Worker body size drives cuticle thickness and intensity of positive reaction for lipids in the leaf-cutting ants Atta laevigata and Atta sexdens

Worker body size drives cuticle thickness and intensity of positive reaction for lipids in the leaf-cutting ants Atta laevigata and Atta sexdens | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Worker body size drives cuticle thickness and intensity of positive reaction for lipids in the leaf-cutting ants Atta laevigata and Atta sexdens Iasmin Frossard, Kênia Aparecida dos Santos Mateus, Karina Dias Amaral, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9546263/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract The body size of insects influences the surface area-volume ratio and can affect the thickness of the cuticle and the amount of cuticular lipids, particularly in eusocial species, such as leaf-cutting ants of the genus Atta , which exhibit pronounced worker polymorphism. In this study, we evaluated the relationship between body size, cuticular lipid intensity, and cuticle thickness in workers of Atta laevigata (Smith, 1858) (Hymenoptera: Formicidae) and Atta sexdens (Linnaeus, 1758) (Hymenoptera: Formicidae). Small, medium, and large workers, defined according to the width of the cephalic capsule, were compared regarding relative cuticle thickness and cuticular lipid intensity. Both traits varied among worker size classes. Small workers showed higher cuticular lipid intensity and greater relative cuticle thickness, while medium and large workers exhibited lower values for both traits, indicating that different cuticle components respond similarly to body size, regardless of the species. Although the intensity of cuticular lipids was not differentiated between species, cuticle thickness was consistently greater in A. sexdens than in A. laevigata in all size classes. Overall, our findings suggest that the different components of the cuticle of A. laevigata and A. sexdens are not determined solely by body size, but also reflect the specific function performed by the workers within the colony. polymorphism subcastes surface-to-volume ratio water balance Figures Figure 1 Figure 2 Figure 3 INTRODUCTION Leaf-cutting ants exhibit a highly organized collective foraging system, in which worker ants explore the environment in search of plant material, cutting and transporting this material to the interior of the colony, where it will be used to cultivate the symbiotic fungus (Della Lucia, 2011 ). The performance of the worker ants during foraging is crucial, as it directly influences the quantity and quality of the collected material and, consequently, the growth of the fungus and the maintenance of the colony (Bass & Cherrett, 1995 ). This performance depends, among other factors, on the body size of the worker ants, which influences both the size of the load they can carry and the speed of movement, as well as determining how these individuals respond to the thermal conditions of the external environment (Kalinkat et al., 2015 ; Kühsel, 2017). The body size of insects directly influences important physiological processes, including thermoregulation, gas exchange, and water balance (Bouchebti, 2015; Buxton, 2021). Smaller insects have a higher surface area to volume ratio, which proportionally increases the body area exposed to the environment (Chown, 2011; Kühsel et al., 2017 ). Consequently, they gain and lose heat more rapidly in response to ambient temperature and humidity. Elevated body temperatures accelerate metabolic activity and gas exchange rates, leading to greater water loss through the cuticle and tracheal spiracles (O'Donnell, 2022). In contrast, larger insects, with a smaller surface area to volume ratio (Kühsel et al., 2017 ), warm up more slowly, perform gas exchange less frequently, and lose proportionally less water, making them less susceptible to dehydration (Bouchebti, 2015; Brückner, 2017). The main barrier against water loss in insects is the cuticle (Chown, 2011), which is composed of three distinct layers. The epicuticle, the outermost layer, is rich in lipids, particularly cuticular hydrocarbons, which form an effective barrier, reducing water loss, protecting against pathogens, and mediating intra- and interspecific communication in social insects (Howard & Blomquist, 2005 ). Below this layer, the exocuticle and endocuticle are composed mainly of chitin and associated proteins, providing both rigidity and flexibility to the exoskeleton (Wigglesworth, 1948 ). The amount of chitin, proteins, and lipids in the insect cuticle is directly related to body size, since larger insects require greater amounts of cuticular material to cover a proportionally larger surface area (Evans & Sanson, 2005 ). Despite this, relatively little is known about how body size influences other cuticle attributes, such as thickness and intensity of cuticular lipids, especially in eusocial species where the size of individuals is often associated with function in the colony, imposing additional selective pressures on the cuticle (Peeters et al., 2017 ). Leaf-cutting ants of the genus Atta constitute an interesting model for studying the interaction between body size and cuticular characteristics. In addition to the differences between reproductive and non-reproductive castes, the worker caste is subdivided into several size categories, associated with different behavioral roles and levels of environmental exposure (Hölldobler & Wilson, 1990; Peeters et al., 2017 ). Medium-sized (≈ 1.4 mm) and large (≈ 2.0–2.2 mm) workers are predominantly involved in foraging and nest defense, activities that expose them to adverse external conditions such as low humidity and high temperatures (Santos, 2022; Püffel et al., 2023 ). Their larger body size reduces the surface area to volume ratio, making them less prone to dehydration (Kühsel et al., 2017 ). In contrast, smaller workers (≈ 0.8–1.0 mm), which primarily perform internal colony tasks, including maintaining the fungus garden, building the nest, and caring for the brood (Santos, 2022; Püffel et al., 2023 ), have a larger surface area to volume ratio and are therefore more vulnerable to water loss (Kühsel et al., 2017 ). We could suggest that larger workers already possess thermal advantages due to greater thermal inertia, making it more advantageous for the colony to redirect cuticular investment to more vulnerable individuals. In this context, this study aimed to evaluate the relationship between body size and cuticular lipid intensity in worker ants of the leaf-cutter ants Atta laevigata (Smith, 1858) and Atta sexdens (Linnaeus, 1758). We investigated both intraspecific variation among small, medium, and large workers and interspecific differences. Given that both species coexist in the same environment but differ in their foraging activity