Transcripts of genes encoding reproductive neuroendocrine hormones and androgen receptor in the brain and testis of goldfish exposed to vinclozolin,flutamide, testosterone,and their combinations

Vinclozolin (VZ) is a pesticide that acts as an anti-androgen to impair reproduction in mammals. However, VZ-induced disruption of reproduction is largely unknown in fish. In the present study, we have established a combination exposure in which adult goldfish were exposed to VZ (30 and 100 μg/L), anti-androgen flutamide (Flu, 300 μg/L), and androgen testosterone (T, 1 μg/L) to better understand effects of VZ on reproductive endocrine system. mRNA levels of kisspeptin (kiss-1 and kiss-2) and its receptor (gpr54), salmon gonadotropin-releasing hormone (gnrh3) and androgen receptor (ar) in the mid-brain, and luteinizing hormone receptor (lhr) in the testis were analyzed and compared with those of control following 10 days of exposure. kiss-1 mRNA level was increased in goldfish exposed to 100 µg/L VZ and to Flu, while kiss-2 mRNA level was increased following exposure to Flu and to combinations of 30 µg/L VZ with Flu, 100 µg/L VZ with T, and Flu with T. gpr54 mRNA level was increased in goldfish exposed to Flu and to combination of 30 µg/L VZ with Flu and 100 µg/L VZ with T. gnrh3 mRNA level was increased in goldfish exposed to 100 µg/L VZ, to Flu, and to combinations of 30 µg/L VZ with Flu, 100 µg/L VZ with T, and Flu with T. The mid-brain ar mRNA level was increased in goldfish exposed to Flu and to combinations of 30 µg/L VZ with Flu, 100 µg/L VZ with T, and Flu with T. Testicular lhr mRNA level was increased in goldfish exposed to Flu and to combination of 30 µg/L VZ with Flu. These results suggest that VZ and Flu are capable of interfering with kisspeptin and GnRH systems to alter pituitary and testicular horonal functions in adult goldfish and the brain ar mediates VZ-induced disruption of androgen production.     

Immunogene expression in head kidney and spleen of common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> L.) following thermal stress and challenge with Gram-negative bacterium, <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis>

To evaluate the effect of thermal and microbial stress on the immune response of common carp (Cyprinus carpio L.), relative mRNA expression level of pro-inflammatory cytokines [tumor necrosis factor alpha (TNF-α) and interleukin (IL)-1β] and other genes related to immune or stress response [inducible nitric oxide synthase (iNOS), heat shock protein 70 (Hsp70), superoxide dismutase one (SOD1), and glucocorticoid receptor (GR)] was measured by quantitative PCR (qPCR). In addition, total protein and total immunoglobulin level in blood plasma of experimental common carp was also assayed. All the above parameters were estimated 24 h post-challenge with Gram-negative bacterium, Aeromonas hydrophila. Common carp (54.89?±?6.90 g) were initially exposed to 20 °C (control group) and 30 °C (thermal stress group) water temperature for 30 days, followed by experimental challenge with 2.29?×?10⁸ colony forming unit/mL (CFU/mL; LD₅₀ dose) of A. hydrophila. Exposure of fish to thermal stress and subsequently challenge with A. hydrophila significantly (P?<?0.05) increases the IL-1β mRNA expression in head kidney and spleen of common carp by ~?39.94 and ~?4.11-fold, respectively. However, TNF-α mRNA expression in spleen decreased ~?5.63-fold in control fish challenged with A. hydrophila. Thermal stress and challenge with bacterium suppresses the iNOS and GR mRNA expression in spleen of common carp. Moreover, significant (P?<?0.05) increase in total protein content of blood plasma (~?43 mg/g) was evident in fish exposed to thermal stress and challenged with A. hydrophila. In conclusion, our study highlights the importance of elevated temperature stress and microbial infection in differential regulation of expression of several immunogenes in common carp.     

