Abstract
Pannychia moseleyi is a deep-sea holothurian found at
depths from approximately 500m to 3000m in the Monterey Bay
Canyon. The bioluminescence in Pannychia moseleyi has a
complex dynamic pattern spread throughout the epithelium, but
absent from the tube feet, papillae, and tentacles. The waves
observed were greatly varied, but can fit into three general
classifications - spiral, circular from a point, and traveling. The
basis of bioluminescence in Pannychia appears to be intracellular
and microscopic, and shows strong evidence for being passed in an
excitable medium such as a nerve net. The strongest evidence for
the coordination by a nerve net comes from transection studies.
Introduction
Pannychia moseleyi (Figure 1) is a deep-sea holothurian
found at depths from approximately 500m to 3000m in the
Monterey Bay Canyon. One of the most interesting characteristics
of Pannychia is the dynamic bioluminescence elicited by tactile
stimulation. Propagation occurs over most of the dorsal surface,
and tends to concentrate near the caudal end. The
bioluminescence travels in complex patterns consisting of spiral,
circular, and traveling waves.
The main purpose of this study was to investigate these
patterns in relation to the morphological characteristics of the
Holothuria. From these studies it appears that the
bioluminescence is coordinated by a nerve net located in the
basiepithelial plexus and that through-conduction is mediated by
a combination of the deep plexus and the radial nerve cords.
Materials & Methods
Collection
Animals were collected using the remotely operated vehicle
(ROV) Ventana on multiple cruises of the MBARI research vessel
Point Lobos in the Monterey Bay. Three specimens were collected
prior to the spring course. Seven more specimens were collected
in on April 6, 1993. Two more specimens were collected in depths
of on April 19,1993. One specimen was collected at a depth of
April 26,1993. The last seven specimens used for research were
collected on May 4, 1993. Three more Pannychia were collected
on May 24, 1993, but were only observed. They were collected in
depths ranging from 550 to 900 meters.
Storage
All Pannychia were placed in Rubbermaid containers with 5
C sea water and a little sediment on board the Point Lobos. Upon
arrival at Hopkins Marine Station, the Pannychia were placed in
the water holding tanks in the Deep Sea Lab. These were kept at
temperatures from 5-7 C. While in the holding tanks, the
Pannychia were maintained in complete darkness except when
the insulated covers were lifted. The animals were divided into
groups based mostly on the date of collection. The original three
became group A. The next seven became B 1-4 and C 1-3. The
remaining Pannychia became D-1 and D-2, E-1, F 1-7, and G 1-3.
The numbers within the group were arbitrary and were assigned
based on the order of use for experimentation.
Natural Sea Water Filming
The animals to be filmed were transferred by hand from
their holding Rubbermaid containers into a Rubbermaid
butterdish filled with 5 C sea water. Bioluminescent activity
during transfer was noted. The animals were then covered,
placed back in the holding tanks, and allowed to recover for 45 to
90 minutes (usually 60 minutes). The whole transfer process was
done under red light.
The filming was done using a Cohu Silicon-Intensified Tube
(SIT) camera. The camera was set on Auto while a bright field
image was taken. The light was switched off and the camera set
on Manual with maximum gain and black contrast at one quarter
for the filming of bioluminescence. All filming was done on the
dorsal surface of the animal (the bivium). (Figure 2) The stimulus
was a gentle tap with a quarter centimeter diameter glass rod
usually near the caudal end of the animal slightly lateral to one of
the bivium radial nerves. The darkness was maintained until the
bioluminescence ceased. Another bright field image was then
taken. This process was then repeated so that for each recording
session an animal had three bright field images and two stimulus
attempts. This procedure would sometimes be altered by adding
a third stimulus attempt to ensure good video footage or by
stopping after one stimulus attempt if bioluminescence failed to
occur. The whole filming process for three animals usually took
10-15 minutes.
After the filming session, the Pannychia were transferred
back to their larger containers and bioluminescence observed was
recorded.
