Moth Brain Research Articles

Integration of the antennal lobe glomeruli and three projection neurons in the standard brain atlas of the moth Heliothis virescens

Digital three dimensional standard brain atlases (SBAs) are valuable tools for integrating neuroimaging data of different preparations. In insects, SBAs of five species are available, including the atlas of the female Heliothis virescens moth brain. Like for the other species, the antennal lobes (ALs) of the moth brain atlas were integrated as one material identity without internal structures. Different from the others, the H. virescens SBA exclusively included the glomerular layer of the AL. This was an advantage in the present study for performing a direct registration of the glomerular layer of individual preparations into the standard brain. We here present the H. virescens female SBA with a new model of the AL glomeruli integrated into the atlas, i.e. with each of the 66 glomeruli identified and labelled with a specific number. The new model differs from the previous H. virescens AL model both in respect to the number of glomeruli and the numbering system; the latter according to the system used for the AL atlases of two other heliothine species. For identifying female specific glomeruli comparison with the male AL was necessary. This required a new male AL atlas, included in this paper. As demonstrated by the integration of three AL projection neurons of different preparations, the new SBA with the integrated glomruli is a helpful tool for determining the glomeruli innervated as well as the relative position of the axonal projections in the protocerebrum.

Neurons associated with the flip‐flop activity in the lateral accessory lobe and ventral protocerebrum of the silkworm moth brain

The lateral accessory lobe (LAL) and the ventral protocerebrum (VPC) are a pair of symmetrical neural structures in the insect brain. The LAL-VPC is regarded as the major target of olfactory responding neurons as well as the control center for olfactory-evoked sequential zigzag turns. Previous studies of the silkworm moth Bombyx mori showed that these turns are controlled by long-lasting anti-phasic activities of the flip-flopping descending neurons with dendrites in the LAL-VPC. To elucidate the neural mechanisms underlying the generation of this alternating activity between the LAL-VPC units of both hemispheres, we first analyzed the detailed neural architecture of the LAL-VPC and identified five subregions. We then investigated the morphology and physiological responses of the LAL-VPC neurons by intracellular recording and staining and morphologically identified three types of bilateral neurons and three types of unilateral neurons. Bilateral neurons showed either brief or cyclic long-lasting responses. At least some neurons of the latter type produced gamma-aminobutyric acid (GABA). Unilateral neurons linking the LAL and VPC, in contrast, showed long-lasting or quick alternating activity. Timing analysis of the activity onset of each neural type suggests that quick reciprocal neural transmission between unilateral neurons would be responsible for the generation of long-lasting activity in one LAL-VPC unit, which lasts for up to a few seconds. Reciprocal inhibition and excitation by the bilateral neurons with long-lasting activities would mediate the alternating long-lasting activity between both LAL-VPC units, which might last for up to 20 seconds.

1TA3-05 構成論的アプローチによる昆虫匂い識別機構の分析(神経回路・脳の情報処理,第47回日本生物物理学会年会)

1TA3-05 構成論的アプローチによる昆虫匂い識別機構の分析(神経回路・脳の情報処理,第47回日本生物物理学会年会)

Representations of odor plume flux are accentuated deep within the moth brain

Odor space, the representation of odor quality in the insect brain, is known to be optimally resolved when lateral inhibitory pathways are functioning normally. A new study published in the Journal of Biology now shows that odor time resolution also depends on the normal functioning of such pathways.

Digital, three-dimensional average shaped atlas of the heliothis virescens brain with integrated gustatory and olfactory neurons

We use the moth Heliothis virescens as model organism for studying the neural network involved in chemosensory coding and learning. The constituent neurons are characterised by intracellular recordings combined with staining, resulting in a single neuron identified in each brain preparation. In order to spatially relate the neurons of different preparations a common brain framework was required. We here present an average shaped atlas of the moth brain. It is based on 11 female brain preparations, each stained with a fluorescent synaptic marker and scanned in confocal laser-scanning microscope. Brain neuropils of each preparation were manually reconstructed in the computer software Amira, followed by generating the atlas using the Iterative Shape Average Procedure. To demonstrate the application of the atlas we have registered two olfactory and two gustatory interneurons, as well as the axonal projections of gustatory receptor neurons into the atlas, visualising their spatial relationships. The olfactory interneurons, showing the typical morphology of inner-tract antennal lobe projection neurons, projected in the calyces of the mushroom body and laterally in the protocerebral lobe. The two gustatory interneurons, responding to sucrose and quinine respectively, projected in different areas of the brain. The wide projections of the quinine responding neuron included a lateral area adjacent to the projections of the olfactory interneurons. The sucrose responding neuron was confined to the suboesophageal ganglion with dendritic arborisations overlapping the axonal projections of the gustatory receptor neurons on the proboscis. By serving as a tool for the integration of neurons, the atlas offers visual access to the spatial relationship between the neurons in three dimensions, and thus facilitates the study of neuronal networks in the Heliothis virescens brain. The moth standard brain is accessible at http://www.ntnu.no/biolog/english/neuroscience/brain

