Behavioural Processes 46 (1999) 57 – 62
Does the Pyrenean salamander Euproctus asper use chemical cues for sex identification and mating behaviour? O. Guillaume Laboratoire souterrain du Centre National de la Recherche Scientifique, F-09200 Moulis, France Received 2 July 1998; received in revised form 7 December 1998; accepted 16 December 1998
Abstract This paper examined the potentiality of chemical cues for sex identification and mating activity in Euproctus asper. The study tested the ability of males and females to distinguish between the odour of animals of their own sex and that of animals of the opposite sex, as it was carried to them by water flowing over another animal. Their ability to distinguish between water flowing over another E. asper and water flowing over a control, where no other animal was present, was also tested. Then, the study tested their ability to distinguish between the diffusing odour of animals of their own sex, that of animals of the opposite sex, and that of a control, where no other animal was present. There was no evidence that males and females identify their mates using chemical cues. Observations of the courtship behaviour were also carried out. Mating seems to be induced by the male’s display of his tail when he captures the female as she passes near him, to form an amplexus, without any obvious preliminary. On the basis of these data, the question whether the mate identification occurs during the amplexus in this species was raised. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Amphibia; Courtship; Euproctus asper; Mating; Salamandridae
1. Introduction The salamander Euproctus asper is endemic to the Pyrenees, living in the mountain streams between 250 and 2400 m in altitude, and in some cave rivers (Astre, 1950; Combes and Knoepffler, 1963; Clergue-Gazeau, 1965). According to Clergue-Gazeau (1971), reproduction in epigean populations occurs from April onwards, on melting of the snow and differs according to the
climate, whereas it occurs throughout the entire year for the cave populations. Chemical communication plays a major role in the reproductive behaviour of some Urodeles (Malacarne and Vellano, 1987; Mathis, 1991; Houck and Sever, 1994; Toyoda et al., 1994; Verrell, 1994; Kikuyama et al., 1995). Courtship behaviour was reported in the genus Euproctus (Despax, 1923; Ahrenfeldt, 1955; Salthe, 1967; Brizzi et al., 1995), but the question of chemical
0376-6357/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 6 - 6 3 5 7 ( 9 9 ) 0 0 0 0 3 - 0
58
O. Guillaume / Beha6ioural Processes 46 (1999) 57–62
cues for sex identification and mating is still under debate. The present study examined this potentiality in Euproctus asper.
2. Materials and methods The 20 specimens of Euproctus asper (ten males and ten females) used in the present study were adults of between 8 and 12 years old, born and bred in the cave-laboratory of the C.N.R.S. (Arie`ge, France). Males were isolated from females 14 days before the experiments. The males then received an injection of 1500 UI of human chorionic gonadotropin (HCG) purchased from Organon S.A., and the females an injection of 2500 UI. During the four experiments presented here, the position of the sources of stimulation and of the animals in the testing apparatus were randomly determined for each trial. The first experiment tested the ability of E. asper to discriminate between the odour of a potential mate from the odour of an animal of its own sex, the odours being carried to the tested animal by water flowing over the others animals. The testing apparatus was a plexis Y-maze. Each arm (15 cm length, 2.1 cm diameter) was connected to a compartment (10 cm diameter, 5 cm height). An opaque perforated wall prevented animals from passing from the compartment into the Y-maze, but allowed the water to flow through the apparatus. Water flow of 5 ml/s drained from a reservoir into the two side arms, via the compartments, each containing one animal (a male in one, a female in the other) serving as the odour source. The tested animal (male or female) was kept in the compartment of the third arm of the Y-maze that received the flow of water from the two side arms. After an acclimatisation period of 30 min, the animal was then given a choice between the two side arms by removing the perforated opaque wall to unblock the passage between the compartment in which it was isolated from other parts of the Y-maze. The time spent in both arms was measured for 30 min, 5 min after the beginning of the test.
