Fine Structure of the Female Genital System in ... - Springer Link

0 downloads 0 Views 42MB Size Report
The vaginal duct opens into the vagina (genital atrium, which is covered ...... coalesce to form the vitelline membrane, its formation would be quite dif- ferent from ...
Experimental and Applied Acarology 25: 525–591, 2001. © 2002 Kluwer Academic Publishers. Printed in the Netherlands.

Fine structure of the female genital system in phytoseiid mites with remarks on egg nutrimentary development, sperm-access system, sperm transfer, and capacitation (Acari, Gamasida, Phytoseiidae) ANTONELLA DI PALMA1 and GERD ALBERTI2,∗ 1 Facoltà di Agraria, Università degli Studi di Foggia, Via Napoli 25, I-71100 Foggia, Italy 2 Zoologisches Institut und Museum, Universität Greifswald, J.-S.-Bach-Str. 11/12, D-17489

Greifswald, Germany (Received 31 May 2001; accepted 3 October 2001)

Abstract. The fine structure of the female genital system is described in two phytoseiid species: Phytoseiulus persimilis Athias-Henriot (mating females) and Typhlodromus rhenanoides Athias-Henriot (overwintering females). The female genital tract is composed of an unpaired gonad, the uterus (oviduct I), and the vaginal duct (oviduct II). The latter leads to the vagina (genital atrium), into which a pair of vaginal glands opens. The gonad (ovary s.l.) has two components: the ovary (s.str.) where germ cells develop and the lyrate organ serving as a nutrimentary compartment. In the ovary (s.str.), somacells and germ cells are observed. The germ cells surround a central tissue, to which they have direct contact with a nutritive cord at least in the previtellogenic phase during oogenesis. In fertilized females, cells likely representing capacitated sperm cells are also found in the ovary. The lyrate organ has two arms that extend anteriorly but join in their posterior part in front of the ovary (s.str.). The lyrate organ is composed of a somatic (supporting) and a nutritive tissue. The nutritive tissue, which is a syncytium, is continuous with the central tissue. The uterus starts from the ventral region of the central tissue. Finally, the ultrastructure of the sperm-access system, composed of paired solenostomes, major and minor ducts, emboli, calyces, and vesicles, is reported and functional aspects are discussed. The minor ducts end in the somatic tissue of the ovary s.str. However, because of its extremely reduced lumen and the peculiar morphology of its beginning, it seems unlikely that the minor duct lumen serves as a simple route for the sperm towards the ovary. Key words: capacitation, female reproductive system, podospermy, sperm-access system, spermatozoa Abbreviations: I – oocyte of stage I; II – oocyte of stage II; III – oocyte of stage III; IV – oocyte of stage IV; al – annulate lamellae; cal – calyx; cc – cup cell; Ch – chelicera; cm – cytoplasmic material; cT – central tissue; cu – cuticle; D – Golgi body (dictyosome); ech – endochorion; emb – embolus; ex – extension of central tissue; EP – epidermis; ER – endoplasmic reticulum; eT – extraovarian nutritive tissue; f – filaments; GO – genital opening; ∗ Author for correspondence: (Tel.: +49-3834-864250; Fax: +49-3834-864252; E-mail: [email protected])

526 gp – genital plate (epigynium); HL – hemolymph; la – lamellae of extracellular material; lo – lyrate organ; lu – lumen; Md – major duct; md – minor duct; MG – midgut; MI – mitochondrium; MT – Malpighian tubule; MU – muscle; N – nucleus; Nc – nucleus of cup cell; nc – nutritive cord; NE – nerve ending; Ne – nerve; Ng – nucleus of vaginal gland cell; Nl – nucleus of lyrate organ; No – nucleus of oocyte; Ns – nucleus of somacell; Nt – nucleus of electron lucent tissue; NU – nucleolus; Nu – nucleus of uterus cell; Pa – pedipalp; Pe – peritreme; res – reservoir of vaginal gland; Ri – ribosomes; SC – somacells; sd – salivary duct; Se – secretion; Sp – sperm cell (putative); ss – salivary stylus; T – electron lucent tissue; U – uterus; V – vesicle; va – vagina; vd – vaginal duct; vE – epithelium of vagina; vg – vaginal gland; Y – yolk droplet.

Introduction Phytoseiidae are more or less specialized predators, which have achieved more and more importance as natural antagonists of serious crop pests, in particular of spider mites, but also of, for example, thrips and aphids. Thus, there are many studies dealing with the biology, ecology, life-history strategies, etc. of species belonging to this mite family (e.g., Helle and Sabelis, 1985; Sabelis and Harmsen, 1992; Sabelis and Janssen, 1994). On the other hand, only few papers have contributed to the knowledge of the anatomy or fine structure (e.g., Jagers op Akkerhuis et al., 1985). Anatomy and histology of the phytoseiid female genital system have been investigated until now by Petrova (1970) light microscopically and by Alberti (1988), Nuzzaci et al. (2001), and Alberti and Di Palma (in press) also electron microscopically. Like in other Dermanyssina, the reproductive system is composed of one region involved in egg development and oviposition and another for sperm reception and, probably, storage and capacitation. Males transfer a sac-like spermatophore using their modified chelicerae via insemination pores into a sperm-access system (spermathecal apparatus), a manner of direct spermatophore transfer called podospermy (e.g., Athias-Henriot, 1968; Alberti and Coons, 1999; Alberti, in press). The spermatophore material appearing within the sperm-access system, is called the endospermatophore (Schulten, 1985; Evans, 1992; Alberti and Coons, 1999). During or shortly after copulation the endospermatophore achieves a distinct wall. Its origin is unclear. It has been suggested that it is formed after insemination from the material injected or that it is the product of an interaction between spermatophore fluid and vesicle contents. The endospermatophore disappears ‘gradually’ from the vesicle within 1–2 days (Schulten, 1985). Nevertheless, although some useful information on the female gonad anatomy and ultrastructure has been acquired, still little is known about it, in particular about the phytoseiid-type sperm-access system, which considerably

527

Figure 1. SEM Ph. persimilis female. (A) Frontal view. Scale bar: 100 µm. (B) Ventral view. Scale bar: 100 µm. Abbr.: gp, genital plate; Pa, pedipalp; Pe, peritreme.

528

Figure 2. Schematic drawing of phytoseiid female genital system. (A) Dorsal view. (B) Sagittal section. Arrows indicate sperm cells in different positions within the electron lucent tissue. Arrowheads point to somatic tissue. Abbr.: I, II, III, IV, oocyte stage I, II, III, or IV; cal, calyx; cc, cup cell; Ch, chelicerae; gp, genital plate; lo, lyrate organ; lu, lumen; Md, major duct; md, minor duct; N, nucleus; nc, nutritive cord; Pa, pedipalp; sd, salivary duct; ss, salivary stylus; T, electron lucent tissue; V, vesicle; va, vagina; vd, vaginal duct; vg, vaginal gland.

differs from the sperm-access systems of the laelapid-type of other Dermanyssina (Evans and Till, 1979; Alberti and Coons, 1999). The available papers on the phytoseiid-type of this system (e.g., Dosse, 1958, 1959; Schuster and Smith, 1960; Fain, 1963; Athias-Henriot, 1971; Wainstein, 1973; Amano and Chant, 1978; Karg, 1982; Lindquist, 1995; Walter and Lindquist, 1997)

529

Figure 2. (continued)

mainly deal with its gross morphology and cuticular components, which are very useful in taxonomy, while the information on functional anatomy, histology, and cytology is still fragmentary and unclear (Alberti, 1988; Nuzzaci et al., 2001; Alberti and Di Palma, in press). Furthermore, there is no information at all regarding sperm capacitation and transfer to the ovary. Similarly, almost no information exists regarding nutritive egg development, which has been described from Varroa jacobsoni1 ) (Dermanyssina) by Akimov and Yastrebtsov (1984), Alberti and Zeck-Kapp (1986), Alberti and Hänel (1986), Akimov et al. (1988), and Steiner et al. (1995). Since there are striking differences in the composition of both types of sperm-access systems, it can be questioned whether the reproductive systems of phytoseiid mites have evolved convergently, a question that affects the systematic concept of the taxon Dermanyssina (Alberti, in press). Hence, it should be very interesting to compare the phytoseiid-type sperm-access system with the type of sperm-access system observed in other Dermanyssina (laelapid-type), whose functional morphology and ultrastructure was described by Alberti and Hänel (1986). A further aspect concerning the reproductive system of Phytoseiidae is the occurrence of a peculiar type of haplodiploidy, that is parahaploidy (pseudoarrhenotoky), meaning that males are produced by fertilized females, but that 1 According to the recent analysis of the Varroa jacobsoni complex published by Anderson

and Trueman (2000) the correct name of the Varroa species should be Varroa destructor Anderson and Trueman, 2000.

