Purinergic signalling displays an anti‐inflammatory profile in the spleen of fish experimentally infected with Aeromonas caviae: Modulation of the immune response

Extracellular adenosine triphosphate (ATP) and its metabolite adenosine (Ado) are recognized as key mediators of immune and inflammatory responses. Depending on its concentration, ATP may act as an immunostimulant or immunodepressant, while Ado levels display an anti‐inflammatory profile. The aim of this study was to evaluate whether splenic purinergic signalling is capable of modulating immune and inflammatory responses in fish experimentally infected with Aeromonas caviae. Triphosphate diphosphohydrolase (NTPDase) and 5′‐nucleotidase activities increased in the spleen of silver catfish (Rhamdia quelen) experimentally infected with A. caviae compared with the uninfected control group. Moreover, splenic Ado levels increased in the infected animals relative to the uninfected control group. Based on these lines of evidence, our findings revealed that adenine nucleotide hydrolysis is modified in the spleen of fish infected with A. caviae attempting to restrict the inflammatory process through the upregulation of NTPDase and 5′‐nucleotidase activities, which occurs in an attempt to hydrolyse the excessive ATP in the extracellular environment and rapidly hydrolyse AMP to form Ado. In summary, purinergic signalling can modulate immune and inflammatory responses during A. caviae infection.     

Survival of Diploid and Triploid Rhamdia quelen Juveniles Under Different Oxygen Concentrations

The objective of this study was to determinate the lethal concentration of dissolved oxygen (DO) over 96 hours of exposure (LC50–96h) for diploid and triploid jundia Rhamdia quelen juveniles. Diploid and triploid fish weighing approximately 4 g were subjected to DO concentrations varying between 0.4 and 1.3 mg O₂ L^?1; water temperature was maintained at 27?°C and pH at 6.3. The LC50–96h for diploids of Rhamdia quelen was 0.535 mg O₂ L^?1, while the value obtained for triploids was 6% greater. These results demonstrated that triploids of Rhamdia quelen juveniles have greater sensitivity to hypoxia compared to diploids.     

Changes in the cerebral phosphotransfer network impair energetic homeostasis in an aflatoxin B<Subscript>1</Subscript>-contaminated diet

The phosphotransfer network system, through the enzymes creatine kinase (CK), adenylate kinase (AK), and pyruvate kinase (PK), contributes to efficient intracellular energetic communication between cellular adenosine triphosphate (ATP) consumption and production in tissues with high energetic demand, such as cerebral tissue. Thus, the aim of this study was to evaluate whether aflatoxin B₁ (AFB₁) intoxication in diet negatively affects the cerebral phosphotransfer network related to impairment of cerebral ATP levels in silver catfish (Rhamdia quelen). Brain cytosolic CK activity decreased in animals fed with a diet contaminated with AFB₁ on days 14 and 21 post-feeding, while mitochondrial CK activity increased, when compared to the control group (basal diet). Also, cerebral AK and PK activity decreased in animals fed with a diet contaminated with AFB₁ on days 14 and 21 post-feeding, similarly to the results observed for cerebral ATP levels. Based on this evidence, inhibition of cerebral cytosolic CK activity is compensated by stimulation of mitochondrial CK activity in an attempt to prevent impairment of communication between sites of ATP generation and ATP utilization. The inhibition of cerebral AK and PK activity leads to impairment of cerebral energy homeostasis, decreasing the brain’s ATP availability. Moreover, the absence of a reciprocal compensatory mechanism between these enzymes contributes to cerebral energetic imbalance, which may contribute to disease pathophysiology.     

Changes in Biochemical Compositions and Quality of White Shrimp (Litopenaeus vannamei) During Storage

ABSTRACT

To understand biochemical characteristics, storage stability, and freshness indicators of white shrimp (Litopenaeus vannamei), changes in extractable nitrogenous compounds, microbial count, and sensory rating of white shrimp during storage at 25 and 4°C were investigated. Free amino acids showed a slow increase during storage at 25°C, but no obvious change was found at 4°C. Adenosine triphosphate (ATP) and adenosine diphosphate (ADP) were found at initial stage and decreased rapidly after storage. Both inosine 5′-monophosphate (IMP) and adenosine monophosphate (AMP) increased and then decreased during storage. Inosine, hypoxanthine, and the K-value gradually increased with time. The levels of total volatile basic nitrogen (TVB-N), NH₃, and trimethylamine (TMA) of white shrimp increased with storage time at 25 and 4°C. The TVB-N, NH₃, TMA, inosine, hypoxanthine, and K-value could be considered as freshness indicators of white shrimp during storage. However, the total plate count did not corroborate the acceptability recommended limits for white shrimp during storage. The sensory evaluation, associated with TVB-N, TMA, and K-value, showed the quality was unacceptable after 6 h storage at 25°C and 7 days at 4°C.     

