Olfactory Basis of Homing Behavior in the Giant Garden Slug, Limax maximus
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Proc. Nat. Acad. Sci. USA
Vol. 71, No. 3, pp. 966-970, March 1974
Olfactory Basis of Homing Behavior in the Giant Garden Slug, Limax maximus
(digitate ganglion/locomotion/orientation/terrestrial pulmonate)
ALAN GELPERIN
Department of Biology, Princeton University, PrincetQn, New Jersey 08540
Communicated by V. G. Dethier, November 14, 1973
ABSTRACT Time lapse photography of slugs living in in the soil and forage over an area extending at least 4.5
an experimental enclosure shows that these animals can meters from the home (14). Time lapse photography of grey
return to a homesite from over 90 cm by a direct route.
Slime trail following and vision are not involved in this field slugs, Agriolimax reticulatus, locomoting on an enclosed
behavior. In the presence of a low velocity wind, homing soil surface shows that the animals often return to the same
occurs upwind. Surgical disconnection of the presumptive hole in the soil from which they emerged earlier in the night
olfactory apparatus (digitate ganglion) from the central (15). The present work documents homing behavior in Limax
nervous system eliminates homing. Neurophysiological maximus and presents initial physiological investigation of
recordings from the receptor surface associated with the
digitate ganglion and the olfactory nerve demonstrate its sensory basis.
the olfactory function of the digitate ganglion. The olfac-
tory acuity and capacity for directed locomotion via olfac- MATERIALS AND METHODS
tory cues are also relevant to studies of slug feeding be-
havior, ecology, and learning ability. The behavioral experiments were done on slugs confined to a
1.5 by 1.7-meter area of moist filter paper bounded by a 2-
"Simple" animals often reveal their possession of sophisticated inch wide border of crystalline NaCl. An inverted clay flower
behavioral machinery when experimental questions are asked pot with four semicircular notches cut in its lip, was centrally
in the proper context. This is nowhere better documented than located and served as the animal's daytime resting site. The
in studies of orientation. The sun-compass orientation mecha- filter paper was kept moist by inverted water reservoirs. Food
nism of bees and ants (1), the ability of noctuid moths to items such as carrot (Daucus carota), potato (Solanum tubero-
steer their flight path away from bats using two sense cells sum). dog food (Ken-L-Ration), or mushroom (Boletus edulis)
(2), and the apparent use of hydrodynamic cues by migrating were supplied in a petri dish at one corner of the arena. Fluo-
lobsters (3) are examples of complex neural mechanisms which rescent room lights provided illumination and were automati-
became apparent when physiological experiments were done cally controlled to produce a cycle of 12 hr of light and 12 hr
in an ethological context. The present experiments on homing of darkness. In some experiments, a plastic covering was used
in Limax maximus were undertaken to probe the complexity to shield the arena from air currents.
of behavior possible in a preparation amenable to cellular A 16-mm camera modified for time lapse operation and
neurophysiological analysis (4). equipped with a wide-angle lens was mounted vertically 2.7
Homing behavior has been documented in a wide variety of meters above the experimental arena. A xenon bulb flash unit
molluscan species. Aristotle described the homing behavior with a flash duration of 2 msec was triggered synchronously
of limpets and experiments to date are still searching for the with the camera shutter. The charging capacitor in the flash
sensory basis of this behavior (5, 6). Octopus can return to its unit was changed from 300 uf to 40 Mf to produce the least
nest after forays covering considerable distances (7). Small intense flash which would give a distinct image oln plus-X
colonies of the intertidal pulmonate Onchidium nest in rock negative film with the lens wide open. A framing rate of 4 per
crevasses and after a period of feeding away from the nest, minute was used. Typically the camera was activated from
all members of a particular colony simultaneously return di- 1700 hr to 0900 hr the following day. No behavioral response
rectly to their nest (8). Capture and release experiments have could be detected to the light pulse emitted by the flash unit.
shown that the garden snail, Helix pomatia, can return to More than 1600 hr of activity were filmed and analyzed using
sites favorable for overwintering with an angular error of less an analytical projector.
