Kareius Research Articles

Seasonal and ontogenetic diet shift in juvenile stone flounder Platichthys bicoloratus

The diet of juvenile (0 year old) stone flounder Platichthys bicoloratus after settlement was investigated in an estuarine habitat. The principal prey items included harpacticoids and palps of polychaetes in February and March and thereafter shifted to siphons of the bivalve Nuttallia olivacea from April to June. This seasonal change in diet occurred irrespective of body size, suggesting that other factors, such as morphological development of the predator or vulnerability of prey, induced the diet shift.

Identification, purification, and immunoassay of stone flounder (Kareius bicolouratus) vitellogenin

Vitellogenin (Vtg), the precursor of egg yolk proteins, serves as nutrients for the developing embryos. Vtg production in male fish has become an excellent biomarker for environmental estrogens. Thus, Vtg synthesis was induced by injection of 17β-estradiol (E2) in stone flounder (Kareius bicolouratus). Plasma from uninduced and E2-treated fish was subjected to native polyacrylamide gel electrophoresis (PAGE) and the gels were stained with Coomassie brilliant blue R250 for proteins, methyl green for phosphates, Sudan black B for lipids, and periodic acid-Schiff’s reagent for carbohydrates. These analyses verified the presence of two phospholipoglycoproteins in the plasma from E2-treated stone flounder, which were tentatively identified as Vtg. Vtg was then isolated by two purification procedures: gel filtration followed by anion exchange chromatography, or selective precipitation combined with anion exchange chromatography. The latter procedure was more effective at isolating Vtg with a high degree of purity. In native PAGE, the apparent molecular masses of Vtgs were determined to be 520 and 550 kDa, respectively. Two major bands with approximate molecular masses of 165 and 106 kDa together with two additional faint bands of lower molecular weights were observed after sodium dodecyl sulfate- PAGE under reducing conditions. These bands exhibited differential affinity for polyclonal antisera against red drum (Sciaenops ocellatus) Vtg in Western blot. The present study established the purification procedure and characterized the electrophoretic and immunological properties of stone flounder Vtg.

Identification, purification, and immunoassay of stone flounder (Kareius bicolouratus) vitellogenin

Identification, purification, and immunoassay of stone flounder (Kareius bicolouratus) vitellogenin

Pseudoalbinism and ambicoloration in hatchery-reared pleuronectids as malformations of asymmetrical formation

Morphological abnormalities in eye location and/or body coloration are commonly observed in hatchery-reared juveniles of many pleuronectid species, and have become one of the most serious problems in juvenile production for stock enhancement. In this study, these morphological abnormalities of six pleuronectid species—barfin flounder Verasper moseri, slime flounder Microstomus achne, stone flounder Platichthys bicoloratus, starry flounder Pl. stellatus, cresthead flounder Pseudopleuronectes schrenki, and marbled sole Ps. yokohamae—were successfully classified into four morphological types [normal, two types of pseudoalbinism (normal-eye position or top-eye position), and ambicoloration], following the same classification scheme proposed for other two pleuronectids. Based on our results, the characteristics of each morphological type are quite similar among species. It is confirmed that the normal type has the same ocular side and blind side characteristics as those of wild fish, not only in eye position and body coloration, but also in scales or dentition. The pseudoalbino types have the blind side characteristics of wild fish on both sides, with top-eye type in all characteristics, and normal-eye type in all other characteristics other than eye position. The ambicoloration type has all the ocular side characteristics of wild fish on both sides. Therefore, the pseudoalbinism and ambicoloration of hatchery-reared juveniles of pleuronectid species can be considered malformations of asymmetrical formation. Since this process normally occurs during metamorphosis, the term “metamorphosis-related malformation” is proposed for the abnormal formation of juveniles in pleuronectid species.

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.

A Novel Cell Line from Liver of Stone Flounder, Kareius bicoloratus, and Its Susceptibility to Different Iridoviruses

A novel cell line (SFL) was established from the liver of stone flounder, Kareius bicoloratus, and its susceptibility to different iridoviruses was evaluated. The SFL cells grew well in Dulbecco's Modified Eagle Medium supplemented with fetal bovine serum, basic fibroblast growth factor, and insulin-like growth factor-II, and have been subcultured over 82 passages. The optimal growth temperature was 22 C. The SFL cells were fibroblastic in morphology and grew at a steady rate, with a population doubling time of 38.8 h. Karyotype analysis showed that SFL cells exhibited chromosomal aneuploidy with a modal chromosome number of 48. The susceptibility evaluation of SFL cells revealed that cytopathic effects (CPE) appeared after infection by different iridoviruses, lymphocystis disease virus (LCDV) and turbot reddish body iridovirus (TRBIV). In addition, a large number of TRBIV and LCDV particles were observed in the infected SFL cells by electron microscope examination. It is suggested that the SFL cell line could be used as a valuable tool for isolation and propagation of different iridoviruses.

