Biochemical responses of fish exposed to a harmful dinoflagellate Cochlodinium polykrikoides

To elucidate the ichthyotoxic mechanisms of a harmful dinoflagellate Cochlodinium polykrikoides, biochemical responses of fish exposed to blooms were investigated. Particularly, based on our finding that oxidative damages of gill were associated with fish mortality (J. Plankton Res. 21 (1999) 2105-2115), dysfunction of ion-transporting enzymes and secretion of gill mucus of fish exposed to this bloom species were examined. The susceptibilities of several fishes to C. polykrikoides were different; the active pelagic fishes such as black scraper Thamnaconus septentrionalis, red sea bream Pagrus major, beakperch Oplegnathus fasciatus and seaperch Malakichthys wakiyae, were more vulnerable than the benthic fishes, flounder Paralichthys olivaceus and rockfish Sebastes inermis. In addition, the higher the algal cell density, the higher the fish mortality. When the test fishes were exposed to C. polykrikoides of 5000 cells ml(-1), the transport-related enzymes, carbonic anhydrase and Na(+)/K(+)-ATPase activities were significantly decreased. The activity of carbonic anhydrase was decreased with increasing algal cell density and exposure time. The quantity of total polysaccharide in gill mucus is higher in the fish exposed to C. polykrikoides than in the control fish; the magnitudes were higher in the pelagic fishes than that of benthic fishes. Moreover, a drop of blood pH and oxygen partial pressure (pO(2)) was also observed in red sea bream and flounder subjected to C. polykrikoides. These results suggest that the inactivation of gill transport-related enzymes activities, the fall in blood pO(2) and abnormal secretion of gill mucus by the C. polykrikoides may be one of the principal causes of fish kill.     

Tissue Distribution, Effects of Salinity Acclimation, and Ontogeny of Aquaporin 3 in the Marine Teleost, Silver Sea Bream (Sparus sarba)

The purpose of the present study was to ascertain the tissue-specific expression of the water channel protein, aquaporin 3 (AQP3), during salinity acclimation and larval development of silver sea bream (Sparus sarba). A cDNA fragment encoding aquaporin 3 (aqp3) from silver sea bream gill was cloned and from the deduced amino acid sequence a polyclonal antibody was prepared. AQP3 was found to be present in gill, kidney, liver, brain, heart, and spleen but not in whole blood. The abundance of AQP3 was significantly highest in gills of hypoosmotic (6 ppt) and isoosmotic (12 ppt) acclimated sea bream when compared to seawater (33 ppt) and hypersaline (50 ppt)- acclimated sea bream. Spleen tissue also displayed significantly high levels of AQP3 protein in hypoosmotic and isoosmotic salinities whereas the AQP3 abundance in brain, liver, heart, and kidney remained unchanged across the range of salinities tested. The ontogenetic profile of AQP3 was also investigated from developing sea bream larvae and AQP3 was first detected at 14 days posthatch (dph) and increased steadily up to 28–46 dph. In conclusion, this study has demonstrated that AQP3 expression is modulated in gill and spleen tissue of salinity acclimated sea bream and that it can be detected relatively early during larval development.     

Ichthyophonus infections in cultured marine fish from Spain

Ichthyophonus sp. is reported for the first time in Mugil capito (thinlip grey mullet) and Li a saliens (leaping grey mullet). The fungus was also found in L. aurata (golden grey mullet), Dicentrarchus labrax (sea bass), Sparus aurata (gilthead sea bream) and Scophthalmus maximus (turbot), whereas Mugil cephalus (grey mullet) was not parasitized. In fish sampled periodically, the highest prevalences were observed in sea bass and the lowest in turbot. Among the fish sampled occasionally, the fungus was found associated to an epizootic in thinlip grey mullet. Ichthyophonus was never found in fish weighing <0·5 g. An increase in the prevalence of infection with the age of turbot and gilthead sea bream was observed. Gilthead sea bream and sea bass showed higher prevalences in a closed system than in open and semi-intensive systems. Multinucleate spherical spores, hyphae and endospores of Ichthyophonus sp. parasitized different organs of thinlip and leaping grey mullets, though infection intensity was maximal in the spleen. In the remaining fish, the fungus was found mainly in the trunk kidney, where it appeared frequently in a necrotic form. Ichthyophonus sp. can be considered a potential threat for marine fish aquaculture, especially in culture conditions which may favour the introduction and transmission of the fungus.     

