Germ cell cluster formation and ovariole structure in
viviparous and oviparous generations of the aphid
GRAZYNA PYKA-FOSCIAK and TERESA SZKLARZEWICZ*
Department of Systematic Zoology, Institute of Zoology, Jagiellonian University, Krakow, Poland
ABSTRACT The developing ovaries of S. quercus contain a limited number of oogonial cells which
undergo a series of incomplete mitotic divisions resulting in the formation of clusters of
cystocytes. Ovaries of viviparous generations contain 6 to 9 clusters, containing 32 cystocytes
each, whereas ovaries of oviparous generations contain 5 clusters containing 45-60 cystocytes.
During further development, clusters become surrounded by a single layer of follicular cells, and
within each cluster the cystocytes differentiate into oocytes and trophocytes (nurse cells).
Concurrently, cysts transform into ovarioles. The anterior part of the ovariole containing the
trophocytes becomes the tropharium, whereas its posterior part containing oocytes transforms
into the vitellarium. The vitellaria of viviparous females are composed of one or two oocytes,
which develop until previtellogenesis. The nuclei of previtellogenic oocytes enter cycles of mitotic
divisions which lead to the formation of the embryo. Ovarioles of oviparous females contain a
single oocyte which develops through three stages: previtellogenesis, vitellogenesis and
choriogenesis. The ovaries are accompanied by large cells termed bacteriocytes which harbor
KEY WORDS: aphids, Stomaphis quercus, oogenesis, ovariole, cystocyte
Ovarioles of insects are categorized into two types: panoistic
and meroistic (for further details see Stys and Bilinski, 1990;
Buning, 1994; Bilinski, 1998). Panoistic ovarioles contain oo-
cytes, which form by mitotic divisions (with complete cytokinesis)
of oogonial cells. In meroistic ovarioles oogonial cells (termed
cystoblasts) undergo divisions with incomplete cytokinesis result-
ing in the formation of the clusters of interconnected cells termed
cystocytes. In each cluster one (in polytrophic ovarioles) or more
(in telotrophic ovarioles) cystocyte(s) differentiate(s) into oocyte(s),
while the remaining cells become the trophocytes (nurse cells).
In aphids, like in other hemipterans, ovaries consist of telotrophic
ovarioles (for description of hemipteran ovaries see Buning,
1994). Each ovariole contains single cluster of germ cells. In
advanced families Aphididae and Drepanosiphidae, the initial
cystoblast undergoes a series of usually five incomplete mitotic
divisions forming cluster of 32 cystocytes (Blackmann, 1978;
Buning, 1985). In contrast to advanced aphids, in the primitive
family Adelgidae, the cystoblast undergoes five or six successive
Int. J. Dev. Biol. 52: 259-265 (2008)
THE INTERNATIONAL JOURNAL OF
*Address correspondence to: Dr. Teresa Szklarzewicz. Department of Systematic Zoology, Institute of Zoology, Jagiellonian University, R. Ingardena 6,
PL-30060 Krakow, Poland. Tel: +4812-663-2437. Fax: +4812-634-3716. e-mail: email@example.com
Published online: 14 February 2008
© UBC Press
Printed in Spain
mitotic cycles followed by the supernumerary divisions of indi-
vidual cystocytes, which result in the formation of the clusters
containing 50-92 cystocytes (Szklarzewicz et al., 2000). In evolu-
tionary advanced families, number of oocytes per cluster is
species specific (e.g. in Drepanosiphum platanoides 16 cystocytes
become oocytes, in Metopolophium dirhodum – 8), whereas in the
evolutionary primitive family Adelgidae this number is not con-
stant and ranges from 14 to 27 (Buning, 1985; Szklarzewicz et al.,
2000). The remaining cystocytes differentiate into trophocytes.
Since the basic function of these cells is to synthesize RNA-s,
their nuclei undergo cycles of endoreplication. Manicardi et al.,
(1995) showed that the DNA content in the trophocyte nuclei of
oviparous generations of an aphid Megoura viciae is equal to
2048C, whereas in viviparous generations to16C.
