Barfin Flounder Research Articles

Evaluation of the Draft Australian and New Zealand Standard Diagnostic Procedure (ANZSDP) for PCR-detection of Betanodaviruses

Several Australian betanodaviruses (NNVs) and other, exotic NNVs were used in a study of the performance of a nested PCR test in common use in Australia and New Zealand. This test for the detection of NNVs, designated the ANZSDP VNN Nested PCR, was compared with a currently available commercial PCR kit for NNV detection. With respect to the endemic viruses, the ANZSDP was pan-specific for the NNVs tested, while the commercial PCR kit failed to detect the South Australian strain of barramundi Lates calcarifer NNV. With respect to the relative sensitivities of the two tests, in general, the ANZSDP appeared more sensitive than the commercial test. The exception was the Australian bass Macquaria novemaculeata NNV isolate, which was detected at lower levels by the commercial test. Among the exotic NNVs, both tests detected red-spotted grouper nervous necrosis virus and barfin flounder nervous necrosis virus, but neither test detected striped jack nervous necrosis virus (SJNNV). Further investigation revealed mismatches between SJNNV sequences and the nested primers used in the ANZSDP.

Profiles of α-melanocyte-stimulating hormone in the Japanese flounder as revealed by a newly developed time-resolved fluoroimmunoassay and immunohistochemistry

Profiles of α-melanocyte-stimulating hormone (α-MSH) in the Japanese flounder were examined by a newly developed time-resolved fluoroimmunoassay (TR-FIA) and immunohistochemistry. A TR-FIA for α-MSH was newly developed, and its levels in the pituitary gland and plasma of Japanese flounder reared in a white or black tank for 5 months were compared. A competitive assay using two antibodies was performed among secondary antibodies in the solid phase, α-MSH antibodies, samples, and europium-labeled Des-Ac-α-MSH. The sensitivity of the assay, defined as twice the standard deviation at a zero dose, was 0.98 ng/ml (49 pg/well). The intra- and interassay coefficients of variation of the assay were 8.8% ( n = 8) and 17.3% ( n = 5), respectively, at about 50% binding. Cross-reactivities of Des-Ac-α-MSH and Di-Ac-α-MSH were about 100%. Cross-reactivities of adrenocorticotropic hormone, salmon gonadotropin-releasing hormone (sGnRH), and chicken GnRH-II were less than 0.2%, and that of melanin-concentrating hormone was less than 2.0% at 50% binding. Displacement curves of serially twofold-diluted hypothalamus extract, pituitary gland extract, and plasma extract of Japanese flounder with the assay buffer were parallel to the α-MSH standard curve. Moreover, displacement curves of serially twofold-diluted hypothalamus and/or pituitary gland extract of masu salmon, goldfish, red seabream, Japanese eel, tiger puffer, and barfin flounder with the assay buffer were also parallel to the α-MSH standard. In Japanese flounder, total immunoreactive (ir)-α-MSH levels in the pituitary gland were lower in the black tank, whereas those in the plasma tended to be higher in the black tank, suggesting that the synthesis and release of α-MSH are higher in the black tank. α-MSH-ir cells were detected in the pars intermedia and a small part of the pars distalis of the pituitary gland. α-MSH-ir cell bodies were located in the basal hypothalamus and α-MSH-ir fibers were distributed not only in the hypothalamus but also in the telencephalon, midbrain, cerebellum, and medulla oblongata, suggesting that α-MSH functions as a neuromodulator in the brain.

Identification of Two Forms of Vitellogenin-derived Phosvitin and Elucidation of Their Fate and Roles During Oocyte Maturation in the Barfin Flounder, Verasper moseri

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.

