Beiping Tan

Effects of dietary chitosan and Bacillus subtilis on the growth performance, non-specific immunity and disease resistance of cobia, Rachycentron canadum

The present study was performed to investigate the effects of various levels of dietary Bacillus subtilis and chitosan on the growth performance, non-specific immunity and protection against Vibrio harveyi infection in cobia, Rachycentron canadum. Fish were fed with the control diet and six different experimental diets containing three graded levels of B. subtilis at 2 × 10(10) CFU g(-1) (0.0, 1.0, 2.0 g kg(-1) diet) for each of two levels of chitosan (3.0 and 6.0 g kg(-1) diet). The results of 8 weeks feeding trial showed that the survival rate ranged from 81.3% to 84.0% with no significant difference (P > 0.05). The SGR (%) in the fish fed with dietary treatments was significantly higher than that of the control fish except diet 6 group with 2.0 g kg(-1)B. subtilis and 3.0 g kg(-1) chitosan. The serum lysozyme activities were significantly higher in 6.0 g kg(-1) chitosan groups and no significant differences were observed among B. subtilis levels. The serum ACP activities were significantly higher in 3.0 g kg(-1) chitosan groups at 0.0 and 1.0 g kg(-1)B. subtilis levels; at low chitosan level, the cobia fed diets with 1.0 g kg(-1)B. subtilis had significantly higher serum ACP activity, but at high chitosan level, the cobia fed diets with 2.0 g kg(-1)B. subtilis had significantly higher serum ACP activity. The phagocytosis and respiratory burst activity in the fish fed with dietary treatments was significantly higher than that of the control fish except diet 3 group with 6.0 g kg(-1) chitosan. Moreover, fish fed the diet containing 2.0 g kg(-1)B. subtilis and 6.0 g kg(-1) chitosan had significantly higher post-challenge survival on the 7th day following V. harveyi infection and post-challenge survival showed clearly the synergistic effect of chitosan and B. subtilis. Based on these results, the combination of 1.0 g kg(-1)B. subtilis and 6.0 g kg(-1) chitosan is optimal for the growth, innate immunity and disease resistance of cobia with an 8-week oral administration.

Isolation of a putative probiotic strain S12 and its effect on growth performance, non-specific immunity and disease-resistance of white shrimp, Litopenaeus vannamei.

The common pathogens in aquaculture are very different from those in terrestrial animals. The objective of this study was to isolate probiotic strain (s) from the digestive tract of healthy white shrimp Litopenaeus vannamei which was effective against aquatic animal pathogens. The putative probiotic strain S12 was identified as Bacillus subtilis based on the morphological and biochemical properties and 16S rDNA gene sequencing. The L. vannamei were fed with five different diets: control (basal diet with no probiotics or antibiotics), antibiotic control (basal diet supplemented with 0.3% florfenicol), basal diet supplemented with 5 × 10(9) cfu kg(-1) , 5 × 10(10) cfu kg(-1) and 5 × 10(11) cfu kg(-1) probiotic S12 (PS1-3). Each diet was randomly fed to quadruplication groups of 40 shrimps (0.4 ± 0.01 g) reared in tanks. After an 8-week feeding, the survival rate of shrimps fed with PS1 and PS3 were the highest among all treatments (P < 0.05). The moisture content of shrimps fed with florfenicol was significantly lower than that of the control group (P < 0.05). The supplement of probiotic S12 decreased the body crude lipid significantly (P < 0.05). The activities of phagocytic rate, lysozyme (LZ), superoxide dismutase phenoloxidase (SOD) and antibacterial activity were significantly higher than those in the control (P < 0.05), and the activities of SOD and the antibacterial activity in PS2 and PS3 were significantly higher than those in antibiotic control (P < 0.05). When infected with Vibrio harveyi at 4-weeks, the mortality was significantly lower (P < 0.05) in PS2 and PS3 groups than that in the control. After being infected with V. harveyi at 8-weeks, the mortality was significantly lower in the probiotic and antibiotic groups than that in the control (P < 0.05). This study suggested that probiotics could be used as an effective immunopotentiator, the optimal dose of the probiotic strain S12 is 5 × 10(10) cfu kg(-1) diet.

