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Elevated carbon dioxide has the potential to impact alarm cue responses in some freshwater fishes

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Freshwater fish behaviors have the potential to be impacted by acidification due to increases in dissolved carbon dioxide (CO2). Recent work in the marine environment suggests that increased CO2 levels due to climate change can negatively affect fishes homing to natal environments, while also hindering their ability to detect predators and perform aerobically. The potential for elevated CO2 to have similar negative impacts on freshwater communities remains understudied. The objective of our study was to quantify the effects of elevated CO2 on the behaviors of fathead minnows (Pimephales promelas) and silver carp (Hypophthalmichthys molitrix) following exposure to conspecific skin extracts (alarm cues). In fathead minnows, their response to conspecific skin extracts was significantly impaired following exposure to elevated CO2 levels for at least 96 h, while silver carp behaviors were unaltered. However, fathead minnow behaviors did return to pre-CO2 exposure in high-CO2-exposed fish following 14 days of holding at ambient CO2 levels. Overall, this study suggests there may be potential impacts to freshwater fishes alarm cue behaviors following CO2 exposure, but these responses may be species-specific and will likely be abated should the CO2 stressor be removed.
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Elevated carbon dioxide has the potential to impact alarm
cue responses in some freshwater fishes
John A. Tix .Caleb T. Hasler .Cody Sullivan .Jennifer D. Jeffrey .Cory D. Susk
Received: 2 May 2016 / Accepted: 6 September 2016
ÓSpringer Science+Business Media Dordrecht 2016
Abstract Freshwater fish behaviors have the poten-
tial to be impacted by acidification due to increases in
dissolved carbon dioxide (CO
2
). Recent work in the
marine environment suggests that increased CO
2
levels due to climate change can negatively affect
fishes homing to natal environments, while also
hindering their ability to detect predators and perform
aerobically. The potential for elevated CO
2
to have
similar negative impacts on freshwater communities
remains understudied. The objective of our study was
to quantify the effects of elevated CO
2
on the
behaviors of fathead minnows (Pimephales promelas)
and silver carp (Hypophthalmichthys molitrix) follow-
ing exposure to conspecific skin extracts (alarm cues).
In fathead minnows, their response to conspecific skin
extracts was significantly impaired following expo-
sure to elevated CO
2
levels for at least 96 h, while
silver carp behaviors were unaltered. However,
fathead minnow behaviors did return to pre-CO
2
exposure in high-CO
2
-exposed fish following 14 days
of holding at ambient CO
2
levels. Overall, this study
suggests there may be potential impacts to freshwater
fishes alarm cue behaviors following CO
2
exposure,
but these responses may be species-specific and will
likely be abated should the CO
2
stressor be removed.
Keywords Acidification Olfaction Climate
change Behavior Fathead minnows Silver carp
Introduction
Fishes have a keen sense of olfaction (Hara 1993),
which is important across a variety of life stages (Lima
and Dill 1990) and for a number of processes,
including predator avoidance (Fuiman and Magurran
1994), kin recognition (Gerlach et al. 2008) and
habitat selection (Dittman and Quinn 1996). Olfaction
is particularly important for fishes within the super-
order Ostariophysi, as it has been shown that chemical
cues used by this group can elicit predator avoidance
behaviors (e.g., refuging, shoaling, darting and freez-
ing) when fish are exposed to injured conspecifics or
heterospecifics, due to alarm cues or pheromones that
they possess within the skin (Pfeiffer et al. 1985;
Chivers and Smith 1998; Brown et al. 2000). As is the
case for many life processes in fish, environmental
stressors, such as reduced pH (Leduc et al. 2013),
Handling Editor: Piet Spaak.
Electronic supplementary material The online version of
this article (doi:10.1007/s10452-016-9598-8) contains supple-
mentary material, which is available to authorized users.
J. A. Tix C. T. Hasler C. Sullivan
J. D. Jeffrey C. D. Susk (&)
Department of Natural Resources and Environmental
Sciences, University of Illinois Urbana-Champaign,
W-503 Turner Hall, 1102 South Goodwin Ave, Urbana,
IL 61801, USA
e-mail: suski@illinois.edu
123
Aquat Ecol
DOI 10.1007/s10452-016-9598-8
runoff (Fisher et al. 2006) or pollutants (Hara et al.
1976), can negatively influence olfaction capabilities
in fish, thereby reducing their ability to sense and/or
respond to information contained within chemical
cues.
One environmental stressor that potentially could
influence olfaction in Ostariophysi is the rise of carbon
dioxide (CO
2
) concentrations in freshwater, which
also has a concomitant effect of reducing pH. Acid-
ification of water in the context of acid rain (i.e., drop
of pH from 8.0 to near 4.0 due to the addition of strong
acids, such as nitric or sulfuric acid) has been shown to
negatively impact olfaction and alarm cue responses in
fishes, largely through two mechanisms (Lemly and
Smith 1985; Brown et al. 2002; Leduc et al.
2004,2009,2013). First, one of the main chemical
components of the alarm pheromone is hypoxan-thine-
3(N)-oxide (H
3
NO), and the structure of this molecule
can be altered in water at a pH of 6.0 making it
undetectable (Brown et al. 2002). Second, olfaction
can be negatively impacted by reduced pH because the
sensitivity or affinity of olfactory receptors may be
reduced at low pH, evidenced by the fact that fathead
minnows showed a reduced feeding response to amino
acids at pH 6.0 (Lemly and Smith 1985). These studies
have shown that acidification of freshwater due to
factors such as acid rain can potentially impact
olfaction, as well as the alarm cue responses of
freshwater fishes.
Recently, research related to ocean acidification in
the context of climate change has shown that not
only can the reduction in pH related to ocean
acidification disrupt the alarm cue responses of fish,
but the rise in dissolved CO
2
can also negatively
impact olfaction (Dixson et al. 2010; Munday et al.
2010). In the marine environment, the partial pres-
sure of CO
2
(pCO
2
) in water can increase due to
many factors (e.g., atmospheric levels of CO
2
, seeps,
upwellings, etc.), and pCO
2
in the oceans has
increased over the past several decades due to
increases in atmospheric CO
2
(Ciais et al. 2013).
The increase in dissolved CO
2
causes respiratory
acidosis in fishes (Heuer and Grosell 2014), and this
acidosis results in a disruption of cellular ionic
gradients. It has been well established that this
change in cellular ionic gradients alters GABA
A
receptor function, which, in turn, impacts fish
behavior (Nilsson et al. 2012; Regan et al. 2016).
More specifically, exposure to elevated pCO
2
has
been found to have a range of negative impacts for
fish including a loss of anti-predator responses (Allan
et al. 2013), changes in auditory preferences (Simp-
son et al. 2011), increased activity levels (Munday
et al. 2010; Ferrari et al. 2011), poor prey detection
and feeding (Cripps et al. 2011) and negatively alters
visual risk assessment (Ferrari et al. 2012). More
importantly, Dixson et al. (2010) showed that
settlement-stage orange clownfish Amphiprion per-
cula larvae were attracted to the smell of a predator
and could not distinguish between a predator and
non-predator odor cues, following exposure to
elevated levels of pCO
2
for a short duration (11 days
post-hatch), which could have important conse-
quences for survival. Laboratory tests have also
shown that marine juvenile damselfish exposed to
CO
2
-acidified water for 4 days displayed impaired
responses to conspecific alarm cues (Ferrari et al.
2011). Despite these findings, behavioral changes
driven by elevated pCO
2
have been found to be
variable across fish species, as no change to behav-
iors such as predator avoidance, lateralization and
swimming kinematics have been observed in some
fish species despite extended exposures to high pCO
2
(Jutfelt and Hedga
¨rde 2013; Maneja et al. 2013;
Sundin and Jutfelt 2015). The rise in CO
2
and
concomitant reduction in pH does inhibit olfaction in
some marine species, and therefore, it may be
possible that similar response exists for freshwater
fishes.
In freshwater, pCO
2
levels can vary across water-
sheds (Cole et al. 1994), as well as on episodic,
seasonal and diel cycles (Maberly 1996; Riera et al.
1999). For example, levels of pCO
2
in freshwater are
naturally variable, and ranged from 107 to 4128 latm.
across 62 lakes measured globally (Cole et al. 1994),
and, within a lake, free CO
2
may increase sevenfold
above atmospheric concentrations in Fall, Winter and
early Spring (Maberly 1996). Furthermore, freshwater
fishes may experience elevated pCO
2
due to a number
of mechanisms including a rise in atmospheric CO
2
(Phillips et al. 2015), increased terrestrial primary
productivity (Arneth et al. 2010), hatchery rearing
(Colt and Orwicz 1991) or the deployment of a non-
physical barrier that use zones of elevated CO
2
gas to
prevent fish movements (Noatch and Suski 2012).
