Molecular cloning and expression profiling of procollagen α1 (I) of cultured Pacific bluefin tuna

Type I collagen is widely distributed in most organs in teleosts. It plays a role not only in intercellular adhesion, but also in molecular signaling. In this study, Pacific bluefin tuna (PBT) procollagen α1 (I) cDNA was cloned and characterized. The nine fragments of a procollagen α1 (I) chain cDNA clone were prepared and spliced together to create the complete coding region. The resulting amino acid sequence was homologous with that of other teleosts. The mRNA expression profile of PBT procollagen α1 (I) in various tissues and the phylogenetic analysis with other vertebrate procollagen α1 (I) chains suggest that PBT procollagen α1 (I) could be a precursor form of the PBT type I collagen α1 chain. In addition, its level of expression in PBT larvae and early juveniles gradually increased with somatic growth. This increase was related to the standard length, wet body weight, and protein content of each individual fish. Therefore, the expression profile of procollagen α1 (I) may be a useful indicator for somatic growth in fish larvae and juveniles.     

Intestinal absorption of amino acids in the Pacific bluefin tuna (Thunnus orientalis): in vitro lysine–arginine interaction using the everted intestine system

The interaction between lysine (Lys) and arginine (Arg) in the proximal intestinal region of Pacific bluefin tuna (Thunnus orientalis) was evaluated using the everted intestine method. This in vitro intestinal system has been shown to be an effective tool for studying the nutrient absorption without the need to handle the tuna fish in marine cages as needed for digestibility and amino acid (AA) absorption. We used a factorial design with two sets of variables: low and high Lys concentration (10 and 75 mM) and four different Arg concentrations (3, 10, 20, and 30 mM). Both amino acids were dissolved in marine Ringer solution with a basal amino acidic composition consisting of a tryptone solution (9 mg mL^?1). No interaction was observed between the absorption of Lys and Arg during the first 10 min of the experiment when low concentration of Lys and Arg was used in the hydrolyzate solution. However, there seemed to be a positive effect on Lys absorption when both amino acids were at high concentrations (30 and 75 mM, respectively). This type of studies will led us to test different formulations and/or additives to better understand the efficiency of AA supplementation as an alternative to in situ studies that are difficult to follow to design with the Pacific Bluefin Tuna.     

Myosin denaturation in “Burnt” Bluefin tuna meat

A quantitative conversion of tuna meat into muscle homogenate made it possible to study myosin denaturation in Bluefin tuna meat. Myosin denaturation was accessed by measuring Ca²⁺-ATPase activity, salt-solubility with and without Mg-ATP, monomeric myosin content, and amount of subfragment-1 (S-1) and rod produced by chymotryptic digestion. Commercially available tuna used in this study showed a pH around 5.4–5.7. Myosin in the meat lost the salt-solubility measured in the absence of Mg-ATP; however, such myosin showed full salt-solubility when released from actin in the presence of Mg-ATP. Incubation of tuna meat at 30 °C for up to 90 min caused obvious myosin denaturation. However, the tuna meat dialyzed against neutral pH buffer showed practically no myosin denaturation by the same heating. It was suggested that exposure to lowered pH to around 5.5 and increased temperature of 30 °C led myosin denaturation. Myosin denaturation in the “Burnt” Bluefin tuna sample was analyzed. A significant myosin denaturation was observed with the part showing the “Burning” symptom, the inner part of the tuna meat near the spine. Myosin in that part showed almost no Ca²⁺-ATPase activity, no salt-solubility even with Mg-ATP, no recovery of monomeric myosin, and almost no production of S-1 by chymotryptic digestion. However, myosin denaturation was not detectable for the meat taken from outer parts of the same tuna near the skin with normal appearance. It was demonstrated that “Burning” of tuna meat occurring in the deep part of the body is accompanied by myosin denaturation. The above results suggested that insufficient cooling of the deep part of body would be the reason for “Burning” of tuna meat.     

Shooting fish in a barrel? Assessing fisher‐driven changes in catchability within tropical tuna purse seine fleets

With constant innovation to find more efficient ways to find, catch and process fish, catchability in wild fisheries can increase. Catchability is a combination of resource abundance, fishing effort and fishing efficiency: any increase in fleet efficiency can lead to undesirable effects not only on stocks, but also on the ability to assess them. When using effort controls as part of management, it is necessary to adjust for the increase in catchability due to the increases in efficiency over time to avoid stock depletion. Accounting for changes in catchability can be problematic for pelagic stocks, due to the changes in fishing behaviour and the continual change in fishing efficiency. This study investigates the success in finding patches of fish for fleets operating within the western and central Pacific purse seine fishery between 1993 and 2012. Three indices, widely used in ecological research, were used to study how spatial variation in fisher behaviour for sets on fish aggregating devices (FADs) and free‐school sets was related to catchability. For free‐school set types, the diversity index was negatively correlated with Katsuwonus pelamis catchability. When this index was low, catch rates were at their highest and there was a reduction in the area fished. In contrast, for FAD sets, catches increase when the patchiness index was low, implying a degree of random behaviour, potentially due to advances in FAD technology. An improved understanding of the spatial allocation of effort can improve catchability estimates widely used for fisheries stock assessments and in indices of global biodiversity.     

