The Comet Assay, a Sensitive Biomarker of Water Quality Improvement Following Adoption of Beneficial Agricultural Practices?

Numerous actions have been undertaken by farmers to attenuate the impact of agricultural activities on aquatic ecosystems. The identification of biomarkers that respond quickly to water quality improvement could facilitate the assessment of adopted alternative practices and help maintain mobilization among stakeholders. We evaluated the potential of the comet assay, a biomarker of genotoxic effects, using a freshwater mussel, Elliptio complanata, as a model animal. The frequency of DNA damage was assessed in hemocytes of mussels collected from a pristine habitat and caged for 8 weeks in the Pot au Beurre River, a tributary of the fluvial Lake St.-Pierre (Quebec, Canada) impacted by agricultural activities. We found that the level of DNA damage naturally induced in mussel hemocytes was low and showed very limited variations over time. Compared with these baseline levels and to laboratory controls, we observed a doubling in DNA alterations in mussels exposed to agricultural runoff in the third branch of the Pot au Beurre River. The genotoxic response was significantly lower in mussels caged in the first branch of the Pot au Beurre River, where longer stretches of shoreline have been restored as buffer strips. Glyphosate, mesotrione, imazethapyr, and metolachlor were the main discriminant pesticides between these two branches. Metolachlor was found in sufficient concentrations to induce DNA damage, but it is more likely that the observed genotoxicity was the result of a "cocktail effect," that is, the cumulative contribution of coexisting genotoxicants including the above-mentioned herbicides and ingredients in their formulation. Our findings suggest that the comet assay is a sensitive tool for the early detection of changes in water toxicity following the adoption of agricultural beneficial practices. Environ Toxicol Chem 2023;42:2201-2214. © 2023 Crown copyright and The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. This article is published with the permission of the Controller of HMSO and the King's Printer for Scotland.

Mitochondrial thermo-sensitivity in invasive and native freshwater mussels.

Climate change is impacting many, if not all, forms of life. Increases in extreme temperature fluctuations and average temperatures can cause stress, particularly in aquatic sessile ectotherms such as freshwater mussels. However, some species seem to thrive more than others in face of temperature-related stressors. Thermal tolerance may, for example, explain the success of invasive species. It is also known that mitochondria can play a key role in setting an ectothermic species' thermal tolerance. In this study, we aimed to characterize the mitochondrial thermo-tolerance in invasive and endemic freshwater mussels. With the use of high-resolution respirometry, we analyzed the mitochondrial respiration of two freshwater bivalve species exposed to a broad range of temperatures. We noticed that the invasive dreissenid Dreissena bugensis possessed a less thermo-tolerant mitochondrial metabolism than the endemic unionid Elliptio complanata This lack of tolerance was linked with a more noticeable aerobic metabolic depression at elevated temperatures. This decrease in mitochondrial metabolic activity was also linked with an increase in leak oxygen consumption as well as a stable maintenance of the activity of cytochrome c oxidase in both species. These findings may be associated both with the species' life history characteristics, as D. bugensis is more adapted to unstable habitats, in which selection pressures for resistance adaptations are reduced. Our findings add to the growing body of literature characterizing the mitochondrial metabolism of many aquatic ectotherms in our changing world.

Cumulative effects of cadmium and natural stressors (temperature and parasite infection) on molecular and biochemical responses of juvenile rainbow trout.

