Male moths rely on olfactory cues to find females for reproduction. Males also use volatile plant compounds (VPCs) to find food sources and might use host-plant odor cues to identify the habitat of calling females. Both the sex pheromone released by conspecific females and VPCs trigger well-described oriented flight behavior toward the odor source. Whereas detection and central processing of pheromones and VPCs have been thought for a long time to be highly separated from each other, recent studies have shown that interactions of both types of odors occur already early at the periphery of the olfactory pathway. Here we show that detection and early processing of VPCs and pheromone can overlap between the two sub-systems. Using complementary approaches, i.e., single-sensillum recording of olfactory receptor neurons, in vivo calcium imaging in the antennal lobe, intracellular recordings of neurons in the macroglomerular complex (MGC) and flight tracking in a wind tunnel, we show that some plant odorants alone, such as heptanal, activate the pheromone-specific pathway in male Agrotis ipsilon at peripheral and central levels. To our knowledge, this is the first report of a plant odorant with no chemical similarity to the molecular structure of the pheromone, acting as a partial agonist of a moth sex pheromone.

Recognition of intra-specific olfactory signals within a complex environment of plant-related volatiles is crucial for reproduction in male moths. Sex pheromone information is detected by specific olfactory receptor neurons (Phe-ORNs), highly abundant on the male antenna. The information is then transmitted to the pheromone processing macroglomerular complex (MGC) within the primary olfactory center, the antennal lobe, where it is processed by local interneurons and projection neurons. Ultimately a behavioral response, orientation toward the pheromone source, is elicited. Volatile plant compounds (VPCs) are detected by other functional types of olfactory receptor neurons (ORNs) projecting in another area of the antennal lobe. However, Phe-ORNs also respond to some VPCs. Female-produced sex pheromones are emitted within a rich environment of VPCs, some of which have been shown to interfere with the detection and processing of sex pheromone information. As interference between the different odor sources might depend on the spatial and temporal features of the two types of stimuli, we investigated here behavioral and neuronal responses to a brief sex pheromone blend pulse in a VPC background as compared to a control background in the male noctuid moth Agrotis ipsilon. We observed male orientation behavior in a wind tunnel and recorded responses of Phe-ORNs and MGC neurons to a brief sex pheromone pulse within a background of individual VPCs. We also recorded the global input signal to the MGC using in vivo calcium imaging with the same stimulation protocol. We found that VPCs eliciting a response in Phe-ORNs and MGC neurons masked responses to the pheromone and decreased the contrast between background odor and the sex pheromone at both levels, whereas α-pinene did not interfere with first order processing. The calcium signal produced in response to a VPC background was tonic, lasting longer than the VPC stimulus duration, and masked entirely the pheromone response. One percent heptanal and linalool, in addition to the masking effect, caused a clear delay in responses of MGC neurons to the sex pheromone. Upwind flight toward the pheromone in a wind tunnel was also delayed but otherwise not altered by different doses of heptanal.

As in vertebrates, the insect steroid hormones, especially 20-hydroxyecdysone (20E), initiate and regulate sexual behavior by acting on the central nervous system. This 20E action is, in part, triggered by transcriptional events mediated through the binding of 20E to a heterodimer comprising the ecdysone receptor (EcR) and ultraspiracle (USP). However, to date, our knowledge about this genomic steroid pathway remains incomplete. In moths, males detect female sex pheromones, eliciting stereotyped sexual behavior. In Agrotis ipsilon males, the behavioral response and the neuronal sensitivity to sex pheromone in the olfactory center, the antennal lobe (AL), increase with age. We recently showed that 20E controlled this age-dependent olfactory plasticity via the activation of an EcR/USP-dependent pathway in the AL. Here, we cloned the gene encoding A. ipsilon synaptotagmin I (AisytI), a presynaptic vesicle protein known to act as a calcium sensor in neurotransmitter release. AisytI was expressed in the AL, where its amount increased with age, whereas its knockdown inhibited the sex pheromone-oriented flight of males. 20E administration to males induced AL AisytI expression in a dose-dependent and time-dependent manner. Moreover, A. ipsilon EcR silencing caused decreases in AL AisytI expression and the behavioral response to sex pheromone. Our results show that the synaptotagmin I gene is a target gene for the genomic steroid signaling that controls the expression of insect sexual behavior by acting on central sex pheromone processing. This study thus represents a significant advance in our understanding of the steroid actions that influence neural functions, and thereby behavioral plasticity, in various organisms. DatabaseThe nucleotide sequence of Agrotis ipsilon synaptotagmin I is available in the DDBJ/EMBL/ GenBank databases under the accession number KJ863735.