ABSTRACT
Few observations have been made on Atolla, a bioluminescent
deep-sea coronate scyphomedusa. With the use of a remotely
operated vehicle and a specially designed plankton-kriesel, Atolla in
the Monterey Bay have been videoed in situ, captured, and observed
in lab to learn more about their behavior. A characteristic response
to stimulation with light is described called the light reflex. The light
reflex consists of a number of quick pulses and/or the medusa
turning belly-up. Once in the belly-up position, Atolla rarely reacts
to any further stimulus. In addition, scanning electron microscopy
and light microscopy were used to take a preliminary look at the
nematocyst composition of the trailer tentacle compared to the many
short tentacles.
e.
INTRODUCTION
In the past, because Atolla have generally been found between
500 and 800 meters depth, they have been relatively inaccessible for
behavioral studies. Even early anatomical descriptions of Atolla
were incomplete because the animals were caught in trawl nets
which ripped off their tentacles. In more recent studies of the
bioluminescent responses of Atolla, Herring noted that:
the observed physiological responses of oceanic animals
that have been caught in trawl nets rarely reflect the
true capabilities of these animals in situ. Better methods
of capture, maintenance and in situ observation are
needed in order to appreciate the full range of responses
which oceanic animals can employ and the circumstances
in which they are normally elicited. (Herring 1990)
Therefore, this study was conducted with a remotely operated
vehicle (Etchemendy and Davis 1991) both to view Atolla of
Monterey Bay in situ and to carefully capture it alive and intact for
further lab studies. These live animals were maintained in a
plankton-kriesel. This study has revealed that Atolla behave
differently in different types of light and that they share a similar
reflex, described as the "off-response" (Singla 1974; Arkett and
Spencer 1986), with many hydromedusa.
MATERIALS AND METHODS
The Monterey Bay Aquarium Research Institute's remotely
operated vehicle (ROV) was used to view Atolla in situ. The ROV
provided video footage of Atolla under bright white lights and in
turbulent waters.
Thirteen Atolla wyvillei Haekel, 1880 and Atolla vanhoeffeni,
Russell, 1957 were brought back. One was collected with the suction
sampler on the ROV, the other twelve were collected in detritus
samplers on the ROV. These animals were collected at depths
ranging from 350 meters to 595 meters. Because of the delicate
structure of these creatures, species identification was difficult and
all specimens were referred to as Atolla. All specimens were kept in
à specially designed plankton-kriesel with a constant current, at 6.5
dégrees Celsius (+ 5 degrees), and with a salinity concentration
maintained at 34.5 parts per thousand (+- 1 part per thousand)
Red Verses White Light
The first two specimens were observed and studied in white
light. The remaining eleven were observed in a room lit with six
one-hundred watt red lightbulbs. The red light was adjusted with a
rheostat and all observations were recorded under two different red-
light conditions; either full intensity or half intensity. When not
being studied, these animals were kept in darkness except when
other people were working in the coldroom. The last eight Atolla
were kept in the same conditions except black plastic was used to
prevent any white light from reaching them unless it was an
intentional part of an experiment.
Thin White Beam With Red Background
To observe reactions of Atolla to white light, specific body parts
were stimulated. These include the trailer tentacle, short tentacles,
bell, and bell margin. This beam originated from two different
flashlights; one averaging 8.86 mE/m2/s and the other averaging
9.48 mE/m2/s when measured twelve centimeters away from the
light source. Each was taped up with many layers of duct tape,
allowing only a pinhole of light to shine through the tape.
Full Flashlight With Red Background
A full white flashlight was sometimes used both directly and
indirectly to observe the behavior of Atolla when moving shadows
were created in the kriesel and to observe how Atolla react to a
sudden, full beam of white light.
Unlit Room
A night vision monocular (Litton M982) was used to gather
information on Atolla kept in the darkness.
Mechanical Stimulation
Mechanical stimulation was used to catalog reactions of Atolla
to stimuli other than light.
Indirect mechanical stimulation: involved altering water conditions
around Atolla. Eddies were created by stirring up the water in the
kriesel. More subtle current changes were generated by using thin
rubber tubing to gently blow bubbles near an animal.
Direct mechanical stimulation; involved using a glass rod to
manipulate Atolla directly. Observations were recorded for all
experiments.
Feeding Experiments
A variety of potential prey items were introduced into the
plankton-kriesel and any interactions were recorded over two to
seven hour intervals spanned over one to four days. Adult and
larval Artemia, Beroe, Aurelia, Nanomia, Pleurobrachia and gelatin
were all items used.
