Characterization of Drosophila Nidogen/entactin reveals roles in basement membrane stability, barrier function and nervous system patterning.

Basement membranes (BMs) are specialized layers of extracellular matrix (ECM) mainly composed of Laminin, type IV Collagen, Perlecan and Nidogen/entactin (NDG). Recent in vivo studies challenged the initially proposed role of NDG as a major ECM linker molecule by revealing dispensability for viability and BM formation. Here, we report the characterization of the single Ndg gene in Drosophila. Embryonic Ndg expression was primarily observed in mesodermal tissues and the chordotonal organs, whereas NDG protein localized to all BMs. Although loss of Laminin strongly affected BM localization of NDG, Ndg-null mutants exhibited no overt changes in the distribution of BM components. Although Drosophila Ndg mutants were viable, loss of NDG led to ultrastructural BM defects that compromised barrier function and stability in vivo Moreover, loss of NDG impaired larval crawling behavior and reduced responses to vibrational stimuli. Further morphological analysis revealed accompanying defects in the larval peripheral nervous system, especially in the chordotonal organs and the neuromuscular junction (NMJ). Taken together, our analysis suggests that NDG is not essential for BM assembly but mediates BM stability and ECM-dependent neural plasticity during Drosophila development.

Sensing of Substrate Vibrations in the Adult Cicada Okanagana rimosa (Hemiptera: Cicadidae).

Detection of substrate vibrations is an evolutionarily old sensory modality and is important for predator detection as well as for intraspecific communication. In insects, substrate vibrations are detected mainly by scolopidial (chordotonal) sense organs found at different sites in the legs. Among these sense organs, the tibial subgenual organ (SGO) is one of the most sensitive sensors. The neuroanatomy and physiology of vibratory sense organs of cicadas is not well known. Here, we investigated the leg nerve by neuronal tracing and summed nerve recordings. Tracing with Neurobiotin revealed that the cicada Okanagana rimosa (Say) (Hemiptera: Cicadidae) has a femoral chordotonal organ with about 20 sensory cells and a tibial SGO with two sensory cells. Recordings from the leg nerve show that the vibrational response is broadly tuned with a threshold of about 1 m/s2 and a minimum latency of about 6 ms. The vibratory sense of cicadas might be used in predator avoidance and intraspecific communication, although no tuning to the peak frequency of the calling song (9 kHz) could be found.

Phonotactic flight of the parasitoid fly Emblemasoma auditrix (Diptera: Sarcophagidae).

The parasitoid fly Emblemasoma auditrix locates its hosts using acoustic cues from sound producing males of the cicada Okanagana rimosa. Here, we experimentally analysed the flight path of the phonotaxis from a landmark to the target, a hidden loudspeaker in the field. During flight, the fly showed only small lateral deviations. The vertical flight direction angles were initially negative (directed downwards relative to starting position), grew positive (directed upwards) in the second half of the flight, and finally flattened (directed horizontally or slightly upwards), typically resulting in a landing above the loudspeaker. This phonotactic flight pattern was largely independent from sound pressure level or target distance, but depended on the elevation of the sound source. The flight velocity was partially influenced by sound pressure level and distance, but also by elevation. The more elevated the target, the lower was the speed. The accuracy of flight increased with elevation of the target as well as the landing precision. The minimal vertical angle difference eliciting differences in behaviour was 10°. By changing the elevation of the acoustic target after take-off, we showed that the fly is able to orientate acoustically while flying.

Vibrational sensitivity of the subgenual organ complex in female Sipyloidea sipylus stick insects in different experimental paradigms of stimulus direction, leg attachment, and ablation of a connective tibial sense organ.

