Coordinated contractions effectively expel water

The Journal of Experimental Biology 210, 3736-3748
Published by The Company of Biologists 2007
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
*Author for correspondence (e-mail:
Accepted 16 July 2007
In response to mechanical stimuli the freshwater sponge
extracellular signal may pass between cells. Bundles of
actin filaments traverse endopinacocytes of the apical
Spongillidae) carries out a series of peristaltic-like
pinacoderm. Actin-dense plaques join actin bundles in
adjacent pinacocytes to form continuous tracts spanning
contractions that is effective in expelling clumps of waste
material from the aquiferous system. Rates of contraction
the whole sponge. The orchestrated and highly repeatable
depend on the region of tissue they are propagating
series of contractions illustrates that cellular sponges are
through: 0.3–1·␮m·s–1 in the peripheral canals, 1–4·␮m·s–1
capable of coordinated behavioural responses even in the
in central canals, and 6–122·␮m·s–1 in the osculum. Faster
absence of neurons and true muscle. Propagation of the
events include twitches of the entire sponge choanosome
events through the pinacocytes also illustrates the presence
and contraction of the sheet-like apical pinacoderm that
of a functional epithelium in cellular sponges. These results
forms the outer surface of the animal. Contraction events
suggest that control over a hydrostatic skeleton evolved
are temporally and spatially coordinated. Constriction of
prior to the origin of nerves and true muscle.
the tip of the osculum leads to dilation of excurrent canals;
fields of ostia in the apical pinacoderm close in unison just
Supplementary material available online at
prior to contraction of the choanosome, apical pinacoderm [high
and osculum. Relaxation returns the osculum, canals and
quality copies of the videos are available from the authors on
the apical pinacoderm to their normal state, and three such
request (]
coordinated ‘inflation–contraction’ responses typically
follow a single stimulus. Cells in the mesohyl arrest
Key words: Ephydatia muelleri, peristalsis, evolution of conduction,
crawling as a wave of contraction passes, suggesting an
Porifera, propagated contraction.
Sponges (phylum Porifera) have a fossil record of over 600
million years (for example, see Conway-Morris, 1993). Despite
their ancient origin, they possess a vast repertoire of genes that
encode regulatory signalling molecules, many of which are
homologous to those in higher animals (Morris, 1993; Müller,
2003; Adell et al., 2007). Recent studies also indicate that
sponge embryogenesis is characterized by a spatio-temporal
pattern of gene expression that structures different regions or
layers of the developing larva (Larroux et al., 2006). Other
research has shown that aspects of the immune response (Wiens
et al., 2004), respiration, maintenance of homeostasis (Zocchi
et al., 2001), and even control of body shape as a result of
changes to the stiffness of the extracellular matrix (Wilkie et al.,
2004), have many features in common with equivalent
physiological processes in higher animals. These examples
illustrate the sponge’s ability to regulate its developmental and
physiological functions; however, coordinated movements of
the whole animal in response to external stimuli – the
quintessential feature of Eumetazoa – are not well known.
In contrast to its molecular and physiological complexity,
the sponge is a structurally simple animal. The sponge body
is composed of at least eight types of cells arranged around an
extensive aquiferous canal system built for filter feeding
(Simpson, 1984). It is often suggested that sponges lack
conventional epithelia, with typical cell–cell junctions and a
basement membrane, which would create sealed internal
compartments (Tyler, 2003). However, sealing junctions,
though not often dense or belt-form, are present in sponge
epithelia (Woollacott and Pinto, 1995; Gonobobleva and
Ereskovsky, 2004). Homoscleromorph sponges have a clear
basement membrane containing type IV collagen, a diagnostic
feature of basal laminae (Boute et al., 1996; Boury-Esnault et
al., 2003), and complexes of extracellular matrix underlying
the epithelium in other demosponges have recently been found
to contain spongin short chain collagen that is functionally
equivalent to Type-IV of basement membranes (Exposito et
al., 1991; Aouacheria et al., 2006). Although sponges lack
typical organs and nervous tissue, they do have contractile
cells called myocytes [or actinocytes (Boury-Esnault and
Coordination in a freshwater sponge 3737
Rutzler, 1997)] that structurally and by pharmacological
manipulation resemble primitive smooth muscle cells,
allowing certain contractile behaviour to occur (Parker, 1910;
Prosser et al., 1962; Bagby, 1965; Prosser, 1967). The extent
of coordination of this behaviour is the question addressed in
the present study.
As a filter-feeder, it is likely that the main problem
encountered by a sponge is intake of unwanted material into
the aquiferous system. Like other filter feeders, sponges have
developed mechanisms to control the feeding current – but
these differ in the two physiologically distinct types of sponges.
Glass sponges (Class Hexactinellida) form syncytial tissues
during early embryogenesis, and this tissue allows them to
arrest their feeding current by propagating action potentials
(Lawn et al., 1981; Lawn, 1982; Mackie et al., 1983; Leys and
Mackie, 1997; Leys et al., 1999). These animals apparently
lack any contractile tissues. In contrast, cellular sponges
(Classes Calcarea and Demospongiae) control their feeding
current by contracting centralized sphincters or cells that line
the aquiferous canal system (Leys and Meech, 2006). The slow
rate of contractions recorded to date suggests there is no
electrical coupling between cells as suggested by Mackie
(Mackie, 1979) and reiterated by Nickel (Nickel, 2004). So far
ultrastructural studies have not identified gap junctions in
cellular sponges (Green and Bergquist, 1979; Garrone et al.,
1980; Lethias et al., 1983), but since proteins immunoreactive
to anti-connexin antibodies were found in penatulaceans and
anemones (Anctil and Carette, 1994; Mire et al., 2000),
innexin- or connexin-like molecules may yet surface from the
current sponge genome project (Joint Genome Institute 20057). Nevertheless, in the presumed absence of such junctions
cellular sponges must possess another mechanism for
coordinating effective responses to stimuli.