The simultaneous presence of natural and anthropogenic stressors in aquatic ecosystems can challenge the identification of factors causing decline in fish populations. These stressors include chemical mixtures and natural abiotic and biotic factors such as water temperature and parasitism. Effects of cumulative stressors may vary from antagonism to synergism at the organismal or population levels and may not be predicted from exposure to individual stressors. This study aimed to evaluate the combined effects of chronic exposure to cadmium (Cd) and elevated water temperature (23 °C) or parasite infection in juvenile rainbow trout (Oncorhynchus mykiss) using a multi-level biological approach, including RNA-sequencing. Fish were exposed to diet-borne Cd (6 µg Cd/g wet feed), individually and in combination with thermal (23 °C) or parasitic stressors, for 28 days. The parasite challenge consisted of a single exposure to glochidia (larvae) of the freshwater mussel (Strophitus undulatus), which encysts in fish gills, fins and skin. Results indicated lower fish length, weight, and relative growth rate in fish exposed to a higher water temperature (23 °C). Body condition and hepatosomatic index of trout were, however, higher in the 23 °C temperature treatment compared to the control fish kept at 15 °C. Exposure to thermal stress or parasitism did not influence tissue Cd bioaccumulation. More than 700 genes were differentially transcribed in fish exposed to the individual thermal stress treatment. However, neither Cd exposure nor parasite infection affected the number of differentially transcribed genes, compared to controls. The highest number of differentially transcribed genes (969 genes) was observed in trout exposed to combined Cd and high temperature stressors; these genes were mainly related to stress response, protein folding, calcium metabolism, bone growth, energy metabolism, and immune system; functions overlapped with responses found in fish solely exposed to higher water temperature. Only 40 genes were differentially transcribed when fish were exposed to Cd and glochidia and were related to the immune system, apoptosis process, energy metabolism and malignant tumor. These results suggest that dietary Cd may exacerbate the temperature stress and, to a lesser extent, parasitic infection stress on trout transcriptomic responses. Changes in the concentrations of liver ethoxyresorufin-o-deethylase, heat shock protein 70 and thiobarbituric acid reactive substances coupled to changes in the activities of cellular glutathione S-transferase and glucose-6-phosphate dehydrogenase were also observed at the cellular level. This study may help understand effects of freshwater fish exposure to cumulative stressors in a changing environment.

Stress-related gene transcription in fish exposed to parasitic larvae of two freshwater mussels with divergent infection strategies.

Freshwater unionoid mussels have a unique life cycle involving a temporary parasitic phase. Their larvae (glochidia) attach to the gills or fins of fish hosts where they remain encysted until metamorphosis into free-living juveniles. The physiological response of fish during the critical period of glochidial attachment is not well understood, but recent work suggests that glochidia retention and survival is enhanced in stressed and cortisol-injected hosts. In this study, the early changes induced by glochidiosis were investigated for the first time at the transcriptional level. In 2 separate experiments, juvenile yellow perch Perca flavescens were inoculated with glochidia of Elliptio complanata (a host generalist) and Lampsilis radiata (a host specialist) following a standardized procedure. The transcriptional levels of 5 genes involved in the fish response to stress were assessed in the host liver and gills 24 h post-infection using quantitative real-time PCR. The number of encysted glochidia did not significantly differ between fish inoculated with E. complanata and L. radiata. Both species induced a 3-fold increase of 70 kDa heat-shock protein gene (hsp70) transcription in host liver. However, only E. complanata influenced the transcription of cortisol-regulated genes, notably glucocorticoid receptor DNA-binding factor 1 (grlf1). This gene, known to modulate tissue responsiveness to cortisol, was downregulated in infected fish compared to controls. Our findings suggest that different glochidia species interact with their fish host in distinct ways. Additional studies are required to address this hypothesis and further investigate the significance of the observed host transcriptional responses.

The effect of cryopreservation on mitochondrial function in freshwater mussel tissue samples (Bivalvia: Unionida).

In non-thermoregulating and sessile organisms, such as the imperiled freshwater mussels (Bivalvia: Unionida), thermal sensitivity of mitochondria is a key factor for survival to global warming. Given the protected status of many unionids, non-destructive biopsies and subsequent cryopreservation are advisable procedures for further investigation of their mitochondrial function. To address whether long-term cryopreservation affects mitochondria in freshwater mussels, the mitochondrial respiration in permeabilized somatic cells of Elliptio complanata has been fully characterized through high-resolution respirometry. Our results indicate that cryopreservation does affect the absolute rate of respiration, which significantly decrease compared to fresh tissues, independently of substrates combination, respiratory state and normalizing factor. However, the negative impact is not reflected at the level of flux control ratios, suggesting that, even in front of a sharp decline in the aerobic capacity, cryopreserved tissues preserve the mitochondrial organization and could be thus employed for the qualitative analysis of mitochondrial function.

Cumulative effects of municipal effluent and parasite infection in yellow perch: A field study using high-throughput RNA-sequencing.

