Regeneration of peripheral nerves to supernumerary legs in cockroaches.

1. Clusters of legs were grown from metathoracic coxae of the cockroach. Legs of a cluster had different segmental origins, sizes, and orientations. 2. Regenerating metathoracic nerves tended to enter the nearest leg, and to a lesser extent of the largest leg, but showed no significant tendency to penetrate metathoracic rather than prothoracic legs, or normally oriented rather than abnormally oriented legs. 3. Movements of legs were evoked by nerve stimulation significantly more often in nearest, largest, and normally oriented legs, but were equally frequent in prothoracic and metathoracic legs. 4. Close proximity of peripheral nerves is not required for the differentiation of the legs, since nerves were visible in the legs only at the later stages of their development, and many of the legs were apparently nerve innervated.     

Mass spectrometric analysis of FMRFamide-like immunoreactive neurons in the prothoracic and subesophageal ganglion of Periplaneta americana

MALDI-TOF mass spectrometry combined with immunocytochemistry and retrograde labeling, was used to study the expression pattern and morphology of Pea-FMRFamide-related peptides in single neurons of the prothoracic ganglion and the subesophageal ganglion (SEG) of the American cockroach Periplaneta americana. In contrast to the postero-lateral cells (PLCs) of the meta- and mesothoracic ganglion, the prothoracic FMRFamide-related peptides expressing neurons not only extend in the posterior median nerve but also in an anterior median nerve, which is described herein. The peptidome of the prothoracic PLCs is identical with that of the meso- and metathoracic neurons, respectively. In this study, we identified a truncated form of Pea-FMRFa-24 which was found to be more abundant than the peptide originally designated as Pea-FMRF-24. FMRFamide-related peptides expressing postero-lateral cells were also detected in the labial neuromere of the SEG. Although their projection could not be solved, mass spectrometric analyses revealed the same peptide complement in these neurons as found in the thoracic postero-lateral cells. In all neurons which we studied no co-localized peptides of other peptide families were observed.     

Axonal transport involvement in long-lasting synaptic modifications in Blatta orientalis

To determine whether axonal transport plays a role in the establishment of long-lasting changes in synaptic transmission, the effects of colchicine on transport and on synaptic modifications induced by hyperactivity were studied in the nerve cord of the cockroach Blatta orientalis. Application of a lead weight on the insect's dorsum, and the consequent exaggerated use of antigravity reflexes, facilitated synaptic transmission along a particular nervous pathway in the metathoracic ganglion. Application of colchicine in the prothoracic ganglion reversibly blocked such synaptic facilitation and temporarily interfered with the transport of proteins along the cord. Five components of axonal transport, moving at 2, 10, 25, 75, and 150 mm/day, were altered by colchicine treatment with a temporal course that coincided with the reversible inhibition of synaptic facilitation. These results were brought about by colchicine acting directly on axonal transport at the level of the prothoracic ganglion, rather than on synaptic transmission measured at the metathoracic ganglion. The temporal correlation observed between the effects of colchicine on axonal transport and on synaptic facilitation strongly suggest that the transport process is essential for long-lasting synaptic modifications to take place.     

Functional morphology and development of tibial organs in the legs I,II and III of the bushcricketEphippiger ephippiger (Insecta,Ensifera)

Summary The anatomy of the complex tibial organs in the pro-, meso- and metathoracic legs of adults and larvae of the bushcricketEphippiger ephippiger is described comparatively. The subgenual organ and the intermediate organ are differentiated in the same way in legs I, II and III; the anatomy of the crista acustica and the tracheal morphology are significantly different. The final number of scolopidia in the tibial organ of each leg is present at the time of hatching. In the subgenual organ, the number of scolopidia is the same in all legs; in the intermediate organ, and especially in the crista acustica, the number of scolopidia decreases from leg I to legs II and III. In the first larval instar, the morphology of the tibia, the course of the trachea and the anatomy of accessory structures are developed in the same way in each leg. The specific differentiations forming the auditory receptor organ in leg I, such as the acoustic trachea, the tympana and tympanal cavities, develop step by step in subsequent instars. The auditory threshold recorded from the tympanal nerve in the prothoracic leg of adults is remarkably lower than in the meso- and metathoracic legs. Morphometrical analyses of structures that are suggested to play a role in stimulus transduction on scolopidia of the crista acustica reveal significant differences in the three legs.     

