/. Embryol. exp. Morph. Vol. 32, 1, pp. 69-79, 1974
69
Printed in Great Britain
Extent and properties of the
regeneration field in the larval legs of cockroaches
{Leucophaea maderae)
II. Confirmation by transplantation experiments
ByHORST BOHN1
From the Zoologisches Institut der Universitat, Miinchen
SUMMARY
The investigation of the regeneration field of the larval legs of cockroaches {Leucophaea
maderae), which commenced in a previous paper with extirpation experiments, has been
continued by transplantation experiments. The extirpation experiments showed that there are
two regions near the leg which are indispensable for leg regeneration: the basal sclerites, and
the membranous region extending behind the leg up to the spiracle of the next segment,
called'leg-inducing membrane' (LIM). TheLIM is followed by 'sclerite-inducing membrane'
(SIM) which, upon contact with sclerites, only allows formation of sclerite structures.
The results of the extirpation experiments have been confirmed by transplantation experiments. When the whole leg including the basal sclerites is removed, no leg regeneration
occurs. The regenerative ability can be restored by implantation of part of the basal
sclerites (for instance, the trochantin), but leg regeneration takes place only when the implantation area is covered by LIM. When the sclerites are transplanted to a region which is
covered by SIM only additional basal sclerites are formed.
Whole sets of basal sclerites have been implanted at different distances behind the uninjured hindleg. Additional legs are regenerated only in the anterior half of the membranous
field extending between the hindleg and the first abdominal segment. Thus, there is a distribution of LIM and SIM in the region of the hindleg, similar to that near the midleg.
Whole sets of basal sclerites have been implanted at various sites on the dorsal or ventral
surfaces of the abdomen. Legs are formed on both surfaces, but only when the transplanted
sclerites contact the intersegmental membranes. This means that the intersegmental membranes of the abdomen also have leg-inducing capacities.
The implantation of a trochantin into a field of LIM is followed by the development of
two regenerates - a normal one at the posterior border of the field, and one with reverse
anterior-posterior polarity at the anterior border. When the trochantin is transplanted
together with the praecoxa in a similar way, only one normal regenerate is formed at the
posterior margin of the trochantin. The praecoxa prevents contact of the anterior margin of
the trochantin with LIM, and contact of the anterior margin of the praecoxa with LIM does
not promote leg regeneration.
INTRODUCTION
In a previous paper on the regeneration field of the legs of cockroaches
(Bohn, 1974a) it was shown that leg regeneration is possible only when at least
1
Author's address: Zoologisches Institut der Universitat, 8000 Miinchen 2, Luisenstr. 14,
Germany.
70
H. BOHN
two parts of the leg surroundings are allowed to interact - namely, part of the
sclerites of the leg base and part of the membranous area extending behind the
coxa of the leg, which was called' leg-inducing membrane' (LIM). A third region,
making up the most anterior part of the ventral surface of a thoracic segment,
when combined with sclerites does not initiate leg formation but only allows
regeneration of sclerites. This membranous region therefore was called 'scleriteinducing membrane' (SIM).
The previous results were obtained by excising various parts of the tissues
surrounding the leg; those results are confirmed by transplantation experiments
presented in the present paper. Either whole sets of basal sclerites or parts
thereof (the trochantin) were transplanted to the desired region. The first series
of experiments (series D) attempted to learn whether regenerative ability, which
was lost after the extirpation of the midleg and its basal sclerites, could be
restored by the implantation of a sclerite. A second series of experiments (E)
was undertaken to analyse the regeneration field of the hindleg. Finally, an
attempt was made (series F) to find tissues with leg-inducing capacities in the
segments of the abdomen, which normally does not bear legs.
MATERIALS AND METHODS
Most experiments have been done with larvae of Leucophaea maderae.
Operations were performed on freshly moulted third instar larvae, which were
observed after operation for at least three further moults. In experimental
series D and F the operations were made in two steps; first, the left midleg,
including its basal sclerites, was removed from third instar larvae. After two
moults, when it was clear that no legs had been regenerated, sclerites of different
type or orientation were transplanted to the site. Thus, the final operation was
performed on fifth instar larvae. The age of the animals from which transplants
were taken differed according to the desirable size of the transplanted tissues;
in most cases third instar larvae were used.
Since experiments of the previous paper will often be referred to in this and
the following paper, it seemed desirable to arrange the experimental series of all
three papers in a continuous order to avoid confusion. Thus the three experimental series of paper I (A-C) (Bohn 1974 a) are continued by the three series
(D-F) in this paper and by three more (G-J) in the one which follows (Bohn.
