/ . Embryol. exp. Morph. Vol. 46, pp. 75-87, 197S
Printed in Great Britain © Company of Biologists Limited 1978
75
Ultrastructural study of the
development of the auditory tympana in the cricket
Teleogryllus commodus (Walker)
By E. E. BALL 1 AND A. N. COWAN 1
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 electrondense 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sounds of adult males (Alexander, 1962; K. G. Hill, unpublished) in spite of the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 interneurons 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 photographed 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
ffl&
Tympanal cuticle
10//m
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 thefirstfew 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 postapolysis 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)/nonlamellate (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 nontympanal 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 electrondense 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 nontympanal 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 redorange. 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 helicoidal 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 endocuticle 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
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 animal used in Fig. 7.
Note helicoidal chitin (he) and helicoidally arranged dense material (dm).
83
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 tympanum 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 Mossbauer 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
4 0 - 6 0 k H z 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 interneurons 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 enlarged. 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 surrounding 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 preparation) 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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PHILOGENE,
{Received 5 December 1977, revised 27 January 1978)