Effects of exogenous non-starch polysaccharide-degrading enzymes in diets containing <Emphasis Type="Italic">Gracilaria lemaneiformis</Emphasis> on white-spotted snapper <Emphasis Type="Italic">Lutjanus stellatus</Emphasis> Akazaki

A feeding experiment was conducted to investigate the effects of exogenous non-starch polysaccharide (NSP)-degrading enzymes in diets containing Gracilaria lemaneiformis (GL) on growth performance and digestive enzyme activities of white-spotted snapper Lutjanus stellatus Akazaki (initial mass 8.0 ± 0.1 g). A basal diet (D0) containing a mixed protein source (fish meal, soybean meal and GL meal) was used as the control. Two diets supplemented with 0.5 g (D1) and 1 g (D2) exogenous NSP-degrading enzymes per kilogram of diet were formulated. Each diet was assigned to triplicate groups of fish in a total of nine floating sea cages (270 fish, 30 fish per cage). After a 60-day feeding trail, significantly higher weight gain, specific growth ratio and feed efficiency ratio were observed in fish fed D2 diet compared to those of control (P < 0.05). Body lipid, moisture and ash contents were not significantly affected by NSP-degrading enzyme supplementation, but significantly higher protein content was noticed in fish fed D2 diet compared to that of control (P < 0.05). The amylase activity in the stomach and intestine was significantly higher in fish fed D2 diet (P < 0.05), but no significant differences were observed in pepsin and lipase activities in the stomach or trypsin and lipase activities in the intestine between all treatments. The results suggested that addition of 1 g kg^?1 NSP-degrading enzymes in diet could efficiently improve seaweed feed utilization and growth performance of white-spotted snapper fish.     

Molecular cloning and expression of the heat shock protein 70 gene in the Kumamoto oyster <Emphasis Type="Italic">Crassostrea sikamea</Emphasis>

Episodes of summer mortality of the Kumamoto oyster Crassostrea sikamea are a major problem for its cultivation. Expression of the heat shock protein 70 (HSP70) is induced by various environmental stresses, including heat. We cloned and sequenced hsp70 complementary DNA from C. sikamea to investigate the relationship between hsp70 expression and heat tolerance in this oyster. Quantitative real-time polymerase chain reaction was performed using gill tissue dissected from oysters before and after heat shock for 1 h. The results showed hsp70 expression was faster and greater in oysters cultured at 20–22 °C than at 10–12 °C, and survival was lower among oysters cultured at 20–22 °C than at 10–12 °C. Moreover, heat tolerance was investigated by a 1-h pre-heat treatment, followed by exposure to heat shock conditions 5 days later. Survival was higher and hsp70 expression was notably lower in oysters that received the pre-heat treatment compared with those that did not. We conclude that a pre-heat treatment of only 1 h may be useful for inducing heat tolerance in C. sikamea, and that a low level of hsp70 expression after heat shock is an important index in selecting for high heat tolerance in these oysters.     

Isolation and characterization of <Emphasis Type="Italic">Vibrio metschnikovii</Emphasis> causing infection in farmed <Emphasis Type="Italic">Portunus trituberculatus</Emphasis> in China

In August 2008, a massive epizootic occurred among Portunus trituberculatus reared together with Palaemon carincauda Holthuis and Sinonovacula constricta in the seawater pond of Ningang farm, Rudong district, Jiangsu province, China. The disease occurred in crabs from juveniles to adults, and the mortality rate reached 30–40%. The diseased crabs exhibited lethargy, hepatopancreas turgidity, and elevated levels of turbid hemolymph. A gram-negative, rod-shaped bacterium (designated as strain LGS-1) was isolated from the ascitic fluid of the diseased and moribund crabs and was confirmed as the causative agent by our infectivity study. We conducted morphological and biochemical characterization of LGS-1, and sequenced the 16S rRNA gene, which led us to identify the bacterium as Vibrio metschnikovii. Drug sensitivity tests showed that this pathogenic bacterium is sensitive to florfenicol, orfloxacin, and SXT, but completely resistant to antibacterial drugs like gentamicin, erythrocin, and acheomycin. This is the first report on Vibrio metschnikovii as a virulent pathogen for Portunus trituberculatus.     

