- Email: [email protected]
Supernumerary regeneration in the large milkweed bug Oncopeltus fasciatus
DEVELOPMENTAL
BIOLOGY
&,
221-230 (19%)
Supernumerary
Regeneration
in the Large Milkweed
Oncopeltus
Bug
fasciatus
VICTORIA K. SHAW AND PETER J. BRYANT Center for Pathobiology,
University Accepted
of California, March
Irvine, Irvine,
California
92664
14, 1975
As an approach to the problem of pattern formation in the insect appendage, various graft combinations were studied in the legs of the large milkweed bug Oncopeltus fasciatus. Metathoracic legs of fourth instar larvae were amputated through the tibia within 24 hr after ecdysis and grafted back onto the stumps. The orientation of the graft was altered by rotation through 90 or 180’ and/or by exchanging right and left stumps and grafts, yielding seven possible orientations in addition to the control. Many of these grafts resulted in the production of one or two supernumerary regenerates of the distal segments, which appeared at the graft junction after the second postoperative ecdysis. When two supernumerary regenerates resulted, one appeared to be produced from the stump and the other from the graft. When one regenerate was present, it appeared to be a composite of material produced from both the stump and the graft. In contrast to the results obtained in cockroaches, the external face of the leg appeared to be the only one capable of giving rise to a supernumerary regenerate. INTRODUCTION
When insects were discovered in nature with triplicated distal leg parts, it was assumed that the extra parts had arisen by supernumerary regeneration. The formation of extra appendages was explained in terms of the wound hypothesis (Przibram, 1921) which proposed that, when an appendage is injured, the more distal part may remain attached, thus creating two wound surfaces. Regeneration of distal parts then occurs at each of these surfaces. Experimental results (Bohn, 1965), obtained by making a V-shaped cut into the appendage, tended to confirm this hypothesis, but only one supernumerary regenerate resulted which appeared to be a composite of tissue regenerated from both wound surfaces. In the cockroaches Periplaneta americana (Bodenstein, 1962), Blabem craniifer (Bullikre, 1970), and Leucophaea maderae (Bohn, 1965; 1972), the walking stick, Carausius morosus (Bar& 1971), the moths Phryganidia californica and Pyrameis cardui (Bodenstein, 1937), and the
earwig Anisolabis maritima (Furukawa, 1940) supernumerary regenerates have been obtained by grafting the distal portion of an appendage onto a proximal stump with changed orientation either by axial rotation through 90 or 180” or by the combination of contralateral stumps and grafts. Usually two supernumerary regenerates appear at the grafting site after the postoperative ecdysis or, in the case of Lepidoptera, after metamorphosis. In the case of contralateral grafts, it is usually found that the two supernumerary regenerates arise from that part of the graft junction where the circumferential levels of graft and stump are maximally out of register (Bulliltre, 1970; Bohn, 1972). Where handedness can be determined, the two supernumeraries are both of the same handedness as the stump following contralateral grafting, but are of opposite handedness to one another in the case of ipsilatera1 grafting with axial rotation (Bohn, 1972). As is pointed out by Bohn (1972) these relations of symmetry can be explained by a modification of Przibram’s (1921) hypothesis. In the simplest form of 221
222
DEVELOPMENTALBIOLOGY
this explanation (see Bull&e, 1970), it is assumed that one of the two supernumerary regenerates arises from the wound surface of the stump and therefore is of stump handedness. The other supernumerary would arise from the wound surface of the graft and, as is usual with proximally directed regeneration, would be a mirrorimage duplicate with opposite handedness to the graft. However, since the results of interspecific transplants showed that most supernumeraries are built up from both graft and stump tissues, Bohn (1972) proposed a modification of Przibram’s hypothesis, to the effect that the two supernumeraries follow the symmetry of the graft and stump, respectively, without necessarily being derived from the two sources respectively. Although it is known that some hemipteran insects can regenerate legs and antennae (Liischer, 1948; Wolsky, 1957; Shaw and Bryant, 1974) the possibility of supernumerary regeneration in this group has not hitherto been explored. Yet many of these insects have short life cycles which make them potentially very useful for such studies and make it feasible to contemplate genetic approaches to the regeneration problem. In this paper we report the production of supernumerary leg regenerates following various graft combinations in the large milkweed bug Orzcopeltus fasciatus, a hemipteran in which several mutations have been isolated (Lawrence, 1970) and in which simple, intercalary and reversed regeneration have already been studied (Shaw and Bryant, 1974; 1975).
VOLUME 45, 1975
gas was used to anesthetize the insects during the grafting operations. The animals were raised on milkweed seeds at a temperature of 27°C and a relative humidity of 66%. Eight orientations of the graft with respect to the stump were studied. Four of the orientations were obtained by grafting right legs onto the right stumps of another set of insects (ipsilateral grafting operations, Figs. 1 and 2) and four of the orientations were produced by recombining right and left stumps and grafts (contralatera1 grafting operations, Figs. 3 and 4). In both series the orientations consisted of: No rotation of the graft with respect to the stump; 180” rotation; 90” rotation of the external surface of the graft towards the anterior surface of the host; and 90” rotation towards the posterior surface of the host. In this paper, the external surface of the tibia refers to the surface that is contiguous with the dorsal surface of the femur, and the internal surface of the tibia is that which adjoins the ventral surface of the femur. In the contralateral grafts with no rotation, the internal-external axes of graft and stump are opposed, and, in the contralateral grafts with 180” rotation, their anterior-posterior axes are opposed. The control operations were ipsilateral grafts with no rotation. These were performed between different animals (homografts) to conform with the experimental series. An extra set of autograft controls, amputation and replacement of the same leg with the correct orientation, were studied to ascertain the effect of the operation on the growth of the tibia and tarsus. After MATERIALS AND METHODS two ecdyses, when the insects had reached the adult stage, the legs were removed and Metathoracic legs of fourth instar larvae studied. of Oncopeltus fasciatus were amputated at the mid-tibia1 level within 24 hr after RESULTS ecdysis and grafted back, with or without Ipsilateral Leg Grafting Operations changed orientation, onto the stumps. AmThe effects of the control operation on putations were performed using iridectomy the growth of the tibia and tarsus are scissors, and a drop of Eastman 910 adheshown in Table 1. The table indicates that sive was placed at the grafting site in order the tibiae and tarsi of the grafted legs were to hold the graft in place. Carbon dioxide
SHAW AND BRYANT
Supernumerary
was rotated 180” with respect to the host animal, so that the internal and external surfaces of the graft and stump corresponded, but the anterior and posterior surfaces were out of register. No rotation was observed in this series, and 48% of the appendages contained two supernumerary regenerates while 10% contained one regenerate. When the graft was rotated 90” towards the anterior of the host or towards the posterior of the host, usually either no correction or a 45” correction was observed. A 90” anterior rotation yielded two supernumerary regenerates in 47% of the insects and no regenerates in 53%. With a 90” posterior rotation, 44% contained two regenerates and 39% contained one regenerate. When no regenerates were obtained after contralateral grafting operations, the area around the grafting site was usually malformed and the bristle pattern was disrupted.
