Characterization of Bacterial Symbionts in Deep-Sea Fauna: Protocols for Sample Conditioning, Fluorescence In Situ Hybridization, and Image Analysis

Duperron, Sébastien

doi:10.1007/8623_2015_73

Cited by 5 publications

(5 citation statements)

References 52 publications

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“…Individual worms were extracted from their tubes by holding down the posterior end of the tubes with a lightweight pair of forceps, and gently squeezing the tube behind the worm with a fine paintbrush. To ensure that host and symbiont based analyses were conducted on the same individuals, a single individual was divided into pieces, each for the different fixations outlined below [38]. The extraction process was difficult, and whole worms or even large pieces were not always obtained.…”

Section: Methodsmentioning

confidence: 99%

Cryptic frenulates are the dominant chemosymbiotrophic fauna at Arctic and high latitude Atlantic cold seeps

et al. 2018

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We provide the first detailed identification of Barents Sea cold seep frenulate hosts and their symbionts. Mitochondrial COI sequence analysis, in combination with detailed morphological investigations through both light and electron microscopy was used for identifying frenulate hosts, and comparing them to Oligobrachia haakonmosbiensis and Oligobrachia webbi, two morphologically similar species known from the Norwegian Sea. Specimens from sites previously assumed to host O. haakonmosbiensis were included in our molecular analysis, which allowed us to provide new insight on the debate regarding species identity of these Oligobrachia worms. Our results indicate that high Arctic seeps are inhabited by a species that though closely related to Oligobrachia haakonmosbiensis, is nonetheless distinct. We refer to this group as the Oligobrachia sp. CPL-clade, based on the colloquial names of the sites they are currently known to inhabit. Since members of the Oligobrachia sp. CPL-clade cannot be distinguished from O. haakonmosbiensis or O. webbi based on morphology, we suggest that a complex of cryptic Oligobrachia species inhabit seeps in the Norwegian Sea and the Arctic. The symbionts of the Oligobrachia sp. CPL-clade were also found to be closely related to O. haakonmosbiensis symbionts, but genetically distinct. Fluorescent in situ hybridization and transmission electron micrographs revealed extremely dense populations of bacteria within the trophosome of members of the Oligobrachia sp. CPL-clade, which is unusual for frenulates. Bacterial genes for sulfur oxidation were detected and small rod shaped bacteria (round in cross section), typical of siboglinid-associated sulfur-oxidizing bacteria, were seen on electron micrographs of trophosome bacteriocytes, suggesting that sulfide constitutes the main energy source. We hypothesize that specific, local geochemical conditions, in particular, high sulfide fluxes and concentrations could account for the unusually high symbiont densities in members of the Oligrobrachia sp. CPL-clade.

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Section: Methodsmentioning

confidence: 99%

Cryptic frenulates are the dominant chemosymbiotrophic fauna at Arctic and high latitude Atlantic cold seeps

et al. 2018

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“…Gills were fixed individually after each experiment using 3.7% formaldehyde in SFSW, rinsed in SFSW, dehydrated in increasing ethanol series, then embedded in Steedman resin as described in Duperron (2015). Sections (8 µm-thick) were cut on a microtome (Thermo, UK) and placed on SuperFrost Plus slides (VWR, France).…”

Section: Specimen Sampling Edu Incubations and Vizualisationmentioning

confidence: 99%

Regionalized cell proliferation in the symbiont-bearing gill of the hydrothermal vent mussel Bathymodiolus azoricus

et al. 2020

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Deep-sea mussels Bathymodiolus spp. harbor high densities of chemosynthetic bacterial symbionts located within their gill epithelial cells. Compared to non-symbiotic coastal mussel relatives of similar size, Bathymodiolus gills are considerably larger, a feature often considered an adaptation to symbiosis because it is related to the presence of intracellular bacteria in epithelial cells located in the lateral zone. In order to document the mechanisms underlying these sizes differences, this study compares gill cell proliferation patterns in Bathymodiolus azoricus and Mytilus edulis using microscopybased approaches. We used incubation experiments with a synthetic nucleotide (5-ethynyl 2'deoxyuridine, EdU), detectable throughout novel cell divisions, and phosphohistone H3 immunolabeling, a marker of mitosis. The results revealed proliferation areas in the ciliated zone and in the bacteria-loaded bacteriocytes located close to the frontal zone of gill filaments, swept by the incurrent sea-waterflow, and also in the dorsal region of gills in B. azoricus. Cell proliferation seems far less intensive in M. edulis. This study overall suggests high cell turnover and fast tissue dynamics in symbiont-bearing mussels.

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“…were performed using generalist probes targeting Eubacteria (EUB338), Gammaproteobacteria (GAM42), and the probe NON338 (antisense of EUB338) as a negative control {Amann, 1990 #159}. All hybridizations were conducted using 30% formamide at 46°C for 3 hours, followed by a 15 minutes rinse in appropriate buffer using the protocol described in (Duperron, 2017). FISH hybridizations were performed on whole specimens of Capitella fixed in paraformaldehyde 4% to visualize epibionts, as well as on 8µm-thick cross sections of specimens that were previously embedded in Steedman Wax as described in (Duperron et al, 2008), using DAPI as a background stain.…”

Section: Fluorescence In Situ Hybridization (Fish) Of Epibiotic Microflora -Fish Experimentsmentioning

confidence: 99%

Investigation of Capitella spp. symbionts in the context of varying anthropic pressures: First occurrence of a transient advantageous epibiosis with the giant bacteria Thiomargarita sp. to survive seasonal increases of sulfides in sediments

Hourdez

Boidin-Wichlacz

Jollivet

et al. 2021

Science of The Total Environment

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HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

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Characterization of Bacterial Symbionts in Deep-Sea Fauna: Protocols for Sample Conditioning, Fluorescence In Situ Hybridization, and Image Analysis

Cited by 5 publications

References 52 publications

Cryptic frenulates are the dominant chemosymbiotrophic fauna at Arctic and high latitude Atlantic cold seeps

Cryptic frenulates are the dominant chemosymbiotrophic fauna at Arctic and high latitude Atlantic cold seeps

Regionalized cell proliferation in the symbiont-bearing gill of the hydrothermal vent mussel Bathymodiolus azoricus

Investigation of Capitella spp. symbionts in the context of varying anthropic pressures: First occurrence of a transient advantageous epibiosis with the giant bacteria Thiomargarita sp. to survive seasonal increases of sulfides in sediments

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