Microbial diversity and potential pathogens in ornamental fish aquarium water

Abstract

Ornamental fishes are among the most popular and fastest growing categories of pets in the United States (U.S.). The global scope and scale of the ornamental fish trade and growing popularity of pet fish in the U.S. are strong indicators of the myriad economic and social benefits the pet industry provides. Relatively little is known about the microbial communities associated with these ornamental fishes or the aquarium water in which they are transported and housed. Using conventional molecular approaches and next generation high-throughput amplicon sequencing of 16S ribosomal RNA gene hypervariable regions, we characterized the bacterial community of aquarium water containing common goldfish (Carassius auratus) and Chinese algae eaters (Gyrinocheilus aymonieri) purchased from seven pet/aquarium shops in Rhode Island and identified the presence of potential pathogens. Our survey identified a total of 30 phyla, the most common being Proteobacteria (52%), Bacteroidetes (18%) and Planctomycetes (6%), with the top four phyla representing >80% of all sequences. Sequences from our water samples were most closely related to eleven bacterial species that have the potential to cause disease in fishes, humans and other species: Coxiella burnetii, Flavobacterium columnare, Legionella birminghamensis, L. pneumophila, Vibrio cholerae, V. mimicus. V. vulnificus, Aeromonas schubertii, A. veronii, A. hydrophila and Plesiomonas shigelloides. Our results, combined with evidence from the literature, suggest aquarium tank water harboring ornamental fish are an understudied source for novel microbial communities and pathogens that pose potential risks to the pet industry, fishes in trade, humans and other species.

Figure 1. Phylum-level Bacterial Diversity.

Phylum-level bacterial diversity as revealed by pyrotag sequencing of the hypervariable V3–V5 region of 16S rRNA genes in our study samples (Table 1). Thirty phyla were detected. A) Relative frequency of phyla as a proportion of total tags. Interstore variance in relative frequency is depicted by color for the two most abundant phyla, Proteobacteria and Bacteroidetes, representing ∼70% of all sequences. B) Interstore variance in relative frequency for the remaining phyla (<30% of total reads) normalized to 100% after subtracting the Proteobacteria and Bacteriodetes.

Figure 2. Alpha Diversity in Pet Shops.

Alpha diversity within retail stores based on A) CatchAll's Best Model analysis with non-rarified data shown with 95% Bonferroni-corrected upper and lower confidence bounds for each estimate. B) Phylogenetic Diversity (PD) - Whole Tree analysis calculated after rarifying samples to equal sequencing depth in QIIME. Both metrics were applied to calculate inter-store alpha diversity by grouping 2 samples from each store for a single analysis.

Figure 3. Beta Diversity Between Samples.

Beta diversity between samples based on A) Morisita-Horn similarity metrics of non-rarified data visualized using Non-Metric Multidimensional Scaling (NMDS). B) Unwieghted UNIFRAC distance calculated after rarifying samples to equal sequence depth in QIIME, visualized using Principal Component Analysis.

Figure 4. Vibrio Reference Tree.

Vibrio reference tree created using sequences of isolates from the SILVA-ARB 16S rRNA gene database. Shown in red are Vibrio OTUs from our FLX run whose GAST taxonomy fell within the Vibrio genus. Red OTUs are labeled with species designations, tag count and the number of samples that contained that OTU.