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Trans. R. ent. SOL. Lond. 126 (4) pp. 447-461, 5 figs. 1973
The relative growth of normal and regenerated legs of the cockroach BZateZZa gerrnanica (L.)
B R I A N R O B E R T S Department of Zoology, University of New England, Armidale, New South Wales 2351"
[Communicated by H.B.N. Hynes]
C O N T E N T S
............................................................ .......................................... I Introduction 447 11 Materials and methods ...................................................................................................... 448
450 111 Results ..........................................................................................
IV Discussion 459 v Summary 461
References 461
....................................................................................................................................
....................................................................... ................................................
...................................................................................................................................
With 5 text-figures
S Y N O P S I S
An account is given of the growth of the normal uninjured metathoracic leg o f Blatella germanica and of growth of the regenerated leg following removal before or after the critical period in the first instar. The presence and effects of two growth gradients found in the regenerated leg are discussed.
I I N T R O D U C T I O N
O'Farrell & Stock (1953, 1958) have shown that the removal of a metathoracic leg from the cockroach Blattella germanica (L.) at the trochantero-femoral articulation can lead to the appearance of a fully differentiated regenerate with a tetramerous tarsus at the next ecdysis, which is delayed by a period roughly equal to the time elapsed between the previous ecdysis and removal of the leg. If a leg is removed after the critical period, a rounded papilla appears at the next ecdysis and the regenerate does not appear until the second ecdysis after the operation, the duration of the instars not being significantly altered. In either situation the regenerated leg is substantially smaller, at its first appear- ance, than the normal uninjured leg.
* Present address: Department of Zoology, Monash University Clayton, Victoria 3 168.
447
448 B. Roberts
I t has been shown by Woodruff & Seamans (1939) in Blattella germanica and Voy (195 I ) in Blatta orientalis L. that the size of a regenerated appendage increases at succes- sive ecdyses until it approximates that of the normal appendage. Little precise information is available, and nothing is known of the differences in growth pattern between regenera- tions initiated before and after the critical period.
In the present work the growth patterns of the normal metathoracic leg and the regenerating metathoracic leg, removed before and after the critical period of the first instar, have been followed through to metamorphosis in both sexes.
I 1 M A T E R I A L S A N D M E T H O D S
Cultures of Blattella germanica were reared according to the methods of O'Farrell & Stock (1953, 1956) at 29' C f 1.0' C with the relative humidity controlled at 7-80 per cent. R.H. These cultures were observed at regular intervals and nymphs of known age were obtained.
In experimental animals the left metathoracic leg was removed by traction on the femur (O'Farrell & Stock, 1953). Such operations were performed on the first and fifth days after hatching, so that the process of regeneration initiated before and after the critical period of the first instar could be studied.
Animals at each required stage of development were sacrificed. The entire meta- thoracic segment was removed, mounted with the anterior surface facing upward, and
Fig. I. Standard points of measurement in the metathoracic leg segments of Bluttellu germunicu (L.). c, coxa; tr, trochanter; f, femur; ti, tibia; ts , tarsus; t l- ts , tarsomeres ' -5 .
Relative growth of normal and regenerated legs in Blatella
00 +a m z z z $ z ; ? ? ? ? ? ? ? ? ? ? 0 0 0 0 0 0 0 0 0 0 +l t ls i+s i t l t i$ I+ i i ~1 + m m r - m - t m r . ~ N N N “?????9 0 0 0 0 0 0 0 0 0 0
N N N O
m m m * w m m m 2% ? ? ? ? ? ? ? ? ? ? t l + + S 1 H S I H + S I S I ? ? ? ? ? F ? ? ? ?
