/ . Embryol. exp. Morph. Vol. 28, 1, pp. 185-208,1972
Printed in Great Britain
185
The origin of the epidermis
in the supernumerary regenerates of triple
legs in cockroaches (Blattaria)1
By HORST BOHN2
From the Zoologisches Institut der Universitdt, Munchen
SUMMARY
An attempt has been made to clarify the origin of the supernumerary regenerates of triple
legs by transplantations between two species of cockroaches, Gromphadorhina portentosa
and Leucophaea maderae. The three ways of combining stump and transplant tissues were:
heteropleural, dorso-dorsal, antero-posterior (I); heteropleural, dorso-ventral, anteroanterior (II); homopleural, dorso-ventral, antero-posterior (III). The transplantations have
been performed at the level of the tibia as well as at the level of the coxa.
(1) The supernumerary regenerates were partly mixed, i.e were composed of a mosaic
of the tissues of both species of cockroach, partly homogeneously built up by the tissues of
only one of the two species. In combination I nearly all regenerates were mixed; in the other
two combinations homogeneous regenerates were relatively numerous, sometimes even
the most numerous.
Both supernumerary regenerates of a triple leg might be mixed; or one was homogeneous
and the second mixed; or they were both homogeneous. In the latter case one regenerate
was built up by Gromphadorhina tissues, the other by Leucophaea tissues, or, more rarely,
both were built by up Gromphadorhina tissues.
(2) The intraspecific transplantations simultaneously done in Leucophaea gave results which
correspond in general with the results of the interspecific transplantations.
(3) From the composition of the supernumerary regenerates it must be concluded that
the four different properties of a cross-section (anterior, posterior, etc.) are not yet determined irreversibly in the tissues of the cockroach legs. Thus, missing properties may be
completed by the other properties to give an entire cross-section.
INTRODUCTION
Biologists have always been very interested in naturally occurring multiple
branched appendages, especially the three-pronged appendages often observed
in arthropods. The symmetric relations of the branches have proved remarkably
uniform (Bateson, 1894). It was this arrangement of the different branches that
induced Przibram (1921) to propose a hypothesis about their origin.
In spite of a great number of investigations in Arthropoda (Balazuc, 1948;
Bart, 1971a, 6; Bodenstein, 1937, 1941; Bohn, 1965; Bulliere, 1970; Furukawa,
1
Dedicated to Professor Dr G. Krause (Wiirzburg) on the occasion of his 65th birthday.
Author's address: Zoologisches Institut der Universitat, 8000 Munchen 2, Luisenstr. 14,
Germany.
2
186
H. BOHN
1937, 1940; Hoarau, 1969; Lheureux, 1970, 1971; Noulin, 1970; Penzlin, 1965)
the participation of the host and transplant tissues in the formation of the
supernumerary branches is not yet clear. The first experiments in this respect
were done by Bodenstein (1937), Bart (1971 a, b), Bulliere (1970) and Lheureux
(1971). Bodenstein, Bulliere and Lheureux on the one hand and Bart on the
other come to different conclusions, which can hardly be due to the different
objects used by them since in most other regeneration phenomena all arthropods
behave remarkably uniformly.
The difficulties of interpretation result from the fact that the leg parts
combined have always been taken from one species, although from different
segments. But often the structural differences of the appendages from
different segments of the body are not great enough to allow identification of
every part of the regenerate without doubt. Transplantations between the two
cockroaches Leucophaea maderae and Gromphadorhina portentosa (Bohn, 1971),
the tissues of which can easily be distinguished by their different pigmentation,
offered a method to solve the problem of the origin of the supernumerary
regenerates definitively.
MATERIALS AND METHODS
Multiple regenerates regularly form when the axial organization of stump
and transplant tissues do not harmonize with one another. In the main there
are three kinds of disharmonic orientation (Fig. 1, I—III). Combinations I
and II are obtained by carrying out the transplantations between the legs of
the left and right side of the body (heteropleural transplantation); in combination II the transplant is in addition turned round the longitudinal axis through
180°. Combination III is obtained by doing transplantations between the legs
of the same body side combined with a 180° rotation of the transplant (homopleural transplantation).
The operations have been performed with two different leg segments: the
experiments of group 1 with the tibia of the midleg cut at a median or distal
level; the experiments of groups 2 and 3 with the coxa of the foreleg and hind leg
cut at a median level. In each of the three groups of experiments all three
possible combinations (I to III) have been done. Only recently moulted larvae
of Gromphadorhina portentosa (first instar) and Leucophaea maderae (second
instar in group 2, third instar in groups 1 and 3) have been used for the operations. The transplantations have been performed either interspecifically (groups 1
and 3) or intraspecifically (group 2, transplantations between foreleg and hind
leg of Leucophaea', see also Table 1).
The supernumerary regenerates appear as a rule after the first (group 1) or
second (groups 2 and 3) postoperative moult. Since the regenerates of groups 2
and 3 are easily lost by autotomy, it was often necessary to wait for further
moults till well-developed triple legs were available.
The tissues of Leucophaea and Gromphadorhina may easily be distinguished
The epidermis in cockroach regenerates
a
a
Heteropleural
dorso-dorsal
intero-anterior
I
Heteropleural
dorso-ventral
antero-posterior
II
187
Homopleural
dorso-ventral
antero-posterioi
III
Fig. 1. The three possible combinations of disharmonic orientation of stump and
transplant. The outer circles symbolize the cross-section of the stump, the inner
circles that of the transplant (looked at from distal). The letters mark the four
symmetry properties: d = dorsal; v = ventral; a = anterior; p — posterior. For
further explanations see text.
by their different colour: the legs of Leucophaea have a yellowish brown, those
of Gromphadorhina a dark brown or even black pigmentation. The characteristics of the foreleg and hind leg of Leucophaea, important for the experiments
of group 2, are specified elsewhere (see p. 194). For characterization of the position of the supernumerary regenerates (anterior, posterior, etc.) I always refer to
the organization of the stump tissues.