The effects of starvation and re-feeding on growth and swimming performance of juvenile black carp (<Emphasis Type="Italic">Mylopharyngodon piceus</Emphasis>)

We investigated the effects of starvation and re-feeding on growth and swimming performance and their relationship in juvenile black carp (Mylopharyngodon piceus). We measured the specific growth rate (SGR), resting metabolic rate (RMR) and constant acceleration test speed (U _CAT, the maximum swimming speed at exhaustion by constant acceleration test with 0.1667 cm s^?2 rate) in a treatment group (21 days of starvation then 21 days of re-feeding) and control group (routine feeding) (n = 20). Starvation resulted in a 17 % decrease in body mass of black carp (P < 0.05). After 21 days of re-feeding, body mass was greater than that of pre-starvation but still less than that of the control group at 42 days. During the re-feeding phase, the SGR of the treatment group was higher than that of the control group (P < 0.05). Starvation resulted in a significant decrease in the RMR and U _CAT. After 21 days of re-feeding, both the RMR and U _CAT recovered to the pre-starvation levels. In the control group, individual juvenile black carp displayed strong repeatability of the RMR and U _CAT across the measurement periods (P ≤ 0.002). In the treatment group, RMR showed significant repeatability between pre-starvation and re-feeding (P = 0.007), but not between pre-starvation and starvation or between starvation and re-feeding. U _CAT showed significant repeatability between pre-starvation and starvation (P = 0.006) and between pre-starvation and re-feeding (P = 0.001), but not between starvation and re-feeding. No correlation or only a weak correlation was found between any two variables of RMR, U _CAT and SGR, whereas the increment of the U _CAT (ΔU _CAT) was negatively correlated with that of SGR during the starvation phase (r = ?0.581, n = 20, P = 0.007) and re-feeding phase (r = ?0.568, n = 20, P = 0.009). This suggested that within individual black carp, there is a trade-off between growth and maintenance (or development) of swimming performance under food-limited conditions.     

Stimulatory effect of dietary taurine on growth performance,digestive enzymes activity,antioxidant capacity,and tolerance of common carp, <Emphasis Type="Italic">Cyprinus carpio</Emphasis> L., fry to salinity stress

The present study was carried out to evaluate the effect of dietary taurine (Tau) on performance, digestive enzymes, antioxidant activity, and resistance of common carp, Cyprinus carpio L., fry to salinity stress. Fish (0.97?±?0.033 g) were fed on different taurine levels of 0.0 (control), 5, 10, 15, or 20 g/kg diet up to satiation twice daily for 8 weeks. At the end of the feeding trial, fish were stressed by exposure to 10 ppt salinity for 3 days during which fish mortality was observed. Fish performance was significantly (P?<?0.05) improved by dietary taurine up to 15 g Tau/kg diet after which fish growth and feed intake were almost the same. Also, taurine supplementation significantly (P?<?0.05) elevated activities of intestinal amylase, lipase, and protease resulting in an improving in feed intake giving better performance. Furthermore, Tau-stimulated antioxidant activity of common carp was observed in a dose-related manner, where activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were significantly (P?<?0.05) higher, but malondialdehyde (MDA) value was significantly (P?<?0.05) lower in Tau-fed fish groups than those fed the control diet. In salinity stress experiment, highest survival rate was observed at fish fed Tau-supplemented diets without significant (P?>?0.05) differences over fish fed the control diet. It appears that taurine could be used as a feed supplement to confer better growth and health of common carp fry with optimal level of 15 g/kg diet.     

Environmental quality and natural food performance at feeding sites in a carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis>) pond

Just like other domesticated animals, common carp (Cyprinus carpio) are able to learn where food can easily be obtained. As a result, carp in semi-intensive polyculture ponds could conceivably restrict their main activity centres to supplemental feeding sites, leading to lowered exploitation of natural food resources (zooplankton and macrozoobenthos) and localised degradation of environmental conditions. At two semi-intensive ponds in southern Moravia (Czech Republic), areas around feeding sites displayed significantly reduced oxygen concentration and saturation (mainly p < 0.001). Likewise, temperature, pH and transparency also decreased significantly at feeding sites (mainly p < 0.05), while turbidity increased. While there was no significant difference in zooplankton biovolume at feeding and non-feeding sites, zoobenthos density and biomass were significantly lower at feeding sites (mainly p < 0.01). The feeding behaviour of carp, therefore, led to significant changes in both abiotic and biotic conditions at supplemental feeding sites.     

