J. Cell Sci. 2, 603-616 (1967)
Printed in Great Britain
603
POLYPLOIDY AND NUCLEAR FUSION IN
THE FAT BODY OF RHODNIUS (HEMIPTERA)
V. B. WIGGLESWORTH
Agricultural Research Council Unit of Insect Physiology,
Department of Zoology, University of Cambridge
SUMMARY
The diploid number in Rhodnius is 22 in both sexes. At hatching the fat-body cells are
tetraploid, with a few octoploid. This polyploidy is presumed to arise by endomitosis in the
egg. In the fully nourished insect this state persists throughout life; and occasional i6« and 32W
nuclei occur. Whether these arise by endomitosis or by nuclear fusion has not been established.
In extreme starvation (e.g. 8 months from the time of moulting in the 4th-stage larva at
26 °C) polyploidy increases greatly. This results from nuclear fusion in interphase. The evidence
is as follows:
(i) All intermediate stages in fusion can be observed.
(ii) Polyploidy develops many months after growth and mitosis have been arrested.
(iii) It occurs mainly in those regions where the food reserves first become exhausted.
(iv) When these starved insects are fed the polyploid cells divide and chromosome counts
include i2n, 2on, 24W, etc., as well as the regular series 472, 8n, i6n, 32W, etc.
Contiguous nuclei which have not fused in interphase often amalgamate their chromosomes
to form a single plate and spindle at metaphase and likewise produce more highly polyploid
daughter cells.
The high incidence of nuclear fusion in Rhodnius is ascribed to the low rate of metabolism
which permits prolonged survival in the starving insect.
INTRODUCTION
In the 1930s it was established by a number of authors that the giant (polytene)
chromosomes in the salivary glands of Diptera arise through a process of repeated
doubling by internal growth without mitosis. On the other hand, Berger (1937, 1938)
showed that in the epithelial nuclei of the ileum in Culex internal divisions lead to
a multiplication of separated chromosomes which may continue until the nuclei
become 32-ploid. Geitler (1937) described even higher grades of polyploidy in many
tissues of Gerris (Hemiptera). He was at first inclined to attribute this to nuclear fusion
or to restitution after incomplete mitosis. But following Berger's observations and his
own further work (Geitler, 1939) he adopted the same explanation: that the most important cause of polyploidy is by what he named 'endomitosis'.
Geitler (1941, 1953) has consistently recognized that there are other ways of attaining polyploidy; but the tendency has been for most authors to assume that the main
cause is endomitosis. The polyploidy that appears in many tissues of Apis, for example,
has recently been attributed, probably with justice, to endomitosis (Mittwoch, Kalmus
& Webster, 1966; compare Risler, 1954). The same is true of the nematocerous
larva Ptychoptera (Risler, 1950) and the beetle Oryzaephilus (Romer, 1964, 1966).
39
Cell Sci. 2
604
V. B. Wigglesworth
During a study of the cytological changes in the fat body of Rhodnius under varied
conditions (Wigglesworth, 1967) it was discovered that after 7-9 months starvation
in the 4th-stage larva, when virtually all the reserves of fat and glycogen have been
consumed, many of the cells fuse with their neighbours to produce cells with two or
more nuclei; and in many of these multinucleate cells the nuclei themselves fuse. The
purpose of the present work was to study this process of fusion systematically. A
preliminary note on the findings has been published elsewhere (Wigglesworth, 1966).
METHODS
Most of the observations have been made on the 4th-stage larva of Rhodnius. The
abdominal tergites with the fat body adhering were dissected off and mounted whole
as already described (Wigglesworth, 1967). Some were fixed in osmium tetroxide and
stained with ethyl gallate (Wigglesworth, 1957); most were fixed with Carnoy's
mixture and stained with Groat's haematoxylin or with pyronin and methyl green.
Chromosome counts at metaphase were done on acetic-orcein squash preparations.
Spectrophotometric measurements of deoxyribonucleic acid (DNA) were made on
Feulgen-stained preparations of the isolated fat body after Carnoy fixation. The
measurements were carried out for me by Dr C. L. Smith of the Department of
Radiotherapeutics, Cambridge.
RESULTS
Normal chromosome numbers
Orcein-acetic acid squash preparations of the developing gonads, of the nuclei of
the intersegmental muscles, which undergo active mitosis in the early stages of
moulting, and of the epidermal cells, show a diploid number (211) of 22 chromosomes
in both sexes.