Physiological investigation on the role of the innervation in the regulation of the prothoracic gland in Periplaneta americana

Under in vitro conditions the prothoracic gland nerve of the last larval instar of Periplaneta americana shows the same efferent nervous activity as under in situ conditions–ie, low activity at the 9th day and high activity at the 20th day of the molting interval. Isolation of the prothoracic ganglion from the subesophageal ganglion provokes an increase in this nerve activity, suggesting an inhibitory effect of the subesophageal ganglion on prothoracic gland nerve activity in vivo. Only in 20-day-old larvae does electrical stimulation of isolated prothoracic glands in vitro via the gland nerve result in a slightly increased release of ecdysteroids from the gland. This effect could not be influenced by different lengths of stimulation periods. Denervation of the prothoracic gland by transection of the gland nerve on the 13th day of the molting interval results in a complete abolition of the first peak of ecdysteroid production in the gland but has no influence on the occurrence and the amount of the main ecdysteroid peak just before the molt. The results suggest the participation of nervous activity in special periods of prothoracic gland regulation in the cockroach.     

A proprioceptor with central cell bodies in the cockroach,Periplaneta americana (Insecta)

Summary A previously undescribed receptor in the coxo-trochantinal region of the metathoracic leg of the cockroach Periplaneta americana was found to have central cell bodies. This cockroach stretch receptor is the second sensory receptor in insects reported to possess somata in the CNS and its remarkable similarity to a locust proprioceptor suggests it to be homologous.     

Ultrabithorax gene expression in Drosophila imaginal discs and larval nervous system

Using a monoclonal antibody and image-processing procedures, the patterns of expression of the Ultrabithorax (Ubx) gene product have been characterized in Drosophila larvae. As reported previously, the metathoracic imaginal discs stain most intensely with anti-Ubx, with some mesothoracic and no prothoracic expression detectable. In the metathoracic discs, the greatest modulation in anti-Ubx staining is along the proximodistal axis. Ubx is generally expressed at higher levels in the posterior regions of metathoracic discs, although relatively high anterior expression is found in some areas. Expression in the mature wing disc is confined to the squamous peripodial membrane cells; in younger wings, Ubx expression fills the posterior half of the peripodial side of the disc. The mesothoracic leg stains with a pattern that is qualitatively similar (but not identical) to that of the metathoracic leg; Ubx is expressed in some anterior regions of the mesothoracic leg, in parasegment 4. Double staining with anti-Ubx and anti-engrailed reveals that discontinuities in Ubx expression that have been suggested to correspond to compartment borders do not coincide with the compartment boundaries in some cases. In the larval ventral ganglion, Ubx expression is greatest in parasegments 5 and 6, as in the embryonic nervous system.     

Transdetermination in leg imaginal discs ofDrosophila melanogaster undDrosophila nigromelanica

Summary The transdetermination capacities of leg discs ofDrosophila melanogaster were examined by mechanically disrupting and kneading whole discs from late third instar larvae and by culturing the resulting tissue mass for 10–14 days in adult female abdomens where the cells continued to divide. The grown implants were then dissected from the abdomens and injected into third instar larvae to undergo metamorphosis.After this treatment, prothoracic leg discs ofDrosophila melanogaster transdetermined with a high frequency (59% of all implants) to wing. Mesothoracic leg discs also transdetermined to wing, but at a very low frequency (4%). Metathoracic leg discs exhibited the same low frequency of transdetermination (4%), but in this case the direction of transdetermination was to haltere (Table 1,D. melanogaster).Very similar results were obtained with leg discs ofDrosophila nigromelanica (Table 1,D. nigromelanica), showing that the peculiar behavior of the three leg discs is not unique forDrosophila melanogaster.The homeotic mutation Polycomb (Pc ³) which partially transforms meso- and metathoracic legs into prothoracic legs did not significantly increase the frequencies of transdetermination in these leg dises and had clearly no effect on the direction of transdetermination (Table 1).We dedicate this publication to the memory of our teacher and advisor, the late Professor Ernst Hadorn, whose enthusiasm and interest stimulated our work     

Leg Regrowth in Blaberus discoidalis (Discoid Cockroach) following Limb Autotomy versus Limb Severance and Relevance to Neurophysiology Experiments

Background

Many insects can regenerate limbs, but less is known about the regrowth process with regard to limb injury type. As part of our neurophysiology education experiments involving the removal of a cockroach leg, 1) the ability of Blaberus discoidalis cockroaches to regenerate a metathoracic leg was examined following autotomy at the femur/trochanter joint versus severance via a transverse coxa-cut, and 2) the neurophysiology of the detached legs with regard to leg removal type was studied by measuring spike firing rate and microstimulation movement thresholds.

