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Aquatic Toxicology xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Aquatic Toxicology

journal homepage: www.elsevier.com/locate/aquatox

Effects of a pesticide and a parasite on neurological, endocrine, and

behavioral responses of an estuarine fish

a,b,∗ b,c d

Violet Compton Renick , Kelly Weinersmith , Doris E. Vidal-Dorsch ,

a

Todd W. Anderson

a

San Diego State University, Department of Biology and Coastal and Marine Institute, 5500 Campanile Drive, San Diego, CA 92182, United States

b

University of California Davis, Department of Environmental Science and Policy, One Shields Avenue, Davis, CA 95616, United States

c

Rice University, BioSciences, 6100 Main Street, Houston, TX 77005, United States

d

Southern California Coastal Water Research Project, 3535 Harbor Blvd. Suite 110, Costa Mesa, CA 92626-1437, United States

a r t i c l e i n f o a b s t r a c t

Article history: In coastal waters, pesticides and parasites are widespread stressors that may separately and interac-

Received 1 June 2015

tively affect the physiology, behavior, and survival of resident organisms. We investigated the effects

Received in revised form

of the organophosphate pesticide chlorpyrifos and the trematode parasite Euhaplorchis californiensis on

11 September 2015

three important traits of California killifish ( parvipinnis): neurotransmitter activity, release of

Accepted 21 September 2015

the stress hormone cortisol, and behavior. Killifish were collected from a population without E. cali-

Available online xxx

forniensis, and then half of the fish were experimentally infected. Following a 30 day period for parasite

maturation, infected and uninfected groups were exposed to four concentrations of chlorpyrifos (sol-

Keywords:

vent control, 1–3 ppb) prior to behavior trials to quantify activity, feeding behavior, and anti-predator

Fundulus parvipinnis

responses. Water-borne cortisol release rates were measured non-invasively from each fish prior to infec-

Euhaplorchis californiensis

Chlorpyrifos tion, one-month post-infection, and following pesticide exposure. Killifish exposed to 3 ppb chlorpyrifos

Parasites exhibited a 74.6 ± 6.8% and 60.5 ± 8.3% reduction in brain and muscle acetylcholinesterase (AChE) activity

Multiple stressors relative to controls. The rate of cortisol release was suppressed by each chlorpyrifos level relative to con-

Behavior

trols. Killifish exposed to the medium (2 ppb) and high (3 ppb) pesticide concentrations exhibited reduced

activity and a decrease in mean swimming speed following a simulated predator attack. Muscle AChE

was positively related to swimming activity while brain AChE was positively related to foraging behav-

ior. No effects of the parasite were observed, possibly because of low metacercariae densities achieved

through controlled infections. We found that sublethal pesticide exposure has the potential to modify

several organismal endpoints with consequences for reduced fitness, including neurological, endocrine,

and behavioral responses in an ecologically abundant fish.

© 2015 Elsevier B.V. All rights reserved.

1. Introduction consequences (McGourty et al., 2009; Dachs and Méjanelle, 2010),

and parasites are common and significant biological stressors

Coastal and marine environments consist of a myriad of chemi- in marine systems (Lafferty, 2013). When considered separately,

cal, biological, and physical stressors that challenge the survival of pollutants and parasites can exert considerable effects on the physi-

resident biota. Among marine and estuarine stressors, pollutants ology, metabolism, reproduction, behavior, and survival of resident

and parasites are ubiquitous and highly influential on the fit- organisms. However, their potential interactive effects on organ-

ness and survival of resident organisms (Marcogliese and Pietrock, isms including fishes remain largely unknown (Sures, 2008a,b). As

2011). Pesticides and heavy metals are carried by run-off into organisms are exposed to both stressors concurrently, it is crucial

coastal urban where they can negatively affect physio- to understand the combined effect of pesticides and parasites on

logical traits of resident organisms and have significant ecological the host phenotype.

Pollutants can disrupt neurological and endocrine function as

well as behavior. Among pollutants, organophosphate pesticides

are one of the most globally pervasive and neurotoxic chemi-

∗

Corresponding author at: Grossmont College, Department of Biology, 8800 cal groups (Saunders et al., 2012). Organophosphate pesticides

Grossmont College Dr., San Diego, CA 92020, United States. can severely modify the neurological and endocrine responses

E-mail address: [email protected] (V.C. Renick).

http://dx.doi.org/10.1016/j.aquatox.2015.09.010

0166-445X/© 2015 Elsevier B.V. All rights reserved.

Please cite this article in press as: Renick, V.C., et al., Effects of a pesticide and a parasite on neurological, endocrine, and behavioral

responses of an estuarine fish. Aquat. Toxicol. (2015), http://dx.doi.org/10.1016/j.aquatox.2015.09.010

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of non-target organisms even at relatively low concentrations 1997; Sures, 2008b; Blanar et al., 2009). A greater understanding

(Grue et al., 1997). Organophosphate pesticides inhibit the acetyl- of the singular and interactive effects of pesticides and parasites is

cholinesterase (AChE) enzyme which regulates acetylcholine, one necessary because of the pervasiveness of both of these stressors in

of the most widely distributed neurotransmitters in vertebrates marine environments and their potential for dramatically reducing

(Behra et al., 2002; Sandahl et al., 2005). Acetylcholine is key the fitness of fishes (Marcogliese and Pietrock, 2011). More specif-

in transmitting neuronal messages to muscle cells in vertebrates ically, the neurological and endocrine mechanisms and behavioral

(Tilton et al., 2011). AChE inhibition results in an accumulation of consequences underlying their interactive effects require further

acetylcholine in the central and peripheral synapses, causing modi- investigation.

