Investigating the effects of biogenic amines on the dominance hierarchy in the small carpenter bee, Ceratina calcarata

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Miller, Sandra M. Rehan, Chelsea N. Cook, Colin S. Brent, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7528609/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Mar, 2026 Read the published version in Insectes Sociaux → Version 1 posted 5 You are reading this latest preprint version Abstract Biogenic amines are highly conserved across animal species and aid in the regulation of movement, behavior, motivation, temperature, blood pressure, and endocrine secretion. Within eusocial species, such as the honey bee Apis mellifera , biogenic amines are responsible for modulating traits associated with higher levels of sociality, such as division of labor. In Ceratina calcarata , a facultatively social bee species, mothers establish a dominance hierarchy over their eldest by providing them with less pollen and less protein, resulting in smaller, more submissive adults referred to as dwarf eldest daughters (DEDs). We hypothesized that this dominance hierarchy is additionally modulated by biogenic amines. To examine this hypothesis, we first compared biogenic amine levels between mothers and daughters and found significantly higher levels of dopamine, octopamine and tyramine in the former group. To quantify the effects of these biogenic amine differences, dopamine was topically administered to C. calcarata DEDs, while the mothers were topically treated with the dopamine blocker cis-(Z)-Flupentixol dihydrochloride. Aggression helps to establish social hierarchies, so mothers and DEDs from the same nest underwent behavioral assays to assess aggressive and tolerant behaviors. When dopamine was blocked in the naturally more aggressive mothers, though there was no difference in the number of aggressive interactions, treated mothers became more tolerant of DEDs, indicating dopamine plays a key role in modulating these behaviors. More work is needed to understand the different roles each biogenic amine plays in the development of a dominance hierarchy, especially in these species on the brink of eusociality. Division of labor dopamine facultatively social bee Apidae Figures Figure 1 Figure 2 Figure 3 Introduction Sociality exists on a spectrum ranging from solitary to eusocial organisms. This complex form of eusociality has evolved convergently multiple times from non-eusocial ancestors across multiple lineages and most frequently in the Hymenoptera (Toth and Rehan 2017 ; Costa 2018 ). Eusocial species form complex social groups characterized by overlapping generations, division of labor, and cooperative brood care (Wilson 1971 ). In contrast, conspecific interactions are limited to mating in solitary organisms (Michener and Brothers 1974 ; Leonhardt et al. 2016 ). Facultatively social species, which are defined by their ability to have a social structure that varies from solitary to social, are excellent models for exploring the underlying mechanisms of sociality due to their plasticity (Séguret et al. 2016 ). Social behavior including dominance interactions are largely regulated by interactions between nestmates (Sasaki et al 2021). Dominance within species often manifests as aggressive exchanges where the dominant individual performs aggressive actions towards subordinates (Syme 1974 ). Subordinates will carry out aggressive interactions towards the higher-ranking individual to challenge them for rank (Amsalem and Hefetz 2010 ; Jandt et al. 2014 ; Withee and Rehan 2016 ). Thus, investigating species in which the social conditions are quite malleable permits close examination of both the physiological and evolutionary constraints of complex social systems (Séguret et al. 2016 ). The stimuli generated during social interactions are processed in the central nervous system (CNS), which interprets the information to drive appropriate short and long-term responses. It has been suggested that the neural process–behavioral output pathway may not be fixed but is instead flexibly selected from existing pathways or from combined multiple pathways in the CNS, and that this selection process is influenced by neural modifiers, such as biogenic amines (Sasaki et al. 2021a ). Previous behavioral interactions or physiological state can both impact circulating neurotransmitter levels, which in turn determine the resultant response to new stimuli (Barbero and Casacci 2025 ). Biogenic amines are signaling molecules that aid in regulating functions such as movement, behavior, emotions, temperature, blood pressure, and endocrine secretion within all animals (Kumar 2003 ; Grimmelikhuijzen et al. 2009 ). They can act as neurotransmitters, neuromodulators and neurohormones in the peripheral nervous system and the CNS (Evans 1980 ; Roeder 2005 ; Lange 2009 ; Sasaki et al. 2021a ). Within social insects, biogenic amines have been shown to play roles in many different social interactions. Dopamine is a biogenic amine neurotransmitter that plays a significant role in regulating the physiology and behavior within all animals (Blenau and Baumann 2001 ), aided by its fast synaptic transmission (Wagener-Hulme et al. 1999 ). In eusocial Hymenoptera, dopamine plays roles in both reproduction and behaviour, including associative learning, ovarian development and mating flight behaviour (Sasaki and Harano 2010 ). Brain dopamine levels have been found to be correlated with reproduction in different species of bumble bees (Bloch et al. 2000 ; Sasaki et al. 2017 ; Sasaki et al. 2021b ), the honey bee Apis mellifera (Harris and Woodring 1995 ; Sasaki and Nagao 2001 ), and the paper wasp Polistes chinensis (Sasaki et al. 2009 ). Furthermore, dopamine has been linked to aggression, though the exact role that this amine serves in modulating aggression is not yet defined because dopaminergic systems influence many behaviors (Huber and Kravitz 2010 ; Alekseyenko et al. 2013 ). Dopamine has been shown to modulate the aggressive behavior status of queenless ants (Cuvillier and Lenoir 2006; Penick et al. 2014 ) and in honeybees, dopamine levels are linked to reproductive behaviors, such as aggression and mating flights (Farkhary et al. 2019 ). A study observing the behavior of mother-offspring groups of primitively eusocial halictid bees suggested that intentional parental manipulation could play an important role in the evolution of eusociality (Michener and Brothers 1974 ). Within the facultatively social species Ceratina calcarata , a dominance hierarchy is seen between the mother and her eldest daughter. Mothers provide smaller pollen balls to their first female offspring during the active brood stage which results in a smaller female (Rehan and Richards 2010b ; Lawson et al. 2016 ). This female, known as the “dwarf eldest daughter” (DED), is relegated to remaining at the parental nest to forage and feed her siblings (Rehan and Richards 2010a ; Rehan et al. 2014 ). Moreover, the DED does not reproduce despite not being biologically sterile, remaining a worker (Rehan and Richards 2013 ). The DED phenotype results from an act of parental manipulation that establishes a dominance hierarchy. Whether the underlying molecular mechanisms that promote this hierarchy are similar to those found in more highly evolved social hymenopteran remains unknown, but such information could have important implications for understanding how complex social systems might evolve. The links between dopamine and aggression in social species, and the insight investigating aggression gives to understanding social structures, gives reason to better understand the role played by biogenic amines in regulating neural activities in a facultatively social species, particularly as they relate to the transition from solitary to eusocial behaviors. The aim of this study is to determine the role biogenic amines play in the establishment of the dominance hierarchy in a facultatively social species. Specifically, we compared amine levels between mothers and daughters either foraging or defending the nest, then we experimentally manipulated amine levels of dopamine in DEDs and mothers to verify the putative functional the role this neurotransmitter plays in mediating the establishment of the hierarchy. Materials and Methods Brain Biogenic Amine Content Mothers and daughter C. calcarata from the same nest were collected from full brood nests throughout