Abstract:
The ability of cephalopods to hear has been controversial for some time (Moynihan
1985; Budelmann and Hanlon 1987). However, the organs specialized to detect tiny water
vibrations characteristic of sound have been identified (Budelmann 1992), and experiments
have been successful in showing that cuttlefish, Sepia officinalis, have the ability to
associate low frequency sounds (below 100 Hz) with negative stimuli such as electric
shock (Packard et al. 1990). In the following experiments, it was determined that
cuttlefish can hear within a frequency range of 9 Hz to 9260 Hz, and that cuttlefish possess
the ability to complete a complex task, combining the cognitive ability to associate a sound
stimuli with the presentation of food.
Introduction:
Sound is generally defined as the transmission of mechanical vibrations through an
elastic medium. In an aqueous environment, such acoustic energy results in rhythmic
waves of compression and rarefaction of water molecules, as well as, particle displacement
near the source (Kinne 1975). For cephalopods, perception of sound in the form of
hearing has been a controversial subject (Moynihan 1985; Budelmann and Hanlon 1987).
in part due to the difficulty in distinguishing between behavioral responses resulting from
tactile vibration and sound perception.
For mammals, the distinction is made simple by defining hearing as "the sense
modality concerned with the perception of sound entering the ears at low and moderate
intensities, as distinct from the perception of touch, vibration, and pain (Budelmann
1992). Such a definition would preclude cephalopods from having the ability to hear
since they lack the developed "ear-like" structure being cited. However, Budelmann and
Bleckmann (1988, see also Figure 1) describe the presence of epidermal lines similar to the
lateral lines of fish and aquatic amphibians. In Sepia officinalis there are eight lateral
lines" which extend from the dorsal surface of the head along the arms and contain
mechanoreceptive hair cells that detect local water displacements. Budelmann also
displayed that even tiny water displacements (2 um peak-to-peak displacement) along the
epidermal head lines generated microphonic potentials indicating that these hair cells are
indeed involved in the detection of water vibrations. However, another possible receptor
involved in sound detection is the statocyst receptor system. Unlike the hair cells, these
receptors are internal and greatly resemble the vertebrate inner ear. Statocysts are
equilibrium receptors and can act as linear accelerometers detecting particle motion, since
the entire animal vibrates with the water column during the production of sound
(Budelmann 1992). A clear possibility is that both systems are involved.
Several experiments have been conducted in which cuttlefish are shown to have the
ability to associate sound with a stimuli. In one instance, Sepia was conditioned to the
stimulus of sound vibrations (1 to 10 Hz) followed by mild electric shock. It was shown
that an association could be made, and in later experimental sessions, fluctuations in
breathing rhythms were observed to occur upon onset of the sound, before a shock was
delivered (Packard et al. 1990).
Although it thus seems that cuttlefish can detect sound stimuli, comparatively little
work has been done testing frequencies above 200 Hz or attempting to condition cuttlefish
to make à positive association between sound and a reward, such as food. In addition.
adaptation or habituation to a particular sound occurs fairly rapidly and results in the loss of
behavioral cues that indicate to the viewer that the sound is being heard. By conditioning
the cuttlefish to associate the sound with a reward, the occurrence of a response becomes
more reliable and frequencies and amplitudes may be altered to determine threshold hearing
ranges. This paper addresses these ideas through three basic questions: (1) Within what
range of frequencies and amplitudes can cuttlefish, Sepia officinalis, hear? (2) Can Sepia
be conditioned to sound? (3) Can Sepia successfully complete a complex behavioral task
that requires a higher level of cognitive association between sound and food?
Materials and Methods:
Animals: The experimental subjects included six cuttlefish, Sepia officinalis, (dorsal
mantle length ranging from 2-7 cm) provided by the National Center of Cephalopods,
Galveston, Texas and a large male cuttlefish (mantle length 23 cm) provided by the
Monterey Bay Aquarium.
