Altered behaviour and reduced survival of juvenile olive flounder, Paralichthys olivaceus, infected by an invasive monogenean, Neoheterobothrium hirame

Neoheterobothriumhirame is a blood feeding monogenean of olive flounder Paralichthys olivaceus. The parasite was first reported in the mid-1990s from the Sea of Japan and became epidemic within cultured and wild flounder populations after several years. Infected fish often suffer from severe anaemia and thus the parasite is thought to have played an important role in the recent depletion of flounder populations in some areas of Japan. However, the causal mechanism underlying the parasite epidemic and decreases in host populations is unclear because apparently N. hirame infection is not fatal to the host. Here, we tested the hypothesis that N. hirame indirectly reduces the survival of wild juvenile flounder by altering their behaviour and making them more susceptible to predation. We conducted a series of experiments to compare behaviours and predation susceptibility between experimentally infected juvenile P. olivaceus and uninfected fish. Results showed that N. hirame infection increases the activity level, alters diel activity and has negative effects on burrowing performance and swimming endurance. When juvenile flounder cohabitated with predators, the survival rate of infected juveniles was approximately 25% less than that of uninfected fish. We believe this is the first empirical evidence linking N. hirame infection to death of the host through predation. Consequences of N. hirame-induced behavioural change for the survival of juvenile flounder in the wild are discussed. We conclude that recent outbreaks of N. hirame are likely to have been a key factor in the decline of flounder populations in Japan.     

Impacts of diclidophorid monogenean infections on fisheries in Japan

Many monogeneans are pathogenic to economically important fish in Japan. However no other monogenean is comparable with the diclidophorids, Heterobothrium okamotoi and Neoheterobothrium hirame, on the scale of impacts they inflict on Japanese fisheries. The shared importance of the two monogenean infections lies in their pathogenicity, fecundity and tolerance to chemical treatment. Heterobothrium okamotoi infects the gills and wall of the branchial cavity of the tiger puffer, Takifugu rubripes (Tetraodontidae), which is widely cultured in western Japan. The main presenting signs of infected fish are anaemia and extensive necrosis caused by adult worms. This monogenean deposits long strings of eggs, which reach lengths of almost 3m. Egg entanglement with the mesh of culture nets increases the chance of hatched larvae encountering susceptible fish. The oncomiracidium maintains infectivity for up to 4 days after hatching. Hydrogen peroxide is the only commercially available chemical able to control the infection, but can only kill immature worms on the gills. Neoheterobothrium hirame infects the gills and wall of the buccal cavity of the Japanese flounder, Paralichthys olivaceus (Paralichthyidae). Since the first known occurrence of this monogenean in 1993, the species has been recorded from almost all areas where the host is distributed. Neoheterobothrium hirame has the potential to produce 781 eggs per day at 20 degreeC. In the western Sea of Japan, wild young-of-the-year flounder became infected in early summer, followed by a sharp increase in prevalence in late summer. By late summer, juvenile flounder have nearly disappeared from the area, strongly suggesting that N. hirame is responsible for mortality of young fish. This is in good agreement with the recent decline in the local flounder population. Neoheterobothrium hirame has also been considered the causative agent of anaemia among wild Japanese flounder since the late 1990s.     

Rapid MALDI biotyper-based identification and cluster analysis of <Emphasis Type="Italic">Streptococcus iniae</Emphasis>

Streptococcus iniae causes severe mortalities among cultured marine species, especially in the olive flounder (Paralichthys olivaceus), which is economically important in Korea and Japan. Recently, there has been growing concern regarding the emergence of S. iniae as a zoonotic pathogen. Here, 89 S. iniae isolates obtained from diseased olive flounders collected from 2003 to 2008 in Jeju Island, South Korea, were characterized using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The results were aligned both with the available Bruker Daltonics data-base and with a new set of S. iniae data entries developed in our laboratory, and the results were compared. When we used the Bruker Daltonics database, the 89 isolates yielded either “no reliable identification” or were incorrectly identified as Streptococcus pyogenes at the genus level. When we used the new data entries from our laboratory, in contrast, all of the isolates were correctly identified as S. iniae at the genus (100%) and species (96.6%) levels. We performed proteomic analysis, divided the 89 isolates into cluster I (51.7%), cluster II (20.2%), and cluster III (28.1%), and then used the MALDI Biotyper software to identify specific mass peaks that enabled discrimination between clusters and between Streptococcus species. Our results suggest that the use of MALDI TOF MS could outperform the conventional methods, proving easier, faster, cheaper and more efficient in properly identifying S. iniae. This strategy could facilitate the epidemiological and taxonomical study of this important fish pathogen.     

