The existence of aspolin and its trimethylamine-<Emphasis Type="Italic">N</Emphasis>-oxide demethylating activity in the muscle of freshwater fish

ABSTRACT:   Aspolin is a polyaspartic acid-like protein, which is originally isolated from walleye pollack Theragra chalcogramma muscle as trimethylamine- N -oxide (TMAO) demethylase. Although carp Cyprinus carpio muscle contains a trace amount of the enzyme substrate, TMAO, aspolin can be extracted and purified by acid treatment, successive chromatographies and polyacrylamide gel electrophoresis, and has twice the amount of that in walleye pollack muscle. Carp aspolin showed a low enzymatic activity in the presence of Fe²⁺ and reductants, and its Km value (100 mM) to TMAO was extremely high. It was a thermostable protein and had an unfolded conformation. The amino acid sequence of carp aspolin 1 deduced from cDNA revealed that it contained a long Asp polymer, an uninterrupted stretch of 138 Asp residues, followed by four amino acid residues, His-Glu-Glu-Leu, in C-terminus. The chain length was shorter by 42 Asp residues than that of its walleye pollack counterpart.     

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.     

Preventive effects of amino acids and glutathione on the formaldehyde-induced denaturation of myofibrillar proteins

ABSTRACT:     Formaldehyde (FA)-induced denaturation of myofibrillar proteins and its prevention were investigated by means of measuring the solubility, adenosine triphosphatase (ATPase) activity, and thermal gel formability of myofibrils and surimi proteins in the presence and absence of free amino acids and glutathione, reduced form. The addition of FA decreased the solubility of myofibrils in 0.5 M NaCl at pH 7.0 and 0°C depending on its concentration and incubation time. The solubility decrease was completely inhibited by the presence of equal, twofold, and threefold amounts of cysteine (Cys), glutathione, and histidine (His) to the amount of FA, respectively. Myofibrillar Ca-ATPase was markedly activated at the initial phase and then decreased later by the addition of FA. The K-ATPase was inactivated with an increase in the amount of FA. The FA-induced changes in both ATPase activities were inhibited in the presence of Cys and His. Thermal gel formability of surimi paste increased only in a short period after the addition of a low concentration of FA. Practically, FA inhibited the thermal gelation and setting effect through the inactivation of transglutaminase. In the presence of Cys, His or glutathione, a strong elastic surimi gel was produced because FA-induced detrimental effects were inhibited.     

TMAOase, trimethylamine-N-oxide demethylase, isa thermostable and active enzyme at 80°C

ABSTRACT:   Purified trimethylamine- N -oxide demethylase (TMAOase)from walleye pollack muscle is a thermostable protein that was notinactivated after heating at 80°C for 30 min.The heated enzyme was electrophoresed in the same manner as fornative enzyme. Circular dichroism (CD) spectra for purified enzymechanged reversibly in the temperature range of 10–80°C.As the enzyme was still active at 80°C, the CD spectralchange did not directly relate to enzyme activity. TMAOase activity inthe myofibrillar fraction decreased sharply above 30°C,but was extracted and recovered from the heated myofibrillar fraction,suggesting that the activity seemed to be interrupted and apparently inactivateddue to the thermal alteration of myofibrillar proteins or some unknownfactors. The complicated profile found in dimethylamine (DMA) formationfrom trimethylamine- N -oxide (TMAO) in walleye pollack muscleduring heating consisted of both enzymic and non-enzymic processes.Most DMA was produced enzymatically below 40°C and interruptedabove 40°C. Therefore, DMA and trimethylamine was formednon-enzymatically at high temperatures regardless of the presenceof native enzyme. A new, simple and easy purification method wasproposed based on the thermostable nature of the enzyme.     

