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Latitudinal variation in egg size and number in anadromous masu salmon Oncorhynchus masou was examined. Relatively greater variation in egg size occurred among rivers than among females within rivers or within females. Egg size was generally greater and egg number generally lower at more northerly latitudes.

Gastrointestinal (GI) peptide hormones, ghrelin (GHRL), cholecystokinin (CCK), and peptide YY (PYY) genes were identified in Atlantic salmon, Salmo salar. Full-length cDNAs encoding two isoforms of GHRL (GHRL-1 and GHRL-2), two isoforms of CCK (CCK-L and CCK-N) and peptide YY (PYY) cDNA were obtained. The GHRL-1 and GHRL-2 genes encoded proteins of 111- and 108-amino acids, respectively. Both types of GHRL were mainly expressed in the stomach, but also weakly expressed in the pyloric caeca, mid-gut, adipose tissue, and testis. The CCK-L and CCK-N genes encoded preproproteins of 132- and 140-amino acids, respectively. Both types of CCK were strongly expressed in the brain and comparatively weakly expressed in other tissues, including the digestive tract. In the digestive tract, CCK-L was mainly expressed in the pyloric caeca and hind-gut, while CCK-N was only expressed in the pyloric caeca. The PYY gene encoded for 97-amino acid residues and was mainly expressed in the brain and anterior part of the intestine, including the pyloric caeca. In an experiment, we demonstrated that 6 days starvation led to, increased GHRL-1 mRNA levels in the GI tract (stomach), while there no significant changes in expression levels for the other hormones in the GI tract. This suggests an orexigenic role for GHRL-1 in Atlantic salmon. These data contribute to elucidate the functional relationships among teleost gastrointestinal peptide hormones.

Occurrence of chimeric sequences and related artifacts in PCR cloning procedures gives us risks of over-estimation of haplotypes or alleles. Recombination among haplotypes occurs through template switching during PCR cycles or through random repair of mismatch sites on heteroduplex DNA by the host cell. To eliminate the chimeric cloning artifacts, we tested two alternative protocols using T7 endonuclease I cleavage of mismatch sites and re-extension of nascent strands. Though T7 endonuclease I effectively eliminated chimeric clones in some cases, it produced many short fragments. Protocol with single re-extension of PCR products successfully recovered non-recombinant clones with fewer short fragments. In spite of the improvement of allelic recovery through these two protocols, there were still a few recombinants that remained in both reaction mixtures, and thus interpretation of the results for haplotype diversity in a PCR-amplified DNA population should be cautionary. Because re-extension in a diluted reaction mixture is quick, inexpensive and effective, it is advisable to use this procedure for recovery of chromosomal alleles with PCR cloning.

This is the first report of the propagation of the toxic dinoflagellate Dinophysis fortii Pavill. under laboratory conditions when fed on the marine ciliate Myrionecta rubra grown with the cryptophyte Teleaulax amphioxeia (W. Conrad) D. R. A. Hill. In contrast, reduced growth of D. fortii (max. of 3-4 divisions) and formation of small cells were observed in the absence of the ciliate or when provided with T. amphioxeia only as prey, showing that D. fortii cannot utilize T. amphioxeia as prey. In the TEM observation of D. fortii cells, which had fully fed on the ciliate prey, well-developed chloroplasts (5-12 μm in length) were seen and three thylakoids were usually arranged in most of the chloroplasts observed, but chloroplasts having two thylakoids were sometimes confirmed. In cells starved for 4 weeks, decrease of chloroplast numbers and disappearance of large chloroplasts were observed, and only a few small chloroplasts (0.5-2 μm in length) remained in the marginal regions. In the observation of the sequestration process of the chloroplasts ingested from M. rubra by D. fortii, within 15 min after D. fortii captured M. rubra, incorporation of almost all of the chloroplasts was observed, while most of the other cell contents still remained in the M. rubra cell. After that, dispersion of the ingested chloroplasts toward the marginal regions was confirmed, suggesting that chloroplasts of M. rubra are ingested and dispersed in D. fortii cells in advance of the ingestion of the other cell contents to prevent them from being digested in food vacuoles. The ingested chloroplasts can also function as kleptoplastids., (© 2008 Phycological Society of America.)

Gillnet fisheries are strongly size-selective and seem to produce changes in size at maturity for exploited fishes. After Word War II, large-scale gillnet fisheries targeted Pacific salmon (Oncorhynchus spp.) in the high seas area of the North Pacific and the Bering Sea, but these fisheries were closed in 1993. To assess the effects of this high seas gillnet fishery (and its closing) on size at maturity, we examined long-term trends in size at 50% probability of maturing (L50) for chum salmon (O. keta) from three populations in Hokkaido, Japan. The L50 trends were statistically different among rivers, but showed similar temporal patterns with decreases in the 1970s and early 1980s and increases after the 1985 brood year. While fishery-induced evolution seemed largely responsible for this temporal change in L50 during the fishing period, natural selection and phenotypic plasticity induced by environmental changes could contribute to the increases in L50 after the relaxation of fishing pressure.

To clarify the natural diet of phyllosoma larvae of the Japanese spiny lobster Panulirus japonicus, the sources of 18S rDNA clones obtained from the hepatopancreas were investigated. Of a total of 1537 clones examined, 160 had different restriction profiles from the host larvae, in which 21 restriction types were observed. Nucleotide sequences of 16 of 21 restriction types were successfully determined and their assignments were investigated by homology search and phylogenetic analysis. From seven late-stage larvae collected in spring to early summer, eukaryote DNA molecules of Teleostei, Oomycetes, Mycetozoa, and Fungi were identified. Exogenous DNA from four younger phyllosoma larvae collected in late autumn could not be recovered. A previous study identified DNAs of cnidarians and urochordates in late-stage phyllosoma larvae of a closely related species collected in winter. This indicates that the phyllosoma larvae are opportunistic carnivores, whose diets correlate with the relative abundance of prey organisms in the ambient water.

The fish egg is surrounded by a thick envelope called the chorion. The fertilizing spermatozoon enters the egg through a canal-like structure in the chorion, the micropyle. Examination of micropyle at fertilization is difficult if eggs are large and have no distinct landmarks surrounding the micropyle, or if they are positively buoyant in water. Eggs of many commercially important fishes (e.g., flounder, sea bream and eel) are buoyant in water or only slightly adhere to solid objects (e.g., sands, rock and water plants), which makes observation of spermatozoa at fertilization difficult. Here, we report that such eggs can be firmly attached to plastic and glass dishes that have been previously coated with poly-L-lysine. These adhering eggs can be fertilized and develop normally on the dishes. Observations of micropyles of three fish species, before and after sperm entry are presented.

