Gonad maturation and spawning of cobia,Rachycentron canadum (Linnaeus, 1766) off the Dungun coast,Malaysia

The reproductive biology of cobia, Rachycentron canadum, from the coastal waters of Dungun, Malaysia was studied from June 2014 to May 2015. From commercial trawls, a total of 201 samples (combined sexes) were collected (fork lengths [FL] 37.5–124.0 cm; body weights 0.5–20.4 g). The overall sex ratio of females to males was 1:0.9, which was not significantly different (χ² = 2.12, df = 1; p < .05). Estimations of length at 50% maturity (L₅₀) showed that both sexes matured at approximately 75 cm FL; estimated spawning frequency was 6 days. Mean batch fecundity (BF) ranged from 0.55 to 4.32 million eggs. The average number of eggs per gram of ovary was from 2,100 to 5,400 eggs, and relative fecundity 147 eggs/g. There was a weak positive correlation (r² = .48) between BF and female FL as well as BF with an ovary‐free body weight (r² = .56), possibly due to females being in a continuous spawning condition and some possibly half‐spent, based on the histological examination of the female gonads. Despite cobia being asynchronous spawners, the gonadosomatic index in both males and females showed peaks in June, November, and particularly March. Based on histological examination, spawning‐capable males were encountered throughout the study period, whereas spawning‐capable females in the late developing subphase were found mostly in March and April. This is the first study on the reproductive aspects of cobia in Malaysian waters.     

Effect of dietary vitamin C on the growth performance and innate immunity of juvenile cobia (Rachycentron canadum)

This study was conducted to evaluate the effects of dietary vitamin C on growth performance, hematologic parameters and innate immune responses in juvenile cobia, Rachycentron canadum. Seven practical diets were formulated to contain 0.0 (as the basal diet), 13.6, 27.2, 54.4, 96.6, 193.4 and 386.5 mg ascorbic acid equivalent kg(-1) diet. Each diet was fed to triplicate groups of juvenile cobia with initial body weight of 5.5 g in 500-L cylindrical fiberglass tank. The results of 8 weeks feeding trial showed that typical vitamin C-deficient signs such as spinal deformation and body nigrescence were observed in the fish fed the basal diet. Fish fed the basal diet had significantly lower weight gain, specific growth rate (SGR), protein efficiency ratio (PER) and feed efficiency (FE) than those fed the diets supplemented with vitamin C, but no significant differences were observed among diets supplemented with vitamin C. However, survival rate was significantly affected by the dietary vitamin C levels, fish fed the basal diet had lower survival rate than those fed the diets supplemented with vitamin C. The ascorbic acid concentration in liver was correlated positively with the dietary vitamin C levels, however, the thiobarbituric acid reactive substances (TBARS) concentrations in liver was not significantly affected by the dietary vitamin C levels, although, fish fed the basal diet had the highest TBARS values among all treatments. The activities of serum lysozyme, superoxide dismutase (SOD), alkaline phophatase (AKP) and total immunoglobulin (Ig) were significantly influenced by the dietary vitamin C levels, fish fed the basal diet had lower lysozyme, SOD, AKP and total Ig than those fed diets supplemented with vitamin C. The serum glucose and triglyceride concentrations were significantly affected by the dietary vitamin C levels. Fish fed the basal diet had lower red blood cell and hemoglobin values than those fed the vitamin C supplemented diets. The challenge experiment with Vibrio harveyi showed that lower cumulative survival was in fish fed the unsupplemented diet, the cumulative survival were significantly increased with increase of the dietary ascorbic acid levels from 13.6 to 96.6 mg kg(-1), while the cumulative survival reached plateau when dietary ascorbic acid levels increased from 96.6 to 386.5 mg kg(-1). These results indicated that dietary vitamin C did significantly influence on growth performance and immune response of juvenile cobia.     

