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; RNAI (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=l32) 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 sug ...