than 300 over distances up to 40 meters (9). The sea hare The 25 slugs used in these experiments were Limax maximus
Aplysia is diurnally active and returns to a particular location and included both locally collected animals and individuals
in its tank of seawater to sleep (10). reared from eggs in the laboratory. No differences in homing
The literature contains scattered suggestions of homing be- ability between these two categories of animals were observed.
havior among slugs. The ability of Limax maximus to show Anatomical studies oln the optic tentacles were done using
homing behavior has been referred to anecdotally by Taylor standard histological techniques to prepare 8-,Am serial sec-
(11), Pilsbry (12), and Frdmming (13). Field observations of tions of whole tentacles stained with Mallory Heidenhain's
the California banana slug, Ariolimax columbianus, suggest triple stain (16). Neural pathways in the digitate (= tentacu-
that animals establish a homesite by excavating a depression lar) ganglion were stained using axonal iontophoresis (17) to
introduce Co++ ions into axon.s. The tissue was then treated
Abbreviation: EOG, electro-olfactogram. with ammonium sulfide, dehydrated with ethanol, and cleared
966Proc. Nat. Acad. Sci. USA 71 (1974) Olfactory Basis of Slug Homing 967
A
B' Ce D
FIG. 1. Track of single slug during one night.
in basic methyl benzoate (18). Ganglia so treated were studied
and photographed as whole mounts.
Electrical recordings from the receptor surface of an optic FIG. 2. Homeward paths of several different slugs. Arrow
tentacle were made with saline-agar filled electrodes of tip provides constant compass direction reference.
diameter 50-100 /Am connected to a neutralized input capaci-
tance dc amplifier. Polyethylene suction electrodes were at- Fig. 2 presents 10 return Ipaths selected to represent the
tached to the olfactory nerve after severing its central con- variation in directness of homing observed in this study. The
nections. Both types of signals were recorded relative to a maximum distance from which homing occurred was 93 cm,
ground electrode in the saline bath. The signals were dis- the outer limit of the arena. The animals used varied in body
played on a multichannel oscilloscope and either photo- length from 7.5 to 16.5 cm, and inter-optic tentacle distance
graphed directly or recorded using an FM tape recorder. varied from 1.5 to 2.5 cm. Fig. 2 also illustrates that the same
The saline used had the following composition in mM: Na slug can use different paths home on successive nights (Paths
70, K 2.5, Ca 3.4, Mg 0.8, Cl 81, glucose 0.6, Tris 50. C, F, J) and that two slugs living together can use different
paths home on the same night (Paths A, I).
RESULTS The nonrandom nature of homing was tested mathemati-
The animals spend the daylight hours in the dark and humid cally in the following way. A set of linear path segments with
environment provided by the homesite. With a latency vary- origins close to the periphery of the arena was selected; only
ing from several minutes to several hours after lights off, they those paths which were linear because the animal moved
emerge and move about the arena at speeds ranging between along the salt barrier were excluded. For the path whose ori-
0.069 cm/sec to 0.26 cm/sec. These travels bring them in con- gin was closest to the home, the flower pot subtended 150 of
tact with the salt barrier, the food dish, other slugs, and ulti- the horizon. I assume, for purposes of this test, that the animal
mately, the homesite. Periods of locomotor activity are inter- selects his direction of travel from a 1800 sector. This assump-
slpersed with periods of sleep, sexual activity, or feeding. The tion yields a probability of contacting the home by chance of
return to the homesite is often quite direct and over virgin 15°/180° or 8.3%. The sample of 41 linear path segments con-
territory. tained 13 (32%) which were homing paths, a clearly non-
A representative tracing of the travels of a slug about the random distribution.
