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Function of mucus secretion by lamellose ormer, Haliotis tuberculata lamellosa, in response to starfish predation
Animal Behaviour 78 (2009) 1189–1194
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Function of mucus secretion by lamellose ormer, Haliotis tuberculata lamellosa, in response to starfish predation Francesco Bancala`* ¨ r Marine Biologie, Isola del Giglio, Italy Institut fu
a r t i c l e i n f o Article history: Received 21 October 2008 Initial acceptance 5 January 2009 Final acceptance 8 August 2009 Available online 10 September 2009 MS. number: 08-00679R
Gastropods subjected to predation from asteroids have a number of different defensive mechanisms to prevent and defend against attacks. I investigated the production and function of mucus in the lamellose ormer in response to attacks by the spiny starfish, Marthasterias glacialis (Asteriidae, Asteroidea). Production of mucus from respiratory pores on the shell helped obscure the direction of escape of the gastropod from the starfish. The mucus also had repellent properties against this predator and there was some evidence of it being an intraspecific alarm. The results suggest that mucus secretion is part of a larger stereotyped escape response, in which the mucus has an olfactory interference role. Ó 2009 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Keywords: asteroid escape response Haliotis tuberculata lamellosa lamellose ormer mucus secretion Marthasterias glacialis spiny starfish stereotyped behaviour
To prevent predation by asteroids, many gastropods have specific types of response (Bullock 1953) with different functions: (1) avoidance (Mackie 1972; Phillips 1975), (2) repulsion (Thompson 1960; Schmitt 1981) or (3) escape (Schiemenz 1895; Margolin 1964). Haliotidae (ormers) in particular perform a specific (Bennet 1927; Bullock 1953; Feder 1963) and well-developed escape behaviour, which is composed of a sequence of different types of recognizable subunits (Parsons & Macmillan 1979) and involves the secretion of mucus, whose function is unclear. Initially the mollusc extends and moves its tentacles (tentacular sweeping), a behaviour that seems to be of detective value (Montgomery 1967). Once in contact with starfish tube feet, the tentacles retract and a limb of the epipodium on the stimulated side is raised and extended to cover part of the shell (covering). If contact continues, the ormer lifts its shell (mushrooming) and turns it violently from one side to the other (twisting) to break the starfish’s grip. Some tube feet can be pulled out by this movement, consequently inducing a temporary retreat of the starfish. Finally, the ormer moves directly away from the predator (running;
* Correspondence and present address: F. Bancala`, Via Lucino 44, 6932 Breganzona, Switzerland. E-mail address: [email protected]
MacGinitie & MacGinitie 1949; Bullock 1953; Montgomery 1967; Parsons & Macmillan 1979). The timing and sequence of these subunits of the escape response are stereotyped and the response depends on stimuli that are characteristic of the different phases of the attack (Bullock 1953; Parsons & Macmillan 1979). Indeed, the response of gastropods such as ormers to contact by foreign objects other than tube feet (e.g. crab claws, seaweeds, snail shells or soft parts, rocks, human finger or implements) is never to mushroom up but to clamp down (Bullock 1953). Moreover, during an attack each subunit of the escape response seems to be less efficient if expressed in the wrong context or order (Parsons & Macmillan 1979). For instance, the twisting of the shell appears to break the hold of the asteroid’s tube feet (Bennet 1927; Montgomery 1967). Consequently, a running response is less efficient if expressed before twisting, when the asteroid’s tube feet are still holding on to its shell, than after breaking the hold with the twisting response. The nature and function of mucus secretion by ormers during the escape response have not been investigated (Montgomery 1967). As a defence against predation, mucus could have (1) an intraspecific alarm function or (2) a repellent effect on starfishes. Indeed, Montgomery (1967) highlighted the fact that the mucus of Haliotis assimilis is enough to induce a full escape response in resting conspecifics. Moreover, although no exhaustive study exists on the chemistry of the mucus secreted by Haliotis, evidence for the
0003-3472/$38.00 Ó 2009 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.anbehav.2009.08.008
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presence of a chemical deterrent in the epipodium of Haliotis ruber has been found, suggesting that characteristics of mucus could be useful to avoid rapid diffusion (and dilution) of the deterrent contained inside it, thus maintaining its efficiency (Day et al. 1995). Finally, (3) Day et al. (1995) suggested that ormers could use the mucus to interfere with the starfish’s olfactory system to obscure their escape direction, in a way analogous to that described by Denny (1989) for squid. Indeed, Day et al. (1995) observed that, during attacks by the starfish Coscinasterias calamaria, the mucus of H. ruber was ejected through one of the respiratory pores and diffused very slowly into the water. Afterwards, the starfish stopped its attack or moved slowly in another direction. This may be explained if the smell of the ormer, associated with the mucus which sticks to the starfish arm in contact with the shell, possibly obscures the actual position of the ormer (Day et al. 1995). Indeed, starfishes have been shown to follow chemical traces, by sensory tentacles situated on the tips of their arms, to find and attack their prey (Rochette et al. 1994). My aim in the present study was (1) to investigate the role of mucus secretion by the lamellose ormer, Haliotis tuberculata lamellosa, during a spiny starfish, Marthasterias glacialis, attack under experimental conditions; (2) to examine the position of mucus secretion within the interaction hierarchy described by Parsons & Macmillan (1979); and (3) to investigate the three main hypotheses for the function of mucus secretion by testing the reaction of ormers to mucus, the reaction of starfishes to mucus and the influence that mucus has on the time that a starfish needs to locate and reach an ormer that cannot move. METHODS Animal Collection and Maintenance Experiments were carried out at Giglio Island, Tuscan Archipelago, Italy, in the Institut fu¨r Marine Biologie (IfMB). Animals were sampled in September 2002 by SCUBA. Twenty H. tuberculata lamellosa were taken from a wild population living on a cone of stones (block size ranging from 1 dm3 to 1 m3), situated between 5 and 15 m depth, around the base of an old pylon (42 21059.0000 N, 10 520 29.3100 E). The shell size of the ormers ranged between 18 and 49 mm (mean 36.7 mm). The 14 individuals of M. glacialis, as well as 14 individuals of Echinaster sepositus, were collected in three other sites (site 1: 42 230 24.4000 N, 10 530 7.6600 E; site 2: 42 200 31.3100 N, 10 530 9.8500 E; site 3: 42 2301.6300 N, 10 520 41.4100 E), at depths between 5 and 40 m, from granite substrate. This species is known to have a certain vertical mobility (Riedl 2005) and consequently it is unlikely that this depth range could have influenced the results. The size of the starfishes ranged between 24 and 42 cm (mean 31.7 cm); the sex and age of the individuals of both species were not determined. Haliotis tuberculata lamellosa were kept in four aquaria (25 25 cm and 40 cm high), each containing five individuals, until required for the experiment. The holding tanks were filled with sea water and provided with filters and oxygenation systems. During capture of the ormers rocks were collected and then placed inside the holding tanks. Algae growing on the rocks provided a food source for the ormers, and were replaced with rocks containing fresh algae at weekly intervals. Water was fully changed every 3 days. The temperature of the water was 20 C and the light:dark regime was 14:10 h. To distinguish between individuals, each ormer was numbered on the shell using a small piece of paper (3 3 mm) fixed with odourless silicon, to avoid the tag influencing the dynamics of capture events. Starfishes were held in a cage (50 50 cm and 100 cm high) in the sea, near the institute at a depth of 6 m. To keep them in the
