Partial characterization and ontogenetic development of pancreatic digestive enzymes in Japanese eel Anguilla japonica larvae

The pancreatic digestive enzymes, trypsin, chymotrypsin, lipase and amylase were partially characterized, and changes in their activities were examined during the initial ontogeny of Japanese eel Anguilla japonica larvae from 5 to 34 days post-hatching (dph). The pH optima of the eel larval enzymes were narrower than those other fish species; trypsin activity was highest at pH 9, chymotrypsin and amylase activities were highest at pH 7 and 8, and lipase activity was highest at pH 8 and 9. In an analysis of thermal profiles, the larval pancreatic enzymes had a high optimal temperature and high thermal stability, which are typical of fish from the tropics. At 12 and 13 dph, lipase activity and gene expression levels of trypsin (-a and -b), lipase and amylase decreased markedly, suggesting a marked change in larval metabolism at that time. These data could be useful in the development of artificial larval diets in Japanese eel.     

Temporal expression analyses of pancreatic and gastric digestive enzymes during early development of the olive flounder (Paralichthys olivaceus)

The olive flounder (Paralichthys olivaceus) is one of the most important aquaculture species in Korea. Previous studies focused on the morphological changes and only analysed digestive enzyme expression patterns of certain digestive systems. A comprehensive analysis of the pancreatic and gastric digestive enzymes of the olive flounder in its early developmental stages has not been performed. In this study, we determined the mRNA expression levels of pancreas and stomach digestive enzymes by extracting total RNA from the whole body of olive flounders from hatching to post‐metamorphosis and conducting real‐time PCR analysis with gene‐specific primers for each isoform. The pancreatic digestive enzymes were initially and strongly expressed from the first feeding period to pre‐metamorphosis. The expression of trypsinogen 3 was detected from the early stage of development to metamorphosis, whereas trypsinogen 2 was significantly increased only in the post‐metamorphosis period. The mRNA expression of pepsinogen was only detected at metamorphosis and corresponded to stomach differentiation. The lipid digestive‐related enzymes had already reached a certain level at the beginning of hatching, then increased during the early developmental stage and gradually decreased before metamorphosis. These results suggest that food ingestion is exclusively digested by pancreatic digestive enzymes and trypsinogen 3 during the early developmental stage until pre‐metamorphosis, and it then fully switches to digestion by pepsinogen and trypsinogen 2 in mid‐metamorphosis. In conclusion, understanding the expression profiles of digestive enzymes in the pancreas and stomach during the early developmental stages is an important consideration when developing formulated larval diets for aquacultured olive flounder.     

Application of microparticle diets for Japanese flounder Paralichthys olivaceus larvae

ABSTRACT:   A feeding trial was conducted with two newly developed microparticle diets (MD-Q and MD-V), which differed in the mixing ratios of two types of casein hydrolysates (C700 and C800) as the protein source. The ratio of C700 and C800 were adjusted to 7:3 in MD-Q and 6:4 in MD-V, respectively. Japanese flounder larvae were fed from 7 days after hatching (d.a.h.) to 32 d.a.h. on live food (LF), MD solely, MD + 1/3 LF (either of the MD and one-third quantity of the live food) and 1/3 LF (one-third quantity of live food alone), respectively. They were then switched to Kyowa diet B from 33 d.a.h. until 40 d.a.h. The larvae fed on MD-Q exclusively had a higher survival rate (36.4%) than those fed on MD-V (24.2%) by 22 d.a.h. Unfed larvae could survive up to only 12 d.a.h. Larvae fed on MD-Q + 1/3 LF also had a significantly higher survival rate (34.2%) than those on MD-V + 1/3 LF (16.3%) and 1/3 LF solely (15.4%) at 32 d.a.h. These results suggest the potential of MD-Q as a part replacement for LF from the early developmental stage in the seed production of Japanese flounder.     

