Sudden exposure to warm water causes instant behavioural responses indicative of nociception or pain in Atlantic salmon

Abstract

Thermal treatment has become the most used delousing method in salmonid aquaculture. However, concerns have been raised about it being painful for the fish. We studied the behavioural response of Atlantic salmon acclimated to 8 °C when transferred to temperatures in the range 0–38 °C. Exposure time was 5 min or until they reached the endpoint of losing equilibrium and laying on their side, a sign of imminent death. At temperatures below 28 °C, none of the fish reached endpoint within the 5-min maximum. At 28 °C four of five fish reached endpoint, and fish reached endpoint more rapidly as temperature increased further. Fish transferred to temperatures above 28 °C had higher swimming speed immediately after transfer and maintained a high swimming speed until just before loss of equilibrium. Their behaviour was from the start characterised by collisions into tank walls and head shaking. Just before loss of equilibrium they started breaking the surface of the water, swimming in a circle pattern and in some instances displayed a side-wise bending of their body. In other words, salmon transferred to temperatures above 28 °C showed instant behavioural responses indicative of nociception or pain. Keywords: Behavioural response, Fish, Heated water, Nociception, Pain, Thermal delousing

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Veterinary and Animal Science
journal homepage: www.elsevier.com/locate/vas
Sudden exposure to warm water causes instant behavioural responses
indicative of nociception or pain in Atlantic salmon
Jonatan Nilsson
a,⁎
, Lene Moltumyr
a
, Angelico Madaro
a
, Tore Sigmund Kristiansen
a
,
Siri Kristine Gåsnes
b
, Cecilie Marie Mejdell
b
, Kristine Gismervik
b
, Lars Helge Stien
a
a
Institute of Marine Research, P.O. Box 1870 Nordnes, NO-5817 Bergen, Norway
b
Norwegian Veterinary Institute, P.O. Box 750, 0106 Oslo, Norway
ARTICLE INFO
Keywords:
Behavioural response
Fish
Heated water
Nociception
Pain
Thermal delousing
ABSTRACT
Thermal treatment has become the most used delousing method in salmonid aquaculture. However, concerns
have been raised about it being painful for the fish. We studied the behavioural response of Atlantic salmon
acclimated to 8 °C when transferred to temperatures in the range 0–38 °C. Exposure time was 5 min or until they
reached the endpoint of losing equilibrium and laying on their side, a sign of imminent death. At temperatures
below 28 °C, none of the fish reached endpoint within the 5-min maximum. At 28 °C four of five fish reached
endpoint, and fish reached endpoint more rapidly as temperature increased further. Fish transferred to tem-
peratures above 28 °C had higher swimming speed immediately after transfer and maintained a high swimming
speed until just before loss of equilibrium. Their behaviour was from the start characterised by collisions into
tank walls and head shaking. Just before loss of equilibrium they started breaking the surface of the water,
swimming in a circle pattern and in some instances displayed a side-wise bending of their body. In other words,
salmon transferred to temperatures above 28 °C showed instant behavioural responses indicative of nociception
or pain.
1. Introduction
As the salmon louse (Lepeophtheirus salmonis) has increasingly de-
veloped resistance against chemotherapeutants, thermal delousing by
exposing fish to heated water (appr. 28–34 °C, sometimes higher) has
become the most used delousing method in salmonid and especially
Atlantic salmon (Salmo salar L.) aquaculture in Norway (Gismervik,
Gåsnes, Nielsen, amp; Mejdell, 2018b;Overton et al., 2018). Thermal
delousing is also used against Caligus elongatus, particularly in Northern
Norway, and Caligus rogercresseyi in Chile (Sitjà-Bobadilla &
Oitmann, 2017). Thermal delousing has been promoted as an en-
vironmentally friendly delousing method, as it only uses heated water.
There is, however, often elevated mortality after thermal delousing
compared to delousing the salmon with chemical baths or mechanical
removal of lice by seawater flushing (Overton et al., 2018). Concerns
have also recently been raised about thermal delousing being experi-
enced as painful by the fish (Poppe, Dalum, Røislien, Nordgreen &
Helgesen, 2018).
