Acetylcholine and its metabolic enzymes in developing antennae of the moth, Manduca sexta.

Antennae of the moth, Manduca sexta, are thickly populated with sensory neurons, which send axons through antennal nerves to the brain. These neurons arise by cell divisions and differentiate synchronously during the 18 days of metamorphosis from pupa to adult. Biochemical studies support the hypothesis that antennal neurons use acetylcholine (ACh) as a neurotransmitter: (1) Antennae incubated with [¹⁴C]choline synthesize and store [¹⁴C]ACh; several other transmitter candidates do not accumulate detectably when appropriate radioactive precursors are supplied; (2) antennae and antennal nerves contain endogenous ACh; and (3) extracts of mature antennae contain choline acetyltransferase (ChAc) and acetylcholinesterase (AChE) with properties similar to those reported for the enzymes from other arthropods. Levels of ACh, ChAc, and AChE begin to increase in antennae soon after the sensory neurons are “born.” Levels rise exponentially for over a week as the neurons differentiate and then reach a plateau, at about the time the neurons reach morphological maturity, that is maintained into adulthood. In contrast, levels of carnitine acetyltransferase, cholinesterase, and soluble protein, presumably not confined to nervous tissue, change little during metamorphosis. Levels of ACh, ChAc, and AChE rise in an intracranial segment of antennal nerve at about the same time as in the antenna, indicating that axons can transport neurotransmitter machinery at an early stage in their development.     

Structure and development of antennae in a moth,Manduca sexta

The antenna of the moth, Manduca sexta, comprises two small basal segments and a long (2 cm) flagellum, which is divided into nearly 80 annuli. The annuli bear cuticular scales and small sensory organs, sensilla. A trachea, a blood vessel, and two nerve trunks run through the lumen of the antenna and into the head. Sensilla are arranged in an orderly pattern that is repeated on each flagellar annulus. Each flagellum bears about 10⁵ sensilla, which contain about 2.5 × 10⁵ primary sensory neurons. Clumps of undifferentiated cells (imaginal disks), present in the larva, form pupal antennae during the larval-pupal molt. During the subsequent metamorphic development of the adult, cell divisions, changes in cell shape, and cellular differentiation transform pupal into adult antennae. Sensilla and scales arise and differentiate in the antenna during metamorphosis; regions in which sensilla and scales will arise can be recognized before overt differentiation occurs. All of the flagellar annuli develop synchronously. The dense innervation and neuronal simplicity of antennal flagella, as well as their synchronous development at a late and accessible stage in the animal's life cycle, suit them for studies of neuronal differentiation.     

Invariant association of ecdysis with increases in cyclic 3′,5′-guanosine monophosphate immunoreactivity in a small network of peptidergic neurons in the hornworm, Manduca sexta

At the end of each molt insects shed their old cuticle by performing the stereotyped behavior of ecdysis. In the moth, Manduca sexta, this behavior is triggered by the neuropeptide eclosion hormone (EH). Insights into the mechanism of action of EH have come from the identification of a small network of peptidergic neurons that shows increased cyclic 3′,5′-guanosine monophosphate (cGMP) immunoreactivity at ecdysis in insects from many different orders. Here we present further evidence that strengthens the association between ecdysis and the occurrence of this cGMP response in Manduca. We found that the cGMP increases occurred at every ecdysis, although some of the neurons that showed a response at larval ecdysis did not participate at pupal and adult ecdysis. Both ecdysis and the cGMP increases only required an intact connection with the brain for the first 30 min after EH injection. Interestingly, ecdysis in debrained animals only occurred if the cGMP response had been initiated, suggesting that the onset of this response marks the time at which the central nervous system is first able to drive ecdysis. Finally, we found that the appearance of sensitivity to EH for triggering the cGMP response coincided with the time at which EH first triggers ecdysis. Accepted: 6 May 1997     