Detection of DNAs homologous to betanodavirus genome RNAs in barfin flounder Verasper moseri and Japanese flounderParalichthys olivaceus

I investigated the presence of DNA homologous to genome RNA1 and RNA2 (RNA1 DNA and RNA2 DNA) of betanodaviruses - the causative agent of viral nervous necrosis (VNN) --in eggs, sperm, ovarian cavity fluid, larvae, and juveniles of barfin flounder Verasper moseri and larvae and juveniles of Japanese flounder Paralichthys olivaceus collected at 6 sites in Hokkaido, Japan, from 1994 to 2001. RNA1 DNA and RNA2 DNA were detected by PCR in 13 and 33 % of barfin flounder samples and 0 and 69% of Japanese flounder samples, respectively. No infectious virus was detected by cell culture or by successive immunoblot against coat protein (genome RNA2 product) using an E-11 cell line, except for a virus present in 1 dead fish collected during an outbreak of VNN in 1995. Nucleotide sequence analysis showed that RNA1 DNA had a 82 to 96 % similarity to betanodavirus genome RNA1, and that RNA2 DNA had a 69 to 98 % similarity to RNA2. The detection rate of RNA2 DNA after intraperitoneal injection of betanodavirus strain HCF-1 into larvae and juveniles of the 2 flounder species was higher in samples from surviving fish than in the uninfected controls, whereas the detection rate of RNA1 DNA did not show a clear trend. Infectious virus was only detected in samples from fish that died subsequent to injection. Transfection assays of the viral genome RNAs into the barfin flounder cell line MK-1 and Japanese flounder cell lines H-1 and H-2 resulted in production of RNA2 DNA in all 3 cell lines. Quantitative measurement by ELISA revealed reverse transcriptase (RTase) activity. These results suggest that the DNA forms are produced and persist in the 2 flounder species as both clinical and subclinical infections, and do not lead to virion production.

Genetic population evaluation of two closely related flatfish species, the rare barfin flounder and spotted halibut, along the Japanese coast

Barfin flounder and spotted halibut have been selected as target species for stock enhancement in Japan. Understanding the genetic condition of the wild stock is a principal requirement in any stock enhancement program. The genetic variability of barfin flounder and spotted halibut, and the population structure of spotted halibut were evaluated using microsatellite DNA markers (msDNA) and the control region of the mitocondrial DNA (mtDNA). Barfin flounder and spotted halibut showed high genetic variability at the msDNA level. Barfin flounder A was 16.7 and H e was 0.860; spotted halibut A n ranged from 7.7 to 10.2 and H e ranged from 0.710 to 0.774. At the mtDNA level, high haplotype (h=0.922) and low nucleotide (π=0.002) diversities were observed for barfin flounder; however, low haplotype and nucleotide diversities (h=0.603–0.620 and π=0.001–0.002), and very low haplotype and nucleotide diversities (h=0.193 and π=0.0003) were observed for spotted halibut in the north and south locations, respectively. Slight genetic differentiation among spotted halibut sampling locations was observed from the msDNA. MtDNA analyses showed genetic differentiation between north and south locations, but not within them. The designation of north-specific and south-specific management units in the future stock enhancement activities of spotted halibut is recommended.

Aquaculture performance of triploid barfin flounder Verasper moseri

To evaluate the aquaculture performance of triploid barfin flounder Verasper moseri, the sex ratio, maturation, growth and the relative proportion of body parts were examined. The sex ratio of triploids was similar to diploids under communal rearing conditions, but the proportion of female diploids was higher than that of triploids under separate rearing conditions. The gonadosomatic index of triploid females was very low even during the spawning season, and the ovaries were rudimentary. These results suggest that triploid barfin flounder females were sterile. In addition, triploid males produced a small quantity of milt containing very few spermatozoa with abnormal shapes. Spermatozoa obtained from triploids were aneuploidies. When normal eggs were fertilized with sperm from triploid males, no fry developed. These results suggest that triploid barfin flounder males were functionally sterile. Triploid males grew more slowly than diploid males, and triploid females showed similar or shower growth than diploid females, whether reared separately (23 months) or communally (35 months). The ratios of visceral weight to the edible parts for triploid males were similar to those for diploid males, but ratios for triploid females were higher than for diploid females during the spawning period. In conclusion, a significant improvement of growth was not found in triploid barfin flounders.