This study investigated seasonal and spatial dynamics of the bacterial community in Gokasho bay with denaturing gradient gel electrophoresis (DGGE) profiles of PCR-amplified 16S rRNA gene fragments. The community structure was related to physico-chemical water conditions in the area examined. The bacterial community clustered into three groups: bacteria collected during January-May; those collected from water at the surface in July and September; and those collected from water at the bottom in July and September and from both depths in November. Canonical correspondence analyses indicated that the seasonal variability in bacterial community was associated with water temperature succession. On the other hand, concentrations of particulate organic matter and nitrite plus nitrate were related to the vertical change in community structure in summer and autumn as well as HNF abundance, suggesting that both top-down and bottom-up control affected the community. The influence of salinity was insignificant though bacterial production was related to salinity. No relationship was observed between the variation in community structure and that in hydrolytic enzyme activity. The results indicate that changes in bacterial activity are not coupled with variation in community structure.

Major yolk protein (MYP), a transferrin superfamily protein that forms yolk granules in sea urchin eggs, is also contained in the coelomic fluid and nutritive phagocytes of the gonad in both sexes. MYP in the coelomic fluid (CFMYP; 180 kDa) has a higher molecular mass than MYP in eggs (EGMYP; 170 kDa). Here we show that MYP has a zinc-binding capacity that is diminished concomitantly with its incorporation from the coelomic fluid into the gonad in the sea urchin Pseudocentrotus depressus. Most of the zinc in the coelomic fluid was bound to CFMYP, whereas zinc in eggs was scarcely bound to EGMYP. Both CFMYP and EGMYP were present in nutritive phagocytes, where CFMYP bound more zinc than EGMYP. Saturation binding assays revealed that CFMYP has more zinc-binding sites than EGMYP. Labeled CFMYP injected into the coelom was incorporated into ovarian and testicular nutritive phagocytes and vitellogenic oocytes, and the molecular mass of part of the incorporated CFMYP shifted to 170 kDa. Considering the fact that the digestive tract is a major production site of MYP, we propose that CFMYP transports zinc, essential for gametogenesis, from the digestive tract to the ovary and testis through the coelomic fluid, after which part of the CFMYP is processed to EGMYP with loss of zinc-binding site(s).

A major challenge for diving birds, reptiles, and mammals is regulating body temperature while conserving oxygen through a reduction in metabolic processes. To gain insight into how these needs are met, we measured dive depth and body temperatures at the core or periphery between the skin and abdominal muscles simultaneously in freely diving Brünnich's guillemots (Uria lomvia), an arctic seabird, using an implantable data logger (16-mm diameter, 50-mm length, 14-g mass, Little Leonardo Ltd., Tokyo). Guillemots exhibited increased body core temperatures, but decreased peripheral temperatures, during diving. Heat conservation within the body core appeared to result from the combined effect of peripheral vasoconstriction and a high wing beat frequency that generates heat. Conversely, the observed tissue hypothermia in the periphery should reduce metabolic processes as well as heat loss to the water. These physiological effects are likely one of the key physiological adaptations that makes guillemots to perform as an efficient predator in arctic waters.

The insulinotropic effects of eighteen L-amino acids, two D-amino acids, and glucose were investigated to evaluate the priority of those as stimulators of insulin secretion in barfin flounder (Verasper moseri). This is also the first step in characterizing the insulinotropin-sensing molecule. After intramuscular injection of amino acids or glucose at doses of 3.50 and 1.75 mmol/kg body weight, plasma was collected periodically to determine plasma insulin level. Twelve amino acids and glucose showed insulinotropic effects. Four L-amino acids (Arg, Ala, Met, Ser) produced significantly higher integrated levels of plasma insulin (12.4-34.8 ng/ml) than glucose (average: 4.7 ng/ml) during 3h after injection. D-Amino acids (Arg, Ala) showed no activity. This indicates that many amino acids have strong insulinotropic activities and supports a classic idea, which is well known but has not been confirmed, that amino acids rather than glucose are the important insulinotropins in fish. This study also indicates that the insulinotropic activity of amino acids is restricted to L-amino acids and establishes which amino acids are the strongest stimulators of the insulinotropin sensor in barfin flounder. Co-injection of insulin and L-Thr, L-Ala, or glucose produced a hypoglycemic and hypoaminoacidemic state, indicating that insulin can lower blood amino acid level as well as blood sugar level. This study suggests that insulin plays a more important role than glucose in the regulation of blood L-amino acid metabolism, at least in flounder.

Several strains of Protoceratium reticulatum, one of the dinoflagellates producing yessotoxins (YTXs), were collected from various shellfish producing areas in Japan. YTXs in the cultured strains were analyzed by liquid chromatography-mass spectrometry (LC-MS). Neutral loss scan monitoring, multiple reaction monitoring (MRM) for more than 20 YTX analogues, and full-scan MS/MS spectra obtained with a hybrid triple quadrupole/linear ion trap mass spectrometer showed that yessotoxin (YTX), 45,46,47-trinoryessotoxin (trinorYTX), 1-homoyessotoxin (homoYTX), and 45,46,47-trinor-1-homoyessotoxin (trinor-1-homoYTX) were the dominant toxins in these strains of P. reticulatum. Enone isomer of 42,43,44,45,46,47,55-heptanor-41-oxoyessotoxin (noroxoYTX enone) was also detected in some strains. Toxin profiles and contents were different among the strains. Some strains produced YTX, trinorYTX, 1-homoYTX, trinor-1-homoYTX, and noroxoYTX enone, whereas other strains produced only YTX or 1-homoYTX. This is the first identification of 1-homoYTX and noroxoYTX enone in P. reticulutum in Japan. Some strains did not produce any detectable YTX analogues.

This study presents linkage maps for the Pacific abalone (Haliotis discus hannai) based on 180 microsatellite DNA markers. Linkage mapping was performed using three F1 outbred families, and a composite linkage map for each sex was generated by incorporating map information from the multiple families. A total of 160 markers are placed on the consolidated female map and 167 markers on the male map. The numbers of linkage groups in the composite female and male maps are 19 and 18, respectively; however, by aligning the two maps, 18 linkage groups are formed, which are consistent with the haploid chromosome number of H. discus hannai. The female map spans 888.1 cM (Kosambi) with an average spacing of 6.3 cM; the male map spans 702.4 cM with an average spacing of 4.7 cM. However, we encountered several linkage groups that show a high level of heterogeneity in recombination rate between families even within the same sex, which reduces the precision of the consolidated maps. Nevertheless, we suggest that the composite maps are of significant potential use as a scaffold to further extend the coverage of the H. discus hannai genome with additional markers.

