Acute exposure to water from the Chicago Area Waterway System induces molecular indices of stress and disturbance in silver carp: implications for deterrence to range expansion

Acute exposure to water from the Chicago Area Waterway System induces molecular indices of stress and disturbance in silver carp: implications for deterrence to range expansion | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Acute exposure to water from the Chicago Area Waterway System induces molecular indices of stress and disturbance in silver carp: implications for deterrence to range expansion Amy E Schneider, Andrew J Bennett, Clark E Dennis, Andrew J Esbaugh, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6221923/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Jul, 2025 Read the published version in Biological Invasions → Version 1 posted 5 You are reading this latest preprint version Abstract Exposure of an animal to sub-optimal habitat can result in impairment, damage or increased energy expenditure to maintain homeostasis, which can direct resources away from reproduction and reduce fitness. Animals may therefore avoid sub-optimal habitats to minimize physiological costs. Silver carp ( Hypophthalmichthys molitrix ) are a prolific invader in the Mississippi River basin. Despite their propensity for spread, the ‘leading edge’ of silver carp at the northern limit of their distribution in the Illinois River has stalled and not advanced for over a decade. Studies have suggested that contaminants in the Chicago Area Waterway System (CAWS) may be deterring upstream range expansion, but this hypothesis has had limited testing to date. The current study sought to quantify linkages between CAWS water constituents and impaired range expansion of silver carp. For this, CAWS water from beyond the current upstream distribution of silver carp was collected and transported downstream to the core of the population. Silver carp from the population core were collected and exposed to either water from their collection location (control), or to CAWS water from upstream of the current distribution, simulating range expansion. Following exposure, olfactory and liver tissue were collected to quantify genetic expression and histological indices of damage. Results showed that silver carp olfactory tissue experienced increased activity of genes related to xenobiotic defense and oxidative stress following exposure to CAWS water from upstream of the current distribution, highlighting costs associated with inhabiting this environment, providing a role for contaminants in CAWS water at deterring further upstream movement. habitat selection range expansion contaminants silver carp gene expression Figures Figure 1 Figure 2 Figure 3 Introduction Selection of optimal habitat is a key aspect of life history for most animals. There are many factors that can contribute to an organism’s choice of habitat, including competition for and availability of food resources, energy conservation, risk of predation, and abiotic factors (Hayes et al. 1996 ; Craig and Crowder 2000 ). Organisms can minimize energy expenditure by selecting optimal environments (Walsberg 1985 ; Huey 1991 ), and this can affect how different species populations are distributed in a system. Indeed, habitat selection can be a result of trade-offs with important processes, such as growth and reproduction (Craig and Crowder 2000 ). Thus, habitat choice can be extremely important for an organism’s survival and fitness. When an organism is forced to occupy sub-optimal habitat, an increase in energy expenditure can occur to combat the negative effects that the external environment may be causing. In turn, this increase in energy use could come at the cost of siphoning energy from biological processes such as foraging, predator avoidance, growth or reproduction as the organism attempts to restore homeostasis (Brown and Chivers 2006 ; Sadoul and Vijayan 2016 ). Additionally, occupying sub-optimal environments, such as hypoxic or polluted environments, can cause changes in gene expression in fish related to growth, immune response, and stress (Ton et al. 2003 ; Richards 2009 ; Tierney 2016 ; Kumar and Denslow 2016 ). To avoid these costly trade-offs, animals may therefore completely avoid sub-optimal habitat. Recent evidence has suggested that silver carp ( Hypophthalmichthys molitrix ) in the Illinois River may be avoiding upstream areas of the Illinois River watershed due to the creation of sub-optimal habitat from contaminants in Chicago Area Waterway System (CAWS). Silver carp are prolific invaders and are expanding throughout the upper Mississippi basin undeterred by the many locks and dams (Camacho et al. 2023 ), and have had a number of negative consequences for aquatic environments (Irons et al. 2007 ; Sass et al. 2014 ; Chick et al. 2020 ). One of these tributaries, the Illinois River, has had an established population of silver carp documented since the 90s (Irons et al. 2011 ; Sass et al. 2014 ). Despite their propensity to invade, the ‘leading edge’ of silver carp in the Illinois River has not advanced upstream in over a decade despite a lack of dams or physical structures preventing further upstream movement (MRRWG 2012). Although this lack of range expansion could be due to several factors, including extensive population suppression downstream (Altenritter et al. 2022 ), past work has also shown that contaminant loads are higher at the leading edge compared to further downstream in the Illinois River where populations are larger (Battaglin et al. 2020 ). Additionally, past research has shown that silver carp at the ‘leading edge’ have higher indices of stress due to contaminants than fish from downstream areas (Jeffrey et al. 2019 ; Curtis-Quick et al. 2021 ), and also that exposure of hatchery-reared silver carp to CAWS water in a laboratory setting causes reduced activity and increased metabolic rate (Schneider et al. 2024 ). Together, these lines of evidence suggest that the presence of bioactive contaminants in the CAWS may be playing a role in the lack of upstream movement of the silver carp population as animals avoid sub-optimal habitats upstream that are created by the presence of contaminants. To date, however, this hypothesis has largely been formulated using correlative, observational sampling of silver carp in the CAWS, precluding us from drawing definitive, causal relationships between water quality and range expansion. Water quality in the Chicago area has been improving over the past several years, and many native fish populations are increasing in size and number (Gibson-Reinemer et al. 2017 ; Happel and Gallagher 2021 ; Happel 2022 ). Therefore, identifying mechanisms that may be preventing further upstream movement of invasive carp is important to predicting conditions that may facilitate movement in the future should water quality continue to improve and a noxious stimulus inadvertently be removed, as eliminating an invasive species after establishment is almost impossible (Leung et al. 2002 ). The objective of this study was to quantify genetic and physiological indices of stress, disturbance and tissue damage in silver carp following an acute exposure to water from the CAWS collected from a point farther upstream their current distribution. More specifically, this study sought to answer 2 questions: (1) Does the expression of genes within rosettes and liver of silver carp change in response to different durations of CAWS water exposure?, and (2) Does the expression of genes within rosette and liver of silver carp change in response to different intensities of CAWS water exposure? To accomplish this goal, silver carp were collected from the population ‘core’ exposed to water collected from the vicinity of the upstream leading edge of their distribution in the CAWS. Following this exposure, the molecular response of liver and olfactory tissue was quantified across several gene families, and changes in the morphology of gill tissue were also assessed. Together, results from this study will identify molecular and physiological responses to acute CAWS water exposure that may be related to the lack of upstream movement of silver carp in the Illinois River. Methods Collection of Water To quantify the genetic and physiological impact that acute exposure to water from the CAWS has on silver carp in the Illinois River, we obtained water from two different sources. First, water from the CAWS was collected from a public boat ramp in Channahon, IL, (41.42448, -88.20854) on June 6th, 2022. This site is in proximity to the “leading edge” of silver carp populations in the Illinois River (MRRWG 2012). While we did not conduct any chemical analyses of the CAWS water used in this study, a number of previous investigations have performed a thorough examination of CAWS water near this collection site (Channahon, IL) (Duncker et al. 2020 ; U.S. Geological Survey (USGS) National Water Information System database at https://doi.org/10.5066/F7P55KJN using site number 05538020) and shown higher concentrations of anthropogenic contaminants near this upstream location relative to downstream locations where population densities of carp are higher (Battaglin et al. 2020 ). Wild silver carp sampled from near this location also show increased evidence of contaminant exposure and energy use in livers relative to more downstream populations (Jeffrey et al. 2019 ; Curtis-Quick et al. 2021 ), and exposure to water collected from this site also caused hatchery-reared silver carp to reduce activity levels and increase metabolic rates (Schneider et al. 2024 ). Together, these lines of evidence suggest that water at this collection point should be exhibiting some degree of noxious stimului that induces avoidance behavior, and therefore has the potential to induce molecular and physiological responses. Collection of water at this site occurred by submerging pumps in the river at a public boat ramp and transferring water into a 473 L liquid storage tank. The CAWS water was then transported by truck to the Illinois River Biological Station (IRBS) in Havana, IL, and stored outdoors in closed liquid storage tanks. The second source of water was from a public boat ramp on the La Grange Pool of the Illinois River in proximity to IRBS (40.304961, -90.066698) on June 7th and June 8th, 2022, using methods identical to that of the CAWS water collection. This site on the Illinois River is approximately 157.9 river km downstream of the upstream collection point, and has had robust populations of silver carp for decades (Irons et al. 2011 ). Water chemistry analyses has shown that a number of chemical compounds found upstream are absent from this site (Battaglin et al. 2020 ), essentially making it a control treatment for the CAWS water exposure assay. Fish Collection Adult silver carp were collected from the La Grange pool of the Illinois River by pulse DC electroshocking on June 7th and June 8th, 2022. Following capture, fish were held onboard the shocking boat in low densities in livewells filled with ambient river water. Once several silver carp had been collected, fish were taken to a nearby boat ramp and then hauled by truck to IRBS. Exposure Assay Assays to quantify genetic and physiological responses in silver carp following acute exposure to CAWS water were performed on June 7th and June 8th, 2022. For this, silver carp were netted from livewell on the collection boat and randomly distributed into one of five different experimental tanks that contained water from one of the five treatments used in this study. The five different treatments were: (1) a 1-hour exposure to water collected from the La Grange Pool of the Illinois River [1hr River], (2) a 1-hour exposure to water collected from the CAWS [1hr CAWS], (3) a 4-hour exposure to water from the La Grange Pool [4hr River], (4) a 4-hour exposure to water from the CAWS [4hr CAWS], and (5) a 1-hour exposure to water that was a mix of CAWS water diluted with equal parts of water from the La Grange Pool [1hr Mix]. Exposure assays for the 1hr River, 1hr CAWS, 4hr River and 4hr CAWS treatments were run on June 7th, while the 1hr Mix treatment (equal parts CAWS water and La Grange Pool water) was performed on June 8th. All experimental tanks were shaded with canopy tents, outfitted with air stones, and consistently monitored for dissolved oxygen (Professional Plus, YSI Inc., Yellow Springs, Ohio, USA), while pH and temperature were also checked intermittently. Water quality parameters were within acceptable limits for fish holding, and we did not observe any mortality during the holding period. The total length and weight of fish did not differ across the 5 treatments (one-way analysis of variance (ANOVA) length , F 1,45 = 0.797, p = > 0.05, one-way ANOVA weight , F 1,45 = 2.340, p = > 0.05) (Online Resource 1). After an exposure period of either 1 hr or 4 hrs, silver carp were removed from the experimental tank, euthanized by cerebral percussion, and measured for total length (cm) and weighed (kg) (Online Resource 1). Liver, gill filaments, and olfactory rosettes used for genetic and physiological analyses were harvested immediately. One olfactory rosette (of two) was stored in 1 mL of RNAlater (Invitrogen, Waltham, Massachusetts, USA) following manufacturer’s protocols for molecular analyses; samples were kept in a cooler of wet ice during collection, held in a 4°C refrigerator overnight, and then transferred to a -20°C freezer for permanent storage. Liver samples used for gene expression analysis (see below) were flash frozen in liquid nitrogen and then stored in a -80°C freezer until use. A section of harvested gill and olfactory tissue were also sampled for histology. This gill tissue was immediately stored in 10% neutral buffer formalin for 48 hours (Fournie et al. 2000 ), after which samples were transferred to 70% ethanol for permanent storage at room temperature (Bieber et al. 2019 ). All fish collection, handling and experimental procedures were approved by the University of Illinois Institutional Animal Care and Use Committee (Protocol #21139). Gene Expression Analyses RNA isolation and cDNA synthesis Total RNA was isolated from liver and rosette samples using the RNeasy Mini Kit (Qiagen, Germantown, Maryland, USA) according to manufacturer’s protocol. Briefly, tissue samples were homogenized using a Bullet Blender Blue (Next Advance, Troy, New York, USA). RNA was extracted and treated with