ZEBRAFISH

Volume 00, Number 00, 2014 Original Article

ª Mary Ann Liebert, Inc.
DOI: 10.1089/zeb.2014.0977

Effects of the Number of Subjects on the Dark/Light

Preference of Zebrafish (Danio rerio)

Bruno de Matos Mansur,1 Bruno Rodrigues dos Santos,1 Cláudio Alberto Gellis de Mattos Dias,2
Marcelo de Sena Pinheiro,1 and Amauri Gouveia Jr.1

Abstract

This research aims to describe the effects of a variable number of Danio rerio fish subjects, ranging from one to
eight, in the light/dark box preference test. Four hundred eighty adult male short-finned phenotype zebrafish
were tested in the light/dark box. There were four groups in this experiment and a different number of subjects
was used in each group: the control group had only one subject, whereas the experimental groups had either
two, four, or eight subjects simultaneously inside the apparatus in every session. The average occurrence (AO)
of subjects in the white side of the aquarium and the first choice average (FC) were recorded. The AO revealed
no difference between the control group and test groups with two and four subjects. The results for the test
group with eight subjects showed significant difference when compared to the control group and from the test
group with two subjects. The FC also showed no difference between the control group and test groups with two
and four subjects. There was significant variation between the control and the test group with eight subjects. The
results reflect a conflict between the animal’s preference for dark places and the innate drive to explore new
environments. Zebrafish are highly social animals, exhibiting preference for swimming in groups and other
patterns of social cohesion. The reduced white avoidance behavior in the test group of eight subjects may
possibly reflect the role of shoaling, which is a defensive behavior, in reducing anxiety and stress. On the other
hand, the absence of difference between the control group and test groups with two and four subjects suggest
that it is feasible to run the light/dark test with up to four subjects, becoming an alternative to streamline and
simplify data collection and test analysis.

Introduction et al.,8 scototaxis can be used as an indicator of stress, fear,

and anxiety in zebrafish, which can be measured and
Z ebrafish (Danio rerio) originated in the valleys of
India and Bangladesh,1 and has been widely used in
behavioral research due to its small size, ease of use, breed-
exploited through the light/dark box test.

Anxiety and the light/dark test

ing, and keeping. It has also shown potential to be used as a
model in social behavior2 research. The zebrafish is well
adapted to life in aquariums.3 Diurnal4 fish live in clean and
clear water in ditches or shallow lakes, and in between veg-
etation and rice paddies.5 Consequently, the behavior of
hiding in dark places to avoid predators is a requirement to
survive in these well-lit environments. Scototaxis, which is
the preference for dark environments as opposed to bright
ones,6 is a natural strategy that promotes the survival of an-
imals in this environment, constituting a particular defensive
pattern of species that exploit concealment or lack of illu-
mination in the environment to avoid predators.7,8 This be-
havior may be an indicator of fear and anxiety in several
species, including zebrafish.9,10 According to Maximino
Tests like the light/dark test are based on novelty paradigms
and are commonly used in behavioral neuroscience to study
fear and anxiety.11 According to McNaughton and Corr,12
anxiety is most often generated by concurrent and equivalent
activation of fear and risk assessment systems. The particular
set of responses depends on the perceived threat.13–16 Thus,
risk assessment behaviors, like thigmotaxis (swimming next to
walls), stretching posture to observe, and different exploratory
distribution in threatening and safe situations are seen in situ-
ations where the risk is low.17 This can be observed in tests like
the open field, the elevated plus maze, and the light/dark box.
The light/dark test has gained behavioral validity for some
fish species (Cardinal-tetras Paracheirodon axelrodi; lambaris

