Ontogeny of digestive and immune system organs of larval and juvenile kelp grouper Epinephelus bruneus reared in the laboratory

ABSTRACT:   A histological examination was made of the ontogenetic development of the digestive and immune systems of the larval and juvenile kelp grouper Epinephelus bruneus reared in the laboratory. The liver, gall bladder, pancreas and the demarcating region between the intestines and rectum were formed within 3 days post-hatch (dph). During the preflexion phase (within 16 dph), revolution of the intestine concluded, and pharyngeal teeth and the mucous cells of the esophagus were differentiated. In the transitional period to the juvenile stage (25 dph), the blind sac of the stomach, gastric glands and pyloric caeca began to form. From the viewpoint of the differentiation phase of the adult-type digestive system, the kelp grouper is similar to Heterosomata, hitherto reported. The primordial thymus, kidney and spleen were present at 12, 1 and 6 dph, and the small lymphocytes in these lymphoid organs appeared at 21, 30 and 33 dph, respectively. The developmental sequence of the lymphoid organs and the appearance ages of the lymphoid organs and small lymphocytes in the lymphoid organs in the kelp grouper are similar to those of other marine fish previously reported, except for the Pacific bluefin tuna Thunnus orientalis .     

四指马鲅淋巴器官发育组织学观察

采用石蜡组织连续切片和HE染色技术,对1~60 dph(Days post hatching)四指马鲅(Eleutheronema tetradactylum)的淋巴器官个体发育进行研究,描述了淋巴器官发育特点及组织学特征。结果显示,在盐度为9.0±0.5,水温为(28±2)℃条件下,3 dph胸腺原基出现,由4~6层未分化干细胞和淋巴母细胞样的细胞组成;胸腺发育迅速,主要由淋巴细胞填充;至25 dph皮质区和髓质区分明显,胸腺发育基本完成。3 dph头肾原基形成,由前肾管和少量造血干细胞组成;5 dph胸腺淋巴细胞向头肾迁移,头肾开始淋巴化;随着鱼体生长,造血干细胞分化成不同类型细胞;18 dph前肾管开始退化,至53 dph完全消失,头肾主要由网状内皮系统支持下的淋巴造血组织构成。7 dph脾脏原基形成,至16 dph开始淋巴化;脾脏内皮系统较头肾发达,但其发育速度较胸腺和头肾慢,淋巴细胞明显少于胸腺和头肾。研究表明,四指马鲅淋巴器官原基出现及淋巴化的顺序是胸腺、头肾、脾脏。免疫淋巴器官结构及功能尚未发育完善,可能是四指马鲅在变态期间幼体死亡率高的主要原因之一。     

四指马泌尿系统胚后发育组织学研究

为了解四指马鲅(Eleutheronema tetradactylum)泌尿系统胚后发育的一般规律,采用石蜡组织连续切片和H-E染色技术,对1~50 dph(days post hatching)的四指马鲅泌尿系统胚后发育进行了研究。结果显示,四指马鲅前肾小管在3 dph已经形成,中间分布少量未分化干细胞;至7 dph前肾小管刷状缘明显,泌尿机能增强;15 dph前肾小管继续分化,管壁细胞结构清晰;20 dph观察到少量前肾小管退化,泌尿机能开始衰退;前肾在发育过程中未观察到肾小体形成。中肾肾小管原基在7 dph开始形成;20~25 dph中肾早期肾小体和肾小管芽形成;30 dph肾小管数量剧增,亚成熟肾小体形成,近端小管和远端小管开始区分;45 dph第一近端小管和第二近端小管开始区分,至50 dph观察到成熟肾小体;中肾有较多黑色素-巨噬细胞中心、淋巴细胞和红细胞分布,兼具造血、免疫及泌尿功能。中肾管为输尿管,始于中肾后端,并行向后合并膨大为输尿管膀胱。     

