/. Embryol. exp. Morph. Vol. 46, pp. 75-87, 197S 75 Printed in Great Britain © Company of Biologists Limited 1978

Ultrastructural study of the development of the auditory tympana in the commodus (Walker)

By E. E. BALL1 AND A. N. COWAN1 From the Department of Neurobiology, Australian National University, Canberra

SUMMARY The cuticle in the tympanal area of immature crickets, Teleogryllus commodus (Walker), is ultrastructurally indistinguishable from that elsewhere on the prothoracic leg. It is only in the pharate adult that changes associated with development of the tympana first appear. In pharate adults and adults the external layer of the tympana consists of a layer of electron- dense material overlying a layer where the electron-dense material is interspersed with cuticle in which the bundles of microfibrils are coarser and more loosely arranged than elsewhere in the leg. The innermost portion of the tympana consists of this same type of cuticle without the electron-dense material. Associated with the appearance of the electron-dense material in the tympana of the pharate adult is a change in the toluidine blue staining properties from blue to deep purple. The reaction of the tympana in acid and base is consistent with their being composed of chitin. There are no major deposits of resilin in the tympana. In the first few days following the imaginal ecdysis the posterior tympanum and underlying trachea come into tight apposition due to the withdrawal of the epidermal cells. The epidermal cells do not withdraw from beneath the anterior tympanum. The surrounding non-tympanal cuticle continues to thicken for several weeks with the result that in the mature adult the posterior tympanum serves as an acoustic window in the thick cuticle of the leg. The functional significance of the anterior tympanum has not been established.

INTRODUCTION An interesting problem in the behavioural physiology of crickets involves the failure of immature to show behavioural responses to the stridulatory sounds of adult males (Alexander, 1962; K. G. Hill, unpublished) in spite of the presence of representatives of each of the five groups of sensilla found in the adult ear as early as five moults before adulthood (Ball & Young, 1974). There are probably several reasons for the failure to respond, but a major contributing factor appears to be that the ears of immature animals are much less sensitive to sound than those of adults. Comparison of the responses of auditory inter- neurons of adults and animals of the preceding two instars to airborne sound established that there is a dramatic increase in sensitivity to sound at the final 1 Authors' address: Department of Neurobiology, Research School of Biological Sciences, Australian National University, P.O. Box 475, Canberra City A.C.T. 2601, Australia. 76 E. E. BALL AND A. N. COWAN ecdysis and that this change is not due to changes in the receptors since their response to direct vibration of the leg shows a much smaller change (E. E. Ball & K. G. Hill, in preparation). One obvious change which occurs at the final moult is the appearance of two auditory tympana on each prothoracic leg, a large posterior tympanum and a smaller anterior tympanum. The external development of these tympana has previously been described (Ball & Young, 1974; Young & Ball, 1974). In brief, the sensilla on the areas where the tympana will later form start to become less abundant at the moult to the third instar before adulthood (A-3) and the resulting bare depressed areas increase in size at the next two moults. At the final moult there is a dramatic change in the appearance of the tympanal area from the brown colour of the surrounding integument to a shining silvery white. In earlier papers (Young & Ball, 1974; Ball & Young, 1974) special attention was paid to the development of the receptors and tympanal changes were examined only at the level of light microscopy and scanning electron microscopy. However, since it now appears that the abrupt increase in the sensitivity of the auditory system at the imaginal moult is due to the development of the tympana (E. E. Ball & K. G. Hill, in preparation) a better understanding of the changes that occur in the tympanal area during development is desirable.

MATERIALS AND METHODS Experimental animals The Teleogryllus commodus used in the present experiments were from near Canberra and were either collected in the field as nymphs or cultured from previously collected stocks. Adults and the two preceding instars (as recognized by wing condition) were used and are here termed adult (A), ultimate instar (A-l) and penultimate instar (A-2). Crickets were checked for moulting on alternate days and were kept in an outdoor greenhouse with partial temperature control, so the post-moult times given are only intended to indicate the relative point between moults. Ages of animals used in these studies are as follows: A-2 post-apolysis, pre-ecdysis (i.e. a pharate A-l) A-l newly ecdysed 7 days post-ecdysis 13 days post-ecdysis post-apolysis, pre-ecdysis (pharate adult) A newly ecdysed 2^4 days post-ecdysis 14 days post-ecdysis 21 days post-ecdysis Development of auditory tympana in Teleogryllus 77

Light and transmission electron microscopy Prothoracic tibiae were cut off into Ribi's fixative (Ribi, 1976) where they were either trimmed close to the tympana or split longitudinally. They were fixed overnight at 5 °C, rinsed in buffer, postfixed in 2 % OsO4 for 2 h and dehydrated through an ethanol series, taken through propylene oxide and a propylene oxide-Araldite mixture and embedded in Araldite. 1 jam sections were cut on glass knives, stained with toluidine blue and examined and photo- graphed on a Zeiss Photomicroscope. Thin sections were cut on a diamond knife, mounted on slot grids covered with Formvar or parlodion and examined unstained or stained with either barium permanganate or uranyl acetate and lead citrate. They were examined and photographed on a JEOL 100C electron microscope.