Leg Regrowth Results

First appearance of leg regrowth was after 5 weeks in the autotomy group and 12 weeks in the coxa-cut group. Moreover, regenerated legs in the autotomy group were 72% of full size on first appearance, significantly larger (p<0.05) than coxa-cut legs (29% of full size at first appearance). Regenerated legs in both groups grew in size with each subsequent molt; the autotomy-removed legs grew to full size within 18 weeks, whereas coxa-cut legs took longer than 28 weeks to regrow. Removal of the metathoracic leg in both conditions did not have an effect on mortality compared to matched controls with unmolested legs.

Neurophysiology Results

Autotomy-removed legs had lower spontaneous firing rates, similar marked increased firing rates upon tactile manipulation of tibial barbs, and a 10% higher electrical microstimulation threshold for movement.

Summary

It is recommended that neurophysiology experiments on cockroach legs remove the limb at autotomy joints instead of coxa cuts, as the leg regenerates significantly faster when autotomized and does not detract from the neurophysiology educational content.     

The structure and function of serially homologous leg motor neurons in the locust. I. Anatomy

Twenty-one prothoracic and 17 mesothoracic motor neurons innervating leg muscles have been identified physiologically and subsequently injected with dye from a microelectrode. A tract containing the primary neurites of motor neurons innervating the retractor unquis, levator and depressor tarsus, flexor tibiae, and reductor femora is described. All motor neurons studied have regions in which their dendritic branches overlap with those of other leg motor neurons. Identified, serially homologous motor neurons in the three thoracic ganglia were found to have: (1) cell bodies at similar locations and morphologically similar primary neurites (e.g., flexor tibiae motor neurons), (2) cell bodies at different locations in each ganglion and morphologically different primary neurites in each ganglion (e.g., fast retractor unguis motor neurons), or (3) cell bodies at similar locations and morphologically similar primary neurites but with a functional switch in one ganglion relative to the function of the neurons in the other two ganglia. As an example of the latter, the morphology of the metathoracic slow extensor tibiae (SETi) motor neurons was similar to that of pro- and mesothoracic fast extensor tibiae (FETi) motor neurons. Similarly the metathoracic FETi bears a striking resemblance to the pro- and the mesothoracic SETi. It is proposed that in the metathoracic ganglion the two extensor tibiae motor neurons have switched functions while retaining similar morphologies relative to the structure and function of their pro- and mesothoracic serial homologues.     

TheUbx syndrome ofDrosophila: the prothoracic transformation (ppx) is independent ofbx,bxd andpbx

Summary If, early in development, theUbx ⁺ gene is removed by mitotic recombination from cells of the meso-and metathoracic leg primordia, theseUbx ^– cells develop as in the posterior prothoracic leg. We show that this transformation, termedpostprothorax, is a discrete genetic function that is independent of other homeotic transformations such asbx, pbx orbxd, which also result from the inactivation of theUbx gene.     

The neuroethology of sound production in tiger moths (Lepidoptera,Arctiidae)

When stimulated either acoustically or tactually, certain species of arctiid moths rhythmically emit trains of clicks from metathoracic tymbals. The purpose of the experiments presented here was to determine the location within the central nervous system (CNS) of the proposed tymbal central pattern generator (CPG) in Cycnia tenera. Motor neuron impulses that underlie tymbal activation were recorded extracellularly from the tymbal nerve while moths were subjected to selective severing of the suboesophageal, prothoracic, pterothoracic and abdominal ganglia connectives. Motor output evoked by either acoustic or tactile stimulation originates from a common CPG because tymbal nerve spikes in both cases are similar in amplitude, waveform and rhythmicity. Our results showed: (1) removal of the CNS posterior of the second abdominal neuromere had no effect, (2) removal of the head decreased the responsiveness of the animal to acoustic stimulation and, (3) severing the connectives between the prothoracic and pterothoracic ganglia abolished responses to acoustic stimuli and diminished responses to tactile stimuli. We conclude that although the minimal circuitry sufficient for activating the tymbals resides in the pterothoracic ganglion, the prothoracic and cephalic ganglia are required for the normal, and in particular, auditory-evoked operation of the tymbal CPG.Abbreviations ASR acoustic startle response - CNS central nervous system - CPG central pattern generator - dB peSPL decibel peak equivalent sound pressure level (rms re 20 Pa) - ISI inter-spike interval     

