Urine makes the difference : chemical communication in fighting crayfish made visible
Fakultät für Biologie, Universität Konstanz, Postfach
5560 (M618), D-78457 Konstanz, Germany
* Present address: Department of Biological Sciences, University of Hull, Hull
HU6 7RX, UK
(e-mail: t.breithaupt{at}hull.ac.uk )
Accepted 11 February 2002
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Summary |
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Key words: urine, visualisation technique, chemical signal, olfaction, dominance, fighting, Decapoda, Crustacea, Astacus leptodactylus, crayfish
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Introduction |
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Crayfish are nocturnal animals with a well-developed sense of olfaction.
Previous studies on a variety of crayfish species suggest that chemical
signals play an important role in various aspects of their life including
courtship, brood care, predator avoidance and agonistic interactions
(Ameyaw-Akumfi and Hazlett,
1975; Blake and Hart,
1993
; Dunham and Oh,
1992
; Little,
1975
; Tierney and Dunham,
1982
; Zulandt Schneider and
Moore, 2000
; Zulandt Schneider
et al., 1999
). In recent years, their agonistic behaviour has
received increasing attention as a model system for the study of mechanisms
underlying complex behaviours (Herberholz
et al., 2001
; Huber and
Delago, 1998
; Issa et al.,
1999
; Listerman et al.,
2000
; Yeh et al.,
1997
). Crayfish frequently engage in fights over resources.
Factors determining fight outcome include size, sex, past experience and who
initiates the fight (Bovbjerg,
1956
; Guiasu and Dunham,
1997
; Issa et al.,
1999
; Rubenstein and Hazlett,
1974
; Scrivener,
1971
; Sinclair,
1977
). Fights between size-matched individuals are longer and
escalate in aggression from simple approach to displays such as meral
spreading (a threat display) and finally to potentially damaging behaviours
such as claw ripping (Bruski and Dunham,
1987
; Huber and Delago,
1998
). Male crayfish (Procambarus clarkii, Orconectes
virilis) also show meral spreading when exposed to tank water from male
conspecifics, suggesting that chemical signals may play a role in agonistic
interactions (Ameyaw-Akumfi and Hazlett,
1975
; Dunham and Oh,
1992
; Hazlett,
1985
). However, the source of the chemical signal and its specific
function remain unclear.
In other decapod crustaceans (lobsters, green crabs and blue crabs), the
chemical signals are mostly urine-borne and elicit specific responses that are
different in males and females (Atema and
Cowan, 1986; Bamber and Naylor,
1997
; Christofferson,
1978
; Eales, 1974
;
Gleeson, 1991
). In lobsters,
catheters consisting of flexible plastic tubing attached to the nephropores
were used to monitor urine release
(Breithaupt et al., 1999
) and
demonstrated the important role of urine signals in the maintenance of
dominance hierarchies (Breithaupt et al.,
1999
; Karavanich and Atema,
1998b
). Dominance is (at least in part) based on the loser's
olfactory recognition of the individual composition of the urine of their
previous winners (Karavanich and Atema,
1998a
). In addition to individual signatures, lobster urine
contains signal components indicating dominance status
(Bushmann and Atema, 2000
). In
lobsters, urine signalling is limited to offensive behaviours and increases
with increasing levels of aggression
(Breithaupt and Atema, 2000
).
However, the use of catheters prevents the transmission of urine to the
opponent. These studies on lobsters could not therefore reveal whether urine
signals have an immediate effect on the behaviour of the receiver during the
fight.
We were interested to know whether crayfish employ urine as an aggressive signal and whether urine signals elicit a response from the receiver during the fight. We were also interested in the message conveyed by urine signals. Does urine contain information about the identity of the signaller? Does it reveal the aggressive motivation of the signaller?
Crayfish aggressive interactions were studied in size-matched male Astacus leptodactylus. The animals were reversibly blindfolded to prevent responses to the visual occurrence of the dye that was used to visualise urine release.
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Materials and methods |
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To study dominance interactions, we used intermoult males of Astacus
leptodactylus (carapace length 40-50 mm, mass 50-90 g) with intact
appendages. After marking them individually, we kept the crayfish in four 250
l tanks containing up to 20 animals. The four groups were separated for more
than 2 weeks to reduce the chance of individual memory being developed between
individuals from different tanks [Karavanich and Atema
(1998a) showed that such
memory does not last 2 weeks in lobsters]. Seventy-two hours before the fight,
individual crayfish were separated and placed in 20 l aquaria at 16°C.
