Kinetics and rhythm of body contractions in the sponge Tethya wilhelma (Porifera: Demospongiae)
Department of Zoology, Biological Institute, Stuttgart University, D-70550 Stuttgart, Germany
e-mail: michael.nickel{at}bio.uni-stuttgart.de
Accepted 15 September 2004
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Summary |
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Key words: contraction, kinetics, diurnal rhythm, time-lapse imaging, coordination, behaviour, sponge, Tethya wilhelma
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Introduction |
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Sponges are generally regarded as sedentary organisms with no striking
degree of behaviour or irritability. Nevertheless, since the time of Aristotle
(384-322 BC) it has been well known, at least among sponge scientists, that
sponges can contract (see Aristotle,
1498; Lieberkühn,
1859
; Schmidt,
1866
; Weissenfels,
1990
), react to external stimuli
(Emson, 1966
;
Leys and Mackie, 1997
;
McNair, 1923
;
Pavans de Ceccatty, 1979
) and
even move (Bond, 1992
;
Bond and Harris, 1988
;
Fishelson, 1981
;
Jones, 1957
;
Kilian, 1967
;
McNair, 1923
). The integration
and coordination of this behaviour has been widely discussed over the last 50
years. The main foci of these discussions have been whether or not sponges
possess a nervous system (Jones,
1962
; Lentz, 1968
;
Mackie, 1979
,
1990
;
Pantin, 1952
; Parker,
1910
,
1919
;
Pavans de Ceccatty, 1960
;
Perovic et al., 1999
) and
other possible mechanisms of integration
(Jones, 1962
; Pavans de
Ceccatty, 1974
,
1979
;
Weyrer et al., 1999
). This
polarizing discussion concluded with the statement that sponges do not possess
a nervous system, but did not explain the mechanisms underlying coordination
in the aneural Porifera. Much more effort is needed on this topic, which is
directly linked to basic questions about the evolution of multicellularity,
and for which experimental model sponge systems are needed.
Recently we described three new species of the genus Tethya
(Sarà et al., 2001),
which have the potential to serve as model organisms for integrative research
on behaviour, signal transduction and the underlying basal molecular
mechanisms. One of these species, T. wilhelma, is especially
interesting as a model system because it displays strong, rhythmical body
contractions and is able to expand and retract body extensions and even to
move (Nickel, 2001
,
2003
; Nickel and Brümmer,
in press). In addition it is possible to cultivate T. wilhelma.
The present report characterizes the contraction behaviour of T. wilhelma, its endogenous short- and long-term rhythms, and detailed kinetics of the contraction cycles, taking into account any endogenous and exogenous triggers involved in contraction regulation. The aim of the study was to assess sponge behaviour precisely for the first time, both qualitatively and quantitatively. We used T. wilhelma as a model system for quantitative physiological and pharmacological studies on signal triggering, transmission and integration from organism level down to tissue, cellular and molecular levels, respectively.
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Materials and methods |
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Experimental manipulations
For the experiments the sponges were allowed to attach on a black plastic
carrier slide for 24-48 h in the aquarium. After attachment they were
transferred to stages inside the observation chambers. For one experiment, two
sponges were allowed to fuse together, and then their contractile behaviours
recorded.
Observation chambers
Long-term observations were either performed directly in the aquarium, or
in open and closed observation chambers. The open glass chamber had a volume
of 3.5 l and was connected to the aquarium by a pump cycle. The closed system
had a total volume of 0.25 l, and consisted of an aerated experimental
reactor, based on the principles of airlift reactor design, connected to a
temperature regulation unit (F25, Julabo, Seelbach, Germany). Oxygen level and
temperature were monitored using a multi-sensor system (P4, WTW, Weilheim,
Germany), controlled by a computer-software (MultiLab Pilot 3.0, WTW). A
built-in optical glass filter (Ø; 49 mm, D.K. Enterprises, India)
allowed proper imaging.