Spatial variation in the abundance and condition of the bivalve Nuttallia olivacea in relation to environmental factors and sublethal predation

Our aim was to assess the spatial heterogeneity in the abundance and condition of the tellinacean bivalve Nuttallia olivacea in estuarine tidal flats, as well as to reveal the relationships between these variables and abiotic and biotic factors. The intensity of sublethal siphon predation by juvenile stone flounder Platichthys bicoloratus was considered as a possible factor affecting the bivalve condition. A 2-scale sampling of stations at 200 to 800 m intervals and 2 to 5 plots at 5 to 20 m intervals on a transect line at each station was conducted in the Natori River estuary, Japan. Abundance and somatic condition greatly varied at both scales. Densities varied from 0 to 4902 ind. m–2 among stations and from 2480 to 6448 ind. m–2 among plots at the station with the highest density. Body mass index varied from 4.0 to 5.0 between stations and from 4.9 to 5.7 between plots at the station with the highest body mass. It was commonly observed that N. olivacea was abundant and that body condition was high at sites with low silt-clay contents and low elevations, as revealed by generalized linear models. A negative influence of siphon predation was detected only on somatic condition, but it accounted for only 1.2% of the variation, whereas environmental variables accounted for 30.2%. These results indicate that environmental variables are key factors affecting bivalve abundance and condition, while sublethal predation is only a potential factor affecting bivalve condition.

Occurrence and Migration of Stone Flounder Larvae in Matsukawaura

Occurrence and Migration of Stone Flounder Larvae in Matsukawaura

Hybrids between olive flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus: karyotype, allozyme and RAPD analyses

The hybrid between olive flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus was produced by artificial insemination of olive flounder eggs with stone flounder sperm. Sinistral and dextral are two types of hybrid progeny after metamorphosis. Karyotypes of both hybrid flounders are the same as those of the two parental species. Of the 22 loci examined from 12 allozymes, 12 confirmed hybridization of the paternal and maternal loci in hybrids and no difference was found in allozyme patterns of sinistral and dextral hybrid fishes. RAPD patterns of these specimens were also studied with 38 primers selected from 104 tested. Among them, the PCR products of 30 primers showed hybridization of the paternal and maternal bands. Genetic variation between hybrids and their parental stocks was analyzed by RAPD using 10 of the above 38 primers. The average heterozygosity and genetic distance were calculated. The results suggested that the filial generation could inherit a little more genetic materials from paternal fish than that from maternal fish.

Intragenomic variability and pseudogenes of ribosomal DNA in Stone flounder Kareius bicoloratus

Molecular study of Stone flounder Kareius bicoloratus revealed a high level of intra-individual polymorphism of ribosomal DNA (18S rDNA, ITS1 and 5.8S rDNA). This polymorphism includes not only multiple functional genes but also putative pseudogenes and recombinants. Three major types (Type I, II and III) of 18S rDNA differing in lengths coexisted in the genome of each studied individual. Type II and Type III have a 15-bp-deletion and Type III has an additional 7-bp-deletion relative to Type I. Expression analyses gave clear evidence that Type I 18S rDNA was abundantly expressed. In contrast, transcription of Type II and III 18S rDNA were not detected. Further sequence analysis revealed that Type I was the expected and functional 18S rDNA in K. bicoloratus, while Type II and Type III were pseudogenes. Phylogeny of the ITS1 sequences formed three groups (Group I, II and III), of which Group I linking with Type I 18S rDNA were functional, whereas the remaining two Groups (II and III) linking with Type II and Type III 18S rDNA were pseudogenes. Groups II and III ITS1 sequences were shorter (685–717 bp in length) than Group I ITS1 (762–776 bp), but had much higher sequence divergence (9.8%) than the later ones did (4.9%). Group II sequences were recombinants between Group I and Group III sequences, providing the possibility that the rDNA of K. bicoloratus is in a transition stage of concerted evolution. In addition, by comparing standard PCR and high-stringency PCR, we concluded that DMSO might prevent functional ITS1 sequences from being amplified instead of enhancing its amplification efficiency. The analysis suggests a non-concerted evolution of rDNA in K. bicoloratus. Features of rDNA functional genes and pseudogenes are described. Possible causes for the lack of homogenization of rDNA paralogues within individuals are discussed.

