Neural Pathways Of Sensing And Encoding Sex Pheromones In Male Helicoverpa Armigera

Olfaction is an ancient chemical sense,with most animals possessing the capacity to discern distinct odors.As one of the most successful animal species in terms of evolution,insects largely rely on their advanced olfactory nervous systems for survival and reproduction.Different odor information plays a crucial role in foraging,mating,group communication,and other insect behaviors,which are primarily controlled by various neuron types in the olfactory nervous system.Consequently,investigating the neural pathways responsible for insect odor information sensing and encoding is instrumental in understanding the underlying principles of insect behavior,providing foundational theoretical knowledge for green pest control technologies based on insect olfaction,but also holds significant implications for neurobiological research in other animals.Lepidopteran sex pheromone components are relatively simple,yet they ensure successful reproduction within species and maintain reproductive isolation among related species.This makes them an ideal model for exploring olfactory neural pathways underlying insect behaviors and a popular topic in chemical ecology research.In this study,we examined the neural pathways of sensing and encoding sex pheromones of a significant pest within the heliothine species,Helicoverpa armigera,utilizing various neuron recording and staining techniques,mass staining,and immunocytochemistry methods.Images were acquired using a confocal laser scanning microscope,and stained neurons and corresponding brain structures were established through three-dimensional reconstruction.The primary results are as follows:(1)Neural pathway encoding the sex pheromone from the antennae to the primary center of H.armigeraThe projection patterns and target regions of olfactory sensory neurons in the antennal lobe,as well as the morphology and function of individual olfactory sensory neurons of male H.armigera,were investigated were investigated using mass staining of trichoid sensilla on the antennae in male H.armigera,combined with single sensillum recording and staining,intracellular recording and staining from the antennal lobe,and immunocytochemistry methods.The morphology of olfactory sensory neurons and target glomeruli in the antennal lobe were established through three-dimensional reconstruction.The results indicate that the axons of olfactory sensory neurons in the trichoid sensilla of male H.armigera predominantly project into the macroglomerular complex of the antennal lobes in the brain.These sensilla can be categorized into three types(A,B,and C)according to their functions,with the majority containing two olfactory sensory neurons with distinct morphologies.Their axons projected to different glomeruli in the antennal lobe,and the axon terminal branches are randomly distributed within a small portion of the outer edge in the target glomeruli.olfactory sensory neurons in type A sensilla sensitive to cis-11-hexadecenal projected to the cumulus,while olfactory sensory neurons in type B sensilla sensitive to cis-9-tetradecenal projected to the anterior dorsomedial glomerulus.Type A and type B sensilla also possess secondary olfactory sensory neuron,which were silent neurons and projected to the glomerulus 49 and posterior dorsomedial glomerulus.One olfactory sensory neuron in type C sensilla sensitive to cis-9-tetradecenal and cis-11-hexadecenol projected to the anterior dorsomedial glomerulus,whereas the other olfactory sensory neuron in type C sensilla sensitive to cis-9-hexadecenal and cis-9-tetradecenal projected to the posterior dorsomedial glomerulus.These findings suggest that the three units of the macroglomerular complex in the antennal lobe of male H.armigera are responsible for receiving pheromone compounds with varying behavioral significance.The cumulus is responsible for receiving the primary pheromone components,and the anterior dorsomedial glomerulus for receiving pheromone antagonists,and the posterior dorsomedial glomerulus for receiving the secondary components.