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Two feeding trials were conducted to evaluate the performance of the earthworm (Eudrilus eugeniae) meal (vermimeal) as a replacement for fish meal in the diet of juvenile mangrove crab (Scylla serrata) in terms of growth, feed efficiency, intermolt duration, carapace width increment, molting success, survival, and incidence of cannibalism. Four experimental diets were formulated with 0, 25, 50, and 75% fish meal protein replaced with earthworm meal protein, designated as D0, D25, D50, and D75, respectively. In the first feeding trial, the experimental diets were fed to crablets with an initial average weight of 0.09±0.007g. Each replicate comprised ten (10) crablets individually stocked in 750ml-capacity plastic containers randomly distributed in a static water bath. Final weight gain, feed efficiency and survival were measured after the 5^th^ molt. In the second feeding trial, 150 crablets with an average weight of 0.07±0.002g were stocked at 30 crablets each in five (5) replicate tanks. Survival and incidence of cannibalism were monitored daily until the end of the culture period of 30 days. The first study showed that the growth of mangrove crab juveniles fed the diet with up to 50% of the fish meal protein replaced by the earthworm meal protein was statistically comparable to the control group. The survival of crablets in the second feeding trial was highest in D25 (33%), followed by D50 (29%), D0 (18%), and lowest in the group fed with D75 (13%). Overall, earthworm meal (Eudrilus eugeniae) can replace as much as 50% of the fish meal protein in juvenile mangrove crab (Scylla serrata) diet without negative effects on growth, carapace width increment, feed efficiency, intermolt duration, molting success, survival, and cannibalism.

Hatchery seed production of mussels has been identified as a sustainable method to address the problem of low production due to insufficient seed supply. We conducted the first hatchery production trials of the Asian green mussel Perna viridis spats in the Philippines to demonstrate the feasibility of producing a sufficient and reliable seed supply for grow-out operations. However, results of small-scale experiments cannot be directly rolled-out commercially unless these are verified in bigger-scale facilities approximating those of commercial hatcheries. Thus, techniques on broodstock collection, spawning, and larval rearing, developed by the project in the laboratory during the experimental trials, were applied and verified in a production run using industry-scale tank facilities. Mature broodstocks collected from traditional mussel growing areas were successfully spawned in the hatchery. Eggs were fertilized, and these developed into D-hinged larvae, pediveliger, and metamorphosed into early spat before fully developing into the spat stage. Successful larval rearing up to the spat stage required the use of appropriate algal species and rates of feeding, close monitoring of larval stages, and water quality management. The survival from eggs to D-hinged larvae, D-hinged larvae to pediveliger, pediveliger to early spat, D-hinged larvae to early spat, and early spat (1 mm) to fully grown spat was 77, 64, 6.4, 3.1, and 72%, respectively. This study has established the feasibility of producing P. viridis in commercial-scale hatchery facilities.

Changes in innate immunity parameters and epinecidin mRNA transcript levels were examined to characterize the non-specific immune response of E. coioides to pathogenic V. harveyi JML1 isolated from affected cage-cultured fish. After fish had been injected with bacteria at a dose causing 30% mortality, blood and tissue samples were collected at 0, 6, 12, 24, 48, 72, 96, 120, and 240 h post-infection (hpi) for assessment of indices such as the oxidative burst (OB) and phagocytic index (PI) of head kidney cells, and lysozyme activity (LYS) and total immunoglobulin (Total Ig) levels of the plasma. The epinecidin mRNA transcript levels (EGE) from skin, gills, liver, kidney, and spleen tissues were also determined by gelbased RT-PCR. Lastly, daily mortality (DM), liver total bacterial load (TBC), and presumptive Vibrio count (TVC) were monitored up to 240 hpi. The results revealed that bacteria proliferated rapidly in fish tissue, reaching peak densities at 24 hpi for both TBC and TVC but was on a downward trend thereafter. The pattern in fish mortality closely correlated with TBC and TVC. Total Ig, OB, and PI in E. coioides were suppressed in the early part of infection when V. harveyi load was high but recovered and later increased as bacterial density declined. LYS and EGE were consistently high and their activities were not hampered by bacterial infection. The study demonstrated that V. harveyi JML1 interacts with E. coioides by transiently inhibiting some immune parameters resulting in mortalities. However, consistently high LYS, upregulated EGE, and resurgent PI, OB and Total Ig conferred resistance and subsequent recovery in the fish. The study provides new insights on the interaction between E. coioides and V. harveyi JML1 that can aid in formulating health management strategies for groupers. Further studies on prophylactic interventions to enhance the innate immune response in grouper during infection with V. harveyi JML1 are suggested.

A 15-week feeding trial was conducted to determine the optimum partial inclusion of mung bean protein in milkfish diet. Six isonitrogenous practical-type diets with mung bean included at 0%, 4%, 8%, 12%, 16%, and 20% of the diet equivalent to 0%, 3%, 7%, 10%, 13%, and 17% of the total dietary protein, respectively, were formulated. Milkfish with average body weight (ABW) of 8.5 ± 0.23g were distributed in eighteen tanks (6 treatments X 3 replications) with 10 fish each. The fish were fed the diets three times daily. Results showed that growth of milkfish was not adversely affected by the inclusion of mung bean protein at any dietary level. Feed conversion ratio (FCR) and protein efficiency ratio (PER) were significantly improved by the inclusion of mung bean at 20% of the diet. Nutrient compositions of the fish carcass were similar in all diets. Furthermore, no detrimental effects attributable to mung bean inclusion were seen in terms of protein retention, hepatosomatic index (HSI), and liver and midgut histology of the fish. Overall, mung bean is a promising protein source for milkfish and can be included up to 20% of the diet contributing as much as 17% of the total dietary protein without detrimental effects on growth, feed performance, PER, protein retention, HSI, and liver and intestinal histology.