Scanning Electron Microscopy: For SEM, one animal (20 mm mantle length) was pinned
with a fine, insulated insect needle in a petri dish and fixed in 2% OsOA (Zetterquist buffer,
pH 7.4) for 1 hour at 4°C. After fixation, the tissue was dehydrated in graded ethanol
solutions, critical point dried in a Balzers CPD 020 unit, gold/palladium coated with a
Hummer Il sputter unit (Technics, Alexandria, Virginia, USA), and viewed with a Hitachi
S240 scanning electron microscope (Pacific Grove, California, USA).
Behavioral Response Testing: In order to determine the frequencies at which cuttlefish are
most responsive, a Lubell Model 98 hydrophone transmitter was lowered into a large
circular tank (180 cm diameter, 57 cm deep, 12'C) along with a hydrophone receiver. As
depicted in Figure 2A, bursts of sinusoidal signals produced by a B+K Precision 3020
Sweep/ Function Generator and amplified by a linear power amplifier were used to drive
the submerged hydrophone. Both the stimulus sine wave and the output from the
underwater hydrophone receiver were inputted into a Hitachi VC-6025 digital storage
oscilloscope for sampling and comparison. The hydrophone output was also directly
recorded onto videotape with a Canon Al Digital 8 mm video camera used for filming
behavioral responses (shutter speed of 1/60th of a second).
Frequencies ranging from 9-20,000 Hz were tested and behavioral responses of the
large cuttlefish were recorded. The hydrophone produced short bursts of sound 1.15
seconds in length which occurred approximately every 2 seconds. Since adaptation to
particular tones was found to rapidly occur, frequencies were varied quite often. An
alteration in behavior upon onset of the tone was considered a behavioral response. Since
the hydrophone was suspended by a rope, reaction to the sound was assumed to result
from fluid motion rather than tank vibration.
Conditioning Experiments: Two classical conditioning paradigms requiring different
cognitive capabilities were designed in order to provide an independent test of hearing
capabilities and to allow determination of threshold levels that would reliably prompt a
specific behavior.
The first conditioning experiment tested for an association between fifteen 180 Hz
pulses of sound followed by the presentation of a crab. Three cuttlefish were placed in a
plastic tank (4Ox 54 x 17 cm) with two 120 V fluorescent lights running along the sides of
the tank. A four inch loud speaker was mounted on the external surface of the back wall of
the tank; bolted and secured in place with silicon. This attachment allowed transmission of
180 Hz vibrations through the tank sides into the seawater. To control for tactile
stimulation resulting from vibration of the tank, trials in which the cuttlefish were touching
the sides or bottom of the tank were disregarded in the analysis.
Each experimental session included three trials in which fifteen 180 Hz pulses 1.15
seconds in length were followed, approximately 5 seconds later, by the presentation of a
crab through a long opaque tube. The entire set-up was enclosed by a black plastic drape
to exclude visual stimuli. Each trial was recorded on video, through remote camera
operation, and the tapes were later analyzed for behavioral responses.
The second type of conditioning experiment had the goal of determining whether
Sepia could associate a complex conditioned response with the presentation of sound.
Whereas in the previous experiment behavioral responses were used as indicators that the
sound was heard; this experiment required the cuttlefish to learn a task, associate it with a
sound, and then carry out the task upon initiation of the sound.
Two animals were used for this test. They were placed in a glass tank (SOx19x15
cm) and maintained at an approximate temperature of 14'C. A clear Plexiglas barrier was
positioned at a 45' angle to the walls in one corner partially sectioning off that corner of the
tank but allowing a passageway between the barrier and one wall (see Figure 2B). Black
plastic netting (0.8 cm mesh size) was glued on the surface of the Plexiglas to provide
texture and a visual cue as to where the barrier ended. The opaque tube used for the
introduction of crabs into the tank was positioned in the comer behind the barrier. The
hydrophone used in this case was made from a four inch stereo speaker secured tightly
within a Tupperware container, and held on the surface of the water.
Initial sessions consisted of fifteen 180 Hz pulses (1.15 seconds in length) being
followed after 5 seconds with the presentation of a crab behind the barrier. During this
period, the crab could be seen through the netted barrier, so the cuttlefish could associate
the sound with the visual stimulus of food and the task of maneuvering around the barrier.