We document the sensitivity to sinusoidal vibrations for chordotonal organs in the stick insect tibia (Sipyloidea sipylus). In the tibia, the scolopidial subgenual organ (~40 scolopidial sensilla), distal organ (~20 scolopidial sensilla), and distal tibial chordotonal organ (~7 scolopidial sensilla) are present. We study the sensitivity of tibial sensory organs in all leg pairs to vibration stimuli as sensory thresholds by recording summed action potentials from Nervus cruris in the femur. The tibia was stimulated with a minishaker delivering vibrational stimuli. Because different experimental procedures may affect the vibration sensitivity, we here analysed possible effects of different experimental conditions: (1) the stimulus direction delivered in either horizontal or vertical direction to the leg; (2) recording responses only from the subgenual organ complex after ablation of the distal tibial chordotonal organ, and (3) the attachment of the leg to the minishaker by plastilin, beeswax-colophony, or freely standing legs. The tibial scolopidial organs give summed responses to vibration stimuli with highest sensitivity between 500 and 1000Hz for all leg pairs. In the different experimental series, we find that (1) thresholds were influenced by stimulation direction with lower thresholds in response to vertical vibrations, (2) ablating the distal tibial chordotonal organ by cutting the distal-most tibia did not change the summed sensory thresholds significantly, and (3) the attachment material between legs and the minishaker (plastilin or beeswax-colophony mixture) did not significant influence the sensory thresholds against free-standing tarsi. The distal tibial chordotonal organ is a connective chordotonal organ attached to a tendon and is likely a proprioceptive organ. These results emphasise that vibrational thresholds are mainly direction-sensitive. Thus, the direction of stimulus delivery during electrophysiological recordings is relevant for comparisons of vibratory sensory thresholds.

The Auditory System of the Dipteran Parasitoid Emblemasoma auditrix (Sarcophagidae).

Several taxa of insects evolved a tympanate ear at different body positions, whereby the ear is composed of common parts: a scolopidial sense organ, a tracheal air space, and a tympanal membrane. Here, we analyzed the anatomy and physiology of the ear at the ventral prothorax of the sarcophagid fly, Emblemasoma auditrix (Soper). We used micro-computed tomography to analyze the ear and its tracheal air space in relation to the body morphology. Both tympana are separated by a small cuticular bridge, face in the same frontal direction, and are backed by a single tracheal enlargement. This enlargement is connected to the anterior spiracles at the dorsofrontal thorax and is continuous with the tracheal network in the thorax and in the abdomen. Analyses of responses of auditory afferents and interneurons show that the ear is broadly tuned, with a sensitivity peak at 5 kHz. Single-cell recordings of auditory interneurons indicate a frequency- and intensity-dependent tuning, whereby some neurons react best to 9 kHz, the peak frequency of the host's calling song. The results are compared to the convergently evolved ear in Tachinidae (Diptera).

Parasitoid flies exploiting acoustic communication of insects-comparative aspects of independent functional adaptations.

Two taxa of parasitoid Diptera have independently evolved tympanal hearing organs to locate sound producing host insects. Here we review and compare functional adaptations in both groups of parasitoids, Ormiini and Emblemasomatini. Tympanal organs in both groups originate from a common precursor organ and are somewhat similar in morphology and physiology. In terms of functional adaptations, the hearing thresholds are largely adapted to the frequency spectra of the calling song of the hosts. The large host ranges of some parasitoids indicate that their neuronal filter for the temporal patterns of the calling songs are broader than those found in intraspecific communication. For host localization the night active Ormia ochracea and the day active E. auditrix are able to locate a sound source precisely in space. For phonotaxis flight and walking phases are used, whereby O. ochracea approaches hosts during flight while E. auditrix employs intermediate landings and re-orientation, apparently separating azimuthal and vertical angles. The consequences of the parasitoid pressure are discussed for signal evolution and intraspecific communication of the host species. This natural selection pressure might have led to different avoidance strategies in the hosts: silent males in crickets, shorter signals in tettigoniids and fluctuating population abundances in cicadas.

Neurons of self-defence: neuronal innervation of the exocrine defence glands in stick insects.

BACKGROUND: Stick insects (Phasmatodea) use repellent chemical substances (allomones) for defence which are released from so-called defence glands in the prothorax. These glands differ in size between species, and are under neuronal control from the CNS. The detailed neural innervation and possible differences between species are not studied so far. Using axonal tracing, the neuronal innervation is investigated comparing four species. The aim is to document the complexity of defence gland innervation in peripheral nerves and central motoneurons in stick insects. RESULTS: In the species studied here, the defence gland is innervated by the intersegmental nerve complex (ISN) which is formed by three nerves from the prothoracic (T1) and suboesophageal ganglion (SOG), as well as a distinct suboesophageal nerve (Nervus anterior of the suboesophageal ganglion). In Carausius morosus and Sipyloidea sipylus, axonal tracing confirmed an innervation of the defence glands by this N. anterior SOG as well as N. anterior T1 and N. posterior SOG from the intersegmental nerve complex. In Peruphasma schultei, which has rather large defence glands, only the innervation by the N. anterior SOG was documented by axonal tracing. In the central nervous system of all species, 3-4 neuron types are identified by axonal tracing which send axons in the N. anterior SOG likely innervating the defence gland as well as adjacent muscles. These neurons are mainly suboesophageal neurons with one intersegmental neuron located in the prothoracic ganglion. The neuron types are conserved in the species studied, but the combination of neuron types is not identical. In addition, the central nervous system in S. sipylus contains one suboesophageal and one prothoracic neuron type with axons in the intersegmental nerve complex contacting the defence gland. CONCLUSIONS: Axonal tracing shows a very complex innervation pattern of the defence glands of Phasmatodea which contains different neurons in different nerves from two adjacent body segments. The gland size correlates to the size of a neuron soma in the suboesophageal ganglion, which likely controls gland contraction. In P. schultei, the innervation pattern appears simplified to the anterior suboesophageal nerve. Hence, some evolutionary changes are notable in a conserved neuronal network.

