Parasites play a critical yet understudied role in Arctic ecosystems, particularly within key subsistence species such as Arctic char (Salvelinus alpinus). As the Arctic undergoes rapid climate-driven change, establishing baseline parasite biodiversity is essential for monitoring ecological shifts and anticipating emerging health and conservation concerns. This study provides the most comprehensive assessment to date of Arctic char parasites in the North American Arctic by integrating a systematic literature review with new empirical data from 378 Arctic char collected across five Nunavut areas (2020–2024). The literature review (30 studies, 1956–2024) documented 67 parasite taxa—48 identified to species—with helminths (nematodes, cestodes, trematodes, acanthocephalans) dominant. Overall, 82

Climate-driven increases in mean water temperature and the frequency of heatwaves affect the thermal tolerance of ectotherms, including fishes. Fishes can increase their thermal tolerance through acclimation, optimizing survival during extreme weather events. Few studies have investigated the exposure duration necessary for individuals to acclimate to warmer conditions or differences in acclimation dynamics among populations, limiting our understanding of how species deal with acute heat stress. Warmer waters can also increase parasite transmission. Although infections can reduce host thermal tolerance, the impact of parasite prevalence and abundance on fish acclimation capacity has not been explored, and may help explain population-level differences in thermal tolerance. We assessed thermal tolerance and acclimation dynamics across three populations of pumpkinseed sunfish (Lepomis gibbosus) from lakes in Quebec, Canada, differing in trematode and cestode infection prevalence. Pumpkinseed were acclimated to 22 °C or 27 °C from 3 h to 60 days before measuring critical thermal maximum (CTmax). CTmax increased with acclimation duration, with detectable increases after only 3 h, indicating rapid induction of acclimatory mechanisms. Fish from all populations appeared to reach full acclimation after 10 days at 27 °C. However, thermal tolerance was consistently highest in the control lake (Lake Triton, no cestodes or trematodes) compared to populations with intermediate (Lake Croche) and high (Lake Cromwell) infection prevalence, despite no relationship with parasite abundance. Although our design does not permit causal inference, these results suggest pumpkinseed rapidly acclimate to higher temperatures, but natural exposure to parasites could contribute to population-level differences in thermal tolerance.

Aquatic environments are often thermally variable, and ectotherms may select thermal habitats based on their physiological state. However, it remains unclear whether immune reactions and sickness behaviours, including behavioural fever or chill, drive temperature preference and what role natural temperature shifts play in thermal choice. We used a shuttle box system to determine temperature preference in wild-caught pumpkinseed sunfish, Lepomis gibbosus, that were: (i) naturally co-infected by parasitic helminths; (ii) experimentally infected with trematodes, or (iii) immune challenged through lipopolysaccharide (LPS) endotoxin injection. We found no significant differences in preferred temperatures or activity levels based on parasite infection intensity, experimental trematode exposure or LPS injection, despite significant among-individual variation in temperature preference. However, LPS-injected fish caught in 2021 preferred higher temperatures compared with those caught in 2022, reflecting warmer water temperatures experienced in June and July of 2021. Additionally, fish from the natural infection experiment captured over three weeks in July 2021 showed increased preferred temperatures that paralleled rising water temperatures in their natural habitat. Our results suggest that variability in temperature preference reflects natural temperature shifts rather than infection status. Ultimately, this finding enhances our understanding of individual microhabitat choice while challenging the concept of behavioural fever/chill as a thermoregulatory strategy in sunfish.This article is part of the theme issue 'Embracing variability in comparative physiology: why it matters and what to do with it'.