Genetic evaluation of interindividual relatedness for broodstock management of the rare species barfin flounder Verasper moseri using microsatellite DNA markers

The barfin flounder has been recently declared to be rare, as the number of wild individuals in Japan has greatly reduced since 1975. The Japanese National Center for Stock Enhancement started the stocking program for this species on 1987. In the present study, microsatellite DNA loci were applied in order to determine the pedigree of the hatchery-produced juveniles for stock enhancement. Additionally, using six polymorphic microsatellite markers, the accuracy to assign dyads to categories of kinship was estimated using the relatedness estimator (R xy ), similarity indexes (individual genetic identity [GI] and proportion of shared alleles [P s ], identification of kin groups using unweighted pair group method with arithmetic mean (UPGMA) clustering based on interindividual genetic distances (1-R xy, 1-P s, and 1-Gl), a likelihood ratio approach, and partition methods to separate individuals into cohorts. The results were compared with the pedigree relationship previously obtained from parentage analysis. Estimation of the kinship relationship between individuals could be better inferred by means of UPGMA dendrogram clustering based on 1-Gl or 1-P s as interindividual genetic distances, or using computer software to perform partitioning of set of individuals.

希少種マツカワの遺伝的多様性の維持に向けた交配技術

希少種マツカワの遺伝的多様性の維持に向けた交配技術

マツカワの種苗生産技術に関する研究

マツカワの種苗生産技術に関する研究

Expression of three proopiomelanocortin subtype genes and mass spectrometric identification of POMC-derived peptides in pars distalis and pars intermedia of barfin flounder pituitary

Proopiomelanocortin (POMC) is a common precursor of adrenocorticotropic hormone (ACTH), melanophore-stimulating hormone (MSH), and endorphin (END). In pituitary gland, POMC receives posttranslational processing by which different peptides are generated in the pars distalis (PD) and pars intermedia (PI). Recently, we cloned three subtypes of the POMC gene in pituitary gland of barfin flounder. The present study was undertaken to elucidate whether the three POMC genes are expressed in both the PD and PI of barfin flounder pituitary, and to identify peptides derived from POMCs in these lobes. We amplified the transcripts of POMC-A, -B and -C in both the PD and PI by the reverse transcription-polymerase chain reaction. In situ hybridization also detected signals for these three subtypes in the PD and PI. These results demonstrated that all three POMC genes are expressed in both the PD and PI of barfin flounder pituitary. By mass spectrometric analyses, ACTH-A, Des-acetyl (Ac)-α-MSH-A/B (amino acid sequence of α-MSH-A is identical to that of α-MSH-B), β-MSH-A, corticotropin-like intermediate lobe peptide (CLIP)-A, and N-terminal peptide ( N-POMC)-A were identified in the PD. Moreover, Des-Ac-α-MSH-A/B, α-MSH-A/B, β-MSH-A and -B, N-β-lipotropin-A, CLIP-A, N-Ac-β-END-A 1–41 (C-terminally truncated form of N-Ac-β-END-A), and N-POMC-A were identified in the PI. Predominant detection of POMC-A-derived peptides indicates the greatest production of POMC-A and no detection of POMC-C-derived peptides indicates the lowest production of POMC-C in both the PD and PI. ACTH-A is specifically produced in the PD, however, the occurrence of Des-Ac-α-MSH-A, CLIP-A, and β-MSH-A shows that the entire POMC-A is further cleaved into small peptides as in the PI. In the PI, some peptides receive modification or truncation as shown by the occurrence of α-MSH-A/B and N-Ac-β-END-A 1–41. These results show differential posttranslational processing of POMC between the PD and PI in barfin flounder pituitary.