1
Universidade Federal do Pará, Belém, Brazil.
2
Universidade Federal do Amapá, Macapá, Brazil.

1
2 MANSUR ET AL.
Astyanax altiparanae; Nile tilapias Oreochromis niloticus;
guppies Poecilia reticulata; and banded-knife fish Gymnotus
carapo), including zebrafish.6 To establish pharmacological
validity, the light/dark test with the Danio rerio8 species used
several drugs, such as lysergic acid diethylamide (LSD),18
acute or chronic fluoxetine, buspirone, benzodiazepines, mo-
clobemide, caffeine, ethanol, and dizocilpine.19 Other vari-
ables, like the effects of the forced exposition to a white
chamber, lightness level (250 and 500 lux),20 effects of alarm
substance,21 methylmercury,22 and the effects of circadian
cycle,23 have also been tested. The results have altogether
demonstrated that this method can be useful to assess anxiety-
like behaviors in Danio rerio, showing consistency in the dark
preference behavioral pattern, which is responsive to test
manipulations. Such findings render the model a large face, use
and predictive validity as an animal model of anxiety for this
species.
Shoal
Zebrafish are highly social and prefer swimming in groups.
Social cohesion in zebrafish is exhibited through social in-
teractions, shoaling, boldness, aggression, and social prefer-
ence.24,25 Being part of a group helps to avoid predators, to
travel efficiently, to acquire food,26 and to reproduce more
successfully.27 According to Spence et al.,4 forming schools
is a natural zebrafish behavior and shoaling initiates subse-
quently to the eggs hatching.1 In addition, fish created in
isolation quickly form schools when placed in the company
of other fish28 and show preference for joining others similar
to the ones with which they were raised, even if they have
different phenotypes, just like in an imprinting phenomenon
of social preference acquisition during early development.29
The shoaling behavior increases in threatening situations
and in response to a pheromone released as a result of injury
to epidermal cells.1,30–32 This shows that shoaling is relevant
in predator avoidance. According to Rehnberg and Smith,33
in the presence of threat there is greater group cohesion, with
agitated swimming or subject’s immobilization close to the
substrate. These behaviors increase the individual’s survival
probability since fish schools are more likely to detect
predators than an isolated fish.34
The shoaling behavior seems to have reinforcing proper-
ties,29 reducing anxiety and stress in fish. This is evident in the
effects of different drugs in behavioral tests. In shoaling tests,
in which the distance between conspecifics are measured,
zebrafish exposed to anxiolytic substances, like LSD18 and
alcohol,35 had the distance between subjects altered. Alcohol is
a stimulating substance in lower doses and an inhibitor in
higher doses. As expected, alcohol reduced the distance be-
tween subjects in lower doses and increased in higher doses.35
LSD was proven to enhance inter-fish distance in this test.18
Meanwhile, the alarm substance, a substance known for its
anxiogenic and stressor effects, has reduced the distance be-
tween subjects.31 Altogether, these results suggest that shoal-
ing behavior reduces anxiety and stress responses in zebrafish,
since social cohesion is an indicator of anxiety.31
Computational simulations in laboratory have been suc-
cessful in forecasting migratory movements in groups and
schools movements based on temperature, salinity, and other
ecological variables.36–38 According to Romey,39 the study of
schools can be done in two ways. One way is to use models of
population dynamics that investigate the size and the group’s
displacement as a whole among the environment. Another
way is to use behavioral models, which investigates the
movement of individuals within the group. This study helps
to elucidate how light variations play a vital role in computer
algorithms/simulations that predict movement.
The factors that contribute to the animal’s decision to initiate
a movement include momentary contingencies and variables,
such as hunger, exposure to predators and temperature, as well
as durable and morphological variables, which include size,
age, lipid levels, parasites, and genetic predisposition.40–46 The
position of each individual in schools, seems to be due to the
relative position of other group members, in an attraction and
repulsion chain, in which an individual is attracted to conspe-
cific members, unless they get too close, and if so, turning
away.39
Behavioral models are used to gather information about
behavior. Moreover, they need to better detect and point out
the changes in brain and behavior. With the study of the light/
dark preference test applied to groups, we expect to verify the
influence of conspecifics on the behavior of isolated indi-
vidual group members. In the absence of significant change in
behavior, group tests become an alternative to streamline and
simplify data collection and test analysis.
Objective
This research aims to describe and analyze the effects of a
variable number of Danio rerio subjects, ranging from one to
eight, on the light/dark preference test. To develop a more
efficient data collection and to achieve an increased
throughput test, we will also verify the feasibility of running
the light/dark test with more than one subject.
Materials and Methods
Subjects
Four hundred eighty adult male short-finned phenotype
zebrafish were acquired from a local pet shop and only ex-
perimentally naive fish were used in the experiments. They
were kept in the laboratory for at least 2 weeks before the
experiments, housed in two collective 30 L tanks (240 sub-
jects per tank), measuring 32 · 34 · 67 cm, with controlled
light exposure (12-h light–12-h dark, starting at 6:30 a.m.).
The average pH was 6.8 and average temperature was held
constant at 24C – 2C. The fish were fed once a day with
chow (Color Tetra Tetra GmbH). The experiment was con-
ducted in the Laboratory of Neuroscience and Behavior at the
Center of Theory and Research of Behavior, Federal Uni-
versity of Pará and the housing conditions met the APA
Guidelines for Ethical Conduct in the Care and Use of Ani-
mals. The experimental procedures were approved by the
ethics committee of UFPA (CEPAE: 138-13).
Test apparatus and general procedure
All the experiments are conducted in rectangular acrylic
opaque tanks (black/white boxes), half side black and half
side white, each side measuring 15 · 10 · 45 cm, with water
column of 10 cm height. The central compartment where the
fish are placed measures 15 · 10 · 10 cm and it is delimited by
sliding doors that can be pulled up. The sliding doors have the
same color and material as the aquarium, one side is black
NUMBER OF SUBJECTS ALTERS DARK/LIGHT PREFERENCE? 3