Over a century of research has explored the intricacies of
sponge responsiveness, but because each study has observed
different structures (ostia, oscula or choanosome) in different
animals, the events have been thought to be localized and
decremental (not propagated); cellular sponges have not been
considered capable of the coordinated behaviours of higher
animals (Jones, 1962; Mackie, 1979; Pavans de Ceccatty, 1979).
However, a fresh look at the activities of species in two
demosponge genera, Tethya and Ephydatia, suggests cellular
sponges are able to propagate contractions both endogenously
and in response to external stimuli. Tethya is an opaque ballshaped sponge that contracts rhythmically, shrinking to one
third of its normal size in 21·min (Pavans de Ceccatty et al.,
1960; Reiswig, 1971); similar contractions can be triggered by
natural stimuli (e.g. touch of a crustacean) (Nickel, 2004) and
by chemical stimuli (Parker, 1910; Emson, 1966; Ellwanger and
Nickel, 2006). In juveniles of Ephydatia muelleri, a transparent
encrusting sponge, waves of contraction travel through the
canals and chambers, taking up to 1·h to entirely encompass the
entire sponge (de Vos and Van de Vyver, 1981; Weissenfels,
1984; Weissenfels, 1990).
The objective of the present study was to determine whether
responses to stimuli amount to a coordinated event; that is, do
the various parts of the sponge – the pinacoderm, ostia, osculum
and aquiferous canals – function together to propagate
contractions in a directional manner throughout the sponge’s
body. This study presents the first characterization of the
inflation (dilation)–contraction behaviour of E. muelleri. Due to
its small size and transparency, and the simplicity of its body
design, E. muelleri offers a practical model for future
physiological studies.
Materials and methods
Collecting and culturing of sponges
Gemmules (reduction bodies) and pieces of the freshwater
sponge Ephydatia muelleri (Lieberkuhn 1955) were scraped
from sunken trees or submerged rocks in Frederick Lake, BC,
Canada (48°47⬘51.7559⬙; 125°2⬘58.5600⬙) at a depth of
0–3·m and stored in unfiltered lake water at 4°C in the dark
until use (Ricciardi and Reiswig, 1993). Bags with sponge
pieces were aerated monthly, and gemmules stored in this
way were viable for at least one year. The gemmules were
removed from the spicule skeleton by gently rubbing sponge
fragments between 2 pieces of wet corduroy. Loose
gemmules were washed in cold distilled water (4°C) to
remove debris, sterilized with a 1% hydrogen peroxide
(H2O2) solution for 5·min, and rinsed with cold distilled water
to remove excess H2O2.
Using sterile pipettes, gemmules were transferred to Petri
dishes containing Strekal’s growth medium (0.9·mmol·l–1
Na2SiO3·9H2O, 0.1·mmol·l KCl) (Strekal and McDiffett,
1974) or M-medium (0.5·mmol·l–1 MgSO4·7H2O, 1·mmol·l–1
CaCl2·2H2O, 0.5·mmol·l–1 NaHCO3, 0.05·mmol·l–1 KCl,
0.25·mmol·l–1 Na2SiO3·9H2O) (Funayama et al., 2005). For
whole-mount preparations, single gemmules were placed on an
ethanol-washed, flamed glass or plastic 22·mm2 coverslip in
Petri dishes. For sandwich preparations, one 18·mm2 coverslip
was mounted with dental wax (Hygenic Corporation, Arkon,
OH, USA) at the corners on a cover slip-bottom culture dish
(Willco Wells B. V., Amsterdam, The Netherlands) that had
been sterilized in 30% H2O2 and rinsed with 100% ethanol prior
to use. Two gemmules were placed at the edge of the raised
coverslip, and dishes were left undisturbed at room temperature
(21°C) in the dark. The growth medium was replaced every
Digital video time-lapse microscopy and image analysis
Time-lapse imaging was carried out using either an inverted
compound microscope (Zeiss Axioskop) or a stereomicroscope
(Olympus SZX-12). Images were captured with digital cameras
(QI-Cam monochrome with color filter, Retiga monochrome
and Sony CCD), which were interchangeable on both
microscopes. Image capture and analysis was carried out using
Northern Eclipse version 7 (Empix Imaging Inc., Mississauga,
ON, Canada) from both live video feed and digitally taped
material. Stimulation of the juvenile sponges consisted of
exposing sponges to water-soluble black calligraphy ink (Sumi
black ink, Delta Art Supplies, Edmonton, AB, Canada) at a
concentration of 1 drop (25·␮l) of 100⫻ diluted ink in 1·ml
culture water (final dilution 4000⫻) or vigorous shaking
(2–4·Hz) of the culture medium over the sponge in the Petri dish
for 1·min [hereafter called agitation, as published elsewhere (de
Vos and Van de Vyver, 1981)]. Images were captured by
Northern Eclipse every 5, 10 or 20·s, as indicated for each
3738 G. Elliott and S. P. Leys
experiment. The use of water jets, pin-pricking or damage to
sponge tissue did not solicit an inflation–contraction cycle; these
stimuli only generated local contractions of tissue.
