Influence of Hatchery Protocols on Mitochondrial DNA Variation in Japanese Flounder Juveniles

The Japanese flounder Paralichthys olivaceus is a commercially important fish that is stocked extensively from hatchery rearing programs in Japan. To examine the genetic variability of hatchery-raised juveniles of the Japanese flounder that are used for stocking into natural waters, we analyzed a portion of the mitochondrial genome. The mtDNA region extending from the 3'half of the cytochrome b gene to the central domain of the control region was PCR amplified and analyzed using 11 restriction endonucleases. We identified 34 polymorphic cleavage sites out of a total of 61 sites, which resulted in 67 different haplotypes in a total of 265 offspring, examined from eight hatchery stocks. Haplotype diversity of offspring at each of the eight hatcheries ranged from 0.49 ± 0.09 (SE) to 0.94 ± 0.03 (SE). Also, we observed 40 polymorphic sites out of a total of 59 sites, which resulted in a total of 50 haplotypes in 60 wild flounder. Haplotype diversity of the wild population was 0.98 ± 0.01 (SE). The use of subcultured fishes as broodstock appears to be one of the most important causes of reduced genetic diversity in hatchery-raised flounder juvenile. Our results suggest that the use of wild fish for broodstock is an effective way to maintain genetic variability in Japanese flounder offspring.     

Predator–prey size relationship in siphon cropping between the juvenile stone flounder Platichthys bicoloratus and the bivalve Nuttallia olivacea

ABSTRACT:   The purpose of the present paper was to examine the size relationship between the juvenile stone flounder Platichthys bicoloratus and the bivalve Nuttallia olivacea , the siphon of which is important prey for the juvenile flounder. Juvenile stone flounder feed mainly on tips of the inhalant siphon of the bivalve. The maximum width of siphon tips in the stomach contents of age-0 fish could not reach that of age-1 fish, although the siphons in age-0 fish became larger as they grew. This size discrepancy indicated a limitation in the size of bivalves available to juveniles. The proportion of total cropping frequency for the siphon of the bivalve by juveniles was estimated according to the bivalve size class. Most bivalves that had cropped siphon tips ranged from 5 to 30 mm in shell length. The total cropping frequency per bivalve was particularly intense on bivalves of 10–25 mm shell length in spite of their small proportion of 24.9% of the total. This frequency intensity indicated that the size of bivalves with cropped siphons by juvenile stone flounder might depend on the ability of juveniles rather than the size composition of the bivalves.     

Changes in growth,higher salinity tolerance and behavioral traits in Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> after repeated cross-breeding

ABSTRACT:     To improve stock enhancement strategies, the influences of repeated cross-breeding on several traits were studied in hatchery-reared juveniles of the Japanese flounder. Ten families (seven caught in the wild and three from cross-bred brood fish) were produced by artificial fertilization. Ninety-four days after hatching, we investigated settlement, burying in the sand and escape behavioral traits, and then measured tolerance of high salinity and six morphological traits. Juveniles from the repeatedly cross-bred brood fish showed significantly higher growth and tolerance than those from the wild-caught brood fish. Among the behavioral traits, a significantly lower settling speed and incidence of burying in the sand were observed in the juveniles from the repeatedly cross-bred families. Heritability ( h ²) of growth traits was more than 0.2 in both origins, while that of other traits was close to zero. These results suggest that repeated cross-breeding of Japanese flounder under hatchery conditions improves growth performance and leads to the loss of certain behavioral traits that are considered essential to survival in wild environments.     

Comparative stomach contents and growth of the juvenile black rockfish Sebastes inermis reared in illuminated and unilluminated cages

ABSTRACT:   The principal objective of the present study was to determine the positive effects of artificial illumination on the juvenile black rockfish Sebastes inermis by comparing stomach contents and growth between juveniles exposed to light and those maintained in the absence of light. The major prey items of juvenile black rockfish in illuminated cages were: copepods (% IRI  = 62.6); amphipods (% IRI  = 36.7); and polychaetes (% IRI  = 0.3), whereas those in unilluminated cages were: copepods (% IRI  = 93.3); amphipods (% IRI  = 6.1); and polychaetes (% IRI  = 0.4). The specific growth rates of the juveniles reared in illuminated cages (0.67%) were significantly higher than those juveniles maintained in the unilluminated cages (0.27%).     

