First isolation of Flavobacterium psychrophilum from wild adult Great Lakes lake whitefish (Coregonus clupeaformis).

Lake whitefish (Coregonus clupeaformis; LWF) is an economically and ecologically valuable native species to the Great Lakes, but recent declines in their recruitment have generated significant concern about their future viability. Although studies have sought to identify factors contributing to declining recruitment, the potential role(s) of infectious diseases has not been thoroughly investigated. In 2018 and 2019, adult LWF were collected from Lakes Superior, Michigan, and Huron for clinical examination and bacteriological analyses. Herein, we describe the first isolation of Flavobacterium psychrophilum, aetiological agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), from systemically infected adult LWF. Bacterial isolates were yellow-orange, Gram-negative, filamentous bacilli that were oxidase and catalase positive, and produced a flexirubin-type pigment in 3% potassium hydroxide. Isolate identity was confirmed via F. psychrophilum-specific PCR, and multilocus sequence typing revealed three new singleton sequence types (STs) that were distinct from all previously described F. psychrophilum STs. The prevalence of F. psychrophilum infections was 3.3, 1.7, and 0.0% in Lakes Superior, Michigan and Huron respectively. Findings illustrate the potential for F. psychrophilum to cause systemic infections in adult LWF and highlight the need for future studies to investigate the bacterium's potential role in declining LWF recruitment.

In Vivo Experiments Provide Evidence That Flavobacterium psychrophilum Strains Belonging to Multilocus Sequence Typing Clonal Complex ST191 Are Virulent to Rainbow Trout.

Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease (BCWD), causes significant economic losses worldwide, particularly in farmed Rainbow Trout Oncorhynchus mykiss. Over the last decade, multilocus sequence typing has revealed >30 clonal complexes (CCs) globally, comprised of >320 F. psychrophilum sequence types (STs). Despite the large number of CCs worldwide, CC-ST10, which is currently the largest CC affecting Rainbow Trout, has been the primary focus of F. psychrophilum virulence studies, leaving the role of other CCs as primary causes of BCWD epizootics unclear. To this end, fingerling Rainbow Trout were experimentally challenged with F. psychrophilum strains belonging to the CC now recognized as the second largest in the world (CC-ST191) alongside CC-ST10 strains. Cumulative percent mortality was 100% in 7-month-old Rainbow Trout and between 27.8% and 61.1% in 8-month-old Rainbow Trout. All examined F. psychrophilum STs were virulent to Rainbow Trout, and no significant differences in virulence between CC-ST10 and CC-ST191 were detected. Due to their wide distribution and high pathogenic potential, both CC-ST191 and CC-ST10 F. psychrophilum strains are excellent candidates for further research aimed at preventing and controlling BCWD.

Host-specific preference of some Flavobacterium psychrophilum multilocus sequence typing genotypes determines their ability to cause bacterial coldwater disease in coho salmon (Oncorhynchus kisutch).

Flavobacterium psychrophilum causes bacterial coldwater disease (BCWD) in salmonids, resulting in significant losses worldwide. Several serotyping and genetic studies of F. psychrophilum have suggested some geno-/serotypes may be either host-specific or generalistic in nature; however, this association has not been adequately explored in vivo using more natural exposure routes. Herein, F. psychrophilum isolate US19-COS, originally recovered from coho salmon (Oncorhynchus kisutch) and belonging to multilocus sequence typing clonal complex (CC) CC-ST9, and isolate US53-RBT, recovered from rainbow trout (Oncorhynchus mykiss) and belonging to CC-ST10, were serotyped via PCR, evaluated for proteolytic activity and utilized to determine their median lethal dose in immersion-challenged coho salmon fingerlings. US19-COS belonged to serotype 0, hydrolysed casein and gelatin but not elastin, led to fulminant multiorgan infections and elicited severe gross and microscopic pathology. In contrast, US53-RBT, belonging to serotype 2, hydrolysed all three substrates, but did not lead to detectable infections, disease signs or mortality in any exposed coho salmon despite proving virulent to rainbow trout in previous experiments. This study provides in vivo evidence for potential host specificity of some F. psychrophilum genotypes that can also be serologically distinct, a matter of importance towards better understanding F. psychrophilum disease ecology and epidemiology.

Temporal variation in total phosphorus concentrations revealed from a multidecadal monitoring program on Big Platte Lake, Michigan.

