germ cell cluster differentiation in polytrophic ovarioles of hanging

71
Zoologica
G E R(2003)
M - C E L L48/1-4:
- I N - O V71-79
ARIOLES-OF-HANGING-FLIES
Poloniae
71
GERM CELL CLUSTER DIFFERENTIATION
IN POLYTROPHIC OVARIOLES OF HANGING-FLIES
(MECOPTERA: BITTACIDAE)
B O¯ENA S IMICZYJEW
Department of General Zoology, Zoological Institute, University of Wroc³aw,
Sienkiewicza 21, 50-335 Wroc³aw, Poland
Abstract. The results of histological, histochemical and ultrastructural studies
on the germ cell cluster formation and differentiation in three bittacids: Bittacus hageni,
B. nipponicus and B. strigosus are presented in comparison with well investigated
panorpid Panorpa communis. The data indicate several similarities between bittacids
and panorpids; the only insignificant differences involve the organization of the nurse
cells. The phylogenetic relationships between hanging-flies and scorpion-flies are discussed in the context of presented results.
INTRODUCTION
Insect ovaries are built of ovarian tubes termed ovarioles. Two basic types
of ovarioles are distinguished: panoistic and meroistic (B RANDT , 1874). The
classification of these ovarioles is based on the ultimate fate of the developing
germ cells. In the panoistic ovary all germ cells can become oocytes while in
meroistic ovaries divisions of gonial cells lead to formation of clusters of sibling
cells which remain interconnected by intercellular bridges forming a syncytium.
The cells within a cluster (cystocytes) diversify into oocyte(s) and nurse cells
(trophocytes). Meroistic ovaries have been classified in two basic categories:
polytrophic and telotrophic (G ROSS , 1903). An ovariole in telotrophic ovary
comprise a single, usually huge, germ cell cluster that consists of several oocytes connected to a common trophic region (tropharium) built of numerous
trophocytes. In polytrophic ovary each ovariole contains a number of clusters
of germ cells that form separate structural and functional units termed egg
chambers (for review: K ING and B ÜNING , 1985; B ÜNING , 1994; B ILIÑSKI , 1998).
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Comparative studies on insect ovaries indicate that the oogenesis and ovariole
structure can be used in phylogenetic considerations. This approach has been
employed to elucidate the interrelationships between or within several insect
groups (B ILIÑSKI , 1993; B ÜNING , 1994; K UBRAKIEWICZ et al., 1998; S IMICZYJEW ,
2002).
Bittacidae, known as hanging-flies, are among the most speciose and most
diverse families of Mecoptera. The group comprises about 145 species belonging to 17 genera. They are widespread in temperate and tropical regions of
Europe, Asia and Africa (P ENNY , 1997). The relationships between bittacids and
other mecopterans have been disscussed by several authors. K ALTENBACH (1978)
classified Bittacidae, together with most mecopteran families, into suborder
Eumecoptera, while WILLMAN (1987) differentiated a separate infraorder Raptipedia
(within the suborder Pistillifera) comprising only hanging-flies. The analysis of
bittacid oogenesis can offer additional arguments to clairfy the position of the
hanging-flies within the mecopterans and support the first or the second taxonomic concept. The polytrophic character of bittacid ovaries has been demonstrated previously (B ILIÑSKI et al., 1998; S IMICZYJEW , 1998), however details of
the formation and differentiation of the germ cell clusters have not been described. In the paper the results of the germ cell cluster differentiation in three
species of Bittacus are presented - to date previously these processess in
mecopterans have been described in detail only in scorpionfly Panorpa communis (S IMICZYJEW , 1996).
MATERIAL AND METHODS
Adult females of three species of Bittacus were collected in different
places in Europe, Asia and North America: B. hagenii (Moravia, Czech Republic), B. strigosus (Cansas, USA) and B. nipponicus, (Hachioji, Japan). Dissected
ovaries of B. hageni and B. strigosus were fixed in 2.5% glutaraldehyde in
0.05M phosphate buffer (pH 7.4) at room temperature. The ovaries of B. nipponicus
were fixed in a mixture of 4% glutaraldehyde and 2% formaldehyde, both in 0.1
M phosphate buffer (pH 7.4).