Effect of replacing fish meal with extruded soybean meal on growth, feed utilization and apparent nutrient digestibility of juvenile white shrimp (Litopenaeus vannamei)

Extruded soybean meal (ESBM) was evaluated as a protein source for partial replacement of fish meal (FM) in diets of juvenile Litopenaeus vannamei. In the control diet (Diet 1), FM protein was replaced with increasing dietary levels of ESBM (4.28%, 8.40%, 12.62%, 16.82%, and 25.26%) at 10%, 20%, 30%, 40%, and 60% levels (Diets 2 to 6, respectively). An eight-week feeding trial was conducted on 720 juvenile shrimp (0.67 g ± 0.01 g mean initial weight), and nutrient digestibility of the six diets was determined. ESBM could replace 20% of FM without causing a significant reduction in growth of shrimp, but other dietary treatments strongly affected whole body composition. Crude protein content of the whole body fed Diet 6 was significantly lower than that fed Diet 2 (P < 0.05), while crude lipid content of the whole body fed Diet 5 or 6 was significantly higher than that fed Diet 2 (P < 0.05). Protein digestibilities of Diets 5 and 6 were significantly lower than that of Diet 1 (P < 0.05). Digestibility of lipids ranged from 96.97% in Diet 6 to 98.34% in Diet 3, whereas dry matter digestibility decreased with increasing replacement level. This study indicates that 20% FM replacement with ESBM in the basic diet containing 40% protein and 30% FM is optimal for juvenile L. vannamei.

Toxic effects of two sources of dietborne cadmium on the juvenile cobia, Rachycentron canadum L. and tissue-specific accumulation of related minerals

In the present study, juvenile cobia, Rachycentron canadum L. were fed diets contaminated by two different sources of cadmium: squid viscera meal (SVM-Cd, organic form) and cadmium chloride (CdCl2-Cd, inorganic form). The Cd concentrations in fish diet were approximate 3.0, 5.0 and 10.0mg Cd kg(-1) for both inorganic and organic forms. In the control diet (0.312mg Cd kg(-1) diet, Cd mainly come from fish meal), no cadmium was added. The experiment lasted for 16 weeks and a statistically significant inverse relationship was observed between specific growth rate (SGR) and the concentration of dietary Cd. The SGR of cobia fed a diet with SVM-Cd increased at the lowest doses and decreased with the increasing level of dietary SVM. Fish fed diet contaminated SVM-Cd had significantly higher SGR than those fed diets contaminated CdCl2-Cd among the high Cd level diets treatments. The dietary Cd levels also significantly affected the survival rate of the fish. Among the hematological characteristics and plasma constituents, glutamic-pyruvic transaminase activities and alkaline phosphatase activities in serum and liver increased and hepatic superoxide dismutase activity decreased with the increasing dietary Cd levels. The cobia fed diet contaminated by high level of CdCl2-Cd had significantly higher ALP activity than cobia fed diet contaminated by high level of SVM-Cd. The results from these studies indicate no differences in toxicity response to dietborne SVM-Cd and CdCl2-Cd at a low level of Cd. However, at a higher level, cobia was more sensitive to dietborne CdCl2-Cd than SVM-Cd. Based on quadratic regression of SGR, The Cd concentrations was 3.617mg kg(-1) in the optimal diet, Cd source was SVM (126mg Cd kg(-1) in SVM) which stimulate the growth of cobia and the added level was determined to be 26.7g kg(-1) diet in the present study. Cd accumulations in the kidney of cobia fed both types of Cd were higher than other tissues, and the order of Cd accumulation in tissues were kidney>liver>intestine>gill>muscle. Iron accumulation in liver and kidney and calcium accumulation in vertebra and scale were also significantly affected by dietary Cd levels.

Effect of dietary potassium on growth, nitrogen metabolism, osmoregulation and immunity of pacific white shrimp (Litopenaeus vannamei) reared in low salinity seawater

An 8 weeks feeding experiment was conducted to determine the effect of dietary potassium on the growth and physiological acclimation of Pacific white shrimp (Litopenaeus vannamei) reared in diluted seawater (salinity 4). Six semi-purified practical diets containing 0.59, 0.96, 1.26, 1.48, 1.74, and 2.17 g potassium K+ per 100 g diet were formulated, respectively. The survival and feed conversion rate did not show significant difference among groups of shrimps given these diets (P>0.05). The shrimps fed the diets containing 0.96–1.48 g K+ per 100 g diet gained the highest weight, specific growth rate, and protein efficiency ratio. Their ammonium-N excretion rate as well as hemolymph concentration of Na+ and Cl− were significantly lower than those of the control (P<0.05), but a reverse trend was observed for their gill Na+/K+-ATPase. Moreover, the shrimps fed with 1.48 g K+ per 100 g diet were the highest in hemolymph urea level, and the phenoloxidase and lysozyme activities were significantly higher than those of the control (P<0.05). The growth and physiological response of the test shrimps suggested that diet containing 1.48 g K+ per 100 g diet improved the growth of L. vannamei in low-salinity seawater, and enhanced the physiological acclimation of the organism.

