There is mounting evidence from many species that physiological state can be reflected in external cues, such as the structure of vocalizations. North American red squirrels (Tamiasciurus hudsonicus (Erxleben, 1777)) produce individually distinct territorial vocalizations, called rattles, containing information relating to both a proximate stressor and reproductive condition. Given the importance of these vocalizations in communicating territory ownership, and their ability to contain cues relating to physiological state, we hypothesized that information regarding individual circulating glucocorticoid concentrations is transmitted between conspecifics within an audible range. We analysed fecal cortisol metabolite (FCM) concentrations from 129 red squirrels, comparing focal individual FCMs to conspecific FCMs within an acoustic neighbourhood. We found support for our hypothesis through a significant positive relationship between the FCM concentrations of focal squirrels and their neighbours independent of local density. These findings demonstrate that individual physiology interacts with conspecific physiology at ecologically relevant spatial scales and suggest that the social relationships between individual red squirrels may play a larger role in dictating individual responses to the environment.

Identifying the factors driving juvenile recruitment is crucial for predicting the response of populations to environmental change. Importantly, how early life conditions carry over to influence recruitment may be highly dependent on the context in which they occur. For example, the effects of challenging early life conditions may be more pronounced under high densities or when young are born late in the season. We examined the ecological factors influencing local recruitment spanning three decades in Savannah sparrows (Passerculus sandwichensis) breeding on Kent Island, NB, Canada. The effect of nestling mass on recruitment depended on both population density and fledging date. At low-population densities or early in the breeding season, nestling mass had little effect on recruitment probability. At high-population densities or later in the breeding season, mass had a stronger effect, with heavier individuals more likely to recruit. Lighter fledglings may have lower recruitment under challenging conditions due to lower competitive ability, lower mobility and greater susceptibility to resource limitation relative to heavier fledglings. Our findings have important implications for life-history evolution and selection on body size in a changing world, highlighting the relationships between population density, timing of breeding and offspring recruitment.

Clinical literature indicates there may be a therapeutic use of cannabidiol (CBD) for stress-related disorders. Preclinical literature remains conflicted regarding the underlying neurobehavioral mechanisms, reporting mixed effects of CBD (increased, decreased, or no effect) on anxiety- and fear-related behaviors. Preclinical data demonstrated that CBD modulates hypothalamus–pituitary–adrenal (HPA) axis gene expression; it is unknown whether CBD changes HPA axis responsivity and how this relates to altered behavior. We aimed to evaluate whether acute or chronic CBD administration would alter physiological and behavioral measures of HPA axis responsivity in male or female mice. C57BL/6 mice of both sexes were injected with vehicle or CBD (30 mg/kg, i.p.) daily for 26 days. Plasma corticosterone (CORT) levels were evaluated following dexamethasone suppression and adrenocorticotropin hormone stimulation tests after acute and chronic CBD exposure. After chronic CBD, mice were tested for anxiety-like behavior using an elevated plus maze (EPM) and associative fear learning and memory using a trace fear conditioning (FC) protocol. Compared to vehicle, CBD induced a state of HPA axis hyperactivation, an effect which was significant in males; it also normalized anxiety-like behavior in female mice classified as having HPA axis hypofunction and primed all female mice for enhanced conditioned responding. Significant sex differences were also detected: females had greater plasma CORT levels and HPA axis responsivity than males, exhibited less EPM anxiety-like behavior, and were more responsive during FC. CBD potentiated physiological and behavioral markers of HPA axis function and normalized anxiety-like behavior in a sex-specific manner. This observation has implications for cannabinoid-based drug development targeting individuals with stress-related disorders involving HPA axis hypofunction pathology.

Many birds produce two broods each year and exhibit significant intra-population variation in the length of time between the hatching of first and second broods (the inter-brood interval). Why within-population variation in inter-brood interval occurs and whether longer inter-brood intervals influence second-brood survival remain key questions in understanding the benefits of producing a second brood. We examined the causes and consequences of variation in inter-brood interval in a wild population of migratory Savannah Sparrows Passerculus sandwichensis breeding on Kent Island, New Brunswick, Canada. Using a 29-year dataset, we show that females tended to have short inter-brood intervals when their first nest was initiated later in the season, when they had a low number of first-brood fledglings to care for, or when they had higher lifetime recruitment (suggesting that they were of higher quality). Males with the shortest and longest inter-brood intervals had low feeding rates. Finally, structural equation modelling provided evidence that longer inter-brood intervals led to later second-brood hatch dates, which in turn led to smaller clutch sizes and fewer recruits. Our results suggest that the length of time between broods is a key factor predicting the first-year survival of second-brood offspring. These long-term data reveal that inter-brood interval reflects the timing of breeding and variation in female quality and that it influences offspring survival and, likely, fitness.