July 2017 in Durham, New Hampshire, USA (43.1339° N, 70.9264° W). Mothers were collected by placing cups over the nest entrance and capturing bees leaving the nest. Daughters were captured in the nest. Immediately following capture, bees were frozen in liquid nitrogen. Nests were collected and dissected in the lab. Mothers and daughters were confirmed by measuring wing wear; mothers have more wing wear from a season of foraging (Rehan and Richards 2010a ). Body size was also used to discriminate castes; mothers and regular daughters are significantly (10%) larger than the dwarf eldest daughter (Rehan and Richards 2010b ; Lawson et al. 2017 ). Relative size determinations were made by measuring across the face from the widest margin of each compound eye, using a Nikon SMZ800 microscope fitted with an ocular micrometer. Bees were stored at − 80°C until dissected on dry ice to preserve amines. For each sample, the brains of five bees were pooled, as is typical for similarly sized insects to maximize amine detectability, yielding eight samples per group (Cook et al. 2019 ). Brains were placed together in 1.5-mL tubes and stored at − 80°C until analysis. Using well-established methods (Brent et al. 2016 ; Cook et al. 2019 ), biogenic amines were extracted by placing the five brains comprising a sample into 20 µL of chilled 0.2M perchloric acid that contained the internal standard dihydroxybenzylamine (DHBA, 87pg/µl; Sigma-Aldritch, St. Louis, MO, USA). The tissue was initially homogenized using plastic pestles, further disrupted for 5 min in a sonicating bath filled with an ice slurry, then kept in the bath for an additional 20 min of incubation to finish amine extraction. The tissue was pelletized by centrifugation at 12,000 RCF for 10 min. The biogenic amine content of 10 µl of supernatant was determined by high-performance liquid chromatography (HPLC). The HPLC (Thermo Fisher Scientific, Waltham, MA, USA) consisted of an ECD-3000RS electrochemical detector with a 2 channel coulometric cell, an ISO-3100BM biocompatible isocratic pump, and a Hypersil GOLD C18 250x4.6 mm 5 um HPLC column. Samples were delivered using a manual injector (Rheodyne 9125, Rohnert Park, CA, USA) with a 10-µl loop. Detector cell voltage was set to 800 mV. To minimize batch effects and daily variation in system performance, samples were run consecutively across all test groups. Standard curves were calculated from external standards (hydrochloride form, Sigma Aldrich) of the 4 biogenic amines: dopamine (DA), octopamine (OA), serotonin (5-HT), and tyramine (TA). Dopamine Manipulation Nests of C . calcarata were collected from staghorn sumac ( Rhus tyhia ) in Hamden, Connecticut, USA (41.420137°N, -72.90059°W) and New Haven, Connecticut, USA (41.286089°N, -72.924381°W) in August 2023. Nests were collected before 08:30am to ensure all nest occupants were present at the time of collection. Nests were opened in lab using a blade, carefully removing the top half of the branch to ensure the nest was exposed without damaging the brood. All individuals of the nest were extracted and placed in a petri dish. Using a stereo microscope, sex was determined by the presence (male) or absence (female) of a yellow t-line on the front of the bee and number of tergal metasomal terga; females have six segments, while males have seven (Rehan and Richards 2010a ). The number of each sex was recorded. Mothers and DEDs were identified as previously described measuring wing wear and size (Rehan and Richards 2010b ; Lawson et al. 2017 ). Mothers and DEDs of the same nest were paired together for behavioral assays. Behavioral trials were performed no more than two hours after nest opening to minimize behavioral changes due to stress (Pabalan et al. 2000 ). A total of 108 individuals from 54 nests were used for the experiments. All paired mothers and DEDs from nests were randomly assigned to the treatment or controls. Mothers and dwarf eldest daughters from the same nest were paired together for treatment and behavioral assays. The treatment for daughters was dopamine (DA; 4 mg/mL; Sigma Aldrich, St. Lous, MO, USA), and the treatment for mothers was cis-(Z)-Flupentixol dihydrochloride (DA antagonist; 4 mg/mL; Santa Cruz Biotechnology, Dallas, TX, USA), both of which were dissolved in dimethylformamide (dMF; Sigma Aldrich, St. Lous, MO, USA), a solvent able to penetrate the cuticle (Barron et al. 2007 ). Vehicle controls received 1 µL of the delivery solvent dMF, and controls received no topical application prior to behavioural assays. Collected bees were immobilized by chilling at -20 ℃ for one minute. Bees were treated topically by applying 1 µL of solution to the thorax with a micropipette. To quantify the behavioral effects of the presence or absence of dopamine, we used circle tube assays to monitor behavioral interactions between mothers and DEDs. Circle tubes provide an artificial environment that allows easy observation of interactions between individuals that are similar to behaviors displayed in their natural environment (Brothers and Michener 1974 ; Breed et al. 1978 ; Packer 2006 ). For each circle tube assay, bees were simultaneously introduced to opposite ends of 30 cm polyethylene tubing with an inner diameter 4 mm, which is twice the average size of C. calcarata head width (Rehan and Richards 2013 ). The ends of the tubing were joined to form a circle. Given that used tubes can contain odors from former bee occupants (Smith and Weller 1989 ), a new tube was used for each trial. Interactions were observed for 20 minutes, beginning once individuals started moving within the tube. Behaviors were recorded once individuals were within one body length of each other (Kukuk 1992 ; Packer 2005 ). Interactions between the two individuals were categorized as aggressive, tolerant, following, and avoidant according to previous studies (Rehan and Richards 2013 ). Aggressive behaviors included nudging, biting, or C-posturing, where a bee curls its abdomen under their thorax resulting in its body forming a C-shape with mandibles and stinger pointed at the other individual. Avoidance behaviors included a bee backing away or reversing themselves 180° to move away from the other individual. Tolerant behaviors included passing, antennae-to-antennae, and head-to-head contact. Following behaviors have been considered cooperative in some communal species (McConnell-Garner and Kukuk 1997 ; Bosei and Polidori 2011), subordinate in eusocial species (Breed et al. 1978 ; Michener 1990 ), and dominant in other species (West-Eberhard 1979 ). Due to the uncertainty of the behavioral categorization of this interaction, following behaviors were categorized separately. We used circle tubes compare the behaviours of untreated mothers versus untreated DEDs, delivery control mothers versus delivery control DEDs, and 5HT blocker treated mothers versus 5HT treated DEDs. Statistical Analysis Data were determined to be non-normally distributed via a Shapiro-Wilk test; thus non-parametric analyses were utilized. A Kruskal-Wallis ANOVA on ranks was used to compare biogenic amine concentrations. The same analysis was performed on the behavioral data, followed by Dunn’s multiple comparison tests. Specifically, the number of interactions for each behavior type (aggressive, avoidant, tolerant and following) by mother and daughter for each treatment group was compared. Additionally, the difference in the number of interactions between mothers and daughters in the treatment groups was compared. All statistical analyses were performed in R version 4.4.2 (R Studio 2020). Results Brain Biogenic Amine Content We compared the concentrations of each of the four biogenic amines in the brains of mothers and daughters. Relative to their daughters, mothers had significantly higher concentrations of octopamine (Kruskal-Wallis ANOVA: H = 18.099, df = 1, p < 0.001), dopamine (H = 20.157, df = 1, p < 0.001), and tyramine (H = 6.99, df = 1, p = 0.008). There was no difference between groups in concentration of serotonin (H = 2.56, df = 1, p = 0.109) (Fig. 1 ). Dopamine Manipulation To examine the behavioral effects of the addition or removal of dopamine, we compared the number of behaviors (aggressive, avoidant, tolerant and following), individual interactions and differences in total frequencies across treatment groups. There was a significant difference in head-to-head interactions across treatment groups with daughters who received the