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Frogs and toads in front of a mirror: lateralisation of response to social stimuli in tadpoles of five anuran species
Behavioural Brain Research 134 (2002) 417 /424 www.elsevier.com/locate/bbr
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Frogs and toads in front of a mirror: lateralisation of response to social stimuli in tadpoles of five anuran species Angelo Bisazza *, Andrea De Santi, Silvia Bonso, Valeria Anna Sovrano Department of General Psychology, University of Padua, Via Venezia 8, 35131 Padova, Italy Received 17 October 2001; received in revised form 11 March 2002; accepted 11 March 2002
Abstract Tadpoles of five anuran species were tested for preferences in the use of the eyes during inspection of their own visual image in a mirror. When tested in a tank with several small mirrors, tadpoles of five different species (Bufo bufo , Bufo viridis , Rana temporaria , Rana esculenta , Bombina variegata ) preferentially approached and positioned themselves with the mirror located on their left side, thus looking at the image with the monocular field of their left eye. Similar results were obtained with tadpoles of R. temporaria tested in a simple task in which they had to choose approaching one or other of two large mirrors located on their left and right side. Control experiment showed that the behavioural asymmetry was not due to motor preferences and that it was independent of morphological asymmetries in the positions of the spiracles. This is the first demonstration of a functional visual lateralisation among juvenile amphibia before metamorphosis. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Lateralisation; Brain asymmetry; Left eye preference; Right hemisphere; Anura; Tadpoles
1. Introduction It seems to be well-established that cerebral lateralisation is present in all vertebrate classes [5,45,48]. There are, however, some issues still unresolved about the evolutionary origins of cerebral lateralisation. One concerns the possible homolog y of the phenomenon across vertebrates. It is likely in fact that there are advantages in having an asymmetric brain [19,34,42] and that cerebral lateralisation may have appeared independently several times during evolution. On the other hand, some authors suggest that cerebral lateralisation was appeared in first chordates and that this primitive asymmetric organization has been inherited, for the most part unchanged, in subsequent vertebrates [48]. Andrew et al. [2], for example, suggested that brain asymmetry of vertebrates has originated from other anatomical asymmetries of early chordates. Research carried out recently seems to suggest a basic homology between different classes of vertebrates, on the basis of similarity among different species in the direction of * Corresponding author. Fax: 39-049-8276600 E-mail address: [email protected] (A. Bisazza).
asymmetries in the same task. For instance sounds production and recognition seems to be lateralized to the left hemisphere in songbirds [28], macaque monkeys [31], frogs [3], catfish [17] and humans [8]. Spatial tasks, on the contrary, seem to be lateralized to the right hemisphere in rats [11 /13], birds [10,39,40,46] and humans [26]. Another important behavioural function that has been widely investigated for lateralisation is social recognition. It has been shown that humans [35], monkeys [20,21,23], sheep [9,30], birds [41,43,44,49] all exhibit a right hemisphere dominance in response to visual stimuli provided by conspecifics. Very recently it has been demonstrated that this asymmetry can affect social interaction in fish [4,16]. Moreover, a preference for using the left eye during inspection of conspecifics has been documented in eight different species of fish [36,37]. While these data are strongly suggestive of an homology in lateralisation across vertebrates, they do not yet constitute a demonstration: cerebral dominance is a two-state character, and if it evolved a few times independently (for example in fishes, mammals and birds), then it may easily have the same direction by chance. To clarify this point it would be necessary to know more about lateralisation of social response in
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reptiles and amphibians. There is evidence for preferential left eye use during agonistic encounters in iguanid lizards [14,15] and toads [32,47], but it could be that lateralisation in these organisms is more related to aggressive behaviour than to social recognition per se. Unfortunately both amphibians and reptiles exhibit relatively poor sociality. Few species show functional aggregations and social interactions are, for the most part, aggressive or sexual in nature. One interesting exception is represented by the juvenile stages of anuran amphibians. Although most tadpoles do not show a true shoaling behaviour like fish, in several species they show aggregative behaviour apparently based on kin or familiarity. Although kin recognition is mainly mediated by chemical cues, visual cues are important for the formation of the aggregates [7,29,50 /54]. The aim of this paper is to provide the first extensive investigation on possible visual lateralisation in tadpoles in response to social stimulation.
2. Experiment 1 The aim of the experiment was to verify whether anurans show asymmetry in the use of their eyes to look at social stimuli, as other vertebrates do. Tadpoles in general do not form shoals and therefore cannot be tested using the same procedures used with fish [36]. However, they tend to adhere to the substrate aggregating with other conspecifics; research has shown that aggregation is not random: tadpoles tend to aggregate on the basis of kin and familiarity [29,50,52 /54]. Apparently, the tendency to aggregate is enhanced by the presence of a predator either visually or olfactorily recognized [33,56]. In Experiment 1 we take advantage of these ethological peculiarities to investigate lateralisation in social aggregation responses.