Screening of eye-position related genes with DD-RT-PCR and RDA in the hybrids between Japanese flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus

Flatfish or flounder moves one eye to change body proportion into vertebral asymmetry during metamorphosis, during which some become sinistral while others dextral. However, the mechanism behinds the eye-position has not been well understood. In this research, hybrids between Japanese flounder(♀) and stone flounder (♂) show mixed eye-location in both dextral type and sinistral type, and thus become good samples for studying the eye-migration. mRNAs from pro-metamorphosis sinistral and dextral hybrids larvae were screened with classical differential display RT-PCR (DD-RT-PCR) and representational difference analysis of cDNA (cDNA-RDA); 30 and 47 putative fragments were isolated, respectively. The cDNA fragments of creatine kinase and trypsinogen 2 precursor genes isolated by cDNA-RDA exhibited eye-position expression patterns during metamorphosis. However, none of the fragments was proved to be related to flatfishes’ eye-position specifically. Therefore, further studies and more sensitive gene isolated methods are needed to solve the problems.

Isolation and characterization of polymorphic microsatellite loci from a repeat-enriched genomic library of stone flounder (Kareius bicoloratus) and cross-species amplification

Stone flounder (Kareius bicoloratus) is a rare fish species in China. Here, we reported 11 polymorphic microsatellite loci isolated from a repeat-enriched genomic library of stone flounder. The number of alleles, observed and expected heterozygosity per locus in 32 individuals ranged from 3 to 6, from 0.1613 to 0.8667 and from 0.1549 to 0.7932, respectively. Two loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium between pairs of loci was found. Cross-species amplification of these microsatellite loci in additional three fish species was performed. These polymorphic microsatellite loci would be useful for investigating genetic population structure and construction of genetic linkage map in Kareius bicoloratus.

Habitat selection of stone and starry flounders in an estuary in relation to feeding and survival

Juveniles of both stone flounder Platichthys bicoloratus and starry flounder Platichthys stellatus utilize estuaries as nursery grounds. To understand their habitat selection and the functions of habitats such as food supply, we defined the seasonal distribution of recently settled fish of these species in shallow nursery areas and investigated their feeding habits in the Natori River estuary, Japan. Distribution of stone flounder was limited to the lower estuary (<3 km upriver from the mouth) and stone flounder were most abundant near the mouth. Recently settled starry flounder were first detected further upstream in areas characterized by low salinity <10 and by the absence of the predatory sand shrimp Crangon uritai. Early juvenile stone and starry flounders consumed mainly siphons of the bivalve Nuttallia olivacea and the mysid Neomysis awatschensis, respectively; however, 1- and 2-yr-old fish of both stone and starry flounders fed mainly on the bivalve siphons. These results indicate that habitat selections of juvenile stone and starry flounders enable utilization of preferred prey and predator avoidance, respectively, and that non-overlap of these species' habitats results in avoidance of inter-specific competition for food between these two species.

Freshwater adaptation during larval, juvenile and immature periods of starry flounder Platichthys stellatus, stone flounder Kareius bicoloratus and their reciprocal hybrids

The freshwater tolerance of starry flounder Platichthys stellatus, stone flounder Kareius bicoloratus and their reciprocal hybrids, produced by artificial insemination, were examined in the larval, juvenile and immature phases. Survival rate on being transferred to fresh water in the pre‐settlement phase was 0% in stone flounder and hybrids and 16·7% in starry flounder. This rate in the post‐settlement phase was elevated to &gt;50% in starry flounder and hybrids but was still 0% in stone flounder and similarly in the immature period starry flounder and hybrids survived in fresh water, although stone flounder did not. The lamella chloride cells of the gill epithelium increased in starry flounder and hybrids in fresh water in all periods. Densities of lamella chloride cells increased from 1·6 ± 0·4 (mean ±s.e. number of cells per 1 mm filament) before the transferral (day 0) to 60·3 ± 6·2 on 14 days after the transfer to fresh water (day 14) in starry flounder in the immature period. These densities in hybrids were 0·6 ± 0·3 and 1·0 ± 0·3 on day 0, and, 35·3 ± 2·8 and 23·2 ± 4·6 on day 14, respectively. Stone flounder did not show a substantial change in chloride cell densities throughout the experimental period. These results suggest that low salinity tolerance was well developed in the settlement period in starry flounder and hybrids, and hybrids were also adapted to fresh water sufficiently regardless of the cross type.

