Group rearing leads to long-term changes in locomotor activity of <i>Drosophila</i> males

doi:10.4236/ojas.2013.34A2004

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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; [email protected]

Received 17 August 2013; revised 21 September 2013; accepted 6 October 2013

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-

A. A. Panova et al. / Open Jou r nal of Animal Sciences 3 (2013) 31-35

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.

A. A. Panova et al. / Open Jou r nal of Animal Sciences 3 (2013) 31-35

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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