Hyperparasitism of trichodinid ciliates on monogenean gill flukes of two marine fish

Two unusual cases of hyperparasitism of trichodinid ciliates on monogenean gill flukes are described from southern Israel (Red Sea). The first case occurred in cultured European sea bass Dicentrarchus labrax infected by Diplectanum aequans, while the second was observed in a feral devil firefish Pterois miles infected by Haliotrema sp. In both cases, the trichodinids heavily co-infested the host fish gills. The flukes were completely coated by the ciliates, which gave them a cobblestone appearance, but no damage to their tegument was apparent. Both cases are most likely a result of accidental hyperparasitism, brought about by perturbed environmental conditions.     

Distribution of peptidase activity in teleost and rat tissues

Peptides play important roles in cell regulation and signaling in many tissues. The actions of peptides are regulated by peptidases. Although the activity of these enzymes has been thoroughly characterized in mammals, little is known about their presence or function in fish. In the present study, we compared the activity of several peptidases in selected tissues (pituitary gland, different brain areas, kidney and gills) of the gilthead sea bream and rainbow trout with that found in similar rat tissues (lungs studied in place of gills). Soluble puromycin-sensitive aminopeptidase showed the highest values in the pituitary gland of the sea bream, whereas the membrane-bound form was found to be more active in the trout kidney. Very high levels of activity of aminopeptidase N were detected in trout and sea bream plasma. In contrast, the highest levels of activity of aminopeptidase B were found in rat tissues, with the exception of the gills of the trout. Aminopeptidase N levels tended to be higher in sea bream tissues with respect to those of trout. In contrast, the level of activity of aminopeptidase B was found to be consistently much higher in trout tissues than in those of the sea bream. Prolyl endopeptidase activity was principally detected in the pituitary gland and in the brain areas of teleosts. These differences between species could be related to different mechanisms of osmoregulation in saltwater- and in freshwater-adapted fish.     

Infection Status of Estuarine Fish and Oysters with Intestinal Fluke Metacercariae in Muan-gun, Jeollanam-do, Korea

The source of human infection with intestinal flukes was surveyed in estuarine fishes, including the dotted gizzard shad, common sea bass, common blackish goby, redlip mullet, black sea bream, and oyster collected from Muan-gun, Jeollanam-do, Korea during August and September 2007. Collected fishes and oysters were artificially digested in pepsin-HCl solution and examined under a stereomicroscope. In 36 shads (Konosirus punctatus) and 20 basses (Lateolabrax japonicus) examined, Heterophyopsis continua metacercariae were found in 58.3% and 100%, and their average numbers were 12.0 and 6.3 per infected fish, respectively. In 34 gobies (Acanthogobius flavimanus) examined, metacercariae of H. continua were detected in 79.4%, Stictodora lari in 97.1%, and Acanthotrema felis in 92.1%, and their average numbers were 45.8, 189.3, and 235.3 per infected fish, respectively. In 37 redlip mullets (Chelon haematocheilus), Heterophyes nocens metacercariae were found in 56.8%, Pygidiopsis summa in 94.6%, and Stictodora fuscata in 45.9%, and the average metacercarial densities were 17.4, 31.3, and 35.1 per infected fish, respectively. In 30 black sea breams (Acanthopagrus schlegeli) and 45 oysters (Crassostrea gigas) examined, no metacercariae were detected. From the above results, it has been confirmed that the dotted gizzard shad, common sea bass, common blackish goby, and redlip mullet from Muan-gun, Jeollanam-do, Korea are infected with the metacercariae of heterophyid flukes.     