The ability of subjective experience, i.e. some level of consciousness
or awareness, is by definition a prerequisite for experiencing pain
IASP (1994), and there is an intense debate in the scientific literature on
whether fish have this ability (see for example Key, 2016 and
Sneddon et al., 2018 and the threads of responses to these articles).
Absolute evidence for subjective experience in fish or other animals are
obviously hard to obtain, and following the precautionary principle fish
are included in European welfare legislation: EU directive 98/58/EC
Article 2 includes ‘fish’in the term ‘animals’, and Article 3 states that
owners or keepers of animals must take all reasonable steps to ensure
the welfare of animals under their care and to ensure that those animals
are not caused any unnecessary pain, suffering or injury. The Norwe-
gian Food Safety Authorities are therefore concerned that thermal de-
lousing violates EU directive 98/58/EC and the Norwegian animal
welfare act stating that keepers of animals, including fish, must ensure
that the animals are treated well and are protected from danger of
unnecessary stress and strains.
Fish have nociceptors for heat (Nordgreen et al., 2009;
Sneddon, Brathwaite & Gentle, 2003), and salmonids exposed to warm
water respond with abnormal behaviour such as jumps from the water,
collisions and sudden swimming bursts (Elliott, 1991;Ineno, Tsuchida,
Kanda & Watabe, 2005). From delousing operations in the industry,
there are anecdotal reports about loud bangs and noises from within the
treatment chambers, and flight reactions during treatment are
https://doi.org/10.1016/j.vas.2019.100076
Received 7 February 2019; Received in revised form 5 July 2019; Accepted 25 September 2019
⁎
Corresponding author.
E-mail addresses: jonatan@hi.no,jonatan.nilsson@imr.no,jonatan@hi.no (J. Nilsson).
Veterinary and Animal Science 8 (2019) 100076
Available online 27 September 2019
2451-943X/ © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/BY/4.0/).
T

suspected to be the cause of many of the injuries often seen (Gismervik
et al., 2018a;Poppe et al., 2018). The water temperatures used for
thermal delousing (28–34 °C) may be lethal within minutes (reviewed
in Elliott & Elliott, 2010). The ultimate lethal temperature, i.e. the
temperature at which fish cannot survive for 10 min, has been found to
be 30–33 °C for Atlantic salmon parr and smolts (Elliott & Elliott, 2010).
The much shorter exposure time of 30 s usually used during thermal
delousing is however not likely to directly kill salmon, but tissue da-
mage after treatment has been observed (Gismervik et al., 2018b;
Poppe et al., 2018) although it is not known if this is due to temperature
per se or other aspects of the treatment.
Coldwater is a new candidate method for thermal delousing
(Overton et al., 2019). Live chilling induces stress in salmon
(Roth, Slinde & Robb, 2006) and temperatures below 0 °C may be lethal
(Elliott & Elliott, 2010). Elliott (1991) observed behavioural responses
to rapidly decreasing temperatures similar to the responses seen for
rapidly increasing temperatures. Thus, although no nociception has
been documented at low temperatures (Ashley, Sneddon & McCrohan,
2007), sudden exposure to very low temperatures appear to be aversive
to salmon.
In the present study, we aimed to investigate if there is a tem-
perature threshold within the range from 0 to 38 °C from where salmon
post-smolts transferred from 8 °C respond with clear behavioural
changes indicative of nociception or pain. The experiment was con-
ducted in accordance with Norwegian regulations on animal experi-
mentation under permit number 15383.
2. Material and method
2.1. Subjects
The subject fish were out-of-season Atlantic salmon (Salmo salar L.)
smolts of the AquaGen strain, hatched 16 January 2017 and transferred
from freshwater to tanks (1.5 m wide, 1200 L) with seawater (salinity
34 ppt, temperature 8–9 °C) on 17 October 2017, held on a natural light
regime and fed dry feed (Skretting Spirit Supreme) during daytime.