The results of all experiments are summarized in Table 1. Statements about
the frequency of distinct cases, if not mentioned in the text, may be looked up
there. An explanation of the half-schematic drawings of Figs. 2, 3, 6, 8 and 11
may be found in the legend of Fig. 2.
RESULTS
(1) Multiple formations of the tibia {group 1)
(a) General phenomenona
In combinations I and II all operated legs with surviving transplant had
two supernumerary regenerates consisting of tarsus and the distal parts of the
tibia. The position of these extra regenerates was in accordance with earlier
results (Bohn 1965). The regenerates as a rule appeared at that point of the
suture, where tissues of different symmetry properties met (Fig. 1), i.e. anterior
and posterior in combination I (Fig. 2 A), dorsal and ventral in combination II
(Fig. 3 A). They had the tendency to move together in a dorsal (comb. I) or posterior (comb. II) direction and even to fuse partly or in full length (Figs. 2B,
3B).
In Exp. l/III complete regenerates occurred rather rarely (Fig. 4C). More
often there were more or less reduced tarsus-like formations (Fig. 4B); often
only a tiny part of the joint region (combining tibia with tarsus) was formed
(Fig. 4 A). In one case regenerates were completely absent. The reduced
I
II
II
II
III
III
III
3
1
2
3
1
2
3
Group of
experiments
1
2
3
1
2
3
1
2
3
I
2
lfcl~
I
1 Ull
1
l VV/11
tion)a
VI
experiments
CiToiin
^ - * *- V/ *-* J-'
oi tnc
transplant
Orientation
2
1
—
16
7
16
—
3
14
Partly or
entirely fused
10
—
—
Incomplete
—
'
1
Juji
unvi
'
1
23
181
10
31
10
14
16
16
1
20
7
Entirely
separated
\, UJ. j.*.wx*
3
6
4
—
—
—
—
—
15
22
14
—
—
—
—
—
8
3
15 + 910"
2f (+l) e
IP
2'(+3)'
2'+10
2
2'
.—g.
28
Consisting only of
one kind of tissues
26 + 27*
16
40
12 + 3*
18
1
5 + 6"
Mixed
4
3
10
15
8
11
5
7
Undeterminable0
4
2
—
—
—
—
—
—
Undeterminable
—
2-3 moults
Composition of the complete regenerates6
3
33
50
18
18
1
16
16
19
28
13
34
28
13
34
OliX VlVlllfi
Orientation of the transplant after
i
Same as
Partly <Completely
furn**/"!
iinet aft*»r
turned
back
transplant operation
back
—
20
—
20
A
/\nimais
with
Qiifvivinff
158
100
50
311
100
60
172
Both branches complete
Present
Supernumerary regenerates
Hind-leg coxa of
Leucophaea
Hind-leg coxa of
Leucophaea
Midleg tibia of Leucophaea
Hind-leg coxa of
Leucophaea
Hind-leg coxa of
Leucophaea
Midleg tibia of Leucophaea
Hind-leg coxa of
Leucophaea
Hind-leg coxa of
Leucophaea
Stump
Midleg tibia of Leucophaea
No. of
operated
animals
Table 1. Compilation of the results
Material used for
Missing
_
—
—
—
—
—
1
6
—
Transplant
Midleg tibia of
Gromphadorhina
Foreleg coxa of
Leucophaea
Foreleg or hind-leg
coxa of
Gromphadorhina
Midleg tibia of
Gromphadorhina
Foreleg coxa of
Leucophaea
Foreleg or hind-leg
coxa of
Gromphadorhina
Midleg tibia of
Gromphadorhina
Foreleg coxa of
Leucophaea
Foreleg coxa of
Gromphadorhina
transplant
(combination)"
I
I
I
II
II
II
III
III
III
Onentatior L
X
25
y—u
fcd
O
or
OC
Ventral
19
11
10
Dorsal
19
12
28
—
2
Anterior
7
3
20
10
1
12
Predominantly
Gromphadorhina tissues
5
40
Posterior
11
Undeterminable
Position of the borderline between Gromphadorhina
and
Leucophaea tissues of the mixed regenerates71
Gromphadorhina and
Leucophaea
tissues of
Predominantly
about equal
Leucophaea
quantities
tissues
16
1
Undeterminable
Amounts of the tissues forming the
two corresponding supernumerary regenerates
6
e
d
6
° See Fig. 1.
Each supernumerary regenerate is listed separately; thus each animal with a triple leg is represented by two values.
This column contains, among others, the completely fused extra regenerates, in which the composition of just one regenerate naturally cannot be stated.
The second number contains such extra regenerates, the organization of which is most likely but not entirely certain as stated.
The regenerates put in brackets have fused at their bases. Only the distally separated segments have been taken into account. Because the border is often
oblique these regenerates cannot be compared with completely separated regenerates.
' Both regenerates belonged to one triple leg; one regenerate consisted entirely of stump tissues, the other of transplant tissues.
0
All regenerates consisted entirely of Gromphadorhina tissues.
h
Corresponding to the two borderlines every supernumerary regenerate is represented by two values. In cases with an oblique borderline it is referred to the
situation at the base of the regenerate.
Orientation
of the
transplant
(combinaGroup of
tion)0
experiments
I
1
I
2
I
3
1
II
II
2
II
3
1
III
III
2
III
3
Table 1 (cont.)
oo
a'
I
190
H. BOHN
Fig. 2 A-G. Exp. I/I, right midleg tibia of Gromphadorhina (dark pigmentation)
rotated 180° round the longitudinal axis and transplanted on to the left midleg tibia
(light pigmentation). Two tarsi develop at the suture, which either are completely
separated (A) arising anteriorly (a) and posteriorly (p), or are partly or wholly fused
(B) and then have a dorsal position. As a rule the extra tarsi are mixed, i.e. composed of Gromphadorhina and Leucophaea tissues (A,.C, D, F, G); in one case (E)
one tarsus consists only of Gromphadorhina tissues, the other only of Leucophaea
tissues (the regenerates in this case exceptionally are arranged one behind the
other, i.e. proximally {prox) and distally (dist) on the posterior surface of the tibia).
