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Social responses of zebrafish induced by different visual stimuli: a cyber-ethological study

Authors:
  • Associazione ETICOSCIENZA
  • Eticoscienza

Abstract

The social behaviour of zebrafish (Danio rerio, Cyprinidae) appears complex and little is known about sex-related shoaling preference and mate choice. The aim of this project is studying the potential influence of various stimuli (e.g. habitat colours, conspecifics-of different number and sex-, and 2D images) on shoaling preferences, with a focus on visual communication. In the present study 40 sexually matured GloFish® danios were involved into two non-invasive behavioural experiments using a multipurpose cross-maze, together with a video-tracking software (EthoVision XT) methodology. These experiments were approved by the ethical commission and no animal was harmed or killed during the trials. In the pre-test period of the first experiment two groups, one for males (n=10) and one for females (n=10), were kept in sex-isolated aquariums for 7 days, with a different habitat colour for each one: green (males) and red (females). After that, each group was maintained separated and 8 behavioural tests were conducted, investigating with focal observations the possible influence of environmental colouring on zebrafish social preferences. In the second experiment, various 2D social stimuli (manipulated by graphics software) were used in 9 behavioural tests to study social and sexual interactions (i.e. mate stimulant attraction and shoaling preference) of other two groups, one for males (n=10) and the other one for females (n=10). We did not observe any clear influence of environmental colouring on social zebrafish choices, but there was a significant difference between males and females in response and selection of shoaling partners. For instance, we found that females showed a general preference in shoaling with conspecifics of the other sex, and avoided other phenotypically similar females. Males spent more time close to another male and less with a female, and they were not choosy for social and reproductive partners. This methodology could be used to study social and sexual preference also in other aquatic species, with several applications on husbandry, animal welfare and fish conservation. Aims Danio rerio (zebrafish) is a tropical freshwater fish, member of the family Cyprinidae. Zebrafish is a very social animal and researchers worldwide are increasingly studying a huge range of behaviors exhibited by this species [1]. Most of the studies on its sociality examined the shoaling behavior, i.e. aggregation in groups. Shoaling plays a key role in foraging, predator avoidance and reproductive success. Experiments on shoaling preference have demonstrated that both male and female zebrafish prefer to spend more time in a social compartment as compared to an empty one [2-3]. In zebrafish, the existence of visual discrimination learning of environmental coloring [4] and prey capture based on early visual system development was demonstrated. Moreover, preference and avoidance towards colors of different wavelengths have been well documented for this species; however, the results are conflicting. Since the use of zebrafish in the biomedical field has increased, several studies have shown the existence of effective methodologies, which involves the use of artificial stimuli, to limit the use of live animals in research [5]. It was observed that the tested individuals spent more time close to compartments with animated 2D images representing conspecifics, in a manner equivalent to what happens with the projection of videos regarding zebrafish in motion or in the presence of real stimuli [6]. According to a study [7] zebrafish did not show preferences between the image of a wild-type individual and that of a shorter and extended individual on the vertical axis (especially in the abdominal region), while they showed the behavior of avoidance compared to the altered image representing an elongated individual. Other works published in recent years have tried to study the behavioral response of this species in front of stylized models or robots. As shown by Ruberto and collaborators (2016), it would seem to be the association "robot + movement" to trigger the social response of the observed individuals [8]. It must be said, however, that the robotic stimulus used, although it was the result of advanced experimental methodologies, would not seem to mimic perfectly the phenotype and the typical colors of a real Danio rerio. This may have influenced the lack of "attraction" to a fixed / immobile stimulus of this type and therefore can not be excluded that, using a phenotypically more refined stimulus, still there is no social response. Background Materials and methods
Social responses of zebrafish induced by different
visual stimuli: a cyber-ethological study
Christian Lenzi1*, Chiara Grasso1, Paolo Peretto2,3, Alexandra Savuca4, Alin Ciobica4,
Stefan-Adrian Strungaru5, Gabriel Plavan4, Mircea Nicoara4