The second experiment tested the ability of E. asper to discriminate between the odour of a potential mate and a control. Tests were performed as in the first experiment, with one of the two side arms not containing any animal as a source of odour. Data from these two first experiments were analysed using the Wilcoxon-matched-pairedsigned-rank-test. The third experiment tested the ability of E. asper to discriminate between the odour diffused by an animal of its own sex, the odour diffused by an animal of the opposite sex, and from a control with any odour source. The testing apparatus was a plastic aquarium (32 cm in diameter) filled with water (3 cm in depth), and divided into three identical compartments. A plastic and opaque perforated box (8 cm in diameter and 5 cm in depth) was placed in each compartment; a female was kept in the first box, a male in the second, and the third remained empty. Each tested animal was introduced into a glass enclosure (8 cm in diameter), that was placed in the center of the aquarium. After an acclimatisation period of 30 min, the enclosure was carefully removed and the time spent by the tested animal (male or female) in each compartment was measured for 30 min. Data from this third experiment were analysed using the Friedman two way analysis of variance by ranks. The fourth experiment attempted to observe the courtship behaviour of ten male-female pairs of E. asper. In an aquarium (50 cm× 30 cm× 20 cm) filled with water (9 cm in depth) and divided into compartments separated by a removable wall, each partner was isolated separately in a confined compartment for 30 min before the observations. The wall was then removed to unblock the passage between the two compartments, and the behaviour of each animal was observed for the first 2 h and was checked during the following 2 days.
3. Results
3.1. Sex recognition In the first experiment, the males preferred the
O. Guillaume / Beha6ioural Processes 46 (1999) 57–62
stream flowing over the male to the stream flowing over the female, but the difference was not however significant (Table 1). In addition, the females spent as much time in the arm draining the water which flowed over the male as in the arm draining the water flowing over the female. There was therefore no evidence that adults of E. asper distinguished their potential mate’s odour carried by water through the Y-maze, from that of the animal of its own sex (whereas, using the same procedure, animals discriminated the water flowing over chironomid larvae to a control water flow, personal observations). In the second experiment, males spent as much time in the arm draining the water flowing over the female as in the arm draining water where no other animal was present (Table 2). Furthermore, females preferred the control water flow to that passing over the male, even though the difference was not significant. The results of this second experiment therefore indicate that the animals do not discriminate between the potential mate’s odour carried by water through the Y-maze from a plain water flow. In the third experiment, males spent as much time in the compartment of the aquarium in which a male was confined in an opaque box as in the compartment in which a female was confined in an identical manner, and as in the compartment in which an empty box was placed (Table 3). The female preferred, though not significantly, the compartment of the testing apparatus containing the box in which a male was isolated to the sector with the box in which a female was kept, and to the one in which a empty box was placed. Thus, data from the third experiment suggest that adults
59
of E. asper do not distinguish between their potential mates’ odour diffusion, the odour of animals of the same sex, and the control.
3.2. Ethogram of the mating beha6iour 3.2.1. Female actions 1. Wandering: the female moves through the environment in no particular direction. 2. Contact: the female touches the males with her snout or the toes of her anterior legs. 3. Fight: the male having caught the female, she arches her body, and writhes with rapid movements. Sometimes, she also tries to bite the male.
3.2.2. Male actions 1. Prospection: the male moves through the environment in no particular direction. 2. Approach: the male moves towards the female when she is stationary or moves towards him. 3. Following: the male moves after the female when she moves away. 4. Touch: the male touches the female with his snout or with the toes of his front leg. 5. Tail raising: the male raises his tail vertically. 6. Capture: the male catches or attempts to catch the female when she remains or passes behind him. By a rapid movement, its raised tail coils up around her body, generally round the pelvic region, thus immobilising her hind legs. 7. Cloaca-seizure: the male having caught the female, he seizes her cloaca between his hind feet.
Table 1 Comparison of the mean time spent by the tested E. asper in the two side arms of the Y-maze, one receiving the male’s odour and the second the female’s odour Tested animal
Duration in percentage Number of trials (n) P level of significance (Wilcoxon’s)
Male
Female
Male odour
Female odour
Male odour
Female odour
42.9 10 NS
11.45
35.5 10 NS
37
60
O. Guillaume / Beha6ioural Processes 46 (1999) 57–62
Table 2 Comparison of the mean time spent by the tested E. asper in the two side arms of the y-maze, one receiving the mate’s odour and the second the plain flow (control) Tested animal
Duration in percentage Number of trials (n) P level of significance (Wilcoxon’s)
Male
Female
Control odour
Female odour
Male odour
Control odour
16.55 10 NS
11.45
22.83 10 NS
42
8. Cloaca-rub: the male having caught the female, he repeatedly rubs his hind feet, simultaneously or only one of them, against her cloaca in an anteroposterior movement.
