Screening of freshwater fish species for their susceptibility to a betanodavirus

Betanodaviruses, the causative agents of viral nervous necrosis in marine fish, have bipartite positive-sense RNA genomes. Because the genomes are the smallest and simplest among viruses, betanodaviruses have been well studied using a reversed genetics system as model viruses. However, studies of virus-host interactions have progressed slowly because permissive hosts for betanodaviruses (basically larvae and juveniles of marine fish) are only available for limited periods of the year and are not suitable for the construction of a genetic engineering system. To obtain a model fish species that are not subject to these problems, 21 freshwater fish species were injected intramuscularly with a betanodavirus (redspotted grouper nervous necrosis virus) and tested for their susceptibility to the virus. Based on their responses, the tested fish were classified into 3 groups: 4 susceptible fish, 10 less susceptible fish, and 7 resistant fish. The susceptible fish, celebes rainbowfish Telmatherina ladigesi, threadfin rainbowfish Iriatherina werneri, dwarf rainbowfish Melanotaenia praecox, and medaka Oryzias latipes, exhibited erratic swimming and eventually died within 10 d post-inoculation. The virus was specifically localized in the brains, spinal cords, and retinas of the infected fish, similar to the pattern of infection in naturally infected marine fish. We believe that these susceptible freshwater fish species could act as good host models for betanodavirus-fish interaction studies.     

Variable region of betanodavirus RNA2 is sufficient to determine host specificity

Betanodaviruses, the causative agents of viral nervous necrosis in marine fish, have bipartite positive-sense RNA genomes. The viruses have been classified into 4 distinct types based on nucleotide sequence similarities in the variable region (the so-called T4 region) of the smaller genomic segment RNA2 (1.4 kb). Betanodaviruses have marked host specificity, although the primary structures of the viral RNAs and encoded proteins are similar among the viruses. We have previously demonstrated, using reassortants between striped jack nervous necrosis virus (SJNNV) and redspotted grouper nervous necrosis virus (RGNNV), that RNA2, which encodes the coat protein, strictly controls host specificity. However, because RNA2 is large, we were unable to propose a mechanism underlying this RNA2-based host specificity. To identify the RNA2 region that controls host specificity, we constructed RNA2 chimeric viruses from SJNNV and RGNNV and tested their infectivity in the original host fish, striped jack Pseudocaranx dentex and sevenband grouper Epinephelus septemfasciatus. Among these chimeric viruses, SJNNV mutants containing the variable region of RGNNV RNA2 infected sevenband grouper larvae in a manner similar to RGNNV, while RGNNV mutants containing the variable region of SJNNV RNA2 infected striped jack larvae in a manner similar to SJNNV. Immunofluorescence microscopic studies using anti-SJNNV polyclonal antibodies revealed that these chimeric viruses multiplied in the brains, spinal cords and retinas of the infected fish, as in infections by the parental viruses. These results indicate that the variable region of RNA2 is sufficient to control host specificity in SJNNV and RGNNV.     

Nutritional condition, starvation status and growth of early juvenile Japanese sea bass (Lateolabrax japonicus) related to prey distribution and feeding in the nursery ground

The nutritional condition and protein growth rates of Japanese temperate bass larvae and juveniles were studied in relation to prey distribution and feeding habits in the nursery grounds in Chikugo estuary, Ariake Sea, Japan. Samples were collected from a wide spatial area covering the nursery grounds of the fish in March and April 2003. Food habits of the fish were analyzed by examining the gut contents. Fish condition was evaluated by using RNA/DNA ratio and other nucleic acid-based indices and protein growth rates. The nucleic acid contents in individually frozen larvae and juveniles were quantified by fluorometric method. Two distinguished feeding patterns, determined by the distribution of prey copepods, were identified along the nursery ground. The first pattern showed the dependency of the fish on the calanoid copepod Sinocalanus sinensis, which was the single dominant prey in low saline upper river areas and the second pattern involved a multi-species dietary habit mainly dominated by Acartia omorii, Oithona davisae and Paracalanus parvus. Values of RNA, DNA, total protein, growth rates and for all the nucleic acid-based indices were higher in upstream areas than in the downstream areas. The proportion of the starving fish was higher in the downstream areas than in the upstream areas. Condition of juvenile sea bass was not equal throughout the nursery grounds; fish in the upper river were in better condition than those in the lower estuary. We speculated that utilization of S. sinensis, which appears a suitable prey item and provide a better foraging environment in the upstream nursery ground, is one of the key factors for early survival and growth of Japanese temperate bass larvae and juveniles in the Chikugo estuary.     