Developmental changes of the antenna and its neurons in the silkworm,Bombyx mori,with special regard to larval-pupal transformation

The larval antenna of Bombyx mori has 13 sensilla and about 52 sensory neurons in its distal portion. The axons form two nerve cords which unite in the cranial hemocoel to supply the brain as the olfactory nerve. The antennal imaginal disc, which is a thick pseudostratified epithelium continuous with the antennal epidermis, thickens markedly during the 5th instar by rapid cell proliferation. At the prepupal stage cell proliferation ceases and the disc everts to form a large pupal antenna. Simultaneously, an extensive cell rearrangement occurs in the antennal epidermis and the disc tissue becomes much thinner because of the abrupt expansion of antennal surface area. The two larval nerve cords thin down markedly by degeneration of axons, but they do not disintegrate totally even after the onset of pupation. The epidermis of the larval antenna forms the distal portion of the pupal antenna, while the imaginal disc forms the more basal portion. Development to the adult antenna occurs almost immediately after the onset of pupation; many adult neurons appear in the simple epidermis facing toward the thick outer side of the newly formed pupal cuticle. By 12 hours after the onset of pupation, these neurons align themselves in many transverse rows which are the first sign of the adult antennal configuration. Addition of these neuronal axons to the once-thinned nerve cords causes resumed thickening of the cords during the first 24 hours and thereafter. Differentiation of adult sensilla begins in the next 24 hours and is almost completed at the third day of pupation, which requires a total of 10 days.     

Juvenile hormone acid synthesis and HMG-CoA reductase activity in corpora allata of Manduca sexta prepupae

Corpora allata (CA) of last instar larvae of Manduca sexta switch from juvenile hormone (JH) to JH acid secretion just before the onset of wandering behavior. JH acid secretion peaked during the prepupal period and ceased prior to pupal ecdysis. HMG-CoA reductase activity also peaked during the prepupal period and then declined. However, substantial enzyme activity was present in pupal and pharate adult glands. Removal of the brain at the wandering stage caused a reduction in JH acid secretion by prepupal CA. The profile of HMG-CoA activity in CA of debrained larvae resembled that of sham-operated larvae except that the prepupal peak was smaller than in control larvae. Addition of brain extracts to CA maintained in vitro neither stimulated not inhibited JH acid secretion and HMG-CoA reductase activity. It is suggested that the brain regulates CA activity in post-wandering stages via intact nerves.     

The development of the sensory neuron pattern in the antennal disc of wild-type and mutant (lz3, ssa) Drosophila melanogaster

The development of the sensory neuron pattern in the antennal disc of Drosophila melanogaster was studied with a neuron-specific monoclonal antibody (22C10). In the wild type, the earliest neurons become visible 3 h after pupariation, much later than in other imaginal discs. They lie in the center of the disc and correspond to the neurons of the adult aristal sensillum. Their axons join the larval antennal nerve and seem to establish the first connection towards the brain. Later on, three clusters of neurons appear in the periphery of the disc. Two of them most likely give rise to the Johnston's organ in the second antennal segment. Neurons of the olfactory third antennal segment are formed only after eversion of the antennal disc (clusters t1-t3). The adult pattern of antennal neurons is established at about 27% of metamorphosis. In the mutant lozenge3 (lz3), which lacks basiconic antennal sensilla, cluster t3 fails to develop. This indicates that, in the wild type, a homogeneous group of basiconic sensilla is formed by cluster t3. The possible role of the lozenge gene in sensillar determination is discussed. The homeotic mutant spineless-aristapedia (ssa) transforms the arista into a leg-like tarsus. Unlike leg discs, neurons are missing in the larval antennal disc of ssa. However, the first neurons differentiate earlier than in normal antennal discs. Despite these changes, the pattern of afferents in the ectopic tarsus appears leg specific, whereas in the non-transformed antennal segments a normal antennal pattern is formed. This suggests that neither larval leg neurons nor early aristal neurons are essential for the outgrowth of subsequent afferents.     