0 0 0 N r n N N
0 0 0 0 0 0 0 0 0 0
m a r . 0 N N m m m m
0 0 0 0 0 0 0 0 0 0
b r . 0 m m m o m b r . - - m - m m m N - N
0 0 0 0 0 0 0 0 0 0 ? ? ? ? ? ? ? ? ? ? $ 1 $ 1 t l $ 1 t l $ i t l $ i $ I S I z 2% m m t - w 0 -to
? N N N ” “? 0 b b o o o o o o o
r - z m a m m m m m * 0 r N N n N n m m ? ? ? ? ? ? ? ? ? ? 0 0 0 0 0 0 0 0 0 0
00 m m o m N N m n m - N N N n N n N m m
0 0 0 0 0 0 0 0 0 0 ? ? ? ? ? ? ? ? ? ? HHH-HHHH-H-H-H
0 0 0 0 0 0 0 0 0 0 N N N N N n N N N N
%cw Y)cw%cw4<
unn
449
450 B. Roberts
measured microscopically with a calibrated micrometer eyepiece. Each leg was measured in a standard manner (fig. I ) .
I 1 1 R E S U L T S
The results were calculated from 20 individual measurements for each age group, except for the uninjured metathoracic leg segments of fifth instar females, where 10 measurements were made.
Table 2. Length (in mm.) 5 standard deviation of the regenerated major metathoracic leg segments
Coxa Trochanter
Instar
I I1
I11 IV V d V ?
VI d VI 0
VII A S VII A
~-
Before Ratio critical per. R/N*
After Ratio critical per. R/N
Operation Papilla o.74fo.026 0 . 8 5 1.13&0.028 0.96 I .44f0'030 I ' O O
I '53&0'023 I ' O O
1 ~ 7 6 ~ 0 ~ 0 2 2 1.00
1.89fo.024 1.00 2 ~ 0 2 ~ 0 ~ 0 1 8 1.00
2 .28+0 .032 1.00
Before Ratio critical per. R/N
Operation
0 ~ 1 4 f o ~ o 1 1 0.93
~~
0 . 3 0 ~ 0 ' 0 2 9 0 . 8 5
0.59&0'015 1'00 0'75&0'0IZ 1'03 0 ~ 8 0 f o ~ 0 1 7 1.00 0 ~ 9 1 ~ 0 ~ 0 2 0 1.00
I ~ O I * O ~ O I I 1'00
1 . 1 6 ~ 0 . 0 2 9 1.00
0'99f0.009 I ' O O
After Rati! critical per. R/N
Operation Papilla 0.39fo.008 0.88 0.58*0.016 0'97
0 ~ 8 0 ~ 0 ~ 0 0 9 1.4 0.75&0.024 1'00
O'92&O0.0I3 I'M) 0.99f0.012 1'00
I ~ O Z + O ~ O I I 1'00 1.17fo.oz1 1.00
* R/N = ratio of regenerated segment to normal uninjured segment.
Table 3. Length (in mm.) i standard deviation of the regenerated metathoracic tarsal s egmen~
Tarsomere I Tarsomere 2
Instar
I I1
I11 IV V d V ?
VI s VI 0
VII AS VIIA
Before critical per.
Operation 0 .36i0 .023 0'51&0'032 0'77&0'032 I '04&0.023 1 ~ 1 3 + 0 ~ 0 z 1 1'3410.035 1 ~ 4 1 f 0 ~ 0 z z 1.68&0.025 1.87fo-022
Ratio R/N*
0.77 0.88 0.99 I '03 1'00 1 '02
1'01
1'02
1'01
After critical per.
Ratio R/N
Operation Papilla 0.46&0.027 0.69&0.052 0.98*0.019 1.09+0.036 1 ~ 3 0 ~ 0 ~ 0 2 0 1.38+0.019 I .67 +o '01 6 1 ~ 8 6 f 0 ~ 0 z z
0.79 0.87 0.98 0.97 1'00
1'00 1'01
1'01
Before critical per.
Operation 0.16*0.042 0 ~ 2 1 + 0 ~ 0 4 1
0 ~ 3 2 f 0 ~ 0 2 1
~~~~ ~ ~
0'44fO'OIO 0'47&0.009 0.5410'013 0.58*0.014 0 ~ 6 0 ~ 0 ~ 0 1 7 0 ~ 7 0 ~ 0 ~ 0 1 8
Ratio R/N
0.88 0.95
1 . 1 5
1.15
1.17 1.14
1.13
1 ' 1 0
1 ' 1 1
After critical per.
