Reduced Cuticular Hydrocarbon Production in the Ant, Nylanderia fulva, Is Associated with Low Desiccation Resistance and Lack of Intraspecific Aggression in Its Invasive Range

Reduced Cuticular Hydrocarbon Production in the Ant, Nylanderia fulva, Is Associated with Low Desiccation Resistance and Lack of Intraspecific Aggression in Its Invasive Range | 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 Reduced Cuticular Hydrocarbon Production in the Ant, Nylanderia fulva, Is Associated with Low Desiccation Resistance and Lack of Intraspecific Aggression in Its Invasive Range Pierre-André Eyer, Anjel M. Helms, Megan N. Moran, Edward L. Vargo This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5362726/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cuticular hydrocarbons (CHCs) are ubiquitous among insects where they form an outer wax layer that helps maintain water balance and prevent desiccation. In social insects, CHCs were subsequently co-opted as semiochemicals in many contexts, including nestmate recognition, which maintains boundaries among competing colonies by ousting non-nestmates. In some ant populations, workers do not discriminate against non-nestmates. This leads to the development of supercolonies, a large network of interconnected nests exchanging unrelated individuals. In this study, we investigate CHC production by workers and their resistance to desiccation in the ant Nylanderia fulva , which exhibits supercolonial behavior within its invasive range in the USA. We found greatly reduced CHC production by workers and increased susceptibility toward desiccation compared to other invasive ants of similar body size. This relative absence of CHCs sheds light on the susceptibility of this species to abiotic stress through desiccation with implications for its potential distribution and its development of large supercolonies in its invasive range by impairing nestmate recognition. Invasive species 2-tridecanone Nestmate recognition Ecological stress Supercolony Figures Figure 1 Figure 2 Figure 3 INTRODUCTION Hydrocarbons are produced by insects and cover their body surface. Their production initially enabled insects to cope with water loss, as an increased production of cuticular hydrocarbons (CHCs) improves desiccation resistance (Gibbs et al. 1997, Gibbs 1998, Ferveur et al. 2018, Wang et al. 2022). In terrestrial species, the acclimation to specific climatic conditions ( e.g. , warm, dry) results in variation in individual chemical profiles, favoring certain classes of CHCs under specific stress conditions (Menzel et al. 2017, Otte et al. 2018, Menzel et al. 2019, Baumgart et al. 2022). Therefore, by producing a high diversity and quantity of CHC compounds, insect species can plastically adjust their CHC profile and improve individual survival through desiccation resistance under changing climatic conditions (Sprenger et al. 2018). CHC compounds have been subsequently used as semiochemicals by insect species (Chung and Carroll 2015). In social insects, CHC compounds are used as recognition cues, allowing individuals to signal various information, such as their species identity, colony of origin, caste, age, health, reproductive status or fertility (Denis et al. 2006, Holman et al. 2010, Baracchi et al. 2012, Kather and Martin 2015, Cappa et al. 2016, Leonhardt et al. 2016, Beani et al. 2019, Sprenger and Mezel 2020, Eyer et al. 2021), and facilitating division of labor and colony organization (Greene and Gordon 2003, Balbuena et al. 2018). In ants and other social insects, CHC compounds also play a major role in nestmate recognition, as they are used as a template to identify colony membership (Reeve 1989, Tsutsui et al. 2003, Blomquist and Bagnères 2010). Distinct species typically produce different blends of chemical compounds, while different colonies within a species usually differ in their relative proportions of a common set of chemical compounds (Lahav et al. 1999, Howard and Blomquist 2005, Martin et al. 2008, Van Zweden et al. 2010, Bos and d'Ettorre 2012, Sturgis and Gordon 2012, di Mauro et al. 2015, Leonhardt et al. 2016). In most cases, workers exhibit avoidance or antagonism against non-nestmate conspecifics, thereby maintaining closed boundaries between distinct nests and enabling colonies to partition resources by excluding conspecific competitors (Hölldobler and Wilson 1990). Invasive ant populations are often characterized by a lack of aggression toward non-nestmates, allowing a free exchange of individuals and resources among a network of interconnected nests, a social structure called supercoloniality (Suarez et al. 1999, Tsutsui et al. 2000, Giraud et al. 2002, Helanterä et al. 2009, Eyer and Vargo 2021, Helanterä 2022). The supercolonial structure enables invasive populations to reach extreme densities and ecological dominance without intraspecific competition and the need to defend territorial boundaries (Holway et al. 2002). The development of such a social structure has been suggested to arise from a homogenization of the gestalt CHC odor of distinct nests and, therefore, homogenization of the recognition threshold accepted by workers (Tsutsui et al. 2003, Vasquez et al. 2008). This lack of chemical distinctiveness between workers from different nests is thought to originate from the reduced genetic differentiation among nests or the absence of polymorphism at genetic loci influencing chemical profiles (Pirk et al. 2001, Giraud et al. 2002, Tsutsui et al. 2003). Alternatively, the development of supercolonies has also been suggested to result from the selection for reduced nestmate recognition to avoid recurrent fights with neighboring nests in densely populated introduced ranges (Holway et al. 1998, Holway 1999, Chapuisat et al. 2005, Jackson 2007, Steiner et al. 2007). Finally, the development of supercolonies has also been suggested to stem from the pre-existence of polydomous and polygynous small supercolonies in the native range (Giraud et al. 2002, Heller 2004), reaching tremendous sizes in the introduced range in the absence of intraspecific competition (Pedersen et al. 2006). Whatever the evolutionary mechanisms underlying the development of supercolonies, the lack of discrimination toward non-nestmates is associated with an absence of behavioral, genetic, and chemical distinctiveness between geographically distant nests within supercolonies (Helanterä et al. 2009, Helanterä 2022). For example, Argentine ant workers from the large Californian supercolony, the large European supercolony, and the Australian supercolony have similar CHC profiles and similar genetic composition (Brandt et al. 2009), suggesting that these ants will recognize and accept each other as colonymates despite their worldwide distribution. The tawny crazy ant ( Nylanderia fulva ) is an invasive species in parts of North and South America that is native to the region of South America from Brazil to Argentina, along with Uruguay and Paraguay (Gotzek et al. 2012). This species was first introduced into Colombia, Peru, and the Caribbean (Zenner-Polania 1990, Wetterer and Keularts 2008), but later spread unintentionally to the USA in the 1950’s. This species was first reported in Florida in the 2000’s and has rapidly dispersed to Mississippi, Louisiana, Alabama, Georgia, and Texas in the last two decades (Meyers and Gold 2008, MacGown and Layton 2010, Wang et al. 2016). Genetic data revealed that the invasion of this ant species is associated with founder event(s) significantly reducing the amount of genetic diversity