best condition and to reduce the risk of automutilation (C. Valentin, unpublished data) the cage was divided into 20 separate boxes, each containing one M. glacialis. They were generally fed once per week with mussels, Mytilus edulis, but the feeding rate could change depending on experimental requirements (e.g. to obtain a starving status the starfish was not fed for 2 weeks). Experimental Procedures The aquaria (N ¼ 10) used for the experiments (25 25 cm and 40 cm high) were scrubbed with ethanol and filled with 20 litres of filtered sea water after each experiment. Each individual tested (both ormer and starfish) was randomly assigned to control or treatment groups with a maximum of one trial per day. Once introduced into the experimental aquarium, each animal (ormer or starfish) was allowed to acclimatize for 30 min. After testing, the individuals were kept in separate tanks, away from untested animals. The time between experiments was generally 1 week. After the study, captive animals were released in the same area where they were collected 2 months earlier. The ormers were exposed to stressful situations (a predator and stimuli of predators) but no individual died during an attack. Manipulation of individuals of M. glacialis involved food limitation and diet modification, following a randomized plan; no asteroids died of hunger. Mucus Sampling For experiments 2a, b and c, I required mucus secreted by H. tuberculata lamellosa. Using a syringe (20 ml) without a needle, I collected mucus from the respiratory pore of each ormer after repeated stimulation with a tube foot of M. glacialis. Stimulation was particularly effective on the mantle or on the respiratory tentacles of the gastropod. To avoid any effect of this prestimulation on the mucus production response, I did this only after testing ormers for the mucus response (experiment 1). Moreover, after the production of the sampled mucus for experiments 2a, b, the ormer was allowed to rest for a day. This was not the case for the olfactory illusion test (experiment 2c), where the use of the same individual that produced mucus was needed for control purposes. In this case, however, ormers were not the subject of the experiment and their behaviour could not influence the results. Mucus was used as the treatment, whereas the control substance was sea water containing the smell of the same ormer. I obtained the control substance by sampling the water of the small tank containing the ormer before starting the stimulation for mucus secretion. The treatment or control was then applied with a syringe (20 ml); its exit flow was roughly 1 ml/s. Mucus Secretion In experiment 1, I investigated mucus secretion by ormers in the presence of a predatory asteroid. I tested 20 individuals of H. tuberculata lamellosa, each of them in both treatment and control tests. Five individuals of M. glacialis (predator) were used as the treatment and five individuals of E. sepositus (nonpredator) as the control. Consequently each starfish was used four times in a randomly defined order. The ormer was introduced into an aquarium 30 min before the experiment to acclimatize; this was also the control period. Once the acclimatization time was passed, an asteroid was introduced into the same aquarium and left there for 30 min. During this time, I recorded how many ormers reacted to attacks with mucus secretion. The prediction was that this number would be greater if the asteroid was predatory than if it was deposit feeding (nonpredatory).
F. Bancala` / Animal Behaviour 78 (2009) 1189–1194
I also noted type, sequence and duration of each subunit of response for each attack and individual. To establish whether a correlation exists between mucus secretion and other subunits, such as twisting and running, I calculated frequencies of mucus ejection after twisting, and before running for each individual:
ft ¼ Mt =T where ft is the frequency of mucus ejection after twisting. Thus, M(t) is the total number of mucus ejections (made after the twisting behaviour) per total number of twisting behaviours, T.
fr ¼ Mr =R where fr is the frequency of mucus ejection before running. Thus, M(r) is the total number of mucus ejections (made before running behaviour) per total number of running behaviours, R. Observed average frequencies were then calculated for both associations and compared to a stochastic frequency of 0.50 using a test of one proportion to verify association between these subunits. Function of Mucus Intraspecific alarm In experiment 2a, I examined the possible function of mucus as a signal of danger, inducing escape responses in other ormers. Each of 20 individuals was used in both treatment and control tests. The control or treatment substance (5 ml) was introduced 1 cm from the anterior side of an ormer resting in an aquarium (N ¼ 10). During the following 2 min I observed the reaction of the ormer. If the ormer did not respond, a negative result was assigned to that individual and interpreted as ‘no detection’ of alarm signal in the substance. If the ormer responded (from rest to movement or escape behaviours), the result was positive. I recorded the number of ormers that moved away from the stimulus. I predicted more positive responses would be observed in the presence of mucus than in the presence of the control substance (sea water). Repellent The objective of experiment 2b was to investigate whether the ormer’s mucus has a repellent effect on spiny starfish. Each of 14 M. glacialis was used in both treatment and control tests. Starfishes have a large number of sensory cells within the epidermis which are the primary sensory receptors and probably function for the reception of light, contact and chemical stimuli (Barnes 1980). They are particularly present on the suckers of tube feet, on the tentacles and along the margins of the ambulacral groove, where up to 70 000 sensory cells/mm2 occur (Barnes 1980). Consequently, some regions of the body, such as the oral perimeter and the arm distal extremity, are more sensitive than others. For this reason, I tested the effect of mucus on these two regions separately. An M. glacialis was introduced into an aquarium. To induce the starfish to rest on the vertical glass (for a standard observation of the tube feet activity) I created a dark zone by placing a small board on the short side of the aquarium. A spiny starfish was then introduced into the aquarium 30 min before the experiment. The starfish tended to rest in the dark zone where I started the experiment. When the individual was at rest, a treatment or control substance (5 ml) was sprayed towards the sensory tube feet on the tip of the arm or on the oral perimeter. To reach the oral perimeter without disturbing the starfish, I slipped a needle mounted on the syringe between the animal and the aquarium pane. I then observed the reaction of the tube feet (organ level) and the reaction of the whole animal (organism level). Reactions were
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classified into three distinct types: positive (movement towards the source of the stimulus), neutral (no significant change in activity) or negative (movement away from the source of the stimulus). I predicted that both the number of individuals that withdrew and the number of individuals whose tube feet retreated from the source of the stimulus would be greater after application of mucus than the control substance (sea water). Olfactory interference The objective of experiment 2c was to investigate whether there was an influence of mucus on the capture time of ormers by starfishes. This was considered to be an indicator of starfishes’ olfactory performance. During this experiment 14 M. glacialis were used for both treatment and control tests following a randomized order. I induced starfishes to rest at a standard location for the beginning of the experiment by creating a dark zone as described above. A spiny starfish was introduced into an aquarium 30 min before the experiment. In the meantime an ormer was introduced into a small container made of metallic net, so as to let its smell diffuse into the tank. The diffusion, which is by definition stochastic (Crank 1975; Lauffer 1989; Price 2005), of odour molecules towards the starfish is very gradual in perfectly still water (Dittmer et al. 1996; Grasso 2001). The container was then placed into the aquarium, on the opposite side to the dark zone, 30 cm from the starfish. At the same time, 5 ml of the control (sea water) or mucus were ejected with the syringe around the starfish arm. To avoid any possible reaction of the starfish caused by contact with the syringe, I ejected the substance 1 cm from the starfish. Timing commenced when the control substance or mucus was applied and stopped when the starfish touched the container holding the ormer. I predicted that the time the starfish took to reach the ormer would be greater in the presence of mucus than in the presence of sea water. Statistical Analysis For statistical analysis I used S-PLUS 2000 (Insightful Corp., Seattle, WA, U.S.A) and SYSTAT (SPSS Inc., Chicago, IL, U.S.A). The McNemar’s test was used to test the hypotheses except for the olfactory illusion experiment (experiment 2c) for which I used a paired t test and an F test for entered parameters. The McNemar’s test tests the hypothesis that two proportions are equal. A common design that uses this analysis is when subjects make two dichotomous responses. For example, each subject is measured once after treatment A and again after treatment B. The response is ‘1’ if the event of interest occurs or ‘0’ otherwise. The assumptions are: (1) the two groups are related or dependent (e.g. each individual may be measured in two different circumstances); (2) every individual is classified according to whether the events of interest occur in both circumstances, one circumstance only, or in neither. The assumptions of the paired t test are: (1) the data are continuous (not discrete); (2) the data (i.e. the differences for the matched pairs) follow a normal probability distribution; (3) the sample of pairs is a simple random sample from the population (each individual in the population has an equal probability of being selected in the sample). The F test for entered parameters was used to test the hypothesis of same variance. All the assumption of the tests were met. All tests were two tailed. RESULTS Mucus Secretion Among 20 tested individuals 15 secreted mucus during at least one attack of M. glacialis. Indeed, M. glacialis stimulated mucus
