/. Embryol. exp. Morph. Vol. 61, pp. 87-101, 1981
Printed in Great Britain © Company of Biologists Limited 1981
87
Pattern re-establishment - transplantation
and regeneration of the leg in the cricket
Teleogryllus commodus (Walker)
By ROBERT J. BIGGIN 1
From the Department of Zoology, University of Melbourne
SUMMARY
Regeneration and grafting experiments were carried out on the prothoracic leg of the
cricket Teleogryllus commodus (Walker) to examine the precision with which surface cuticular
structures and internal epidermal derivatives are reformed. By comparing regenerated and
grafted limbs with normal limbs it was found that the three-dimensional structure of epidermal derivatives is not restored. This is despite the fact that regenerated and grafted limbs
appear similar in their external morphology to normal limbs. The implication of these
results are discussed in the context of theories of pattern formation.
INTRODUCTION
Regeneration has interested biologists for many years. Recently developmental biologists have been using regeneration, together with experiments involving grafting as a way of gaining an understanding into the events occurring
in development. The result of these studies has been the general concept of
positional information (Wolpert, 1969, 1972), that is, the concept that cells
have access to information about their position in a developing field and differentiate accordingly.
One model using this concept is the polar co-ordinate or 'clockface model'
(French, Bryant & Bryant, 1976). In this model, positional information and
therefore the resultant pattern is held to be specific in two dimensions (French
et al. 1976; Carlson, 1975). Structures having a three-dimensional shape emerge
via folding and shaping of a two-dimensional cell sheet, similar to the process
occurring in gastrulation.
Insect sensory structures offer an opportunity to study a three-dimensional
arrangement in which positional information is held to be specified in two
dimensions. One class of insect sensory structures, chordotonal organs, although
internal in position, are derived from epidermal cell derivatives which become
internalised during development (Moulins, 1976; Wigglesworth, 1953).
1
Author's address: Department of Zoology, University of Melbourne, Parkville, Victoria,
3052, Australia.
88
R. J. B I G G I N
In regeneration experiments involving either amputation of a limb or removal
of a strip of epidermis, the normal cuticular pattern is restored at subsequent
moults (French, 1978). In view of the epidermal origin of chordotonal organs,
one might expect that they would also regrow the normal three-dimensional
arrangement.
This paper compares the three-dimensional arrangement of the tibial sensory
complex (Eibl, 1978) with that of the cuticular surface in both regenerated and
grafted limbs in the Australian field cricket Teleogryllus commodus (Walker).
MATERIALS AND METHODS
Animals
Crickets Teleogryllus commodus (Walker) were collected in the field and were
kept in culture in the laboratory. Cultures were supplied with water and commercial rat food, and kept under a 12 h day/night cycle at 25 °C. Under these
conditions post-embryonic development took approximately 7-8 weeks.
Experimental animals were placed in individual containers (10 x 6 x 6 cm),
supplied with food and water. All operated crickets were examined periodically.
Those which showed graft rejection were discarded (Table 1).
Table 1. Details of experiments
Experiment*
Non-congruent graft-host junction
Congruent graft-host junction
Leg removal and regeneration
Number
attempted
Percentage
rejectionf
100
72
43
48%
42%
N.A.
Number
Number
surviving
examined
till adult histologically§
30J
31
20
17
15
12
* The age at which experiment was done is indicated in Figure legends after type of graft,
e.g. congruent grafts (Instar 1).
t Rejection is expressed as the percentage of animals dying or losing the graft due to
a premature moult in the first 10 days after experiment. Other losses of animals are spread
over a considerable time period. Not all those crickets surviving to adult produced the
required result (Table 1).
% Of these only 17 were successful in producing multiple outgrowths.
§ The number examined histologically met criteria for examination (with the exception of
non-congruent grafts where only 8 limbs met criteria - see Results).
Grafts
Two types of grafting experiments, using nymphal crickets, were carried out
by transplanting a prothoracic coxa (Table 1). These were:
Pattern re-establishment in cricket leg
89
(a) Non-congruent graft-host junction
Either a left or right (L or R) coxa was grafted onto a R or L host coxa
respectively in order to reverse the anterior-posterior (A-P) polarity.