Scanning electron microscopy For scanning electron microscopy prothoracic legs were cut off, mounted on paper triangles, coated with carbon or carbon followed by gold, and examined and photographed on a Hitachi HHS-2R scanning electron microscope.

Tests to identify the biochemical nature of the changes in the tympanal cuticle of pharate adults and adults To investigate the biochemical nature of adult tympanal cuticle the following tests were carried out: (1) Fluorescence test for resilin following the methods of Scott (1970). This test was carried out on unstained wax sections and on frozen sections in which it was possible to separate tympanal tissue from the underlying trachea. Locust wing hinge, with its known deposits of resilin (Anderson & Weis-Fogh, 1964), was used as a control to be sure the test was working properly. (2) Staining of unfixed material in toluidine blue and light green - the 'simple colour test' for resilin of Anderson & Weis-Fogh (1964). Locust wing hinge was again run as a control. (3) Mallory's Triple Stain (Pantin, 1946). (4) Masson's Trichrome Stain (Pantin, 1946). (5) To test for the presence of chitin and/or protein, portions of prothoracic tibiae containing a tympanum were placed in (a) 1 N-NaOH, and (b) 5 N-HC1 in an oven at 60 °C and examined periodically over the next 96 h.

RESULTS Normal development Changes in the external morphology of the tympana during development have previously been described (Ball & Young, 1974; Young & Ball, 1974). Aside from the lack of sensilla on the tympanal region, tympanal and non-tympanal

6 E M B 46 78 E. E. BALL AND A. N. COWAN Tympanal Non-tympanal Tympanal Non-tympanal

Pharate A

Pharate A-l

A, new A-l, new

A-l, 7 days

Epicuticle and dense exocuticle

Exocuticle

Endocuticle

1 Epidermis 10//m ffl& Tympanal cuticle A, 21 days Fig. 1. Summary of the changes occurring in tympanal cuticle and in non-tympanal cuticle from the same part of the tibia during the last two pre-adult instars and adulthood. Differences between the two types of cuticle first become apparent in the pharate adult. During the firstfe w weeks of adulthood epidermal tissue withdraws from between the tympanal cuticle and the underlying trachea of the posterior tympanum while the non-tympanal cuticle continues to thicken. Development of auditory tympana in Teleogryllus 79 cuticle are identical throughout the last two pre-adult instars (Fig. 1). The post- apolysis A-2 cuticle shown in Fig. 2 is typical of pre-adult cuticle, with a thin epicuticle, a thicker finely lamellate layer of electron-dense exocuticle and a wide, finely lamellate layer of electron-lucent exocuticle penetrated by pore canals. Beneath this lies the endocuticle which is made up of non-lamellate layers alternating with narrower lamellate layers. The number of lamellae in a lamellate layer varies from two to four. The greatest number of lamellate (night)/non- lamellate (day) growth layers found in our immature crickets was five. The developmental changes in both tympana are similar so all statements refer to both unless otherwise noted. The differences between tympanal and non- tympanal cuticle first become apparent in the pharate adult (Figs. 3-5), when the tympanal cuticle becomes electron dense. Beneath an apparently solid layer of electron-dense material is a layer where chitin microfibrils and the electron- dense material are laid down in a helicoidal pattern (Figs. 4, 7, 8). The difference between tympanal and non-tympanal cuticle of pharate adults and adults is also clearly shown in toluidine blue stained 1 [im Araldite sections where tympanal cuticle stains deep purple, in contrast to non-tympanal cuticle and tympanal cuticle of pre-adults, which stain a lighter bluish-purple (Fig. 6). The surface of the epidermis underlying both tympanal and non-tympanal cuticle is thrown into folds or microvilli (Figs. 3, 4, 5B) between which abundant vesicles are found (Figs. 4, 5 A, 5B). Similar vesicles are sometimes seen in the pore canals of non-tympanal cuticle (Fig. 5A, 5B). Adult tympanal cuticle is shown in Figs. 7-9. Within a few days after the final ecdysis the epidermal cells withdraw from between the posterior tympanum and the trachea, leaving them tightly apposed (Fig. 9). Once the epidermal tissue has withdrawn the tympanal thickness remains constant while the non- tympanal cuticle continues to grow in thickness for at least 2 weeks (Fig. 1). The epidermal cells do not withdraw from beneath the anterior tympanum. The adult tympana appear smooth and silvery to the naked eye but scanning electron microscopy reveals that, in contrast to non-tympanal cuticle, they are covered with microtrichia (Fig. 10A, B). We have been unable positively to identify the material of which the bulk of the adult tympanum is composed. Results of the tests listed in Materials and Methods are given below: (1) Fluorescence - the tympana in whole legs fluoresced blue, but most of this fluorescence originated from the underlying tracheae. Locust wing hinge resilin fluoresced bright blue as expected. (2) Staining of unfixed material in toludine blue and light green - most of the tympanal cuticle remained transparent except for a narrow turquoise-staining rim around the periphery. Locust wing hinge stained sapphire blue as expected. (3) Mallory's Triple Stain - non-tympanal cuticle stained orange. Tympanal cuticle stained deeper orange than non-tympanal but there was no differentiation within the tympanum itself. 6-2 80 E. E. BALL AND A. N. COWAN