Time-lapse imaging
Digital images of the sponges were taken at a resolution of 2048x1536
pixels at regular intervals of 30-200 s, depending on the experiment. A Nikon
Coolpix 990E digital camera in manual macro focus and exposure mode was used
to acquire greyscale images. The camera was connected to a Nikon SB 24 flash
unit, set to manual mode (24 mm, output 1/16). The camera was controlled by a
PC, using USB connection cable and the software DC_RemoteShutter V 2.3.0 in
conjunction with DC_TimeTrigger V. 1.0
(Madson, 2003). Images were
downloaded, saved on the PC and erased on the CF-card of the camera
immediately after being taken. A reference image including a scale bar placed
next to the sponge was taken for each experimental series, for scaling. In all
cases a black background was used to maximize contrast.
Image analysis and statistics software
Image analysis was performed using ImageJ 1.30 and 1.31 (NIH, Washington,
USA), based on built in functions (Rasband, 1997-2004;
http://rsb.info.nih.gov/ij/).
Excel 2000 (Microsoft, Redmond, USA) was used to prepare activity diagrams.
The SPSS software package (V. 11.5, SPSS Inc, Chicago, USA) was used to
perform statistical analysis.
Projected area measurement
The projected area measurement was based on the contrast difference between
sponge (whitish) and background (black). All images were scaled using the
reference image. A threshold value between 50 and 90 was applied to the 8-bit
images and the absolute projected area of the sponge was measured using
ImageJ's built-in measurement tool. All time-lapse series were loaded as image
stacks into ImageJ. A macro was programmed to measure semi-automatically.
Measurement results were written to a text file and further computed using
Excel 2000. For the aquarium-based time-lapse series a manual image control
and correction was performed in ImageJ prior to measurement. In this way false
measurements were avoided in cases where errant organisms (snails, polychaets
or amphipods) were crawling on the sponge surface.
For the measurement of two fused sponges, the method described was slightly modified. The outer half of each sponge was used for projected area measurement. A compensation of the shift of the areas during contraction was applied manually. For projected area calculations the values of the measurements were doubled to estimate the area of each individual.
Segment measurement
A measurement method based on image segmentation was developed to obtain
temporally resolved information on contraction waves running over the sponge
body. A grid of 1 mm2 fields was projected over the image and
selected fields were measured separately, as described for the whole sponge
projected area above.
Contraction kinetics
For calculation of the kinetics of an average contraction cycle, values of
12 sequential cycles of a time-lapse series were analysed. For maximum
contraction (area minimum), time was set to zero. For each cycle, relative
contraction values were calculated by setting the starting non-contracted
state (area maximum) of each cycle to 1. The relative projected area values of
each cycle were calculated in relation to the antecedent maximum. In this way
the influence of changes in the body extension on the projected area was
minimized. The average contraction including standard deviation
(S.D.) was calculated for each relative time point. Contraction
kinetic diagrams were plotted for two independent datasets. Subcontractions,
small but incomplete events, were not used when calculating contraction cycle
kinetics.
Statistical analysis
For statistical analysis of the long-term rhythm, cycle durations were
measured as time between maximum contractions (area minimum). A
t-test (Welch-test; assuming equal distribution, but no equal
variances; N=80) was performed on two alternative hypotheses:
H0: no difference in cycle length between day and night,
vs H1: difference in cycle length between day and night.
Since two datasets (N=36 and N=44) were combined for this
analysis, the same test was applied to make sure there was no significant
difference between them.
For comparison of different contraction states (full vs partial contractions) the contraction extent was compared using relative projected area values (see above). A t-test (Welch-test; assuming equal distribution, but no equal variances; N=53) was performed on two alternative hypotheses: H0: only one class of contraction extent, vs H1: two classes of contraction extent.
