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Calcification in the planula and polyp of the hydroid Hydractinia symbiolongicarpus (Cnidaria, Hydrozoa)

Constance L. Rogers and Mary Beth Thomas^*

Department of Biology, The University of North Carolina at Charlotte, Charlotte, NC 28223, USA

View larger version (93K): [in a new window]   Fig.1. Location of crystals in the planulae and polyps. (A,B) Comparison of the same live planula (slightly different because of movement) viewed by (A) brightfield and (B) polarized light microscopy. The crystals are located in the endoderm (En); the ectoderm (Ec) is devoid of crystals. The white bars in B indicate the margin of the ectoderm. Scale bar, 100µm. (C) Posterior end of planula as seen with DIC microscopy. The mesoglea (Mg) forms the boundary between the endoderm (En) and ectoderm (Ec). The nucleus (n) of the gastrodermal cell (GDC; cell area indicated by a dashed line) is suspended in a large vacuole (v) by thin strands of cytosol. Arrows indicate crystals within the large vacuoles. The position of crystals do not correspond to those of nematocysts (nc). Scale bar, 10µm. (D,E) A living polyp from a colony growing on a slide, viewed by (D) polarized light and (E) DIC microscopy. The mouth, located at the tip of the hypostome (H), opens into the gastrovascular cavity (GvC). Crystals are located in the endodermal cells of the body column (BC) and stolons (S) of the polyps; few, however, are located in the hypostome or tentacles (T). The ectoderm (Ec) is devoid of crystals. Scale bar, 200µm.

View larger version (115K): [in a new window]   Fig.2. Stolons and stolonal mat of established colonies. (A) Section of a new stolon from a portion of a colony growing on a slide. The stolon is a tubular structure composed of an outer layer of ectoderm (Ec) surrounding an inner layer of endoderm (En) that lines the gastrovascular tube (GvT). The crystals (arrows) are confined to the endoderm and are intracellular rather than free in the gastrovascular tube. The dashed box encloses two crystals that are out of the plane of focus. Scale bar, 20µm. (B) New stolons growing from a portion of a colony recently transplanted to a microscope slide. New stolonal tubes (S) are separated by a layer consisting only of ectoderm (Ec). The crystals (arrows) are located within the endodermal cells that form the walls of the tube; the ectodermal cells that separate the tubes are devoid of crystals. The lower part of the section shows a crystalline mat (CM) below the living stolons, just out to the plane of focus. Scale bar, 200µm. (C,D) Comparison of the same section of the stolonal mat, grown on a coverglass and viewed from the underside by (C) brightfield and (D) polarized light microscopy. What appear to be dead, hardened stolon tubes (arrows) in C run throughout the stolonal mat. The dead tubes are refractile when observed by polarized light (arrows in D correspond to arrows in C). Scale bar, 200µm.

View larger version (83K): [in a new window]   Fig.3. Crystals of stolonal mat and a portion of the skeleton of a colony. (A) Evidence of inorganic precipitation of crystals in the crystalline mat. Scale bar, 50µm. (B) Skeleton of a dead colony of H. symbiolongicarpus. Arrows indicate mineralized remains of polyps on a shell of a hermit crab. Bar, 250µm.

View larger version (19K): [in a new window]   Fig.4. Analysis of crystalline mat and commercially available calcium carbonate by infrared (IR) spectrometry. The infrared spectra were obtained for the crystalline mat (red line) and commercially available calcium carbonate (black line). The graph shows transmittance (arbitrary units) at each wavenumber. By comparing the IR spectra obtained in this study with those of calcium carbonate polymorphs obtained in previous studies (Adler and Kerr, 1962; Kikuchi and Tamiya, 1984), the crystalline mat was determined to be aragonite, whereas, the commercially available calcium carbonate was determined to be a mixture of aragonite and calcite, accounting for the wide and double headed V3 band. The peaks in transmittance are due primarily to the CO32- radical of calcium carbonate. V3, the characteristic transmittance peak for calcium carbonate, represents doubly degenerate asymmetric stretching of the CO32- radical. V2 corresponds to out-of-plane bending and V4 to doubly degenerate planular bending. Both sample have other notable peaks (asterisk) found in the spectra of aragonite. The peak in the range of approximately 3250–3750 wavenumbercm-1 is due to the presence of water (H2O) in the samples.

View larger version (14K): [in a new window]   Fig.5. The effects of extracellular calcium levels on crystal formation in planulae. Planulae were exposed to calcium-free SW (0mmoll-1; N=15), 9mmoll-1 Ca2+ SW (9mmoll-1; N=17) and 50mmoll-1 Ca2+ SW (50mmoll-1; N=10). *Significant difference compared with 9mmoll-1 Ca2+ SW (for comparison of 0mmoll-1 Ca2+ SW with 9mmoll-1 Ca2+ SW, P<0.001; for 50mmoll-1 Ca2+ SW with 9mmoll-1 Ca2+ SW, P<0.05). Values given as mean ± S.E.M.

View larger version (12K): [in a new window]   Fig.6. The effects of treatments with pharmacological agents on crystal formation in planulae. Artificial sea water (ASW; N=17) and 1% dimethyl sulfoxide in ASW (DMSO; N=9) serve as controls. Diltiazem (Dz; 0.001mmoll-1; N=14), nifedipine (Nif; 0.1mmoll-1; N=10) and verapamil (Vp; 0.001mmoll-1; N=16) are inhibitors of L-type calcium channels. Ruthenium Red (RR; 0.1mmoll-1; N=12) is an inhibitor of Ca2+-ATPases. Caffeine (Caff; 0.1mmoll-1; N=15), procaine (Proc; 0.001mmoll-1; N=12), and ryanodine (Ry; 0.001mmoll-1; N=11) are modulators of ryanodine-sensitive calcium stores. Acetazolamide (AZ; 0.01mmoll-1; N=13) is an inhibitor of carbonic anhydrase, and DIDS (0.001mM; N=13) is an inhibitor of anion transport. Crystal production in the presence of inhibitors is compared with that in ASW (N=17) and 1% DMSO (N=9) controls. *Significant difference compared to ASW; **significant difference compared to ASW and 1% DMSO for treatments requiring DMSO. Values are means ± S.E.M.