Prior to isolation, they were blindfolded by wrapping opaque tape around the
eyestalks and rostrum and fixing the tape to the carapace with cyanoacrylate
glue. The blindfold served to exclude possible reactions to visual
disturbances including those associated with the release of Fluorescein from
the nephropores.
To eliminate the influence of body size on the intensity and outcome of a
fight, the opponents were size-matched (carapace length differences less than
5%, chelae length differences less than 6%) [for the effect of size
differences on lobster fights, see Scrivener
(1971)]. Combatants were taken
from separate communal tanks, thus ensuring that they could not remember the
opponent from previous encounters
(Karavanich and Atema,
1998a
).
Urine visualisation technique
We tried several methods (oral application, injection into muscles and
heart) of administering four dyes (Methylene Blue, Indigo Carmine, Phenol Red
and sodium Fluorescein; Merck KGaA, Darmstadt, Germany). Of these, only
injection of Fluorescein into the bloodstream worked reliably.
A solution of 0.1% sodium Fluorescein dissolved in crayfish saline
(Van Harrefeld and Verwey,
1936) was injected at a dose of 2-6 µg g-1 body mass
into the heart/pericardium region of the crayfish approximately one-third of a
carapace length rostral to the caudal edge of the carapace. The dye was
injected using a 250 µl glass syringe (Unimetrics Corp, Folsom, Germany)
and 45-gauge needle (Luer-Lok 0.45 mmx13 mm; B. Braun, Melsungen,
Germany). After injection, the hole in the carapace was quickly sealed with
beeswax and tape to avoid loss of haemolymph. Release of dye was observed
starting 30-60 min after injection (Fig.
1). Close-up video recordings of the frontal area of crayfish
(Astacus leptodactylus and Procambarus clarkii) confirmed
that the dye was released from the nephropore. Dye was released intermittently
over the next 4-8 h. Dye release occurred after feeding, after social contact
or spontaneously. The technique was successful in visualising urine in almost
all individuals of the species tested: 20 Procambarus clarkii, 48
Astacus leptodactylus, two Astacus astacus, eight
Astropotamobis torrentium, two Pacifastacus leniusculus and
two Orconectes limosus. Four small crayfish (Procambarus
clarkii, 9 g body mass) died after receiving a high dose (6 µg
g-1) of Fluorescein. Lower concentrations caused no obvious
damage.
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General procedure for fights
One to three hours prior to the fight, two size-matched crayfish
(Astacus leptodactylus) were injected with 0.1% Fluorescein solution
(2 µg g-1 body mass). Interactions were filmed with two video
recorders (top view, Sony Hi8 CCD-VX1E; front view, Panasonic S-VHS AG-450;
recording at 50 frames s-1) in a glass aquarium (70 cmx40
cmx50 cm) with the floor and three side walls covered with a black
velvet lining to provide good background contrast for filming Fluorescein
release. Bright light was provided by two 250 W slide projectors. The two
recordings were combined (Panasonic Digital AV Mixer WJ-AVE7) and viewed on a
video monitor with split screen. Fights were generally started after a 10 min
acclimation period by lifting a polyvinylchloride divider that had separated
the animals. In eight cases, urine release was recorded for 60 min prior to
lifting the divider. Interactions were recorded for 30 min. To discriminate
between effects caused by individual or by dominance recognition, we conducted
repeated fights with either familiar or unfamiliar opponents.
Fights against familiar opponents (24 fights)
Two consecutive fights were initiated between the same pairs of crayfish
(24 fights). The loser of the first fight was re-matched with the winner of
the previous fight after a 24 h isolation period.
Fights against unfamiliar opponent (36 fights)
The losers of initial fights (N=18) were re-matched (after 24 or
48 h of isolation) against unfamiliar dominants that had previously won one
(N=12 of which seven were without Fluorescein injection) or two
(N=6) fights.
Analysis of fights and of urine release during fights
The visualisation technique allowed us to analyse the probability of urine
release during the fight but not the volume of urine released. During each 5 s
interval, we noted whether or not stained urine was released and whether the
anterior body appendages (maxillipeds and antennules) were active. During each
interval, we also assigned both crayfish an agonistic level
(Table 1) (see also
Atema and Voigt, 1995;
Breithaupt and Atema, 2000
).