The developmental expression of serotonin-immunoreactivity in the brain of the pupal honeybee

The biogenic amine serotonin is a neurotransmitter and modulator in both vertebrates and invertebrates. In the CNS of insects, serotonin is expressed by identifiable subsets of neurons. In this paper, we characterize the onset of expression in the brain and suboesophageal ganglion of the honeybee during pupal development. Several identified serotonin-immunoreactive neurons are present in the three neuromeres of the suboesophageal ganglion the dorsal protocerebrum, and the deutocerebrum at pupal ecdysis. Further immunoreactive neurons are incorporated into the developing pupal brain in two characteristic developmental phases. During the first phase, 5 days after pupal ecdysis, serotonin immunoreactivity is formed in the protocerebral central body, the lamina and lobula, and the deutocerebral antennal lobe. During the second phase, 2 days later, immunoreactivity appears in neurons of the protocerebral noduli of the central complex, the medulla, and the pedunculi and lobes of the mushroom bodies. Three novel serotonin-immunoreactive neurons that innervate the central complex and the mushroom bodies can be individually identified.     

Metamorphic development of locusta-tachykinin immunoreactive neurons of the antennal lobes of the beetle Tenebrio molitor and the effect of fenvalerate application

The effects of the widely used neurotoxic pyrethroid insecticides on neuronal development or plasticity are unclear. To expand knowledge about the influence of the pyrethroid fenvalerate on neuronal development, metamorphic remodelling of the primary olfactory neuropil of the beetle Tenebrio molitor has been studied. The antennal lobe is subdivided into distinct glomeruli before metamorphosis. This is in contrast to that which occurs in other well-studied holometabolous insects such as the moth Manduca sexta and the honeybee. As an indicator of antennal lobe interneurons, locusta-tachykinin immunoreactive neurons have been used. They project into the antennal lobes and form tufted arbors in larval and adult stages within glomeruli throughout the neuropil. These glomerular structures are invaded by glomerular sensory afferent axons and are surrounded by processes of glia cells. With pupation, the glomerulization is lost and no locusta-tachykinin or substance P immunoreactivity is visible in the antennal lobe. The immunoreactivity reappears during metamorphosis, starting with diffusely branched arbors that later become tufted. Application of the neurotoxic insecticide fenvalerate at pupation in sublethal concentrations resulted in a loss or reduction of glomerular pattern formation by neurons and glia cells during metamorphosis. Labelling of antennal sensory axons revealed that the olfactory neuropil was not deafferented, and also that the sensory axons were not organized into a normal glomerular pattern. In addition to the morphological differences, fenvalerate treatment caused locusta-tachykinin immunoreactivity to reappear prematurely during metamorphosis. Possible reasons for fenvalerate-induced alterations in antennal lobe development and their implications for normal development are discussed.     

Reorganization of the ventral nerve cord in the moth Manduca sexta (L.) (Lepidoptera : Sphingidae)

Morphology of the ventral nerve cord of the hawkmoth, Manduca sexta (Lepidoptera : Sphingidae), changes at the larval-pupal transition as several separate larval ganglia fuse to form single ganglia characteristic of the adult. We examined in detail the time course of ganglionic fusion. Changes in the relative positions of the ganglia were studied by staining the tissue with methylene or toluidine blue. Alterations in the positions and structure of individual neurons were studied by filling neurons with a cobalt-lysine complex. The first gross morphological change, anterior movement of the first abdominal ganglion, is visible within the first 24 hr after pupal ecdysis. Adult ventral nerve cord morphology is recognizable 6 days later, approximately 12 days before the adult will emerge. The sequence in which the individual ganglia fuse is invariant. During ganglionic fusion, the neuronal cell bodies and associated neuropil move out of their former ganglionic sheath and through the sheath covering the connectives. Axons between the fusing ganglia form loops in the shortening connectives. The presence of looping axons is a morphological feature that identifies the boundaries between ganglia during intermediate stages of fusion. Some individual adult neurons also show looped axons at the boundaries of fused ganglia. These axonal loops may be a valuable morphological marker by which neurons can be characterized as conserved neurons.     