In most cases the amounts of both tissues forming one pair of supernumerary
regenerates are more or less equal (C, D, E), more rarely the Gromphadorhina
tissues predominate (G); only in one case do Leucophaea tissues slightly predominate (F). A, Dorsal view; B, posterior view. In C-G, as in Figs. 3, 6, 8 and 11,
the correlated regenerates of triple legs are put together by pairs, a (anterior),
p (posterior), d (dorsal), and v (ventral) indicate from which surface of a triple leg
the supernumerary regenerate arises. The names are always used with reference
to the axial organization of the stump. The regenerates are delineated as if cut up
longitudinally on the ventral surface and stretched out in a plain. The positions of
the four surfaces of a leg are specified in Ca. Light parts: Leucophaea tissues;
dotted: Gromphadorhina tissues; hatched (only in Figs. 3F, 8E and 11 A): segments
lacking or borderline not identifiable.
The epidermis in cockroach regenerates
191
B
Fig. 3 A-F. Exp. l/II, right midleg tibia of Gromphadorhina transplanted on to the left
midleg tibia of Leucophaea without rotation. The two extra regenerates develop
dorsally and ventrally (A, d and v), sometimes they fuse together and then lie
posteriorly (B). AH regenerates are mixed (A, C, D, F) with the exception of one
pair of homogeneously built tarsi, one of which consists of Gromphadorhina, the
other of Leucophaea tissues (E). Mostly the Gromphadorhina tissues predominate (C),
more rarely the amounts of both tissues are of equal size (D, E); only in one case
do the Leucophaea tissues predominate (F). A, Posterior view; B, ventral view.
B
Fig. 4 A-C. Exp. l/III, left midleg tibia of Gromphadorhina rotated 180° round the
longitudinal axis and transplanted on to the left midleg tibia of Leucophaea. The
rotated transplant mostly turns back to its normal position (A; only partly in B). Two
fully developed regenerates appeared only in two cases (C); in the other cases the
tarsi are incomplete and have fused together longitudinally (B); often only a socket
and condyle (g in A) are present or extra regenerates are missing. The amounts
of the two kinds of tissues forming the regenerates are, except for the leg shown in
C, approximately of equal size. All legs in posterior view.
192
H. BOHN
tendency to form regenerates in this combination is due to the fact that the
disharmonic orientation may be harmonized by a subsequent movement of the
transplant. In most cases the transplants have turned back to their original
position by a rotation of 180° round the longitudinal axis (Fig. 4 A, B).
(b) The composition of the supernumerary regenerates
In each of the three combinations (I—III) one triple leg was found, the one
tarsus of which was formed only by Gromphadorhina tissues, the other by
Leucophaea tissues (Figs. 2E, 3E). In four cases with partly fused regenerates
one of the distally free branches consisted only of Gromphadorhina tissues, the
other being mixed (Fig. 4C). The supernumerary regenerates of all other
animals were made up of a mixture of the tissues of both species.
Combination I. The borderline between the tissues of both species of cockroach, marked by the different pigmentation, is often approximately in the
middle of the dorsal and ventral side of the tarsus (Fig. 2 Ca, Cp, Da, Fa).
The anterior half of the anterior tarsus consisted of Leucophaea tissues, the
posterior half of Gromphadorhina tissues. The other supernumerary tarsus of
the same triple leg was composed inversely. Thus the two accessory tarsi in this
respect were symmetric to each other (Fig. 2C). In other cases one of the
borders was medio-ventral (Fig. 2 Gp) or medio-dorsal (Fig. 2 Ga), the second
border, however, was more anterior or posterior. Rarely both borderlines
deviated considerably from the median (Fig. 2 Dp).
The borderline was not always exactly parallel to the longitudinal axis of the
tarsus. Especially at the posterior surface of the tarsus the border was often
rather oblique (Fig. 2 Dp, Fp, Ga). Thus, the composition of the tarsus at the
base could be different from that at the top.
Adding the portions of each kind of tissue participating in both supernumerary
regenerates of a triple leg gives, as a rule, a relation of 1:1 (Fig. 2C, D, E).
More rarely the Gromphadorhina tissues predominate considerably (Fig. 2G).
in only one case did the Leucophaea tissues show a weak predominance (Fig. 2F).
Combination II. As in comb. I, one of the borderlines was often approximately medio-dorsal or medio-ventral: the other borderline, however, was in
most cases at the posterior surface of the tarsus (Fig. 3D). Thus, the amounts
of the two different tissues forming a supernumerary regenerate were very
unequal. But even if the portions from the two correlated regenerates are
taken together the amounts of both tissues are rarely equal (Fig. 3 D, E). In most
cases the Gromphadorhina tissues predominate (Fig. 3C); in only one case was
there slightly more Leucophaea than Gromphadorhina tissues (Fig. 3F).
Combination III. In this combination only two triple legs occurred with fully
developed extra tarsi. In one case one tarsus consisted completely of Gromphadorhina tissues, the second of Leucophaea tissues. In the other case (Fig. 4C)
both tarsi fused together in segments one and two. The free distal segments of
the one tarsus were built up only by Gromphadorhina tissues, the other tarsus
The epidermis in cockroach regenerates
193
pReg
aReg
Fig. 5 A-E. Exp. 2/1, right foreleg coxa rotated 180° round the longitudinal axis and
transplanted on to the left hind-leg coxa {Leucophaea only). A, General view of
a triple leg; the mostly mixed extra regenerates lie anteriorly (aReg) and posteriorly
(pReg, last tarsal segment missing); B-E, the three branches of the same triple leg
separately shown. B, Foreleg transplant (Tra); C, the bend of the tibia and tarsus of
the anterior regenerate is an indication for its mixed composition; D, E, posterior
regenerate, the foreleg structure of the femur and the tibia (arrows) can clearly be
recognized on the anterior surface (E). The posterior surface (D) consists of
hind-leg tissues as indicated by the folds on the tibia and the first tarsal segment. D,
Posterior view; in all other cases anterior view.
was mixed, 1/3 consisting of Leucophaea tissues. The incomplete tarsi as a rule
were composed half and half by tissues of Leucophaea and Gromphadorhina
(Fig. 4B).