in the invasive range in the USA compared to the native range (Eyer et al. 2018). Behavioral and population genetic analyses showed that N. fulva displays a multicolonial social structure in its native range, with separate nests maintaining strict boundaries (Eyer et al. 2018, LeBrun et al. 2019). In contrast, this species exhibits a supercolonial structure in its USA invasive range, with no clear boundaries between geographically separated nests (Eyer et al. 2018). Within the southern USA, nests are not genetically differentiated from each other. This lack of genetic differentiation is associated with an absence of nestmate recognition. The loss of nestmate discrimination leads to an absence of aggression toward non-nestmates, even from nests separated by hundreds of kilometers, with sharing of individuals and resources between neighboring nests (Eyer et al. 2018, LeBrun et al. 2019, Lawson and Oi 2020, Kjeldgaard et al. 2022). In addition, each invasive nest is headed by many queens, up to hundreds in number. Overall, these findings show that the entire invasive USA range of N. fulva comprises a single massive supercolony, extending over more than 2000 km (Eyer et al. 2018, LeBrun et al. 2019), greatly enhancing its ecological dominance in invaded areas. Despite its ecological success, invasive populations of this species often exhibit boom-bust population dynamics, with frequent collapses of populations leading to a high rate of local extinction. The frequent collapse of populations has recently been attributed to the presence of a microsporidian pathogen (LeBrun et al. 2022); however, other factors may also contribute to the fluctuating survival of N. fulva populations. Here, we investigate the production of CHCs by workers of the tawny crazy ant ( N. fulva ) within an unicolonial population in its invasive range and we assess the resistance of N. fulva workers toward desiccation through survival assays. We report a surprisingly low quantity of CHC in this species, and we further show that the CHC-poor chemical profiles of N. fulva increase their susceptibility to desiccation, potentially contributing to population collapse in this species. METHODS AND MATERIALS Data collection procedures and chemical analyses . In spring 2021, 40 workers of N. fulva were collected in the invasive range of this species in Bryan, TX, USA. Individuals were cooled down for one minute on ice before cuticular chemical compounds were extracted by placing each worker into an individual vial containing 200 µL hexane for two minutes. Extracts were prepared and analyzed using similar techniques as those previously performed on other ant species (e.g., Brandt et al. 2009, Blumenfeld et al. 2022). Extracts were evaporated under a stream of high-purity nitrogen and resuspended in 5 µL hexane including 25 ng of octadecane (C18) used as an internal standard. The solution was transferred to a 100 µL glass conical insert in a 1.5 mL autosampler vial and was analyzed using a 7890B Agilent Gas chromatograph (GC) and 5977B Agilent Mass Spectrometer (MS). A sample volume of 2 µL was injected in splitless mode using a 7693 Agilent autosampler into a HP-5MS UI column (30 m × 0.250 mm internal diameter × 0.25 µm film thickness; Agilent) with ultrahigh-purity helium as the carrier gas (16.1 psi constant flow rate). The GC temperature increased from 50 to 320°C at 10°C/min after an initial step of 1 min at 50°C and a final hold at 320°C for 10 min. Chemical compounds were ionized by electron impact ionization at 70 eV and mass spectra were obtained by scanning from 40 to 550 m/z at 2.9 scans/s. In addition, cuticular chemical compounds were extracted from 52 workers of N. terricola , 32 workers of Solenopsis invicta , and 37 workers of Linepithema humile , all collected from Bryan, TX, USA, as well as four workers of Tapinoma sessile collected in Boulder, CO, USA, following the same procedure. Workers of Nylanderia fulva, N. terricola , T. sessile , and L. humile exhibit a small and continuous size polymorphism, with workers of N. fulva being the largest (Wild 2004, Gotzek et al. 2012). Workers were randomly chosen for those species. However, S. invicta workers exhibit a large variation in body size, with the largest workers being 2 to 3-fold larger than the smallest workers (Tschinkel 2013). Care was taken to only use smaller workers of S. invicta , whose sizes were similar to those of N. fulva workers. Overall, the reduction of cuticular chemical compounds in N. fulva (see Results) unlikely results from an absence of detection, as workers of this species were similar in size or even larger than those of the other species studied (Figure S1 ). The abundance of chemical compounds in the chemical profile was inferred from the known amount of internal standard in the sample. The total abundance of chemical compounds was compared between species using the posthoc Dunn test following the Kruskal-Wallis test using the R package PMCMR . The P -values were adjusted for multiple comparisons between each pair of species using a Bonferroni correction. For N. fulva and N. terricola , the distribution of individuals in the overall quantity of chemical compounds produced was investigated using the ggridges package. All statistical analyses have been performed on R v.3.6.2. Desiccation analyses . In spring 2021, 3 additional nests of N. fulva , as well as 3 colonies of N. terricola , and 3 colonies of S. invicta were collected in Bryan, TX, US and maintained in the lab for two weeks before the start of desiccation analyses. The colonies were kept under standard rearing conditions (26 ± 2°C, 80 ± 10% relative humidity with LD cycles of 12 h:12 h and fed ad libitum with sugar water and cockroaches). For desiccation analyses, individual ants were placed in Petri dishes with sides coated with Fluon (4.5 cm in diameter). Petri dishes were stacked in covered plastic boxes, above a layer of fully dehydrated Drierite (W.A. Hammond Drierite Co. Ltd., Xenia, OH), decreasing the relative humidity inside the plastic boxes to approximately 0–2%. As a control, Petri dishes with individual ants were placed in plastic boxes without Drierite in 100% humidity chambers. Two experiments were performed. The first experiment only compared desiccation tolerance between N. fulva and N. terricola. One hundred individual ants were used from each species under desiccation conditions and 40 individuals from each species were used under control conditions. Worker condition was determined hourly, recording the time to death for each individual. The second experiment compared desiccation tolerance between all three species, 60 individuals were used for S. invicta , and 24 individuals were used for each of the two Nylanderia species. Worker condition was assessed at 16, 24, 40, 48, 64 and 72h, recording the time to death for each individual. In each experiment, the difference in desiccation tolerance between species was visualized using Kaplan-Meier curves and tested using Cox proportional-hazard models (Therneau 2011). Similar to chemical analyses, we only used workers of similar size for the three species studied, because worker resistance to desiccation is known to be associated with worker body mass (Golian et al. 2022, Ostwald et al. 2023). RESULTS We found that the quantity of chemical compounds in cuticular extracts of workers of the tawny crazy ant N. fulva and its congener N. terricola are drastically reduced compared to levels found in other invasive ants, including small workers of the fire ant Solenopsis invicta or