The ovariole of adult aphids has four well-defined regions: a
terminal filament, trophic chamber (tropharium), vitellarium, and
ovariole stalk (pedicel) that joins the ovariole with the lateral
oviduct (Blackmann, 1978; Buning, 1985; Szklarzewicz et al.,
2000). The trophic chamber contains the trophocytes and the
early previtellogenic oocytes termed arrested oocytes. The cen-
260 G. Pyka-Fosciak and T. Szklarzewicz
tral area of the trophic chamber, termed a trophic core, is free of
cells and is filled with the bundles of microtubules. The trophic
chamber is surrounded by an inner epithelial sheath of flat
somatic cells. In the vitellarium usually two oocytes develop. The
vitellarial oocytes are surrounded by a single layer of follicular
Comparative studies on ovaries of five aphid species, repre-
sentatives of family Aphididae and Drepanosiphidae, showed that
besides the difference in the level of the polyploidy of trophocyte
nuclei, viviparous and oviparous generations differ in the function-
ing of the ovaries (Buning, 1985). In viviparous generations the
previtellogenic growth of the oocytes is shortened and vitellogen-
esis as well as choriogenesis are completely blocked.
In the present studies we examined the differences in the
development of ovarioles in the generations of an aphid Stomaphis
quercus (the life cycle of S. quercus is described in Material and
Methods), a representative of the family Lachnidae.
In all the generations of Stomaphis quercus, when the embryo
completes the formation of the blastoderm, the paired ovaries
form at the posterior pole of the embryo (Fig. 1A). Each ovary
contains several oogonial cells (Fig. 1A). Simultaneously, the
embryos are invaded by endosymbiotic microorganisms (Fig.
1A). Somewhat later, the oogonial cells enter the cycles of mitotic
divisions with incomplete cytokineses resulting in a formation of
clusters of cells termed cystocytes connected by intercellular
bridges (Fig. 1B). The ovaries of viviparous generations form 6 to
9 clusters, while the oviparous generation – form 5 clusters. The
lumen of the intercellular bridges is packed with parallel arranged
microtubules and small, smooth vesicles (Fig. 1C). In the next
step of cyst development, the cystocytes arrange into a rosette
and form a spherical cyst (Fig. 1D). Cysts become covered with
a single layer of flat follicular cells (Fig. 1D). Clusters of viviparous
generations (fundatrix, V1, V2, sexupara) are composed of 32
cystocytes, and clusters of oviparous generation (sexualis) con-
tain 45 to 60 cystocytes. All cystocytes in the cluster are morpho-
logically similar. They are cone-shaped (Fig. 1D) and their nuclei
are large and spherical (Fig. 1D, E). The nuclei in viviparous and
in oviparous generations measure about 6 µm in diameter. The
cystocyte cytoplasm is packed with ribosomes and mitochondria
(Fig. 1E). In the perinuclear cytoplasm accumulations of electron-
dense “nuage” material are present (Fig.1E). Cystocytes open via
intercellular bridges into the centre of the rosette (Fig. 1F). In
contrast to other hemipterans, the centre of the rosette of Stomaphis
quercus does not contain the polyfusome (Fig. 1F). During further
development, cystocytes differentiate into oocytes and trophocytes
(Fig. 2 A, B). Analysis of serial sections of 10 clusters from each
generation showed that the clusters of fundatrix, V1, V2 and
sexupara generations contain 8 oocytes, whereas the clusters of
sexualis generation contain 5 to 8 oocytes. In next step of
development the cysts elongate and transform into young ovari-
oles (Fig. 2C, D). The oocytes occupy the posterior pole of the
ovariole, while the trophocytes are localized in its anterior part
(Fig. 2C, D). Simultaneously, in the centre of the cluster a trophic
core develops (Fig. 2E). During further development, trophocyte
nuclei enlarge. In viviparous generations their diameter increases
to about 10 µm, while in oviparous generation to about 30 µm. The
trophocyte nucleoli become prominent (Figs 2C, D, E, 3A, B, C,
4A). Additionally, the trophocyte nuclei of oviparous generation
change their shape from spherical (Fig. 2B) to irregular (Fig. 2D).
The follicular cells differentiate into two groups: (1) the cells of an
inner epithelial sheath and (2) the proper follicular cells (Fig. 2C,
D). The inner epithelial sheath envelops the anterior pole of the
ovariole containing the trophocytes (Fig. 2C, D). The proper
follicular cells surround the posterior pole of the ovariole which
contains the oocytes (Fig. 2C, D). During further development, the
anterior part of the ovariole transforms into the tropharium and the
posterior part into the vitellarium (Fig. 2C).