Molecular cloning of glucose transporter 1 in grouper Epinephelus coioides and effects of an acute hyperglycemia stress on its expression and glucose tolerance

The glucose transporter family proteins play pivotal roles in glucose metabolism. In this study, we successfully cloned the orange spotted grouper (Epinephelus coioides) glucose transporter 1 (EcGlut1) gene (GenBank accession: JQ623903). The full-length EcGlut1 cDNA was 2126xa0bp with a 1476xa0bp ORF, a 437bp5′-UTR and 223bp3′-UTR. EcGlut1 is predicted to encode a 491 amino acid protein with a MW of 53.9xa0kDa, a pI of 8.66 and a Pfam domain. Bioinformatics analysis revealed that EcGlut1 was evolutionally conserved between fishes with 80–89xa0% amino acid identities. EcGlut1 was expressed predominantly in heart and liver and at lower levels in muscle, intestine, stomach and brain. We also investigated the effect of acute hyperglycemia stress on EcGlut1 expression. In glucose tolerance test, changes in EcGlut1 mRNA expression in response to glucose injection and glucose metabolism-related indictors were assessed at the same time. Glucose injection significantly suppressed EcGlut1 mRNA expression in liver at 12xa0h and in brain at 24xa0h postinjection (Pxa0<xa00.05). EcGlut1 mRNA levels in heart were increased at 6xa0h (Pxa0<xa00.05). Plasma glucose level increased significantly and reached its maximum at 3xa0h postinjection (Pxa0<xa00.05). The spatiotemporal expression of EcGlut1 and glucose metabolism suggested that orange spotted grouper might rely on fat anabolism to reduce acute hyperglycemia stress and the delayed transcription of EcGlut1 gene might be one reason for glucose intolerance in E. coioides.

Effects of supplemental coated or crystalline methionine in low-fishmeal diet on the growth performance and body composition of juvenile cobia Rachycentron canadum (Linnaeus)

We evaluated the effects of supplemental coated and crystalline methionine (Met) on the growth performance and feed utilization of juvenile cobia (Rachycentron canadum Linnaeus) in a 60-d feeding trial. Fish groups were fed one of six isonitrogenous and isolipidic diets: 1) fishmeal control; 2) un-supplemented experimental (low-fish-meal diet deficient in Met); or 3) one of four Met diets supplemented with crystalline L-Met, cellulose-acetate-phthalate coated L-Met, acrylic-resin coated L-Met, or tripalmitin-polyvinyl alcohol coated L-Met. The test diets were fed to triplicate groups of cobia (initial body weight 5.40±0.07 g) twice a day. The weight gain and specific growth rate of the fish fed the RES diet were highest among the Met-supplemented groups and were 23.64% and 7.99%, respectively, higher than those of the fish fed with the un-supplemented experimental diet (P<0.05). The protein efficiency ratio of the fish fed the MET diet was significantly higher than that of the fish fed the un-supplemented experimental diet and the fish in the other methionine supplementation groups (P<0.05). Our results suggest that supplementation of crystalline Met in low-fish-meal diets promotes the growth performance of juvenile cobia.

Molecular cloning, characterization and expression analysis of Wnt4, Wnt5, Wnt6, Wnt7, Wnt10 and Wnt16 from Litopenaeus vannamei.

