physiological and behavioral responses of house sparrows to ...submitted 2 march 2018 accepted 21...

Submitted 2 March 2018Accepted 21 May 2018Published 6 June 2018

Corresponding authorBrenna MG Gormallybrennagormallytuftsedu

Academic editorAngelo Piato

Additional Information andDeclarations can be found onpage 17

DOI 107717peerj4961

Copyright2018 Gormally et al

Distributed underCreative Commons CC-BY 40

OPEN ACCESS

Physiological and behavioral responsesof house sparrows to repeated stressorsBrenna MG Gormally Jessica Wright-Lichter J Michael Reed andL Michael RomeroDepartment of Biology Tufts University Medford MA United States of America

ABSTRACTDespite decades of research we still lack a complete understanding of what factorsinfluence the transition of the necessary and adaptive acute stress response to what hasbecome known as chronic stress This gap in knowledge has illuminated the necessityfor studies that examine the thresholds between these two sides of the stress responseHere we determine how repeated exposure to acute stressors influences physiologicaland behavioral responses In this repeated measures study house sparrows (Passerdomesticus) were exposed to a chronic stress protocol We took physiological andbehavioral measurements before during and after the protocol Blood samples wereused to assess four aspects of hypothalamic-pituitary-adrenal (HPA) axis functionbaseline corticosterone stress-induced corticosterone negative feedback and themaximal capacity to secrete corticosterone We also assessed bacterial killing capacityand changes in uric acid concentration Neophobia trials were used to assess behavioralchanges throughout the protocol We found no significant changes in HPA axisregulation in any of the four aspects we tested However we found that uric acidconcentrations and neophobia significantly decreased after only four days of the chronicstress protocol while bacterial killing capacity did not decrease until after eight days ofexposure These results indicate that different components of the stress response canbe impacted by chronic stress on different timescales Our results further indicate theimportance of assessing multiple aspects of both physiology and behavior in order tounderstand how exposure to chronic stress may influence ability to cope with futurechallenges

Subjects Animal Behavior Ecology Neuroscience ZoologyKeywords Chronic stress Corticosterone Immune capacity Reactive scope AllostasisNeophobia

INTRODUCTIONIn recent decades the stress response has become widely studied across many disciplinesincluding ecology (eg Bonier et al 2009 Brearley et al 2012 Crespi et al 2013)conservation biology (eg Fefferman amp Romero 2013 Partecke Schwabl amp Gwinner 2006Walker Dee Boersma ampWingfield 2006) and field endocrinology (eg Bauer et al 2014Love et al 2016 Schoech Bowman amp Reynolds 2004) Many of these studies focus on howphysiological and behavioral systems work in tandem to enable an animal to cope withstressful stimuli (Bennett et al 2016) Other studies consider the negative implicationsof chronic stress which can occur when the very systems that help an organism survive

How to cite this article Gormally et al (2018) Physiological and behavioral responses of house sparrows to repeated stressors PeerJ6e4961 DOI 107717peerj4961

become overworked (Cyr et al 2007 McCormick Shea amp Langkilde 2015 Pavlides Nivoacutenamp McEwen 2002) For example rapid acute increases in sympathetic nervous systemactivity via catecholamines is crucial for the upregulation of essential survival systemsduring a stressor (Nephew amp Romero 2003) however chronic elevations can lead tohypertension stroke insulin resistance and hypertriglyceridemia (Rupp 1999) Despitethe wide array of studies we still lack a complete understanding of how these responsesinteract and what factors influence the transition of acute beneficial stress responses tothose characterized as chronic or harmful (Romero et al 2015) To begin teasing apart thesefactors researchers need to better understand whether identifiable thresholds exist betweenthese types of responses The purpose of this study was to push house sparrows (Passerdomesticus) to this threshold in order to identify key transition points in physiological andbehavioral indices

The stress response involves several behavioral and physiological systems (egRomero ampWingfield 2016 Sapolsky Romero amp Munck 2000) We focused on four of those responsesin this studymdashhypothalamic-pituitary-adrenal (HPA) axis regulation immune killingcapacity metabolism and neophobic behavior The HPA axis is the primary focus ofmany stress studies with particular attention paid to the glucocorticoids cortisol andcorticosterone (CORT) (Romero amp Wingfield 2016) With receptors in nearly every tissue(Lattin et al 2015) CORT has extremely wide-reaching effects (Ballard et al 1974 Lattinet al 2012b) Acute activation of the HPA axis and elevation of CORT induces thetranscription of proteins that broadly upregulate essential and suppress nonessentialsurvival systems (Sapolsky Romero amp Munck 2000) What has become increasinglyobvious however is that CORT concentrations do not have a clear relationship withchronic stress symptoms of chronic stress do not necessarily develop in tandem withelevated CORT concentrations (Dickens amp Romero 2013) Other aspects of HPA axisregulation are also important including total physiological capacity of CORT productionas well as the shut-down of CORT release via negative feedback (Dallman amp Bhatnagar2010 Dickens Delehanty amp Romero 2010)

CORT helps regulate downstream effects of the stress response including metabolicimmunological and behavioral effects (Romero amp Wingfield 2016) CORT is primarilyrecognized for the metabolic processes it either directly or permissively promotes Theseinclude gluconeogenesis (Romero amp Wingfield 2016) and glycogenolysis (Nonogaki 2000)These catabolic processes increase the breakdown of proteins and thus elevations inboth blood glucose and uric acidmdashthe major end product of nitrogen catabolismmdashare seen following acute CORT increases (Lin Decuypere amp Buyse 2004a) Uric acidis also an important antioxidant (Ames et al 1981) and itrsquos therefore been suggestedthat stress-induced changes may be related to maintaining redox balance (CohenKlasing amp Ricklefs 2007) In addition to these metabolic roles CORT is hypothesizedto both augment and inhibit immunity and inflammation on different timescales acuteactivation can enhance immunity while chronic activation can lead to immunosuppressiveeffects (Dhabhar 2002 Dhabhar amp McEwen 1999 Dhabhar amp McEwen 1997) FinallyCORT can influence the expression of several behaviors including elevated activity

Gormally et al (2018) PeerJ DOI 107717peerj4961 224

(Breuner Greenberg amp Wingfield 1998) survival behavior (Astheimer Buttemer ampWingfield 1992) and neophobiamdashthe fearavoidance of novel objects (Lendvai Boacutekony ampChastel 2011)

While acute activation of the stress response is important chronic activation eitherthrough exogenous CORT administration or psychological or physical stress has beenshown to dysregulate many of these functions HPA axis function itself can become alteredduring periods of chronic stress Baseline CORT can become elevated (Dickens amp Bentley2014 Lattin Pechenenko amp Carson 2017 Lattin et al 2012a Love Lovern amp DuRant 2017)as well as reduced (Cyr et al 2007 Rich amp Romero 2005) while stress-induced levels tendto decrease (Cyr et al 2007 Lattin et al 2012a) during laboratory-induced chronic stressRegulation of the HPA axis can also be affected as reflected in weakened (Dickens Earleamp Romero 2009b) or strengthened (Lattin et al 2012a) negative feedback but sustainedor strengthened maximum capacity (eg Dickens Delehanty amp Romero 2009a Lattin etal 2012a) Many of these effects are dependent on crucial factors including species andlife history stage Chronic stress can also cause significant weight loss (Bartolomuccia et al2004 Cyr et al 2007) Uric acid has been shown to increase under chronic corticosteroneadministration in broiler chickens (Gallus gallus domesticus) (Lin Decuypere amp Buyse2004b) but remain unchanged under chronic psychological stress conditions in Europeanstarlings (Sturnus vulgaris) (Awerman amp Romero 2010) Additionally the immune systemhas been shown to be suppressed by chronic stress in some studies (Dhabhar amp McEwen1997DuRant et al 2016aMartin et al 2012) while other studies have foundmaintenanceof immune function (DuRant et al 2016b Love Lovern amp DuRant 2017) Finally chronicstress has been shown to decrease activity in open field tests (Katz Roth amp Carroll 1981)and increase neophobia (Wisłowska-Stanek et al 2016) in rodents As is clear from theabove list of previous studies physiological and behavioral parameters rarely change inconsistent ways across different studies and species However few studies have measuredmultiple parameters concurrently In the present study we chose to measure indices ofHPA axis regulation metabolism immune function and behavior in an attempt to reacha more comprehensive and conclusive phenotype of chronic stress in a single species

