Sequence and organization of the complete mitochondrial genomes of spotted halibut (Verasper variegatus) and barfin flounder (Verasper moseri).

In this work, the mitochondrial genomes for spotted halibut (Verasper variegatus) and barfin flounder (Verasper moseri) were completely sequenced. The entire mitochondrial genome sequences of the spotted halibut and barfin flounder were 17,273 and 17,588 bp in length, respectively. The organization of the two mitochondrial genomes was similar to those reported from other fish mitochondrial genomes containing 37 genes (2 rRNAs, 22 tRNAs and 13 protein-coding genes) and two non-coding regions (control region (CR) and WANCY region). In the CR, the termination associated sequence (ETAS), six central conserved block (CSB-A,B,C,D,E,F), three conserved sequence blocks (CSB1-3) and a region of 61-bp tandem repeat cluster at the end of CSB-3 were identified by similarity comparison with fishes and other vertebrates. The tandem repeat sequences show polymorphism among the different individuals of the two species. The complete mitochondrial genomes of spotted halibut and barfin flounder should be useful for evolutionary studies of flatfishes and other vertebrate species.     

Microsatellite markers for a rare species of right-eye flounder <Emphasis Type="Italic">Verasper variegatus</Emphasis> (Pleuronectiformes,Pleuronectidae)

In the context of stock enhancement for aquatic animals, the risk of negative genetic impacts of releasing hatchery-produced seedlings upon the genetic diversity of indigenous recipient populations becomes a growing concern. This issue is particularly relevant to threatened or rare species such as a member of right-eye flounder, the spotted halibut Verasper variegatus. We present here 29 microsatellite markers for spotted halibut, expecting that these markers could serve as a molecular tool of choice to address a wide range of conservation issues in this species.     

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).     

Development of polymorphic microsatellite markers in barfin flounder (<Emphasis Type="Italic">Verasper moseri</Emphasis>) and spotted halibut (<Emphasis Type="Italic">Verasper variegatus</Emphasis>) 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.     

Development of polymorphic microsatellite markers from barfin flounder (Verasper moseri) and their cross-species amplification

Barfin flounder (Verasper moseri) is a rare fish species in the world. Here, we reported 10 polymorphic microsatellite loci isolated from a dinucleotide-enriched genomic library of barfin flounder (Verasper moseri). The number of alleles, observed, and expected heterozygosity per locus in a test population ranged from 2 to 6, from 0.3333 to 1.0000, and from 0.4866 to 0.7774, respectively. One locus significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium was found between pairs of loci. Cross-species amplification of these microsatellite loci in additional five fish species was performed. These polymorphic microsatellite loci would be useful for investigating genetic population structure and construction of genetic linkage map in Verasper moseri. Gui-Dong Miao and Chang-Wei Shao Contributed equally to this work.     

Genetic linkage maps of barfin flounder (<Emphasis Type="Italic">Verasper moseri</Emphasis>) and spotted halibut (<Emphasis Type="Italic">Verasper variegatus</Emphasis>) 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 F₁ 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.     

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

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).     

Morphology of unfertilized mature and fertilized developing marine pelagic eggs in four types of multiple spawning flounders

Starry flounder Platichthys stellatus, spotted halibut Verasper variegates, turbot Scophthalmus maximus, and Japanese flounder Paralichthys olivaceus are four commercially cultivated multiple spawning flounders that spawn pelagic eggs. Through appropriate light and scanning electron microscope processing, the shape and surface structures (such as micropyle, pores, pore density, and paten) of unfertilized mature and fertilized developing eggs of the four species were observed and measured. First, individual or intraspecific comparisons of the surface structures of eggs at different developmental stages were made. Second, interspecific differences among the four species at the same developmental stage of unfertilized mature eggs were statistically computed and analyzed through one-way analysis of variance and hierarchical cluster analysis. Eggs of the same species collected at different stages of development tend to be different in morphology. Smoothing of the convoluted egg envelope surface and closure of the micropyle to serve as a final step of the polyspermy-preventing reaction are common after fertilization. Based on detailed morphology of micropyle of just-mature fertilizable eggs, turbot, starry flounder, and Japanese flounder each have a micropyle with a long canal but no distinct micropylar vestibule, type III of Riehl and Götting (Arch Hydrobiol 74:393–402, 1974). In contrast, spotted halibut has a micropyle with a distinct flat micropylar vestibule and a long canal, type II. Envelope surface microstructures, especially those in the micropyle region, are useful characters for egg identification among the four species. Cluster analysis using selected egg characters indicated the highest similarity between turbot and Japanese flounder and that starry flounder is obviously more similar to turbot and Japanese flounder than to spotted halibut.     

