In insects, olfaction is the primary sensory modality that drives several key behaviors. For eusocial Hymenoptera, chemical communication among individuals in a colony influences key social behaviors such as nestmate recognition and reproductive division of labor (1). An important class of chemicals that mediates these social interactions is cuticular hydrocarbons (CHCs), mixtures of long-chain alkanes and alkenes which coat the hydrophobic epicuticle of terrestrial insects and other arthropods (2). CHCs primarily serve as protective barriers against desiccation and abrasion in many terrestrial arthropods, but eusocial Hymenoptera and other social insect taxa use CHC profiles to signal task allocation, discriminate nestmates from non-nestmates, and suppress ovary development in sterile workers (3–6).

In the Indian jumping ant, Harpegnathos saltator, the roles and detection of CHCs have been previously examined at several levels. Harpegnathos saltator is a primitively eusocial ant species in the subfamily Ponerinae that has relatively small colony sizes (65 ± 40 workers) and a unique social structure defined by reproductive plasticity (7). When a queen’s fecundity decreases, workers in the colony can transition to a reproductive caste, termed a gamergate, and take over egg-laying duties. During the worker-to-gamergate transition, their CHC profiles shift to longer chain hydrocarbons which resemble those of reproductive queens (8). Notably, 13,23-dimethylheptatriacontane (13,23-DiMeC37) is the largest CHC by mass found in the species and is only present in reproductive queens and gamergates (8). It is hypothesized that 13,23-DiMeC37 and a subset of other CHCs constitute a fertility signal which serves to suppress ovarian activity and gamergate transition in workers.

CHCs in ants are detected by basiconic sensilla on the antennae. These sensilla are only present in female castes, can house an abundance of olfactory receptor neurons, and are increasingly abundant in the distal segments of the antennae (9, 10). In H. saltator and ants in the Camponotus genus, electrophysiological analysis of basiconic sensilla revealed that they are responsive to a wide range of CHCs (11–13). Additionally, H. saltator gamergates showed decreased sensitivity to several long-chain CHCs compared to workers, suggesting that an olfactory shift accompanies their increase in reproductive activity (11).

To understand the molecular basis of CHC detection, attention has turned to the odorant receptors (ORs) in eusocial insects. Genomic analyses in ants have revealed greatly expanded OR families in ants and other eusocial Hymenoptera compared to other insects, with >300 ORs identified across several species (14–17). Notably, the 9-exon subfamily of ant ORs, which represents roughly a third or more of the entire family, is highly enriched in antennal transcriptomes, and is under significant positive selection (16–19). Recent studies using heterologous expression in Drosophila melanogaster olfactory receptor neurons (ORNs) have shown that H. saltator ORs (HsOrs) are sensitive to HCs, both individually and as mixtures in cuticular extracts. The 9-exon subfamily HsOrs showed variable tuning to HCs, with some broadly responsive to several HCs and others sensitive to only a single or a few structurally similar HCs (20). Interestingly, HC sensitivity was also observed in several other HsOr clades, suggesting that despite the prominence of 9-exon HsOr genes, HC detection may rely on combinatorial coding that extends beyond this expansive subfamily (21).

In this study, our objective was to explore HsOr tuning to HCs broadly across the 381-gene HsOr family, specifically in the smaller subfamilies that have yet to be characterized. Using Drosophila, we functionally expressed 23 HsOrs from across 16 different subfamilies. Of these subfamilies, 12 did not contain a previously characterized HsOr. Each HsOr was screened against a panel of HCs, many of which are found in H. saltator cuticular extracts (8). We then extended our analyses to include all existing HsOr functional data to observe trends in coding capacity relative to HsOr subfamily and HC chain length. With a total of 70 HsOrs screened across this HC panel, this work provides insights into the combinatorial coding that can facilitate chemical communication across social insects.