Diploid nuclei are absent in the fat body. The predominant type of nucleus is
constant throughout all the instars. It measures about 7 /.i in mean diameter in the
fasting state and gives a count of 44 chromosomes (4*1) at metaphase. A larger type of
fat-body nucleus, about 11 /t in mean diameter, is much less numerous but is to be
found at all stages. At metaphase it gives a count of 88 chromosomes (8»). Nuclei with
higher degrees of polyploidy occur sporadically; notably 1611 (176 chromosomes) with
a mean diameter about 13 fi.
Fat-body cells on hatching from the egg
First-stage larvae were fed within 2 or 3 days of hatching and the tergites and fat
body mounted at once. At this time there are no diploid nuclei in the fat body. The
majority of the nuclei are tetraploid. Usually there are a few octoploid nuclei (Fig. 5);
but in some specimens these are very scarce or possibly absent.
This almost uniform polyploidy therefore arises during embryonic development.
It is presumed to be the result of endomitosis at some stage—but no observations
have been made on the embryo.
Polyploidy in fat body of Rhodnius
605
Effects of starvation in ist-stage larvae
About 60 newly hatched larvae were starved at 26 °C. At known intervals groups
were allowed to take a small feed of blood (in order to facilitate dissection) and were
then immediately dissected and fixed.
The unfed ist-stage larva cannot survive starvation for more than 21-26 days. By
this time all the lipid reserves in the fat body have been consumed. A very few fat-body
cells have fused to form binucleate cells, but no detectable nuclear fusion has taken
place. There has been no obvious increase in the incidence of polyploid nuclei.
Effects of prolonged starvation in each instar
A large batch of first-stage larvae was divided into three groups:
Group A were kept at 28 °C and reared with the minimum starvation. They were
fed within a few days after hatching and fed within a few days after each succeeding
moult. {Rhodnius takes only a single large meal of blood in each instar.) Under these
circumstances they reached the 4th stage in 7 weeks and were fed and examined soon
afterwards, making a total of 9 weeks from the time of hatching.
Group B were kept at 28 °C in each instar without feeding until the larvae were
beginning to die of starvation. They were starved 6 weeks in the 2nd stage; 18 weeks
in the 3rd stage. Fourth-stage larvae which have been well nourished in the earlier
stages will survive starvation for 9 months or more; but the time of survival is greatly
reduced if the larvae have been subjected to prolonged starvation in each of the preceding stages. In group B they were beginning to die off at 19 weeks after moulting,
a total of 47 weeks after hatching.
Group C were starved in the same way as group B but during this time they were
kept at room temperature (18-20 °C). This resulted in longer periods of survival in
each instar. They were starved 6 weeks in the 2nd stage; 21 weeks in the 3rd stage;
and the 4th-stage larvae were beginning to die off at 27 weeks after moulting, making
a total of 61 weeks since the time of hatching. It was thought that maintenance at the
lower temperature might influence the development of polyploidy; but there was no
detectable difference between series B and C. The results are therefore presented
together.
Series A. In the 4th instar the vast majority of nuclei are \n (Fig. 6) but a
variable number of 8ra nuclei occur (Fig. 7). These are often in pairs or small groups.
Sometimes there are quite large patches of these %n nuclei; and also occasional cells
with higher degrees of ploidy. It is not uncommon to see adjacent cells in the fat
body with no visible plasma membrane between; they appear to be binucleate cells
(Fig. 8).
These results suggest that 8M nuclei present in the ist-stage larva give rise to subsequent growing generations of octoploid nuclei. It is possible that a small amount of
nuclear fusion, following the breakdown of the plasma membrane of contiguous cells,
may be occurring at mitosis even in the unstarved insect. But it is not possible to
exclude the development of polyploidy, in these unstarved larvae, either by endomitosis
or by the restitution of nuclei after mitosis without cytokinesis.
39-2
606
V. B. Wigglesworth
Series B and C. Larvae starved in each of the earlier instars, and starved 24-28 weeks
in the 4th instar, show:
(a) Greatly increased incidence of polyploidy as compared with series A, apparent
both in the far greater number of 8M nuclei and in the greater numbers of nuclei
showing higher grades of ploidy.