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Results |
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Long-term rhythm of contraction
The contraction rhythms of two specimens were recorded inside the aquarium
(Fig. 2A) and an open chamber
(Fig. 2B) for 177 h and 169 h,
respectively, under a light:dark cycle of 12 h:12 h. When conditions in the
aquarium were natural-like, T. wilhelma displayed 44 contraction
cycles during the observation period, compared to 36 in the open chamber,
where there were no other organisms and very stable current conditions. The
duration of the contraction cycles varied between 73.3 min (2.3
x10-4 Hz) and 609.0 min (
2.7 x10-5
Hz).
|
The average relative contraction extent, measured as relative reduction of
the projected sponge area, differed significantly between the two sponges. In
contrast no significant difference could be found for the average duration of
the contraction cycles between the two datasets. Consequently both datasets
were combined for analysis of a day-night cycle. During the day, sponges
contracted every 215.7±92 min(7.7 x10-5±2
x10-4 Hz; N=44), in comparison to a cycle length of
274.5±149 min (
6.1 x10-5±2
x10-4 Hz; N=36). A significant difference was found
between the average cycle durations of day and night (P=0.042,
=0.05; Fig. 3).
|
In addition to the regular full contraction (movie S1 in supplementary
material) several subcontractions were observed, especially in the aquarium
specimen (Fig. 2A, movie S2 in
supplementary material). The subcontractions led to a reduction in the
relative projected sponge area by 0.158±0.04 (N=9) in
comparison to 0.442±0.06 (N=44) for regular full contractions
in the same experiment. The maximum reduction of projected area observed was
0.58, with minimum 0.30, representing the extremes for regular contractions.
The difference in contraction extent between full contraction and
subcontractions was highly significant (P<0.00001,
=0.05).
In aquarium conditions, several periods of irregular contractions occurred that are a direct reaction of the sponge to organisms such as snails, polychaets and crustaceans crawling on its surface or even feeding on the sponge or epibiontic algae. In one case, T. wilhelma showed a series of strong, irregular contractions when attacked by an amphipod (Fig. 2A, movie S3 in supplementary material). Comparable irregular contraction patterns were not observed in the open chamber, which was virtually free of errant organisms.
Kinetics of contraction
Two consensus diagrams were calculated and plotted, from two independent
datasets (N=12; Fig.
4). The principle kinetics are the same in both cases, though
there is variability in extent of contraction, depending on the sponge
specimen. Maximum contraction differs by means of relative projected area in
the range of 0.15 (0.71±0.03, N=12 vs
0.56±0.03, N=12). The maximum relative rates of contraction
() calculated by change in
projected area per unit time varies between -42x10-3
s-1 and -63x10-3 s-1, respectively,
whereas the maximum speed of expansion calculated as change in projected area
per unit time is very similar in both cases, 19 x10-3
s-1 and 22 x10-3 s-1, respectively. The
general kinetics of the contraction cycle of T. wilhelma can be
subdivided into four phases (Fig.
5): contraction (size reduction), contracted state, expansion
(size increase) and expanded state. Contraction is a faster process than
expansion (
>
).
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Local contractions, spreading
In time-lapse movies prepared from the image series the propagation and
spreading of local contractions over the sponge surface can be observed (movie
S4 in supplementary material). By measuring the local projected area changes
of two sponge sectors of 2 and 3 mm2, respectively, at a distance 3
mm apart, the speed of propagation was quantified. The local contraction
spread along the pinacoderm at a rate of 750 µm min-1 (=12.5
µm s-1; Fig. 6).
The first local contractions may propagate faster
(Fig. 6B), but the main
contraction clearly represents a wave running over the sponge.
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Contraction in fused sponge specimens
Since T. wilhelma reproduces mainly by budding, most specimens in
our aquariums are clones, which may be fused to form larger sponge masses.
Sponges undergo a characteristic morphological reorganisation during fusion,
resulting in the final loss of individual skeletal structures of the
progenitor sponges by forming one larger sponge. In the early phase of
reconstruction the progenitors can still be recognised as individuals by their
contraction pattern, which is not synchronised at the beginning. In a fusion
experiment this individuality could be demonstrated (movie S5 in supplementary
material). By measuring the projected area of the outer parts of each
progenitor the contraction pattern of both parts of the fused sponge could be
monitored independently (Fig.