Monitoring of the genetic variability of the hatchery and recaptured fish in the stock enhancement program of the rare species barfin flounder Verasper moseri

In relation to the stock enhancement program for barfin flounder, hatchery juveniles produced in 2001 were genotyped using microsatellite DNA markers (msDNA) and then released to natural waters. Subsequently, recaptured individuals, designated as ‘tentative recaptured’, were examined using msDNA. In order to evaluate the effectiveness of the stock enhancement program, pairwise F ST and genic differentiation tests were used to estimate the genetic divergence between the wild samples, the hatchery broodstock, and the tentative recaptured samples. Analysis showed significant differentiation among these three groups. Pedigree determination by msDNA was used to establish the origin of the tentative recaptured individuals, in order to elucidate whether they were hatchery produced, ‘real recaptured’, or wild specimens. Wild individuals were not found. The effective population size of the real recaptured stock was very low (N e=16.6). Equal family survivability was observed between the released and real recaptured stocks, indicating that the genetic variability of the released stock was maintained in the natural environment. Future broodstock management, breeding designs, and family contribution equalization of the offspring to be released will be required in this rare species to avoid unintended genetic differentiation between the wild population and the hatchery broodstock.

Development of Non-Radioisotopic Immunoassay Systems for Measuring Flounder IGF-I

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.

Effects of tank color on melanin-concentrating hormone levels in the brain, pituitary gland, and plasma of the barfin flounder as revealed by a newly developed time-resolved fluoroimmunoassay

A pleuronectiform fish, the barfin flounder Verasper moseri, reared in a white tank had a smaller ratio of pigmented area of the skin on non-eyed side, grew faster, and had greater melanin-concentrating hormone (MCH)-immunoreactive cell bodies and MCH gene expression in the brain than in the black tank, indicating that synthesis and release of MCH are higher in fish from a white tank. In the present study, a time-resolved fluoroimmunoassay for MCH was developed. MCH levels were assessed in the brain, pituitary gland, and plasma of barfin flounders reared in a white or black tank. A competitive assay using two antibodies was performed among secondary antibodies in the solid phase, MCH antibodies, samples, and europium-labeled MCH. Displacement curves of serially diluted extracts (brain, pituitary gland, and plasma) of the barfin flounder paralleled that of the MCH standard. MCH levels in the brain and plasma were higher in fish reared in the white tank for 5 months than in the black tank. These results suggest that synthesis and secretion of MCH are enhanced with the white background and that MCH is involved in both somatic growth and the skin pigmentation in the barfin flounder.

Nucleotide sequence and expression of three subtypes of proopiomelanocortin mRNA in barfin flounder

Melanophore-stimulating hormone (MSH) has been shown to be associated with food intake in addition to body color change in teleosts. MSH is encoded by a proopiomelanocortin (POMC) gene together with endorphin (END). To assess the significance of MSH to biological activities, we determined the structure and evaluated the expression of POMC mRNA in barfin flounder (bf), Verasper moseri, a member of a group of teleosts, Pleuronectiformes. Three subtypes of POMC cDNAs (A, B, and C) were amplified from bf pituitary glands. These bfPOMCs contained segments for N-POMC, α-MSH, β-MSH, and β-END as do other teleost POMCs, while POMC-C showed remarkable variations in the segments corresponding to N-POMC and β-END. A phylogenetic tree of ray-finned fish POMCs constructed by the neighbor joining method revealed that the three POMC subtypes may have appeared as a result of duplication events occurring at least twice during the course of bf evolution. The first duplication may have generated the lineage leading to an ancestor of bfPOMC-A and -B and that leading to bfPOMC-C, and then the lineage of bfPOMC-A may have diverged from that of bfPOMC-B. All peptides flanked by processing signals excluding N-POMC-C (1–14) were identified in a single pituitary extract by mass spectrometry, and the cDNAs of three POMCs were amplified from a single pituitary by reverse transcription polymerase chain reaction. These results demonstrated that the three POMC genes are expressed in a single individual. While the bfPOMC-A gene was exclusively expressed in the pituitary, the bfPOMC-B and -C genes were expressed in non-pituitary tissues such as brain, gill, heart, spleen, liver, stomach, intestine, testis, muscle, blood, and skin in addition to the pituitary. The expression levels of the POMC-A, -B, and -C genes in pituitary neurointermediate lobe were greater in the fish reared with a black background than the fish reared with a white background, indicating that MSH derived from all of the three bfPOMC genes was associated with body color change. No difference was observed in the expression levels of bfPOMC-C in the brain in response to feeding status.