Magnesium Chloride Filming
Animals were transferred in a method similar to above into
Rubbermaid Butterdishes containing different ratios of 5 C
Isotonic MgCl2 (110 mM MgCl2 added to Natural Sea Water for
160 mM MgCl2 total) and natural sea water and allowed to sit for
45 minutes before filming. The ratios ranged from 1 part
isotonic/2 part natural sea water to 1 part isotonic/7 parts natural
sea water. Filming was performed with the same protocol as in
natural sea water; three bright field images with two
bioluminescent attempts interspersed. Äfter filming the animals
were transferred by hand back into their normal Rubbermaid
containers. The level of bioluminescent activity was noted on
both transfers into and out of the experimental solution.
Computer Analysis
Video footage was analyzed using the Megavision in Dr.
Stuart Thompson's laboratory at Hopkins Marine Station. The
footage was first transferred to Optical Memory Disc Recording
(OMDR) to allow for easier manipulation and a sharp image for
processing. Using a combination of functions, measurements were
made of conduction velocity of a bioluminescent wave, maximum
intensity of a wave front, time lit of an area during each wave
front, and period between wave fronts.
Localization of the luminescence on the Pannychia was
confirmed on Megavision as well. A video segment was
accumulated and then overlaid on a bright field image from the
same video session. Annihilation was also observed in slow
motion on the Megavision.
Transection Studies
Various transections were performed on the Pannychia and
the effects were observed and the bioluminescence was recorded
on the SIT camera. These included a single cut of one radial nerve
approximately three centimeters from the caudal end of the
animal (E-1,F-1,F-6,F-3), a double cut of one radial nerve
approximately three and four centimeters from the caudal end (F¬
7), and complete removal of the caudal tip (C-3, C-1). The
transected animals were allowed to sit for one hour before
filming, while the amputated animals were allowed to sit for 24-
36 hours before filming.
Stimulation varied from study to study, but followed the
general pattern of alternating bright field and stimulus attempts.
Usually the first stimulus was ipsilateral and rostral to the cut(s),
the second was near the caudal tip of the animal, and the third
was contralateral and rostral to the cut(s). In the case of
amputation, stimulation was done as with the Natural sea water
experiments.
Results
General Trends
Some general patterns of bioluminescence could be
observed. Ninety per cent of the time the bioluminescence would
appear caudal to the stimulus (up to 1-2 cm) and propagate in the
caudal third of the animal, usually lasting longest at the caudal tip.
Ten per cent of the time, however, the bioluminescence would
travel in both directions from the point of stimulus, thus lighting
up a greater portion of the animal. In these cases, the central
section of the animal would usually cease bioluminescence 3-8
seconds before the caudal section. With repeated stimulation, the
Pannychia would eventually fatigue to the stimulus, and would
fail to produce luminescence. In such cases, luminescence could
be elicited by lifting the animal during transfer back to its main
container. This bioluminescence tended to cover the whole
surface with bioluminescence again lasting longest near the caudal
end.
Wave Types and Characteristics
The waves observed were greatly varied, but can fit into
three general classifications - spiral, circular from a point, and
traveling. (Figures 3, 4, and 5) The traveling waves tended to
show up with the gentle stimulus of the glass rod. They would
travel along the radial cord and sometimes cross to another radial
cord. An isolated traveling wave was not common, but was
probably often present in more complex patterns. Spiral and
circular waves tended to show up in both point stimulus and
animal transfer stimulus. They were often both present on the
same animal and resulted in complex patterns running in
repetitive cycles on the surface of the specimen. Within these
complex patterns annihilation was often observed. Annihilation is
defined as when two intense waves propagate towards each other,
and upon collision result in a loss of luminescent activity. (Figures
6a, 6b, and 6c) Waves of luminescence are also bi-directional. and
there are specific instances of a wave reversing direction in the
course of one stimulation.