Do drifting and anchored Fish Aggregating Devices (FADs) similarly influence tuna feeding habits? A case study from the western Indian Ocean

Anchored and drifting Fish Aggregating Devices (FADs) are intensively used in tropical tuna fisheries. In both small-scale and industrial fisheries, skipjack (Katsuwonus pelamis) and yellowfin tuna (Thunnus albacares) are the main targets. The increasing development of this fishing practice by industrial purse seiners has raised the question of the impact of FADs on tuna communities, as they might act as an ecological trap. This study investigated the feeding habits of skipjack and yellowfin tuna associated with anchored and drifting FADs in the western Indian Ocean. The diet of 352 tunas was analysed taking into account the type of FAD, ontogenetic variations, and the resources richness of the area. Poor-food and rich-food areas were defined according to the abundance of stomatopod Natosquilla investigatoris, the main prey of tunas, on the fishing sites. Diet composition was expressed through functional groups of prey. Significant dietary differences were found between both FAD types, as well as an effect of individual size. Around anchored FADs tuna preyed on diverse assemblages of coastal fish and crustacean larvae and juveniles, whereas a low diversity of epipelagic prey dominated the tuna diet associated with drifting FAD. Compared to anchored FADs, the frequency of empty stomachs was significantly higher and the stomach content mass significantly lower among skipjack and small yellowfin tunas caught around drifting FADs. This was magnified in poor-food areas, where drifting FADs often evolved, suggesting that these FADs could negatively impact the growth of skipjack and small yellowfin tuna. Larger yellowfin tuna exhibited differences in their dietary habits between anchored and drifting FADs, and between poor-food and rich-food areas. However, drifting FADs did not impact them as strongly as juveniles of yellowfin or skipjack tunas. Our study gives new highlights on possible detrimental effects of FAD on tunas, and this has to be considered in future sustainable management strategies of tuna fisheries.     

Just a FAD? Ecosystem impacts of tuna purse‐seine fishing associated with fish aggregating devices in the western Pacific Warm Pool Province

The western and central Pacific Ocean supports the world's largest tuna fisheries. Since the 1990s, the purse‐seine fishery has increasingly fished in association with fish aggregating devices (FADs), which has increased catches of juvenile bigeye and yellowfin tunas and vulnerable bycatch species (e.g., sharks). This has raised concerns regarding the sustainability of these species’ populations and the supporting ecosystem, but may provide improved food security of Pacific Island nations through utilisation of FAD‐associated byproduct species (e.g., wahoo). An ecosystem model of the western Pacific Warm Pool Province was used to explore the potential ecological impacts of varying FAD fishing effort (±50% or 100%) over 30 years. The ecosystem has undergone a significant change in structure since 1980 from heavy exploitation of top predators (e.g., tunas) and “fishing up the food web” of high‐trophic‐level non‐target species. The ecosystem appeared resistant to simulated fishing perturbations, with only modest changes (<10%) in the biomass of most groups, although some less productive shark bycatch species decreased by up to 43%, which had a subsequent positive effect on several byproduct species, the prey of sharks. Reduction of FAD effort by at least 50% was predicted to increase the biomass of tuna species and sharks and return the ecosystem structure to a pre‐industrial‐fishing state within 10 years. Spatial disaggregation of the model and integration of economic information are recommended to better capture ecological and economic changes that may result from fishing and/or climate impacts and to develop appropriate management measures in response.     

Supercritical CO<Subscript>2</Subscript> extraction and concentration of n−3 polyunsaturated fatty acid ethyl esters from tuna cooking juice

Cooking is an important step in the tuna canning process; it usually produces functional solubilized proteins and lipids. The objective of this study was to recover and increase the concentration of functional n−3 fatty acids from the lipids in cooking juice. The lipids contained 12.98% eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) after ethyl esterification and increased to 37.4% with subsequent urea adduct fractionation. Supercritical carbon dioxide extraction was further used to increase the concentration of EPA and DHA ethyl esters. The ratio of EPA+DHA ethyl esters (high-molecular-weight components) to that of C₁₆ and C_18∶1 fatty acid ethyl esters (low-molecular-weight components) was used as a separation index for evaluating the process variables. Experimental results indicated that a high CO₂ density caused a low separation factor. At 1500 psig and 328.2 K, the extraction collected over 600 L CO₂, displaying an accepted concentration factor of EPA+DHA ethyl esters herein. About 80% yield of EPA and DHA was obtained and the ethyl esters increased from 37.4% to 54.3%.     