Asymmetry of the interrenal gland in two flounder species in the spawning period

Significant differences in the area of cell nuclei of the left and right lobes of the interrenal glad have been revealed within the spawning period in two flounder species—the the barfin plaice Liopsetta pinnifasciata and the Korean flounder Glyptocephalus stelleri, which testifies to the different functional activities of these lobes and, accordingly, about the functional asymmetry of the interrenal glad. Individuals with a functionally more active right lobe of the gland prevail for the barfin plaice, whereas with the Korean flounder the number of individuals with a more active left lobe is equal to the number of individuals with a more active right lobe.     

Biological features of common fish species in Olyutorsky-Navarin region and the adjacent areas of the Bering Sea: 3. Righteye flounders (Pleuronectidae)

Biological features of the seven abundant commercial species—the Pacific halibut Hippoglossus stenolepis, the Greenland halibut Reinhardtius hippoglossoides, the Alaska plaice Pleuronectes quadrituberculatus, the northern rock sole Lepidopsetta polyxystra, the yellowfin sole Limanda aspera, the flathead sole Hippoglossoides elassodon, and the Bering flounder H. robustus (Pleuronectidae)—have been studied for a 20-year period (1995?2015). These species are present in the northwestern Bering Sea in the summer–autumn season; they form the gatherings in Olyutorsky-Navarin region. The size-weight spectra of the fish caught by different fishing gear has been analyzed, the peculiarities of the linear growth and the weight gain, as well as the spawning period and scale and the spawning conditions, have been described. The largest halibut specimens have been registered in the bottom setlines and gill nets, while flounders were in snurrevad catches; the smallest specimens have been observed in trawl catches. The abundant year-class in most of the studied species is seen well on the long-term plots of the fish size spectra and is tracked by the decrease of their biological parameters. The species that demand vast growing grounds (Pacific halibut, Alaska plaice, northern rock sole, and yellowfin sole) are characterized by a smaller average body size of the fish sampled in the coastal waters due to the prevalence of the young specimens in this area.     

Melanophore comparisons in different forms of ambicoloration in the flatfish Pseudopleuronectes americanus and Reinhardtius hippoglossoides

The melanophores associated with contrasting manifestations of ambicoloration in two species, winter flounder ( Pseudopleuronectes americanus ) and Greenland halibut ( Reinhardtius hippoglossoides ), from the Pleuronectidae are compared microscopically. Winter flounder is a benthic inshore species, whilst Greenland halibut is a deep water species considered to spend the greater part of its life away from the sea floor, although the relatively thick blind-side epidermis associated with a benthic life occurs in both species. Various forms of abnormal ambicoloration occur in some specimens of winter flounder. Melanophores from the flounder blind side are morphologically similar to those from the ocular side in the different forms of ambicoloration, although there are differences in melanophore densities. In Greenland halibut, ambicoloration is a normal characteristic which is considered to be secondarily acquired. The melanophores of the ocular side of Greenland halibut are similar to the melanophores of other Pleuronectidae, whereas those of the blind side are very different in morphology and size.     

Two species of parasitic copepods (Caligidae) new to Japan

Two species of parasitic copepods (Siphonostomatoida: Caligidae), Caligus sclerotinosus Roubal, Armitage & Rohde, 1983, parasitic on red seabream Pagrus major (Temminck & Schlegel), and Lepeophtheirus longiventralis Yü & Wu, 1932, parasitic on spotted halibut Verasper variegatus (Temminck & Schlegel), are redescribed based on material found on their respective hosts cultured in Japan. Both species are new to Japan. Preliminary observation on the occurrence of L. longiventralis indicates that the larval development takes place on the body surface of the host and only the post-mated female migrates into the host's gill-cavities. Whilst the occurrence of L. longiventralis in Japan can be considered as due to natural causes, the occurrence of C. sclerotinosus is likely due to anthropogenic activities.     

Zoogeographical classification of the macroparasite faunas of four flatfish species from the northeastern Atlantic

The macroparasite faunas of four righteye flounder species (flounder Platichthys flesus, witch flounder Glyptocephalus cynoglossus, American plaice Hippoglossoides platessoides and Atlantic halibut Hippoglossus hippoglossus) caught off the northern Norwegian coast at 70°N, were classified zoogeographically. The parasite faunas of all four host species were dominated by boreal parasites. This appears to be the situation with the entire area of distribution of these flatfish species. The relationship between the extension of the range of the host species towards the Arctic and the proportion of parasites belonging to the arctic group is indicated.     