and the other white. Two lamps illuminate the apparatuses,
each of them cautiously positioned one meter above the ex-
tremities of the aquarium to keep a uniform illumination,
avoiding shadow formation. After each session, the tanks are
rotated 180 to prevent effects of spatial orientation (Fig. 1).
Procedure
The first subject is placed in the central compartment of the
black/white box for a 5-min period (300 s) of habituation, to
diminish effects of manipulation of the subjects. After this
period the sliding doors are carefully removed at the same
time and the subject can freely explore both compartments of
the tank. The subject’s behavior is recorded with a video
camera during 15 min (900 s). Afterward, the video files were
analyzed and the occurrence of subjects on each side of the
aquarium was registered every 15 s, resulting in 60 occur-
rences per session. Furthermore, the first side chosen by each
subject was also recorded.
There were four groups in this experiment and a different
number of subjects was used in each group: the control group
had only one subject inside the apparatus during the test,
whereas the experimental groups had 2, 4, or 8 subjects
placed simultaneously inside the apparatus on every session
(As shown on Table 1). Each group was tested 32 times. To
control day-to-day variations, each group had three sessions a
day, totaling 12 sessions per day. All group types were tested
each day. The tests were always performed between 8 and 11
a.m. and the subjects were not used in more than one test. No
animals were hurt or sacrificed as a result of this experiment.
Measurement methods
The total time spent by each subject on both sides of the
tank is the main standard variable used to analyze dark/light
preference. In this study the number of occurrence of subjects
on each side of the aquarium was registered every 15 s. To
validate this method, 16 sessions of the control group were
analyzed using both techniques (total time and number of
occurrences) for further comparison.
Statistical analysis
To verify if the dark/light preference pattern changes with
the number of subjects in the tank, the average occurrence
Table 1. Subjects Per Session Shows How Many
Subjects Were Included in the Apparatus on Each
Session; Subjects Per Group Shows the Total Number
of Subjects on Each Group and Number of Sessions
Shows the Number of Sessions for Each Group
Subjects Subjects Number
per session per group of sessions
Ctrl 1 32 32
2 64 32
4 128 32
8 256 32
(AO) of subjects in each side of the aquarium and the first
choice average was calculated and compared between
groups. Since normality was not assumed for the data, the
one-way analysis of variance Kruskal–Wallis test was used
followed by Dunn’s post test. A Pearson correlation test was
performed to determine if measuring the number of occur-
rences every 15 s is as accurate as measuring the total time of
permanence.
Results
Validation of measurement methods
Preliminary descriptive statistics was run to compare the
average total time spent by subjects in the white side of the
apparatus and the average number of occurrences of subjects
in the white side of the apparatus every 15 s, revealing similar
results (as shown in Table 2 and Fig. 2).
The Pearson correlation test comparing the two analysis
methods was statistically significant, with a p-value of
0.000000000858. Furthermore, a very strong positive corre-
lation coefficient r = 0.968 was found.
Dark preference
After data collection and transcription it was possible to
assess and compare the AO of subjects in the white side of the
apparatus in each group. The Kruskal–Wallis test revealed
significant variation between groups [H(3) = 18.838,
p = 0.001] and the Dunn’s post test showed that the test group
with eight subjects differs significantly from the control
group and from the test group with two subjects (Fig. 3).
A linear regression test was conducted to verify whether
the AO of subjects has a linear relationship between groups.
The linear regression test has shown a slightly positive linear
relationship. R = 0.370; R2 = 0.137; Coefficient = 2.753;
Standard Error = 0.615; t = 4.474; P = 0.001 (Fig. 4).

