William A. Dew

Contaminant-specific targeting of olfactory sensory neuron classes: Connecting neuron class impairment with behavioural deficits

The olfactory system of fish comprises several classes of olfactory sensory neurons (OSNs). The odourants L-alanine and taurocholic acid (TCA) specifically activate microvillous or ciliated OSNs, respectively, in fish. We recorded electro-olfactograms (EOG) in fathead minnows (Pimephales promelas; a laboratory-reared model species) and wild yellow perch (Perca flavescens) whose olfactory chambers were perfused with either L-alanine or TCA to determine if OSN classes were differentially vulnerable to contaminants, in this case copper or nickel. Results were consistent in both species and demonstrated that nickel targeted and impaired microvillous OSN function, while copper targeted and impaired ciliated OSN function. This result suggests that contaminant-specific effects observed in model laboratory species extrapolate to wild fish populations. Moreover, fathead minnows exposed to copper failed to perceive a conspecific alarm cue in a choice maze, whereas those exposed to nickel could respond to the same conspecific cue. These results demonstrate that fathead minnows perceive conspecific, damage-released alarm cue by ciliated, but not microvillous, OSNs. Fish living in copper-contaminated environments may be more vulnerable to predation than those in clean lakes owing to targeted effects on ciliated OSNs.

Olfactory recovery of wild yellow perch from metal contaminated lakes.

Fish depend on their sense of smell for a wide range of vital life processes including finding food, avoiding predators and reproduction. Various contaminants, including metals, can disrupt recognition of chemical information in fish at very low concentrations. Numerous studies have investigated metal effects on fish olfaction under controlled laboratory conditions. However, few have measured olfactory acuity using wild fish in source water. In this study, we used electro-olfactography (EOG) to measure the olfactory acuity of wild yellow perch (Perca flavescens) from a clean lake (Geneva Lake) and two metal contaminated lakes (Ramsey and Hannah lakes) from Sudbury, ON, in their own lake water or in water from the other lakes. The results showed that fish from the clean lake had a greater olfactory acuity than those from metal contaminated lakes when fish were tested in their own lake water. However, when fish from the clean lake were held for 24h in water from each of the two contaminated lakes their olfactory acuity was diminished. On the other hand, fish from the contaminated lakes held for 24h in clean lake water showed a significant olfactory recovery relative to that measured in their native lake water. These results show that although fish from a clean lake demonstrated impaired olfaction after only 24h in metal-contaminated water, fish from metal contaminated lakes showed a rapid olfactory recovery when exposed to clean water for only hours.

Effects of Continuous Copper Exposure and Calcium on the Olfactory Response of Fathead Minnows

The current gill-based Biotic Ligand Model (gbBLM) is an acute-toxicity model used to predict site-specific safe copper (Cu) concentrations. Recent effort to develop a chronic BLM has focused on the olfactory epithelium. To further this effort, the current study looked at the effect of varying Cu concentration and exposure duration on Cu-induced olfactory dysfunction, and whether calcium (Ca) protected against Cu-induced impairment as it does at the gill. Fathead minnows (Pimephales promelas) were treated with five Cu concentrations for varying exposure durations in hard and soft water. A neurophysiological technique, electro-olfactography (EOG), was employed to determine the level of olfactory dysfunction. At the low, ecologically relevant Cu concentrations tested there was significant inhibition of EOG function; however, over time there was at least a partial recovery of olfactory function, despite the continuous Cu exposure. Calcium did not appear to protect against Cu-induced olfactory dysfunction; and even alone, Ca appeared to interfere with the olfactory response to the amino acid L-arginine. Safe copper concentrations as predicted by the gbBLM, chemosensory-based BLMs, the USEPA BLM, and hardness-adjustment equations based on the exposure waters were not entirely protective against olfactory dysfunction.

Biological effects and toxicity of diluted bitumen and its constituents in freshwater systems

Approximately 50 billion cubic meters of bitumen resides within the oil sands region of Alberta, Canada. To facilitate the transport of bitumen from where it is extracted to where it is processed, the bitumen is diluted with natural gas condensate (‘dilbit’), synthetic crude from hydrocracking bitumen (‘synbit’), or a mixture of both (‘dilsynbit’). A primary consideration for the effects of diluted bitumen products on freshwater organisms and ecosystems is whether it will float on the water surface or sink and interact with the stream or lake sediments. Evidence from a spill near Kalamazoo, MI, in 2010 and laboratory testing demonstrate that the nature of the spill and weathering of the dilbit, synbit or dilsynbit prior to and during contact with water will dictate whether the product floats or sinks. Subsequent toxicological data on the effects of dilbit and other diluted bitumen products on freshwater organisms and ecosystems are scarce. However, the current literature indicates that dilbit or bitumen can have significant effects on a wide variety of toxicological endpoints. This review synthesizes the currently available literature concerning the fate and effects of dilbit and synbit spilled into freshwater, and the effects of bitumen and bitumen products on aquatic organisms and ecosystems. Dilbit is likely to provide ecological impacts that are similar to and extend from those that follow from exposure to lighter crude oil, but the prospect of bitumen settling after binding to suspended sediments elevates the risk for benthic impacts in streams and lakes. Copyright

Smelling salt: calcium as an odourant for fathead minnows

Calcium plays an essential role in olfactory sensory neuron function. Studies with fish have indicated that in addition to being involved in olfactory signalling, calcium is itself an odourant. In this study we used fathead minnows (Pimephales promelas) and employed two different techniques; electro-olfactography (EOG), a neurophysiological technique that measures olfactory acuity at the olfactory epithelium, and a behavioural choice assay using a trough maze. The results demonstrate that calcium and a known odourant L-arginine are cross-adaptive, that calcium induces an EOG response in a concentration-dependent manner, and that calcium induces a strong avoidance behaviour. The behavioural avoidance was also demonstrated to be olfactory-dependent. Taken together, the results demonstrate that calcium is a potent odourant for fathead minnows. Being able to smell calcium may represent an ability to sense and avoid areas with significant changes in ionic strength, thereby avoiding physiological stress.

