Studies on the control measures of viral nervous necrosis (VNN) in seed production process of marine fish

Stock enhancement to increase fishery production has been recognized worldwide as the most useful fishery-management program. For almost 50 years in Japan, government-and prefecture-based marine hatcheries have played a key role in production of seed (juveniles of fish and shellfish) to ensure a source for release to the marine environment. However, mass mortalities have occurred in produced larvae and juveniles at high frequency, and particularly microbial infection has been regarded as a major hindrance to the stable production of seeds. The present study focused on viral nervous necrosis (VNN), which has caused a great deal of damage in the seed production process for the last three decades worldwide. The causative agent, piscine nodavirus (genus Betanodavirus, family Nodaviridae), is non-enveloped and icosahedral in shape (about 25 nm in diameter) with two positive-sense single-stranded RNAs; RNA1 (3.1 kb) encodes the replicase and RNA2 encodes the coat protein. Currently, betanodaviruses are classified into four genotypes: SJNNV, RGNNV, TPNNV, and BFNNV. SJNNV (the type species of the genus Betanodavirus) was originally isolated from diseased striped jack (Pseudocaranx dentex) larvae and RGNNV has been most frequently isolated from diseased warm-water fishes. In the present study, I examined VNN in fish species attracting attention as new targets at marine hatcheries and aquaculture facilities in Japan, with special reference to potential control measures for the disease. These fish include red spotted grouper (Epinephelus akaara), red tilefish (Branchiostegus japonicus), bluefin tuna (Thunnus orientalis), and striped jack. Chapter 1: Firstly, I described activities of marine fish farming and aquaculture in Japan as a background of this study, and then summarized major disease problems caused by a variety of viruses, bacteria, fungi, and parasites that severely afflicted the seed production activity. Particular attention was paid to VNN and the major detrimental impact on fish production, with previously reported findings on control measures of the disease. Chapter 2: In this chapter, I first analyzed the status of the seed production in target species based on the data in the annual reports from 1984 to 2009 by the Japan Sea Farming Association (JASFA). The association was recently integrated with Japan's Fisheries Research and Education Agency (FRA). During this period, the total seed production amounts began to decline in 2000 and onwards, but the number of target species did not substantially change and more than 1 million individual juveniles were produced in 16 species of fish, 8 species of crustaceans, and 18 species of other shellfish. Next, I summarized mass mortality cases from 2000 to 2009, based on reports by JASFA and the council collecting information about disease outbreaks in national and prefectural hatcheries. Viral, bacterial, fungal, and parasitic diseases accounted for 26%, 37%, 8%, and 12% of the reported cases, respectively, with 17% of unknown cause. While the incidence of viral and fungal diseases decreased compared with those in the previous period (1989 to 1999), bacterial and parasitic diseases increased particularly in newly targeted fish species for aquaculture. Diseases such as VNN, red sea bream iridoviral disease, gliding bacterial disease, bacterial abdominal swelling, vibriosis, bacterial enteritis, scuticociliatosis, and crustacean fungal diseases were reported continuously, as were in 1989 to 1999. Among them, VNN outbreaks have occurred in 21 fish species of 5 orders and particularly for over 10 years in redspotted grouper, kelp grouper (Epinephelus moara), striped jack, and Japanese flounder (Paralichthys olivaceus). Thus, VNN is considered as a serious menace to stable seed production. Chapter 3: Control measures against VNN in redspotted grouper was examined. Redspotted grouper is an important species in the stock enhancement program of marine fish in Japan because of its migration behavior within relatively limited area. In this species, VNN outbreaks have been reported for over 11 years. Incidence of VNN at the larval stage decreased by disinfecting the fertilized eggs with iodine and/or ultraviolet radiation-treated seawater. However, subsequent mortalities at juvenile and older stages before release to the open water were not entirely prevented, mainly due to lack of knowledge on the infection route. In the present study, I detected the betanodavirus (RGNNV) gene by RT-PCR (reverse transcription-polymerase chain reaction) in apparently healthy juveniles of redspotted grouper that survived after VNN occurrence at a hatchery (Tamano Laboratory, Okayama pref., FRA). Furthermore, retina and brain samples of adult redspotted grouper (n=132) collected from four Japanese sea waters were 4.5% and 33.3% positive for a betanodavirus RGNNV by RT-PCR and nested PCR, respectively, although the detection rates of virus varied fairly depending on captured waters. This