Ultrastructural Investigation of the Oogenesis in ...

Entomological Review, Vol. 78, No. 7,1998, pp. 808-821. Translated from Entomologicheskoe Original Russian Text Copyright © 1998 by Filimonova, Brodskaya. English Translation Copyright © 1998 by MAHK "Наука/lnterperiodica " (Russia).

Obovenie,

Vol. 77, No. 4,1998,

pp.

737-752.

Ultrastructural Investigation of the Oogenesis in Bloodsucking Biting Midges of the Genus Culicoides Latr. (Diptera, Ceratopogonidae) S. A. Filimonova and N. K. Brodskaya Zoological Institute, Russian Academy of Sciences, St. Petersburg,

Russia

Received September 20, 1998 Abstract—Ultrastructural changes in the ovarian follicles of adult Culicoides punctatus and C. grisescens are de scribed. In both species, each follicle consists of an oocyte, 7 nurse cells, and the surrounding follicular epithelium. A characteristic feature of the previtellogenic development is an extremely early differentiation of the oocyte and nurse cells, which occurs long before the follicle separation. The structures of the synaptonemal complex are ob served in the oocyte nucleus till the end of previtellogenesis. Yolk accumulation begins only 10-12 hours after bloodmeal and is associated with the pinocytic complexes formed in the peripheral ooplasm. The nurse cells appear not to enter meiosis; they undergo several endoreplication cycles and form large polymorphic nucleoli. Materials produced by their nuclei are transported into the oocyte through cytoplasmic bridges till termination of oocyte growth. The nurse cells degenerate when the vitellogenesis is completed. The eggshell formation has been investi gated only in C. punctatus. The mature oocyte is covered with a vitelline membrane, thin lamellar membrane, and complex endochorion consisting of 3 sublayers: inner floor, middle part with pillars, and a very thin roof. No exochorion was observed. All these eggshell layers are produced by follicular epithelium. The secretion of the vitelline membrane granules occurs simultaneously with vitellogenesis. The formation of the outer layer is followed by ul trastructural changes in the follicular cells. Degenerative processes in follicular epithelium begin immediately after the eggshell formation terminates. Characteristic features of the culicoid oogenesis are discussed with reference to other dipteran species.

The gonotrophic harmony, characteristic of blood sucking biting midges, has been clearly demonstrated in quite a number of species from different geographic zones (Glukhova, 1989). Published data on the internal morphology of Ceratopogonidae mostly refer to this particular biological aspect (Glukhova, 1958; Amosova, 1959; Linley, 1965; Mullens and Schmidtmann, 1982). Biting midges, like all Diptera, have polytrophic ovaries, with each follicle containing, besides the oocyte, specialized nurse cells. Examination of indi viduals dissected at different stages of gonad devel opment showed that oogenesis of anautogenous spe cies corresponds, with slight modifications, to a scheme proposed for mosquitoes (Christophers, 1911; Mer, 1936). According to this scheme, the entire process of ovary development includes 2 stages of follicle formation in the germarium and 5 stages of its subsequent growth in the vitellarium (Glukhova, 1958; Amosova, 1959; Linley, 1965). In general, the oogenesis in biting midges resembles that in other representatives of Diptera (Cummings and King, 1969; Anderson and Spielman, 1971; Mahowald, 1972; Chia and Morrison, 1972; Clements and Boo808

cock, 1984). However, in the absence of data on cel lular mechanisms of oogenesis, no up-to-date com parative morphological analysis can be performed. Consequently, biting midges are not covered at all by reviews dealing with the relevant aspects of entomol ogy (Aizenshtadt, 1977; Margaritis, 1985; Raikhel and Dhadialla, 1992, etc.). At the same time, the family Ceratopogonidae may represent a very interesting group, not only in terms of comparison with closely related dipteran groups, but also owing to the possible variability of morphophysiological mechanisms of their oogenesis: the high degree of gonotrophic harmony in bloodsucking spe cies and complete disharmony in non-bloodsucking ones (Glukhova, 1989). In addition, an investigation of oogenesis in obligatory bloodsucking species may be significant for better understanding of some biological aspects of biting midges as objects of epidemiological importance. This work was aimed at detailed ultrastructural in vestigation of the egg follicles at all stages of oogene sis in 2 species of bloodsucking biting midges of the large genus

Culicoides.

ULTRASTRUCTURAL INVESTIGATION OF THE OOGENESIS

MATERIALS AND METHODS Females of Culicoides

punctatus

(Mg.) and C. gris-

escens Edw. were investigated; these species are known to have anautogenous ovary development (Glukhova, 1989). Biting midges were collected from cattle-shed win dows in Sebezh District (Pskov Province). Unfed fe males were selected, of which some were fixed imme diately, and some were allowed to feed on a human. For further experiments only fully-engorged females were used, they were kept in laboratory at 18-20°C, with sugar syrup as food. Fixations for light and elec tron microscopy were performed simultaneously, 15 min, 1, 3, 6, 10-12, and 24 hours after bloodmeal, and every 12 hours during the following 4 days. To reveal possible changes in the follicle structure in the interval of time between oviposition and the subsequent bloodmeal, C. punctatus females were fixed 2, 12, 24, and 60 hours after complete oviposi tion. In addition, C. punctatus females were fixed 1 and 2 days after emergence. The material for light microscopy was fixed in a mixture of alcohol, formalin, and acetic acid ( 9 : 3 : 1). Paraffin sections were stained with haematoxylineosin; PAS reaction was performed to reveal the car bohydrate component of the yolk. For electron microscopy, dissected abdomina of biting midges were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), then in 1% O S 0 in the same buffer, and embedded in Epon-812. Thin sections were examined using Tesla-BS-500 electron microscope. 4