addition of dopamine and mothers who received a dopamine blocker having an increased number of tolerant head-to-head behaviors relative to untreated controls (N = 114; H = 11.46, df = 3, p = 0.04; Fig. 2 b). There were no significant differences found with any other tolerant behavioral interactions. Total frequencies of behavior did not differ among treatment groups: aggressive (N = 114; H = 7.62, df = 5, p = 0.18), avoidant (N = 114; H = 4.13, df = 5, p = 0.53), tolerant (N = 114; H = 6.45, df = 5, p = 0.26), or following (N = 114; H = 2.32, df = 5, p = 0.80) (Fig. 3 ). Discussion Understanding the proximate mechanisms involved in the development of social hierarchies in facultatively social species is critical to the understanding of the evolution of more complex social behaviors. Ceratina calcarata is an ideal species for such studies because of the unique social structure in the species, where the mother serves as the reproductive for the nest and the eldest daughter serves as the forager for the nest at a cost of her own reproduction. Previous research has shown the mothers can manipulate the body size of her offspring by providing differing quantities (Johnson 1988 ; Rehan and Richards 2010b ) and quality (Lawson et al. 2016 ) of pollen and nectar to each brood cell. Nutritional deprivation results in smaller, less aggressive eldest daughters (Lawson et al. 2017 ). Whether the behavioral repertoires associated with these divergent phenotypes are the result of induced changes to circulating neurotransmitter levels was unknown. In other social species, biogenic amines have been shown to play an important role in the development of dominance hierarchies (Barbero et al. 2023 ). Previous research has shown an associative link between biogenic amine concentrations and reproductive status in C. calcarata (Cook et al. 2019 ). We found that reproductive mothers have significantly higher levels of dopamine, octopamine and tyramine in their brains compared to non-reproductive daughters (Fig. 1 ). Because the levels of dopamine differed the most between mother and daughter, we first explored the role of this biogenic amine on the development of social hierarchies by examining the effects of increased dopamine on daughters and decreased dopamine in mothers. Given that higher levels of dopamine are often associated with the dominant/aggressive individuals in other Hymenopteran species (Penick et al. 2014 ; Sasaki et al. 2009 ; Cuvillier and Lenoir 2006; Penick et al. 2014 ), it was expected that dopamine-treated DEDs would exhibit a decrease in tolerant behaviors and an increase in aggressive interactions. We observed that mothers treated with a dopamine antagonist and daughters treated with dopamine had more head-to-head interactions than the controls (Fig. 2 b), rather than the fewer interactions that were expected. However, no other individual tolerant behavior showed a significant shift. Tolerant behaviors can be rather subtle, therefore, any shifts induced by treatment could have been missed. As with most tolerant behaviors, those categorized as aggressive (biting, nudging), also did not respond to dopamine manipulation (Fig. 3 ). Such interactions are easier to spot than tolerant behaviors, therefore, it is unlikely that they were miscounted. This result was unexpected when considering previous studies investigating the link of biogenic amines to behaviors within Hymenoptera. In workers of Formica polycenta , dopamine treatment stimulated more threatening behaviors and overall aggression (Szczuka et al. 2013 ). However, in that study they utilized the injection delivery method instead of topical application, which could explain the difference in their results compared to this study. It is also possible that the concentration of dopamine blocker may have been insufficient to induce a string behavioral response. Honeybees treated with various concentrations of the blocker flupentixol had widely varied responses depending on dose (Farkhary et al. 2017 ), which could indicate that the effect of blocking the dopamine receptors in C. calcarata could have various effects on aggressive behaviors. It was expected that mothers treated with the dopamine blocker would increase avoidant behaviors compared to untreated mothers. Receiving a dopamine blocker was expected to make mothers less aggressive while daughters treated with DA were expected to be more aggressive, creating increased opportunities for mothers to be avoidant. However, there was no significant difference found in the frequency of avoidant behaviors across treatment groups, likely due to the lack of observable change in daughter aggression. The lack of impact that dopamine manipulation had on aggressive behavior in C. ceratina , may also indicate that another neurotransmitter may be responsible for modulating such behaviors. Octopamine and tyramine were also elevated in mothers compared to their daughters. Octopamine seems the likeliest candidate, given that is has been implicated in the aggressive behaviors and motivational states in a number of invertebrate species. Within invertebrates, octopamine has the ability to induce and modulate signal transduction pathways like norepinephrine in vertebrates (Farooqui 2012 ). Octopamine levels in the hemolymph have been shown to trigger a “fight or flight” reaction in locusts (Malamud et al. 1988 ; Adamo et al. 1995 ). High octopamine concentrations are accompanied by an aggressive behavior, resulting in dominance (Roeder et al. 2003 ) and influencing division of labor in Apis mellifera (Schulz et al. 2001). Future work investigating the role octopamine plays in modulating behaviours within C. calcarata would be an insightful next step in understanding the underlying influence biogenic amines have on social behaviours. Conclusions In summary, C. calcarata mothers have significantly elevated levels of multiple biogenic amines (octopamine, dopamine and tyramine) compared to daughters. When examining the role dopamine plays in the dominance hierarchy of C. calcarata mothers and DEDs, there was an observable shift in head-to-head tolerant behaviors within the treatment group. Further work is needed to determine if the carrier also had an impact on these behaviors or biogenic amine alone. This study provides evidence for a role of the relationship between biogenic amines and dominance behaviors within a facultatively social species and future studies on the effects of other biogenic amines and other facultatively social species is needed. Declarations Conflict of interest: The authors declare no conflict of interest. Funding: Funding was made possible by internal grants from Quinnipiac University. The funding source had no role in study design, data collection, data analysis, data interpretation, or writing of the manuscript. Author contributions: All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by RM and SL. The first draft of the manuscript was written by RM and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Acknowledgement: We would like to thank Lisa Kaplan, Tom Torello and Alexandre de Lencastre for their helpful insights on experimental design and feedback on the manuscript. RM was supported by the Quinnipiac University Interdisciplinary Program for Research and Scholarship (QUIP-RS), which provided feedback and support. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer. Data availability: The data that supported these findings has been uploaded to Figshare: https://figshare.com/s/1364e85987a50496b52c References Adamo SA, Linn CE, Hoy RR (1995) The role of neurohormonal octopamine during fight or flight behavior in the field cricket Gryllus bimaculatus . 