Evolutionary diversification of the auditory organ sensilla in Neoconocephalus katydids (Orthoptera: Tettigoniidae) correlates with acoustic signal diversification over phylogenetic relatedness and life history.

Neoconocephalus Tettigoniidae are a model for the evolution of acoustic signals as male calls have diversified in temporal structure during the radiation of the genus. The call divergence and phylogeny in Neoconocephalus are established, but in tettigoniids in general, accompanying evolutionary changes in hearing organs are not studied. We investigated anatomical changes of the tympanal hearing organs during the evolutionary radiation and divergence of intraspecific acoustic signals. We compared the neuroanatomy of auditory sensilla (crista acustica) from nine Neoconocephalus species for the number of auditory sensilla and the crista acustica length. These parameters were correlated with differences in temporal call features, body size, life histories and different phylogenetic positions. By this, adaptive responses to shifting frequencies of male calls and changes in their temporal patterns can be evaluated against phylogenetic constraints and allometry. All species showed well-developed auditory sensilla, on average 32-35 between species. Crista acustica length and sensillum numbers correlated with body size, but not with phylogenetic position or life history. Statistically significant correlations existed also with specific call patterns: a higher number of auditory sensilla occurred in species with continuous calls or slow pulse rates, and a longer crista acustica occurred in species with double pulses or slow pulse rates. The auditory sensilla show significant differences between species despite their recent radiation, and morphological and ecological similarities. This indicates the responses to natural and sexual selection, including divergence of temporal and spectral signal properties. Phylogenetic constraints are unlikely to limit these changes of the auditory systems.

Embryonic development of the central projection of auditory afferents (Schistocerca gregaria, Orthoptera, Insecta).

The auditory system of Schistocerca gregaria is a well investigated sensory network in the adult grasshopper. Here we present a first study on the embryonic development of this neuronal network. Focussing on the auditory receptor cells we show that they differentiate axonal processes at around 45% of embryonic development. These axons fasciculate with the intersegmental nerve and enter the central nervous system by 45-50% of development. First collaterals sprout into the major arborization area, the frontal auditory projection area of the metathoracic ganglion by 60%. This projection increases in density until an adult-like morphology is established by 90% of development. Furthermore, by the end of embryogenesis all three types of receptor fiber projections can be distinguished. This development is independent of a hearing ability, which develops much later during postembryonic life. The auditory projection co-develops with the fusion of neuromeres to the metathoracic ganglion, the formation of the target neuropile areas and the expression of the synapse associated molecule synapsin. Fasciclin I and Lachesin, both potential axon-guidance molecules, are expressed strongly on both, peripheral and central auditory pathways and, although much weaker, within the synaptic target area.

Auditory behaviour of a parasitoid fly (Emblemasoma auditrix, Sarcophagidae, Diptera).

Females of the parasitoid fly Emblemasoma auditrix find their host cicada (Okanagana rimosa) by its acoustic signals. In laboratory experiments, fly phonotaxis had a mean threshold of about 66 dB SPL when tested with the cicada calling song. Flies exhibited a frequency dependent phonotaxis when testing to song models with different carrier frequencies (pulses of 6 ms duration and a repetition rate of 80 pulses s(-1)). However, the phonotactic threshold was rather broadly tuned in the range from 5 kHz to 11 kHz. Phonotaxis was also dependent on the temporal parameters of the song models: repetition rates of 60 pulses s(-1) and 80 pulses s and pulse durations of 5-7 ms resulted in the highest percentages of phonotaxis performing animals coupled with the lowest threshold values. Thus, parasitoid phonotaxis is adapted especially to the temporal parameters of the calling song of the host. Choice experiments revealed a preference of a song model with 9 kHz carrier frequency (peak energy of the host song) compared with 5 kHz carrier frequency (electrophysiologically determined best hearing frequency). However, this preference changed with the relative sound pressure level of both signals. When presented simultaneously, E. auditrix preferred 5-kHz signals, if they were 5 dB SPL louder than the 9-kHz signal.

Pathfinding, target recognition, and synapse formation of single regenerating fibers in the adult grasshopper Schistocerca gregaria.

After lesion of the peripheral tympanal nerve of the adult locust (Schistocerca gregaria), sensory axons regenerate into their original target areas. We examined the individual behavior of single regenerating auditory afferents during pathway and target selection by intracellularly recording and labeling them at different times postlesion. During axotomy, spontaneous activity is not increased in either the distal or proximal part of the cells. Stimulus response properties of lesioned cells with or without regenerating axons are not influenced. Surprisingly, only 55% of sensory neurons regenerate through the lesion site and often give rise to more than one axonal fiber. Within the central nervous system, 70% of regenerated axons consistently follow an incorrect pathway to reach the correct target region. Often, one of two processes formed by a cell chooses the correct pathway, and the other the incorrect one. In the target region, regenerated axons reconstitute somatotopically ordered projections and form synapses that resemble those of intact fibers in number and structure. The regeneration process does not induce a detectable expression of antigens that are known to be expressed during neural development in these neurons. Our study clearly demonstrates that precise synaptic regeneration is possible in adult animals within a completely differentiated central nervous system, although pathfinding and formation of arborizations are disturbed in a particular and probably system-related manner. The results strongly suggest that accurate pathfinding is unlikely to be a decisive factor in target area recognition and synaptogenesis.

