Effect of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> K-C2 on the growth performance,amino acid content and gut microflora of amberjack <Emphasis Type="Italic">Seriola dumerili</Emphasis>

This study aimed to evaluate the effect of Lactococcus lactis K-C2 on the growth performance, microbial diversity, and release of free amino acids in the intestinal tract and the edible parts of young amberjack, Seriola dumerili. Fish were fed a diet with or without strain K-C2 (2?×?10¹⁰ cfu/g feed) for 25 days. The results indicated that the growth performance of fish in the treated group was significantly higher than those in the control group (p?<?0.05). The amount of five amino acids (aspartate, sarcosine, taurine, alanine, and arginine) in the gut content and 13 of 21 amino acids in the edible parts of fish in the treated group were significantly higher (p?<?0.05) than those in the control group. Sphingomonas, Propionibacterium, and Mycobacterium were observed in gut microflora of fish in both the control and treated groups. Staphylococcus and Kocuria were detected in one sample from the control and treated groups; Acinetobacter and Acidobacteria were found in one sample from the control group. L. lactis was only found in one sample in the treated group. In conclusion, the dietary administration of probiotic L. lactis stimulated growth, reduced feed consumption, and improved the nutritional value of cultured amberjack.     

<Emphasis Type="Italic">Virgibacillus proomii</Emphasis> and <Emphasis Type="Italic">Bacillus mojavensis</Emphasis> as probiotics in sea bass (<Emphasis Type="Italic">Dicentrarchus labrax</Emphasis>) larvae: effects on growth performance and digestive enzyme activities

This study examined the effects of two probiotics (Virgibacillus proomii and Bacillus mojavensis) on the digestive enzyme activity, survival and growth of Dicentrarchus labrax at various ontogenetic stages in three separate experiments. These probiotics were incorporated as single or mixed into fish feed for a period of 60 days. The growth parameters, proximate composition of whole body, digestive enzymes and gut microbiology were monitored at regular. The increments in length and weight and the survival were significantly higher (P < 0.05), and the values of food conversions were significantly lower (P < 0.05) in fishes fed the probiotic. The administration of V. proomii and B. mojavensis in diet resulted in an increase (P > 0.05) in body ash and protein content and in the specific activity of phosphatase alkaline and amylase in the digestive tract of all the fishes. V. proomii and B. mojavensis persisted in the fish intestine and in the feed in high numbers during the feeding period (group 1: 5.8 × 10⁴ CFU/ml, group 2: 9.6 × 10⁴ CFU/ml, and group 3: 9.8 × 10⁴ CFU/ml day 60). The two probiotics V. proomii and B. mojavensis were adequate for improved growth performance and survival and for healthy gut microenvironment of the host.     

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.     

Demographic and competition studies on <Emphasis Type="Italic">Brachionus ibericus</Emphasis> and <Emphasis Type="Italic">Proales similis</Emphasis> in relation to salinity and algal (<Emphasis Type="Italic">Nannochloropsis oculata</Emphasis>) density

We isolated the rotifers Brachionus ibericus and Proales similis from the sediment of shrimp tanks and studied their individual demographic characters and competition between them at two food levels (0.25?×?10⁶, 1.00?×?10⁶ cells ml^?1 of Nannochloropsis oculata at 25 °C) and salinities ranging from 10 to 30‰. Our hypothesis was that growth rates would be higher with increasing food levels and salinities. Observations were taken twice a day for life table studies and daily once for population growth experiments. Using survivorship and fecundity data, we derived various life history variables. Although the average life span (7.6?±?0.4 days) and gross reproductive rate (33.8?±?2.9 neonate female^?1 day^?1) of B. ibericus were higher than those of P. similis (average life span 5.4?±?0.6 days and gross reproductive rate 13.0?±?0.6 neonate female^?1 day^?1), the population growth experiments showed that P. similis had higher r values (0.32?±?0.005 day^?1) than B. ibericus (0.23?±?0.002 day^?1) at 1.0?×?10⁶ cells ml^?1 of N. oculata. The rotifer P. similis was more adversely affected due to the presence of B. ibericus than vice versa. The data are important for developing techniques for a large-scale culture of these rotifers as food in aquaculture.     

