Antennal Sensilla Basiconica Responses to Pheromones and General Odorants in Red Imported Fire Ants, Solenopsis invicta

Simple Summary

Ants, like many other social insects, rely on complex chemical signals to organize their colonies, coordinate tasks, and respond to threats. Their sense of smell plays a key role in detecting these chemical cues. In this study, we measured how the red imported fire ant’s antenna responds to 62 different odor cues using a highly sensitive recording method. We found that a type of sensory hair on the antenna can detect a broad range of odors. Importantly, worker ants and winged females showed different patterns of odor detection, suggesting that each caste has smell-based abilities suited to its role in the colony. These findings may help guide the development of new attractants or repellents for fire ant management.

Abstract

The red imported fire ant, Solenopsis invicta Buren, is a eusocial insect that relies on a sophisticated chemical communication system for colony organization and function. Its olfactory system is vital for detecting semiochemicals in the environment. This study utilized single sensillum recording (SSR) to assess the olfactory neuronal responses of female alates and workers from basiconica sensilla exposed to a panel of 62 individual pheromones and general odorants, including terpenes, terpenoids, pyrazines, pyridines, ketones, aldehydes, alcohols, acids, aliphatic and aromatic acetates, benzoates, benzyl esters, and three essential oils. Basiconica sensilla, which contain multiple olfactory receptor neurons (ORNs), exhibited moderate to strong responses to most of the tested compounds, demonstrating a broad sensitivity to all odorants elevated. Comparative analysis of the two castes revealed that ORNs had similar responses to 47 odorants; however, workers showed stronger responses to nine specific compounds, while female alates responded more strongly to six others. These differences underscore the caste-specific olfactory tuning, likely reflecting their distinct roles within the colony. This study presents the first comprehensive mapping of basiconica sensilla responses to general odorants in S. invicta female alates and workers, enhancing our understanding of the S. invicta chemical ecology and potentially contribute to more effective fire ant management strategies.

1. Introduction

In eusocial insects, chemical communication is essential for social organization and colony maintenance [1]. Semiochemicals, compounds that transmit information from various sources, including animals, plants or microbes, play a pivotal role in this communication. Pheromones, a specific class of semiochemicals produced by organisms for intraspecific communication, are used by ants in various contexts. For instance, they release alarm pheromones in response to danger or death to alert nestmates, lay chemical trails to food sources, and emit colony-specific blends of cuticular hydrocarbons (CHCs) to differentiate nestmates from non-nestmates [2]. Identifying semiochemicals is a key objective in insect chemical ecology research, though distinguishing between behaviorally active and inactive compounds within complex samples, such as headspace collections, remains significant challenges [3].

The insect olfactory system plays a critical role in detecting semiochemicals in their environment. Olfactory detection is mediated by specialized cuticular structures known as sensilla, primarily located on the antennae. These sensilla contain olfactory receptor neurons (ORNs) immersed in sensillar lymph, with each ORN expressing specific olfactory receptors (ORs) on its dendritic membranes. Together with the obligate co-receptor Orco, ORs form ligand-gated ion channels that convert chemical stimuli into electrical signals, measurable using electrophysiological techniques such as electroantennography (EAG) and single sensillum recording (SSR) [4,5]. The sensory output of the antenna is largely determined by the identity and number of ORs activated within each sensillum [6,7]. While most insect species possess fewer than 100 OR genes, hymenopterans, including ants, bees, and social wasps, exhibit a significant expansion of OR gene families, reflecting their reliance on sophisticated chemical communication [5].

The red imported fire ant, Solenopsis invicta Buren, an important invasive pest, was introduced into the southeastern United States from South America between 1930s. Since then, it has spread throughout Puerto Rico and across much of the southern and western United States, from Maryland to southern California [8]. As an eusocial insect, S. invicta displays considerable diversity in morphological and sexual phenotypes [9]. Mature colonies consist of one or more reproductive queens, female and male alates, and numerous sterile female workers, which carry out the tasks essential for colony survival [10]. Like other social insects, S. invicta relies on complex pheromonal communication for colony cohesion, social regulation, and defense.

