/. Embryol. exp. Morph. Vol. 32, 1, pp. 81-98. 1974
Printed in Great Britain
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
ByHORST BOHN1
From the Zoologisches Institut der Universitat, Munchen
SUMMARY
Previous papers dealing with the regeneration field of cockroach {Leucophaea maderae)
legs have shown that two elements of the leg surroundings are indispensable for regeneration
of a leg: the basal sclerites and the 'leg-inducing membrane' (LIM). The experiments of this
paper partly clarify the way in which these two tissues interact; the origin of the regenerative
tissues and the determination of polarity and symmetry in the regenerated legs were studied.
By combination of sclerites and LIM of different species or segments it has been shown
that each of the tissues makes up half of a leg regenerate - the sclerite tissues the anterior
longitudinal half, the LIM the posterior longitudinal half.
The trochantin was implanted in four different orientations (normal, turned 90° clockwise
or anti-clockwise, turned 180°) into a normally orientated field of LIM. The anteriorposterior and medio-lateral axes of the regenerates had the same orientation as those of the
sclerites. Therefore, the sclerites alone determine symmetry of the regenerate; the LIM has
no influence in this respect. Neither of the two axes is fixed irreversibly in the cells of the
LTM; the cells seem able to accept any polarity which is forced upon them by the sclerites.
The symmetry properties are more strongly fixed in the cells of the sclerites, but an inversion
of polarity (through 180°) is possible for both axes.
Regeneration of a leg from the level of the trochantin seems to be initiated by or dependent
on a small medial region of the trochantin. A tiny fragment of the medial wedge of the trochantin, when transplanted to a field of LIM, can form a complete leg regenerate; a lateral
half of the trochantin never does. As long as this small medial part of the trochantin is prevented from making contact with LIM by a praecoxa fragment, no regeneration takes
place at the anterior border of the trochantin, even when the greatest part of the border has
contact with LTM.
The regenerative capacities of the praecoxal sclerite are different at the anterior and
posterior cut surfaces, irrespective of the level of the cut and the orientation of the sclerite. An
anterior cut surface never forms a leg regenerate, but a posterior cut surface does.
1
Author's address: Zoologisches Institut der Universitat, 8000 Munchen 2, Luisenstr. 14,
Germany.
EM B 32
82
H. BOHN
INTRODUCTION
In the larvae of cockroaches, complete extirpation of a leg (including the
coxa) is followed, as a rule, by regeneration of a new leg. The prerequisite of a
successful regeneration is the presence of two kinds of tissue normally surrounding the leg-the basal sclerites, which lie anterior to the coxa, and the 'leginducing membrane' (LIM) adjoining the leg posteriorly. The interaction of
these two tissues is necessary for leg regeneration (Bohn, 1914 a, b), but the
nature of the interaction during regeneration is unknown. The goal of the
experiments presented in this paper was to clarify at least part of this problem.
One question was the origin of the tissues of the regenerates. The question
could be answered by experiments (series H) in which sclerites and LIM from
different segments or different species were combined. Another related question
was whether the two co-operating tissues were of equal importance for the
development of the regenerate; which of the two tissues, for instance, determines the symmetry properties of the new leg ? Sclerites and LIM were combined
in divergent orientations (series J); observation of the orientation and symmetry of the legs developing under such conflicting conditions permitted
analysis of the respective influence of the two tissues on symmetry determination
during regeneration. A third problem arose from the transplantation experiments described in Bohn (19746). It seemed that the capacities of different parts
of the basal sclerites to regenerate legs upon contact with LIM were different;
therefore, basal sclerites in various combinations were transplanted to a LIM
to test their regenerative capacities (series G).
MATERIALS AND METHODS
The animals (larvae of Leucophaea maderae) and the experimental procedures were the same as in previous papers (Bohn, 1974 a, b). In one experiment
(H2) another species of cockroach (Gromphadorhina portentosa) was used in
addition to Leucophaea to allow combination of sclerites and LIM from two
different species. The three experimental series (G, H, J) of this paper continue
the six series (A-F) of the preceding papers.
EXPERIMENTS AND RESULTS
Series G (Table 1)
Experimental series G was undertaken to clarify some contradictory results
of the preceding experimental series, concerning the regenerative capacity of the
basal sclerites. When a trochantin was implanted into a field covered by LIM,
two legs were regenerated; when a complete set of basal sclerites (trochantin
plus praecoxa) was transplanted in a similar way, only one leg developed,
always at the posterior margin of the trochantin. There were a few exceptions, in
which a second leg was formed at the anterior margin of the transplant; it
seemed that the medial part of the praecoxa had disappeared in these cases.