fied physiological and neuroendocrine processes (Behra et al., 2002; In this study, we investigated the neurological, endocrine, and

Sandahl et al., 2005; Tilton et al., 2011). Such physiological modi- behavioral effects of the organophosphate pesticide chlorpyrifos

fications can subsequently lead to changes in behaviors, including and the trematode parasite E. californiensis in California killifish

reduced swimming performance (Levin et al., 2004; Almeida et al., to provide a better understanding of their interactive effects on

2010; Yang et al., 2011), altered social behavior (Khalil et al., 2013), the behavior and survival of estuarine fishes. We based our study

reduced foraging (Sandahl et al., 2005), and greater predation risk on this parasite-host system due to the relatively high numerical

(Carlson et al., 1998). abundance and ecological importance of both species in Southern

The endocrine stress response is particularly sensitive to natural California estuaries, which is well documented in a rich source of

and anthropogenic stressors including pollutant exposure (Bisson literature detailing the ecological and physiological effects of par-

and Hontela, 2002; Jobling and Tyler, 2003; Brar et al., 2010). In fish, asitism by E. californiensis on California killifish (Shaw et al., 2010).

this response is characterized primarily by the release of the cor- Many other species of parasites can infect California killifish, but

ticosteroid cortisol by the hypothalamo–pituitary–interrenal (HPI) E. californiensis is typically the most abundant and prevalent para-

axis. However, links between sublethal organosphosphate expo- site found in this host species (Lafferty, 2008). Moreover, the larval

sure and the endocrine stress response have rarely been quantified cercariae of E. californiensis are easily shed in the laboratory, mak-

in fishes (Oruc¸ , 2010). These links may be important because alter- ing them a reliable and relevant source for experimental infection

ations in cortisol release rates produce acute and chronic effects (Shaw et al., 2009). We measured the activity of AChE, release of the

on an organism’s biochemistry and physiology (including impacts stress hormone cortisol, and behavioral modifications caused by

on osmoregulation and energy metabolism; Øverli et al., 2002; both stressors. We asked the following questions: (1) Are there sep-

Ezemonye and Ikpesu, 2013). Furthermore, changes to rates of arate and interactive effects of pesticide exposure and on

cortisol release may have important implications for behavior, as AChE activity and rates of cortisol hormone release in killifish? (2)

cortisol is often strongly linked to activity and locomotion, social- What are the behavioral responses of killifish exposed to either or

ity, reproduction, and foraging (Gregory and Wood, 1999; Øverli both stressors? (3) What are the relationships between the phys-

et al., 2002, 2005). iological responses (AChE activity and cortisol release rates) and

Parasites can also significantly impact the physiology, behavior, altered behaviors of killifish?

and survival of their hosts (Moore, 2002; Cézilly et al., 2013; Santos

and Santos, 2013). Parasites that are transmitted trophically can

2. Materials and methods

be particularly effective at exerting changes to host phenotype, as

they can manipulate the physiology and behavior of their hosts

2.1. Fish collection and acclimation

to increase predation by a definitive host species (Adamo, 2013;

Lafferty and Shaw, 2013). For instance, the California killifish, Fun-

California killifish were collected by beach seine from a small

dulus parvipinnis, infected with trematode parasite Euhaplorchis ◦  

isolated lagoon at Camp Pendleton, California, USA (33 15 48.1 N,

californiensis metacercariae (the larval stage which encysts on ◦  

−117 26 20.4 W). This collection site was chosen because it his-

the surface of host brain tissue) display conspicuous behaviors

torically lacked the trematode E. californiensis and the parasite’s

including surfacing, flashing, and body contortions 3–4 times more

first intermediate host (the California black horn snail, Cerithidea

frequently than do uninfected killifish. Parasitized killifish are 10 to

californica). Prior to initiating the experiment, a subset of killi-

30 times more likely to be consumed by avian predators, the defini-

fish were euthanized and dissected to confirm that experimental

tive hosts of E. californiensis (Lafferty and Morris, 1996). California

fish were not already infected with E. californiensis. Other parasitic

killifish infected with E. californiensis have altered serotonergic and

trematodes were occasionally observed in dissected killifish, but

dopaminergic neurotransmitter activity (Shaw et al., 2009; Shaw

their possible effects on behavior or physiology were controlled for

and Øverli, 2012), and such changes in neurochemistry may be

through experimental infections in an environment free of infec-

an explanatory factor behind the increased conspicuous behaviors

tious stages of other parasite species. Killifish were transported in

of infected killifish. Serotonergic activity has downstream effects

aerated coolers to the San Diego State University (SDSU) Coastal and

on cortisol synthesis (Winberg et al., 1997; Lim et al., 2013), and

Marine Institute Laboratory (CMIL), in San Diego, CA. Fish were held

thus E. californiensis may also influence cortisol release. In addition ◦

in 37.5 L aquaria in filtered seawater (0.45 ␮m, 20 ± 1 C, salinity:

to potential manipulation of cortisol release rates by the para-

∼34 ppt) for four wk under a 14:10 h light:dark photoperiod. Fish

site, the stress of infection alone may increase the rate of cortisol

were fed frozen Artemia brine shrimp (San Francisco Bay Brand, San

release. Studies of the European eel, Anguilla anguilla, infected with

Francisco, CA, USA) ad libitum once daily.

the swim-bladder parasite Anguillicola crassus and simultaneously

exposed to pollutants (cadmium and PCB-126) indicate that the

stress of a parasite infection could exacerbate the cortisol response 2.2. Cortisol collection and processing

to other stressors such as pesticides (Sures et al., 2006). Interactions

between E. californiensis and stressors such as pesticides have yet Cortisol is released from the gills of fishes, where it can be

to be explored in California killifish. extracted from the surrounding water (Scott et al., 2008). In var-