3.2.3. Temporal organisation of mating The mating of E. asper can be divided into two distinct and consecutive phases: orientation and amplexus. Orientation consists of the preliminary stage during which the male becomes aware of a potential mate. At first, both animals randomly prospected the environment and seemed generally unaware of their partners. During their movement, touching and contact could occur. If the male touched the female, she generally fled, whereas the female continued her wandering if she had induced contact. Sometimes, during his prospection, and having met the female, the male approached or followed her. This behaviour could be discontinued as the female fled. Orientation came to an end when the male fixed, raised vertically his tail and waited until the female passed or remained behind him to capture her. The duration of this phase was very variable (median = 56 min, range= 10–105 min). The amplexus consists of the male capturing the female, followed by spermatophore release. The time elapsing between tail-raising and capture (or attempt to do so) was variable (median= 0.45 min, range =0.05–3 min). Capture was followed immediately by fighting, interrupted at regular intervals by quiet periods. During this time, the male seized and rubbed the cloaca of the female. This latter behaviour was generally concomitant with the releasing of successive spermatophores which were therefore pushed toward the female cloacal orifice, and sometimes concomitant with
the expulsion of an egg by the female. All of the spermatophores (mean of the number of spermatophore released per male = 2.8, range = 1–5) released during the experiment fell to the aquarium floor during the mating, and did not lodge inside the female’s cloaca. The time between capture and spermatophore release was very variable (median= 31.5 min, range= 2–68 min). After 48 h, all the pairs were still embraced, which suggests that the duration of the amplexus may exceed several days.
4. Discussion Results of this work indicate first that E. asper does not use chemical cues carried by water, in order to meet its mate. These experimental data agree with fieldwork observations. Indeed, E. asper lives in rapid running streams which form an unfavourable environment for transmitting such cues. From an other source, experimental and ecological data show that E. asper does not exhibit a site attachment, but continuously travels inside its home range, finding new refuges as and when it moves, and does not use chemical marks on the substrate or use faecal pellets to communicate either (Guillaume and Cavagnara, 1999). These cues, would indeed be inefficient if the emitter of these messages (which only have a very local impact), does not come back to the place where it had released them to find out the aimed reciever’s response (faecal pellets are further likely to be washed away by the water current). Thus, all the data converge to indicate that E. asper does not use chemical cues for finding its mate.
O. Guillaume / Beha6ioural Processes 46 (1999) 57–62
On the other hand, the present study shows further that mating is initiated only by the male. Indeed, the male fixes in tail-raised posture, and waits until the female, which could be detected by mechanical stimuli and visual stimuli in photic conditions, passes behind him to capture her. During this orientation phase, the female is apparently indifferent to the male’s presence or looking to avoid him, and so does not exhibit any behaviour that could be interpreted as a releaser or a response to the male behaviour (the tailraising included). Then, the capture produces an amplexus, to which the female answers by the fight. Thus, mating occurs only from the male’s instigation, without any obvious persuasion on the part the female, or any behavioural interaction characteristical of the classical courtship of salamanders. These results show then clearly that E. asper does not use any behavioural signal to identify its mate before the mating. In addition, aberrant amplexus male – male, and male – young, sometime involving several individuals, often occurs in breeding, and in nature, where amplexus with sympatrics like Triturus hel6eticus, are also frequent (personal observations). Such behaviours were also observed by Ahrenfeldt (1955) whom reported referring to the males of the genus Euproctus: ‘‘The clasping reflex in these newts is, without any doubt, extremely strong, and in absence of female, is applied indiscriminately to the males of the same species, newts of a different genus, and other animate and inanimate objects.’’, and Despax (1923): ‘‘... la fre´quence des accouplements homosexuels entre maˆles... e´taient presque aussi fre´quents que les accouplements normaux et cependant... j’avais 30 femelles pour 45 maˆles; il est donc impossible d’expliquer ici la fre´quence de
