The three-spined stickleback as a model for behavioural neuroscience
The three-spined stickleback as a model for behavioural neuroscience
William H. J. Norton 0 1
He? ctor Carre?o Guti e?rrezID 0 1
0 Editor: Stephan C.F. Neuhauss, University Zu ?rich , SWITZERLAND
1 Department of Neuroscience , Psychology and Behaviour , College of Life Sciences, University of Leicester , Leicester , United Kingdom
The three-spined stickleback (Gasterosteus aculeatus) is a small teleost fish that is ubiquitous across the Northern Hemisphere. Among the behaviours that have been characterised in this species is ritualized courtship, aggressiveness and parental behaviour. Whereas three-spined sticklebacks have been used for ecological, evolutionary, parasitological and toxicological research, its complex behavioural repertoire and experimental advantages have not been exploited for basic neuroscience research. The aim of the present study is to describe some innate behaviours of laboratory bred three-spined sticklebacks by using a battery of tests that have been developed and validated to model some aspects of human psychiatric disorders in zebrafish. We recorded mirror induced aggression, novel object boldness, shoaling, and anxiety-like behaviour using both the novel tank diving and the black-white preference tests. We show that behaviour of three-spined sticklebacks in these standard tests is remarkably similar to that of zebrafish and other species and can be altered by fluoxetine and buspirone. These findings highlight the potential of using three-spined sticklebacks for cross-species and translational studies.
Data Availability Statement; All relevant data are within the manuscript
Funding: This research received funding by the
National Centre for the Replacement, Refinement
and Reduction of Animals in Research under grant
agreement no NC/R001049/1. The funder had no
role in study design, data collection and analysis,
decision to publish, or preparation of the
Competing interests: The authors have declared
that no competing interests exist.
The three-spined stickleback (Gasterosteus aculeatus) is a small teleost fish that is ubiquitous
across the Northern Hemisphere. Originally from marine environments, they colonised diverse
fresh water systems thousands of years ago, which has made them ideal species to study
evolutionary biology, adaptation and ecology [
]. Three-spined stickleback are easy to find in rivers,
lakes and ponds as well as coastal regions. They are also easy to breed and maintain in the lab at
low cost and the genome sequence is available (www.ensembl.org). The ethology of this species
was extensively studied during the 20th century thanks to the pioneering work of Nico
]. Three-spined stickleback have also been used to study behavioural syndromes such as
aggression-boldness and their implication for evolution [
]. Among the behaviours better
characterised in this species are ritualized courtship, aggressiveness and parental behaviour.
Sexually mature males develop a bright red throat and fore-belly called nuptial coloration.
During the breeding season territorial males become aggressive and dominant. A male carefully
builds a nest which he actively protects from conspecifics and also other species. When two
males encounter they display several agonistic behaviours, including circling with the spines
erected, charges and chases, bites to the opponents? body and the eventual flee of the
subordinate . If a ripe female with a prominent silver belly approaches the male?s territory courtship
proceeds following a chain of events. After a zig-zagging dance, the male leads the female to the
nest and he shows the position of the entrance. Once the female is inside the nest the male
stimulates spawning by quivering the female?s tail. After spawning the female leaves the nest and the
male quickly fertilises the eggs [
]. When several clutches have been collected the parental phase
starts. The male stays close to the nest to look after the offspring, fanning the eggs and
protecting the newly hatched fish for several days [
One of the experimental advantages of these animals over other fish species such as
zebrafish is the ease of tagging individuals and measuring consistent individual differences in
behaviour over time and across different contexts [
]. In addition to inter-individual variation
within a population, differences in behaviour have been reported between populations from
different habitats. Differences in predation pressure in different environments affect
physiological and behavioural responses [
]. Marine and benthic populations differ in schooling
], and fish from streams and ponds show differences in boldness [
the presence of water pollutants such as endocrine disruptors has an impact on behaviour [
Parasitism is another well-described source of variation in behavioural responses.
Threespined sticklebacks are the intermediate host of the tape-worm Schistocephalus solidus.
Infected fish become bolder and therefore more likely to be eaten be predators allowing the
parasite to complete its life cycle [
]. Uninfected fish also differ in their responses to
predators, either real or simulated, with some individuals taking more risks than others .
Some of these behavioural studies have used lab bred animals, whereas the majority of them
used fish caught from natural populations in harbours or freshwater systems. These fish are
transferred to the lab where they are kept under controlled conditions that vary, with
temperature and day-night cycle simulating natural conditions or adjusted as the study requires.
Housing conditions also differ as fish are kept in different types of tanks with different kinds of
enrichment. Some studies have been carried out using groups of fish, and others on single
previously-isolated individuals. In some experiments, sexually mature adult fish have been tested
(usually males) whereas in other cases sub-adults or fry have been used. The different methods
and tests used by different labs have been described in great detail in scientific publications.
However, there is a need for standardisation of procedures and behavioural tests in order to
compare results across labs and design future studies based upon previous findings. Whereas
three-spined sticklebacks have successfully been used for ecological, evolutionary,
parasitological and toxicological research, its complex behavioural repertoire and experimental advantages
have not been exploited in basic neuroscience research. This species has the potential to be
used alongside other popular models to gain insight into the mechanisms underlying some
diseases affecting behaviour. Studying the behaviour of the three-spined stickleback in standard
tests that are relevant for human diseases, with good face and predictive validity, would be a
starting point for cross-species and translational studies.
The aim of the present study is to describe some innate behaviours of laboratory bred
three-spined sticklebacks by using a battery of tests that have been developed to model some
aspects of human psychiatric disorders in zebrafish [
]. We have performed assays to
measure mirror-induced aggression, novel object boldness, shoaling and anxiety-like
behaviour, taking advantage of automated video tracking to generate the data. We discuss the
similarities and differences in behaviour of the animals tested in our study with that reported by
previous studies using similar paradigms. We also compare behaviour of sticklebacks and
zebrafish highlighting some striking similarities.
2 / 18
Materials and methods
Animals and husbandry
In the present study we used three-spined sticklebacks (Gasterosteus aculeatus) that were the F3
of a freshwater population captured in the river Soar (Leicester, Leicestershire, UK) in 2014. The
fish used in this study were randomly taken from a large population obtained by in vitro
fertilisation in July 2017. Forty fish were housed in a 27 L tank on a circulating system (Xenoplus
systems, Techniplast) in a dedicated fish facility at the University of Leicester. Flow rate was set at 2
tank changes an hour. Fish were reared under temperature and light-dark conditions simulating
natural seasonal variation until March 2018 (S1 Table). From this point fish were kept in
nonbreeding conditions at 12 ? 1?C on a 12:12 h light:dark cycle (i.e. March conditions) for the
duration of the experiments, that were conducted in June 2018. This permitted us to measure
behaviour in non-reproductive individuals to avoid the confounding factors associated with
territorial and courtship behaviours. As the fish we used had not reached sexual maturity males
and females were visually identical. One week before the behavioural recordings fish were
randomly caught and split in three 13.4 L tanks: two tanks containing 15 fish each and one tank
containing 10 fish. These tanks were located in the central shelf of a large rack, so that all three
received similar illumination and were surrounded by other tanks on the sides. No enrichment
was provided. Fish were fed daily ad libitum with defrosted Chironomid larvae each afternoon
at the end of the experiments. Fish were kept in accordance with institute guidelines for animal
welfare. All work was conducted under a UK Home Office licence and was approved by a local
Animal Welfare and Ethical Review Body (AWERB) committee at the University of Leicester.