Given that double-brooding (rearing two broods within a season) can increase annual fecundity, it is unclear why some females in multi-brooded populations rear only one brood per season. The Quality Hypothesis proposes that double-brooded females are high quality and, thus, have sufficient energetic resources available to bear the costs of rearing two broods per season. Glucocorticoids - endocrine hormones that have a critical role in energy regulation - could reflect female quality, and, therefore, also have the potential to indicate whether a female will rear a second brood. Using 12 years of reproductive data on migratory Savannah sparrows (Passerculus sandwichensis) from a population in eastern Canada, we explored whether baseline corticosterone concentrations were correlated with measures of female quality (body condition and fat score) and whether a female's baseline corticosterone concentrations during her first brood would predict whether she attempted a second. We found weak evidence that baseline corticosterone was negatively correlated with female body condition and found strong evidence that baseline corticosterone was negatively correlated with fat score. There was weak evidence for a positive relationship between double-brooding and baseline corticosterone in females sampled during the first brood incubation stage. Additionally, there was moderate evidence to suggest that the probability of double-brooding was negatively related to baseline corticosterone in females sampled during the first brood nestling stage. Our results provide evidence that corticosterone can reflect female condition in the context of double-brooding and demonstrate the importance of considering breeding stage when assessing corticosterone concentrations in parents.

In birds, rearing multiple broods per season can substantially increase the annual number of fledglings produced. However, the contribution of double-brooding to lifetime fitness is unclear because the number of recruits arising from single- and double-brooded females is rarely measured. Poor estimates of fitness also make it challenging to document potential trade-offs between double-brooding and survival or future reproductive output. To understand the contribution of double-brooding to lifetime fitness and whether double-brooding was associated with life-history trade-offs, we used 30 years of reproductive data on female Savannah sparrows (Passerculus sandwichensis) breeding on Kent Island, New Brunswick. Estimates of fitness included an analysis of recruitment of both F1 (first generation) and F2 (second generation) offspring from females that did and did not raise a second brood. We detected no net costs of double-brooding. Double-brooded females had higher annual apparent survival rates than single-brooded females and F1 offspring from first broods of double-brooded females were more likely to recruit into the population than F1 offspring from single-brooded females. Double-brooding also improved lifetime fitness. Recruitment of F1 offspring was positively related to the number of seasons that a female double-brooded and, as a result, there was a higher number of F2 recruits from F1 offspring arising from double-brooded females than from F1 offspring arising from single-brooded females. Our results provide strong evidence that double-brooding is a beneficial reproductive strategy for Savannah sparrows and suggests that double-brooding females are likely high-quality individuals capable of rearing two broods a season with no net fitness costs.

The evolution of mating systems reflects a balance of the often-conflicting interests of males and females. Polygyny, a mating system in which males have multiple mates, presents a fitness benefit to males, but the consequences for females are less clear. Females with polygynous social mates may suffer reduced fitness, especially secondary females who typically receive less male support. We used 32 yr of detailed reproductive data on a population of Savannah sparrows (Passerculus sandwichensis) breeding on Kent Island, NB, Canada, to investigate the effects of females' social mating status on 6 indices of female fitness: survival, clutch size, fledging success, number of fledglings produced per nest and annually, and recruitment of offspring. Secondary females produced fewer fledglings per nest and annually than did monogamous or primary females, and their young were less likely to recruit into the breeding population. Yearling secondary females also had lower survival rates than older secondary females. Combined with higher rates of partial brood loss among secondary females, our results suggest that secondary females are unable to provide enough care to consistently fledge all nestlings in their broods, likely due to reduced male provisioning. Given that the sex ratio of breeders in the population is female-biased, we suggest that polygyny persists despite its fitness costs because some females must mate polygynously to "make the best of a bad situation." Our study demonstrates the value of detailed, long-term population monitoring data for understanding mating systems and using multiple indices of fitness to analyze the costs of polygyny.