Comparison of low-salinity adaptability and morphological development during the early life history of five pleuronectid flatfishes, and implications for migration and recruitment to their nurseries

The nursery habitats of flatfishes range from offshore areas to rivers, with salinity conditions varying significantly between the habitats selected by larvae and juveniles. Morphological characteristics of larvae also contribute to the successful recruitment to specific nursery areas. In the present study, ontogenetic developments of low-salinity tolerance and morphology of five pleuronectid flatfishes were examined, and compared with their previously described ontogenetic migrations. Species examined were starry flounder Platichthys stellatus, stone flounder Platichthys bicoloratus, spotted halibut Verasper variegatus, marbled flounder Pseudopleuronectes yokohamae and slime flounder Microstomus achne. The first three were categorised as ‘cross-shelf species’, marbled flounder as ‘demersal-egg species’ and slime flounder as ‘offshore species’. Their typical nurseries were rivers, estuaries, tidal flats, coastal areas and offshore areas, respectively. Low-salinity tolerance from hatching to juvenile stage was examined by survival 48 h after transfer (A to I stage) and seven days after transfer (I stage) from seawater to various salinities (0, 1, 2, 4, 8, 16 and 32 ppt). High survival in 4 ppt was found in all yolk-sac larvae, but decreased in mid-larval development. During metamorphosis, starry flounder, stone flounder and spotted halibut developed strong low-salinity tolerance and juveniles were able to survive in 0, 1 and 2 ppt, respectively. In contrast, marbled flounder and slime flounder did not show these clear developments and juveniles could only survive in 4 and 8 ppt, respectively. In the morphological development, differences in standard length (SL) and relative body depth (BD) to SL (%BD/SL, an indicator of locomotive adaptation of flatfish larvae showing tilt swimming behaviour during the eye translocation) increased with larval development, and the greatest SL and %BD/SL were observed at the metamorphic climax H stage in the order of slime flounder (24.0 mm, 61.7% at 110 days after hatching, DAH), spotted halibut (14.5 mm, 42.1% at 50 DAH), stone flounder (9.5 mm, 41.1% at 34 DAH), starry flounder (7.0 mm, 34.8% at 28 DAH) and marbled flounder (6.9 mm, 28.5% at 24 DAH). ‘Cross-shelf species’ are characterised by strong inshore migration and settlement in shallower nurseries, ‘demersal-egg species' by a small habitat shift from coastal spawning to nursery areas with faster settlement of larvae with small size and slender shape, and ‘offshore species’ are characterised by gradual settlement in deep areas by locomotive adaptations of larvae with a large body surface area. So the development of low-salinity tolerance and morphology closely reflect the known ontogenetic migrations of the five species along the nearshore-offshore gradients. Especially, inverse relationships between low-salinity tolerance and metamorphic size in the three cross-shelf species, together with the general patterns of lower predator densities in lower salinity environments, suggest that low-salinity adaptability is critical for the survival strategy of species using shallow, nearshore areas as nurseries.

Tissue expression and stock variation of isozymes of stone flounder (Kareius bicoloratus)

Tissue expression and stock variation of isozymes of stone flounder (Kareius bicoloratus) were analyzed with horizontal starch gel electrophoresis. For the fourteen enzymes assayed, 31 loci were recorded. The results indicated that all the isozymes examined were obviously tissue-specific. The expressions of SOD *, GDH *, G3PDH-2 * and ADH-2 * were detected only in liver, SDH-1 *, MDH-1 * and ADH-1 * only in muscle, and LDH-B * and LDH-C * only in eyes. In comparison, MDH-2 *, GPI-3 * and SDH-2 * were detected in all tissues examined. Other loci examined were detected in a variety of tissues. Muscle and liver were selected to detect the isozyme variation of the two geographic stocks of Qingdao and Weihai, Shandong Province, China. The percentages of polymorphic loci (P 0.99) were 29.17% and 25.00%, the observed heterozygosities (H 0) were 0.028 ± 0.014 and 0.040 ± 0.019, and the expected heterozygosities (He) were 0.039 ± 0.017 and 0.052 ± 0.022 in Qingdao and Weihai stock, respectively. The coefficient of gene differentiation (F st) and genetic distance (D) between the two stocks was 0.012 and 0.0011, respectively, indicating that the genetic differentiation is low between them. Compared with other species of Pleuronectiformes, both the percentage of polymorphic loci and the mean heterozygosity of K. bicoloratus were at a middle level.