19746).
EXPERIMENTS AND RESULTS
Series D (Fig. 1, Table 1)
When the left midleg including all basal sclerites and part of the anterior
membranous area was removed, a leg was seldom regenerated (Expt. A6,
Bohn, 1974a). The absence of regenerative capabilities could be due either to the
severe disturbance of the tissues by the large wound area, or to the complete
Regeneration field of cockroach legs
I. Extirpation of
left midleg
II. Transplantation
of trochantin
71
Results
Fig. 1. Design of experimental series D. The experiment was performed in two
steps. First (I) the left midleg was removed completely. The figure at the left shows
a situation where only the distal parts of the leg were removed (cross-hatched area:
cut surface). In addition to this, the basal sclerites and part of the anterior membranous area were also cut out (heavily dotted area). After two moults (II), during
which time no leg regeneration occurred, the trochantin (heavily dotted) of a left
midleg of another animal was implanted in one of two different positions (D t 2,
indicated by the heavily dotted outlines) into the membranous area covering the
former leg area. The results are seen at the right. When the trochantin is implanted
far anteriorly, i.e. near to the spiracle (Dj), only basal sclerites are regenerated; a
double set of basal sclerites is formed. When the trochantin is implanted more
posteriorly, one or two legs are formed. The results confirm that leg formation
depends on the presence of part of the basal sclerites and of a suitable membranous
region, the 'leg-inducing membrane', which normally extends between the posterior
border of the coxa and the spiracle of the next segment. It is reached by the transplant only in Expt. D 2 (for further explanation see text and Fig. 11). Lightly dotted
area, membranous parts; clear area, sclerotized elements, consisting of sternites
(in the middle), the legs and their basal parts; longitudinally hatched area, wing
anlagen.
absence of sclerites. To answer this question, part of the basal sclerites, namely
the trochantin of the left midleg of another animal, was transplanted into the
membranous, experimentally leg-free area between fore- and hindleg. Two
positions were selected for the transplants (Fig. 1). In Expt. D x the trochantin
was implanted at the level of the spiracle of the mesothoracic segment - a
region which is considered to belong to the SIM (Bohn, 1974a). In Expt. D 2 ,
the trochantin was implanted in the middle of the field between the spiracles
of the meso- and metathoracic segments, which is expected to be covered by
the LIM.
72
H. BOHN
Table 1. Compilation of the results of experimental series D and E
{see Figs. 1 and 8)
Legs regenerated
Experimental
series
No. of
operated
animals
Surviving
No leg
formed
Dx
D,
Ex"
E2
E3
E4
E5
E6
17
42
41
20
20
20
20
75
11
34
37
19
19
20
15
42
11
16
11
12
18
20
14
42
f
1 leg
—
10
26
7
1
—
—
—
•
2 legs
8
——
—
—
1*
—
* The sclerites had come into contact with the intersegmental membrane between abdominal segments .1 and 2. Medial part of the praecoxa missing.
No legs were regenerated in Expt. D l5 but there was regeneration of basal
sclerites; anteriorly, a praecoxa was added, posteriorly the whole set of basal
sclerites was reduplicated symmetrically (Fig. 2). Expt. D 2 showed three types of
results. About half of the animals had structures similar to those resulting from
Expt. Dj and no leg regenerates; in the other animals legs had been regenerated.
In the latter case, either a complete leg was formed, the basal sclerites also being
completed by the formation of a praecoxa and epimeron (Fig. 3), or the completion of the basal sclerites did not take place; instead, a second, symmetrical,
trochantin and leg were formed anteriorly (Fig. 4).
F I G U R E S 2-7
Figs. 2-4. Examples of the main results of experimental series D.
Fig. 2. Only a double set of basal sclerites (p, praecoxa; /, trochantin; broken line,
axis of symmetry) is formed after transplantation of the trochantin close to the
spiracle (sp) (Expt. DJ. When the trochantin is transplanted to a more posterior
region (Expt. D2) a complete normal leg (Fig. 3) or two symmetrical legs (Fig. 4)
are formed, c, Coxa of the unaffected right midleg.
Figs. 5-7. Examples of results for experimental series E.
Fig. 5. Reduplicated basal sclerites (p, praecoxa; e, epimeron; t, trochantin) after
transplantation of sclerites near the first abdominal sternite (a). The parts below the
broken line (axis of symmetry) represent the transplant, those above the line the
regenerate.
