Silver carp experience metabolic and behavioral changes when exposed to water from the Chicago Area Waterway; implications for upstream movement

doi:10.21203/rs.3.rs-3068489/v1

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Silver carp experience metabolic and behavioral changes when exposed to water from the Chicago Area Waterway; implications for upstream movement

https://doi.org/10.21203/rs.3.rs-3068489/v1

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published 25 Oct, 2024

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One of the hallmarks of invasive species is their propensity to spread. Removing an invasive species after establishment is virtually impossible, and so considerable effort is invested in preventing the range expansion of invaders. Silver carp were discovered in the Mississippi River in 1981 and have spread throughout the basin. Despite their propensity to expand, the ‘leading edge’ in the Illinois River has stalled south of Chicago, and has remained stable for a decade. Studies have suggested that pollutants in the Chicago Area Waterway System (CAWS) may be contributing to the lack of upstream movement, but this hypothesis has not been tested. This study used a laboratory setting to quantify the role of pollutants in deterring upstream movement of silver carp within the CAWS. For this, water was collected from the CAWS near the upstream edge of the distribution and transported to a fish culture facility. Silver carp and one native species were exposed to CAWS water, and activity, behavior, avoidance and metabolic rates were quantified. Results showed that silver carp experience an elevated metabolic cost in CAWS water, along with reductions in swimming behavior. Together, results suggest a role for components of CAWS water at deterring range expansion.

Earth and environmental sciences/Ecology/Invasive species

Biological sciences/Ecology/Ecophysiology

Biological sciences/Physiology/Metabolism

Biological sciences/Zoology/Animal behaviour

Biological sciences/Zoology/Animal physiology

Habitat selection is a key factor influencing the abundance, distribution, and fitness of organisms. The selection of a habitat by an animal results from a complex series of choices that involve tradeoffs between energy acquisition, energy expenditure, predation risk and reproductive opportunities. Ultimately, organisms seek to occupy habitats that help minimize energetic costs and maximize energetic gains^1,2 and can therefore influence how individuals are distributed in their respective environments^3–5. More importantly, habitat selection can have a pronounced impact on survival, reproduction and fitness if the balance between energy input and energy expenditure impairs energy available for growth or gamete production^6,7. Thus, choices related to habitat selection by organisms is a key component of survival, reproduction and fitness.

Circumstances can arise where animals are exposed to sub-optimal habitat, which can result in a number of physiological, energetic and behavioral consequences. In particular, fish have a finite energy budget such that increased energetic costs, or reduced energetic intake, due to the occupation of sub-optimal habitat can translate to reduced energy availability for reproduction, which, in turn, can impair fitness⁷. For fish, inhabiting habitat that is sub-optimal, such as water that is too warm or has reduced oxygen or contains contaminants, can result in an upregulation of the stress response in an effort to combat sub-optimal habitat and restore homeostasis, which can direct energy away from biological processes, such as foraging or reproduction⁸. Changes in swimming behavior in response to an environmental stressor has also been documented, as altered behavior can be an indicator of decreased performance in individuals⁹. In extreme cases, in an effort to avoid increased energetic costs concomitant with sub-optimal habitat, fish may choose to simply swim away and avoid certain areas, exhibiting behavioral avoidance¹⁰. Specifically, aquatic organisms will avoid deleterious environmental conditions and move to a more optimal location, with some species having different environmental tolerances than others¹¹. Thus, extended occupation of sub-optimal habitats by fish can translate to reductions in individual fitness, and fish may employ behavioral responses to sub-optimal habitat such as avoidance to conserve energy.

The presence of silver carp (Hypophthalmichthys molitrix) in the Illinois River, USA, is a unique case of an invasive species that appear to be experiencing restrictions in range expansion due to sub-optimal habitat. Silver carp are prolific planktivores, that have become the most abundant fish in certain parts of North America, and their presence has had negative impacts on native fish due through their consumption of phytoplankton and zooplankton¹². Furthermore, silver carp are able to pass through locks and dams, with their range expansion not impaired by physical barriers^13,14. Interestingly, the “invasion front” of the silver carp population in the Illinois River has not expanded beyond its current location for over 10 years, despite the fact that there are no locks, dams or physical structures to deter further upstream movement¹⁵. One hypothesis to explain why the invasion front of silver carp has not expanded further upstream is that they are being deterred by the presence of anthropogenic bioactive contaminants (i.e., pollutants or contaminants) coming from the Chicago Area Waterway System (CAWS), and these contaminants are reducing habitat quality that silver carp are avoiding. There are a few pieces of evidence that support this theory, including the fact that there is a higher concentration of bioactive contaminants upstream of the invasion front compared to downstream¹⁶, along with studies showing that carp at the ‘leading edge’ in the Illinois River have an upregulation of genes related to contaminant exposure¹⁷ along with evidence of reduced energy stores in liver¹⁸, both of which were absent from silver carp at the population core. Thus, correlative evidence to date suggests that further upstream range expansion of silver carp in the Illinois River is being deterred due to the presence of anthropogenic contaminants from the CAWS that is reducing habitat quality. To date, this hypothesis has not been tested directly, impairing us from defining a mechanism to explain the curious lack of upstream movement of this prolific invader that has been expanding throughout the Mississippi Basin. Knowledge of how water quality is influencing habitat choice is a key piece of information that may aid in the management and control of this injurious group of fishes.