the supercolonial ant species Linepithema humile (Figs. 1 a, S1; Supplemental information). Although workers of both Nylanderia species produced low levels of chemical compounds (X ± SD = 79.0 ± 59.2 ng for N. terricola and 74 ± 56.3 ng for N. fulva ), the total amount present in individuals followed a normal distribution in N. terricola , but those in N. fulva followed a bimodal distribution (Fig. 1 b). In N. fulva , half of the individuals produced a quantity of chemical compounds close to zero (Fig. 2 a,b,c), while the larger quantities found in the other half were mostly, or totally, the result of the production of a single compound (2-tridecanone; Retention time = 12.82 min; Figs. 2 d,e). We further showed that the impoverished cuticular chemical profiles of N. fulva (containing mostly the non-CHC compound 2-tridecanone) were associated with reduced resistance toward desiccation in a low humidity chamber (0–2% RH; Supplemental information). We found that after eight hours in a low humidity chamber, only ~ 20% of N. fulva individuals survived, while 90% of N. terricola workers were still alive (Fig. 3 b). We confirmed that this difference in mortality is primarily explained by desiccation stress, as 39 out of 40 workers of N. fulva (38 out of 40 for N. terricola ) survived when kept in high humidity chambers for the same time period (control: 100% RH; Fig. 3 b). We next demonstrated that the lower production of cuticular chemical compounds by Nylanderia workers leads to a reduced survival toward desiccation compared to the compound-abundant species S. invicta (Fig. 3 a; S2). All N. fulva individuals and ~ 70% of N. terricola workers died after 16h, while ~ 90% of S. invicta individuals were still alive at this time. DISCUSSION Our results reveal that chemical profiles of N. fulva workers are mostly made of 2-tridecanone, which is not a cuticular hydrocarbon. This compound is a methyl ketone encountered in the Dufour’s gland of many ant species (Regnier and Wilson 1968, Attygalle and Morgan 1984) and is abundantly found in N. fulva (10x more than the closely related species, Paratrechina longicornis ; Chen et al. (2013)). Coupled with formic acid, 2-tridecanone mostly serves as a defensive compound. The large presence of 2-tridecanone in cuticular extracts of N. fulva workers suggests that it could stem from their frequent and ritualized cuticular washes, which are used, among other things, to detoxify S. invicta venom (LeBrun et al. 2014, Li et al. 2021). Workers of N. fulva grab their acidopore ( i.e. , a specialized exocrine-gland duct at the end of the gaster, connected to the Dufour’s gland) with their mandibles and groom themselves vigorously with formic acid and potentially 2-tridecanone. Although 2-tridecanone is likely not directly involved in S. invicta venom detoxification, the frequent washes of N. fulva workers may explain the large abundance of 2-tridecanone found on their cuticle. However, it is important to note that the N. fulva workers used in this study were not in contact with S. invicta workers in the laboratory before performing chemical assays. Moreover, it remains to be determined whether N. fulva workers produce other cues not detected by our chemical analyses or secrete CHC compounds potentially depleted during frequent acidopore grooming. By analyzing different body parts in 17 species, Sprenger et al. (2021) reported that recognition cues are not homogeneously present across the insect body. Our study extracted whole, intact ants to measure the total quantities of cuticular compounds. We therefore cannot rule out that different cuticular chemical profiles are found on different body parts of N. fulva workers, whereby CHC compounds used for nestmate recognition are restricted to few body parts, therefore diluting their detection in whole-body analyses. Interestingly, low quantities of cuticular hydrocarbons were also reported in other social species, usually in the context of chemical mimicry and camouflage (Dettner and Liepert 1994). For example, workers of the ant Ectatomma ruidum perform intraspecific cleptobiosis, whereby workers of one colony infiltrate a neighboring colony to intercept food brought by non-nestmate foragers, then carry it to its own colony (Breed et al. 1992, 1999, 2012). Thief workers are able to infiltrate foreign colonies without inducing specific aggressiveness through chemical camouflage (producing lower total quantities of cuticular compounds than non-thieves; Breed et al. 1992, Jeral et al. 1997). Similarly, in the social parasites Vespa dybowskii and Polistes atrimandibularis , queens hide themselves and their eggs from heterospecific host workers through a form of chemical concealment, whereby hydrocarbon profiles of the parasites represent a reduced proportion of the profiles of the host species (Bagnères and Lorenzi 2002, Martin et al. 2008). As a defensive compound, 2-tridecanone affords minimal protection against desiccation, which is usually provided by long linear alkanes (Wagner et al. 2001, Sprenger and Menzel 2020, Ostwald et al. 2023). Similarly, 2-tridecanone provides limited information for nestmate recognition (mostly based on methyl-branched alkanes and alkenes;(mostly based on methyl-branched alkanes and alkenes; Sprenger and Menzel 2020). Therefore, because the chemical profile of N. fulva workers mainly consists of a single, information-poor compound, it is unlikely to allow for robust nestmate discrimination. The lack of a strong template to discern non-nestmates may have contributed to the development of a supercolonial structure in the introduced range of this species, enhancing its ecological dominance through resource monopolization (Eyer et al. 2018). This result stands in sharp contrast with other supercolonial invasive ants, such as the Argentine ant L. humile , where workers from different nests within a supercolony have similar CHC profiles but there is still sufficient diversity and quantity of compounds to provide a sufficient signature to identify workers from different supercolonies (Brandt et al. 2009). Interestingly, within supercolonial populations of L. humile , the total amounts of n- alkanes and n- alkenes are positively correlated with temperature, but negatively with precipitation, suggesting they play a role in waterproofing and desiccation resistance (Buellesbach et al. 2018). In N. fulva , the variable aggression levels observed among native nests (LeBrun et al. 2019) may suggest that the information-poor chemical profiles of the workers have acted in concert with a reduction of genetic diversity during its invasion in its invasive range to allow the entire invasive population in the USA to form a single large supercolony (Eyer et al. 2018). More studies are needed to investigate the chemical cues underlying nestmate recognition in the native range of this species. The humid and subtropical origin of N. fulva (Ward 2000) and its nesting habitat in the leaf litter may have lowered the selective pressure toward desiccation stress, therefore relaxing the need for waterproofing efficiency in this species. Evaluating whether the loss of an CHCs in N. fulva is correlated with changes in physiology (Ozaki et al. 2005, Gellert et al. 2022, Watanabe et al. 2023) or in the expression of genes associated with CHC production and/or detection (Wen et al. 2020, Sprenger et al. 2021, Ward and Moehring 2021), and relaxed selective pressure on these genes certainly merits further investigation. Future work should also quantify CHC levels among populations