(b) All stages of nuclear fusion, as follows:
(i) Cells with 2, 3 or more discrete nuclei.
(ii) Cells with such nuclei in immediate contact, but with the nuclear membranes
still intact between them.
Fig. 1. Examples of nuclear fusion in severely starved 4-th stage larva of series B
(see text): a, normal tetraploid; b, binuclear cell with two tetraploid nuclei; c-e,
stages in the fusion of pairs of tetraploid nuclei; /, fusion of three tetraploid nuclei;
g, normal octoploid; h, fusion of two octoploid nuclei; j , fusion of (apparently) two
octoploid nuclei below and two tetraploid nuclei above.
(iii) Cells with the nuclear membrane no longer visible at the points of contact of
the nuclei, but with the constituent nuclei that are in process of fusion readily
distinguishable.
(iv) Very large nuclei of bizarre shapes, apparently formed by the fusion of an
indeterminate number of cells, often seemingly a mixture of 4W, 8ra and more highly
polyploid nuclei.
(v) Cells with large nuclei with evenly rounded or oval outline. It is impossible to
decide from inspection whether these have arisen by nuclear fusion or by endomitosis.
(But see 'Time of mitosis' below.)
Examples of these diverse forms are illustrated in the line drawing, Fig. 1, and in the
photomicrographs, Figs. 9-18.
In extreme starvation some of the fat-body cells die. The nuclei of such cells are
Polyploidy in fat body of Rhodnius
607
readily distinguishable from those of the living cells, if they are fixed and stained
with the osmium/ethyl gallate method. Whereas the normal nuclei fixed with neutral
osmium tetroxide have a homogeneous palely staining nuclear sap, the dead or dying
nuclei have a granular appearance and stain much more deeply. It is not uncommon
for these dying cells to fuse with living cells, which thus contain both a normal
nucleus and a darkly staining nucleus in varying stages of autolysis (Figs. 9-11).
These autolysing nuclei contribute to the conspicuous cytolysomes in the fat-body
cells of starved Rhodnius larvae (Wigglesworth 1967).
Fig. 12 is an example of a binucleate cell resulting from the fusion of two living
cells in extreme starvation, likewise stained with osmium and ethyl gallate. Figs. 13
and 14 illustrate various stages in the fusion of multiple nuclei, similarly stained.
Figs. 15-18 show similar examples of fusing nuclei after fixation in Carnoy's
mixture and staining with haematoxylin or with pyronin and methyl green. Fig. 16
shows five recognizable tetraploid nuclei in process of fusion. This same figure and
Fig. 18 show also large, evenly rounded nuclei the origin of which cannot be inferred
from inspection.
Starvation in $th-stage larva and adult
Fifth-stage larvae derived from series A (reared without starvation) and from series
B and C (starved in all stages) showed the same results: mainly tetraploid nuclei in
series A; extensive polyploidy with many very large and distorted nuclei after extreme
starvation of insects derived from series B and C.
Adult insects reared without starvation were likewise subjected to varying degrees
of starvation in the adult stage. Those mounted soon after moulting showed mainly
tetraploid, with some octoploid nuclei. Those starved for 12 weeks, by which time the
lipid reserves in the fat body are almost exhausted, show extensive fusion of nuclei,
sometimes leading to gigantic structures (Fig. 19).
Time of mitosis
As pointed out above, it is not possible from inspection to decide whether polyploid nuclei have arisen by endomitosis or from nuclear fusion. But mitosis in the
fat-body cells takes place only during a restricted period. In the 4th-stage larva,
mitosis is limited to a period between 3 and 10 days after the single blood meal. It is
completely at an end some days before moulting, which occurs in the 4th-stage larva
at 14-15 days after feeding. Thereafter there is no further mitosis in the fat body until
the insect feeds again, which may be 7 or even 9 months later.
It is in these late stages of starvation, when no mitosis is occurring in the fat body,
that polyploid nuclei arise in large numbers.
Likewise in the adult; no mitosis occurs in the adult fat body. But, as we have seen,
extensive nuclear fusion and polyploidy develop in advanced starvation.
6o8
V. B. Wigglesworth
Numbers and distribution of polyploid nuclei
The fat body in the abdomen of Rhodnius is a lace-like structure made up of a single
layer of cells flattened between the blood-filled stomach and the integument. Only
at the lateral margins of the abdomen are the fat-body lobes more than one cell thick.