When considered in combination, pesticides and parasites may ious different species, cortisol release rates mirror concentrations

act additively or synergistically to reduce the survival of exposed of cortisol in the plasma, allowing for non-invasive and repeated

fishes (Sures, 2008a). However, few studies in ecotoxicology incor- measurements of cortisol and other steroid hormones (Ellis et al.,

porate the effects of parasites, and there remain many gaps in 2013). The relationship between cortisol release rates and plasma

knowledge surrounding their potential interactive effects (Lafferty, cortisol is also observed in California killifish (Weinersmith, unpub-

Please cite this article in press as: Renick, V.C., et al., Effects of a pesticide and a parasite on neurological, endocrine, and behavioral

responses of an estuarine fish. Aquat. Toxicol. (2015), http://dx.doi.org/10.1016/j.aquatox.2015.09.010

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lished data). To initiate cortisol collection, individual fish were first in groups of three to 10 L tanks for a 2 day acclimation period prior

captured from their home tank using a hand net and gently placed to exposure to sublethal concentrations of chlorpyrifos for 4 days.

into a clean 500 mL glass jar filled with 400 mL of artificial seawater Following exposure, the final cortisol collection (T2) was conducted

®

(distilled water and Instant Ocean , Blacksburg, VA, USA; salinity: before fish were placed in experimental aquaria for behavioral

∼33 ppt). The glass jars and hand net were cleaned with 95% ethanol observations. Killifish were euthanized with an overdose of tricaine

and rinsed with deionized water prior to use. Handling time was methanesulfonate (MS-222), measured for length and weight, and

recorded as the time between capture of the fish in the aquarium dissected for tissue collection.

and depositing of the fish into the glass jar. Only one fish was placed

in each jar, which was visually isolated from external stimuli by a lid 2.4. Experimental parasite infections

and opaque siding. Individuals remained in the glass jar for 1 h, after

which they were removed by gently pouring the contents through a California horn snails were collected from marshes within San

clean net into a secondary clean jar. Total time in the experimental Diego Bay and placed into cool, dark, and damp 12 L containers at

jar was recorded. CMIL for a minimum of 3 days. To induce cercariae release, snails

Each water sample was filtered using Whatman filter paper were placed into small beakers of warm seawater under a heat

(Grade 1, 24 cm diameter) and passed through a C18 Sep–Pak lamp. Cercariae were identified to species (Martin, 1950a,b) and

column (3 cc, 500 mg; Waters, Milford, MA, USA) using a vac- those of E. californiensis were pooled for experimental infection.

uum manifold. Prior to use, the column was cleaned with 4 mL of Five 1 mL samples of cercariae were later counted from a homoge-

methanol and primed with 4 mL of distilled water. The columns nized and pooled sample to determine the density and variability

◦

were stored at −20 C before being shipped to the University of of parasite abundance for each replicate. The average abundance of

◦

Alabama (Tuscaloosa, AL, USA) on dry ice and again stored at −20 C. larval cercariae used to infect killifish was 6062.0 cercariae per mL

Free hormones were then eluted with 4 mL ethyl acetate into (±690.2 SD).

◦

13 mm × 100 mm borosilicate vials and stored at 4 C. Ethyl acetate California killifish were removed from their 37.5 L acclimation

◦

was later evaporated by placing vials in a water bath (38.7 C) and aquarium and divided equally and randomly into two 5 L aquaria

running a gentle stream of nitrogen over each sample. Cortisol and given 1 h to recover from handling stress. Cercariae were gently

was re-suspended by adding 40 ␮L of 100% ethanol, vortexing for released by glass pipette into one of the aquaria for a one hour infec-

1 min, adding 760 ␮L Enzyme Immunoassay (EIA) buffer, and then tion period, while the second group was exposed only to salt water

vortexing again for 10 min. Cortisol samples were assayed in dupli- released by pipette as a procedural control. Infected fish were then

cate using EIA kits according to instructions provided with the kits removed by hand nets, rinsed with clean seawater to remove any

(Cayman Chemicals, Ann Arbor, MI, USA). Plates were read on a external cercariae, and combined with uninfected fish into their

405 nm BioTek plate reader (model ELx808, Winooski, VT, USA). original acclimation aquarium. Uninfected fish were subjected to

Twenty-five samples were further diluted and run again to bring the same handling treatments as the procedural control. All killifish

their values closer to the provided standard curve to increase mea- were subsequently given 27–28 days before further measurement

surement accuracy. All cortisol samples were standardized by fish to allow cercariae to mature and encyst on the brain (following

mass (pg/g/h). Shaw et al., 2009). At the termination of the experiment, metacer-

Water-borne cortisol pools were created by pipetting 100 ␮L cariae were counted from brain homogenates under a compound

from each vortexed sample into a 15 mL Falcon tube. The pooled microscope.

◦

sample was stored at 4 C and then vortexed prior to pipetting onto

each plate. The cortisol pool was used to calculate intra-assay coef- 2.5. Pesticide exposure and AChE measurements

ficients of variation (range: 1.1–7.3%, median: 4.6%) and inter-assay

coefficients of variation (12.1%). Following the second cortisol measurement (T1), all experimen-

tal fish were matched for size and placed into 10 L glass aquaria

2.3. Experimental design to conduct experimental tests. Killifish were housed in groups of

three and were given 2 days to acclimate in experimental aquaria

Following acclimation of 4–5 days, an initial water-borne cor- to decrease handling stress and possible increased mortality due

tisol measurement (T0) was collected from individual killifish to pesticide exposure. A 4 day chlorpyrifos exposure was initi-