patterns, we expect that smaller workers of both species exhibit thicker cuticles and a higher intensity of positive reaction to lipids in the cuticle than medium and large workers, due to their higher surface area-volume ratio and greater susceptibility to water loss. We hypothesize that A. laevigata exhibits greater cuticle thickness and higher cuticular lipid content than A. sexdens , reflecting its predominantly diurnal foraging behavior. MATERIALS AND METHODS Collection of ant workers Workers from three colonies of A. laevigata and three colonies of A. sexdens were collected from nests located in an area of shaded native vegetation, in the municipality of Viçosa, Minas Gerais state, Brazil (20°45′ S, 42°52′ W). The sizes of A. laevigata nests were 21.3 m², 34.0 m², and 40.6 m² of loose soil, whereas the A. sexdens nests measured 20.5 m², 23.8 m², and 42.4 m². Size of workers A total of 24 workers per size class were selected for each species, comprising eight workers from each of the three colonies. Thus, 24 small, medium, and large workers were analyzed per species workers were photographed using a camera coupled to a Zeiss Stemi 2000 C stereomicroscope. The images were used to measure head capsule width, defined as the maximum distance across the head, using ImageJ software, which was adopted as the criterion for size classification (Wilson, 1980 ). Workers with head capsule widths between 0.8 and 1.0 mm were classified as small, those between 1.4 and 1.6 mm as medium, and those between 2.0 and 2.2 mm as large, following Wilson ( 1980 ) and Santos (2022). Intensity of cuticular lipids and cuticle thickness For each species, 24 workers per size class (small, medium, and large) were selected, comprising eight workers from each of the three colonies. The pronotum was carefully removed using scissors and forceps. The pronotum is commonly used in studies of ant cuticle thickness because it represents a morphologically uniform region, allowing for reliable comparisons among individuals (Peeters et al., 2017 ). After dissection, the pronotum was transferred to 4% paraformaldehyde in 0.15 M sodium phosphate buffer (pH 7.2) fixative solution for 24 h. The samples were then rinsed in the same buffer, dehydrated through a graded ethanol series (70%, 80%, 90%, and 95%), and embedded in glycol methacrylate historesin (Leica Biosystems Inc.) according to the manufacturer’s instructions. Sections 2 µm thick were obtained using a Leica RM2255 motorized microtome and stained with Nile blue for the detection of total lipids (Bancroft & Gamble, 2008 ). The sections were mounted, examined, and photographed using an Olympus BX60 light microscope equipped with a Q-Color digital camera. All images were obtained using 20x and 0.40 numerical aperture objective lens under identical light intensity and exposure settings to ensure consistency among samples. Morphometry Images of pronotum slices from each worker were analyzed using ImageJ software to quantify cuticular lipid intensity, following a standardized protocol based on the intensity of the Nile blue histochemical reaction. Briefly, images of the pronotum cuticle were converted to 32-bit grayscale and inverted to negative. In each image, five non-overlapping areas with standardized dimensions (64 × 64 pixels) were selected in equivalent regions of the cuticle. The mean gray value obtained from these regions was used to calculate the average lipid reaction intensity for each individual, serving as a proxy for cuticular lipid intensity (Urstadt, 2022 ). Cuticle thickness was measured in the same images by drawing a line perpendicular to the cuticular surface, from the outer edge to the basal boundary of the cuticle (Fig. 1 ). To allow comparisons among workers of different sizes, both cuticle thickness and relative cuticular lipid intensity were standardized by body size. Values were divided by head capsule width (Whyte, 2023), used as a proxy for body length (Weiser, 2006 ). This procedure yielded estimates of relative cuticle thickness (µm of cuticle per mm of head capsule) and relative cuticular lipid intensity. Statistical Analyses To evaluate the effects of worker size and species on relative cuticular lipid intensity and relative cuticle thickness, the data were analyzed using linear mixed-effects models (LMMs). In the models, worker size and species were treated as fixed effects, while nest identity was included as a random effect to account for spatial dependence and intrinsic autocorrelation in sampling units from the same colony. Models were fitted using the lme4 package, and the significance of fixed effects was assessed using Satterthwaite’s approximation for degrees of freedom implemented in the lmerTest package. Model assumptions were evaluated through the analysis of simulated residuals using the DHARMa package, ensuring compliance with assumptions of normality and homoscedasticity. This modeling framework allows the biological effects of interest to be isolated from environmental or genetic variation shared within colonies. All statistical analyses were conducted in R version 4.5.0 (R Core Team, 2025 ), adopting a significance level of 0.05. RESULTS The relative cuticular lipid intensity did not differ between species (F₁,₁₃₈ = 0.0157, p = 0.90), but varied between worker size classes in both species (F₂,₁₃₈ = 38.17, p = 6.41 × 10⁻¹⁴). Small workers of both species exhibited a higher intensity of cuticular lipids than medium and large workers, which did not differ from each other (Fig. 2 ). The relative cuticle thickness varied between species (F₁,₁₃₈ = 85.363, p < 2.2 × 10⁻¹⁶) and between worker size classes (F₂,₁₃₈ = 74.857, p < 2.2 × 10⁻¹⁶). In all size classes, Atta laevigata showed lower relative cuticle thickness values ​​than Atta sexdens (p < 0.0001). Furthermore, in both species, the relative cuticle thickness decreased with worker size, with smaller workers showing the highest values, followed by medium and large workers, all differing significantly from each other (Fig. 3 ). DISCUSSION Worker ants of both species exhibited higher relative cuticular lipid intensity and greater relative cuticle thickness in smaller workers. This pattern is potentially associated with their lower thermal inertia and higher susceptibility to water loss (Kühsel et al., 2017 ). The increased amount of cuticular lipids may reduce cuticle permeability, thereby mitigating the greater water loss expected in smaller body sizes (Gibbs, 2010 ; O’Donnell, 2022 ). Similarly, a thicker cuticle in small workers may further contribute to water conservation by increasing the diffusion pathway of water through the exoskeleton (Hadley, 1994 ). Smaller workers of Atta cephalotes have been shown to possess relatively thicker cuticles than larger ones (Peeters et al., 2017 ; Constantino, 2021), supporting the hypothesis that