Thyroid hormone deficiency in abnormal larvae of the Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

ABSTRACT:   In large-scale rearing of juveniles of the Japanese flounder Paralichthys olivaceus, a certain morphological abnormality occurred spontaneously in 4% of the fish. These fish showed a slight but clearly different appearance from any developmental stage of this species, and did not settle when all the other juveniles in the same tank completed metamorphosis and had settled. From comparisons of external and internal structures between the normal and the abnormal fish, the abnormality was attributed to unbalanced progress of metamorphosis, mainly due to metamorphic stasis. The thyroid of the abnormal fish was apparently activated morphologically. In addition, serum thyroxine (T4) concentrations in the abnormal fish were reduced to less than 1/10 of that of normal fish. After 14 days of T4 treatment (0.1 p.p.m) of the abnormal fish, all the abnormal characteristics disappeared, and the fish recovered to normal, suggesting normal responsiveness to thyroid hormones in peripheral tissues, whereas thiourea treatment (30 p.p.m., 14 days) further delayed metamorphosis. These results suggest that these abnormal fish were suffering from thyroid hormone deficiency, and were unable to secrete a sufficient amount of thyroid hormone to complete metamorphosis.     

Effect of dietary taurine and lipid contents on conjugated bile acid composition and growth performance of juvenile Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

ABSTRACT:   The effects of dietary taurine levels and lipid contents on the conjugated bile acid composition and growth performance of juvenile Japanese flounder Paralichthys olivaceus were investigated. Six types of diet (three different levels of taurine at two different levels of lipid) were fed to juveniles (average body weight, 0.04 g). Fishmeal that was washed with 70% ethanol to remove taurine was used as the sole protein source. Feeding experiments were carried out at 20°C for 6 weeks. At the end of the experiments, fish were weighed and analyzed for free amino acids in the body and the composition of the conjugated bile acids. The body weight and percent weight gain of the juveniles were improved by the dietary taurine supplementation. The taurine contents of the whole body and tissues increased with the increase of the dietary taurine contents. The conjugated bile acids in the gall bladder consisted of taurocholic acid and taurochenodeoxycholic acid, which increased with the increase of the dietary taurine level. Taurocholic acid accounted for more than 95% of the total conjugated bile acids. This indicates that taurine is the sole amino acid to conjugate bile acid in Japanese flounder.     

Parental Contributions in a Japanese Flounder Hatchery Inferred from Mitochondrial DNA Haplotypes

We analyzed a portion of the mitochondrial genome of hatchery Japanese flounder Paralichthys olivaceus to compare the genetic characteristics between maternal parents and their offspring. The maternal parents consisted of 11 subcultured fish (broodstock taken from hatchery-raised fish) and 41 wild fish. A 1098 base pair region extending from the 3'half of the cytochrome b gene to the central domain of the control region of mtDNA was amplified by PCR and analyzed using 11 restriction endonucleases. We also analyzed the number of copies of tandem repeats in the control region from the sizes of the PCR products. Each female parent had a unique haplotype, but only 31 of them were observed among the 66 offspring examined. From these results, we estimated that about 60% of the maternal parents in the hatchery contributed to the offspring we surveyed. The distribution of the family sizes of the wild fish was Poisson, which is consistent with expectation from a randomly mating population. In contrast, the distribution of family sizes of the subcultured fish deviated from a Poisson distribution. One of the sources of the deviation was that none of the six oldest fishes contributed to offspring production, which is consistent with observation that the spawning peaks later in the spawning season for older fish in flounder hatcheries. Uneven contribution of the female flounders in the hatchery has the potential to reduce substantially the genetic diversity in the offspring. Our results suggest that the misuse of subcultured fish as broodstock is an important cause of reduced genetic diversity in hatchery-raised flounder juvenile.     

Detection of Japanese flounder-specific DNA from gut contents of potential predators in the field

ABSTRACT:   Predation by invertebrates may be one of the major factors influencing the mortality of released flounder juveniles in the field. We developed a polymerase chain reaction (PCR)-based technique for the detection of Japanese flounder material from the gut contents of potential predators in the field. The PCR primers used here are flounder specific and encompass a 153 bp region from the right domain of the mitochondrial control region. With these oligonucleotide primers, PCR reactions yielded specific amplification products from the gut contents of some putative predators, including Matuta lunaris , Portunus gladiator , Sepioteuthis lessoniana , Suggrundus meerdervoorti , Platycephalus sp. and Rhyncopelates oxyrhynchus . Many animals with gut contents of unidentified fish material showed indication of predation on the flounder after release of hatchery reared flounder juveniles. The technique developed in the present study is useful not only for the survey of predation on the flounder by known species, but also for the search for novel potential predatory species in the field.     