The data analysis followed the accepted scheme of oogenesis (see Introduction; Fig. 1). The quantitative characteristics of follicle growth in C. punctatus (ta ble) obtained in our work well agree with data for C. obsoletiformis (Amosova, 1959), allowing com parison of oogenesis in different culicoid species. The structure and formation of eggshells were studied in C. punctatus only. RESULTS The gonads of culicoid females comprise numerous ovarioles, the number of which varies even within a species (Glukhova, 1989). Each ovariole has a struc ture common to insects, consisting of a terminal fila ment, germarium, vitellarium, and a pedicle connect ing it to one of the paired lateral oviducts. The lateral ENTOMOLOGICAL REVIEW Vol. 78 No. 7 1998

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oviducts form dilatable canals within the ovary and unite outside it to form an unpaired common oviduct, through which mature eggs leave the ovary. The terminal filament is about 13 um long and con sists of 4 - 5 very small cells with irregularly-shaped nuclei, occupying a major part of their volume and containing aggregations of condensed chromatin gran ules typical of interphase cells (Fig. 3). The terminal filament fixes the ovariole on the gonad sheath con sisting of a single circular layer of cross-striated mus cles. Additionally, each ovariole has a sheath of its own, which also includes muscle elements. All parts of the ovarioles contain rickettsiae, espe cially numerous in somatic tissues—terminal filament cells, ovariole sheaths, and the follicular epithelium. Several cell clusters, representing different stages of follicle formation, can be discerned in the apical and middle parts of a germarium. This stage of oogenesis is commonly termed No (Fig. 3). At stage No, each cluster is surrounded by prefollicular tissue cells which are connected via long and thin cytoplasmic processes without forming complete epithelial layer. Such a process separates the germarium from cells of the terminal filament; no other partition or septa was observed in this place in the culicoid species studied. Free ribosomes, rickettsiae, and various residual bodies abound in cells of the prefollicular tissue; small Golgi fragments and separate small cisternae of rough endoplasmic reticulum irer) are occasionally observed in their cytoplasm. Cystocytes (germ cells making up a cluster) have denser cytoplasm owing to a higher con centration of free ribosomes. These cells are connected by well-discernible cytoplasmic bridges, resulting from incomplete cytotomy of the initial cystoblast. Mitotic figures, rarely observed among cystocytes in the apical germarium part (Fig. 4), suggest that forma tion of clusters continues in ovarioles in the adult stage as well. The same is indicated by the increasing number of cells in a cluster, as its size and distance from the terminal filament increase. The number of clusters in a germarium at stage No varies from 1 to 3 between individuals. The clusters also vary with re spect to the number of constituent cells. In the most complete pattern, the apical part of germarium con tains a cluster of 2 identical cells. Their nuclei com prise particles of condensed chromatin and no nucle oli. Electron-dense substances, transported into the cytoplasm through the nuclear envelope, form the so-

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FILIMONOVA, BRODSKAYA

Fig. 1. Diagram of the egg follicle development in biting midges, based on light microscopy. I-V—stages of oogenesis; ch—chorion; fe—follicular epithelium; nc—nurse cells; nf—nucleus of follicle cell; no—nucleus of oocyte; nu—nucleolus; oc—oocyte; vm—vitelline membrane; у—yolk granules. called perinuclear bodies adjoining the outer surface of the nucleus (Gruzova, 1977). In some females, larger clusters containing as many as 3-4 cells per section were observed just near the terminal filament (Fig. 3). Among the clustered cells, the oocyte can be easily distinguished by a regularly rounded nucleus with electron-lucent nucleoplasm in which chromosomes are discerned as rather large fluffy threads (Fig. 5). Synaptonemal complexes (SC) are visible in the chromosome material. When an oo

cyte enters meiosis, numerous electron-dense granules 60 nm in diameter appear in its nucleus. Such granules are a characteristic feature of an oocyte in both culicoid species, being present during most part of oo genesis (Fig. 6). No such changes are observed in the nuclei of the nurse cells. Their chromatin is distributed over the entire nucleus in the form of distinct, comparatively electron-dense lumps; a small central nucleolus ap pears in the nucleus. ENTOMOLOGICAL REVIEW Vol. 78 No. 7 1998


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Fig. 2. Diagram of the fine structure of the follicular epithelium in biting midges at different stages of oogenesis. I-V—stages of oogene sis; ae—amorphous matrix of endochorion; bl—basal lamina; /—"floor" of endochorion; G—Golgi complex; /—lipids; Im—lamellar membrane; m—mitochondrion; mv—microvilli; pe—pillars of endochorion; pv—pinocytic vesicles; r—ribosomes; rb—residual body; re—"roof of endochorion; rer—rough endoplasmic reticulum; rk—rickettsiae; sf—secretoryfields;sg—secretory granules. Other desig nations as in Fig. 1. The basal part of germarium always contains a sin gle cluster of a larger size, which represents a wellformed follicle surrounded by flat cells of follicular epithelium (stage N) (Fig. 3; table). Analysis of semithin sections showed that in both the species studied the follicle contains an oocyte and 7 nurse cells. At stage N, the cytoplasmic bridges connecting the adjacent cells in the cluster occupy a central posi tion, forming a rosette-like figure typical of polytroENTOMOLOGICAL REVIEW Vol. 78 No. 7 1998

phic ovarioles (Fig. 5). The chromosome material of the oocyte is represented by still more condensed threads including SC, which probably corresponds to the pachytene (Fig. 5). In the nurse cells, the nucleolus increases in size and the chromatin is "split" into smaller fragments. The terminal follicle in the germarium remains at stage N until the only developing follicle in the vitel-

