Inducible Tolerance to Agrochemicals Was Paved by Evolutionary

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Inducible tolerance to agrochemicals was paved by evolutionary responses to predators Devin K. Jones, William D Hintz, Matthew S Schuler, Erika K Yates, Brian M Mattes, and Rick A. Relyea Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b03816 • Publication Date (Web): 31 Oct 2017 Downloaded from http://pubs.acs.org on November 4, 2017

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Title: Inducible tolerance to agrochemicals was paved by evolutionary responses to predators Authors: Devin K. Jones,*,† William D. Hintz,† Matthew S. Schuler,† Erika K. Yates,† Brian M. Mattes,† and Rick A. Relyea† Affiliations: † Darrin Fresh Water Institute, Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, New York 12180, USA * Address correspondence to: Devin K. Jones Department of Biological Sciences Rensselaer Polytechnic Institute 1W14 Jonsson-Rowland Science Center 110 Eight Street Troy, NY 12180-3590 E-mail: [email protected] Phone: (724) 681-2084 Conflict of interest: The authors declare no conflicts of interest.


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Abstract Recent research has reported increased tolerance to agrochemicals in target and non-

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target organisms following acute physiological changes induced through phenotypic plasticity.

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Moreover, the most inducible populations are those from more pristine locations, far from

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agrochemical use. We asked why do populations with no known history of pesticide exposure

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have the ability to induce adaptive responses to novel agrochemicals? We hypothesized that

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increased pesticide tolerance results from a generalized stressor response in organisms, and

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would be induced following sublethal exposure to natural and anthropogenic stressors. We

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exposed larval wood frogs (Lithobates sylvaticus) to one of seven natural or anthropogenic

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stressors (predator cue (Anax spp.), 0.5 or 1.0 mg carbaryl/L, road salt (200 or 1000 mg Cl-/L),

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ethanol-vehicle control, or no-stressor control) and subsequently tested their tolerance to a lethal

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carbaryl concentration using time-to-death assays. We observed induced carbaryl tolerance in

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tadpoles exposed to 0.5 mg/L carbaryl and also in tadpoles exposed to predator cues. Our results

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suggest that the ability to induce pesticide tolerance likely arose through evolved anti-predator

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responses. Given that anti-predator responses are widespread among species, many animals

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might possess inducible pesticide tolerance, buffering them from agrochemical exposure.



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Introduction Anthropogenic activities have modified nearly every environment worldwide.1 Novel

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environmental conditions can act as selective forces, leading to evolutionary responses in many

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species.2-4 However, it remains unknown whether contemporary evolutionary responses to

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anthropogenic activities have natural precursors.4,5 For example, evolved responses to predators

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include changes in the physiology, morphology, or behavior of numerous prey species.6,7 Similar

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responses have been reported in organisms following exposure to anthropogenic activities.4,8,9

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Therefore, one could posit that novel evolutionary responses to human-caused stressors were

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forged from responses to natural stressors.4,8

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Freshwater ecosystems are increasingly threatened by multiple anthropogenic

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contaminants.10-12 Run-off and atmospheric deposition following the application of

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anthropogenic chemicals carries contaminants of concern, including agrochemicals and road

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salts, into adjacent systems.13,14 For example, the annual application of over 24.5 million metric

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tons of road salt (e.g., NaCl) in the United States is associated with the salinization of freshwater

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ecosystems.15,16 Aquatic organisms are highly sensitive to chemical contaminants, often at levels

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below concentrations found in affected systems.17-19 Thus, it is important to understand how

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species respond to these novel chemical stressors.

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The paradigm for evolutionary responses of organisms to anthropogenic contaminants

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has been selection over many generations.20-21 However, many organisms induce morphological,

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behavioral, or physiological responses to human-induced environmental changes within a single

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generation through the use of phenotypic plasticity—the ability of a single genotype to produce

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multiple phenotypes in response to their immediate environment.4,22 Populations that are affected

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by anthropogenic contaminants likely use multiple evolutionary responses.4,23,24 For example,


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amphibian populations located close to agriculture (a proxy for frequency of exposure to

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pesticides) that display high constitutive tolerance to pesticides, rarely show changes in their

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tolerance to pesticides following a sublethal pesticide exposure.25,26 In contrast, populations far

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away from agriculture that are highly susceptible to pesticide exposure exhibit inducible

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tolerance to pesticides following short-term sublethal pesticide exposure. Due to the relatively

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recent development of synthesized chemicals, it is unclear how organisms induce adaptive

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responses to stressors never experienced throughout their evolutionary history.

