Abstract

A Florida manatee unusual mortality event (UME) in Brevard County was declared in 2013 due to undermined causes and has not been closed to date. A dramatic reduction of seagrass due to long-term, non-toxic phytoplankton blooms in the Indian River Lagoon preceded the UME. A dietary change due to little or no seagrass availability was a hypothesized contributor to the manatee deaths. To investigate the gut microbiota and diet consumed by manatees in the area affected by the UME, a high-throughput metagenomic barcoding approach was implemented. The Illumina MiSeq next-generation platform was used to generate non-overlapping 2x300 bp paired-end DNA barcode sequences to provide comprehensive and rapid indexing of species. A targeted sequencing approach based on the bacteria 16S rRNA genes was employed to PCR amplify loci of available microbial species present in the gut. Manatee fecal material (n=20) from UME-associated animals was compared to ‘control’ samples (n=10) killed by boat strikes from the same area. The MiSeq runs produced millions of sequences in total with a minimum Phred score of 20 on trimmed reads. This represents a minimum base call accuracy of approximately 99%, providing high confidence in the quality of DNA submitted. Operational taxonomic units (OTUs) observed richness of species diversity (α-diversity), and differences between control and experimental samples (β-diversity) were assessed to determine differences in bacterial species associated with the control and UME samples. Microbiota from over 500 genera and nearly 1800 bacterial species were identified in the gut, and the UME samples had slightly altered microorganism communities with higher read counts of some common and potentially pathogenic species. No single bacterial species was exclusive to either group. Sequencing the gut microbiome can help to identify diagnostic bacterial species, such as toxic gut microbiota, and irregular community assemblages signifying a physiological change. This high-throughput approach can add to the tools used to investigate this and future UMEs in manatees.

*Presenting author