Barfin Flounder Research Articles

Participating in VER-IPT: is it worth the effort?

Interlaboratory proficiency tests (IPTs) are an essential tool to maintain quality standards and performances of diagnostic laboratories at a high level. In 2022 the VER IPT reached its 4th edition. The panel consisted of 10 ampoules that contained all known genotypes of Betanodavirus, the causative agent of Viral Encephalopathy and Retinopathy. Detecting the presence or absence of the target in the samples by real time or conventional RT-PCR was the main target of the IPT. Species identification to be performed by sequencing or by any other method available at the participant’s laboratory was also encouraged. Twenty-four laboratories out of 25 participants completed the exercise on time. Thirteen out of 24 (54.16% of participants) obtained the maximum score, while the remaining 11 produced a percentage of correct results ranging between 70%-90%, achieving an overall good outcome. Notably, in this IPT edition the BFNNV (barfin flounder nervous necrosis virus) was included for the first time and did not pose any particular problems in its detection. Viral species identification gave results far below the expectations and only 50% of the participants completed this part of the exercise, scoring quite well; however, laboratories should make greater efforts in improving their capacity to correctly identify the genotype.

Emergence of Reassortment between a New and Reported Types of Betanodavirus in Shellfish.

Recently, three types of betanodavirus including red spotted grouper nervous necrosis virus (RGNNV), barfin flounder nervous necrosis virus (BFNNV), and Korean shellfish nervous necrosis virus (KSNNV) (proposed as a new fifth type) have been detected in shellfish in the marine environment around Korea. To investigate the presence of reassortment between betanodavirus types, the type based on the RNA2 segment of betanodaviruses carried in 420 domestic shellfish (n = 306) and finfish (n = 35), as well as imported shellfish (n = 79), was compared with the type identified by reverse-transcriptase polymerase chain reaction (RT-PCR) for RNA1 segment. Only five samples carrying reassortant betanodaviruses were found, appearing as RG/KSNNV (n = 2), KS/RGNNV (n = 1), and SJ/RGNNV (n = 2) types. From these samples, we successfully isolated two reassortant strains from Korean and Chinese shellfish in E-11 cells and called them KG1-reKS/RG and CM1-reRG/KS, respectively. In the full genome sequences, each RNA segment of the reassortant strains exhibited the same gene length and high sequence homology (≥98%) with the reference strains corresponding to the type of each segment. Both these reassortant strains induced high mortality to sevenband grouper (Epinephelus septemfasciatus) larvae with high viral concentrations in the body (109 viral particles/mg) and severe vacuolation in the retina and brain. These are the first results showing the involvement of the KSNNV type in the reassortment of RNA segments in the reported types of betanodavirus, which could represent a new potential risk in fish.

Morphological and Molecular Phylogenetic Analyses of an Ectoparasitic Trichodinid Ciliate, <i>Trichodina hokkaidoensis</i> n. sp., Infecting Artificially Reared Barfin Flounder <i>Verasper moseri</i>

Infections by ectoparasitic trichodinid ciliates cause epidermal damages to artificially reared juvenile barfin flounder Verasper moseri of mariculture fisheries in Hokkaido, northern Japan. ​In the present study, morphological observation and molecular phylogenetic analysis using the small subunit ribosomal RNA gene (SSU rDNA) were performed to identify the trichodinid ciliates. ​Morphological analysis of denticles and adoral ciliary spiral demonstrated that the trichodinid ciliate belongs to the genus Trichodina within ten genera of Trichodinidae. ​Denticles of the trichodinid ciliate were clearly distinguishable from those of other Trichodina species by small denticle span, and extensive elongation of the central conical part and anterior ray apophysis. ​All the isolated ciliate had the same 1,699 base pair SSU rDNA nucleotide sequences, which revealed that the trichodinid ciliates comprised a single species. ​The SSU rDNA of the trichodinid ciliate showed the highest homology with that of T. pectenis (98.5%) among 30 species of trichodinid ciliates, and the two species formed a single clade in the phylogenetic tree. ​However, no identical sequence with the present trichodinid ciliate was found in the database. ​We propose Trichodina hokkaidoensis n. sp. for the ciliate infecting artificially reared juvenile barfin flounder, based on both morphological and molecular phylogenetic analyses.

