Hormones and Behavior 66 (2014) 696–704

Contents lists available at ScienceDirect

Hormones and Behavior
journal homepage: www.elsevier.com/locate/yhbeh

A fighter's comeback: Dopamine is necessary for recovery of aggression
after social defeat in crickets
Jan Rillich a, Paul A. Stevenson b,⁎

Institute for Neurobiology, Free University of Berlin, Koenigin-Luise-Str. 28-30, 14195 Berlin, Germany
Institute for Biology, Leipzig University, Talstr. 33, 04103 Leipzig, Germany

a r t i c l e

i n f o

Article history:
Received 23 May 2014
Revised 27 August 2014
Accepted 23 September 2014
Available online 28 September 2014
Agonistic behavior
Social behavior
Loser effect
Amine depletion
Behavioral depression
Subordinate behavior
Animal conflict
Aggressive motivation

a b s t r a c t
Social defeat, i.e. losing an agonistic dispute with a conspecific, is followed by a period of suppressed aggressiveness in many animal species, and is generally regarded as a major stressor, which may play a role in psychiatric
disorders such as depression and post-traumatic stress disorder. Despite numerous animal models, the mechanisms underlying loser depression and subsequent recovery are largely unknown. This study on crickets is the
first to show that a neuromodulator, dopamine (DA), is necessary for recovery of aggression after social defeat.
Crickets avoid any conspecific male just after defeat, but regain their aggressiveness over 3 h. This recovery
was prohibited after depleting nervous stores of DA and octopamine (OA, the invertebrate analogue of noradrenaline) with α-methyl-tyrosine (AMT). Loser recovery was also prohibited by the insect DA-receptor (DAR) antagonist fluphenazine, but not the OA-receptor (OAR) blocker epinastine, or yohimbine, which blocks receptors for
OA's precursor tyramine. Conversely, aggression was restored prematurely in both untreated and amine depleted
losers given either chlordimeform (CDM), a tissue permeable OAR-agonist, or the DA-metabolite homovanillyl
alcohol (HVA), a component of the honeybee queen mandibular pheromone. As in honeybees, HVA acts in
crickets as a DAR-agonist since its aggression promoting effect on losers was selectively blocked by the DARantagonist, but not by the OAR-antagonist. Conversely, CDM's aggression promoting effect was selectively
blocked by the OAR-antagonist, but not the DAR-antagonist. Hence, only DA is necessary for recovery of aggressiveness after social defeat, although OA can promote loser aggression independently to enable experience
dependent adaptive responses.
© 2014 Elsevier Inc. All rights reserved.

Intra-specific aggression is common throughout the animal kingdom and a major hallmark of this behavior is that its expression is influenced by a wide variety of experiences (Huntingford and Turner, 1987;
Archer, 1988; Hsu et al., 2006, 2009). In mammals, for example, experiences as diverse as physical exertion (Raichlen et al., 2011), the possession of a resource (Fuxjager et al., 2010) and winning (Hsu et al., 2006,
2009) can each promote the aggressiveness of an individual, whereas
losing leads to a pronounced reduction in the expression of aggressive
behavior (Hsu et al., 2006, 2009). Investigations of the consequences
of social defeat and its importance for understanding social behavior
and psychiatric disorders such as post-traumatic stress disorder are receiving increasing amounts of attention (Huhman, 2006). Comparatively
little, however, is known about the neuronal control mechanisms underlying the effects of experiences such as social defeat on aggressive

⁎ Corresponding author at: Institute for Biology-II, Leipzig University, Talstr. 33,
D-04103 Leipzig, Germany. Fax: +49 341 9736848.
E-mail address: stevenson@rz.uni-leipzig.de (P.A. Stevenson).

0018-506X/© 2014 Elsevier Inc. All rights reserved.

Our work on male crickets (Gryllus bimaculatus de Geer) has shown
that the physical experiences such as flying (Stevenson et al., 2005),
winning (Rillich and Stevenson, 2011) and possession of a shelter
(Rillich et al., 2011) all lead to a state of enhanced aggression which is
mediated by the biogenic amine octopamine (OA; reviews: Stevenson
and Rillich, 2012; Stevenson and Schildberger, 2013; Simpson and
Stevenson, 2014), the invertebrate analogue of noradrenaline (Pflueger
and Stevenson, 2005). The mechanisms underlying depressed aggression and its subsequent recovery in crickets and indeed other animals
are practically not known. Most behavioral theories agree that information gathered from agonistic signals exchanged during fighting is somehow assessed to time the decision to flee, and our work on crickets
shows that agonistic signals act to suppress aggression of the opponent
(Rillich et al., 2007; for review see Elwood and Arnott, 2012).
Neuromodulators are likely to play key roles, for example serotonin
and nitric oxide, which can suppress aggression in both mammals
(Nelson and Trainor, 2007) and insects (Iwasaki et al., 2007; see also
Edwards and Spitzer, 2006 on serotonin and social dominance in
In this paper we specifically address the possibility that OA and/or
dopamine (DA), are involved in the recovery of aggression after social
defeat. As in many animals, crickets that have just lost a fight behave

J. Rillich, P.A. Stevenson / Hormones and Behavior 66 (2014) 696–704

submissively towards previous victors (Stevenson and Rillich, 2013)
and even unfamiliar opponents (Rillich et al., 2007), but slowly regain
their aggressiveness after 3 h of social isolation (Stevenson and Rillich,
2013), after which they will readily engage and fight any conspecific
adult male. The involvement of OA in recovering from this so-called
loser effect is suggested by the finding that losers rapidly regain their
aggressiveness after treatment with the insecticide chlordimeform
(CDM, Stevenson et al., 2005), a tissue permeable OA-receptor (OAR)
agonist (Roeder, 1995) with low affinity for other amine receptors
(Hiripi et al., 1999). Here we show that aggression is also restored in
losers by treatment with homovanillyl alcohol (HVA), a catecholamine
metabolite, that is structurally related to DA and acts as a DA-receptor
(DAR) agonist in honeybees (Beggs and Mercer, 2009). Taken together
our experiments provide evidence that DA and OA can independently
promote aggression in defeated crickets, but while OA is sufficient,
DA alone is necessary for the normal recovery of aggressiveness
after social defeat. This adds firm support to recent findings in fruit
flies (Alekseyenko et al., 2013) and ants (Szczuka et al., 2013) suggesting that DA may play some role in insect aggression, and provides general insights into how amines operate in controlling social behavior,
perhaps even in mammals (cf. O'Connell and Hofmann, 2011).
Experimental animals
Mature, 2–3 week old, adult male Mediterranean field crickets,
G. bimaculatus (de Geer) were taken from a breeding stock maintained
under constant standard conditions at Leipzig University (22–24 °C,
relative humidity 40–60%, 12 h:12 h light:dark regime daily feeding
on bran and fresh vegetables) and kept isolated in individual glass jars
for at least 24 h prior to all experiments. After this time all known effects
of previous social interactions on the expression of aggressive behavior
have abated (Stevenson and Rillich, 2013), and we refer to the animals
as being "socially naive." All animal treatments complied with the Principles of Laboratory Animal Care and the German Law on the Protection
of Animals (Deutsches Tierschutzgesetz).
Evaluation of aggression
Aggressive behavior was evaluated in dyadic contests (cf. Stevenson
et al., 2005) between pairs of previously isolated adult male crickets that
were matched for body mass (b5% mass difference). The opponents
were placed at opposite ends of a small, Perspex-glass rectangular fighting arena (l·w·h: 16 × 9 × 7 cm) having a sand-covered floor and divided halfway along its length by an opaque sliding door. On removing the
door the animals generally interact with each other within seconds.
Their interactions follow an escalating sequence of stereotyped motor
performances (Stevenson et al., 2000, 2005), which do not differ significantly from fights that occur in the field as part of their normal behavioral repertoire (Alexander, 1961). The intensity of aggressiveness was
scored on a scale of 0–6 (Hofmann and Stevenson, 2000; Stevenson
et al., 2000) denoting the level to which a fight escalates before the
winner is established by the retreat of one contestant: Level 0: mutual
avoidance without aggression. Level 1: one cricket attacks, the other
retreats. Level 2: antennal fencing, whereby the contestants face each
other and lash each other's antennae. Level 3: one contestant spreads
its mandibles in a threat display. Level 4: mandible spreading by both
crickets. Level 5: mandible engagement, whereby the two opponents
interlock their mandibles. Level 6: grappling, an all-out fight involving
repeated mandible engagements with biting, and body flipping. A contest may be concluded at any of the levels when one opponent retreats,
leaving the established winner, which becomes hyper-aggressive and
generally generates the characteristic rival song and body jerking movements (Rillich and Stevenson, 2011). The losers normally actively avoid
contact to any conspecific male for 1–3 h (Stevenson and Rillich, 2013).