ABSTRACT The size and shape of an animal’s breeding territory are dynamic features influenced by multiple intrinsic and extrinsic factors and can have important implications for survival and reproduction. Quantitative studies of variation in these territory features can generate deeper insights into animal ecology and behavior. We explored the effect of age, breeding strategy, population density, and number of neighbors on the size and shape of breeding territories in an island population of Passerculus sandwichensis (Savannah Sparrow). Our dataset consisted of 407 breeding territories belonging to 225 males sampled over 11 years. We compared territory sizes to the age of the male territorial holder, the male’s reproductive strategy (monogamy vs. polygyny), the number of birds in the study population (population density), and the number of immediate territorial neighbors (local density). We found substantial variation in territory size, with territories ranging over two orders of magnitude from 57 to 5,727 m2 (0.0057–0.57 ha). Older males had larger territories, polygynous males had larger territories, territories were smaller in years with higher population density, and larger territories were associated with more immediate territorial neighbors. We also found substantial variation in territory shape, from near-circular to irregularly shaped territories. Males with more neighbors had irregularly shaped territories, but shape did not vary with male age, breeding strategy, or population density. For males that lived 2 years or longer, we found strong consistent individual differences in territory size across years, but weaker individual differences in territory shape, suggesting that size has high repeatability whereas shape has low repeatability. Our work provides evidence that songbird territories are highly dynamic, and that their size and shape reflect both intrinsic factors (age and number of breeding partners) and extrinsic factors (population density and number of territorial neighbors).

Animals use colouration to serve diverse functions including camouflage, thermoregulation, and communication. Recent research has revealed that many anurans exhibit drastic colour changes and growing evidence supports that these changes are sexually selected signals. Male yellow toads, Incilius luetkenii, exhibit dynamic sexual dichromatism, changing from mud -brown to lemonyellow during their brief breeding events. Toads darken when isolated in captivity, which is hypothesized to be a stress response, although the mechanisms driving this change have yet to be experimentally investigated. We confned breeding toads to small terrariums for four hours and predicted that colour and corticosterone levels would change in isolation. We found that toads darkened during isolation, but that corticosterone levels did not change with colour. Our correlational results suggest that corticosterone is not the main driver of colour change in yellow toads and highlight avenues for future research that may enhance our understanding of colour change in anurans.

Female advertisement of reproductive state and receptivity has the potential to play a large role in the mating systems of many taxa, but investigations of this phenomenon are underrepresented in the literature. North American red squirrels (Tamiasciurus hudsonicus) are highly territorial and engage in scramble competition mating, with males converging from spatially disparate territories to engage in mating chases. Given the narrow estrus window exhibited in this species, the ubiquitous use of vocalizations to advertise territory ownership, and the high synchronicity of males arriving from distant territories, we hypothesized that female vocalizations contain cues relating to their estrous state. To test this hypothesis, we examined the spectral and temporal properties of female territorial rattle vocalizations collected from females of known reproductive condition over 3 years. While we found no distinct changes associated with estrus specifically, we did identify significant changes in the spectral characteristics of rattles relating to both female body mass and reproductive state relative to parturition. To the best of our knowledge, this is the first evidence of changes in vocal characteristics associated with late pregnancy in a nonhuman mammal. Despite no evidence that vocal cues relating to estrus are communicated in the North American Red Squirrel, we found that female vocal characteristics showed significant variation related to both reproductive state and body mass. To our knowledge, this is the first evidence of vocal changes associated with pregnancy in a nonhuman mammal. Graphical Abstract

Host-associated bacterial microbiomes can facilitate host acclimation to seasonal environmental change and are hypothesized to help hosts cope with recent anthropogenic environmental perturbations (e.g., landscape modification). However, it is unclear how recurrent and recent forms of environmental change interact to shape variation in the microbiome. The majority of wildlife microbiome research occurs within a single seasonal context. Meanwhile, the few studies of seasonal variation in the microbiome often restrict focus to a single environmental context. By sampling urban and exurban eastern grey squirrel populations in the spring, summer, autumn, and winter, we explored whether seasonal rhythms in the grey squirrel gut microbiome differed across environments using a 16S amplicon sequencing approach. Differences in the microbiome between urban and exurban squirrels persisted across most of the year, which we hypothesize is linked to anthropogenic food consumption, but we also observed similarities in the urban and exurban grey squirrel microbiome during the autumn, which we attribute to engrained seed caching instincts in preparation for the winter. Host behaviour and diet selection may therefore be capable of maintaining similarities in microbiome structure between disparate environments. However, the depletion of an obligate host mucin glycan specialist (Akkermansia) during the winter in both urban and exurban squirrels was among the strongest differential abundance patterns we observed. In summary, urban grey squirrels showed different seasonal patterns in their microbiome than squirrels from exurban forests; however, in some instances, host behaviour and physiological responses might be capable of maintaining similar microbiome responses across seasons.