812


Figs. 3-6. Characteristic features of culicoid previtellogenesis. (5) Apical part of ovariole of C. punctatus (No, N—follicles in the corre sponding stages of development); (4) mitosis in germarium of C. punctatus; (5) stage N in germarium of C. punctatus; (6) oocyte nucleus of C. punctatus at stage IIA (1 h after bloodmeal). chr—chromatin;/*—fusome; pb—perinuclear bodies; sc—structures of synaptonemal complex; tf—terminalfilament.Other designations as in Figs. 1, 2. Arrows point at intranuclear granules of the oocyte.

larium stops growing and chorion formation begins (stage IV B: Fig. 1). Then, a next follicle detaches from the germarium and enters stage I of oogenesis (see the table; Figs. 1-3). Despite the transfer of the follicle to the vitellarium, stage I must be considered part of previtellogenesis, because all components of the follicle grow without any considerable internal

transformations. Judging from its nuclear morphology, the oocyte remains in the pachytene. Stage I can be divided into 2 phases. During the first phase (Stage I A), the follicle, having just separated from the germarium, remains rounded and no more than 25-27 um in diameter. This structure is typical of basal follicles found in females soon after emergence, ENTOMOLOGICAL REVIEW Vol. 78 No. 7 1998


Characteristics of egg follicles at different stages of oogenesis in Culicoides Stage

Proportion Start, h of oocyte in follicle

813

punctatus

Size (um) and characteristic features follicle

nuclei

oocyte oocyte

follicle cells

Main characteristic

nurse cells*

Before bloodmeal N

-

'/5

13x13

4-5

3.5, SC

4

Flat

IA

-

V5

26x26

5-7

3.5, SC

6-8

//

Follicle separated from germarium

IB

-

'/4

28x35

7-10, SC

4.2

9-10

IIA

1

'/3

30x40

9-12

6.0, SC

11-12

7-8

Cubical, rer

Follicle separated from germarium

IIВ

10-12

>/2

38x54

25-27

8.5

12-14

8

Columnar

Beginning of vitellogenesis

24

42, /3

III В

48

2

Vitellogenesis and synthesis of vm

IV A

60

3

IV В

84

V

108

III A

Follicle in ger marium

After bloodmeal

2

9

50x70

35^10, у

9.0

13-15

9

Cubical, sg

3

75x120

70x80, у

13.0

13-15

10-11

Flat, sg

4

/ 4 , /5

80x160

75x120, Y

13.0

16-23

12-14

Flat, sg, I, v

/ l 0 and higher

55x350

55x350, Y

13.0

1

55x350

55x350, Y

-

/3, /4

Degenerate

Flattened Beginning of degeneration

Formation of vm completed Formation of la mellar membrane and chorion

Notes: v—Vacuoles with amorphous content; vm—vitelline membrane; у—yolk granules with crystalline structure; Y—mature yolk granules; /—lipids; sg—secretory granules, precursors of vitelline membrane. * Beginning with stage II A, the nurse cells are subdivided into small and large ones.

or not later than 12 h after oviposition. After this peri od of time (or after 24 h for newly-emerged females), the follicles reach stage I B; they become oval and in crease considerably in size to about 40 um. The growth of nurse cells is the most noticeable: the dia meter of their nuclei increases nearly twofold by the end of stage I (see the table). As in most insects, the nurse cells probably undergo endoreplication cycles (Aizenshtadt, 1977). At the same time, very large po lymorphous nucleoli including considerable fibrillar component appear in their nuclei. By the end of stage I, a gap appears between the oocyte and the follicular epithelium; in some places, small protrusions are formed on the oocyte surface, which later give rise to microvilli. In this phase, the follicle development is interrupted, to be resumed after the bloodmeal (see the table). As shown by experiments, no changes in the follicle structure are observed 15-20 min after bloodmeal. One hour later, the follicle enters stage II A, charac terized, in addition to drastic increase in size (see the table), by a number of qualitative changes. ENTOMOLOGICAL REVIEW Vol. 78 No. 7 1998

This is manifested primarily in the surpassing growth of the oocyte, which already occupies about h the follicle volume. The nurse cells are subdivided into larger ones, adjacent to the oocyte, and smaller ones, being in contact with the follicular epithelium (Fig. 1). Electron microscopy showed these cells to have about the same structure. The chromatin is even more dif fusely distributed in the nuclei than it is at the pre ceding stages; the nucleoli are capable of fragmenta tion and contain a considerable amount of granular material which is transported into the cytoplasm via the nuclear pores. The nurse cell cytoplasm is thick with free ribosomes and numerous small mitochon dria; these organelles seem to be transported into the growing oocyte, being mainly observed near the cyto plasmic bridges connecting the nurse cells with the oocyte. Mitochondria concentrate in the ooplasm near the nurse cells. At the same time, both rer and Golgi bodies are virtually absent in the nurse cells at this and all the following stages. Later, these cells undergo no changes except for growth of nuclei and nucleoli (see the table). x

814


Figs. 7-9. Characteristic features of culicoid vitellogenesis. (7) General view of the follicular epithelium of C. grisescens 24 h after bloodmeal (stage III A); (S) secretory granules: precursors of vitelline membrane in the follicle cells of C. punctatus 3 days after bloodmeal (stage IV A); (9) vacuoles with amorphous content in the follicular epithelium of C. punctatus 3 days after bloodmeal (stage IV A). Designations as in Figs. 1, 2.