7). The resulting contraction pattern is not as precise as in the
case of non-fused sponges, since contraction of one progenitor always
indirectly influences the measurement area of the other progenitor. It is
methodologically impossible to compensate for this completely. Nevertheless
the independence of contraction of both parts can be monitored. It becomes
obvious that two asynchronous contraction pattern are overlain, resulting in
the mixture of four possible states: both expanded, one contracted and the
other expanded (and vice versa), and both contracted. The contraction
patterns of both sponges influence each other. In many cases a contraction of
one sponge is followed by one of the other within 15 min. However, neither of
the two sponges attains a consistent leading position, triggering the
contraction of the other sponge.
|
In contrast to body contraction the formation and retraction of filaments is synchronised (movie S5 in supplementary material). It is unclear whether one of the sponges or both sponges together trigger the signal for extension and retraction of the filaments.
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Discussion |
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The rhythm or frequency of contraction cycles is not necessarily disturbed if subcontractions occur. In some cases subcontractions replace regular contractions (Fig. 1), even though the majority of subcontractions occur irregularly. Distinct contraction types in sponges have not been reported before, and this is the first time that different contraction behaviours have been discovered in a sponge.
The frequency of contraction is usually quite variable over a restricted
range in long-term experiments (Fig.
2). Nevertheless, for periods of hours or even days the
contraction cycle frequency can be very stable
(Fig. 1), raising the question
of an intrinsic, endogenous timing or triggering mechanism. Similar regular
repeated contractions have been shown for oscules of Spongia
officinalis (Pavans de Ceccatty,
1971) and for whole individuals of the marine species Tethya
crypta (Reiswig, 1971
) as
well as for the freshwater sponge Ephydatia fluviatilis
(Kilian and Wintermann-Kilian,
1979
; Weissenfels,
1984
,
1990
). Even though the
existance of an endogenous rhythm in E. fluviatilis has been
questioned (De Vos and Van De Vyver,
1981
), re-examination of this data points towards endogenous
periodicity (Weissenfels,
1990
).
Two independent long-term experiments show a significant difference in the
contraction cycle frequency between day and night
(Fig. 3), suggesting a diurnal
rhythm, as has also been proposed for the related species T. crypta
(Reiswig, 1971). In addition,
Reiswig found that artificial illumination during darkness disturbs the
rhythm, a result that seems not to be same for T. wilhelma. However,
further experiments need to be performed under constant darkness and constant
light conditions, in order to determine if the diurnal rhythm is endogenous or
coupled to light sensitivity.
In T. wilhelma the difference in contraction cycle duration is
only given by the variable duration of the fully expanded phase. Variabilities
in the durations of the contraction, the contracted phase and the subsequent
expansion are negligible (Table
1). Contraction kinetics calculated from two independent datasets
(Fig. 4), revealed variation
only in the amplitude of the contraction, and not in the duration of the cycle
phases. The contracted state itself lasts only a few minutes. The schematic
representation demonstrates that the contraction phase is shorter than the
expansion phase (Fig. 5),
indicating a difference in the mechanism. This is underlined by the fact that
the absolute value of the maximum contraction rate
is higher than that of
maximum expansion rate
.
An active expansion mechanism by contraction of antagonistic mesohyle cells
has been discussed (Wilson,
1910
), but seems unlikely to occur in light of the difference
between contraction and expansion kinetics in T. wilhelma.
Furthermore, histological details of the appearance of dilating tissue and
canals are not consistent with an active expansion mechanism
(Jones, 1962
). It seems more
likely that expansion follows an increasing hydrostatic pressure inside the
aquiferous system.