(2) Multiple formations at the level of the coxa {groups 2 and 3)
{a) General phenomena
The results are very similar to those of group 1, except the supernumerary
regenerates which consist of tarsus, tibia, femur, and the distal half of the coxa
(Figs. 5, 7, 9). The extra regenerates have an anterior and posterior (comb. I) or
dorsal and ventral position (comb. II). In combination III there was often a
re-rotation of the rotated transplant. But complete regenerates now appeared
more often as compared with group 1.
The more complex structure of the accessory regenerates makes it possible
to determine their axial organization. In the combinations I and II both regenerates are in accordance with the stump in this respect, i.e. if one transplants
a part of a right leg on to a stump of a left leg two supernumerary left legs are
formed. In contrast one left and one right leg is formed in combination III in
which a left leg in reverse position is transplanted on to a stump of a leg of the
same side of the body.
13
E M B 28
194
H. BOHN
(b) The composition of the supernumerary regenerates
Combination I. In group 2 parts of a foreleg and hind leg of Leucophaea have
been combined. So it is necessary to describe briefly the different structures
of these legs. The foreleg differs from the hind leg in having a longitudinal row
of relatively robust bristles at the antero-ventral edge of the femur. Besides this
there are differences in the number and pattern of the bristles, especially at the
anterior surface of the tibia. The tibia and the first tarsal segment in the foreleg
are considerably shorter than in the hind leg (Fig. 5B). Naturally, these rather
scanty differences do not allow absolutely clear identification of every part of
a regenerate, all the more so as these structural characteristics often are only
weakly developed in the regenerates. Nevertheless, they are sufficient for elucidating the approximate composition of at least a part of the supernumerary
regenerates.
The supernumerary regenerate lying posteriorly is in nearly all cases (26 of 31)
clearly mixed (Fig. 5D, E). The anterior surface of the regenerate consisted of
tissues of the foreleg, indicated by the bristles on femur and tibia (Fig. 5E). The
posterior surface was formed by tissues of the hind leg marked by the folds of
the epidermis of the tibia and the first tarsal segment (Fig. 5D) and by the
pronounced bending of these segments in a posterior direction (the convex side
being posterior). Both phenomena are due to the different length of those
segments in the foreleg and hind leg. The different length of both sides of the
segments cannot be compensated by the bend alone; therefore the epidermis
gets creased.
Likewise, the other extra regenerate lying anteriorly was often considerably
bent, but in the opposite direction (the convex side being anterior, Fig. 5C).
So it might be suggested that the tissues of the hind leg mainly participate in
forming the anterior side of the leg, whereas the posterior side is built up by
foreleg tissues. Since characteristic patterns are lacking at the leg's posterior
side, this composition is not completely certain. Therefore the corresponding
regenerates are listed separately in Table 1.
The composition of the regenerates is much easier to identify, when the
tissues of Gromphadorhina and Leucophaea are combined (group 3). The results
confirm the conclusions of the preceding group of experiments. The extra
regenerates consist approximately half-and-half of Gromphadorhina and Leucophaea tissues. The anterior half of the posterior leg and the posterior half of
the anterior leg is built up by Gromphadorhina tissues, the remaining parts
consist of Leucophaea tissues. In most cases the border between the two tissues
lies approximately medio-dorsal and/or medio-ventral (Fig. 6A, Ba, Bp, Da;
Cp partly) sometimes posterior (Fig. 6 Ca; Cp partly). The ratio of the two
kinds of tissues forming the two correlated regenerates is nearly 1:1 (Fig. 6B, C);
but in some cases the Gromphadorhina tissues predominate considerably.
Examples of the last case are those two legs, the posterior regenerates of which
The epidermis in cockroach regenerates
195
D
Fig. 6 A-D. Exp. 3/1, right foreleg or hind-leg coxa of Gromphadorhina rotated
180° round the longitudinal axis and transplanted on to the left hind-leg coxa of
Leucophaea. As a rule both accessory regenerates are mixed (A, B, C, Da), only
rarely is one of the supernumerary legs formed completely by Gromphadorhina
tissues (Dp). The amounts of Gromphadorhina and Leucophaea tissues forming
the extra regenerates of a triple leg are mostly of equal size (B, C), sometimes the
Gromphadorhina tissues predominate (D). A, Dorsal view.
Fig. 7 A-E. Exp. 2/II, right foreleg coxa transplanted on to a left hind-leg coxa
without rotation (Leucophaea only). A, General view of a triple leg; B-E, the three
branches of the same triple leg separately shown. The extra regenerates develop
ventrally (vReg) and dorsally (dReg) with respect to the position of the transplant
(Tra). The dorsal regenerate in most cases is clearly mixed. The anterior surface (E)
shows foreleg structures (arrows), the posterior surface (D) consists of hind-leg
tissues as indicated by the folds on the tibia. The ventral regenerate (C) is more or
less a pure foreleg. D, Posterior view; in all other cases anterior view.
13-2
196
H. BOHN
are completely built up by Gromphadorhina tissues (Fig. 6 Dp). Pure Leucophaea
regenerates were never observed.
Combination II. Group 2. In contrast to the preceding combination the supernumerary regenerates mostly lie dor sally and ventrally (Fig. 7 A). In a great
number of cases especially the dorsal regenerates clearly show a mixed composition, the anterior side having foreleg structures, the posterior surface those of
the hind leg (Fig. 7D, E). The ventral regenerate in most cases seems to consist
uniformly of foreleg material, but minimal participation of hind-leg material
cannot be excluded. In some cases the mixed composition is certain, but the
stripe with hind-leg structures on the anterior or ventral surface of the leg is
always very small. One animal has two uniformly structured regenerates: the
ventral regenerate is a foreleg, the dorsal regenerate a hind leg.