Understanding the mechanism behind potential
increases in pCO
2
, coupled with a reduction in pH,
is essential for predicting consequences of elevated
Aquat Ecol
123
pCO
2
on the behavior of freshwater fish (Hasler et al.
2016).
Based on this background, the objectives of this
study were to (1) determine how exposure to elevated
of pCO
2
would change olfactory predator avoidance
behaviors of fathead minnows (Pimephales promelas)
and silver carp (Hypophthalmichthys molitrix) and (2)
if impaired olfaction behavior occurred, determine
whether ‘normal’ behaviors re-establish after fish are
returned to ambient conditions. To accomplish these
goals, fathead minnows and silver carp were exposed
to one of three different levels of CO
2
(ambient, low,
high) for at least 4 days and were then exposed to
conspecific skin extracts. Naı
¨ve fish in holding tanks
were returned to ambient pCO
2
for at least 11 days
prior to undergoing the same behavioral trials.
Materials and methods
Experimental animals
Adult fathead minnows were obtained from Logan
Hollow Fish Farm (Murphysboro, IL) and transported
to the University of Illinois Aquatic Research Facility
for experimentation (Urbana, IL; travel time 3.25 h),
while experiments with hatchery-reared silver carp
took place at the Upper Midwest Environmental
Sciences Center (UMESC; La Crosse, WI). Fathead
minnows were placed in a 3 w/v% salt (NaCl) bath for
30 s to disinfect and promote fish health upon arrival
at the aquatic facility (Swann and Fitzgerald 1991).
Fathead minnows were then divided into three groups
of &200 and held in separate 379 l plastic holding
tanks supplied with oxygen through an air stone
attached to an air blower and water from a 0.04 ha,
earthen pond. About 1.2 g/l of salt was added and
manually flushed out each day for the first 2 days of
laboratory acclimation to further reduce stress and
promote fish health (Swann and Fitzgerald 1991).
Fathead minnows were given a total of 5 days to
recover from transport and acclimate to laboratory
conditions prior to the onset of experiments. Waste
was siphoned, and 50 % water changes occurred one
to three times daily to ensure ammonia levels
remained low (measured using: Hach Company, kit
224100, Loveland, CO, USA). Silver carp at UMESC
were collected from a common holding tank, sepa-
rated into groups of 20, and placed into re-circulating
flow-through 230 l tanks supplied with well water.
For both fathead minnow and silver carp, water
quality was monitored daily for the duration of the
experiment: temperature, dissolved oxygen (DO)
(YSI, 550A Yellow Springs Instruments, Irvine, CA,
USA), total alkalinity (TA) (Hach Company, Titrator
model 16,900 and kit 94399, Loveland, CO, USA) and
pH (WTW pH 3310 m with a SenTix 41 probe,
Germany); the pH probe was calibrated daily during
this study (Moran 2014). In addition, pCO
2
was
measured daily during silver carp trials using an
infrared CO
2
sensor (Vaisala, Carbon Dioxide Trans-
mitter Series GMT220, Finland) wrapped in a semi-
permeable polytetrafluoroethylene cover (Johnson
et al. 2010; Munday et al. 2014) (Supplementary
Table S1). During fathead minnow trials, pCO
2
was
quantified by entering temperature, pH and alkalinity
data into CO2Calc (Robbins et al. 2010;http://pubs.
usgs.gov/of/2010/1280/) using all other parameters as
constants. All fish were fed commercial pellet feed
until satiation every day.
pCO
2
exposure treatments
In the treatment tanks (379 l for fathead minnows and
227 l for silver carp), fish were exposed to one of three
different CO
2
treatments: control (ambient) (&750),
low pCO
2
(&1500) and high pCO
2
(&7500 latm.;
Supplementary Table S1). These treatment levels were
chosen because (a) Kates et al. (2012) found that short-
term exposure to 70 mg/l of CO
2
(&150,000 latm.)
altered ventilation rates and caused behaviors indica-
tive of ‘stress’ (e.g., surface ventilations, coughing,
loss of equilibrium) suggesting that a holding level
below 150,000 latm. would prevent such conse-
quences, (b) many marine acidification studies that
have demonstrated an impact of CO
2
exposure on
olfactory responses targeted &1500 latm. for high
CO
2
exposure, which is a future projection of pCO
2
in
marine ecosystems (Forsgren et al. 2013; Jutfelt et al.
2013) and (c) Heuer and Grosell (2014) indicated that
the use of multiple CO
2
levels within a single study can
help define mechanisms of CO
2
impacts. Even though
no previous ocean acidification studies on fish have
used pCO
2
levels as high as 7000 latm. as an
experimental treatment, pCO
2
in freshwater ecosys-
tems fluctuates widely and can experience higher
levels of pCO
2
than marine ecosystems (Leduc et al.
2013; Hasler et al. 2016), making 7000 latm. a
Aquat Ecol
123
relevant and valuable level for holding. Target pCO
2
levels were held constant using a Pinpoint pH Regu-
lator Kit (American Marine Inc., Ridgefield, CT, USA)
(Munday et al. 2012; Allan et al. 2013) adjusted to add
CO
2
to the water when water pH rose above a set level
(The pH set level to target the corresponding pCO2
levels for fathead minnows was low =8.20, high =7.25
and for silver carp was low =7.35, high =7.05; Gattuso
et al. 2010). A homogenous mixture of CO
2
was
achieved in the holding tanks by using an air stone
connected to a 1.80 amp air compressor (Sweetwater,
Aquatic Eco-Systems, Apopka, FL, USA), which also
prevented hypoxia. Fathead minnows were held in the
treatment tanks for 4–12 days, while silver carp were
held for 4–10 days prior to commencing behavioral
testing. This exposure duration was chosen based on
previous work that has shown the potential for
olfactory behavioral impairments in fish to occur
following 96 h of continuous exposure to elevated
pCO
2
(Munday et al. 2010; Ferrari et al. 2011).
Following the behavioral tests (described below), all
holding tanks were returned to ambient pCO
2
for
11–14 days for fathead minnows and 14–17 days for
silver carp by replacing CO
2
-rich water in the tank with
water at ambient levels. Ambient pCO
2
levels varied
and were higher than many marine studies, as pCO
2
levels in freshwater typically experience both daily and
seasonal fluctuations (Maberly 1996). For example,
pCO
2
in a productive lake was shown to be depleted to
almost zero during the day and replenished during the
night causing an overall shift in pH of 1.8 units
(Maberly 1996).
Alarm cue extraction
Alarm cue stimuli preparation methods were adapted
from Mathis and Smith (1993). Stimuli were prepared
from 90 fathead minnows and 45 silver carp that had a
mean fork length of 5.04 ±0.70 standard error (SE)
and 11.67 ±2.05 cm, respectively. Breeding-condi-
tioned males and females, identified by the presence of
gametes, were not used as breeding males prohibit the
production of alarm cues (Smith 1973). Donor fathead
minnows and silver carp were euthanized by snipping
off their heads with scissors, and skin from both sides
of each fish was removed using a scalpel. The length
and width of each skin sample were measured, and the
total area of skin collected was &274.4 and 891.6 cm
2
for fathead minnows and silver carp, respectively.
Skin samples were immediately placed in 600 ml of
chilled ultra-pure water (&5°C) and homogenized
with a disperser (T18 Basic Ultra-Turrax, IKA,
Germany). The homogenate was filtered through glass
wool to remove scales and other solid particles and
then was further diluted by the addition of 1800 ml of
ultra-pure water (total volume was 2400 ml) and
stored at -20 °C in 30 ml aliquots until use. Addi-
tionally, 30 ml aliquots of ultra-pure water were stored
at -20 °C and used as a control (Little et al. 2011).
Behavioral trials
To quantify behavioral responses of fathead minnows
and silver carp to skin extracts, a flume channel
(Choice Tank, Loligo Systems, Denmark; Jutfelt and
Hedga
¨rde 2013) containing a 32 940 cm arena with a
water depth of 15 cm was used. Two 208 l vertical
header tanks, outfitted with an air stone to facilitate
aeration, as well as a small fountain pump to facilitate
mixing of water, were attached to the flume and water
flowed from the header tanks into the choice channel
by gravity. One header tank was identified as a
‘control’ tank, while the second tank was identified as
the ‘treatment’ tank, and the treatment tank received
skin extracts (skin extracts were always added to the
same ‘treatment’ tank to prevent contamination of the
‘control’ tank). A valve downstream of the header
tanks allowed the flume to receive water from either
header tank with minimal interruption to water flow.
Both vertical header tanks were filled with equal
amounts of ambient freshwater (pond water for
fathead minnows and well water for silver carp) as
effects of CO
2
on fish behavior is not altered by
different experimental test water (Munday et al. 2016).
At the commencement of the ‘acclimation’ period
and prior to a fish being placed into the arena, one
30-ml aliquot of ultra-pure water (described above)
was added to the control tank and given 10 min to mix.
