Changes in marine turbot (Scophthalmus maximus) epidermis and skin mucus composition during development from bilateral larvae to juvenile flat fish

Alike other flat fish, marine turbot has the particularity that changes from larvae with bilateral symmetry to adult with asymmetry, in terms of the position of the eyes. As expected, the skin configuration of this species is also affected by the development and transformation suffered by fish during metamorphosis. In this context, changes in the epidermis of marine turbot were studied using conventional staining and histochemical techniques using six lectins (UEA-I, PNA, RCA-I, WGA, Con A and SBA). During development from larvae to juvenile (3–300 days post-hatching), the epidermis increased in both thickness and the number of cell layers. In fact, the simple cuboidal epithelium observed in larvae at day 3 already became stratified at days 10–12, which sequentially increase in thickness with fish development. Turbot epidermis is composed basically of four cell types: epithelial and mucous or secretory cells that are present through the development, and pigmented cells and a type that the authors described as club-like cells that appear during and post-metamorphosis. The Alcian blue-periodic acid Schiff (AB-PAS) histochemical method revealed the presence of neutral glycoconjugates in mucous and club-like cells at post-metamorphic stages of fish. Accordingly, lectin analysis showed mucous cells containing glycoproteins rich in fucose (UEA-I labelling) and glycoconjugates rich in the sequence galactose-N-acetyl galactosamine (PNA and RCA-I labelling) when this cell type appears. Interestingly, melanophores were observed in the dorsal epidermis of post-metamorphic juveniles. This type of cell contains a black-to-brown pigment that provides the skin the typical colour of this fish species. Changes in mucous coat composition were observed during fish development, which was attributed to different roles of the glycoconjugates.     

Developmental change in RNA: DNA ratios of fed and starved laboratory-reared Japanese flounder larvae and juveniles, and its application to assessment of nutritional condition for wild fish

Starved Japanese flounder Paralichthys olivaceus larvae were characterized by relatively lower levels of RNA content throughout their early life stages. Significant differences in the RNA: DNA ratios were found between fed and starved fish, and appeared to increase as starvation proceeded. Ontogenetic changes in RNA: DNA ratios were clearly observed during metamorphosis, especially decreasing during the period from the late-metamorphic to postmetamorphic stages. The criteria established from these laboratory experiments, were applied to the nutritional condition of wild larvae and juveniles collected in Wakasa Bay, Sea of Japan in 1994 and 1995 by measuring RNA and DNA content. Starved fish were mainly found in stage I (settling stage) fish during the late season of settlement in 1995. This suggests that starvation could be associated with settlement in Japanese flounder.     

Comparison of growth and RNA: DNA ratios in three populations of juvenile turbot reared at two salinities

Salinity significantly influenced growth and RNA: DNA ratios in three populations (Iceland. Norway and France) of juvenile turbot Scophthalmus maximus as these variables were higher at 15 than at 33.5‰ salinity. Furthermore, growth and RNA: DNA ratios of the two high latitude populations (Iceland and Norway) were higher than those of the low latitude population (France): the Norwegian and the Icelandic populations had RNA: DNA ratios of 6.028 and 5.91, respectively, whereas the ratio of the French population was 5.01. It is concluded that in the upper temperature range for turbot, growth can be improved by rearing at intermediate salinities at least during the first year.     

