Functional map of the macroglomerular complex of male Helicoverpa armigera.

The mechanism of sex pheromone reception in the male cotton bollworm Helicoverpa armigera has been extensively studied because it has become an important model system for understanding insect olfaction. However, the pathways of pheromone processing from the antenna to the primary olfactory center in H. armigera have not yet been clarified. Here, the physiology and morphology of male H. armigera olfactory sensory neurons (OSNs) were studied using single sensillum recording along with anterograde filling and intracellular recording with retrograde filling. OSNs localized in type A sensilla responded to the major pheromone component cis-11-hexadecenal, and the axonal terminals projected to the cumulus (Cu) of the macroglomerular complex (MGC). The OSNs in type B sensilla responded to the behavioral antagonist cis-9-tetradecenal, and the axonal terminals projected to the dorsomedial anterior (DMA) unit of the MGC. In type C sensilla, there were 2 OSNs: one that responded to cis-9-tetradecenal and cis-11-hexadecenol with the axonal terminals projecting to the DMA, and another that responded to the secondary pheromone components cis-9-hexadecenal and cis-9-tetradecenal with the axonal terminals projecting to the dorsomedial posterior (DMP) unit of the MGC. Type A and type B sensilla also housed the secondary OSNs, which were silent neurons with axonal terminals projected to the glomerulus G49 and DMP. Overall, the neural pathways that carry information on attractiveness and aversiveness in response to female pheromone components in H. armigera exhibit distinct projections to the MGC units.

Serotonergic Neurons in the Brain and Gnathal Ganglion of Larval Spodoptera frugiperda.

The fall armyworm Spodoptera frugiperda (S. frugiperda) (Lepidoptera: Noctuidae) is a worldwide, disruptive, agricultural pest species. The larvae of S. frugiperda feed on seedling, leave, and kernel of crops with chewing mouthparts, resulting in reduced crop yields. Serotonin is an important biogenic amine acting as a neural circuit modulator known to mediate lots of behaviors including feeding in insects. In order to explore the serotonergic neural network in the nervous system of larval S. frugiperda, we performed immunohistochemical experiments to examine the neuropil structure of the brain and the gnathal ganglion with antisynapsin and to examine their serotonergic neurons with antiserotonin serum. Our data show that the brain of larval S. frugiperda contains three neuromeres: the tritocerebrum, the deutocerebrum, and the protocerebrum. The gnathal ganglion also contains three neuromeres: the mandibular neuromere, the maxillary neuromere, and the labial neuromere. There are about 40 serotonergic neurons in the brain and about 24 serotonergic neurons in the gnathal ganglion. Most of these neurons are wide-field neurons giving off processes in several neuropils of the brain and the gnathal ganglion. Serotonergic neuron processes are mainly present in the protocerebrum. A pair of serotonergic neurons associated with the deutocerebrum has arborizations in the contralateral antennal lobe and bilateral superior lateral protocerebra. In the gnathal ganglion, the serotonergic neuron processes are also widespread throughout the neuropil and some process projections extend to the tritocerebrum. These findings on the serotonergic neuron network in larval S. frugiperda allow us to explore the important roles of serotonin in feeding and find a potential approach to modulate the feeding behavior of the gluttonous pest and reduce its damage.

Identification of Odorant-Binding and Chemosensory Protein Genes in Mythimna separata Adult Brains Using Transcriptome Analyses.

Large numbers of chemosensory genes have been identified in the peripheral sensory organs of the pest Mythimna separata (Walker) to increase our understanding of chemoreception-related molecular mechanisms and to identify molecular targets for pest control. Chemosensory-related genes are expressed in various tissues, including non-sensory organs, and they play diverse roles. To better understand the functions of chemosensory-related genes in non-sensory organs, transcriptomic analyses of M. separata brains were performed. In total, 29 odorant-binding proteins (OBPs) and 16 chemosensory proteins (CSPs) putative genes were identified in the transcriptomic data set. The further examination of sex- and tissue-specific expression using RT-PCR suggested that eight OBPs (OBP5, -7, -11, -13, -16, -18, -21, and -24) and eight CSPs (CSP2-4, -8, CSP10-12, and -15) genes were expressed in the brain. Furthermore, bands representing most OBPs and CSPs could be detected in antennae, except for a few that underwent sex-biased expression in abdomens, legs, or wings. An RT-qPCR analysis of the expression profiles of six OBPs (OBP3-5, -9, -10, and -16) and two CSPs (CSP3 and CSP4) in different tissues and sexes indicated that OBP16 was highly expressed in male brain, and CSP3 and CSP4 were female-biased and highly expressed in brain. The expression levels of OBP5 and OBP10 in brain were not significantly different between the sexes. The findings expand our current understanding of the expression patterns of OBPs and CSPs in M. separata sensory and non-sensory tissues. These results provide valuable reference data for exploring novel functions of OBPs and CSPs in M. separata and may help in developing effective biological control strategies for managing this pest by exploring novel molecular targets.

Neuronal architecture and functional mapping of the taste center of larval Helicoverpa armigera (Lepidoptera: Noctuidae).