Fishes of the order Cypriniformes are almost completely restricted to freshwater bodies and number > 3400 species placed in 5 families, each with poorly defined subfamilies and/or tribes. The present study represents the first attempt toward resolution of the higher-level relationships of the world's largest freshwater-fish clade based on whole mitochondrial (mt) genome sequences from 53 cypriniforms (including 46 newly determined sequences) plus 6 outgroups. Unambiguously aligned, concatenated mt genome sequences (14,563 bp) were divided into 5 partitions (first, second, and third codon positions of the protein-coding genes, rRNA genes, and tRNA genes), and partitioned Bayesian analyses were conducted, with protein-coding genes being treated in 3 different manners (all positions included; third codon positions converted into purine [R] and pyrimidine [Y] [RY-coding]; third codon positions excluded). The resultant phylogenies strongly supported monophyly of the Cypriniformes as well as that of the families Cyprinidae, Catostomidae, and a clade comprising Balitoridae + Cobitidae, with the 2 latter loach families being reciprocally paraphyletic. Although all of the data sets yielded nearly identical tree topologies with regard to the shallower relationships, deeper relationships among the 4 major clades (the above 3 major clades plus Gyrinocheilidae, represented by a single species Gyrinocheilus aymonieri in this study), were incongruent depending on the data sets. Treatment of the rapidly saturated third codon-position transitions appeared to be a source of such incongruities, and we advocate that RY-coding, which takes only transversions into account, effectively removes this likely "noise" from the data set and avoids the apparent lack of signal by retaining all available positions in the data set.

A new method for visualizing small and multiple phosvitins (Pvs) in oocytes from a marine teleost was developed by a combination of gel filtration, alkaline phosphatase treatment, and SDS-PAGE followed by silver staining. Three distinct Pv polypeptides having molecular masses of 15 kDa, 8 kDa, and 7 kDa were visualized in vitellogenic follicle extract of barfin flounder, Verasper moseri. N-terminal amino acid sequencing identified two different N-termini that fell into the PvA (7 kDa) and PvB (15 kDa and 8 kDa) groups, which were derived from two forms of vitellogenin (Vg), VgA and VgB, respectively. Analysis of time-course change in phosphorus-rich peaks of gel chromatography fractions of follicle extracts from different maturational stages demonstrated a rapid degradation of Pvs during mid-phase of oocyte maturation. Quantitative analysis of free amino acids in maturing follicles revealed an increment of serine content but not of phosphoserine, indicating the occurrence of dephosphorylation concomitant with Pv degradation. Measurement of phosphatase activity in follicles and eggs at different maturational stages demonstrated a significant activation of phosphatase especially under acidic conditions. This suggested that Pv degradation and dephosphorylation are regulated by changes in ooplasm pH during oocyte maturation. Our results also suggested that the Pvs in barfin flounder vitellogenic oocytes bind to much lower amounts of calcium and magnesium than those of masu salmon, Oncorhynchus masou. This indicates that the Pvs in the barfin flounder, a marine teleost spawning its eggs in seawater, do not play a role in the transport and deposition of calcium and magnesium into oocytes.

In many benthic organisms with a planktonic larval stage, local populations have different morphology. Such difference may arise from some of the following proximate mechanisms. "Local recruitment (LR)": no larvae move between local populations, and segregated populations possess alleles coding for locally adaptive morphology. "Intragenerational selection (IS)": larvae move between local populations, and individuals with alleles for locally adaptive morphology survive after recruitment. "Phenotypic plasticity (PP)": larvae move between local populations and show phenotypic plasticity to adapt to a locality after recruitment. We examined which mechanism explains our finding that a planktonic developer Turbo coronatus coronatus (Gastropoda) had significantly longer spines on its shell on more exposed shores at scales of < 2 km. Experiments at Ishigaki Island, Okinawa, Japan, showed the following results. (a) Shorter- and longer-spined populations occurring within 2 km showed non-significant low psi(ST) values (-0.0040 to 0.00095) for the mitochondrial DNA COI region. This suggests no segregation of the local populations, supporting the mechanisms IS and PP. (b) T. c. coronatus generated significantly longer spines 70 days after being transplanted to the habitat of a longer-spined population, supporting IS and PP. (c) Individuals caged in the sea for 79 days generated longer spines than individuals in the laboratory, supporting PP. In conclusion, shore-specific morphology of T. c. coronatus arises most likely from phenotypic plasticity and possibly from intragenerational selection.

We present novel microsatellite markers of the Japanese abalone (Haliotis discus hannai) for general mapping studies in this species. A total of 75 microsatellite markers were developed, and the allele-transmission patterns of these markers were studied in three families generated by pair crosses. For allele scoring, we employed the 5'-tailed primer polymerase chain reaction (PCR) technique, which substantially reduces the cost for fluorescent labeling of primers. Of the 225 possible marker-family combinations (75 markers x 3 families), 18 cases of informative null-allele segregation were inferred. When such null-allele segregations were allowed, more than 70% of the 75 markers in the families turned out to be markers with an expected segregation ratio of 1:1:1:1, allowing maximal exploitation of the codominant nature of microsatellite markers. There were 16 instances of segregation distortion at the 5% significance level. The test for independence of segregation assigned the 75 markers into 17 linkage groups, which is in close agreement with the haploid chromosome number of H. discus hannai (n = 18). Six markers could not be placed into any linkage group. We suggest that these markers could help construct a H. discus hannai linkage map.

Mass mortalities of the pen shell Atrina pectinata occurred in the fishing grounds of Ariake Bay, in southwestern Japan, during late spring and summer in 2003 and 2004. Histological examination revealed extensive necrosis in the epithelial cells of the kidney and gill, and impairment of the endothelial cells of the mantle arteria. Although cestode larvae belonging to the genus Tylocephalum were found in the mantle, adductor muscle, kidney, and digestive gland, their prevalence and the intensity of infection were low. Examinations of moribund pen shells for Haplosporidium spp. infection using PCR analysis and for Perkinsus spp. infection using Ray's fluid thioglycollate medium were negative. Unenveloped virus-like particles were detected by transmission electron microscopy in the cytoplasm of affected kidney and gill cells of moribund pen shells. They were icosahedral spherical and 50 to 55 nm in diameter. These virus-like particles found in moribund pen shells are different from those described in other marine mollusks, and may be the causative agent of the mass mortalities of pen shells.

Body size is widely believed to affect the occurrence of sexual maturation. Recent studies have used changes in the age-specific body size at which the probability of maturing is 50%, a feature of probabilistic reaction norms, to quantify purported evolution of life histories. However, body size results from a combination of growth rates during successive developmental stages. Therefore, to understand the evolution of the maturation schedule, it is necessary to comprehend the relationships among body size, growth history, and maturation schedule. We examined the relationships among body size, previous growth history, and maturation probability in chum salmon (Oncorhynchus keta). In this study, previous growth history was estimated from yearly specific growth increments that provide information describing body size. Previous growth history was found to be more closely linked to maturation probability than body size. The most recent growth condition was the most important factor affecting whether a fish matured during the subsequent breeding season. Because individuals of similar body size and same age can have different growth histories, the relationship between body size and maturation probability could be plastically modified by growth history. This may violate an assumption required to infer evolution, namely that size-related maturation trends in probabilistic reaction norms are immune to growth history.