Effect of Blood Removal and Aging at Low Temperature on Quality of Meat of Matsukawa(Verasper moseri)

Effect of Blood Removal and Aging at Low Temperature on Quality of Meat of Matsukawa(<i>Verasper moseri</i>)

Daily otolith ring formation and the relationship between water temperature and the onset of ring formation of barfin flounder Verasper moseri larvae and juveniles

Daily otolith ring formation and the relationship between water temperature and the onset of ring formation of barfin flounder <i>Verasper moseri</i> larvae and juveniles

Amino acidic substitutions in the polymerase N-terminal region of a reassortant betanodavirus strain causing poor adaptation to temperature increase

Nervous necrosis virus (NNV), Genus Betanodavirus, is the causative agent of viral encephalopathy and retinopathy (VER), a neuropathological disease that causes fish mortalities worldwide. The NNV genome is composed of two single-stranded RNA molecules, RNA1 and RNA2, encoding the RNA polymerase and the coat protein, respectively. Betanodaviruses are classified into four genotypes: red-spotted grouper nervous necrosis virus (RGNNV), striped jack nervous necrosis virus (SJNNV), barfin flounder nervous necrosis virus (BFNNV) and tiger puffer nervous necrosis virus (TPNNV). In Southern Europe the presence of RGNNV, SJNNV and their natural reassortants (in both RNA1/RNA2 forms: RGNNV/SJNNV and SJNNV/RGNNV) has been reported. Pathology caused by these genotypes is closely linked to water temperature and the RNA1 segment encoding amino acids 1–445 has been postulated to regulate viral adaptation to temperature. Reassortants isolated from sole (RGNNV/SJNNV) show 6 substitutions in this region when compared with the RGNNV genotype (positions 41, 48, 218, 223, 238 and 289). We have demonstrated that change of these positions to those present in the RGNNV genotype cause low and delayed replication in vitro when compared with that of the wild type strain at 25 and 30 °C. The experimental infections confirmed the impact of the mutations on viral replication because at 25 °C the viral load and the mortality were significantly lower in fish infected with the mutant than in those challenged with the non-mutated virus. It was not possible to challenge fish at 30 °C because of the scarce tolerance of sole to this temperature.

Effects of green light on the growth of spotted halibut, Verasper variegatus, and Japanese flounder, Paralichthys olivaceus, and on the endocrine system of spotted halibut at different water temperatures

We have previously shown that the somatic growth of barfin flounder, Verasper moseri, was promoted by green light. The present study was undertaken to elucidate whether growth-promoting effect of green light can be observed in other flatfishes and to understand the roles of endocrine systems in green light-induced growth. Herein, we demonstrated facilitation of growth by green light in the spotted halibut, Verasper variegatus, and Japanese flounder, Paralichthys olivaceus. Blue and blue-green light showed potencies that were similar to that of green light, while the potencies of red and white light were equivalent to that of ambient light (control). We also examined the effects of green light on growth and endocrine systems of V. variegatus at various water temperatures. Growth of the fish was facilitated by green light at four different water temperatures examined; the fish were reared for 31 days at 12 and 21 °C, and 30 days at 15 and 18 °C. Increase in condition factor was observed at 15 and 18 °C. Among the genes encoding hypothalamic hormones, expression levels of melanin-concentrating hormone 1 (mch1) were enhanced by green light at the four water temperatures. Expression levels of other genes including mch2 increased at certain water temperatures. No difference was observed in the expression levels of pituitary hormone genes, including those of growth hormone and members of proopiomelanocortin family, and in plasma levels of members of the insulin family. The results suggest that green light may generally stimulate growth of flatfishes. Moreover, it is conceivable that MCH, production of which is stimulated by green light, is a key hormone; it augments food intake, which is intimately coupled with somatic growth.

A microplate incubation method for assessing egg quality of the barfin flounder: effects of well size and rearing medium on larval viability

In microplate incubation, fish eggs and larvae are individually reared in small wells. This technique is useful for assessing hatchability, larval survival, and larval abnormality. Nevertheless, the optimal well size (water volume) and rearing media required to ensure full larval viability vary among species. In this study, we examined the effects of well size and rearing medium on hatchability and larval viability of the barfin flounder Verasper moseri and attempted to optimize the incubation conditions for this species. Fertilized eggs were individually stocked in 24-, 48-, and 96-well plates filled with 2, 1, and 0.25 mL seawater containing antibiotics (penicillin G potassium and streptomycin sulfate) and bovine serum albumin (BSA), both of which are required in the rearing media for other species. The eggs were incubated without replacing the medium. Hatch timing and rate were similar in all plate types. Completion of yolk resorption was delayed only in the 96-well plates. In the 24- and 48-well plates, the larvae survived more than 2 weeks after complete yolk resorption until they were starved to death. When the antibiotics were removed from the media, larval mortality increased. In contrast, removal of BSA from the media did not increase yolk sac stage larval mortality for barfin flounder unlike that reported for other species. These results indicate that the use of 24- and 48-well microplates filled with seawater containing antibiotics is optimal for barfin flounder larval viability beyond the completion of yolk resorption.