Multiple metabolic, immune and reproductive effects have been reported in fish residing in effluent-impacted sites. Natural stressors such as parasites also have been shown to impact the responses of organisms to chronic exposure to municipal effluent in the St. Lawrence River (Quebec, Canada). In order to comprehensively evaluate the cumulative impacts of anthropogenic and natural stressors on the health of yellow perch, differential mRNA transcription profiles were examined in juvenile females collected from effluent-impacted and upstream sites with low or high infection levels of the larval trematode Apophallus brevis. Transcriptomics was used to identify biological pathways associated with environmental exposure. In total, 3463 isoforms were differentially transcribed between sites. Patterns reflecting the combined effects of stressors were numerically dominant, with a majority of downregulated transcripts (68%). The differentially expressed transcripts were associated with 27 molecular and cellular functions ranging from cellular development to xenobiotic metabolism and were involved in the development and function of 13 organ systems including hematological, hepatic, nervous, reproductive and endocrine systems. Based on RNA-seq results, sixteen genes were measured by qPCR. Significant differences were observed for six genes in fish exposed to both stressors combined, whereas parasites and effluent individually impacted the transcription of one gene. Lysozyme activity, lipid peroxidation, retinol-binding protein and glucose-6-phosphate dehydrogenase were selected as potential biomarkers of effects to study specific pathways of interest. Lipid peroxidation in perch liver was different between sites, parasite loads, and for combined stressors. Overall, results indicated that juvenile yellow perch responded strongly to combined parasite and effluent exposure, suggesting cumulative effects on immune responses, inflammation and lipid metabolism mediated by retinoid receptors. The present study highlight the importance of using a comprehensive approach combining transcriptomics and endpoints measured at higher levels of biological organization to better understand cumulative risks of contaminants and pathogens in aquatic ecosystems.

Enigmatic decline of a common fish parasite (Diplostomum spp.) in the St. Lawrence River: Evidence for a dilution effect induced by the invasive round goby.

As they integrate into recipient food webs, invasive exotic species may influence the population dynamics of native parasites. Here we assess the potential impact of the Eurasian round goby (Neogobius melanostomus) on the abundance of eyeflukes of the genus Diplostomum, which are common parasites in fishes of the St. Lawrence River (Canada). Analyses of data collected over nearly two decades revealed that the infection levels in three native fish [spottail shiner (Notropis hudsonius), golden shiner (Notemigonus crysoleucas) yellow perch (Perca flavescens)] declined sharply throughout the St. Lawrence River after the introduction of the goby. At two sites where data were collected at regular time intervals, declines of Diplostomum spp. in spottail shiners occurred within two years of the goby's first recorded appearance, with prevalence dropping as much as 77-80% between pre-invasion and post-invasion periods. Furthermore, in localities where gobies remained scarce, infection in native species did not change significantly over time. Altogether, these observations suggest that gobies play a role in the eyefluke collapse. The decline in populations of the main definitive host (ring-billed gulls, Larus delawarensis) and changes in hydrology during periods of parasite recruitment were not strongly supported as alternate explanations for this phenomenon. Since other snail-transmitted trematodes with similar life cycles to Diplostomum spp. did not show the same decreasing pattern, we conclude that eyeflukes did not decline as a result of snail depletion due to goby predation. Rather, we suggest that gobies acted as decoys, diluting the infection. As Diplostomum spp. occurred at lower abundance in gobies than in native fish hosts, the replacement of native fish with exotic gobies in the diet of gulls might have played a part in reducing parasite transmission. In contrast to the typically negative impact of invasions, the goby-induced decline of this pathogen may have beneficial effects for native fishes.

Integrated spatial health assessment of yellow perch (Perca flavescens) populations from the St. Lawrence River, Quebec, Canada) part A: physiological parameters and pathogen assessment.

A multi-disciplinary approach was used to evaluate the health of yellow perch (Perca flavescens) in the St. Lawrence River (Quebec, Canada), which is experiencing a severe population decline in the downstream portion of the river. Physiological parameters, liver alterations, trace metal concentrations, parasite prevalence and abundance, stable isotope composition, and the presence/absence of the viral hemorragic septicemia virus (VHSV) were evaluated in perch collected at six sites along the river: Lake St. François, Lake St. Louis (north and south), Beauregard Island, and Lake St. Pierre (north and south). Trace metal concentrations in surface water were higher in Lake St. Louis and downstream of a major urban wastewater treatment plant discharge, indicating that this effluent was a significant source of Cu, As, Ag, Zn, and Cd. Levels of Pb in surface water exceeded thresholds for the protection of aquatic life in Lake St. Louis and were negatively correlated with body condition index in this lake. In Lake St. Pierre, Cu, Ag, and Cd bioaccumulated significantly in perch liver and lower body condition index and greater liver damage were observed compared to upstream sites. Parasite analyses indicated a higher abundance of metacercariae of the trematodes Apophallus brevis and Diplostomum spp. in Lake St. Louis, and VHSV was not detected in the liver of yellow perch for all studied sites. Overall, results suggested that the global health of yellow perch from Lake St. Pierre is lower compared to upstream studied sites, which could contribute to the documented population collapse at this site.