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secretion in H. tuberculata lamellosa significantly more than the control species E. sepositus (McNemar’s test: P < 0.01). In addition, each individual showed the ability to produce this substance during stimulation with an isolated tube foot of M. glacialis. However, contact with a starfish tube foot was not enough to evoke immediate mucus secretion by the ormer. Indeed, the mucus was rarely expelled during the first contact with M. glacialis and usually many attacks were necessary to stimulate a reaction. Moreover, during the experiment I observed that the stimulus was particularly efficient if the tube foot touched the respiratory tentacles, more so than any other part of the ormer. In fact, mucus was usually secreted once the starfish tube feet had already been fixed onto the ormer’s shell and it is possible that some tube feet touched one or more respiratory tentacles during that step of the attack. Mucus was always ejected from one or two respiratory pores of the shell. No ormer (0/20) secreted mucus during experiments using E. sepositus. Other ‘escape’ manoeuvres (tentacle activity, covering, mushrooming, running and twisting), already described in other gastropods (e.g. H. rufescens, H. assimilis), were observed in each individual of H. tuberculata lamellosa when stimulated by M. glacialis, whereas E. sepositus elicited only covering responses (8/20), and very rarely some twisting responses (3/20). Finally, no mucus production was observed during the acclimatization period. The average frequency of mucus ejection after twisting was 0.85 and before running was 0.77 Both averages are significantly different (one proportion test: after twisting: Z ¼ 1.84, N ¼ 18, P ¼ 0.033; before running: Z ¼ 1.93, N ¼ 21, P ¼ 0.026) from a stochastic average of 0.50, indicating the existence of the associations ‘twisting–mucus’ and ‘mucus–running’. Function of Mucus Intraspecific alarm Of the 20 ormers 15 made escape responses in the mucus treatment group. Only one of the ormers responded positively when the control stimulus was applied. The difference is highly significant (McNemar’s test: P < 0.01). However, the reaction time observed was very variable (3–118 s). Repellent No repulsion effect was observed when the arm extremities of M. glacialis were stimulated, either in the treatment or in the control groups, both at the organ level and at the whole organism level (Fig. 1a, b). In contrast, tube feet (13/14 individuals) and individuals (8/14) showed an attraction to mucus (Fig. 1a, b). This reaction occurred nine and four times, respectively, when ormer-smelling sea water was used. Indifference to mucus was observed once at the organ level and six times at the organism level, while when the control substance was used, it was recorded five and 10 times, respectively (Fig. 1a, b). However, differences between treatments were not significant (two-tailed McNemar’s test: tube feet: P ¼ 0.10; individuals: P ¼ 0.10). On the other hand, stimulation of the oral region produced a negative reaction at the tube feet level (7/14 individuals) and at the whole animal level (5/14; Fig. 1c, d). No similar reactions were observed during the control treatment (Fig. 1c, d). Attraction to mucus appeared four times both at the organ level and at the organism level; the same reaction was observed eight and 11 times, respectively, in the presence of the control substance (Fig. 1c, d). Indifference to mucus was shown three times by tube feet and five times by whole individuals, while it occurred six and three times, respectively, when ormer-smelling sea water was used (Fig. 1c, d). The difference between test and control is highly significant (two-tailed McNemar’s test: tube feet: P < 0.01; individual:
P < 0.01). The difference in mucus stimulation response between arm extremities and oral region is also highly significant (two-tailed McNemar’s test: P < 0.01). Olfactory interference Treatment with mucus significantly increased (paired t test: t1 ¼ 5.58, P < 0.01) the time M. glacialis spent capturing an ormer, nearly doubling it (Fig. 2). Moreover, the standard error changed from the control group (54 s) to the test group (215 s) and the difference in variance is highly significant (F test for entered parameters: F ¼ 3.98, N ¼ 14, P ¼ 0.02). DISCUSSION Experiment 1 provided evidence that H. tuberculata lamellosa secretes mucus when it is attacked by the predatory starfish M. glacialis but not in the presence of a nonpredatory starfish. That kind of behaviour has already been described for other Haliotidae (Montgomery 1967; Parsons & Macmillan 1979; Day et al. 1995), as well as for other gastropods (Rice 1985). As multiple predatory contacts were needed, the stimulation threshold for the secretion of mucus in other parts of the body could be lower. That might be because of a higher density of the mucus or a different kind of receptor, more sensitive to predatory starfish ‘odours’, such as the saponin M2 (Mackie et al. 1968, Leroy 1987). If that is the case, the production of mucus may have a certain cost to the ormer and may be secreted only when it is really needed. In fact, the production of defensive substances in gastropods is limited by their utilization, which means that they can run out of defensive substance if they secrete it too many times in a short period (Thompson 1960; Day et al. 1995). Moreover, if mucus of males contains sperm (Montgomery 1967) the cost of production could be even higher. The results of experiment 2a indicate that mucus of H. tuberculata lamellosa is recognized as a sign of danger among conspecifics. That effect has already been observed in other Haliotidae by Montgomery (1967). However, alarm may not be the principal function of the mucus. If alarm was the primary function, one would predict (1) a more frequent usage of mucus, (2) the secretion to start as soon as the predator has been noticed and (3) the alarm signal should be contained in a substance much more diffusible than mucus. Rochette et al. (1998) showed that the bivalve Buccinum undatum is more sensitive to predatory asteroids in the presence of odours of stressed, wounded or dead conspecifics. Thus, if this characteristic is also valid for H. tuberculata lamellosa, sensitivity to mucus could reveal important information about the intensity of prey–predator relationships in any ormer population that is under the predatory pressure of carnivorous asteroids. Experiment 2b highlighted a difference in response to mucus between the oral region and the arms extremities, a significant repellent effect on M. glacialis being detected only at the oral level. However, the retreat of the tube feet as well as of the entire individual could be caused by an unpleasant taste rather than a response to harmful chemicals contained within the mucus. That appears to be confirmed by the negative test on the arm tips. Indeed, the starfish would be expected to move away from the mucus if it is chemically aggressive, whereas the opposite happened. Rice (1985) observed that when the gastropod Trimusculus reticulatus (Pulmonatae) is attacked by the starfish Pisaster ochraceus, it secretes whitish mucus, which is able to anaesthetize the tube feet of P. ochraceus temporarily; but the starfish did not suffer a permanent effect, even after prolonged or repeated exposure to the mucus. Additionally, no sign of illness or injury was seen in individuals that ate T. reticulatus. That too may be the case in the present study although no anaesthetic effect was
F. Bancala` / Animal Behaviour 78 (2009) 1189–1194
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Figure 1. Number of individuals of M. glacialis reacting with repulsion (white bar), indifference (grey bar) and attraction (black bar) to stimulation with mucus or a control (sea water) (a) local response of tube feet stimulated on arm extremity; (b) response of the whole individual stimulated on arm extremity; (c) local response of tube feet stimulated on oral region; (d) response of the whole individual stimulated on oral region.