Feeding habits of stone flounder Platichthys bicoloratus larvae in Mutsu Bay, Japan

To clarify the feeding strategy of pelagic larvae of stone flounder in Mutsu Bay, the dietary composition and pray size was investigated from February to April during 1989–1999. Diets were compared with the numerical and volumetric compositions and frequency of occurrence of each prey species. Mensuration formulae were applied to estimate individual prey volume in the diet, while the length of planktonic species was measured from net and water samples. Prey shapes were assumed as sphere, cylinder, ellipsoid, pyramid, two elliptical cones, or a combination of ellipsoid and cylinder. Prey-size range increased as the larvae grew. Preflexion larvae fed mainly on copepod nauplii. Flexion and postflexion larvae ingested primarily appendicularians, with a suggestion that these larvae might depend on some parts of the microbial food web. Low frequencies of flexion and postflexion larvae with empty guts (1.7 and 1.4%, respectively) might be derived from feeding on slow-swimming appendicularians. From a size comparison between ‘house’-like organ length and trunk length of the appendicularian Oikopleura sp., almost all house-like organs with trunks in the larval diet were nonexpanded ‘house rudiments’, not expanded ‘houses’. Thus, stone flounder larvae may not chew the houses, but swallow the house rudiments with trunks.

Interactive effects of sublethal predation and body size on siphon production of the bivalve Nuttallia olivacea

This paper is intended to reveal effects of siphon cropping on siphon production of a tellinacean bivalve Nuttallia olivacea, an important prey for juvenile stone flounder. We carried out a two-way field experiment in which bivalves of three treatments (3-times siphon removal, 1-time removal, no removal) and four size classes (9–50 mm shell length) were caged and placed in the field for 3 mo. Growth of repeatedly-removed bivalves was inhibited, indicating reduced siphon growth (natural increase of siphon size according to somatic growth). However, siphon production of removed bivalves was larger than non-removed bivalves, possibly because of siphon regeneration. Juveniles ( N. olivacea < 20 mm shell length) showed high growth performance. Their siphon growth was greater than their siphon regeneration. In all bivalves except juveniles, siphon regeneration was greater than siphon growth and engendered high siphon production. Siphon growth was dependent on bivalve size and was only slightly reduced by siphon loss, but siphon regeneration seemed to be dependent mostly on the extent of siphon loss. Greater siphon removal enhanced larger siphon production. These results indicate that intensive siphon cropping by juvenile stone flounder induces high siphon production without serious impact on N. olivacea.

Feeding and growth of juvenile stone flounder in estuaries: generality and the importance of sublethal tissue cropping of benthic invertebrates

We investigated the feeding habits and growth of juvenile stone flounder (Platichthys bicoloratus) in several estuarine nurseries in Sendai Bay, Japan. Prey abundance and composition and juvenile diet varied largely among sites. However, polychaete palps (mostly Pseudopolydora kempi) and bivalve siphons (mostly Nuttallia olivacea) were positively selected and frequently consumed by juveniles, indicating the generality of sublethal feeding of juvenile stone flounder in estuarine nurseries. Recent growth rates determined by otolith microstructure analyses were dependent on juvenile body size and water temperature, but independent of juvenile density and food abundance. Growth rates were nearly maximal from March to May, suggesting that food conditions are nearly optimal in these estuarine nurseries. Sublethal tissue cropping of benthic invertebrates is thought to contribute largely to these high growth rates.

Regeneration of lost siphon tissues in the tellinacean bivalve Nuttallia olivacea

The inhalant siphon of the tellinacean bivalve Nuttallia olivacea is an important prey item for juvenile stone flounder Platichthys bicoloratus in estuaries in Japan. We examined quantitative siphon regeneration of N. olivacea in rearing experiments of siphon-removed bivalves (> 30 mm shell length) both in the laboratory and in their natural habitat. Under laboratory conditions, siphon-removed bivalves regenerated lost tissues quantitatively at 15 and 25 °C 1 mo after siphon removal, although regeneration was incomplete. A 3-mo caging experiment in the field showed that great regeneration occurred in siphon-removed bivalves. However, the siphon weight of removed bivalves was significantly smaller than that of non-amputated bivalves, suggesting the incomplete regeneration. In a 1-mo caging experiment, bivalves that had approximately 15% of their siphons amputated were selected at some intervals to illustrate the quantitative regeneration process. Estimated daily siphon production was remarkably high only a few days after amputation. It decreased greatly thereafter, but regeneration was not completed within 30 d. These results indicate that bivalves regenerate siphons rapidly just after losing siphon tissues and then regeneration is slowed down before it is completed.