Age and Task Modulate Olfactory Sensitivity in the Florida Carpenter Ant Camponotus floridanus.

Age-related changes in behavior and sensory perception have been observed in a wide variety of animal species. In ants and other eusocial insects, workers often progress through an ordered sequence of olfactory-driven behavioral tasks. Notably, these behaviors are plastic, and workers adapt and rapidly switch tasks in response to changing environmental conditions. In the Florida carpenter ant, smaller minors typically perform most of the work needed to maintain the colony, while the larger majors are specialized for nest defense and rarely engage in these routine tasks. Here, we investigate the effects of age and task group on olfactory responses to a series of odorant blends in minor and major worker castes. Consistent with their respective roles within the colony, we observed significant age-associated shifts in the olfactory responses of minors as they transitioned between behavioral states, whereas the responses of majors remained consistently low regardless of age. Furthermore, we have identified a unitary compound, 3-methylindole, which elicited significantly higher responses and behavioral aversion in minor nurses than in similarly aged foragers suggesting that this compound may play an important role in brood care. Taken together, our results suggest that age- and task-associated shifts in olfactory physiology may play a critical role in the social organization of ant colonies.

Cup and Pan Behavioral Assays for Assessing Anopheles coluzzii Larval Volatile Responses.

Larval stage Anopheles coluzzii are highly reliant on their olfactory system to locate food sources and to avoid predators and less advantageous microenvironments within their aqueous habitats. The major larval chemosensory appendage, the antenna, is a complex organ with multiple sensory components that is responsible for both gustation and olfaction, thereby facilitating the detection and of both soluble and volatile compounds of biological relevance. Such compounds include food sources, predators, and a range of environmental toxicants. Unlike other mosquitoes, Anopheles coluzzii often position themselves parallel and just under the surface of their aqueous habitats, where they can detect and respond to volatile stimuli. We describe two assays for assessing the behavioral responses of larval anophelines in response to volatile chemicals. The first is a dual-choice, water-surface, inverted-cup assay designed to behaviorally characterize the response valences (attraction, neutral, and repulsion) of anopheline larvae by monitoring and recording the distribution of larvae proximate to chemical volatiles relative to solvent controls. Second, an aqueous-based larval pan behavior assay is designed to assess the responses of mosquito larvae to soluble compounds (as well as potential headspace volatiles) that are released from a point source within larval water. Here, the response valence (attractive, neutral, and repulsive) of mosquito larvae is assessed by quantifying the numbers of larvae in predefined zones proximate to chemical sources.

Physiological and Behavioral Characterization of Larval Olfaction in the Malaria Vector Mosquito Anopheles coluzzii.

When viewed from both academic and vector-control perspectives, the chemosensory biology of larval-stage anopheline mosquitoes is both enigmatic and paradoxical. As is true for all mosquito species, anopheline larvae are free-swimming organisms that use complex sensory processes to both locate nutrients and avoid predators. Because of their obligatory and therefore restrictive aquatic habitats, mosquito larvae are the most easily sampled and targeted mosquito life stage and as such they have been the focus of the majority of vector control strategies used to date. Although this might reasonably have resulted in the accumulation of a robust body of knowledge of the natural and molecular biology of larval-stage chemosensory processes, there is, instead, a paucity of such information relative to adults. Here, we describe two relatively simple laboratory-based bioassays that allow for the characterization of larval chemosensory-driven behaviors as well as an electrophysiological approach to examine the responses of larval peripheral neurons to volatile odorant stimuli. Taken together, these approaches provide a road map for the study of the chemosensory biology and chemical ecology during this important stage in the life cycle of anophelines that transmit malaria.

Examining Peripheral Electrophysiological Responses of the Larval Antennal Sensory Cone of Anopheles coluzzii to Volatile Odorants.

Anopheline larvae rely on their antennae to respond to a complex suite of stimuli with which they navigate their aquatic environment, search for food, and avoid predators and pollution. Chemosensory signaling initiates on dendrites innervating the sensory peg and sensory cone, which are adjacently located at the distal (apical) end of the larval antennae. These structures are the primary sites for the detection of both soluble and volatile semiochemicals, which are biologically relevant chemical signals (typically unitary or blends of compounds) released by one organism that affect the behavior of another. The sensory neurons housed in the larval antennal sensory cone are responsible for the signal transduction processes that initiate responses to a range of volatile stimuli. To investigate the mosquito larval olfactory neuronal response to volatile odorants, we developed this method to record, extracellularly, the electrophysiological responses of sensory cone neurons to a range of chemical stimuli. This method provides an in vivo demonstration of how mosquito larvae perceive volatile semiochemicals in their environment.