This arrangement makes it possible to count the number of cells in the fat body.
It is not feasible to count the entire fat body; a standard area has been selected. This
is represented in Fig. 2. It overlies one half of the fourth tergite of the abdomen. It is
bounded medially by the free margin of the fat body adjoining the heart; in front and
behind by the intersegmental boundaries; and laterally by a line drawn through the
middle of the insertions of the innermost row of dorso-ventral muscles, which lie
just behind each intersegmental membrane.
1 mm
2 mm
Fig. 2. To the right, numbered abdominal tergites of 4th-stage larva showing the heart
in the mid-line and the insertions of the dorso-ventral muscles. The cross-hatched
portion of the fourth segment marks the standard area, over which the fat-body
nuclei are counted, divided into inner, middle and outer zones. To the left, detail of
the fat body over the standard area of the opposite side.
In the ist-stage larva (Fig. 3) this standard area contains about 50 fat-body cells,
mostly tetraploid, sometimes with a few octoploid. In the 2nd stage the number is
about 200; in the 3rd stage about 450; and in the 4th stage about 1200.
Counts from some selected 4th-stage larvae, based on camera lucida drawings, are
set out in Table 1. These are made up as follows. No. 1 was a typical example from
series A (reared with minimum starvation). This is an example with more octoploid
nuclei than some specimens but with fewer than others. Nos. 2, 3 and 4 are all from
series B or C (prolonged starvation in each instar). No. 2 was a specimen showing a
moderate degree of polyploidy. In no. 3 polyploidy was more extreme but was by no
means the most extreme in the series. No. 4 was approaching the last stages of starvation. Many of the nuclei had broken down and disappeared; others were undergoing
pyknosis.
Polyploidy in fat body of Rhcdnius
609
In no. 2 and no. 3 the nuclei are classed as '4.11' ,' ?8n' and ' ?i6w and over'. These
values are based on inspection. We have seen that in these insects An and 8M and some
higher categories of nuclei are fusing with one another. This will give a mixture of
An, 8n, i2w, i6w, zon, 24M, 28re, 32M, etc. Since the nuclei fuse with their nearest
neighbours, which will tend to show the same degree of ploidy, the regular series
An, 8n, I6M, 32W will predominate. But in Table 1, 12M nuclei will have been included
some in the ' ?8»' class and some in the ' ?I6M and over' class.
The individual variation is such that the figures in Table 1 cannot usefully be
analysed more deeply. The cell total in no. 4 has clearly been reduced by nuclear
0-1 mm
Fig. 3. Standard area on the fourth abdominal tergite of a recently hatched ist-stage
larva (immediately after feeding) showing 54 fat-body cells. Of these one is octoploid,
the remainder tetraploid.
breakdown. No. 2 and no. 3 show less reduction in the total number of nuclei than
was to be expected on the basis of the degree of nuclear fusion that has occurred. But
this nuclear fusion has probably occurred over several moulting stages and has
perhaps been compensated for by increased mitosis. In many places the mucopolysaccharide sheaths of the fat-body strands appear almost empty, with just 1 or 2 large
polyploid nuclei widely separated.
As pointed out in an earlier paper (Wigglesworth, 1967), during starvation the
exhaustion of lipid reserves in the fat body first becomes apparent in the inner region
alongside the heart. The depletion of reserves is progressively less evident in the more
lateral regions of the fat body. In no. 3 (Table 1) the standard area was subdivided
into 3 longitudinal zones of approximately equal area (Fig. 2). As can be seen, the
incidence of polyploidy runs parallel with the degree of starvation as evidenced by
the depletion of reserves and becomes progressively greater towards the inner zone.
6io
V. B. Wigglesworth
Table i. Numbers and percentages of polyploid nuclei in fat-body cells over
equivalent areas of fourth abdominal segment of \th-stage larvae
p8w
4«
'
No.
of total
1197
(i) Reared without
starvation
(2) Prolonged starvation
975
in each stage
(moderate polyploidy)
(3) Prolonged starvation
794
in each stage (extensive polyploidy)
(a) Inner third of area
138
(6) Middle third of
219
area
(c) Outer third of area 437
No.
z and over
A
1
As%
of total
As %
of total
No.