Experimental infection of a periodical cicada (Magicicada cassinii) with a parasitoid (Emblemasoma auditrix) of a proto-periodical cicada (Okanagana rimosa).

BACKGROUND: The proto-periodical cicada Okanagana rimosa is subject to infection by the acoustically orientating parasitoid fly Emblemasoma auditrix. Furthermore, it is also the only known host of E. auditrix. Here we test the question, whether the highly adapted parasitoid can also infect other cicadas, like the periodical cicada (Magicicada cassinii) and which steps of the parasitization process can be completed. The experiments might also reveal whether such a parasitoid could hypothetically have been involved in the evolution of periodicity. RESULTS: The hearing threshold of E. auditrix matches with the spectrum of the calling song of M. cassinii, indicating potential host localization. Behaviourally, host localization is possible by the parasitoid as it approaches a loudspeaker broadcasting the buzz part of the calling song of M. cassinii. Magicicada cassinii is readily accepted as host and for host infection the parasitoid uses the same behavioural sequence as for its host O. rimosa. A larva is deposited into the timbal of the cicada. By contrast to O. rimosa the development of the fly larva is delayed and eventually suppressed in M. cassinii. CONCLUSIONS: The host range of E. auditrix is mainly determined by acoustic parameters. This filter is important, as other sensory cues seem not to be involved in the host selection process and larva will not develop in unsuited host. Although the recent parasitoid-host system seems to be stable in terms of coexistence of both species, an acoustically hunting parasitoid could have been a selective force during evolution of prime numbered periodicity in cicadas.

Morphological and physiological regeneration in the auditory system of adult Mecopoda elongata (Orthoptera: Tettigoniidae).

Orthopterans are suitable model organisms for investigations of regeneration mechanisms in the auditory system. Regeneration has been described in the auditory systems of locusts (Caelifera) and of crickets (Ensifera). In this study, we comparatively investigate the neural regeneration in the auditory system in the bush cricket Mecopoda elongata. A crushing of the tympanal nerve in the foreleg of M. elongata results in a loss of auditory information transfer. Physiological recordings of the tympanal nerve suggest outgrowing fibers 5 days after crushing. An anatomical regeneration of the fibers within the central nervous system starts 10 days after crushing. The neuronal projection reaches the target area at day 20. Threshold values to low frequency airborne sound remain high after crushing, indicating a lower regeneration capability of this group of fibers. However, within the central target area the low frequency areas are also innervated. Recordings of auditory interneurons show that the regenerating fibers form new functional connections starting at day 20 after crushing.

Sounds, behaviour, and auditory receptors of the armoured ground cricket, Acanthoplus longipes.

The auditory sensory system of the taxon Hetrodinae has not been studied previously. Males of the African armoured ground cricket, Acanthoplus longipes (Orthoptera: Tettigoniidae: Hetrodinae) produce a calling song that lasts for minutes and consists of verses with two pulses. About three impulses are in the first pulse and about five impulses are in the second pulse. In contrast, the disturbance stridulation consists of verses with about 14 impulses that are not separated in pulses. Furthermore, the inter-impulse intervals of both types of sounds are different, whereas verses have similar durations. This indicates that the neuronal networks for sound generation are not identical. The frequency spectrum peaks at about 15 kHz in both types of sounds, whereas the hearing threshold has the greatest sensitivity between 4 and 10 kHz. The auditory afferents project into the prothoracic ganglion. The foreleg contains about 27 sensory neurons in the crista acustica; the midleg has 18 sensory neurons, and the hindleg has 14. The auditory system is similar to those of other Tettigoniidae.