Variation in Parasite Communities in Spottail Shiners (Notropis hudsonius) Linked with Precipitation.

The Richelieu River, Quebec, is a highly-regulated waterway subject to numerous anthropogenic influences from municipal effluents and agricultural activities. Parasite communities in 234 spottail shiners (Notropis hudsonius) were examined from 4 localities in late spring 2003 and 2004. Component species richness varied between 15 and 18 species in 2003 but declined to 9 to 13 in 2004, while total parasite abundance was consistently lower in 2003. Parasite component community similarity among localities could not be directly linked to available upstream water quality measurements or anthropogenic activity and was best explained by precipitation. Total precipitation in May 2003 was approximately 40% more than in May 2004, presumably altering patterns of runoff, river flow rates, and water quality. This study suggests that fish parasite species composition and richness in the Richelieu River are influenced by environmental parameters which in turn ultimately are driven by a combination of climatic conditions and anthropogenic activities in the watershed.

Correlation of parasites with growth of yellow perch.

The possible influence of parasites on the short-term and long-term growth and condition of yellow perch Perca flavescens was examined by investigating correlations between parasite abundance and specific growth variables. The following parasites were enumerated in age-1 yellow perch collected from Lake St. Pierre in June 2008: Apophallus brevis, Diplostomum spp., Ichthyocotylurus spp., Tylodelphys scheuringi, Phyllodistomum superbum, and Raphidascaris acus. Short-term growth was estimated using RNA/DNA ratios and long-term growth via the total length and condition as measured by the Fulton index. No correlation was found between parasite abundance and short-term growth, but a negative influence of combined infections of T. scheuringi and P. superbum on long-term growth was detected. In addition, the abundance of Ichthyocotylurus spp. was positively correlated with the condition of the yellow perch. Together these results suggest that limited or subtle pathogenic effects in juvenile fish are not discernable in recent growth but only in long-term growth indices. Furthermore, in future studies examination of parasite effects on fish growth should account for multiple infections.

Impact of municipal effluents and hydrological regime on myxozoan parasite communities of fish.

Increased productivity from sewage effluents can enhance species richness locally. Results from a study of spottail shiners (Notropis hudsonius) in 1999 showed that prevalence and the mean number of myxozoan parasite species per host were higher downstream of the wastewater outflow from the Island of Montreal than upstream in the St. Lawrence River, Quebec, Canada. This was attributed to organic enrichment of the sediments which presumably lead to increased densities of oligochaetes, the alternate hosts, downstream of Montreal. Spottail shiners subsequently were collected every August/early September in 2001-2004 to examine the stability and repeatability of these patterns. Prevalence and mean number of myxozoan species per fish typically were again higher downstream of the sewage source each year compared to upstream, although there was no significant difference in these measurements pooled across years between localities immediately upstream and downstream of the effluent outflow. Density of the oligochaete Limnodrilus hoffmeistereri, a common alternate host of myxozoans, was much higher at two downstream localities than at an upstream one. At a larger spatial scale, mean myxozoan infracommunity richness across sites in the St. Lawrence River was negatively correlated with mean water levels measured in the 3 months prior to fish sampling. Results suggest that on a local scale, variations in prevalence and diversity among localities are influenced by municipal effluents, but that at a landscape scale annual variations across sites are affected by the hydrological regime and climate. In effect, water level fluctuation had a landscape-wide impact that was superimposed over pollution-induced local variations.

Gill and head kidney antioxidant processes and innate immune system responses of yellow perch (Perca flavescens) exposed to different contaminants in the St. Lawrence River, Canada.