Protein A from orange-spotted grouper nervous necrosis virus triggers type I interferon production in fish cell

Family Nodaviridae consists of two genera: Alphanodavirus and Betanodavirus, and the latter is classified into four genotypes, including red-spotted grouper nervous necrosis virus, tiger puffer nervous necrosis virus, striped jack nervous necrosis virus, and barfin flounder nervous necrosis virus. Type I interferons (IFNs) play a central role in the innate immune system and antiviral responses, and the interactions between IFN and NNV have been investigated in this study. We have found that the RNA-dependent RNA polymerase (RdRp) from orange-spotted nervous necrosis virus (OGNNV), named protein A, was capable of activating IFN promoter in fathead minnow (FHM) cells. Transient expression of protein A was found to induce IFN expression and secretion, endowing FHM cells with anti-tiger frog virus ability. Protein A from SJNNV can also induce IFN expression in FHM cells but that from Flock House virus (FHV), a well-studied representative species of genus Alphanodavirus, cannot. RdRp activity and mitochondrial localization were shown to be required for protein A to induce IFN expression by means of activating IRF3 but not NFκB. Furthermore, DsRNA synthesized in vitro transcription and poly I:C activated IFN promoter activity when transfected into FHM cells, and dsRNA were also detected in NNV-infected cells. We postulated that dsRNA, a PAMP, was produced by protein A, leading to activation of innate immune response. These results suggest that protein As from NNV are the agonists of innate immune response. This is the first work to demonstrate the interaction between NNV protein A and innate immune system, and may help to understand pathogenesis of NNV.

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

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

Lack of in vivo cross-protection of two different betanodavirus species RGNNV and SJNNV in European sea bass Dicentrachus labrax

Viral encephalopathy and retinopathy (VER) is a severe infective disease characterized by neuropathological changes in several fish species associated with high mortality. The etiological agent is a virus belonging to the Nodaviridae family, genus Betanodavirus. To date, four different betanodavirus species have been officially recognized by International Committee on Taxonomy of Viruses (ICTV), namely the red-spotted grouper- (RGNNV), the striped jack- (SJNNV), the barfin flounder- (BFNNV) and the tiger puffer nervous necrosis virus (TPNNV). Moreover, two reassortants RGNNV/SJNNV and SJNNV/RGNNV have been described.Betanodaviruses can be classified into three different serotypes (A, B and C) that are antigenically different, so none (between serotype A and C) or partial (between serotype B and C) cross-immunoreactivity has been detected in vitro.In this study we investigated the in vivo cross-protection of the two main betanodavirus species (RGNNV and SJNNV), which belong to distinct serotype, by immunizing intraperitoneally (IP) juvenile sea bass with formalin inactivated RGNNV and SJNNV vaccines, followed by a challenge with RGNNV.Fish IP vaccinated with inactivated RGNNV showed a high protection value (85%). Serological analyses highlighted a great specific anti-NNV immunoglobulin M (IgM) production against the homologous virus, while a good seroconversion with low neutralization property was highlighted against the heterologous virus. In fish IP vaccinated with inactivated SJNNV the protection recorded was equal to 25%, significantly lower respect to the one provided by RGNNV IP vaccine. ELISA test detected good IgM production against the homologous virus, and a lower, but still detectable IgM production against the heterologous one. By contrast, serum neutralization test highlighted a poorly detectable antibody production unable to neutralize either the homologous or the heterologous virus.These results confirm that the two serotypes are not cross-protective in vivo. According to these findings, the production of multivalent formulation, or at least the provision of different types of vaccines based on both fish and virus species requirement, should be recommended in order to broaden the range of protection.

High prevalence of betanodavirus barfin flounder nervous necrosis virus as well as red-spotted grouper nervous necrosis virus genotype in shellfish.