The nest environment may limit the ability of nest-bound birds to hear sounds from the outside world. In vocal learning species, such as humans and songbirds, it is vital for young animals to hear the voices of conspecific animals early in life. In songbirds, nest structure varies considerably across species, and the resulting impact on sound transmission may have consequences for vocal learning in nestlings. In this study, we tested the hypothesis that the nest environment attenuates song of Savannah Sparrows (Passerculus sandwichensis), grassland songbirds that build nests on the ground under cover of dense vegetation. We recorded live Savannah Sparrows singing at variable distances from 21 nests. We recorded songs using one microphone inside the nest (the typical position of a nestling) and another placed 1 m directly above the nest (a typical position of an adult). We found a substantial reduction in signal-to-noise ratio, where songs recorded inside the nest were an average of 11 dB lower than songs recorded directly above the nest. We estimate that the attenuation imposed by the nest reduced the maximum acoustic environment from 117.7 m (for recordings above the nest), to 78.6 m (for recordings within the nest), which is analogous to listening from a position 39.1 m farther away from the singer. Previous research estimated that song transmits up to 100 m in this species, so any adult male within 100 m of a young bird was previously considered a potential vocal tutor. By reducing the nestling acoustic environment from 100 m to 78.6 m, the number of male tutors available to nestlings is reduced by an average of 27%. Given the growing evidence that song learning begins very early in life, future research on vocal development should account for how the structural properties of the natal environment affect the songs that nestlings hear.

Social communication often involves vocal learning, whereby young animals learn their vocalizations early in life by imitating the sounds of adults. In animals that learn their vocalizations, it is common to find patterns of geographical variation known as 'vocal dialects', acoustic features shared within a cluster of animals that differ from the vocalizations of animals in nearby clusters. Dialects may form when animals learn their vocalizations early in life and then disperse short distances, or when they modify their vocalizations to match local vocal patterns after dispersal. Dialects are typically studied at a regional or continental scale, but they may also persist at smaller scales in so-called 'microgeographical song dialects' or 'microdialects'. Microdialects have received little research attention. In this study, we investigate microdialects and dispersal distance in song-learning Savannah sparrows, Passerculus sandwichensis, through a 4-year study of birds living on three islands in the Bay of Fundy, Canada. Our analyses yield evidence of microgeographical variation: birds showed higher acoustic similarity to their neighbours than to faraway individuals in the same population. When we classified songs on the basis of their highly variable middle sections, we found that particular song types formed spatial clusters of similar-sounding individuals. Therefore, acoustic variation in Savannah sparrow song appears to show dialects across small geographical distances. In an analysis of dispersal from natal sites to breeding sites, we found a median distance of 189 m, consistent with previous findings. Our results suggest that limited dispersal distances, combined with the social processes of vocal learning (overproduction and selective attrition), likely contribute to the presence of microdialects. (c) 2022 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Young songbirds draw the source material for their learned songs from parents, peers, and unrelated adults, as well as from innovation. These learned songs are used for intraspecific communication, and have well-documented roles for such functions as territory maintenance and mate attraction. The songs of wild populations differ, forming local “dialects” that may shift over time, suggesting that cultural evolution is at work. Recent work has focused on the mechanisms responsible for the cultural evolution of bird songs within a population, including drift, learning biases (such as conformity and rare-form copying), and selection (including sexual selection). In many songs or song repertoires, variability is partitioned, with some songs or song segments being stable and consistent, while others vary within the population and across time, and still others undergo population-wide transitions over time. This review explores the different mechanisms that shape the cultural evolution of songs in wild populations, with specific reference to a long-term investigation of a single population of philopatric Savannah sparrows. Males learn a single four-segment song during their 1st year and sing the same song thereafter. Within this song, the buzz segment is a population marker, and may be stable for decades – variant forms occur but eventually disappear. In contrast, the middle segment is highly variable both within the population and over time; changes in relative prevalence of different forms may be due to cultural drift or a rare-form learning bias. Within the introductory segment, a high note cluster was replaced by a click train between 1982 and 2010, following an S-shaped trajectory characteristic of both selective sweeps in population genetics and the replacement of one form by another in human language. In the case of the Savannah sparrows, this replacement may have been due to sexual selection. In subsequent generations, the number of clicks within trains increased, a form of cultural directional selection. In contrast to the narrowing of a trait's range during directional selection in genetic systems, variation in the number of clicks in a train increased as the mean value shifted because improvisation during song learning allowed the range of the trait to expand. Thus, in the single short song of the Savannah sparrow, at least four different mechanisms appear to contribute to three different types of cultural evolutionary outcomes. In the future, it will be import to explore the conditions that favor the application of specific (and perhaps conditional) learning rules, and studies such as the ongoing song seeding experiment in the Kent Island Savannah sparrow population will help in understanding the mechanisms that promote or repress changes in a population's song.

