A female-specific odorant receptor mediates oviposition deterrence in the moth Helicoverpa armigera.

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 the 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 the 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 elucidate the intriguing biological phenomenon of repulsion to conspecific eggs during oviposition and contribute new insight into a female-specific olfactory pathway linked to reproduction.

Organization of the parallel antennal-lobe tracts in the moth.

The olfactory pathways of the insect brain have been studied comprehensively for more than 40 years, yet the last decade has included a particularly large accumulation of new information relating to this system's structure. In moths, sharp intracellular recording and staining has been used to elucidate the anatomy and physiology of output neurons from the primary olfactory center, the antennal lobe. This review concentrates on the connection patterns characterizing these projection neurons, which follow six separate antennal-lobe tracts. In addition to highlighting the connections between functionally distinct glomerular clusters and higher-order olfactory neuropils, we discuss how parallel tracts in the male convey distinct features of the social signals released by conspecific and heterospecific females. Finally, we consider the current state of knowledge regarding olfactory processing in the moth's protocerebrum and make suggestions as to how the information concerning antennal-lobe output may be used to design future studies.

Brain Investigation on Sexual Dimorphism in a Gynandromorph Moth.

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.

Distinct protocerebral neuropils associated with attractive and aversive female-produced odorants in the male moth brain.

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, that is, 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' widespread 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.

Revisiting the Labial Pit Organ Pathway in the Noctuid Moth, Helicoverpa armigera.

Lepidopteran species detect CO2 via a specialized organ located on the peripheral segment of the labial palps, the labial palp pit organ (LPO). Based on tracing of LPO sensory neurons targeting one distinct antennal-lobe glomerulus, Kent and her colleagues described the projections originating from the LPO in the sphinx moth as "an accessory olfactory pathway in Lepidoptera" already in the 1980 ties. In spite of similar reports from studies of other lepidopteran species, however, it has been an unresolved issue whether additional termination areas of the labial nerve, such as the gnathal ganglion (GNG) and the ventral nerve cord, are actually output sites of LPO neurons. Since the previous studies have interpreted slightly differently about the projection pattern occurring from the classical mass staining, we performed selective mass staining from the inside of the pit and from the outer surface of the peripheral palp. The results demonstrated that the LPO sensory neurons project exclusively to the LPO glomerulus (LPOG), whereas the non-LPO sensory neurons target the GNG and the ventral nerve cord. Additional iontophoretic staining of individual LPO sensory neurons, performed from the LPO and the LPOG, showed three morphological neuron types: one bilateral targeting the LPOG in both antennal lobes, one unilateral targeting the ipsilateral LPOG only, and one contralateral targeting the LPOG in the other antennal lobe. Finally, to explore putative differences in the projection pattern of neurons housed by two previously reported sensillum types in the pit, i.e., hair-shaped sensilla located distally and club-shaped sensilla located proximally, we performed mass staining from two different levels of the peripheral palp. We found a projection pattern implying stronger innervation of the ipsi- than the contralateral LPOG in both staining experiments.

Antennal-lobe tracts in the noctuid moth, Heliothis virescens: new anatomical findings.

As in other insects, three main tracts in the moth brain form parallel connections between the antennal lobe and the protocerebrum. These tracts, which consist of the antennal-lobe projection-neuron axons, target two main areas in the protocerebrum, the calyces of the mushroom bodies and the lateral horn. In spite of the solid neuroanatomical knowledge already established, there are still unresolved issues regarding the antennal-lobe tracts of the moth. One is the proportion of lateral-tract neurons targeting the calyces. In the study presented here, we have performed both retrograde and anterograde labeling of the antennal-lobe projection neurons in the brain of the moth, Heliothis virescens. The results from the retrograde staining, obtained by applying dye in the calyces, demonstrated that the direct connection between the antennal lobe and this neuropil is maintained primarily by the medial antennal-lobe tract; only a few axons confined to the lateral tract were found to innervate the calyces. In addition, these staining experiments, which allowed us to explore the arborization pattern of labeled neurons within the antennal lobe, resulted in new findings regarding anatomical arrangement of roots and cell body clusters linked to the medial tract. The results from the anterograde staining, obtained by applying dye into the antennal lobe, visualized the total assembly of axons passing along the antennal-lobe tracts. In addition to the three classical tracts, we found a transverse antennal-lobe tract not previously described in the moth. Also, these staining experiments revealed an organized neuropil in the lateral horn formed by terminals of the four antennal-lobe tracts.

Morphological and physiological characteristics of the serotonin-immunoreactive neuron in the antennal lobe of the male oriental tobacco budworm, Helicoverpa assulta.

We have characterized, by intracellular recording and staining combined with immunocytochemistry, a serotonin-immunoreactive neuron in the central olfactory pathway of the male moth Helicoverpa assulta. The neuron joins the unique category of so-called SI antennal-lobe neurons, previously described in several insect species. In similarity with that originally discovered in the sphinx moth Manduca sexta, the neuron identified here has a large soma located posteriorly in the lateral cell cluster of the antennal lobe and an unbranched neurite projecting into the ipsilateral protocerebrum via the inner antennocerebral tract. After bypassing the central body, the axon crosses the midline and extends through the corresponding antennocerebral tract to the contralateral antennal lobe where it innervates the entire assembly of glomeruli including the male-specific macroglomerular complex. The neuron arborizes into several fine branches in bilateral protocerebral regions anterior to the calyces of the mushroom bodies, particularly on the contralateral side. The physiology of the neuron revealed 2 distinctly different spiking amplitudes, 1 small showing a relatively high spontaneous activity and 1 large showing low activity. The small-amplitude spikes displayed increased frequency when pheromones and plant odors were blown over the antenna. The large-amplitude spikes, which had an unusually long duration, showed no observable responses.