Despite their vital role in ecosystem health and biodiversity, parasites are often overlooked or misunderstood by the public, scientists, and managers. One misconception is that parasites should be eradicated rather than studied or conserved. Thus, our understanding of many parasite species, including their impacts on hosts and ecosystems, is limited, making it difficult to predict how infection dynamics may shift in a warming world. This lack of knowledge is particularly concerning in Canadian aquatic systems, where fishes, both culturally and economically important species, are increasingly threatened. Synthesizing knowledge of host–parasite interactions in Canadian fishes can help identify areas in greater need of investigation and establish a baseline for monitoring disease dynamics over time and space. Focusing on freshwater fishes in Quebec, this perspective assesses the current state knowledge of metazoan fish parasites and highlights species and geographic locations in need of further research. We also explore how emerging threats—including climate change, invasive species, range shifts, and disease spillover—may impact host–parasite relationships. This work offers a foundation for similar studies across Canada and beyond.

Literature syntheses link knowledge across study systems and regions. However, specific details on the biological systems of the included publications may not always be explicitly outlined because of communication gaps (a.k.a. The Curse of Knowledge) and/or knowledge gaps. These gaps can lead to inappropriate comparisons across studies if researchers unfamiliar with the systems make inaccurate assumptions about aspects of study design or natural history. In the context of movement ecology, a growing number of publications explore how host infection and host migration intersect, work that is critical for predicting how global change will impact both animal movement and parasite transmission. Given the complexity of host and parasite life histories, these studies may be particularly vulnerable to knowledge and communication gaps. To minimize communication gaps and highlight knowledge gaps, we developed a checklist with 15 specific elements separated into three categories about migration, parasites, and sampling that should be addressed within a publication to provide a more complete picture of the study system for a non-specialist. We validate our checklist in three ways. First, we use two worked examples to show the information each element reveals. Second, we apply our checklist to a database of 36 publications on migrating infected hosts to quantify which details are typically missing from the published literature. Third, we create a try-it-yourself example based on a fictional study system to illustrate the challenges associated with extracting information on an unknown system. All but one of the publications assessed were missing information on at least one checklist element and 11 were missing more than half. Our checklist is therefore a tool that researchers can use to broaden the impact of their studies and facilitate cross-species comparisons and syntheses. We hope this work stimulates interest in the value of reporting guidelines and encourages researchers to apply this approach to other aspects of animal ecology that might similarly suffer from the Curse of Knowledge.

Climate change is shifting the aerobic capacity of aquatic ectotherms, affecting their ability to move efficiently through their environment. Rising temperatures also alter host-parasite interactions, yet how these stressors interact to impact locomotion remains unclear. This is especially relevant for infections that disrupt streamlining and fin function, with implications for wild fish populations, aquaculture, and fisheries. Pumpkinseed sunfish (Lepomis gibbosus (Linnaeus, 1758)), a popular recreational fishing species, are naturally co-infected with trematodes, forming rigid cysts on fins and body, and cestode tapeworms, infecting the liver and digestive tract. We tested whether pumpkinseed swimming performance is affected by drag from cysts by measuring critical swimming speed (Ucrit) and aerobic metabolic traits in naturally infected fish and fish treated to remove cestodes. Individuals with more cysts had lower Ucrit, maximum metabolic rate (MMR) and aerobic scope, likely due to increased drag. Next, we acclimated wild-caught, co-infected fish to 20, 25, and 30 degrees C and measured Ucrit and MMR. Warmer temperatures increased both metrics, and internal parasites were related to reduced MMR and Ucrit. Overall, infections can impair swimming by increasing drag and through physiological effects, but warming does not appear to exacerbate these effects in species not living near their thermal limits.

Abstract Critical thermal limits, commonly quantified as CTmax (maximum) or CTmin (minimum), are core metrics in the thermal biology of aquatic ectotherms. CTmax, in particular, has recently surged in popularity due to its various applications, including understanding and predicting the responses of animals to climate warming. Despite its growing popularity, there is a limited literature aimed at establishing best practices for designing, running and reporting CTmax experiments. This lack of standardisation and insufficiently detailed reporting in the literature creates challenges when designing CTmax studies or comparing results across studies. Here, we provide a comprehensive, practical guide for designing and conducting experiments to measure critical thermal limits, with an emphasis on CTmax. Our recommendations cover 12 topic areas including apparatus design, masking (blinding), warming rates, end points, replication and reporting. We include diagrams and photos for designing and building critical thermal limit arenas for field or lab applications. We also provide a reporting checklist as a reference for researchers when carrying out experiments and preparing manuscripts. Future studies incorporating critical thermal limits would benefit from transparent reporting of warming/cooling rates (raw data, supplementary graphs) and photo/video evidence showing arena designs and critical thermal limit end points. We also provide directions for empirical research that will help further inform the measurement of critical thermal limits, including biotic factors like stress and digestion, warming/cooling rates, the effects of body mass on heat transfer and the physiological mechanisms underlying thermal tolerance.