observed. Day et al. (1995) observed that the mucus of H. ruber did not induce any repulsion response in Coscinasterias calamaria, and its mucus had a pH close to that of sea water (7–8.3). Conversely, the defensive secretions described by Thompson (1960) among
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Figure 2. Mean þ SD capture time of 14 individuals of M. glacialis tested with mucus (black bar) and with sea water (white bar).
three families of gastropods (Pleurobranchidae, Philinidae, Lamellariidae) were acidic with a pH close to 1. Although other studies document the repellent properties of many gastropods, they deal with passive characteristics (Schmitt 1981; Faulkner & Ghiselin 1983), which do not involve the secretion of mucus. Protein components, as well as sulphuric acid or mucopolysaccharides, are among the substances secreted by gastropods (Nicole 1964; Barnard 1983). These substances are produced by different organs (Day et al. 1995) and it is possible that cosecretion of these components is necessary for chemical defence to be successful (Thompson 1960). Therefore, it is possible that in my experiments factors other than mucus could have been present, which possibly lowered the repellent effect of the mucus. However, the attractive rather than the repellent effect of mucus on the arm tips could be easily explained if considered in the context of another function of the mucus: an olfactory decoy, which by definition has to be attractive. Indeed, this study is the first to show that mucus appears to impede or confound olfactory reception of a predatory starfish (experiment 2c). The confounding effect also appeared in experiment 2b. Starfishes were attracted by mucus when this was ejected onto arm extremities, and tended to follow the direction of the arm coated with mucus, which was generally different from the escape direction of the ormer. This mechanism is easier to understand if we consider that mucus secretion has a hierarchical position within a behavioural sequence, as confirmed by the results of experiment 1, whose final goal is to allow escape at an advanced stage of attack. As soon as the starfish reaches the shell of the ormer with its arm(s), it fixes its tube feet onto the ormer. During this stage, the starfish prevents the ormer escaping and tries to remove it from the substratum. The escape reaction of the ormer would dramatically lower its grip on the substratum, thus increasing the probability of removal by the starfish. Twisting would enable the ormer to loosen
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the tube feet from its shell, mucus secretion (from pores on the shell) would coat the predator’s arm(s), keeping the smell of the prey fixed on the predator’s primary olfactory sensors and provide the ormer some seconds for running. During the experiments I noted that the ormers moved faster than M. glacialis, and were thus able to escape. Mucus produced by ormers and the ink produced by cephalopods both appear to distract the predator, thus increasing capture time. The clear advantage of a defence that increases the time spent in capture, consumption and digestion is pronounced when many prey are present (Jeschke & Tollrian 2000). Thus, such defences are more efficient in environments with abundant and diverse prey, as a predator would probably be dissuaded from pursuing a difficult prey if it can quickly find an easier one. Conversely, this defence strategy is less effective if the ormer lives in an environment with a high density of predatory starfishes (Day et al. 1995) where the production of mucus is presumably quickly exhausted (Thompson 1960; Day et al. 1995). The differences in capture time between the treatment group (mucus) and the control group (sea water), observed during experiment 2c, may be caused by several factors. First, because the mucus used in the experiments was always produced by different individuals, its quality and quantity were less standardized than sea water, possibly producing a higher interindividual variance. Indeed, depending on the timing of mucus expulsion and the ormer that produced it, the mucus could vary in density and it could contain a different quantity of smell or of other substances which might increase or decrease the effect on M. glacialis. Finally, because of the method of application of the mucus, human error may have influenced the outcome of the experiments. However, the differences between test groups and control groups remained significant, and a refinement of the standard conditions would increase the confidence level of the results. In conclusion, although the aim of this study was to investigate whether H. tuberculata lamellosa secretes mucus only if attacked by a predatory starfish, as in other species of the genus Haliotis, the principal result was the identification of the main function of mucus release as an olfactory decoy for asteroid predators. Similar to a lizard that leaves its tail behind to occupy the predator while the prey escapes, the ormer secretes its sticky and attractive mucus on the starfish’s body, thus fooling the asteroid’s detection system. However, the release of this substance is not an isolated escape manoeuvre, but a behavioural subunit that occupies a specific position within a larger and complex sequence of defensive behaviours. The complexity and length of this sequence usually increase as the success of its early defensive subunits decreases, and mucus release is one of the last manoeuvres used by ormers to escape from predatory starfish. This is also why the use of mucus as an intraspecific alarm seems to be only of secondary importance for the individual that releases it, although I have shown in this study that it can be easily and usefully recognized by nearby ormer as a sign of danger.
Acknowledgments I thank the following for their useful help during the organization of the experiments and for comments on the manuscript: Professor J. Hausser, Professor P. Heeb, Dr C. Valentin, Associate Professor G. Skilleter and Professor R. Martin. Moreover, special
thanks are dedicated to the Socie´te´ Acade´mique Vaudoise for the financial support of this work.
References Barnard, C. J. 1983. Animal Behaviour. Ecology and Evolution. London: Croom Helm. Barnes, R. D. 1980. Invertebrate Zoology. Philadelphia: Saunders College. Bennet, E. W. 1927. Notes on some New Zealand seastars and on autonomous reproduction. Canterbury Museum Records, 3, 125–149. Bullock, T. H. 1953. Predator recognition and escape response of some intertidal gastropods in presence of starfish. Behaviour, 5, 130–140. Crank, J. 1975. The Mathematics of Diffusion. Oxford: Oxford University Press. Day, R., Dowel, A., Sant, G., Klemke, J. & Shaw, C. 1995. Patchy predation: foraging behavior of Coscinasterias calamaria and escape response of Haliotis rubra. Marine and Fresh Water Behaviour and Physiology, 26, 11–33. Denny, M. W. 1989. Invertebrate mucous secretion: functional alternatives to vertebrate paradigms. In: Mucus and Related Topics (Ed. by E. Chantler & A. A. Ratcliffe), pp. 337–366. Cambridge: Society for Experimental Biology. Dittmer, K., Grasso, F. W. & Atema, J. 1996. Obstacles to flow produce distinctive patterns of odor dispersal on a scale that could be detected by marine animals. Biological Bulletin, 191, 313–314. Faulkner, D. J. & Ghiselin, M. T. 1983. Chemical defense and evolutionary ecology of dorid nudibranchs and some other opisthobranch gastropods. Marine Ecology Progress Series, 13, 295–301. Feder, H. M. 1963. Gastropod defensive response and their effectiveness in reducing predation by starfishes. Ecology, 44, 505–512. Grasso, F. W. 2001. Invertebrate-inspired