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First published online October 19, 2007
Journal of Experimental Biology 210, 3736-3748 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.003392

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Coordinated contractions effectively expel water from the aquiferous system of a freshwater sponge

Glen R. D. Elliott and Sally P. Leys^*

Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada

View larger version (56K): [in this window] [in a new window]   Fig. 1. Fracture (A) and schematic diagram (B) illustrating the principal features of Ephydatia muelleri: the apical pinacoderm (apd), sub-dermal cavity (sdc), choanocytes (ch), and basal pinacoderm (bpd). The apical pinacoderm consists of an inner layer of endopinacocytes (enp) and and outer layer of exopinacocytes (exp); porocytes (p), which form the ostia (os), are sandwiched between the two layers. The choanosome contains incurrent (in) and excurrent (ex) aquiferous canals, choanocyte chambers (cc) and spicule tracts (sp) that support the apical pinacoderm. A thin collagenous middle region (mesohyl, me) houses mobile cells. Prosopyles (pp), the entrance to chambers are formed by perforate `sieve'-like cells. Apopyles (ap) vent water from chambers. Scale bar, 20 µm.

View larger version (70K): [in this window] [in a new window]   Fig. 2. The response of E. muelleri to mechanical agitation. (A–D) Light micrographs illustrating the changes to the excurrent aquiferous system (black arrows) during one inflation–contraction cycle. Choanosome (ch), excurrent canals (ex), gemmule (g), incurrent canals (in), and osculum (osc). Scale bar, 1 mm. (A) Initial contraction of the osculum: immediately after stimulation the base of the osculum contracts but the tip remains slightly open. (B) Inflation phase: excurrent canals dilate (black arrows); the base of the osculum begins to dilate, but the tip remains constricted (white arrows); hollow arrows indicate the locations of peripheral (p), middle (m) and central (c) canals. (C) Contraction phase: excurrent canals contract (black arrows) and the base of the osculum dilates (white arrow). (D) Contraction of the osculum (arrow) and return of canals to their original diameter. A–D correspond to phases a–d, respectively, in (E–G) below. (E–G) Changes in diameter of the largest excurrent canal and osculum (E) during the inflation–contraction cycle, and of all canals on the right (F) and left (G) sides of the sponge. R1–R4 and L1–L4 in D indicate locations of measurements plotted in F and G. (See Movie 1 in supplementary material.)

View larger version (8K): [in this window] [in a new window]   Fig. 3. The duration of the inflation–contraction cycle depends on the resting diameter of the largest excurrent canals. Responses to agitation were measured in five sponges with the same sized (diameter) choanosome, but with varying sizes (1–5) of excurrent canals: 64.9, 76.9, 103.52, 154.13 and 213.35 µm, respectively. Sponges with larger excurrent canal diameters have a longer inflation and contraction period (500, 899, 1399, 2988, 2052 s, respectively), but the rate of propagation of contractions along incurrent and excurrent canals was not significantly different (in: 2.80±0.26 µm s–1, N=5; ex: 3.30±0.45 µm s–1, N=5, P=0.23). Bars denote ± s.e.m. of three measurements from one sponge (see supplementary material Fig. S2).

View larger version (70K): [in this window] [in a new window]   Fig. 4. Analysis of the kinetics of the contraction of incurrent (A,B) and excurrent (C,D) canals shows that waves of contraction propagate along and across canals. (A,C) Sponges grown as sandwich preparations were stimulated by addition of inedible ink. Measurements of incurrent (A) and excurrent (C) canal diameters over time are plotted in (B) and (D), respectively. (B,D) In B the wave of contraction propagated between sites 1 and 2 (100 µm apart) in 300 s, a rate of 0.33 µm s–1. The wave of contraction reached site 3 with a delay of 150 s. Cells crawling through the mesohyl (indicated by white and black stars on A and B) arrest movement for approximately 10 min (B, white arrows) while the wave of contraction passes. In D the wave of contraction propagated between sites 3 and 4 (300 µm apart) in 940 s, rate of 0.32 µm s–1. in, incurrent; ex, excurrent; arrows indicate direction of water flow in the canal. Scale bars, 100 µm (A); 300 µm (C). (Movie 3 in supplementary material.)