Genome-wide characterization of microsatellites in cobia Rachycentron canadum (Linnaeus, 1766): Survey and analysis of their abundance and diversity

The cobia Rachycentron canadum, mainly distributed in the warm waters of tropical and subtropical regions around the world, remains a fish of considerable economic importance. Detailed diversity and the number of microsatellite sequences in the cobia genome are still unintelligible. The primary aim of this work was to identify and quantify the miscellaneous SSR sequences in the cobia genome. More than 280,000 sequences were sequenced and screened using next-generation sequencing technology and microsatellite identification. Perfect mononucleotide repeats, dinucleotide microsatellites, and trinucleotide microsatellites contain (A)₁₀/(T)₁₀, (AC)₆/(TG)₆, and (AAT)_5–32 as the largest number of motifs in each type of microsatellite, respectively. The tetranucleotide and pentanucleotide microsatellites (TTM and PTM) consist of the largest number of motifs of both (ATCT)_5–32 and (TCAT)_5–31 in TTMs, and (CTCTC)_5–9 in PTMs, whereas the hexanucleotide microsatellites are rarely observed in the cobia genome. All c. 38000 sequences of composite microsatellites are extremely diverse, including compound (11.71%), interrupted compound (71.77%), complex (0.45%), and interrupted complex (16.07%). In this study, we developed a convenient and useful recording system for writing down and categorizing diverse composite microsatellite types. This system will provide great support for exploring repeat origins, evolutionary mechanisms, and the application of polymorphic microsatellites.     

饲料中不同水平n-3 HUFA对军曹鱼生长及脂肪酸组成的影响

以鱼粉、豆粕和酪蛋白为蛋白源,通过调节饲料中的牛油和鱼油水平,使饲料中高度不饱和脂肪酸（n-3HU-FA）的含量分别达到0．60、0．96、1.24、1．56、1．77和2．11％,配制出6种等氮等能的实验饲料,并以此饲料投喂军曹鱼幼鱼（8．3±0、5g）8周。实验结果显示饲料中n-3HUFA水平对实验鱼成活率没有显著影响,但却显著影响了军曹鱼的特定生长率（SGR）。随着饲料中n-3HUFA水平的升高,SGR相应提高,但当饲料中n-3HUFA水平高于1．24％时,各处理之间的生长没有出现显著差异。随着饲料中n-3HUFA水平的升高,肌肉和肝脏中18：1n-9的含量逐渐下降,而22：6n-3的水平相应升高。根据实验结果,当以特定生长率为评价指标时,用折线模型估计出军曹鱼对饲料中n-3HUFA的需求量约为1．49％。     

盐度对军曹鱼胚胎和仔鱼发育的影响

观察比较了不同盐度梯度(20、23、26、29、32、35、38、41和44)下军曹鱼受精卵的沉浮性、孵化率和畸形率以及测定了不同盐度梯度(10、14、18、22、26、30、34、38、42和44)下仔鱼不投饵存活系数SAI值。结果表明,受精卵在海水盐度32以上为完全浮性,26以下为完全沉性。军曹鱼受精卵孵化的最适盐度范围为29～38,适宜盐度范围为26～41,其中23～26和41～44分别视为受精卵孵化在低盐区和高盐区的两个临界区域,在此盐度上下,军曹鱼受精卵孵化率大幅度下降而仔鱼畸形率大幅度上升。试验盐度范围内军曹鱼仔鱼SAI值为2.32～16.24,仔鱼生长和存活的最适盐度范围为26～34,适宜盐度范围为22～38,而盐度18～22及38～42可分别视为军曹鱼仔鱼存活率在低盐区和高盐区的两个临界区域。     

Identification and expression analysis of cobia (Rachycentron canadum) Toll-like receptor 9 gene