Using two serially executed PCRs, the discriminative multiplex two-step RT-PCR (DMT-2 RT-PCR) following the detection seminested two-step RT-PCR (DSN-2 RT-PCR), we found a high frequency presence of BFNNV genotype as well as RGNNV in various domestic and imported shellfish. This was definitely different from the previous reports of outbreaks and asymptomatic infection only by the RGNNV genotype in cultured finfish in Korea. Cultivation of NNV entrapped in shellfish was performed successfully by a blind passage. Thus, in an attempt to elucidate the epidemiology of betanodavirus, experiments conducted on 969 shellfish samples concluded that (i) distribution of NNV genotype, especially BFNNV, in shellfish is clearly different from that found in finfish of the world; (ii) unlike RGNNV, which showed a high rate in summer, BFNNV showed no seasonal variation and this result suggests BFNNVs in the marine environment remain fairly constant throughout the year; and (iii) the entrapped virus in shellfish was alive and culturable invitro. These results are the first report of high level prevalence of invitro culturable NNV in shellfish, for both BFNNV and RGNNV, which may present a potential risk in transmitting nodaviruses to host species in a marine environment.

High nervous necrosis virus (NNV) diversity in wild wrasse (Labridae) in Norway and Sweden.

Wild goldsinny wrasse Ctenolabrus rupestris, corkwing wrasse Symphodus melops and ballan wrasse Labrus bergylta were collected at 8 sampling sites in Sweden and Norway during summer 2014. Brain tissue from 466 wrasses were analyzed for nervous necrosis virus (NNV) infections by real-time RT-PCR, and positive samples were subjected to sequencing and phylogenetic analysis of partial segments of the RNA2 and RNA1 genes. This study shows that NNV is present in wild ballan, corkwing and goldsinny wrasse along the coastline of Sweden and Norway. The overall prevalence in the sampled labrids was 6.7%. Prevalence was 6.4% in goldsinny, 6.3% in corkwing and 18% in ballan wrasse. The wrasse RNA2 NNV sequences revealed high genetic variability and were divided into 3 clusters within the cold water barfin flounder NNV (BFNNV) and warm water cluster red-spotted grouper NNV (RGNNV) genogroups. Within the BFNNV genogroup, wrasse NNVs clustered in 2 sub-genogroups, with grey mullet NNV (GMNNV) and with Atlantic halibut NNV (AHNNV). These groups were previously dominated by virus originating from Atlantic cod Gadus morhua and Atlantic halibut Hippoglossus hippoglossus from the northeast Atlantic. The presence of NNV in wild wrasse and the surprising high genetic variability observed in this study should be considered before moving wild-caught wrasse between geographically distant sites. The results show that use of wild-caught wrasse as brood fish in wrasse farming represents a risk of introducing NNV into aquaculture.

Effects of different green light intensities on the growth performance and endocrine properties of barfin flounder Verasper moseri

We previously reported that the somatic growth of barfin flounder, Verasper moseri, was effectively stimulated by the green light compared to the blue and red lights. Herein, we report the effects of different green light intensities on the growth and endocrine system of the fish. Fish were reared in a dark room with light from a light-emitting diode (LED) at a peak wavelength of 518nm under controlled photoperiod (10.5:13.5h, light:dark cycle; 06:00–16:30, light) with three levels of photon flux density (PFD)—2 (low), 7 (medium), or 21 (high) μmol·m−2·s−1 at the water surface. The average water temperature was 10.2°C, and the fish were fed until satiety. The fish reared under high PFD of green light showed the highest specific growth rates, followed by the medium PFD group. Under high PFD, the fish showed the highest amount of melanin-concentrating hormone mRNA in their brains and insulin in plasma, while the lowest amount of growth hormone was observed in their pituitary glands. These results suggest that the green light stimulated the growth of barfin flounders in a light intensity-dependent manner in association with their central and peripheral endocrine systems. However, when the fish were reared in an ordinary room where they received both ambient and green LED lights, the fish under LED and ambient light grew faster than those under ambient light only (control). Moreover, no difference was observed in the specific growth rate of the fish reared under the three different green LED light intensities, suggesting that the growth was equally stimulated by the green light within a certain range of intensities under ambient light.

Genomewide analysis of copy number variants in alopecia areata in a Central European cohort reveals association with MCHR2.