Fight duration, from first contact until conclusion, was measured to the
nearest second with a stopwatch. Very occasionally, the animals appeared
to lose contact with each other so that fighting paused for a brief period
before resuming when contact was regained. As in our previous studies,
we chose to deduct the duration of these pauses in the few cases they
occur in order to give a more representative measure of the actual
time spent fighting.
Pharmacological treatments
Various pharmacological treatments were performed to evaluate the
role of amines in loser recovery. To minimize variations due to random
differences in daily performances, we took the precaution of evaluating
single pairs of crickets from control and test groups in parallel and
accumulated data from multiple experimental sessions (three groups
per session, test sequence changed at each). The numbers of cricket
pairs for each test group is given in tables and figures.
Unless stated otherwise, all drugs were obtained from Sigma Aldrich
(Deisenhofen, Germany). Their effects were tested by injecting 10 μl solutions into the hemocoel via the pronotal shield using a micro-syringe
(Hamiliton®, Bonaduz, Switzerland). The most effective concentrations
that induced noticeable changes in aggressive behavior, but without
having any obvious detrimental effect on general motility were determined in pilot investigations.
Hydrochloride salts of native OA and DA were found to have no
obvious effect on aggression at concentrations as high as 20 mM,
which is due we suspect to the permeability barrier of the ganglion
sheath (cf. Schofield et al., 1984). Contrasting this, the tissue permeable
OAR-agonist chlordimeform hydrochloride (CDM; cf. Roeder, 1995) and
the DAR-agonist homovanillyl alcohol (HVA: Beggs and Mercer, 2009)
were both effective at 1 mM in saline solution containing 1% DMSO
(dimethylsulfoxide; saline components in mmol L−1: NaCl 140, KCl
10, CaCl2 7, NaHCO3 8, MgCl2 1, N-trismethyl-2-aminoethanesulfonic
acid 5, D-trehalose dihydrate, pH 7.4). The following amine receptor
antagonists were dissolved in the same solvent (saline, 1% DMSO) and
applied at the same concentration (10 mM): epinastine hydrochloride,
a selective OAR-blocker (Roeder et al., 1998), fluphenazine dihydrochloride
a D1/D2 dopamine receptor (DAR) blocker in insects (Degen et al.,
2000) and yohimbine hydrochloride an insect tyramine receptor
(TAR) blocker (Roeder, 2005). Previous experiments have shown that
aminergic drugs injected into the hemocoel require about 30 min to
become effective, and that their effects last for up to 4 h (Stevenson
et al., 2005; Rillich et al., 2011; Rillich and Stevenson, 2011).
The roles of the amines OA, DA and TA in loser recovery were also
evaluated by applying the competitive synthesis inhibitor α-methyltyrosine (AMT). We have previously shown that a comparatively high
dosage is required in order to effectively deplete OA and DA from the
cricket central nervous system as determined by immunocytochemistry
(Stevenson et al., 2000) and we followed the same protocol in this
study. Briefly, each animal received two successive injections of
1.5 mg AMT in 20 μl saline administered at 48 h intervals, and evaluated
aggressive behavior 48 h after the last injection. Controls received two
successive injections of saline only. Since AMT inhibits the conversion
of tyrosine to OA's precursor TA, this amine is probably also depleted
in addition to OA and DA by this treatment.
Experimental procedure
All experiments were performed during daylight hours, avoiding
times when aggression tends to be depressed (just after midday and
on generally dreary days; cf. Dixon and Cade, 1986; Stevenson et al.,
2000). Fights were first staged in an initial fight to establish clear winners
and losers and the hierarchical relationship verified by re-matching the
same opponents 1 min later to assure that the designated loser retreated
immediately from the designated winner. To chart the time course of
loser recovery, the same opponents were re-matched 15, 30, 60 or


J. Rillich, P.A. Stevenson / Hormones and Behavior 66 (2014) 696–704

180 min after the initial fight, whereby different groups of animals were
tested for each time slot. In our current experiments all tested crickets
were drug treated 30–60 min before any social interactions, to circumvent any effects of handling the losers, and their aggression was evaluated up until maximally 4 h after the injection. Since the effect of losing on
aggression is maximal 15–30 min after defeat, this gave us ample time to
evaluate the effects of aminergic drugs on loser aggression. Although
both contestants were drug-treated, our experimental design still allows
conclusions concerning loser behavior. Firstly, the level and duration of a
fight are determined primarily by the subordinate, which normally
retreats, while winners are always aggressive (Rillich et al., 2007).
Secondly, even though aminergic receptor blockers, for example, can
reduce the aggressiveness of winners (Rillich and Stevenson, 2011),
rank reversals, in which the designated winners retreat from the
designated loser were less than 2% in our experiments (see also Rillich
et al., 2007), so that the scored level and duration of aggression still
reflected the loser's decision to fight or flee.
Data analysis
All statistical tests were performed using standard commercial software (Prism 5, GraphPad Software Inc., La Jolla, CA, USA) running on a
Macintosh computer (Apple Computers, Cupertino, CA, USA). The median and the interquartile range (IQR) were calculated for non-parametric
data sets. Non-parametric tests were also performed on duration since
the data sets failed D'Agostino and Pearson omnibus normality tests,
even after log transformations. To evaluate whether loser recovery

was evident for a given pharmacological treatment, we applied the
Kruskal–Wallis test in order to compare the aggressive scores for the
15, 30, 60 and 180 min time slots, which each comprised data from
different individual animals. To determine whether or not recovery
was complete, we applied the Wilcoxon signed rank test for paired
data to compare the aggressive score at the initial fight with the score
at 180 min. The Mann–Whitney U test was used to compare the aggressive scores at specific time slots for independent pharmacological test
groups. In one experiment three groups were compared (Figs. 1 and
4A: vehicle compared to OAR- and DAR-agonist), so we applied the
Bonferroni correction of alpha for multiple comparisons in order to
avoid type I errors (alpha is given in legends).
OAR- and DAR-agonists restore loser aggression
As shown for untreated naive crickets (Rillich and Stevenson, 2011),
the initial fights of vehicle treated (1% DMSO in saline) naive crickets
typically escalate to mandible engagement (median level 5, IQR 2.5–5,
Fig. 1, n = 93) and last several seconds (median duration 7 s, IQR 4–
10). After the initial fight, the vehicle treated losers avoided their previous opponents for up to 30 min after defeat (median level 1, IQR 1–2,
n = 24, Tables 1 and 2, Fig. 1), but exhibited low levels of aggression
after 60 min (median 2.5, IQR 1–5, n = 24; U tests compared to
30 min: p-level = 0.0046, p-duration = 0.013; Tables 1 and 2, Fig. 1A),
and fully regained their aggressiveness after 180 min (median level 5,