Development of microparticle diets for Japanese flounder Paralichthys olivaceus larvae

ABSTRACT:   Two newly designed microparticle diets (MD), with two kinds of peptide (C700 and C800) as a protein source were developed. Microparticle diet Q (MD-Q) contained C700 (molecular weight ∼30 000 Da) and C800 (molecular weight 1000–2000 Da); in contrast, microparticle diet T (MD-T) contained C700 only. Two separate trials, representing larvae from different hatches, were conducted. Japanese flounder Paralichthys olivaceus larvae were fed newly designed MD or a combination of them with live food (LF) between 3 and 10 days after hatching (d.a.h.) in experiment I, or between 11 and 20 d.a.h. in experiment II, and compared them to LF and a commercial diet (CD) feeding groups. The growth and survival rates in both experiments were the highest in the LF treatment. But in the microparticle treatment larvae fed MD-Q had a higher survival rate and better growth than those fed MD-T, either alone or in combination with LF. First-feeding larvae fed on MD-Q had a 20.5% survival rate and 1.12 mm gain by 10 d.a.h. Unfed larvae died within 6 d.a.h. These results indicate that using a mixture of different molecular weight peptides is good protein sources and also this type of microparticle diet can be applied to flounder from larval to juvenile stages.     

Ontogeny of pancreatic enzymes in larval red drum Sciaenops ocellatus

The growth, survival and trypsin, lipase and amylase activities of red drum larvae were measured in two experiments. For the first trial, a group was fed live prey only (L) and another group was fed a combination of a microparticulate diet (MPD) and live food (L-MP). For the second growth trial a group fed the MPD only (MP) and a starvation group (ST) were examined in addition to the L and L-MP treatments. Enzyme activities of live prey were measured to estimate their possible contribution to larval digestion. No significant ( P  > 0.05) differences in final size and survival were observed between treatments L and L-MP. Larvae subjected to starvation or fed the MPD diet alone were smaller than treatments fed live prey and did not survive past days 5 and 14, respectively. Trypsin, lipase and amylase activities were detectable at hatching. No significant differences ( P  > 0.05) in total enzyme activities among treatments were observed before day 14. Specific activity of trypsin, lipase and amylase peaked on day 3 (prior to first feeding) and subsequently decreased. For trypsin, the percentage of enzyme activity potentially attributable to ingested prey increased with age to a maximum of 17%. For lipase and amylase this fraction was less than 5% throughout the study, except on day 8 (12% and 24%, respectively). The lack of significant differences observed in the activity of digestive enzymes among treatments suggests that dietary regime, availability of prey and possible effects of exogenous enzymes did not significantly influence enzyme activity. Therefore, the lower growth rate observed in the L-MP, MP and starved treatments cannot be attributed to low digestive enzyme production of the enzymes measured. It is more likely that the MPD failed to supply the required nutrients for adequate development.     

克氏原螯虾幼体发育时期消化酶活力及氨基酸含量研究

采用生物化学方法对克氏原螯虾各期幼体消化酶活力与氨基酸组成进行了分析。实验结果显示,在克氏原螯虾幼体发育过程中,五种消化酶活力(胃蛋白酶、类胰蛋白酶、淀粉酶、纤维素酶和脂肪酶)表现出两种变化模式:胃蛋白酶和类胰蛋白酶在幼体发育Ⅱ龄幼体期和Ⅳ龄幼体期活力较高,其中Ⅳ龄幼体期该两种酶活力最高;淀粉酶、纤维素酶和脂肪酶活力为先增高后降低,在Ⅲ龄幼体期,三种酶的活力达到最高,但与Ⅳ龄幼体期的酶活力比较无显著差异。总氨基酸含量在幼体发育早期逐渐降低,幼体发育至Ⅲ龄幼体期,其氨基酸含量最低,Ⅳ龄幼体期又有所增加。在测定的所有氨基酸中谷氨酸含量最高,必需氨基酸中赖氨酸含量最高,单个必需氨基酸含量与必需氨基酸总量的比值(A/E)在整个胚胎发育过程中的变化趋于一致。     