Antibacterial abilities of intestinal bacteria from larval and juvenile Japanese flounder against fish pathogens

The present study was undertaken to examine the antibacterial abilities of intestinal bacteria isolated from juveniles and larvae of Japanese flounder Paralichthys olivaceus . Newly hatched larvae of flounder were held in a 100 L plastic circular tank and fed rotifiers, Artemia nauplii and commercial feeds, depending on the developmental stage of the fish. Genera Aeromonas , Moraxella and Vibrio were predominantly isolated from the intestinal tracts of Japanese flounder at larval and juvenile stages, whereas Aeromonas , Bacillus , coryneforms, Moraxella , Pseudomonas and Vibrio were detected at high densities in live diets and artificial feeds. Antibacterial bacteria accounted for 1.7–24.3% of the intestinal isolates against Lactococcus garvieae , Pasteurella piscicida , Vibrio anguillarum and V. vulnificus . In particular, as much as 53.3% of Vibrio spp. other than Vibrio -swarmer isolated from 197-day-old juveniles inhibited the growth of P. piscicida . These results suggest that intestinal bacteria having antibacterial activity play a role in the prevention of infectious diseases.     

Can fermented soybean meal and squid by‐product blend be used as fishmeal replacements for Japanese flounder (Paralichthys olivaceus)?

An experiment was carried out to evaluate fermented soybean meal and squid by‐product blend (1:1) (FP) as replacement of fishmeal (FM) for Japanese flounder, Paralichthys olivaceus. Five isocaloric (19 kJ g^?1) diets were prepared by replacing 0 (FP0), 12 (FP12), 24 (FP24), 36 (FP36) and 48% (FP48) FM protein with FP. Triplicate groups of juveniles (mean weight of 3.9 g) were delivered the test diets for 8 weeks in a flow‐through sea water system. The results showed that there were no significant differences (P > 0.05) among the growth rates of fish fed FP0, FP12, FP24 and FP36 diets. Growth and nutrient utilization parameters were significantly reduced in fish fed FP48 diet. Although, whole body proximate composition of fish were not significantly affected by the dietary treatments compared to the control; methionine and phenylalanine contents were significantly decreased in FP48 group. Protein retention was also significantly decreased in the similar group of fish. Dietary treatments did not alter most of the plasma metabolites, while some of the health parameters were improved in the replacement groups. Results suggested that FP is a potential candidate for alternative protein ingredient in aquafeed and can replace 36% FM protein in the diet of Japanese flounder.     

Food organisms and feeding habits of larval and juvenile Japanese flounder Paralichthys olivaceus at Ohama Beach in Hiuchi-Nada, the central Seto Inland Sea, Japan

ABSTRACT:   The abundance of food organisms and feeding habits of larval and juvenile Japanese flounder were examined during the period from May to August in 1999, 2000 and 2001 at the sandy Ohama beach, the central Seto Inland Sea. The food organisms collected with a sledge net consisted of 40 families from 18 orders, dominated by mysids, crangonids and gammarids. The mean densities of mysids, crangonid shrimp ( Crangon spp.), gammarids and fish were 2.74, 6.74, 2.91 and 0.15 individuals/m², respectively. The main prey of the flounder ( n  = 202; range of total length 9.80–75.95 mm) was mysids and small crangonid shrimp (<14 mm in body length). Prey fish availability was low, as the density of fish was low. The small crangonid shrimp was abundant, and the large crangonid shrimp, which could prey on larval flounder, was not abundant. The crangonid shrimp was important not as a predator for the flounder but as prey. The flounder preferred epifaunal mysids, Nipponomysis ornata and Anisomysis ijimai , to sand-burrowing mysids, Iiella oshimai , and avoided crangonid shrimp.     

Effects of fish size and water temperature on the acute toxicity of boron to Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> and red sea bream <Emphasis Type="Italic">Pagrus major</Emphasis>

ABSTRACT:     The acute toxicities of boron were examined for Japanese flounder Paralichthys olivaceus and red sea bream Pagrus major in terms of fish size and water temperature. Japanese flounder of 0.1–70.0 g and red sea bream of 0.6–20.3 g were exposed to different concentrations of boron for 96 h at 20°C under semi-static conditions. In both fish species, the median lethal concentration (LC₅₀) for 96 h of boron increased linearly with increasing fish size, ranging from 108 to 252 mg B/L for the flounder, and from 97 to 172 mg B/L for the sea bream. The effect of water temperature on acute toxicity was examined for Japanese flounder of 0.6 and 1.5 g at 10, 15, 20 and 25°C, and for red sea bream of 0.6 and 2.4 g at 12, 15, 20 and 25°C. The toxicity of boron for the flounder increased linearly with increasing water temperature. The 96 h LC₅₀ values ranged from 299 to 108 mg B/L for the 0.6 g flounder and from 350 to 113 mg B/L for the 1.5 g flounder. A similar trend was shown for the 2.4 g red sea bream; however, the relationship for the 0.6 g red sea bream was not significant.     