Fig. 1. OAR- and DAR-agonist restore loser aggression. Bar graphs giving the level of aggression (i, top) and fight duration (ii, bottom) for fights between pairs of socially naive crickets and
the resulting losers against their respective winners 15, 30, 60 and 180 min after defeat (different animals for each time slot; n is given above the x-axis in i). The animals received the
following treatments before the initial fight: A. Vehicle (1% DMSO in saline, white bars); B. OAR-agonist (CDM, blue bars); C. DAR-agonist (HVA, red bars). Circles give the medians
and boxes the interquartile ranges. The p(KW) gives error probabilities from Kruskal–Wallis analyses for differences in the four loser vs. winner test periods as an indicator of loser recovery.
Asterisks indicate significant differences between each test groups and vehicle for each time slot (U test, Bonferroni correction to alpha for three comparisons: *p b 0.025; **p b 0.005,
***p b 0.0005).


J. Rillich, P.A. Stevenson / Hormones and Behavior 66 (2014) 696–704

Table 1
The level of aggression (median and interquartile range, n in parenthesis) for fights between socially naive crickets and the subsequent losers versus winners 15, 30, 60 and 180 min
after defeat for the following experimental groups: saline, DMSO, CDM, HVA, AMT, yohimbine, epinastine and fluphenazine. Transient recovery of loser aggression is indicated by the
p(KW)-value that gives the error probabilities from Kruskal–Wallis analyses for differences in the four loser vs. winner test periods (p N 0.05, not significant for AMT and fluphenazine).
Test group

Initial fight

15 min

30 min

60 min

180 min



5, 3–5
5, 2.5–5

1, 1–2
1, 1–1.75
5, 1–5
1.5, 1–4
1, 1–1
1, 1–2
1, 1–3
1, 1–4

1, 1–2
1, 1–2
5, 3.5–5
1, 1–1
1, 1–4.75
1, 1–2
1, 1–3.75

3, 1–5
2.5, 1–5
1, 1–1
5, 1.75–5
3, 1–5
1, 1–3.5

5, 3–5
5, 2.5–5
1, 1–1
5, 1.75–5
5, 2–5
1, 1–5

p b 0.001


IQR 2.5–5, median duration 6 s, IQR 3.5–8, n = 25, differences to initial
fights insignificant, Wilcoxon signed rank test). We also applied the
Kruskal–Wallis test to compare loser–winner interactions for all time
groups, and this gave highly significant differences for both the level
and duration of the interactions (p-level and p-duration b 0.001), as a
further indicator of loser recovery.
Supporting our earlier findings (Stevenson et al., 2005) fights between pairs of crickets treated with the OAR-agonist chlordimeform
(CDM) were fiercer and longer at the initial fight (median level 6,
IQR 5–6, median duration 15 s, IQR 7–31, n = 70; U test compared to
vehicle: p-level and p-duration b 0.001), and the losers regained their aggressiveness more quickly. In our current experiments, and in intended
contrast to our earlier study, different groups of CDM (1 mM) treated
winners and losers were tested at different times after the initial fight
(Fig. 1B, Tables 1 and 2). The Kruskal–Wallis test still indicates a transient increase in level of aggression of losers against winners over
time (p = 0.0085), but no change in fight duration (p = 0.201, n.s.).
Furthermore, even as early as 15 min after the initial defeat, the CDMtreated losers interacted significantly more aggressively with their
previous winners than vehicle-treated crickets in physical fights that
escalated to level 5 (median, IQR 1–5, n = 23) and lasted 6 s (median,
IQR 0–9; U test compared to vehicle: p-level = 0.0002, p-duration b
0.0001). In contrast to CDM (1 mM), the natural agonist OA (n = 12)
produced no significant change in aggression at the highest dosage tested compared to vehicle (saline, n = 12; U-tests: initial fight, p-level =
0.8132, p-duration = 1.00; losers vs. winners, 15 min: p-level = 0.791,
p-duration = 1.00, data not illustrated; see also Stevenson et al., 2005).
The putative DAR-agonist homovanillyl alcohol HVA (1 mM) had no
obvious effect on aggression at the initial encounter (U-tests compared

p b 0.001
p = 0.009
p = 0.009
p = 0.968
p b 0.001
p b 0.001
p = 0.853

to vehicle: p-level = 0.694 and p-duration = 0.712), and there was still
a clear transient increase of loser–winner aggression over the observation period of 180 min (Fig. 1C, Tables 1 and 2; Kruskal–Wallis tests:
p-level = 0.0092; p-level = 0.0022). Nonetheless, as found for CDM,
HVA also led to an earlier loser recovery. For example, 15 min after
defeat losers interacted more aggressively with their previous winners
(median 1.5, IQR 1–4, n = 28) than the vehicle-treated group in fights
that lasted significantly longer (median 1 s, IQR 0–4.75; U test: plevel = 0.016, p-duration = 0.019). As for OA, DA itself (n = 12) produced no significant changes in aggression at the highest dosage tested
(10 μl, 20 mM) compared to vehicle (saline: n = 12, U-tests: initial
fight: p-level = 0.9465, p-duration = 0.6665; losers vs. winners,
15 min: p-level = 0.5912, p-duration = 0.5669, data not illustrated).
OA/DA depletion reversibly blocks loser recovery
Extending our earlier findings (Stevenson et al., 2000), treatment
with α-methyl-tyrosine (AMT) to selectively deplete OA and DA (and
probably TA) from the cricket nervous system reduced aggression at
the initial fight (AMT, n = 80, U test compared to saline control:
p-level b 0.001, p-duration b 0.001, Fig. 2), and completely blocked
the recovery of aggression throughout the 180 min observation period
(Kruskal–Wallis test: p-level = 0.967, p-duration = 0.9428; Fig. 2B).
This effect of AMT was reversible and hence selective rather than generally detrimental. First, 24 h after defeat AMT-treated losers fought their
previous winners as aggressively and as long as at their initial fights
(Wilcoxon signed rank tests: p-level = 0,545, p-duration = 0.129,
Fig. 1B). Second, additional administration of either the OAR-agonist
CDM or DAR-agonist HVA to AMT-treated crickets, each effectively