shorter than the normal segments in both the fifth instar and adult stages, but no other abnormalities were observed. The results from ipsilateral grafts are shown in Table 2 and illustrated in Figs. 1 and 2. In all cases in which the graft was not rotated, the leg appeared normal in the adult. When the graft was rotated 180” with respect to the stump so that none of the surfaces coincided, the majority of the grafts underwent a 90” correctional rotation either anteriorly or posteriorly. Thirtythree percent of the grafted appendages showed two supernumerary regenerates, and 12.5% had one supernumerary regenerate. When the graft was rotated through 90” towards the anterior or towards the posterior of the stump, usually no supernumerary regenerates were produced, but the area around the grafting site was malformed and the bristle pattern was disrupted. Correctional rotation occurred frequently in these experiments, and in Table 2 these cases are divided into those showing no correction and those showing approximately 45 and 90” correction. Contralateral
Leg Grafting
Supernumerary
of Morphology Regenerates
Operations
TABLE
1
EFFECT OF THE CONTROL OPERATION ON GROWTH OF THE TIBIA Length of tarsus (mm + SE) Normal Fourth instar
1.00 i 0.01
Fifth instar Adult
1.6 + 0.1 2.23 f 0.04
Leg
The grafting site on control legs showed a normal bristle pattern (Fig. 51, whereas the bristle pattern was altered and supernumerary regenerates were present on most of the experimental legs. The supernumerary regenerates were characterized by the presence of an intersegmental membrane separating a tiny segment from the rest of the appendage, and many of the regenerates contained tarsal claws (Figs. 6 and 7). When two supernumerary regenerates were produced, one regenerate was more proximal on the leg and was usually located adjacent to the external surface of the stump. The other regenerate was more
The results from contralateral grafting operations are shown in Table 3 and Figs. 3 and 4. When the graft was not rotated with respect to the host animal, so that the anterior and posterior surfaces of the graft and stump coincided, while the internal and external surfaces were out of register, the grafts usually did not undergo rotation after the operation. In 50% of the appendages two supernumerary regenerates resulted, and in 9% one regenerate resulted. In another series of experiments the graft
Stage
223
Regeneration
Grafted 1.12 - 0.03 1.44 * 0.04
AND TARSUS
Length of tibia (mm * SE) Normal
Grafted
1.97 * 0.03
-
3.26 * 0.05 4.6 i 0.1
2.86 i 0.04 4.15 * 0.02
224
DEVELOPMENTALBIOLOGY TABLE
2
IPSILATERAL.GRAITING OPERATIONS’ Operation No rotation
Number of operations Successful operations No. % No regenerates No. % One regenerate No.
% Two regenerates No.
% No correction No.
% 45” Anterior correction No. % 45” Posterior correction No. % 90” Anterior correction No. % 96” Posterior correction No.
% No determination possible No.
%
160” rotation
96” anterior rotation
90” Posterior rotation
35
49
35
29
16 46
24 49
24 69
15 52
16 loo
13 54
21 87.5
13 a7
0 0
3 13
3 13
1 7
0 0
a
0
33
0
1 7
16 166
0 0
5 21
4 27
0 0
2 a
0 0
10 67
0 0
0 0
9 38
0 0
0 0
5 21
0 0
1 7
0 0
12 50
10 42
0 0
0 0
5 21
0 0
0 0
LIAnterior and posterior rotation refer to the direction of displacement surface of the graft.
and correction of the external
distal on the leg and was usually adjacent to the external surface of the graft (Fig. 8). In the appendages where only one regenerate resulted, it was produced either at the
VOLUME 45. 1975
external surface of the host or of the graft, or it lay in between these two positions. The regenerate often contained a region, lighter in appearance, which divided it longitudinally into two halves (Fig. 9). When no supernumerary regenerates were present on the experimental combinations, the malformed regions between stump and graft often contained unsegmented protuberances (Fig. 10). The positions of the protuberances generally corresponded to the positions of supernumerary regenerates on other grafted legs with similar orientation. Grafting
Experiments
on the Antenna
In order to determine whether supernumerary regenerates are produced after altering orientation within the antenna, ipsilateral and contralateral graft combinations were performed on the third proximal segment. No supernumerary regenerates resulted and only a few antennae showed a slight bristle pattern disturbance or small protuberance. In many cases the second antenna1 segment was longer than the normal segment, and in several cases it contained a partial intersegmental membrane yielding an antenna with one more segment than normal (Fig. 11). DISCUSSION
When metathoracic legs of the milkweed bug are amputated and the distal portions are grafted back onto the stumps, wound healing must occur. This wound healing, which involves a reorganization process at the grafting site, retards the growth of the grafted leg. This stunted growth of the tibia and tarsus is the only effect of the control grafting operation. When the orientation of the graft with respect to that of the stump is altered in ipsilateral and contralateral grafting operations, two phenomena may occur: There may be an axial rotation of the graft with respect to the stump, and supernumerary regenerates may be produced at the graft junction.