Neuron differentiation and axon growth in the developing wing of Drosophila melanogaster

Sensory neurons in the wing of Drosophila originate locally from epithelial cells and send their axons toward the base of the wing in two major bundles, the L1 and L3 nerves. We have estimated the birth times of a number of identified wing sensory neurons using an X-irradiation technique and have followed the appearance of their somata and axons by means of an immunohistochemical stain. These cells become immunoreactive and begin axon growth in a sequence which mirrors the sequence of their birth times. The earliest ones are born before pupariation and begin axonogenesis within 1 to 2 hr after the onset of metamorphosis; the last are born and differentiate some 12 to 14 hr later. The L1 and L3 nerves are formed in sections, with specific neurons pioneering defined stretches of the pathways during the period between 0 and 4 hr after pupariation (AP), and finally joining together around 12 hr AP. By 16 hr AP the adult complement of neurons is present and the adult peripheral nerve pattern has been established. Pathway establishment appears to be specified by multiple cues. In places where neurons differentiate in close proximity to one another, random filopodial exploration followed by axon growth to a neighboring neuron soma might be the major factor leading to pathway construction. In other locations, filopodial contact between neighboring somata does not appear to occur, and axon pathways joining neural neighbors by the most direct route are not established. We propose that in these cases additional factors, including veins which are already present at the time of axonogenesis, influence the growth of axons through non-neural tissues.     

Dramatic changes in patterning gene expression during metamorphosis are associated with the formation of a feather-like antenna by the silk moth, Bombyx mori

Many moths use sex pheromones to find their mates in the dark. Their antennae are well developed with lateral branches to receive the pheromone efficiently. However, how these structures have evolved remains elusive, because the mechanism of development of these antennae has not been studied at a molecular level. To elucidate the developmental mechanism of this type of antenna, we observed morphogenesis, cell proliferation, cell death and antennal patterning gene expression in the branched antenna of the silk moth, Bombyx mori. Region-specific cell proliferation and almost ubiquitous apoptosis occur during early pupal stages and appear to shape the lateral branch cooperatively. Antennal patterning genes are expressed in a pattern largely conserved among insects with branchless antennae until the late 5th larval instar but most of them change their expression dramatically to a pattern prefiguring the lateral branch during metamorphosis. These findings imply that although antennal primordium is patterned by conserved mechanisms before metamorphosis, most of the antennal patterning genes are reused to form the lateral branch during metamorphosis. We propose that the acquisition of a new regulatory circuit of antennal patterning genes may have been an important event during evolution of the sensory antenna with lateral branches in the Lepidoptera.     

Dynamics and metamorphosis of an identifiable peptidergic neuron in an insect

Eclosion hormone (EH) is a 7000 Da peptide that triggers ecdysis behavior in insects. In the moth, Manduca sexta, EH is found in two pairs of ventromedial (VM) cells in the brain which send their axons down the ventral nerve cord to a neurohemal site in the proctodeal nerve in the larva and pupa. During adult development, these cells send axon collaterals to the corpora cardiaca where they form a new release site used for adult eclosion. Studies of bioassayable peptide during the 5th larval instar and the larval-pupal transformation revealed that after depletion at ecdysis, the VM cells showed a transient increase in EH found in their cell bodies and axons. By contrast, their terminals in the proctodeal nerve showed a gradual accumulation of peptide followed by a release of over 90% of the stored material at pupal ecdysis. In situ hybridization analysis on whole mounts of the brains showed that the VM cells always contained EH mRNA with increased accumulation during the larval and pupal molting periods with a slight decline just before ecdysis. High levels of EH mRNA were found in brains of diapausing pupae. During the first two-thirds of adult development, mRNA accumulated to high levels, then slowly declined until ecdysis. EH mRNA levels up to 3 days after adult eclosion. At no time was EH mRNA found in the lateral neurosecretory cell cluster previously reported to produce EH for adult eclosion. 1994 John Wiley & Sons, Inc.     