Localization of Bioluminescence
The bioluminescence tends to be localized on the bivium and
does not occur on the tube feet, the papillae, or the tentacles
surrounding the oral opening. Bioluminescence does not appear to
occur on the trivium between the two rows of tube feet. Within
the bivium, there are dark unexcitable spots which correspond to
the base of the papillae. (Figure 7) In less intense bioluminescent
waves, these spots can appear connected giving the overall
appearance of parallel dark unexcitable bands. (Figure 8) The
actual photocytes of bioluminescence are below the resolution of
the Megavision, which puts them in the range of less than 100
microns (based on one pixel at 9 pixels/mm). On examination of
one animal in particular(F-7), it appears that units of
bioluminescence are arranged in a regular fashion, separated by
approximately 500-800 microns. These units have a large range
in size, and thus appear to be amalgamations of individual
photocytes.
It can be seen that the photocytes do not fatigue during
multiple wave stimulations as they can reach the same intensity
throughout. For example, in bioluminescence seen in C-2, the
maximum intensity scale of 255 (out of 255) was reached on
seven successive wave fronts passing through the same point.
This is seen in the other specimens that had multiple wave fronts
passing through a single point as well.
Transection Studies
Transection studies were performed to investigate the
pathways involved in bioluminescence. Upon transection of a
single radial nerve, the bioluminescent response is altered
markedly. While the bioluminescence can travel past the point of
the cut in both ipsilateral and contralateral stimulation, the
bioluminescence does not travel as far towards the caudal end in
ipsilateral stimulation as would be expected from observations of
normal animals. When transferred, the bioluminescence is normal
except immediately around the cut, where a disturbance in the
wave pattern results in a diffuse glow. (Figure 9) Upon double
transection of a single radial nerve, the bioluminescent response is
altered even more. Ipsilateral stimulation bioluminescence stops
at the double transection in a sharp line and tip stimulation
bioluminescence stops at a similar sharp line on the other side of
the double transection. (Figures 10 and 11) In both cases, the
wave front reaches the cut, and instead of annihilating or
dissipating, changes direction and runs along the cut, as if looking
for a new path. Contralateral stimulation bioluminescence travels
down the contralateral side and lights the whole caudal area.
When transferred, the bioluminescence is normal except for
diffuse glows around the immediate cut area, even without
stimulation in the area between the two cuts. (Figure 12) In both
sets of transections, the specimens can be seen to reseal the cut
and start the regeneration of the damaged area.
Amputations
Removal of the caudal tip served two purposes, to
investigate the recovery of the animal and to see if
bioluminescence could be produced in isolated sections of the
animal. Upon removal of the caudal tip (Icm), the specimens
healed their caudal ends and within 24-36 hours gave
bioluminescent responses that were similar to earlier responses,
except that they were longer. The animals were healthy and
recovered well from their amputations. The isolated pieces could
not be made to bioluminesce - even with treatment with KCl,
Nacl, and prodding with forceps.
Magnesium Chloride
Different ratios of MgCl2 were employed to investigate the
neural properties of the bioluminescence in relation to calcium
channels. The 1 part isotonic MgCl2/ 2 part natural sea water
solution had the ability to knock out bioluminescence completely,
even upon major stimulus. At lower ratios of isotonic MgCl2, the
animals had a raised threshold for bioluminescence. This led to
multiple taps being needed to elicit bioluminescence and short
total lengths of bioluminescent propagation.
Mathematical Data
From analysis on the Megavision, some mathematical
characteristics of the waves have been found. The conduction
velocity varies within each specimen and across the population.
has a range of 5.2-47.6 cm/sec with an average conduction
velocity of 17.6 cm/sec. (Figure 13). The velocity in transection
studies has a range of 14.2-18.1 cm/sec and an average of 15.54
cm/sec. Thus it appears that near a transection, both the faster
and slower pathways are obliterated. The period between
successive wave fronts ranges from .33 s to .69 s and has an
average of 5 s and a standard deviation of .1 s. Also, the velocity
and period are inversely related. (Figure 14).