RNase-free DNase (Qiagen, Germantown, Maryland, USA) to remove any residual DNA from the sample. RNA was then quantified using a Qubit 3.0 Fluorometer (Fisher Scientific, Hampton, New Hampshire, USA). RNA was stored at -80°C. Prior to cDNA synthesis, RNA integrity was quantified at the Roy J. Carver Biotechnology Center at the University of Illinois at Urbana-Champaign using either an Agilent 2100 Bioanalyzer or an AATI fragment analyzer (Agilent, Santa Clara, California, USA). Samples with RNA integrity numbers below 5 were excluded due to high sample degradation (Fleige and Pfaffl 2006 ). Synthesis of cDNA was performed using a QuantiTect Reverse Transcription Kit (Qiagen, Germantown, Maryland, USA) using 1 µg of total RNA for a reaction volume of 20 µL. For most samples, a final reaction concentration of 50 ng/µL was created. However, some samples were created to reaction concentrations of either 12.5 or 25 ng/µL due to low initial sample volume or low initial RNA concentration. An Eppendorf Mastercycler Pro thermal cycler (Eppendorf, Hamburg, Germany) was used to facilitate the cDNA synthesis reaction, and all reactions were performed following the manufacturers’ instructions: 1) 42°C for 15 min and 2) 95°C for 3 min. cDNA was stored at -20°C. qPCR primers Gene specific qPCR primers for silver carp were obtained either a) from previous studies or b) by designed using Primer 3 (Koressaar and Remm 2007 ; Untergasser et al. 2012 ) using sequences available in the GenBank database and divided into functional groups found in Table 1 . Primer sequences and fragment lengths are shown in Table 2 . To verify specificity of each qPCR primer (i.e., generation of only one product), we performed PCR using TAQ5X PCR Master Mix (New England Biolabs, Ipswich, Massachusetts, USA), the forward and reverse qPCR primers for each gene (Integrated DNA Technologies, Coralville, Iowa, USA), cDNA, and RNase-free water. The PCR reaction was performed on an Eppendorf Mastercycler Pro thermal cycler (Eppendorf, Hamburg, Germany) under the following conditions: 1) 95°C for 30 sec; 2) 40 cycles of (a) 95°C for 30 sec, (b) 55°C for 30 sec, and (c) 68°C for 30 sec; followed by 3) a final cycle at 68°C for 5 min. PCR products were then run on a 3% agarose gel containing SYBR Safe Dye (Invitrogen, Waltham, Massachusetts, USA) to confirm the amplicon was the correct length and the only product generated by the reaction. qPCR gene expression analysis In total, 3 reference genes ( β-actin , ef1α , and 18s ) and 20 target genes (Table 1 ) were analyzed for all tissue sample using a microfluidics qRT-PCR platform (two 96.96 Dynamic Array TM IFC, Standard Biotools, San Francisco, California, USA). The 20 target genes chosen contained a selection of genes related to oxidative stress, xenobiotic defense, growth and energy storage, cell death and repair, and general stress (Table 1 ). Each cDNA sample was run in triplicate. A pre-amplification step on the cDNA was conducted prior to gene expression analysis under the following conditions: 1) 95° C for 10 min, followed by 14 cycles of 2) 95° C for 15 sec and 3) 60° C for 4 min. An exonuclease treatment was then applied to each PCR reaction to remove access primer. Samples were then loaded into the microfluidic plates and run on a BioMark HD automated, high-performance qPCR system (Standard Biotools, San Francisco, California, USA). qPCR was performed under the following conditions: 1) thermal mix stage at 70° C for 40 min, 2) 60° C for 30 sec, 3) a hot start cycle at 95° C for 1 min, 4) 30 cycles of 96° C for 5 sec and 55° C for 20 sec, and 5) a melting stage run at 55° C for 3 sec and then ramped up by 1° C every 3 sec to 95° C. Gene expression data were extracted using the Standard BioTools Real-Time PCR Analysis Software (Standard Biotools, San Francisco, California, USA) using C t thresholds set manually for each gene. Each gene assay included a no template control (NTC) that needed to be negligibly expressed compared to the cDNA samples (i.e., not detected or > 5 C t from sample cDNA). Amplification and melting curves for each sample were also visually inspected and spurious samples were removed (i.e., no sigmoid curve, multiple products observed on melting curve). To quantify the efficiency of our target and housekeeping genes, cDNA from RNA extractions of pooled liver and rosette samples were serially diluted in 7 dilutions of 1:4 (1 to 1:4096). PCR efficiencies for each gene were calculated using (10 1/slope – 1) × 100, for which the slope was estimated by plotting the C t over the serial dilutions of cDNA. Sample gene expression was normalized with the ΔΔC t method and correction for PCR efficiency according to Pfaffl ( 2001 ). Expression of target genes were normalized to the expression of the housekeeping gene, ef1α , which was found to be the most suitable tested reference gene (e.g., 18s and β-actin were too highly expressed) and there was no significant difference in gene expression (C t ) of ef1α between our 5 treatments (one-way ANOVA liver , F 1,39 = 1.098, p > 0.05; one-way ANOVA rosette , F 1,38 = 1.464, p > 0.05). Gene expression data for each target gene was calculated as a relative expression ratio relative to the average expression levels observed in silver carp exposed to 1hr of river water. Histology All gill samples were embedded and stained with methylene blue by the Histology Laboratory at the College of Veterinary Medicine at the University of Illinois at Urbana-Champaign. Slides were imaged using a Nanozoomer 2.0-HT Slide Scanner (Hamamatsu Photonics, Hamamatsu, Japan) at the Carl R. Woese Institute for Genomic Biology at the University of Illinois at Urbana-Champaign. To quantify histological indices of damage, two random filaments were chosen, while ten lamellae from each filament were randomly selected to be measured(Blair et al., 2016 ). Each lamella was measured for length, width, and interlamellar cell mass (ILCM) height between lamellae(Blair et al., 2016 ; Ong et al., 2007 ). Additionally, instances of aneurysm and clubbing were also recorded(Strzyzewska et al., 2016 ). All slides were assigned a number using a random number generator to allow for blind scoring by the reader. Measurements were taken using the open-source software Fiji (Schindelin et al. 2012 ). Statistical Analysis Gene expression Because we did not have a perfectly balanced statistical design and only had a single sampling time point for our 1hr Mix treatment, the response of silver carp to CAWS water exposure was conducted using two separate statistical models that aligned with the two questions of our study. The first statistical model was associated with our first question and identified changes in gene expression due to the duration of CAWS water exposure (1hr vs 4hr). This model consisted of a two-way ANOVA with the main effects of water source (CAWS water vs water downstream from Havana), exposure time (1hr vs 4hr) and their interaction (water source × exposure time). The second statistical model addressed our second research question and was focused on the response of tissues to the intensity of CAWS water exposure. This statistical model consisted of a one-way ANOVA comparing the three treatments that exposed silver carp to 1hr of water either from Havana (1hr River), CAWS water (1hr CAWS) or the 50% dilution of CAWS water with Havana River water (1hr Mix). To account for the fact that some treatments were used twice in separate analyses, and to reduce the Type I error rate, a conservative α of 0.025 was used as a threshold of statistical significance for all models (VanderWeele and Mathur 2019 ). To meet the assumptions of ANOVAs (i.e., normal distribution, homogenous variances), relative gene expression ratio data were rank transformed (Conover and Iman 1981 ; Potvin and Roff 1993 ). To improve data interpretation in figures, gene expression ratio data were log 2 transformed, which presents upregulated genes relative to the reference (control) treatment (i.e., 1hr River) as positive values and downregulated genes relative to the control treatment as negative values(Franks et al. 2018 ). Gene expression ratio data within the results are presented as fold-change (e.g., gene expression ratio of 0.25 = 2-fold decrease in gene expression relative to the control treatment) to facilitate data interpretation. Histology Analyses for lamellar length, lamellar width, and ILCM height, used a similar statistical approach to gene expression analysis that included both two-way and one-way models aligned with our two questions, but with a few small differences. More specifically, histology analyses used a linear mixed effects model approach with fish identification number as a random effect as multiple measurements were collected from each individual fish due to the use of multiple lamellae from an individual (Lindstrom and Bates 1990 ; Crawley 2012 ). Additionally, generalized linear mixed models with binomial error distributions were run to analyze instances of clubbing and aneurysms as these are count data (Crawley 2012 ). Models were interpreted using the anova() function from the ‘lmerTest’ package (Kuznetsova et al. 2017 ). If any of the main effects our ANOVA models were significant, or if the interaction was significant, we then conducted the appropriate post-hoc test (t-test or Tukey HSD test) to separate means and identify differences within and across groups. If a significant interaction was found in a model, main effects were ignored. Normality was assessed visually using a normal Q-Q plot of model residuals along with a formal Shapiro-Wilk normality test, while homogenous variances were assessed visually using a residuals vs fitted values plot along with a formal Levene’s test (Tabachnick 2007 ; Dean et al. 2017 ). All statistical analyses were performed in R version 4.3.0 (2022). Results Question 1: Does the expression of genes within rosette and liver of silver carp change in response to different durations of CAWS water exposure? Rosettes A total of 4 genes within the rosettes of silver carp changed their expression following exposure to CAWS water, but the duration of the CAWS water exposure (1 hour or 4 hours) did not alter the pattern of gene expression (Fig. 1 ; Online Resource 2). More specifically, we observed increased mRNA expression of a xenobiotic defense gene, CYP1A (Fig. 1 A), and an oxidative stress gene, GST (Fig. 1 C), in the rosettes of silver carp exposed to CAWS water compared to River water (Online Resource 2). In contrast, GADD45 (Fig. 1 B), a gene related to DNA repair, and MCL 1 (Fig. 1 D), a gene relative to the activation of cell death, were downregulated in silver carp exposed to CAWS water compared to fish exposed to River water (Online Resource 2). We also observed 4 genes change expression following longer holding/exposure to water either from the CAWS or Havana (River) water. Three genes decreased their mRNA expression following 4 hours of holding relative to silver carp in the 1hr treatments: two general stress genes (CJUN and HIF1a; Online Resource 2, Online Resource 3) and one xenobiotic defense gene (CYP1A; Fig. 1 A; Online Resource 2). PHD3, an oxidative stress gene, was the only mRNA transcript to increase expression due to a longer holding period (2-fold increase at 4hr vs. 1hr; Online Resource 2, Online Resource 3). All other target genes did not change mRNA expression due to water source (CAWS or River) or exposure time (1hr or 4hr) (Online Resource 2, Online Resource 3). Liver None of the target genes within liver tissue altered mRNA expression levels when exposed to CAWS water for either 1 hour or 4 hours (Online Resource 4, Online Resource 5). Silver carp mRNA expression did change for 3 genes following longer holding/exposure to either CAWS or River water. HIF1α, a gene related to general stress, and PHD3, an oxidative stress gene, were significantly upregulated following 4 hours of holding relative to silver carp in the 1hr treatments (Online Resource 4, Online Resource 5). In contrast, we observed a 3-fold decrease in the relative expression of GST mRNA, another oxidative stress gene, within silver carp livers at the 4-hour exposure treatment relative to the 1-hour exposure (Online Resource 4, Online Resource 5). All other target genes did not change across the different exposure times (Online Resource 4, Online Resource 5). Question 2: Does the expression of genes within rosettes and liver of silver carp change in response to different intensities of CAWS water exposure? Rosette Six genes within the rosettes of silver carp changed their expression based on the intensity of CAWS water exposure (i.e., 1hr River vs 1hr Mix vs 1hr CAWS). After a 1hr exposure to 50% CAWS and River water, silver carp displayed a 2-fold decrease in CYP1A mRNA expression (a xenobiotic defense gene transcript) relative to the 1hr River treatments (Fig. 1 A, Online Resource 1), which was also significantly different from the 1hr CAWS treatment that was upregulated following exposure to CAWS water. In contrast, we observed an increase in two oxidative stress genes, GST (Fig. 1 C) and CAT (Fig. 2 A), and a different xenobiotic defense gene, CYP3A137 (Fig. 2 C), following a 1hr exposure to the Mix treatment relative to the 1hr River treatment (Online Resource 2). A greater than 2-fold decrease in mRNA expression was observed for two general stress genes, CJUN (Fig. 2 B) and HSP70 (Fig. 2 D), in the rosettes of silver carp exposed to the 1hrMix treatment relative to fish exposed to river water (Online Resource 2). All other target genes examined in the rosettes of silver carp did not differ in expression based on the intensity of CAWS water (Online Resource 4, Online Resource 5). Liver Silver carp downregulated two genes in liver in response to a 1hr exposure to a 50–50 mix of CAWS/River water: xenobiotic defense gene CYP3A137 was 2-fold lower expressed in the 1hr Mix treatment than the 1hr River or 1hr CAWS treatments (Fig. 3 A; Online Resource 4), and the mRNA expression of general stress gene HSP70 decreased 3-fold in the 1hr Mix treatment relative to fish in the 1hr CAWS treatment (Fig. 3 B; Online Resource 4). In contrast, we observed a significant 2-fold increase in GADD45 mRNA expression, an indicator of DNR repair, in the liver of fish exposed to the 1hr Mix treatment relative to fish exposed to full-strength CAWS water (Fig. 3 C; Online Resource 5). All other target genes examined in the liver of silver carp did