Now showing items 1-5 of 5

    • Conference paper

      Development of a simple, rapid, cost-effective diagnostic kit for WSSV 

      PMT Arabit, ADD Nicolasora, PEZ Go, CMA Caipang & MBB Maningas - In MRR Romana-Eguia, FD Parado-Estepa, ND Salayo & MJH Lebata-Ramos (Eds.), Resource Enhancement and Sustainable Aquaculture Practices in Southeast Asia: Challenges in Responsible Production … International Workshop on Resource Enhancement and Sustainable Aquaculture Practices in Southeast Asia 2014 (RESA), 2015 - Aquaculture Department, Southeast Asian Fisheries Development Center
      Shrimp aquaculture is one of the most important sources of income and livelihood in the Philippines. For the past two decades, the white spot syndrome virus (WSSV) has adversely affected the production of the Philippine shrimp industry resulting to losses in revenue. Shrimps infected by the virus experience up to 100% mortality, 3 to 10 days post-infection. One way of controlling the disease is early detection, which remains to be too complicated and inaccessible to shrimp farmers. Being a DNA virus, the first step to WSSV diagnosis is the isolation of high-quality DNA suitable for polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP). Using readily available and affordable reagents, a DNA extraction protocol has been especially developed for rapid WSSV-detection; DNA has been successfully extracted from the pleopods of shrimps and the results were comparable with that of commercially available kits from Promega and Zymoresearch. LAMP has been optimized for WSSV detection in the temperature range of 55°C to 68°C and was shown to be faster and ten times more sensitive than conventional PCR. This study together with a locally fabricated machine, offers a more convenient, practical and efficient way of detecting WSSV, with the advantage of using non-invasive means of obtaining shrimp tissue therefore not losing any shrimp meat in the process.
    • Conference paper

      First record of Laem-Singh virus in black tiger shrimp (Penaeus monodon) in the Philippines 

      CAM Cruz, PC dela Cruz, PCD Alcala, FGM Tagle, ES Santos, MD Santos & MBB Maningas - In MRR Romana-Eguia, FD Parado-Estepa, ND Salayo & MJH Lebata-Ramos (Eds.), Resource Enhancement and Sustainable Aquaculture Practices in Southeast Asia: Challenges in Responsible Production … International Workshop on Resource Enhancement and Sustainable Aquaculture Practices in Southeast Asia 2014 (RESA), 2015 - Aquaculture Department, Southeast Asian Fisheries Development Center
      Laem-Singh Virus (LSNV), a single-stranded RNA virus that causes growth retardation in Penaeus monodon, is also known as Monodon Slow-Growth Syndrome (MSGS) virus. Black Tiger shrimps afflicted with this virus exhibit unusual dark color, a weight gain of less than 0.1 g in 1 to 2 weeks, unusual yellow markings, bamboo-shaped abdominal markings and brittle antennae. It was first detected in Thailand and the virus quickly spread to neighboring Asian countries such as Malaysia and Singapore. The shrimp economy of countries where infections have occurred experienced losses in the export of live shrimps and broodstocks. An earlier study in 2009 reported that LSNV was not present in the Philippines. However, since no follow-up researches were done in the succeeding years, this study was conducted to detect the presence of virus in selected sites of Luzon. Results based on biased sampling method and RT-PCR data indicated that LSNV is indeed present in the country. This is further supported by DNA sequence data, showing 100% identity with LSNV India isolate. Phylogenetic analysis showed that the Philippine isolate clustered closely with other LSNV isolates. The outcome of this study might have implications in the current practices in the Philippine shrimp aquaculture industry.
    • Conference paper

      Shrimp metabolism: The roles of lactate dehydrogenase (c31), glycogen phosphorylase (c34) and protein kinase (PK) as revealed by RNA interference 

      MVR Tare, H Kondo, I Hirono & MBB Maningas - In MRR Romana-Eguia, FD Parado-Estepa, ND Salayo & MJH Lebata-Ramos (Eds.), Resource Enhancement and Sustainable Aquaculture Practices in Southeast Asia: Challenges in Responsible Production … International Workshop on Resource Enhancement and Sustainable Aquaculture Practices in Southeast Asia 2014 (RESA), 2015 - Aquaculture Department, Southeast Asian Fisheries Development Center
      Energy metabolism is well-studied in vertebrate systems, providing insights on the genes and mechanisms involved in different pathways necessary for the survival of an organism. Yet, such studies are still lacking in invertebrate systems much more in shrimp. An earlier study has showed several contigs from the black tiger shrimp to be homologous to white spot syndrome virus (WSSV), a devastating pathogen in shrimp, including contig 31-WSSVORF82 (c31) and contig 34-WSSVORF21 (c34). This study aims to unveil the roles of three genes: c31, c34 and protein kinase (PK) in the shrimp system and its possible role in WSSV-infection. Rapid amplification of cDNA ends-polymerase chain reaction or RACE-PCR was used to obtain the full-length sequence of c31 and c34, followed by in vivo gene silencing using RNAi technology, and intramuscularly injecting dsRNA to WSSVchallenged Macrobrachium rosenbergii and Penaeus (Marsupenaeus) japonicus. Gene expression followed for healthy shrimps and dsRNA-treated shrimps.

