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In JoVE (1)
Other Publications (11)
- Diseases of Aquatic Organisms
- Aquatic Toxicology (Amsterdam, Netherlands)
- Environmental Toxicology and Chemistry / SETAC
- Aquatic Toxicology (Amsterdam, Netherlands)
- Toxicology
- Aquatic Toxicology (Amsterdam, Netherlands)
- Environmental Science & Technology
- Aquatic Toxicology (Amsterdam, Netherlands)
- Environmental Toxicology and Chemistry / SETAC
- Biochimica Et Biophysica Acta
- Environmental Toxicology and Chemistry / SETAC
Articles by Keith B. Tierney in JoVE
JoVE General
Swimming Performance Assessment in Fishes
Department of Biological Sciences, University of Alberta
The lives of the majority of fish are predicated on swimming. This protocol describes techniques for capturing a range of swimming modes available to individual and schooling fish, and includes metrics associated with swimming physiology and behaviour.
Other articles by Keith B. Tierney on PubMed
Subclinical Listonella Anguillarum Infection Does Not Impair Recovery of Swimming Performance in Rainbow Trout Oncorhynchus Mykiss
This study examines whether injections of the commonly used bacterial-challenge pathogen Listonella anguillarum (formerly Vibrio anguillarum) negatively impact the ability of rainbow trout Oncorhynchus mykiss Walbaum to perform repeat swimming trials. Fish were given intraperitoneal injections of either a sub-lethal (10(5) colony forming units; CFUs) or a lethal (10(7) CFUs) dose of L. anguillarum, held for 48 h, and then given 2 successive ramp critical swimming speed (Ucrit) tests separated by 45 min. Compared with saline-injected control fish, the low-dose injection did not significantly impair swimming performance and recovery. Similarly, Ucrit and re-performance for fish surviving the high-dose injection were comparable to control (2 of 6 fish died after injection and before testing). In contrast, a positive control test of seawater challenge did impair recovery of swimming performance. In view of these results and common use of L. anguillarum as a challenge pathogen for toxicological studies, it seems unlikely that the consequences of pathogenesis impact the important cardiorespiratory changes associated with exercise.
The Alarm Reaction of Coho Salmon Parr is Impaired by the Carbamate Fungicide IPBC
To determine whether the carbamate fungicide IPBC alters the olfactory-mediated behavioral and physiologic alarm responses of coho salmon parr (Oncorhynchus kisutch), groups of coho were exposed to skin extract (an alarm pheromone source) under a variety of conditions. In the 3min following skin extract exposure, freezing behavior was significantly increased (In the 3 min following skin extract exposure, freezing behavior was significantly increased under darkness (IR lighting) but not ambient lighting (25.3+/-2.6% and 7.5+/-5.7%, respectively; Delta calculated as: [(time (s) after/time (s) before)-1]x100%), and so IR was used for further experiments. Physiologically, following skin extract exposure, plasma cortisol concentration was increased at 0.5h (58.1+/-14.6ng/ml versus 4.32+/-1.31ng/ml, exposed versus control), hematocrit (Hct) was increased at 2h (50.4+/-1.0% versus 41.7+/-1.6%), and leucocrit (Lct) was decreased at 0.5 and 2h (0.534+/-0.114 and 0.13+/-0.01% versus 1.23+/-0.20%). After 0.5h exposures to 0, 1, 10 and 100microg/l IPBC and skin extract, the time spent dashing (>5cm/s) increased significantly (323+/-118%) in the first minute after skin extract exposure, but was absent in IPBC-exposed coho. Freezing behavior increased after skin extract exposure with control and 1microg/l IPBC exposures (11.0+/-3.0% and 17.7+/-11.0%, respectively), but was absent after 10microg/l and decreased after 100microg/l IPBC. Physiologically, Hct and plasma lactate concentration were significantly increased above controls after 1microg/l IPBC exposure (Hct: 45.7+/-1.6% versus 34.0+/-1.6%, lactate: 12.8+/-1.2mM versus 3.30+/-1.2mM). After 10microg/l exposure, IPBC alone elicited a stress response similar to skin extract. However in the 100microg/l treatment group the stress parameters were not different from controls. These findings suggest that the behavioral and physiologic alarm responses of juvenile salmonids may be impaired by acute exposure to > or =1microg/l IPBC.
