METAGENOMIC ANALYSIS OF PROBIOTIC EFFECT ON SHRIMP MICROBIAL COMMUNITY

Becky L. Rivoire*, Susan E. Knudson, Jeremy Ellis, and Lucas S. Keeton
 
Keeton Industries, Inc. 
1520 Aquatic Drive
Wellington, CO  80521
Luke@Keetonaqua.com
 

Introduction: Culturable bacteria from aquatic environments is estimated to be less than 0.1% of the living bacteria present.  A high-resolution account of the complexity and diversity of bacteria in their natural environments is now possible using metagenomics. This relatively new technology provides an opportunity to phylotype and quantitate culture-independent microbial communities.  Metagenomics was used to evaluate the biodiversity of bacterial communities in shrimp ponds when treated or not treated with probiotics during the most stressful period of the shrimp lifecycle.  

Materials/Methods: Four adjacent five-acre shrimp ponds in the USA were used for this study; two were treated with probiotics and two were untreated.  Pond water samples were collected during the last three months of shrimp grow-out, days 74, 85, and 114 post-seeding with juvenile shrimp.  Samples were held at 4 °C for less than three days before centrifugation at 10,000 x g for 30 minutes.  Pellets were frozen at - 40 °C and sent to Transgenada Enterprises, Inc. (Mesa, AZ) for gDNA extraction and 16s rRNA gene sequencing.  Conventional bacterial culturing for Vibrio spp. and non-Vibrio organisms was carried out and water chemistry performed.

Findings: Twelve samples resulted in over 3.3 million reads.  rRNA phylotyping showed three novel organisms, which diverge from their closest reference sequence by more than 2% (Table 1). Correlation analysis is underway to assess community composition, quantity, and shifts in prominent bacteria, probiotics, and Vibrio species.  

Results: Preliminary analysis of these data show a very unique ecosystem with enriched novel bacteria in the water column at different times of the shrimp production cycle.  With further analysis, we expect to be able to delineate the ebb and flow of major microbial players and Vibrio spp. populations relative to the presence/absence of probiotics.