SHAWAND
225
Supernumerary Regeneration
BRYANT
P
Fro. 1. Graft combinations for two orientations (no rotation and 180” rotation) in the ipsilateral grafting operations. Smaller circles represent graft surfaces and larger circles represent stump surfaces. a, Anterior; p, posterior; i, internal; and e, external. The results found in the adult are also shown.
NO
900
Rotation
Rotation
0 .
i
P
0
ii
*e 0
P
NO
P
Corr*ction
a
P e
f D
0
(I
e
i
P
0. 900 P 0 Correction
ip
@
e 0
P
0
Correction
a*
i
P
Correction
P a i
a
P
0
e
e*
P 0
NO
e
D
. Q
a*
450
0
P
Por.
0
0 0
e
0
0
900
0 a
P
a
Ant.
1800
P
900 Correction
0
P
450 Correction
Fro. 2. The four orientations in the ipsilateral grafting operations and the most frequent results found in the adults. The protuberances drawn on some of the circles indicate the production of supernumerary regenerates at the corresponding surfaces. Symbols and abbreviations as in Fig. 1.
In our experiments, correctional rotation occurred to varying extents in most casesof rotated ipsilateral grafts (Fig. 21, although it was not as complete as it is in cockroaches (Bohn, 1965; Bull&e, 1970). The
effect of such rotation was to bring the anterior, posterior, internal and external surfaces of the graft into closer register with the corresponding surfaces of the stump. However, in the cases of contralat-
226
DEVELOPMENTALBIOLOGY
TABLE 3 CONTRALATERAL GRAFTING OPERATION@ Operation
Number of operations Successful operations No. % No regenerates No. % One regenerate No. % Two regenerates No. % No correction No. % 45” Anterior correction No. % 45” Posterior correction No. % 90” Anterior correction No. % 90” Posterior correction No. % No determination possible No. %
No rotation
180” rotation
90” Anterior rotation
90” Posterior rotation
30
30
31
34
22 73
21 70
17 55
18 53
9 41
2 10
8 47
3 17
2 9
9 43
2 12
7 39
11 50
10 48
7 41
8 44
14 64
21 100
7 41
6 33
0 0
0 0
1 6
8 44
4 18
0 0
8 47
1 6
0 0
0 0
0 0
3 17
1 5
0 0
1 6
0 0
3 14
0 0
0 0
0 0
a Anterior and posterior rotation refer to the direction of displacement surface of the graft.
and correction of the external
era1 grafts, no amount of correctional tion can bring all surfaces of the graft closer register with those of the stump, we found a much lower frequency of
rotainto and rota-
VOLUME 45, 1975
tion in these experiments. However, in those cases where such a rotation did occur, it was clear that its effect was to bring the internal and external surfaces of the graft and stump into closer register while at the same time causing increased displacement of the anterior and posterior surfaces. If correctional rotation is to be interpreted as the result of the tendency of homologous surfaces to approach each other, then this tendency must be stronger for internal and external surfaces than for anterior and posterior surfaces. Nothing is known of the force responsible for correctional rotation, although the physical pressures from muscle attachments and from walking movements may contribute to rotation in grafted legs. However, the discovery of a similar phenomenon in grafted pieces of abdominal epidermis (Bohn, 1974; Lawrence, 1974; Niibler-Jung, 1974) indicates that the main force is probably generated by the epidermal cells themselves. In all orientations produced by the ipsilateral and contralateral grafting operations, except for control grafts (ipsilateral, no rotation), supernumerary regenerates have been found. In most of these cases two supernumerary regenerates are present and they are separated from the rest of the appendage by intersegmental membranes. Since they often bear tarsal claws, the regenerates must contain tarsal segments, but we were unable to identify clearly any more proximal segments. In the antenna there is a developmental response to these kinds of graft combinations, but in this case the stimulated growth is intercalary in position, producing elongation of the appendage rather than supernumerary regeneration. In many of our experiments, the graft and stump do not fuse as completely as they do in cockroaches, and the position of the graft junction is usually evident from the disturbance of bristle patterns at that level. This often makes it possible to deduce the origin of the supernumerary re-
SHAW AND BRYANT
No
Supernumerary
Regeneration
Rotation
FIG. 3. Graft combinations and results for two orientations, no rotation contralateral grafting operations. Symbols and abbreviations as in Fig. 1.
1800
90~
and 180” rotation,
Ant.
900
Rotation
in the
Pas.
Rotation a
0
0 1p
00
0
0 *
‘p 0
0
00
I0 0
P
NO Corrsctlon
P
NO Correction
P 0
0 P
*
I
P
0
0
0 i
0
c3 P
FIG. 4. The four orientations in the contralateral grafting the adults. Symbols and abbreviations as in Fig. 1.
generates, since one supernumerary regenerate usually forms proximally to the graft junction and one distally to it. It seems reasonable to conclude that the proximal supernumeraries originate from the stump and the distal ones from the graft. The
*
*
0 ASO
Correction
operations
45’
io
and the most frequent
results found in
orientation of claws on the regenerates supports this conclusion: On the proximal regenerate the claw orientation conforms to the orientation of the stump, and on the distal regenerate the claw orientation is symmetrical to that of the graft.