In the field of conservation physiology, there is often a trade off between conducting research in controlled laboratory settings or in inherently variable field environments. However, this belief sets up a false dichotomy where laboratory experiments are perceived as providing precise, mechanistic understanding with low variability at the cost of environmental realism while field studies are ecologically relevant but criticized for generating inconsistent evidence that is difficult to interpret and replicate. Despite the perceived binary view, these approaches are not in opposition to one another, but rather form a continuum along increasing ecological complexity. Here, we argue that it is possible to mindfully and purposefully design studies and develop integrative collaborations in conservation physiology that span the lab-field continuum to address pressing environmentally-relevant questions that can be used to inform policy and practice. We first outline the advantages and disadvantages of different approaches to knowledge generation. We then highlight ways to bridge the lab-field divide though leveraging the advantages provided by different approaches to build a more comprehensive understanding of the natural world, including how recent technological advances can help connect lab- and field-based research. Next, we discuss the importance of partnership and collaboration across sectors for informing our understanding of ecological patterns and physiological processes. Finally, we reflect on how to best translate physiological research into action and the reciprocal role that environmental practitioners can have in driving research questions in conservation physiology.

The need for data rescue. Data are dying all around us: when they are stored in inaccessible or fragile repositories, in proprietary formats, on defunct media or without complete metadata. In environmental science, such data loss has high societal and scientific costs. For example, failure to archive ecological data focused on the Exxon Valdez oil spill represents an estimated loss of >100 million USD (Bledsoe et al. 2022). Loss of environmental data can also represent erasure of historic baselines that can never be recollected. The Living Data Project ( LDP ). This award-winning program helps scientists and organizations archive valuable “legacy” datasets, making them permanently open and accessible (Bledsoe et al. 2022). We pair data custodians with graduate students who we train in reproducible data management and provide mentoring by postdoctoral researchers and faculty and assistance by undergraduates. By training Canada’s next generation of scientists, we aim to ensure that data are never lost again. Over the last five years, we have trained 305 students, across 93 internships, rescuing > 2000 years of data. LDP student working groups then analyze archived data to investigate pressing biodiversity questions and develop open access teaching resources. Benefits of data rescue. LDP-rescued datasets, many stretching back into the last century, provide missing historical baselines that enable the detection of environmental change – such as change in lake ice formation and melt over the last 150 years. Rescue of historical datasets, like surveys of urban birds or plant communities collected five decades ago, sets the stage for contemporary re-surveys. Combining rescued data with contemporary data can also yield discoveries such as legacy effects of pesticides on stream health (Sugden et al. 2025). LDP rescue of classic studies in ecological and evolutionary theory (Darwin’s Finches, Serengeti ungulates, and freshwater sticklebacks) enables new analyses that were computationally impossible at the time. LDP projects help environmental organizations archive data over broad spatial and temporal scales, critical for metrics such as the Watershed Reports or Living Planet Index. LDP also assists non-professionals to archive data; e.g., helping communities archive water quality data on DataStream enables easy comparison to national standards. Data rescue can knit together disparate datasets in relational databases, such as LDP compilation of fish, invertebrate, phytoplankton, and limnology data from the Canadian government’s Turkey Lakes Watershed research. The benefits of data rescue will pay incalculable dividends for decades to come. However, to ensure successful data rescue, we recommend the following: Rules for success: Prevent scope creep. When non-essential data is included in the initial scope of the project or the scope broadens during the rescue process, concluding the project is challenging. It is better to successfully archive a subset of the data than fail to archive any. Involve data collectors and custodians throughout. They provide important contextual knowledge, help decipher obscure data codes and can suggest constraints to values, facilitating validation. Formalizing their knowledge in the metadata ensures future usability. Continually champion projects until the dataset DOI is minted. It is easy for projects to stall in the final stages. Delaying archiving to add more years or variables or to publish one more paper increases the risk of no data being archived. Encourage the use of versioning or embargo periods, available for many repositories, to deposit promptly the core data while enabling later additions and public release. Manage adaptively. Problems like contradictory data, inconsistent use of terms, and heterogeneous collection methods may only be apparent after working with the data. At the LDP, we use high-level checks of progress throughout each rescue project to adaptively manage the scope and personnel needed, informed by our experience in a wide variety of projects. Prevent scope creep. When non-essential data is included in the initial scope of the project or the scope broadens during the rescue process, concluding the project is challenging. It is better to successfully archive a subset of the data than fail to archive any. Involve data collectors and custodians throughout. They provide important contextual knowledge, help decipher obscure data codes and can suggest constraints to values, facilitating validation. Formalizing their knowledge in the metadata ensures future usability. Continually champion projects until the dataset DOI is minted. It is easy for projects to stall in the final stages. Delaying archiving to add more years or variables or to publish one more paper increases the risk of no data being archived. Encourage the use of versioning or embargo periods, available for many repositories, to deposit promptly the core data while enabling later additions and public release. Manage adaptively. Problems like contradictory data, inconsistent use of terms, and heterogeneous collection methods may only be apparent after working with the data. At the LDP, we use high-level checks of progress throughout each rescue project to adaptively manage the scope and personnel needed, informed by our experience in a wide variety of projects. Summary. Environmental data rescue has many benefits, including establishing baselines and change in the environment, enabling new analyses and composite indices, connecting disparate data, facilitating community science, and training the next generation of researchers in open science. However, data rescue projects requires continued and careful management to ensure success, which we defined as the deposition of data in open, accessible and permanent repositories.