卵形鲳够免疫器官的早期发育

研究了卵形鲳鲹（Trachinotus ovatus）仔、稚鱼期间3种免疫器官的发生和发育过程。结果表明,3种免疫器官原基出现的先后顺序是头肾、脾脏和胸腺,出现时间分别为2日龄、3日龄和3日龄。其中,头肾的早期发育过程可分为原基形成期、造血功能发展期和泌尿功能退化期。进入稚鱼期后头肾的肾小管分布相对减少,表明头肾已不是主要的泌尿功能区。6-8日龄仔鱼脾脏中造血干细胞向红血细胞分化并产生大量未成熟红细胞。22日龄时脾脏组织体积进一步增大,内部血细胞参与血液循环。胸腺原基出现后胸腺细胞增多、体积增大,细胞嗜碱性不强。10-13日龄时胸腺细胞的嗜碱性增强,淋巴细胞开始增多,此时胸腺开始分区。20-22日龄稚鱼期结束时胸腺的内区和外区进一步分化,分区更为明显,表明胸腺进一步成熟。未发现胸腺和头肾之间淋巴细胞迁移的现象。     

中华鲟免疫器官的早期发育

为了解中华鲟(Acipenser sinensis)免疫器官早期发育的形态和组织学特征,实验以1~300 dph中华鲟子二代的仔鱼、稚鱼和幼鱼为研究对象,使用连续石蜡切片技术和显微观察方法对免疫器官(头肾、胸腺和脾脏)的发育过程进行观察和记录。结果显示,培育水温为12.9~22.6℃时,中华鲟免疫器官原基出现的先后顺序为头肾(3 dph)、胸腺(7 dph)和脾脏(9 dph);免疫器官淋巴化先后顺序为胸腺(12 dph)、头肾(15 dph)和脾脏(33 dph)。仔鱼发育至15 dph可见头肾和胸腺间有淋巴细胞“桥”连接现象, 180 dph胸腺内可见哈氏小体结构,头肾、胸腺和脾脏内含有黑色素细胞或黑色素巨噬细胞中心(MMCs)。总体而言,中华鲟免疫器官发育具有原基出现时间早、发育速度慢和发育周期长的特点。由于仔稚幼鱼免疫系统发育不完善,建议在其培育过程中加强病害防治和早期疫苗的开发。     

Mass Production of Juveniles of the Fat Snook Centropomus parallelus in Brazil

A pilot‐scale trial to rear fat snook Centropomus parallelus through larval, weaning and nursery phases was conducted in Florianópolis, Brazil. Eggs (96% fertilization) from captive broodstock, induced to spawn using 50 μg/kg LHRHa were stocked in two 4,000‐L cylindrical fiberglass tanks at a mean density of 19.2 eggs/L. Nannochloropsis oculata was stocked with the eggs and maintained at a mean density of 0.5–1.0 ± 10⁶ cells/mL up to 31 dph (31 dph). Hatching averaged 90%. Larvae were fed rotifers Brachionus rotundiformis enriched with an oil emulsion from 3 dph to 36 dph (30–40 rotifers/mL) and Artermia meta‐nauplii enriched with Selco from 22 dph to 60 dph (mean 2.9 meta‐nauplii/mL). Weaning began at 45 dph with an artificial dry diet NRG (50% protein), supplied together with concentrated and enriched Artemia meta‐nauplii. No critical period of mortality was observed during larval rearing. During the 43 days of weaning and nursery, less than 1% mortality was recorded. Food conversion rate during nursery was 1.17, with a change in the coefficient of variation of mean total length of 1.3%. Specific total growth rate in weight was 13.0 %/d and mean growth in total length and total weight were 0.65 mm/d and 24.0 mg/d over the whole rearing trial respectively. Mean total length and total weight of juveniles were 57.6 ± 0.1 mm and 2.11 ± 0.12 g, respectively, and the length‐weight relationship was W = 8.29931 ± 10–⁵ TL^3.049607 (r= 0.9986). on 88 dph when the trial was terminated. The condition factor on 88 dph was 1.104. On 88 dph a total of 35,000 juveniles were harvested, overall survival was 25.5% with mean final density of 4.4 fishn and biomass of 6.9 kg/m³. The present trial demonstrated the feasibility of mass production of fat snook. Possible improvements necessary for commercial cultivation of fat snook C. parallelus are discussed.     