The objective of this study was to define the role of anthropogenic contaminants in the Chicago Area Waterway System at deterring the upstream movement of silver carp. To accomplish this goal, hatchery-reared silver carp were given an acute exposure to water from the CAWS in a laboratory, and three different assays were performed: an avoidance assay to quantify if silver carp would avoid CAWS water, the behavior of silver carp was quantified after they were transferred into CAWS water, and the metabolic rates of silver carp were quantified while they were held in CAWS water. We also conducted identical assays with golden shiners (Notemigonus crysoleucas) as a representative native species commonly found in the CAWS to quantify potential differences between silver carp and native species. Together, results from this study will help define a possible mechanism responsible for the lack of upstream movement of silver carp in the Illinois River that has been observed in the past decade.

Collection of CAWS Water

Water from the Chicago Area Waterway was collected from a public boat ramp in Channahon, IL (41.420501, -88.215916) on January 19th, 2021. This site in proximity to the “leading edge” of carp populations in the Illinois River^15,19, and has previously been shown to have higher concentrations of anthropogenic contaminants than downstream locations where population densities of carp are higher¹⁶, suggesting that water conditions at this site should be exhibiting some degree of deterrence to carp movement. Further downstream of this site, the concentration of deterrents in the water are lower and may not be representative of conditions that may be deterring upstream movements, while locations upstream of this collection point have never contained carp populations, suggesting the presence of a strong negative stimulus, thereby highlighting the appropriateness of this collection point. Water was collected using pumps submerged in the CAWS that passed through a 600-micron filter before being transferred into 1463 L intermediate bulk containers (IBC totes). Totes were transported to the Upper Midwest Environmental Science Center (UMESC), in La Crosse, WI, and stored indoors in a biosecure room. One aliquot of CAWS water needed for a day’s worth of experiments was collected from holding totes the evening before and moved to an environmental chamber used for testing so that the temperature of the CAWS water was able to equilibrate to the temperature of the chamber.

Study Site and Organisms

Juvenile silver carp and golden shiners were cultured by the United States Geological Survey (USGS) in La Crosse, Wisconsin. Both silver carp and golden shiners were held at temperatures ranging from 12-13°C for the duration of the study. All fish were fed daily, with silver carp fed #0 Starter Feed (Skretting, Tooele, Utah) and golden shiners fed #1 Starter Feed (Skretting, Tooele, Utah). Prior to use in experiments, all fish were fasted for 24 hours.

Avoidance Assay

Assays to quantify avoidance following acute exposure to CAWS water took place between January 24 and February 16, 2022, in an environmental chamber set to 12°C. Assays were run using two shuttle box choice tanks (Loligo, Viborg, Denmark). Each system has two circular tanks (40 cm diameter × 20 cm depth) connected by a narrow chamber (10 cm × 6.5 cm × 20 cm). Water was drawn from each chamber of the shuttle box into an external buffer tank (20 cm × 20 cm × 50 cm) using two Eheim 300 recirculating pumps (Eheim, Deizisau, Germany), and then gravity-fed back into the shuttle box at approximately 38.5 mL/sec. Fish activity within the shuttle box was recorded by a camera mounted directly overhead (1080p Pro Stream Webcam, (Lolitech, Newark California); GoPro Hero 3 (GoPro, San Mateo California, USA)). Light was shone indirectly on the shuttle box to create contrast to help locate the fish within the tank. Before each trial, shuttle boxes were filled with 30 L of water from the aquatic facility, identical to the water used to hold fish. Fish (n = 8) were transferred into one side of the shuttle box (determined by a random number generator) and allowed to acclimate for 15 minutes. Preliminary trials showed that fish became settled in the shuttle box after about 10 minutes, indicated by a reduction in movement, and acclimation times of 15 minutes are common for these kinds of studies^20,21.