草地螟滞育幼虫的蛋白和核酸含量变化

为了阐明草地螟Loxostege sticticalis 滞育的分子调控基础, 本研究应用Trizol法、 DNA和蛋白质定量试剂盒、 SDS-PAGE电泳技术分别对进入滞育1, 2, 3和4个月、 解除滞育以及非滞育草地螟老熟幼虫中的核酸含量、 总蛋白含量和组分的变化进行了测定。结果表明： 滞育不同月份幼虫的总RNA含量显著低于非滞育幼虫的总RNA含量（P＜0.05）; 解除滞育幼虫的总RNA含量显著高于滞育2, 3和4个月的幼虫, 但仍显著低于非滞育的对照组。滞育不同月份、 非滞育以及解除滞育幼虫的总DNA含量没有显著差异（P＞0.05）。RNA/DNA比值随滞育的开始而显著下降, 随着滞育的结束而显著上升。滞育不同月份的幼虫总蛋白含量显著高于非滞育幼虫的总蛋白含量（P<0.05）, 但解除滞育与非滞育幼虫的总蛋白含量无显著差异。利用SDS-PAGE电泳分析发现滞育幼虫体内存在滞育关联蛋白, 蛋白条带在24 kDa左右, 但非滞育和已经解除滞育的幼虫则没有该蛋白条带。这些结果表明, 总RNA含量的降低、 RNA/DNA比值下降、 总蛋白含量的升高, 以及24 kDa蛋白的存在是草地螟幼虫滞育的主要生理特征。     

Histological changes in starved turbot larvae (Scophthalmus maximus) quantified by digital image analysis

In turbot larvae, Scophthalmus maximus , deprived of food for 24 h there was a significant increase in the specific surface of the epithelium and the corresponding microvillous border of the foregut accompanied by slight cellular degeneration. Following 48 h starvation Sarvae showed severe tissue degeneration in the foregut mucosa, progressing to extensive mucosal desquamation and cellular sloughing. Intracellular vacuolation of the epithelium and loss of microvilli was also extensive and the ability of the gut to absorb food must be severely impaired, with further starvation probably resulting in larval death. There was no evidence of parasitic infection in any of the larvae sampled and all observed alterations are attributable to food deprivation.     

Colonization of Vibrio pelagius and Aeromonas caviae in early developing turbot (Scophthalmus maximus L.) larvae

Polyclonal antisera made in rabbits against whole washed cells of Vibrio pelagius and Aeromonas caviae were used for detection of these bacterial species in the rearing water and gastrointestinal tract of healthy turbot ( Scophthalmus maximus ) larvae exposed to V. pelagius and/or Aer. caviae . The results demonstrated that this method is suitable for detection of V. pelagius and Aer. caviae in water samples and larvae at population levels higher than 10³ ml⁻¹ and 10³ larva⁻¹. Populations of aerobic heterotrophic bacteria present in the gastrointestinal tract of turbot larvae, estimated using the dilution plate technique, increased from approximately 4 × 10² bacteria larva⁻¹ on day 3 post-hatching to approximately 10⁵ bacteria fish⁻¹ 16 days post-hatching. Sixteen days after hatching, Vibrio spp. accounted for approximately 3 × 10⁴ cfu larva⁻¹ exposed to V. pelagius on days 2, 5 and 8 post-hatching. However, only 10³ of the Vibrio spp. belonged to V. pelagius . When larvae were exposed to Aer. caviae on day 2 post-hatching, the gut microbiota of 5-day old larvae was mainly colonized by Aeromonas spp. (10⁴ larva⁻¹), of which 9 × 10³ belonged to Aer. caviae . Later in the experiment, at the time when high mortality occurred, 9 × 10⁵ Aer. caviae were detected. Introduction of V. pelagius to the rearing water seemed to improve larval survival compared with fish exposed to Aer. caviae and with the control group. It was therefore concluded that it is beneficial with regard to larval survival to introduce bacteria ( V. pelagius ) to the rearing water.     

Structural Studies of Heterochiral DNA/DNA,RNA/RNA,AND DNA/RNA Duplexes

Using DNA and RNA heptanucleotides containing an unnatural L-nucleotides as well as the complementary strands, effects of the introduction of an L-nucleotide on the structure of DNA/DNA, RNA/RNA, and DNA/RNA duplexes were investigated by circular dichroism experiments and RNase H-mediated RNA strand cleavage reaction. The results suggested that the substitution of the central D-nucleotide with an L-nucleotide in the duplexes causes the significant structural alterations as the duplex structures change to conformations with more B-form similarities.     