Effect of nitrite exposure on oxygen-carrying capacity and gene expression of <Emphasis Type="Italic">NF-κB</Emphasis>/<Emphasis Type="Italic">HIF-1α</Emphasis> pathway in gill of bighead carp (<Emphasis Type="Italic">Aristichthys nobilis</Emphasis>)

Nitrite (NO₂^?) contamination of water can severely impact the health of aquatic life and is a major concern for commercial aquaculture. In order to study the effect of nitrite on Aristichthys nobilis, we investigated the oxygen-carrying capacity, NF-κB/HIF-1α pathway, and the gill tissue structure under nitrite stress. In the current study, bighead carp (initial weight 180.05?±?0.092 g) were exposed to nitrite (48.634 mg/L) for 96 h and then for 96 h recovery test. After nitrite exposure for 6 h, hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-κB (NF-κB) mRNA expression increased significantly in the gill of bighead carp (P?<?0.05). After nitrite exposure for 12 h, hemoglobin (Hb) and methemoglobin reductase (MHBR) content in blood decreased significantly (P?<?0.05); TLR4 mRNA expression increased significantly (P?<?0.05). After nitrite exposure for 24 h, methemoglobin (MetHb) content increased significantly (P?<?0.05). After recovery test, all the indicators except TLR4 mRNA expression level recovered to initial level. In conclusion, nitrite exposure can affect hemoglobin dynamics, as oxidization of nitrite by hemoglobin results in the reduction of Hb to MetHb leading to hypoxia and nitrite exposure can also result into gill tissue damage. In the face of nitrite exposure, NF-κB and HIF-1α mRNA expression level increased immediately to protect the body from oxidative damage and eased hypoxic condition caused by nitrite. It was also observed that nitrite damage is recoverable in Aristichthys nobilis, but it may be need more than 96 h.     

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.     

Molecular mobility of fish flesh measured by low-field nuclear magnetic resonance (LF-NMR) relaxation: effects of freeze–thaw cycles

In this study, the molecular mobility of fish flesh was measured by low field nuclear magnetic resonance (LF-NMR) relaxation. Sardine, tuna and mackerel were frozen at ?40 °C and stored for 1 day (24 h); and then these samples were thawed at room temperature (20 °C). The relaxation of water protons in fish flesh was measured for fresh (i.e., before freezing) and multi-cycle freeze–thaw samples (i.e., up to 12 times). Three domains from different pools of protons (i.e., low-mobile, medium-mobile and high-mobile) were identified from the relaxation curve. The T _2b (low-mobile), T ₂₁ (medium-mobile) and T ₂₂ (high-mobile) indicated the proton populations in the protein molecules, strongly bound water molecules, and weakly bound water molecules, respectively. In all cases, the relaxation time (T _2b: sardine r = 0.736 and p < 0.01, tuna r = 0.857 and p < 0.001, mackerel r = 0.904 and p < 0.001; and T ₂₂: sardine r = 0.956 and p < 0.0001, tuna r = 0.927 and p < 0.0001, mackerel r = 0.890 and p < 0.0001) increased with the freeze–thaw cycles and it reached a nearly constant value after 6 freeze–thaw cycles. The increased relaxation time (i.e., higher mobility) up to 6 freeze–thaw cycles could be due to the increase in proton mobility. However, relaxation time (T ₂₁: sardine r = ?0.510 and p > 0.05, tuna r = 0.162 and p > 0.5, mackerel r = 0.513 and p > 0.01) showed insignificant change with the increase of freeze–thaw cycles, which indicated minimal change in the medium-mobile protons. The results in this study revealed that the changes in proton mobility in the fish flesh during freeze–thaw cycles could be identified using T _2b and T ₂₂ relaxation of LF-NMR.     