After 25 sessions it was clear that Sepia had mastered this task, and the barrier was
covered with black, opaque plastic. Any resulting movement by the Sepia around the
barrier upon initiation of the sound could, therefore, only be a result of the sound stimulus
and the learning associated with it.
Results:
Scanning Electron Microscopy: By using scanning electron microscopy to examine the
dorsal head and arm region of the cuttlefish, the epidermal head lines characterized by
Budelmann and Bleckmann (1988) were viewed. Each line consisted of a groove
containing hair cells that possessed many kinocilia (up to 12 um in length). The hair cells
did not form a continuous line; single hair cells and groups of hair cells occurred in
alternation (Sundermann 19883).
Behavioral Responses to Sound Presentation: Although a range of frequencies from 9 Hz
to 20,000 Hz was tested (limited by the capabilities of the hydrophone), responses were
only observed for frequencies between 9 and 9260 Hz. All responses were divided into
two categories; startle and non-startle behavioral responses. Startle responses include color
flashes, head retractions, or increasing fin beats and occur immediately (within 3-8 video
frames) at the onset of the first sound pulse. Non- startle behavioral responses are
distinguishable from startle responses in that the reactions are more prolonged, appear to be
controlled, and are not subject to habituation. Such responses include rotation or motion
toward the source of sound production, fin beating in synchrony with sound pulses,
avoidance responses, and color changes.
High amplitude tones over a large frequency range were successful in eliciting
startle responses (see Figure 3), and a "threshold" level exists at an intensity of
approximately 5.5 V, above which all responses occur in connection with a startle
response. The most common reaction to such high amplitude sounds was a slight drawing
in of the tentacles, flaring of the fin, and retraction of the head.
Low amplitude signals produced startle responses only occasionally, but a variety
of continuous behavioral responses were produced, again with sounds of a wide frequency
range. An unusual reaction was produced to a stimulation of 9 Hz. On six separate
occasions, the cuttlefish turned or proceeded in the direction of the hydrophone upon
initiation of the 9 Hz tone.
Conditioning Experiments: The first conditioning experiment attempted to determine
whether Sepia would display excitatory behavior during sound production in anticipation
of food. The resulting responses are summarized in Figure 4 and correspond to the
reactions that preceeded the presentation of the crab. Color changes were the first
continuous behavioral responses to be observed and were normally seen as a darkening of
the chromatophores throughout the body or tentacles, but the incidence of color change
decreased with the number of trials. Fin beating, defined as an increase in beating rate or
beating in synchrony with the sound pulses, also was regularly displayed after
approximately 10 trials. Rotation or movement toward the tube before the introduction of a
crab increased with the number of trials. The final factor used to deduce anticipatory
behavior was the positioning in an attention posture (Messenger 1973), with two tentacles
raised. This behavior is normally seen during preparation for prey attack. It was virtually
absent during the first fifteen trials, after which its occurrence became more regular.
Figure 5 shows the percentage of trials, out of the total number of trials, in which each
benavioral response occurred. Changes in the rate of fin beating was seen most frequently,
in 51 percent of the observations.