HsOr functional responses were assessed against a panel of both commercially available alkanes and synthesized methyl-branched and unsaturated HCs as previously described (20, 21). In this panel, 17 of the total 39 HCs naturally occur on the H. saltator cuticle, with the others serving to demonstrate the molecular receptive range of HsOr binding pockets (8). Additionally, some of these HCs may be ecologically relevant for H. saltator, as CHCs from small arthropod prey can be encountered during both capture and consumption. A total of 23 HsOr genes were either cloned from antennal cDNA or synthesized based on sequences from antennal transcriptomes (Figure 1A) (16). These HsOr genes were chosen to cover a broad range of HsOr genes from the 20 subfamilies outside of the expansive 9-exon subfamily that was previously characterized (20). During the selection process, priority was given to HsOr genes with higher transcript abundance in worker antennae relative to males (Figures 1A, B). We also included two genes from very small subfamilies – HsOr62 from subfamily M and HsOr219 from subfamily C, even though their expression was not female-biased. This was done in order to include members from every unexplored HsOr subfamily in our study. Finally, we included HsOr16 to compare it to the very closely related HsOr13, even though HsOr16 also did not show female-biased expression.

Heterologous expression of HsOr genes in ORNs of D. melanogaster was achieved by generating a UAS-HsOr transgenic fly and crossing it with a robust Orco-GAL4 driver line (Bloomington Drosophila Stock Center #23292). Here, the expressed HsOr protein forms a functional odorant receptor complex by oligomerizing with the endogenous D. melanogaster Orco (DmOrco). Due to the demonstrated functional conservation of Orco, HsOr responses with DmOrco are expected to be similar to those when paired with the native HsOrco (22). As done previously, HsOr function was assessed using single-sensillum recordings (SSR) from the ab2A neuron and a screening panel of 39 HCs, of which 17 have been found in H. saltator cuticular extracts (Figure 1C) (8, 20, 21).

From this dataset of 897 distinct HC-HsOr pairs, we found that eleven of the 23 HsOrs responded to at least one HC above 30 Δspikes/s, a threshold set six times higher than the native ab2A spontaneous firing rate as done previously (Figures 2, 3) (23). All of these >30 Δspikes/s responses were elicited by HCs with a chain length of C21 or longer, indicating a slightly lower detection range relative to the previously characterized HsOrs where no responses were found below C27 (20, 21). Six HsOrs (HsOr240, HsOr191, HsOr213, HsOr70, HsOr157, HsOr139) demonstrated broad tuning with >30 Δspikes/s responses to seven or more HCs on the panel. Additionally, 6 subfamilies not previously characterized had HsOrs with HC responses >30 Δspikes/s. Twelve HsOrs did not show a >30 Δspikes/s to any HC on the panel, with four (HsOr219, HsOr187, HsOr165, HsOr129) having generally weak negative responses across the panel where the HCs inhibited the firing rate of HsOr-expressing ab2A neuron.

One receptor, HsOr152 of the V subfamily, was found to be narrowly tuned to C28, with a mean response of 87.5 Δspikes/s (Figures 1C, 2, 3). Notably, two other V subfamily receptors, HsOr129 and HsOr165, displayed largely inhibitory responses to many HCs on the panel, with several pairs that resulted in -10 Δspikes/s or greater inhibition of the ab2A spontaneous firing rate. With the exception of HsOr152, most receptors characterized in this study showed overall weaker and broader tuning to HCs relative to the previously decoded 9-exon HsOrs (20, 21).

To further explore the HC sensitivity, we performed dose-response assays for the most efficacious HsOr-ligand pairs for the 11 HsOrs with a >30 Δspikes/s response (Figure 4). Dose response curves showed that HC detection occurred in a concentration-dependent manner (Figure 4). Interestingly, several HsOrs had notably larger (HsOr196, HsOr191, HsOr157) or smaller (HsOr240, HsOr152) responses to 20 nmol of HC when compared to the same dose in the screening panel, suggesting that the repeated HC stimulation during the screen may positively or negatively affect receptor response dynamics. Additionally, no HsOrs showed any responses above 10 Δspikes/s at or below 2 nmol of HC.