530 part of the genome is immobilized during subsequent development. Resulting males are thus functionally haploid (Helle et al., 1978; Norton et al., 1993). The morphological or fine-structural alterations occurring during this process of genome immobilization are not known. Thus, the aim of the present study was to clarify unknown aspects in order to improve the knowledge of these mites and of the general aspects mentioned above. Furthermore, such improved knowledge will likely contribute to enhancing the effectiveness of using them as biological control of several pests. Finally, these results should improve phylogenetic interpretations as well as clarifying systematical concepts of Gamasida (see also Alberti, in press). Materials and Methods Overwintering females of Typhlodromus rhenanoides Athias-Henriot, collected from Cotoneaster horizontalis Decne fruits in Bari, southern Italy, and active females of Phytoseiulus persimilis Athias-Henriot bought from a commercial supplier (Neudorff company, Germany) were used for this study. Several females of P. persimilis were fixed at different times after mating. T. rhenanoides females were dissected and prefixed in Karnovsky’s (1965) solution, while P. persimilis females were dissected and prefixed in glutaraldehyde (3.5% in phosphate buffer at pH 7.4). After 2 h of fixation, specimens were rinsed in buffer solution, subsequently post-fixed in buffered osmiumtetroxide solution, rinsed again in buffer solution, dehydrated with graded ethanols, and finally embedded in Araldite using propyleneoxide as an intermedium. Polymerization occurred at 65◦ C. Semithin sections were stained according to Richardson et al. (1960) and used for general orientation during ultrathin sectioning. Ultrathin sections were cut with a Leica ultracut UCT microtome using a diamond knife (Diatome), and observations were performed with a Zeiss EM 10 CR electron microscope. Phytoseiulus females were macerated in lactic acid, subsequently dehydrated in graded ethanols, transferred into liquid CO2 using amylacetate as an intermedium and dried according to the critical-point method, and glued on Al-stubs. Then, the dorsal shield was removed and the specimens were sputtered with Palladium-gold using a Polaron SC 7640 coating apparatus. Other males and females were simply collected and processed accordingly for scanning electron-microscopy. A Zeiss DSM 940A scanning electron microscope was used during the study. Finally, slides for light microscopy were prepared from females and males that were fixed while mating. These specimens were mounted in Hoyer’s medium (Krantz, 1978). Observations and micrographs were performed with an Olympus BX50 light microscope.

531 Results In the present study, overwintering as well as active females (Figure 1) were used in order to compare tissue structures in both physiological states. The general anatomy of the reproductive system is the same in both species and physiological situations (Figure 2). The female genital tract is composed of an unpaired gonad, located posteriorly in the body. There is an unpaired oviduct composed of two sections, the uterus (oviduct I) and the vaginal duct (oviduct II). The vaginal duct opens into the vagina (genital atrium, which is covered by the epigynium (genital plate). A pair of vaginal glands opens into the vagina. Furthermore, a sperm-access system is present, which is composed of paired elements: insemination pores (= sperm induction pores or solenostomes), major ducts, emboli, minor ducts, calyces, and vesicles (terminology according to Fain, 1963; Karg, 1982). Gonad As already reported by Alberti (1988) and Nuzzaci et al. (2001), the gonad is composed of a central dome-shaped region (ovary s. str., germinative part) and the lyrate organ (nutritive part), extending anteriorly into two arms (Figure 2). In both species, the gonad shows an arrangement of germ cells that surround a central tissue into which the nutritive tissue of the lyrate organ merges (Figures 3, 4). Furthermore, the uterus starts from this region. Germ cells and the nutritive tissue are embedded in a somatic tissue (also called supporting tissue regarding the lyrate organ) that is continuous with the electron lucent tissue of the sperm-access system. Ovary (s.str.) The ovary is composed of a somatic tissue surrounding oogonia and oocytes; in mated females, also peculiar cells, assumed to be capacitated spermatozoa, occur in the ovary (Figure 3–8). (a) General organization: The somatic tissue differs structurally in the various parts of the ovary or due to different stages of egg development. Early stages of oogenesis (stage 1, see below) are surrounded by small somacells with relatively large nuclei that show a distinct nucleolus. The cytoplasm is only poorly developed. In later stages, somacells close to the developing eggs increase in size and get a dense cytoplasm rich in rough endoplasmic reticulum and containing many mitochondria. The nuclei are sometimes rather dark (Figure 3–10). In addition to these cells (cup cells), there are somacells which contribute to a complex supporting tissue that is likely continuous with the supporting tissue of the lyrate organ (see below) and the electron lucent

532

Figure 3. TEM aspect close to central region of the female gonad (Ph. persimilis) showing oocytes of stages I–III, lyrate organ, and electron lucent tissue. Scale bar: 2 µm. Abbr.: I, II, III, oocyte of stage I, II, or III; cc, cup cells; lo, lyrate organ; Nl, nucleus of lyrate organ; No, nucleus of oocyte; Ns, nucleus of somacell; Nt, nucleus of electron lucent tissue; SC, somacell; T, electron lucent tissue.

533

Figure 4. TEM aspect of the central region of the female gonad (Ph. persimilis). Oocytes of stages II–IV, electron lucent tissue containing putative sperm cells. Central tissue containing bundles of dark filaments. Scale bar: 2 µm. Abbr.: II, III, IV, oocytes of stage II, III, or IV; cc, cup cell; cT, central tissue; Nc, nuleus of cup cell; SC, somacell; Sp, sperm cell.

534

Figure 5. TEM detail showing germinative part of the gonad (ovary s.str.) with oocytes I forming a row parallel to the electron-lucent tissue containing a putative sperm cell sectioned tangentially (Ph. persimilis; compare Figures 17(A), (B), 23, 34(D)). Scale bar: 2 µm. Abbr.: lo, lyrate organ; MG, midgut; Nl, nucleus of lyrate organ; No, nucleus of oocyte; Ns, nucleus of somacell; SC, somacell; Sp, sperm cell; T, electron lucent tissue.

535

Figure 6. TEM detail of ovary with oocytes I (Ph. persimilis). One (indicated with an arrowhead) transforms into a later stage. Note electron lucent tissue at the bottom containing one putative sperm cell. Scale bar: 2 µm. Abbr.: Nc, nucleus of cup cell; No, nucleus of oocyte; Ns, nucleus of somacell; Sp, sperm cell; T, electron lucent tissue.

tissue of the sperm-access system (see below). This tissue represents a compartment in the ovary where peculiar cells, interpreted as putative capacitated sperm cells, arrange themselves close to the oocytes in inseminated females (Figures 3–6, 8). This component of the somatic tissue contains nuclei that are rather small, of varying shape, with a well-defined nucleolus, and conspicuous patches of heterochromatin. At least over large areas, no cell boundaries

536

Figure 7. TEM of further developed oocyte (Ph. persimilis). Note adjacent electron lucent tissue. Scale bar: 1 µm. Abbr.: cc, cup cell; No, nucleus of oocyte; T, electron lucent tissue; Y, yolk.

537

Figure 8. TEM details from ovary (Ph. persimilis). Stage II oocyte with cup cells and stage IV oocyte in contact with electron lucent tissue containing putative sperm cells. Scale bar: 1 µm. Abbr.: II, IV, oocyte of stage II or IV; No, nucleus of oocyte; Nt, nucleus of electron lucent tissue; Sp, sperm cells; T, electron lucent tissue.

were recognized between adjacent nuclei. Hence this tissue may represent a syncytium. Components in the cytoplasm of the somatic tissue differ in appearance according to their position within the gonad, as will be shown below.

538 (b) Oogenesis: In the females of Phytoseiulus, four egg developmental stages are detected, which are mainly defined by cell growth and yolk production. In overwintering females of Typhlodromus, no vitellogenic oocytes are seen. In Phytoseiulus females fixed during or soon after mating, no oocytes belonging to the third and fourth stages are observed, while an egg is sometimes present in the uterus. Stage 1 In Phytoseiulus, a number of peculiar cells are frequently seen that are characterized by rather large nuclei with heterochromatin in a star-shaped configuration. The nuclei are surrounded by only little cytoplasm. These cells, perhaps oogonia or early oocytes, are surrounded by moderate-densely staining somacells (Figures 3, 5, 6). Rarely, cells are seen that have enlarged and show dark inclusions in the cytoplasm which could represent first indications of yolk. The nucleus still shows peculiarly shaped heterochromatin (Figure 7). Stage 2 Oocytes belonging to this stage are larger and characterized by an electrondense cytoplasm, very rich in free ribosomes, rough endoplasmic reticulum, and roundish or irregularly shaped mitochondria arranged in clusters at the periphery of the cell. Their cytoplasm thus very much resembles the cytoplasm observed in the nutritive tissue of the lyrate organ, but contains some heterogeneous inclusions, perhaps precursors of droplets of yolk (Figures 3, 4, 8). The nucleus is less dense than in the previous stage and irregularly shaped. It lacks the star-shaped heterochromatin. Oocytes in this stage show a special relationship to a peculiar type of somacells. These cells (cup-cells), in the sections usually in numbers of two (but, more likely, there are three; Figure 11(B)), are located in close contact with one oocyte pole. They form a cone on this pole from which they extend surrounding the oocyte (Figures 3, 8–10). These cup-cells, which flatten as the oocyte grows, have an ovoidal nucleus provided with a rather large nucleolus (Figures 9, 10). The cells are separated from each other, the oocyte, and the surrounding somatic tissue by their distinct cell membranes. Their cytoplasm is slightly more electron dense than that of the oocyte and is rich in ribosomes, rough endoplasmic reticulum, and mitochondria. Towards the oocyte, these cells show a slightly folded surface (Figures 9, 10). In the cytoplasm of the oocyte, close to the cup cells, a peculiar half-moonshaped region is frequently observable (Figures 3, 8–10, 11(A)). This is an electron-lucent area that is only found at this pole of the oocyte. A rather thin process extends from the oocyte, passing through the cup cells and apparently connecting the oocyte to another tissue. This process, very likely a nutritive cord, in contrast to the half-moon region within the oocyte, is rich in dense

539

Figure 9. TEM of an oocyte II with nutritive cord almost reaching the tissue characterized by filament bundles and straight processes (Ph. persimilis). Note homogeneous material in an half moon shaped area (x) in the oocyte and ribosomes in the nutritive cord. Arrowheads indicate straight processes (some are transversely sectioned). Scale bar: 2 µm. Abbr.: II, oocyte of stage II; cc, cup cell; cT, central tissue; Nc, nucleus of cup cell; nc, nutritive cord.

