Dietary supplementation of yeast (<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>) improves growth,stress tolerance,and disease resistance in juvenile Nile tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>)

The yeast Saccharomyces cerevisiae is one of the commonest probiotics incorporated in aquafeeds. An 84-day feeding trial was conducted to evaluate the effects of varying dietary inclusions of S. cerevisiae, 0% (control), 3% (YF3), 5% (YF5), and 7% (YF7), on growth, stress tolerance, and disease resistance in juvenile (body mass ~?21 g) Nile tilapia (Oreochromis niloticus). Fish were randomly distributed in groups of 20 into 12 1-m³ hapas and fed isoenergetic (~?17 kJ g^?1 gross energy) and isonitrogenous (~?300 g kg^?1 crude protein) diets at 3% of their bulk weight daily. Specific growth rates were significantly higher for the yeast-fed fish (0.77–0.78% day^?1) than for the control fish (0.60% day^?1) and resulted in significantly higher mean final weights for the yeast-treated groups. Protein and lipid retention efficiencies were also significantly higher in the yeast-fed fish than in the control group. In subsequent stress challenge trials, the yeast-fed fish had greater tolerance to acute heat as well as hypoxia exposure than the control fish. Survival rates of the yeast-treated groups following sudden exposure to elevated water temperature (40 °C) ranged from 82.5 to 100% compared to 15% for the control. Mean survival rates following a 24-h hypoxia exposure were also generally significantly higher for the yeast-fed fish. The probiotic groups recorded a relative percent survival (RPS) of 75% after a 14-day Aeromonas hydrophila infection challenge. The results of this study indicate that S. cerevisiae as an additive in Nile tilapia diets has beneficial impacts on growth, stress tolerance, and disease resistance.     

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.     

Characterization of <Emphasis Type="Italic">kiss2</Emphasis> and <Emphasis Type="Italic">kissr2</Emphasis> genes and the regulation of kisspeptin on the HPG axis in <Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>

Reproduction allows organisms to produce offspring. Animals shift from immature juveniles into mature adults and become capable of sexual reproduction during puberty, which culminates in the first spermiation and sperm hydration or ovulation. Reproduction is closely related to the precise control of the hypothalamic–pituitary–gonadal (HPG) axis. Kisspeptin peptides are considered as the important regulator of HPG axis in mammalian. However, the current understanding of kisspeptin in flatfish is not comprehensive. In this study, we cloned and analyzed the kiss2 and kissr2 genes in Cynoglossus semilaevis. Interesting alternative splicing in the 5′-untranslated regions (UTR) of the Cskissr2 gene was found. The expression profiles of Cskiss2 and Cskissr2 showed relative high messenger RNA (mRNA) levels at the late gastrula stage during embryonic development, at total length = 40 mm during early gonadal differentiation, and in the brains and gonads of all investigated tissues. These results suggested that the kisspeptin system participated in embryogenesis and in the regulation of gonadal differentiation and development. Considering that the control and regulatory mechanisms of kisspeptin in the central reproductive axis are still unclear, we documented that the intramuscular injection of kisspeptin caused different sGnRH and cGnRH mRNA levels in a dose- and tissue-dependent manner. The mRNA expressions of FSH and LH were stimulated in the ovary and were inhibited in the testis under the kisspeptin treatments. These results provided foundations for understanding the roles of kisspeptin in the neuroendocrine system in fish. The manipulation of the kisspeptin system may provide new opportunities to control the gonadal development and even reproduction in fish.     

Digestive enzymes and in-vitro digestibility of different species of phytoplankton for culture of the freshwater pearl mussel, <Emphasis Type="Italic">Hyriopsis</Emphasis> (<Emphasis Type="Italic">Hyriopsis</Emphasis>) <Emphasis Type="Italic">bialatus</Emphasis>

Hyriopsis (Hyriopsis) bialatus has been cultured during the mussel life cycle from glochidia to the adult stage with a low total survival of 6% up to 130-day-old juveniles. The main digestive enzymes (amylase and proteinases) were not detectable in one-day-old juveniles, and increased during development. The stomach, including digestive glands, was the major digestive organ for both carbohydrate and protein. The optimum conditions for amylase activity were 40°C and pH 7; for acidic proteinases they were 60°C and pH 5. Two main alkaline proteinases were found in the intestine, with optimum conditions of 30°C and pH 8 and 60°C and pH 8. To improve mussel survival by finding suitable phytoplankton species and age as food for juveniles and adults, an in-vitro digestibility test was performed on ten algal species three and seven days old using amylase and proteinases in crude enzyme extracts from different mussel life stages. Among the phytoplankton selected, the three most efficiently digested by juveniles were seven-day-old Chlorella sp.2, seven-day-old Chlorococcum sp. and seven-day-old Kirchneriella incurvata, in the ratio 1:1:3 for 30-day-old juveniles and 3:1:1 for 130-day-old juveniles. For the adult mussel, three-day-old Chlorella sp.2, seven-day-old Coccomyxa sp., and seven-day-old Monoraphidium sp., in the ratio 3:1:1, were the most digestible phytoplankton. Levels of in-vitro digestibility were related to the quality (not the concentrations) of carbohydrate and protein in the phytoplankton mixtures, and protein digestibility seemed to be the key factor determining food quality for the mussel.     