Alopecia areata (AA) is a common hair loss disorder of autoimmune aetiology, which often results in pronounced psychological distress. Understanding of the pathophysiology of AA is increasing, due in part to recent genetic findings implicating common variants at several genetic loci. To date, no study has investigated the contribution of copy number variants (CNVs) to AA, a prominent class of genomic variants involved in other autoimmune disorders. Here, we report a genomewide- and a candidate gene-focused CNV analysis performed in a cohort of 585 patients with AA and 1340 controls of Central European origin. A nominally significant association with AA was found for CNVs in the following five chromosomal regions: 4q35.2, 6q16.3, 9p23, 16p12.1 and 20p12.1. The most promising finding was a 342.5-kb associated region in 6q16.3 (duplications in 4/585 patients; 0/1340 controls). The duplications spanned the genes MCHR2 and MCHR2-AS1, implicated in melanin-concentrating hormone (MCH) signalling. These genes have not been implicated in previous studies of AA pathogenesis. However, previous research has shown that MCHR2 affects the scale colour of barfin flounder fish via the induction of melanin aggregation. AA preferentially affects pigmented hairs, and the hair of patients with AA frequently shows a change in colour when it regrows following an acute episode of AA. This might indicate a relationship between AA, pigmentation and MCH signalling. In conclusion, the present results provide suggestive evidence for the involvement of duplications in MCHR2 in AA pathogenesis.

Behaviourally mediated thermal experience in relation to final oocyte maturation by free-swimming barfin flounder (Verasper moseri)

The reproductive performance of barfin flounder (Verasper moseri), a multi-batch spawner spawning during February-April (boreal winter) in the wild, is particularly sensitive to temperature perturbations. Final oocyte maturation (FOM) of captive flounder occurred only when post-vitellogenesis fish experienced a slowly-elevated water temperature regime (SETR) (from 2°C to 6°C over a month). We used electronic tags to show how post-vitellogenesis flounder experience SETR during its pre-spawning migration, during their southward migration, to a location about 750km from the summer feeding grounds in the coastal waters of Hokkaido (CWH; 40.2–43.4°N). From 2010 through 2012, we tagged a total of 208 V. moseri in early December at the feeding grounds of the CWH, during in which vitellogenin incorporation into oocyte was almost completed. One hundred and two tags were recovered and the data from 98 were downloaded. All of the tagged females moved offshore by the end of January. Fish spent more time in cold and deeper waters (3–4°C, 351–380m) in January than they did in December (6–8°C, 33–59m), but experienced warmer temperatures (5.1–5.7°C, 367–396m) in February and March around the spawning period. All tagged fish experienced SETR and then experienced the onset of SETR in January, although SETR of tagged fish have large variations among individuals. A comparison between depth-temperature data recorded by data-logger and oceanic horizontal temperature conditions suggests that fish did not experience warm surface water of the Tsugaru Warm Current, a branch of the Kuroshio that flows into the Pacific Ocean via the Tsugaru Strait, and instead were in the colder Oyashio water by migrating deep and finally experiencing warmer water in February and March. In conclusion, SETR appears to be one of the exogenous factors that induces FOM in the wild.

Identification and evaluation of the pathogenicity of a scuticociliate isolated from diseased barfin flounder Verasper moseri and sensitivity of a pathogen isolate to vinegar and tea extract solutions in sea water

Identification and evaluation of the pathogenicity of a scuticociliate isolated from diseased barfin flounder Verasper moseri and sensitivity of a pathogen isolate to vinegar and tea extract solutions in sea water

Molecular Basis for Antigenic Diversity of Genus Betanodavirus.

