Abundance and Multilocus Sequence Analysis of Vibrio Bacteria Associated with Diseased Elkhorn Coral (Acropora palmata) of the Florida Keys

Abstract

The critically endangered elkhorn coral (Acropora palmata) is affected by white pox disease (WPX) throughout the Florida Reef Tract and wider Caribbean. The bacterium Serratia marcescens was previously identified as one etiologic agent of WPX but is no longer consistently detected in contemporary outbreaks. It is now believed that multiple etiologic agents cause WPX; however, to date, no other potential pathogens have been thoroughly investigated. This study examined the association of Vibrio bacteria with WPX occurrence from August 2012 to 2014 at Looe Key Reef in the Florida Keys, USA. The concentration of cultivable Vibrio was consistently greater in WPX samples than in healthy samples. The abundance of Vibrio bacteria relative to total bacteria was four times higher in samples from WPX lesions than in adjacent apparently healthy regions of diseased corals based on quantitative PCR (qPCR). Multilocus sequence analysis (MLSA) was used to assess the diversity of 69 Vibrio isolates collected from diseased and apparently healthy A. palmata colonies and the surrounding seawater. Vibrio species with known pathogenicity to corals were detected in both apparently healthy and diseased samples. While the causative agent(s) of contemporary WPX outbreaks remains elusive, our results suggest that Vibrio spp. may be part of a nonspecific heterotrophic bacterial bloom rather than acting as primary pathogens. This study highlights the need for highly resolved temporal sampling in situ to further elucidate the role of Vibrio during WPX onset and progression.IMPORTANCE Coral diseases are increasing worldwide and are now considered a major contributor to coral reef decline. In particular, the Caribbean has been noted as a coral disease hot spot, owing to the dramatic loss of framework-building acroporid corals due to tissue loss diseases. The pathogenesis of contemporary white pox disease (WPX) outbreaks in Acropora palmata remains poorly understood. This study investigates the association of Vibrio bacteria with WPX.

Keywords: Acropora palmata; Caribbean; Vibrio; bacterial bloom; coral disease; pathobiome; pathobiont; white pox.

FIG 1

The abundance of TCBS cultivable Vibrio spp. (mean ± standard error of the mean [SEM] CFU per milliliter) for A. palmata mucus and seawater collected from Looe Key Reef, FL. Bars with the same letters designate significant groupings based on Tukey's multiple-comparison tests following one-way ANOVA for each sampling period. Abbreviations: H, mucus from apparently healthy colonies with no WPX signs; DPX, mucus from WPX lesions; DH, mucus from asymptomatic areas on colonies with WPX lesions; reef H₂O, seawater from approximately 1 m above the A. palmata colonies; surface H₂O, seawater from the first 10 cm of the sea surface.

FIG 2

The Vibrio relative abundance index (VAI) of A. palmata mucus samples collected from Looe Key Reef, FL, during August 2014. The VAI (mean ± SEM) was calculated as the ratio of Vibrio species genome equivalents to total bacteria genome equivalents measured by qPCR. Bars with the same letters designate significant groupings assessed by Tukey's multiple-comparison tests following one-way ANOVA. Abbreviations: H, mucus from apparently healthy colonies with no WPX signs; DPX, mucus from WPX lesions; DH, mucus from asymptomatic areas on colonies with WPX lesions.

FIG 3

Maximum likelihood phylogenetic analysis based on eight gene loci. Boldface type indicates study isolates. Brackets indicate Vibrio clades containing isolates from A. palmata colonies. Bootstrap values (≥70) for 500 iterations are shown. Abbreviations for isolation source: H, mucus from apparently healthy A. palmata colonies with no WPX signs; DPX, mucus from WPX lesions; DH, mucus from asymptomatic areas on colonies with WPX lesions; reef H₂O, seawater from approximately 1 m above the A. palmata colonies; surface H₂O, seawater from the first 10 cm of the sea surface.

FIG 4

Maximum likelihood phylogenetic analysis based on eight gene loci for the Harveyi clade. Boldface type indicates study isolates. Bootstrap values (≥70) for 500 iterations are shown. Abbreviations for isolation source: H, mucus from apparently healthy A. palmata colonies with no WPX signs; DPX, mucus from WPX lesions; DH, mucus from asymptomatic areas on colonies with WPX lesions; reef H₂O, seawater from approximately 1 m above the A. palmata colonies; surface H₂O, seawater from the first 10 cm of the sea surface.