Group 3. Most accessory regenerates are mixed, but a certain percentage is
built up completely by Gromphadorhina tissues. In five cases the ventral regenerate is homogeneous, whereas the corresponding dorsal regenerate is mixed
(Fig. 8D, E); in three cases even both extra regenerates are completely built up
by Gromphadorhina tissues (Fig. 8F). The position of the border between the
two tissues in the mixed regenerates is not chiefly median as in combination I,
but seems to be regularly distributed to all four sides. The amounts of transplant
and host tissues rarely are equal (Fig. 8B); in most cases the Gromphadorhina
tissues predominate (Fig. 8 A, C, D, E, F).
Combination III. Group 2. The position of the supernumerary regenerates,
provided they are formed at all, is not as constant as in the preceding combinations. Very often the stump seems to pass on directly into one of the regenerates
(Fig. 9 A, Zx). The coxa of the transplant mostly arises near the ventral face of
the coxa of the stump, from which it is separated by a slight constriction. The
second regenerate (Z2) arises directly from or at least near the coxa of the transplant. Z x is a left leg and as a rule only consists of hind-leg tissues (Fig. 9C);
Z 2 is a homogeneous right foreleg (Fig. 9D). In some cases, however, one of the
regenerates clearly shows mixed features; in some other cases the mixed structure seems rather likely, but cannot be proved with complete certainty.
As in all former experiments multiple formations with more than two
accessory regenerates were never observed. The multiple formation shown in
Fig. 10 A, B seems to be an exception to this rule. It is composed of two branches.
Fig. 8 A-F. Exp. 3/II, right foreleg or hind-leg coxa of Gromphadorhina transplanted on to the left hind-leg coxa of Leucophaea without rotation. Some of the
supernumerary regenerates are partly mixed (A, B, C, Dd, Ed), some consist completely of Gromphadorhina tissues (Dv, Ev, F). The amounts of Gromphadorhina and
Leucophaea tissues forming the regenerates are rarely of equal size (B), mostly the
Gromphadorhina tissues predominate (A, C, D, E), in some cases even both
regenerates are formed only by Gromphadorhina tissues (F). Av, Ventral view;
Ad, dorsal view. The extra regenerates shown in B exceptionally lie anteriorly (a)
and posteriorly (p).
The epidermis in cockroach regenerates
^ ^
197
198
H. BOHN
Fig. 9 A-D. Exp. 2/III, left foreleg coxa rotated 180° round the longitudinal axis
and transplanted on to the left hind-leg coxa (Leucophaea only). A, General view
of a triple leg; B-D, the three branches of the same triple leg separately shown.
The rotated transplant mostly turns back to its normal position (Tra, A). Mostly one
of the extra regenerates possesses hind-leg features (Zl9 C), the other foreleg features
(Z2, D). The two regenerates have different axial organization: the hind leg is a left
leg, the foreleg a right leg. D, Posterior view; in all other cases anterior view.
The ventral branch is a right leg with mainly foreleg structures (Z2). The second
branch is a compound formation, which is the result of the fusion of more than
two legs, because there are three ventral parts of a femur (Tra + ZJ. The development of this multiple formation may explain its composition. Originally
(after the second postoperative moult) it consisted as usual of three completely
separated branches, i.e. the transplant and two accessory regenerates. By an
accident this leg was damaged rather severely, as a result of which large parts of
the transplant and of one extra regenerate were destroyed. In the course of the
succeeding regeneration the abnormal formation described above developed
out of the wound area. So, the compound formation consists of the transplant,
a supernumerary regenerate, and the ventral parts of an additional supernumerary leg. This third supernumerary regenerate is not formed by the original
host-transplant combination, but by a secondary wound. In a similar way it is
possible to get a supernumerary although incomplete branch by wounding a
normal leg (Bohn, 1965).
Group 3. Only seven animals had well-developed extra regenerates, which in
most cases are mixed. Some of the regenerates forming right legs are homogeneously built up by Gromphadorhina tissues. Regenerates consisting only ofLeuco-
The epidermis in cockroach regenerates
199
Fig. 10 A, B. The only case with apparently more than two supernumerary regenerates (Exp. 2/HI). Z2 extra regenerate with predominant foreleg features. The
second extra regenerate has fused with the transplant (Tra + ZJ. The three light
stripes on the femur (arrows) suggest that at least the ventral parts of a third extra
regenerate had participated in this leg. A, Posterior view; B, anterior view.
Fig. 11 A, B. Exp. 3/III, left foreleg coxa of Gromphadorhina rotated 180° round
the longitudinal axis and transplanted on to the left hind-leg coxa of Leucophaea.
One of the two supernumerary regenerates is a right leg (/-), the other is a left leg (/).
The left leg always was mixed (A/, B/); the right leg was mixed (Ar) or consisted only
of Gromphadorhina tissues (Br).
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H. BOHN
phaea tissues could not be observed. The amounts of the two tissues forming
a pair of extra regenerates are either nearly equal (1 case, Fig. 11 A) or the
Gromphadorhina tissues predominate (3 cases, Fig. 11B). Because of the small
number of triple legs with completely developed supernumerary regenerates
nothing generally valid can be said about the position of the border between
the two tissues.
Summary of the results
(a) The composition of the supernumerary regenerates
In combination I the results of the three groups of experiments are more or
less uniform. In nearly all cases the supernumerary regenerates are mixed.
Regenerates consisting only of Gromphadorhina or Leucophaea tissues are
exceptions. In group 2 homogeneously formed regenerates were not observed;
but this does not mean that such regenerates cannot occur at all in this group,
for it cannot be excluded that one or the other of the 41 regenerates classified
as not unequivocally identifiable consisted nevertheless of only one sort of
tissues.
The borderline between stump and transplant tissues mostly lay mediodorsally and medio-ventrally; so the anterior longitudinal half of a regenerate
consisted of one, the posterior half of the other sort of tissue. The amounts of
both sorts of tissues forming the two correlated accessory regenerates were
approximately equal; the transplant (= Gromphadorhina) tissues predominated
rather rarely.
In combination II, too, the mixed regenerates are the most numerous, especially
in group 1. In group 3 a considerable part of the extra regenerates was not
mixed. The position of the borderline was preferably dorsal and posterior.