Anti-oxidative functions of <Emphasis Type="Italic">mt2</Emphasis> and <Emphasis Type="Italic">smtB</Emphasis> mRNA expression in the gills and brain of zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) upon cadmium exposure

There were not any past studies about metallothionein isoforms (smtB and mt2) having anti-oxidative functions on zebrafish after Cd²⁺ exposure. On the other hand, the anti-oxidative enzymatic factors such as superoxide dismutase (sod), glutathione peroxidase (gpx1a), and catalase (cat) are used as references to investigate whether the smtB and mt2 have anti-oxidative responses on the gills and brain of zebrafish after 1–6 h of 0 and 1.78 μM Cd²⁺ exposure. The anti-oxidative system such as sod, cat, and gpx1a mRNA expressions demonstrated a cascade response upon Cd²⁺-induced oxidative stress in the present study. Interestingly, the smtB mRNA expression levels increased by 3.2- to 6.1-fold, and mt2 raised by 4.1- to 11.3-fold in gills at 1 and 3 h after exposure to Cd²⁺, respectively. On the other hand, the smtB mRNA levels increased by 10.6- to 58.6-fold, but mt2 mRNA levels increased by 2.3- to 11.1-fold in brain at 1 and 3 h after exposure to Cd²⁺, respectively. In addition, both tissues showed increased apoptosis levels at 3 h, and recovery after 6 h of Cd²⁺ exposure. From the results, we suggest that both mt2 and smtB play a role in anti-oxidation responses within 6 h after exposure to Cd²⁺. In conclusion, the smtB mRNA levels have a higher response than mt2 in the brain, but both mRNA expressions appear to have a similar pattern in the gill. We suggest that smtB plays an important role to defend oxidative stress in the brain of adult zebrafish upon acute Cd²⁺ exposure.     

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.     

Pre-sedation and transport of <Emphasis Type="Italic">Rhamdia quelen</Emphasis> in water containing essential oil of <Emphasis Type="Italic">Lippia alba</Emphasis>: metabolic and physiological responses

The effects of transporting silver catfish (Rhamdia quelen) for 6 h in plastic bags containing 0 (control), 30 or 40 µL/L of essential oil (EO) from Lippia alba leaves were investigated. Prior to transport, the fish in the two experimental groups were sedated with 200 µL/L of EO for 3 min. After transport, dissolved oxygen, carbon dioxide, alkalinity, water hardness, pH, temperature and un-ionized ammonia levels in the transport water did not differ significantly among the groups. However, total ammonia nitrogen levels and net Na⁺, Cl^? and K⁺ effluxes were significantly lower in the groups transported with EO of L. alba than those in the control group. PvO₂, PvCO₂ and HCO₃ ^? were higher after transporting fish in 40 µL/L of EO of L. alba, but there were no significant differences between groups regarding blood pH or hematocrit. Cortisol levels were significantly higher in fish transported in 30 µL/L of EO of L. alba compared to those of the control group. The metabolic parameters (glycogen, lactate, total amino acid, total ammonia and total protein) showed different responses after adding EO to the transport water. In conclusion, while the EO of L. alba is recommended for fish transport in the conditions tested in the present study because it was effective in reducing waterborne total ammonia levels and net ion loss, the higher hepatic oxidative stress in this species with the same EO concentrations reported by a previous study led us to conclude that the 10–20 µL/L concentration range of EO and lack of pre-sedation before transport are more effective.     