Different effects of the biogenic amines dopamine,serotonin and octopamine on the thoracic and abdominal portions of the escape circuit in the cockroach

1. The escape behavior of the cockroach, Periplaneta americana, is known to be modulated under various behavioral conditions (Camhi and Volman 1978; Camhi and Nolen 1981; Camhi 1988). Some of these modulatory effects occur in the last abdominal ganglion (Daley and Delcomyn 1981a, b; Libersat et al. 1989) and others in the thoracic ganglia (Camhi 1988). Neuromodulator substances are known to underlie behavioral modulation in various animals. Therefore, we have sought to determine whether topical application of putative neuromodulators of the escape circuit enhance or depress this circuit, and whether these effects differ in the last abdominal vs. the thoracic ganglia. 2. Topical application of the biogenic amines serotonin and dopamine to the metathoracic ganglion modulates the escape circuitry within this ganglion; serotonin decreases and dopamine enhances the response of leg motoneurons to activation of interneurons in the abdominal nerve cord by electrical or wind stimulation. 3. The neuropil of the thoracic ganglia contains many catecholamine-histofluorescent processes bearing varicosities, providing a possible anatomical substrate for dopamine release sites. 4. Topical application of octopamine to the terminal abdominal ganglion enhances the response of abdominal interneurons to wind stimulation of the cerci. In contrast, serotonin and dopamine have no effect at this site. 5. It is proposed that release of these biogenic amines may contribute to the known modulation of the cockroach escape response.     

Parasitoid wasp sting: a cocktail of GABA, taurine, and beta-alanine opens chloride channels for central synaptic block and transient paralysis of a cockroach host

The wasp Ampulex compressa injects venom directly into the prothoracic ganglion of its cockroach host to induce a transient paralysis of the front legs. To identify the biochemical basis for this paralysis, we separated venom components according to molecular size and tested fractions for inhibition of synaptic transmission at the cockroach cercal-giant synapse. Only fractions in the low molecular weight range (<2 kDa) caused synaptic block. Dabsylation of venom components and analysis by HPLC and MALDI-TOF-MS revealed high levels of GABA (25 mM), and its receptor agonists beta-alanine (18 mM), and taurine (9 mM) in the active fractions. Each component produces transient block of synaptic transmission at the cercal-giant synapse and block of efferent motor output from the prothoracic ganglion, which mimics effects produced by injection of whole venom. Whole venom evokes picrotoxin-sensitive chloride currents in cockroach central neurons, consistent with a GABAergic action. Together these data demonstrate that Ampulex utilizes GABAergic chloride channel activation as a strategy for central synaptic block to induce transient and focal leg paralysis in its host.     

The three leg imaginal discs of Drosophila: "Vive la différence"

The imaginal discs of Drosophila are the larval primordia for the adult cuticular structures of the adult fly. Fate maps of different discs have been generated that show the localization of prospective adult structures. Even though the three legs differ in their morphology, only the fate map for the T1 (prothoracic) leg disc has been generated. Here we present fate maps for the T2 (meso-) and T3 (metathoracic) leg discs. We show that there are many similarities to the map of the T1 leg disc. However, there are also significant differences in the contributions of each disc to the thorax, in the morphology of joints connecting the legs to the thorax, in bristle patterns, and in the positioning of some sensory organs. We also tested the developmental potential of disc fragments and observed that T2 and T3 leg discs have more limited plasticity and are unable to transdetermine. The differences in the cuticle patterns between legs are robust and conserved in many species of dipterans. While most previous analyses of imaginal disc development have not distinguished between the different leg discs, we believe that the underlying differences of the three leg discs demonstrated here cannot be ignored when studying leg disc development.     

The secretion and metabolism of alpha-ecdysone by cockroach (Leucophaea maderae) tissues in vitro

Hemolymph β-ecdysone levels are high (～1.6 μg/ml) in late last instar cockroach ($\text{Leucophaea}$$\text{maderae}$) nymphs; the level of α-ecdysone (～0.1 μg/ml) is evidently subphysiological. Cultured leg regenerates, target organs of ecdysone, are capable of slowly converting α- to β-ecdysone. Cultured prothoracic glands secrete α-ecdysone, which was identified by complete mass spectrometry. These results are consistent with the view that α-ecdysone, secreted by the prothoracic gland, functions as a prohormone which is converted into the active moulting hormone, β-ecdysone, in other tissues.     

