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The function of trail following in the pulmonate slug, Limax pseudoflavus
Anita. Behav., 1992, 43, 813-821
The function of trail following in the pulmonate slug, Limax pseudoflavus ANTHONY COOK Department of Biological and Biomedical Sciences, University of Ulster, Coleraine, Co. Londonderry BT52 1SA, U.K.
(Received 20 February 1991; initial acceptance 24 April 1991; final acceptance 4 October 1991; MS. number: 3732)
Abstract. Trail following in Limaxpseudoflavus Evans is easily observable under laboratory conditions but it is less frequent in the field and its function remains unclear. In experiments allowing the choice between trails and other environmental cues slugs tended to follow trails most frequently when those trails were going in a direction close to that dictated by the other cue. Similarly trails were more often followed when approached at a narrow angle. Trail following was observed as a prelude to courtship. It is concluded that trails provide low priority directional cues and that they are only followed in the absence of other cues or when their direction closely coincides with those other cues. Trail following is a component of food finding, homing and courtship behaviour in the field, but in each case its role may be subordinate to that of airborne odours. Measurements of the quantity of mucus in the trails show that there is no reduction in the secretion of mucus while trail following. Trail following cannot therefore be seen as a mucus-saving tactic.
Mucous trail following is a common feature of the behaviour of gastropods and has been observed, both experimentally and in the field, in a variety of species from a variety of taxa. For some of these species the function of the behaviour is clear (Table I). For other species trail following has been demonstrated in laboratory trials but the possible function of the behaviour in the field remains obscure (e.g. Ilyanassa, Achatina). Limax pseudoflavus is a large slug growing to about 15 cm in length. In common with other large slugs it homes consistently to day-time resting sites which are labelled chemically and in which large groups of animals can be found, often closely packed together. The involvement of trail following in the homing of this slug has been demonstrated in laboratory experiments (Cook 1979) and in the field (Cook 1980) but the frequency and facility with which trail following occurs in the artificial conditions of a laboratory (Cook 1977) do not correspond with the comparative rarity of its occurrence in the field. In the closely related species Limacus (= Limax) flavus L., Chelazzi et al. (1988) have shown that trail following is infrequent when a trail is approached at 90 ~ (about 13~ of contacts resulted in trail following) and that, when allowed a choice between a trail and the smell of conspecifics in a Y-maze, this species oriented with respect to the airborne odour rather than the trail. 0003 3472/92/050813+09 $03.00/0
Clearly trail following is a component of the behavioural repertoire of slugs, but its role in the ecology of these species is not yet fully understood. In this paper I describe a sequence of experiments and observations which have been designed to examine more thoroughly the possible roles of trail following in the life of Limax pseudoflavus. I address the following questions. (1) Do slugs follow trails at random or do they only follow those going roughly in the same direction as the slug is already travelling? (2) When faced with a number of conflicting directional cues, what priority is given to trail following? (3) How frequently and under what circumstances do slugs follow trails in the field? (4) Can slugs make any savings in the amount of mucus deposited during trail following? METHODS
Slugs were collected in the field in the Coleraine area and maintained in lunch boxes, the bases of which were lined with moist tissue. Containers were regularly watered and slugs provided with Readybrek breakfast cereal ad libitum. Trail Choice
The methods used to examine which trails a slug follows were based on those of Cook (1977).
9 1992 The Association for the Study of Animal Behaviour 813
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Animal Behaviour, 43, 5
Table I. A summary of the gastropod species known to follow mucous trails Taxon
Function
Authority
Natiea lllyanassa
Prey finding ?
Patella
Homing
Collisella Lottia Monodonta Littorina
Feeding and homing ? Trail blazing
Gonor (1967) Crisp (1969) Trott & Dimock (1978) Cook et al. (1969) Funke (1968) Connor (1986)
Opisthobranch
Navanax Bursatella
Prey capture Aggregation
Pulmonate
Physa Biomphalaria Siphonaria spp. Helix Limax Aehatina Onchidium Euglandina
Prosobranch
Genus
Deroeeras Limaeus
? ? Homing ? Homing ? Homing Prey capture ' Courtship Courtship ?
Briefly, experiments, which were conducted on polythene sheets, consisted of allowing slugs to wander one at a time over an open arena. The trails were then visualized by immersion in a suspension of powdered calcium carbonate. The frequency with which trails were superimposed for more than 5 cm and the angle between the original trail and that of the superimposed trail were measured. Unlike the practice of Cook (1977) angles were measured irrespective of the direction in which the trail was laid, rather than as the angle through which the following slug would have to turn in order to follow the trail in the direction in which the trail had originally been laid. Thus angles range from 0 to 90 ~ The Priority Given to Trail Following Experiments were performed in a cubical booth lined with black polythene. The front face was open and illuminated by a 60-W bulb above it. Inside the booth there was a polythene-covered, Perspex disc of 76 cm diameter inclined at an angle of 45 ~ to the front face. A marker slug placed at the base tended to move upwards and away from the light, into the darker interior of the booth. The disc was mounted on a support which allowed it to be rotated by
Wells & Buckley(1972) Hall (1973) Paine (1963) Lowe & Turner (1976) Wells & Buckley(1972) Townsend (1974) Cook & Cook (1975) Bailey (1989) Cook (1977, 1980) Chase et al. (1978) McFarlane (1980) Cook (1985) Wareing (1986) Chelazzi et al. (1988)
known angles around its centre. A slug (the marker) was placed at the bottom of the disc and allowed to move up the polythene sheet. Slugs that made circular movements at the foot of the disc were removed and the experiment restarted. When the marker slug had traversed the diameter of the disc it was removed, the disc rotated if necessary, and a second slug (the tracker) placed at the start point of the marker slug. After the tracker had left the disc, the length of both the marker and the tracker trails and the length of coincident trail were measured. Experiments were performed at 5~intervals until no trail following occurred (25~ A minimum of 10 replicates were performed at each angle. Experimental data were compared with control data generated by superimposing 'sham trackers' (Cook 1977) on marker trails. Sham trackers are animals moving from the same start point but in the absence of a marker trail. Field Observations of Trail Following Previous observations of trail following in the field (Cook 1980) concentrated on the behaviour in a comparatively small area around the home. Very few instances of trail following were observed. Those that did occur were associated with homing
Cook: The function of trail following in slugs
815
56 I1.,1_1~Followed Not followed 50 24 g g ~8 12
0
0-15 o
16-50 o
51-45 ~
46-60 ~
61-75 o
76-90 ~
Angle of approach
Figure 1. A comparison between the distributions of the angles of approach that led to following with those that did not. The two distributions differ significantly (Zz = 45.34, df= 5, P < 0.001). downwind. My objective in the current observations was to attempt to observe trail following in a broader field of view in a more heterogeneous environment. An A u t o m a x 16CC4 1 6 m m time lapse camera fitted with a Vivitar 238 flashgun set to minimum power was set up 2-8 m vertically above a tiled area on which slugs were known to be active. Frames of K o d a c h r o m e 40 were taken every 2 min during the hours of darkness. I recorded the position of slugs from the film using a Specto projector.