Cobia culture is hindered by bacterial infection (Photobacterium damselae subsp. piscicida) and in order to study the effect of P. damselae subsp. piscicida challenge and CpG ODN stimulation on cobia Toll like receptor 9 (RCTLR9), we used PCR to clone RCTLR9 gene and qRT-PCR to quantify gene expression. The results indicated that RCTLR9 cDNA contains 3141 bp. It encodes 1047 amino acids containing 16 typical structures of leucine-rich repeats (LRRs) including an LRRTYP, LRRCT and a motif involved in PAMP binding was identified at position 240–253 amino acid. Broad expression of RCTLR9 was found in larval, juvenile and adult stages irrespective of the tissues. In larval stage, RCTLR9 mRNA expression decreased at 5 d and then increased at 10 dph. At juvenile stage cobia, the expression was significantly high (p < 0.05) in spleen and intestine compared to gill, kidney, liver and skin. However, at adult stage, the significant high expression was found in gill and intestine. Cobia challenged with P. damselae subsp. piscicida showed significant increase in RCTLR9 expression at 24 h post challenge in intestine, spleen and liver, while in kidney the expression was peak at 12 h and later it decreased at 24 h. The highest expression was 40 fold increase in spleen and the lowest expression was ∼3.6 fold increase in liver. Cobia stimulated with CpG oligonucleotides showed that the induction of these genes was CpG ODN type and time dependent. In spleen and liver, CpG ODNs 1668 and 2006 injected group showed high expression of RCTLR9, IL-1β, chemokine CC compared to other groups. Meanwhile, CpG ODN 2006 has induced high expression of IgM. The CpG ODNs 2395 have induced significant high expression of Mx in spleen and liver. These results demonstrates the potential of using CpG ODN to enhance cobia resistance to P. damselae subsp. piscicida infection and use as an adjuvant in vaccine development.     

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.     

摄食不同淀粉含量饲料对军曹鱼血清生化指标的影响

实验旨在研究经过10周投喂不同淀粉含量的饲料的养殖实验后, 禁食1d后再投喂对170 g军曹鱼血清生化指标的影响. 饲料以鱼粉为蛋白源, 鱼油、豆油、大豆卵磷脂为脂肪源, 分别添加0%(对照组)、6%、12%、18%、24%和30%的小麦淀粉(以微晶纤维素调平), 养殖实验期间每天饱食投喂2次. 结果表明, 饥饿再投喂饲料后各处理组军曹鱼血糖含量均在(0-5)h升高, 在5h时达到峰值, 并显著高于其他时间组, 摄食5h后开始下降, 7h时基本恢复到投喂前水平; 血清总蛋白含量在不同处理组以及同一处理组的不同时间点上均没有显著差异; 血清中甘油三酯含量在(0-5)h显著升高, 5h后其含量开始下降, 7h时基本恢复到投喂前水平. 其中0%、6%和12%组, 摄食5h后其甘油三酯含量显著高于摄食前与摄食后7h和24h, 而在18%、24%和30%组中, 其含量无显著差异; 0%淀粉组血清中胆固醇含量在(0-2)h显著升高, 2h时达到峰值, 在(2-7)h呈下降趋势, 之后趋于平稳. 其他处理组都是在(0-5)h升高, 5h后开始下降, 在7h时基本恢复到投喂前水平. 18%-30%组, 摄食后5h血清中胆固醇含量显著高于摄食前与摄食后24h. 军曹鱼血清中低密度脂蛋白-胆固醇在(0-5)h显著升高, 摄食5h后其含量开始下降, 7h时基本恢复到投喂前水平, 但各组不同时间点间均无显著性差异. 而血清中高密度脂蛋白-胆固醇含量除在24%添加组中, 摄食后5h显著高于摄食前(0h)及投喂结束后24h处理组, 其他处理之间无显著差异. 综上所述: 170 g军曹鱼禁食再摄入不同含量的小麦淀粉后其血清中血糖含量先升高后降低, 随着饲料中糖添加量的增加, 其峰值先增加, 后趋于平稳. 军曹鱼血清甘油三酯和胆固醇的含量会升高, 对其血清中低密度脂蛋白-胆固醇及高密度脂蛋白-胆固醇影响不显著.       

