J. Embryol. exp. Morph. Vol. 32, 2, pp. 533-555, 1974
Printed in Great Britain
533
The relation between larval and
adult abnormalities in the abdominal segmentation
of Calliphora erythrocephala (Diptera)
ByM. J. P E A R S O N 1
From Department of Zoology Cambridge
SUMMARY
Early embryonic stages of Calliphora erythrocephala were X-irradiated to produce abdominal abnormalities of segmental pattern. Abnormal patterns were examined in the
puparial stage: 75 (Series AA) larvae with dorsal or lateral abnormality were classified and
the larval abnormality compared with the pattern of abdominal segmentation in the emerging
adult fly; another 75 (Series aa) larvae were examined in dorsal, lateral and ventral aspect
with respect to segmental abnormality and again in relation to abnormality in the adult.
It was observed (i) that the pattern of abdominal intersegmental membranes is not
necessarily maintained through metamorphosis and may regulate to normal; (ii) that tergal
abnormalities in the adult result from a failure of correct bilateral fusion of corresponding
contralateral hemitergites; (iii) that reduction or displacement of scleral structures in an
adult abdominal segment is correlated with deficiency or displacement of epidermal muscle
insertions, and hence presumably with the histoblast anlagen which are normally associated
with them. In the case of dorsal abnormalities, hemitergite differentiation is normal.
Larval abdominal abnormality patterns point to the mode of bilateral morphogenesis
of the germbands in embryogenesis. The relation of adult to larval abnormalities suggests
some similar independence of contralateral hemisegments at metamorphosis.
The possible developmental origin of abdominal abnormalities produced by X-irradiation
of embryos is discussed, and the conclusions of the analysis of the relation between larval
and adult abnormalities in Calliphora are set against the propositions of previous students
of such abnormalities in Drosophila.
INTRODUCTION
It was noticed that in normal laboratory cultures of Calliphora erythrocephala
occasional larvae display an abnormal pattern of segmentation. Similar abnormalities have been described in Calliphora by Fraenkel & Harrison (1938),
and in adult Drosophila (see Sobels, 1952). Fraenkel & Harrison (1938) also
found by breeding experiments that the abdominal irregularity in their stock
of Calliphora was hereditary, and probably involved several genes of low
penetration. However, it is also possible to produce phenotypically identical
abnormalities in Drosophila by heat shock (Maas, 1948) or X-irradiation
(Ulrich, 1951) during sensitive periods of embryonic development, or at the
end of the third larval instar.
1
Author's address: Biology Building, University of Sussex, Falmer, Brighton BN19QG,
U.K.
534
M. J. PEARSON
tli
abd
Fig. 1. Third instar acephalous maggot larva of Calliphora erythrocephala. The
head segment with tanned mouthparts (m) is retractable, followed by 3 thoracic
segments (th) and 8 abdominal segments (abd).
Dorsal
Ventral
V* %J
A
B
Fig. 2. Correspondence between abdominal segmentation in (A) larval and (B)
adult female Calliphora erythrocephala. In the adult preabdomen, tergites 1 and
2 are fused; 3, 4 and 5 correspond with the larval segments from whose anlagen
they derive. In the female postabdomen, 6 and 7 dorsal tergal structures similarly
correspond with larval segmentation. Separate sternal structures of abdominal
segments 1 to 7 are also evident. In the adult male, post-abdominal morphology
differs in its relation of scleral elements to segmentation.
The gross pattern of the acephalous 'maggot' larva of higher Diptera is
shown in Fig. 1, and the correspondence between abdominal segmentation in
larva and adult (Crampton, 1942) in Fig. 2. The larval abdomen comprises
seven segments with histoblast anlagen followed by a terminal eighth segment
which carries the genital discs. The replacement of larval epidermal cells in
abdominal segments by the invasive migration of histoblasts at metamorphosis
has been described by Bautz (1971) and Pearson (1972), but it is not clear
whether the corresponding adult epidermal segment is defined by either (i) the
pattern of larval intersegmental membranes; (ii) the larval pattern of histo-
Abnormalities
in segmentation
of Calliphora
535
blast anlagen ; and/or (iii) some underlying property of the organism whether
larval or adult. This report describes the correlation between the bilateral
abnormalities in larval and adult Calliphora after X-irradiation during a
sensitive period in early embryogenesis, and is aimed toward new insight
into the bilateral and segmental morphogenesis of Diptera.
MATERIALS AND METHODS
Segmental abnormalities (abnormal abdomen) occurred in normal cultures
with a frequency of about 0-5 %. Temperature shocks of \ h at 38-40 °C were
ineffectual on embryos older than 2 h, and from 0 to 2 h killed up to 90 %
embryos while producing abnormalities in 10-20 % of survivors. X-irradiation
during this early period, however, did produce a higher percentage of abnormalities. Embryos of \-\\ h were irradiated at a dose rate of 500 rad/minute
(220 kV at 15 mA, 1 mm aluminium filter, distance of 5 mm) and a final dose
of 60-70 rad, with the following results :
Embryos died
40-60 %
Larvae normal
30-50 %
Larvae abnormally segmented 10-15 %
Doses above 100 rad were found to be invariably lethal at this early stage.
Larvae from irradiated eggs were cultured until pupariation. Once immobile,
and with the larval segmentation evident on the puparium (Fig. 18), it is an
easy matter to identify abnormal larvae and to compare the abnormality with
any corresponding abnormality in the subsequently emerging adult fly.