Tissue origin and symmetry in cockroach leg regenerates
83
Table 1. Experimental series G and H: types of operation and results
The transplant (dotted area) in series G was taken from the basal sclerites of the
right (Expts. G ^ ) or left (Expts. G7i8) midleg and was implanted immediately
behind the basal sclerites of the left hindleg (clear area in third column) after
removal of the distal parts of the leg. In Expts. G^6 the transplant was rotated
through 180° before it was implanted. In Expt. H± the basal sclerites of a left
foreleg were transplanted in the same way as in series G. In Expt. H2 the basal
sclerites of Leucophaea were implanted at the site of the extirpated hindleg of
Gromphadorhina (Gr. tissues indicated by cross-hatching).
Expt.
G1
Source
of
transplant
Orientation
of
No. of
trans- operated
plant
animals
Medial part of
precoxa
Normal
leg
regenerated
Missing
Surviving
No leg
formed
20
19
17
20
18
12
11
20
16
16
16
20
18
15
20
18
12
20
16
16
16
20
15
11
20
17
17"
20
9
Very
small
20
* All legs mixed, the anterior half consisting of foreleg tissue, the posterior half of hindleg
tissue.
f Eight of the nine regenerated legs clearly mixed: anterior half Leucophaea tissue,
posterior half Gromphadorhina tissue; character of one leg indeterminable.
Therefore it was desirable to analyse the regenerative ability of a trochantin in
combination with different parts of the praecoxa. For this purpose an experimental arrangement was used which gave a higher yield of successful regenerates
as compared to series D and J. The left hindleg was removed at the base of the
6-2
84
H. BOHN
Tissue origin and symmetry in cockroach leg regenerates
85
coxa, and the sclerites to be tested were implanted into this wound area, immediately adjacent to the trochantin of the hindleg. Posteriorly they had contact
with the LIM of this leg. When the capacities of anterior wound surfaces of the
praecoxa and the trochantin were studied (Expts. Gx_&), the sclerites were implanted with inverted anterior-posterior polarity to allow contact with the LIM.
To avoid complications by divergent medio-lateral polarity of host and transplant tissues, the basal sclerites of right midlegs were used. When posterior
wound surfaces were tested, basal sclerites of left midlegs were used and implanted in normal orientation (Expts. G 7 8 ).
Experiments G^Q. The basal sclerites, from which different parts of the
praecoxa had been removed, were implanted with reversed anterior-posterior
polarity. Host and transplant sclerites grew together without any signs of
regeneration (Fig. 1), but eventually a leg regenerated at the former anterior
border of the implanted basal sclerites, now directed to the rear end of the
animal. A leg with a normally orientated trochantin just behind the transplanted
trochantin was regenerated in all cases when only a trochantin was transplanted
(Expt. G 3 ; Fig. 3) or when at least the medial part of the trochantin had contact
with the LIM (Expt. G6). But no legs were regenerated when the LIM was
combined with the following wound surfaces of the basal sclerites: anterior
margin of the praecoxa (Expt. G1; Fig. 1), intermediate level of the praecoxa
(Expt. G2), lateral half (Expt. G^) or lateral two-thirds of the posterior
margin of the trochantin (Expt. G5). When a regenerate formed in these experiments, at least part of the transplant had disappeared; either the medial half
or third of the praecoxa was absent (Fig. 2; column 8 of Table 1) or, if present,
it was reduced to a very thin sclerite (last column of Table 1). In all cases without
leg regeneration, the sclerites were completed.
Experiments G7_8. Part of the base of the left midleg (part of SIM + complete
praecoxa in Expt. G 7 ; part of SIM + anterior half of praecoxa in Expt. G8) were
transplanted in normal orientation into the wound field. A third set of basal
sclerites with inverted anterior-posterior polarity was regenerated between host
and transplant sclerites (compare with Fig. 5 of Expt. H4), except in two animals
which had regenerated a normal leg at the posterior border of the host trochantin. In all experiments, legs were formed at the posterior wound borders
of the transplants.
FIGURES 1-3
Examples from experimental series G. When a complete set of basal sclerites of a
right leg is combined in reverse anterior-posterior orientation with the basal
sclerites of a left hind-leg, no leg regeneration takes place (Fig. 1). The arrow
indicates the border between host (above) and transplant (below) tissues. But when
the praecoxa of the transplant was obliterated after transplantation (Fig. 2) or
had been removed before transplantation (Fig. 3) a leg was regenerated at the
posterior border of the transplant. In these cases a third trochantin (/) formed
between the transplanted trochantin and the coxa of the regenerated leg.