Branchial osmoregulatory response to salinity in the gilthead sea bream, Sparus auratus

The branchial osmoregulatory response of gilthead sea bream (Sparus auratus L.) to short-term (2-192 hr) and long-term (2 weeks) exposure to different environmental salinities (5 per thousand, 15 per thousand, 25 per thousand, 38 per thousand and 60 per thousand) was investigated. A "U-shaped" relationship was observed between environmental salinity and gill Na+,K+ -ATPase activity in both long- and short-term exposure to altered salinity, with the increase in activity occurring between 24 and 96 hr after the onset of exposure. Plasma osmolality and plasma ions (sodium, chloride, calcium and potassium) showed a tendency to increase in parallel with salinity. These variables only differed significantly (P<0.05) in fish adapted to 60 per thousand salinity with respect to fish adapted to full-strength sea-water (SW). Plasma glucose remained unchanged whereas plasma lactate was elevated at 5 per thousand and 60 per thousand. Muscle water content (MWC) was significantly lower in fish adapted to 60 per thousand. Chloride cells (CC) were only present on the surface of the gill filaments and absent from the secondary lamellae. CC distribution was not altered by external salinity. However, the number and size of CC were significantly increased at salinity extremes (5 per thousand and 60 per thousand), whereas fish exposed to intermediate salinities (15 per thousand and 25 per thousand) had fewer and smaller cells. Furthermore, the CC of fish exposed to diluted SW became rounder whereas they were more elongated in fish in full-strength and hypersaline SW. This is consistent with previous reports indicating the existence of two CC types in euryhaline fish. At likely environmental salinities, gilthead sea bream show minor changes in plasma variables and the effective regulation of gill Na+,K+ -ATPase. However, at very low salinities both haemodilution and up-regulation of gill Na+,K+ -ATPase predict a poor adaptation most likely related to deficiency or absence of specific components of the CC important for ion xuptake.     

Mast cell responses to Ergasilus (Copepoda), a gill ectoparasite of sea bream

Immunocytochemical, light microscopy and ultrastructural studies were conducted on gill of sea bream, Sparus aurata L., naturally parasitized with the important parasitic copepod Ergasilus sp. to assess pathology and cellular responses. Thirty-seven S. aurata were examined from a fish farm; 26 (70%) were parasitized, with infection intensity ranging from 3 to 55 parasites per fish. Hosts were divided into two groups, lightly infected fish (<15 parasites per fish) and heavily infected fish (>15 parasites per fish). In histological sections, the copepod encircled gill lamellae with its second antennae, compressed the epithelium, provoked hyperplasia and hemorrhage, occluded arteries and often caused lamellar disruption. Fusion of the secondary lamellae due to epithelial hyperplasia was common in all infected fish; heavily infected fish showed more intense branchial inflammation. In both healthy and infected fish, mast cells (MCs) were free within the connective tissue inside and outside the blood vessels of the primary lamellae and made close contact with vascular endothelial cells, mucous cells and rodlet cells (RCs). MCs were irregular in shape with a cytoplasm filled by numerous electron-dense, membrane-bound granules. Immunostaining of primary and secondary gill filaments with an antibody against the antimicrobial peptide (AMP) piscidin 3 (anti-piscidin 3 antibody, anti-HAGR) revealed a subpopulation of MCs that were positive. These MCs were more abundant in gills of heavily infected fish than in either lightly infected or uninfected fish (ANOVA, P<0.05). Our report documents the response of gill to ectoparasite infection and provides further evidence that mast cells and their AMPs may play a role in responding to branchial ectoparasite infections.     