Adult neurogenesis in a moth brain

In both vertebrates and invertebrates, neurogenesis not only occurs during development but also persists throughout adult life. So far, adult neurogenesis has been detected in a few insect orders but not in Lepidoptera. In the moth, Agrotis ipsilon, the sensitivity of antennal lobe interneurons of males to sex pheromone is age- and juvenile hormone-dependent in accordance with changes in the behavioral response. As a first step to understand this neuronal plasticity, we tested the hypothesis that adult neurogenesis could occur in the moth brain using 5-bromo-2'-deoxyuridine injections on newly born and sexually mature moths. Our results show that persistent neurogenesis occurs in mushroom bodies of adult males and females of A. ipsilon. Two clusters of one to four neuroblasts in each brain hemisphere continue to divide in adult moths and give rise to small clusters of Kenyon cells in the cortex of the mushroom body calyces. Neurogenesis was observed in both newly born and sexually mature males. There was a clear increase in the number of newly born cells in brains as the time increased after the treatment that was performed soon after emergence. When treatments were performed in mature 3-day-old adults, neurogenesis was still detected in brains dissected 3 hours after treatment but was hardly visible 2 days later. Adult neurogenesis was also detected in the optic lobes but not in the antennal lobes. We hypothesize that the newly born neurons could play a role in the central nervous plasticity of olfactory processing in the adult moth A. ipsilon.

Projections to higher olfactory centers from subdivisions of the antennal lobe macroglomerular complex of the male silkmoth.

The macroglomerular complex (MGC) is the first-order center for synaptic processing of olfactory information about the female sex pheromone in the male moth brain. We have investigated the MGC subdivisions of the male silkmoth Bombyx mori by use of three-dimensional reconstruction of the MGC from sequential series of confocal slice images. The B. mori MGC consists of three subdivisions similar to those of Manduca sexta: the cumulus, toroid and horseshoe. Intracellular recording and staining revealed that responses of MGC projection neurons to pheromonal stimulation correlate with their dendritic arborizations in the subdivisions of the MGC (the cumulus, toroid and horseshoe) and each subdivision specific projection neuron transmits information to different regions in the calyces of the mushroom body and the inferior lateral protocerebrum. We revealed that major pheromone component information is transferred to the medial part of the inferior lateral protocerebrum through three different antennocerebral pathways. Although it is generally accepted that the calyces of the mushroom body and the inferior lateral protocerebrum are the target sites for pheromone information from the MGC in moths, our results suggest that the medial part of the inferior lateral protocerebrum may be a more important processing site for major pheromonal information in B. mori.

Digital atlases of the antennal lobe in two species of tobacco budworm moths, the Oriental Helicoverpa assulta (male) and the American Heliothis virescens (male and female).

The antennal lobe of the moth brain is the primary olfactory center processing information about pheromones and plant odors. We present here a digital atlas of the glomerular antennal lobe structures in the male of Helicoverpa assulta and the male and female of Heliothis virescens, based on synaptic antibody staining combined with confocal microscopy. The numbers of the glomeruli in the three specimens were similar, 65, 66, and 62, respectively. Whereas the male antennal lobe has a macroglomerular complex consisting of three and four units in the two species, the female lobe has two enlarged glomeruli at a corresponding position, near the entrance of the antennal nerve. Another large glomerulus, showing homology in the three specimens, is ventrally located. The small size of the heliothine moths is advantageous for confocal microscopy because the entire brain can be visualized as a single image stack. The maps are freely accessible on the internet, and the digital form of the data allows each atlas to be rotated and sectioned at any angle, providing for the identification of glomeruli in different preparations.