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Although the exact processes by which species respond to stressful novel chemicals, like

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pesticides, have largely remained elusive, contaminants induce a wide array of physiological

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responses in affected organisms.27-29 Induced responses, such as the increase in stress hormone

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concentration, have also been observed in species exposed to predator cues.29 As many species

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induce anti-predator responses, they may possess the ability to induce adaptive responses to

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anthropogenic contaminants. Our objective was to determine if tolerance to an anthropogenic

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contaminant is linked with the evolutionary response to natural stressors. We tested the induced

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tolerance of wood frogs (Lithobates sylvaticus) tadpoles to the carbamate insecticide carbaryl

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following the exposure to one of seven sublethal anthropogenic or natural stressors. We

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hypothesized that inducible pesticide tolerance represents a generalized stressor response shared

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among natural and anthropogenic stressors. We predicted that tadpoles exposed to predator cues,

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carbaryl, or road salts would induce increased tolerance to carbaryl.

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Materials and Methods Model system Amphibians are an excellent model organism to investigate evolved responses to natural and anthropogenic stressors.30 Their use of aquatic systems to breed and oviposit and in larval



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developmental stages might expose individuals to a variety of stressors, including predators,6

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agrochemicals,14,31 and road salt (i.e., sodium chloride).32-34 Wood frog tadpoles in particular

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have been shown to induce highly plastic morphological and behavioral responses to predation.6

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Although agrochemicals are relatively novel in an evolutionary sense, wood frog populations

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close to agriculture have evolved tolerance to a number of organophosphate and carbamate

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pesticides.26,35 Moreover, wood frog tadpoles have been shown to induce increased tolerance to

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carbaryl within a single generation following sublethal exposure to carbaryl in an earlier life

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stage.25,26 Lastly, the rapid salinization of freshwater systems due to the application and runoff of

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road salt provides yet another anthropogenic stressor for wood frogs, as they are one of the most

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sensitive amphibian species to increasing chloride concentrations.17,32

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Pesticide background

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We chose to use the carbamate insecticide carbaryl (CAS 63-25-2) for the sublethal and

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lethal pesticide exposures. Carbaryl has been historically applied in the agricultural sector to

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control pest species and increase yields of crops such as tomatoes and apples, but has become

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heavily applied (1.8-2.7 million kg active ingredient used in 2006) in the U.S. home and garden

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sector as a broad-spectrum insecticide for numerous pest species.36 Though the half-life of

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carbaryl in aquatic environments is 10.5 d at a pH of 7, it is often observed in freshwater systems

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at concentrations ≤ 1.5 mg/L.37,38 Furthermore, the aerial drift of carbamates in to freshwater

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systems is linked to amphibian population declines.14 Carbaryl is an acetylcholine esterase

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inhibitor that reversibly binds to the active site of acetylcholine esterase, inhibiting the removal

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of acetylcholine from nerve cell receptors. Consequently, overstimulation of nerve cells results in

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sporadic spasms and ultimately death in affected organisms.

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Experimental design


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We conducted the experiment at the Rensselaer Aquatic Laboratory at Rensselaer

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Polytechnic Institute (Troy, NY, USA). We collected 10 newly oviposited wood frog egg masses

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on 14 March 2016 from a nearby pond (42°37'37.3"N, 73°33'54.9"W) far from agriculture (>

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1,000 m). Collected egg masses were placed in outdoor 500-L plastic pools filled with 400 L of

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aged tap water where they developed under ambient conditions. Hatchling tadpoles were fed

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rabbit chow (Bunny 16; Blue Seal, Muscatine, IA, USA) ad libitum.

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We employed a two-phase experiment similar to Hua et al.25 In Phase 1, tadpoles were

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exposed to one of seven sublethal stressors including a common insecticide, road salt, and

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predator cues. In Phase 2, the exposed tadpoles were placed in either a no-insecticide control or

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lethal insecticide treatment using time-to-death (TTD) assays to determine if sublethal exposure

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to stressors increased their pesticide tolerance.

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Phase 1: Sublethal exposure to natural and anthropogenic stressors

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The goal of Phase 1 was to expose tadpoles to sublethal anthropogenic and biotic

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stressors. We exposed tadpoles to predator cue, 200 or 1000 mg Cl-/L of road salt (NaCl), 0.5 or

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1.0 mg carbaryl/L, an ethanol-vehicle control, or a no-stressor control. Sodium chloride

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concentrations used were representative of concentrations found in freshwater ponds and

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wetlands receiving runoff following road salt applications.17,32 Likewise, the carbaryl

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concentrations employed were below concentrations found in freshwater systems following

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direct overspray and runoff,37,38 and are known to induce increased pesticide tolerance in gray

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treefrogs (Hyla versicolor) and wood frogs.25,26,39 Each treatment was replicated five times for a

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total of 35 experimental units.