Changes in diameter of the canals for every first, fifth, tenth
or 20th image of the aquiferous canals, ostia, osculum and
apical pinacoderm were measured in triplicate using Northern
Eclipse, and data were logged to MS Excel 2003. In whole
preparations, measurements of the aquiferous canals were
taken at the center (diameter 217.73±14.93·␮m), middle
(diameter 107.38±3.75·␮m), and peripheral canals (diameter
40.16±1.39·␮m). In sandwich preparations, the inner
diameter of the canals was measured at two locations (100
and 300·␮m apart) along a single canal. For area
measurements, images of ink-fed sponges were converted to
greyscale with Adobe Photoshop, two regions of 1450·␮m by
1350·␮m (those occupied by canals) were thresholded from 0
to 130 and the black area (that occupied by canals) was
calculated and expressed as a proxy for the contraction of
Fixation for fluorescence and confocal microscopy
Juvenile sponges on glass coverslips (Fisher no. 1, Ottawa,
ON, Canada) were placed directly into a mixture of 3.7%
paraformaldehyde and 0.3% gluteraldehyde in phosphatebuffered saline (PBS; 100·mmol·l–1) for 24·h at 4°C. After
fixation, preparations were washed in cold buffer and incubated
in 1% sodium borohydride for 5·min to remove autofluorescent
free aldehyde groups. Sponge tissues were permeabilized with
0.2% Triton-X100 in PBS for 2·min and washed in cold PBS.
To label the actin cytoskeleton, coverslips were inverted onto a
drop of solution containing Bodipy 591 Phalloidin, Alexa 594
Phalloidin or Bodipy 505 FL Phallacidin (Molecular
Probes–Invitrogen, Carlsbad, CA, USA) in PBS with 10%
bovine serum albumin (BSA). A 300·␮l depression was made
in a ParafilmTM-covered Petri dish to prevent damage to the soft
tissue by the gemmule. After 3·h at room temperature,
preparations were rinsed three times in cold PBS. For mounting,
sponges were incubated in a 50:50 v/v glycerine:PBS solution,
and mounted in 100% glycerine or in Mowiol with Dabco
(antifade reagent; Polysciences, Warrington, PA, USA), and
allowed to harden overnight. For best results slides were stored
at 4°C. Preparations were viewed with a Zeiss Axioskop
epifluorescence microscope or a Leica 2 photon confocal
Fixation for scanning electron microscopy
Juvenile sponges on plastic or glass coverslips were fixed in
a cocktail consisting of 1% OsO4, 2% gluteraldehyde in
0.45·mol·l–1 sodium acetate buffer (pH·6.4) with 10% sucrose
for 24·h at 4°C (Harris and Shaw, 1984). The following day,
preparations were washed with cold distilled water and
dehydrated in cold 70% ethanol for 24·h at 4°C. Sponges on
glass coverslips were desilicified in 4% HF in 70% ethanol for
2·h at 4°C. Once the sponge had lifted off the coverslip, it was
placed into a new Petri dish with fresh 4% HF in 70% ethanol
at 4°C until spicules were dissolved. After desilicification, the
loose sponges were dehydrated to 100% ethanol and, while still
in the vial of ethanol, fractured in liquid nitrogen. Sponges on
plastic coverslips and fractured pieces of loose sponge were
critical point dried, mounted on aluminium stubs with silver
paste or nail polish, gold coated, and viewed in a field emission
scanning electron microscope (SEM).
Description of juvenile sponge
Gemmules of the freshwater sponge Ephydatia muelleri
hatched in sterile culture dishes at room temperature (18–23°C)
in the laboratory within 2–4 days of plating. Within 4 days of
hatching, the apical pinacoderm (surface epithelium),
choanocyte chambers, canal system and incipient osculum had
begun to develop, and by 7–10 days, a filtering juvenile sponge
was formed. 7–10-day-old sponges typically had a single
osculum arising from two large excurrent canals that bifurcated
around the gemmule and branched successively into finer canals
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·␮m.
Coordination in a freshwater sponge 3739
lined by choanocyte chambers. In other specimens, the osculum
was positioned directly over the gemmule and arose from a
highly branched network of smaller excurrent canals
Description of the inflation–contraction behaviour
The response triggered by stimulation of the sponge, either
by adding ink to the water or by agitation of the dish, consisted
of three phases (Fig.·2A–E; Movie·1 in supplementary
material): an inflation phase, in which the major excurrent
canals dilated; a plateau phase, involving dilation of smaller
diameter canals (this phase was most pronounced in larger
specimens); and a contraction phase, in which the excurrent
canals constricted and there was a rapid contraction of the
osculum. The sequence of events following either stimulus was
similar, but changes in the morphology were more readily
measured in the absence of ink.
Every response consisted of eight identifiable events: (1)
initial contraction of the osculum and apical pinacoderm
(Fig.·2A); (2) Inflation phase: expansion of the sub-dermal
cavity raising the apical pinacoderm and dilation of the
excurrent canals travelling from the base of the osculum back
along the aquiferous canal system; (3) dilation of the smallest
peripheral canals; (4) contraction of porocytes (closure of ostia)
in the apical pinacoderm; (5) Contraction phase: lowering of the
apical pinacoderm forcing the water into the aquiferous canals;
(6) a peristaltic-like wave of contraction that travelled along the
excurrent canals from the distal edge of the sponge to the base
of the osculum and caused the contraction of the choanocyte
chambers (Fig.·2B); (7) a rapid contraction propagating from the
base to the tip of the osculum (Fig.·2C); and (8) relaxation of
the canals to their original diameter and extension of the
osculum back to its original length (Fig.·2D). This sequence
followed a predictable time course, and up to three such
sequences occurred after a single stimulus, each separated by a
recovery period.