Copper transporter 1, metallothionein and glutathione reductase genes are differentially expressed in tissues of sea bream (Sparus aurata) after exposure to dietary or waterborne copper

The high affinity copper transporter 1 (Ctr1), metallothionein (MT) and glutathione reductase (GR) are essential for copper uptake, sequestration and defense respectively. Following rearing on a normal commercial diet (12.6+/-0.2 mg kg(-1) Cu), sea bream were fed an experimental control diet lacking mineral mix (7.7+/-0.3 mg kg(-1) Cu), an experimental diet enhanced with Cu (135+/-4 mg kg(-1) Cu) or an experimental diet (7.7+/-0.3 mg kg(-1) Cu) whilst exposed to Cu in water (0.294+/-0.013 mg L(-1)). Fish were sampled at 0, 15 and 30 days after exposures. Fish fed the Cu-enhanced experimental diet showed lower levels of expression of Ctr1 in the intestine and liver compared to fish fed control experimental diets, whilst Ctr1 expression in the gill and kidney was unaffected by excess dietary Cu exposure. Waterborne-Cu exposure increased Ctr1 mRNA levels in the intestine and the kidney compared to experimental controls. Excess dietary Cu exposure had no effect on levels of metallothionein (MT) mRNA, and the only effect of dietary excess Cu on glutathione reductase (GR) mRNA was a decrease in the intestine. Both MT mRNA and GR were increased in the liver and gill after waterborne-Cu exposure, compared to levels in fish fed experimental control low Cu diets. Thus, Ctr1, MT and GR mRNA expression in response to excess Cu is dependent on the route of exposure. Furthermore, the tissue expression profile of sea bream Ctr1 is consistent with the known physiology of copper exposure in fish and indicates a role both in essential copper uptake and in avoidance of excess dietary and waterborne copper influx.     

Experimental transmission of Enteromyxum leei to freshwater fish

The myxosporean Enteromyxum leei is known to infect a wide range of marine fish hosts. The objective of the present study was to determine whether freshwater fish species are also receptive hosts to this parasite. Seventeen species of freshwater fish were experimentally fed E. leei-infected gut tissue from donor gilthead sea bream Sparus aurata obtained from a commercial sea bream cage farm. Four of the tested species, tiger barb Puntius tetrazona, zebra danio Danio rerio, oscar Astronotus ocellatus and Mozambique tilapia Oreochromis mossambicus, were found to be susceptible with prevalences ranging from 53 to 90%. The course of infection and pathology was limited to the gut mucosa epithelium and was similar to that observed in marine hosts. Little is known of the differences in physiological conditions encountered by a parasite in the alimentary tract of freshwater vs. marine teleost hosts, but we assume that a similar osmotic environment is maintained in both. Parasite infectivity may be influenced by differences in the presence or absence of a true stomach, acidic gastric pH and digestive enzyme activity both in the stomach and intestine. Variability in susceptibility among species may also stem from differences in innate immunity. Dimensions of spores produced in the donor sea bream and recipient freshwater species are variable in size, as previously observed in other captive marine host species.     

Pathogenicity of Vibrio alginolyticus for Cultured Gilt-Head Sea Bream (Sparus aurata L.)

The in vivo and in vitro pathogenic activities of whole cells and extracellular products of Vibrio alginolyticus for cultured gilt-head sea bream were evaluated. The 50% lethal doses ranged from 5.4 × 10⁴ to 1.0 × 10⁶ CFU/g of body weight. The strains examined had the ability to adhere to skin, gill, and intestinal mucus of sea bream and to cultured cells of a chinook salmon embryo cell line. In addition, the in vitro ability of V. alginolyticus to adhere to mucus and skin cells of sea bream was demonstrated by scanning electron microscopy. The biological activities of extracellular products of V. alginolyticus were hydrolytic activities; the products were able to degrade sea bream mucus. V. alginolyticus was cytotoxic for fish cell lines and lethal for sea bream. Moreover, the extracellular products could degrade sea bream tissues. However, experiments performed with the bath immersion inoculation technique demonstrated that V. alginolyticus should be considered a pathogen for sea bream only when the mucus layer is removed and the skin is damaged.     