Binding profiles and cytokine-inducing effects of fish rhamnose-binding lectins on Burkitt’s lymphoma Raji cells

Rhamnose-binding lectin (RBL) is one of the animal lectin categories which take part in the innate immune responses of fish. Osmerus lanceolatus lectin (OLL) from shishamo smelt eggs is an RBL composed of two tandem-repeated domains, both of which are considered to be a carbohydrate-recognition domain. SAL, catfish (Silurus asotus) egg RBL composed of three domains, binds to Burkitt’s lymphoma Raji cells through globotriaosylceramide (Gb3) carbohydrate chain and to reduce cell size and growth by altering membrane composition without causing cell death. In this experiment, we tried to compare the binding effects of these two RBLs on Raji cells. Flow cytometric and fluorescence microscopic analyses revealed that OLL also directly bound to and shrunk Raji cells with ten times less reactivity than SAL but reduced cell growth with decreasing cell viability. Anti-Gb3 antibody completely blocked the binding of SAL to Raji cells but not that of OLL. In addition, the direct bindings of OLL and SAL to Raji cells were comparably inhibited by melibiose, but lactose was more effective inhibitor for the binding of OLL than that of SAL. These results suggest that OLL has slightly different cell-binding property compared with SAL and binds not only to Gb3 but also to the other carbohydrate receptor-bearing β-galactoside chains. The quantitative RT-PCR analysis revealed that SAL induced the expression of TNF-α but not of IFN-γ, IL-1β, and IL-10. Thus, SAL-induced cytostatic effect on Raji cells might be partially caused by TNF-α-mediated signaling pathway.     

The role of cAMP in regulating the β-adrenergic response of rainbow trout (Oncorhynchus mykiss) red blood cells

The -adrenergic response of teleost red blood cells (RBCs) enables the fish to maintain or even enhance the oxygen affinity of haemoglobin during various stress situations. The role of CAMP in the pronounced -adrenergic response of hypoxic rainbow trout RBCs was studied. Rainbow trout RBCs were incubated with three different -agonists (noradrenaline, adrenaline and isoproterenol, 10^–9 - 10^–4 M) at two oxygen tensions (P_{O 2}, 155 and 8 mmHg), and thereafter cAMP accumulation and cellular water content were measured.The cAMP concentration of non-stimulated trout RBCs was ca. 1200 nmol/kg dw. Of the three -agonists used, isoproterenol was the most effective in formation of cAMP, followed by noradrenaline and adrenaline. Oxygen tension affected the accumulation of cAMP in two ways. At physiological catecholamine levels (1–100 nM) there was either no difference between normoxic and hypoxic cells or a slight increase in the normoxic ones. At high catecholamine concentrations the accumulation of cAMP was greater in the hypoxic than in the normoxic cells. Oxygen tension also affected the magnitude of cell swelling but had no effect on the catecholamine concentrations causing half-maximal swelling (EC₅₀-values). The results indicate that, at physiological catecholamine levels, the -adrenergic response of rainbow trout RBCs is mainly regulated on the level of the Na⁺/H⁺ exchange.     

Enteroendocrine profile of α-transducin immunoreactive cells in the gastrointestinal tract of the European sea bass (Dicentrarchus labrax)

In vertebrates, chemosensitivity of nutrients occurs through the activation of taste receptors coupled with G-protein subunits, including α-transducin (G_αtran) and α-gustducin (G_αgust). This study was aimed at characterising the cells expressing G_αtran immunoreactivity throughout the mucosa of the sea bass gastrointestinal tract. G_αtran immunoreactive cells were mainly found in the stomach, and a lower number of immunopositive cells were detected in the intestine. Some G_αtran immunoreactive cells in the stomach contained G_αgust immunoreactivity. Gastric G_αtran immunoreactive cells co-expressed ghrelin, obestatin and 5-hydroxytryptamine immunoreactivity. In contrast, G_αtran immunopositive cells did not contain somatostatin, gastrin/cholecystokinin, glucagon-like peptide-1, substance P or calcitonin gene-related peptide immunoreactivity in any investigated segments of the sea bass gastrointestinal tract. Specificity of G_αtran and G_αgust antisera was determined by Western blot analysis, which identified two bands at the theoretical molecular weight of ~45 and ~40 kDa, respectively, in sea bass gut tissue as well as in positive tissue, and by immunoblocking with the respective peptide, which prevented immunostaining. The results of the present study provide a molecular and morphological basis for a role of taste-related molecules in chemosensing in the sea bass gastrointestinal tract.     

