Molecular characterization of microalgae used in aquaculture with biotechnology potential

Microalgae are the main component of first tropic level in aquatic food chain; it is for this reason that they are used as food in aquaculture. Also due to its biotechnological potential properties, they are used in the production of diverse components, dyes, antioxidants, enzymes, emulsifiers, etc. The extended ways of microalgae applications require physiologically and genetically stable cultures as well as correctly identified organisms to guarantee reproducibility and reliability. But the variety of species and the morphological similarity between some of them make difficult the identification of some microalgae. The use of molecular markers has supplied a very useful tool for identification of microalgae in fast mode, such as in classification. The present study has worked on the molecular characterization of main species of microalgae used in aquaculture in base of the molecular markers 18S rRNA and 16S rRNA. Microalgae DNA has been amplified and sequenced, and the resultant sequences were analyzed and reflected in phylogenetic trees. The phylogenetic analyses obtained reflect as both molecular markers allow to differentiate the main genus used in aquaculture.     

Mitochondrial NAD(P)-dependent malic enzyme from herring testicular tissue: Purification,kinetic behaviour and regulatory properties

Mitochondrial NAD(P)-dependent malic enzyme [EC 1.1.1.39, L-malate: NAD⁺ oxidoreductase (decarboxylating)] was purified from herring testicular tissue to a specific activity of 26.4 μmol NADH/min/mg protein. Herring testicular tissue is one of the most abundant sources of this enzyme. The purification procedure involved differential centrifugation of mitochondria and then chromatography on DEAE-Sephacel, Red Agarose and Sephacryl S-300. This enzyme catalyzes the oxidative decarboxylation of malate in the presence of Mn²⁺ and either NAD or NADP. Under V_max conditions the ratios for the rate of NAD/NADP reduction was 1.8. A study of the reductive carboxulation reaction indicated that this enzyme reaction is reversible; at pH 7.0 the reverse reaction exhibited 22% of the activity of forward reaction. Some kinetic characteristics of the enzyme were determined. ATP was found to be a competitive inhibitor with respect to malate. Fumarate reversed ATP inhibition. Regulation of NAD(P)-dependent malic enzyme from herring testicular tissue mitochondria could respond to changing levels of mitochondrial ATP and fumarate in vivo.     

Effects of dietary selenium on growth performance and oxidative stress in juvenile grass carp Ctenopharyngodon idellus

An 8‐week feeding trial was conducted to investigate the effect of dietary selenium (Se) on feed intake, weight gain and antioxidant activity in juvenile grass carp (11.2 ± 0.03 g). Six Se levels (0.13, 0.41, 0.56, 1.12, 2.18 and 4.31 mg/kg) of semi‐purified diets were assayed in triplicate. The maximum weight gain, specific growth rate and feed intake were obtained in fish fed with 1.12 mg Se/kg diet. Hepatic glutathione peroxidase activity was markedly increased when dietary Se ≤1.12 mg/kg diet and reached a plateau when dietary Se ≥1.12 mg/kg diet. Hepatic superoxide dismutase and serum catalase activities in juvenile grass carp fed with 0.56, 1.12 and 2.18 mg Se/kg diets were all significantly higher than those in the other groups. The malondialdehyde content in liver and serum was firstly decreased and then increased with increasing dietary Se content, and the lowest content was observed in fish fed with 1.12 mg Se/kg diet. With the increase in Se level, the activities of serum alanine aminotransferase and aspartate aminotransferase were reduced. In addition, serum alkaline phosphatase activity and albumin content were highest in fish fed with 1.12 mg Se/kg diet. This study indicated that both the Se deficiency and excess of Se caused negative effect on the oxidative stress in juvenile grass carp and suggested that the health‐giving concentration of dietary inorganic Se was 1.12 mg/kg diet. Moreover, based on the broken‐line regression analysis of weight gain, the optimal concentration of dietary inorganic Se was 0.83 mg/kg for juvenile grass carp.     

Assessing the effects of line capture and barotrauma relief procedures on post-release survival of key tropical reef fish species in Australia using recreational tagging clubs

Abstract  Common coral trout, Plectropomus leopardus Lacepède, crimson snapper, Lutjanus erythropterus Bloch, saddletail snapper, Lutjanus malabaricus (Bloch & Schneider), red emperor, Lutjanus sebae (Cuvier), redthroat emperor, Lethrinus miniatus (Schneider) and grass emperor, Lethrinus laticaudis Alleyne & Macleay, were tagged to determine the effects of barotrauma relief procedures (weighted shot-line release and venting using a hollow needle) and other factors on survival. Release condition was the most significant factor affecting the subsequent recapture rate of all species. Capture depth was significant in all species apart from L. malabaricus and L. miniatus , the general trend being reduced recapture probability with increasing capture depth. Recapture rates of fish hooked in either the lip or mouth were generally significantly higher than for those hooked in the throat or gut. Statistically significant benefit from treating fish for barotrauma was found in only L. malabaricus , but the lack of any negative effects of treating fish indicated that the practices of venting and shot-lining should not be discouraged by fisheries managers for these species.     

Inactivation of infectious pancreatic necrosis virus for vaccine use

Abstract. Formalin and β-propiolactone (BPL) were compared for efficacy in inactivating infectious pancreatic necrosis virus (IPNV) for vaccine use. Incubation with a 1:200 dilution of formalin at 20°C for four days or longer, or a 1:200 dilution of BPL at 4°C for six days or longer, completely inactivated the virus infectivity. However, whereas treatment with formalin caused only a slight reduction in anti-genicity as titrated by in vitro tests, treatment with BPL destroyed over 50% of the antigenicity of the virus. Virus inactivated with formalin also proved highly effective at inducing high titres of neutralizing antibody in trout.     

