Introduction

Habitat selection is a key factor influencing the abundance, distribution, and fitness of organisms1,2,3. 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 succeed best in habitats that help minimize energetic costs and maximize energetic gains1,2 and can therefore influence how individuals are distributed in their respective environments3,4,5. More importantly, habitat selection can affect survival, reproduction, and fitness if the balance between energy input and energy expenditure impairs energy available for growth or gamete production6,7. Thus, choices related to habitat selection by organisms are key components of survival, reproduction, and fitness.

Circumstances can arise where animals are exposed to sub-optimal habitat, which can result in several 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. 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, directing energy away from biological processes, such as foraging or reproduction8. Changes in swimming behavior in response to an environmental stressor have also been documented and can be an indicator of decreased fitness in individuals9. In extreme cases, to avoid increased energetic costs concomitant with sub-optimal habitat, fish may choose to simply swim away once a specific threshold of a stressor has been reached10, exhibiting behavioral avoidance11. Additionally, environmental tolerance can vary by species12. Regardless of species, extended occupation of sub-optimal habitats by fish can translate to reductions in individual fitness.

The presence of silver carp (Hypophthalmichthys molitrix) in the Illinois River, USA, is a unique case of an invasive species that appears 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 effects on native fish due through their consumption of phytoplankton and zooplankton13. Furthermore, silver carp easily pass through locks and dams, with their range expansion not impaired by physical barriers14,15. Of note, 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 the population has been able to advance upstream until that point16. Several possible hypotheses can explain this lack of upstream movement, including a scarcity of suitable habitat, low availability of food resources, or low population densities at the leading edge due to harvest efforts farther downstream17. An additional, alternate hypothesis to explain why the invasion front of silver carp has not expanded farther upstream is that they are being deterred by the presence of anthropogenic bioactive contaminants coming from the Chicago Area Waterway System (CAWS), and these contaminants are reducing habitat quality. To date, this hypothesis has not been tested directly, hindering 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. Anecdotal evidence supports this hypothesis, including the presence of a higher concentration of bioactive contaminants upstream from the invasion front compared to downstream18,19. Furthermore, carp at the ‘leading edge’ in the Illinois River have an upregulation of genes related to contaminant exposure20 along with evidence of reduced energy stores in liver21, both of which were absent from silver carp at the population core. 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 in 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: (1) a behavioral assay to determine if silver carp would avoid CAWS water, (2) the behavior of silver carp was monitored upon transfer from untreated control water into CAWS water, and (3) 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.

Methods

Study site and organisms

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

Collection, transport, and storage of CAWS water

Water from the Chicago Area Waterway was collected from a public boat ramp in Channahon, Illinois (41.420501, − 88.215916) on January 19, 2022. This site is in proximity to the “leading edge” of carp populations in the Illinois River16,22, and has previously been shown to have higher concentrations of anthropogenic contaminants than downstream locations where population densities of carp are higher18,19, indicating that water conditions at this site could be exhibiting some degree of deterrence to carp movement. Farther downstream from this site, the concentration of contaminants in the water are lower and may not be representative of conditions that may be deterring upstream movements, while locations upstream from this collection point have never contained carp populations, suggesting the presence of a strong negative stimulus, thereby highlighting the appropriateness of the chosen collection point; a chemical analysis of water that was collected as well as historical data are available from the U.S. Geological Survey (USGS) National Water Information System database at https://doi.org/10.5066/F7P55KJN using site number 05538020. 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 USGS Upper Midwest Environmental Sciences Center (UMESC), in La Crosse, Wisconsin, 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. Note that for all experiments, the control treatment consisted of the USGS UMESC water in which the animals were cultured. Ideally comparisons of CAWS water would have been made to a downstream site; however, the logistical challenges associated with transporting two water types to the animal husbandry site could not be overcome. That being said, waters from both areas are broadly similar with respect to baseline hardwater-type chemical composition.

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). A shuttle box schematic can be viewed in Tucker and Suski 201923. 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/s. Fish activity within the shuttle box was recorded by a camera mounted directly overhead (1080p Pro Stream Webcam, (Lolitech, Newark, California, USA); 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 well water from the test 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 min. Preliminary trials showed that fish became settled in the shuttle box after about 10 min, indicated by a reduction in movement, and acclimation times of 15 min are common for fish behavior studies24,25.

