Behavioral and hormonal responses to urbanization in odorous house ants (Tapinoma sessile)

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Neumann, Liam Hoeferlin, Saieshwar Chikoti, Evan Frank, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8963511/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 5 You are reading this latest preprint version Abstract Urbanization has profound effects on biological communities. Many organisms cannot persist in anthropogenic environments, while others may adapt to urban conditions. Behavioral traits can facilitate this adaptation and predict how species might respond to urbanization. We studied the behavior of the odorous house ant ( Tapinoma sessile ) which is common in both natural (i.e. forests) and urban areas. Relative to natural environments, colonies in urban areas are typically more aggressive and have many more workers and queens. To examine how this variation may influence other behaviors, we compared the exploratory behavior of T. sessile workers and colonies from natural and urban environments. We found repeatable variation in exploratory behavior, suggesting workers have distinct behavioral types. Additionally, colonies from natural environments had higher exploration and foraging activity relative to urban colonies. Activity also varied among ants with different behavioral roles - workers that were foraging were more exploratory than workers taken from the nest or that were engaged in a defensive role (i.e. recruited to the location of a different colony). Finally, we identified a potential proximate mechanism that might be influencing activity. Treatment with the neuromodulator octopamine led to increased levels of individual exploration and colony level foraging activity for colonies from both habitat types. However, natural variation in worker octopamine levels did not vary between environments. Together, these results suggest that exploratory behavior plays a role in adaptation to urbanization. Furthermore, octopamine may be a key driver for exploratory and foraging behavior in odorous house ants. exploration foraging octopamine neuromodulator personality Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Urbanization presents various challenges to organisms, especially through the modification of habitat structure, resource availability, and microclimate (Wong & Candolin 2015 ). In response, animals have adopted a wide range of behavioral strategies to deal with these changes, and in some cases, even take advantage of the opportunities presented by urbanization (Chapple et al. 2012 ; Sol et al. 2013 ; Wrensford et al., 2025). Individuals in urban populations may have increased levels of exploration, boldness, or neophilia. For example, ground beetles (Carabidae) in more urbanized areas are more exploratory (Schuett et al. 2018 ), and Anolis lizards from urban areas are bolder and more exploratory compared to those from forest populations (Lapiedra et al. 2017 ; Avilés-Rodríguez & Kolbe 2019 ). Additionally, foraging behaviors often differ between urban and non-urban populations because of novel resources present in urban habitats (Lato et al. 2021 ). For example, foraging activity increases in urban populations of the stingless bee Tetragonula carbonaria (Apidae), with altered resource availability from gardens likely driving this change (Kaluza et al. 2016 ). In addition to documenting behavioral changes among urban and non-urban populations, it is important to understand the proximate hormonal mechanisms underlying these differences. House sparrows ( Passer domesticus ) near the edge of a range expansion exhibit increased exploration and higher corticosterone levels (Liebl & Martin 2012 ). In dark eyed juncos ( Junco hyemalis ), there were correlated changes in parental care and testosterone expression associated with the colonization of a coastal, urban habitat from nearby temperate forests (Atwell et al. 2014 ). However, more information across different taxa and environments is needed to tease apart the relationship between hormones, behavior, and phenotypic divergence between urban and natural populations. Behaviors also vary based on their context in relation to the task an individual is performing (e.g., foraging, defending territory, socializing, courting mates; Madrzyk & Pinter-Wollman 2022 ; Trigos-Peral et al. 2023 ). For instance, boldness in grey mouse lemurs ( Microcebus murinus ) increases during foraging, and the willingness to take risks was dependent on the level of predation risk (Dammhahn & Almeling 2012 ). In cooperatively breeding cichlids ( Neolamprologus pulcher ), exploration is related to territoriality, with exploratory individuals actively defending the territory (Bergmüller & Taborsky 2007 ). Taking social context into consideration is particularly important with eusocial organisms which exhibit pronounced division of labor and tasks are divided among individuals (Robinson 1992 ). In Myrmica rubra ants, workers focusing on within-nest tasks like brood care were less aggressive and exploratory relative to workers focusing on tasks outside the nest, like foraging or nest defense (Pamminger et al. 2014 ). This also underscores the importance of quantifying the repeatability of behavior, as differences in behavior across contexts could be due to plasticity within individuals or consistent differences across individuals (Garrison et al. 2018 ). Ants are a strong model for exploring behavioral variation in response to disturbance as they are abundant in both natural and urban environments (Buczkowski et al. 2023 ). In urban areas, dominant, aggressive ant species often exclude native species or may be better at colonizing disturbed habitats (Suarez et al. 1999 ; King & Tschinkel 2008 ; Neumann & Pinter-Wollman 2019 ). Furthermore, ants exhibit division of labor among workers, allowing for comparison of behavior across different social contexts (e.g. caring for brood in the nest, searching for food or nests sites, or defending the colony from threats) (Modlmeier et al. 2014 ). Finally, many hormones are relatively well-studied in ants, providing candidate targets and methods for further exploration (Starkey & Tamborindeguy 2023 ; Ye et al. 2025 ). Understanding the role of behavior in how ants respond to urbanization can elucidate which species may be ‘winners’ or ‘losers’ in the face of global change (Salyer et al. 2014 ). This is particularly important given the diverse roles they play in ecosystems as seed dispersers, scavengers, and soil engineers (Folgarait 1998 ; Del Toro & Pelini 2012). The odorous house ant ( Tapinoma sessile ) is an emerging model organism for examining differences in biology between urban and natural populations (Menke et al. 2010 ; Buczkowski 2010 ; Blumenfeld et al. 2022 ). Odorous house ants are found across North America in a variety of natural environments, including forests, wetlands, and prairies, and disturbed environments such as cities and agricultural landscapes. Urban populations of T. sessile are associated with shifts in their social structure and negative impacts on native ant species richness (Buczkowski & Bennett 2008; Salyer et al. 2014 ). Colonies from natural environments tend to have fewer workers and queens relative to urban colonies which are highly polygynous (multiple queens) and polydomous (occupy multiple nests) and can have hundreds of thousands of workers (Buczkowski 2010 ). Odorous house ants exhibit high levels of interspecific aggression in urban populations and have been identified as a potential threat to become an introduced species given the suite of characteristics they share with known invasive ants, including high levels of interspecific aggression, large, rapidly dispersing colonies, and generalist habitat and foraging preferences (Buczkowski & Krushelnycky 2012 ). While aggressive behavior in T. sessile is relatively well studied (Buczkowski & Bennett 2008), we know less about how other behaviors vary between natural and urban populations. Exploratory behavior is a potentially important behavior to explore further because of its relevance to various worker tasks including identifying food sources, territorial defense, and searching for new nest sites (Nonacs 1991 ; Barbani 2002 ; Page et al. 2018 ). Furthermore, exploration and foraging activity is higher in other urban ant species (Jacquier et al. 2023 ), and thus could be contributing to the success of T. sessile in urban habitats. The hormonal mechanisms underlying variation in behavior between urban and natural populations of T. sessile have not been explored. In ants and other invertebrates, one hormone of interest is the neurotransmitter and neuromodulator octopamine. Octopamine is similar in structure to norepinephrine, the “flight or fight” hormone in vertebrates (Adamo et al. 1995 ). It has broad function across invertebrates but is generally related to activity, aggression, and sensory systems (Roeder, 1999 ; Wada-Katsumata et al., 2011 ; Kamhi et al., 2015 ). Thus, octopamine could be a potential mechanism of exploratory behavior. Felden and colleagues ( 2018 ) found invasive Argentine ants ( Linepithema humile ) treated with octopamine had increased foraging activity, although the degree of response was not related to the source environment (e.g. native or introduced populations). Additionally, octopamine is important for tracking olfactory cues, which may be related to exploratory or foraging behavior (Wissink & Nehring 2021 ). We examined the behavior of odorous house ants from natural and urban environments to answer the following: (1) Does individual exploratory behavior and/or collective foraging activity differ among workers from natural vs. urban colonies? (2) Is exploratory behavior related to a worker’s behavioral state (foraging, brood care, or nest defense)? (3) Do standing levels of octopamine differ between natural and urban colonies, and (4) What is the effect of octopamine on individual exploration and collective foraging activity? We predicted that individual exploration and collective foraging activity would be higher in urban colonies. Additionally, we predicted foragers would have higher levels of exploration relative to workers from within the nest. Finally, we predicted that octopamine levels would be positively correlated with individual exploration and collective foraging activity, and thus, standing octopamine levels would be higher in urban colonies. METHODS Ant collection and care In June 2023, we collected one colony from each of eight locations in Lafayette, IN, USA; four urban environments (on or near the Purdue University campus) and four natural environments (mixed hardwood forests) (Buczkowski et al. 2023 ). For urban colonies, we took a subset of larger, polygynous, polydomous ‘supercolonies’, approximately 10,000–15,000 workers, 40–50 queens, and numerous brood. Natural colonies contained approximately 3,000–5,000 workers, 10–20 queens, and numerous brood. Prior work found high levels of genetic differentiation and aggression between workers from these same populations, suggesting that they are distinct populations (Blumenfeld et al. 2022 ). Colonies were housed in the lab at the University of Illinois Urbana-Champaign in plastic containers coated with Fluon (60x40cm), with plastic tubes covered in tinfoil as artificial