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Insectes Soc 52:139–146. https://doi.org/10.1007/s00040-004-0784-7 Penick CA, Brent CS, Dolezal K, Liebig J (2014) Neurohormonal changes associated with ritualized combat and the formation of a reproductive hierarchy in the ant Harpegnathos saltator . J Exp Biol 217(9):1496–1503. https://doi.org/10.1242/jeb.098301 RStudio T, RStudio (2020) PBC, Boston, MA Rehan SM, Berens AJ, Toth AL (2014) At the brink of eusociality: transcriptomic correlates of worker behaviour in a small carpenter bee. BMC Evol Biol 14:260. https://doi.org/10.1186/s12862-014-0260-6 Rehan SM, Richards MH (2013) Reproductive aggression and nestmate recognition in a subsocial bee. Anim Behav 85(4):733–741. https://doi.org/10.1016/j.anbehav.2013.01.010 Rehan SM, Richards MH (2010a) Nesting biology and subsociality in Ceratina calcarata (Hymenoptera: Apidae). Can Entomol 142(1):65–74. https://doi.org/10.4039/n09-056 Rehan SM, Richards MH (2010b) The influence of maternal quality on brood sex allocation in the small carpenter bee, Ceratina calcarata . Etho 116(9):876–887. https://doi.org/10.1111/j.1439-0310.2010.01804.x Roeder T, Seifert M, Kähler C, Gewecke M (2003) Tyramine and octopamine: Antagonistic modulators of behavior and metabolism. Arch Insect Biochem Physiol 54:1–13. https://doi.org/10.1002/arch.10102 Roeder T (2005) Tyramine and octopamine: ruling behavior and metabolism. Ann Rev Entomol 50:447–477. https://doi.org/10.1146/annurev.ento.50.071803.130404 Sasaki K, Harano KI (2010) Multiple regulatory roles of dopamine in behavior and reproduction of social insects. Trends Entomol 6:1–13 Sasaki K, Okada Y, Shimoji H, Aonuma H, Miura T, Tsuji K (2021a) Social evolution with decoupling of multiple roles of biogenic amines into different phenotypes in Hymenoptera. Front Ecol Evol 9:659160. https://doi.org/10.3389/fevo.2021.659160 Sasaki K, Matsuyama H, Morita N, Ono M (2017) Caste differences in the association between dopamine and reproduction in the bumble bee Bombus ignitus . J Insect Physiol 103:107–116. https://doi.org/10.1016/j.jinsphys.2017.10.013 Sasaki K, Nagao T (2001) Distribution and levels of dopamine and its metabolites in brains of reproductive workers in honeybees. J Insect Physiol 47(10):1205–1216. https://doi.org/10.1016/S0022-1910(01)00105-6 Sasaki K, Yamasaki K, Tsuchida K (2009) Gonadotropic effects of dopamine in isolated workers of the primitively eusocial wasp, Polistes chinensis . Sci Nat 96:625–629. https://doi.org/10.1007/s00114-009-0510-4 Sasaki K, Yokoi K, Toga K (2021b) Bumble bee queens activate dopamine production and gene expression in nutritional signaling pathways in the brain. Sci Rep 11:5526. https://doi.org/10.1038/s41598-021-84992-2 Schulz DJ, Robinson GE (2001) Octopamine influences division of labor in honey bee colonies. J Comp Physiol A 187(1):53–61. https://doi.org/10.1007/s003590000177 Séguret A, Bernadou A, Paxton RJ (2016) Facultative social insects can provide insights into the reversal of the longevity/fecundity trade-off across the eusocial insects. Curr Opin Insect Sci 16:95–103. https://doi.org/10.1016/j.cois.2016.06.001 Smith BH, Weller C (1989) Social competition among gynes in halictine bees: The influence of bee size and pheromones on behavior. J Insect Behav 2:397–411. https://doi.org/10.1007/BF01068064 Syme GJ (1974) Competitive orders as measures of social dominance. Anim Behav 22:931–940. https://doi.org/10.1016/0003-3472(74)90016-5 Szczuka A, Korczyńska J, Wnuk A, Symonowicz B, Gonzalez Szwacka A, Mazurkiewicz P, Kostowski W, Godzińska EJ (2013) The effects of serotonin, dopamine, octopamine and tyramine on behavior of workers of the ant Formica polyctena during dyadic aggression tests. Acta Neurobiol Exp (Wars) 73(4):495–520. https://doi.org/10.55782/ane-2013-1955 Toth AL, Rehan SM (2017) Molecular evolution of insect sociality: An eco-evo-devo perspective. Annu Rev Entomol 62:419–442. https://doi.org/10.1146/annurev-ento-031616-035601 Wagener-Hulme C, Kuehn JC, Schulz DJ, Robinson GE (1999) Biogenic amines and division of labor in honey bee colonies. J Comp Physio A 184(5):471–479. https://doi.org/10.1007/s003590050347 West-Eberhard MJ (1979) Sexual selection, social competition, and evolution. Proc Am Philos Soc–51:222–234 Wilson E (1971) The Insect Societies. Belknap Press of Harvard University, Cambridge, Massachusetts Withee JR, Rehan SM (2016) Cumulative effects of body size and social experience on aggressive behaviour in a subsocial bee. Behaviour 153(12):1365–1385. https://doi.org/10.1163/1568539X-00003382 Cite Share Download PDF Status: Published Journal Publication published 16 Mar, 2026 Read the published version in Insectes Sociaux → Version 1 posted Editorial decision: Major Revisions Needed 31 Oct, 2025 Reviewers agreed at journal 12 Sep, 2025 Reviewers invited by journal 05 Sep, 2025 Editor assigned by journal 04 Sep, 2025 First submitted to journal 03 Sep, 2025 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-7528609","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":511015487,"identity":"66892ec3-b7b5-4157-8f40-f1aef8c38837","order_by":0,"name":"Riya N. Miller","email":"","orcid":"","institution":"York University","correspondingAuthor":false,"prefix":"","firstName":"Riya","middleName":"N.","lastName":"Miller","suffix":""},{"id":511015488,"identity":"23745baf-52ab-4460-9402-bf9aea1d0c88","order_by":1,"name":"Sandra M. Rehan","email":"","orcid":"","institution":"York University","correspondingAuthor":false,"prefix":"","firstName":"Sandra","middleName":"M.","lastName":"Rehan","suffix":""},{"id":511015489,"identity":"570ba4c4-4603-4cf3-ab00-d95cb4c6f568","order_by":2,"name":"Chelsea N. Cook","email":"","orcid":"","institution":"Marquette University","correspondingAuthor":false,"prefix":"","firstName":"Chelsea","middleName":"N.","lastName":"Cook","suffix":""},{"id":511015490,"identity":"87d9c9b8-4bc1-4891-a2be-5977bcad8919","order_by":3,"name":"Colin S. Brent","email":"","orcid":"","institution":"USDA-ARS: USDA Agricultural Research Service","correspondingAuthor":false,"prefix":"","firstName":"Colin","middleName":"S.","lastName":"Brent","suffix":""},{"id":511015491,"identity":"0e2f81ec-2c12-4c98-8333-eda2803947ff","order_by":4,"name":"Sarah P Lawson","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-8014-6960","institution":"Quinnipiac University","correspondingAuthor":true,"prefix":"","firstName":"Sarah","middleName":"P","lastName":"Lawson","suffix":""}],"badges":[],"createdAt":"2025-09-03 15:30:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7528609/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7528609/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00040-026-01086-w","type":"published","date":"2026-03-16T15:59:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91369930,"identity":"5ebe1dd5-aab2-4dca-b463-797d998ad910","added_by":"auto","created_at":"2025-09-15 18:37:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":112422,"visible":true,"origin":"","legend":"\u003cp\u003eDifferences in biogenic amine concentrations in the brains of \u003cem\u003eC. ceratina\u003c/em\u003e mothers and daughters. The y-axis was log base 10 transformed given the high levels of dopamine compared to other amines. Shown are the medians, interquartile ranges, 90th and 10th percentiles (whiskers), and any outlier data points exceeding these outer bounds (•). An asterisk indicates a significant difference between mother and daughter (K-W ANOVA on ranks, α = 0.05). It was found mothers had significantly higher concentrations of octopamine (p \u0026lt; 0.001), dopamine (p \u0026lt; 0.001), and tyramine (p = 0.008).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7528609/v1/662c8dbbdee58b3930d852c7.png"},{"id":91369627,"identity":"6faf47e3-c2fe-41d8-afd2-3096c9f1aba4","added_by":"auto","created_at":"2025-09-15 18:29:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":122557,"visible":true,"origin":"","legend":"\u003cp\u003eTotal number of different types of tolerant behaviors observed between mothers and dwarf eldest daughters. Because tolerant interactions involve both the mother and daughter only treatment groups are compared. a) Average number of passing interactions. b) Average number of head-to-head interactions. c) Average number of antennation. \u0026nbsp;Boxes depict the medians and lower and upper quartiles, whiskers signify the maximum and minimum observations, and points outside of the whiskers signify outliers. Test groups included: untreated control (Control), solvent control (DMF), and dopamine manipulated (Treatment). \u0026nbsp;An asterisk indicates a significant difference across treatment groups (K-W ANOVA on ranks, α = 0.05).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7528609/v1/78d4d693537c481dd7770c02.png"},{"id":91368893,"identity":"3257d8f4-7ee7-4166-a693-264ab685c103","added_by":"auto","created_at":"2025-09-15 18:21:12","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":303268,"visible":true,"origin":"","legend":"\u003cp\u003eTotal number of behavioral interactions for both mothers (M) and dwarf eldest daughters (D) across treatment groups. Boxes depict the medians and lower and upper quartiles, whiskers signify the maximum and minimum observations, and points outside of the whiskers signify outliers. None (controls) received no topical application, DMF received topical application of solvent only, and treated daughters received topical application of dopamine dissolved in DMF, while treated mothers received topical application of dopamine blocker dissolved in DMF. No significant differences were found for any of the behavioral categories (K-W ANOVA on ranks, α = 0.05).