Dietary docosahexaenoic acid decreased lipid accumulation via inducing adipocytes apoptosis of grass carp, <Emphasis Type="Italic">Ctenopharygodon idella</Emphasis>

The purpose of this study was to explore the mechanism of by which docosahexaenoic acid (DHA) inhibit the accumulation of adipose tissue lipid in grass carp (Ctenopharyngodon idella). We therefore designed two semi-purified diets, namely DHA-free (control) and DHA-supplemented, and fed them to grass carp (22.19 ± 1.76 g) for 3 and 6 weeks. DHA supplementation led to a significantly lower intraperitoneal fat index (IPFI) than that in the control group by reducing the number of adipocytes but significantly higher adipocyte size (P < 0.05). In the intraperitoneal adipose tissue, the DHA-fed group showed significantly higher peroxisome proliferator-activated receptor (PPAR)γ, CCAAT enhancer-binding protein (C/EBP)α, and sterol regulatory element-binding protein (SREBP)1c mRNA expression levels at both 3 and 6 weeks (P < 0.05). However, the ratio of the expression levels of B cell leukemia 2 (Bcl-2) and Bcl-2-associated X protein (Bax) was significantly lower in the DHA-fed group than in the control group (P < 0.05), and the protein expression levels of the apoptosis-related proteins caspase 3, caspase 8, and caspase 9 were also significantly higher (P < 0.05). Overall, although DHA promotes lipid synthesis, it is more likely that DHA could suppress the lipid accumulation in adipocytes of grass carp by inducing adipocyte apoptosis.     

Feeding,somatic condition and survival of sand lance <Emphasis Type="Italic">Ammodytes</Emphasis> sp. larvae in Mutsu Bay,Japan

To clarify the recruitment process of sand lance Ammodytes sp., we investigated larval condition factor, relative gut fullness (%GF), prey abundance and oceanographic structure in Mutsu Bay, Japan, during 1999–2001. Ammodytes sp. larvae, which were collected by horizontal hauls of Motoda nets and a ring net at depths of 1, 10, 20, 30 and 40 m, were mainly distributed at 10–30 m. Larvae at the first feeding time until 12 mm in body length (BL) fed predominantly on copepod nauplii, whereas large larvae with BL of 12.1–14.0 mm fed on a mixture of copepod nauplii, copepodites and appendicularians from late February to April. A path analysis showed that difference in water density between 35- and 5-m depths negatively affected naupliar abundance at 10–30-m depth (standardised path coefficient β = ?0.71, p = 0.005 for 3.3–8.0-mm BL larvae and β = ?0.78, p < 0.001 for 8.1–12.0-mm BL larvae). Naupliar abundance positively affected the %GF of Ammodytes sp. larvae (β = 0.75, p < 0.001 for 3.3–8.0-mm BL larvae and β = 0.66, p < 0.001 for 8.1–12.0-mm BL larvae), whereas it was negatively affected by water temperature (β = ?0.45, p = 0.008 for 3.3–8.0-mm BL larvae and β = ?0.56, p = 0.002 for 8.1–12.0-mm BL larvae), and the temperature effect was weak compared with that of naupliar abundance. In turn, %GF positively affected larval somatic weight (β = 0.91, p < 0.001 for 6.0-mm BL larvae and β = 0.70, p = 0.005 for 10.0-mm BL larvae). The recruitment failure in 1999 was likely caused by a reduced condition factor, which resulted from low naupliar abundance. In contrast, the abundance of nauplii and Oithona similis copepodites was high in 2000 and 2001. It is possible that the higher recruitment success in 2001 was because of the higher water temperatures in Mutsu Bay, sustaining faster growth of the larvae than in 2000 under the high-prey abundance conditions.     

Effects of lipoic acid on growth and biochemical responses of common carp fed with carbohydrate diets