The Wnt (Wg-type MMTV integration site) signaling represents as the negative regulator of virus-induced innate immune responses. Wnt genes act as ligands to activate the Wnt signaling. To know more about the information of Wnt genes in invertebrates, Litopenaeus vannamei Wnt genes (LvWnts) were identified and characterized. In this study, Six Wnt genes (LvWnt4, LvWnt5, LvWnt6, LvWnt7, LvWnt10 and LvWnt16) were obtained in L.xa0vannamei. The complete cDNAs open reading frames (ORF) of LvWnt4, LvWnt5, LvWnt6, LvWnt7, LvWnt10 and LvWnt16 were 1077xa0bp, 1107xa0bp, 1350xa0bp, 1047xa0bp, 1509xa0bp and 1158xa0bp (GenBank accession no. KU169896, KU169897, KU169898, KU169899, KU169900 and KU169901), encoding 358, 368, 449, 348, 502 and 385 amino acid (aa) residues respectively. All the six members of LvWnts contain a Wnt1 domain, which is considered as an important feature of Wnt gene family. ClustalW analysis with amino acid sequences revealed that the proportion of identity with other species was more than 48% for all the LvWnts except LvWnt10 (36-41%). The phylogenetic relationship analysis illustrated that different subtype of Wnts formed their own separate branches and were placed in branch of invertebrates respectively with strong bootstrap support. The constitutive expressions of LvWnts were confirmed by RT-PCR in all the examined five developmental stages and eleven tissues of L.xa0vannamei with different express patterns. LvWnt4, LvWnt5 and LvWnt10 were expressed highest in nerve while LvWnt6, LvWnt7 and LvWnt16 were expressed highest in intestine, stomach and gill, respectively. In addition, all the LvWnts were regulated by white spot syndrome virus (WSSV) challenges at different levels in hepatopancreas, gill and hemocytes, suggesting that Wnt genes may play a role in the defense against pathogenic virus infection in innate immune of L.xa0vannamei.

Effect of various Na/K ratios in low-salinity well water on growth performance and physiological response of Pacific white shrimp Litopenaeus vannamei

To investigate the influence of sodium to potassium (Na/K) ratios on the growth performance and physiological response of the Pacific white shrimp (Litopenaeus vananmei), various concentrations of KCl were added to low-salinity well water (salinity 4) in an 8-week culture trial. Six treatments with Na/K ratios of 60:1, 42:1, 33:1, 23:1, 17:1, and 14:1 were replicated in triplicate. The highest weight-gain rate (3 506±48)% and survival rate (89.38±0.88)% was observed in well water with Na/K ratios of 23:1 and 42:1, respectively, while the feed conversion ratio (1.02±0.01), oxygen consumption, and ammonia-N excretion rate was the lowest in the medium with a Na/K ratio of 23:1. Gill Na+-K+-ATPase activity, as an indicator of osmoregulation, peaked in the treatment where the Na/K ratio was 17:1. The total hemocyte count, respiratory burst, and immune-related enzyme activities (ALP, LSZ, PO, and SOD) of L. vananmei were affected significantly by Na/K ratios (P<0.05). After challenged with Vibrio harveyi, the cumulative mortality of shrimp reared in a Na/K ratio of 23:1 (30±14.14)% was significantly lower than the control (75±7.07)%. In conclusion, the addition of K+ to low-salinity well water in L. vannamei cultures is feasible. Na/K ratios ranging from 23:1 to 33:1 might improve survival and growth. Immunity and disease resistance are also closely related to the Na/K ratio of the low-salinity well water. The findings may contribute to the development of more efficient K + remediation strategies for L. vananmei culture in low-salinity well water.

Molecular cloning, characterization and expression analysis of ARMC6, ARMC7, ARMC8 from Pacific white shrimp, Litopenaeus vannamei

Armadillo repeat-containing proteins (ARMCs) comprise a large family that is widely distributed in eukaryotes and plays prominent roles in cell-cell adhesion, intracellular signaling, and cytoskeletal regulation. In this study, three ARMC genes, termed LvARMC6, LvARMC7 and LvARMC8, were identified and characterized from Litopenaeus vannamei. The complete cDNAs open reading frames (ORF) of LvARMC6, LvARMC7, and LvARMC8 (GenBank accession no. MG735126, MG728109 and KX058562) were 1410u202fbp, 570u202fbp and 2046u202fbp, encoding 469, 189, and 681 amino acids, respectively. Topology analysis indicated that three ARM domains were present in LvARMC6, one in LvARMC7 and six in LvARMC8. The identities of all the three LvARMCs with other species were between 50% and 71%. Phylogenetic analysis illustrated that different subtype of ARMCs formed their own separate branches and LvARMCs were placed in branch of invertebrates respectively with strong bootstrap support. The constitutive expressions of LvARMCs were confirmed by real-time quantitative PCR. LvARMC6, LvARMC7 and LvARMC8 were expressed highest in heart, gills and epithelium, respectively. After challenge with either white spot syndrome virus (WSSV), Vibrio parahemolyticus, or Staphylococcus aureus, all of the LvARMCs demonstrated differential expression profiles in hemocytes, hepatopancreas, intestine and gills. Taken together, our results suggest that LvARMCs may play a role in the innate immune defense against pathogenic viral and bacterial infections of L. vannamei.