The goal of this study was to expose house sparrows to repeated acute stressors overthe course of several days to push them just to the edge of experiencing serious symptomsof chronic stress Prior studies have shown that different aspects of HPA axis functionand regulation are significantly altered after eight to ten days of simulated chronic stressin laboratory and field settings (Cyr et al 2007 Cyr amp Romero 2007 Dickens Earle ampRomero 2009b Rich amp Romero 2005) While we know that CORT becomes affected onthis timescale we do not yet know how other components of the stress response areaffected Furthermore it has become clear that CORT is not the best indicator of whenan animal might be a risk of experiencing negative symptoms of chronic stress (Dickensamp Romero 2013) Therefore in this study we sought to test how repeated exposure tostressors influences not only HPA axis regulation but also immune capacity metabolismand behavior in house sparrows This was accomplished by exposing house sparrowsto eight days of stressorsmdashfour days of one stimulus followed by four days of anotherstimulus (Fig 1) We hypothesized that the effects of exposure over the eight-day period


Capture

Acclimationgt2weeks

StressorA StressorB

Pre-StressControl(Sample1)

4days(Sample2)

8days(Sample3)

4days 4days10days

Figure 1 Experimental design of the chronic stress protocol Birds were randomly assigned to one oftwo treatment groups One group received stressor A (cage rolling) for four days followed by stressor B forfour days (cage tapping) The other group was exposed to these stressors in the opposite order Three setsof physiological and behavioral samples (denoted by the asterisks) were taken before (Pre-Stress Control)during (four days) and after (eight days) the stress period

Full-size DOI 107717peerj4961fig-1

would become amplified over time In other words the initial four-day exposure wouldreduce the birdsrsquo ability to cope with future challenges (the final four-day exposure)Therefore we expected to see more serious changes in the measured indices at the finalsample point relative to that taken in the middle of the protocol We predicted thatbased on previous studies referenced above this would not be reflected in altered HPAaxis regulation (reduced baseline and stress-induced CORT disrupted negative feedbackdecreased capacity) but would be reflected in immunosuppression elevated uric acid andenhanced neophobic responses

MATERIALS AND METHODSExperimental designTo test the physiological and behavioral effects of repeated exposure to stressors 22 wildfree-living house sparrows (12 females 10 males) were caught June 1ndash3 2016 in MedfordMA USA Individual birds were randomly assigned to one of two treatment groups andhoused in cages in separate rooms according to this distinction Randomization occurredsuch that each group contained both males and females and such that no group containedbirds that were all captured on the same day Birds were housed in pairs andor in view ofother birds in cages (45 cmtimes 37 cmtimes 33 cm) and kept on a natural 15L9D light cycle Anacclimation period occurred for at least 2 weeks during which birds were disturbed onlyfor routine husbandry This period was determined based on previous research involvinghouse sparrows which showed hormonal acclimation occurring after 2 weeks in captivity(Fischer Wright-Lichter amp Romero 2018)

This study was a repeated measures design in which the responses of individual birdsduring the experiment were compared to pre-stress samples At the end of the initial 2-weekacclimation period a pre-experiment blood sample was taken (lsquolsquoPre-Stress Controlrsquorsquo inFig 1) This sample served as the control to which the responses to chronic stress werecompared We did not include a separate control group of birds that did not experience thechronic stress treatment because prior research has shown that no significant hormonalchanges occur to house sparrows following acclimation (Dickens et al 2009c Lattin amp


Romero 2014 Rich amp Romero 2001) The chronic stress protocol commenced 10 daysafter the end of the acclimation period Birds in treatment group A received stressor A forfour days followed by four days of stressor B while birds in treatment group B receivedthe stressors in the opposite order (Fig 1) This was to ensure that the results were notstimulus or order-dependent Stressor A (cage rolling) involved the cages being placed onlab carts and being gently rolled back and forth for 30 min twice per day During stressorB (cage tapping) one person entered the experimental room every 25 min and tapped onthe cages this persisted for 30 min twice per day The birds experienced these stressorsat unpredictable times throughout the day but always twice per day Repeated variableapplication of acute stressors has been shown used to elicit physiological and behavioralchanges consistent with chronic stress inmany species (egCyr et al 2007Rich amp Romero2005) including using both of these stimuli in previous studies involving house sparrows(DuRant et al 2016a) Each stressor was presented for 4 days a time period chosen basedon previous studies that suggested physiological changes indicative of chronic stress occurover eight to ten days of stimulus presentation (Cyr et al 2007 Rich amp Romero 2005)

Animals were collected under aMassachusetts state collection permit and all experimentswere conducted with the approval of the Tufts Institutional Animal Care and UseCommittee and in compliance with the Guidelines for Use of Wild Birds in Research(Fair Paul amp Jones 2010)

Plasma samplingBlood samples were taken at three time points to assess HPA axis functioning immunecapacity and changes in uric acid concentrations (Fig 1) The control sample was taken 10days prior to exposure to the experimental stressors and at least two weeks after initiationof captivity We decided on this separation between the control sample and the startof the stress protocol to remain under the ethical requirement for volume of blood forsampling A second sample (lsquolsquo4 daysrsquorsquo in Fig 1) was taken after four complete days of thefirst stressor and a third and final sample (lsquolsquo8 daysrsquorsquo in Fig 1) was taken after the entireeight-day protocol On each of these days a series of blood samples was taken startingat 900am Baseline samples were taken within 3 min of entering the room (Romero ampReed 2005) Birds were then placed in an opaque cloth bag for 30 min after which astress-induced sample was taken At this time 1 mgkg dexamethasone (125 microL DEXPhoenix Pharmaceuticals Inc St Joseph MO USA) a synthetic glucocorticoid wasinjected into the pectoralis muscle (30 G BF PrecisionGlideTM Needle Becton Dickinsonand Company Franklin Lakes NJ USA) to assess efficacy of negative feedback in the HPAaxis Birds were sampled again 90 min after DEX injection then 100 IUkg ACTH (10 microLSigma Aldrich St Louis MO USA Catalog No A6303) was injected as before to assess themaximum capacity of the HPA axis The final blood sample was taken after an additional15 min of restraint All blood samples were taken from the alar vein and collected inheparinized capillary tubes Samples were stored on ice until processing which occurredno more than 5 h after sampling Centrifugation at sim1200 g for 10 min (Centrific Model225 Fisher Scientific Pittsburgh PA USA) was used to separate plasma which was frozen


atminus20 C until assay Separate aliquots were created from the baseline samples in order toconduct CORT bacterial killing and uric acid assays

Corticosterone assaysRadioimmunoassays (RIA) were used to quantify CORT concentrations in the baselinestress-induced negative feedback and Max plasma samples (Wingfield Vleck amp Moore1992) Briefly steroids were extracted from plasma samples (133 plusmn 59 microL) usingdichloromethane dried under N2 gas and then rehydrated with 550 microL phosphate-buffersaline with gelatin A standard curve was created and the RIA was run using the antibodyB3-163 (Esoterix Calabasas Hills CA USA) Assay sensitivity was 1 ngml baseline andnegative feedback samples were often below the level of detection (20 samples) and thereforeassigned this floor value The inter and intra-assay CVs were 76 and 35 respectively