High permissivity of the fish cell line SSN-1 for piscine nodaviruses.

Seventeen isolates of piscine nodavirus from larvae or juveniles of 13 marine fish species affected with viral nervous necrosis (VNN) were examined for their infectivity to a fish cell line SSN-1. Based on cytopathic effects (CPE) and virus antigen detection by fluorescent antibody technique (FAT) after incubation at 25 degrees C, the infectivity of these virus isolates was divided into 4 groups. Group 1, including 9 virus isolates from 4 species of grouper, 2 species of sea bass, barramundi, rock porgy, and Japanese flounder showed CPE characterized by rounded, granular cells with heavy cytoplasmic vacuoles within 3 d post-incubation (p.i.), and the monolayer partially or completely disintegrated over 3 to 6 d p.i. Scattered FAT-positive cells appeared at 3 h p.i. and spread through the cell sheet with an increasing fluorescence signal over 24 h p.i. Group 2, consisting of 3 virus isolates from striped jack, induced CPE with thin or rounded, granular, refractile cells without conspicuous vacuole formation, and extensive FAT-positive reaction was observed in a time course similar to that of Group 1. Cells inoculated with Group 3 (1 isolate from tiger puffer) developed no distinct CPE but viral infection was evidenced by localized FAT-positive cells. There were no FAT-positive cells in Group 4, which included 4 isolates from Japanese flounder, Pacific cod and Atlantic halibut. However, when incubation was performed at 20 degrees C, the SSN-1 cells inoculated with the Group 3 isolate showed CPE similar to that of Group 1 and extensive FAT-positive reaction. Evidence of virus proliferation at 20 degrees C was also obtained in Group 4 isolates. The virus titers in the infected fish varied from 10(11) to 10(16) tissue culture infectious dose (TCID50) g(-1) of fish. There is a good correlation between these infectivities to the SSN-1 cells and the coat protein gene genotypes of the isolates. The present results indicate that SSN-1 cells are useful for propagating and differentiating genotypic variants of piscine nodavirus.     

Trypsin activity and physiological aspects in larval rearing of European sea bass (Dicentrarchus labrax) using live prey and compound diets

The aim of this study was to evaluate the potential of a compound diet as a live prey substitute for feeding European sea bass larvae ( Dicentrarchus labrax L.). The effect of a commercial diet (Nippai ML feed) and live prey ( Artemia nauplii) on tryptic enzyme activity, protein content, growth (standard length) and survival rates of sea bass larvae were tested during a 27-day rearing experiment. Sea bass larvae were divided into two groups. The live food group (control group) was fed exclusively on newly hatched Artemia nauplii (Inve AF grade), the test group was fed exclusively with the compound diet from day 15 onwards. As trypsin has been demonstrated to be a useful indicator for evaluating digestibility of food and the nutritional condition of fish larvae, individual tryptic enzyme activity was determined in both feeding groups. Larvae older than 14 days after hatching and fed on live food showed a significantly higher tryptic enzyme activity than larvae fed the compound diet. A similar relationship between tryptic activity and standard length in both test groups was detected only in small larvae (standard length < 7 mm). The usefulness of proteolytic enzyme activity measurements in larval fish research, as well as its use in aquaculture nutrition research, was confirmed. Protein content, increase in length and survival rates of the sea bass larvae were additionally determined in order to evaluate an influence on the diet. The protein content of larvae fed the Artemia nauplii was higher and the growth of larvae fed the compound diet was reduced. Larval mortality was not affected by the diet given.     

Sea bass Dicentrarchus labrax nervous necrosis virus isolates with distinct pathogenicity to sea bass larvae.