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.)
3740 G. Elliott and S. P. Leys
Table·1. Duration of the phases in the inflation–contraction cycle in response to different stimuli
⌬t I (min:s)
⌬t Ch-C (min:s)
⌬t Os-C (min:s)
⌬t I-C (min:s)
Ink ‘fed’
11:30±4:43 (8)
8:31±1:38 (12)
15:55±3:19 (8)
10:43±1:34 (12)
1:43±0:24 (5)
0:41±0:19 (3)
30:45±2:01 (8)
19:09±2:45 (12)
Values are means ± s.e.m.; N values are given in parentheses.
⌬t I Duration of inflation cycle; ⌬t Ch-C, duration of choanosome contraction; ⌬t Os-C, duration of oscular contraction.
Although the velocity of the propagated contraction varied as
it progressed from the periphery of the sponge towards the
osculum, both the left and right hand sides of the sponge inflated
and contracted equally (Fig.·2F,G).
Response to the addition of inedible ink particles
The inflation–contraction cycle was first recorded in response
to the addition of inedible ink to the culture dish. Ink taken into
the sponge became lodged in the choanocyte chambers, making
them black. Addition of the ink into the culture medium resulted
in several events. The first was identical to the orchestrated
series of responses triggered by agitation of the sponge as
described above, but resulted in ejection of clumps of ink
(supplementary material Fig.·S1A–E; Movie·2). Additional
responses followed minutes to hours later; up to three additional
peristaltic-like waves traversed the entire sponge over a 48·h
The ink treatment also generated brief contractions that
occurred simultaneously in different parts of the choanosome
(like twitches), as well as short waves of contraction that
propagated across portions of the choanosome in a linear
direction: ripples. There were also local non-propagating
inflations and contractions: local events. Addition of too little
ink to the dish did not trigger the full ‘inflation–contraction
behaviour’, but twitches and ripples still occurred. Similarly,
too little agitation of the dish failed to trigger a full
inflation–contraction cycle, but twitches, ripples and local
events were common after any amount of agitation.
Attempts to trigger the full inflation–contraction cycles by
focal tactile stimuli (pin pricks) and electrical stimuli have so
far been unsuccessful.
The kinetics of the inflation–contraction cycle
Comparison of the duration of the entire cycle from start of
inflation to end of contraction for ink ‘fed’ (30:45±2:1·min:s;
N=8) and shaken sponges (19:9±2:45·min:s; N=12) suggests
that addition of ink slows down the process (Table·1).
Estimates of rates at which the events occur in different regions
of the sponge (across the choanosome, along a canal, and up
the osculum) depended upon the type of preparation (whole
mount or sandwich) and type of stimulus applied. Contractions
of the osculum were fastest, but contractions propagated
across the choanosome more slowly during a full
‘inflation–contraction’ cycle (2–5·␮m·s–1) than during ripples
that occurred between cycles (4–11·␮m·s–1) (Table·2). During
an ‘inflation–contraction’ cycle, contractions usually
propagated across all tissues from the periphery of the sponge
to the base of the osculum; in some cases the waves travelled
along the long axis of canals, but in others an entire canal
expanded in unison as the wave propagated across it. All told,
the rates appeared to be very dependent on the resulting effect
of the contraction.
The full inflation–contraction behaviour had stereotypical
‘inflation’, ‘plateau’ and ‘contraction’ phases, but the duration
of the entire event varied depending on the initial (resting)
diameter of the aquiferous canals: sponges with larger resting
canal diameter (e.g. 64.9, 76.9, 103.52, 154.13 and 213.35·␮m)
had a longer overall inflation–contraction phases (500, 899,
1399, 2052, 2988·s; Fig.·3 and supplementary material Fig.·S2),
extending the duration of the entire cycle from 15 to 40·min.
Otherwise, the events occurred almost identically in sponges
Table·2. Rates of contraction in different regions of E. muelleri juveniles
Contraction rate
2.80±0.26 (5)
3.30±0.45 (5)
Canals (full cycle; ink; sandwich preparation)
Incurrent canal (inflation phase)
Excurrent canal (contraction phase)
1.68±0.78 (6)
0.49±0.13 (7)
Canals (ripple; agitation; whole mount
7.09±0.95 (7)
71.85±32.4 (3)
17.68±8.26 (5)
Region (type of response; stimulus and preparation)
Canals (full cycle; agitation; whole mount)
Incurrent canal (inflation phase)
Excurrent canal (contraction phase)
Osculum (full cycle; agitation; whole mount)
(full cycle; ink; whole mount)
Values are means ± s.e.m.; N values are given in parentheses.
Coordination in a freshwater sponge 3741
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·␮m, 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·␮m·s–1,
N=5; ex: 3.30±0.45·␮m·s–1, N=5, P=0.23). Bars denote ± s.e.m. of
three measurements from one sponge (see supplementary material
Fig. S2).
Diameter of canals (μm)
1800 2400
Time (s)
with quite different patterns of canals. The rates of dilation and
contraction of the large excurrent canals were similar
(2.80±0.26·␮m·s–1, N=5 and 3.30±0.45·␮m·s–1, N=5, P=0.23,
respectively; Table·2). Interestingly, the rate of the peristalticlike contraction, measured from preparations in which specific
points on the canals could be accurately tracked at high
resolution, depended upon which region of the aquiferous
system it moved through. In the peripheral canals it traveled at
0.03–1·␮m·s–1, in the central canals at 1–4·␮m·s–1, and up the
osculum at 6–122·␮m·s–1.