86
H. BOHN
M..
FIGURES 4-6
Examples from experimental series H, dealing with the origin of regenerate tissues.
Fig. 4. Ventral view of the first and second thoracic segments, showing the structural differences between the coxa of fore- and mid- (or hind-) legs. The left legs
were removed at the trochanter-femur joint. The meron (m) is very narrow in the
foreleg, but broad in the midleg and hindleg. There is a sharp linear edge on the
coxa of the foreleg, which is bent and not very prominent in the midleg and hindleg
(arrows).
Fig. 5. Mixed leg which developed after the combination of basal sclerites of a foreleg
with the LIM of a hindleg. Anteriorly (upper surface in the figure) there are foreleg
structures (the arrow points to the sharp edge characteristic of the foreleg), posteriorly there are hindleg structures (broad meron (w) as in the hindleg). A third
set of basal sclerites (t = trochantin, p = praecoxa) has regenerated between host
and transplant sclerites.
Fig. 6. Mixed leg which developed after the combination of basal sclerites of Leucophaea with LIM of Gromphadorhina. The anterior half of the leg consists of
Leucophaea tissues (light), the posterior half of Gromphadorhina tissues (dark). The
leg was cut between femur (/) and tibia (ti) and within the tarsus to allow better
photography. The claw segment (c/) is shown from the dorsal surface, the other parts
from the ventral surface.
Tissue origin and symmetry in cockroach leg regenerates
87
Series H (Table 1)
In analysis of the respective roles of basal sclerites and LIM during regeneration it was desirable to clarify the origin of the cellular material composing the
regenerates. Does it come from the LIM, from the sclerites, or from both
tissues ? The method for approaching this problem was to combine sclerites and
LIM from different segments (Expt. Hj) or different species (Expt. H2).
Experiment Hx. The experimental procedure was very similar to that of
Expts. G 7 8 . The complete set of basal sclerites of the left foreleg was implanted
behind the basal sclerites of the left hindleg, which had been previously removed.
Thus, the sclerites of a foreleg were combined with the LIM of a hindleg. All
regenerates which formed at the posterior margin of the trochantin had a composite structure, with foreleg features at their anterior surface (straight at the
anterior surface of the coxa, etc.) and hindleg features at the posterior surface
(broad meron at the lateral border of the coxa, etc.) (Figs. 4, 5). The exact
boundary between the two kinds of tissue was usually not visible, since the
recognizably differing structures make up only a part of the leg, but it seemed
that the anterior longitudinal half of the leg was formed by the sclerite (foreleg)
tissues, and the posterior half by the LIM (hindleg) tissues. As in Expt. G 7 8 , a
third set of basal sclerites with inverted anterior-posterior polarity was
regenerated between host and transplant sclerites (p and t in Fig. 5).
Experiment H2. The left hindleg, including its basal sclerites, was extirpated
in larvae of Gromphadorhina portentosa and replaced by the basal sclerites of the
left hindleg of Leucophaea maderae. Thus the Leucophaea basal sclerites came
into contact with the Gromphadorhina LIM. All animals with successful transplants regenerated a leg. Host and transplant tissues were easily distinguished
by their different pigmentation; transplanted Gromphadorhina tissues are nearly
black, Leucophaea tissues have a light brownish colour. All legs were partly
Gromphadorhina tissue (posterior half) and partly Leucophaea tissue (anterior
half). The border between the two tissues ran fairly linearly at the mid-ventral
and mid-dorsal surfaces over the whole length of the leg (Fig. 6).
Series J (Table 2; Fig. 7)
In the preceding series it was shown that each of the two interacting tissues,
the sclerites and the LIM, contribute about equal amounts of material to the
developing regenerate. This does not necessarily mean that both tissues are of
equal importance in all respects and during all phases of regeneration. One
possible means of separating the contributions of the two different tissues was
to create a conflicting situation between sclerites and LIM, and see which of the
two had more influence on the character of the developing regenerate; therefore
a conflict of polarity and orientation between sclerites and LIM was produced
(Expts. Ji_5). In the same series I attempted to settle another problem which
arose from the results of series F. It seemed that the different parts of the
H. BOHN
Table 2. Experimental series J: types of operation and results
The figures in the second column represent the basal sclerites of the left (Expts.
