as a source of transglutaminase (TGase) because it has extremely high TGase activity compared with other tissues. The enzyme was purified using successive chromatographies of Sephacryl S-300 and hydroxyapatite columns. The yield and purification-fold of the enzymatic activity was 12.6% and 14.1-fold, respectively. The molecular mass of the purified enzyme was estimated to be 94 kDa by using sodium dodecylsulfate–polyacrylamide gel electrophoresis analysis. Enzyme activity was enhanced 15-fold with an increase in NaCl concentration. Although the activity was dependent on Ca²⁺ concentration, it was not sufficiently activated even by 50 mM CaCl₂ in the absence of NaCl, but could be fully activated with 10 mM CaCl₂ in 0.7 M NaCl. However, in the absence of substrates, the enzyme was rapidly inactivated. The pH and temperature optima of the enzyme were approximately pH 8.0 and 20°C, respectively. It was stable in the absence of Ca²⁺ at pH 7.5–9.0 and had a rate constant (K _D ) of 1.6 × 10^–5 s^–1 for thermal inactivation at 50°C. These results in which squid gill TGase could be activated at higher concentrations of Ca²⁺ and NaCl than at a physiological concentration, suggest that contact with seawater or body fluid seems to activate the enzyme if the tissue is disrupted.     

A rapid and inexpensive method to assay transport of short chain peptides across intestinal brush-border membrane vesicles from the European eel (Anguilla anguilla)

Membrane potential depolarization due to electrogenic peptide transport activity was examined in eel ( Anguilla anguilla ) intestinal brush-border membrane vesicles (BBMV) by monitoring the fluorescence quenching of the voltage-sensitive dye 3,3'-diethylthiadicarbocyanine iodide. Our experimental approach consisted of generating an internal negative membrane potential mimicking in vivo conditions and measuring membrane potential depolarization due to different extravesicular dipeptides. Peptide-dependent membrane potential depolarization was observed in both the presence and absence of extravesicular Na⁺ and was inhibited by diethylpyrocarbonate, which is consistent with the involvement of electrogenic, Na⁺-independent, H⁺-dependent peptide transport activity. Kinetic analysis indicated that peptide-dependent membrane potential depolarization is a saturable process ( K _m,app ∼ 1.5 mmol L⁻¹) and that within the 0.1–10 mmol L⁻¹ peptide range a single carrier system is involved in the transport process. Our results suggest that a peptide transport activity, kinetically resembling the PepT1(Slc15A1)-type-mediated H⁺/peptide cotransport action, can be monitored in eel intestinal BBMV using an easy and inexpensive fluorescence assay.     

Influence of dietary levels of soybean meal on the performance and gut histology of gilthead sea bream (Sparus aurata L.) and European sea bass (Dicentrarchus labrax L.)

The present study was performed to determine the effect of soybean meal (SBM) on the performance and gut histology of gilthead sea bream and European sea bass. Three isonitrogenous and isolipidic extruded diets (crude protein, 470 g kg⁻¹ diet; crude fat, 200 g kg⁻¹ diet) were formulated containing 0 (0 SBM), 180 (180 SBM) and 300 (300 SBM) g kg⁻¹ diet SBM and tested on both species in two separate experiments. Fish at an initial average weight of around 18 g were randomly allocated to 800 L square tanks connected to a closed recirculating system. The trials lasted 80 days for sea bream and 89 days for sea bass. Fish were hand-fed to apparent satiation. Increasing the level of SBM had no significant effects on the specific growth rate, feed intake and feed conversion rate in both the species. In the sea bream distal intestine, lamina propria was moderately and diffusely expanded in some fish due to an increase in cell infiltration represented by mononuclear cells, this finding being more frequent in animals fed diet 300 SBM. No other morphological alterations in intestinal folds, enterocytes or other inflammation signs were noticed in the sea bream distal intestine of any group. No histological differences were found in sea bass in any experimental group.