nests. They were fed sugar water and dried crickets ad libitum . Colonies were given one week to acclimate to lab conditions prior to data collection. Individual exploration We measured exploratory behavior using an open-field assay. A worker was gently placed into the center of a plastic container (60x40cm) coated with Fluon, with a grid placed underneath the container separating it into twelve equally sized sections. We observed for 5 minutes and recorded the number of times the worker moved between sections. Containers were cleaned with ethanol before the next trial. We repeated this for 25 workers from each of the eight colonies. All behavioral measurements occurred between 11:00 am and 4:00 pm. Furthermore, to assess the consistency of individual behavior, we repeated this assay for 10 workers from each colony (e.g. the same individual was measured twice), waiting 24 hours between measurements. Between measurements, workers were kept in a mesh enclosure that was placed back into the colony, allowing us to keep them exposed to other workers while maintaining their identity. To minimize observer bias, here and in all of the following section, blinded methods during analysis of behavioral data. Collective foraging activity To measure collective foraging activity, we first moved a colony fragment consisting of 1,000 workers, 5 queens, and numerous brood to a new nest box coated with fluon (40x20 cm). Colonies were fed 20% sugar water during an acclimation period of 72 hrs. Colonies were starved of protein over this time to motivate exploration, as ants require a consistent protein source to produce new brood. Then, we uncovered an opening to a 1m tube that connected to another box (10x10 cm) containing a protein source (dried crickets) and took a photo of this box every 20 min for 12 hrs (10:00AM to 10:00PM) using an AKASO Brave 8 camera. Collective foraging activity was quantified by counting the number of workers present in the box at each time point. We repeated this on two different colony fragments per population, for a total of 16 replicates. Relationship between individual exploration and division of labor To examine if exploration varied based on the worker’s behavioral role in the nest (foraging, nest care or defense), we designed a three-chambered arena, consisting of a nest box (containing an artificial nest), a foraging box, and a competition box (Fig. 1 ). We aspirated a colony fragment − 1,000 workers, 5 queens, and numerous brood and placed them into the nest box. The foraging and competition boxes were not initially accessible to the experimental colony. The experimental colony was given time to move into their new nest and then starved for 24 hrs after which the rest of the arena was opened for exploration. The foraging arena had a tube of sugar water and a dried cricket, while the competition arena had a mesh container with approximately 100 foreign T. sessile workers collected from the University of Illinois campus. Workers could detect the foreign T. sessile workers through the mesh but could not directly engage in any fights or aggressive behaviors, allowing us to better control for the effect of the competitor on behavior across trials. The experimental colony was then given another 24 hrs to explore the arena. We returned and collected 30 workers in total, 10 workers from each box (foraging box, nest box, and competition box). For the nest box, workers were collected only from within the nest chamber to reduce the chances of sampling a worker that had just returned from the foraging or competition boxes. Workers were sampled by gently picking them up with a paintbrush when they were not directly next to another worker, to limit the impact of collection on nearby workers. We measured exploratory behavior of all workers as previously described. We repeated this process three times for colonies from each of the eight locations. Workers that were measured for behavior were not returned to their original nest box to prevent any worker from being tested in multiple trials. These methods were replicated three times for each source colony. Relationship between octopamine and behavior Standing variation in octopamine in field colonies We explored the relationship between octopamine, behavior, and habitat (urban vs. natural) in Tapinoma sessile . We first compared standing levels of octopamine within workers across the eight populations. We aspirated 90 workers from each location, immediately placing them on dry ice after collection, and then stored them at -80° C. We also examined seasonal differences in octopamine levels between colonies from natural and urban habitats and differences in octopamine levels between workers either sampled from foraging trails (e.g. foragers) or workers sampled from the nest entrance. For this additional sampling, we identified three urban colonies and three natural, forest dwelling colonies near Champaign, IL, USA. For each site, ten workers were sampled from the entrance to the nest site and another ten workers were sampled along a foraging trail at least one meter from the nest entrance. When possible, we sampled workers from the same nest entrance each time. If a nest entrance from a previous visit was not occupied, we instead sampled from a nearby nest entrance. T. sessile are polydomous, allowing us to be confident we were sampling from the same colony. This sampling process was repeated twice a month from June through August 2025, during the peak season of activity for T. sessile . Thus, there were 72 pooled samples of 10 workers per sample: 6 time points for each of 6 colonies, from two locations (nest and foraging trail) for each colony. All samples were immediately put on dry ice until processing. For sample preparation, we first dissected workers’ heads over dry ice, removing the antennae. Each sample was pooled with 10 heads, for a total of nine samples from each of the eight locations. The heads were homogenized in 100 µl of ice cold 0.1 M formic acid with 5 ng of 3,4-di-hydroxybenzylamine (DHBA) as an internal standard, and then vortexed. Samples were centrifuged for 20 minutes at 13,500 rpm and 4° C. Then, 100 µl of a 1:3 isopropanol/chloroform solution was added, and the samples were centrifuged for another 5 minutes under the same conditions. We collected 50 µl of supernatant per sample and stored them at -80° C. All sample preparation methods were consistent with prior work (Wada-Katsumata et al. 2011 ; Hojo et al. 2015 ; Mannino et al. 2018 ). Following sample preparation, we performed liquid chromatography-mass spectrometry (LC-MS) using a SYNAPT G2-Si Mass Spectrometer in collaboration with the University of Illinois School of Chemical Sciences’ Mass Spectrometry Lab. LC-MS data was processed and concentrations of octopamine were quantified using Mass Lynx v4.2 (Waters Corp., Milford, USA). We only quantified relative concentrations of octopamine rather than actual measurements because this made the LC-MS methods more straightforward and relative concentrations were sufficient to answer our research questions. This did limit our ability to compare the presence of octopamine in T. sessile to other species or consider the role of octopamine in specific biochemical pathways. Effect of octopamine on individual exploration We explored the effect of octopamine on individual exploration. We took 160 workers (20 from each colony, 80 per colony type (i.e., the habitat the colony came from)) and assigned them to either an octopamine treatment or a control treatment, with all workers in same-colony groups of 10. Workers were starved for 24 hrs and then given 24 hrs to feed ad libitum on either 20% sugar water with 4 mg/mL octopamine or a control solution of 20% sugar water (as in Felden et al. 2018 ). Next, the individual exploration of each worker was tested using the individual exploration assay described above. To verify that these methods were effective in altering octopamine level, we quantified the relative concentrations of octopamine in a subset of workers across the two treatments using the same procedures. Effect of octopamine on collective foraging activity We also examined the effect of octopamine on the collective foraging of colonies. To do this, we repeated the same assay for collective foraging as described above, but with colonies being fed a solution of 20% sugar water with 4 mg/mL octopamine. We repeated the assay on two different colony fragments from each population for a total of 16 replicates. The colonies described in the Collective foraging activity section, which were fed just 20% sugar water, acted as a control. Data analysis To compare individual exploration across colony types (urban vs. natural), we ran a generalized linear mixed model (GLMM) with a negative binomial family, the number of sections explored as the response variable, colony type as a fixed effect, and population as a random effect, using the lme4 package in R (Bates et al. 2015 ). Here and in all following models, the significance of fixed effects was assessed by conducting Type II Wald chi-square tests using the Anova function in the car package (Fox & Weisberg, 2019 ). Additionally, overdispersion of this and the following GLMMs was tested using the testDispersion function in the DHARMa package (Hartig 2025 ). To measure the consistency of exploration, we computed the adjusted repeatability for each behavior, which considers variation due to factors (e.g., population) through mixed modeling approaches (Nakagawa & Schielzeth 2010 ). We ran linear mixed models (LMMs) with a log transformation of the number of sections explored as the response variable, colony type (natural vs. urban) as a fixed effect and ant ID as a random effect, using the rptR package (Stoffel et al. 2017 ). We calculated 95% confidence intervals and p-values with parametric bootstrapping (n = 10,000 iterations) and p-values from permutation tests (n = 10,000 iterations). To analyze the relationship between individual exploration and the worker’s behavioral role in the colony, we ran a generalized linear mixed model with a negative binomial family, number of sections explored as the response variable, behavioral role (foraging, nest, or competition) and an interaction term between role and colony type as fixed effects, and population as a random effect. We also included replicate as a random effect to account for repeated measures from each population. We made post-hoc comparisons for the effect of social context using the emmeans package in R (Lenth 2022 ). To compare standing levels of octopamine across colony types, we ran a linear mixed model with relative concentration of octopamine as a response variable, colony type as a fixed effect, and population as a random effect. There were nine individuals that had either extremely low concentrations or no octopamine detected. We removed these individuals from analysis as we could not rule out the possibility that these results were due to methodological error. The interpretation of the results did not change after removing these individuals. To analyze the seasonal field data, we ran a generalized linear model (Gamma family) with relative octopamine concentration as the response variable and time, location (nest vs. foraging trail) and habitat (urban vs. natural) as fixed effects. The significance