Mucus Production During Trail Following An array of clean glass microscope slides was placed on a support mounted at approximately 45 ~ to the vertical in the booth described above and a slug allowed to walk up it. Each slug was allowed to walk up the slides three times. The first time was to provide slides on which there was a single trail. The second time was to lay a marker trail and the final time was to act as a tracker on its own previously laid marker trail. Thus the same animal was used as both marker and tracker. Slides were collected on which there was either a single trail or one that had been followed. The slides were stained in toluidine blue which is known to stain strongly the neutral mucopolysaccharide secreted by the suprapedal gland into the trail (Cook & Shirbhate 1983). The optical density of the stain was measured for both single and double trails using an L K B 2202 Ultroscan laser densitometer. Repeated scans were taken across the width of the trail.
RESULTS
Trail Choice In 44 experiments there were 286 contacts between a slug and a previously laid trail. Each of these contacts represents an opportunity to follow a trail. Following for a distance greater than 5 cm occurred on 92 occasions (32.2%), a frequency similar to that previously reported (Cook 1977). Approaches to marker trails that subsequently led to trail following were made at an angle (X_+ sE) of 41 _+3~ whereas those that did not result in trail following were made at a mean angle of 62 + 2 ~ This is a significant difference ( M a n n - W h i t n e y U-test: U=4649, P<0.001). Figure 1 shows the distribution of angles of approach between those occasions on which following ensued and those on which it did not. It is clear that when a slug approaches a trail at a narrow angle there is a significantly greater chance that it will follow the trail than if it approached the trail at a greater angle.
The Priority Given to Trail Following Trail superimpositions o f less than 5 cm (an approximate body length) were discounted. Comparison of the control (using sham trackers) and experimental data shows that there is a significant degree of trail following only when the marker trail had been rotated through angles of 20 ~ or less. This is true of both the frequency of following and the distance followed (Table II).
Animal Behaviour, 43, 5
816
Table II. The effect of rotating marker trails from the vertical on measures of trail following Angle rotated
% Followed
Distance followed (X_+SE,cm)
0 5 I0 15 20 25 Sham at 0~
86 100 90 50 20 0 23
28 _+3'2 14+2.4 20_+6.1 10_+1.5 7, 17 -13_+3.7
N 45 12 9 5 2 8
*Sham trackers at other angles showed no trail superimpositions greater than 5 cm.
It may be concluded that the slugs follow trails that deviate only a little from the direction in which the slug would travel in the absence of the trail.
Field Observations of Trail Following Figure 2 shows the area of view of the camera. Slugs emerged from a drain and normally foraged on the algae growing on the paved area. During these observations additional food (cucumber slices) were provided at point F 1 (Fig. 2) or at point F2 (Fig. 3). On most nights so many slugs were active that a comprehensive view of trail superimposition was impossible to gain. Further it is likely that with most slugs making journeys between the home and the food, trail superimposition is likely to occur by chance more frequently than would occur in the random movements seen in laboratory observations. On several dry nights the mucus remained as highly refective silvery trails which formed a radiating mass around the food. Individual trails were difficult to discern. Under these circumstances the reporting of trail following must be restricted to nights on which relatively few slugs were active. Figure 2 also shows a selection of the routes taken by slugs on a single night. It is clear that there is no consistency in the behaviour of individual slugs. Some followed the line of the gully, others navigated without apparent reference to trails and yet others made extensive use of previously laid trails. Wind direction during these observations is unknown, but the pattern of flow around the area is likely to be complex owing to the proximity of walls.
Figure 3a shows the details of movements of individual slugs in a smaller area. Most movements did not involve trail following. The movement of slug D, however, involved following the trail of two slugs (A and B). Slug D left the trail of slug B at the point at which that trail was no longer travelling in the direction of the food. I observed 18 slugs moving between the home and the food between 2100 and 2400 hours on a single night. Of the 69 contacts observed between trails, 17 (24%) resulted in trail superimpositions of more than a body length of the following slug. All these instances of trail following were associated with slugs moving towards food. Homing occurred later at night and a similar pattern of movements was observed. Courtship and copulation were observed on three occasions and each time it was preceded by trail following (Fig. 3b). Copulation was extremely brief, lasting no more than two frames (4 rain). The marker slug was not necessarily the individual involved in subsequent copulation (Fig. 3b). During courtship one slug is the trail layer while the other is the trail follower. Although sperm exchange is reciprocal the roles of the two animals involved are not identical. On the three occasions that copulation was observed on film the trail layer turned back on itself to meet the trail follower and copulation took place in a head to head position. After copulation the trail layer executed a loop (see inset in Fig. 3b) before remaining at rest in the vicinity of the site of copulation at which a mucous mat is normally visible.
Mucus Production During Trail Following The quantity of mucus laid in the trail varies greatly. The stained trail is extremely streaky, probably indicating that mucus is redistributed by the foot after being deposited from the suprapedal gland. Density measurements taken across the width of a trail, however, integrate this streakiness and the integrated measures are remarkably constant for any one animal during the course of a single experiment. Five animals completed sufficient of the experiment to give useful measurements. Density readings of single and joint trails were made and are shown in Table III. During trail following by slug 3 (Table III) instances occurred in which two separate trails were deposited on the same microscope slide before and
Cook: The function of trailJbllowing in slugs
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Figure 2. The area of activity of slugs in the field. The squares are quarry tiles (approximately 23 x 23 cm) bordered to the left by a wall and at the bottom by a gully leading to a drain at the bottom left hand corner in which most slugs had their day-time resting sites, Food was placed at F 1. In later observations food was placed at F2. Selected tracks of slugs are shown from a single night. Trails were laid in more or less alphabetical order. A ( - - -) A direct journey from home to the food. B ( ) A return from the food to the home almost entirely along the outward trail of A. C G . . -) A brief excursion not involving trail following. D (. 9.) A slug making an extensive journey after leaving the food. E G . . . . . -) A slug making a return to home from outside the field of view, along a gully and making circular movements involving retracing its own trail. F ( - - - ) A second slug making a return journey from outside the field of view along the gully and following the trails of slugs E (forwards) and C (backwards). G ~ ~ A slug returning from the food following the trails laid by E and F (forwards) and C (forwards). H (...) A slug crossing the trails of other slugs but not interacting with them.
after trail following. These trails were treated together in the densitometer a n d gave a j o i n t reading o f 28.4_+2.1 (.~_+SE, N = 14) c o m p a r e d with a density o f 28.1-t-3.6 ( N = 10) when the trails were
superimposed. Clearly the quantities o f m u c u s deposited during trail following a n d d u r i n g n o r m a l m o v e m e n t are similar over the short periods o f trail following observed d u r i n g these experiments.