圆斑星鲽及相关种类线粒体DNA控制区结构分析

采用PCR产物直接测序法测定了圆斑星鲽(Verasper variegatus)的24个个体的线粒体控制区(Control region)核苷酸全序列, 并进行了结构分析。结果表明, 圆斑星鲽线粒体控制区核苷酸全序列具有长度多态性, 得到4种长度单元型, 主要表现为控制区中的串联重复序列的长度不同。对鲽形目鱼类如鲽科的条斑星鲽(Verasper moseri)、黄盖鲽(Limanda feruginea)、马舌鲽 (Reinhardtius hippoglossoides), 美洲拟庸鲽(Heppoglossoides platessoides )和鲆科的牙鲆(Paralichthys olivaceus)以及鳎科的欧洲鳎(Solea solea)、塞内加尔鳎(S. senegalensis)和沙鳎(S. lascari)的控制区的比较研究发现, 鲽形目鱼类的线粒体控制区均存在相似的结构, 即线粒体控制区可分为终止相关序列区(ETAS)、中央保守区(包括CSB-A、CSB-B、CSB-C、CSB-D、CSB-E、CSB-F)以及保守序列区(CSB1、CSB2、CSB3)和重复序列区(Repeat region)4个区域。通过与脊椎动物各个纲线粒体控制区序列的比较分析, 发现只有鲽形目(包括鲆、鲽类和鳎类)鱼类和两栖纲的无尾类在CSB-3之后存在相似的串联重复序列。     

Green‐shifting of SWS2A opsin sensitivity and loss of function of RH2‐A opsin in flounders,genus Verasper

We identified visual opsin genes for three flounder species, including the spotted halibut (Verasper variegatus), slime flounder (Microstomus achne), and Japanese flounder (Paralichthys olivaceus). Structure and function of opsins for the three species were characterized together with those of the barfin flounder (V. moseri) that we previously reported. All four flounder species possessed five basic opsin genes, including lws, sws1, sws2, rh1, and rh2. Specific features were observed in rh2 and sws2. The rh2‐a, one of the three subtypes of rh2, was absent in the genome of V. variegatus and pseudogenized in V. moseri. Moreover, rh2‐a mRNA was not detected in M. achne and P. olivaceus, despite the presence of a functional reading frame. Analyses of the maximum absorption spectra (λ_max) estimated by in vitro reconstitution indicated that SWS2A of M. achne (451.9 nm) and P. olivaceus (465.6 nm) were blue‐sensitive, whereas in V. variegatus (485.4 nm), it was green‐sensitive and comparable to V. moseri (482.3 nm). Our results indicate that although the four flounder species possess a similar opsin gene repertoire, the SWS2A opsin of the genus Verasper is functionally green‐sensitive, while its overall structure remains conserved as a blue‐sensitive opsin. Further, the rh2‐a function seems to have been reduced during the evolution of flounders. λ_max values of predicted ancestral SWS2A of Pleuronectiformes and Pleuronectidae was 465.4 and 462.4 nm, respectively, indicating that these were blue‐sensitive. Thus, the green‐sensitive SWS2A is estimated to be arisen in ancestral Verasper genus. It is suggested that the sensitivity shift of SWS2A from blue to green may have compensated functional reduction in RH2‐A.     

Involvement of host recognition by oncomiracidia and post-larval survivability in the host specificity of Heterobothrium okamotoi (Monogenea: Diclidophoridae)

Heterobothrium okamotoi, a monogenean gill parasite, shows high host specificity for tiger puffer, Takifugu rubripes. In the present study, in vivo and in vitro experimental infections were conducted using various fish species, including T. rubripes, to understand the mechanisms of specificity. In in vivo experiments, T. rubripes, grass puffer, Takifugu niphobles, olive flounder, Paralichthys olivaceus, and red sea bream, Pagrus major, were exposed to oncomiracidia of H. okamotoi labelled with a fluorescent dye, 5- (and -6) carboxyfluorescein diacetate succinimidyl ester, and the numbers of parasites on the gills and skin were recorded at intervals. Oncomiracidia were attached to gills and skin of all the experimental fish species immediately after exposure, and the infection intensity on T. rubripes was higher than that on T. niphobles and much higher than those on the other two species. After 2 days, the attached parasites remained on the gills of T. rubripes, but disappeared from the other hosts. During in vitro experiments, gill filaments excised from seven different fish species (four fish species used in the in vivo experiments and panther puffer, Takifugu pardalis, southern flounder, Paralichthys lethostigma and spotted halibut, Verasper variegates) were exposed to oncomiracidia and the attachment to each fish species and subsequent larval behaviour was observed. The percentage of post-larvae that attached to T. rubripes was slightly higher than those which attached to congeneric fish species and much higher than those of non-tetraodontid fish species. In vivo and in vitro experiments demonstrated that oncomiracidia of H. okamotoi have an affinity for their natural host, T. rubripes, and congeneric fish species. The disappearance of attached post-larvae from 'alien' hosts within 2 days during in vivo experiments suggested that host recognition by oncomiracidia and subsequent post-larval survivability are involved in the host specificity of H. okamotoi.