Reproduction of nodavirus disease was performed by experimental infection of sea bass eggs during fertilization or at larval stage 4 with 2 genetically distinguishable nodavirus strains (Sb1 and Sb2) isolated from sea bass collected along the Atlantic and Mediterranean French coast. The pathogenicity of the virus strains was assigned after detection of the virus by ELISA and immunohistochemistry (IHC). The Atlantic (Sb1) strain was more pathogenic than the Mediterranean (Sb2) strain during the fertilization step whilst both strains were pathogenic following experimental exposure of 4 d old larvae. Virus lesions developed in the brain 4 to 6 d following experimental exposure. Experimental ELISA proved very sensitive for detecting the nodavirus in Sb1 or Sb2 experimentally infected larvae, as well as in naturally infected sea bass larvae collected in French hatcheries or in barramundi larvae reared in the Pacific area. The development of an ELISA specific for the 2 nodavirus strains isolated from the sea bass should be useful for the detection of the virus, in addition to other techniques recommended by the Office International des Epizooties (OIE).     

Estuarine colonization, population structure and nursery functioning for 0-group sea bass (Dicentrarchus labrax), flounder (Platichthys flesus) and sole (Solea solea) in a mesotidal temperate estuary

The function of the Mondego estuary as a fish nursery habitat was investigated from June 2003 to June 2004 by comparing the timing of estuarine colonization with juveniles of sea bass Dicentrarchus labrax, flounder Platichthys flesus, and sole Solea solea, while also analysing their population structure, growth and diet composition. Differences in the onset of estuarine colonization were observed, since sole juveniles were the first to enter the estuary (in January), followed by flounder in April and sea bass in June. The estuarine population of these species consisted of several age‐groups, although the majority of individuals belonged to age‐groups 0 and 1. The growth rates determined for 0‐group fish were within the range of those reported for other European estuarine systems. Some differences were also recognized regarding the timing of estuarine colonization and the length of the growing season. Diet of 0‐group sea bass consisted mainly of Crustacea, Polychaeta and Mollusca. Flounder juveniles fed chiefly on Amphipoda (especially Corophium spp.), with Polychaeta, Isopoda and Decapoda also being common prey. The diet of 0‐group sole was dominated by Polychaeta, with Amphipoda, Mollusca and Decapoda ranking highest, with other important benthic organisms also being present. Dietary overlap among these species was relatively low.     

Viral encephalopathy and retinopathy outbreak in freshwater fish farmed in Italy

Viral encephalopathy and retinopathy (VER), otherwise known as viral nervous necrosis (VNN), is a neuropathological condition affecting > 40 species of fish. Although VER affects mainly marine fish, the disease has also been detected in certain species reared in freshwater environments. There are relatively few reports concerning the disease in freshwater species, and there is not much information on clinical signs. Nevertheless, the most common clinical findings reported from affected freshwater species are consistent with the typical signs observed in marine species. In this paper we describe the main clinical signs and the laboratory results associated with the detection of a betanodavirus in hybrid striped bass x white bass (Morone saxatilis x Morone chrysops) and largemouth bass Micropterus salmoides, reared in a freshwater environment. We also detected the virus by real-time PCR and isolated it in cell culture from a batch of pike-perch Sander lucioperca farmed in the same system.     

Detection of SJNNV and RGNNV genotypes using a relative quantification RT‐PCR assay

Viral Encephalopathy and Retinopathy (VER), is caused by a nodavirus included within the Betanodavirus genus of the Nodaviridae family. This disease affects more than 30 marine fish species worldwide and has been a major obstacle in the aquaculture industry; control of the disease is based on virus detection, essentially in carrier specimens. This study describes a real time PCR procedure for viral nervous necrosis virus detection from several organs of sea bass, Senegalese sole, and gilt‐head sea bream, from fish displaying either clinical symptoms or asymptomatic cases. The sensitivity of this technique was about 10⁶‐fold higher than that of the conventional RT‐PCR. The newly designed primers detected nodavirus isolates belonging to the RGNNV and SJNNV genotypes.     