Following this acclimation period, 8 L of water was simultaneously added to both external buffer tanks, allowing this water to be gravity-fed into the shuttle box chambers. For control trials, 8 L of laboratory water was added to both buffer tanks. For treatment trials, 8 L of CAWS water was added to the buffer tank corresponding to the side of the shuttle box that contained the fish, and 8 L of lab water was simultaneously added to the opposite side. The use of this technique allowed fish in the shuttle box to receive an acute exposure to CAWS water in a way that minimized splashing or sudden dumping of water that could startle them, and the order of trials (control vs. treatment) was determined with a random number generator. After the addition of water, fish were allowed to move within their chamber of the shuttle box, and also to move between sides of the shuttle box, over a ten-minute period. This monitoring period was chosen because preliminary trials with food coloring revealed that the shuttle box system will begin to show some mixing across the two sides around the ten-minute mark, such that the side of the shuttle box with CAWS water started to become diluted, potentially reducing its efficacy at inducing avoidance. Immediately following this 10-minute period, the experiment ended, temperature and dissolved oxygen measurements within the shuttle box were taken using a handheld oxygen meter (Professional Plus, YSI Inc., Yellow Springs, Ohio, USA), and fish were removed, weighed, and measured. Fish length did not differ between treatments for both species (one-way analysis of variance (ANOVA)_{silver carp}, F_1,16 = 1.963, p > 0.05, one-way ANOVA_{golden shiner}, F_1,16 = 1.198, p > 0.05, Appendix I).

Activity Assay

Assays to quantify fish activity following acute exposure to CAWS water were run from February 11, until February 15, 2022, using four 19-liter opaque white buckets placed on top of acrylic sheets above 4 light emitting diode (LED) work lights (Home Depot, HDX 1000 Lumen Portable LED Work Lights). These lights were pointed upwards such that they illuminated the bottom of the bucket and generated contrast that facilitated identifying fish location. Video cameras (1080p Pro Stream Webcam, (Lolitech, Newark California); Sony Handycam CX405 (Sony Corp., Japan); GoPro Hero 3 (GoPro, San Mateo California, USA)) were mounted above each bucket to record fish position and activity. Approximately 3.5 L of water was added to each bucket at the beginning of each trial. This volume of water was sufficiently deep to allow fish to move comfortably in the bucket, but not so high that they could change depth. For control trials, buckets were filled with water from the aquatic facility identical to that used for fish holding. For treatment trials, CAWS water was used. Fish (n = 9) were collected from their holding tank and gently transferred into a bucket with either laboratory water or CAWS water, with the placement determined by a random number generator. Fish activity was recorded over a ten-minute period after they were placed in the buckets. Immediately following this 10-minute period, the experiment ended, temperature and dissolved oxygen measurements within each bucket were taken using a handheld oxygen meter, and fish were removed, weighed, and measured. Fish length did not differ between treatments for both species (one-way ANOVA_{silver carp}, F_1,18 = 1.031, p > 0.05, one-way ANOVA_{golden shiner}, F_1,18 = 1.756, p > 0.05, Appendix I).

Metabolic rate

Standard metabolic rate (SMR) was measured for silver carp and golden shiners in either laboratory water (control) or 100% CAWS water to quantify the energetic cost of inhabiting CAWS water^22–24. Data collection occurred from January 24 through February 10, 2022, and was performed in a 92.075 cm diameter bath containing approximately 50.74 L of water. Four 270 mL glass chambers were immersed in this bath filled with either laboratory water or CAWS water. Each chamber was connected to a recirculating pump via 16 mm inside diameter vinyl tubing. This allowed for a closed system during testing as well as allowing the system to flush between measurement periods²². An oxygen probe was fitted into each chamber to record dissolved oxygen concentration during a 20-minute measurement period using respirometry software (Autoresp v 2.2.2, Loligo, Denmark). The measurement period for silver carp was set to a 5-minute flush period, a 1-minute wait period, and a 14-minute measurement period. For golden shiners, the measurement period was a 17-minute period to account for the smaller size of this species. Fish (n = 12; n = 11 for control treatment for golden shiners) were weighed and loaded in these chambers in the afternoon and held throughout the night until the following morning. After this, fish were removed from the chambers and total lengths were taken. All control trials with laboratory water were ran for both species prior to trials with CAWS water to eliminate possible cross-contamination or residual contaminants that could influence metabolic rates of fish in control trials. Additionally, chambers were ran empty before and after each trial to account for background respiration present within each system^23,24. Mass did not differ between treatments for both species (one-way ANOVA_{silver carp}, F_1,24 = 0.661, p > 0.05, one-way ANOVA_{golden shiner}, F_1,23 = 2.588, p > 0.05). Fish length also did not differ between treatments for both species (one-way ANOVA_{silver carp}, F_1,24 = 0.661, p > 0.05, one-way ANOVA_{golden shiner}, F_1,23 = 1.058, p > 0.05, Appendix I).

