Abstract
Pannychia moseleyi is a commonly found benthic organism
living at depths greater than 400 m in the Monterey Bay canyon.
Observations in the laboratory have shown that, upon tactile
stimulation, Pannychia produces blue-green spiral and quasi-circular
waves of bioluminescence over the entire body surface except the
podia, papillae and tentacles. Video analysis showed dispersion in
traveling waves, i.e. the wave velocity at a point depends on the time
elapsed since the previous wave passed that point. Annihilation was
observed to occur when waves collided. This leads to the conclusion
that the mechanism of bioluminescence behaves as an excitable
medium with a refractory period of § 0.5 seconds. There appear to
exist regions on the body surface which are incapable of
bioluminescing. These banded unexcitable zones are arranged
diagonally with respect to the horizontally-oriented animal.
Introduction
Pannychia moseleyi Théel, 1882 is broadly distributed along
the coasts of the Pacific from Australia and Indonesia to Canada and
Peru, and off the Hawaiian Islands. It is found at depths between
212 m and 2598 m (Hansen, 1975). Pannychia (Fig. 1) is a benthic
sea cucumber which can be found laying in flat, open spaces as well
as clinging to ledges. There is an extensive amount of variation in
the species with regard to coloration, number of tube feet (Hansen,
1975), and dorsal papillae size.
Herring (1974) and Robison (1992) examined bioluminescence
in holothurians, including another member of Pannychia's family,
Laetmogone violacea. In most of these species, the bioluminescence
was described as a scattering of points of light on the body surface
and the tips of tentacles, tube feet, and papillae.
Materials and Methods
In situ observations, video photography, and collection were
conducted using the remotely-operated vehicle (ROV) 'Ventana' on
cruises of the research vessel Point Lobos' in the Monterey bay. Six
specimens, collected on 12 March 1992, were taken from depths of
458 m to 470 m. Six more, collected on 9 April 1992, were taken
from depths of 517 m to 555 m. One specimen, not taken using the
ROV, was collected on the outside of a black cod trap at 1116 m.
Upon being brought aboard ship, the animals were kept in
darkened containers of sea water at approximately 5°C before being
transferred to chilled-water tanks at the Monterey Bay Aquarium,
Monterey, CA. Each March-collected specimen was placed in its own
5-gal. tank with deep-sea mud as the substrate. Two of the April¬
collected specimens and the 'cod trap' specimen were also placed in
5-gal. tanks. The four remaining April-collected specimens were put
in one 2' x 3' x 2' tank. The substrate for this tank was mud taken
from the Elkhorn Slough at Moss Landing, CA. All 5-gal. tanks were
kept in the dark, while the large tank was kept in light.
Preliminary Investigation
The animals were stimulated to bioluminesce by lifting them
from their rest position, by hand, and allowing them to sink to the
bottom of the tank. The pattern and duration of the light were
noted. Following stimulation, animal length measurements were
taken.
Spectral Analysis
Bioluminescence spectra were taken from tissue samples of
unpreserved specimens kept in cold sea water. The tissue was
stimulated by immersing it in fresh water and prodding it with a
metal rod to increase the light output. Spectra were measured with
an EG&G Princeton Applied Research Model 1215 optical
multichannel analyzer (ÖMA) using a linear array detector consisting
of 700 intensified silicon photodiodes. A 1-mm entrance slit was
used. Details of calibration and operation have been described
previously (Widder et al, 1983). Two spectra taken consecutively
from a single tissue sample were summed to increase the signal-to¬
noise ratio. The resulting waveform was corrected for background
noise and smoothed.
Primary Investigation
Animals were stimulated by gently rubbing the dorsal surface
with a fingertip. Video recordings of bioluminescent activity were
taken with a Cohu Model 5000 silicon-intensified tube (SIT) camera.
Stimulation was maintained until bioluminescence ceased. Traces
were drawn directly from the video monitor at successive frame
intervals to study bioluminescent wave propagation. Wave velocities
were calculated by measuring the distance of the wave front from a
given point 0.1 s before reaching that point.
Results
Preliminary Investigation
Only a mild correlation between animal length and duration of
bioluminescent activity was observed (Fig. 2). While there is a high
degree of scatter in the data, the larger specimens were able to
sustain light longer in general.
Eight of the thirteen specimens bioluminesced in a combination
of pinwheel and quasi-circular waves (Fig. 3). Typically, the entire
main surface area of the body (excluding tube feet, papillae, and
tentacles) would light up almost at once. The luminous activity
would continue for a time before dying out. The mid-section would
die out first, leaving only the anterior and posterior ends still
bioluminescing. In one of these eight, a soft glow near the anterior
and posterior ends followed the period of dynamic, luminous activity.
Three of the specimens produced rectangular bands of order 1
cm length travelling along both ventro-lateral sides. These were
relatively short episodes (3.1 s, 6.9 s, and 7.0 s.) All three of these
animals had been kept in the large tank. Two simply emitted points
of light distributed over most of the body surface.
Spectral Analysis
The emission spectrum for Pannychia bioluminescence is
shown in figure 4. The maximum intensity occured at approximately
480 nm while the full width at half maximum (FWHM) value was
about 80 nm (450 nm to 530 nm). There is a slight asymmetry
toward longer wavelengths.
Primary Investigation