Stage П A is characterized by enlarged gap between the oocyte and the follicular epithelium; small micro villi occur on the oocyte and follicle cell surfaces fac ing the gap. As a result of growth and reproduction of flat follicle cells, they acquire a cubical shape (Fig. 2; table). Rare rer cisternae appear in their cytoplasm; no secretory granules are present at this stage. Chromatin

threads are no longer observed in the oocyte nucleus, even though the SC figures are still discernible, ar ranged in a certain pattern at the ucleus periphery, which suggests that they remain attached to partly separated diplotene chromosomes (Fig. 6). A small number of lipid granules appear in the ooplasm. Their formation cannot be attributed to any ENTOMOLOGICAL REVIEW Vol. 78 No. 7 1998


815

Figs. 10-12. Eggshell formation in biting midges. (10) Formation of lamellar membrane 3.5 days after bloodmeal (stage IV B); (11) oocyte surface 4.5 days after bloodmeal (stage V); (12) pillar of endochorion. Designations as in Fig. 2.

particular type of organelles. Females of C. grisescens, fixed 1 and 3 h after bloodmeal, showed some increase in the number of rer cisternae and mitochondria. First yolk granules in their oocytes are only observed 10-12 h after bloodmeal. During this period, the oo cyte occupies nearly half the follicle volume, which corresponds to stage II В according to the commonly accepted system. In addition to the oocyte growth, the size of its nucleus also noticeably increases (see the table). Materials produced in the oocyte nucleus are transported into the ooplasm via numerous nuclear pores, similarly to what is observed in the nurse cells. In both culicoid species, the oocyte nucleus no longer reveals SC figures or any structures analogous to nu cleoli. As regards the chromosome material, the ENTOMOLOGICAL REVIEW Vol. 78 No. 7 1998

authors have more than once observed structures re sembling the karyosphere of other insects during vitellogenesis (Gruzova, 1977); however, these data need further verification in a separate research. Yolk granules are small and most numerous in peripheral ooplasm. They are formed by fusion many pinocytic vesicles originating at bases of microvilli. The number of lipid granules also creases.

the of the in

At this stage, the follicle cells surrounding the oo cyte are cubical or columnar (Figs. 1, 2). The cells are separated basally, forming conical intercellular spaces filled with an amorphous matrix (Fig. 7). That part of the follicular epithelium which surrounds the nurse

816


cells is virtually not involved in these modifications: the cells remain flat, with their cytoplasm full of free ribosomes. 24 h after bloodmeal all females had follicles al ready in stage Ш (see the table; Figs. 1, 2). This is the period of the most active vitellogenesis; pinocytic vesicles and lipid and yolk granules in the oocyte be come more numerous. Farther from the oocyte surface, the yolk granules are larger and their content acquires crystal structure. Intercellular spaces in the follicular epithelium be come even larger; the internal structure of follicle cells continues to change in relation to the beginning of their secretory activity. A complicated network of interconnected rer canals is formed, occupying the entire cytoplasm volume (Figs. 2, 8). Golgi cisternae increase in number; several small cisternae, typical of the preceding stages, are transformed into an aggrega tion of numerous vesicles 70-90 nm in diameter. The surrounding cytoplasm is less electron-dense and con tains no ribosomes, so that a considerable secretory field is formed (Figs. 2, 7, 8). A similar type of Golgi complex has been described in cells of the follicular epithelium of various insects (Anderson and Spielman, 1971; Cummings, 1972; Chia and Morrison, 1972; Clements and Boocock, 1984; Filimonova, 1996) and appears to be very typical of the follicle cells during the vitelline membrane synthesis. The secretory fields contain granules 90-150 nm in diameter, whose con tent becomes denser as the granule size increases. Condensation of the secretory product continues after granules leave the secretory field, so they increase in diameter from 150 to 250 nm and become more elec tron-dense as they approach the apical membrane. Mature granules are secreted by typical merocrine mechanism into the space between the oocyte and the follicle cells, where their content forms larger secre tory droplets (Fig. 8). During the vitellogenesis, yolk granules in the oo plasm increase in size; by the second half of stage Ш, most granules reach their maximum size, remaining crystalline. The follicle cells continue to secrete the vitelline membrane material. The process of secretion is ac companied by increasing the volume of secretory fields and rer. About 2-2.5 days after bloodmeal, a group of follicle cells appears between the oocyte and nurse cells (stage Ш В: see the table; Fig. 1). Spread ing over the oocyte surface, these cells also give rise to a vitelline membrane layer.

Stage ГУ of the culicoid oogenesis is commonly subdivided into 2 phases: the follicle, oval in stage IV A, rapidly elongates and assumes its definitive shape (resembling a long and slightly curved cigar) in stage ГУ В (Fig. 1). This transformation appears to be very rapid, since no intermediate forms were observed. In C. punctatus

and C. grisescens,

the follicles en

tered stage IV A 2-2.5 days after bloodmeal (see the table). By this time, the oocyte occupies as much as about / 4 the follicle area on a section; the oocyte cy toplasm is uniformly filled by large yolk granules and smaller lipid inclusions. The yolk granules complete maturation and have dense homogenous content, di vided into parts by narrow bands of electron-lucent material (Fig. 2). The oocyte nucleus retains its central position and amoeboid shape. This stage is character ized by disappearance of the most part of intranuclear granules 60 nm in diameter, typical of the preceding stages of oogenesis. Aggregation of substances on the outer surface of the nuclear envelope, indicating the nucleus-to-cytoplasm transport activity, cannot be observed, either. 3