Operation Papilla 0.18*0.047 0.28+0.048 0.41 &0.014 0 ~ 4 6 ~ 0 ~ 0 1 2 0'53&0'020 0.55 +0'029 0.62f0.008 0 ~ 7 1 & 0 ~ 0 0 6
Ratio R/N
0.82 0.96
I ' I 5 1 - 1 3
1 . 1 3
1 . 1 4
1 ' 1 1
1'10.
* R/N = ratio of regenerated segment to normal uninjured segment. t Ratio of regenerated tarsomere 3 to normal tarsomere 4 $ Ratio of regenerated tarsomere-4 to normal tarsomere 5.
Relative growth of normal and regenerated legs in Blatella 45' Growth of the uninjured metathoracic leg
The mean length standard deviation of the metathoracic leg segments of both sexes for all developmental stages is shown in Table I.
Measurements showed no sexual differentiation in larval instars I to IV, but differ- ences were apparent in all segments in the later instars, when all measurements of females were distinctly larger than those of similarly aged males.
Growth of the regenerated metathoracic leg
Tables 2 and 3 show the mean lengths of the regenerated metathoracic leg segments and the ratio of the regenerated segments to the normal uninjured leg segments for all
B.gwmanica
mur Tibia
ore ical per.
Ratio R / N
0.85 0.93 1'00 1'00
1'00 1'00
1'00 1'00
1'00
Ratio R/N
Before critical per.
Ratio R / N
After critical per.
Ratio R/N
0.82 0.96 1'00 1'00 1'00 1'00
1'00
1'00
0.80 0.87 0.98 1'00
1'00
1'00 1'00
1'00 1'00
0.77 0.92 0.96 0.94 1'00 1'00 1'00
1'00
3.gerntanica.
rsomere 3 Tarsomere 4
ore After Before Ratio After Ratio ical per. Ratio R/N critical per. Ratio R/N critical per. R/N critical per. R/N
eration Operation Operation Operation 7i0.004 0.58 or 71.05 Papilla
1f0.024 0 3 8 1 . 1 0 0.11fo-042 0.58 1.00 0.38+0.012 1.00 0.39&0.008 1.00
2 ~ 0 ~ 0 0 9 0.48 1.00 o.1z-10.020 0.49 1.00 0'47fO'OIO 1.00 0 ~ 4 6 ~ 0 ~ 0 0 9 1.00 250.028 0.46 1.00 0 .1z io .105 0.46 1.00 O . ~ ~ & O . O I Z IOO 0*48+0.013 1.00
~.
0.21fo.046 $0.91 Papilla 8fo.011 0'53 1'14 0 ~ 0 6 ~ 0 ~ 0 0 7 0.40or to.86 0'27zkO*O43 0.97 O.ZZ&O.Ogg $0'79
3+0'025 0.46 1'00 0 '13 i0 '024 0.45 I ' O O 0.54fO.009 I ' O O O ~ 5 5 ~ o ' o o 6 1.00 4f0.032 0'45 I ' O O 0.14&0*027 0.45 I ' O O 0'59&0'011 I ' O O 0.58&0.026 I ' O O
5 i0 .009 0'44 I ' O O O ~ I ~ f o ~ o 1 6 0.44 I ' O O 0.57&0'011 I ' O O 0.57*0.011 I ' O O 650.013 0.41 1.00 0.16&0.013 0.41 1 - 0 0 0-62~ t0 .015 1.00 0.6250.017 1.00
452 B.Roberts
developmental stages, following removal of the left metathoracic leg before and after the critical period in the first instar.