Ontogeny of the digestive tract in shi drum (Umbrina cirrosa L.) reared using the mesocosm larval rearing system

Histological changes of the digestive tract were studied in shi drum (Umbrina cirrosa) from hatching until 41 days post hatching (dph), when the fry had a mean (±S.D.) total length (TL) of 32 ± 2 mm and wet weight (WW) of 0.42 ± 0.07 g. Larvae were reared using the mesocosm technique, the most natural among commercially employed rearing methods for marine larvae. Shi drum opened their mouth at 2 dph (2.78 ± 0.09 mm TL), at which time 90% of the larvae already had an inflated swim bladder. The differentiation of the digestive tract into buccopharynx, esophagus, and anterior and posterior intestine was completed by 3 dph (2.82 ± 0.07 mm TL), 1 day after the onset of exogenous feeding. The alimentary canal started coiling and formed its first loop at 2 dph, while the pancreas and liver were differentiated at 3 dph. Yolk sac reserves lasted until 7 dph (4.3 ± 0.1 mm TL), suggesting a brief period of endogenous and exogenous feeding. The first esophageal goblet cells appeared at 7 dph containing acid mucins and at 8 dph taste buds appeared on the buccopharyngeal epithelium. The stomach was morphologically differentiated at 9 dph (5.5 ± 0.1 mm TL) when gastric glands became abundant in the cardiac region, and the first pyloric caeca appeared at 14 dph (10.1 ± 0.9 mm TL). Supranuclear eosinophilic vacuoles were observed in the posterior intestine between 3 and 11 dph (6.3 ± 0.9 mm TL). Their number decreased as the stomach differentiated, suggesting a change in the protein digestion mechanism. The results of the study suggest a rapid development of shi drum and its digestive system and underline the possibility of weaning larvae to artificial feed even earlier than the 12 dph employed in the present study.     

Morphological development and allometric growth of yellowtail kingfish Seriola lalandi V. larvae under culture conditions

Morphological development and allometric growth patterns of Seriola lalandi larvae were assessed to characterize normal growth patterns under culture conditions. Early ontogenetic stages of yellowtail kingfish exhibited an exponential growth in terms of standard length as a function of age. Five development stages were characterized from hatching to the juvenile stage: larval stage I (0–2 days post hatch, dph) with endogenous feeding, characterized by a small yolk sac, unpigmented eyes, primordial finfold surrounding the body and a closed mouth; larval stage II (2–15 dph) characterized by mouth opening, complete pigmentation of eyes and the beginning of the exogenous feeding; subsequently, in the larval stage III (15–25 dph) the posterior tip of notochord of the larvae bended upward and the first rays appeared in fins, concomitant with a change in swimming behaviour; thereafter, larval stage IV (post‐flexion stage; 25–30 dph) began when larvae resembled in morphology to a juvenile organism; however, caudal and dorsal fins were not completely development. Lastly, the juvenile stage was reached 30 dph characterized by a morphology and fin structures similar to those of the adults. Growth and development of structures and organs associated with vital functions such as feeding, sensorial and breathing systems seemed to be more critical previous to 23 dph, which was reflected with a positive allometric growth of head and eyes during this period. The results from this study can be used as a tool‐guide to assess normal development in larval research with S. lalandi to improve existing rearing protocols in hatchery production.     

Nile tilapia broodfish fed high‐protein diets: Digestive enzymes in eggs and larvae

The aim of this study was to assess the activity of gastric, pancreatic and intestinal digestive enzymes during the embryonic and larval development of Nile tilapia (Oreochromis niloticus) GIFT strain Aqua America^® 1 obtained from a broodstock fed two levels of crude protein (CP). A total of 72 females and 24 males, 10 hapas, two CP levels (32% and 38%) and six phases of embryonic (cleavage, blastula, gastrula) and larval (hatching, 7 and 10 days post hatch, dph) stages were used. The eggs were collected in cleavage, blastula and gastrula stages, 300 mg was collected, and kept in cryogenic tubes in liquid nitrogen. For the samples at larval stage, the remaining eggs were separated according to crude protein level and kept in hatcheries and samples were collected on 7 and 10 dph the same way as before. A total of 48 samples were collected: at each protein level (32% and 38% CP), four samples were collected in each phase of embryonic and larval development. Statistical differences were not observed during embryonic development for acid proteases, trypsin, amylase and lipase activity at both levels of crude protein (32 and 38% CP). Differences for acid proteases were noticed on 7 dph; trypsin and amylase on 7 dph and 10 dph. Significant differences on blastula and 7 dph for protease; as for aminopeptidase, there was significant difference on 7 dph. The data indicated early appearance of digestive enzymes in Nile tilapia broodfish receiving 32% CP taking into account the rapid growth and development of this species.     

