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First published online March 17, 2006
Journal of Experimental Biology 209, 1355-1363 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02126
The effects of social experience on the behavioral response to unexpected touch in crayfish
1 Department of Biology, Georgia State University, Atlanta, GA 30303,
USA
2 Department of Psychology, University of Maryland, College Park, MD 20742,
USA
* Author for correspondence (e-mail: dedwards{at}gsu.edu)
Accepted 26 January 2006
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Summary |
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 TOP Summary IntroductionMaterials and methodsResultsDiscussionReferences |
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Key words: Procambarus clarkii, Crustacea, dominance hierarchy, behavior, touch, social condition
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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;
Livingstone et al., 1980;
Bruski and Dunham, 1987;
Krasne et al., 1997;
Figler et al., 1999).
Dominance status affects non-social behaviors as well as social behaviors.
Herberholz et al. found that burrow digging increased among new social
dominants and was inhibited in new social subordinates
(Herberholz et al., 2003).
Similarly, an unexpected touch of the tailfan elicited an avoidance reaction
from low-ranked or small crayfish and an immediate, oriented and aggressive
reaction from high-ranked or large crayfish
(Bovbjerg, 1953;
Nagayama et al., 1986). Under
the threat of unexpected attack, dominant and subordinate crayfish rely on
different types of escape circuitries that produce the escape response, the
tailflip (Krasne et al.,
1997).
To study the effect of social context on the crayfish's response to
unexpected touch more completely, we examined the behavioral responses of
socially inexperienced and experienced crayfish in three different social
conditions: while socially isolated for a prolonged period, while briefly
isolated from a dominant or subordinate partner and while in the presence of a
dominant or subordinate partner. We found that the behavioral responses of a
crayfish to an unexpected touch depended on both its social status and on the
social context at the time of the touch. Part of this study was previously
presented in abstract form (Song et al.,
2000).
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Materials and methods |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Preparation of socially `inexperienced' and experienced animals
Twenty-eight animals were isolated in their divided aquaria for 4 weeks
without disturbance to eliminate the influences of previous social history
(Yeh et al., 1996;
Yeh et al., 1997)
(Fig. 1A). Eight animals
(`Isolates' in Fig. 1) remained
isolated thereafter with their opaque dividers intact
(Fig. 1B), while members of 10
pairs (`Social Group' in Fig.
1) were then allowed to interact and form a dominance hierarchy by
removing their dividers for 30 min day–1 for two weeks. One
of the isolated animals died during molting and two subordinate animals died
or were severely injured during pairing, leaving seven isolates and eight
pairs for analysis. The eight pairs were monitored continuously during their
daily 30 min periods of interaction. Six pairs retained the same
dominant–subordinate relationship throughout the period of pairing;
however, two pairs experienced status reversal. Status reversals did not occur
during the period of experiments. Social status was determined during each 30
min period of interaction by scoring the numbers of attacks, approaches,
retreats and tailflips (escapes) of both members of a pair. The animal that
made a higher dominance score (attack, +2; approach, +1; retreat, –1;
tailflip, –2) was identified as the dominant, and its opponent as the
subordinate. Dominance score (D) was calculated for all pairs during
the pairing period:
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Experiments on Day 1
On the next morning, all animals were tested over a 3-h period
(Fig. 1D). Isolates and paired
animals remained isolated by a closed divider during testing. A minimum of
five manual touches was delivered in alternation to each side of the first
abdominal segment with a fine brush by an experimenter working under dim red
light and ignorant of the social status or experience of the animal undergoing
tests. Each touch stimulus was delivered to minimize disturbances of the
water, which would alert the animal to the approach of the brush, so that the
animal was unexpectedly touched. Responses were recorded on videotape
(Panasonic, WVBP500; 30 frames s–1) for later analysis. The
interval between touches was 10 min. Tests were performed when the animals
were stationary and not interacting. Following the experiment, the opaque
dividers were replaced to separate the dominant and subordinate for
approximately 15 h overnight (Fig.
1E).
Experiments on Day 2
The opaque dividers between the pairs were removed to allow the dominant
and subordinate animals to interact freely
(Fig. 1F). After 30 min of
interaction, these animals were tested for 2–3 h while together, whereas
the Isolates were tested while remaining isolated
(Fig. 1G). After the initial
tests, open dividers were placed between the dominant and subordinate animals
for 30 min to minimize physical contact between them while permitting chemical
signaling (Fig. 1H). The
animals were then tested with the open dividers still in place
(Fig. 1I). After these tests,
the open dividers were replaced with opaque dividers to re-isolate the paired
animals for 30 min (Fig. 1J).
