J. Embryol. exp. Morph. Vol. 67, pp. 153-165, 1982
Printed in Great Britain © Company of Biologists Limited 1982
153
The regeneration of supernumerary cockroach
antennae
By VERNON FRENCH1 AND JOHN DOMICAN2
From the Zoology Department, University of Edinburgh
SUMMARY
Grafting experiments performed on the basal scape segment of the antenna of Blabera
craniifer give very similar results to those of corresponding grafts done on the legs of cockroaches and many other insects. If the antenna is amputated and replaced on the stump, it
heals, while a 180°-rotated graft de-rotates and sometimes forms a symmetrical partial
supernumerary. If the antenna is grafted on to the contralateral stump, one transverse axis
of the graft is reversed relative to the host, and this results in the regeneration of a supernumerary antenna with host orientation from each of the two points of maximum incongruity
between graft and host. Sometimes one double supernumerary forms midway between these
points, and its orientation suggests that it results from a secondary fusion of the two supernumeraries.
The similarity of these results to those of leg grafts suggests that legs and antennae have a
similar general organisation of positional information and similar rules for cellular behaviour. Further, the two appendages may have the same set of positional values but have
evolved different ways of interpreting it. Preliminary attempts to test this idea directly (by
grafting between leg and antenna) were rather unsuccessful since only a few control grafts
healed (poorly) and all rotated and contralateral grafts were eliminated.
INTRODUCTION
During each larval instar the single layer of epidermal cells of hemimetabolous
insects separates from the cuticle, grows and secretes a new cuticle bearing a
precise pattern of structures such as bristles and spines. Many grafting experiments have been performed on the legs of larval cockroaches (Bohn, 1965,
1970, 1972; Bulliere, 1970, 1971; French, 1976, 1978) and other insects (Bart,
1971a; French, 1981; Shaw & Bryant, 1975; Balazuc, 1948; Bodenstein, 1937),
to analyse the role of cellular interactions in controlling growth and the formation of pattern.
Many other appendages of insects are known to regenerate after amputation
but there have been few grafting experiments to see whether their cells will
interact to form intercalary or supernumerary regenerates. The results of grafting the forceps of earwigs (Furukawa, 1940) and, especially, the cerci of
1
Author's address: Zoology Department, University of Edinburgh, Kings Buildings, West
Mains Road, Edinburgh EH9 3JT, U.K.
2
Author's present address: Department of Zoology, Kings College, University of London,
Strand, London WC2R 2L8, U.K.
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V. FRENCH AND J. DOMICAN
crickets (Palka & Schubiger, 1975) suggests that the abdominal appendages and
the legs respond in the same way to confrontation of normally non-adjacent
cells. This paper reports several grafting experiments on the basal scape segment
of the larval cockroach antenna. Since the results are comparable with those of
similar grafts performed on the leg, we argue that the two developing appendages have a similar spatial organization.
MATERIALS AND METHODS
Fourth to sixth instar larvae of the cockroach, Blabera craniifer, were taken
from mass cultures (see French, 1978) 2-4 days after moulting, and anaesthetized
with CO2. Amputations and grafts were performed at precise levels in the
antenna, using fine forceps, spring scissors and knives made from fragments of
razor blade. Operated animals were kept in groups of 20-30 and were killed
after the first (rarely), second or third post-operative moult. The heads were
fixed in alcohol and examined.
RESULTS
Structure of the antenna
The cockroach antenna (see Fig. 1) consists of three proximal segments - the
scape, the pedicel and the third segment - and a long segmented flagellum. It
articulates within the antennal socket which is positioned on the head capsule
anterior and medial to the compound eye, and it contains a major articulation
between the scape and the pedicel. During larval life growth occurs in the
proximal segments, particularly the third segment, which forms about 15 new
flagellum segments, in distal-to-proximal sequence, during an instar. During the
next instar each of these primary flagellum segments splits into two secondary
segments (Haas, 1955).
The transverse axes of the antenna are labelled anterior/posterior (A/P) and
medial/lateral (M/L); this corresponds only roughly to their orientation on the
head, but corresponds to the axes of the leg. The scape is slightly convex on the
medial side, but the only circumferential marker structures reliably formed on
regenerated antennae are the articulatory structures of the socket and the
distinct A and P hinges between scape and pedicel (see Fig. 1B, C).