Effective water quality management depends on enactment of appropriately designed monitoring programs to reveal current and forecasted conditions. Because water quality conditions are influenced by numerous factors, commonly measured attributes such as total phosphorus (TP) can be highly temporally varying. For highly varying processes, monitoring programs should be long-term and periodic quantitative analyses are needed so that temporal trends can be distinguished from stochastic variation, which can yield insights into potential modifications to the program. Using generalized additive mixed modeling, we assessed temporal (yearly and monthly) trends and quantified other sources of variation (daily and subsampling) in TP concentrations from a multidecadal depth-specific monitoring program on Big Platte Lake, Michigan. Yearly TP concentrations decreased from the late 1980s to late 1990s before rebounding through the early 2000s. At depths of 2.29 to 13.72 m, TP concentrations have cycled around stationary points since the early 2000s, while at the surface and depths ≥ 18.29 concentrations have continued declining. Summer and fall peaks in TP concentrations were observed at most depths, with the fall peak at deeper depths occurring 1 month earlier than shallower depths. Daily sampling variation (i.e., variation within a given month and year) was greatest at shallowest and deepest depths. Variation in subsamples collected from depth-specific water samples constituted a small fraction of total variation. Based on model results, cost-saving measures to consider for the monitoring program include reducing subsampling of depth-specific concentrations and reducing the number of sampling depths given observed consistencies across the program period.

Laboratory investigation into the role of largemouth bass virus (Ranavirus, Iridoviridae) in smallmouth bass mortality events in Pennsylvania rivers.

BACKGROUND: Mortality episodes have affected young-of-year smallmouth bass (Micropterus dolomieu) in several river systems in Pennsylvania since 2005. A series of laboratory experiments were performed to determine the potential role of largemouth bass virus (Ranavirus, Iridoviridae) in causing these events. RESULTS: Juvenile smallmouth bass experimentally infected with the largemouth bass virus exhibited internal and external clinical signs and mortality consistent with those observed during die-offs. Microscopically, infected fish developed multifocal necrosis in the mesenteric fat, liver, spleen and kidneys. Fish challenged by immersion also developed severe ulcerative dermatitis and necrotizing myositis and rarely panuveitis and keratitis. Largemouth bass virus-challenged smallmouth bass experienced greater mortality at 28 °C than at 23 or 11 °C. Co-infection with Flavobacterium columnare at 28 °C resulted in significant increase in mortality of smallmouth bass previously infected with largemouth bass virus. Aeromonas salmonicida seems to be very pathogenic to fish at water temperatures < 23 °C. While co-infection of smallmouth bass by both A. salmonicida and largemouth bass virus can be devastating to juvenile smallmouth bass, the optimal temperatures of each pathogen are 7-10 °C apart, making their synergistic effects highly unlikely under field conditions. CONCLUSIONS: The sum of our data generated in this study suggests that largemouth bass virus can be the causative agent of young-of-year smallmouth bass mortality episodes observed at relatively high water temperature.

Detection accuracy of Renibacterium salmoninarum in Chinook salmon, Oncorhynchus tshawytscha (Walbaum) from non-lethally collected samples: Effects of exposure route and disease severity.

Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum, threatens salmonid populations throughout the Northern hemisphere. Many fishery regulatory authorities require ongoing disease monitoring in hatcheries and spawning runs prior to gamete collection to prevent BKD outbreaks and spread. According to diagnostic protocols of the American Fisheries Society-Fish Health Section, monitoring for R. salmoninarum generally consists of lethal sampling of visceral organs from fish. However, non-lethal sampling would be preferable, especially for valuable broodstock or endangered species. In this study, non-lethal sampling methods were evaluated for their ability to detect R. salmoninarum in Chinook salmon (Oncorhynchus tshawytscha) that were experimentally infected via two different routes (e.g., intraperitoneal injection and waterborne immersion) to mimic acute and chronic disease courses. Non-lethal (e.g., blood, mucus, and a urine/feces mixture) and lethal (e.g., kidney and spleen homogenate) samples were collected from challenged and mock-challenged Chinook salmon and the presence of R. salmoninarum was assessed by culture on modified kidney disease medium, nested polymerase chain reaction (nPCR), and semi-quantitative enzyme-linked immunosorbent assay (ELISA). Sensitivity, specificity, and accuracy of lethal and non-lethal samples in detecting R. salmoninarum were calculated using receiver operating characteristic (ROC) analyses. For ROC analyses, true disease status was evaluated under two different assumptions: 1) that lethal samples represented the true disease status and 2) that all experimentally challenged fish were truly infected. We found that sensitivity and specificity of non-lethal samples depended upon time of sampling after experimental infection, sample type, and R. salmoninarum exposure route. Uro-fecal samples had the greatest potential as non-lethal samples compared to mucus and blood. In terms of future monitoring, combining lethal samples tested by ELISA assay with uro-fecal samples tested by nPCR could be the best strategy for detecting R. salmoninarum prevalence in a population as it reduces the overall number of fish required for sampling.