Rare serotype occurrence and PFGE genotypic diversity of Streptococcus agalactiae isolated from tilapia in China

Previously, we reported 10 PEGE types of 85 tilapia Streptococcus agalactiae(GBS), which shifted from Streptococcus iniae in China, by using PEGE method. Presently, larger and more representative tilapia GBS were isolated, for the ?rst time in China, to characterize their serotypes and genetic diversities more precisely than had done before. 168 GBS strains were distributed in ?ve provinces of China, in which Guangdong, Guangxi and Hainan were the major ones, holding36.9%(62/168), 37.5%(63/168) and 19.6%(33/168), respectively. Serotypes, Ia, Ib and III, were observed in these strains and the most predominant one was Ia(95.2%), which mainly distributed in Guangdong, Guangxi and Hainan. Ia initially occurred in 2009, it shoot up to 32.1% in 2010,but decreased to 16.1% in 2011 before went up to 45.2% in 2012. Ib sporadically occurred during2007–2011, III onlyoccurred in 2012. 14 different PFGE types, including 4 new types(N, O,P and Q), were observed, in which B, D, F and G were the predominant types, holding 83.9%(141/168) of the total GBS strains. Ia corresponded to 11 PFGE types(A–H, N–P), in which type D predominated(51%). Ib represented 3 genotypes(I, J and Q) and III harbored only 2genotypes(N and F). Type N and Fsynchronously presented in Ia and III. In summary, the genetic diversity of tilapia GBS varied by serotypes and changed with geographical locations and years.Although Iastillpredominated, new rareserotypeIII alreadyoccurred in China.     

Growth hormone (GH) and reproduction: a review

Interaction between growth and reproduction occurs in many vertebrates and is particularly obvious at certain stages of the life cycle in fish. Endocrine interactions between the gonadotropic axis and the somatotropic axis are described, the potential role of GH being emphasised. A comparative analysis of these phenomena in mammals, amphibians and fish, suggests a specific role of GH in the physiology of puberty, gametogenesis and fertility. It also shows the original contribution made by studies on the fish model in this field of investigations.     

Purification and partial characterization of GtHs (cfLH and cfFSH) from Indian walking catfish (Clarias batrachus) (L.) and development of a homologous ELISA for cfLH

Two gonadotropins (GtH; Qa and Qb) were purified by gel filtration and ion exchange chromatography from the pituitaries of Indian walking catfish (Clarias batrachus). The presence of GtH during purification was assessed by in vitro oocyte maturation and in vivo steroidogenic activity, and their identities were determined by elution profiles, molecular weight, biological activities and yield. The molecular weights of Qa and Qb were 37 and 42 kDa, respectively, and composed of distinct subunits (Qa: 20 and 14 kDa and Qb: 26 and 18 kDa). Polyclonal antibodies raised against Qa immunostained Qa, Qb and pituitary GtH cells. A competitive Qa‐ELISA was developed whose sensitivity was 6.25 ng mL^?1 (1.25 ng well^?1) with intra‐ (3.5%) and inter‐ (12.4%) assay coefficients of variation. Displacement curves parallel to the standard were obtained with plasma and pituitary extracts of catfish, Qb and carp GtHII. The assay was validated by measuring the plasma Qa levels after LHRH treatment and in relation to ovarian growth in the female catfish during different reproductive phases. Based on the results, Qa and Qb corresponded to fish LH and FSH respectively. The findings will increase the knowledge of the mechanisms controlling fish reproduction and identification of sensitive phases in fish in captivity for hormonal manipulation.     

Controlled infection of Poecilia reticulata Peters (guppy) with Tetrahymena by immersion and intraperitoneal injection

Tetrahymena is a protozoan parasite, which infects guppy, Poecilia reticulata Peters, and causes substantial economical losses in commercial farms worldwide. Studies of guppy infected by Tetrahymena require standardized infection protocols. The LD50 for Tetrahymena infection of guppies by intraperitoneal (IP) injection was calibrated, and the level obtained was 946 parasites per fish. Guppy infection with Tetrahymena by immersion, imitating the natural route of infection via the integument, was studied under normal or stress conditions. Exposure to cold and netting (CNI) and to cold only (CI) followed by immersion exposure to 10 000 Tetrahymena per mL resulted in 22.5% and 19.2% mortality, respectively, as compared to 14.2% and 10% in groups that were netted only (NI) or non‐stressed (I). Histopathology revealed that immersion infection resulted in a systemic infection. Lysozyme levels, measured 3 weeks after infection, were significantly higher in the CNI group (288 μg per mg protein) compared with CI‐, NI‐ and I‐treated groups (94.5, 64 and 62.3 μg mg^?1, respectively). There was no evident parasite immobilization activity in body homogenates, suggesting no development of acquired immunity. Re‐infection by IP injection revealed no increase in protection in any of the treatment groups, mortality range of 56.3–75%, higher than in the non‐exposed control (40.6% mortality).     