Data Acquisition and Analysis

A total of 4 metrics to quantify avoidance in the shuttle box were scored manually by a single observer: (1) a binary response of whether or not a fish ‘shuttled’ (i.e., moved to the opposite chamber in the shuttle box) after water was added to the buffer chambers, (2) a count of the total number of shuttles between the two sides of the shuttle box that occurred following the addition of water, (3) the latency (time in s) required for the initial shuttle to occur, and (4) the total duration of time spent by a fish on each side of the shuttle box during a trial (control vs. treatment side). Because all metrics were objective, blind scoring did not occur. Statistical analyses were conducted in R version 4.2.1 (R Core Team 2021) using a one-way ANOVA. The binary response of whether a fish shuttled was run using a generalized linear model with a binomial error distribution, while count data (e.g., number of shuttles) were run with a generalized linear model with a Poisson error distribution²⁵. Continuous variables (e.g., total duration spent on the treatment side) were run with a Gaussian distribution²⁵. All avoidance models were validated using standard techniques that included generating quantile-quantile plots to quantify normality, fitting residuals versus fitted values to verify homogeneity, and examining residuals versus each explanatory variable to check for independence²⁶. The presence of potential influential data points was also assessed²⁶. Analyses for golden shiners and silver carp were run separately.

Data for activity assays from fish in buckets were scored using the program Ethovision XT (Version 16.1) (Noldus, Leesburg, Virginia) to objectively quantify and track movement^27,28. Metrics that were measured encompassed aspects of swimming activity as well as swimming behavior, and included (1) total distance (cm) moved during the trial, (2) swimming velocity (m/s), (3) the proportion of time that animals spent wall hugging (defined as the area closest to the wall that is half a body length of each individual fish), (4) the number of rotations that occurred (where a rotation was defined as the center body point has a cumulative turn angle of 360° in either the clockwise or counterclockwise direction), and (5) average turn angle (measured in degrees).

Prior to analyses, activity assay data were checked for collinearity²⁹, and distance, velocity, and rotations were found to be correlated (Pearson correlation coefficient, r, > 0.95 for all three metrics for both species). We therefore distilled these variables using principal components analyses (PCA), which generated a single PC with all variables loading positively, representing movement behaviors of fish (Appendix II). Suitability of the data for use in a PCA was confirmed using a Kaiser-Meyer-Olkin test and Bartlett’s test for sphericity³⁰ using the ‘psych’ package³¹ (Version 2.2.9).

Behavior data from the 10-minute trial were grouped into one minute bins to better visualize the response of fish to CAWS water, the persistence of any response, and to identify potential habituation³². Data were then analyzed using a two-way linear mixed model (main effects treatment, time bin and a treatment × time bin interaction) using the ‘lme4’ package³³ (Version 1.1.30), with fish identification included as a random effect to account for multiple measurements from each individual over time^25,34. If a significant difference in main effects was detected in the mixed model, the ‘emmeans’ package^35,36 (Version 1.8.1.1) was used to separate means and identify significant differences; if a significant interaction was detected, main effects were ignored, and means were separated for the interaction term³⁷. All models were validated as described above. Upon examination of residuals, PC1 and wall hugging time were found to violate model assumptions and were rank transformed prior to re-running models, while turn angle was log transformed for both species.

Data for metabolic rates were inspected and any measurement interval with a coefficient of determination (r²) value below 0.9 was excluded²². Next, background respiration was accounted for by using the background respiration from before and after the trial and assuming a linear increase for each fish. Following this, values for SMR, defined as the mean of the lowest 10% of data generated during each overnight measurement period²², were compared across treatments using a one-way ANOVA with standard metabolic rate set as the dependent variable, and the independent variables were treatment (either control or CAWS water) and fish mass. Previous work has shown that SMR and mass have a non-linear relationship in fish, necessitating the use of a log-log transformation in data analysis³⁸. However, this non-linear relationship was less clear in our study (Appendix I, Appendix V), likely due to the small size ranges of fish used³⁸, making the use of log-transformed data less clear. To acknowledge this uncertainty, both log-log transformed and untransformed data were analyzed and reported. If a significant difference was detected in the model, Tukey multiple comparison tests were performed using estimated marginal means (least-squares means) with the ‘emmeans’ package^35,36 (Version 1.8.1.1). Analyses for golden shiners and silver carp were run separately, and all models were again validated by examination of residuals as described above²⁹.

All model results were interpreted using the Anova() function from the ‘car’ package (Fox et al. 2007), the level of significance (α) was set at 0.05, and all means are shown as ± standard error (SE) where appropriate. All models were initially run with trial date and fish size as fixed effects, but no significant date or size effects were detected, so these variables were subsequently removed from final analyses.