Many breeding birds produce conspicuous sounds, providing tremendous opportunities to study free-living birds through acoustic recordings. Traditional methods for studying population size and demographic features depend on labor-intensive field research. Passive acoustic monitoring provides an alternative method for quantifying population size and demographic parameters, but this approach requires careful validation. To determine the accuracy of passive acoustic monitoring for estimating population size and demographic parameters, we used autonomous recorders to sample an island-living population of Savannah Sparrows (Passerculus sandwichensis) over a 6-year period. Using the individually distinctive songs of males, we estimated the male population size as the number of unique songs detected in the recordings. We analyzed songs across 6 years to estimate birth year, death year, and longevity. We then compared the estimates with field data in a blind analysis. Estimates of male population size through passive acoustic monitoring were, on average, 72% of the true male population size, with higher accuracy in lower-density years. Estimates of demographic rates were lower than true values by 29% for birth year, 23% for death year, and 29% for longevity. This is the first investigation to estimate longevity with passive acoustic monitoring and adds to a growing number of studies that have used passive acoustic monitoring to estimate population size. Although passive acoustic monitoring underestimated true population parameters, likely due to the high similarity among many male songs, our findings suggest that autonomous recorders can provide reliable estimates of population size and longevity in a wild songbird. Lay Summary center dot Ornithologists can study free-living birds through passive acoustic recordings of their songs, although these bioacoustic methods require careful assessment and validation. center dot Based on 6 years of data, we estimated the size of a population of Savannah Sparrows (Passerculus sandwichensis) based on the number of unique songs detected in passive acoustic recordings, and we compared our estimates to measurements of population size collected through in-person field studies. center dot We also estimated the longevity of birds by comparing songs recorded across subsequent years, again comparing our estimates to measurements of longevity from in-person field studies. center dot We found that bioacoustic recordings provided predictable and reliable estimates of population size and longevity, although the recordings underestimated true population size and true longevity. center dot Although our acoustic data underestimated population parameters, likely owing to the high similarity among many male songs, our findings reveal that bioacoustic recordings can provide reliable estimates of population size and longevity in wild birds.

The effect of the social environment on individual state or condition has largely focused on glucocorticoid levels (GCs). As metabolic hormones whose production can be influenced by nutritional, physical, or psychosocial stressors, GCs are a valuable (though singular) measure that may reflect the degree of "stress" experienced by an individual. Most work to date has focused on how social rank influences GCs in group-living species or how predation risk influences GCs in prey. This work has been revealing, but a more comprehensive assessment of the social environment is needed to fully understand how different features of the social environment influence GCs in both group living and non-group living species and across life history stages. Just as there can be intense within-group competition among adult conspecifics, it bears appreciating there can also be competition among siblings from the same brood, among adult conspecifics that do not live in groups, or among heterospecifics. In these situations, dominance hierarchies typically emerge, albeit, do dominants or subordinate individuals or species have higher GCs? We examine the degree of support for hypotheses derived from group-living species about whether differential GCs between dominants and subordinates reflect the "stress of subordination" or "costs of dominance" in these other social contexts. By doing so, we aim to test the generality of these two hypotheses and propose new research directions to broaden the lens that focuses on social hierarchies and GCs.

Environmental change and biodiversity loss are but two of the complex challenges facing conservation practitioners and policy makers. Relevant and robust scientific knowledge is critical for providing decision-makers with the actionable evidence needed to inform conservation decisions. In the Anthropocene, science that leads to meaningful improvements in biodiversity conservation, restoration and management is desperately needed. Conservation Physiology has emerged as a discipline that is well-positioned to identify the mechanisms underpinning population declines, predict responses to environmental change and test different in situ and ex situ conservation interventions for diverse taxa and ecosystems. Here we present a consensus list of 10 priority research themes. Within each theme we identify specific research questions (100 in total), answers to which will address conservation problems and should improve the management of biological resources. The themes frame a set of research questions related to the following: (i) adaptation and phenotypic plasticity; (ii) human-induced environmental change; (iii) human-wildlife interactions; (iv) invasive species; (v) methods, biomarkers and monitoring; (vi) policy, engagement and communication; (vii) pollution; (viii) restoration actions; (ix) threatened species; and (x) urban systems. The themes and questions will hopefully guide and inspire researchers while also helping to demonstrate to practitioners and policy makers the many ways in which physiology can help to support their decisions.