(b) Congruent graft-host junction
As a control, a donor coxa was removed and transplanted onto a like-handed
prothoracic stump. In some cases the same animal was used as both donor and
host. In such cases all axes were congruent.
In both types of grafts both host and donor nymphs were matched by means
of a staging system developed by Ball & Young (1974). All grafts were performed
on cold-immobilized animals. To ensure grafts remained in position until the
haemolymph hardened, a strip of insect wax (25 % violin resin, 75 % bees wax)
was applied to the graft-host junction using a micro-soldering iron. Crickets
were allowed to recover at room temperature.
Regeneration
Either a R or L prothoracic leg was cut off at mid-coxal level and allowed to
regenerate. In all cases the contralateral leg was allowed to grow normally.
Criteria for examination of tibial sensory complex
In order to facilitate comparisons between the two types of graft, leg regeneration and the normal condition, one must have some consistent criteria
for comparing animals after these different treatments. Otherwise, differences
could be due to different stages of development. In order to overcome this
problem, only those duplicated and regenerated limbs showing full segmentation
and having a tibial length 70 % or greater compared to the contralateral limb
were included in the examination. Contralateral limbs were normal.
Histology
Immediately after the last moult, the tibial segment was removed and placed
in fixative (alcoholic Bouin) for 2 h. Tibial segments were then dehydrated in
graded alcohols, passed through xylene and vacuum embedded in paraffin wax
(m.p. 56 °C). Sections were stained with Ehrlich's haematoxylin and Eosin or
Baker's modification of Masson's trichrome stain (Pantin, 1946). Some material
was embedded in glycol methacrylate (Polysciences) and stained with Lee's
methylene blue-basic fuchsin (Bennet, Wynick, Lee & McNeil, 1976). Sections
were photographed with a Leitz Orthomat microscope/camera system using
Kodak Pan F or Ilford FP4 film.
External cuticular pattern
External cuticular anatomy of both normal and regenerated and graft limbs
was studied by scanning electron microscopy. Material was either critical-point
90
R. J. BIGGIN
CS
SoSc
TO
(b)
TN
SN
PN
DN
E
SoN
Fig. 1. Line drawing of tibial sensory complex in a normal limb. Tn (a) the position
of the complex is shown in relation to the tibia (Ti) and femur (Fe) while in (b) the
sensory complex is shown in detail. The tibia has been drawn as if the cuticle (C)
and epidermis (E) on its anterior face has been removed exposing the internal
structures. Muscle has been eliminated for clarity. The right-hand side of the figure
corresponds to the distal portion of tibia. Note tympanal organ (TO) in association
with expansion of anterior (Atr) and posterior trachea (Ptr). Its two groups of
neurones a proximal (PN) and distal (DN) are indicated. The proximal neurones
are in close association with the neurones of subgenual organ (SON). The subgenual organ scolopoles (SoSc) are also shown. The campaniform sensilla (CS) lie
on the dorsal cuticle. Modified from Young & Ball (1974). PT, posterior tympanum;
SN, subgenual nerve; TN, tympanal nerve. Calibration = 0-1 mm.
dried or air dried, gold coated and examined using a Cambridge Stereoscan
electron microscope. A total of 33 normal limbs from various instars was
examined, while 8 regenerated and transplanted limbs were examined also. Some
limbs were photographed with a Zeiss-Tessovar microscope/camera system.
RESULTS
(1) General morphology of experimental limbs
Congruent grafts and regenerates produced single outgrowths. In contrast,
the non-congruent grafts produced multiple outgrowths. Of the 100 noncongruent grafts attempted 17 were successful in producing multiple outgrowths (Table 1). Of these 17, eight produced triple outgrowths consisting of
two laterals from the sites of axial incompatibility and a regenerate lying
between the two as expected (Bohn, 1965). The remaining nine produced only
a single fully segmented lateral; the other lateral was either reduced to a small
unsegmented bud or was absent altogether.
Pattern re-establishment in cricket leg
91
(a)
Fig. 2. Line drawing of tympanal organ in experimental cases (a) regenerate
(Tnstar 1) and (6) congruent graft (Instar 1). Note neurones (N) contained in an
envelope located between epidermis (E) and trachea (Tr). Scolopales and attachment cells are absent. Top margin of drawing corresponds to dorsal side of tibia,
the left hand corresponds to anterior side of tibia. Drawings traced from 10 /im
transverse wax sections stained with haematoxylin and eosin. The cuticle has been
lost in sectioning. Calibration = 25 fim.