When the leg was removed before the critical period the coxa, trochanter and femur regained normal length at the third ecdysis, and the tibia1 segment at the fourth ecdysis, after the operation; when removed after the critical period the coxa, trochanter and femur required four stadia and the tibia five to regain normal length after the operation. The dimensions of the uninjured legs in experimental animals were not affected by the presence of a regenerating leg.
The tetramerous condition of the regenerated tarsus was complicated. From Table 3 it is seen that the regenerated third tarsomere approximated to the normal uninjured fourth tarsomere. Consequently it was assumed that the “missing” segment was the equivalent of the normal third tarsomere. When the leg had been removed before the critical period the regenerated first, second and fourth tarsomeres were smaller than the normal uninjured corresponding segments, but regained normal length five, four and four stadia respectively after the operation. The two proximal tarsomeres, especially tarsomere two, became much larger than the normal. In the early stages of development the regenerated third tarsomere was longer than the normal uninjured fourth tarsomere,
Males Females
t i
f
C
t r
I l l I I I i r r 1 1 I I 1 I I I I l l V V V I V l l A I I 1 I l l IV V V I V l l A
l n s t a r D u r a t i o n
Fig. 2. Growth curves of the normal and regenerated major leg segments following removal before the critical period in the first instar. c , coxa; tr, trochanter; f, femur; ti, tibia. - normal leg; . . . . . . . . . regenerated leg.
Relative growth of normal and regenerated legs in Blatella 453 M a l e s Females
t i
f
C
t r
5 .0
4.0
3.0
2.0
1 .o
Log. L e n g t h
(rnms.)
0.5
0.2
/- I l l I I I I
/ I I I I I I 1 . . , . . .
I 1 1 1 1 1 I v V V I V l l A I I I Ill I v v V I V l l A
l n s t a r Durat ion
Fig. 3. Growth curves of the normal and regenerated major leg segments following removal after the critical period in the first instar. c, coxa; tr, trochanter; f, femur; ti, tibia. - normal leg ; . . . . . . . . . regenerated leg.
but at the fourth ecdysis it was of equal length and in the ensuing instars was always the same length. At no stage of development did the regenerated fourth tarsomere exceed the length of the normal uninjured fifth tarsal segment. Removal of the leg after the critical period produced a different situation in the regenerated tarsus (Table 3). Both regenerated distal tarsomeres were of normal length after the third ecdysis. The regener- ated third tarsomere was smaller than the normal uninjured fourth tarsomere and later increased in length to normal proportions. At no stage of further development did the two distal regenerated tarsomeres exceed the length of the uninjured distal tarsomeres. Tarsomeres I and 2 regained the normal length after the fifth and fourth ecdyses. In the ensuing developmental stages increased length, particularly in the regenerated second tarsomere, was recorded.
Growth curves of normal and regenerated metathoracic leg segments
Figures 2, 3, 4 and 5 show the logarithmic length of the normal and regenerated meta- thoracic leg segments plotted against the duration (Murray, 1967) of all larval instars. Growth curves for males and females are given separately but as stated above there were no sexual differences in size over the first four instars.
454 %.Roberts
Males Females
2.0
1.0
0.5
0.1
0.a
0.0
r t I t l
.- - I I I I l l I V V V I V l l A I I I I l l I V V V I VllA
lns tar Durat ion
Fig. 4. Growth curves of the normal and regenerated tarsal segments following removal before the critical period in the first instar. ti-t5, normal tarsomeres I to 5 ; rti-rt4, regenerated tarsomeres I to 4. ___ normal segments ; . . . . . . . . . regenerated segments.
Each major leg segment possessed its own normal growth curve (figs. z and 3). The smallest segment, the trochanter, was characterized by a depressed curve, and all other major segments, especially the tibia, possessed growth curves of a high gradient. The curves following the removal of a leg both before (fig. 2) and after the critical period (fig. 3) were of a markedly steeper gradient until the normal segment length was regained.
The tarsomeres (figs. 4 and 5 ) also showed their own characteristic growth curves. The growth curves of the regenerated tarsomeres were complex, owing to the absence of a segment and the increased length of the two proximal tarsomeres.
