COMPARISON OF MORPHOLOGY, PHYSIOLOGY AND DEVELOPMENT OF THE RECEPTOR ORGANS

I

In bushcrickets, auditory–vibratory communication plays an important role in reproductive behaviour, agonistic interactions, detection of predators and for general acoustic orientation in the environment. The most important and physiologically dominant receptor organs of the bimodal auditory–vibratory sensory system are the complex tibial organs of all six legs. Complex tibial organs are present in the pro metathoracic, mesometathoracic, and metathoracic legs. In each leg, the complex tibial organs consist of three scolopale organs, e.g. the subgenual organ (SO), the

intermediate organ (IO), and the crista acustica (CA). Only in the forelegs the tibial organs are specialised as tympanal organs, where the crista acustica and the distal parts of the intermediate organ serve as auditory receptors (Schumacher, 1979). In tettigoniids, the prothoracic spiracles are the main input for airborne sound. The acoustic trachea transmits sound to the tympanal organs in the proximal tibiae of the forelegs; the vibrations of the tympana are caused by sound acting on the inner surface of the tympanum (Lewis, 1974; Michelsen and Larsen, 1978; Heinrich et al., 1993).

The presence of the sound transmitting system and the anatomical differentiation within the auditory receptor organs in the forelegs allow sensitivity to airborne sound. However, the absolute sensitivity and the frequency tuning of the auditory threshold can vary significantly between species.

In spite of the absence of tympana and spiracle, the receptor complexes of the subgenual organ, the intermediate organ and the CA are also fully developed in the mid- and hind legs. However, the CA in the mid- and hind legs consists of a smaller number of receptor cells than in the forelegs. Some structural differences at the receptor-cell level are present (Lin et al., 1994). Schumacher (1979) referred to the organs in the mid- and hind legs as atympanate organs. Some success has been achieved in elucidating the function of the tibial organs of the forelegs (Autrum, 1941; Rheinla¨nder, 1975; Kalmring et al., 1978; Zhantiev and Korsunovskaya, 1978; Oldfield, 1982; Lin et al., 1993). During embryonic development, the tibial organs differentiate in all legs from an invagination of ectodermal cells of the tibia (Meier and Reichert, 1990). The authors postulate that the auditory organs in Ensifera evolved from a serially reiterated group of leg- associated mechano-receptors, which in the prothoracic leg became specialised for the perception of airborne sound.

Receptor cells of the complex tibial organs in the meso- and metathoracic legs also respond to low frequency airborne sound at high intensities (Autrum, 1941). However, in contrast to the prothoracic organs, the sensitivity in the mesothoracic and metathoracic tibiae is not sufficient for the perception of the natural song. The real function of the CA in the mid- and hind legs is still unknown.

Sound can reach the receptor cells of the forelegs in two different ways, via the tympana and via the acoustic tracheal system which consists of the large spiracle, the vesicle and the long tapering trachea. There are many published controversial opinions concerning the function and importance of this stimulus transducing system. It is not that the spiracle is the main site of sound entry (Lewis, 1974; Nocke, 1975; Seymour et al., 1978; Hill and Oldfield, 1981; Larsen, 1981; Oldfield, 1984, 1985; Heinrich et al., 1993; Kalmring et al., 1993; Lin et al., 1993; Shen, 1993), but there is considerable disagreement upon the transmission properties of the acoustic trachea and its contribution to the frequency selectivity of the auditory receptors.

Some detailed physiological investigations exist on the function of the auditory receptor cells of the CA and the IO of the foreleg (Rheinla¨nder, 1975; Kalmring et al., 1978; Zhantiev and Korsunovskaya, 1978; Ro¨mer, 1983; Oldfield, 1985). Individual receptor cells differ in their tuning curves, but the whole receptor population of one tympanal organ covers a frequency range from at least 2 kHz up to 70 kHz. There is much overlapping of the frequency–intensity response characteristics of these cells. Attempts to correlate function and structure of the receptor cells of the CA by recording directly from the cells within the organs (Zhantiev and Korsunovskaya, 1978; Oldfield, 1982, 1984) have failed to give a satisfactory explanation of the mechanical stimulus transformation process within the tympanal organ. It is difficult to investigate the structures of the receptor organs which are involved in stimulus transduction, biophysically and physiologically. These structures are located inside the leg and can only be exposed for experimental means by dissecting the cuticle. Such procedures are expected to alter the biophysical and physiological properties of the organ.

Insect Sounds and Communication: Physiology, Behaviour, Ecology and Evolution 36

The critical structures for mechanical stimulus-transforming processes inside the organs are probably:

(a) The dorsal wall of the acoustic trachea on which the receptor and satellite cells are located

(b) The tectorial membrane covering the receptor and satellite cells situated in the haemolymph channel

(c) Possibly the dividing wall separating the acoustic trachea inside the receptor organ into two chambers

Mechanical displacement of these structures induced by sound stimuli via the motion of the tympana (Bangert et al., 1998) could directly or indirectly evoke the specific activity of the different auditory receptor cells.