Neuroanatomy and physiology of the complex tibial organ of an atympanate ensiferan, Ametrus tibialis (Brunner von Wattenwyl, 1888) (Gryllacrididae, Orthoptera) and evolutionary implications.

We investigated the neuroanatomy and physiology of the complex tibial organ of an atympanate ensiferan, the Gryllacridid Ametrus tibialis. This represents the first analysis of internal mechanoceptors in Gryllacridids. The complex tibial organ is tripartite consisting of a subgenual organ, intermediate organ and a homologue organ to the crista acustica of tympanate ensiferan taxa of Tettigoniidae, Haglidae, and Anostostomatidae. The crista homologue contains 23 +/- 2 receptor neurons in the foreleg. It is associated with the leg trachea and found serially in all three thoracic leg pairs. Central projections of the sensory nerve of the complex tibial organ bifurcate in two lobes in the prothoracic ganglion, which do not reach the midline. The axonal endings project into the mVAC, the main vibratory-auditory neuropile of Ensifera. Recordings of the tibial nerve show that the tibial organ is sensitive to vibrational stimuli with a minimum threshold of 0.02 to 0.05 ms(-2) at 200-500 Hz, but rather insensitive to airborne sound. The main function of the tibial organ is therefore vibration sensing, although the specific function of the crista homologue remains unclear. The presence of the crista acustica homologue is interpreted in phylogenetic context. Because ensiferan phylogeny is unresolved, two alternative scenarios can be deduced: (a) the crista homologue is a precursor structure which was co-opted as an auditory system and represent a morphologically highly specialized structure before acquisition of its new function; (b) a previously functional tibial ear is evolutionary reduced but the neuronal structures are maintained. Based on comparison of neuroanatomical details, the crista acustica homologue of A. tibialis could present the neuronal complement of an ear evolutionary precursor structure, which was successively made sensitive to airborne sound by elaboration of cuticular tympana, auditory spiracle and trachea for sound propagation.

Independent suboesophageal neuronal innervation of the defense gland and longitudinal muscles in the stick insect (Peruphasma schultei) prothorax.

This study investigates the neuroanatomy of the defense gland and a related muscle in the stick insect Peruphasma schultei with axonal tracing and histological sections. The gland is innervated by three neurons through the Nervus anterior of the suboesophageal ganglion (SOG), the ipsilateral neuron (ILN), the contralateral neuron (CLN) and the prothoracic intersegmental neuron (PIN). The ILN has a large soma which is typical for motoneurons that cause fast contraction of large muscles and its dendrites are located in motor-sensory and sensory neuropile areas of the SOG. The CLN might be involved in the coordination of bilateral or unilateral discharge as its neurites are closely associated to the ILN of the contralateral gland. Close to the ejaculatory duct of the gland lies a dorsal longitudinal neck muscle, musculus pronoto-occipitalis (Idlm2), which is likely indirectly involved in gland discharge by controlling neck movements and, therefore, the direction of discharge. This muscle is innervated by three ventral median neurons (VMN). Thus, three neuron types (ILN, CLN, and PIN) innervate the gland muscle directly, and the VMNs could aid secretion indirectly. The cytoanatomy of motorneurons innervating the defense gland and neck muscle are discussed regarding the structure and functions of the neuropile in the SOG. As a basis for the neuroanatomical study on the defense gland we assembled a map of the SOG in Phasmatodea.

Morphology and physiology of the prosternal chordotonal organ of the sarcophagid fly Sarcophaga bullata (Parker).

The anatomy and the physiology of the prosternal chordotonal organ (pCO) within the prothorax of Sarcophaga bullata is analysed. Neuroanatomical studies illustrate that the approximately 35 sensory axons terminate within the median ventral association centre of the different neuromeres of the thoracico-abdominal ganglion. At the single-cell level two classes of receptor cells can be discriminated physiologically and morphologically: receptor cells with dorso-lateral branches in the mesothoracic neuromere are insensitive to frequencies below approximately 1 kHz. Receptor cells without such branches respond most sensitive at lower frequencies. Absolute thresholds vary between 0.2 and 8m/s(2) for different frequencies. The sensory information is transmitted to the brain via ascending interneurons. Functional analyses reveal a mechanical transmission of forced head rotations and of foreleg vibrations to the attachment site of the pCO. In summed action potential recordings a physiological correlate was found to stimuli with parameters of leg vibrations, rather than to those of head rotation. The data represent a first physiological study of a putative predecessor organ of an insect ear.