of fixed effects was assessed by conducting Type II Wald chi-square tests. For the lab experiment, we verified that our octopamine treatment did lead to increased levels of octopamine compared to the control group with a Kruskal-Wallis test. To identify if octopamine had an impact on individual exploration, we ran a generalized linear mixed model with a negative binomial family, number of sections explored as the response variable, colony type and treatment (octopamine vs. control) as fixed effects, and population as a random effect. Finally, to compare collective foraging activity across colony types and treatments (octopamine vs. control), we ran a generalized linear mixed model with a Poisson family, number of foragers present as the response variable, colony type, treatment, time, and interaction terms between time and colony type and time and treatment as fixed effects, and population and replicate as random effects. This work followed all Guidelines for the Use of Animals in Research. Tapinoma sessile is not a threatened species and there were no licenses or permits required for this work. All conditions in the laboratory were in line with conditions T. sessile face in their natural environment. For lethal sampling, individuals were sacrificed as quickly as possible by being placed on dry ice. RESULTS Variation and repeatability of individual exploration Workers from natural colonies visited more sections in an open field than workers from urban colonies (GLMM: Chi-sq = 12.18, df = 1, N = 200, P < 0.001; Fig. 2 ). Furthermore, exploration was repeatable within individuals, suggesting that there are relatively stable behavioral types (LMM permutations: adjusted repeatability = 0.41 (0.21–0.58), N = 80, P < 0.001). Collective foraging activity In line with the individual behavioral results, colonies from natural environments had higher foraging rates compared to urban colonies (GLMM: Chi-sq = 4.46, df = 1, N = 24, P = 0.035; Fig. 3 ). There was not a significant effect of the interaction between colony type and time, suggesting that this pattern was consistent across the trial (GLMM: Chi-sq = 0.021, df = 1, N = 24, P = 0.64; Fig. 3 ). Relationship between individual exploration and division of labor Workers sampled from the foraging box were more exploratory than workers sampled from the competition box or the nest box (GLMM: Chi-sq = 42.21, df = 2, N = 720, P < 0.0001; Fig. 4 ). This pattern was consistent across both urban and natural colonies (GLMM: Chi-sq = 0.87, df = 2, N = 720, P = 0.65). Standing variation in octopamine across colonies Octopamine was present in the field colonies, and there was considerable variation among individuals in standing octopamine levels. However, there was not a significant difference in octopamine concentrations between natural and urban colonies (LMM: Chi-sq = 0.022, df = 1, N = 63, P = 0.96; Fig. 5 ). Seasonal variation in octopamine between foragers and nest workers. Workers sampled from the foraging trail had marginally higher levels of octopamine than those sampled from the nest entrance (GLM: Chi-sq = 3.23, df = 1, N = 72, P = 0.073; Figure S1 ). However, there was not a significant effect of time (GLMM: Chi-sq = 0.028, df = 1, N = 72, P = 0.86) or habitat (natural versus urban; GLM: Chi-sq = 0.29, df = 1, N = 72, P = 0.59) (Figure S1 ). Effect of octopamine on individual exploration The octopamine treatment led to increased levels of octopamine in workers, thus validating that our methods to manipulate octopamine levels were effective (Kruskal-Wallis chi square = 5.33, df = 1, N = 8, P = 0.021). Furthermore, the octopamine treatment led to significantly higher individual exploration scores (GLMM: Chi-sq = 8.14, df = 1, N = 160, P = 0.0043; Fig. 6 ). However, there was no difference in exploration across urban and natural colonies (GLMM: Chi-sq = 1.78, df = 1, N = 160, P = 0.18). Effect of octopamine on collective foraging activity In line with the individual behavior results, octopamine-treated colonies had a significantly higher foraging rate than control colonies (GLMM: Chi-sq = 98.13, df = 1, N = 24, P < 0.0001; Fig. 7 ) and this pattern was consistent over time (GLMM: Chi-sq = 0.032, df = 1, N = 24, P = 0.85; Fig. 7 ). However, there was no difference in collective foraging activity between urban and natural colonies. (GLMM: Chi-sq = 1.46, df = 1, N = 24, P = 0.34). DISCUSSION Anthropogenic disturbance affects the abundance, distribution, and fitness of animals by altering abiotic and biotic conditions. Studying the behavior of animals in this context is crucial as behavior can facilitate or constrain adaptation to novel environments. Here, we identified differences in exploratory and foraging behavior of T. sessile across urban and forest habitats. Furthermore, we uncovered hormonal and social mechanisms that are driving these behaviors. Individual exploration and collective foraging activity were higher among workers from colonies found in natural environments, which was the opposite of our prediction. It is possible that the type and quantity of resources in urban environments actually favor decreased exploration. Highly polydomous species, like T. sessile , often move their nests closer to resources (Toennisson et al. 2020 ). Urban colonies could therefore establish next to reliable human-derived food sources (i.e., trash can, compost pile, gardens), reducing the need to explore for new resources (Penick et al. 2015). Similar patterns have been seen in other systems, as herring gulls ( Larus argentatus ) from less urban colonies had a greater diversity of foraging habitats compared to those from more urban colonies (Fuirst et al. 2018 ). It is also possible that differences in foraging strategy (e.g., solitary vs. recruitment-based foraging) could explain variation across species. Further research would be needed comparing T. sessile with other ant species like Temnothorax nylanderi that exhibit increased exploration in urban environments (Jacquier et al. 2023 ). Finally, there may also be more competition for resources in natural sites, where ant diversity tends to be higher, resulting in workers having to explore a larger area for resources. Indeed, it is a common pattern in urban areas for a few, dominant species, like T. sessile , to control resources (Shochat et al. 2010 ). We then assessed two possible proximate mechanisms for variation in behavior between sites: hormones and the behavioral role of individual workers. Octopamine-treated ants had higher individual exploration and octopamine-treated colonies had higher collective foraging activity, suggesting that octopamine may be influencing worker behavior in T. sessile . This aligns with work on ants and other invertebrates, including lobsters, cockroaches, and bees, where octopamine is associated with activity, aggression, exploration, and other sensory responses (Antonsen & Paul 1997 ; Roeder 1999 ; Felden et al. 2018 ; Yakovlev 2018 ). The effect of octopamine on collective behavior suggests that it may play a role in communication or social interactions. Indeed, octopamine is associated with social networks in Drosophila (Certel et al., 2010 ), trophallaxis in carpenter ants ( Camponotus fellah ; Boulay et al. 2000 ), and communication during the waggle dance in honeybees (Barron et al. 2007 ). Based on these results, we expected that standing levels of octopamine would be higher in colonies from natural environments, but there was no difference in octopamine levels in workers sampled from urban or natural sites. It is possible that there are other neurohormones or neuromodulators that are associated with exploratory behavior besides octopamine; for instance, dopamine (Friedman et al. 2018 ) juvenile hormone (Norman & Hughes 2016 ), or serotonin (Muscedere et al. 2012 ). Alternatively, there may still be differences in octopamine across environments that we were not able to quantify through our methods. For instance, there could be differences in the regulation of octopamine or its receptors that would not necessarily be present in overall levels (O’Connell & Hofmann 2011 ). Indeed, population divergence in metabolic rate between marine- and stream-dwelling stickleback is driven by cis-regulatory differences at the locus associated with thyroid stimulating hormone (Kitano et al. 2010 ). While we did not find any differences among colony types, we did find a trend for higher levels of octopamine in workers sampled from foraging trails compared to those sampled at the nest entrance. This is the first study examining the effects of octopamine on foraging behavior in T. sessile . Future work comparing the relationship between octopamine and other behaviors, like aggression, and its interaction with other neurohormones and neuromodulators, is warranted. Exploratory behavior varied among workers taken from different subpopulations within the nest where they performed different roles. Workers collected while foraging were more exploratory than workers who were within the nest, presumably caring for queens and brood, or that had been recruited to the presence of another colony, presumably in a defensive role. However, it remains unclear whether this variation reflects relatively long-lasting differences in behavioral state associated with division of labor, or if it is an immediate reaction to their environment (i.e. collecting food vs being near competitors). For example, does the act of foraging make an individual more exploratory, or are more exploratory individuals more likely to become foragers? This distinction could translate to colony level differences in behavior among environments if, for example, colonies maintain a larger proportion of foragers in urban environments versus natural environments. We also found that individual exploration was repeatable, suggesting workers have consistent behavioral types, at least in the short term. Previous work with T. sessile found that workers sampled near a food source were less aggressive than those sampled near a potential nest site (Neumann & Pinter-Wollman 2022 ). In a similar study on Argentine ants ( Linepithema humile ), workers that were sampled from food sources were more exploratory than other workers (Hui & Pinter-Wollman 2014 ). Our findings add to evidence from other taxa suggesting that individual behavioral differences can contribute to the functioning of animal groups (Loftus et al. 2021). For instance, variation in exploratory behavior allowed groups of great tits ( Parus major ) to exploit food sources more efficiently (Aplin et al. 2014). There are a few important caveats to consider in the context of this study. First, we fit colonies into a binary habitat type of natural or urban. However, even within these habitats there is considerable variation in environmental characteristics including microclimate, resource availability and abundance, and/or competition. Our replication at the colony level was also limited, and it is possible that increased sampling would pick up on more variation that is present in these environments. Finally, because T. sessile workers do not typically have significant variation in body size, we did not look at brain or body size effects in this study. It is possible that population differences in these traits could have had an impact on our findings. Overall, this