540

Figure 10. TEM details from the ovary (Ph. persimilis). An oocyte II is seen with its nutritive cord extending from the half moon shaped area (x) of the oocyte. The nutritive cord is surrounded by cup cells. An extension from the central tissue with dense filaments points to the nutritive cord. Arrowhead points to small process of the oocyte surface. Scale bar: 2 µm. Abbr.: cT, central tissue; ex, extension of central tissue; Nc, nuclei of cup cells.

particles, presumably free ribosomes or ribosome precursors (Figures 9, 11). Rarely, also mitochondria were seen. The central tissue, to which the process presumably connects the oocyte and in which no nuclei were observed, shows a cytoplasm rich in ribosomes and mitochondria, similar to the cytoplasm of the nutritive tissue of the lyrate organ (it is likely an extension of the nutritive tissue). Moreover, this tissue is characterized by its bundles of densely staining filaments and a peripheral cell membrane, folded in a peculiar way and showing very straight, filament-containing processes that are sometimes quite

541

Figure 11. TEM details from the ovary Ph. persimilis. (A) Half-moon shaped homogeneous area (x) at the pole of an oocyte from which the nutritive cord extends. Note numerous free ribosomes within the oocyte and in the cord. The cell membranes are dense because of desmosomal properties. Scale bar: 0.5 µm. (B) Cross section through nutritive cord and three adjacent cup cells. Scale bar. 0.5 µm. Abbr.: I, II, III, three cup cells surrounding nutritive cord; nc, nutritive cord.

long. These processes reach into the surrrounding tissue, which represents the somatic tissue of the ovary (Figures 4, 9, 10, 12(A), (B)). Stage 3 In this stage, the oocyte is quite large, dorsally located in the ovary, bulging into the haemocoel and tightly surrounded by the adjacent tissues. Only one oocyte belonging to this stage is present in the ovary. The nucleus is of a round shape, electron lucent, with a very uniform matrix, and with a similar lucent area surrounding the nuclear envelope (Figures 8, 13(A)). The cytoplasm is less electron-dense than in the earlier stage. It is still rich in ribosomes and mitochondria, but these are less densely packed than in the previous stage. Moreover, some dense droplets of lipid yolk start to be present. Peculiar small protrusions of the cell membrane, are observed. The surface of the oocyte is covered by a very thin homogeneous layer (Figure 13(B)). Stage 4 Only one large vitellogenic oocyte is present in Phytoseiulus females. It is located dorsally and posteriorly in the ovary and laterally to the one of stage 3.

542

Figure 12. TEM details from the ovary (Ph. persimilis). (A) The central tissue characterized by dense filament bundles. Note straight process (arrowhead) extending into surrounding somatic tissue. Sale bar: 2 µm. (B) Detail of such a process (arrowhead). Scale bar: 1 µm. Abbr.: cT, central tissue.

The cell has grown considerably due to the increase of groundcytoplasm and yolk. In the peripheral layer of the cytoplasm, large electron-lucent vesicles appear at the cell periphery, which shows an irregular border of very short microvilli (Figures 4, 8, 14(A), (B), 15(A)–(C)). These large vesicles, likely developed from small dark droplets observed in the deeper parts of the

543

Figure 13. TEM details from the ovary (Ph. persimilis). (A) The nuclear region of an oocyte II. Scale bar: 2 µm. (B) Periphery of an oocyte III. Note small protrusions. Scale bar: 0.5 µm. Abbr.: MI, mitochondrium; No, nucleus of oocyte.

cytoplasm, are located just under the cell membrane, occupying the entire cell periphery except for the oocyte region close to the nutritive cord. The large lucent vesicles open towards the extracellular space. Vesicles adjacent to each other fuse (Figure 15(C)). In the cytoplasm, large yolk granules, protein and lipid droplets, are abundant. Irregularly shaped mitochondria, organized in clusters, free ribosomes and extensive smooth and rough endoplasmic reticulum are present (Figures 14(A), (B)). The nucleus, less electron-dense than the cytoplasm and irregular in outline, shows many nuclear pores. The nucleolus presents a central, roundish electron-dense region and some diffuse, less electron-dense material (Figure 14(A)). The nucleus is still surrounded by an electron-lucent area. Finally, in one female of Phytoseiulus, a sac-like structure in the posterior region of the ovary, ventral to the vitellogenic oocyte, is observed.

544

Figure 14. TEM details from the ovary (Ph. persimilis). (A) Nuclear region of oocyte IV. Note nucleus with large nucleolus and homogeneous area around the nuclear envelope. Scale bar: 2 µm. (B) Slightly later stage. Note large yolk droplets and mitochondria. Scale bar: 1 µm. Abbr.: MI, mitochondria; No, nucleus of oocyte; Y, yolk droplet.

545

Figure 15. TEM of oocyte IV (Ph. persimilis). (A) The almost mature egg has pressed aside almost all other organs and is close to the body surface. Scale bar: 1 µm. (B) Periphery showing few small microvilli-like processes and lucent vesicles that open to the exterior (arrowhead). Scale bar: 0.25 m. (C) Tangential section showing fusion of adjacent lucent vesicles. Scale bar: 0.25 µm. Abbr.: IV, oocyte of stage IV; cu, cuticle; EP, epidermis; Y, yolk droplet.

546

Figure 16. TEM of a sac-like structure that likely represents an empty pouch from which the mature oocyte has moved into the uterus (Ph. persimilis). Note extracellular material composed of strongly folded multilayered lamellae. The sac contains destroyed cytoplasmic material. Scale bar: 2 µm. Abbr.: cm, destroyed cytoplasmic material; la, extracellular lamellae.

Its thick wall seems to be composed of highly folded membranous material (Figure 16), which could previously have been stretched to surround an egg already laid. The contents of this sac resemble destroyed tissue. (c) Peculiar cells in the ovary of inseminated females: There are several cells in the ovary of inseminated females that are difficult to interpret. Only in Phytoseiulus we have observed cells that likely represent modified, that is, capacitated sperm cells since they are strikingly different from sperm cells observed in the male (cf. Alberti, 1988; Alberti and Coons, 1999; Alberti and Di Palma, in prep.) and quite certainly do not represent female germ cells or somacells. They are frequently seen arranged in a tissue close

547 to early oocytes (Figures 3–6, 8, 17(A), (B)). Apparently this tissue is a continuation of the putative syncytium that extends from the end of the minor duct (see below) near the ovary (see below). The putative sperm cells have a roundish aspect and contain a densely staining material, likely chromatin. Their surface is provided with peculiar, thin pseudopodal processes reaching deeply into the surrounding somatic tissue and provided with microtubules (Figures 18(A)–(C)). In this stage, sperm cells seem to be closest to the oocytes, with their pseudopodal processes approaching the cup cells or even the young oocyte (Figure 18(B)). Thus, it appears that these putative sperm cells, starting from the region close to the minor duct end (see below) at the anterior region of the ovary, move inside the ovary (i.e., within the somatic tissue) towards the oocytes; moreover, in the posterior part of the ovary, sperm cells with pseudopodal processes are more frequent than in the anterior part. There are further small cells that are even more difficult to interpret at the present state of investigation. These are cells in Phytoseiulus which are different from the early oocytes as well as from the putative capacitated sperm cells (Figures 19(A), (B)). They might either represent intermediate stages of oocyte differentiation or of sperm cell modification. Even more difficult are cells found in the ovary of Typhlodromus which show peculiar patterns of heterochromatin in their relatively large nuclei. They may represent stored sperm or oocytes kept in a peculiar stage of development during the overwintering phase (Figures 19(C), (D)). These relatively large cells frequently show parallel membrane structures within the cytoplasm that resemble septate junctions. Lyrate organ The two arms of the lyrate organ, arising ventro-laterally from the ovary and extending dorsally and anteriorly, are composed of a supporting tissue and a syncytial nutritive tissue (Figures 2–5, 20, 22, 23). In both species, the nutritive tissue is embedded in a supporting tissue that reaches the periphery of the organ and the basal lamina that surrounds it. The branching cells of the supporting tissue present nuclei of differing shapes and provided with a prominent nucleolus and irregularly condensed chromatin. In Typhlodromus the branches of the supporting tissue are relatively broad and electron-lucent in contrast to Phytoseiulus where this tissue shows only very narrow and dense strands. The nuclei of the nutritive tissue in Phytoseiulus are very large, with large nucleoli and scattered patches of heterochromatin. The nuclear envelope presents many pores. The nuclei are surrounded by a homogeneous, electron-lucent area (Figure 21(A)). Moreover, nuclei in the nutritive tissue are interconnected and show numerous nuclear pores. Annulate lamellae, often close to the nuclei of the nutritive tissue, but also free in the cytoplasm,

548

Figure 17. TEM of putative sperm cells (Ph. persimilis). (A) Electron lucent tissue with two sperm cells. Note heterogeneous cytoplasm of the tissue. Scale bar: 1 µm. (B) Sperm cells containing dense, perhaps nuclear material and showing thin pseudopodal processes. Scale bar: 1 µm. Abbr.: I, oocyte of stage I; sp, sperm cell; T, electron lucent tissue.

549

Figure 18. TEM of putative sperm cells (Ph. persimilis). (A) A thicker process extending into thinner ones. The thick process contains microtubules. Another section through a process is close by (arrowhead). Scale bar: 0.5 µm. (B) Pseudopodal processes of putative sperm cells approaching a oocyte III. Scale bar: 0.5 µm. (C) One sperm cell from which thin processes (arrowheads) invaginate into the surrounding tissue. Scale bar: 0.25 µm. Abbr.: III, oocyte of stage III; Sp, sperm cell.

550

Figure 19. TEM of cells in the ovary that could not be interpreted with certainty. (A)–(B) Ph. persimilis. (A) Cell with relatively large nucleus with conspicuous nucleolus. Arrowhead indicates intranuclear lamellae attached to the nuclear envelope. Scale bar: 0.5 µm. (B) Two adjacent cells with paralle lamellae (arrowhead) at the cell periphery. Scale bar: 0.5 µm. (C)–(D) T. rhenanoides. (C) Peculiar cells showing conspicuous dense material (heterochromatin?) and parallel lamellae (arrowheads). Scale bar: 1 µm. (D) Parallel lamellae in higher magnification. Scale bar: 0.2 µm. Abbr.: N, nucleus; Ns, nucleus of somacell; NU, nucleolus.