cuticular traits scale adaptively with body size in leaf-cutter ants. In addition, an excessively thick cuticle may increase body mass and consequently raise the mechanical costs of locomotion and load transport. One study demonstrated that the mechanical work required for movement and load carrying increases with body mass in the harvester ant Messor barbarus (Merienne et al., 2021 ). These costs may be particularly relevant for medium- and large-sized workers, which routinely perform external tasks such as foraging and material transport (Santos, 2022; Püffel et al., 2023 ). On the other hand, cuticle thickness does not always follow this pattern across different ant species. In Dorylus orientalis (Dorylinae), Pheidole noda (Myrmicinae), and Cataglyphis bombycina (Formicinae), larger workers possess thicker cuticles (Peeters et al., 2017 ). Notably, C. bombycina forages during the hottest periods of the day in the Sahara Desert, and its larger workers, which are more active under these extreme conditions, exhibit higher thermal tolerance, allowing prolonged foraging activity (Willot, 2018). Although smaller workers of Atta spp. predominantly perform tasks inside the nest, they may occasionally engage in external activities and, due to their higher surface area-to-volume ratio, are inherently more vulnerable to water loss (Kühsel et al., 2017 ). This greater susceptibility likely favors increased investment in cuticular lipids and thicker cuticles, both of which function as effective barriers against desiccation. In contrast, larger workers are mainly associated with foraging and nest defense, activities that expose them to harsh environmental conditions such as low humidity and high temperatures (Helanterä, 2008 ; Püffel et al., 2023 ). However, their lower surface area-to-volume ratio reduces susceptibility to desiccation (Kühsel et al., 2017 ), which may explain the reduced investment in cuticle thickness and cuticular lipids in these workers of both studied species. Regarding interspecific differences, it was expected that A. laevigata would exhibit higher relative cuticular lipid intensity across all worker sizes compared to Atta sexdens , reflecting greater investment in cuticular lipids, as consequence of its predominantly diurnal foraging activity. However, despite being more active during the day, A. laevigata forages preferentially during milder periods, such as early morning and late afternoon (Viana et al., 2004), thereby reducing prolonged exposure to extreme environmental conditions. This activity pattern may decrease the need for increased investment in cuticular lipids, consequently reducing the metabolic costs associated with cuticle synthesis. Although the cuticle plays a central role in limiting water loss, it is a metabolically costly structure, as its formation requires substantial nitrogen investment for the synthesis of chitin and structural proteins derived from nitrogen-rich amino acids (Mullins et al., 2025 ). Therefore, the absence of interspecific differences in cuticular lipid content may reflect a resource allocation strategy in which metabolic resources are conserved or redirected to other physiological functions of the cuticle. In contrast, A. sexdens exhibited greater cuticle thickness across all size classes. Given the high metabolic cost associated with cuticle synthesis, it is plausible that the greater thickness observed in A. sexdens reflects higher resource availability at the colony level, allowing greater investment in metabolically expensive structures. In addition, although A. laevigata is more active during the day, it forages preferentially during milder periods such as early morning and late afternoon (Viana et al., 2004), thereby reducing prolonged exposure to extreme environmental conditions. This activity pattern may decrease the need for increased investment in cuticular lipids, consequently reducing the metabolic costs associated with cuticle synthesis. Moreover, its relatively thinner cuticle suggests that other cuticular attributes, such as ventilation patterns (Lighton, 1990, 1992; Perl & Niven, 2018; Oladipuppo et al., 2022), epicuticular lipid composition (Hood & Tschinkel, 1990), cuticular hydrocarbon profiles (Gibbs, 2002; Johnson & Gibbs, 2004; Ajayi et al., 2020), pilosity, sculpture (Buxton et al., 2021 ), or abrasion level (Johnson et al., 2011), may contribute to protection against water loss and other environmental pressures. The contrasting cuticular patterns observed across worker size classes and between the two species in the present study and in other ant species (Peeters et al., 2017 ; Willot, 2018; Birkenfeld, 2024) reinforce the hypothesis that cuticular protection in ants is not determined by a single morphological trait. Instead, it results from an integrated adjustment between cuticle thickness, lipid composition, and body size, which together function as adaptive solutions to the diverse environmental challenges faced by different species. CONCLUSION Small workers exhibit thicker cuticles and greater amounts of cuticular lipids than medium and large ones, suggesting an adaptive investment against water loss, even though they primarily perform internal tasks. Larger workers, despite being more exposed during foraging and nest defence, show lower susceptibility to desiccation due to their lower surface area-to-volume ratio, justifying reduced investment in cuticle and lipids. At the interspecific level, A. sexdens showed greater cuticle thickness across all size classes, possibly reflecting greater resource availability for the synthesis of metabolically costly structures, while A. laevigata appears to compensate for water balance protection through foraging behavior in milder periods of the day. These patterns indicate that cuticular morphology in Atta spp. is shaped by both physiological constraints and behavioral strategies, with ecological implications for task allocation, tolerance to extreme environmental conditions, and efficiency in resource exploitation. Declarations Ethical Approval Not applicable. Competing Interests The authors declare no conflict of interest. The authors confirm that there are no disputes over the ownership of the data presented in this article and that all contributions have been appropriately attributed. Data availability statement The data that support the findings of this study are available from the corresponding author upon reasonable request. Author Contributions Conceptualization, R.Z. and I.G.F.; methodology, J.E.S., R.Z. and I.G.F.; resources, R.Z. and J.E.S.; supervision, J.E.S. and R.Z.; investigation, I.G.F. and K.D.A.; formal analysis, I.G.F., K.A.S.M. and J.J.S.; data curation, C.L.J. and I.G.F.; writing—original draft preparation, I.G.F.; writing—review & editing, K.D.A., D.S.S., K.A.S.M., C.L.J., R.Z. and J.E.S.; visualization, E.B.A., K.A.S.M. and J.J.S. All authors have read and agreed to the published version of the manuscript.All authors read and approved the final version of the manuscript. Declarations Not applicable Funding This study was funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES/PROEX), grant number 1852/2023, process number 88881.893116/2023-01. References Bancroft JD, Gamble M (eds) (2008) Theory and Practice of Histological Techniques. Elsevier Health Sciences Bass M, Cherrett JM (1995) Fungal hyphae as a source of nutrients for the leaf-cutting ant Atta sexdens. 