Monitoring of <Emphasis Type="Italic">Francisella noatunensis</Emphasis> subsp. <Emphasis Type="Italic">orientalis</Emphasis> in farmed Nile tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>) in Brazil

Francisella noatunensis orientalis is a bacterium that causes emerging bacteriosis in Nile tilapia (Oreochromis niloticus) in many parts of the world, including Brazil. It is a non-motile, Gram-negative, strictly aerobic, facultative intracellular coccobacillus. This species of bacteria is responsible for low to high mortality in fish farms, causing economic losses for fish farmers. This study aimed to detect the presence of F. noatunensis orientalis using qPCR (real-time polymerase chain reaction) and to describe lesions caused by the bacterium in O. niloticus in Brazilian aquaculture. For this purpose, 360 fish from six fish farms (30 per farm) were sampled at two time points (n = 180 per sampling). Necropsies and histopathology were performed for lesion observation, in addition to qPCR and sequencing for detection and identification of Francisella species. Environmental data were collected using a multiparameter sonde YSI EXO2. All measured limnological variables were within the optimum range for cultivation of Nile tilapia. The major lesions present were melanization of the skin, splenomegaly, granulomas, and inflammatory cell responses. The prevalence of francisellosis varied from 0 to 86.66% between time periods and fish farms analyzed, and an outbreak was observed during the second sampling period. This study describes the prevalence of francisellosis in O. niloticus and reports that the lesions found are not exclusively associated with this bacterial disease.     

Anesthesia induces stress in Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis>), Atlantic cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>) and Atlantic halibut (<Emphasis Type="Italic">Hippoglossus hippoglossus</Emphasis>)

Stress in response to anesthesia with benzocaine, MS-222, metomidate and isoeugenol was studied in Atlantic salmon (Salmo salar), Atlantic halibut (Hippoglossus hippoglossus), and Atlantic cod (Gadus morhua) with no concomitant stress from handling or confinement in association with anesthesia or sampling. All of the anesthetics tested induced a stress response in all species, displayed by a release of cortisol to the water. MS-222 anesthesia elicited the highest cortisol release rates, reaching maximum levels 0.5 h post-exposure and returning to basal levels after 3–4 h. Benzocaine anesthesia caused a bimodal response where the initial peak in cortisol release rate was followed by a second increase lasting towards the end of the trial (6 h). This bimodality was more profound in Atlantic salmon than in Atlantic halibut and Atlantic cod. Metomidate anesthesia induced the lowest release of cortisol of the agents tested in both Atlantic halibut and Atlantic cod, but resulted in a bimodal response in Atlantic salmon where the initial increase in cortisol release was followed by a larger increase peaking at 2–2.5 h post exposure before returning to basal levels after 5 h. The stress induced in Atlantic salmon by isoeugenol anesthesia resembled that of MS-222, but did not reach the same elevated level. Overall, the cortisol release was most profound in Atlantic salmon followed by Atlantic halibut and Atlantic cod.     

Low-salinity tolerance of juvenile fugu <Emphasis Type="Italic">Takifugu rubripes</Emphasis>

ABSTRACT:   To study the adaptability of juvenile fugu Takifugu rubripes to low-salinity environments, fish were transferred from full-strength seawater (100% SW) to freshwater (FW) and 25, 50, 75 and 100% SW, and checked for mortality over 3 days. No mortality was observed in 25–100% SW, whereas all fish died in FW. In fish transferred to 25–100% SW, blood osmolality was maintained within a physiological range. To further explore the lower limit of salinity that fugu could tolerate, fish were transferred from 100% SW to FW and 1, 5, 10, 15 and 25% SW. All fish survived in 5–25% SW, but fish died in FW and 1% SW. In fish surviving transfer to FW and 1 and 5% SW, blood osmolality was decreased to a near sublethal level of approximately 300 mOsm/kg·H₂O. Therefore, the lower limit of salinity tolerance is estimated to lie between 5 and 10% SW. Preacclimation in 25% SW for 7 days did not essentially affect the survival salinity range. Although survival rates and blood osmolality were slightly improved by preacclimation in 25% SW, blood osmolality was markedly decreased in salinities less than 10% SW, as was seen in the direct transfer. Neither chloride cell morphology nor sodium-potassium adenosinetriphosphatase activity in the gills showed a significant change following transfer to low salinities. These findings indicate that fugu can be adapted to hypoosmotic environments to some extent, exerting hyperosmoregulatory ability, although chloride cells are less likely to absorb ions in hypoosmotic environments.     