Four weeks before the start of the experiment, on 6 April 2018, 125 fish
were distributed into three tanks that were located next to the experi-
mental tank to minimize air exposure time and handling during transfer
to the experimental tank (see below for tank description). The fish were
stocked at ~8 kg m
–3
, well within the 17 kg m
−3
upper limit for good
welfare defined in the RSPCA welfare standards for farmed Atlantic
salmon (RSPCA, 2018). The fish were starved for two days before the
experiment started. The mean ± S.D. length and weight of the fish
during the experiment were 279 ± 23 mm and 234 ± 52 g, respec-
tively.
2.2. Experimental tank
The experimental tank where fish were exposed to seawater (34 ppt)
of various temperatures (Table 1) was of the same type as the stock
tanks: squared with rounded corners, 150 cm wide and light grey of
colour, but the water depth in this tank was restricted to 15 cm depth
(volume around 300 L) in order to facilitate temperature regulation.
The lid of the tank was partly lifted and a camera (GoPro™, San Mateo,
CA, USA) was attached to the lid so that it recorded the entire water
volume from above. Temperature (Testo 176T2, Testo Ltd., UK) and
oxygen (Handy Polaris 2, OxyGuard Inc., Denmark) were logged im-
mediately before a new fish was transferred to the tank. Water tem-
perature of 0 °C was obtained by adding blocks of frozen seawater to the
tank. Remaining ice was removed before fish were transferred to the
tank. Water temperature of 4 °C was thereafter obtained by adding
seawater of ambient temperature (8 °C). For the control group, which
was exposed to water of ambient temperature, i.e. exactly the same
temperature as they were transferred from, all water was replaced be-
fore transfer of fish. For the remaining groups, which were exposed to
higher temperatures than the ambient, water was regulated by adding
heated seawater. The exposure temperature never deviated more than
0.5 °C from the group mean for any individual fish.
2.3. Humane endpoints
The endpoint for taking an individual out of the experiment was
when it lost equilibrium and laid on the side for 2 s. Fish that reached
this endpoint was immediately netted out and euthanized by and
overdose of tricaine methanesulfonate (Finquel, Ayerst Laboratories,
New York). The rational for subjecting fish for both longer and higher
temperatures than the current industry standard was that we were
uncertain if the acute response to transfer itself could be distinguished
from the response to temperature within 30 s. Furthermore, it is im-
portant to study the “safety margins”in terms of temperature and ex-
posure time, especially as higher temperatures and longer treatment
times may be attempted if the salmon louse develop resistance to warm
water (Ljungfeldt, Quintela, Besnier, Nilsen & Glover, 2017).
2.4. Procedure
Single fish were netted out of the holding tank and immediately
released into the experimental tank. Each fish was left in the tank for
maximum 5 min or until it reached the endpoint. The fish was then
netted out and euthanized in an overdose of tricaine methanesulfonate
(Finquel, Ayerst Laboratories, New York). Temperature was controlled
and regulated by adding cold or warm seawater and then removing
excess water (if needed) before a new fish was transferred. The beha-
viour of the fish was recorded on video by the GoPro camera. From 0 to
24 °C the temperature was increased with 4 °C for each new tempera-
ture group, and from 24 °C with 2°C, in order to increase the resolution
in the higher temperature interval where the temperature threshold was
expected to be. Up to 36 °C each temperature group included six fish,
but only four fish were exposed to 38 °C as we from subjective esti-
mation of the behavioural response during exposure judged this tem-
perature to be unacceptably high. Instead of increasing the temperature
further, we exposed four fish to 37 °C to increase the resolution at high
temperatures. For practical reasons with heating and cooling of the
water the sequence of tests became: Day 1: 0, 4, 8, 20, 16, 12; Day 2: 28,
30, 32, 34, 36, 38; Day 3: 24, 26.
Four video files for the 0–36 °C groups were corrupt and could not
be opened and viewed, probably due to camera malfunction or mistakes
by the operator. The number of fish analysed in each temperature was
therefore only 5 for some of the groups (Table 1). All four videos were
analysed in each of the 37 and 38 °C groups (Table 1).