Antibacterial effect of medium-chain fatty acid: caprylic acid on gnotobiotic <Emphasis Type="Italic">Artemia franciscana</Emphasis> nauplii against shrimp pathogens <Emphasis Type="Italic">Vibrio harveyi</Emphasis> and <Emphasis Type="Italic">V. parahaemolyticus</Emphasis>

The present study was carried out to determine the antibacterial effect of caprylic acid in the culture system of Artemia franciscana nauplii inoculated with shrimp pathogens Vibrio harveyi and V. parahaemolyticus. To begin with, the antibacterial effect of different concentrations (1, 10 and 100 mM) of caprylic acid against V. harveyi and V. parahaemolyticus was assessed through bacterial growth study. This indicated that at 100 mM concentration, the growth of the pathogens was completely inhibited within 5 h, whereas, in 1.0 and 10 mM concentrations, effective inhibition was observed with the extension of time. Subsequently, the influence of pH variation on the growth inhibitory effect of 10 mM caprylic acid against V. harveyi and V. parahaemolyticus at different pH (5–7) was also made through bacterial growth study. At pH 5, the pathogen growth was very less, compared with pH 6 and 7. The Artemia nauplii (instar II) reared in 10 mM caprylic acid supplemented medium were challenged with V. harveyi and V. parahaemolyticus, and the mortality was recorded at an interval of 6 h up to 60 h. In this study, the mortality of Artemia nauplii reduced to 20.61 and 16.30% in V. parahaemolyticus and V. harveyi challenged groups, respectively. The present results provide evidence for the potential antibacterial activity of caprylic acid in aquaculture against luminescent vibrios.     

Epidemiological cut-off values for disc diffusion data generated by standard test protocols from <Emphasis Type="Italic">Edwardsiella tarda</Emphasis> and <Emphasis Type="Italic">Vibrio harveyi</Emphasis>

The susceptibility of 52 isolates of Edwardsiella tarda and 54 isolates of Vibrio harveyi to 12 antibiotics was established using internationally standardised disc diffusion protocols. Normalised resistance interpretation was employed to generate statistically valid epidemiological cut-off values for these susceptibility data. For three of these 24 species/antibiotic data sets, there were insufficient fully sensitive strains to allow analysis and for an additional two there were sufficient fully susceptible isolates to allow only a provisional estimate to be made. The data sets for a further two species/antibiotic combinations were considered too diverse to allow the calculation of a valid cut-off value. Valid epidemiological cut-off values were, therefore, calculated for 17 species/antibiotic combinations. It is argued that these cut-off values will greatly facilitate the performance of studies designed to monitor the consequence of antibiotic use in aquaculture. They will also facilitate the prudent and rational choice of antibiotics to be applied therapeutically in commercial farming of aquatic animals.     

Induced thermotolerance and expression of heat shock protein 70 in sea cucumber <Emphasis Type="Italic">Apostichopus japonicus</Emphasis>

ABSTRACT:   Thermal limits, induced thermotolerance and the expression of heat shock protein 70 (Hsp70) in an echinoderm Apostichopus japonicus were studied. The sublethal and lethal temperatures for the juveniles were 30 and 34°C, respectively; a previous sublethal heat shock exposure (30°C, 2 h) could increase the survival rates of the sea cucumbers when they were exposed to 34°C. This induced thermotolerance could last for at least 2 days. Levels of Hsp70 increased substantially after sublethal heat shock exposure and linearly decreased with time. This result indicated that a close relationship existed between the induction of thermotolerance and the levels of Hsp70 in A. japonicus.     