A valve on the control tank was then opened, and the
choice area received water at a flow rate of 6.7 l/min
(verified with a flow meter; 807 series Rotameter,
Georg Fischer, Schaffhausen, Switzerland). The out-
flowing water from the arena was captured at the outlet
in a 49.2 l plastic tub and returned to the header tank
via a 124 W submersible pump, thus creating a re-
circulating system. A single fish was carefully netted
from one of the selected holding tanks (treatment was
selected randomly using a random number generator),
Aquat Ecol
123
placed into the arena and allowed 1 h to acclimate. A
1-h acclimation period was chosen as preliminary
trials indicated that this period of time was sufficient to
reduce increased freezing and darts behaviors follow-
ing introduction to the arena, and for the fish to begin
exploring the choice area; previous studies have also
used a similar 1-h acclimation period (e.g., De
Robertis et al. 2003). A single fish was tested at a
time (as opposed to testing multiple individuals
concurrently) to obtain a response not influenced by
conspecifics (Lawrence and Smith 1989), the entire
arena was surrounded by dark plastic wrapping, and
noise level in the immediate area of the arena was
limited to reduce the potential for external stimuli to
influence fish behavior. During the final 10 min of the
1-h acclimation period, fish position, behavior and
activity were recorded using a camera (iDS uEye
1480-C camera, iDS, Obersulm, Germany) (Little
et al. 2011; Poulsen et al. 2014). Two fish that
remained stationary during the acclimation period
were removed from the arena and excluded from the
study (Munday et al. 2010).
After the acclimation period, one 30-ml aliquot of
prepared skin extract was added to the ‘treatment’
header tank and was allowed to mix for 10 min. Water
with the skin extract was then allowed to flow from the
header tank into the arena (also at a rate of 6.7 l/min).
Once water containing skin extracts entered the choice
arena (determined to be 30 s using a preliminary dye
test), fish were again recorded for 10 min (Little et al.
2011; Poulsen et al. 2014). Following this 10-min
recording period, the fish was removed from the
choice tank and measured for total length (mm) and
weight (g) (Supplementary Table S2). This procedure
was repeated for 27 fish until a sample size of N=9
for each treatment was achieved for both fathead
minnows and silver carp, and fish were only used once
and then were euthanized. Gender was unknown for
both species. Note that, during the skin extract
exposure period, water was not returned to the header
tank using the submersible pump, and between trials,
the tank was thoroughly rinsed.
To quantify the potential for changed behaviors to
return to ‘normal’, fathead minnows were held for an
additional 11–14 days in water at ambient pCO
2
(&400 latm.) using protocols outlined above, and
behavioral trials were repeated for 29 naı
¨ve fish (i.e.,
previously assessed fish were not re-used). Similarly,
silver carp were allowed to recover for 14–17 days in
ambient pCO
2
water (&950 latm.), and behavior
trials were repeated for 27 naı
¨ve fish. This duration of
recovery was chosen as Hamilton et al. (2014) found
that anxiety behaviors of juvenile California rockfish
(Sebastes diploproa) altered by exposure to increased
levels of pCO
2
returned to normal after returning to
ambient seawater for 12 d.
Data acquisition and statistical analyses
Analyses of total distance travelled, velocity and
active time were generated using videos with the
program Lolitrack (Loligo Systems, Denmark; Lawr-
ence and Smith 1989; Poulsen et al. 2014). Total
distance travelled and velocity were transformed into
body lengths (BL) and BL/s, respectively, to stan-
dardize metrics across fish lengths. In addition, each
video was manually analyzed for darts (rapid move-
ment lasting at least 1 s) and freezes ([30 s motion-
less) using protocols defined by Chivers and Smith
(1994), and these two metrics were then summed
together to generate irregular activities. In addition to
darts and freezes, jumps were also quantified as part of
silver carp irregular activities, as jumps are known to
be a fright response in silver carp (Kolar et al. 2007).
To determine whether elevated pCO
2
had an effect
on the response to skin extract, generalized linear
mixed models (GLMMs) were performed, with appro-
priate error, distributions and link-functions. For the
GLMMs, a Poisson distribution was used only for
count data (Quinn and Keough 2002) (i.e., total
irregular activities). For fathead minnows, data were
parsed by pCO
2
treatment, and therefore, activity,
velocity, and total distance travelled were analyzed
using GLMMs with normal distributions. These four
metrics (irregular activities, activity, distance trav-
elled and velocity) were entered as response variables,
exposure (acclimation or stimulus levels) and treat-
ment period (CO
2
or recovery) were included as fixed
effects, and fish ID was included as a random effect for
each treatment. The use of a random effect (a repeated
measures design) was necessary because multiple
measurements were taken from each fish across trials
(acclimation and stimulus), meaning that each mea-
surement was not independent and potentially corre-
lated within an individual across treatments (i.e., due
to inherent inter-individual differences, some fish may
be more active or freeze more, than others, which
Aquat Ecol
123
needs to be considered across treatments) (Laird and
Ware 1982; Lindstrom and Bates 1990).
For silver carp, data were not parsed by pCO
2
treatment due to obtaining normality in residuals, and
therefore, the models included the four metrics entered
as response variables, exposure (acclimation or stim-
ulus levels) and treatment (control, low, high) were
included as fixed effects with fish ID as a random
effect. Including pCO
2
treatment as a fixed effect was
done because of poorly distributed residuals when the
fixed effect was not included.
GLMMs for continuousresponse variables were fitted
using the ‘glmer’ function from the ‘lme4’ library in R
(Venables and Ripley 2002; Bates 2010), and, for models
with count response variables, which were also over-
dispersed, the ‘glmmPQL’ function from the ‘MASS’
library was used (Bolker et al. 2009). A visual analysis of
fitted residuals using a normal probability plot was used
to assess normality (Anscombe and Tukey 1963), and
visual inspection of the distribution of residuals was used
to assess homogeneity of variance. If expectations of
normality or homogeneity of variance were not met, a
log transformation of the response variables (i.e., a log-
linear model) was used to adjust residuals and achieve
normality (Keene 1995). For models of count variables
using GLMMs, significance was tested at the 95 % level.
For the GLMMs containing continuous variables, to
define the importance of fixed effects, the sim function
(‘arm’ package in R) was used to generate N=1000
posterior simulations of each fixed effect. The resulting
posterior distribution of effect estimates was assessed to
determine significance of the effec ts (i.e., distributions of
fixed effects whose 95 % credible intervals did not
overlap0weresaidtobesignicant).Tocomplete
multiple comparisons between levels of significant
factors, changes in means and 95 % credible intervals
of simulated changes in model intercepts were com-
pared. All data are reported as mean ±standard error,
SE, where appropriate. Treatment duration for each
individual was initially included as a covariate in all
analytical models, but was not significant for both
fathead minnows and silver carp and was therefore
excluded in final models (Engqvist 2005).
Results
Both fathead minnows and silver carp responded to the
skin extracts of conspecifics. Specifically, for fathead
minnows held at ambient pCO
2
, the number of
irregular activities (e.g., darts and freezes) increased
4.5- and 2.4-fold during the treatment and recovery
periods after being exposed to skin extracts, respec-
tively (Fig. 1a; Supplementary Table S3). Similarly,
silver carp responded to skin extracts with increased
irregular activities (e.g., darts, freezes and jumps) by
1.6-fold during the stimulus relative to the acclimation
period in both the exposure and recovery periods
(Fig. 2; Supplementary Table S3). In addition to
responding with increased irregular activities, fathead
minnows held at ambient pCO
2
also had faster
swimming velocity and greater distance travelled, as
fish swam 0.34 ±0.11 BL/s faster (Fig. 5a; Supple-
mentary Table S4) and travelled 138 ±46 BL more
(Table 1; Fig. 4a; Supplementary Table S4), respec-
tively, following exposure to skin extracts.
Exposure to elevated pCO
2
resulted in changes to
the responses of fathead minnows to skin extracts, but
not silver carp. More specifically, unlike fish held at
control conditions (600 latm.), fathead minnows
treated with high pCO
2
(7000 latm.) displayed no
irregular responses to skin extract exposure (Fig. 1c;
Supplementary Table S3). Fathead minnows exposed
to low pCO
2
(800 latm.) still displayed a response to
conspecific skin extracts in the form of irregular
activities; however, these appeared to be lower than
the increases observed in fish held at control condi-
tions (2.4–4.5-fold increase) as the number of irregular
activities increased by only 1.1–2.3-fold relative to the
acclimation period when exposed to skin extracts
(Fig. 1b; Supplementary Table S3). Furthermore, the
changes in distance travelled (Table 1; Fig. 4b, c;
Supplementary Table S4) and swimming velocity
(Fig. 4b, c; Supplementary Table S4) that was
observed for fathead minnows held at control condi-
tions were no longer visible when fish were exposed to
both levels of elevated CO
2
(800 and 7000 latm.). For
silver carp, all behavior metrics did not differ relative
to the acclimation period following exposure to skin
extracts, regardless of pCO
2
treatment (Table 2;
Supplementary Tables S3, S4).