Gonadal morphogenesis and sex differentiation in cultured chub mackerel,Scomber japonicus

The timing of primordial germ‐cell (PGC) migration with regard to the gonadal anlagen, gonad formation and sex differentiation was examined histologically in the chub mackerel (Scomber japonicus) at 5–190 days post hatching (dph). At 5 dph, PGCs appeared on the peritoneal epithelium surface or in the mesentery, on the dorsal side of the abdominal cavity. By 10 dph, stromal cells around the PGCs proliferated. The gonadal primordium was formed by 15 dph. The gonadosomatic index was 0.01% at 30 dph and increased thereafter (0.32% in females and 0.04% in males at 160 dph). Ovarian differentiation occurred at 30–40 dph, indicated by ovarian cavity formation (elongation and fusion of the upper and lower ovarian edges). Meiosis was subsequently initiated. A few meiotic oocytes surrounded the cavity at 50 dph; most were in the perinucleolus stage at 60 dph and attained a diameter of 60–70 μm at 190 dph. Testicular differentiation occurred at 30 dph, indicated by the formation of the sperm duct primordium. Spermatogonia gradually proliferated, developing into spermatocytes at the chromatin–nucleolus stage (after 90 dph) and subsequently into spermatids and spermatozoa (160 dph). These data could aid the development of seeding and cell‐engineering technologies for scombrid fish.     

草鱼jam-a分子在胚胎幼鱼期及受GCRV感染PSF细胞的表达分析

草鱼呼肠孤病毒(Grass carp reovirus, GCRV)可引发草鱼(Ctenopharyngodon idellus)出血病,导致高死亡率。草鱼吻端成纤维细胞(Grass carp snout fibroblast cells, PSF)是GCRV的敏感细胞系。JAM-A (Junctional adhesion molecule A)为免疫球蛋白超家族成员,是多种病毒的细胞受体。本研究在前期克隆到草鱼3种jam-a基因,命名为gcjam-a1,gcjam-a2和gcjam-a3。在获取ORF序列的基础上,利用qRT-PCR分析了3种gcjam-a在草鱼胚胎及幼鱼不同发育时期及PSF细胞中受GCRV(GD108株)感染前后的表达模式。结果显示,检测的13个胚胎及幼鱼发育时期中,gcjam-a1在未受精卵中高表达,在受精卵至出膜前的胚胎表达水平均较低;从出膜后1~3 d表达量开始上升;出膜6~15 d均呈高水平表达。gcjam-a2与gcjam-a3在草鱼胚胎及幼鱼发育各阶段表达水平较低。在无病毒感染的PSF细胞中,gcjam-a只有少量表达。受GCRV-GD108感染后,病毒S7基因在PSF细胞中的拷贝数随时间呈显著上调趋势,gcjam-a的表达量也有不同程度的上调,mRNA上调水平为gcjam-a1>gcjam-a2>gcjam-a3。本研究证实了3种gcjam-a基因在PSF细胞中的表达均与GCRV-GD108感染相关,其中,gcjam-a1的表达水平受GCRV-GD108感染影响最大,同时,它在孵化出膜后表达上升,推测它可能与病毒的感染更相关。gcjam-a1可作为下一步GCRV与宿主互作研究中的候选分子。     

Development of digestive system and swim bladder of larval nase (Chondrostoma nasus L.)