Regeneration after amputation
Antennae were amputated at the proximal/distal levels used in the grafting
operations. Amputation in the proximal scape (42 animals), the mid scape
(25 animals) or the distal scape (42 animals) was always followed by distal
regeneration. The small antenna present after the first moult was fairly normal
in structure, but had a reduced number of flagellum segments and sometimes
had abnormalities in the segmentation of the flagellum. In subsequent instars
Regeneration of supernumerary cockroach antennae
155
D
Fig. 1. The right antenna of a seventh instar Blabera larva, shown in camera-lucida
drawings (A-C) and schematic distal view (D). A, P, M, L, anterior, posterior,
medial and lateral faces of the antenna; Sc, Pe, Th, fx and f2, scape, pedicel, third
segment, primary and secondary flagellum segments of the antenna.
(A) View of head and medial face of antenna. Stippling denotes articulatory
membranes in the antennal socket, between scape and pedicel, and between flagellum
segments. The compound eye is marked by dashed lines.
(B, C) Posterior and anterior views of the base of the antenna, showing the
cuticular process (m) projecting from the medial rim of the antenna socket, and the
distinct posterior (p) and anterior (a) articulations between scape and pedicel shading denotes heavily sclerotised cuticle.
(D) Schematic distal view of antenna. Proximal is peripheral and distal is central,
so the outer circle represents the base of the scape and the inner circle the junction
between scape and pedicel. The medial process (m), posterior (p) and anterior (a)
articulations are shown.
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V. FRENCH AND J. DOMICAN
Bii
Fig. 2. Graft 1, right scape grafted to right scape, control orientation. (A) Schematic
view of operation. (B) Anterior (i) and posterior (ii) views of the result after two
moults, showing simple healing.
the regenerate developed a completely normal structure, including the distinct
hinges between scape and pedicel.
Amputation in the antennal socket was also followed by regeneration
(32 animals), while Urvoy (1963) found that removal of the socket plus the
surrounding region of head capsule prevents regeneration. The antenna does
not regenerate from an amputation surface in the flagellum, but increases the
rate of formation of new segments from the third segment (Haas, 1955).
Antennal graft
Grafts were performed between the right antennae of different animals of the
same instar (grafts 1 and 4) or between left and right antennae of the same
animal (grafts 2 and 3). The donor antenna was cut in the proximal third of the
Fig. 3. Graft 2, left scape grafted to right scape, M/L axis reversal. (A) Schematic
view of operation. (B-D) Results after two or three moults. (B) Posterior (i) and
anterior (ii) views of antenna with separate supernumeraries arising in medial (SM)
and lateral (SL) locations, with anterior (a) and posterior (p) articulations in anterior
and posterior positions relative to the host. (C) Medial (i) and posterior (ii) views
of antenna with a double supernumerary (S) arising posteriorly, with anterior
articulations (a) in medial and lateral positions, and posterior articulations (p) in
posterior/distal and posterior/proximal positions. (D) Medial view of antenna with
double supernumerary (S) in anterior location, with posterior articulations (p) in
medial and lateral positions.
Regeneration of supernumerary cockroach antennae
157
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V. FRENCH AND J. DOMICAN
Regeneration of supernumerary cockroach antennae
159
scape (see Fig. 1) and the graft was telescoped with the appropriate orientation
into the host antenna amputated in the distal third of the scape. This choice of
levels ensured a good fit and the graft was secured by dried haemolymph.
(a) Graft 1: right scape grafted to right scape: control orientation
All 85 successful control grafts healed (often with a slight bulge) with no
change in orientation of the graft and no formation of supernumerary structures
(Fig. 2).
(b) Graft 2: left scape grafted to right scape: M/L axis reversal
As shown in Fig. 3, the M/L axis of the grafted scape was reversed relative
to that of the host and, in 46/51 successful cases, this orientation of the graft
was retained. The remaining five mis-orientated grafts may have resulted from
errors in grafting or from subsequent rotation, and they will be considered
below, together with similar mis-orientated results from Graft 3. The 46
correctly orientated grafts produced two separate supernumerary antennae
(40 cases) or one supernumerary double-antenna (6 cases).
Separate supernumeraries usually arose in M and L positions (33/40 cases)
and their A/P axes were orientated like that of the host (Fig. 3B). In all but
one case they originated from the level of the graft/host junction in the scape
(often one was slightly more distal in origin than the other) and consisted of
distal scape, pedicel and flagellum. Other positions of origin were AM and AL
(3 cases), PM and PL (3 cases), and M and PL (1 case), and all these supernumeraries were also orientated like the host.
Supernumerary double-antennae usually arose from a P position (5/6 cases)
on the graft/host junction. They were very large and were clearly double
structures since, at the scape/pedicel articulation, they had two A hinges (in M
and L positions relative to the host) and two P hinges (Fig. 3C). The remaining
double-antenna arose from an A position and also had two hinges and two P
hinges (in M and L positions), as shown in Fig. 3D.