Effects of potential probiotic <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis> on digestive enzymatic activities of live feed <Emphasis Type="Italic">Artemia franciscana</Emphasis>

In the present study, cellulase, protease, lipase and amylase activities were performed to investigate the effects of Lactococcus lactis subsp. lactis CF4MRS bioencapsulation of Artemia franciscana. Our results show that cellulase activities (total cellulase—FPase activity, exoglucanase and endoglucanase—CMCase activity) were significantly higher (P < 0.05) in A. franciscana tissue homogenates compared to those in the control group after 8 h of L. lactic bioencapsulation. Notably, an exception case was found in β-D-glucosidase activity, whereby the cellulase activity was not significantly different (P > 0.05) compared to the control group. Administrations of L. lactis at cell concentration of 10⁸ CFU mL^?1 showed considerable improvement of other important enzymatic activities such as amylase, protease and lipase in A. franciscana. The amylase/protease ratio in probiotic-treated A. franciscana was recorded at 0.343, approximately two times higher than those without probiotic administration (0.184). In contrary, amylase/lipase ratio showed half of a reduction (0.330) in L. lactis-administrated A. franciscana compared to the control (0.614). Our study suggests that important digestive enzymes, e.g., cellulase, amylase, protease and lipase, can be enhanced through bioencapsulation of A. franciscana with L. lactis subsp. lactis, which could in turn lead to further stimulation of endogenous enzymes in the fish and shrimp larvae.     

Effect of <Emphasis Type="Italic">Cratoxylum formosum</Emphasis> on innate immune response and disease resistance against <Emphasis Type="Italic">Streptococcus agalactiae</Emphasis> in tilapia <Emphasis Type="Italic">Oreochromis niloticus</Emphasis>

The effect of aqueous extract of Cratoxylum formosum on innate immune response and disease resistance in tilapia Oreochromis niloticus was investigated. The fish were fed diets containing 0% (control), 0.5% (diet 1), 1% (diet 2), and 1.5% (diet 3) of C. formosum aqueous extract for 30 days. At the end of the experimental period, parameters of innate immune response including phagocytosis of blood leukocytes, lysozyme activity in plasma, and respiratory bust activity were examined. Feeding the fish with diet 2 and diet 3 for 20 days enhanced phagocytic activity and for 30 days stimulated lysozyme and respiratory burst activities. Diet 1 increased the phagocytic activity at 30 days, but did not affect the other measured parameters. All parameters were not significantly changed (P > 0.05) in the control group throughout the experiment. Following 30 days of feeding, fish were infected with S. agalactiae. The cumulative mortalities of bacterial-infected tilapia that were fed diet 1, diet 2, and diet 3 were 56, 12, and 10%, respectively, compared with 85% in the control group. These results indicate that the aqueous extract of C. formosum may elevate the innate immune response and enhance disease resistance in tilapia.     

Dietary polysaccharide from <Emphasis Type="Italic">Angelica sinensis</Emphasis> enhanced cellular defence responses and disease resistance of grouper <Emphasis Type="Italic">Epinephelus malabaricus</Emphasis>

The purpose of this study was to determine the effect of Angelica sinensis polysaccharide (ASP) supplemented in diet on the innate cellular immune response and disease resistance in grouper, Epinephelus malabaricus. Fish were fed diets containing different doses of ASP (0, 500 and 3,000 mg kg⁻¹ diet) for 12 weeks. After 12 week feeding, the respiratory burst activity, phagocytic activity, and leukocytes proliferation in head kidney were assayed. The functional immunity in terms of cumulative mortality was also assessed by a challenge with live Edwardsiella tarda. Results showed that the respiratory burst activities in ASP-supplemented groups were increased significantly. The respiratory burst index was the highest in fish-fed 3000 mg kg⁻¹ diet and the lowest in control. The phagocytic activities in ASP-supplemented groups were significantly higher than that of control. No significant difference in phagocytic activity was observed between ASP-supplemented groups. ASP stimulated the head kidney leukocytes proliferation significantly, despite the absence of lipopolysaccharide (LPS) or not. The cumulative mortalities of fish fed with 3000 mg ASP kg⁻¹ diet were significantly lower than those fed with 500 mg ASP kg⁻¹ diet and control diet after 96 h of challenge. In conclusion, dietary ASP enhanced some cellular immune parameters and disease resistance against E. tarda in grouper.     

