Verasper Variegatus Research Articles

Habitat utilization, feeding, and growth of wild spotted halibut Verasper variegatus in a shallow brackish lagoon: Matsukawa-ura, northeastern Japan

Habitat utilization, feeding, and growth of a rare pleuronectid flatfish, spotted halibut Verasper variegatus, were examined in a brackish lagoon in northeastern Japan: Matsukawa-ura. The distribution and date–length data of spotted halibut collected mainly from beam-trawl samplings during 1983–2008 indicated that age-0 juveniles [n = 25, 6.0–18.0 cm total length (TL)] and older spotted halibut (n = 71, 13.8–43.0 cm TL) inhabited almost the entire northern part of Matsukawa-ura. Comparative distribution surveys of spotted halibut, stone flounder, and marbled flounder during 1985–1989 revealed similar distribution patterns of these flatfishes; the highest densities were found around the central part of Matsukawa-ura, although significantly lower abundance was detected for spotted halibut. Linear growth equations of age-0 juveniles demonstrated that spotted halibut were able to achieve high growth after June, probably because abundant prey (e.g., mysids and gammarids) and suitable physical conditions (warmer water temperature and lower salinity) were present. The main prey items shifted from various crustaceans including mysids, caridean shrimps, and anomurans to brachyura Hemigrapsus spp. (≥20 cm TL). Our study shows that spotted halibut use the shallow brackish lagoon as an important nursery for juveniles, and also as a feeding ground for young and adults.

Major histocompatibility complex class IIA and IIB genes of the spotted halibut Verasper variegatus: genomic structure, molecular polymorphism, and expression analysis

The major histocompatibility complex (MHC) is a large genomic region characterized by extremely high polymorphism and its association with resistance/susceptibility to disease in vertebrates. In this study, the full lengths of MHC IIA and IIB cDNA were obtained from spotted halibut (Verasper variegates) by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The genomic structure, molecular polymorphism, and expression patterns were examined to study MHC II gene functions in fish. As in other teleosts, the genomic structure of the spotted halibut MHC IIA contained 4 exons and 3 introns. The deduced amino acid sequence of the class IIA molecule shared 28-79% similarity with those of teleosts and mammals. Nine class IIA alleles were identified from five individuals. Three alleles originating from a single individual suggested the existence of at least two class IIA loci in the genome. Six exons and 5 introns were identified from spotted halibut MHC IIB, and the deduced amino acid sequence shared 33-79% similarity with those of teleosts and mammals. Twelve alleles were identified, among which five were observed in a single individual, which suggested at least three class IIB loci. Quantitative real-time PCR analysis revealed the presence of class IIA and IIB transcripts in nine normal tissues with high expression level in kidney and gill. Furthermore, MHC IIA and IIB are probably two candidates of immune molecules involved in the acute-phase response in spotted halibut, because their transcriptional levels were significantly up-regulated in blood and liver after bacterial challenge.

Staging of initial pepsinogen and chitinase expression and complete gastric gland development within the larval stomach of Japanese flounder, spotted halibut, seven-band grouper and greater amberjack

The gastric glands of marine aquaculture fish species are known to start secretion of pepsinogens (Pep) during larval development, far after first feeding. Although characterization of gastric gland development is considered useful for managing larval feeding, the timing of initial Pep synthesis by the gastric gland has only been reported for a few species. Consequently, this study employed in situ hybridization (ISH) to characterize the onset of expression of genes encoding the gastric enzymes Pep and Chitinase (Chi), as well as the complete development of gastric glands in the larval stomach of Japanese flounder ( Paralichthys olivaceus), spotted halibut ( Verasper variegatus), seven-band grouper ( Epinephelus septemfasciatus) and greater amberjack ( Seriola dumerili). The expression of both Chi and Pep was observed to begin in the cardiac region of the larval stomach at 25 days post-hatching (dph) in Japanese flounder, 36 dph in spotted halibut, 38 dph in seven-band grouper and 13 dph in greater amberjack. The gastric glands of these species were fully developed in the stomach approximately 5–12 days after the initiation of Pep expression. Development of gastric glands was observed to coincide with external manifestations of metamorphosis, such as the development of adult coloration in Japanese flounder and spotted halibut of the Pleuronectiformes, while it finished prior to adult-type coloration in seven-band grouper and greater amberjack of the Perciformes.