饵料系列对军曹鱼仔鱼生长、消化酶活力和体成分的影响

饵料是鱼类早期发育最重要的条件之一,并且随着个体发育,饵料类别不断变化。为了评定各种饵料的效果并筛选得到军曹鱼仔鱼早期发育阶段适宜的饵料组合,进行了本实验。军曹鱼仔鱼在750L桶中孵出并暂养2d,然后在水泥池中培育4d（3—6日龄）,并投喂轮虫;在第7日龄,将240尾仔鱼随机平均分配至12个70L桶中,共分4组,每组3桶;7—15日龄,4组仔鱼分别投喂轮虫、卤虫无节幼体、桡足类和桡足类投;16—42日龄,分别投喂轮虫、卤虫无节幼体、桡足类和鱼糜。第42日龄,将全部仔鱼取样、称重并于-80℃保存备用。鱼体匀浆、离心后的上清液作为粗酶液用以测定蛋白酶、淀粉酶和脂肪酶等消化酶活力,还测定了粗蛋白、粗脂肪等主要体成分,并统计了生长指标。结果表明：（1）组IV的生长表现最佳;（2）组III的成活率最高;（3）饵料对三种酶的活力和体成分都有极显著的影响;（4）组III的蛋白酶活力、组I的淀粉酶活力和脂肪酶活力最高,尤其是组I的淀粉酶活力比其他各组高出6.8—11.9倍;（5）各组鱼体的粗蛋白含量均较相应饵料的低,而粗脂肪含量则比饵料的高。因此,可以认为：仔鱼长期饥饿时,消化酶可能存在补偿性分泌,并优先利用糖类和脂肪作为能源;军曹鱼仔鱼对脂肪的沉积效率较蛋白高。     

The effect of feeding rate on growth of pectoral fin spine of juvenile great sturgeon Huso huso (Linnaeus, 1758)

The effect of two feeding rates (0.5 and 1.5% of total body weight) was assessed on the growth of pectoral fin spines of captive juvenile great sturgeon, Huso huso, after second year of life. The fish received Oxytetracycline (OTC) twice in the first and second years of their lives under basic diet. During the 5‐month experimental period, juveniles (mean 1,187.4 g, 0.1 standard deviation [SD]), n = 50) were reared with two feeding rates under similar conditions in 10 fiberglass tanks (1.5 m³). The fish were fed manually with a commercial diet twice a day (35% Biomar, Nersac, France) throughout the experiment. The OTC marks were distinguished in all pectoral fin spine sections under ultraviolet light. The means of the first and second annular radii were 806.6 µm (27.2 SD) and 2,246.5 µm (50.2 SD), respectively. The marginal increment analysis beyond the second OTC mark revealed a significantly smaller marginal increment for low feeding rate treatment (143.9, 11.2 SD) as compared to the high feeding rate one (269.0, 14.6 SD). The results indicate the slower growth rate in the fish fed the low feeding treatments seen in the pectoral fin spine formation, which can be used as an indicator of recent feeding history in sturgeon juveniles. The best daily feeding rate for great sturgeon of 2,460 g was determined to be 1.5% body weight/day in this study.     

军曹鱼(Rachycentron canadum)早期发育阶段的摄食及其影响因子*

研究了军曹鱼(Rachycentron canadum)仔、稚鱼的日摄食节律和摄食强度,以及饵料密度、温度、盐度对其摄食的影响。结果表明,发育8d仔鱼(摄食轮虫)、23d稚鱼(摄食卤虫无节幼体)和38d稚鱼(摄食鳗鱼粉状饵料)均表现为明显的昼夜摄食节律,摄食主要在白天进行,白天摄食强度占日摄食强度的68%以上;在白天的摄食中,又以早晨6:00-8:00和傍晚16:00-18:00摄食强度最大:夜间摄食较少或基本不摄食,所以军曹鱼早期幼体的摄食习性属白天摄食且偏于晨昏性类型。在不同饵料密度梯度的试验中,发育8d-11d仔鱼和23d-26d稚鱼摄食的适宜饵料密度范围分别为15ind·mL^-1-20ind·mL^-1和7ind·mL^-1-2ind·mL^-1。温度和盐度对发育21d-24d稚鱼日摄食强度的影响均表现为抛物线型的变化曲线,摄食的适宜水温范围为27-31℃,适宜盐度范围为28‰-32‰。结果还表明,幼休摄食强度与饵料密度、水温、盐度的关系均适合于用二次多项式来定量描述。     

Ontogeny of the gastrointestinal tract and selected digestive enzymes in cobia Rachycentron canadum (L.)