The immune modulatory effect of oregano (<Emphasis Type="Italic">Origanum vulgare</Emphasis> L.) essential oil on <Emphasis Type="Italic">Tilapia zillii</Emphasis> following intraperitoneal infection with <Emphasis Type="Italic">Vibrio anguillarum</Emphasis>

The current study aimed to evaluate the possible effect of Origanum essential oil on innate immune parameters as well as the hematological profiles of Tilapia zillii following challenge with Vibrio anguillarum. Fifty-four of Tilapia zillii weighing 180?±?10.2 g were randomly distributed into three identical closed recirculating seawater systems. The study included three groups (G1, G2, and G3) repeated in triplicates. Fish of the first two groups were fed on a basal diet without herbs, whereas fish of the last group were fed on a basal diet supplemented with Origanum essential oil at concentration 1 g kg^?1 for 15 days. Subsequently, fish of G2 and G3 subjected to a peritoneal inflammation by intraperitoneally injecting V. anguillarum (5.5?×?10⁵ CFU mL^?1), whereas fish of G1 injected with saline and served as control. Fish of all groups were then sampled at 4, 12, and 24 h post injection. No mortalities were observed in both basal and Origanum fed groups. However, some specimens of fish fed basal diet showed dorsal fin erosions, eroded mouth, and detached skin. Although the kinetics of RBCs, WBCs, Hb, and differential leukocyte values remained unchanged in fish fed different diets at the beginning of the trial, significant increases in those values were observed in fish fed Origanum essential oil at 12 and 24 h post injection. Similarly, an augmentation of plasma proteases, antiproteases, and lysozyme activities were recorded in fish fed Origanum essential oil at the same particular sampling points. A significant enhancement in plasma bactericidal capacity was only recorded in fish fed Origanum essential oil at 12 and 24 h post challenge compared to those fed basal diet. In conclusion, Origanum essential oil had a pronounced influence on the innate immunity and increased the fish resistance to V. anguillarum. These data gave insight into the potential use of Origanum in prophylactic strategies against threatening pathogens.     

Study of growth and body composition of red snapper <Emphasis Type="Italic">Lutjanus erythropterus</Emphasis> fed diets containing <Emphasis Type="Italic">Escherichia coli</Emphasis> expressing recombinant tilapia insulin-like growth factor-I

ABSTRACT:   To examine the effects of insulin-like growth factor (IGF)-I on the growth performance of fish, juvenile red snapper Lutjanus erythropterus with an initial body weight of approximately 12 g were fed diets containing different amounts of IGF-I containing Escherichia coli BL21 powder (BL21-tIGF-I). The growth of fish was enhanced by lower doses (0.25, 0.5 and 1%), but deteriorated at higher amounts (2.5 and 5%). The best results were obtained with 0.5% BL21-tIGF-I supplementation, which resulted in 154 and 85% weight gain and feed efficiency, respectively. In addition, muscle proteins appeared to be elevated, but muscle lipids were reduced except for in the 1% BL21-tIGF-I group. In contrast, liver lipids were reduced at 0.25, 0.5 and 1%, but increased at 2.5 and 5% BL21-tIGF-I. Furthermore, to examine the effects of BL21-tIGF-I on later-stage red snapper, we selected fish of approximately 23–24 g weight and fed them a diet either with or without 0.5% BL21-tIGF-I for 12 weeks. Results showed that the growth performance of later-stage fish was better with BL21-tIGF-I than those without supplementation after 6 weeks of feeding. The average body weight of fish that did not receive BL21-tIGF-I during the first 6 weeks, but were fed BL21-tIGF-I in weeks 7–12, was significantly higher than those fed without BL21-tIGF-I for the entire experiment. These results suggest that a continuous supply of BL21-tIGF-I may be beneficial for the growth performance of red snapper.     