Ethics Statements

This study was carried out in accordance with relevant guidelines and regulations and approved by the University of Illinois IACUC # 21139. All methods were reported in accordance with ARRIVE guidelines.

When silver carp were held in a shuttle box that received an acute input of CAWS water, 4 out of 8 individuals ‘shuttled’ to the opposite chamber of the shuttle box, while 5 of 8 silver carp exposed to laboratory (control) water shuttled, and this difference was not statistically significant (Table 1, Appendix III). Silver carp spent 6.1% more time in CAWS water than silver carp in the control treatment, delayed approximately 37% longer than fish in control water before shuttling to the opposite chamber of the shuttle box, and conducted approximately one-third fewer shuttles than control-treatment fish over the 10-minute trial, but differences across treatments were not significantly different (Table 1, Appendix III).

Table 1

Results of statistical tests comparing avoidance behaviors and standard metabolic rate of silver carp and golden shiners that received an acute exposure to water from the Chicago Area Waterway (CAWS) relative to individuals that were exposed to laboratory (control) water. Avoidance assays following CAWS exposure were quantified in a shuttle box, and standard metabolic rate was generated using intermittent flow respirometry. Binary data were analyzed with a binomial or Poisson error distribution and therefore show a Χ² statistic, while continuous data were analyzed using a Gaussian error distribution and show an F-value. Significant differences across treatments are shown in bold.
Silver Carp
Assay	Metric	Df	F-value or LR Χ²	P-value
Avoidance Assay	Did fish shuttle? (yes/no)	1	0.255	0.614
	Count of number of shuttles	1	1.995	0.158
	Latency to first shuttle (s)	1	1.000	0.334
	Time spent on receiving side (s)	1	0.115	0.740
Metabolic rate (untransformed)	Treatment Mass	1	3.472	0.0765
Metabolic rate (untransformed)	Treatment Mass	1	6.793	0.016
Metabolic rate (log transformed)	Treatment	1	4.866	0.039
Metabolic rate (log transformed)	Mass	1	5.250	0.032
Golden Shiner
Avoidance Assay	Did fish shuttle?	1	1.011	0.315
	Number of shuttles	1	30.805	< 0.01
	Latency to first shuttle (s)	1	2.006	0.179
	Time spent on receiving side (s)	1	0.156	0.699
Metabolic rate (non-transformed)	Treatment Mass	1	2.835	0.108
Metabolic rate (non-transformed)	Treatment Mass	1	1.297	0.268
Metabolic rate	Treatment	1	3.064	0.095
(log transformed)	Mass	1	1.047	0.318

Golden shiners that received an acute exposure to CAWS water shuttled 5 out of 8 times, while 3 out of the 8 fish tested in lab water shuttled, and this difference was not significant across treatments (Table 1, Appendix IV). Golden shiners in lab water delayed shuttling approximately 56% longer than fish in CAWS water. Furthermore, golden shiners exposed to CAWS water spent approximately 8% less time on the receiving side than fish exposed to lab water. None of these metrics showed p-values less than 0.05 (Table 1, Appendix IV). However, shuttle frequency was found to be a little over four times higher for golden shiners in CAWS water (Table 1, Appendix IV).

Silver carp displayed a reduction in movement behaviors (PC1, a composite of distance travelled, velocity and number of rotations) within 1 minute after being placed in CAWS water and this reduction in movement relative to the start of the trial remained present until the trial ended (Fig. 1, Table 2). In contrast, silver carp in lab water did not show a change in movement behaviors until 9 minutes after being placed in buckets (Fig. 1, Table 2). Silver carp did not show any significant differences in the proportion of time spent wall hugging or average turn angle during the activity assay over the trial across treatments with lab vs. CAWS water (Table 2).

Table 2

Results of statistical tests following the activity assay for silver carp and golden shiners that received an acute exposure to water from the Chicago Area Waterway (CAWS) relative to individuals that were exposed to laboratory (control) water. Assays were performed in buckets. Data were analyzed by a two-way linear mixed model (main effects treatment, time bin and a treatment × time bin interaction). Significant differences across variables are shown in bold. R² values were calculated from r.squaredGLMM() from the MuMin package.
Silver Carp
Metric	Variable	Df	LR or Χ²	P-value	Marginal r² for mixed models	Conditional r² for mixed models
PC1	Treatment	1	0.278	0.598	0.137	0.816
	Time bin	9	85.088	< 0.01
	Treatment × Time bin	9	17.373	0.0431
Wall-hugging time (s)	Treatment	1	0.321	0.570	0.044	0.653
	Time bin	9	14.073	0.120
	Treatment × Time bin	9	8.769	0.459
Turn angle (degrees)	Treatment	1	0.228	0.633	0.054	0.681
	Time bin	9	15.854	0.070
	Treatment × Time bin	9	5.193	0.817
Golden Shiner
PC1	Treatment	1	0.446	0.504	0.035	0.628
	Time bin	9	13.144	0.156
	Treatment × Time bin	9	4.658	0.863
Wall-hugging time(s)	Treatment	1	0.322	0.570	0.070	0.547
	Time bin	9	6.832	0.655
	Treatment × Time bin	9	3.705	0.930
Turn angle (degrees)	Treatment	1	5.771	0.0163	0.235	0.733
	Time bin	9	12.718	0.176
	Treatment × Time bin	9	1.330	0.998