Table 2. Descriptive Statistics Comparing

the Techniques of Registering Behavior
(Total Time and Number of Occurrences)
Standard Standard
Variable n Mean deviation error
Total time 16 17,264 13,478 3369
Number of 16 17,111 14,228 3557
occurrences
FIG. 1. Dark/light box setup.
4 MANSUR ET AL.
FIG. 2. Descriptive statistics graphic comparing the aver-
age percentages of total time spent in the white side and
average occurrence of subjects in the white side on every 15 s.
First choice
It was also assessed and compared the average of subjects
that have first chosen the white side of the apparatus within
each group. The Kruskal–Wallis test revealed significant
variation between groups [H(3) = 10.167, p = 0.017] and the
Dunn’s post test showed difference between control and the
group with eight subjects (Fig. 5).
A linear regression test was conducted to verify if the av-
erage of subjects that first chose the white side of the apparatus
has a linear relationship between groups. The linear regression
test did not show any positive or negative relationship between
these variables. R = 0.0416; R2 = 0.00173; Coefficient = 0.364;
Standard Error = 0.779; t = 0.468; P = 0.641 (Fig. 6).
Discussion
The data indicates no significant differences in the light/dark
preference test when carried out with as many as four subjects,
FIG. 3. Average occurrence (plus standard error) of sub-
jects in the white side of the apparatus measured on every
15 s. **Differs from control and from group 2, assuming
that p < 0.05. The average occurrence is calculated for each
group adding up the percentage of occurrence of subjects in
the white side of all sessions (32 per group) and then di-
viding the result by the number of sessions (32).
FIG. 4. Linear regression line and data points of the av-
erage occurrence of subjects in the white side of the appa-
ratus.
meanwhile, the test group with eight subjects showed statis-
tical differences, when compared with the control group and
the test group with two subjects. This indicates that the light/
dark test may be done with up to four subjects in the same tank,
during the same session, without affecting the light/dark
preference behavior of individual subjects.
The white side of the apparatus is an aversive stimulus to
the fish. Given the significant results obtained with the eight
subjects group, it is possible to deduce that the shoaling be-
havior reduces stress and anxiety.31 In the light/dark box
experiment with more than one subject, there are some
competing stimuli that induce the animal to move to the white
side or to the dark side. The animal’s natural tendency to seek
protection in dark places, avoiding bright environments, and
the anxiety caused by new environments contribute to the
preference for dark places. On the other hand, the innate drive
to explore new places added to a possible anxiolytic effect
caused by the presence of other subjects inside the apparatus
contributed to the subject’s displacement to the white side.
Zebrafish are a highly social animal, exhibiting preference for
swimming in groups and many other patterns of social co-
hesion. The reduced white avoidance behavior in the group of
eight subjects reflects the role of shoaling, which is a defense
FIG. 5. Average of subjects (plus standard error) that have
first chosen the white side of the apparatus. *Differs from
control, assuming that p < 0.05. The average occurrence is
calculated for each group adding up the subjects that have
first chosen the white side within the 32 sessions and then
dividing this number by the number of sessions.
NUMBER OF SUBJECTS ALTERS DARK/LIGHT PREFERENCE?
FIG. 6. Linear regression line and data points of the av-
erage of subjects that have first chosen the white side of the
apparatus. The numbers beside the dots represent frequency
of the events.
mechanism, in reducing anxiety and stress. However, the test
results show that four subjects inside the apparatus is not
enough to cause the anxiolytic effects of shoaling in the light/
dark test.
Figure 4 shows that the regression line has a mild positive
tendency for the AO of subjects in the white side of the ap-
paratus; it might reflect a slight decrease in white avoidance
even in the group of four subjects. In the novel tank test, for
example, four subjects are enough to form a shoal with con-
sistent performance in the test.47 Perhaps only four subjects in
a school is not enough to elicit all the benefits observed in
larger schools, such as food location, efficient displacement,
food acquisition, reproductive improvement and, particularly,
predator detection and avoidance. It reflects the subject’s
performance on the test, since there is no significant reduction
in white avoidance behavior in the groups with two and four
subjects. These findings support future test with more than one