61
ces rapprochements par la pe´nurie des femelles; il faut donc en chercher la cause dans le comportement du maˆle en rut, tel que je viens de le de´crire: place´ en position d’attente, le maˆle saisit tout animal qui passe a` sa porte´, sans distinction de sexe, son mouvement de capture est un pur reflexe et n’implique aucun discernement.’’ These aberrant amplexus seem however to last for a shorter time than those occurring with the females, and do not induce the release of spermatophores (personal observations). All the data converge to indicate that the male does not identify immediately the ‘partner’ he captures, whereas the release of spermatophores depends on interaction with the female during amplexus. Consequently, the hypothesis where the male recognises the female during amplexus, thus triggering spermatophore release, must be retained. It is possible that this unilateral recognition occurs when the male seizes the female cloaca, specifically through tactile recognition of the female cloaca whose conical shape is characteristic, and/or by inducing the release of molecules by the cloacal glands, which are know to be sources of the production of sexual pheromones in certain urodeles (Malacarne and Vellano, 1987; Kikuyama et al., 1995). The anterior ventral glands, by secreting on the epidermis of the cloacal cone, seem likely to be the potential source of a sexual pheromone released during cloacal-seizure and cloacal-rub (in preparation). The results of the present study also indicate that the male pushes the spermatophores towards the female’s cloacal area during cloacal-rub. These spermatophores are not introduced into the female’s cloaca, as is classically the case with urodeles, usually under the female’s instigation,
Table 3 Comparison of the mean time spent by the tested E. asper in the three sectors of the container, one containing the box in which a male was kept, the second with a box in which a female was kept, and the third with a box remaining empty Tested animal
Male
Female
Odour
Male
Female
Empty
Male
Female
Empty
Duration in percentage Number of trials (n) P level of significance (Friedman’s)
36.8 10 P= 1
31
32.2
41.5 10 P = 0.355
32.5
26
62
O. Guillaume / Beha6ioural Processes 46 (1999) 57–62
but are dispersed in the surrounds during amplexus. These observations are in accordance with studies by Despax (1923) who reported: ‘‘La femelle reste entie`rement passive pendant toute la dure´e du rapprochement sexuel, c’est le maˆle luimeˆme qui assure le transport des spermatophores au voisinage de l’ouverture ge´nitale de la femelle et qui y fait pe´ne´trer les spermatozoı¨des, il se sert pour cela de ses pattes poste´rieures et ses orteils jouent, dans une certaine mesure, le roˆle d’organe d’intromission.’’ and by Salthe (1967), who reported: ‘‘... the male moves the spermatophore with his feet toward the female cloaca and that it may stuff the sperm cap or loose spermatozoa (these having no capsule around them and being motile in the cap) between her cloacal lips’’. Moreover, the spermatophores released during amplexus are able to fertilise ‘virgin’ female’s eggs with which they come into contact (personal observations). Data therefore seem to indicate that fertilisation occurs when, during cloaca-rub, the male pushes his spermatophores with his hind legs, thus releasing the spermatozoa near the female’s cloacal orifice. The spermatozoa of E. asper, motile in the spermatophore, and which have an undulating membrane, are perfectly capable of moving around in a liquid environment (in preparation), and of thus gaining the cloacal orifice of the immobilised female. In conclusion, the results of this study indicate that E. asper does not use chemical cues as an aid in the recognition of sexual partners, and it does not develop any courtship behaviour. Consequently, the recognition between sexes may take place during amplexus and lead to the release of spermatophores which are not introduced into the female’s cloaca. Rather, the spermatozoa may be released near the female’s cloacal orifice which they may then swim to.
Acknowledgements This study was supported by the ‘Communaute´ de Travail des Pyre´ne´es – Conseil Re´gional de Midi-Pyre´ne´es’. I wish to thank Dr J. Durand for critically reading the manuscript, anonymous re.
