Progression of staining-type hypermelanosis on the blind side in normally metamorphosed juveniles and pigmentation progression in pseudoalbino juveniles of the Japanese flounder Paralichthys olivaceus using individual identification

Abnormal flounder coloration frequently occurs in flounder hatcheries and diminishes the commercial value of the fish. To understand hypermelanosis, the progression of staining-type hypermelanosis in normally metamorphosed juveniles and ocular-side pigmentation in pseudoalbino juveniles were examined in the Japanese flounder Paralichthys olivaceus. Sixty-five days post hatching, juveniles (total length, 6 cm) were individually identified by color-marker implantation, and the darkened area of the body surface was examined for 10 weeks by image analysis of digital photographs of the fish taken from above or below the transparent tank. Staining was observed to mainly begin at the upper and lower bases of the tail fin, expanded anteriorly along the peripheral part of the trunk, and ceased after 2 months. The individuals in which staining occurred earlier expressed severe staining and small body size by the end of the experiment. Further, pigmentation of the ocular side in pseudoalbino juveniles ceased after 2 months, but the order of pigmentation was different from that on the blind side. In this case, darkening began from the posterior, but expanded from the center to the periphery of the trunk. Even at the end of the experiment, ctenoid scales were exclusively found within the darkened area, together with cycloid scales.     

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...     

An effective and practical method of net settings in rearing tank to suppress hypermelanosis in Japanese flounder

In Japanese flounder aquaculture, hypermelanosis occurs widely on the blind side. Rearing flounders in a net-lined tank was recently reported to prevent hypermelanosis. To effectively apply this method to larger tanks for aquaculture farming, the net setting method was examined. Juvenile flounders without darkened areas on the blind side [total length (TL) 13 cm] were selected, and reared for 6 months (TL 32 cm). In the control tank without a net, the median value of darkened area ratio (darkened area to blind side area) was 46%. By only covering the tank bottom with slack net, the darkened area ratio was suppressed to 8%, less than 1/5 of that in the control tank. At the end of experiment, bottom coverage ratios of 0%, 10%, 30%, 50%, and 100% revealed a negative correlation between bottom net coverage and darkened area ratio. In this experiment, the darkening area in the tank with 50% bottom net coverage decreased to 1/3 of the control. Although the occurrence of hypermelanosis differs depending on the production lot, these results are expected to serve as a reference for selecting the suitable net size to meet the level of clearness of the blind side.

     

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.     

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.     

牙鲆胚胎玻璃化冷冻及解冻过程中的温度因子与玻璃化率

为避免鱼类胚胎玻璃化冷冻过程中脱玻璃化现象发生,本文对冷冻过程中温度变化及解冻方法进行了研究.在室温13℃下,将含有玻璃化液FVS3的麦管直接投入到-196℃的液氮中,麦管内温度达到-196℃时,所用的时间为15.46±1.38s,降温速率为800℃/min.对玻璃化麦管在35～45℃水浴中解冻时间进行测定.结果显示,解冻所需时间在6.17～7.94s之间,麦管从-196℃升高到13℃的解冻速率为1500～2000℃/min.当水浴温度从37℃逐步升高到72℃时,对水浴表面蒸气温度的变化进行了测定,相应水温下蒸气温度从25.47℃升高到38.6℃.在麦管中加入不同浓度的玻璃化液FVS1、FVS2、FVS3和FVS4进行玻璃化冷冻,然后将麦管分别在37℃水浴中和28.6℃的蒸气中解冻时玻璃化率进行研究.结果显示,在蒸气中解冻时的玻璃化率明显高于水浴中解冻;麦管玻璃化率在水浴中解冻时不稳定,在蒸气中解冻时表现稳定,玻璃化率随着玻璃化液浓度的升高有逐渐升高的趋势.对含有牙鲆胚胎的玻璃化冷冻麦管分别在37℃水浴和28.6℃蒸气中解冻,结果表明,蒸气中解冻时的玻璃化率同样明显高于水浴中解冻.     

Undulated flooring in the rearing tank decreases hypermelanosis in Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

In the aquaculture of the Japanese flounder Paralichthys olivaceus, hypermelanosis, a malpigmentation condition in which the scales of a significant area on the blind side express the characteristics of those on the ocular side, remains a major concern. Since introducing sand into the rearing tank effectively suppresses hypermelanosis, the inhibitory effects of various characteristics of the surface of the tank floor were investigated. Although an inhibitory effect was observed in both tanks with a sand-image floor and a sand-pasted floor, the strongest effect was found in tanks with a dimpled floor. In addition, covering the inner surface of the tank with net also inhibited hypermelanosis. Using a commercially available corrugated plate, the inhibition of hypermelanosis was confirmed with a noted increase in this effect when combined with light coloration. Juveniles tended to situate themselves in the valley portions of the corrugated plate, suggesting that floor contact with the blind side may contribute to hypermelanosis inhibition. Further observations on the floor contact area with various floor configurations suggested that hypermelanosis is locally suppressed in the areas with floor contact. Therefore, suppression by an undulated (both dimpled and corrugated) floor is likely due to an increase in floor contact area with the blind side.     