Table 2
The fight duration in s (median and interquartile range, n as in Table 1) for fights between socially naive crickets and the subsequent losers versus winners 15, 30, 60 and 180 min after
defeat for the following experimental groups: saline, DMSO, CDM, HVA, AMT, yohimbine, epinastine and fluphenazine. Transient recovery of loser aggression is indicated by the p(KW)value that gives the error probabilities from Kruskal–Wallis analyses for differences in the four loser vs. winner test periods (p N 0.05, not significant for CDM, AMT and fluphenazine).
Test group

initial fight

w-l (s)
15 min

w-l (s)
30 min

w-l (s)
60 min

w-l (s)
180 min



6.5, 3–10.75
7, 4–10
3, 1–5

0, 0–3
0, 0–1.5
6, 0–9
1, 0–4.75
0, 0–0
0, 0–2
0, 0–1
0, 0–4

0, 0–2
0, 0–1
6, 3.5–8.25
5.5, 0–8.75
0, 0–0
0, 0–5.75
0, 0–1
0, 0–3.5

3, 0–5
3, 0–7
6, 4–30.75
7, 4.75–9.25
0, 0–0
4.5, 0.75–9.5
3.5, 0–6.5
0, 0–3

6, 3.5–8.5
6, 3.5–8
12, 5–28
9, 7.25–12
0, 0–0
5.5, 1.5–8
6.5, 0.75–9
0, 0–5


b 0.001
b 0.001
= 0.201
= 0.002
= 0.943
b 0.001
b 0.001
= 0.883


J. Rillich, P.A. Stevenson / Hormones and Behavior 66 (2014) 696–704

Fig. 2. OA/DA depletion reversibly blocks loser recovery. Bar graphs, as in Fig. 1, giving the level of aggression (i, top) and fight duration (ii, bottom) for fights between naive crickets and
losers versus winners 15, 30, 60 and 180 min after defeat (different animals for each time slot; n is given above the x-axis in i) for the following treatments: A. Vehicle (saline, white bars);
B. α-Methyl-tyrosine (AMT, black bars), which depletes the CNS of DA and OA; C. AMT plus the OAR-agonist CDM (OARag, blue hatched bars) or the DAR-agonist HVA (DARag, red hatched
bars). Circles give the medians and boxes the interquartile ranges. The p(KW) gives error probabilities from Kruskal–Wallis analyses for differences in the four loser vs. winner test periods.
Asterisks indicate significant differences between test and vehicle groups for the same time slot (U test: **p b 0.01, ***p b 0.001).

Fig. 3. DAR- but not OAR-antagonist blocks loser recovery. Bar graphs, as in Fig. 1, giving the level of aggression (i, top) and fight duration (ii, bottom) for fights between naive crickets and
losers versus winners 15, 30, 60 and 180 min after defeat (different animals for each time slot; n is given above the x-axis in i) for the following treatments: A. Epinastine (OAR-blocker,
blue hatched bars); B. Fluphenazine (DAR-blocker, red hatched bars); C. Yohimbine (TAR-blocker, yellow hatched bars). Circles give the medians and boxes the interquartile ranges. The
p(KW) gives error probabilities from Kruskal–Wallis analyses for differences in the four loser vs. winner test periods. Asterisks indicate significant differences between the interactions of
naive crickets, and interactions of the same animals as losers and winners 180 min after fighting (Wilcoxon signed rank test, *p b 0.05, **p b 0.01, n.s., not significant).

J. Rillich, P.A. Stevenson / Hormones and Behavior 66 (2014) 696–704


restored aggression of losers against winners when tested 15 min
after defeat (U-tests compared to AMT: AMT + CDM, n = 11: plevel b 0.001, p-duration b 0.001; AMT + HVA, n = 11: p-level =
0.0097, p-duration = 0.0042; Fig. 2C).
DAR- but not OAR-antagonist blocks loser recovery
Against our expectations, the OAR-antagonist epinastine (Roeder
et al., 1998) did not prohibit the transient increase and recovery of
aggression after losing at the maximum dosage tested (10 μl, 10 mM).
As shown earlier, the level and duration of aggression at the initial
fight were somewhat reduced compared to controls (Tables 1 and 2,
compared to DMSO, U test: p-level = 0.034, p-duration b 0.001,
Fig. 3A), but there was still a clear gradual increase in aggression of
losers against winners over the 180 min observation period (Kruskal–
Wallis tests: p-level b 0.001, p-duration b 0.001; Tables 1 and 2) so
that the losers fully regained the aggressiveness exhibited at their initial
fights within the observation period (Wilcoxon signed rank test:
p-level = 0,324, p-duration = 0.447).
While OA does not seem to be obligate for the losers to recover,
experiments with DAR-antagonist fluphenazine (Degen et al., 2000)
suggests that DA is neccessary. Although this DAR-antagonist (10 μl,
10 mM) had no effect on the initial fights (U-tests compared to vehicle:
p-level = 0.0901, p-duration = 0.3379), the subsequent losers failed
to regain their aggressiveness towards their previous winners over the
3 h observation period (Kruskal–Wallis test: p-level = 0.8532, pduration = 0.8827, Tables 1 and 2, Fig. 3B). Hence, 180 min after losing,
both the level and duration of aggression were still reduced compared
to the initial fights (n = 21; Wilcoxon signed rank test: p-level =
0.009, p-duration = 0.0128).
Since AMT competitively inhibits the conversion of tyrosine to OA's
precursor tyramine (TA), this amine, an established neuromodulator
in insects (Kononenko et al., 2009; reviews: Lange, 2009; Farooqui,
2012), is probably also depleted by AMT. As an additional control we accordingly tested the effects of yohimbine, a potent insect TAR-blocker
(Roeder, 2005), but found no effects on cricket aggression at the tested
dosage (10 μl, 10 mM, Fig. 3C).
Selectivity of agonists and antagonists
In order to establish whether or not the tested aminergic drugs are
selectively targeting their designated receptors, we evaluated the efficacy of the OAR- and DAR-antagonists to selectively block the aggression
promoting effects of the agonists (antagonists were applied 1 h and agonists 30 min before evaluation). As summarized in Fig. 4, loser–winner
interactions evaluated 15 min after the initial fight are significantly
more aggressive for crickets treated with either the OAR-agonist CDM
or the DAR-agonist HVA compared to vehicle-treated controls (Fig. 4A
data from Fig. 1A, B, C). However, the enhancing effect of the OARagonist was abolished in the presence of the OAR-antagonist (median
level: 1, IQR 1–1, n = 15), which notably did not influence the effect
of the DAR-agonist (U-tests: p-level = 0.015, p-duration = 0.0101).
Conversely, the aggression enhancing effect of the OAR-agonist CDM
was still evident in the presence of the designated DAR-antagonist
fluphenazine (median level: 4, IQR 2–5, n = 15), but blocked in crickets
that received the DAR-agonist HVA (median level: 1, IQR 1–1, n = 15;
U-tests: p-level b 0.001, p-duration b 0.001). These data demonstrate
that the chosen drugs target their designated receptors selectively,
and hence support the notion that OA and DA can each promote aggression independently.
Fig. 5 summarizes the influences of all tested drugs on the time
course of aggressive recovery after social defeat, plotted as a percentage
of the level of aggression exhibited at the initial fight. This illustrates
that both saline and DMSO-treated crickets fully regain their aggressiveness after 3 h (DMSO 97%, saline 103% of initial fight performance) as
shown elsewhere for untreated crickets (Stevenson and Rillich, 2013).