The sense of taste plays a crucial role in herbivorous insects by discriminating nutrients from complex plant metabolic compounds. The peripheral coding of taste has been thoroughly studied in many insect species, but the central gustatory pathways are poorly described. In the present study, we characterized single neurons in the gnathal ganglion of Helicoverpa armigera larvae using the intracellular recording/staining technique. We identified different types of neurons, including sensory neurons, interneurons, and motor neurons. The morphologies of these neurons were largely diverse and their arborizations seemingly covered the whole gnathal ganglion. The representation of the single neurons responding to the relevant stimuli of sweet and bitter cues showed no distinct patterns in the gnathal ganglion. We postulate that taste signals may be processed in a manner consistent with the principle of population coding in the gnathal ganglion of H. armigera larvae.

Brain Organization of Apolygus lucorum: A Hemipteran Species With Prominent Antennal Lobes.

The anatomical organization of distinct regions in the insect brain often reflects their functions. In the present study, the brain structure of Apolygus lucorum was examined by using immunolabeling and three-dimensional reconstruction. The results revealed the location and volume of prominent neuropils, such as the antennal lobes (AL), optic lobes (OL), anterior optic tubercles (AOTU), central body (CB), lateral accessory lobes (LAL), mushroom lobes, and distinct tritocerebral neuropils. As expected, this brain is similar to that of other insects. One exception, however, is that the antennal lobes were found to be the most prominent neuropils. Their size relative to the entire brain is the largest among all insect species studied so far. In contrast, the calyx, a region getting direct input from the antennal lobe, has a smaller size relative to the brain than that of other species. These findings may suggest that olfaction plays an essential role for A. lucorum.

Distribution of Serotonin-Immunoreactive Neurons in the Brain and Gnathal Ganglion of Caterpillar Helicoverpa armigera.

Serotonin (5-hydroxytryptamine, 5-HT) is an important biogenic amine that acts as a neural circuit modulator. It is widespread in the central nervous system of insects. However, little is known about the distribution of serotonin in the nervous system of the cotton bollworm Helicoverpa armigera. In the present study, we performed immunohistochemical experiments with anti-serotonin serum to examine the distribution of serotonin in the central nervous system of H. armigera larvae. We found about 40 serotonin-immunoreactive neurons in the brain and about 20 in the gnathal ganglion. Most of these neurons are wide-field neurons giving rise to processes throughout the neuropils of the brain and the gnathal ganglion. In the central brain, serotonin-immunoreactive processes are present bilaterally in the tritocerebrum, the deutocerebrum, and major regions of the protocerebrum, including the central body (CB), lateral accessory lobes (LALs), clamps, crepine, superior protocerebrum, and lateral protocerebrum. The CB, anterior ventrolateral protocerebrum (AVLP), and posterior optic tubercle (POTU) contain extensive serotonin-immunoreactive process terminals. However, the regions of mushroom bodies, the lateral horn, and protocerebral bridges (PBs) are devoid of serotonin-immunoreactivity. In the gnathal ganglion, the serotonin-immunoreactive processes are also widespread throughout the neuropil, and some process projections extend to the tritocerebrum. Our results provide the first comprehensive description of the serotonergic neuronal network in H. armigera larvae, and they reveal the neural architecture and the distribution of neural substances, allowing us to explore the neural mechanisms of behaviors by using electrophysiological and pharmacological approaches on the target regions.

Central Projections of Antennal and Labial Palp Sensory Neurons in the Migratory Armyworm Mythimna separata.

The oriental armyworm, Mythimna separata (Walker), is a polyphagous, migratory pest relying on olfactory cues to find mates, locate nectar, and guide long-distance flight behavior. In the present study, a combination of neuroanatomical techniques were utilized on this species, including backfills, confocal microscopy, and three-dimensional reconstructions, to trace the central projections of sensory neurons from the antenna and the labial pit organ, respectively. As previously shown, the axons of the labial sensory neurons project via the ipsilateral labial nerve and terminate in three main areas of the central nervous system: (1) the labial-palp pit organ glomerulus of each antennal lobe, (2) the gnathal ganglion, and (3) the prothoracic ganglion of the ventral nerve cord. Similarly, the antennal sensory axons project to multiple areas of the central nervous system. The ipsilateral antennal nerve targets mainly the antennal lobe, the antennal mechanosensory and motor center, and the prothoracic and mesothoracic ganglia. Specific staining experiments including dye application to each of the three antennal segments indicate that the antennal lobe receives input from flagellar olfactory neurons exclusively, while the antennal mechanosensory and motor center is innervated by mechanosensory neurons from the whole antenna, comprising the flagellum, pedicle, and scape. The terminals in the mechanosensory and motor center are organized in segregated zones relating to the origin of neurons. The flagellar mechanosensory axons target anterior zones, while the pedicular and scapal axons terminate in posterior zones. In the ventral nerve cord, the processes from the antennal sensory neurons terminate in the motor area of the thoracic ganglia, suggesting a close connection with motor neurons. Taken together, the numerous neuropils innervated by axons both from the antenna and labial palp indicate the multiple roles these sensory organs serve in insect behavior.