      Mrc31 was revealed to be the enzyme lactase dehydrogenase (LDH), commonly released during tissue damage and is a marker for disease. The most parsimonious tree pictured Mrc31 to be sister clades to LDH of other shrimp species, Penaeus monodon and P. vannamei, supported with 100% and 72% bootstrap values, respectively. Mrc34 was highly homologous to the glycogen phosphorylase (GP) enzymes of other organisms including that of another shrimp, M. japonicus, bearing a bootstrap value of 99%. For PK, phylogenetic analysis revealed that the three open reading frames (ORFs) from P. monodon, M. rosenbergii and P. japonicus have 30% homology to WSSV-PK supported by a 98% bootstrap value. Mortality data from dsRNA-treated and WSSV-infected shrimps showed that treatment with dsRNA-LDH, GP and PK had significantly higher survival rates compared to that of the controls, Phosphate Buffered Saline (PBS) and Green Fluorescent Protein (GFP). Silencing the three genes in the shrimp has rendered some protective effect against the virus. Gene expression showed that all three genes are present in immune-related organs such as the gills, hepatopancreas and hemocyte. This study is the first to report the possible identities and functions of contigs 31, 34 and PK providing valuable data on the shrimp's genome.
    • Article

      Subtracted transcriptome profile of tiger shrimp (Penaeus monodon) that survived WSSV challenge 

      BA Maralit, MFH Ventolero, MBB Maningas, EC Amar & MD Santos - Dataset Papers in Science, 2014 - Hindawi Publishing Corporation
      There is increased interest in the development of virus-resistant or improved shrimp stock because production is currently hindered by outbreaks and limited understanding of shrimp defense. Recent advancement now allows for high-throughput molecular studies on shrimp immunity. We used next-generation sequencing (NGS) coupled with suppression subtractive hybridization (SSH) to generate a transcriptome database of genes from tiger shrimp that survived White spot syndrome virus (WSSV) challenge. A total of 9,597 unique sequences were uploaded to NCBI Sequence Read Archive with accession number SRR577080. Sixty-five unique sequences, 6% of the total, were homologous to genes of Penaeus monodon. Genes that were initially related to bacterial infection and environmental stress such as 14-3-3 gene, heat shock protein 90, and calreticulin were also found including a few full-length gene sequences. Initial analysis of the expression of some genes was done. Hemocyanin, ferritin, and fortilin-binding protein exhibited differential expression between survivor and control tiger shrimps. Furthermore, candidate microsatellite markers for brood stock selection were mined and tested. Four trinucleotide and one dinucleotide microsatellites were successfully amplified. The study highlights the advantage of the NGS platform coupled with SSH in terms of gene discovery and marker generation.
    • Conference paper

      Targeting essential gene utilizing RNA interference to protect the ailing shrimp/prawn industry against WSSV 

      JMS Lazarte & MBB Maningas - In MRR Romana-Eguia, FD Parado-Estepa, ND Salayo & MJH Lebata-Ramos (Eds.), Resource Enhancement and Sustainable Aquaculture Practices in Southeast Asia: Challenges in Responsible Production … International Workshop on Resource Enhancement and Sustainable Aquaculture Practices in Southeast Asia 2014 (RESA), 2015 - Aquaculture Department, Southeast Asian Fisheries Development Center
      The white spot syndrome virus (WSSV) remains to be the most widespread and devastating infectious agent that has hit particularly the marine shrimp aquaculture industry worldwide. To date, there are no known effective strategies that can combat WSSV infection. This study aimed to elucidate host-pathogen interaction through the functional study of host - gene. Utilizing RNA Interference, the function of contig23 (c23) in the shrimp genome, identified to have high homology with WSSVORF-325, was determined. Three set-ups were prepared for treatment of c23-, GFP-dsRNA, and PBS using Macrobrachium rosenbergii freshwater prawns. Each treatment group was challenged with WSSV and survival rate was recorded. C23-, and GFP-dsRNA injected prawns showed a significant survival rate of 100%, in contrast to 20% of the PBS injected prawns at 10 days post-infection (dpi). Results showed that injection of c23- and GFP-dsRNA prior to challenge with WSSV, delayed and reduced mortality in contrast to PBS-treated prawns, which showed high mortality. Gene expression analysis showed silencing of both WSSV and c23 at day 3 post-WSSV challenge. This study proved that c23-dsRNA has a protective effect on WSSVchallenged prawns and highlights its involvement in the infectivity of WSSV in M. rosenbergii.