Full-length cDNAs encoding three forms of vitellogenin (Vg) were obtained from a liver cDNA library of estrogen-treated red seabream, Pagrus major. Two of the three Vg sequences had high homology with type-A and -B Vgs (VgA and VgB) of other teleosts. The third red seabream Vg was classified as a type-C or phosvitinless (Pvl) Vg due to its lack of a phosvitin (Pv) domain. Two Vg preparations (610 and 340 kDa) from blood serum of estradiol-treated fish were biochemically characterized. Analyses of precursor-product relationships by examination of N-terminal amino acid sequences verified cleavage of the 610 kDa Vg into a 540 kDa lipovitellin (Lv) and a 32 kDa beta'-component. Each of these yolk preparations comprising both VgA- and VgB-derived polypeptides. The 340 kDa Vg, which was immunologically verified to be a PvlVg, was accumulated by vitellogenic oocytes with no alterations to its native molecular mass. During oocyte maturation, the VgA- and VgB-derived yolk proteins were differentially processed, presumably to generate a pool of free amino acids for oocyte hydration or for allocation of specific types of nutrients, amino acids, and proteins, to the developing embryo. Conversely, the 340 kDa Vg-derived yolk protein is unlikely to contribute to oocyte hydration or diffusible nutrients since the molecule underwent only minor proteolytic nicking during oogenesis. The present study elucidates for the first time specific functions of three different forms of Vg and their product yolk proteins in a higher taxonomic group of marine teleosts that spawn pelagic eggs., (Copyright 2006 Wiley-Liss, Inc.)

A time-resolved fluoroimmunoassay system (TR-FIA) for measuring flounder insulin-like growth factor-I (IGF-I) was developed using biotinylated flounder IGF-I, anti-fish IGF-I antiserum and europium-avidin conjugate. The detection limit per well was <5 pg/well corresponding to <0.5 ng/ml in a basic procedure for sample of 10 microl/well and to <0.08 ng/ml in a procedure modified for high volume samples (up to 70 microl/well). Specificity of the assay was validated using various IGF-Is and insulins. All IGFs except seabream IGF-I showed very low or no crossreactivity. Binding inhibition curves for flounder and seabream IGF-Is were completely identical to each other. Intra- and interassay variations ranged from coefficients of variations of 3.9% to 7.2%. Recovery tests using barfin flounder plasma varied from 82.7 to 101.6% in the added range from 20 to 160 ng/ml. This assay system was applied for measuring total plasma IGF-I in barfin flounder injected porcine growth hormone (GH). A group injected with GH at the dose of 0.05 IU/gBW showed a significant increase of total plasma IGF-I compared with those of albumin-injected (control) and initial groups. In addition, I was able to substitute time-resolved fluorometric detection in this assay system with enzymatic fluorometric detection (FIA). Binding inhibition curve for flounder IGF-I in this substituted assay system showed equal performance with that of the TR-FIA system. Correlation of IGF-I levels between TR-FIA and FIA was high (r(2)=0.957) in plasma samples from barfin flounders in various physiological conditions. Thus, the present study shows precision and efficiency of two non-radioisotopic immunoassay systems for measuring flounder IGF-I.

We previously demonstrated the presence of three forms of vitellogenin (Vg), two 600 kDa Vgs (600Vg; VgA and VgB) and a 400 kDa Vg (400Vg; phosvitinless Vg) in plasma from maturing female viviparous mosquitofish, Gambusia affinis. For further quantitative elucidation of the accumulation and utilization of the multiple Vg-derived yolk proteins, two sandwich enzyme-linked immunosorbent assays (ELISA) were developed using antisera against 600Vgs and a 400 kDa yolk protein (400Yp; derived from 400Vg), respectively. Contents of 560 kDa yolk protein (560Yp; lipovitellins derived from 600Vg) and 400Yp measured by the ELISAs increased in accordance with the growth of vitellogenic oocytes, keeping their proportional ratio (mol/mol) at about 4:1. A similar ratio obtained for plasma Vgs suggests that the proportional accumulation of the multiple Vg-derived yolk proteins is regulated by the hepatic synthesis and secretion of their precursor Vgs. When egg homogenate was analyzed by gel chromatography, three peaks, consisting of 560Yp, 400Yp and 28 kDa native beta'-component, were observed. The elution profile showed no change until embryos reached the early neurula stage, however, the relative height of the 560Yp peak as compared to the 400Yp one decreased after retinal pigmentation. Results from measurements of 560Yp and 400Yp at each embryonic stage supported the occurrence of unequal utilization of the two yolk proteins. The proportional ratios (mol/mol) of 560Yp content versus 400Yp content gradually decreased from 4.1 fold in early neurula embryo to 1.4 fold in larva just before parturition. The present study thus demonstrated unequal utilization of the multiple Vg-derived yolk proteins in developing embryos of mosquitofish.

We investigated the assimilation efficiency (AE) of a piscivorous alcid, Rhinoceros Auklet (Cerorhinca monocerata), chicks when fed Japanese anchovy (Engraulis japonicus) and Japanese sand lance (Ammodytes personatus), which are their main prey species. The assimilation efficiency corrected for nitrogen retention (NR) of the chicks fed sand lance (81.6%) was significantly higher than those fed anchovy (78.0%). The values of assimilation efficiencies for both fish species are similar to those of fish-fed seabird adults and fledglings.

The vertical distribution of dioxins in a sediment core was investigated to elucidate historical trends of dioxins discharged into Sendai Bay, Japan. The dioxin concentration was 410 pg/g dry weight (dw) in sediments deposited in the mid-1930s and 3870 pg/g dw in those deposited in the mid-1980s. Dioxin fluxes increased from the mid-1930s and then reached a maximum in the mid-1980s. 1,3,6,8-TeCDD+1,3,7,9-TeCDD, OCDD, and Co-PCB concentrations were 110, 140, and 26 pg/g dw, respectively, in mid-1930s sediments, and reached maximums of 1800, 1100, and 200 pg/g dw, respectively, in mid-1980s sediments. Shipments to Miyagi Prefecture of CNP and PCP products, the major sources of 1,3,6,8-TeCDD+1,3,7,9-TeCDD and OCDD, were highest in 1975 (4700t) and 1970 (3100t), respectively; and in Japan, the amount of PCBs, the major source of Co-PCB congeners, used was highest (11,100t) in 1970. Thus, the period for which the maximum concentrations of 1,3,6,8+1,3,7,9-TeCDD, OCDD, and Co-PCBs were measured in the sediment core (mid-1980s) did not correspond to the time of maximum use of CNP, PCP, or PCB products, but lagged behind by more than 10 years. We attributed this time lag to the time required for the movement of dioxins from Miyagi Prefecture to Sendai Bay.

This report deals with an unusual mode of mitochondrial gene introgression between Cobitis hankugensis (C. sinensis) and C. longicorpus which is mediated by a unisexual hybridogenetic system of diploid-triploid C. hankugensis-longicorpus complex. Mitochondrial DNA sequences of 3329-3330bp encompassing from upstream ND6 to 12S rDNA indicated that mitochondrial genomes from the diploid hybrids, triploid hybrids, and their parental species are almost identical. Because triploid hybrids produce haploid ova with C. hankugensis chromosome set, normal diploid C. hankugensis regenerates upon insemination with C. hankugensis sperm. If the hybrid carries C. longicorpus mitochondrial genome, the regenerated C. hankugensis is a nucleo-cytoplasmic hybrid, thus accomplishing the unusual mode of mitochondrial gene introgression.