818
Animal Behaviour, 43, 5
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DISCUSSION Trail following appears to be a low priority behaviour for most slugs. Chelazzi et al. (1988) found that the airborne odours o f a conspecific were preferred to trails, when slugs were offered a choice and I have previously (Cook 1980) found that trails were used for homing only when the individuals were isolated from airborne odours as a result of approaching home from a down-wind direction. The measure of the angle turned by slugs onto a trail used by Cook (1977) gives a false impression of the deviation that slugs make at the commencement of trail following since it records the change in direction necessary to follow in the same direction as the individual laying the trail. The measure used here of the angle at which the slug approaches the
trail (Fig. 1) clearly shows that the slugs follow trails approached at low angles more frequently than those approached at right angles. This concurs closely with the results of Chelazzi et al. (1988) who showed a low frequency of following at angles around 90 ~ but that, on crossing a trail there is a deflection in the direction of travel towards the trail, even though this does not always result in trail following. Such a deflection at small angles of approach could result in trail superimposition. In Fig. 3a slug D turns towards the trail of slug C after crossing it but does not subsequently follow it. It almost immediately encounters the trail of slug A and another turn results in trail following. It might be that turning on crossing a trail allows a sampling process to occur which results in the following slug orienting gradually with respect to a series of
Cook: The function o f trailfollowing in slugs
819
(b)
F2
9
.r4
l
Figure 3b Figure 3. Movement of slugs (a) to the food and (b) prior to copulation. Slugs are labelled with letters. The numbers refer to the frame number (at 2-min intervals) appropriate to that location of each slug. The precise locations of slugs have been slightly displaced during trail following for clarity. (a) Details of the movements of four slugs between the gully and the food at F2. Slugs A and C moved direct from the food to the gully. Slug B made a short loop which involved a short length of trail following before coming to rest from frame 6 onwards. Slug D'sjourney towards the food overlapped with the trails laid by A (backwards), branched onto that laid by B (backwards) before leaving that trail to travel directly to the food. (b) Details of the movements of three slugs involved in courtship and copulation. Slug A moved to point X where it stayed for 30 min (15 frames). Slug B moved from the food and contacted the rear of slug A which then started to move (frame 6). Slug C closely followed slug B for most of its journey and then followed slug A with which it then mated (frame 12) at point Y. Inset: a sketch of the routes taken after copulation. Slug B continued on its way while slug A remained at rest after executing a short loop.
similarly oriented trails r a t h e r t h a n just one. Could this be the slug version o f the o p i n i o n poll in which a n u m b e r of trails influence the decision to follow just one? T h e response to gravity (geotaxis) has been d e m o n s t r a t e d in a n u m b e r o f gastropods (Agriolimax (=Deroceras): W o l f 1927; Arianta arbustorum: B a u r & Gosteli 1986; Lirnax: Crozier &
Federighi 1925). In the present experiments the slugs were given a n u p w a r d incline a n d a d a r k area to a p p r o a c h a n d a trail t h a t deviated by a varying angle from t h a t direction. Trail following occurred at a m u c h higher frequency w h e n all three o f these orienting cues were acting in the same direction with between 80 a n d 9 0 % o f tracker trails being followed w h e n the m a r k e r trail was r o t a t e d by less
Animal Behaviour, 43, 5
820
Table III. Mean integrated densitometer readings (in arbitrary units) across the trail width (_+SE(N)) for fiveslugs Slug no. 1 2 3 4 5
Double trail 86.1+5.7(26) 65.9_+5.6(23) 28.6_+3.6(10) 37.9+__1.8(25) 50.8_+3.9(26)
Single trail
Ratio double:single
27.2+3.9(13) 27.6_+3.0(22) 14.2+1.1(28) 18.0_+1.7(26) 28.1_+4.1(25)
3.16 2.39 2.02 2.10 1.81
Double trails are those that had been followed, single trails are those not showing trail following. The double: singleratio should be 2 if no change in mucus production during trail following occurred. than 10~ When the trail deviated by more than this from the vertical both the frequency and the distance followed fell until, by angles of 25 ~ no trail following was observed at all. Again it may be concluded that trail following occurs only if the trail encountered is going in roughly the same direction as the following slug. The field observations of the movements of slugs reveal that trail superimposition can be more frequent than previously reported (Cook 1980). These previous observations involved relatively few slugs moving over a restricted area. Whether trail superimposition at the density of movements sometimes observed in the field is always equivalent to trail following is open to doubt. The density of trails around the food in particular must make trail superimposition inevitable. Nevertheless measurements taken at the beginning of a night on which relatively few slugs were active show that 24% of contacts between trails resulted in subsequent trail following. This compares with the 32% observed under laboratory conditions. No consistent directionality in general trail following was noted. When copulation occurred after trail following, however, the trails were followed for relatively long distances and were always followed towards the laying slug. The directionality in trail following preceding copulation has been noted in other slugs (Wareing 1986). Whether this directionality indicates that the trail contains directional information or that there is a second, directional, perhaps airborne, cue is not known. It may be significant, however, that on one occasion in the present observations, the trail layer was not eventually involved in the ensuing copulation (Fig. 3b). On this occasion the directional cue might have
