Damsels in distress: Oil exposure modifies behavior and olfaction in

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Ecotoxicology and Human Environmental Health

Damsels in distress: Oil exposure modifies behavior and olfaction in bicolor damselfish (Stegastes partitus) Lela Schlenker, Megan J. Welch, Tricia L. Meredith, Edward Mager, Ebrahim Lari, Elizabeth A. Babcock, Greg G. Pyle, Philip Munday, and Martin Grosell Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.9b03915 • Publication Date (Web): 26 Aug 2019 Downloaded from pubs.acs.org on August 29, 2019

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Environmental Science & Technology

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TITLE

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Damsels in distress: Oil exposure modifies behavior and olfaction in bicolor damselfish

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(Stegastes partitus)

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AUTHOR NAMES

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Lela S. Schlenker1*, Megan J. Welch2, Tricia L. Meredith3, Edward M. Mager1,4, Ebrahim

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Lari5§, Elizabeth A. Babcock1, Greg G. Pyle5, Philip L. Munday2, Martin Grosell1

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AUTHOR ADDRESSES

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1 Department

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and Atmospheric Sciences, 4600 Rickenbacker Causeway Miami, FL 33149 USA

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2ARC

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Australia 4811

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3 Florida

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4Department

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University of North Texas, 1511 W. Sycamore Street, Denton, Texas 76203 USA

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5Department

of Marine Biology and Ecology, University of Miami, Rosenstiel School of Marine

Centre of Excellence for Coral Reef Studies, James Cook University, Townsville, QLD

Atlantic University, 777 Glades Road, Boca Raton, FL 33431 USA of Biological Sciences and Advanced Environmental Research Institute,

of Biological Sciences, University of Lethbridge, Lethbridge, AB T1K 3M4 Canada

ACS Paragon Plus Environment

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ABSTRACT

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In fishes, olfactory cues evoke behavioral responses that are crucial to survival; however, the

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receptors, olfactory sensory neurons, are directly exposed to the environment and are susceptible

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to damage from aquatic contaminants. In 2010, 4.9 million barrels of crude oil was released into

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the northern Gulf of Mexico from the Deepwater Horizon disaster, exposing marine organisms to

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this environmental contaminant. We examined the ability of bicolor damselfish (Stegastes

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partitus) exposed to the water accommodated fraction (WAF) of crude oil to respond to chemical

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alarm cue (CAC) using a two-channel flume. Control bicolor damselfish avoided CAC in the

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flume choice test, whereas WAF-exposed conspecifics did not. This lack of avoidance persisted

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following eight days of control water conditions. We then examined the physiological response

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to CAC, brine shrimp rinse, bile salt, and amino acid cues using the electro-olfactogram (EOG)

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technique and found that WAF-exposed bicolor damselfish were less likely to detect CAC as an

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olfactory cue but showed no difference in EOG amplitude or duration compared to controls.

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These data indicate that a sub-lethal WAF exposure directly modifies detection and avoidance of

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CAC beyond the exposure period and may suggest reduced predator avoidance behavior in oil-

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exposed fish in the wild.


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INTRODUCTION The olfactory sensory neurons (OSNs) of marine organisms are directly exposed to the

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environment, which allows for rapid detection of olfactory cues essential for navigation1, 2,

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settlement3, social interactions4, spawning behavior5, assessing habitat quality6, and evading

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predation7. Olfactory cues provide information critical to survival and can be detected over great

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distances, at low concentrations, and in conditions that can make other senses ineffective8.

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However, while this direct connection between the OSNs and the environment is essential, it also

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exposes OSNs to aquatic contaminants such as crude oil9.

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In 2010 the Deepwater Horizon (DWH) blowout released approximately 4.9 million

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barrels of crude oil into the northern Gulf of Mexico over an 87-day period, resulting in the

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largest documented oil spill in United States history10, 11. While some of the oil evaporated at the

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sea surface, formed surface slicks, or was mechanically removed or burned, as much as 31% of

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the total oil spilled is estimated to have ended up in shallow and deep-water sediments12, 13.