RESULTS
(1) Mortality among abnormally segmented animals
After X-irradiation 40-60 % eggs fail to hatch. Many of these appear opaque
white and show no evident sign of embryonic development. Another percentage
of eggs undergo embryogenesis and show, through the chorion, the differentiation
of segments which is evident in normal embryos only 1-2 h before hatching.
However, the pattern of segmentation is often grossly abnormal, and such
embryos fail to hatch. Death occurs at or shortly before hatching. Of the
150 puparia examined, a further 20 % died at the pupal stage, and a few
more died as pharate adults.
(2) The types of larval abnormality
In a first study 78 animals (series AA) were examined in dorsal and lateral
aspect. There were few ventral abnormalities. In a second (series aa) group
of animals both dorsal, lateral and ventral abnormalities were considered. A
total (AA + aa) of 150 abnormal larvae were examined during the prepupal
period.
536
M. J. P E A R S O N
Lateral aspect
Normal
Dorsal aspect
Class I
LI
Class
Class III
L2
L3
Class IV
ntermediate
^C
LR
Fig. 3
Fig. 4
Fig. 3. The classes of dorsal abnormality in the abdominal segmentation of
Calliphora larvae after X-irradiation during early embryogenesis.
Fig. 4. Classes of lateral abnormality of larval abdominal segmentation. Both
lateral and dorsal aspect of each class are shown diagrammatically. Only one
L3 abnormality was found, in a multiply abnormal larva.
(a) Dorsal abnormalities can be recognized as three main types (Fig. 3). It
is possible to relate all classes of segmental abnormality to the normal pattern
and to each other by referring the segmental pattern to the hemisegments of
each side; that is, the intersegmental membranes may be considered as bilaterally matched boundaries between co-lateral hemisegments, morphogenetically independent either side of the larva. In the abnormal case, a
hemisegmental boundary fails properly to join in the median line with the corresponding contralateral boundary. The first type is a median break, failure of
corresponding boundaries to fuse mid-dorsally. The second irregularity results
from a fusion of a hemisegmental boundary (x) with the non-corresponding
contralateral boundary (x +1), leaving two open hemisegmental membranes.
Abnormalities
in segmentation
of Calliphora
537
The third type is a mid-dorsal convergence of co-lateral hemisegmental
boundaries.
These aberrations in bilateral correspondence of contralateral membranes
give four common classes of segmentation pattern (Fig. 3), recognizable in
the pattern of intersegmental cuticular spines as well as the line of insertion
of intersegmental muscles visible on the puparium. Class I larvae, corresponding to Fraenkel & Harrison's (1938) 'broken' type, show dorsal breaks;
class II shows a single intersegmental spiral ; while class III - the ' spiral '
class of Fraenkel & Harrison, shows repetition of the same abnormality giving
rise to local spiral segmentation. Fraenkel & Harrison (1938) also describe
an 'irregular class' where hemisegmental misalignment ran across several
segments; this was observed only in one larva which died at metamorphosis.
The fourth class (IV) presents the appearance of a mid-dorsal cross where
consecutive hemisegmental membranes of both sides converge. Occasional
larvae of classes II and III show a difference bilaterally in type of abnormality
(Fig. 3), which serves to emphasize the hemisegmental basis of segmentation ;
and larvae carrying multiple abnormalities are of particular interest.
(b) Segmental abnormalities in ventral aspect are less frequent than dorsal.
No larvae were observed abnormal in ventral aspect which were not abnormal
dorsally; of aa puparia screened for such ventral abnormalities the following
types were apparent; one with ventral cruciform pattern (class IV); three
class I ventral breaks; and five similar class I breaks associated with ventrolateral membrane reduction (LR). Three further animals are ventrally abnormal due to lateral fusion or obliteration of hemisegments. These animals
involving lateral abnormality are discussed in that context.
About 10 % dorsally abnormal larvae thus show ventral abnormalities which
are all simple failures of normal intersegmental membrane formation and
never of class II or III contralateral non-correspondence.
(c) Lateral abnormalities are of 4 types (Fig. 4): a break in the hemisegmental membrane (LI); an obliteration of a hemisegmental region by the
fusion over some length of co-lateral membranes (L2) ; or a reduction (LR - or
in the extreme case L3) of a hemisegmental membrane dorsally or ventrally
which necessarily incurs a dorsal or ventral break.
(3) Segmental abnormalities after metamorphosis
Maas (1948) first showed the relation between larval and adult patterns of
segmentation in Drosophila after larval abnormalities had been induced by
heat shock during the embryonic period. It was found that the segmental
abnormalities induced by X-irradiation during early embryogenesis of Calliphora may also be expressed in the segmentation of the adult, but that in
many cases regulation occurs at metamorphosis.
The types of dorsal adult abnormality have been loosely classed A, B, C
(Fig. 5) and D. The more extensive classification of Löbbecke (1958), who
538
M. J. PEARSON
Class A
g
I =sa
Class B
Tfl
!
Class C
(CI)
(C2)
-
!
-
Fig. 5. Classes of dorsal abdominal segmental abnormality in adult flies which
develop from abnormally segmented larvae. A, Medio-dorsal fusion between
adjacent tergites; B, fusion of one or both hemitergites with tergite (or hemitergite)
from adjacent level, but not with its contralateral hemitergite; C, partial or total
failure of contralateral fusion.
analysed morphologically the patterns of abnormal abdomina in adult Drosophila, encompasses these types, but a more detailed descriptive classification
than that proposed here may simply obscure the similarity underlying A, B,
C and the dorsal abnormalities of the larvae. It is, however, not acceptable
to use the same categories as were employed to describe larval abnormalities,
since in that case the morphological evidence was the pattern of intersegmental
boundaries. In the adult, the intersegmental membranes are not morphologically evident except by contrast with the scleral plates - in the dorsal case,
the hemitergites - and it is these segmental structures which provide the basis
of descriptive classification.