Isolation of a rhabdovirus during outbreaks of disease in cyprinid fish species at fishery sites in England

A virus was isolated during disease outbreaks in bream Abramis brama, tench Tinca tinca, roach Rutilis rutilis and crucian carp Carassius carassius populations at 6 fishery sites in England in 1999. Mortalities at the sites were primarily among recently introduced fish and the predominant fish species affected was bream. The bream stocked at 5 of the 6 English fishery sites were found to have originated from the River Bann, Northern Ireland. Most fish presented few consistent external signs of disease but some exhibited clinical signs similar to those of spring viraemia of carp (SVC), with extensive skin haemorrhages, ulceration on the flanks and internal signs including ascites and petechial haemorrhages. The most prominent histopathological changes were hepatocellular necrosis, interstitial nephritis and splenitis. The virus induced a cytopathic effect in tissue cultures (Epithelioma papulosum cyprini [EPC] cells) at 20 degrees C and produced moderate signals in an enzyme immunoassay (EIA) for the detection of SVC virus. The virus showed a close serological relationship to pike fry rhabdovirus in both EIA and serum neutralisation assays and to a rhabdovirus isolated during a disease outbreak in a bream population in the River Bann in 1998. A high degree of sequence similarity (> or = 99.5% nucleotide identity) was observed between the English isolates and those from the River Bann. Experimental infection of juvenile bream, tench and carp with EPC cell-grown rhabdovirus by bath and intraperitoneal injection resulted in a 40% mortality of bream in the injection group only. The virus was re-isolated from pooled kidney, liver and spleen tissue samples from moribund bream. The field observations together with the experimental results indicate that this rhabdovirus is of low virulence but may have the potential to cause significant mortality in fishes under stress.     

Morphology and distribution of blood fluke eggs and associated pathology in the gills of cultured Pacific bluefin tuna, Thunnus orientalis

Infestations of blood flukes of the genus Cardicola have been observed in juvenile Pacific bluefin tuna (PBT) cultured in Japan. Infected fish harbor large numbers of parasite eggs in their gills. Although the link between blood fluke infection and juvenile mortality is not clear, accumulation of parasite eggs appears to be pathogenic to the fish. We investigated the origins, general morphology/distribution, and histopathology of these eggs in artificially produced 0 yr old PBT. Dead and live fish were sampled on several occasions from two culture facilities in Wakayama prefecture, Japan. The number of eggs in each gill filament was enumerated under a microscope. In addition, we estimated the total number of eggs by dissolving the gills in a weak NaOH solution. We observed two morphologically distinct egg types in the gill filaments, smaller, oval shaped eggs in the gill lamellae and larger, crescent shaped eggs that occurred primarily in the filamentary arteries. Based on the ITS2 sequence, the ovoid and crescent shaped eggs were identified as C. orientalis and C. opisthorchis, respectively. Eggs of the former species were more abundant (maximum: 6400 per filament) than the latter (maximum: 1400), but the number was highly variable among filaments. The eggs of the latter species were relatively evenly distributed among the filaments. In a heavily infected individual, we estimated a total of >4.5 million eggs were present in the gills on one side of the fish. The number of eggs from the two species was positively correlated to each other and the dead fish tended to harbor more eggs than the live fish. Histological observation revealed host responses around the eggs, including encapsulation by fibroblasts and nodule formation, as seen in response to other aporocotylid eggs. In addition, we observed widespread fusion of gill lamellae and blockage of the filamentary arteries in some instances. Our results provide information that can be used for routine diagnosis of Cardicola blood flukes in cultured tuna and suggest they represent a risk to juvenile PBT.     

Red sea bream iridoviral disease

The first outbreak of red sea bream iridoviral disease caused by red sea bream iridovirus (RSIV) was recorded in cultured red sea bream Pagrus major in Shikoku Island, Japan in 1990. Since 1991, the disease has caused mass mortalities of cultured marine fishes not only red sea bream but also many other species. The affected fish were lethargic and exhibited severe anemia, petechiae of the gills, and enlargement of the spleen. The causative agent was a large, icosahedral, cytoplasmic DNA virus classified as a member of the family Iridoviridae and was designated as red sea bream iridovirus (RSIV). The genome of RSIV is liner dsDNA and considered to be circularly permitted and terminally redundant like other iridoviruses. The length of physical map of RSIV genome is 112,415bp. An indirect immunofluorescence test with a monoclonal antibody and PCR are commonly used for the rapid diagnosis of RSIV infected fish in the field. For the control of this disease, a formalin-killed vaccine against red sea bream iridoviral disease was developed and now commercially available.     