2.1.2. Apparatus and procedure Sixty tadpoles were tested in the open (in a well outside of the laboratory) with natural lighting, while the others were tested in a laboratory room with artificial lighting. Testing was performed in a glass tank (45 /45 /24 cm3) on a small table 80 cm high. Into the tank 40 small mirrors (12.5 /1 cm2) were positioned, 8 forming the angles of the tank, while the others were placed in pairs (attached between them with reflecting surface outside and perpendicularly to the side of the tank) every 8 cm on each side of the tank (Fig. 1). In this way each side of the tank was divided in five segments, each of them 8 cm in length and delimited by two reflecting surfaces. Outside of the tank, on each side, millimetered paper was attached in order to subdivide the five segments in 16 vertical sectors (0.5 cm in length). Brown cardboard was inserted below the tank in order to simulate the colour of the gravel into rearing tanks. The apparatus was completely surrounded by white plastic material † (Poliplak , 46 /46 /90 cm3) with lateral small rectangular openings (20 /3 cm2) to allow the experimenter to observe the animal without being seen. For the test performed in the laboratory, artificial lighting was provided by four fluorescent lamps (15 W) mounted on a white Poliplak screen located above the test apparatus. The water in the apparatus was composed for 3/4 of the water of the tadpoles tank’s and for 1/4 of water of a Lepomis gibbosus tank’s. This was done because gregarious behaviour in tadpoles is facilitated by detection of biological substances emitted by predators [22,33,38]. This water was renewed for 1/4 at each test. The apparatus was cleaned up every day before starting the experiment. Testing was performed between 11:00 and 14:00 h, which is known to be the period of more pronounced activity and gregarious behaviour in tadpoles [33]. Each tadpole was tested singly, by placing it in the center of the apparatus and recording its behaviour (by
2.1. Materials and methods
2.1.1. Subjects We used 84 tadpoles of Bufo bufo coming from a wild population in Valsanzibio, Galzignano, Padua, North Italy, captured a week after hatching and brought to our laboratory. All tests were performed between the second and the fifth week of life; thus tadpoles were maintained in the laboratory for at least 1 week before the experiment. Tadpoles were kept in white plastic tanks (120 l) with gravel on the floor (1 cm) and with thick vegetation useful as protection. They were daily fed with † commercial tropical fish food (SERA GVG-mix ). At the end of the experiment all tadpoles were brought back to the place of capture.
Fig. 1. Schematic representation of the experimental apparatus used in Experiments 1 and 2.
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direct observation) for 10 min. Tadpole position was then scored every 15 s. The scoring was limited to the period in which the tadpole remained attached to the walls of the apparatus; periods in which tadpoles remained on the floor were discarded from the 10 min of the observation. Two measures were considered: the percentage of the number of times the tadpole located itself with the closest mirror to the left (total observations) irrespective of the distance, and the percentage of the number of times in which the whole body of the animal was within 1 cm from the mirror located to the left (close-distance observations). As can be seen in Fig. 1, the placement of the tadpoles with respect to the mirrors reflected the use of the left or the right to inspect their images, i.e. when the tadpole was close to the left mirror it used its left eye, when it was close to the right mirror it used its right eye. To check for changes with time, the first 5 min and the second 5 min of the test were analyzed separately.
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2.1.3. Results and discussion The results are shown in Fig. 2. Data were analyzed by analysis of variance (ANOVA) with testing conditions (natural lighting vs. artificial lighting) as a between-subjects factor, and time (first 5 min vs. second 5 min of test) as a within-subjects factor. The ANOVA for total observations (Fig. 2a) revealed a close to significance effect of time (F (1, 82) /3.44, P /0.067); there were no significant effect of testing conditions (F (1, 82) /0.19, n.s.) and interaction (F (1, 82) /0.04, n.s.). The ANOVA for close-distance observations (Fig. 2b) did not reveal any statistically significant effects (lighting conditions: F (1, 82) /0.07; time: F (1, 82) / 1.44; interaction: F (1, 82) /0.005). Overall, there was a significant preference for using the left eye for total observations (one-sample two-tailed t -test: t(83) /2.15, P /0.03), whereas there were no significant preferences for close-distance observations (t(83) /0.99, n.s.). During the first 5 min of test tadpoles showed no left /right preferences (total observations: t (83) /0.26, n.s.; closedistance observations: t(83) /0.09, n.s.). On the contrary during the second 5 min there was a preference to use the left eye (total observations: t (83) /3.24, P B/ 0.002; close-distance observations: t(83) /1.9, P / 0.064).
3. Experiment 2 Results of the first experiment showed that B. bufo tadpoles used the left eye to inspect their own visual image in the mirror. The aim of the second experiment was to check whether the same behavioural asymmetry could be observed in other species of anurans. 3.1. Materials and methods 3.1.1. Subjects We used 34 Bufo viridis tadpoles collected near Asiago, North /East Italy, 30 Rana temporaria tadpoles collected near Asiago, North/East Italy, and 25 Rana esculenta tadpoles collected near Spinea, Venezia, North /East Italy. Rearing conditions were the same as in the previous experiment. 3.1.2. Apparatus and procedure All tests were carried out in a corridor in the open air, thus under natural lighting, located in the main building of the Department. Apparatus and procedure were the same as in Experiment 1.
Fig. 2. Percentage tadpoles’ preference (group means with SEM are shown) for locating themselves with the mirror to their right or to their left side in animals tested under natural or artificial lighting conditions. (a) Total observations; (b) Close-distance observations.
3.1.3. Results and discussion The results are shown in Fig. 3. The ANOVA (including also the data for B. bufo of the previous experiment) did not reveal any significant difference between species (total observations: F (3, 169) /0.28;
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ference rather than to an eye preference. For instance if tadpoles have a tendency to swim counter-clockwise in the tank, then they should encounter the mirrors on the left more frequently. The aim of the following experiment was to evaluate this hypothesis. 4.1. Materials and methods 4.1.1. Subjects We used 46 R. temporaria tadpoles not used in previous experiment. Age and rearing conditions were the same as in Experiment 2. 4.1.2. Apparatus and procedure The experimental apparatus was the same as in the previous experiment, except that four transparent glasses were inserted in the tank and placed perpendicular to the mirrors (and parallel to the walls of the tank) to prevent tadpoles from seeing their images in the mirrors while maintaining the visual aspect of the apparatus similar to that of the previous experiment (Fig. 4). In order to measure the tadpoles’ clockwise and counter-clockwise direction of swimming, four lines were drawn on the floor (Fig. 4) and the number of crossings of these lines in clockwise and counter-clockwise direction was recorded. A laterality index was calculated as percentages of clockwise rotations. Even in this case all testings were carried out in the corridor in the open air under natural lighting. Fig. 3. Percentage tadpoles’ preference (group means with SEM are shown) for locating themselves with the mirror to their right or to their left side. (a) Total observations; (b) Close-distance observations.