Chemical communication in ants is not only central to colony organization but also highly relevant to applied pest management. Because many invasive ant species rely heavily on olfactory cues for foraging, recruitment, and nestmate recognition, synthetic odorants and pheromone-based lures have become important tools for monitoring and controlling pest populations. A detailed understanding of semiochemical detection at the level of individual sensilla is valuable for identifying behaviorally relevant olfactory active compounds. However, progress in these applications has been limited by the lack of detailed electrophysiological data linking odorant structure to sensory neuron responses. Establishing the response properties of sensilla therefore provides not only fundamental insight into fire ant olfaction but also a foundation for designing more targeted and efficient chemical control strategies.

In S. invicta, several antennal sensillum types, including sensilla trichodea, basiconica, chaetica, ampullacea, and coeloconica, have been described in previous morphological studies, providing a structural foundation for understanding fire ant olfaction. Although basiconic sensilla have been shown to respond to general odorants in several other ant species using SSR [11,12], no single-sensillum electrophysiological studies have been conducted in S. invicta, leaving their functional properties entirely unknown. This gap limits our understanding of how fire ants detect semiochemicals and how their expanded olfactory receptor repertoire is deployed at the level of individual sensilla. To address this, we asked whether sensilla basiconica in S. invicta exhibit broad or chemically selective responses to ecologically relevant odorants, and whether workers and female alates differ in their sensory tuning. Because basiconic sensilla house broadly tuned olfactory receptor neurons that respond to a wide range of ecologically relevant volatile compounds, including many of the odorants tested here, they represent the most informative starting point for characterizing odor detection in S. invicta. Their broad sensitivity, combined with their accessible position on the antenna, makes them particularly well suited for SSR. For these reasons, we focused on basiconic sensilla in the present study, with SSR analyses of trichoid and coeloconic sensilla planned for future work. The objectives of this study were therefore to: (1) use SSR to characterize the response profiles of basiconic sensilla in workers and female alates, (2) evaluate their sensitivity to a diverse panel of pheromones and general odorants, and (3) identify patterns of chemical selectivity that may underlie semiochemical detection in S. invicta.

2. Materials and Methods

2.1. Insects

Solenopsis invicta colonies were collected from Washington County, Mississippi. These colonies were maintained in Fluon-coated trays and provided with 15% sucrose solution and frozen house crickets. They were kept at 26 °C, ~70% relative humidity, and a 16:8 h dark: light photoperiod. Colony social form was determined using PCR amplification of Gp-9 alleles, following the protocol of Valles and Porter (2003) [13]. All ants used in this study originated from monogyne colonies.

2.2. Scanning Electron Microscopy

Individual female alates or major workers were decapitated, and the antennae were excised from the antennal sockets under a stereomicroscope (SMZ 1500, Nikon, Tokyo, Japan). Antennae were then mounted dorsally, ventrally, or laterally onto aluminum stubs using carbon-coated double-sided tape and subsequently sputter-coated with gold (EMX 550X auto sputter coater, Carl Zeiss, Jena, Germany). Samples were examined using an EVO 50 scanning electron microscope (Carl Zeiss, Jena, Germany), and micrographs were taken of the most distal flagellar segments of both workers and female alates.