Molecular characterisation and biological activity of a novel CXC chemokine gene in rock bream (Oplegnathus fasciatus)

Chemokines are chemoattractant cytokines defined by the presence of four conserved cysteine residues. In mammals, these cytokines can be divided into four subfamilies depending on the arrangement of the first two conserved cysteines in the sequence, and include the CXC(α), CC(β), C(γ), and CX3C(δ) classes. We identified CXC chemokine cDNA, designated RbCXC, isolated using expressed sequence tag analysis of a lipopolysaccharide (LPS)-stimulated rock bream liver cDNA library. The full-length RbCXC cDNA (742 bp) contained an open reading frame of 342 bp encoding 114 amino acids. Results from phylogenetic analysis showed that RbCXC was strictly separated into a distinct clade compared to other known CXC chemokine subgroups. RbCXC was significantly expressed in the trunk kidney, liver, spleen, gill, peripheral blood leukocytes (PBLs), and head kidney. Rock bream PBLs were stimulated with several mitogens, including LPS and polyinosinic-polycytidylic acid (poly I:C), which significantly induced the expression of RbCXC mRNA. RbCXC mRNA expression was examined in several tissues under conditions of bacterial and viral challenge. Experimental challenges revealed that all examined tissues from fish infected with Edwardsiella tarda and red sea bream iridovirus showed significant increases in RbCXC expression compared to the control. In the case of Streptococcus iniae infection, RbCXC mRNA expression was markedly upregulated in the kidney, spleen, and liver. In addition, a maltose binding protein fusion recombinant RbCXC (～53 kDa) was produced in an Escherichia coli expression system and purified. Subsequently, the addition of purified recombinant RbCXC (rRbCXC) to kidney leukocytes was examined to investigate the impact of proliferative and chemotactic activity. The rRbCXC induced significant kidney leukocyte proliferation and attraction at concentrations ranging from 10 to 300 μg/mL, suggesting that it can be utilised as an immune stimulant and/or molecular adjuvant to enhance the immunological effects of vaccines.     

Involvement of host recognition by oncomiracidia and post-larval survivability in the host specificity of Heterobothrium okamotoi (Monogenea: Diclidophoridae)

Heterobothrium okamotoi, a monogenean gill parasite, shows high host specificity for tiger puffer, Takifugu rubripes. In the present study, in vivo and in vitro experimental infections were conducted using various fish species, including T. rubripes, to understand the mechanisms of specificity. In in vivo experiments, T. rubripes, grass puffer, Takifugu niphobles, olive flounder, Paralichthys olivaceus, and red sea bream, Pagrus major, were exposed to oncomiracidia of H. okamotoi labelled with a fluorescent dye, 5- (and -6) carboxyfluorescein diacetate succinimidyl ester, and the numbers of parasites on the gills and skin were recorded at intervals. Oncomiracidia were attached to gills and skin of all the experimental fish species immediately after exposure, and the infection intensity on T. rubripes was higher than that on T. niphobles and much higher than those on the other two species. After 2 days, the attached parasites remained on the gills of T. rubripes, but disappeared from the other hosts. During in vitro experiments, gill filaments excised from seven different fish species (four fish species used in the in vivo experiments and panther puffer, Takifugu pardalis, southern flounder, Paralichthys lethostigma and spotted halibut, Verasper variegates) were exposed to oncomiracidia and the attachment to each fish species and subsequent larval behaviour was observed. The percentage of post-larvae that attached to T. rubripes was slightly higher than those which attached to congeneric fish species and much higher than those of non-tetraodontid fish species. In vivo and in vitro experiments demonstrated that oncomiracidia of H. okamotoi have an affinity for their natural host, T. rubripes, and congeneric fish species. The disappearance of attached post-larvae from 'alien' hosts within 2 days during in vivo experiments suggested that host recognition by oncomiracidia and subsequent post-larval survivability are involved in the host specificity of H. okamotoi.