兴凯湖红鳍原鲔消化酶活力和肌肉氨基酸组成的季节变化

研究了不同季节体质量为107．824-22．97g的兴凯湖野生红鳍原鲐（Cultrichthys erythropterus）肝胰脏、肠道消化酶活力及夏季昼夜变化和肌肉氨基酸种类组成、含量及其变化,探讨其营养生态特性及变化规律。结果显示：不同季节红鳍原铂的蛋白酶、淀粉酶和脂肪酶的比活性显著不同（P〈0．05）,夏、秋季高于春、冬季,同一季节蛋白酶比活均显著高于淀粉酶比活（P〈0．05）;红鳍原鲐蛋白酶、淀粉酶和脂肪酶比活的昼夜变化为：19：00～21：00、19：00和16：00～19：00时值最高,7：00、7：00和21：00值最低。红鳍原鲴肌肉由18种氨基酸组成,冬季缬氨酸、蛋氨酸、异亮氨酸、苯丙氨酸和赖氨酸五种必需氨基酸的含量显著低于春、秋季（P〈0．05）,而非必需氨基酸的含量无明显的季节变化;不同季节谷氨酸含量均最高,最低为胱氨酸。春、夏季氨基酸总量和必需氨基酸总量较高,秋、冬季较低;秋季鲜味氨基酸含量最低。研究表明,野生红鳍原鲐消化生理具有不同季节和日变化特点,氨基酸组成的季节变化提示：冬季鱼体代谢活动对肌肉组织中必需氨基酸的依赖。     

Effect of cortisol and triiodothyronine bath treatments on the digestive enzyme profile and growth of Catla catla larvae during ontogenic development

Digestive enzyme profile is a good indicator of the nutritional and health status of the fish. The present investigation aims to evaluate the effect of exogenous bath treatment of hormones, cortisol and triiodothyronine, on the digestive enzyme activities and growth of carp Catla catla (Ham.) during ontogenic development. Catla larvae (4 days old) were given bath treatment with cortisol (hydrocortisone, 0.2 mg L^?1), 3,5,3′‐triiodothyronine (T3, 2.5 mg L^?1) and a combination of cortisol and T3 for 30 min. Digestive enzyme profile was recorded on every third day and was continued for 30 days. Larvae were fed with live food for initial 14 days and then weaned to mix feeding of live food and prepared diet. Significantly (P < 0.05) higher amylase, total protease, trypsin, chymotrypsin, lipase, chitinase and chitinobiase activities were found in the hormone‐treated groups compared to the control one during ontogenic development. Among the treated groups, amylase activity was highest in cortisol‐treated larvae. Total protease, trypsin, chymotrypsin, lipase, chitinase and chitinobiase activities were significantly (P < 0.05) higher in larvae exposed at combined treatment of cortisol and T3 compared to the other two groups in most sampling days. Average length, weight and specific growth rate of treated larvae were higher compared to the control one. The combined bath treatment of cortisol and triiodothyronine influenced the digestive enzyme activities of catla larvae and thereby enhanced the growth at early developmental stage. This helps the larvae to overcome the problems associated with early developmental stage.     

Deficiency of dietary niacin decreases digestion and absorption capacities via declining the digestive and brush border enzyme activities and downregulating those enzyme gene transcription related to TOR pathway of the hepatopancreas and intestine in young grass carp (Ctenopharyngodon idella)

To investigate the effects of niacin on growth, digestion and absorption capacity, and the potential mechanism for digestive and brush border enzyme activities, grass carp (Ctenopharyngodon idella) (256 ± 0.41 g) were fed diets containing 3.95 (basal diet group), 14.92, 24.98, 35.03, 44.97 and 55.01 mg niacin kg^?1 diet for 8 weeks. Results indicated that percentage weight gain (PWG), feed intake and feed efficiency were the lowest in basal group (P < 0.05). Similarly, niacin deficiency decreased hepatopancreas trypsin, chymotrypsin, lipase and amylase activities (P < 0.05), intestinal Na⁺, K⁺‐ATPase, alkaline phosphatase, γ‐glutamyl transpeptidase and creatine kinase (CK) activities, the cholecystokinin (CCK) content in proximal intestine (PI) and growth hormone content in serum (P < 0.05). Furthermore, niacin deficiency downregulated gene expression of hepatopancreas trypsinogen 1, trypsinogen 2, chymotrypsinogen and amylase, intestinal Na⁺, K⁺‐ATPase alpha subunit isoform 1, Na⁺, K⁺‐ATPase alpha subunit isoform 8 and CK, and target of rapamycin (TOR) and S6 kinase 1 (S6K1) of hepatopancreas and intestine (P < 0.05), whereas upregulated eIF4E‐binding protein (4EBP) gene expression (P < 0.05). The niacin requirement for young grass carp (256–689 g) based on PWG, hepatopancreas trypsin activity and Na⁺, K⁺‐ATPase in PI was 34.01, 35.10 and 42.08 mg kg^?1 diet, respectively.     