Fig. 1. Formation of clusters of germ cells. (A) Longitudinal section
through the young embryo. The ovaries are filled with oogonial cells
(encircled). The posterior pole of the embryo is invaded by endosymbiotic
microorganisms (arrow). Methylene blue; bar, 20 µm. (B) Cross section
through the ovary showing the dividing cystocytes (arrows) and clusters
of cystocytes forming the rosettes (encircled). Methylene blue; bar, 20
µm. (C) Cross section through the intercellular bridge connecting two
neighboring cystocytes (C) showing the microtubules (arrows), the ring
canal rim (arrowhead) and vesicles (encircled). TEM; bar, 1 µm. (D) Cysts
filled with cystocytes. Follicular cells (arrows) surround each cyst. Meth-
ylene blue; bar, 20 µm. (E) Fragment of the cluster of cystocytes during
rosette formation. The intercellular bridges (asterisks), and the accumu-
lations of “nuage” material (arrows) in the perinuclear cytoplasm are
visible. TEM; bar, 2 µm. (F) Central part of the fully grown cyst with the
intercellular bridges (asterisks). TEM; bar, 2 µm. B1, B2, bacteriocytes
containing two different types of endosymbiotic bacteria; BL, blasto-
derm; C, cystocyte; CN, cystocyte nucleus; CNU, cystocyte nucleolus; F,
follicular epithelium; M, mitochondria; OV, lateral oviduct.
Germ cell clusters in aphids 261
In oviparous females ovaries are composed of 5 ovarioles.
Usually, 1-4 of them degenerate during previtellogenesis. As a
rule, in females collected in September the number of degenerat-
ing ovarioles is smaller than in females collected at the end of
October. In viviparous females number of ovarioles per ovary is
not constant and ranges between 6 and 9. Each ovariole contains
long, inconspicuous terminal filament (not shown), trophic cham-
ber (Figs 3A, B, 4A), vitellarium (Fig. 3A) and pedicel (not shown).
Terminal filaments of all ovarioles are joined together into a
suspensory ligament that anchors the ovary to the fat body (not
shown). The tropharium is sphere-shaped with diameter about 65
µm in viviparous females and about 300 µm in oviparous females.
It is composed of trophocytes and arrested oocytes (Figs 3A, 4A,
F). The latter are always located at the base of tropharium (Figs
3A, 4A, F). The trophic chamber is covered with an inner epithelial
sheath, which consists of a single layer of flat somatic cells with
slender projections (Fig. 3C). The centre of the trophic chamber
is occupied by a common cytoplasmic area, termed a trophic core
(Figs 3B, 4A). The trophic core of oviparous females is much
larger than in viviparous females. Its diameter measures about 70
µm, while in viviparous females 20 µm. The core is filled with
bundles of microtubules (not shown). The trophocytes form pro-
cesses which extend into the trophic core (Figs 3B, 4A). The
trophocyte membranes in the vicinity of the trophic core are folded
and intertwined (not shown). Trophocytes possess large nuclei
with giant, single nucleoli (Figs 3A, B, C, 4A). Numerous pores
perforate the nuclear envelope (Fig. 4E). The perinuclear cyto-
plasm contains accumulations of electron-dense “nuage” mate-
rial (Figs 3C, 4E). In all the generations “nuage” is accompanied
by groups of mitochondria (Figs 3C, 4E). The remaining cyto-
plasm is filled with ribosomes and mitochondria (Fig. 3C). In
addition, the trophocyte cytoplasm contains numerous rod-shaped
endosymbiotic microorganisms (Fig. 4G). Arrested oocytes have
large, spherical nuclei with decondensed chromatin (Figs 3A, 4F).
