/. 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 cricket Teleogryllus 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immature animals to show behavioural responses to the stridulatory sounds of adult males (Alexander, 1962; K. G. Hill, unpublished) in spite of the presence of representatives of each of the five groups of sensilla found in the adult ear as early as five moults before adulthood (Ball &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 first few 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.