Vol.3, No.4B, 31-35 (2013) Open Journal of Animal Sciences
http://dx.doi.org/10.4236/ojas.2013.34A2004
Group rearing leads to long-term changes in
locomotor activity of Drosophila males
Anna A. Panova, Julia V. Bragina, Larisa V. Danilenkova, Natalia G. Besedina,
Elena A. Kamysheva, Sergey A. Fe dotov, Nikolay G. Kamyshev
Pavlov Institute of Physiology, Russian Academy of Sciences, St. Petersburg, Russia;
Received 17 August 2013; revised 21 September 2013; accepted 6 October 2013
Copyright © 2013 Anna A. Panova et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Previous social experience may affect subse-
quent behavior. It was shown by other authors
that Drosophila melanogaster males kept indi-
vidually are more aggr essiv e and se xually activ e
than males kept in a group. In the present study,
we tested the locomotor activity of individual
males and females previously reared either in-
dividually, or in a group. We found that keeping
20 young males for three days together led to a
strong long-term (up to 5 days) reduction in their
further locomotor activity as individuals. Rear-
ing of young males in groups of other sizes (2, 5,
10, and 30) produced a smaller or no after-effect.
At the same time, we have not found any differ-
ence in subsequent behavior of individual fe-
males previously kept either individually, or in a
group. We suppose that in a group, flies learned
to suppress their locomotor activity to prevent
unpleasant contacts with other animals (operant
learning). It seems that in males this learning is
more efficient because of the higher level of ag-
gression producing the stronger negative rein-
forcement.
Keyw ords: Drosophila; Social Experience; Group
Situation; Locomotor Activity
1. INTRODUCTION
It is known that previous social experience can modify
further behavior. It is true not only for the social animals
but for animals without developed sociality, like Droso-
phila, too. Today little is known about social interactions
and their consequences in subsequent Drosophila behav-
ior. In addition to better understanding of animal behave-
ior, such studies may have implications for creation of
animal models of human psychiatric disorders, where
social isolation or interaction is a contributing factor.
The main interactions between Drosophila individuals
are presented by courtship, aggressive and aggregation
behaviors. Flies cooperate into aggregation being at-
tracted by volatile pheromone cis-vaccenyl acetate [1-2].
It was shown in Drosophila mutants that keeping flies in
a group leads to significant life extension [3]. But it is
not still clear what is more stressful for Drosophila:
staying alone or in a group.
It was shown that each fly prefers to maintain individ-
ual distance free from other flies [4]. Close contacts are
accompanied by kicks, wing threats and jumps [5-6].
Also, in a group situation flies spent more time in preen-
ing, this behavior was considered as a visual signal pre-
venting contacts [7]. Previously, we found that in a group
situation, Drosophila melanogaster females showed two
unconditioned responses: 1) running from the fly break-
ing the individual distance, that increased their run fre-
quency, and 2) stopping when continuation of the run
threatened collision with another female, that decreased
their run duration. By the trial and error method (operant
learning) females learn to decrease their locomotor activ-
ity that results in reduction of unpleasant contacts with
other individuals [8]. After isolation from a group situa-
tion, females restored their level of activity very rapidly
showing fast extinction of operant conditioning. How-
ever, the classical conditioning in heterogeneous envi-
ronment was found, when threat from other flies it was
associated with context stimuli, and further presentation
of context in absence of other flies was sufficient to in-
hibit activity [8].
One more attempt to understand the process of coop-
eration of flies in a group was made by Tinette group. [9].
They suggest that flies may act in a cooperative manner
to perform food searching, namely that there are two
types of flies: “primers” and “followers”. The former in-
vestigate food substrate and choose the best one. The
latter, “followers”, move to that food source and aggre-

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32
gate there. In other studies it was shown that keeping
males in a homogenous group leads to a long-term de-
pression of their aggressive and sexual behaviors [10-
14,6]. Females, if they have a choice between a male
previously kept in a group and a male kept individually,
choose the more active one, i. e . kept individually. Also it
was found that isolation of males from conspecifics led
to increase of homosexual behavior. The probable reason
is the modification of cuticle pheromone composition. In
two studies it was shown that chemical signaling is af-
fected, besides light-dark cycle and genotype, by the
social environment [15-17].
The problem of influence of social context and social
experience on behavior is widely studied in laboratory
animals [18-22].