Fig. 3a; close-distance observations: F(3, 169) /2.56; Fig. 3b). There was a significant effect of time for total observations (F (1, 169) /6.06, P /0.015), but not for close-distance observations (F (1, 169) /0.78, n.s.). The interaction was not significant (total observations: F (3, 169) /0.81: close-distance observations: F (3, 169) / 0.62). Overall there was a significant preference for using the left eye during the second 5 min of test (total observations t(172) /4.68, P B/0.001; close-distance observations t(172) /4.06, P B/0.001). During the first 5 min of test there was a tendency for a similar left eye preference for close-distance observations (total observations t(172) /0.77, n.s.; close-distance observations t(172) / 1.94, P /0.054). The results thus showed that four different species of anurans exhibited similar left eye preferences during inspection of their own mirror image.
4.1.3. Results and discussion The results are shown in Fig. 5. The ANOVA revealed that there were no significant differences between the first and the second 5 min of test (F (1, 45) /0.23, n.s.).
4. Experiment 3 We were concerned with the possibility that the behavioural asymmetry was due to a rotational pre-
Fig. 4. Schematic representation of the experimental apparatus used in Experiment 3.
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Fig. 5. Percentage tadpoles’ preference for clockwise rotation (group means with SEM are shown).
The one-sample t -test revealed that there were no significant rotational preferences (t (45) /0.62, n.s.).
5. Experiment 4 As a further confirmation of the hypothesis that the behavioural asymmetry was due to an eye rather than a rotational preference, we performed another experiment using a different method and apparatus. 5.1. Materials and methods 5.1.1. Subjects We used 104 R. temporaria tadpoles not used in previous experiment. Age and rearing conditions were the same as in Experiment 2. 5.1.2. Apparatus and procedure Testing was performed within a glass tank (30 /30 / 20 cm3) lit from above with two fluorescent lamps (8 W) and closed on top by a black cardboard; water was 10 cm deep. Within the tank there were two mirrors (29 / 30 cm2) angled 21.58. Each tadpole was inserted singly in a funnel-shaped structure made of plastic material through which it could enter in the experimental chamber where the mirrors were located, one on the left and the other on the right wall of the tank (Fig. 6). Choices for the left or the right mirror were recorded in a series of three consecutive trials. We considered a choice valid when a tadpole approached a mirror within an area of at least 3 cm. Two measures were considered: the mirror chosen in the first trial and the mirror most chosen in the three trials. Sixty-four tadpoles were used as the experimental group. Forty tadpoles were used as a control group. Tadpoles in the control group were tested in the same conditions as those of the experimental group, except
Fig. 6. Schematic representation of the experimental apparatus used in Experiment 4.
that in this case the mirrors were covered by two opaque † green sheets of plastic material (Poliplak ). All testings were carried out in the laboratory under artificial lighting. 5.1.3. Results and discussion Results revealed a significant heterogeneity between the experimental and the control group (x 2(1) /4.65 P /0.031). In the experimental group 44 out of 64 tadpoles chose most often in the three trials the mirror located on the left (x 2(1) /9.00, P B/0.003); in the control group 19 tadpoles chose the mirror on the left and 21 tadpoles chose the mirror on the right (x 2(1) / 0.1, n.s.). Similar results were obtained when considering the first choice. There was a significant overall heterogeneity between groups (x 2(1) /4.99, P /0.025). In the experimental group 43 out of 64 tadpoles chose the mirror on the left (x 2(1) /7.56, P /0.006); in the control group 18 out of 40 chose the mirror on the left (x 2(1) /0.4, n.s.).
6. Experiment 5 All the tadpoles species we have considered until now have a gross external asymmetry, namely a single sinistral spiracle through which water that enters the mouth is expelled from the body [55,58]. In order to check whether this morphological asymmetry can be related to the asymmetry in eye use we observed, we
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tested another species, Bombina variegata , in which the morphological asymmetry is absent (the spiracle is located centrally). 6.1. Materials and methods 6.1.1. Subjects Subjects were 34 tadpoles of B. variegata collected near Asiago, North /East Italy. Rearing conditions were the same as in the previous experiments. 6.1.2. Apparatus and procedure The apparatus and experimental procedures were the same as in Experiments 1 and 2. 6.1.3. Results and discussion The results are shown in Fig. 7. The ANOVA revealed a close to significant effect of time for total observations (F (1, 33) /3.69, P /0.06), but not for close-distance observations (F (1, 33) /1.70, n.s.). Overall there was a significant preference for using the left eye for total observations (t (33) /2.34, P /0.025) and a close to significance preference for the use of the left eye for close-distance observations (t(33) /1.95, P /0.06). Separate analyses for the first and second 5 min of test revealed that in the second 5 min of test tadpoles preferentially used the left eye (total observations t(33) /3.23, P /0.003; close-distance observations t(33) /2.52, P /0.018). There were no significant eye preferences during the first 5 min of test (total observations t (33) /0.37, n.s.; close-distance observations t(33) /0.74, n.s.). We also performed an overall analysis with the five species studied in the same apparatus (B. bufo , B. viridis , R. temporaria , R. esculenta , B. variegata , N /207 overall). In the second 5 min there was a highly significant preference for the left eye (total observations
Fig. 7. Percentage B. variegata tadpoles’ preference (group means with SEM are shown) for locating themselves with the mirror to their right or to their left side. (a) Total observations; (b) Close-distance observations.