Bacterial killing assaysBaseline plasma samples were also used for in vitro analyses of bacterial killing capacityAll samples were assayed within 10 days of sampling as longer periods of freezing havebeen shown to dramatically affect killing (Liebl amp Martin 2009) This assay assesses theproficiency of plasma antibodies and proteins to combat a pathogen and it has beenvalidated for house sparrows (Liebl amp Martin 2009) Briefly 15 microL of plasma was dilutedwith 345 microL PBS and was incubated with 125 microL of Escherichia coli (104 bacteriamLATCC Rcopy 8739) at 37 C for 30 min Then 250 microL of tryptic soy broth (TSB) was added toeach tube followed by an additional 12-hour incubation at 37 C Positive controls withonly E coli and TSB were also created Finally a NanoDrop spectrophotometer (NanoDrop2000 Thermo Fisher Scientific Waltham MA USA) was used to quantify absorbance ofeach sample at 300 nm Killing capacity was calculated as 1 ndash(absorbance of sample absorbance of positive control) All samples were run in triplicate when possible only 4samples out of all those that were assayed had to be run in duplicate The inter-assay CVbased on the positive controls was 115

Uric acid fluorometric assayBaseline plasma samples were used to quantify uric acid using the Amplex Rcopy Red UricAcidUricase Assay Kit (Molecular Probes Eugene OR USA) This assay relies on theconversion of uric acid to hydrogen peroxide which subsequently reacts with Amplex Rcopy Redreagent in the presence of horseradish peroxidase (HRP) to form a red-fluorescent productBriefly plasma samples were diluted with 200 microL of the provided reaction buffer and a uricacid standard curve was generated using the provided reagents To have enough reagentsto run all samples the Mastermix was slightly altered to include 150 microL of Amplex Rcopy Redreagent and 85 microL of the reaction buffer To account for this change spectrophotometricreadings were taken at 30 and 45 min at 560 nm No statistical differences were foundbetween these samples and therefore all further analysis was completed using the 30-minutesample A negative control lacking uric acid and a maximum-reading positive controlwere also included on the 96-well plates Four separate assays were completed and sampleswere run in triplicate The inter-assay CV from the positive controls was 73


Neophobia and perch hopping trialsIn addition to the physiological data gathered from plasma sampling changes in behaviorwere assessed by measuring the birdsrsquo responses to novel objects Three neophobia trialswere conducted prior to any stimuli after five days of the chronic stress protocol and afterseven days of the chronic stress protocol Because blood and behavioral samples couldnot be taken the same day we were limited which days the neophobia trials could occurBeginning five days prior to the first neophobia trial food dishes were removed each night(within 1 h before lights off) and replaced each morning (within 1 h after lights on) toacclimate the birds At this time the cage liners were also replaced to remove any excessfood was removed from the bottom of each cage this was to ensure that birds wouldreliably return to their food in the morning

On the night directly before a trial food dishes were removed and opaque blinders wereplaced between each cage to prevent the birds from seeing the neighboring trial whichcould alter their own behavior (Fryday amp Greig-Smith 1994) The following morningfood was replaced with one of three possible novel objectsmdasha colored plastic egg in thedish a red ribbon across the dish or a food dish painted red All objects had been usedpreviously in studies involving avian species and shown to elicit a neophobic response(Fischer amp Romero 2016) Behavior was recorded for 20 min after initial food presentationand then the novel objects and blinders were removed Each individualrsquos latency to feedwas recorded Feed latency was defined as the time between replacing the food and the birdvisibly feeding Additionally perch hopping was calculated during 15 min of the same trialThe first 5 min were discounted to avoid activity that occurred due to the presence of theexperimenter replacing the food dish A perch hop was defined as movement throughoutthe cage typically between the perch water dish cage andor ground

Three control trials were also conducted (one each day) prior to each neophobia trial toensure that the stress treatments did not affect the birdsrsquo normal feeding behavior Duringthese control trials food dishes were returned in the morning without a novel objectNeophobia and control trials were conducted on days separate from blood sampling

Data analysisAll statistical analyses were conducted in RStudio (RStudio Team 2015) Before performingany linear mixed effects models or ANOVAs the data were checked for heteroscedasticityusing Bartlettrsquos test In the event of a violation data were log-transformed this was thecase with baseline and stress-induced CORT as well as with perch hopping

Group and sex effectsBefore conducting further analyses we tested whether there was an effect of group or sexin any of the datasets This was done in order to check if differences in sex or stimulusorder (whether cage rolling or cage tapping was experienced first) caused significant effectsto physiological or behavioral responses We used linear mixed effects models (LMM)with individual bird identity as a random effect and group and sample as main effects inseparate models (lme4 package Bates et al 2015) The lsquoAnovarsquo function in the car package(Fox amp Weisberg 2011) generated results for Type III Wald F-tests with Kenward-Roger


adjusted degrees of freedom Interactions were checked using the same method but withan interaction term between group or sex and sample

Hormone dataFour baseline samples (n= 62) and three negative feedback samples (n= 63) did notcontain enough plasma to be run One sample from the stress-induced data was droppedbecause that bird did not mount an acute stress response to the restraint stimulusand a second sample did not contain enough plasma (n= 64) The different CORTresponses measured here (baseline stress-induced negative feedback and maximalcapacity) play complicated activation inhibition permissive and preparative rolesand interact with different physiological systems (Romero 2004 Sapolsky Romero ampMunck 2000) Because these are distinct physiological responses often regulated bydifferent receptors four separate models were used to analyze changes in each typeof CORT sample across each of the three sampling points However bonferonniadjustments were used if necessary to account for non-independence of the dependentvariables The efficacy of negative feedback was calculated as the percent decreasein CORT from stress-induced levels to those after injection of DEX LMMs withindividual bird identity as a random effect were used to analyze baseline stress-inducednegative feedback and maximum CORT responses (lme4 package Bates et al 2015)All models were visually checked for model assumption violations using residual plots

Finally for each bleed type the lsquoAnovarsquo function was used to run ANOVAs with sampleas a main effect and bird ID as a random effect In the event of significant result Tukeyrsquosmultiple comparison post-hoc tests were run using the lsquoglhtrsquo function in the multcomppackage (Hothorn Bretz amp Westfall 2008)

Immune capacity and uric acid concentrationsOne sample did not contain enough plasma to be run in the BKA (n= 65) while twosamples did not contain enough plasma to be run in the uric acid assay (n= 64) A singleLMM was used to assess changes in killing capacity and uric acid concentrations overthe course of the chronic stress protocol again individual bird identity was included as arandom effect Tukeyrsquos multiple comparison post-hoc analyses were conducted as indicatedabove

Behavioral dataBirds that failed to approach the novel object in the allotted time were assigned a ceilingvalue of 20 min Interactions between feed latency and novel object were assessed withseparate LMMs and a final LMM was run to compare feed latencies across the samplingprotocol Perch hops were counted during the last 15 min of the neophobia videos Thefirst 5 min were discounted to avoid confounding effects of experimenter presence Allvideos were coded by the same person The movements of two birds were obscured fromwhere the camera was placed and one sample from an additional bird was removed fromanalysis because they did not display any perch hopping in the allotted time making logtransformation impossible (n= 59) Interactions between total perch hops and group sexand object were checked with individual LMMs and again a separate LMM was completed


to compare activities across sampling periods Separate models were used to assess changesin the control trials for both the feed latency and perch hopping data sets