551

Figure 20. TEM of lyrate organ (Ph. persimilis). Detail showing interconnected nuclei, abundant free ribosomes and mitochondria. The connection between two nuclei is indicated by an arrow. Note only thin extensions of supportive (somatic) tissue (arrowheads). Scale bar: 5 µm. Abbr.: MI, mitochondria; Nl, nucleus of lyrate organ; NU, nulceolus; Ri, ribosomes.

552

Figure 21. TEM of lyrate organ (Ph. persimilis). (A) Periphery of nucleus with electron lucent ‘halo’. Note densely packed ribosomes. Arrowhead indicates nuclear pore. Scale bar: 0.5 µm. (C) Annulate lamellae. Scale bar: 0.5 µm. Abbr.: al, annulate lamellae; Nl, nucleus of lyrate organ; Ri, ribosomes.

are observed (Figure 21(B)). The cytoplasm is rich in free ribosomes and mitochondria. Rough endoplasmic reticulum is less abundant (Figure 20). In Phytoseiulus, the two arms of the lyrate organ extend far anteriorly into the body, reaching and being appressed to the vesicles (receptacula seminis, see below) at the level of the endosternite. They are much more enlarged laterally and dorsoventrally than in Typhlodromus, occupying almost the entire body cavity at the level of the ovary. In Typhlodromus, the two arms of the lyrate organ do not reach so far anteriorly. In the region where the two arms of the lyrate organ join the ovary, the tissues are strongly interconnected, and supporting cells are observed to extend into the ovary (Figures 3, 4, 22). Hence, the supporting tissue of the lyrate organ is continuous with the somatic tissue of the ovary (s.str.). Uterus (Oviduct I) The unpaired uterus is located ventrally to the ovary and directed anteriorly, opening into the vaginal duct (oviduct II) (Figure 2). It starts from a

553

Figure 22. TEM of center of the ovary occupied by filament rich central tissue to which an oocyte IV is attached via its cup cells (Ph. persimilis). Note further cell types. Scale bar: 5 µm. Abbr.: I, II, IV, oocyte of stage I, II, or IV; cc, cup cell; cT, central tissue; ER, endoplasmic reticulum; lo, lyrate organ; Nl, nucleus of lyrate organ; SC, somacell; Sp, sperm cell; Y, yolk droplet.

central region underneath the central tissue where the two branches of the lyrate organ and the ovary (s.str.) connect (Figures 22, 23(A)). In this region cylindrical epithelial cells connect the central tissue of the ovary and uterus (Figure 23(B)). A lumen is hardly detectable. More anteriorly, in Typhlodromus, the uterus shows a flat lumen, which is difficult to recognize; no egg is

554

Figure 23. TEM close to center of ovary from which the uterus starts, that is sectioned probably slightly above or in front of the section shown in Figure 22 (Ph. persimilis). (A) The branches of the lyrate organ merge. Note in the upper part a row of oocytes I paralleled by electron lucent tissue. At the bottom part of the connection with the uterus is seen. Scale bar: 5 µm. (B) Figure adjacent to the lower part of Figure (A) but in higher magnification. Note the columnar cells of connection to the uterus. A lumen is not apparent. Scale bar: 2 µm. Abbr.: I, oocyte of stage I; lo, lyrate organ; MG, midgut; Nl, nucleus of lyrate organ; Nu, nucleus of uterus cell; T, electron lucent tissue; U, uterus.

555 present inside. In contrast, in some Phytoseiulus females, a very large lumen is filled with a large mature egg. In these cases, the uterus occupies almost the entire ventral body cavity, reaching the medial region while the ovary is pushed dorsally. Here, the cells of the uterus epithelium are irregularly shaped and flattened. The nuclei are located basally, showing a roundish nucleolus and dispersed chromatin. In the cytoplasm surrounding the nucleus, free ribosomes, numerous roundish mitochondria, lipid droplets, and dark inclusions are present (Figures 24(A)–(C)). Muscles are beneath the uterus epithelium. Only one egg is present in the uterus. It is very large and roundish in shape, even though artificially shrunken. An external electron-lucent, homogeneous thin endochorion layer surrounds the egg, which contains very electron-dense material. It is not possible to distinguish any cellular or tissue structure inside, likely due to endochorion impermeability to fixatives and embedding media (Figure 24(B)). Vaginal duct (Oviduct II) and Vagina (Genital atrium) The rather short vaginal duct is lined with cuticle (Figure 24(D)). The cuticle thickness increases gradually towards the vagina, into which it continues without a distinct border. We define the end of the vaginal duct and the beginning of the vagina by the presence of the epigynium (genital plate) (Figures 25(A), 28(A), (B)). From this region onwards, the genital duct is no longer a completely closed tube, but rather opened laterally. The vaginal duct and vagina is flattened and folded, much wider than high. The distal part shows plicated cuticular walls. Epithelial cells are flat, interdigitated in a complicated way, provided with elongated nuclei showing scattered chromatin, scarce endoplasmic reticulum, and a few mitochondria. Moreover, strong muscles, connected to the distal part of the oviduct and the genital plate, are observed. They are likely involved in oviposition. Vaginal glands A pair of vaginal glands is located above the vaginal duct and vagina (Figures 2, 25(A)). Both glands open separately into the vagina dorsolaterally, apparently via rather long slits (Figure (27)). Each gland is composed of at least eight large cells, which contain large nuclei with conspicuous nucleoli. The cytoplasm of these cells is full of rough endoplasmic reticulum, whose cisternae are mostly inflated and contain an electron-lucent material (Figure 26(A)). Golgi bodies are present, but rather inconspicuous. They consist of a number of electron-lucent vesicles; cisternae are not evident. The glandular cells narrow towards their extrusion poles. Here, they are provided with a

556

Figure 24. TEM of oviduct (Ph. persimilis). (A): Detail of the uterus. Note small dense vesicles in the cell apex (arrowheads). Arrow points to basal lamina. Scale bar: 1 µm. (B) Uterus cell with dense vesicles (arrowheads) and small detail of an egg showing tight endochorion and dark contents of the egg. Scale bar: 1 µm. (C) Basal part of uterus epithelium with flattened nucleus. Scale bar: 1 µm. D: Detail of vaginal duct with thin cuticle underlain by very flat cells. Scale bar: 1 µm. Abbr.: ech, endochorion; lu, lumen; MI, mitochondria; Nu, nucleus of uterus cell.

557

Figure 25. TEM of vagina and vaginal glands. (A)–(B) Ph. persimilis. (A) Overview of cross section showing vagina and the two reservoirs of the vaginal glands. Scale bar: 2 µm. (B) Detail of reservoir filled with secretion. Note thin cuticle (arrowheads) and bundles of filaments in the epithelium. Scale bar: 0.5 µm. (C) Reservoir of vaginal gland of T. rhenanoides. Note that the reservoir is empty in this overwintering specimen. Arrowheads indicate thin cuticle of reservoir. Scale bar: 0.5 µm. Abbr.: eT, extraovarian nutritive tissue; f, filaments; HL, hemolymph; Ne, nerve; res, reservoir of vaginal gland; Se, secretion; va, vagina.

558

Figure 26. TEM of vaginal gland (Ph. persimilis). (A) Detail of gland cell showing nuclear region with abundant rough endoplasmic reticulum with swollen cisternae. Inconspicuous Golgi bodies (dictyosomes) produce light vesicles that fuse to larger secretory droplets. Scale bar: 0.5 µm. (B) Extrusion pole with small microvilli and a nerve ending with dense synaptic vesicles close by. Arrowhead points to neurotubules. Scale bar: 0.5 µm. Abbr.: D, Golgi body; ER, rough endoplasmic reticulum; NE, nerve ending; Ng, nucleus of gland cell; Se, secretion.

559

Figure 27. TEM of vaginal gland (Ph. persimilis). The reservoir opens into the vagina (arrowhead). Note different appearance of cuticle in reservoir, duct and vagina. Scale bar: 0.5 µm. Abbr.: cu, cuticle of gland opening; res, reservoir; va, vagina.

few irregular microvilli (Figure 26(B)). The secretory vesicles discharge their contents into a lumen that continues into a system of short collecting tubes, which ends in a common reservoir filled with dispersed material. Semithin sections showed metachromasy of the secretion when stained according to Richardson et al. (1960) with Toluidin blue and appeared light red. Collecting tubes and reservoir are provided with a thin cuticle (Figures 25(A)–(C), 27). The reservoir opens into the vagina through an undulating canal or slit, which is provided with a thicker cuticle (Figure 27). The gland is strongly innervated. There are numerous nerve endings containing dense vesicles (Figure 26(B)). Thin and thick muscles are close to the gland. However, it

560 is not clear whether they belong to the gland or are muscles attached to the vaginal duct or vagina and involved in oviposition. Genital opening The genital opening is in principle a transverse (rather, an inverted U-like) slit bordering the epigynium (genital plate; Figures 1(B), 28(A), (B)). The epigynium is a ventral duplication arising from behind. It contains, besides the epithelial, muscular and nervous tissues, a strongly developed extraovarian nutritive tissue. Extraovarian nutritive tissue A peculiar tissue consisting of large cells (Figures 29(A)–(C)) that are already conspicuous when sections are obeserved with the light microscope is found in the body periphery immediately beneath the thin epidermal layer. It replaces part of the region occupied by fat body cells. The extraovarian nutritive tissue is strongly developed within the epigynium, but is probably most prominent in the dorsal body between the caeca of the gut system. The cells of the extraovarian nutritive tissue are almost completely filled with narrow cisternae of a strongly developed rough endoplasmic reticulum. Sometimes, clusters of droplets are seen (Figure 29(C)). Nuclei are quite large and provided with roundish and distinct nucleoli and patches of heterochromatin (Figures 29(A), (B)). A few mitochondria are present in the cytoplasm that are frequently destroyed artificially, however (Figure 29(A)). Similar cells, though less conspicuous, were observed in Typhlodromus. Sperm-access system In both species, the general anatomy of the sperm-access system is the same, even though some differences in the gross morphology are present, hence the taxonomic value of this structure. The system is composed of paired elements: solenostomes, major and minor ducts, emboli, calyces and vesicles (terminology according to Fain, 1963; Karg, 1982) (Figures 2, 30(A)–(E)). Solenostome The sperm-access system opens by two solenostomes (insemination pores, sperm induction pores), on each ventral side of the body, located close to but in front of coxae IV (according to Evans and Till, 1979, they open into the arthrodial membrane of coxae III). These pores are simple invaginations of the cuticle connected to the major duct. A cuticular circular fold surrounds the solenostome (Figure 31(A)).