Fig. 6. A leg (r) is regenerated when the basal sclerites are implanted near the coxa
of the hindleg. The coxa of the regenerate is mostly hidden beneath the proximal
parts of the hindleg (hi) whose distal parts were removed for photography.
Fig. 7. Exceptional case with leg regenerates (c, coxae of the two regenerated
legs) after implantation of the basal sclerites near the first abdominal sternite. By
obliteration of part of the first abdominal sternite (a) the transplant had come into
contact with the intersegmental membrane between the first and second abdominal
sternite (arrow).
Regeneration field of cockroach legs
73
Series E (Fig. 8, Table 1)
The distribution of the two different kinds of membranous tissues (SIM and
L1M) covering the ventral surface of the mesothoracic segment, as determined
by extirpation experiments (series A-C) was shown in Bonn (1974a, Fig. 1).
This has been further confirmed by the results of series D. Series E was designed
to study the possibility of a similar distribution behind the hindleg. Complete
sets of basal sclerites of midlegs were implanted at varying distances from the
left hindleg, which was left intact (Fig. 8). Since regenerates only occurred in
transplants of this type at the posterior border of the trochantin (see also Expt.
Gx in Bohn, 19746) the sclerites had to be implanted with reversed anterior-
74
H. BOHN
Midleg,
~
5\ 2\ \l
leg regeneration
LIM
SIM
3/4/V'
Fig. 8. Design of experimental series E. The basal sclerites of the left or right midleg
(lower figure, heavily dotted area) were implanted at different distances (heavily
dotted outlines in upper figures) behind the left hindleg, in normal or anteriorposterior reversed position. For drawing, the left hindleg was turned anteriorly to
allow full view of the implantation area. The left midleg was left intact but is drawn
only up to the base of the coxa (broken line). Leg regeneration only occurs when the
cut surface of the transplanted trochantin is lying in the anterior half of the membranous region between the hindleg and the first abdominal sternite; in the other
cases (below the horizontal line) only a symmetrical reduplication of the basal
sclerites occurs. Therefore, similar to the situation at the base of the midleg, the
membranous field behind the hindleg consists of two areas with different morphogenetic capacities: anteriorly, a 'leg-inducing membrane' (LIM), posteriorly a
'sclerite-inducing membrane' (SIM). Lightly dotted area, membranous parts; clear
area, sclerotized parts; longitudinally hatched area, wing anlagen.
posterior polarity in some of the experiments (Expt. EJL 2 and E5) to allow investigation of the regenerative capacities of the membranous field in the immediate proximity of the hindleg. In these cases the basal sclerites of the right legs
were used as grafts, and by rotation, the medto-lateral polarity of the transplant came into accordance with that of the host tissues.
When the sclerites were transplanted to the membranous area near the hindleg
an additional leg was often regenerated (Expt. E 1 2 ; Fig. 6). But the frequency
of leg regeneration rapidly decreased with increasing distance from the hindleg
(Expt. E3_6). The legs regenerated in E lt 2 had reversed anterior-posterior polarity,
as was to be expected from the polarity of the sclerites. As usual, when no legs
were regenerated, the sclerites had been reduplicated symmetrically (Fig. 5).
Therefore the posterior part of the regeneration field of the hindleg seems to be
similar to that of the other legs. An area with LIM is followed posteriorly by a
membranous field which only allows regeneration of sclerites (SIM). In Expt.
E 5 there was one exception - an animal which had regenerated two legs (Fig. 7).
Obviously there had been an enlargement of the wound surface. The sternite of
the first abdominal segment had been split into two parts. Because of this gap
the transplant came into contact with the intersegmental membrane of the next
segment, which seems to have leg-inducing capacities (as will be seen in series F).
75
Regeneration field of cockroach legs
Table 2. Compilation of the results of experimental series F.
Transplantation of basal sclerites to abdominal segments
Site of implantation
Experimental
series
Fx
F;
F4
Legs regenerated
Surface of
abdomen
Membrane
or sclerite
area
No. of
operated
animals
Surviving
No leg
formed
Ventral
Ventral
Dorsal
Dorsal
membrane
sclerite
membrane
sclerite
45
131
40
80
21
57
20
44
18
57
18
44
A
1 leg
2 legs
2
1*
2
—
Medial part of the praecoxa missing.