228
DEVELOPMENTALBIOLOGY
VOLUME
45, 1975
FIG. 5. Region of the grafting site in the adult from a control operation (ipsilateral, no rotation). FIG. 6. Two supernumerary regenerates containing tarsal claws (C) produced from a contralateral grafting operation with no rotation. The external surfaces (e) of the stump and graft are indicated as well as the presence of intersegmental membranes (IS). FIG. 7. Two supernumerary regenerates produced from an ipsilateral operation with a 90” rotation toward the posterior. FIG. 8. Two supernumerary regenerates located at the external surfaces of the graft and stump which resulted from a contralateral operation, no rotation. FIG. 9. One regenerate located at the external surface of the stump and graft which resulted from a contralateral operation with 180” rotation. FIG. 10. An unsegmented protuberance resulting from an ipsilateral operation with 180” rotation.
The supernumerary regenerates produced in cockroaches usually arise from that part of the graft junction where the graft and stump surfaces are maximally out of register. Thus, in contralateral graft combinations with no rotation, the supernumerary regenerates arise from the regions where internal and external surfaces are apposed, and, in contralateral combinations with 180" rotation, the supernumeraries arise from the points where anterior and posterior surfaces are apposed (Bohn, 1965; Bulliere, 1970). However, in our experiments, the external surface of the stump or graft appears to be the only region that gives rise to supernumerary
regenerates. In the cases where no surfaces are in register (ipsilateral rotated grafts) and where the external-internal axes are opposed, the supernumerary regenerates arise at the external surfaces of the stump and the graft. Even in those cases where only the anterior-posterior axes are opposed (contralateral grafts with 180” rotation) the supernumerary regenerates arise at or near the external surfaces of the stump and the graft. Often only one supernumerary regenerate is produced, and its appearance indicates that it is probably a composite of two regenerates. The lighter region, which separates the segment into distal and proximal sides, appears to be
SHAW AND BRYANT
Supernumerary
229
Regeneration
pared with the cockroaches Bryant, 1974).
(Shaw
and
This investigation was supported by Grant No. HD 06082 from the National Institutes of Health. REFERENCES
FIG. 11. A control antenna (a) compared with an antenna (b) resulting from a contralateral grafting operation, 180” rotation, in the third proximal segment. The latter contains a partial intersegmental membrane (IS) in the second proximal segment. GJ, graft junction.
intersegmental membrane. Bohn (1972) found a similar phenomenon in cockroaches, and he interpreted this as resulting from a tendency of supernumerary regenerates, which were originally initiated at the antero-posterior graft junctions to move together and fuse in a dorsal (i.e., external) direction. Of course, we cannot exclude the possibility of such movement in our experiments, but if it occurs it must always be directed towards an external surface. In Oncopeltus, the production of supernumerary regenerates at the external surface, as well as the strong tendency to correctional rotation when external and internal surfaces are out of register indicates that the external leg surface is especially sensitive to the presence of adjacent nonhomologous tissue. Alternatively, it is conceivable that only this section of the circumference is capable of regenerative growth, in the graft combinations and even after complete amputations. If the entire regenerate had to be built up from a limited section of the stump surface, this might account for the rather limited leg regeneration shown by Oncopeltus as com-
BART, A. (1971). MorphogenBse surnumeraire au niveau de la patte du phasme Carausius morosus Br. Wilhelm Roux Arch. Entwicklungsmech. Organismen 166, 331-364. BODENSTEIN, D. (1937). Beintransplantationen an Lepidopterenraupen. IV. Zur Analyse experimentell erzeugter Beinmehrfachbildungen. Wilhelm Roux Arch. Entwicklungsmech. Organismen 136, 745-785. BODENSTEIN, D. (1962). &generation in insects. Symp. Genet. Biol. 9, l-19. BOHN, H. (1965). Analyse der Regenerationsftihigkeit der Insektenextremitlt durch Amputations-und Transplantationsversuche an Larven der Afrikanischen Schabe (Leucophaea maderae Fabr.). II. Mitteilung: Achsendetermination. Wilhelm Roux Arch. Entwicklungsmech. Organismen 156, 449-503. BOHN, H. (1972). The origin of the epidermis in the supernumerary regenerates of triple legs in cockroaches (Blattaria). J. Embryol. Exp. Morphol. 28, 185-208. BOHN, H. (1974). Pattern reconstitution in abdominal segment of Leucophaea maderae (Blattaria). Nature (London) 248,608-609. BWLIBRE, D. (1970). InterprCtation des &g&rats multiples chez les Insectes. J. Embryol. Exp. Morphol. 23, 337-357. FURUKAWA, H. (1940). Transplantation experiments on appendages of Anisolabis maritima (Dermaptera). III. Transplantation of forceps. Jap. J. Zool. 8, 510-535. LAWRENCE, P. A. (1970). Some new mutants of the large milkweed bug Oncopeltus fasciatus Dall. Genet. Res. 15, 347-350. LAWRENCE, P. A. (1974). Cell movement during pattern regulation in Oncopeltus. Nature (London) 248, 609-610. LUSCHER, M. (1948). The regeneration of legs in Rhodnius prolixus (Hemiptera). J. Exp. Biol. 25, 334-343. NDBLER-JUNG, K. (1974). Cell migration during pattern reconstitution in the insect segment (Dysdercus intermedius Dist., Heteroptera). Nature (London) 248, 610-611. PRZIBRAM, H. (1921). Die Bruchdreifachbildung im Tierreiche. Wilhelm Roux Arch. Entwicklungsmech. Organismen 48, 205-244. SHAW, V. K., and BRYANT, P. J. (1974). Regeneration
230
DEVELOPMENTALBIOLOGY
of appendages in the large milkweed bug, Oncopeltus fasciatus. J. Insect Physiol. 20, 1849-1857. SHAW,V. K., and BRYANT,P. J. (1975). Intercalary leg regeneration in the large milkweed bug Oncopeltus
VOLUME 45, 1975
fasciatus. Develop. Biol. 45, 187-191. WOLSKY,A. (1957). “Compensatory hyper-regeneration” in the antennae of Hemiptera. Nature (London) 180, 1144-1145.
