IDENTIFICATION OF BACTERIA ASSOCIATED WITH DINOFLAGELLATES (DINOPHYCEAE) ALEXANDRIUM SPP. USING TYRAMIDE SIGNAL AMPLIFICATION–FLUORESCENT IN SITU HYBRIDIZATION AND CONFOCAL MICROSCOPY¹

Abstract

In the marine environment, phytoplankton and bacterioplankton can be physically associated. Such association has recently been hypothesized to be involved in the toxicity of the dinoflagellate genus Alexandrium. However, the methods, which have been used so far to identify, localize, and quantify bacteria associated with phytoplankton, are either destructive, time consuming, or lack precision. In the present study we combined tyramide signal amplification–fluorescent in situ hybridization (TSA‐FISH) with confocal microscopy to determine the physical association of dinoflagellate cells with bacteria. Dinoflagellate attached microflora was successfully identified with TSA‐FISH, whereas FISH using monolabeled probes failed to detect bacteria, because of the dinoflagellate autofluorescence. Bacteria attached to entire dinoflagellates were further localized and distinguished from those attached to empty theca, by using calcofluor and DAPI, two fluorochromes that stain dinoflagellate theca and DNA, respectively. The contribution of specific bacterial taxa of attached microflora was assessed by double hybridization. Endocytoplasmic and endonuclear bacteria were successfully identified in the nonthecate dinoflagellate Gyrodinium instriatum. In contrast, intracellular bacteria were not observed in either toxic or nontoxic strains of Alexandrium spp. Finally, the method was successfully tested on natural phytoplankton assemblages, suggesting that this combination of techniques could prove a useful tool for the simultaneous identification, localization, and quantification of bacteria physically associated with dinoflagellates and more generally with phytoplankton.

Aerobic Degradation of Petroleum Components by Microbial Consortia

“…However, they do not exist in isolation, and their phycosphere (loosely defined as the zone around algal cells in which bacteria feed on algal products) constitutes an important habitat that is colonised by an abundant and diverse community of heterotrophic bacteria [206,207].…”

Intriguing diversity among diazotrophic picoplankton along a Mediterranean transect: a dominance of rhizobia

“…Prior to hybridizations, filters containing fixed cells were covered with agarose to avoid cell loss during hybridization steps, according to Biegala and Raimbault (2008), with slight modification in Le Moal and Biegala (2009), where 0.4% agarose was used instead of 0.1% for the >10 µm size fraction. Hybridizations were done according to the protocol of Biegala et al (2002), modified in Biegala and Raimbault (2008). Briefly, unicellular diazotrophic cyanobacteria were hybridized with the Horse Radish Peroxidase-labeled 16S rDNA Nitro821 probe (Thermo, Germany, 5 -CAA GCC ACA CCT AGT TTC-3 ) which is specific for the UCYN lineage (Mazard et al, 2004), and subsequently stained in green with FITC (Fluorescein IsoThioCyanate, TSA-Kit Perkin Elmer, France).…”

Erratum to Bacterial diversity in toxic Alexandrium tamarense blooms off the Orkney Isles and the Firth of Forth

“…Of the many natural factors thought to play an important role in HAB dynamics, algal-bacterial interactions are among the components increasingly cited as potentially important regulators of algal growth and toxicity (Doucette et al 1998;Töbe et al 2001). Up to now there have been some studies on bacteria associated with cultured toxic A. tamarense strains (Ogata et al 1990;Franca et al 1996;Gallagher et al 1997;Doucette et al 1998;Hold et al 2001;Biegala et al 2002). The bacteria were identified as Proteobacteria predominantly related to the genera Roseobacter, Alteromonas and Pseudomonas (Kopp et al 1997;Gallacher and Smith 1999).…”