Ji-3}e) or right (Expts. J4> 5) midleg. Dotted area: part of the sclerite used as transplant. The third column shows orientation of the transplant just after operation:
in Expts. J2>3 the transplant was turned through 90° anti-clockwise or clockwise,
respectively; in the other experiments there was no turning before implantation.
The site of implantation was the membranous field, which is outlined by heavy dots
in II of Fig. 7.
Orientation of leg
regenerates
Source
of
transExpt. plant
Orientation
of
No. of
trans- operated Surplant animals viving
21
15
24
20
20
15
39
29
Animals with
0
1
2
regenerated legs
10
Same Between
as
trans- Same
trans- plant
as
plant and host host
—
4
29
—
—
Fig. 7. Plan of the main experiments of series J. The experiment was performed in
two steps. First (I) the left hindleg was removed completely. The figure at the left
shows a situation where only the distal parts of the leg had been removed (crosshatched area: cut surface). In addition to this the basal sclerites and large parts of
the anterior membranous area (SIM) had also been cut out (heavily dotted area).
The midleg had not been cut, but is shown only up to the base of the coxa (broken
line). After two moults (II), during which time no leg regeneration occurred,
sclerites were implanted into the membranous field covering the former leg area.
This area of implantation (encircled with heavily dotted outline in the right top
figure) mostly consisted of LIM of the meso- and metathoracic segments. The
lower part of the figure shows the origin of the transplant in the left column (transplant heavily dotted) and the orientation in which it was implanted, in the centre
column. The thick black arrow indicates the medio-lateral polarity of the transplant
and of the resulting regenerate (m, medial; 1, lateral). Two symmetrically arranged
legs are formed, whose orientation mostly coincides with that of the transplant.
Lightly dotted area: membranous parts; clear area: sclerotizedparts; longitudinally
hatched area: wing anlagen.
Tissue origin and symmetry in cockroach leg regenerates
1. Extirpation of
left hind leg
89
. II. Transplantation of
basal sclerites
Area of
implantation
Source of
transplant
Orientation
of transplant
Results
Fig. 7. For legend see opposite.
trochantin had different capacities for regeneration; therefore, either lateral or
medial parts of the trochantin were implanted into an area covered by LIM
(Expts. J4_6).
The operation for preparing an implantation area was similar to that of
series D. The left hindleg was removed, together with all basal sclerites. In order
FIGURES
8-11
Examples from experimental series J. Regeneration of differently orientated double legs after the implantation of a trochantin
in various orientations into a field of LIM.
Fig. 8. Normal orientation, knees (k) pointing laterally.
Fig. 9. Trochantin turned 90° anti-clockwise, knees pointing anteriorly.
Fig. 10. Trochantin turned 90° clockwise, knees pointing posteriorly.
Fig. 11. Trochantin of a right leg implanted without turning (corresponds to a 180° turning), knees pointing medially.
do
O
Tissue origin and symmetry in cockroach leg regenerates
91
to have a large area covered only by LIM, the SIM in front of the hindleg was
also removed. Thus the LIM of mid- and hindleg together formed the area to
which sclerites could be transplanted (Fig. 7).
Experiments Jx_^. The trochantin of the left midleg was implanted in various
orientations: normal orientation (Expt. Jj), turned 90° anti-clockwise (Expt. J2)
or clockwise (Expt. J3). In experiment J4 the medial half of the trochantin of
the right midleg was implanted without turning it. This situation was nearly
identical to a 180° turning of a left trochantin. In most cases, two legs were
formed, as would be expected when a trochantin is used as transplant. Only the
double legs were used for analysing the orientation of regenerates; orientation
of the single legs was difficult to evaluate since they were often abnormally bent.
The orientation of regenerated legs mostly coincided with that of the transplant
(Figs. 7-11), but in some cases it seemed to lie between the orientation of the
host tissues and that of the transplant's original position.
Experiments / 5j6 . A rectangular piece comprising about one-sixth of the
whole area of the trochantin, cut out of the medial third of the trochantin of the
right hindleg, was used in Expt. J5. In experiment J6 the lateral half of the trochantin of the left or right hindleg was transplanted. Implanted lateral halves of
the trochantin were not able to elicit a leg regenerate, whereas medial halves did
so (see experiment J 4 ); even pieces as small as those used in experiment J6, if
taken from the medial half, could initiate leg regeneration. Half of the regenerates
of experiment J5 had transplant orientation, the other half had an orientation
which was somewhere between host and transplant orientation.