been provided by one individual and the trail by another. If the following of trails is of such a low priority in normal behaviour why follow them at all? One possibility is that the slug may make use of the mucus that has already been laid to aid its locomotion. Denny (1980) reported that as much as 35% of the energy costs involved in adhesive locomotion in slugs is a direct result of the need to secrete mucus. Davis et al. (1990) working with limpets, Patella vulgata, arrived at a similarly high figure for the cost of mucus production (23 % of the energy ingested is used in mucus secretion). Trail following could be viewed as a mucus-saving tactic if following slugs secreted less mucus than those that laid the original trail. The measures made of the density of stained mucopolysaccharides in the trails before and after trail following show that double trails stain roughly twice as intensely as single trails (Table III). This indicates that no savings have been made by the following slug. While some savings of other components cannot be ruled out, it appears that the major component of the trail is not husbanded during trail following. The current experiments involve relatively short lengths of trails being followed and it might be that the control over mucus production cannot be accomplished over such a short time. Since trail following is observed only over relatively short lengths of marker trail in most gastropods it appears that mucus saving is not a major consideration. Pulmonates are hermaphrodites and these slugs exchange spermatophores during copulation. Details of courtship have not been reported for L. pseudoflavus and the frame frequency of one per 2 min does not allow a detailed description here, except to confirm that courtship and copulation are terrestrial rather than aerial as in L. maximus L. (Chace 1953). With such infrequent observations it is not even certain that copulation was completed, and comments on the apparent brevity of copulation in this species are therefore premature. The asymmetry of the behaviour of the two animals after copulation is curious. Clearly the animals have different roles initially, one being the trail layer while the other is the follower. Why the differentiation in roles should persist after copulation when the trail layer remains close to the mucous mat and the other slug moves rapidly away is not known. In conclusion it is clear that, while trail following can be demonstrated in the laboratory in
Cook: The function o f trail following in slugs L. pseudoflavus, it is not a high priority behaviour in natural circumstances. Turning to follow a trail that is going roughly in the same direction as the following slug will bring the follower closer to the source or the destination of the trail layer. The decision to follow a particular trail may be influenced by a variety of other factors such as encounters with previous trails and other directional stimuli such as airborne odours and the directions of illumination, gravity and wind. Thus trail following may contribute an additional cue to the position of a h o m e or food and add a necessary precision to the location of a mate. It should not be surprising if slugs possess a variety of orienting mechanisms that are invoked in a hierarchical manner since the ability to home and aggregate is a key feature in the behavioural control of water balance (Cook 1981) and the management of water dominates much of the biology of this type of animal. These abilities will be especially important in large slugs with restricted access to sufficiently large day-time resting sites. In addition, as with all animals, suitable food and mates need to be reliably located in a variety of conditions.
ACKNOWLEDGMENTS I am grateful to Douglas F o r d and Stephen Porter who assisted in the collection of some of the data reported in this paper.
REFERENCES Baur, B. & Gosteli, M. 1986. Between and within population differences in geotactic responses in the land snail Arianta arbustorum L.) (Helicidae). Behaviour, 97, 147-160. Bailey, S. E. R. 1989. Daily cycles of feeding and locomotion in Helix aspersa. Haliotis, 19, 23 31. Chace, L. 1953. The aerial mating of the great slug. Discovery, 13, 356-357. Chase, R., Pryer, K., Baker, R. & Madison, D. 1978. Responses to conspecific chemical stimuli in the terrestrial snail Achatina fuliea (Pulmonata: Sigmurethra). Behav. Biol., 22, 30~315. Chelazzi, G., Le Voci, G. & Parpagnoli, D. 1988. Relative importance of airborne odours and trails in group homing of Limaeus flavus (Linnaeus) (Gastropoda, Pulmonata). J. moll. Stud., 54, 173-180. Connor, V. M. 1986. The use of mucous trails by intertidal limpets to enhance food resources. Biol. Bull. mar. biol. lab. Woods Hole, 171,548--564.
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Cook, A. 1977. Mucus trail following by the slug Limax grossui Lupu. Anim. Behav., 25, 774-781. Cook, A. 1979. Homing by the slug Limaxpseudoflavus. Anim. Behav, 27, 545-552. Cook, A. 1980. Field studies of homing in the pulmonate slug Limax pseudoflavus Evans. J. moll. Stud., 46, 100-105. Cook, A. 1981. Huddling and the control of water loss by the slug Limax pseudoflavus Evans. Anim. Behav., 29, 289-298. Cook, A. 1985. Functional aspects of trail following in the carnivorous snail, Euglandina rosea Ferussac. Malacologia, 26, 173-181. Cook, A., Bamford, O. S., Freeman, J. D. B. & Teideman, D. J. 1969. A study of the homing habit of the limpet. Anim. Behav., 17, 330-339. Cook, A. & Shirbhate, R. 1983. The mucus producing glands and the distribution of cilia of the pulmonate slug Limax pseudoflavus. J. Zool., Lond., 201, 97-116. Cook, S. B. & Cook, C. B. 1975. Directionality in the trail following response of the pulmonate limpet Siphonaria alternata. Mar. Behav. Physiol., 3, 147 155. Crisp, M. 1969. Studies on the behaviour of Nassarius obseletus (Say) (Mollusca:Gastropoda). Biol. Bull. mar. biol. lab. Woods Hole, 136, 335-373. Crozier, W. J. & Federighi, H. 1925. The locomotion of Limax II. Vertical ascension with added loads. J. gen. Physiol., 7, 415 419. Davies, M. S., Hawkins, S. J. & Jones, H. D. 1990. Mucus production and physiological energetics in Patella vulgata L. J. moll. Stud., 56, 49%503. Denny, M. 1980. Locomotion: the cost of gastropod crawling. Science, 208, 1288-1290. Funke, W. 1968. Heimfindevermogen und Ortstreue bei Patella L. (Gastropoda:Prosobranchia). Oeeologia (Berl.), 2, 139-t42. Gonor, J. J. 1967. Predator-prey reactions between two marine prosobranch gastropods. Veliger, 7, 228 232. Hall, J. R. 1973. Intraspecific trail following in the marsh periwinkle Littorina irrorata. Veliger, 16, 72-75. Lowe, E. F. & Turner, R. L. 1976. Aggregation and trail following in juvenile Bursatella leachiipleii. Veliger, 19, 153 155. MeFarlane, I. D. 1980. Trail-following and trailsearching behaviour in homing of the intertidal gastropod mollusc, Onchidium verruculatum. Mar. Behav. Physiol., 7, 95 108. Paine, R. T. 1963. Food recognition and predation on opisthobranchs by Navanax inermis (Gastropoda: Opisthobranchia). Veliger, 6, 1-9. Trott, T. J. & Dimock, R. V. 1978. Intraspecific trail following by the mud snail Ilyanassa obseleta. Mar. Behav. Physiol., 5, 91-101. Townsend, C. R. 1974. Mucus trail following by the snail Biomphalaria glabrata (Say). Anim. Behav., 22, 171 178. Wareing, D. R. 1986. Directional trail following in Deroceras reticulatum (Muller). J. moll. Stud., 52, 256-258. Wells M. J. & Buckley S. K. L. 1972. Snails and Trails. Anim. Behav, 20, 345-355. Wolf, E. 1927. Geotropism of Agriolimax. J. gen. Physiol., 10, 757-765.