The behavioural experiments were conducted inside the fish facility to avoid the potential stress
of transferring the animals into a different room. Recordings were conducted between 10:00
and 16:00 to minimise possible diel influences. The 13.4 L housing tank containing the 15 fish
designated as the control group was taken out of the system and placed onto a trolley for 30 min
before the experiments began. Single fish were netted and placed into the testing tank. After the
experiment fish were collected in an intermediate tank and then placed back into the same 13.4
L housing tank and retested for a different behaviour 2 days later. Drug treatments were applied
to fish located in the other two 13.4 L housing tanks. The one with 15 fish was used for
buspirone treatment and the one with 10 fish for fluoxetine treatment. Fluoxetine hydrochloride (Cat.
No. F4780, LKT Laboratories) was applied by immersion at a concentration of 5 mg/L for 3
hours before measuring behaviour [
]. Buspirone hydrochloride (Cat. No. 0962/100,
Tocris) was applied by immersion at a concentration of 25 ?M for 30 min before measuring
behaviour . The fish treated with either fluoxetine or buspirone were not reused for other
tests to avoid any potential alteration of the brain neurochemistry that could affect behaviour.
The number of sticklebacks tested (n = 10?15) was calculated using power analysis based upon
pilot experiments that we carried out in three-spined sticklebacks and published studies in
]. Recordings were performed using FlyCapture2 126.96.36.199 software and two digital
cameras from Point Grey Research. Ethovision XT8 video tracking software (Noldus) and were
used to analyse most of the behavioural endpoints. The latency to the first attack, number of
attacks, time spent biting, time spent freezing at the bottom of a novel tank, time spent in the
white compartment and number of crosses in a black-white preference test were manually
scored after replaying the videos in Windows Media Player (Microsoft). To avoid any bias, the
videos were renamed and manually scored in a blind manner. We conducted the following
experiments using tests and paradigms that have been established for zebrafish research.
3 / 18
Adult stickleback aggression was measured using mirror-induced stimulation as described for
zebrafish in [
]. Single fish were placed in the middle of a glass tank (30 ? 10 ? 15 cm, L ? W
? H) filled with 10 cm water depth and immediately recorded from above for 5 min. Three
walls of this setup were covered in a white opaque material. A mirror offset at an angle of 22.5?
was placed outside the transparent wall to elicit aggression. One of the control fish got trapped
in the net when it was being transferred into the testing tank and so we discarded it, meaning
that we used 14 control fish and 10 fluoxetine treated fish. The time biting or pushing against
the mirror and thrashing the tail fin was manually scored as time spent biting. We also
manually quantified the latency to the first bite and the number of aggressive attacks as the number
of times the fish approached the mirror from a distance larger than one body length to bite it.
The total distance swum was quantified with Ethovision. Ten sub-adult fish of 3 months of age
were simultaneously tested in the setup described in [
]. Single fish were placed in small
tanks (9 ? 4 ? 4 cm, L ? W ? H) arranged in groups of four with three walls opaque and the
fourth transparent to allow the interaction with the mirrors. Locomotion was also measured in
this setup by covering the mirrors with an opaque divider. Fish were recorded from the top for
5 min and the analysis was automatically obtained with an extension of ZebraLab software
(VpCore2) developed by ViewPoint Behavior Technology. Aggression and locomotion are
expressed as aggression units and locomotion units. These are arbitrary units that correlate
well with time spent in aggressive display (i.e. directing bites to the mirror image and thrashing
the tail, quantified manually) and with total distance swum (quantified using Ethovision) as
described for juvenile zebrafish in [
]. We also measured aggression manually and calculated
its correlation with the automatic score.
Novel object boldness
Novel-object boldness was measured using the setup described in [
]. The testing tank
(40 ? 25 ? 15 cm, L ? W ? H) had a 15 ml Falcon tube filled with dark blue and yellow
modelling clay suspended midway in the water column at one end used as the object. This setup was
filled with 10 cm of water and the walls were surrounded by white opaque material to
minimise reflection. Single fish were placed into the setup and recorded from the top for 5 min. We
used Ethovision to quantify the time spent within one body length of the novel object, the
latency to the first approach, and the total distance swum. We used 15 fish.
Shoaling was measured in 6 groups of 5 individuals swimming in a large opaque plastic tank
(80 ? 60 ? 60 cm, L ? W ? H) filled with water to a depth of 10 cm. A group was placed in the
tank a left to explore the tank for 20 min. After this period of habituation the fish were
recorded from the top for 10 min. The nearest neighbour and inter-individual distances were
measured using VpCore2 (ViewPoint Life Sciences).
Novel tank diving test (NTT)
We performed the NTT to measure anxiety-like behaviour in a standard 3.5 L trapezoid tank
(27.9 ? 22.5 ? 11.5 ? 15 cm, L (top) ? L (bottom) ? W ? H) similar to the 1.5 L trapezoid
widely used for zebrafish [
]. The only difference was that our tank was slightly wider
accounting for the larger size of sticklebacks. Single fish were placed in this setup and recorded
from the side for 5 minutes. The tank was divided in three zones?bottom, middle and top?for
the analysis of the videos with Ethovision. We automatically measured the amount of time
4 / 18
spent on the top third of the tank and total distance swum. We manually quantified freezing as
cessation of swimming while at the bottom of the tank, as three-spined sticklebacks can hover
in the middle of the water column. We used 15 fish as controls and 15 fish treated with
Black-white preference test
This setup consisted of a transparent plastic tank (30 ? 20 ? 12 cm, L ? W ? H) with the
bottom and walls surrounded by two removable opaque plastic covers that divided the tank into
two zones: black and white. This set is also used for zebrafish behavioural studies [
water depth was 10 cm. Single fish were transferred into the white half and recorded from
above for 5 min. We manually quantified the total time spent in the white zone and the
number of crosses from the black to the white zone. We considered a cross when the pectoral fins
were in the white half of the tank. We used 15 fish as controls and 15 fish treated with
Data from Ethovision and manual analysis were collected in Excel (Microsoft) and statistical
analyses were carried out with GraphPad Prism7. The graphs show the mean and the standard
error of the mean. Each dot represents one fish. Data were assessed for normality using
D?Agostino & Pearson normality test. Homocedasticity was tested using Levene?s test. We
used unpaired Student?s t-tests or Mann Whitney U tests as appropriate for comparison of
drug treated fish and controls. We also used the Pearson?s correlation coefficient. The
significance level was set at 5%.
We tested aggression in 10 sub-adult fish of 3 months of age in the setup described in [
where multiple individual can be recorded simultaneously in small arenas increasing the
throughput of the experiment (Fig 1A). This setup is connected to software that automatically
quantifies aggression. All the fish interacted with their own mirror image and displayed
aggressive postures. The average aggression levels were 289.6 ? 65.66 aggression units (Fig 1B).
The manual quantification of these videos revealed a good correlation with the values obtained
by the software (Pearson?s r: 0.8259, p = 0.0032), yielding 100.71 ? 21.33 s, which is very similar
to the values obtained in adults (Fig 1G). The software also measures the distance swum by the
fish in the testing tanks before the mirror is presented as a readout of general locomotor
activity (Fig 1C). This protocol to measure aggression automatically in young fish can be used to
screen for different drugs that modify this behaviour [
We used the mirror-induced aggression test to quantify aggressive behaviour of adult fish
in the mirror induced aggression test as described in [
] (Fig 1D). When single fish were
placed in the testing tank some on them immediately approached the mirror and started biting
and chasing their own reflection (Fig 1E). Although a few fish had a higher latency to
approach, all of the 14 fish tested responded to their own mirror image attacking as if there
was an opponent present. The repeated biting was accompanied by pushes towards the mirror
and thrashes of the caudal fin resulting in undulating body movements. The total duration of
this aggressive display was 108.2 ? 11.85 s (mean ? SEM) (Fig 1F), split into several attacks
alternating with exploration of the rest of the tank. From the middle and back of the tank fish
directed an average of 10.21 ? 1.17 short fast attacks to the mirror that ended with a bite
5 / 18
Fig 1. Aggression test. (a) Set-up to measure mirror induced aggression in sub-adult fish. (b) Aggression units and (c) locomotion units measured in 3 month
old fish. (d) Mirror induced aggression test for adult fish. (e) Latency to first bite, (f) total time spent biting, (g) number of attacks, (h) time spent near the mirror,
(i) time spent biting when swimming near the mirror and (j) total distance swum by control and fluoxetine treated fish in this set-up. p < 0.01, p < 0.001,
p < 0.0001.