A number of investigations have shown the impor-
tance of social environment for rodents. It was revealed
that rearing in social isolation during pubertal period
could lead to hyperactivity, increased aggressive behave-
ior, reduced habituation, novel object recognition and
resulted in reduction of medial prefrontal cortex volume,
cytoskeletal alteration in hippocampus and changes in
CREB expression and dopamine and serotonin turnover
in different parts of brain [23].
2. MATERIALS AND METHODS
2.1. Flies
Drosophila melanoga ster wild-type line Canton-S was
used. Males and females were collected during 3 hours
after eclosion by aspiration. All flies were kept in 25 ×
95 mm vials containing standard yeast-semolina-raisin-
sugar medium under 12:12 light-dark cycle at 25˚C and
relative humidity of 60% ± 5%. Collected males and
females were kept either individually (control), or in
groups (experiment) for 3 days. Then experimental males
were separated from each other. To examine the influ-
ence of group size (2, 5, 10, 20, or 30 individuals per vial)
on subsequent locomotor activity of experimental males
they were tested individually immediately after separa-
tion. Experimental males used in investigation of the
after-effect duration were kept in groups of 20. The con-
trol and experimental males were tested daily for 8 days
(independent samples for each day). Experimental fe-
males after keeping in groups of 20 were tested individu-
ally immediately after isolation.
2.2. Locomotion Analysis
All experiments were made from 11 a.m. to 5 p.m.
Males were introduced individually by aspiration into
chambers (15 mm diameter, 5 mm height) made in a
plexiglass plates. The plates were positioned under two
web cameras, watching behavior of 20 flies simultane-
ously, using the original program “Drosophila tracks” (©
N. G. Kamyshev) allowing to monitor and to analyze the
time course of various parameters of locomotion. The
main parameter is activity index, i.e. the percent of time
spent in locomotion. In case of examination of group size
effect in males and experiments with females, registra-
tion lasted for 1 hour with statistical analysis of 12 suc-
cessive 5-min periods. When duration of the after-effect
of group keeping was examined, daily registration time
was 5 hours with statistical analysis of 10 successive
30-min periods (wet filer paper was placed into experi-
mental chambers in this case). Also we computed the
joint activity index for last 5 periods (2.5 hours) of
5-hours testing period.
3. RESULTS AND DISCUSSION
3.1. Dependence of Subsequent Locomotor
Activity in Individuals, Previously Kept
in a Group, on Group Size
First of all, we checked the influence of group size on
further locomotor activity of males separated from a
group to choose the one with maximum after-effect
(Figure 1). We kept Drosophila males alone or in a
group of 2, 5, 10, 20 or 30 flies for 3 days after eclosion.
Then we tested locomotor activity in individuals sepa-
rated from a group. The largest effect of previous social
experience was found for flies kept in groups of 20 indi-
viduals.
Reduction of locomotor activity in line 1→5→10→20
flies is quite good predictable. The more flies, the more
frequent collisions between them. That, in turn, increases
the magnitude of changes in their behavior.
Figure 1. Influence of group size on further locomotor activity
of males separated from a group. Effect of group keeping for 3
days depends on group size. 20-flies group showed the largest
difference from the control (flies kept individually). Y axis:
activity index (percent of time spent in locomotion). X axis:
5-min periods of observation. Vertical bars show 95% confi-
dence intervals. N = 28 − 31 for each point.

A. A. Panova et al. / Open Jou r nal of Animal Sciences 3 (2013) 31-35 33
One exclusion from this rule is no or even opposite
after-effect of group keeping, when a group consists only
of two males. When two males meet in a vial, it can be
assumed, they prefer to stay separately. Probably, every
accidental meeting stimulates them to run away. As flies
do not encounter many obstacles, they need not stop run-
ning. As a result we can see a slight activation of activity
in comparison to males kept individually. In contrast, the
group density of 5 (in given experimental conditions) is
already sufficient to provide inhibition of locomotor ac-
tivity in a group situation.
The most surprising are the meanings of locomotor ac-
tivity of flies kept in 30-individuals group. Although at
the beginning of observation these flies show no differ-
ence from control, by the end of 1 hour their locomotor
activity became less than activity of individual ones.
Nevertheless, they are more active than flies kept in
20-flies group.