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Demersal reef fishes in the northern Gulf of Mexico, such as the bicolor damselfish (Stegastes

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partitus), were heavily impacted by the spill and experienced severe population declines14-16,

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likely due in part to their close association with the benthos, affinity to a specific territory, and

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consumption of benthic algae and prey17, 18. In addition to the outright morality that occurred

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following the oil spill15, 16, transcriptomic and morphological data from marine teleosts exposed

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to sublethal concentrations of crude oil have suggested sensory and nervous system

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impairment19-21. However, despite the expansive range of research completed following the spill,

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and the link between physiology, behavior, and ecological processes, very little is known about

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the effects of crude oil exposure on olfactory physiology and associated impacts on behavior.


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To assess the lasting implications of the 2010 spill on marine fishes, it is essential to

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understand the impacts of crude oil on key sensory systems, like the olfactory system, and

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behavior of marine teleosts. In vertebrates, the process of olfactory detection begins when

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olfactory molecules in the water bind to G-protein-coupled receptor proteins on olfactory sensory

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neurons (OSNs) covering the olfactory epithelium (OE)22. Binding releases second messengers

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that open gated ion channels which indirectly leads to cell depolarization and the increased

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likelihood of action potentials22, 23. If they occur, resulting action potentials are transmitted to the

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olfactory bulb (OB) where signal processing takes place before being relayed to secondary

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neurons and higher brain centers22. Therefore, behavioral disruptions that may occur as a result

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of an exposure to a toxicant24-26, could result from either impairment peripherally at the OE or an

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effect on higher order central nervous system (CNS) processing. The link between olfaction and

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behavior has been demonstrated for fish exposed to contaminants9, 27 and both metrics are critical

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predictors of fitness in marine fishes28, 29.

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Exposure to crude oil or individual polycyclic aromatic hydrocarbons (PAHs), the

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primary toxic constituents of crude oil, are known to be detrimental to the health of OSNs and

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the OE30-33. Pink salmon (Oncorhynchus gorbuscha) exposed to sublethal concentrations of

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benzene showed an 83% reduction in OE cilia and increased mucus production compared to

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control fish30. Killifish (Fundulus heteroclitus) exposed to naphthalene31 developed necrotic

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areas throughout the OE, a condition also observed in conjunction with OE hyperplasia in inland

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silversides (Menidia beryllina) and hogchoker (Trinectes maculatus) exposed to crude oil33.

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Similarly, Atlantic silversides (Menidia menidia) exposed to crude oil exhibited lesions and

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hyperplasia of the OE32.


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Chemical alarm cue (CAC) released from injured conspecifics has long been shown to

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elicit behavioral responses in teleost fishes 34, 35 and has been established as an olfactory cue in

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marine and freshwater teleosts36-38. Several species of damselfish are known to display an innate

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avoidance of CAC29, 35, a behavior that is hypothesized to have an evolutionary role as a

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protective mechanism against predation29. Fish rely on the detection of CAC as an olfactory cue

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to avoid predation39; therefore, impairment of OSNs that would interfere with CAC detection has

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the potential to have direct effects on the survival of wild fish40, 41.

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Recent studies have separately demonstrated behavioral and olfactory impacts to marine

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fishes briefly exposed to the water accommodated fraction (WAF) of crude oil from the DWH

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spill. WAF-exposed coral reef fish display reduced settling to an experimental reef, less shoaling,

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increased boldness, and a 2.7-fold increase in mortality as a result of predation24. Similarly,

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WAF-exposed juvenile red drum (Sciaenops ocellatus) were found to be more likely to be

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subordinate than controls in dyad experiments25. Use of the electro-olfactogram (EOG) technique

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to measure the odor-evoked extracellular field potential, an indirect measurement of the OE

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generator potential, has shown that WAF-exposed Atlantic stingrays (Hypanus sabinus)

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demonstrate an average 46% reduction in response magnitude when stimulatory amino acids are

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introduced28. Additionally, WAF-exposed Atlantic stingrays had slower EOG response onset and

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longer EOG response duration compared to unexposed animals28.