Light and transmission electron microscopy
The material was rinsed and postfixed in 1% osmium tetroxide in the same
buffer. After dehydration in a series of ethanol, the material was embedded in
Epon 812 (Serva, Heidelberg, Germany). Semithin sections were stained with 1%
methylene blue in 1% borax and examined in an Olympus microscope. Ultrathin
sections were contrasted with uranyl acetate and lead citrate and examined in
a Zeiss 900 electron microscope at accelerating voltage of 80 kV.
Fluorescence microscopy
The ovaries were rinsed in PBS for 30 minutes. After dehydration in
ethanol series the material was embedded in acrylic resin Histocryl (Agar,
Stanstead, UK). Semithin sections (1.5 µm) were stained with 4',6 diamidino-2
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phenylindole dihydrochloride (DAPI, 0.2 µg/ml; Sigma Chemical) and propidium
iodide (0.5 mg/ml; Serva, Heidelberg, Germany) and examined in Olympus BHS
fluorescence microscope equipped with appropriate filters.
RESULTS AND DISCUSSION
General morphology of the ovarioles
The ovaries of the studied bittacids are composed of several meroisticpolytrophic ovarioles like in mecopterans from the families: Apteropanorpidae,
Choristidae, Eomeropidae, Meropeidae, Panorpidae and Panorpodidae. Two remaining mecopteran families: Boreidae and Nannochoristidae possess panoistic
ovaries (B ILIÑSKI et al., 1998; S IMICZYJEW , 2002). Each ovariole is differentiated
into a terminal filament, germarium, vitellarium and ovariole pedicel. The germarium
contains germ cell clusters during their formation and somatic prefollicular cells
(Figs 1, 3), while the vitellarium comprises egg chambers in a linear arrangement.
Each egg chamber is built of an oocyte and three nurse cells, surrounded by a
follicular epithelium (Figs 7, 9, 10). Within the vitellarium of an adult ovariole
previtellogenic, vitellogenic and choriogenic egg chambers can be observed.
Germarium
Germarium of all the studied species is conspicuously elongated. Within
germarium three zones can be distinguished: (1) the apical part that comprises
dividing cystoblasts and cystocytes (not shown); (2) the middle region with
numerous undifferentiated germ cell clusters (Fig. 1) and (3) the basal part
containing differentiating germ cell clusters separated by somatic prefollicular
cells (Fig. 3). The mitotic division of the cystoblast in the anterior region of the
germarium results in two sibling cells (cystocytes) which then undergo synchronous mitotic divisions to give a cluster of 4 cystocytes. In other studied
mecopterans with polytrophic ovaries a germ cell cluster contains 4 cells, too
(S IMICZYJEW , 1996; B ILIÑSKI et al., 1998; S IMICZYJEW , 2000). As a consequence of
incomplete cytokinesis the cystocytes become interconnected by intercellular
bridges. After each division, the lumen of the forming intercellular bridge is
filled with a mass of material, termed fusome. Fusomes of neighbouring bridges
merge with each other to form a prominent polyfusome, well visible on semithin
sections stained with methylene blue (Fig. 1). The presence of fusomes and
polyfusomes during the germ cell cluster formation in bittacids under study was
observed also in germarium of the scorpionfly (S IMICZYJEW , 1996) and many other
insects with meroistic ovaries (B ÜNING , 1994; K UBRAKIEWICZ , 1997; LIN et al.,
1994). In the studied bittacids all cystocytes of the cluster enter the prophase
of meiosis and form synaptonemal complexes during pachytene (Fig. 2). This
mode of the germ cell cluster differentiation is evidently connected with a small
number of cystocytes within the cluster and was demonstrated also in P. communis (S IMICZYJEW , 1996), and-besides- in psocopterans (BÜNING and S OHST , 1990).