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 laboratory water (well water used by UMESC in the fish holding facility) 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 versus 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 to move between sides of the shuttle box, over a 10-min 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 10-min 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-min period, the experiment ended, temperature and dissolved oxygen were measured in both chambers of the shuttle box using a handheld oxygen meter in order to control for any differences that might affect the behavior of the fish (Professional Plus, YSI Inc., Yellow Springs, Ohio, USA) (Appendix VII–VIII), 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, F1,16 = 1.963, p > 0.05, one-way ANOVAgolden shiner, F1,16 = 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-L 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, USA); 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 high enough to allow vertical movement in the water column. For control trials, buckets were filled with water from the test 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 10-min period after they were placed in the buckets. Immediately following this 10-min period, the experiment ended, temperature and dissolved oxygen within each bucket were measured using a handheld oxygen meter (Appendix VII–VIII), and fish were removed, weighed, and measured. Fish length did not differ between treatments for both species (one-way ANOVAsilver carp, F1,18 = 1.031, p > 0.05, one-way ANOVAgolden shiner, F1,18 = 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 water26,27,28. 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. A respirometry schematic can be viewed at Killen et al. 202128. 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 periods26. An oxygen probe was fitted into each chamber to record dissolved oxygen concentration during a 20-min measurement period using respirometry software (Autoresp v 2.2.2, Loligo, Denmark). The measurement period for silver carp was set to a 5-min flush period, a 1-min wait period, and a 14-min measurement period. For golden shiners, the measurement period was a 17-min 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 measured. 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 flushed before and after each trial to account for background respiration present within each system27,28. Mass did not differ between treatments for both species (one-way ANOVAsilver carp, F1,24 = 0.661, p > 0.05, one-way ANOVAgolden shiner, F1,23 = 2.588, p > 0.05). Fish length also did not differ between treatments for both species (one-way ANOVAsilver carp, F1,24 = 0.661, p > 0.05, one-way ANOVAgolden shiner, F1,23 = 1.058, p > 0.05, Appendix I).

Data acquisition and analysis

A total of four 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 4.2.129 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 distribution30. Continuous variables (e.g., total duration spent on the treatment side) were run with a Gaussian distribution30. 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 independence31. The presence of potential influential data points was also assessed31. 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, USA) to objectively quantify and track movement32,33. 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 (cm/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 collinearity34, 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 principal component (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 sphericity35 using the ‘psych’ package36 (Version 2.2.9).

Behavior data from the 10-min trial were grouped into 1-min bins to better visualize the response of fish to CAWS water, the persistence of any response, and to identify potential habituation37. 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’ package38 (Version 1.1.30), with fish identification included as a random effect to account for multiple measurements from each individual over time30,39. If a significant difference in main effects was detected in the mixed model, the ‘emmeans’ package40,41 (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 term42. 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 excluded, relating milligrams of O2 by time in hours26. 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 period26, 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 mass43. 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 analysis42. However, this non-linear relationship was less clear in our study (Appendix I, Appendix V), likely due to the small size ranges of fish used42, making the use of log-transformed data less clear. To acknowledge this uncertainty, both log–log transformed and untransformed data were analyzed and reported for both species (Table 1, Appendix III–VI). 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’ package40,41 (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 above34.

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.
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All model results were interpreted using the Anova() function from the ‘car’ package44, 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 conducted 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.

Results

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 into the opposite chamber. This difference was not statistically significant (Table 1, Appendix III). Silver carp spent 6.1% more time in CAWS water than they did in the control treatment. The silver carp exposed to CAWS water delayed shuttling to the opposite chamber approximately 37% longer than fish in control water did. The silver carp exposed to CAWS water also conducted approximately one-third fewer shuttles to the opposite chamber than control-treatment fish over the 10-min trial, but differences across treatments were not significantly different (Table 1, Appendix III).

Golden shiners that received an acute exposure to CAWS water shuttled to the opposite chamber 5 out of 8 times, while 3 out of the 8 fish tested in laboratory water shuttled, and this difference was not significant across treatments (Table 1, Appendix IV). Golden shiners in laboratory water delayed shuttling to the opposite chamber approximately 56% longer than fish in CAWS water. Furthermore, golden shiners exposed to CAWS water spent approximately 8% less time in the CAWS water chamber than fish exposed to laboratory water. None of these metrics showed p-values less than 0.05 (Table 1, Appendix IV). However, the frequency of shuttles across chambers 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 min after being placed in CAWS water and this reduction in movement remained present until the trial ended (Fig. 1, Table 2). In contrast, silver carp in laboratory water did not show a change in movement behaviors until 9 min 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 laboratory versus CAWS water (Table 2).

Fig. 1
figure 1

Box plots showing principal component (PC) scores for silver carp held in either laboratory (control) or Chicago Area Waterway System (CAWS) water over each minute for the length of the 10-min trial. Data were generated from the activity assay conducted at 12 °C. Thick horizontal line shows the median and the diamond shows the mean for each treatment. Asterisks indicate significant differences between a given time point relative to minute one of a trial for silver carp in CAWS water. Plus signs indicate significant differences for a time point relative to distance traveled at 1 min of the trial for fish in laboratory water. Results from statistical tests are given in Table 1, and fish sizes are shown in Appendix I.

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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.
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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, Supplement Appendix III). The standard metabolic rate of golden shiners in CAWS water was similar to animals held in laboratory water (Table 1).

Fig. 2
figure 2

Box plots showing the standard metabolic rate (SMR) (mg O2/hr) for golden shiners and silver carp held in either laboratory (control) or Chicago Area Waterway System (CAWS) water overnight. Data were generated using intermittent flow respirometry conducted at 12 °C. Thick horizontal line shows the median and the diamond shows the mean for each treatment. Asterisks indicate significant p-values. Results from statistical tests are given in Table 1, and fish sizes are shown in Appendix I.