Role of cellular response in elimination of the monogenean Neoheterobothrium hirame in Japanese flounder Paralichthys olivaceus

Adult worms of the blood-feeding monogenean parasite Neoheterobothrium hirame, which cause anemia in the Japanese flounder Paralichthys olivaceus, attach to the host fish by embedding their posterior part deeply into the host tissue. To investigate the possibility that cellular responses of the host fish can eliminate N. hirame, flounder were experimentally infected with N. hirame larvae and reared in either fed or starved conditions. Mature parasites were identified on the buccal cavity wall of the fish 33 d post-infection (Day 33). Monocytes/macrophages and granulocytes increased rapidly in the blood and infected sites after the appearance of mature parasites. These cells adhered to the tegument of the parasites. In addition, a few cells with large electron-dense granules (DGCs) were observed in the inflammatory foci. On Day 47, the tegument of some parasites collapsed partially and were phagocytosed by the infiltrated host cells. Some infiltrated cells adhered directly to the inner tissues of the parasites. On Day 54, in the fed fish group, the loss of the tegument led to damage of the parasites' inner tissue by a large number of infiltrated cells. In this group, the elimination of the parasites was noted from Day 47 to 54. These observations probably suggest that the cellular response of the host fish destructed the parasite's posterior part embedded in the tissue, thereby eliminating the parasites. On the other hand, a high mortality was observed in the starved group. The starved fish developed much more severe anemia than the fed fish, and the elimination of the parasites was not observed in this group. The results of the present study suggest that flounder can eliminate N. hirame if they are fed sufficiently.     

Rapid fluctuation in infection levels of Neoheterobothrium hirame (Monogenea) in Japanese flounder Paralichthys olivaceus in the Joban area, Japan

Neoheterobothrium hirame a haematophagous monogenean parasite on Japanese flounder Paralichthys olivaceus occurred at high prevalences (>70%) from 1999 to 2002 but thereafter declined and remained low (<20%) from 2003 to 2005 in the Joban area, Japan. In 2006, N. hirame became abundant again reaching a prevalence of 63%. Generalized linear models explained these rapid fluctuations in infection in relation to inshore water temperature, which affected the reproductive success of the parasite, and also the encounter rate between host and parasite through changes in their respective population densities. Severely anaemic fish were few, 2·4% even in 1999–2002 and 2006, suggesting that the effect of N. hirame infection on the P. olivaceus population was small in this area.     

Phylogenetic analysis of the major capsid protein gene of iridovirus isolates from cultured flounders Paralichthys olivaceus in Korea

In 2003, 13 isolates of iridovirus were obtained from cultured flounders Paralichthys olivaceus during epizootics in Korea. The full open reading frames (ORFs) encoding the major capsid protein (MCP) (1362 bp) from the 13 flounder iridoviruses (FLIVs) were sequenced and the deduced amino acid sequences were phylogenetically analyzed. Phylogenetic analysis of the MCP revealed that all 13 FLIVs were the same species as rock bream iridovirus (RBIV), red sea bream iridovirus (RSIV), and infectious spleen and kidney necrosis virus (ISKNV), and were grouped into an unknown genus which was different from the 2 genera known to infect fish, Ranavirus and Lymphocystivirus. This is the first report on the isolation and phylogenetic analysis of the iridovirus of unknown genus from flounders during epizootics.     

Phylogenetic analysis of betanodaviruses isolated from cultured fish in Korea

Since the publication of the first report on fish nodaviruses in Korea in 1998, fish nodaviruses have caused widespread epizootic events among various fish species in Korea. However, the genotypes of fish nodaviruses in Korea have not yet been determined due to a lack of information about their nucleotide sequences. In this study, we isolated 5 fish nodaviruses from 4 fish species cultured in 4 different regions in Korea: rock bream Oplegnathus fasciatus, Japanese flounder Paralichthys olivaceus, sevenband grouper Epinephelus septemfasciatus, and grey mullet Mugil cophalus. The full open-reading frame (ORF) encoding the coat protein (1017 nt) was sequenced from each of the 5 fish nodaviruses and the nucleotide sequences were phylogenetically analyzed. Results showed that even though their sequences were not identical, all 5 Korean isolates were clustered in the RGNNV genotype. This is the first report on the phylogenetic analysis of fish nodaviruses from cultured fish in Korea.     