Fig. 4. Selectivity of used agonists and antagonists. Bar graphs giving the level of aggression (i, top) and fight duration (ii, bottom) for fights between losers and winners
15 min after defeat (different animals for each time slot; n is given above the x-axis in
i) for the treatments: A. Vehicle (DMSO in saline, white bar), the OAR-agonist CDM
(blue bars) or the DAR-agonist HVA (red bars) alone, with no antagonist (no blocker);
B. OAR-blocker epinastine followed by treatment with CDM (OARag, blue bars) or HVA
(OARag, red bars); C. DAR-blocker fluphenazine followed by OARag (blue bars) or OARag
(red bars). The circles give the medians and the boxes the interquartile ranges. The
p(KW) gives error probabilities from Kruskal–Wallis analyses for differences in the four
loser vs. winner test periods. Asterisks indicate significant differences between HVA
and CDM for the same pretreatment with either epinastine or fluphenazine (U test:
*p b 0.05, ***p b 0.001, except for A where *p b 0.025; **p b 0.005, ***p b 0.0005 to accommodate Bonferroni correction to alpha for three comparisons).

While recovery begins earlier in crickets treated with the agonists
CDM and HVA, recovery is abolished following depletion of OA and DA
using AMT and by treatment with the DAR-antagonist fluphenazine.
Antagonists for OAR and TAR, in contrast, have no effect on the course
of recovery.
Conspecific males of essentially all species that compete aggressively
exhibit a prolonged condition of submissive behavior towards conspecifics after social defeat (Hsu et al., 2006; Earley et al., 2013), but very
little is known of its causes. In our experiments we demonstrated this
loser effect in crickets by re-matching losers against their previous victors at different times after the initial contest. It is important to stress
in our case that the reduced aggressiveness after social defeat is not
due to the subordinates simply retreating from familiar dominants.
Several independent studies have shown that defeated crickets retreat
from any approaching conspecific male, and that they do not retain
information from encounters which causes them to respond differently
in subsequent encounters with the same individual (Alexander, 1961;
Khazraie and Campan, 1999; Hofmann and Stevenson, 2000; Iwasaki
et al., 2006; Rillich et al., 2007; Stevenson and Rillich, 2013; review:
Simpson and Stevenson, 2014). In this paper we provide several lines
of evidence that DA and OA can each act independently to restore


J. Rillich, P.A. Stevenson / Hormones and Behavior 66 (2014) 696–704

Fig. 5. Charts summarizing effects of amine agonists, depletion and antagonists on the recovery of aggression of losers matched against their previous winners. Each chart plots the mean
level of aggression (level 1 fights were scored here as 0) exhibited by losers towards their previous winners as a percentage of the mean level at the initial fight at selected times after losing
on a log2 scale. The animals received the following treatments: A. Vehicle (1% DMSO with saline, white circles), the OAR-agonist CDM (OAR-ag, blue triangle) or the DAR-agonist
HVA (DAR-ag, red triangles); B. Vehicle (saline, white circle) or the semi-selective amine-depleting agent AMT (black circle); C. The OAR-blocker epinastine (OAR-bl, green circle), the
DAR-blocker fluphenazine (DAR-bl, red circle) and the TAR-blocker yohimbine (TAR-bl, yellow circle).

aggressiveness in subordinate crickets, and that DA, but not OA, is actually even necessary for loser recovery. Hence our data suggest that DA
and OA have different roles in the control of aggression, even though
their net effects appear to be the same.
OA/DA depletion prohibits loser recovery
While the depletion of biogenic amines from the nervous system
leads to reduced levels of general excitability, escape responses and
aggression (Stevenson et al., 2000), it cannot completely prevent the
expression of aggressive behavior in fight inexperienced (naive) individuals. Extending an earlier study (Stevenson et al., 2005), we now
show that selective depletion of OA and DA (and probably TA) using
the competitive synthesis inhibitor AMT (cf. Sloley and Orikasa, 1988)
reversibly prohibits the recovery of aggression after social defeat for at
least 3 h (Fig. 2), which is the time span normally required for complete
recovery (Stevenson and Rillich, 2013). Experiments with selective
agonists and antagonist for DAR and OAR illustrate that this effect of
the monoamine synthesis inhibitor AMT on loser recovery is entirely
due to loss of DA rather than OA.
DA is necessary for loser recovery
The main problem in "whole-animal" behavioral pharmacological
studies such as ours, especially when the effects of closely related
amines are concerned, is to ensure selectivity of the drugs used. In honeybees, for example, DAR and OAR subtypes linked to calcium signaling
are phylogenetically and pharmacologically closely related (Beggs et al.,
2011), and hence their functions are difficult to tease apart. The problem
is exacerbated by the orthopteran ganglion sheath, a formidable blood–
brain barrier even to the passage of ions (Schofield et al., 1984), so that
exogenously applied native amines first become effective only after
prolonged perfusion or at relatively high concentrations (Stevenson
and Kutsch, 1987; Stevenson et al., 2005; Buhl et al., 2008; Rillich
et al., 2013). It is hence not so surprising that injections of up to
20 mM DA (or OA) had no discernible effect on the expression of aggression as analyzed by us. In contrast, the structurally related DA-metabolite
homovanillyl alcohol (HVA) caused a significant increase in the aggressive behavior of losers after defeat, and also restored aggressiveness
in amine depleted (AMT-treated) losers at the comparatively low
dosage of 1 mM (10 μl). Given a total hemolymph volume of around
500 μl, the effective concentration would be about 50 μM, i.e. thus
just within the physiological range for natural amine effects in insects
(cf. Evans, 1985).

We propose that the action of HVA on aggression in crickets is due to
selective activation of DARs. To date, HVA is known to act as a DARagonist only in honeybees, where it is a key component of the queen
mandibular pheromone (Beggs et al., 2007; Beggs and Mercer, 2009)
that can elicit aggressive behavior in honeybees at high concentrations
(Vaitkevicienè and Budrienè, 1999). In fact, metabolites of DA, such as
HVA are generally considered to be pharmacologically inactive, and only
recent evidence suggests that metabolites such as 3-methoxytyramine
can modulate trace amine associated receptors in mice (Sotnikova et al.,
2010). The claim that HVA activates cricket DARs is supported by our
finding that its promoting effect on loser aggression is fully blocked by
fluphenazine (Fig. 4), an established DAR-antagonist in honeybees
(Degen et al., 2000; Mustard et al., 2003) and crickets (Unoki et al.,
2005; Mizunami and Matsumoto, 2010), but not by the OAR-blocker
epinastine. Furthermore, and as found for AMT, this DAR-antagonist
fully suppressed the recovery of aggression in losers, whereas the OAR
blocker epinastine did not (Fig. 3). Although epinastine seems able to
block both DAR and OAR in honeybees (Beggs et al., 2011), our finding
that it fails to block dopaminergic function in crickets is supported by
several other studies on crickets (Unoki et al., 2005, 2006; Mizunami
et al., 2009; Rillich and Stevenson, 2011). We hence conclude that
epinastine is a selective OAR blocker in crickets, as proposed by Roeder
et al. (1998). Finally, the finding that the insect TAR-antagonist
yohimbine (Roeder, 2005) had no influence on the aggressive behavior
of subordinates, further supports our suggestion that DA alone is necessary for loser recovery.
To our knowledge, this is the first time that a specific neuromodulator,
namely DA, has been shown to be actually necessary for the recovery of
aggression after social defeat in any animal. In mammals, pharmacologically induced DA is generally associated with increased aggression and
appears to be involved in signaling reward (O'Connell and Hofmann,
2011; Gil et al., 2013), i.e. similar to OA in insects (Stevenson and
Rillich, 2012; Stevenson and Schildberger, 2013), but its specific role
in mediating aggression still remains unclear (Nelson, 2006; Barron
et al., 2010; O'Connell and Hofmann, 2011). Recently, the stress of social
defeat was found in rats to reduce dopaminergic function (Watt et al.,
2014), but it is not known whether DA signaling is necessary for loser
depression as shown here for crickets. In insects, DA is mostly associated with arousal and enhanced general responsiveness (van Swinderen
and Andretic, 2011). With respect to insect aggression, dopaminergic
neurons have been found that both enhance or suppress it in fruit flies
(Alekseyenko et al., 2013), while in ants (Harpegnathos saltator) DA
levels are known to rise in aggressive workers at the start of a tournament, but decline in workers that were policed by their nest-mates
(Penick et al., 2014). Notably in our experiments neither HVA nor