(2)Neural pathway encoding the sex pheromones from the primary center to the higher center of H.armigeraThe projection tracts and target regions of the projection neurons from the antennal lobe to the protocerebrum,as well as the morphology and function of individual projection neurons conveyed by the macroglomerular complex of male H.armigera,were investigated utilizing mass staining of the antennal lobe,combined with intracellular recording and staining from the antennal lobe,and immunocytochemistry methods.The morphologies of projection neurons and corresponding brain structures were established by three-dimensional reconstruction.The results demonstrated the presence of three primary tracts of projection neurons leading to higher brain centers,designated as the medial antennal lobe tract,the mediolateral antennal lobe tract,and the lateral antennal lobe tract.Projection neurons along these diverse tracts target to the calyx of the mushroom body and lateral protocerebrum,which includes the lateral horn,superior lateral protocerebrum,superior intermediate protocerebrum,and ventrolateral protocerebrum.Based on the glomeruli innervated by projection neurons’ dendrites,labeled individual projection neurons can be classified into five types,namely,uniglomerular projection neurons with dendrites distributed in the cumulus,anterior dorsomedial glomerulus,posterior dorsomedial glomerulus,and multiglomerular projection neurons with dendrites distributed in the macroglomerular complex as well as both macroglomerular complex and some ordinary glomeruli.Dendritic branches of uniglomerular projection neurons nearly innervate entire glomeruli,and the majority of their functions correspond to the olfactory sensory neurons that innervate the same glomeruli.The dendritic branches of multiglomerular projection neurons are irregularly distributed in the innervated glomeruli,and their functions are diverse,tending to resemble the function of a specific type of uniglomerular projection neuron,though their functions appear uncorrelated with the density of dendritic branches.The number of projection neurons projecting along the medial antennal lobe tract was the greatest,with their axons projecting to the lateral horn,superior lateral protocerebrum,superior intermediate protocerebrum,and ventrolateral protocerebrum;followed by the number of projection neurons projecting along the lateral antennal lobe tract,with their axons projecting to the superior intermediate protocerebrum;and the smallest number of projection neurons projecting along the mediolateral antennal lobe tract,with their axons projecting to the superior lateral protocerebrum,superior intermediate protocerebrum,and ventrolateral protocerebrum.The results indicate that the extensive projections of projection neurons are organized according to behavioral significance,encompassing spatial separation and integration of signals.The primary pheromone component is processed in the superior lateral protocerebrum and superior intermediate protocerebrum,while the secondary components and antagonists are processed in the lateral horn,and the ventrolateral protocerebrum processes all pheromone components,playing a crucial role in combinatorial encoding and parallel processing.(3)Neural pathway encoding the sex pheromones in the higher center of H.armigeraThe projection patterns and target regions of the lateral protocerebrum neurons in the higher center of the protocerebrum,as well as the morphology and function of individual lateral protocerebrum neurons in male H.armigera,were investigated using mass staining of the lateral protocerebrum,combined with intracellular recording and staining from the lateral protocerebrum,and immunocytochemistry methods.The morphology of lateral protocerebrum neurons and corresponding brain were established by three-dimensional reconstruction.The results indicate that lateral protocerebrum neurons exhibit a broad neurite distribution in the protocerebrum without forming a tract.The highest number of neurons connected with the lateral horn,superior lateral protocerebrum,superior intermediate protocerebrum,ventrolateral protocerebrum,and superior medial protocerebrum of the ipsilateral hemisphere,and some nerve branches of neurons crossed the midline,extending to the superior medial protocerebrum,superior intermediate protocerebrum,superior lateral protocerebrum and ventrolateral protocerebrum of the contralateral hemisphere;the nerve branches of a few lateral protocerebrum neurons also reached the medial lobe of the mushroom body and superior clamp in the ipsilateral hemisphere.Individual lateral protocerebrum neurons display a range of morphologies and can be classified into input neurons,local neurons,and output neurons based on neurite distribution and terminal appearance of neurons in the brain.Most lateral protocerebrum neurons,unlike olfactory sensory neurons and projection neurons,exhibit broad odor-tuned characteristics and have excitatory responses to all pheromone component stimuli.Some also respond to mechanosensory stimuli.These results suggest that lateral protocerebrum neurons function as integrators to process multimodal sensory inputs.In addition,the lateral protocerebrum may serve to distinguish different types of olfactory information.The specific role of lateral protocerebral neurons requires the further validation.