Pseudocohnilembus persalinus (Ciliophora: Scuticociitida) is an additional species causing scuticociliatosis in olive flounder Paralichthys olivaceus

In the present study, Pseudocohnilembus persalinus was first reported as a species causing scuticociliatosis in olive flounder Paralichthys olivaceus. Based on the stained specimens, P. persalinus was clearly differentiated from Uronema marinum, which has been shown to be a cause of scuticociliatosis in farmed olive flounder in Korea from its characteristic oral infraciliature structure. The 1754 bp small subunit ribosomal RNA (SS rRNA) gene sequence of P. persalinus showed 95% homology with the partial sequence of P. hargisi SS rRNA. Moreover, multiplex PCR based on the species-specific amplification of the SS rRNA gene sequence enabled us to distinguish P. persalinus from U. marinum in a simple and rapid manner. P. persalinus was clearly differentiated from U. marinum even when the host was infected simultaneously with both species. These data suggest that the multiplex PCR procedure would make it possible to avoid the cumbersome and time-consuming procedures of morphological analysis for the definitive identification of ciliates.     

Morphology and biology of parasite responsible for scuticociliatosis of cultured olive flounder Paralichthys olivaceus.

Based on the arrangement and shape of the buccal structure, scuticociliates isolated from cultured olive flounder Paralichthys olivaceus belonged to the family Uronematidae and showed many characteristics of Uronema marinum. There was variation in the morphometry of clinical isolates taken from different organs of infected flounder. However, the isolates did not show any significant difference in morphometry under cultured conditions. The ciliates were easily maintained in in vitro medium to which antibiotic agents had been added and which had been enriched with the raw brain tissue of a healthy olive flounder. The ciliates propagated in a wide range of both temperature (6 to 30 degrees C) and salinity (10 to 35 ppt).     

Genetic diversity and differentiation of the Korean starry flounder (Platichthys stellatus) between and within cultured stocks and wild populations inferred from microsatellite DNA analysis

The Korean starry flounder, Platichthys stellatus, is economically valuable coastal resident fish species. However, the annual catch of this fish has fluctuated and suffered major declines in Korea. We examined the genetic diversity and population structure for four wild populations and three hatchery stocks of Korean starry flounder to protect its genetic integrity using nine microsatellites. A group of 339 genotypes belonging to seven populations were screened. High degrees of polymorphism at the microsatellite loci were observed within both the wild and hatchery populations. Compared to the wild populations, genetic changes, including reduced genetic diversity and highly significant differentiation, have occurred in cultured stocks. Significant population differentiation was also observed in wild starry flounder populations. Similar degrees of inbreeding and significant Hardy–Weinberg equilibrium deviations were detected in both the wild and the hatchery populations. The genetic connectivity pattern identified four distinct metapopulations of starry flounder in Korea by clustering in the phylogenetic tree, Bayesian analyses, molecular variance analysis, PCA and multidimensional scaling analysis. A pattern of isolation-by-distance was not significant. This genetic differentiation may be the result of the co-effects of various factors, such as historic dispersal, local environment or anthropogenic activities. These results provide useful information for the genetic monitoring of P. stellatus hatchery stocks, for the genetic improvement of this species by selective breeding and for designing suitable management guidelines for the conservation of this species.     