J. Rillich, P.A. Stevenson / Hormones and Behavior 66 (2014) 696–704

fluphenazine influenced the fighting behavior of naive crickets, and
HVA did not prevent them from actually losing. Thus, DA appears to
have a clear, context-specific effect on the aggressive behavior expressed
only by subordinate crickets, indicating that the event of social defeat
may be a prerequisite for DA to exert its promoting influence. Interestingly, social defeat in lobsters leads to changes in the expression of
serotonin receptors to a pattern more appropriate for the new status
(Edwards and Spitzer, 2006), so that corresponding changes in the
insect dopaminergic system are plausible.
Subordinate behavior is controlled by multiple modulators
While it is conceivable that losing induces a transient depression or
delayed promotion of dopaminergic signaling, the mechanism controlling subordinate behavior is likely to be far more complex, involving differential recruitment of different receptor subtypes and interactions
with multiple modulator systems. Serotonin for example, can promote
or suppress aggression depending on the receptor subtype involved
(mammals: Nelson and Trainor, 2007; fruit flies: Johnson et al., 2009;
see Dyakonova and Krushinsky, 2013 for effects in crickets and Edwards
and Spitzer, 2006 for data on lobsters). Furthermore, aggression in mammals is also suppressed by the gaseous modulator nitric oxide (NO), partly
via influences on serotonergic signaling (Nelson and Trainor, 2007). In
crickets, blocking the NO/cGMP pathway promotes aggression in losers
(Iwasaki et al., 2007), while ongoing work suggests that NO is actually
required for the decision made by naive crickets to flee (Stevenson
and Rillich, 2012; Stevenson and Rillich, submitted for publication).
Our current experiments, discussed below, illustrate that OA also has
a selective role in controlling aggression in subordinate crickets.
OA promotes loser recovery
While experiments with the selective OAR blocker epinastine show
that OA is not necessary for loser recovery, OA can clearly promote the
expression of aggression in subordinates. Supporting earlier studies
(Stevenson et al., 2005), aggression in losers is also restored by the
OAR-agonist CDM. Its effect in losers is even more pronounced than
that of HVA (e.g. 15 min after defeat, Fig. 1) and, in contrast to HVA,
CDM also increased aggression in naive crickets. This probably reflects
the optimized tissue permeability and almost irreversible OAR binding
capacity of this insecticide (cf. Evans, 1985). The fact that CDM's promoting effect on loser aggression is abolished by the OAR-antagonist
epinastine, but not by the DAR-blocker fluphenazine firmly supports
binding studies suggesting that CDM is a selective OAR agonist
(Roeder, 1995; Hiripi et al., 1999). Hence, activation of OAR can promote
aggression in subordinate crickets independent of DA. We have previously shown that subordinate crickets in possession of a resource,
such as an artificial burrow, become highly aggressive, and that this
residency effect is OA dependent (Rillich et al., 2011). Hence, although
OA is not necessary for the normal course of loser recovery, it can
mediate the effect of aggression promoting experiences on aggression,
and thus enable adaptive aggressive responses.
Amines—mediators of rewarding and aversive experiences in insect
In insects OA is reputed to convey reward in appetitive learning
(honeybees: Hammer and Menzel, 1995; fruit flies: Schwaerzel et al.,
2003; crickets: Mizunami et al., 2009) and value of food sources in foraging honeybees (Barron et al., 2010). Intriguingly, OA neurons of the
type found to mediate reward in honeybees (Hammer, 1993) appear
to correspond to those important for expressing aggression in fruit
flies (Zhou et al., 2008). Accordingly, we recently proposed that OA
may also signal events evaluated as being rewarding in cricket
aggression (Stevenson and Rillich, 2012), since it mediates the enhancing effects of a wide variety of experiences that span the extremes of