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To more completely understand the physiological and ecological implications of the 2010

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DWH oil spill it is important to examine behavior and olfactory responses in WAF-exposed

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individuals of the same species. The present study is the first to examine both the olfactory

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physiology and behavior of a marine teleost exposed to the WAF of crude oil. The primary goals

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of this study were, first, to examine the behavioral responses of control and WAF-exposed


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bicolor damselfish to CAC using a two-channel flume behavioral test, and second, to evaluate

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whether control and WAF-exposed bicolor damselfish were able to detect CAC, a food cue, a

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bile salt cue, and amino acid cues at the olfactory epithelium using the EOG technique. By

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studying both behavior and olfaction within a species we can begin to probe into the mechanisms

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through which oil-exposure affects marine teleosts.

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MATERIALS AND METHODS

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Test animals

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Bicolor damselfish were hand netted from Emerald reef (2540’27.0 N, 8005’55.2 W)

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east of Key Biscayne, Florida (SAL-18-303-SR), by divers using SCUBA. Collections occurred

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on September 29, 2015 for flume choice experiments and on August 13, 2018 for EOG

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experiments. Fish were transported to the University of Miami Rosenstiel School of Marine and

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Atmospheric Science and maintained in flow-through seawater aquaria for one to eight weeks

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before use in experiments. Temperature and salinity were measured daily with a ProODO optical

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probe (YSI, Inc., Yellow Springs, OH) and a refractometer, respectively, in order to monitor

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water chemistry. Damselfish were fed daily ad libitum until transfer into 24-hour treatment or

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control exposures where they were fasted. Photoperiod was maintained with 16 hours of light

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and 8 hours of darkness in all holding conditions. Seawater used in all experiments matched the

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temperature, pH, and salinity profiles in which fish were housed.

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For both two-channel flume and EOG experiments, bicolor damselfish were transferred

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to static 24-hour exposures of either control seawater or the WAF of crude oil, one day prior to

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experimentation. During static 24-hour treatments, initial and final water chemistry

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measurements were recorded for the following: temperature and dissolved oxygen using a


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ProODO optical probe (YSI, Inc., Yellow Springs, OH), pH measured with a PHM201 meter

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(Radiometer, Copenhagen, Denmark), and salinity determined with a refractometer (Table 1). To

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evaluate ammonia concentrations and to ensure they did not accumulate above toxic thresholds

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in static exposures, the final ammonia concentration of each tank was assessed using

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Micromodified Indophenol Blue with a colorimetric assay (Table 1)42. All experiments on

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bicolor damselfish were performed under the University of Miami IACUC protocol # 15-019.

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Oil preparation

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Slick oil collected from surface skimming operations during the DWH spill (sample ID:

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OFS-20100719-Juniper-001 A00884) was used to prepare a high-energy water accommodated

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fraction (HEWAF) of crude oil by mixing 1 g of oil per liter of seawater at low speed for 30 s in

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a Waring CB 15 blender (Torrington, CT). The blended oil and seawater mixture was

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immediately transferred to a glass separatory funnel, allowed to settle for 1 hour, and the lower

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90% was drained and retained as the 100% WAF. This WAF was then diluted with control

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seawater to make a 6% static exposure treatment and damselfish were immediately introduced

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into either control or WAF-exposure treatment tanks. A 6% WAF exposure was chosen because

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it is within the range of concentrations measured in the Gulf of Mexico following the DWH

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disaster43 and because it is typically sublethal, but sufficient to cause detrimental effects on

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morphology and/or physiology in other marine teleosts24.