In contrast to comb. I a distinct disproportion could be observed between
the tissues of Gromphadorhina and Leucophaea, not only in a single regenerate,
but also in almost every pair of supernumerary regenerates. The Gromphadorhina tissues predominated in most cases, in some cases even quite considerably.
In combination III the great number of homogeneously formed regenerates of
Exp. 2/III is remarkable, whereas the regenerates of Exp. 3/III predominantly
are mixed. On the whole, the Gromphadorhina tissues provide the main part of
the material for the supernumerary regenerates.
(b) Number, position and symmetry of the supernumerary regenerates
After heteropleural transplantation (combinations I and II) two extra regenerates are formed invariably, if the transplant survived. They arise from those
regions of the suture between stump and transplant, where tissues of different
symmetry properties meet together. In combination I this situation is anterior
and posterior, in combination II dorsal and ventral. Both regenerates have the
same axial structure as the stump, i.e. left legs are regenerated on a left side
stump. The supernumerary regenerates, especially those arising from the tibia,
may move together (in dorsal (I) or posterior (II) direction) and fuse partly or
The epidermis in cockroach regenerates
201
in full length. This fusion is not accompanied by any regression of the fused
regenerates.
After homopleural transplantation (comb. Ill) the rotated transplant very
often turns back to its normal position. At most two extra regenerates are
formed, which often have a tendency to fuse. In contrast to the heteropleural
transplantation this fusion is accompanied by a regression of the regenerates. In
the extreme, only traces of the distal following joint region are to be found (in
group 1, for instance, the socket and condyle of the tibio-tarsal joint, in groups 2
and 3 traces of the coxa-trochanter joint). Even though rudimental the regenerated structures are always developed twice, i.e. symmetrically. Some of the
animals had no regenerates at all; in those cases the transplant had always
turned back to its normal position. The two extra regenerates have different
axial structures: one is in accordance with the stump, the other with a leg of
the opposite body side. Thus one left and one right leg is formed in all cases.
DISCUSSION
(1) The composition of the supernumerary regenerates
The experiments described above attempt to elucidate the origin of the
supernumerary regenerates by using leg tissues with different structures as
stump and transplant. Regenerate parts having the same structure as stump or
transplant tissues were assumed to arise from the corresponding stump and
transplant tissues. This is only permissible if the tissues do not lose their
special features during regeneration, which is accompanied by dedifferentiation
and cell division. In a recent paper (Bohn, 1971) it has been shown that the
features used (differences in pigmentation between Gromphadorhina and Leucophaea; structural differences between foreleg and hind-leg tissues) are firmly
determined in this respect and yet can manifest themselves autonomously in
a foreign environment.
The experiments show that both possibilities of composition of the extra
regenerates occur: in each group there are legs with mixed accessory regenerates
as well as uniform regenerates. It is remarkable that the Gromphadorhina
tissues, i.e. the transplant tissues, predominate in the regenerates of combinations I and II. This raises the question whether the rules found in the combinations of Gromphadorhina and Leucophaea tissues prove correct in intraspecific
combinations, too. The results of Exp. 2/II show that there are, at least in this
respect, no great differences between interspecific and intraspecific transplantations. For in Exp. 2/11 again usually the transplant tissues seem to predominate:
the dorsal regenerate was clearly mixed; the ventral regenerate seemed to be
uniformly built up by foreleg tissues or contained only small amounts of hind-leg
tissues. Thus, the accessory regenerates are composed of transplant and stump
tissues in the ratio of about 2 (or more): 1,
In comparing Exps. 2/III and 3/III there are beyond question certain
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H. BOHN
differences between intraspecific and interspecific transplantations. In the first
experiment uniformly composed regenerates predominate, in the latter mixed.
But this difference is only quantitative and without importance with respect to
our further conclusions.
In the following our results relating to the composition of the extra regenerates are compared with the results of other authors.
The experiments of Bodenstein (1937) with legs of Lepidoptera {Pyrameis
cardui) are comparable with combination III. He transplanted parts of the first
or second leg on to a stump of the third leg. One of the supernumerary
regenerates consisted of host tissues, the other of transplant tissues. His results
are partly in accordance with ours in so far as just in this combination (for
instance in Exp. 2/III) often triple legs occur having one transplant and one
stump regenerate; mixed regenerates formed considerably more rarely. Since
Bodenstein had only four cases with well-developed triple legs, the occurrence of
mixed regenerates cannot be excluded in Lepidoptera.
Bulliere (1970), in his experiments, used differently structured legs of the
same species (Blabera craniifer). But the clearly identifiable structures are limited
to a few parts of the leg only, and are not sufficient to allow identification of
mixed regenerates in every case (see p. 194). The mere presence of the specific
foreleg structures, which are restricted only to the anterior surface of the leg,
does not suffice to take such a leg for a pure foreleg; conversely, the absence of
these structures does not mean that the leg is a hind leg. Bulliere concludes
from his experiments that one regenerate consists of host and the other of
transplant tissues. This conclusion, in my opinion, is only correct to the extent
that triple legs may occur in Blabera in the same way as in Leucophaea, which
are built up in this way. But the figures presented by Bulliere argue against
his own opinion. In Fig. 3B, C, D (Bulliere, 1970, p. 344) both supernumerary
regenerates show the same bend as our mixed regenerates and thus make it
likely that they are composed of both, foreleg and hind-leg tissues.
The same objections may be raised to the experiments of Lheureux (1971).
The legs used as stump and transplant (pedipalps respectively hind legs of the
spider Tegeneria saeva) are distinguished more or less only by their distal ends
having two or three claws respectively. This is not enough to allow clear
identification of mixed structures like those presented in Fig. 8 Ad and 8Cv. So
it remains undecided, whether or not mixed extra regenerates may be found in
spiders.