On the contrary, the nurse cell nuclei reach the maximum size and appear very active at this stage (table; Fig. 1). The electron-dense material is being transported into the cytoplasm via the nuclear pores and accumulates there as perinuclear bodies lying around the entire nucleus and observable also in the cytoplasm of the nurse cells, being usually surrounded there by mitochondria. The nurse cells remain con nected to the oocyte via cytoplasmic bridges. As the oocyte continues growing, the follicle cells flatten (Fig. 2). By the end of stage IV A, expanded spaces between them disappear; the cells attain regu lar-hexagonal shape (as it can be seen on tangential sections) and their lateral surfaces come into close contact. Pinocytic vesicles on the oocyte surface dis appear. On the whole, these phenomena can be re garded as the completion of vitellogenesis. The synthesis of the vitelline membrane is also nearly complete. At this stage, the membrane is repre sented by large fragments occupying the entire space between the oocyte and the follicular epithelium (Fig. 2). Each fragment, produced by a single follicle cell, increases in size and finally repeats the shape of the follicle cell. As can be seen on paraffin sections, the vitelline membrane becomes slightly folded by the end of stage ГУ A (Fig. 1), which possibly provides for further expansion of the oocyte surface. ENTOMOLOGICAL REVIEW Vol. 78 No. 7 1998


The completion of the vitelline membrane synthesis affects the chemical composition of the follicle cells. When stained with Azure II at pH 6.8 in semithin sec tions, the previously blue follicular epithelium turns violet. The ultrastructure of these cells also changes (Figs. 2, 9): the nucleoli decrease in size; rer some times forms piles of parallel cisternae. Numerous lipid inclusions appear in the cytoplasm. The morphology of the secretory fields of follicle cells changes: some of these areas are probably transformed into rather large (up to 1 um) inclusions with amorphous, moder ately electron-dense content, which may be to a large extent removed from sections by reagents used in ТЕМ processing; for this reason, some of the vacuoles appear empty. The vacuoles are usually surrounded by rer fragments (Fig. 9). Secretion of these vacuoles could not be observed; however, they decrease in number during the subsequent oogenetic phases and disappear completely during stage V. When the vitelline membrane fragments merge to form a complete layer about 1 um thick, the follicle rapidly elongates and enters stage IV В (Figs. 1, 2; table). This stage is characterized by degeneration of the nurse cells, which starts with pycnotic changes of nuclei in large cells adjacent to the oocyte. The re mainders of the nurse cells are partly utilized by addi tional follicle cells which probably migrate into the region. In the ooplasm, the space between the yolk spheres is filled with lipid droplets and small aggregations of glycogen p-granules. The oocyte nucleus shifts from the central to a basilateral position. The microvilli of the oocyte degenerate, and their remnants can be ob served as irregular-shaped vesicles in the narrow space separating the oocyte from the follicular epithelium. The same process is observed at the follicle cell sur face, but in this case fragments of microvilli probably participate in the formation of the next eggshell layer, the lamellar membrane (Fig. 10). At this phase, the follicular epithelium cells show no indication of synthetic activity: their nucleoli are no more revealed; rer decreases in volume; the secretory fields and most of vacuoles with amorphous content typical of the preceding stage disappear. The concen tration of lipid inclusions noticeably decreases; these inclusions probably participate in the formation of the lamellar membrane or the chorion. After complete degeneration of the nurse cells and utilization of their remnants, the oocyte occupies vir ENTOMOLOGICAL REVIEW Vol. 78 No. 7 1998

817

tually the entire follicle volume. Its nucleus is no more observed, indicating the completion of meiotic pro phase I. The follicle thus enters stage V, during which the eggshell layers are completed. The lamellar membrane appears as an electronlucent layer no more than 30-50 nm thick, immedi ately adjacent to the vitelline membrane (Figs. 2, 11, 12). Its lamellar structure can be revealed at high mag nifications only. Outside the lamellar membrane, a complex endochorion is formed by the follicular epi thelium. In C. punctatus, the endochorion consists of a basal sublayer or "floor," a thick middle sublayer with pillars, and a thin " r o o f (Figs. 11, 12). The basal sublayer consists of small overlapping arched plates, each formed by 2 electron-dense layers separated by an electron-lucent one. The thickness of the "floor" does not exceed 50 nm. The middle sublayer is about 1 um thick and consists of pillars embedded in an amor phous matrix (Fig. 12) and connecting the basal sub layer and the " r o o f of chorion. There are small spherical cavities within the pillars; the lining of these cavities displays the same pattern of alternating bands with different electron densities as that observed in the basal sublayer, suggesting a common chemical compo sition of the endochorion "floor" and pillars. As their material probably serve moderately electron-dense granules 40 nm in diameter, secreted by cells of the follicular epithelium. The granules are formed in the Golgi complex, which comes back to its classical for mat during stage V of the oogenesis (Fig. 2). The rer fragments are few in number and mostly spherical. Autophagous vacuoles and lamellar bodies are quite common in the cell cytoplasm, increasing in number as the eggshell synthesis is completed. At the end of stage V, disintegration of the follicu lar epithelium begins, manifested by formation of thin cytoplasmic outgrowths, detaching from the cell and undergoing fragmentation. The oocyte surface shows no indication of exochorion typical of most insects (Margaritis, 1985); therefore, the endochorion " r o o f remains the outermost layer. The " r o o f is thinner than the basal sublayer and consists of small units separated by small electron-lucent areas, resulting in a dotted pattern on sections. The " r o o f is somewhat less elec tron-dense than the endochorion pillars. DISCUSSION The use of electron microscopy significantly im proved our interpretation of different oogenetic stages in bloodsucking Ceratopogonidae. The oogenesis of