The function of trail following in the pulmonate slug, Limax pseudoflavus ANTHONY COOK Department of Biological and Biomedical Sciences, University of Ulster, Coleraine, Co. Londonderry BT52 1SA, U.K.
(Received 20 February 1991; initial acceptance 24 April 1991; final acceptance 4 October 1991; MS. number: 3732)
Abstract. Trail following in Limaxpseudoflavus Evans is easily observable under laboratory conditions but it is less frequent in the field and its function remains unclear. In experiments allowing the choice between trails and other environmental cues slugs tended to follow trails most frequently when those trails were going in a direction close to that dictated by the other cue. Similarly trails were more often followed when approached at a narrow angle. Trail following was observed as a prelude to courtship. It is concluded that trails provide low priority directional cues and that they are only followed in the absence of other cues or when their direction closely coincides with those other cues. Trail following is a component of food finding, homing and courtship behaviour in the field, but in each case its role may be subordinate to that of airborne odours. Measurements of the quantity of mucus in the trails show that there is no reduction in the secretion of mucus while trail following. Trail following cannot therefore be seen as a mucus-saving tactic.
Mucous trail following is a common feature of the behaviour of gastropods and has been observed, both experimentally and in the field, in a variety of species from a variety of taxa. For some of these species the function of the behaviour is clear (Table I). For other species trail following has been demonstrated in laboratory trials but the possible function of the behaviour in the field remains obscure (e.g. Ilyanassa, Achatina). Limax pseudoflavus is a large slug growing to about 15 cm in length. In common with other large slugs it homes consistently to day-time resting sites which are labelled chemically and in which large groups of animals can be found, often closely packed together. The involvement of trail following in the homing of this slug has been demonstrated in laboratory experiments (Cook 1979) and in the field (Cook 1980) but the frequency and facility with which trail following occurs in the artificial conditions of a laboratory (Cook 1977) do not correspond with the comparative rarity of its occurrence in the field. In the closely related species Limacus (= Limax) flavus L., Chelazzi et al. (1988) have shown that trail following is infrequent when a trail is approached at 90 ~ (about 13~ of contacts resulted in trail following) and that, when allowed a choice between a trail and the smell of conspecifics in a Y-maze, this species oriented with respect to the airborne odour rather than the trail. 0003 3472/92/050813+09 $03.00/0
Clearly trail following is a component of the behavioural repertoire of slugs, but its role in the ecology of these species is not yet fully understood. In this paper I describe a sequence of experiments and observations which have been designed to examine more thoroughly the possible roles of trail following in the life of Limax pseudoflavus. I address the following questions. (1) Do slugs follow trails at random or do they only follow those going roughly in the same direction as the slug is already travelling? (2) When faced with a number of conflicting directional cues, what priority is given to trail following? (3) How frequently and under what circumstances do slugs follow trails in the field? (4) Can slugs make any savings in the amount of mucus deposited during trail following? METHODS
Slugs were collected in the field in the Coleraine area and maintained in lunch boxes, the bases of which were lined with moist tissue. Containers were regularly watered and slugs provided with Readybrek breakfast cereal ad libitum. Trail Choice
The methods used to examine which trails a slug follows were based on those of Cook (1977).
9 1992 The Association for the Study of Animal Behaviour 813
814
Animal Behaviour, 43, 5
Table I. A summary of the gastropod species known to follow mucous trails Taxon
Function
Authority
Natiea lllyanassa
Prey finding ?
Patella
Homing
Collisella Lottia Monodonta Littorina
Feeding and homing ? Trail blazing
Gonor (1967) Crisp (1969) Trott & Dimock (1978) Cook et al. (1969) Funke (1968) Connor (1986)
Opisthobranch
Navanax Bursatella
Prey capture Aggregation
Pulmonate
Physa Biomphalaria Siphonaria spp. Helix Limax Aehatina Onchidium Euglandina
Prosobranch
Genus
Deroeeras Limaeus
? ? Homing ? Homing ? Homing Prey capture ' Courtship Courtship ?
Briefly, experiments, which were conducted on polythene sheets, consisted of allowing slugs to wander one at a time over an open arena. The trails were then visualized by immersion in a suspension of powdered calcium carbonate. The frequency with which trails were superimposed for more than 5 cm and the angle between the original trail and that of the superimposed trail were measured. Unlike the practice of Cook (1977) angles were measured irrespective of the direction in which the trail was laid, rather than as the angle through which the following slug would have to turn in order to follow the trail in the direction in which the trail had originally been laid. Thus angles range from 0 to 90 ~ The Priority Given to Trail Following Experiments were performed in a cubical booth lined with black polythene. The front face was open and illuminated by a 60-W bulb above it. Inside the booth there was a polythene-covered, Perspex disc of 76 cm diameter inclined at an angle of 45 ~ to the front face. A marker slug placed at the base tended to move upwards and away from the light, into the darker interior of the booth. The disc was mounted on a support which allowed it to be rotated by
Wells & Buckley(1972) Hall (1973) Paine (1963) Lowe & Turner (1976) Wells & Buckley(1972) Townsend (1974) Cook & Cook (1975) Bailey (1989) Cook (1977, 1980) Chase et al. (1978) McFarlane (1980) Cook (1985) Wareing (1986) Chelazzi et al. (1988)
known angles around its centre. A slug (the marker) was placed at the bottom of the disc and allowed to move up the polythene sheet. Slugs that made circular movements at the foot of the disc were removed and the experiment restarted. When the marker slug had traversed the diameter of the disc it was removed, the disc rotated if necessary, and a second slug (the tracker) placed at the start point of the marker slug. After the tracker had left the disc, the length of both the marker and the tracker trails and the length of coincident trail were measured. Experiments were performed at 5~intervals until no trail following occurred (25~ A minimum of 10 replicates were performed at each angle. Experimental data were compared with control data generated by superimposing 'sham trackers' (Cook 1977) on marker trails. Sham trackers are animals moving from the same start point but in the absence of a marker trail. Field Observations of Trail Following Previous observations of trail following in the field (Cook 1980) concentrated on the behaviour in a comparatively small area around the home. Very few instances of trail following were observed. Those that did occur were associated with homing
Cook: The function of trail following in slugs
815
56 I1.,1_1~Followed Not followed 50 24 g g ~8 12
0
0-15 o
16-50 o
51-45 ~
46-60 ~
61-75 o
76-90 ~
Angle of approach
Figure 1. A comparison between the distributions of the angles of approach that led to following with those that did not. The two distributions differ significantly (Zz = 45.34, df= 5, P < 0.001). downwind. My objective in the current observations was to attempt to observe trail following in a broader field of view in a more heterogeneous environment. An A u t o m a x 16CC4 1 6 m m time lapse camera fitted with a Vivitar 238 flashgun set to minimum power was set up 2-8 m vertically above a tiled area on which slugs were known to be active. Frames of K o d a c h r o m e 40 were taken every 2 min during the hours of darkness. I recorded the position of slugs from the film using a Specto projector.