Returning fathead minnows to water at ambient
pCO
2
for 11–14 days caused some behavioral impair-
ments induced by CO
2
exposure to abate. During the
recovery period, fathead minnows previously exposed
to high pCO
2
demonstrated a 2.7-fold increase in
irregular activities after exposure to skin extract
(Fig. 1; Supplementary Table S3). Fathead minnows
Aquat Ecol
123
exposed only to control values of pCO
2
were also
monitored and showed a response to skin extracts
during the recovery period too, as activity decreased
0
2
4
6
8
10
12
CO 2Recovery
c
Irregular Activities
0
2
4
6
8
10
12
b
0
2
4
6
8
10
12
Acclimation
Stimulus
a<
<
Fig. 1 Number of irregular activities (i.e., darts and freezes) for
fathead minnows (P. promelas) during the acclimation and
stimulus periods following CO
2
exposure and for separate
fathead minnows during recovery following CO
2
exposure.
Fathead minnows were exposed to either acontrol (600 latm.),
blow (800 latm.) or chigh (7000 latm.) CO
2
levels for
4–12 days and then held for an additional 11–14 days at
ambient (750 latm. CO
2
) conditions. Data are presented as
mean ±SE (N=9–10). For a,bthe gray and black boxes with
aless than symbol represent a significant effect of monitoring
period between the acclimation and stimulus period (GLMM,
see supplementary Table S3). For c, a significant interactive
effect of stimulus and recovery was detected, but multiple
comparisons did not reveal the source of the significant
difference (GLMM, see supplementary Table S3)
Fig. 2 Number of irregular activities (darts, freezes, and
jumps) for silver carp (H. molitrix) during acclimation and
stimulus periods following CO
2
exposure and recovery. Silver
carp were aexposed to either ambient (1000 latm.), low
(3000 latm.) or high (8000 latm.) CO
2
levels for 4–10 days
and then bheld for an additional 11–14 days at ambient
(750 latm. CO
2
) conditions. Data are presented as mean ±SE
(N=9). The gray and black boxes with the less than symbol
represent a significant effect of monitoring period between
acclimation and stimulus (GLMM, see supplementary Table S3)
Table 1 Changes in the intercept estimate for response vari-
ables with a significant interactive effect (see Supplementary
Table S4) for control fathead minnows during the CO
2
expo-
sure and recovery period
Response Treatment Mean 95 % Credible interval
Log (activity) CO
2
0.61 -0.02, 1.24
Recovery -0.98 -1.62,-0.35
Log (distance) CO
2
1.11 0.21,2.06
Recovery -0.71 -1.58, 0.17
Both mean and 95 % credible intervals were calculated from
estimates obtained using posterior simulations of each fixed
effect. Significance was determined if the 95 % credible
intervals did not overlap zero and are bolded
Aquat Ecol
123
by about double during the stimulus period (Table 1;
Fig. 3; Supplementary Table S4).
Discussion
Some alarm cue behaviors in fathead minnows
exposed to conspecific skin extracts were altered
when fish were held in water with elevated pCO
2
for
4–12 days. Specifically, fathead minnows exposed to
high pCO
2
(7000 latm.) did not display changes in
irregular activities, velocity or distance travelled when
exposed to skin extracts (Table 1; Figs. 1,4,5;
Supplementary Tables S3, S4). No changes in velocity
or distance travelled were observed for low
(800 latm.) pCO
2
-treated fathead minnows (Table 1;
Figs. 1,4,5; Supplementary Tables S3, S4) following
exposure skin extracts as well. These results are
similar to what has been found in marine ecosystems
where marine fishes are unable to detect predator
olfactory cues following exposure to elevated pCO
2
(Munday et al. 2009; Dixson et al. 2010). For example,
Munday et al. (2010) discovered that clownfish larvae
(A.percula) were attracted to, rather than repelled by,
predator odors after just 2 days at 850 latm. pCO
2
.
One possible explanation to why fish exposed to
elevated pCO
2
have a reduced alarm cue response may
be due to reduced sensitivity of the olfactory receptors.
Specifically, with respect to acidification, mucus can
increase on the olfactory epithelium in low pH and can
disrupt olfaction capabilities (Lemly and Smith 1987;
Klaprat et al. 1988). Olfactory receptor alterations and
increased mucus production may have caused the
decrease in fathead minnow’s ability to respond to the
alarm cue. Previous research has also shown that
rainbow trout (Oncorhynchus mykiss), Atlantic sal-
mon (Salmo salar) and fathead minnows exposed to a
pH of 6.0 for 30 min to 72 h were unable to respond to
amino acids and ovulated female urine, suggesting that
olfactory receptors were inhibited by reduced pH
(Lemly and Smith 1985; Royce-Malmgren and Wat-
son 1987; Moore 1994). However, with some of these
studies, the cause of no response was most likely due
degradation of the alarm cue (Leduc et al. 2013). Our
testing was completed in ambient freshwater and not
acidified water as the previous stated research has been
conducted. Similar results to ours were found where
alarm cue testing in pH 7.54, similar to ours, Pacific
salmon could no longer avoid conspecific skin extracts
in a two-choice flume, and anxiety behaviors were
reversed with the treatment of gabazine suggesting
GABA
A
receptors are being impacted by pCO
2
exposure (Ou et al. 2015). Nilsson et al. (2012) and
Regan et al. (2016) showed function of the GABA
A
Table 2 Total activity (s), total distance travelled in body lengths (BL) and velocity (BL/s) for silver carp during acclimation and
stimulus periods following CO
2
exposure and recovery
Procedure Treatment Monitoring period Activity (s) Total distance (BL) Velocity (BL/s)
CO
2
Control Acclimation 315.8 ±34.5 106.4 ±18.5 0.32 ±0.03
Stimulus 329.1 ±27.0 109.8 ±15.7 0.32 ±0.03
Low Acclimation 289.4 ±28.2 93.2 ±18.3 0.30 ±0.03
Stimulus 338.0 ±31.0 121.7 ±17.9 0.34 ±0.03
High Acclimation 396.6 ±36.0 151.9 ±21.8 0.36 ±0.03
Stimulus 366.6 ±28.4 128.1 ±16.3 0.34 ±0.02
Recovery Control Acclimation 310.4 ±24.8 105.7 ±16.3 0.33 ±0.02
Stimulus 315.2 ±39.5 112.7 ±18.3 0.33 ±0.02
Low Acclimation 284.7 ±31.8 88.1 ±15.0 0.29 ±0.02
Stimulus 257.1 ±38.9 75.8 ±15.6 0.27 ±0.02
High Acclimation 344.9 ±29.0 115.9 ±15.8 0.32 ±0.02
Stimulus 333.3 ±25.5 110.0 ±13.9 0.32 ±0.02
Silver carp were exposed to either ambient (1000), low (3000) or high (8000 latm.) CO
2
levels for 4–10 days and then held for an
additional 11–14 days at ambient (750 latm. CO
2
) conditions. Data are presented as mean ±SE (N=9). No significant effect of
monitoring period or treatment were detected within the CO
2
or recovery treatment periods (GLMM, see Supplementary Tables S3,
S4)
Aquat Ecol
123
receptors, a major inhibitory neurotransmitter recep-
tor, was reversed causing it to become excitatory
(efflux of anions) rather than inhibitory (normal influx
of anions). These abrupt changes in ion gradients
result in changes in behaviors of fishes and could
explain why fathead minnows were unable to respond
to conspecific alarm cues following CO
2
exposure
especially with no degradation of the alarm cue.
Together these results clearly demonstrate that in
fathead minnows, some conspecific alarm cue
responses are affected by elevations in pCO
2
.
Fathead minnows may be able to recover and
respond to conspecific skin extracts after pCO
2
exposure and subsequent return to ambient freshwater.
Fathead minnows that showed behavioral impairments
after exposure to &7000 latm. displayed a threefold
increase in irregular activities during the recovery
period (Fig. 1c; Supplementary Table S3). It has been
shown that when an environmental stressor is
removed, fish physiology and behavior may return to
pre-exposure levels. Some examples of this include
recovery following changes in temperature (Galloway
and Kieffer 2003), dissolved oxygen (Suski et al.
0
100
200
300
400
Activity (s)
0
100
200
300
400
0
100
200
300
400
Acclimation
Stimulus
CO2
*
Recovery
a
b
c
Fig. 3 Mean activity (s) for fathead minnows (P. promelas)
during the acclimation and stimulus periods following CO
2
exposure and recovery. Fathead minnow were exposed to either
aambient (600 latm.), blow (800 latm.) or chigh
(7000 latm.) CO
2
levels for 4–12 days and then held for an
additional 11–14 days at ambient (400 latm. CO
2
) conditions.