study investigated how exploratory and foraging behavior, and possible mechanisms underlying them, may contribute to the exploitation of urban environments by odorous house ants. Understanding how behavioral variation may mitigate responses to environmental change is important as ecosystems continue to be altered globally. Species in urban areas may face increased temperatures due to the “urban heat island” effect, where cities are hotter than nearby, non-urban areas (Diamond & Martin 2020). Odorous house ants fit within this framework as a species that rapidly colonizes urban habitats and can survive at higher temperatures than other urban ant species (Harris et al. 2024), and has shown the potential to be an invasive species (Buczkowski & Krushelnycky 2012 ). Whether the phenotypic changes associated with urbanization in T. sessile result from adaptation or plasticity remains unknown. We suggest that future work explore genetic variation associated with these traits, including behavior, to address this gap. Researchers could also consider applying our approach to other systems by exploring the influence of both habitat differences and hormones on ecologically relevant behaviors like exploration or foraging. STATEMENTS AND DECLARATIONS ACKNOWLEDGMENTS We thank all members of the Suarez lab for feedback on the analysis and manuscript. We also thank Furong Sun, Xiuli Mao, and all members of the University of Illinois School of Chemical Sciences Mass Spectrometry Lab for their support in hormone analysis. DATA AVAILABILITY STATEMENT All data and code for analysis is uploaded to Dryad (https://doi.org/10.5061/dryad.m37pvmdff). We have no conflicts of interest to declare. FUNDING This work was supported by National Science Foundation DGE 21-46756 to KMN and USDA-NIFA 2023-10089 to AVS. AUTHOR CONTRIBUTIONS KMN: Conceptualization, Methodology, Formal analysis, Funding acquisition, Writing - original draft, Writing - review & editing. LH: Conceptualization, Methodology, Formal analysis, Writing - original draft, Writing - review & editing. SC: Conceptualization, Methodology, Formal analysis, Writing - original draft, Writing - review & editing. EF: Conceptualization, Methodology, Formal analysis, Writing - original draft, Writing - review & editing. JP: Conceptualization, Methodology, Formal analysis, Writing - original draft, Writing - review & editing. GB: Conceptualization, Formal analysis, Project Administration, Writing - original draft, Writing - review & editing. AVS: Conceptualization, Formal analysis, Funding acquisition, Project Administration, Writing - original draft, Writing - review & editing. 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Front Insect Sci. 5. https://doi.org/10.3389/finsc.2025.1581307 Supplementary Files supplementarymaterials.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Major Revisions Needed 09 Apr, 2026 Reviewers agreed at journal 04 Mar, 2026 Reviewers invited by journal 02 Mar, 2026 Editor assigned by journal 25 Feb, 2026 First submitted to journal 24 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8963511","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":599326423,"identity":"77990b38-e569-4e2f-aa1b-ad31fbde26ca","order_by":0,"name":"Kevin M. 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After acclimation and 24 hours without food, tubes connecting the nest box to the other two boxes were opened. After another 24 hours, 30 workers were collected from each the foraging box, the nest box and the competition box, and individually tested for exploratory behavior. The nest box contained a covered, humid nest site; the foraging box contained sugar water and dried crickets, and the competition box contained a different \u003cem\u003eT. sessile\u003c/em\u003e colony held inside a mesh box which allowed for interaction but no direct fights. Figure not to scale\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8963511/v1/7442f220103254a079723ff7.png"},{"id":103982399,"identity":"1712865d-c952-4f21-a231-ff39db5ea0b3","added_by":"auto","created_at":"2026-03-05 09:43:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":47177,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of sections explored by workers in an open field, by colony type. Points represent individual workers. Boxes, here and in all following figures, signify first quartile, median, and third quartile\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8963511/v1/b97b8a48331a2d4b3c0dc629.png"},{"id":104402382,"identity":"6733bc49-40c7-46a3-a2c5-bc45d8f2ae8b","added_by":"auto","created_at":"2026-03-11 12:15:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":118039,"visible":true,"origin":"","legend":"\u003cp\u003eMean number of workers found in foraging box with dried crickets after being starved of protein, over time and by colony type. Individual points represent mean values across all colonies. Shaded area represents +/- one standard error\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8963511/v1/f210b592263290bed74641a9.png"},{"id":103982402,"identity":"0e9acfc8-464c-4fc0-8c93-2f2495d4b8a0","added_by":"auto","created_at":"2026-03-05 09:43:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":136045,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of sections explored by workers in an open field, separated by the location they were in (foraging, nest, or competition box; see Figure 1). Workers from natural colonies are on the left and from urban colonies are on the right\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8963511/v1/e3402d8da9220b4651a390e9.png"},{"id":104402653,"identity":"1c091772-b89b-44c4-8511-e08df28a5268","added_by":"auto","created_at":"2026-03-11 12:16:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":55377,"visible":true,"origin":"","legend":"\u003cp\u003eRelative concentration of octopamine, by colony type. Points represent pooled samples of 10 workers. Values are log transformed for visualization\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8963511/v1/169fee29bb4b42dd6058ad86.png"},{"id":103982406,"identity":"11652f0a-7570-484b-9f59-34759e6897ce","added_by":"auto","created_at":"2026-03-05 09:43:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":211339,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of sections explored by workers in an open field, by treatment (control = 20% sugar water solutions; octopamine = 20% sugar water solution + 4 mg/mL octopamine). Points represent individual workers\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8963511/v1/ee5b62e23645f5ed876d3c98.png"},{"id":103982403,"identity":"6bb17a57-029d-4a48-a1ec-6a2355c1842b","added_by":"auto","created_at":"2026-03-05 09:43:36","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":128651,"visible":true,"origin":"","legend":"\u003cp\u003eMean number of workers found in foraging boxes with dried crickets after being starved of protein, over time and by treatment (octopamine solution or control solution). Individual points represent mean values across all colonies. Shaded area represents +/- one standard error\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8963511/v1/5b438f0e4a2f88c3a6279f31.png"},{"id":104408350,"identity":"8fb76441-a789-4fba-b523-0a6e01111102","added_by":"auto","created_at":"2026-03-11 12:42:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1383767,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8963511/v1/8dfa8312-3440-47b6-bbc0-7256322f4e9b.pdf"},{"id":103982405,"identity":"865cb568-c51d-4bbe-b3dc-960a4d453230","added_by":"auto","created_at":"2026-03-05 09:43:36","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":142479,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-8963511/v1/c8f4476203432b90bb739406.docx"}],"financialInterests":"","formattedTitle":"Behavioral and hormonal responses to urbanization in odorous house ants (Tapinoma sessile)","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eUrbanization presents various challenges to organisms, especially through the modification of habitat structure, resource availability, and microclimate (Wong \u0026amp; Candolin \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In response, animals have adopted a wide range of behavioral strategies to deal with these changes, and in some cases, even take advantage of the opportunities presented by urbanization (Chapple et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Sol et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Wrensford et al., 2025). Individuals in urban populations may have increased levels of exploration, boldness, or neophilia. For example, ground beetles (Carabidae) in more urbanized areas are more exploratory (Schuett et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), and \u003cem\u003eAnolis\u003c/em\u003e lizards from urban areas are bolder and more exploratory compared to those from forest populations (Lapiedra et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Avil\u0026eacute;s-Rodr\u0026iacute;guez \u0026amp; Kolbe \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Additionally, foraging behaviors often differ between urban and non-urban populations because of novel resources present in urban habitats (Lato et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). For example, foraging activity increases in urban populations of the stingless bee \u003cem\u003eTetragonula carbonaria\u003c/em\u003e (Apidae), with altered resource availability from gardens likely driving this change (Kaluza et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition to documenting behavioral changes among urban and non-urban populations, it is important to understand the proximate hormonal mechanisms underlying these differences. House sparrows (\u003cem\u003ePasser domesticus\u003c/em\u003e) near the edge of a range expansion exhibit increased exploration and higher corticosterone levels (Liebl \u0026amp; Martin \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In dark eyed juncos (\u003cem\u003eJunco hyemalis\u003c/em\u003e), there were correlated changes in parental care and testosterone expression associated with the colonization of a coastal, urban habitat from nearby temperate forests (Atwell et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). However, more information across different taxa and environments is needed to tease apart the relationship between hormones, behavior, and phenotypic divergence between urban and natural populations.