In other insect species the process is very diversified. Within each cluster of
Drosophila ovary only two of 16 germ cells undergo meiotic divisions (KING ,
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Fig. 1. Middle part of the germarium in B. hageni. The undifferentiated cystocytes (C)
are visible. Note prominent polyfusomes (arrows). Epon, methylene blue.
400x.
Fig. 2. Cystocyte in the middle part of the germarium. B. hageni . Note the
synaptonemal complex (arrow) in cystocyte nucleus (CN). TEM. 8400x.
Fig. 3. Basal region of a germarium in B. nipponicus. The differences between the
pro-oocytes (O) and the nurse cells (NC) can be clearly seen. Arrows indicate
prefollicular cells. Histocryl, DAPI and propidium iodide staining. 400x.
Fig. 4. Chromatin aggregations (asterisks) in the karyoplasm of nurse cell nucleus
(basal part of the germarium). B. nipponicus. TEM. 14300x.
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1970; B Ü N I N G , 1994). In the hymenopteran, Coleocentrotus soldanskii,
8 of 32 cystocytes in each cluster, enter meiosis, although finally only one
differentiates into the oocyte (K LAG and B ILIÑSKI , 1994). In the basal region of
the germarium the cystocytes within the cluster become diversified (Fig. 3). One
of the central cells of the cluster differrentiates into an oocyte while the remaining cystocytes become nurse cells. The nurse cells undergo polyploidization,
their nuclei become large and show numerous heterochromatin clumps (Figs 3, 4).
Vitellarium
The vitellarium is filled with the descendants of germ cell clusters surrounded by follicular epithelium (egg chambers). In the early previtellogenic egg
chamber the oocytes are small with centrally located, relatively large nuclei
(germinal vesicles)(not shown) while the nurse cells grow gradually (Fig. 7). In
the late previtellogenic stage the nurse cells reach their maximum volume and
contain polyploid, roughly spherical nuclei (Figs 9, 10) contrary to the situation
found in panorpids (Panorpa and Neopanorpa) where the nurse cells contain
huge, highly polyploid, irregular nuclei (S IMICZYJEW , 1996; B ILIÑSKI et al., 1998).
The cytoplasm of the nurse cells comprises numerous ribosomes, mitochondria,
elements of rough endoplasmic reticulum and a few accumulations of nuage
material (Fig. 5, 6). In the nurse cells of the studied bittacids only one type of
nuage material was found. The accumulations of nuage material were demonstrated also in representatives of Panorpidae but in the latter case it is particularly abundant and diversified into two types: irregular material in the perinuclear region and large clumps, located peripherally (S IMICZYJEW , 1996). Recently obtained data indicate that the latter structures contain Vasa protein and
splicing factors of pre-mRNA (snRNPs) (B ATALOVA and P ARFENOV , 2003). The
differences in the nurse cells organization between hanging-flies and scorpionflies may reflect variable rate of the synthetic activity and/or dynamics of a
macromolecular transport from the nurse cells to the ooplasm. In the advanced
previtellogenic stage, the oocytes are relatively large (Figs 9, 10). Typically for
other mecopterans with polytrophic ovaries in the late previtellogenesis their
germinal vesicles contain the chromosomes condensed into a karyosphere. Besides,
in bittacid species under study, a few, spherical nuclear bodies (NB) differing
in size occur: small, homogenous NB and large NB, possessing heterogenous
structure (Fig. 11). Cytochemical detection of nucleic acids indicates that these
structures do not contain DNA and RNA but were found AgNOR-positive (Figs
12, 13). The detailed studies on oocyte nuclear structures in Panorpa communis
indicates also the presence of different nuclear bodies. Based on immunochistochemical studies two of them were found to be homologues of the ßsnurposomes, the third one shows characteristic features of coiled (Cajal) bodies (CBs) (B ATALOVA and T SVETKOV , 1998). CBs are complex nuclear domains
containing more than 30 components, including small nuclear RNAs engaged in
splicing. Morphological and cytochemical data presented in this paper allow to
speculate that the large NB in the oocytes of the hanging-flies are also Cajal
bodies.