Effect of enrofloxacin and emodin on heat-shock proteins’ expression in hepatic cells of grass carp (Ctenopharyngodon idellus)

The aim of the present study was to compare the expression pattern of heat-shock protein 60 (HSP60), 70 (HSP70), and 90 (HSP90) mRNA in hepatic cells of grass carp exposed to enrofloxacin and emodin concentrations. The expression pattern of different genes encoding heat-shock proteins in hepatic cells of grass carp was exposed to graded levels of enrofloxacin (12.5, 25, 50, 100, and 200 μg/ml) or emodin (0.04, 0.2, 1, 5, and 25 μg/ml) for 24 h and were investigated by real-time quantitative PCR. When cells were exposed to up 50 μg/ml enrofloxacin, both HSP60 and HSP70 mRNA levels firstly were increased and thereafter significantly dropped at high concentrations (P < 0.05), while HSP90 decreased with increasing enrofloxacin concentration. Besides, HSP60 and HSP70 expressions were significantly inhibited in the high-concentration groups. In addition, the HSP90 mRNA levels in the treatment exposed to 100 and 200 μg/ml enrofloxacin were significantly lower than those of the control group (P < 0.05). Furthermore, when cells were exposed to graded levels of emodin, HSPs (HSP60, 70, and 90) mRNA levels significantly increased in the groups exposed to 5 and 25 μg/ml of emodin. The different expression pattern of HSPs implied that enrofloxacin could inhibit the expression levels of HSPs, while optimal level of emodin could trigger higher expression levels of HSPs in hepatic cell of grass carp to protect against further damage.     

cDNA microarray analysis of interleukin-1β-induced Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> kidney cells

ABSTRACT:   Interleukin-1β (IL-1β) cDNA of Japanese flounder was found to consist of 1329 bp, encoded 247 amino acid residues. Among the fish IL-1β in the databases, the one with the highest identity of Japanese flounder IL-1β was that of seabass (62% identity). The expression of IL-1β was induced by treatment with concanavalin A (ConA)/phorbol myristate acetate (PMA) and lipopolysaccharide. The copy number of IL-1β mRNA was increased 30-fold after stimulation with ConA/PMA. Of 871 cDNA on a microarray, 93 genes (10.7%) were up-regulated or down-regulated by IL-1β at 1, 3 and 7 days post-injection. The induced gene expression was highest on day 1 followed by day 3 and day 7. A total of 7% of known and 3.7% of unknown genes of the 871 tested genes were differentially expressed. Of the genes tested, 7.4% were up-regulated and 3.3% were down-regulated. Cytokine genes such as tumor necrosis factor, granulocyte colony stimulating factor and chemokine receptor A were induced in response to IL-1β. Cell surface antigens such as IgM, MHC class I and CD20 receptor were up-regulated. Signal transduction genes such as Toll-like receptor 1 and SH3P2 were also up-regulated. The glucocorticoid receptor and cAMP early repressor were down-regulated in our microarray analysis.     

Effects of low environmental salinity on the cellular profiles and expression of Na+, K+-ATPase and Na+, K+, 2Cl? cotransporter 1 of branchial mitochondrion-rich cells in the juvenile marine fish Monodactylus argenteus

The goal of this study was to determine the osmoregulatory ability of a juvenile marine fish, silver moony (Monodactylus argenteus), for the purpose of developing a new experimental species for ecophysiological research. In this study, M. argenteus was acclimated to freshwater (FW), brackish water (BW), or seawater (SW). The salinity tolerance of this euryhaline species was effective, and the fish survived well upon osmotic challenges. The largest apical surface of mitochondrion-rich cells was found in the FW individuals. Immunohistochemical staining revealed that Na(+), K(+)-ATPase immunoreactive (NKA-IR) cells were distributed in the interlamellar region of the gill filaments of the silver moony in all experimental groups. In addition to the filaments, NKA-IR cells were also found in the lamellae of the FW individuals. The number of NKA-IR cells in the gills of the FW individuals exceeded that of the BW and SW individuals. The NKA-IR cells of FW and SW individuals exhibited bigger size than that of BW fish. The NKA activities and protein expression of the NKA α-subunit in the gills of the FW individuals were significantly higher than in the BW and SW groups. Additionally, the relative amounts of Na(+), K(+), 2Cl(-) cotransporter 1 (NKCC1) were salinity-dependent in the gills. Immunofluorescent signals of NKCC1 were localized to the basolateral membrane of NKA-IR cells in all groups. In the gills of the FW individuals, however, some NKA-IR cells did not exhibit a basolateral NKCC1 signal. In conclusion, the present study illustrated the osmoregulatory mechanisms of this easy- and economic-to-rear marine teleost with euryhaline capacity and proved the silver moony to be a good experimental animal.