Developmental constraint of insect audition.

BACKGROUND: Insect ears contain very different numbers of sensory cells, from only one sensory cell in some moths to thousands of sensory cells, e.g. in cicadas. These differences still await functional explanation and especially the large numbers in cicadas remain puzzling. Insects of the different orders have distinct developmental sequences for the generation of auditory organs. These sensory cells might have different functions depending on the developmental stages. Here we propose that constraints arising during development are also important for the design of insect ears and might influence cell numbers of the adults. PRESENTATION OF THE HYPOTHESIS: We propose that the functional requirements of the subadult stages determine the adult complement of sensory units in the auditory system of cicadas. The hypothetical larval sensory organ should function as a vibration receiver, representing a functional caenogenesis. TESTING THE HYPOTHESIS: Experiments at different levels have to be designed to test the hypothesis. Firstly, the neuroanatomy of the larval sense organ should be analyzed to detail. Secondly, the function should be unraveled neurophysiologically and behaviorally. Thirdly, the persistence of the sensory cells and the rebuilding of the sensory organ to the adult should be investigated. IMPLICATIONS OF THE HYPOTHESIS: Usually, the evolution of insect ears is viewed with respect to physiological and neuronal mechanisms of sound perception. This view should be extended to the development of sense organs. Functional requirements during postembryonic development may act as constraints for the evolution of adult organs, as exemplified with the auditory system of cicadas.

Embryonic development of pleuropodia of the cicada, Magicicada cassini.

In many insects the first abdominal segment possesses embryonic appendages called pleuropodia. Here we show the embryogenesis of pleuropodial cells of the periodical cicada, Magicicada cassini (Fisher 1851) (Insecta, Homoptera, Cicadidae). An antibody, anti-horseradish perioxidase (HRP), that is usually neuron-specific strongly marked the pleuropodial anlagen and revealed their ectodermal origin shortly after limb bud formation. Thereafter the cells sank into the epidermis and their apical parts enlarged. A globular part protruded from the body wall. Filamentous structures were marked at the stem region and into the apical dilation. In later embryonic stages the pleuropodia degenerated. Despite the binding of anti-HRP the cells had no morphological neuronal characters and cannot be regarded as neurons. The binding indicates that glycosylated cell surface molecules contribute to the adhesion between the presumably glandular pleuropodial cells. In comparison, anti-HRP does not mark the pleuropodia of Orthoptera.

How many mechanosensory organs in the bushcricket leg? Neuroanatomy of the scolopidial accessory organ in Tettigoniidae (Insecta: Orthoptera).

The subgenual organ and associated scolopidial organs are well studied in Orthoptera and related taxa. In some insects, a small accessory organ or Nebenorgan is described posterior to the subgenual organ. In Tettigoniidae (Ensifera), the accessory organ has only been noted in one species though tibial sensory organs are well studied for neuroanatomy and physiology. Here, we use axonal tracing to analyse the posterior subgenual organ innervated by the main motor nerve. Investigating seven species from different groups of Tettigoniidae, we describe a small group of scolopidial sensilla (5-9 sensory neurons) which has features characteristic of the accessory organ: posterior tibial position, innervation by the main leg nerve rather than by the tympanal nerve, orientation of dendrites in proximal or ventro-proximal direction in the leg, and commonly association with a single campaniform sensillum. The neuroanatomy is highly similar between leg pairs. We show differences in the innervation in two species of the genus Poecilimon as compared to the other species. In Poecilimon, the sensilla of the accessory organ are innervated by one nerve branch together with the subgenual organ. The results suggest that the accessory organ is part of the sensory bauplan in the leg of Tettigoniidae and probably Ensifera.

Nitric oxide/cyclic guanosine monophosphate signaling in the central complex of the grasshopper brain inhibits singing behavior.