Immunocytochemical localization and ontogenic development of ?-melanocyte-stimulating hormone (?-MSH) in the brain of a pleuronectiform fish, barfin flounder

Alpha-melanocyte-stimulating hormone (alpha-MSH) is a pituitary hormone derived by post-translational processing from proopiomelanocortin and is involved in background adaptation in teleost fish. It has also been reported to suppress food intake in mammals. Here, we examined the immunocytochemical localization of alpha-MSH in the brain and pituitary of a pleuronectiform fish, the barfin flounder (Verasper moseri), as a first step in unraveling the possible function of alpha-MSH in the brain. The ontogenic development of the alpha-MSH system was also studied. In the pituitary, alpha-MSH-immunoreactive (ir) cells were preferentially detected in the pars intermedia. In the brain, alpha-MSH-ir neuronal somata were located in the nucleus tuberis lateralis of the basal hypothalamus, and alpha-MSH-ir fibers were located mainly in the telencephalon, hypothalamus, and midbrain. Alpha-MSH-ir neuronal somata did not project their axons to the pituitary. The alpha-MSH-ir neurons differed from those immunoreactive to melanin-concentrating hormone. Alpha-MSH cells in the pituitary and alpha-MSH-ir neuronal somata in the brain were first detected 1 day and 5 days after hatching, respectively. The distribution of alpha-MSH-ir cells, neuronal somata, and fibers showed a pattern similar to that in adult fish 30 days after hatching. These results indicate that the functions of alpha-MSH in the brain and pituitary are different and that alpha-MSH plays physiological roles in the early development of the barfin flounder.

White background reduces the occurrence of staining, activates melanin-concentrating hormone and promotes somatic growth in barfin flounder

A pleuronectiform fish, the barfin flounder, Verasper moseri, is promising for aquaculture and resource enhancement in Northern Japan due to its high commercial value. A major problem in the aquaculture is staining (pigmentation of the skin on non-eyed side) under culture conditions since it diminishes the commercial value of the fish. Melanin-concentrating hormone (MCH) is a peptide hormone produced in the hypothalamus which turns the body of teleosts pale. Here, we examined the employment of MCH to prevent staining. Bolus injection of MCH (1 μg/g body weight (BW)) in barfin flounder turned the stained area pale. The occurrence of staining was suppressed by injection of MCH (1 μg/g BW, once a week, total 10 times). These results indicated that MCH affected the non-eyed side color in addition to the physiological color change on the eyed side. Then, we prepared juvenile fish having no staining by rearing with a tank containing Microceramic on the bottom and transferred them to white, yellow, or black tanks without Microceramic. Eight months later, the percent area of each fish that is stained in white or yellow tanks was significantly smaller than that of fish reared in a black tank. The number of MCH-immunoreactive cell bodies in the brain was significantly greater in the white tank fish than that of the black tank fish, suggesting that white background enhanced the secretion of endogenous MCH, by which occurrence of staining was prevented. Interestingly, both the total length and body weight of the white-reared fish were greater than those of the black-reared fish, respectively. The enhanced production of MCH might augment the food intake of barfin flounders as in mammals, so that the somatic growth of the fish is promoted.

Production of monoclonal antibodies against grouper nervous necrosis virus (GNNV) and development of an antigen capture ELISA

Five (2 IgG, 3 IgM) monoclonal antibodies (MAbs) against the G9508KS strain of grouper nervous necrosis virus (GNNV) were produced and characterized. All 5 MAbs showed positive signals in the retina of GNNV-infected grouper larvae and in the cytoplasm of GNNV-infected GF-1 cells using immunohistochemistry staining. Two MAbs reacted with the denatured capsid protein derived from GNNV-infected GF-1 cells in Western blot analysis, but did not react with the GNNV recombinant capsid protein expressed by E. coli in an indirect immnunosorbent assay (ELISA). All 5 MAbs were able to neutralize GNNV, tiger puffer NNV (TPNNV) and barfin flounder NNV (BFNNV), while only 2 of the MAbs neutralized striped jack NNV (SJNNV). A capture ELISA system based on the use of MAbs for capture and a rabbit polyclonal antibody for detection was developed. When absorbance values higher than 0.5 were judged to be positive, the sensitivity of the capture ELISA system was 2.5 ng per well of purified GNNV protein or 6.5 x 10(4) TCID50 per well of GNNV supernatant from culture cells. This capture ELISA system could become a more specific and sensitive tool for NNV diagnosis in the field and in routine laboratories.