The 23 HsOrs characterized in this study demonstrate that HC detection is not limited to the greatly expanded 9-exon subfamily, but extends to several additional HsOrs found in the other 20 subfamilies, in agreement with a previous study (21). HC response profiles varied greatly across and within subfamilies but overall showed broader tuning and relatively weaker responses for most HsOrs in this study compared to the 9-exon study (excluding HsOr152). Although some HC-evoked responses were below our 30 Δspikes/s threshold, it is still possible that they may play a role in the combinatorial coding of HCs in the H. saltator antennae, where a single HC can elicit responses from multiple chemosensory receptors. While this 30 Δspikes/s threshold does not directly indicate biological relevance, it is a useful measure for comparing HsOr response data across HsOrs tested and consistent with previous research (21). The characterized HsOrs displayed a preferential tuning to longer chain HCs above C21, which can be attributed to their ecological relevance as several of these have been found in cuticular extracts from H. saltator (8). The panel used in this study did not contain all the CHCs found in H. saltator cuticular extracts due to the high cost and technical difficulty of custom synthesis of methyl-branched long-chain hydrocarbons. Thus, as with all OR screening studies, it is likely that our panel may not contain all the true ligands of the characterized receptors.

The narrow ligand specificity of some of these HsOrs was evidenced by the strong response of HsOr152 to C28 but weak response to C27, C29, and 2-MeC28. Similarly, a one-carbon difference in HC length between C29 and C30 resulted in either a lack of response (3.8 Δspikes/s) or a substantial excitatory response (33.0 Δspikes/s), respectively, in HsOr115. This discriminatory power indicates that these HsOrs can distinguish small structural differences between HCs, suggesting that a deeper exploration of additional methyl-branched and unsaturated HCs, preferably those found in cuticular extracts, may yield additional ligand-receptor pairs. Additionally, a systematic exploration of the effect of methyl branch position on HsOr responses could help further define the molecular receptive range of HC receptors.

Furthermore, four HsOrs (HsOr219, HsOr187, HsOr165, HsOr129) showed broad inhibitory responses to most HCs on the screening panel. Of these, HsOr219 is notable for its transcript abundance in male antennae (Figure 1B). This inhibition may play an important role in olfactory system modulation at the receptor level of the periphery, as previously observed in Drosophila where important odorants from overripe fruit can inhibit the CO₂ receptor (24). Here, the presence of salient HCs could reduce the ability to detect environmental odorants through receptor-mediated inhibition. This mechanism could favor the detection of social cues through other receptors while turning down the “gain” of environmental stimuli.

Notably, the V subfamily showed a high degree of diversification and specialization with receptors being a combination of narrowly tuned (HsOr152), inhibited (HsOr129 and HsOr165) and broadly excited (HsOr157 and HsOr139) by HCs. Specifically, many of the >30 Δspikes/s responses of HsOr157 were evoked by odd-numbered carbon chain lengths, reflecting a similar pattern found in H. saltator CHC extracts, which are strongly biased towards odd-numbered chains (8). This prevalence reflects their biosynthesis, which consists of the sequential attachment of two-carbon acyl-CoA units, and culminating in a decarboxylation to give the odd-numbered chains, though even-numbered CHCs are also present in lower abundances (2, 8). The wide functional variation within the V subfamily HsOrs aligns with the reported rapid expansion and diversification observed at the genetic level of the ant ORs, allowing them to display strikingly different ligand affinity and efficacy (18).

Many of the dose-response curves (Figure 4) showed less potent responses to the tested HCs when compared to the previously tested 9-exon HsOrs (20). Specifically, 2 nmol of the most efficacious ligand did not elicit a notable response. Additionally, some responses from the dose response data are higher or lower than those from the full screening panel. This could be due to inherent variability in heterologous in vivo expression, but it is also possible that sequential stimulation with the full HC panel could cause HsOr-expressing ORNs to be sensitized or desensitized, as has been observed in other insects, after exposure to structurally related HC ligands (25–27). Furthermore, including previously tested 9-exon HsOrs in future characterization efforts should address potential experimental variability.