Table 1
Temperature groups, group sizes (n), measured exposure temperatures
(mean ± S.D.) and oxygen saturation (mean ± S.D., percent of air saturation)
in the experimental groups.
Group n Temperature ( °C) O
2
(%)
0 6 0.5 ± 0.3 86 ± 3
4 6 4.1 ± 0.1 95 ± 0
8 5 8.5 ± 0.0 99 ± 1
12 6 12.2 ± 0.2 100 ± 1
16 6 16.1 ± 0.2 99 ± 1
20 5 19.9 ± 0.2 100 ± 1
24 5 24.1 ± 0.2 109 ± 3
26 6 26.1 ± 0.2 101 ± 2
28 5 28.1 ± 0.3 108 ± 3
30 6 30.1 ± 0.2 102 ± 1
32 6 32.0 ± 0.2 99 ± 1
34 6 34.0 ± 0.2 100 ± 1
36 6 36.1 ± 0.2 97 ± 1
37 4 36.9 ± 0.1 95 ± 1
38 4 38.0 ± 0.1 96 ± 1
J. Nilsson, et al. Veterinary and Animal Science 8 (2019) 100076
2

2.5. Analysis of behaviour
2.5.1. Estimation of swimming speed
Swimming speed was estimated using a script created in MATLAB
R2018b (The MathWorks, Inc., USA). Input to the script was which
movie to analyse, and how many seconds since start of exposure the
analysis should be performed. The script then opened the movie at the
given time, and extracted four subsequent image frames, 1/3 of a
second apart (0, 1/3, 2/3 and 3/3 into the second). The script then
displayed the four images, and the position of the snout of the fish was
manually marked using the mouse. Euclidian distance between these
four consecutive points was then calculated as an estimation of swim-
ming speed per second at the given time and normalised to the length of
the fish in the images to standardise the speed into body length per
second (BL s
–1
). Since this analysis was relatively laborious, it was done
every second for the 10 first seconds of the exposures to get high re-
solution immediately after transfer, then every 5 s from 10 to 60 s of the
exposures, every 10 s from 60 to 200 s of the exposures, and finally only
every 20 s from 200 to 300 s of the exposures when behaviour had
stabilised.
2.5.2. Behavioural events
In addition to surface breaks and collisions as described by
Elliot (1991), three random video clips from high temperatures
(34–38 °C) was studied in order to find other easily countable abnormal
behaviours, e.g. behaviours assumed to reflect responses to high tem-
perature. These were then defined for use when behaviour of all tem-
perature groups was analysed and are described in Table 2. Video clips
showing examples of the behavioural categories are available in the
online supplementary material. During these analyses, the temperature
was blinded to the observer in order to avoid observation bias. An open
source software (CowLog 3.0.2, Pastell, 2016) was used to analyse the
videos for the behavioural categories. In this program, behavioural
categories defined by the user are programmed with keyboard shortcuts
that are pressed when a behaviour is observed. Frequency and latency
of each behavioural event is then the output from the analysis.
2.6. Statistics
Although some of the data could have been analysed using non-
linear parametric methods, all data are analysed using the same non-
parametric methods for consistency. The results are presented as
median [25-, 75-percentile] observed time to endpoint, median [25-,
75-percentile] swimming speed, or median [25-, 75-percentile] fre-
quency of behaviour event. Since the output data typically looked like
step functions (Crawley, 2007), non-parametric conditional inference
trees were used to objectively find thresholds and classify temperature
intervals according to swimming speed and behaviour frequencies (R
package party, function ctree, Hothorn, Hornik & Zeileis, 2006).
Spearman rank correlation was used to test if the observed values in-
creased or decreased with temperature (function cor.test,
method = "spearman", R version 3.5, R Core Team 2018).
3. Results
3.1. Time to endpoint
At 28 °C four of five fish reached endpoint (i.e. lost equilibrium and
laid on the side for >2 s) before end of the pre-set maximum exposure
time of 300 s. Median time of endpoint for these four fish was 251 [237,
266] s, or if including the fifth fish as a 300 s observation, the median
endpoint was 266 [238, 267] s. While none of the fish exposed to
<28 °C reached the endpoint, all the fish that were exposed to tem-
peratures >28 °C did so, with time to endpoint decreasing with in-
creasing temperature (r
s
= 0.96, p= 0.002, Fig. 1).