Discrete roles of Ir76b ionotropic coreceptor impact olfaction, blood feeding, and mating in the malaria vector mosquito Anopheles coluzzii.

Anopheline mosquitoes rely on their highly sensitive chemosensory apparatus to detect diverse chemical stimuli that drive the host-seeking and blood-feeding behaviors required to vector pathogens for malaria and other diseases. This process incorporates a variety of chemosensory receptors and transduction pathways. We used advanced in vivo gene-editing and -labeling approaches to localize and functionally characterize the ionotropic coreceptor AcIr76b in the malaria mosquito Anopheles coluzzii, where it impacts both olfactory and gustatory systems. AcIr76b has a broad expression pattern in female adult antennal grooved pegs, coeloconic sensilla, and T1 and T2 sensilla on the labellum, stylets, and tarsi, as well as the larval sensory peg. AcIr76b is colocalized with the Orco odorant receptor (OR) coreceptor in a subset of cells across the female antennae and labella. In contrast to Orco and Ir8a, chemosensory coreceptors that appear essential for the activity of their respective sets of chemosensory neurons in mosquitoes, AcIr76b−/− mutants maintain wild-type peripheral responses to volatile amines on the adult palps, labellum, and larval sensory cone. Interestingly, AcIr76b−/− mutants display significantly increased responses to amines in antennal grooved peg sensilla, while coeloconic sensilla reveal significant deficits in responses to several acids and amines. Behaviorally, AcIr76b mutants manifest significantly female-specific insemination deficits, and although AcIr76b−/− mutant females can locate, alight on, and probe artificial blood hosts, they are incapable of blood feeding successfully. Taken together, our findings reveal a multidimensional functionality of Ir76b in anopheline olfactory and gustatory pathways that directly impacts the vectorial capacity of these mosquitoes.

Transcriptome profiles of Anopheles gambiae harboring natural low-level Plasmodium infection reveal adaptive advantages for the mosquito.

Anopheline mosquitoes are the sole vectors for the Plasmodium pathogens responsible for malaria, which is among the oldest and most devastating of human diseases. The continuing global impact of malaria reflects the evolutionary success of a complex vector-pathogen relationship that accordingly has been the long-term focus of both debate and study. An open question in the biology of malaria transmission is the impact of naturally occurring low-level Plasmodium infections of the vector on the mosquito's health and longevity as well as critical behaviors such as host-preference/seeking. To begin to answer this, we have completed a comparative RNAseq-based transcriptome profile study examining the effect of biologically salient, salivary gland transmission-stage Plasmodium infection on the molecular physiology of Anopheles gambiae s.s. head, sensory appendages, and salivary glands. When compared with their uninfected counterparts, Plasmodium infected mosquitoes exhibit increased transcript abundance of genes associated with olfactory acuity as well as a range of synergistic processes that align with increased fitness based on both anti-aging and reproductive advantages. Taken together, these data argue against the long-held paradigm that malaria infection is pathogenic for anophelines and, instead suggests there are biological and evolutionary advantages for the mosquito that drive the preservation of its high vectorial capacity.

Pleiotropic Odorant-Binding Proteins Promote Aedes aegypti Reproduction and Flavivirus Transmission.

Insect odorant-binding proteins (OBPs) are small soluble proteins that have been assigned roles in olfaction, but their other potential functions have not been extensively explored. Using CRISPR/Cas9-mediated disruption of Aedes aegypti Obp10 and Obp22, we demonstrate the pleiotropic contribution of these proteins to multiple processes that are essential for vectorial capacity. Mutant mosquitoes have impaired host-seeking and oviposition behavior, reproduction, and arbovirus transmission. Here, we show that Obp22 is linked to the male-determining sex locus (M) on chromosome 1 and is involved in male reproduction, likely by mediating the development of spermatozoa. Although OBP10 and OBP22 are not involved in flavivirus replication, abolition of these proteins significantly reduces transmission of dengue and Zika viruses through a mechanism affecting secretion of viral particles into the saliva. These results extend our current understanding of the role of insect OBPs in insect reproduction and transmission of human pathogens, making them essential determinants of vectorial capacity. IMPORTANCE Aedes aegypti is the major vector for many arthropod-borne viral diseases, such as dengue, Zika, and chikungunya viruses. Previous studies suggested that odorant-binding proteins (OBPs) may have diverse physiological functions beyond the olfactory system in mosquitoes; however, these hypothesized functions have not yet been demonstrated. Here, we have used CRISPR/Cas9-based genome editing to functionally delete (knock out) Obp10 and Obp22 in Aedes aegypti. We showed that disruption of Obp10 or Obp22 significantly impairs female and male reproductive capacity by adversely affecting blood feeding, oviposition, fecundity and fertility, and the development of spermatozoa. We also showed that disruption of Obp10 or Obp22 significantly reduces the transmission of dengue and Zika viruses through a mechanism affecting secretion of viral particles into the saliva. Thus, our study is not only significant in understanding the functions of OBPs in mosquito biology, but also shows that OBPs may represent potent flavivirus transmission-blocking targets. Our study is in this regard particularly timely and important from a translational and public health perspective.