t (206) /5.61, P /0.001; close-distance observations t (206) /4.77, P /0.001) and a similar preference could be observed even in the first 5 min for the close-distance observations (t(206) /2.07, P /0.04), but not for total observations (t (206) /0.58, n.s.). To check for changes with time we also performed an analysis with overall data set lumping together the data every 2 min. The results are shown in Fig. 8. The ANOVA showed that there was a significant increase of the preference for the left eye with time (ANOVA: F(4, 768) /3.59, P B/0.07; linear trend F (1, 188) /8.66, P B/ 0.04) and such an increase of the bias occurred similarly in all species (interaction F (4, 188) /1.02, n.s.).
7. General discussion The results of Experiments 1, 2 and 4 clearly showed that tadpoles of five different species (of both Rana and Bufo genus) prefer to have the (mirror) image of a conspecific on their left side, thus looking at the image with their left eye. Although the measure of the eye preference was obtained in a somewhat indirect way, Experiment 3 showed that motor asymmetries (i.e. rightward or leftward rotational preferences) cannot account for the phenomenon; moreover, Experiment 5 showed that the eye preference was unrelated to asymmetries in the location of the spiracle: B. variegata exhibited the very same left eye preference of the species that have the spiracle located to the left, in spite of the fact that it has not any spiracle positioning asymmetry [55,58]. Experiment 1 also demonstrated that the left eye preference was unaffected by the lighting conditions used during test (rotational preferences in fish are deeply affected by sun-compass mechanisms, see [6]).
Fig. 8. Percentages of tadpoles’ preference (group means with SEM are shown) for locating themselves with the mirror to the right or to the left side as a function of time in all the five species studied. (Data were lumped together every 2 min; only close-distance observations were considered.)
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In general, the preference for the left eye is quite reduced during the first minutes of test and increase progressively with time of testing. It is likely that this reflects initial adaptation to the novel environment, but it is interesting to note that in fish a reverse pattern is usually observed [36,37]. It is unclear at present whether this reflect general behavioural differences between species or something more specifically related to lateralisation. In adult anurans nearly all ganglion cell axons cross at the optic chiasma with few (B/5%) ipsilateral retinal projections implicated in binocular vision [18,57]. Ipsilateral projections begin to develop at metamorphosis when the eye move dorsofrontally giving rise to binocular vision and thus in the tadpole virtually all ganglion cells send axon contralaterally [24,25]. As a consequence, like in most vertebrates with laterally placed eyes, visual information processed by one side of the brain is primarily received from the contralateral retina. Our results seem thus to imply that amphibians exhibit a right hemisphere dominance in response to visual stimuli provided by a conspecific. As mentioned in Section 1, there is now evidence for teleost fish, birds and mammals of a dominance of the right hemisphere in the control of visual processing and storing of information related to social stimuli and social interaction ([42,45] for reviews). Although this evidence would support the hypothesis that lateralisation appeared early in the evolution of vertebrates, it is not enough to prove that the trait is truly homologous in all vertebrates. In fact, there are data available for one species of bird and for few species of mammals (with one exception, they all belong to the order primates). Teleost fishes provide a larger sample (eight) of species studied, but still they represent few orders, none of which closely related to the lineage that some 400 million years ago gave rise to the early tetrapods [27]. Assuming that lateralisation of social recognition processes evolved independently in the three classes, it could possibly present the same direction by chance. The data shown here constitute the very first extensive evidence that tadpoles exhibit a left eye preference very similar to that shown by fish and birds (and, with reference to the left visual hemifield, to mammals too, see [30]). The hypothesis of an ancient and possibly unique origin of brain lateralisation in a common chordate ancestor ([1,2,45]) appears substantiated by these data.
Acknowledgements We thank Giorgio Vallortigara and Richard J. Wassersug for comments on the manuscript. The research was supported by a grant from the Italian
Ministry of (M.I.U.R.).