In vitro and in vivo evaluation of probiotic properties of <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> in Kenyi cichlid, <Emphasis Type="Italic">Maylandia lombardoi</Emphasis>

In this study, Enterobacter cloacae bacterium was isolated from curd and its antibacterial potential against the pathogen Plesiomonas shigelloides was evaluated using the freshwater ornamental fish Kenyi cichlid (Maylandia lombardoi). Among the bacterial isolates, E. cloacae exhibited tolerance to acidic pH 2 and demonstrated the highest antibacterial activity against P. shigelloides in various in vitro assays. Dietary supplementation of E. cloacae with prebiotic supplement 2% mannan oligosaccharide improved the growth performance and reduced the toxic metabolites such as nitrite in culture tank water of Kenyi cichlid. In vivo application of E. cloacae with mannan oligosaccharide significantly elevated (p?<?0.05) white blood cell counts (88.47?±?2.15 10³ mm^?3) and respiratory burst activity (0.243?±?0.007) of Kenyi cichlid when challenged with P. shigelloides. Intestinal morphology of Kenyi cichlid fish treated with probiotic and prebiotic combinations showed improved intestinal architectures. The present findings confirm that the isolated bacterium E. cloacae is a potential probiotic and can be used effectively to prevent infection of P. shigelloides in freshwater ornamental fish culture.     

A study of the damage of the intestinal mucosa barrier structure and function of <Emphasis Type="Italic">Ctenopharyngodon idella</Emphasis> with <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis>

The aim of this study is to explore the effect of Aeromonas hydrophila on the intestinal mucosal barrier structure and intestinal permeability in grass carp (Ctenopharyngodon idella). Histopathological examinations showed that A. hydrophila induced severe intestinal lesions, including inflammatory cell infiltration and intestinal villus fusion and swelling. Messenger RNA (mRNA) expression of the inflammatory cytokines TNF-α, IL-1β, IL-8, IL-10 and MyD88 was significantly increased after infection with A. hydrophila. The permeability of intestinal mucosa was determined using Evans blue (EB) and d-lactic acid. The results indicated that the levels of EB and serum d-lactic acid were significantly increased after infection with A. hydrophila (p < 0.05). Our results also indicated that the intestinal mucosal barrier injury induced by A. hydrophila infection was closely associated with the expression of the tight junction (TJ) protein zonula occludens-1 (ZO-1), occludin, claudin b and claudin c as well as the activity of Na⁺, K⁺-ATPase and Ca²⁺, Mg²⁺-ATPase. Lower mRNA levels of occludin and lower Na⁺, K⁺-ATPase and Ca²⁺, Mg²⁺-ATPase activity in the intestines were observed after challenge. ZO-1 and claudin c were significantly increased 24 h after infection with A. hydrophila. The most interesting finding was that claudin b also significantly increased 24 h after challenge and then decreased to lower levels at 72, 120 and 168 h post-infection compared to the PBS-treated control group. The results demonstrated that grass carp infection with A. hydrophila induced intestinal inflammation and impaired the structure and function of the intestinal mucosal barrier.     

Growth and fatty acid composition of <Emphasis Type="Italic">Octopus vulgaris</Emphasis> paralarvae fed with enriched <Emphasis Type="Italic">Artemia</Emphasis> or co-fed with an inert diet

The rearing of Octopus vulgaris paralarvae during its planktonic life stage is a major challenge, as mortality is currently very high and unpredictable. In this study, we examined the survival and growth rates, as well as the fatty acid composition, of O. vulgaris paralarvae fed on three different dietary treatments: group ArDHA was offered juvenile Artemia enriched with a lipid emulsion (Easy DHA-Selco^®); group ArMA was fed with juvenile Artemia enriched with a mixture of microalgae (Rhodomonas lens and Isochrysis galbana); and group ArMA+ID received the same Artemia as group ArMA complemented with an inert diet. Dietary treatments were tested in triplicate with homogenous groups of paralarvae (25 individuals l^?1) established in 50-l tanks, and the experiment was conducted for 15 days. The survival rate of 15-day post-hatch (-dph) paralarvae from groups ArMA (20 ± 8%) and ArMA+ID (17 ± 4%) tended to be higher than in group ArDHA (13 ± 5%), though these differences were not statistically different. The dry weight (DW) of 15-dph paralarvae increased by almost 60% in groups ArMA and ArMA+ID, and nearly 40% in group ArDHA, with respect to hatchlings. The fatty acid (FA) composition of paralarvae revealed a remarkable drop of docosahexaenoic acid (22:6n-3, DHA) from hatchlings to 15-dph paralarvae of all groups (P < 0.05). However, paralarvae from group ArDHA contained higher levels of DHA than those from ArMA and ArMA+ID (P < 0.05). Despite Artemia enriched with DHA-Selco^® contained three-times more DHA than Artemia enriched with microalgae, no beneficial effects of this dietary treatment were observed on the performance of paralarvae.     