towards its own reflection (Fig 1G). Aggression can be modulated by drugs that interact with
5-HT neurotransmitter signalling in zebrafish [
]. The selective 5-HT reuptake inhibitor
fluoxetine decreases aggression in this species [
]. We treated three-spined sticklebacks with
this drug and recorded their behaviour in the mirror induced aggression test hypothesizing
that it would decrease aggression. Fluoxetine did not alter the latency to the first bite (Mann
Whitney test: U = 70, p > 0.9999; Fig 1E) or the total time spent biting the mirror and
thrashing the tail (Student?s t-test: t(22) = 1.717, p = 0.1000; Fig 1F). However, the number of attacks
was reduced in comparison with the control untreated fish (Mann Whitney test: U = 1.5,
p < 0.0001; Fig 1G). Interestingly, fluoxetine treated fish spent more time near the mirror (i.e.
within on body length) than control fish (Mann Whitney test: U = 18, p = 0.0014; Fig 1H).
However, the percentage of time spent displaying overt aggression when swimming near the
mirror was reduced in the fluoxetine treated group (Student?s t-test: t(22) = 4.178, p = 0.0004;
6 / 18
Fig 1I) suggesting a decrease in aggression. The total distance swum was also smaller upon
fluoxetine treatment (Student?s t-test: t(22) = 4.027, p = 0.0006; Fig 1J).
Novel object boldness
We tested 15 fish in the setup described in [
] where a 15 ml centrifuge tube filled with clay is
suspended in the water at one side of a tank (Fig 2A). The latency to first approach the novel
object was 32.17 ? 9.143 s (Fig 2B) and was followed by an average of 6 ? 0.676 approaches
(Fig 2C). The total time spent within 1 body length of the object was 39.99 ? 5.071 s in a 5 min
experiment (Fig 2D). The time spent near the novel object could be a result of random
exploratory activity across the whole tank, with more active fish swimming more time close to the
object, without a specific motivation (i.e. novel object approach or avoidance). We measured
the total distance swum in the testing tank (1012 ? 72.35 cm (Fig 2E)) and calculated the
correlation between general locomotor activity and time spent close to the object. Interestingly,
there was no correlation between these two parameters (Pearson?s r = 0.1183, p = 0.6745).
There was also no correlation between the time spent near the object and the time spent in an
area opposite the object (Pearson?s r = -0.0244, p = 0.9312).
Several studies have quantified the tendency of a focal three-spined stickleback to approach
and stay close to a group of conspecifics placed behind a transparent barrier in different
]. However, to our knowledge there are no studies measuring shoaling in free
swimming sticklebacks by calculating the distances between the members of the group.
Nearest neighbour (NND) and inter-individual (IID) distances are two measures of shoal cohesion.
Fig 2. Boldness and shoaling tests. (a) Novel object boldness test. (b) Latency to first approach to the novel object (b), number of inspections (c), total time
spent close to the object (d) and total distance swum (e) by adult fish tested in this setup. (f) Shoaling test. (g,h) Nearest neighbour- and inter-individual distance
measured in this tank. Each dot on the scatter plots represents one fish.
7 / 18
We tested 6 shoals of 5 fish each placing them in a large tank (Fig 2F). After a 20 min
habituation period where the fish explored the tank we recorded from the top for 5 min. We used
ViewPoint software to automatically track the individuals and determine the distances
between them. The average NND was 7.965 ? 0.656 cm and remained stable over time (Fig
2G). The average IID was 28.32 ? 3.383 cm and was also quite stable (Fig 2H).
Novel tank diving test
We used the novel tank diving test to measure anxiety-like behaviour (Fig 3A). This set-up has
been extensively validated in zebrafish by applying drugs that are known anxiolytic or
anxiogenic. The first drug used was the 5-HT receptor 1A partial agonist buspirone, a well-known
anxiolytic compound which strongly reduces anxiety-like behaviour in zebrafish in a dose
dependent manner [
]. In a non-treated group of fish the NTT elicited a strong geotaxic
response, where fish immediately dived to the bottom third of the tank. Eleven out of 15 fish
showed freezing behaviour, which is a cessation of movement except for the opercula and the
mouth. The average duration of these freezing episodes was 91.04 ? 24.07 s (Fig 3B). When
fish were freezing they also erected their dorsal fins and bent their tails upwards in a J-shape.
After the initial bout of freezing, fish gradually explored of the top third of the tank. The total
time spent swimming on the top was 42.24 ? 15.15 s in a 5 min experiment (Fig 3C). Freezing
and geotaxis are indicators of stress responses and anxiety in a potentially threatening context.
Using video-tracking we can also automatically quantify the total distance swum (Fig 3D) as a
readout of exploratory activity. To test the face validity of this test in three-spined
sticklebacks?i.e. its ability to measure anxiety?we applied 25 ?M buspirone by immersion for 30
Fig 3. Novel tank diving and black white preference tests. (a) Novel tank diving setup. (b) Time spent on the top third of the tank, (c) freezing at the bottom
and (d) total distance swum in control and buspirone treated fish. (e) Black white preference test. (f) Number of crosses from black to white and (g) total time
spent in the white zone of control and buspirone treated fish in this set-up. Each dot on the scatter plots represents one fish. p < 0.05, p < 0.01,
p < 0.0001.
8 / 18
min. We hypothesised that this anxiolytic drug would cause a decrease in the anxiety-like
behaviour measured in this test. Only 4 out of 15 fish displayed freezing upon buspirone
treatment, a significant decrease compared to untreated fish (Mann-Whitney test: U = 54,
p = 0.0089; Fig 3B). Buspirone-treated fish spent 251.9 ? 16.13 swimming at the top of the
tank, which is a dramatic increase compared to the untreated fish (Mann-Whitney test: U = 5,
p < 0.0001; Fig 3C). The strong decrease in freezing and geotaxis indicate a sharp reduction of
anxiety-like behaviour. Total distance swum was slightly reduced (Mann-Whitney test:
U = 64, p = 0.0367; Fig 3D).
Black-white preference test
We further characterised anxiety-like behaviour using the black-white preference test (Fig
3E). This test has been used in some fish species, including zebrafish, to measure the
preference for a black zone when an individual experiences the conflict of choosing between black
or white [
]. It has also been validated with a number of drugs such as buspirone. We
placed individual fish in the white zone and measured the latency to enter the black zone,
the number of transitions from black to white and the time spent in the white zone. The
number of transitions was on average 6.33 ? 1.71 (Fig 3F). Most of these transitions were
short bouts of rapid swimming into the white side followed by a turn back into the black
side. However, some fish spent long periods of time in the white zone immobile. The total
time spent in the white zone was 35.65 ? 10.12 s (Fig 3G) indicating a strong preference for
staying in the black side or scototaxis, suggesting anxiety-like behaviour. We treated the fish
with 25 ?M buspirone in system water for 30 min reasoning that this could increase the
number of transitions into the white side or the amount of time spent in this area. However,
there were no differences between treated and untreated fish in the number of transitions
(Mann-Whitney test: U = 74; p = 0.1006; Fig 3F) or the time spent in the white zone
(MannWhitney test: U = 101, p = 0.6420; Fig 3G).
In this paper we have described the behaviour of laboratory bred three-spined sticklebacks
using paradigms that are relevant to model some aspects of human psychiatric disorders in
other species. We have described aggressive behaviour using the mirror-induced aggression
set-up. We have also tested novel object boldness and measured group cohesion within a
shoal. Finally, we measured anxiety-like behaviour using both the novel tank diving and the
black-white preference tests. All these tests have been validated for zebrafish, a popular model
for behavioural neuroscience, and can be used to measure behaviour across fish species.