The video analysis confirmed the existence of clockwise¬
turning spiral waves and pulsing quasi-circular waves emanating.
radially outward, from source points. The period of the pulsing
sources was approximately 0.5 s. The quasi-circular waves would
change their mode of propagation and become cycling spirals.
Lechleiter (1991) noticed this phenomenon in his observations of
propagating waves of Ca2+ release in Xenopus laevis oocytes. (The
ventro-lateral band pattern was not seen at this stage of the
investigation.) Annihilation was observed to occur when traveling
waves of any type collided with one another. High intensity colliding
waves underwent what I termed a strong wave annihilation (Fig. 5).
In this scenario, an intermediate point source is formed as the high
intensity waves approach each other. The point source grows while
the high intensity waves close in on it. All three collide and are
annihilated.
The wave dispersion relation (Fig. 6) shows the dependence of
wave velocity on the time spacing of the waves. The speed of a wave
passing through a particular point depends on the amount of time
elapsed since the previous wave passed that point; the larger the
spacing, the higher the wave velocity. Observations showed that the
velocity was tending toward some maximum.
As the waves moved over the body surface, it was clear that
some regions were never illuminated. These regions were arranged
along the entire body length in diagonal bands with respect to the
horizontally-oriented animal. These banded unexcitable zones,
(Fig. 7), do not travel with the wave, but remain in a fixed position
on the body surface as the waves pass over them. The banded
unexcitable zones are the gray bands shown in the figure.
Discussion
Pannychia bioluminescence has been seen to occur only on the
body surface, unlike most of the other members of the Elasipod order
which display luminescence on the tentacles, papillae, and/or tube
feet (Herring, 1974). In addition, the dynamic nature of Pannychia's
bioluminescence is unlike that of any of the holothurians studied by
Herring (1974, 1978) or Robison (1992).
Although a good correlation has not been demonstrated
between animal size and duration of bioluminescent activity, this can
not be ruled out. During the collection process, the animals are
subjected to varying degrees of stress and physical injury. Thus, if a
smaller specimen is in better condition than a larger specimen, it is
possible that its bioluminescence would last longer. The tendency for
the mid-section of an animal to stop bioluminescing before the
posterior and anterior ends correlates with Smith's (1965) assertion
that the middle region of holothurians is the least sensitive to
mechanical stimulation.
The emission maximum and half bandwidth values (480, 80) of
Pannychia bioluminescence fall approximately in the middle of the
range of the two other members of the Laetmogonidae family which
are known to bioluminesce (Herring, 1983). Laetmogone violacea has
the values (470, 70). Taking the average of the four measurements
reported by Herring, one obtains the values for Benthogone rosea,
(480, 90). Two of the four measurements, however, indicated that
there were shoulders at 505 nm on an otherwise unimodal spectrum
which implies that the emission from B. rosea has a substantial green
component. This fact becomes interesting when one considers the
depth ranges in which these species are found (Fig. 8).
Bathymetrically speaking, Pannychia is found between Laetmogone
and Benthogone (Hansen, 1975). This indicates that there may be
some correlation between depth and emission spectral properties.
would be necessary to study other families containing
bioluminescent members to determine if this is a general trend.
Perhaps the most significant finding is that the bioluminescent
mechanism found in Pannychia behaves as an excitable medium; i.e.,
a system which when excited above a critical point by a stimulus
requires a refractory period to recover its excitability (Lechleiter et
al, 1991). Undamaged cardiac muscles are an important example of
excitable media (Winfree, 1989). Some characteristics of excitable
media include rotating spiral waves, dispersion effects and wave
annihilation (Gerhardt et al, 1990), all of which were observed to
occur in in vivo Pannychia bioluminescence.
The spiral waves were observed to rotate only in the clockwise
direction. It is not clear whether this is a general rule. Epstein
(1991) notes that a pacemaker nucleus is required to generate
periodic supercritical perturbations. The pulsing period of 0.5 s for
the quasi-circular waves appears to be an upper limit for and
possibly very close to the actual refractory period of the the medium.
Nicol (1955) found that the refractory period for the bioluminescence
mediated in a nerve net in Renilla köllikeri was around 0.2-0.5 s
indicating that 0.5 s for Pannychia places it in a reasonable
physiological range. From figure 6 one can see that the wave
velocity approaches a limiting value of about 4 cm/s which is again a
reasonable velocity for conduction of signals through the nervous
system. (Nicol (1955) found a value of 7.8 cmls in Renilla.)
The strong wave annihilation is an interesting phenomenon
about which I propose the following to describe its mechanism. The
wave of luminescence travels at the same velocity as the stimulus
(assumed to be neuronal activity), but lags behind slightly because of
the finite amount of time required to excite the medium. In a strong
wave there is a strong stimulus which, when it encounters another
strong stimulus traveling towards it, excites an area between the two
strong waves more quickly than if there had only been one stimulus
to excite the area. The area is illuminated before either wave has
reached it. This point source grows until all three collide and are
