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D. biwae
D. biwae, after Yang and Shen, 2005

Difflugia biwae Kawamura, 1918

Diagnosis: Shell in lateral view fusiform in outline with a straight neck, a conspicuous great collar with a ragged margin and a long aboral horn. Aperture circular. Nucleus ovular with many nucleoli.

Dimensions: Length including the horn 165-306 µm (mean 227 µm); width 53-69 µm; collar diameter 63-101 µm; aperture 27-45 µm; horn 29-153 µm; nucleus 25-30 μm; n=100 (Yang and Shen, 2005).

Ecology: Freshwater. Distribution of D. biwae is limited, as far as it is known at present, to only some deep lakes in Japan and China.

Description: (From Yang and Shen, 1905) The shell is brown, fusiform, 2.1-3.7 collar diameters in body length. Surrounding the circular oral aperture is a conspicuous low-funnel-shaped (95°-125°) collar with a ragged margin; the body is circular in cross section, narrowest below the collar and gradually swelling to broadest in the position of the posterior 0.20-0.25 of the body length, then narrowing abruptly towards the aboral horn, its greatest diameter 0.60-0.98 collar diameter; the aboral horn is stout, tubular and somewhat curved, variable in length, 0.16-0.50 of the total length, and gradually tapering to a blunt tip. One to seven colourless long finger-like pseudopodia from the protoplasmic body may protrude through the aperture. A gas vacuole was often observed in living specimens. Such specimens can right themselves either by extrusion of the long pseudopodia attaching to the substrate, or by a combination of the pseudopodia and gas vacuole formation. The cytoplasm commonly occupies the greater part of the shell cavity and usually attaches to the internal walls by one or more threads of ectoplasm; sometimes the cytoplasm can extend into the aboral horn.
Reproduction was sometimes encountered, but the evidence (two shells joined at their oral apertures, could not be used to determine if it was sexual or asexual because the high optical density of the shells obscured details of the nuclei in these specimens. Although the shell is only semitransparent or opaque, the fluorescent light micrograph stained with DAPI clearly shows that single spherical nucleus is generally located in the posterior of the protoplasmic body and a few tiny algae are sometimes attached to the surface of the shell. The nucleus is ovular with many nucleoli and has a diameter range of 25-30 μm.
Study under light microscopy reveals the shell wall is thin, uniform in thickness, composed of fine sand granules with flattish pieces of quartz and muddy particles to produce a smooth surface. It has been illustrated by scanning electron microscopy that shells are covered with variously shaped particles, apparently of exogenous origin as determined from the irregularity of the shapes of included particles. However, the details of the surface of the shell are visibly different along the anterior body, the posterior body and the aboral horn. The reason is because the particles constituting the anterior portion of the shell covering are small to medium, angular and polymorphic to give a smooth appearance; the posterior region is composed of small angular particles and medium flattish pieces of quartz to give a smoother appearance; the aboral horn is made of small flattish pieces of quartz to give the smoothest appearance. It appears that Difflugia biwae is able to select and arrange the building material according to size and shape to construct a species-specific shell. This is in good agreement with that reported by Meisterfeld (2000) in the genus Difflugia. No cement structures are recognizable in the scanning electron microscope.

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