RESULTSGroup and sex effectsThere was nomain effect of group in the datasets for stress-induced negative feedback andmaximum capacity CORT bacterial killing uric acid or neophobia datasets (pgt 010 forall tests) There were also no significant interactions between group and sample (pgt 054for all tests) Therefore stimulus order was removed from any further analyses of thosedatasets We conducted the same check for sex effects and interactions and found none(pgt 006 for all main effects tests and pgt 028 for all interaction tests) sex was thereforeremoved from any further analyses in all datasets except for the perch hopping data wherewe did detect a significant effect of sex

Corticosterone responsesGroupmdashthe order that the stimuli were experienced inmdashsignificantly affected baselineCORT (F160 = 573 p= 003 Fig 2A) Birds in group A (experienced cage rolling first)had higher baseline CORT at each sample point However even when analyzed separatelywe did not detect statistically significant differences in either group A (F227 = 118p= 033) or group B (F229= 157 p= 023) across the chronic stress protocol We alsodid not detect any statistically significant differences in CORT levels at any of the otherthree bleed types (stress-induced F261= 142 p= 025 negative feedback F260= 007p= 093 maximum capacity F263= 124 p= 030) over the course of the chronic stressprotocol (Figs 2Bndash2D)

Immune capacityThe killing capacity of samples taken at the final time point was significantly lower thanthose for those at the control and second time point (F262= 197 plt 00005 Fig 3)

Uric acidUric acid significantly decreased once the chronic stress protocol was initiated and remainedlow (F261= 128 plt 00005 Fig 4)

NeophobiaWe found no significant interactions between object type (red dish red ribbon plastic egg)and sample (F461= 058 p= 068) There was however a significant main effect of object(F263 = 394 p= 003) Regardless of object type feed latency decreased significantlyafter 4 days of the chronic stress protocol (F263 = 403 p= 003 Fig 5) Feed latenciesalso significantly decreased during control trials as the chronic stress protocol progressed(F263= 259 plt 000001 Fig 5 white bars)

Perch hoppingPerch hopping was not significantly affected by neophobic object (F256= 102 p= 037)nor were there significant interactions between object and perch hops (object F454= 037


ABaseline

CNegativefeedback

BStress-induced

GroupAB

DMaximumcapacity

Figure 2 CORT responses before (Pre-Stress Control) during (4 days) and after (8 days) the chronicstress protocol (A) Baseline samples were taken within 3 min of entering the experimental room Whitebars represent group A (birds experienced cage rolling first) and grey bars represent group B (birds experi-enced cage tapping first) While there was a significant main effect of sex (p= 003) neither group A (p=033) nor group B (p= 023) experienced significantly changes baseline CORT (B) Stress-induced CORTsamples were taken 30 min after restraint in an opaque cloth bag and did not significantly change (LMMp= 025) (C) Efficacy of negative feedback remained constant throughout the protocol (LMM p= 093)(D) Physiologically maximum levels of CORT taken after injection of ACTH did not change (LMM p =03) Error bars representplusmnSEM Note the different y-axes


p= 083) Perch hopping significantly decreased after the initiation of the stress protocoland remained reduced through the remainder of the protocol (F256= 1116 plt 00005Fig 6A)

Perch hopping remained constant during the control trials We did detect a significantmain effect of sex females were more active than males (F157= 623 p= 002) howeverneither females (F230 = 027 p= 077) nor males (F223 = 016 p= 086) significantlychanged their activity during these control trials We also found a significant interactionbetween group and perch hopping (F652= 752 plt 0005) during the control trials whenno novel objects were present Further inspection of graphs revealed that this interactionwas driven by birds in group A (experienced cage rolling first) increased their perchhopping at the final sample point while those in group B (experienced cage tapping first)decreased their perch hopping (Fig 6B)


Figure 3 In vitro bacterial killing capacity of plasma samples from birds before (Pre-Stress Control)during (four days) and after (eight days) the chronic stress protocol Killing capacity remained un-changed after four days of the chronic stress protocol but became significantly reduced after eight daysTriple asterisk denotes sample 3 different from samples 1 and 2 with a significance of p lt 00001 Errorbars representplusmn SEM


DISCUSSIONThis study examined the physiological and behavioral changes that occurred in housesparrows in response to a series of repeated acute stressors simulating a chronic stressprotocol The findings indicate that symptoms of chronic stress can appear at threedifferent timescales when measured in four different indices While eight days was not longenough to elicit significant changes in HPA axis function and regulation birds experiencedsubstantial changes to uric acid levels and behavior after only four days of the chronicstress protocols and to immune capacity after eight days These data are significant becausethey further indicate that CORT may not always the best indicator of when an animal isentering homeostatic overload

Effects of chronic stress on HPA axis regulationWe found no significant changes in any of the four measurements of HPA axis regulationand responsiveness across the three sampling time points (Fig 2) The lack of change inbaseline CORT levels was not entirely surprising as a previous study using house sparrowsand a rotating repeated psychological stress protocol found no significant changes (Lattinamp Romero 2014) Interestingly there was a main effect of group on baseline CORTDespite randomizing the birds into groups A and B group A birds tended to have higher


Figure 4 Uric acid concentrations before (Pre-Stress Control) during (four days) and after (eightdays) Baseline uric acid significantly decreased after four days of chronic stress Double asterisk denotessignificance at plt 0001 Error bars representplusmn SEM


baseline CORT even prior to experiencing any stimuli (Fig 2A) Regardless when analyzedseparately we did not detect a statistically significant change in CORT over the course of theexperiment however both groups appeared to exhibit a similar downward trend by day 8(Fig 2A) Additionally stress-induced CORT levels were also trending down relative to thecontrol levels at the start of the experiment it is likely that had the experiment continuedwe would have found statistically significant decreases as shown in prior studies (Cyret al 2007 Cyr amp Romero 2007 Dickens Earle amp Romero 2009b Rich amp Romero 2005)We want to stress that our primary goal was not to induce significant changes in HPA axisfunction and regulation but rather to push animals to the edge of when these changesmightbegin Thus this protocol was effective at pushing animals to the edge of experiencinghomeostatic overload at least as measured by metrics of the HPA axis It is also unlikelythat these results suggest acclimation or habituation to the chronic stress protocol since thestressors were encountered at random and unpredictable times throughout the day (seeRich amp Romero 2005)

Surprisingly we found no changes in negative feedback strength or maximum CORTproduction throughout the course of the experiment (Fig 2) We expected that negativefeedback strength and production capacity would change as wear-and-tear increased


A

B B

Pre-StressControl

4days 8days

NovelObject

NoObject

TrialType

A

B B

Figure 5 Time to feed when novel objects are present before (Pre-Stress Control) during (four days)and after (eight days) the chronic protocol Feed latency decreased significantly after 4 days of chronicstress White bars indicate control samples (no novel object present) taken within two days of neophobiatrials Control and neophobia trials were analyzed independently Asterisk denotes plt 005 Different let-ters indicate significance at plt 00001 Error bars representplusmnSEM


throughout the protocol Previous studies involving avian species have reported changesin both these measurements due to different types of chronic stress For example captivitycombined with translocation of chukar (Alektoris chukar) significantly reduced feedbackstrength (Dickens Delehanty amp Romero 2009a) In addition HPA axis maximum capacityincreased in house sparrows due to initial transference to captivity during two lifehistory stages (late breeding and molt) (Lattin et al 2012a) These differences suggestthat experimental conditions play a role in the physiological changes that occur due tochronic stress Though translocation captivity and a rotating repeating psychologicalstress protocol are each considered long-term lsquolsquochronicrsquorsquo stressors animals likely interpretthese stressors differently it is therefore plausible that the physiological changes that resultfrom these stimuli are unique

Immunological effects of chronic stressOur results indicate that immune capacity was severely affected in chronically stressedbirds While birds maintained an in vitro immune response after the first bout ofstressors the building wear-and-tear throughout chronic stress protocol exposure inducedimmunosuppression following the second bout (Fig 3) This finding was consistent withour predictions based on evidence that chronic stress leads to immunosuppression while