(n = 128) to assess their cortisol release rate prior to pesticide or ated within experimental aquaria. A chlorpyrifos stock solution,

parasite exposure. Immediately following hormone collection, all O,O-diethyl O-3,5,6-trichloropyridin-2-yl phosphorothioate (Chem

fish were uniquely marked with visible implant elastomer (VIE) Service, West Chester, PA, USA, 99.5% purity) was made using

tags (Northwest Marine Technologies, Inc., Shaw Island, WA, USA) acetone as a solvent. Test concentrations were chosen based on

to allow for individual recognition throughout the experimental previous sublethal exposures of juvenile killifish to chlorpyrifos for

period. Tags were made using combinations of four colors injected 4 days known to cause behavioral abnormalities (Renick, unpub-

in four locations on the caudal peduncle (two tags on each of lished data) and to represent a range of environmentally relevant

the lateral sides). Approximately 2–4 days after preliminary hor- concentrations of chlorpyrifos measured in the field (Phillips et al.,

mone collection, half of all collected killifish were experimentally 2012). Final nominal concentrations included a solvent control

infected (see Section 2.4) with the larval form of the E. californien- (hereafter referred to simply as control) and a low (1 ppb), medium

sis parasite (cercariae) and allowed to recover and acclimate in the (2 ppb), and high (3 ppb) concentration, and 70% of tank seawater

laboratory for 27–28 days. E. californiensis metacercariae are able was renewed daily.

to infect avian hosts after 14 days (Martin, 1950a), and parasite- At the termination of the experiment, brain and lateral mus-

associated changes in neurochemistry have been observed 28 days cle tissue of frozen killifish were homogenized to quantify AChE

post-infection (Shaw et al., 2009). Following this secondary accli- activity in response to pesticide exposure following a modified

mation period, a second round of water-borne cortisol collection Ellman assay (Ellman et al., 1961; Sandahl and Jenkins, 2002).

(T1) was conducted to measure changes in cortisol response to Tissues were weighed and blended using a tissue homogenizer

®

parasite infection. Next, parasitized and control individuals were (Fisher PowerGen Model 125) at a ratio of 1:20 w/v with 1 mM

randomly assigned to one of four sublethal chlorpyrifos pesticide PBS and 1% Triton X-100. Samples were centrifuged at 6000 rpm

◦

treatments (control, 1–3 ppb) in a fully orthogonal factorial design for 10 min at 4 C. Sample supernatant was collected and incu-

(n = 128; 8 replicates; 16 fish/treatment). Killifish were transferred bated in 0.7 mM DTNB (Sigma–Aldrich Chemical Co., St. Louis, MO,

Please cite this article in press as: Renick, V.C., et al., Effects of a pesticide and a parasite on neurological, endocrine, and behavioral

responses of an estuarine fish. Aquat. Toxicol. (2015), http://dx.doi.org/10.1016/j.aquatox.2015.09.010

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◦

USA) for 5 min at 25 C. Preliminary studies included simultaneous

incubation with the selective butylcholinesterase (BChE) inhibitor

iso-OMPA (tetraisopropyl pyrophosphoramide), but no significant

BChE expression was observed and this step was discontinued.

Acetylthiocholine iodide (3 mM) was used as the enzyme substrate

and was added to the samples in a 96-well microtiter plate. Each

96-well plate contained tissue and substrate blanks. Samples were

assayed in triplicate and averaged to provide a single value. Acetyl-

◦

cholinesterase enzymatic activity was determined at 25 C using a

microplate reader (Metertech M965/965+) with absorbance moni-

tored at 412 nm. A Pierce BCA Protein Assay Kit (Rockford, IL, USA)

was used to determine the total protein concentration in each sam-

ple.

2.6. Behavioral observations

Fig. 1. Proportional inhibition of AChE in killifish brain and muscle tissue relative

Behavioral trials were conducted in a 75.7 L glass aquarium to controls after a 4 day chlorpyrifos exposure. Chlorpyrifos concentrations: con-

trol, low (1 ppb), medium (2 ppb), and high (3 ppb). Each bar represents the mean

× ×

(61 33 43 cm) immediately after the 4 day pesticide exposure.

proportion ± SE of AChE activity relative to the control mean for each tissue type.

The aquarium consisted of a sandy bottom with two pieces of PVC

Letters above bars denote significant differences among pesticide concentrations for

(2.5 cm diameter) at each end of the aquarium. Blinds covered the

both tissue types (p < 0.01; n = 113 and 125 across 8 replicates for brain and muscle

sides of each aquarium to minimize the influence of observer activ- tissue, respectively).

ity on behavior, and a digital camera (Canon Powershot A2200)

was placed in a small opening in the blind to record fish behavior.

start of the experiment. AChE, cortisol, and behavioral responses

Individuals were removed from their pesticide exposure tank and

were analyzed using two-factor analysis of variance with pesticide

placed into the experimental tank for 30 min of acclimation.

concentration (categorical variable) and parasite intensity (contin-

Individuals were subjected to three consecutive behavioral

uous variable) as the main factors. When results were significant,

assays: activity and water column position (5 min), foraging

Tukey’s pairwise multiple comparison tests were used to determine

(5 min), and response to a simulated predator attack (10 min). In

differences among treatments. Handling time and its interaction

the first assay, the swimming activity (distance traveled), average

with metacercariae intensity were included as covariates in each

swimming speed, and position of each fish relative to the surface

model, but these terms did not significantly influence model out-

was quantified (see below). In the second assay, 0.5 mL of thawed

come and were excluded from further tests. Relationships between

adult Artemia was dropped from a tube held above the aquarium.