of N. fulva in its native range to determine whether lower CHC production is common and/or preceded its spread. Whatever the reason for the relative lack of CHCs, our results have implications for the potential expansion of the invasive range of this species. A climate niche model predicted the potential distribution of this species includes much of present-day southeastern U.S., northern South America, tropical areas of Africa and Asia, and eastern Australia (Kumar et al. 2015). The main predictive climatic factors in the model were temperature and precipitation in the driest quarter, which corresponds with our results showing high susceptibility to desiccation. As temperatures continue to warm in northern latitudes, the potential distribution of this species can be expected to also shift northward as long as there is sufficient moisture to sustain it. Declarations Author Contribution Author contributions. P.A.E.: conceptualization, data curation, formal analysis, methodology, visualization, writing—original draft, writing—review and editing; A.M.H: conceptualization, formal analysis, methodology, writing—review and editing, funding acquisition; M.N.M.: methodology, writing—review and editing; E.L.V.: conceptualization, methodology, writing—review and editing, funding acquisition, project administration Acknowledgement We thank Alexander Blumenfeld for assistance in collecting samples and Kuan-Ling Liu for help with chemical analysis. 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Zool Sci 37:371-381. https://doi.org/10.2108/zs190138 Wetterer JK, Keularts JLW (2008) Population explosion of the hairy crazy ant, Paratrechina pubens (Hymenoptera: Formicidae), on St. Croix, US Virgin Islands. Fla Entomol 91:423-427. https://doi.org/10.1653/0015-4040(2008)91[423:Peothc]2.0.Co;2 Wild AL (2004) Taxonomy and distribution of the Argentine ant, Linepithema humile (Hymenoptera : Formicidae). Ann Entomol Soc Am 97:1204-1215. https://doi.org/10.1603/0013-8746(2004)097[1204:Tadota]2.0.Co;2 Zenner-Polania I (1990) Biological aspects of the “hormiga loca,” Paratrechina ( Nylanderia ) fulva (Mayr), in Colombia. In: R. K. V. Meer, K. Jaffe, A. Cedeno and C. O. Boulder (ed) Applied Myrmecology: A World Perspective. Westview Press, USA, pp290-297 Additional Declarations No competing interests reported. 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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-5362726","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":377744575,"identity":"431acebb-8b63-4615-ab95-e0ee1bc51dce","order_by":0,"name":"Pierre-André Eyer","email":"","orcid":"","institution":"Texas A\u0026M University","correspondingAuthor":false,"prefix":"","firstName":"Pierre-André","middleName":"","lastName":"Eyer","suffix":""},{"id":377744576,"identity":"35a7f69f-74db-4c7e-8ec9-eafa57d60d5a","order_by":1,"name":"Anjel M. Helms","email":"","orcid":"","institution":"Texas A\u0026M University","correspondingAuthor":false,"prefix":"","firstName":"Anjel","middleName":"M.","lastName":"Helms","suffix":""},{"id":377744577,"identity":"73da24ee-ac55-4e49-b1ca-023251fd46f7","order_by":2,"name":"Megan N. Moran","email":"","orcid":"","institution":"Texas A\u0026M University","correspondingAuthor":false,"prefix":"","firstName":"Megan","middleName":"N.","lastName":"Moran","suffix":""},{"id":377744578,"identity":"5ce0c744-b167-40e3-bd99-b53bd211b55b","order_by":3,"name":"Edward L. Vargo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxklEQVRIiWNgGAWjYFCCBAjFD+UyNhCtRbKNZC0Gx4jVws+ewCbxc0+tnPH95qebeRhsZDccIKBFsucBm2TPs+PGZsfYzG7zMKQZE9RicCOB2YDnwLHEbccYQFoOJxLUYg/UYvgHqGVzG/s3oJb/hLUYSCQwPuY5UJO4gY0HZMsBwlokzjxsfCxz4ICxxLGcsptzDJKNZxLSwt+efODgmwN1cvzNx7fdeFNhJ9tHSAs0Ig7D3ElQORzUEa90FIyCUTAKRh4AAJJsRjWsRi+fAAAAAElFTkSuQmCC","orcid":"","institution":"Texas A\u0026M University","correspondingAuthor":true,"prefix":"","firstName":"Edward","middleName":"L.","lastName":"Vargo","suffix":""}],"badges":[],"createdAt":"2024-10-30 17:23:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5362726/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5362726/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":69812893,"identity":"c88244e6-c9de-49d1-95a7-5199a52166ca","added_by":"auto","created_at":"2024-11-25 12:55:07","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":267485,"visible":true,"origin":"","legend":"\u003cp\u003eTotal amount of cuticular compounds extracted from individuals of different ant species (\u003cem\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/em\u003e). Lowercase letters indicate significant differences among species. Distribution in the amount of cuticular compounds produced among workers of \u003cem\u003eN. fulva\u003c/em\u003e and \u003cem\u003eN. terricola \u003c/em\u003e(\u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e). Bold solid lines represent the means, dotted lines indicate the 1\u003csup\u003est\u003c/sup\u003e and 3\u003csup\u003erd\u003c/sup\u003e quartiles for each species.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5362726/v1/7e67fcb5320b46b67dbf8ac6.jpeg"},{"id":69812892,"identity":"ca00e60f-3473-44d3-a19a-0303459c20f7","added_by":"auto","created_at":"2024-11-25 12:55:07","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":540870,"visible":true,"origin":"","legend":"\u003cp\u003eExample of cuticular profiles of workers of \u003cem\u003eNylanderia terricola\u003c/em\u003e (\u003cem\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e)\u003c/em\u003e, as well as profiles of \u003cem\u003eN. fulva\u003c/em\u003e without cuticular compounds (\u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e), or with almost only 2-tridecanone compound (\u003cem\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003c/em\u003e). Peaks colored in green and labeled with an asterisk represent the amount of octadecane (C18; internal standard) within samples. Comparison of the MS fragmentation pathway of the chemical compound found in the \u003cem\u003eN. fulva\u003c/em\u003e profile at retention time 12.82 min (upper panel) with the expected fragmentation pathway of 2-tridecanone (lower panel) (\u003cem\u003e\u003cstrong\u003ed\u003c/strong\u003e\u003c/em\u003e). Distribution of the proportion of 2-tridecanone in the overall profile of \u003cem\u003eN. fulva\u003c/em\u003e workers (\u003cem\u003e\u003cstrong\u003ee\u003c/strong\u003e\u003c/em\u003e). Bold solid lines represent the means, dotted lines indicate the 1\u003csup\u003est\u003c/sup\u003e and 3\u003csup\u003erd\u003c/sup\u003e quartiles for each species.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5362726/v1/69af58edbc80af386a87bd63.jpeg"},{"id":69812891,"identity":"fe2b631f-43bf-41ce-8e29-9eece8a7a0d1","added_by":"auto","created_at":"2024-11-25 12:55:07","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":205255,"visible":true,"origin":"","legend":"\u003cp\u003eKaplan-Meier survival distributions of workers of \u003cem\u003eNylanderia fulva,\u003c/em\u003e \u003cem\u003eN. terricola\u003c/em\u003e and \u003cem\u003eSolenopsis invicta\u003c/em\u003e for 72 hours under desiccation conditions. Kaplan-Meier survival distributions of workers of \u003cem\u003eN. fulva\u003c/em\u003e and \u003cem\u003eN. terricola\u003c/em\u003e for eight hours under desiccation conditions (\u003cem\u003ei.e.,\u003c/em\u003e \u003cem\u003eDesi\u003c/em\u003e.; solid lines) and under control conditions (\u003cem\u003ei.e.,\u003c/em\u003e \u003cem\u003eCont\u003c/em\u003e.; dotted lines) (\u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5362726/v1/dfccd18691a6ed156ea9020c.jpeg"},{"id":81216606,"identity":"ea567815-1bbd-4c92-94bc-3dbb4bf1bbf7","added_by":"auto","created_at":"2025-04-23 14:23:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1473397,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5362726/v1/542fda7b-dd8e-4bae-980b-00dbf8ac46bb.pdf"},{"id":69812885,"identity":"c3dcffba-6b0f-44a0-9a89-dd0afc4fc464","added_by":"auto","created_at":"2024-11-25 12:55:03","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":218382,"visible":true,"origin":"","legend":"","description":"","filename":"NfulvProcBSupplemental.