DISCUSSION
The regenerative capacity of praecoxa and trochantin
It has been repeatedly shown that the regeneration field of the cockroach leg
consists of two different types of tissue which are both necessary for initiating
leg regeneration - the basal sclerites of the leg and the 'leg-inducing membrane'
(LIM). Contact between the two tissues is sufficient to induce leg regeneration
even if no leg has been removed (Expts. C3_6; E ^ ) . But the regenerative
capacity of different parts of the basal sclerites appears to be different. When,
for instance, a trochantin is transplanted to a LIM, two legs are usually regenerated, one at the posterior margin of the transplant and one at the anterior
margin (Expts. D 2 ; Ji_4). The regenerate formed at the anterior margin has
reversed anterior-posterior polarity. These two legs and their basal sclerites form
exact mirror images of each other (Fig. 7). Therefore, after the implantation of
a trochantin, the regenerate at the anterior margin also begins with a trochantin,
symmetrical to the implanted one. In Expt. G3, where only the anterior margin
(which is facing posteriorly because of rotation of the trochantin) had contact
with the LIM only one regenerate (with normal polarity) is formed. Again the
regenerate begins with a trochantin, symmetrical to the implanted one. A
92
H. BOHN
similar situation is found in Expts. C4) 6, except that the anterior margin of the
trochantin is pointing anteriorly and the regenerate therefore has inverted
anterior-posterior polarity.
The praecoxa never forms a leg regenerate at its anterior margin when combined with LIM, irrespective of whether the margin is facing anteriorly (Expt.
E3) or posteriorly (Expts. E l5 Gx). When the praecoxa is cut at an intermediate
level, the success of regeneration after contact with LIM depends on which of
the two cut surfaces is used. An anterior cut surface of the praecoxa never forms
a leg regenerate, irrespective of the actual orientation of the sclerite (compare,
for instance, Expts. C7 8 with Expt. G2), but a posterior cut surface does (Expts.
A3_5; G8). The posterior margin of the praecoxa upon contact with LIM forms
a normal regenerate (Expts. A2, G7). It is not clear why anterior and posterior
cut surfaces of the praecoxa, although of the same intermediate level, behave so
differently. One could assume that the tissues of the praecoxa are not able to
reverse their polarity, since, as in the case of the anterior margin of the trochantin, a leg with reverse anterior-posterior polarity should develop. But this
cannot be true. For when a similar wound surface was combined with sclerites,
a second set of basal sclerites with reverse polarity could be regenerated (Expt.
B4). So the cells of the praecoxa when exposed to an anterior cut surface are
able to reverse their anterior-posterior polarity, but they are not able to form a
leg regenerate upon contact with LIM. The developmental meaning of this is
not clear; a possible biological meaning could be that the inability of an
anterior cut surface of the praecoxa to elicit leg regenerates might reduce the
chance of formation of a presumably hindering extra leg after accidental
damage in the region between the sclerites and the LIM of the preceding leg (see
experimental series C) without reducing the capacity for leg regeneration following the loss of a leg and large parts of its basal sclerites.
But even within the trochantin the capacities for leg regeneration are not
evenly distributed. Lateral halves of the trochantin were not able to elicit a leg
regenerate, but pieces as small as one-sixth the size of the trochantin could
initiate leg regeneration if taken from the medial part of the trochantin (Expts.
J4_6). When a whole trochantin was transplanted, regeneration only occurred
if this medial part of the trochantin came into direct contact with the LIM. The
coverage of only the medial one-third of the trochantin with a praecoxal
sclerite was sufficient to inhibit leg regeneration (Expts. G 4 5 ), whereas regeneration occurred in all cases when the medial half of the trochantin was free and the
lateral half was protected by the praecoxa (Expt. G6). These results seem to be
in contrast to an earlier experiment in which it was possible to get complete leg
regenerates from lateral halves of the basal sclerites (Expt. A 9 ); but in this case
the missing parts of the basal sclerites, and thus also the important medial
halves of the trochantin, had been regenerated. One could imagine that leg
regeneration only started after the regeneration of the medial part of the
trochantin. Such regeneration does not seem to be possible when the lateral
Tissue origin and symmetry in cockroach leg regenerates
93
halves of the trochantin are implanted into a field of LTM (Expt. J6) and therefore no leg regenerate is formed.
Restriction of the leg-promoting capacities of the trochantin to a small
medial region seems to be responsible for the fact that the triangular trochantin
only forms two leg regenerates when transplanted to a LIM, though there are
three cut surfaces in contact with LIM.