Lipoic acid (LA) is an antioxidant that also favors glucose uptake in mammals. Until now, there are no studies evaluating the potential effect of this molecule on glycemic control in fish. It was evaluated LA effects on glucose uptake in common carp Cyprinus carpio fed with carbohydrate diets from two carbohydrate sources: glucose (GLU) and starch (STA), and supplemented or not with LA, being the diets: +GLU/?LA (GLU); +GLU/+LA (GLU + LA); +STA/?LA (STA); and +STA/+LA (STA + LA). Carp juveniles (6.5 ± 0.1 g) were fed with each diet ad libitum 4 times a day, during 68 days. Muscle glycogen concentration was higher (p < 0.05) in GLU and GLU + LA than in STA and STA + LA groups. On fish fed with starch, muscle cholesterol and triglyceride concentrations were higher (p < 0.05) in fish fed diets supplemented with LA. Muscle protein levels were higher in fish fed with LA, independent of the diet carbohydrate source. Lipid peroxidation was significantly reduced (p < 0.05) in fish muscle on fish fed the STA + LA diets when compared with the STA diet. Our findings indicate that LA modulates lipid, proteins and carbohydrate metabolism together with the well-known antioxidant effect. Also, LA showed to enhance starch utilization taking into account muscle cholesterol and triglyceride levels.     

Astaxanthin degradation and lipid oxidation of Pacific white shrimp oil: kinetics study and stability as affected by storage conditions

The kinetics of astaxanthin degradation and lipid oxidation in shrimp oil from hepatopancreas of Pacific white shrimp (Litopenaeus vannamei) as affected by storage temperature were studied. When shrimp oil was incubated at different temperatures (4, 30, 45 and 60 °C) for 16 h, the rate constants (k) of astaxanthin degradation and lipid oxidation in shrimp oil increased with increasing temperatures (p < 0.05). Thus, astaxanthin degradation and lipid oxidation in shrimp oil were augmented at high temperature. When shrimp oils with different storage conditions (illumination, oxygen availability and temperature) were stored for up to 40 days, astaxanthin contents in all samples decreased throughout storage (p < 0.05). All factors were able to enhance astaxanthin degradation during 40 days of storage. With increasing storage time, the progressive formation of primary and secondary oxidation products were found in all samples as evidenced by the increases in both peroxide values (PV) and thiobarbituric acid reactive substances (TBARS) (p < 0.05). Light, air and temperatures therefore had the marked effect on astaxanthin degradation and lipid oxidation in shrimp oils during the extended storage.     

Silymarin inhibits adipogenesis in the adipocytes in grass carp <Emphasis Type="Italic">Ctenopharyngodon idellus</Emphasis> in vitro and in vivo

In this study, two experiments were performed to explore the function of silymarin in adipogenesis in grass carp (Ctenopharyngodon idellus) using in vitro and in vivo models. In experiment 1, differentiated grass carp pre-adipocytes were treated with silymarin for 6 days. Treatment with 100 μg mL^?1silymarin (SM100 group) significantly reduced triglyceride accumulation at day 6. The adipogenic gene expression levels of PPARγ, C/EBPα, SREBP1c, FAS, SCD1, and LPL, and the protein expression level of PPARγ were significantly down-regulated in the SM100 group. Additionally, the SM100 group had significantly lower reactive oxygen species production and reduced glutathione contents compared with the control in vitro. In experiment 2, the juvenile grass carp (mean body weight= 27.4 ± 0.17 g) were fed six isonitrogenous and isocaloric diets in a factorial design containing 0, 100, or 200 mg kg^?1 silymarin (SM0, SM100, SM200) associated with either 4 or 8% lipid levels (low lipid, LL, and high lipid, HL, respectively) for 82 days. The results demonstrated that dietary silymarin supplementation significantly reduced the elevated intraperitoneal fat index in grass carp fed with high-lipid diets, and the gene expression of adipogenesis (PPARγ, FAS) when supplemented with dietary silymarin was notably lower than when no silymarin was supplemented under the high-lipid diets. Thus, our data suggest that silymarin suppressed lipid accumulation in grass carp both in vitro and in vivo, and the effect might be due to an influence on the expression of adipogenesis factors and ROS production partly associated with effects on antioxidant capability.     

