Hakim JA et al. (2015), An abundance of Epsilonproteobacteria revealed ...

ECB-ART-44325

Front Microbiol 2015 Oct 13;6:1047. doi: 10.3389/fmicb.2015.01047.

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An abundance of Epsilonproteobacteria revealed in the gut microbiome of the laboratory cultured sea urchin, Lytechinus variegatus.

Hakim JA , Koo H , Dennis LN , Kumar R , Ptacek T , Morrow CD , Lefkowitz EJ , Powell ML , Bej AK , Watts SA .

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In this study, we have examined the bacterial community composition of the laboratory cultured sea urchin Lytechinus variegatus gut microbiome and its culture environment using NextGen amplicon sequencing of the V4 segment of the 16S rRNA gene, and downstream bioinformatics tools. Overall, the gut and tank water was dominated by Proteobacteria, whereas the feed consisted of a co-occurrence of Proteobacteria and Firmicutes at a high abundance. The gut tissue represented Epsilonproteobacteria as dominant, with order Campylobacterales at the highest relative abundance (>95%). However, the pharynx tissue was dominated by class Alphaproteobacteria. The gut digesta and egested fecal pellets had a high abundance of class Gammaproteobacteria, from which Vibrio was found to be the primary genus, and Epsilonproteobacteria, with genus Arcobacter occurring at a moderate level. At the class level, the tank water was dominated by Gammaproteobacteria, and the feed by Alphaproteobacteria. Multi-Dimensional Scaling analysis showed that the microbial community of the gut tissue clustered together, as did the pharynx tissue to the feed. The gut digesta and egested fecal pellets showed a similarity relationship to the tank water. Further analysis of Campylobacterales at a lower taxonomic level using the oligotyping method revealed 37 unique types across the 10 samples, where Oligotype 1 was primarily represented in the gut tissue. BLAST analysis identified Oligotype 1 to be Arcobacter sp., Sulfuricurvum sp., and Arcobacter bivalviorum at an identity level >90%. This study showed that although distinct microbial communities are evident across multiple components of the sea urchin gut ecosystem, there is a noticeable correlation between the overall microbial communities of the gut with the sea urchin L. variegatus culture environment.

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Genes referenced: LOC100887844 LOC583082

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	Figure 1. Stacked column bar graph depicting the relative abundances and distribution of the most highly abundant resolved taxa across the 10 samples of this study. The gut microbiome consisted mainly of Phylum Proteobacteria, whereas the sea urchin feed was dominated by both Firmicutes and Proteobacteria. At the highest resolution, order Campylobacterales was determined to be the most abundant taxa in the gut tissue. In the gut digesta and egested fecal pellets, Vibrio, Arcobacter, and Agarivorans were observed. Relative abundances were performed through QIIME (v1.7.0), and graphs were generated using Microsoft Excel software (Microsoft, Seattle, WA). UR1, sea urchin 1; UR2, sea urchin 2.
	Figure 2. Oligotype distributions for the 10 samples used in this study. The relative abundance of each oligotype within the total Campylobacterales diversity for each sample is presented in stacked column bar graphs (bottom), and the proportion of the relative abundance of total Campylobacterales within all bacterial diversity for each sample is shown with light gray bars (top). Oligotyping analyses were performed using the open-source pipeline for oligotyping, available at http://oligotyping.org. The stacked column bar graphs were generated using Microsoft Excel software (Microsoft, Seattle, WA). UR1, sea urchin 1; UR2, sea urchin 2.
	Figure 3. 2D multidimensional scaling (MDS) graph generated through PRIMER-6 (www.primer-e.com). Overlay of similarity clusters were produced according to Bray–Curtis Similarity values, set at 10% intervals from 20% to 50%. The pharynx tissue and sea urchin feed sample microbial ecologies clustered with a similarity greater than 40%. The tank water, gut digesta, and egested fecal pellet samples also clustered together at a similarity greater than 20%. The gut tissue samples from the two sea urchins showed a divergent cluster pattern, illustrating a reduced degree of similarity to the other samples of the study. UR1, sea urchin 1; UR2, sea urchin 2. Similarity = Bray–Curtis Similarity (scaled to 100).
	Figure 4. Heatmap of microbial compositions at the order level. The rows represent the bacterial taxa and the columns represent the 10 samples used in this study. Both dendrograms were created using hierarchical clustering (complete linkage) of the compositional data. The heatmap was generated using the “heatmap.2” function in R package (available at http://CRAN.R-project.org/package=gplots). UR1, sea urchin 1; UR2, sea urchin 2.

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