<Emphasis Type="Italic">Curcuma longa</Emphasis> as additive in the diet for <Emphasis Type="Italic">Astyanax aff. bimaculatus</Emphasis>

The aim of the present study was to evaluate the effect of turmeric (Curcuma longa) as additive in the diet for Astyanax aff. bimaculatus. Fish (0.83 ± 0.04 g) were fed, for 60 days, with six diets containing 0.0, 20.0, 40.0, 60.0, 80.0, and 100.0 g turmeric kg^?1 feed. There was an increasing linear effect of turmeric on the thickness of the muscular layer, and height and width of the folds of the intestine. In the liver, a quadratic effect was observed of turmeric on the percentage of hepatocyte cytoplasm and a decreasing linear effect on the percentage of sinusoid capillaries. A quadratic effect was also observed of turmeric on the liver glycogen. There was no effect of turmeric on the antioxidant activity in the liver, carcass composition or productive performance of the fish. Thus, we concluded that Curcuma longa has trophic effects on the epithelium and the muscular layer of the intestine of A. aff. bimaculatus. Additionally, low levels of Curcuma longa cause increased deposition of liver glycogen and high levels cause reduction.     

Influence of the dietary inclusion of <Emphasis Type="Italic">Gracilaria cornea</Emphasis> and <Emphasis Type="Italic">Ulva rigida</Emphasis> on the biodiversity of the intestinal microbiota of <Emphasis Type="Italic">Sparus aurata</Emphasis> juveniles

Partial substitutions of fish meal by 5, 15, or 25 % of Gracilaria cornea or Ulva rigida in experimental diets were evaluated to study their effects on biodiversity of intestinal microbiota composition in gilthead seabream (Sparus aurata) juveniles. The diets were offered to duplicate groups of 15 juvenile fish (14.0 ± 0.5 g) for 70 days, and at the end of the experiment the intestinal microbiota from four specimens of each treatment was analysed by denaturing gel gradient electrophoresis. Results showed that the substitution of fish meal by algae meal induced important modifications in the intestinal microbiota community, as a big reduction of the biodiversity when the highest percentage (25 %) of U. rigida was included. On the contrary, an increase on the number of species was detected when a 15 % of algae was included. Various Lactobacillus delbrueckii subspecies were selectively stimulated when G. cornea was included in the feed, and other bacterial species, such as those included in the Vibrio genus, were reduced.     

Effects of dietary <Emphasis Type="Italic">myo</Emphasis>-inositol on growth,chemical composition and plasma chemistry of Amur sturgeon <Emphasis Type="Italic">Acipenser schrenckii</Emphasis>

The present study was conducted to demonstrate the dietary myo-inositol requirement and its effects on the growth, proximate composition and blood chemistry of Amur sturgeon (Acipenser schrenckii). Triplicate groups of 30 fish (initial weight 11.90?±?0.12 g) were fed different diets containing graded levels of myo-inositol (28.75, 127.83, 343.83, 565.81, 738.15 and 936.28 mg kg^?1) until satiation for 56 days. The fish were weighed after a 24-h fast, and six fish were used for whole body composition analysis. Further, the liver and muscle were sampled from another six fish for lipid analysis. The blood and liver were sampled from the remaining six fish for haematology and fatty acid analysis. The weight gain of fish increased with myo-inositol content, from the 28.75- to 343.83-mg kg^?1 myo-inositol treatment groups, and then stabilised. The liver lipid content and hepatosomatic index decreased significantly from 21.91 to 19.14% and from 3.20 to 2.76% with increased dietary myo-inositol supplementation, respectively. The whole body lipid content generally decreased from 6.33 to 5.55%. The content of liver-saturated fatty acids decreased significantly (28.13%) in the 936.28-mg kg^?1 treatment group. The content of plasma non-esterified fatty acids increased with the increase in dietary myo-inositol supplementation from 0.77 to 1.17 mmol L^?1, whereas the content of triglycerides significantly decreased from 4.62 to 3.28 mmol L^?1. In conclusion, the optimum myo-inositol requirement was found to be 336.1 mg kg^?1, based on weight gain in a two-slope quadratic broken-line model.     