In oviparous generation, arrested oocytes have small amount of
cytoplasm (Fig. 4F). In viviparous generations arrested oocytes
Fig. 2 (Left). Transformation of sphere-shaped cysts into young
ovarioles. (A, B) Cystocytes differentiate into oocytes (O) and trophocytes
(T). The centre of the cluster is occupied by the trophic core (TC). Clusters
are surrounded by a one-layered follicular epithelium (arrows). (A) Vivipa-
rous generation, (B) Oviparous generation. (A, B) Methylene blue; bar, 20
µm. (C, D) Young ovarioles. Follicular cells are differentiated into the cells of an inner epithelial sheath (IS) which surround the trophocytes (T) and
the proper follicular cells which surround the oocytes (O). (C) Viviparous generation, (D) Oviparous generation. (C, D) Methylene blue; bars: (C) 20 µm;
(D) 50 µm. (E) Central part of the young ovariole of viviparous generation. Note a characteristic labyrinth of trophocyte membranes. TEM; bar, 2 µm.
AO, arrested oocyte; B1, bacteriocytes; F, follicular cells; IS, inner epithelial sheath; O, oocyte; ON, oocyte nucleus; T, trophocyte; TC, trophic core;
TN, trophocyte nucleus; TNU, trophocyte nucleolus.
Fig. 3 (Right). Ovariole of viviparous female. (A, B) Longitudinal section through the ovariole. Nutritive cord (arrow), broad processes of trophocytes
(arrowheads). (A, B) Methylene blue; bar, 20 µm. (C) Fragment of tropharium. Arrows indicate accumulations of “nuage” material. TEM; bar, 5 µm.
(D) Fragment of follicular epithelium (F) and oocyte (O) during early previtellogenesis. TEM; bar, 2 µm. (E) Fragment of the embryo. Methylene blue;
bar, 20 µm. (F) Ovariole containing two embryos (E1 and E2). Methylene blue; bar, 50 µm. F, follicular epithelium; FN follicular celll nucleus; IN, inner
epithelial sheath cell nucleus; IS, inner epithelial sheath cell; L, lipid droplets; O, oocyte; ON, oocyte nucleus; T, trophocyte; TC, trophic core; TN,
trophocyte nucleus; TNU, trophocyte nucleolus; TR, tropharium.
262 G. Pyka-Fosciak and T. Szklarzewicz
are capable of further development, whereas in oviparous females
they degenerate. In the vitellaria of viviparous generations two
oocytes develop, while in oviparous females only one. The vitellarial
oocyte is connected to the core by the broad nutritive cord (Figs 3A,
4A). The nutritive cord is filled by parallel-arranged microtubules
and ribosomes (Fig. 4I). The nutritive cords of older ovarioles also
contain numerous mitochondria and rod-shaped endosymbiotic
bacteria (not shown). In viviparous generations, oocyte surface is
usually smooth and the microvilli are rare and short (Fig. 3D). The
oocytes of viviparous females do not accumulate yolk and do not
become covered with egg envelopes. The only reserve substances
which are accumulated in these oocytes, are lipid droplets (Fig.
3A). Ultrastructure of follicular cells surrounding the oocyte indi-
cates that these cells are not active synthetically (Fig. 3D). Thus,
the period of the oocyte growth is very short and stops at the stage
of previtellogenesis. Immediately after previtellogenesis, the oo-
cyte nucleus starts to divide to form the embryo (Fig. 3E, F). The
embryos are surrounded by closely adhering follicular cells (Fig.
3E, F). When the formation of blastoderm is completed, the
follicular cells covering the posterior pole of the embryo separate
from each other generating wide opening that enables entry of
endosymbiotic microorganisms (Fig. 1A).
Within the body of embryos that develop in fundatrix, V1 and V2
females, the next generations of embryos arise (Fig. 3F). Thus, the
body of fundatrix, V1 and V2 females contains two generations of
embryos. In contrast, the embryos developing in sexupara females
develop into males and females of last generation (sexualis).
In oviparous females, oocyte undergoes three stages of growth:
previtellogenesis, vitellogenesis and choriogenesis. The onset of
vitellogenesis is manifested by many morphological changes within
the oocyte: the oolemma starts to form microvilli and numerous
endocytotic vesicles form in the cortical ooplasm (not shown).