2.3. Single Sensillum Recording (SSR)

Major workers and female alates were randomly selected for SSR analysis. Each ant was anesthetized on ice for 2–3 min and secured to a microscope slide (76 × 26 mm) with double-sided tape. Modeling clay was used to stabilize the head and body, and one antenna was positioned and affixed to the tape using fine human hair. A small section of abdominal cuticle was removed to facilitate the insertion of a reference electrode. The most distal flagellar segments were placed under a Nikon Eclipse FN1 microscope (×1000) to clearly visualize caste-specific basiconic sensilla. Tungsten microelectrodes were electrolytically sharpened in 10% KNO₂ at 5–8 V to produce ~1 μm tip diameters. The reference electrode, connected to ground, was inserted into the abdomen, while the recording electrode, connected to a preamplifier probe (INR-II; Syntech^®, Buchenbach, Germany), was inserted into the base of the targeted basiconic sensillum using motorized patch-clamp micromanipulators (Burleigh PCS-6000, Victor, NY, USA).The preamplifier was connected to an IDAC4 data acquisition controller (Syntech, Buchenbach, Germany), which interfaced with a computer for real-time signal visualization and recording. Electrophysiological signals were recorded for 20 s, starting 10 s prior to odor stimulation. Action potentials were counted offline over a 500 ms window before and after stimulation. Net spike activity was calculated by subtracting pre-stimulation spike counts from post-stimulation counts and multiplying by two to obtain the change in firing rate (spikes/s) for a single sensillum.

2.4. Chemical Delivery and Stimuli

A panel of 62 odorants representing diverse chemical groups—terpenes, terpenoids, pyrazines, pyridines, ketones, aldehydes, alcohols, acids, aliphatic and aromatic acetates, benzoates, benzyl esters, lactones, and three essential oils—was tested (Table S1). Several of these compounds are known ant pheromones, attractants, or repellents, and some have been previously examined using SSR in other ant species, including 2,3-butanedione, 2-heptanone, 6-methyl-5-hepten-2-one, 1-hexanol, ethyl acetate, geranyl acetate, 1-octen-3-ol, methyl salicylate, and isopentyl acetate [12,14,15,16]. Additional odorants were selected based on their demonstrated activity in EAG studies of S. invicta, such as 2-ethyl-3,5(6)-dimethylpyrazine [17], 2,4,6-trimethylpyridine [18], prenyl acetate, benzyl acetate [19], several structurally diverse acetate esters [20], and ylang-ylang oil. Some of tested compounds have also been identified directly in S. invicta samples [21].

Each odorant was prepared as a 100 µg/µL stock solution in paraffin oil (Sigma, molecular grade), distilled water (for formic, acetic, and lactic acids), or pentane (Sigma, 99% purity) (for springene and squalene). Ten-fold serial dilutions were made, and a single concentration (10 µg/µL) was used for all tests. This dose is widely used in electrophysiological studies of social insects [12,14,16], and in our preliminary trials, consistently elicited robust and measurable responses without saturating or suppressing neuronal activity. A 10 µL aliquot of each solution was applied to a Whatman filter paper strip (3 × 40 mm). After the solvent was evaporated, the paper strip was then inserted into a glass Pasteur pipette (Fisher Scientific, Pittsburgh, PA, USA). Filter paper treated with solvent alone served as the control. The pipette tip was inserted into a side port of a glass tube (130 mm long, 12 mm diameter) positioned approximately 5 mm from the antennal preparation. A continuous stream of humidified air was directed through the tube, and odor stimuli were delivered as 0.5 s air puffs using an air stimulus controller (CS-55; Syntech^®, Buchenbach, Germany). All odorants were tested on individual sensilla, with at least six biological replicates per chemical. Spike counts for each odorant were averaged across all replicates. Sensilla producing < 25 spikes/s in response to stimulation were classified as nonresponders [22]. SSRs were obtained from at least 6 individuals of each caste (worker and female alates). Because it is not feasible to screen all 62 odorants on a single sensillum (due to physiological constraints and recording stability), we typically used a subset of 4–5 special odorants to assign each sensillum to a functional subtype (SBI–SBIII). Once classified, the remaining odorants in the panel were tested across representative sensilla of each subtype.

2.5. Data Analysis

Data are presented as mean ± SE. Unpaired Student’s t-tests were employed for comparisons between two groups. Tuning curves were generated by plotting odorants on the x-axis according to the magnitude of the response elicited by each sensillum type. Odorants generating the strongest responses were positioned near the center of the plot, while weaker odorants were placed toward the edges. Because response strengths differ across sensillum types, the order of odorants varies between tuning curves. The K value, a statistical index of response “peakedness”, is shown in the upper right corner of each plot. K values were calculated following standard methods for quantifying the tuning curve peakedness. For each sensillum type, responses to all odorants were first normalized to the maximum response. The K value was then computed as the kurtosis of the normalized response distribution, where higher K values indicate narrower, more sharply peaked tuning and lower K values indicate broader tuning.

