ABSTRACT
Chemical cues from macrocystis and mysid are capable of
triggering behaviors in the nudibranch mollusc, Melibe leonina
The behaviors ellicited are appropriate to the stimulus. (Le.
Melibe locomote in response to macrocystis, and exhibit feeding
behavior in response to mysid.) These behaviors are not seen
when the animals are in an isolated environment or are
stimulated solely by touch or vision. Furthermore, Melibe exhibit
the ability to integrate chemical cues. while neither brine shrimp
nor kelp alone will inspire them to feed, brine shrimp combined
with the smell of kelp provides the threshold stimulus necessary
to begin feeding. Rhinophore ablation indicates that the
rhinophores are the chemoreceptive organs responsible for the
detection of these cues.
INTRODUCTION
Chemoethology is the effect of chemical stimuli on behayjor. The
two principal forms of chemoreception are olfaction and
gustation. There are numerous ways of differentiating between
the two: necessary concentrations for detection, solubility.
volatility, organs for detection, and distance. In this paper.
distance is the criterion used to distinguish between the two.
Smell is distance chemoreception, while taste is touch
chemoreception.
Underwater, olfaction is one of the most useful senses. Highly
volatile compounds such as amino acids, which on land can only be
tasted, are water soluble. This allows them to impart important
chemical messages in the water. Chemical stimuli are especjally
useful because they occur in large quantities, can be detected in
either light or dark, and can impart highly specific messages,
despite needing simple receptors. (For review see Croll, 1983
and Ache, 1987) Chemoreception has been shown to play a role in
initiating many behaviors in molluscs. Chemoreception has been
shown to be important in homing (Gelperin, 1973), feeding (Frings
and Frings, 1965), and localization of conspecifics (Audesirk and
Audesirk, 1977).
Much of the research done with chemoreception has used amino
acids as stimuli. (Agersbourg, 1922; Bicker et al, 1982; Jahan¬
Parwar, 1972) However, for the purpose of this experiment,
used stimuli that are found in the natural environment of the
Melibe. This allowed for the targeting of behaviors which are
triggered by natural stimuli. Although amino acids are the most
probable source of smell, the ability of an animal to detect both
the unique combination of amino acids and concentrations of
amino acids in a specific stimulus provides more insight into the
role of smell in the natural environment than would experiments
employing isolated and or increased concentrations of amino
acids. Behavioral studies were performed to determine which
stimuli to study and to determine which behaviors can be¬
triggered by chemoreception.
The rhinophore is the principal organ which has been indicated in
chemoreception in numerous nudibranchs. In Melibe leonina, a
rhinophore of between Amm and 6mm in length is located within a
small pocket of tissue on the tip of each of the rhinophore bearing
processes (rbp). (Figure 1) The rhinophore is highly convoluted;
therefore, it has a large surface area. This allows for an
increased area with which it can contact different water borne
molecules. In an active animal the rhinophore is exposed;
howeyer, when an animal is stressed it retracts the rhinophore
into the pocket. The rhinophore is innervated by a nerve (C4)
which leads to the tentacular lobe of the central ganglion. (Hurst,
1968). Unlike the other three cerebral nerves which innervate the
oral hood, C4 has no interconnections with other cerebral nerves.
This implies that information from the rhinophore is carried
directly to the brain without integrating information from other
systems. This paper seeks to define specific behaviors which are
triggered by chemical cues, and to locate the anatomical basis for
the detection of these cues.
MATERIALS AND METHODS
Collection and Maintenance
Melibe leonina were collected from the kelp forest near Del Monte
Beach in Monterey, California at depths between 10 and 35 feet.
The Melibe were contained in tanks with an open flowing sea
water system at 100-15°C. Macrocystis and a variety of snails
were added to provide the Melibe with a natural and clean
environment. Melibe were fed either mysids collected by dip net
in the Hopkins Marine Life Refuge or commercially grown brine
shrimp every other day.
Chemical detection
Melibe were placed in specially designed tanks to test
chemoreceptive ability. (Figure 2) The tanks were 15-liter
Plexiglass“ tanks. Plexiglass"" dividers were caulked in the
center to divide the tanks into two compartments. Two holes of
6.3cm in diameter were drilled in the dividers, and Nytex screen
was caulked over the holes to allow passage of water and
chemical stimuli through the tank. Seawater between 110 and
15°C flowed freely into the compartment in which the stimulus
was contained. Melibe were placed in the other compartment and
were allowed to acclimate for at least six hours before a
stimulus was added. The water drained from the tank through a
standpipe in the same compartment as the Melibe. Only one
animal was placed in each tank for each trial, and the tanks were
washed with fresh water between trials.