Sandwich preparations allowed clear observations of the
waves of peristaltic-like contraction, and use of the ink stimulus
provided a clear marker for incurrent and excurrent aquiferous
canals. This preparation revealed that during the inflation phase,
dilation of the excurrent canals was caused by a wave of
contraction travelling along the incurrent canals. Ink entered
incurrent canals rapidly filling choanocyte chambers
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·␮m apart) in 300·s, a rate of 0.33·␮m·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·␮m apart) in 940·s, rate of 0.32·␮m·s–1. in, incurrent; ex, excurrent;
arrows indicate direction of water flow in the canal. Scale bars, 100·␮m (A); 300·␮m (C). (Movie 3 in supplementary material.)
3742 G. Elliott and S. P. Leys
(supplementary material Fig.·S3, Movie 3). Contraction of the
incurrent canals condensed the ink in the chambers and incurrent
canals and even forced some ink-filled water through the
choanocyte chambers into the excurrent canals during the plateau
phase. During the contraction phase, a wave of contraction
propagated along the excurrent canals so as to cause the dilation
of the incurrent canals. As the excurrent canals contracted, the
flow of water (as seen by movement of ink) briefly reversed
direction, and then remained stationary for up to 6·min. After one
inflation–contraction cycle the sponge returned to a relaxed state.
This type of preparation also illustrated that the wave of
contraction propagated along two vectors, both along and across
the incurrent and excurrent canals (Fig.·4A; supplementary
material Movie 3). Contractions traveled across canals that were
310·␮m apart at a delay of 300·s (approximately 1·␮m·s–1).
Furthermore, cells crawling through the mesohyl arrested
forward motion for about 10·min (approximately 600·s) as the
wave of contraction passed by (Fig.·4B). The two cells tracked
here were 1053·␮m apart, and they arrested with a delay of 600·s.
In sandwich preparations stimulated with ink, the
contractions propagated along the incurrent canals slightly
faster than along the excurrent canals (Table·2; Fig.·4C,D).
Time-lapse images of these events in sandwich preparations
suggest that cells in the mesohyl between two canals shorten,
causing the choanocyte chambers to compress. These images
also show that cells crawling through the mesohyl stop moving
as the waves of contraction pass over them (supplementary
material Movie 3).
Immediately after agitation or addition of ink, the osculum
contracted downwards. Then, as the aquiferous canals
contracted, the base of the osculum dilated to become almost
balloon-like. Only when the entire choanosome had completely
contracted did a wave of contraction run from the base to the
tip of the osculum (Fig.·5A–D). The final oscular contraction
took 71.85±32.4·␮m·s–1 (N=3) in agitated sponges and
17.68±8.7·␮m·s–1 (N=5) in ink-fed sponges (range
6–122·␮m·s–1), and was always followed by a slow extension
(supplementary material Movie 4). Because precise changes in
diameter of the osculum were difficult to track in ink-fed
animals, measurements for those animals present a conservative
estimate of the duration of the contraction event.
Apical pinacoderm
Upon agitation the apical pinacoderm contracted down
towards the choanosome, lowering 50–200·␮m within 60·s.
This contraction occurred after the initial response of the
osculum, but before the inflation of the canals. The apical
pinacoderm moved as a single unit, like a diaphragm, reducing
the volume of the sub-dermal space. During the inflation phase,
the apical pinacoderm moved back to its relaxed position
(Fig.·5A,B), and just before the excurrent canals contracted, it
lowered again. For sponges with a diameter of 3–5·mm, these
waves of contraction traveled at 50–80·␮m·s–1 propagating from
the periphery of the sponge to the base of the osculum. In some
instances a series of twitches occurred across the entire surface
of the sponge just before the main wave of contraction that
lowered the entire apical pinacoderm (supplementary material
Movie 4).
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·␮m, in 12·s, a rate of 122.8·␮m·s–1. Bars denote
± s.e.m. of an average of three measurements from one sponge. (Movie
4 in supplementary material.)
In relaxed sponges, fields of porocytes – flat cells that formed
the ostia, incurrent openings for water – littered the apical
pinacoderm (Fig.·6). The margin of each cell was anchored in
a collagenous extracellular matrix between the inner and outer
epithelia of the apical pinacoderm. Each porocyte was
surrounded by 3–4 plate-like exopinacocytes. In a relaxed
sponge there were 10–12·porocytes·mm–2 of apical pinacoderm
(Fig.·6A). After stimulus by agitation, fields of up to 35 ostia
closed synchronously (Fig.·6A–C; supplementary material
Movie 5). Individual ostia took approximately 40·s to close, and
a field of ostia closed just before the contraction of excurrent
canals in the choanosome. Ostia re-opened as canals relaxed
In ink-fed sponges the ostia also closed just before canals
contracted, and remained closed until the contractions had
finished. Use of the ink as a stimulus revealed that in all cases
Coordination in a freshwater sponge 3743
Area II
1450 μm
Area I
1350 μm
Projected area (%)
Area I
Area II
Event 1
Event 2
150 200 250 300
Time (s)
400 450
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·␮m
apart. (Movie 2 in supplementary material.)