长江口水域夏季鱼卵和仔稚鱼年间变化

基于2005年、2008年、2009年和2011年8月(夏季)在长江口水域(30°30'—31°45'N,121°15'—123°10'E)4个航次的浮游生物拖网资料,分析了长江口水域鱼卵和仔稚鱼的种类组成、数量分布特征及其年间变化。结果表明:4个航次采集的鱼卵和仔稚鱼鉴定到种的种类有17种,隶属于8目13科,以鲈形目种类最多,11种,其次是鲱形目,5种,其他各目种类均小于5种;种类数存在明显年间差异,2005年种类数最多(鱼卵3种,仔稚鱼8种),其次是2009年和2011年,2008年种类数最少(鱼卵1种,仔稚鱼5种)。优势种年间更替明显,长蛇鲻(Saurida elongata)、虾虎鱼(Gobiidae spp.)和中华小公鱼(Stolephorus chinensis)在2005年是优势种,2008年优势种为鳀鱼(Engraulis japonicus),2009年优势种为鳀鱼、寡鳞飘鱼(Pseudolaubuca engraulis)、虾虎鱼等,2011年虾虎鱼和小公鱼(包括小公鱼属未定种Stolephorus spp.和中华小公鱼Stolephorus chinensis)成为优势种。2005年鱼卵和仔稚鱼数量分布的密集区在嵊泗列岛附近水域,2008年鱼卵和仔稚鱼出现较少,未出现明显的数量密集区;2009年鱼卵数量较少,仔稚鱼数量较多,密集区主要分布在在长江口以外123°E附近水域;2011年鱼卵主要分布在在长江北支口门外附近水域,仔稚鱼在调查区内分布相对均匀。     

两种鳜病毒的共感染现象及引起感染细胞的超微变化

借助细胞培养和电镜技术,揭示了鳜球形病毒(Siniperca chuatsi spherical virus,SCSV)与鳜弹状病毒(Siniperca chuatsi rhabdovirus,SCRV)在草鱼鳍细胞(Grass carp fins,GCF)中共感染的现象。在筛选到敏感鱼类细胞系和建立了 鳜病毒体外增殖系统的基础上,取息典型病毒感染出血症的鳜组织,制备组织悬液,接种到GCF细胞中传代培养, 在攻毒后间隔不同时间收集细胞,对攻毒细胞的超薄切片进行电镜观察。揭示两种形态的鳜病毒可在同一个GCF 细胞中增殖,并描述和分析了病毒复制引起感染细胞的超微病变。本研究结果有助于阐明鱼类重要病毒病害的发 生过程及致病机理。     

Differences in the pattern of antipredator behaviour between hatchery‐reared and wild European sea bass juveniles

Shoals of hatchery‐reared and wild sea bass juveniles Dicentrarchus labrax were tested for differences in their antipredator responses towards a potential live predator, the eel Anguilla anguilla . Eight experimental shoals ( i.e . replicates), each composed of 15 individuals from the same stock of juveniles ( i.e . wild or hatchery), were video recorded for 5 min before and after predator exposure. A set of behavioural variables were measured during the pre‐stimulus and stimulus phases of each test and compared between the two groups of replicates. Results showed that in both hatchery‐reared and wild juveniles predator exposure elicited a significant increase in the mean level of shoal cohesiveness and mean shoal distance from the predator, and a significant decrease in the mean shoal distance from the bottom. Shoals of wild juveniles, however, aggregated more quickly and reached higher shoal cohesiveness within the first 20 s of the stimulus period than shoals of hatchery‐reared fish. During this period, the wild fish also reached the highest peak in shoal cohesiveness, which then decreased gradually towards the levels observed before predator exposure. Another component of the antipredator response, the predator inspection behaviour, was fully developed in both wild and hatchery fish. Wild fish, however, tended to inspect the predator at a closer distance than hatchery fish.     

Preparation and Characterization of Alginate Microparticles Containing a Model Protein for Oral Administration in Gnotobiotic European Sea Bass (<Emphasis Type="Italic">Dicentrarchus labrax</Emphasis>) Larvae

Aquaculture is the fastest growing animal production sector. However, the production of marine fish is still hampered by the high mortality rate in the first few weeks after hatching. Mortality in larvae is often caused by microbial infections. Today, the incorporation of immunostimulants into microparticles provides us new tools to enhance disease resistance in marine larviculture. In this study, we prepared alginate microparticles loaded with the model antigen fluorescein isothiocyanate conjugated-bovine serum albumin. Optimum concentrations of alginate and CaCl₂, the correct alginate viscosity and the appropriate preparatory conditions led to the creation of desirable microparticles with the correct size for oral feeding in gnotobiotic European sea bass larvae. The prepared alginate microparticles were stable in sea water and were successfully ingested by gnotobiotic sea bass larvae at day after hatching 7 without causing any negative effects. Results suggest the suitability of this drug delivery system for targeting the innate immune system of fish larvae in order to enhance disease resistance and thus reduce mortality in larviculture.     