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Neotropical Entomology , 33, 391–393. https://doi.org/10.1590/S1519-566X2004-000300019 Weiser MD, Kaspari M (2006) Ecological morphospace of New World ants. Ecol Entomol 31(2):131–142 Whyte BA, Sandidge R, Buellesbach J, Cash EI, Scheckel KJ, Gibson JD, Tsutsui ND (2023) The role of body size and cuticular hydrocarbons in the desiccation resistance of invasive Argentine ants (Linepithema humile). J Exp Biol 226(16):jeb245578 Wigglesworth VB (1948) The insect cuticle. Biol Rev 23(4):408–451. https://doi.org/10.1111/j.1469-185X.1948.tb00566 Wilson EO (1980) Caste and division of labor in leaf-cutter ants (Hymenoptera: Formicidae: Atta) II. The ergonomic optimization of leaf cutting. Behav Ecol Sociobiol 7(2):157–165 Willot Q, Mardulyn P, Defrance M, Gueydan C, Aron S (2018) Molecular chaperoning helps safeguarding mitochondrial integrity and motor functions in the Sahara silver ant Cataglyphis bombycina . 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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-9546263","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":635643836,"identity":"b02d40dc-79c2-492f-8b2e-2b82e5339c05","order_by":0,"name":"Iasmin Frossard","email":"data:image/png;base64,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","orcid":"https://orcid.org/0009-0008-6362-9590","institution":"Universidade Federal de Lavras Departamento de Entomologia","correspondingAuthor":true,"prefix":"","firstName":"Iasmin","middleName":"","lastName":"Frossard","suffix":""},{"id":635643837,"identity":"36649a9f-3a21-4884-bd72-6a676af9af86","order_by":1,"name":"Kênia Aparecida dos Santos Mateus","email":"","orcid":"","institution":"UFLA: Universidade Federal de Lavras","correspondingAuthor":false,"prefix":"","firstName":"Kênia","middleName":"Aparecida dos Santos","lastName":"Mateus","suffix":""},{"id":635643838,"identity":"5a98f121-c208-45d4-a0b0-66e7f9168856","order_by":2,"name":"Karina Dias Amaral","email":"","orcid":"","institution":"UFV: Universidade Federal de Vicosa","correspondingAuthor":false,"prefix":"","firstName":"Karina","middleName":"Dias","lastName":"Amaral","suffix":""},{"id":635643839,"identity":"f1bdf163-c7f0-44dc-b701-29193d75c727","order_by":3,"name":"Diego dos Santos Souza","email":"","orcid":"","institution":"UFV: Universidade Federal de Vicosa","correspondingAuthor":false,"prefix":"","firstName":"Diego","middleName":"dos Santos","lastName":"Souza","suffix":""},{"id":635643840,"identity":"1ca5b8d9-1b2b-4bd8-8521-afbc1beb0fd1","order_by":4,"name":"Jéssica Josefa Sanches","email":"","orcid":"","institution":"UFLA: Universidade Federal de Lavras","correspondingAuthor":false,"prefix":"","firstName":"Jéssica","middleName":"Josefa","lastName":"Sanches","suffix":""},{"id":635643841,"identity":"d69113c3-8ac3-43e2-ba46-bf0218b3b79c","order_by":5,"name":"Catarina de Lima","email":"","orcid":"","institution":"UFLA: Universidade Federal de Lavras","correspondingAuthor":false,"prefix":"","firstName":"Catarina","middleName":"","lastName":"de Lima","suffix":""},{"id":635643842,"identity":"eda7c30c-3b38-41ea-82c3-e12ab9e830cd","order_by":6,"name":"Elane Borba Alves","email":"","orcid":"","institution":"UFV: Universidade Federal de Vicosa","correspondingAuthor":false,"prefix":"","firstName":"Elane","middleName":"Borba","lastName":"Alves","suffix":""},{"id":635643843,"identity":"8b9b45c2-1aeb-42d4-967a-a39ebd1f5315","order_by":7,"name":"José Eduardo Serrão","email":"","orcid":"","institution":"UFV: Universidade Federal de Vicosa","correspondingAuthor":false,"prefix":"","firstName":"José","middleName":"Eduardo","lastName":"Serrão","suffix":""},{"id":635643844,"identity":"5fa988e0-3317-40db-b409-f4e40a62b6d1","order_by":8,"name":"Ronald Zanetti","email":"","orcid":"","institution":"UFLA: Universidade Federal de Lavras","correspondingAuthor":false,"prefix":"","firstName":"Ronald","middleName":"","lastName":"Zanetti","suffix":""}],"badges":[],"createdAt":"2026-04-27 22:05:59","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9546263/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9546263/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109317638,"identity":"34ae08ce-85ef-4269-aa96-1438278e8f2c","added_by":"auto","created_at":"2026-05-15 12:50:47","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":97002,"visible":true,"origin":"","legend":"\u003cp\u003eTransverse histological sections of the pronotum of small, medium, and large workers of \u003cem\u003eAtta laevigata\u003c/em\u003e (A–C) and \u003cem\u003eAtta sexdens\u003c/em\u003e (D–F). (A, D) large workers; (B, E) medium workers; (C, F) small workers. The images indicate the region used for measuring cuticle thickness and Cuticular lipid intensity. Cuticle thickness was determined by drawing a line perpendicular to the cuticular surface, from the external edge to the base of the cuticle. Relative cuticular lipid intensity was quantified in standardized areas of the same histological section. Scale bars = 50 µm.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9546263/v1/c1a4cc03b37b1666959663d2.jpg"},{"id":109317637,"identity":"341be72a-f39d-4946-b293-401930885b50","added_by":"auto","created_at":"2026-05-15 12:50:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":70249,"visible":true,"origin":"","legend":"\u003cp\u003eRelative cuticular lipid intensity (values ​​proportional to head capsule size) of \u003cem\u003eAtta laevigata\u003c/em\u003e (dark gray) and \u003cem\u003eAtta sexdens\u003c/em\u003e (light gray) workers in three size classes (small, medium, and large). Bars represent means ± standard errors. Different capital letters indicate significant differences between worker size classes within each species (multiple comparison test, p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9546263/v1/07b9f61edfc4a99bd045b723.png"},{"id":109317639,"identity":"49da481e-f65f-4e4e-846f-85ac43d16895","added_by":"auto","created_at":"2026-05-15 12:50:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":32836,"visible":true,"origin":"","legend":"\u003cp\u003eRelative cuticle thickness (values proportional to head capsule size) of \u003cem\u003eAtta laevigata \u003c/em\u003eand\u003cem\u003e Atta sexdens\u003c/em\u003e workers in three size classes (small, medium, and large). The bars represent the means ± standard errors. Capital letters indicate significant differences between sizes within each species; small letters indicate differences between species within the same size (multiple comparison test, p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9546263/v1/3d71971f91bbfb3d8a2c7c75.png"},{"id":109317641,"identity":"33ff3b29-b08f-4324-90c5-8525320bc123","added_by":"auto","created_at":"2026-05-15 12:50:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":375240,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9546263/v1/c018c514-2872-4c48-a5ae-d766f1c9dc6a.pdf"}],"financialInterests":"","formattedTitle":"Worker body size drives cuticle thickness and intensity of positive reaction for lipids in the leaf-cutting ants Atta laevigata and Atta sexdens","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eLeaf-cutting ants exhibit a highly organized collective foraging system, in which worker ants explore the environment in search of plant material, cutting and transporting this material to the interior of the colony, where it will be used to cultivate the symbiotic fungus (Della Lucia, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The performance of the worker ants during foraging is crucial, as it directly influences the quantity and quality of the collected material and, consequently, the growth of the fungus and the maintenance of the colony (Bass \u0026amp; Cherrett, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). This performance depends, among other factors, on the body size of the worker ants, which influences both the size of the load they can carry and the speed of movement, as well as determining how these individuals respond to the thermal conditions of the external environment (Kalinkat et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; K\u0026uuml;hsel, 2017).\u003c/p\u003e \u003cp\u003eThe body size of insects directly influences important physiological processes, including thermoregulation, gas exchange, and water balance (Bouchebti, 2015; Buxton, 2021). Smaller insects have a higher surface area to volume ratio, which proportionally increases the body area exposed to the environment (Chown, 2011; K\u0026uuml;hsel et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Consequently, they gain and lose heat more rapidly in response to ambient temperature and humidity. Elevated body temperatures accelerate metabolic activity and gas exchange rates, leading to greater water loss through the cuticle and tracheal spiracles (O'Donnell, 2022). In contrast, larger insects, with a smaller surface area to volume ratio (K\u0026uuml;hsel et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), warm up more slowly, perform gas exchange less frequently, and lose proportionally less water, making them less susceptible to dehydration (Bouchebti, 2015; Br\u0026uuml;ckner, 2017).\u003c/p\u003e \u003cp\u003eThe main barrier against water loss in insects is the cuticle (Chown, 2011), which is composed of three distinct layers. The epicuticle, the outermost layer, is rich in lipids, particularly cuticular hydrocarbons, which form an effective barrier, reducing water loss, protecting against pathogens, and mediating intra- and interspecific communication in social insects (Howard \u0026amp; Blomquist, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Below this layer, the exocuticle and endocuticle are composed mainly of chitin and associated proteins, providing both rigidity and flexibility to the exoskeleton (Wigglesworth, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1948\u003c/span\u003e). The amount of chitin, proteins, and lipids in the insect cuticle is directly related to body size, since larger insects require greater amounts of cuticular material to cover a proportionally larger surface area (Evans \u0026amp; Sanson, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Despite this, relatively little is known about how body size influences other cuticle attributes, such as thickness and intensity of cuticular lipids, especially in eusocial species where the size of individuals is often associated with function in the colony, imposing additional selective pressures on the cuticle (Peeters et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLeaf-cutting ants of the genus \u003cem\u003eAtta\u003c/em\u003e constitute an interesting model for studying the interaction between body size and cuticular characteristics. In addition to the differences between reproductive and non-reproductive castes, the worker caste is subdivided into several size categories, associated with different behavioral roles and levels of environmental exposure (H\u0026ouml;lldobler \u0026amp; Wilson, 1990; Peeters et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Medium-sized (\u0026asymp;\u0026thinsp;1.4 mm) and large (\u0026asymp;\u0026thinsp;2.0\u0026ndash;2.2 mm) workers are predominantly involved in foraging and nest defense, activities that expose them to adverse external conditions such as low humidity and high temperatures (Santos, 2022; P\u0026uuml;ffel et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Their larger body size reduces the surface area to volume ratio, making them less prone to dehydration (K\u0026uuml;hsel et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In contrast, smaller workers (\u0026asymp;\u0026thinsp;0.8\u0026ndash;1.0 mm), which primarily perform internal colony tasks, including maintaining the fungus garden, building the nest, and caring for the brood (Santos, 2022; P\u0026uuml;ffel et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), have a larger surface area to volume ratio and are therefore more vulnerable to water loss (K\u0026uuml;hsel et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). We could suggest that larger workers already possess thermal advantages due to greater thermal inertia, making it more advantageous for the colony to redirect cuticular investment to more vulnerable individuals.\u003c/p\u003e \u003cp\u003eIn this context, this study aimed to evaluate the relationship between body size and cuticular lipid intensity in worker ants of the leaf-cutter ants \u003cem\u003eAtta laevigata\u003c/em\u003e (Smith, 1858) and \u003cem\u003eAtta sexdens\u003c/em\u003e (Linnaeus, 1758). We investigated both intraspecific variation among small, medium, and large workers and interspecific differences. Given that both species coexist in the same environment but differ in their foraging activity patterns, we expect that smaller workers of both species exhibit thicker cuticles and a higher intensity of positive reaction to lipids in the cuticle than medium and large workers, due to their higher surface area-volume ratio and greater susceptibility to water loss. We hypothesize that \u003cem\u003eA. laevigata\u003c/em\u003e exhibits greater cuticle thickness and higher cuticular lipid content than \u003cem\u003eA. sexdens\u003c/em\u003e, reflecting its predominantly diurnal foraging behavior.