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7528609/v1/ac9a68a4085a54335009de57.jpeg"},{"id":105223674,"identity":"4971fcc1-aba6-43f4-917e-413519d1c608","added_by":"auto","created_at":"2026-03-23 16:08:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":985918,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7528609/v1/3fdfe03d-5b3e-4a95-88a5-5b3cb9f8d9df.pdf"}],"financialInterests":"","formattedTitle":"Investigating the effects of biogenic amines on the dominance hierarchy in the small carpenter bee, Ceratina calcarata","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSociality exists on a spectrum ranging from solitary to eusocial organisms. This complex form of eusociality has evolved convergently multiple times from non-eusocial ancestors across multiple lineages and most frequently in the Hymenoptera (Toth and Rehan \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Costa \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Eusocial species form complex social groups characterized by overlapping generations, division of labor, and cooperative brood care (Wilson \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e1971\u003c/span\u003e). In contrast, conspecific interactions are limited to mating in solitary organisms (Michener and Brothers \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1974\u003c/span\u003e; Leonhardt et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Facultatively social species, which are defined by their ability to have a social structure that varies from solitary to social, are excellent models for exploring the underlying mechanisms of sociality due to their plasticity (S\u0026eacute;guret et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSocial behavior including dominance interactions are largely regulated by interactions between nestmates (Sasaki et al 2021). Dominance within species often manifests as aggressive exchanges where the dominant individual performs aggressive actions towards subordinates (Syme \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e1974\u003c/span\u003e). Subordinates will carry out aggressive interactions towards the higher-ranking individual to challenge them for rank (Amsalem and Hefetz \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Jandt et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Withee and Rehan \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Thus, investigating species in which the social conditions are quite malleable permits close examination of both the physiological and evolutionary constraints of complex social systems (S\u0026eacute;guret et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe stimuli generated during social interactions are processed in the central nervous system (CNS), which interprets the information to drive appropriate short and long-term responses. It has been suggested that the neural process\u0026ndash;behavioral output pathway may not be fixed but is instead flexibly selected from existing pathways or from combined multiple pathways in the CNS, and that this selection process is influenced by neural modifiers, such as biogenic amines (Sasaki et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e). Previous behavioral interactions or physiological state can both impact circulating neurotransmitter levels, which in turn determine the resultant response to new stimuli (Barbero and Casacci \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBiogenic amines are signaling molecules that aid in regulating functions such as movement, behavior, emotions, temperature, blood pressure, and endocrine secretion within all animals (Kumar \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Grimmelikhuijzen et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). They can act as neurotransmitters, neuromodulators and neurohormones in the peripheral nervous system and the CNS (Evans \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1980\u003c/span\u003e; Roeder \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Lange \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Sasaki et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e). Within social insects, biogenic amines have been shown to play roles in many different social interactions. Dopamine is a biogenic amine neurotransmitter that plays a significant role in regulating the physiology and behavior within all animals (Blenau and Baumann \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), aided by its fast synaptic transmission (Wagener-Hulme et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e1999\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn eusocial Hymenoptera, dopamine plays roles in both reproduction and behaviour, including associative learning, ovarian development and mating flight behaviour (Sasaki and Harano \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Brain dopamine levels have been found to be correlated with reproduction in different species of bumble bees (Bloch et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Sasaki et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Sasaki et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e), the honey bee \u003cem\u003eApis mellifera\u003c/em\u003e (Harris and Woodring \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Sasaki and Nagao \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), and the paper wasp \u003cem\u003ePolistes chinensis\u003c/em\u003e (Sasaki et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Furthermore, dopamine has been linked to aggression, though the exact role that this amine serves in modulating aggression is not yet defined because dopaminergic systems influence many behaviors (Huber and Kravitz \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Alekseyenko et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Dopamine has been shown to modulate the aggressive behavior status of queenless ants (Cuvillier and Lenoir 2006; Penick et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and in honeybees, dopamine levels are linked to reproductive behaviors, such as aggression and mating flights (Farkhary et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eA study observing the behavior of mother-offspring groups of primitively eusocial halictid bees suggested that intentional parental manipulation could play an important role in the evolution of eusociality (Michener and Brothers \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1974\u003c/span\u003e). Within the facultatively social species \u003cem\u003eCeratina calcarata\u003c/em\u003e, a dominance hierarchy is seen between the mother and her eldest daughter. Mothers provide smaller pollen balls to their first female offspring during the active brood stage which results in a smaller female (Rehan and Richards \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2010b\u003c/span\u003e; Lawson et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). This female, known as the \u0026ldquo;dwarf eldest daughter\u0026rdquo; (DED), is relegated to remaining at the parental nest to forage and feed her siblings (Rehan and Richards \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2010a\u003c/span\u003e; Rehan et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Moreover, the DED does not reproduce despite not being biologically sterile, remaining a worker (Rehan and Richards \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The DED phenotype results from an act of parental manipulation that establishes a dominance hierarchy. Whether the underlying molecular mechanisms that promote this hierarchy are similar to those found in more highly evolved social hymenopteran remains unknown, but such information could have important implications for understanding how complex social systems might evolve. The links between dopamine and aggression in social species, and the insight investigating aggression gives to understanding social structures, gives reason to better understand the role played by biogenic amines in regulating neural activities in a facultatively social species, particularly as they relate to the transition from solitary to eusocial behaviors.