Targeted ablation of CCAP neuropeptide-containing neurons of Drosophila causes specific defects in execution and circadian timing of ecdysis behavior

Insect growth and metamorphosis is punctuated by molts, during which a new cuticle is produced. Every molt culminates in ecdysis, the shedding of the remains of the old cuticle. Both the timing of ecdysis relative to the molt and the actual execution of this vital insect behavior are under peptidergic neuronal control. Based on studies in the moth, Manduca sexta, it has been postulated that the neuropeptide Crustacean cardioactive peptide (CCAP) plays a key role in the initiation of the ecdysis motor program. We have used Drosophila bearing targeted ablations of CCAP neurons (CCAP KO animals) to investigate the role of CCAP in the execution and circadian regulation of ecdysis. CCAP KO animals showed specific defects at ecdysis, yet the severity and nature of the defects varied at different developmental stages. The majority of CCAP KO animals died at the pupal stage from the failure of pupal ecdysis, whereas larval ecdysis and adult eclosion behaviors showed only subtle defects. Interestingly, the most severe failure seen at eclosion appeared to be in a function required for abdominal inflation, which could be cardioactive in nature. Although CCAP KO populations exhibited circadian eclosion rhythms, the daily distribution of eclosion events (i.e., gating) was abnormal. Effects on the execution of ecdysis and its circadian regulation indicate that CCAP is a key regulator of the behavior. Nevertheless, an unexpected finding of this work is that the primary functions of CCAP as well as its importance in the control of ecdysis behaviors may change during the postembryonic development of Drosophila.     

Retrograde BMP signaling controls Drosophila behavior through regulation of a peptide hormone battery

Retrograde BMP signaling in neurons plays conserved roles in synaptic efficacy and subtype-specific gene expression. However, a role for retrograde BMP signaling in the behavioral output of neuronal networks has not been established. Insect development proceeds through a series of stages punctuated by ecdysis, a complex patterned behavior coordinated by a dedicated neuronal network. In Drosophila, larval ecdysis sheds the old cuticle between larval stages, and pupal ecdysis everts the head and appendages to their adult external position during metamorphosis. Here, we found that mutants of the type II BMP receptor wit exhibited a defect in the timing of larval ecdysis and in the completion of pupal ecdysis. These phenotypes largely recapitulate those previously observed upon ablation of CCAP neurons, an integral subset of the ecdysis neuronal network. Here, we establish that retrograde BMP signaling in only the efferent subset of CCAP neurons (CCAP-ENs) is required to cell-autonomously upregulate expression of the peptide hormones CCAP, Mip and Bursicon β. In wit mutants, restoration of wit exclusively in CCAP neurons significantly rescued peptide hormone expression and ecdysis phenotypes. Moreover, combinatorial restoration of peptide hormone expression in CCAP neurons in wit mutants also significantly rescued wit ecdysis phenotypes. Collectively, our data demonstrate a novel role for retrograde BMP signaling in maintaining the behavioral output of a neuronal network and uncover the underlying cellular and gene regulatory substrates.     