\u003c/p\u003e \u003cp\u003eBehaviors also vary based on their context in relation to the task an individual is performing (e.g., foraging, defending territory, socializing, courting mates; Madrzyk \u0026amp; Pinter-Wollman \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Trigos-Peral et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). For instance, boldness in grey mouse lemurs (\u003cem\u003eMicrocebus murinus\u003c/em\u003e) increases during foraging, and the willingness to take risks was dependent on the level of predation risk (Dammhahn \u0026amp; Almeling \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In cooperatively breeding cichlids (\u003cem\u003eNeolamprologus pulcher\u003c/em\u003e), exploration is related to territoriality, with exploratory individuals actively defending the territory (Bergm\u0026uuml;ller \u0026amp; Taborsky \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Taking social context into consideration is particularly important with eusocial organisms which exhibit pronounced division of labor and tasks are divided among individuals (Robinson \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). In \u003cem\u003eMyrmica rubra\u003c/em\u003e ants, workers focusing on within-nest tasks like brood care were less aggressive and exploratory relative to workers focusing on tasks outside the nest, like foraging or nest defense (Pamminger et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). This also underscores the importance of quantifying the repeatability of behavior, as differences in behavior across contexts could be due to plasticity within individuals or consistent differences across individuals (Garrison et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAnts are a strong model for exploring behavioral variation in response to disturbance as they are abundant in both natural and urban environments (Buczkowski et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In urban areas, dominant, aggressive ant species often exclude native species or may be better at colonizing disturbed habitats (Suarez et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; King \u0026amp; Tschinkel \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Neumann \u0026amp; Pinter-Wollman \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Furthermore, ants exhibit division of labor among workers, allowing for comparison of behavior across different social contexts (e.g. caring for brood in the nest, searching for food or nests sites, or defending the colony from threats) (Modlmeier et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Finally, many hormones are relatively well-studied in ants, providing candidate targets and methods for further exploration (Starkey \u0026amp; Tamborindeguy \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Ye et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Understanding the role of behavior in how ants respond to urbanization can elucidate which species may be \u0026lsquo;winners\u0026rsquo; or \u0026lsquo;losers\u0026rsquo; in the face of global change (Salyer et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). This is particularly important given the diverse roles they play in ecosystems as seed dispersers, scavengers, and soil engineers (Folgarait \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Del Toro \u0026amp; Pelini 2012).\u003c/p\u003e \u003cp\u003eThe odorous house ant (\u003cem\u003eTapinoma sessile\u003c/em\u003e) is an emerging model organism for examining differences in biology between urban and natural populations (Menke et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Buczkowski \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Blumenfeld et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Odorous house ants are found across North America in a variety of natural environments, including forests, wetlands, and prairies, and disturbed environments such as cities and agricultural landscapes. Urban populations of \u003cem\u003eT. sessile\u003c/em\u003e are associated with shifts in their social structure and negative impacts on native ant species richness (Buczkowski \u0026amp; Bennett 2008; Salyer et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Colonies from natural environments tend to have fewer workers and queens relative to urban colonies which are highly polygynous (multiple queens) and polydomous (occupy multiple nests) and can have hundreds of thousands of workers (Buczkowski \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Odorous house ants exhibit high levels of interspecific aggression in urban populations and have been identified as a potential threat to become an introduced species given the suite of characteristics they share with known invasive ants, including high levels of interspecific aggression, large, rapidly dispersing colonies, and generalist habitat and foraging preferences (Buczkowski \u0026amp; Krushelnycky \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile aggressive behavior in \u003cem\u003eT. sessile\u003c/em\u003e is relatively well studied (Buczkowski \u0026amp; Bennett 2008), we know less about how other behaviors vary between natural and urban populations. Exploratory behavior is a potentially important behavior to explore further because of its relevance to various worker tasks including identifying food sources, territorial defense, and searching for new nest sites (Nonacs \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Barbani \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Page et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Furthermore, exploration and foraging activity is higher in other urban ant species (Jacquier et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and thus could be contributing to the success of \u003cem\u003eT. sessile\u003c/em\u003e in urban habitats.\u003c/p\u003e \u003cp\u003eThe hormonal mechanisms underlying variation in behavior between urban and natural populations of \u003cem\u003eT. sessile\u003c/em\u003e have not been explored. In ants and other invertebrates, one hormone of interest is the neurotransmitter and neuromodulator octopamine. Octopamine is similar in structure to norepinephrine, the \u0026ldquo;flight or fight\u0026rdquo; hormone in vertebrates (Adamo et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). It has broad function across invertebrates but is generally related to activity, aggression, and sensory systems (Roeder, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Wada-Katsumata et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Kamhi et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Thus, octopamine could be a potential mechanism of exploratory behavior. Felden and colleagues (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) found invasive Argentine ants (\u003cem\u003eLinepithema humile\u003c/em\u003e) treated with octopamine had increased foraging activity, although the degree of response was not related to the source environment (e.g. native or introduced populations). Additionally, octopamine is important for tracking olfactory cues, which may be related to exploratory or foraging behavior (Wissink \u0026amp; Nehring \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe examined the behavior of odorous house ants from natural and urban environments to answer the following: (1) Does individual exploratory behavior and/or collective foraging activity differ among workers from natural vs. urban colonies? (2) Is exploratory behavior related to a worker\u0026rsquo;s behavioral state (foraging, brood care, or nest defense)? (3) Do standing levels of octopamine differ between natural and urban colonies, and (4) What is the effect of octopamine on individual exploration and collective foraging activity? We predicted that individual exploration and collective foraging activity would be higher in urban colonies. Additionally, we predicted foragers would have higher levels of exploration relative to workers from within the nest. Finally, we predicted that octopamine levels would be positively correlated with individual exploration and collective foraging activity, and thus, standing octopamine levels would be higher in urban colonies.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnt collection and care\u003c/h2\u003e \u003cp\u003eIn June 2023, we collected one colony from each of eight locations in Lafayette, IN, USA; four urban environments (on or near the Purdue University campus) and four natural environments (mixed hardwood forests) (Buczkowski et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). For urban colonies, we took a subset of larger, polygynous, polydomous \u0026lsquo;supercolonies\u0026rsquo;, approximately 10,000\u0026ndash;15,000 workers, 40\u0026ndash;50 queens, and numerous brood. Natural colonies contained approximately 3,000\u0026ndash;5,000 workers, 10\u0026ndash;20 queens, and numerous brood. Prior work found high levels of genetic differentiation and aggression between workers from these same populations, suggesting that they are distinct populations (Blumenfeld et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Colonies were housed in the lab at the University of Illinois Urbana-Champaign in plastic containers coated with Fluon (60x40cm), with plastic tubes covered in tinfoil as artificial nests. They were fed sugar water and dried crickets \u003cem\u003ead libitum\u003c/em\u003e. Colonies were given one week to acclimate to lab conditions prior to data collection.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIndividual exploration\u003c/h3\u003e\n\u003cp\u003eWe measured exploratory behavior using an open-field assay. A worker was gently placed into the center of a plastic container (60x40cm) coated with Fluon, with a grid placed underneath the container separating it into twelve equally sized sections. We observed for 5 minutes and recorded the number of times the worker moved between sections. Containers were cleaned with ethanol before the next trial. We repeated this for 25 workers from each of the eight colonies. All behavioral measurements occurred between 11:00 am and 4:00 pm. Furthermore, to assess the consistency of individual behavior, we repeated this assay for 10 workers from each colony (e.g. the same individual was measured twice), waiting 24 hours between measurements. Between measurements, workers were kept in a mesh enclosure that was placed back into the colony, allowing us to keep them exposed to other workers while maintaining their identity. To minimize observer bias, here and in all of the following section, blinded methods during analysis of behavioral data.\u003c/p\u003e\n\u003ch3\u003eCollective foraging activity\u003c/h3\u003e\n\u003cp\u003eTo measure collective foraging activity, we first moved a colony fragment consisting of 1,000 workers, 5 queens, and numerous brood to a new nest box coated with fluon (40x20 cm). Colonies were fed 20% sugar water during an acclimation period of 72 hrs. Colonies were starved of protein over this time to motivate exploration, as ants require a consistent protein source to produce new brood. Then, we uncovered an opening to a 1m tube that connected to another box (10x10 cm) containing a protein source (dried crickets) and took a photo of this box every 20 min for 12 hrs (10:00AM to 10:00PM) using an AKASO Brave 8 camera. Collective foraging activity was quantified by counting the number of workers present in the box at each time point. We repeated this on two different colony fragments per population, for a total of 16 replicates.