Tilapia protein hydrolyzate improves growth performance,protein absorption and antioxidant status in Silver catfish (Rhamdia quelen)

This study evaluated the substitution of fish meal with tilapia protein hydrolyzate (TPH) in diets for Silver catfish (Rhamdia quelen) by verifying the effects on growth, body composition, blood and biochemical parameters, and biomarkers of oxidative stress. Five isoproteic and isoenergetic diets (37% crude protein, 3,800 kcal/kg) were produced with different substitution levels of TPH (0%, 5%, 10%, 15% and 20%) for fishmeal. The hydrolyzate was obtained by enzymatic hydrolysis from carcasses of Nile tilapia (Oreochromis niloticus), which were exposed to swine pepsin enzyme at 36°C for four hours. The experimental design was completely randomised with five treatments and four replications. The inclusion of 20% TPH in diets for silver catfish increased the growth of Silver catfish juveniles. At levels above 5%, there was an increase in body protein deposition and the efficiency of oxidative metabolism.     

The parameters of energetic status of the wild Atlantic salmon (Salmo salar L.) spermatozoa

The energetic status of fresh sperm of wild Atlantic salmon (Salmo salar L.) was presented in selected males (n=10) with motility rate ≥80%. Purine and pyridine nucleotides: adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), guanosine triphosphate, guanosine diphosphate, guanosine monophosphate, inosine monophosphate, nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate; nucleosides: adenosine, guanosine and inosine; bases: hypoxanthine, xanthine and uric acid were determined using the high‐performance liquid chromatography method. The average value of adenylate energy charge in the group was 0.77 ± 0.10, and the mean total adenine nucleotide content (TAN) was 65.1 ± 18.3 pmol × 10^?6 spermatozoa. The mean concentrations of ATP, ADP and AMP were 43.5, 11.8 and 9.9 pmol × 10^?6 spermatozoa respectively. The concentrations of the other energetic nucleotides studied were lower. In all males studied (n=23) with a motility rate from 0% to 95%, no statistically significant correlation between the per cent of motile sperm and ATP concentration was found (R_s=0.35), whereas the correlation between the per cent of motile sperm and ADP was statistically significant (R_s=0.60). A negative correlation was found between hypoxanthine and the per cent of motile spermatozoa (R_s=?0.44). Our results suggest that hypoxanthine is the final product of salmon spermatozoa of adenine nucleotide catabolism.     

AMP-deaminase from goldfish white muscle: regulatory properties and redistribution under exposure to high environmental oxygen level

AMP-deaminase was partially purified from white skeletal muscle of goldfish, Carassius auratus. The enzyme was highly stable, showing virtually no change in activity at 1 month following the purification process when stored in 1 M KCl at 2–4°C. The specific activity of the purified enzyme was 130–150 U/mg protein, with a pH optimum of about pH 6.5. AMP-aminohydrolase (AMPD) showed non-Michaelis–Menten kinetics, with a S_0.5 (half saturation by the substrate) for AMP of 0.73 ± 0.03 mM, a Hill coefficient of 2.01 ± 0.26, and a V _max (maximum velocity) of 176 ± 46 U/mg protein. Both sodium and potassium ions activated goldfish AMPD at low concentrations, with maximal activation at about 80 mM of each chloride salt, whereas higher concentrations became inhibitory. Magnesium and calcium ions also inhibited goldfish muscle AMPD, as did phosphate and fluoride; at a concentration of 8 mM, each anion reduced activity by about 66%. ADP and ATP were strong activators and both demonstrated concentration-dependent activation, with maximal effects at 0.5–1.5 mM. Fish exposure to a high concentration of oxygen (18–20 mg/l against 5–6 mg/l in the control) and recovery to the initial level induced a redistribution of AMPD between free and bound forms in goldfish white muscle and brain in a tissue-dependent manner. A spatial–temporal redistribution may be among the mechanisms regulating enzyme operation in vivo. Possible regulatory mechanisms of AMP-deaminase function in fish muscle are discussed.     