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Discussion

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’ survival45,46. Quantifying behaviors of fishes in response to contaminants is common, as it is sensitive to environmental variation47, a reliable indicator of stress, and, more importantly, alterations in behavior can have negative consequences for responses such as reproduction, foraging and survival45,48. For example, both Little et al. (1990)49, and Little and DeLonay (1996)48, showed that exposure to several different chemicals resulted in reduced activity in rainbow trout (Oncorhynchus mykiss), while Fernandez-Vega et al. (2002)50 reported that European eels (Anguilla anguilla) exposed to thiobencarb, an herbicide, had reduced motility and increased respiratory frequency. Similarly, Da Rosa et al. (2016)51 showed zebrafish (Danio rerio) decreased rotation number when exposed to gamma-Hydroxybutyric acid, or GHB. In our study, upon being placed in water collected from the 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 1 min following exposure to CAWS water, while fish held in laboratory water did not show a decrease in movement until the final minute of the 10-min trial. 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 use a reactive coping style typically adopt an immobile strategy to conserve energy during times of acute stress52,53,54,55,56. Zeng et al. (2019)56, 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)57 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)58 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, our results indicate 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 conditions59,60, and has been used to compare thermal tolerance, hypoxia, and exposure of foreign compounds61,62. 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 challenges7,63. For example, McKenzie et al. (2007)59 showed that resting metabolic rates of European chub (Leuciscus cephalus) increased by about 50% when exposed to contaminated 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 contaminated river sites relative to clean river sites. Du et al. (2019)64 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)65 found that orange-spotted spinefoot (Siganus guttatus) exposed to linear alkylbenzene sulfonate (LAS) had a significantly higher SMR than a control group in contaminant-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 duress7 and will have less energy available for other critical functions, such as reproduction and growth66. Second, the stress-induced increase in standard metabolic rate of a fish 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 simultaneously67,68,69. Ackerly and Esbaugh (2020)61 found that a combination of hypoxic conditions and exposure to oil resulted in decreases in aerobic scope in red drum (Sciaenops ocellatus). 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 farther upstream that might be more contaminated69. The data from the current study indicate that in the laboratory, juvenile silver carp in water from the CAWS have higher standard metabolic rates relative to animals held in control water. This higher SMR 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 avoided the CAWS water by seeking out untreated water, frequency of avoidance, latency to first avoidance, and time spent in the side of the shuttle box that received an input of CAWS water during the avoidance assay. Organisms can show several possible responses when confronted with a deleterious change in their environment10,45, with one possible response being a choice between different environments based on physiological preferences11. 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’11. In the past, shuttle boxes have been used to quantify avoidance of noxious stimuli such as salinity, CO2 gas, and temperature70,71,72,73. For example, Dennis et al. (2015)71 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)10 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, to try to minimize 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)71, 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 stimuli 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 stimuli24,74. 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, the response to a common stressor can be different. Jacobsen et al. (2014)75 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)76 reported that congeneric chinook salmon (Oncorhynchus tshawytscha) were more susceptible to physiological effects of copper exposure than 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 few decades indicating that populations are healthy and robust77. 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 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. Together, this evidence implies that carp may be impaired by inhabiting CAWS water and are likely not moving upstream from the avoidance front 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 downstream20,21. Water quality in the CAWS has been improving thanks to federal regulations like the Clean Water Act78, as well as actions from local agencies. This has resulted in improved diversity in native fish communities in the CAWS77,78,79. Thus, current improvements to water quality may continue to 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.

While results from this study demonstrate clear responses from silver carp to acute exposure to CAWS water, there are a number of opportunities for future work that can provide additional insights for mechanisms behind the observed responses. For example, additional work should be conducted to 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 if that stimulus is removed, ideally comparing water from the ‘leading edge’ with water closer to the core of the sliver carp population downstream where populations are robust17. Future studies should also quantify the response of fishes to longer-term exposure to CAWS water that may influence habituation, acclimatization or other mechanisms that can improve tolerance to current conditions or promote further upstream movement. Finally, continuing to conduct analyses that directly compare water from the leading edge of silver carp distributions with water from further downstream will help define alternate mechanisms that may be deterring range expansion, such as the attraction of carp to water from downstream locations. Together, these suggestions can help overcome the limitations in the current laboratory study, and enhance our understanding of potential mechanisms that have deterred the upstream range expansion of silver carp over the past decade.

Conclusion

Throughout nature, organisms that occupy habitat that minimizes energetic costs while maximizing energetic gains achieve greater fitness as a whole. 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 contaminants and chemicals in certain aquatic systems. The leading edge of the silver carp population in the Illinois River has not moved upstream from its current location in over a decade, and correlative data indicate that the presence of contaminants in the water may be contributing to the lack of upstream range expansion. Results from the current study showed 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 could provide information on why silver carp might move upstream from the currently stalled out invasion front if the river’s water quality improves in the future.