Genetic linkage maps of barfin flounder (Verasper moseri) and spotted halibut (Verasper variegatus) based on AFLP and microsatellite markers

Barfin flounder (Verasper moseri) and spotted halibut (Verasper variegatus) are two economically important marine fish species for aquaculture in China, Korea and Japan. Construction of genetic linkage maps is an interesting issue for molecular marker-assisted selection (MAS) and for better understanding the genome structure. In the present study, we constructed genetic linkage maps for both fish species using AFLP and microsatellite markers based on an interspecific F(1) hybrid family (female V. moseri and male V. variegatus). The female genetic map comprised 98 markers (58 AFLP markers and 40 microsatellite markers), distributing in 27 linkage groups, and spanning 637 cM with an average resolution of 8.9 cM. Whereas the male genetic map consisted of 86 markers (48 AFLP and 38 microsatellite markers) in 24 linkage groups, covering a length of 625 cM with an average marker spacing of 10 cM. The expected genome length was 1,128 cM in female and 1,115 cM in male, and the estimated coverage of genome was 56% for both genetic maps. Moreover, five microsatellite markers were observed to be common to both genetic maps. This is the first time to report the genetic linkage maps of V. moseri and V. variegatus that could serve as the basis for genetic improvement and selective breeding, candidate genes cloning, and genome structure research.

Development of polymorphic microsatellite markers in barfin flounder (Verasper moseri) and spotted halibut (Verasper variegatus) by the cross-species amplification

Barfin flounder (Verasper moseri) and spotted halibut (Verasper variegatus) are two commercially important flatfish species in the Northeast Asia. In the present study, we reported polymorphic microsatellite markers in V. moseri and V. variegatus by the cross-species amplification of microsatellite primers developed previously in two other related marine fish species. A total of 244 polymorphic microsatellite markers were selected for cross-species amplification in V. moseri and V. variegatus, of which 182 markers deriving from Atlantic halibut (Hippoglossus hippoglossus) and 62 markers deriving from Japanese flounder (Paralichthys olivaceus). A sample of 10 individuals were detected. As a result, a total of 67 loci showed polymorphisms in V. moseri, and 62 loci showed polymorphisms in V. variegatus, with the observed number of alleles per locus ranging from two to five in V. moseri, and from two to seven in V. variegatus, respectively. This paper provided more candidate microsatellite markers which could be useful for construction of genetic linkage maps, evaluation of population genetic structure and stock management of V. moseri and V. variegatus.

Genetic structure in species with shallow evolutionary lineages: a case study of the rare flatfish Verasper variegatus

We examined the genetic population divergence of the spotted halibut Verasper variegatus. A previous report suggested two conservation units for this species along the Japanese Pacific coast. Extending the coverage of the genomes (29 microsatellites and three mitochondrial DNA segments) revealed hitherto-undetected genetic population boundaries. We screened population samples from the major habitats along the Japanese coast and the Yellow Sea coast (East Asian Continent). Significant genetic differentiation was found in every comparison between the habitats. In most cases, the nuclear and mitochondrial population divergences were incongruent, most likely caused by differences between the two genomes in the effects of genetic drift after recent population isolation and bottleneck events. We discuss the ecological and evolutionary mechanisms of the genetic structure as well as the units of conservation. The present study illustrates the merits of wider coverage of genomes in genetic population analysis especially for species with a shallow population history.

Successful stocking of a depleted species, spotted halibut Verasper variegatus, in Miyako Bay, Japan: evaluation from post-release surveys and landings

Post-release adaptation and stocking effectiveness of a highly depleted pleuronectid flatfish, spotted halibut Verasper variegatus, were assessed in Miyako Bay, Japan. In early July 2004 and 2005, 22 000 and 5000 hatchery-reared juveniles (mean 81.2 and 89.8 mm total length, respec- tively) were released in shallow waters in the inner bay area. Results from surveys with a 2 m beam- trawl conducted over a 2 mo post-release period, laboratory predation experiments using potential predators (age-1 Japanese flounder), and a market census continued until December 2008, suggested that released juveniles, which had a size refuge from predators, could quickly adapt to brackish water habitats where there were suitable physical conditions (temperature, salinity and sediments) and abundant prey organisms. Juveniles selectively preyed on epibenthic gammarids and cumaceans, and had full stomachs 1 wk after release. Some released fish grew to >40 cm by age-1+, feeding exclusively on larger crustaceans (e.g. isopods, carideans and brachyurans). Market census revealed that released fish showed >99% contribution to catches, and recapture rates for fish released in 2004 (7.6%) and 2005 (15.7%) were higher than for releases in 2000 to 2003 (0.7 to 5.0%), possibly due to the development and application of improved release strategies (i.e. release habitat, size and season was appropriate in 2004 and 2005). Our study demonstrates that (1) local depleted stocks can be effectively restored by hatchery releases and that (2) development of release strategies based on the analysis of ecological interactions between released juveniles and the target ecosystem can largely contribute to the successful restocking of depleted species.