energy consumption (flying: Stevenson et al., 2005; winning: Rillich
and Stevenson, 2011; resource possession: Rillich et al., 2011).
Notwithstanding recent studies demonstrating that OA-dependent
appetitive memory also requires DA-signaling (Burke et al., 2012), DA
is generally associated with conveying punishment signals in aversive
learning paradigms (fruit flies: Heisenberg, 2003; Schwaerzel et al.,
2003; crickets: Unoki et al., 2005, 2006; honeybees: Vergoz et al.,
2007a), and also in forgetting stored memories (fruit flies: Berry et al.,
2012). Interestingly, the dopamine metabolite HVA, shown by us to
restore aggression in subordinate crickets, apparently both promotes
aggression (Vaitkevicienè and Budrienè, 1999) and blocks aversive
memory in honeybees (Vergoz et al., 2007b). We speculate that the
experience of losing may be evaluated as being aversive, and that
loser recovery represents the forgetting of social subjugation. This
opens the intriguing possibility that the actions of amines during
aggressive interactions use common mechanisms for integrating potential aversive and rewarding stimuli as used in appetitive and aversive
learning and memory. Now more work is needed to reveal how DA
interacts with other transmitter systems in the control of aggression.
Author contributions
Conceived and designed the experiments: JR PAS. Performed the
experiments: JR PAS. Analyzed the data: JR PAS. Contributed reagents/
materials/analysis tools: PAS JR. Wrote the paper: PAS JR.
We are very grateful to Prof. Dr. K. Schildberger, Leipzig for use
of facilities at his disposal, Prof. Dr. H.-J. Pflüger, FU-Berlin for
numerous suggestions and the anonymous referees for their constructive critic. Funding by the German Research Council (Deutsche
Forschungsgemeinschaft) is gratefully acknowledged (DFG Research
Group 1363, grant STE 714/4-1). The funders exercised no influence
on the conception and writing of this study.
Alekseyenko, O.V., Chan, Y.B., Li, R., Kravitz, E.A., 2013. Single dopaminergic neurons that
modulate aggression in Drosophila. Proc. Natl. Acad. Sci. U. S. A. 110, 6151–6156.
Alexander, R.D., 1961. Aggressiveness, territoriality, and sexual behaviour in field crickets
(Orthoptera: Gryllidae). Behaviour 17, 130–223.
Archer, J., 1988. The Behavioural Biology of Aggression. Cambridge University Press,
Cambridge, New York.
Barron, A.B., Sovik, E., Cornish, J., 2010. The roles of dopamine and related compounds in
reward-seeking behavior across animals phyla. Front. Behav. Neurosci. 4, 1–9.
Beggs, K.T., Mercer, A.R., 2009. Dopamine receptor activation by honey bee queen
pheromone. Curr. Biol. 19, 1206–1209.
Beggs, K.T., Glendining, K.A., Marechal, N.M., Vergoz, V., Nakamura, I., Slessor, K.N.,
Mercer, A.R., 2007. Queen pheromone modulates brain dopamine function in worker
honey bees. Proc. Natl. Acad. Sci. U. S. A. 104, 2460–2464.
Beggs, K.T., Tyndall, J.D., Mercer, A.R., 2011. Honey bee dopamine and octopamine
receptors linked to intracellular calcium signaling have a close phylogenetic and
pharmacological relationship. PLoS One 6, e26809.
Berry, J.A., Cervantes-Sandoval, I., Nicholas, E.P., Davis, R.L., 2012. Dopamine is required for
learning and forgetting in Drosophila. Neuron 74, 530–542.
Buhl, E., Schildberger, K., Stevenson, P.A., 2008. A muscarinic cholinergic mechanism
underlies activation of the central pattern generator for locust flight. J. Exp. Biol.
211, 2346–2357.
Burke, C.J., Huetteroth, W., Owald, D., Perisse, E., Krashes, M.J., Das, G., Gohl, D., Silies, M.,
Certel, S., Waddell, S., 2012. Layered reward signaling through octopamine and
dopamine in Drosophila. Nature 492, 433–437.
Degen, J., Gewecke, M., Roeder, T., 2000. The pharmacology of a dopamine receptor in the
locust nervous tissue. Eur. J. Pharmacol. 396, 59–65.
Dixon, K.A., Cade, W.H., 1986. Some factors influencing male male-aggression in the field
cricket Gryllus integer (time of day, age, weight and sexual maturity). Anim. Behav.
34, 340–346.
Dyakonova, V.E., Krushinsky, A.L., 2013. Serotonin precursor (5-hydroxytryptophan)
causes substantial changes in the fighting behavior of male crickets, Gryllus
bimaculatus. J. Comp. Physiol. A. 199, 601–609.
Earley, R.L., Lu, C.K., Lee, I.H., Wong, S.C., Hsu, Y., 2013. Winner and loser effects are
modulated by hormonal states. Front. Zool. 10, 6.
Edwards, D.H., Spitzer, N., 2006. Social dominance and serotonin receptor genes in
crayfish. Curr. Top. Dev. Biol. 74, 177–199.


J. Rillich, P.A. Stevenson / Hormones and Behavior 66 (2014) 696–704

Elwood, R.W., Arnott, G., 2012. Understanding how animals fight with Lloyd Morgan's
canon. Anim. Behav. 84, 1095–1102.
Evans, P.D., 1985. Octopamine. In: Kerkut, G.A., Gilbert, L.I. (Eds.), Comprehensive Insect
Physiology Biochemistry and Pharmacology. Pergamon, Oxford, pp. 499–530.
Farooqui, T., 2012. Review of octopamine in insect nervous systems. Open Access Insect
Physiol. 4, 1–17.
Fuxjager, M.J., Forbes-Lorman, R.M., Coss, D.J., Auger, C.J., Auger, A.P., Marler, C.A., 2010.
Winning territorial disputes selectively enhances androgen sensitivity in neural
pathways related to motivation and social aggression. Proc. Natl. Acad. Sci. U. S. A.
107, 12393–12398.
Gil, M., Nguyen, N.-T., McDonald, M., Albers, H.E., 2013. Social reward: interactions with
social status, social communication, aggression, and associated neural activation in
the ventral tegmental area. Eur. J. Neurosci. 38, 2308–2318.
Hammer, M., 1993. An identified neurone mediates the unconditioned stimulus in
associative olfactory learning in honeybees. Nature 366, 59–63.
Hammer, M., Menzel, R., 1995. Learning and memory in the honeybee. J. Neurosci. 15,
Heisenberg, M., 2003. Mushroom body memoir: from maps to models. Nat. Rev. Neurosci.
4, 266–275.
Hiripi, L., Nagy, L., Hollingworth, R.M., 1999. In vitro and in vivo effects of formamidines in
locust (Locusta migratoria migratoriodes). Acta Biol. Hung. 50, 81–87.
Hofmann, H.A., Stevenson, P.A., 2000. Flight restores fight in crickets. Nature 403, 613.
Hsu, Y., Earley, R.L., Wolf, L.L., 2006. Modulation of aggressive behaviour by fighting experience: mechanisms and contest outcomes. Biol. Rev. Camb. Philos. Soc. 81, 33–74.
Hsu, Y.Y., Lee, I.H., Lu, C.K., 2009. Prior contest information: mechanisms underlying
winner and loser effects. Behav. Ecol. Sociobiol. 63, 1247–1257.
Huhman, K., 2006. Social conflict models: can they inform us about human psychopathology? Horm. Behav. 50, 640–646.
Huntingford, F.A., Turner, A.K., 1987. Animal conflict. Chapman and Hall, London,
New York.
Iwasaki, M., Delago, A., Nishino, H., Aonuma, H., 2006. Effects of previous experience on
the agonistic behaviour of male crickets, Gryllus bimaculatus. Zool. Sci. 23, 863–872.
Iwasaki, M., Nishino, H., Delago, A., Aonuma, H., 2007. Effects of NO/cGMP signaling on
behavioral changes in subordinate male crickets, Gryllus bimaculatus. Zool. Sci. 24,
Johnson, O., Becnel, J., Nichols, C.D., 2009. Serotonin 5-HT(2) and 5-HT(1A)-like receptors
differentially modulate aggressive behaviors in Drosophila melanogaster. Neuroscience 158, 1292–1300.
Khazraie, K., Campan, M., 1999. The role of prior agonistic experience in dominance
relationships in male crickets Gryllus bimaculatus (Orthoptera: Gryllidae). Behav.
Processes 44, 341–348.
Kononenko, N.L., Wolfenberg, H., Pflueger, H.J., 2009. Tyramine as an independent transmitter and a precursor of octopamine in the locust central nervous system: an immunocytochemical study. J. Comp. Neurol. 512, 433–452.
Lange, A.B., 2009. Tyramine: from octopamine precursor to neuroactive chemical in
insects. Gen. Comp. Endocrinol. 162, 18–26.
Mizunami, M., Matsumoto, Y., 2010. Roles of aminergic neurons in formation and recall of
associative memory in crickets. Front. Behav. Neurosci. 4, 172.
Mizunami, M., Unoki, S., Mori, Y., Hirashima, D., Hatano, A., Matsumoto, Y., 2009. Roles of
octopaminergic and dopaminergic neurons in appetitive and aversive memory recall
in an insect. BMC Biol. 7, 46.
Mustard, J.A., Blenau, W., Hamilton, I.S., Ward, V.K., Ebert, P.R., Mercer, A.R., 2003. Analysis
of two D1-like dopamine receptors from the honey bee Apis mellifera reveals agonistindependent activity. Brain Res. Mol. Brain Res. 113, 67–77.
Nelson, R.J., 2006. Biology of aggression. Oxford University Press, Oxford, New York.
Nelson, R.J., Trainor, B.C., 2007. Neural mechanisms of aggression. Nat. Rev. Neurosci. 8,
O'Connell, L.A., Hofmann, H.A., 2011. The vertebrate mesolimbic reward system and social
behavior network: a comparative synthesis. J. Comp. Neurol. 519, 3599–3639.
Penick, C.A., Brent, C.S., Dolezal, K., Liebig, J., 2014. Neurohormonal changes associated
with ritualized combat and the formation of a reproductive hierarchy in the ant
Harpegnathos saltator. J. Exp. Biol. 217, 1496–1503.
Pflueger, H.J., Stevenson, P.A., 2005. Evolutionary aspects of octopaminergic systems with
emphasis on arthropods. Arthropod. Struct. Dev. 34, 379–396.
Raichlen, D.A., Foster, A.D., Gerdeman, G.L., Seillier, A., Giuffrida, A., 2011. Wired to run:
exercise-induced endocannabinoid signaling in humans and cursorial mammals
with implications for the ‘runner's high’. J. Exp. Biol. 215, 1331–1336.
Rillich, J., Stevenson, P.A., 2011. Winning fights induces hyperaggression via the action of
the biogenic amine octopamine in crickets. PLoS One 6, e28891.