Changes in Juvenile Coho Salmon Electro-olfactogram During and After Short-term Exposure to Current-use Pesticides
For anadromous salmonids, olfaction is a critical sense, enabling return migration. In recent years, several pesticides have been identified that interfere with salmonid olfaction at concentrations in the microg/L range; thus, they may pose a risk to species longevity. In the present study, we investigated the acute effects of five agricultural pesticides on juvenile coho salmon (Oncorhynchus kisutch) olfaction using the electro-olfactogram (EOG), a measure of odorant-evoked field potentials. Electro-olfactogram responses to the odorant L-serine were measured during and following a 30-min exposure of the left olfactory rosette to chlorothalonil, endosulfan, glyphosate acid, iodocarb (IPBC), trifluralin, and 2,4-dichlorophenoxyacetic acid. With the relatively insoluble pesticides endosulfan and trifluralin, decreases in EOG amplitude were only apparent at relatively high concentrations (100 and 300 microg/L, respectively) following 20 min of exposure and were absent for chlorothalonil (1 mg/L). With the water-soluble herbicide glyphosate, significant EOG reductions occurred within 10 min of exposure to 1 mg/L and more rapidly with higher concentrations. Recovery of EOG post-glyphosate exposure was concentration-dependent, and complete recovery was not observed with some concentrations at 60 min postexposure. Dichlorophenoxyacetic acid only affected EOG at high concentration (100 mg/L), where it eliminated EOG within 2 min of exposure. With IPBC, EOG was decreased at 25 min of exposure to 1 microg/L; higher concentrations caused decreases to occur more rapidly. Excluding IPBC and glyphosate, all EOG reductions occurred at concentrations greater than the current Canadian water-quality guidelines and reported 96-h lethality values. Our results show that olfactory neurons can be impaired rapidly by some current-use pesticides, even at exposures in the low-microg/L range.
Relating Olfactory Neurotoxicity to Altered Olfactory-mediated Behaviors in Rainbow Trout Exposed to Three Currently-used Pesticides
Odor-evoked neurophysiological responses can form the basis for behavioral responses. Here we first characterized olfactory-mediated behavioral and neurophysiological responses of juvenile rainbow trout to the amino acid l-histidine, then looked at whether there were similar responses to the carbamate antisapstain IPBC and the herbicides atrazine and Roundup, and lastly explored how exposures to these pesticides modified the l-histidine responses. Trout were behaviorally attracted to 10(-7)M l-histidine (as assayed in a counter-current olfactometer), but this preference behavior switched to indifference with higher histidine concentrations. Neurophysiologically, the summed electrical responses of peripheral olfactory neurons, as measured using electro-olfactogram (EOG), was 0.843+/-0.252 mV to 10(-7)M l-histidine. Of the pesticides, only Roundup evoked EOGs, indicating the amino acid-based pesticide may have acted as an odorant, and generated a behavioral response: it was avoided at active ingredient [AI; glyphosate isopropyl amine] concentrations > or =10 mg/l. With 30 min pesticide exposures, 10(-7)M l-histidine preference behavior was eliminated following exposure to 1 microg/l IPBC and atrazine, and 100 microg/l AI Roundup. Similarly, 10(-7)M l-histidine-evoked EOGs were significantly reduced by exposure to 1 microg/l IPBC, 10 microg/l atrazine, and 100 microg/l AI Roundup. When combined together, the results demonstrate that typical preference behavior can be abolished when neurophysiological responses are reduced by >60% of control. This asymmetry in response thresholds suggests that behavioral responses may be more sensitive toxicological endpoints than neurophysiological responses.
Linuron and Carbaryl Differentially Impair Baseline Amino Acid and Bile Salt Olfactory Responses in Three Salmonids
For salmon, amino acid and bile salt detection form the basis for important behaviors including predator evasion and conspecific recognition, respectively. For this reason, decreases in olfactory sensory neuron responses to the amino acid l-serine and the bile salt taurocholic acid (TChA) have been used in studies as indicators of acute olfactory pesticide toxicity to environmental contaminants such as metals and pesticides. In this study, we first compare baseline responses to these two odorant classes across three salmonids, and then explore how two currently used pesticides alter these responses. We found baseline differences in electro-olfactogram (EOG) responses and their sensitivity to pesticide exposure between rainbow trout, coho and sockeye salmon. For example, rainbow trout had lower baseline EOGs than either coho or sockeye (e.g. 10(-5)M TChA EOGs of 1.34+/-0.17 versus 2.57+/-0.46 and 2.72+/-0.43 mV, respectively). At 15 min after exposure to 10 microg/L of the herbicide linuron, rainbow l-serine-evoked EOGs were 49.6% of control versus 78.5 and 69.8% for sockeye and coho, indicating rainbow were more sensitive to linuron. In contrast, at 30 min of exposure to 100 microg/L carbaryl, l-serine-evoked EOGs of sockeye were 49.7% of control versus 60.3 and 62.3% for rainbow and coho, suggesting sockeye were more sensitive to carbaryl. In all species the l-serine-evoked EOGs did not return to baseline by 15 min after 100 microg/L carbaryl exposure, suggesting persisting impairment of amino acid detection. The TChA-evoked EOGs were less affected by carbaryl exposure (i.e. EOGs were 83.3, 84.9 and 66.0% of control 15 min after exposure) and not affected at all by 100 microg/L linuron exposure. Species-specific differences in pesticide sensitivity may limit extrapolation of toxicity across salmonids while the generally greater sensitivity of amino acid olfaction may lead to selective impairment of behaviors such as predator evasion.