experimental arena is shown in Fig. 1. Three of the trips are Experiments were then directed to the question of the sen-
short and in the immediate vicinity of the pot. These short sory cue providing direction to the homeward path. The ani-
trips predominate during the first night in the apparatus mals are not following slime trails home, although they can
when the pot and filter paper are clean. Two of the trips are follow slime trails and do so routinely to locate sexual part-
more extensive and have terminal segments which clearly ners. The use of visual cues is possible but unlikely. The op-
suggest a directed locomotion back to the homesite. All of the tical system and fine structure of the slug eye suggest poor
activity occurred during the dark period; the animal made its visual acuity (19) and light was available for only 2 msec
final return home 2.2 hr before light onset. We do not know every 15 sec. The use of vision in homing was tested in two
when the animal actually decided to return home, but the ways. During the dark period, slugs were removed from the
final segments of the two long trips demonstrate the animal's home, placed at the periphery of the arena, and allowed to
ability to return home in a direct I)ath from the outer limits return home in complete darkness, which they did. Two ani-
of the arena over previously untraveled territory. mals in which the optic nerves were successfully sectioned968 Physiology: Gelperin Proc. Nat. Acad. Sci. USA 71 (1971)
FIG. 3. Distribution of homeward path directions relative to
direction of prevailing wind. Arrow indicates wind direction.
bilaterally also retained the ability to return home by a direct
path if removed from the home and placed at the periphery
of the arena. :.:K-
Two kinds of experiments suggest that olfaction may be the
key stimulus for homing in these exl)eriments. If a very gentle
wind is set up across the arena, the animals show a distinct
tendency to move against the wind. Fig. 3 shows the distri-
bution of 42 homeward paths in relation to the wind direction
over the app)aratus. Five times as many returns occur from
the two downwind sectors as from the two upwind sectors
(42%o versus 8%7). Application of the x2 one-sample test to the
distribution shown in Fig. 3 indicates a significant deviation
from the expectation that returns are uniformly distributed
around the home (P < 0.001). A distribution of returns based FIG. 4. Digitate ganglion after CoCl2 was iontophoresed
on the hypothesis that the two downwind sectors receive be- toward the ganglion via axons in the olfactory nerve and after
tween 4 and 8 times as many returns as the two upwind sec- cobalt was precipitated with ammonium sulfide. Dark areas out
tors is not significantly different from the observed distribu- of focus at top are pigment cells in sensory epithelium. Three
tion (0.05 < P < 0.1). neuronal somata in the ganglion are indicated by short arrows.
The digitate ganglion, located at the distal end of the The majority of the fibers in the olfactory nerve terminate in the
olfactory nerve in the dorsal or optic tentacles, has been ganglion. scale bar = 500 um.
assigned an olfactory function based on behavioral (20-22)
and histological (23-25) observations. To test the idea that el)ithelial lpad or sensory zone (26) at the end of the tentacle.
the digitate ganglion is important in homing behavior, slugs Presumptive sensory neurons located in and under the sensory
were subjected to bilateral olfactory nerve section. They were el)ithelium send lprocesses into the digitate ganglion, as do
anesthetized with CO2 and an incision made in the lateral large numbers of 5-7 Azm cells located in the distal extensions
body wall of the head. The olfactory nerves were cut where of the ganglion (24). A mantle of neurosecretory cells sur-
they emerge from the cerebral ganglia. Control slugs received rounds the ganglion (27). Cobalt backfills of the olfactory
the same operative procedure except the nerves were not cut. nerve reveal six to eight large cells in the body of the ganglion
Operations were verified by autopsy. The six slugs success- and several fiber tracts ending within it (Fig. 4). This suggests
fully operated on, never homed again whereas the controls that in fact the majority of the axons in the olfactory nerve
continued to do so. The excursions of the operated animals are second order processes.
were of normal extent and at normal speed. However, even To test the olfactory function of the digitate ganglion
though they occasionally passed within 2 cm of home, the physiologically, a preparation of the sensory el)ithelium,
operated slugs did not return to it. digitate ganglion, and olfactory nerve was isolated from the
Several behavior patterns exhibited by normal slugs are animal and set up in vitro so that the sensory surface was ex-
suggestive of an olfactory sensitivity. Locomotion of any tyele l)osed to the air while the ganglion and nerve were immersed
is always accompanied by movements of the optic tentacles in saline. An agar-filled electrode recorded from the sensory
which sweep through an arc of 15-20° on either side of the surface (28, 29) and a suction electrode monitored the olfac-
midline. During homeward locomotion, animals often exhibit tory nerve. Filter paper discs were saturated with odorants
a characteristic "head-waving" behavior during which the and l)laced in a Swinny adapter ('Millipore Corp.) mounted
on a 1-cc syringe. The syringe was mounted so that the tip of
anterior end of the body is lifted above the substrate and the
head moved from side to side with a frequency of approxi- the adapter was I cm from the receptor surface. Stimuli were
mately 1-2 per sec. Paths toward odorous foods, particularly delivered by hand.
