Effect of artemia nauplii enriched with vitamin A palmitate on hypermelanosis on the blind side in juvenile Japanese flounder Paralichthys olivaceus

ABSTRACT:   The effect of Artemia nauplii enriched with different level of vitamin A (VA) palmitate (1 µg = 1 IU) on the occurrence of hypermelanosis on the blind side of Japanese flounder Paralichthys olivaceus was determined. Artemia were enriched with 0, 1, 2, 5 or 10 mg VA palmitate/L (control group, and 1-, 2-, 5-, and 10-mg groups). The enriched Artemia were fed to the larvae from 27 to 31 days post hatching (dph) corresponding to the F–G stage. VA palmitate, retinol and retinoic acid (RA) contents of Artemia were correlatively elevated with increasing VA palmitate in the culture medium. RA was detected in Artemia enriched with 5 mg and 10 mg, and a significantly high frequency of hypermelanosis on the blind side was observed in these groups at 65 dph ( P  < 0.05). These results suggest that RA synthesized from VA palmitate in Artemia could induce hypermelanosis on blind side of flounder when Artemia are enriched with more than 5 mg VA palmitate/L.     

Formation process of staining-type hypermelanosis in Japanese flounder juveniles revealed by examination of chromatophores and scales

Staining-type hypermelanosis, defined as blind-side melanosis occurring after completion of metamorphosis, reduces commercial value in hatchery-produced flatfishes. Detailed characterization was performed on the stained area of juvenile Japanese flounder Paralichthys olivaceus to physiologically understand this phenomenon. From 80 to 120 days after hatching, juveniles were reared in sandy and sandless tanks. By classifying the staining degree into 7 levels, about 2 times higher occurrence of middle-level staining was reconfirmed in sandless tank (about 80 %) than in sandy tank (about 40 %). In the stained area, we found 3 types of chromatophores (melanophore, xanthophore, and iridophore) and ctenoid scales, which would be typically observed on the normal ocular side. Detailed examination on the melanophores revealed further similarity between the stained area and the normal ocular side, in terms of the distribution at 2 layers (shallower and deeper than scale), and the densities in both layers (about 1000 cells/mm² above scale and 200 cells/mm² beneath scale). These results strongly suggest that the staining is a status change in the body surface conditions from the blind side to that on the ocular side, and not a simple darkening caused by disordered proliferation of melanophores on the blind side.     

Effects of time and duration of rearing with bottom sand on the occurrence and expansion of staining-type hypermelanosis in the Japanese flounder Paralichthys olivaceus

We previously reported that the progression of staining-type hypermelanosis spontaneously ceased at a specific time and area in Japanese flounder Paralichthys olivaceus. To examine whether time is a limiting factor in the spontaneous cessation of staining, we experimentally controlled the initiation and duration of staining by manipulating the bottom substrate condition in the fish tanks. At 151 days post hatching (DPH; 11 weeks), spontaneous cessation of staining was observed in fish reared in tanks without a sandy substrate. However, staining resumed (or was initiated) in tanks where sand was removed from 11 weeks, indicating a strong but temporary effect of bottom sand and the absence of time limitation in the staining progression by 151 DPH. Extended duration of the inhibitory period of hypermelanosis expansion (9 weeks or more) aided in only a 20 % reduction of the final staining area because of the increased rate of staining expansion. The bottom sandy substrate decreased the visibility of the staining area in individuals, but this was observed only before the completion of the staining expansion. These findings are discussed in relation to possible presence of area limitation of future staining, as well as the fundamental nature of staining.     

Prevention of hypermelanosis by rearing Japanese flounder Paralichthys olivaceus in net-lined tanks

Fisheries Science - In artificially reared flatfish, especially the Japanese flounder Paralichthys olivaceus, pigmented skin (hypermelanosis) frequently appears on the fish’s blind side after...     