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[40] Tommasi L, Andrew RJ, Vallortigara G. Eye use in search is determined by the nature of the task in the domestic chick. Behav Brain Res 2000;112:119 /26. [41] Vallortigara G. Right hemisphere advantage for social recognition in the chick. Neuropsychologia 1992;30:761 /8. [42] Vallortigara G. Comparative neuropsychology of the dual brain: a stroll through left and right animals’ perceptual worlds. Brain Lang 2000;73:189 /219. [43] Vallortigara G, Andrew RJ. Lateralisation of response to change in a model partner by chicks. Anim Behav 1991;41:187 /94. [44] Vallortigara G, Andrew RJ. Differential involvement of right and left hemisphere in individual recognition in the domestic chick. Behav Process 1994;33:41 /58. [45] Vallortigara G, Bisazza A. How ancient is brain lateralisation? In: Andrew RJ, Rogers LJ, editor. Comparative Vertebrate Lateralisation. Cambridge: Cambridge University Press 2002; 9 /69. [46] Vallortigara G, Regolin L, Bortolomiol G, Tommasi L. Lateral asymmetries due to preferences in eye use during visual discrimination learning in chicks. Behav Brain Res 1996;74:135 /43. [47] Vallortigara G, Rogers LJ, Bisazza A, Lippolis G, Robins A. Complementary specializations of the right and left hemifield for predatory and agonistic behaviour in toads. NeuroReport 1998;9:3341 /4. [48] Vallortigara G, Rogers LJ, Bisazza A. Possible evolutionary origins of cognitive brain lateralisation. Brain Res Rev 1999;30:164 /75. [49] Vallortigara G, Cozzutti C, Tommasi L, Rogers LJ. How birds use their eyes: opposite left /right specialization for the lateral and frontal visual hemifield in the domestic chick. Curr Biol 2001;11:29 /33. [50] Waldman B. Kin recognition and sibling association among wood frog (Rana sylvatica ) tadpoles. Behav Ecol Sociobiol 1984;14:171 /80. [51] Waldman B, Adler K. Toad tadpoles associate preferentially with siblings. Nature 1979;282:611 /3. [52] Wassersug RJ. Aspects of social behavior in anuran larvae. In: Vial JL, editor. Evolutionary biology of the anurans. Columbia: University of Missouri Press, 1973:273 /97. [53] Wassersug RJ, Hessler CM. Tadpole behaviour: aggregation in larval Xenopus laevis . Anim Behav 1971;19:386 /9. [54] Wassersug RL, Lum AM, Potel MJ. An analysis of school structure for tadpoles (Anura: Amphibia). Behav Ecol Sociobiol 1981;9:15 /22. [55] Wassersug RJ, Naitoh T, Yamashita M. Turning bias in tadpoles. J Erpet 1999;33:543 /8. [56] Watt PJ, Nottingham SF, Young S. Toad tadpole aggregation behaviour: evidence for a predator avoidance function. Anim Behav 1997;54:865 /72. [57] Wye-Dvorak J, Straznicky C, Toth P. Retinal projections in the cane toad, Bufo marinus . Brain Behav Evol 1992;39:208 /18. [58] Yamashita M, Naitoh T, Wassersug RJ. Startle response and turning bias in Microhyla tadpoles. Zool Sci 1999;17:185 /9.
Research report
Frogs and toads in front of a mirror: lateralisation of response to social stimuli in tadpoles of five anuran species Angelo Bisazza *, Andrea De Santi, Silvia Bonso, Valeria Anna Sovrano Department of General Psychology, University of Padua, Via Venezia 8, 35131 Padova, Italy Received 17 October 2001; received in revised form 11 March 2002; accepted 11 March 2002
Abstract Tadpoles of five anuran species were tested for preferences in the use of the eyes during inspection of their own visual image in a mirror. When tested in a tank with several small mirrors, tadpoles of five different species (Bufo bufo , Bufo viridis , Rana temporaria , Rana esculenta , Bombina variegata ) preferentially approached and positioned themselves with the mirror located on their left side, thus looking at the image with the monocular field of their left eye. Similar results were obtained with tadpoles of R. temporaria tested in a simple task in which they had to choose approaching one or other of two large mirrors located on their left and right side. Control experiment showed that the behavioural asymmetry was not due to motor preferences and that it was independent of morphological asymmetries in the positions of the spiracles. This is the first demonstration of a functional visual lateralisation among juvenile amphibia before metamorphosis. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Lateralisation; Brain asymmetry; Left eye preference; Right hemisphere; Anura; Tadpoles
1. Introduction It seems to be well-established that cerebral lateralisation is present in all vertebrate classes [5,45,48]. There are, however, some issues still unresolved about the evolutionary origins of cerebral lateralisation. One concerns the possible homolog y of the phenomenon across vertebrates. It is likely in fact that there are advantages in having an asymmetric brain [19,34,42] and that cerebral lateralisation may have appeared independently several times during evolution. On the other hand, some authors suggest that cerebral lateralisation was appeared in first chordates and that this primitive asymmetric organization has been inherited, for the most part unchanged, in subsequent vertebrates [48]. Andrew et al. [2], for example, suggested that brain asymmetry of vertebrates has originated from other anatomical asymmetries of early chordates. Research carried out recently seems to suggest a basic homology between different classes of vertebrates, on the basis of similarity among different species in the direction of * Corresponding author. Fax: 39-049-8276600 E-mail address: [email protected] (A. Bisazza).
asymmetries in the same task. For instance sounds production and recognition seems to be lateralized to the left hemisphere in songbirds [28], macaque monkeys [31], frogs [3], catfish [17] and humans [8]. Spatial tasks, on the contrary, seem to be lateralized to the right hemisphere in rats [11 /13], birds [10,39,40,46] and humans [26]. Another important behavioural function that has been widely investigated for lateralisation is social recognition. It has been shown that humans [35], monkeys [20,21,23], sheep [9,30], birds [41,43,44,49] all exhibit a right hemisphere dominance in response to visual stimuli provided by conspecifics. Very recently it has been demonstrated that this asymmetry can affect social interaction in fish [4,16]. Moreover, a preference for using the left eye during inspection of conspecifics has been documented in eight different species of fish [36,37]. While these data are strongly suggestive of an homology in lateralisation across vertebrates, they do not yet constitute a demonstration: cerebral dominance is a two-state character, and if it evolved a few times independently (for example in fishes, mammals and birds), then it may easily have the same direction by chance. To clarify this point it would be necessary to know more about lateralisation of social response in
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reptiles and amphibians. There is evidence for preferential left eye use during agonistic encounters in iguanid lizards [14,15] and toads [32,47], but it could be that lateralisation in these organisms is more related to aggressive behaviour than to social recognition per se. Unfortunately both amphibians and reptiles exhibit relatively poor sociality. Few species show functional aggregations and social interactions are, for the most part, aggressive or sexual in nature. One interesting exception is represented by the juvenile stages of anuran amphibians. Although most tadpoles do not show a true shoaling behaviour like fish, in several species they show aggregative behaviour apparently based on kin or familiarity. Although kin recognition is mainly mediated by chemical cues, visual cues are important for the formation of the aggregates [7,29,50 /54]. The aim of this paper is to provide the first extensive investigation on possible visual lateralisation in tadpoles in response to social stimulation.