Betanodaviruses are the causative agents of viral nervous necrosis (VNN), a devastating disease for the Mediterranean mariculture. Four different betanodavirus species are recognized, Striped jack-, Redspotted grouper-, Tiger puffer-, and Barfin flounder nervous necrosis virus (SJNNV, RGNNV, TPNNV and BFNNV), but there is little knowledge on their antigenic properties. In order to describe the serological relationships among different betanodavirus genotypes, serum neutralization assays were performed using rabbit polyclonal antisera against eight fish nodaviruses that cover a wide species-, temporal-, spatial- and genetic range. The results indicate that the SJNNV and RGNNV are antigenically distinct, constituting serotypes A and C, respectively. The TPNNV and BFNNV, the latter representing cold-water betanodaviruses, are antigenically related and cluster within serotype B. The reassortant viruses RGNNV/SJNNV and SJNNV/RGNNV group within serotypes A and C, respectively, indicating that the coat protein encoded by RNA2 acts as major immunoreactivity determinant. Immunostaining of in vitro expressed wild type and chimeric capsid proteins between the RGNNV and the SJNNV species indicated that the C-terminal part of the capsid protein retains the immunoreactive portion. The amino acid (aa) residues determining RGNNV and SJNNV antigenic diversity were mapped to aa residues 217–256 and aa 257–341, respectively. Neutralization of reverse genetics derived chimeric viruses indicated that these areas determine the neutralizing epitopes. The data obtained are crucial for the development of targeted serological tests for the diagnosis of VNN, and informative for development of cross-protective vaccines against various betanodavirus genotypes.

Elucidating the spawning migration and core reproductive duration of male flatfish using sperm duct volume as an index for better fishery advice and management

The conservation and management of heavily exploited fish stocks require reliable data on various aspects of reproduction. In particular, information about spawning migration and spawning period of both sexes is essential for protecting important spawning habitats. However, studies that focus on male spawning ecology are very limited. We elucidated the male spawning migration, core duration of reproduction, and seasonal changes in the maturity of the barfin flounder Verasper moseri, an important target flatfish for stock enhancement programs in Japan. We conducted histological observations of the testes and utilized the sperm duct volume index to facilitate accurate determination of the male spawning period. Fish before spermiation were mainly observed off Hokkaido and partially off northern Tohoku (40.2–43.4°N) during August–December. However, fish undergoing spermiation and sperm release were not found near these areas and were observed only off southernmost Tohoku (35.8–37.5°N approximately 300m deep) from late January to April. Very few spent fish were caught off southern Tohoku, but were frequently observed off Hokkaido during May–June. These results demonstrate that male V. moseri repeat widespread spawning migrations over 700km between feeding grounds off Hokkaido and spawning grounds off southern Tohoku. Moreover, a combined analysis related to seasonal variations in the sperm duct volume and the proportion of spawning adults identified that the core duration of male reproduction was during March. Until now, reproduction of the next generation in the wild has not been confirmed, despite the continual release of seedlings and local fishery restrictions around Hokkaido. Our results underscore the necessity of a more widespread fishery management plan for V. moseri that encompasses the waters off Hokkaido and Tohoku to more effectively enhance reproduction.

Dimerization of melanocortin receptor 1 (MC1R) and MC5R creates a ligand-dependent signal modulation: Potential participation in physiological color change in the flounder

Vertebrates produce α-melanocyte-stimulating hormone (α-MSH), which contains an N-terminal acetyl group, and desacetyl-α-MSH, which does not contain an N-terminal acetyl group. In teleosts and amphibians, α-MSH-related peptides stimulate pigment dispersion via melanocortin receptors 1–5 (MC1R–MC5R), which are members of the G-protein-coupled receptor (GPCR) family. We previously reported an interesting phenomenon associated with physiological color changes in the skin of a flatfish, barfin flounder (bf). Specifically, pigments in xanthophores expressing only the bfMC5R gene were dispersed by both α-MSH and desacetyl-α-MSH, whereas those in melanophores expressing both the bfMC1R and bfMC5R genes were dispersed by desacetyl-α-MSH, but not by α-MSH. In this study, we examined whether heterodimers of bfMC1R and bfMC5R can act as significant inhibitory receptors for the N-terminal acetylation of α-MSH in mammalian Chinese hamster ovary cells. Immunofluorescence analyses showed that bfMC1R and bfMC5R were localized together at the plasma membrane when expressed in the same cells. Indeed, after coexpression of Flag-bfMC1R and HA-bfMC5R, immunoprecipitation with anti-Flag antibodies resulted in the presence of anti-HA immunoreactivity in the precipitate, and vice versa. Importantly, cyclic AMP assays showed that cotransfection of bfMC1R with bfMC5R inhibited the cyclic AMP accumulation induced by α-MSH to a greater extent than that observed after transfection of bfMC1R alone. Of note, this inhibitory response was not caused by desacetyl-α-MSH. Thus, we show a ligand-dependent signaling through functional heterodimerization of MC1R and MC5R in mammalian cells. The ligand-selective receptor complex also provide the first mechanistic implication that may play a role in the control of color change in teleosts.