Data are presented as the mean ±SE. An asterisk represents a
significant interaction between acclimation and stimulus during
the recovery period (GLMM, see Tables 1, S4)
0
100
200
300
400
Acclimation
Stimulus
Total Distance (BL)
0
100
200
300
400
0
100
200
300
400
*
a
b
c
CO2Recovery
Fig. 4 Mean total distance (BL) for fathead minnows (P.
promelas) during the acclimation and stimulus periods follow-
ing CO
2
exposure and recovery. Fathead minnow were exposed
to either aambient (600 latm.), blow (800 latm.) or chigh
(7000 latm.) CO
2
levels for 4–12 days and then held for an
additional 11–14 days at ambient (400 latm. CO
2
) conditions.
Data are presented as the mean ±SE. An asterisk represents a
significant interaction between acclimation and stimulus during
the recovery period (GLMM, see Tables 1, S4)
Aquat Ecol
123
2006) and ammonia (Suski et al. 2007). Previous work
has also shown that behaviors altered by exposure to
elevated pCO
2
can be reversed following removal of
the CO
2
and pH stressors, but this potential for
recovery has not been well studied. For example,
Munday et al. (2010) showed predator avoidance
behaviors were re-established in larval P.wardi after
returning them to ambient seawater for 2 days
following exposure to elevated pCO
2
. Other work
has also shown that when tested in pH \6.0 using
sulfuric acid, fathead minnow olfaction was impaired,
but, when re-tested in ambient freshwater (pH 8.0),
olfactory impairments were abolished (Brown et al.
2002). The most likely explanation for recovery of fish
response that were previously exposed to elevated
pCO
2
is a return of GABA
A
receptors to normal
functioning (i.e., inhibitory rather than excitatory) as
Nilsson et al. (2012) observed. To better understand
the mechanisms underlying changes in the alarm cue
responses of freshwater fishes in response to elevated
pCO
2
and the recovery of such responses, future
studies should include GABA
A
receptor antagonist
such as gabazine (Nilsson et al. 2012), as well as
methods to isolate pH and elevated pCO
2
as indepen-
dent contributors to altered responses.
Interestingly, silver carp showed no impairment to
alarm cue responses despite 4–10 days of exposure to
low or high pCO
2
(3000 and 8000 latm., respectively;
Fig. 2; Supplementary Tables S3, S4). Other fish
species have demonstrated conservative responses to
alarm cues following exposure to environmental
stressors. For example, juvenile Atlantic cod (Gadus
morhua) were still able to avoid a predator odor
following 6 weeks of exposure to 1000 latm. (Jutfelt
and Hedga
¨rde 2013). Species like silver carp, which
may live in environments where variations in pH and
pCO
2
occur regularly, may be more adapted to
elevated pCO
2
and reduced pH than species that live
in more stable environments such as large bodies of
water (Hirata et al. 2003; Melzner et al. 2009).
Species, like cardinal tetras (Cheirodon axelrodi),
have acclimated to have physiological tolerances to
acidified water of pH 3.1 and survived for 5 weeks
(Dunson et al. 1977). Similarly, Gonzalez and Dunson
(1987) showed Enneacanthus obesus had no change in
body sodium concentration after 5 weeks in a pH of
4.0. Another possible explanation of why silver carp
still responded to the skin extracts following elevated
pCO
2
exposure may have been due to either a higher
concentration of alarm cue used relative to fathead
minnows, or possibly a higher concentration of alarm
pheromone relative to other previous studies with
silver carp (i.e., over-stimulation of process related to
sensing pheromones in the water) (Little et al. 2011).
Species-specific thresholds that result in responses to
skin extracts have not been identified in silver carp,
and we did not quantify the concentration of alarm
0.0
0.2
0.4
0.6
0.8
1.0
1.2 Acclimation
Stimulus
Velocity (BL s
-1)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
<
a
b
c
CO2Recovery
Fig. 5 Mean swimming velocity (BL/s) for fathead minnows
(P. promelas) during the acclimation and stimulus periods
following CO
2
exposure and recovery. Fathead minnows were
exposed to either aambient (600 latm.), blow (800 latm.) or
chigh (7000 latm.) CO
2
levels for 4–12 days and then held for
an additional 11–14 days at ambient (400 latm. CO
2
) condi-
tions. Data are presented as the mean ±SE. The gray and black
boxes with the less than symbol represent a significant effect of
monitoring period between acclimation and stimulus (GLMM,
see supplementary Table S4)
Aquat Ecol
123
pheromone used in this study, precluding our ability to
quantitatively compare concentrations across species.
However, because the purported mechanism for the
impaired response to olfactory cues is based on
changes to GABA
A
receptors (Nilsson et al. 2012;
Regan et al. 2016), it is likely that the continued
response of silver carp to the skin extracts may
indicate that the GABA
A
receptors were likely not
depolarized following CO
2
exposure, as has been
shown for other species (Jutfelt and Hedga
¨rde 2013;
Maneja et al. 2013; Jutfelt and Hedga
¨rde 2015). Some
freshwater species, like silver carp, may be able to
adapt to elevated pCO
2
and still be able to respond
appropriately to conspecific alarm cues, and thus,
there may be minimal impacts to populations and
overall mortality. Results from this study support the
idea that impacts to conspecific alarm cues may be
species dependent to exposures to elevated pCO
2
.
A reduced alarm cue response after exposure to
elevated CO
2
has many implications for management
and the ecology of freshwater fish species. Freshwater
fishes may be exposed to elevated pCO
2
due to natural
environmental variation (reviewed by Hasler et al.
2016), climate change (Phillips et al. 2015), hatchery
rearing (Colt and Orwicz 1991) and zones of elevated
pCO
2
deployed as fish barriers (Kates et al. 2012;
Noatch and Suski 2012). If fathead minnows were
subjected to an increase in pCO
2
concentrations, they
may lose their ability to appropriately respond to
conspecific alarm cues such as skin extracts from a
predator event. Thus, this impaired alarm cue response
may have implications to mortality and population
dynamics (Leduc et al. 2013) and could potentially
alter community composition (Chown and Gaston
2015). However, if the CO
2
stimulus was removed,
behaviors such as darts and freezes may return to
normal. Interestingly, silver carp appear to be more
robust to changes in environmental pCO
2
, and eleva-
tions in pCO
2
may have minimal effects on their
ability to detect and respond to alarm cues. Additional
research is needed to define the mechanisms underly-
ing these differences in the responses of freshwater
fish species to elevated pCO
2
. Together, these results
provide information about the possible consequences
and responses of freshwater fish to environmental
changes such as elevations in pCO
2
and acidification.
Acknowledgments This work was supported by the Illinois
Department of Natural Resources through funds provided by the
US Environmental Protection Agency’s Great Lakes Restora-
tion Initiative, as well as the Illinois Chapter of the American
Fisheries Society. The Upper Midwest Environmental Sciences
Center (UMESC) provided laboratory space and silver carp for
experiments. All work performed in this study conformed to
guidelines established by the Institutional Animal Care and Use
Committee (IACUC) of the University of Illinois (Protocol
#14168).
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... Tras la obtención de un genoma completo de alta calidad, múltiples herramientas pueden ser utilizadas para el ensamblaje y la identificación de los marcos de lectura abierto; Spades (19), 100 Unicycler (Wick et al., 2017), y Velvet (Zerbino 2010), basados en Gráfico de Bruijn, son ejemplos de ensambladores. GeneMark (Besemer & Borodovsky 2005), Glimmer (Delcher et al., 2007), y ...
... However, species-specific differences in response have been shown. For example, high CO2 levels do not alter the behavior of silver carp Hypophthalmichthys molitrix (19), which might be due in part to an adaptation to low environmental oxygen conditions seen in the habitat of this species (20). ...
... (1) pathogen antagonism by competitive exclusion or antimicrobial production and stimulation of host immune response; (2) metabolite production; (3) nutritional substance production; (4) survival and colonisation in the host digestive tract (DT) by adhesion; (5) storage stability; (6) safety; (7) origin from animal source [12,16]. The use of beneficial organisms or probiotics offers wide opportunities for shellfish aquaculture [17,18] because of their antimicrobial properties [19], particularly against different species of pathogenic Vibrio spp. [20]. ...