suggests that wild redspotted grouper as broodstock candidates are subclinically infected with RGNNV at high rates. Experimental pathogenicity test demonstrated that RGNNV isolates from the wild fish were highly pathogenic to juvenile redspotted grouper. Based on these findings, I proposed the following measures to prevent VNN of redspotted grouper at hatchery; broodstock candidates should be introduced from sea area where fish were betanodavirus-free or at lower infection rates, and reared for short period and spawned under less stressful conditions. Electrolizer-treated seawater is preferable to disinfect the fertilized eggs. Chapter 4: VNN of Japanese red tilefish, which is a particularly important species in coastal fishery because of its very high commercial value, was targeted in this study. In winter of 2004, juveniles produced in a hatchery (Miyazu Laboratory, Kyoto pref., FRA) exhibited abnormal swimming behavior, circling either at the surface of the water or the bottom prior to death. Based on histopathological, immunological and virological examinations of the affected fish, it was concluded that this was caused by betanodavirus RGNNV infection; this is the first record of VNN in red tilefish. An epidemiological examination to determine the infection source of the virus was performed using the PCR-based methods and revealed that wild-caught red tilefish were highly infected with the virus, suggesting that these broodstocks are the most probable source of infection into the produced juveniles. For the prevention of VNN, PCR-negative broodstocks were selected for artificial insemination, and the fertilized eggs were disinfected with electrolyzer-treated seawater and fish were reared in the treated water. As the result, RGNNV was not detected from produced larvae and juveniles by PCR, and no VNN occurred in red tilefish seed productions from 2005 to 2009 at the hatchery. Chapter 5: Pacific bluefin tuna is a species that has attracted interest in view of both stock enhancement and aquaculture worldwide. In this species, however, seed production technology has not yet been fully established; thus, there is substantial depletion in juveniles. Amami Laboratory (Kagoshima pref.) of FRA has experienced mass mortalities at larval stages of this species in the process of seed production since around 2000. In some cases of the mortalities, but not all cases, the diseased fish showed abnormal swimming behavior characteristic to VNN and a betanodavirus (RGNNV genotype) was detected in the affected fish, suggesting that VNN can be a cause of larval mortality of Pacific bluefin tuna. This is the first record of VNN in larval Pacific bluefin tuna. In epidemiological investigations, RGNNV was detected by PCR in wild juveniles, aquaculture broodstocks, fertilized eggs, and larvae, suggesting a vertical viral transmission from broodstocks. Because adult bluefin tuna are extremely large to handle, they cannot be subjected to virus screening by PCR-based methods as other fish species. Therefore, I concentrated my research on practical methods for disinfection of fertilized eggs and rearing waters, and showed that use of electrolyzer-treated water decreased VNN occurrences at larval stages and increased the number of produced juveniles. As a future subject, improvement of spawning methods is required to reduce viral propagation in fish. Chapter 6: VNN of larval striped jack has long been successfully controlled by the established methods; elimination of virus-carrying broodstocks and disinfection of fertilized eggs and rearing waters. However, a VNN case of striped jack larvae happened in a hatchery (Kamiura Laboratory, Oita pref., FRA) where the broodstocks, previously proved to be betanodavirus-free, were reared using disinfected seawater, but frozen wild fish were routinely used as supplementary feed for the broodstocks. Epidemiological investigations to estimate the infection route of this VNN case revealed that a betanodavirus SJNNV was detected in 55% of frozen samples of wild Japanese jack mackerel (Trachurus japonicus) kept as feed for broodstocks. A virus isolate (05SaiJJM-3) from feed fish exhibited almost same pathogenicity as a representative SJNNV (SJNag93) from diseased larval striped jack against larvae of both Japanese jack mackerel and striped jack which had been artificially produced in the Kamiura Laboratory. These results suggest that wild Japanese jack mackerel was a virus source to striped jack larvae. However, phylogenetic analysis on RNA2 (T4 region) showed that 05SaiJJM-3 was clustered differently from SJNag93 and other SJNNV strains including European type SJNNVs. Thereafter, in seed productions of striped jack without use of wild fish as a supplementary feed, no VNN cases were not encountered in the facility. This means that a special attention should be paid to infection via wild fish as feed for aquaculture. Chapter 7: Finally, considering all the above-mentioned findings, I discussed further practical procedures to control VNN in the process of seed production of marine fish from various aspects.