Class A abnormalities show fusion of consecutive tergites in the dorsal
midline.
Class B abnormalities result from the fusion of a hemitergite with the tergite
Abnormalities in segmentation of Calliphora
539
Table 1. The relation between larval and adult abdominal abnormalities
Larvae with thoracic or thoracic/abdominal segmental abnormalities are not
included in Table 1. All gave normal adults.
Adults
Abnormal larvae
Dorsal
Class
Total
A
i
Normal
A
B
CI
C2
28
12
—
8
2
—
2
5
—
2
—
—
3
1
—
5
7
1
1
4
1
—
(+2
4 double B)
—
2
—
2
11
4
I
48
II
27
'Irregular'
1
II over 2 segments
III
19
IV
12
Lateral
Total
LI
L2
6
8 (1 in
multiple class)
3 (2 in
multiple class)
10
L3
LR
Died
at
metamorphosis
Normal
hemisclerite
Reduced/missing
hemisclerite
Died
(See Table 2 for analysis of aa LR abnormalities)
Ventral
Break (I)
Total
Normal
Abnormal sternite
3
—
3
(3 other ventral breaks associated with LR abnormality. See Table 2)
Cruciform
1
—
—
(IV)
Multiple
Total
Normal
Abnormal
22
3
11-see
text and figures
Died
—
1
Died
8
or contralateral hemitergite of adjacent level, but not with its opposite (contralateral) hemitergite.
Class C abnormalities show incomplete (CI) or no fusion (C2) of contralateral hemitergites.
Class D refers to the reduction or absence of a hemitergite.
(4) The relation between larval and adult segmental abnormality
A summary of adult abnormalities derived from abdominal larvae is given
in Table 1. These data refer to those animals whose irregularities occur in the
preabdomen, including the 5/6 boundary. In a few cases, not included in
Table 1, segmental abnormalities involve (i) thoracic intersegmental boundaries,
540
M. J. P E A R S O N
(h)
(«)
un
(c)
Incomplete fusion
of hemitergites 3
Tergites
Abnormal
larval boundary
Larva
aa71
aal 9
AA43
Fig. 6. Abnormal segmentation in larval and adult Calliphora erythrocephala.
(a) larval class I abnormalities give rise to (b) class A adults; but also (c) class B
and (d) class C adults. 55 % class I larvae gave normally segmented adults.
(«)
0»
U)
Incomplete fusion
in tergite 4; joins
tergite 5,
Abnormal
larval boundaries
2/3/4
3/4/5
Larva
A A31
AA26
<</)
3/4/5
aa53
Fig. 7. Adult abnormalities deriving from (a) larval class II abnormalities. (6) Adult
class A, (c) class A, (d) class B. 43 % cases regulated to normal.
and (ii) the thoracic/abdominal 1 boundary. The latter case is of interest because
it brings into question the nature of the thoracic/abdominal boundary which,
while not obviously different from intra-abdominal boundaries in the larva, is
profoundly important during metamorphosis and separates in the adult form
two tagmata derived from developmentally different stocks of imaginai cells.
These larvae fell into classes I and II, and in all cases, the adult showed no
segmental abnormality.
(i) Larval and segmental abnormalities do not necessarily correspond
Class I larval abnormalities correspond to the adult class A, and more than
50 % of abnormal flies from class I larvae in fact show class A abnormalities.
However, class B^and C flies also derive from class I (Fig. 6).
Abnormalities in segmentation of Calliphora
(a)
(b)
(c)
(c)
Hemitergite L3
reduced width
Tergites
,_
aal 8
AA15
541
id)
Incomplete
fusion
aa35
Fig. 8. Adult abnormalities deriving from (a) class III larval abnormalities, (b) In
AA15 the adult class B abnormality is confined to segments 4 and 5. (c) In aal8,
an adult class B abnormality over segments 2 and 3 derives from a larval class I
break (2/3); whereas a class III larval abnormality from 4/5 to 7/8 leaves in the
adult a C2 partial failure of hemitergite fusion in segment 5 only, (d) aa35 shows
in the adult a double type of class B abnormality resulting from a 2/3 to 5/6 larval
class III.
(«)
(0
(h)
Hemitergites
5 do not fuse
Larval abnormal
boundaries
.4/5/6
AA56
4/5/6
Fusion of
hemitergites 5
does not cover
full length of
segment 5
AA65
Fig. 9. Adult abnormalities deriving from (a) larval class IV abnormalities. 50 %
of these surviving larval abnormalities gave normal adults, and 50 % class C
(CI e.g. AA56, and C2 e.g. AA65) adults.
In adult derivatives of class II it is apparent that a segment which is open
in the larva may be normal in the adult (Fig. 7). In AA31 (Fig. 7) boundaries
2/3 and 3/4 are irregular; whereas the adult shows a defect in the dorsal
midline of boundary 3/4 only. AA26 shows a similar regulation of the anterior
of 2 defective consecutive boundaries. In aa53 however, it is the posterior
boundary which is corrected to give a class B abnormality.