牙鲆盾纤毛虫病及防治技术研究

从牙鲆(Paralichthys oliuaceus)患病鱼体的溃烂组织中分离出一种盾纤毛虫,通过活体观察、碳酸银法和电镜观察,对其进行了形态学研究分析。鉴定该盾纤毛虫为贪食迈阿密虫(Miamiensis avidus Thompson & Moewus,1964)。这是国内关于贪食迈阿密纤毛虫感染养殖牙鲆的首次报道。同时本文在防治方法方面进行了初步研究,通过离体药敏实验表明:淡水、甲醛、双氧水对该纤毛虫有较好杀灭作用,而中草药的杀灭效果不理想。     

Bottom feeding and net chasing improve foraging behavior in hatchery-reared Japanese flounder Paralichthys olivaceus juveniles for stocking

We examined the effect of bottom feeding and net chasing as means to improve the maladaptive off-bottom swimming of hatchery-reared Japanese flounder Paralichthys olivaceus juveniles for stock enhancement. Three treatments were tested: (1) a bottom feeding treatment in which fish were fed near the bottom; (2) a net chasing treatment in which fish were chased by a hand net two to four times a day, and (3) a surface feeding treatment in which fish were fed from the surface (control treatment). Foraging behavior at the surface with surface feeding was analyzed 1 day before initiating these treatments and 2 weeks thereafter. A comparison of the pre- and post-treatments revealed that off-bottom swimming was less in the bottom feeding treatment and net chasing treatments. A comparison of off-bottom swimming in the bottom feeding treatment between the pre- and post treatments revealed that bottom fed fish showed less off-bottom swimming than surface fed fish. These findings suggest that fish behavior can be manipulated during the rearing period and that such manipulations can be used to improve the behavior of fish for release.     

有丝分裂雌核发育牙鲆的微卫星鉴定

通过静水压抑制卵裂方法获得多个牙鲆(Paralichthys olivaceus)有丝分裂雌核发育家系,在仔鱼阶段经历大批死亡后,将各个家系残存的个体混合饲养,24个月后获得1批存活个体。利用11对微卫星分子标记,对173尾有丝分裂雌核发育牙鲆进行分析,结果表明,其中111尾在11位点全部纯合,62尾在部分位点杂合,平均杂合子比例为0.2338。11个位点的等位基因数为4～8,平均等位基因数为5.0336,有效等位基因数为2.0421～5.1268,平均有效等位基因数为3.2815。采用NJ法对111尾纯合牙鲆的亲缘关系进行聚类分析,可分为4群。微卫星标记鉴定结果表明,通过静水压抑制卵裂法可以获得完全纯合的牙鲆,但有部分杂合个体出现。分析其原因,可能是由于某些卵子发育不同步,静水压抑制其第二极体排出而非全部抑制卵裂,导致减数分裂雌核发育所致。     

牙鲆消化道发育的组织学观察

牙鲆（Paralichthys olivaceus）肉质鲜美、营养价值高,是我国重要的海水养殖鱼类之一,同时,牙鲆二次变态显著,是研究硬骨鱼类变态发育很好的模式生物。在其变态过程前后,外型上最大的变化是其右眼的移位,内部器官如脑颅骨、骨骼肌等都发生结构和功能上的改变,而消化道的发育和分化,特别是其胃在变态前后有显著的变化。消化系统的发育关系到饵料的选择,与仔鱼成活率有直接关系,有较为重要的研究价值。[第一段]     

褐牙鲆♀×大西洋牙鲆♂杂交育种研究

为探讨褐牙鲆和大西洋牙鲆杂交育种的可行性,通过繁殖调控使褐牙鲆和大西洋牙鲆同步性成熟,采取正反交试验,获得了褐牙鲆(♀)×大西洋牙鲆(♂)杂交F1代——花美牙鲆(Paralichthys olivaceus♀×P.dentatus♂)。花美牙鲆受精卵卵径平均1.01mm;孵化适宜温度15～21℃,盐度25～35,孵化率平均71.3%,初孵仔鱼全长2.5～2.7mm;仔鱼在16～20℃下36d后伏底变态,变态个体全长平均11mm,育成率32.5%;反交受精率和孵化率极低,未获得杂交后代。简述了其发育时序和形态特征,探讨温度、盐度对花美牙鲆胚胎和仔鱼发育的影响,比较亲本与杂交鱼早期生长特征,初步分析反交不成功的原因。     