viewers that provided useful comments and the staff of the cave-laboratory in Moulis for their technical assistance. References Ahrenfeldt, R.H., 1955. Mating behaviour of Euproctus asper in captivity. Br. J. Herpetol. 2, 194 – 197. Astre, G., 1950. La station la plus basse d’Euproctes des Pyre´ne´es au pays de Luchon. Revue Comminges 63, 123 – 128. Brizzi, R., Calloni, C., Delfino, G., Tantari, G., 1995. Notes on the male cloacal anatomy and reproductive biology of Euproctus montanus (Amphibia: Salamandridae). Herpetologica 51 (1), 8 – 18. Clergue-Gazeau, M., 1965. Etude comparative de L’Euprocte des lacs et de l’Euprocte cavernicole. Ann. Spe´le´ol. 20 (2), 301 – 316. Clergue-Gazeau, M., 1971. L’Euprocte Pyre´ne´en. Conse´quence de la vie cavernicole sur son de´veloppement et sa reproduction. Ann. Spe´le´ol. 26 (4), 55 – 960. Combes, C., Knoepffler, L.-P., 1963. Euproctus asper dans les Pyre´ne´es orientales. Vie Milieu 14 (1), 149 – 154. Despax, R., 1923. Contribution a` l’e´tude anatomique et biologique des Batraciens Urode`les du groupe des Euproctes et spe´cialement de l’Euprocte des Pyre´ne´es Triton (Euproctus) asper Duge`s. Bull. Soc. Hist. Nat. Toulouse 51, 185 – 440. Guillaume, O., Cavagnara, F., 1999. Spatial and temporal distribution of Euproctus asper from hypogean and epigean populations: experimental and fieldwork studies. Me´m. Biospe´ol. 25, in press. Houck, L.D., Sever, D.M., 1994. Role of the skin in reproduction and behaviour. In: Heatwale, H., Barthalamus, G.T. (Eds.), Amphibian Biology, vol. 1. Surrey Beatty, Chipping Norton, NSW, pp. 351 – 381. Kikuyama, S., Toyoda, F., Ohimiya, Y., Tanaka, S., Hayashi, H., 1995. Sodefrin: a female-attracting peptide pheromone in newt cloacal glands. Science 267, 1643 – 1645. Malacarne, G., Vellano, C., 1987. Behavioural evidence of a courtship pheromone in the crested newt, Triturus cristatus carnifex Laurenti. Copeia 1987 (1), 245 – 247. Mathis, A., 1991. Territories of male and female terrestrial salamanders: costs, benefits, and intersexual spatial associations. Oecologia 86, 433 – 440. Salthe, S.E., 1967. Courtship patterns and the phylogeny of the Urodeles. Copeia 1967 (1), 100 – 117. Toyoda, F., Tanaka, S., Matsuda, K., Kikuyama, S., 1994. Hormonal control of response to and secretion of the sex attractants in Japanese newts. Physiol. Behav. 55 (3), 569 – 576. Verrell, P.A., 1994. The courtship behaviour of the Apalachicola dusky salamander, Desmognathus apalachicolae Means and Karlin (Amphibia, Caudata, Plethodonthidae). Ethol. Ecol. Evol. 6, 497 – 506.
Does the Pyrenean salamander Euproctus asper use chemical cues for sex identification and mating behaviour? O. Guillaume Laboratoire souterrain du Centre National de la Recherche Scientifique, F-09200 Moulis, France Received 2 July 1998; received in revised form 7 December 1998; accepted 16 December 1998
Abstract This paper examined the potentiality of chemical cues for sex identification and mating activity in Euproctus asper. The study tested the ability of males and females to distinguish between the odour of animals of their own sex and that of animals of the opposite sex, as it was carried to them by water flowing over another animal. Their ability to distinguish between water flowing over another E. asper and water flowing over a control, where no other animal was present, was also tested. Then, the study tested their ability to distinguish between the diffusing odour of animals of their own sex, that of animals of the opposite sex, and that of a control, where no other animal was present. There was no evidence that males and females identify their mates using chemical cues. Observations of the courtship behaviour were also carried out. Mating seems to be induced by the male’s display of his tail when he captures the female as she passes near him, to form an amplexus, without any obvious preliminary. On the basis of these data, the question whether the mate identification occurs during the amplexus in this species was raised. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Amphibia; Courtship; Euproctus asper; Mating; Salamandridae
1. Introduction The salamander Euproctus asper is endemic to the Pyrenees, living in the mountain streams between 250 and 2400 m in altitude, and in some cave rivers (Astre, 1950; Combes and Knoepffler, 1963; Clergue-Gazeau, 1965). According to Clergue-Gazeau (1971), reproduction in epigean populations occurs from April onwards, on melting of the snow and differs according to the
climate, whereas it occurs throughout the entire year for the cave populations. Chemical communication plays a major role in the reproductive behaviour of some Urodeles (Malacarne and Vellano, 1987; Mathis, 1991; Houck and Sever, 1994; Toyoda et al., 1994; Verrell, 1994; Kikuyama et al., 1995). Courtship behaviour was reported in the genus Euproctus (Despax, 1923; Ahrenfeldt, 1955; Salthe, 1967; Brizzi et al., 1995), but the question of chemical
0376-6357/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 6 - 6 3 5 7 ( 9 9 ) 0 0 0 0 3 - 0
58
O. Guillaume / Beha6ioural Processes 46 (1999) 57–62
cues for sex identification and mating is still under debate. The present study examined this potentiality in Euproctus asper.