physiological responses (i.e., cortisol release rates and AChE activ-

Latency to begin foraging and the number of foraging strikes were

ity in brain and muscle tissue) and behaviors were examined using

quantified. For the third and final assay, a pulley system positioned

Pearson product-moment correlation coefficients. When more than

above the aquarium was used to remotely control a model of a

one physiological variable was correlated with a particular behav-

heron that simulated an avian predator attack. Additional thawed

ior, partial correlation analyses were conducted to adjust for the

Artemia (0.5 mL) were released into the tank and once a fish began

effect of each physiological variable independent of the others. All

to forage, or after 2 min if foraging did not occur, the model preda-

statistical analyses were conducted using Systat (ver.12).

tor struck once in the center of the tank. The speed and distance

traveled by each fish in the 10 s immediately following attack were

3. Results

quantified (see below). Experimental aquaria were emptied and

refilled following each trial to minimize potentially confounding

Final experimental sample size consisted of a total of 128 killifish

chemical cues, possible pesticide residues, and variability in tem-

across eight replicates (30 ± 0.2 mm [mean ± SE] standard length

perature or dissolved oxygen content. Individual fish, identified by

(SL); 0.6 ± 0.02 g [mean ± SE] wet weight). Four cortisol samples

their tag, were measured (weight, standard length [SL]) and euth-

were lost, while 15 brain samples and 3 muscle samples were

anized using an overdose of MS-222.

excluded from AChE measurement due to a prohibitively small

To quantify activity and swimming behaviors, photographic

tissue mass. An average of 49 ± 3.4 metacercariae cysts per brain

stills were scan-sampled from every second of each video using

(range: 5–150) were quantified in killifish infected with the E. cal-

the open-source program VirtualDub (v.1.10.4). Images were then

iforniensis parasite.

analyzed using Image J Analysis (National Institutes of Health,

Bethesda, MD). The x–y coordinates of each fish were measured and

used to calculate distance traveled and swimming speed. Latency to 3.1. Inhibition of AChE in brain and muscle tissue

forage and the number of foraging strikes were visually quantified

from videos using Etholog 2.25 (Ottoni, 2000). AChE activity was inhibited by chlorpyrifos exposure in kil-

lifish brain (F3,109 = 9.591, p < 0.001; Fig. 1) and muscle tissue

2.7. Statistical analyses (F3,121 = 9.770, p < 0.001; Fig. 1). AChE activity in control brain tis-

sue was higher than AChE activity in the brain of fish exposed to

All data were first graphed and analyzed for normality and medium and high concentrations of chlorpyrifos, but was not dif-

heteroscedasticity using Kolmogorov-Smirnov goodness-of-fit and ferent from the lowest concentration. Brain AChE activity of fish

Levene’s homogeneity of variances tests. Data that did not con- exposed to the lowest chlorpyrifos concentration was higher than

form to these assumptions were either square-root transformed AChE in the highest concentration, but was not different from the

or arcsin square-root transformed to meet the assumptions for medium concentration. Muscle AChE activity patterns mirrored

parametric tests, after which the assumptions were met. Cortisol those of brain tissue, where control fish AChE activity was higher

response was calculated as the percentage change in individual than the medium and high concentrations, but was not different

cortisol release rate from T0 to T1, and from T0 to T2, in order from the low concentration. Muscle AChE activity in the low con-

to account for individual differences in cortisol secretion at the centration was also higher than the medium concentration and the

Please cite this article in press as: Renick, V.C., et al., Effects of a pesticide and a parasite on neurological, endocrine, and behavioral

responses of an estuarine fish. Aquat. Toxicol. (2015), http://dx.doi.org/10.1016/j.aquatox.2015.09.010

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is, cortisol decreased significantly after exposure to the pesticide

even at the lowest concentrations tested. There was no significant

effect of the parasite (F1,109 = 2.316, p = 0.131), nor of its interaction

with pesticide concentration (F3,109 = 0.275, p = 0.844) on cortisol

expression from T0 to T2.

3.3. Behavioral responses

Several killifish behaviors were altered by chlorpyrifos expo-

sure, but none were singularly or interactively affected by E.

californiensis infection (Table 1). Swimming activity (measured as

distance traveled) decreased with pesticide exposure (Fig. 3a), but

Fig. 2. Proportional change (mean ± SE) in cortisol release with chlorpyrifos expo- only in the medium concentration (2 ppb) was it significantly lower

sure from T0 (baseline) to T2 (post-pesticide exposure) time points. Chlorpyrifos than the controls (p = 0.028). Foraging behavior and water column

concentrations: control, low (1 ppb), medium (2 ppb), and high (3 ppb). Different

position did not change significantly with pesticide exposure or

letters above bars denote significant differences among pesticide concentrations

parasite infection (Fig. 3b). Mean swimming speed and maximum

(p < 0.05; n = 124 across 8 replicates).

swimming speed following a simulated predator attack (Fig. 3c

& d) both decreased significantly with pesticide exposure. These

high concentration AChE. Relative to control brain and muscle tis-

behaviors were significantly reduced in only the highest concentra-

sues, respectively, chlorpyrifos exposure resulted in 89.3 ± 11.5%

tion (3 ppb) exposure treatment relative to the controls (p = 0.023,

±

and 76.5 6.8% activity at the low concentration, 44.4 ± 9.0% and

p = 0.005, respectively).

38.0 ± 6.9% activity at the medium concentration, and 25.4 ± 6.8%

and 39.5 ± 8.3% activity in the high concentration (Fig. 1).