Grasshopper sound production, in the context of mate finding, courtship, and rivalry, is controlled by the central body complex in the protocerebrum. Stimulation of muscarinic acetylcholine receptors in the central complex has been demonstrated to stimulate specific singing in various grasshoppers including the species Chorthippus biguttulus. Sound production elicited by stimulation of muscarinic acetylcholine receptors in the central complex is inhibited by co-applications of various drugs activating the nitric oxide/cyclic guanosine monophosphate (cGMP) signaling pathway. The nitric oxide-donor sodium nitroprusside caused a reversible suppression of muscarine-stimulated sound production that could be blocked by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxaline-1-one (ODQ), which prevents the formation of cGMP by specifically inhibiting soluble guanylyl cyclase. Furthermore, injections of both the membrane-permeable cGMP analog 8-Br-cGMP and the specific inhibitor of the cGMP-degrading phosphodiesterase Zaprinast reversibly inhibited singing. To identify putative sources of nitric oxide, brains of Ch. biguttulus were subjected to both nitric oxide synthase immunocytochemistry and NADPH-diaphorase staining. Among other areas known to express nitric oxide synthase, both procedures consistently labeled peripheral layers in the upper division of the central body complex, suggesting that neurons supplying this neuropil contain nitric oxide synthase and may generate nitric oxide upon activation. Exposure of dissected brains to nitric oxide and 3-(5'hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) induced cGMP-associated immunoreactivity in both the upper and lower division. Therefore, both the morphological and pharmacological data presented in this study strongly suggest a contribution of the nitric oxide/cGMP signaling pathway to the central control of grasshopper sound production.

Phonotaxis of the female parasitoid Emblemasoma auditrix (Diptera, Sarcophagidae) in relation to number of larvae and age.

The dipteran parasitoid Emblemasoma auditrix locates its host acoustically. Analysis showed that phonotactic female flies usually carry fully developed larvae within their uteri. The mean number of larvae per female at the beginning of the season was 37.9 (range from 10 to 50). The number of larvae decreased rapidly with increasing singing activity of the host cicada (Okanagana rimosa). In high-density host populations the parasitoid is likely to become egg-limited. A possible selective phonotactic responsiveness depending on the number of larvae or the age of the female was tested with song models. Phonotaxis depended on both the temporal structure and the frequency content, but in the field no correlation was found between the number of larvae and the preferences for the acoustic signal. Experiments in the laboratory showed that flies without host contact broadened their phonotactic stimulus range with age.

Position-dependent hearing in three species of bushcrickets (Tettigoniidae, Orthoptera).

A primary task of auditory systems is the localization of sound sources in space. Sound source localization in azimuth is usually based on temporal or intensity differences of sounds between the bilaterally arranged ears. In mammals, localization in elevation is possible by transfer functions at the ear, especially the pinnae. Although insects are able to locate sound sources, little attention is given to the mechanisms of acoustic orientation to elevated positions. Here we comparatively analyse the peripheral hearing thresholds of three species of bushcrickets in respect to sound source positions in space. The hearing thresholds across frequencies depend on the location of a sound source in the three-dimensional hearing space in front of the animal. Thresholds differ for different azimuthal positions and for different positions in elevation. This position-dependent frequency tuning is species specific. Largest differences in thresholds between positions are found in Ancylecha fenestrata. Correspondingly, A. fenestrata has a rather complex ear morphology including cuticular folds covering the anterior tympanal membrane. The position-dependent tuning might contribute to sound source localization in the habitats. Acoustic orientation might be a selective factor for the evolution of morphological structures at the bushcricket ear and, speculatively, even for frequency fractioning in the ear.

Infection behavior of a parasitoid fly, Emblemasoma auditrix, and its host cicada Okanagana rimosa.

Males of the cicada Okanagana rimosa (Homoptera: Cicadidae) that produce calling songs are parasitised by the parasitoid fly Emblemasoma auditrix (Diptera: Sarcophagidae). An ethogram of the infection behavior was extracted from videotaped experiments with tethered hosts. The infection behavior can be divided into three phases, each involving different sensory cues: long-range host detection via acoustic signals, visual short-range orientation, and a contact phase with tactile/chemical cues. After phonotaxis by flight, the fly lands on or near the host cicada. It walks around the host to identify the caudal end and squeezes underneath the wings (with a 64-67% preference of the left side). Finally, E. auditrix cuts into the timbal and deposits a larva into the sound producing organ of O. rimosa. This highly specific behavior restricts the host range to cicadas, only two species of which occur simultaneously with the parasitoid. During the infection behavior, the fly does not discriminate between male and female hosts. However, females were not successfully infected during the experiments. The host O. rimosa shows only weak defense behavior (mainly flapping their wings) and seems to rely on parasitoid avoidance. Production of the protest song does not prevent infection.