\u003c/p\u003e\n\u003ch3\u003eRelationship between individual exploration and division of labor\u003c/h3\u003e\n\u003cp\u003eTo examine if exploration varied based on the worker\u0026rsquo;s behavioral role in the nest (foraging, nest care or defense), we designed a three-chambered arena, consisting of a nest box (containing an artificial nest), a foraging box, and a competition box (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe aspirated a colony fragment\u0026thinsp;\u0026minus;\u0026thinsp;1,000 workers, 5 queens, and numerous brood and placed them into the nest box. The foraging and competition boxes were not initially accessible to the experimental colony. The experimental colony was given time to move into their new nest and then starved for 24 hrs after which the rest of the arena was opened for exploration. The foraging arena had a tube of sugar water and a dried cricket, while the competition arena had a mesh container with approximately 100 foreign \u003cem\u003eT. sessile\u003c/em\u003e workers collected from the University of Illinois campus. Workers could detect the foreign \u003cem\u003eT. sessile\u003c/em\u003e workers through the mesh but could not directly engage in any fights or aggressive behaviors, allowing us to better control for the effect of the competitor on behavior across trials.\u003c/p\u003e \u003cp\u003eThe experimental colony was then given another 24 hrs to explore the arena. We returned and collected 30 workers in total, 10 workers from each box (foraging box, nest box, and competition box). For the nest box, workers were collected only from within the nest chamber to reduce the chances of sampling a worker that had just returned from the foraging or competition boxes. Workers were sampled by gently picking them up with a paintbrush when they were not directly next to another worker, to limit the impact of collection on nearby workers. We measured exploratory behavior of all workers as previously described. We repeated this process three times for colonies from each of the eight locations. Workers that were measured for behavior were not returned to their original nest box to prevent any worker from being tested in multiple trials. These methods were replicated three times for each source colony.\u003c/p\u003e\n\u003ch3\u003eRelationship between octopamine and behavior\u003c/h3\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStanding variation in octopamine in field colonies\u003c/h2\u003e \u003cp\u003eWe explored the relationship between octopamine, behavior, and habitat (urban vs. natural) in \u003cem\u003eTapinoma sessile\u003c/em\u003e. We first compared standing levels of octopamine within workers across the eight populations. We aspirated 90 workers from each location, immediately placing them on dry ice after collection, and then stored them at -80\u0026deg; C.\u003c/p\u003e \u003cp\u003eWe also examined seasonal differences in octopamine levels between colonies from natural and urban habitats and differences in octopamine levels between workers either sampled from foraging trails (e.g. foragers) or workers sampled from the nest entrance. For this additional sampling, we identified three urban colonies and three natural, forest dwelling colonies near Champaign, IL, USA. For each site, ten workers were sampled from the entrance to the nest site and another ten workers were sampled along a foraging trail at least one meter from the nest entrance. When possible, we sampled workers from the same nest entrance each time. If a nest entrance from a previous visit was not occupied, we instead sampled from a nearby nest entrance. \u003cem\u003eT. sessile\u003c/em\u003e are polydomous, allowing us to be confident we were sampling from the same colony.\u003c/p\u003e \u003cp\u003eThis sampling process was repeated twice a month from June through August 2025, during the peak season of activity for \u003cem\u003eT. sessile\u003c/em\u003e. Thus, there were 72 pooled samples of 10 workers per sample: 6 time points for each of 6 colonies, from two locations (nest and foraging trail) for each colony. All samples were immediately put on dry ice until processing.\u003c/p\u003e \u003cp\u003eFor sample preparation, we first dissected workers\u0026rsquo; heads over dry ice, removing the antennae. Each sample was pooled with 10 heads, for a total of nine samples from each of the eight locations. The heads were homogenized in 100 \u0026micro;l of ice cold 0.1 M formic acid with 5 ng of 3,4-di-hydroxybenzylamine (DHBA) as an internal standard, and then vortexed. Samples were centrifuged for 20 minutes at 13,500 rpm and 4\u0026deg; C. Then, 100 \u0026micro;l of a 1:3 isopropanol/chloroform solution was added, and the samples were centrifuged for another 5 minutes under the same conditions. We collected 50 \u0026micro;l of supernatant per sample and stored them at -80\u0026deg; C. All sample preparation methods were consistent with prior work (Wada-Katsumata et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Hojo et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Mannino et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Following sample preparation, we performed liquid chromatography-mass spectrometry (LC-MS) using a SYNAPT G2-Si Mass Spectrometer in collaboration with the University of Illinois School of Chemical Sciences\u0026rsquo; Mass Spectrometry Lab. LC-MS data was processed and concentrations of octopamine were quantified using Mass Lynx v4.2 (Waters Corp., Milford, USA). We only quantified relative concentrations of octopamine rather than actual measurements because this made the LC-MS methods more straightforward and relative concentrations were sufficient to answer our research questions. This did limit our ability to compare the presence of octopamine in \u003cem\u003eT. sessile\u003c/em\u003e to other species or consider the role of octopamine in specific biochemical pathways.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEffect of octopamine on individual exploration\u003c/h3\u003e\n\u003cp\u003eWe explored the effect of octopamine on individual exploration. We took 160 workers (20 from each colony, 80 per colony type (i.e., the habitat the colony came from)) and assigned them to either an octopamine treatment or a control treatment, with all workers in same-colony groups of 10. Workers were starved for 24 hrs and then given 24 hrs to feed ad libitum on either 20% sugar water with 4 mg/mL octopamine or a control solution of 20% sugar water (as in Felden et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Next, the individual exploration of each worker was tested using the individual exploration assay described above. To verify that these methods were effective in altering octopamine level, we quantified the relative concentrations of octopamine in a subset of workers across the two treatments using the same procedures.\u003c/p\u003e\n\u003ch3\u003eEffect of octopamine on collective foraging activity\u003c/h3\u003e\n\u003cp\u003eWe also examined the effect of octopamine on the collective foraging of colonies. To do this, we repeated the same assay for collective foraging as described above, but with colonies being fed a solution of 20% sugar water with 4 mg/mL octopamine. We repeated the assay on two different colony fragments from each population for a total of 16 replicates. The colonies described in the \u003cem\u003eCollective foraging activity\u003c/em\u003e section, which were fed just 20% sugar water, acted as a control.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eTo compare individual exploration across colony types (urban vs. natural), we ran a generalized linear mixed model (GLMM) with a negative binomial family, the number of sections explored as the response variable, colony type as a fixed effect, and population as a random effect, using the lme4 package in R (Bates et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Here and in all following models, the significance of fixed effects was assessed by conducting Type II Wald chi-square tests using the Anova function in the car package (Fox \u0026amp; Weisberg, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Additionally, overdispersion of this and the following GLMMs was tested using the testDispersion function in the DHARMa package (Hartig \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo measure the consistency of exploration, we computed the adjusted repeatability for each behavior, which considers variation due to factors (e.g., population) through mixed modeling approaches (Nakagawa \u0026amp; Schielzeth \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). We ran linear mixed models (LMMs) with a log transformation of the number of sections explored as the response variable, colony type (natural vs. urban) as a fixed effect and ant ID as a random effect, using the rptR package (Stoffel et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). We calculated 95% confidence intervals and p-values with parametric bootstrapping (n\u0026thinsp;=\u0026thinsp;10,000 iterations) and p-values from permutation tests (n\u0026thinsp;=\u0026thinsp;10,000 iterations).\u003c/p\u003e \u003cp\u003eTo analyze the relationship between individual exploration and the worker\u0026rsquo;s behavioral role in the colony, we ran a generalized linear mixed model with a negative binomial family, number of sections explored as the response variable, behavioral role (foraging, nest, or competition) and an interaction term between role and colony type as fixed effects, and population as a random effect. We also included replicate as a random effect to account for repeated measures from each population. We made post-hoc comparisons for the effect of social context using the emmeans package in R (Lenth \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo compare standing levels of octopamine across colony types, we ran a linear mixed model with relative concentration of octopamine as a response variable, colony type as a fixed effect, and population as a random effect. There were nine individuals that had either extremely low concentrations or no octopamine detected. We removed these individuals from analysis as we could not rule out the possibility that these results were due to methodological error. The interpretation of the results did not change after removing these individuals. To analyze the seasonal field data, we ran a generalized linear model (Gamma family) with relative octopamine concentration as the response variable and time, location (nest vs. foraging trail) and habitat (urban vs. natural) as fixed effects. The significance of fixed effects was assessed by conducting Type II Wald chi-square tests. For the lab experiment, we verified that our octopamine treatment did lead to increased levels of octopamine compared to the control group with a Kruskal-Wallis test.\u003c/p\u003e \u003cp\u003eTo identify if octopamine had an impact on individual exploration, we ran a generalized linear mixed model with a negative binomial family, number of sections explored as the response variable, colony type and treatment (octopamine vs. control) as fixed effects, and population as a random effect.\u003c/p\u003e \u003cp\u003eFinally, to compare collective foraging activity across colony types and treatments (octopamine vs. control), we ran a generalized linear mixed model with a Poisson family, number of foragers present as the response variable, colony type, treatment, time, and interaction terms between time and colony type and time and treatment as fixed effects, and population and replicate as random effects.\u003c/p\u003e \u003cp\u003eThis work followed all Guidelines for the Use of Animals in Research. \u003cem\u003eTapinoma sessile\u003c/em\u003e is not a threatened species and there were no licenses or permits required for this work. All conditions in the laboratory were in line with conditions \u003cem\u003eT. sessile\u003c/em\u003e face in their natural environment. For lethal sampling, individuals were sacrificed as quickly as possible by being placed on dry ice.