Neuronal odor coding in the larval sensory cone of Anopheles coluzzii: Complex responses from a simple system.

Anopheles mosquitoes are the sole vectors of malaria. Although adult females are directly responsible for disease transmission and accordingly have been extensively studied, the survival of pre-adult larval stages is vital. Mosquito larvae utilize a spectrum of chemosensory and other cues to navigate their aquatic habitats to avoid predators and search for food. Here we examine larval olfactory responses, in which the peripheral components are associated with the antennal sensory cone. Larval behavior and sensory cone responses to volatile stimuli in Anopheles coluzzii demonstrate the sensory cone is particularly tuned to alcohols, thiazoles, and heterocyclics, and these responses can be assigned to discrete groups of sensory cone neurons with distinctive profiles. These studies reveal that the anopheline larvae actively sample volatile odors above their aquatic habitats via a highly sophisticated olfactory system that is sensitive to a broad range of compounds with significant behavioral relevance.

Ammonium transporter AcAmt mutagenesis uncovers reproductive and physiological defects without impacting olfactory responses to ammonia in the malaria vector mosquito Anopheles coluzzii.

Anopheline mosquitoes are the sole vectors of malaria and rely on olfactory cues for host seeking in which ammonia derived from human sweat plays an essential role. To investigate the function of the Anopheles coluzzii ammonium transporter (AcAmt) in the mosquito olfactory system, we generated an AcAmt null mutant line using CRISPR/Cas9. AcAmt-/- mutants displayed a series of novel phenotypes compared with wild-type mosquitoes including significantly lower insemination rates during mating and increased mortality during eclosion. Furthermore, AcAmt-/- males showed significantly lower sugar consumption while AcAmt-/- females and pupae displayed significantly higher ammonia levels than their wild-type counterparts. Surprisingly, in contrast to previous studies in Drosophila that revealed that the mutation of the ammonium transporter (DmAmt) induces a dramatic reduction of ammonia responses in antennal coeloconic sensilla, no significant differences were observed across a range of peripheral sensory neuron responses to ammonia and other odorants between wild-type and AcAmt-/- females. These data support the existence in mosquitoes of novel compensatory ammonia-sensing mechanisms that are likely to have evolved as a result of the importance of ammonia in host-seeking and other behaviors.

Advances in the Study of Olfaction in Eusocial Ants.

Over the past decade, spurred in part by the sequencing of the first ant genomes, there have been major advances in the field of olfactory myrmecology. With the discovery of a significant expansion of the odorant receptor gene family, considerable efforts have been directed toward understanding the olfactory basis of complex social behaviors in ant colonies. Here, we review recent pivotal studies that have begun to reveal insights into the development of the olfactory system as well as how olfactory stimuli are peripherally and centrally encoded. Despite significant biological and technical impediments, substantial progress has been achieved in the application of gene editing and other molecular techniques that notably distinguish the complex olfactory system of ants from other well-studied insect model systems, such as the fruit fly. In doing so, we hope to draw attention not only to these studies but also to critical knowledge gaps that will serve as a compass for future research endeavors.

Mutagenesis of the orco odorant receptor co-receptor impairs olfactory function in the malaria vector Anopheles coluzzii.