The mean daily percentage increment in length during each instar
The mean daily percentage increment per unit of existing length during each instar is shown in Tables 4 and 5 . These values are obtained by taking the differences between mean lengths of segments in a specific instar and those of the previous instar, expressed as a percentage increment, and dividing by a factor based on the average duration for
Relative growth of normal and regenerated legs in Blatella 455 M a l e s Females
2.0
1.0
0.5
0.’
0.1
0.1 -- I I I I l l I V V V I V l l A I I I I l l I V V V I V l l A
l n s t a r D u r a t i o n
Fig. 5. Growth curves of the normal and regenerated tarsal segments following removal after the critical period in the first instar. ti-ts, normal tarsomeres I to 5 ; Z k Z t 4 ,
regenerated tarsomeres I to 4. ~ normal segments ; . . . . . . . . . regenerated segments.
each instar as calculated by Murray (1967). The quotient expresses “average daily growth” based on total changes in size visible at each ecdysis.
In the normal uninjured leg (Table 4, Type A), all major leg segments showed an initial high percentage increase during the first instar, with maximal daily increases in the third instar in males and the third and fourth instars in females. In the later developmental stages the daily increases were considerably smaller, being directly related to the shape of the growth curves (figs. 2 and 3) of the individual segments. The three uninjured proximal tarsomeres (Table 5, Type A) increased their length most markedly during the first and third instars in males and the first, third and fourth instars in females. The two distal tarsomeres showed small daily increases in the first instar and a high daily increase in length during the third instar in both sexes.
Removal of a leg before the critical period (Tables 4 and 5 , Type B), resulted in higher daily increases in all segments of the regenerated leg in nymphal instars in which the regenerated segments were smaller than those of the uninjured segments. The pattern was similar to that of the normal leg, with the highest daily increases in the third instar. The same pattern of daily increase resulted from the removal of a leg after the critical
29
Tab
le 4
. T
he m
ean
daily
per
cent
age
incr
emen
t in
leng
th o
f th
e m
ajor
met
atho
raci
c le
g se
gmen
ts o
f B
.ger
mun
icu.
A, u
nope
rate
d co
ntro
l le
g se
gmen
ts. B
, reg
ener
ated
leg
segm
ents
follo
win
g re
mov
al b
efor
e th
e cr
itica
l pe
riod
in
inst
ar I
. C, r
egen
erat
ed l
eg s
egm
ents
follo
win
g re
mov
al a
fter
the
cri
tical
per
iod
in i
nsta
r I.
Mal
es
Fem
ales
Mea
n da
ily p
erce
ntag
e in
crem
ent i
n le
ngth
dur
ing
each
inst
ar
Mea
n da
ily p
erce
ntag
e in
crem
ent i
n le
ngth
dur
ing
each
inst
ar
Segm
ent
Typ
e I
2
34
56
I
23
45
6
Cox
a A
4'
95
4'03
5-
15
3'35
3'
04
1.38
4'
95
4'03
s'
I.5
4'
33
3.22
1.
89
B 5.
83
6.89
3.
35
3.04
1.
38
5.83
6.
89
4.33
3.
22
1.89
C
8.
53
3.89
3'
04
1.38
8-
53
4.98
3.
22
1.89
T
roch
ante
r A
3.
39
3.95
5'
09
3.82
3'
09
0'92
3'
39
3'95
5'
09
5.0
1
3'23
1.
62
B
5.63
6.
55
3.82
3.
09
0.92
5.
63
6.55
5.
01
3.23
1.
62
C
7'27
4'
13
3.09
0'92
7'27
5'
34
3'23
1.
62
Fem
ur
A
4.24
3.
74
5.16
3.
62
3.46
2.
34
4'24
3.
74
5.16
4.
56
3'52
2'52
B
5.40
6.
67
3.62
3'
46
2.34
5-
40
6.67
4.
56
3.52
2
'52
C
8.