not differ in expression based on the intensity of CAWS water (Online Resource 4, Online Resource 5). Histology There was no significant effect of treatment on lamellae length, although lamellae length showed a nearly 20% decrease between the 1hr and 4hr CAWS treatments, as well as a 6% decrease between the 1hr and 4 hr River treatments (Online Resource 6, Online Resource 7). Lamellae width and interlamellar cell height in the selected filaments also showed no change between CAWS treatments or durations (Online Resource 6, Online Resource 7). Additionally, there was no statistical difference between the number of instances of lamellar aneurysms or clubbing between treatments or durations (Online Resource 6). Discussion Acute exposure to water from the Chicago Area Waterway System for either 1 or 4 hours increased the expression of genes associated with xenobiotic defense and oxidative stress within the rosette tissue of silver carp. Specifically, we observed that acute exposure to water collected from upstream of the current distribution of silver carp caused an upregulation in genes related to xenobiotic exposure (CYP1A) and oxidative stress (GST). When animals are exposed to toxicants and contaminants in the environment, outcomes such as sensory disruption and neurological dysfunction can occur (Kennedy 2011 ; Sopinka et al. 2010 ). Because olfactory rosettes are directly exposed to the external environment, they are vulnerable to disruptions in homeostasis from xenobiotics. Damage to rosettes caused by environmental contaminants can be severe (Tierney et al., 2010 ) resulting in outcomes such as impaired olfaction and improper perception of environmental information (Tierney et al. 2007 ), and, in extreme cases can result in the transport of contaminants to the brain along the olfactory nerve (Sloman 2011 ; Tierney et al. 2010 ). To prevent these negative outcomes, several changes can occur at the molecular level soon after a contaminant is experienced, including the upregulation of genes related to protection processes. For example, CYP enzymes (including the CYP1A subfamily) have been shown to be important for the metabolism and biotransformation of foreign compounds, making them critical early responders in the of detoxification of environmental contaminants (Sarasquete and Segner 2000 ). Additionally, Espinoza et al. ( 2012 ) found that the rosettes of coho salmon ( Oncorhynchus kisutch ) increased expression of nine GST isoforms following 8, 24, and 48 hour exposures to cadmium, with this upregulation of GST isoforms serving to detoxify foreign compounds and protect cells from damage. In the current study, when silver carp were exposed to water from the CAWS, the observed upregulation of CYP1A and GST genes in rosettes likely occurred with the intention of protecting tissues from damage associated with exposure to bioactive compounds within the water (Battaglin et al. 2020 ). Thus, the exposure of silver carp to water upstream of their current distribution within the CAWS resulted in an upregulation of genes related to protection from contaminants in rosettes, highlighting that CAWS water acts as a noxious stimulus for silver carp. In addition to the activation of genes related to detoxification, acute exposure of silver carp from the CAWS also resulted in a downregulation of genes in rosettes suggesting a disruption of certain cellular functions. More specifically, a 1- or 4-hour exposure to water upstream of the current distribution of silver carp resulted in a downregulation of genes related to cell death regulation (MCL1) and DNA repair (GADD45). While contaminant exposure can induce the activation of protective mechanisms at the cellular level, in extreme cases, animals may divert energy away from important bodily and cellular processes to aid in the detoxification of foreign contaminants (Jeffrey et al., 2019 ). For example, Hong et al. ( 1999 ) saw a downregulation of MCL1 proteins in a Chinook salmon embryonic cell line within 8 hours of being infected with pancreatic necrosis virus (IVPN), along with a negative correlation between MCL1 expression and viral replication. Similarly, Higashi et al. ( 2006 ) found that GADD45 mRNA showed a ten-fold decrease in expression in human lung tumor tissues compared to normal lungs. More importantly, silver carp sampled from the leading edge of their distribution in the Illinois River, close to the collection point of water used in the current study, displayed both a downregulation in several genes involved in DNA repair in liver tissue (Jeffrey et al. 2019 ), as well as a decrease in metabolites related to cellular-level protection in liver (Curtis-Quick et al. 2021 ), demonstrating consistency between wild-caught fish and the experimental treatments used in the current study. Thus, acute exposure of silver carp to water from the Chicago Area Waterway System results in a downregulation of genes related to protection of olfactory tissues from bioactive compounds in CAWS water. Although our study observed multiple effects of acute exposure of CAWS water to silver carp olfactory tissue, the same exposure did not alter the genetic expression of any of the genes analyzed within liver tissue. The liver plays a major role in vertebrates in the detoxification of harmful contaminants, and thus is a target for studies seeking to quantify the effect of pollutants on organisms (Anadon 1996 ; Kime 1998 ). Indeed, exposure to pollutants can induce negative morphological changes in the liver such as decreased glycogen and degeneration of cell membranes (Kime 1998 ), as well as an increase in gene activity due to possible health effects (Wirgin and Waldman 1998 ; Jordan-Ward et al. 2024 ). Additionally, past work with silver carp has shown that livers from animals samples from the Illinois River at the leading edge of their range displayed an upregulation in genes related to xenobiotic defense, metabolism, and apoptosis compared to core populations downstream, demonstrating an impact of CAWS water on toxicological pathways in livers (Jeffrey et al. 2019 ). We feel there could be three possible explanations for why we did not see changes in the gene expression patterns of silver carp following our experimental treatments. First, one reason that this study did not see changes in genetic expression in silver carp livers following exposure to CAWS water may be because our exposure durations were not sufficiently long to induce a response. In a study performed by Momoda et al. ( 2007 ), for example, rainbow trout ( Oncorhynchus mykiss ) were exposed to a simulation of low water conditions across multiple timepoints, and the upregulation of genes related to stress did not occur until 3 hours into the experiment, while Craig et al. ( 2007 ) exposed zebrafish ( Danio rerio ) to multiple concentrations of copper and showed an increase in gene expression after 4 hours at the highest concentration of copper, while other genes either took longer to show expression differences demonstrating a delayed response in the liver to external stressors. A second possible explanation for why we did not see an upregulation of genes in the liver of silver carp acutely exposed to CAWS water is that the concentration of contaminants could have been too low, and the expression of genes could have been influenced by a dose-dependent response. For example, work by Cai et al. ( 2012 ) showed that bigheaded carp ( Hypophthalmichthys nobilis ) experienced elevated expression of the cellular metabolic maintenance gene CDA in livers when injected with higher concentrations of the cyanotoxin microcystin-LR (MCLR) compared to lower concentrations. Finally, a third explanation for why there were no changes in the activity of genes in the livers of silver carp following acute exposure to CAWS water is that the genes selected simply did not change in response to this external environmental pressure. To perform RT-qPCR, it is necessary to know the gene sequence of interest before performing analyses, particularly if researchers are interested in a non-model gene (Costa et al. 2013 ). However, because some sequences have multiple primer pair options to choose from, it’s possible to pick primer pairs within the sequence that can bind on themselves instead of the cDNA product that the qPCR process is attempting to amplify. Therefore, the chosen primer pair from within the gene sequence may not be conducive for the RT-qPCR process (Costa et al. 2013 ). Together, our study found no evidence of changes in genetic expression in the livers of silver carp following acute exposure to CAWS water despite seeing up- and down-regulation of genes across several different pathways in rosettes, and targeting several known toxicological pathways previously shown to change following toxicant exposure in other fish species. An acute exposure to a 50–50 mix of CAWS and River water resulted in a variety of changes in gene expression in both liver and rosette tissues, but no strong pattern or signature of CAWS water exposure. For example, we found genes related to general stress in both rosette and liver tissue were downregulated in the mix treatment compared to the control and 1-hour treatments, but we also saw both upregulation and downregulation of genes related to xenobiotic defense, oxidative stress, and cell death and cell repair in rosette and liver tissue. By exposing silver carp to diluted CAWS water, we assumed we would see a linear dose-response curve in expression relative to patterns observed when silver carp were exposed to full-strength CAWS water. However, this traditional linear dose-response model has been shown to be unreliable in predicting organism responses to low-dose exposures (Agathokleous and Calabrese 2020 ). Indeed, some organisms will experience hormesis, or multi-level dose response curves, in which a toxin may induce beneficial effects at low concentrations, which may then be reversed as the dosage increases (Morkunas et al. 2018 ; Agathokleous and Calabrese 2020 ). Additionally, it is important to note that analysis of water from the CAWS has documented hundreds of different bioactive contaminants in the water, and no specific chemical or chemicals has been determined to be causing this molecular reaction in silver carp (Battaglin et al. 2020 ; Duncker et al. 2020 ). Thus, when exposed to a lower concentration of water from the CAWS, the lack of discernable trends in the molecular response in tissues likely occurred as the different concentration of contaminants in this treatment resulted in an upregulation of some molecular processes, and a downregulation of other molecular processes due to hormesis. Additionally, our study saw no change in morphology of gills following CAWS water exposure of either duration. Specifically, lamellar length, lamellar width, and interlamellar cell mass height did not change in silver carp gills between our treatments that varied the intensity or exposure duration to CAWS water. Histological changes in tissue can be effective markers for contaminant and pollution exposure (Yancheva 2016 ), and gills are susceptible to changes caused by pollutants, making them a strong general biomarker for damage and stress and an effective tool for monitoring the response of fish to environmental pollution (Mallatt 1985 ; Au 2004 ; Yancheva 2016 ). Despite this potential, we did not see any evidence of histological changes in gill tissue following exposure to CAWS water at either the 1-hour or 4-hour timepoint. One reason for this could be that the exposure timepoints explored in this study were not long enough to illicit any structural changes in gill tissues. Work by Guo et al. ( 2021 ) showed that white shrimp ( Litopenaeus vannamei ) exposed to nonylphenol experienced changes in gill morphology 3 hours after exposure initiation. Additionally, another reason changes in gill morphology were not observed in our study could be because the concentration of bioactive contaminants in CAWS water was not high enough to cause a physical reaction in the gills. A study by Santos et al. ( 2011 ) exposed juvenile Florida pompano ( Trachinotus carolinus ) to naphthalene, a component of petroleum, at multiple concentrations and showed a positive correlation between concentration of naphthalene exposure and degree of gill tissue damage. Ultimately, no evidence of histological gill damage was found in silver carp following acute exposure to CAWS water. Results from this study contribute to the growing body of evidence that contaminants from Chicago are influencing the upstream movement of silver carp ( Jeffrey et al. 2019 ; Curtis-Quick et al. 2021 ). Past work with the ‘leading edge’ of carp has shown an increase in stress and disturbance in silver carp at this location relative to downstream populations ( Jeffrey et al. 2019 ; Curtis-Quick et al. 2021 ), and laboratory exposure of hatchery-reared silver carp to CAWS water upstream of their distribution resulted in reduced activity and increased metabolic rate (Schneider et al. 2024 ). Results from the current study demonstrate that exposing silver carp to water from the Chicago Area Waterway system results in an upregulation of stress genes in olfactory tissue. Water quality in the CAWS has continued to improve since the implementation of federal actions like the Clean Water Act (Gibson-Reinemer et al. 2017 ). This, and other actions, has resulted in an increase of native fish biodiversity in the Chicago area waterways ( Happel and Gallagher 2021 ; Happel 2022 ). Efforts are also underway to continue improving water quality and make improvements to water treatment facilities in the Chicago area ( https://mwrd.org/stormwater ). While these improvements are both valuable and important, they may inadvertently induce upstream movement of silver carp if they are no longer deterred by an inadvertent removal of noxious stimuli in the water that are responsible for inducing molecular or behavioral responses. Therefore, furthering research regarding how silver carp movement is affected by contaminants from the CAWS is of continued interest to researchers and managers. Conclusion Fish select habitats that minimize energy expenditure and maximize growth and reproduction. However, if a fish is forced to occupy sub-optimal habitat, homeostasis may be negatively influenced, resulting in an increase in pathways and processes that can increase energetic costs due to stress and disturbance in an effort to maintain