Mucus Production During Trail Following An array of clean glass microscope slides was placed on a support mounted at approximately 45 ~ to the vertical in the booth described above and a slug allowed to walk up it. Each slug was allowed to walk up the slides three times. The first time was to provide slides on which there was a single trail. The second time was to lay a marker trail and the final time was to act as a tracker on its own previously laid marker trail. Thus the same animal was used as both marker and tracker. Slides were collected on which there was either a single trail or one that had been followed. The slides were stained in toluidine blue which is known to stain strongly the neutral mucopolysaccharide secreted by the suprapedal gland into the trail (Cook & Shirbhate 1983). The optical density of the stain was measured for both single and double trails using an L K B 2202 Ultroscan laser densitometer. Repeated scans were taken across the width of the trail.
RESULTS
Trail Choice In 44 experiments there were 286 contacts between a slug and a previously laid trail. Each of these contacts represents an opportunity to follow a trail. Following for a distance greater than 5 cm occurred on 92 occasions (32.2%), a frequency similar to that previously reported (Cook 1977). Approaches to marker trails that subsequently led to trail following were made at an angle (X_+ sE) of 41 _+3~ whereas those that did not result in trail following were made at a mean angle of 62 + 2 ~ This is a significant difference ( M a n n - W h i t n e y U-test: U=4649, P<0.001). Figure 1 shows the distribution of angles of approach between those occasions on which following ensued and those on which it did not. It is clear that when a slug approaches a trail at a narrow angle there is a significantly greater chance that it will follow the trail than if it approached the trail at a greater angle.
The Priority Given to Trail Following Trail superimpositions o f less than 5 cm (an approximate body length) were discounted. Comparison of the control (using sham trackers) and experimental data shows that there is a significant degree of trail following only when the marker trail had been rotated through angles of 20 ~ or less. This is true of both the frequency of following and the distance followed (Table II).
Animal Behaviour, 43, 5
816
Table II. The effect of rotating marker trails from the vertical on measures of trail following Angle rotated
% Followed
Distance followed (X_+SE,cm)
0 5 I0 15 20 25 Sham at 0~
86 100 90 50 20 0 23
28 _+3'2 14+2.4 20_+6.1 10_+1.5 7, 17 -13_+3.7
N 45 12 9 5 2 8
*Sham trackers at other angles showed no trail superimpositions greater than 5 cm.
It may be concluded that the slugs follow trails that deviate only a little from the direction in which the slug would travel in the absence of the trail.
Field Observations of Trail Following Figure 2 shows the area of view of the camera. Slugs emerged from a drain and normally foraged on the algae growing on the paved area. During these observations additional food (cucumber slices) were provided at point F 1 (Fig. 2) or at point F2 (Fig. 3). On most nights so many slugs were active that a comprehensive view of trail superimposition was impossible to gain. Further it is likely that with most slugs making journeys between the home and the food, trail superimposition is likely to occur by chance more frequently than would occur in the random movements seen in laboratory observations. On several dry nights the mucus remained as highly refective silvery trails which formed a radiating mass around the food. Individual trails were difficult to discern. Under these circumstances the reporting of trail following must be restricted to nights on which relatively few slugs were active. Figure 2 also shows a selection of the routes taken by slugs on a single night. It is clear that there is no consistency in the behaviour of individual slugs. Some followed the line of the gully, others navigated without apparent reference to trails and yet others made extensive use of previously laid trails. Wind direction during these observations is unknown, but the pattern of flow around the area is likely to be complex owing to the proximity of walls.
Figure 3a shows the details of movements of individual slugs in a smaller area. Most movements did not involve trail following. The movement of slug D, however, involved following the trail of two slugs (A and B). Slug D left the trail of slug B at the point at which that trail was no longer travelling in the direction of the food. I observed 18 slugs moving between the home and the food between 2100 and 2400 hours on a single night. Of the 69 contacts observed between trails, 17 (24%) resulted in trail superimpositions of more than a body length of the following slug. All these instances of trail following were associated with slugs moving towards food. Homing occurred later at night and a similar pattern of movements was observed. Courtship and copulation were observed on three occasions and each time it was preceded by trail following (Fig. 3b). Copulation was extremely brief, lasting no more than two frames (4 rain). The marker slug was not necessarily the individual involved in subsequent copulation (Fig. 3b). During courtship one slug is the trail layer while the other is the trail follower. Although sperm exchange is reciprocal the roles of the two animals involved are not identical. On the three occasions that copulation was observed on film the trail layer turned back on itself to meet the trail follower and copulation took place in a head to head position. After copulation the trail layer executed a loop (see inset in Fig. 3b) before remaining at rest in the vicinity of the site of copulation at which a mucous mat is normally visible.