When we detected more than one agonistic level within one time interval, we
declared on overall level for that interval on the basis of the following
ranking: agonistic levels 5, 4 and 3 outranked (>) levels 2, 1, 0, -1 and
-2; level 5>4>3>2>1; level -2 outranked level -1, and both levels
-2 and -1 outranked levels 2, 1 and 0. We analysed only sequences in which
animals fought (bouts) and/or released urine including an additional 10 s
before and 10 s after the episode.
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To analyse the duration of repeated fights between familiar and unfamiliar opponents, we evaluated only those combats in which the first fight exceeded 1 min and contained a bout that was longer than 45 s. Similarly, to evaluate the risk and effectiveness of urine signals, we included only bouts that exceeded 30 s. These criteria excluded from analysis those fights in which the loser provided little resistance.
Lag sequential analysis
This analysis identifies non-random sequences of behaviour occurring during
social interactions (Sacket,
1979; Waas,
1991b
). Changes in the relative frequency of selected behaviours
are quantified following a behaviour of interest (`criterion'). We used this
analysis to identify changes in the behaviour of the receiver caused by the
chemical signal of the sender (`effectiveness of the urine signal') and in the
behaviour of the sender following its signal (`predictive value of the
signal').
Analysis of the effectiveness of the urine signal
We selected two criteria and analysed differences in the response to these
criteria: (a) `offensive urine release', offensive behaviour
(agonistic levels 2-5) accompanied by urine release; (b) `offensive
behaviour', offensive behaviour without urine release. For these criteria, we
determined (in a data set comprising 40 fights and 36 individuals) the
frequency of the opponent's defensive (levels -2 and -1), neutral (levels 0,
A) and offensive (levels 1-5) behavioural acts in the current time intervals
(lag 0) and in the time intervals preceding (lag -1) and following (lag 1, lag
2) the criteria. Differences in the relative frequency of defensive, neutral
or offensive behaviour in the subsequent intervals compared with the preceding
interval were used as a measure of the change in response to the opponent's
signal. For a valid application of parametric analysis to our data, relative
frequencies were arcsine-transformed (Zar,
1999) to meet the assumption of normal distribution. A
multivariate analysis of variance (ANOVA) was used to identify those
behavioural changes in the eventual loser that were significantly affected by
offensive urine release by the eventual winner compared with offensive
behaviour without urine release. Since we found no effect of crayfish identity
on the behavioural response, we pooled the data of all 36 animals for the
analysis.
Analysis of the predictive value of urine signals
We selected four criteria and analysed changes in the behaviour of the
actor following these criteria: agonistic levels 2 and 3 with urine release
(criteria A and B respectively) and without urine release
(criteria C and D respectively). We then compared the
relative frequency of behavioural acts of higher, the same or lower agonistic
level in the subsequent time interval (lag 1, lag 2) with the agonistic level
of the respective criterion (lag 0). We analysed differences in the response
to the two criteria using a multivariate ANOVA.
Other statistical procedures
We used parametric statistics (multi-way ANOVA, repeated-measure design)
(Zar, 1999) to test for
possible differences in urine release between winners and losers and also for
the effect of `experience' on urine release. A measure of urine output was
derived for each combatant from the proportion (%) of total time of urine
release in each 30 min interaction. Proportions were arcsine-transformed to
meet the requirements for parametric statistics.
Logistic regression analysis of the original data set was used to analyse
the dependence between urine release probability and agonistic levels
(Breithaupt and Atema, 2000).
Previous urine release can influence probability of current urine output.
Therefore, individual data points adjacent in time are not independent of each
other. For example, when urine is released from a filled bladder, the release
would be expected to last longer than 5 s. To take this autocorrelation into
account, we included time-lagged series of the urine release data as
independent variables in the analysis. We allowed for variations among
individuals by including crayfish identity. Probabilities attributing to
agonistic levels (see Fig. 3)
were calculated from parameter estimates of the logistic regression analysis.
The logistic regression analysis also tested for significant differences of
these parameter estimates from the mean overall agonistic levels.
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Multivariate ANOVA (Zar,
1999) was used to test for differences in the use of the anterior
body appendages (maxillipeds) between offensive and spontaneous urine release.
For each 30 min interaction, we calculated (i) the proportion of time that
appendage movements accompanied urine release during fights and (ii) the
proportion of time that appendage movements occurred during spontaneous urine
release. For the test, we used arcsine-transformed proportions. Since we found
no effect of crayfish identity (i.e. no individual differences among animals)
on the behavioural response, we pooled the data from all animals for the
analysis.