Mosquitoes rely heavily on their olfactory systems for host seeking, selection of oviposition sites, and avoiding predators and other environmental dangers. Of these behaviors, the preferential selection of a human blood-meal host drives the vectorial capacity of anthropophilic female Anopheles coluzzii mosquitoes. Olfactory receptor neurons (ORNs) are dispersed across several appendages on the head and express an obligate odorant receptor co-receptor (Orco) coupled with a "tuning" odorant receptor (OR) to form heteromeric, odor-gated ion channels in the membrane of these neurons. To examine the mechanistic and functional contributions of Orco/OR complexes to the chemosensory processes of An. coluzzii, we utilized CRISPR/Cas9 gene editing to create a line of homozygous, Orco-knockout, mutant mosquitoes. As expected, orco-/- ORNs across both adult and larval stages of An. coluzzii display significantly lower background activity and lack nearly all odor-evoked responses. In addition, blood-meal-seeking, adult female, orco-/- mutant mosquitoes exhibit severely reduced attraction to human- and non-human-derived odors while gravid females are significantly less responsive to established oviposition attractants. These results reinforce observations in other insects that Orco is crucial in maintaining the activity of ORNs. In that light, it significantly influences a range of olfactory-driven behaviors central to the anthropophilic host preference that is critical to the vectorial capacity of An. coluzzii as a primary vector for human malaria.

Gene editing reveals obligate and modulatory components of the CO2 receptor complex in the malaria vector mosquito, Anopheles coluzzii.

The sensitivity to volatile carbon dioxide (CO2) produced by humans and other animals is a critical component in the host preference behaviors of the malaria vector mosquito Anopheles coluzzii. The molecular receptors responsible for the ability to sense CO2 are encoded by three putative gustatory receptor (Gr) genes (Gr22,23,24) which are expressed in a distinctive array of sensory neurons housed in maxillary palp capitate peg sensilla of An. coluzzii. Despite the identification of these components and subsequent studies, there is a paucity of understanding regarding the respective roles of these three GRs in the mosquito's CO2 transduction process. To address this, we have used CRISPR/Cas9-based gene editing technique combined with in vivo electrophysiological recordings to directly examine the role of Gr22,23,24 in detecting CO2 in An. coluzzii. These studies reveal that both Gr23 and Gr24 are absolutely required to maintain in vivo CO2 sensitivity while, in contrast, Gr22 knock out mutants are still able to respond to CO2 stimuli albeit with significantly weaker sensitivity. Our data supports a model in which Gr22 plays a modulatory role to enhance the functionality of Gr23/24 complexes that are responsible for CO2 sensitivity of mosquitoes.

Heterogeneous expression of the ammonium transporter AgAmt in chemosensory appendages of the malaria vector, Anopheles gambiae.

Ammonia is one of the principal kairomones originating from human and other animal emanations and in that context, plays an essential role in the host-seeking behaviors of the malaria vector mosquito Anopheles gambiae. Nevertheless, despite its importance in directing host-seeking, the mechanisms underlying ammonia detection in the mosquito olfactory system remains largely unknown. In addition to ongoing efforts to identify and characterize the molecular receptors that underlie ammonia sensitivity, previous studies have revealed a prominent role for ammonium transporters (Amt) in modulating antennal and behavioral responses in Drosophila melanogaster and An. gambiae. In the former, localization of DmAmt in antennal sensilla to auxiliary cells surrounding the ammonia sensory neurons led to the hypothesis that its role was to clear excess ammonium ions in the sensillar lymph. In the latter, RT-PCR and heterologous expression have been used to examine the expression and functional characteristics of the An. gambiae ammonium transporter, AgAmt. We now employ advanced transgenic tools to comprehensively examine AgAmt spatial localization across the peripheral chemosensory appendages in larvae and adult female An. gambiae. In the larval antennae, AgAmt appears localized in both neuronal and auxiliary cells. In contrast to D. melanogaster, in the adult antennae, AgAmt-derived signals are observed in both non-neuronal auxiliary cells and in sensory neurons in ammonia-responsive basiconic and coeloconic sensilla. In the maxillary palps, labella, and tarsi, AgAmt appears restricted to sensory neurons. We have also characterized the responses to ammonia of adult antennal coeloconic sensilla and maxillary palp capitate pegs revealing a correlation between sensillar AgAmt expression and ammonia sensitivity. Taken together, these data suggest that AgAmt may play heterogeneous roles in the adult and larval chemosensory apparatus and potentially broad utility as a supra-receptor target in mosquito control.

Odor coding of nestmate recognition in the eusocial ant Camponotus floridanus.