sensory-motor systems and autonomous, olfactory-guided exploration. Biological Bulletin, 200, 160–168. Jeschke, J. M. & Tollrian, R. 2000. Density-dependent effects of prey defense. Oecologia, 123, 391–396. Lauffer, M. A. 1989. Motion in Biological Systems. New York: A. R. Liss. Leroy, E. 1987. L’Univers de l’Odorat Animal. Paris: Boube´e´. MacGinitie, G. E. & MacGinitie, N. 1949. Natural History of Marine Animals. New York: McGraw-Hill. Mackie, A. M. 1972. Escape response of marine invertebrates to predatory starfish. In: European Marine Biology Symposium (fifth) (Ed. by B. Battaglia), pp. 269–274. Padova: Piccin. Mackie, A. M., Lasker, R. & Grant, P. T. 1968. Avoidance reactions of mollusc Buccinum undatum to saponin-like surface-active substances in extracts of the starfish Asterias rubens and Marthasterias glacialis. Comparative Biochemistry and Physiology, 26, 415–428. Margolin, A. S. 1964. A running response of Acmea to seastars. Ecology, 45, 191–193. Montgomery, D. H. 1967. Response of two haliotid gastropods (Mollusca), Haliotis assimilis and Haliotis rufescens, to the forcipulate asteroids (Echinodermata), Pycnopodia heliantoides and Pisaster ochraceus. Veliger, 9, 359–368. Nicole, J. A. 1964. Special effectors: luminous organs, chromatophores, pigments, and poison glands. In: Physiology of Mollusca. Vol. 1 (Ed. by K. M. Wilbur & C. M. Yonge), pp. 353–381. New York: Academic Press. Parsons, D. W. & Macmillan, D. L. 1979. The escape response of abalone (Mollusca, Prosobranchia, Haliotidae) to predatory gastropods. Marine Behavior and Physiology, 6, 65–82. Phillips, D. W. 1975. Distance chemoreception-triggered avoidance behavior of the limpets Acmaea (Collisella) limatula and Acmaea (Notoacmea) scutum to the predatory starfish Pisaster ochraceus. Journal of Experimental Zoology, 191, 199–210. Price, W. S. 2005. Application of pulse gradient spin-echo NMR diffusion measurements to solution dynamics and organization. Diffusion Fundamentals, 2, 112.1–112.19. Rice, S. H. 1985. An anti-predator chemical defense of the marine pulmonate gastropod Trimusculus reticulatus. Journal of Experimental Marine Biology and Ecology, 93, 83–89. Riedl, R. 2005. Flora e Fauna del Mediterraneo. Montereggio: F. Muzzio. Rochette, R., Hamel, J. F. & Himmelman, J. H. 1994. Foraging strategy of the asteroid Leptasterias polaris. Role of prey odours, current and feeding status. Marine Ecology Progress Series, 106, 93–100. Rochette, R., Arsenault, D. J., Justome, B. & Himmelman, J. H. 1998. Chemically mediated predator-recognition learning in marine gastropod. Ecoscience, 5, 353–360. Schiemenz, P. 1895. Wie o¨ffen die Seesterne Austern? Mitteilungen des Deutschen Seefischerievereins, 12, 102–118. Schmitt, R. J. 1981. Contrasting anti-predator defence of sympatric gastropods (family Trochidae). Journal of Experimental Marine Biology and Ecology, 54, 251–263. Thompson, T. E. 1960. Defensive acid-secretion in marine gastropods. Journal of the Marine Biological Association of the United Kingdom, 39, 115–122.
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Animal Behaviour journal homepage: www.elsevier.com/locate/anbehav
Function of mucus secretion by lamellose ormer, Haliotis tuberculata lamellosa, in response to starfish predation Francesco Bancala`* ¨ r Marine Biologie, Isola del Giglio, Italy Institut fu
a r t i c l e i n f o Article history: Received 21 October 2008 Initial acceptance 5 January 2009 Final acceptance 8 August 2009 Available online 10 September 2009 MS. number: 08-00679R
Gastropods subjected to predation from asteroids have a number of different defensive mechanisms to prevent and defend against attacks. I investigated the production and function of mucus in the lamellose ormer in response to attacks by the spiny starfish, Marthasterias glacialis (Asteriidae, Asteroidea). Production of mucus from respiratory pores on the shell helped obscure the direction of escape of the gastropod from the starfish. The mucus also had repellent properties against this predator and there was some evidence of it being an intraspecific alarm. The results suggest that mucus secretion is part of a larger stereotyped escape response, in which the mucus has an olfactory interference role. Ó 2009 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Keywords: asteroid escape response Haliotis tuberculata lamellosa lamellose ormer mucus secretion Marthasterias glacialis spiny starfish stereotyped behaviour
To prevent predation by asteroids, many gastropods have specific types of response (Bullock 1953) with different functions: (1) avoidance (Mackie 1972; Phillips 1975), (2) repulsion (Thompson 1960; Schmitt 1981) or (3) escape (Schiemenz 1895; Margolin 1964). Haliotidae (ormers) in particular perform a specific (Bennet 1927; Bullock 1953; Feder 1963) and well-developed escape behaviour, which is composed of a sequence of different types of recognizable subunits (Parsons & Macmillan 1979) and involves the secretion of mucus, whose function is unclear. Initially the mollusc extends and moves its tentacles (tentacular sweeping), a behaviour that seems to be of detective value (Montgomery 1967). Once in contact with starfish tube feet, the tentacles retract and a limb of the epipodium on the stimulated side is raised and extended to cover part of the shell (covering). If contact continues, the ormer lifts its shell (mushrooming) and turns it violently from one side to the other (twisting) to break the starfish’s grip. Some tube feet can be pulled out by this movement, consequently inducing a temporary retreat of the starfish. Finally, the ormer moves directly away from the predator (running;
* Correspondence and present address: F. Bancala`, Via Lucino 44, 6932 Breganzona, Switzerland. E-mail address: [email protected]
MacGinitie & MacGinitie 1949; Bullock 1953; Montgomery 1967; Parsons & Macmillan 1979). The timing and sequence of these subunits of the escape response are stereotyped and the response depends on stimuli that are characteristic of the different phases of the attack (Bullock 1953; Parsons & Macmillan 1979). Indeed, the response of gastropods such as ormers to contact by foreign objects other than tube feet (e.g. crab claws, seaweeds, snail shells or soft parts, rocks, human finger or implements) is never to mushroom up but to clamp down (Bullock 1953). Moreover, during an attack each subunit of the escape response seems to be less efficient if expressed in the wrong context or order (Parsons & Macmillan 1979). For instance, the twisting of the shell appears to break the hold of the asteroid’s tube feet (Bennet 1927; Montgomery 1967). Consequently, a running response is less efficient if expressed before twisting, when the asteroid’s tube feet are still holding on to its shell, than after breaking the hold with the twisting response. The nature and function of mucus secretion by ormers during the escape response have not been investigated (Montgomery 1967). As a defence against predation, mucus could have (1) an intraspecific alarm function or (2) a repellent effect on starfishes. Indeed, Montgomery (1967) highlighted the fact that the mucus of Haliotis assimilis is enough to induce a full escape response in resting conspecifics. Moreover, although no exhaustive study exists on the chemistry of the mucus secreted by Haliotis, evidence for the
0003-3472/$38.00 Ó 2009 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.anbehav.2009.08.008