2. Materials and methods The 20 specimens of Euproctus asper (ten males and ten females) used in the present study were adults of between 8 and 12 years old, born and bred in the cave-laboratory of the C.N.R.S. (Arie`ge, France). Males were isolated from females 14 days before the experiments. The males then received an injection of 1500 UI of human chorionic gonadotropin (HCG) purchased from Organon S.A., and the females an injection of 2500 UI. During the four experiments presented here, the position of the sources of stimulation and of the animals in the testing apparatus were randomly determined for each trial. The first experiment tested the ability of E. asper to discriminate between the odour of a potential mate from the odour of an animal of its own sex, the odours being carried to the tested animal by water flowing over the others animals. The testing apparatus was a plexis Y-maze. Each arm (15 cm length, 2.1 cm diameter) was connected to a compartment (10 cm diameter, 5 cm height). An opaque perforated wall prevented animals from passing from the compartment into the Y-maze, but allowed the water to flow through the apparatus. Water flow of 5 ml/s drained from a reservoir into the two side arms, via the compartments, each containing one animal (a male in one, a female in the other) serving as the odour source. The tested animal (male or female) was kept in the compartment of the third arm of the Y-maze that received the flow of water from the two side arms. After an acclimatisation period of 30 min, the animal was then given a choice between the two side arms by removing the perforated opaque wall to unblock the passage between the compartment in which it was isolated from other parts of the Y-maze. The time spent in both arms was measured for 30 min, 5 min after the beginning of the test.
The second experiment tested the ability of E. asper to discriminate between the odour of a potential mate and a control. Tests were performed as in the first experiment, with one of the two side arms not containing any animal as a source of odour. Data from these two first experiments were analysed using the Wilcoxon-matched-pairedsigned-rank-test. The third experiment tested the ability of E. asper to discriminate between the odour diffused by an animal of its own sex, the odour diffused by an animal of the opposite sex, and from a control with any odour source. The testing apparatus was a plastic aquarium (32 cm in diameter) filled with water (3 cm in depth), and divided into three identical compartments. A plastic and opaque perforated box (8 cm in diameter and 5 cm in depth) was placed in each compartment; a female was kept in the first box, a male in the second, and the third remained empty. Each tested animal was introduced into a glass enclosure (8 cm in diameter), that was placed in the center of the aquarium. After an acclimatisation period of 30 min, the enclosure was carefully removed and the time spent by the tested animal (male or female) in each compartment was measured for 30 min. Data from this third experiment were analysed using the Friedman two way analysis of variance by ranks. The fourth experiment attempted to observe the courtship behaviour of ten male-female pairs of E. asper. In an aquarium (50 cm× 30 cm× 20 cm) filled with water (9 cm in depth) and divided into compartments separated by a removable wall, each partner was isolated separately in a confined compartment for 30 min before the observations. The wall was then removed to unblock the passage between the two compartments, and the behaviour of each animal was observed for the first 2 h and was checked during the following 2 days.
3. Results
3.1. Sex recognition In the first experiment, the males preferred the
O. Guillaume / Beha6ioural Processes 46 (1999) 57–62
stream flowing over the male to the stream flowing over the female, but the difference was not however significant (Table 1). In addition, the females spent as much time in the arm draining the water which flowed over the male as in the arm draining the water flowing over the female. There was therefore no evidence that adults of E. asper distinguished their potential mate’s odour carried by water through the Y-maze, from that of the animal of its own sex (whereas, using the same procedure, animals discriminated the water flowing over chironomid larvae to a control water flow, personal observations). In the second experiment, males spent as much time in the arm draining the water flowing over the female as in the arm draining water where no other animal was present (Table 2). Furthermore, females preferred the control water flow to that passing over the male, even though the difference was not significant. The results of this second experiment therefore indicate that the animals do not discriminate between the potential mate’s odour carried by water through the Y-maze from a plain water flow. In the third experiment, males spent as much time in the compartment of the aquarium in which a male was confined in an opaque box as in the compartment in which a female was confined in an identical manner, and as in the compartment in which an empty box was placed (Table 3). The female preferred, though not significantly, the compartment of the testing apparatus containing the box in which a male was isolated to the sector with the box in which a female was kept, and to the one in which a empty box was placed. Thus, data from the third experiment suggest that adults
59
of E. asper do not distinguish between their potential mates’ odour diffusion, the odour of animals of the same sex, and the control.
3.2. Ethogram of the mating beha6iour 3.2.1. Female actions 1. Wandering: the female moves through the environment in no particular direction. 2. Contact: the female touches the males with her snout or the toes of her anterior legs. 3. Fight: the male having caught the female, she arches her body, and writhes with rapid movements. Sometimes, she also tries to bite the male.
3.2.2. Male actions 1. Prospection: the male moves through the environment in no particular direction. 2. Approach: the male moves towards the female when she is stationary or moves towards him. 3. Following: the male moves after the female when she moves away. 4. Touch: the male touches the female with his snout or with the toes of his front leg. 5. Tail raising: the male raises his tail vertically. 6. Capture: the male catches or attempts to catch the female when she remains or passes behind him. By a rapid movement, its raised tail coils up around her body, generally round the pelvic region, thus immobilising her hind legs. 7. Cloaca-seizure: the male having caught the female, he seizes her cloaca between his hind feet.