A

TrialType

NovelObject

NoObject

Pre-StressControl

4days 8days

Group

NoObjectPre-StressControl

NoObject4days

NoObject8days

B

GroupAB

Figure 6 Changes in perch hopping during the chronic stress protocol (A) Number of perch hopswhen novel objects are present before (Pre-Stress Control) during (four days) and after (eight days)chronic stress protocol Perch hopping significantly decreased after four days of the chronic stress proto-col White bars indicate control samples (no novel object present) taken within two days of novel objecttrials Control and neophobia trials were analyzed independently (B) A significant interaction was foundbetween group (whether the birds received stressor A or B first) and sample This was driven by birds ingroup A increasing and those in group B decreasing perch hopping at the final sample point Double aster-isk denotes plt 0001 Error bars representplusmnSEM


acute stress can lead to immunoenhancement (Dhabhar 2002 Glaser et al 1987 Martin2009McEwen 1998) Furthermore the data presented here are consistent with a previousstudy in which elevating allostatic load through topical CORT application slowed housesparrowsrsquo healing of a superficial wound (DuRant et al 2016a) What we find particularlyintriguing about these results is that four days of the chronic stress protocol is not enough toinduce changes in immune capacity as it is in the case of uric acid and behavior The initialmaintenance of bacterial killing capacity is possibly indicative of an initial enhancement ofimmune function It is possible that during the initial lsquolsquoshort-termrsquorsquo 4 days of the chronicstress protocol immune function is actually enhanced By the time the second sample istaken however immune function is returning to original levels before falling even furtherupon exposure to the second set of stressors Prior studies have indicated the complexbalance between stress and immunity with short-term exposure enhancing and long-termexposure suppressing (Dhabhar amp McEwen 1999 Martin 2009)

We believe that this final decrease in in vitro immune function is representative of thebirds transitioning into a lsquolsquochronically stressedrsquorsquo state or what the reactive scope modelterms homeostatic overload (Romero Dickens amp Cyr 2009) The fact that this is happeningafter approximately 8 days of the chronic stress protocol supports prior findings thathave investigated the physiological changes during chronic stress (Cyr et al 2007 Cyr ampRomero 2007 DuRant et al 2016a Rich amp Romero 2005) While we recognize that this is


a single measure of immune function and relies on only one kind of pathogen the BKAhas been found to be correlated with other immune parameters and assays in avian speciesincluding lipopolysaccharide challenge phagocytosis assay and acute phase proteins (Milletet al 2007)

Metabolic changes during chronic stressInterestingly uric acid concentrations decreased immediately during the chronic stressprotocol following the initial stimulus (Fig 4) This was contrary to predictions basedon previous studies that indicated that chronic corticosterone exposure or psychologicalor physical stress lead to elevations in uric acid in avian species (Azad et al 2010 LinDecuypere amp Buyse 2004a Lin Decuypere amp Buyse 2004b) While a prior survey of nearly100 bird species found that uric acid concentrations predominately fall following the acutestress response (Cohen Klasing amp Ricklefs 2007) fewer studies have examined uric acidchanges due to chronic stress independent of CORT administration We predicted thatbasal uric acid concentrations would increase over time because as birds experienced thechronic stress protocol their metabolism would increase thus leading to more uric acid asa byproduct of purine metabolism However these previous studies specifically relied onelevations of CORT to induce elevations in uric acid whereas our stress protocol resultedin no changes in baseline CORT Furthermore previous studies report that uric acid is animportant and potent antioxidant in avian species one study specifically indicated thatreduced levels of uric acid correlate with increased oxidative stress (Klandorf et al 2001)In the present study reduced uric acid concentrations could be an indication that the redoxbalance becomes upset within four days of the chronic stress protocol In other words thechronic stress protocol caused the house sparrows to use up their uric acid stores whichare not replenished or at least not replenished fast enough to compensate for ongoing uricacid catabolism This change suggests that birds may not have the same antioxidant bufferunder chronic stress conditions as they do under lsquolsquonormalrsquorsquo conditions

We find it interesting that this decrease in uric acid concentrations does not point tothe same time frame for transitioning into chronic stress as the immune data While theimmune data do not show significant changes until after eight days of the chronic stressprotocol uric acid significantly decreases within the first four days This finding emphasizesthat different physiological metrics can suggest alternate time frames for when the shiftinto chronic stress occurs

Behavioral effects of chronic stressFinally behavioral tests show that birds approached novel objects faster and reduced theiractivity as they progressed through the stress protocol Note that a faster approach isunlikely to reflect habituation to the novel object because each bird was exposed to anunexperienced object for each trial which in an earlier study elicited equivalent neophobicresponses for each trial (Fischer amp Romero 2016) Interestingly in this study object type didhave a significant main effect on feed latency Feed latency when the red dish and ribbonwere present elicited similar declines over the course of the protocol whereas responsesto the plastic egg did not vary Importantly object type did not have a main effect in the


perch hopping data Therefore we feel that focusing on the overall pattern across all objecttypes reflects the accurate biological response

Faster approaches and lower activity were contrary to our original prediction whichwas that neophobic and anxious behaviors would increase with chronic stress Thisprediction was again based on studies that suggested associations between CORT titersand fearavoidance behavior (eg Korte 2001 Skoacuterzewska et al 2006) The reducedneophobic behaviors may be a result of this experiment not inducing increased CORTconcentrations as in these other studies Similarly perch hopping frequency decreasedthrough the chronic stress protocol suggesting that overall activity decreased Thereforethe faster feed times were not a result of random flighty activity within the cage but ratherdeliberate movements towards the novel object and food dish

Interestingly feed latencies during the no-object control trials significantly decreasedafter the initial trial (Fig 5 white bars) This suggests that the chronic stress protocol causedthe birds to approach their food dish faster in general This does not lessen the significanceof the pattern we see in the neophobia trials during which birds also approach the novelobjects faster Birds still took about seven times longer to feed during the neophobia timesrelative to the no-object controls Therefore the neophobic response did not disappearbirds were simply likely to more quickly approach the food dish regardless of there a novelobject was present

We found a significant interaction between group and perch hopping in the controlsamples (when no novel object was present) (Fig 6B) Birds that experienced cage rollingfirst decreased their activity while those experiencing cage tapping first increased theiractivity by the end of the chronic stress protocol It is not clear why order was related toperch hopping but this kind of interaction effect was not apparent in any of the otherphysiological or behavioral data

Although our original hypotheses for the behavioral measurements were based on priorstudies connecting CORT and behavior there is an alternative interpretation arising fromthe reactive scope model that could explain the decreases The data suggest that chronicstress induced house sparrows to take additional risk when foraging Rather than delayingtheir approach towards a novel potentially dangerous object these birds more readilyrisked potential harm to forage In certain circumstances this could be quite dangerousparticularly in the wild where novel objects might not be so innocuous as a colored fooddish ribbon or plastic egg

Many behavioral ecology studies have attempted to link CORT to broader indices ofbehavior including explorative behavior and foraging activity (eg Atwell et al 2012Crino et al 2017) The results of this study indicate that at least in some instances chronicstress-induced changes to CORT and behavior happen at different time scales This couldbe particularly important to consider in field-based studies when the stimuli might be ona much larger context including urbanization and predator presence Finally we found itinteresting that during the no-object control trials females tended to be much more activethan males It is particularly intriguing that this sex difference is absent in the neophobiatrials when novel objects are present While the response to novel objects is not affected bysex underlying sex differences appear to change baseline activity