Survival and growth of silver catfish larvae exposed to different water pH

The objective of this study was to determine the influence ofwater pH (5.5, 6.0, 7.0, 8.0, and 8.5) on survival and growth ofsilver catfsh (Rhamdia quelen) larvae. Larvae were obtained frominduced spawning in November and maintained under controlledconditions of 25 (1) °C using a water re-use system incontinuously aerated 40 L polyethylene tanks (350 larvae/tank).Larvae were fed until satiation six times a day. At days 0, 7, 14,and 21 and after yolk absorption, 10 larvae from each group wererandomly chosen to evaluate length, weight, and specific growthrate (SGR). Length, weight, survival, and biomass at the end of 21days were significantly higher at pH 8.0 and 8.5. As expected,specific growth rate decreased with time. These results suggest that 8.0–8.5 isthe best pH range for survival and growth of the larvae of thisspecies.     

Aeromonas caviae inhibits hepatic enzymes of the phosphotransfer network in experimentally infected silver catfish: Impairment on bioenergetics

Several studies have been demonstrated that phosphotransfer network, through the adenylate kinase (AK) and pyruvate kinase (PK) activities, allows for new perspectives leading to understanding of disease conditions associated with disturbances in energy metabolism, metabolic monitoring and signalling. In this sense, the aim of this study was to evaluate whether experimental infection by Aeromonas caviae alters hepatic AK and PK activities of silver catfish Rhamdia quelen. Hepatic AK and PK activities decreased in infected animals compared to uninfected animals, as well as the hepatic adenosine triphosphate (ATP) levels. Also, a severe hepatic damage was observed in the infected animals due to the presence of dilation and congestion of vessels, degeneration of hepatocytes and loss of liver parenchyma architecture and sinusoidal structure. Therefore, we have demonstrated, for the first time, that experimental infection by A. caviae inhibits key enzymes linked to the communication between sites of ATP generation and ATP utilization. Moreover, the absence of a reciprocal compensatory mechanism between these enzymes contributes directly to hepatic damage and for a severe energetic imbalance, which may contribute to disease pathophysiology.     

Cellular responses to temperature stress in steelhead trout (Onchorynchus mykiss) parr with different rearing histories

This study compared the heat-shock response and metabolic energy status in hatchery-raised and two groups of wild-caught steelhead trout parr collected from the Navarro River watershed, California. Wild parr were from coastal and inland sites with different thermal regimes. Fish were exposed in the laboratory to 25 ± 0.2°C for 2-h and the heat-shock response was assessed in muscle tissue via induction of heat-shock proteins (hsps) 63, 72, 78, and 89. Metabolic measurements included muscle phosphocreatine (PCr), ATP, ADP, and AMP, and hepatic glycogen. Inland and coastal fish overlapped considerably with regard to their hsp responses and energetic endpoints, but hatchery fish were distinct in the biochemical patterns they exhibited. Hsp expression levels after temperature shock were significantly lower in hatchery than in wild fish. Hatchery fish also had significantly lower hepatic glycogen and higher muscle ADP, ATP, and PCr concentrations than wild fish. Coastal and inland steelhead did not differ significantly with regard to peak hsp72 and hsp89 levels or to concentrations of energy metabolites. However, fish from the warm-water, inland site expressed significantly less hsp63, maintained higher basal levels of hsp72, and induced hsp89 more slowly than fish from the cold-water, coastal site. Discriminant function analysis revealed that hatchery fish can be distinguished from wild Navarro River fish with 84.9% certainty using the following function: f(x) = − 43.6 + 0.14(Gly) + 4.1(PCr) + 186.4(AMP) + 80.8(ADP) − 0.14(hsp63) + 0.005(hsp72). This study demonstrates that within a single species, rearing conditions or genetic variation can influence an organism’s disposition and cellular response to thermal stress. Extrapolation of results from laboratory studies on hatchery fish to wild fish may therefore not be possible, and caution must be used when interpreting hsp data obtained for wild fish with different thermal histories.     