中国近海日本鳗鲡玻璃鳗体组织营养成分及氨基酸、脂肪酸组成的比较

为了解日本鳗鲡玻璃鳗的早期营养需求，分别对中国近海6个不同地点玻璃鳗体组织一般营养成分、氨基酸和脂肪酸含量进行了分析。结果显示，野生玻璃鳗体组织中水分含量为78.62%±2.03%，干重基础下粗蛋白质含量为69.08%±0.64%、粗脂肪含量为11.96%±1.33%、粗灰分含量为11.45%±1.30%。在台州、舟山、南通和东台4个采样点的样品中，水分含量无显著差异，而粗蛋白含量以舟山点最高、粗脂肪含量最低，并且舟山点样品肠道内发现可见内容物。玻璃鳗样品中氨基酸含量以谷氨酸、天门冬氨酸、赖氨酸、亮氨酸最高，半胱氨酸最低；除舟山点外，各采样点氨基酸含量无明显差异。不同洄游地点玻璃鳗组织中脂肪酸含量均以C16:0、 C18:1n9、 C20:5n3(EPA)、 C22:5n3、 C22:6n3(DHA)为主要成分，其中EPA和DHA共占脂肪酸总量的31.59%±1.00%，不同洄游地点样品必需脂肪酸含量差异明显。研究表明，必需氨基酸、必需脂肪酸对于玻璃鳗的正常生长存活具有重要意义。此外，对比不同地点营养物质组成及稚鱼饵料理论需求量分析，推测舟山点海洋环境中包含更适合玻璃鳗稚鱼摄食的食物，...     

Ontogeny of the digestive tract of thick lipped grey mullet (Chelon labrosus) larvae reared in “mesocosms”

This work describes the ontogeny of the digestive tract in thick lipped grey mullet (Chelon labrosus) larvae reared until day 36 post-hatching with the semi-extensive technology in mesocosms. Diet was constituted by live preys, rotifers, Artemia and wild zooplankton, then compound diet was added from day 20 (p. h.). Linear growth, weight growth and digestive enzymes specific activities were studied during larval ontogeny. Pancreatic enzymes (trypsin and amylase) and intestinal enzymes (leucine-alanine peptidase “Leu-ala”, aminopeptidase N “AN” and alkaline phosphatase “AP”) were assayed in larvae sampled throughout the rearing trial to evaluate gastrointestinal maturation along the development.The trypsin specific activities were very high during the first two weeks and then declined as observed in marine fish species. A following increase in trypsin specific activity from day 20 was attributed notably to ingestion of particle compound diet. In contrast to the pattern generally described in fish larvae, amylase specific activity showed a continuous increase. This could be attributed to the fact that C. labrosus is an omnivorous species and suggests that the fish might be able to use efficiently diets containing higher levels of starch or other carbohydrates since the end of larval development.Relative expression of intestinal brush border membrane enzymes (AP and AN) and cytosolic enzyme (Leu-ala), showed an abrupt increase of both AP/leu-ala and AN/leu-ala ratios at day 8 (p. h.), indicating that maturation of intestinal tract in C. labrosus larvae is particularly precocious. It is assumed that larvae of C. labrosus might support early co-feeding and weaning strategies, which could reasonably be initiated since mouth opening.     