Mucus Production During Trail Following The quantity of mucus laid in the trail varies greatly. The stained trail is extremely streaky, probably indicating that mucus is redistributed by the foot after being deposited from the suprapedal gland. Density measurements taken across the width of a trail, however, integrate this streakiness and the integrated measures are remarkably constant for any one animal during the course of a single experiment. Five animals completed sufficient of the experiment to give useful measurements. Density readings of single and joint trails were made and are shown in Table III. During trail following by slug 3 (Table III) instances occurred in which two separate trails were deposited on the same microscope slide before and
Cook: The function of trailJbllowing in slugs
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Figure 2. The area of activity of slugs in the field. The squares are quarry tiles (approximately 23 x 23 cm) bordered to the left by a wall and at the bottom by a gully leading to a drain at the bottom left hand corner in which most slugs had their day-time resting sites, Food was placed at F 1. In later observations food was placed at F2. Selected tracks of slugs are shown from a single night. Trails were laid in more or less alphabetical order. A ( - - -) A direct journey from home to the food. B ( ) A return from the food to the home almost entirely along the outward trail of A. C G . . -) A brief excursion not involving trail following. D (. 9.) A slug making an extensive journey after leaving the food. E G . . . . . -) A slug making a return to home from outside the field of view, along a gully and making circular movements involving retracing its own trail. F ( - - - ) A second slug making a return journey from outside the field of view along the gully and following the trails of slugs E (forwards) and C (backwards). G ~ ~ A slug returning from the food following the trails laid by E and F (forwards) and C (forwards). H (...) A slug crossing the trails of other slugs but not interacting with them.
after trail following. These trails were treated together in the densitometer a n d gave a j o i n t reading o f 28.4_+2.1 (.~_+SE, N = 14) c o m p a r e d with a density o f 28.1-t-3.6 ( N = 10) when the trails were
superimposed. Clearly the quantities o f m u c u s deposited during trail following a n d d u r i n g n o r m a l m o v e m e n t are similar over the short periods o f trail following observed d u r i n g these experiments.
818
Animal Behaviour, 43, 5
(a)
All
~V
~
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1
A f-Figure 3a
DISCUSSION Trail following appears to be a low priority behaviour for most slugs. Chelazzi et al. (1988) found that the airborne odours o f a conspecific were preferred to trails, when slugs were offered a choice and I have previously (Cook 1980) found that trails were used for homing only when the individuals were isolated from airborne odours as a result of approaching home from a down-wind direction. The measure of the angle turned by slugs onto a trail used by Cook (1977) gives a false impression of the deviation that slugs make at the commencement of trail following since it records the change in direction necessary to follow in the same direction as the individual laying the trail. The measure used here of the angle at which the slug approaches the
trail (Fig. 1) clearly shows that the slugs follow trails approached at low angles more frequently than those approached at right angles. This concurs closely with the results of Chelazzi et al. (1988) who showed a low frequency of following at angles around 90 ~ but that, on crossing a trail there is a deflection in the direction of travel towards the trail, even though this does not always result in trail following. Such a deflection at small angles of approach could result in trail superimposition. In Fig. 3a slug D turns towards the trail of slug C after crossing it but does not subsequently follow it. It almost immediately encounters the trail of slug A and another turn results in trail following. It might be that turning on crossing a trail allows a sampling process to occur which results in the following slug orienting gradually with respect to a series of
Cook: The function o f trailfollowing in slugs
819
(b)
F2
9
.r4
l
Figure 3b Figure 3. Movement of slugs (a) to the food and (b) prior to copulation. Slugs are labelled with letters. The numbers refer to the frame number (at 2-min intervals) appropriate to that location of each slug. The precise locations of slugs have been slightly displaced during trail following for clarity. (a) Details of the movements of four slugs between the gully and the food at F2. Slugs A and C moved direct from the food to the gully. Slug B made a short loop which involved a short length of trail following before coming to rest from frame 6 onwards. Slug D'sjourney towards the food overlapped with the trails laid by A (backwards), branched onto that laid by B (backwards) before leaving that trail to travel directly to the food. (b) Details of the movements of three slugs involved in courtship and copulation. Slug A moved to point X where it stayed for 30 min (15 frames). Slug B moved from the food and contacted the rear of slug A which then started to move (frame 6). Slug C closely followed slug B for most of its journey and then followed slug A with which it then mated (frame 12) at point Y. Inset: a sketch of the routes taken after copulation. Slug B continued on its way while slug A remained at rest after executing a short loop.
similarly oriented trails r a t h e r t h a n just one. Could this be the slug version o f the o p i n i o n poll in which a n u m b e r of trails influence the decision to follow just one? T h e response to gravity (geotaxis) has been d e m o n s t r a t e d in a n u m b e r o f gastropods (Agriolimax (=Deroceras): W o l f 1927; Arianta arbustorum: B a u r & Gosteli 1986; Lirnax: Crozier &
Federighi 1925). In the present experiments the slugs were given a n u p w a r d incline a n d a d a r k area to a p p r o a c h a n d a trail t h a t deviated by a varying angle from t h a t direction. Trail following occurred at a m u c h higher frequency w h e n all three o f these orienting cues were acting in the same direction with between 80 a n d 9 0 % o f tracker trails being followed w h e n the m a r k e r trail was r o t a t e d by less
Animal Behaviour, 43, 5
820
Table III. Mean integrated densitometer readings (in arbitrary units) across the trail width (_+SE(N)) for fiveslugs Slug no. 1 2 3 4 5
Double trail 86.1+5.7(26) 65.9_+5.6(23) 28.6_+3.6(10) 37.9+__1.8(25) 50.8_+3.9(26)
Single trail
Ratio double:single
27.2+3.9(13) 27.6_+3.0(22) 14.2+1.1(28) 18.0_+1.7(26) 28.1_+4.1(25)
3.16 2.39 2.02 2.10 1.81