Coincident stimulation with pheromone components improves temporal pattern resolution in central olfactory neurons.

Male moths must detect and resolve temporal discontinuities in the sex pheromonal odor signal emitted by a conspecific female moth to orient to and locate the odor source. We asked how sensory information about two key components of the pheromone influences the ability of certain sexually dimorphic projection (output) neurons in the primary olfactory center of the male moth's brain to encode the frequency and duration of discrete pulses of pheromone blends. Most of the male-specific projection neurons examined gave mixed postsynaptic responses, consisting of an early suppressive phase followed by activation of firing, to stimulation of the ipsilateral antenna with a blend of the two behaviorally essential pheromone components. Of 39 neurons tested, 33 were excited by the principal (most abundant) pheromone component but inhibited by another, less abundant but nevertheless essential component of the blend. We tested the ability of each neuron to encode intermittent pheromonal stimuli by delivering trains of 50-ms pulses of the two-component blend at progressively higher rates from 1 to 10 per second. There was a strong correlation between 1) the amplitude of the early inhibitory post-synaptic potential evoked by the second pheromone component and 2) the maximal rate of odor pulses that neuron could resolve (r = 0.92). Projection neurons receiving stronger inhibitory input encoded the temporal pattern of the stimulus with higher fidelity. With the principal, excitatory component of the pheromone alone as the stimulus, the dynamic range for encoding stimulus intermittency was reduced in nearly 60% of the neurons tested. The greatest reductions were observed in those neurons that could be shown to receive the strongest inhibitory input from the second behaviorally essential component of the blend. We also tested the ability of these neurons to encode stimulus duration. Again there was a strong correlation between the strength of the inhibitory input to a neuron mediated by the second pheromone component and that neuron's ability to encode stimulus duration. Neurons that were strongly inhibited by the second component could accurately encode pulses of the blend from 50 to 500 ms in duration (r = 0.94), but that ability was reduced in neurons receiving little or no inhibitory input (r = 0.23). This study confirms that certain olfactory projection neurons respond optimally to a particular odor blend rather than to the individual components of the blend. The key components activate opposing synaptic inputs that enable this subset of central neurons to copy the duration and frequency of intermittent odor pulses that are a fundamental feature of airborne olfactory stimuli.

Preparation of primary cultures and acute slices of the nervous system of the moth Manduca sexta

Interactions among receptor neurons, glial cells and neurons intrinsic to the antennal lobe of the moth underlie the formation of olfactory glomeruli. To isolate these interactions, as well as to understand the effect of a variety of humoral agents on differentiation of the neurons and glia, we generate primary cultures of neurons or glia. These methods are described. In addition, we describe a protocol for producing slice preparations of the developing moth brain that we are using to study the biophysical and morphological development of glial cells. This technique allows us to examine a class of glial cells associated with the glomeruli that otherwise are nearly inaccessible using standard intracellular recording techniques. It also preserves the 3-dimensional arrangement of glia that may strongly influence the development of glomeruli.

Glial cells stabilize axonal protoglomeruli in the developing olfactory lobe of the mothManduca sexta

Odor information is processed in spherical structures called glomeruli, which in all animals with differentiated olfactory systems are sites of densely spaced synaptic contacts between olfactory sensory axons and target central nervous system (CNS) neurons. Glomerulus development in the antennal (olfactory) lobe of the moth brain, which is initiated by the arrival of antennal receptor axons, requires interaction among three elements: glial cells, receptor axons, and their targets, the antennal-lobe neurons. Receptor axons form an array of protoglomeruli that become surrounded by glia and serve as a template for mature glomeruli. Previous experiments showed that when the number of glial cells is sharply reduced during development either by irradiation or by an anti-mitotic agent, receptor axons form protoglomeruli, but in the mature lobes, glomeruli are absent and central neurons lack the characteristic glomerular tufting of their arbors. The current investigation was conducted to determine which cellular events in the process of glomerulus formation are disrupted by severe reduction in glial-cell number. The branching patterns of receptor axons and antennal-lobe neurons were examined in animals that had been irradiated to produce glia-deficient antennal lobes at stages during which glomeruli normally would develop. We found that the receptor axons did form protoglomeruli, but that the protoglomeruli quickly disintegrated in glia-deficient antennal lobes; the receptor axons branched diffusely, except where several neighboring glia survived irradiation and together formed a wall of processes that appeared to block the passage of neuronal processes. Multi-glomerular antennal-lobe neurons never developed tufted arbors even at early stages. These results suggest that maintenance of protoglomeruli depends on the border of glia that forms around each protoglomerulus and that the subsequent tufting of antennal-lobe neurons depends on maintenance of the protoglomerular template during the period of dendritic growth.