BIOLOGY
&,
221-230 (19%)
Supernumerary
Regeneration
in the Large Milkweed
Oncopeltus
Bug
fasciatus
VICTORIA K. SHAW AND PETER J. BRYANT Center for Pathobiology,
University Accepted
of California, March
Irvine, Irvine,
California
92664
14, 1975
As an approach to the problem of pattern formation in the insect appendage, various graft combinations were studied in the legs of the large milkweed bug Oncopeltus fasciatus. Metathoracic legs of fourth instar larvae were amputated through the tibia within 24 hr after ecdysis and grafted back onto the stumps. The orientation of the graft was altered by rotation through 90 or 180’ and/or by exchanging right and left stumps and grafts, yielding seven possible orientations in addition to the control. Many of these grafts resulted in the production of one or two supernumerary regenerates of the distal segments, which appeared at the graft junction after the second postoperative ecdysis. When two supernumerary regenerates resulted, one appeared to be produced from the stump and the other from the graft. When one regenerate was present, it appeared to be a composite of material produced from both the stump and the graft. In contrast to the results obtained in cockroaches, the external face of the leg appeared to be the only one capable of giving rise to a supernumerary regenerate. INTRODUCTION
When insects were discovered in nature with triplicated distal leg parts, it was assumed that the extra parts had arisen by supernumerary regeneration. The formation of extra appendages was explained in terms of the wound hypothesis (Przibram, 1921) which proposed that, when an appendage is injured, the more distal part may remain attached, thus creating two wound surfaces. Regeneration of distal parts then occurs at each of these surfaces. Experimental results (Bohn, 1965), obtained by making a V-shaped cut into the appendage, tended to confirm this hypothesis, but only one supernumerary regenerate resulted which appeared to be a composite of tissue regenerated from both wound surfaces. In the cockroaches Periplaneta americana (Bodenstein, 1962), Blabem craniifer (Bullikre, 1970), and Leucophaea maderae (Bohn, 1965; 1972), the walking stick, Carausius morosus (Bar& 1971), the moths Phryganidia californica and Pyrameis cardui (Bodenstein, 1937), and the
earwig Anisolabis maritima (Furukawa, 1940) supernumerary regenerates have been obtained by grafting the distal portion of an appendage onto a proximal stump with changed orientation either by axial rotation through 90 or 180” or by the combination of contralateral stumps and grafts. Usually two supernumerary regenerates appear at the grafting site after the postoperative ecdysis or, in the case of Lepidoptera, after metamorphosis. In the case of contralateral grafts, it is usually found that the two supernumerary regenerates arise from that part of the graft junction where the circumferential levels of graft and stump are maximally out of register (Bulliltre, 1970; Bohn, 1972). Where handedness can be determined, the two supernumeraries are both of the same handedness as the stump following contralateral grafting, but are of opposite handedness to one another in the case of ipsilatera1 grafting with axial rotation (Bohn, 1972). As is pointed out by Bohn (1972) these relations of symmetry can be explained by a modification of Przibram’s (1921) hypothesis. In the simplest form of 221
222
DEVELOPMENTALBIOLOGY
this explanation (see Bull&e, 1970), it is assumed that one of the two supernumerary regenerates arises from the wound surface of the stump and therefore is of stump handedness. The other supernumerary would arise from the wound surface of the graft and, as is usual with proximally directed regeneration, would be a mirrorimage duplicate with opposite handedness to the graft. However, since the results of interspecific transplants showed that most supernumeraries are built up from both graft and stump tissues, Bohn (1972) proposed a modification of Przibram’s hypothesis, to the effect that the two supernumeraries follow the symmetry of the graft and stump, respectively, without necessarily being derived from the two sources respectively. Although it is known that some hemipteran insects can regenerate legs and antennae (Liischer, 1948; Wolsky, 1957; Shaw and Bryant, 1974) the possibility of supernumerary regeneration in this group has not hitherto been explored. Yet many of these insects have short life cycles which make them potentially very useful for such studies and make it feasible to contemplate genetic approaches to the regeneration problem. In this paper we report the production of supernumerary leg regenerates following various graft combinations in the large milkweed bug Orzcopeltus fasciatus, a hemipteran in which several mutations have been isolated (Lawrence, 1970) and in which simple, intercalary and reversed regeneration have already been studied (Shaw and Bryant, 1974; 1975).
VOLUME 45, 1975
gas was used to anesthetize the insects during the grafting operations. The animals were raised on milkweed seeds at a temperature of 27°C and a relative humidity of 66%. Eight orientations of the graft with respect to the stump were studied. Four of the orientations were obtained by grafting right legs onto the right stumps of another set of insects (ipsilateral grafting operations, Figs. 1 and 2) and four of the orientations were produced by recombining right and left stumps and grafts (contralatera1 grafting operations, Figs. 3 and 4). In both series the orientations consisted of: No rotation of the graft with respect to the stump; 180” rotation; 90” rotation of the external surface of the graft towards the anterior surface of the host; and 90” rotation towards the posterior surface of the host. In this paper, the external surface of the tibia refers to the surface that is contiguous with the dorsal surface of the femur, and the internal surface of the tibia is that which adjoins the ventral surface of the femur. In the contralateral grafts with no rotation, the internal-external axes of graft and stump are opposed, and, in the contralateral grafts with 180” rotation, their anterior-posterior axes are opposed. The control operations were ipsilateral grafts with no rotation. These were performed between different animals (homografts) to conform with the experimental series. An extra set of autograft controls, amputation and replacement of the same leg with the correct orientation, were studied to ascertain the effect of the operation on the growth of the tibia and tarsus. After MATERIALS AND METHODS two ecdyses, when the insects had reached the adult stage, the legs were removed and Metathoracic legs of fourth instar larvae studied. of Oncopeltus fasciatus were amputated at the mid-tibia1 level within 24 hr after RESULTS ecdysis and grafted back, with or without Ipsilateral Leg Grafting Operations changed orientation, onto the stumps. AmThe effects of the control operation on putations were performed using iridectomy the growth of the tibia and tarsus are scissors, and a drop of Eastman 910 adheshown in Table 1. The table indicates that sive was placed at the grafting site in order the tibiae and tarsi of the grafted legs were to hold the graft in place. Carbon dioxide
SHAW AND BRYANT
Supernumerary
was rotated 180” with respect to the host animal, so that the internal and external surfaces of the graft and stump corresponded, but the anterior and posterior surfaces were out of register. No rotation was observed in this series, and 48% of the appendages contained two supernumerary regenerates while 10% contained one regenerate. When the graft was rotated 90” towards the anterior of the host or towards the posterior of the host, usually either no correction or a 45” correction was observed. A 90” anterior rotation yielded two supernumerary regenerates in 47% of the insects and no regenerates in 53%. With a 90” posterior rotation, 44% contained two regenerates and 39% contained one regenerate. When no regenerates were obtained after contralateral grafting operations, the area around the grafting site was usually malformed and the bristle pattern was disrupted.