Leg regeneration is only possible when parts of the sclerites and part of the
LIM are present and have the chance to interact. These two types of tissues
seem to contain two different principles which are necessary for regeneration.
It was shown in experiments H 1 2 that each of the two tissues make up half of
the leg regenerate; the anterior half of the leg is formed by the sclerites, the
posterior half by the LIM. The question was whether these two principles were
still present in the respective tissues of more distal segments of the leg. This
problem was touched by Expts. C3j 5 in which anterior coxal tissues had contact
with LIM; in these cases legs were regenerated. The results could be confirmed
by transplantation experiments (Bohn, unpublished results); parts of the
anterior and posterior surfaces of the coxa were cut out and transplanted to a
LIM. Only the tissues of the anterior surface in combination with LIM were
able to elicit leg regenerates. So it becomes clear that the tissues of the anterior
surface of the coxa, when in contact with LIM, behave in the same way as the
basal sclerites from which they are derived, and therefore they may have the
same morphogenetic principles.
In this connexion it is interesting to see whether there are correlations between
the conditions of leg regeneration at the base and at more distal levels. It has
often been observed that extra leg regenerates are formed when leg parts are combined in such a way that the polarities of the transverse axes do not harmonize
in host and transplant tissues. The accessory regenerates normally sprout at
the sites of the greatest incompatibility between host and transplant tissues.
(Bohn 1965; Bart, 1971) but an anterior-posterior disharmony is as potent in
creating additional regenerates as c dorso-ventral disharmony. Therefore it is
not possible to directly correlate the results of experiments at the base and at
more distal levels of the leg. On the other hand, Leucophaea and most other
insects (Przibram, 1921; Bodenstein, 1937; Bulliere, 1970; Bohn, 1972; but
see also Bart, 1971) never form more than two additional legs, even under
conditions which should allow the formation of multiple legs. It seems that
the development of regenerates at more distal levels of the leg is based on a
dualistic principle similar to that observed in leg regeneration at the base.
Nevertheless, one basic difference remains: when a leg is wounded on only one
side, an incomplete distal regenerate may develop, containing only the side
qualities of the wounded surface (Bohn, 1965; Bart, 1970); at the base, on the
contrary, either a complete leg is formed or no distal parts are formed at all.
Additional experiments are necessary to find a more satisfactory concept which
eventually could explain both phenomena by a common principle.
94
H. BOHN
Origin of the tissues and determination of polarity and
symmetry of the regenerates
The establishment of a leg regenerate is dependent on the interaction of
sclerites and LIM; the nature of the interaction is still largely unknown. Experimental series H and J were designed to elucidate at least part of these
problems, namely, the origin of the cellular material composing the regenerate,
and the respective roles of sclerites and LIM in determining polarity of the
regenerate.
The first problem was studied by combining sclerites and LIM of different
segments (Expt. Hx) or different species (Expt. H2). All experiments showed the
same results - both participants contributed equal amounts of cellular material
to the regenerate. The anterior longitudinal half of the regenerate was formed
by material derived from the sclerites, the posterior half by material proliferated
by the LIM. In this connexion it should be emphasized once more that neither
the LIM nor the sclerite is able to form its respective half of a leg without the
other tissue.
In series F, legs were formed on the abdomen after the implantation of basal
sclerites. It would be interesting to see whether the abdominal tissues in this
case had also provided half of the material for the regenerates; interspecific
transplantation experiments are necessary to answer this question.
The problem of polarity has already been touched upon in series C, D and E,
in which eventually legs developed with inverted anterior-posterior polarity.
Reversal of polarity in these series occurred when the anterior margin of the
trochantin or coxa was contacted by LIM. The problem of polarity was more
directly attacked by experimental series J. In Expts. J2 3 the trochantin had been
rotated through 90° clockwise or anti-clockwise before it was implanted into a
field of normally orientated LIM. Thus, the orientation of the anterior-posterior
axes of transplant and host tissues were at right angles. As a rule two legs were
formed, one at the original posterior margin and one at the former anterior
margin of the trochantin. The first had the same anterior-posterior polarity as
the transplant, while the polarity of the latter was inverted through 180° with
respect to the transplant's polarity, as is usual for regenerates developing at the
anterior margin of the trochantin (see also Expts. G 3 6 ).