3.4. Relationships between physiological and behavioral

3.2. Cortisol response

responses

The mean baseline rate of cortisol release was

Swimming activity (i.e., distance traveled) was positively cor-

6514.5 ± 368.9 pg/g/h. Mean cortisol release across all treat-

related with AChE activity in muscle tissue (r = 0.263, p = 4.004)

ment groups increased at T1 (post-parasite exposure) to

but not with brain AChE (r = 0.143, p = 0.132; Table 2). Swimming

7382 ± 342.6 pg/g/h, and then fell slightly at T2 (post-pesticide

activity was also positively correlated with cortisol (T2, r = 0.213,

exposure) to 6587 ± 390.8 pg/g/h. The percentage change in

p = 0.018). However, when cortisol was adjusted for AChE, it became

cortisol of killifish infected with E. californiensis from T0 to T1

less strongly associated with activity (r = 0.155, p = 0.092) while

was not significantly different from that of uninfected killifish

AChE adjusted for cortisol was still significantly correlated with

(F1,115 = 2.572, p = 0.112).

activity (r = 0.216, p = 0.018). Additionally, brain AChE was posi-

The proportional change in cortisol response from T0 to T2

tively correlated with foraging behavior (r = 0.190, p = 0.045). No

indicated that cortisol was suppressed in killifish exposed to all

anti-predator behaviors were related to AChE activity in brain or

concentrations of chlorpyrifos (F3,109 = 6.706, p < 0.001; Fig. 2). Cor-

muscle tissue (Table 2). Behaviors related to foraging and anti-

tisol released by killifish in each pesticide treatment group was

predator responses, however, were not significantly correlated

reduced relative to the controls (p < 0.01), but the cortisol release

with cortisol concentration (Table 2).

was not significantly different among the treatment groups. That

Table 1

Effects of sublethal chlorpyrifos exposure and E. californiensis infection on killifish behaviors. Results are generated from a two-factor ANOVA. Bolded values indicate a factor

had a significant effect on behavior (p < 0.05). The direction of each significant effect is indicated by a negative sign (decrease) or positive sign (increase).

Variable Factor df MS F-ratio p-value Effect

Distance from surface Parasite 1 2229.377 0.784 0.378 n/a

(mm) Pesticide 3 2015.933 0.709 0.548

Parasite × pesticide 3 2536.46 0.893 0.447

Error 120 2841.868

Distance traveled (mm) Parasite 1 1087.83 0.923 0.339 (−)

Pesticide 3 3344.55 2.837 0.041

Parasite × pesticide 3 387.35 0.329 0.805

Error 119 1178.883

Foraging: Number of Parasite 1 1.102 0.427 0.515 n/a

strikes Pesticide 3 2.575 0.999 0.396

Parasite × pesticide 3 0.902 0.35 0.789

Error 120 2.578

Swimming speed after Parasite 1 87.736 0.296 0.587 (−)

predator attack (mm/s) Pesticide 3 866.028 2.926 0.037

Parasite × pesticide 3 153.231 0.518 0.671

Error 119 295.966

Max swimming speed Parasite 1 785.666 0.081 0.776 (−)

after predator attack Pesticide 3 39488.22 4.074 0.009

(mm/s) Parasite × pesticide 3 9189.046 0.948 0.42

Error 119 9692.672

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Fig. 3. Behavioral responses of killifish to sublethal chlorpyrifos exposure. Mean ± SE of (a) swimming activity, measured as the distance traveled (mm) for five minutes,

(b) mean number of foraging strikes, (c) mean swimming speed (mm/s) over 10 s following a simulated predator attack, and (d) maximum swimming speed (mm/s) over

10 s immediately following a simulated predator attack. Different letters above bars denote significant differences among pesticide concentrations (p < 0.05; n = 128 across 8 replicates).

Table 2

Pearson product-moment correlation coefficients between physiological measurements (cortisol, brain AChE, and muscle AChE) and killifish behaviors. In the case where

two variables were both correlated with one behavior, partial correlation analyses were conducted to adjust for the effect of either variable; these values are indicated by

the presence of a (*). Fish are pooled from all treatments (n = 128). Cortisol measurements (pg/g/hr) were collected from individuals following a 4 days exposure to sublethal

chlorpyrifos immediately before behavior trials. Bolded values denote statistical significance (p < 0.05).

Cortisol Brain AChE Muscle AChE

Behavior r p r p r p

Distance from surface (mm) 0.047 0.602 0.070 0.468 0.049 0.595

Distance traveled (mm) 0.213 0.018 0.143 0.132 0.263 0.004

0.155* 0.092* 0.216* 0.018*

Number of strikes 0.100 0.268 0.190 0.045 0.055 0.548

Distance traveled after predator attack (mm) 0.086 0.341 0.077 0.422 0.056 0.541

Average swimming speed after predator attack (mm/s) 0.046 0.613 0.104 0.279 0.092 0.313

4. Discussion muscle tissues, respectively, of fishes exposed to the highest con-

centration (3 ppb) relative to control fish (Fig. 1). Interestingly,

In this study, we provide evidence that an estuarine fish the medium and high concentrations of chlorpyrifos resulted in

exposed to sublethal concentrations of the organophosphate similar rates of AChE inhibition, indicating a possible threshold

pesticide chlorpyrifos exhibits changes in neurotransmitter activ- of AChE inhibition that has been reached after exposure to 2 ppb

ity and hormone release rates. California killifish exposed to of chlorpyrifos for this species. These inhibition rates demon-

chlorpyrifos exhibited significantly inhibited AChE activity and a strate the potent neurotoxicity caused by this pesticide in sublethal

suppressed stress hormone response. Additionally, we observed doses. Similar rates of inhibition due to chlorpyrifos exposure

several changes in activity and antipredator behavior following have been found in the east-coast congener Fundulus heteroclitus

pesticide exposure, and activity changes were correlated with the (Thirugnanam and Forgash, 1977). Comparable rates of AChE inhi-

observed changes in muscle AChE. Interestingly, no singular or bition after organophosphate pesticide exposure have also been

interactive effects of infection by the trematode parasite E. cali- shown for other fishes such as zebrafish, Danio rerio, (Tilton et al.,

forniensis were observed. 2011) and coho salmon, Oncorhynchus kisutch (Sandahl et al., 2005)

following exposure to chlorpyrifos, and have been observed in rain-

bow trout, Oncorhynchus mykiss (Beauvais et al., 2000; Brewer et al.,

4.1. AChE activity

2001) exposed to diazinon and malathion.

