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How to quantify endosymbionts in the gills of the hydrothermal vent mussel Bathymodiolus azoricus? 3D-FISH versus qPCR
Abstracts / Comparative Biochemistry and Physiology, Part A 151 (2008) S37–S43
Hydrothermal vents along the oceanic dorsales axes are characterised by time and space-scale variable extreme physical and chemical conditions. Hydrothermal ecosystem is developed in the mixing zone between the hydrothermal fluid, very hot and rich in sulfur and metals and anoxic, and the surrounding sea-water, cold and aerobic. Actually, only few prokaryotes are known to be able to live at temperatures above 100 °C when eukaryotes appear to be more susceptible to high temperatures and mainly live in environmental temperature below 55 °C. In this context, the thermotolerance of marine species has been widely discussed in hydrothermal environment despite the fact that most of species are leaving at temperatures below 30 °C. Among annelids, Alvinellidae represent a important part of the hydrothermal annelid fauna by occupying various ecological niches characterized by variable environmental pressures. Two main genus are found in hydrothermal vents, Alvinella and Paralvinella, both strictly endemic to Pacific vents. Alvinella pompejana and Paralvinella sulfincola are able to resist to extreme temperature when other Paralvinella species are living in colder environments such as. P. grasslei that is living in the thermal moderate part of the chimney where the mineral charge is low. In order to study the response to temperature challenge, transcriptomic tools (cdNA library sequencing and quantitative PCR) have been developed and used to identify new genes regulated by temperature in P. grasslei. First results are presented and discussed. doi:10.1016/j.cbpa.2008.05.157 20. Development of a micro cell culture analogue for evaluation of chemical toxicity, called Tox Cell Chip C. Vanparys, K. Niescierowicz (University of Antwerp, Belgium); H. Xu, M. Shuler (Cornell University, USA); W. De Coen, R. Blust, J. Robbens (University of Antwerp, Belgium) Recently, a promising new techniques, called micro cell culture analog (µCCA), has been developed to implement biological processes as metabolisation and accumulation into in vitro systems. This µCCA is a silicon chip (2 × 2 cm) with different cell compartments interconnected with a microfluidic channelling system. By seeding different cell lines in the different compartments, the functionalities of the different cell types are in constant relation to each other. For instance, liver cells can represent metabolism while fat cells represent bioaccumulation. By means of a circulation system in micro-channels,
S43
a chemical can migrate between the different compartments and subsequently be subjected to biological processes inherent to the different cell types. In this way, a more accurate evaluation of the toxicity of a chemical can be achieved. At this moment, preliminary proof-of-concept experiments have been performed to evaluate the potential of this technique in a toxicological context, but to a rather limited extent. Only robust parameters, as survival of cells and glutathione depletion, were measured. In the future, this TCC will be further optimised and evaluated with more comprehensive ‘omics’ techniques (microarray and real-time PCR) to further explore the use of this promising and innovative technique in toxicity evaluation of chemicals. doi:10.1016/j.cbpa.2008.05.158 21. How to quantify endosymbionts in the gills of the hydrothermal vent mussel Bathymodiolus azoricus? 3D-FISH versus qPCR I. Boutet, A. Tanguy, F.H. Lallier, (Station Biologique de Roscoff, France); S. Halary, S. Duperron, F. Gaill (Univ. Paris 06, France) The mussel Bathymodiolus azoricus forms dense colonies around the deep sea hydrothermal vents of the mid-Atlantic ridge. They derive most if not all of their metabolic needs from the two types of endosymbiotic chemoautotrophic bacteria they harbour in their gills. The two types of gamma-proteobacteria, a methanotroph and a thiotroph, are found in variable amounts in the bacteriocytes, apparently as a function of methane and sulfide availability in the mussel environment. Here we compare two methods aiming at quantifying each type of bacteria. The first one (3D-FISH) measures volumes occupied by each type of symbiont in bacteriocyte sections from a vent mussel gill filament using fluorescence in situ hybridization with 16S rRNA-based specific probes coupled to three dimensional microscopy and image analysis carried out by a dedicated software. The other method (qPCR) uses the same probes and some others targeting specific metabolic genes to measure the relative expression of these genes in gill extracts. Qualitatively, the two methods give congruent results and confirm the impact of local environmental parameters on symbiont abundances. doi:10.1016/j.cbpa.2008.05.184