Twelve HsOrs did not respond to any of the HCs on the screening panel (<30 Δspikes/s). Given that these HsOrs are expressed in H. saltator antennae and have maintained open reading frames in their sequence, some possible explanations for the lack of a response to our HC panel could be that these HsOrs may detect non-HC ligands, be narrowly tuned to a HC not in our screening panel, be difficult to express in our heterologous Drosophila expression system, or even no longer bind a ligand. An expanded screening panel or expression in cell culture-based heterologous systems could address some of these possibilities.

With the addition of these 23 HsOrs, a total of 70 HsOrs have now been tested against a largely overlapping HC panel. To make these 70 HsOr response profiles accessible, we have developed a web application, the HsOr Response Database (https://pask-lab.shinyapps.io/HsOrResponseDatabase/) to serve as a data repository. Overall, the functional data from across the broader HsOr family shows similar trends of generally weaker responses and broader tuning as compared to those in the 9-exon HsOrs (20, 21). Combining all functional data by HsOr subfamily, we saw the greatest excitatory responses to straight-chain, long HCs between C28-37 (Figure 5). This mirrors the CHC profiles previously identified in H. saltator cuticular extracts, where 66 of 71 CHCs have a 28–37 carbon chain length (8). Given the CHC diversity in this range, it is possible that the plethora of methylated, dimethylated, and unsaturated HCs not in our screening panel may elicit greater responses from our characterized HsOrs, and these would be interesting to test in our heterologous synthesis in future studies. It is also important to note that the seemingly reduced responses to the methyl-branched alkanes from HsOrs outside the 9-exon subfamily, specifically the E, H, L, and V subfamilies, can be a result of 22 out of 70 HsOrs only being tested against straight chain alkanes (as shown in Figure 6A). Additionally, the two unsaturated HCs in the panel, Z9-C29:1 and Z9-C31:1, though present in H. saltator cuticular extracts, have yet to elicit notable responses from the screened 70 HsOrs. Inhibitory responses were present in a few representative subfamilies, with more widespread inhibition observed for short-chain HCs (C10-C17). It is also possible that the true ligands for these receptors have yet to be identified and may come from non-HC general odorant chemical classes, similar to previously studied HsOrs (16, 21).

We generated a heat map of all the 70 HsOr responses to the HC panel and then calculated the average receptor response for each HC (Figures 6A, B). The HCs with the six largest average responses were, in order, C35, 15-MeC31, C32, 13,23-DiMeC37, 13-MeC31, and 5-MeC31. We then generated a “receptivity” value for each HsOr-HC response by multiplying the Δspikes/s by each receptor’s previously reported expression value from a worker antennal transcriptome, as has been previously done with heterologous mosquito OR response data (Figure 6C) (16, 28). When weighting these responses and averaging the receptivity for each HC, the top six receptivity values were 15-MeC31, C35, C32, 5-MeC31, 13,23-DiMeC37, and 13-MeC29. Previous electrophysiological recordings from basiconic sensilla of H. saltator workers showed responses to both straight-chain and methyl-branched HCs, but single-unit resolution is not possible given the >50 neurons housed in the basiconic sensilla (9, 11). In the combined neuronal responses of basiconic sensilla, C10, C11, C23, C25, C28, and C32 elicited the six largest responses. While C28 and C32 elicited some large responses in our HsOr functional data, the other hydrocarbons did not activate any of the 70 characterized HsOrs, suggesting that other HsOrs in the odorant receptor family are responsive to these shorter chain HCs.

Overall, these functional data provide further evidence for the combinatorial coding involved in CHC detection by eusocial insects, where HsOrs can be either broadly or narrowly tuned to CHC ligands, and an individual CHC can bind to several HsOrs. We suggest that H. saltator can serve as a eusocial insect model for understanding the molecular basis of olfactory communication and its role in shaping social behaviors in a colony environment. While recent and further studies, such as the knockout of Orco in two ant species, are integral for a better understanding of the neurobiological mechanisms and behavioral outputs related to ant olfactory communication, functional characterization of these HsOrs can provide further insights into the HsOr-CHC interactions (29, 30). Recent advances in cryo-EM 3D structures of insect ORs and protein modeling platforms can explore the observed specificity and/or flexibility of CHC-binding pockets with ant ORs (31–34).