818

the investigated species is largely similar to that ob served in other representatives of Diptera. The folli cles are formed by cluster formation typical of polytrophic ovarioles. The bridges between the oocyte and the nurse cells persist until the end of the vitellogene sis. Yolk accumulation in the oocyte occurs simulta neously with the vitelline membrane synthesis by fol licle cells. All these features are present in most dipteran species studied (Cummings and King, 1969; Anderson and Spielman, 1971; Mahowald, 1972; Clements and Boocock, 1984; Pollard et al., 1986). At the same time, a number of peculiarities can be noted. PREVITELLOGENESIS The biting midges studied demonstrate a relatively early differentiation of egg follicles within the ger marium. Clusters of cystocytes are surrounded by cells of the prefollicular tissue since the very beginning of their formation. The number of clusters at stage No varies between females, probably depending on their physiological age; this phenomenon requires further investigation. It can be suggested that the mitotic fig ures, infrequently observed among gonocytes, are re stricted to younger females in which formation of new clusters continues. The ovarioles containing only large clusters in apical position probably belonged to older females. The oocyte is distinguished within a cluster already at stage No and appears to enter meiotic prophase I immediately after the cystocyte divisions are com pleted. Structures of the synaptonemal complex are never observed in the nurse cell nuclei, suggesting that these cells do not enter meiosis at all, as it was shown for the fly oogenesis (Gruzova, 1977). According to Aizenshtadt (1977), reduction of early meiotic stages during the nurse cell development is an adaptation toward earlier functional specialization. It is highly probable that biting midges, like most insects with polytrophic ovarioles, also have polyploid nurse cells. Modifications of their chromosome material start al ready in the germarium and continue during the entire previtellogenesis, with their dynamics corresponding to that observed in Drosophila melanogaster oogene sis (Cummings, King, 1969). A nucleolus is formed in a culicoid nurse cell al ready at stage No; later, it grows and undergoes frag mentation and finally occupies a major part of nucleo plasm. It is probably the material of the nucleolus that is the main component of the substance secreted via the nuclear pores into the cytoplasm of nurse cells and

observed there as perinuclear bodies during all func tional stages of these cells. The synthesis-associated organelles are completely absent in the nurse cell cy toplasm, with only ribosomes and mitochondria being observed in the cytoplasmic bridges. All these facts indicate a narrow specialization of the nurse cells, mainly related to supply of the ribosomal components to the oocyte. The oocytes of both the species studied do not participate in the rRNA synthesis, having nc nucleoli during the entire oogenesis. The numerous intranuclear granules 60 nm in diameter should be considered a kind of intranuclear bodies, described foi oocytes and somatic tissues of various animals and presently believed to participate in the processing ol certain classes of RNA (Gall, 1991). Disappearance ol these granules by the end of vitellogenesis in Culicoides correlates with the end of transport of the electrondense material via the nuclear pores. In addition tc these granules, several less permanent kinds of intra nuclear bodies were observed in both the culicoid spe cies studied; their detailed investigation is beyond the scope of the present work and requires adequate tech niques. Preliminary observation showed, however, thai these bodies have considerably different structure ir C. punctatus

and С

grisescens.

The basal part of germarium always contains a sin gle more mature follicle. The development of its oo cyte is arrested in the early pachytene until the onlj oocyte in the vitellarium completes vitellogenesis anc reaches stage ГУ В. This fact agrees with Linley'i (1965) data on Leptoconops bequaerti and may repre sent a general rule for most anautogenous species According to published data (Glukhova, 1958; Amo sova, 1959), a similar arrest of development occurs before emergence. In our experiments, the follicles ol newly emerged females reached the next resting stage (I B) already 24 h after emergence. In the case of re peated gonotrophic cycles, maturation to this stag* takes only 12 h, because the follicles reach stage I A already at the moment of oviposition. During most part of previtellogenesis, the oocyte remains in pachytene, as indicated by the presence о condensed chromosomes connected by the SC struc tures. This is a characteristic feature of the culicoic species studied, since in most insects the mentionec period generally corresponds to diplotene. In both culicoid species, the oocyte enters diploten< only after bloodmeal, probably in stage П A; conse quently, the second resting stage of oogenesis alsc occurs at pachytene. The arrest of the oocyte devel ENTOMOLOGICAL REVIEW Vol. 78 No. 7 1991


