Functional morphology of chloride cells in killifish Fundulus heteroclitus, a euryhaline teleost with seawater preference

ABSTRACT:   Recent advances in the functional morphology of chloride cells in killifish Fundulus heteroclitus , a euryhaline teleost with seawater (SW) preference, were reviewed. Immunocytochemical detection of chloride cells with anti-Na⁺/K⁺-ATPase revealed transitional processes of the chloride cell distribution during early life stages. Chloride cells first appear in the yolk-sac membrane at an early embryonic stage, followed by their appearance in the body skin in the later embryonic stages. The principal site for the chloride cell distribution then shifts from the yolk-sac membrane and body skin during embryonic stages to the gills and opercular membrane in larval and later developmental stages. Morphologically distinct SW- and freshwater (FW)-type chloride cells were identified in adult killifish adapted to SW and FW, respectively. Both types of chloride cells are equally active in the two environments, but exert different ion-transporting functions. Following direct transfer of killifish from SW to FW, SW-type chloride cells were transformed into FW-type cells as a short-term response, followed by the promotion of chloride cell replacement as a long-term response. In killifish acclimated to low NaCl (0.1 mM) FW, intense immunoreactivity for vacuolar-type proton pump (V-ATPase) was detected in the basolateral membrane of FW-type chloride cells, whereas the immunoreactivity was much weaker in fish acclimated to FW with higher NaCl concentrations (1.0 and 10.0 mM). These results suggest the occurrence of active ion absorption in FW-type chloride cells and the involvement of V-ATPase in ion-absorbing mechanisms. In view of recent advances in this field, future chloride cell research should be considered in relation to the functional diversity of chloride cells.     

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).     

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⁺.     

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.     

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.     

Thermal denaturation and autolysis profiles of myofibrillar proteins of mantle muscle of jumbo squid Docidicus gigas

ABSTRACT:    Jumbo squid was very similar to Japanese common squid in terms of myofibrillar Ca²⁺-, Mg²⁺- and K⁺(EDTA)-ATPase activities. Myofibrils of jumbo squid were significantly stabilized upon addition of Ca²⁺ and destabilized by increasing KCl concentration for heating. Incubation of muscle homogenate of jumbo squid induced a selective cleavage of myosin into two major fragments and the cleavage was inhibited by EDTA. Autolysis was prominent at and above 0.3 M NaCl where myosin filaments dissolve. The enzyme involved in the autolysis was proved to be unstable showing maximal autolysis rate at 25°C. Washing the homogenate partially reduced the autolysis activity.     

Effects of urea and trimethylamine-N-oxide on ATPase of requiem shark myofibril and its constituents

ABSTRACT: The effects of trimethylamine- N -oxide (TMAO) on the urea-resistibility of requiem shark myofibrils were investigated, using Ca²⁺- and Mg²⁺-ATPase activities as a parameter. Both activities were hardly changed or activated up to 0.6 M urea. In contrast, the two activities both decreased to less than 50% in the presence of TMAO up to 0.5 M. When measured at a 2 : 1 molar ratio of urea and TMAO, Ca²⁺- and Mg²⁺-ATPase activities were similar to those in the presence of TMAO alone, indicating that TMAO reduced the urea-resistibility of myofibrils. Myosin, the most abundant protein in myofibrils, from requiem shark exhibited the effects of urea and TMAO on its Ca²⁺-ATPase activity, which was primarily similar to those of myofibrils. However, Ca²⁺-ATPase activities in the coexistence of urea and TMAO for actomyosin reconstituted from requiem shark myosin and chicken F-actin were approximately average of those measured independently in the presence of either urea or TMAO alone. Carp myofibrils, reconstituted actomyosin and myosin, which were used as teleost references, all showed a tendency in the effects of urea and TMAO on Ca²⁺-ATPase activities that was similar to those of requiem shark counterparts.     

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.     

Comparison of characteristics of actomyosin from white, pink, and red muscle fiber types in cultured carp

ABSTRACT: The present study compared and examined the characteristics of actomyosin among white (W), pink (P), and red (R) muscle fiber types in carp (cultured). Both the superprecipitation reaction and the Mg²⁺-ATPase activity of actomyosin became higher with increased Ca²⁺ concentration (pCa 7.0–pCa 5.0) and with decreased adenosine triphosphate (ATP) concentration (3.0–0.5 mM) in all three muscle fiber types. A comparison of the three fiber types shows that the superprecipitation reaction of actomysoin was lower in the order of W < P < R and, in contrast, was higher for Mg²⁺-ATPase activity in the order of W > P > R. A significantly positive correlation between both values was found for each of the three muscle fiber types, but these correlations were clearly different among the three muscle fiber types, and the superprecipitation reaction of actomyosin was lower in the order of W < P < R when Mg²⁺-ATPase activity was at the same level. In conclusion, the characteristics of actomyosin were remarkably different among white, pink, and red muscle fiber types.     