561

Figure 28. SEM of female genital structures in Ph. persimilis. (A) Ventral view of genital plate (epigynium). Scale bar: 20 µm. (B) Oblique fronto-ventral view of genital plate showing that the genital plate is over a large part a free tongue-like duplication (arrowheads) protruding from the posterior. The insemination pores to be expected between coxae III and IV (arrow) are not visible because they are located deeply in the arthrodial region. Scale bar: 20 µm. Abbr.: gp, genital plate.

562

Figure 29. TEM of extraovarian tissue (Ph. persimilis). (A) Overview. Note that the cells are almost completely occupied by rough endoplasmic reticulum. Scale bar: 2 µm. (B) Nuclear region. Scale bar: 1 µm. (C) Small group of droplets. Scale bar: 1 µm. Abbr.: ER, rough endoplasmic reticulum; HL, hemolymph; N, nucleus; va, vagina; vE, epithelium of vagina.

563

Figure 30. SEM of sperm access-system (Ph. persimilis). Structures are shown from the inner side of the specimens after removing tissues with lactic acid. (A) Detail showing the cuticular components of the sperm-access system. Major duct, calyx and vesicle are seen. The origin of minor duct that is torn off is indicated. Scale bar: 10 µm. (B) Detail showing the narrow part of the calyx. Scale bar: 2 µm. (C) Transition between calyx and vesicle, the latter with folded wall. Scale bar: 2 µm. (D) Origin of minor duct (embolus). Scale bar: 2 µm. (E) Origin of minor duct. Note that the minor duct is deeply inserted into this region corresponding to the embolus (see Figures 31(C), 33(A), (B). Scale bar: 1 µm. Abbr.: cal, calyx; emb, embolus; Md, major duct; md, minor duct; V, vesicle.

564

Figure 31. TEM of T. rhenanoides sperm-access system. (A) Solenostome (insemination pore). Scale bar: 1 µm. (B) Major duct close to solenostome. Note cuticular folds. Scale bar: 1 µm. (C) Embolus with folded major duct and minor duct (arrowhead). Scale bar: 1 µm.

565 Major duct This duct presents thick cuticular walls. In both species, it is short and extends inside the body, directed dorsally and a slightly posteriorly. Its lumen is sometimes filled with very electron-dense material (Figures 31(B), 32). Shortly behind the solenostome, the cuticular wall of the major duct of Typhlodromus shows peculiar cuticular folds, while in Phytoseiulus it remains rather smooth (Figures 31(B), (C), 32, 36(A)). The major duct cuticle is multi-layered, and its epithelium is flat, showing endoplasmic reticulum, roundish mitochondria, and Golgi bodies. Nuclei are irregular in outline with scattered chromatin. Atrium and embolus The embolus is a modified region in the major duct, from which the so-called minor duct starts. Here, the lumen of the major duct (or atrium) is very much reduced by a strong cuticular protrusion or plug. The lumen appears sickleshaped in cross section. The so-called minor duct starts from the center of the protrusion (Figures 31(C), 33(A), (B)). Scanning electron-microscopic

Figure 32. TEM of Ph. persimilis sperm-access system. Major duct containing dense material and with rather smooth cuticle. Scale bar: 0.5 µm.

566

Figure 33. TEM of Ph. persimilis sperm-access system. (A)–(B) Embolus sectioned at different levels. Note very narrow lumen of major duct and almost invisible lumen of minor duct (arrowheads). Scale bars: 0.5 µm.

567 observations of the macerated system from the inner (tissue) side revealed a kind of depression that showed several rings in the cuticle and the thin minor duct arising from the center of this depression (Figures 30(D), (E)). After the beginning of the minor duct, the major duct widens to produce the calyx (Figure 30(A)). Minor duct The minor duct connects to the major duct in the embolus as described above. At its beginning, the minor duct shows a barely visible lumen, surrounded by a relatively thick and uniform cuticle. The lumen is represented, in longitudinal sections of the duct, merely by a darker or lucent line (depending on the preparation) in the cuticle itself and, in cross sections, by a fine spot (Figures 31(C), 33(A), (B)). More posteriorly, the lumen becomes slightly wider. Nonetheless, it is still fine and looks smaller in Typhlodromus (30 nm) than in Phytoseiulus (40 nm). In the latter species we observed that the lumen slighlty increases in diameter along its course (100 nm); moreover, in Typhlodromus, it was always empty, while in Phytoseiulus some diffuse, not identified material, was sometimes present in the duct (Figures 34(A)–(C)). The minor duct is appressed to or even sunken into the epithelial tissue that accompanies the calyx and vesicle (Figures 39–43). The flat cells show a few roundish mitochondria and endoplasmic reticulum, while the irregularly shaped nucleus presents diffuse chromatin. At its beginning, the minor duct is mainly directed dorsad, as is shown by cross sections, in which it is cut tangentially, and in horizontal sections, where it is cut in a transverse manner. The duct then runs posteriad, close to the walls of the calyx and vesicle (Figures 39–43). In the following part, it extends towards the dorsally located lyrate organ. During its path, it is further observed ventral to the Malpighian tubules and then appressed to the medioventral surface of the lyrate organ. The minor duct ends in the region where the lyrate organ meets the ovary. Close to its end, the cuticular wall becomes thinner, more irregular, and finally ends with many folds, the lumen becomes larger (Figures 35(A), (B)). But neither an opening towards the haemocoel nor a direct connection to the ovary or the lyrate organ was observed. The end of the minor duct is occupied and surrounded by an electron-lucent tissue adjacent to the basal lamina of the lyrate organ (Figures 35(A), (B)). This electron-lucent tissue exends transversely along the inner (anterior) side of the ovary and reaches the corresponding tissue coming from the other side. Thus a kind of transverse belt is formed (Figures 5, 23(A), 34(D)). Within this tissue, some electron-lucent nuclei and many lysosome-like and heterogeneous structures are present (Figures 35(A), (B)), while cell borders were observed quite rarely. The

568

Figure 34. TEM of sperm-access system. (A) Minor duct (arrowhead) of T. rhenanoides near its beginning at the embolus sectioned almost transversely. Scale bar: 0.25 µm. (B)–(D) Ph. persimilis. (B) Minor duct (arrowhead) in cross section close to its beginning at the embolus. Scale bar: 0.5 µm. (C) Oblique sections through the minor duct (arrowheads) deeper in the animal. Note that the lumen is slightly wider. Scale bar: 0.5 µm. (D) TEM showing electron lucent tissue forming a bridge from one side to the other through the ovary paralleling the row of oocytes I (the figure has been orientated vertically for technical reasons). Scale bar: 2 µm. Abbr.: MG, midgut; No, nucleus of oocyte; Ns, nucleus of somacell; Nt, nucleus of electron lucent tissue; T, electron lucent tissue.

569

Figure 35. TEM of minor ducts of Ph. persimilis. (A) Minor duct sectioned transversely close to its proximal (inner) end. Note that the duct is surrounded by the electron lucent tissue. Scale bar: 1 µm. (B) End of minor duct within electron lucent tissue. Arrowheads indicate extensions of the cuticle of the minor duct end. Scale bar: 0.5 µm. Abbr.: md, minor duct; T, electron lucent tissue.

570

Figure 36. TEM of calyx of T. rhenanoides. (A) The calyx close to the embolus still shows the cuticular folds. Arrowhead points to minor duct. Scale bar: 1 µm. (B) More interior in the body the calyx gets a smooth cuticle. Note the different layers in the cuticle. Scale bar: 1 µm.

571

Figure 37. TEM of calyx of Ph. persimilis. (A) Transverse section through the thin portion of the calyx (compare Figure 30(B)). Scale bar: 0.5 µm. (B) Wider part of calyx. Note dense contents. Scale bar: 0.5 µm.

572

Figure 38. TEM of calyx-vesicle continuation (Ph. persimilis). (A) Note highly folded vesicle and the large vacuoles (x) within the underlying epithelium of the calyx. Note dense material within calyx (compare Figure 37(B)). The vesicle cuticle is composed of at least two layers, a more dense inner layer and a less dense outer layer. Scale bar: 1 µm. (B) Note rather strong cuticle in the calyx region containing a plug of dense material. The vesicle part is apparently more flexible. The inner cuticle of the latter is rather thick and is folded (see also Figure 38(A)). Scale bar: 1 µm. Abbr.: cal, calyx; V, vesicle.

573

Figure 39. TEM of calyx-vesicle continuation (Ph. persimilis). The vesicle is much folded. Note the large vacuoles in the vesicle and calyx epithelium (x). Arrowhead points to minor duct. Scale bar: 2 µm. Abbr.: cal, calyx; lo, lyrate organ; MU, muscle; Nl, nucleus of lyrate organ; V, vesicle.