48
4'45
3'
46
2'34
8.
48
5.35
3.
52
2.52
Tib
ia
A
4.56
4'
27
4.86
4.
34
3.92
3.
47
4-56
4'
27
4.86
5.
70
3.64
3'
47
B
5'45
7.
82
4.63
3'
92
3'47
5.
45
7.82
6.
08
3.64
3.
47
C
8.98
4.
82
4-67
3.
47
8.98
6.
75
5.48
3.
47
Tab
le 5
. T
he m
ean
daily
per
cent
age
incr
emen
t in
len
gth
of t
he m
etat
hora
cic
tars
al s
egm
ents
in B
.qw
man
ica.
For
exp
lana
tion
of A
, B
and
C se
e T
able
4.
Mal
es
Fem
ales
Mea
n da
ily p
erce
ntag
e inc
rem
ent i
n le
ngth
dur
ing
each
inst
ar
Mea
n da
ily p
erce
ntag
e inc
rem
ent i
n le
ngth
dur
ing
each
inst
ar
Segm
ent
Typ
e I
23
45
61
2 3
45
6
Tar
som
ere
I A
5'
15
4'35
4.
81
4'15
3'
93
2'59
5'
15
4'35
4.
81
6-14
3'
30
3'25
B
6.
40
7'59
4'
95
3'94
2.
40
6.40
7
59
6.
58
3.38
3.
07
C
7.46
5.
91
4-46
2.28
7.46
8.
15
3.64
3.
28
Tar
som
erez
A
4.
68
4.28
4.
47
3-75
3.
25
1.60
4.
68
4.28
4.
47
5.15
3-
34
2.04
B
4.
80
7.82
5.
28
3-11
1-
05
4.80
7.
82
6.59
3.
20
1.95
C
8.28
6.53
3.
67
1.59
8.
28
9'04
2.
68
2'74
T
arso
mer
e 3
A
4.05
3.
78
3.89
3.
76
1-74
1-
86
4.05
3.
78
3.89
5
.00
1.
67
2.89
T
arso
mer
e4
A
2.24
3.
71
6.42
2
.05
0.
93
1.72
2.
24
3.71
6.
42
2.59
1
-1
2
1.2
8
c.f.
B
2.2
0
5.60
2.
05
0.93
1.
72
2.2
0
5.60
2-
59
1.1
2
1.28
Tar
som
ere
3 T
arso
mer
es
A
2.34
2.
61
5.86
2.
89
2.04
0
.52
2.
34
2.61
5.
86
3-61
2
.52
0.
65
c.f.
B
4.40
6.
07
2.89
2.
04
0.5
2
4.40
6.
07
3.61
2
.52
0.
65
rege
nera
ted
C
11-5
3 2.
89
2'04
0
'52
11
.53
3.61
2
.52
0.
65
Tar
som
ere
4
rege
nera
ted
C
12'4
3 2
.05
0.
93
1.72
12
.43
2.59
1
'12
1-
28
a 3 E
R a R
Tab
le 6.
Gro
wth
rat
ios
of n
orm
al a
nd r
egen
erat
ed m
etat
hora
cic
leg
segm
ents
of
B.g
errn
anic
a. A
, leg
seg
men
ts o
f un
oper
ated
con
trol
s.
B, l
eg s
egm
ents
fol
low
ing
rem
oval
bef
ore
the
criti
cal
peri
od i
n in
star
I.
C, r
egen
erat
ed l
eg s
egm
ents
fol
low
ing
rem
oval
aft
er t
he
criti
cal
peri
od i
n in
star
I.
Mal
es
Fem
ales
S
egm
ent
rt3
rt
4
Seg
men
t rt
3 rt
4 E
cdys
is
Typ
e c
tr
f ti
t1
tz
tg
t4
t5
c
tr
f ti
tl
t2
t3
t4
t5
1.14
1.
17
I '2
9
1'39
I
'41
"77
I .2
6 I
.26
I .2
6 1.