annihilated.
The origin of the banded unexcitable zone pattern is
speculative. Perhaps it is simply caused by the papillae obstructing
the view of the light or perhaps the luminous cells are organized in
banded patterns. I was unable to determine the location of luminous
cells in Pannychia. Herring (1974) found circumstantial evidence for
the location of epidermal luminous cells in L. violacea and B. rosea,
but did not report on how they were distributed in the tissue.
The nature of Pannychia bioluminescence with regards to its
nervous system is still unclear. Smith (1965) reports that strong
localized stimuli in holothurians causes reactions to spread to
neighboring areas. This may imply transmission through a nerve
net. Although the pathways for conduction are not known for
certain, Smith (1965) postulates that certain phenomena are
transmitted through "afferent and efferent paths connected with the
nerve cord." Facilitation, he adds, is required in the neuronal
synaptic junctions.
The function of bioluminescence in Pannychia is a subject for
debate. Although holothurians are assumed to have body surfaces
which are sensitive to light (Hyman, 1955), I have not been able to
show this conclusively in Pannychia. Since bioluminescence is
initiated upon tactile stimulation, it is reasonable to conclude that its
primary function is predator evasion. Morin (1983) has given a
thorough description of behavioral functions of bioluminescence.
may have a temporary blinding effect on a dark-adapted predator,
which Morin (1983) refers to as the "flash bulb effect." Also, since
Pannychia is able to sustain its light for several seconds, the
bioluminescence may serve to warn the predator that its prey is
invulnerable to attack or would be a distasteful food item (Morin,
1983). The sustained light may also serve to attract an organism
which preys on Pannychia's predator; this is the so-called burglar
alarm effect put forth first by Burkenroad (1943). The latter
functions seems to be the most appropriate since Pannychia is unable
to swim or move quickly away from a predator once the predator
attacks. Robison (1992) notes that the glowing skin of the
holothurian Enypniastes eximia is adherent and might serve to
temporarily mark its aggressor in such a way that a predator of the
aggressor would be able to find the aggressor. Studies of Pannychia
behavior in the presence of possible predator species should prove
fruitful in determining the functional role of its bioluminescence.
Acknowledgements
I would like to thank Karen Light of the Monterey Bay
Aquarium for the use of the animals under her care. (May they live
long and prosper.)
Much thanks to Steve Haddock at UC Santa Barbara for giving
up a day to do spectra. (We have to play two-man volleyball
sometime.)
Also, thanks to Chuck Baxter for wise counsel, Stuart Thompson
for videography and ideas, and David Epel for convincing me to take
this class. (Otherwise l'd be studying for four finals right now.)
Thanks to Chris Patton for help with photography and Teri Nicholson
for endless hours of behind-the-scene work.
Thanks to Bruce Robison of MBARI and the entire crew of the
"Point Lobos." May you find continued joy in sailing the high seas.
Thanks to David Bracher for your kindness and willingness to help
"Tanks" to Kim Reisenbichler for technical support.
Ultimate thanks to God for constant support and love throughout the
quarter.
Literature Cited
Epstein, I. R., 1991. Spiral waves in chemistry and biology. Science.
252, 67
Gerhardt, M., Schuster, H., and Tyson, J. J., 1990. A cellular automaton
model of excitable media including curvature and dispersion.
Science, 247
Hansen, B., 1975. Scientific results of the Danish deep-sea expedition
round the world 1950-1952. Galathea Report, 13, 73-75, 210
Herring, P. J., 1974. New observations on the bioluminescence of
echinoderms. Journal of Zoology, 172, 409-417
Herring, P. J., 1978. Bioluminescence of invertebrates other than
insects. In Bioluminescence in Action, 235
Herring, P. J., 1983. The spectral characteristics of luminous marine
organisms. Proceedings of the Royal Society of London, 220,
198
Hyman, L. H., 1955. Echinodermata, The invertebrates, 4, 144
Lechleiter, J., Girard, S., Peralta, E., and Clapham, D., 1991. Spiral
wave propagation and annihilation in Xenopus laevis oocytes.
Science, 252, 124
Morin, J. G., 1983. Coastal bioluminescence: patterns and functions.
Bulletin of Marine Science, 33
Nicol, J. A. C., 1955. Nervous regulation of luminescence in the sea
pansy Renilla köllikeri. Experimental Biology, 32, 627
Robison, B. H., 1992. Bioluminescence in the benthopelagic
holothurian Enypniastes eximia. Journal of Marine Biolegy, 72
33
in379
Smith, J. E., 1965. Echinodermata. In Bullock, T. H., Horridge, G. A.,
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353
Figure Legend
Sketch of Pannychia moseleyi.
Duration of bioluminescent activity following mechanical
stimulation vs. animal size.
Typical wave patterns seen in Pannychia bioluminescence.
Smoothed spectrum of Pannychia bioluminescence.
Time lapse sketch of the annihilation of two high intensity
waves and the associated point source which develops as they
approach each other.
Dispersion relation for bioluminescent waves. The points are
measurements taken directly from the screen. The bars are
errors in measuring velocity due to the uncertainty of the
actual location of the wave front.
Banded Unexcitable Zones in bioluminescent waves. White
areas are regions of bioluminescence in this cut-away view of
the animal. The waves are propagating from left to right, while
the banded unexcitable zones remain stationary.
Bathymetric distribution of three bioluminescent members of
the Laetmogonidae family. A summary of their spectrum
characteristics is also shown.
Figures
Papilla