Inhibitory neurotransmitter serotonin and excitatory neurotransmitter dopamine both decrease food intake in Chinese perch (<Emphasis Type="Italic">Siniperca chuatsi</Emphasis>)

Aminergic neurotransmitters play important roles in the regulation of food intake. However, their effects on feeding in fish have been less explored and still unclear. In the present study, the effects of serotonin (5-HT) and dopamine (DA) on food intake were evaluated through intraventricular (ICV) administration in Chinese perch (Siniperca chuatsi) and the mRNA expression levels of neuropeptide Y (NPY), agouti gene-related protein (AgRP), and pro-opiomelanocortin (POMC) were detected. At 1 h post-injection, 5-HT significantly decreased food intake in a dose-dependent manner. The mRNA expression of NPY and AgRP were significantly decreased (p < 0.05), whereas the mRNA expression of POMC was significantly increased (p < 0.05), suggesting the involvement of NPY, AgRP, and POMC in inhibitory action of 5-HT on food intake in Chinese perch. DA significantly decreased (p < 0.05) food intake and AgRP mRNA expression at 1 h post-injection, indicating the inhibitory effect of DA on food intake might be mediated through AgRP. This might shed new light on the regulation of food intake in Chinese perch.     

Toxicological and hematological effect of <Emphasis Type="Italic">Terminalia arjuna</Emphasis> bark extract on a freshwater catfish, <Emphasis Type="Italic">Heteropneustes fossilis</Emphasis>

Increasing demand for eco-friendly botanical piscicides and pesticides as replacements for harmful synthetic chemicals has led to investigation of new sources of plant materials. Stem bark of Terminalia arjuna, which has been used as a popular folk medicine since ancient time, was examined for its piscicidal activity. This study aims to determine toxicity of ethanol extract of T. arjuna bark on fresh water stinging catfish (Heteropneustes fossilis), along with evaluation of changes in hematological parameters of the fishes exposed to a lethal concentration. The percent mortality of fishes varied significantly in response to concentrations of the extract and exposure times (between exposure time F = 36.57, p < 0.001; between concentrations F = 39.93, p < 0.001). The lethal concentrations (LC₅₀) of ethanol extract were found to be 12.7, 8.94, 5.63 and 4.71 mg/l for 24, 48, 72 and 96 h, respectively. During acute toxicity test, blood samples of treatment fishes showed significant decreases in the red blood cells count, hematocrit content, hemoglobin concentration, mean corpuscular hemoglobin concentration and plasma protein level when compared to those of the control group, while there were significant increases in the mean corpuscular volume, mean corpuscular hemoglobin, white blood cells count and plasma glucose concentration. These results suggest that T. arjuna bark extract could be considered as a potent piscicide due to its toxic effect on fish, particularly fish hematology.     

Growth performance and biochemical composition of juvenile pearl oyster <Emphasis Type="Italic">Pinctada martensii</Emphasis> fed on artificial diets

The effects of three artificial diets (S1, S2 and S3) on survival, growth and biochemical composition of one-year-old pearl oyster Pinctada martensii were investigated. Six experimental groups (EG1, EG2, EG3, EG4, EG5 and EG6) and one control group (CG) were set up. EG1, EG2 and EG3 were solely fed on S1, S2 and S3, respectively. EG4, EG5 and EG6 were fed on mixed diets, as follows: S1 and Platymonas subcordiformis; S2 and P. subcordiformis; and S3 and P. subcordiformis, respectively. CG was fed on only P. subcordiformis. All groups were continuously fed for 60 days. Survival, growth and biochemical composition of soft tissues were compared across the groups. Results showed that survival rate, the absolute growth rate (AGR) and relative growth rate (RGR) of shell length did not differ significantly across the groups (p > 0.05). The AGR and RGR of total weight differed significantly among the groups (p < 0.05). AGR and RGR of shell length and total weight were the highest in EG5 and the lowest in EG1. Gross fat content showed insignificant differences among the groups (p > 0.05). However, gross protein content and ash content showed significant differences across the groups (p < 0.05). The gross protein of the groups solely fed on artificial diets was lower than those of the groups fed on mixtures of artificial diets and microalgae or single microalgae. The contents of other amino acids, total amino acids (TAAs) and essential amino acids (EAAs) showed significant differences across the groups (p < 0.05). TAA, EAA and delicious amino acids of the groups fed solely on artificial diets were lower than those of the groups fed on mixtures of artificial diets and microalgae or single microalgae. Results indicated that the artificial diet (S2) can serve as substitutes of microalgal diets for P. martensii.     

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.