Cytological Observations of the Large Symbiotic Foraminifer <i>Amphisorus kudakajimensis</i> Using Calcein Acetoxymethyl Ester

<div><p>Large benthic foraminifera are unicellular calcifying reef organisms that can form symbiotic relationships with a range of different microalgae. However, the cellular functions, such as symbiosis and calcification, and other aspects of cellular physiology in large benthic foraminifera are not fully understood. <i>Amphisorus kudakajimensis</i> was used as a model to determine the detailed cellular characteristics of large benthic foraminifera. We used calcein acetoxymethyl ester (calcein AM) as a fluorescent indicator for live confocal imaging. We demonstrated that calcein AM is a useful fluorescent indicator to stain the fine network of reticulopodia and the cytoplasm in living <i>A</i>. <i>kudakajimensis</i>. We showed that at least two types of reticulopodia exist in <i>A</i>. <i>kudakajimensis</i>: the straight bundle of reticulopodia that spreads from the aperture and the fine reticulopodia along the surface of the aperture and chamber walls. The cytoplasm in outer chambers was highly branched and contained a few dinoflagellates. In contrast, the inner chamberlets contained condensed cytoplasm and many dinoflagellates, suggesting that the cytoplasm of <i>A</i>. <i>kudakajimensis</i> performs different functions based on its location within the large test. Our confocal detailed image analysis provides real-time cellular morphology and cell physiology of living foraminifera.</p></div

Confocal images of the intratest cytoplasm and reticulopodia covering the test surface of <i>A</i>. <i>kudakajimensis</i>.

<p><b>(A)</b> Superimposed macro-confocal and bright-field images (Z-stack image at a depth of approximately 150 μm within the area of interest). The boxed areas in (A) correspond to the enlargement image in (B), (D) and (F). Four compartments were defined in <i>A</i>. <i>kudakajimensis</i>: outer chamber, shown in (B) and (C); intermediate and inner chambers, shown in (D) and (E); and inner chamber and embryonic apparatus, shown in (F). Scale bar = 1 mm. <b>(B)</b> The outer chambers consisted of approximately 6–8 rows from the periphery. The white arrows indicates fine and radically spreading reticulopodia extending from the aperture. Scale bar = 50 μm. <b>(C)</b> High-magnification image of the outer chambers from the dotted-line box shown in (B). The white encircled area indicates vacuoles. Cytosolic streaming is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165844#pone.0165844.s005" target="_blank">S3 Movie</a>. Scale bar = 50 μm. <b>(D)</b> High-magnification image of the intermediate to inner chambers from the boxed area shown in (A). Z-stack image of the intermediate chambers at a depth of approximately 50 μm. <b>(E)</b> High-magnification image of the boxed region shown in (D). The white arrow shows the fine reticulopodial network covering the test surface. The yellow arrow indicates the yellowish-green autofluorescence from dinoflagellates. Time-lapse imaging was performed in a single focal plane to visualize cytoplasmic streaming (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165844#pone.0165844.s006" target="_blank">S4 Movie</a>). Scale bar = 50 μm. <b>(F)</b> Z-stack image of the inner chambers and embryonic apparatus at a depth of approximately 150 μm. Reticulopodia can be seen emerging at the base of the inner chambers (white arrows). Scale bar = 200 μm.</p

Cytoplasm and reticulopodia of <i>Amphisorus kudakajimensis</i> observed using bright-field and confocal imaging with calcein AM.

<p>An example of the discrimination between live and dead <i>A</i>. <i>kudakajimensis</i> with calcein AM staining. <b>(A, B)</b> Living specimens stained with calcein AM (10 μM). <b>(C, D)</b> Living specimens without calcein AM. <b>(E, F)</b> Dead <i>A</i>. <i>kudakajimensis</i> stained with calcein AM (10 μM). <b>(A, C, E)</b> Bright-field images. <b>(B, D, F)</b> Confocal images. The blue arrows indicate the spreading reticulopodia, and white arrow indicates the cytoplasm in chamberlets. All scale bars = 200 μm.</p

Bright-field and confocal images of <i>A</i>. <i>kudakajimensis</i> with spreading reticulopodia.

<p><b>(A, B, C)</b> Transmitted bright-field images, <b>(D, E, F, G, H)</b> Confocal images. <b>(A)</b> Low-magnification images of <i>A</i>. <i>kudakajimensis</i> with spreading reticulopodia. Orange arrows point to excrement. Scale bar = 400 μm. <b>(B)</b> Image of the same specimen from (A) in a vertical position. The base of the specimen (aperture) was shaded under trans-illumination. Scale bar = 100 μm. <b>(C)</b> High-magnification image of the box shown in (B). Orange arrows indicate excrement. Scale bar = 50 μm. <b>(D)</b> Confocal image corresponding to the bright-field image in (A). The reticulopodia and cytoplasm are shown in green, and the dinoflagellates are shown in red. White arrows indicate bright spots in the reticulopodia that are attached to the glass substrate. Time-lapse images show streaming reticulopodia (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165844#pone.0165844.s003" target="_blank">S1 Movie</a>). Scale bar = 400 μm. <b>(E)</b> High-magnification image of the aperture area corresponding to the image in (B). Scale bar = 100 μm. <b>(F)</b> High-magnification image of the reticulopodia corresponding to the image in (C). Scale bar = 50 μm. <b>(G)</b> Three-dimensional image (depth = 120 μm) of reticulopodia and the cytoplasm at the outermost part of the test. Scale bar = 400 μm. 3D rotation is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165844#pone.0165844.s004" target="_blank">S2 Movie</a>. <b>(H)</b> High-magnification three-dimensional image of the box shown in (G). Thick reticulopodia (white arrows) and fine reticulopodia (blue arrows) emerged from the marginal apertures. The ring-shape reticulopodial network around the marginal apertures was indicated with yellow arrows. Scale bar = 200 μm.</p