Mechanisms and functions of contractions
There have been many discussions concerning the nature of the contractile
tissue in sponges (Jones,
1962). The two hypothetical principles are: (1) the contraction of
the mesohyle is due to a contractile cell type called myocytes
(Bagby, 1966
; Pavans de
Ceccatty, 1960
,
1974
;
Sollas, 1888
) or more recently
actinocytes (Boury-Esnault and
Rützler, 1997
); (2) the contraction of the pinacoderm is due
to the pinacocytes themselves (Bagby,
1970
; Pavans de Ceccatty,
1986
; Wilson,
1910
). Neither of the two hypotheses can be excluded on the basis
of present knowledge, but many observations point towards the latter
mechanism, or both mechanisms working in conjunction. The presence of
actinocytes in the mesohyle of the cortex has been shown in T.
wilhelma (Nickel, 2001
),
but there is no direct evidence for their contractile nature. Both cell types
contain actin filaments and networks (Bagby,
1966
,
1970
;
Matsuno et al., 1988
;
Pavans De Ceccatty, 1981
) and
myosin has also been demonstrated in actinocytes and other sponge cells
(Lorenz et al., 1996
;
Nickel, 2001
). Hence it can be
assumed that contraction of sponge cells is mediated by an actin-myosin
mechanism.
The results presented here strongly support the contractile pinacoderm
mechanism. Contractile waves on the surface of the sponge can be recorded
(Fig. 6), indicating that
direct contraction of the pinacoderm occurs. The extent of contraction
discussed above is another piece of evidence: the cortex of T.
wilhelma is very rich in endopinacoderm (canals and lacuna), whereas the
mesohyle of the cortex is of low cellular density
(Nickel, 2001;
Sarà et al., 2001
). The
extent of contraction can be easily attributed to the distinctive
endopinacoderm, taking into account the physiological and ecological value of
an increased water exchange due to a contraction of the endopinacocytes. The
main volume change affects the volume of the aquiferous system (canal and
lacuna) and not the volume of the mesohyle. The volume of the mesohyle is not
necessarily reduced during contraction in this model, only the shape of the
mesohyle, which is possible due to the loose organization of the cells and the
extracellular matrix. In this case, every contraction cycle is accompanied by
an enormous exchange of water whereby nutrient- and oxygendepleted water,
which may also be loaded with waste products, is discarded. Regular rhythmic
contraction is therefore a concomitant factor in the continuous water exchange
provided by the currents produced by choanocytes.
Experiments simulating strong sedimentation events, similar to those in the
natural environment of many tropical Tethya species (i.e. the reef
top or shallow lagoons), indicate that contraction plays an additional
ecological role in unloading sediment from the sponge body (data not shown).
This is in conjunction with the observation that sponges can use reverse
currents for `backwashing' of blocked canals
(Simpson, 1984;
Storr, 1979
). Since the flow
direction during contraction has not yet been determined for T.
wilhelma, we cannot exclude the occurrence of backwashing during
contraction.
For other sponge groups, e.g. Aplysina, Spongia or
Tedania, contractions are usually thought to be limited to the
oscular region (Pavans de Ceccatty,
1971; Prosser et al.,
1962
). By applying digital time-lapse imaging to other sponges, it
can be demonstrated that at least the whole sponge cortex or exopinacoderm
region is able to contract to various degrees (data not shown), depending on
the morphology of the sponge species. The oscular regions of many sponges are
less rigid and resemble more-or-less the lacunar cortex of T.
wilhelma: they are characterized by a low density of framework-building
spicules, a distinct mesohyle part, a high degree of canals and cavities, and
therefore a dominant number of pinacocytes. Subsequently, I assume that
oscular contractions in most, if not all sponges follow the same mechanism
than body contraction in T. wilhelma.
Coordination of contraction
In 350 BC, in his history of the animals, Aristotle wrote in chapter 1 of
book one and in chapter 16 of book five that sponges are animals endowed with
a certain sensibility (Aristotle,
1498). It has taken more than 2000 years to accept his view that
sponges are true animals (Müller and
Müller, 2003
) but whether they are able to react directly to
mechanical stimuli is still a question of debate. Our results clearly show
that T. wilhelma directly responds to external stimuli, e.g. the
attack of an amphipod (Fig. 2A,
movie S3 in supplementary material). The contraction helps the sponge to
protect its tissue from mechanical damage. A dense layer of tylasters can be
found close to the surface in sections of T. wilhelma
(Sarà et al., 2001
).
Contraction condenses this layer of micrasters, consequently enhancing its
mechanical stability, resulting in a robust, but flexible protective coating,
resembling chain mail.
Many authors have discussed the controversial question of whether sponges
possess a nervous system (Jones,
1962; Lentz, 1968
;
Mackie, 1979
,
1990
;
Pantin, 1952
; Parker,
1910
,
1919
; Pavans de Ceccatty,
1974
,
1979
). The question itself
seems to be more of philosophical quality than of biological evidence.
Obviously, sponges do not possess exactly what we call a nervous system in
higher organisms, since no true neurons have yet been found in sponges. The
far more interesting question is: which elements of the nervous systems of
higher animals can be found in sponges? Some elements of nervous systems, like
neurotransmitters and their specific receptors, have been reported from
unicellular Protozoa (Walker et al.,
1996
; Walker and Holden Dye,
1991
), so we can also expect to find such elements in the
Porifera, which evolved early in the lineage of the Metazoa. Indeed, there
have been many hints that sponges react to neuromodulating substances or
possess elements of their accompanying signal transduction pathways, such as
neurotransmitters, enzymes needed for their synthesis or degradation, or their
specific receptors (Emson,
1966
; Jones, 1962
;
Lendenfeld, 1889
;
Lentz, 1966
;
Pavans de Ceccatty, 1971
;
Perovic et al., 1999
;
Weyrer et al., 1999
). Even
electrical propagation and action potentials have been shown, though so far
only in Hexactinellida (Leys and Mackie,
1997
,
1999
;
Leys et al., 1999
;
Mackie et al., 1983
). On the
other hand all these results remain sketchy and patchy: no conclusive,
comprehensive hypothetical model on coordination in sponges has been
developed; no model sponge has been used to test the hypothesis that sponges
are capable of integrating a variety of signals, both chemical and
electro-chemical, through several differentiated signalling pathways. However,
there are at least two sponge models suitable for comprehensive studies, and
these could combine behavioural, physiological, pharmacological, histological,
cell and molecular biological studies: the freshwater sponges, which have been
used for many investigations (De Vos and
Van De Vyver, 1981
; Kilian and
Wintermann-Kilian, 1979
;
McNair, 1923
;
Wintermann, 1951
) and the
marine sponges of the genus Tethya, especially T. wilhelma
as reported here. Both systems exhibit contractions that are triggered
endogenously and by external events (e.g. mechanical stimulation). The results
from fused individuals of T. wilhelma, reported here, indicate that
the coordination system in sponges must have reached a certain complexity:
while the contraction patterns of the two individuals are not synchronized at
first, the expansion and retraction of body extensions are. In conclusion, at
least two independent means of triggering and controlling these behaviours are
necessary. Moreover, contractile waves were observed, spreading at 12.5 µm
s-1 over the sponge surface, implying that contractions are
triggered locally and spread consequently, following a diffusing (possibly
chemical) signal. The existence of subcontractions, significantly weaker than
regular contractions, again indicates that the sponge is able to control this
behaviour by means of integrating various internal (physiological) and
external (environmental) information. Preliminary results of our ongoing
research indicate that several neuroactive substances are involved in the
coordination of contraction in T. wilhelma
(Ellwanger et al., 2004
).
The detailed analysis of the kinetics and rhythm of T. wilhelma
reported here is unique for sponges, allowing for the first time a
differential, quantitative characterization of sponge behaviour. These results
provide the basis for establishing a new sponge model system for the
investigation of the integration signals and coordination of behaviours in an
aneural organism. Since it has been pointed out that early Eumetazoa were also
probably aneural (Mackie,
1990), studies on T. wilhelma and other sponge models may
provide valuable information about the early evolution of metazoan signalling
systems. Further work on this topic is in process, including the development
of a tissue-culture system for experiments on the cellular level, as well as
molecular work.
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Acknowledgments |
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Footnotes |
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Supplementary material available online at http://jeb.biologists.org/cgi/content/full/207/26/4515/DC1
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References |
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