Monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV) remain as two of the serious pathogens that infect black tiger shrimp (Penaeus monodon) in the Philippines. It is, therefore, necessary to prevent the spread and occurrence of such pathogens, especially in shrimp hatcheries where postlarvae (PLs) stocked in culture ponds are usually obtained. One effective means is to establish rapid and reliable diagnostic techniques for these viruses. This research was undertaken mainly to develop monoclonal antibodies (MAbs) against MBV and HPV. Consequently, these MAbs will be used as primary reagents to establish an appropriate immunologically-based detection method for MBV and HPV. Immunologically-based diagnostic methods have proven to be rapid, sensitive and quite specific. Furthermore, they could be performed in the field using very simple set-up. Four main studies were conducted for this research. The first involves the development of infection model of HPV in P. monodon. Series of experiments showed that HPV could be horizontally-transmitted in postlarval P. monodon through the oral route. This allowed the availability of infected materials to be used as immunogen and for screening of antibodies. The infection model also provided a method by which the pathogen could be further studied, including its control in hatchery conditions. The second study describes the development of MAbs against HPV and MBV, including the optimization of an immunofluourescent antibody test (IFAT), as a screening technique. The third study involves the production of polyclonal antibodies (PAbs) against MBV and HPV wherein IFAT, immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA) were optimized and used to screen the antibodies as to their usefulness in MBV and HPV diagnosis. The fourth set of experiments were undertaken to determine the efficiency of chlorine as a disinfectant in inhibiting HPV infection in P. monodon PLs. The results of each study are discussed in this report.

Research is the main component of the Regional Fish Disease Project supported by the Government of Japan (GOJ) Trust Fund. When the project started in 2000, research was undertaken by scientists of the SEAFDEC Aquaculture Department only. Subsequently, scientists from three research institutions under the Department of Fisheries in Thailand and those from the SEAFDEC Marine Fisheries Research Department (MFRD) in Singapore joined the project in 2001 and 2002, respectively. During the period from 2000 to 2004, a total of 29 research studies were conducted in the following five categories:

• Establishment and standardization of diagnostic methods
• Biology and pathogenesis of disease pathogens
• Disease prevention and control
• Establishment of evaluation methods for residual chemicals in aquaculture products
• Epizootiology and prevention of koi herpesvirus disease

We developed a specific competitive enzyme-linked immunosorbent assay (ELISA) for yellowtail kingfish (Seriola lalandi) follicle stimulating hormone (FSH). We previously produced a full-length single chain recombinant yellowtail kingfish FSH using the Pichia pastoris expression system. We used the same method to produce the β subunit of the hormone, against which polyclonal antibodies were raised in rabbits. We first confirmed immunoreactivity of the polyclonal antibodies with the recombinant full length FSH and FSHβ as well as plasma and pituitary FSH of sexually immature and mature yellowtail kingfish by Western blot analysis. We then developed a precise and reproducible ELISA for yellowtail kingfish FSH and validated the assay in plasma and pituitary extracts. The intra- and inter-assay coefficients of variation was <2.2% and 10.2%, respectively. The sensitivity of the assay was 78 pg/ml. For further validation of the assay, we measured the plasma FSH in immature yellowtail kingfish treated with increasing doses (blank, 50, 100 and 150 µg/kg) of kisseptin2-10 peptide from a previous study. The dose response observed in treated females was not significant, however the increased plasma FSH levels coincided with the significantly higher estradiol levels we previously reported in the treated groups. We assessed the applicability of the assay in measuring circulating FSH in other species. We observed parallelism between the linearized FSH standard curve and displacement curves of serially diluted plasma from Atlantic bluefin tuna (Thunnus thynnus) and tilapia (Oreochromis niloticus). We also observed similar parallelism with full length recombinant giant grouper (Epinephelus lanceolatus) FSH. The ELISA we developed for yellowtail kingfish FSH will be useful in understanding the reproductive biology of the species as well as enhancing its aquaculture.