Later, the first yolk granules are formed (Fig. 4H). As vitellogenesis
progresses, the size of the ooocyte increases through the accumu-
lation of yolk granules and lipid droplets in its cytoplasm (Fig. 4D,
J). Follicular cells surrounding the previtellogenic and early
vitellogenic oocytes are cylindrical and closely adhere to each
other (Fig. 4A, B, C). The cytoplasm of follicular cells contains
ribosomes and mitochondria (Fig. 4C). During mid-vitellogenesis
follicular cells separate from each other, generating wide spaces
between neighboring cells (Fig. 4D). During advanced vitellogen-
esis these cells diversify into two subpopulations: the main body
cells and cells surrounding the posterior pole of the oocyte. Both
subpopulations are morphologically similar, but the main body
cells start to produce precursors of egg envelopes much earlier
than the latter (Fig. 4J, arrows). Concurrently, the endosymbiotic
bacteria invade the posterior pole of the oocyte (Fig. 4J). These
microorganisms migrate between neighboring follicular cells (Fig.
4K), enter the perivitelline space, traverse the oolemma and enter
the ooplasm (Fig. 4J). When the migration of endosymbionts is
completed, the posterior cells start to produce precursors of egg
The large cells termed the bacteriocytes accompany the ovaries
of viviparous and oviparous females (Figs. 1 A,B,D, 2B). In
Stomaphis there are two types of bacteriocytes: bacteriocytes
containing large, spherical bacteria belonging to the genus Buchnera
aphidicola (Figs 1A, B, D, 2B, 3F) and bacteriocytes housing
smaller, coccoid bacteria (Fig. 1A). Apart from endosymbionts that
are harbored in the cytoplasm of the bacteriocytes, the rod-shaped,
small bacteria are also present in the cytoplasm of the germ cells
(i.e. cystocytes, trophocytes and oocytes) (Fig. 4G).
Morphology of the ovaries
Present and previous studies have shown that the general
organization of ovaries is similar in different families of aphids
Fig. 4. Ovariole of oviparous female. (A) Longitudinal section through
the ovariole. Broad processes of trophocytes (arrowheads), arrested
oocyte (arrow). Methylene blue; bar, 20 µm. (B,C) Fragment of early
vitellogenic oocyte (O) and follicular epithelium (F). (B) Methylene blue;
bar, 20 µm. (C) TEM; bar, 2 µm. (D) Fragment of oocyte (O) and follicular
epithelium (F) during mid-vitellogenesis. Note the presence of wide
spaces between neighboring cells (arrowheads). Methylene blue; bar, 20
µm. (E) Fragment of trophocyte nucleus and perinuclear cytoplasm.
Asterisk indicates accumulation of “nuage” material associated with
mitochondria (M). TEM; bar, 1 µm. (F) Cross section through the basal
part of tropharium. Methylene blue; bar, 20 µm. (G) Fragment of tropho-
cyte cytoplasm filled with endosymbiotic microorganisms. TEM; bar, 2
µm. (H) Fragment of follicular cell (F) and cortical ooplasm (O). Endocy-
totic vesicles (arrows), microvilli (asterisks), yolk granule (encircled).
TEM; bar, 1 µm. (I) Fragment of the nutritive cord packed with microtu-
bules (arrows). TEM; bar, 1 µm. (J) Posterior pole of the oocyte.
Incomplete egg envelopes (arrows), endosymbiotic bacteria (encircled).
Methylene blue; bar, 20 µm. (K) Endosymbiotic bacterium (encircled)
during migration between neighboring follicular cells. TEM; bar, 2 µm.
AO, arrested oocytes; F, follicular epithelium; FN, follicular cell nucleus;
NC, nutritive cord; O, oocyte; ON, oocyte nucleus; T, trophocyte; TC,
trophic core; TN, trophocyte nucleus.
Germ cell clusters in aphids 263
(Blackmann, 1978; Buning, 1985, Szklarzewicz et al., 2000). There
are, however, differences in ovary structure in advanced versus
primitive families as well as in viviparous females versus oviparous
females. In advanced families such as Aphididae and
Drepanosiphidae (Buning, 1985), the ovarioles contain 32 germ
cells, whereas in the primitive family Adelgidae (Szklarzewicz et
al., 2000) number of germ cells is much larger and varies from 50
to 92. These indicate that during the phylogeny of aphids, like in
phylogeny of scale insects, the reduction of the number of germ
cells per cluster took place (Szklarzewicz et al., 2000). Buning
(1985) studied ovaries of five aphid species belonging to families
Aphididae and Drepanosiphidae and found that in viviparous
generations there is a shortening of the previtellogenic growth of
the oocyte, vitellogenesis and choriogenesis are absent, and the
trophocyte nuclei have a lower level of ploidy than those in
oviparous generation. Our studies showed that ovaries of vivipa-
rous and oviparous females of Stomaphis quercus, a member of
family Lachnidae, differ in the number of ovarioles, number of germ
cells per ovariole and course of oogenesis. In oviparous females
ovaries contain 5 ovarioles, whereas in viviparous females the
number of ovarioles is larger and variable. Since Leather et al.,
(1988) made similar observations for the representatives of five
families of aphids, it is possible that the variability in ovariole
numbers is typical for the viviparous generations.