subject, since shoal cohesion remains stable and practically
unaltered and only stimuli of considerable magnitude are able
to alter its natural condition.48 Therefore, the subjects are
prone to behave accordingly to the scototaxis behavior.
The variables commonly measured in the light/dark box
test are the total time in each side of the box; number of
midline crossings; latency for the first choice; first choice of
compartment and thigmotaxis. The measurement of the time
of permanence is related to dark preference/white avoidance
behavior and can be used as a measure of anxiety, whereas
thigmotaxis and first choice are measures of locomotor be-
havior and fear.6,8 From the above mentioned variables, this
research tested only for first choice behavior. The total time
of permanence of subjects in each side of the apparatus would
be a very difficult task to perform, since there were too many
subjects being observed at the same moment. Therefore, in-
stead of the total time of permanence of each subject in each
side of the apparatus, this research measured the occurrence
of subjects in each side of the apparatus every 15 s. This
measure has proven to be efficient and just as reliable as the
measurement of total time (as shown in Table 2 and Fig. 2).
Accordingly to this, the present model with enhanced num-
bers of subjects has the potential to provide faster outcomes
and new possibilities for the light/dark box test. However,
researchers are advised to consider the limitations of this
method. Although faster and more convenient, in this varia-
5
tion of the test, it would be difficult to measure the total time
of permanence and thigmotaxis.
The results of the research may also have been influenced by
two outside variables, social behavior and/or shoaling, and the
preference for dark places. Social behavior is presented by
the Danio rerio species, which has an established hierarchy in
the shoal, where the subordinated subjects follow the dominant
subjects. In this study, fish of Danio rerio species were placed
to swim in a 15 · 10 · 45 cm dimensions light/dark box with a
10 cm water column, containing 4.5 L. According to Aoki,49
there is a regular distance between individuals in a shoal,
which rises due to the maintenance of a private space for each
individual. Our tests were made successfully with up to four
subjects in the same apparatus. The inefficacy in the test with
eight subjects was possibly due to the lack of space inside the
test aquarium that lead the researchers to investigate whether
the scototaxic behavior is altered or supplanted by a tendency
to maintain the distance between subjects. According to
Westerfield,50 zebrafish is a shoaling fish and can be main-
tained at high concentrations such as 20 subjects per 3 L of
water at 3 months of age. In our experiment the highest con-
centration of fish was eight subjects per 6.75 m3 of water.
However, studies using light/dark boxes of different sizes and
with different water volumes should be further conducted.
The test still needs to undergo pharmacological validation
to verify the effectiveness of utilizing more than one subject
in the presence of drugs, provided that some drugs can
modify both fish and shoal behavior,47,51 resulting in cogni-
tive impairments, lowering or altering social behavior. It is
possible that such deficit in the subject’s ability to follow
conspecifics may harm the group’s movement cohesion,
causing the absence of shoaling behavior. Nevertheless, even
without this kind of behavior, it is likely that the results in the
light/dark test remain unmodified in the presence of any
drugs, if the fish scototaxis behavior remains intact, because
the white avoidance behavior would most likely keep the
subjects in the dark side. This study does not attempt to de-
termine if the results are due to social behavior or scototaxis,
therefore, further studies with anxiolytic drugs are necessary
to investigate this matter.
Conclusion
The reduced white avoidance behavior in the group of
eight subjects seems to reflect the role of shoaling, which is a
defense mechanism in reducing anxiety and stress. On the
other hand, the absence of significant difference between the
control group and the test groups with two and four subjects
suggest feasibility to run the light/dark test with up to four
subjects, becoming an alternative to streamline and to sim-
plify the data collection and test analysis. Pharmacological
tests are still required to determine whether the effect of drugs
alters the model with enhanced number of subjects.
Acknowledgments
This research was supported by grants from CAPES (Co-
ordination for the Improvement of Higher Level or Education
Personnel).
Disclosure Statement
No competing financial interests exist.
6 MANSUR ET AL.
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