Axonal degeneration with tympanal nerve of Schistocerca gregaria.

This study describes time course and ultrastructural changes during axonal degeneration of different neurones within the tympanal nerve of the locust Schistocerca gregaria. The tympanal nerve innervates the tergit and pleurit of the first abdominal segment and contains the axons of both sensory and motor neurones. The majority of axons (approx. 97%) belong to several types of sensory neurones: mechano- and chemosensitive hair sensilla, multipolar neurones, campaniform sensilla and sensory cells of a scolopidial organ, the auditory organ. Axons of campaniform sensilla, of auditory sensory cells and of motor neurones are wrapped by glial cell processes. In contrast, the very small and numerous axons (diameter <1 microm) of multipolar neurones and hair sensilla are not separated individually by glia sheets. Distal parts of sensory and motor axons show different reactions to axotomy: 1 week after separation from their somata, distal parts of motor axons are invaded by glial cell processes. This results in fascicles of small axon bundles. In contrast, distal parts of most sensory axons degenerate rapidly after being lesioned. The time to onset of degeneration depends on distance from the lesion site and on the type of sensory neurone. In axons of auditory sensory neurones, ultrastructural signs of degeneration can be found as soon as 2 days after lesion. After complete lysis of distal parts of axons, glial cell processes invade the space formerly occupied by sensory axons. The rapid degeneration of distal auditory axon parts allows it to be excluded that they provide a structure that leads regenerating axons to their targets. Proximal parts of severed axons do not degenerate.

Tracing of a neuronal network in the locust by pressure injection of markers into a synaptic neuropil.

Central neuronal circuits of vertebrates have often been investigated using injection of markers into synaptic neuropils, whereas similar techniques have rarely been applied in invertebrates. In this study we tested several neuroanatomical tracers for their ability to mark central neuronal circuits in insects, using the well described auditory network of the locust, Locusta migratoria. After physiological localization of an auditory neuropil various tracers were pressure injected. Horseradish peroxidase, dextrans (3 and 10 kDa) and especially biocytin and neurobiotin were effectively incorporated by auditory interneurons, which resulted in their extensive labeling. Postsynaptic regions turned out to be the major, if not exclusive sites of uptake of injected markers, which is deduced from two lines of evidence: (i) for labeling of identified auditory neurons it was necessary to apply the tracer to postsynaptic sites of the neuron; (ii) only a few non-auditory neurons were labeled (probably by lesioning axons during electrode impalement). No evidence could be found for an activity dependent uptake. We conclude that pressure injection of certain tracers into synaptic areas can be used to identify central nervous circuits in insects.

Stimulus transmission in the auditory receptor organs of the foreleg of bushcrickets (Tettigoniidae) I. The role of the tympana.

The auditory organs of the tettigoniid are located just below the femoral tibial joint in the forelegs. Structurally each auditory organ consists of a tonotopically organized crista acustica and intermediate organ and associated sound conducting structures; an acoustic trachea and two lateral tympanic membranes located at the level of the receptor complex. The receptor cells and associated satellite structures are located in a channel filled with hemolymph fluid. The vibratory response characteristics of the tympanic membranes generated by sound stimulation over the frequency range 2-40 kHz have been studied using laser vibrometry. The acoustic trachea was found to be the principal structure through which sound energy reached the tympana. The velocity of propagation down the trachea was observed to be independent of the frequency and appreciably lower than the velocity of sound in free space. Structurally the tympana are found to be partially in contact with the air in the trachea and with the hemolymph in the channel containing the receptor cells. The two tympana were found to oscillate in phase, with a broad band frequency response, have linear coherent response characteristics and small time constant. Higher modes of vibration were not observed. Measurements of the pattern of vibration of the tympana showed that these structures vibrate as hinged flaps rather than vibrating stretched membranes. These findings, together with the morphology of the organ and physiological data from the receptor cells, suggest the possibility of an impedance matching function for the tympana in the transmission of acoustic energy to the receptor cells in the tettigoniid ear.

Lectin histochemistry of the metathoracic ganglion of the locust Schistocerca gregaria before and after axotomy of the tympanal nerve.

