JEB003392 Supplementary Material
Files in this Data Supplement:
- Supplemental Figure S1
Fig. S1. Uptake and clearing of inedible ink by a 7-day-old sponge. (A) Prior to addition of ink the choanosome (cho) is white, the yellow gemmule husk (g) is in the centre of the sponge, and the osculum (osc) is to the right. (B) 5 min after the addition of ink. (C) 1 h after ink was added the sponge has undergone one inflation−contraction cycle. (D) 24 h after ink was added, 4−6 inflation−contraction cycles have removed some of the ink. (E) 48 h after ink was added the choanosome is cleared of all ink particles. Scale bars, 1 mm.
- Supplemental Figure S2
Fig. S2. Stereomicrographs showing the sponges. osc, osculum. Scale bars, 1 mm. Insets show enlargements of the regions of the principal canals, measured in Fig. S3.
- Supplemental Figure S3
Fig. S3. Contraction of incurrent and excurrent canals in a sandwich preparation. Incurrent (in, white arrows) and excurrent (ex, black arrows) canals can be identified by the movement of ink along the incurrent canals in (B). Scale bar, 100 μm. (A−H) Inflation phase: Uptake of ink into the canals and chambers triggers contraction of the incurrent canals dilating excurrent canals. The wave of contraction moves from right to left along each canal and across canals from bottom to top of the image. At maximum contraction of the incurrent canals, some ink is forced through the choanocyte chambers (black arrowheads, E). (F−H) Contraction phase: Excurrent canals begin to contract, dilating the incurrent canals. Water is stagnant for a period of 6 min (F,G), and as the incurrent canals dilate, some ink flushes backwards along the incurrent canals (double black arrows, G). (H) Excurrent canals continue to contract. (see also Movie 3 in supplementary material).
- Supplemental Figure S4
Fig. S4. Morphology of the aquiferous canals and choanocyte chambers studied by scanning electron microscopy of sponges preserved when relaxed (A,B), and after stimulus when contracted (C,D). When relaxed the canals are broad, endopinacocytes (enp) form a thin layer lining the canals and choanocyte chambers (chc) are round, with well-spaced choanocytes (ch, arrows) and broad apopyles (ap). In contrast, canals in contracted specimens are narrower, endopinacocytes are slightly thicker (double arrows) and choanocyte chambers are compressed such that the flagella (fl) project out of the apopyle into the excurrent canal (C,D). pp, prosopyles; ex, excurrent canal. Scale bars, 10 μm (A), 5 μm (B−D).
- Movie 1
Movie. 1. (Fig. 2). Response of Ephydatia muelleri to 1 min of agitation (2−3.Hz) of the Petri dish.
The �inflation−contraction� behavior of the sponge consists of three phases − inflation (0−1140 s), plateau (1150−1880 s) and contraction (1890−3240 s). Upon stimulation, the sponge constricts the osculum (lower centre), contracts the incurrent canals that dilates the excurrent canals and then contracts the excurrent canals venting all the water out of the osculum. At the final oscular contraction (2600−2740 s) the water is expelled out of the osculum. The choanosome is white, and the empty gemmule from which the sponge hatched is yellow. Duration: 61.9 min; time interval: 10 s; and frame rate: 10 frames s−1.
- Movie 2
Movie. 2. (Fig. 7). Response of Ephydatia muelleri to the addition of inedible ink particles.
The �inflation−contraction� cycle (840−2240 s) lasts approximately 30 min, successfully expelling unwanted ink. During this cycle back-flushing occurs through a few open ostia in the apical pinacoderm (1260−2180 s). Note that during the �inflation-contraction cycle the osculum remains open expelling inedible ink. At 6400 s, a twitch occurs across the entire choanosome. Duration: 164.7 min; time interval: 20 s; and frame rate: 10 frames s−1.
- Movie 3
Movie. 3. (Fig. 4 and Fig. S3). Sandwich preparation of Ephydatia muelleri showing the response of the sponge to the addition of inedible ink.
The �inflation−contraction� behaviour consists of three phases: inflation (590−1490 s), plateau (1500−1790 s) and contraction (1800−3290 s). Cells crawling in the mesohyl arrest as the incurrent canals contract during the inflation and plateau phases (600−1700 s). During the inflation phase, back-flushing occurs through the choanocyte chambers (1200−1850 s) and water in the incurrent canals stops flowing for a more than 10 min (2390−3340 s) as indicated by the static ink front. During the �inflation phase�, the canals contract from right to left along the canals and from the bottom to top between canals; contractions run in the opposite directions during the �contraction phase�. Duration: 74.5 min; time interval: 10 s; and frame rate: 6 frames s−1.
- Movie 4
Movie. 4. (Fig. 5). Lateral view of the final contraction of the osculum in Ephydatia muelleri in response to agitation (2−3 Hz) of the Petri dish (530−610 s).
Trembling of apical pinacoderm occurs in advance of a second �inflation−contraction� cycle (1625−2995 s). The apical pinacoderm contracts initially (prior to the inflation phase); then it begins to raise as the canals inflate. Duration: 49.9 min; time interval: 5 s; and frame rate: 10 frames s−1.
- Movie 5
Movie. 5. (Fig. 6). Contraction of ostia in the apical pinacoderm of Ephydatia muelleri in response to agitation (2−3 Hz) of the Petri dish.
Arrows indicate position of ostia. Ostia close in unison just before the choanosome (darker tissue on the right) contracts. As the choanosome expands again the ostia re-open. Duration: 49.9 min; time interval: 5 s; and frame rate: 6 frames s−1.
- Movie 6
Movie. 6. Contraction of ostia in the apical pinacoderm after stimulation by Sumi Ink in Ephydatia muelleri
Ink is taken into the choanosome stimulating a series of quick contractions and a final massive contraction that expels ink in clumps over the sponge. The ostia contract in unison twice, always just before the contraction of the choanosome (the dark region located to the top left). Arrows indicate ostia that close in unison; a small amount of ink squeezes back through two ostia that remain open near the choanosome (between 5810−6190 s). Duration: 166.4 min; time interval: 20 s; and frame rate: 6 frames s−1.
Note: High quality versions of the videos can be obtained at this link: