Multiple photoreceptor systems control the swim pacemaker activity in box jellyfish

Abstract

Like all other cnidarian medusae, box jellyfish propel themselves through the water by contracting their bell-shaped body in discrete swim pulses. These pulses are controlled by a swim pacemaker system situated in their sensory structures, the rhopalia. Each medusa has four rhopalia each with a similar set of six eyes of four morphologically different types. We have examined how each of the four eye types influences the swim pacemaker. Multiple photoreceptor systems, three of the four eye types, plus the rhopalial neuropil, affect the swim pacemaker. The lower lens eye inhibits the pacemaker when stimulated and provokes a strong increase in the pacemaker frequency upon light-off. The upper lens eye, the pit eyes and the rhopalial neuropil all have close to the opposite effect. When these responses are compared with all-eye stimulations it is seen that some advanced integration must take place.

Full text

3951
INTRODUCTION
The presence of a central nervous system (CNS) and the level of
neuronal processing in cnidarians have long been matters of dispute.
In most zoological textbooks their nervous system is presented as
a simple nerve net without condensations, implying only little
processing (e.g. Lesh-Laurie and Suchy, 1991). Still, during the last
decades Mackie and coworkers have convincingly shown that
hydromedusae have a complex nervous system with a CNS
performing several types of processing and integration (Mackie,
1971; Passano, 1976; Mackie and Meech, 1995a; Mackie and
Meech, 1995b; Mackie and Meech, 2000; Mackie et al., 2003;
Mackie, 2004). The main part of this CNS is a double ring nerve
that encircles the bell-shaped body and it is here that several
subsystems can be distinguished by their physiology and
neurotransmitter profiles (Mackie, 2004). From our earlier
neuroanatomical studies we have shown that another cnidarian
group, the cubozoans (or box jellyfish) also have condensations in
their nervous system, which probably qualify as a CNS (Garm et
al., 2006; Skogh et al., 2006; Garm et al., 2007b). The CNS of box
jellyfish differs from that of hydrozoans because there is only a
single ring nerve and an additional part in each of their four sensory
clubs, the rhopalia. This rhopalial nervous system is directly
connected to the ring nerve making the box jellyfish CNS one
coherent system (Garm et al., 2006). Still, so far it has not been
shown what kind of processing and integration takes place in the
box jellyfish CNS.
One way to evaluate to what level processing takes place in the
CNS is by looking at the complexity of the sensory input and
behavioural output. Here box jellyfish stands out in Cnidaria with
their advanced visual system, comprising of 24 eyes of four
morphologically distinct types (Berger, 1898; Yamasu and Yoshida,
1976). Further, two of the eye types are structurally similar to
camera-type eyes of vertebrates (Laska and Hündgen, 1982; Nilsson
et al., 2005). The rhopalial nervous system lies in direct connection
with these eyes, and subsystems seem to interconnect the different
types of eyes and the two sides of the bilateral symmetric rhopalium
(Parkefelt et al., 2005; Skogh et al., 2006; Parkefelt and Ekström,
2009). What exact information the eyes register and how it is being
processed by the rhopalial nervous system is largely unknown
though.
Concerning box jellyfish behaviour, more and more evidence
points towards the presence of an elaborate repertoire. It has been
shown that at least some species have internal fertilisation of their
eggs, which includes a proper mating behaviour (Werner, 1973;
Lewis and Long, 2005). Another well-documented cubozoan
behaviour is obstacle avoidance (Hamner et al., 1995; Matsumoto,
1995) and it has been shown that this behaviour is visually guided
and involves true spatial vision (Garm et al., 2007a). A major
part of these behaviours is the swim speed of the medusa, which
is largely controlled by the rate of bell contractions. The bell
contractions are in turn controlled by a central pattern generator
situated in the rhopalial nervous system (Yatsu, 1917; Satterlie,
1979). This swim pacemaker system has a one-to-one relationship
with the swim pulses (Satterlie, 1979) and is influenced by the
visual input (Garm and Bielecki, 2008). Under constant light
intensities the pacemaker frequency stays constant but if the
rhopalium experiences a sudden increase or decrease in light
intensity it has great impact on the pacemaker. A decrease in
intensity induces a so-called shadow response with a steep
increase in pulse frequency for a limited period of time (Garm
and Bielecki, 2008), a behaviour also known from hydromedusae
(Yoshida and Ohtsu, 1973; Arkett, 1985; Arkett and Spencer,
1986a; Arkett and Spencer, 1986b). An increase in the light
intensity inhibits the pacemaker making the jellyfish sink and this
is an important part in optimising the feeding behaviour for
Tripedalia cystophora (Buskey, 2003; Garm and Bielecki, 2008).
Because the pacemaker system is involved in several of the box
jellyfish behaviours it seems to be the ideal system to investigate
how the visual input influences these behaviours. Among other
things, it offers the possibility to examine if parts of the rhopalial
nervous system integrate information from several eye types or if
the modulations of the pacemaker are governed by a single eye type
only. In the present study we examine the visual control of the swim
pacemaker of the Caribbean box jellyfish T. cystophora. We record
The Journal of Experimental Biology 212, 3951-3960
Published by The Company of Biologists 2009
doi:10.1242/jeb.031559
Multiple photoreceptor systems control the swim pacemaker activity in box jellyfish
A. Garm* and S. Mori
Section of Aquatic Biology, University of Copenhagen, Denmark
*Author for correspondence (algarm@bio.ku.dk)
Accepted 9 September 2009
SUMMARY
Like all other cnidarian medusae, box jellyfish propel themselves through the water by contracting their bell-shaped body in
discrete swim pulses. These pulses are controlled by a swim pacemaker system situated in their sensory structures, the rhopalia.
Each medusa has four rhopalia each with a similar set of six eyes of four morphologically different types. We have examined how
each of the four eye types influences the swim pacemaker. Multiple photoreceptor systems, three of the four eye types, plus the
rhopalial neuropil, affect the swim pacemaker. The lower lens eye inhibits the pacemaker when stimulated and provokes a strong
increase in the pacemaker frequency upon light-off. The upper lens eye, the pit eyes and the rhopalial neuropil all have close to
the opposite effect. When these responses are compared with all-eye stimulations it is seen that some advanced integration must
take place.
Key words: cubomedusae, eyes, pacemaker, swim pulse,
Tripedalia
.
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3952
the pacemaker signals while stimulating one eye at a time with white
light of a range of different intensities. The results demonstrate that
stimulating the upper lens eye (ULE), the pit eyes (PE) or the
neuropil (NP) has similar effects whereas stimulation of the lower
lens eye (LLE) has the opposite effect.
MATERIALS AND METHODS
Animals
Adult medusae (7–9mm in bell diameter) of Tripedalia cystophora
Conant 1897 were obtained from our cultures at the University of
Copenhagen, Denmark. In the cultures the medusae are kept in a
200l tank with circulating seawater at 25‰ and about 28°C and
fed SELCO (INVE Technologies, Dendermonde, Belgium)-enriched
Artemia daily. They reach adult size in 2–3 months.
Electrophysiology
A rhopalium was cut off approximately midway along the stalk with
a pair of fine scissors and transferred using a pipette to a small Petri
dish containing seawater. The seawater was kept at a temperature
of 28±0.5deg. using a Peltier element. In the Petri dish the rhopalium
was held by a micropipette at the area of the crystal, which allowed
for orienting the rhopalium such that there was access to all four
eye types. Under a dissection microscope a glass suction electrode
[for electrode details, see Derby (Derby, 1995)] was applied to the
cut surface of the rhopalial stalk in the area of the epidermal nerve.
The suction electrode was moved around until the regular activity
pattern of the pacemaker was seen where after the rhopalium was
left to dark adapt for 3min.
A Linos microbench system was used for light stimulation.
Light from an ultra bright white LED (Luxeon III star, Philips,
San Jose, CA, USA) was focused into a quartz light guide, 50m
in diameter. The light guide was arranged in the Petri dish such
that the light shone directly into either one of the PE, the ULE,
the LLE or one of the slit eyes (SE). Due to the small diameter
of the light guide, direct light could be limited to the eye type of
interest only. The maximum intensity was 1.1⫻105Wsr–1m–2
when integrated between 350 and 750nm and measured at the tip
of the light guide (ILT900W spectroradiometer, International
Lights Technologies, Peabody, MA, USA). The LED was
controlled via a NI6229 A/D converter (National Instruments,
Austin, TX, USA) and a custom made program for LabView 8.5
(National Instruments).
The electrophysiological experiments tested the response to
sudden changes in light intensities covering a range of
approximately 1.1 log units (from 8.7⫻103Wsr–1m–2 to
1.1⫻105Wsr–1m–2) in five steps. To ensure maximum health of
the preparation, only one or two rhopalia were used from each
medusa and only one eye from each rhopalium. The protocol for
each eye contained five consecutive recordings and only data from
preparations that lasted a full protocol were used. Each recording
started with 1min of darkness (<1⫻10–3Wsr–1m–2) followed by
3min of light and then 3min of darkness, giving the protocol a
total duration of 35min. Due to some indications of long-term
adaptations half of the trials were started from the low intensity
end and the other half from the high intensity end. All four eye
types were tested (N10 for each eye type). Control recordings
A. Garm and S. Mori
ABC
EFG
PE
ULE
LLE SE
PE
SE
PE
ULE
LLE
SE
ULE
PE
ST
*
*
100 µm
D
ST
Fig.1. Stimulus accuracy. The accuracy of the light stimulus varied with the different eyes. When working with the ULE, PE or NP the rhopalium was held by
a micropipette at the back of the crystal (A) but in the case of the LLE and SE the micropipette was attached to the side of the crystal (E). When the 50m
light guide was aimed at the ULE it is seen that here the pigment screen is not light proof and the NP is therefore also illuminated (B, asterisk). Illumination
of the PE also allows light to reach the NP, because of its incomplete pigment screen and small diameter (C). When stimulating the NP at the base of the
stalk (arrow) light scattered throughout most of the NP (D). The pigment screen of the LLE is completely light proof and stimulating this eye leaves the rest
of the rhopalium in complete darkness (F). As for the PE, the small size and incomplete pigment screen of the SE causes the neuropil and the back side of
the PE (G, asterisk) to be illuminated. LLE, lower lens eye; PE, pit eye; NP, neuropil; SE, slit eye; ST, stalk; ULE, upper lens eye. Scale bar in A applies to
all subfigures.
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3953Pacemaker control in box jellyfish
were done (i) with the light guide aimed at the neuropil from above
besides the stalk base (no eye was stimulated directly), which
resulted in light scattered throughout most of the NP (Fig.1D) and
(ii) in 35min of darkness. The neuropil was chosen as a control
because leakiness of the PE and the ULE and the small size of
the SE and PE result in illumination of the neuropil when working
with these eyes (Fig. 1). For organisation of the NP see Skogh et
al. (Skogh et al., 2006). A possible concern is heating of the
preparation during the 3min of stimulation but because we used
a LED and light guide system heat transmission is minimal.
Further, heating caused by photon absorption should be less than
what they will experience under natural conditions where they are
exposed to close to full sunlight (Stewart, 1996).
The recorded signals were amplified 1000 times (1700 differential
AC amplifier from A-M systems, Carlsborg, WA, USA) and
filtered through high- and low-pass filters in the amplifier (0.1 and
1000Hz, respectively). The amplifiers 50Hz notch filter was also
used. All recordings lasted 7min and were stored and analysed on
a laptop using the NI6229 A/D converter and the program Igor Pro
6.03A (WaveMetrcs Inc., Lake Oswego, OR, USA) with a
NeuroMatic add-on.
RESULTS
Characteristics of the pacemaker signal
When recording from the rhopalial stalk the pacemaker signal is
not represented by discrete action potentials but rather by complex
signals of long duration as described earlier (Garm and Bielecki,
2008). This is evident from the highly variable amplitude seen in
Fig.2A. In constant darkness the pacemaker has a mean frequency
close to 1Hz when measured over minutes. This frequency is rather
variable and at times the activity occurs in bursts (Fig.2B).
Dark control
The pacemaker activity was recorded in darkness for 35min in 7min
slots during control experiments. As said above the activity pattern
changed and at times it was highly regular (Fig.2A) but at other
times the pacemaker would fire in bursts (Fig.2B). Still, when the
mean frequency was taken from 10 rhopalia and measured in 10s
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0–
0.25
0.25–
0.5
0.5–
0.75
0.75–
1
1–
1.25
1.25–
1.5
1.5–
1.75
1.75–
2
2–
2.25
2.25–
2.5
2.5–
2.75
2.75–
3
3–
3.25
3.25–
3.5
3.5–
3.75
3.75–
4
4–
4.25
4.25–
4.5
4.5–
4.75
4.75–
5
>5
Dark 0–7 min
Dark 7–14 min
Dark 14–21 min
Dark 21–28 min
Dark 28–35 min
B
C
(V)
0.5
1
1.5
2
Pulse frequency (Hz)
Time interval (s)
0
–0.2
–0.4
(V)
0
–0.2
–0.4
A
0–10 410–420200–210100–110 300–310
0–7 min
7–14 min
14–21 min
21–28 min
28–35 min
Interpulse intervals (s)
Frequency of occurrence
D
0 100 200 300 400
Time (s)
0 100 200 300 400
Fig.2. Dark reference. The pacemaker
activity was recorded in darkness
(<1⫻10–3Wsr–1 m–2) for 35min (total
time of the experimental protocol) in five
slots of 7min. Under these conditions the
activity varied. In some cases the
pacemaker showed a regular activity
pattern (A) but in other cases strong
bursting was seen (B). When the
frequency is measured in 10s intervals it
is seen that the frequency tends to be
highest, and most stable, in the first 7min
(C). The mean frequency is close to 1Hz,
the black line indicates the means ±
s.e.m. When the stability is examined
using the inter-pulse interval no significant
differences were found between the five
different time slots (D).
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3954
intervals the frequency stayed between 0.5 and 1.6Hz (Fig.2C).
When the five slots of 7min are compared it is seen that there is a
tendency for the pacemaker to be most active during the first 7min.
The mean frequency during the first 7min was approximately
1.25Hz whereas during the last 7min frequency was approximately
0.9Hz but this difference was not significant [one-way analysis of
variance (ANOVA), F4,202.703, P>0.05]. The mean of the five
7min slots is therefore used for comparison with the frequencies
obtained under the different experiments conditions (Fig.2C).
To examine if the regularity of the pacemaker activity is
influenced by the time spent in darkness the inter-pulse intervals
were measured in bins of 250ms and compared between the five
7min slots (Fig.2D). This showed that the distribution of inter-pulse
intervals does not differ between the different time periods spent in
darkness.
The lower lens eye
When the LLE was stimulated by the light guide it had major effect
on the pacemaker activity (Fig.3). The light-on had a strong and
close to immediate inhibitory effect on the pacemaker (Fig.3B). In
the most extreme cases the pacemaker became almost silent during
the 3min of light stimulation (Fig.3A). For all intensities the
frequency during light was significantly lower than the dark
reference (one-way ANOVA, F5,102206, P<0.0001, Tukey HSD
post hoc P<0.001). The light-off response was the opposite and had
an immediate and strong stimulatory effect on the pacemaker
(Fig.3C). This off-response not only restored the pacemaker
frequency to the initial level but transiently overshot it (Fig.3D,
two tailed t-test, P<0.0024). In the strongest responses the frequency
reached 2.4Hz in the first 10s after light-off. The light-off response
lasted only for 10s where after the frequency dropped below the
dark reference (Fig.3D). This drop was long lasting and the
pacemaker frequency did not return to the level of the dark reference
until about 2min after light-off.
The upper lens eye
Stimulation of the ULE had the opposite effect on the pacemaker
as when stimulating the LLE. At light-on a fast and strong increase
in the pacemaker frequency was seen (Fig.4A,B). The initial effect
typically exceeded 1.8Hz but was brief and lasted for 10s only.
Over the next approximately 30s the frequency declined to about
1.5Hz, where it remained until light-off (Fig.4D). For all intensities
the frequency during light was significantly higher than the dark
reference (one-way ANOVA, F5,10229.2, P<0.0001, Tukey HSD
post hoc P<0.0001). During the light period the pacemaker activity
is less variable, which is seen by the, in general, smaller standard
error (Fig.4D). At light-off the pacemaker activity immediately falls
back to the level of the dark reference (Fig.4C,D, one-way ANOVA,
F5,304.8, P<0.0024, Tukey HSD post hoc 0.15<P<1).
The pit eye and the neuropil
Similar effects were seen in the pacemaker activity when
stimulating either one of the PE (Fig.5) or the NP at the base of
the stalk (Fig.6) although with slightly different magnitudes. These
effects were again similar to what was seen for the ULE (compare
Fig.4 and Fig.5). At light-on an increase in the pacemaker
frequency was obtained but this did not peak until 10–20s after
light-on. A maximum of 1.9–2.1Hz was typically seen for the NP
A. Garm and S. Mori
0
5
10
15
20
25
0-10 10-20 20-3030-40 40-50 50-60 60-70 70-80 80-90 90-100 100-110 110-120 120-130130-140 140-150 150-160 160-170 170-180 180-190 190-200 200-210 210-220 220-230230-240 240-250 250-260 260-270 270-280 280-290 290-300 300-310 310-320 320-330330-340 340-350 350-360 360-370 370-380 380-390 390-400 400-410 410-420
Time (s)
D
Dark Dark
Light
Time (s)
0 100 200 300 400
*
0
–0.2
–0.3
–0.1
(V) (V)
0
–0.2
–0.1
A
B
0
–0.2
–0.3
–0.1
40 50 60 70 80 220 230 240 250 260
0–10 200–210 410–420300–310100–110
Time interval (s)
2
1.5
1
0.5
Pulse frequency (Hz)
C
Fig.3. Pacemaker activity when
stimulating the lower lens eye (LLE).
The pacemaker frequency is strongly
influenced by stimulation of the LLE
alone. Light-on causes a sudden
decrease in the frequency and, in the
most extreme cases, an almost
complete stop of the pacemaker (A,B).
Light-off, by contrast, causes an
increase in the frequency (A,C). In both
cases the response is almost immediate
(B,C). The light-on response is long
lasting whereas the light-off is transient
and lasts for about 10·s (D). After the
transient light-off response the
frequency again drops below the level
of the dark reference (black line in D).
Note the very small error bars during
light-on. The red line in A, B, C and
under D indicates the stimulus pattern.
The black line in D is the mean of the
dark recordings and the error bars
indicate ± s.e.m. The asterisk in A
indicates activity of other non-
pacemaker cells. Light
intensity1.1⫻105Wsr–1 m–2 in A–C and
8.7⫻103Wsr–1 m–2 in D.
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3955Pacemaker control in box jellyfish
and 1.7–1.9Hz for the PE but the difference was not significant
(two tailed t-test, P0.17). As for the ULE the pacemaker
frequency would drop to 1.4–1.6Hz during the rest of the stimulus
period. For all intensities the frequency during light was
significantly higher than the dark reference (one-way ANOVA,
F5,10239.9, P<0.0001, Tukey HSD post hoc P<0.0001). At light-
off, the frequency decreased to about 0.3–0.6Hz in the case of the
PE and stayed lower than the dark reference during the 3min of
darkness (Fig. 5D, one-way ANOVA, F5,10252.4, P<0.0001,
Tukey HSD post hoc P<0.0001). For the NP the light-off response
takes the pacemaker to 0.5–0.9Hz (Fig.6A,C,D). For both the NP
and PE the light-off caused some long term effects in the
pacemaker frequency, which resulted in low pre-stimulus
frequencies (Figs5 and 6, one-way ANOVA, F5,3012.1,
P<0.0001, Tukey HSD post hoc 0.0001<P<0.029).
The slit eyes
The pacemaker response when stimulating the SE was more variable
than when performing the other stimulations. Still, the average effect
resembled that of stimulating the ULE, PE or NP (Fig.7). Light-on
in general caused an increase in the pacemaker frequency, normally
peaking in the first 10s (Fig.7A,B,E). For all intensities the
frequency during light was significantly higher than the dark
reference (one-way ANOVA, F5,10228.3, P<0.0001, Tukey HSD
post hoc P<0.0001). Light-off caused the frequency to fall back to
about the level of the dark control (Fig.7A,C,E). But in some of
the trials a distinct off-response was missing (Fig.7D). When
compared with the ULE, PE or NP the magnitude of the pacemaker
response was, in general, smaller when working with the SE (Fig.8).
The pacemaker activity never exceeded 1.7Hz and after the initial
light-on response the frequency would typically settle around 1.3Hz
until light-off.
Effects of the light intensity
All of the five different types of stimulations (four different eye
types and NP) were tested with five different light intensities
covering about 1.1 log units (Fig.8). When stimulating the ULE,
PE or SE no significant correlation was found between light
intensity and magnitude of the light-on response, when measured
as the mean frequency during the first 20s after the stimulus onset
(Fig.8B,C,E, linear regression, R20.07, 0.76 and 0.68,
respectively). The light-on responses from these areas therefore
seem to be all-or-nothing responses at least within the examined
intensity range. Here it should be noted that saturation might have
occurred and it could have been advantageous to have used a
broader range of intensities to investigate this. A typical eye has
a dynamic range of 2–2.5 log units when not taking adaptations
into account. When working with the NP or the LLE there was a
significant change with changing intensity (Fig.8A,D). The higher
the intensity the higher the mean swim pacemaker frequency in
the first 20s after light-on (linear regression, R20.96 for NP and
R20.92 for LLE). This means for the LLE that the higher the
light intensity the less the inhibition of the pacemaker. In the pre-
stimulus situation there is some variation in the pacemaker
frequency for the LLE. There is a trend for this variation to follow
the intensity but this is not significant (linear regression, R20.51).
If, instead, the response is measured as the mean pacemaker
frequency during the full 3min of light a significant positive
0
5
10
15
20
25
0-10 10-20 20-3030-40 40-50 50-60 60-70 70-80 80-90 90-100 100-110 110-120 120-130130-140 140-150 150-160 160-170 170-180 180-190 190-200 200-21 0 21 0-220 220-230230-240 240-250 250-260 260-270 270-280 280-290 290-300 300-310 310-320 320-330330-340 340-350 350-360 360-370 370-380 380-390 390-400 400-4 1 0 41 0-420
D
0.5
1
1.5
2
Pulse frequency (Hz)
0 100 200 300 400
Time ( s)
(V)
0
–0.8
–0.4
A
(V)
0
–0.8
–0.4
0
–0.8
–0.4
C
Time ( s)
40 50 60 70 80 220 230240 250 260
*
*
0–10 200–210 410–420
300–310
100–110
Time interval (s)
Dark Dark
Light
B
Fig.4. Pacemaker activity when
stimulating the upper lens eye (ULE).
Stimulating the ULE causes effects on
the pacemaker that are opposite to what
was seen when stimulating the lower
lens eye (LLE) (A and Fig. 2). At light-on
the pacemaker frequency increases
(A,B,D). This increase is immediate and
culminates 0–10s after the onset of the
stimulus (2.1Hz at 7.1⫻104Wsr–1 m–2 as
shown in D) where after it stabilises at
about 1.5Hz for 2min until light-off. At
light-off the frequency falls back to the
level of the dark reference (D). The red
line in A, B, C and under D indicates the
stimulus pattern. The asterisks in C
indicate activity of other non-pacemaker
cells. The black line in D is the mean of
the dark recordings and the error bars
indicate ± s.e.m. Light
intensity7.1⫻104Wsr–1 m–2 for all
subfigures.
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3956
correlation is seen with NP and PE (linear regression, R20.92 for
NP and R20.85 for PE).
When considering the light-off response the situation is similar
to the light-on response. With the LLE, ULE, SE and PE there is
no significant correlation between the magnitude of the response
and the light intensity when the magnitude is measured either as
the mean pacemaker frequency in the first 20s after light-off or as
the mean frequency during the full 3min after light-off (R2<0.64).
With NP there was a positive correlation between the intensity and
the mean pacemaker frequency in the first 20s after light-off
(R20.86). Interestingly, this again shows that the larger the relative
change in intensity the less the inhibition.
DISCUSSION
There are many interesting questions concerning vision in
cubomedusae. How did this complex visual system evolve? Why
are so many different eye types needed? What visual cues do they
pick up? What visually guided behaviours do they support? How
is the visual information translated into these behaviours? Answers
are beginning to appear to some of these questions and in the present
paper we have looked further into how the visual input to individual
eyes is integrated in the rhopalial nervous system and influences
one of the important behavioural parameters of the medusae; their
rate of swim contractions.
Multiple effectors
Interestingly, the results we present indicate that all four eye types
are involved in modifying the swim pacemaker frequency. To our
surprise not only stimulating the eyes but also stimulating the general
NP of the rhopalium influenced the pacemaker. The question arises
whether some of the results are artefacts caused by light escaping
the stimulated eye and possibly reaching other photosensitive areas.
Unfortunately this cannot be ruled out. As shown in Fig.1
imperfection of the pigment screens along with very small eye size
has the effect that it was not possible to stimulate the ULE, PE, SE
and NP without having stray light affecting the others or at least
the NP. Only in the case of the LLE will no light escape the eye.
With respect to this it is also noteworthy that the ULE, PE, SE and
NP provoke similar responses from the swim pacemaker. One way
to evaluate which of these are true responses, and which might be
caused by problems with the stimulation, is to look for differences
in the response magnitudes and unique characteristics in the detailed
responses.
The influence by ULE is probably valid because the light-on
response seen here has a very distinct peak in the first 10s, which
was significantly higher than the following 10s (two-tailed t-test
for paired observations, P<0.01 for 8.7⫻103and 9.6⫻104Wsr–1m–2,
P<0.1 for the other three intensities). This difference was not seen
for any of the other stimulations (two-tailed t-test for paired
observations, 0.27<P<0.87). The PE is probably also its own
effector, because when stimulating this eye, the strongest and most
consistent off-response is seen resulting in significantly lower
frequencies for this eye at all of the tested intensities than for ULE,
SE or NP (one-way ANOVA, F3,6815.7–70.5, P<0.0001, Tukey
HSD post hoc P<0.0005). Further, the light-on response caused by
stimulating the NP in general has the largest amplitude (although
not statistically significant but in a few cases) and shows the clearest
intensity dependence (highest R2values, see above), which in our
A. Garm and S. Mori
D
Pulse frequency (Hz)
Time interval (s)
Dark Dark
Light
Time ( s)
0 100 200 300 400
(V)
0
–0.4
–0.6
–0.2
A
(V)
0
–0.4
–0.6
–0.2
B
0
–0.4
–0.6
–0.2
C
Time ( s)
–0.8
40 50 60 70 80 220 230240 250 260
1
2
1.5
0.5
0–10 200–210 410–420
300–310
100–110
Fig. 5. Pacemaker activity when
stimulating a pit eye (PE). Stimulating a
PE has a large impact on the
pacemaker frequency similar to when
stimulating the upper lens eye (ULE) (A
and Fig. 3). At light-on a strong increase
is seen (A,B) whereas light-off has a
strong and immediate inhibitory effect on
the pacemaker (A,C). The light-on
response has the strongest effect
10–20s after light-on after which it
decreases and stabilises at 1.4–1.5Hz
throughout the remainder of the light-on
period (D). The light-off response causes
a large drop in the frequency to about
1/3 of the dark level and this lasts for at
least 3min (D). The long-term influence
of the light-off response also has the
effect that the pre-stimulus frequency is
lower than the dark reference. The red
line in A, B, C and under D indicates the
stimulus pattern. The black line in D is
the mean of the dark recordings and the
error bars indicate ± s.e.m. Light
intensity1⫻105Wsr–1 m–2 for all
subfigures.
THE JOURNAL OF EXPERIMENTAL BIOLOGY

3957Pacemaker control in box jellyfish
opinion authenticate stimulations in this area. Stimulating the SE,
however, produced some more dubious results. The responses were
highly variable and did not appear to have any unique features. Also,
the response amplitude, measured as the amount of change in the
swim pacemaker frequency, was the smallest for this eye. We
therefore believe that the areas that modify the pacemaker upon
light stimulation are the LLE, ULE and PE and probably also NP.
The effects seen from the SE are artefacts caused by stray light
stimulating the ULE, PE, NP or a combination of them.
The above conclusion also matches what is known about the
visual fields of the different eyes. When comparing the data
available on the optics of T. cystophora (Nilsson et al., 2005; Garm
et al., 2008) it becomes clear that the ULE and PE must have vastly
overlapping visual fields and the same goes for the LLE and the
SE. It makes good sense, therefore, that the effects of stimulating
either the ULE or the PE are similar, because it is close to
impossible under natural conditions to stimulate the one eye type
without stimulating the other. The LLE and SE also have vastly
overlapping visual fields. If the effects seen from the SE are not
artefacts, these eyes will have more or less directly opposite effects
on the pacemaker counteracting each other constantly, which would
seem like an inappropriate and unlikely arrangement.
Extraocular photoreception
The putative effects mediated by the NP came as a surprise and are
admittedly not easily explained. Using the same logic as above there
will be a constant conflict between the input to the swim pacemaker
system provided by the LLE and the NP, because a large part of
the light illuminating the NP will originate from within the large
visual field of the LLE. Further, the effects mediated by NP are
similar to those mediated by the ULE and the PE. Why have rather
similar effects located in three different places? We do not have
any good answers at this stage but the fact that the strongest intensity
dependency is found for the NP causes us to believe that the effects
are real.
Extraocular photoreceptors in the nervous system are commonly
found throughout the animal kingdom, not least in cnidarians (Ohtsu,
1982; Arendt et al., 2004; Taddei-Ferretti et al., 2004). In Hydra
such photoreceptors also modify a pacemaker system controlling
body contractions (Taddei-Ferretti et al., 2004). One of the
interesting things about extraocular photoreceptors is that they have
a tendency to make use of other photopigments than those
conventionally used in vision. In vertebrates melanopsin is such an
extraocular photopigment (Kumbalasiri and Provencio, 2005), and
a special ‘cnidops’ opsin clade has been found in cnidarians
(Plachetzki and Oakley, 2007). Recently, two research groups have
characterised opsins from two different species of cubomedusae, T.
cystophora and Carybdea rastonii (Koyanagi et al., 2008; Kozmic
et al., 2008). In both cases only a single opsin was found and
interestingly they were only found in direct association with the
retinas of the two lens eyes. Such an expression pattern is supported
by our own immunofluorescence data (Ekström et al., 2008). This
could be taken as evidence for a lack of photoreception in the NP
but because these studies also failed to find any opsins in connection
with the PE and SE we believe that additional undiscovered
photopigments are at play. The fact that the effects on the swim
pacemaker mediated by the PE and NP are intensity-dependent will
be used in the near future to determine the spectral sensitivity of
0
5
10
15
20
25
1234567891011121314 15 16 17 18 19 20 21 22 2324 25 26 27 28 29 30313233 343536373839404142
D
0
0.5
1
1.5
2
Pulse frequency (Hz)
0 100 200 300 400
Time ( s)
(V)
0
–0.2
–0.1
A
(V)
0
–0.2
–0.1
0
–0.2
–0.1
C
Time ( s)
40 50 60 70 80 220 230240 250 260
**
0–10 200–210 410–420
300–310
100–110
Time interval (s)
Dark Dark
Light
B
Fig.6. Pacemaker activity when stimulating
the neuropil (NP). Stimulating the NP has
a large impact on the pacemaker similar to
when stimulating a pit eye (PE) (compare
with Fig. 5). At light-on the strongest
increase is seen (A,B) whereas light-off
has a strong and immediate inhibitory
effect on the pacemaker (A,C). Like for PE
the light-on response peaks 10–20s after
light-on after which it stabilises at
1.4–1.5Hz (D). The light-off response
causes a drop in the frequency to just
below the dark level (D). Similar to the PE
long-term influence of the light-off response
causes a pre-stimulus frequency lower
than the dark reference. The red line in A,
B, C and under D indicates the stimulus
pattern. The black line in D is the mean of
the dark recordings and the error bars
indicate ± s.e.m. The asterisks in B
indicate activity of other non-pacemaker
cells. Light intensity8.7⫻103Wsr–1 m–2 in
A–C 8 and 1.1⫻105Wsr–1 m–2 in D.
THE JOURNAL OF EXPERIMENTAL BIOLOGY

3958
the underlying photoreceptors and thereby shed light on the nature
of their photopigments.
Complex swim pacemaker control
Being cnidarians cubomedusae belong to the group of animals first
in evolution to possess a true nervous system. Even though their
CNS holds several thousand nerve cells the number of computational
units is probably a lot lower due to redundancy (Skogh et al., 2006;
Garm et al., 2007b). It is therefore of great interest how such a
relatively sparse CNS is able to handle the information provided by
the far from simple visual system. Earlier work has suggested that
one mechanism used is strong filtering in the periphery, which
reduces the amount of information passed on to the CNS (Nilsson
et al., 2005; O’Connor et al., 2009). Still, the neuroanatomy
suggests that fairly complex information processing and integration
does happen in the rhopalial nervous system (Parkefelt et al., 2005;
Parkefelt and Ekström, 2009).
The results presented here support the idea that visual integration
takes place in the rhopalial nervous system. Three of the four eye
types and probably also the NP have impact on the pacemaker
activity and all with at least slightly different characteristics. Recent
work showed that applying a light stimulus to the entire rhopalium
resulted in light-on and light-off responses resembling what is found
here when stimulating the LLE alone (Garm and Bielecki, 2008).
There are important differences, though, because the light-on
response for the LLE alone is much stronger (the pacemaker activity
decreases more) and the light-off is more brief and bi-phasic. This
demonstrates that the visual control of the pacemaker is not a mere
hierarchy between the stimulated eyes or a sum of their individual
inputs. What kind of interactions is taking place we cannot say at
this point. To look further into this it is necessary to identify which
cells in the rhopalial nervous system make up the swim pacemaker
and map how they connect with the different eye types.
Visual ecology of the swim pacemaker control
The swim pacemaker frequency is an important part of controlling
the swim speed of the medusae and therefore the swim pacemaker
control is behaviourally important. One of the central behaviours
here is the so-called shadow response or shadow reflex, which is
known from both cubomedusae and hydromedusae (Yoshida and
A. Garm and S. Mori
0
5
10
15
20
25
0-10 10-20 20-3030-40 40-50 50-60 60-70 70-80 80-90 90-100 100-110 110-120 120-130130-140 140-150 150-160 160-170 170-180 180-190 190-200 200-210 210-220 220-230230-240 240-250 250-260 260-270 270-280 280-290 290-300 300-310 310-320 320-330330-340 340-350 350-360 360-370 370-380 380-390 390-400 400-410 410-420
Time ( s)
Time ( s)
0 100 200 300 400
0 100 200 300 400
(V)
0
–0.1
–0.15
–0.05
A
(V)
0
–0.1
–0.15
–0.05
B
0
–0.1
–0.15
–0.05
C
Time ( s)
40 50 60 70 80 220 230240 250 260
(V)
0
–0.4
–0.2
D
Dark Dark
Light
0–10 200–210 410–420
300–310
100–100
Time interval (s)
E
Pulse frequency (Hz)
2
1.5
1
0.5
Fig.7. Pacemaker activity when
stimulating a slit eye (SE). Stimulating a
SE has similar effect on the pacemaker
as stimulating the upper lens eye (ULE) or
the pit eye (PE) but more variable (A,D).
Light-on stimulates the pacemaker
frequency (A,B,E). Light-off has an
inhibitory effect in some cases (A,C) but
in other cases it has but little effect (D). In
A and B a spontaneous burst started
about 15s before the stimulus and the
onset of the stimulus caused a further
increase in the activity. The light-on effect
culminates in the first 10s and at light-off
the pacemaker frequency falls back to the
level of the dark reference (E). The red
line in A–D and under E indicates the
stimulus pattern. The black line in E is the
mean of the dark recordings and the error
bars indicate ± s.e.m. Light
intensity1.1⫻105Wsr–1 m–2 for all
subfigures.
THE JOURNAL OF EXPERIMENTAL BIOLOGY

3959Pacemaker control in box jellyfish
Ohtsu, 1973; Arkett, 1985; Arkett and Spencer, 1986a; Garm and
Bielecki, 2008). Upon a sudden drop in light intensity the
medusae increase the pulse frequency for a shorter
(hydromedusae) or longer period of time (cubomedusae). In
hydromedusae this may function as predator avoidance or control
of the diurnal migration (Anderson and Mackie, 1977; Arkett and
Spencer, 1986a) but in cubomedusae it has been shown to help
optimise their feeding behaviour (Stewart, 1996; Garm and
Bielecki, 2008). What we have shown here is that the eye type
mainly governing this behaviour is the LLE. The swim pacemaker
is also involved in the feeding behaviour by slowing down when
experiencing an increase in light intensity, which prolongs the
time the medusae stay in the light shaft where they feed (Garm
and Bielecki, 2008). From our results it is again evident that this
part of the feeding behaviour is largely controlled by the LLE.
Hence, all of the cubozoan behaviours so far proven to be visually
guided are controlled by the LLE (present results) (Garm et al.,
2007a). To better understand the cubozoan visual system it is now
important to find out what roles the other eye types play in the
behavioural control of the medusae. The obvious place to start is
to reveal the behavioural significance of the swim pacemaker
control by the ULE and PE shown here.
0
5
10
15
20
25
0-10 10-20 20-3030-40 40-50 50-60 60-70 70-80 80-90 90-100 100-110 110-120 120-130130-140 140-150 150-160 160-170 170-180 180-190 190-200 200-210 210-220 220-230230-240 240-250 250-260 260-270 270-280 280-290 290-300 300-310 310-320 320-330330-340 340-350 350-360 360-370 370-380 380-390 390-400 400-410 410-420
0
5
10
15
20
25
0-10 10-20 20-3030-40 40-50 50-60 60-70 70-80 80-90 90-100 100-110 110-120 120-130130-140140-150 150-160 160-170 170-180 180-190 190-200 200-210 210-220 220-230230-240 240-250 250-260 260-270 270-280 280-290 290-300 300-310 310-320 320-330330-340 340-350 350-360360-370 370-380 380-390 390-400 400-410 410-420
lle 2,5v
lle 1,9v
lle 1 ,3v
lle 0,7v
lle 0,1v
0
5
10
15
20
25
0-10 10-20 20-3030-40 40-50 50-60 60-70 70-80 80-90 90-100 100-110110-120 120-130130-140 140-150 150-160 160-170 170-180 180-190 190-200 200-210 210-220 220-230230-240 240-250 250-260 260-270 270-280 280-290 290-300 300-310 310-320 320-330330-340 340-350 350-360 360-370 370-380 380-390 390-400 400-410 410-420
0
5
10
15
20
25
0-10 10-20 20-3030-40 40-50 50-60 60-70 70-80 80-90 90-100 100-110110-120 120-130130-140 140-150 150-160 160-170 170-180 180-190 190-200 200-210 210-220 220-230230-240 240-250 250-260 260-270 270-280 280-290 290-300 300-310 310-320 320-330330-340 340-350 350-360 360-370 370-380 380-390 390-400 400-410 410-420
0
5
10
15
20
25
0-10 10-20 20-3030-40 40-50 50-60 60-70 70-80 80-90 90-100 100-110110-120 120-130130-140 140-150 150-160 160-170 170-180 180-190 190-200 200-210 210-220 220-230230-240 240-250 250-260 260-270 270-280 280-290 290-300 300-310 310-320 320-330330-340 340-350 350-360 360-370 370-380 380-390 390-400 400-410 410-420
0–10 200–210 410–420
300–310
100–110
Time interval (s)
Dark Dark
Light
0.5
1
1.5
2.0
B
C
D
E
0.5
1
1.5
2.0
Pulse frequency (Hz)
0.5
1
1.5
2.0
0.5
1
1.5
2.0
0.5
1
1.5
2.0
ALower lens eye
Upper lens eye
Pit eye
Neuropil
Slit eye
52
1.1x10 W/sr/m
52
1.0x10 W/sr/m
42
9.6x10 W/sr/m
42
7.1x10 W/sr/m
32
8.7x10 W/sr/m
Fig.8. Effects of different light intensities.
In all five different types of stimulations
five different intensities were tested
covering ~1.1 log units. When the LLE or
NP was stimulated the response
magnitude during light was correlated
with the intensity of the light stimulus
(A,D). The stronger the light the higher
the mean pacemaker frequency during
the first 20s after light-on. In the case of
the PE, ULE and SE, no such correlation
is seen (B,C,E, see text for details). In
the case of the entire light-on period a
positive correlation between amplitude
and intensity was found for the NP and
PE. The situation is similar for the light-
off response where a correlation between
the frequency change and stimulus
intensity is again seen for the NP. LLE,
lower lens eye; PE, pit eye; NP, neuropil;
SE, slit eye; ULE, upper lens eye.
THE JOURNAL OF EXPERIMENTAL BIOLOGY

3960
The authors appreciate the constructive comments offered by several members of
the Vision Group, Lund University and we truly thank Assoc. Prof. Poul Bennekou,
University of Copenhagen, for the generosity with his equipment. A.G. would also
like to acknowledge the grant # 272-07-0163 from the Danish Research Council
(FNU).
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