Who conducts biological research, where they do it and how results are disseminated vary among geographies and identities. Identifying and documenting these forms of bias by research communities is a critical step towards addressing them. We documented perceived and observed biases in movement ecology, a rapidly expanding sub-discipline of biology, which is strongly underpinned by fieldwork and technology use. We surveyed attendees before an international conference to assess a baseline within-discipline perceived bias (uninformed perceived bias). We analysed geographic patterns in Movement Ecology articles, finding discrepancies between the country of the authors' affiliation and study site location, related to national economics. We analysed race-gender identities of USA biology researchers (the closest to our sub-discipline with data available), finding that they differed from national demographics. Finally, we discussed the quantitatively observed bias at the conference, to assess within-discipline perceived bias informed with observational data (informed perceived bias). Although the survey indicated most conference participants as bias-aware, conversations only covered a subset of biases. We discuss potential causes of bias (parachute-science, fieldwork accessibility), solutions and the need to evaluate mitigatory action effectiveness. Undertaking data-driven analysis of bias within sub-disciplines can help identify specific barriers and move towards the inclusion of a greater diversity of participants in the scientific process.

Developing non-lethal techniques to estimate parasite infection is critical for studying disease ecology in wild animals. We tested the effectiveness of coelomic ultrasonographic examination and plasma enzyme markers to detect liver infection with bass tapeworms Proteocephalus ambloplitis (Leidy 1887) as well as the effectiveness of ultrasound in predicting fish sex based on gonad imaging ante mortem in two populations of pumpkinseed sunfish Lepomis gibbosus (L. 1758). We also conducted cytopathological and histopathological analyses on a small subset of fish to investigate the potential for these techniques to detect signs of infection and liver disease. We found that fish sex was correctly identified by ultrasound in 87% of fish screened. There was no statistically significant relationship between parasite density and plasma enzyme concentration in infected fish. However, there were clinical differences between individuals from uninfected and infected populations in the enzymes creatine kinase and alanine transaminase. Histopathology and cytopathology assays confirmed the presence of macrophages and clear signs of inflammation within the liver of infected fish. Our results demonstrate that ultrasound, while useful for sex determination, was not effective in detecting infection in small species like sunfish. However, techniques such as blood analysis and potentially cytopathology are promising tools for parasitic detection in L. gibbosus and warrant further investigation, especially for use in other larger species.