褐牙鲆♀×大西洋牙鲆♂杂交育种研究

为探讨褐牙鲆和大西洋牙鲆杂交育种的可行性，通过繁殖调控使褐牙鲆和大西洋牙鲆同步性成熟，采取正反交试验，获得了褐牙鲆（♀）×大西洋牙鲆（♂）杂交F1代——花关牙鲆（Paralichthysolivaceus早×Pdentatus6）。花关牙鲆受精卵卵径平均1．01mm；孵化适宜温度15—21℃，盐度25～35，孵化率平均71．3％，初孵仔鱼全长2．5～2．7mm；仔鱼在16—20℃下36d后伏底变态，变态个体全长平均11mm，育成率32．5％；反交受精率和孵化率极低，未获得杂交后代。简述了其发育时序和形态特征，探讨温度、盐度对花关牙鲆胚胎和仔鱼发育的影响，比较亲本与杂交鱼早期生长特征，初步分析反交不成功的原因。     

Effects of arachidonic acid on pigmentation and fatty acid composition of Japanese flounder, Paralichthys olivaceus (Temminck and Schlegel)

The dietary effect of arachidonic acid (20:4n-6, AA) on the composition of the head and the body, and on the pigmentation of Japanese flounder, Paralichthys olivaceus (T. & S.), was studied by means of microbound diets. Increasing levels of this essential fatty acid produced higher growth and pigmentation success in juveniles after 45 days of feeding. The role played by AA in the composition of phosphatidylinositol and eicosanoid synthesis is discussed in relation to the normal function of the neural system and the production of hormones involved in the metamorphosis and pigmentation of flatfish.     

Distribution of immunoglobulin-positive cells in the spleen and kidney of Japanese flounder Paralichthys olivaceus

SUMMARY: The distribution of surface immunoglobulin-positive (sIg⁺) cells in the spleen, the head-kidney, and the trunk-kidney of adult Japanese flounder Paralichthys olivaceus was investigated by a flow cytometric analysis using a monoclonal antibody FB17 against flounder immunoglobulin M. Although the percentage of sIg⁺ cells varied among organs, a good correlation was revealed between the percentage in the head-kidney and that in the trunk-kidney ( r = 0.996, P < 0.001). In contrast, no correlation was obtained between the percentage of sIg⁺ cells in the spleen and the head-kidney or trunk-kidney. The head-kidney and the trunk-kidney contained cells bearing fewer sIg that were stained dimly by FB17 in addition to brightly stained sIg⁺ cells regarded as mature B cells, while the spleen contained only sIg⁺ cells stained brightly. Moreover, the immunocytochemical analyses showed that the head-kidney contains plasma cells with strongly stained cytoplasm in addition to B cells with stained cell surface, which suggests that the cells with fewer sIg detected by flow cytometric analysis are presumed to be at a stage during the processes of differentiation to plasma cells. These results suggest that there are some differences between the spleen and kidney in the role of the immune system and it is considered that differentiation of B cells to plasma cells occurs in the kidney.     

Importance of bottom type and background color for growth and blind-side hypermelanosis of the olive flounder,Paralichthys olivaceus

When flatfishes are cultured at high-density in an artificial facility, the bright background color and the carpeted bottom substratum (sand or gravel) help significantly to enhance growth and prevent blind-side hypermelanosis. However, how the bright background and carpeted gravel enhance growth and prevent the hypermelanosis is unknown. Thus, we performed three experiments using the olive flounder, Paralichthys olivaceus. In the first, the acupressure of gravel to blind skin and the burrowing behavior of flounder were assessed to determine which eventually suppressed hypermelanosis. In the second experiment, we assessed whether the color of the carpeted gravel affected flounder hypermelanosis and growth. In the final experiment, we assessed whether hypermelanosis of flounder reared on a gravel bottom was continuously inhibited after removal of the gravel substratum, and whether tank color affected the hypermelanosis and fish growth. The results showed that the burrowing of flounder into gravel was more important than the stimulus of rough gravel on blind-skin to prevent blind-side hypermelanosis. The color of the gravel and tanks did not affect hypermelanosis. Removal of the gravel substratum resulted in enhancement of blind-side hypermelanosis. However, fish growth was enhanced by a bright-colored tank but not a bright-colored bottom substratum. In conclusion, these results confirm that the absence of shelter in which flounder can burrow or hide induces blind-side hypermelanosis, and that flounder consistently burrow beneath the substratum to prevent malpigmentation. Additionally, our results show that a bright-colored tank resulted in increased growth, indicating that flounder should be reared in a bright-background to enhance growth.     