In tettigoniids the neuropiles of the anterior Ring Tract (aRT) (Tyrer and Gregory, 1982) are the projection areas of the auditory receptor cells of the CA and the IO in the forelegs (Oldfield, 1985; Ro¨mer et al., 1988; Ahi et al., 1993). The projection of the auditory and auditory–vibratory receptors is restricted to the ipsilateral hemiganglion. Receptor cells of the subgenual organ as well as those of other vibro-receptors of the forelegs (e.g. campaniform sensilla, femoral and tibial chordotonal organs) project to lateral parts of the related hemiganglion (Mu¨cke, 1991). The same receptor organs as in the forelegs are found also in the mid- and hind legs. The receptor outline of the CA and the IO is similarly constructed, with the decisive difference of lacking a sound transmitting system (spiracle, acoustic trachea and tympana are not developed in the mid- and hind legs) (Schumacher, 1973, 1975). The function and projection of the receptors of the tibial organs there are not yet known. The same is true for the synaptic connection of their receptor input onto auditory–vibratory central neurons.

The projection of some auditory receptors of the forelegs in tettigoniids has been already described (Oldfield, 1983; Ro¨mer, 1983, 1985; Ro¨mer et al., 1988); this includes the projection

pattern and targets of identified receptor cells within a small part of the aRT (up to 50mm

parasagittal to the midline) and a counter clockwise tonotopic organisation across the aRT of the prothoracic ganglion. A first comparative quantitative analysis of the central projections of auditory receptor cells was carried out in three closely related species by Ahi et al. (1993).

Since the investigations by Cˇokl et al. (1977) and Silver et al. (1980) it is well known that the auditory system of the ventral nerve cord in locusts and bushcrickets is a bimodal sensory system. Most of the auditory neurons in the ventral nerve cord, which ascend to the supraesophageal ganglion, receive inputs from both the auditory receptors and the different vibration receptors of all six legs (intermediate organs, subgenual organs, chordotonal organs of the femur and tibia and campaniform sensilla). Therefore, it is better referred to as a combined auditory–vibratory system.

The function, and in part the morphology, of the bimodal auditory–vibratory ventral cord neurons ascending to the head ganglia have been described (Cˇokl et al., 1977; Silver et al., 1980; Kalmring and Ku¨hne, 1980; Boyan, 1984). The same is true for the auditory interneurons of the thoracic ventral nerve cord intercalating the auditory receptor cells with the bimodal central neurons (Ro¨mer, 1983; Ro¨mer et al., 1988; Lakes et al., 1990).

For bushcrickets, so far nothing is known about the neuronal connections between the vibratory receptor cells of all six legs and the auditory–vibratory central neurons ascending to the brain. There is the possibility that information from the receptors of one leg could be collected by one or several interneurons, which transmit this information to ascending neurons of the same segment or of different thoracic segments. It is still unclear if the ascending bimodal ventral cord neurons possess only one or more than one dendritic region for the vibratory and auditory input. In the latter case, the dendritic regions may be distributed on several segments, modulating the response at different positions along the neurons.

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O

Location and Morphology

The tibial organs are the main receptor organs for the detection of acoustic and vibratory signals used in the intraspecific communication of bushcrickets. In each leg the tibial organs are composed of three scolopale organs: the subgenual organ (SO), the IO and the CA, which all lie inside the proximal tibia in the haemolymph channel in close connection with the dorsal wall of the large leg trachea or one of its branches (Figure 3.1).

Similar systems are found in several other tettigoniid species (Figure 3.2) (Ro¨ssler, 1992a, 1992b; Lin et al., 1993, 1994). The overall arrangement of the scolopidia in the SO, the IO and the CA show similarities in the three legs, but the differences in the tracheal morphology and size of the structures are evident.

The number of scolopidia in the SO varies between 20 and 25 in each leg. In the intermediate organ, and especially in the CA, the number of scolopidia decreases from the prothoracic to the meso- and metathoracic legs.