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eVariation and repeatability of individual exploration\u003c/h2\u003e \u003cp\u003eWorkers from natural colonies visited more sections in an open field than workers from urban colonies (GLMM: Chi-sq\u0026thinsp;=\u0026thinsp;12.18, df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eN\u003c/em\u003e\u0026thinsp;=\u0026thinsp;200, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Furthermore, exploration was repeatable within individuals, suggesting that there are relatively stable behavioral types (LMM permutations: adjusted repeatability\u0026thinsp;=\u0026thinsp;0.41 (0.21\u0026ndash;0.58), \u003cem\u003eN\u003c/em\u003e\u0026thinsp;=\u0026thinsp;80, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCollective foraging activity\u003c/h2\u003e \u003cp\u003eIn line with the individual behavioral results, colonies from natural environments had higher foraging rates compared to urban colonies (GLMM: Chi-sq\u0026thinsp;=\u0026thinsp;4.46, df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eN\u003c/em\u003e\u0026thinsp;=\u0026thinsp;24, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.035; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). There was not a significant effect of the interaction between colony type and time, suggesting that this pattern was consistent across the trial (GLMM: Chi-sq\u0026thinsp;=\u0026thinsp;0.021, df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eN\u003c/em\u003e\u0026thinsp;=\u0026thinsp;24, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.64; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRelationship between individual exploration and division of labor\u003c/h2\u003e \u003cp\u003eWorkers sampled from the foraging box were more exploratory than workers sampled from the competition box or the nest box (GLMM: Chi-sq\u0026thinsp;=\u0026thinsp;42.21, df\u0026thinsp;=\u0026thinsp;2, \u003cem\u003eN\u003c/em\u003e\u0026thinsp;=\u0026thinsp;720, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This pattern was consistent across both urban and natural colonies (GLMM: Chi-sq\u0026thinsp;=\u0026thinsp;0.87, df\u0026thinsp;=\u0026thinsp;2, \u003cem\u003eN\u003c/em\u003e\u0026thinsp;=\u0026thinsp;720, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.65).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStanding variation in octopamine across colonies\u003c/h2\u003e \u003cp\u003eOctopamine was present in the field colonies, and there was considerable variation among individuals in standing octopamine levels. However, there was not a significant difference in octopamine concentrations between natural and urban colonies (LMM: Chi-sq\u0026thinsp;=\u0026thinsp;0.022, df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eN\u003c/em\u003e\u0026thinsp;=\u0026thinsp;63, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.96; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eSeasonal variation in octopamine between foragers and nest workers.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eWorkers sampled from the foraging trail had marginally higher levels of octopamine than those sampled from the nest entrance (GLM: Chi-sq\u0026thinsp;=\u0026thinsp;3.23, df\u0026thinsp;=\u0026thinsp;1, N\u0026thinsp;=\u0026thinsp;72, P\u0026thinsp;=\u0026thinsp;0.073; Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). However, there was not a significant effect of time (GLMM: Chi-sq\u0026thinsp;=\u0026thinsp;0.028, df\u0026thinsp;=\u0026thinsp;1, N\u0026thinsp;=\u0026thinsp;72, P\u0026thinsp;=\u0026thinsp;0.86) or habitat (natural versus urban; GLM: Chi-sq\u0026thinsp;=\u0026thinsp;0.29, df\u0026thinsp;=\u0026thinsp;1, N\u0026thinsp;=\u0026thinsp;72, P\u0026thinsp;=\u0026thinsp;0.59) (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eEffect of octopamine on individual exploration\u003c/h2\u003e \u003cp\u003eThe octopamine treatment led to increased levels of octopamine in workers, thus validating that our methods to manipulate octopamine levels were effective (Kruskal-Wallis chi square\u0026thinsp;=\u0026thinsp;5.33, df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eN\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.021). Furthermore, the octopamine treatment led to significantly higher individual exploration scores (GLMM: Chi-sq\u0026thinsp;=\u0026thinsp;8.14, df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eN\u003c/em\u003e\u0026thinsp;=\u0026thinsp;160, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0043; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). However, there was no difference in exploration across urban and natural colonies (GLMM: Chi-sq\u0026thinsp;=\u0026thinsp;1.78, df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eN\u003c/em\u003e\u0026thinsp;=\u0026thinsp;160, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.18).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eEffect of octopamine on collective foraging activity\u003c/h2\u003e \u003cp\u003eIn line with the individual behavior results, octopamine-treated colonies had a significantly higher foraging rate than control colonies (GLMM: Chi-sq\u0026thinsp;=\u0026thinsp;98.13, df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eN\u003c/em\u003e\u0026thinsp;=\u0026thinsp;24, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) and this pattern was consistent over time (GLMM: Chi-sq\u0026thinsp;=\u0026thinsp;0.032, df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eN\u003c/em\u003e\u0026thinsp;=\u0026thinsp;24, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.85; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). However, there was no difference in collective foraging activity between urban and natural colonies. (GLMM: Chi-sq\u0026thinsp;=\u0026thinsp;1.46, df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eN\u003c/em\u003e\u0026thinsp;=\u0026thinsp;24, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.34).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eAnthropogenic disturbance affects the abundance, distribution, and fitness of animals by altering abiotic and biotic conditions. Studying the behavior of animals in this context is crucial as behavior can facilitate or constrain adaptation to novel environments. Here, we identified differences in exploratory and foraging behavior of \u003cem\u003eT. sessile\u003c/em\u003e across urban and forest habitats. Furthermore, we uncovered hormonal and social mechanisms that are driving these behaviors.\u003c/p\u003e \u003cp\u003eIndividual exploration and collective foraging activity were higher among workers from colonies found in natural environments, which was the opposite of our prediction. It is possible that the type and quantity of resources in urban environments actually favor decreased exploration. Highly polydomous species, like \u003cem\u003eT. sessile\u003c/em\u003e, often move their nests closer to resources (Toennisson et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Urban colonies could therefore establish next to reliable human-derived food sources (i.e., trash can, compost pile, gardens), reducing the need to explore for new resources (Penick et al. 2015). Similar patterns have been seen in other systems, as herring gulls (\u003cem\u003eLarus argentatus\u003c/em\u003e) from less urban colonies had a greater diversity of foraging habitats compared to those from more urban colonies (Fuirst et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). It is also possible that differences in foraging strategy (e.g., solitary vs. recruitment-based foraging) could explain variation across species. Further research would be needed comparing \u003cem\u003eT. sessile\u003c/em\u003e with other ant species like \u003cem\u003eTemnothorax nylanderi\u003c/em\u003e that exhibit increased exploration in urban environments (Jacquier et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Finally, there may also be more competition for resources in natural sites, where ant diversity tends to be higher, resulting in workers having to explore a larger area for resources. Indeed, it is a common pattern in urban areas for a few, dominant species, like \u003cem\u003eT. sessile\u003c/em\u003e, to control resources (Shochat et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe then assessed two possible proximate mechanisms for variation in behavior between sites: hormones and the behavioral role of individual workers. Octopamine-treated ants had higher individual exploration and octopamine-treated colonies had higher collective foraging activity, suggesting that octopamine may be influencing worker behavior in \u003cem\u003eT. sessile\u003c/em\u003e. This aligns with work on ants and other invertebrates, including lobsters, cockroaches, and bees, where octopamine is associated with activity, aggression, exploration, and other sensory responses (Antonsen \u0026amp; Paul \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Roeder \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Felden et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Yakovlev \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The effect of octopamine on collective behavior suggests that it may play a role in communication or social interactions. Indeed, octopamine is associated with social networks in \u003cem\u003eDrosophila\u003c/em\u003e (Certel et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), trophallaxis in carpenter ants (\u003cem\u003eCamponotus fellah\u003c/em\u003e; Boulay et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), and communication during the waggle dance in honeybees (Barron et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBased on these results, we expected that standing levels of octopamine would be higher in colonies from natural environments, but there was no difference in octopamine levels in workers sampled from urban or natural sites. It is possible that there are other neurohormones or neuromodulators that are associated with exploratory behavior besides octopamine; for instance, dopamine (Friedman et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) juvenile hormone (Norman \u0026amp; Hughes \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), or serotonin (Muscedere et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Alternatively, there may still be differences in octopamine across environments that we were not able to quantify through our methods. For instance, there could be differences in the regulation of octopamine or its receptors that would not necessarily be present in overall levels (O\u0026rsquo;Connell \u0026amp; Hofmann \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Indeed, population divergence in metabolic rate between marine- and stream-dwelling stickleback is driven by cis-regulatory differences at the locus associated with thyroid stimulating hormone (Kitano et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). While we did not find any differences among colony types, we did find a trend for higher levels of octopamine in workers sampled from foraging trails compared to those sampled at the nest entrance. This is the first study examining the effects of octopamine on foraging behavior in \u003cem\u003eT. sessile\u003c/em\u003e. Future work comparing the relationship between octopamine and other behaviors, like aggression, and its interaction with other neurohormones and neuromodulators, is warranted.