Sublethal exposure to chlorpyrifos for 4 days resulted in a max-

imum AChE activity of 25.4 ± 6.8% and 39.5 ± 8.3% in brain and

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4.2. Cortisol release rates exposed to the medium (2 ppb) and high (3 ppb) concentrations

relative to control fish. A reduction in swimming activity may lead

Exposure to all of the sublethal concentrations of chlorpyrifos to the inability to forage efficiently, find mates, avoid predators,

tested resulted in a suppression of cortisol release rates relative and form social aggregations. For this reason, as well as its rela-

to control fish (Fig. 2). Endocrine disrupting effects of chlorpyrifos tive ease of quantification, modification of swimming activity and

and other organophosphate pesticides have been found in mam- performance is one of the most commonly investigated behavioral

malian systems (Grue et al., 1997), yet relatively few studies have endpoints for fishes exposed to sublethal pesticides. Reductions

demonstrated such effects in fishes (Oruc¸ , 2010). Different classes in swimming activity occur for European seabass, Dicentrarchus

of pollutants, such as heavy metals, organic pesticides, and mix- labrax, exposed to sublethal fenitrothion (Almeida et al., 2010),

tures of chemicals can suppress cortisol release (Scott, 2003; Miller adult zebrafish exposed to chlorpyrifos (Tilton et al., 2011), and

et al., 2009; Firat et al., 2011). For instance, a decrease in blood larval rainbow trout exposed to malathion and diazinon (Beauvais

serum cortisol occurs in adult Oreochromis niloticus exposed to et al., 2000; Brewer et al., 2001). Although many studies have found

the organochlorine pesticide endosulfan (Ezemonye and Ikpesu, significant reductions in swimming behavior, it should be noted

2013). Similar relationships have been found for yellow perch, that the nominal test concentrations are typically much higher than

Perca flavescens, and northern pike, Esox lucius, from sites pol- those used in this study (Beauvais et al., 2000; Brewer et al., 2001;

luted by polycyclic aromatic hydrocarbons (PAHs), polychlorinated Almeida et al., 2010; Tilton et al., 2011). Thus, our study highlights

biphenyls (PCBs), and mercury (Hontela et al., 1992). the importance of considering the impacts of even low concentra-

Although the mechanisms behind cortisol suppression by pol- tions of pesticides on exposed organisms.

lutants are not fully understood, two main hypotheses have The anti-predator response of pesticide-exposed killifish was

been suggested. First, certain chemicals such as organochlorine also altered by exposure to sublethal chlorpyrifos, indicating the

pesticides may exert their endocrine disrupting effects by impair- potential increased risk of predation for fish in polluted habitats.

ing adrenocortical cell function in inter-renal tissue, leading to Exposed killifish displayed a reduction in average and maximum

a reduced production of cortisol (Bisson and Hontela, 2002). swimming speeds immediately following a simulated avian preda-

Organochlorine and organophosphate pesticides share chemical tor attack (Fig. 3). Few studies have demonstrated effects of

similarities, therefore this mechanism may be a possible driv- sublethal exposure to organophosphate pesticides on anti-predator

ing force behind cortisol suppression in this study. Second, it is behaviors. Existing evidence includes a reduction in the escape

possible that exposed fish first experienced an acute increase in behavior and subsequent increase in predation risk of medaka,

cortisol release, which ultimately led to negative feedback inhibi- Medaka medaka, exposed to chlorpyrifos (Carlson et al., 1998) and

tion (Hontela et al., 1992). Regardless of the mechanism, chronic a decrease in survival of rainbow trout exposed to the insecticides

suppression of a normal stress response has been linked to the parathion and carbamate carbaryl (Little et al., 1990). Interest-

depletion of energy reserves, immune suppression, and a reduc- ingly, although parasitism did not change anti-predator behavior in

tion in energy-mobilizing capacity which could ultimately impact this study, Lafferty and Morris (1996) note that infection increased

individual fitness and survival (Hontela et al., 1992). predation risk for infected fish by 10–30 times that relative to unin-

Contrary to predictions, cortisol release was not modified by fected fish. This increase in predation rate is likely attributed to

infection with E. californiensis. It is possible that our controlled parasite-induced conspicuous behaviors that draw the attention

infections failed to achieve physiologically relevant infection of predatory birds. In infected populations that are also exposed

intensities. Controlled infections yielded an average of 49 ± 3.4 to chlorpyrifos, killifish may display both conspicuous behaviors

metacercariae per fish, while naturally infected fish of a similar size that attract predators due to infection and exhibit less effective

would typically harbor hundreds or perhaps even a thousand par- responses to the presence of the predator due to pesticide exposure.

asites by this age (Shaw et al., 2010). A threshold parasite intensity Thus, the combination of parasites and pesticides could dramati-

may be necessary before physiological or endocrine responses due cally increase predation risk for killifish. Future studies are needed

to infection become apparent. Additionally, recent observations to tease apart how these two factors interact to determine killifish

suggest that E. californiensis may not reach full size until ∼42 days predation risk.