2. Experiment 1 The aim of the experiment was to verify whether anurans show asymmetry in the use of their eyes to look at social stimuli, as other vertebrates do. Tadpoles in general do not form shoals and therefore cannot be tested using the same procedures used with fish [36]. However, they tend to adhere to the substrate aggregating with other conspecifics; research has shown that aggregation is not random: tadpoles tend to aggregate on the basis of kin and familiarity [29,50,52 /54]. Apparently, the tendency to aggregate is enhanced by the presence of a predator either visually or olfactorily recognized [33,56]. In Experiment 1 we take advantage of these ethological peculiarities to investigate lateralisation in social aggregation responses.
2.1.2. Apparatus and procedure Sixty tadpoles were tested in the open (in a well outside of the laboratory) with natural lighting, while the others were tested in a laboratory room with artificial lighting. Testing was performed in a glass tank (45 /45 /24 cm3) on a small table 80 cm high. Into the tank 40 small mirrors (12.5 /1 cm2) were positioned, 8 forming the angles of the tank, while the others were placed in pairs (attached between them with reflecting surface outside and perpendicularly to the side of the tank) every 8 cm on each side of the tank (Fig. 1). In this way each side of the tank was divided in five segments, each of them 8 cm in length and delimited by two reflecting surfaces. Outside of the tank, on each side, millimetered paper was attached in order to subdivide the five segments in 16 vertical sectors (0.5 cm in length). Brown cardboard was inserted below the tank in order to simulate the colour of the gravel into rearing tanks. The apparatus was completely surrounded by white plastic material † (Poliplak , 46 /46 /90 cm3) with lateral small rectangular openings (20 /3 cm2) to allow the experimenter to observe the animal without being seen. For the test performed in the laboratory, artificial lighting was provided by four fluorescent lamps (15 W) mounted on a white Poliplak screen located above the test apparatus. The water in the apparatus was composed for 3/4 of the water of the tadpoles tank’s and for 1/4 of water of a Lepomis gibbosus tank’s. This was done because gregarious behaviour in tadpoles is facilitated by detection of biological substances emitted by predators [22,33,38]. This water was renewed for 1/4 at each test. The apparatus was cleaned up every day before starting the experiment. Testing was performed between 11:00 and 14:00 h, which is known to be the period of more pronounced activity and gregarious behaviour in tadpoles [33]. Each tadpole was tested singly, by placing it in the center of the apparatus and recording its behaviour (by
2.1. Materials and methods
2.1.1. Subjects We used 84 tadpoles of Bufo bufo coming from a wild population in Valsanzibio, Galzignano, Padua, North Italy, captured a week after hatching and brought to our laboratory. All tests were performed between the second and the fifth week of life; thus tadpoles were maintained in the laboratory for at least 1 week before the experiment. Tadpoles were kept in white plastic tanks (120 l) with gravel on the floor (1 cm) and with thick vegetation useful as protection. They were daily fed with † commercial tropical fish food (SERA GVG-mix ). At the end of the experiment all tadpoles were brought back to the place of capture.
Fig. 1. Schematic representation of the experimental apparatus used in Experiments 1 and 2.
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direct observation) for 10 min. Tadpole position was then scored every 15 s. The scoring was limited to the period in which the tadpole remained attached to the walls of the apparatus; periods in which tadpoles remained on the floor were discarded from the 10 min of the observation. Two measures were considered: the percentage of the number of times the tadpole located itself with the closest mirror to the left (total observations) irrespective of the distance, and the percentage of the number of times in which the whole body of the animal was within 1 cm from the mirror located to the left (close-distance observations). As can be seen in Fig. 1, the placement of the tadpoles with respect to the mirrors reflected the use of the left or the right to inspect their images, i.e. when the tadpole was close to the left mirror it used its left eye, when it was close to the right mirror it used its right eye. To check for changes with time, the first 5 min and the second 5 min of the test were analyzed separately.
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2.1.3. Results and discussion The results are shown in Fig. 2. Data were analyzed by analysis of variance (ANOVA) with testing conditions (natural lighting vs. artificial lighting) as a between-subjects factor, and time (first 5 min vs. second 5 min of test) as a within-subjects factor. The ANOVA for total observations (Fig. 2a) revealed a close to significance effect of time (F (1, 82) /3.44, P /0.067); there were no significant effect of testing conditions (F (1, 82) /0.19, n.s.) and interaction (F (1, 82) /0.04, n.s.). The ANOVA for close-distance observations (Fig. 2b) did not reveal any statistically significant effects (lighting conditions: F (1, 82) /0.07; time: F (1, 82) / 1.44; interaction: F (1, 82) /0.005). Overall, there was a significant preference for using the left eye for total observations (one-sample two-tailed t -test: t(83) /2.15, P /0.03), whereas there were no significant preferences for close-distance observations (t(83) /0.99, n.s.). During the first 5 min of test tadpoles showed no left /right preferences (total observations: t (83) /0.26, n.s.; closedistance observations: t(83) /0.09, n.s.). On the contrary during the second 5 min there was a preference to use the left eye (total observations: t (83) /3.24, P B/ 0.002; close-distance observations: t(83) /1.9, P / 0.064).