Digestive tract and swim bladder differentiation during nase (Chondrostoma nasus L.) larval development was evaluated using histological methods. The mouth, pharynx and oesophagus were impervious at the time of hatching. The straight tubular intestine consisted of undifferentiated cells and was situated over the yolk sac. The intestine, lined with a single layer of columnar epithelial cells, opened on 2 day posthatching (dph). First evidence of protein digestion and absorption by the enterocytes was observed on the sixth day, while lipid digestion and absorption began on the ninth day of larval development. Intestinal mucous cells developed and became active by the fourth day. The posterior chamber of the swim bladder inflated on the third day, while the anterior one filled 12 dph. Complete yolk sac resorption took place on the ninth day.     

Anti-oxidant status in embryonic,post-hatch and larval stages of Asian seabass (<Emphasis Type="Italic">Lates calcarifer</Emphasis>)

The concentrations of anti-oxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and selenium-dependent glutathione peroxidase (SeGPx), and low molecular weight free-radical scavengers such as reduced glutathione (GSH) and ascorbic acid (vitamin C) were evaluated during the period from gastrulation (GS) to 25 days post-hatch (dph) in the larvae of Asian Seabass, Lates calcarifer. Oxidative damage due to lipid peroxidation (LPO) was also assessed, by evaluation of the formation of malondialdehyde (MDA). All the three anti-oxidant enzymes, SOD, CAT and GPx, showed high activities during gastrulation, suggesting an increased metabolic rate during the period of embryonic development. Though the SOD activity apparently decreased progressively during 3–20 dph of larval development, the difference was not significant. CAT showed high activity during gastrulation and remained constant up to 3 dph, suggesting an increased need to metabolise hydrogen peroxide (H₂O₂) and organic peroxides. In contrast, SeGPx activity increased progressively from 5 dph to 25 dph during larval development, indicating an increased need to detoxify lipid peroxides. This is evident from the observation of increased lipid peroxidation from 10 dph to 25 dph during larval development. GSH levels were low at gastrulation, indicating increased metabolic rate and formation of lipid radicals during this period, corresponding to the decrease in the level of ascorbic acid, which is consumed for regeneration of GSH.     

Fine structure and differentiation of the alimentary canal in captive-bred Japanese eel Anguilla japonica preleptocephali

ABSTRACT:   The fine structure of the alimentary canal in preleptocephali produced by artificially matured Japanese eel was examined. At 1 day posthatch (dph), the alimentary canal was found only above the dorsal side of the yolk mass, and the epithelium was composed of a single layer of epithelial cells. By 5 dph, the alimentary canal was divided into three segments based on the structure of the epithelial cells: foregut, midgut and hindgut, corresponding to the future esophagus, intestine and rectum, respectively. After 7 dph, the epithelium in the foregut was surrounded by a circular muscle layer, suggesting a role in the transportation of food materials. The epithelial cells of the midgut exhibited well-developed membranous structures, which are deduced to be invaginations of the cytoplasmic membrane. Pinocytotic invaginations and vacuoles were observed in the epithelial cells of the hindgut; this observation suggests that this region is involved in the uptake of food. Significant changes in morphological features of the epithelial cells in each segment were observed until 7 dph; however, these were not evident between 7 dph and 13 dph. Consequently, the differentiation of the alimentary canal was completed by 7 dph, and preleptocephalus had developed the ability to absorb food by 7 dph.     

Effects of early weaning strategies on growth,survival and digestive enzyme activities in cobia (<Emphasis Type="Italic">Rachycentron canadum</Emphasis> L.) larvae

The effects of weaning strategies of cobia (Rachycentron canadum L.) larvae to commercial microdiets, either from rotifers or from Artemia, on growth, survival and enzymatic digestive capacity, were investigated. In the first experiment, cobia larvae were weaned from rotifers by co-feeding with a microdiet (Otohime) from 8, 13 or 20 days post-hatching (dph). The larvae in the control treatment were fed rotifers (2–12 dph), Artemia nauplii from 7 dph, and co-fed with the microdiet from 20 dph. In the second experiment, the larvae were weaned from Artemia, which was fed to the larvae from 7 dph, by co-feeding with a microdiet (NRD) from 8, 13 or 18 dph. The larvae in control treatment were fed rotifers, then Artemia to the end of the experiment (28 dph). Weaning of cobia larvae onto a microdiet directly from rotifers significantly reduced growth, survival and digestive capacity of the larvae and did not lead to larval acceptance of the microdiet, compared to those weaned from Artemia in the first experiment. Early weaning of cobia larvae onto NRD microdiet (on 8 or 13 dph) from Artemia in the second experiment also reduced growth, survival rate and gut maturation index, compared to those fed live feed. With available microdiets, weaning of cobia larvae could start from Artemia at around 18 dph in order to obtain comparable growth, survival and gut maturation to larvae fed live feed.     