In eusocial ants, aggressive behaviors require the ability to discriminate between chemical signatures such as cuticular hydrocarbons that distinguish nestmate friends from non-nestmate foes. It has been suggested that a mismatch between a chemical signature (label) and the internal, neuronal representation of the colony odor (template) leads to aggression between non-nestmates. Moreover, a definitive demonstration that odorant receptors are responsible for the processing of the chemical signals that regulate nestmate recognition has thus far been lacking. To address these issues, we have developed an aggression-based bioassay incorporating highly selective modulators that target odorant receptor functionality to characterize their role in nestmate recognition in the formicine ant Camponotus floridanus Electrophysiological studies were used to show that exposure to either a volatilized antagonist or an agonist eliminated or dramatically altered signaling, respectively. Administration of these compounds to adult workers significantly reduced aggression between non-nestmates without altering aggression levels between nestmates. These studies provide direct evidence that odorant receptors are indeed necessary and sufficient for mediating aggression towards non-nestmates. Furthermore, our observations support a hypothesis in which rejection of non-nestmates depends on the precise decoding of chemical signatures present on non-nestmates as opposed to the absence of any information or the active acceptance of familiar signatures.

Olfactory receptor gene abundance in invasive breast carcinoma.

Expression of olfactory receptors (ORs) has been reported in many human tissues outside the nasal epithelium. ORs have been validated as biomarkers in prostate cancer. In breast cancer, however, the expression and role of OR genes remain understudied. We examined the significance of OR transcript abundance in a large invasive breast carcinoma population and identified two OR genes, OR2W3 and OR2B6 to be potentially correlated to breast cancer progression. 960 breast invasive tumors and 56 human breast cancer cell lines were assessed for OR gene expression and 21 OR genes were highly abundant among 198 cases. Our transcriptome analysis discovered three significantly abundant OR genes among three sub-populations of invasive breast carcinoma patients. OR2W3 was correlated with invasion genes and basal-like subtype whereas OR2B6 was correlated with proliferation genes and luminal A subtype. Analyzing the OR gene upregulation among breast cancer cell lines showed that OR2B6 and OR2W3 were abundant similar to invasive breast tumors. Our study suggests that specific OR genes may be correlated with breast cancer characteristics, making ORs potential new diagnostic, and/or treatment markers. This study suggests future directions for the exploration of a role for ORs in the mechanisms of breast cancer proliferation and progression.

Antennal Olfactory Physiology and Behavior of Males of the Ponerine Ant Harpegnathos saltator.

In comparison to the large amount of study on the communication abilities of females in ant societies and their associated chemical ecology and sensory physiology, such study of male ants has been largely ignored; accordingly, little is known about their olfactory sensory capabilities. To address this, we explored peripheral odor sensitivities in male Harpegnathos saltator by measuring the electrophysiological activity of olfactory sensory neurons within antennal trichoid and coeloconic sensilla using an extracellular recording technique. In an initial trial of 46 compounds, sensilla trichodea responded strongly to two alarm pheromone components, while a limited number of non-hydrocarbon odorants elicited strong responses in sensilla coeloconica. Both sensillar types responded indifferently to 31 cuticular hydrocarbons (CHCs) and synthetic long-chain hydrocarbons (HCs) typically found on insect cuticle. In a search for sensilla responding to CHCs and other compounds, we found some sensilla that responded to synthetic HCs and CHCs from virgin queen postpharyngeal glands that are potentially used in close range mate recognition. Olfactometer bioassays of male ants to 15 non-HCs correlated sensory responsiveness to the respective behavioral responses. Comparing olfactory responses between H. saltator males and females, we found that sensilla coeloconica and basiconica of workers showed greater responses and broader selectivity to all compounds. The rarity of CHC-responding trichoid sensilla in Harpegnathos males suggests a more specific role in sexual communication compared to that in females, which use CHCs in a broader communication context.

Characterization of Chemosensory Responses on the Labellum of the Malaria Vector Mosquito, Anopheles coluzzii.

Anopheles gambiae coluzzii (An. coluzzii) uses olfaction to modulate a range of critical behaviors that are essential for survival and reproduction; most notably, host preference and selection underlie its vectorial capacity for human malaria. As is the case for all mosquitoes, An. coluzzii has three specialized peripheral olfactory appendages-the antennae, maxillary palps and labella-which are used to detect and orient in response to a large variety of olfactory cues. Of these, neither the molecular nor the physiological significance of the labellum have been thoroughly characterized despite suggestions that labial-derived odorant reception is critical for close-range host attraction. Here we report global chemoreceptor transcriptome profiles together with a systematic electrophysiological analysis of labial T2 sensilla, and associated behavioral responses of female An. coluzzii. Single sensillum recordings of the T2 sensilla revealed robust responses to odorants previously associated with human sweat and oviposition sites and identified a 10-component blend that elicited attraction in a dual-choice landing bioassay designed to mimic host seeking in which non-blood fed females were significantly more attracted to the labial-responsive odorant blend as compared to gravid females. Taken together, these data suggest that, in An. coluzzii, olfactory responses derived from the labellum contribute to host-seeking.

