Experiment-1

In group 1, number of distress calls was compared in repeated sessions of single condition. In group 2, the companion chick was replaced by another wild-type unfamiliar chick, which the subject had never been reared together. In group 3, a mirror replaced the companion chick. A 9 × 9 cm² metal-plated glass mirror was placed upright in the experimental chamber. In group 4 (Fig. 2D), an albino chick replaced the companion chick. As shown in Fig. 1C, albino chicks had yellow plumage and red eyes, but lacked the characteristic pattern of the wild-type plumage. Fertilized eggs of the albino line (AWE strain) were kindly supplied by the Nippon Institute for Biological Science (Yamanashi, Japan), and incubated in our laboratory until hatching. On post-hatch day-2, the albino chicks (5.81±0.14 g; n=16) weighed slightly, but significantly, less than the wild-type chicks (6.40 ±0.16 g, n=10) (mean±s.e.m.; Mann-Whitney's U-test, p = 0.040). Furthermore, albino chicks seemed less active in the experimental chamber than the wild-type chicks in their spontaneous walking / pecking behaviors.

Fig. 2

Effects of different behavioral conditions on the distress calls in socially isolated quail chicks. A: Single condition. B: Unfamiliar chick. C: Mirror image. D: albino chick. The number of distress calls emitted during 5 min was plotted for 5 successive trials. Mean values± s.e.m. are shown, and n denotes the number of chicks in each group. The “paired” indicates that the subject chick was paired with the companion chick, while the “single” indicates that the subject chick was left alone in the chamber. Asterisks denote that chicks showed significantly more distress calls than in the paired conditions; *: p<0.05, **: p<0.01, Wilcoxon signed rank test of corresponding data. Data from the 4th trial were also compared with data from the 2nd single trial using the same statistical test. NS indicates no significant difference above p>0.05.

Experiment-2

In group 5 and group 6, the companion chick was replaced by video images on a thin film transistor (TFT) liquid crystal color display (LC-84TV1, 640×480 pixels; Sharp Co.). The size of single pixel was 0.32 mm (width)×0.27 mm (height). It has been reported that TFT images are superior to CRT images in eliciting behavioral responses in finches, presumably because the visual system of birds has a high temporal resolution. For detailed information about the physical properties of the TFT, refer to the analysis in that previous study by Ikebuchi and Okanoya (1999). In group 5 and group 7, we used a pair of speakers (SC-C37, Active Speaker System; Aiwa Co.) for reproduction of simultaneously recorded calls. For video replay, we selected a part of a recording (8 min long) of a non-subject wild-type chick, which emitted distress calls at a rate of 26 calls / min in social isolation. The size of the chick images on the TFT display was adjusted to be almost the same as that of a real chick. The sound pressure was roughly adjusted to the level of a live chick calling in the cage. In other groups where TFT display was not used, the surface of the display was covered with a Styrofoam plate. In group 8, instead of replacing the companion chick, the subject chick was separated from the companion by using a transparent Plexiglas plate (2 mm thick, 10 cm high). In this condition, the subject chick could recognize the companion by either visual or acoustic cues, although direct tactile stimuli were not available. Since the chamber was not separated into two airtight partitions, the subject could also have recognized the companion by ol-factory cues, even if attenuated to some extent.

Experiment-3

In group 9, the companion chick was replaced by a wild-type chick stained in green. A green acrylic paint (type X-28, water-soluble acrylic paint; Tamiya Co., Japan) was used. The plumage of 2 wild-type chicks was carefully stained using a brush, then immediately dried in a moisturized incubator for 30 min at 37°C. These stained chicks walked, called, and pecked at food and water normally, without any behavioral signs of physical or mental stress. In group 10, an anesthetized chick of wild-type plumage replaced the companion chick. A wild-type chick was anesthetized by subcutaneous injection of Ketaral (0.15 ml; containing 11.5 mg ketamine per 1 ml of saline) immediately before test. The anesthetized chick lay down with its side on the floor, and continued stable respiratory movements without any active movements until recovery (for 20–30 min). In group 11 and group 12, we separated the subject from the companion with a half-mirror. The cage was divided into two by a Plexiglas plate (2 mm thick), on which half-mirror film was attached. One partition was illuminated as usual, while the other was not. In this condition, the subject chick in the dark partition could see the companion chick in the illuminated partition, while the companion could not see the subject. The companion chick faced its own mirror image, and then emitted distress calls to some extent. The number of calls emitted by the companion was not counted. In group 11 and group 12, we used different half-mirror films; half-mirror 1 (type VM-S101, transmissivity: 22%; IKC Co.) in group 11, and half-mirror 2 (type WF-301, transmissivity: 19%; Wako Co.) in group 12. The illumination in the subject partition was turned off, so that the subject chick could see the companion chick in the illuminated partition. On the other hand, the companion chick faced its own mirror image, and did not see the subject. The half-mirror 1 degraded the fine details of the visual image, whereas the half-mirror 2 simply attenuated the luminance (see Fig. 4C, and D). Due to fine cross-stripe patterns (0.4 mm×0.4 mm), images viewed through half-mirror 1 were much more vague than those viewed through half-mirror 2.