Molecular cloning,characterization and expression analysis of p53 from turbot Scophthalmus maximus and its response to thermal stress

The tumor suppressor protein, p53 plays a crucial role in protecting genetic integrity. Once activated by diverse cell stresses, p53 reversibly activates downstream target genes to regulate cell cycle and apoptosis. However, few studies have investigated the effects of thermal stress in turbot, specifically the p53 signaling pathway. In this study, the rapid amplification of cDNA ends was used to obtain a full-length cDNA of the turbot p53 gene (Sm-p53) and perform bioinformatics analysis. The results showed that the cDNA of the Sm-p53 gene was 2928 bp in length, encoded a 381 amino acid protein, with a theoretical isoelectric point of 6.73. It was composed of a DNA binding and a tetramerization domain. Expression of Sm-p53 in different tissues was detected and quantified by qRT-PCR, and was highest in the liver. We also investigated the expression profiles of Sm-p53 in different tissue and TK cells after thermal stress. These result suggested that Sm-p53 plays a key role, and provides a theoretical basis for Sm-p53 changes in environmental stress responses in the turbot.     

Selection and identification of autochthonous potential probiotic bacteria from turbot larvae (Scophthalmus maximus) rearing units

The purpose of this study was to select, identify and characterise bacteria as a disease control measure in the rearing of marine fish larvae (turbot, Scophthalmus maximus). Thirty-four out of 400 marine bacterial strains exhibited in vitro anti-bacterial activity against three fish larval pathogens. Two strains originated from culture collections and thirty two strains were isolated directly from turbot larvae rearing units using a pre-selection procedure to facilitate detection of antagonists. Approximately 8,500 colonies from colony-count plates were replica-plated on agar seeded with Vibrio anguillarum, and 196 of them caused zones of clearing in the V. anguillarum agar layer. Of these, 32 strains exhibited reproducible antibacterial properties in vitro when tested against the fish pathogens V. anguillarum 90-11-287, V. splendidus DMC-1 and a Pseudoalteromonas HQ. Seventeen antagonists were identified as Vibrio spp. and four of twelve tested were lethal to yolk-sac larvae. The 15 remaining strains were identified as Roseobacter spp. based on phenotypic criteria and 16S rDNA gene sequence analysis of two strains representing the two major RAPD groups. Most of the remaining 164 strains selected in the initial replica plating were identified as Vibrionaceae or Pseudoalteromonas. Roseobacter spp. were not lethal to egg yolk sac turbot larvae and in two of three trials, the mortality of larvae decreased (p > 0.001) in treatments where 10(7) cfu/ml Roseobacter sp. strain 27-4 was added, indicating a probiotic potential.     

Ontogenetic changes in RNA,DNA and protein contents of Chinese loach,Paramisgurnus dabryanus (Dabry de Thiersant, 1872), larvae and juveniles

The changes in nucleic acid‐based indices and protein variables of Chinese loach, Paramisgurnus dabryanus, larvae and juveniles from hatching to 60 days after hatching (DAH) were conducted to assess its growth potential. The nucleic acid contents were analysed using a UV‐based method (n = 3, rearing temperature 24.4 ± 0.4°C, dissolve oxygen 7.1 ± 0.5 mg L^?1, pH 7.9 ± 0.4). Ribonucleic acid (RNA) concentration significantly decreased from 2 to 5 DAH, then increased rapidly until 10 DAH, declining slightly thereafter. Deoxyribonucleic acid (DNA) concentration increased 2–5 DAH, decreased until 9 DAH, slightly increased again around 26 DAH, and then declined to a relatively stable level. Both RNA‐DNA and protein‐DNA ratios showed a statistically obvious relationship with growth rates. A significantly positive relationship was found between RNA‐DNA ratio and growth rates during the early life stage of Chinese loach. According to the results, growth of Chinese loach is characterized by rapid hyperplasia from hatching through completion of the yolk‐sac stage followed by continued rapid hyperplasia combined with increasing hypertrophy after feeding commences. The stage preceding 17 DAH of Chinese loach P. dabryanus is presumed to be critical for its survival and growth at 24°C.     