Fig. 4. Graft 3, left scape grafted to right scape, A/P axis reversal. (A) Schematic
view of operation. (B, C) Results after three or four moults. (B) Medial (i) and
posterior (ii) and anterior (iii) views of antenna with separate supernumeraries
arising in anterior (SA) and posterior (Sp) locations, with anterior (a) and posterior
(p) articulations in anterior and posterior positions relative to the host. (C) Anterior
(i) and posterior (ii) views of antenna with a double supernumerary (S) arising
laterally, with anterior articulations (a) in proximal LA and distal LP positions
(relative to host axes), and posterior articulations (p) in distal LA and proximal
LP positions.
6-2
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V. FRENCH AND J. DOMICAN
(c) Graft 3: left scape grafted to right scape: A/P axis reversal
As shown in Fig. 4, the A/P axis of the graft was reversed relative to the host
and, in 67/77 successful cases, this orientation was retained. The remaining
10 mis-orientated grafts are considered separately below. The 67 A/P reversed
grafts produced two separate supernumeraries (57 cases), one supernumerary
(5 cases) or one supernumerary double-antenna (5 cases).
The two separate supernumeraries arose from the graft/host junction in A
and P positions (53/57 cases) or in A and PM positions, and their A/P axes
were orientated like that of the host (except for a few broken or rudimentary
structures where the axis could not be identified). In the 5 cases where only one
supernumerary was formed, this arose in a P position and had host orientation.
Supernumerary double-antennae usually arose from a M position (4/5 cases)
and bore two A hinges (in distal MP and proximal MA positions) and two P
hinges. The remaining double-antenna arose from a L position and also had
two A hinges (in distal LP and proximal LA positions) and two P hinges (Fig.
4C).
The 15 mis-orientated cases from grafts 2 and 3 can be considered together.
In 4 cases the A face of the graft was orientated medially, corresponding to a
reversal of the MP/LA axis of the graft relative to the host. In all 4 cases
separate supernumeraries of host orientation were formed in approximately
MP and LA positions. In 11 cases the A face of the graft was orientated laterally, reversing the MA/LP axis of the graft. Eight of these grafts formed
separate supernumeraries in approximately MA and LP positions while, in the
remaining 3 cases, a double-antenna was formed in a MP position.
(d) Graft 4: right scape grafted to right scape: 180° rotation
As shown in Fig. 5, both transverse axes of the graft were reversed relative to
the host and, in all 29 successful cases, this orientation was changed by derotation of the graft. De-rotation in either clockwise or anti-clockwise direction
brought the graft back into alignment with the host by the second post-operative
moult. In 22/29 cases, graft and host levels, healed together with the formation
of no extra structures (Fig. 5B), but in 4 cases a partial supernumerary was
formed, consisting of distal scape, articulatory membrane and a tiny lobe.
The partial supernumeraries could arise from any circumferential position,
could be identified in 2 cases as symmetrical structures (Fig. 5 C) and did not
regenerate further in subsequent instars. In 2 cases graft and host levels independently regenerated distal structures and fused at the level of the pedical
(Fig. 5D). The remaining grafted antenna formed supernumerary regenerates
from A and P positions on the pedicel.
Regeneration of supernumerary cockroach antennae
Fig. 5. Graft 4, right scape grafted to right scape, reversal of both axes. (A) Schematic view of operation. (B-C) results after three or four moults showing graft de-rotated into alignment with host. (B) Posterior view of antenna with no supernumerary
structures. (C) Posterior view of antenna with a symmetrical partial supernumerary
(S) consisting of 2 posterior articulations (p) and a small lobe. (D) Posterior (i) and
and anterior (ii) views of antenna on which graft and host independently regenerated
scape apex, articulations and part of the predicel (Pe).
161
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V. FRENCH AND J. DOMICAN
Fig. 6. Formation of a double supernumerary antenna after reversal of the M/L
axis of the graft. A i, B i, Formation of separate supernumeraries (stippled) from
the graft/host junction (heavy line) at points of maximum incongruity between
graft and host. These supernumeraries will have the handedness and orientation of
the host (see Fig. 3B). Supernumeraries form by local growth under the old cuticle
and if they come into contact (arrows) on the posterior (A ii) or anterior (B ii) side,
they may fuse (heavy dashed line), to form one double-supernumerary. If the
double supernumerary forms posteriorly (A ii), posterior articulations (p) w ill lie
on the line of fusion, in distal and proximal posterior positions (see Fig. 3 C). If
fusion occurs anteriorly (B ii) posterior articulations (p) will lie medially and laterally
(see Fig. 3 D).