Field study suggests that sex determination in sea lamprey is directly influenced by larval growth rate.

Sex determination mechanisms in fishes lie along a genetic-environmental continuum and thereby offer opportunities to understand how physiology and environment interact to determine sex. Mechanisms and ecological consequences of sex determination in fishes are primarily garnered from teleosts, with little investigation into basal fishes. We tagged and released larval sea lamprey (Petromyzon marinus) into unproductive lake and productive stream environments. Sex ratios produced from these environments were quantified by recapturing tagged individuals as adults. Sex ratios from unproductive and productive environments were initially similar. However, sex ratios soon diverged, with unproductive environments becoming increasingly male-skewed and productive environments becoming less male-skewed with time. We hypothesize that slower growth in unproductive environments contributed to the sex ratio differences by directly influencing sex determination. To the best of our knowledge, this is the first study suggesting that growth rate in a fish species directly influences sex determination; other studies have suggested that the environmental variables to which sex determination is sensitive (e.g. density, temperature) act as cues for favourable or unfavourable growth conditions. Understanding mechanisms of sex determination in lampreys may provide unique insight into the underlying principles of sex determination in other vertebrates and provide innovative approaches for their management where valued and invasive.

DNA Vaccination Partially Protects Muskellunge against Viral Hemorrhagic Septicemia Virus (VHSV-IVb).

A DNA vaccine containing the glycoprotein (G) gene of the North American viral hemorrhagic septicemia virus (VHSV) genotype IVb was developed to evaluate the immune response of fish following vaccination and evaluate its efficacy in protecting a susceptible species, the Muskellunge Esox masquinongy, against VHSV-IVb challenge. Seven weeks (539 degree-days) following vaccination with 10 µg of either pVHSivb-G or a control plasmid, Muskellunge were challenged by immersion with 105 plaque-forming units (pfu)/mL of VHSV-IVb. Fish vaccinated with pVHSivb-G had a relative percent survival (RPS) of 45%. Vaccinated fish also had significantly lower mean viral titers in tissues (4.2 × 102 pfu/g) and viral prevalence (4%) than fish receiving the plasmid control vaccine (3.3 × 105 pfu/g; 82%). Neutralizing antibodies were detected 28 d (308 degree-days) postchallenge (11 weeks postvaccination) in 100% of Muskellunge vaccinated with pVHSivb-G compared with only 12% of plasmid-control-vaccinated Muskellunge, suggesting robust induction of a secondary, adaptive immune response. In addition, pVHSivb-G-vaccinated Rainbow Trout Oncorhynchus mykiss challenged 7 d (100 degree-days) postvaccination with the heterologous novirhabdovirus, infectious hematopoietic necrosis virus (IHNV), experienced an RPS of 61%, compared to control fish, suggesting induction of an early and transient nonspecific antiviral immune response. This study provides an important starting point for VHSV-IVb vaccine development and useful information about the antiviral immune response elicited by DNA vaccination in a nondomesticated fish species. Received May 1, 2016; accepted September 1, 2016.

Assessment of dam effects on streams and fish assemblages of the conterminous USA.