Supply and demand affect the values of goods exchanged in cooperative trades where high demand typically leads to a higher price. An exception has been described in the marine cleaning mutualism involving the cleaner fish Labroides dimidiatus and its variety of ‘client’ coral reef fishes. Cleaner fish feed on clients’ ectoparasites but prefer eating clients’ mucus instead, which constitutes cheating. Here, we provide field observations, followed by a set of laboratory experiments with real clients and Plexiglas feeding plates as surrogates for clients. In the field and in three experiments with real clients, we found that satiated cleaner fish were more cooperative, even though low hunger levels should make them less dependent on cleaning interactions. Similarly, the more abstract version of the experiments using Plexiglas plates offering two food types mimicking client ectoparasites and mucus showed that satiation led cleaner fish to feed more against their preferences – an indicator of cooperative behaviour. However, this outcome occurred only if the temptation to eat the preferred food was low. When the temptation to cheat was high, cleaners did so. We provide further general support to these findings with a game-theoretic model. Many mutualisms involve food as a commodity. Thus, identifying foraging decision rules will enhance our understanding of how individuals adjust to real-time market conditions rather than playing evolved strategies adapted to the average market conditions.

Climate warming with associated heat waves presents a concerning challenge for ectotherms such as fishes. During heatwaves, the ability to rapidly acclimate can be crucial for survival. However, surprisingly little is known about how different species and life stages vary in their acclimation dynamics, including the magnitude of change in thermal tolerance through acclimation (i.e. acclimation capacity; also known as the acclimation response ratio, ARR), the duration needed for the novel acclimation temperature to significantly alter thermal tolerance from the initial level (which we term the response induction time, tinduction), or the duration needed to achieve the new acclimation steady state (which we term the time to full acclimation, tsteady). To shed light on this knowledge gap, we studied the acclimation dynamics of three wild-caught fishes (goldsinny wrasse, threespined stickleback and European flounder) by assessing upper thermal tolerance (CTmax) after different periods of time acclimating to a warmed environment. We also measured both CTmax and lower thermal tolerance (CTmin) in juvenile and adult lab-bred zebrafish acclimated to a warmed environment. Upper thermal tolerance of zebrafish and sticklebacks significantly increased after a 3 h exposure to a warm treatment, while tinduction took six and 24 h in the wrasse and flounder, respectively. Goldsinny wrasse had the highest ARR, and did not reach full acclimation of CTmax within the duration of the study (10 days). All other species fully acclimated within 4-10 days. Juvenile zebrafish showed similar acclimation dynamics to adults for both upper and lower thermal tolerance, but had a higher CTmin for all acclimation durations. Our results demonstrate that acclimation dynamics of thermal tolerance vary across species, but can be similar between life stages within species. Understanding species-specific thermal plasticity is important for accurately modeling the projected impacts of climate change.

Exposure to environmental stressors can induce physiological responses in organisms, which can lead to population-level differences in physiological traits. However, the mechanisms underlying these responses both within individuals and among populations are often unknown. Parasite infections, in particular, are important biotic stressors that can induce a range of effects in hosts, including altered whole-organism metabolism. However, few studies have explored sub-cellular alterations to metabolic performance across both an individual infection gradient and population-level differences in infection prevalence. We compared mitochondrial enzyme activities across five distinct populations of pumpkinseed sunfish ( Lepomis gibbosus ) differing in the prevalence of cestode and trematode parasites. Overall, we found that enzymes from the OXPHOS pathway (cytochrome c oxidase, NADH dehydrogenase and coenzyme Q : cytochrome c oxidoreductase), the anaerobic pathway (lactate dehydrogenase), the TCA cycle (citrate synthase) and lipid metabolism (carnitine palmitoyl transferase) were positively correlated with parasite density with trends depending on the organ, parasite species and host population studied. Our results suggest complex and nuanced relationships among infections and host physiological performance and provide strong evidence of parasite-induced alterations to mitochondrial metabolism which may explain previously reported whole-organism responses to infections. ### Competing Interest Statement The authors have declared no competing interest. NSERC, 2018-05692, BESC D-589480-2024 FRQNT, 2023-2024-B1X-326015 Groupe de recherche interuniversitaire en limnologie (GRIL), GRIL-PCR-22A07 Canada Research Chairs, Mitochondrial Evolutionary Biology, Eco-Evolution of Host-Parasite Interactions