Molecular characterization of two trypsinogens in the orange-spotted grouper, Epinephelus coioides, and their expression in tissues during early development

In this study, we cloned two trypsinogens of the orange-spotted grouper, Epinephelus coioides, and analyzed their structure, expression, and activity. Full-length trypsinogen complementary (c)DNAs, named T1 and T2, were 900 and 875 nucleotides, and translated 242 and 244 deduced amino acid peptides, respectively. Both trypsinogens contained highly conserved residues essential for serine protease catalytic and conformational maintenance. Results from isoelectric and phylogenetic analyses suggested that both trypsinogens were grouped into trypsinogen group I. Both trypsinogens had similar expression patterns of negative relationship with body weight; expression was first detected at 1 day post-hatching (DPH) and exhibited steady-state expression during early development at 1–25 DPH. Both expression and activity levels significantly increased after 30 DPH due to metamorphosis. Grouper larval development is very slow with insignificant changes in total length and body weight before 8 DPH. The contribution of live food to an increase in the trypsin activity profile may explain their importance in food digestion and survival of larvae during early larval development.     

Modeled transport of winter flounder larvae spawned in coastal waters of the Gulf of Maine

Winter flounder, Pseudopleuronectes americanus, from southern New England and the Gulf of Maine were historically considered to be obligate estuarine spawners. However, recent experiments and observations document that winter flounder in the Gulf of Maine also utilize coastal waters for spawning. An individual‐based modeling approach was used to investigate the transport of winter flounder larvae from three hypothesized coastal spawning grounds in the Gulf of Maine. Transport success rates were greatest for larvae released from Ipswich Bay, intermediate for Stellwagen Bank and least successful for those released from Jeffreys Ledge. There was substantial interannual variability in larval transport and geographic patterns of potential connectivity. Furthermore, the date of spawning had an important influence on transport success. Model results suggest that certain coastal spawning grounds used by winter flounder may serve as an important source of larvae to estuaries and nearshore nursery areas. The potential influx of coastal spawned larvae could have implications for the resilience, productivity and gene flow in local populations. Model results provide further support for the conclusion that winter flounder in the Gulf of Maine may not be solely dependent upon estuaries for spawning. Results also suggest that coastal spawning groups should be considered explicitly in the management of winter flounder, and protected under Essential Fish Habitat regulations.     

牙鲆NR4A1基因在甲状腺素诱导仔鱼变态中的表达调控分析

NR4A1基因是一个重要的转录因子,在信号转导早期通过对靶基因的特异性转录调控发挥着重要的作用。从牙鲆(Paralichthys olivaceus)中克隆了NR4A1基因c DNA序列,检测了其在牙鲆变态中和成鱼各组织中的表达模式,分析了其在外源甲状腺素(TH)以及硫脲(TU)处理仔鱼中的表达变化。结果表明,牙鲆NR4A1 c DNA全长3264 bp,编码568个氨基酸;牙鲆NR4A1基因与其它物种具有很高的同源性,在系统进化树中与鱼类聚为一支;牙鲆NR4A1基因在成鱼心、肌肉和鰓中高表达;在变态过程中,NR4A1水平逐渐升高,且在25 dph达到最高,之后逐渐降低;TH组中的NR4A1基因水平在变态早期和高峰期显著低于正常组,TU组的水平在变态中后期和结束期显著高于正常组;TU组中变态被抑制的仔鱼在正常海水和0.1 mg·L~(-1)TH海水中饲养6 d后,能够顺利变态,且NR4A1基因在拯救组(TU抑制变态仔鱼在正常海水和0.1 mg·L~(-1)TH海水中饲养)中的表达水平与TU对照组相比显著降低,与正常组中同时期的水平一致。结果表明,牙鲆NR4A1基因可能在仔鱼变态调控中扮演着非常重要的角色。     

胭脂鱼仔鱼的生长和消化酶活力变化

对胭脂鱼(Myxocyprinus asiaticus)孵化后1～37 d仔鱼的体长、体重、RNA/DNA比率、胰蛋白酶、脂肪酶和淀粉酶的酶活力变化进行了测定。结果显示:胭脂鱼仔鱼1～37 d体长稳定增长。体重增加呈阶段性,在1～21 d体重增长缓慢,21～29 d体重增加较快,29～37 d体重快速增加。仔鱼从7 d开始摄食,1～6 d,RNA/DNA比率变化很小,蛋白含量逐渐降低;7～21 d,RNA/DNA比率波动较大,蛋白含量增长缓慢;21～37 d,RNA/DNA比率和蛋白质均增长迅速。生长率(GR)与RNA/DNA比率的回归方程为:GR=1.01RNA/DNA+1.6077(R2=0.8516)。从2、5和7 d开始分别检测到明显的胰蛋白酶、脂肪酶和淀粉酶活力,之后,消化酶活力随着发育先快速地上升然后波动性地下降到一个稳定水平。结果表明:胭脂鱼仔鱼从内源性到外源性营养转化过程中,不同的消化酶能够检测到明显酶活力的时间不同,到仔鱼开始摄食时已经具备消化蛋白质、脂肪和淀粉的能力;RNA/DNA比率可以作为判定仔鱼生长率的可靠指标。     

