Functional unification of sex pheromone-receptive glomeruli in the invasive Turkestan cockroach derived from the genus Periplaneta.

Female Periplaneta americana cockroaches emit two cooperatively working pheromone components, periplanone-B (PB) as a long-range attractant and periplanone-A (PA) as a short-range arrestant, and males develop enlarged glomeruli for processing them separately in the first-order olfactory center. Using intracellular recordings and neuronal labelings, we found that the Turkestan cockroach, Blatta lateralis, which is phylogenetically close to P. americana but having adapted to inground habitats, has an extraordinary large glomerulus. This is caused by drastic enlargement of the PB-responsive glomerulus but not the PA-responsive glomerulus during the late nymphal stage. The output neuron from the macroglomerulus is sensitive to both PA and PB, at a dose of only 0.1 fg. Nevertheless, B. lateralis males never exhibited courtship rituals in response to the presentation of periplanones or natural sex pheromone but exhibited courtship rituals in response to antennal contact with females. Our findings indicate that the unique behavioral ecology and habitats of B. lateralis are related to the functional unification of the pheromone processing system, opposite to the functional differentiation that often underlies species diversification.

Spatial Receptive Fields for Odor Localization.

Animals rely on olfaction to navigate through complex olfactory landscapes, but the mechanisms that allow an animal to encode the spatial structure of an odorous environment remain unclear. To acquire information about the spatial distribution of an odorant, animals may rely on bilateral olfactory organs and compare side differences of odor intensity and timing [1-6] or may perform spatial and temporal signal integration of subsequent samplings [7]. The American cockroach can efficiently locate a source of sex pheromone even after the removal of one antenna, suggesting that bilateral comparison is not a prerequisite for odor localization in this species [8, 9]. Cognate olfactory sensory neurons (OSNs) originating from different locations on the flagellum, but bearing the same olfactory receptor, converge onto the same glomerulus within the antennal lobe, which is thought to result in a loss of spatial information. Here, we identified 12 types of pheromone-responsive projection neurons (PNs), each with spatially tuned receptive field. The combination of (1) the antennotopic organization of OSNs terminals and (2) the stereotyped compartmentalization of PNs' dendritic arborization within the macroglomerulus (MG), allows encoding the spatial position of the pheromone. Furthermore, each PN type innervates a different compartment of the mushroom body, providing the means for encoding spatial olfactory information along the olfactory circuit. Finally, MG PNs exhibit both excitatory and inhibitory spatial receptive fields and modulate their responses based on changes in stimulus geometry. In conclusion, we propose a mechanism for encoding information on the spatial distribution of a pheromone, expanding both our understanding of odor coding and of the strategies insects adopt to localize a sexual mate.

Group-housed females promote production of asexual ootheca in American cockroaches.

BACKGROUND: Facultative parthenogenesis, seen in many animal phyla, is a reproductive strategy in which females are able to generate offspring when mating partners are unavailable. In some subsocial and eusocial insects, parthenogenesis is often more prevalent than sexual reproduction. However, little is known about how social cooperation is linked to the promotion of parthenogenesis. The domiciliary cockroach Periplaneta americana is well-suited to addressing this issue as this species belongs to the superfamily Blattoidea, which diverged into eusocial termites and shows facultative parthenogenesis. RESULTS: We studied environmental factors that influence asexual production of ootheca using behavioral assays in P. americana. When more than three virgin females immediately after the imaginal molt were kept together in a small sealed container, they tended to produce egg cases (oothecae) via parthenogenesis earlier than did isolated females, resulting in apparent synchronization of ootheca production, even among females housed in different containers. In contrast, virgin females housed with genitalia-ablated males or group-housed females with antennae ablated did not significantly promote ootheca production compared to isolated females. Daily addition of the primary sex pheromone component to the container did not promote ootheca production in isolated females. Another line of study showed that grouped females make parthenogenesis more sustainable than previously known; a founder colony of 15 virgin females was sufficient to produce female progeny for a period of more than three years. CONCLUSIONS: Group-housed females promote and stabilize asexual ootheca production compared to isolated females, and that this promotion is triggered by female-specific chemosensory signals (other than sex pheromone) primarily detected by antennae. Promotion of ootheca production between females is likely to be an early stage of social cooperation, reminiscent of the foundation and maintenance of a colony by female pairs in the eusocial termite Reticulitermes speratus.

Complete identification of four giant interneurons supplying mushroom body calyces in the cockroach Periplaneta americana.