Phenotypic plasticity allows organisms to adjust the timing of life-history events in response to environmental and demographic conditions. Shifts by individuals in the timing of breeding with respect to variation in age and temperature are well documented in nature, and these changes are known to scale to affect population dynamics. However, relatively little is known about how organisms alter phenology in response to other demographic and environmental factors. We investigated how pre-breeding temperature, breeding population density, age, and rainfall in the first month of life influenced the timing and plasticity of lay date in a population of Savannah Sparrows (Passerculus sandwichensis) monitored over 33 yr (1987-2019). Females that experienced warmer pre-breeding temperatures tended to lay eggs earlier, as did older females, but breeding population density had no effect on lay date. Natal precipitation interacted with age to influence lay date plasticity, with females that experienced high precipitation levels as nestlings advancing lay dates more strongly over the course of their lives. We also found evidence for varied pace of life; females that experienced high natal precipitation had shorter lifespans and reduced fecundity, but more nesting attempts over their lifetimes. Rainfall during the nestling period increased through time, while population density and fecundity declined, suggesting that increased precipitation on the breeding grounds may be detrimental to breeding females and ultimately the viability of the population as a whole. Our results suggest that females adjust their laying date in response to pre-breeding temperature, and as they age, while presenting new evidence that environmental conditions during the natal period can affect phenological plasticity and generate downstream, population-level effects.

Neonicotinoid insecticides are used to reduce crop damage caused by insect pests, but sublethal levels could affect development and reproduction in nontarget insects, such as monarch butterflies (Danaus plexippus) (Lepidoptera: Nymphalidae). To investigate the impact of field-realistic concentrations of the neonicotinoid clothianidin on monarch butterflies, we grew swamp milkweed (Asclepias incarnata) (Apocynaceae) in either low (15 ng/g of soil) or high (25 ng/g of soil) levels of clothianidin, or in a control (0 ng/g), then raised monarchs on the milkweed. Morphological traits of monarch caterpillars were measured during development and, once they eclosed, were mated as adults to quantify egg size and mass and the number of eggs laid. Although the effects of the treatment had complex effects on caterpillar length, width and volume of late-instar caterpillars were negatively affected. Fifth-instar caterpillars from the high-dose insecticide treatment had lower mass than other groups. Adult monarch butterflies raised on treated milkweed were larger than controls, but clothianidin exposure did not affect the number of eggs laid or egg size. Although the magnitude of the effect depends on clothianidin concentration, our results suggest that exposure to clothianidin during early life can impact monarch caterpillar development but is unlikely to reduce female reproductive output.

Migratory insects use a variety of innate mechanisms to determine their orientation and maintain correct bearing. For long-distance migrants, like the monarch butterfly (Danaus plexippus), these journeys could be affected by exposure to environmental contaminants. Neonicotinoids are synthetic insecticides that work by affecting the nervous system of insects resulting in impairment of their mobility, cognitive performance and other physiological and behavioural functions. To examine how neonicotinoids might affect the ability of monarch butterflies to maintain a proper directional orientation on their ∼4,000 km migration, we grew swamp milkweed (Asclepias incarnata) in soil that was either untreated (0 ng/g: control) or mixed with low (15 ng/g of soil), or high (25 ng/g of soil) levels of the neonicotinoid clothianidin. Monarch caterpillars were raised on control or clothianidin-treated milkweed and, after pupation, either tested for orientation in a static flight simulator or radio-tracked in the wild during the fall migration period. Despite clothianidin being detectable in milkweed tissue consumed by caterpillars, there was no evidence that clothianidin influenced the orientation, vector strength (i.e., concentration of direction data around the mean), or the rate of travel of adult butterflies and nor was there evidence that morphological traits (i.e., mass and forewing length), testing time, wind speed, or temperature impacted directionality. While samples sizes for both flight simulator and radio-tracking tests were limited, our preliminary results suggest that clothianidin exposure during early caterpillar development does not affect the directed flight of adult migratory monarch butterflies or influence their orientation at the beginning of migration.