The ontogeny of the digestive system of cobia Rachycentron canadum from hatching to 22 days post-hatch (dph) (20·1 mm standard length) was examined with light microscopy. The activities of selected pancreatic enzymes were also determined during this period in order to optimize current rearing methods for this species. At hatching (3·6 mm), the digestive tract consisted of a relatively undifferentiated, straight tube positioned dorsally to the yolk sac. The major morphological changes in the digestive tract primarily occurred over the first 1–4 dph (3·6–4·4 mm). During this time, larvae began exogenous feeding (3 dph) and the digestive tract differentiated into five histologically distinct regions: buccopharynx, oesophagus, stomach anlage, anterior intestine and posterior intestine. Yolk reserves were exhausted by 5 dph (4·5 mm) and the oil globule began rapidly decreasing in size disappearing entirely by 9–10 dph (6·3–6·8 mm). Gastric glands differentiated at this time, and by 12 dph (8·1 mm) surface mucous cells of the stomach anlage stained positive for neutral mucosubstances. By 16 dph (11·6 mm), the blind sac (fundic region) of the stomach formed as did the pyloric caecae which initially appeared as a single protrusion of the anterior intestine just ventral to the pyloric sphincter. Generally, enzyme activities (U larva⁻¹) for amylase (0·0–1·8), chymotrypsin (0·0–7902·4), trypsin (0·2–16·6) and lipase (9·3–1319·0) were measurable at or soon after hatching and increased steadily from c. 8–22 dph (5·7–20·1 mm). The results of this study are discussed in terms of current and future weaning practices of this species.     

急性盐度胁迫对军曹鱼稚鱼渗透压调节的影响

研究了环境盐度急性胁迫对军曹鱼(Rachycentron canadum)稚鱼鳃Na⁺-K⁺ATPase(NKA)活性及血清渗透压、Na⁺、K⁺和Cl^-离子调节的影响.结果表明:将稚鱼从盐度37中直接转移至盐度0、5、15、25、37（对照）和45的水体中,12 h后仅盐度0处理出现死亡(死亡率100％).各处理鳃NKA活性和血清渗透压在最初3 h内出现一定波动,随后变化平稳.试验结束时(12 h), NKA活性与盐度梯度呈“U”型分布,盐度5处理酶活性显著高于其它处理(P<0.05),盐度15处理活性最低,而各处理的血清渗透压大小(293～399 mOsmol·kg^-1)与盐度呈正相关;在3～12 h内稚鱼血清Na⁺和Cl^-浓度随盐度升高而升高,但增幅较小,血清K⁺浓度则与盐度呈负相关;12 h稚鱼的等渗点为328.2 mOsm·kg^-1,相当于盐度11.48,而Na⁺、K⁺和Cl^-等离子点分别为155.2、6.16和137.1 mmol·L^-1,分别相当于盐度10.68、20.44及8.41.军曹鱼在生理上具有广盐性鱼类的“低渗环境高NKA活性”特征,有较强及迅速的渗透压和离子调节与平衡能力.     

Ecological niche models reveal the potential zones of invasion of the cobia (Rachycentron canadum) in the Eastern Pacific Ocean

Hydrobiologia - Cobias (Rachycentron canadum) are large fish that live in all the tropical oceans of the world, except for the Eastern Pacific Ocean (EPO). During 2015, a cobia cage culture was...     

Purification of a toxic metalloprotease produced by the pathogenic Photobacterium damselae subsp. piscicida isolated from cobia (Rachycentron canadum)

The aim of the present study was to purify and characterize a toxic protease secreted by the pathogenic Photobacterium damselae subsp. piscicida strain CP1 originally isolated from diseased cobia (Rachycentron canadum). The toxin isolated by anion exchange chromatography, was a metalloprotease, inhibited by L-cysteine, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA), 1,10-phenanthroline, N-tosyl-L-phenylalanine-chloromethyl ketone (TPCK), and N-alpha-p-tosyl-L-lysine-chloromethyl ketone (TLCK), and showed maximal activity at pH 6.0-8.0 and an apparent molecular mass of about 34.3 kDa. The toxin was also completely inhibited by HgCl2, and partially by sodium dodecyl sulfate (SDS) and CuCl2. The extracellular products and the partially purified protease were lethal to cobia with LD50 values of 1.26 and 6.8 microg protein/g body weight, respectively. The addition of EDTA completely inhibited the lethal toxicity of the purified protease, indicating that this metalloprotease was a lethal toxin produced by the bacterium.