Global inhibition is a fundamental physiological mechanism that has been proposed to shape odor representation in higher-order olfactory centers. A pair of mushroom bodies (MBs) in insect brains, an analog of the mammalian olfactory cortex, are implicated in multisensory integration and associative memory formation. With the use of single/multiple intracellular recording and staining in the cockroach Periplaneta americana, we succeeded in unambiguous identification of four tightly bundled GABA-immunoreactive giant interneurons that are presumably involved in global inhibitory control of the MB. These neurons, including three spiking neurons and one nonspiking neuron, possess dendrites in termination fields of MB output neurons and send axon terminals back to MB input sites, calyces, suggesting feedback roles onto the MB. The largest spiking neuron innervates almost exclusively the basal region of calyces, while the two smaller spiking neurons and the second-largest nonspiking neuron innervate more profusely the peripheral (lip) region of the calyces than the basal region. This subdivision corresponds well to the calycal zonation made by axon terminals of two populations of uniglomerular projection neurons with dendrites in distinct glomerular groups in the antennal lobe. The four giant neurons exhibited excitatory responses to every odor tested in a neuron-specific fashion, and two of the neurons also exhibited inhibitory responses in some recording sessions. Our results suggest that two parallel olfactory inputs to the MB undergo different forms of inhibitory control by the giant neurons, which may, in turn, be involved in different aspects of odor discrimination, plasticity, and state-dependent gain control. J. Comp. Neurol. 525:204-230, 2017. © 2016 Wiley Periodicals, Inc.

Divergent and convergent projections to the two parallel olfactory centers from two neighboring, pheromone-receptive glomeruli in the male American cockroach.

Many animals utilize sex pheromone for detecting conspecific mates. Sex pheromone is usually a blend of two or more components with similar chemical compositions. The pheromone receivers are equipped with localized olfactory glomeruli in the first-order olfactory center for specifically processing these pheromone components. In the American cockroach, Periplaneta americana, either periplanone A or periplanone B emitted by virgin females evokes identical sexual behaviors in males. The antennal lobes of adult male cockroaches have enlarged, neighboring A- and B-glomeruli, which preferentially process periplanones A and B, respectively. By using intracellular recording and staining of neurons in the same preparations, we provide the first detailed projection maps of output neurons (projection neurons; PNs) from the A-glomerulus and the B-glomerulus. Although both PNs project to the mushroom body calyces and the lateral horn, their proximities in the two centers largely differ: in the calyces, the axon terminals of the A-PN were located more predominantly in the periphery compared with those of the B-PN, whereas axon terminals of both PNs were highly congruent in the anteromedial region of the lateral horn. These results suggest that pheromone component signals are dispersed in the mushroom body for specific odor discrimination but are integrated in the lateral horn for generating behaviors common to the pheromone components. Stimulation of the ipsilateral antenna with various odors showed that the odor specificity of A-PN is higher than that of B-PN. The different developmental lineages of A- and B-PNs suggested by these results are discussed.

Visual and olfactory input segregation in the mushroom body calyces in a basal neopteran, the American cockroach.

The cockroach Periplaneta americana is an evolutionary basal neopteran insect, equipped with one of the largest and most elaborate mushroom bodies among insects. Using intracellular recording and staining in the protocerebrum, we discovered two new types of neurons that receive direct input from the optic lobe in addition to the neuron previously reported. These neurons have dendritic processes in the optic lobe, projection sites in the optic tracts, and send axonal terminals almost exclusively to the innermost layer of the MB calyces (input site of MB). Their responses were excitatory to visual but inhibitory to olfactory stimuli, and weak excitation occurred in response to mechanosensory stimuli to cerci. In contrast, interneurons with dendrites mainly in the antennal lobe projection sites send axon terminals to the middle to outer layers of the calyces. These were excited by various olfactory stimuli and mechanosensory stimuli to the antenna. These results suggest that there is general modality-specific terminal segregation in the MB calyces and that this is an early event in insect evolution. Possible postsynaptic and presynaptic elements of these neurons are discussed.

Pheromone detection by a pheromone emitter: a small sex pheromone-specific processing system in the female American cockroach.

Many animals depend on pheromone communication for successful mating. Sex pheromone in insects is usually released by females to attract males. In American cockroaches, the largest glomerulus (B-glomerulus) in the male antennal lobe (first-order olfactory center) processes the major component of sex pheromone. Using intracellular recordings combined with fine neuroanatomical techniques, we provide evidence that the female homolog of the male B-glomerulus also acts as a sex pheromone-specific detector. Whereas ordinary glomeruli that process normal environmental odors are innervated by single projection neurons (PNs), the B-glomerulus in both sexes is innervated by multiple PNs, one of which possesses a thicker axon, termed here B-PN. Both soma size and axon diameter were smaller on B-PNs from females compared with B-PNs from males. The female B-PNs also produce fewer terminal arborizations in the protocerebrum than male B-PNs. Termination fields in the lateral protocerebrum of the female B-PN are mostly segregated from those formed by other uniglomerular PNs innervating ordinary glomeruli. Female B-PN activity was greatest in response to sex pheromone but lower than that in the male B-PN. This specific detection system suggests that sex pheromone affects the behavior and/or endocrine system of female cockroaches.

Cuticular lipids and odors induce sex-specific behaviors in the male cricket Gryllus bimaculatus.