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Water samples were collected in 250 mL amber bottles directly from each tank at the

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beginning and end of 24-hour exposures to quantify PAHs. Samples were shipped overnight on

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ice to ALS Environmental (Kelso, WA) for gas chromatography/mass spectrometry—selective

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ion monitoring (GC/MS—SIM; based on EPA method 8270D). Reported PAH values are the


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sum of 50 individual PAHs averaged using the geometric mean of initial and 24-hour samples.

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Both experiments exposed fish to a 6% WAF exposure, but due to the inherent variability of

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preparing crude oil, the 24-hour exposures used in behavior experiments (50 PAH 31.7 

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4.31g/L) were more concentrated than those used in EOG experiments (50 PAH 11.22  5.55

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g/L). Control exposures had a negligible quantity of PAHs present (50 PAH 0.02917 g/L). A

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summary of all bicolor damselfish sizes, PAH initial and final concentrations, and a

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representative PAH profile are available in Supplementary Information (Figure S1; Table S1,

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S2, S3, S4).

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Behavioral experiments Behavioral experiments were designed to test the hypothesis that exposure to the WAF of

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crude oil would modify behavioral responses to CAC. Immediately following static exposures,

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control (n=15) and WAF-exposed (n=12) bicolor damselfish were tested in a two-channel flume

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(13 cm X 4 cm) divided down half of the length so that the two water streams, CAC and

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untreated seawater, remained separate44. An additional test with control bicolor damselfish (n=8)

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used untreated seawater on both sides of the flume to test for any possible side bias in the test. A

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previous study demonstrated near laminar flow and separation of flows between the two sides in

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this exact flume35. Water flow to both sides of the flume was gravity fed at a constant flow rate

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of 200 mL per minute. Water flow was maintained and monitored throughout experiments using

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a Dwyer Mini-Master Flowmeter (MMA-38, Michigan City, Indiana).

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Following established protocol35, 45, conspecific CAC was produced by euthanizing a

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control bicolor damselfish with a quick blow to the head, making six superficial cuts on the

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lateral sides of the fish, and rinsing each side of the fish with 15 mL of seawater. This rinse water


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was added to 5 L of seawater, mixed, and added to one side of the gravity flow system

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immediately prior to the start of each flume test. The side of the flume to initially hold CAC was

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alternated between fish. One donor damselfish was used to make CAC for each experimental

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damselfish trial.

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For each trial, a fish was placed directly in the center of the downstream side of the flume

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and given two minutes to habituate. After the habituation period, the position of the fish in the

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flume (CAC side or untreated seawater side) was noted every five seconds for two minutes. The

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side on which the CAC and the untreated seawater were located was switched during a one-

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minute rest period to eliminate the confounding effect of a potential side-bias. The fish was re-

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centered at the end of the rest period, and the entire habituation and trial process were repeated

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with the cue on the opposite side of the flume. The flume choice test observer was not blinded to

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the treatment group.

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Following the completion of two-channel flume experiments, all damselfish were

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returned to clean seawater and were fed daily ad libatum. WAF-exposed damselfish were re-

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tested in the same manner as described above three- and eight-days following exposure and were

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again fasted for the 24-hours prior to experimentation. The identity of individual WAF-exposed

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fish was tracked, but in tests performed three- and eight-days post-exposure, the observer was

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blinded to individual fish identification. Experiments were halted once the WAF-exposed fish

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had been tested three times.

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Electro-olfactogram experiments EOG experiments were designed to test the hypothesis that observed reduced behavioral responses of WAF-exposed bicolor damselfish were associated with diminished sensitivity of


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OSNs to CAC and other olfactory cues. All EOG recordings for control (n=14) and WAF-

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exposed (n=11) bicolor damselfish were performed blind such that the researcher did not know

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whether the fish was a control or WAF-exposed individual. Underwater EOG experiments

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commenced immediately following 24-hour exposures. Bicolor damselfish were anesthetized

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with 0.2 g/L MS-222 (tricaine methanesulfonate, Western Chemical, Inc., Ferndale, WA),

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buffered with NaHCO3 (Sigma Aldrich, St. Louis, MO), and transferred to a submerged

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experimental chamber where they were ventilated with aerated control seawater containing 0.1

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g/L MS-222. To access the OE, the septum that separates the anterior and posterior nares was

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surgically removed and a recording electrode was placed on the longest lamellae of the anterior

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dorsal section of the OE and a reference electrode was placed directly posterior to the eye.