574 aspect of the cytoplasm resembles the one found in the somatic tissue invaded by the pseudopodal sperm cells (Figures 4–6, 8, 17, 22, 23). Calyx The major duct widens to form the stalk of the calyx, which is also cuticlelined. In both species, the calyx is quite long, in contrast to the rather short major duct. The calyx is directed dorsally and a bit posteriorly and continues into the vesicle (Figure 30(A)). At its beginning, it is slightly curved, the calyx then extends dorsad and its diameter becomes smaller for a short distance (Figures 30(A)–(C), 37(A), (B)). It then widens again to continue into the vesicle. In cross sections through the calyx, it usually has a circular shape, which is sometimes also deformed by pressures from adjacent tissues. In Typhlodromus, the wall of the calyx close to the embolus still shows the peculiar cuticular folds described above (Figure 36 (A)). Moreover, the cuticle is composed of three layers. There is a thin, compact and uniform external and a similar internal layer; these are separated by a thick, less compact layer (Figures 36(A), (B)). More interiorly in the body, the cuticle becomes thinner and is thus only represented by a single homogeneous ring in transverse section. In Phytoseiulus, the cuticle is more uniform. The lumen of the calyx of some specimens contains some heterogeneous material, which is sometimes very electron-dense (Figures 37(B), 38(A), (B)). Vesicle The calyx continues into the vesicle, which is also cuticle lined. In this region, the cuticle is rather conspicuous, but less sclerotized than in the calyx, hence it frequently is very folded (Figures 30(A), (C), 38(A), (B), 39, 40). In Typhlodromus females, the vesicle always presents folded walls (Figure 41), while in some specimens of Phytoseiulus, especially if just mated, at least one vesicle is present with a roundish shape, presumably containing remnants of the endospermatophore (Figures 42, 43). The vesicle wall is difficult to understand. It may be that its different appearances depend on the mating state. In Typhlodromus, the folded wall is composed of an electron-dense cuticle-like material of varying thickness and the underlying flat epithelium shows a very irregular apical region with many microvilli-like protrusions (Figure 41). In Phytoseiulus, the vesicle cuticle seems to be thinner, and the epithelium varies. Sometimes it is very flat and inconspicuous (Figures 43, 44(B)), in other specimens it is at least in some regions thick with extensive dark inclusions or vacuoles, together with some endoplasmic reticulum, free ribosomes, mitochondria and an irregularly shaped nucleus (Figures 38–40). The cytoplasm above the vacuoles, that is adjacent to the lumen of the vesicle, is sometimes very thin or is even lacking

575

Figure 40. TEM of another aspect of a vesicle that is rather full (Ph. persimilis). At the upper part a portion of the calyx cuticle is still seen. Note that the vesicle cuticle is slightly folded and is composed of a dense inner part and a less conspicuous outer part. The latter cuticular layer may be very thin or even lacking (see lower part of the Figure). Again large vacuoles (x) are seen in the epithelium. These vacuoles contain some dense material. Arrowhead indicates minor duct. Scale bar: 1 µm. Abbr.: cal, calyx; lo, lyrate organ; MT, epithelium of an adjacent Malpighian tubule; MU, muscle; V, vesicle.

(Figures 40, 44(A)–(C)). The contents of the vesicles also vary and are difficult to interpret. In Typhlodromus, it is an unstructured material of varying electron density. In Phytoseiulus, the contents are very much more complex, apparently due to the presence or absence of an endospermatophore that may also be of varying age. Thus, we found a vesicle that contained an electron-

576

Figure 41. TEM of a vesicle of T. rhenanoides. Note the strongly folded cuticle-like contents and the obvious microvilli. Arrowheads indicate minor duct. Scale bar: 1 µm. Abbr.: Mv, microvilli; V, vesicle.

lucent material surrounded by a thick, multilayered envelope (Figure 44(A)). Another one included dense material surrounded by a thick, rather homogeneous wall (Figure 44(B)). In another female, there was a very heterogeneous material surrounded by a very thin layer (Figure (43)). We never could distinguish sperm cells inside the vesicle, although we found very densely staining structures that might represent strongly condensed nuclear material (Figure (43)).

Discussion The phytoseiid female reproductive system follows the general pattern of Dermanyssina, showing a differentiation into a region involved in egg development and oviposition and a region (the sperm-access system) for sperm reception (Alberti and Coons, 1999 with further literature). Furthermore, there is an unpaired oviduct differentiated into a uterus and a vaginal duct as in other taxa showing neospermy (i.e., Parasitina and Dermanyssina; Alberti and Coons, 1999; Alberti, in press). The gonad is differentiated into a

577

Figure 42. LM of a Ph. persimilis female killed after copulation and macerated. Note that one vesicle is round in shape containing an endospermatophore whereas the other one is empty and shrunken. Scale bar: 20 µm. Abbr.: cal, calyx; emb, embolus; GO, genital opening, V, vesicle.

germinative part (ovary s.str.) and a nutritive part (lyrate organ). The ovary is the region where growth and fertilization of oocytes take place (Akimov and Yastrebtsov, 1984; Alberti and Hänel, 1986; Alberti and Zeck-Kapp, 1986; Akimov et al., 1988; Nuzzaci et al., 2001; Alberti and Di Palma, in press). Its solid structure is due to the presence of somacells surrounding and holding the oocytes in position. These somacells occur in at least two types, one type immediately adjacent to the oocytes (cup cells) and another type establishing the tissue between these cells and continuing into the supportive tissue of the lyrate organ. Thus, the general histology of the gonad is quite similar to that observed in Varroa jacobsoni, in which also two types of somacells were observed (somacells I and II) with somacells II surrounding the oocyte and the nutritive cord. Like in Varroa there are apparently three somacells (cup cells) surrounding the nutritive cord in Phytoseiulus (Alberti and Hänel, 1986; Alberti and Zeck-Kapp, 1986). An ovarian lumen is absent, as in Varroa jacobsoni and probably all Dermanyssina and Parasitina (Alberti et al., 1999). This situation creates a

578

Figure 43. TEM of a vesicle perhaps recently filled with an endospermatophore (Ph. persimilis). Note its spherical appearance, very thin epithelium and the heterogeneous contents with conspicuous dense material. Arrowhead points to minor duct. Scale bar: 1 µm.

problem, since the routes of spermatozoa towards the oocytes thus become more complicated. Due to the solid character of the ovary in Parasitina and Dermanyssina, which is probably a consequence of differentiation of the ovary into a germinative part and a nutritive part, sperm cells are not able to move up through the oviducts and reach a tubular ovary to fertilize oocytes,

579

Figure 44. TEM details of vesicle (Ph. persimilis). (A) This vesicle shows a wall like structure composed of numerous fibrillar elements surrounding a lucent central material. Note a vacuole within the epithelium which is almost open towards the vesicle contents. Scale bar: 1 µm. (B) In this vesicle a structure is included which shows a conspicuous multilayered wall. The epithelium is very flat and includes a lucent vacuole (x). Arrowhead points to basal lamina. Scale bar: 0.5 µm. (C) Part of a vesicle wall with large dense vacuole which almost is continuous with the vesicle contents. Arrowhead indicates basal lamina. Scale bar: 0.5 µm. Abbr.: V, vesicle lumen.

580 as in early derivative anactinotrichid mites, but are forced to penetrate female somatic tissues (Alberti and Hänel, 1986; Alberti, 1988, 1991, 2000; Alberti and Coons, 1999; Alberti et al., 2000; Alberti, in press). Unfortunately, we could not yet follow the routes of spermatozoa after copulation. We also could not distinguish sperm cells within the vesicles of the sperm-access system, though they are likely to be expected in the heterogeneous contents of inflated vesicles after mating. However, we observed peculiar cells of different appearances in the ovary. These cells, which we consider, at least some of them, to represent modified (capacitated) sperm cells, were frequently found embedded in the somatic tissue in a way that suggests a somatic tissue path towards the oocytes. The interpretation of active penetration of spermatozoa through the ovary is also supported by the presence of a double membrane surrounding each sperm cell. However, female cells are not disrupted; hence, the penetration seems to be a controlled one, resulting from an interaction between male and female cells. This is similar to what has been observed in Parasitina (Alberti et al., 2000). On the other hand, the putative transformation of sperm cells (capacitation) must be a presumably rapid process that lead to spermatozoa, which are very much different from those observed in the Phytoseiulus male (Alberti, 1988; Alberti and Coons, 1999) being simpler in their cytological structure, in particular regarding their cell surface. A similar profound and even extremely complex modification of sperm structure during capacitation was also described from ticks (e.g., Feldman-Muhsam and Filshie, 1979; Oliver, 1982; Coons and Alberti, 1999). A transformation from complex not-capacitated sperm cells (also called prospermia) is also known from a species belonging to the Dermanyssina, for example, Varroa jacobsoni (Alberti and Hänel, 1986; Akimov et al., 1988; Alberti and Coons, 1999). Moreover, this capacitation process provides spermatozoa with structures (e.g., pseudopodal processes in Parasitina; Alberti et al., 2000) that are apparently very effective for moving within the female genital tract (ticks) or among ovarian somatic tissue. Fertilization likely occurs in the early stages of oogenesis by penetration of these capacitated spermatozoa into the oocytes. Another type of cells, present in higher numbers in Typhlodromus, but absent in Phytoseiulus, may represent a resting sperm stage capable of being stored in the overwintering female. But this needs further investigation. The anatomical shape and fine structure of the lyrate organ closely resembles that of the only other species investigated until now of another family of Dermanyssina, Varroa jacobsoni (Alberti and Hänel, 1986; Alberti and Zeck-Kapp, 1986; Steiner et al., 1995; see also Nuzzaci et al., 2001 regarding Typhlodromus). Ultrastructurally, the lyrate organ is also similar to the nutritive tissue of Parasitina (Alberti et al., 1999). In all these taxa, the nutritive

