Next generation sequencing uncovers unexpected pathogens associated with early mortality syndrome (EMS) in shrimp Kallaya Sritunyalucksana1, Anuphap Prachumwat1, Sissades Tongsima2, Siripong Thitamadee3, Timothy W. Flegel4 1 Shrimp-virus Interaction Laboratory (ASVI), National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok 10400, Thailand 2 Biostatistics and Informatics Laboratory, Genome Institute National Center for Genetic Engineering and Biotechnology, Pathum Thani 12120, Thailand 3 Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand 4 National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathum Thani 12120, Thailand E-mail: [email protected] _________________________________________________________________________________ ABSTRACT The most threatening new problem in Asian shrimp aquaculture since 2009 has been serious mortality within approximately the first 35 days after stocking shrimp ponds with post larvae. The problem is commonly called early mortality syndrome (EMS) and started at the early of 2012 in Thailand and has caused Thai shrimp production to reduce 54% in 2013. The cause of EMS was originally unknown. We carried out a metagenomic analysis to determine whether any unique bacteria (culturable or unculturable) might be present in EMS ponds when compared to normal ponds. DNA was extracted from pooled HP tissue samples from normal and EMS shrimp ponds and subjected to PCR amplification of small subunit ribosomal RNA (ssu rRNA) gene fragments using universal bacterial primers. After primer and multiplex adapter sequence trimming, the remaining 59.7 million bases from 163,102 reads with lengths of at least 100 bp were used to construct unique sequences (operational taxonomic units, OTUs). Since no OTU were found to be exclusive to EMS ponds, we compared the proportion of reads for OTU between EMS ponds and normal ponds. This comparison revealed 32 OTUs of six genera from four algorithms that had a significantly higher proportion of reads in EMS ponds than in normal ponds. This comparison revealed OTU with high identity to expected Vibrio species (e.g., V. parahaemolyticus, V. harveyi and V. campbellii) and with high identity to other bacteria known from aquatic environments, but never previously reported as shrimp pathogens. The latter included the genera Delftia and Ralstonia from the order Berkholderiales and Leifsonia and Rhodococcus from the Order Actinomycetales. Preliminary studies with bioassay confirmed that these bacteria could be direct shrimp pathogens. Besides the bacterial pathogens, we also found the concurrent problems in shrimp associated with EMS including the occurrence of aggregated transformed microvilli (ATM). Preliminary study using the next generation sequencing and bioinformatic analysis suggested that there might be the pathogen involved. Further investigations on these pathogens are urgently required in order to design the appropriate strategy to control EMS in shrimp industry. Keywords: early mortality syndrome (EMS), aggregated transformed microvilli (ATM), metagenomic, next generation sequencing ACKNOWLEDGEMENTS: We thank National Research Council of Thailand (NRCT), National Center for Genetic Engineering and Biotechnology (BIOTEC) and Agricultural Research Development Agency (ARDA) for grant support for the study on shrimp EMS. Next Generation Sequencing for Genetic and Genomic Studies (NGS2014) 29 - 30 กรกฎาคม 2557 ณ โรงแรมวินเซอร สวีทส สุขุมวิท กรุงเทพฯ -6-