Evaluation of a Fast Method Based on the Presence of Two Restriction Sites in the Mitochondrial ND5 (mt ND5) Gene for the Identification of Scomber Species

The purpose of this work was to evaluate the suitability of a method based on the presence of two restriction sites (for Hae III and Hindf I) in the mitochondrial NADH dehydrogenase subunit 5 (mt ND5) gene to identify Scomber species. The evaluation was performed on 144 reference and market samples by sequencing of the entire 505-bp fragment of the mt ND5 gene and of a 464-bp fragment of the Kocher fragment of the cytochrome b gene (mt Cytb). Sequence analysis of any of the two fragments allows the identification of each of the four Scomber species, but S. japonicus and S. colias had the same restriction sites at the ND5 amplicon and would not have been differentiated by this analysis. Similarly, loss of the Hae III site in some S. scombrus individuals would have misidentified them as not being Scomber. All the market products were correctly labeled except one acquired in Spain labeled as originating in the Atlantic and containing S. japonicus.     

Imported ornamental fish are colonized with antibiotic‐resistant bacteria

There has been growing concern about the overuse of antibiotics in the ornamental fish industry and its possible effect on the increasing drug resistance in both commensal and pathogenic organisms in these fish. The aim of this study was to carry out an assessment of the diversity of bacteria, including pathogens, in ornamental fish species imported into North America and to assess their antibiotic resistance. Kidney samples were collected from 32 freshwater ornamental fish of various species, which arrived to an importing facility in Portland, Oregon from Colombia, Singapore and Florida. Sixty‐four unique bacterial colonies were isolated and identified by PCR using bacterial 16S primers and DNA sequencing. Multiple isolates were identified as bacteria with potential to cause disease in both fish and humans. The antibiotic resistance profile of each isolate was performed for nine different antibiotics. Among them, cefotaxime (16% resistance among isolates) was the antibiotic associated with more activity, while the least active was tetracycline (77% resistant). Knowing information about the diversity of bacteria in imported ornamental fish, as well as the resistance profiles for the bacteria will be useful in more effectively treating clinical infected fish, and also potential zoonoses in the future.     

Effect of iodophor disinfection of non‐hardened Salmo trutta eggs on their bacterial and fungus load

This study investigated the efficiency of iodophor disinfection (135 ppm active iodine for 15–30 min) of non‐hardened Salmo trutta eggs against different groups of bacteria and against fungus. Egg samples were taken from non‐disinfected and from disinfected eggs, microorganisms were cultured on specific nutrient media and their mass was measured by turbidimetric methods. Bacteria and fungus mass of non‐hardened eggs could be reduced but not eliminated by iodophor disinfection with 135 ppm active iodine for 15 min. The extent of reduction was 47–65% (Experiment 1). The efficiency of disinfection increased with disinfection time as the reduction in bacteria and fungus mass was 40–55% after 15 min and 58–74% after 30 min (Experiment 2). Disinfection efficiency of iodophor solution diluted in water (reduction 49–57%) and of iodophor solution diluted in sodium chloride solution iso‐osmolar to the oocytes (reduction 52–61%) was similar (Experiment 3). The reduction in bacteria and fungus mass was persistent as it was 39–72% lower in embryos deriving from disinfected eggs than in embryos deriving from non‐disinfected ones (Experiment 4). In conclusion, the tested disinfection method is inadequate to eliminate pathogens completely but it could positively influence immune defence of eggs and embryos.     