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·␮m. (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
a few ostia near the base of the osculum remained open,
allowing a small amount of water to flush back out through the
sub-dermal cavity (observed as puffs of ink in supplementary
material Movie 6).
Kinetics of twitches, ripples and local contractile events
In between sequential inflation–contraction cycles, brief
propagating and non-propagating contractions took place. In
many experiments, waves of contraction rippled across portions
of the sponge choanosome at a rate of 7.09±0.95·␮m·s–1 (N=7);
these contractions did not travel towards the osculum and
occurred without periodicity. Local contractile events also
occurred in the interval between major inflation–contraction
cycles. Here, a small region of the choanosome, usually no more
than several hundred ␮m in diameter, inflated and contracted
independently of any other activity of the sponge. In some
experiments, the entire sponge choanosome contracted rapidly
and apparently simultaneously like a twitch. These quick, global
contractions of less than 20·s duration occurred nearly
simultaneously (<5·s difference) in very different regions of the
choanosome (Fig.·7A,B; supplementary material Movie 2).
Typically, an unstimulated sponge exhibited occasional
ripples, twiches and local inflation–contraction events;
however, only one full inflation–contraction cycle occurred
during every 8·h of 48·h of observation.
The contractile apparatus of the sponge
Phalloidin-labelled sponges revealed dense tracts of actin in
3744 G. Elliott and S. P. Leys
endopinacocytes of the apical pinacoderm, canals and the
osculum. In the apical pinacoderm, 2–3 bundles of filamentous
actin traversed individual pinacocytes (Fig.·8A,B). Contacts
between neighbouring cells labelled brightly, like adhesion
plaques, and actin bundles in adjacent cells continued in the
same direction so as to form tracts that were continuous for up
to 3·mm (Fig.·8B). These tracts of actin stretched across the
apical pinacoderm, around the perimeter of the sponge, and
from the perimeter of the sponge to the top of the gemmule
converging at the pinnacle of shafts of spicules that supported
the overlying apical pinacoderm. Endopinacocytes lining the
canals labelled much less intensely with phalloidin, and fine
tracts of actin were visible only in sandwich preparations in
which sponges grew in a 50·␮m thick space between two
coverslips (Fig.·8C). In unstimulated sponges, excurrent canals
were lined by thin (1–3·␮m) endopinacocytes, and choanocyte
chambers were spherical (30·␮m in diameter) (supplementary
material Fig.·S4A,B). In sponges fixed in a contracted state,
endopinacocytes lining the excurrent canals were thicker
(5–7·␮m and choanocyte chambers were compressed to the
extent that their flagella projected out through the apopyle
(supplementary material Fig.·S4C,D).
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·␮m. (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·␮m. (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·␮m.
Relative change in diameter
In response to a mechanical stimulus Ephydatia muelleri
initiates a series of slow contractions (summarized in Fig.·9) that
effectively expel water and wastes from the aquiferous system.
The periodicity of contractions seen in unstimulated sponges,
and reported in other species and genera, suggests this behaviour
may also function to assist the sponge feeding and/or respiratory
activity (Weissenfels, 1990; Nickel, 2004). There is growing
evidence that sponges, like other metazoans, possess a broad
repertoire of signaling molecules (e.g. Perovic et al., 1999;
Nichols et al., 2006; Adell et al., 2007; Sakarya et al., 2007) and
experiments have demonstrated that many of these substances
trigger contractile responses in a variety of sponges (Emson,
1966; Prosser, 1967; Ellwanger and Nickel, 2006; Ellwanger et
al., 2007). The exact nature of the contractile response has
nevertheless been rather unclear, largely due to the difficulty of
watching the animal at the cellular level. The small size and
transparent tissues of E. muelleri, however, allow high
magnification observations of specific regions of the sponge,
which illustrate that it is not the speed of contraction but rather
the temporal and spatial coordination of all the events that
allows the sponge canal system to form an effective peristaltic-
Apical pinacoderm
Time (s)
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.
Coordination in a freshwater sponge 3745
like pump. Given the basal position of sponges within Metazoa,
and the absence of nerves and true muscle in this group (Pavans
de Ceccatty, 1989) (see references above), it can be inferred that
what we see in sponges today represents a coordination system
that predated the evolution of neuromuscular systems observed
in higher Metazoa.
Rates of contraction
Rates of waves of contraction reported in cellular sponges are
several orders of magnitude slower than electrically controlled
contractile systems (for reviews, see Mackie 1979; Mackie et
al., 1983). Contractions tend to be slightly slower in freshwater
than marine sponges (presumably due to the lower calcium
available), but rates of endogenous contractions reported in the
literature largely depend on what region of a sponge was
observed. For example, waves of endogenous contractions cross
the surface (including the choanosome) of marine sponges
(Tethya wilhelma, Tethya lyncurium, Euspongia officinalis) at
12–30.5·␮m·s–1 (Pavans de Ceccatty, 1969; Pavans de Ceccatty,
1971; Nickel, 2004), and freshwater sponges (Ephydatia
fluviatilis, Eunapius fragilis, Spongilla lacustris) at
8–11·␮m·s–1 (de Vos and Van de Vyver, 1981; Weissenfels,
1990). Electrical stimuli applied to the base or tip of the osculum
of Ephydatia fluviatilis triggered faster waves of contractions up
and down (170 and 350·␮m·s–1, respectively) the osculum
(McNair, 1923); Prosser (Prosser et al., 1962) reports a similarly
quick contraction of the oscula (1–5·s for 1·mm diameter oscula)
in several marine species. Furthermore, precise measurements
of rates are difficult to calculate from video recordings or a
series of still images. For example, in T. wilhelma, periodic
contractions have been documented by measuring the decrease
in area of a projection of the sponge (Nickel, 2004).