In viviparous females all ovarioles develop, while in oviparous
females some of them degenerate. Moreover, because the num-
ber of degenerating ovarioles is much larger in late autumn than in
early autumn females, the late autumn females lay less eggs. It
seems probable that the number of degenerating ovarioles (and
laid eggs) is regulated by the availability of nutrients (i.e. quality of
phloem sap). This hypothesis corresponds well with the observa-
tions of Wiktelius and Chiverton (1985) that the bird cherry-oat
aphids, Rhopalosiphum padi feeding on low-quality hosts have
lower number of ovarioles.
Analysis of serial sections showed that the ovarioles of ovipa-
rous females of Stomaphis quercus contain more trophocytes
than those of viviparous generations. In addition, the trophocytes
of oviparous females are much larger than those in viviparous
females. This fact indicates that the level of ploidy of trophocytes
is much higher in oviparous than in viviparous females. Manicardi
et al., (1995) have demonstrated, using cytofluorometric analysis,
that in an aphid Megoura viciae, the enlargement of trophocyte
nuclei is related to the overreplication of the whole genome.
These authors have experimentally proved that the initiation of
endoreplication is correlated with the formation of ommatidia in
the embryo. This finding strongly supports an earlier hypothesis
that the degree of DNA overreplication in trophocyte nuclei of
aphids is influenced by photoperiod (Lees, 1973). In addition, the
transition from viviparous generations to oviparous generation
correlates with the shortening of day length (Lees, 1960; Dixon,
In viviparous and in oviparous females of Stomaphis quercus,
the trophocytes exhibit all characteristics typical for the trophocytes:
large nuclei containing massive nucleoli, accumulation of “nuage”
material in the close neighborhood of nuclei and enormous
number of ribosomes in the cytoplasm. Such an ultrastructure is
related to the basic function of trophocytes, i.e. synthesis of RNA-
s and subsequent transport of ribosomes, mitochondria and
endosymbiotic microorganisms via nutritive cords to the develop-
ing oocytes. Like in other hemipterans, nutritive cords of Stomaphis
are tightly packed with microtubules that are responsible for the
transport of macromolecules as well as organelles from trophic
chamber into developing oocytes (Stebbings, 1988).
The main differences in the functioning of ovaries between
viviparous and oviparous females concern the length and mode
of oocyte growth. In oviparous females, oocytes accumulate yolk
(during vitellogenesis) and become covered with egg envelopes
(during choriogenesis), whereas in viviparous females oocytes
stop developing at the end of previtellogenesis. In viviparous and
in oviparous females oocytes are surrounded by a single layer of
follicular cells. However, ultrastructural observations showed that
follicular cells are synthetically active in the oviparous generation
only. In this generation, follicular cells diversify into two subpopu-
lations - the main body cells and posterior cells. The former start
to synthesize precursors of egg envelopes much earlier than the
posterior cells which results in the temporary lack of egg enve-
lopes in the region of the posterior pole of the oocyte. The
incomplete egg coverings enable the entry of endosymbiotic
bacteria from the body cavity into the ovaries (see below). Such
phenomenon is very rare within insects and has been described
previously only in the scale insect Palaeococcus fuscipennis
(Szklarzewicz et al., 2006).
The existence of the “embryo in embryo” is of special interest.
Moran (1992) called this strategy of development as “a conse-
quent telescoping of generations”. According to this author, a
beginning of development before the mother is born results in the
shortening of postnatal developmental period and leads to higher
rate of reproduction.