\u003c/p\u003e\u003cp\u003eThe aim of this study is to determine the role biogenic amines play in the establishment of the dominance hierarchy in a facultatively social species. Specifically, we compared amine levels between mothers and daughters either foraging or defending the nest, then we experimentally manipulated amine levels of dopamine in DEDs and mothers to verify the putative functional the role this neurotransmitter plays in mediating the establishment of the hierarchy.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eBrain Biogenic Amine Content\u003c/h2\u003e\u003cp\u003eMothers and daughter \u003cem\u003eC. calcarata\u003c/em\u003e from the same nest were collected from full brood nests throughout July 2017 in Durham, New Hampshire, USA (43.1339\u0026deg; N, 70.9264\u0026deg; W). Mothers were collected by placing cups over the nest entrance and capturing bees leaving the nest. Daughters were captured in the nest. Immediately following capture, bees were frozen in liquid nitrogen. Nests were collected and dissected in the lab. Mothers and daughters were confirmed by measuring wing wear; mothers have more wing wear from a season of foraging (Rehan and Richards \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2010a\u003c/span\u003e). Body size was also used to discriminate castes; mothers and regular daughters are significantly (10%) larger than the dwarf eldest daughter (Rehan and Richards \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2010b\u003c/span\u003e; Lawson et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Relative size determinations were made by measuring across the face from the widest margin of each compound eye, using a Nikon SMZ800 microscope fitted with an ocular micrometer. Bees were stored at \u0026minus;\u0026thinsp;80\u0026deg;C until dissected on dry ice to preserve amines. For each sample, the brains of five bees were pooled, as is typical for similarly sized insects to maximize amine detectability, yielding eight samples per group (Cook et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Brains were placed together in 1.5-mL tubes and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until analysis.\u003c/p\u003e\u003cp\u003eUsing well-established methods (Brent et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Cook et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), biogenic amines were extracted by placing the five brains comprising a sample into 20 \u0026micro;L of chilled 0.2M perchloric acid that contained the internal standard dihydroxybenzylamine (DHBA, 87pg/\u0026micro;l; Sigma-Aldritch, St. Louis, MO, USA). The tissue was initially homogenized using plastic pestles, further disrupted for 5 min in a sonicating bath filled with an ice slurry, then kept in the bath for an additional 20 min of incubation to finish amine extraction. The tissue was pelletized by centrifugation at 12,000 RCF for 10 min. The biogenic amine content of 10 \u0026micro;l of supernatant was determined by high-performance liquid chromatography (HPLC). The HPLC (Thermo Fisher Scientific, Waltham, MA, USA) consisted of an ECD-3000RS electrochemical detector with a 2 channel coulometric cell, an ISO-3100BM biocompatible isocratic pump, and a Hypersil GOLD C18 250x4.6 mm 5 um HPLC column. Samples were delivered using a manual injector (Rheodyne 9125, Rohnert Park, CA, USA) with a 10-\u0026micro;l loop. Detector cell voltage was set to 800 mV. To minimize batch effects and daily variation in system performance, samples were run consecutively across all test groups. Standard curves were calculated from external standards (hydrochloride form, Sigma Aldrich) of the 4 biogenic amines: dopamine (DA), octopamine (OA), serotonin (5-HT), and tyramine (TA).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDopamine Manipulation\u003c/h3\u003e\n\u003cp\u003eNests of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ecalcarata\u003c/em\u003e were collected from staghorn sumac (\u003cem\u003eRhus tyhia\u003c/em\u003e) in Hamden, Connecticut, USA (41.420137\u0026deg;N, -72.90059\u0026deg;W) and New Haven, Connecticut, USA (41.286089\u0026deg;N, -72.924381\u0026deg;W) in August 2023. Nests were collected before 08:30am to ensure all nest occupants were present at the time of collection. Nests were opened in lab using a blade, carefully removing the top half of the branch to ensure the nest was exposed without damaging the brood. All individuals of the nest were extracted and placed in a petri dish. Using a stereo microscope, sex was determined by the presence (male) or absence (female) of a yellow t-line on the front of the bee and number of tergal metasomal terga; females have six segments, while males have seven (Rehan and Richards \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2010a\u003c/span\u003e). The number of each sex was recorded. Mothers and DEDs were identified as previously described measuring wing wear and size (Rehan and Richards \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2010b\u003c/span\u003e; Lawson et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Mothers and DEDs of the same nest were paired together for behavioral assays. Behavioral trials were performed no more than two hours after nest opening to minimize behavioral changes due to stress (Pabalan et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). A total of 108 individuals from 54 nests were used for the experiments.\u003c/p\u003e\u003cp\u003eAll paired mothers and DEDs from nests were randomly assigned to the treatment or controls. Mothers and dwarf eldest daughters from the same nest were paired together for treatment and behavioral assays. The treatment for daughters was dopamine (DA; 4 mg/mL; Sigma Aldrich, St. Lous, MO, USA), and the treatment for mothers was cis-(Z)-Flupentixol dihydrochloride (DA antagonist; 4 mg/mL; Santa Cruz Biotechnology, Dallas, TX, USA), both of which were dissolved in dimethylformamide (dMF; Sigma Aldrich, St. Lous, MO, USA), a solvent able to penetrate the cuticle (Barron et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Vehicle controls received 1 \u0026micro;L of the delivery solvent dMF, and controls received no topical application prior to behavioural assays. Collected bees were immobilized by chilling at -20 ℃ for one minute. Bees were treated topically by applying 1 \u0026micro;L of solution to the thorax with a micropipette.\u003c/p\u003e\u003cp\u003eTo quantify the behavioral effects of the presence or absence of dopamine, we used circle tube assays to monitor behavioral interactions between mothers and DEDs. Circle tubes provide an artificial environment that allows easy observation of interactions between individuals that are similar to behaviors displayed in their natural environment (Brothers and Michener \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1974\u003c/span\u003e; Breed et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1978\u003c/span\u003e; Packer \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). For each circle tube assay, bees were simultaneously introduced to opposite ends of 30 cm polyethylene tubing with an inner diameter 4 mm, which is twice the average size of \u003cem\u003eC. calcarata\u003c/em\u003e head width (Rehan and Richards \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The ends of the tubing were joined to form a circle. Given that used tubes can contain odors from former bee occupants (Smith and Weller \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e1989\u003c/span\u003e), a new tube was used for each trial. Interactions were observed for 20 minutes, beginning once individuals started moving within the tube. Behaviors were recorded once individuals were within one body length of each other (Kukuk \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Packer \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Interactions between the two individuals were categorized as aggressive, tolerant, following, and avoidant according to previous studies (Rehan and Richards \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Aggressive behaviors included nudging, biting, or C-posturing, where a bee curls its abdomen under their thorax resulting in its body forming a C-shape with mandibles and stinger pointed at the other individual. Avoidance behaviors included a bee backing away or reversing themselves 180\u0026deg; to move away from the other individual. Tolerant behaviors included passing, antennae-to-antennae, and head-to-head contact. Following behaviors have been considered cooperative in some communal species (McConnell-Garner and Kukuk \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Bosei and Polidori 2011), subordinate in eusocial species (Breed et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1978\u003c/span\u003e; Michener \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1990\u003c/span\u003e), and dominant in other species (West-Eberhard \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e1979\u003c/span\u003e). Due to the uncertainty of the behavioral categorization of this interaction, following behaviors were categorized separately. We used circle tubes compare the behaviours of untreated mothers versus untreated DEDs, delivery control mothers versus delivery control DEDs, and 5HT blocker treated mothers versus 5HT treated DEDs.