3.2. Swimming speed
All temperature groups had an initial peak in swimming speed im-
mediately after being released into the treatment tank (Fig. 2A). The
ctree-algorithm identified two distinct increasing steps for initial
swimming speed (first 10 s), one threshold between 28 and 30 °C
(p<0.001), and one threshold between 34 and 36 °C (p<0.001)
(Fig. 2B). After the first 10 s, the fishes in all groups reduced their
swimming speed (Fig. 2A), but the fishes subjected to 28–38 °C main-
tained a higher swimming speed than the fishes subjected to the lower
temperatures (Fig. 2C), until the speed dropped as they approached
their endpoint (Fig. 2A and Fig. 1).
3.3. Behaviour events
The ctree-algorithm identified a clear step in observed ‘Direction
change’per minute of observation time between 28 and 30 °C
(p<0.001, Fig. 3A). However, ‘Direction change’occurred frequently
during the first 100 s of the exposure at all temperatures (Fig. 4A), but
the lower frequency later in the observation period resulted in different
“per minute of observation time”levels (Fig. 3A). ‘Collision’mainly
occurred above 30 °C, where the ctree-algorithm identified a clear step
(p<0.001, Fig. 3B). However, directly comparing collisions at 28 °C
with observed collisions for the controls gives significant more colli-
sions compared to controls also for this relatively low temperature
(0.00 [0.00, 0.00] vs. 0.45 [0.00, 1.26] obs. min
−1
,r
s
= 0.64,
p= 0.045). The fish started colliding immediately after transfer to ex-
posure temperatures and continued to do so until they reached the
endpoint (Fig. 4B). ‘Head shake’was not observed below 20 °C, from
where the ctree-algorithm identified a minor step (p= 0.004), and two
Table 2
Behavioural events counted during video analyses.
Behaviour Description
Direction change The fish changes swimming direction with >90° within 1
body length
Circling The fish swims in a circle (>270°)
Head shake The fish makes rapid shakes with the head
Collision Uncontrolled collision with the tank wall
Surface break The fish breaks the surface with its head
Bend The fish bends the body sideways >90° without moving
forward
Fig. 1. Median time to endpoint (laying motionless on the side for >2 s) at
different temperatures. The error bars represent the 25- ant 75-percentiles. No
fish reached the endpoint within the maximum exposure time of 300 s at
temperatures below 28 °C. Point colouring is given for easy identification of
temperatures.
J. Nilsson, et al. Veterinary and Animal Science 8 (2019) 100076
3

more clear steps between 24 and 26 °C (p<0.001) and after 34 °C
(p<0.001, Fig. 3C). At 20 and 24 °C the majority of the head shakes
occurred during the first 30 s (Fig. 4C). At 26 and 28 °C head shakes
occurred mainly in the beginning and the end of the observation period,
while at higher temperatures they occurred throughout the observation
period (Fig. 4C). ‘Circling’was only observed at 26 °C or higher, with
the highest rate at the highest temperatures (Fig. 3D), and usually
started to occur shortly (~1 min) before the endpoint (Fig. 4D). Fish
performing ‘Circling’typically swam partly with the side facing up-
wards but still moved relatively fast. ‘Surface break’occurred at highest
rates at the higher temperatures, with a clear threshold between 26 and
28 °C (p<0.001, Fig. 3E), but there were also random occurrences at
the lower temperatures (Fig. 4E). As with ‘Circling’,‘Surface break’was
generally most frequent during the last minute before the fish reached
their endpoint (Fig. 4E). ‘Bend’was never observed at temperatures
below 30 °C. In contrast, 47% of the fish from 30 °C and above per-
formed ‘Bend’, but with high variation between individuals (1–35
bends) (Fig. 3F). ‘Bend’typically occurred just before the fish reached
the endpoint (Fig. 4F), with median occurrence of first ‘Bend’15.2 [9.5,
25.0] s before the endpoint.