1 Associazione ETICOSCIENZA, Turin, Italy; 2 Department of Life Sciences and Systems Biology (DBIOS), University of Turin, Turin 10123, Italy; 3 Neuroscience Institute Cavalieri Ottolenghi (NICO), Orbassano 10043, Italy; 4 “Alexandru
Ioan Cuza” University of Iasi, Department of Biology, Faculty of Biology, Bd. Carol I, 20A, 700505, Iasi, Romania; 5 “Alexandru Ioan Cuza” University of Iasi, Department of Research, Faculty of Biology, Romania
*christianlenzi.eticoscienza@gmail.com
Abstract
The social behaviour of zebrafish (Danio rerio, Cyprinidae) appears complex and little is known about sex-related shoaling preference and mate choice. The aim of this project is studying the potential influence of
various stimuli (e.g. habitat colours, conspecifics - of different number and sex -, and 2D images) on shoaling preferences, with a focus on visual communication. In the present study 40 sexually matured GloFish®
danios were involved into two non-invasive behavioural experiments using a multi-purpose cross-maze, together with a video-tracking software (EthoVision XT) methodology. These experiments were approved by
the ethical commission and no animal was harmed or killed during the trials. In the pre-test period of the first experiment two groups, one for males (n=10) and one for females (n=10), were kept in sex-isolated
aquariums for 7 days, with a different habitat colour for each one: green (males) and red (females). After that, each group was maintained separated and 8 behavioural tests were conducted, investigating with focal
observations the possible influence of environmental colouring on zebrafish social preferences. In the second experiment, various 2D social stimuli (manipulated by graphics software) were used in 9 behavioural tests
to study social and sexual interactions (i.e. mate stimulant attraction and shoaling preference) of other two groups, one for males (n=10) and the other one for females (n=10). We did not observe any clear influence of
environmental colouring on social zebrafish choices, but there was a significant difference between males and females in response and selection of shoaling partners. For instance, we found that females showed a
general preference in shoaling with conspecifics of the other sex, and avoided other phenotypically similar females. Males spent more time close to another male and less with a female, and they were not choosy for
social and reproductive partners. This methodology could be used to study social and sexual preference also in other aquatic species, with several applications on husbandry, animal welfare and fish conservation.
Aims
Danio rerio (zebrafish) is a tropical freshwater fish, member of the family
Cyprinidae. Zebrafish is a very social animal and researchers worldwide are
increasingly studying a huge range of behaviors exhibited by this species [1].
Most of the studies on its sociality examined the shoaling behavior, i.e.
aggregation in groups. Shoaling plays a key role in foraging, predator
avoidance and reproductive success.
Experiments on shoaling preference have demonstrated that both male and
female zebrafish prefer to spend more time in a social compartment as
compared to an empty one [2-3].
In zebrafish, the existence of visual discrimination learning of environmental
coloring [4] and prey capture based on early visual system development was
demonstrated. Moreover, preference and avoidance towards colors of different
wavelengths have been well documented for this species; however, the results
are conflicting.
Since the use of zebrafish in the biomedical field has increased, several
studies have shown the existence of effective methodologies, which involves
the use of artificial stimuli, to limit the use of live animals in research [5]. It
was observed that the tested individuals spent more time close to
compartments with animated 2D images representing conspecifics, in a
manner equivalent to what happens with the projection of videos regarding
zebrafish in motion or in the presence of real stimuli [6]. According to a study
[7] zebrafish did not show preferences between the image of a wild-type
individual and that of a shorter and extended individual on the vertical axis
(especially in the abdominal region), while they showed the behavior of
avoidance compared to the altered image representing an elongated
individual. Other works published in recent years have tried to study the
behavioral response of this species in front of stylized models or robots. As
shown by Ruberto and collaborators (2016), it would seem to be the
association "robot + movement" to trigger the social response of the observed
individuals [8]. It must be said, however, that the robotic stimulus used,
although it was the result of advanced experimental methodologies, would not
seem to mimic perfectly the phenotype and the typical colors of a real Danio
rerio. This may have influenced the lack of "attraction" to a fixed / immobile
stimulus of this type and therefore can not be excluded that, using a
phenotypically more refined stimulus, still there is no social response.