\u003c/p\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eData were determined to be non-normally distributed via a Shapiro-Wilk test; thus non-parametric analyses were utilized. A Kruskal-Wallis ANOVA on ranks was used to compare biogenic amine concentrations. The same analysis was performed on the behavioral data, followed by Dunn\u0026rsquo;s multiple comparison tests. Specifically, the number of interactions for each behavior type (aggressive, avoidant, tolerant and following) by mother and daughter for each treatment group was compared. Additionally, the difference in the number of interactions between mothers and daughters in the treatment groups was compared. All statistical analyses were performed in R version 4.4.2 (R Studio 2020).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eBrain Biogenic Amine Content\u003c/h2\u003e\u003cp\u003eWe compared the concentrations of each of the four biogenic amines in the brains of mothers and daughters. Relative to their daughters, mothers had significantly higher concentrations of octopamine (Kruskal-Wallis ANOVA: H\u0026thinsp;=\u0026thinsp;18.099, df\u0026thinsp;=\u0026thinsp;1, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), dopamine (H\u0026thinsp;=\u0026thinsp;20.157, df\u0026thinsp;=\u0026thinsp;1, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and tyramine (H\u0026thinsp;=\u0026thinsp;6.99, df\u0026thinsp;=\u0026thinsp;1, p\u0026thinsp;=\u0026thinsp;0.008). There was no difference between groups in concentration of serotonin (H\u0026thinsp;=\u0026thinsp;2.56, df\u0026thinsp;=\u0026thinsp;1, p\u0026thinsp;=\u0026thinsp;0.109) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eDopamine Manipulation\u003c/h2\u003e\u003cp\u003eTo examine the behavioral effects of the addition or removal of dopamine, we compared the number of behaviors (aggressive, avoidant, tolerant and following), individual interactions and differences in total frequencies across treatment groups. There was a significant difference in head-to-head interactions across treatment groups with daughters who received the addition of dopamine and mothers who received a dopamine blocker having an increased number of tolerant head-to-head behaviors relative to untreated controls (N\u0026thinsp;=\u0026thinsp;114; H\u0026thinsp;=\u0026thinsp;11.46, df\u0026thinsp;=\u0026thinsp;3, p\u0026thinsp;=\u0026thinsp;0.04; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). There were no significant differences found with any other tolerant behavioral interactions. Total frequencies of behavior did not differ among treatment groups: aggressive (N\u0026thinsp;=\u0026thinsp;114; H\u0026thinsp;=\u0026thinsp;7.62, df\u0026thinsp;=\u0026thinsp;5, p\u0026thinsp;=\u0026thinsp;0.18), avoidant (N\u0026thinsp;=\u0026thinsp;114; H\u0026thinsp;=\u0026thinsp;4.13, df\u0026thinsp;=\u0026thinsp;5, p\u0026thinsp;=\u0026thinsp;0.53), tolerant (N\u0026thinsp;=\u0026thinsp;114; H\u0026thinsp;=\u0026thinsp;6.45, df\u0026thinsp;=\u0026thinsp;5, p\u0026thinsp;=\u0026thinsp;0.26), or following (N\u0026thinsp;=\u0026thinsp;114; H\u0026thinsp;=\u0026thinsp;2.32, df\u0026thinsp;=\u0026thinsp;5, p\u0026thinsp;=\u0026thinsp;0.80) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eUnderstanding the proximate mechanisms involved in the development of social hierarchies in facultatively social species is critical to the understanding of the evolution of more complex social behaviors. \u003cem\u003eCeratina calcarata\u003c/em\u003e is an ideal species for such studies because of the unique social structure in the species, where the mother serves as the reproductive for the nest and the eldest daughter serves as the forager for the nest at a cost of her own reproduction. Previous research has shown the mothers can manipulate the body size of her offspring by providing differing quantities (Johnson \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Rehan and Richards \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2010b\u003c/span\u003e) and quality (Lawson et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) of pollen and nectar to each brood cell. Nutritional deprivation results in smaller, less aggressive eldest daughters (Lawson et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Whether the behavioral repertoires associated with these divergent phenotypes are the result of induced changes to circulating neurotransmitter levels was unknown. In other social species, biogenic amines have been shown to play an important role in the development of dominance hierarchies (Barbero et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Previous research has shown an associative link between biogenic amine concentrations and reproductive status in \u003cem\u003eC. calcarata\u003c/em\u003e (Cook et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). We found that reproductive mothers have significantly higher levels of dopamine, octopamine and tyramine in their brains compared to non-reproductive daughters (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBecause the levels of dopamine differed the most between mother and daughter, we first explored the role of this biogenic amine on the development of social hierarchies by examining the effects of increased dopamine on daughters and decreased dopamine in mothers. Given that higher levels of dopamine are often associated with the dominant/aggressive individuals in other Hymenopteran species (Penick et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Sasaki et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Cuvillier and Lenoir 2006; Penick et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), it was expected that dopamine-treated DEDs would exhibit a decrease in tolerant behaviors and an increase in aggressive interactions. We observed that mothers treated with a dopamine antagonist and daughters treated with dopamine had more head-to-head interactions than the controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), rather than the fewer interactions that were expected. However, no other individual tolerant behavior showed a significant shift. Tolerant behaviors can be rather subtle, therefore, any shifts induced by treatment could have been missed.\u003c/p\u003e\u003cp\u003eAs with most tolerant behaviors, those categorized as aggressive (biting, nudging), also did not respond to dopamine manipulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Such interactions are easier to spot than tolerant behaviors, therefore, it is unlikely that they were miscounted. This result was unexpected when considering previous studies investigating the link of biogenic amines to behaviors within Hymenoptera. In workers of \u003cem\u003eFormica polycenta\u003c/em\u003e, dopamine treatment stimulated more threatening behaviors and overall aggression (Szczuka et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). However, in that study they utilized the injection delivery method instead of topical application, which could explain the difference in their results compared to this study. It is also possible that the concentration of dopamine blocker may have been insufficient to induce a string behavioral response. Honeybees treated with various concentrations of the blocker flupentixol had widely varied responses depending on dose (Farkhary et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), which could indicate that the effect of blocking the dopamine receptors in \u003cem\u003eC. calcarata\u003c/em\u003e could have various effects on aggressive behaviors.