10
5
,
Figs. 9, 10. Examples of results for experimental series F. Legs may be regenerated
after transplantation of basal sclerites to abdominal segments (r, Fig. 10) provided
that the sclerites are combined with the intersegmental membrane. In most cases,
however, only symmetrical basal sclerites are formed (Fig. 9). Broken line, axis
of symmetry; above the line, transplant; below the line, regenerate.
76
H. BOHN
Experiment
Sclerite- inducing
membrane
(SIM)
•
™ _
_™™
$
Leg-inducing
membrane
(LIM)
Fig. 11. Interpretation of the results of experiments Dx and D2 (compare with
Fig. 1). The type of regenerate depends on the site of implantation of the trochantin
(triangular structure). In Expt. Dx all transplants (1) were lying within the region
of the SIM, therefore only sclerites were regenerated. The site of implantation in
Expt. D2 was in the region of the boundary between SIM and LIM, so the trochantin happened to lie either completely within the SIM (2), or within the LIM (4),
or in between (3). Therefore in case (2) only basal sclerites were formed, as in
Expt. Dx (1); in case (3) sclerites were regenerated at the anterior (here upper)
margin of the trochantin; but a leg was regenerated (indicated by the arrow) at its
posterior margin which had contact with the LIM. Because of the contact of the
trochantin with the LIM at both margins, two legs developed in case (4), one at the
anterior and one at the posterior margin.
Two legs were regenerated in this case because of the obliteration of the medial
part of the praecoxa (see series G in part III).
Series F (Table 2)
This series of experiments attempted to discover whether leg-inducing
capacities were restricted to the corresponding membranes in the thoracic
segments, or whether there were other tissues - for instance, on the abdomen which in combination with basal sclerites would allow regeneration of legs.
These experiments were suggested by an exceptional result of experiment E5, in
which legs had been regenerated by a trochantin which had come into contact
with the intersegmental membrane between the first and second abdominal
segments.
Complete sets of hindleg basal sclerites were transplanted in normal orientation to the ventral (Expt. F 1; 2) or dorsal (Expt. F 3t 4) surface of the abdomen.
The posterior margin of the trochantin either bordered the anterior margin of
the intersegmental membrane (Expt. F 1 3 ) or a sternite or tergite (Expt. F 2)4 ).
When no leg was regenerated, there was a reduplication of the basal sclerites
(Fig. 9). In 10-14% of cases where the posterior margin of the trochantin
bordered the anterior margin of the intersegmental membrane, legs were formed irrespective of whether the sclerites had been transplanted to the dorsal or the
ventral surface (Fig. 10).
Regeneration field of cockroach legs
11
DISCUSSION
The conclusions regarding the regeneration field of cockroach legs reached
in a previous paper (Bohn, 1974a) have now been confirmed by transplantation experiments. After the loss of regenerative ability following an extirpation
of leg plus sclerites, the regenerative capacities could be restored by implantation
of sclerites - for instance, the trochantin (series D). But the success of the implantation depended on the site of implantation. When the trochantin had been
implanted far anteriorly (Expt. Dx) only sclerites were regenerated. At a more
posterior position (Expt. D2), either sclerites, or one or two legs were formed
(Fig. 1). These results are interpreted as follows (see Fig. 11): the sclerite parts of
the legs are bordered by membranous areas, anteriorly by the sclerite-inducing
membrane (SIM), and posteriorly by the leg-inducing membrane (LIM). After
extirpation of the leg including all basal sclerites, the former leg area is covered
by a homogeneous membrane which should partly consist of LIM and partly of
SIM. The results of the implantation experiments suggest an approximate
boundary between these two kinds of tissue. In Expt. D x the trochantin obviously had been implanted within a region covered by SIM, therefore sclerites
were regenerated at its anterior and posterior margins (position 1 in Fig. 11).
The site of implantation in Expt. D 2 seemingly was in the region of the boundary
between SIM and LIM. When the trochantin happened to lie within the SIM
(position 2, Fig. 11) only sclerites were regenerated, as in Expt. D x . The cases
where one complete leg was regenerated, can easily be explained by an intermediate position (position 3 in Fig. 11) of the trochantin; anteriorly it bordered
the SIM, therefore the basal sclerites were completed; posteriorly it had contact
with the LIM, so a leg was regenerated. The third possibility was that the
trochantin was completely surrounded by LIM (position 4, Fig. 11); in this
case two legs were regenerated, a normal one at the posterior margin of the
trochantin and another at the anterior margin, with reverse anterior-posterior
polarity.