9S6
19
16
79'5
208
17-0
39
73'2
163
15-0
128
46-4
78-8
72
24-2
88
29-5
34
12-5
25
90
57
11-2
15
2-9
85-8
811 and over
\
(4) As (2) and (3) with
extreme starvation
?i&
_A
^A
Total
0
1216
3'2
1222
n-8
1085
0
298)
278
1085
5°9 i
Pyknotic
A
No.
As %
of total
No.
of total
No.
Total
659
75-5
214
24-5
44
873
As%
in 4th stage
Thus the number of ' ?I6M and over' increases from 2-9 % in the outer zone, to 9-0 %
in the middle zone, and to 29-5 % in the inner zone.
Fate of polyploid cells on feeding
Squash preparations of 4th-stage larvae in advanced starvation (32 weeks after
moulting) showed the same conditions as the whole mounts described above. The
fat-body nuclei were no longer evenly separated by cytoplasm. Many of them were
contiguous and showed all stages of fusion. In these severely starved larvae, growth
and mitosis are delayed; but by 8 or 9 days after feeding, fat-body cells at all stages
of mitosis can commonly be seen. They are sufficiently numerous for squash preparations to yield a fair number of countable metaphases (Figs. 23-27).
Plenty of metaphase plates containing 44 (411), 88 (8w) and 176 (i6n) chromosomes
have been counted precisely. About half a dozen metaphase plates in which the
number of chromosomes was estimated by counting them over a measured area in
relation to the whole, with a camera lucida or with an ocular graticule, gave values
in the region of 350: presumably 32M (= 362).
Several evenly rounded metaphase plates with approximately 132 chromosomes
(i2w) (Fig. 4#) and 264 chromosomes (24W) (Fig. 46) were counted; these had presumably arisen from the fusion of tetraploid and octoploid nuclei. In addition a
number of metaphase plates have been seen in which the chromosomal components
Polyploidy in fat body of Rhodnius
611
of several nuclei were still distinguishable. These may take the form of two contiguous metaphase plates (Fig. 4 h) or plates in which there are distinct zones corresponding to the nuclei from which the chromosomes have been derived (Fig. 4/,/, /)•
For example, an 8n and a 47? zone (Fig. 4 / ) ; two Sn zones (Fig. 4/); two 8n zones and
a 4« zone (Fig. 4/) and so forth. There seems no reason to doubt that these mitoses
would have gone forward to produce viable daughter nuclei: i2«, i6n, and zon.
In extreme starvation, as already noted, many nuclei undergo pyknosis and disappear. It may be that this affects predominantly the large polypioid nuclei. But there
is no evidence that large polypioid nuclei do break down after feeding. Among the
\?352
Jo
P
%
c
»S%fA
"e'gaooi*' e
« a j
'
?88
Fig. 4. Acetic-orcein squash preparations from 4th-stage larva starved for 31 weeks,
8 days after feeding, showing chromosomes in metaphase. The numbers indicate
chromosome counts, a, zn nucleus from epidermis; b~j, are fat-body nuclei; b, 411;
c, Sn; d, 1611; e, probably 32ft;f, probably raw, formed by fusion of 4?? and 8n nuclei;
g, as / ; h, two Sn metaphase plates in process of fusion; j , two 8« nuclei at more
advanced stage of fusion; k, probably 24«, fusion complete; /, multiple metaphase
consisting apparently of one 47? nucleus and two 8n nuclei.
normal mitoses there are commonly a few polypioid nuclei which show abnormal
mitosis with multiple spindles, often giving rise to triradiate metaphase plates (Figs.
22, 27). In one of the squash preparations there was a large nucleus of this type in
which the main metaphase plate was estimated to be 32/2 and the accessory plate to
be about i6n. It seems likely that these abnormal mitoses appear when division has
begun before nuclear fusion was sufficiently far advanced. The ultimate fate of such
nuclei is not known.
6i2
V. B. Wigglesworth
Spectrophotometric estimations of DNA
An attempt was made to demonstrate the presence of intermediate nuclei, that is,
of the triploid series i2w, 24W, and perhaps of 2ow and 28M, by photometric estimations
of DNA. Such measurements are commonly made on cells at various stages of DNA
replication and the results assessed statistically. For the present purpose it was
necessary to know the relative DNA content of single nuclei. It was assumed that all
the nuclei in the starved insect, which are in a resting interphase condition, would be
at the low point of DNA content; and that comparison of the larger nuclei in the fat
body with the abundant 471 nuclei would give a direct measure of the degree of polyploidy, with the usual 1 1 % standard error.