Deciphering the olfactory repertoire of the tiger mosquito Aedes albopictus.

BACKGROUND: The Asian tiger mosquito Aedes albopictus is a highly invasive species and competent vector of several arboviruses (e.g. dengue, chikungunya, Zika) and parasites (e.g. dirofilaria) of public health importance. Compared to other mosquito species, Ae. albopictus females exhibit a generalist host seeking as well as a very aggressive biting behaviour that are responsible for its high degree of nuisance. Several complex mosquito behaviours such as host seeking, feeding, mating or oviposition rely on olfactory stimuli that target a range of sensory neurons localized mainly on specialized head appendages such as antennae, maxillary palps and the mouthparts. RESULTS: With the aim to describe the Ae. albopictus olfactory repertoire we have used RNA-seq to reveal the transcriptome profiles of female antennae and maxillary palps. Male heads and whole female bodies were employed as reference for differential expression analysis. The relative transcript abundance within each tissue (TPM, transcripts per kilobase per million) and the pairwise differential abundance in the different tissues (fold change values and false discovery rates) were evaluated. Contigs upregulated in the antennae (620) and maxillary palps (268) were identified and relative GO and PFAM enrichment profiles analysed. Chemosensory genes were described: overall, 77 odorant binding proteins (OBP), 82 odorant receptors (OR), 60 ionotropic receptors (IR) and 30 gustatory receptors (GR) were identified by comparative genomics and transcriptomics. In addition, orthologs of genes expressed in the female/male maxillary palps and/or antennae and involved in thermosensation (e.g. pyrexia and arrestin1), mechanosensation (e.g. piezo and painless) and neuromodulation were classified. CONCLUSIONS: We provide here the first detailed transcriptome of the main Ae. albopictus sensory appendages, i.e. antennae and maxillary palps. A deeper knowledge of the olfactory repertoire of the tiger mosquito will help to better understand its biology and may pave the way to design new attractants/repellents.

An Engineered orco Mutation Produces Aberrant Social Behavior and Defective Neural Development in Ants.

Ants exhibit cooperative behaviors and advanced forms of sociality that depend on pheromone-mediated communication. Odorant receptor neurons (ORNs) express specific odorant receptors (ORs) encoded by a dramatically expanded gene family in ants. In most eusocial insects, only the queen can transmit genetic information, restricting genetic studies. In contrast, workers in Harpegnathos saltator ants can be converted into gamergates (pseudoqueens) that can found entire colonies. This feature facilitated CRISPR-Cas9 generation of germline mutations in orco, the gene that encodes the obligate co-receptor of all ORs. orco mutations should significantly impact olfaction. We demonstrate striking functions of Orco in odorant perception, reproductive physiology, and social behavior plasticity. Surprisingly, unlike in other insects, loss of OR functionality also dramatically impairs development of the antennal lobe to which ORNs project. Therefore, the development of genetics in Harpegnathos establishes this ant species as a model organism to study the complexity of eusociality.

Specialized odorant receptors in social insects that detect cuticular hydrocarbon cues and candidate pheromones.

Eusocial insects use cuticular hydrocarbons as components of pheromones that mediate social behaviours, such as caste and nestmate recognition, and regulation of reproduction. In ants such as Harpegnathos saltator, the queen produces a pheromone which suppresses the development of workers' ovaries and if she is removed, workers can transition to a reproductive state known as gamergate. Here we functionally characterize a subfamily of odorant receptors (Ors) with a nine-exon gene structure that have undergone a massive expansion in ants and other eusocial insects. We deorphanize 22 representative members and find they can detect cuticular hydrocarbons from different ant castes, with one (HsOr263) that responds strongly to gamergate extract and a candidate queen pheromone component. After systematic testing with a diverse panel of hydrocarbons, we find that most Harpegnathos saltator Ors are narrowly tuned, suggesting that several receptors must contribute to detection and discrimination of different cuticular hydrocarbons important in mediating eusocial behaviour.Cuticular hydrocarbons (CHC) mediate the interactions between individuals in eusocial insects, but the sensory receptors for CHCs are unclear. Here the authors show that in ants such as H. saltator, the 9-exon subfamily of odorant receptors (HsOrs) responds to CHCs, and ectopic expression of HsOrs in Drosophila neurons imparts responsiveness to CHCs.