3. Results

3.1. Scanning Electron Micrographs (SEM) of Antennal Sensilla of S. invicta

The antenna of both S. invicta workers and female alates consists of a scape, a pedicel, and ten flagellar segments. Based on morphological characteristics, sensilla on the tenth flagellar segment were categorized into seven types: coelocapitular, coeloconic, ampullaceal, basiconic, trichoid-I, trichoid-II, and chaetic sensilla (Figure 1). Among these, basiconic sensilla are relatively short and thick, thumb-like in shape, and separated from the antennal cuticle by a distinct basal gap (Figure 1). In this study, SSRs were performed exclusively on basiconic sensilla, which are present in both workers and female alates but absent in males. No noticeable morphological differences in basiconic sensilla were observed between workers and female alates.

3.2. Distinctive Response Profiles of ORNs to General Odorants

A panel of 62 semiochemicals was tested on basiconic sensilla of S. invicta workers and female alates. Multiple spike amplitudes were recorded from each sensillum, corresponding to the activity of ORNs housed within a single basiconic sensillum. For illustrative SSR traces, 3-octanone and benzyl acetate were selected because they reliably evoked clear, representative responses across sensillum types and castes (Figure 2). 3-Octanone is a common component of alarm pheromones in other ant species, whereas benzyl acetate is a widespread floral ester. The responses to these compounds may therefore represent two key aspects of fire ant chemical communication: defense and foraging. Because individual ORNs could not be functionally distinguished, the total summed spike activity of all ORNs in each sensillum was analyzed. Cluster analysis of the summed neuronal response profiles grouped all tested basiconic sensilla into three distinct functional classes: SBI, SBII, and SBIII (Figure 3; Table S2). SBIII sensilla were not included in illustrative SSR trace (Figure 2), because their responses were generally weaker and exhibited higher variability, making them less suitable for representative trace visualization; however, all SBIII data are fully incorporated into the quantitative analyses. Odorants were organized according to the response strength, and the resulting heatmaps revealed distinct patterns for each sensillum class (Figure 3). Across the distal flagellar segments of workers, 25 sensilla were classified as SBI, 11 as SBII, and 16 as SBIII (n = 52 total). In female alates, 15 SBI, 8 SBII, and 11 SBIII sensilla were identified (n = 34 total) (Figure 4). SBI sensilla produced strong responses to most odorants, SBII sensilla showed intermediate responses, and SBIII sensilla exhibited weak or no excitatory responses (Figure 2 and Figure 3; Table S2). In workers, 60 of the 62 odorants (exceptions: ethyl phenylacetate and hexyl benzoate) elicited significant responses (≥25 Δ spikes/s) in SBI sensilla. SBII sensilla responded significantly to fifty-eight odorants, and SBIII to forty-three. In female alates, fifty-eight odorants (exceptions: ethyl phenylacetate, hexyl benzoate, ethyl acetate, and benzyl benzoate) generated significant responses in SBI sensilla, followed by fifty-five in SBII sensilla and thirty-nine in SBIII sensilla (Figure 4; Table S2). Response magnitude was categorized as: very strong (++++): ≥100 Δ spikes/s; strong (+++): 75–100 Δ spikes/s; moderate (++): 50–75 Δ spikes/s; weak (+): 25–50 Δ spikes/s; no response (.): <25 Δ spikes/s. More than 70% of the odorants at 10 µg/µL elicited strong responses (≥75 Δ spikes/s) in SBI sensilla of workers. Odorant tuning curves for both workers and female alates displayed broad response distributions, as reflected by low kurtosis values in workers: SBI (k = 0.305), SBII (k = −0.363), and SBIII (k = −0.371); and in female alates: SBI (k = 1.493), SBII (k = −0.447), and SBIII (k = −0.338). These low k values indicate that all three sensillum classes exhibit broadly tuned, generalist odorant response profiles (Figure 4).