shorter than the normal segments in both the fifth instar and adult stages, but no other abnormalities were observed. The results from ipsilateral grafts are shown in Table 2 and illustrated in Figs. 1 and 2. In all cases in which the graft was not rotated, the leg appeared normal in the adult. When the graft was rotated 180” with respect to the stump so that none of the surfaces coincided, the majority of the grafts underwent a 90” correctional rotation either anteriorly or posteriorly. Thirtythree percent of the grafted appendages showed two supernumerary regenerates, and 12.5% had one supernumerary regenerate. When the graft was rotated through 90” towards the anterior or towards the posterior of the stump, usually no supernumerary regenerates were produced, but the area around the grafting site was malformed and the bristle pattern was disrupted. Correctional rotation occurred frequently in these experiments, and in Table 2 these cases are divided into those showing no correction and those showing approximately 45 and 90” correction. Contralateral
Leg Grafting
Supernumerary
of Morphology Regenerates
Operations
TABLE
1
EFFECT OF THE CONTROL OPERATION ON GROWTH OF THE TIBIA Length of tarsus (mm + SE) Normal Fourth instar
1.00 i 0.01
Fifth instar Adult
1.6 + 0.1 2.23 f 0.04
Leg
The grafting site on control legs showed a normal bristle pattern (Fig. 51, whereas the bristle pattern was altered and supernumerary regenerates were present on most of the experimental legs. The supernumerary regenerates were characterized by the presence of an intersegmental membrane separating a tiny segment from the rest of the appendage, and many of the regenerates contained tarsal claws (Figs. 6 and 7). When two supernumerary regenerates were produced, one regenerate was more proximal on the leg and was usually located adjacent to the external surface of the stump. The other regenerate was more
The results from contralateral grafting operations are shown in Table 3 and Figs. 3 and 4. When the graft was not rotated with respect to the host animal, so that the anterior and posterior surfaces of the graft and stump coincided, while the internal and external surfaces were out of register, the grafts usually did not undergo rotation after the operation. In 50% of the appendages two supernumerary regenerates resulted, and in 9% one regenerate resulted. In another series of experiments the graft
Stage
223
Regeneration
Grafted 1.12 - 0.03 1.44 * 0.04
AND TARSUS
Length of tibia (mm * SE) Normal
Grafted
1.97 * 0.03
-
3.26 * 0.05 4.6 i 0.1
2.86 i 0.04 4.15 * 0.02
224
DEVELOPMENTALBIOLOGY TABLE
2
IPSILATERAL.GRAITING OPERATIONS’ Operation No rotation
Number of operations Successful operations No. % No regenerates No. % One regenerate No.
% Two regenerates No.
% No correction No.
% 45” Anterior correction No. % 45” Posterior correction No. % 90” Anterior correction No. % 96” Posterior correction No.
% No determination possible No.
%
160” rotation
96” anterior rotation
90” Posterior rotation
35
49
35
29
16 46
24 49
24 69
15 52
16 loo
13 54
21 87.5
13 a7
0 0
3 13
3 13
1 7
0 0
a
0
33
0
1 7
16 166
0 0
5 21
4 27
0 0
2 a
0 0
10 67
0 0
0 0
9 38
0 0
0 0
5 21
0 0
1 7
0 0
12 50
10 42
0 0
0 0
5 21
0 0
0 0
LIAnterior and posterior rotation refer to the direction of displacement surface of the graft.
and correction of the external
distal on the leg and was usually adjacent to the external surface of the graft (Fig. 8). In the appendages where only one regenerate resulted, it was produced either at the
VOLUME 45. 1975
external surface of the host or of the graft, or it lay in between these two positions. The regenerate often contained a region, lighter in appearance, which divided it longitudinally into two halves (Fig. 9). When no supernumerary regenerates were present on the experimental combinations, the malformed regions between stump and graft often contained unsegmented protuberances (Fig. 10). The positions of the protuberances generally corresponded to the positions of supernumerary regenerates on other grafted legs with similar orientation. Grafting
Experiments
on the Antenna
In order to determine whether supernumerary regenerates are produced after altering orientation within the antenna, ipsilateral and contralateral graft combinations were performed on the third proximal segment. No supernumerary regenerates resulted and only a few antennae showed a slight bristle pattern disturbance or small protuberance. In many cases the second antenna1 segment was longer than the normal segment, and in several cases it contained a partial intersegmental membrane yielding an antenna with one more segment than normal (Fig. 11). DISCUSSION
When metathoracic legs of the milkweed bug are amputated and the distal portions are grafted back onto the stumps, wound healing must occur. This wound healing, which involves a reorganization process at the grafting site, retards the growth of the grafted leg. This stunted growth of the tibia and tarsus is the only effect of the control grafting operation. When the orientation of the graft with respect to that of the stump is altered in ipsilateral and contralateral grafting operations, two phenomena may occur: There may be an axial rotation of the graft with respect to the stump, and supernumerary regenerates may be produced at the graft junction.
