Finding ideal oviposition sites is a task of vital importance for all female insects. To ensure optimal conditions for their progeny, females of herbivorous insects detect not only odors of a relevant host plant but also chemicals released by eggs, named oviposition-deterring pheromones (ODPs). It is reported that such chemicals play critical roles in suppressing female oviposition behavior, however, the molecular mechanism underlying detection of egg derived ODPs remains elusive. Here, we have identified three specific fatty acid methyl esters from the surface of eggs of Helicoverpa armigera serving as ODPs - methyl oleate (C18:1ME), methyl palmitate (C16:0ME), and methyl stearate (C18:0ME). We demonstrated that these ODPs are detected by the receptor, HarmOR56, exclusively expressed in sensilla trichodea on female antennae. To assess the significance of this receptor, we disrupted HarmOR56 in H. armigera using CRISPR/Cas9 and found that mutant females did not respond to the ODPs neither in behavioral nor in electrophysiological tests. We therefore conclude that HarmOR56, is indispensable for identifying the ODPs. This study explores, for the first time, how a female-specific odorant receptor detects chemicals from conspecific eggs. Our data elucidates the intriguing biological phenomenon of repulsion to conspecific eggs during oviposition and contributes with new insight into a female-specific olfactory pathway linked to reproduction.

Simple Summary The noctuid moth, Helicoverpa armigera, is one of the globally most damaging agricultural pest insects. Generally, exploration of the male- and female-specific neural architecture underlying its reproductive behavior is crucial for developing biological and environment-friendly alternatives to the traditional pest control management. In this study, we utilized the opportunity to uncover putative sex differences in H. armigera by comparing details in the brain anatomy between the male and female hemispheres in one gynandromorphic individual. The methods included synapsin immunostaining, confocal microscopy, and the digital reconstruction of several brain areas involved in processing input about odor and vision, respectively. The results demonstrated sex-specific arrangements applying to distinct olfactory neuropils, including not only the primary olfactory center, the antennal lobe, but also higher order levels involved in odor-associated memory formation. Abstract The present study was dedicated to investigating the anatomical organization of distinct neuropils within the two brain hemispheres of a gynandromorphic moth of the species Helicoverpa armigera. High quality confocal imaging of a synapsin immuno-stained preparation combined with three-dimensional reconstructions made it possible to identify several brain structures involved in processing odor input and to measure their volumes in the male and female hemispheres. Thus, in addition to reconstructing the antennal lobes, we also made digital models of the mushroom body calyces, the pedunculus, and the vertical and medial lobes. As previously reported, prominent sexual dimorphism was demonstrated in the antennal lobes via the identification of a male-specific macroglomerular complex (MGC) and a female-specific complex (Fc) in each of the two brain hemispheres of the gynandromorph. Additionally, sex-specific differences were found in volume differences for three other neuropil structures—the calyces, pedunculus, and vertical lobe. The putative purpose of larger volumes of three mushroom body neuropils in females as compared to males is discussed.

The pheromone system of heliothine moths is an optimal model for studying principles underlying higher-order olfactory processing. In Helicoverpa armigera, three male-specific glomeruli receive input about three female-produced signals, the primary pheromone component, serving as an attractant, and two minor constituents, serving a dual function, i.e. attraction versus inhibition of attraction. From the antennal-lobe glomeruli, the information is conveyed to higher olfactory centers, including the lateral protocerebrum, via three main paths – of which the medial tract is the most prominent. In this study, we traced physiologically identified medial-tract projection neurons from each of the three male‑specific glomeruli with the aim of mapping their terminal branches in the lateral protocerebrum. Our data suggest that the neurons’ wide-spread projections are organized according to behavioral significance, including a spatial separation of signals representing attraction versus inhibition – however, with a unique capacity of switching behavioral consequence based on the amount of the minor components.

The pheromone system of heliothine moths is an optimal model for studying principles underlying higher-order olfactory processing. In Helicoverpa armigera, three male-specific glomeruli receive input about three female-produced signals, the primary pheromone component, serving as an attractant, and two minor constituents, serving a dual function, i.e. attraction versus inhibition of attraction. From the antennal-lobe glomeruli, the information is conveyed to higher olfactory centers, including the lateral protocerebrum, via three main paths - of which the medial tract is the most prominent. In this study, we traced physiologically identified medial-tract projection neurons from each of the three male specific glomeruli with the aim of mapping their terminal branches in the lateral protocerebrum. Our data suggest that the neurons' wide-spread projections are organized according to behavioral significance, including a spatial separation of signals representing attraction versus inhibition - however, with a unique capacity of switching behavioral consequence based on the amount of the minor components.

Many insects possess the ability to detect fine fluctuations in the environmental CO2 concentration. In herbivorous species, plant-emitted CO2, in combination with other sensory cues, affect many behaviors including foraging and oviposition. In contrast to the comprehensive knowledge obtained on the insect olfactory pathway in recent years, we still know little about the central CO2 system. By utilizing intracellular labeling and mass staining, we report the neuroanatomy of projection neurons connected with the CO2 sensitive antennal-lobe glomerulus, the labial pit organ glomerulus (LPOG), in the noctuid moth, Helicoverpa armigera. We identified 15 individual LPOG projection neurons passing along different tracts. Most of these uniglomerular neurons terminated in the lateral horn, a previously well-described target area of plant-odor projection neurons originating from the numerous ordinary antennal-lobe glomeruli. The other higher-order processing area for odor information, the calyces, on the other hand, was weakly innervated by the LPOG neurons. The overlapping LPOG terminals in the lateral horn, which is considered important for innate behavior in insects, suggests the biological importance of integrating the CO2 input with plant odor information while the weak innervation of the calyces indicates the insignificance of this ubiquitous cue for learning mechanisms.