The role of various sensory inputs in establishing social hierarchies in crayfish

Crayfish form social hierarchies through agonistic interactions. During formation of social hierarchies, individual crayfish establish dominance by signalling status through olfaction, vision and touch. Our study investigated which of these three sensory modalities played the most important role in establishing dominance in rusty crayfish (Orconectes rusticus). Olfaction, vision and touch were systematically impaired in staged triadic and dyadic agonistic interactions to determine the relative contribution of each sensory input. Our results suggest that olfaction is the most important sensory modality during the initial formation of dominance hierarchies in rusty crayfish. Using olfaction alone, crayfish were capable of communicating social status with sensory competent crayfish; without full olfactory ability crayfish were unable to effectively establish dominance. Vision and touch were also found to play practical roles in reducing unnecessary risk; with antennae for touch, functionally reducing the number of fight initiations, and vision allowing a crayfish under imminent attack to ready itself, strike first, or retreat.

Electroantennogram measurement of the olfactory response of Daphnia spp. and its impairment by waterborne copper

In this study an electroantennogram (EAG) method was developed for use on live daphniids. The EAG response of Daphnia magna and Daphnia pulex to a variety of amino acids was measured. The strongest response measured was elicited by L-arginine and was shown to induce a concentration-dependent response indicating the response is olfactory in nature. Subsequent exposures of D. magna to a low, ecologically-relevant concentration of copper (7.5 μg/L) showed a disruption in EAG function. This study utilizes the development of an EAG method for measuring olfactory acuity of live daphniids and demonstrates that at ecologically-relevant concentrations, the olfactory dysfunction caused by copper can be detected. The EAG technique is a useful tool for investigating the olfactory response of daphniids to odourants at the cellular level and detecting the effects of toxicants on the olfactory acuity of daphniids.

Effects of Lampricide on Olfaction and Behavior in Young-of-the-Year Lake Sturgeon (Acipenser fulvescens)

The lampricide, 3-trifluoromethyl-4-nitrophenol (TFM), is a primary component to sea lamprey control in the Laurentian Great Lakes. Though the lethal effects of TFM are well-known, the sublethal effects on fishes are virtually unknown. Here we studied the effects of TFM on the olfactory capabilities and behavior of young-of-the-year (YOY) lake sturgeon (Acipenser fulvescens). At ecologically relevant concentrations of TFM there was reduced olfactory response to all three cues (l-alanine, taurocholic acid, food cue) tested, suggesting that TFM inhibits both olfactory sensory neurons tested. Sturgeon exposed to TFM also showed a reduced attraction to the scent of food and reduced consumption of food relative to unexposed fish. Exposed fish were more active than control fish, but with slower acceleration. Fish were able to detect the scent of TFM, but failed to avoid it in behavioral trials. The connection between neurophysiological and behavioral changes, and the commonality of habitats between sturgeon and lamprey ammocoetes, suggests that there may be effects at the ecosystem level in streams that undergo lamprey control treatments.

Cadmium-induced olfactory dysfunction in rainbow trout: Effects of binary and quaternary metal mixtures.

A functioning olfactory response is essential for fish to be able to undertake essential behaviors. The majority of work investigating the effects of metals on the olfactory response of fish has focused on single-metal exposures. In this study we exposed rainbow trout to cadmium, copper, nickel, zinc, or a mixture of these four metals at or below the current Canadian Council of Ministers of the Environment guidelines for the protection of aquatic life. Measurement of olfactory acuity using an electro-olfactogram demonstrated that cadmium causes significant impairment of the entire olfactory system, while the other three metals or the mixture of all four metals did not. Binary mixtures with cadmium and each of the other metals demonstrated that nickel and zinc, but not copper, protect against cadmium-induced olfactory dysfunction. Testing was done to determine if the protection from cadmium-induced olfactory dysfunction could be explained by binding competition between cadmium and the other metals at the cell surface, or if the protection could be explained by an up-regulation of an intracellular detoxification pathway, namely metallothionein. This study is the first to measure the effects of binary and quaternary metal mixtures on the olfactory response of fish, something that will aid in future assessments of the effects of metals on the environment.

Chemosensory mediated behaviors and gene transcription profiles in wild yellow perch (Perca flavescens) from metal contaminated lakes.

The olfactory system of fish is sensitive to the toxic effects of low concentrations of contaminants. To investigate the effects of long-term metal exposure on olfaction in wild yellow perch (Perca flavescens), fish from one clean (Geneva Lake) and two metal-contaminated lakes (Ramsey and Hannah lakes) were collected in and around the metal-mining district of Sudbury, ON. Two different techniques were used to measure the effects of exposure to environmental contamination: (i) behavioral responses were recorded in response to conspecific skin extract and (ii) gene transcription differences in olfactory rosettes were characterized using a novel, 1000-candidate gene yellow perch microarray. Behavioral assays performed on fish from the clean lake demonstrated avoidance of a conspecific skin extract, while fish from metal contaminated lakes showed no avoidance response. A total of 109 out of the 1000 genes were differentially transcribed among the lakes. Most of the differentially transcribed genes were between the two metal contaminated lakes relative to either of the contaminated lakes and the reference lake. No genes were differentially expressed between Geneva Lake (clean) and Hannah Lake (metal contaminated). These results demonstrated that even though the different populations of fish from both Hannah and Ramey lakes were affected at the behavioral level, the impairment of olfaction was not measurable using gene transcriptional changes in olfactory rosettes.