Ecological Archives E093-169-A1

Nicolas Toupoint, Lisandre Gilmore-Solomon, François Bourque, Bruno Myrand, Fabrice Pernet, Frédéric Olivier, and Réjean Tremblay. 2012. Match/mismatch between the Mytilus edulis larval supply and seston quality: effect on recruitment. Ecology 93:1922–1934. http://dx.doi.org/10.1890/11-1292.1

Appendix A. Methodological technical details for fatty acids and flow cytometry analysis.

 1. Fatty acids analysis

Filters were stored under nitrogen at -80 °C in dichloromethane-methanol (2:1, v/v) containing BHT antioxidant (3,5-Di-ter-butyl-4-hydroxytoluene; 0.1 %, w/v). Fatty acid methyl esters (FAME) were extracted in hexane through direct transesterification (Lepage and Roy 1984), i.e., without prior extraction or purification, using sulfuric acid–methanol (2:98, v/v) and toluene–methanol (2:3, v/v). In 2007, FAME were run on Varian CP3900 gas chromatograph equipped with a fused silica Omegawax 320 capillary column (Supelco Inc., Bellefonte, PA, USA); chromatograms were analyzed using the Star Chromatography Workstation v.5.51 software (Varian Inc., Palo Alto, CA, USA). In 2008, FAME were analyzed in MSMS scan mode (ionic range: 60 – 650 m/z) on a Polaris Q ion trap coupled to a Trace GC (Thermo Finnigan, Mississauga, ON, Canada) equipped with a Valcobond VB-5 capillary column (VICI Valco Instruments, Broakville, ON, Canada); data were treated using Xcalibur v.1.3 software (Thermo Scientific, Mississauga, ON, Canada). For both years, FAME were identified by comparing retention times with known standards (Supelco 37 Component FAME Mix and menhaden oil; Supleco Inc., Belfonte, PA, USA).

2. Flow cytometry

Samples (4.5 mL) of pre-filtered surface water (20 µm square mesh) were fixed with glutaraldehyde (0.1 % final v/v) and analyzed using an Epic Altra flow cytometer (Beckman Coulter, Fullerton, BC, Canada) fitted with a 488 nm laser operated at 15 mW, with a flow rate set to 60 microliters per minute. Data were analyzed with the Expo32 v.1.2b software (Beckman Coulter Inc., Fullerton, BC, Canada). Heterotrophic bacteria were quantified in diluted samples stained with SYBR Green I nucleic acid bounder (Molecular Probes Inc., OR, USA), and they were separated according to their nucleic acid content following Belzile et al. (2008; LNA and HNA for low and high nucleic acid, respectively). Pigmented cells (eukaryotes and cyanobacteria) were separated from other cells through their natural fluorescence; the pico (0.2–2 µm) and nano (2–20 µm) size classes were considered by using green-fluorescent microspheres (Polyscience Inc., PA, USA) as an 2 µm internal size standard (Tremblay et al. 2009).

LITERATURE CITED

Belzile, C., S. Brugel, C. Nozais, Y. Gratton, and S. Demers. 2008. Variations of the abundance and nucleic acid content of heterotrophic bacteria in Beaufort Shelf waters during winter and spring. Journal of Marine Systems 74:946–956.

Lepage, G., and C. C. Roy. 1984. Improved recovery of fatty acid through direct transesterification without prior extraction or purification. Journal of Lipid Research 25:1391–1396.

Tremblay, G., C. Belzile, M. Gosselin, M. Poulin, S. Roy, and J. E. Tremblay. 2009. Late summer phytoplankton distribution along a 3500 km transect in Canadian Arctic waters: strong numerical dominance by picoeukaryotes. Aquatic Microbial Ecology 54:55–70.


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