Tertidal sites on the Canadian Atlantic and BL-8040MedChemExpress TF14016 Pacific coasts of North America. All collection sites were unrestricted public access areas and given that only small volumes (< 2 ml) of seawater or sediments were involved for eachPLOS ONE | DOI:10.1371/journal.pone.0141150 October 20,4 /Auxosporulation in Paraliasample, no specific permissions were required for these activities, and no endangered or protected species were involved. Single chains of Paralia were isolated from these samples to establish monoclonal cultures as described in [33]. All cultures were grown in f/2 growth media [34] at 12?6 at an irradiance of 40?0 mol photons m-2 s-1, 12:12 Dark:Light (D:L) cycle, and inoculated into fresh media approximately every 6 weeks. The three clones examined were: West1C2 (from Aberdeen, WA, USA; 46?8'31.34"N, 123?8'56.59"W), GP1 and GP3 (both from Grove's Point, Cape Breton, Canada; 46?3'N, 60?0'W).Fixation, staining and light microscopyTo visualize nuclei during auxosporulation, cells were fixed with 2.5 (v/v) glutaraldehyde in seawater (final concentration) and stained with DAPI (4', 6-diamidino-2-phenylindole) according to [35] with minor modifications. Chloroplasts were bleached with 10 ml 99 methanol followed by 5 ml Tris buffer. Following bleaching, 0.1 l DAPI (10 g/ml) was added and incubated in darkness at 2? for another 24 hours prior to examination. Brightfield and DAPI fluorescence light microscopy was performed using two Zeiss microscopes as required (Carl Zeiss, G tingen, Germany). A Zeiss Axioskop 2 Plus fitted with a cooled AxioCam color camera, HBO 100 fluorescence illuminator and Filter Set 01 was used for reconnaissance work, while a Zeiss AxioImager.Z2 microscope with a cooled AxioCam MRm monochrome camera, Colibri LED fluorescence illuminator (365 nm LED) and Filter Sets 62HE and 49 was used for in-depth investigation. Monochrome fluorescence images presented here were pseudo-colored appropriately based on the filter used for acquisition.SEM imaging and energy dispersive X-ray spectroscopy (EDS)Auxospores and frustules from each of the cultures were subjected to SEM examination on flat wcs.1183 polycarbonate filter as in [36] or a grooved LP substrate as in [37], as appropriate. Specimens were examined using a JEOL JSM-5600 SEM (JEOL USA, Peabody, MA, USA) at the Digital Microscopy Facility, Mount Allison University, operating at 10 kV and 8?0 mm working distance. Diameters of cells in auxosporulating cultures were measured using dmfMeasure software [38]. A complete metric data set for the clones is available in [32]. Valve structure terminology followed [33] and [39] while the structures associated with auxosporulation were named following [7]. EDS was performed with the same instrument equipped with an Oxford Inca Energy 200 EDS system and at 20 mm working distance. Since the only element of interest in this j.jebo.2013.04.005 study was silicon (Si-K, X-ray energy 1.74 keV), an accelerating voltage of 10 keV provided sufficient Pan-RAS-IN-1 site overvoltage for efficient X-ray excitation. Spectra were acquired for 100 s (dead time corrected) at 0.1 nA beam current, energy range 0?0 keV into 1024 channels. The EDS spectra were collected from intact and unobstructed structures and/or auxospores. Spectra from the polycarbonate support filter adjacent to the auxospores were also routinely taken and showed no remote excitation from neighboring siliceous components (if present) at distances as close as 3 m.ResultsAuxosporulation was examined in three clones of two.Tertidal sites on the Canadian Atlantic and Pacific coasts of North America. All collection sites were unrestricted public access areas and given that only small volumes (< 2 ml) of seawater or sediments were involved for eachPLOS ONE | DOI:10.1371/journal.pone.0141150 October 20,4 /Auxosporulation in Paraliasample, no specific permissions were required for these activities, and no endangered or protected species were involved. Single chains of Paralia were isolated from these samples to establish monoclonal cultures as described in [33]. All cultures were grown in f/2 growth media [34] at 12?6 at an irradiance of 40?0 mol photons m-2 s-1, 12:12 Dark:Light (D:L) cycle, and inoculated into fresh media approximately every 6 weeks. The three clones examined were: West1C2 (from Aberdeen, WA, USA; 46?8'31.34"N, 123?8'56.59"W), GP1 and GP3 (both from Grove's Point, Cape Breton, Canada; 46?3'N, 60?0'W).Fixation, staining and light microscopyTo visualize nuclei during auxosporulation, cells were fixed with 2.5 (v/v) glutaraldehyde in seawater (final concentration) and stained with DAPI (4', 6-diamidino-2-phenylindole) according to [35] with minor modifications. Chloroplasts were bleached with 10 ml 99 methanol followed by 5 ml Tris buffer. Following bleaching, 0.1 l DAPI (10 g/ml) was added and incubated in darkness at 2? for another 24 hours prior to examination. Brightfield and DAPI fluorescence light microscopy was performed using two Zeiss microscopes as required (Carl Zeiss, G tingen, Germany). A Zeiss Axioskop 2 Plus fitted with a cooled AxioCam color camera, HBO 100 fluorescence illuminator and Filter Set 01 was used for reconnaissance work, while a Zeiss AxioImager.Z2 microscope with a cooled AxioCam MRm monochrome camera, Colibri LED fluorescence illuminator (365 nm LED) and Filter Sets 62HE and 49 was used for in-depth investigation. Monochrome fluorescence images presented here were pseudo-colored appropriately based on the filter used for acquisition.SEM imaging and energy dispersive X-ray spectroscopy (EDS)Auxospores and frustules from each of the cultures were subjected to SEM examination on flat wcs.1183 polycarbonate filter as in [36] or a grooved LP substrate as in [37], as appropriate. Specimens were examined using a JEOL JSM-5600 SEM (JEOL USA, Peabody, MA, USA) at the Digital Microscopy Facility, Mount Allison University, operating at 10 kV and 8?0 mm working distance. Diameters of cells in auxosporulating cultures were measured using dmfMeasure software [38]. A complete metric data set for the clones is available in [32]. Valve structure terminology followed [33] and [39] while the structures associated with auxosporulation were named following [7]. EDS was performed with the same instrument equipped with an Oxford Inca Energy 200 EDS system and at 20 mm working distance. Since the only element of interest in this j.jebo.2013.04.005 study was silicon (Si-K, X-ray energy 1.74 keV), an accelerating voltage of 10 keV provided sufficient overvoltage for efficient X-ray excitation. Spectra were acquired for 100 s (dead time corrected) at 0.1 nA beam current, energy range 0?0 keV into 1024 channels. The EDS spectra were collected from intact and unobstructed structures and/or auxospores. Spectra from the polycarbonate support filter adjacent to the auxospores were also routinely taken and showed no remote excitation from neighboring siliceous components (if present) at distances as close as 3 m.ResultsAuxosporulation was examined in three clones of two.