Complete nucleotide sequences of two forms of vitellogenin (Vg) cDNA in Japanese common goby were determined from a liver cDNA library of E(2)-treated male fish. These two Vg cDNAs contained complete open reading frames encoding 1664 and 1238 amino acid residues including signal peptides, respectively. From comparison of the deduced amino acid sequences of both Vgs and the partial amino acid sequences of the yolk proteins, the longer sequence was concluded to be cDNA of the Vg-530 and the shorter one was that of the Vg-320 of the Japanese common goby which were reported in our previous paper. The deduced sequence of Vg-530 without signal peptide was arranged by lipovitellin heavy-chain (LvH), phosvitin (Pv), lipovitellin light-chain (LvL), and beta'-component beta'-c) domains from the N-terminus, and showed a range of 40-45% sequence identity to those of other fish. Furthermore, the deduced sequence of Vg-320 showed no obvious Pv domain, has a shortened C-terminal coding region after the LvH domain, and showed a close similarity to the phosvitin-less Vg of zebrafish. Moreover, biochemical analysis of the yolk proteins verified that Vg-530 cleaves into the Lv-Pv complex (molecular mass: 470 kDa) and beta'-c (33 kDa), while Vg-320 showed no change when incorporated into oocytes. The present study demonstrated the existence of the two different forms of Vgs at both the cDNA and protein level, and showed molecular alteration of the two Vgs during vitellogenesis. Two Vg sequence data will aid in designing nucleotide probes for detecting Vg gene expressions as a biomarker of environmental estrogens.

The concentrations of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), non-ortho polychlorinated biphenyls (non-ortho PCBs), and mono-ortho polychlorinated biphenyls (mono-ortho PCBs) in Japanese flounder (Paralichthys olivaceus, body length 10.4-36.6 cm) collected from Sendai Bay, Japan, were determined using high-resolution gas chromatography-mass spectrometry. The relationship between the concentrations of these compounds (dioxins) and the body length of the Japanese flounder was examined. The total PCDD and total PCDF concentrations did not correlate with body length (both r(2) < 0.1, both p > 0.05), whereas the total non-ortho PCB and total mono-ortho PCB (coplanar polychlorinated biphenyls, Co-PCBs) concentrations were significantly correlated (r(2)= 0.8, p < 0.05 and r(2)= 0.63, p < 0.05, respectively). The bioaccumulation properties of PCDD/Fs in Japanese flounder differed from those of Co-PCBs. Toxicity equivalency quotient (TEQ) values derived from the Co-PCBs made up 46.3%-63.7% of the total TEQ value for all the dioxins. Although the concentrations of non-ortho PCBs were lower than those of mono-ortho PCBs, the TEQ value for non-ortho PCBs was higher than that for mono-ortho PCBs. The TEQ value for non-ortho PCBs increased more with increasing body length than did the values for PCDDs, PCDFs, and mono-ortho PCBs. These results show that from the standpoint of risk management, non-ortho Co-PCBs are the most important of the dioxins in Japanese flounder.

An unisexual species (either parthenogenetic or gynogenetic form) often coexists sympatrically with a closely related anisogamous sexual species, forming a unisexual complex. This is puzzling because, all else being equal, a sexual population with the two-fold cost of sex (or the cost of producing males) cannot coexist with a unisexual population. This suggests that some ecological processes are at work to make possible the coexistence of the unisexual complex. Field and laboratory studies suggest that parasitism in a gynogenetic complex of the Japanese crucian carp (Carassius auratus) may play an important role in realizing the coexistence by giving frequency-dependent benefit to sexual population. Here, we study the simple dynamics of host-parasite interactions in which non-specific immune reaction of a sexual host is more effective than that of an unisexual host. We simply assume that the infective individuals are sterile. Stable coexistence of a unisexual species with their sexual relative is possible if pathogens are virulent and if the susceptibility of the unisexual form is more than twice as high as that of the sexual form. The coexistence is more difficult, when the fertility of males is relatively low in gynogenetic complexes. This implies that the coexistence of gynogenetic complexes is more difficult than that of parthenogenetic complexes in which parthenogenesis has no constraint on males. We conclude that parasitism is a promising candidate mechanism for the coexistence of unisexual complexes.

The dinoflagellate Dinophysis acuta has been associated with various incidents of diarrhetic shellfish poisoning. A sample of Dinophysis acuta collected from New Zealand waters in 2002 was previously found to contain high levels of pectenotoxins, but only a very low level of the diarrhea-inducing okadaic acid (OA). After hydrolysis under basic conditions, however, the concentration of OA increased substantially, indicating the presence of conjugated forms of OA. Using various liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) techniques, a number of OA esters were detected in the original extract. The principal compound was identified as a C8 diol-ester of OA (OA-D8), which had been identified previously in another dinoflagellate, Prorocentrum lima. The retention time, as well as positive and negative ion MS, MS/MS and UV spectra of the D. acuta compound, matched exactly those of OA-D8 isolated from P. lima. In addition to OA-D8, several other novel OA esters were detected in the D. acuta but these have not yet been identified. This is the first report identifying the presence of OA esters in Dinophysis species., (Copyright 2004 Crown in the right of Canada. Published by John Wiley & Sons, Ltd.)

The concentrations of PCDD/F and Co-PCB congeners in seawater, sediment, Pacific oyster, Japanese anchovy, marbled sole, and Japanese flounder samples from Sendai Bay, Japan, were analyzed. The compositions of total PCDD/F and total Co-PCB concentrations in sediment and Pacific oyster reflected that in seawater--the compositions of total PCDD, PCDF, and Co-PCB concentrations were approximately 60, 10, and 30%, respectively. The compositions in Japanese anchovy, marbled sole, and Japanese flounder were different from those in seawater and sediment-the ratio of total Co-PCB concentration to total PCDD/F plus Co-PCB concentrations in Japanese anchovy, marbled sole, and Japanese flounder was above 90%. Tetrachlorinated PCDDs (T4CDDs), such as 1,3,6,8- and 1,3,7,9-T4CDD, were the predominant congeners in seawater and sediment; the total T4CDD concentrations in seawater and sediment were approximately 46 and 48% of the total PCDD concentration. Furthermore, shipments of the herbicide 1,3,5-trichloro-2-(4-nitrophenoxy)benzene to Miyagi Prefecture, the so-called granary of Japan, were the highest in Japan over the last 12 years. The major sources of PCDD/Fs and Co-PCBs in Sendai Bay, which is in Miyagi Prefecture, are impurities in chlorinated herbicides. The order of concentration of PCDD/Fs was Pacific oyster > Japanese anchovy = marbled sole > Japanese flounder; the concentration in Japanese flounder, which is a higher-trophic-level consumer in the marine food web, was lower than that in shellfish (Pacific oyster) and Japanese anchovy, which are lower-trophic-level consumers. The order of concentration of Co-PCBs was Pacific oyster < Japanese anchovy = marbled sole < Japanese flounder; the concentrations in the higher-trophic-level consumers were higher than the concentrations in the lower-trophic-level consumers. Different PCDD/F congeners tended to bioaccumulate in different organisms. On the other hand, all species of Co-PCB congener tended to bioaccumulate in all organisms.