Characterization of fast skeletal myosin from white croaker in comparison with that from walleye pollack

ABSTRACT:   Enzymatic and structural properties of white croaker fast skeletal muscle myosin were determined and compared with those of walleye pollack counterpart. Ca²⁺-ATPase activity of white croaker myosin was decreased to approximately 70% of the original activity during 1 day of storage at 0°C and pH 7.0 in 0.5 M KCl and 0.1 mM dithiothreitol, whereas that of walleye pollack was decreased to approximately 20% under the same condition. The activation energy ( E _a) for inactivation of white croaker myosin calculated by the Arrhenius plot for inactivation rate constant (K_D) was 1.2-fold higher than that of walleye pollack. While Ca²⁺-ATPase showed a similar KCl-dependency for the two species, the maximal activity was observed at pH 6.2 and 6.3 for white croaker and walleye pollack, respectively. Actin-activated myosin Mg²⁺-ATPase activity of white croaker was approximately half that of walleye pollack at 0.05 M KCl and pH 7.0, although the two myosins showed a similar affinity to F-actin with K _m of 1.7 and 1.4, respectively. Limited proteolysis with α-chymotrypsin cleaved heat-denatured white croaker myosin mainly at heavy meromyosin/light meromyosin (HMM/LMM) junction, whereas walleye pollack myosin was cleaved at several sites in LMM as well as at the HMM/LMM junction.     

Seasonal variation in osmoregulatory and metabolic parameters in earthen pond-cultured gilthead sea bream Sparus auratus

Seasonal variations in osmoregulatory and metabolic parameters were assessed in juvenile gilthead sea bream ( Sparus auratus ) cultured in earthen ponds under a natural photoperiod and temperature. Specimens were sampled, and the plasma, gill, kidney and liver were collected during winter 2005 and 2006 (January), spring 2005 (April), summer 2005 (July) and autumn 2005 (October). Plasma osmoregulatory parameters showed higher values in summer, while metabolic parameters presented different patterns of variations. Gill Na⁺,K⁺-ATPase activity decreased significantly in winter, while gill metabolite levels showed different patterns of variations among seasons. The enzymatic activities tested did not present a clear pattern of variation [(glutamate dehydrogenase (EC 1.4.1.2) (GDH) and hexokinase (EC 2.7.1.11) (HK)] or significant differences along seasons [glucose 6-phosphate dehydrogenase (EC 1.1.1.49)]. Kidney Na⁺,K⁺-ATPase activity decreased during summer and autumn. Different patterns of variation were observed in kidney metabolite levels while all the enzymatic activities assessed [lactate dehydrogenase-oxidase (EC 1.1.1.27) (LDH-O), HK and GDH] presented the highest values during summer. In the liver, metabolite levels and enzymatic activities did not show significant variations or present clear patterns of variation along different seasons. These results indicated seasonal variations in the osmoregulatory and metabolic parameters of different organs (blood, gill, kidney and liver) in earthen pond-cultured gilthead sea bream ( S. auratus ), which could be mainly attributed to seasonal changes in temperature.     

Preparation of heavy meromyosin from the autolyzed squid mantle muscle homogenate

ABSTRACT:   Incubation of squid mantle muscle homogenate caused a selective cleavage of myosin into heavy meromyosin (HMM) and light meromyosin (LMM). HMM was isolated from the incubated homogenate by using ammonium sulfate fractionation. The purified HMM retained two types of light chain components. Its Mg²⁺-ATPase activity with or without F-actin showed a Ca-sensitivity. HMM was cleaved into subfragment-1 and subfragment-2 upon chymotryptic digestion with or without Ca²⁺, possessing different light chain composition. Two types of light chain component were kept intact when digested in the presence of Ca²⁺. Ca²⁺ stabilized HMM especially in a bound form to F-actin.     

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.     

Urine properties in carp, Cyprinus carpio L., with sekoke disease

Abstract. Changes in the properties of urine and blood of carp with sekoke disease were studied. With the progress of this disease, the parameters of blood and particularly urine changed significantly. After 120 days, urine flow, osmolarity and concentrations of inorganic ions (K⁺, Ca²⁺, Mg²⁺, Cl⁻) and organic compounds (ammonium, creatine and creatinine, protein, glucose) in the experimental group showed higher values than those of the controls. Even after 60 days, when clinical signs of sekoke disease were not evident, abnormally high concentrations of ammonium, protein and glucose in the urine were found in the experimental group.     

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.     

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.     

Changes in enzymatic and structural properties of grass carp fast skeletal myosin induced by the laboratory-conditioned thermal acclimation and seasonal acclimatization

ABSTRACT:   Myosins were prepared from fast skeletal muscles of grass carp thermally acclimated to 10, 20 and 30°C in the laboratory as well as from those seasonally acclimatized and collected in January (winter) 2003 and May (spring), August (summer) and November (autumn) 2002. The maximal initial velocities ( V _max) of actin-activated Mg²⁺-ATPase activity for myosins from the 10°C-acclimated and winter grass carp were 1.7–1.8-fold as high as those from the 30°C-acclimated and summer fish. The inactivation rate constant ( K _D) of Ca²⁺-ATPase for myosin from the 10°C-acclimated grass carp was three to fourfold higher than those for myosins from the fish acclimated to 20°C and 30°C, whereas myosin from winter grass carp was about sevenfold as high as that for myosin from summer fish. Myosins from spring and autumn fish showed K _D values comparable to those of the fish acclimated to 30°C and 10°C, respectively. In differential scanning calorimetry analysis, the transition temperature ( T _m) was observed near 38°C and 45–46°C with most myosins. However, the lowest T _m at 32–33°C was given as one of the major endotherms in myosins from the 10°C-acclimated, autumn and winter fish. These responses of grass carp to changed environmental temperatures were almost similar to those for common carp reported previously.