As it turned out, the measurements were too variable to give convincing conclusions. In onefieldof one preparation, for example, the results shown in Table 2 were
obtained on twenty nuclei.
Table 2. Relative DNA contents of single nuclei
The ploidy, as judged from microscopic appearance of the nuclei,
is shown in parentheses.
(i6n)
15
25
4i
45
63
93
101
141
141
84
116
n6
141
141
25
3°
66
66
68
106
147
i47
151
158
These and similar DNA values from other preparations are not definite enough to
afford evidence of the degree of ploidy in single cells. To determine whether this
amount of scatter is due to technical error in the preparation of the material or
whether the resting nuclei are indeed at different levels of DNA replication, would
need much further study.
DISCUSSION
Polyploidy arising by endomitosis is regarded by Geitler (1941) as an important
element in tissue differentiation, although its physiological significance remains
unknown. The basic polyploidy of the fat body in Rhodnius, the cells of which are
mainly tetraploid throughout life, is of this type.
The sporadic polyploidy considered in this paper is comparable rather with the
polyploidy in the mammalian liver. Here the incidence of binucleate and polyploid
cells increases with age (Alfert & Geschwind, 1958) and is exaggerated during regeneration (Beams & King, 1942; Geshwind, Alfert & Schooley, 1958). It has been variously
attributed to nuclear fusion in interphase or in mitosis (Beams & King, 1942) or to
endomitosis (Swartz, 1956; Wilson & LeDuc, 1948). It has not been possible to obtain
decisive evidence for any one of these explanations. Recently Nadal & Zajdela (1966)
have produced good evidence that most of the polyploidy in regenerating liver cells
of the rat results from the amalgamation of the two sets of chromosomes from the
dividing nuclei of binucleate cells, which form a single plate and spindle at metaphase.
Polyploidy in fat body of Rhodnius
613
The sporadic polyploidy in the fat body of Rhodnius is here shown to result mainly
from nuclear fusion during interphase. The evidence is as follows:
(i) Polyploidy can be induced by extreme starvation, which leads first to cellular
fusion to produce binucleate or multinucleate cells and then to fusion of the nuclei.
The nuclei involved in fusion are different combinations of 4M, 8ra, i6«, 32M.
(ii) This process occurs predominantly in those regions of the fat body in which
the food reserves are first exhausted during starvation.
(iii) It occurs many months after growth and mitosis in the fat body have been
arrested. Mitosis in the fat body takes place only during the period from about 3 to
10 days after feeding (in the 4th-stage larva). Nuclear fusion takes place in the starved
insect many months later.
(iv) When these starved insects are fed, chromosome counts in the dividing polyploid cells show that they fall not only into the series 4M, 8ra, i6«, 32W, but include i2«,
2ow, 24«, etc. These results cannot be explained by endomitosis or by the restitution
of nuclei after mitosis.
The possibility that some of this sporadic polyploidy may arise by endomitosis
or by nuclear restitution during mitosis cannot be excluded, and it is certain that many
nuclei which have not completely fused during interphase will amalgamate their
chromosomes into a single spindle at metaphase (Fig. 4) as demonstrated by Nadal &
Zajdela (1966) in the liver of the rat.
Extensive nuclear fusion has been described in the trophocytes (nurse cells) in the
ovaries of the milkweed bug Oncopeltus (Bonhag, 1955). These fused nuclei, however,
are not viable structures; they are in process of disintegration and incorporation into
the yolk of the oocyte. But fusion of nuclei to give viable polyploid cells, as in Rhodnius,
was described by Eilers(i925)in the fat body of Melasoma (Coleoptera, Chrysomelidae).
On the basis of chromosome counts Nur (1966) has suggested that in cells believed
to be oenocytes of the mealy bug Planococcus citri both endomitosis and nuclear
fusion may be taking place at the same time.
