Fig 1.
Periodic pauses in motility coincide with cessation of ciliary movements.
(A). A Trichoplax on a glass substrate viewed with partially polarized transmitted light on a confocal microscope. Invaginations along the edge (arrows) are folds and dark particles throughout the interior are inclusions in fiber cells. The bright particles near the edge are birefringent crystals in crystal cells [11]. (B). Ventral cilia near the edge of a gliding Trichoplax. Tips of beating cilia (arrowheads) contact the substrate. The animal was moving toward the bottom of this field. Differential interference contrast (DIC) on a widefield microscope. (C). Five consecutive DIC images (46.5 ms intervals) from the animal illustrated in (B) showing cilia beating asynchronously. (D). Second DIC sequence (46.5 ms interval) during a pause showing arrest of ciliary beat. (E). Time lines track pauses in red and motility between pauses in blue. The top set of lines shows Trichoplax in dishes without food. These animals rarely paused (see text; only pausing animals are included in this figure) and those that did pause spend most of the time moving (mean time moving 71%; n = 6). Pauses are more frequent when algae are present (lower set of timelines) and the percent of time moving is smaller (43%; n = 7). Numbers at ends of bars represent concentrations of algae on substrate. Scale bars: A—100 μm; B—10 μm; C-D—5 μm; E—1 min.
Fig 2.
Trichoplax feeding on Rhodamonas salina microalgae.
Algae appear as tiny red specks on the substrate due to their content of fluorescent phycoerythrin. At 8 to 24 sec groups of algae under the paused Trichoplax precipitously release their contents apparent as contiguous domains of bright red phycoerythrin. The fluorescence has spread further and diminished at 112 sec. At 408 sec the animal has changed shape and a separate group of algae now covered by the paused Trichoplax release their contents at 440 sec. Merged transmitted light and fluorescence images (543 nm illumination) from a confocal microscope. Scale bar—200 μm.
Fig 3.
(A). Field of lipophil granules (large orange spheres) near a clump of algae and algae debris (yellow-green) prior to and following granule secretion (cyan circles; time interval 308 ms). Secreted granules (yellow arrows) enlarged and became greener due to uptake of FM1-43 dye. (B-D). Sequential images (61.5 ms intervals) showing secretion of individual granules (yellow arrows). Granules appeared as red/green doublets during secretion because the granules were moving and red/green channels were captured sequentially. After secretion, the contents of the granule began to disperse. Lipophil granules were stained with Lysotracker Red (visible in both red and green channels). FM1-43 (green) was added to the seawater to stain the contents of the secreted granules. Sequential two-channel, single focal plane time series collected with a spinning disk confocal microscope. Scale: A- 20 μm, B- D 10 μm.
Fig 4.
Frequency histograms of measurements of the timing (red) and positioning (blue) of individual secretory events as evident by the abrupt extrusion of a lipophil granule.
Time measurements, in seconds, started when the first granule was released (N = 107 granules from eight time lapse sequences). Distance measurements show how far in microns each granule was from the nearest fluorescent algal cell or algae debris.
Fig 5.
(A). Electron micrographs of long axes along lipophil cells prepared by freezing and freeze substitution. They are packed with extending granules from the cell bodies (nucleus) to the ventral surface where a large ventral granule lies closely apposed to the plasma membrane. Granules vary in their contents throughout the lipophil. (B). Deep in the cell body (nucleus) granules are produced by the Golgi apparatus (lettering indicates probable order). (C). Unfixed frozen section through a lipophil granule shows that these granules actually have uniformly dense contents. (D). Live cell imaging of a lipophil isolated from a Trichoplax showing that granules are transported from the cell body anterogradely down processes. Scale bars: A B, D—2 μm, Scale bar C -1 μm.