Nitrogenous waste excretion and accumulation of urea and ammonia inChalcalburnus tarichi (Cyprinidae), endemic to the extremely alkaline Lake Van (Eastern Turkey)

The endemic, anadromous cyprinidChalcalburnus tarichi is the only fish species known to occur in alkaline Lake Van (Eastern Anatolia, Turkey). EightC. tarichi were maintained individually in Lake Van water (17 – 19°C; pH 9.8; 153 mEq·I^–1 total alkalinity; 22 total salinity) and tank water samples analyzed for 24 h in 2 to 4 h intervals. At zero time, < 1µM ammonia was present and urea was undetectable in the tank water; at 24 h, total ammonia and urea made up 114±32 and 35±25µM, respectively. Over the experimental period, ammonia-N and urea-N excretion averaged 1041±494 and 607±169moles·kg^–1 fish·h^–1, respectively. The extent of urea excretion was highly variable between specimens. Uric acid excretion was not detectable.Urea was present at high concentrations in all tissues and plasma (25 – 35moles·g^–1·ml^–1) of freshly caughtC. tarichi; total ammonia content of the tissues was by a factor of 1.9 (liver) to 3.0 (brain) lower. High arginase activity (2.4±0.2 U·min^–1·g^–1) was detected in the liver ofC. tarichi but ornithine carbamoylphosphate transferase, a key enzyme of the ornithine-urea-cycle, was absent. Ureagenesis is likely through degradation of arginine and/or uricolysis. High glutamine synthetase activity (11±0.6 U·min^–1·g^–1) and low ammonia content in brain suggest that, like other teleosts,C. tarichi has an efficient ammonia detoxification in the brain, but in no other tissue.Nitrogenous waste excretion at alkaline pH is discussed. The ability ofC. tarichi to excrete high levels of ammonia at extremely alkaline pH is unique among teleosts studied so far. The mechanism of ammonia excretion under Lake Van conditions remains to be elucidated.     

Influence of the antibacterial herbs, Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia on the survival, growth and bacterial load of Penaeus monodon post larvae

The indiscriminate use of antibiotics and chemicals in shrimp hatcheries has led to biomagnification and that in turn could lead to rejection of a whole consignment. The application of the bioencapsulation technique as a tool for curative treatment in shrimp larvae was investigated. Herbs having antibacterial properties such as Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia (methanolic extracts) were bioencapsulated in Artemia and fed to Penaeus monodon post larvae PL 1–25. The post larvae were reared in a medium inoculated with pathogenic bacteria such as Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi and Vibrio sp. Post larvae reared in the non-inoculated water and fed with non-enriched Artemia exhibited 90% survival, highest specific growth rate (12.43%) and reduced bacterial load. P. monodon reared in the bacterial inoculated water and fed with the non-enriched Artemia exhibited the lowest survival (10–30%), specific growth rate (8.42–9.1%) and increased bacterial load (2.86 × 10³ to 3.76 × 10⁵ cfu/g). The methanolic extracts of the herbs helped to increase survival and specific growth rate and reduced bacterial load in the P. monodon culture system. Among the three herbal extracts, P. corylifolia enriched Artemia fed post larvae showed the tendency to higher survival (>50%), growth rate (11.5 averaged) and low bacterial load (1.12 × 10⁵ cfu/g). This revised version was published online in August 2006 with corrections to the Cover Date.     

Effects of cortisol on hepatic carbohydrate metabolism and responsiveness to hormones in the sea raven,Hemitripterus americanus

The sea raven, Hemitripterus americanus, is a sit-and-wait, low metabolic rate, marine teleost. The objective of this study was to determine i) whether cortisol implantation (50 mg. kg^-1) for 7 days altered hepatocyte metabolism, and hepatocyte responsiveness to epinephrine, glucagon and insulin, and ii) whether 8 weeks of food-deprivation modified the above response. Cortisol implantation significantly increased hepatocyte total glucose production and oxidation from alanine compared to the sham group. There was no cortisol effect on glycogen breakdown, suggesting that the activation of other pathways, including gluconeogenesis, are required to account for the increased glucose production. Epinephrine-mediated (10^-5M) glycogen breakdown and insulin-mediated (10^-8M) total glucose production were enhanced in hepatocytes of cortisol implanted sea ravens, but there were no change in any glucagon (10^-7M) effects. The enhanced glycogen breakdown in the absence of similar increases in total glucose production with epinephrine indicates mobilization of carbohydrate reserves for endogenous use by the liver. Food-deprivation for 8 weeks significantly decreased condition factor, plasma cortisol concentration and liver glycogen content in the sea raven, but had no effect on plasma glucose concentration. Hepatocyte total glucose production and flux rates from alanine increased significantly with food-deprivation. Moreover, food-deprivation increased responsiveness of total hepatocyte glucose production to the actions of glucagon and insulin, but not to epinephrine; none of these effects were modified by cortisol implantation. Our results indicate that cortisol in the sea raven exerts both a direct and an indirect or permissive effect on hepatocyte metabolism by modifying hepatocyte responsiveness to epinephrine and insulin stimulation. Cortisol implantation did not modify the effects of glucagon or food-deprivation in this species.     