Discussion
Unlike the bioluminescence of other holothurians studied by
Robison (1992) and Herring (1974), the bioluminescence in
Pannychia moseleyi has a complex dynamic pattern spread
throughout the epithelium, but absent from the tube feet, papillae,
and tentacles. This dynamic bioluminescence appears to be
located in the basiepithelial plexus where it is under a nerve net
control. Through-conduction is via the polarized deep plexus and
connects to the central nervous system of the radial nerve cord
for more distant transfers. (Smith, 1965)
Similar to Enypniastes eximia (Robison, 1992), the basis of
bioluminescence in Pannychia appears to be intracellular and
microscopic. The individual photocytes could not be resolved with
video imaging, which gives them a maximum size of 100 microns
(Robison measured 35 microns for Enypniastes eximia). Evidence
for the intracellular nature comes from the short time lit of .05-.4
seconds, which is similar to the 1 second found for Ptychordia
flava by Baxter and Pickens (1964) and much too short for the
discharge of luminous secretions which last minutes (Baxter and
Pickens, 1964). Also, the photocytes show no fatigue in maximum
intensity as a set of waves propagate through, which would not be
expected in an extracellular system (Baxter, 1964).
The bioluminescence of Pannychia shows strong evidence
for it being passed in an excitable medium such as a nerve net.
The strongest evidence comes from annihilation as shown in
figures 6a, 6b and 6c. Annihilation is a characteristic property of
excitable media, and is prevalent in the complex waves of
Pannychia.. The presence of spiral waves also points to an
excitable medium, as the spiral results from the refractory period
which defines this type of medium. The inverse correlation
between velocity and period is not typical of the excitable media
models presented in the literature (Gerhardt, 1990). This does not
show that it is not an excitable medium, as the physiological
system of a nerve net does not perfectly align with a chemical
reaction or other models.
The effects that Magnesium Chloride has on the
bioluminescence point towards a neural system which involves
muscarinic channels. At high enough concentration (1 part
isotonic MgCl2 / 2 parts natural sea water) the Mg++ ions are
prevalent enough to anesthetize the animal and halt
bioluminescence. At lower concentrations, it is likely that the
threshold increase is due to competition between Mgtt and Catt
ions at muscarinic channels. This leads to the shorter
bioluminescence and the need for multiple stimulations to elicit
the bioluminescence. Similarly, it has been observed in
Coelenterate nerve nets that Mg+ ions depress the response
without preventing conduction (Bullock, 1965).
There is considerable evidence for the coordination of
bioluminescence by a nerve net localized in the basiepithelial
plexus. The nervous systems of the Holothuria contain features of
both primitive and advanced design. Similar to coelenterates,
they have a fiber system with a net arrangement located in the
epithelial layer and composed of small-celled interneurons. In
addition they have a central nervous system composed of the five
radial nerve cords and the nerve ring. (Smith, 1965). These two
systems, interacting via polarized specializations for through¬
conduction in the deep plexus, allow for a complex system of
control of bioluminescence.
The strongest evidence for the coordination by a nerve net
comes from the transection studies. With a single cut of the radial
nerve bioluminescence can diffuse around the cut and propagate
towards the caudal end, however the through -conducting system
is interrupted and the signal travels less than expected in a
normal animal and becomes a more localized response. With a
double cut of a radial nerve, bioluminescence is halted and fails to
propagate past the disrupted area. In both cases, the bigger
stimulus of lifting the animal causes bioluminescence to spread
over the whole surface, with slight disturbances near the cuts.
The lack of annihilation and the change of direction when the
wave front reaches the edge of the cut gives evidence for multiple
possible conduction pathways. These results show how there is a
diffuse conduction of nervous excitation that has a tolerance for
incomplete cuts. This is a definition of a nerve net (Bullock,
1965).
The transection studies point towards interneural facilitation
which is a common aspect of nerve net conduction (Smith, 1965;
Pantin, 1935a). The single cut allows transmission to pass because
the signal can find a more diffuse path through the nerve net and
circumvent the obliterated area. The double cut causes more
problems and halts transmission because the need for facilitation
prevents the more circuitous routes from succeeding.
The transection studies also shed light on the through-
conducting system located in the polarized deep plexus. Since the
through-conducting system receives its stimulation from the
basiepithelial plexus, a disturbance of the nerve net will lead to a
subsequent disturbance of the deep plexus. This is seen in the
single cut in which the bioluminescence can spread past the cut,
but it remains as a localized luminescence due to the degradation
of the signal in the deep plexus. The larger stimulus presents
enough activity in the nerve net to stimulate the through-
conducting system and spread the bioluminescence over the
whole body regardless of transections.