3. Experiment 2 Results of the first experiment showed that B. bufo tadpoles used the left eye to inspect their own visual image in the mirror. The aim of the second experiment was to check whether the same behavioural asymmetry could be observed in other species of anurans. 3.1. Materials and methods 3.1.1. Subjects We used 34 Bufo viridis tadpoles collected near Asiago, North /East Italy, 30 Rana temporaria tadpoles collected near Asiago, North/East Italy, and 25 Rana esculenta tadpoles collected near Spinea, Venezia, North /East Italy. Rearing conditions were the same as in the previous experiment. 3.1.2. Apparatus and procedure All tests were carried out in a corridor in the open air, thus under natural lighting, located in the main building of the Department. Apparatus and procedure were the same as in Experiment 1.
Fig. 2. Percentage tadpoles’ preference (group means with SEM are shown) for locating themselves with the mirror to their right or to their left side in animals tested under natural or artificial lighting conditions. (a) Total observations; (b) Close-distance observations.
3.1.3. Results and discussion The results are shown in Fig. 3. The ANOVA (including also the data for B. bufo of the previous experiment) did not reveal any significant difference between species (total observations: F (3, 169) /0.28;
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ference rather than to an eye preference. For instance if tadpoles have a tendency to swim counter-clockwise in the tank, then they should encounter the mirrors on the left more frequently. The aim of the following experiment was to evaluate this hypothesis. 4.1. Materials and methods 4.1.1. Subjects We used 46 R. temporaria tadpoles not used in previous experiment. Age and rearing conditions were the same as in Experiment 2. 4.1.2. Apparatus and procedure The experimental apparatus was the same as in the previous experiment, except that four transparent glasses were inserted in the tank and placed perpendicular to the mirrors (and parallel to the walls of the tank) to prevent tadpoles from seeing their images in the mirrors while maintaining the visual aspect of the apparatus similar to that of the previous experiment (Fig. 4). In order to measure the tadpoles’ clockwise and counter-clockwise direction of swimming, four lines were drawn on the floor (Fig. 4) and the number of crossings of these lines in clockwise and counter-clockwise direction was recorded. A laterality index was calculated as percentages of clockwise rotations. Even in this case all testings were carried out in the corridor in the open air under natural lighting. Fig. 3. Percentage tadpoles’ preference (group means with SEM are shown) for locating themselves with the mirror to their right or to their left side. (a) Total observations; (b) Close-distance observations.
Fig. 3a; close-distance observations: F(3, 169) /2.56; Fig. 3b). There was a significant effect of time for total observations (F (1, 169) /6.06, P /0.015), but not for close-distance observations (F (1, 169) /0.78, n.s.). The interaction was not significant (total observations: F (3, 169) /0.81: close-distance observations: F (3, 169) / 0.62). Overall there was a significant preference for using the left eye during the second 5 min of test (total observations t(172) /4.68, P B/0.001; close-distance observations t(172) /4.06, P B/0.001). During the first 5 min of test there was a tendency for a similar left eye preference for close-distance observations (total observations t(172) /0.77, n.s.; close-distance observations t(172) / 1.94, P /0.054). The results thus showed that four different species of anurans exhibited similar left eye preferences during inspection of their own mirror image.
4.1.3. Results and discussion The results are shown in Fig. 5. The ANOVA revealed that there were no significant differences between the first and the second 5 min of test (F (1, 45) /0.23, n.s.).
4. Experiment 3 We were concerned with the possibility that the behavioural asymmetry was due to a rotational pre-
Fig. 4. Schematic representation of the experimental apparatus used in Experiment 3.
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Fig. 5. Percentage tadpoles’ preference for clockwise rotation (group means with SEM are shown).
The one-sample t -test revealed that there were no significant rotational preferences (t (45) /0.62, n.s.).
5. Experiment 4 As a further confirmation of the hypothesis that the behavioural asymmetry was due to an eye rather than a rotational preference, we performed another experiment using a different method and apparatus. 5.1. Materials and methods 5.1.1. Subjects We used 104 R. temporaria tadpoles not used in previous experiment. Age and rearing conditions were the same as in Experiment 2. 5.1.2. Apparatus and procedure Testing was performed within a glass tank (30 /30 / 20 cm3) lit from above with two fluorescent lamps (8 W) and closed on top by a black cardboard; water was 10 cm deep. Within the tank there were two mirrors (29 / 30 cm2) angled 21.58. Each tadpole was inserted singly in a funnel-shaped structure made of plastic material through which it could enter in the experimental chamber where the mirrors were located, one on the left and the other on the right wall of the tank (Fig. 6). Choices for the left or the right mirror were recorded in a series of three consecutive trials. We considered a choice valid when a tadpole approached a mirror within an area of at least 3 cm. Two measures were considered: the mirror chosen in the first trial and the mirror most chosen in the three trials. Sixty-four tadpoles were used as the experimental group. Forty tadpoles were used as a control group. Tadpoles in the control group were tested in the same conditions as those of the experimental group, except
Fig. 6. Schematic representation of the experimental apparatus used in Experiment 4.
that in this case the mirrors were covered by two opaque † green sheets of plastic material (Poliplak ). All testings were carried out in the laboratory under artificial lighting. 5.1.3. Results and discussion Results revealed a significant heterogeneity between the experimental and the control group (x 2(1) /4.65 P /0.031). In the experimental group 44 out of 64 tadpoles chose most often in the three trials the mirror located on the left (x 2(1) /9.00, P B/0.003); in the control group 19 tadpoles chose the mirror on the left and 21 tadpoles chose the mirror on the right (x 2(1) / 0.1, n.s.). Similar results were obtained when considering the first choice. There was a significant overall heterogeneity between groups (x 2(1) /4.99, P /0.025). In the experimental group 43 out of 64 tadpoles chose the mirror on the left (x 2(1) /7.56, P /0.006); in the control group 18 out of 40 chose the mirror on the left (x 2(1) /0.4, n.s.).