If to agree (see Introduction and Conclusion) that in-
hibition of locomotor activity after group rearing results
from learning, this may imply that group density of 30
flies per vial leads to cognitive disfunctions. The reasons
for that may consist in the worsening of life conditions
due to great number of negative interactions including
loss in concurrency for food resources.
3.2. Previous Group Keeping Leads to
Changes of Male, but not Female
Locomotor Activity
To verify the previous data about the lack of after-ef-
fect of group keeping in females [8], we compared lo-
comotor activity in females kept for 3 days after eclosion
either individually, or in a 20-flies group (Figure 2). Our
results confirm the fact that females do not change their
behavior after group keeping. The effects of previous
social experience, is yet poorly investigated. In rodents,
it was shown that rearing in social isolation during pu-
bertal period led to hyperactivity, reduced habituation,
novel object recognition and floating time in forced
swim tests [23]. But all these effects concern both males
and females. Our results show that consequences of so-
cial experience in Drosophila are quite different for two
sexes. It may be assumed that Drosophila females are
less aggressive than males and provoke collisions with
other flies less frequently (normally, they do not show
homosexual behavior).
3.3. Keeping Drosophila Males in a Group
Leads to a Long-Term Locomotion
Inhibition
To investigate the duration of behavioral changes after
group rearing we kept males in 20-flies groups during 3
days. Then we isolated them from each other and tested
experimental and control flies immediately and for next
7 days, each day for 5 hours. Because the most signify-
cant difference between experimental and control flies
were observed during last 2.5 hours, the joint activity
index for this time interval was analyzed (Figure 3).
There is a great difference between males kept indi-
vidually and males kept in a group, which retains until 5
days after isolation of experimental males. Importantly,
these behavioral changes are temporary. This excludes
the possibility that they are based on alteration of brain
structures that was found in rodents [24,25]. According
to our previous investigations [8] we suppose that this
locomotor suppression is the result of operant learning
Figure 2. Absence of after-effect of group keeping in Droso-
phila females. In contrast to Drosophila males, females do not
change their locomotor activity after 3-days keeping in a group.
Y axis: activity index (percent of time spent in locomotion). X
axis: 5-min periods of observation. Vertical bars show 95%
confidence intervals. N = 40 − 50 for each point.
Figure 3. Group keeping leads to long-term changes in Droso-
phila male locomotor activity. Y axis: activity index calculated
for the last 2.5 hours of 5-h observation. X axis: days after iso-
lation from a group. Vertical bars show 95% confidence inter-
vals. N = 40 − 50 for each point.

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occurred in a group. Being in a group situation, flies
learn to avoid contacts with other individuals because
they are accompanied with threats and fighting playing a
role of negative reinforcement. As a result, flies prefer to
stay in rest than being active.
4. CONCLUSIONS
Previous studies have shown that keeping Drosophila
males in a group leads to increase of aggression and de-
crease of sexual activity in comparison with males kept
individually [10-14,6]. In our research we showed that
previous social experience is extremely important for
further behaviour. Keeping males together for 3 days in
groups of 20 flies leads to a long-term strong reduction
of their locomotor activity maintained for 5 days after
separation. This may be a result of operant learning
similar to learning described earlier [Kamyshev et al.
2002] in females. It was shown that in a group the flies
try to avoid close unpleasant contacts accompanied by
kicks and wing threats from other insects. Therefore,
they run from the individuals, which came too near, and
stop their running to prevent meeting with another fe-
male. Trial and error learning forces them to suppress
their locomotor activity and stay at rest; as a result they
have a less number of unpleasant contacts. But in the
case of females this learning leads to no after-effects in
absence of the appropriate contextual cues. Males, in
contrast, possibly due to more intensive aggression, re-
tain the altered behavior for a long time independently of
the context, in which they experienced aggression from
other individuals.
Ascertainment of underlying physiological and mo-
lecular mechanisms of these phenomena may illuminate
the basic processes of Drosophila social interactions.
These results will help to standardize laboratory proto-
cols of Drosophila housing. Also, the fruit fly, as well as
rodents, can be used as a model for investigation of so-
cial stress and its effects on development of neuropsy-
chiatric disorders.
5. ACKNOWLEDGEMENTS
This study was supported by grants No. 13-04-02153 and No. 13-04-
12030 from Russian Foundation for Basic Researches, by Programs of
Presidium of the Russian Academy of Sciences No. 7 and No. 30.
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