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presence of a chemical deterrent in the epipodium of Haliotis ruber has been found, suggesting that characteristics of mucus could be useful to avoid rapid diffusion (and dilution) of the deterrent contained inside it, thus maintaining its efficiency (Day et al. 1995). Finally, (3) Day et al. (1995) suggested that ormers could use the mucus to interfere with the starfish’s olfactory system to obscure their escape direction, in a way analogous to that described by Denny (1989) for squid. Indeed, Day et al. (1995) observed that, during attacks by the starfish Coscinasterias calamaria, the mucus of H. ruber was ejected through one of the respiratory pores and diffused very slowly into the water. Afterwards, the starfish stopped its attack or moved slowly in another direction. This may be explained if the smell of the ormer, associated with the mucus which sticks to the starfish arm in contact with the shell, possibly obscures the actual position of the ormer (Day et al. 1995). Indeed, starfishes have been shown to follow chemical traces, by sensory tentacles situated on the tips of their arms, to find and attack their prey (Rochette et al. 1994). My aim in the present study was (1) to investigate the role of mucus secretion by the lamellose ormer, Haliotis tuberculata lamellosa, during a spiny starfish, Marthasterias glacialis, attack under experimental conditions; (2) to examine the position of mucus secretion within the interaction hierarchy described by Parsons & Macmillan (1979); and (3) to investigate the three main hypotheses for the function of mucus secretion by testing the reaction of ormers to mucus, the reaction of starfishes to mucus and the influence that mucus has on the time that a starfish needs to locate and reach an ormer that cannot move. METHODS Animal Collection and Maintenance Experiments were carried out at Giglio Island, Tuscan Archipelago, Italy, in the Institut fu¨r Marine Biologie (IfMB). Animals were sampled in September 2002 by SCUBA. Twenty H. tuberculata lamellosa were taken from a wild population living on a cone of stones (block size ranging from 1 dm3 to 1 m3), situated between 5 and 15 m depth, around the base of an old pylon (42 21059.0000 N, 10 520 29.3100 E). The shell size of the ormers ranged between 18 and 49 mm (mean 36.7 mm). The 14 individuals of M. glacialis, as well as 14 individuals of Echinaster sepositus, were collected in three other sites (site 1: 42 230 24.4000 N, 10 530 7.6600 E; site 2: 42 200 31.3100 N, 10 530 9.8500 E; site 3: 42 2301.6300 N, 10 520 41.4100 E), at depths between 5 and 40 m, from granite substrate. This species is known to have a certain vertical mobility (Riedl 2005) and consequently it is unlikely that this depth range could have influenced the results. The size of the starfishes ranged between 24 and 42 cm (mean 31.7 cm); the sex and age of the individuals of both species were not determined. Haliotis tuberculata lamellosa were kept in four aquaria (25 25 cm and 40 cm high), each containing five individuals, until required for the experiment. The holding tanks were filled with sea water and provided with filters and oxygenation systems. During capture of the ormers rocks were collected and then placed inside the holding tanks. Algae growing on the rocks provided a food source for the ormers, and were replaced with rocks containing fresh algae at weekly intervals. Water was fully changed every 3 days. The temperature of the water was 20 C and the light:dark regime was 14:10 h. To distinguish between individuals, each ormer was numbered on the shell using a small piece of paper (3 3 mm) fixed with odourless silicon, to avoid the tag influencing the dynamics of capture events. Starfishes were held in a cage (50 50 cm and 100 cm high) in the sea, near the institute at a depth of 6 m. To keep them in the
best condition and to reduce the risk of automutilation (C. Valentin, unpublished data) the cage was divided into 20 separate boxes, each containing one M. glacialis. They were generally fed once per week with mussels, Mytilus edulis, but the feeding rate could change depending on experimental requirements (e.g. to obtain a starving status the starfish was not fed for 2 weeks). Experimental Procedures The aquaria (N ¼ 10) used for the experiments (25 25 cm and 40 cm high) were scrubbed with ethanol and filled with 20 litres of filtered sea water after each experiment. Each individual tested (both ormer and starfish) was randomly assigned to control or treatment groups with a maximum of one trial per day. Once introduced into the experimental aquarium, each animal (ormer or starfish) was allowed to acclimatize for 30 min. After testing, the individuals were kept in separate tanks, away from untested animals. The time between experiments was generally 1 week. After the study, captive animals were released in the same area where they were collected 2 months earlier. The ormers were exposed to stressful situations (a predator and stimuli of predators) but no individual died during an attack. Manipulation of individuals of M. glacialis involved food limitation and diet modification, following a randomized plan; no asteroids died of hunger. Mucus Sampling For experiments 2a, b and c, I required mucus secreted by H. tuberculata lamellosa. Using a syringe (20 ml) without a needle, I collected mucus from the respiratory pore of each ormer after repeated stimulation with a tube foot of M. glacialis. Stimulation was particularly effective on the mantle or on the respiratory tentacles of the gastropod. To avoid any effect of this prestimulation on the mucus production response, I did this only after testing ormers for the mucus response (experiment 1). Moreover, after the production of the sampled mucus for experiments 2a, b, the ormer was allowed to rest for a day. This was not the case for the olfactory illusion test (experiment 2c), where the use of the same individual that produced mucus was needed for control purposes. In this case, however, ormers were not the subject of the experiment and their behaviour could not influence the results. Mucus was used as the treatment, whereas the control substance was sea water containing the smell of the same ormer. I obtained the control substance by sampling the water of the small tank containing the ormer before starting the stimulation for mucus secretion. The treatment or control was then applied with a syringe (20 ml); its exit flow was roughly 1 ml/s. Mucus Secretion In experiment 1, I investigated mucus secretion by ormers in the presence of a predatory asteroid. I tested 20 individuals of H. tuberculata lamellosa, each of them in both treatment and control tests. Five individuals of M. glacialis (predator) were used as the treatment and five individuals of E. sepositus (nonpredator) as the control. Consequently each starfish was used four times in a randomly defined order. The ormer was introduced into an aquarium 30 min before the experiment to acclimatize; this was also the control period. Once the acclimatization time was passed, an asteroid was introduced into the same aquarium and left there for 30 min. During this time, I recorded how many ormers reacted to attacks with mucus secretion. The prediction was that this number would be greater if the asteroid was predatory than if it was deposit feeding (nonpredatory).
F. Bancala` / Animal Behaviour 78 (2009) 1189–1194
I also noted type, sequence and duration of each subunit of response for each attack and individual. To establish whether a correlation exists between mucus secretion and other subunits, such as twisting and running, I calculated frequencies of mucus ejection after twisting, and before running for each individual:
ft ¼ Mt =T where ft is the frequency of mucus ejection after twisting. Thus, M(t) is the total number of mucus ejections (made after the twisting behaviour) per total number of twisting behaviours, T.