Baseline behavioral observations were taken with no stimuli to
determine the behavior of the Melibe in a sterile environment.
Macrocystis and mysids, the stimuli used, were chosen because
they serve as the preferred location and food source,
respectively. To study the chemoeffective properties of
macrocystis, two fronds of macrocystis, each with a length of
55-65cm, were added to the stimulus compartment of the tank.
For the mysid trials, approximately thirty mysids were added to
the stimulus compartment. The animals were then observed for a
30 minute period. To control for variations in water flow and
light due to the presence of the stimuli, trials were conducted in
the same manner using macrocystis shapes made from, black
plastic to control for the presence of macrocystis or brine shrimp
to control for the presence of mysids.
Anatomy
The pouch containing the rhinophore was excised from a conscjous
animal using irridectomy scissors. The animals were then tagged
by injecting them with 1cc of either methyl blue or fast green
dye. In controls a comparably sized piece of tissue was removed
in the same manner from the area adjacent to the rhinophore on
control animals. To guard against infection, all surgically altered
animals were individually isolated for twenty-four hours in tanks
which for ten minutes every two hours received an inflow of UV
and heat sterilized seawater filtered to 5u. Trials were
conducted in the same manner as for the normal animals. Stimuli
studied were macrocystis, control macrocystis, and mysids.
RESULTS
The behaviors that were observed during Melibe observation are
defined as follows:
resting foot and hood resting on the substratum tentacles folded
into the hood. Tentacles may or may not be visible.
scanning: continuous head motion while body remains still.
walking locomotion along the substratum.
treading back and forth movement of the body while the foot is
still attached to the substratum
swimming: complete removal of foot from the substratum with
consequent locomotion through water.
open hood an extension of the hood beyond the point where
tentacles are extended.
The baseline behavioral data obtained, when the Melibe were
observed for thirty minutes with no stimuli indicates a low level
of activity. The only behavior that occurred was scanning. Three
of the ten Melibe tested performed this behavior. The remaining
seven Melibe remained in the resting position for the entire test
period. (Table 1)
Response to Macrocystis
When normal Melibe were exposed to macrocystis, all 13 animals
tested came out of the resting state. Behaviors included scanning
(11/13), walking (12/13), treading (11/13), swimming (7/13),
and hood opening 7713. (Table 2) All the animals tested
performed one of the locomotive behaviors: walking, treading, or
swimming.
When the simulated macrocystis was used as the stimulus the
only behavior observed was scanning. This behavior was seen in
six of nineteen animals tested. The remaining animals stayed in
the resting state. (Table 2)
Äfter rhinophore ablation, fewer animals came out of the resting
state in response to macrocystis stimulus. (Table 3) Both the
scanning and open hood behaviors were observed (2710), (1710).
There was no appreciable difference between the behavior of the
normal animals and those which had a small piece of rbp removed.
Response to Mysids
When normal Melibe were exposed to mysid thirteen of thirteen
animals came out of the resting state. The oberved behaviors
were scanning (12/13), walking (4/13), treading (2/13), amd
swimming (1/13). (Table 4)
The only behavior observed when normal Melibe were exposed to
the smell of brine shrimp was scanning (3/13). (Table 4)
Furthermore, when Melibe were exposed directly to brine shrimp,
only two of the thirteen began to feed. However, under these
conditions, introduction of macrocystis into the stimulus
compartment of the tank initiated feeding in all but one of the
animals. (Figure 3)
0
Animals with their rhinophores removed did not respond to the
presence of mysid. In the ten trials, only one animal came out of
the resting state. This animal both scanned and opened its hood
(Table 3)
DISCUSSION
The major finding of this study is that Melibe leonina perform
specific behaviors as a result of olfactory information detected
by the rhinophore organ. The data indicate that Melibe alter their
behavior based on chemical cues from their environment.
Introduction of macrocystis in the stimulus compartment causes
a transition from a resting state into a motile state.
Introduction of mysid causes a transition from a resting state
into a feeding state. The indication that the cue is olfactory, as
opposed to visual or tactile, is that the behavioral transition is
not accomplished with plastic macrocystis shapes or with brine
shrimp alone. (Figures 4 and 5) However, macrocystis and brine
shrimp presented together are sufficient to evoke feeding
behavior. These data indicate that the Melibe world is olfactory
as well as tactile.