Two vitellogenins (Vgs) were detected in serum from estradiol-17beta (E(2))-injected Japanese common goby (Acanthogobius flavimanus). Vitellogenins with molecular masses of 530 kDa (Vg-530) and 320 kDa (Vg-320) were purified, and used to raise specific antisera in rabbits. Sandwich enzyme-linked immunosorbent assays (ELISAs) for Vg-530 and Vg-320 were developed using the antisera and the isolated Vgs. The sensitivity ranges of these ELISAs were 1.25-160 ng/ml for Vg-530 and 0.26-66 ng/ml for Vg-320, and very low cross-reactivity was found with the alternate Vg in each assay. Treatment of male gobys with E(2) by injection and immersion induced both Vgs in sera in a dose-dependent manner. The mean concentrations of the Vgs increased from 10 ng/L E(2) exposure for three weeks. Serum concentrations of the two Vgs in field-collected maturing females increased in accordance with increment of E(2) level and ovarian development, and the mean concentrations of Vg-530 were higher than those of Vg-320 in maturing female. These results indicate that the sandwich ELISAs for Vg-530 and Vg-320 developed in the present study is useful as an assay system for surveys of estrogenic activity in coastal areas of Japan.

We document the potential of novel microsatellites as a genetic tool in furthering our understanding of the Crassostrea gigas genetic structure. From the microsatellite-enriched libraries we constructed, 123 repeat regions that had sufficient sequence information to design polymerase chain reaction primer sets were isolated. From these, 9 primer pairs were screened in a C. gigas population of 67 individuals to evaluate the genetic variability. All but 1 of the 9 loci showed allelic variation (number of alleles, 2-20; observed heterozygosity, 0.119-0.925; unbiased expected heterozygosity, 0.139-0.914). Considerable discrepancy of genotypic proportions from the Hardy-Weinberg equilibrium was observed at 1 locus with an apparent heterozygote deficiency. Several loci were successfully amplified in 3 other related species with the appropriate allele size: 6 loci in C. sikamea, 4 loci in C. ariakensis, and 5 loci in C. nippona.

The acid-catalyzed inter-conversion of spiroketal isomers of pectenotoxins PTX1, PTX6 and PTX2 were studied by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-MS-MS). Using a C8-silica reversed-phase column and a mobile phase of aqueous acetonitrile containing 2 mM ammonium formate and 50 mM formic acid, the known spiroketal stereoisomers of PTX1 eluted in order of PTX1, PTX4 and PTX8, while those of PTX6 eluted in the order PTX6, PTX7 and PTX9. Acid treatment of PTX2 yielded two novel spiroketal stereoisomers, which have been named PTX2b and PTX2c. LC-MS-MS spectra obtained for the [M+NH4]- ions of PTX2, PTX2b and PTX2c were essentially identical. As an application of the LC-MS-MS methodology, a sample of the toxic dinoflagellate Dinophysis acuta collected from the coast of New Zealand was analyzed for pectenotoxins. PTX2 and a new pectenotoxin, which has been named PTX11, were detected as the most predominant compounds. Novel PTX2 and PTX11 isomers were also found in the D. acuta although the levels of these compounds were low.

Ostariophysi is the second largest superorder within Teleostei. It contains five orders: Gonorynchiformes, Cypriniformes, Characiformes, Siluriformes, and Gymnotiformes. Resolving the higher-level relationships among ostariophysan and related fishes will aid in resolving basal teleostean divergence and provide basis to historical biogeographic analysis of major freshwater fish groups. In this study, we report the complete mitochondrial (mt) DNA sequences for eleven ostariophysan fishes and the results of phylogenetic analyses including these species plus four other ostariophysan and nine non-ostariophysan teleostean fishes. Maximum likelihood and maximum parsimony analyses reconfirmed clupeiforms as the closest relatives of ostariophysans. However, gonorynchiforms were closer to clupeiforms than to otophysans (ostariophysan groups excluding gonorynchiforms), thus raising a question over the current definition of Ostariophysi. The lack of clarity in otocephalan (ostariophysans + clupeiforms) basal relationships implies that such divergence took place over a short period of time. The monophyly of cypriniforms, characiphysans (characiforms, siluriforms, and gymnotiforms), and orders or superorders outside the ostariophysans examined here were conceivably reconstructed. The phylogenetic hypothesis suggests a Pangean origin of otophysans. Within characiphysans, gymnotiforms and siluriforms have independent evolutionary origins and evolutionary histories comparable to or older than that of characiforms. This helps to explain the present geographic distribution of characiphysans.

The large race of the Cobitis striata complex is a bisexual polyploid population. It is difficult to distinguish this polyploid loach morphologically from a sympatric diploid C. striata, the Biwa small race, indicating the close relationship between these two populations. The polyploid loach does have striata-specific satellite DNAs, but it also harbors C. biwae-related mtDNA. The large race has 2n=4x=98 chromosomes with 56 acrocentric chromosomes. Diploid C. striata has 2n=50 and C. Biwae 2n=48 chromosomes, suggesting the karyotype of polyploid striata as a combination of these two genomes. Meiotic figures showed a few quadrivalent formations besides many bivalents. Many of the quadrivalents were of metacentric chromosomes. Some were, however, made of four acrocentric or of two acro- plus two metacentric chromosomes. Chiasmata were visible in some quadrivalent association. Quadrivalent formation with chiasmata indicates the presence of homologous segments capable of crossing-over between two genomes. Thus, the general polyploid model is also applicable to this case of polyploidy.

We previously produced four monoclonal antibodies to testicular proteins of a teleost, the Nile tilapia. One of the monoclonal antibodies, TAT(Testicular Antigen of Tilapia)-10, recognizes a Mr=27,000 protein (27 kD protein), which is present in A and early B type spermatogonia, spermatids, and spermatozoa in testis. In order to clarify the function of this protein, molecular cloning was conducted. The cDNA for the 27 kD protein contains a complete open reading frame encoding 220 amino acid residues. The predicted amino acid sequence of the 27 kD protein was homologous to those of the ubiquitin carboxy-terminal hydrolases (UCH) reported in mammals. The measurement of the ubiquitin-releasing activity of the recombinant 27 kD protein revealed that the protein is the active form of UCH. Northern blot analysis showed that the UCH mRNA was expressed in ovary and brain in addition to the testis. Immunohistochemical study showed that, in brain, UCH was localized especially on the olfactory organ including the olfactory bulb and olfactory epithelium in olfactory rosetta, suggesting the involvement of the protein in chemoreceptive function. In the Tilapia ovary, UCH localized especially in pre-vitellogenic oocytes, suggesting that the enzyme activity could be important in oocyte growth. This is the first report for the cDNA cloning and cellular localization of UCH in fish. J. Exp. Zool. 293:368-383, 2002., (Copyright 2002 Wiley-Liss, Inc.)