This study also examined the olfactory responses of S. invicta basiconic sensilla to one monoterpene (ocimene), one diterpene (springene), one triterpene (squalene), one sesquiterpene (E-β-farnesene), and eleven monoterpenoids. SBI sensilla in both workers and female alates displayed strong neuronal responses (≥75 spikes/s) to all four terpenes and to eight monoterpenoids: 1,4-cineole, cineole, citronellal, camphor, menthol, geraniol, citral, and linalool. SBI sensilla showed moderate responses (≥50 spikes/s) to terpineol, (+)-terpinen-4-ol, and (–)-terpinen-4-ol (Figure 3 and Figure 5; Table S2). SBII sensilla in workers responded at similar or lower levels to most terpenes and monoterpenoids, except for (+)-terpinen-4-ol, which elicited stronger activity (Figure 3 and Figure 5; Table S2). SBIII sensilla in both workers and female alates exhibited only weak responses to all tested terpenes and terpenoids, with the exception of ocimene in workers, none of the terpene or monoterpenoid stimuli evoked firing rates above 50 spikes/s in SBIII sensilla. Caste-related differences were also evident. In the SBI class, worker sensilla displayed significantly stronger responses to ocimene than female alates, whereas SBII sensilla in workers exhibited stronger responses to E-β-farnesene. Conversely, female alates showed greater responses to (+)-terpinen-4-ol in SBI sensilla. We also tested two pyrazines: 2-ethyl-3,5(6)-dimethylpyrazine and tetramethylpyrazine, and one pyridine, 2,4,6-trimethylpyridine. SBI sensilla exhibited very strong responses to both pyrazines, firing at 127 and 106 spikes/s, respectively. Both SBI and SBII sensilla responded robustly to 2,4,6-trimethylpyridine, with firing rates of 121 and 104 spikes/s. No significant differences in SSR responses to these compounds were detected between workers and female alates (Figure 3 and Figure 5; Table S2).

Seven ketones and five aldehydes were also evaluated. SBI sensilla elicited strong olfactory responses to five out of seven ketones and four out of five aldehydes, with the firing rates exceeding 100 spikes/s. Slightly lower responses were observed for 2-tridecanone, 2-undecanone and propanal, with firing rates of 54, 65 and 92 spikes/s, respectively. SBII showed strong neuronal responses to trans-2-hexenal and benzaldehyde, with a firing rate of 92 and 100 spikes/s, respectively. Additionally, SBI sensilla in worker exhibited a significant stronger response to 3-octanone (140 spikes/s) compared with female alate (77 spikes/s) (Figure 3 and Figure 5; Table S2). SBI in workers also exhibited stronger responses to the tested alcohols and acids, with the firing rate exceeding 100 spikes/s. In contrast, SBI in female alate showed reduced responses to 3-octanol, formic and acetic acids, with firing rates of 77, 73 and 77 spikes/s, respectively. Compared with SBI in worker, SBII showed similar responses to 4-methyl-3-heptanol, 1-octen-3-ol, 1-octanol, 1-hexanol and lactic acid, but weaker responses to 3-octanol, formic acid and acetic acid. Notably, none of the neuronal responses recorded from SBIII sensilla in either workers or female alates exceeded 50 spikes/s for any alcohol or acid tested alate (Figure 3 and Figure 5; Table S2).