Experiment-1

In 4 experimental groups with different conditions, we tried to confirm that distress call is consistently suppressed when accompanied by another conspecific chick with wild-type plumage.

In group 1, subject chicks emitted relatively few distress calls when paired with their companion, while they emitted distress calls at a high frequency when left alone (Fig. 2A). In both of the “single” sessions (2nd and 4th), subject chicks emitted significantly more calls than in the “paired” sessions (1st, 3rd, and 5th). No significant differences were found in a comparison of the 2nd and the 4th, indicating little habituation in the distress calling in repeated sessions of social isolation. In the following groups, therefore, subject chicks were exposed to a variety of different conditions in the 4th session, and the number of calls was compared with data from the single and paired sessions.

In group 2, even an unfamiliar chick was effective in completely suppressing the distress calls (Fig. 2B). With an unfamiliar chick, the number of calls by the subject chick did not significantly differ from the “paired” session. Slight differences in the individual cues, visual or non-visual if any, do not seem to matter.

In group 3, mirror image was effective in reducing the number of distress calls (Fig. 2C). When confronted with their own image on the mirror, chicks emitted significantly fewer calls than in the 2nd session of the “single” condition. However, the number of calls was still significantly greater than the average of the 3rd and the 5th sessions (paired). It is thus suggested that chicks recognized the mirror image similar, but not identical, to the companion chick. Taking into account the fact that the subjects did not concern the differences in individual appearance (group 2), the unusually coherent behavior of the mirror image might have impaired the suppressive effects.

In group 4, similarly, an albino chick partially suppressed the distress call (Fig. 2D). This also demonstrates that subject chicks discriminate between the wild-type and the albino chicks. The incomplete suppression could have been due, either to the obvious difference in the external appearances, or to the slightly less active nature of the albino chicks in their behaviors.

These results suggest that the appearance of conspecific wild-type individual is critical in suppressing the distress calls. What aspects of the conspecific individuals could be responsible for the effects? In the next set of experimental groups, we tried to identify critical modality of the conspecific chick as call suppressor.

Experiment-2

Conspecific chicks may suppress the distress call by way of visual, acoustic, olfactory, tactile cue, or a combination of multiple cues. In the following 4 groups, we tried to identify the modality of the most critical cue. In groups 5 and 6, the companion chick was replaced by video images with or without replay of calling sound.

In group 5, video images and simultaneously recorded sound (mostly the distress calls) of another wild-type chick were replayed using a TFT display and a pair of speakers (Figs. 1A, 3A). In group 6, video images were given without the sound (Fig. 3B). In group 7, recorded sound was replayed without video images (Fig. 3C). In both groups 5 and 6, distress calls were suppressed, but not completely as in the “paired” session. In group 7, on the other hand, the calling sound per se did not suppress the distress calls at all. As shown in group 8, distress call was suppressed even without physical contacts. We can therefore eliminate the possibility of calling sound and direct tactile stimuli as significant cues, and reasonably assume that visual stimuli gives some critical cues. Olfactory cues, if any, might not be relevant, since it was absent in the TFT (group 6) and the Speaker (group 7) conditions and present in the separated (group 8) condition; no obvious correlations between the possible olfactory cues and the call suppression. Visual cues thus proved critical.

Fig. 3

Effects of video images and sound replay on the distress calls. A: Video images of a single chick on a TFT monitor with sound via a couple of speakers. B: TFT monitor alone. C: Replay of call sound alone. D: Separation by a transparent Plexiglas. In D (physical separation), the subjects emitted significantly less calls than in the “single” condition (*: p<0.05).

Experiment-3

For suppressing the distress calls, visual TFT images were effective but not completely. What aspects of the TFT image caused the suppression, and why the suppression was incomplete? Several possibilities can be considered about critical sub-modalities of the visual cues which the wild-type companion chicks could give rise to. These are; exact coloration, natural movements, patterning of the plumage at a high spatial resolution, or behavioral correspondence between the subject and the companion. In other words, we can argue that the TFT image differs from the wild-type companion in coloration. Otherwise, the chick on the TFT seems to move in an unnatural way for the subject. Or, the TFT image does not have a sufficient spatial resolution, while chicks need fine details of the visual images. Or, chick on the TFT does not respond to the subject, thus fails to give some behavioral signals necessary for call suppression. The following 4 experimental groups were designed in order to examine these possibilities.