Process of Final Oocyte Maturation and Ovulation Cycle in Captive Spotted Halibut, Verasper variegatus

Process of Final Oocyte Maturation and Ovulation Cycle in Captive Spotted Halibut, Verasper variegatus

Present Status and Problems of Seedling Production of Spotted halibut, Verasper variegatus

Present Status and Problems of Seedling Production of Spotted halibut, Verasper variegatus

Effects of Dietary Protein and Energy Levels on Growth, Feed Conversion and Body Composition of Spotted Halibut Verasper variegatus

Effects of Dietary Protein and Energy Levels on Growth, Feed Conversion and Body Composition of Spotted Halibut Verasper variegatus

Artificial gynogenesis and assessment of homozygosity in meiotic gynogens of spotted halibut (Verasper variegatus)

In the present study, methodology of gynogenetic induction in spotted halibut were developed and optimized; the sex ratio of putative meiotic gynogenetic diploids was determined using AFLP-based molecular sexing technique; the homozygosity of gynogenetic population was assessed as opposed to cultivated population. The results showed that high percentage of meiotic gynogenetic diploids were generated when the eggs fertilized with irradiated heterologous sea perch frozen sperm (30–50 mJ cm−2) were cold shocked in sea water of −1°C for 40–75 min at 5 min after fertilization. About 15,200 diploid gynogenetic larvae were achieved and they exhibited normal morphology similar to diploid control. The gynogenetic diploids were 100% female, which first confirmed the female homogamete (XX/XY) sex determination in spotted halibut. The genetic analysis showed that the average H O was, respectively, 0.404 and 0.724 in gynogenetic population and cultivated population, indicating an increase of homozygosity in gynogenetic population.

Isolation of sex-specific AFLP markers in Spotted Halibut (Verasper variegatus)

Spotted Halibut (Verasper variegatus) is a commercially important marine fish species. In the present study, to isolate sex-specific markers in Spotted Halibut, we screened the genomes of Spotted Halibut by AFLP technique with 64 different primer combinations. Two primer combinations, MseI-CAG/EcoRI-ACC and MseI-CAT/EcoRI-AGG, produced a female-specific fragment in all females (n = 88) and in no males (n = 60, except 3 individuals), respectively. Both fragments were excised from the gel, cloned and characterized. The first fragment (named VevaF533) was 533 bp long, while the length of the second one (named VevaF218) was 218 bp. The two sequences showed no similarity to each other, and to the known sequences existing in the GenBank database using BLASTn. Cross-species amplification showed that the marker VevaF218 is a species-specific marker which is present in Spotted Halibut females but absent in Barfin Flounder (Verasper moseri). So this marker could be used for discriminating unambiguously between Spotted Halibut females and Barfin Flounder. Examination of the patterns of inheritance of VevaF218 in an interspecific hybrid family (V. variegatus ♂×V. moseri ♀) showed a female-specific pattern of inheritance from mother to daughter, implying that the marker VevaF218 is located on the female sex chromosome. This study provides a reliable AFLP-based genetic sexing of Spotted Halibut that could be useful for genetic mapping of the sex chromosomes and identification of sex-linked genes.