In previous work, several ester compounds elicited significant EAG responses in S. invicta workers and female alates [20]. Here, we further examined additional esters representing four categories: aliphatic acetates, aromatic acetates, benzoates, and benzyl esters. SBI sensilla exhibited strong neuronal responses to prenyl acetate, isopentyl acetate, geranyl acetate, dodecyl acetate, and 2-ethoxyethyl acetate, and moderate responses to pentyl acetate, hexyl acetate, and heptyl acetate, but showed only weak responses to ethyl acetate in workers. SBII sensilla generally exhibited similar or weaker responses to these aliphatic acetates, except for hexyl acetate and heptyl acetate, which elicited relatively stronger activity. SBIII sensilla produced responses comparable or weaker than SBII and showed no respond to several esters, including hexyl acetate, heptyl acetate, ethyl acetate, geranyl acetate, and dodecyl acetate (<25 spikes/s). Notably, geranyl acetate evoked strong responses in SBI sensilla but elicited no response in SBII or SBIII sensilla in either workers or female alates. SBI sensilla in workers displayed response magnitudes similar to those of female alates for most aliphatic acetates but responded more strongly to ethyl acetate. In contrast, SBI sensilla in female alates responded more strongly to pentyl acetate, hexyl acetate, and heptyl acetate. Among the four aromatic acetates tested, sensilla basiconica exhibited strong responses to β-phenethyl acetate, moderate responses to benzyl acetate and p-cresyl acetate, and no response to ethyl phenylacetate in either caste. SBI sensilla in female alates showed stronger responses to benzyl acetate and p-cresyl acetate compared with workers. For benzoates and benzyl esters, SBI sensilla in workers exhibited strong responses to methyl benzoate, methyl salicylate, ethyl benzoate, benzyl formate, and benzyl butyrate, but weak or no responses to benzyl benzoate and hexyl benzoate in both workers and female alates. SBII sensilla in workers displayed stronger responses to benzyl benzoate and benzyl butyrate, while SBII sensilla in female alates responded more strongly to methyl benzoate (Figure 3 and Figure 5; Table S2).

Three essential oils—ylang-ylang oil, Japanese mint oil, and orange oil—were also evaluated. Sensilla basiconica exhibited strong neuronal responses to ylang-ylang oil and moderate responses to Japanese mint oil and orange oil in both workers and female alates. Additionally, both SBI and SBII sensilla showed strong responses to mellein in workers, with firing rates of 109 and 107 spikes/s, respectively, while female alates showed slightly weaker responses of 80 and 78 spikes/s (Figure 3 and Figure 5; Table S2). Overall, our study investigated the responsiveness of ORNs housed in sensilla basiconica to 62 general odorants and pheromone-related compounds. Except for twelve odorants that elicited low or negligible responses (<50 spikes/s), ORNs associated with SBI and SBII sensilla in both workers and female alates displayed moderate to strong responses to the remaining 50 odorants. Workers and female alates exhibited similar response patterns to 47 odorants; however, workers responded more strongly to nine odorants, whereas female alates responded more strongly to six odorants (Figure 5; Table S2). SBI sensilla in workers exhibited stronger responses to alcohols and acids, with firing rates exceeding 100 spikes/s. In female alates, responses to 3-octanol, formic acid, and acetic acid were reduced (77, 73, and 77 spikes/s, respectively). SBII sensilla showed similar responses to 4-methyl-3-heptanol, 1-octen-3-ol, 1-octanol, 1-hexanol, and lactic acid, but significantly lower responses to 3-octanol, formic acid, and acetic acid compared with workers. SBIII sensilla in both castes produced weak responses (<50 spikes/s) to all alcohols and acids tested (Figure 3 and Figure 5; Table S2).

4. Discussion

The structures and distribution of antennal sensilla in S. invicta observed in this study were consistent with previous reports for this species [23] and with descriptions from other ants, such as Camponotus japonicus [24]. Seven sensillum types were identified in both workers and female alates: coelocapitular, coeloconic, ampullaceal, basiconic, trichoid-I, trichoid-II, and chaetic sensilla. Sensilla basiconica in both castes exhibited broad sensitivity to the odorant panel, with workers and female alates responding to 60 and 58 of the 62 odorants, respectively. Multiple spike amplitudes were detected in individual basiconic sensilla, indicating activation of numerous ORNs and suggesting that each sensillum houses dendrites from a large ORN population. This is consistent with findings that hymenopteran social insects possess some of the largest known odorant receptor (Or) gene families among insects, including major lineage-specific expansions [18]. For instance, S. invicta workers express 333 OR genes [25], and each basiconic sensillum of C. japonicus contains over 130 ORNs [24].