Effect of dietary taurine and lipid contents on conjugated bile acid composition and growth performance of juvenile Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

ABSTRACT:   The effects of dietary taurine levels and lipid contents on the conjugated bile acid composition and growth performance of juvenile Japanese flounder Paralichthys olivaceus were investigated. Six types of diet (three different levels of taurine at two different levels of lipid) were fed to juveniles (average body weight, 0.04 g). Fishmeal that was washed with 70% ethanol to remove taurine was used as the sole protein source. Feeding experiments were carried out at 20°C for 6 weeks. At the end of the experiments, fish were weighed and analyzed for free amino acids in the body and the composition of the conjugated bile acids. The body weight and percent weight gain of the juveniles were improved by the dietary taurine supplementation. The taurine contents of the whole body and tissues increased with the increase of the dietary taurine contents. The conjugated bile acids in the gall bladder consisted of taurocholic acid and taurochenodeoxycholic acid, which increased with the increase of the dietary taurine level. Taurocholic acid accounted for more than 95% of the total conjugated bile acids. This indicates that taurine is the sole amino acid to conjugate bile acid in Japanese flounder.     

Digestive Enzyme Activity during Larval Development of Black Snook,Centropomus nigrescens

Black snook, Centropomus nigrescens, have been identified as a promising candidate for aquaculture although, like many of the Centropomid species, high mortality associated with early larval stages presents a significant bottleneck to their commercialization. The digestive capacity of black snook larvae throughout the first 37 d after hatch (d.a.h.) was evaluated by quantifying digestive enzyme activities using biochemical techniques. Results showed that black snook larvae have alkaline proteases at hatching, which are known to be important during the first days of feeding for digestion. Toward the end of the study, acid proteases concentration increased (37 d.a.h.). Enzymes for lipid digestion, pancreatic lipase and bile salt‐activated lipase, were already present in the larvae before exogenous feeding commenced, and their activity increased with age and growth (length). Intracellular digestion, measured as the activity of leucine‐alanine peptidase, was high early on (5 d.a.h.) and decreased as development progressed (next 32 d). In contrast, alkaline phosphatase activity was lowest at first feeding and subsequently increased with age. Overall patterns in enzyme activity suggest the possibility of live feed weaning before 32 d.a.h. if artificial diets can be properly balanced.     

Hen egg yolk and skinned krill as possible foods for rearing leptocephalus larvae of Anguilla japonica Temminck & Schlegel

Usual diets for rearing leptocephalus larvae of Japanese eel Anguilla japonica include eggs of the endangered spiny dogfish Squalus acanthias (SE). We investigated the effects of alternative food materials, hen egg yolk (HEY) and exoskeleton‐free (skinned) Antarctic krill (SAK), on the growth and survival of eel larvae. We found that feed comprising whole krill including exoskeleton (WAK) containing higher levels of fluoride (37.89 mg kg^?1) was acutely toxic to eel larvae exposed to this alone. In contrast, extract from SAK containing lower concentrations of fluoride (4.25 mg kg^?1) showed no apparent adverse effects. Growth of larvae fed a mixture of SE and SAK in a feed trial of 58 days [mean body weight (BW), 6.0 mg] was about twofold higher than that of larvae fed a mixture of SE and WAK (3.2 mg) (P < 0.01). A mixture of HEY and SAK also had some dietary benefits for eel larvae, enabling them to survive for up to 58 days and to grow significantly (mean BW, 2.4 mg), compared with their initial weight (mean BW, 0.2 mg) (P < 0.001). Although additional nutritional improvements are needed, the present results suggest that combination diet HEY and SAK may be a good alternative to SE as an effective diet for eel larvae.