In all cases the outgrowths showed segmentation developing in a proximodistal order, i.e. tarsal segments were last to be differentiated.
(2) Comparing the experimental series
In order to make comparisons of the end point of development two criteria
were chosen (Materials/Methods). Though arbitrary, such criteria are useful
because a normal limb at a similar morphological stage contains the full complement of sensilla in the subgenual organ and the tympanal organ is almost
complete (Ball & Young, 1974). The campaniform sensilla pattern is also nearly
complete.
(3) Tympanal organ
The structure of the normal tympanal organ has been described earlier, so
has its post-embryonic development (Ball & Young, 1974; Young & Ball, 1974).
It consists of 70 sensilla (scolopidia) which can be divided into five distinct
anatomical groups (Fig. 1). These sensilla, like the others in the tibial complex,
must arise de novo in regenerates and grafts, since the distal segments of the leg
are removed during the initial operation.
In the course of normal development a limb of 70 % of adult tibial length
would contain between 50 and 60 of the total complement of sensilla. All the
five anatomical groups would be represented. In contrast in only five experimental cases (four regenerates, one congruent graft) could a structure homologous with the tympanal organ be identified. The homology was based on
similar proximo-distal and circumferential positions within the tibia, being
distal to and separate from the subgenual organ. In four out of the five
92
R. J. BIGGIN
(a)
(b)
Fig. 3. Line drawing of tympanal organ in (a) most advanced experimental case
(regenerate, Instar 1) and for comparison in a normal limb at the same level. Note
both cases contain neurones (N), scolopales (SC) and attachment cells (ac) while
each differ in mode of attachment to epidermis (E) and number of sensilla. Top of
drawing corresponds to dorsal portion tibia, the right side the anterior side of the
tibia. Drawings traced from 5 fim transverse glycol methacrylate sections stained
with Lee's methylene blue/basic Fuschin. Atr, anterior trachea; C, cuticle; Ptr,
posterior trachea. Calibration = 50 /tm.
cases this structure contained five neurones or less, with no associated scolopales
or attachment cells. These neurones were in close association with the epidermis
(Fig. 2).
In the remaining case, using a newly emerged instar 1 (regeneration), the
regenerate tympanal organ in the adult was considerably more advanced, consisting of a dorsal mass of attachment cells, ten neurones and associated scolopales (Fig. 3). These sensilla however could not be allocated to any of the groups
described by Young & Ball (1974). No similar structure either of only neurones
or sensilla was seen in non-congruent grafts. Thus a regenerate limb and congruent graft showed the rudiments of a tympanal organ internally while a noncongruent graft did not, even though both were similar in external morphology.
(4) Subgenual organ
The subgenual organ is a fan-like chordotonal organ located in the dorsal
haemolymph space. It lies midway between the campaniform sensilla and the
tympanal organ (Figs. 1, 4b). Its normal adult structure has been described in
detail elsewhere (Young & Ball, 1974; Eibl, 1978). The organ is supplied by two
nerves, the tympanal nerve which also supplies the tympanal organ sensilla and
the subgenual nerve which also supplies the campaniform sensilla (Young &
Ball, 1974). It contains about 20 sensilla, the neurones of which are divided into
Pattern re-establishment in cricket leg
93
so
TON
SO
PtN
TN
(a)
(b)
Fig. 4. Line drawing of tibial sensory complex in (a) regenerate Instar (1) and for
comparison a normal limb (b). In each the tibia has been drawn as if the cuticle (C)
and epidermis (£) on the dorsal surface has been removed. The underlying trachea
and muscle has been omitted for clarity. Right-hand side corresponds to posterior
side of tibia, the top of the figure corresponds to the distal portion of tibia. In (b)
the distal portion of tympanal organ has also been omitted. Note presence of subgenual organ (SO) in both normal and regenerated limbs while tympanal organ is
only present in regenerated limbs as a small group of neurones (TON), unlike the
normal limb in which distinct proximal (PN) and distal neurones (DN) are present.