18
1.1
8
1.1
8
I '0
7 I
'07
I '0
7
c, c
oxa;
tr,
tro
chan
ter;
f, f
emur
; ti
, ti
bia;
tl,
tz, t
3, t4
, t5
, tar
som
eres
1-5
; rt
3,
rt4
, re
gene
rate
d ta
rsom
eres
3 a
nd 4
Relative growth of normal and regenerated legs in Blatella 459
period (Tables 4 and 5, Type C). The third instar showed an extremely high daily increase superimposed on the normally high level of the uninjured leg, this being most obvious in the two regenerated distal tarsomeres.
Growth ratios for all phases of development
Growth ratios for every segment of the metathoracic leg were calculated from the mean values of specific segments of a known instar divided by the means of the same segment of the previous instar. The results are shown in Table 6.
In the normal uninjured metathoracic leg (Table 6, Type A), excluding subimaginal development, the coxa, trochanter, femur, tibia and first and second tarsomeres were found to have growth ratios of the same general order, but those of tibia1 and tarsal segments were highest. Ratios for the third tarsal segment were of slightly smaller value. The minimal growth ratios of the entire leg occurred in the fourth tarsomere, except at the second and third ecdyses when high specific growth ratios for the segment were recorded. Usually a second but less obvious high ratio was observed for the fifth tarsomere. At the final ecdysis the highest growth ratios were found in the tibia, first tarsomere and the femur, and the smallest ratios in the trochanter and fifth tarsomere. All other segments presented intermediate values.
Growth ratios for the nymphal phases of development of the regenerated leg follow- ing operation before the critical period (Table 6, Type B) showed the same trends des- cribed for the normal uninjured leg. The ratios of all segments were higher until the normal length had been regained. The high growth ratios of the two proximal tarsomeres, the most distal fourth tarsomere and the minimal ratios of the regenerated third tar- somere were more pronounced. Growth ratios of the regenerated leg at the final ecdysis were similar to those described for the normal leg.
When a leg was removed after the critical period (Table 6, Type C), the growth ratios for the third.nympha1 ecdysis showed that the highest ratios were in the two distal tarsomeres. All other leg segments presented ratios of the same general order. At the later nymphal ecdyses and the final ecdysis the pattern was the same as that of the uninjured leg.
I V D I S C U S S I O N
In the present experiments the timing of the removal of the left metathoracic leg was specifically chosen, in light of the findings of O’Farrell & Stock (1953, 1958), so as to avoid altering the duration of the first or later instars.
The linear measurements of the metathoracic leg segments provide a means of recognising the different instars of B.germanica, if sexual differences are taken into account. These became obvious only at and after the fourth ecdysis and may possibly be due to a hormonal effect on growth related to development of the gonads (Wiggles- worth, 1964). Roth & Willis (1954) have shown that in B.gernzanica the testes produce spermatozoa as early as the second instar but the ovaries are dormant until the fourth instar. Perhaps the greater length of the leg in females is related to ovarian development.
Without exception, the regenerated segments equalled and often exceeded the normal size by the time of metamorphosis. The most rapid increase in length was in the early stages of regrowth, most noticeable with operations performed after the critical
460 B. Roberts
period, where the onset of regeneration was postponed until after the subsequent moult. In this situation the blastema does not differentiate until the following instar (O’Farrell and Stock, 1958) and then within a substantially larger coxal stump.
Growth ratio calculations based on comparison of one instar with another do not correctly depict the relative changes in size involved in normal growth and regeneration, as the time factor is largely ignored (Richards, 1949). Woodruff & Seamans (1939) concluded that growth ratios of regenerated legs are somewhat inaccurate, since the initial measurement is usually larger than for the normal leg; this is especially true of post-critical period operations in B.germanica, when the appearance of the regenerate is postponed by one stadium. The growth ratios (Table 6) underemphasise the early rapid growth and exaggerate the constantly decreasing growth rate characteristic of normal tissues. Tables 4 and 5 give the potential mean daily increment in length for each nymphal instar and, as time is now a considered factor, the high initial and later diminishing growth rates are shown in truer perspective. These calculations ignore the fact that most, if not all, of the growth occurs only after the critical period when the moulting hormone is released. The early stages in regeneration then represent an enor- mous acceleration of growth by comparison with the normal uninjured leg for the same period of time.