4. Discussion
When transferred to the experimental tank salmon responded with
elevated swimming speed and frequent changes of swimming direction
at all temperatures, although the responses were stronger at the highest
temperatures. However, while fish exposed to low and intermediate
temperatures calmed down within half a minute, fish exposed to tem-
peratures above 28 °C sustained a high swimming speed, showed be-
haviour such as ‘Collision’,’Surface break’and ‘Bend’, before they lost
equilibrium and eventually stopped moving. The behavioural responses
occurred earlier the higher the temperature.
To our knowledge this is the first study of salmon behaviour after
direct transfer into high temperatures. Peterson and Anderson (1969)
exposed salmon parr to rapid (~30 min) changes in temperature from
6 °C to maximum 21 °C, and from 18 °C to maximum 27 °C and
minimum 6 °C. The fish responded during the temperature changes with
marked increases in activity level, which then quickly died down once
the temperature had stabilised, except for the fish on 28 °C which
maintained high activity also after the change was complete.
Elliott (1991) exposed salmon parr to temperatures up to 36 °C, but did
so by gradually elevating the temperature 1 °C h
–1
rather than by a
sudden transfer as in the present study. The study by Elliott focused on
thermal tolerance in terms of feed intake and survival and gives only a
brief description of the behavioural responses. Still, the responses de-
scribed by Elliott, which included ‘sudden bursts of activity with fre-
quent collisions with the tank sides, rolling and pitching’, followed by
‘short bursts of weak swimming’, and the maintained high activity on
27 °C described by Peterson and Anderson, correspond well to the re-
sponses described in the present study. Elliott also noted that in the last
phase before death, movements were restricted to the opercula, pec-
toral fins and eyes, but such detailed observations were not possible in
the present study.
There was little response to cold water and no differences from the
control (8 °C) was found. Nociceptors do not respond to low tempera-
tures (Ashley et al., 2007), and strong behavioural responses were
therefore not expected. Elliott (1991) reports that for salmon parr,
thermal stress due to falling temperature resulted in sudden bursts of
activity followed by a coma-like state. This coma-like state was also
seen in the cold-water delousing experiment by Overton et al. (2019),
but the temperature drop was here from 15 °C, and not from 8 °C as in
the present study. In the present study, no fish lost equilibrium within
Fig. 2. Swimming speed per temperature
group. A) Measured swimming speeds from
start of exposure till endpoint, or maximum
300 s of exposure. B) Median swimming speed
first 10 s. C) Median swimming speed in the
period 10–30 s after exposure. The error bars
represent the 25- and 75-percentiles. *p<
0.05, **p< 0.01, ***p< 0.001 indicate sig-
nificance of the thresholds found by the ctree-
algorithm. Point colouring is given for easy
identification of temperatures. (For inter-
pretation of the references to colour in this
figure legend, the reader is referred to the web
version of this article.)
J. Nilsson, et al. Veterinary and Animal Science 8 (2019) 100076
4

5 min at the lower temperatures, and swimming speed was classified as
being in the same group as the control by the ctree-algorithm.
Temperature is one of the main drivers for spatial distribution of
caged salmon, and salmon actively avoid temperatures above 18 °C
(reviewed by Oppedal, Dempster & Stien, 2011). Still, signs of alarm in
terms of frequent collisions, surface breaks and increased swimming
speed were not significant when salmon were transferred directly from
the 8 °C acclimation temperature to tanks holding up to 26 °C. At 28 °C
initial median swimming speed did not differ from the control, but the
variation was high with some individuals swimming fast, and the fish at
28 °C maintained high median swimming speed throughout. Also, the
identified step thresholds for the frequencies of the different beha-
vioural events varied to start between 26 and 28 °C (‘Circling’,‘Surface
break’), between 28 and 30 °C (‘Direction change’,‘Bend’) or between
30 and 32 °C (‘Collisions’), except for ‘Head shake’which started al-
ready at 20–24 °C. All in all, and also considering that comparing col-
lisions at 28 °C revealed significant difference from the control tem-
perature, makes it reasonable to set an overall thresholds for when
temperature is acutely aversive around 28 °C. This was also the lowest
temperature at which fish reached the endpoint within the 5-min ob-
servation period.