Immunomodulatory effects of <Emphasis Type="Italic">Rhodomyrtus tomentosa</Emphasis> leaf extract and its derivative compound,rhodomyrtone, on head kidney macrophages of rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)

Rhodomyrtus tomentosa is a medicinal plant that shows biological effects including immunomodulatory activity on human and other mammals but not in fish. In this study, we evaluated the in vitro immunomodulatory effects of R. tomentosa leaf extract and its active compound, rhodomyrtone, on the immune responses, using rainbow trout (Oncorhynchus mykiss) head kidney (HK) macrophages as a model. The tested immune functions included the expression of genes involved in innate immune and inflammatory responses and the production of reactive oxygen species (ROS). Gene expression was evaluated after exposure to 10 μg mL^?1 of R. tomentosa and 1 μg mL^?1 of rhodomyrtone for 4 and 24 h. R. tomentosa and rhodomyrtone induced changes in the expression of pro-inflammatory cytokines (il1β, il8, and tnfα), anti-inflammatory cytokines (il10 and tgfβ), inducible enzymes (inos, cox2, and arginase), and an antioxidant enzyme (gpx1). Co-exposure of R. tomentosa with LPS resulted in a prominent reduction in the expression of genes related to an inflammatory process (il1β, il8, tnfα, inos, saa, hepcidin, and gpx1), suggesting anti-inflammatory effects. Similarly, co-exposure of rhodomyrtone with LPS led to a downregulation of inflammation-related genes (il1β, inos, saa, and hepcidin). In addition, exposure to both natural plant products caused a reduction in cellular ROS levels by HK macrophages. The present results indicate that R. tomentosa and rhodomyrtone exerted immunostimulatory and anti-inflammatory effects on fish macrophages, thus opening up the possibility of using these natural products to further develop immunostimulants for health management in aquaculture.     

Characterization of <Emphasis Type="Italic">pax3a</Emphasis> and <Emphasis Type="Italic">pax3b</Emphasis> genes in artificially induced polyploid and gynogenetic olive flounder (<Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>) during embryogenesis

Although chromosome set manipulation techniques including polyploidy induction and gynogentic induction in flatfish are becoming increasingly mature, there exists a poor understanding of their effects on embryonic development. PAX3 plays crucial roles during embryonic myogenesis and neurogenesis. In olive flounder (Paralichthys olivaceus), there are two duplicated pax3 genes (pax3a, pax3b), and both of them are expressed in the brain and muscle regions with some subtle regional differences. We utilized pax3a and pax3b as indicators to preliminarily investigate whether chromosome set manipulation affects embryonic neurogenesis and myogenesis using whole-mount in situ hybridization. In the polyploid induction groups, 94 % of embryos in the triploid induction group had normal pax3a/3b expression patterns; however, 45 % of embryos in the tetraploid induction group showed abnormal pax3a/3b expression patterns from the tailbud formation stage to the hatching stage. Therefore, the artificial induction of triploidy and tetraploidy had a small or a moderate effect on flounder embryonic myogenesis and neurogenesis, respectively. In the gynogenetic induction groups, 87 % of embryos in the meiogynogenetic diploid induction group showed normal pax3a/3b expression patterns. However, almost 100 % of embryos in the gynogenetic haploid induction group and 63 % of embryos in the mitogynogenetic diploid induction group showed abnormal pax3a/3b expression patterns. Therefore, the induction of gynogenetic haploidy and mitogynogenetic diploidy had large effects on flounder embryonic myogenesis and neurogenesis. In conclusion, the differential expression of pax3a and pax3b may provide new insights for consideration of fish chromosome set manipulation.     

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.     

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.     

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.