Glycogen in the central neurons of insects: Massive aggregations induced by anoxia or axotomy

Large nerve cell bodies in the metathoracic ganglion of the cockroach, Periplaneta americana were examined for glycogen content. Periodic Acid Schiff (PAS) staining of frozen sections, together with electron microscopic evidence, confirmed the low glycogen content in normal neuron somata. Subjecting animals to a variety of stressful factors including axotomy and CO₂ or N₂ anaesthesia resulted in a rapid and substantial increase in neuronal glycogen. The glycogen appeared as large aggregates 1–3 μm in diameter closely associated with numerous mitochondria. These structures were principally restricted to the trophospongial regions of the nerve cell cytoplasm. Glycogen was rarely encountered in the trophospongial glia either in normal or experimental animals. The glycogen response, which persisted in large nerve cell bodies for at least 6.25 days, is discussed in relation to fluctuations in circulating trehalose resulting from the rapid mobilization of fat body glycogen.     

The effect of temperature changes on the spontaneous activity in the neural ganglia of the cockroach, Periplaneta americana

1. 1.|The effect of localized heating or protocerebrum (bp), the prothoracic (t₁), metathoracic (t₃) and the sixth abdominal (a₆) ganglion on the spontaneous neuronal firing rate in these ganglia was investigated in Periplaneta americana.

2. 2.|In almost every case heating the ganglion increased the firing rate. The most heat sensitive were bp, t₃ and t₁, a₆ was much less so.

3. 3.|In bp and t₁ the firing rate stayed on an increased level even 90 s after the thermal stimulus was over.

4. 4.|The differentiated thermosensitivity of the tested ganglia is discussed in terms of thermoregulatory behaviour of the cockroach.

Properties of the trochanteral hair plate and its function in the control of walking in the cockroach.

1. The physiological properties of the group of long hair sensilla of the trochanteral hair plate in the cockroach metathoracic leg were studied. The sensilla were divided into type I and type II according to their responses to imposed displacements. 2. Type I hair sensilla responded to dynamic displacements whereas type II hair sensilla responded to both dynamic and static displacements. The hair sensilla are normally excited by phasic flexion movements of the femur near the end of leg protraction. 3. Activity in the trochanteral hair plate afferents had a short latency excitatory effect on the motoneurone producing slow extension movements of the femur and an inhibitory effect on the femur flexor motoneurones. 4. Removal of the trochanteral hair plate in one leg caused overstepping of that leg in a walking animal due to exaggerated flexion of the femur. This change in leg movement can be explained by the removal of the inhibitory influence from the hair plate afferents to the femur flexor motoneurones. 5. We conclude that one function of the trochanteral hair plate is to limit femur flexion during a step cycle.     

Comparative morphology of the prothoracic leg in heliconian butterflies: Tracing size allometry,podite fusions and losses in ontogeny and phylogeny

Prothoracic legs of heliconian butterflies (Nymphalidae, Heliconiinae, Heliconiini) are reduced in size compared to mesothoracic and metathoracic legs. They have no apparent function in males, but are used by females for drumming on host plants, a behavior related to oviposition site selection. Here, taking into account all recognized lineages of heliconian butterflies, we described their tarsi using optical and scanning electron microscopy and searched for podite fusions and losses, and analyzed allometry at the static, ontogenetic and phylogenetic levels. Female tarsi were similar, club-shaped, showing from four to five tarsomeres, each bearing sensilla chaetica and trichodea. Male tarsi were cylindrical, formed from five (early diverging lineages) to one (descendant lineages) either partially or totally fused tarsomeres, all deprived of sensilla. Pretarsi were reduced in both sexes, in some species being either vestigial or absent. Tarsal lengths were smaller for males in almost all species. An abrupt decrease in size was detected for the prothoracic legs during molting to the last larval instar at both histological and morphometric levels. In both sexes, most allometric coefficients found at the population level for the prothoracic legs were negative compared to the mesothoracic leg and also to wings. Prothoracic tarsi decreased proportionally in size over evolutionary time; the largest and smallest values being found for nodes of the oldest and youngest lineages, respectively. Our results demonstrate that evolution of the prothoracic leg in heliconian butterflies has been based on losses and fusions of podites, in association with negative size allometry at static, ontogenetic and phylogenetic levels. These processes have been more pronounced in males. Our study provided further support to the hypothesis that evolution of these leg structures is driven by females, by changing their use from walking to drumming during oviposition site selection. In males the leg would have been selected against due to absence of function and thus progressively reduced in size, in association with podites fusions and lost.