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Abstract

The zebrafish has become increasingly utilized in behavioral neuroscience, psychopharmacology, and behavior genetics research. However, little attention has been paid to the potential effects of environmental conditions fish are exposed to before and during behavioral testing. One such important factor is temperature, more specifically, the difference in temperature between holding and test tanks. In the current study, we bred and raised zebrafish of the AB strain in 28°C water for 7 days until they were free swimming. On the 7 th day we placed each experimental subject singly into the testing well with water set to 24°C, 28°C or 30°C for 15 minutes and recorded the behavior of the subjects. We found significant and behavior-specific idiosyncratic effects of the employed temperature changes. For example, lowering the temperature decreased speed, but increasing the temperature did not alter it. Increased temperature reduced duration of immobility and reduced absolute turn angle, but lowered temperature did not alter these behaviors. While lowered temperature, and to a lesser degree also increased temperature, reduced intraindividual temporal variance of absolute turn angle. Furthermore, we found no change in thigmotaxis and frequency of immobility by either temperature change. Our results demonstrate the importance of temperature in behavioral studies with zebrafish and suggest that equating water temperature between holding and testing tanks is required to enhance reproducibility and replicability of results with this species.
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Physiology & Behavior
journal homepage: www.elsevier.com/locate/physbeh
The effects of small but abrupt change in temperature on the behavior of
larval zebrafish
Amira Abozaid
a
, Benjamin Tsang
b
, Robert Gerlai
a,b,
a
Department of Cell & System Biology, University of Toronto
b
Department of Psychology, University of Toronto Mississauga
ARTICLE INFO
Keywords:
Zebrafish
Fear and anxiety
Locomotor behavior
Video-tracking
Environmental factors
Temperature
Replicability
ABSTRACT
The zebrafish has become increasingly utilized in behavioral neuroscience, psychopharmacology, and behavior
genetics research. However, little attention has been paid to the potential effects of environmental conditions
fish are exposed to before and during behavioral testing. One such important factor is temperature, more spe-
cifically, the difference in temperature between holding and test tanks. In the current study, we bred and raised
zebrafish of the AB strain in 28 °C water for 7 days until they were free swimming. On the 7th day we placed each
experimental subject singly into the testing well with water set to 24 °C, 28 °C or 30 °C for 15 min and recorded
the behavior of the subjects. We found significant and behavior-specific idiosyncratic effects of the employed
temperature changes. For example, lowering the temperature decreased speed, but increasing the temperature
did not alter it. Increased temperature reduced duration of immobility and reduced absolute turn angle, but
lowered temperature did not alter these behaviors. While lowered temperature, and to a lesser degree also
increased temperature, reduced intra-individual temporal variance of absolute turn angle. Furthermore, we
found no change in thigmotaxis and frequency of immobility by either temperature change. Our results de-
monstrate the importance of temperature in behavioral studies with zebrafish and suggest that equating water
temperature between holding and testing tanks is required to enhance reproducibility and replicability of results
with this species.
1. Introduction
Temperature is a crucial factor that influences biochemical reactions
and physiological responses, and thus may affect all biological pro-
cesses, including brain function and behavior of organisms.
Poikilothermic (ectothermic) species, e.g. fish, which are unable to
maintain a constant body temperature against environmental tem-
perature fluctuations may be particularly affected by temperature de-
pendent changes in biological processes [1]. Temperature effects may
be especially robust in organisms whose body surface to volume ratio is
large. This ratio increases with decreasing body size. Zebrafish is one of
the most frequently employed vertebrate laboratory organisms in bio-
medical research [2]. It is a small fish with a body length of 4 cm when
fully grown, and thus, it has large body surface to volume ratio. Fur-
thermore, the majority of zebrafish studies are conducted with larval
zebrafish [28] whose body surface to volume ratio is even larger.
Therefore, one may expect juvenile/larval zebrafish to be even more
affected by temperature variations in their environment.
The consequences of long-term developmental exposure to different
temperatures in zebrafish and other species are well appreciated with
studies documenting changes in a wide spectrum of phenotypical ef-
fects ranging from metabolism through immune function and sex de-
termination to a variety of behaviors [3–7]. Nevertheless, we know
much less about how short-term, abrupt, changes in temperature may
affect zebrafish, although pioneering studies have revealed effects of
acute temperature change on gene expression [8], nitrogen cycle and
metabolism [9], as well as circadian clock function [10]. This is an
important problem, because most experimental studies with zebrafish
involve removing the fish from their home (holding) tank and placing
them in an experimental apparatus, a test tank or well. Only rarely are
the temperatures of the water in the holding and experimental tanks
measured and reported, and even more rarely are they equalized. As-
suming that an abrupt temperature change is irrelevant may be in-
appropriate, especially for studies concerned with neuroscience, psy-
chopharmacology, or behavior genetics of zebrafish. Furthermore, such
potential temperature changes affect not only Central Nervous System
mechanisms but a variety of other processes too. Thus, given the rapidly
accelerating use of the zebrafish in practically every subfield of biology,
https://doi.org/10.1016/j.physbeh.2020.113169
Received 6 July 2020; Received in revised form 20 August 2020; Accepted 8 September 2020
Corresponding author: Robert Gerlai, Department of Psychology, Rm CCT4004, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, Ontario,
Canada, L5L 1C6.
Physiology & Behavior 227 (2020) 113169
Available online 10 September 2020
0031-9384/ © 2020 Elsevier Inc. All rights reserved.
T
... Immobility here is defined as fewer than 20% of the number of pixels corresponding to the total visible surface area of the subject changing from one video-frame to the next (with 30 frame per s temporal resolution). Although intuitively total distance travelled and immobility duration may seem to be redundant, negatively correlating measures, empirical data have suggested that they represent independent behavioral responses [41,44,71,75]. Frequency of immobility (number of immobility episodes) was also quantified as it may represent a behavioral strategy independent of the duration of immobility. ...
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