Development of clusters of germ cells
Our observations showed that in viviparous as well as in
oviparous generations of Stomaphis quercus, development of
clusters of germ cells occurs through the similar pathway, and the
main differences lie in the number of germ cells present in the
clusters. In contrast to representatives of family Aphididae and
Drepanosiphidae (Buning, 1985) in which all the generations
have the same number (32) of cystocytes, the clusters of ovipa-
rous females of Stomaphis are composed of larger number of
cystocytes (i.e. 45-60) than those in viviparous females (i.e. 32).
Moreover, clusters in oviparous females do not obey Giardina’s
rule, which states that the cystocyte number (N) is given by the
formula: N= 2n, where “n” defines the number of cystoblast
divisions. Since we never observed symptoms of degeneration of
cystocytes, we postulate that their irregular number is the conse-
quence of asynchrony of their divisions. Thus, the initial cystoblast
undergoes a series of five divisions and some of the cystocytes
enter the sixth division. Similar situation has been described in
polytrophic ovaries of neuropterans (Kubrakiewicz, 1997) and
beetles (Jaglarz, 1998) and in telotrophic ovaries of scale insects
(Szklarzewicz and Bilinski, 1995; Szklarzewicz, 1997, 1998a, b)
and primitive aphids (Szklarzewicz et al., 2000).
Our detailed studies of development of clusters clearly show
that each cyst transforms into single ovariole. Thus, the individual
ovariole contains single cluster of interconnected germ cells.
Buning (1985) suggested that in aphids, each ovariole-anlage
contains several germ cells. Then, only one of them starts to
divide, whereas the remaining cells degenerate. However, our
analysis of serial sections has shown that in Stomaphis, the
264 G. Pyka-Fosciak and T. Szklarzewicz
number of oogonial cells is limited and these cells do not exhibit
any symptoms of degeneration, which suggests that each
oogonial cell develops into the cluster.
The results of present study and Buning’s (1985) observa-
tions of developing ovaries in Aphididae and Drepanosiphidae,
indicate that the most characteristic feature of developing
clusters is a lack of recognizable fusomal material inside the
intercellular bridges and consequently lack of a polyfusome
extending through the ring canals into all cystocytes. It is
generally accepted that the polyfusome is responsible for the
coordination of cystocyte divisions, i.e. their synchronization,
restriction of their number, arrest of cystocyte cytokinesis and
the differentiation of germ cells (see Storto and King, 1989; de
Cuevas et al.,1997 for further details). The presence of
polyfusomes has been observed in scale insects (sister group
of aphids) (Szklarzewicz, 1997, 1998a, b) as well as in remain-
ing hemipterans (Buning, 1994). According to Buning (1985),
the role of polyfusome in aphid clusters may be substituted by
persisting microtubules. Our observations (see Fig. 1C) strongly
support this hypothesis. Buning (1998) suggested also that the
aphids might contain fusome but it is underdeveloped or short-
lived which makes it hard to visualize. For the same reasons
also the presence of polyfusome may be overlooked. Similar
situation has been described in two other closely related groups,
in snakeflies and alderflies. Jedrzejowska and Kubrakiewicz
(2004) detected polyfusome in developing clusters of the
snakefly Raphidia, whereas the alderfly Sialis lacks the
polyfusome (Buning, 1979). Jedrzejowska and Kubrakiewicz
(2004) postulated that the polyfusome in alderflies might be
easily overlooked due to the short period of its occurrence.
We observed that the lumen of intercellular bridges of S.
quercus contains numerous, small, and ribosome deficient
vesicles. Similar structures have been found inside the bridges
connecting cystocytes in Drosophila melanogaster (Koch and
King, 1966; Mahowald, 1971). Until recently, the nature and
origin of these vesicles were unknown, but Snapp and co-
workers (2004) using live cell imaging techniques found them to
be elements of the continuous endoplasmic reticulum which
extends through all the cystocytes constituting the cluster.
According to these authors, common endoplasmic reticulum
may be responsible for the synchronization of mitotic divisions.