6. Experiment 5 All the tadpoles species we have considered until now have a gross external asymmetry, namely a single sinistral spiracle through which water that enters the mouth is expelled from the body [55,58]. In order to check whether this morphological asymmetry can be related to the asymmetry in eye use we observed, we
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tested another species, Bombina variegata , in which the morphological asymmetry is absent (the spiracle is located centrally). 6.1. Materials and methods 6.1.1. Subjects Subjects were 34 tadpoles of B. variegata collected near Asiago, North /East Italy. Rearing conditions were the same as in the previous experiments. 6.1.2. Apparatus and procedure The apparatus and experimental procedures were the same as in Experiments 1 and 2. 6.1.3. Results and discussion The results are shown in Fig. 7. The ANOVA revealed a close to significant effect of time for total observations (F (1, 33) /3.69, P /0.06), but not for close-distance observations (F (1, 33) /1.70, n.s.). Overall there was a significant preference for using the left eye for total observations (t (33) /2.34, P /0.025) and a close to significance preference for the use of the left eye for close-distance observations (t(33) /1.95, P /0.06). Separate analyses for the first and second 5 min of test revealed that in the second 5 min of test tadpoles preferentially used the left eye (total observations t(33) /3.23, P /0.003; close-distance observations t(33) /2.52, P /0.018). There were no significant eye preferences during the first 5 min of test (total observations t (33) /0.37, n.s.; close-distance observations t(33) /0.74, n.s.). We also performed an overall analysis with the five species studied in the same apparatus (B. bufo , B. viridis , R. temporaria , R. esculenta , B. variegata , N /207 overall). In the second 5 min there was a highly significant preference for the left eye (total observations
Fig. 7. Percentage B. variegata tadpoles’ preference (group means with SEM are shown) for locating themselves with the mirror to their right or to their left side. (a) Total observations; (b) Close-distance observations.
t (206) /5.61, P /0.001; close-distance observations t (206) /4.77, P /0.001) and a similar preference could be observed even in the first 5 min for the close-distance observations (t(206) /2.07, P /0.04), but not for total observations (t (206) /0.58, n.s.). To check for changes with time we also performed an analysis with overall data set lumping together the data every 2 min. The results are shown in Fig. 8. The ANOVA showed that there was a significant increase of the preference for the left eye with time (ANOVA: F(4, 768) /3.59, P B/0.07; linear trend F (1, 188) /8.66, P B/ 0.04) and such an increase of the bias occurred similarly in all species (interaction F (4, 188) /1.02, n.s.).
7. General discussion The results of Experiments 1, 2 and 4 clearly showed that tadpoles of five different species (of both Rana and Bufo genus) prefer to have the (mirror) image of a conspecific on their left side, thus looking at the image with their left eye. Although the measure of the eye preference was obtained in a somewhat indirect way, Experiment 3 showed that motor asymmetries (i.e. rightward or leftward rotational preferences) cannot account for the phenomenon; moreover, Experiment 5 showed that the eye preference was unrelated to asymmetries in the location of the spiracle: B. variegata exhibited the very same left eye preference of the species that have the spiracle located to the left, in spite of the fact that it has not any spiracle positioning asymmetry [55,58]. Experiment 1 also demonstrated that the left eye preference was unaffected by the lighting conditions used during test (rotational preferences in fish are deeply affected by sun-compass mechanisms, see [6]).
Fig. 8. Percentages of tadpoles’ preference (group means with SEM are shown) for locating themselves with the mirror to the right or to the left side as a function of time in all the five species studied. (Data were lumped together every 2 min; only close-distance observations were considered.)
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In general, the preference for the left eye is quite reduced during the first minutes of test and increase progressively with time of testing. It is likely that this reflects initial adaptation to the novel environment, but it is interesting to note that in fish a reverse pattern is usually observed [36,37]. It is unclear at present whether this reflect general behavioural differences between species or something more specifically related to lateralisation. In adult anurans nearly all ganglion cell axons cross at the optic chiasma with few (B/5%) ipsilateral retinal projections implicated in binocular vision [18,57]. Ipsilateral projections begin to develop at metamorphosis when the eye move dorsofrontally giving rise to binocular vision and thus in the tadpole virtually all ganglion cells send axon contralaterally [24,25]. As a consequence, like in most vertebrates with laterally placed eyes, visual information processed by one side of the brain is primarily received from the contralateral retina. Our results seem thus to imply that amphibians exhibit a right hemisphere dominance in response to visual stimuli provided by a conspecific. As mentioned in Section 1, there is now evidence for teleost fish, birds and mammals of a dominance of the right hemisphere in the control of visual processing and storing of information related to social stimuli and social interaction ([42,45] for reviews). Although this evidence would support the hypothesis that lateralisation appeared early in the evolution of vertebrates, it is not enough to prove that the trait is truly homologous in all vertebrates. In fact, there are data available for one species of bird and for few species of mammals (with one exception, they all belong to the order primates). Teleost fishes provide a larger sample (eight) of species studied, but still they represent few orders, none of which closely related to the lineage that some 400 million years ago gave rise to the early tetrapods [27]. Assuming that lateralisation of social recognition processes evolved independently in the three classes, it could possibly present the same direction by chance. The data shown here constitute the very first extensive evidence that tadpoles exhibit a left eye preference very similar to that shown by fish and birds (and, with reference to the left visual hemifield, to mammals too, see [30]). The hypothesis of an ancient and possibly unique origin of brain lateralisation in a common chordate ancestor ([1,2,45]) appears substantiated by these data.
Acknowledgements We thank Giorgio Vallortigara and Richard J. Wassersug for comments on the manuscript. The research was supported by a grant from the Italian
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