Purification and characterization of a cysteine-like protease from the body wall of the sea cucumber <Emphasis Type="Italic">Stichopus japonicus</Emphasis>

The sea cucumber (Stichopus japonicus) is able to undergo autolysis in response to a variety of environmental and mechanical cues. Within the framework of a long-term study of this phenomenon we have purified a protease from the body wall of the sea cucumber by means of ion-exchange chromatography with DE-52 cellulose and gel filtration chromatography with Sephadex G-100. The final enzyme preparation was nearly homogeneous on polyacrylamide gel electrophoresis, and its molecular weight was estimated to be approximately 35.5 kDa. The purified enzyme exhibited a maximum activity for the hydrolysis of casein at pH 7.0 and 50°C and a remarkable stability at pH 4.0–7.0 and 40–60°C. Based on the inhibition and activation profiles obtained with numerous specific protease inhibitors and an activator, the protease purified from the body wall of the sea cucumber was defined to be a cysteine-like protease.     

High water-temperature tolerance in photosynthetic activity of <Emphasis Type="Italic">Zostera marina</Emphasis> seedlings from Ise Bay,Mie Prefecture,central Japan

Photosynthetic activities of seedlings of Zostera marina were successively measured using a gas volumeter for 6 days at seven light (0–400 μmol photons/m² per s) and 11 water temperature conditions (5–35°C). The seedlings were collected from mature plants (Ise Bay, central Japan), and stored and cultured in incubators accurately controlled at each test temperature. The maximum gross photosynthesis (P _maxg) was recorded at an optimal water temperature of 29°C after 0 days. After 6 days, P _maxg appeared at 25°C and most plants cultured at 29–30°C bleached and withered after the drastic increase of light compensation point (I _c). On the contrary, at 5–28°C, the photosynthetic activities either changed little (5–25°C) or recovered after a temporal reduction (26–28°C); seedlings survived and looked healthy after being cultured for 6 days. The recovery was thought to be an acclimation to tolerate higher water temperature. As a result, the critical upper water temperature for Z. marina seedlings was proposed as 28°C. The temperature was consistent with the previously reported maximum water temperature in habitats around the southern boundary of Z. marina in the northern hemisphere.     

DNA barcoding reveals blend of silver catfish Rhamdia species from fish farms in Southern Brazil

Rhamdia quelen is the most produced native freshwater fish in fish farms in South Brazil. Until recently, Rhamdia branneri and Rhamdia voulezi were synonyms of R. quelen, and all the species are commercialized as silver catfish (locally called jundiá or bagre sapo) by the aquaculture industry. In fact, because these species are morphologically very similar, interspecific crosses easily might occur in fish farming. We employed standard DNA barcoding to identify jundiá molecular operational taxonomic units (MOTUs) in fish cultivated and commercialized in the industry and in possible escapees in the natural environment in southern Brazil. We analysed 48 individuals from six fish farms and 48 individuals from three rivers (Uruguay, Benedito Novo and Itapocu Rivers) using the mitochondrial cytochrome oxidase subunit I (COI). Four MOTUs were identified based on the estimated optimum threshold (OT = 0.77), and these MOTUs were concordant with Bayesian Inferece (BI) and Neighbour‐Joining (NJ) trees. Our results support the existence of at least three species in our dataset: R. branneri, R. voulezi, and R. quelen 1 and R. quelen 2. The interspecific genetic divergence ranged from 1.1% to 5.1% (mean = 3.5%), and the intraspecific distance ranged from 0% to 1.4% (mean = 0.24%). The presence of cultivated fish in the Uruguay and Benedito Novo Rivers provides evidence of genetic contamination in native populations. These results show the need to regulate aquaculture activities and to characterize the species and commercial lineages of silver catfish that are cultivated in South Brazil.     

The effect of temperature on the energy budget of the Manila clam, <Emphasis Type="Italic">Ruditapes philippinarum</Emphasis>

In this study, the energy budget of the Manila clam, Ruditapes philippinarum, was evaluated after one-week acclimation periods at 5, 10, 15, 20, and 25°C. Small clams (151 ± 12 mg DW) and large clams (353 ± 16 mg DW) were fed with the microalgae, Isochrysis galbana. Filtration rate, ingestion rate, assimilation efficiency, oxygen-consumption rate, and ammonia excretion rate were measured. Both filtration rate and ingestion rate of small and large clams were found to be related to temperature. The highest Q ₁₀ values were measured in the range 15–20°C for both small and large clams. Assimilation efficiency of both small and large clams was not significantly influenced by temperature, although the maximum mean values were detected at 20°C. Oxygen consumption rate and ammonia excretion rate of small and large clams were found to be related directly to temperature over the entire range, with a maximum being detected at 25°C. The highest Q ₁₀ value was estimated in the range 10–15°C with regard to oxygen consumption rate, and in the range of 15–20°C with regard to ammonia excretion rate. Scope for growth (SFG) was positive at all temperatures, achieving a maximum value at 20°C in both small and large clams, primarily as a consequence of the enhanced ingestion rate which offset the concomitant elevation in the metabolic rate. In this study we have estimated the thermal optimum for this species at 20°C.     