Fishes can acclimate to a range of temperatures. However, the signalling factors controlling thermal acclimation are not well understood. Here, in two experiments, we examined the putative roles of plasma-borne factors (e.g. hormones) and skin thermoreception in the acclimation process. In experiment 1, 16°C-acclimated Atlantic cod (Gadus morhua) were subjected to a transfusion treatment by injecting plasma from 8°C (cold), 16°C (control) or 21°C (warm) acclimated cod, 10 times over four days. Plasma was collected from donor cod that were 24 h into their acclimation. In experiment 2, 16°C-acclimated goldsinny wrasse (Ctenolabrus rupestris) were exposed to an immersion treatment consisting of 10 s immersions in an 8°C (cold), 16°C (control) or 24°C (warm) water bath, repeated five times daily for five days. These brief immersions allowed for changes to skin temperature but not deeper tissues. Following these treatments, we measured the critical thermal maximum (CTmax) of all fish and the standard metabolic rate (SMR) in cod. Neither the immersions nor transfusions affected fish CTmax. However, the SMR was elevated in cod receiving plasma from cold-acclimated donors, suggesting that circulating molecules transferred from donors had initiated metabolic compensation in recipients. Thyroid hormone plasma levels were not different amongst acclimated donors and thus appear not to have been involved in the metabolic compensation. Our experiments found no evidence that brief, repeated cutaneous exposures to temperature changes can trigger acclimation, but do demonstrate a potential role of haematological endocrine control in metabolic acclimation, although further experiments will be required to investigate this process.

The characteristics of infected animals, including their odour, appearance, behaviour and sound, greatly differ from those of uninfected conspecifics. These differences can serve as cues to recognize and avoid infected individuals and minimize the risk of infection. Avoidance of infected conspecifics is a risksensitive behaviour that can be influenced by various factors, including sensory cues, which are poorly understood in fish. We investigated the ability of two populations of wild-caught pumpkinseed sunfish, Lepomis gibbosus, to distinguish between conspecifics infected with parasitic worms and uninfected individuals in two-choice experiments using visual and chemical cues separately. One population was from a lake without parasites (parasite-na & iuml;ve), whereas the second population originated from a lake with a high prevalence of trematode and cestode worms (parasite-experienced). Both populations preferred to be with conspecifics, regardless of their infection level, over being alone when given visual cues but avoided conspecifics and preferred to be alone when given chemical cues, suggesting that visual and chemical cues are not redundant. Fish from neither population showed a preference between infected and uninfected conspecifics when given visual cues. However, there was a large interindividual variation in preference for chemical cues: some fish preferred uninfected conspecifics, whereas others preferred infected fish. On average, na & iuml;ve fish avoided infected conspecifics, whereas experienced fish did not show any preferences, suggesting that fish from lakes with high prevalence of infection are habituated to infection cues. We suggest that pumpkinseeds use chemical rather than visual cues to discriminate between infected and uninfected conspecifics during shoaling decisions. Our study highlighted the importance of considering different sensory cues as well as environmental parasite abundance when studying avoidance and shoaling behaviours, especially considering the growing impacts of global changes on the sensory landscape and parasite abundance in freshwater ecosystems. (c) 2024 The Authors. Published by Elsevier Ltd on behalf of The Association for the Study of Animal Behaviour. This is an open access article under the CC BY license (http://creativecommons.org/licenses/ by/4.0/).