Metamorphic pitx2 expression in the left habenula correlated with lateralization of eye‐sidedness in flounder

The bilateral symmetry of flounder larvae changes through the process of morphogenesis to produce external asymmetry at metamorphosis. The process is characterized by the lateral migration of one eye and pigmentation at the ocular side. Migration of the left or right eye to produce either dextral or sinistral forms, respectively, is usually fixed within a species. Here we propose a mechanism for the mediation of lateralization by the nodal-lefty-pitx2 (NLP) pathway in flounders, in which pitx2, the final left-right determinant of the NLP pathway, is re-expressed in the left habenula at pre-metamorphosis. After the initiation of left-sided pitx2 re-expression, the eye commences migration, when the habenulae shift their position on the ventral diencephalon rightwards in sinistral flounder (Paralichthys olivaceus) and leftwards in dextral flounder (Verasper variegatus). In addition, the right habenula increases in size relative to the left habenula in both species. Loss of pitx2 re-expression induces randomization of eye-sidedness, manifesting as normal, reversed or bilateral symmetry, with laterality of the structural asymmetry of habenulae being entirely inverted in reversed flounders compared with normal ones. Thus, flounder pitx2 appears to be re-expressed in the left habenula at metamorphosis to direct eye-sidedness by lateralizing the morphological asymmetry of the habenulae.

Isolation and characterization of 21 novel microsatellite markers from spotted halibut (Verasper variegatus)

Isolation and characterization of 21 novel microsatellite markers from spotted halibut (Verasper variegatus)

Development of 40 microsatellite markers in spotted halibut (Verasper variegatus) and the cross‐species amplification in barfin flounder (Verasper moseri )

indicus). Conversely, the allele frequency of D12 was extremely low compared with other breeds (ranging from 0.14 in German Brown to 0.74 in Japanese Holstein). Moreover, we observed four haplotypes in Chinese yak with respect to both indel loci, and the haplotype frequency of D23–D12 was very low. Interestingly, although the frequency distributions of allele, genotype and haplotype showed no statistical difference among the surveyed populations, the distribution patterns in Chinese yak were significantly different from those in BSE-affected cattle (P < 0.03 in each comparison).

Isolation and characterization of 31 polymorphic microsatellite markers in barfin flounder (Verasper moseri) and the cross-species amplification in spotted halibut (Verasper variegatus)

The construction of a genetic linkage map and evaluation of population genetic diversity both require large numbers of polymorphic molecular markers. In the present study, we report 31 polymorphic microsatellite markers, of which 28 were isolated from two repeat-enriched libraries constructed from genomic DNA, and three were detected in two genes (MCH-R1 and MCH-R2) of barfin flounder (Verasper moseri). A total of 94 alleles were detected with an average of 3.0 alleles per locus. The number of alleles, observed and expected heterozygosity per locus ranged from two to six, from 0.30 to 1.00 and from 0.33 to 0.78, respectively. Three loci significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction (P < 0.0016) and no significant linkage disequilibrum between pairs of loci was found. Cross-species amplification of these markers was evaluated using the closely related species spotted halibut (Verasper variegatus). This study will potentially be useful for stock management, constructing of a genetic linkage map, mapping economically important quantitative trait loci (QTL), and studying the population genetic diversity of barfin flounder (Verasper moseri).

Adult‐type pigment cells, which color the ocular sides of flounders at metamorphosis, localize as precursor cells at the proximal parts of the dorsal and anal fins in early larvae

Flounders form left-right asymmetry in body coloration during metamorphosis through differentiation of adult-type melanophores and xanthophores on the ocular side. As the first step in investigating the formation of flounder body coloration asymmetry, in this study, we aimed to determine where the precursors of adult-type chromatophores distribute in larvae before metamorphosis. In Paralichthys olivaceus and Verasper variegatus, GTP cyclohydrolase 2 (gch2), a common marker of melanoblasts and xanthoblasts, was found to be transiently expressed in cells located along the bilateral skeletal muscles at the basal parts of the dorsal and anal fins of premetamorphic larvae. When V. variegatus larvae were fed with a strain of Artemia collected in Brazil, this gch2 expression was abolished and the differentiation of adult-type melanophores was completely inhibited, while the density of larval melanophores was not affected. In a cell trace test in which the cells at the basal part of the dorsal fin were labeled with DiI at the premetamorphic stage, adult-type melanophores labeled with DiI were found in the skin on the ocular side after metamorphosis. These data suggest that, in flounder larvae, adult-type melanophores are distributed at the basal parts of the dorsal and anal fins as unpigmented precursor cells.