For nearly all the feeding experiments, only one type of
possible food was offered at a time. However, one experiment was
conducted on four Atolla in which both Pleurobrachia and larval
Artemia were allowed in the kriesel simultaneously. Twelve hours
later a tiny Beroe was added. Twenty-seven hours after the Beroe
entered, clear gelatin was added to the kriesel. Nearly another day
passed, and then more larval Artemia were added. After a few
hours, ten adult Artemia were added. Live animals were used as
prey and they were placed in the kriesel at least seven hours after
Atolla were caught. In some cases, Atolla would remain in captivity
many days before ever having an opportunity to feed.
Scanning Electron Microscopy (SEM)
Preparation of tentacle for SEM:
Small portions of
tentacles (about 1 cm long) were cut from a live, newly captured
Atolla. These specimens were preserved in three percent
gluteraldehyde and kept refrigerated. Further processing took place
within thirty-six hours. The tentacles were then washed with
seawater for five minutes, after which they were placed in 1%
osmium tetroxide for one hour. No buffers were used. Dehydration
with progressively increasing concentrations of ethanol began by
doing two rinses at 30% for five minutes each and then continuing
with washes of 40%, 50%, 60%, up to 70% at which point the
specimens could be refrigerated for up to twenty-four hours.
Dehydration continued at 80%, 90%, 95% and ending with two 100%
washes. The tentacle samples were mounted on stubs, coated with
gold, and then viewed with the Scanning Electron Microscope (Hitachi
S450, 15,000 volts).
Nematocyst Isolation
Protocol (derived from Weber 1987): Whole tentacles or large
portions of tentacles were severed from live jellyfish and washed in
cold distilled water. Keeping each tentacle in separate containers,
excess water was removed from them before they were frozen.
Further processing occurred within thirty-six hours. Each tentacle
was then thawed and homogenized with a pasteur pipette in a cold
solution of distilled water containing fifty percent Percoll. These
homogenates were placed on ice for thirty minutes and then
centrifuged for ten minutes at 5,000 rpm. The liquid, debris and
fragments were removed from each container and the nematocyst
pellets (which were often too small to see with the naked eye) were
frozen for less than thirty-six hours before being resuspended with a
drop or two of distilled water.
Light Microscopy
Squash preparations of the tentacles, nematocyst isolations, and
debris from the isolations were examined with an inverted
compound microscope (Axiovert 10) at 100, 200, and 400 times
magnification.
RESULTS
Video Footage
When viewed by video with the ROV, Atolla generally seemed
to pulse rapidly, often actively swimming away from the ROV. Atolla
was also documented to stop pumping, and would roll over into what
is designated as the "belly-up position. In this position the jellyfish
rarely pumps, but rather drifts with the current and allows its
tentacles to pile on top of its belly (Figure 1).
Collections
Out of the thirteen Atolla captured, seven were red and six
were white. The red jellyfish tended to be more active and to react
more vigorously to different stimuli. In captivity, the red jellyfish
lived longer than the white jellyfish (Figure 2). Also, the red jellyfish
tended to lose their pigment gradually from the moment of capture
until death.
=-Lab Observatioms=-
White Light
When viewed in constant white light, Atolla consistently cycles
around the kriesel in the belly-up position (Figure 3). If kept in the
full white light long enough (about twenty minutes) Atolla may
partially spread out its tentacles (Figure 4).
Red Light
When viewed in red light Atolla tend to stay upright,
alternating between actively swimming and drifting in circles around
the tank (Figure 5). Only animals in extremely poor condition, which
have been in captivity many days (over seven days), drift in the
belly-up position when viewed in red light.
Unlit Room
When viewing the jellyfish in an unlit room with a night-vision
monocular, Atolla behaved in the same way as it did under the red
light.
Thin White Beam
Shining a thin white beam on different parts of Atolla produced
varying responses. When the thin beam was directed at the bell
margin and tentacle-base region of an up-right Atolla, the jellyfish
would tend to turn belly-up (Figure 6) . Prior to assuming this belly
up position, Atolla would perform a series of actions such as rapidly
pumping, swimming away from the light source, darting all over the
kriesel, and finally rolling over. I have referred to this series as "the
light reflex, and have summarized it as "a number of quick pulses
and/or turns belly-up." Two out of the twenty-nine times
stimulated, a ring around the margin of Atolla bioluminesced before
turning belly-up. The light reflex occurs at different intensities for
different individuals. The red Atolla was more vigorous in all its
responses compared to the white. The white jellyfish was never
observed to bioluminesce and rarely darted around the kriesel.
When the beam was directed at the tentacles, the tentacles
were pulled in towards the bell and/or Atolla turned belly-up,
twelve out of twenty-four times (Figure 7). A number of quick
pulses were never associated with the thin beam directed at the
tentacles. The red Atolla reacted in this way more than twice as
often as the white Atolla. Shining the thin beam on any of the other
body parts did not seem to result in any consistent reactions.
