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.     

双列杂交分析比较两个养殖群体杂交牙鲆抗病力

采用双列杂交设计对牙鲆Paralichthys olivaceus两个养殖群体：日本群体（J）和中国抗病选育群体（C）进行群体间杂交和群体内自繁,获得C（♀）×C（♂）、J（♀）×J（♂）、J（♀）×C（♂）和C（♀）×J（♂）4个交配组合的子一代。针对受精率、孵化率、白化率和存活率4个性状进行了组合间的比较,计算了各项性状的杂种优势率,对组间各性状进行了方差分析,并进行了相关分析。结果表明,杂交组在受精率、孵化率及存活率上存在着不同程度的杂种优势,其中C（♀）×J（♂）后代在孵化率和存活率两个性状上的杂种优势率分别为33.99%和31.37%。对4个性状的表型相关分析表明,受精率和孵化率的相关系数为0.657,受精率和存活率相关系数为0.432,分别达到了极显著水平（P〈0.01）。分析结果认为,两个不同群体间的种内杂交是牙鲆优良品种培育的有效途径。     

mPRα在性成熟雌性牙鲆(Paralichthys olivaceus)不同组织中的定性定量表达特征

通过荧光实时定量PCR方法(qRT-PCR)检测雌性性成熟牙鲆(Paralichthys olivaceus)第Ⅴ时期不同时相的卵母细胞中膜孕激素受体(mPRα)的表达,同时采用原位杂交、免疫组化和Western blotting方法研究性成熟雌性牙鲆mPRα mRNA和蛋白在各组织中的分布和表达.qRT-PCR结果显示,在牙鲆卵巢成熟时期的第Ⅴ时相卵母细胞,mPRα mRNA的表达量最高,表明mPRα介导孕激素参与了牙鲆卵母细胞成熟的调控.组织定量定位结果显示,在牙鲆的卵巢、脑、头肾、肾、肝脏组织中均有mPRα mRNA和蛋白表达存在,且在脑、卵巢中表达量较高,表明mPRα在牙鲆不同组织中都发挥着一定的作用,且在内分泌相关组织如脑和卵巢中的作用更明显.原位杂交和免疫组化结果显示,mPRα mRNA和蛋白表达明显定位在牙鲆卵母细胞膜上,且分布在其他组织的管腔结构.本研究为进一步探究牙鲆mPRα信号转导机制提供了理论基础和形态学依据.     

闽南池塘养殖牙鲆和舌鳎试验

本试验采用来自山东乳山人工培育,平均体长为19．8cm的牙鲆鱼苗4566尾和漳埔海区野生,体长10～13cm的舌鳎鱼苗1280尾,在水温10～28℃,盐度27‰～30‰,pH值7．8～8．5的条件下,进行为期180d的池塘混养和人工驯养。结果表明,总成活率为69.2％,总产量1209kg,牙鲆冬季南移养殖及与舌鳎混养是可行的,且舌鳎海区野生苗人工驯养初获成功。此外,本文还就影响牙鲆和舌鳎养殖效果和经济效益的因素进行了讨论。     

Effects of the Dietary Fermented Tuna By‐product Meal on Growth,Blood Parameters,Nonspecific Immune Response,and Disease Resistance in Juvenile Olive Flounder,Paralichthys olivaceus

This study evaluated the effects of dietary fermented tuna by‐product meal (FTBM) in juvenile olive flounder, Paralichthys olivaceus. Five diets were formulated to replace fishmeal (FM) with FTBM at 0% (FTBM₀), 12.5% (FTBM_12.5), 25.0% (FTBM₂₅), 37.5% (FTBM_37.5), or 50% (FTBM₅₀). After 8 wk, weight gain, specific growth rate, and feed efficiency of fish fed FTBM₀ and FTBM_12.5 diets were significantly higher than fish fed the other diets (P < 0.05). Also, mean cumulative survival rates (%) of fish fed the FTBM₀ and FTBM_12.5 diets were significantly higher than those fed FTBM₅₀ diet at Day 9 postchallenge with Edwardsiella tarda (P < 0.05). Protein efficiency ratio of fish fed FTBM₀ and FTBM_12.5 diets was significantly higher (P < 0.05) than fish fed diets FTBM_37.5 and FTBM₅₀. Broken‐line regression analysis of weight gain showed an optimal FM replacement level of 10.65% with FTBM. Therefore, the optimal dietary inclusion of FTBM in juvenile olive flounder diets could be greater than 10.65% but less than 12.5% without any adverse physiological effects on fish health.     

牙鲆变态发育过程中epigen基因的空间表达分布

细胞分裂在鲽形目鱼类变态过程中发挥了重要作用。Epigen作为表皮分裂原,通过激活MAPK通道来调控有丝分裂。以前的研究利用定量PCR表明,epigen在变态前期表达量升高,而变态后期表达量下降,表明其与变态发育相关。为了进一步调查epigen与牙鲆变态发育的关系,利用RNA整体原位杂交技术检测了epigen在变态期牙鲆体内的空间表达分布,发现在变态期的各个阶段,epigen基因表达分布均比较广泛,表皮、鳃、肝脏、肠道、鳍条、支鳍骨、肌节等组织都有表达,在牙鲆变态前和变态早期(E期)信号较强,而在变态高峰期(F期和G期)和变态后期(H期)信号逐渐变弱。Epigen空间表达分布模式提示,其作为表皮分裂原,可能广泛参与牙鲆变态发育早期事件,尤其可能与肝脏、肠道、支鳍骨和鳍条的发育有关。     

Induction of diploid gynogenesis in turbot <Emphasis Type="Italic">Scophthalmus maximus</Emphasis> with left-eyed flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> sperm

Turbot Scophthalmus maximus exhibits sexually dimorphic growth, with females growing faster and reaching larger adult sizes than males. Thus, development of techniques for preferentially producing females is necessary to optimize production of these species. In this paper, gynogenetic diploids of turbot were induced by activating egg development with ultraviolet (UV)-irradiated left-eyed flounder Paralichthys olivaceus sperm combined with cold shock to prevent extrusion of the second polar body. The results of UV irradiation experiments showed that survival, motility, and duration of activity of P. olivaceus sperm generally decreased with increase in UV dose. The typical Hertwig’s effect was observed after fertilized turbot eggs with UV-irradiated P. olivaceus sperm and the optimal UV dose for gynogenetic haploid production was 36,000 erg mm⁻². At 15°C, appropriate timing of cold shock for retention of the second polar body in turbot eggs was at 6 min after fertilization. Results of different combinations of two shock temperatures (1 or 3°C) and four shock durations (15, 25, 35 or 45 min) at 6 min after fertilization demonstrated that shock of 25 min at 1°C gave the highest production of diploid gynogens (39.58% relative to its diploid control). The results of this study reveal that the use of UV-irradiated P. olivaceus sperm for activation of turbot eggs and cold shock for polar body retention is an effective method to produce gynogenetic offspring.