Aggressive behaviour in territorial males has been documented decades ago. When a male
establishes its territory during the breeding season it defends its boundaries to keep intruders
away by chasing and biting them [
]. A similar aggressive response can be experimentally
elicited by placing an intruder confined in a transparent container into a tank occupied by a
territorial male [
]. An alternative approach to test fish aggressiveness is quantifying the
responses of an experimental fish to its own mirror image.
Mirror stimulation has been extensively used during the last decades in many species such
as the Siamese fighting fish [
], Astatotilapia burtoni [
], coho salmon [
], rainbow trout and guppies [
], Mozambique tilapia [
] and zebrafish [
Despite the biological differences between evolutionary and ecologically distant fishes, they all
display aggressive postures and direct bites to their mirror images indicating that aggression is
a highly conserved behaviour among teleost fishes. However, the differences in set-ups, scoring
9 / 18
methods and analysis reported in different studies makes it challenging to compare data
Some recent studies have already described mirror induced aggression in male three-spined
sticklebacks. A study by [
] showed that isolated territorial males that were actively building
their nests approached and attacked a mirror placed in their tanks. When individual fish were
placed in large tanks with three mirrors covering the walls they also displayed aggression,
although some fish did not respond to this stimuli [
]. Kellner and colleagues have also used
the approach of lowering a mirror into a tank to measure attack behaviour in adult females
]. In a later study they tested adults of both sexes prior to sexual maturation by lowering a
mirror into a tank occupied by single fish, although some fish did not discover the mirror and
were omitted [
We demonstrate here that the set-up routinely used in zebrafish research, with three opaque
walls and an inclined mirror outside the tank, is a suitable and reliable method to measure
three-spined stickleback aggression. The opaque walls in our setup ensure that all fish see the
mirror stimulus without the need of exploring a large area and the presence of other
confounding stimuli in the tank. Moreover, we have observed in our lab that when a mirror is presented
outside of a plastic transparent tank fish respond less to this stimulus and bite the walls instead
(unpublished observations). In our experiment all the individuals?sub-adults and
adultsresponded to the mirror stimulus by approaching and displaying attack behaviour.
The feasibility of testing sub-adult fish in this setup coupled to automatic quantification
] can be used to perform screens to test the effect of drugs or mutations on aggression in
both zebrafish and three-spined sticklebacks. When manually scoring adult fish we quantified
the total amount of time the fish spent in active aggressive display, biting and thrashing their
tail (i.e. time spent biting) [
]. We favour this method instead of counting individual bites or
attacks as previous studies have done as bites are difficult to quantify and attacks do not
represent the duration of the aggressive interaction. Zebrafish are more active and so they attack the
mirror more intensively, whereas three-spined sticklebacks swim slower, which affects the
intensity of the aggressive interaction, although we cannot quantify this at present. However,
the total time that our sticklebacks spent in aggressive display was slightly higher than those
reported in zebrafish studies [
When applied acutely, the specific 5-HT reuptake inhibitor fluoxetine reduces zebrafish
aggression both in the mirror test [
] and in a real opponent paradigm [
]. Acute treatment
with fluoxetine also reduces aggression directed against dummy fish [
] or to the mirror
] in male Siamese fighting fish. Both acute and chronic treatment with fluoxetine
also decreases aggression in the bluehead wrasse [
]. Thus, the decrease in aggression upon
fluoxetine treatment that we report in the present study is in line with previous findings in
other fishes and supports the role of this drug in controlling aggression. Other substances such
as deprenyl [
], ethanol [
] or lithium carbonate [
] also decrease aggression in zebrafish
and can be tested in three-spined sticklebacks. Understanding the genetic and pharmacological
basis of this behaviour in different species may shed light on conserved mechanisms
underpinning pathological levels of aggression.
Boldness is the propensity to engage in risk taking behaviour as opposed to shyness.
Aggression and boldness are key components of the aggression-boldness behavioural syndrome,
which was described in three-spined sticklebacks some decades ago [
]. Boldness has been
extensively characterised in sticklebacks using different paradigms. One approach is to test a
fish?s responses to the presence or the attack of a predator?either real or simulated [
this method, some studies have measured the latency to resume eating after an attack [
the amount of time freezing after an attack [
] or the number of inspections and orienting to
a predator [
]. The tendency to leave a shoal is also an indicator of boldness [
10 / 18
An alternative and easy to perform approach is to measure the response of a fish to a novel
object present in the tank. The behavioural endpoints used to score this type of behaviour
include recording the position of the fish 2.5 times per second for 10 minutes to obtain the
mean distance to a little statue [
]; manually recording the time spent freezing when a glass
flask is introduced into the tank [
]; measuring the distance to a plastic toy every 20 seconds
during 10 minutes [
]; or quantifying the time spent in 3 concentric circles surrounding a
plastic toy pig [
]. These methodologies are all based on manual quantification which implies
lengthy analysis and less accurate measurements than those obtained by video tracking.
Moreover, fish used in different studies were caught from different habitats (i.e. a freshwater pond, a
creek or a harbour) which adds potentially confounding factors since predator pressure in
different habitats may impact behavioural responses [
]. The different methodologies and
behavioural endpoints recorded, and the different populations studied, hamper the comparison of
results between different labs.
In this paper we have tested boldness as the amount of time spent near a novel object as
previously described in zebrafish studies [
]. Quantifying the actual time the fish spends near
the object by automated video tracking gives an accurate endpoint that is easy to compare
between laboratories. Furthermore, recording the total distance swum in this set up gives a
readout of the general exploratory activity of the fish in this test. The non-existent correlation
between the time spent near the object and the time spent in an area of equal size opposite to
the object suggests that the behaviour of the fish is driven by the object; bold fish spend more
time close to the object than in the empty area at the other end of the tank. In line with this,
the absence of a correlation between time spent near the object and total distance swum
suggests that exploration of the novel object is not simply due to higher locomotor activity in the
whole tank. The novel object boldness test can complement other tests used to characterise
Shoaling is the tendency of fish to form groups of conspecifics. This aggregation provides
benefits for their members such as reduced predation risk and increased foraging efficiency
and mating opportunities [
]. In three-spined sticklebacks this behaviour is typically
measured in tanks with three compartments separated by transparent walls, where a focal fish in
the centre shows preference to swim close to conspecifics located in one of the lateral
]. The same type of attraction is elicited if the conspecifics are confined in a
glass flask placed in the tank with the focal fish [
]. When a shoal is moving synchronously
and becomes polarized so that its members swim in the same direction it is named a school
. An ingenious paradigm has been implemented to manually quantify the tendency of a
focal three-spined stickleback fish to follow a group of model conspecifics that swim in circles
in a large tank forming a school [
]. However, these kind of tests do not actually measure
the cohesion of the group members and do not reflect the natural characteristics of a free
Nearest neighbour (NND) and inter-individual distance (IID) are two commonly used
parameters to measure social cohesion in zebrafish [
]. NND is the average of the
distance between any fish of the shoal and its closest neighbour. IID is the average of all the
distances between the members of a shoal. Whereas IID precisely estimates the cohesion of a
group as it considers all the distances it is affected by group size, and this must be accounted
for if groups of different sizes are compared. IID can also be substantially modified by a single
fish that leaves the shoal for some time. If a large group of fish splits into two or more
subgroups during the behavioural recordings this will affect the average IID; however the NND
will remain similar as long as there is cohesion in the subgroups. Therefore, these two
measures are complementary but not inter-changeable.