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.     

微粒饲料中以微藻粉替代鱼油对牙鲆(Paralichthys olivaceus)稚鱼生长存活和脂肪酸组成的影响

以18日龄的牙鲆(Paralichthys olivaceus)稚鱼为研究对象,通过11 d 的生长实验,研究了添加不同比例的微藻粉替代鱼油对牙鲆稚鱼生长、存活率和脂肪酸组成的影响。以鱼油组(FO)为对照组,以裂壶藻粉(Schizochytrium sp.)、微绿球藻粉(Nannochloropsis sp.)和橄榄油替代不同比例的鱼油,配制成5组等氮等能的实验饲料,分别命名为鱼油组(FO),50%混合替代组(M50)、100%混合替代组(M100)、100%裂壶藻橄榄油替代组(S100)、100%微绿球藻橄榄油替代组(N100)。结果显示,微藻粉替代鱼油对牙鲆稚鱼的生长无显著影响;含有裂壶藻的各饲料组(M50、M100、S100)成活率显著高于 FO 组和 N100组(P?0.05);微藻粉替代鱼油不影响牙鲆稚鱼主要脂肪酸的组成;Person相关性分析发现,C14：0、C16：1n-7、C18：2n-6、C20：0、C18：3n-3、C22：0、C20：4n-3、EPA、C22：5n-6和 DHA 的百分含量均与其饲料中的百分含量呈显著正相关(P<0.05);总饱和脂肪酸、总单不饱和脂肪酸、n-3多不饱和脂肪酸的百分含量以及 DHA/EPA 比率均与其饲料组成表现出显著正相关(P<0.05)。综上所述,微藻作为脂肪源替代鱼油完全可以满足牙鲆稚鱼的生长和发育,各种脂肪酸均可以被牙鲆稚鱼充分消化和吸收,并且添加两种微藻后提高了稚鱼的 DHA 含量和 DHA/EPA 比率,与鱼油对照组相比显著提高了牙鲆稚鱼的成活率。因此,以微藻替代鱼油在牙鲆稚鱼的培育中是可行的。     

Effects of hypoxia on principal prey and growth of flathead flounder Hippoglossoides dubius in Funka Bay, Japan

ABSTRACT:   Effects of hypoxia on the principal prey and growth of flathead flounder Hippoglossoides dubius were studied in Funka Bay. Of the three dominant year-classes that occurred in recent years, the 1995 year-class was small in total length at age ≥ 3 and low in condition factor at age ≥ 2. Ophiuroids (almost Ophiura sarsi ), which were the dominant prey in the 1980s, were of little importance and instead, crustaceans such as mysids, natant decapods and pelagic amphipods, bivalves and fish were important prey items for H. dubius in 2000–2001. In addition, the feeding intensity of H. dubius in 2000–2001 was lower than that in the 1980s. These facts are closely related to a reduction of prey abundance, particularly ophiuroids. It seems that the hypoxia that occurred in the central part of the bay during the summer and autumn of 1995–1997 caused the poor food supply and low growth rate at ages 2–4 of the 1995 year-class.     

Occurrence and distribution of larval and juvenile Japanese flounder Paralichthys olivaceus at sandy beaches in eastern Hiuchi-Nada, central Seto Inland Sea, Japan

ABSTRACT:   The occurrence and distribution of larval and juvenile Japanese flounder Paralichthys olivaceus and major demersal fish from May to September were examined at seven sandy beaches at the central Seto Inland Sea. The flounder appeared from late May to August and were mainly collected at a depth of 2–5 m. Settlement of larval flounder was seen from late May to late June. A total of 6412 individuals of 17 species representing 12 families were collected in the study area. The dominant fish species were Favonigobius gymnauchen (56.0%), Tarphops oligolepis (8.1%), Rudarius ercodes (7.8%), P. olivaceus (7.3%), Repomucenus spp. (7.0%) and Heteromycteris japonica (3.3%). The catches of flounder differed among beaches and months. The number of flounder in June at Ohama Beach, where the most flounder were collected, was 22.6 individuals per haul (400 m²). The density of the flounder correlated negatively to that of F. gymnauchen .     