post-infection (Weinersmith, personal observation), and potential No effects of sublethal chlorpyrifos were found on the foraging

trade-offs between the cost of manipulating host physiology and or surfacing behaviors of California killifish at the concentrations

parasite growth may result in parasites refraining from manipula- tested in this study. It is likely that the concentrations used in

tion until their full size is achieved. However, E. californiensis has this study were too low to inhibit foraging or alter surfacing

been shown to affect the serotonergic and dopaminergic responses behavior of killifish. Few studies have examined the effects of sub-

of killifish experimentally infected with low densities of metacer- lethal organophosphate pesticide exposure on foraging or feeding;

cariae on timescales similar to those used in this study (Shaw et al., reductions have been observed in coho salmon exposed to chlor-

2009). It is interesting to note that although we did not observe a pyrifos (Sandahl et al., 2005), bream, Abramis brama, exposed to

significant effect of parasite infection on the cortisol response of the organophosphate dichlorvos (Pavlov et al., 1992), and rain-

killifish, a trend of higher cortisol excretion was observed in par- bow trout exposed to the organosphosphate parathion (Little et al.,

asitized fish. It is possible that this response could be due to the 1990). Similarly, very few studies have examined the effects of sub-

physiological stress associated with parasite infection (Trubiroha lethal organophosphate pesticides on surfacing behavior (Saglio

et al., 2010). To definitively determine the effect of E. californien- and Trijasse, 1998; Patil and David, 2008; Halappa and David, 2009).

sis on killifish cortisol excretion, future studies should employ Interestingly, Fredensborg and Longoria (2012) found an increase

repeated controlled infections to achieve higher parasite intensities in surfacing behavior of longnose killifish, Fundulus similis, natu-

and possibly should allow more time for parasites to mature. rally infected with metacercariae of Euhaplorchis sp. A—a relative

of E. californiensis. However, the observed parasite intensities were

4.3. Behavioral responses generally much higher (55–549 metacercariae) than those achieved

by experimental infection in our study. Surfacing behavior merits

Several different behaviors were modified by exposure to sub- further investigation because it may be modified by parasite infec-

lethal concentrations of chlorpyrifos, but none were significantly tion, it may be common strategy to deal with increased respiratory

altered by parasite exposure. Killifish exposed to chlorpyri-

fos exhibited decreased swimming activity, particularly for fish

Please cite this article in press as: Renick, V.C., et al., Effects of a pesticide and a parasite on neurological, endocrine, and behavioral

responses of an estuarine fish. Aquat. Toxicol. (2015), http://dx.doi.org/10.1016/j.aquatox.2015.09.010

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8 V.C. Renick et al. / Aquatic Toxicology xxx (2015) xxx–xxx

distress due to altered metabolic processes after pesticide expo- Conflicts of interest

sure, and may lead to increased predation.

The authors declare that they have no conflicts of interest.

Ethical approval

4.4. Relationships between physiological and behavioral

responses

All applicable international, national, and institutional guide-

lines for the care and use of animals were followed. All fish were

We observed a significant relationship between muscle AChE

handled according to SDSU Institutional Animal Care and Use pro-

and swimming activity and between brain AChE and foraging

tocols (#11-04-010A). This article does not contain any studies with

behavior. This lends further evidence to the role of the AChE

human participants performed by any of the authors.

biochemical pathway in mediating neurobehavioral responses.

These results are supported by other studies that have demon-

strated correlations with AChE activity and swimming metrics for Acknowledgements

fishes exposed to organophosphate pesticides (Heath et al., 1997;

Beauvais et al., 2000; Brewer et al., 2001; Sandahl et al., 2005).

We thank L. Wiborg, J. Long, M. Edwards, S. Morgan and G.

However, other conflicting studies have found weaker relation-

Cherr for thoughtful comments and discussion. M. Rouse aided in

ships between swimming behaviors and AChE (Tilton et al., 2011).

coordinating the collection of killifish from Camp Pendleton, Cal-

Additionally, few studies have demonstrated links between AChE

ifornia. A. Mena, T. Moniz, C. Bayne, S. Rodriguez, R. Sandsness,

and ecological behaviors such as foraging or anti-predator behav-

C. McNally aided with fish collection and husbandry, laboratory

iors (Sandahl et al., 2005). Thus, there remains a need to further

experiments, and behavioral observations. R. Earley (University of

investigate different behaviors which might be controlled by AChE

Alabama) graciously provided resources and laboratory equipment

pathways in different species, as well as the role of other non-AChE

to analyze cortisol samples. Funding to V.C.R. was provided through

mechanisms (Scott and Sloman, 2004; Xing et al., 2012).

the San Diego State University and University of California, Davis

Cortisol was also a positive predictor of swimming activity, but

Joint Doctoral Program in Ecology, and K.L.W. was funded by an

when adjusted for its correlation with AChE was only weakly asso-

American Association of University Women Dissertation Fellow-

ciated with this behavior. However, this lends some evidence to

ship and Block Grants from the Graduate Group in Ecology at the

the possible role of both physiological pathways in influencing

University of California, Davis. V.C.R. performed the laboratory and

activity rate. No significant relationships were observed between

behavioral experiments and V.C.R. and K.W. performed cortisol

cortisol and foraging or anti-predator behaviors. The role of cortisol

measurements. All authors contributed equally to the writing of

in influencing activity rate is still unclear and context-dependent

the manuscript. This is #42 in the SDSU CMIL contribution series.

(Gregory and Wood, 1999; Ellis et al., 2012). For instance, chronic

diet-administered cortisol inhibits locomotory activity in juvenile

rainbow trout, while short-term cortisol exposure, such as that References

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Please cite this article in press as: Renick, V.C., et al., Effects of a pesticide and a parasite on neurological, endocrine, and behavioral

responses of an estuarine fish. Aquat. Toxicol. (2015), http://dx.doi.org/10.1016/j.aquatox.2015.09.010