In most insects, pheromone-sensitive ORNs are typically located in trichoid sensilla, ORNs detecting food-related volatiles are found in basiconic sensilla, and acids and amines are primarily detected by ORNs in coeloconic sensilla [26,27]. Our findings suggest that S. invicta basiconic sensilla detect a wide variety of food- and flower-derived volatiles, consistent with the ecological relevance of many tested odorants, which commonly occur in nectar, plant materials, and insect prey [8]. Interestingly, S. invicta basiconic sensilla also responded robustly to 2-ethyl-3,5(6)-dimethyl pyrazine, which contains ~50% 2-ethyl-3,6-dimethyl pyrazine, the fire ant’s alarm pheromone. This suggests that pheromone detection in S. invicta may not be restricted to trichoid sensilla. However, whether basiconic sensilla contribute directly to alarm signaling remains to be tested behaviorally. Several acids, including formic, acetic, and lactic acids also elicited strong responses from basiconic sensilla in both workers and female alates. Responsiveness to formic acid is expected, as it is a major defensive compound used by many formicine ants. Encounters between S. invicta and formicine species are common in natural habitats, including interactions with invasive competitors such as tawny crazy ants (Nylanderia fulva) [28]. Detection of formic acid could provide adaptive value in interspecific interactions, although behavioral assays will be required to determine its functional significance in S. invicta.

Broadly tuned basiconic sensilla appear to be widespread among ants, with similar patterns reported in Camponotus laevigatus, Camponotus floridanus, and Harpegnathos saltator, where basiconic sensilla also detect pheromones and acids [11,12]. Ant sensilla may generally show broader response profiles than those of non-social insects because they house far more ORNs. For example, in the black garden ant Lasius niger, alarm pheromones can be detected not only by trichoid curvata sensilla but also by ORNs in coeloconic sensilla [29,30]. These findings collectively suggest that ant alarm pheromones are broadly tuned odorant stimuli capable of activating multiple OR classes. Because the present study focused exclusively on basiconic sensilla, future work will examine other sensillum types to determine their contributions to semiochemical detection.

The S. invicta basiconic sensilla examined in this study could be classified into three functional groups—SBI, SBII, and SBIII—based on their distinct neuronal response profiles, likely reflecting differences in receptor expression between subtypes. Overall, worker basiconic sensilla exhibited a slightly broader response spectrum and generally higher sensitivity than those of female alates. SBI sensilla in workers responded to 97% of tested odorants, compared with 94% in female alates. Moreover, worker SBI sensilla produced moderate to strong responses to 70% of odorants, whereas female alates responded similarly to only 53% of the compounds.

The responsiveness of SBI to a range of sesquiterpenes, diterpenes, triterpenes, and numerous monoterpenoids indicates that these classes of compounds may play important roles in S. invicta chemical communication. The strong response to E-β-farnesene, an alarm pheromone of many aphid species, is particularly intriguing given the widespread mutualistic interactions between ants and aphids [31]. Notably, many of the same terpenes and terpenoids that activated S. invicta sensilla were also reported to elicit strong neuronal responses in H. saltator [16]. Both SBI and SBII sensilla also exhibited robust responses to a broad range of aldehydes, ketones, alcohols, and acids. In contrast, responses to aliphatic and aromatic acetates, were more odorant-specific. Prenyl acetate, 2-ethoxyethyl acetate, methyl benzoate, benzyl formate, and isopentyl acetate elicited strong neuronal responses, whereas ethyl acetate and ethyl phenylacetate produced weak or no responses. The strong activity of ylang-ylang oil is consistent with its chemical composition which includes several constituents previously shown to evoke strong SSR responses.