漠斑牙鲆仔鱼、稚鱼和幼鱼对低盐度的耐受力和淡水驯化技术的研究

研究了漠斑牙鲆仔鱼、稚鱼和幼鱼对低盐度的耐受力和漠斑牙鲆的淡水驯化技术。初孵仔鱼在盐度为5的水中最多存活5d,5d以后全部死亡。30日龄稚鱼在4h10min淡水组全部死亡,对淡水的耐受力较低,而盐度为5～25的各组72h的存活率都在95.00%以上。90日龄幼鱼经缓慢淡水驯化9d后存活率可达98%以上,对漠斑牙鲆幼鱼进行淡水驯化的最佳年龄为90日龄。淡水驯化影响幼鱼初期的摄食,以后则逐渐趋于正常;急性淡水驯化对幼鱼的伤害比缓慢淡水驯化大;漠斑牙鲆对低盐的耐受力随生长和发育逐渐增加,年龄是影响幼鱼对低盐度耐受力的主要因素。     

编码金属标签对牙鲆(Paralichthys olivaceus)苗种标记的效果

为了开发适宜牙鲆(Paralichthys olivaceus)苗种放流的标志技术,利用编码金属标签(CWT)对不同规格牙鲆苗种进行了标记实验,并从标记死亡率、脱标率、适宜标记鱼规格等方面评价了CWT的标记效果.结果显示,CWT标记3种规格的牙鲆苗种后,脱标均发生在标记后的4d内,其后未再发生脱标现象.小规格苗种[全长为(5.92±0.41)cm]死亡率较高(13％),中规格苗种[全长(8.92±0.36) cm]死亡率为2％,大规格苗种[全长为(12.06士0.62) cm]死亡率为1％.小规格、中规格和大规格苗种脱标率分别为3.3％、2.4％和0.7％.建立了标记死亡率(M)与苗种全长(TL)、体厚度(BT)的关系模型:M=0.7254 TL2-15.3220TL+79.4260 (R2=0.9601);M=1.3627 BT2-15.5610 BT+ 44.4330 (R2=0.9645),为适宜苗种规格选择与标记效果评价提供了依据.今后利用CWT标签进行牙鲆苗种标志放流时,建议选择全长6 cm以上的苗种进行背部肌肉标记,标记对苗种游泳行为和生长无影响,表明CWT是一种适宜在牙鲆大规模标志放流中应用的理想标志方式.     

Note on occurrence and the food of juvenile kachlan (Trachinotus ovatus,Linnaeus) (Pisces,Carangidae) from the Mediterranean

Juvenile kachlan (Trachinotus ovatus, Linnaeus) were collected with beach seines from the Mediterranean coast of Israel. Length frequency distributions of 1 539 fish were used to indicate the arrival of juveniles. The estimated time of arrival is July–August. Stomach contents of 153 juveniles were determined. The fish ranged from 19 to 90 mm in total length. Percent frequency occurrence of diet contents was determined. Juvenile kachlan ate polychaetes, cladocera, copepods, isopods, amphipods, insects and small fish.     

Feeding habits of Pacific pomfret Brama japonica inthe transition zone of the central North Pacific

ABSTRACT:   We examined the diets of three size groups of the Pacific pomfret Bramajaponica collected in April and May 2000 in the transition zoneof the central North Pacific. Amphipods, cephalopods and fish werethree of the most common prey groups of pomfret. Of the three size groups,small pomfret (11.0–19.9 cm standard length (SL))preyed mainly on amphipods and occasionally on cephalopods (mainlytransitional species Onychoteuthis borealijaponica ) and fish.(These prey amphipods and fish could not be identified to lowertaxonomic levels because they were too digested.) Medium and largepomfret (20.0–30.9 and 31.0–40.9 cm SL,respectively) preyed mainly on cephalopods (mainly O. borealijaponica ,but also on two subarctic species, namely Berryteuthis anonychus and Gonatopsisborealis ), suggesting that these cephalopod species ensure energyfor the northward migration of these two size groups in the transitionzone in the spring. The next most common prey for medium pomfretwas amphipods (mainly subtropical species Phronima sedentaria and Platyscelusovoides ), followed by fish (mainly Bathylagus sp.). However,in the case of large pomfret, fish (mainly Paralepididae sp. 'A')were the next most common prey, followed by amphipods (mainly P.sedentaria and P. ovoides ), indicating that the importanceof fish as prey increases with growth for medium and large pomfret.