Aggressive behaviour and cannibalism in greater amberjack,Seriola dumerili : effects of stocking density,feeding conditions and size differences

Aggressive behaviour, assessed by 24‐h observations, and survival rates, determined in a 2‐week rearing experiment, were examined in greater amberjack, Seriola dumerili, subjected to single and multiple factors (high density, restricted feeding and diverse size). Aggressive behaviour frequency in the group subjected to both restricted feeding and size diversity (FS) was significantly higher than that in the other groups (Tukey, n=4, P<0.05). In the FS group, aggressive behaviour increased with the starvation time from 19 days post hatching (dph) to 23 dph (two‐way analysis of variance, Tukey, n=4, P<0.05), but decreased gradually thereafter to 46 dph. In the rearing experiment, the survival rates for the diverse size and FS groups were lower than those of the control or restricted feeding groups (Kaplan–Meier, log‐rank test, n=100, P<0.01). Daily mortality in all groups was the highest at 24 dph; although dead fish were usually small, disappearance due to cannibalism was not observed during the rearing period. The results indicate that aggressive behaviour in greater amberjack is induced by hunger and size diversity in the early juvenile stages (23 dph). Mass mortality during seedling production was mainly caused by injury and the death of small fish due to aggressive behaviour/cannibalism.     

斑点鳟仔、稚、幼鱼的形态发育

以斑点鳟(Oncorhynchus mykiss)发眼卵为材料,进行了发眼卵的人工孵化及仔鱼的人工培育研究。对斑点鳟仔稚鱼的形态发育进行了系统观察,描述了各发育期的形态特征,并测定生长参数。斑点鳟早期生活史的分期如下:初孵仔鱼全长14.25±0.45 mm,体重85±5 mg,肛门未开口,营内源性营养,脊柱末端向上弯曲;10 dph开始上浮,投喂卤虫;12 dph开口摄食,开始营内外源混合性营养;14 dph鳍膜消失,各鳍独立;16dph开始投喂配合饵料;24 dph卵黄囊吸收完毕,营完全外源性营养;34 dph,体侧形成8～9个幼鲑斑;44 dph各鳍的鳍条发育健全;60 dph幼鱼外形为纺锤形,与成鱼相同。研究表明,斑点鳟早期生长发育过程中全长、肛门前长、水平眼径和头高的生长情况基本相同,0～16 dph生长速度较快,16～44 dph生长速度较慢,44 dph后生长速度再次变快。     

Larval development in European hake (Merluccius merluccius L.) reared in a semi-intensive culture system

Eggs of European hake (Merluccius merluccius L.) were stripped from fish caught at sea. Larvae were kept under semi‐intensive conditions at around 12°C. In addition, eggs were incubated in single wells at 9.2, 12.7 and 14.5°C, where hatching, development and survival were closely examined. During the larval stage, a total of 299 larvae were sampled to follow development and growth. In addition a small number of juveniles were sampled. Larvae hatched approximately 4 days after fertilization, and were 2.9 mm in total length (TL). At 6‐day post hatching (dph), the larvae were 4.1 mm (TL), the jaw apparatus was developed, and the larvae had started to feed. Most of the growth during the early larval period is restricted to the head, and there is almost no increase in length for the first 3–4 weeks post hatching. Teeth and pelvic fins appear at 25 dph. Development of unpaired fins at approximately 30 dph marks the start of the larval–juvenile transition. Weaning to formulated feed was accomplished 50 dph, when external morphology was similar to that of adult hake.