Background Materials and methods
Results
Discussion and conclusions
A total of 40 (n=20 males; n=20 females) sexually mature red GloFish®
zebrafish were used for this study (ethical statement: §).
For both the experiments male and female zebrafish were randomly extracted
from a lab population and kept separately in two groups. Each group was
housed in a 5 L (19.5x30x17 cm) tank and all the variables (i.e. T, salinity,
acidity, and pH) were controlled and maintained within a limited range for the
entire experimental period.
For the first experiment, each group was sex-isolated for 7 days in separated
tanks, with different environmental colouring: green for males and red for
females. Since the absence of a natural preference between the red and green
settings was previously documented in this species, we assigned randomly
one habitat color per group.
For the second experiment, graphically manipulated non-moving 2D stimuli,
representing one or more Zebrafish individuals, were used (Fig.2). The
photographic images were manipulated using the Adobe Photoshop CC 2017
graphics processing software. Basically, only the abdominal region (female),
the caudal fin (male), the size, and the body colour were extended or
modificated, maintaining the general morphometric proportions unchanged.
Different non-invasive behavioural tests (8 for the first experiment and 9 for
the second one) were performed using a multi-purpose cross-maze made from
transparent Plexiglas that was adapted to a T-maze.
Zebrafish behavior was recorded and analyzed by EthoVision XT 11.5
video-tracking software (Noldus Information Technology, Netherlands). A
digital camera, fixed above the center of the cross-maze, was directly
connected to the video-tracking software inside the computer. An infrared
radiation slab was located under the cross-maze in order to obtain high
contrast video-images (Fig.1).
§ Ethical statement: The animals were strictly maintained and treated
according to EU Commission Recommendation and Directive 2010/63/EU.
The local committee approved the testing. No animal was harmed, killed or
suffered during the trials.
First experiment: we observed that environmental coloring does not have an “imprinting” effect on adult zebrafish. In fact, males
preferred an empty arm instead of a green zone and females showed a higher preference for the green arm than the red one. Moreover,
habitat colors in general did not influence the sociality of zebrafish when collocated alone in one arm of the cross-maze. Since
environmental coloring does not seem to have a definitive role also in zebrafish sociality, we wanted to examine the sex-related shoaling
differences by comparing the social stimuli preferences in different behavioral tests. We found significant differences when we compared
the findings of “test home color vs. other sex 1” and “test home color vs. same sex 1”. The females showed an evident preference for the
“other sex 1” stimulus with regarding to the cumulative duration (Student’s t-test, t=5.655, N1 =30, N2 =30, p<0.001), while males had an
equal preference between the two stimuli (Student’s t-test, t=-0.172, N1 =30, N2 =30, P=0.864). Comparing the results of “test home color
vs. other sex 3” and “test home color vs. other sex 1”, we found that female fish shoaled more with one male fish and less with 3 males
(Student’s t-test, t=-2.733, N1 =30, N2 =30, P=0.013). Again, the males did not show a significant preference (Student’s t-test, t=0766, N1
=30, N2 =30, p=0.453). Lastly, we compared the mean duration scores for the social stimuli zones for “test home color vs. other sex 3” and
“test home color vs. same sex 3”. Females showed a stronger preference for the “other sex 3” (Wilcoxon signed-rank test, W=110,
Z=2.146, N1 =30, N2 =30, p=0.032) zone, and males for the “same sex 3” zone (Student’s t-test, t=-2.362, N1 =30, N2 =30, P=0.029).
Second experiment: significant sex-related differences were noted regarding the time spent near certain stimuli compared to others. In the
second behavioural test, "test big female vs. big red female", the females showed a greater cumulative duration for the "big red female"
area (Student's t-test, t = -2.267, N1 = 10, N2 = 10, p = 0.049). In the third behavioural test, "test big female - red vs. blue vs. green",
females show a significant preference in terms of cumulative duration for the "big blue female" stimulus compared to the "big red female"
stimulus (Student's t-test , t = -4.595, N1 = 10, N2 = 10, p = 0.001).