11 / 18
Both the NND and IID can be calculated using software that allows automated tracking of
multiple individuals. Miller and Gerlai developed the first kind of this software which opened
up the possibility of measuring different parameters to assess changes in shoal density over
]. Since then, different software and refined approaches are being created to
accurately measure the dynamics of a group. For example, Ethovision offers software to track
multiple fish , and more sophisticated tools such as idtracker (www.idtracker.es/idsocial) can
measure in great detail the interactions between fish swimming together whilst identifying
each individual within a group [
]. In the present study we have used ViewPoint software
(VpCore2) to measure NND and IID in groups of 5 fish swimming in a large tank. As
expected, both parameters can be obtained in three-spined sticklebacks. We observed that
sometimes one or two fish leave the group for a few seconds to explore the tank. In a group of
5 fish, when one individual leaves the group it alters 4 of the total 10 inter-individual distances
contributing to 40% of the IID. If two individuals leave, they will account for 70% of the IID.
This could explain the greater fluctuations in IID compared to the more stable NND.In
zebrafish, shoaling can be impaired by some drugs such as nicotine or ethanol , phencyclidine
(PCP), 3,4-methylenedioxymethamphetamine (MDMA), or reserpine [
], MK-801 [
], or scopolamine [
]. Both pharmacological manipulations and genetic studies
can potentially be implemented in three-spined sticklebacks, making these species a promising
tool for comparative and translational neuroscience.
Novel tank diving is a robust test used to measure anxiety-like behaviour in zebrafish
]. In this paradigm fish prefer to stay at the bottom of a novel tank and avoid the top,
which is perceived as threatening. A recent study measured bottom preference or geotaxis in
adult female three-spined sticklebacks captured in a harbour [
]. The novel tank had three
walls covered with black plastic and was divided into a bottom and top by a line. Behavioural
parameters such as time spent in the top half, latency to first entry on top, time spent freezing
and general locomotion were scored manually [
]. We suggest that there could be several
issues with this setup. First, the large dimensions of the testing tank are likely to elicit
horizontal exploratory swimming and thigmotaxis. A narrower tank would create a greater conflict
between swimming on the potentially threatening top versus staying in the safer bottom.
Second, the authors used a period of 20 min of habituation in a small container prior the
recording in the novel tank. This confinement is potentially stressful and could affect the response of
the fish by adding a stressor prior to testing. Moreover, we consider that the division of the
tank into thirds instead of halves gives more accurate results, since some anxious or stressed
individuals may cross the middle line and yet never swim on the top (close to the surface).
Recording behaviour in three areas increases the power to detect subtle differences in anxiety
between individuals. In line with this, video tracking of the fish would allow to better assess
locomotor activity and speed up the analysis of all the behavioural endpoints.
We performed the novel tank diving test in a narrow tank without previous habituation
and found a strong scape response. Fish immediately dived to the bottom where they swam
and froze. Bottom preference and freezing often co-vary and are indicators of stress reactivity
or anxiety-like behaviour. As the experiment progresses and fish become habituated to the
tank they tend to gradually spend more time in the top third, a response typically observed in
]. Although the swimming pattern differ in these two species the preference for
the bottom of the novel tank is remarkably similar. Three-spined sticklebacks tend to hover in
the middle of the tank and move vertically. However, freezing should be only scored as lack of
movement at the bottom of the tank (see Materials and Methods). Diving responses, bottom
dwelling and freezing have also been observed in other species such as guppies [
Endler guppies .
12 / 18
Zebrafish studies have demonstrated that anxiety-like behaviour is controlled by 5-HT.
Drugs targeting different components of the 5-HTergic pathway modulate behaviour in these
two tests [
]. One of these drugs is the 5-HT receptor 1a partial agonist buspirone, which
acts as anxiolytic in zebrafish decreasing geotaxis [
]. We have found that this drug
strongly decreases geotaxis in three-spined sticklebacks, and reduces both the time spent
freezing and the distance swam, which indicates a reduction of anxiety-like responses in agreement
with zebrafish studies.
Scototaxis is an innate behaviour that has been observed in some fishes including zebrafish
by using the black-white preference test [
]. Zebrafish strongly avoid the white compartment
and stay in the black side most of the time, although some differences in this behavioural
pattern have been reported in fishes from other taxa [
]. Scototaxis has been recently described
in adult three-spined sticklebacks of mixed, unknown age and sex, caught in a freshwater
]. In this study the authors used a grey open tank with two small white- and
blackbottomed areas at one end and counted the number of times a single fish entered into each.
Fish entered more often into the black area, demonstrating scototaxis in this species. However,
there are two weaknesses in this set-up. First, fish can also choose not to enter any of these
areas. Secondly, instantaneous recording of the number of counts every ten seconds does not
accurately inform about the actual time spent in each area. The same study measured
behaviour in a new combined diving and scototaxis test [
]. In this set-up there is a divider with a
gap between the white and black half, adding an extra conflict. Thus, this test measures
geotaxis, scototaxis, and also emergence and exploratory activity making the results difficult to
Whereas both the novel tank diving and the black-white preference tests are widely used to
measure anxiety in zebrafish, they are complementary and measure different aspects of
]. This could also be the case for three-spined sticklebacks and it is a possibility worth
investigating in future studies. Here we have used a black-white test in which fish have to
respond to the conflict between taking the risk of exploring the white half where they are more
exposed or hide in the black area where they can camouflage. The strong scototaxis response
we observed in our set-up could be explained by the dark colour of this species, especially in
the dorsal part. Some individuals showed freezing behaviour, which is indicative of anxiety
]. Thus, there is a striking similarity in the response of these two species in the black-white
test. However it has been reported that scototaxis differs between species. For example, strong
black preference is shown by guppies and cardinal-tetras, whereas no preference has been
observed in the Nile tilapia or mosquitofish [
We applied buspirone expecting to see a decrease in scototaxis in line with the anxiolytic
effect observed in the novel tank diving test. Moreover, buspirone has been shown to decrease
scototaxis and geotaxis in zebrafish [
]. However, we did not observed any effect on
scototaxis. It Is possible that a higher concentration of drug or a longer treatment is required to
increase the time spent in the white zone. Alternatively, it may be that buspirone does not
affect scototaxis in the same way as it affects diving responses in three-spined sticklebacks,
because these behaviours are controlled by different 5-HT receptors or pathways.
Some serotonergic drugs do not have the same anxiolytic or anxiogenic effect on zebrafish
in both tests. For example para-chlorophenylalanine (pCPA) is anxiogenic in the novel tank
diving test and anxiolytic in the black-white preference test, and fluoxetine has the opposite
effect in these two tests [
]. This emphasises the need to consider these tests as
complementary due to their different sensitivity to the same pharmacological manipulations. The novel
tank diving and the black-white preference tests elicit escape responses to perceived potential
threats, such as predator attacks, in the form of bottom and black preference, in both zebrafish
and three-spined sticklebacks. Interestingly, the same context (i.e. testing tank) elicits similar
13 / 18
responses in both species. Further pharmacological validation of these tests is likely to make
them powerful tools for biomedical research and drug discovery.
Three-spined sticklebacks and zebrafish exhibit species-specific features and biological
differences due to their evolutionary distance. The zebrafish is a tropical species that actively swims
at regular speed by using the thrust of its caudal fin. Three-spined sticklebacks live in cooler
habitats, are less active, and alternate bursts of swimming using its pectoral fins with episodes
of immobility in the middle of the water column. However, both are amenable for behavioural
testing in the lab and display very similar innate behaviours in the same tests. The availability
of three-spined stickleback as a new fish model to perform comparative studies and
translational research represents a valuable tool to further our knowledge about brain malfunction
leading to neurobiological diseases.
The wealth of knowledge about stickleback behaviour obtained in the past decades, the
consistent individual differences in behaviour and the availability of standard and easy to perform
tests can be readily used to study human psychiatric disorders and search for novel treatments.