The role of parasites in maintaining consistent inter-individual differences in behavior (ie personality) is the subject of increasing study and debate. While behavioral differences may expose individuals differently to parasites, parasite infection can itself change host behavior, sometimes favoring the parasite's own transmission. Furthermore, parasites can alter the functioning of energetically costly organs like the brain, thus affecting cognitive performance. However, relationships among infection, cognition, and behavior can be complex and difficult to interpret, especially in wild populations where individual health status is unknown. The inclusion of body condition as a fitness proxy may help reveal relationships between parasites and host traits that are otherwise masked. We examined relationships among host body condition, personality (ie exploration, boldness), cognition (ie aversive learning) and parasite density in wild-caught pumpkinseed sunfish (Lepomis gibbosus) naturally infected with endoparasites. Exploration in an open field test was repeatable in sunfish. Boldness, assessed using a shelter test, was not repeatable, but was correlated with exploration. Host exploration decreased with both increasing parasite density and decreasing body condition. Only individuals in relatively lower body condition displayed a negative relationship between parasite density and exploration, suggesting a pathologic effect of the parasites on the sunfish. Aversive learning was not influenced by co-infection. Our results show that body condition is important to consider when studying wild populations as some patterns observed between parasite density and host behavior were only revealed when body condition was taken into consideration.

Parasites can impair host performance including alterations to host metabolism. Since mitochondria are responsible for cellular energy production, it is likely that disruptions in cellular metabolism contribute to changes at the whole-organism level. However, studies investigating parasite-induced alteration do not take the presence of parasites in the target tissues into account. It is critical to assess the potential interference of parasite contamination in biochemical assays of host tissues to have confidence in the results measured from infected animals. Our objectives were to (1) develop and validate a protocol for extracting parasites from hepatic tissues of pumpkinseed sunfish (Lepomis gibbosus Linnaeus, 1758) infected with cestodes (Proteocephalus ambloplitis Leidy, 1887) and (2) to evaluate the extent to which parasite contamination impacts estimates of enzyme activities. We tested five treatments: uninfected lake, infected livers that were cleaned, infected livers with parasites (repopulated), uninfected livers from an infected lake and parasites alone. We compared the activity of metabolic enzymes among groups and assessed contamination through PCR tests. Enzyme activities of cleaned livers and repopulated livers were similar despite PCR tests revealing contamination in cleaned livers suggesting that studying the organs of heavily parasitized individuals is possible. Nevertheless, we recommend that our cleaning protocol is applied to infected organs.

Parasite occurrence and infection estimates vary through time and space, making understanding the underlying drivers highly complex. Comparative studies based on empirical data must consider the factors of variation involved in estimating infection metrics in natural populations to make appropriate and reliable comparisons. Using a multi‐scale approach, we explored the sources of variation in the estimation of infection prevalence, focusing on black spot disease in littoral freshwater fish communities sampled across 15 lakes in Québec, Canada. Method‐related sampling biases led to significant variations in prevalence estimates and spatial patterns of disease occurrence. Our results also indicated that low sampling efforts tend to overestimate the prevalence of infection in the landscape, with minimum sampling effort required to estimate an accurate infection prevalence depending on the sampling method employed. Our results showed that infection prevalence is spatially heterogeneous across the landscape with evidence of infection hotspots and coldspots. Physico‐chemical characteristics of the sites and local fish community structure were found to be the best drivers of infection at smaller spatial scales. Furthermore, our results suggest dilution effects, due to physical obstruction and compatibility barriers, limit the survival of the free‐living cercaria parasite life stage. Several relationships between infection prevalence and environmental drivers revealed non‐linearity, suggesting complex interactions. Examining infection prevalence data at various spatial scales revealed method‐induced biases, sampling effort effect, and environment‐driven relationships underscoring the importance of context‐dependencies and scale‐dependencies in empirical studies on host–parasite interactions.