Most of the previous experiments for the study of regeneration fields in the
legs of Leucophaea (Bohn, 1974a) had involved the midlegs. It was not considered likely that there were great differences in the regenerative fields of the
different legs; nevertheless, it seemed desirable to make at least one trial experiment with the field of another leg - for instance, of the hindleg. Instead of
extirpating tissues, basal sclerites were implanted at different distances from the
hindleg (series E). Regeneration only occurred in the anterior half of the membranous field which extends between the hindleg and the first abdominal segment. Therefore it seems that - as in the case of the midleg - the coxa of the
hindleg is followed posteriorly by a strip of LIM, which then is followed by an
area consisting of SIM.
In series E there was one exceptional case (Expt. E5, Fig. 7) with a leg regenerate arising from a very posterior transplant, in a region which was thought to
78
H. BOHN
be covered by SIM. However, in this animal the implantation of the transplant
obviously was followed by extended necrosis of the surrounding host tissues.
The enlarged wound surface caused a splitting of the sternite of the first abdominal segment into two parts. By means of this gap the transplant was able to
come into contact with the intersegmental membrane between the first and
second abdominal segments. The simplest explanation for the exceptional leg
regenerate is that there is a leg-inducing capacity in the intersegmental membranes of the abdomen. This assumption was tested in the last experimental
series (F). Basal sclerites were transplanted either to a membrane region or to a
sclerite region of both ventral and dorsal surfaces of the abdomen. Legs were
regenerated on both surfaces, but only when the sclerites had been combined
with the tissues of the intersegmental membranes. Therefore, leg-inducing
capacities are not restricted to the surroundings of a leg, but are also present in
the intersegmental membranes of the abdominal segments, which normally have
no legs. The rather low incidence of regenerates (10-14%) could indicate that,
as in the case of the thoracic segments, only part of the membranous area has
leg-inducing capacities.
When the three experimental series of this paper are compared with respect
to the number of regenerated legs per transplant, a striking difference appears
between those experiments in which only the trochantin had been transplanted
(series D) and the experiments where a complete set of basal sclerites (i.e.
trochantin plus praecoxa; series E and F) was used as the transplant. In series
D, 44% of the animals with leg regenerates had double legs, while in the two
series E and F only 5 % (two cases) had double legs. The development of double
legs seems to be caused by contact of the trochantin with LIM both at its
posterior and at its anterior margin; one leg is formed at each margin. In series
E and F, only the posterior margin of the trochantin has a free border which
can be brought into contact with LIM; the anterior margin is covered by the
other basal sclerites which, despite their contact with LIM, do not form leg
regenerates. A similar situation with similar results was described in Bohn
(1974a) (series C), where the basal sclerites of an intact leg had been brought
into contact with the LIM of the previous segment. Leg regeneration only
occurred after removal of the praecoxa, when the trochantin had contact with
the LIM. The simplest conclusion which could be drawn from these results that the praecoxa has no capacity for leg regeneration - is not valid, since
regeneration occurred from a posterior cut surface of the praecoxa (Expt.
A2_4) with high frequency. The difference in reaction seems to be due to the
different orientation of the cut surface of the praecoxa. In Expt. A2_4 it was a
posterior cut surface; in series C, D and F it was an anterior cut surface. The
reason for the different capacities of differently orientated cut surfaces is not
clear. However, the two cases with double legs in series E and F need a satisfactory
explanation. The second leg had formed although the greater part of the praecoxa
was present, and prevented the trochantin from extensive contact with the
Regeneration field of cockroach legs
79
LIM. But it seemed that the medial-most wedge of the praecoxa was missing.
Possibly the contact of the trochantin with the LIM over such a restricted
length was still sufficient to elicit leg regeneration. Therefore, the question
arises: to what extent and at which point must the trochantin be exposed to
the LIM to allow regeneration at the anterior margin ? Experiments of this kind
have been performed and are presented in Bohn (19746).
REFERENCES
H. (1974a). Extent and properties of the regeneration field in the larval legs of cockroaches {Leucophaea maderae). I. Extirpation experiments. /. Embryol. exp. Morph. 31,
557-572.
BOHN, H. (19746). Extent and properties of the regeneration field in the larval legs of cockroaches {Leucophaea maderae). III. Origin of the tissues and determination of symmetry
properties in the regenerates. /. Embryol. exp. Morph. 32, 81—98.
BOHN,
{Received 12 September 1973, revised 8 January 1974)