Our previous study shows that seminal plasma of a teleost, the Nile tilapia, contains a glycoprotein Mr = 120,000 named as SPP (Seminal plasma glycoprotein)120 which forms a homopolymer that has sperm immobilizing activity. In order to elucidate the mechanisms of the formation of the homopolymer and the immobilization of sperm, molecular cloning of SPP120 was conducted. The cDNA for SPP120 contains a complete open reading frame encoding 797 amino acid residues with 14 potential N-glycosylation sites. The predicted amino acid sequence of SPP120 contains a partial von Willebrand factor type D domain and a zona pellucida domain, that are involved in protein-protein adhesion that form filamentous structures in various kinds of cells. This result suggests that SPP120 forms a homopolymer via these domains in seminal plasma and probably interacts with spermatozoa. Northern blotting reveals that the gene is also expressed in ovary, even in ovulated eggs. The results of in situ hybridization indicate that in testis the gene is expressed in Sertoli cells and epithelial cells of sperm ducts, and the localization corresponds to that of the protein analyzed by immunohistochemistry. In the ovary, the gene is expressed at the perinucleolus stage of oocytes; however, the protein is not detected in any cells other than oocytes., (Copyright 2002 Wiley-Liss, Inc.)

A time-resolved fluoroimmunoassay (TR-FIA) system was developed to quantify insulin levels in the barfin flounder. This TR-FIA system is a solid-phase assay based on competition of unlabeled insulins and biotinylated barfin flounder insulin-II against an anti-barfin flounder insulin-II antibody. The minimum detectable level of barfin flounder insulin-I and -II in this TR-FIA was 10 pg/well which corresponded to 1.0 ng/ml, and insulin-II showed slightly higher crossreactivity than insulin-I. The accuracy of this TR-FIA was assured by specificity test, validation test, and recovery test using plasma added insulin-II. The results indicated the high specificity and sufficient accuracy of this assay system for insulin level measurement. This system was applied to the measurement of plasma insulin levels of fed and fasted barfin flounders. Plasma insulin levels (average +/- SEM) in fed flounders reached a maximum 2 h (9.3 +/- 1.7 ng/ml) and decreased gradually thereafter, while those in fasted flounders remained at low levels (1.1 +/- 0.1-2.0 +/- 0.2 ng/ml) during the experiment. After removing proteins by acidification and subsequent gel filtration, plasma samples taken from fed and fasted flounders at 2 h after feeding were fractionated separately by reversed-phase HPLC. In fed flounders, insulin immunoreactivity was detected in fractions corresponding to those of insulin-I or -II. The ratio of integrated insulin immunoreactivities of each peak was 0.378 +/- 0.044 (average +/- SD). This value was in good agreement with those (0.355 +/- 0.019) of absorbance areas of each insulin from Brockmann body extracts of the barfin flounder on reversed-phase HPLC. In fasted flounders, very weak insulin immunoreactivities were observed at retention times corresponding to those of insulin-I and -II. These results indicated that both insulin-I and -II were secreted into the blood being induced by feeding stimulation with approximately the same ratio as that of the quantities harbored in the Brockmann body., ((C)2002 Elsevier Science (USA).)

Mussels, Mytilus galloprovincialis, were contaminated by paralytic shellfish poisoning (PSP) toxins by being fed with the toxic dinoflagellate Alexandrium tamarense. Temporal variations in the toxin content and the profile of mussels during the feeding experiment were monitored by high-performance liquid chromatography (HPLC). The toxin profile of mussels was compared with that of A. tamarense to clarify the mechanism of uptake of toxins in mussels. The prominent toxins in mussels and A. tamarense were N-sulfocarbamoyl toxins (C1,2) and carbamate toxins, gonyautoxin-1,4 (GTX1,4). The toxin profiles of both mussels and A. tamarense were almost constant throughout the experimental period. There were no remarkable differences in the toxin proportion between mussel and A. tamarense. These results indicate that mussels do not selectively accumulate particular toxins.

We examined population genetic structure by means of microsatellite analysis among 7 Japanese flounder (Paralichthys olivaceus) populations collected from coastal sea areas around Japan. As was expected, all of the 11 microsatellite loci examined were variable in all populations (number of alleles per locus, 15.2-18.2; average of expected heterozygosity, 0.74-0.78). Eleven population pairs in 21 possible pairwise comparisons showed significant genetic heterogeneity associated with allele frequency distributions or fixation index (F(ST)). Modified Cavalli-Sforza chord distance (D(A)) and Nei's standard genetic distance (D(ST)) ranged from 0.051 to 0.090, and from 0.000 to 0.025, respectively. There was evidence that the populations assessed in this study were not drawn from a single panmictic population; however, it appears that Japanese flounder populations around Japan are not well-structured, as an estimate of the fixation index value among the 7 localities was very low (F(ST) = 0.0025).

Ninety-six-hour toxicity tests of five organic solvents (methanol, ethanol, acetone, N,N-dimethylformamide [DMF], and dimethyl sulfoxide [DMSO]) on nine species of marine microalgae (Bacillariophyceae; Skeletonema costatum, and Chaetoceros calcitrans, Prasinophyceae; Tetraselmis tetrathele, Haptophyceae; Isochrysis galbana and Pavlova lutheri, Dinophyceae; Prorocentrum minimum, Raphidophyceae; Heterosigma akashiwo, Euglenophyceae; Eutreptiella sp., Chlorophyceae; Dunaliella tertiolecta) were conducted. For D. tertiolecta, T. tetrathele, I. galbana, and P. lutheri, methanol was the least toxic solvent. For S. costatum, C. calcitrans and Eutreptiella sp., DMSO was the least toxic solvent. For Heterosigma akashiwo, DMF was the least toxic solvent. In general, solvent toxicity was ethanol > methanol or acetone > DMF > DMSO. In relation to sensitivity of the microalgae to solvents, D. tertiolecta and T. tetrathele were lower in sensitivity, and Eutreptiella sp. and H. akashiwo were higher in sensitivity than the other species. The no observed effect concentration (NOEC) of acetone, DMF, and DMSO were higher than for ethanol and methanol. Acetone, DMF, and DMSO were suitable for carrier solvents used in toxicity test of water-insoluble chemicals. Excellent regression between logEC(50) and logNOEC was obtained (logNOEC = 1.09 x logEC(50) - 0.84, R(2) = 0.91). This equation indicated that higher toxicity was higher application factor (EC(50)/NOEC) than lower toxicity. All application factors were higher than 0.1.