Class B adult flies are also the predominant type of abnormal derivatives
542
M. J. P E A R S O N
(«)
Adult,
(ventral)
aa03
Displaced
hemistemite
3'
Irregular
hemistemite
4
(h)
aalO
Fusion
of
hemistemite
3R with
stemite 4
(c)
aal 2
Fig. 10. Ventral abnormalities among aa stock (see Results). A ventral break
(of the type seen in class I dorsal abnormalities) in the larva leads to a failure of
hemistemite fusion in the mid-ventral line in the adult. There may be (aa03, aal2)
a fusion with an adjacent sternite.
of class III larvae (Fig. 8), where a greater (AA15) or lesser degree of intersegmental regulation is found, both of posterior and/or anterior irregular
boundaries.
Class IV larvae correspond to C adults, and all abnormalfliesfrom class IV
larvae show C abnormality (Fig. 9).
(ii) Regulation at metamorphosis of abnormal larval segmentation
In addition to the examples discussed above where correction of one or
more abnormal larval intersegmental boundary has occurred at metamorphosis,
about 50 % of adult survivors were perfectly normally segmented flies. It is
clear from Table 1 that the frequency of total regulation is greater where the
larval irregularity is less extensive-is higher, for example, in class I than
Abnormalities
in segmentation
of Calliphora
543
—^7,
Intersegmental •.;/''
cuticular
'•'•,
spines
Histoblast
anlagen
Dorso-ventral
muscles.
Dorsal midline
I
"^:
Hi'
(til
I
Fig. 11. The position of abdominal histoblast anlagen, with respect to intersegmental
membranes and insertions of the bilateral dorso-ventral segmental muscles, in a
class II abnormal larva. Whole mount. D, dorsal; PD, postero-dorsal; V, ventral.
class III - among the dorsal classes. Of class LI larvae, 100 % gave normal
adults, whereas L2 abnormalities (the obliteration of a lateral region of a
segment by fusion of co-lateral membranes) do not regulate.
(iii) Ventral
A single larva with class IV cruciform ventral abnormality died in metamorphosis.
Abnormal larvae with a mid-ventral class I break in the intersegmental
membrane - either accompanied or not by a lateral reduction (LR) of a hemisegmental membrane - invariably showed sternal irregularity in the adult.
There was no evidence, in this small sample, of ventral regulation. In adult
flies aa03 (Fig. 10), aal3, aa24 and aa38 the hemisternites remain separated
by an untanned ventral cuticle and/or displaced; in aalO and aal2 there is
diagonal fusion of hemisternites between the two adjacent segments (Fig. 10).
There is no correlation between this intersegmental fusion and the presence/
absence of LR membrane reduction associated with the larval break.
(5) The histoblast anlagen and lateral abnormalities
It is possible that tergal abnormalities could result from a disturbed organization of the histoblast anlagen in abnormal larvae (Lübbecke, 1958). Three
larvae of both classes I and II were dissected at 72 h, and Feulgen whole
35
E M B 32
544
M. J. PEARSON
Table 2. The relation between abnormalities of dorsal and ventral larval muscle
insertions and adult scleral abnormalities in abdominal segments of aa Calliphora erythrocephala
The relation of histoblast anlagen to normal insertions is shown in Fig. 11.
(1) Muscle insertion missing
aa larva (segment)
1
D
PD
V
Lateral
segmental
abnormality
aaOl (2)
—
—
L2(l/3)
aa25(4)
—
—
L2(3/5)
—
LR (3/4)
aa42 (4)
aa52 (3)
—
—
L2(2/4)
—
—
aa50 (4)
aa27 (2)
L2 0/3)
LR (3/4)
Adult
scleral abnormality
Small scleral plate latero-ventrally;
no dorsal hemitergite 2
Small scleral plate latero-ventrally;
no dorsal hemitergite 4
Small scleral plate latero-ventrally ;
no dorsal hemitergite 4
Small scleral plate latero-ventrally;
no dorsal hemitergite 3
No hemisternite 2. Tergite normal
Hemitergite reduced dorsally with
abnormal macrochaete pattern
(2) Muscle insertions abnormal
Displaced insertion aa larva
D
PD
V
Lateral
segmental
abnormality
—
aa03 (3)
LR (3/4)
—
—
aa38 (3)
aa52 (4)
LR (3/4)
L2 (2/4)
Adult
scleral abnormality
Laterally displaced hemisternite 3,
also abnormal sternite 4
Laterally displaced hemisternite 3
Laterally displaced hemisternite 4
(3) Normal insertions associated with LR abnormality
LR abnormality in larva
1
Dorsal
Ventral
aa05
aa31
aa34
aal 2
Adult abnormality
Class BÏ
Class A f hemitergites normal
Class BJ
fusion of hemisternites between
adjacent segments
D, dorsal; PD, postero-dorsal; V, ventral.
Abnormalities
in segmentation
of Calliphora
545
Fig. 12. In aa52 and L2 lateral abnormality in the larva (a) has resulted in obliteration
of the dorsal left hemisegment 3. (b) The ventral muscle insertions (x ) are normal,
but the antero-dorsal insertion with which the dorsal histoblast is normally
associated, is absent, (c) In the adult only a small scleral structure (sc) lateral
to sternite 3 represents the ventro-lateral margin of the hemitergite. In segment 4
the left hemitergite is displaced in correspondence with the displacement of the
anläge in the larva.
Fig. 13. aa27 shows a correlation between lack of muscle insertion - with which
is normally associated the ventral anläge - in right hemisegment 2 and missing
hemisternite in the adult ( x ).
mounts prepared. In each case, the histoblast anlagen were histologically
normal, with a normal population, and situated in normal respect to the
dorso-ventral muscle insertions (Fig. 11).