581 tissue is a syncytium composed of a cytoplasmic component that is very rich in free ribosomes, rough endoplasmic reticulum, mitochondria and interconnected nuclei. Thus, quite likely a nutritive function can also be assumed for the lyrate organ in phytoseiid mites (see also Alberti, 1988). Moreover, the presence of nutritive cords connecting oocytes to the lyrate organ (via the central tissue) and the high number of ribosomes present in both nutritive tissue and nutritive cords support this hypothesis. On the other hand, the observed extraovarian nutritive tissue could be involved in supporting yolk production in the oocytes. The larger size of the lyrate organ in Phytoseiulus compared with Typhlodromus could be simply due to species-specifity. However, it could also be a consequence of different physiological states. Whereas Typhlodromus females were inactive overwintering females, Phytoseiulus females were caught in the mating season. Hence, a higher activity of the gonad could be expected and may be realized by the numerous nuclear pores, annulate lamellae, and interconnected nuclei observed in Phytoseiulus, demonstrating a high activity most likely in producing ribosomes or ribosome precursors. It may be that the nutritive tissue enlarges at the beginning of the mating season by endomitosis and subsequent cytoplasmic growth. Likewise, the extraovarian tissue is very much evident in Phytoseiulus, while it is not so in Typhlodromus. However, in the latter, some large cells located in the same region and also showing an extensive rough endoplasmic reticulum are present. These cells may become more evident during the reproductive season. Finally, the syncytial structure of the nutritive tissue, the localization in one part of the gonad only and the presence of supporting cells suggest the analogy with telotrophic meroistic ovarioles of insects, as already pointed out by Alberti and Zeck-Kapp (1986) in Varroa. Younger and more numerous oocytes (stages 1, 2) are located centrally in the ovary, while the older (stages 3, 4) – possibly because of their larger size – are located dorsally in the ovary, bulging into the haemocoel. Oocytes are first completely surrounded by cup cells. Later, when rapid vitellogenesis occurs, these cells are predominantly present in the region where the nutritive cord connects the oocyte to the central tissue and the oocyte is at least partly exposed to the hemolymph (see Varroa; Alberti and Zeck-Kapp, 1986; Steiner et al., 1995). Cup cells, in contrast to the follicular cells described in Pergamasus barbarus Berlese (Witaliñski, 1987a), were not observed to take part in perivitelline space formation. Moreover, we observed a process, mainly due to oocyte activity, that may lead to the formation of an external envelope. In oocytes that already show some yolk droplets, this process starts by producing small membrane protrusions, which result in a small irregular border of microvilli on the oocyte surface located under a very thin basal lamina. In a later step, a high number of electron-lucent vesicles is found,

582 located around the entire oocyte surface below the microvilli border. It is only absent at the oocyte pole where the nutritive cord starts. Unfortunately, we could not observe the following steps of the process. If these vesicles coalesce to form the vitelline membrane, its formation would be quite different from vitelline-membrane formation in other Acari (or even Arachnida) (e.g., Witaliñski 1986, 1987a, 1988, 1993; Alberti and Coons, 1999). In any case, for some time the vitelline membrane is lacking in the region of the nutritive cord or egg stalk (also termed funicle). This region was considered a transitory micropyle by Brinton and Oliver (1971) and Witaliñski (1987b). The vitelline membrane is later, according to the nomenclature of Witaliñski (1986, 1987a), transformed into the endochorion. An endochorion was observed surrounding an egg in the uterus and appeared to be very tight. Uterus cells contain electron-dense granules, possibly of secretory nature, but the influence of substances produced by the oviduct walls can only be suspected. It is still not clear how oocytes move through the ovary to reach the uterus lumen, also considering their large size. In females producing eggs, some roundish areas of destroyed tissue containing fragmentated material and partly surrounded by a sac-like structure were observed. These areas could have been the area where a mature oocyte was located before being transferred towards the uterus. The sac-like structure may represent remnants of the material surrounding the growing oocyte (cf. Alberti and Coons, 1999; Coons and Alberti, 1999). Perhaps the peculiar central tissue characterized by the bundles of dense filaments may represent the route by which the mature egg reaches the uterus. This tissue is seen in the center of the ovary (Figure 4) and is connected to the oocyte on the one hand and, likely, to the uterus on the other hand. The destroyed cytoplasm seen in the sacs may represent the remnants of this tissue. What forces the egg into the uterus? There are no muscles surrounding the almost mature egg. Only the very thin basal lamina forms a sac that holds the egg in contact with the ovary. At least two mechanisms have been suggested to explain the egg movement: increase of hemolymphic pressure or contractions and other activities of stalk cells involving cytoskeletal elements (Coons and Alberti, 1999). However, none of these can really explain this event sufficiently. We suggest a further mechanism that takes into account the considerably different appearance of the basal lamina in the expanded state (surrounding the egg) and after ovulation: it may be that a molecular rearrangement of the materials that provide this sac creates the force needed to push the egg out. Since this type of ‘external’ vitellogenesis (i.e., oocytes exposed to the fluids in the body cavity; e.g., the hemolymph during vitellogenesis) is found in taxa of very distant systematical positions and hence represents a wide-spread phenomenon (e.g., Priapulida: Alberti and Storch, 1989; Onychophora: Storch and Ruhberg, 1991;

583 Chelicerata: Alberti, 1974, Alberti and Zeck-Kapp, 1986; Alberti and Coons, 1999; Coons and Alberti, 1999; Fahrenbach, 1999; Farley, 1999; Felgenhauer, 1999), the suggested mechanism may be worth consideration. Somewhat puzzling is also the appearance of nutritive cords, very distinct in Varroa (Alberti and Hänel, 1986; Alberti and Zeck-Kapp, 1986) and similarly also present in phytoseiids as shown here. Though not yet observed in Parasitina electron microscopically it seems likely that nutritive cords are also present in this group (Alberti et al., 1999). However, cords were not found in Euryparasitus emarginatus (Ologamasidae) by Witaliñski (1987b). Instead spherical evaginations were described from this species projecting against the central cell of the stalk (funicle) that connects the growing egg to the center of the ovary. The stalk was described as being composed of several central cells and peripheral parietal cells. The central cells and parietal cells contain many microtubules besides other organells. May be that the evaginations correspond to the nutritive cord and the parietal cells to the somacells (cup cells) observed in Varroa and the phytoseiids. The central cells of Euryparasitus probably are equivalent to the central tissue in phytoseiids and may provide the path of the egg into the uterus. No such cells were yet observed in Varroa. Nothing is known about the fate of the nutritive cord in the late phases of oogenesis. Probably it is withdrawn by the late oocyte prior to its moving through the stalk/central tissue into the uterus. The vaginal duct seems to be very short and shows no peculiarities, in contrast to, for example Pergamasus crassipes (Parasitina). A sphincter region and ventral and latero-medial glandular complexes are lacking, as in Varroa jacobsoni (Alberti and Hänel, 1986; Alberti et al., 1999). The vagina is also quite simple when compared with Parasitina. This may be due to the simple function as an egg path in Phytoseiidae, in contrast to Parasitina where the vagina also acts as a spermatophore-storing and probably controlling organ (Alberti et al., 1999). In contrast to Varroa jacobsoni, in which no vaginal glands have been observed, phytoseiids possess these glands like Parasitina and probably at least some other Dermanyssina (e.g., Dermanyssus gallinae: Pound and Oliver, 1976; Alberti and Coons, 1999). It is not clear if the gland contents are passively pressed out of the reservoirs during egg passage or discharged by means of muscle action. These glands likely produce a surface layer that is added to the endochorion during oviposition and may provide a waterprooflayer to the eggs. The metachromasy of the secretion observed in phytoseiids after staining with Toludin blue (Richardson et al., 1960) may indicate an acid mucopolysaccharide nature, though this needs to be more specifically proven (Boeck, 1984). Like in Pergamasus, the glands are innervated (Alberti et al., 1999). The apparent absence of these glands in Varroa jacobsoni may be a

584 consequence of the peculiar reproductive biology of this bee parasite. Oviposition in Varroa occurs in the sealed cell of the bee and, hence, desiccation is not a problem (e.g., Ritter, 1981). The action of strong muscles connected to the vagina may force the egg out. From light-microscopic investigations, it is known that endospermatophores are transferred into the vesicle via the major duct by means of the male chelicerae, which are provided with a peculiar structure, the spermatodactyl (Michael, 1892; Dosse, 1959; Amano and Chant, 1978; Krantz and Wernz, 1979; Evans, 1992; Alberti and Coons, 1999). A complex terminology has been developed to describe the various parts of the sperm-access system that has become an important character set in taxonomy (see Table 1). In addition to terms used in this contribution, two further terms are in use: The atrium is simply the inner end of the major duct. It is connected to the minor duct and to the calyx that leads into the vesicle. The term cervix is unfortunately used with different meanings. Some authors use it as a synonym of the term calyx whereas others refer to the cervix (= neck) as the connection between atrium and calyx. In the species investigated here both respective areas are not distinctive structures. According to Amano and Chant (1978), in Phytoseiulus females that were fixed during copulation or soon after, the endospermatophore was still present inside the vesicle. The endospermatophore disappears ‘gradually’ during the next 1–2 days (Schulten, 1985). In our Typhlodromus overwintering females, vesicles always show the original folded shape of their walls and the vesicles appear to be largely empty. However, endospermatophores have also been observed in overwintering females of certain species (see Schulten, 1985). The mechanism of sperm transfer and the fate of the spermatophore and sperm within the female in phytoseiids is still enigmatic. Considering the small lumen of both the solenostome and major duct as well as the cuticular plug at the level of the minor-duct insertion (embolus) that narrows the major-duct lumen, a dilatation must be possible to allow endospermatophore introduction into the vesicle. On the other hand, in all mating or just mated females, no spermatozoa were recognized inside the vesicle with certainty. Only a very electron-dense or heterogeneous material, of differing arrangement and likely representing the endospermatophore, was detectable. Dosse (1959) pointed already out that this structure is strongly ‘chitinized’ and not even destroyed during maceration of the entire mite in lactic acid with heat (see also Amano and Chant, 1978; Schulten, 1985; Alberti and Coons, 1999). The minor duct leads to the ovary. However, it seems very improbable, considering its very narrow lumen, in which sperm has never been observed, that it could serve to transport spermatozoa to the ovary. This is even more difficult to believe considering that, at its beginning (embolus), the minor

Ductus major

Major duct

Karg (1982)

Chant (1985) (based on Wainstein (1973)

Calyx (cervix) and vesicle (sacculus) together are also called the spermatheca.