SHAWAND
225
Supernumerary Regeneration
BRYANT
P
Fro. 1. Graft combinations for two orientations (no rotation and 180” rotation) in the ipsilateral grafting operations. Smaller circles represent graft surfaces and larger circles represent stump surfaces. a, Anterior; p, posterior; i, internal; and e, external. The results found in the adult are also shown.
NO
900
Rotation
Rotation
0 .
i
P
0
ii
*e 0
P
NO
P
Corr*ction
a
P e
f D
0
(I
e
i
P
0. 900 P 0 Correction
ip
@
e 0
P
0
Correction
a*
i
P
Correction
P a i
a
P
0
e
e*
P 0
NO
e
D
. Q
a*
450
0
P
Por.
0
0 0
e
0
0
900
0 a
P
a
Ant.
1800
P
900 Correction
0
P
450 Correction
Fro. 2. The four orientations in the ipsilateral grafting operations and the most frequent results found in the adults. The protuberances drawn on some of the circles indicate the production of supernumerary regenerates at the corresponding surfaces. Symbols and abbreviations as in Fig. 1.
In our experiments, correctional rotation occurred to varying extents in most casesof rotated ipsilateral grafts (Fig. 21, although it was not as complete as it is in cockroaches (Bohn, 1965; Bull&e, 1970). The
effect of such rotation was to bring the anterior, posterior, internal and external surfaces of the graft into closer register with the corresponding surfaces of the stump. However, in the cases of contralat-
226
DEVELOPMENTALBIOLOGY
TABLE 3 CONTRALATERAL GRAFTING OPERATION@ Operation
Number of operations Successful operations No. % No regenerates No. % One regenerate No. % Two regenerates No. % No correction No. % 45” Anterior correction No. % 45” Posterior correction No. % 90” Anterior correction No. % 90” Posterior correction No. % No determination possible No. %
No rotation
180” rotation
90” Anterior rotation
90” Posterior rotation
30
30
31
34
22 73
21 70
17 55
18 53
9 41
2 10
8 47
3 17
2 9
9 43
2 12
7 39
11 50
10 48
7 41
8 44
14 64
21 100
7 41
6 33
0 0
0 0
1 6
8 44
4 18
0 0
8 47
1 6
0 0
0 0
0 0
3 17
1 5
0 0
1 6
0 0
3 14
0 0
0 0
0 0
a Anterior and posterior rotation refer to the direction of displacement surface of the graft.
and correction of the external
era1 grafts, no amount of correctional tion can bring all surfaces of the graft closer register with those of the stump, we found a much lower frequency of
rotainto and rota-
VOLUME 45, 1975
tion in these experiments. However, in those cases where such a rotation did occur, it was clear that its effect was to bring the internal and external surfaces of the graft and stump into closer register while at the same time causing increased displacement of the anterior and posterior surfaces. If correctional rotation is to be interpreted as the result of the tendency of homologous surfaces to approach each other, then this tendency must be stronger for internal and external surfaces than for anterior and posterior surfaces. Nothing is known of the force responsible for correctional rotation, although the physical pressures from muscle attachments and from walking movements may contribute to rotation in grafted legs. However, the discovery of a similar phenomenon in grafted pieces of abdominal epidermis (Bohn, 1974; Lawrence, 1974; Niibler-Jung, 1974) indicates that the main force is probably generated by the epidermal cells themselves. In all orientations produced by the ipsilateral and contralateral grafting operations, except for control grafts (ipsilateral, no rotation), supernumerary regenerates have been found. In most of these cases two supernumerary regenerates are present and they are separated from the rest of the appendage by intersegmental membranes. Since they often bear tarsal claws, the regenerates must contain tarsal segments, but we were unable to identify clearly any more proximal segments. In the antenna there is a developmental response to these kinds of graft combinations, but in this case the stimulated growth is intercalary in position, producing elongation of the appendage rather than supernumerary regeneration. In many of our experiments, the graft and stump do not fuse as completely as they do in cockroaches, and the position of the graft junction is usually evident from the disturbance of bristle patterns at that level. This often makes it possible to deduce the origin of the supernumerary re-
SHAW AND BRYANT
No
Supernumerary
Regeneration
Rotation
FIG. 3. Graft combinations and results for two orientations, no rotation contralateral grafting operations. Symbols and abbreviations as in Fig. 1.
1800
90~
and 180” rotation,
Ant.
900
Rotation
in the
Pas.
Rotation a
0
0 1p
00
0
0 *
‘p 0
0
00
I0 0
P
NO Corrsctlon
P
NO Correction
P 0
0 P
*
I
P
0
0
0 i
0
c3 P
FIG. 4. The four orientations in the contralateral grafting the adults. Symbols and abbreviations as in Fig. 1.
generates, since one supernumerary regenerate usually forms proximally to the graft junction and one distally to it. It seems reasonable to conclude that the proximal supernumeraries originate from the stump and the distal ones from the graft. The
*
*
0 ASO
Correction
operations
45’
io
and the most frequent
results found in
orientation of claws on the regenerates supports this conclusion: On the proximal regenerate the claw orientation conforms to the orientation of the stump, and on the distal regenerate the claw orientation is symmetrical to that of the graft.