Table 1 Comparison of the mean time spent by the tested E. asper in the two side arms of the Y-maze, one receiving the male’s odour and the second the female’s odour Tested animal
Duration in percentage Number of trials (n) P level of significance (Wilcoxon’s)
Male
Female
Male odour
Female odour
Male odour
Female odour
42.9 10 NS
11.45
35.5 10 NS
37
60
O. Guillaume / Beha6ioural Processes 46 (1999) 57–62
Table 2 Comparison of the mean time spent by the tested E. asper in the two side arms of the y-maze, one receiving the mate’s odour and the second the plain flow (control) Tested animal
Duration in percentage Number of trials (n) P level of significance (Wilcoxon’s)
Male
Female
Control odour
Female odour
Male odour
Control odour
16.55 10 NS
11.45
22.83 10 NS
42
8. Cloaca-rub: the male having caught the female, he repeatedly rubs his hind feet, simultaneously or only one of them, against her cloaca in an anteroposterior movement.
3.2.3. Temporal organisation of mating The mating of E. asper can be divided into two distinct and consecutive phases: orientation and amplexus. Orientation consists of the preliminary stage during which the male becomes aware of a potential mate. At first, both animals randomly prospected the environment and seemed generally unaware of their partners. During their movement, touching and contact could occur. If the male touched the female, she generally fled, whereas the female continued her wandering if she had induced contact. Sometimes, during his prospection, and having met the female, the male approached or followed her. This behaviour could be discontinued as the female fled. Orientation came to an end when the male fixed, raised vertically his tail and waited until the female passed or remained behind him to capture her. The duration of this phase was very variable (median = 56 min, range= 10–105 min). The amplexus consists of the male capturing the female, followed by spermatophore release. The time elapsing between tail-raising and capture (or attempt to do so) was variable (median= 0.45 min, range =0.05–3 min). Capture was followed immediately by fighting, interrupted at regular intervals by quiet periods. During this time, the male seized and rubbed the cloaca of the female. This latter behaviour was generally concomitant with the releasing of successive spermatophores which were therefore pushed toward the female cloacal orifice, and sometimes concomitant with
the expulsion of an egg by the female. All of the spermatophores (mean of the number of spermatophore released per male = 2.8, range = 1–5) released during the experiment fell to the aquarium floor during the mating, and did not lodge inside the female’s cloaca. The time between capture and spermatophore release was very variable (median= 31.5 min, range= 2–68 min). After 48 h, all the pairs were still embraced, which suggests that the duration of the amplexus may exceed several days.
4. Discussion Results of this work indicate first that E. asper does not use chemical cues carried by water, in order to meet its mate. These experimental data agree with fieldwork observations. Indeed, E. asper lives in rapid running streams which form an unfavourable environment for transmitting such cues. From an other source, experimental and ecological data show that E. asper does not exhibit a site attachment, but continuously travels inside its home range, finding new refuges as and when it moves, and does not use chemical marks on the substrate or use faecal pellets to communicate either (Guillaume and Cavagnara, 1999). These cues, would indeed be inefficient if the emitter of these messages (which only have a very local impact), does not come back to the place where it had released them to find out the aimed reciever’s response (faecal pellets are further likely to be washed away by the water current). Thus, all the data converge to indicate that E. asper does not use chemical cues for finding its mate.