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.     

Use of a <Emphasis Type="Italic">Noctiluca</Emphasis>-killing bacterium <Emphasis Type="Italic">Marinobacter salsuginis</Emphasis> strain BS2 to reduce shrimp mortality caused by <Emphasis Type="Italic">Noctiluca scintillans</Emphasis>

The ability of an algicidal bacterium Marinobacter salsuginis strain BS2, isolated from shrimp pond water, to reduce shrimp mortality was investigated under laboratory conditions. When two species of shrimp (Penaeus monodon and Litopenaeus vannamei) (body length 1.5–1.8 cm) were cultured together with the dinoflagellate Noctiluca scintillans, nearly 80 % of the shrimps died within 7 days. However, when bacterial strain BS2 was also added to the culture, N. scintillans was killed within 48 h, and shrimp survival rates on the 7th day improved from 23 to 87 % for both P. monodon and L. vannamei. The bacterium BS2 alone had no effect on shrimp condition. Under conditions of increased dissolved oxygen, the effect of using BS2 was greater, and shrimp survival rates improved even more dramatically, from 26 to 98 %. These studies provide the first evidence that the use of killing bacteria, isolated from shrimp culture water, can suppress harmful algal blooms (HABs) and thus restore the efficiency of shrimp production. The control of HABs in this way in shrimp culture farms would be a major benefit for shrimp production.     

Growth and survival of grouper <Emphasis Type="Italic">Epinephelus</Emphasis><Emphasis Type="Italic">coioides</Emphasis> (Hamilton) larvae fed free-living nematode <Emphasis Type="Italic">Panagrellus redivivus</Emphasis> at first feeding

The free-living nematode, Panagrellus redivivus, was tested as live food for grouper Epinephelus coioides larvae during the first feeding stage. A series of experiments were conducted to determine the acceptability of the free-living nematodes in grouper larvae at first feeding, the optimum nematode density and the response of the larvae to nutritionally enriched nematode. All experiments were conducted in 200-L conical tanks filled with 150-L filtered seawater and stocked at 15 larvae L⁻¹. Duration of feeding experiments was up to day 21 (experiment 1) and 14 days (experiment 2 and 3). Brachionus plicatilis and Artemia (experiment 1) and Brachionus plicatilis alone (experiment 2 & 3) was used as the control treatment. Observations indicated that the grouper larvae readily fed on free-living nematodes as early as 3 days posthatching, the start of exogenous feeding. Optimum feeding density for the larvae was 75 nematodes ml⁻¹. The enrichment of cod liver oil or sunflower oil influenced the total lipids and n-3 highly unsaturated fatty acids of P. redivivus, which in turn influenced those of the grouper larvae, however, growth and survival of the larvae were not affected (P > 0.05). The results from this investigation showed that the nematode, P. redivivus, can be used as first live food for grouper larvae from the onset of exogenous feeding until they could feed on Artemia nauplii.     

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.     

Immune response of sea bass <Emphasis Type="Italic">Dicentrarchus labrax</Emphasis> to <Emphasis Type="Italic">Tenacibaculum maritimum</Emphasis> antigens

ABSTRACT:   Seawater fishes are affected by a pathology commonly called 'myxobacteriosis', caused by Tenacibaculum maritimum (formerly Flexibacter maritimus ). The disease is characterized by fin erosion and necrotic ulcers of skin and muscle, and by low but constant mortality in cultured marine fish; in Italy is one of the most important and widely spread diseases affecting sea bass Dicentrarchus labrax , gilthead seabream Sparus aurata , sharp-snouted bream Diplodus puntazzo , white bream Diplodus sargus , and six-tooted bream Dentex dentex . In order to obtain an effective vaccine against the disease, formalin killed cells (FKC), extracellular products (ECPs) and crude lipopolysaccharide (LPS) preparations were obtained from the T. maritimum strain SPVId and injected intraperitoneally twice into the sea bass. The fish immune response to the preparations was studied: agglutinating antibody titer and in vitro phagocytosis were determined after the first and second injection in order to evaluate whether the preparations are immunogenic or not and if the booster effect took place. The results show that FKC and LPS preparations increased the antibody titer after the first injection when compared to the control sea bass. Moreover, all the preparations stimulated a secondary (booster) response. In vitro phagocytosis of the total blood was significantly higher for all the preparations when compared to the controls, but the crude LPS immunized sea bass showed the highest activity.