Book
Full-text available
Índice / Index Dr. Albert Tacon (Aquahana LLC, Hawaii, USA) El papel de la nutrición de peces en la mejoría de la salud humana y la seguridad mundial The role of fish nutrition in improving human health and global good security. Page 7 Oscar Monroig (Instituto de Acuicultura Torre de la Sal, España); Últimos avances en el estudio de la biosíntesis de ácidos grasos omega-3 de cadena larga en invertebrados acuáticos. Latest advances in the study of the biosynthesis of long-chain omega 3 fatty acids in aquatic invertebrates. Page 25 David Celdrán Sabater (Bioaquafloc, Costa Rica); Acuicultura Simbiótica como nueva técnica productiva. Symbiotic aquaculutre as a new productive technique of Bioaquafloc. Page 28 Joseph Selvin (University of Pondicherry, India); Application of Poly-ß-hydroxybutyrate in Shrimp Health Management. Aplicación de poli- ß-hydroxybutyrato en el manejo de la salud del camarón. Page 35 Emmanuel Martínez (Universidad Politécnica de Sinaloa, México); Proteínas y Péptidos de Residuos Líquidos Pesqueros: Obtención, Bioactividad y Uso en la Alimentación Acuícola. Proteins and peptides from liquid fishery residues: obtention, bioactivity and its us in aquaculture feed. Page 50 Eduardo Quiroz (Centro de Investigaciones Biológicas del Noroeste, S.C. La Paz, B.C.S. México); Bacteriófagos: Herramientas de Control Biológico para una Acuicultura Sostenible. Bacteriophages: biological control tolos for a sustainable aquaculture. Page 71 Helene Volkoff University of Newfoundland, Canada); Effects of nutritional status and environmental factors on the endocrine regulation of feeding in freshwater fish. Efectos del estado nutricional yu factores ambientales sobre la regulación endocrina de la alimentación en peces teleósteos de agua dulce seleccionados. Page 113 Francisco J. Toledo S. (Universidad de Ciencias y Artes de Chiapas, México); El titarro (Lathyrus cicera L.) como una alternativa sustentable para el remplazo de harina de soya en alimentos de Oncorhynchus mykiss. Titarro (Lathyrus cicera L.) as a sustainable alternative to replace soybean meal in Oncorhynchus mykiss feeds. Page 118 Madison Powell (University of Idaho, USA); Correlation of expression and enzyme activity among stress-related genes in Salmonids. Correlación de expresión y actividad enzimática entre genes relacionados con el estrés en salmónidos. Page 137 Vikas Kumar (University of Idaho, India); Nutrigenomic approaches improve the efficiency of soybean meal utilization in salmonids Aquaculture. Nutrigenomic approaches improve the efficiency of soybean meal utilization in salmonids aquaculture. Page 156 Yutaka Haga (Tokyo University of Marine Science and Technology, Japón); Effect of taurine precursor on growth and taurine content of marine fish. Efecto del precursor de taurine sobre el crecimiento y contenido de taurina en peces marinos. Page 158 Francisco Javier Alarcón (Universidad de Almería, España); Develando el potencial de las algas para la elaboración de piensos para peces de acuicultura. Unvelling the potential of algae for the production of feed for aquaculture fish. Page 176 Crisantema Hernandez (Centro de Investigación en Alimentación y Desarrollo, A.C. Unidad Mazatlán, México); Estrategias Nutricionales y Productos de soya para la Alimentación de Juveniles de Róbalo blanco del pacífico (Centropomus viridis): hacia la Rentabilidad de su Cultivo Nutritional Strategies and Soy Products for the Feeding of Juvenile Pacific White Bass (Centropomus viridis): Towards the Profitability of its Cultivation Page 217 Ángel Campa (Centro de Investigaciones Biológicas del Noroeste, La Paz, B.C.S., México); Bacteria and microalgae interaction on rearing Kumamoto oyster Crassostrea sikamea spat / Interacción bacteria microalga en el cultivo de semilla del ostión Kumamoto Crassostrea sikamea. Page 223 Luis Martínez (Universidad de Sonora, México); Alternativas de acuacultura sostenible: aspectos nutricionales. Sustainable aquaculture alternatives: nutritional aspects. Page 245 Martín Arenas (Universidad Nacional Autónoma de México, UMDI Sisal, México); Avances en la nutrición de Centropomus undecimalis. Advances in the nutrition of Centropomus undecimalis.. Page 263 Luis Hernández (Universidad Nacinal Autónoma de México, FES Iztacala, México); Avances en nutrición del langostino Macrobrachium acanthurus. Advances in nutrition of the shrimp Macrobrachium acanthurus. Page 284 Alberto Peña (Centro de Investigaciones Biológicas del Noroeste, S.C. La Paz, B.C.S. México); Valorización de macroalgas para su uso como alimento acuícola. Valorization of macroalgae for use as aquaculture feed. Page 294 Grecia Montalvo (Universidad Nacional Autónoma de México, UMDI Sisal, México); Respuesta antioxidante y del sistema inmune de machos reproductores de Litopeneaus vannamei Boone (1931) alimentados con dietas suplementadas con vitamina E. Antioxidant and immune system response of breeding male Litopenaeus vannamei Boone (1931) fed diets supplemented with vitamina E. Page 316 Omar Mendoza (Commonwealth Scientific and Industrial Research Organisation, CSIRO Australia), Development of high-throughput omics resources for aquaculture nutrition. Desarrollo de recursos ómicos de alto rendimiento para la nutrición acuícola. Page 343 Artur Rombenso (Commonwealth Scientific and Industrial Research Organisation, CSIRO Australia); Aquaculture nutrition research at CSIRO: Maintaining productivity and competitiveness in challenging times. Aquaculture nutrition research at CSIRO: Maintaining productivity and competitiveness in challenging times. Page 361 Claudia Maytorena (Universidad Juárez Autónoma de Tabasco, México); Efecto de manano-oligosacáridos en el crecimiento, actividad de enzimas digestivas y expresión de genes relacionados a la mucosa intestinal de larvas de pejelagarto Atractosteus tropicus. Effect of mannan-oligosaccharides on growth, digestive enzymes activity, and expression of genes related to the intestinal mucosa of Atractosteus tropicus larvae. Page 394 Addison Lawrence (Texas A&M University, Texas A&M AgriLife Research, USA); Review of bacterial and yeast base single cell protein ingredients as attractants and fish meal replacements in diets for Litopenaeus vannamei. Revisión de ingredientes de proteínas unicelulares con base en bacterias y levaduras como atrayentes y reemplazos de harina de pescado en dietas para Litopenaeus vannamei. Page 413 Marcelo Tesser (Universidade Federal do Rio Grande-FURG Brasil); Uso de Alimentos no Convencionales en Acuicultura: Estudios Realizados em la Universidad Federal De Rio Grande -FURG. Use of non-conventional feeds in aquaculture: Studies carried out at the Federal University of Rio Grande-FURG. Page 415 Andressa Teles (Universidad Juárez Autónoma de Tabasco, México); Bioencapsulación de levadura probiótica para larvas de Seriola rivoliana. Bioencapsulation of probiotic yeast for Seriola rivoliana larvae. Page 432 Carmen Monroy (Universidad Autónoma Metropolitana, México); Alimentos funcionales y su aplicación en organismos acuáticos. Functional foods and their application in aquatic organisms. Page 455 María Celia Portella (São Paulo State University, Brasil); FLOCponics: The integration of biofloc technology with plant prodution and the possibiity to reduce the protein level of tilapia juveniles diet. La integración de la tecnología biofloc con la producción de plantas y la posibilidad de reducir el nivel de proteína en la dieta de juveniles de tilapia. Page 472
... Tras la obtención de un genoma completo de alta calidad, múltiples herramientas pueden ser utilizadas para el ensamblaje y la identificación de los marcos de lectura abierto; Spades (19), 100 Unicycler (Wick et al., 2017), y Velvet (Zerbino 2010), basados en Gráfico de Bruijn, son ejemplos de ensambladores. GeneMark (Besemer & Borodovsky 2005), Glimmer (Delcher et al., 2007), y ...
... However, species-specific differences in response have been shown. For example, high CO2 levels do not alter the behavior of silver carp Hypophthalmichthys molitrix (19), which might be due in part to an adaptation to low environmental oxygen conditions seen in the habitat of this species (20). ...
... (1) pathogen antagonism by competitive exclusion or antimicrobial production and stimulation of host immune response; (2) metabolite production; (3) nutritional substance production; (4) survival and colonisation in the host digestive tract (DT) by adhesion; (5) storage stability; (6) safety; (7) origin from animal source [12,16]. The use of beneficial organisms or probiotics offers wide opportunities for shellfish aquaculture [17,18] because of their antimicrobial properties [19], particularly against different species of pathogenic Vibrio spp. [20]. ...
Chapter
El langostino Macrobrachium acanthurus es una especie con el potencial para ser cultivado y en el Laboratorio de Producción Acuícola de la UNAM FES Iztacala se ha trabajado en la determinación de los requerimientos nutricionales en diferentes estadios de desarrollo de esta especie. Por ello, a continuación, presentamos algunos de los avances que hemos obtenido en los requerimientos de proteína, lípidos y carbohidratos en postlarvas, de vitaminas en las larvas, así como el uso de algunos aditivos en juveniles. Se ha logrado establecer la combinación de inclusión de 35% proteína, 15-120% lípidos y un máximo de 15% de carbohidratos. La inclusión de las vitaminas A y C permiten aumentar el porcentaje de supervivencia durante la etapa larvaria, mientras que el uso de prebióticos (fructooligosacaridos y mananoligosacaridos) podrían tener un efecto positivo en la mad uración sexual de hembras.