O. Guillaume / Beha6ioural Processes 46 (1999) 57–62
On the other hand, the present study shows further that mating is initiated only by the male. Indeed, the male fixes in tail-raised posture, and waits until the female, which could be detected by mechanical stimuli and visual stimuli in photic conditions, passes behind him to capture her. During this orientation phase, the female is apparently indifferent to the male’s presence or looking to avoid him, and so does not exhibit any behaviour that could be interpreted as a releaser or a response to the male behaviour (the tailraising included). Then, the capture produces an amplexus, to which the female answers by the fight. Thus, mating occurs only from the male’s instigation, without any obvious persuasion on the part the female, or any behavioural interaction characteristical of the classical courtship of salamanders. These results show then clearly that E. asper does not use any behavioural signal to identify its mate before the mating. In addition, aberrant amplexus male – male, and male – young, sometime involving several individuals, often occurs in breeding, and in nature, where amplexus with sympatrics like Triturus hel6eticus, are also frequent (personal observations). Such behaviours were also observed by Ahrenfeldt (1955) whom reported referring to the males of the genus Euproctus: ‘‘The clasping reflex in these newts is, without any doubt, extremely strong, and in absence of female, is applied indiscriminately to the males of the same species, newts of a different genus, and other animate and inanimate objects.’’, and Despax (1923): ‘‘... la fre´quence des accouplements homosexuels entre maˆles... e´taient presque aussi fre´quents que les accouplements normaux et cependant... j’avais 30 femelles pour 45 maˆles; il est donc impossible d’expliquer ici la fre´quence de
61
ces rapprochements par la pe´nurie des femelles; il faut donc en chercher la cause dans le comportement du maˆle en rut, tel que je viens de le de´crire: place´ en position d’attente, le maˆle saisit tout animal qui passe a` sa porte´, sans distinction de sexe, son mouvement de capture est un pur reflexe et n’implique aucun discernement.’’ These aberrant amplexus seem however to last for a shorter time than those occurring with the females, and do not induce the release of spermatophores (personal observations). All the data converge to indicate that the male does not identify immediately the ‘partner’ he captures, whereas the release of spermatophores depends on interaction with the female during amplexus. Consequently, the hypothesis where the male recognises the female during amplexus, thus triggering spermatophore release, must be retained. It is possible that this unilateral recognition occurs when the male seizes the female cloaca, specifically through tactile recognition of the female cloaca whose conical shape is characteristic, and/or by inducing the release of molecules by the cloacal glands, which are know to be sources of the production of sexual pheromones in certain urodeles (Malacarne and Vellano, 1987; Kikuyama et al., 1995). The anterior ventral glands, by secreting on the epidermis of the cloacal cone, seem likely to be the potential source of a sexual pheromone released during cloacal-seizure and cloacal-rub (in preparation). The results of the present study also indicate that the male pushes the spermatophores towards the female’s cloacal area during cloacal-rub. These spermatophores are not introduced into the female’s cloaca, as is classically the case with urodeles, usually under the female’s instigation,
Table 3 Comparison of the mean time spent by the tested E. asper in the three sectors of the container, one containing the box in which a male was kept, the second with a box in which a female was kept, and the third with a box remaining empty Tested animal
Male
Female
Odour
Male
Female
Empty
Male
Female
Empty
Duration in percentage Number of trials (n) P level of significance (Friedman’s)
36.8 10 P= 1
31
32.2
41.5 10 P = 0.355
32.5
26
62
O. Guillaume / Beha6ioural Processes 46 (1999) 57–62
but are dispersed in the surrounds during amplexus. These observations are in accordance with studies by Despax (1923) who reported: ‘‘La femelle reste entie`rement passive pendant toute la dure´e du rapprochement sexuel, c’est le maˆle luimeˆme qui assure le transport des spermatophores au voisinage de l’ouverture ge´nitale de la femelle et qui y fait pe´ne´trer les spermatozoı¨des, il se sert pour cela de ses pattes poste´rieures et ses orteils jouent, dans une certaine mesure, le roˆle d’organe d’intromission.’’ and by Salthe (1967), who reported: ‘‘... the male moves the spermatophore with his feet toward the female cloaca and that it may stuff the sperm cap or loose spermatozoa (these having no capsule around them and being motile in the cap) between her cloacal lips’’. Moreover, the spermatophores released during amplexus are able to fertilise ‘virgin’ female’s eggs with which they come into contact (personal observations). Data therefore seem to indicate that fertilisation occurs when, during cloaca-rub, the male pushes his spermatophores with his hind legs, thus releasing the spermatozoa near the female’s cloacal orifice. The spermatozoa of E. asper, motile in the spermatophore, and which have an undulating membrane, are perfectly capable of moving around in a liquid environment (in preparation), and of thus gaining the cloacal orifice of the immobilised female. In conclusion, the results of this study indicate that E. asper does not use chemical cues as an aid in the recognition of sexual partners, and it does not develop any courtship behaviour. Consequently, the recognition between sexes may take place during amplexus and lead to the release of spermatophores which are not introduced into the female’s cloaca. Rather, the spermatozoa may be released near the female’s cloacal orifice which they may then swim to.
Acknowledgements This study was supported by the ‘Communaute´ de Travail des Pyre´ne´es – Conseil Re´gional de Midi-Pyre´ne´es’. I wish to thank Dr J. Durand for critically reading the manuscript, anonymous re.
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