At the endpoint when a fish had stopped moving and lay on the side
it was removed from the tank and euthanized, except for one individual
that was returned to an 8 °C tank for several minutes. This fish did not
regain any signs of motion and had no opercular movements, it was
dead. Severe tissue damage, including gill- and brain bleeding, was
found on a subset of fish that were examined for tissue damage after
exposure to 34–38 °C (Gismervik et al., 2019). Although no systematic
attempts were made to study the probability to survive after reaching
the endpoint, we therefore assume this state to be life threatening.
Elliot (1991) found, by elevating temperature relatively slowly (1 °C
h
–1
), that 31 °C was the highest temperature at which salmon parr ac-
climated to 10 °C survived for 10 min, while fish in the present study
reached the endpoint and were likely doomed within 5 min at 28 °C,
and for 38 °C already within 1.5 min. With different methods and life
stages in these two studies comparisons should be made with caution,
but it is possible that the rapid onset of loss of equilibrium and probable
death in the current experiment can be explained by the temperature
shock at direct transfer to the exposure temperature. Fish size may also
play a role for the temperature tolerance. Intuitively one might expect
that larger fish, with a lower surface-area-to-volume ratio, are more
resistant to high temperatures (Stevens & Fry, 1970). However,
Huntsman (1942), studying thermal death of wild salmon in a river
during hot summers, reported that at 29.5 °C the largest salmon died
first, and that ‘of at least 12 grilse in the vicinity only 4 died and not a
single parr’. Decreasing temperature tolerance with increasing size was
subsequently confirmed experimentally (Huntsman, 1942).
Ashley et al. (2007) showed that mechanothermal nociceptors in
rainbow trout had a thermal threshold at ~29 °C, while the polymodal
nociceptors had a thermal threshold at ~33 °C. These receptors are in
the skin, explaining the instant reaction to temperatures above 28 °C,
before internal physiological processes can have been affected and
Fig. 3. Median observed frequency of behavioural events per minute before end of exposure (300 s) or laying on the side for the fish in each temperature group
0–38 °C. A) Direction change, B) Collison, C) Head shake, D) Circling, E) Surface break, F) Bend. The error bars represent the 25- and 75-percentiles. *p< 0.05, **p<
0.01, ***p< 0.001 indicate significance of the thresholds found by the ctree-algorithm. Point colouring is given for easy identification of temperatures.
J. Nilsson, et al. Veterinary and Animal Science 8 (2019) 100076
5

caused behavioural impairment. The further increase in swimming
speed identified between 34 and 36 °C seen in Fig 2B could reflect that
also the polymodal receptors respond to the highest temperatures. The
salmons’behavioural reactions to high temperatures were in-
stantaneous, not only with fast swimming, but also with collisions into
tank walls and head shaking (Fig 3B and C, Fig 4B and C), suggesting a
response with loss of control of their behaviour.
The temperature threshold for strong behavioural responses, i.e.
28–30 °C, corresponds with the lethal temperature threshold. A similar
notion was made by Ashley et al. (2007) for rainbow trout (Oncor-
hynchus mykiss), where the heat threshold for nociceptors to respond
(29–33 °C) was within the range of short-term lethal temperature of
30 °C (Ineno et al., 2005). It is therefore reasonable to assume that the
behavioural responses to high temperatures reflect nociception, and
that the nociceptors have evolved to respond at temperatures that may
lead to tissue damage and ultimately death. Although
Ashley et al. (2007) found the mean thresholds for the mechanothermal
and the polymodal nociceptors at respectively ~29 °C and ~33 °C, they
also report some measurements down to 20 °C for the polymodal and
down to 22 °C for the mechanothermal. There were, however, little
evidence that such early responses of nociceptors were intense enough
to elicit significant behavioural responses in the current study.