Subcontractions (equivalent to ripples) propagate at 12.5·␮m·s–1
over the surface of the sponge, yet full contractions take
20–50·min to encompass the entire sponge; relaxation
(inflation) takes somewhat longer. Although it is proposed that
the contraction travels through the pinacoderm, because Tethya
is an opaque sphere, the route that the contractile wave travels
cannot be easily determined.
We encountered similar difficulty in determining precisely
when contractions initiate at two points 100·␮m apart along a
canal. Cells in the mesohyl around the canal begin to change
shape long before changes to the diameter of the canal are
evident. Also, in some instances entire canals seemed to widen
uniformly along their entire length, such that no ‘rate’ of
propagation could be measured. In general, however,
contractions propagated very slowly through the canals at the
periphery of the sponge (0.3–1·␮m·s–1), slightly faster through
the large exhalent canals (1–4·␮m·s–1), and even faster up the
osculum (6–122·␮m·s–1); these were part of the overall
‘inflation–contraction’ behaviour, while ripples and twitches
occurred separately. Thus our study indicates that the actual
speed of propagation of a contraction depends on the function
of the contractile tissue (the effector). From this we infer that
because each region comprises part of a hydrostatic skeleton
whose function is to expel water from the aquiferous system,
the rates observed indicate control of the body of water rather
than the absolute ability to propagate a signal. The individual
rates observed result from coordination of these regions.
Coordination of effectors
Coordination of the series of effectors is seen most acutely in
the synchronous closure of fields of ostia independently of, and
usually just before, the contraction of the apical pinacoderm. It
has long been known that individual porocytes contract (Emson,
1966; Kilian and Wintermann-Kilian, 1979), but this is the first
data showing that whole fields of porocytes contract and relax
in unison. Synchronous closure of ostia is a remarkable event.
The contraction of each porocyte sphincter takes some 60·s, but
the fact that up to 50 ostia close over the same time frame, and
just before the choanosome contracts, points either to some
fairly rapid coordinating signal traversing the apical
pinacoderm, or suggests that inflation of the entire sponge
stretches the apical pinacoderm, triggering simultaneous closure
of ostia (presumably by entry of calcium into each porocyte). It
is interesting to note that in all experiments a few ostia remained
open around the base of the osculum, allowing ink to be flushed
back and out of the sub-dermal cavity. Reversal of flow by
sponges has only been described by Storr (Storr, 1964) and
likely refers to a similar back-flushing event during a periodic
(cyclical) contraction event.
Contraction of inhalant and exhalant canals also demonstrates
coordination of effectors. We initially thought that dilation of
the exhalent canals occurred by passive inflation when the
osculum closed in response to the initial stimulus. However,
careful observation of videos shows that the osculum is never
entirely closed – the tip constricts, but a fast stream of water
continues to flow from it at all times (e.g. ink flows from the
constricted osculum prior to explusion of ink from the
choanosome, see supplementary material Movie 2). Sponges
treated with cytochalasin B did not inflate the choanosome
(dilate the incurrent or excurrent canals), even though the
osculum did a small initial contraction when the dish was
vigorously shaken; thus passive inflation of the choanosome is
unlikely (data not shown). Because videos of sandwich cultures
show that cells in the mesohyl bridging adjacent exhalent canals
contract during the inflation period, we suggest that dilation of
the exhalent canals seems to be at least partly due to the active
contraction of inhalant canals. These observations explain why
the rates of inflation and contraction are very similar regardless
of the diameter of the canal (Table·1). What can also be seen is
that water is absolutely stagnant for some part of the plateau
phase (the ink front in the excurrent canal remains completely
stationary for up to 6·min in one instance; supplementary
material Movie 3), i.e. the sponge uses contractions to control
the movement of water in its canals. This observation is the first
precise visual demonstration that cellular sponges can stop their
feeding current.
Evidence for effector tissue and signal propagation
Most studies suggest that endopinacocytes (the cells that line
the inside of the sponge) are responsible for propagated
contractions (de Vos and Van de Vyver, 1981; Pavans de
Ceccatty, 1986; Nickel, 2004), but in sponges with a denser
mesohyl, it is implied that either myocytes (cells in the mesohyl)
or pinacocytes form sphincters that constrict flow through
canals (Parker, 1910; Pavans de Ceccatty, 1960; Jones, 1962;
Prosser et al., 1962; Bagby, 1965; Pavans de Ceccatty et al.,
1970). The contractile apparatus has been difficult to pin down.
3746 G. Elliott and S. P. Leys
The actin cytoskeleton is only known from stationary
basoendopinacocytes of freshwater sponges (Pavans de
Ceccatty, 1986; Wachtmann and Stockem, 1992), and from
myocytes in one marine sponge (Microciona prolifera). In
basendopinacocytes, the cytoskeleton is much like that of a
fibroblast in which microfilaments form stress fibres across and
around the cell. Actin filaments are slightly denser between
neighboring cells, and between cells adhesion plaques
reminiscent of early stage desmosomes in fish embryos (Lentz,
1966) can be seen in freeze–fracture electron micrographs
(Pavans de Ceccatty, 1986). In contrast, myocytes in sphincters
in the canals are well endowed with both thick and thin
filaments (Bagby, 1965).