Biomarkers of oxidative stress metabolism and the innate immune response were examined in gill and head kidney tissue of wild-caught yellow perch (Perca flavescens) collected from four sites ranging in type and degree of metal pollution in the St. Lawrence River, Quebec, Canada. Sites were ranked as follows: Ile Dorval

Short-term seasonal changes in parasite community structure in northern leopard froglets (Rana pipiens) inhabiting agricultural wetlands.

Parasite community structure can change seasonally with shifts in host habitat and in diet. However, anthropogenic activity may influence the natural changes in transmission dynamics of different parasite species. Effects of seasonal and agricultural activity on the parasite communities of newly metamorphosed northern leopard frogs (Rana pipiens) were investigated in July and September 2001 in 5 wetlands, 3 of which were exposed to pesticide runoff from surrounding agriculture. Nineteen parasite taxa were found. Component community richness was consistently high at the pristine reference wetland, whereas the communities at a managed reference wetland remained depauperate. Infracommunity richness increased throughout the season, but more so in frogs resident in agricultural wetlands. Digeneans using frogs as intermediate hosts dominated the communities, although many species were much lower in abundance in September, suggesting mortality of heavily infected frogs. Mean abundance of Haematoloechus spp. was positively related to that of odonate naiads in the frog diet, which appeared to reflect differential second intermediate host availability between reference and agricultural wetlands. Although virtually absent from wetlands in July just after frog metamorphosis, monoxenous nematodes were more prevalent and abundant at agricultural wetlands as the season progressed. Our results suggest that agricultural activity may further facilitate the transmission of monoxenous nematodes as frogs become more terrestrial.

Joint effects of parasitism and pollution on oxidative stress biomarkers in yellow perch Perca flavescens.

Yellow perch Perca flavescens were collected from a contaminated site and a reference site in the St. Lawrence River, Quebec, Canada. Fish were assessed for oxidative stress (lipid peroxidation and reduced glutathione levels) and parasitism by the nematode Raphidascaris acus and metacercariae of the digenean Apophallus brevis. Lipid peroxidation is not only considered a measure of oxidative stress, but of stress in general, and thus serves as an indicator of fish health. Fish from the contaminated site exhibited higher levels of lipid peroxidation than those from the reference site, independent of parasitic infections. However, fish infected with R. acus at the contaminated site tended to have higher levels of lipid peroxidation than uninfected fish at the same site, whereas no difference was observed between infected and uninfected fish at the reference site. Yellow perch infected with > 10 metacercariae of A. brevis expressed higher levels of lipid peroxidation than those infected with < 10 metacercariae at both the contaminated and the reference sites. No differences were found in levels of reduced glutathione in liver or muscle in relation to site or either parasite species. Results support the use of lipid peroxidation as a biomarker of water contamination. They further suggest that lipid peroxidation may be used as a biomarker of pathological effects caused by parasitism. Most importantly, results demonstrate that contaminants and parasites occurring together exacerbate oxidative stress in fish, suggesting that parasitized fish in polluted environments are in a poorer state of health than uninfected fish.

Effects of agricultural pesticides on the immune system of Rana pipiens and on its resistance to parasitic infection.

In the past 30 years, many amphibian species have suffered population declines throughout the world. Mass mortality have been frequently reported, and in several instances, infectious diseases appear to be the cause of death. The role that contaminants could play in these die-offs through immunotoxic effects has been poorly investigated. In this study, juvenile leopard frogs (Rana pipiens) were exposed for 21 d to a mixture of six pesticides (atrazine, metribuzin, aldicarb, endosulfane, lindane, and dieldrin) and subsequently challenged with a parasitic nematode, Rhabdias ranae. Exposure to the mixture at environmentally realistic concentrations significantly reduced lymphocyte proliferation. Three weeks after the end of the exposure, lymphocyte proliferation had recovered and was stimulated in frogs challenged with parasites with the exception of those previously exposed to the highest concentration. No pesticide effects on phagocytosis and splenocyte numbers were detectable at the end of the exposure period, but these two parameters were diminished 21 d after the infection challenge in frogs previously exposed to the highest levels of pesticides. In these animals, the prevalence of lung infection by R. ranae also tended to be higher. These results suggest that agricultural pesticides can alter the immune response of frogs and affect their ability to deal with parasitic infection.