CONCLUSIONWe have shown that both physiology and behavior can significantly change over the courseof a short chronic stress protocol Most importantly there are different timescales at whichthe effects of chronic stress are being reflected in the different indices measured hereAdditionally birds became less neophobic and more active These results suggest thatthe overall physiology of the birds is entering a new state perhaps indicative of lsquolsquochronicstressrsquorsquo It is possible that these metrics indicate that these birds are closer to the overloadthreshold of the reactive scope framework Interestingly eight days of the chronic stressprotocol did not appear sufficient to significantly alter HPA function although a longerperiod of stress may have elicited an effect Furthermore these results provide importantsupport for the idea that CORT is not the best indicator of chronic stress particularlyconsidering its complicated relationship with so many other physiological and behavioralsystems Despite decades of research on stress we still lack the ability to predict underwhat circumstances pathological symptoms may develop We hope that these data provideinitial steps towards being able to better interpret results pertaining to the vertebrate stressresponse and their relation to overall individual animal health and fitness

ACKNOWLEDGEMENTSWe thank M Henry and E Bowers for help in the collection of these data In additionwe extend gratitude to C P Fischer for significant statistical assistance and to the TuftsUniversity Animal Care staff for animal care

ADDITIONAL INFORMATION AND DECLARATIONS

FundingThis study was conducted with support from NSF REU grant DBI 1560380 to Dr PhilStarks and NSF IOS1655269 to L Michael Romero The funders had no role in studydesign data collection and analysis decision to publish or preparation of the manuscript

Grant DisclosuresThe following grant information was disclosed by the authorsNSF REU DBI 1560380NSF IOS1655269

Competing InterestsThe authors declare there are no competing interests

Author Contributionsbull Brenna MG Gormally conceived and designed the experiments performed theexperiments analyzed the data contributed reagentsmaterialsanalysis tools preparedfigures andor tables authored or reviewed drafts of the paper approved the final draftbull Jessica Wright-Lichter performed the experiments analyzed the data contributedreagentsmaterialsanalysis tools prepared figures andor tables authored or revieweddrafts of the paper approved the final draft


bull J Michael Reed analyzed the data contributed reagentsmaterialsanalysis toolsauthored or reviewed drafts of the paper approved the final draftbull L Michael Romero conceived and designed the experiments analyzed the datacontributed reagentsmaterialsanalysis tools authored or reviewed drafts of the paperapproved the final draft

Animal EthicsThe following information was supplied relating to ethical approvals (ie approving bodyand any reference numbers)

Tufts University Institutional Animal Care and Use Committee provided approval forthis study

Field Study PermissionsThe following information was supplied relating to field study approvals (ie approvingbody and any reference numbers)

Massachusetts Division of Fisheries and Wildlife provided approval for this study

Data AvailabilityThe following information was supplied regarding data availability

The raw data are provided in the Data S1

Supplemental InformationSupplemental information for this article can be found online at httpdxdoiorg107717peerj4961supplemental-information

REFERENCESAmes BN Cathcart R Schwiers E Hochstein P 1981 Uric acid provides an antioxidant

defense in humans against oxidant- and radical-caused aging and cancer a hypoth-esis Proceedings of the National Academy of Sciences of the United States of America786858ndash6862 DOI 101073pnas78116858

Astheimer LB ButtemerWAWingfield JC 1992 Interactions of corticosterone withfeeding activity and metabolism in passerine birds Ornis Scand 23355ndash365DOI 1023073676661

Atwell JW Cardoso GCWhittaker DJ Campbell-Nelson S Robertson KW KettersonED 2012 Boldness behavior and stress physiology in a novel urban environmentsuggest rapid correlated evolutionary adaptation Behavioral Ecology 23960ndash969DOI 101093behecoars059

Awerman JL Romero LM 2010 Chronic psychological stress alters body weight andblood chemistry in European starlings (Sturnus vulgaris) Comparative Biochemistryand Physiology Part A Molecular amp Integrative Physiology 156136ndash142DOI 101016jcbpa201001010

AzadMAK KikusatoMMaekawa T Shirakawa H ToyomizuM 2010Metaboliccharacteristics and oxidative damage to skeletal muscle in broiler chickens exposed


to chronic heat stress Comparative Biochemistry and Physiology Part A Molecular ampIntegrative Physiology 155401ndash406 DOI 101016jcbpa200912011

Ballard PL Baxter JD Higgins SJ Rousseau GG Tomkins GM 1974 General presenceof glucocorticoid receptors in mammalian tissues Endocrinology 94998ndash1002DOI 101210endo-94-4-998

Bartolomuccia A Pederzani T Sacerdote P Panerai AE Parmigiani S Palanza P2004 Behavioral and physiological characterization of male mice under chronicpsychosocial stress Psychoneuroendocrinology 29899ndash910DOI 101016jpsyneuen200308003

Bates D Maechler M Bolker BWalker S 2015 Fitting linear mixed-effects modelsusing lme4 Journal of Statistical Software 671ndash48

Bauer CM Hayes LD Ebensperger LA Romero LM 2014 Seasonal variation inthe degu (Octodon degus) endocrine stress response General and ComparativeEndocrinology 19726ndash32 DOI 101016jygcen201311025

Bennett AM Longhi JN Chin EH Burness G Kerr LR Murray DL 2016 Acute changesin whole body corticosterone in response to perceived predation risk a mechanismfor anti-predator behavior in anurans General and Comparative Endocrinology22962ndash66 DOI 101016jygcen201602024

Bonier F Martin PR Moore ITWingfield JC 2009 Do baseline glucocorticoids predictfitness Trends in Ecology amp Evolution 24634ndash642 DOI 101016jtree200904013

Brearley G McAlpine C Bell S Bradley A 2012 Influence of urban edges on stress in anarboreal mammal a case study of squirrel gliders in southeast Queensland AustraliaLandscape Ecology 271407ndash1419 DOI 101007s10980-012-9790-8

Breuner CW Greenberg ALWingfield JC 1998 Noninvasive corticosterone treat-ment rapidly increases activity in Gambelrsquos white-crowned sparrows (Zonotrichialeucophrys gambelii) General and Comparative Endocrinology 111386ndash394DOI 101006gcen19987128

Cohen A Klasing K Ricklefs R 2007Measuring circulating antioxidants in wild birdsComparative Biochemistry and Physiology Part B Biochemistry amp Molecular Biology147110ndash121 DOI 101016jcbpb200612015

Crespi EJ Williams TD Jessop TS Delehanty B 2013 Life history and the ecology ofstress how do glucocorticoid hormones influence life-history variation in animalsFunctional Ecology 2793ndash106 DOI 1011111365-243512009

Crino OL Buchanan KL Trompf L MainwaringMC Griffith SC 2017 Stressreactivity condition and foraging behavior in zebra finches effects on boldnessexploration and sociality General and Comparative Endocrinology 244101ndash107DOI 101016jygcen201601014

Cyr NE Earle K Tam C Romero LM 2007 The effect of chronic psychological stresson corticosterone plasma metabolites and immune responsiveness in Europeanstarlings General and Comparative Endocrinology 15459ndash66DOI 101016jygcen200706016


Cyr NE Romero LM 2007 Chronic stress in free-living European starlings reducescorticosterone concentrations and reproductive success General and ComparativeEndocrinology 15182ndash89 DOI 101016jygcen200612003

DallmanMF Bhatnagar S 2010 Chronic stress and energy balance role of thehypothalamo-pituitary-adrenal axis In Comprehensive physiology BethesdaAmerican Physiological Society

Dhabhar FS 2002 A hassle a day may keep the doctor away stress and the augmentationof immune function Integrative and Comparative Biology 42556ndash564DOI 101093icb423556

Dhabhar FS McEwen BS 1997 Acute stress enhances while chronic stress suppressescell-mediated immunity in vivo a potential role for leukocyte trafficking BrainBehavior and Immunity 11286ndash306 DOI 101006brbi19970508