Our images show that a substantial actin network exists in the
cells that form the lower portion of the apical pinacoderm, the
endopinacocytes. Bundles of actin filaments form tracts
traversing endopinacocytes, and each tract connects to another
in neighboring cells through a dense plaque of actin; together
these form the longest semi-continuous tracts known in sponges
(1–3·mm). Continuity of the cytoskeleton in the apical
pinacoderm is presumably necessary for the entire tent-like
structure to lower in a single diaphragm-like movement in less
than 60·s. Actin microfilaments appear as ‘rings’ around the
circumference of the aquiferous canals; in both cases tracts
connect to others in neighboring cells, as in the apical
Earlier researchers favored mechanical tugging of one cell on
another as the explanation of contractile waves (Parker, 1910;
Pavans de Ceccatty et al., 1960; Emson, 1966; Pavans de
Ceccatty, 1969). This hypothesis is difficult to test because
damage to any portion of the sponge disrupts flow and interrupts
contractions throughout the sponge. Furthermore, although
mechanical ‘tugging’ might explain how waves of contraction
propagate along canals, it does not readily explain how the
waves propagate across canals or between completely distinct
regions of the sponge as during twitches. It is possible that a
change in pressure could result in signals being transmitted to
a distant site, but how ink building up in the chambers could
generate a pressure wave causing the osculum to constrict (the
first event to occur) is unclear. Moreover, how pressure waves
could orchestrate the spatio-temporal coordination of
contractions in different regions is difficult to imagine.
Recent evidence that diffusible chemical messengers
including amino acids (glutamate and GABA), biogenic amines
and short-lived gases (e.g. nitric oxide) trigger or modulate
contractions in Tethya wilhelma strongly suggest that signals
travel through the mesohyl in a paracrine-like manner or
through the aquiferous system (Ellwanger and Nickel, 2006;
Leys and Meech, 2006; Ellwanger et al., 2007). Perhaps the
most definitive evidence that a diffusible chemical messenger
is involved in contractions in Ephydatia is that cells crawling
through mesohyl stop moving as contractions pass by (Fig.·4),
as also noted by other authors (de Vos and Van de Vyver, 1981).
Since these cells are wandering through the mesohyl, not in
contact with pinacocytes, it can be inferred that a signal passes
through the mesohyl at least at 1.75·␮m·s–1 (a distance of
1053·␮m in 600·s). It is quite possible that chemical and
mechanical signalling function together to coordinate the
propagation of contractions. Nevertheless, the rapid lowering of
the apical pinacoderm and rapid contraction of the osculum are
faster events than can be explained by calcium signalling, which
is generally up to 20·␮m·s–1 (Nedergaard, 1994).
Comparison with other contractile systems
We describe contractions in E. muelleri as ‘peristaltic-like’,
but this is the first time the term peristalsis would be applied to
an animal that lacks muscle. Peristalsis is usually considered to
involve neurogenic modulation of myogenic contraction to
propel a fluid through a tube (Randall et al., 2002). In the sponge
the canals behave as a single motor complex, in which a period
of dilation is followed by a propagated contraction that squeezes
the water forward towards the osculum. Except that neuronal
modulation is absent, the system does not appear much different
from those composed of multi-unit smooth muscles (Randall et
al., 2002).
Peristalsis seems to be a central feature of body plans in all
animals. It is involved in moving fluid for nutrient transfer in the
gastrovascular cavity (GVC) of the sea pansy Renilla koellikeri
(Anctil, 1994), for burrowing by anemones, nemerteans,
polychaetes and bivalves (Ansell and Trueman, 1968). Peristalsis
is also involved in the contraction of the heart in tunicates and
amphioxus (Holland et al., 2003). In each of these instances
control is thought to be myogenic, although the role of nerves is
not well understood. It is interesting to note that while
contractions of the GVC of Renilla propagate at 1–1.3·␮m·s–1
(Anctil, 1994; Anctil et al., 2005), contractions of the body wall
of the sessile anemone Metridium senile propagate at
~500·␮m·s–1 (Batham and Pantin, 1950) and even slower in the
tiny burrowing starlet anemone Nematostella vectensis (at
3–20·␮m·s–1) (S.P.L., unpublished observation). Cnidarians
have the advantage of both muscle (epitheliomyocytes) and
neurons, yet contractions are still slow. As previously suggested
(Batham and Pantin, 1950), this is presumably due to the load
the muscle acts against rather than intrinsic limitations, because
when stimulated electrically, the same region of the body wall
can contract much faster. Our observations suggest this is also
true for sponges. In order to expel water, the tissues contract in
a controlled and coordinated manner; but when water is not being
pushed out of the aquiferous system faster contractions are
possible, as when ripples run across portions of the sponge or
the osculum contracts down in response to mechanical agitation.
Evidently sponges have, without nerves or true muscle, evolved
a way of coordinating contractions of cells to generate an
effective mechanism of controlling water flow.
The next step is to determine what signal or mechanism
controls each type of contraction. Because of its small size and
transparency, the freshwater sponge promises to be an excellent
model system for further study of the role of signalling
molecules in inducing, controlling, and modulating behaviour
in these ‘simple animals’.
We gratefully acknowledge the assistance of the Director
and staff at the Bamfield Marine Sciences Centre where
animals were collected and Ikenna Ejieke for help with the
analysis of Fig.·4. George Mackie, A. Richard Palmer and
three anonymous reviewers provided helpful suggestions on
earlier drafts of the manuscript. This research was funded by an
NSERC Discovery Grant to S.P.L.
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