Spatial learning-induced <Emphasis Type="Italic">egr</Emphasis>-<Emphasis Type="Italic">1</Emphasis> expression in telencephalon of gold fish <Emphasis Type="Italic">Carassius auratus</Emphasis>

The immediate-early gene (egr-1) expression was used to examine the neuron’s response in telencephalon of goldfish during spatial learning in small space. Fishes were pre-exposed in the experimental apparatus and trained to pick food from the tray in a rectangular-shaped arena. The apparatus was divided into identical compartments comprising three gates to provide different spatial tasks. After the fish learned to pass through the gate one, two more gates were introduced one by one. Fish made more number of attempts and took longer time (P < 0.05) to pass through the first gate than the gate two or three. This active learning induces the expression of egr-1 in telencephalon as established by western blot analysis. Subsequently, the fish learn quickly to cross the similar type of second and third gate and make fewer errors with a corresponding decline in the level of egr-1 expression. As the fish learned to pass through all the three gates, third gate was replaced by modified gate three. Interestingly, the level of egr-1 expression increased again, when the fish exhibit a high exploratory behavior to cross the modified gate three. The present study shows that egr-1 expression is induced in the telencephalon of goldfish while intensively acquiring geometric spatial information to pass through the gates.     

Molecular characterization and expression analysis of heat shock proteins 40, 70 and 90 from kuruma shrimp <Emphasis Type="Italic">Marsupenaeus japonicus</Emphasis>

Heat shock proteins (HSPs) are proteins that are expressed more strongly when the cells are exposed to physiological and stressful conditions. In this study, the full-length cDNAs of heat shock proteins 40 (MjHSP40), 70 (MjHSP70) and 90 (MjHSP90) were cloned from kuruma shrimp Marsupenaeus japonicus. The open reading frames (ORFs) of the cDNA clones have lengths of 1,191, 1,959 and 2,172 bp and encode 396, 652 and 723 amino acid residues, respectively. The predicted MjHSP40 amino acid sequence contains a J domain, a glycine/phenylalanine-rich region, and a central domain containing four repeats of a CxxCxGxG motif, indicating that it is a type I HSP40 homolog. The signature sequences of the HSP70 and HSP90 gene families are conserved in the MjHSP70 and MjHSP90 amino acid sequences. The deduced amino acid sequences of MjHSP70 and MjHSP90 share high identity with previously reported shrimp HSP70s and HSP90s, respectively. The expression of MjHSP90 mRNA increased at 32°C. Additionally, the expressions of MjHSP40, MjHSP70 and MjHSP90 mRNAs increased in defense-related tissues (i.e., hemocytes and lymphoid organ) when the shrimp were challenged with white spot syndrome virus.     

Cloning and characterization of <Emphasis Type="Italic">Bax1</Emphasis> and <Emphasis Type="Italic">Bax2</Emphasis> genes of <Emphasis Type="Italic">Ctenopharyngodon idellus</Emphasis> and evaluation of transcript expression in response to grass carp reovirus infection

Multidomain proapoptotic Bcl-2-associated X (Bax) protein is an essential effector responsible for mitochondrial outer membrane permeabilization, resulting in cell death via apoptosis. In this study, two Bax genes of grass carp (Ctenopharyngodon idellus), designated as CiBax1 and CiBax2, were isolated and analyzed. The obtained CiBax1 cDNA is 2058 bp long, with a 579 bp open reading frame (ORF) coding a protein of 192 amino acid residues. The full-length cDNA of CiBax2 is 1161 bp, with a 618 bp ORF coding 205 amino acids. Both CiBax1 and CiBax2 are typical members of Bcl-2 family containing conserved Bcl and C-terminal domains, and they share conserved synteny with zebrafish Bax genes despite the grass carp Bax mapping to different linkage groups. Phylogenetic analysis showed that CiBax1 was clustered with Bax from most teleost fish, and CiBax2 was close to Bax2 from teleost fish but far separated from that of Salmo salar. Quantitative real-time PCR analysis revealed broad expression of CiBax1 and CiBax2 in tissues from healthy grass carp, but the relative expression level differed. The mRNA expression of CiBax1 and CiBax2 was both upregulated significantly and peaked in all examined tissues at days 5 or 6 post-infection with grass carp reovirus. Subcellular localization indicated that CiBax1 protein was localized in both nucleus and cytosol, while CiBax2 protein only in cytosol. Moreover, CiBax2, but not CiBax1 was colocalized with mitochondrion under normal condition. Taken together, the findings would be helpful for further understanding of the function of Bax in teleost fish.     