Transplantation of pigmented and non-pigmented scales into the ocular and blind sides of the Japanese flounder Paralichthys olivaceus,suggesting the presence of ocular-side characteristic inducer in pigmented scales

After metamorphosis, both eyes of the Japanese flounder Paralichthys olivaceus are located on its left side, with only the ocular side becoming pigmented. Staining, or hypermelanosis, occurs on the blind side at 2–3 months after metamorphosis, thereby lowering the market price of the fish. To understand the pigmentation expansion process, we performed scale transplantation between the blind and ocular sides of an individual. About 40 % of transplanted scales were successfully engrafted, regardless of donor or recipient site. When blind-side scales were transplanted to the ocular side, they became pigmented after 2 weeks, while no change was observed when the scales were transplanted to the blind side. Ocular-side scales did not lose pigment, regardless of the recipient site. However, after removal of transplanted ocular-side scales, pigmented scales regenerated after 3 weeks, even at blind-side sites. Identical results were obtained when the stained area on the blind side was used as the recipient location. When an ocular-side scale with skin tissue was inserted under blind-side scales, the scales immediately above the transplanted area became pigmented, whereas ocular-side scales stripped of tissue did not induce pigmentation. These results strongly suggest the presence of an ocular-side characteristic inducer in pigmented scale tissues.     

石鲽、牙鲆精子冷冻保存研究及其在人工杂交中的应用

对石鲽(Kareius bicoloratus )和牙鲆(Paralichthys olivaceus)精子冷冻保存技术进行了研究,筛选到一个优良的稀释液MPRS,用MPRS冷冻保存石鲽、牙鲆精子,冻后成活率在70%以上。在石鲽冻精和牙鲆卵的杂交实验中,杂交组受精率是 28 4%±4 55(n=3),孵化率是 42 7%±7 35(n=3),胚胎发育正常;5 000尾杂交鱼苗有102尾成功度过变态期。在牙鲆冻精和大菱鲆卵的杂交实验中,杂交组的受精率只有2 7%,部分受精卵虽能孵化出膜,但仔鱼全为畸形。这两个杂交实验,特别是石鲽冻精和牙鲆卵的杂交实验,证实了冷冻保存的精子完全可以应用在杂交育种工作中。     

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.     

Rearing of flounder (Platichthys flesus) juveniles in semiextensive systems

A low-technology rearing system was implemented for rearing juvenile flounder for stock enhancement in a Danish fjord, the Limfjord. Each year during 1996–2002, between 13,000 and 153,000 juveniles were reared from the yolk-sac stage until metamorphosis in outdoor ponds relying on phyto- and zooplankton blooms as their main food source. In contrast to other similar systems, the blooms in this system are closely monitored and, to a certain extent, regulated. The zooplankton blooms consisted mainly of calanoid copepods, dominated by the species Temora longicornis and Centropages hamatus. Most juveniles produced (>99.5%) were normally pigmented with average yearly survival rates from hatch to metamorphosis varying from 7±9% to 48±18%, lowest in the first years of production.     

cDNA cloning and characterization of two gelatinases from Japanese flounder

SUMMARY: Toughness is one of the most important elements that define the commercial value of the raw meat of fish. Degradation of the extracellular matrix is thought to be a cause of postmortem tenderization of fish meat. A previous study has suggested that this tenderization is caused mainly by metalloproteinases. The present study seeks to identify the proteinase(s) involved in tenderization; hence, cloned cDNA of two gelatinases from Japanese flounder, which showed high homology with mammalian matrix metalloproteinase (MMP)-2 and MMP-9, were designated as jfMMP-2 and jfMMP-9 , respectively. Northern blot analysis revealed that jfMMP-2 mRNA was expressed almost ubiquitously in adult tissues including the brain, muscle, gill, heart, gut, kidney, spleen, testis, and ovary. In contrast, the expression of jfMMP-9 mRNA was observed in those tissues which were abundant in blood cells, such as kidney, spleen, heart, and gill. Both recombinant proteins (jfMMP-2 and jfMMP-9) produced with the COS-7 cell system exhibited gelatin-degrading activity that was sensitive to 1,10-phenanthroline, a typical metalloproteinase inhibitor.     

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。     

Influence of density and background color to stress response, appetite, growth, and blind-side hypermelanosis of flounder, Paralichthys olivaceus

To study the relevance of density and background color to stress response, appetite, and growth in olive flounder, Paralichthys olivaceus, we reared two duplicate groups of juveniles (total length 4.46 ± 0.06 cm, body weight 0.77 ± 0.03 g) in flat-bottom aquaria with dark-green (control) and white backgrounds for 120 days. We measured cortisol and glucose levels in blood and calculated the daily food intake, food conversion efficiency, survival rate, and growth rate. To study the relevance of density and background color to malpigmentation (hypermelanosis) on the blind side, we also compared malpigmented ratios and prepro-melanin-concentrating hormone mRNA activities in the brain between the dark-green and white background groups, as well as between a relatively lower density (60 days) and higher density (120 days). Although we measured relatively higher levels of cortisol and glucose in the white background group and over 200 % of coverage area [PCA]), the bright background failed to induce an acute stress response of more than 20 ng/ml cortisol and 40 mg/dl glucose both in 60 days and 120 days, but did enhance appetite and growth. Also, a bright background color delayed hyperpigmentation only at a low density below 200 % PCA, but did not inhibit malpigmentation at a high density of more than 200 % PCA. In addition, below 200 % PCA, expression of MCH mRNA was significantly higher in the white group, but the level was reversed and was lower in the white group at more than 200 % PCA. In conclusion, although did not induce a high stress response over 200 % PCA, the bright background color resulted in a moderate increasing of cortisol level in blood below 20 ng/ml and enhanced appetite and growth. Moreover, at a density below 200 % PCA, the bright color inhibited hypermelanosis with high MCH mRNA activity, but at more than 200 % PCA did not inhibit malpigmentation, and the fish showed low MCH mRNA activity, indicating that the inhibitory effect of a bright background color on hypermelanosis is density dependent.