Further validation of these tests using pharmacological approaches and measuring
physiological responses, characterisation of complex behaviours such as learning and memory, attention,
impulsivity or cognition, and neuroanatomical studies describing brain areas and circuits
controlling behaviour will open up new avenues for cross species studies and translational
S1 Table. Light-dark and temperature conditions under which fish were reared until the
We are grateful to Carl Breaker and Ceinwen Tilley for three-spined stickleback care and
technical support and all members of the Norton lab for discussions about this data. We also thank
Iain Barber for his comments on this manuscript.
Conceptualization: He?ctor Carre?o Gutie?rrez.
Data curation: He?ctor Carre?o Gutie?rrez.
Formal analysis: He?ctor Carre?o Gutie?rrez.
Methodology: He?ctor Carre?o Gutie?rrez.
Project administration: William H. J. Norton.
Supervision: William H. J. Norton.
Writing ? original draft: He?ctor Carre?o Gutie?rrez.
Writing ? review & editing: William H. J. Norton.
14 / 18
15 / 18
16 / 18
17 / 18
1. Barber I , Nettleship S ( 2010 ) From 'trash fish' to supermodel: The rise and rise of the three-spined stickleback in evolution and ecology . Biologist 57 : 15 - 21 .
2. Von Hippel F ( 2010 ) Tinbergen's Legacy in Behaviour: Sixty Years of Landmark Stickleback Papers: Brill.
3. Wootton RJ , Wootton RJ ( 1976 ) The biology of the sticklebacks : Academic Press.
4. Huntingford FA ( 1976 ) The relationship between anti-predator behaviour and aggression among conspecifics in the three-spined stickleback , Gasterosteus Aculeatus. Anim Behav 24 : 245 - 260 .
5. Bell AM ( 2005 ) Behavioural differences between individuals and two populations of stickleback (Gasterosteus aculeatus) . J Evol Biol 18 : 464 - 473 . https://doi.org/10.1111/j.1420- 9101 . 2004 . 00817 . x PMID : 15715852
6. Dingemanse NJ , Wright J , Kazem AJ , Thomas DK , Hickling R , et al. ( 2007 ) Behavioural syndromes differ predictably between 12 populations of three-spined stickleback . J Anim Ecol 76 : 1128 - 1138 . https:// doi.org/10.1111/j.1365- 2656 . 2007 . 01284 . x PMID : 17922709
7. Iersel JJAv ( 1953 ) An analysis of the parental behaviour of the male three-spined stickleback (Gasterosteus aculeatus L.) . Leiden: Brill.
8. Tinbergen N ( 1951 ) The study of instinct . New York, NY, US: Clarendon Press/Oxford University Press. xii, 237-xii, 237 p.
9. Ward AJW , Thomas P , Hart PJB , Krause J ( 2004 ) Correlates of boldness in three-spined sticklebacks (Gasterosteus aculeatus) . Behavioral Ecology and Sociobiology 55 : 561 - 568 .
10. Bell AM , Henderson L , Huntingford FA ( 2010 ) Behavioral and respiratory responses to stressors in multiple populations of three-spined sticklebacks that differ in predation pressure . J Comp Physiol B 180 : 211 - 220 . https://doi.org/10.1007/s00360-009 -0395-8 PMID: 19705129
11. Wark AR , Greenwood AK , Taylor EM , Yoshida K , Peichel CL ( 2011 ) Heritable differences in schooling behavior among threespine stickleback populations revealed by a novel assay . PLoS One 6 : e18316 . https://doi.org/10.1371/journal.pone. 0018316 PMID: 21464914
12. Alvarez D , Bell AM ( 2007 ) Sticklebacks from streams are more bold than sticklebacks from ponds . Behav Processes 76 : 215 - 217 . https://doi.org/10.1016/j.beproc. 2007 . 05 .004 PMID: 17583445
13. Bell AM ( 2001 ) Effects of an endocrine disrupter on courtship and aggressive behaviour of male threespined stickleback,Gasterosteus aculeatus . Anim Behav 62 : 775 - 780 .
14. Barber I , Hoare D , Krause J ( 2000 ) Effects of parasites on fish behaviour: a review and evolutionary perspective . Reviews in Fish Biology and Fisheries 10 : 131 - 165 .
15. Barber I ( 2013 ) Sticklebacks as model hosts in ecological and evolutionary parasitology . Trends Parasitol 29 : 556 - 566 . https://doi.org/10.1016/j.pt. 2013 . 09 .004 PMID: 24145060
16. Kalueff AV , Stewart AM , Gerlai R ( 2014 ) Zebrafish as an emerging model for studying complex brain disorders . Trends Pharmacol Sci 35 : 63 - 75 . https://doi.org/10.1016/j.tips. 2013 . 12 .002 PMID: 24412421
17. Stewart AM , Ullmann JF , Norton WH , Parker MO , Brennan CH , et al. ( 2015 ) Molecular psychiatry of zebrafish . Mol Psychiatry 20 : 2 - 17 . https://doi.org/10.1038/mp. 2014 .128 PMID: 25349164
18. Homberg JR , Kyzar EJ , Nguyen M , Norton WH , Pittman J , et al. ( 2016 ) Understanding autism and other neurodevelopmental disorders through experimental translational neurobehavioral models . Neurosci Biobehav Rev 65 : 292 - 312 . https://doi.org/10.1016/j.neubiorev. 2016 . 03 .013 PMID: 27048961
19. Theodoridi A , Tsalafouta A , Pavlidis M ( 2017 ) Acute Exposure to Fluoxetine Alters Aggressive Behavior of Zebrafish and Expression of Genes Involved in Serotonergic System Regulation . Frontiers in neuroscience 11 : 223 - 223 . https://doi.org/10.3389/fnins. 2017 .00223 PMID: 28487628
20. Norton WH , Stumpenhorst K , Faus-Kessler T , Folchert A , Rohner N , et al. ( 2011 ) Modulation of Fgfr1a signaling in zebrafish reveals a genetic basis for the aggression-boldness syndrome . J Neurosci 31 : 13796 - 13807 . https://doi.org/10.1523/JNEUROSCI.2892- 11 . 2011 PMID: 21957242
21. Bencan Z , Sledge D , Levin ED ( 2009 ) Buspirone, chlordiazepoxide and diazepam effects in a zebrafish model of anxiety . Pharmacol Biochem Behav 94 : 75 - 80 . https://doi.org/10.1016/j.pbb. 2009 . 07 .009 PMID: 19643124
22. Carreno Gutierrez H , O'Leary A , Freudenberg F , Fedele G , Wilkinson R , et al. ( 2017 ) Nitric oxide interacts with monoamine oxidase to modulate aggression and anxiety-like behaviour . Eur Neuropsychopharmacol https://doi.org/10.1016/j.euroneuro. 2017 . 09 .004 PMID: 28951000
23. Carreno Gutierrez H , Vacca I , Pons AI , Norton WHJ ( 2018 ) Automatic quantification of juvenile zebrafish aggression . J Neurosci Methods 296 : 23 - 31 . https://doi.org/10.1016/j.jneumeth. 2017 . 12 .012 PMID: 29274793
24. Egan RJ , Bergner CL , Hart PC , Cachat JM , Canavello PR , et al. ( 2009 ) Understanding behavioral and physiological phenotypes of stress and anxiety in zebrafish . Behav Brain Res 205 : 38 - 44 . https://doi. org/10.1016/j.bbr. 2009 . 06 .022 PMID: 19540270
25. Cachat J , Stewart A , Grossman L , Gaikwad S , Kadri F , et al. ( 2010 ) Measuring behavioral and endocrine responses to novelty stress in adult zebrafish . Nat Protoc 5 : 1786 - 1799 . https://doi.org/10.1038/ nprot. 2010 .140 PMID: 21030954
26. Parker MO , Brock AJ , Millington ME , Brennan CH ( 2013 ) Behavioural phenotyping of casper mutant and 1-pheny-2-thiourea treated adult zebrafish . Zebrafish 10 : 466 - 471 . https://doi.org/10.1089/zeb. 2013 .0878 PMID: 23869690
27. Maximino C , Marques de Brito T , Dias CA , Gouveia A Jr., Morato S ( 2010 ) Scototaxis as anxiety-like behavior in fish . Nat Protoc 5 : 209 - 216 . https://doi.org/10.1038/nprot. 2009 .225 PMID: 20134420
28. Herculano AM , Puty B , Miranda V , Lima MG , Maximino C ( 2015 ) Interactions between serotonin and glutamate-nitric oxide pathways in zebrafish scototaxis . Pharmacol Biochem Behav 129 : 97 - 104 . https://doi.org/10.1016/j.pbb. 2014 . 12 .005 PMID: 25536532
29. Norton WHJ ( 2018 ) Screening for drugs to reduce aggression in zebrafish . Neuropharmacology https:// doi.org/10.1016/j.neuropharm. 2018 . 10 .023.