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCollection of ant workers\u003c/h2\u003e \u003cp\u003eWorkers from three colonies of \u003cem\u003eA. laevigata\u003c/em\u003e and three colonies of \u003cem\u003eA. sexdens\u003c/em\u003e were collected from nests located in an area of shaded native vegetation, in the municipality of Vi\u0026ccedil;osa, Minas Gerais state, Brazil (20\u0026deg;45\u0026prime; S, 42\u0026deg;52\u0026prime; W). The sizes of \u003cem\u003eA. laevigata\u003c/em\u003e nests were 21.3 m\u0026sup2;, 34.0 m\u0026sup2;, and 40.6 m\u0026sup2; of loose soil, whereas the \u003cem\u003eA. sexdens\u003c/em\u003e nests measured 20.5 m\u0026sup2;, 23.8 m\u0026sup2;, and 42.4 m\u0026sup2;.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSize of workers\u003c/h3\u003e\n\u003cp\u003eA total of 24 workers per size class were selected for each species, comprising eight workers from each of the three colonies. Thus, 24 small, medium, and large workers were analyzed per species workers were photographed using a camera coupled to a Zeiss Stemi 2000 C stereomicroscope. The images were used to measure head capsule width, defined as the maximum distance across the head, using ImageJ software, which was adopted as the criterion for size classification (Wilson, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1980\u003c/span\u003e). Workers with head capsule widths between 0.8 and 1.0 mm were classified as small, those between 1.4 and 1.6 mm as medium, and those between 2.0 and 2.2 mm as large, following Wilson (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1980\u003c/span\u003e) and Santos (2022).\u003c/p\u003e\n\u003ch3\u003eIntensity of cuticular lipids and cuticle thickness\u003c/h3\u003e\n\u003cp\u003eFor each species, 24 workers per size class (small, medium, and large) were selected, comprising eight workers from each of the three colonies. The pronotum was carefully removed using scissors and forceps. The pronotum is commonly used in studies of ant cuticle thickness because it represents a morphologically uniform region, allowing for reliable comparisons among individuals (Peeters et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). After dissection, the pronotum was transferred to 4% paraformaldehyde in 0.15 M sodium phosphate buffer (pH 7.2) fixative solution for 24 h. The samples were then rinsed in the same buffer, dehydrated through a graded ethanol series (70%, 80%, 90%, and 95%), and embedded in glycol methacrylate historesin (Leica Biosystems Inc.) according to the manufacturer\u0026rsquo;s instructions. Sections 2 \u0026micro;m thick were obtained using a Leica RM2255 motorized microtome and stained with Nile blue for the detection of total lipids (Bancroft \u0026amp; Gamble, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The sections were mounted, examined, and photographed using an Olympus BX60 light microscope equipped with a Q-Color digital camera. All images were obtained using 20x and 0.40 numerical aperture objective lens under identical light intensity and exposure settings to ensure consistency among samples.\u003c/p\u003e\n\u003ch3\u003eMorphometry\u003c/h3\u003e\n\u003cp\u003eImages of pronotum slices from each worker were analyzed using ImageJ software to quantify cuticular lipid intensity, following a standardized protocol based on the intensity of the Nile blue histochemical reaction. Briefly, images of the pronotum cuticle were converted to 32-bit grayscale and inverted to negative. In each image, five non-overlapping areas with standardized dimensions (64 \u0026times; 64 pixels) were selected in equivalent regions of the cuticle. The mean gray value obtained from these regions was used to calculate the average lipid reaction intensity for each individual, serving as a proxy for cuticular lipid intensity (Urstadt, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCuticle thickness was measured in the same images by drawing a line perpendicular to the cuticular surface, from the outer edge to the basal boundary of the cuticle (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). To allow comparisons among workers of different sizes, both cuticle thickness and relative cuticular lipid intensity were standardized by body size. Values were divided by head capsule width (Whyte, 2023), used as a proxy for body length (Weiser, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). This procedure yielded estimates of relative cuticle thickness (\u0026micro;m of cuticle per mm of head capsule) and relative cuticular lipid intensity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eStatistical Analyses\u003c/h3\u003e\n\u003cp\u003eTo evaluate the effects of worker size and species on relative cuticular lipid intensity and relative cuticle thickness, the data were analyzed using linear mixed-effects models (LMMs). In the models, worker size and species were treated as fixed effects, while nest identity was included as a random effect to account for spatial dependence and intrinsic autocorrelation in sampling units from the same colony. Models were fitted using the \u003cem\u003elme4\u003c/em\u003e package, and the significance of fixed effects was assessed using Satterthwaite\u0026rsquo;s approximation for degrees of freedom implemented in the \u003cem\u003elmerTest\u003c/em\u003e package. Model assumptions were evaluated through the analysis of simulated residuals using the \u003cem\u003eDHARMa\u003c/em\u003e package, ensuring compliance with assumptions of normality and homoscedasticity. This modeling framework allows the biological effects of interest to be isolated from environmental or genetic variation shared within colonies. All statistical analyses were conducted in R version 4.5.0 (R Core Team, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), adopting a significance level of 0.05.