homeostasis. If the reason for the sub-optimal habitat is the presence of contaminants, fish may need to divert energy from important energetic processes to filter toxins in the water or upregulate protective mechanisms. The leading edge of silver carp in the Illinois River have not moved upstream in over a decade despite the absence of any physical barrier that could prevent further range expansion. This lack of movement could be due to several reasons, but past studies suggest that contaminant exposure from the CAWS could be playing a role in the population’s stalling behavior. Data from this study indicate that silver carp experienced an upregulation in the activity of genes associated with protective mechanisms in their olfactory tissue when exposed to CAWS water, consistent with evidence provided by past field and laboratory research that silver carp at the upstream edge of their distribution are experiencing increased levels of stress associated with contaminant exposure. As water quality in the CAWS improves, there may be a facilitation in movement of the silver carp population. Therefore, identifying the mechanism that is causing the leading edge of the population to stall may provide insight into why they may move in the future, protecting valuable aquatic ecosystems from future invasions. Statements & Declarations Acknowledgements Funding for this study was provided by the U.S. Geological Survey (USGS) WRRI Aquatic Invasive Species (AIS) Competitive Grants Program (Award G21AP10174-01), 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. Additional funding was provided through the Illinois AFS Larimore Grant, and the Illinois Muskie Tournament Trail Scholarship. We would like to acknowledge all researchers from the Illinois River Biological Station for providing a space to work, collecting river water for the control treatments, and collecting all fish used in the experiments. Additional thanks goes to Qihong Dai, Joseph Reinhofer, Scarlett Hoffer, and John Bieber for assistance with CAWS water collection, experimental setup and data collection. Lastly, we would like to acknowledge Jonathan Johnson, who provided major support in RNA extraction. Funding Funding for this study was provided by the U.S. Geological Survey (USGS) WRRI Aquatic Invasive Species (AIS) Competitive Grants Program (Award G21AP10174-01), 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. Additional funding was provided through the Illinois AFS Larimore Grant, and the Illinois Muskie Tournament Trail Scholarship. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author Contributions A.J.E. and C.D.S. conceived the study design, and all authors contributed to planning field and laboratory experiments. A.E.S. and C.E.D performed data collection, all data analysis and generation of figures with additional guidance from A.J.B. All authors contributed to writing and editing the final manuscript. Data Availability Data from this study are available through the University of Illinois Data Bank (https://databank.illinois.edu/) at https://doi.org/10.13012/B2IDB-0347483_V1). References Agathokleous E, Calabrese EJ (2020) Environmental toxicology and ecotoxicology: How clean is clean? Rethinking dose-response analysis. 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Mutat Res Mol Mech Mutagen 399:193–219. https://doi.org/10.1016/S0027-5107(97)00256-X Yancheva V (2016) Histological biomarkers in fish as a tool in ecological risk assessment and monitoring programs: a review Appl Ecol Environ Res 14:47–75. https://doi.org/10.15666/aeer/1401_047075 Tables Tables 1 to 2 are available in the Supplementary Files section Supplementary Files Tables.docx OnlineResource.docx Cite Share Download PDF Status: Published Journal Publication published 24 Jul, 2025 Read the published version in Biological Invasions → Version 1 posted Reviewers agreed at journal 02 Apr, 2025 Reviewers invited by journal 25 Mar, 2025 Editor invited by journal 17 Mar, 2025 Editor assigned by journal 14 Mar, 2025 First submitted to journal 13 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6221923","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":433823721,"identity":"88d40796-6015-4991-bf2a-92acac1c8d64","order_by":0,"name":"Amy E Schneider","email":"","orcid":"","institution":"University of Illinois Urbana-Champaign","correspondingAuthor":false,"prefix":"","firstName":"Amy","middleName":"E","lastName":"Schneider","suffix":""},{"id":433823722,"identity":"89ff6443-b998-4662-b0dc-6b605a6efa66","order_by":1,"name":"Andrew J Bennett","email":"","orcid":"","institution":"University of Illinois Urbana-Champaign","correspondingAuthor":false,"prefix":"","firstName":"Andrew","middleName":"J","lastName":"Bennett","suffix":""},{"id":433823723,"identity":"310400f2-c6eb-4155-9dd9-29568f3fc6b6","order_by":2,"name":"Clark E Dennis","email":"","orcid":"","institution":"University of Illinois Urbana-Champaign","correspondingAuthor":false,"prefix":"","firstName":"Clark","middleName":"E","lastName":"Dennis","suffix":""},{"id":433823724,"identity":"1f8f80fe-94b0-4fd1-9c9f-6d573700e9c2","order_by":3,"name":"Andrew J Esbaugh","email":"","orcid":"","institution":"The University of Texas at Austin","correspondingAuthor":false,"prefix":"","firstName":"Andrew","middleName":"J","lastName":"Esbaugh","suffix":""},{"id":433823725,"identity":"088e8d89-8d6c-4f3b-b959-3bc8b5a8e21e","order_by":4,"name":"James T Lamer","email":"","orcid":"","institution":"University of Illinois Urbana-Champaign Illinois Natural History Survey","correspondingAuthor":false,"prefix":"","firstName":"James","middleName":"T","lastName":"Lamer","suffix":""},{"id":433823726,"identity":"c30246fc-85df-4659-80e6-d738406a496c","order_by":5,"name":"Cory David Suski","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwUlEQVRIiWNgGAWjYHACZoaECgkZPmYgMwHEP0CUljMSPGykaWFsY+Bhg/MJaZGfkf7Y4OE8Cx42dt6DDx78YZDju5GAX4vBjRzjhMRtIIfxJRsktjEYSxLUIpHDfACihcdMIrGBIXEDIS0ghx1InAPVkvCHoZ6gFoYbCUCHNcC0sDEkGBB02Jk3xgYJx8BajIF+kTCceeYBAYe1pz+W/FFTJ8fPf8bw4Y8/NvJ8xwk5DA1IkKZ8FIyCUTAKRgF2AABZwzo64ecjMwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-8280-873X","institution":"University of Illinois Urbana-Champaign","correspondingAuthor":true,"prefix":"","firstName":"Cory","middleName":"David","lastName":"Suski","suffix":""}],"badges":[],"createdAt":"2025-03-13 17:01:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6221923/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6221923/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10530-025-03633-1","type":"published","date":"2025-07-24T15:57:27+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79893070,"identity":"41288a77-50e9-4845-9ed0-5997ea5beb18","added_by":"auto","created_at":"2025-04-04 08:13:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":82917,"visible":true,"origin":"","legend":"\u003cp\u003eGenes within the rosettes of silver carp that displayed a significant change in mRNA expression following exposure to CAWS water: (A) CYP1α, (B) GADD45, (C) GST, and (D) MCL1.\u0026nbsp; Relative gene expression data were analyzed using two separate models: 1) exposure duration to CAWS water (boxplots to the left of the vertical dashed line) and 2) intensity of CAWS water (boxplot to the right of the vertical dashed line).\u0026nbsp; Exposure duration is presented on the x-axis, while water source is presented as different colors on the box plots (e.g., blue = Havana River water; red = CAWS water; orange = a mix of 50% River and 50% CAWS water).\u0026nbsp; For model 1, significant differences in gene expression observed for fish exposed to water from the CAWS (1hr CAWS and 4hr CAWS) compared to the River (1hr River and 4hr River) are denoted by uppercase roman letters, while significant differences in gene expression caused by holding duration (1hr vs 4hr treatment) are denoted by lowercase Greek letters.\u0026nbsp; For model 2, significant differences in gene expression due to the intensity of CAWS water exposure was denoted using lowercase roman letters.\u0026nbsp; Relative gene expression data are presented in box-and-whisker plots such that the horizontal line in the middle of the box plots denotes the median of a treatment; the upper and lower horizontal lines represent the 75th and 25th percentiles, respectively; the vertical lines represent data that are 1.5 × the interquartile range above or below the 75th or 25th percentiles; and the actual data points are shown with filled circles overlayed on the boxplot.\u0026nbsp; For clarity, data are expressed relative to the mean mRNA expression of each gene during the 1hr River treatment and then log\u003csub\u003e2\u003c/sub\u003e transformed to clearly denote upregulation (values \u0026gt; 0) and downregulation (values \u0026lt; 0).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6221923/v1/13d3be0473dc32e8ecc08f58.png"},{"id":79892384,"identity":"1912e7cd-8fc2-4e4e-a48f-c4b1df25e62a","added_by":"auto","created_at":"2025-04-04 08:05:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":80365,"visible":true,"origin":"","legend":"\u003cp\u003eGenes within the rosettes of silver carp that displayed a significant change in mRNA expression following a 1hr exposure to 50% River – 50% CAWS water (1hr Mix): (A) CAT, (B) CJUN, (C) CYP3α, and (D) HSP70.\u0026nbsp; Relative gene expression data were analyzed using two separate models: 1) exposure duration to CAWS water (boxplots to the left of the vertical dashed line) and 2) intensity of CAWS water (boxplot to the right of the vertical dashed line).\u0026nbsp; Exposure duration is presented on the x-axis, while water source is presented as different colors on the box plots (e.g., blue = Havana River water; red = CAWS water; orange = a mix of 50% River and 50% CAWS water).\u0026nbsp; For model 1, significant differences in gene expression observed for fish exposed to water from the CAWS (1hr CAWS and 4hr CAWS) compared to the River (1hr River and 4hr River) are denoted by uppercase roman letters, while significant differences in gene expression caused by holding duration (1hr vs 4hr treatment) are denoted by lowercase Greek letters.\u0026nbsp; For model 2, significant differences in gene expression due to the intensity of CAWS water exposure was denoted using lowercase roman letters.\u0026nbsp; Relative gene expression data are presented in box-and-whisker plots such that the horizontal line in the middle of the box plots denotes the median of a treatment; the upper and lower horizontal lines represent the 75th and 25th percentiles, respectively; the vertical lines represent data that are 1.5 × the interquartile range above or below the 75th or 25th percentiles; and the actual data points are shown with filled circles overlayed on the boxplot.\u0026nbsp; For clarity, data are expressed relative to the mean mRNA expression of each gene during the 1hr River treatment and then log\u003csub\u003e2\u003c/sub\u003e transformed to clearly denote upregulation (values \u0026gt; 0) and downregulation (values \u0026lt; 0).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6221923/v1/338e5f1ffb25a7cf65fd92d6.png"},{"id":79893073,"identity":"69c279f3-0769-4286-aa93-09c5e31349bc","added_by":"auto","created_at":"2025-04-04 08:13:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":61861,"visible":true,"origin":"","legend":"\u003cp\u003eGenes within the liver of silver carp that displayed a significant change in mRNA expression following a 1hr exposure to 50% River – 50% CAWS water (1hr Mix): (A) CYP3α, (B) GADD45, and (C) HSP70.\u0026nbsp; Relative gene expression data were analyzed using two separate models: 1) exposure duration to CAWS water (boxplots to the left of the vertical dashed line) and 2) intensity of CAWS water (boxplot to the right of the vertical dashed line).\u0026nbsp; Exposure duration is presented on the x-axis, while water source is presented as different colors on the box plots (e.g., blue = Havana River water; red = CAWS water; orange = a mix of 50% River and 50% CAWS water).\u0026nbsp; For model 2, significant differences in gene expression due to the intensity of CAWS water exposure was denoted using lowercase roman letters.\u0026nbsp; Relative gene expression data are presented in box-and-whisker plots such that the horizontal line in the middle of the box plots denotes the median of a treatment; the upper and lower horizontal lines represent the 75th and 25th percentiles, respectively; the vertical lines represent data that are 1.5 × the interquartile range above or below the 75th or 25th percentiles; and the actual data points are shown with filled circles overlayed on the boxplot.\u0026nbsp; For clarity, data are expressed relative to the mean mRNA expression of each gene during the 1hr River treatment and then log\u003csub\u003e2\u003c/sub\u003e transformed to clearly denote upregulation (values \u0026gt; 0) and downregulation (values \u0026lt; 0).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6221923/v1/88b87ce26e12be40ed76db78.png"},{"id":87756793,"identity":"995b609d-7d0a-4b41-9145-d2a23e4abaff","added_by":"auto","created_at":"2025-07-28 16:09:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":833153,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6221923/v1/17054c5c-f40b-4af5-a624-e6a38b6771b1.pdf"},{"id":79893293,"identity":"e239dd8c-74dc-4b20-9231-470af986a888","added_by":"auto","created_at":"2025-04-04 08:21:00","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":40808,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6221923/v1/477ff8e0a393777218f3e842.docx"},{"id":79892387,"identity":"269f7846-2baf-425c-a0d0-c09456801dd2","added_by":"auto","created_at":"2025-04-04 08:05:00","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":89140,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineResource.docx","url":"https://assets-eu.researchsquare.com/files/rs-6221923/v1/6987c00755aa28d6858989a8.docx"}],"financialInterests":"","formattedTitle":"Acute exposure to water from the Chicago Area Waterway System induces molecular indices of stress and disturbance in silver carp: implications for deterrence to range expansion","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSelection of optimal habitat is a key aspect of life history for most animals. There are many factors that can contribute to an organism\u0026rsquo;s choice of habitat, including competition for and availability of food resources, energy conservation, risk of predation, and abiotic factors (Hayes et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Craig and Crowder \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Organisms can minimize energy expenditure by selecting optimal environments (Walsberg \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Huey \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1991\u003c/span\u003e), and this can affect how different species populations are distributed in a system. Indeed, habitat selection can be a result of trade-offs with important processes, such as growth and reproduction (Craig and Crowder \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Thus, habitat choice can be extremely important for an organism\u0026rsquo;s survival and fitness.