opment at the end of previtellogenesis is very typical of many insects, including the vast majority of blood sucking dipterans, whose vitellogenesis starts after bloodmeal only (Aizenshtadt, 1977). According to published data, the arrest of oogenesis in biting midges occurs during stage II A (Glukhova, 1958; Amosova, 1959; Linley, 1965) or already during stage I (Camp bell and Kettle, 1975; Mullens and Schmidtmann, 1982). The differences refer not only to the general follicle size, but also to the relative size of the oocyte in its content and the presence of yolk granules. VITELLOGENESIS The electron microscopic data show that the arrest of follicle development in C. punctatus and C. grisescens occurs during stage I B, characterized by elonga tion of the follicle and formation of a gap between the oocyte and the nurse cells, where microvilli start to appear. The same characters are typical of the resting stage of previtellogenic follicles in most of other in sects (Aizenshtadt, 1977). In mosquitoes, the pinocytic complex is formed at the oocyte surface during this period (Anderson and Spielman, 1971). Contrariwise, in the culicoid species studied the pinocytic vesicles appear in the oocyte only after bloodmeal; a similar picture is observed in flies switching to a protein diet (Cummings and King, 1970; Mahowald, 1972). The beginning of pinocytosis is accompanied by a complex of changes in follicle structure, including separation of follicle cells and further surface differentiation of the oocyte and the follicular epithelium. All these changes occur during stage II A, which is preparatory to the beginning of vitellogenesis. It is interesting that the pinocytic complex formation in previtellogenic oo cytes of mosquitoes also does not rule out the same preparatory stage. In most species, the vitellogenesis is observed only 7-10 h after bloodmeal and also coin cides with the appearance of broad intercellular spaces in the follicular epithelium (Tadkowski and Jones, 1979; Clements and Boocock, 1984). At the same time, oocytes of some dipteran species have been shown to produce some quantities of endogenous yolk prior to the beginning of vitellogenesis (Mahowald, 1972; Tadkowski and Jones, 1979). According to our data, mass accumulation of yolk granules in culicoid oocytes starts only 10-12 h after bloodmeal, as the follicle enters stage П B. All the data demonstrate that these granules are primarily formed from exogenous vitellogenins. In addition, occasional lipid droplets appear in the oocyte cytoplasm of both ENTOMOLOGICAL REVIEW Vol. 78 No. 7 1998

819

C. grisescens and C. punctatus from the first hours after bloodmeal. It is not improbable that the relative importance of the endogenous synthesis of both lipid and yolk inclusions may vary to a considerable extent among culicoid species. In this case, the yolk aggrega tions observed in oocytes of some biting midges dis sected prior to bloodmeal may be of endogenous ori gin, especially because they usually lie in the perinu clear area (Glukhova, 1958; Linley, 1965). The vitellogenesis in biting midges continues during the major part of the egg maturation. The formation of the yolk granules is most active during stage III and is accompanied by a decrease of the pinocytic activity. Stage IV A is the last stage of the vitellogenesis, dur ing which pinocytic vesicles on the oocyte surface and intercellular areas between follicle cells disappear. The synthesis of the vitelline membrane, which starts in the follicular epithelium at the beginning of vitello genesis, is also completed at this stage. EGGSHELL FORMATION The general structure of eggshells in biting midges resembles that in mosquitoes (Pollard et al., 1986; Sahlen, 1990). In both cases, a mature egg is covered by a relatively thick layer of the vitelline membrane, with the lamellar membrane and the chorion lying outside. Unfortunately, published data provide no complete understanding of the formation of all these layers. The most obscure is the synthesis of the vitel line membrane, which is probably accomplished in a similar way in very different insects (Margaritis, 1985). Vast areas, including the Golgi complex and aggregated secretory granules with different electron densities and referred to as "secretory fields" in the present work, are formed in the follicle cell's cyto plasm. The secretion of granules starts soon after the beginning of vitellogenesis and becomes more inten sive as the pinocytic activity of the oocyte gradually decreases. In most insects, including some representatives of Diptera (Mahowald, 1972; Pollard et al., 1986), the beginning of chorion synthesis by the follicle cells has no considerable effect on the morphology of their Golgi complex and even on the structure of the secre tory product. In biting midges, the internal transfor mation of follicle cells occurs already during the com pletion of the vitelline membrane synthesis: the se cretory fields disappear, the rer decreases in volume, and large granules with amorphous content and nu merous lipid inclusions appear in the cytoplasm. These


820

inclusions probably participate in the formation of the lamellar membrane, becoming noticeably less abun dant in cells after the formation of this membrane is completed. Microvilli of the follicle cells also play some role in this process, being fragmented into small vesicles of moderate electron density. Similar events have been described for lamellar membrane formation in Drosophila

melanogaster

(Quattropani and Ander

son, 1969), where, as in the present work, no secretory product was revealed in the cell cytoplasm. The secre tion probably does not involve granule formation. Large vacuoles with amorphous content, similar to those observed in the present work, are often revealed in the follicular epithelium of various insects after the completion of the vitelline membrane synthesis. They are usually surrounded by rer cisternae (Quattropani and Anderson, 1969; Cummings, 1972; Matthew and Rai, 1975; Margaritis, 1986; Filimonova, 1996). Se

cretion of these granules is observed very rarely; they most probably supply material for the amorphous ma trix between the endochorion pillars (Cummings, 1972; Matthew and Rai, 1975; Filimonova, 1996). The

pillars themselves and the endochorion "floor" appear to be formed from the contents of small moderately electron-dense granules, observable in close contact with Golgi areas in follicle cells during choriogenesis. The change of the secretory product of the follicle cells is accompanied by structural modifications of their Golgi complex. Unfortunately, no information is presently available concerning the formation of the endochorion "roof." This thin outer layer is probably secreted very rapidly and does not involve accumulation of abundant secre tory product, because of which no corresponding in clusions can be revealed in the follicle cells. Also ob scure is the problem of the exochorion, which was not observed in C. punctatus.

Further investigations are

necessary to find out whether the absence of this structure is characteristic of this species only, or repre sents a feature common to all biting midges. REFERENCES 1. Aizenshtadt, T.B., Oocyte Growth and Vitellogenesis, Sovremennye problemy oogeneza (Current Problems of Oogenesis), Moscow: Nauka, 1977, pp. 5-50. 2. Amosova, I.S., Gonotrophic Relations in Biting Midges of the Genus Culicoides (Diptera, Heleidae), Entom. Obozr., 1959, vol. 38, no. 4, pp. 774-779. 3. Anderson, W.A. and Spielman, A., Permeability of the Ovarian Follicle of Aedes aegypti Mosquitoes, J. Cell Biol, 1971, vol. 50, no. 1, pp. 201-221.