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eThe relative cuticular lipid intensity did not differ between species (F₁,₁₃₈ = 0.0157, p\u0026thinsp;=\u0026thinsp;0.90), but varied between worker size classes in both species (F₂,₁₃₈ = 38.17, p\u0026thinsp;=\u0026thinsp;6.41 \u0026times; 10⁻\u0026sup1;⁴). Small workers of both species exhibited a higher intensity of cuticular lipids than medium and large workers, which did not differ from each other (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe relative cuticle thickness varied between species (F₁,₁₃₈ = 85.363, p\u0026thinsp;\u0026lt;\u0026thinsp;2.2 \u0026times; 10⁻\u0026sup1;⁶) and between worker size classes (F₂,₁₃₈ = 74.857, p\u0026thinsp;\u0026lt;\u0026thinsp;2.2 \u0026times; 10⁻\u0026sup1;⁶). In all size classes, Atta laevigata showed lower relative cuticle thickness values ​​than Atta sexdens (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Furthermore, in both species, the relative cuticle thickness decreased with worker size, with smaller workers showing the highest values, followed by medium and large workers, all differing significantly from each other (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eWorker ants of both species exhibited higher relative cuticular lipid intensity and greater relative cuticle thickness in smaller workers. This pattern is potentially associated with their lower thermal inertia and higher susceptibility to water loss (K\u0026uuml;hsel et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The increased amount of cuticular lipids may reduce cuticle permeability, thereby mitigating the greater water loss expected in smaller body sizes (Gibbs, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; O\u0026rsquo;Donnell, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Similarly, a thicker cuticle in small workers may further contribute to water conservation by increasing the diffusion pathway of water through the exoskeleton (Hadley, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Smaller workers of \u003cem\u003eAtta cephalotes\u003c/em\u003e have been shown to possess relatively thicker cuticles than larger ones (Peeters et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Constantino, 2021), supporting the hypothesis that cuticular traits scale adaptively with body size in leaf-cutter ants. In addition, an excessively thick cuticle may increase body mass and consequently raise the mechanical costs of locomotion and load transport. One study demonstrated that the mechanical work required for movement and load carrying increases with body mass in the harvester ant \u003cem\u003eMessor barbarus\u003c/em\u003e (Merienne et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These costs may be particularly relevant for medium- and large-sized workers, which routinely perform external tasks such as foraging and material transport (Santos, 2022; P\u0026uuml;ffel et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). On the other hand, cuticle thickness does not always follow this pattern across different ant species. In \u003cem\u003eDorylus orientalis\u003c/em\u003e (Dorylinae), \u003cem\u003ePheidole noda\u003c/em\u003e (Myrmicinae), and \u003cem\u003eCataglyphis bombycina\u003c/em\u003e (Formicinae), larger workers possess thicker cuticles (Peeters et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Notably, \u003cem\u003eC. bombycina\u003c/em\u003e forages during the hottest periods of the day in the Sahara Desert, and its larger workers, which are more active under these extreme conditions, exhibit higher thermal tolerance, allowing prolonged foraging activity (Willot, 2018).\u003c/p\u003e \u003cp\u003eAlthough smaller workers of \u003cem\u003eAtta\u003c/em\u003e spp. predominantly perform tasks inside the nest, they may occasionally engage in external activities and, due to their higher surface area-to-volume ratio, are inherently more vulnerable to water loss (K\u0026uuml;hsel et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This greater susceptibility likely favors increased investment in cuticular lipids and thicker cuticles, both of which function as effective barriers against desiccation. In contrast, larger workers are mainly associated with foraging and nest defense, activities that expose them to harsh environmental conditions such as low humidity and high temperatures (Helanter\u0026auml;, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; P\u0026uuml;ffel et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, their lower surface area-to-volume ratio reduces susceptibility to desiccation (K\u0026uuml;hsel et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), which may explain the reduced investment in cuticle thickness and cuticular lipids in these workers of both studied species.\u003c/p\u003e \u003cp\u003eRegarding interspecific differences, it was expected that \u003cem\u003eA. laevigata\u003c/em\u003e would exhibit higher relative cuticular lipid intensity across all worker sizes compared to \u003cem\u003eAtta sexdens\u003c/em\u003e, reflecting greater investment in cuticular lipids, as consequence of its predominantly diurnal foraging activity. However, despite being more active during the day, \u003cem\u003eA. laevigata\u003c/em\u003e forages preferentially during milder periods, such as early morning and late afternoon (Viana et al., 2004), thereby reducing prolonged exposure to extreme environmental conditions. This activity pattern may decrease the need for increased investment in cuticular lipids, consequently reducing the metabolic costs associated with cuticle synthesis. Although the cuticle plays a central role in limiting water loss, it is a metabolically costly structure, as its formation requires substantial nitrogen investment for the synthesis of chitin and structural proteins derived from nitrogen-rich amino acids (Mullins et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Therefore, the absence of interspecific differences in cuticular lipid content may reflect a resource allocation strategy in which metabolic resources are conserved or redirected to other physiological functions of the cuticle.\u003c/p\u003e \u003cp\u003eIn contrast, \u003cem\u003eA. sexdens\u003c/em\u003e exhibited greater cuticle thickness across all size classes. Given the high metabolic cost associated with cuticle synthesis, it is plausible that the greater thickness observed in \u003cem\u003eA. sexdens\u003c/em\u003e reflects higher resource availability at the colony level, allowing greater investment in metabolically expensive structures. In addition, although \u003cem\u003eA. laevigata\u003c/em\u003e is more active during the day, it forages preferentially during milder periods such as early morning and late afternoon (Viana et al., 2004), thereby reducing prolonged exposure to extreme environmental conditions. This activity pattern may decrease the need for increased investment in cuticular lipids, consequently reducing the metabolic costs associated with cuticle synthesis. Moreover, its relatively thinner cuticle suggests that other cuticular attributes, such as ventilation patterns (Lighton, 1990, 1992; Perl \u0026amp; Niven, 2018; Oladipuppo et al., 2022), epicuticular lipid composition (Hood \u0026amp; Tschinkel, 1990), cuticular hydrocarbon profiles (Gibbs, 2002; Johnson \u0026amp; Gibbs, 2004; Ajayi et al., 2020), pilosity, sculpture (Buxton et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), or abrasion level (Johnson et al., 2011), may contribute to protection against water loss and other environmental pressures.