30. Harcourt JL , Sweetman G , Johnstone RA , Manica A ( 2009 ) Personality counts: the effect of boldness on shoal choice in three-spined sticklebacks . Anim Behav 77 : 1501 - 1505 .
31. Kellner M , Porseryd T , Hallgren S , Porsch-Hallstrom I , Hansen SH , et al. ( 2016 ) Waterborne citalopram has anxiolytic effects and increases locomotor activity in the three-spine stickleback (Gasterosteus aculeatus) . Aquat Toxicol 173 : 19 - 28 . https://doi.org/10.1016/j.aquatox. 2015 . 12 .026 PMID: 26827268
32. Rystrom TL , Clement VF , Rick IP , Bakker TCM , Mehlis M ( 2018 ) Shoal sex composition and predation risk influence sub-adult threespine stickleback shoaling decisions . Behav Processes https://doi.org/10. 1016/j.beproc. 2018 . 06 .009 PMID: 29933058
33. Maaswinkel H , Zhu L , Weng W ( 2013 ) Assessing social engagement in heterogeneous groups of zebrafish: a new paradigm for autism-like behavioral responses . PLoS One 8 : e75955 . https://doi.org/10. 1371/journal.pone. 0075955 PMID: 24116082
34. Bakker TCM ( 1986 ) Aggressiveness in Sticklebacks (Gasterosteus Aculeatus L .) : a Behaviour-Genetic Study . Behaviour 98 : 1 - 144 .
35. Tinbergen N ( 1953 ) Social behaviour in animals: with special reference to vertebrates . Oxford, England: Wiley.
36. Verbeek P , Iwamoto T , Murakami N ( 2007 ) Differences in aggression between wild-type and domesticated fighting fish are context dependent . Anim Behav 73 : 75 - 83 .
37. Desjardins JK , Fernald RD ( 2010 ) What do fish make of mirror images? Biol Letters .
38. Swain DP , Holtby LB ( 1989 ) Differences in Morphology and Behavior between Juvenile Coho Salmon (Oncorhynchus kisutch) Rearing in a Lake and in its Tributary Stream . Canadian Journal of Fisheries and Aquatic Sciences 46 : 1406 - 1414 .
39. Earley RL , Hsu Y , Wolf LL ( 2000 ) The Use of Standard Aggression Testing Methods to Predict Combat Behaviour and Contest Outcome in Rivulus marmoratus Dyads (Teleostei: Cyprinodontidae) . Ethology 106 : 743 - 761 .
40. Holmberg A , Fogel J , Albertsson E , Fick J , Brown JN , et al. ( 2011 ) Does waterborne citalopram affect the aggressive and sexual behaviour of rainbow trout and guppy? Journal of Hazardous Materials 187 : 596 - 599 . https://doi.org/10.1016/j.jhazmat. 2011 . 01 .055 PMID: 21300431
41. Ros AF , Becker K , Oliveira RF ( 2006 ) Aggressive behaviour and energy metabolism in a cichlid fish, Oreochromis mossambicus . Physiol Behav 89 : 164 - 170 . https://doi.org/10.1016/j.physbeh. 2006 . 05 . 043 PMID: 16828128
42. Gerlai R , Lahav M , Guo S , Rosenthal A ( 2000 ) Drinks like a fish: zebra fish (Danio rerio) as a behavior genetic model to study alcohol effects . Pharmacol Biochem Behav 67 : 773 - 782 . PMID: 11166068
43. Sebire M , Elphinstone Davis J , Hatfield R , Winberg S , Katsiadaki I ( 2015 ) Prozac affects stickleback nest quality without altering androgen, spiggin or aggression levels during a 21-day breeding test . Aquat Toxicol 168 : 78 - 89 . https://doi.org/10.1016/j.aquatox. 2015 . 09 .009 PMID: 26453812
44. Kleszczynska A , Sokolowska E , Kulczykowska E ( 2012 ) Variation in brain arginine vasotocin (AVT) and isotocin (IT) levels with reproductive stage and social status in males of three-spined stickleback (Gasterosteus aculeatus) . Gen Comp Endocrinol 175 : 290 - 296 . https://doi.org/10.1016/j.ygcen. 2011 . 11 . 022 PMID: 22137910
45. Kellner M , Porseryd T , Porsch-Hallstrom I , Borg B , Roufidou C , et al. ( 2018 ) Developmental exposure to the SSRI citalopram causes long-lasting behavioural effects in the three-spined stickleback (Gasterosteus aculeatus) . Ecotoxicology 27 : 12 - 22 . https://doi.org/10.1007/s10646-017 -1866-4 PMID: 29058178
46. Kohlert JG , Mangan BP , Kodra C , Drako L , Long E , et al. ( 2012 ) Decreased aggressive and locomotor behaviors in Betta splendens after exposure to fluoxetine . Psychol Rep 110 : 51 - 62 . https://doi.org/10. 2466/02.13. PR0.110.1 . 51 -62 PMID: 22489377
47. Perreault HAN , Semsar K , Godwin J ( 2003 ) Fluoxetine treatment decreases territorial aggression in a coral reef fish . Physiology & Behavior 79 : 719 - 724 .
48. Nery LR , Eltz NS , Martins L , Guerim LD , Pereira TC , et al. ( 2014 ) Sustained behavioral effects of lithium exposure during early development in zebrafish: involvement of the Wnt-beta-catenin signaling pathway . Prog Neuropsychopharmacol Biol Psychiatry 55 : 101 - 108 . https://doi.org/10.1016/j.pnpbp. 2014 . 04 .011 PMID: 24813569
49. Grecias L , Valentin J , Aubin-Horth N ( 2018 ) Testing the parasite mass burden effect on alteration of host behaviour in the Schistocephalus-stickleback system . J Exp Biol 221 .
50. Bell AM , Backstrom T , Huntingford FA , Pottinger TG , Winberg S ( 2007 ) Variable neuroendocrine responses to ecologically-relevant challenges in sticklebacks . Physiol Behav 91 : 15 - 25 . https://doi.org/ 10.1016/j.physbeh. 2007 . 01 .012 PMID: 17321556
51. Thompson RR , Paul ES , Radford AN , Purser J , Mendl M ( 2016 ) Routine handling methods affect behaviour of three-spined sticklebacks in a novel test of anxiety . Behav Brain Res 306 : 26 - 35 . https:// doi.org/10.1016/j.bbr. 2016 . 03 .015 PMID: 26965568
52. McGhee KE , Pintor LM , Bell AM ( 2013 ) Reciprocal behavioral plasticity and behavioral types during predator-prey interactions . Am Nat 182 : 704 - 717 . https://doi.org/10.1086/673526 PMID: 24231533
53. Brydges NM , Boulcott P , Ellis T , Braithwaite VA ( 2009 ) Quantifying stress responses induced by different handling methods in three species of fish . Appl Anim Behav Sci 116 : 295 - 301 .