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5362726/v1/95ed2c95b34503c99bb16be2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Reduced Cuticular Hydrocarbon Production in the Ant, Nylanderia fulva, Is Associated with Low Desiccation Resistance and Lack of Intraspecific Aggression in Its Invasive Range","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eHydrocarbons are produced by insects and cover their body surface. Their production initially enabled insects to cope with water loss, as an increased production of cuticular hydrocarbons (CHCs) improves desiccation resistance (Gibbs et al. 1997, Gibbs 1998, Ferveur et al. 2018, Wang et al. 2022). In terrestrial species, the acclimation to specific climatic conditions (\u003cem\u003ee.g.\u003c/em\u003e, warm, dry) results in variation in individual chemical profiles, favoring certain classes of CHCs under specific stress conditions (Menzel et al. 2017, Otte et al. 2018, Menzel et al. 2019, Baumgart et al. 2022). Therefore, by producing a high diversity and quantity of CHC compounds, insect species can plastically adjust their CHC profile and improve individual survival through desiccation resistance under changing climatic conditions (Sprenger et al. 2018).\u003c/p\u003e \u003cp\u003eCHC compounds have been subsequently used as semiochemicals by insect species (Chung and Carroll 2015). In social insects, CHC compounds are used as recognition cues, allowing individuals to signal various information, such as their species identity, colony of origin, caste, age, health, reproductive status or fertility (Denis et al. 2006, Holman et al. 2010, Baracchi et al. 2012, Kather and Martin 2015, Cappa et al. 2016, Leonhardt et al. 2016, Beani et al. 2019, Sprenger and Mezel 2020, Eyer et al. 2021), and facilitating division of labor and colony organization (Greene and Gordon 2003, Balbuena et al. 2018).\u003c/p\u003e \u003cp\u003eIn ants and other social insects, CHC compounds also play a major role in nestmate recognition, as they are used as a template to identify colony membership (Reeve 1989, Tsutsui et al. 2003, Blomquist and Bagn\u0026egrave;res 2010). Distinct species typically produce different blends of chemical compounds, while different colonies within a species usually differ in their relative proportions of a common set of chemical compounds (Lahav et al. 1999, Howard and Blomquist 2005, Martin et al. 2008, Van Zweden et al. 2010, Bos and d'Ettorre 2012, Sturgis and Gordon 2012, di Mauro et al. 2015, Leonhardt et al. 2016). In most cases, workers exhibit avoidance or antagonism against non-nestmate conspecifics, thereby maintaining closed boundaries between distinct nests and enabling colonies to partition resources by excluding conspecific competitors (H\u0026ouml;lldobler and Wilson 1990).\u003c/p\u003e \u003cp\u003eInvasive ant populations are often characterized by a lack of aggression toward non-nestmates, allowing a free exchange of individuals and resources among a network of interconnected nests, a social structure called supercoloniality (Suarez et al. 1999, Tsutsui et al. 2000, Giraud et al. 2002, Helanter\u0026auml; et al. 2009, Eyer and Vargo 2021, Helanter\u0026auml; 2022). The supercolonial structure enables invasive populations to reach extreme densities and ecological dominance without intraspecific competition and the need to defend territorial boundaries (Holway et al. 2002). The development of such a social structure has been suggested to arise from a homogenization of the gestalt CHC odor of distinct nests and, therefore, homogenization of the recognition threshold accepted by workers (Tsutsui et al. 2003, Vasquez et al. 2008). This lack of chemical distinctiveness between workers from different nests is thought to originate from the reduced genetic differentiation among nests or the absence of polymorphism at genetic loci influencing chemical profiles (Pirk et al. 2001, Giraud et al. 2002, Tsutsui et al. 2003). Alternatively, the development of supercolonies has also been suggested to result from the selection for reduced nestmate recognition to avoid recurrent fights with neighboring nests in densely populated introduced ranges (Holway et al. 1998, Holway 1999, Chapuisat et al. 2005, Jackson 2007, Steiner et al. 2007). Finally, the development of supercolonies has also been suggested to stem from the pre-existence of polydomous and polygynous small supercolonies in the native range (Giraud et al. 2002, Heller 2004), reaching tremendous sizes in the introduced range in the absence of intraspecific competition (Pedersen et al. 2006). Whatever the evolutionary mechanisms underlying the development of supercolonies, the lack of discrimination toward non-nestmates is associated with an absence of behavioral, genetic, and chemical distinctiveness between geographically distant nests within supercolonies (Helanter\u0026auml; et al. 2009, Helanter\u0026auml; 2022). For example, Argentine ant workers from the large Californian supercolony, the large European supercolony, and the Australian supercolony have similar CHC profiles and similar genetic composition (Brandt et al. 2009), suggesting that these ants will recognize and accept each other as colonymates despite their worldwide distribution.\u003c/p\u003e \u003cp\u003eThe tawny crazy ant (\u003cem\u003eNylanderia fulva\u003c/em\u003e) is an invasive species in parts of North and South America that is native to the region of South America from Brazil to Argentina, along with Uruguay and Paraguay (Gotzek et al. 2012). This species was first introduced into Colombia, Peru, and the Caribbean (Zenner-Polania 1990, Wetterer and Keularts 2008), but later spread unintentionally to the USA in the 1950\u0026rsquo;s. This species was first reported in Florida in the 2000\u0026rsquo;s and has rapidly dispersed to Mississippi, Louisiana, Alabama, Georgia, and Texas in the last two decades (Meyers and Gold 2008, MacGown and Layton 2010, Wang et al. 2016). Genetic data revealed that the invasion of this ant species is associated with founder event(s) significantly reducing the amount of genetic diversity in the invasive range in the USA compared to the native range (Eyer et al. 2018). Behavioral and population genetic analyses showed that \u003cem\u003eN. fulva\u003c/em\u003e displays a multicolonial social structure in its native range, with separate nests maintaining strict boundaries (Eyer et al. 2018, LeBrun et al. 2019). In contrast, this species exhibits a supercolonial structure in its USA invasive range, with no clear boundaries between geographically separated nests (Eyer et al. 2018). Within the southern USA, nests are not genetically differentiated from each other. This lack of genetic differentiation is associated with an absence of nestmate recognition. The loss of nestmate discrimination leads to an absence of aggression toward non-nestmates, even from nests separated by hundreds of kilometers, with sharing of individuals and resources between neighboring nests (Eyer et al. 2018, LeBrun et al. 2019, Lawson and Oi 2020, Kjeldgaard et al. 2022). In addition, each invasive nest is headed by many queens, up to hundreds in number. Overall, these findings show that the entire invasive USA range of \u003cem\u003eN. fulva\u003c/em\u003e comprises a single massive supercolony, extending over more than 2000 km (Eyer et al. 2018, LeBrun et al. 2019), greatly enhancing its ecological dominance in invaded areas. Despite its ecological success, invasive populations of this species often exhibit boom-bust population dynamics, with frequent collapses of populations leading to a high rate of local extinction. The frequent collapse of populations has recently been attributed to the presence of a microsporidian pathogen (LeBrun et al. 2022); however, other factors may also contribute to the fluctuating survival of \u003cem\u003eN. fulva\u003c/em\u003e populations.