Transovarial transmission of endosymbiotic bacteria
The ovaries of Stomaphis, like of most aphids, are accompa-
nied by large cells termed bacteriocytes. The cytoplasm of
these cells is tightly packed with endosymbiotic bacteria. It is
generally known that presence of endosymbionts (bacteria or
yeasts) in the insect body is related to their restricted diet, i.e.
diet deficient in some essential substances (see Douglas,
1989, 1998; Bauman, 2005, for further details). Since aphids
feed on phloem poor in essential amino acids, their endosym-
bionts are responsible for the amino acid synthesis and their
delivery to the host insect (Douglas and Prosser, 1992). Numer-
ous studies have shown the most aphids contain the endosym-
biotic bacterium Buchnera aphidicola (Baumann et al., 1995;
Douglas, 1998; Sabater et al., 2001; Baumann, 2005). Besides
Buchnera, aphids usually harbor additional endosymbionts
(called secondary endosymbionts) (Buchner, 1965; Sabater et
al., 2001; Baumann, 2005). The bacterium Buchnera, like
primary endosymbionts of other insects, is essential for growth
and reproduction of the host insect, whereas the role of second-
ary endosymbionts is still under discussion (Baumann, 2005).
Primary endosymbionts are transmitted from one generation to
the next transovarially (vertically), i.e. through the female ga-
metes. In contrast, secondary endosymbionts may be inherited
vertically and/or horizontally. Our studies have shown that in
the body of Stomaphis quercus harbors Buchnera and two
types of secondary endosymbionts. In Stomaphis, both Buchnera
and secondary endosymbionts are transmitted transovarially.
Although endosymbioses (i.e. types of microorganisms, their
distribution, function and evolution) in aphids have been exten-
sively studied, the mode of endosymbiont transmission is poorly
known. Buchner (1965) described the process of egg infection
in several aphid species. Miura et al. (2003) and Braendle et al.
(2003) observed the origin of bacteriocytes in Acyrthosiphon
pisum, a member of family Aphididae. However, there is no
ultrastructural data concerning this process. In vivipaparous
generations of Stomaphis quercus bacteria invade embryos,
whereas in oviparous generation oocytes are infested. In all the
generations, the beginning of the invasion is correlated with the
development of the germ cells. Embryos formed in viviparous
generations are invaded at the time the germ cells start to form
ovaries. In oviparous generation bacteria invade choriogenic
oocytes. Thus, our observations support earlier hypothesis that
the migration of endosymbionts into ovaries is induced by an
unknown factor released from the ovaries (Eberle and Mc Lean,
1982; Zelazowska and Bilinski, 1999; Szklarzewicz and Moskal,
2001; Szklarzewicz et al., 2006).
Materials and Methods
A Stomaphis quercus life cycle consists of five yearly generations
(Lorenz and Scheurer,1998). The first four generations (i.e. fundatrix,
V1, V2, sexupara) are viviparous and reproduce parthenogenetically.
The last, bisexual generation (i.e. sexualis) appears in the autumn.
Sexualis females are oviparous. These females lay fertilized eggs that
overwinter. In the spring these eggs hatch into larvae that develop into
the first viviparous generation (i.e. fundatrix).
Adult females of Stomaphis quercus (L.) were collected from stems
of Betula verrucosa and Quercus sessilis in Beskid Sadecki Mountains
and Ojcow National Park (south Poland). The females of viviparous
generations have been collected from May to September. The females
of oviparous generation have been collected in September and Octo-
Light and electron microscopy
Dissected embryos and ovaries were fixed in 2.5% glutaraldehyde
in 0.1 M phosphate buffer (pH 7.4) at room temperature. After being
washed in phosphate buffer, the material was postfixed in 1% osmium
tetroxide in 0.1 M phosphate buffer (pH 7.4), dehydrated in a series of
alcohol and acetone, and embedded in epoxy resin Epox 812 (Fullam
Inc., Latham, N.Y., USA). Semithin sections were stained with 1%
methylene blue in 1% borax and photographed in a Jenalumar (Zeiss
Jena) microscope. Ultrathin sections were stained with lead citrate and
uranyl acetate and examined using a JEM 100 SX electron microscope
at 80 kV.
We would like to express our gratitude to Professor Szczepan
Bilinski for suggesting this species for examination and providing first
Germ cell clusters in aphids 265
specimens. We also thank to M.Sc. Wladyslawa Jankowska and Dr.
Beata Szymanska for their skilled technical assistance. This work was
supported by funds from the research grant from Ministry of Science
and Informatization: 2 PO4C 004 27.
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