It is further possible, however, that lateral abnormalities - including those
among the multiple abnormal class - where reduced or absent tergal or sternal
structures result at metamorphosis, might involve histoblast anläge defects.
Table 2 sets out the correlation between the type of irregularity of the relevant
muscle insertion associated with the position of the histoblast anlagen in
larvae with lateral abnormalities, and the type of adult abnormality. There
is no direct histological evidence to show conclusively that anlagen are absent
when the muscle insertion is missing. However, Table 2 shows that where a
dorsal insertion is missing, although no hemitergite is properly formed in the
adult, a small scleral structure corresponding to the ventro-lateral tergite
margin is present in each case (Fig. 12). Since extirpation and transplant
experiments indicate that this marginal tergite region is formed either by ventral
histoblast derivatives (Emmert, 1972), or by the spiracle anläge (Bhaskaran,
1973), this result strongly suggests specific dorsal histoblast anläge deficiency
35-2
546
M. J. PEARSON
aa45
aa59
aa69
Fig. 14. Multiple abnormalities in the segmental pattern which, in each case, regulated
at metamorphosis to give a normal adult.
in these larvae. Similarly, where a ventral insertion is missing the corresponding
hemisternite is missing in the adult (Fig. 13).
Fig. 12 also shows how a muscle insertion - and by inference, the associated
anläge - may be shifted by an intersegmental abnormality. In LR cases where
muscle insertions are morphologically normal in spite of lateral membrane
reduction, larvae behave at metamorphosis as class I dorsal breaks and produce
types A and B adult abdomina (Fig. 11).
DISCUSSION
In adult Calliphora, a preabdomen of five segments is morphologically
distinct from a postabdomen (Fig. 2) derived from the histoblasts of segments
6 and 7 and the genital discs (Emmert, 1972). The segmental character of the
postabdomen - as distinct from the segmental disposition of the anlagen in
the larva - is not clear, but preabdominal segments with their separate scleral
structures articulating by untanned intersegmental membranes, on both the
evidence of comparative morphology (Crampton, 1942) and the results of
operational experiments (Emmert, 1972), correspond with the first five abdominal segments of the larva.
Where adult abnormalities do not correspond precisely with abnormalities
in the larva, we may look for any consistency in alterations or regulation of the
abnormality which might point to factors involved in setting up the adult
segments at metamorphosis. Maas (1948) attempted to show that the 'anterior
margin ' of the larval segmental border solely constituted the segmental pattern
which, at metamorphosis, directs the organization of histoblasts and their
differentiation to give a normal tergite. Maas implies that the segment behaves
as a field under the influence of an organizer, the anterior margin of its
posterior intersegmental membrane, whose role in the whole pattern of abdominal segmentation remains constant through metamorphosis. Several
abnormal animals discussed above demonstrate that (a) an abnormal intersegmental membrane in the larva may be rectified at metamorphosis (e.g. see
Abnormalities in segmentation of Calliphora
547
AA34
— LR and
reduced
hemisegment'
3
AA35
LR and
reduced
hemisegment
3
Reduced hemitergite
3R
Failure of
dorsal fusion
Slightly reduced
hemitergite 3
AA45
aa67
Fig. 15. Multiple abnormalities. Although patterns of tergal differentiation in the
adult do not correspond precisely with the larval irregularities, in terms of the
failure of contralateral interhemisegmental boundaries to fuse normally, the same
points along the mid-dorsal axis produce segmental abnormalities in both larva
and adult. In aa67, abnormal hemitergites 2 and 3 derive from highly abnormal
anläge and boundary patterns in the larva.
Fig. 14); and (b) that a larval segment whose posterior boundary is abnormal
may differentiate a normal, whole adult tergite. For example, AA34 (Fig. 15)
is an abnormal larva with double class I breaks in intersegmental membranes
2/3 and 3/4. In the adult, tergite 2 is normal, whereas contralateral hemitergites of segments 3 and 4 are misaligned (Fig. 15). Axiom 1: the pattern of
abdominal intersegmental membranes is not necessarily maintained through metamorphosis.
548
M. J. PEARSON
The normal adult tergite is formed by epidermal cells derived from the
histoblast anlagen of each hemisegment (Emmert, 1972; Pearson, 1972;
Bhaskaran, 1973). If the anlagen of one hemisegment are extirpated, the
contralateral hemitergite is normal, and the pattern of hairs differentiates in
the pharate stage, reaching clearly to the dorsal midline. In a normal fully
tanned tergite the midline of fusion of the hemitergites is apparent. In all but
one instance of simple dorsal abnormality (Fig. 8 c), hemitergite differentiation
is normal except in the region of medio-dorsal fusion; the pattern of microchaetes and the posterior row of macrochaetes is normal. Axiom 2: the observed
tergal abnormalities result from a failure of correct bilateral fusion of corresponding contralateral hemitergites.
The dorsal abdominal abnormalities thus reflect in some way the bilateral
morphogenesis of the insect segment. It is not the precise pattern of the dorsal
irregularities (the class of abnormality) which is carried through metamorphosis,
but an underlying instability in bilateral correspondence present during both
larval and adult morphogenesis. In Drosophila, Santamaria & Garcia-Bellido
(1972) produced abnormalities in the organization of adult tergites, corresponding to the classes A, B and C described in the present study, by epidermal
cautery in the larva. These contralateral abnormalities were most frequently
the result of cautery in the mid-dorsal region; co-lateral fusions of hemisegments resulted from cautery of the corresponding regions delimiting larval
hemisegments co-laterally. In these cases, apparently specific damage to
the larval epidermis in hemisegmental boundary regions leads to boundary
abnormalities of adult hemisegments, and hence to abnormal segmental
patterns.