Biochemical and pharmacological characterization of adenosine A1 receptors in eel (Anguilla anguilla) brain

N⁶-cyclohexyl[³H]adenosine ([³H]CHA) was used to label adenosine A1 receptors in membranes prepared from male and female eel whole brain. The A1 receptor agonist [³H]CHA bound saturably, reversibly and with high affinity (K_d = 0.91 ± 0.12 nM; B_max = 120.36 ± 5.2 fmol mg⁻¹ protein). In equilibrium competition experiments, the adenosine agonists and antagonists all displaced [³H]CHA from high-affinity binding sites with the rank order of potency in displacing, characteristics of an A1 adenosine receptor. Mg²⁺ dramatically increased the affinity of [³H]CHA without modifying the maximal binding capacity. The specific binding was inhibited by guanosine 5′-triphosphate (K_i = 2.54 ± 0.98 μM). The [³H]CHA binding sites are ubiquitously distributed with a maximum in cerebellum and a minimum in olfactory bulb. No difference was observed between male and female brain. In eel brain, synaptosomes (P2), stimulation of adenosine 3′,5′-monophosphate (cyclic AMP) accumulation with 10⁻⁵ M forskolin was markedly reduced (45.5%) by treatment with the adenosine A1 receptor agonist CHA (10⁻⁴ M), and the reduction was reversed in presence of the selective A1 receptor antagonist 8-cyclopentyltheophylline (10⁻⁵ M). In superfused eel cerebellar synaptosomes, K⁺ stimulated the release of adenosine in a partially Ca²⁺-dependent manner. The findings, taken together, suggest the hypothesis that adenosine A1 receptors present in eel brain could modulate synaptic transmission, as A1 receptors do in other vertebrates.     

Hematologic and serum biochemical values for jundiá (<Emphasis Type="Italic">Rhamdia quelen</Emphasis>)

The determination of hematologic parameters for jundiá Rhamdia quelencultivated in ponds is an easy and useful tool for the control of their sanitary and nutritional conditions. In the present work hematologic indices (RBC, WBC, Ht, Hb, MCV, MCH, MCHC), electrolytes (Cl, Na, K, Ca, Mg, P), metabolic products (creatinine, urea, ammonia, glucose, total protein, triglycerides, cholesterol, bilirubin) and enzymes (alanine and aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase) were measured in a population of jundiá. The mean ± SEM and range were established.     

Plasma levels of cortisol and glucose in response to capture and tank transference in Rhamdia quelen (Quoy & Gaimard), a South American catfish

Males and females of Rhamdia quelen (Quoy & Gaimard) were subjected to capture and tank transference in order to evaluate the stress response. This process provoked a characteristic stress response in both sexes, with cortisol values reaching a peak in one hour after stress. High levels of cortisol found in females were explained by the increase of energy demand and the mobilization in the vitellogenic phase. Several previous studies have shown that cortisol is strongly correlated to these periods in another catfish.     

Temperature characteristics in seed germination and growth of <Emphasis Type="Italic">Zostera japonica</Emphasis> Ascherson &; Graebner from Ago Bay,Mie Prefecture,central Japan

The optimal water temperature in seed germination and the upper critical water temperature in seedling growth were determined for Zostera japonica collected from Ago Bay, Japan. The relationship between the seed germination rates and seed storage period (0, 30, and 60 days) at 0°C was also examined. The optimal water temperature in seed germination was in the range 15–20°C regardless of the storage period, in which germination rates were up to 14%. Seedlings, grown from seeds up to 10 cm in total length, were cultured for 1 week at various water temperatures to measure their relative growth rates. The optimal water temperature in early growth was in the range 20–25°C; relative growth rates ranged from 3.8 to 4.2%. Seedlings could survive up to a water temperature of 29°C, but most seedlings withered at 30 or 35°C. The optimal water temperatures for seed germination and seedling growth were related to the seasonal changes of water temperature in the sampling site. Although seedlings were hardly observed in Ago Bay in summer, Z. japonica might extend its distribution as far as where the summer water temperature is lower than 29°C.