Identification of two differentially expressed forms of progranulin mRNA in the teleost fish Paralichthys olivaceus

Two forms of progranulin mRNA were isolated from kidney and spleen cDNA libraries of the olive flounder, Paralichthys olivaceus. The 3'-untranslated regions (3'-UTRs) of these flounder progranulin (f-pgrn) mRNAs differed in a 20-nucleotide sequence element (5'-AACTGATTACGTTCAACAAC-3') that was present in one mRNA (designated f-pgrn type II) and not in the other (designated f-pgrn type I). Both mRNA sequences contained an open reading frame encoding a 289-amino-acid polypeptide of approximately 33 kDa. Southern blot analysis of the P. olivaceus flounder genome using an f-pgrn cDNA probe and a PCR-based approach identified a single copy of f-pgrn corresponding to the type II mRNA. The expression profiles of the two types of f-pgrn mRNA differed from each other and were tissue- and condition-dependent. The type II mRNA was detected abundantly in studied tissues (gill, kidney, spleen, and intestine) of non-stimulated healthy flounders. The type I mRNA was rarely expressed in any tissues of healthy flounders, but it was continuously increased in the examined tissues of flounders after the intraperitoneal injection of lipopolysaccharide. On the other hand, the expression of type II mRNA was decreased in inverse proportion to the type I mRNA in the LSP-stimulated flounders. These results suggest that type I and type II f-pgrn mRNA are translated into different proteins with different activities in the immune system of flounder.     

Philasterides dicentrarchi, a histophagous ciliate causing scuticociliatosis in olive flounder, Philasterides dicentrarchi--histopathology investigations

Philasterides dicentrarchi is a histophagous scuticociliate infecting Korean olive flounder farms in Jeju Island, South Korea, where it causes significant economic losses. However, the route of entry of these parasites in olive flounder is currently unknown. In the present study, we attempted experimental infection with different doses (concentrations) of P. dicentrarchi by intraperitoneal (IP) injection; maximum cumulative mortality of 95% was recorded on the administration of 2.1×10(8) ciliates/ml. In small size group (3 cm length, 24±2 gwt) the ciliates were found in connective tissues of muscle, fins, and nervous tissue while in large size group (5 cm length, 47±3 gwt), the ciliates were predominantly occurred with associated symptoms of liquefaction in the central nervous system. The major clinico-pathological manifestations were loss of scales, appearance of bleached spots that coalesced to form brownish patches, hemorrhagic, and severe dermal necrotic lesion. In addition there was degeneration of muscle fibers, necrosis of the hepatic parenchyma, and severe edema of the intestinal walls, extensive fouling, necrotic degeneration and hyperplasia in the gill bronchial epithelium. In this study, many ciliates were found in the blood vessel, brain, dorsal fins, muscle, kidney, and vertebra of infected flounder. The histopathological investigations are of significant importance in view of possible routes of entry into the host and doses of ciliates that trigger fast infection for potential utility of drugs as a strategy for the control of P. dicentrarchi in farmed olive flounder.     

Sequence conservation in the internal transcribed spacers and 5.8S ribosomal RNA of parasitic scuticociliates Miamiensis avidus (Ciliophora, Scuticociliatia)

The scuticociliate Miamiensis avidus is a histophagous parasite that causes high mortality in cultured marine fishes, with clinical signs of severe ulcers and hemorrhages in the skeletal muscle. The internal transcribed spacer (ITS) region, which is widely used in taxonomy and molecular phylogeny because of a high degree of variation, was compared for 21 cloned strains of M. avidus (Ciliophora, Scuticociliatia). These strains were isolated from olive flounder Paralichthys olivaceus, ridged-eye flounder Pleuronichthys cornutus and spotted knifejaw Oplegnathus fasciatus in Korea and Japan. The ITS1 (140 bp), ITS2 (236 bp) and 5.8S (119 bp) regions from 21 strains were identical, indicating that these regions are highly conserved in M. avidus. Phylogenic analysis of ITS2 shows that the ciliate should be included in the Philasterida with a close relationship to Pseudocohnilembus hargisi. This study exhibits the first detailed analysis of the ITS1, 5.8 S and ITS2 rRNA regions of M. avidus.     

Occurrence of scuticociliatosis in olive flounder Paralichthys olivaceus by Phiasterides dicentrarchi (Ciliophora: scuticociliatida)

In the course of identifying scuticociliates recently obtained from systemically infected olive flounder Paralichthys olivaceus in Korea, we found a scuticociliate species whose small subunit ribosomal RNA (SS rRNA) gene was not amplified by species-specific primers previously designed for Uronema marinum and Pseudocohnilembus persalinus. By studying morphological characteristics of wet-mounted and stained specimens, we identified the species as Philasterides dicentrarchi, which has been reported to cause systemic infection in the European sea bass Dicentrarchus labrax and turbot Scophthalmus maximus. In this study, we compared morphological characteristics of our specimens with previously reported Philasterides species, including P. dicentrarchi, and sequenced the SS rRNA gene in order to design P. dicentrarchi specific primers. This is the first report on scuticociliatosis caused by P. dicentrarchi from marine fish in Asia.     