Dietary supplementation of curcumin augments heat stress tolerance through upregulation of <Emphasis Type="Italic">nrf-2</Emphasis>-mediated antioxidative enzymes and <Emphasis Type="Italic">hsps</Emphasis> in <Emphasis Type="Italic">Puntius sophore</Emphasis>

Heat stress is one of the major environmental concerns in global warming regime and rising temperature has resulted in mass mortalities of animals including fishes. Therefore, strategies for high temperature stress tolerance and ameliorating the effects of heat stress are being looked for. In an earlier study, we reported that Nrf-2 (nuclear factor E2-related factor 2) mediated upregulation of antioxidative enzymes and heat shock proteins (Hsps) provide survivability to fish under heat stress. In this study, we have evaluated the ameliorative potential of dietary curcumin, a potential Nrf-2 inducer in heat stressed cyprinid Puntius sophore. Fishes were fed with diet supplemented with 0.5, 1.0, and 1.5% curcumin at the rate 2% of body weight daily in three separate groups (n = 40 in each group) for 60 days. Fishes fed with basal diet (without curcumin) served as the control (n = 40). Critical thermal maxima (CTmax) was determined for all the groups (n = 10, in duplicates) after the feeding trial. Significant increase in the CTmax was observed in the group fed with 1.5% curcumin- supplemented fishes whereas it remained similar in groups fed with 0.5%, and 1% curcumin-supplemented diet, as compared to control. To understand the molecular mechanism of elevated thermotolerance in the 1.5% curcumin supplemented group, fishes were given a sub-lethal heat shock treatment (36 °C) for 6 h and expression analysis of nrf-2, keap-1, sod, catalase, gpx, and hsp27, hsp60, hsp70, hsp90, and hsp110 was carried out using RT-PCR. In the gill, expression of nrf-2, sod, catalase, gpx, and hsp60, hsp70, hsp90, and hsp110 was found to be elevated in the 1.5% curcumin-fed heat-shocked group compared to control and the basal diet-fed, heat-shocked fishes. Similarly, in the liver, upregulation in expression of nrf-2, sod, catalase, and hsp70 and hsp110 was observed in 1.5% curcumin supplemented and heat shocked group. Thus, this study showed that supplementation of curcumin augments tolerance to high temperature stress in P. sophore that could be attributed to nrf-2-induced upregulation of antioxidative enzymes sod, catalase, gpx, and the hsps.     

Potential of cinnamon (<Emphasis Type="Italic">Cinnamomum verum</Emphasis>) oil to control <Emphasis Type="Italic">Streptococcus iniae</Emphasis> infection in tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>)

In this study, four essential oils—cinnamon oil, leech lime oil, lemongrass oil, and turmeric oil—were examined for their antimicrobial activities against Streptococcus iniae, a bacterium that is pathogenic in fish, in which it causes streptococcosis. Cinnamon oil was the most potent antimicrobial agent among these oils, with a minimal inhibitory concentration (MIC) of 40 μg/ml. By using gas chromatography–mass spectrometry (GC–MS), it was found that the major components of cinnamon oil were cinnamaldehyde (90.24), limonene (2.42%), cinnamyl acetate (2.03%), linalool (1.16%), and α-terpineol (0.87%). Of these compounds, only cinnamaldehyde exhibited antimicrobial activity against S. iniae, with an MIC of 20 μg/ml. In an in vivo trial, no mortality was apparent in fish fed on fish diets supplemented with 0.4% (w/w) of cinnamon oil and with 0.1% (w/w) of oxytetracycline 5 days prior to infection with S. iniae. These results indicate that cinnamon oil had a protective effect on experimental S. iniae infection in tilapia, and thus has the potential to replace the antibiotics used to control this disease.     