Despite the prevalence of damming as a global disturbance to river habitats, detailed reach-based assessments of the ecological effects of dams are lacking, particularly across large spatial extents. Using data from nearly 50,000 large dams, we assessed stream network fragmentation and flow alteration by large dams for streams of the conterminous USA. We developed 21 dam metrics characterizing a diversity of dam influences operating at both localized (e.g., distances-to-dams) and landscape scales (e.g., cumulative reservoir storage throughout stream networks) for every stream reach in the study region. We further evaluated how dams have affected stream fish assemblages within large ecoregions using more than 37,000 stream fish samples. Streams have been severely fragmented by large dams, with the number of stream segments increasing by 801% compared to free-flowing streams in the absence of dams and a staggering 79% of stream length is disconnected from their outlet (i.e., oceans and Great Lakes). Flow alteration metrics demonstrate a landscape-scale disturbance of dams, resulting in total upstream reservoir storage volumes exceeding estimated annual discharge volumes of many of the nation's largest rivers. Further, we show large-scale changes in fish assemblages with dams. Species adapted to lentic habitats increase with dams across the conterminous USA, while rheophils, lithophils, and intolerant fishes decrease with dams. Overall, fragmentation and flow alteration by dams have affected fish assemblages as much or more than other anthropogenic stressors, with dam effects generally increasing with stream size. Dam-induced stream fragmentation and flow alteration are critical natural resource issues. This study emphasizes the importance of considering dams as a landscape-scale disturbance to river habitats along with the need to assess differential effects that dams may have on river habitats and the fishes they support. Together, these insights are essential for more effective conservation of stream resources and biotic communities globally.

A DNA vaccine encoding the viral hemorrhagic septicemia virus genotype IVb glycoprotein confers protection in muskellunge (Esox masquinongy), rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trutta), and lake trout (Salvelinus namaycush).

BACKGROUND: The viral hemorrhagic septicemia virus (VHSV) is one of the most serious fish pathogens. In 2003, a novel sublineage (genotype IVb) of this deadly virus emerged in the Great Lakes basin causing serious fish kills. We have previously demonstrated that a DNA plasmid (pcDNA), containing a cytomegalovirus (CMV) promoter and the viral hemorrhagic septicemia virus (VHSV) genotype IVb glycoprotein (G) gene insert (designated pVHSivb-G) confers moderate protection in muskellunge (Esox masquinongy), a highly susceptible species upon challenge. In order to achieve optimal protection, we investigated a number of factors including the incubation time [i.e. the number of degree days (° days)] before challenge, and viral challenge dose and route. Additionally, we tested if pVHSivb-G provides protection against VHSV-IVb to less susceptible salmonids such as rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trutta) and lake trout (Salvelinus namaycush). RESULTS: An increase in the period lapsed between vaccination and challenge to 1880° days resulted in 95% relative percent protection (RPS) in muskellunge following a single administration of the pVHSivb-G plasmid and viral challenge. An RPS of 100% for muskellunge was achieved with a longer incubation period (2400° days) and in conjunction with a booster dose of the plasmid. The pVHSivb-G vaccine also elicited significant protection in all three salmonid species, reaching 100% RPS in lake trout following an incubation period of 1001° days prior to viral challenge. Vaccination with pVHSivb-G was also associated with the development of significant levels of circulating VHSV-binding antibodies in muskellunge as measured by indirect ELISA, which reached peak levels 6-7 weeks post-vaccination. Viral shedding in vaccinated survivors was minimal and of transient nature. CONCLUSIONS: The study shows that the pVHSivb-G plasmid can elicit a protective response against the wild virus strain in a range of species important in recreational and commercial Great Lakes fisheries.

Does Herd Immunity Exist in Aquatic Animals?

Viral hemorrhagic septicemia virus genotype IVb (VHSV-IVb) is presently found throughout the Laurentian Great Lakes region of North America. We recently developed a DNA vaccine preparation containing the VHSV-IVb glycoprotein (G) gene with a cytomegalovirus (CMV) promoter that proved highly efficacious in protecting muskellunge (Esox masquinongy) and three salmonid species. This study was conducted to determine whether cohabitation of VHSV-IVb immunized fishes could confer protection to non-vaccinated (i.e., naïve) fishes upon challenge. The experimental layout consisted of multiple flow-through tanks where viral exposure was achieved via shedding from VHSV-IVb experimentally infected muskellunge housed in a tank supplying water to other tanks. The mean cumulative mortality of naïve muskellunge averaged across eight trials (i.e., replicates) was significantly lower when co-occurring with immunized muskellunge than when naïve muskellunge were housed alone (36.5% when co-occurring with vaccinated muskellunge versus 80.2% when housed alone), indicating a possible protective effect based on cohabitation with vaccinated individuals. Additionally, vaccinated muskellunge when co-occurring with naïve muskellunge had significantly greater anti-VHSV antibody levels compared to vaccinated muskellunge housed alone suggesting that heightened anti-VHSV antibodies are a result of cohabitation with susceptible individuals. This finding could contribute to the considerably lower viable VHSV-IVb concentrations we detected in surviving naive muskellunge when housed with vaccinated muskellunge. Our research provides initial evidence of the occurrence of herd immunity against fish pathogens.