\u003c/p\u003e \u003cp\u003eWhen an organism is forced to occupy sub-optimal habitat, an increase in energy expenditure can occur to combat the negative effects that the external environment may be causing. In turn, this increase in energy use could come at the cost of siphoning energy from biological processes such as foraging, predator avoidance, growth or reproduction as the organism attempts to restore homeostasis (Brown and Chivers \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Sadoul and Vijayan \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Additionally, occupying sub-optimal environments, such as hypoxic or polluted environments, can cause changes in gene expression in fish related to growth, immune response, and stress (Ton et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Richards \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Tierney \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Kumar and Denslow \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). To avoid these costly trade-offs, animals may therefore completely avoid sub-optimal habitat.\u003c/p\u003e \u003cp\u003eRecent evidence has suggested that silver carp (\u003cem\u003eHypophthalmichthys molitrix\u003c/em\u003e) in the Illinois River may be avoiding upstream areas of the Illinois River watershed due to the creation of sub-optimal habitat from contaminants in Chicago Area Waterway System (CAWS). Silver carp are prolific invaders and are expanding throughout the upper Mississippi basin undeterred by the many locks and dams (Camacho et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and have had a number of negative consequences for aquatic environments (Irons et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Sass et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Chick et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). One of these tributaries, the Illinois River, has had an established population of silver carp documented since the 90s (Irons et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Sass et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Despite their propensity to invade, the \u0026lsquo;leading edge\u0026rsquo; of silver carp in the Illinois River has not advanced upstream in over a decade despite a lack of dams or physical structures preventing further upstream movement (MRRWG 2012). Although this lack of range expansion could be due to several factors, including extensive population suppression downstream (Altenritter et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), past work has also shown that contaminant loads are higher at the leading edge compared to further downstream in the Illinois River where populations are larger (Battaglin et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Additionally, past research has shown that silver carp at the \u0026lsquo;leading edge\u0026rsquo; have higher indices of stress due to contaminants than fish from downstream areas (Jeffrey et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Curtis-Quick et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and also that exposure of hatchery-reared silver carp to CAWS water in a laboratory setting causes reduced activity and increased metabolic rate (Schneider et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Together, these lines of evidence suggest that the presence of bioactive contaminants in the CAWS may be playing a role in the lack of upstream movement of the silver carp population as animals avoid sub-optimal habitats upstream that are created by the presence of contaminants. To date, however, this hypothesis has largely been formulated using correlative, observational sampling of silver carp in the CAWS, precluding us from drawing definitive, causal relationships between water quality and range expansion. Water quality in the Chicago area has been improving over the past several years, and many native fish populations are increasing in size and number (Gibson-Reinemer et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Happel and Gallagher \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Happel \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, identifying mechanisms that may be preventing further upstream movement of invasive carp is important to predicting conditions that may facilitate movement in the future should water quality continue to improve and a noxious stimulus inadvertently be removed, as eliminating an invasive species after establishment is almost impossible (Leung et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe objective of this study was to quantify genetic and physiological indices of stress, disturbance and tissue damage in silver carp following an acute exposure to water from the CAWS collected from a point farther upstream their current distribution. More specifically, this study sought to answer 2 questions: (1) Does the expression of genes within rosettes and liver of silver carp change in response to different \u003cem\u003edurations\u003c/em\u003e of CAWS water exposure?, and (2) Does the expression of genes within rosette and liver of silver carp change in response to different \u003cem\u003eintensities\u003c/em\u003e of CAWS water exposure? To accomplish this goal, silver carp were collected from the population \u0026lsquo;core\u0026rsquo; exposed to water collected from the vicinity of the upstream leading edge of their distribution in the CAWS. Following this exposure, the molecular response of liver and olfactory tissue was quantified across several gene families, and changes in the morphology of gill tissue were also assessed. Together, results from this study will identify molecular and physiological responses to acute CAWS water exposure that may be related to the lack of upstream movement of silver carp in the Illinois River.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eCollection of Water\u003c/h2\u003e\n \u003cp\u003eTo quantify the genetic and physiological impact that acute exposure to water from the CAWS has on silver carp in the Illinois River, we obtained water from two different sources. First, water from the CAWS was collected from a public boat ramp in Channahon, IL, (41.42448, -88.20854) on June 6th, 2022. This site is in proximity to the \u0026ldquo;leading edge\u0026rdquo; of silver carp populations in the Illinois River (MRRWG 2012). While we did not conduct any chemical analyses of the CAWS water used in this study, a number of previous investigations have performed a thorough examination of CAWS water near this collection site (Channahon, IL) (Duncker et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e; U.S. Geological Survey (USGS) National Water Information System database at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.5066/F7P55KJN\u003c/span\u003e\u003c/span\u003e using site number 05538020) and shown higher concentrations of anthropogenic contaminants near this upstream location relative to downstream locations where population densities of carp are higher (Battaglin et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). Wild silver carp sampled from near this location also show increased evidence of contaminant exposure and energy use in livers relative to more downstream populations (Jeffrey et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e; Curtis-Quick et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e), and exposure to water collected from this site also caused hatchery-reared silver carp to reduce activity levels and increase metabolic rates (Schneider et al. \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e). Together, these lines of evidence suggest that water at this collection point should be exhibiting some degree of noxious stimului that induces avoidance behavior, and therefore has the potential to induce molecular and physiological responses. Collection of water at this site occurred by submerging pumps in the river at a public boat ramp and transferring water into a 473 L liquid storage tank. The CAWS water was then transported by truck to the Illinois River Biological Station (IRBS) in Havana, IL, and stored outdoors in closed liquid storage tanks. The second source of water was from a public boat ramp on the La Grange Pool of the Illinois River in proximity to IRBS (40.304961, -90.066698) on June 7th and June 8th, 2022, using methods identical to that of the CAWS water collection. This site on the Illinois River is approximately 157.9 river km downstream of the upstream collection point, and has had robust populations of silver carp for decades (Irons et al. \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e). Water chemistry analyses has shown that a number of chemical compounds found upstream are absent from this site (Battaglin et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e), essentially making it a control treatment for the CAWS water exposure assay.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eFish Collection\u003c/h3\u003e\n\u003cp\u003eAdult silver carp were collected from the La Grange pool of the Illinois River by pulse DC electroshocking on June 7th and June 8th, 2022. Following capture, fish were held onboard the shocking boat in low densities in livewells filled with ambient river water. Once several silver carp had been collected, fish were taken to a nearby boat ramp and then hauled by truck to IRBS.\u003c/p\u003e\n\u003ch3\u003eExposure Assay\u003c/h3\u003e\n\u003cp\u003eAssays to quantify genetic and physiological responses in silver carp following acute exposure to CAWS water were performed on June 7th and June 8th, 2022. For this, silver carp were netted from livewell on the collection boat and randomly distributed into one of five different experimental tanks that contained water from one of the five treatments used in this study. The five different treatments were: (1) a 1-hour exposure to water collected from the La Grange Pool of the Illinois River [1hr River], (2) a 1-hour exposure to water collected from the CAWS [1hr CAWS], (3) a 4-hour exposure to water from the La Grange Pool [4hr River], (4) a 4-hour exposure to water from the CAWS [4hr CAWS], and (5) a 1-hour exposure to water that was a mix of CAWS water diluted with equal parts of water from the La Grange Pool [1hr Mix]. Exposure assays for the 1hr River, 1hr CAWS, 4hr River and 4hr CAWS treatments were run on June 7th, while the 1hr Mix treatment (equal parts CAWS water and La Grange Pool water) was performed on June 8th. All experimental tanks were shaded with canopy tents, outfitted with air stones, and consistently monitored for dissolved oxygen (Professional Plus, YSI Inc., Yellow Springs, Ohio, USA), while pH and temperature were also checked intermittently. Water quality parameters were within acceptable limits for fish holding, and we did not observe any mortality during the holding period. The total length and weight of fish did not differ across the 5 treatments (one-way analysis of variance (ANOVA)\u003csub\u003elength\u003c/sub\u003e, F\u003csub\u003e1,45\u003c/sub\u003e = 0.797, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u0026gt;\u0026thinsp;0.05, one-way ANOVA\u003csub\u003eweight\u003c/sub\u003e, F\u003csub\u003e1,45\u003c/sub\u003e = 2.340, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Online Resource 1).\u003c/p\u003e\n\u003cp\u003eAfter an exposure period of either 1 hr or 4 hrs, silver carp were removed from the experimental tank, euthanized by cerebral percussion, and measured for total length (cm) and weighed (kg) (Online Resource 1). Liver, gill filaments, and olfactory rosettes used for genetic and physiological analyses were harvested immediately. One olfactory rosette (of two) was stored in 1 mL of RNAlater (Invitrogen, Waltham, Massachusetts, USA) following manufacturer\u0026rsquo;s protocols for molecular analyses; samples were kept in a cooler of wet ice during collection, held in a 4\u0026deg;C refrigerator overnight, and then transferred to a -20\u0026deg;C freezer for permanent storage. Liver samples used for gene expression analysis (see below) were flash frozen in liquid nitrogen and then stored in a -80\u0026deg;C freezer until use. A section of harvested gill and olfactory tissue were also sampled for histology. This gill tissue was immediately stored in 10% neutral buffer formalin for 48 hours (Fournie et al. \u003cspan class=\"CitationRef\"\u003e2000\u003c/span\u003e), after which samples were transferred to 70% ethanol for permanent storage at room temperature (Bieber et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). All fish collection, handling and experimental procedures were approved by the University of Illinois Institutional Animal Care and Use Committee (Protocol #21139).\u003c/p\u003e\n\u003ch3\u003eGene Expression Analyses\u003c/h3\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eRNA isolation and cDNA synthesis\u003c/h2\u003e\n \u003cp\u003eTotal RNA was isolated from liver and rosette samples using the RNeasy Mini Kit (Qiagen, Germantown, Maryland, USA) according to manufacturer\u0026rsquo;s protocol. Briefly, tissue samples were homogenized using a Bullet Blender Blue (Next Advance, Troy, New York, USA). RNA was extracted and treated with RNase-free DNase (Qiagen, Germantown, Maryland, USA) to remove any residual DNA from the sample. RNA was then quantified using a Qubit 3.0 Fluorometer (Fisher Scientific, Hampton, New Hampshire, USA). RNA was stored at -80\u0026deg;C. Prior to cDNA synthesis, RNA integrity was quantified at the Roy J. Carver Biotechnology Center at the University of Illinois at Urbana-Champaign using either an Agilent 2100 Bioanalyzer or an AATI fragment analyzer (Agilent, Santa Clara, California, USA). Samples with RNA integrity numbers below 5 were excluded due to high sample degradation (Fleige and Pfaffl \u003cspan class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eSynthesis of cDNA was performed using a QuantiTect Reverse Transcription Kit (Qiagen, Germantown, Maryland, USA) using 1 \u0026micro;g of total RNA for a reaction volume of 20 \u0026micro;L. For most samples, a final reaction concentration of 50 ng/\u0026micro;L was created. However, some samples were created to reaction concentrations of either 12.5 or 25 ng/\u0026micro;L due to low initial sample volume or low initial RNA concentration. An Eppendorf Mastercycler Pro thermal cycler (Eppendorf, Hamburg, Germany) was used to facilitate the cDNA synthesis reaction, and all reactions were performed following the manufacturers\u0026rsquo; instructions: 1) 42\u0026deg;C for 15 min and 2) 95\u0026deg;C for 3 min. cDNA was stored at -20\u0026deg;C.