FIGURE 3.1 Polysarcus denticauda. (a) Semischematic lateral view of the acoustic trachea of the foreleg. It starts with a large oval opening in the pleurite of the prothoracic segment, the spiracle (sp), which is connected to a funnel-shaped air sac, the vesicle (v), inside the prothorax. From there the trachea continues through the proximal parts of the leg as a gradually narrowing conical tube until it reaches the tibial organ (to). (b) Semischematic view of the acoustic trachea in the prothorax and the forelegs in a frontal view. (c) An adult male. (d) Three-dimensional reconstruction of the complex tibial organs derived from a series of transverse sections. Anterior-dorsal view of these organs in the fore-, mid- and hind legs (I, II, III), respectively, after removing the cuticle. The position of the complex tibial organs in all three legs is indicated (c.f. C). at/pt, anterior/posterior branch of the acoustic trachea; pro/dis, proximal/distal part of the IO; SO, subgenual organ; IO, intermediate organ; CA, crista acustica; tm, tectorial membrane; sb, supporting band. (After Sickmann, T., Kalmring, K., and Mu¨ller A., Hear. Res., 104, 155–166, 1997. With permission.)

Insect Sounds and Communication: Physiology, Behaviour, Ecology and Evolution 38

Another striking difference is the morphology of the trachea. The two large branches of the acoustic trachea in the foreleg are only separated by a thin wall (named the central membrane after Lewis (1974), the dividing wall after Nocke (1975) or the partition after Schumacher (1975)). The smaller tracheal branches in the mid- and hind legs run with greater separation and are connected to each other by the tracheal epithelium. In the foreleg, the volume of the anterior branch is somewhat larger than the posterior one; in the mid- and hind legs, the conditions are reversed. The tectorial membrane covering the CA and the IO is developed in each leg. Supporting bands on both sides of the cap cells are very marked in the foreleg CA only poorly developed in the midleg tibia and completely missing in the hind leg CA.

The detailed morphology of the complex tibial organ in the forelegs of bushcrickets is shown (Figure 3.3) Additionally, a composed transverse section from the middle part of the CA in bushcrickets with (as typical for Decticinae, Tettigoniinae, Ephippigerinae and Conocephalinae) and without tympanal covers (as typical for Phaneropterinae and Meconema- tinae) is inserted.

Figure 3.4 shows transverse sections of the proximal tibia of the three legs at the plane of the SO, the IO and the CA in Ephippiger ephippiger.

FIGURE 3.2 Adult structure of the complex tibial organs in the pro metathoracic (I), meso metathoracic (II), and metathoracic legs (III) of the bushcricket Gampsocleis gratiosa (G.g.), Ephippigerida taeniata (E.t.), and Ephippiger ephippiger (E.e.). Three-dimensional reconstructions from a series of transverse sections showing the organs in an anterior-dorsal view; the integument and the content of the nerve–muscle channel is removed. Abbreviations as in Figure 3.1. (After Ro¨ssler et al., 1992b; Lin et al., 1993. With permission.)

The size and shape of the SO is nearly the same in the fore- and midlegs; the SO of the hind leg is slightly larger especially in the anterioposterior direction. The arrangement of dendrites in the proximal group of scolopidia in the IO is comparable in all legs. At the plane of the CA, the absence of tympana, tympanal cavities and covers, and slits in the mid- and hind legs are obvious. In addition, the arrangement and location of scolopidia within the CA is different in the three legs because of the different size and course of the tracheal branches. In each case, the scolopidia are located on the anterior branch of the trachea. The acoustic trachea in the foreleg divides into two branches at the plane of the proximal part (in other species not earlier than at the middle) of the CA. The two branches are separated by the thin dividing wall. In the midleg, the trachea is divided at the beginning of the proximal part of the IO. In the hind leg, the trachea is divided into a small anterior and a larger posterior branch before the SO. At the plane of the IO and CA, the size of the haemolymph channel differs in the three legs. The volume of the haemolymph channel is significantly smaller in the foreleg than in the mid- and hind leg because of the large volume of the acoustic trachea.

Despite of these similarities in structure, it is only in the foreleg that the CA and the IO serve as sensitive detectors of airborne sound (Kalmring et al., 1994). This is because these are associated

FIGURE 3.3 Morphology of the tibial organ in the forelegs of bushcrickets. (a) Semischematic drawing of the complex tibial organ. For better insight the cuticle is removed at the anterior-dorsal side of the proximal tibia. tr, trachea; ip, inner plate of the tympanum; SO, subgenual organ; IO, intermediate organ; CA, crista acustica; tyn, tympanal nerve; ln, leg nerve; ty, tympanum. (b) A composite transverse section of the tibia at the plane of the middle CA; (left) P. denticauda with exposed tympana and (right) the tympanum protected by covers with slits, as T. viridissima. The morphology of the middle part (“general”) is almost the same in all bushcrickets. Note, the tympana extend from the middle of the haemolymph channel down to the ventral end of the tracheal chambers. atr, anterior tracheal branch; aty, anterior tympanum; cc, cap cells; cu, cuticle; epi, epidermis; hc, haemolymph channel; mf, muscle fibre; n, nerve; nmc, nerve–muscle channel; ptr, posterior tracheal branch; pty, posterior tympanum; sl, slits; so, soma of a receptor cell; tc, tympanal cover; tm, tectorial membrane, tra, trachea in the nerve muscle channel. (After Bangert, M., Kalmring, K., Sickmann, T., Stephen, R., Jatho, M., and Lakes, R., Hear. Res., 115, 27–38, 1998. With permission.)