\u003c/p\u003e \u003cp\u003eHere, we investigate the production of CHCs by workers of the tawny crazy ant (\u003cem\u003eN. fulva\u003c/em\u003e) within an unicolonial population in its invasive range and we assess the resistance of \u003cem\u003eN. fulva\u003c/em\u003e workers toward desiccation through survival assays. We report a surprisingly low quantity of CHC in this species, and we further show that the CHC-poor chemical profiles of \u003cem\u003eN. fulva\u003c/em\u003e increase their susceptibility to desiccation, potentially contributing to population collapse in this species.\u003c/p\u003e"},{"header":"METHODS AND MATERIALS","content":"\u003cp\u003e \u003cem\u003eData collection procedures and chemical analyses\u003c/em\u003e. In spring 2021, 40 workers of \u003cem\u003eN. fulva\u003c/em\u003e were collected in the invasive range of this species in Bryan, TX, USA. Individuals were cooled down for one minute on ice before cuticular chemical compounds were extracted by placing each worker into an individual vial containing 200 \u0026micro;L hexane for two minutes. Extracts were prepared and analyzed using similar techniques as those previously performed on other ant species (e.g., Brandt et al. 2009, Blumenfeld et al. 2022). Extracts were evaporated under a stream of high-purity nitrogen and resuspended in 5 \u0026micro;L hexane including 25 ng of octadecane (C18) used as an internal standard. The solution was transferred to a 100 \u0026micro;L glass conical insert in a 1.5 mL autosampler vial and was analyzed using a 7890B Agilent Gas chromatograph (GC) and 5977B Agilent Mass Spectrometer (MS). A sample volume of 2 \u0026micro;L was injected in splitless mode using a 7693 Agilent autosampler into a HP-5MS UI column (30 m \u0026times; 0.250 mm internal diameter \u0026times; 0.25 \u0026micro;m film thickness; Agilent) with ultrahigh-purity helium as the carrier gas (16.1 psi constant flow rate). The GC temperature increased from 50 to 320\u0026deg;C at 10\u0026deg;C/min after an initial step of 1 min at 50\u0026deg;C and a final hold at 320\u0026deg;C for 10 min. Chemical compounds were ionized by electron impact ionization at 70 eV and mass spectra were obtained by scanning from 40 to 550 m/z at 2.9 scans/s. In addition, cuticular chemical compounds were extracted from 52 workers of \u003cem\u003eN. terricola\u003c/em\u003e, 32 workers of \u003cem\u003eSolenopsis invicta\u003c/em\u003e, and 37 workers of \u003cem\u003eLinepithema humile\u003c/em\u003e, all collected from Bryan, TX, USA, as well as four workers of \u003cem\u003eTapinoma sessile\u003c/em\u003e collected in Boulder, CO, USA, following the same procedure. Workers of \u003cem\u003eNylanderia fulva, N. terricola\u003c/em\u003e, \u003cem\u003eT. sessile\u003c/em\u003e, and \u003cem\u003eL. humile\u003c/em\u003e exhibit a small and continuous size polymorphism, with workers of \u003cem\u003eN. fulva\u003c/em\u003e being the largest (Wild 2004, Gotzek et al. 2012). Workers were randomly chosen for those species. However, \u003cem\u003eS. invicta\u003c/em\u003e workers exhibit a large variation in body size, with the largest workers being 2 to 3-fold larger than the smallest workers (Tschinkel 2013). Care was taken to only use smaller workers of \u003cem\u003eS. invicta\u003c/em\u003e, whose sizes were similar to those of \u003cem\u003eN. fulva\u003c/em\u003e workers. Overall, the reduction of cuticular chemical compounds in \u003cem\u003eN. fulva\u003c/em\u003e (see Results) unlikely results from an absence of detection, as workers of this species were similar in size or even larger than those of the other species studied (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe abundance of chemical compounds in the chemical profile was inferred from the known amount of internal standard in the sample. The total abundance of chemical compounds was compared between species using the posthoc Dunn test following the Kruskal-Wallis test using the R package \u003cem\u003ePMCMR\u003c/em\u003e. The \u003cem\u003eP\u003c/em\u003e-values were adjusted for multiple comparisons between each pair of species using a Bonferroni correction. For \u003cem\u003eN. fulva\u003c/em\u003e and \u003cem\u003eN. terricola\u003c/em\u003e, the distribution of individuals in the overall quantity of chemical compounds produced was investigated using the \u003cem\u003eggridges\u003c/em\u003e package. All statistical analyses have been performed on R v.3.6.2.\u003c/p\u003e \u003cp\u003e \u003cem\u003eDesiccation analyses\u003c/em\u003e. In spring 2021, 3 additional nests of \u003cem\u003eN. fulva\u003c/em\u003e, as well as 3 colonies of \u003cem\u003eN. terricola\u003c/em\u003e, and 3 colonies of \u003cem\u003eS. invicta\u003c/em\u003e were collected in Bryan, TX, US and maintained in the lab for two weeks before the start of desiccation analyses. The colonies were kept under standard rearing conditions (26\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 80\u0026thinsp;\u0026plusmn;\u0026thinsp;10% relative humidity with LD cycles of 12 h:12 h and fed \u003cem\u003ead libitum\u003c/em\u003e with sugar water and cockroaches).\u003c/p\u003e \u003cp\u003eFor desiccation analyses, individual ants were placed in Petri dishes with sides coated with Fluon (4.5 cm in diameter). Petri dishes were stacked in covered plastic boxes, above a layer of fully dehydrated Drierite (W.A. Hammond Drierite Co. Ltd., Xenia, OH), decreasing the relative humidity inside the plastic boxes to approximately 0\u0026ndash;2%. As a control, Petri dishes with individual ants were placed in plastic boxes without Drierite in 100% humidity chambers. Two experiments were performed. The first experiment only compared desiccation tolerance between \u003cem\u003eN. fulva and N. terricola.\u003c/em\u003e One hundred individual ants were used from each species under desiccation conditions and 40 individuals from each species were used under control conditions. Worker condition was determined hourly, recording the time to death for each individual. The second experiment compared desiccation tolerance between all three species, 60 individuals were used for \u003cem\u003eS. invicta\u003c/em\u003e, and 24 individuals were used for each of the two \u003cem\u003eNylanderia\u003c/em\u003e species. Worker condition was assessed at 16, 24, 40, 48, 64 and 72h, recording the time to death for each individual. In each experiment, the difference in desiccation tolerance between species was visualized using Kaplan-Meier curves and tested using Cox proportional-hazard models (Therneau 2011). Similar to chemical analyses, we only used workers of similar size for the three species studied, because worker resistance to desiccation is known to be associated with worker body mass (Golian et al. 2022, Ostwald et al. 2023).