Analysis of bacterial communities associated with the benthic amphipod Diporeia in the Laurentian Great Lakes Basin.

Bacterial communities play important roles in the biological functioning of crustaceans, yet little is known about their diversity, structure, and dynamics. This study was conducted to investigate the bacterial communities associated with the benthic amphipod Diporeia, an important component in the Great Lakes foodweb that has been declining over the past 3 decades. In this study, the combination of 16S rRNA gene sequencing and terminal restriction fragment length polymorphism revealed a total of 175 and 138 terminal restriction fragments (T-RFs) in Diporeia samples following treatment with the endonucleases HhaI and MspI, respectively. Relatively abundant and prevalent T-RFs were affiliated with the genera Flavobacterium and Pseudomonas and the class Betaproteobacteria. T-RFs affiliated with the order Rickettsiales were also detected. A significant difference in T-RF presence and abundance (P = 0.035) was detected among profiles generated for Diporeia collected from 4 sites in Lake Michigan. Comparison of profiles generated for Diporeia samples collected in 2 years from lakes Superior and Michigan showed a significant change in diversity for Lake Superior Diporeia but not Lake Michigan Diporeia. Profiles from one Lake Michigan site contained multiple unique T-RFs compared with other Lake Michigan Diporeia profiles, most notably one that represents the genus Methylotenera. This study generated the most extensive list of bacteria associated with Diporeia and sheds useful insights on the microbiome of Great Lakes Diporeia that may help to reveal potential causes of the decline of Diporeia populations.

Spatio-temporal dynamics of parasites infecting Diporeia spp. (Amphipoda, Gammaridae) in southern Lake Michigan (USA).

Since the 1990s, populations of the benthic amphipod Diporeia spp. (Diporeia) have sharply declined across much of the Laurentian Great Lakes. This study was undertaken to identify contemporary and historical community composition, structure, and dynamics of parasites infecting Diporeia collected from nine sites in the southern basin of Lake Michigan, where declines of the amphipod have been well documented over the past 20years. An additional aim of this study was to assess whether infection dynamics and dreissenid densities could explain the declines in Diporeia densities that have occurred. We found that Diporeia were host to eight groups of uni- and multicellular pathogens. Of the 3082 amphipods analyzed, 1624 individuals (52.7%) were infected with at least one type of parasite. Ciliophora was the most prevalent parasite (50.08% prevalence of infection), followed by Gregarinasina (2.79%), Microsporidia (0.68%), Cestoda (0.45%), Acanthocephala (0.36%), Haplosporidia (0.23%), Yeast (0.32%), and filamentous Fungi (0.10%). Considerable spatial and temporal variability were observed in parasite prevalences, with prevalences frequently appearing to cycle between low and high values. Parasite species belonging to Microsporidia and Haplosporidia were associated with tissue alteration and host inflammatory response; however, parasite prevalences explained very little in terms of Diporeia density declines at assessed sites. Despite these findings, we do not discount the possibility that parasitic infections may have played a role in declining Diporeia densities in the Great Lakes, as the cyclical prevalences that were observed are possibly suggestive of parasitic outbreaks that are followed by die-offs at affected sites. This study suggests that if parasites have affected Diporeia densities in the Laurentian Great Lakes, then the relationship may be a complicated one.

Detection of viral hemorrhagic septicemia virus-IVb antibodies in sera of muskellunge Esox masquinongy using competitive ELISA.

A competitive enzyme-linked immunosorbent assay (cELISA) was developed for the detection of antibodies to viral hemorrhagic septicemia virus genotype IVb (VHSV-IVb) in fish sera. Assay conditions were standardized using known negative and positive muskellunge Esox masquinongy. A positive-negative threshold of 14.6% inhibition was established based on analysis of sera of 60 muskellunge with no previous exposure to VHSV-IVb. The cELISA was then used to investigate immune responses of wild muskellunge sampled from 5 water bodies in Michigan and Wisconsin, USA, between 2005 and 2012. Antibodies were detected in fish from Lake St. Clair, Michigan, and Lower Fox River/Green Bay, Wisconsin. Both water systems were considered enzootic for VHSV-IVb. Additionally, antibodies were detected in muskellunge from Thornapple Lake, a Michigan inland lake previously considered negative for VHSV-IVb based on virus isolation methods. Muskellunge populations from Lake Hudson, Michigan, and Butternut Lake, Wisconsin, lacked evidence of an immune response to VHSV-IVb. When results of the cELISA were compared to the 50% plaque neutralization test for several groups of fish, there was 78.4% agreement between the tests for antibody presence. The cELISA is a rapid and efficient test for the detection of binding antibodies to VHSV-IVb and will be a useful non-lethal tool for monitoring the spread of this serious pathogen.