\u003c/p\u003e \u003cp\u003eThe contrasting cuticular patterns observed across worker size classes and between the two species in the present study and in other ant species (Peeters et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Willot, 2018; Birkenfeld, 2024) reinforce the hypothesis that cuticular protection in ants is not determined by a single morphological trait. Instead, it results from an integrated adjustment between cuticle thickness, lipid composition, and body size, which together function as adaptive solutions to the diverse environmental challenges faced by different species.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eSmall workers exhibit thicker cuticles and greater amounts of cuticular lipids than medium and large ones, suggesting an adaptive investment against water loss, even though they primarily perform internal tasks. Larger workers, despite being more exposed during foraging and nest defence, show lower susceptibility to desiccation due to their lower surface area-to-volume ratio, justifying reduced investment in cuticle and lipids. At the interspecific level, \u003cem\u003eA. sexdens\u003c/em\u003e showed greater cuticle thickness across all size classes, possibly reflecting greater resource availability for the synthesis of metabolically costly structures, while \u003cem\u003eA. laevigata\u003c/em\u003e appears to compensate for water balance protection through foraging behavior in milder periods of the day. These patterns indicate that cuticular morphology in \u003cem\u003eAtta\u003c/em\u003e spp. is shaped by both physiological constraints and behavioral strategies, with ecological implications for task allocation, tolerance to extreme environmental conditions, and efficiency in resource exploitation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest. The authors confirm that there are no disputes over the ownership of the data presented in this article and that all contributions have been appropriately attributed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, R.Z. and I.G.F.; methodology, J.E.S., R.Z. and I.G.F.; resources, R.Z. and J.E.S.; supervision, J.E.S. and R.Z.; investigation, I.G.F. and K.D.A.; formal analysis, I.G.F., K.A.S.M. and J.J.S.; data curation, C.L.J. and I.G.F.; writing—original draft preparation, I.G.F.; writing—review \u0026amp; editing, K.D.A., D.S.S., K.A.S.M., C.L.J., R.Z. and J.E.S.; visualization, E.B.A., K.A.S.M. and J.J.S. All authors have read and agreed to the published version of the manuscript.All authors read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study was funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES/PROEX), grant number 1852/2023, process number 88881.893116/2023-01.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBancroft JD, Gamble M (eds) (2008) Theory and Practice of Histological Techniques. Elsevier Health Sciences\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBass M, Cherrett JM (1995) Fungal hyphae as a source of nutrients for the leaf-cutting ant Atta sexdens. 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Sci Rep 8(1):9220\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":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"neotropical-entomology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nent","sideBox":"Learn more about [Neotropical Entomology](https://www.springer.com/journal/13744)","snPcode":"13744","submissionUrl":"https://www.editorialmanager.com/nent/default2.aspx","title":"Neotropical Entomology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"polymorphism, subcastes, surface-to-volume ratio, water balance","lastPublishedDoi":"10.21203/rs.3.rs-9546263/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9546263/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe body size of insects influences the surface area-volume ratio and can affect the thickness of the cuticle and the amount of cuticular lipids, particularly in eusocial species, such as leaf-cutting ants of the genus \u003cem\u003eAtta\u003c/em\u003e, which exhibit pronounced worker polymorphism. In this study, we evaluated the relationship between body size, cuticular lipid intensity, and cuticle thickness in workers of \u003cem\u003eAtta laevigata\u003c/em\u003e (Smith, 1858) (Hymenoptera: Formicidae) and \u003cem\u003eAtta sexdens\u003c/em\u003e (Linnaeus, 1758) (Hymenoptera: Formicidae). Small, medium, and large workers, defined according to the width of the cephalic capsule, were compared regarding relative cuticle thickness and cuticular lipid intensity. Both traits varied among worker size classes. Small workers showed higher cuticular lipid intensity and greater relative cuticle thickness, while medium and large workers exhibited lower values for both traits, indicating that different cuticle components respond similarly to body size, regardless of the species. Although the intensity of cuticular lipids was not differentiated between species, cuticle thickness was consistently greater in \u003cem\u003eA. sexdens\u003c/em\u003e than in \u003cem\u003eA. laevigata\u003c/em\u003e in all size classes. Overall, our findings suggest that the different components of the cuticle of \u003cem\u003eA. laevigata\u003c/em\u003e and \u003cem\u003eA. sexdens\u003c/em\u003e are not determined solely by body size, but also reflect the specific function performed by the workers within the colony.\u003c/p\u003e","manuscriptTitle":"Worker body size drives cuticle thickness and intensity of positive reaction for lipids in the leaf-cutting ants Atta laevigata and Atta sexdens","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-15 12:50:43","doi":"10.21203/rs.3.rs-9546263/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-05-06T16:24:59+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-05-06T15:45:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-29T12:15:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Neotropical Entomology","date":"2026-04-28T09:38:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"neotropical-entomology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nent","sideBox":"Learn more about [Neotropical Entomology](https://www.springer.com/journal/13744)","snPcode":"13744","submissionUrl":"https://www.editorialmanager.com/nent/default2.aspx","title":"Neotropical Entomology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"309a1b8b-c6b7-4574-b9bc-f03745c9d124","owner":[],"postedDate":"May 15th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"","date":"2026-05-06T16:24:59+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-05-06T15:45:25+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-15T12:50:43+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-15 12:50:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9546263","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9546263","identity":"rs-9546263","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}