Double trails are those that had been followed, single trails are those not showing trail following. The double: singleratio should be 2 if no change in mucus production during trail following occurred. than 10~ When the trail deviated by more than this from the vertical both the frequency and the distance followed fell until, by angles of 25 ~ no trail following was observed at all. Again it may be concluded that trail following occurs only if the trail encountered is going in roughly the same direction as the following slug. The field observations of the movements of slugs reveal that trail superimposition can be more frequent than previously reported (Cook 1980). These previous observations involved relatively few slugs moving over a restricted area. Whether trail superimposition at the density of movements sometimes observed in the field is always equivalent to trail following is open to doubt. The density of trails around the food in particular must make trail superimposition inevitable. Nevertheless measurements taken at the beginning of a night on which relatively few slugs were active show that 24% of contacts between trails resulted in subsequent trail following. This compares with the 32% observed under laboratory conditions. No consistent directionality in general trail following was noted. When copulation occurred after trail following, however, the trails were followed for relatively long distances and were always followed towards the laying slug. The directionality in trail following preceding copulation has been noted in other slugs (Wareing 1986). Whether this directionality indicates that the trail contains directional information or that there is a second, directional, perhaps airborne, cue is not known. It may be significant, however, that on one occasion in the present observations, the trail layer was not eventually involved in the ensuing copulation (Fig. 3b). On this occasion the directional cue might have
been provided by one individual and the trail by another. If the following of trails is of such a low priority in normal behaviour why follow them at all? One possibility is that the slug may make use of the mucus that has already been laid to aid its locomotion. Denny (1980) reported that as much as 35% of the energy costs involved in adhesive locomotion in slugs is a direct result of the need to secrete mucus. Davis et al. (1990) working with limpets, Patella vulgata, arrived at a similarly high figure for the cost of mucus production (23 % of the energy ingested is used in mucus secretion). Trail following could be viewed as a mucus-saving tactic if following slugs secreted less mucus than those that laid the original trail. The measures made of the density of stained mucopolysaccharides in the trails before and after trail following show that double trails stain roughly twice as intensely as single trails (Table III). This indicates that no savings have been made by the following slug. While some savings of other components cannot be ruled out, it appears that the major component of the trail is not husbanded during trail following. The current experiments involve relatively short lengths of trails being followed and it might be that the control over mucus production cannot be accomplished over such a short time. Since trail following is observed only over relatively short lengths of marker trail in most gastropods it appears that mucus saving is not a major consideration. Pulmonates are hermaphrodites and these slugs exchange spermatophores during copulation. Details of courtship have not been reported for L. pseudoflavus and the frame frequency of one per 2 min does not allow a detailed description here, except to confirm that courtship and copulation are terrestrial rather than aerial as in L. maximus L. (Chace 1953). With such infrequent observations it is not even certain that copulation was completed, and comments on the apparent brevity of copulation in this species are therefore premature. The asymmetry of the behaviour of the two animals after copulation is curious. Clearly the animals have different roles initially, one being the trail layer while the other is the follower. Why the differentiation in roles should persist after copulation when the trail layer remains close to the mucous mat and the other slug moves rapidly away is not known. In conclusion it is clear that, while trail following can be demonstrated in the laboratory in
Cook: The function o f trail following in slugs L. pseudoflavus, it is not a high priority behaviour in natural circumstances. Turning to follow a trail that is going roughly in the same direction as the following slug will bring the follower closer to the source or the destination of the trail layer. The decision to follow a particular trail may be influenced by a variety of other factors such as encounters with previous trails and other directional stimuli such as airborne odours and the directions of illumination, gravity and wind. Thus trail following may contribute an additional cue to the position of a h o m e or food and add a necessary precision to the location of a mate. It should not be surprising if slugs possess a variety of orienting mechanisms that are invoked in a hierarchical manner since the ability to home and aggregate is a key feature in the behavioural control of water balance (Cook 1981) and the management of water dominates much of the biology of this type of animal. These abilities will be especially important in large slugs with restricted access to sufficiently large day-time resting sites. In addition, as with all animals, suitable food and mates need to be reliably located in a variety of conditions.
ACKNOWLEDGMENTS I am grateful to Douglas F o r d and Stephen Porter who assisted in the collection of some of the data reported in this paper.
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Cook, A. 1977. Mucus trail following by the slug Limax grossui Lupu. Anim. Behav., 25, 774-781. Cook, A. 1979. Homing by the slug Limaxpseudoflavus. Anim. Behav, 27, 545-552. Cook, A. 1980. Field studies of homing in the pulmonate slug Limax pseudoflavus Evans. J. moll. Stud., 46, 100-105. Cook, A. 1981. Huddling and the control of water loss by the slug Limax pseudoflavus Evans. Anim. Behav., 29, 289-298. Cook, A. 1985. Functional aspects of trail following in the carnivorous snail, Euglandina rosea Ferussac. Malacologia, 26, 173-181. Cook, A., Bamford, O. S., Freeman, J. D. B. & Teideman, D. J. 1969. A study of the homing habit of the limpet. Anim. Behav., 17, 330-339. Cook, A. & Shirbhate, R. 1983. The mucus producing glands and the distribution of cilia of the pulmonate slug Limax pseudoflavus. J. Zool., Lond., 201, 97-116. Cook, S. B. & Cook, C. B. 1975. Directionality in the trail following response of the pulmonate limpet Siphonaria alternata. Mar. Behav. Physiol., 3, 147 155. Crisp, M. 1969. Studies on the behaviour of Nassarius obseletus (Say) (Mollusca:Gastropoda). Biol. Bull. mar. biol. lab. Woods Hole, 136, 335-373. Crozier, W. J. & Federighi, H. 1925. The locomotion of Limax II. Vertical ascension with added loads. J. gen. Physiol., 7, 415 419. Davies, M. S., Hawkins, S. J. & Jones, H. D. 1990. Mucus production and physiological energetics in Patella vulgata L. J. moll. Stud., 56, 49%503. Denny, M. 1980. Locomotion: the cost of gastropod crawling. Science, 208, 1288-1290. Funke, W. 1968. Heimfindevermogen und Ortstreue bei Patella L. (Gastropoda:Prosobranchia). Oeeologia (Berl.), 2, 139-t42. Gonor, J. J. 1967. Predator-prey reactions between two marine prosobranch gastropods. Veliger, 7, 228 232. Hall, J. R. 1973. Intraspecific trail following in the marsh periwinkle Littorina irrorata. Veliger, 16, 72-75. Lowe, E. F. & Turner, R. L. 1976. Aggregation and trail following in juvenile Bursatella leachiipleii. Veliger, 19, 153 155. MeFarlane, I. D. 1980. Trail-following and trailsearching behaviour in homing of the intertidal gastropod mollusc, Onchidium verruculatum. Mar. Behav. Physiol., 7, 95 108. Paine, R. T. 1963. Food recognition and predation on opisthobranchs by Navanax inermis (Gastropoda: Opisthobranchia). Veliger, 6, 1-9. Trott, T. J. & Dimock, R. V. 1978. Intraspecific trail following by the mud snail Ilyanassa obseleta. Mar. Behav. Physiol., 5, 91-101. Townsend, C. R. 1974. Mucus trail following by the snail Biomphalaria glabrata (Say). Anim. Behav., 22, 171 178. Wareing, D. R. 1986. Directional trail following in Deroceras reticulatum (Muller). J. moll. Stud., 52, 256-258. Wells M. J. & Buckley S. K. L. 1972. Snails and Trails. Anim. Behav, 20, 345-355. Wolf, E. 1927. Geotropism of Agriolimax. J. gen. Physiol., 10, 757-765.