Macrocystis evokes locomotion indicating that chemical stimuli
appears to trigger behavior that is useful in the natural
environment of the Melibe. Macrocystis is the location of a
number of important behaviors: egg laying, food finding, and
location of conspecifics. Macrocystis has a characteristic
secretion, providing Melibe with an olfactory stimulus
underwater. Although Melibe are sensitive to changes in light and
in water flow, experiments with simulated macrocystis indicate
that the chemical attributes of the macrocystis, not other
factors, trigger an increase in activity. This ability is important
because it increases the probability that the Melibe relocates the
macrocystis if it is detached. However, when Melibe are attached
to macrocystis, they move infrequently. A possible explanation
for this is that there is a tactile inhibition by macrocystis which
overides the olfactory input. Another explanation is that the
animals habituate to the smell of the macrocystis.
Mysids also evoke heightened activity in the Melibe. In contrast
to macrocystis, this chemical stimulus triggers hood opening.
This serves two purposes. It allows for an increased surface area
for tactile stimulation. Furthermore, once they receive the
tactile stimulus, all the melibe must do is close their mouths
around their prey. The nature of the behavior makes the chemical
trigger essential. Because of water currents, a tactile stimulus
is probably less useful under water. More importantly, Melibe
move relatively slowly. If they were to wait for a tactile
stimulus to begin feeding, their prey would escape before they
could catch it.
The experiments with brine shrimp indicate that changes in water
flow alone can not account for the hood opening behavior in
response to mysid. Furthermore, the experiments allowing
tactile stimulation by brine shrimp indicate that tactile
stimulation to a resting animal is not enough to trigger feeding in
a sterile environment. However, when macrocystis smell is
introduced into the system, feeding behavior is initiated. One
explanation of the integration of these two stimuli to yield
feeding is that the concomitant relaxation of the hood to allow
stimulation of the tentacles initiates the feeding behavior.
Although the mechanism for this phenomena is not understood at
present, this experiment does indicate that Melibe are capable of
integrating chemical cues.
The rhinophore ablation experiments indicate that the rhinophores
are the principal chemosensory organs for the passage of
afferent information regarding the smell of macrocystis and
mysid. There is a negligible difference in the responses of the
rhinophorectomized animals exposed to chemoeffective stimuli,
those exposed to control stimuli, and normal animals exposed to
control stimuli. (Figure 6) This contrasts with the
Pleurobranchaea californica in which it has been shown that the
rhinophores, tentacles and oral veil are all receptive to the same
chemical stimuli. (Bicker et al, 1982) while there is no indication
of a lack of sensitivity of other organs of the Melibe to
macrocystis and mysid, the removal of the rhinophores yields a
decrease in behavioral responses to the comparable levels of an
unstimulated control animal. This indicates that the rhinophore
may be the sole organ responsible for the detection of these
stimuli.
Rhinophores may potentially detect many other stimuli besides
mysids and macrocystis. Furthermore, they may be sensitive to
variations in quantities of these stimuli. The independence of C4
may be indicative of the complexity of the rhinophore. he
interconnecting pathways of the other cerebral neurons allows
for reflex actions and short circuits without involving the central
ganglion. (Hurst 1968). In contrast, nerve impulses from C4 must
form synapses on the central ganglia. This may be necessary for
the decoding of olfactory information from the rhinophore, and
subsequent translation into evoked behavjors.
ACKNOWLEDGEMENTS
1 would like to thank my advisor, Stuart Thompson, for his help and
enthusiasm. I would also like to thank Mary Lucero for sharing her
vast knowledge (and reprints) on olfaction. I am grateful to Karla
Palmeri for the use of the tanks, space, and for answering all my
questions about animal care, Chris Patton for his help with
photography, and John Lee for all his mechanical help and an endless
supply of caulk. Chris Mathes deserves thanks for a great deal of
encouragement and for forcing my to put things in perspective. Thanks
to Sam Wang for always taking the time to discuss science, (Sam..., do
you have a minute?") and for vast amounts of constructive criticism.
Thanks to everyone at Hopkins for being helpful and friendly. I'd like
to express my gratitude to the spring class, especially to Agnieszka
Rawa for always asking questions, William Nathaniel Timmins for
providing cookies and the answer to the ultimate question about
gustation of Melibe, and Amanda Schivell for her boundless enthusiasm
and open ears during the Melibe and mysid hunts.
Literature Cited
Ache, Barry W. 1987. Chemoreception in Invertebrates. Pp. 39¬
64. In: T.E. Finger & W.L. Silver (eds.), Neurobiology of Taste and
Smell. John Wiley & Sons, Inc: New York.
Agersborg, H.P.K. 1921. Some observations on qualitative
chemical and physical stimulations in nudibranchiate mollusks
with special reference to the role of the 'rhinophores. 1921.