The thoracic ganglia of insects exhibit a highly ordered organization. It seems possible that the information underlying the emergence of this order during development and its maintenance throughout insect life is given via a distinct pattern of molecules distributed within the ganglion. The question we asked was whether the adult insect ganglion is subdivided by the distribution of specific carbohydrates and furthermore whether or not this distribution changes during degeneration and regeneration of neurons. In order to determine the normal carbohydrate distribution, we stained sections of the intact metathoracic ganglion of the locust Schistocerca gregaria with fluorescence-coupled lectins. We succeeded in labeling three sensory neuropil areas with either peanut agglutinin (PNA): Phaseolus vulgaris erythrolectin (PVE), soybean agglutinin, wheat germ agglutinin (WGA), or Vicia villosa agglutinin. Apart from this, PNA, WGA, and succinylated WGA also selectively labeled some neuronal cell bodies, including dorsal unpaired median neurons. Datura stramonium lectin (DSL), Griffonia simplicifolia lectin II, and Solanum tuberosum lectin (STL) bound to glial cells or glia surrounding extracellular matrix. A few lectins stained all structures within the ganglion; some showed no binding at all. In the second part of our study, we tested whether carbohydrates were differentially regulated during transient deafferentation after the axotomy of the tympanal nerve. Binding of PNA and PVE within the auditory neuropil did not change. However, binding of the two glia-associated markers, DSL and STL, clearly differed from that found in intact animals; they bound transiently (day 3-4 until day 10-20 post-surgery) to axonal tracts and neuropils of the axotomized sensory afferents.

Birth times of neurons in labellar taste sensilla of the blowfly Phormia regina.

We studied the birth times of neurons of labellar taste sensilla in blowflies using incorporation of the thymidine analogue 5-bromodeoxyuridine (BrdU) as an indicator of birth time. We found that one of the two main sensillum types, the taste papillae, arise according to a clear spatial gradient of birth times, whereas the other sensillum type, taste hairs, arise without any apparent spatial ordering. Within each sensillum type, there was a strong tendency for either all or none of the neurons to have incorporated BrdU. Among those rare sensilla in which only some of the neurons incorporated BrdU, there were clear patterns of the distribution of labeled and unlabeled neurons per sensillum. These results suggest that subsets of the neurons of a sensillum are siblings, and thus argue against the possibility that the several neurons of a sensillum arise from a single stem cell precursor through repeated asymmetrical divisions.

Interneurons with inhibitory effects on stridulation in grasshoppers exhibit GABA-like immunoreactivity.

Three identified descending interneurons (SOG-1, -2, -3) of the suboesophageal ganglion are described that have inhibitory effects on stridulatory movements in the grasshoppers Chorthippus mollis and Omocestus viridulus. No major anatomical or physiological difference has been found between the interneurons of both species. The SOG-1 neuron has a median cell body in the labial neuromere, bilateral symmetrical dendrites and a contralateral descending axon. The SOG-2 and SOG-3 neurons lie in the maxillary neuromere and differ anatomically from each other in an anterior dendritic branch which is present in the SOG-3 neuron. Depolarization of each of the three cells result in a decrease in amplitude or total cessation of the stridulation movement. After the intracellular recordings the neurons were injected with Lucifer yellow and subsequently processed for anti-GABA immunocytochemistry. Each of the neurons shows GABA-like immunoreactivity as revealed on the same section used to identify the location of the cell body.

From embryo to adult: anatomy and development of a leg sensory organ in Phormia regina Meigen (Insecta: Diptera). I. Anatomy and physiology of a larval "leg" sensory organ.

Neurons within the precursor of the adult leg, the imaginal disc, innervate a larval sense organ, Keilin's organ. Electron microscopical investigations of first instar larvae show that five dendrites end at the organ: three insert at the bases of the three hairs of the organ and two end against the cuticle, without any apparent cuticular specialization. In third instar larvae, the imaginal leg discs invaginate into the body cavity, and only four of the dendrites (the outer segments of which become greatly elongated) remain in contact with Keilin's organ. The axons of the neurons that supply Keilin's organ project into a ventral neuropile region of the central nervous system, with a pattern that resembles the projections of other larval sensilla. Electrical activity can be recorded from neurons of the imaginal disc in response to mechanical stimulation.

From embryo to adult: anatomy and development of a leg sensory organ in Phormia regina, Meigen (Insecta: Diptera). II. Development and persistence of sensory neurons.

The imaginal leg disc of Phormia regina contains eight neurons that arise during embryogenesis. Five of the neurons are associated with Keilin's organ, and of these five, two persist to the adult fly. Two new neurons arise at about the time of pupariation and flank each of these persisting neurons, forming two triplets of cells. Both triplets can be followed throughout metamorphosis; in the late pupa they are situated anteriorly and posteriorly at the tip of the fifth tarsomere. Two triplets of cuticular specializations are found at corresponding positions in the adult fly, each consisting of two campaniform sensilla and a trichoid hair. The central member of each set of sensilla, a campaniform sensillum, is associated with the persisting cell.