‘Head shake’was performed by no fish below 20 °C, by one in-
dividual at 20 °C and by most individuals at 24 °C and above. At 20 and
24 °C ‘Head shake’was only performed early during the observation
period, at 26 and 28 °C they were performed early and then subsided,
and performed again late in the observation period, while at 30 °C and
above ‘Head shake’was performed throughout the period until they
Fig. 4. Behaviour map showing the proportion of fish at each exposure temperature performing a behavioural response in each 10-s interval of the observation
period, as indicated by the colour scale. White fields indicate that all fish at that temperature have reach the endpoint. A) Direction change, B) Collison, C) Head
shake, D) Circling, E) Surface break, F) Bend.
J. Nilsson, et al. Veterinary and Animal Science 8 (2019) 100076
6

approach the endpoint. Observations of head shakes was difficult for
fish with rapid swimming, surface breaks etc., and thus the occurrence
of ‘Head shake’was likely underestimate at the higher temperatures. It
is difficult to know exactly why the fish were shaking their heads.
Roberts, Johnson and Casten (2004) observed that rainbow trout with
bullae on the gills from parasites would shake their heads as a response
to the irritation. As the response subsided at 20–24 °C it may be that the
fish initially experienced some irritation that was lost when they had
overcome the transition shock, while at higher temperatures the irri-
tation returned (26–28 °C) or remained (30 °C and above).
Shortly before the fish exposed to the higher temperatures reached
the endpoint, they changed behaviour and lost swimming control,
moved in circle patterns and splashed in the surface, and some fish even
showed an abnormal bending of their body. If these abnormal beha-
viours arise because of increasing nociception or pain in the skin,
possible nociception or pain from organ failure (‘deep pain’),or if they
rather reflect neurological and/or physiological impairment and loss of
muscle control, or a combination of these, cannot be judge by visual
observations alone. However, it can be argued that both temperature
itself and possible breakdown of body functions cause nociception or
pain, and therefore that the experience of alarm increases with tem-
perature and exposure time.
The struggling behaviour during exposure to warm water would
increase the risk for mechanical damage, which may contribute to the
relatively high mortality associated with thermal delousing
(Overton et al., 2018). In the present study, the threshold for nocifen-
sive or pain behaviour was around 28 °C. However, all individuals were
acclimated to 8.5 °C. Acclimation temperature matters for critical
temperature for survival, with the critical temperature around 3 °C
higher when salmon parr were acclimated to 20 °C than to 5 °C
(Elliott, 1991). Anttila et al. (2014) found an even larger effect of ac-
climation temperature on cardiac capacity. While temperature induced
death is usually the result of inner physiological impairment such as
cardiac collapse (Anttila et al., 2014), the acute behaviour response is
more likely due to nociceptors, and we do not know if the threshold for
these nociceptors to respond is affected by acclimation temperature.
A criticism of the study may be that for practical reasons the order
of the temperature tests where not randomised, but often incremental
due to practical considerations when adding buckets of cooled or he-
ated seawater to regulate water temperature between tests. This meant
that water was only changed to a limited degree between fish, and
especially between fish tested on the same temperature. However,
plotting median initial swimming speed per fish on day 2 of the test
procedure with temperature test sequence 28, 30, 32, 34, 36, 38, 37°
reveals completely random patterns between fish within the same
temperatures (Fig. 5). This supports that the observed behaviours of the
fish are responses to changes in temperatures, and not responses to any
residues left in the water from the previous fish.
In conclusion, the present study suggests, based on behavioural
observations, that temperatures above ~28 °C are acutely aversive to
salmon and results in nocifensive or pain responses within seconds.
Ethical statement
The experiment was conducted in accordance with Norwegian
regulations on animal experimentation under permit number 15383.
Declarations of interest
None
Declaration of Competing Interest
All authors are employees at independent national research in-
stitutes and have no affiliations with or involvement in any organiza-
tion or entity with any financial or non-financial interest in the subject
matter or materials discussed in this manuscript.
Acknowledgements and funding
This work was supported by Institute of Marine Research and
Norwegian Veterinary Institute in connection with research based
governmental support ordered by the Norwegian Food Safety
Authorities. We would like to thank the staffat Matre Research station
for technical assistance.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.vas.2019.100076.
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