The role of the through-conducting systems manifests itself
in other ways as well. The general trend of the spreading towards
the caudal end when stimulated in the caudal third is probably a
result of the directional nerves of the deep plexus. The ability of
the bioluminescence to start caudal to the actual point of stimulus
could be the result of the sensory cells transmitting directly to the
through- conducting system before starting bioluminescence in
the nerve net. This would be a highly specialized response, which
agrees with Pantin's idea that through-conducting systems are
specializations, developed differently in different species (Pantin,
1935b).
Another piece of evidence for the nerve net coordination of
bioluminescence is the property of after-discharge, in which
animals continue flashing after the stimulus has ceased. This is
common in Pannychia, as bioluminescent waves will often move
on the body and enter new areas long after the initial gentle tap
has ended. Pantin described this as an unpredictable property
that gave an arbitrary element to nerve net responses (Pantin,
1935c). On his research in Renilla, Nicol observed a similar effect
and concluded that this is obviously a condition dependent on
excitation of the nerve net (Nicol, 1955).
A generally slow conduction velocity is another
characteristic of nerve nets. This is largely because the level of
facilitation needed and the conduction velocity are inversely
related (Pantin, 1935b). The conduction velocities in Pannychia fit
this description. The range from 5.2-47.6 cm/s is similar to the
observed ranges for other nerve net systems. For example,
Pantin found three ranges depending on direction of 5-10 cm/s,
9-20 cm/s, and 4-120 cm/s in Calliactis and Nicol found a range of
6.66-10.15 cm/s in Renilla (Pantin, 1935b, Nicol 1955). This
points to a nerve net in which conduction velocity is low in
response to a high interneural facilitation. The speeds in
Pannychia are much slower than the 55cm/s observed in the
radial nerve cord of the brittlestar Ophiapturis papillae (Yee,
Burkhardt, Gilly, 1987). Also, the range of conduction velocities is
not unheard of, as Brehm reported a range of 10-85 cm/s within a
single animal for bioluminescence along an ophiuroid radial nerve
(Brehm, 1977). One explanation for the varying speeds could be
varying levels of interneural facilitation and through-conduction
interacting to form bioluminescence.
Another characteristic common to nerve nets is unpolarized
synapses. This was observed in video footage as a reversal of
wave direction within a single bioluminescent set. This is
indicative of a bi-directional synapse system which allows for
multiple diffuse conduction pathways (Bullock, 1965).
Overall, the bioluminescence seems to follow complex
predetermined general patterns which could be inherent to the
nerve net (Bullock, 1965). The bioluminescence of the nerve net
seems to spread throughout the basiepithelial plexus, except at
the base of papillae (figure 7). Here the wave disappears and
then reappears on the other side. It seems as if the nerve net
continues through the base of the papillae, but that photocytes are
lacking in these areas. This explains the dark unexcitable spots.
The origin of the dark unexcitable bands seems to stem from an
apparent connection of the dark unexcitable spots. This is
probably due to the complex wiring of the nerve net in relation to
the distribution of papillae. All of these factors help to contribute
to each animal having a distinctive pattern, sometimes enough to
be identified based solely on a bioluminescent wave.
There are many theories as to the actual function of
bioluminescence in oceanic and coastal marine organisms. These
range from the burglar alarm effect in which the prey tries to
attract the predators of its predators to the lure effect in which
the bioluminescence is meant to attract prey (Young, 1983; Morin,
1983) Since the bioluminescence is initiated purely by
stimulation in Pannychia, this limits the functions to defense
mechanisms. Because of the paucity of information about the
predators of Pannychia, it is hard to guess as to which defense
theory is most appropriate. Based purely on observations of the
waves and localization of bioluminescence in Pannychia, it seems
that the whole body flash could be a warning to other Pannychia
of danger in the area or an attempt to startle the predator and
gain freedom. The tendency to bioluminesce near the tail and the
amputation experiments point to the idea that this limited
bioluminescence might attract the predator to the dispensable
part of the body, and thus preserve life as a whole.