Lernanthropus kroyeri infections in farmed sea bass Dicentrarchus labrax: pathological features

Twenty sea bass Dicentrarchus labrax L. from a fish farm (floating cage) in Greece were examined for the presence of parasites. The gills of 7 (35%) fish were infected with adult female specimens of the parasitic copepod Lernanthropus kroyeri van Beneden, 1851, and the intensity of infection ranged from 1 to 24 parasites per host. The most infected portion of the gills appeared to be the primary lamellae. Erosion, desquamation and necrosis of the secondary lamellae were noticed near the site of copepod attachment; furthermore, the terminal claw of the second antennae lacerated tissue and vessels of infected gill. Parasitism by L. kroyeri affected the host's condition factor (mean +/- SE in uninfected vs parasitized; 1.88 +/- 0.04 vs 1.66 +/- 0.12; p < 0.05).     

Efficacy of a Listonella anguillarum (syn. Vibrio anguillarum) vaccine for juvenile sea bass Dicentrarchus labrax

The efficacy of a commercial bivalent Listonella anguillarum (serotype 01 and 02) vaccine (MICROViB, Microtek International) was tested on prime- and booster-immersion vaccinated sea bass Dicentrarchus labrax juveniles. We carried out 2 challenge tests on the prime-vaccinated fish, 50 and 90 d after initial vaccination. A second group of fish received a booster vaccination 60 d after the prime vaccination, and were tested with a single challenge 30 d later. Relative percent survival (RPS) was 92 and 84% (both p < 0.01) among the prime-vaccinated fish on the first and second challenges, respectively. The RPS of the booster-vaccinated sea bass was 100% (p < 0.01). Antibody titres were tested only among 10 prime-vaccinated and 10 unvaccinated (control) sea bass, 60 d post-immunisation, and were found to rise to 1/32 in the vaccinated fish. Our results demonstrate that MICROViB immersion vaccine can effectively protect juvenile sea bass from L. anguillarum infection.     

Habitat choice and recruitment of tropical fishes on temperate coasts of Japan

Tropical reef corals are expanding on Japanese temperate coasts in response to rising sea surface temperatures, and many tropical fish juveniles have been observed routinely in these coral habitats. The present study explored how offshore tropical fish larvae locate coral habitat on the temperate coasts of Japan. Settlement-stage larvae were sampled between July and October 2009–2011 with light traps anchored on coral-replete and coral-free habitats (rocky habitats) at two-level distance (distance between each habitat type was 6 km and 500 m, respectively). Larval abundance was significantly higher on the coral-dominated habitat than that on the rocky habitat at both short and long distance sites, suggesting that coral habitats attract offshore tropical fish larvae. In underwater visual survey, Chaetodontidae and Pomacentridae juveniles were more abundant in coral habitats than in rocky habitats at both the sites, and a laboratory habitat choice experiment demonstrated that these larvae showed a preference for corals rather than rocks. In contrast, densities of juvenile Mullidae did not differ between the coral and rocky habitats, and the larvae did not show a substrate preference in the habitat choice experiment. These observations suggest that habitat choice at settlement possibly accounts for the differences in settlement patterns of tropical fishes between the two habitats. Taken together, our results showed that most tropical fish larvae colonize their settlement coast at a scale of ~0.5 km, and that they may locate coral habitats after reaching a reef. Moreover, the results suggest that coral habitat expansion on temperate coasts will lead to an increase in coral-associated tropical fishes and will change assemblage structures of fishes on temperate coasts.     

神经坏死病毒在赤点石斑鱼组织中的分布

研究采用地高辛原位杂交和免疫荧光检测技术分别检测病毒RNA2和衣壳蛋白在患病赤点石斑鱼Epinephelus akaara稚鱼中分布。地高辛检测结果表明病毒RNA2主要分布在脑、脊髓、视网膜和鳃上; 免疫荧光检测结果和地高辛检测结果一致, 表明病毒靶器官主要也是脑、脊髓、视网膜和鳃。肠道中几乎检测不到阳性信号, 可能不是病毒的靶器官。因此可以推测神经坏死病毒感染赤点石斑鱼的主要途径是鳃, 而不是肠道。