View larger version (64K): [in this window] [in a new window]   Fig. 5. Stereomicrographs (A–D) and changes in diameter (E) of the tip and base of the osculum (at positions indicated by arrows in A) during contraction of the osculum. (A) Immediately after stimulation by agitation, (B) during the contraction phase when its base is fully inflated, (C) fully contracted; and (D) when relaxed at the end of the cycle; insets show enlargements of the position of the apical pinacoderm. Scale bar, 1 mm. A–D correspond to phases a–d, respectively, in (E). Between A and D the canals inflate and the apical pinacoderm raises. The wave of contraction propagates from base to tip, a distance of 1473 µm, in 12 s, a rate of 122.8 µm s–1. Bars denote ± s.e.m. of an average of three measurements from one sponge. (Movie 4 in supplementary material.)

View larger version (73K): [in this window] [in a new window]   Fig. 6. Closure of a field of porocytes in the apical pinacoderm correlates with contraction of the choanosome after stimulus by agitation. (A–C) Stereo microscope images show that individual ostia (arrows) close within 50 s, and a field of 20 porocytes closes over a period of 83 s. Scale bar, 100 µm. (D) Constriction (closing) of eight ostia in the field shown in A is correlated with the contraction of the choanosome (broken line). Shortly after the sponge was stimulated the ostia closed and the choanosome contracted. A few ostia opened briefly at 1200 s during an expansion of the choanosome, but these closed again as the choanosome contracted. The field opened again with the expansion of the choanosome at t=1500 s (here approximately 22 min later), and again closed just prior to contraction of the choanosome at 2400 s.

View larger version (17K): [in this window] [in a new window]   Fig. 7. Rapid contractions (`twitches') occur simultaneously in distinct regions of the choanosome after uptake of inedible ink. (A) Projected area (density of the tissue) was measured as a proxy for the extent of contraction of the tissue; a contraction was observed as a decrease in the area occupied by tissue (a decrease in blackness). (B) The change in projected area of each region shows that two contraction events occur within seconds of each other in Areas I and II of A, 700 µm apart. (Movie 2 in supplementary material.)

View larger version (103K): [in this window] [in a new window]   Fig. 8. Actin distribution and morphology of pinacocytes. (A) Scanning electron microscopy shows that endopinacocytes (enp) are elongated cells that form the underside of the apical pinacoderm. Dotted lines indicate cell boundaries. Scale bar, 10 µm. (B) Epifluorescence microscopy of Bodipy 505 FL Phallacidin-labelled tissue shows extensive tracts of actin in endopinacocytes of the apical pinacoderm, a region equivalent to that shown in A. Actin is brightly labeled in focal adhesion plaques between cells (arrows). Dotted lines indicate cell boundaries, demonstrating that the actin tracts continue in adjacent cells. Scale bar, 50 µm. (C) In cells lining excurrent canals of a sandwich preparation, actin tracts (black arrows) are much less brightly labelled. The preparation was fixed as a wave of contraction passed through the field of view. Dense packing of choanocyte chambers (cc) indicates that the lower canal was contracted. Scale bar, 100 µm.

View larger version (12K): [in this window] [in a new window]   Fig. 9. Summary diagram illustrating the temporal coordination of contractions by the aquiferous canals, apical pinacoderm, ostia and osculum, during a single inflation–contraction event in E. muelleri. During the inflation phase, the apical pinacoderm, canals and osculum gradually dilate. The ostia contract for the duration of the inflation phase. The contraction of the apical pinacoderm and canals lead to the full inflation of the osculum and its rapid contraction. Ostia open only after all other components have relaxed.