Golden shiners did not display any significant differences in any behavioral parameter between control or CAWS treatments measured during the activity assay, displaying a statistically similar PC1, proportion of time spend wall-hugging, and average turn angle across the time bins (Table 2).

The standard metabolic rate for silver carp held overnight in CAWS water was nearly 30% higher compared to animals held in laboratory water (Fig. 2, Table 1, Appendix III). The standard metabolic rate of golden shiners in CAWS water was similar to animals held in CAWS water (Table 1).

Upon being placed in water collected from the Chicago Area Waterway System (CAWS), silver carp showed a reduction in movement that occurred sooner than conspecifics held in control water. Specifically, decreases in PC scores for silver carp occurred after only one minute following exposure to CAWS water, while fish held laboratory water did not show a decrease in movement until the final minute of the 10-minute trial. Behavior is defined as observable physical activities performed by an organism that are informed by genetics, environmental stimuli, and physiology that assist in that organisms’ survival^39,40. Quantifying behaviors of fishes in response to pollutants is common, as it is easy, sensitive to environmental variation⁴¹, a reliable indicator of stress, and, more importantly, alterations in behavior can have negative consequences for responses such as reproduction, foraging and survival^39,42. For example, both Little et al. (1990)⁴³, and Little and DeLonay (1996)⁴², showed that exposure to a number of different chemicals resulted in reduced activity in rainbow trout (Oncorhynchus mykiss), while Fernandez-Vega et al. (2002)⁴⁴ saw that European eels (Anguilla anguilla) exposed to thiobencarb, an herbicide, reduced motility and increased respiratory frequency. Similarly, Da Rosa et al. (2016)⁴⁵ showed zebrafish (Danio rerio) decreased rotation number when exposed to gamma-Hydroxybutyric acid, or GHB. The reason for the decline in activity shown by silver carp exposed to CAWS water in the current study has not been clearly identified but could result from one of two possible mechanisms. The first possible explanation for a reduction in activity following exposure to contaminants relates to coping style and the possibility that silver carp may be displaying a reactive coping style in response to a stressor. Coping style is defined as an individual organism’s response to certain adverse situations that is consistent over time, and animals that employ a reactive coping style typically adopt an immobile strategy to conserve energy during times of acute stress^46–50. Zeng et al. (2019)⁵⁰, for example, explored coping styles in the marine olive flounder (Paralichthys olivaceus) when transferred to freshwater, and saw decreased swimming activity consistent with a passive coping style upon encountering this stressor. Alternatively, the reduction in activity displayed by silver carp after being exposed to CAWS water may have resulted from altered brain biochemistry, which, in turn, can lead to behavioral alterations. Brewer et al. (2001)⁵¹ showed that rainbow trout exposed to malathion experienced a reduction in brain acetylcholinesterase concentration, which, in turn, resulted in reductions in distance travelled and swim speed, while Nichols et al. (1984)⁵² showed that exposure of coho salmon (Oncorhynchus kisutch) to arsenic trioxide reduced concentrations of plasma thyroxine, as well as a reduction in outmigration activity, thereby providing a link between environmental pollution, physiology, and reductions in activity. Regardless of the mechanism, results from the current study clearly show that silver carp showed reductions in activity metrics when acutely transferred to CAWS water relative to fish in laboratory water.