Regulation
The most impressive cases of dorsal regulation were seen in multiple abnormal
larvae aa45, aa59, aa69 (Fig. 14, cf. Fig. 15) where axial series of bilateral
non-correspondence are rectified to give normal adult abdomina.
If a mediodorsal instability was latent along the whole animal although
manifest in the larva only at single or occasional intersegmental membranes, it
might be expected to find new points of bilateral irregularity appearing at
metamorphosis, as well as regulation of larval abnormalities. In general this
is not the case, although in one animal, aa36, an adult contralateral noncorrespondence does arise where the larval 3/4 membrane was bilaterally
matched. Even here, however, although there is correspondence in the larva,
the membrane is slightly irregular in the midline (Figs. 16 and 22). It appears
rather that instabilities at metamorphosis are specific for those intersegments
which show larval irregularity.
Contralateral hemisegments and hemisegmental boundaries then retain some
morphogenetic independence during metamorphosis. This degree of inde-
Abnormalities
in segmentation
of Calliphora
549
Failure of
hemitergites 4 to
fuse dorsally
aa34
aa36
Fig. 16. (a) Larval aa34 shows a class I break between thorax and first abdominal
segment. In the adult, the tagmata are morphologically normal. A double abdominal
class I break 2/3 and 3/4 produces in the adult bilateral misalignment in segments 3
and 4 but not in segment 2, i.e. the bilateral fusion is not necessarily directed by the
posterior boundary of that larval segment, (b) aa36 shows a larval class I break
2/3, which regulates in the adult, and a slightly irregular fusion 3/4. This was the
only observation of a (more or less) normal larval correspondence leading to
irregular (class C) adult tergite. See also Fig. 22.
pendence in post-embryonic development explains both the regulative ability
and the appearance of an adult abnormality different from the larval type.
The patterns or classes of dorsal abnormality are perhaps not in themselves
important.
Hemisegmental boundaries and the histoblast anlagen
Extirpation experiments (Emmert, 1972) show that the ventral sternite is
formed exclusively by ventral histoblast derivatives, and histoblasts from the
dorsal anlagen are largely responsible for tergite formation. In experimental
situations at least, there may be a ventral anläge contribution to the ventrolateral tergite margin, although according to Bhaskaran (1973), who employed
more elegant transplant techniques, it is the imaginai cells from the spiracle
anläge which form this tergal region. Histological observation of whole mounts
during larval and metamorphic stages indicates that intersegmental membranes
of the abdomen in Calliphora are also reconstituted by the derivatives of the
segmental anlagen (Pearson, 1972). There are no evident intersegmental anlagen.
Lawrence (1973) has shown in the embryogenesis of Oncopeltus (Hemimetabola)
that the segmental margins do not have a discrete lineage.
An analysis of tergite patterns in adult flies produced by X-irradiation during
metamorphosis (Lübbecke, 1958) obviously cannot examine the relation
between abnormal larval and adult patterns of intersegmental boundaries ; and
cannot therefore properly distinguish the roles in the control of tergite formation
of the segmental histoblasts and/or pre-existing intersegmental boundaries -
550
M. J. P E A R S O N
Muscle I
1
insprtionl
ƒ
missing \
7
\
y ^ ^ . Reduced hemitergite
\Js
R5
AA7I
Fig. 17. AA71 adult shows reduction dorsally of right hemitergite 5. There is no
LR abnormality of the larva but longitudinal reduction of the hemisegment and
concomitant absence of antero-dorsal muscle insertions.
whether normal or abnormal. Löbbecke suggested that the aberrant flies he
obtained (with abnormal abdominal segmentations very like those described
in this study) were results of histoblast damage during irradiation. It is unlikely
in the present case, however, where the abnormalities arise in embryogenesis,
that the adult abnormalities, or the relation of adult to larval abnormality,
should arise in the histoblast anlagen. The anlagen in those class I and II
larvae examined in whole mounts were of normal appearance; and in abnormal
adults the formation of the hemitergites appears normal.
The case of lateral reductions and fusions (L2 and LR) is different. Sobels
(1952) noted in Drosophila that where a hemisegmental boundary is reduced i.e. the intersegmental boundary is not only interrupted mid-dorsally but on
one side ends short laterally - a reduction is found of the anterior tergite in
the adult. This is shown, in Calliphora, in AA35; but in AA45 it is the posterior
hemitergite which is reduced (Fig. 15). In both cases, as in AA71 where there
is no lateral reduction in the larva, the hemitergite reduction may rather be
correlated with an antero-posterior shortening of the hemisegment in the
region of the dorsal histoblast anlagen (Fig. 17). The question is whether
(a) tergite reduction results from membrane reduction with consequent loss
of some information for tergite formation (Sobels, 1952), or whether (b) in
the larval stages of these animals, a corresponding anläge (dorsal and/or
postero-dorsal) is affected. In AA71, where there is no lateral membrane
reduction, the former cannot be the case. Indeed, it is clear from Table 2 that
if the absence of the appropriate muscle insertion indicates absence of a particular
anläge in the larva, then the cases of tergite or sternite loss (or reduction) in
aa flies derived from laterally (L) abnormal larvae corroborate the second
proposition (b): that reduction or absence of sclerites in the adult arises
through histoblast deficiencies. Clearly, in LI larval abnormalities an interruption laterally in the intersegmental membrane does not 'over the same
area inhibit the outgrowth of the corresponding histoblast' (Sobels, 1952),
since LI larvae gave 100 % normal adults.