Energy Intake Affects the Biotransformation Rate, Scope for Induction, and Metabolite Profile of Benzo[a]pyrene in Rainbow Trout
The metabolic conversion of benzo[a]pyrene (B[a]P) by rainbow trout (Oncorhynchus mykiss) hepatocytes was not significantly different between any group of fed fish (fed one of three isoenergetic diets that varied in protein and lipid content at full satiation levels or half rations), however at 12 weeks, fasted fish exhibited significantly reduced B[a]P biotransformation rates (by 58%). Alterations in metabolite profiles were also seen: fasted fish produced significantly more Phase I metabolites, higher levels of both glucuronide and sulphate conjugates, and lower levels of presumptive glutathione conjugates, compared to fed fish. When fish were fasted, higher proportions of phenols were produced, with lower proportions of quinones, triols and tetrols. Inducing metabolism (using beta-naphthoflavone) increased metabolic scope for B[a]P by 2-fold, regardless of each diet's baseline metabolic rate. However, the balance between Phase I and II reactions was altered with induction and fasting: higher proportions of Phase I metabolites were found, with lower glutathione conjugates and higher proportions of triols/tetrols. Fasting-mediated reductions in glutathione conjugation, and increased induction of oxidation vs. conjugating enzymes, can explain altered metabolite profiles. These results suggest that in contaminated habitats, where pollution-induced reductions in food quantity or quality are combined with the presence of toxic compounds and inducers, detoxification rates can be diminished.
Salmon Olfaction is Impaired by an Environmentally Realistic Pesticide Mixture
Many of the salmon-producing waterways of the world contain pesticides known to harm olfactory sensory neurons (OSNs) that are critically important throughout the salmon lifecycle. The ability of OSNs to retain functionality after exposure to complex pesticide mixtures remains unknown. Here we show that a 96-h exposure to an environmentally realistic concentration of a mixture made from the ten most frequently occurring pesticides in British Columbia's Nicomekl River reduced the OSN responses of rainbow troutto a behaviorally relevant odorant. Odor-evoked responses were not altered by exposure to one-fifth of the realistic concentration, and this may have been due an upregulation in detoxification enzymes, since glutathione-S-transferase activity reached a maximum (> 32% above control) at this concentration. Mixture exposure did not help to prevent OSN impairment from a second, brief (5 min) exposure to a higher (20 x) concentration of the mixture, suggesting longer-term, low-concentration exposures may not prevent damage from brief, high-concentration pulse exposures. This study demonstrates that environmentally observed pesticide mixtures can injure salmon olfactory tissue, and by extension, contribute to the threatened and endangered status of many salmon stocks.