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Recording and reference electrodes were built identically with non-polarizable Ag-AgCl

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electrodes (Warner Instruments, Hamden, CT) fitted with a 1.5 mm glass capillary tube (Warner

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Instruments), pulled to a fine tip, and filled with 3 M potassium chloride (Sigma Aldrich).

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In addition to CAC the following olfactory cues were used to assess the effect of WAF

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exposure on detection of a variety of cue types: a brine shrimp rinse (brine shrimp; San Francisco

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Bay Brand, Newark, CA), a ten-fold dilution of the brine shrimp rinse (10% brine shrimp), 10-1

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M L-alanine (Ala 10-1; Sigma Aldrich), 10-2 M L-alanine (Ala 10-2), and 10-3 M taurocholic acid

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(TCA; Sigma Aldrich). Brine shrimp rinse was made by soaking 0.5 g of frozen brine shrimp in

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200 mL of control seawater for 30 minutes and filtering it through a 100 m mesh. CAC for

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EOG experiments was made as described for flume choice experiments with the modification

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that the rinse water was added to 20mL of control seawater. This concentration of CAC was

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selected for EOG work following initial trials that showed a consistent, measurable EOG

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response to this dilution of CAC. CAC was made immediately following a successful recording


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for each individual and one control donor fish was used for each experimental fish. Cues were

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selected to represent both natural cues, e.g., CAC and a concentrated and dilute brine shrimp

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rinse, as well as standard olfactory cues of varying concentrations, e.g., Ala 10-1, Ala 10-2, and

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TCA. L-alanine cues were used to examine the effect of WAF exposure on microvillous OSNs

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and represent food cues while TCA was used to evaluate the effect of WAF exposure on ciliated

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OSNs and represents an alarm cue27.

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All cues were delivered for three-seconds using an eight-channel perfusion valve control

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system (Warner Instruments) that allowed seamless transitions from OE perfusion, to cue

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delivery, and back to OE perfusion without interruption in flow or change in flow volume.

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Seawater was used to perfuse the OE for 90-seconds between cues and was also used as a

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negative control cue. Perfusate, as well as all cues, contained 0.1 g/L MS-222 to match the

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submerged experimental chamber and ventilation water. Cues were administered in a

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randomized order with each cue delivered three times per experiment with a paired negative

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control cue following each cue. At the conclusion of the experiment fish were euthanized by

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pithing.

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During EOG experiments the output from the recording and reference electrodes were

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differentially amplified 1000x (DP-311, Warner Instruments), filtered (0.1Hz-0.1kHz, 50/60Hz;

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DP-311, Warner Instruments), digitized and filtered (60 Hz, Power Lab, AD Instruments,

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Dunedin, NZ), and recorded (Chart Software v. 8.1.3, AD Instruments, Colorado Springs, CO).

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For each EOG response, amplitude (mV), duration (s), integral (mV·s), mean (mV), average

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slope (mVs-1), and maximum slope (mVs-1) were measured, and the corresponding negative

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control response was subtracted. When the negative control response was greater than the

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corresponding cue response the cue was considered to be a non-detection.


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Statistical analysis All data were analyzed in R46 and all linear models were performed with the “lme4”

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package47. Akaike’s Information Criterion (AIC)48, 49 was used to discriminate among competing

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formulations when multiple models were assessed.

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In behavior trials, percent time spent in the CAC was arcsine transformed for all analyses.

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Treatment (WAF or control) was used in a linear model to predict the time that bicolor

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damselfish spent in the CAC with individual fish identity added as a random effect and a

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Bonferroni correction for pairwise comparisons (Table S5). A Student t-test was used to compare

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the time that negative control fish (seawater on both sides of the flume) spent on one side of the

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flume to 50% time on a side to evaluate whether any side bias occurred.