All statistical analyses were performed with JMP 4.02 (SAS Institute). We used the standard error of the mean (S.E.M.) to indicate deviations from the mean.
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Results |
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Prior to the fight, crayfish released urine spontaneously once or twice per
hour (eight individuals tested). After lifting the divider separating two
crayfish, urine release occurred almost exclusively during aggressive
interactions at or above agonistic level 2 (see
Table 1) but rarely
spontaneously (Fig. 3). The
eventual winner of the fight released urine with significantly higher
probability than the loser. Urine release rate decreased slightly in repeated
fights (Fig. 3). However, we
found no significant difference in urine release rate between first, second
and third fights and no difference between fights between unfamiliar or
familiar opponents (data not shown). During a fight, the eventual winner
showed mostly offensive behaviours (Fig.
4A), with agonistic level 3
(Table 1; physical contact,
claws not used to grasp) occurring most often, followed by level 4 (claws used
to grasp) and level 2 (threat displays). The fights rarely advanced to the
highest aggressive level (level 5: unrestrained use of claws including claw
snapping and claw ripping; =tail-flipping while keeping a firm hold on the
opponent, `offensive tail-flipping'; Fig.
4A) (Herberholz et al.,
2001). The behaviour of the eventual loser was dominated by
defensive behaviour (level -1, avoidance;
Fig. 4B) followed by offensive
behaviour (level 3) and inactivity (level 0). In both winners and losers, the
probability of urine release increased with increasing levels of offensive
behaviour (levels 1-5; Fig.
4C,D). In losers, urine was rarely released during defensive
behaviour (Fig. 4D). Winners,
in contrast, released urine in 12 % (level -1) and 38 % (level -2) of
defensive behaviours (Fig. 4D).
Since winners only rarely exhibited defensive behaviour (on average only 3 s
per fight at level -1 and 1.3 s at level -2;
Fig. 4A) and always showed
offensive behaviours thereafter, these retreats and escapes of the winner may
be interpreted as tactical offensive manoeuvres (e.g. repositioning) rather
than as defensive acts.
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During agonistic interactions of Astacus leptodactylus, urine was
directed towards the opponent, probably carried by the gill currents
(Atema, 1985). The direction of
urine signals changed when urine was released spontaneously. Fanning activity
of the flagella of the mouthparts (the exopodites of the three maxillipeds)
(Breithaupt, 2001
) directed
the urine stream laterally. Fanning occurred almost exclusively during
spontaneous release but rarely during offensive urine release
(Fig. 5). Spontaneous urine
release was further accompanied by flicking and downward-pointing of the
antennules (the chemosensory appendages of crustaceans)
(Fig. 5). These behaviours have
been interpreted as enhancing olfaction in crustaceans
(Schmitt and Ache, 1979
). They
rarely occurred during fights (Figs
2,
5). Offensive urine release was
accompanied by upward extension of the large endopodites of the third
maxillipeds, which then covered the exopodites, thus inactivating and
protecting the fan organs (Figs
2,
5).
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Fight duration for familiar and unfamiliar combatants
Previous studies (Breithaupt and Atema,
2000; Karavanich and Atema,
1998a
,
b
) on lobsters suggested that
a subordinate animal recognising the individual urine scent of a familiar
dominant individual maintains dominance and avoids escalated fights. The
evidence for this interpretation was that, between familiar lobsters, but not
between unfamiliar lobsters, second fights are generally shorter and less
aggressive than first fights (Karavanich
and Atema, 1998a
). We measured fight duration in crayfish as the
sum of the duration of individual bouts. Fights between familiar opponents
decreased significantly in duration from 263±32.7 s in the first
encounter to 135±40 s in the second encounter on another day (means
± S.E.M.; N=10; P<0.01, paired t-test).
Even if paired with an unfamiliar opponent, the loser of a previous day's
fight gave up earlier: second fights were significantly shorter
(68.5±8.2 s) than first fights (205.5±32.2 s; N=10;
P<0.01, paired t-test). We found no difference in
duration of either first or second fights between familiar and unfamiliar
crayfish (P=0.88; two-way ANOVA). This suggests that there is no
individual recognition or that recognition is not a significant factor for the
avoidance of repeated fights in the crayfish Astacus
leptodactylus.