The results of Bart (1971 b) are in good agreement with ours. Bart did not
transplant whole legs, but only sectors thereof, and in most cases got only one
accessory leg. This regenerate was clearly mixed at least in those cases or parts
that allowed unequivocal identification. Naturally, Bart could not show an
exact borderline, since he combined the parts of legs of the same species (Carausius morosus). But he supposes that the adjacent tissues of the stump and
transplant extend into the supernumerary regenerate; thus, each forms one
The epidermis in cockroach regenerates
203
longitudinal half of the regenerate. Our experiments have shown that this
supposition is correct as far as concerns comb. I, but in the other combinations (II and III) this situation is only exceptionally realized. Besides such
clearly mixed regenerates Bart also gets regenerates which seem to consist only
of host tissues. This assumption could be confirmed by interspecific transplantation experiments (A. Bart, personal communication). Most curiously, there are
never supernumerary regenerates, which consist only of transplant tissues. In
phasmids, contrary to the case in cockroaches, the host tissues seem to
predominate.
(2) The number, position and axial structure of the supernumerary regenerates
Most authors (Bodenstein, 1937, 1941; Bohn, 1965; Bulliere, 1970; Lheureux,
1970, 1971) agree with respect to number, position and orientation of the accessory regenerates. The re-rotation of rotated transplants in comb. Ill, previously reported in Leucophaea (Bohn, 1965) has also been established for
Blabera (Bulliere, 1970). In Carausius on the contrary there seems to be no
re-rotation (Bart, 1971a). Bart is the only one who gets three supernumerary
regenerates in several cases after homopleural transplantation. In our experiments there was only one similar case (Fig. 10 A, B). But the development of
this multiple leg clearly indicates that originally there had been only two supernumerary regenerates. The third incomplete branch has developed owing to
an untypical wounding. Likewise Furukawa (1940) finds a leg with three
accessory regenerates as an exception among his multiple legs. In the only
figure Bart presents of such a quadruple leg, two of the three supernumerary
regenerates have fused together at their bases and are only separated at their
distal ends. It is possible that secondary effects have led to these formations,
and in the legend of this figure Bart does say that there has been a partial
necrosis of the axial regenerate. Nevertheless, it cannot be excluded that
Carausius behaves quite differently in this respect from all other arthropods,
especially since Bart did not observe any re-rotation of the rotated transplant.
(3) The causes for the development of supernumerary regenerates
The extra regenerates always appear after disharmonic orientation of transplants. Remarkably they always develop at that site where different symmetry
properties of stump and transplant (anterior-posterior, dorsal-ventral) meet.
Bart (1971 a) therefore concludes that in those places' morphogenetic centres' are
established which give rise to the development of regeneration blastemas. This
hypothesis seems plausible as far as combinations I and II are concerned.
According to Bart's hypothesis, however, one should get four extra regenerates
in the experiments of combination III, since there are disharmonic situations
at all four sides of the leg. But Bart never gets more than three accessory
regenerates; mostly (75%) he only gets two. Other insects never show more
than two accessory regenerates.
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H. BOHN
Undoubtedly the disharmonic orientation is of great importance for the
induction of regenerative processes (Bodenstein, 1937; Furukawa, 1940; limbs
of Amphibia see Mangold, 1929); but it seems questionable to reduce the problem of the origin of the extra regenerates to the simple formula: regenerates
always develop in accordance with position and number of sites with opposite
orientation. Some of Bart's own experiments argue against such an interpretation. For instance, he gets supernumerary regenerates, when he combines
a dorsal part of the scape with a posterior part of the coxa.
In my opinion Bart uses the different qualities anterior, posterior, etc. much
too formally. In fact, these names only represent a topographic characterization.
Beyond this they may also imply that the surface of an appendage is asymmetrically organized. But instead of distinguishing only four different qualities
one could arrange the cross-section into a two-dimensional coordinate
system, the axes of which coincide with the antero-posterior and dorso-ventral
axis. In this way it becomes much more evident that in reality every cell of a cut
surface is different from any other (according to the position within the
system), even though lying within the same sector of the section, i.e. anteriorly,
posteriorly, etc. The sites bearing the extra regenerates in combinations I and II
are thus sites in which these differences are greatest. In the combination III the
degree of disharmonic orientation is equal at every point. Thus, contrary to
Bart's opinion there are more than four regions with disharmonic orientation.
The correspondence of the number of accessory regenerates with the number
of existing cut surfaces is striking. This fact is in good agreement with the
hypothesis of Przibram (1921) about the formation of the 'Bruchdreifachbildungen'. According to this hypothesis each cut surface forms one regenerate:
a normal distal regenerate sprouts from the amputation surface of the stump and
a so-called proximal regenerate form the proximal wound surface of the transplant. The axial organization of the extra regenerates is in good agreement with
this assumption. Taking a left leg as host the supernumerary regenerates
formed in combinations I and II are two left legs, but one left and one right
leg is formed in combination III. Naturally, the conclusions of Przibram's hypothesis are not valid with respect to the material used for the regenerates because it
may happen that both supernumerary regenerates are built up only by material
of the transplant. In modifying the hypothesis of the 'Bruchdreifachbildung'
one could say: by combining two cut surfaces with divergent orientations each
cut surface is stimulated to induce or organize one regenerate, the material
for which may come partly or as a whole from the other amputation
surface.
Nevertheless, this conception is not completely satisfying because, firstly,
the phenomenon of the regular arrangement of the regenerates still rests
unsolved unless it is assumed that the points of greatest disharmony are
favoured above others in developing blastemas; secondly, it seems possible to
The epidermis in cockroach regenerates
205
get complete regenerates even if only a part of a cross-section had been
exposed by a wound (Bart, 1971a, b). Further experiments must be done before
these seemingly contradictory phenomena are resolved under a single hypothesis
which accounts for them all.
(4) The determination of symmetry properties
An important problem already discussed by Bart (1971 b) is that of the
determination of the symmetry properties. Are cells of a distinct side (for
instance anterior) able to transform into or to proliferate cells of another side
(for example posterior) ? The experiments with partly severed legs done so far
(Bonn, 1965; Bart, 1970) argue against such a transformation, for the distal
regenerates arising from such wounds only possess those properties that had
been exposed by the wound.