Among the odorants tested, many were SSR-active in other ant species [12,14,15,16], and several had been previously shown to elicit positive EAG responses in S. invicta workers and female alates [17,19,20] (Table S1), including 2-ethyl-3,5(6)-dimethyl pyrazine, pentyl acetate, benzyl acetate, methyl benzoate, and ylang-ylang oil [19,20]. Notably, SSR and EAG responses were not always concordant. For instance, E-β-farnesene failed to elicit significant EAG responses [17], but produced clear SSR responses in the present study. Conversely, compounds such as ethyl phenylacetate and hexyl benzoate elicit no responses in both EAG and SSR assay. Overall, our findings are consistent with studies in other ant species showing that terpenes, terpenoids, aldehydes, and alcohols are among the most effective odorant stimuli [11,12,16]. Several compounds previously identified from S. invicta, including ocimene, β-springene, squalene, and mellein [32], also generated strong SSR responses, supporting their potential pheromonal or semiochemical roles in this species. The broad chemical diversity represented in this panel underscores its utility for identifying candidate attractants, repellents, and pheromones for fire ant management, while providing a valuable foundation for future studies on S. invicta olfactory coding.

Considering the similar distribution of antennal sensilla and their broadly overlapping response profiles, it is likely that S. invicta workers and female alates share a conserved general organization of sensillar function and olfactory information processing. However, because these castes perform distinct roles within the colony, differences in sensitivity to certain odorants were expected—and our results confirm this prediction. Workers exhibited significantly stronger responses to nine odorants, including ocimene, E-β-farnesene, 3-octanone, 3-octanol, formic acid, acetic acid, ethyl acetate, benzyl benzoate, and benzyl butyrate. These odorants represent promising candidates for worker-specific behavioral roles, but their involvement in foraging, defense, or interspecific interactions will require confirmation through behavioral experiments. In contrast, female alates showed stronger responses to only six odorants: (+)-terpinen-4-ol, pentyl acetate, benzyl acetate, ρ-cresol acetate, heptyl acetate, and methyl benzoate, raising the possibility that these compounds could be relevant to alate-specific behaviors. Behavioral assays will be necessary to determine whether they influence mate recognition or mating flight. Several odorants tested here, including the alarm pheromone 2-ethyl-3,5(6)-dimethyl pyrazine, 6-methyl-5-hepten-2-one, 2,3-butanedione, 2-heptanone, and 1-hexanol, have also elicited strong olfactory responses in other ant species [11,12]. This cross-species responsiveness indicates that these compounds are broadly detected by ant olfactory systems, although their specific behavioral roles may differ between species and remain to be experimentally validated.

It is important to note that SSR responses reflect the physiological sensitivity of peripheral olfactory neurons but do not, on their own, establish behavioral relevance. The odorants identified here should therefore be viewed as candidates for future behavioral assays aimed at determining their roles in communication, foraging, defense, or reproductive behaviors in S. invicta. Our electrophysiological results provide a foundation for such work by identifying compounds that reliably activate specific sensillum subtypes. Since amplitude differences were not sufficiently distinct or stable across recordings to reliably separate individual ORNs within a sensillum, and given that each basiconic sensillum contains multiple ORNs with overlapping spike amplitudes, these data provide a practical means of identifying functional response patterns but do not resolve neuron-specific contributions. Therefore, the SBI–SBIII categories should be interpreted as functional groupings rather than definitive anatomical or neuronal classifications.

5. Conclusions

Using SSR, we examined the responses of S. invicta ORNs housed in basiconic sensilla to a panel of 62 general odorants. Both workers and female alates responded to most of compounds tested, reflecting the broad tuning and high ORN diversity characteristic of ant sensilla basiconica. Nevertheless, caste-specific differences were evident for several odorants, underscoring functional specialization within the S. invicta olfactory system. This study provides foundational insight into the chemical ecology of S. invicta, offering the first comprehensive map of basiconic sensillar responses to a wide range of odorants. Understanding these peripheral olfactory mechanisms is critical for deciphering how ants detect semiochemicals and how these cues regulate ecologically important behaviors. Given the global pest status of the red imported fire ant and the environmental concerns associated with conventional insecticides, the identification of behavior-modifying chemicals such as attractants, repellents, and pheromones offer promising avenues for developing targeted, environmentally safe management tools. The odorants characterized here represent valuable candidates for future research aimed at improving fire ant monitoring and control strategies.