Secondly, the evidence of abnormal animals where lateral reduction of an
Abnormalities
in segmentation
of Calliphora
551
interhemisegmental membrane is associated with abnormally placed muscle
insertions in the larva - indicating abnormally located anlagen - suggests that
the hemisegmental position of the anlagen may be important in the development
of the normal scleral structures at metamorphosis (see Fig. 12). In each of
four cases involving ventral anlagen, the differentiation of an irregular and
displaced sternal structure resulted. It appears that in Calliphora lateral
reduction of intersegmental membranes may be of special morphogenetic
significance only so far as it is accompanied by deficiencies or displacement of
histoblast anlagen.
The developmental origin of the abdominal abnormality produced by X-irradiation
of early embryos
At 21 °C, the blastoderm of Calliphora forms 1^-2 h after oviposition
(Davis, Krause & Krause, 1968). In the present study, the effective period for
X-irradiation was up to 1 | h after oviposition at room temperature (20 °C ± 2 °C) :
that is, at, or before blastoderm formation. It is possible, therefore, that the
causal basis of the asymmetric abnormalities seen in the larvae could lie in
a disturbance of either (i) an ooplasmic reaction system which is responsible
for the determination of segmental pattern; or (ii) nuclei whose cell lineage
will subsequently participate in the movements and growth of the germ band,
and the differentiation of segments. The definitive larval segments are delimited, in antero-posterior succession, beginning at about 6 h at 21 °C (Davis
et al 1968).
There is at present no evidence to deal with the first suggestion; it would
not easily fit a purely 'quantitative' model of an axial gradient phenomenon
(Herth & Sander, 1973). If, however, the effect of X-irradiation is to damage
(with respect to subsequent division) a number of nuclei in cleavage or early
blastoderm stage such that, at segment determination, the initial cell populations
are unequal in contralateral germbands, then the larval expression of asymmetry
in segmentation could result from bilaterally unequal growth and development
of contralateral hemisegments. This interpretation accords with the character
of insect segmentation in temporal and spatial aspect. In Cyclorrhapha, the
first intersegmental boundaries to form are between mandibular/maxillary/labial
segments, and then in posterior sequence (Anderson, 1962). However, the rate
of development of each segment is not constant along the insect axis (Bock,
1939; Counce, 1973) and increases posteriorly, such that, as the hemisegmental
primordia of the embryo grow up from the ventral side to meet in the dorsal
midline (dorsal closure), ' the consequence of the differential rates of segment
differentiation is that dorsal organs such as the aorta, form almost simultaneously
in all segments' (Counce, 1973). Clearly, interference with the dorsal development of contralateral hemisegments will ensure that the temporal pattern of
closure is disturbed. Such temporal dislocation could introduce bilateral
asymmetries of spatial pattern. For example, if a right hemisegment reaches
552
M. J. PEARSON
'18
'19
20
21
Fig. 18. aa72 shows a class I dorsal break between segments 4 and 5.
Fig. 19. aa60 shows dorsal abnormality between segments 4, 5 and 6. The left
membranes 4/5 and 5/6 converge to give an 'intermediate' abnormality differing
only trivially from a double class I abnormality.
Fig. 20. aa30 shows a class II dorsal abnormality 4/5/6.
Fig. 21. aa35 shows a class III dorsal abnormality 3/4/5/6/7. See Fig. 8.
Fig. 22. aa36 is shown diagramatically in Fig. 16. Although interhemisegmental
membranes 3/4 have fused mediodorsally, hemitergites 4 in the adult did not fuse.
This was the only observed instance of an abnormally differentiated adult tergite
which did not obviously correlate with a defect of larval intersegmental membranes.
Fig. 23. Larval aa26, where mid-dorsal intersegmental membranes fail to differentiate
down the entire abdominal axis until 7/8. Right and left hemisegments show a
consistent bilateral displacement.
Abnormalities
in segmentation
of Calliphora
553
the dorsal midline before the corresponding left hemisegment, closure may
result with an adjacent (anterior or posterior) left hemisegment, giving a class
II irregularity in the larval pattern. Such a bilateral difference of closure point
along two or more segments could produce class III abnormalities.
In any case, it is suggested that this type of dorsal abnormality is the
consequence of abnormal dorsal closure, which results from a dislocation of
the processes of growth and differentiation in the ventro-dorsal and in the
longitudinal axes.
In aa larvae, hemisegmental misalignment of the class II or class III kind
were not found ventrally; contralateral hemisegmental anlagen correspond at
the stage of segment initiation. However, in 10 % dorsally abnormal larvae, a
failure of normal intersegmental membrane differentiation is evident midventrally, often with lateral reduction of the membrane of one side. The origin
of these class I defects, both ventral and dorsal, is clearly not the result of
any hemisegmental misalignment. In either case, closure of the segmental
epidermis has occurred, but boundaries have failed to develop during the
closure. (It is implicit in the manner of insect gastrulation that hemisegmental
anlagen are bilaterally separated by mesoderm invagination, necessitating a
ventral as well as the more obvious dorsal closure.)