fungi, are also often quite direct over distances up to 80 cm. A puff of air containing an odorant such as amyl acetate
The digitate ganglion is situated within the distal end of elicits a large compound action l)otential in the olfactory
the cylindrical tentacle retractor muscle. The finger-like nerve (Fig. 5. A) and a negative electro-olfactogram (EOG)
processes emanating from the ganglion innervate a distinctive (Fig. 5, A1,). Several small single unit responses are evident inProc. Nat. Acad. Sci. USA 71 (1974) Olfactory Basis of Slug Homing 969
Al preceding and following the compound action potential.
The single unit responses typically lasted for the duration of
the active phase of the EOG. A puff of moist air produces no
response (Fig. 5, B1, B2). The EOG is not recorded from epi-
thelium outside the sensory patch. The size of the compound
action potential and EOG can be increased by more rapid ap-
plication of a given volume of odorant-laden air. Similar re-
sponses were obtained to aqueous extracts of carrot and po-
tato. These data demonstrate the olfactory function of the
digitate ganglion.
DISCUSSION
The results presented here demonstrate the homing ability of
Limax maximus. The maximum distance over which Limax
can forage and still return home is unknown. Estimates of
this distance require knowledge of the chemical species used
as an olfactory cue and information on the behavioral thresh-
old to this chemical (30). The behavioral threshold could be FIG. 5. Al and B1 are recordings from olfactory nerve; A2 and
very low since insect and vertebrate olfactory systems can B2 are recordings from receptor surface. Al and A2 were recorded
trigger a behavioral response to a few molecules of odorant simultaneously in response to 0.5 cc of amyl acetate vapor.
(30). If the initial behavioral response triggered by the odor B1, B2 are the responses to 0.5 cc moist air. Calibration bar
is movement ul)willd, a detectable gradient is unnecessary and applies to A and B and indicates 1 sec., 200 ,uV for Al, B1 and
the odor will be effective in promoting homing even at the 400 ,AV for A2, B2.
fringes of its "active space". Lirnax can show positive anemo-
taxis to gentle, l)resumably odor-free winds (31). The data presented here are very useful in designing experi-
Olfactory stimuli are also important in locating food. Slugs ments to probe plasticity of behavior in the slug. Olfaction,
normally eat a variety of plants, including fungi (13). Re- taste, and vibration sensitivity (42) are the dominant senses
moval of the optic tentacles containing the digitate ganglion and learning paradigms must accommodate these facts.
reduces the distance at which slugs detect the stinkhorn Closely related snails have been trained to avoid previously
(Phallus impudicus) from 120 cm to 20 cm (20). A similar re- attractive plant odors by shocking them in the presence of the
sult was obtained testing the response of A griolimax reticulatus odor (43). The aversive taste of quinine was effective in modi-
to potato before and after optic tentacle removal (21). Both fying the climbing behavior of Helix (44). Snails can also
of these exl)erimelits indicated a residual olfactory sensitivity learn to keep an optic tentacle retracted to avoid an aversive
after optic tentacle removal attributed to the smaller an- stimulus (45). The recent demonstration of operant condi-
terior tentacles. However, this l)henomenon was iiot al)l)arent tioning in Octopus (46) has further extended our knowledge of
in my exl)eriments. The olfactory sensitivity and capacity the capabilities of molluscan brains.
for directed locomotion via olfactory cues are also relevant
to ecological studies of slug distribution in relation to food Note Added in Proof. Recent experiments (47) demonstrate
plant abundcance (32, 33). that using olfactory cues, Limax can rapidly learn to avoid
The neurophysiological data obtained to date demonstrate new foodplants if aversive stimulation is paired with ingestion
the olfactory function of the digitate ganglion in the slug. A of the new foodplant.
negative EOG in response to attractive plant odors has been I thank D. Giesker for collecting some of these data. Supported
recorded from snail tentacles (28), whereas methanol and by N.S.F. Grant GB 20762.
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