Axons of olfactory receptor cells of transsexually grafted antennae induce development of sexually dimorphic glomeruli in Manduca sexta

The influence of olfactory receptor cell (ORC) axons from transsexually grafted antennae on the development of glomeruli in the antennal lobes (ALs), the primary olfactory centers, was studied in the moth Manduca sexta. Normally during metamorphic adult development, the pheromone-specific macroglomerular complex (MGC) forms only in the ALs of males, whereas two lateral female-specific glomeruli (LFGs) develop exclusively in females. A female AL innervated by ORC axons from a grafted male antenna developed an MGC with three glomeruli, like the MGC of a normal male AL. Conversely, a male AL innervated by ORC axons from a grafted female antenna lacked the MGC but exhibited LFGs. ORC axons from grafted male antenna terminated in the MGC-specific target area, even in cases when the antennal nerve (AN) entered the AL via an abnormal route. Within ectopic neuromas formed by ANs that had become misrouted and failed to enter the brain, male-specific axons were not organized and formed terminal branches in many areas. The results suggest the presence of guidance cues within the AL for male-specific ORC axons. Depending on the sex of the antennal innervation, glial borders formed in a pattern characteristic of the MGC or LFGs. The sex-specific number of projection neurons (PNs) in the medial group of AL neurons remained unaffected by the antennal graft, but significant changes occurred in the organization of PN arborizations. In gynandromorphic females, LFG-specific PNs extended processes into the induced MGC, whereas in gynandromorphic males, PNs became restricted to the LFGs. The results indicate that male-and female-specific ORC axons play important roles in determining the position, anatomical features, and innervation of sexually dimorphic glomeruli.     

Projection of sensory neurons from a homeotic mutant appendage,Antennapedia, in Drosophila melanogaster

Central projections of sensory neurons from homeotic mutant appendages (Antennapedia) of Drosophila melanogaster were compared with those of wild-type antennae and wild-type legs by means of degeneration and cobalt backfilling methods. Sensory axons originating from wild-type thoracic legs terminate within the ventral ipsilateral half of the corresponding neuropile segment and do not project to the brain. Sensory fibers from the third antennal segment (AIII) of wild-type animals project into the ipsilateral antennal glomerulus (AG) and to a lesser extent into the contralateral AG, whereas those from the second antennal segment terminate principally within the ipsilateral posterior antennal center. The sensory terminals of femur, tibia, and tarsi of the homeotic leg show a distribution very similar to that of the homologous wild-type antennal segment AIII, differing to a minor degree only in the size and precise localization of terminals within the antennal glomeruli. No degenerating axons were evident in ultrastructural examination of neck connectives after removal of homeotic legs. It is thus very improbable that any sensory fibers of the homeotic leg project to normal leg projection areas in the thoracico-abdominal ganglion. Several alternative explanations are offered for the apparent retention of antennal specificity by axons from the transformed appendage.     

Disturbance of adult eclosion by fenoxycarb in the silkworm,Bombyx mori

Fenoxycarb treatment before and after pupal ecdysis of Bombyx mori disturbed adult eclosion and the animals were unable to escape from the pupal exuviae. This effect of fenoxycarb was dose and time dependent with the highest sensitivity around the pupal ecdysis. The sensitivity rapidly diminished within 20 hours of pupal ecdysis. Twenty-hydroxyecdysone (20E) produced similar effects. Fenoxycarb injection at the pupal ecdysis induced higher ecdysteroid production by the prothoracic glands and higher PTTH-secretory activity in the brain-corpora cardiaca-corpora allata complexes. As a result, the fenoxycarb treated pupae contained higher ecdysteroid titres in the haemolymph. Both the fenoxycarb and the 20E treatments resulted in the lack of development of the rectum in pharate adults. This was the main cause of high ecdysteroid titres in the pharate adult stage. This effect was mimicked by either removal of the rectum early in the pharate adult stage or a surgical extirpation of the hindgut at the time of pupal ecdysis. These results suggest that the disturbance of adult eclosion by fenoxycarb is due in part to the inability of the formation of the rectum in the pharate adult stage.     

