Short communication Experimental evidence of horizontal transmission of Betanodavirus in hatchery-produced Asian seabass, Lates calcarifer and brown-marbled grouper, Epinephelus fuscoguttatus fingerling Benny Obrain Manin, Julian Ransangan ⁎ Microbiology and Fish Disease Laboratory, Borneo Marine Research Institute, Universiti Malaysia Sabah, Jalan UMS 88400, Kota Kinabalu, Sabah, Malaysia abstract article info Article history: Received 22 June 2011 Received in revised form 17 August 2011 Accepted 23 August 2011 Available online 31 August 2011 Keywords: Betanodavirus Horizontal transmission Asian seabass Brown-marbled grouper In the present study we report the experimental evidence of horizontal transmission of betanodavirus in hatchery-produced Asian seabass (Lates calcarifer) and brown-marbled grouper (Epinephelus fuscogutattus) fingerling. The experiment was conducted by incubating fish fingerlings in aerated sterile natural seawater inoculated with tissue homogenate of betanodavirus infected fish fingerling at 28° C for 30 min and after which they are transferred into the aquarium. Dead fish were collected daily for 10 days and subjected to RT-PCR and histological examinations. The cDNA of coat protein gene of betanodavirus from positive fish specimens were subjected to RFLP-PCR and DNA sequencing analyses. The result showed high fish mortality in treatment than in the control experiment. The RT-PCR and histological analyses showed all fish specimens in treatment groups except for D2 and E2 were successfully infected with Betanodavirus. In contrast, all fish specimens in control groups remained uninfected. The result of the present study indicate that mixing of fish fingerlings obtained from different sources and the use of surplus fish eggs for feeding supplement can poten- tially promote the horizontal transmission of Betanodavirus in hatchery. This can threaten the sustainability of aquaculture industry in Malaysia. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Asian seabass, Lates calcarifer and brown-marbled grouper, Epinephelus fuscoguttatus are two of the most extensively cultured marine fish species in Malaysia. This is driven by high demand and market price of these fish especially during festive seasons (Othman, 2008). The aquaculture of these species is carried out in net cages installed in the coastal waters (Othman, 2008). Most of the seeds are produced in hatcheries (Ransangan and Manin, 2010). However, seeds are also imported from neighboring countries. Due to space limitations in most hatcheries, seeds of the same fish species but obtained from dif- ferent sources are mixed in the same nursery tank. In addition, surplus eggs are sometimes used as feed supplement during the larval rearing. These practices could have been the reasons for many outbreaks of viral nervous necrosis (VNN) in Malaysia (Ransangan et al., 2011; Ransangan and Manin, 2010). Viral nervous necrosis (VNN) is caused by a RNA virus called the Betanodavirus under the family Nodaviridae. The virus can be transmitted both in horizontal and vertical fashions (Boonyaratpalin et al., 1996; Castric et al., 2001; Oh et al., 2002; Watanabe et al., 2000). The hori- zontal transmission of the virus is known to occur from infected fish, contaminated trash fish and contaminated water supply (Chérif et al., 2009; Gomez et al., 2008, 2010; Watanabe et al., 2000). The cannibalistic nature of Asian seabass and brown-marbled grouper fingerlings may also facilitate the horizontal transmission of virus in the culture tank. This study was conducted to experimentally verify the horizontal transmis- sion of Betanodavirus in Asian seabass and brown-marbled grouper seeds through culture water contaminated with tissue homogenate of Betanodavirus-infected fish fingerlings. 2. Methods and materials 2.1. Fish specimens Asian seabass (10dph, 26dph and 40dph) and brown-marbled grouper (10dph) fingerlings were obtained from a hatchery in Tuaran, Sabah. The seeds were maintained in the aquarium (capacity 100 L) with re-circulated sea water and aerated throughout the ex- periment. The fish specimens were fed twice a day with artemia or/ and commercial pellet. A day before the experiment, three pools (20 individuals) of Asian seabass (10dph and 26dph), brown-marbled grouper (10dph) and three individuals of Asian seabass (40dph) fin- gerlings were first randomly checked for Betanodavirus infection using RT-PCR and histopathological methods. Subsequently, only fish specimens from negative batches were then used in the subsequent experiments. Aquaculture 321 (2011) 157–165 ⁎ Corresponding author. Tel.: + 60 88 320000; fax: + 60 88 320261. E-mail address: liandra@ums.edu.my (J. Ransangan). 0044-8486/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2011.08.021 Contents lists available at SciVerse ScienceDirect Aquaculture journal homepage: www.elsevier.com/locate/aqua-online