Glial cells stabilize axonal protoglomeruli in the developing olfactory lobe of the moth Manduca sexta

Odor information is processed in spherical structures called glomeruli, which in all animals with differentiated olfactory systems are sites of densely spaced synaptic contacts between olfactory sensory axons and target central nervous system (CNS) neurons. Glomerulus development in the antennal (olfactory) lobe of the moth brain, which is initiated by the arrival of antennal receptor axons, requires interaction among three elements: glial cells, receptor axons, and their targets, the antennal-lobe neurons. Receptor axons form an array of protoglomeruli that become surrounded by glia and serve as a template for mature glomeruli. Previous experiments showed that when the number of glial cells is sharply reduced during development either by irradiation or by an anti-mitotic agent, receptor axons form protoglomeruli, but in the mature lobes, glomeruli are absent and central neurons lack the characteristic glomerular tufting of their arbors. The current investigation was conducted to determine which cellular events in the process of glomerulus formation are disrupted by severe reduction in glial-cell number. The branching patterns of receptor axons and antennal-lobe neurons were examined in animals that had been irradiated to produce glia-deficient antennal lobes at stages during which glomeruli normally would develop. We found that the receptor axons did form protoglomeruli, but that the protoglomeruli quickly disintegrated in glia-deficient antennal lobes; the receptor axons branched diffusely, except where several neighboring glia survived irradiation and together formed a wall of processes that appeared to block the passage of neuronal processes. Multi-glomerular antennal-lobe neurons never developed tufted arbors even at early stages. These results suggest that maintenance of protoglomeruli depends on the border of glia that forms around each protoglomerulus and that the subsequent tufting of antennal-lobe neurons depends on maintenance of the protoglomerular template during the period of dendritic growth. © 1996 Wiley-Liss, Inc.

Isolation and kinetic properties of the larval brain phosphofructokinase from the gypsy moth, Lymantria dispar

1. 1. Phosphofructo-1-kinase (PFK) was isolated from gypsy moth larval brain by affinity chromatography using ATP-Sepharose. 2. 2. Gypsy moth brain PFK is a tetrameric enzyme composed of a single type of subunit. 3. 3. The insect brain PFK is inactive in the absence of allosteric activators fructose 2,6-bisphosphate or AMP. Citrate antagonized the activation of PFK by cyclic-AMP but was a relatively poor inhibitor of the moth PFK. 4. 4. The Michaelis constants for ATP (71 μM) and fructose-6-phosphate (55 μM) were determined at pH 8.0 in the presence of fructose 2,6,-bisphosphate. 5. 5. The enzyme displayed co-operativity with respect to substrate and adenine nucleotides.

The effect of insecticides on (Ca 2+ + Mg 2+)-ATPase and the ATP-dependent calcium pump in moth brain synaptosomes and synaptosome membrane vesicles from the bertha armyworm, Mamestra configurata Wlk

The cyclodienes chlordane, heptachlor, aldrin, and toxaphene inhibited both (Ca 2+ + Mg 2+)-ATPase activity and ATP-dependent Ca 2+ transport in brain synaptosome membrane vesicles from the moth, Mamestra configurata, under identical incubation conditions in a similar dose-dependent manner. These cyclodienes appear to affect the outward-directed ATP-dependent calcium pump in moth brain synaptic sites in a specific manner. 1,1,1-Trichloro-2,2-bis-( p-chlorophenyl)ethane (DDT) inhibited (Ca 2+ + Mg 2+)-ATPase activity but had no corresponding inhibitory effect on ATP-dependent calcium transport and, at high concentrations (>10 −5 M), strongly stimulated calcium transport. DDT appears to affect calcium movement in brain synaptosomal preparations of M. configurata in a way that may not involve the ATP-dependent calcium pump. The pyrethroids permethrin and decamethrin were weak inhibitors of moth brain (Ca 2+ + Mg 2+)-ATPase. Allethrin was an effective inhibitor of enzyme activity and a potent inhibitor of calcium transport. Inhibition of the ATP-dependent calcium pump may contribute to the action of some pyrethroids in insects. Organophosphates, carbamates, and formamidines were weak inhibitors of the moth brain (Ca 2+ + Mg 2+)-ATPase. Changes in the activity of synaptosomal (Ca 2+ + Mg 2+)-ATPase in the brains of moths after in vivo poisoning with chlordane, DDT, and allethrin were consistent with the effects of these insecticides in vitro.