The animals were then tested for a final time while isolated
(Fig. 1K). Each set of tests
took 2–3 h to perform.
Quantification of behavioral responses
Video images containing the positions of an animal before and after each
manual touch (stimulus) were captured using Scion image (Scion Image, NIH)
(Fig. 2). Behavioral responses
were examined and categorized into `Orienting', `Avoidance' and `No Response'
groups. Orienting responses were movements immediately following the touch
that reoriented the animal to face the stimulus source
(Fig. 2A). The category
includes three behaviors: a turn towards the stimulus source, a brief walk
backward while turning to face the stimulus source, or a short backward jump
with a brief abdominal flexion to face the stimulus source. Avoidance
responses were movements away from the stimulus source. These included five
behavioral responses: the animal rapidly walked forward away from the site of
the touch (Fig. 2B), it walked
backward away, it walked sideways away, it rotated the body axis away from the
stimulus source without walking or it tailflipped away. The No Response
behavior included all those in which the animal remained in the same position.
Movements or responses that were initiated two seconds or more after the
manual touch were not considered.
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(the angle between the body axis
and the HP vector; see Fig.
2C,D) were obtained. The HP distance decreased when an animal
moved towards the stimulus probe (Fig.
2C), whereas it increased when an animal moved away from the
stimulus probe (Fig. 2D). The
angle tended to decrease either when the animal turned to orient
towards the probe (Fig. 2Ci) or
moved a body length or more backward to avoid the probe
(Fig. 2Di). The angle
was large when an animal did not move, turned toward the stimulus probe and
moved forward, beyond the probe position
(Fig. 2Cii), or moved forward
(Fig. 2Dii).
Analysis of behavioral responses to touch
The behavioral response patterns of the isolate, dominant and subordinate
animals were compared using the Kruskal–Wallis test (nonparametric
one-way ANOVA). When an overall significant difference was found in the
behavioral response patterns of the three social types of animals, the
Mann–Whitney test (nonparametric two-tailed t-test) was used to
identify which type of animals differed. The Friedman test was used to examine
the overall difference among the response patterns of the dominant and
subordinate animals in the four consecutive social conditions; separated,
paired, semi-separated and separated. The Wilcoxon signed rank test was used
for pair-wise comparison only when there was an overall significant difference
throughout the four different behavioral response patterns.
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Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Initial responses to unexpected touch
Isolates were tested while isolated; members of pairs in Social Group were
tested when separated from their partners by an opaque divider
(Fig. 1D). Unexpected lateral
touch stimuli were delivered to animals in both groups under dim red light so
that the animals could not see the approach of the brush. The response
patterns of the Isolates and dominant and subordinate members of Social Group
were similar. The predominant response was to orient towards the stimulus:
average frequencies fell between 68 and 80% for the three social classes
(Isolates, dominants and subordinates of Social Group;
Fig. 4A). The frequencies of
Avoidance and No Responses were approximately equal in each class, and no
statistical difference was found between the three classes' response patterns
(Kruskal–Wallis test, P=0.4570 for Orienting Responses,
P=0.7133 for Avoidance Responses, P=0.6163 for No
Responses).
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Test on social pairs separated by an open divider, Day 2
To determine whether the change in the subordinate animals' responses
depended on being able to interact freely with the dominants, the animals were
tested again after being separated for 30 min by an open divider, again under
dim red light. During this period, the subordinate animals usually stayed away
from the open divider, whereas the dominants would often approach it. Both
animals, especially the subordinates, often moved along the surrounding walls,
touching them with their claws. When the subordinates touched the holes of the
open divider, the dominants often tried to attack them, but the plastic mesh
prevented serious physical interactions from occurring. The responses of the
dominants were as before, with nearly two-thirds being the Orienting Responses
(Fig. 4C). However, the
subordinate animals displayed the Orienting and the Avoidance Responses at
similar frequencies (Fig. 4C),
a pattern that newly emerged in subordinates after pairing experience.
Moreover, the frequencies of the Orienting Responses of the subordinate
animals in a semi-separated condition were significantly different from each
of the response frequencies recorded earlier
(Fig. 4E), first when the
subordinates were isolated on Day 1 (Wilcoxon test, P=0.0313), and
then when they were paired on Day 2 (Wilcoxon test, P=0.0156).