Journal of Experimental Zoology 36: 423-444.
Audesirk, T.E. & G.J. Audesirk. 1977. Chemoreception in Aplysia
californica. II. Electrophysiological evidence of detection of the
odor of conspecifics. Comparitive Biochemistry and Physiology
564:267-270.
Bicker Gerd, W.J. Davis and EM. Matera. 1982. Chemoreception
and mechanoreception in the gastropod mollusc Pleurobranchia
californica. Journal of Comparitive Physiology 149:221-234.
Croll, Roger P. 1983. Gastropod Chemoreception. Biological
Review 58:293-319.
Frings, Hubert and Carl Frings. 1965. Chemosensory bases of
food-finding and feeding in Aplysia juliana (Mollusca,
Opistobranchia). Biological Bulletin 128:211-217.
Gelperin, Alan. 1974. Olfactory basis of homing behavior in the
giant garden slug, Limax maximus. Proceedings of the National
Academy of Sciences (USA) 71:66-70.
Hurst, Anne. 1968. The Feeding Mechanism and Behaviour of the
Opistobranch Melibe leonina. Symposium of the Zoological
0.
Society of London zz:151-166.
Jahan-Pawar, Behrus. 1972. Behavioral and electrophysiological
studies on chemoreception in Aplysia. American Zoologist
2:525-537.
FIGURE LEGEND
1. Dorsal view of Melibe leonina.
2. Sketch of chemical detection tanks.
3. Tank set-up for brine shrimp experiment. Animals observed for
thirty minutes after introduction of the stimulant.
a. brine shrimp in stimulus compartment evokes no response
from melibe
b. brine shrimp in Melibe compartment evokes no reponse
from melibe
c. kelp in stimulus compartment and brine shrimp in Melibe
compartment evokes a feeding response.
4. Comparison of the percentage of Melibe tested performing
specific behaviors in response to smell of kelp and control kelp
within 30-minute period. The behavioral totals may exceed 100%
because individual Melibe may perform more than one behavior
during the observation period. At least 50% of the animals
performed each behavior in response to kelp. In response to the
control, only 328 of the animals were responsive and only one
behavior was performed.
5. Comparison of the percentage of Melibe tested performing
specific behaviors in response to smell of mysid and brine shrimp
within a 30-minute period. All tested animals opened their hoods
in response to mysid, and many animals exhibited other behaviors
as well. In contrast, none of the Melibe opened their hoods in
response to brine shrimp smell and only 25% of tested animals
came out of the resting state.
6. Comparison of rhinophorectomized Melibe exposed to
chemoeffective stimuli and normal Melibe exposed to control
stimuli during a thirty minute period. Rhinophorectomized
animals exposed to chemoeffective stimuli display similar levels
of behavior to normal animals exposed to control stimuli.
Table Legend
1. Observed behaviors of Melibe when no stimulus was introduced
into the tanks. Each row represents the behavior of an individual
animal during the thirty minute observational period.
2. Observed behaviors of Melibe during a thirty minute period
after macrocystis or plastic was introduced into the stimulus
compartment of the tanks. Each row represents the behavior of
an individual animal during the observational period. In response
to kelp all animals performed at least one of the locomotive
behaviors (i.e. walking, treading, and swimming)
3. Observed behavior of rhinophorectomized Melibe in response to
plastic, macrocystis, or mysid. Each row represents the behavior
of an individual animal during the observational period.
4 Observed behaviors of Melibe in response to mysid or brine
shrimp. Each row represents the behavior of an individual animal
during the observational period.
O
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RESPONSES TO MACROCYSTIS OR CONTROL
DURING 30 MINUTE INTERVAL
NORMAL ANIMALS
MACROCYSTIS (N=13)
100%
E CONTROL (N=19)
80%
40%
20%
0%
SCAN
WALK
TREAD
SWIM OENT
BEHAVIOR
100%
80%
60%
40%
20%
RESPONSES TO MYSID OR BRINE SHRIMP
DURING 30 MINUTE INTERVAL
NORMAL ANIMALS
SMYSID (N=13)
□ BRINE SHRIMP (N=12)
8
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NORMAL ANIMALS' RESPONSES TO CONTROL
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MACROCYSTIS (N=10)
□ CONTROL (N=13)
80%
60%
40%
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RHINOPHORECTOMIZED ANIMALS' RESPONSES
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STIMULUS
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MACROCYSTIS
MACROCY.
MACROCYSTIS
MACROCYS
MACROCY
MACROCY:
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MACROCYSTIS
MACROCY.
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MACROCYSTIS +
MACROC
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MACROCYSTIS
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