In addition to reductions in activity, the standard metabolic rate of silver carp held in CAWS water was 30% higher than fish held in laboratory water. Metabolic rate can be used to compare the energetic costs of inhabiting different environmental conditions^53,54, and has been used to compare thermal tolerance, hypoxia, and exposure of foreign compounds^55,56. Following exposure to an environmental stressor, fish can experience an increase in metabolic rate as part of the secondary stress response, which functions to re-establish homeostasis in the face of environmental challenges^7,57. McKenzie et al. (2007)⁵³, for example, showed that resting metabolic rates of European chub (Leuciscus cephalus) increased by about 50% when exposed to polluted river site relative to two cleaner rivers. In the same study, common carp (Cyprinus carpio) showed around a 20% increase in resting metabolic rates when exposed to a different set of polluted river sites relative to clean river sites. Du et al. (2019)⁵⁸ showed that wild-caught bluegill (Lepomis macrochirus) exposed to sewage outflows of wastewater treatment plants had resting metabolic rates around 45% higher than wild-caught bluegill exposed to clean water. Similarly, Baum et al. (2016)⁵⁹ found that orange-spotted spinefoot (Siganus guttatus) exposed to linear alkylbenzene sulfonate (LAS) had a significantly higher SMR than a control group in pollutant-free water. The elevated SMR observed for silver carp in CAWS water relative to control fish can have two main consequences. First, fish will expend more energy to maintain homeostasis during periods of duress (Sadoul and Vijayan 2016)⁷ and will have less energy available for other critical functions, such as reproduction and growth⁶⁰. Second, the increase in standard metabolic rate of a fish during periods of stress can reduce aerobic scope. Aerobic scope is the difference between the minimum and maximum metabolic rate for an organism and can dictate the number of metabolic processes that an organism can perform simultaneously^61,62,63. Ackerly and Esbaugh (2020)⁵⁵ found that a combination of hypoxic conditions and exposure to oil resulted in decreases in aerobic scope in red drum. This reduction in aerobic scope also makes it difficult for organisms to properly respond to external stressors or changing environmental conditions. Thus, the increased SMR experienced by silver carp inhabiting CAWS water may play a role in a lack of the upstream expansion if is too energetically costly for animals to allocate energy to reproduction in areas further upstream⁶³. Additional work on the quantity and quality of food resources within the CAWS available to silver carp would be required to corroborate this hypothesis. Together, results from the current study clearly show that silver carp in CAWS have higher standard metabolic rates relative to animals held in control water, which, in turn, can have consequences for energy availability for essential processes such as growth or reproduction, and may be contributing to a lack of upstream movement.

Silver carp did not show active avoidance of CAWS water following acute exposure. More specifically, there was no significant difference between the CAWS and control treatments for metrics including whether or not the fish shuttled, frequency of shuttles, latency to first shuttle, and time spent the side of the shuttle box that received an input of CAWS water during the avoidance assay. Organisms can show a number of possible responses when confronted with a deleterious change in their environment^39,64, with one possible response being a choice between different environments based on physiological preferences¹⁰. Many organisms exhibit environmental preferences in direct response to possible negative physiological effects that could occur while occupying sub-optimal habitat². One tool that can be used to quantify avoidance or preference of differing environments in a laboratory setting is a ‘shuttle box’¹⁰. In the past, shuttle boxes have been used to quantify avoidance of noxious stimuli such as salinity, CO₂ gas, and temperature^65–68. For example, Dennis et al. (2015)⁶⁶ found that multiple fish species elicited an avoidance response in a shuttle box following acute exposure to hypercarbia. In the current study, the lack of an avoidance response from silver carp receiving an acute exposure to CAWS water could have occurred from one of three possible explanations. First, the CAWS water may not have been a strong enough stressor to induce avoidance in silver carp. Tierney (2016)⁶⁴ indicated that many stimuli have threshold-based avoidance, or a positive correlation between avoidance concentration and a stimulus, such that, at low concentration, avoidance might not have occurred. Second, the concentration of CAWS water may have been too dilute, and any strong noxious stimulus that could have induced avoidance was at a low concentration. The design we used in this study was to add CAWS water to the external buffer tanks of the shuttle box rather than a direct application into the chamber with the fish, in hopes of minimizing a startle response. This resulted in an unavoidable dilution of CAWS water, which might have reduced the concentration required to induce avoidance. Dennis et al. (2015)⁶⁶, for example, found that there was a threshold of CO₂ concentrations that induced an avoidance response in multiple fish species. Finally, many shuttle box applications add a stimulus like increased temperature or dissolved oxygen to the system while keeping water volume constant. The change in volume of water in the external buffer tanks used in the current study could have influenced behavior and been a stronger stimulus than the presence of CAWS water, thereby masking potential avoidance responses from contaminants in the water. Flow-through behavior tanks that provide continuous exposure to two different water types are one option that could be used for similar avoidance studies with CAWS water in the future, as these flow-through tanks have been used for measuring preference in other environmental stimuli ^20,69. Overall, results from this study did not provide strong evidence that silver carp are engaging in immediate avoidance behavior following acute exposure to CAWS water.