Leg I antenna grafts
Grafts were performed between the leg tarsus and the antenna by telescoping
the prothoracic tarsus (cut at proximal level) into the distal scape of the right
antenna of the same aninral. Grafts were done with control orientation (62 cases)
180° rotation (62 cases), or reversal of the M/L axis (62 cases) or the A/P axis
(62 cases). At the first moult a few grafts were still attached by a thin neck of
tissue to the host scape, but no graft survived to the second moult.
Grafts between the leg tibia and the antenna were performed by telescoping
Regeneration of supernumerary cockroach antennae
163
the prothoracic tibia (cut at proximal level) of a second-instar donor into the
distal scape of the host animal. Of 80 grafts with control orientation, most were
rejected by the first moult and only 4 were retained at the second moult. The
graft remained more or less aligned with the host but was poorly healed. Grafts
were done to reverse the A/P axis (160 cases) or the M/L axis (92 cases) of the
graft, but all these grafts were rejected by the second moult.
DISCUSSION
The most obvious feature of the results of the antenna grafts is their similarity
to the results of corresponding grafts done on the cockroach leg. While a control
graft simply heals (graft 1), when a grafted scape is rotated 180° so that both
transverse axes are reversed relative to the host (graft 4), it de-rotates, sometimes
forming a symmetrical partial supernumerary at the graft/host junction. When
one transverse axis of the grafted scape is reversed by a contralateral graft,
supernumerary regenerates are reliably formed (grafts 2 and 3). Typically, a
complete antenna regenerates from each of the points of maximum incongruity
between graft and host, and its A/P axis (the only one which can be identified)
is orientated like that of the host. Sometimes only one supernumerary structure
is formed, midway between the points of maximum incongruity, but it is a
double structure and its orientation suggests that it arises from secondary fusion
of the two supernumeraries growing in the confined space beneath the old
cuticle (French, 1976). This is illustrated for the M/L axis reversal in Fig. 6.
These results suggest that the spatial organization and the rules for cellular
behaviour are similar in the insect leg and in the scape segment of the antenna.
There is no evidence yet relating to the more distal antennal segments, but the
different mode of growth and response to amputation suggest there may be
different rules for cellular behaviour in the 3rd segment (or blastema) and the
numerous segments of the flagellum.
Insects are metamerically segmented and are assumed to have evolved from
annelid-like forms which were not divided into distinct head, thorax and
abdomen, but had a sequence of body segments bearing similar jointed appendages (see Snodgrass, 1935; Manton, 1977). During evolution the appendages
of different segments have been lost or adapted for locomotory, feeding or
sensory functions. As pointed out by Wolpert (1971) and others, changes in
morphology may occur because of changes in the stimuli (positional information, hormone concentrations, etc.) which developing cells receive from their
surroundings, or changes in the ways in which cells respond to a set of stimuli.
Indirect evidence for the existence of similar sets of positional values in the
insect antenna and leg comes from two situations where chimeric appendages are
formed: heteromorphic regeneration of the stick insect antenna (Brecher, 1924;
Urvoy, 1970) and homoeotic transformations in Drosophila (Postlethwaite &
Schneiderman, 1971). Numerous grafts between pro- and metathoracic legs
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V. FRENCH AND J. DOMICAN
(Bohn, 1970,1972; Bulliere, 1970; Bart, 1971 b; French, 1980,1981) suggest that
legs differ considerably in size and details of structure because of differences in
cellular response, rather than positional information. A similar approach has
reached the same conclusion concerning the different cuticular patterns formed
on different abdominal segments (Stumpf, 1968).
The present axial grafts between the leg tarsus or tibia and the scape were an
attempt to extend this approach. Healing of control grafts, derotation of rotated
grafts and production of supernumeraries from A/P or M/L reversed grafts
would have suggested that the same positional values run around leg and antenna.
Unfortunately almost all grafts were rejected. It is a common finding in insect
grafting experiments that epidermis from different body regions tends to reduce
contact, isolating a graft from the supply of haemolymph and leading to graft
death and rejection. The basis of this response is not understood: presumably it
results from the surface properties of the different populations of cells (Nardi,
1977) but perhaps not those properties related to position within a segment or an
appendage. If the cells will not heal together, they cannot interact to produce
movement, growth or regeneration.
Cases of de-rotation (Bohn, 1965) and supernumerary formation (Urvoy,
1963) from.other rotated leg/antenna grafts suggest that the two appendages
may be capable of interacting and may have common positional values. The same
conclusion is also suggested by the results of grafts of the cricket cercus to the leg
stump (Schubiger, personal communication; French, unpublished), where the
graft is often rejected, but a rotated graft can de-rotate, and reversal of one
transverse axis of the graft tends to produce supernumerary structures.
This work is supported by the Science Research Council.
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