Acknowledgments
Kim, thank you for tolerating me.
Stuart, thank you for being a great project advisor. We still
need to use your Hobie some time.
Molly, rubbing your head cures all.
your advice and gifts of animals were fabulous
Chuck,
Jim&a Rafe, you made 1129 Piedmont more than a house, you
made it a legend.
Sam, thank you for being their late at night and for all your
conversations.
Chris, your help with my talk and cheery demeanor were
great.
Jay & Ildiko & Adiwia, you made the lab a fun place to be.
175h classmates, you made this quarter fun and exciting.
Susan & Alan, you made the library fun, which is no small
task.
Overall, what a long, strange trip its been!
Literature Cited
Baxter, Charles H. and Pickens, Peter E. (1964). Control of
luminescence in hemichordates and some properties of a
nerve net system. J. Exp. Biol. 41.
Brehm, Paul. (1977). Electrophysiology and luminescence of an
Ophiuroid radial nerve. J. Exp. Biol. 71.
Bullock, T.H. (1965). Coelenterates and Ctenophora, in "Structure
and Function in the Nervous System of Invertebrates, Vol. 1'
(T.H. Bullock and G.A. Horridge), 460-466.
Gerhardt, Martin, Schuster, Heike, Tyson, John J. (1990). A cellular
automaton model of excitable madia including curvature and
dispersion. Science 247.
Herring, Peter J. (1974). New observations on the bioluminescence
of echinoderms. J. Zool. London 172.
Morin, James 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 kollikeri. Exp. Biol. 32.
Pantin
C.F.A. (1935a(. The nerve net of the Actinozoa. I.
Facilitation. J. Exp. Biol. 12.
Pantin, C.F.A. (1935b). The nerve net of the Actinozoa. II. Plan of
the nerve net. J. Exp. Biol. 12.
Pantin, C.F.A. (1935c). The nerve net of the Actinozoa. III. Polarity
and after-discharge. J. Exp. Biol. 12.
Robison, Bruce H. (1992). Bioluminescence in the benthopelagic
holothurian Enypniastes eximia. J. Mar. Biol. Ass. U.K. 72.
Smith, J.E. (1965). Echinodermata, in "Structure and Function in
the Nervous System of Invertebrates, Vol. 2" (T.H. Bullock
and G.A. Horridge), 1520-1531, 1548-1551.
Yee, Audrey, Burkhardt, James, and Gilly, W.F. (1987).
Mobilization of a coordinated escape response by giant axons
in the Ophiuroid, Ophiopteris papillosa. J. Exp. Biol. 128.
Young, Richard Edward (1983). Oceanic bioluminescence : an
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Figure Legend
Artistic rendition of Pannychia moseleyi
Schematic cross-section of Pannychia moseleyi.
Spiral wave, single frame.
Circular wave, single frame.
Traveling wave, accumulation of 45 frames.
Annihilation
a. Wave fronts of bioluminescence traveling
towards each other.
Wave fronts of bioluminescence intersecting.
c. Bioluminescence extinguished.
Unexcitable spots in bioluminescent wave.
Accumulation of 20 frames.
Unexcitable bands in bioluminescent wave.
Accumulation of 20 frames.
Single transection of a radial nerve. Green indicates
normal bioluminescence, red indicates disturbed
bioluminescence, x' indicates spot of
stimulation, and the bar indicates the
transection.
a. Ipsilateral stimulation.
b. Contralateral stimulation.
c. Transport stimulation.
10. Ipsilateral stimulation with double transection of a
radial nerve, single frame.
11. Contralateral stimulation with double transection of a
radial nerve, single frame. The cut is evident at
the bottom of the animal in the figure.
12. Double transection of a radial nerve. The legend is the
same as for figure 9.
a. Ipsilateral stimulation.
b. Tip stimulation.
c. Contralateral stimulation.
d. Transport stimulation.
13. Histogram of conduction velocity in normal sea water.
14. Correlation between conduction velocity and period.
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