Full Flashlight
When Atolla were flashed with bright white light for a few
seconds, they would often perform the light reflex; either turn belly¬
up or dart away from the light source and then turn belly-up (Figure
8). This response was recorded so often and was so predictable that
Atolla could be "steered" to any specific area in the kriesel by
"chasing" it with the beam.
Moving Shadows
When Atolla was subjected to shadows created by white light,
it nearly always responded by performing the "light reflex" (Figure
9).
Sudden "Lights Out'
Many of the times that Atolla did not react to the flashlight
shining on them, they would react when the white flashlight was
turned off again. The animals would portray the characteristic "light
reflex," when the light turned off rather than when it was turned on
(Figure 10). Three out of the thirteen stimulations resulted in Atolla
bioluminescing around its margin.
Manipulations Once Animal Is Belly-up
If Atolla was already in the belly-up position it would not alter
its behavior unless stimulated to an unusually high degree. Stirring
the water in the kriesel was almost invariably followed by more
active swimming unless Atolla was already belly-up before the
disturbance began. Sometimes the jellyfish would turn belly-up in
response to stirred waters. Blowing bubbles through thin tubing also
resulted in more active swimming and pumping by Atolla, but was
never associated with the belly-up position. Touching the animal
often produced no noticeable response, unless the rod was touching
the area at the base of the tentacles by the bell margin. In this case,
three out of three times the jellyfish would pump away quickly and
turn belly-up. Furthermore, using the rod to move Atolla through
the water ended with the jellyfish turning belly-up. None of these
mechanical experiments resulted in bioluminesence. Recovery back
to active, up-right swimming took a variable amount of time and
required an end to all extra stimuli. This recovery occurred more
rapidly for Atolla in good condition than for Atolla in poor condition.
In fact, once these jellyfish have been in captivity for five or six days
and are beginning to appear unhealthy, they tend to remain in the
belly-up position more often than the up-right position.
Feeding
Atolla was never seen definitely using its tentacles to feed.
Two adult Artemia did drift into the mouth of one Atolla who
proceeded to swallow them. Additionally, at least three different
Atolla were seen putting one to four tentacles in their mouths when
larval Artemia were also in the kriesel. It could not be determined,
however, whether there were any larval Artemia on the tentacles
being used. Atolla were never seen placing their tentacles in their
mouths when larval Artemia were not in the kriesel. Other than
these observations, the jellyfish did not react noticeably to contact
with the other items offered.
Scanning Electron Microscopy
Differences could be discerned between the trailer tentacle and
the short tentacles when viewed under 400 to 10,000 times
magnification. A fine connective net tissue covered both types of
tentacle. Nematocysts were intertwined within that tissue, one of
which was found to have discharged (Figure 11). The connective
tissue and nematocysts on the trailer tentacle were finer and smaller
than the short tentacles, requiring more magnification to discern the
same amount of detail
Nematocyst Isolation
Both discharged and non-discharged nematocysts were
examined after isolation. Nematocysts averaging 10.5 to 11.0
microns diameter occurred in the long tentacle (Figure 12). In
contrast, nematocysts from the short tentacle were in much higher
concentration and consisted of both the 10.5 to 11.0 micron size
range and the 23 micron size range (Figure 13). They can be
identified as anacrophores and holotrichous isorhizas, respectively
(Purcell 1984).
DISCUSSION
These results demonstrate that Atolla has a stereotypical
response to both light and mechanical stimulation, namely pulsing
quickly a number of times and/or turning belly-up. Possible
reasons for behaving this way may be associated with interactions
between other deep-sea bioluminescing animals. For instance,
pushing a non-bioluminescing Beroe into the tentacles of Atolla
produced no response in Atolla three out of three times. However,
on the fourth manipulation, Beroe bioluminesced as it contacted
Atolla, and Atolla responded very vigorously with the entire series of
the characteristic light reflex, which included bioluminescing. It is
possible that mechanical manipulation of Beroe induced its
bioluminescent response as an alarm reflex, which in turn caused
Atolla to also perform an alarm reflex. Thus, the bioluminescence of
one creature may serve as a signal or warning to other light-
sensitive creatures that danger may be near. Further studies in this
area are needed before any solid conclusions can be drawn.
Results from the thin beam experiment show that the bell¬
margin region and the tentacles were the most sensitive to light.
This correlates well with Arkett and Spencer's study on
hydromedusae which suggests that the ocelli, structures located near
the bell margin at the base of each tentacle, are the primary
photoreceptors (Arkett and Spencer 1986).
The results from the sudden "lights off" experiments
demonstrate that Atolla responds predictably by quickly pulsing a
number of times and/or turning belly-up. Whenever there was a
reaction, more active pumping was involved; however, only a small
percent of the reactions involved the belly-up position. This
response to rapidly diminishing light intensity suggests that deep¬
sea scyphomedusae, such as Atolla, share a similar reflex with many
shallow water hydromedusae, such as Polyorchis penicillatus , which
have a predictable "off-response" (Singla 1974). This "off-response
consists of an initial swimming contraction, followed by a series of
rapid successive tentacle contractions, finishing with several more
swimming contractions (Arkett and Spencer 1986). Such behavior in
shallow water medusae can be interpretted as a shadow reflex.