Cryopreservation of spotted halibut ( Verasper variegatus) sperm

Spotted halibut ( Verasper variegatus) is now regarded as being endangered in China because of its continuous decline in the last three decades. Preservation and protection of its genetic resources were demanded in order to protect this rare fish species. In this project, cryopreservation technique for sperm of spotted halibut was developed. The effects of various extenders and cryoprotectants on motility of frozen–thawed sperm were examined. The motility of frozen–thawed sperm in TS-2 was higher than that in ASW and MPRS ( p < 0.05) and was not significantly different from that of fresh sperm ( p > 0.05). While the motility of frozen–thawed sperm cryopreserved with 13.3% EG (ethylene glycol), 13.3% glycerol, 13.3% MeOH (methanol) and 13.3% DMF (dimethylformamide) was less than 5%, no significant differences were observed in the motility between fresh sperm and frozen–thawed sperm cryopreserved with 13.3% DMSO (dimethyl sulfoxide), 13.3% PG (propylene glycol) ( p > 0.05). Using the above method, we cryopreserved spotted halibut semen with extender TS-2 and 13.3% DMSO or 13.3% PG. As a result, the fertilization rate (34.52 ± 10.92%) and hatching rate (23.53 ± 11.80%) of frozen–thawed sperm were not significantly different from that of fresh sperm ( p > 0.05). Motility and time delay in the activation of frozen–thawed sperm activated by artificial seawater at different salinity were different. Low salinity (low osmolality) could delay the activation of frozen–thawed sperm. The highest motility was observed with artificial seawater of 30‰ ( p < 0.05). The most suitable temperature of seawater to activate spotted halibut frozen sperm was determined to be 18 °C ( p < 0.05). However, temperature of seawater has no significant effect on time delay in the activation of frozen–thawed sperm ( p > 0.05).

Comparison of behavioural development between Japanese flounder (Paralichthys olivaceus) and spotted halibut (Verasper variegatus) during early life stages

Summary Behavioural development was compared between two flatfish species (Japanese flounder and spotted halibut) from hatching to settlement (juvenile stage) in order to speculate on the ecology of their early life stages and to provide fundamental knowledge for improving seedling production techniques for stock enhancement. Fish were cultured under identical rearing conditions (500-L tank maintained at 17.8 ± 0.4°C, 34 ppt, 10L : 14D light regime and an initial stocking density of 20 larvae L−1). Behavioural observations were conducted at about 4-day intervals from hatching to the juvenile stage. Fish were sampled randomly from the rearing tank, and one fish was transferred into a 250-ml observation container. Behaviour was video-recorded for 5 min without food and for an additional 5 min with live feed (rotifer or Artemia). All behavioural data were sorted according to eight developmental stages and compared among developmental stages and between species. The average standard length of the spotted halibut was significantly greater than that of the Japanese flounder in all developmental stages, while the development of Japanese flounder was faster than that of the spotted halibut. For Japanese flounder, feeding, swimming and Ohm-posture (typical shivering behaviour observed during early life stages in flatfishes) frequency were highest before metamorphosis (mean ± SD; 1.0 ± 2.0 attacks min−1, 24.0 ± 9.6 actions min−1, 1.1 ± 1.1 counts min−1, respectively). Spotted halibut expressed feeding behaviour frequently from the beginning of metamorphosis (3.6 ± 5.2 attacks min−1), had relatively low swimming activity during all developmental stages, and showed a peak of Ohm-posture frequency during the flexion stage (2.6 ± 1.0 counts min−1).

Effects of heavy oil in the developing spotted halibut, Verasper variegatus

It is well known that heavy oil (HO) on the sea surface causes serious problems in the aquatic environment. In particular, some species of teleosts which develop on the sea surface are thought to be affected by the HO which flows out from tankers or coastal industry. However, the toxicological effects of HO are not fully understood. We performed exposure experiments using the Pleuronectiformean fish, spotted halibut ( Verasper variegatus), which is an important fishery resource in Japan. In course of the development, HO-exposed embryos showed remarkable delay in developmental processes including somite formation. We further observed abnormal development of the head morphology. Notably, treated embryos had relatively small eyes and craniofacial structures. These findings strongly suggest that HO seriously affects the cell proliferation and differentiation of the embryo. In addition, HO-exposed embryos showed abnormal neuronal development. We also performed the exposure in the larval stage. Treatment of post-hatching larvae with HO resulted in significantly greater mortality compared with controls. Through these observations, we finally conclude that HO is strongly toxic to halibut in their early life stages.