High affinity (Ca 2+ + Mg 2+)-ATPase in adult brain synaptosomes of the bertha armyworm, Mamestra configurata Wlk.

Ca 2+ + Mg 2+)-ATPase in synaptosomes prepared from the adult brain of Mamestra configurata was strictly dependent on ATP and had a single, high-affinity binding site for ATP ( K m = 122 μM). The enzyme was stimulated by Mg 2+ ( K m = 85 μM) and was maximally active in the presence of 40 mM K +. The enzyme was insensitive to ouabain, an inhibitor of (Na + + K +)-ATPase. Moth brain synaptosomal (Ca 2+ + Mg 2+)-ATPase was stimulated by low concentrations of Ca free 2+ and appeared to have two kinetically distinct binding sites for calcium ion, one with a very high affinity for Ca free 2+ at nanomolar concentrations and the other with a lower affinity for Ca free 2+ at micromolar concentrations. The lower-affinity binding site of the moth brain synaptosomal (Ca 2+ + Mg 2+)-ATPase appears to correspond to the Ca 2+ binding site of (Ca 2+ + Mg 2+)-ATPase in mammalian brain synaptosomes. The high-affinity binding site of the moth brain enzyme for Ca free 2+ ( K m = 0.11 nM) has not been described previously in the literature and conceivably may represent a unique class of high-affinity binding sites for calcium in insect brain. Moth brain (Ca 2+ + Mg 2+)-ATPase displays the same high affinity for Ca free 2+ ( K m = 0.11 nm) as described earlier for ATP-dependent Ca 2+ transport ( K m = 0.1–0.2 nM) in synaptosome membrane vesicles, indicating that the two processes are functionally coupled in the synaptic membrane and contribute to the maintenance of calcium homeostasis in the brain of M. configurata.

Properties of an ATP‐dependent Ca2+ pump and (Ca2+ + Mg2+)‐ATPase in brain synaptosome membrane vesicles from the bertha armyworm, Mamestra configurata Wlk

AbstractBiochemical and kinetic properties under identical substrate and reaction conditions were obtained for an ATP‐dependent Ca2+ pump and (Ca2+ + Mg2+)‐ATPase in synaptosome membrane vesicles prepared from the brain of the moth, Mamestra configurata. Both the ATP‐dependent Ca2+ pump and (Ca2+ + Mg2+)‐ATPase had single, high‐affinity binding sites for ATP (Km = 14 and 116 μM, respectively), Ca2+free (Km = 0.13 nM and 0.072 nM, respectively), and Mg2+ (Km = 1.1 mM and 0.07 mM, respectively). Both systems were relatively little affected by K+ and were insensitive to ouabain, an inhibitor of (Na+ + K+)‐ATPase. The results indicate that the ATP‐dependent Ca2+ pump and (Ca2+ + Mg2+)‐ATPase are functionally coupled in synaptic membranes and constitute a mechanism for Ca2+ transport in the brain of M. configurata.Although moth brain (Ca2+ + Mg2+)‐ATPase is maximally active at nanomolar concentrations of free calcium ion, the enzyme retains at least one‐half of its maximal activity at micromolar calcium concentrations, indicating either that the enzyme has two binding sites for calcium (a high‐affinity site at nanomolar Ca2+free and a low‐affinity site at micromolar Ca2+free), or that there are two enzymes with high and low affinity for calcium, respectively. Calcium extrusion from brain neurones of M. configurata may operate in a two‐stage, concentration‐dependent process in which a first stage, low‐affinity pump reduces intraneuronal calcium to a concentration at which a second stage, high‐affinity pump becomes activated.