Hydrothermal vents along the oceanic dorsales axes are characterised by time and space-scale variable extreme physical and chemical conditions. Hydrothermal ecosystem is developed in the mixing zone between the hydrothermal fluid, very hot and rich in sulfur and metals and anoxic, and the surrounding sea-water, cold and aerobic. Actually, only few prokaryotes are known to be able to live at temperatures above 100 °C when eukaryotes appear to be more susceptible to high temperatures and mainly live in environmental temperature below 55 °C. In this context, the thermotolerance of marine species has been widely discussed in hydrothermal environment despite the fact that most of species are leaving at temperatures below 30 °C. Among annelids, Alvinellidae represent a important part of the hydrothermal annelid fauna by occupying various ecological niches characterized by variable environmental pressures. Two main genus are found in hydrothermal vents, Alvinella and Paralvinella, both strictly endemic to Pacific vents. Alvinella pompejana and Paralvinella sulfincola are able to resist to extreme temperature when other Paralvinella species are living in colder environments such as. P. grasslei that is living in the thermal moderate part of the chimney where the mineral charge is low. In order to study the response to temperature challenge, transcriptomic tools (cdNA library sequencing and quantitative PCR) have been developed and used to identify new genes regulated by temperature in P. grasslei. First results are presented and discussed. doi:10.1016/j.cbpa.2008.05.157 20. Development of a micro cell culture analogue for evaluation of chemical toxicity, called Tox Cell Chip C. Vanparys, K. Niescierowicz (University of Antwerp, Belgium); H. Xu, M. Shuler (Cornell University, USA); W. De Coen, R. Blust, J. Robbens (University of Antwerp, Belgium) Recently, a promising new techniques, called micro cell culture analog (µCCA), has been developed to implement biological processes as metabolisation and accumulation into in vitro systems. This µCCA is a silicon chip (2 × 2 cm) with different cell compartments interconnected with a microfluidic channelling system. By seeding different cell lines in the different compartments, the functionalities of the different cell types are in constant relation to each other. For instance, liver cells can represent metabolism while fat cells represent bioaccumulation. By means of a circulation system in micro-channels,
S43
a chemical can migrate between the different compartments and subsequently be subjected to biological processes inherent to the different cell types. In this way, a more accurate evaluation of the toxicity of a chemical can be achieved. At this moment, preliminary proof-of-concept experiments have been performed to evaluate the potential of this technique in a toxicological context, but to a rather limited extent. Only robust parameters, as survival of cells and glutathione depletion, were measured. In the future, this TCC will be further optimised and evaluated with more comprehensive ‘omics’ techniques (microarray and real-time PCR) to further explore the use of this promising and innovative technique in toxicity evaluation of chemicals. doi:10.1016/j.cbpa.2008.05.158 21. How to quantify endosymbionts in the gills of the hydrothermal vent mussel Bathymodiolus azoricus? 3D-FISH versus qPCR I. Boutet, A. Tanguy, F.H. Lallier, (Station Biologique de Roscoff, France); S. Halary, S. Duperron, F. Gaill (Univ. Paris 06, France) The mussel Bathymodiolus azoricus forms dense colonies around the deep sea hydrothermal vents of the mid-Atlantic ridge. They derive most if not all of their metabolic needs from the two types of endosymbiotic chemoautotrophic bacteria they harbour in their gills. The two types of gamma-proteobacteria, a methanotroph and a thiotroph, are found in variable amounts in the bacteriocytes, apparently as a function of methane and sulfide availability in the mussel environment. Here we compare two methods aiming at quantifying each type of bacteria. The first one (3D-FISH) measures volumes occupied by each type of symbiont in bacteriocyte sections from a vent mussel gill filament using fluorescence in situ hybridization with 16S rRNA-based specific probes coupled to three dimensional microscopy and image analysis carried out by a dedicated software. The other method (qPCR) uses the same probes and some others targeting specific metabolic genes to measure the relative expression of these genes in gill extracts. Qualitatively, the two methods give congruent results and confirm the impact of local environmental parameters on symbiont abundances. doi:10.1016/j.cbpa.2008.05.184