endo

dig

3 I Development of auditory tympana in Teleogryllus 81 (4) Masson's Trichrome Stain - non-tympanal cuticle stained bright red- orange. Tympanal cuticle had a thin red-orange layer outside a thicker green layer. (5) (a) In 1 N-NaOH tympanal cuticle was still present and was not obviously changed, (b) In 5 N-HC1 tympanal cuticle disappeared.

DISCUSSION 1. Structural considerations Neville (1967, 1975) reports finding daily growth layers in adult Gryllus (Acheta) domesticus and Gryllus bimaculatus; but we never found more than five layers in immature crickets which required 12-18 days for the penultimate instar and 13-26 days for the ultimate instar. So, assuming these are daily layers, they cannot be laid down continuously throughout the intermoult period. In adult Teleogryllus endocuticle continues to grow thicker for at least 2-3 weeks. It is difficult to correlate our electron microscopic findings with the light microscope histochemistry of Philogene & McFarlane (1967) on the cuticle of Acheta domesticus. Without the aid of the electron microscope we would have mistaken the thin layer of dense exocuticle beneath the epicuticle for part of the epicuticle and this mistake would have been even more likely in their thicker wax sections. It therefore seems probable that their 'thickest layer of epicuticle', which they characterize as 'being made up of protein material', was in fact the dense outer exocuticle, with the true epicuticle lying external to this. Helicoidal patterning is more obvious and coarser in the internal part of the tympanum (Fig. 7, 8) than elsewhere in the cuticle (e.g. Figs. 2, 5). This heli- coidal arrangement may also be present in the external layer of the tympanum but, if so, it is masked by the electron-dense material there. The surface of the epidermis underlying the cuticle appears microvillar in our material, but Caveney & Podgorski (1975) have established that in Tenebrio the apparent microvilli are, in fact, randomly oriented folds and this may also be

FIGURES 2-3 Fig. 2. A-2 cuticle (which has separated from the newly formed underlying A-l cuticle) showing how the various cuticular layers are arranged. From the exterior (top of figure) these layers are epicuticle (epi) dense exocuticle (d exo) normal exocuticle {exo) containing pore canals (pc), and endocuticle (endo). The endo- cuticle is divided into wide, non-lamellate, layers (/?/) and narrower lamellate layers (/). The number of the latter laid down per night appears to vary. At the inside edge of the cuticle is a zone (dig) where the endocuticle is being digested by moulting fluid. This zone is bounded by the ecdysial membrane (em). Fig. 3. Tympanal cuticle (tc) forming in a pharate adult. At the base of the new cuticle is a zone of folds (/)or microvilli. The epidermal cells contain mitochondria (M), and abundant rough endoplasmic reticulum (er), only two areas of which are labelled. The epidermis is bounded basally by the trachea and a tracheal rib (tr) is shown. 82 E. E. BALL AND A. N. COWAN

Fig. 4. Posterior tympanum of a pharate adult. The chemical composition of the outer layer of electron-dense material is unknown. Beneath the solid electron-dense material (dm) is a zone of helicoidal cuticle (he). The outer margin of the epidermal cells is folded (/) and between the folds are pockets of vesicles (v). Fig. 5. (A) Non-tympanal tibial cuticle in a pharate adult. Note epicuticle (epi), dense exocuticle (d exo), normal exocuticle (exo), pore canals (pc) and vesicles (v), which are found both at the epidermal margin and within the pore canals {pc). (B) Enlarged view of vesicles in the pore canals. Labels as in Fig. 5 A. Development of auditory tympana in Teleogryllus 83