Release of prothoracicotropic hormone and potentiation of developmental ability during diapause in the bollworm, Heliothis zea

In Heliothis zea, pupal diapause is not due to a deficiency of the prothoracicotropic hormone (PTTH), as it is in many other insects. However, PTTH is essential for diapause termination and adult development. Removal of the pupal brain 4 hr after larval-pupal ecdysis blocks the insect's ability to initiate adult development. Transplantation of brain neurosecretory cells restores this ability, whereas other tissues such as corpora allata have no effect. In the diapausing pupa, PTTH is released from the brain within 24 hr after larval-pupal ecdysis. Subsequent removal of the brain fails to block the ability for diapause termination, because PTTH potentiates the ability for adult development. Since diapause termination is suppressed in a temperature of 21°C, the bollworm retains the ability to initiate development in 27°C whereas it remains in diapause in 21°C. Diapause continues even though pupae are supplied with additional PTTH via neurosecretory cell transplantation.Ecdysone injection and prothoracic gland-ablation experiments indicate that the prothoracic glands are the source of the prohormone α-ecdysone, and that diapause is maintained by an α-ecdysone deficiency. This evidence, in conjunction with the above results, suggests that PTTH release potentiates prothoracic gland function in the diapausing pupa which is then regulated by a temperature dependent process.     

Light and electron microscopic studies on the neurosecretory control of diapause incidence in Pieris rapae crucivora

The incidence of diapause was shown to be determined humorally during the larval-pupal ecdysis by means of brain extirpation experiments.On the basis of this observation, light and electron microscopic changes in the neurosecretory type II cells in the pars intercerebralis-corpus cardiacum system during pharate pupal and early pupal stages were examined in insects reared under long day-length (non-diapause individuals) and in insects reared under short day-length (diapause individuals). In the diapause individuals, neurosecretory granules in NS-II cells increased during the pupal instar and large aggregates of granules packed the cytoplasm. Thereafter, inclusion bodies showing cytoplasmic breakdown of the granules appeared.In the non-diapause individuals, on the contrary, electron micrographs suggesting the release of neurosecretory material from axon terminals were obtained just after the pupal ecdysis. There were very few granules, with many Golgi bodies and much rough ER 8 to 12 hr after the ecdysis.It is concluded that adult development is determined by the release of neurosecretory material from the axon terminals of NS-II cells at the larval-pupal ecdysis. If release does not occur, the pupae enter diapause. It is also thought that differences in day-length during the larval stages influence the activities of NS-II cells before pupation.     

Morphogenesis of the antenna of the male silkmoth, Antheraea polyphemus. V. Development of the peripheral nervous system

The imaginal antenna of the male silkmoth Antheraea polyphemus is a feather-shaped structure consisting of about 30 flagellomeres, each of which gives off two pairs of side branches. During the pupal stage (lasting for 3 weeks), the antenna develops from a leaf-shaped, flattened epidermal sac ('antennal blade') via two series of incisions which proceed from the periphery towards the prospective antennal stem. The development of the peripheral nervous system was studied by staining the neurons with an antibody against horseradish peroxidase as well as by electron microscopy. The epithelium is subdivided in segmentally arranged sensillogenic regions alternating with non-sensillogenic regions. Immediately after apolysis, clusters consisting of 5 sensory neurons each and belonging to the prospective sensilla chaetica can be localized at the periphery of the antennal blade in the sensillogenic regions. During the first day following apolysis, the primordia of ca. 70 000 olfactory sensilla arise in the sensillogenic regions. Axons from their neurons are collected in segmentally arranged nerves which run towards the CNS along the dorsal as well as the ventral epidermis and are enveloped by a glial sheath. This 'primary innervation pattern' is completed within the second day after apolysis. A first wave of incisions ('primary incisions') subdivide the antennal blade into segmental 'double branches' without disturbing the innervation pattern. Then a second wave of incisions ('secondary incisions') splits the double branches into single antennal branches. During this process, the segmental nerves and their glial sheaths are disintegrated. The axons are then redistributed into single branch nerves while their glial sheath is reconstituted, forming the 'secondary', or adult, innervation pattern. The epidermis is backed by a basal lamina which is degraded after outgrowth of the axons, but is reconstituted after formation of the single antennal branches.