Feeding trials were conducted to determine the optimum partial replacement level of soybean meal (SBM) with fermented copra meal (FCM). Isonitrogenous and isocaloric diets containing 0, 5, 10, 15, 20, and 25% of the locally produced FCM partially replacing SBM protein by 0, 12, 27, 41, 56, and 71%, respectively and fully replacing copra meal were formulated. The diets were fed to the fish with an initial weight of 2.83±0.14 g for 12 weeks. Thereafter, the best diet was further tested in a preliminary feeding trial in brackishwater grow-out ponds to verify the performance of the formulated diet against a commercial milkfish feed in an outdoor grow-out system. The results of the indoor tank feeding trial indicated that weight gain of the fish was significantly better in the group fed diet 2, with 5% dietary FCM but further increase in the FCM inclusion level up to 20% of the diet did not exhibit statistical differences against the control. Moreover in the preliminary pond feeding trial, growth and feed conversion ratio (FCR) of the fish fed the FCM diet were significantly higher than the commercial control diet. Survival and nutrient composition of the fish carcass were not adversely affected by the treatments. Hence, optimum dietary FCM inclusion level was determined at 5% of the milkfish diet replacing 100% copra meal and 12% SBM protein. However, in terms of economics, up to 20% FCM can be included in the diet replacing 56% SBM protein may be possible with growth comparable to the FCM-less control.

An eight-week feeding trial was conducted to evaluate the effects of combined supplementation of peptidoglycan and mannan oligosaccharide (MOS) in tiger shrimp, Penaeus monodon. Tiger shrimp (0.29 ± 0.02 g) were fed diets supplemented with different levels of peptidoglycan + (MOS) as immunostimulants for six (6) and eight (8) weeks. Four (4) experimental diets were formulated to contain 0, 0.1, 0.2, and 0.4% peptidoglycan + MOS. The feeding trial was conducted in 250 L capacity concrete circular tanks (replicated four (4) times) with 20 shrimp per tank. Growth, survival, respiratory burst activity, total hemocyte count (THC), and in vivo resistance to WSV infection were evaluated. Weight gain of the shrimp was significantly higher in the immunostimulant-fed groups compared to the control. However, different levels of the immunostimulants did not differ in their effect on the the growth of the shrimp. On the other hand, respiratory burst activity and total haemocyte count (THC) were significantly higher in the group supplemented with 0.2% peptidoglycan + MOS than the rest of the treatments. Likewise, survival after infection with White Spot Virus (WSV) was significantly increased in the 0.2% peptidoglycan + MOS compared to the other groups. The present results demonstrated that using peptidoglycan and MOS together at 0.2% of the diet improves growth, activates immune responses such as respiratory burst activity and THC in P. monodon and give better protection to the shrimp against WSV infection.

The aim of this study was to standardize a loop-mediated isothermal amplification (LAMP) assay for the detection of Vibrio harveyi , the causative agent of vibriosis in Asian seabass, Lates calcarifer. The dnaJ gene of the bacterial pathogen was used as the target gene for the LAMP assay. It was optimized at an incubation time of 1 h at 63°C. The assay was highly specific for V. harveyi and did not cross-react with other bacterial pathogens offish. However, the assay was able to detect V. harveyi that was isolated from infected shrimps. The limit of detection of the LAMP assay was 40 pg of DNA mL-1 or 40 fg of the genomic DNA per LAMP reaction and was 10 times more sensitive than conventional PCR in detecting the bacterial pathogen from infected samples. The LAMP products can be quantified spectrophotometrically using hydroxynaphthol blue (HNB) dye and showed positive correlation with the amount of the pathogen. These results demonstrated that LAMP is a simple and sensitive detection technique that has potential application for routine diagnosis of vibrosis caused by V. harveyi in Asian seabass and other aquatic species.

Partial sequence of the dnaJ gene of Vibrio harveyi, which was isolated from diseased juvenile Asian seabass, Lates calcarifer was identified. The partial sequence of dnaJ gene of V. harveyi was 447 bp and shared at least 77% identity at the nucleotide level with the dnaJ gene of other Vibrios. It was distinct from the dnaJ gene of other Vibrios but was closely related with the dnaJ gene of V. rotiferianus and V. campbellii having at least 90% nucleotide identity. PCR primers targeting this gene were designed to detect the pathogen in Asian seabass. The assay was specific to V. harveyi and the limit of detection was 100 pg of genomic DNA ml-1 or 100 fg of bacterial genomic DNA in a PCR reaction. This corresponded to a sensitivity of approximately 20 genome equivalents (GE) of V. harveyi. These results indicate that the dnaJ gene is a good candidate to develop primers for the PCR assay in detecting V.harveyi in fish.