54. Wright D , Rimmer LB , Pritchard VL , Krause J , Butlin RK ( 2003 ) Inter and intra-population variation in shoaling and boldness in the zebrafish (Danio rerio) . Naturwissenschaften 90 : 374 - 377 . https://doi.org/ 10.1007/s00114-003 -0443-2 PMID: 12955228
55. Krause J , Ruxton GD ( 2002 ) Living in Groups: OUP Oxford .
56. Modarressie R , Rick IP , Bakker TC ( 2006 ) UV matters in shoaling decisions . Proc Biol Sci 273 : 849 - 854 . https://doi.org/10.1098/rspb. 2005 .3397 PMID: 16618679
57. Mehlis M , Thunken T , Bakker TC , Frommen JG ( 2015 ) Quantification acuity in spontaneous shoaling decisions of three-spined sticklebacks . Anim Cogn 18 : 1125 - 1131 . https://doi.org/10.1007/s10071- 015-0884-y PMID: 26099740
58. Laskowski KL , Bell AM ( 2014 ) Strong personalities, not social niches, drive individual differences in social behaviours in sticklebacks . Anim Behav 90 : 287 - 295 . https://doi.org/10.1016/j.anbehav. 2014 . 02 .010 PMID: 25076789
59. Miller N , Gerlai R ( 2012 ) From schooling to shoaling: patterns of collective motion in zebrafish (Danio rerio) . PLoS One 7 : e48865 . https://doi.org/10.1371/journal.pone. 0048865 PMID: 23166599
60. Pearish S , Hostert L , Bell AM ( 2017 ) A standardized method for quantifying consistent individual differences in schooling behaviour . J Fish Biol 90 : 443 - 450 . https://doi.org/10.1111/jfb.13176 PMID: 27766651
61. Buske C , Gerlai R ( 2011 ) Shoaling develops with age in Zebrafish (Danio rerio) . Prog Neuropsychopharmacol Biol Psychiatry 35 : 1409 - 1415 . https://doi.org/10.1016/j.pnpbp. 2010 . 09 .003 PMID: 20837077
62. Buske C , Gerlai R ( 2012 ) Maturation of shoaling behavior is accompanied by changes in the dopaminergic and serotoninergic systems in zebrafish . Dev Psychobiol 54 : 28 - 35 . https://doi.org/10.1002/dev. 20571 PMID: 21656763
63. Miller NY , Gerlai R ( 2008 ) Oscillations in shoal cohesion in zebrafish (Danio rerio) . Behav Brain Res 193 : 148 - 151 . https://doi.org/10.1016/j.bbr. 2008 . 05 .004 PMID: 18573546
64. Miller N , Gerlai R ( 2011 ) Redefining membership in animal groups . Behav Res Methods 43 : 964 - 970 . https://doi.org/10.3758/s13428-011-0090-z PMID: 21491173
65. Miller N , Gerlai R ( 2007 ) Quantification of shoaling behaviour in zebrafish (Danio rerio) . Behav Brain Res 184 : 157 - 166 . https://doi.org/10.1016/j.bbr. 2007 . 07 .007 PMID: 17707522
66. Hamilton TJ , Morrill A , Lucas K , Gallup J , Harris M , et al. ( 2017 ) Establishing zebrafish as a model to study the anxiolytic effects of scopolamine . Scientific Reports 7 : 15081 . https://doi.org/10.1038/ s41598-017-15374-w PMID: 29118373
67. Perez-Escudero A , Vicente-Page J , Hinz RC , Arganda S , de Polavieja GG ( 2014 ) idTracker: tracking individuals in a group by automatic identification of unmarked animals . Nat Methods 11 : 743 - 748 . https://doi.org/10.1038/nmeth.2994 PMID: 24880877
68. Hinz RC , de Polavieja GG ( 2017 ) Ontogeny of collective behavior reveals a simple attraction rule . Proceedings of the National Academy of Sciences 114 : 2295 .
69. Miller N , Greene K , Dydinski A , Gerlai R ( 2013 ) Effects of nicotine and alcohol on zebrafish (Danio rerio) shoaling . Behav Brain Res 240 : 192 - 196 . https://doi.org/10.1016/j.bbr. 2012 . 11 .033 PMID: 23219966
70. Stewart AM , Nguyen M , Wong K , Poudel MK , Kalueff AV ( 2014 ) Developing zebrafish models of autism spectrum disorder (ASD) . Prog Neuropsychopharmacol Biol Psychiatry 50 : 27 - 36 . https://doi.org/10. 1016/j.pnpbp. 2013 . 11 .014 PMID: 24315837
71. Zimmermann FF , Gaspary KV , Leite CE , De Paula Cognato G , Bonan CD ( 2015 ) Embryological exposure to valproic acid induces social interaction deficits in zebrafish (Danio rerio): A developmental behavior analysis . Neurotoxicol Teratol 52 : 36 - 41 . https://doi.org/10.1016/j.ntt. 2015 . 10 .002 PMID: 26477937
72. Stewart A , Wu N , Cachat J , Hart P , Gaikwad S , et al. ( 2011 ) Pharmacological modulation of anxiety-like phenotypes in adult zebrafish behavioral models . Progress in Neuro-Psychopharmacology and Biological Psychiatry 35 : 1421 - 1431 . https://doi.org/10.1016/j.pnpbp. 2010 . 11 .035 PMID: 21122812
73. Hallgren S , Volkova K , Reyhanian N , Olsen KH , Hallstrom IP ( 2011 ) Anxiogenic behaviour induced by 17alpha-ethynylestradiol in male guppies (Poecilia reticulata) . Fish Physiol Biochem 37 : 911 - 918 . https://doi.org/10.1007/s10695-011-9488 -x PMID : 21537944
74. Volkova K , Reyhanian N , Kot-Wasik A , Olsen H , Porsch-Hallstrom I , et al. ( 2012 ) Brain circuit imprints of developmental 17alpha-Ethinylestradiol exposure in guppies (Poecilia reticulata): persistent effects on anxiety but not on reproductive behaviour . Gen Comp Endocrinol 178 : 282 - 290 . https://doi.org/10. 1016/j.ygcen. 2012 . 05 .010 PMID: 22687331
75. Olsen KH , Ask K , Olsen H , Porsch-Hallstrom I , Hallgren S ( 2014 ) Effects of the SSRI citalopram on behaviours connected to stress and reproduction in Endler guppy, Poecilia wingei . Aquat Toxicol 148 : 113 - 121 . https://doi.org/10.1016/j.aquatox. 2013 . 12 .032 PMID: 24473162
76. Maximino C , Puty B , Benzecry R , Araujo J , Lima MG , et al. ( 2013 ) Role of serotonin in zebrafish (Danio rerio) anxiety: relationship with serotonin levels and effect of buspirone, WAY 100635, SB 224289, fluoxetine and para-chlorophenylalanine (pCPA) in two behavioral models . Neuropharmacology 71 : 83 - 97 . https://doi.org/10.1016/j.neuropharm. 2013 . 03 .006 PMID: 23541719
77. Maximino C , Marques T. , Dias F. , Cortes F. , Taccolini I. , Pereira P. ( 2007 ) A Comparative Analysis of the Preference for Dark Environments in Five Teleosts . International Journal of Comparative Psychology 20 .
78. Blaser RE , Rosemberg DB ( 2012 ) Measures of Anxiety in Zebrafish (Danio rerio): Dissociation of Black/ White Preference and Novel Tank Test . PLOS ONE 7 : e36931. https://doi.org/10.1371/journal.pone. 0036931 PMID: 22615849
79. Blaser RE , Chadwick L , McGinnis GC ( 2010 ) Behavioral measures of anxiety in zebrafish (Danio rerio) . Behav Brain Res 208 : 56 - 62 . https://doi.org/10.1016/j.bbr. 2009 . 11 .009 PMID: 19896505