Tentacles

1cm
Figure 1. Pannychia moseleyi



Dde feet
Bioluminescence Duration
30 -
25
3 20
5
10 +
5 -


— —
Ottta-

8 10 12 14
16 18 20 22 24 26 28
Specimen Length (cm)
Figure 2. Duration of bioluminescent activity
Spin Direction
Wave Propagation Direction
Spiral
"Circular'
1cm
Figure 3. Typical wave patterns seen in Pannychia bioluminescence
Spectral Distribution for Pannychia Bioluminescence
1000
900 +
800-
2700 ++
3 600 +
- 500
2 400+
300-
200
100-

400
450
350
500
550
600
Wavelength (nm)
S 480 nm
Ama
FWHM s 80 nm
Figure 4. Smoothed spectrum of Pannychia bioluminescence
650
—
High Intensity Waves
Annihilation
Figure 5. Annihilation of high intensity wave
Wave Dispersion Relation


2.5
2

0.45
0.55
0.6
0.5
0.65 0.7 0.75 0.8
Wave Spacing (s)
Figure 6. Dispersion relation for bioluminescent waves
Cut-away view of animal
Banded Unexcitable Zone
&a
1cm
Wave Front
Figure 7. Banded unexcitable zones appearing in bioluminescent traveling
waves.
s.
500++
1000-
470,70
480,
1500+

2000+
480*
2500-
* Shoulder at 505 nm
3000-L
Figure 8. Depth distribution for three bioluminescent members
of Laetmogonidae family. Emission maxima and half
bandwidths are given.