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For EOG data, the amplitude and duration of the response were chosen as the variables of

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interest out of the subset measured, as they were not highly correlated with one another and a

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principle components analysis demonstrated they were conveying different types of information

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(Figure S2). For both EOG amplitude and duration, linear models were used to assess the effect

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of the olfactory cue, treatment (WAF or control), and their interaction on the EOG amplitude and

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duration with individual fish as a random effect. The “multcomp” package in R50 was used post-

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hoc to examine differences in responses to particular cues between control and WAF-exposed

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bicolor damselfish.

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For EOG cues that resulted in both detections and non-detections, individual fish were

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defined as “detectors” of a particular cue if they successfully detected the cue at least 33% of the

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time, while individual fish were conversely defined as “non-detectors” if they did not meet that

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criteria. A linear model using individual fish as a random effect was performed to determine


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whether a fish was a detector for a particular cue was predicted by WAF exposure. To estimate

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confidence intervals for these models, the Monte Carlo method was used to examine the fixed51

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effects for a typical fish.

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RESULTS AND DISCUSSION

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CAC is a biologically relevant and evolutionarily significant cue for fish species. The

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present study utilized a paired approach to examine both the behavior and detection capacity of

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control and WAF-exposed bicolor damselfish when they were presented with CAC. Our results

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demonstrate that WAF-exposed bicolor damselfish do not avoid CAC in a two-channel flume as

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control fish do, and that this exposure reduced the probability of CAC detections at the OE.

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Neither the detection probability of the other cues used in this study, nor the amplitude or

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duration of the EOG responses to any cue, were affected by WAF exposure. Our results suggest

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that modifications to behavior that occur following an environmentally relevant 24-hour

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exposure to WAF may be driven in part by reductions in neuronal signaling that could be

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specific to particular cues.

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When faced with a predator, prey may respond to a variety of cues including auditory

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distress calls52, visual detection of a predator or prey fleeing52, and detection of CAC39. Chemical

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cues often provide an early warning over visual or auditory cues and have been shown to

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increase survival time in experimental interactions between predator and prey fish53. Avoidance

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of CAC has also been shown across a range of animal taxa from flatworms54 to marine teleost

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embryos55 to mammals56, demonstrating the evolutionary significance of this cue. Control fish in

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our experiment that were tested with CAC spent 17% of their time on the side of the flume with

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CAC demonstrating that under control conditions bicolor damselfish will avoid CAC (Fig. 1). In

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contrast, a 24-hour WAF exposure (50 PAH 31.7  4.31g/L) significantly increased the


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amount of time that bicolor damselfish spent in the CAC, a normally deterrent olfactory cue,

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compared to control individuals (p0.05), and 10% dilution brine shrimp rinse and taurocholic acid 10-3 M (p>0.05).


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2.0 1.5

Control

0.5

1.0

Oil−exposed

0.0

Duration EOG Response (s)

2.5

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ALA−1

ALA−2

CAC

BS

10% BS

TCA−3

Cue

445 446 447 448 449 450 451 452 453 454 455 456

Figure 3. Bicolor damselfish EOG response duration (s) with negative control cue subtracted for 24-hour control (n=14) and oil-exposure (n=11, 50 PAH 11.22  5.55 g/L) treatments. A representative EOG response is shown to indicate the measured parameter. ALA-1= L-alanine 10-1 M, ALA-2= L-alanine 10-2 M, CAC=chemical alarm cue, BS=brine shrimp rinse, 10% BS= 10% dilution brine shrimp rinse, TCA-3=taurocholic acid 10-3 M. Means are plotted with standard error. No significant differences were found between oil-exposed and control individuals (p>0.05). Across treatment groups the duration of the response to the brine shrimp rinse cue was significantly different than the response duration to L-alanine 10-1 M (p

Damsels in distress: Oil exposure modifies behavior and olfaction in

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