The effectiveness and predictive value of urine signals
We analysed changes in the relative frequency of agonistic behaviours of
both the signaller and the receiver in response to urine release and offensive
behaviours (see Lag sequential analysis) to determine (i) the effect
of offensive urine release (i.e. offensive behaviour accompanied by urine
release) on the agonistic behaviour of the opponent (effectiveness of urine
signals) and (ii) whether offensive urine release had any predictive value
about the next act of the signaller that could inform the receiver about the
offensive intention of the signaller.
Effectiveness of offensive urine release
We compared the relative frequency of the receiver's agonistic levels
concurrent with offensive behavioural acts and urine release by the signaller
with the receiver's preceding behaviour. The frequency of defensive
behavioural acts (levels -1 and -2) by the receiver increased by more than 10%
and offensive behavioural acts (levels 1-5) decreased in response to offensive
behaviour acts (levels 1-5) decreased in response to offensive behaviour by
the signaller accompanied by urine release
(Fig. 6, open columns). No
change in offensive or defensive behaviour by the receiver was recorded in
response to offensive behaviour by the signaller not accompanied by urine
release (filled columns). Neutral behaviour by the receiver remained unchanged
under all conditions. The response lasted for at least 15 s, as inferred from
analysis including subsequent time intervals (5 s delay, 10 s delay). Hence,
urine signals were effective in reducing the aggression of the opponent.
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Predictive value of offensive urine release
Urine release could be a threat signal if, subsequent to release, the
signaller increased its aggression level, leading to an increased risk of
injury for the opponent. We studied changes in the relative frequency of
agonistic behaviours following an initial low-level offensive behaviour with
or without urine release (initial level 2, see
Fig. 7; initial level 3, data
not shown). This was to investigate whether urine release in conjunction with
offensive behaviour provides predictable information about the subsequent
activities of the signaller and if this differed from that of offensive
behaviour without urine release. We determined the relative frequency of
behavioural acts of higher (levels 3, 4 or 5), the same (level 2 or 3) or
lower (<level 2 or 3) agonistic level in the subsequent time intervals (lag
1, lag 2). We compared these frequencies with those of behaviours concurrent
with the respective criterion (lag 0). After having performed level 2
aggression, the animals showed agonistic levels higher than the initial level
more frequently than lower agonistic levels (P<0.01, contrast
analysis, multivariate ANOVA, Fig.
7). Following initial level 3, lower levels occurred more
frequently than higher levels (P<0.01; data not shown). Urine
release concurrent with level 2 or 3 aggression reduced the agonistic level of
subsequent behaviours to the initial level or to lower than the initial level
(Fig. 7). However, it did not
influence the likelihood of fight escalation (i.e. the frequency of higher
agonistic levels). Therefore, offensive urine release does not reveal the
intention of the signaller to escalate the fight.
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Discussion |
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Contradictory findings about the use of pheromones in mate attraction and
agonistic behaviours have generated a debate about the significance of
pheromones in crayfish (Hazlett,
1984; Thorp,
1984
). Itagaki and Thorp concluded from their studies of
Procambarus clarkii that crayfish do not communicate their sex or
agonistic state chemically (Itagaki and
Thorp, 1981
; Thorp,
1984
; Thorp and Itagaki,
1982
).
Using lag sequential analysis, we identified non-random sequences of
behaviour during the social interactions. This analysis revealed that urine
signals make offensive behaviours more effective in reducing an opponent's
aggressiveness. The relative frequency of the opponent's defensive behaviours
is increased at the expense of its offensive behaviours
(Fig. 6). Offensive behaviour
without urine signals does not change the behaviour of the opponent
(Fig. 6). Hence, chemical
signals appear to be more important than other offensive displays and signals
for settling a fight, at least under visual blackout conditions. In our
experiments, the animals were blindfolded to avoid reactions to the visual
image of the Fluorescein cloud. Blindfolding the animals may have changed
their fighting behaviour. Indeed, Bruski and Dunham
(1987) found, by comparing
fights of Orconectes rusticus in the light and in the dark, that the
duration of individual bouts and the frequency of highly aggressive behaviours
(corresponding to our levels 3-5) are increased in the dark while the
frequency of visual threat displays remains unchanged. Thus, crayfish may need
to fight longer and more vehemently to settle a fight when they cannot see
each other. Blocking the release of urine in visually intact Orconectes
rusticus had a similar effect: it increased both the duration and the
intensity of fights (Zulandt Schneider et
al., 2001
). This suggests that, in daylight, urine signals play a
similarly important role as they do in the dark in reducing the aggression of
the receiver. In summary, these studies indicate that, despite previous doubts
about their behavioural significance in crayfish
(Itagaki and Thorp, 1981
;
Thorp, 1984
;
Thorp and Itagaki, 1982
),
chemical signals have a major impact on the outcome of fights that equal the
effects of visual signals in the daytime and dominate other signals at night.