Synaptosomes and synaptosome membrane vesicles from the brain of Mamestra configurata: Application to voltage-dependent and ATP-dependent Ca 2+ ion transport studies

High yields of relatively pure, morphologically well-preserved, functionally competent synaptosomes were prepared from brains of moths of Mamestra configurata using a modified microscale Ficoll flotation technique. Typical preparations yielded 10 mg of synaptosomal protein per gram of moth brains. The moth brain synaptosomes were virtually free of endoplasmic reticulum and mitochondrial contaminants as judged from marker enzyme studies and electron microscopy. Voltage-dependent Ca 2+ ion transport was studied using the moth brain synaptosome preparations. Synaptosomes took up radioactive 45Ca 2+ from the incubation medium. The rate of uptake was increased up to three-fold when the synaptosomes were incubated in a depolarizing, high [K +] medium. Time course studies indicated that voltage-dependent Ca 2+ uptake was composed of an early (<2 sec) fast phase and a late (>10 sec) slow phase. ATP-dependent Ca 2+ ion transport was studied in moth brain synaptosome membrane vesicles prepared from synaptosomes by osmotic shock and purified on a second Ficoll gradient. The inside-out synaptosome membrane vesicles contained an ATP-dependent calcium ion pump which transported 45Ca 2+ from the incuation medium into the interior of the vesicle in the presence of ATP. The calcium ionophore A23187 rapidly released accumulated 45Ca 2+ from the vesicles. The maximal rate of ATP-dependent Ca 2+ transport occurred at a [Ca 2+ free] of 0.1 to 0.2 nM, indicating that the transport process has a very high affinity for Ca 2+ ions.

Descending protocerebral neurons related to the mating dance of the male silkworm moth

Descending protocerebral neurons in the male silkworm moth brain responding to the sexual pheromone (Bombykol) were identified. The neurons responded well with a tonic type of response and the high-frequency spikes evoked continued even after the end of the stimulus. Characteristics of the dose-response curves of the neurons to the pheromone remarkably resembled those of the wing vibration which is one of the mating behavioral components of the male moths.

Identification of the cerebral neurosecretory cells that contain eclosion hormone in the moth Manduca sexta

Eclosion hormone (EH) is an insect neuropeptide that is released at the end of metamorphosis from the CNS and triggers the stereotyped motor program of adult emergence. Using three distinct experimental approaches, we have identified a discrete set of neurosecretory cells in the brain of the moth Manduca sexta that contains and releases EH. By isolating the neurosecretory somata and testing them with a sensitive behavioral bioassay, we identified a cluster of ipsilaterally projecting cells (Group Ia) that contain EH. Intracellular stimulation of individual cells within this group induced the release of bioactive EH into the hemolymph surrounding the neurohemal organs of the brain, whereas stimulation of cells in the other cerebral neurosecretory clusters did not. We also developed a polyclonal antiserum against purified EH that precipitated all bioactive material from samples containing the peptide. This antiserum selectively stained 5 of the Group Ia cells on either side of the brain, as well as their central and terminal processes. Preincubation of the serum with EH dramatically reduced its ability to bind the peptide subsequently. The combined application of these physiological and immunological techniques has led to the unequivocal identification of the EH neurons in the moth brain.

Trans-sexually grafted antennae influence development of sexually dimorphic neurones in moth brain

Observations in insects reveal sexual differences in the central nervous system (CNS), associated with sexually dimorphic patterns of reproductive behaviour1–9. For example, in certain species of moths, including the sphinx moth Manduca sexta, only males fly towards a sexually receptive female or towards a source of the female sex pheromone10,11. In Manduca, specialized olfactory receptor cells found only on male antennae12,13 respond sensitively and selectively to the female sex pheromone (unpublished experiments with K.-E. Kaissling and R. J. O'Connell). Their axons project into the macroglomerular complex (MGC), which is characteristic of male, but not female, antennal lobes (ALs; Fig. 1b, d)1–3,5,8,9,14. These afferents to the MGC presumably synapse with male-specific AL neurones8,15 to begin the processing of phenomonal information. We have now devised a surgical procedure for producing antennal gynandromorphs of Manduca in which one of the two ALs receives sensory innervation from an antenna formed by a transplanted imaginal disk of the opposite sex. We report here that in these gynandromorphs, the physiological and morphological properties of certain AL neurones are influenced by the gender of the antennal sensory axons contacting them. In particular, neurones resembling the male-specific AL neurones appear in female ALs innervated by sensory axons from a grafted male antenna.