Dhabhar FS McEwen BS 1999 Enhancing versus suppressive effects of stress hormoneson skin immune function Proceedings of the National Academy of Sciences of theUnited States of America 961059ndash1064 DOI 101073pnas9631059

Dickens MJ Bentley GE 2014 Stress captivity and reproduction in a wild bird speciesHormones and Behavior 66685ndash693 DOI 101016jyhbeh201409011

Dickens MJ Delehanty DJ Romero LM 2009a Stress and translocation alterationsin the stress physiology of translocated birds Proceedings of the Royal Society BBiological Sciences 2762051ndash2056 DOI 101098rspb20081778

Dickens MJ Delehanty DJ Romero LM 2010 Stress an inevitable component of animaltranslocation Biological Conservation 1431329ndash1341DOI 101016jbiocon201002032

Dickens MJ Earle KA Romero LM 2009b Initial transference of wild birds to captivityalters stress physiology General and Comparative Endocrinology 16076ndash83DOI 101016jygcen200810023

Dickens MJ Romero LM 2013 A consensus endocrine profile for chronically stressedwild animals does not exist General and Comparative Endocrinology 191177ndash189DOI 101016jygcen201306014

Dickens MJ Romero LM Cyr NE Dunn IC Meddle SL 2009c Chronic stress altersglucocorticoid receptor and mineralocorticoid receptor mRNA expression inthe European starling (Sturnus vulgaris) brain Journal of Neuroendocrinology21832ndash840 DOI 101111j1365-2826200901908x

DuRant SE Arciniega ML Bauer CM Romero LM 2016a A test of reactive scopereducing reactive scope causes delayed wound healing General and ComparativeEndocrinology 236115ndash120 DOI 101016jygcen201607013

DuRant SE De Bruijn R TranMN Romero LM 2016bWound-healing ability isconserved during periods of chronic stress and costly life history events in a wild-caught bird General and Comparative Endocrinology 229119ndash126DOI 101016jygcen201603009

Fair J Paul E Jones J (eds) 2010 Guidelines to the use of wild birds in research InOrnithological council Washington DC Ornithological Council


Fefferman NH Romero LM 2013 Can physiological stress alter population persistenceA model with conservation implications Conservation Physiology 1cot012ndashcot012

Fischer CP Romero LM 2016 The use of α- or β-blockers to ameliorate the chronicstress of captivity in the house sparrow (Passer domesticus) Conservation Physiology4(1)cow049 DOI 101093conphyscow049

Fischer CPWright-Lichter J Romero LM 2018 Chronic stress and the introductionto captivity how wild house sparrows (Passer domesticus) adjust to laboratoryconditions General and Comparative Endocrinology 25985ndash92

Fox J Weisberg S 2011 An R companion to applied regression Second editionThousand Oaks Sage

Fryday SL Greig-Smith PW 1994 The effects of social learning on the food choice ofthe house sparrow (Passer domesticus) Behaviour 128281ndash300DOI 101163156853994X00299

Glaser R Rice J Sheridan J Fertel R Stout J Speicher C Pinsky D Kotur M Post ABeckM Kiecolt-Glaser J 1987 Stress-related immune suppression health implica-tions Brain Behavior and Immunity 17ndash20 DOI 1010160889-1591(87)90002-X

Hothorn T Bretz F Westfall P 2008 Simultaneous inference in general parametricmodels Biometrical Journal 50346ndash363 DOI 101002bimj200810425

Katz RJ Roth KA Carroll BJ 1981 Acute and chronic stress effects on open field activityin the rat implications for a model of depression Neuroscience and BiobehavioralReviews 5247ndash251 DOI 1010160149-7634(81)90005-1

Klandorf H Rathore DS Iqbal M Shi X Van Dyke K 2001 Accelerated tissue agingand increased oxidative stress in broiler chickens fed allopurinol ComparativeBiochemistry and Physiology Part C Toxicology amp Pharmacology 12993ndash104DOI 101016S1532-0456(01)00186-7

Korte S 2001 Corticosteroids in relation to fear anxiety and psychopathology Neuro-science and Biobehavioral Reviews 25117ndash142 DOI 101016S0149-7634(01)00002-1

Lattin CR Bauer CM De Bruijn R Romero LM 2012aHypothalamusndashpituitaryndashadrenal axis activity and the subsequent response to chronic stress differ dependingupon life history stage General and Comparative Endocrinology 178494ndash501DOI 101016jygcen201207013

Lattin CR Keniston DE Reed JM Romero LM 2015 Are receptor concentrationscorrelated across tissues within individuals A case study examining glucocortiocidand mineralocorticoid receptor binding Endocrinology 1561354ndash1361

Lattin CR Pechenenko AV Carson RE 2017 Experimentally reducing corticosteronemitigates rapid captivity effects on behavior but not body composition in a wildbird Hormones and Behavior 89121ndash129 DOI 101016jyhbeh201612016

Lattin CR Romero LM 2014 Chronic stress alters concentrations of corticosteronereceptors in a tissue-specific manner in wild house sparrows (Passer domesticus)Journal of Experimental Biology 2172601ndash2608

Lattin CRWaldron-Francis K Richardson JW De Bruijn R Bauer CM Breuner CWRomero LM 2012b Pharmacological characterization of intracellular glucocorticoid


receptors in nine tissues from house sparrow (Passer domesticus) General andComparative Endocrinology 179214ndash220 DOI 101016jygcen201208007

Lendvai AacuteZ Boacutekony V Chastel O 2011 Coping with novelty and stress in free-livinghouse sparrows Journal of Experimental Biology 214821ndash828

Liebl AL Martin LB 2009 Simple quantification of blood and plasma antimicrobialcapacity using spectrophotometry Functional Ecology 231091ndash1096DOI 101111j1365-2435200901592x

Lin H Decuypere E Buyse J 2004a Oxidative stress induced by corticosterone ad-ministration in broiler chickens (Gallus gallus domesticus) 2 Short-term effectComparative Biochemistry and Physiology Part B Biochemistry amp Molecular Biology139745ndash751 DOI 101016jcbpc200409014

Lin H Decuypere E Buyse J 2004b Oxidative stress induced by corticosterone ad-ministration in broiler chickens (Gallus gallus domesticus) 1 Chronic exposureComparative Biochemistry and Physiology Part B Biochemistry amp Molecular Biology139737ndash744 DOI 101016jcbpc200409013

Love AC Foltz SL Adelman JS Moore IT Hawley DM 2016 Changes in corticos-terone concentrations and behavior duringMycoplasma gallisepticum infection inhouse finches (Haemorhous mexicanus) General and Comparative Endocrinology23570ndash77 DOI 101016jygcen201606008

Love AC LovernMB DuRant SE 2017 Captivity influences immune responses stressendocrinology and organ size in house sparrows (Passer domesticus) General andComparative Endocrinology 25218ndash26 DOI 101016jygcen201707014

Martin LB 2009 Stress and immunity in wild vertebrates timing is everything Generaland Comparative Endocrinology 16370ndash76 DOI 101016jygcen200903008

Martin LB Brace AJ Urban A Coon CAC Liebl AL 2012 Does immune suppressionduring stress occur to promote physical performance Journal of ExperimentalBiology 2154097ndash4103 DOI 101242jeb073049

McCormick GL Shea K Langkilde T 2015How do duration frequency and intensityof exogenous CORT elevation affect immune outcomes of stress General andComparative Endocrinology 22281ndash87 DOI 101016jygcen201507008

McEwen BS 1998 Protective and damaging effects of stress mediators New EnglandJournal of Medicine 338171ndash179 DOI 101056NEJM199801153380307

Millet S Bennett J Lee KA HauM Klasing KC 2007 Quantifying and comparingconstitutive immunity across avian species Developmental and ComparativeImmunology 31188ndash201 DOI 101016jdci200605013