The results allow the following conclusions: when a trochantin is implanted
into a field of LIM, leg regeneration occurs. The trochantin determines the
number and site of the developing regenerates. Irrespective of the shape of the
trochantin, i.e. the number of cut surfaces and the orientation of the cut surfaces, only two regenerates are formed, anteriorly and posteriorly with respect
to the original anterior-posterior axis of the sclerite. The trochantin also
determines the anterior-posterior polarity of the regenerates. That the polarity
of the anterior regenerate is inverted is a consequence of the reversal of the
normal order: anterior sclerites, posterior LIM.
Tissue origin and symmetry in cockroach leg regenerates
95
The situation is very similar if we consider medio-lateral polarity. In Expts.
J2_5 the orientation of the medio-lateral axis of the transplant was different
from that of the LIM; the two axes formed an angle of 90° (Expts. J2 3) or 180°
(Expt. J4). The developing regenerates had the same medio-lateral polarity as
the sclerites. In this respect again the LIM had no influence on the polarity of
the regenerates; the sclerite alone determined the medio-lateral polarity of the
regenerates.
The results of the experiments concerned with polarity of the regenerates
allow some conclusions concerning the stability of the symmetry properties in
the reacting tissues. It is assumed that as in normal regeneration (Expts. H1) 2)
each of the two participating tissues provides half of the tissue of the regenerate.
(This assumption was confirmed by an experiment in which sclerites of the
foreleg were implanted in incorrect medio-lateral orientation into a hindleg
field of LIM; H. Bohn, unpublished observation.) From this it follows that
neither the anterior-posterior nor the medio-lateral polarity is strongly fixed in
the cells of the LIM; they may change their polarity and accept any other that
is forced upon them by the sclerites. In the tissues of the sclerites, though the
axes of symmetry are more stable, some change is possible. Though no change
in the orientation of the two axes of symmetry was observed, at least the polarity
of both could be reversed through 180°. The regenerates which develop at the
anterior margin of the trochantin have inverted anterior-posterior polarity
(with respect to the trochantin's polarity). There was also one example of an
inversion of the medio-lateral polarity. In Expt. A9 all parts of a leg had been
extirpated with the exception of the lateral half of the basal sclerites. Between
these remaining sclerites and a newly formed complete set of basal sclerites
another lateral half of basal sclerites developed; these latter had reversed mediolateral polarity (see Fig. 5, Bohn, 1974a).
There are some cases in Expts. J2_5 in which the orientation of the regenerate
is somewhat between that of the host tissues and that of the original position of
the implanted sclerite. This could mean that both tissues have partly changed
their polarity and that the regenerate represents this compromise, but there are
other, more likely, possibilities. Firstly during an operation and the subsequent
healing period, a transplant may well assume an orientation slightly different
from the desired one. Secondly, an active re-rotation of the whole implant
towards a condition where its medio-lateral polarity is in accordance with that
of the surroundings must be taken into account. There is strong evidence for
similar correcting movements of rotated pieces of integument on tergites of
Leucophaea (Bohn, 1974 c). If such a re-rotation happened, it would indicate
that LIM exerts some influence (if only indirectly) on the orientation of the
regenerate.
96
H. BOHN
Leg induction
The ventral surface of the thoracic segment may be divided into four different
zones (see Bohn, 1974a, fig. 1): most anteriorly the SIM, then the basal
sclerites of the leg, the leg itself, and behind the leg the LIM. The anterior and
posterior membranous areas are called sclerite-inducing membrane and leginducing membrane because additional sclerites or legs are formed when these
membranes are combined with basal sclerites. These terms were at first used
only for practical reasons. The question is whether we are justified in the use of
the term 'inducing' in this connexion, in the sense that embryologists normally
speak of induction. Unfortunately, it is not possible to make the decisive control
experiment, i.e. isolation of the corresponding structures. The tissues under
study always have contact with other tissues. Therefore, it is not possible to
decide why a combination of basal sclerites and SIM cannot form leg regenerates, while a combination of the same sclerites and LIM can. Either the solerites
need an inductive impulse, which can only be provided by the LIM, or the
sclerites are prevented by the SIM from regenerating distal leg tissues, as they
normally would do. The same holds true for the LIM itself, which in combination with the sclerites forms part of the leg: in combination with SIM (Expt.
A5) only membranous tissues are formed. There is still another possibility:
the sclerites could have leg-inducing capacities, but SIM might not be competent to react to this inductive stimulus; only LIM is competent, and for its
part induces the sclerites to form leg regenerates (without response from the
LIM they could only form additional sclerites).