Drawings reconstructed from 10 fim wax sections cut in a horizontal longitudinal
plane. Sections stained with haematoxylin and eosin. (a) Is a composite reconstruction from five regenerated limbs. AtN, anterior neurones; N, neurones; PtN,
posterior neurones; Sc, scolopales; SN, subgenual nerve; TN, tympanal nerve.
Calibration = 01 mm.
two distinct groups - a larger anterior group containing about 15 neurones and
smaller posterior group containing about five neurones (Fig. 4). Unlike the
neurones the scolopales are not divided into two distinct groups and it is unclear
which scolopales correspond to the posterior neurones. In both graft experiments and regenerates, scolopidia are differentiated in two distinct groups:
a posterior and anterior group (Fig. 4). Posterior and anterior are taken with
respect to stump axes. However, the precise ordered structure of the normal
adult subgenual organ was never re-established even using instar 1 animals for
experiments (Fig. 4). Cell counts from three experiments (two non-congruent
laterals, one regenerate) show that the posterior group contains between five
and nine scolopodia, while the anterior group has between three and seven
scolopodia. Each group receives separate innervation by nerves considered to
be equivalent to the subgenual and tympanal nerves respectively. Grafts using
older instars indicate that these anterior and posterior groups of scolopodia
originated from different regions of epidermis.
EMB 6l
94
R. J. B I G G I N
Fig. 5. Photomicrographs ot normal and regenerated tibia (Instar 1) viewed from
(a) posterior surface and (b) anterior surface showing larger posterior and smaller
anterior tympana, respectively. These stand out against the background of much
darker leg cuticle. In each case the normal limb is located on the right. In (a) note
the slightly smaller size at regenerated posterior tympana. In (b) the regenerated
anterior tympana is present only as an area of slightly lighter cuticle (arrowed).
Calibration = 0.5 mm.
\
\
(a)
Fig. 6. Photomicrographs of experimental tibia to show tympana. Same orientation
as in previous figure, (a) posterior tympana; regenerate (Instar 3). Note that it runs
to a point distally, around which the leg cuticle is devoid of hairs; (b) anterior
tympana congruent graft (Instar 1). Note its irregular shape compared to normal
anterior tympana in previous figure. Calibration = 0-1 mm
95
Pattern re-establishment in cricket leg
(b)
(O
i
id)
Fig. 7. Schematic line drawing of successive transverse sections through region
of tibia normally occupied by tibial sensory complex showing different patterns of
tracheal growth in normal limbs and experimental limbs. The outer circle represents the outline of the limb cuticle, while the inner circles represent tracheal
profiles. Arrows indicate position of tympana. The right-hand side of each figure
corresponds to posterior side of limb. The top of each figure corresponds to the
dorsal side of tibia, (a) normal limb; (b) non-congruent graft-anterior lateral
(Tnstar 1); (c) congruent graft (Instar 2); (d) regenerate (Instar 3). The normal
tracheal pattern is consistent from specimen to specimen (compare with Fig. 1).
In contrast the experimental limbs show considerable variability in tracheal pattern.
Calibration = 0-3 mm.
(5) Tympana
A normal limb has two tympana, a larger posterior and smaller anterior one
(Fig. 5). Tympana were formed in all experimental series. In the majority of
experiments only the posterior tympanum differentiated and an area of lighter
cuticle was formed at the site where the anterior tympanum would normally be
found. The posterior tympanum approached the normal condition in terms of
size and shape (Fig. 5), whereas the anterior tympanum, if present, was far more
variable (Figs. 5, 6). To form a tympanum under regeneration conditions
nymphs instar 4 or earlier were required, while graft experiments had to be done
at earlier instars to achieve a similar result.