Rillich, J., Schildberger, K., Stevenson, P.A., 2007. Assessment strategy of fighting crickets
revealed by manipulating information exchange. Anim. Behav. 74, 823–836.
Rillich, J., Schildberger, K., Stevenson, P.A., 2011. Octopamine and occupancy—an
aminergic mechanism for intruder-resident aggression in crickets. Proc. R. Soc. B
278, 1873–1880.
Rillich, J., Stevenson, P.A., Pflueger, H.J., 2013. Flight and walking in locusts -cholinergic
co-activation, temporal coupling and its modulation by biogenic amines. PLoS One
8, e62899.
Roeder, T., 1995. Pharmacology of the octopamine receptor from locust central nervous
tissue (OAR3). Brit. J. Pharmacol. 114, 210–216.
Roeder, T., 2005. Tyramine and octopamine: ruling behavior and metabolism. Annu. Rev.
Entomol. 50, 447–477.
Roeder, T., Degen, J., Gewecke, M., 1998. Epinastine, a highly specific antagonist of insect
neuronal octopamine receptors. Eur. J. Pharmacol. 349, 171–177.
Schofield, P.K., Swales, L.S., Treherne, J.E., 1984. Quantitative analysis of cellular and
paracellular effects involved in the disruption of the blood brain barrier of an insect
by hypertonic urea. J. Exp. Biol. 109, 333–340.
Schwaerzel, M., Monastirioti, M., Scholz, H., Friggi-Grelin, F., Birman, S., Heisenberg, M.,
2003. Dopamine and octopamine differentiate between aversive and appetitive olfactory memories in Drosophila. J. Neurosci. 23, 10495–10502.
Simpson, S.J., Stevenson, P.A., 2014. Neuromodulation of Social Behavior in Insects. Oxford
University Press, Oxford, New York.
Sloley, B.D., Orikasa, S., 1988. Selective depletion of dopamine, octopamine and 5hydroxytryptamine in the nervous tissue of the cockroach (Periplaneta americana).
J. Neurocytochem. 51, 535–541.
Sotnikova, T.D., Beaulieu, J.M., Espinoza, S., Masri, B., Zhang, X., Salahpour, A., Barak, L.S.,
Caron, M.G., Gainetdinov, R.R., 2010. The dopamine metabolite 3-methoxytyramine
is a neuromodulator. PLoS One 5, e13452.
Stevenson, P.A., Kutsch, W., 1987. A reconsideration of the central pattern generator
concept for locust flight. J. Comp. Physiol. A. 161, 115–129.
Stevenson, P.A., Rillich, J., 2012. The decision to fight or flee - insights into underlying
mechanism in crickets. Front. Neurosci. 6, 118.
Stevenson, P.A., Rillich, J., 2013. Isolation associated aggression–a consequence of
recovery from defeat in a territorial animal. PLoS One 8, e74965.
Stevenson, P.A., Rillich, J., 2014. Adding up the odds – NO/cGMP mediates opponent assessment for the decision to flee in fighting crickets, (submitted for publication).
Stevenson, P.A., Schildberger, K., 2013. Mechanisms of experience dependent control of
aggression in crickets. Curr. Opin. Neurobiol. 23, 318–323.
Stevenson, P.A., Hofmann, H.A., Schoch, K., Schildberger, K., 2000. The fight and flight
responses of crickets depleted of biogenic amines. J. Neurobiol. 43, 107–120.
Stevenson, P.A., Dyakonova, V., Rillich, J., Schildberger, K., 2005. Octopamine and experiencedependent modulation of aggression in crickets. J. Neurosci. 25, 1431–1441.
Szczuka, A., Korczynska, J., Wnuk, A., Symonowicz, B., Gonzalez Szwacka, A.,
Mazurkiewicz, P., Kostowski, W., Godzinska, E.J., 2013. The effects of serotonin,
dopamine, octopamine and tyramine on behavior of workers of the ant Formica
polyctena during dyadic aggression tests. Acta Neurobiol. Exp. (Wars.) 73, 495–520.
Unoki, S., Matsumoto, Y., Mizunami, M., 2005. Participation of octopaminergic reward
system and dopaminergic punishment system in insect olfactory learning revealed
by pharmacological study. Eur. J. Neurosci. 22, 1409–1416.
Unoki, S., Matsumoto, Y., Mizunami, M., 2006. Roles of octopaminergic and dopaminergic
neurons in mediating reward and punishment signals in insect visual learning. Eur. J.
Neurosci. 24, 2031–2038.
Vaitkevicienè, G., Budrienè, A., 1999. Age-related changes in response to queen pheromone and retrocerebral complex of Apis mellifera l. workers. Pheromones 6, 39–46.
Van Swinderen, B., Andretic, R., 2011. Dopamine in Drosophila: setting arousal thresholds
in a miniature brain. Proc. Biol. Sci. 278, 906–913.
Vergoz, V., Roussel, E., Sandoz, J.C., Giurfa, M., 2007a. Aversive learning in honeybees
revealed by the olfactory conditioning of the sting extension reflex. PLoS One 2, e288.
Vergoz, V., Schreurs, H.A., Mercer, A.R., 2007b. Queen pheromone blocks aversive learning
in young worker bees. Science 317, 384–386.
Watt, M.J., Roberts, C.L., Scholl, J.L., Meyer, D.L., Miiller, L.C., Barr, J.L., Novick, A.M., Renner,
K.J., Forster, G.L., 2014. Decreased prefrontal cortex dopamine activity following
adolescent social defeat in male rats: role of dopamine D2 receptors. Psychopharmacology 231, 1627–1636.
Zhou, C., Rao, Y., Rau, Y., 2008. A subset of octopaminergic neurons are important for
Drosophila aggression. Nat. Neurosci. 11, 1059–1067.