北京油葫芦卵子发生中DNA及RNA动态变化研究

用孚尔根及甲基绿 -派洛宁组织化学染色法了解北京油葫芦Teleogryllusmitratus(Burmeister)卵子发生各时期阶段中卵内DNA及RNA动态变化规律。在卵子发生的最初阶段 ,核中DNA的合成和复制最活跃 ,以后便慢慢减弱 ;而RNA则在第 2阶段合成最旺盛。在卵子发生各个阶段 ,滤泡细胞中DNA ,RNA均为阳性反应 ,并在卵细胞的卵黄形成期活动旺盛 ,为卵母细胞卵黄蛋白形成提供物质基础。卵子发生第 4～ 6阶段 ,滤泡细胞开放时期 ,血淋巴内一些物质可能直接或间接通过滤泡细胞间隙进入卵母细胞内 ,参与卵母细胞的发育和构建。研究表明卵子发生初期卵母细胞的发育和物质构建主要以内源性合成积累为主 ,中后期则有外源性物质的参与。     

Diet and niche overlap of southern populations of brill Scophthalmus rhombus and turbot Scophthalmus maximus

The diets of adult brill Scophthalmus rhombus and turbot Scophthalmus maximus from the Portuguese coast relied mostly on fishes. There was a higher diversity of food items compared to their northern counterparts, and several of the identified prey are the first records of these species, including a brown alga, echinoderms, nematodes, oligochaetes, gastropods, bivalves and various fish species. The diet of the two species was significantly different and niche overlap was low.     

RNA/DNA ratio as a growth indicator of stream periphyton

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Effects of hyposmotic stress on exocytosis in isolated turbot, Scophthalmus maximus, hepatocytes

The effect of hyposmotic shock on exocytosis was examined in isolated hepatocytes of turbot, a marine flatfish, using the molecular probe FM1-43. Sudden exposure to a reduced osmolality caused an increase in cell exocytic activity related to the osmotic gradient between intra- and extracellular fluids. Cytoskeletal microtubules could contribute to this hyposmotic-induced exocytosis since colchicine inhibited the process. Protein kinase C, phosphatidylinositol-3 kinase, phospholipases A2, C and D could constitute key enzymes in the mechanism since their inhibition by specific agents altered the hyposmotic-induced exocytic activity. Moreover, arachidonic acid and derivates from the 5-lipoxygenase pathway as well as calcium could participate in the process. As regulatory volume decrease (RVD) exhibited by turbot hepatocytes following hyposmotic stimulation involves similar features, a potential role of exocytosis in volume regulation is suggested. In particular, exocytosis could serve RVD by contributing to ATP release since this latter process similarly appeared to be phospholipase D-dependent and related to the osmotic gradient. This study provides the first evidence of a volume-sensitive exocytosis that could aim at volume constancy in a marine teleost fish cell type.     

大菱鲆仔稚幼鱼消化系统发育的组织学研究

大菱鲆(Scophthalmus maximus) 是目前欧洲重要的海水养殖良种之一, 在中国北方沿海大菱鲆的养殖规模也在不断扩大。       

Ovulatory rhythms and over-ripening of eggs in cultivated turbot, Scophthalmus maximus L.

The aim of this study was to investigate ageing of ovulated eggs retained in the ovarian lumen of captive turbot, Scophthalmus maximus L., prior to handstripping. The ovulation times of egg-batches were determined by catheterization, handstripping and plotting the percentage fertilizations and hatches of resulting eggs against time. The catheterization experiments showed that eggs age and change appearance rapidly after ovulation. The stripping method demonstrated that freshly-ovulated eggs showed greater than 90% fertilization and up to 97% hatch, but ovulated eggs retained in the ovary lumen for 1 day before stripping gave 0% hatch. The turbot showed precise ovulatory rhythms. The time between ovulations varied between individuals but was constant for any one female, making it possible to predict future ovulation times. Three of the females studied had ovulatory periods of alternating length.     