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The presented results indicate several similarities in germ cell cluster formation and differentiation in bittacids and panorpids, while only minor differences were found to concern subcellular structure of the nurse cells.
Phylogenetic considerations
The Mecoptera is a small insect order comprising about 600 known species
classified in 9 families (P ENNY , 1997). Panorpidae and Bittacidae comprise most
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Fig. 5. Nurse cell in B. hageni. Accumulations of nuage material in perinuclear
region (arrows). Nurse cell nucleus (N). Epon, methylene blue. 420x.
Fig. 6. Accumulation of nuage material (NG) in the cytoplasm of the nurse cell in
B. hageni. Note numerous ribosomes, mitochondria (M) and elements of rough
endoplasmic reticulum (arrow). TEM. 16300x.
Fig. 7. Early previtellogenic egg chambers. B. hageni. Nurse cells (NC), oocyte (O),
follicular epithelium (F). Epon, methylene blue. 360x.
Fig. 8. Intercellular bridge connecting nurse cell with the oocyte (empty arrow).
B. hageni. Note nuage material (white arrow) in the ooplasm (O) that was descended
from the nurse cell. Nurse cell nucleus (N). Epon, methylene blue. 420x.
Figs 9, 10. Egg chambers in late previtellogenesis. B. hageni. Nurse cells (NC),
oocyte (O), follicular epithelium (F). Fig. 9. Epon, methylene blue. 400x. Fig. 10.
Histocryl, DAPI. 400x.
Figs 11, 12, 13. The oocyte nucleus in late previtellogenic stage in B. nipponicus.
Note two types of nuclear bodies: small (arrows) and large (asterisks). Fig. 11.
Epon, methylene blue. 1000x. Fig. 12. Histocryl, AgNOR method. Note positive
silver staining of nuclear bodies. 1000x. Fig. 13. Histocryl, DAPI. Note that the
karyosphere (K) is the only DNA-containing structure. 1000x.
of the known mecopteran species. The relationships between the mecopteran
families are not well established (B YERS , 1991). M ICKOLEIT (1978 ) suggested the
basal position of Bittacidae within the Mecoptera, while K ALTENBACH (1978)
included Bittacidae, together with most mecopteran families, into suborder
Eumecoptera. According to W ILLMAN 's (1987) classification Bittacidae are members of the suborder Pistillifera which comprises 2 infraorders: Raptipedia with
Bittacidae only and Opisthogonophora including the remaining mecopteran families
except for Nannochoristidae. The results of cytological studies on the germ cell
cluster differentiation in three species of hanging-flies indicate similarities between bittacids and panorpids, suggesting a close relationship between these
mecopteran groups. These findings contradict Willman's concept, and support
the traditional classification of K ALTENBACH (1978). Phylogenetic analyses of
molecular data made by W HITING (2002), also suggest a sister group relationship
between Panorpidae and Bittacidae.
ACKNOWLEDGEMENTS
I am grateful to Dr. Janusz Kubrakiewicz for critical reading of the manuscript. This work was supported by Research Grants 6P04C 052 19 and 1018/S/
IZ/03 from the State Committee for Scientific Research
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RÓ¯NICOWANIE GRONA KOMÓREK P£CIOWYCH
W POLITROFICZNYCH OWARIOLACH BUGARÓW
(MECOPTERA: BITTACIDAE).
STRESZCZENIE
Przeprowadzono badania histologiczne, histochemiczne i ultrastrukturalne
formowania i ró¿nicowania gron komórek p³ciowych u Bittacus hageni,
B. nipponicus i B. strigosus (Mecoptera: Bittacidae). Wyniki badañ porównano
z danymi dotycz¹cymi przebiegu tego procesu u wojsi³ki z rodziny Panorpidae
(Panorpa communis). Stwierdzono wiele podobieñstw (w tworzeniu grona komórek
p³ciowych oraz struktury j¹dra oocytu), sugeruj¹cych bliskie pokrewieñstwo
Bittacidae i Panorpidae. Nieznaczne ró¿nice dotycz¹ organizacji komórek
od¿ywczych.
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Received 08.08.2003