Experiments carried out using older instars consistently lacked an anterior
tympanum, and the posterior tympanum was reduced in size. Its shape, unlike
the normal oval configuration, ran to a point distally. The cuticle around the
4-2
96
R. J. BIGGIN
Fig. 8. Scanning electron micrographs showing successive stages in formation of
the normal pattern of campaniform sensilla (a) Instar 1 (left leg); (b) Instar 4
(right leg); (c) Instar 7 (left leg); (d) adult (left leg). In each micrograph the top
corresponds to the distal portion of the tibia. Note in (a) the distally pointing protuberance on a proximal sensillum (arrowed). In (b) the larger posterior sensillum of the intermediate group is arrowed. In the adult condition (d) the Proximal
Group (PG) and Distal Group (DG) sensilla are indicated. Between them lie the
intermediate group. Magnification: (a) 1500 x : (b) 1200 x ; (c) 700 x ; (d) 820 x
distal margin of the posterior tympanum, unlike the rest of the leg cuticle was
devoid of hairs (Fig. 6). In all cases the circumferential and proximo-distal
positions conformed to the host axes.
(6) Trachea
In contrast to the consistent pattern of tracheal growth seen in normal limbs
(Ball & Young, 1974) tracheal growth was far more variable and never conformed to the adult pattern (Fig. 7).
In experimental limbs the only consistent pattern was the appearance of two
Pattern re-establishment in cricket leg
97
Fig. 9. Scanning electron micrographs of experimental limbs showing campaniform
sensilla pattern. Same orientation as in previous figure, (a) congruent graft (Instar \);
(b) non-congruent graft-middle limb (Instar 1). Note presence of a distally pointing
protuberance, arrowed in (a) indicating each sensillum preserved its intrinsic polarity.
By comparing this figure with the previous one it can be seen that approximately
the same numbers of sensilla are present but in experimental limbs the precise
ordered pattern is not reformed. Magnification: (a) 900 x ; (b) 900 x .
main trachea. Their size and shape, unlike the normal situation, was variable
between experimental limbs. In addition, a number of small trachea was present.
Tympana were associated with internal expansion of the tracheal system,
although no causal relationship can be inferred from this study.
(7) Campaniform sensilla
A group of 13-15 campaniform sensilla is located in a mid-dorsal position on
the tibia in all three pairs of legs. In the prothoracic leg, they lie in the proximal
part of the tibia 0-3-0*4 mm from the femoro-tibial joint (Fig. 1). The sensilla
are normally found in a distinct pattern (Fig. 8d). The normal development and
the pattern arising after regeneration was examined in order to compare
cuticular differentiation. Within the normal pattern, three groups of sensilla
can be designated; a proximal group of the two largest sensilla and a distal
group of three slightly smaller sensilla arranged in the form of a triangle, the
apex of which points towards the femur-tibia joint. Located between these two
groups is an intermediate group of eight to ten sensilla arranged in an arc which
is open distally (Fig. 8). Within this intermediate group the size of the sensilla
varies in a systematic way: the largest are located on the posterior side of the
arc and their size decreases toward the anterior side (Fig. Sb). Hence the arrangement of campaniform sensilla in the adult has both proximo-distal polarity
designated by the proximal and distal groups, and circumferential polarity
(anterior-posterior) due to size differences in the intermediate sensilla. Each
98
R. J. BIGGIN
sensillum has its own intrinsic polarity due to the presence of a distally pointing
protuberance (Fig. 8a). In normal development the two proximal, three distal
and the most posterior (largest) of the intermediate sensilla are present at
hatching (Fig. 8a). At successive instars the other intermediate sensilla are
added in a strict sequence around the arc until the adult condition is achieved
(Fig. 86, c,d).
Five experimental limbs (two regenerates, two congruent, one non-congruent
grafts) were examined. Although in these experimental limbs approximately
the same numbers of sensilla as normally found differentiated, the precise
ordered adult pattern was never reformed (Fig. 9). This was despite the fact
that sensilla preserved their intrinsic polarity in such cases and that the three
size classes corresponding to proximal, distal, and intermediate sensilla appeared
to be present also.
DISCUSSION
Close examination of regenerated and grafted limbs which appear superficially
normal show that pattern formation is never perfect. The sensory structures
reveal a marked disparity between the observed results and what one would
expect to find in a normally growing limb of similar dimensions. In the case of
the two chordotonal organs, the subgenual and tympanal organs, clearly regeneration does not re-establish the adult condition even though these structures
are epidermal derivatives.