Effects of photoperiod on gonadotropin-releasing hormone levels in the brain and pituitary of underyearling male barfin flounder

: A pleuronectiform fish, the barfin flounder Verasper moseri, expresses three gonadotropin-releasing hormone (GnRH) forms in the brain: salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II) and seabream GnRH (sbGnRH). To clarify the effects of photoperiod on GnRH systems, changes in brain and pituitary GnRH peptide levels were examined using time-resolved fluoroimmunoassays. In experiment 1, 5-month-old male barfin flounder (mean total length 9.0 cm, body weight 11.0 g) were divided into short (8:16 h light : dark [L:D] cycle; lights on 08.00–16.00 hours) and long photoperiod (16:8 h L:D cycle; lights on 04.00–20.00 hours) groups in mid September and maintained until November under natural water temperature (19.3–15.2°C). Brain sGnRH concentrations were significantly higher in the 16:8 h L:D group than in the 8:16 h L:D group, whereas no significant differences were observed in total length, body weight, plasma testosterone concentration, brain cGnRH-II concentration and pituitary sbGnRH content. In experiment 2, 7-month-old male barfin flounder (mean total length 16.5 cm, body weight 76.8 g) were divided into short and long photoperiod groups in mid December and maintained until February under natural water temperature (12.5–6.6°C). Total length, body weight and condition factor were significantly greater in the 16:8 h L:D group than in the 8:16 h L:D group, whereas no significant differences were observed in plasma testosterone concentration and GnRH levels in the brain and pituitary. These results indicate that levels of sGnRH in barfin flounder are influenced by photoperiodic treatment dependent on water temperature and/or body size.

Genomic classification of new betanodavirus isolates by phylogenetic analysis of the coat protein gene suggests a low host-fish species specificity.

Viral encephalopathy and retinopathy is a devastating disease that causes neurological disorders and high mortality in a large number of cultivated marine fish species around the world. It is now established that several viral strains classified in the genus Betanodavirus of the family Nodaviridae are the aetiological agents of this disease. Betanodaviruses can be classified into four genotypes based on the coat protein gene sequence. Here, the coat protein genes of the three major strains isolated from sea bass (Dicentrarchus labrax) in France were found to be different. In addition, 21 novel strains of betanodavirus from several fish species from France, Spain, Tunisia and Tahiti were classified by using phylogenetic analysis of a partial sequence (383 nt) of the coat protein gene. Most of the isolates were grouped in the red-spotted grouper nervous necrosis virus type, which was subdivided into two subtypes, one of them containing only French isolates. Furthermore, an isolate obtained from sea bass during an outbreak at low temperature (15 degrees C) was classified as the barfin flounder nervous necrosis virus type. This is the first reported isolation from sea bass of such a strain, which is known to infect several cold-water marine fish species. In addition, a betanodavirus belonging to the striped jack nervous necrosis virus type was detected in Senagalese sole (Solea senegalensis) farmed in Spain, which is the first indication of the presence of this genotype outside Japan. These findings suggest that the different genotypes can infect a variety of fish species and thus have a low host-fish species specificity.

Possible involvement of melanin-concentrating hormone in food intake in a teleost fish, barfin flounder

We investigated the involvement of MCH in food intake in barfin flounder. The structure of barfin flounder MCH was determined by cDNA cloning and mass spectrometry. In fasted fish, the MCH gene expression and the number of MCH neurons in the brain were greater than controls. In white-reared fish, the MCH gene expression and the number of MCH neurons in the brain were greater than black-reared fish. Furthermore, white-reared fish grew faster than black-reared fish. These results indicate that a white background stimulated production of MCH and MCH, in turn, enhanced body growth, probably by stimulating food intake.