Effect of three diets on the gametogenic development and fatty acid profile of Paracentrotus lividus (Lamarck, 1816) gonads

In this study, the effects of three diets were investigated to enhance Paracentrotus lividus production for commercial purposes. P. lividus were fed ad libitum for 80 days with: diet A—fresh Codium tomentosum Stackhouse, 1797; diet B—formulated using a jellified mix of macroalgae and vegetables, including C. tomentosum (20%), Coralina sp. Linnaeus, 1758 (17%), cabbage Brassica oleracea var. capitata Linnaeus, 1753 (30%), carrot Daucus carota Linnaeus, 1753 (30%) and agar (3%) as a gelling agent. Diet C consisted of maize Zea mays Linnaeus, 1753 (56%) and New Zealand spinach Tetragonia tetragonoides (Pallas, 1781) Kuntze, 1891 (44%). Their effects on the gonadal and somatic growths, gonadosomatic index (GI) and gametogenesis were evaluated, as well as on the total lipid content and fatty acid composition of sea urchin's gonads. Diet A provided high values of eicosapentaenoic acid (EPA). Gonads of sea urchins fed with diet A were found mostly in growth and maturation stages of gametogenesis and showed the lowest lipid content. Sea urchins fed with diet B presented their gonads in the reabsorption stage and had the highest values of omega‐3 polyunsaturated fatty acids (PUFAs). Sea urchins fed with diet C were in the early stages of gametogenesis and had the highest values of lipid content, plus omega‐6 PUFAs. Once as an ingredient in a balanced mix with vegetables, C. tomentosum can be a key factor to the development of new promising high‐quality and low‐cost feed for P. lividus roe enhancement.     

Haemolymph metabolic variables in relation to eyestalk ablation and gonad development of Pacific white shrimp Litopenaeus vannamei Boone

The present study analyses some biochemical variables in haemolymph proposed as predictive indicators of the maturation capability following eyestalk ablation. Haemolymph of captive females was obtained before and 8 days after eyestalk ablation, and levels of haemocyanin, total proteins, glucose, lactate, cholesterol, triacylglycerides and vitellogenin were determined. Biochemical variables were also analysed in the hepatopancreas at the end of the experiment. Females were grouped as immature (previtellogenic stage) and mature (vitellogenic and cortical stages) based on histological analysis done 8 days after eyestalk ablation. To analyse haemolymph variables before eyestalk ablation in relation to maturation capability, immature females were classified as those with a low maturation capability and mature females as those with a high maturation capability. Females of high maturation capability had significantly higher vitellogenin levels before eyestalk ablation than females of low maturation capability. No significant differences were found for the other biochemical variables. Vitellogenin was also higher in mature than in immature females at the end of the experiment. These results indicate that vitellogenin levels in haemolymph could be used as possible predictive criteria of maturation capability, possibly because they reflect the degree of ovarian development at the time of eyestalk ablation.     

Effects of temperature and lipid droplet adherence on mortality of hatchery-reared southern hake Merluccius australis larvae

Effects of exogenous (water temperature) and endogenous (lipid droplet adherence) factors were experimentally tested on early survival of southern hake Merluccius australis reared under controlled conditions. Experiments to determine the effect of temperature (10, 12 and 14 °C) on larval growth rates and yolk-sac absorption rates of unfed southern hake were carried out under laboratory conditions. There was no significant differences in growth rates at the temperature range tested (ANCOVA, F = 0.164, p > 0.25), but yolk-sac absorption rates and mortality increased with temperature (ANCOVA, F = 53.84, p < 0.001). A high percentage (between 31 and 81%) of hake eggs showed a lipid droplet not adhered (i.e., freely moving in the yolk, and not located in the posteriormost portion of the yolk-sac). In a second experiment, fed southern hake larvae with the lipid droplet not adhered during embryonic development did not survive after yolk-sac absorption. This study provides the first data on the influence of the lipid droplet absorption on larval survival of cultured hake, and can be used as an early indication of the quality of the batch.     

Epidermal proteases of the Japanese eel

A fluorescent-sensitive assay was used to demonstrate the protease activity in the dorsal skin of Japanese eel (Anguilla japonica). Two distinct extracts were separately prepared from skin mucus and epidermal cell layers, with no mutual contamination. The epidermal extract was sensitive to various substrates, whereas there was no, or only marginal, susceptibility to the same substrates for the mucous extract. Optimum hydrolysis pHs of the epidermal extract was variable and below pH 7.0, and the optimum hydrolysis temperatures were between 40 and 50 °C. In addition, Tos-Phe-Ch₂Cl, chymostatin, CdCl₂, CuCl₂, HgCl₂ and ZnCl₂ inhibited protease activities to different extents. Several other reagents specifically affected the protease activities, and their induced effects were useful for the identification of epidermal proteases. The findings indicate that a proteolytic factor, exhibiting various enzymological specificities, is retained within epidermal cell layers of Japanese eel. This factor is composed of 4 distinct proteases, such as cathepsins L and B-like proteases, a serine protease and an aminopeptidase.     

Mycoplasma contamination in fish cell lines: an evaluation of detection by differential incorporation of 3H-uridine and 14C-uracil

Abstract. Differential incorporation of uridine and uracil was used to assay for mycoplasma contamination in five fish cell lines: bluegill fry (BF-2), chinook salmon embryo (CHSE-214), epithelioma papillosum cyprini (EPC), fathead minnow (FHM) and rainbow trout gonad (RTG-2). The method was not suitable for monitoring BF-2, CHSE-214, FHM, and RTG-2 cell lines because they incorporated uracil. Differential incorporation of uridine and uracil may be applicable for screening EPC cells because only this cell line could distinguish cultures experimentally infected with Mycoplasma orale from cultures known to be free from microbial contaminants.