Similarly, the Avoidance Responses of the subordinate animals in a
semi-separated condition were significantly reduced compared with those
recorded earlier when they were paired on Day 2 (Wilcoxon test,
P=0.0156).
Tests on re-isolated social pairs, Day 2
To determine whether the subordinate animals' change in behavior depended
on the near presence of the dominant, as communicated through the open
divider, we tested the animals again when they were completely separated by an
opaque divider. The behavior patterns of both the subordinate and dominant
animals (Fig. 4D) were little
changed from when the animals were separated by the open divider
(Fig. 4C). The behavioral
responses of the subordinates were neither significantly different from those
of dominants (Fig. 4D;
Mann–Whitney test, P=0.2345 for Orienting Responses,
P=0.3282 for Avoidance Responses) nor different from those recorded
earlier when the subordinates were in a semi-separated condition
(Fig. 4E; Wilcoxon test,
P=0.5625 for Orienting Responses, P=0.8125 for Avoidance
Responses). However, the subordinates when isolated on Day 2
(Fig. 4D) displayed the
Orienting Responses less frequently compared with when they were isolated on
Day 1 (Fig. 4E; Wilcoxon test,
P=0.0313) but more frequently compared with when they were paired on
Day 2 (Fig. 4E; Wilcoxon test,
P=0.0156). These results indicate that both the presence of the
dominant animal and the lingering effects of their interactions during the
test period changed the behavioral response of the subordinate animals to
unexpected touch.
There was no difference in the behavioral response of the dominant animals in the four different agonistic conditions tested (Friedman test, P=0.0979 for Orienting Responses, P=0.09042 for Avoidance Responses, P=0.2992 for No Responses), indicating that the behavioral responses of the dominant animals were not dependent on the presence of their subordinate partners. Thus, it is either the opportunity to interact with the dominant or the interactions themselves that led to a shift in the response patterns of the subordinate animals from Orienting to Avoidance.
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between the animal's body
axis and the direction from the body center to the site of the probe
contact.
No Responses were characterized by low HP values and angles between
90 and 180°, whereas Orienting Responses were associated with similar HP
values and a broader range of angles, between 0 and 180°
(Fig. 5). Avoidance Responses
displayed much larger HP values and angles between 90 and
180°.
Isolate, dominant and subordinate animals displayed similar responses on
Day 1 when tested while isolated (Fig.
4A). Their patterns of movement were also similar, clustering
around low HP values and angles between 90 and 180°
(Fig. 5A). A few animals of
each social type displayed Avoidance or Orienting Responses characterized by
low values. The movement pattern of the subordinates reflected the
change in their responses when paired on Day 2
(Fig. 4B). Subordinates
displayed Avoidance Responses (red squares) with much larger HP values,
extending to more than three body lengths
(Fig. 5B). Most Avoidance
Responses also had angles near 180°, indicating that the animal
had moved forward, away from the probe. A smaller number had low
values, indicating that the animal moved backward. When the animals were
semi-separated by an open divider, the responses of the social dominants did
not change significantly (Figs
4C,
5C), whereas the Avoidance
responses of the subordinates were again characterized by large HP values and
large angles, indicating forward movement away from the probe.
Separation of the subordinates brought partial recovery of the original
movement patterns; the HP distribution of Avoidance Responses was reduced from
its highest values but still extended to greater values than when the
subordinates were tested initially (Fig.
5D).
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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; Yeh et al.,
1997; Krasne et al.,
1997; Teshiba et al.,
2001), and crayfish display agonistic behaviors typical of their
social status (Goessmann et al.,
2000; Herberholz et al.,
2001). Dominant and subordinate animals also differ in their
willingness to engage in other non-social behaviors, such as shelter
construction, in the presence of a social partner
(Herberholz et al., 2003).
Subordinate animals are inhibited from digging in the presence of a dominant
partner, while the dominant's burrowing activity is increased. The inhibition
was conditional upon the near presence of the dominant but also appeared to
linger after the subordinate had been isolated.