Moreover, as nocturnal animals, crayfish might rely more strongly on chemical
than on visual cues for settling fights.
What is communicated by urine signals?
Urine appears to be a threat signal because it is effective in deterring
the opponent (Fig. 6). Does the
signal reveal information about subsequent activities? Previous studies of
bird agonistic interactions and accompanying cost/benefit models indicate that
the effectiveness of aggressive displays (i.e. in deterring opponents)
correlates with the risk of performing this display (the risk of being injured
by one's opponent) (Enquist,
1985; Enquist et al.,
1985
; Popp, 1987
;
Waas, 1991a
,
b
). This `risk/benefit'
approach suggests that an animal reveals a strong motivation to escalate a
fight by using a display that places both itself and its opponent in a
potentially dangerous situation. Such displays appear to contain information
about the subsequent activities of the signaller.
In accordance with these predictions, we found that the signaller increased its aggression level (i.e. increased risk) following agonistic level 2 (`threat display', Fig. 7). However, we found that it reduced its aggression after agonistic level 3. Urine signals accompanying aggressive behaviour did not alter the likelihood of escalation. The predictive value of urine signals is therefore low and cannot be responsible for the reaction of the opponent.
Alternatively, urine signals may allow the receiver to assess the current
physiological and aggressive states of the signaller. Urine signals contain
metabolic breakdown products of the hormones that are effective during
fighting behaviour. There is evidence that in decapod crustaceans aggression
may be modulated by hormones such as serotonin, octopamine and dopamine
(Antonsen and Paul, 1997;
Huber and Delago, 1998
;
Huber et al., 1997
;
Kravitz, 2000
;
Sneddon et al., 2000
).
Injection of the biogenic amines into the haemolymph results, in the American
lobster (Homarus americanus), in the squat lobster (Munida
quadrispina) and in crayfish (Astacus astacus, Procambarus
clarkii), in agonistic postures and in changes in agonistic behaviours
(Antonsen and Paul, 1997
;
Huber et al., 1997
;
Livingstone et al., 1980
). The
relative levels of serotonin, octopamine and dopamine in the blood of the
shore crab Carcinus maenas appear to be linked to fighting ability
(Sneddon et al., 2000
).
Although the specific role of some biogenic amines (e.g. serotonin) in
settling conflicts under natural conditions is still controversial
(Peeke et al., 2000
), their
general impact on the aggressive motivation of crustaceans is undisputed.
Since the metabolites of biogenic amines are found in the excretory green
gland and in the urine of crayfish and lobsters
(Hoeger, 1990
;
Huber et al., 1997
), this
information about aggressive state is provided to the receiver by the release
of urine.
Like lobsters (Breithaupt and Atema,
2000), crayfish couple the release of urine to offensive
behaviours (Fig. 3), thereby
reinforcing the message of the aggressive acts. This combination adds
reliability to the chemical message for the receiver. `Dishonest signallers'
that cannot back up their chemical signals with physical aggressive acts may
not be effective in deterring an opponent and may suffer from the escalated
fight. Conversely, offensive behaviour alone was not effective in deterring
the opponent when not accompanied by the chemical message
(Fig. 6). A positive winning
experience or maintained fighting motivation may result in a specific mixture
of hormone metabolites that is broadcast with the release of urine.
Possible information about the individual identity of the signaller is
either not present in the urine or does not seem to be a significant factor in
crayfish fights. In contrast, Karavanich and Atema
(1998a,
b
) found that in lobsters
dominance is maintained by individual recognition of the urine scent of
familiar dominant individuals. In the crayfish Astacus leptodactylus,
we found no difference in duration between familiar and unfamiliar fights,
suggesting that subordinate animals do not recognise the individual identity
of previous winners. Similar results were obtained from Orconectes
rusticus (Zulandt Schneider et al.,
2001
). Therefore, individual recognition of previously fought
dominant individuals does not seem to be responsible for the observed
reactions of the opponent to urine signals.