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eqPCR primers\u003c/h2\u003e\n \u003cp\u003eGene specific qPCR primers for silver carp were obtained either a) from previous studies or b) by designed using Primer 3 (Koressaar and Remm \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e; Untergasser et al. \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e) using sequences available in the GenBank database and divided into functional groups found in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Primer sequences and fragment lengths are shown in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. To verify specificity of each qPCR primer (i.e., generation of only one product), we performed PCR using TAQ5X PCR Master Mix (New England Biolabs, Ipswich, Massachusetts, USA), the forward and reverse qPCR primers for each gene (Integrated DNA Technologies, Coralville, Iowa, USA), cDNA, and RNase-free water. The PCR reaction was performed on an Eppendorf Mastercycler Pro thermal cycler (Eppendorf, Hamburg, Germany) under the following conditions: 1) 95\u0026deg;C for 30 sec; 2) 40 cycles of (a) 95\u0026deg;C for 30 sec, (b) 55\u0026deg;C for 30 sec, and (c) 68\u0026deg;C for 30 sec; followed by 3) a final cycle at 68\u0026deg;C for 5 min. PCR products were then run on a 3% agarose gel containing SYBR Safe Dye (Invitrogen, Waltham, Massachusetts, USA) to confirm the amplicon was the correct length and the only product generated by the reaction.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eqPCR gene expression analysis\u003c/h3\u003e\n\u003cp\u003eIn total, 3 reference genes (\u003cem\u003e\u0026beta;-actin\u003c/em\u003e, \u003cem\u003eef1\u0026alpha;\u003c/em\u003e, and \u003cem\u003e18s\u003c/em\u003e) and 20 target genes (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) were analyzed for all tissue sample using a microfluidics qRT-PCR platform (two 96.96 Dynamic Array TM IFC, Standard Biotools, San Francisco, California, USA). The 20 target genes chosen contained a selection of genes related to oxidative stress, xenobiotic defense, growth and energy storage, cell death and repair, and general stress (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Each cDNA sample was run in triplicate. A pre-amplification step on the cDNA was conducted prior to gene expression analysis under the following conditions: 1) 95\u0026deg; C for 10 min, followed by 14 cycles of 2) 95\u0026deg; C for 15 sec and 3) 60\u0026deg; C for 4 min. An exonuclease treatment was then applied to each PCR reaction to remove access primer. Samples were then loaded into the microfluidic plates and run on a BioMark HD automated, high-performance qPCR system (Standard Biotools, San Francisco, California, USA). qPCR was performed under the following conditions: 1) thermal mix stage at 70\u0026deg; C for 40 min, 2) 60\u0026deg; C for 30 sec, 3) a hot start cycle at 95\u0026deg; C for 1 min, 4) 30 cycles of 96\u0026deg; C for 5 sec and 55\u0026deg; C for 20 sec, and 5) a melting stage run at 55\u0026deg; C for 3 sec and then ramped up by 1\u0026deg; C every 3 sec to 95\u0026deg; C.\u003c/p\u003e\n\u003cp\u003eGene expression data were extracted using the Standard BioTools Real-Time PCR Analysis Software (Standard Biotools, San Francisco, California, USA) using C\u003csub\u003et\u003c/sub\u003e thresholds set manually for each gene. Each gene assay included a no template control (NTC) that needed to be negligibly expressed compared to the cDNA samples (i.e., not detected or \u0026gt;\u0026thinsp;5 C\u003csub\u003et\u003c/sub\u003e from sample cDNA). Amplification and melting curves for each sample were also visually inspected and spurious samples were removed (i.e., no sigmoid curve, multiple products observed on melting curve). To quantify the efficiency of our target and housekeeping genes, cDNA from RNA extractions of pooled liver and rosette samples were serially diluted in 7 dilutions of 1:4 (1 to 1:4096). PCR efficiencies for each gene were calculated using (10\u003csup\u003e1/slope\u003c/sup\u003e \u0026ndash; 1) \u0026times; 100, for which the slope was estimated by plotting the C\u003csub\u003et\u003c/sub\u003e over the serial dilutions of cDNA. Sample gene expression was normalized with the \u0026Delta;\u0026Delta;C\u003csub\u003et\u003c/sub\u003e method and correction for PCR efficiency according to Pfaffl (\u003cspan class=\"CitationRef\"\u003e2001\u003c/span\u003e). Expression of target genes were normalized to the expression of the housekeeping gene, \u003cem\u003eef1\u0026alpha;\u003c/em\u003e, which was found to be the most suitable tested reference gene (e.g., \u003cem\u003e18s\u003c/em\u003e and \u003cem\u003e\u0026beta;-actin\u003c/em\u003e were too highly expressed) and there was no significant difference in gene expression (C\u003csub\u003et\u003c/sub\u003e) of \u003cem\u003eef1\u0026alpha;\u003c/em\u003e between our 5 treatments (one-way ANOVA\u003csub\u003eliver\u003c/sub\u003e, F\u003csub\u003e1,39\u003c/sub\u003e= 1.098, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05; one-way ANOVA\u003csub\u003erosette\u003c/sub\u003e, F\u003csub\u003e1,38\u003c/sub\u003e = 1.464, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Gene expression data for each target gene was calculated as a relative expression ratio relative to the average expression levels observed in silver carp exposed to 1hr of river water.\u003c/p\u003e\n\u003ch3\u003eHistology\u003c/h3\u003e\n\u003cp\u003eAll gill samples were embedded and stained with methylene blue by the Histology Laboratory at the College of Veterinary Medicine at the University of Illinois at Urbana-Champaign. Slides were imaged using a Nanozoomer 2.0-HT Slide Scanner (Hamamatsu Photonics, Hamamatsu, Japan) at the Carl R. Woese Institute for Genomic Biology at the University of Illinois at Urbana-Champaign. To quantify histological indices of damage, two random filaments were chosen, while ten lamellae from each filament were randomly selected to be measured(Blair et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). Each lamella was measured for length, width, and interlamellar cell mass (ILCM) height between lamellae(Blair et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e; Ong et al., \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e). Additionally, instances of aneurysm and clubbing were also recorded(Strzyzewska et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). All slides were assigned a number using a random number generator to allow for blind scoring by the reader. Measurements were taken using the open-source software Fiji (Schindelin et al. \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical Analysis\u003c/h2\u003e\n \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\n \u003ch2\u003eGene expression\u003c/h2\u003e\n \u003cp\u003eBecause we did not have a perfectly balanced statistical design and only had a single sampling time point for our 1hr Mix treatment, the response of silver carp to CAWS water exposure was conducted using two separate statistical models that aligned with the two questions of our study. The first statistical model was associated with our first question and identified changes in gene expression due to the duration of CAWS water exposure (1hr vs 4hr). This model consisted of a two-way ANOVA with the main effects of water source (CAWS water vs water downstream from Havana), exposure time (1hr vs 4hr) and their interaction (water source \u0026times; exposure time). The second statistical model addressed our second research question and was focused on the response of tissues to the intensity of CAWS water exposure. This statistical model consisted of a one-way ANOVA comparing the three treatments that exposed silver carp to 1hr of water either from Havana (1hr River), CAWS water (1hr CAWS) or the 50% dilution of CAWS water with Havana River water (1hr Mix). To account for the fact that some treatments were used twice in separate analyses, and to reduce the Type I error rate, a conservative \u0026alpha; of 0.025 was used as a threshold of statistical significance for all models (VanderWeele and Mathur \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). To meet the assumptions of ANOVAs (i.e., normal distribution, homogenous variances), relative gene expression ratio data were rank transformed (Conover and Iman \u003cspan class=\"CitationRef\"\u003e1981\u003c/span\u003e; Potvin and Roff \u003cspan class=\"CitationRef\"\u003e1993\u003c/span\u003e). To improve data interpretation in figures, gene expression ratio data were log\u003csub\u003e2\u003c/sub\u003e transformed, which presents upregulated genes relative to the reference (control) treatment (i.e., 1hr River) as positive values and downregulated genes relative to the control treatment as negative values(Franks et al. \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). Gene expression ratio data within the results are presented as fold-change (e.g., gene expression ratio of 0.25\u0026thinsp;=\u0026thinsp;2-fold decrease in gene expression relative to the control treatment) to facilitate data interpretation.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eHistology\u003c/h2\u003e\n \u003cp\u003eAnalyses for lamellar length, lamellar width, and ILCM height, used a similar statistical approach to gene expression analysis that included both two-way and one-way models aligned with our two questions, but with a few small differences. More specifically, histology analyses used a linear mixed effects model approach with fish identification number as a random effect as multiple measurements were collected from each individual fish due to the use of multiple lamellae from an individual (Lindstrom and Bates \u003cspan class=\"CitationRef\"\u003e1990\u003c/span\u003e; Crawley \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e). Additionally, generalized linear mixed models with binomial error distributions were run to analyze instances of clubbing and aneurysms as these are count data (Crawley \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e). Models were interpreted using the \u003cem\u003eanova()\u003c/em\u003e function from the \u0026lsquo;lmerTest\u0026rsquo; package (Kuznetsova et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). If any of the main effects our ANOVA models were significant, or if the interaction was significant, we then conducted the appropriate post-hoc test (t-test or Tukey HSD test) to separate means and identify differences within and across groups. If a significant interaction was found in a model, main effects were ignored. Normality was assessed visually using a normal Q-Q plot of model residuals along with a formal Shapiro-Wilk normality test, while homogenous variances were assessed visually using a residuals vs fitted values plot along with a formal Levene\u0026rsquo;s test (Tabachnick \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e; Dean et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). All statistical analyses were performed in R version 4.3.0 (2022).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eQuestion 1: Does the expression of genes within rosette and liver of silver carp change in response to different durations of CAWS water exposure?\u003c/span\u003e \u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRosettes\u003c/h2\u003e \u003cp\u003eA total of 4 genes within the rosettes of silver carp changed their expression following exposure to CAWS water, but the duration of the CAWS water exposure (1 hour or 4 hours) did not alter the pattern of gene expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Online Resource 2). More specifically, we observed increased mRNA expression of a xenobiotic defense gene, CYP1A (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), and an oxidative stress gene, GST (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), in the rosettes of silver carp exposed to CAWS water compared to River water (Online Resource 2). In contrast, GADD45 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), a gene related to DNA repair, and MCL 1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), a gene relative to the activation of cell death, were downregulated in silver carp exposed to CAWS water compared to fish exposed to River water (Online Resource 2). We also observed 4 genes change expression following longer holding/exposure to water either from the CAWS or Havana (River) water. Three genes decreased their mRNA expression following 4 hours of holding relative to silver carp in the 1hr treatments: two general stress genes (CJUN and HIF1a; Online Resource 2, Online Resource 3) and one xenobiotic defense gene (CYP1A; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA; Online Resource 2). PHD3, an oxidative stress gene, was the only mRNA transcript to increase expression due to a longer holding period (2-fold increase at 4hr vs. 1hr; Online Resource 2, Online Resource 3). All other target genes did not change mRNA expression due to water source (CAWS or River) or exposure time (1hr or 4hr) (Online Resource 2, Online Resource 3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eLiver\u003c/h2\u003e \u003cp\u003eNone of the target genes within liver tissue altered mRNA expression levels when exposed to CAWS water for either 1 hour or 4 hours (Online Resource 4, Online Resource 5). Silver carp mRNA expression did change for 3 genes following longer holding/exposure to either CAWS or River water. HIF1α, a gene related to general stress, and PHD3, an oxidative stress gene, were significantly upregulated following 4 hours of holding relative to silver carp in the 1hr treatments (Online Resource 4, Online Resource 5). In contrast, we observed a 3-fold decrease in the relative expression of GST mRNA, another oxidative stress gene, within silver carp livers at the 4-hour exposure treatment relative to the 1-hour exposure (Online Resource 4, Online Resource 5). All other target genes did not change across the different exposure times (Online Resource 4, Online Resource 5).