CONCLUSION
This analysis of the relation between larval and adult abnormalities in
Calliphora erythrocephala leads to a different view from that proposed by
Maas (1948) and by Sobels (1952) for Drosophila. The latter author concludes
'the larval segmental borders constitute the determinative pattern which governs
the differentiation of hypodermal histoblasts to form normal tergites. When
part of a segmental border is missing in the mid region of the animal, as a
rule the development of the corresponding tergite over the same area is
inhibited. When lateral reductions of the segment borders occur, reduction
of the tergite over the same distance as well as complete reduction of the
corresponding half of a tergite may be the result.' The results of this study
do not lead to firm conclusions on the relative roles of intersegmental boundaries
and histoblast anlagen in the determination of bilateral segmental pattern in
the adult abdomen. It is assumed (i) that in embryogenesis, as in Oncopeltus
(Lawrence, 1973), intersegmental boundaries are morphogenetically consequent
upon the development of the hemisegmental primordia ; it is suggested (ii) that
the bilateral independence of hemisegmental development until closure is
reflected, in abnormal cases, by non-differentiation of intersegmental membranes in the midline, or by dorsal misalignment, to give the observed abdominal abnormality patterns of intersegmental membranes; (iii) that the axial
points of abnormal segmental closure are also evident at metamorphosis though in some cases they regulate - but that the pattern of the abnormality
554
M. J. PEARSON
is not primarily significant; (iv) that in dorsal abnormalities the differentiation
of hemisegments is normal except for closure; and (v) that lateral reduction
of segmental borders does not lead to reduction of sclerites in the adult except
where displacement or deficiency of histoblast anlagen are involved. In lateral
abnormalities, the correlation between absence/displacement of muscle insertion with which anlagen are normally associated, and reduction/displacement
of sclerite in the adult, suggests that not all the information needed for normal
differentiation of adult segmental pattern is provided by the segmental borders.
The fascinating questions remain of the relationship between larval and adult
primordia in early embryogenesis and the precise manner of determination
of pattern in the adult abdominal segments at metamorphosis.
I am very grateful to Peter Lawrence for helpful advice with the manuscript, and for
years of carping personal criticism; to Rae Fairholm who helped produce the figures; and
in particular to Denis Weaver for financial support.
REFERENCES
ANDERSON, D. T. (1962). The embryology of Dacus tryoni (Frogg). [Diptera, Trypetidae
( = Tephritidae)], the Queensland fruit-fly. / . Embryol. exp. Morph. 10, 248-292.
BHASKARAN, G. (1973). Developmental bshaviour of the abdominal histoblasts in the
housefly. Nature, Lond. 241, 94-96.
BAUTZ, A. M. (1971). Chronologie de la mise en place de l'hypoderme imaginai de l'abdomen
de Calliphora erythrocephala Meigen. (Insecte Diptère, Brachycère). Archs Zool. exp.
gén. 112, 157-178.
BOCK, E. (1939). Bildung und Differenzierung der Keimblatter bei Chrysopa perla (L.).
Z. f. Morph. Ökol. Tiere 35, 615-703.
COUNCE, S. J. (1973). The causal analysis of insect embryogenesis. In Developmental
Systems; Insects, vol. II, pp. 1-156. London and New York: Academic Press.
CRAMPTON, G. C. (1942). The external morphology of the Diptera. Bull. Conn. St geol.
nat. Hist. Sur v. 64, 10-165.
DAVIS, C. W. C., KRAUSE, J. & KRAUSE, G. (1968). Morphogenese movements and seg-
mentation of posterior egg fragments in vitro {Calliphora erythrocephala Meig., Diptera).
Wilhelm Roux Arch. EntwMech. Org. 161, 209-240.
EMMERT, W. (1972). Entwicklungsleistungen abdominaler Imaginalscheiben von Calliphora
erythrocephala (Insecta, Diptera). Experimentelle Untersuchungen zur Morphologie des
Abdomens. Wilhelm Roux Arch. EntwMech. Org. 169, 87-133.
FRAENKEL, G. & HARRISON, J. L. (1938). Irregular abdomina in Calliphora erythrocephala
(Mg.). Proc. R. ent. Soc. Lond. (A) B, 95-96.
HERTH, W. & SANDER, K. (1973). Mode and timing of body pattern formation (régionalisation)
in the early embryonic development of cyclorrhaphic dipterans (Protophormia, Drosophila).
Wilhelm Roux Arch. EntwMech. Org. Ill, 1-27.
LAWRENCE, P. A. (1973). A clonal analysis of segment development in Oncopeltus (Hemiptera).
J. Embryol. exp. Morph. 30, 681-699.
LÖBBECKE, V. E. A. (1958). Über die Entwicklung der imaginalen Epidermis des Abdomens
von Drosophila, ihre Segmentierung und die Determination der Tergite. Biol. Zbl. 11,
209-237.
MAAS, A. H. (1948). Über die Auslosbarkeit von Temperaturmodifikationen wahrend der
Embryonalentwicklung von Drosophila melanogaster. Biol. Zbl. 50, 541-554.
PEARSON, M. J. (1972). Developmental Studies on the Abdominal Epidermis of Calliphora
erythrocephala {Diptera). Ph.D. Thesis. University of Cambridge.
Abnormalities in segmentation of Calliphora
555
SANTAMARIA, P. & GARCIA-BELLIDO, A. (1972). Localization and growth pattern of the
tergite Anlage of Drosophila. J. Embryol. exp. Morph. 28, 397-417.
SOBELS, F. H. (1952). Genetics and morphology of the genotype 'asymmetric'. Genetica
26, 117-279.
ULRICH, H. (1951). Sensitive periods and egg regions in production of the modification
'abnormal abdomen' by X-raying eggs of Drosophila melanogaster. Drosoph. Inf. Serv.
25, 131.
{Received 22 March 1974, revised June 1974)