Nephew BC Romero LM 2003 Behavioral physiological and endocrine responsesof starlings to acute increases in density Hormones and Behavior 44222ndash232DOI 101016jyhbeh200306002

Nonogaki K 2000 New insights into sympathetic regulation of glucose and fatmetabolism Diabetologia 43533ndash549 DOI 101007s001250051341

Partecke J Schwabl I Gwinner E 2006 Stress and the city urbanization and itseffects on the stress physiology in European blackbirds Ecology 871945ndash1952DOI 1018900012-9658(2006)87[1945SATCUA]20CO2


Pavlides C Nivoacuten LG McEwen BS 2002 Effects of chronic stress on hippocampal long-term potentiation Hippocampus 12245ndash257 DOI 101002hipo1116

Rich EL Romero LM 2001 Daily and photoperiod variations of basal and stress-inducedcorticosterone concentrations in house sparrows (Passer domesticus) Journal ofComparative Physiology B Biochemical Systemic and Environmental Physiology171543ndash547 DOI 101007s003600100204

Rich EL Romero LM 2005 Exposure to chronic stress downregulates corticosteroneresponses to acute stressors American Journal of Physiology Regulatory Integrativeand Comparative Physiology 288R1628ndashR1636 DOI 101152ajpregu004842004

Romero LM 2004 Physiological stress in ecology lessons from biomedical researchTrends in Ecology amp Evolution 19249ndash255 DOI 101016jtree200403008

Romero LM Dickens MJ Cyr NE 2009 The reactive scope modelmdasha new modelintegrating homeostasis allostasis and stress Hormones and Behavior 55375ndash389DOI 101016jyhbeh200812009

Romero LM Platts SH Schoech SJ Wada H Crespi E Martin LB 2015 Understandingstress in the healthy animalmdashpotential paths for progress Stress 18491ndash497DOI 1031091025389020151073255

Romero LM Reed JM 2005 Collecting baseline corticosterone samples in the fieldis under 3 min good enough Comparative Biochemistry and Physiology Part AMolecular amp Integrative Physiology 14073ndash79 DOI 101016jcbpb200411004

Romero LMWingfield JC 2016 Tempests poxes predators and people stress in wildanimals and how they cope New York Oxford University Press

RStudio Team 2015 RStudio integrated development for R Boston RStudio IncAvailable at httpwwwrstudiocom

Rupp H 1999 Excess catecholamine syndrome pathophysiology and therapy Annals ofthe New York Academy of Sciences 881430ndash444DOI 101111j1749-66321999tb09391x

Sapolsky RM Romero LMMunck AU 2000How do glucocorticoids influence stressresponses Integrating permissive suppressive stimulatory and preparative actionsEndocrine Reviews 2155ndash89

Schoech SJ Bowman R Reynolds SJ 2004 Food supplementation and possible mecha-nisms underlying early breeding in the Florida Scrub-Jay (Aphelocoma coerulescens)Hormones and Behavior 46565ndash573

Skoacuterzewska A Bidziński A Lehner M Turzyńska DWisłowska-Stanek A SobolewskaA Szyndler J Maciejak P Taracha E Plaźnik A 2006 The effects of acute andchronic administration of corticosterone on rat behavior in two models of fearresponses plasma corticosterone concentration and c-Fos expression in the brainstructures Pharmacology Biochemistry and Behavior 85522ndash534DOI 101016jpbb200610001

Walker BG Dee Boersma PWingfield JC 2006Habituation of adult MagellanicPenguins to human visitation as expressed through behavior and corticosteronesecretion Conservation Biology 20146ndash154 DOI 101111j1523-1739200500271x


Wingfield JC Vleck CMMooreMC 1992 Seasonal changes of the adrenocorticalresponse to stress in birds of the Sonoran desert Journal of Experimental Zoology264419ndash428 DOI 101002jez1402640407

Wisłowska-Stanek A Lehner M Skoacuterzewska A Krząścik P Płaźnik A 2016Behavioral effects and CRF expression in brain structures of high- and low-anxiety rats after chronic restraint stress Behavioural Brain Research 31026ndash35DOI 101016jbbr201605001


become overworked (Cyr et al 2007 McCormick Shea amp Langkilde 2015 Pavlides Nivoacutenamp McEwen 2002) For example rapid acute increases in sympathetic nervous systemactivity via catecholamines is crucial for the upregulation of essential survival systemsduring a stressor (Nephew amp Romero 2003) however chronic elevations can lead tohypertension stroke insulin resistance and hypertriglyceridemia (Rupp 1999) Despitethe wide array of studies we still lack a complete understanding of how these responsesinteract and what factors influence the transition of acute beneficial stress responses tothose characterized as chronic or harmful (Romero et al 2015) To begin teasing apart thesefactors researchers need to better understand whether identifiable thresholds exist betweenthese types of responses The purpose of this study was to push house sparrows (Passerdomesticus) to this threshold in order to identify key transition points in physiological andbehavioral indices

The stress response involves several behavioral and physiological systems (egRomero ampWingfield 2016 Sapolsky Romero amp Munck 2000) We focused on four of those responsesin this studymdashhypothalamic-pituitary-adrenal (HPA) axis regulation immune killingcapacity metabolism and neophobic behavior The HPA axis is the primary focus ofmany stress studies with particular attention paid to the glucocorticoids cortisol andcorticosterone (CORT) (Romero amp Wingfield 2016) With receptors in nearly every tissue(Lattin et al 2015) CORT has extremely wide-reaching effects (Ballard et al 1974 Lattinet al 2012b) Acute activation of the HPA axis and elevation of CORT induces thetranscription of proteins that broadly upregulate essential and suppress nonessentialsurvival systems (Sapolsky Romero amp Munck 2000) What has become increasinglyobvious however is that CORT concentrations do not have a clear relationship withchronic stress symptoms of chronic stress do not necessarily develop in tandem withelevated CORT concentrations (Dickens amp Romero 2013) Other aspects of HPA axisregulation are also important including total physiological capacity of CORT productionas well as the shut-down of CORT release via negative feedback (Dallman amp Bhatnagar2010 Dickens Delehanty amp Romero 2010)

CORT helps regulate downstream effects of the stress response including metabolicimmunological and behavioral effects (Romero amp Wingfield 2016) CORT is primarilyrecognized for the metabolic processes it either directly or permissively promotes Theseinclude gluconeogenesis (Romero amp Wingfield 2016) and glycogenolysis (Nonogaki 2000)These catabolic processes increase the breakdown of proteins and thus elevations inboth blood glucose and uric acidmdashthe major end product of nitrogen catabolismmdashare seen following acute CORT increases (Lin Decuypere amp Buyse 2004a) Uric acidis also an important antioxidant (Ames et al 1981) and itrsquos therefore been suggestedthat stress-induced changes may be related to maintaining redox balance (CohenKlasing amp Ricklefs 2007) In addition to these metabolic roles CORT is hypothesizedto both augment and inhibit immunity and inflammation on different timescales acuteactivation can enhance immunity while chronic activation can lead to immunosuppressiveeffects (Dhabhar 2002 Dhabhar amp McEwen 1999 Dhabhar amp McEwen 1997) FinallyCORT can influence the expression of several behaviors including elevated activity






Capture

Acclimationgt2weeks

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4days(Sample2)

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4days 4days10days




































ABaseline

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GroupAB

DMaximumcapacity
















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4days 8days

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ABaseline

CNegativefeedback

BStress-induced

GroupAB

DMaximumcapacity
















A

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4days 8days

NovelObject

NoObject

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A

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NoObject4days

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ABaseline

CNegativefeedback

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DMaximumcapacity
















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physiological and behavioral responses of house sparrows to ...submitted 2 march 2018 accepted 21...

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