Olfactory Toxicity in Fishes
Olfaction conveys critical environmental information to fishes, enabling activities such as mating, locating food, discriminating kin, avoiding predators and homing. All of these behaviors can be impaired or lost as a result of exposure to toxic contaminants in surface waters. Historically, teleost olfaction studies have focused on behavioral responses to anthropogenic contaminants (e.g., avoidance). More recently, there has been a shift towards understanding the underlying mechanisms and functional significance of contaminant-mediated changes in fish olfaction. This includes a consideration of how contaminants affect the olfactory nervous system and, by extension, the downstream physiological and behavioral processes that together comprise a normal response to naturally occurring stimuli (e.g., reproductive priming or releasing pheromones). Numerous studies spanning several species have shown that ecologically relevant exposures to common pollutants such as metals and pesticides can interfere with fish olfaction and disrupt life history processes that determine individual survival and reproductive success. This represents one of the pathways by which toxic chemicals in aquatic habitats may increasingly contribute to the decline and at-risk status of many commercially and ecologically important fish stocks. Despite our emerging understanding of the threats that pollution poses for chemical communication in aquatic communities, many research challenges remain. These include: (1) the determination of specific mechanisms of toxicity in the fish olfactory sensory epithelium; (2) an understanding of the impacts of complex chemical mixtures; (3) the capacity to assess olfactory toxicity in fish in situ; (4) the impacts of toxins on olfactory-mediated behaviors that are still poorly understood for many fish species; and (5) the connections between sublethal effects on individual fish and the long-term viability of wild populations. This review summarizes and integrates studies on fish olfaction-contaminant interactions, including metrics ranging from the molecular to the behavioral, and highlights directions for future research.
Evidence for Behavioral Preference Toward Environmental Concentrations of Urban-use Herbicides in a Model Adult Fish
Fish live in waters of contaminant flux. In three urban, fish-bearing waterways of British Columbia, Canada, we found the active ingredients of WeedEx, KillEx, and Roundup herbicide formulations (2,4-D, dicamba, glyphosate, and mecoprop) at low to high ng/L concentrations (0.26 to 309 ng/L) in routine conditions, i.e., no rain for at least one week. Following rain, these concentrations increased by an average of eightfold, suggesting runoff as a major route of herbicide introduction in these waterways. To determine whether fish might be able to limit point-source exposures through sensory-driven behaviors, we introduced pulses of representative herbicide mixtures to individual adult zebrafish (a model species) in flow-through tanks. Fish did the opposite of limit exposure; they chose to spend more time in pulses of herbicide mixtures representative of those that may occur with rain events. This attraction response was not altered by a previous 4-d exposure to lower concentrations of the mixtures, suggesting fish will not learn from previous exposures. However, previous exposures did alter an attraction response to an amino acid prevalent in food (L-alanine). The present study demonstrates that fish living within urban waterways may elect to place themselves in herbicide-contaminated environments and that these exposures may alter their behavioral responses to cues necessary for survival.
Behavioural Assessments of Neurotoxic Effects and Neurodegeneration in Zebrafish
Altered neurological function will generally be behaviourally apparent. Many of the behavioural models pioneered in mammalian models are portable to zebrafish. Tests are available to capture alterations in basic motor function, changes associated with exteroceptive and interoceptive sensory cues, and alterations in learning and memory performance. Excepting some endpoints involving learning, behavioural tests can be carried out at 4 days post fertilization. Given larvae can be reared quickly and in large numbers, and that software solutions are readily available from multiple vendors to automatically test behavioural responses in 96 larvae simultaneously, zebrafish are a potent and rapid model for screening neurological impairments. Coupling current and emerging behavioural endpoints with molecular techniques will permit and accelerate the determination of the mechanisms behind neurotoxicity and degeneration, as well as provide numerous means to test remedial drugs and other therapies. The emphasis of this review is to highlight unexplored/underutilized behavioural assays for future studies. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.
Environmental Concentrations of Agricultural-use Pesticide Mixtures Evoke Primary and Secondary Stress Responses in Rainbow Trout
The present study sought to determine whether environmentally realistic mixtures of agriculturally important pesticides are stressful to fish. Juvenile rainbow trout were exposed for 96 h to concentrations of a pesticide mixture found in a waterway that is the focus of salmon restoration efforts (Nicomekl River, BC, Canada). This mixture contained organochlorine, organophosphorus, phenylurea, and triazine classes of pesticides. Fish given a realistic mixture exposure (total concentration, 1.01 µg/L) had increased plasma cortisol concentration, packed red cell volume, hematocrit (Hct), as well as decreased white cell volume, leukocrit (Lct). Similar changes in Hct and Lct were apparent after exposure to a lower concentration (0.186 µg/L). Interestingly, no changes in plasma cortisol concentration, Hct, or Lct were noted after exposure to a higher concentration (13.9 µg/L). This suggests that the exposure likely impaired the mechanisms enabling the stress response. Across all exposures, plasma glucose concentration was related to plasma cortisol concentration, not to pesticide mixture concentration. This suggests that a secondary stress response may be more related to variability in individual primary stress response than to differences in pesticide exposure concentrations. In summary, the present study indicates that salmon living in agrichemical-contaminated waterways may be experiencing stress, and this may pose a threat to their survival.