Distribution of tachykinin-related peptides in the brain of the tobacco budworm Heliothis virescens.

Invertebrate tachykinin-related peptides (TKRPs) comprise a group of signaling molecules having sequence similarities to mammalian tachykinins. A growing body of evidence has demonstrated the presence of TKRPs in the central nervous system of insects. In this investigation, we used an antiserum against locustatachykinin-II to reveal the distribution pattern of these peptides in the brain of the moth Heliothis virescens. Immunolabeling was found throughout the brain of the heliothine moth. Most of the roughly 500 locustatachykinin-II immunoreactive cell bodies, that is, ca. 400, were located in the protocerebrum, whereas the rest was distributed in the deutocerebrum, tritocerebrum, and the gnathal ganglion. Abundant immunoreactive processes were located in the same regions. Labeled processes in the protocerebrum were especially localized in optic lobe, central body, lateral accessory lobe, superior protocerebrum, and lateral protocerebrum, while those in the deutocerebrum were present exclusively in the antennal lobe. In addition to brain interneurons, four pairs of median neurosecretory cells in the pars intercerebralis with terminal processes in the corpora cardiaca and aorta wall were immunostained. No sexual dimorphism in immunoreactivity was found. Comparing the data obtained here with findings from other insect species reveals considerable differences, suggesting species-specific roles of tachykinin-related peptides in insects.

A global-wide search for sexual dimorphism of glomeruli in the antennal lobe of female and male Helicoverpa armigera.

By using immunostaining and three-dimensional reconstruction, the anatomical organization of the antennal lobe glomeruli of the female cotton bollworm Helicoverpa armigera was investigated. Eighty-one glomeruli were identified, 15 of which were not previously discovered. The general anatomical organization of the AL of female is similar to that of male and all glomeruli were classified into four sub-groups, including the female-specific glomerular complex, posterior complex, labial-palp pit organ glomerulus, and ordinary glomeruli. A global-wide comparison on the complete glomerular map of female and male was performed and for the first time the quantitative difference in volume for each individual homologous glomerulus was analyzed. We found that the sexual dimorphism includes not only the sex-specific glomeruli but also some of the other glomeruli. The findings in the present study may provide a reference to examine the antennal-lobe organization more in detail and to identify new glomeruli in other moth species. In addition, the complete identification and global-wide comparison of the sexes provide an important basis for mapping the function of distinct glomeruli and for understanding neural mechanisms underlying sexually dimorphic olfactory behaviors.

Central Projection of Antennal Sensory Neurons in the Central Nervous System of the Mirid Bug Apolygus lucorum (Meyer-Dür).

The mirid bug Apolygus lucorum (Meyer-Dür), a polyphagous pest, is dependent on olfactory cues to locate various host plant species and mates. In this study, we traced the projection pathway of the antennal sensory neurons and visualized their projection patterns in the central nervous system of A. lucorum through confocal microscopy and digital reconstructions. We also examined the glomerular organization of the primary olfactory center of the brain, the antennal lobe, and created a three-dimensional model of the glomeruli. We found that the axons of the sensory neurons project into the brain via the ipsilateral antennal nerve, and descend further into the gnathal ganglion, prothoracic ganglion, mesothoracic ganglion, and metathoracic ganglion, and reach as far as to the abdominal ganglion. Such a projection pattern indicates that antennal sensory neurons of A. lucorum may be potentially directly connected to motor neurons. The antennal lobe, however, is the major target area of antennal sensory neurons. The antennal lobe is composed of a large number of glomeruli, i.e. 70-80 glomeruli in one AL of A. lucorum. The results of this study which provide information about the basic anatomical arrangement of the brain olfactory center of A. lucorum, are important for further investigations of chemosensory encoding mechanisms of the mirid bug.

Glomerular identification in the antennal lobe of the male moth Helicoverpa armigera.

This study investigates anatomical organization of the antennal lobe (AL) glomeruli of the male cotton bollworm Helicoverpa armigera by synaptic antibody staining combined with three-dimensional reconstruction. To identify all glomeruli, their boundaries were accurately determined by means of several additional staining techniques visualizing the neuron categories forming the characteristic spherical neuropils. In total, 78-80 glomeruli were identified in the male H. armigera. The number of glomeruli was considerably larger than that previously reported in this species. Thus, compared with previous studies, we identified 15 new glomeruli, G63-G77. Most of them are located in the posterior part of the AL, which was previously considered to be a part of the protocerebrum. From the general anatomical organization of the AL glomeruli of H. armigera, we classified these neuropil structures into four groups, the macroglomerular complex, posterior complex, labial-palp pit organ glomerulus, and ordinary glomeruli. The complete identification of glomeruli is important for future studies seeking to explore further the coding mechanisms residing within the primary olfactory center of the moth brain. J. Comp. Neurol. 524:2993-3013, 2016. © 2016 Wiley Periodicals, Inc.