Buruli ulcer disease prevalence in Benin, West Africa: associations with land use/cover and the identification of disease clusters.

BACKGROUND: Buruli ulcer (BU) disease, caused by infection with the environmental mycobacterium M. ulcerans, is an emerging infectious disease in many tropical and sub-tropical countries. Although vectors and modes of transmission remain unknown, it is hypothesized that the transmission of BU disease is associated with human activities in or around aquatic environments, and that characteristics of the landscape (e.g., land use/cover) play a role in mediating BU disease. Several studies performed at relatively small spatial scales (e.g., within a single village or region of a country) support these hypotheses; however, if BU disease is associated with land use/cover characteristics, either through spatial constraints on vector-host dynamics or by mediating human activities, then large-scale (i.e., country-wide) associations should also emerge. The objectives of this study were to (1) investigate associations between BU disease prevalence in villages in Benin, West Africa and surrounding land use/cover patterns and other map-based characteristics, and (2) identify areas with greater and lower than expected prevalence rates (i.e., disease clusters) to assist with the development of prevention and control programs. RESULTS: Our landscape-based models identified low elevation, rural villages surrounded by forest land cover, and located in drainage basins with variable wetness patterns as being associated with higher BU disease prevalence rates. We also identified five spatial disease clusters. Three of the five clusters contained villages with greater than expected prevalence rates and two clusters contained villages with lower than expected prevalence rates. Those villages with greater than expected BU disease prevalence rates spanned a fairly narrow region of south-central Benin. CONCLUSION: Our analyses suggest that interactions between natural land cover and human alterations to the landscape likely play a role in the dynamics of BU disease. For example, urbanization, potentially by providing access to protected water sources, may reduce the likelihood of becoming infected with BU disease. Villages located at low elevations may have higher BU disease prevalence rates due to their close spatial proximity to high risk environments. In addition, forest land cover and drainage basins with variable wetness patterns may be important for providing suitable growth conditions for M. ulcerans, influencing the distribution and abundance of vectors, or mediating vector-human interactions. The identification of disease clusters in this study provides direction for future research aimed at better understanding these and other environmental and social determinants involved in BU disease outbreaks.

Quantitative identification of disturbance thresholds in support of aquatic resource management.

The identification of disturbance thresholds is important for many aspects of aquatic resource management, including the establishment of regulatory criteria and the identification of stream reference conditions. A number of quantitative or model-based approaches can be used to identify disturbance thresholds, including nonparametric deviance reduction (NDR), piecewise regression (PR), Bayesian changepoint (BCP), quantile piecewise constant (QPC), and quantile piecewise linear (QPL) approaches. These methods differ in their assumptions regarding the nature of the disturbance-response variable relationship, which can make selecting among the approaches difficult for those unfamiliar with the methods. We first provide an overview of each of the aforementioned approaches for identifying disturbance thresholds, including the types of data for which the approaches are intended. We then compare threshold estimates from each of these approaches to evaluate their robustness using both simulated and empirical datasets. We found that most of the approaches were accurate in estimating thresholds for datasets with drastic changes in responses variable at the disturbance threshold. Conversely, only the PR and QPL approaches performed well for datasets with conditional mean or upper boundary changes in response variables at the disturbance threshold. The most robust threshold identification approach appeared to be the QPL approach; this method provided relatively accurate threshold estimates for most of the evaluated datasets. Because accuracy of disturbance threshold estimates can be affected by a number of factors, we recommend that several steps be followed when attempting to identify disturbance thresholds. These steps include plotting and visually inspecting the disturbance-response data, hypothesizing what mechanisms likely generate the observed pattern in the disturbance-response data, and plotting the estimated threshold in relation to the disturbance-response data to ensure the appropriateness of the threshold estimate.