In group 9, experimentally altered coloration did not impair the ability of the wild-type chick to suppress the distress call (Fig. 4A). The green chick, however, perfectly suppressed the calls just as the companion chick of normal plumage. Exact coloration thus does not appear to be critical, though we did not try other colors, e.g., reddish or blue chicks. In group 10, where an anesthetized chick of wild-type plumage was presented, distress calls were not suppressed at all, and the subject behaved as if no chicks accompanied (Fig. 4B). Visual images of normally behaving chick are definitely needed.

Fig. 4

Effects of altered / degraded visual images on the suppression of distress calls. A: chick stained in green. B: anesthetized chick. C: obscured image of a wild-type chick through one-way half-mirror 1. D: clear image of a wild-type chick through half-mirror 2. Photographs on the right show a stained chick (A), a normal wild-type chick (B), and visual image of a wild-type chick viewed through the half-mirror 1 (C) and 2 (D), respectively.

Further experiments using half-mirrors suggested the importance of high spatial resolution in the visual images. In group 11 (Fig. 4C), vague images viewed through the half-mirror 1 partially suppressed the calling, whereas clear images through the half-mirror 2 completely suppressed the calling in group 12 (Fig. 4D). A high spatial resolution of the chick image is thus important. It should be emphasized again that the companion chick was placed in the illuminated partition. The subject was therefore paired with a companion chick that did not see the subject, similarly to the case with the TFT images. Mutual behavioral correspondence, if any, thus is not relevant.

Spatial resolution

A high spatial resolution seems to be critical, as was shown in the series of experiments using the TFT monitor (groups 5 and 6) and the coarse half-mirror 1 (group 11). The size of single pixels on the TFT (0.27 mm×0.32 mm) was finer, but almost comparable to the size of cross-stripes of the half-mirror (0.4 mm×0.4 mm). Visual stimuli obtained through these apparatus should have been filtered, so that components of high spatial frequency above 3–4 cycles / mm were eliminated. In general, birds can see objects at a high spatial resolution despite their relatively small eyes. For example, pigeons can detect up to 18 cycles / degree (Hodos, 1993). The value of 18 cycles / degree corresponds to ca. 20 cycles / mm if viewed at a distance of 50 mm. Though comparable data are not available for quail chick vision, we can assume that the TFT display and half-mirror 1 did not have sufficiently high spatial resolution.

Coloration

Several studies have reported that chicks can precisely memorize and discriminate colors (Aoki et al., 2000; Osorio et al., 1999). Also, from a study of sexual imprinting, it was reported that mate choice in sexually mature quails is dependent on plumage coloration (Bateson, 1978, 1982). Male quails showed the highest preference for those females who had slightly different colors from the companions to which the subject males had been exposed as juveniles, a phenomenon known as optimal out-breeding. In this study, however, chicks stained a greenish color (group 9, Fig. 4) proved to be similarly effective to normal companions in suppressing the distress calls. It is therefore supposed that chicks might have recognized the green chick, but were not concerned with the strange coloration as far as distress calling was concerned.

Olfactory cues

It has been documented that chicks depend on olfaction for food selection (Turro et al., 1994) and discrimination of familiar objects (Vallortigara and Andrew, 1994). Although we did not examine olfactory cues in this study, they would seem irrelevant for the suppression of distress calls. Separation by a transparent plate (group 8, Fig. 3D) or half mirror 2 (group 12, Fig. 4D) failed to increase, in fact decreased, the number of distress calls, and such separation if anything would have obscured the olfactory cues. Experiments using TFT display (groups 5 and 6, Fig. 3A and B) also support the idea that chicks do not depend on the smell of conspecifics.

Behavioral correspondence

Two-way behavioral correspondence, or social interactions, between chicks may not significantly influence the distress calls. In group 12 where half-mirror 2 was used (Fig. 4D), the companion did not see the subject in the darker partition; visual interactions were one-way. Interactions by way of vocalization and other auditory cues can also be disregarded, because the sound replay via speakers (group 7, Fig. 3 C) was not effective.

Neural substrates for controlling distress calls

The neural substrates for distress calling may be linked with cognitive / mnemonic functions, as has been pointed out in the INTRODUCTION. Several research strategies should be pursued in the near future. By searching for brain regions where localized lesions cause disinhibition of the distress calls, or “prosopagnosia” in chicks, one might be able to localize the brain regions specifically involved in the recognition of conspecific chicks. Single unit recordings in freely behaving chicks (Yanagihara et al., 2001) might also be adopted in these brain regions in the search for neurons coding visual images of chicks. The archistriatal complex (Davies et al., 1997; Lowndes et al., 1994) is one among the most plausible candidate regions where the emotional state meets the cognitive process, thus favoring the establishment of imprinting (ten Cate, 1986).