A recombinant giant grouper Luteinizing Hormone (LH) consisting of tethered beta and alpha subunits was produced in a yeast expression system. The giant grouper LH β-subunit was also produced and administered to rabbits for antibody development. The recombinant LH and its antibody were used to develop an Enzyme Linked Immunosorbent Assay (ELISA). This ELISA enabled detection of plasma LH levels in groupers at a sensitivity between 391 pg/ml and 200 ng/ml. Different species of grouper were assayed with this ELISA in conjunction with gonadal histology and body condition data to identify links between circulating LH levels and sexual development. We found that circulating levels of LH decreased when oocytes began to degenerate, and sex-transition gonadal characteristics were apparent when LH levels decreased further. When circulating LH levels were related to body condition (body weight/ body length), transitioning-stage fish had relatively high body condition but low plasma LH levels. This observation was similar across multiple grouper species and indicates that plasma LH levels combined with body condition may be a marker for early male identification in the protogynous hermaphrodite groupers.

This study was conducted to evaluate 8 commercial probiotic products being marketed for the grow-out culture of the tiger shrimp Penaeus monodon and the “SEAFDEC Indigenous Probiotic”. Bacterial parameters investigated consisted of: 1) inherent bacterial population, 2) Total Bacterial Count (TBC), 3) effect on Vibrio harveyi, and 4) presumptive Vibrio populations. Results showed that upon activation, the TBCs of Probiotics A, B, C, F, G and H were precisely as claimed in its product brochure at equal or +/- 1 log difference. In contrast, Probiotics D and E yielded TBCs that were 3 and 2 logs lower than stated in their respective product brochures. At 24 h after pond water inoculation, Probiotics A and E caused an almost 2 logs increased TBCs, Probiotic F a one log increase while no apparent differences from the control were observed in the TBCs of the other test probiotics. The “SEAFDEC Indigenous Probiotic”, Probiotics A, G and H induced the inhibition of luminescent Vibrio by 100% for 48 h exposure, 90% in 24 h, 84-63% in 5-6 days and 77-81% in 5-6 days, respectively. However, such inhibitory effect was not sustained on extended periods. In contrast, the other test Probiotics did not inhibit luminescent Vibrio. On the contrary, Probiotics C, E and F even resulted in a 2 and 1 log increase of the V. harveyi population. For the presumptive Vibrio counts, Probiotics B, E and F had 1-2 logs higher numbers of green colonies than controls while Probiotics A and E yielded higher numbers of yellow colonies. This study showed that inoculation of the shrimp pond waters with Probiotics A, C, D, E and F caused at least one log increase in TBCs. The TBCs of the Probiotics in the presence of ammonia, nitrite and phosphate were affected by the presence or absence of aeration.

This podcast provides information on how an alternate-day feeding strategy can be used to reduce costs for culturing milkfish in ponds or marine cages.

Milkfish (Chanos chanos, or bangus) culture is the largest finfish aquaculture industry in the Philippines and continues to be a top aquaculture commodity primarily because it is easy to culture and can be grown in a wide range of environments. Milkfish thrives in freshwater, brackishwater, marine, and even hypersaline habitats. Milkfish production is increasing rapidly with much of the production moving away from the traditional culture in brackishwater ponds to fish cages in coastal marine waters. To provide a general view of the milkfish culture industry, brief descriptions on breeding, hatchery production, and nursery operation are included in this manual. This manual is meant to serve as a guide for those interested to venture into marine cage farming of milkfish. Milkfish culture has been practiced in the Philippines, Indonesia, and Taiwan for centuries and has been the focus of aquaculture research in a number of institutions for several decades. Innovations in culture practices as a result of information from research data and from farmer’s experiences have been adopted by industry practitioners and these are documented in the recently published book entitled Milkfish Aquaculture in Asia (Liao and Leano, 2010). The recent concern about the rapid growth of aquaculture and its possible environmental impacts, and risks that can threaten the sustainability of this expanding industry has also been the subject of many conferences to develop good management practices in aquaculture. Principles and guidelines on better management practices in marine finfish culture had been drafted and included in this manual.