To understand why urine messages are so successful in deterring an opponent, further studies on the chemical composition of urine and the behavioural significance of specific components are needed to verify that they contain information about the physiological and, thus, aggressive state of the signaller.
When is the best time to send urine signals?
Timing is a critical component of signalling with urine. Urine signals may
reveal information about the motivational state of the sender and, therefore,
the receiver could exploit these signals. For example, a crayfish receiving
signals of low aggressive motivation from an opponent may decide to fight to
gain dominance, even if it is smaller or weaker than the signaller. Our
analysis of urine signals during crayfish agonistic interactions supports
previous findings from American lobsters (Homarus americanus) that
crustaceans adjust the timing of urine release to circumvent exploitation by
the receiver (Breithaupt and Atema,
2000). Living in fresh water, crayfish encounter a higher passive
water inflow (and hence have to discharge more urine) than marine crustaceans
such as lobsters. Urine accumulates in the bladder and, in crayfish, may
represent 2-4% of the body mass (Mantel
and Farmer, 1983
) and is released once or twice per hour in
isolated animals. Marine lobsters in isolation release urine much less
frequently on average only every third hour
(Breithaupt et al., 1999
). In
the presence of a conspecific, crayfish release urine more frequently but
restrict the release to physical interactions and rarely release it
spontaneously. They link it to offensive behaviours and increase the release
rate with increasing aggression.
Storing urine in a bladder prevents it from leaking into the environment
and providing nearby receivers with information about the motivational state
of the sender. The bladder allows urine to be released voluntarily at times
favourable to the sender. Recent theories and studies of animal communication
(for a review, see Bradbury and
Vehrencamp, 1998) have shown that receivers are sceptical and only
respond to signals that are reliable
(Grafen, 1990
;
Zahavi and Zahavi, 1997
).
Honesty can be ensured by the costs of signalling, e.g. the incidental costs
when a dishonest signaller suffers from the increased aggression of the
receiver (Enquist et al.,
1985
; Popp, 1987
).
Breithaupt and Atema (2000
)
suggest that, by coupling urine release to offensive behaviours and increasing
urine release rate with increasing level of aggression, lobsters add
reliability to the chemical signal. Our study shows that crayfish use the same
strategy as lobsters. Reliability of aggressive chemical signals is ensured by
releasing urine under the increased risk of being injured during the
fight.
By restricting urine release to offensive behaviours, crayfish also optimise the detectability of the chemical signal. During offensive behaviours, animals are in close proximity, often facing each other, and the urine signals are directed at the antennal chemoreceptors of the opponent, providing an increased signal-to-noise ratio with respect to other ambient chemicals.
How are urine signals transported towards the opponent?
Observations of the Fluorescein dispersal pattern during fights showed that
urine is transported frontally towards the opponent (T. Breithaupt and P.
Eger, personal observation). The narrow-clawed crayfish Astacus
leptodactylus seems to employ forward-directed gill currents for chemical
signalling during fights. The fan organs are not active during agonistic
interactions (Fig. 5). Other
crayfish species, e.g. the red swamp crayfish (Procambarus clarkii)
and the cambarid crayfish Orconectes limosus, use the fan organs
(exopodites of the mouthparts) and not the gill currents to carry urine
signals towards an opponent (T. Breithaupt and P. Eger, unpublished data)
(Breithaupt, 2001). In
contrast to the urine released during fights, urine released spontaneously is
carried laterally by fanning the exopodites of the mouthparts
(Fig. 5) (Breithaupt, 2001
). These
findings reveal that crayfish actively use their own currents to disperse
chemical signals and that they can either send them towards a receiver or
`hide' them from a receiver. The mechanisms of urine dispersal are similar in
lobsters (Atema, 1985
) (T.
Breithaupt, unpublished data). Using either the gill currents or the currents
generated by fan organs, urine is most effectively carried to the
chemoreceptors on the first antennae of the opponent. The active transport of
urine signals between closely spaced crayfish by water currents may even allow
communication in a river because the currents may prevent urine from being
carried away by the surrounding flow.
Are chemical signals important for the maintenance of dominance
hierarchies in crayfish?