Evolutional conservation of molecular structure and antiviral function of a viral RNA receptor, LGP2, in Japanese flounder, Paralichthys olivaceus

LGP2 is an important intracellular receptor that recognizes viral RNAs in innate immunity. To understand the mechanism of viral RNA recognition, we cloned an LGP2 cDNA and gene in Japanese flounder (Paralichthys olivaceus). Viral hemorrhagic septicemia virus-induced expressions of LGP2 mRNA were evaluated in vivo and in vitro by quantitative real-time PCR (Q-PCR) using primers based on the clone sequences. The expression of LGP2 mRNA in the kidney dramatically increased at 3 d postinfection. The expression of LGP2 mRNA also increased in the head kidney leukocytes stimulated with artificial dsRNA (polyinosin-polycytidylic acid) in vitro. To evaluate the antiviral activity of the flounder LGP2, three expression constructs containing pcDNA4-LGP2 (full-length), pcDNA4-LGP2ΔRD (regulatory domain deleted), and pcDNA4-Empty (as a negative control) were transfected into the hirame (flounder) natural embryo (hirame natural embryo) cell line. Forty-eight hours after transfection, the transfected cells were infected with ssRNA viruses, viral hemorrhagic septicemia virus, or hirame rhabdovirus. The cytopathic effects of the viruses were delayed by the overexpression of Japanese flounder LGP2. The Q-PCR demonstrated that mRNA expression levels of type I IFN and IFN-inducible genes (Mx and ISG15) in the hirame natural embryo cells overexpressing LGP2 were increased by polyinosin-polycytidylic acid and viral infections. These results suggest that Japanese flounder LGP2 plays an important role in the recognition of both viral ssRNA and dsRNA to induce the antiviral activity by the production of IFN-stimulated proteins.     

Epidermal characteristics of reproductive subsets in an inshore population of winter flounder, Pseudopleuronectes americanus

The winter flounder, Pseudopleuronectes americanus (Walbaum), population off Newfoundland can include a 'non-reproductive post-mature' subset at any time together with 'reproductive' and 'immature' subsets. Skin samples were taken from male and female specimens from each subset in the winter, after reproductive flounder have normally undergone a pre-winter phase of gonadal recrudescence, and also during the spring prespawning period and in the summer following spawning. Results for immature flounder demonstrate that fish size influences epidermal thickness in this species. The epidermis of reproductive flounders is thick, representing a secondary sexual characteristic, during gonadal recrudescence. A thick epidermis is most pronounced on the blind-side, particularly in prespawning males. Although the epidermis of non-reproductive post-mature fish was thinner than in prespawning males and females, it was thicker than in immature flounders. Also, the epidermal thickness of non-reproductive post-mature individuals can still display some seasonal change although gonadal recrudescence has not been sustained. There is also a relationship between condition factor and epidermal thickness which is manifested particularly clearly in immature and non-reproductive post-mature flounder.     

On the Morphology of Arrowhead Flounders of the Genus Atheresthes in the Pacific Waters of the Northern Kuril Islands and Southeastern Kamchatka

This paper presents the results of studies on the morphology of arrowhead flounders, the Kamchatka flounder Atheresthes evermanni (35 specimens), and the American arrowtooth flounder A. stomias (37 specimens) collected in Pacific waters of the northern Kuril Islands and southeastern Kamchatka in 1998–1999. The morphology of the discussed species is compared with that of arrowtooth flounders from other areas. From this comparison, available information on the sharp increase in the density of the American arrowtooth flounder in the surveyed area (since 1997), and similarities in size compositions of the fish in Kuril and Aleutian waters, a conclusion has been made about the penetration of this species from Aleutian Pacific waters into the area of the northern Kuril Islands and southeastern Kamchatka. This is a result of expansion of the geographical range of this species due to general water warming in the northwestern Pacific during the second half of the 1990s.