Subgenual organ
In the case of the regenerated and graft subgenual organ, polarity is preserved in
the sense that the neurones are located proximally with proximal running axons,
and scolopales distally. In addition two sensilla populations, an anterior and
posterior one, were present. In the case of duplicated limbs, the middle limb
consistently had the larger sensilla grouping on the anterior side. On this
criterion, together with a posteriorly located larger tympana such regenerates
appear of stump handedness, indicating the anterior-posterior axis with
respect to these structures is capable of reversal, a result not predicted by the
polar co-ordinate model (French et al. 1976). Unfortunately, epidermal markers
do not exist for other epidermal structures to confirm this result generally.
Tympanal organ
The tympanal organ was in the normal position in terms of circumferential
and proximo-distal position within the tibia. The very poor and somewhat
variable representation of the tympanal organ may be due to two factors.
Firstly, it normally develops considerably later than the subgenual organ (Ball
& Young, 1974) and so it seems reasonable that the same should occur during
regeneration. Hence insufficient time may have existed for it to differentiate.
Secondly, the apparent variable nature of the presence or absence of the
tympanal organ itself might have been due to an inability to identify the small
Pattern re-establishment in cricket leg
99
population of tympanal organ sensilla. In view of the close anatomical association between subgenual organ sensilla and proximal sensilla of the tympanal
organ in normal legs (Ball & Young, 1974) a slight change in proximo-distal
position might have led to fusion of the two groups and an inability to detect
the tympanal organ sensilla. In grafts and regenerates differentiation of the
tympana appears to be independent of the tympanal organ itself since a nearly
normal posterior tympanum and the beginnings of the anterior tympanum
were evident in cases where internal differentiation of tympanal organ sensilla
was apparently absent. The timing and degree of tympanal differentiation
was similar to that found in other studies involving regeneration and transplantation in the cricket (Ball, 1979).
Campaniform sensilla
Unlike the two chordotonal organs the campaniform sensilla do not become
internalized during development but remain in an epidermal position. As such,
they can be treated ideally as being on a two-dimensional cylinder and can be
fitted more easily into previous work on pattern formation (French et al. 1976).
Clearly each regenerated sensillum preserved its intrinsic polarity since the
distinct protuberance always pointed distally. However, the anterior/posterior
polarity produced by the size class arrangement within the intermediate group
was lost in regeneration. This was because the precise spatial pattern of the
adult condition was not re-established. However, the number and diversity of
sensilla in terms of the three broad size classes appeared to be retained. This
particular system indicates to some degree the independence of polarity and
spatial pattern. This system requires further study to establish how the precise
normal pattern is lost in regeneration. In normal development the sequential
appearance of sensilla of the intermediate group probably controls both the size
and distribution of sensilla by mechanisms previously proposed (Lawrence,
1973). What is required is a study of the time sequence appearance of sensilla
during various sorts of experimental treatments.
Regeneration and normal development
The assumption implicit and indeed the rationale in most regeneration
studies is that they in some way duplicate the events occurring during normal
development. This approach is understandable in view of the difficulties in
working with embryos, especially where experimental interference is required.
However, some recent evidence suggests that a regenerating system does not
re-establish the adult condition, at least in insects. O'Farrell & Stock (1954)
noted that tracheation in regenerated cockroach limbs is different from that
normally found.
In this study tracheation in experimental limbs was variable. The reasons for
this variability are unclear. In normal limbs tracheal patterns in the region of
subgenual and tympanal organ are consistent from specimen to specimen. In
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R. J. BIGGIN
graft and regenerated limbs the variability may be due to different responses to
functional demand. However, this is difficult to reconcile with the consistent
pattern found in normal limbs.
Differences in muscle development, its biochemical properties and nerve root
branching pattern found in cockroach regenerated limbs led Denberg and
Whitington (1978) to suggest that regeneration may not be a good model
system in which to examine normal development. This study confirms this conclusion, showing that the three-dimensional arrangement of chordotonal organs
is lost and the two-dimensional pattern of the campaniform sensilla is only
partly restored, while the gross morphology of the limb appears normal.
I am grateful to Dr David Young for his advice and criticism. Thanks to Daphne Hards
and Linda Crosby for technical assistance. Jane Doolan and Ralph MacNally commented
on the manuscript. Support was provided by the Commonwealth Postgraduate Research
Award (CPRA) and Victorian Education Department Studentship.
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{Received 26 November 1979, revised 12 August 1980)