\u003c/p\u003e \u003cp\u003eExploratory behavior varied among workers taken from different subpopulations within the nest where they performed different roles. Workers collected while foraging were more exploratory than workers who were within the nest, presumably caring for queens and brood, or that had been recruited to the presence of another colony, presumably in a defensive role. However, it remains unclear whether this variation reflects relatively long-lasting differences in behavioral state associated with division of labor, or if it is an immediate reaction to their environment (i.e. collecting food vs being near competitors). For example, does the act of foraging make an individual more exploratory, or are more exploratory individuals more likely to become foragers? This distinction could translate to colony level differences in behavior among environments if, for example, colonies maintain a larger proportion of foragers in urban environments versus natural environments.\u003c/p\u003e \u003cp\u003eWe also found that individual exploration was repeatable, suggesting workers have consistent behavioral types, at least in the short term. Previous work with \u003cem\u003eT. sessile\u003c/em\u003e found that workers sampled near a food source were less aggressive than those sampled near a potential nest site (Neumann \u0026amp; Pinter-Wollman \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In a similar study on Argentine ants (\u003cem\u003eLinepithema humile\u003c/em\u003e), workers that were sampled from food sources were more exploratory than other workers (Hui \u0026amp; Pinter-Wollman \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Our findings add to evidence from other taxa suggesting that individual behavioral differences can contribute to the functioning of animal groups (Loftus et al. 2021). For instance, variation in exploratory behavior allowed groups of great tits (\u003cem\u003eParus major\u003c/em\u003e) to exploit food sources more efficiently (Aplin et al. 2014).\u003c/p\u003e \u003cp\u003eThere are a few important caveats to consider in the context of this study. First, we fit colonies into a binary habitat type of natural or urban. However, even within these habitats there is considerable variation in environmental characteristics including microclimate, resource availability and abundance, and/or competition. Our replication at the colony level was also limited, and it is possible that increased sampling would pick up on more variation that is present in these environments. Finally, because \u003cem\u003eT. sessile\u003c/em\u003e workers do not typically have significant variation in body size, we did not look at brain or body size effects in this study. It is possible that population differences in these traits could have had an impact on our findings.\u003c/p\u003e \u003cp\u003eOverall, this study investigated how exploratory and foraging behavior, and possible mechanisms underlying them, may contribute to the exploitation of urban environments by odorous house ants. Understanding how behavioral variation may mitigate responses to environmental change is important as ecosystems continue to be altered globally. Species in urban areas may face increased temperatures due to the \u0026ldquo;urban heat island\u0026rdquo; effect, where cities are hotter than nearby, non-urban areas (Diamond \u0026amp; Martin 2020). Odorous house ants fit within this framework as a species that rapidly colonizes urban habitats and can survive at higher temperatures than other urban ant species (Harris et al. 2024), and has shown the potential to be an invasive species (Buczkowski \u0026amp; Krushelnycky \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Whether the phenotypic changes associated with urbanization in \u003cem\u003eT. sessile\u003c/em\u003e result from adaptation or plasticity remains unknown. We suggest that future work explore genetic variation associated with these traits, including behavior, to address this gap. Researchers could also consider applying our approach to other systems by exploring the influence of both habitat differences and hormones on ecologically relevant behaviors like exploration or foraging.\u003c/p\u003e"},{"header":" STATEMENTS AND DECLARATIONS ","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank all members of the Suarez lab for feedback on the analysis and manuscript. We also thank Furong Sun, Xiuli Mao, and all members of the University of Illinois School of Chemical Sciences Mass Spectrometry Lab for their support in hormone analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data and code for analysis is uploaded to Dryad (https://doi.org/10.5061/dryad.m37pvmdff).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe have no conflicts of interest to declare.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by National Science Foundation DGE 21-46756 to KMN and USDA-NIFA 2023-10089 to AVS.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKMN: Conceptualization, Methodology, Formal analysis, Funding acquisition, Writing - original draft, Writing - review \u0026amp; editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLH: Conceptualization, Methodology, Formal analysis, Writing - original draft, Writing - review \u0026amp; editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSC: Conceptualization, Methodology, Formal analysis, Writing - original draft, Writing - review \u0026amp; editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEF: Conceptualization, Methodology, Formal analysis, Writing - original draft, Writing - review \u0026amp; editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eJP: Conceptualization, Methodology, Formal analysis, Writing - original draft, Writing - review \u0026amp; editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGB: Conceptualization, Formal analysis, Project Administration, Writing - original draft, Writing - review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eAVS: Conceptualization, Formal analysis, Funding acquisition, Project Administration, Writing - original draft, Writing - review \u0026amp; editing.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAdamo SA, Linn CE, Hoy RR (1995) The role of neurohormonal octopamine during \u0026ldquo;fight or flight\u0026rdquo; behaviour in the field cricket Gryllus bimaculatus. J Exp Biol. 198(Pt 8):1691\u0026ndash;1700. https://doi.org/10.1242/jeb.198.8.1691\u003c/li\u003e\n \u003cli\u003e\u0026nbsp; Antonsen BL, Paul DH (1997) Serotonin and octopamine elicit stereotypical agonistic behaviors in the squat lobster Munida quadrispina (Anomura, Galatheidae). J Comp Physiol A. 181(5):501\u0026ndash;510. https://doi.org/10.1007/s003590050134. https://doi.org/10.1007/s003590050134\u003c/li\u003e\n \u003cli\u003e\u0026nbsp; Atwell JW, Cardoso GC, Whittaker DJ, Price TD, Ketterson ED (2014) Hormonal, Behavioral, and Life-History Traits Exhibit Correlated Shifts in Relation to Population Establishment in a Novel Environment. The American Naturalist. 184(6):E147\u0026ndash;E160. https://doi.org/10.1086/678398\u003c/li\u003e\n \u003cli\u003e\u0026nbsp; \u0026nbsp;Avil\u0026eacute;s-Rodr\u0026iacute;guez KJ, Kolbe JJ (2019) Escape in the city: urbanization alters the escape behavior of Anolis lizards. 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Front Insect Sci. 5. https://doi.org/10.3389/finsc.2025.1581307\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"insectes-sociaux","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"inso","sideBox":"Learn more about [Insectes Sociaux](http://link.springer.com/journal/40)","snPcode":"40","submissionUrl":"https://www.editorialmanager.com/inso/default2.aspx","title":"Insectes Sociaux","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"exploration, foraging, octopamine, neuromodulator, personality","lastPublishedDoi":"10.21203/rs.3.rs-8963511/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8963511/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUrbanization has profound effects on biological communities. Many organisms cannot persist in anthropogenic environments, while others may adapt to urban conditions. Behavioral traits can facilitate this adaptation and predict how species might respond to urbanization. We studied the behavior of the odorous house ant (\u003cem\u003eTapinoma sessile\u003c/em\u003e) which is common in both natural (i.e. forests) and urban areas. Relative to natural environments, colonies in urban areas are typically more aggressive and have many more workers and queens. To examine how this variation may influence other behaviors, we compared the exploratory behavior of \u003cem\u003eT. sessile\u003c/em\u003e workers and colonies from natural and urban environments. We found repeatable variation in exploratory behavior, suggesting workers have distinct behavioral types. Additionally, colonies from natural environments had higher exploration and foraging activity relative to urban colonies. Activity also varied among ants with different behavioral roles - workers that were foraging were more exploratory than workers taken from the nest or that were engaged in a defensive role (i.e. recruited to the location of a different colony). Finally, we identified a potential proximate mechanism that might be influencing activity. Treatment with the neuromodulator octopamine led to increased levels of individual exploration and colony level foraging activity for colonies from both habitat types. However, natural variation in worker octopamine levels did not vary between environments. Together, these results suggest that exploratory behavior plays a role in adaptation to urbanization. Furthermore, octopamine may be a key driver for exploratory and foraging behavior in odorous house ants.\u003c/p\u003e","manuscriptTitle":"Behavioral and hormonal responses to urbanization in odorous house ants (Tapinoma sessile)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-05 09:43:29","doi":"10.21203/rs.3.rs-8963511/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revisions Needed","date":"2026-04-09T14:29:18+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2026-03-04T14:09:53+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-02T13:00:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-26T02:55:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Insectes Sociaux","date":"2026-02-25T00:41:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"insectes-sociaux","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"inso","sideBox":"Learn more about [Insectes Sociaux](http://link.springer.com/journal/40)","snPcode":"40","submissionUrl":"https://www.editorialmanager.com/inso/default2.aspx","title":"Insectes Sociaux","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"26464907-d3f0-4eb5-88b0-e3ff8c2f5b20","owner":[],"postedDate":"March 5th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-04-09T18:34:57+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-05 09:43:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8963511","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8963511","identity":"rs-8963511","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}