In the fourth behavioural test, "test big female vs. 3 small females", significant preference was found towards the "big female" stimulus, in
terms of cumulative duration, both for males (Student's t-test, t = -2.279, N1 = 10, N2 = 10, p = 0.048) that for females (Student's t-test, t =
-2.304, N1 = 10, N2 = 10, p = 0.046). In the fifth behavioural test, "test big female vs. 3 big females", both males and females spent more
time near the "3 big females" stimulus, but only in terms of cumulative duration (males: Student's t-test, t = -2.696, N1 = 10, N2 = 10, p =
0.024, females: Student's t-test, t = -2.466, N1 = 10, N2 = 10, p = 0.036). In the ninth behavioural test, "test big male vs. small male", the
females showed a preference, within their sample, for the "big male" stimulus in terms of cumulative duration (Student's t-test, t = -2.304,
N1 = 10, N2 = 10, p = 0.047).
References
[1] Kalueff AV, Gebhardt M, Stewart AM,Cachat JM,Brimmer M,Chawla JS. Towards a Comprehensive Catalog of Zebrafish Behavior 1.0 and
Beyond. Zebrafish. 2013;10(1):70-86
[2] Etinger A, Lebron J, Palestis BG. Sex-assortative shoaling in zebrafish (Danio rerio). Bios. 2009;80(4):153-8
[3] Snekser JL, Ruhl N, Bauer K, McRobert SP. The Influence of Sex and Phenotype on Shoaling Decisions in Zebrafish. Intern J Comp Psychol.
2010;23:70-81
[4] Colwill RM, Raymond MP, Ferreira L, Escudero H. Visual discrimination learning in zebrafish (Danio rerio). Behav Process. 2005;70(1):19-31
[5] Ladu F, Bartolini T, Panitz SG, Chiarotti F, Butail S, Macrì S, Porfiri M. 2015. Live predators, robots, and computer-animated images elicit
differential avoidance responses in zebrafish. Zebrafish. 12(3):205-14. doi: 10.1089/zeb.2014.1041
[6] Qin M, Wong A, Seguin D, Gerlai R. 2014. Induction of Social Behavior in Zebrafish: Live Versus Computer Animated Fish as Stimuli. Zebrafish.
11, 185-197
[7] Saverino C, Gerlai R. 2008. The social zebrafish: Behavioral responses to conspecific, heterospecific, and computer animated fish. Behav Brain
Res. 191, 77-87
[8] Ruberto T, Mwaffo V, Singh S, Neri D, Porfiri M. 2016. Zebrafish response to a robotic replica in three dimensions. R. Soc. open sci. 3, 1-14
In this work we investigated the role of environmental coloring in shoaling
preference of zebrafish (first experiment). In particular, our focus was to
determine whether zebrafish show a color preference for their habitat after a
sex-isolation period, and whether the environmental coloring can influence
the response to several fish social stimuli that differ for sex and number
composition.
Moreover, we wanted to investigate the behavioural responses to non-moving
artificial stimuli (second experiment). Specifically, we wanted to test whether
the presence of 2D images of a female or male zebrafish (phenotypically
altered by a graphics software) could influence the social behaviour of this
species.
Lastly, the goal was also to develop new methodologies, minimizing the use
of animals in research, in line with the provisions of the "3R framework".
We found significant sex-related differences between zebrafish responses to the different behavioral tests. Male
zebrafish shoaled more with an individual of the same sex rather than with a female. Females were more active but
no significant difference between the two sex-stimulus zones was shown for females. Similar to what was
documented in previous investigations, both males and females preferred social behavior instead of a zone without
individuals and only with environmental coloring. The role of environmental coloring on shoaling preference
remains unclear. Moreover, as shown by the second experiment, females shoaled more with visual stimuli
representing individuals of bigger size and large abdomen, and seem to prefer conspecifics of the same sex but
phenotypically distant from them. However the effective behavioural response to non-moving 2D stimuli is not
clarified and further studies are needed to deepen this issue.
Fig.2 Non-moving 2D stimuli used for
the second experiment.
Fig.1 Cross-maze used during the
behavioural tests for both the
experiments. The colours represent the
movements of a single individual: in
the red zone the animal spent more
time, and less in the blue one.
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