Microscopic analyses

Selected specimens were fixed with 2.5% glutaraldehyde (TAAB, England, UK) in ASW for 3 h at 4°C. Foraminiferal tests were then decalcified with 0.1 M EDTA for 36 h. After 5 washings with ASW, foraminiferal specimens were post-fixed with 1% osmium tetroxide (OsO₄; EMS, Hatfield, PA) in ASW for 2 h at room temperature (RT). Following 5 washings, specimens were dehydrated in a graded series of ethanol baths, from 50% to 100% and immersed in propylene oxide (EMS, Hatfield, PA) twice, each for 10 minutes. Subsequently, they were embedded in epoxy resin by using increasing concentration of resin (Durcupan Araldite, SIGMA, UK). Foraminifera were ultimately sectioned using an ultramicrotome (LKB, 2088 UltrotomeV). Thick sections of 1 μm were stained with 1% toluidine blue in distilled water at 60°C to provide light-microscope-level overviews of whole sections. Thin sections (100 nm), collected on 300-mesh nickel grids, were stained with uranyl acetate and lead citrate and finally observed with a Philips CM10 transmission electron microscope at 80 KV [20]. Lipid was quantified by calculating the area of 200 lipid vesicles, on average, distributed in the last whorl excluding the final two chambers (last two formed; i.e., youngest two) from 3–6 selected specimens (200–350 μm in diameter) for the two Hg concentrations, plus controls, at both time points. The Mann-Whitney U test, a nonparametric test, was used to check for significant differences between mean lipidic dimension among experimental conditions (concentration and time).

Selected specimens of A. parkinsoniana were incubated with Nile Red (NR) or Acridine Orange (AO) at T1 and T2 and then analyzed with CLSM.

Nile Red is a phenoxazine dye used on living and fixed cells to localize and quantify neutral and polar lipids [21–22]. The absorption and fluorescence properties of NR are known to be sensitive to environmental factors such as polarity. Polar lipids (i.e., phospholipids), which are mostly present in membranes, fluoresce red (emission > 590 nm) whereas neutral lipids (esterified cholesterol and triglycerides), which are present in LD, fluoresce yellow (570–590 nm) [23–24]. NR was used on specimens of A. parkinsoniana to detect membranous vesicles and on whole specimens to compare the lipidic distributions of untreated and Hg-treated conditions by confocal microscopy. For NR microscopy, A. parkinsoniana specimens were fixed in 2% paraformaldehyde for 2 hours, then washed in ASW and decalcified with EDTA (0.1 M) for 48 hours to remove the foraminiferal test. Following decalcification, specimens were rinsed in ASW, transferred to MatTek glass bottom chambers (MatTek Corporation, Ashland, MA) and NR was added at the final concentration of 3 μg/ml for 40 min at RT. Using CLSM, specimens were subject to blue excitation (488 nm) and analyzed separately for yellow and red emissions. Quantitative analyses of NR Mean Fluorescence Intensity (MFI) were performed using ImageJ software (NIH, Bethesda, MD), imaging the specimens at the same magnification, and determining the MFI of all the selected pixels. Subsequently, yellow MFI values were converted to arbitrary units (A.U.) setting the first image of the first control specimen at 100. The fluorescence emission of LD was calculated in the yellow channel of 3–5 selected specimens (200–350 μm in diameter) for each concentration.

The pH-sensitive dye acridine orange (AO) was used to detect and quantify acidic vesicular organelles in A. parkinsoniana specimens by confocal microscopy [25–26]. AO is a cell-permeable fluorescent dye that labels DNA and cytoplasm bright green whereas RNA and acidic vacuoles appear red. It can also enter acidic compartments and organelles, such as lysosomes and autolysosomes, where it becomes protonated and sequestered [25]. When AO is bound to acid compartments, such as lysosomes and acidic vacuoles, it emits red fluorescence (>650 nm) with intensity proportional to the acidity degree. For confocal live imaging, A. parkinsoniana specimens were transferred to MatTek glass bottom chambers (MatTek corporation, Ashland, MA) and then stained with AO (150 ng/ml) and incubated for 40 minutes at RT. Green and red fluorescence emissions illuminated with blue (488 nm) excitation light were analyzed by confocal microscopy. Because AO must be used on living specimens and A. parkinsoniana tests may interfere with dye uptake, only peripheral signals were observed (cytoplasm in green and acidic vesicles in red). Quantitative analyses of AO red MFI were performed on 3–5 specimens (200–350 μm in diameter) for control and 100 ppm treatments. Image analyses were carried out by determining the red MFI of all the selected pixels of the imaged specimens. Subsequently, the red MFI was converted to arbitrary units (A.U.) setting the first image of the first control specimen at 100. Epifluorescence and bright field (BF) microscopies were performed using a CLSM (Leica TCS SP5 II confocal microscope, Leica Microsystems) with 488-, 543- and 633-nm illumination and oil-immersion objectives.

Embedded specimens used for TEM were observed, as a whole, with an environmental scanning electron microscope (FEI ESEM, Quanta 200) to qualitatively characterize the presence of Hg. The ESEM, coupled with energy dispersive X-ray spectrometry (EDS), was used to assess the elemental composition of particles in the cytoplasm. The EDS is a technique employed to collect and determine the energy and the number of X-rays that are given off by atoms in a material [27–28]. Observations were conducted in low vacuum (0.2–1.2 Torr) at 10-mm working distance using secondary and backscattered electron modes with energy varying from 12 to 25 kV. A live counting time of 100 seconds, with spots’ mode from 3 to 5, was used for elemental mapping.

Additionally, thin sections (~100 nm) were mounted onto freshly peeled mica adhered to an SEM stub with double stick tape. The sections were then coated with about 10 nm of carbon using a Cressington 208 carbon coater. The sample was then imaged and elemental maps were obtained using a Hitachi S4800 FEG-SEM at 20 keV equipped with an EDS at the Oak Ridge National Laboratory. The upper secondary detector was used for image acquisition at a working distance of 7–8 mm. EDS maps were obtained using 64 sweeps with a 200 ms dwell time per pixel at a resolution of 256×200 pixels. Maps were plotted using the net intensities of the selected regions of interest with the nearest 3 × 3 pixels averaged to improve signal to noise in the element map images.