\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eWe found that the quantity of chemical compounds in cuticular extracts of workers of the tawny crazy ant \u003cem\u003eN. fulva\u003c/em\u003e and its congener \u003cem\u003eN. terricola\u003c/em\u003e are drastically reduced compared to levels found in other invasive ants, including small workers of the fire ant \u003cem\u003eSolenopsis invicta\u003c/em\u003e or the supercolonial ant species \u003cem\u003eLinepithema humile\u003c/em\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, S1; Supplemental information). Although workers of both \u003cem\u003eNylanderia\u003c/em\u003e species produced low levels of chemical compounds (X\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u0026thinsp;=\u0026thinsp;79.0\u0026thinsp;\u0026plusmn;\u0026thinsp;59.2 ng for \u003cem\u003eN. terricola\u003c/em\u003e and 74\u0026thinsp;\u0026plusmn;\u0026thinsp;56.3 ng for \u003cem\u003eN. fulva\u003c/em\u003e), the total amount present in individuals followed a normal distribution in \u003cem\u003eN. terricola\u003c/em\u003e, but those in \u003cem\u003eN. fulva\u003c/em\u003e followed a bimodal distribution (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). In \u003cem\u003eN. fulva\u003c/em\u003e, half of the individuals produced a quantity of chemical compounds close to zero (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea,b,c), while the larger quantities found in the other half were mostly, or totally, the result of the production of a single compound (2-tridecanone; Retention time\u0026thinsp;=\u0026thinsp;12.82 min; Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed,e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further showed that the impoverished cuticular chemical profiles of \u003cem\u003eN. fulva\u003c/em\u003e (containing mostly the non-CHC compound 2-tridecanone) were associated with reduced resistance toward desiccation in a low humidity chamber (0\u0026ndash;2% RH; Supplemental information). We found that after eight hours in a low humidity chamber, only\u0026thinsp;~\u0026thinsp;20% of \u003cem\u003eN. fulva\u003c/em\u003e individuals survived, while 90% of \u003cem\u003eN. terricola\u003c/em\u003e workers were still alive (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). We confirmed that this difference in mortality is primarily explained by desiccation stress, as 39 out of 40 workers of \u003cem\u003eN. fulva\u003c/em\u003e (38 out of 40 for \u003cem\u003eN. terricola\u003c/em\u003e) survived when kept in high humidity chambers for the same time period (control: 100% RH; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). We next demonstrated that the lower production of cuticular chemical compounds by \u003cem\u003eNylanderia\u003c/em\u003e workers leads to a reduced survival toward desiccation compared to the compound-abundant species \u003cem\u003eS. invicta\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea; S2). All \u003cem\u003eN. fulva\u003c/em\u003e individuals and ~\u0026thinsp;70% of \u003cem\u003eN. terricola\u003c/em\u003e workers died after 16h, while\u0026thinsp;~\u0026thinsp;90% of \u003cem\u003eS. invicta\u003c/em\u003e individuals were still alive at this time.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOur results reveal that chemical profiles of \u003cem\u003eN. fulva\u003c/em\u003e workers are mostly made of 2-tridecanone, which is not a cuticular hydrocarbon. This compound is a methyl ketone encountered in the Dufour\u0026rsquo;s gland of many ant species (Regnier and Wilson 1968, Attygalle and Morgan 1984) and is abundantly found in \u003cem\u003eN. fulva\u003c/em\u003e (10x more than the closely related species, \u003cem\u003eParatrechina longicornis\u003c/em\u003e; Chen et al. (2013)). Coupled with formic acid, 2-tridecanone mostly serves as a defensive compound. The large presence of 2-tridecanone in cuticular extracts of \u003cem\u003eN. fulva\u003c/em\u003e workers suggests that it could stem from their frequent and ritualized cuticular washes, which are used, among other things, to detoxify \u003cem\u003eS. invicta\u003c/em\u003e venom (LeBrun et al. 2014, Li et al. 2021). Workers of \u003cem\u003eN. fulva\u003c/em\u003e grab their acidopore (\u003cem\u003ei.e.\u003c/em\u003e, a specialized exocrine-gland duct at the end of the gaster, connected to the Dufour\u0026rsquo;s gland) with their mandibles and groom themselves vigorously with formic acid and potentially 2-tridecanone. Although 2-tridecanone is likely not directly involved in \u003cem\u003eS. invicta\u003c/em\u003e venom detoxification, the frequent washes of \u003cem\u003eN. fulva\u003c/em\u003e workers may explain the large abundance of 2-tridecanone found on their cuticle. However, it is important to note that the \u003cem\u003eN. fulva\u003c/em\u003e workers used in this study were not in contact with \u003cem\u003eS. invicta\u003c/em\u003e workers in the laboratory before performing chemical assays. Moreover, it remains to be determined whether \u003cem\u003eN. fulva\u003c/em\u003e workers produce other cues not detected by our chemical analyses or secrete CHC compounds potentially depleted during frequent acidopore grooming. By analyzing different body parts in 17 species, Sprenger et al. (2021) reported that recognition cues are not homogeneously present across the insect body. Our study extracted whole, intact ants to measure the total quantities of cuticular compounds. We therefore cannot rule out that different cuticular chemical profiles are found on different body parts of \u003cem\u003eN. fulva\u003c/em\u003e workers, whereby CHC compounds used for nestmate recognition are restricted to few body parts, therefore diluting their detection in whole-body analyses.\u003c/p\u003e \u003cp\u003eInterestingly, low quantities of cuticular hydrocarbons were also reported in other social species, usually in the context of chemical mimicry and camouflage (Dettner and Liepert 1994). For example, workers of the ant \u003cem\u003eEctatomma ruidum\u003c/em\u003e perform intraspecific cleptobiosis, whereby workers of one colony infiltrate a neighboring colony to intercept food brought by non-nestmate foragers, then carry it to its own colony (Breed et al. 1992, 1999, 2012). Thief workers are able to infiltrate foreign colonies without inducing specific aggressiveness through chemical camouflage (producing lower total quantities of cuticular compounds than non-thieves; Breed et al. 1992, Jeral et al. 1997). Similarly, in the social parasites \u003cem\u003eVespa dybowskii\u003c/em\u003e and \u003cem\u003ePolistes atrimandibularis\u003c/em\u003e, queens hide themselves and their eggs from heterospecific host workers through a form of chemical concealment, whereby hydrocarbon profiles of the parasites represent a reduced proportion of the profiles of the host species (Bagn\u0026egrave;res and Lorenzi 2002, Martin et al. 2008).