4. Campbell, H.M. and Kettle, D.S., Oogenesis in Culicoi des brevitarsis Kieffer (Diptera: Ceratopogonidae) and the Development of a Plastron-Like Layer on the Egg, Austr. J. Zool., 1975, vol. 23, pp. 203-218. 5. Chia, W.K. and Morrison, P.E., Autoradiographic and Ultrastructural Studies on the Origin of Yolk Protein in the House Fly Musca domestica, Can. J. Zool., 1972, vol. 50, no. 12, pp. 1569-1581. 6. Clements, A.N. and Boocock, M.R., Ovarian Develop ment in Mosquitoes: Stages of Growth and Arrest and Follicular Resorption, Physiol. Ent., 1984, vol. 9, no. 1, pp. 1-8. 7. Cummings, M.R., Formation of the Vitelline Membrane and Chorion in Developing Oocytes of Ephestia kuhniella,Z. Zellforsch., 1972, vol. 127, pp. 175-188. 8. Cummings, M.R. and King, R.C., The Cytology of the Vitellogenic Stages of Oogenesis in Drosophila mela nogaster. I. General Staging Characteristics, J. Morphol, 1969, vol. 128, no. 4, pp. 427^142. 9. Cummings, M.R. and King, R.C., The Cytology of the Vitellogenic Stages of Oogenesis in Drosophila mela nogaster. II. Ultrastructural Investigations on the Origin of Protein Spheres, J. Morphol., 1970, vol. 130, pp. 467^178. 10. Filimonova, S.A., Electron-Microscopic Investigation of Oogenesis in the Flea Xenopsylla cheopis, Tsitologiya, 1996, vol. 38, no. 7, pp. 703-717. 11. Gall, J.G., Spliceosomes and Snurposomes, Science, 1991, vol. 252, pp. 1499-1500. 12. Glukhova, V.M., Gonotrophic Cycle in Biting Midges of the Genus Culicoides (Diptera, Heleidae) in Karelian ASSR, Parazitol. Sbornik Zool. Inst. Akad. Nauk SSSR, 1958, vol. 18, pp. 239-253. 13. Glukhova, V.M., Bloodsucking Biting Midges of the Genera Culicoides and Forcipomyia (Ceratopogonidae), Fauna SSSR. Nasekomye dvukrylye (Fauna of the USSR: Diptera), Leningrad: Nauka, 1989, vol. 5A, no. 3. 14. Gruzova, M.N., Nucleus in Oogenesis (Structural and Functional Aspects), Sovremennye problemy oogeneza (Current Problems of Oogenesis), Moscow: Nauka, 1977, pp. 51-98. 15. Linley, J.R., The Ovarian Cycle in Culicoides barbosai Wirth & Blanton and C. furens (Poey) (Diptera, Cerato pogonidae), Bull. Ent. Res., 1966, vol. 57, pp. 1-17. 16. Mahowald, A.P., Ultrastructural Observations on Oo genesis in Drosophila, J. Morphol, 1972, vol. 187, no. 1, pp. 29-48. 17. Margaritis, L.H., Structure and Physiology of the Egg shell, Compreh. Insect Physiol. Biochem. Pharmacol, Kerkut, G.A. and Gilbert, L.I., Eds., Pergamon Press, 1985, pp. 153-230. 18. Matthew, G. and Rai, K.S., Structure and Formation of the Egg Membrane in Aedes aegypti (Diptera: Culicidae), Int. J. Insect Morphol Embryol, 1975, vol. 4, pp. 369-380. ENTOMOLOGICAL REVIEW Vol. 78 No. 7

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9. Mer, G.G., Experimental Study on the Development of the Ovary in Anopheles elutus etc., Bull. Ent. Res., 1936, vol. 27, no. 3, pp. 351-359. 10. Mullens, B.A. and Schmidtmann, E.T., The Gonotro phic Cycle of Culicoides varipennis (Diptera: Ceratopo gonidae) and Its Implications in Age-Grouping Field Populations in New York State, USA, J. Med. Ent., 1982, vol. 19, no. 3, pp. 340-349. 11. Pollard, S.R., Motara, M.A., and Cross, R.H.M., The Ultrastructure of Oogenesis in Culex theileri, South Afr. J. Zool., 1986, vol. 21, no. 3, pp. 217-223. 2. Quattropani, S.L. and Anderson, E., The Origin and Structure of the Secondary Coat of the Egg of Droso-

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phila melanogaster, Z. Zellforsch., 1969, vol. 95, pp. 495-510. 23. Raikhel, A.S. and Dhadialla, T.S., Accumulation of Yolk Proteins in Insect Oocytes, Annu. Rev. Ent., 1992, vol. 87, pp. 217-251. 24. Sahlen, G , Egg Raft Adhesion and Chorion Structure in Culex pipiens L. (Diptera: Culicidae), Int. J. Insect Morphol. Embriol, 1990, vol. 19, nos. 5-6, pp. 3 0 7 314. 25. Tadkowski, T.M. and Jones, J.C., Changes in the Fat Body and Oocytes During Starvation and Vitellogenesis in a Mosquito, Aedes aegypti (L.), J. Morphol, 1979, vol. 159, pp. 185-204.