The data for the regenerated leg, removed either before or after the critical period, indicate the presence of two growth gradients : a proximo-distal growth gradient affecting the growth of the leg proximal to the second tarsal segment, and a disto-proximal growth gradient in the two most distal tarsomeres. From growth ratio calculations (Table 6) these growth gradients do not appear to be present in the normal development of the uninjured leg. In regeneration following the removal of a leg before the critical period, the proximo-distal growth gradient was dominant in the initial phase of regrowth, as the more proximal segments were the first to regain the normal length. The disto- proximal growth gradient was less conspicuous in the initial phase of regeneration, but at the two subsequent moults became as evident as the proximo-distal gradient. Where regeneration followed operations performed after the critical period, the disto-proximal growth gradient was initially more conspicuous, as the two distal tarsomeres required only one ecdysis to regain the normal length after the delayed appearance of the appen- dage. However, the proximo-distal growth gradient still produced a growth pattern in the proximal portion of the leg much the same as in a leg regenerating after removal before the critical period. The actual rates of growth were similar in both types of regen- eration, the patterns being altered only by the differing effects of the two opposing growth gradients, which may be related to the findings of O’Farrell & Stock (1958). They reported that the blastema differentiates proximo-distally following operations before the critical period and disto-proximally following the removal of a leg after the critical period. In the former, the blastema differentiates in the instar in which the leg was removed, under the influence of a modified hormonal situation produced by the opera- tion (O’Farrell et al., 1959), whereas in the latter case the onset of differentiation is delayed until the commencement of a new, probably unmodified, moulting cycle.
The high degree of accuracy in regaining the normal length is shown in Tables I ,
2 and 3. At metamorphosis only tarsomeres one and two exceed the normal length, presumably growth compensating for the lack of a tarsal segment. The meeting of the proximo-distal and disto-proximal growth gradients within the tarsus may be respon- sible for the absence of the tarsal segment in the regenerated leg.
Relative growth of normal and regenerated legs in Blatella 461
V S U M M A R Y
The linear length of uninjured metathoracic leg segments provide a means of recognising the instars of Blattella germanica (L.), if sexual differences are taken into account at and after the fourth larval ecdysis. The process of regeneration following the removal of a leg before or after the critical period in the first instar does not appear to influence the normal growth rate of the uninjured leg.
Without exception the regenerated segments equalled and often exceeded the normal size by the time of metamorphosis, the most rapid increase in length being the early stages of regrowth. The growth of the regenerated leg is controlled by a proximo-distal growth gradient present in segments proximal to the second tarsomere and a disto- proximal growth gradient affecting growth only in the two most distal tarsomeres. The proximo-distal growth gradient is dominant in the initial phase of regrowth of a leg removed before the critical period and the disto-proximal growth gradient is initially more conspicuous in regeneration of a leg removed after the critical period.
I t is suggested that the meeting of the two growth gradients may be responsible for the absence in the regenerated leg of one tarsal segment, which is assumed to be the equivalent of the normal third tarsomere.
The suggestions and assistance of Professor A.F.O’Farrel1 and Professor A.Stock regarding the preparation of this manuscript are gratefully acknowledged.
REFERENCES
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(L.). I. Single regeneration initiated during the first instar. Aust. J. biol. Sci. 6 : 485-500. O’FARRELL A.F. & STOCK A. 1958. Some aspects of regeneration in cockroaches. Proc. X Int.
Cong. Ent. (Montreal) 2 : 253-259. O’FARRELL A.F., STOCK A. & MORGAN J.A. 1956. Regeneration and the moulting cycle in Blattella
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Manuscript received 29th February I 972