\u003c/p\u003e\u003cp\u003eIt was expected that mothers treated with the dopamine blocker would increase avoidant behaviors compared to untreated mothers. Receiving a dopamine blocker was expected to make mothers less aggressive while daughters treated with DA were expected to be more aggressive, creating increased opportunities for mothers to be avoidant. However, there was no significant difference found in the frequency of avoidant behaviors across treatment groups, likely due to the lack of observable change in daughter aggression. The lack of impact that dopamine manipulation had on aggressive behavior in \u003cem\u003eC. ceratina\u003c/em\u003e, may also indicate that another neurotransmitter may be responsible for modulating such behaviors. Octopamine and tyramine were also elevated in mothers compared to their daughters. Octopamine seems the likeliest candidate, given that is has been implicated in the aggressive behaviors and motivational states in a number of invertebrate species. Within invertebrates, octopamine has the ability to induce and modulate signal transduction pathways like norepinephrine in vertebrates (Farooqui \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Octopamine levels in the hemolymph have been shown to trigger a \u0026ldquo;fight or flight\u0026rdquo; reaction in locusts (Malamud et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Adamo et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). High octopamine concentrations are accompanied by an aggressive behavior, resulting in dominance (Roeder et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) and influencing division of labor in \u003cem\u003eApis mellifera\u003c/em\u003e (Schulz et al. 2001). Future work investigating the role octopamine plays in modulating behaviours within \u003cem\u003eC. calcarata\u003c/em\u003e would be an insightful next step in understanding the underlying influence biogenic amines have on social behaviours.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, \u003cem\u003eC. calcarata\u003c/em\u003e mothers have significantly elevated levels of multiple biogenic amines (octopamine, dopamine and tyramine) compared to daughters. When examining the role dopamine plays in the dominance hierarchy of \u003cem\u003eC. calcarata\u003c/em\u003e mothers and DEDs, there was an observable shift in head-to-head tolerant behaviors within the treatment group. Further work is needed to determine if the carrier also had an impact on these behaviors or biogenic amine alone. This study provides evidence for a role of the relationship between biogenic amines and dominance behaviors within a facultatively social species and future studies on the effects of other biogenic amines and other facultatively social species is needed.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflict of interest:\u003c/h2\u003e\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eFunding was made possible by internal grants from Quinnipiac University. The funding source had no role in study design, data collection, data analysis, data interpretation, or writing of the manuscript.\u003c/p\u003e\u003ch2\u003eAuthor contributions:\u003c/h2\u003e\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by RM and SL. The first draft of the manuscript was written by RM and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement:\u003c/h2\u003e\u003cp\u003eWe would like to thank Lisa Kaplan, Tom Torello and Alexandre de Lencastre for their helpful insights on experimental design and feedback on the manuscript. RM was supported by the Quinnipiac University Interdisciplinary Program for Research and Scholarship (QUIP-RS), which provided feedback and support. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.\u003c/p\u003e\u003ch2\u003eData availability:\u003c/h2\u003e\u003cp\u003eThe data that supported these findings has been uploaded to Figshare: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://figshare.com/s/1364e85987a50496b52c\u003c/span\u003e\u003cspan address=\"https://figshare.com/s/1364e85987a50496b52c\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdamo SA, Linn CE, Hoy RR (1995) The role of neurohormonal octopamine during fight or flight behavior in the field cricket \u003cem\u003eGryllus bimaculatus\u003c/em\u003e. J Exp Biol 198:1691\u0026ndash;1700\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlekseyenko OV, Chan Y, Li R, Kravitz EA (2013) Single dopaminergic neurons that modulate aggression in \u003cem\u003eDrosophila\u003c/em\u003e. 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Behaviour 153(12):1365\u0026ndash;1385. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1163/1568539X-00003382\u003c/span\u003e\u003cspan address=\"10.1163/1568539X-00003382\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"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":"Division of labor, dopamine, facultatively social bee, Apidae","lastPublishedDoi":"10.21203/rs.3.rs-7528609/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7528609/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBiogenic amines are highly conserved across animal species and aid in the regulation of movement, behavior, motivation, temperature, blood pressure, and endocrine secretion. Within eusocial species, such as the honey bee \u003cem\u003eApis mellifera\u003c/em\u003e, biogenic amines are responsible for modulating traits associated with higher levels of sociality, such as division of labor. In \u003cem\u003eCeratina calcarata\u003c/em\u003e, a facultatively social bee species, mothers establish a dominance hierarchy over their eldest by providing them with less pollen and less protein, resulting in smaller, more submissive adults referred to as dwarf eldest daughters (DEDs). We hypothesized that this dominance hierarchy is additionally modulated by biogenic amines. To examine this hypothesis, we first compared biogenic amine levels between mothers and daughters and found significantly higher levels of dopamine, octopamine and tyramine in the former group. To quantify the effects of these biogenic amine differences, dopamine was topically administered to \u003cem\u003eC. calcarata\u003c/em\u003e DEDs, while the mothers were topically treated with the dopamine blocker cis-(Z)-Flupentixol dihydrochloride. Aggression helps to establish social hierarchies, so mothers and DEDs from the same nest underwent behavioral assays to assess aggressive and tolerant behaviors. When dopamine was blocked in the naturally more aggressive mothers, though there was no difference in the number of aggressive interactions, treated mothers became more tolerant of DEDs, indicating dopamine plays a key role in modulating these behaviors. More work is needed to understand the different roles each biogenic amine plays in the development of a dominance hierarchy, especially in these species on the brink of eusociality.\u003c/p\u003e","manuscriptTitle":"Investigating the effects of biogenic amines on the dominance hierarchy in the small carpenter bee, Ceratina calcarata","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-15 18:21:07","doi":"10.21203/rs.3.rs-7528609/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revisions Needed","date":"2025-10-31T21:05:01+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-09-12T08:43:55+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-06T02:42:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-04T08:18:29+00:00","index":"","fulltext":""},{"type":"submitted","content":"Insectes Sociaux","date":"2025-09-03T11:28:27+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":"c9bf37e7-c52e-4328-a9a2-b1737d76980c","owner":[],"postedDate":"September 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-23T16:05:59+00:00","versionOfRecord":{"articleIdentity":"rs-7528609","link":"https://doi.org/10.1007/s00040-026-01086-w","journal":{"identity":"insectes-sociaux","isVorOnly":false,"title":"Insectes Sociaux"},"publishedOn":"2026-03-16 15:59:00","publishedOnDateReadable":"March 16th, 2026"},"versionCreatedAt":"2025-09-15 18:21:07","video":"","vorDoi":"10.1007/s00040-026-01086-w","vorDoiUrl":"https://doi.org/10.1007/s00040-026-01086-w","workflowStages":[]},"version":"v1","identity":"rs-7528609","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7528609","identity":"rs-7528609","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}