Male crickets display sex-specific (e.g., mating and agonistic) behaviors towards conspecific individuals. One of the key signals for these behaviors is the chemical substance on the cricket body surface. In the present study, we analyzed female and male cuticular substances in behavioral assays. Antennal contact stimulation using female forewings elicited a mating behavior in males, while that using male forewings elicited an agonistic behavior in males. Thin-layer-chromatographic and other techniques analysis showed that saturated cuticular lipids were present in both female and male cuticles and that unsaturated lipids were present only in the male cuticle. Filter papers soaked with saturated or unsaturated cuticular lipids were applied to antennae of male crickets. Males showed mating behavior in response to stimulation with saturated lipids from both females and males but showed avoidance behavior in response to stimulation with male unsaturated lipids. Because cuticular lipids did not induce agonistic behavior in males, we collected odors from male crickets and found that these odors induced agonistic behavior in males. Therefore, we concluded that the key signals for mating, avoidance and agonistic behaviors of male crickets are comprised of at least three different components, saturated and unsaturated cuticular lipids and male odors, respectively.

Effects of NO/cGMP signaling on behavioral changes in subordinate male crickets, Gryllus bimaculatus.

After a loss against an opponent, the aggressiveness of a male cricket is significantly reduced for up to 30 minutes. This depression of aggressiveness is an important factor in the establishment and maintenance of dominance between individuals. In the present study, we investigated the functional roles of nitric oxide (NO) signaling in the depression of aggressiveness in subordinate male crickets. Pairs of male crickets, pre-injected with various NO-related reagents, were allowed to establish dominant/subordinate relationships in dyadic encounters. Opponents were separated for 15 minutes and then paired again. In second encounters, subordinate crickets pre-injected with PTIO (NO scavenger) showed agonistic behavior towards former dominant opponents. A similar effect was observed in crickets pre-injected with L-NAME (NO synthase inhibitor) or ODQ (soluble guanylate cyclase inhibitor). The effects of the latter two drugs were canceled by co-injection of NOR3 (NO donor) with L-NAME or by co-injection of 8-Br-cGMP (cGMP-analog) with ODQ. Injection of NOR3 alone prolonged the inhibition of agonistic behavior in subordinate crickets from 30 minutes to 3 hours. Our results suggest that the change in agonistic behavior observed in subordinate male crickets is closely linked to NO-mediated cGMP signaling.

Morphological and physiological development of anterior thoracic stretch receptors in two isopods, Armadillidium vulgare and Ligia exotica.

Abdominal muscle receptor organs (MROs) monitor the position and movement of abdomen in crustaceans. Thoracic segments of decapods are fused and immovable. It is speculated that MROs had retrograded simple shape, N-cells that lost receptor muscles, a receptor cell and accessory nerves. We focused on the effect of segmental movement in respect to thoracic N-cells and MROs in isopods that have movable thoracic segments. Armadillidium vulgare rolled up its body segments. Ligia exotica swam by quick movement of the posterior thoracic segments. Both isopods possessed N-cells and MROs in the thorax. N-cells were a simple structure, but N-cells from the second and third thoracic segments of A. vulgare had a muscle strand. MROs(T3-T4) (from the third and fourth thoracic segments) of A. vulgare had two receptor muscles. MROs(T3-T4) of L. exotica had one long receptor muscle. N-cells of both species and MROs of A. vulgare showed slowly adapting stretch-activated discharges. MROs of L. exotica showed both slowly and rapidly adapting discharges. The stretch-activated responses of N-cells and MROs inhibited each other. N-cells or MROs in the thorax of isopods are not related to the segmental structure. The morphology and physiology of N-cells and MROs are specialized to species-specific behaviors.

Effects of previous experience on the agonistic behaviour of male crickets, Gryllus bimaculatus.

Male solitary animals frequently enter aggressive interactions with conspecific individuals to protect their territory or to gain access to females. After an agonistic encounter, the loser (subordinate individual) changes its behaviour from aggression to avoidance. We investigated agonistic interactions between pairs of male crickets to understand how dominance is established and maintained. Two naïve males readily entered into agonistic interactions. Fights escalated in a stereotyped manner and were concluded with the establishment of dominance. If individuals were isolated after the first encounter and placed together 15 minutes later, subordinate crickets tended to avoid any further contact with the former dominant opponent. Moreover, subordinate males also avoided unfamiliar dominant and naïve opponents. They displayed aggressive behaviour only towards unfamiliar subordinate opponents. This suggests that the subordinate male change their behaviour depending on the dominance status of the opponent. Dominant crickets, in contrast, displayed aggressive behaviour towards familiar as well as unfamiliar opponents. If the interval between the first and second encounter was longer than 30 minutes, the former subordinate male showed aggressive behaviour again. However, if the subordinate cricket was paired with the same opponent three consecutive times within 45 minutes, it avoided the former dominant opponent for up to 6 hours following the third encounter. Our results suggest that the maintenance of dominance in male crickets depends largely on the behavioural change of subordinate individuals. Possible mechanisms to maintain dominance are discussed.