Simulation and quantification of enrichment and retention processes in the southern Benguela upwelling ecosystem

Important environmental processes for the survival and recruitment of early life stages of pelagic fishes have been synthesized through Bakun's fundamental triad as enrichment, concentration and retention processes (A. Bakun, 1996, Patterns in the Ocean. Ocean Processes and Marine Population Dynamics. San Diego, CA, USA: University of California Sea Grant). This conceptual framework states that from favourable spawning habitats, eggs and larvae would be transported to and/or retained in places where food originating from enrichment areas would be concentrated. We propose a method for quantifying two of the triad processes, enrichment and retention, based on the Lagrangian tracking of particles transported within water velocity fields generated by a three‐dimensional hydrodynamic model. We apply this method to the southern Benguela upwelling ecosystem, constructing putative maps of enrichment and retention. We comment on these maps regarding main features of the circulation in the region, and investigate seasonal variability of the processes. We finally discuss the results in relation to available knowledge on the reproductive strategies of two pelagic clupeoid species abundant in the southern Benguela, anchovy (Engraulis encrasicolus) and sardine (Sardinops sagax). Our approach is intended to be sufficiently generic so as to allow its application to other upwelling systems.     

Status of aquatic and riparian biodiversity in artificial lake ecosystems with and without management for recreational fisheries: Implications for conservation

1. Humanity is facing a biodiversity crisis, with freshwater-associated biodiversity in a particularly dire state. Novel ecosystems created through human use of mineral resources, such as gravel pit lakes, can provide substitute habitats for the conservation of freshwater and riparian biodiversity. Many of these artificial ecosystems are subject to a high intensity of recreational use, however, which may limit their biodiversity potential.
2. The species richness of several taxa (plants, amphibians, dragonflies, damselflies, waterfowl, and songbirds) was assessed and a range of taxonomic biodiversity metrics were compared between gravel pit lakes managed for recreational fisheries (n = 16) and unmanaged reference lakes (n = 10), controlling for non-fishing-related environmental variation.
3. The average species richness of all the taxa examined was similar among lakes in both lake types and no substantial differences in species composition were found when examining the pooled species inventory. Similarly, there were no differences between lake types in the presence of rare species and in the Simpson diversity index across all of the taxa assessed.
4. Variation in species richness among lakes was correlated with woody habitat, lake morphology (surface area and steepness), and land use, but was not correlated with the presence of recreational fisheries. Thus, non-fishing-related environmental variables had stronger effects on local species presence than recreational fisheries management or the presence of recreational anglers.
5. Collectively, no evidence was found that anglers and recreational fisheries management constrain the development of aquatic and riparian biodiversity in gravel pit lakes in the study region; however, the conservation of species diversity in gravel pit lakes could benefit from an increasing reliance on habitat enhancement activities.

     

Epizootics of Streptococcus agalactiae infection in captive rays from Queensland,Australia

The aim of this study was to describe two epizootics of high mortalities from infection with Streptococcus agalactiae, occurring in captive rays held in a marine display aquarium in south‐east Queensland, Australia, in 2009 and 2010. Five different species of rays were affected, including mangrove whiprays (Himantura granulata), estuary rays (Dasyatis fluviorum), eastern shovelnose rays (Aptychotrema rostrata), white‐spotted eagle rays (Aetobatus narinari) and blue‐spotted mask rays (Neotrygon kuhlii). This report describes the history of both epizootics including collection, quarantine and husbandry of rays, the disease epizootics, clinico‐pathological features of the disease, antimicrobial therapy, autogenous vaccine production, and laboratory studies including clinical and histopathology, bacteriology, PCR, molecular serotyping and sequencing of the bacterium S. agalactiae.