Allele segregating patterns of microsatellite DNA loci in 5 experimental families of Japanese flounder Paralichthys olivaceus and genotype frequencies for deviation from Hardy-Weinberg expectations (HWE) in natural P. olivaceus populations were studied to assess inheritability. Of the 12 microsatellite loci examined, 1 locus had a possibility of scoring errors of heterozygous individuals caused by unreproducible polymerase chain reaction amplifications of a particular allele. At the remaining 11 loci, almost all of alleles were segregated according to Mendelian transmission, and observed genotype frequencies in natural populations were consistent with HWE. The results demonstrated here would provide useful information supporting the suitability of these microsatellite loci as inheritable P. olivaceus genetic markers.

A field survey was carried out in early spring to investigate the growth physiology and efficiency of cyst formation of Alexandrium tamarense in low water temperatures. A bloom of A. tamarense occurred in a stratified water column, formed by river inflow. The in situ growth rate estimated from daily cell abundance was high, 0.33 divisions day(-1), at 7.5-9 degrees C. New cysts began to be observed during the late growth phase. Maximum cyst flux (600 cysts cm(-2) day(-1)) was observed just after maximum cell abundance occurred. PO(4)-P Chl a(-1) gradually decreased and reached extremely low levels beyond the mid-growth phase of A. tamarense. As sinking cysts were also recognized at that time, it suggests cyst formation may have been induced by depletion of phosphorus source. The incidence of cyst formation (C.I) was 30%. The number of C.I was the same as reported previously for batch cultures under conditions suitable for vegetative growth. These results indicate that A. tamarense grows with considerably higher growth rate and transforms to cysts in high numbers, in low water temperatures in the field.

Okadaic acid (OA) homologues in toxic dinoflagellate Dinophysis fortii, scallops Patinopecten yessoensis and mussels Mytilus galloprovincialis collected at the same site in Mutsu Bay, Japan were determined by high-performance liquid chromatography fluorescence detection (HPLC-FLD) as their 9-anthryldiazomethane (ADAM) derivatives. Prominent toxin in the scallops and the mussels was esterified dinophysistoxin-1 (DTX3) and dinophysistoxin-1 (DTX1), respectively, although only DTX1 was detected in D. fortii. Toxin contents in the mussels were significantly higher than those in the scallops, indicating that mussels have higher potential to accumulate OA homologues than scallops.

Five molecular forms of glucagon and two molecular forms of insulin were characterized from the kaluga sturgeon. Substitutions occurred at two to thirteen internal amino acid residues among the five molecular forms of glucagons, indicating that these glucagons were encoded by five distinct genes. The amino acid sequences of two insulins from the kaluga sturgeon were identical to those of paddlefish insulin-II and Russian sturgeon insulin except that kaluga sturgeon insulin-I had an extension of five residues at the B-chain N-terminus. This is the first demonstration that more than two molecular forms of glucagon have been characterized from a single animal species.

We have isolated and characterized several highly repetitive DNA elements from two species of Chinese bitterlings, Rhodeus atremius suigensis and R. ocellatus ocellatus. They comprise a partly interspersed and partly tandem repetitive family of about 1.0 to 1.3 kb in length. Individual elements showed considerable length variation, but genomic Southern blotting revealed two major length groups. Their restricted presence of these elements among related species and relative copy number differences indicated rapid change of genome structure in this group of fish. The isolated elements may be useful landmarks for further chromosomal studies.

We cloned and sequenced the complete mitochondrial genome of Japanese flounder (Paralichthys olivaceus). A circular 17,090 bp mitochondrial genome from the flounder contains 37 structural genes as in other vertebrates so far reported. This is the first report of the complete mitochondrial sequence from a higher teleostean fish (Acanthopterygii). The organization including gene order is quite similar to that of other teleostean fishes as well as placental mammals. The putative control region of the Japanese flounder mitochondrial genome contains a length variable region of about a 74 bp tandem repeat cluster. As a preliminary study we adopted the maximum likelihood and neighbor-joining inference methods to examine phylogenetic relationships among teleostean and related fishes. Comparisons of amino acid sequences of protein-coding genes and nucleotide sequences of tRNA genes resolved some middle to deep branches among some teleostean fishes. The flounder mitochondrial genome does not show an indication of evolutionary rate difference among teleosts leading to difficulty in phylogenetic analyses, and our data is useful for future evolutionary studies dealing with higher teleostean fishes.

Determination of diarrhetic shellfish-poisoning (DSP) toxins, okadaic acid (OA), dinophysistoxin-1 (DTX1) and pectenotoxin-6 (PTX6) was carried out by liquid chromatography (LC) followed by on-line atmospheric pressure electrospray ionization-mass spectrometric (ESI-MS) detection with a heated capillary interface. Mass spectra of authentic OA, DTXI and PTX6 standards exhibited abundant [M-H] at m/z 803, 817 and 887, respectively. Linearity of peak area obtained by selected-ion monitoring (SIM) for [M-H]- of each toxin was confirmed over a wide range of concentrations from 10 pg to 30 ng. LC-ESI-MS analysis of OA, DTX1 and PTX6 in scallops and mussels, collected at the same site (Mutsu Bay, Japan), was carried out. Scallops and mussels collected at the same site showed different toxin profiles. Although PTX6 was detected from scallops, it was not detected from mussels.

Two forms of vitellogenin (Vg), Vg A and Vg B, were identified in serum from estrogen-treated barfin flounder (Verasper moseri). Structural changes of lipovitellins (Lvs) derived from the two Vgs were examined during vitellogenesis and oocyte maturation. Two Lvs, vLv A and vLv B, were identified electrophoretically and immunologically in postvitellogenic oocytes. Each appeared to be composed of distinct heavy chains (vLvH A, M(r) 107,000, and vLvH B, M(r) 94,000) and light chains (vLvL A, M(r) 30,000, and vLvL B, M(r) 28,000) when analyzed by SDS-PAGE. Results from N-terminal amino acid sequencing and Western blotting using antisera to vLvH A and vLvH B verified that there are two Vg polypeptides in serum from estrogen-treated fish, Vg A (M(r) 168,000) and Vg B (M(r) 175,000), which give rise to vLvH A-vLvL A and vLvH B-vLvL B, respectively. N-terminal sequencing revealed two sequences for both phosvitin and beta'-component, supporting the concept of duality for all three classes of Vg-derived yolk proteins. During oocyte maturation, native dimeric vLv B was dissociated into a native M(r) 170,000 monomer (oLv B). Meanwhile, vLv A was extensively cleaved including complete degradation of vLvH A into free amino acids. We propose that the quantitative ratio of vLv A to vLv B in postvitellogenic oocytes regulates the buoyancy of the spawned pelagic eggs by controlling availability of free amino acids which function as osmotic effectors during oocyte hydration. The vLv A/vLv B ratio likely also controls the proportional availability of different types of nutrients, free amino acids versus Lv, for use during embryonic development., (Copyright 1999 Academic Press.)