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Experimental units were 14-L opaque plastic containers filled with 10 L of UV-

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irradiated, carbon-filtered water on 2 May 2016 and acclimated to laboratory conditions



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overnight (19.1°C). Each experimental unit contained an inverted 500-mL plastic cup covered

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with mesh screen, which was used to either house dragonfly larvae (Anax junius) or remain

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empty. Such cages are commonly used to expose prey to predator cues without allowing the

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predators to kill any focal prey. On 3 May 2016 (day 0), we added 25 tadpoles to each

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experimental unit (mean mass ± 1 SE = 117 ± 3 mg; mean Gosner40 stage ± 1 SE = 26.8 ± 0.1)

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from a mixture of all ten egg masses. In addition, 20 tadpoles were placed in 10 L of carbon-

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filtered, UV-irradiated water and fed ground rabbit chow ad lib to assess 24-hr survival

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following handling. Survival after 24 hrs was 100%.

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We then applied the sublethal treatments to each experimental unit. We exposed tadpoles

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to predatory stress by feeding each caged dragonfly larvae 301 ± 7 mg of live wood frog biomass

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every 24 hr. Wood frog prey were cultured in outdoor 500-L pools (as described above) and

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were haphazardly selected from a mixture of individuals from all clutches to limit response bias

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of experimental animals. Wood frog behavioral and morphological responses to predation

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plateau when predators consume > 300 mg of prey biomass.41 Dragonfly larvae were fed every

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24 hrs to offset the rapid breakdown of kairomones.41,42 To obtain the nominal chloride

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concentrations of 200 and 1000 mg/L, we added 2.67 and 15.83 g of ground NaCl, respectively

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(Solar Salt; Morton, Chicago, IL, USA). To create the two sublethal carbaryl treatments, we first

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made a carbaryl stock solution by dissolving technical grade carbaryl (Pestanal® analytical

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standard; Sigma-Aldrich, St. Louis, MO, USA) in ethanol. To obtain the nominal concentrations

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of 0.5 and 1.0 mg carbaryl/L, we applied 250 and 500 µL of the stock solution, respectively.

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Previous research has shown little sorption of carbaryl by plastic experimental units.43 Lastly, we

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created the ethanol vehicle and no-stressor controls by applying 500 µL of ethanol and water,

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respectively, matching the highest volume of liquid added to the sublethal carbaryl treatments.


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We did not conduct water exchanges or renew pesticide concentrations over the entire 91-hr

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sublethal exposure period.

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Fasting amphibian larvae during short-term laboratory tests (i.e., 96-hr LC50) is standard

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practice in toxicological studies.44 However, we extended sublethal exposure to 91 hr, and

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expected tadpoles to be exposed to lethal carbaryl concentrations in Phase 2 for an additional 48

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hr. Therefore, we fed tadpoles 290 mg of ground rabbit chow per experimental unit, representing

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a ration that is 10% of total tadpole mass, after 72 hr in the sublethal exposures of Phase 1.

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We measured physiochemical parameters in each experimental unit on 5 May (day 2)

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using a calibrated, multi-probe meter (YSI Incorporated, Yellow Springs, OH, USA). Data

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collected indicated suitable dissolved oxygen concentrations (9.01-10.38 mg/L) and pH levels

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(7.6-7.9) for amphibian larvae. Mean specific conductance (µS/cm) was between 181.5 (± 2.7)

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and 191.9 (±7.5) in the no-stressor control, ethanol vehicle, predator cue, and carbaryl

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treatments, and 664.5 (± 22.4) and 2865.8 (± 41.2) in the 200 and 1,000 mg Cl-/L treatments,

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respectively.

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Phase 2: Time-to-death experiments to assess induction of increased pesticide tolerance

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To determine if sublethal exposure to anthropogenic and biotic stressors induced

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increased pesticide tolerance, we used time-to-death (TTD) assays on animals exposed in Phase

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1. Time-to-death assays use lethal concentrations to assess the relative tolerance of organisms

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following exposure to a given treatment or stressor and are commonly used in toxicological

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studies that investigate survivorship over time when 100% mortality is not expected.45 To discern

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the extent of an organism’s relative tolerance, lethal concentrations used in TTD assays

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commonly exceed concentrations found in natural systems. However, variation in tolerance to a

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lethal concentration is thought to translate to a variety of effects on organisms exposed to



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environmentally relevant concentrations.45,46 We crossed the seven Phase 1 sublethal treatments

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with a no-carbaryl control (water) and a lethal carbaryl concentration (18 mg/L). We chose 18

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mg/L carbaryl for the lethal concentration based on previous toxicological studies25,26 and our

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own pilot data. The 14 treatment combinations were replicated 8 times for a total of 112

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experimental units.