Ontogeny of enzymatic activities in fed and fasting turbot, Scophthalmus maximus L.

The presence and localization of acid and alkaline phosphatase, non-specific proteases, aminopeptidase, amylase, non-specific esterase and lipase was investigated by histoenzymologic methods in fed and fasting turbot from day 1 to day 40 post-hatching and compared with published data. Alkaline phosphatase and aminopeptidase activities were delected at day 1 in the distal region of the developing digestive tube. At day 3 (opening of the mouth) aminopeptidase and alkaline phosphatase activities were found all along the intestine. Sites of non-specific esterase and protease activities became apparent in the digestive tract at days 2 and 3 respectively. Amylase was present in the exocrine pancreas at day 3 and in the lumen of the intestine at day 4. Acid phosphatase was active in the cellular structure surrounding the yolk stores and in the lipid droplets at day 1 and in the intestinal epithelium at day 3. Lipase was found at day 15 when the larvae metamorphose into juveniles.
All the investigated enzymes were detected in fasting animals, except for lipase. However, the intensities of the enzymatic activities were weaker in the fasting specimens relative to the fed specimens between days 7 and 10.     

Evaluating the nutritional condition of individual whitefish (Coregonus spp.) larvae by the RNA/DNA ratio

Larvae of Coregonus spp. were reared in the laboratory under different temperature (4, 6 and 8°C) and feeding conditions ( ad libitum , limited, with starvation intervals). Their RNA/DNA ratios were determined with a highly sensitive fluorescence technique. After resorption of their yolk reserves (about 2 weeks after hatching), well fed larvae (RNA/DNA >2.5) could be significantly distinguished from larvae reared under limited food supply (RNA/DNA < 2.5), both at the 6 and 8°C levels. At 4°C, no differences due to the feeding regime were found. During a second series of experiments, larvae were affected by an intestinal disease, which was caused by the ingestion of unsuitable copepod plankton. This disease provoked high mortality, decreased growth and RNA/DNA ratios which were almost as low as in temporarily starved larvae from the first series. Coregonid larvae sampled in Lake Constance during spring 1990 showed RNA/DNA ratios which were unexpectedly low when judged on the basis of mean body length and average ambient temperature. It was obvious from macroscopic observations that some of these wild larvae were severely attacked by the intestinal disease. The low RNA/DNA ratios in field samples are, therefore, interpreted as a sublethal result of this disease.     

Effects of dietary polyunsaturated fatty acid deficiencies on mortality, growth and gill structure in the turbot, Scophthalmus maximus

The preparation of fish oil concentrates containing only ( n -3) polyunsaturated fatty acids (PUFA) with different ratios of 20:5 ( n -3)/22:6 ( n -3) is described. Three groups of turbot were maintained on different diets containing: 1, 10% of the dry weight of the diet as natural fish oil, equivalent to 2.5% ( n -3) PUFA and 0–23% ( n -6) PUFA; 2, 10% of the dry weight of the diet as palmitic acid, i.e. no PUFA; 3, 8–7% palmitic acid and 1–3% of the dry weight as ( n -3 PUFA and negligible ( n -6) PUFA. Only the fish on the diet containing natural fish oil showed significant growth over a 15-week period. In addition there were high mortalities on the two experimental diets (2 and 3). Changing the ratio of 20:5 ( n -3)/22:6 ( n -3) from 13–8 to 2–2 in the diet containing 1 3% (n-3) PUFA and negligible ( n -6) PUFA markedly decreased the mortalities. Fish fed the two experimental diets (2 and 3) developed gross changes in gill structure involving the disappearance of chloride cells, a 'sloughing off' of the epithelium along the primary and secondary filaments and an accumulation of cellular material in the inter-lamellar spaces. The tissue ultimately disintegrated to leave a skeleton of connective tissue and a mass of cellular material in the inter-lamellar spaces. It is concluded that 22:6 (n-3) is an essential fatty acid for turbot and that the gill epithelium is a sensitive indicator of this deficiency in this species.