Previous reports indicated that the response of a crayfish to an unexpected
touch depended on the animal's size
(Nagayama et al., 1986) or
dominance status (Bovbjerg,
1953). The types of escape reflex circuitries to be activated were
dependent on social status of crayfish under the threat of unexpected attack,
while the excitability of the lateral giant escape reflex was independent of
social status when the threat was removed
(Krasne et al., 1997). Here,
we have found that a crayfish's response to an unexpected touch depends on the
animal's social context as well as its social status. The response of a social
subordinate in the presence of its dominant partner differed from its response
when alone. When kept apart from their partner by a closed barrier, the social
status or experience of the animal had no effect on its responses, which were
identical to those of socially isolated animals. Isolates and separated
dominants and subordinates all turned to confront the source of the unexpected
touch (Figs 4A,
5A,B). However, when
subordinates were tested while in partial contact with their dominant partners
(i.e. semi-separated), their patterns of responses differed from when tested
alone (Figs 4C,E,
5C). These differences
increased dramatically when the subordinates were tested in the presence of
their dominant partner: instead of turning to confront the unexpected touch,
they moved away (Figs 4B,E,
5B). It is apparent, therefore,
that as in the case of burrowing behavior
(Herberholz et al., 2003), the
social context of being subordinate in the presence of the dominant partner
determined the change in the subordinate's response to the stimulus.
Subsequent tests showed that the change in the subordinate's response brought about by being tested while paired persisted to a small degree for some hours (Fig. 4E). Tests while semi-separated (Figs 4C, 5C) and while alone (Figs 4D, 5D) revealed that the subordinates' behavior was not restored to that of the earlier isolated condition (Figs 4A, 5A) five hours after the tests while paired.
These results suggest that the effect of the paired context in which the unexpected touch was received lingers for some hours after the event. This effect may be a state change, similar to fear, or it may be a memory (i.e. a specific association) of having received the earlier unexpected touches in the presence of the dominant. When the subordinates were tested while semi-separated from their dominant partners, this state or memory may have been strengthened by olfactory stimuli from the dominant. When the subordinates were finally tested while isolated from their dominant partners, the response pattern showed some influence of the earlier paired context in which the animals were tested.
The persistence of the change in the subordinate's behavior when the
dominant was absent is similar to the lingering inhibitory effect of the
dominant on the subordinate crayfish's burrowing activity
(Herberholz et al., 2003). In
both these cases, it is not clear whether this persistence represents a
specific memory of a more generalized change in behavior, like fear. Crayfish,
like other crustaceans, can learn specific associations
(Krasne, 1973). Several
studies have demonstrated the ability of crayfish to learn to recognize
crayfish in the process of dominance hierarchy formation and maintenance
(Bovbjerg, 1953) – for
individual recognition (Lowe,
1956), recognition of aggressive state
(Copp, 1986) and status
recognition (Zulandt-Schneider et al.,
1999) – although what is recognized and when the animal
learns are not completely understood.
The complete neural mechanisms of the different behavioral responses to
unexpected lateral touch are unknown, but some elements have been identified.
Bilateral pairs of serotonergic neurons in the abdomen and thorax of crayfish
receive both excitatory and inhibitory inputs in response to a lateral touch
of the rostral portion of the abdomen, but the mix of excitation and
inhibition depends on the social status of the animal
(Drummond et al., 2002). The
ipsilateral 5-hydroxytryptamine (5-HT) neurons of Isolate and dominant
crayfish were excited by a unilateral touch while the contralateral 5-HT
neurons were inhibited. The same neurons in subordinate crayfish were
symmetrically excited in several crayfish and inhibited in as many others;
asymmetric responses did not occur. These 5-HT neurons have been found to
modulate walking leg reflexes, which also display differences between dominant
and subordinate animals (F. A. Issa, D. H. Cattaert and D. H. Edwards,
unpublished observations). It is tempting to link the asymmetric neuronal
responses of Isolate and dominant crayfish to their orienting responses
described here, and the symmetric excitatory or inhibitory neuronal responses
of subordinate crayfish to their symmetric avoidance responses. Future studies
will determine whether these correlations between neuronal responses and
behavior are indicative of underlying causal mechanisms.
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Acknowledgments |
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15 h) to prevent
further agonistic interactions. (D) On experimental Day 1, Isolates and the
socially paired animals were tested while separated. (E) Isolates and socially
paired animals remained separated overnight. (F) On Day 2, Isolates remained
separated while social pairs were allowed to interact freely with their
partners for 30 min. (G) All animals were tested, Isolates while separated and
social pairs while in the presence of their partner. (H) Social pairs were
kept apart by an open divider for 30 min and then (I) tested over a 2–3
h period. (J) Social pairs were then re-isolated by a closed divider for 30
min and then (K) tested again over 2–3 h while isolated.