Nutrition and feeding research in the spotted rose snapper (<Emphasis Type="Italic">Lutjanus guttatus</Emphasis>) and bullseye puffer (<Emphasis Type="Italic">Sphoeroides annulatus</Emphasis>), new species for marine aquaculture

The spotted rose snapper (Lutjanus guttatus) and bullseye puffer (Sphoeroides annulatus) are fish species from the tropical Eastern Pacific for which controlled production of larvae and juveniles has been accomplished in recent years. Diverse topics relating to their biology and aquaculture production are currently under study, in particular the nutrition and feeding aspects required to formulate practical feeds and rearing protocols. Improvements in larval growth and survival are possible by feeding live food organisms with natural or enhanced essential fatty acids content and highly digestible artificial microdiets. The ontogeny of the digestive tract and the expression and activity of digestive enzymes have been described for S. annulatus larvae. The effect of various protein and lipid levels on growth and feed utilization has been studied in juvenile and on-growing fish. Both species have carnivorous feeding habits and require high levels of protein in their diets, from 40% to 45% (dry weight) in spotted rose snapper and above 50% in bullseye puffer, with the younger stages requiring the highest protein levels. Encouraging results have been obtained in feeding experiments with different sources of dietary protein from animal and plant origin to evaluate their suitability as feed ingredients in practical diets. Optimization of fish culture practices through feeding management has also been investigated. Trials with various fish densities and feeding frequencies in intensive culture systems are providing information to improve feed utilization and growth in on-growing fish. Further research is underway to evaluate factors in broodstock nutrition which have an impact on egg and larval quality, and into the use of various commercially available oil sources in on-growing diets. In this paper, the results on nutrition and feeding research with both species are reviewed and research needs to support their commercial production in the region are discussed.     

Molecular cloning and expression analysis of <Emphasis Type="Italic">Megalobrama amblycephala</Emphasis> transferrin gene and effects of exposure to iron and infection with <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis>

Transferrin (Tf) plays an important function in iron homeostasis and metabolism of organisms. In this study, we identified and characterized the Tf gene in Megalobrama amblycephala and evaluated its expression in basal conditions as well as after iron overload and experimental infection with Aeromonas hydrophila. Furthermore, we studied the iron binding properties of recombinant Tf. The full-length M. amblycephala Tf complementary DNA (cDNA) (GenBank accession no.: KX698308) of 2245 bp was cloned and contained a 1953 bp open reading frame (ORF) encoding 650 amino acid residues and flanked by a 68 bp 5′ and a 204 bp 3′ untranslated regions (UTR). Predicted conservative structure illustrated that M. amblycephala Tf consisted of two conservative Tf domains. Amino acid sequence alignment revealed that M. amblycephala Tf had high similarity with that of cyprinids deposited in Genbank, and phylogenetic analysis showed that M. amblycephala Tf clustered with Ctenopharyngodon idella and Hypophthalmichthys molitrix. Tissue expression pattern analyses demonstrated that the liver was the main Tf mRNA expressing organ, being significantly higher than other tissues (p < 0.05). In the liver, Tf mRNA expression in fish artificially injected with the pathogenic bacteria A. hydrophila was significantly upregulated, reaching a peak at 12 h post injection (hpi) and then decreasing afterward. The expression in FeCl₃-injected fish showed a similar tendency, but reached a peak at 8 hpi. Meanwhile, fish serum iron significantly decreased following A. hydrophila injection, but increased to peak at 4 hpi and then decreased in FeCl₃-injected fish. The recombinant M. amblycephala Tf showed iron binding capacity using CAS analysis. These results are helpful to understand the structure and regulation of expression of Tf, as well as the specific function of Tf for both immune responses and iron homeostasis.