Another phenomenon which must be taken into account has been previously
discussed (Bohn, 1974a)-that is, the tendency in the segmented animals to avoid
discontinuities of the pattern. It is not necessary that all given structures of
a segment should be present, but it is indispensable to have an uninterrupted
continuity between those structures which are present. At least some aspects of
the regeneration process may be explicable in terms of this principle. In series G,
for instance, there was no regeneration at the cut surface of host (or transplant)
trochantin when it was combined with tissues of the same 'level' (Expts.
Gi_6). In Expts. G7_8, H l9 where the same surface was combined with tissues of
a more anterior level (SIM), the 'gap' was bridged by a third set of basal
sclerites with reverse anterior-posterior polarity (Fig. 5). Similarly, a third,
normally orientated trochantin developed behind the transplanted trochantin
when leg regeneration occurred at the former anterior borderof the trochantin
in experiments G ^ (Figs. 2,3 ). But the principle is not very helpful for our
main question, namely: why is there leg regeneration in one combination and
no leg regeneration in the other combination ? Though there is no exception to
this principle in our experiments it cannot explain all phenomena of leg
regeneration without additional assumptions.
At this time there is no possibility of answering the question of whether
Tissue origin and symmetry in cockroach leg regenerates
97
'induction' really does take place. Therefore it would be better to avoid the
term induction in this connexion. When I, despite this reservation, make use of
the word, it is because I think the terms SIM and LIM are more informative
than neutral expressions like 'anterior membrane' and 'posterior membrane'.
Comparison of the regeneration field of Leucophaea and of other animals
When features of the regeneration fields of other animals are compared with
those of Leucophaea, one characteristic difference becomes very pronounced.
In amphibia, for instance, one may elicit the regeneration of supernumerary
legs by deviation of nerves to any point in the regeneration field. It seems that
there are no qualitative differences in the different regions of the field; i.e. the
cells lying dorsally or ventrally, anteriorly or posteriorly from the leg are
equally qualified to build up a whole appendage, with its different surfaces. On
the contrary, in Leucophaea there are the two principles represented by the
sclerites and the LIM which only by a mutual interaction can produce leg
regenerates. Neither of them alone is able to generate the structures formed after
this interaction.
The experiments of Noulin (1970, 1971) on Porcellio seem to indicate similar
phenomena in Crustacea. Noulin transplanted pieces of the dorsal integument to the ventral surface of the abdomen. On two sides of the transplant,
laterally and medially, supernumerary pleurepimers and pereiopods developed.
Though the experiments are similar to ours, in so far as two different kinds of
tissues are necessary for leg regeneration, they are not completely comparable.
The incompatability of the combined ventral and dorsal tissues seems to
initiate regeneration processes by which the missing tissues between host and
implant are intercalated. Thus the pleural region is formed on both sides of the
transplant and of course also the legs originating in this area. Therefore the
experiments of Noulin concern the regeneration of the pleural region rather
than the local conditions for leg regeneration.
Some time ago Lender 8c Grobocopateli (1967) published a preliminary
paper dealing with the regeneration field of the legs of Tenebrio molitor (Coleoptera) which unfortunately has not since been confirmed. According to the authors,
the regeneration field includes major parts of the thoracic sternite. Even fragments of this regeneration field comprising only a quarter of the whole field
are able to elicit a leg regenerate when transplanted to the second abdominal
segment, irrespective of whether the pieces are taken from the anterior, posterior,
medial or lateral region of the regeneration field. They also say that abdominal
epidermis participates in leg regeneration when it is implanted into the regeneration field after extirpation of the leg, but this has not been proved.
Recently, Bart & Browaeys-Poly (1973) reported experiments on the regeneration field of the wings of the orthopteran insect Sipyloidea. In this insect the
regenerative capacity of the wing environment seems to be restricted to the
latero-anterior region of the wing base. When this area is removed together with
7
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98
H. BOHN
the wing, no wing is regenerated; when the area is transplanted to an abdominal
segment, a whole wing develops and, as it seems, without participation of the
abdominal tissues. Contrary to the requirements for leg regeneration in Leiicophaea, this restricted area of the wing environment is sufficient for wing development; no interaction with another tissue seems to be necessary to allow the
formation of the two surfaces of the wing.
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BART, A. (1971). Modalites de formation et de developpement d'un centre morphogenetique
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BART, A., & BROWAEYS-POLY, E. (1973). Mise en evidence d'un territoire responsable de la
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(Received 12 September 1973, revised 8 January 1974)