The second conditioning experiment was more complex and required the cuttlefish
to not only associate the sound with a reward (crab), but also to complete a task in order to
obtain the reward. The task was considered to be successfully completed only if the
cuttlefish displayed preparatory behavior by moving behind the barrier before the crab was
introduced. To discount the possibility of reaction towards other stimuli (including
vibration caused by the entrance of the crab into the water, chemical stimuli, etc.), trials in
which such movement occurred after the crab was dropped into the tube were not
considered successes. Figure 6 shows that the cuttlefish became fairly competent in
obtaining the crabs early in the training period. This was accomplished by either moving
around the barrier after the crab was presented or waiting until the crab had moved into
view in the main tank compartment. Successful task completion did not occur until the
fourteenth trial, but occurred rather frequently thereafter (7 out of 19 trials)
Discussion:
Despite testing over a wide range of frequencies between 9 and 20,000 Hz at
amplitudes up to 8.5 V, no responses of any kind were seen above 10,000 Hz, and
responses above 5,500 Hz were rare. This indicates that, while the hearing range is
broader than previously known (Packard et al. 1990), the highest occurrence of responses
occurred at relatively low frequencies. In humans, sensitivity is much greater for
frequencies that correspond to the human voice (approximately 1000 Hz). Similarly, in
cuttlefish, there may be an adaptive value of hearing low frequencies characteristic of prey
or predator tail beats or other sounds associated with movement. In addition, sensitivity to
low frequencies could be useful for communication. Plainfin midshipman fish, Porichthys
notatus, produce low frequency sounds by contraction of the intrinsic muscles of the
swimbladder (Cohen and Winn 1967). A specific frequency range of 98-108 Hz has been
shown to elicit male-searching behavior in gravid females (Ibara et al. 1988), indicating
that production and reception of such sounds is essential in communication. Figure 3
displays a fairly distinct behavioral response line at 5.5 V, above which all non-startle
behavioral responses occurred in association with a startle response. Thus, an amplitude
of 5.5 V could possibly represent the threshold for physiologically relevant sound
intensities in nature, suggesting that intensities above this point are not normally
experienced. To determine whether such an assumption is true, studies would have to be
undertaken which examine the sound intensities and frequencies characteristically produced
by oceanic species.
Sepia also displayed the ability to locate the source of sound production. In the
set-up, two foreign objects, the hydrophone and the hydrophone receiver, were introduced
into the tank. On nine separate occasions, the cuttlefish displayed movement or rotation
towards the hydrophone upon initiation of sound. However, there were no similar
reactions toward the hydrophone receiver suggesting that cuttlefish can detect the
directionality of sound production and can quite accurately locate its source. In addition,
the fact that Sepia displays a particular sensitivity to low frequencies and can associate
sound with food raises the question of whether such an ability is useful during prey
capture. For this to be true, the cuttlefish would have to associate a particular frequency
with the prey. The results of this experiment clearly display that such an association is
possible. Referring once again to Figure 4, the increasing incidence of preparatory
activities such as rotation or movement toward the tube or assumption of the attention
position, with tentacles raised, indicates successful learning; associating sound with prey.
A somewhat unusual result was the decline in frequency of color changes. Since visual
color displays are used for intraspecific communication in Sepia (Tinbergen 1939), a
possible explanation for this decrease in color changes arises from the fact that the three
animals resided within one tank. There was, therefore, competition for the food since a
single crab was presented during each trial. Color changes not only indicate to the viewer
that the sound was heard, but could also conceivably arouse the interest of the other
animals. Therefore, it is in the best interest of the cuttlefish to control such blatant
responses, increasing their own chance of obtaining the prey. A single animal in a tank
would be a useful control in answering such a question.
The goal of the final experiment was to determine whether cuttlefish could master a
task demanding higher learning capabilities which required sound discrimination.
Proficiency in completing the task required a level of cognition beyond the simple
association between sound and food displayed in the previous experiment. For successful
execution of the task, the cuttlefish had to make the connection between sound, food, and
movement around the barrier. As seen in Figure 6, motivation, defined as the desire to
obtain food either by procession around the barrier before or after crab presentation or by
waiting for the crab to escape, remained fairly constant throughout the experiment. This is
important in showing that, although the interest was present at the start of the experiment.
fourteen trials were required before the cuttlefish learned to successfully complete the task.
The relatively high number of successful responses after trial fourteen signifies a clear
ability to leam a complex task while making the association between sound and food.
Thus, the questions posed at the start of the investigation were answered.
Cuttlefish can hear within a wide range of frequencies, displaying particular sensitivity to
low amplitude, low frequency sounds. In addition, cuttlefish possess the ability to leam a
complex task through association of sound with a reward.
Acknowledgments:
T am grateful to Dr. William Gilly, Professor of Neurobiology at Hopkins Marine Station,
for being supportive throughout the project, for calming my frustrations, and for being an
inspiration as a creative research scientist. My thanks, also, to Dr. Thomas Preuss for his
enduring enthusiasm for the project, constant support, and numerous trips to the hardware store.