Viable nuclear fusions have been induced in mammalian cells in culture, even
between cells from different animal species, by exposure to certain viruses (Okada &
Tadokoro, 1963; Okada & Murayama, 1965). Even inactivated virus will induce such
fusions with the formation of hybrid cells of mouse and man which continue to undergo
normal mitosis (Harris, Watkins, Ford & Schoefl, 1966). IntheRhodnius fat body it seems
to be the nutritive deficiencies in extreme starvation which lead to cellular and nuclear
fusions. But it is impossible to be certain that there is not some latent virus present.
This phenomenon in Rhodnius is not regarded as having any special physiological
significance. It probably comes about because of the extremely low rate of metabolism.
In most insects, death from starvation occurs so rapidly that there is no time for the
slow process of cellular and nuclear fusion to take place. In the ist-stage larva of
Rhodnius, in which death from starvation takes place in about 3 weeks, there may be
a little cellular fusion with the formation of binucleate cells, but there has not been
sufficient time for nuclear fusion to occur.
The fusion of nuclei appears to be a slow process. Two or more nuclei in close
contact, with no visible membrane separating the components, may retain an irregular
614
V. B. Wigglesworth
form; the approximate outline of each nucleus persists for some time. Complete fusion,
to form an evenly rounded nucleus is sometimes delayed until the nuclear membrane
is dispersed at mitosis. It follows therefore that if, as is usually supposed, the nuclear
sap has a fluid consistency, the nuclear membrane must be sufficiently rigid to maintain the form of the nucleus.
I am indebted to Dr C. L. Smith for carrying out the photometric measurements of DNA.
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(Received 15 May 1967)
6i6
Fig. 5. Fat body of recently hatched ist-stage larva (Carnoy; haematoxylin) showing
one octoploid nucleus, remainder tetraploid.
Fig. 6. Fat body of newly fed 4th-stage larva (series A, see text, unstarved; Carnoy;
haematoxylin) showing uniform tetraploid nuclei.
Fig. 7. As Fig. 6, showing one octoploid nucleus.
Fig. 8. As Fig. 6, showing two pairs of tetraploid nuclei closely approximated.
Figs. 9-11. Fat-body cells of 4th-stage larva in extreme starvation, 30 weeks (osmium/
ethyl gallate), showing dead nucleus (white arrow) and living nucleus (black arrow)
in the same cell. Granules in fat-body cells are mitochondria and cytolysomes;
granules in epidermal cells seen in the background are mostly pigment granules.
Journal of Cell Science, Vol. 2, No. 4
V. B. WIGGLESWORTH
(Facing p. 616)
Fig. 12. Fat-body cells of 4th-stage larva starved 31 weeks (osmium/ethyl gallate),
showing binucleate cell (centre right) and fusing nuclei (bottom left). Cytoplasm
shows mitochondrial granules, minute vacuoles, and a few darkly stained lipid
droplets.
Fig. 13. As Fig. 12, showing tetraploid nucleus at middle right with fusing nuclei
above and below; large rounded nuclei top left.
Fig. 14. As Fig. 12, showing two cells, each with at least three nuclei in process of
fusion.
Fig. 15. Fat-body cells of 4th-stage larva after prolonged starvation (series B and C
in text; Carnoy; haematoxylin), showing all stages of nuclear fusion.
Fig. 16. As Fig. 15 (stained pyronin/methyl green), showing in the middle 5 tetraploid
nuclei in process of fusion.
Figs. 17, 18. As Fig. 15.
Fig. 19. Fat-body cells of adult starved 11 weeks from the final moult (Carnoy;
haematoxylin), showing tetraploid nuclei above and giant nucleus below.
Journal of Cell Science, Vol. 2, No. 4
V, B. WIGGLESWORTH
Fig. 20. Fat-body cells of severely starved 4th-stage larva 8 days after feeding (Carnoy;
haematoxylin), showing 471 nucleus in metaphase.
Fig. 21. The same with probable 32W nucleus in metaphase.
Fig. 22. The same, showing triradiate metaphase.
Fig. 23. Acetic-orcein squash preparation of fat-body cells from 4th-stage larva
(32 weeks starved) at 8 days after feeding; \n nucleus in metaphase.
Fig. 24. The same: 8n nucleus in metaphase.
Fig. 25. The same: i6w nucleus in metaphase.
Fig. 26. The same: 32M nucleus in metaphase.
Fig. 27. The same: triradiate metaphase with about 300 countable chromosomes.
Journal of Cell Science, Vol. 2, No. 4
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