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eQuestion 2: Does the expression of genes within rosettes and liver of silver carp change in response to different intensities of CAWS water exposure?\u003c/span\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eRosette\u003c/h2\u003e \u003cp\u003eSix genes within the rosettes of silver carp changed their expression based on the intensity of CAWS water exposure (i.e., 1hr River vs 1hr Mix vs 1hr CAWS). After a 1hr exposure to 50% CAWS and River water, silver carp displayed a 2-fold decrease in CYP1A mRNA expression (a xenobiotic defense gene transcript) relative to the 1hr River treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Online Resource 1), which was also significantly different from the 1hr CAWS treatment that was upregulated following exposure to CAWS water. In contrast, we observed an increase in two oxidative stress genes, GST (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) and CAT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), and a different xenobiotic defense gene, CYP3A137 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), following a 1hr exposure to the Mix treatment relative to the 1hr River treatment (Online Resource 2). A greater than 2-fold decrease in mRNA expression was observed for two general stress genes, CJUN (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and HSP70 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), in the rosettes of silver carp exposed to the 1hrMix treatment relative to fish exposed to river water (Online Resource 2). All other target genes examined in the rosettes of silver carp did not differ in expression based on the intensity of CAWS water (Online Resource 4, Online Resource 5).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eLiver\u003c/h2\u003e \u003cp\u003eSilver carp downregulated two genes in liver in response to a 1hr exposure to a 50\u0026ndash;50 mix of CAWS/River water: xenobiotic defense gene CYP3A137 was 2-fold lower expressed in the 1hr Mix treatment than the 1hr River or 1hr CAWS treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA; Online Resource 4), and the mRNA expression of general stress gene HSP70 decreased 3-fold in the 1hr Mix treatment relative to fish in the 1hr CAWS treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB; Online Resource 4). In contrast, we observed a significant 2-fold increase in GADD45 mRNA expression, an indicator of DNR repair, in the liver of fish exposed to the 1hr Mix treatment relative to fish exposed to full-strength CAWS water (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC; Online Resource 5). All other target genes examined in the liver of silver carp did not differ in expression based on the intensity of CAWS water (Online Resource 4, Online Resource 5).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eHistology\u003c/h2\u003e \u003cp\u003eThere was no significant effect of treatment on lamellae length, although lamellae length showed a nearly 20% decrease between the 1hr and 4hr CAWS treatments, as well as a 6% decrease between the 1hr and 4 hr River treatments (Online Resource 6, Online Resource 7). Lamellae width and interlamellar cell height in the selected filaments also showed no change between CAWS treatments or durations (Online Resource 6, Online Resource 7). Additionally, there was no statistical difference between the number of instances of lamellar aneurysms or clubbing between treatments or durations (Online Resource 6).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAcute exposure to water from the Chicago Area Waterway System for either 1 or 4 hours increased the expression of genes associated with xenobiotic defense and oxidative stress within the rosette tissue of silver carp. Specifically, we observed that acute exposure to water collected from upstream of the current distribution of silver carp caused an upregulation in genes related to xenobiotic exposure (CYP1A) and oxidative stress (GST). When animals are exposed to toxicants and contaminants in the environment, outcomes such as sensory disruption and neurological dysfunction can occur (Kennedy \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Sopinka et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Because olfactory rosettes are directly exposed to the external environment, they are vulnerable to disruptions in homeostasis from xenobiotics. Damage to rosettes caused by environmental contaminants can be severe (Tierney et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) resulting in outcomes such as impaired olfaction and improper perception of environmental information (Tierney et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), and, in extreme cases can result in the transport of contaminants to the brain along the olfactory nerve (Sloman \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Tierney et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). To prevent these negative outcomes, several changes can occur at the molecular level soon after a contaminant is experienced, including the upregulation of genes related to protection processes. For example, CYP enzymes (including the CYP1A subfamily) have been shown to be important for the metabolism and biotransformation of foreign compounds, making them critical early responders in the of detoxification of environmental contaminants (Sarasquete and Segner \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Additionally, Espinoza et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) found that the rosettes of coho salmon (\u003cem\u003eOncorhynchus kisutch\u003c/em\u003e) increased expression of nine GST isoforms following 8, 24, and 48 hour exposures to cadmium, with this upregulation of GST isoforms serving to detoxify foreign compounds and protect cells from damage. In the current study, when silver carp were exposed to water from the CAWS, the observed upregulation of CYP1A and GST genes in rosettes likely occurred with the intention of protecting tissues from damage associated with exposure to bioactive compounds within the water (Battaglin et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Thus, the exposure of silver carp to water upstream of their current distribution within the CAWS resulted in an upregulation of genes related to protection from contaminants in rosettes, highlighting that CAWS water acts as a noxious stimulus for silver carp.\u003c/p\u003e \u003cp\u003eIn addition to the activation of genes related to detoxification, acute exposure of silver carp from the CAWS also resulted in a downregulation of genes in rosettes suggesting a disruption of certain cellular functions. More specifically, a 1- or 4-hour exposure to water upstream of the current distribution of silver carp resulted in a downregulation of genes related to cell death regulation (MCL1) and DNA repair (GADD45). While contaminant exposure can induce the activation of protective mechanisms at the cellular level, in extreme cases, animals may divert energy away from important bodily and cellular processes to aid in the detoxification of foreign contaminants (Jeffrey et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). For example, Hong et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) saw a downregulation of MCL1 proteins in a Chinook salmon embryonic cell line within 8 hours of being infected with pancreatic necrosis virus (IVPN), along with a negative correlation between MCL1 expression and viral replication. Similarly, Higashi et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) found that GADD45 mRNA showed a ten-fold decrease in expression in human lung tumor tissues compared to normal lungs. More importantly, silver carp sampled from the leading edge of their distribution in the Illinois River, close to the collection point of water used in the current study, displayed both a downregulation in several genes involved in DNA repair in liver tissue (Jeffrey et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), as well as a decrease in metabolites related to cellular-level protection in liver (Curtis-Quick et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), demonstrating consistency between wild-caught fish and the experimental treatments used in the current study. Thus, acute exposure of silver carp to water from the Chicago Area Waterway System results in a downregulation of genes related to protection of olfactory tissues from bioactive compounds in CAWS water.\u003c/p\u003e \u003cp\u003eAlthough our study observed multiple effects of acute exposure of CAWS water to silver carp olfactory tissue, the same exposure did not alter the genetic expression of any of the genes analyzed within liver tissue. The liver plays a major role in vertebrates in the detoxification of harmful contaminants, and thus is a target for studies seeking to quantify the effect of pollutants on organisms (Anadon \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Kime \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Indeed, exposure to pollutants can induce negative morphological changes in the liver such as decreased glycogen and degeneration of cell membranes (Kime \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1998\u003c/span\u003e), as well as an increase in gene activity due to possible health effects (Wirgin and Waldman \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Jordan-Ward et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Additionally, past work with silver carp has shown that livers from animals samples from the Illinois River at the leading edge of their range displayed an upregulation in genes related to xenobiotic defense, metabolism, and apoptosis compared to core populations downstream, demonstrating an impact of CAWS water on toxicological pathways in livers (Jeffrey et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). We feel there could be three possible explanations for why we did not see changes in the gene expression patterns of silver carp following our experimental treatments. First, one reason that this study did not see changes in genetic expression in silver carp livers following exposure to CAWS water may be because our exposure durations were not sufficiently long to induce a response. In a study performed by Momoda et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), for example, rainbow trout (\u003cem\u003eOncorhynchus mykiss\u003c/em\u003e) were exposed to a simulation of low water conditions across multiple timepoints, and the upregulation of genes related to stress did not occur until 3 hours into the experiment, while Craig et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) exposed zebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e) to multiple concentrations of copper and showed an increase in gene expression after 4 hours at the highest concentration of copper, while other genes either took longer to show expression differences demonstrating a delayed response in the liver to external stressors. A second possible explanation for why we did not see an upregulation of genes in the liver of silver carp acutely exposed to CAWS water is that the concentration of contaminants could have been too low, and the expression of genes could have been influenced by a dose-dependent response. For example, work by Cai et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) showed that bigheaded carp (\u003cem\u003eHypophthalmichthys nobilis\u003c/em\u003e) experienced elevated expression of the cellular metabolic maintenance gene CDA in livers when injected with higher concentrations of the cyanotoxin microcystin-LR (MCLR) compared to lower concentrations. Finally, a third explanation for why there were no changes in the activity of genes in the livers of silver carp following acute exposure to CAWS water is that the genes selected simply did not change in response to this external environmental pressure. To perform RT-qPCR, it is necessary to know the gene sequence of interest before performing analyses, particularly if researchers are interested in a non-model gene (Costa et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). However, because some sequences have multiple primer pair options to choose from, it\u0026rsquo;s possible to pick primer pairs within the sequence that can bind on themselves instead of the cDNA product that the qPCR process is attempting to amplify. Therefore, the chosen primer pair from within the gene sequence may not be conducive for the RT-qPCR process (Costa et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Together, our study found no evidence of changes in genetic expression in the livers of silver carp following acute exposure to CAWS water despite seeing up- and down-regulation of genes across several different pathways in rosettes, and targeting several known toxicological pathways previously shown to change following toxicant exposure in other fish species.\u003c/p\u003e \u003cp\u003eAn acute exposure to a 50\u0026ndash;50 mix of CAWS and River water resulted in a variety of changes in gene expression in both liver and rosette tissues, but no strong pattern or signature of CAWS water exposure. For example, we found genes related to general stress in both rosette and liver tissue were downregulated in the mix treatment compared to the control and 1-hour treatments, but we also saw both upregulation and downregulation of genes related to xenobiotic defense, oxidative stress, and cell death and cell repair in rosette and liver tissue. By exposing silver carp to diluted CAWS water, we assumed we would see a linear dose-response curve in expression relative to patterns observed when silver carp were exposed to full-strength CAWS water. However, this traditional linear dose-response model has been shown to be unreliable in predicting organism responses to low-dose exposures (Agathokleous and Calabrese \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Indeed, some organisms will experience hormesis, or multi-level dose response curves, in which a toxin may induce beneficial effects at low concentrations, which may then be reversed as the dosage increases (Morkunas et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Agathokleous and Calabrese \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Additionally, it is important to note that analysis of water from the CAWS has documented hundreds of different bioactive contaminants in the water, and no specific chemical or chemicals has been determined to be causing this molecular reaction in silver carp (Battaglin et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Duncker et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Thus, when exposed to a lower concentration of water from the CAWS, the lack of discernable trends in the molecular response in tissues likely occurred as the different concentration of contaminants in this treatment resulted in an upregulation of some molecular processes, and a downregulation of other molecular processes due to hormesis.