In addition, I would like to thank Dr. Jim Watanabe for use of his hydrophone and for his undying
dedication to our class as a whole, Dr. Mark Denny for his help in coming up with creative
solutions for an underwater sound device, John Lee for his electrical expertise in building the
sound devise, and the Weissmann lab for our use of their tanks.
References:
Budelmann, B.-U. and Hanlon, Robert T. February 1987. Why Cephalopods Are
Probably Not "Deaf". The American Naturalist. Vol. 129; No. 2; p. 312-317.
Budelmann and Bleckmann. 1988. A lateral line analogue in cephalopods: water waves
generate microphonic potentials in the epidermal head lines of Sepia and
Lolliguncula. Journal of Comparative Physiology A. Vol. 164; p. 1-5.
Budelmann, Bernd U. 1992. Hearing in Nonarthropod Invertebrates. The Evolutionary
Biology of Hearing. Springer-Verlag, New York. Ch. 10; p. 141-155.
Cohen, M.J. and Winn, H.E. 1967. Electrophysiological observations on hearing and
sound production in the fish, Porichthys notatus. Journal of Experimental
Zoology. Vol. 165; p. 355-370.
Ibara, Richard M., Larry T. Penny, Alfred W.Ebeling, Gilbert van Dykhuizen, and Gregor
Cailliet. 1983. The mating call of the plainfin midshipman fish, Porichthys
notatus. Predators and prey in fishes. p. 205-212.
Kinne, O. 1975. Marine Ecology: A Comprehensive, Integrated Treatise on Life in
Oceans and Coastal Waters. John Wiley and Sons; Vol. II, Part 2; p. 740-742.
Messenger, J.B. 1973. Learning in the cuttlefish, Sepia. Animal Behavior. Vol. 21;
p. 801-826.
Moynihan, Martin. March 1985. Why are Cephalopods Deaf? The American Naturalist.
Vol. 125; No. 3; p. 312-317.
Packard, A.; Karlsen, H.E.; Sand, O. 1990. Low frequency hearing in cephalopods.
Journal of Comparative Physiology A. Vol. 166; p. 501-505.
Sundermann, G. 1988. The fine structure of epidermal lines on arms and head of
postembryonic Sepia officinalis and Loligo vulgaris (Mollusca, Cephalopoda).
Cell Tissue Res. Vol. 232; p. 669-677.
e
Tinbergen, L. 1939. Zur Fortpflanzungsethologie von Sepia officinalis. L. Archives
Neerlandaises de Zoologie. Vol. 3; p. 323-364.
Figure 1:
A. Scanning electron micrograph of the dorsal head and arms of the cuttlefish,
Sepia officinalis. The lateral lines containing hair cells can be seen running from the head,
down the arms.
B. A single ciliated hair cell within the lateral line channel.
Figure 2:
A. Set-up used in determining the frequency and amplitude ranges for cuttlefish,
Sepia officinalis.
B. Set-up used in the second conditioning experiment, displaying the barrier that
the cuttlefish had to proceed around in order to obtain the food.
Figure 3:
Responses to frequencies between 9 and 10,000 Hz and amplitudes of 8V to
8.2V. Startle responses refer to those reactions that occur during the first burst of sound.
Behavioral responses involve more sophisticated reactions to sound including; rotation or
movement toward the source, fin beating in synchrony with sound pulses, escape
responses, or long term color changes.
Figure 4:
Distribution of responses during conditioning to 180 Hz. Color changes, increased
or synchronous fin beating, rotation or movement toward the tube and assumption of the
attention position, with two tentacles raised, were all considered behavioral responses to
the sound pulses. Rotation or movement toward the tube and raising of the tentacles are
the clearest indicators that an association between sound and the presentation of food has
been made.
Figure 5:
The percentage of trials in which particular behavioral responses to 180 Hz sound
pulses were observed.
Figure 6:
c
T'ime course for learning experiment. Successful task completion involves only
those trials in which Sepia traveled around the barrier before the crab was presented.
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