... The novel object approach test has been used to examine the effects of aquatic acidification on fish boldness in only a few previous studies, which have all reported different outcomes: decreased boldness (Jutfelt et al., 2013), no effect (Tix et al., 2017), or increased boldness (Ou et al., 2015). These differences might be due to the specific pCO 2 levels that were tested, the relation between the preferred pCO 2 level of each fish species and the experimental pCO 2 levels, or unknown stochastic variables. ...
... Many studies on freshwater fish species have shown negative effects including impaired growth, altered olfaction and feeding (reviewed in Hasler et al., 2018), and behaviour (Ou et al., 2015;Ikuta et al., 2003). However, other studies have found minimal or no behavioural alterations (Vossen et al., 2016;Tix et al., 2017;Midway et al., 2017). As pointed out by our study, these discrepancies might be explained by the different pCO 2 levels used in each study and putative ensuing nonlinear effects. ...
Article
CO2-induced aquatic acidification is predicted to affect fish neuronal GABAA receptors leading to widespread behavioural alterations. However, the large variability in the magnitude and direction of the responses suggest substantial species-specific CO2 threshold differences, life history and parental acclimation effects, experimental artifacts, or a combination of these factors. As an established model organism, zebrafish (Danio rerio) can be reared under stable conditions for multiple generations, which may help control for some of the variability observed in wild-caught fishes. Here, we used two standardized tests to investigate the effect of 1-week acclimatization to four pCO2 levels on zebrafish anxiety-like behaviour, exploratory behaviour, and locomotion. Fish acclimatized to 900 μatm CO2 demonstrated increased anxiety compared to control fish (~480 μatm), however, the behaviour of fish exposed to 2200 μatm CO2 was indistinguishable from that of controls. In addition, fish acclimatized to 4200 μatm CO2 had decreased anxiety-like behaviour; i.e. the opposite response than the 900 μatm CO2 treatment. On the other hand, exploratory behaviour did not differ among any of the pCO2 exposures that were tested. Thus, zebrafish behavioural responses to elevated pCO2 are not linear; with potential important implications for physiological, environmental, and aquatic acidification studies.
... Several studies have started to characterize fish behavior during CO 2 exposure. Choice-chamber experiments in indoor laboratories found that invasive fishes moved away from CO 2 treated tanks in favor of untreated tanks (Dennis et al., 2016(Dennis et al., , 2015Kates et al., 2012;Tix et al., 2017;Tucker et al., 2019). Avoidance responses were relatively similar across species, life stages, and water temperatures (Cupp et al., 2017a(Cupp et al., , 2017cDennis et al., 2015Dennis et al., , 2016Tix et al., 2018), and fish generally did not acclimate with prolonged or repeated exposures to a CO 2 stimulus Tix et al., 2017). ...
... Choice-chamber experiments in indoor laboratories found that invasive fishes moved away from CO 2 treated tanks in favor of untreated tanks (Dennis et al., 2016(Dennis et al., , 2015Kates et al., 2012;Tix et al., 2017;Tucker et al., 2019). Avoidance responses were relatively similar across species, life stages, and water temperatures (Cupp et al., 2017a(Cupp et al., , 2017cDennis et al., 2015Dennis et al., , 2016Tix et al., 2018), and fish generally did not acclimate with prolonged or repeated exposures to a CO 2 stimulus Tix et al., 2017). Similar responses were observed in pond studies where fish temporarily moved away from the CO 2 source until the pond was completely mixed (Donaldson et al., 2016) and passages through the injection site were reduced (Cupp et al., 2017a). ...
Article
Full-text available
Carbon dioxide (CO 2) mixed into water is being explored as a possible management strategy to deter the upstream movements of invasive carps through navigation locks and other migratory pinch-points. This study used two-dimensional acoustic telemetry to assess the effectiveness of dissolved CO 2 as a chemosensory deterrent to two carp species in a large U-shaped pond. Free-swimming movements of telemetered bighead carp (Hypophthalmichthys nobilis) and grass carp (Ctenopharyngodon idella) were documented 24 h before treatment and 24 h during treatments at 60, 121 and 213 mg/L CO 2 (mean concentrations in pond water). Several behavioral endpoints were then quantified and compared to evaluate deterrence efficacy. In general, results showed that both carp species responded similarly to CO 2 treatments. Carps consistently relocated into areas away from the injection site and made fewer attempts to re-enter CO 2 treated areas. On average, CO 2 treatments reduced mid-line crosses between untreated and treated sides of the pond by 58% at 121 mg/L CO 2 and 78% at 213 mg/L CO 2 relative to normal swimming movements recorded before treatment. Fish swim speeds increased significantly when inside the CO 2 plume during treatments during 213 mg/L CO 2 trials relative to swim speeds outside the plume, possibly indicative of active searching and avoidance responses. Overall, this study found that CO 2 altered the behavior of bighead carp and grass carp. Natural resource agencies could consider the CO 2 concentrations identified in this study to inform future applications to deter invasive carps from locations where they are at-risk to move upstream. Published by Elsevier B.V. on behalf of International Association for Great Lakes Research.
... The most commonly discussed schreckstoff, hypoxanthine-3-N-oxide, undergoes irreversible structural changes at pH levels below 6.0 (C), which prevents its detection by fish (Brown et al., 2002). However, the associated reduction in behavioral response (G) appears to be specific to certain fish species (Tix et al., 2017). ...
Article
Full-text available
Chemical communication via infochemicals plays a pivotal role in ecological interactions, allowing organisms to sense their environment, locate predators, food, habitats, or mates. A growing number of studies suggest that climate change‐associated stressors can modify these chemically mediated interactions, causing info‐disruption that scales up to the ecosystem level. However, our understanding of the underlying mechanisms is scarce. Evidenced by a range of examples, we illustrate in this opinion piece that climate change affects different realms in similar patterns, from molecular to ecosystem‐wide levels. We assess the importance of different stressors for terrestrial, freshwater, and marine ecosystems and propose a systematic approach to address highlighted knowledge gaps and cross‐disciplinary research avenues. Chemical communication plays an essential role in ecosystems as it enables organisms to sense their environment, locate predators, food, habitats, or mates and interact with each other. We show that climate change can affect every single step of this fundamental way of communicating from the molecules to ecosystem‐wide interactions. Combined examples from terrestrial, freshwater and marine systems provide an overview across different realms and reveal universal patterns of impact, which help to identify the key aspects that we urgently need to understand to grow our ability to predict future effects and reliably inform ecosystem management efforts.
... For example, trace quantities of nickel and copper released from mining tailings selectively impair, respectively, microvillous and ciliated olfactory receptors, where ciliated olfactory neurons are used for detection of chemical alarm cues (Dew et al. 2014). Atmospheric NO x and SO x emissions create acidic rain and snow precipitation, lower pH of freshwater systems near point sources, and suppress behavioral responses to alarm cues in ostariophysans (minnows, characins, silurids), salmonids Smith 1985, 1987;Leduc et al. 2010Leduc et al. , 2013Tix et al. 2017), and coral reef fishes (Dixson et al. 2010;Ferrari et al. 2011; see also Clark et al. 2020;Munday et al. 2020). ...
Article
Full-text available
Hypoxia is a seasonally recurring environmental condition in small temperate lakes during summer thermal stratification and under ice cover in winter. Anthropogenic eutrophication contributes significantly to hypoxia by increasing primary production of organic materials that subsequently decompose in the hypolimnion. Greenhouse gas emissions that increase global temperatures will reduce the capacity of water to hold dissolved gases such as oxygen and increase the duration of thermal stratification and thus increase the severity and duration of hypoxic conditions in temperate lakes. How hypoxia may impact assessment of predation risk is under-studied. Here, we present a test of the effect of low dissolved oxygen (~ 1 ppm) on antipredator behavioral responses to conspecific alarm cues by fathead minnows. When alarm cues derived from conspecific epidermal tissues were introduced, fish under normoxic conditions (~ 9 ppm) reduced activity and moved out of the water column to spend more time at the bottom. These behaviors serve to reduce the probability of predation. At low dissolved oxygen levels, fathead minnows engaged in aquatic surface respiration, i.e., “drinking” the surface film of oxygen-rich water. When alarm cues were introduced, minnows left the surface waters to dash briefly downward but soon returned to the surface and did not reduce their activity. Taken together, these data indicate that under oxygen stress, minnows engage in truncated antipredator responses and remain near the surface where they would be more vulnerable to avian predators or air-breathing invertebrates such as belastomatids. Through decreased availability of dissolved oxygen, global climate change is likely to disrupt predator–prey interactions in temperate lakes.