Our finding that offensive behaviour is only effective in discouraging an
opponent when accompanied by urine release clearly shows that urine signals
are important in establishing dominance. Are they also important in
maintaining dominance hierarchies? Second fights between both familiar and
unfamiliar animals were found to be up to 50% shorter than first fights,
indicating that dominance is maintained in crayfish. In lobsters, individual
recognition is important for the maintenance of dominance hierarchies
(Karavanich and Atema, 1998a).
In contrast to lobsters, crayfish do not seem to recognise the identity of
previous opponents. If identity is not recognised, they may recognise the
general dominance status or aggressive state of an opponent, as suggested by
(Copp, 1986
) and described for
cockroaches (Moore et al.,
1997
). If chemical signals are important for the maintenance of
the dominance hierarchies in crayfish, we would expect dominant animals to
increase their rate of signalling in repeated fights in order to be recognised
by subordinates and to avoid the risks associated with extended fights. Our
data do not support this proposal because the probability of urine release did
not increase, but instead dropped, in repeated fights
(Fig. 3). Blocking urine
release in Orconectes rusticus did not affect the duration of second
fights (Zulandt Schneider et al.,
2001
). Irrespective of urine signals, dominance was maintained in
this species. These findings in Astacus leptodactylus and
Orconectes rusticus suggest that urine signals are not important for
the maintenance of dominance hierarchies in crayfish. Other mechanisms, e.g.
self-reinforcing effects of fight success
(Goessmann et al., 2000
), may
be more important than chemical signals for maintaining dominance hierarchies
in crayfish.
The adaptive value for the different strategies of maintaining dominance
between pairs of lobsters and crayfish may be explainable by taking into
account the structure and dynamics of natural populations. In lobsters,
dominance secures access to shelters and courtship. They have a high site
fidelity, returning to the same shelter for up to 9 months
(Karnofsky et al., 1989). They
use their activity period primarily to update their knowledge of their
physical and social environment (Atema and
Voigt, 1995
). In this social environment, individual recognition
allows them to observe the activity of other nearby residents that are
potential competitors for food, mates and shelters. Unfortunately, we have no
such detailed knowledge about the social structure of crayfish populations.
However, marking and recapture experiments indicate a low degree of residency
in the crayfish Astacus astacus
(Abrahamsson, 1966
) and
Austropotamobius torrentium (Renz
and Breithaupt, 2000
). Given that the probability of encountering
the same individual repeatedly is low, a more general agonistic strategy may
be more successful than learned individual recognition in reducing the costs
of extended fights. The encounter probability of familiar animals and the
degree of residency may be key factors determining the mechanism of
maintaining dominance in animal communities.
Why do crayfish employ chemical signals in fights?
Our study shows that chemical signals released during offensive behaviour
are effective in reducing the aggression of an opponent. Non-chemical
offensive behaviours are not effective in changing the behaviour of an
opponent. This raises questions about the adaptive value of the urine signals
during fights. Why do visual and tactile agonistic manoeuvres alone have no
greater impact on the course of the fight? The answer to this question may be
that urine signals provide `uncheatable' information about the aggressive
motivation of the signaller. This information may serve as a back-up providing
honesty where other signals may cheat. Mantis shrimps when newly moulted were
found to bluff opponents by producing meral spreading displays, even though
their soft cuticle prevented them from either delivering or withstanding blows
(Adams and Caldwell, 1990). The
metabolic products of moulting hormones detected by a receiver would betray
the bluff. Similarly, breakdown products of other hormones may inform a
receiver about aggressive state, arousal, sex and species. Zulandt Schneider
et al. (1999
) found that the
odour of dominant Procambarus clarkii attracted males and females but
elicited aggressive reactions only in males.
Chemical signals appear to play a major role during nocturnal interactions
between crayfish. In the natural environment, dominance fights can secure
access to and defence of shelters
(Vorburger and Ribi, 1999).
Shelters are important resources for crayfish since they significantly reduce
predation risk (Söderbäck,
1994
). During nocturnal shelter competitions, chemical signals may
gradually replace visual signals or even tactile displays when visual
conditions are poor or when the resident animal is hidden in the shelter. In
crayfish, aggressive behaviour occurs not only within but also between
sympatric species (Vorburger and Ribi,
1999
). It remains to be determined whether different crayfish
species use the same chemical components for aggressive signalling and how
these components relate to the internal state of the signaller. The
exploration of the chemical nature of aggressive signal promises insight into
these still unsolved questions of crustacean agonistic behaviour.
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