These medusae are normally exposed to sunlight but when an animal
swims above them a shadow is created. This type of shadow could
be caused by approaching predators and may be why the shallow
water jellyfish respond to rapidly diminishing light intensity.
However, this behavior is not so easily explained for deep-sea
medusae. Generally, only minimal amounts of light penetrate to 500
meters. Still, that may be enough light for the ocelli of Atolla to
detect. In that case, Atolla would be sensitive to very subtle changes
in light intensity. Possibly, the reasons for an off-response in deep-
sea jellyfish is based on different principals than the shallow water
jellyfish. Atolla may be reacting to alarm signals given by other
bioluminescent animals. Further research on this topic could lead to
a better understanding of the off-response in deep-sea medusae.
All these experiments combined, reveal that once in the belly
up position, Atolla rarely reacts with any further stimulus. This
suggests that the belly-up position may be a stress or alarm
response. By tucking in its tentacles and pulsing very infrequently,
Atolla avoids disturbing the surrounding waters, thus supplying
near-by creatures with fewer clues of its location. Additionally, dead
animals and debris are constantly drifting down through the water
column. Atolla may appear more like a dead animal or sinking
debris when assuming the belly-up position. This could deter
possible predators from choosing to eat Atolla.
The condition of the animal has been shown to be important in
that unhealthy animals or animals stressed by remaining in captivity
too long respond less often and less predictably than animals in good
condition. Because red Atolla respond more often and more
predictably to stimuli than the white Atolla, there may be a
correlation between the color of the jellyfish and its health. Atolla
which were red upon capture were noted to gradually lose their
pigment while in captivity. Further research in this area would be
very helpful.
Closer studies of the tentacles and of isolated nematocysts with
SEM and light microscopy revealed differences in the nematocyst
populations of the trailing tentacle as compared to the short
tentacles. Further research distinguishing differences between the
two tentacles and the significance of the trailer are much needed.
Though special care was taken to minimize stress to these
animals and to simulate a deep-sea environment as well as possible
methods could still be improved. When observing and collecting
Atolla with an ROV, red lights should be tried. Their sensitivity to
white light and their fragility suggest the importance of transfering
jellyfish into their kriesel with as little handling and disturbance as
possible.
ACKNOWLEDGMENTS
First and foremost I must thank George Matsumoto for taking
me under his wing: spending hours of his time guiding me in lab
techniques, loaning source material, and advising me on my
presentation. Warm thanks to Annie Reese, my jelly-twin, who
shivered by me as we observed our animals in the coldroom and
kept me up when Raison and Pickle were getting me down. My
advisors Chuck Baxter and Bruce Robison, have been a tremendous
support and source of inspiration. Hey, Robie, what more must I do
to go down in Deep Rover? I also need to thank Kim Reisenbichler
for putting up with us in the coldroom he designed. Thanks to Chris
Patton and Tom Schroeder for help with SEM. Gil VanDykheusen and
Fraya Sommer, I appreciate your gelatinous gifts. Thanks to Lynn
Lewis and the entire staff at MBARI. Additional thanks to Alan
Baldridge, Mark Denny and the entire Hopkins staff. This project
could not have happened without the help and humor of Brian

Ackerman, Dave Bracher, Jon Consiglio, Craig "1.C. Darve, Steve
Etchemendy, Chris Grech, Roger Hayes, Greg Maudlin, Jim McFarlane,
Mark VandenBerg and Bill Wardle. Moral support from my
housemates (all six of them) was fuel for this project.
LITERATURE CITED
Arkett SA, Spencer AN (1986) Neuronal mechanisms of a
hydromedusan shadow reflex. I. Identified reflex components and
sequence events. J Comp Physiol A 159: 201-213
Etchemendy S, Davis D (1991) Designing an ROV for oceanographic
research. Monterey Bay Aquarium Research Institute
converted an oil-field ROV into a scientifically usable tool. Sea
Technology: 21-24
Herring PJ, (1990) Bioluminescent Responses of Atolla. Mar Biol 106
(3): 413-417
Purcell JE, (1984) The functions of nematocysts in prey capture by
epipelagic siphonophores (Coelenterata, Hydrozoa). Biol Bull
166: 310-327
Singla CL (1974) Ocelli of hydromedusae. Cell Tissue Res 149: 413-
429
Weber (1987) Some physical and chemical properties of purified
nematocysts of Hydra attenuata Pall. (Hydrozoa, Cnidaria). Comp
Biochem and Physiol 88B: 855-862
THE BELLYUP POSTTION
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