Such a transformation cannot yet be concluded, as Bart (1971 b) does, by the
fact that there are triple legs with one extra branch consisting entirely of stump
tissues and the other branches built up entirely by transplant tissues. Naturally,
a supernumerary regenerate develops within a restricted area at one surface of the
wound. But this does not mean that only cells of this restricted region had participated in forming the regenerate. Cells from all regions of a cross-section
could have assembled at that position. Thus, it is not necessary to assume
transformation of symmetry properties in order to allow the development of
complete regenerates. Some of Bart's own experiments are far more more convincing. He implanted a piece of tissue taken from one surface of a leg into the
opposite surface and got a complete regenerate. If the wound areas of each, the
transplant and the implantation zone, really included only a quarter of a complete cut surface it must be concluded that it is possible to get a complete
regenerate with all symmetry properties from a wound containing only two of
four existing sides. But, nevertheless, some doubt remains. It cannot be
excluded that there had been some necrosis following as well as deeper wounds
during operation. By this cells of the other symmetry properties could have
been exposed and could have participated in regeneration.
The experiments with Leucophaea and Gromphadorhina show that transformations of symmetry properties in fact occur much more frequently than has been
supposed. What would the supernumerary regenerates look like if such
transformations were not possible? The amounts of the Leucophaea tissues of
both extra regenerates of one triple leg taken together should make up just one
complete cross-section. Apart from numerous cases in which the Gromphadorhina tissues clearly predominate (Figs. 2G, 3C, 6D, 8E) or even occasionally
yield all of the material for both regenerates (Fig. 8F), this cannot even be found
exactly in those cases where the relation of both tissues is approximately 1:1.
In Fig. 2D, for instance, the postero-ventral part of the first tarsal segment of
the accessory tarsi consists of Gromphadorhina tissues, in Fig. 2F the anterior
claw and anterior part of the arolium is represented twice by Leucophaea tissues,
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H. BOHN
etc. Thus, it must be concluded that regenerates including more than one crosssection, even two in the extreme, may develop from a single cross-section.
There is a further complication as far as the supernumerary regenerates of
combination III is concerned. One left and one right leg is formed, the former
corresponding to the organization of the stump's amputation surface, the latter
by regeneration from the transplant's amputation surface in a proximal direction.
There are several cases with mixed extra regenerates. This means that tissues
originally representing parts of a right leg have been fitted into a left leg, and
the reverse. This insertion apparently happened 'ortsrichtig' since such mixed
legs do not show any anomalies in their patterns. So, in addition to or in
connexion with a change of the symmetry properties there is a second change:
the axial organization apparently is not yet fixed irreversibly and may be fitted
to the conditions obtaining at the time.
Change or transformation of the symmetry properties or of the axial organization does not mean, for example, that a greater part of dorsal tibia tissues may
be transformed to ventral tissues as a whole. A great number of experiments,
for example the formation of triple legs after disharmonic orientation, argues
against such a possibility. It is much more likely that the missing parts will be
added to the edges of an incomplete cross-section by proliferation, i.e. may be
a kind of lateral regeneration. In this way there could be a doubling of crosssections without any cell changing its determination. Therefore, I would prefer
to speak of completion of missing symmetry properties instead of using the
term transformation. Thus it remains open whether this completion is attained
by an extensive transformation of symmetry properties or not.
According to the results of our experiments it seems to be fairly certain that
completion of symmetry properties does occur. Nevertheless, some doubt
remains. For simplicity, consider one extreme where two complete regenerates
have been formed by a single cross-section. This result could have been achieved
by a splitting of the blastema perpendicular to the longitudinal axis, by a kind
of cross-section. Such a process does not seem to be very likely, but cannot be
excluded. These doubts could be settled by transplanting parts of a cross-section
interspecifically. The formation of a complete regenerate by such a sector
including only a part of a whole cross-section irrevocably would demonstrate
the ability of the leg tissues to complete missing symmetry properties.
(5) Cell lineage during regeneration
The organization of the mixed regenerates may help to clarify the problem of
cell lineage and growth in the regenerating leg. As indicated above, the borderline between the two different tissues almost always runs in a longitudinal
direction. This means that, after the establishment of the blastema, either
there had been only longitudinal divisions or that if divisions in the transversal
direction had occurred at all they must have occurred to the same extent at
every level of the longitudinal axis. But the same holds true even for the
The epidermis in cockroach regenerates
207
establishment of the blastema itself. The completion of missing parts of a crosssection as referred to above is only imaginable if there are divisions perpendicular to the longitudinal axis of the leg, unless, as seems unlikely, rather large areas
of tissues may change their side qualities as a whole. So, divisions must have
occurred in such a way that sectors with strongly radial borders had formed
in a presumably dome-like blastema.
On the other hand, the borderline is not always exactly parallel to the
longitudinal axis. It has been mentioned above (see p. 194) that there are legs with
slightly but distinctly oblique border lines (Figs. 2 Dp, Fp, Ga; 3Cv; 8Cv);
narrow stripes of foreign tissues may even taper away (Figs. 3Cv, Fd; 8 Ad, O ) .
These cases are a further indication of the fact that cells are not yet determined
irreversibly with respect to a distinct position within the cross-section. A cell
or at least the descendants of a cell may alter this position.
Studies on cell lineage and related problems have been made recently by
Bryant (Bryant & Schneiderman, 1969; Bryant, 1970) in imaginal wing and leg
discs of Drosophila. Their results, especially in the leg discs, are quite similar to
ours. The clonally related cells produced by X-ray-induced somatic crossing
over become arranged in longitudinal stripes. But the stripes are not always
strictly parallel to the longitudinal axis nor of equal width at every level; moreover there are partly overlapping patches. This means that the cells of an
imaginal leg disc of Drosophila, like those in larval legs of Leucophaea and
Gromphadorhina, are not yet determined, at least until the end of the second
instar, with respect to their position within the cross-section.
I thank Dr D. A. Ede for polishing the style of the English translation and Dr P. A.
Lawrence for helpful criticism.
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BRYANT,
{Manuscript received 22 November 1971,
English translation received 16 February 1972)