fr ¼ Mr =R where fr is the frequency of mucus ejection before running. Thus, M(r) is the total number of mucus ejections (made before running behaviour) per total number of running behaviours, R. Observed average frequencies were then calculated for both associations and compared to a stochastic frequency of 0.50 using a test of one proportion to verify association between these subunits. Function of Mucus Intraspecific alarm In experiment 2a, I examined the possible function of mucus as a signal of danger, inducing escape responses in other ormers. Each of 20 individuals was used in both treatment and control tests. The control or treatment substance (5 ml) was introduced 1 cm from the anterior side of an ormer resting in an aquarium (N ¼ 10). During the following 2 min I observed the reaction of the ormer. If the ormer did not respond, a negative result was assigned to that individual and interpreted as ‘no detection’ of alarm signal in the substance. If the ormer responded (from rest to movement or escape behaviours), the result was positive. I recorded the number of ormers that moved away from the stimulus. I predicted more positive responses would be observed in the presence of mucus than in the presence of the control substance (sea water). Repellent The objective of experiment 2b was to investigate whether the ormer’s mucus has a repellent effect on spiny starfish. Each of 14 M. glacialis was used in both treatment and control tests. Starfishes have a large number of sensory cells within the epidermis which are the primary sensory receptors and probably function for the reception of light, contact and chemical stimuli (Barnes 1980). They are particularly present on the suckers of tube feet, on the tentacles and along the margins of the ambulacral groove, where up to 70 000 sensory cells/mm2 occur (Barnes 1980). Consequently, some regions of the body, such as the oral perimeter and the arm distal extremity, are more sensitive than others. For this reason, I tested the effect of mucus on these two regions separately. An M. glacialis was introduced into an aquarium. To induce the starfish to rest on the vertical glass (for a standard observation of the tube feet activity) I created a dark zone by placing a small board on the short side of the aquarium. A spiny starfish was then introduced into the aquarium 30 min before the experiment. The starfish tended to rest in the dark zone where I started the experiment. When the individual was at rest, a treatment or control substance (5 ml) was sprayed towards the sensory tube feet on the tip of the arm or on the oral perimeter. To reach the oral perimeter without disturbing the starfish, I slipped a needle mounted on the syringe between the animal and the aquarium pane. I then observed the reaction of the tube feet (organ level) and the reaction of the whole animal (organism level). Reactions were
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classified into three distinct types: positive (movement towards the source of the stimulus), neutral (no significant change in activity) or negative (movement away from the source of the stimulus). I predicted that both the number of individuals that withdrew and the number of individuals whose tube feet retreated from the source of the stimulus would be greater after application of mucus than the control substance (sea water). Olfactory interference The objective of experiment 2c was to investigate whether there was an influence of mucus on the capture time of ormers by starfishes. This was considered to be an indicator of starfishes’ olfactory performance. During this experiment 14 M. glacialis were used for both treatment and control tests following a randomized order. I induced starfishes to rest at a standard location for the beginning of the experiment by creating a dark zone as described above. A spiny starfish was introduced into an aquarium 30 min before the experiment. In the meantime an ormer was introduced into a small container made of metallic net, so as to let its smell diffuse into the tank. The diffusion, which is by definition stochastic (Crank 1975; Lauffer 1989; Price 2005), of odour molecules towards the starfish is very gradual in perfectly still water (Dittmer et al. 1996; Grasso 2001). The container was then placed into the aquarium, on the opposite side to the dark zone, 30 cm from the starfish. At the same time, 5 ml of the control (sea water) or mucus were ejected with the syringe around the starfish arm. To avoid any possible reaction of the starfish caused by contact with the syringe, I ejected the substance 1 cm from the starfish. Timing commenced when the control substance or mucus was applied and stopped when the starfish touched the container holding the ormer. I predicted that the time the starfish took to reach the ormer would be greater in the presence of mucus than in the presence of sea water. Statistical Analysis For statistical analysis I used S-PLUS 2000 (Insightful Corp., Seattle, WA, U.S.A) and SYSTAT (SPSS Inc., Chicago, IL, U.S.A). The McNemar’s test was used to test the hypotheses except for the olfactory illusion experiment (experiment 2c) for which I used a paired t test and an F test for entered parameters. The McNemar’s test tests the hypothesis that two proportions are equal. A common design that uses this analysis is when subjects make two dichotomous responses. For example, each subject is measured once after treatment A and again after treatment B. The response is ‘1’ if the event of interest occurs or ‘0’ otherwise. The assumptions are: (1) the two groups are related or dependent (e.g. each individual may be measured in two different circumstances); (2) every individual is classified according to whether the events of interest occur in both circumstances, one circumstance only, or in neither. The assumptions of the paired t test are: (1) the data are continuous (not discrete); (2) the data (i.e. the differences for the matched pairs) follow a normal probability distribution; (3) the sample of pairs is a simple random sample from the population (each individual in the population has an equal probability of being selected in the sample). The F test for entered parameters was used to test the hypothesis of same variance. All the assumption of the tests were met. All tests were two tailed. RESULTS Mucus Secretion Among 20 tested individuals 15 secreted mucus during at least one attack of M. glacialis. Indeed, M. glacialis stimulated mucus
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secretion in H. tuberculata lamellosa significantly more than the control species E. sepositus (McNemar’s test: P < 0.01). In addition, each individual showed the ability to produce this substance during stimulation with an isolated tube foot of M. glacialis. However, contact with a starfish tube foot was not enough to evoke immediate mucus secretion by the ormer. Indeed, the mucus was rarely expelled during the first contact with M. glacialis and usually many attacks were necessary to stimulate a reaction. Moreover, during the experiment I observed that the stimulus was particularly efficient if the tube foot touched the respiratory tentacles, more so than any other part of the ormer. In fact, mucus was usually secreted once the starfish tube feet had already been fixed onto the ormer’s shell and it is possible that some tube feet touched one or more respiratory tentacles during that step of the attack. Mucus was always ejected from one or two respiratory pores of the shell. No ormer (0/20) secreted mucus during experiments using E. sepositus. Other ‘escape’ manoeuvres (tentacle activity, covering, mushrooming, running and twisting), already described in other gastropods (e.g. H. rufescens, H. assimilis), were observed in each individual of H. tuberculata lamellosa when stimulated by M. glacialis, whereas E. sepositus elicited only covering responses (8/20), and very rarely some twisting responses (3/20). Finally, no mucus production was observed during the acclimatization period. The average frequency of mucus ejection after twisting was 0.85 and before running was 0.77 Both averages are significantly different (one proportion test: after twisting: Z ¼ 1.84, N ¼ 18, P ¼ 0.033; before running: Z ¼ 1.93, N ¼ 21, P ¼ 0.026) from a stochastic average of 0.50, indicating the existence of the associations ‘twisting–mucus’ and ‘mucus–running’. Function of Mucus Intraspecific alarm Of the 20 ormers 15 made escape responses in the mucus treatment group. Only one of the ormers responded positively when the control stimulus was applied. The difference is highly significant (McNemar’s test: P < 0.01). However, the reaction time observed was very variable (3–118 s). Repellent No repulsion effect was observed when the arm extremities of M. glacialis were stimulated, either in the treatment or in the control groups, both at the organ level and at the whole organism level (Fig. 1a, b). In contrast, tube feet (13/14 individuals) and individuals (8/14) showed an attraction to mucus (Fig. 1a, b). This reaction occurred nine and four times, respectively, when ormer-smelling sea water was used. Indifference to mucus was observed once at the organ level and six times at the organism level, while when the control substance was used, it was recorded five and 10 times, respectively (Fig. 1a, b). However, differences between treatments were not significant (two-tailed McNemar’s test: tube feet: P ¼ 0.10; individuals: P ¼ 0.10). On the other hand, stimulation of the oral region produced a negative reaction at the tube feet level (7/14 individuals) and at the whole animal level (5/14; Fig. 1c, d). No similar reactions were observed during the control treatment (Fig. 1c, d). Attraction to mucus appeared four times both at the organ level and at the organism level; the same reaction was observed eight and 11 times, respectively, in the presence of the control substance (Fig. 1c, d). Indifference to mucus was shown three times by tube feet and five times by whole individuals, while it occurred six and three times, respectively, when ormer-smelling sea water was used (Fig. 1c, d). The difference between test and control is highly significant (two-tailed McNemar’s test: tube feet: P < 0.01; individual:
P < 0.01). The difference in mucus stimulation response between arm extremities and oral region is also highly significant (two-tailed McNemar’s test: P < 0.01). Olfactory interference Treatment with mucus significantly increased (paired t test: t1 ¼ 5.58, P < 0.01) the time M. glacialis spent capturing an ormer, nearly doubling it (Fig. 2). Moreover, the standard error changed from the control group (54 s) to the test group (215 s) and the difference in variance is highly significant (F test for entered parameters: F ¼ 3.98, N ¼ 14, P ¼ 0.02). DISCUSSION Experiment 1 provided evidence that H. tuberculata lamellosa secretes mucus when it is attacked by the predatory starfish M. glacialis but not in the presence of a nonpredatory starfish. That kind of behaviour has already been described for other Haliotidae (Montgomery 1967; Parsons & Macmillan 1979; Day et al. 1995), as well as for other gastropods (Rice 1985). As multiple predatory contacts were needed, the stimulation threshold for the secretion of mucus in other parts of the body could be lower. That might be because of a higher density of the mucus or a different kind of receptor, more sensitive to predatory starfish ‘odours’, such as the saponin M2 (Mackie et al. 1968, Leroy 1987). If that is the case, the production of mucus may have a certain cost to the ormer and may be secreted only when it is really needed. In fact, the production of defensive substances in gastropods is limited by their utilization, which means that they can run out of defensive substance if they secrete it too many times in a short period (Thompson 1960; Day et al. 1995). Moreover, if mucus of males contains sperm (Montgomery 1967) the cost of production could be even higher. The results of experiment 2a indicate that mucus of H. tuberculata lamellosa is recognized as a sign of danger among conspecifics. That effect has already been observed in other Haliotidae by Montgomery (1967). However, alarm may not be the principal function of the mucus. If alarm was the primary function, one would predict (1) a more frequent usage of mucus, (2) the secretion to start as soon as the predator has been noticed and (3) the alarm signal should be contained in a substance much more diffusible than mucus. Rochette et al. (1998) showed that the bivalve Buccinum undatum is more sensitive to predatory asteroids in the presence of odours of stressed, wounded or dead conspecifics. Thus, if this characteristic is also valid for H. tuberculata lamellosa, sensitivity to mucus could reveal important information about the intensity of prey–predator relationships in any ormer population that is under the predatory pressure of carnivorous asteroids. Experiment 2b highlighted a difference in response to mucus between the oral region and the arms extremities, a significant repellent effect on M. glacialis being detected only at the oral level. However, the retreat of the tube feet as well as of the entire individual could be caused by an unpleasant taste rather than a response to harmful chemicals contained within the mucus. That appears to be confirmed by the negative test on the arm tips. Indeed, the starfish would be expected to move away from the mucus if it is chemically aggressive, whereas the opposite happened. Rice (1985) observed that when the gastropod Trimusculus reticulatus (Pulmonatae) is attacked by the starfish Pisaster ochraceus, it secretes whitish mucus, which is able to anaesthetize the tube feet of P. ochraceus temporarily; but the starfish did not suffer a permanent effect, even after prolonged or repeated exposure to the mucus. Additionally, no sign of illness or injury was seen in individuals that ate T. reticulatus. That too may be the case in the present study although no anaesthetic effect was
F. Bancala` / Animal Behaviour 78 (2009) 1189–1194
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Figure 1. Number of individuals of M. glacialis reacting with repulsion (white bar), indifference (grey bar) and attraction (black bar) to stimulation with mucus or a control (sea water) (a) local response of tube feet stimulated on arm extremity; (b) response of the whole individual stimulated on arm extremity; (c) local response of tube feet stimulated on oral region; (d) response of the whole individual stimulated on oral region.
observed. Day et al. (1995) observed that the mucus of H. ruber did not induce any repulsion response in Coscinasterias calamaria, and its mucus had a pH close to that of sea water (7–8.3). Conversely, the defensive secretions described by Thompson (1960) among
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Sea water Treatment
Figure 2. Mean þ SD capture time of 14 individuals of M. glacialis tested with mucus (black bar) and with sea water (white bar).
three families of gastropods (Pleurobranchidae, Philinidae, Lamellariidae) were acidic with a pH close to 1. Although other studies document the repellent properties of many gastropods, they deal with passive characteristics (Schmitt 1981; Faulkner & Ghiselin 1983), which do not involve the secretion of mucus. Protein components, as well as sulphuric acid or mucopolysaccharides, are among the substances secreted by gastropods (Nicole 1964; Barnard 1983). These substances are produced by different organs (Day et al. 1995) and it is possible that cosecretion of these components is necessary for chemical defence to be successful (Thompson 1960). Therefore, it is possible that in my experiments factors other than mucus could have been present, which possibly lowered the repellent effect of the mucus. However, the attractive rather than the repellent effect of mucus on the arm tips could be easily explained if considered in the context of another function of the mucus: an olfactory decoy, which by definition has to be attractive. Indeed, this study is the first to show that mucus appears to impede or confound olfactory reception of a predatory starfish (experiment 2c). The confounding effect also appeared in experiment 2b. Starfishes were attracted by mucus when this was ejected onto arm extremities, and tended to follow the direction of the arm coated with mucus, which was generally different from the escape direction of the ormer. This mechanism is easier to understand if we consider that mucus secretion has a hierarchical position within a behavioural sequence, as confirmed by the results of experiment 1, whose final goal is to allow escape at an advanced stage of attack. As soon as the starfish reaches the shell of the ormer with its arm(s), it fixes its tube feet onto the ormer. During this stage, the starfish prevents the ormer escaping and tries to remove it from the substratum. The escape reaction of the ormer would dramatically lower its grip on the substratum, thus increasing the probability of removal by the starfish. Twisting would enable the ormer to loosen
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the tube feet from its shell, mucus secretion (from pores on the shell) would coat the predator’s arm(s), keeping the smell of the prey fixed on the predator’s primary olfactory sensors and provide the ormer some seconds for running. During the experiments I noted that the ormers moved faster than M. glacialis, and were thus able to escape. Mucus produced by ormers and the ink produced by cephalopods both appear to distract the predator, thus increasing capture time. The clear advantage of a defence that increases the time spent in capture, consumption and digestion is pronounced when many prey are present (Jeschke & Tollrian 2000). Thus, such defences are more efficient in environments with abundant and diverse prey, as a predator would probably be dissuaded from pursuing a difficult prey if it can quickly find an easier one. Conversely, this defence strategy is less effective if the ormer lives in an environment with a high density of predatory starfishes (Day et al. 1995) where the production of mucus is presumably quickly exhausted (Thompson 1960; Day et al. 1995). The differences in capture time between the treatment group (mucus) and the control group (sea water), observed during experiment 2c, may be caused by several factors. First, because the mucus used in the experiments was always produced by different individuals, its quality and quantity were less standardized than sea water, possibly producing a higher interindividual variance. Indeed, depending on the timing of mucus expulsion and the ormer that produced it, the mucus could vary in density and it could contain a different quantity of smell or of other substances which might increase or decrease the effect on M. glacialis. Finally, because of the method of application of the mucus, human error may have influenced the outcome of the experiments. However, the differences between test groups and control groups remained significant, and a refinement of the standard conditions would increase the confidence level of the results. In conclusion, although the aim of this study was to investigate whether H. tuberculata lamellosa secretes mucus only if attacked by a predatory starfish, as in other species of the genus Haliotis, the principal result was the identification of the main function of mucus release as an olfactory decoy for asteroid predators. Similar to a lizard that leaves its tail behind to occupy the predator while the prey escapes, the ormer secretes its sticky and attractive mucus on the starfish’s body, thus fooling the asteroid’s detection system. However, the release of this substance is not an isolated escape manoeuvre, but a behavioural subunit that occupies a specific position within a larger and complex sequence of defensive behaviours. The complexity and length of this sequence usually increase as the success of its early defensive subunits decreases, and mucus release is one of the last manoeuvres used by ormers to escape from predatory starfish. This is also why the use of mucus as an intraspecific alarm seems to be only of secondary importance for the individual that releases it, although I have shown in this study that it can be easily and usefully recognized by nearby ormer as a sign of danger.
Acknowledgments I thank the following for their useful help during the organization of the experiments and for comments on the manuscript: Professor J. Hausser, Professor P. Heeb, Dr C. Valentin, Associate Professor G. Skilleter and Professor R. Martin. Moreover, special
thanks are dedicated to the Socie´te´ Acade´mique Vaudoise for the financial support of this work.
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