Fig. 6. A 1 /tm Araldite section stained with toluidine blue which clearly shows the difference in staining properties between non-tympanal cuticle (c) and tympanal cuticle (tc). Fig. 7. Transverse section of tympanal cuticle in an adult 14 days post-ecdysis. Note outer layer of dense material (dm), the way that the dense material appears to follow the pore canals (arrows) and the helicoidal arrangement of the bundles of chitin microfibrils (he) in the inner part of the tympanum. Fig. 8. Oblique section of tympanal cuticle from the same used in Fig. 7. Note helicoidal chitin (he) and helicoidally arranged dense material (dm). 84 E. E. BALL AND A. N. COWAN

10B Fig. 9. Tympanum of an adult cricket showing the tympanum and underlying trachea in tight apposition. Note epicuticle (epi), dense material (dm), the helicoidal layer (he) with its patches of dense material, and the underlying trachea with its tracheal rib (tr). Fig. 10 (A) Low power scanning electron micrograph of adult posterior tympanum. The adult tympanum is covered with microtrichia in contrast to non-tympanal cuticle. (B) Higher power scanning micrograph of the microtrichia. Development of auditory tympana in Teieogryllus 85 the case in Teieogryllus. The nature of the vesicles between the epidermal folds (or microvilli) of both tympanal and non-tympanal cuticles (Figs. 4, 5 A, 5B) is unknown although it seems likely that their contents may be involved in cuticle formation. Similar-appearing vesicles are sometimes seen in the pore canals (Fig. 5 A, 5B), especially during periods when cuticle is being laid down. The strongly metachromatic staining of the tympanal cuticle by toluidine blue indicates the presence of acid groups (Sumner & Sumner, 1969). Since these staining properties and the electron-dense material appear at the same time during development we assume that they are related. The behaviour of tympanal cuticle in acid and base is consistent with the presence of chitin there. It was formerly stated (Young & Ball, 1974) that the posterior tympanal trachea of Teieogryllus was not directly apposed to the inner surface of the tympanum but was separated from it by a layer of hypodermis and tracheal epithelium. This statement is true for the newly moulted adults used in the former study, but, as described here, later in adulthood the posterior tympanum and trachea do become apposed. The loss of underlying epithelium from beneath the posterior tympanum and its retention beneath the anterior tym- panum are in agreement with the findings of Schwabe (1906) in Acheta domesticus and Michel (1974) in Gryllus bimaculatus.

2. Functional considerations The properties of the tympanum could, in theory, play an important role in both the tuning and sensitivity of the auditory system. Studies using the Moss- bauer effect (Johnstone, Saunders & Johnstone, 1970) and optical heterodyne spectroscopy (Dragsten, Webb, Paton & Capranica, 1974; Paton, Capranica, Dragsten & Webb, 1977) are in agreement that the peak mechanical response of the posterior tympanum of all cricket species examined is in the range 40-60kHz and Johnstone et al. (1970) have suggested that the tympanic membrane 'is mechanically tuned to a rather narrow spectrum of frequencies * and that, 'the resonant properties of the drum account very likely for the tuning curves reported for single units'. The latter hypothesis was, however, attacked by Loftus-Hills, Littlejohn & Hill (1971) who pointed out that both the calling song frequency and the optimal auditory sensitivity of T. commodus (one of the species investigated by Johnstone et al) fell outside the range of maximal tympanal vibration. Hill (1974) demonstrated that loading the larger posterior tympanum with Vaseline reversibly decreased the sensitivity of auditory inter- neurons in T. commodus by 15-25 dB, but did not comment on any effect on tuning. Paton et al. (1977) did the same experiment on other crickets, found a similar effect on sensitivity, and made the additional observation that the frequency of maximal sensitivity showed little change. Paton et al. (1977) also made a hole in the large tympanic membrane which they then gradually en- larged. As they did this the sensitivity peak in the tuning curve became less 86 E. E. BALL AND A. N. COWAN pronounced. Nocke (1972) detached the posterior tympanum from its surround- ing cuticle while leaving it connected to the underlying trachea and found that the sensitivity of the system was reduced by about 30 dB. Ball & Hill (in pre- paration) have demonstrated that the frequency of peak auditory sensitivity is the same in immature animals which have yet to form a tympanum as it is in adults. From all of these results it is apparent that the tympanum acts as an acoustic window in the wall of the tibia but that its resonant properties play little or no role in the tuning of the ear. The continuity of the tympanum and the surrounding cuticle is, however, important to auditory sensitivity. Judging by their structural differences it would seem that the mechanical properties of tympanal and non-tympanal cuticle should differ. However, due to the difficulty of obtaining adequate amounts of pure material it has not been possible to analyse such properties.

We thank Drs B. Filshie and R. Hackman for advice and discussion during the work and Mr G. Boyan, Dr J. Edwards, Dr B. Filshie, Dr K. Hill and Dr S. Reynolds for comments on the manuscript.

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{Received 5 December 1977, revised 27 January 1978)