Insect Sounds and Communication: Physiology, Behaviour, Ecology and Evolution 40

with structures suitable for conducting, filtering and amplifying airborne sound, acoustic spiracles, an acoustic trachea and tympana have developed exclusively in the foreleg (Figure 3.1a, b and Figure 3.3).

The tuning of the individual receptor cells to particular frequencies is probably due to the acousto-mechanical properties of the receptors in combination with their accessory cells, the dorsal wall of the trachea and the tectorial membrane. For instance, the bushcricket species Decticus albifrons, D. verrucivorus and Pholidoptera griseoaptera belong to the same subfamily Decticinae but differ significantly in size (Figure 3.5). In spite of the great differences in the dimensions of the forelegs, the most sensitive range of hearing lies from 6 to 25 kHz in each species. Only in the frequency range from 2 to 5 kHz and .25 kHz are significant differences present. Figure 3.5 shows that, despite substantial differences in the overall dimensions of the leg, the size of the dorsal wall of the anterior trachea is very similar.

The anatomy of the auditory receptor organs was quantitatively investigated using techniques of semithin sectioning and computer guided morphometry (Ro¨ssler and Kalmring, 1994). The overall number of scolopidia and the length of the CA differ in the three species, but the relative

FIGURE 3.4 Transverse sections of the proximal tibia in the pro metathoracic (I), meso metathoracic (II) and metathoracic legs (III) of Ephippiger ephippiger at the plane of the SO, proximal part of the IO and CA. at, anterior tracheal branch; cc, cap cell of the scolopidium; hc, haemolymph channel; m, muscles; n, tibial and tarsal nerve; pt, posterior tracheal branch; pty, posterior tympanum; sli, slit; tm, tectorial membrane. Anterior to the right. (Modified from Ro¨ssler, W., Zoomorphology, 112, 181–188, 1992b. With permission.)

distribution of scolopidia along the CA is very similar. Additionally, the scolopidia and their attachment structures (tectorial membrane, dorsal tracheal wall and cap cells) are of equal sizes at equivalent relative positions along the CA. The results indicate that the constant relations and dimensions of corresponding structures within the CA of the three species are most likely responsible for the similarities in the tuning of the auditory thresholds (Figure 3.6).

Postembryonic Development

The postembryonic (larval) development of the anatomy, morphology and physiology of the complex tibial receptor organs was investigated in all three pairs of legs in E. ephippiger (Ro¨ssler, 1992a). All the receptor cells in the three parts of the complex tibial organ (the SO, IO, and the crista acustica) are present from the first larval instar (Figure 3.7). The sound transmitting structures of the foreleg tympanal organ, the acoustic trachea, the tympana, the tympanal covers and the acoustic spiracle develop step by step in subsequent instars. The acoustic trachea inside the tibial organ of the foreleg resembles the adult structure for the first time in the fourth instar, although its volume is still small.

In E. ephippiger, the auditory threshold curves recorded from the tympanal nerve of the foreleg in instars four, five and six exhibit the same frequency maxima as those in the adult. Similarly, in Tettigonia cantans the overall sensitivity increases step by step (by about 10 to 20 dB) with each moult (Figure 3.8b) (Unrast, 1996). In contrast to the responses to airborne sound, sensitivity to vibratory stimuli was already high at larval instar three and did not increase in subsequent instars, including the adult. Both neurophysiological findings were confirmed in behaviour experiments.

The larval development of structures within the CA which are probably involved in stimulus transduction and in frequency tuning have been analysed. The dorsal wall of the anterior tracheal

FIGURE 3.5 Morphology of the proximal tibiae of the forelegs of three bushcricket species, Decticus albifrons (D.a.), Decticus verrucivorus (D.v.) and Pholidoptera griseoaptera (P.g.). Upper, left: scale- drawings of the tibiae. The arrows indicate the position of the transverse sections shown below. Lower: scale- drawings from transverse sections at the plane of the scolopidium in the middle of the cristae acusticae. Upper, right: the anterior tracheal branches are superimposed. Anterior is to the right. at, anterior tracheal branch; aty, anterior tympanum; cc, cap cell of scolopidium; dw, dorsal wall of anterior trachea; fe, femur; hc, haemolymph channel; nmc, nerve muscle channel; pt, posterior tracheal branch; pty, posterior tympanum; sli, slit; ti, tibia. (After Ro¨ssler, W. and Kalmring, K., Hear. Res., 80, 191–196, 1994. With permission.)

Insect Sounds and Communication: Physiology, Behaviour, Ecology and Evolution