... Additionally, this study did not observe acoustic and stroboscopic deterrents in isolation but, rather, deterrents in opposition to elevated CO 2 . Exposure to CO 2 may have altered fish avoidance behavior, as CO 2 has been observed to alter freshwater fish behavior in other contexts, such as movement velocity and alarm cue response (Tix et al. 2017a(Tix et al. , 2017b. Previous acoustic and stroboscopic deterrent studies have deployed the same stimuli toward Common Carp, without the addition of CO 2 , and the fish were observed to express mild avoidance responses in both lab (Bzonek et al. 2020) and field (Bzonek et al. 2021a(Bzonek et al. , 2021b trials. ...
Article
Full-text available
Biological invasions erode ecosystem functioning and occur more frequently in freshwater ecosystems than terrestrial environments. Non‐physical deterrents may be used to limit invasive fish dispersal without altering the streamflow or connectivity of a watershed. Little is currently known about how behavioural variation among individuals may effect deterrent efficacy, although such variation has been shown to affect fish dispersal in other contexts, such as range expansion. Furthermore, deterrent effectiveness is rarely tested when fish are motivated to disperse. Across a control, CO2, and CO2 + deterrent treatment, we quantified the avoidance response of invasive Common Carp (Cyprinus carpio) to a combined acoustic‐stroboscopic deterrent. In the CO2 treatment, we motivated individuals to enter a novel environment by degrading the home chamber of a choice arena with a continuous infusion of CO2. In the CO2 + deterrent treatment we introduced acoustic and stroboscopic stimuli to delay fish departure and evaluate deterrent efficacy. Finally, we tested a subset of the fish multiple times to determine if fish consistently responded to the same concentration of CO2. We found that the acoustic and stroboscopic stimuli could detain fish in an increasingly unfavorable environment. Common Carp only took 195 and 131 seconds to swim between chambers during the control and CO2 treatment, but took an average of 596 seconds in the CO2 + deterrent treatment. High CO2 concentrations in the CO2 + deterrent treatment led to most fish eventually dispersing towards the deterrent stimuli. Avoidance behaviour varied widely within the CO2 + deterrent treatment, and Common Carp expressed repeatable differences in the tank‐inflow CO2 concentrations observed during chamber departure. Such inter‐individual variation in deterrent avoidance indicates that some individuals within a given species are more likely to move past a deterrent than others.
... Increased levels of pCO 2 have been shown to impair physiological or sensory capacities Ou et al., 2015;Hasler et al., 2016Hasler et al., , 2017Midway et al., 2017;Tix et al., 2017aTix et al., , 2017bHasler et al., 2018;Kowalewska et al., 2020) in a range of taxa. ...
Article
Increased carbon dioxide from fossil fuel combustion results in an enrichment of CO2 in the global carbon cycle. Recent evidence indicates that rising atmospheric CO2 impacts the partial pressure of carbon dioxide (pCO2) in freshwaters. This affects freshwater biota by disrupting chemical communication between predator and prey. One such well-described predator–prey interaction is the phantom midge larva Chaoborus preying on the freshwater crustacean Daphnia pulex. To counter Chaoborus predation, D. pulex develops defensive neckteeth in response to chemical cues. The strength of neckteeth expression is reduced when D. pulex experience elevated pCO2 levels. This is discussed to directly impair predator perception and results in reduced defence expression. However, it is not known whether there are also long-term effects associated with continuous elevated pCO2. Here, we investigated the effect of long-term exposure of D. pulex to elevated pCO2 levels in a life-table experiment over three generations. Using a flow-through system, we continuously exposed D. pulex to cues released by the predatory larva Chaoborus and control or elevated pCO2 levels. We determined morphological defence expression in the 2nd juvenile instar and the number of neonates as a measure for life-history traits over three successive generations. We detected that elevated pCO2 significantly reduces the expression of predator-induced morphological defences (i.e. neckteeth) and life-history parameters (i.e. number of neonates) in successive generations. Our data clearly show that at least three generations become more vulnerable to predation without indications of transgenerational acclimation. As Daphnia is a keystone grazer of freshwater ecosystems, this may destabilise population growth rates. In conclusion, long-term effects of pCO2-induced reduction of predator-induced plasticity may significantly affect trophic interactions.
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Currently, the acidification of freshwater ecosystems is an overlooked public concern. In this chapter, a review on the origin, threats, impacts, and recent advances related to the CO2-induced acidification in the freshwater ecosystems is presented and discussed.
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Pioneering studies into the effects of elevated CO2 on the behaviour of reef fishes often tested high-CO2 reared fish using control water in the test arena. While subsequent studies using rearing treatment water (control or high CO2) in the test arena have confirmed the effects of high CO2 on a range of reef fish behaviours, a further investigation into the use of different test water in the experimental arena is warranted. Here, we used a fully factorial design to test the effect of rearing treatment water (control or high CO2) and experimental test water (control or high CO2) on antipredator responses of larval reef fishes. We tested antipredator behaviour in larval clownfish Amphiprion percula and ambon damselfish Pomacentrus amboinensis, two species that have been used in previous high CO2 experiments. Specifically we tested if: 1) using control or high CO2 water in a two channel flume influenced the response of larval clownfish to predator odour, and 2) using control or high CO2 water in the test arena influenced the escape response of larval damselfish to a startle stimulus. Finally, 3) because the effects of high CO2 on fish behaviour appear to be caused by altered function of the GABA-A neurotransmitter we tested if antipredator behaviours were restored in clownfish treated with a GABA antagonist (gabazine) in high CO2 water. Larval clownfish reared from hatching in control water (496 uatm) strongly avoided predator cue whereas larval clownfish reared from hatching in high CO2 (1022 uatm) were attracted to the predator cue, as has been reported in previous studies. There was no effect of testing fish using control or high CO2 water in the flume. Larval damselfish reared for 4 days in high CO2 (1051 uatm) exhibited a slower response to a startle stimulus, slower escape speed and a shorter escape distance compared with fish reared in control conditions (464 uatm). There was no effect of test water on escape responses. Treatment of high-CO2 reared clownfish with 4 mg l-1 gabazine in high CO2 seawater restored the normal response to predator odour, as has been previously reported with fish tested in control water. Our results show that using control water in the experimental trials did not influence the results of previous studies on antipredator behaviour of reef fishes and also supports the results of novel experiments conducted in natural reef habitat at ambient CO2 levels.
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Recent studies suggest that projected rises of aquatic CO2 levels cause acid-base regulatory responses in fishes that lead to altered GABAergic neurotransmission and disrupted behaviour, threatening fitness and population survival. It is thought that changes in Cl- and HCO3- gradients across neural membranes interfere with the function of GABA-gated anion channels (GABAA receptors). So far, such alterations have been revealed experimentally by exposing species living in low-CO2 environments, like many oceanic habitats, to high levels of CO2 (hypercapnia). To examine the generality of this phenomenon, we set out to study the opposite situation, hypothesizing that fishes living in typically hypercapnic environments also display behavioural alterations if exposed to low CO2 levels. This would indicate that ion regulation in the fish brain is fine-tuned to the prevailing CO2 conditions. We quantified pH regulatory variables and behavioural responses of Pangasianodon hypophthalmus, a fish native to the hypercapnic Mekong River, acclimated to high-CO2 (3.1 kPa) or low-CO2 (0.04 kPa) water. We found that brain and blood pH was actively regulated and that the low-CO2 fish displayed significantly higher activity levels, which were reduced after treatment with gabazine, a GABAA receptor blocker. This indicates an involvement of the GABAA receptor and altered Cl- and HCO3- ion gradients. Indeed, Goldman calculations suggest that low levels of environmental CO2 may cause significant changes in neural ion gradients in P. hypophthalmus. Taken together, the results suggest that brain ion regulation in fishes is fine-tuned to the prevailing ambient CO2 conditions and is prone to disruption if these conditions change.
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Rising atmospheric carbon dioxide (CO2 ) has caused a suite of environmental issues, however, little is known about how the partial pressure of CO2 (pCO2 ) in freshwater will be affected by climate change. Freshwater pCO2 varies across systems and is controlled by a diverse array of factors, making it difficult to make predictions about future levels of pCO2 . Recent evidence suggests that increasing levels of atmospheric CO2 may directly increase freshwater pCO2 levels in lakes, but rising atmospheric CO2 may also indirectly impact freshwater pCO2 levels in a variety of systems by affecting other contributing factors such as soil respiration, terrestrial productivity and climate regimes. Although future freshwater pCO2 levels remain uncertain, studies have considered the potential impacts of changes to pCO2 levels on freshwater biota. Studies to date have focused on impacts of elevated pCO2 on plankton and macrophytes, and have shown that phytoplankton nutritional quality is reduced, plankton community structure is altered, photosynthesis rates increase and macrophyte distribution shifts with increasing pCO2 . However, a number of key knowledge gaps remain and gaining a better understanding of how freshwater pCO2 levels are regulated and how these levels may impact biota, will be important for predicting future responses to climate change.
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A guide to using S environments to perform statistical analyses providing both an introduction to the use of S and a course in modern statistical methods. The emphasis is on presenting practical problems and full analyses of real data sets.