\u003c/p\u003e \u003cp\u003eAdditionally, our study saw no change in morphology of gills following CAWS water exposure of either duration. Specifically, lamellar length, lamellar width, and interlamellar cell mass height did not change in silver carp gills between our treatments that varied the intensity or exposure duration to CAWS water. Histological changes in tissue can be effective markers for contaminant and pollution exposure (Yancheva \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), and gills are susceptible to changes caused by pollutants, making them a strong general biomarker for damage and stress and an effective tool for monitoring the response of fish to environmental pollution (Mallatt \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Au \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Yancheva \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Despite this potential, we did not see any evidence of histological changes in gill tissue following exposure to CAWS water at either the 1-hour or 4-hour timepoint. One reason for this could be that the exposure timepoints explored in this study were not long enough to illicit any structural changes in gill tissues. Work by Guo et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) showed that white shrimp (\u003cem\u003eLitopenaeus vannamei\u003c/em\u003e) exposed to nonylphenol experienced changes in gill morphology 3 hours after exposure initiation. Additionally, another reason changes in gill morphology were not observed in our study could be because the concentration of bioactive contaminants in CAWS water was not high enough to cause a physical reaction in the gills. A study by Santos et al. (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) exposed juvenile Florida pompano (\u003cem\u003eTrachinotus carolinus\u003c/em\u003e) to naphthalene, a component of petroleum, at multiple concentrations and showed a positive correlation between concentration of naphthalene exposure and degree of gill tissue damage. Ultimately, no evidence of histological gill damage was found in silver carp following acute exposure to CAWS water.\u003c/p\u003e \u003cp\u003eResults from this study contribute to the growing body of evidence that contaminants from Chicago are influencing the upstream movement of silver carp ( Jeffrey et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Curtis-Quick et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Past work with the \u0026lsquo;leading edge\u0026rsquo; of carp has shown an increase in stress and disturbance in silver carp at this location relative to downstream populations ( Jeffrey et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Curtis-Quick et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and laboratory exposure of hatchery-reared silver carp to CAWS water upstream of their distribution resulted in reduced activity and increased metabolic rate (Schneider et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Results from the current study demonstrate that exposing silver carp to water from the Chicago Area Waterway system results in an upregulation of stress genes in olfactory tissue. Water quality in the CAWS has continued to improve since the implementation of federal actions like the Clean Water Act (Gibson-Reinemer et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This, and other actions, has resulted in an increase of native fish biodiversity in the Chicago area waterways ( Happel and Gallagher \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Happel \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Efforts are also underway to continue improving water quality and make improvements to water treatment facilities in the Chicago area (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://mwrd.org/stormwater\u003c/span\u003e\u003cspan address=\"https://mwrd.org/stormwater\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). While these improvements are both valuable and important, they may inadvertently induce upstream movement of silver carp if they are no longer deterred by an inadvertent removal of noxious stimuli in the water that are responsible for inducing molecular or behavioral responses. Therefore, furthering research regarding how silver carp movement is affected by contaminants from the CAWS is of continued interest to researchers and managers.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eFish select habitats that minimize energy expenditure and maximize growth and reproduction. However, if a fish is forced to occupy sub-optimal habitat, homeostasis may be negatively influenced, resulting in an increase in pathways and processes that can increase energetic costs due to stress and disturbance in an effort to maintain homeostasis. If the reason for the sub-optimal habitat is the presence of contaminants, fish may need to divert energy from important energetic processes to filter toxins in the water or upregulate protective mechanisms. The leading edge of silver carp in the Illinois River have not moved upstream in over a decade despite the absence of any physical barrier that could prevent further range expansion. This lack of movement could be due to several reasons, but past studies suggest that contaminant exposure from the CAWS could be playing a role in the population\u0026rsquo;s stalling behavior. Data from this study indicate that silver carp experienced an upregulation in the activity of genes associated with protective mechanisms in their olfactory tissue when exposed to CAWS water, consistent with evidence provided by past field and laboratory research that silver carp at the upstream edge of their distribution are experiencing increased levels of stress associated with contaminant exposure. As water quality in the CAWS improves, there may be a facilitation in movement of the silver carp population. Therefore, identifying the mechanism that is causing the leading edge of the population to stall may provide insight into why they may move in the future, protecting valuable aquatic ecosystems from future invasions.\u003c/p\u003e"},{"header":"Statements \u0026 Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunding for this study was provided by the U.S. Geological Survey (USGS) WRRI Aquatic Invasive Species (AIS) Competitive Grants Program (Award G21AP10174-01), 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. Additional funding was provided through the Illinois AFS Larimore Grant, and the Illinois Muskie Tournament Trail Scholarship. We would like to acknowledge all researchers from the Illinois River Biological Station for providing a space to work, collecting river water for the control treatments, and collecting all fish used in the experiments. Additional thanks goes to Qihong Dai, Joseph Reinhofer, Scarlett Hoffer, and John Bieber for assistance with CAWS water collection, experimental setup and data collection. Lastly, we would like to acknowledge Jonathan Johnson, who provided major support in RNA extraction.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eFunding\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eFunding for this study was provided by the U.S. Geological Survey (USGS) WRRI Aquatic Invasive Species (AIS) Competitive Grants Program (Award G21AP10174-01), 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. Additional funding was provided through the Illinois AFS Larimore Grant, and the Illinois Muskie Tournament Trail Scholarship.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eCompeting Interests\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eAuthor Contributions\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eA.J.E. and C.D.S. conceived the study design, and all authors contributed to planning field and laboratory experiments. A.E.S. and C.E.D performed data collection, all data analysis and generation of figures with additional guidance from A.J.B. All authors contributed to writing and editing the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eData Availability\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eData from this study are available through the University of Illinois Data Bank (https://databank.illinois.edu/) at https://doi.org/10.13012/B2IDB-0347483_V1).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAgathokleous E, Calabrese EJ (2020) Environmental toxicology and ecotoxicology: How clean is clean? Rethinking dose-response analysis. Sci Total Environ 746:138769. https://doi.org/10.1016/j.scitotenv.2020.138769\u003c/li\u003e\n \u003cli\u003eAltenritter ME, DeBoer JA, Maxson KA, Casper AF, Lame, JT (2022) Ecosystem responses to aquatic invasive species management: A synthesis of two decades of bigheaded carp suppression in a large river. 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Mutat Res Mol Mech Mutagen 399:193\u0026ndash;219. https://doi.org/10.1016/S0027-5107(97)00256-X\u003c/li\u003e\n \u003cli\u003eYancheva V (2016) Histological biomarkers in fish as a tool in ecological risk assessment and monitoring programs: a review Appl Ecol Environ Res 14:47\u0026ndash;75. https://doi.org/10.15666/aeer/1401_047075\u003cbr\u003e\u0026nbsp;\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 2 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biological-invasions","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"binv","sideBox":"Learn more about [Biological Invasions](https://www.springer.com/journal/10530)","snPcode":"10530","submissionUrl":"https://submission.nature.com/new-submission/10530/3","title":"Biological Invasions","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"habitat selection, range expansion, contaminants, silver carp, gene expression ","lastPublishedDoi":"10.21203/rs.3.rs-6221923/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6221923/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eExposure of an animal to sub-optimal habitat can result in impairment, damage or increased energy expenditure to maintain homeostasis, which can direct resources away from reproduction and reduce fitness. Animals may therefore avoid sub-optimal habitats to minimize physiological costs. Silver carp (\u003cem\u003eHypophthalmichthys molitrix\u003c/em\u003e) are a prolific invader in the Mississippi River basin. Despite their propensity for spread, the \u0026lsquo;leading edge\u0026rsquo; of silver carp at the northern limit of their distribution in the Illinois River has stalled and not advanced for over a decade. Studies have suggested that contaminants in the Chicago Area Waterway System (CAWS) may be deterring upstream range expansion, but this hypothesis has had limited testing to date. The current study sought to quantify linkages between CAWS water constituents and impaired range expansion of silver carp. For this, CAWS water from beyond the current upstream distribution of silver carp was collected and transported downstream to the core of the population. Silver carp from the population core were collected and exposed to either water from their collection location (control), or to CAWS water from upstream of the current distribution, simulating range expansion. Following exposure, olfactory and liver tissue were collected to quantify genetic expression and histological indices of damage. Results showed that silver carp olfactory tissue experienced increased activity of genes related to xenobiotic defense and oxidative stress following exposure to CAWS water from upstream of the current distribution, highlighting costs associated with inhabiting this environment, providing a role for contaminants in CAWS water at deterring further upstream movement.\u003c/p\u003e","manuscriptTitle":"Acute exposure to water from the Chicago Area Waterway System induces molecular indices of stress and disturbance in silver carp: implications for deterrence to range expansion","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-04 08:04:55","doi":"10.21203/rs.3.rs-6221923/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-04-02T21:23:09+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-25T14:32:06+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Biological Invasions","date":"2025-03-18T01:29:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-14T13:13:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biological Invasions","date":"2025-03-13T13:00:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biological-invasions","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"binv","sideBox":"Learn more about [Biological Invasions](https://www.springer.com/journal/10530)","snPcode":"10530","submissionUrl":"https://submission.nature.com/new-submission/10530/3","title":"Biological Invasions","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c02e4ac1-0f52-42b0-b1cb-1cb5e90f8ada","owner":[],"postedDate":"April 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-28T16:05:16+00:00","versionOfRecord":{"articleIdentity":"rs-6221923","link":"https://doi.org/10.1007/s10530-025-03633-1","journal":{"identity":"biological-invasions","isVorOnly":false,"title":"Biological Invasions"},"publishedOn":"2025-07-24 15:57:27","publishedOnDateReadable":"July 24th, 2025"},"versionCreatedAt":"2025-04-04 08:04:55","video":"","vorDoi":"10.1007/s10530-025-03633-1","vorDoiUrl":"https://doi.org/10.1007/s10530-025-03633-1","workflowStages":[]},"version":"v1","identity":"rs-6221923","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6221923","identity":"rs-6221923","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}