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Phase 2 experimental units were 100-mL glass Petri dishes filled with 70 mL of water or

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carbaryl-treated water. We first created a carbaryl stock solution on 7 May (day 4) by dissolving

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120 mg of technical grade carbaryl in 6 mL of ethanol, and then applied 5.85 mL of the stock

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solution to 6.5 L of carbon-filtered, UV-irradiated water to obtain a nominal concentration of 18

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mg carbaryl/L. We prepared the no-carbaryl control by mock-dosing 6.5 L of carbon-filtered,

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UV-irradiated water with 5.85 mL of water. We first distributed carbaryl-treated water to each

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respective petri dish to avoid contamination of no-carbaryl control replicates. We pooled

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individuals in each of the sublethal treatments of Phase 1 and then haphazardly added seven

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tadpoles from each treatment to each of the eight replicates of Phase 2 treatments, resulting in

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the use of 56 individuals per TTD treatment and 784 total tadpoles. Survival checks were

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conducted every hour for 12 hrs. Tadpoles were not fed during the TTD assay. Surviving

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individuals were euthanized using MS-222 overdose approved by the Rensselaer Polytechnic

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Institute’s Institutional Animal Care and Use Committee protocol #REL-001-15.

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Pesticide analysis

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To confirm pesticide concentrations used in Phase 1 and Phase 2, we extracted two

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samples from each pesticide treatment following carbaryl application. We first added 2 mL of

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methylene chloride preservative (CAS 75-09-2; Fisher Scientific, Waltham, MA, USA) to each

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pre-cleaned glass, amber jar (VWR International, Radnor, PA, USA), added 500 mL of either the


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no-carbaryl control or carbaryl-treated water, and refrigerated each sample at 3ºC. The no-

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carbaryl control water and the 0.5 and 1.0 mg carbaryl/L treatments were sampled on 3 May (day

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0). We sampled the lethal carbaryl treatment on 7 May (day 4). One sample of each treatment

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was then transported on ice to the University of Connecticut’s Center for Environmental Science

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and Engineering (Storrs, CT, USA) for analysis on 10 May (day 7). Analysis of the four samples

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showed no detectable levels of carbaryl in the control, but detected 0.48, 0.93, and 17.01 mg

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carbaryl/L for the nominal concentrations of 0.5, 1.0, and 18 mg carbaryl/L. Given that actual

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carbaryl concentrations fell within 7% of nominal concentrations, we will refer to carbaryl

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concentrations using their nominal values hereafter.

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Statistical analysis

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To examine how the exposure to one of seven sublethal stressors modified wood frog

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tolerance to carbaryl, we compared the survival curves of tadpoles exposed to a lethal carbaryl

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concentration in Phase 2. Specifically, we compared the 12-hr survival curves of tadpoles that

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had been previously exposed to a sublethal stressor in Phase 1 (0.5 or 1.0 mg carbaryl/L, 200 or

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1,000 mg Cl-/L, predator cue, or ethanol vehicle) to the survival curve of tadpoles that had not

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been previously exposed to stressors (i.e., no-stressor control)(Table 1). Cox’s47 proportional

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hazard model was used to analyze the survival data among lethal carbaryl treatments (IBM SPSS

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Statistics; Version 22).

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We then used a second Cox’s proportional hazard model to compare the magnitude of

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induced tolerance among Phase 1 treatments that significantly differed (p < 0.05) from the no-

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stressor control. Specifically, we compared the 12-hr survival curves of tadpoles from Phase 1

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treatments to the survival data of tadpoles that had been previously exposed to 0.5 mg/L



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carbaryl, as it induced the highest magnitude of increased tolerance to carbaryl. The survival data

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of tadpoles from all Phase 1 sublethal treatments were included in both models.

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Results In Phase 1, tadpole survival was high across all sublethal exposure treatments (96-100%).

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In Phase 2, tadpoles that were assigned to the no-carbaryl control experienced 100% survival,

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regardless of sublethal Phase 1 treatment. In contrast, tadpoles that were assigned to the lethal

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concentration of carbaryl experienced 38-86% survival, depending on their earlier Phase 1

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treatment. In Phase 2, tadpoles previously exposed to 0.5 mg carbaryl/L in Phase 1 exhibited

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increased tolerance compared to the tolerance of tadpoles with no previous exposure history (p

Inducible Tolerance to Agrochemicals Was Paved by Evolutionary

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