\u003c/p\u003e \u003cp\u003eAs a defensive compound, 2-tridecanone affords minimal protection against desiccation, which is usually provided by long linear alkanes (Wagner et al. 2001, Sprenger and Menzel 2020, Ostwald et al. 2023). Similarly, 2-tridecanone provides limited information for nestmate recognition (mostly based on methyl-branched alkanes and alkenes;(mostly based on methyl-branched alkanes and alkenes; Sprenger and Menzel 2020). Therefore, because the chemical profile of \u003cem\u003eN. fulva\u003c/em\u003e workers mainly consists of a single, information-poor compound, it is unlikely to allow for robust nestmate discrimination. The lack of a strong template to discern non-nestmates may have contributed to the development of a supercolonial structure in the introduced range of this species, enhancing its ecological dominance through resource monopolization (Eyer et al. 2018). This result stands in sharp contrast with other supercolonial invasive ants, such as the Argentine ant \u003cem\u003eL. humile\u003c/em\u003e, where workers from different nests within a supercolony have similar CHC profiles but there is still sufficient diversity and quantity of compounds to provide a sufficient signature to identify workers from different supercolonies (Brandt et al. 2009). Interestingly, within supercolonial populations of \u003cem\u003eL. humile\u003c/em\u003e, the total amounts of \u003cem\u003en-\u003c/em\u003ealkanes and \u003cem\u003en-\u003c/em\u003ealkenes are positively correlated with temperature, but negatively with precipitation, suggesting they play a role in waterproofing and desiccation resistance (Buellesbach et al. 2018). In \u003cem\u003eN. fulva\u003c/em\u003e, the variable aggression levels observed among native nests (LeBrun et al. 2019) may suggest that the information-poor chemical profiles of the workers have acted in concert with a reduction of genetic diversity during its invasion in its invasive range to allow the entire invasive population in the USA to form a single large supercolony (Eyer et al. 2018). More studies are needed to investigate the chemical cues underlying nestmate recognition in the native range of this species.\u003c/p\u003e \u003cp\u003eThe humid and subtropical origin of \u003cem\u003eN. fulva\u003c/em\u003e (Ward 2000) and its nesting habitat in the leaf litter may have lowered the selective pressure toward desiccation stress, therefore relaxing the need for waterproofing efficiency in this species. Evaluating whether the loss of an CHCs in \u003cem\u003eN. fulva\u003c/em\u003e is correlated with changes in physiology (Ozaki et al. 2005, Gellert et al. 2022, Watanabe et al. 2023) or in the expression of genes associated with CHC production and/or detection (Wen et al. 2020, Sprenger et al. 2021, Ward and Moehring 2021), and relaxed selective pressure on these genes certainly merits further investigation. Future work should also quantify CHC levels among populations of \u003cem\u003eN. fulva\u003c/em\u003e in its native range to determine whether lower CHC production is common and/or preceded its spread.\u003c/p\u003e \u003cp\u003eWhatever the reason for the relative lack of CHCs, our results have implications for the potential expansion of the invasive range of this species. A climate niche model predicted the potential distribution of this species includes much of present-day southeastern U.S., northern South America, tropical areas of Africa and Asia, and eastern Australia (Kumar et al. 2015). The main predictive climatic factors in the model were temperature and precipitation in the driest quarter, which corresponds with our results showing high susceptibility to desiccation. As temperatures continue to warm in northern latitudes, the potential distribution of this species can be expected to also shift northward as long as there is sufficient moisture to sustain it.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthor contributions. P.A.E.: conceptualization, data curation, formal analysis, methodology, visualization, writing\u0026mdash;original draft, writing\u0026mdash;review and editing; A.M.H: conceptualization, formal analysis, methodology, writing\u0026mdash;review and editing, funding acquisition; M.N.M.: methodology, writing\u0026mdash;review and editing; E.L.V.: conceptualization, methodology, writing\u0026mdash;review and editing, funding acquisition, project administration\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Alexander Blumenfeld for assistance in collecting samples and Kuan-Ling Liu for help with chemical analysis.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data have been deposited in the electronic supplementary material depository Open Science Framework (DOI: 10.17605/OSF.IO/ZKTFV).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAttygalle AB, Morgan ED (1984) Chemicals from the glands of ants. Chem Soc Rev 13:245-278. https://doi.org/10.1039/CS9841300245\u003c/li\u003e\n \u003cli\u003eBagn\u0026egrave;res AG, Lorenzi MC (2002) Concealing identity and mimicking hosts: A dual chemical strategy for a single social parasite? (\u003cem\u003ePolistes atrimandibularis\u003c/em\u003e, Hymenoptera: Vespidae). Parasitol 125:507-512. https://doi.org/10.1017/S003118200200238X\u003c/li\u003e\n \u003cli\u003eBalbuena MS, Gonz\u0026aacute;lez A, Farina WM (2018) Characterization of cuticular hydrocarbons according to colony duties in the stingless bee \u003cem\u003eTetragonisca angustula\u003c/em\u003e. Apidologie 49:185-195. https://doi.org/10.1007/s13592-017-0539-x\u003c/li\u003e\n \u003cli\u003eBaracchi D, Fadda A, Turillazzi S (2012) Evidence for antiseptic behaviour towards sick adult bees in honey bee colonies. 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Westview Press, USA, pp290-297\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Invasive species, 2-tridecanone, Nestmate recognition, Ecological stress, Supercolony","lastPublishedDoi":"10.21203/rs.3.rs-5362726/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5362726/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCuticular hydrocarbons (CHCs) are ubiquitous among insects where they form an outer wax layer that helps maintain water balance and prevent desiccation. In social insects, CHCs were subsequently co-opted as semiochemicals in many contexts, including nestmate recognition, which maintains boundaries among competing colonies by ousting non-nestmates. In some ant populations, workers do not discriminate against non-nestmates. This leads to the development of supercolonies, a large network of interconnected nests exchanging unrelated individuals. In this study, we investigate CHC production by workers and their resistance to desiccation in the ant \u003cem\u003eNylanderia fulva\u003c/em\u003e, which exhibits supercolonial behavior within its invasive range in the USA. We found greatly reduced CHC production by workers and increased susceptibility toward desiccation compared to other invasive ants of similar body size. This relative absence of CHCs sheds light on the susceptibility of this species to abiotic stress through desiccation with implications for its potential distribution and its development of large supercolonies in its invasive range by impairing nestmate recognition.\u003c/p\u003e","manuscriptTitle":"Reduced Cuticular Hydrocarbon Production in the Ant, Nylanderia fulva, Is Associated with Low Desiccation Resistance and Lack of Intraspecific Aggression in Its Invasive Range","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-25 12:54:37","doi":"10.21203/rs.3.rs-5362726/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"95a5a34a-0a72-434d-b517-df9d4a4bc2d7","owner":[],"postedDate":"November 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-26T19:23:11+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-25 12:54:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5362726","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5362726","identity":"rs-5362726","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}