Golden shiners did not show overt avoidance or metabolic changes when exposed to CAWS water. Specifically, there was no significant difference between the CAWS and control treatments for any of the metrics in the metabolic and activity assays. Shuttle frequency was the only metric that was significantly different between treatments in the avoidance assay, being significantly higher in CAWS water. When we look across fish species, there can be differences in the response to a common stressor. Jacobsen et al. (2014)⁷⁰ found that habitat selection and swimming activity differed between multiple fish species in response to recreational boating. Specifically, common roach (Rutilus rutilus) increased their occurrence in the central part of the lake following boating disturbances, while European perch (Perca fluviatilis) and northern pike (Esox lucius) did not show a difference in habitat use. Hansen et al. (1999)⁷¹ saw that congeneric chinook salmon (Oncorhynchus tshawytscha) were more susceptible to physiological effects of copper exposure then rainbow trout (Oncorhynchus mykiss). Golden shiners are currently found throughout the CAWS, and the catch-per-unit-effort of golden shiners has increased over the last couple decades suggesting that populations are healthy and robust⁷². Golden shiners therefore may not have shown a strong response to CAWS water in the current study due to a reduced sensitivity and/or increased tolerance to environmental contaminants relative to silver carp, evidenced by their presence throughout the CAWS. Combined over all three assays, results did not show a strong behavioral or metabolic responses of golden shiners to CAWS water.

Management implications

Data from this study show that silver carp are not exhibiting abrupt, pronounced or exaggerated avoidance behaviors when exposed to CAWS water that could be preventing upstream movements. However, this study does show that exposure to CAWS water results in the reduction of movement of silver carp, such as reduced activity and fewer rotations, coupled with an increased energetic cost, implying that carp are impaired by inhabiting CAWS water and are likely not moving closer to Chicago to avoid energetic costs imposed by reduced water quality. This result is in agreement with past work showing that carp at the ‘leading edge’ have increased indices of stress and energy use due to exposure to xenobiotics, as well as increased energetic demands, relative to conspecifics downstream^17,18. Water quality in the CAWS has been improving thanks to federal regulations like the Clean Water Act, as well as actions from local agencies, which has resulted in improved fish communities^73,74. Future improvements to water quality would have important benefits to local ecosystems, but may inadvertently remove stimuli in the water that are contributing to the lack of upstream movement of invasive carp. Additional work should therefore be conducted to further define which aspect of CAWS water is motivating behavioral and metabolic changes for carp to allow managers to be better prepared for possible range expansion should that stimulus be removed.

Throughout nature, organisms choose to occupy habitat that minimizes energetic costs while maximizing energetic gains in an effort to maximize fitness. Exposure to sub-optimal habitats can upset this energetic balance, resulting in physiological or behavioral changes, and increased energetic costs that can come at the expense of reproduction. One example of stressor that could create sub-optimal habitat is the presence of pollutants and chemicals in certain aquatic systems. The leading edge of the silver carp population in the Illinois River has not moved upstream of its current location in over a decade, and correlative data suggest that the presence of contaminants in the water may be contributing to the lack of upstream range expansion. Results from the current study showed for the first time that silver carp display decreased swimming activity and increased energy consumption when exposed to CAWS water, which was not apparent for animals held in control water, providing a putative, energetic mechanism for the lack of range expansion of silver carp within the CAWS. Defining this mechanism responsible for the lack of upstream movement of carp within the CAWS provides information on why silver carp might move in the future.

Acknowledgements

Funding for this study was provided by the United States Geological Survey WRRI Aquatic Invasive Species (AIS) Competitive Grants Program (Award G21AP10174-00), the Department of Natural Resources and Environmental Sciences at the University of Illinois at Urbana-Champaign, and the USDA National Institute of Food and Agriculture, Hatch program project ILLU-875-940. We would like to acknowledge Aaron Cupp, Rachel Nelson, Steve Redman and Justin Smerud with the Upper Midwest Environmental Science Center in La Crosse, WI, for providing a space to work, fish to use, and assistance with data and water collection. Additional thanks go to Qihong Dai and John Bieber for providing influence for study design, as well as Jim Duncker and David Fazio of the USGS Central Midwest Water Science Center for analyzing water samples. Scarlett Hoffer aided with the analysis of behavioral videos.

Data Availability Statement

All files will be made available through the University of Illinois Data Bank (https://databank.illinois.edu/) upon acceptance. Contact Amy Schneider or Cory Suski to request additional information from this study.

Competing Interests

The authors declare that they have no conflict of interest.

Author Contributions

AJE and CDS conceived the study design, while AES planned experiments performed the data collection. AES performed all data analysis and generation of figures. All authors contributed to writing and editing the final manuscript.

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No competing interests reported.

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Silver carp experience metabolic and behavioral changes when exposed to water from the Chicago Area Waterway; implications for upstream movement

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