228
DEVELOPMENTALBIOLOGY
VOLUME
45, 1975
FIG. 5. Region of the grafting site in the adult from a control operation (ipsilateral, no rotation). FIG. 6. Two supernumerary regenerates containing tarsal claws (C) produced from a contralateral grafting operation with no rotation. The external surfaces (e) of the stump and graft are indicated as well as the presence of intersegmental membranes (IS). FIG. 7. Two supernumerary regenerates produced from an ipsilateral operation with a 90” rotation toward the posterior. FIG. 8. Two supernumerary regenerates located at the external surfaces of the graft and stump which resulted from a contralateral operation, no rotation. FIG. 9. One regenerate located at the external surface of the stump and graft which resulted from a contralateral operation with 180” rotation. FIG. 10. An unsegmented protuberance resulting from an ipsilateral operation with 180” rotation.
The supernumerary regenerates produced in cockroaches usually arise from that part of the graft junction where the graft and stump surfaces are maximally out of register. Thus, in contralateral graft combinations with no rotation, the supernumerary regenerates arise from the regions where internal and external surfaces are apposed, and, in contralateral combinations with 180" rotation, the supernumeraries arise from the points where anterior and posterior surfaces are apposed (Bohn, 1965; Bulliere, 1970). However, in our experiments, the external surface of the stump or graft appears to be the only region that gives rise to supernumerary
regenerates. In the cases where no surfaces are in register (ipsilateral rotated grafts) and where the external-internal axes are opposed, the supernumerary regenerates arise at the external surfaces of the stump and the graft. Even in those cases where only the anterior-posterior axes are opposed (contralateral grafts with 180” rotation) the supernumerary regenerates arise at or near the external surfaces of the stump and the graft. Often only one supernumerary regenerate is produced, and its appearance indicates that it is probably a composite of two regenerates. The lighter region, which separates the segment into distal and proximal sides, appears to be
SHAW AND BRYANT
Supernumerary
229
Regeneration
pared with the cockroaches Bryant, 1974).
(Shaw
and
This investigation was supported by Grant No. HD 06082 from the National Institutes of Health. REFERENCES
FIG. 11. A control antenna (a) compared with an antenna (b) resulting from a contralateral grafting operation, 180” rotation, in the third proximal segment. The latter contains a partial intersegmental membrane (IS) in the second proximal segment. GJ, graft junction.
intersegmental membrane. Bohn (1972) found a similar phenomenon in cockroaches, and he interpreted this as resulting from a tendency of supernumerary regenerates, which were originally initiated at the antero-posterior graft junctions to move together and fuse in a dorsal (i.e., external) direction. Of course, we cannot exclude the possibility of such movement in our experiments, but if it occurs it must always be directed towards an external surface. In Oncopeltus, the production of supernumerary regenerates at the external surface, as well as the strong tendency to correctional rotation when external and internal surfaces are out of register indicates that the external leg surface is especially sensitive to the presence of adjacent nonhomologous tissue. Alternatively, it is conceivable that only this section of the circumference is capable of regenerative growth, in the graft combinations and even after complete amputations. If the entire regenerate had to be built up from a limited section of the stump surface, this might account for the rather limited leg regeneration shown by Oncopeltus as com-
BART, A. (1971). MorphogenBse surnumeraire au niveau de la patte du phasme Carausius morosus Br. Wilhelm Roux Arch. Entwicklungsmech. Organismen 166, 331-364. BODENSTEIN, D. (1937). Beintransplantationen an Lepidopterenraupen. IV. Zur Analyse experimentell erzeugter Beinmehrfachbildungen. Wilhelm Roux Arch. Entwicklungsmech. Organismen 136, 745-785. BODENSTEIN, D. (1962). &generation in insects. Symp. Genet. Biol. 9, l-19. BOHN, H. (1965). Analyse der Regenerationsftihigkeit der Insektenextremitlt durch Amputations-und Transplantationsversuche an Larven der Afrikanischen Schabe (Leucophaea maderae Fabr.). II. Mitteilung: Achsendetermination. Wilhelm Roux Arch. Entwicklungsmech. Organismen 156, 449-503. BOHN, H. (1972). The origin of the epidermis in the supernumerary regenerates of triple legs in cockroaches (Blattaria). J. Embryol. Exp. Morphol. 28, 185-208. BOHN, H. (1974). Pattern reconstitution in abdominal segment of Leucophaea maderae (Blattaria). Nature (London) 248,608-609. BWLIBRE, D. (1970). InterprCtation des &g&rats multiples chez les Insectes. J. Embryol. Exp. Morphol. 23, 337-357. FURUKAWA, H. (1940). Transplantation experiments on appendages of Anisolabis maritima (Dermaptera). III. Transplantation of forceps. Jap. J. Zool. 8, 510-535. LAWRENCE, P. A. (1970). Some new mutants of the large milkweed bug Oncopeltus fasciatus Dall. Genet. Res. 15, 347-350. LAWRENCE, P. A. (1974). Cell movement during pattern regulation in Oncopeltus. Nature (London) 248, 609-610. LUSCHER, M. (1948). The regeneration of legs in Rhodnius prolixus (Hemiptera). J. Exp. Biol. 25, 334-343. NDBLER-JUNG, K. (1974). Cell migration during pattern reconstitution in the insect segment (Dysdercus intermedius Dist., Heteroptera). Nature (London) 248, 610-611. PRZIBRAM, H. (1921). Die Bruchdreifachbildung im Tierreiche. Wilhelm Roux Arch. Entwicklungsmech. Organismen 48, 205-244. SHAW, V. K., and BRYANT, P. J. (1974). Regeneration
230
DEVELOPMENTALBIOLOGY
of appendages in the large milkweed bug, Oncopeltus fasciatus. J. Insect Physiol. 20, 1849-1857. SHAW,V. K., and BRYANT,P. J. (1975). Intercalary leg regeneration in the large milkweed bug Oncopeltus
VOLUME 45, 1975
fasciatus. Develop. Biol. 45, 187-191. WOLSKY,A. (1957). “Compensatory hyper-regeneration” in the antennae of Hemiptera. Nature (London) 180, 1144-1145.