偏分离条件下牙鲆生长性状QTL的主成分定位

本研究利用有丝分裂雌核发育建立牙鲆(Paralichthys olivaceus)双单倍体(Doubled Haploids,DH)群体,对牙鲆体重、全长、背鳍长、腹鳍长、体高、尾柄高、头高和躯干长共8组表型性状标准化处理后,进行主成分分析,得到可解释全部性状90.4%的表型主成分性状。然后,基于JoinMap 4、MapDisto、JoinMap 4-DistortedMap、MapDisto-DistortedMap构建4个连锁图谱,用偏分离标记矫正数量性状位点(Quantitative Trait Locus,QTL)基因型条件概率,采用Bayesian模型选择方法定位牙鲆表型主成分性状的加性QTL和上位性QTL。结果表明,用不同方法构建的遗传图谱和表型主成分性状QTL定位结果均有所不同。在图谱构建中,与基于JoinMap 4构建的图谱相比,基于MapDisto构建的图谱中偏分离标记的相对位置和遗传距离都发生了变化,甚至有5个偏分离标记没有被定位到相应的连锁群上;相对于基于JoinMap 4和MapDisto构建的图谱,经DistortedMap校正后的图谱中偏分离标记的相对位置没有发生变化,但是偏分离标记间遗传距离发生了变化。另外,在4个图谱中都检测到3个加性QTL,分别位于6号连锁群上,可解释表型变异的12.95%、14.85%、11.56%和11.76%,9号、22号连锁群上,具有负向加性效应;9号连锁群上,可解释表型变异的13.86%、13.27%、11.17%和11.25%,具有负向加性效应;22号连锁群上,可解释表型变异的5.68%、4.36%、4.97%和3.58%,具有正向加性效应。同时,分别检测到28对、19对、29对和20对上位性QTL,主要分布在6号、7号、9号、17号、20号和22号连锁群上,可解释表型主成分性状变异的2.19%~17.62%、2.40%~22.26%、2.08%~26.0%、3.16%~22.05%。研究结果表明,基于MapDisto软件构建、经DistortedMap软件包矫正后的图谱,定位结果更加准确,但仍需进一步验证。     

牙鲆身体纵轴生长相关性状QTL定位

以经紫外线灭活的真鲷(Pagrosomus major)精子作为异源精子,诱导牙鲆(Paralichthys olivaceus)卵子进行有丝分裂雌核发育,通过静水压方法获得165个双单倍体作为实验材料.测量牙鲆身体纵轴6个生长相关性状(体长、头长、背鳍基长、腹鳍基长、尾柄长和尾长),并用SPSS 19.0对其进行正态分布及相关性分析.运用MapQTL4.0中多座位QTL 模型(MQM)对控制6个性状的QTL进行定位及遗传效应分析.结果表明,所检测6个性状均符合正态分布,并且相互之间呈极显著相关(P<0.01).在全部24个连锁群上检测到22个控制相关性状的QTL,分布在10个连锁群上.其中与体长相关的QTL有4个,头长相关的QTL为3个,控制背鳍基长的QTL为4个,与腹鳍基长相关的QTL为3个,与尾柄长相关QTL 7个,控制尾长的QTL仅有1个.各QTLs的LOD 值在2.01~3.68之间,可解释3.7%~12.6%的表型变异.控制体长和尾长的 QTL存在共定位,控制体长的 QTL 还与控制背鳍基长及腹鳍基长的QTL存在共定位.本研究结果可为今后进一步辅助育种和改良性状提供参考.