Atrium Minor duct starting including from embolus embolus

Alberti and Solenostome Major duct Coons (1999) (= insemination and this paper pore)

Minor duct starting from atrium

Ductus minor starting from atrium

Tr`es fin canalicule partant de l’atrium

Atrium Minor duct starting including from embolus embolus

Atrium

Atrium

Atrium

Minor duct

Feiner ductus

Major duct

Evans (1992) Solenostome

Canal distale

Fain (1963)

Sperm induction pore

Major duct

Schuster and Smith 1960

Atrium

Aufhängeband Hals

Vesicle

Beutel oder Sack

Cervix

Calix

Calyx

Vesicle

Vesicle

Vesicle

Vesiculus

Calice Spermath`eque ou (ou cervix) réceptacle s´eminal

Cervix

Schenkel

Cervix (= neck Calyx = connection between atrium and calyx)

Cervix

Components of sperm-access system (= spermathecal apparatus = insemination apparatus)

Dosse (1958)

Author

Table 1. Terminologies used by various authors to describe the components of the sperm-access system

585

586 duct shows a very fine lumen, if any. In fact, we never observed a pore in the center of the plug. We can hardly imagine that intact spermatozoa could find a putative pore and pass through it. Furthermore, if the minor duct would serve as a passage towards the ovary (s. str.) and if the sperm-containing endospermatophore is deposited within the vesicle, sperm cells would need to move back and find the pore (if present) and would have to follow a very narrow path (of 30–40 nm diameter close to the embolus). Sperm of Phytoseiulus found in the testes have a diameter of approx. 7 µm; Alberti, 1988; Alberti and Coons, 1999). Since no muscle layer is present in the walls of the entire sperm-access system, sperm cells would need to move actively along the calyx and minor duct. Remarkably, several light microscopical studies on species belonging to another family, Ascidae, with the phytoseiid-type of sperm-access system, have described the minor duct possessing a rather wide lumen, equal to the major duct. The end of the minor duct may be widened and truncated (Lindquist, 1995) slightly widened, fimbriated (Walter and Lindquist, 1989) or flower-like (Walter and Lindquist, 1997). This area certainly conforms with the end of the minor duct observed in the present study showing a widened lumen and a folded cuticle. Since we never observed sperm cells in this area and because of the problems the morphology of the system offers (see above) we presently cannot support the suggestion of previous authors that this area might represent a sperm reservoir (e.g., Fain et al., 1977; Walter and Lindquist, 1989, 1997; Lindquist, 1994). Interestingly, these authors have shown that the whole sperm-access system might be developed to strikingly different degrees, from unusually large (e.g., Lasioseius kinikik) via minute and perhaps vestigial (e.g., Lasioseius youcefi) (Walter and Lindquist, 1989) to not discernable (e.g., Lasioseius zaluckii) (Walter and Lindquist, 1997). Furthermore, the minor duct in some species might be thicker, even as thick as the major duct. It is persuasive to conclude that this different morphologies (also in the details) might be correlated with different modes of reproduction, for example parahaploidy or thelytoky (see also Norton et al., 1993). Evidently, further studies are needed to elucidate this. Regarding the peculiar electron-lucent tissue surrounding the minor-duct end, its function is also still unclear. However, it is important to stress that it joins the corresponding end coming from the other side. In this respect, the phytoseiid-type sperm-access systems of both body sides would also represent a continuous structure as the laelapid-type (Alberti and Di Palma, in press). The ducts (so-called rami) leading to the spermatheca close to the ovary in Varroa jacobsoni also seem to be formed by a syncytial tissue (Alberti and Hänel, 1986). Considering these and the even more obvious similarities in shape and fine structure of the lyrate organ and oviduct, we

587 think it most likely that the sperm-access systems of the phytoseiid type and laelapid type are basically homologous. In Varroa and Parasitina, it is already known that spermatozoa reach the ovary by penetrating through a cell bridge which connects the spermatheca with the ovary, in the former (Alberti and Hänel, 1986), and by a haemocoelic route in the latter (Alberti, 1988; Alberti et al., 2000). Witaliñski (1987b) observed frequently spermatozoa in the ovary of Euryparasitus emarginatus close to the stalk, that is close to the transitory micropyle. In any case, it is necessary to point out that, in Parasitina, spermatozoa are introduced into the (primary) genital opening and that, in Varroa, the spermatheca apparently lacks a cuticular intima. In contrast, both in Phytoseiulus and Typhlodromus, vesicle walls are cuticle-lined. It is quite difficult to imagine how spermatozoa could pass through these walls. Furthermore, sperm cells have neither been observed in the haemocoel at the region surrounding vesicle walls nor in other areas of the body, which would be expected if vesicle-wall penetration had occurred. However, one should keep in mind that, in a quite distant taxon, the spider mites (Tetranychidae, Actinotrichida), sperm penetrates the specialized wall of the receptacle (Alberti and Storch, 1976). In Acaridida, in which all species have a peculiar sperm-access system, a rather complex organ has developed that leads the sperm to the ovary via solid transitory cones (Witaliñski et al., 1990). Furthermore, the passage of sperm from the cuticle-lined sperm duct of Varroa jacobsoni to the spermatheca is also not known. In this respect, we consider the alterations in the vesicle epithelium and its cuticular lining in Phytoseiulus quite remarkable. Certainly, they reflect processing of the vesicle contents after deposition of the endospermatophore. It seems very likely that material is resorbed from the vesicle lumen via the vesicle epithelium (see below: cuticular plug). Moreover, if spermatozoa move so fast to the ovary and are not stored in the vesicle, as they were assumed to be, this organ could play a role in sperm reception, but not in storage, which could, however, be realized in the ovary, as probably shown by Typhlodromus females (see above). This is a different functional aspect compared to the laelapid-type sperm-access system, where the capacitation process and spermatozoa storage is realized in the spermatheca. Spermatozoa have always been observed in the ovary of Phytoseiulus, no matter how long the partners had been copulating. This may indicate, as already discussed, that the sperm reach the ovary rather quickly. Hence, the long mating period reported for Phytoseiulus persimilis (Amano and Chant, 1978: 131.07 ± 20.83 min) could be a mechanism of letting sperm reach the ovary and preventing another male from reproductive success (securing sperm priority by prolonged mating; Thomas and Zeh, 1984). The same aim could

588 be performed by the ‘cuticular plug’ present inside the calyx and the vesicle, after transfer of the endospermatophore, and that takes almost 24 h to dissolve (Amano and Chant, 1978). Finally, we must admit that some important questions regarding the functional morphology of the female genital system of phytoseiid mites are still open. Spermatozoa routes inside the females are still incompletely known. Also, the function of the sperm-access system, especially the role played by the complex embolus and minor duct, is not clear and needs further investigation. Perhaps a completely new interpretation is necessary. This system might provide the female with a means of mate control (see Alberti, in press; Alberti and Di Palma, in press). An ultrastructural study of the male’s modified chelicerae (spermatodactyl) and of the spermatophore could also be useful in achieving a better understanding of the mating process and sperm transfer. Acknowledgements This study was financially supported by a grant from the Deutsche Forschungsgemeinschaft to G. A. (Al 138/4-1,2). Both authors also gratefully acknowledge the support by the VIGONI-program (Deutscher Akademischer Austauschdienst, DAAD, and Conferenza dei Rettori delle Università Italiane, CRUI). We are very much grateful for the technical and scientific support from the Department of Agro-Forestry and Environmental Biology and Chemistry, Sec. of Entomology and Zoology, University of Bari, where A. D. P. was working over many years. We also wish to thank Mrs. Ch. Putzar, S. Schade, E. Kreibich, and Mr. H. Fischer, and P. Michalik for their careful assistance. Dr. D. Russell kindly corrected the English. Finally, we express thanks to the referees for their improving suggestions. References Akimov, I.A., Piletskaya, I.V. and Yastrebtsov, A.V. 1988. Morpho-functional age changes in the reproductive system of female Varroa jacobsoni. Vestnik Zool. 6: 48–55. Akimov, I.A. and Yastrebtsov, A.V. 1984. Reproductive system of Varroa jacobsoni. 1. Female reproductive system and oogenesis (in Russian, English summary). Vestnik Zool. 6: 61–68. Alberti, G. 1974. Fortpflanzungsverhalten und Fortpflanzungsorgane der Schnabelmilben (Acarina: Bdellidae, Trombidiformes). Z. Morph. Tiere 78: 111–157. Alberti, G. 1988. Genital system of Gamasida and its bearing on phylogeny. In: Progress in Acarology, G.P. ChannaBasavanna and C.A. Viraktamath (eds), Vol. 1, pp. 197–204. Oxford & IBH Publishing, New Delhi. Alberti, G. 1991. Spermatology in the Acari: systematic and functional implications. In: The Acari-Reproduction, Development and Life-History Strategies, R. Schuster and P.W. Murphy (eds), pp. 77–105. Chapman & Hall, London.

589 Alberti, G. 2000. Chelicerata. In: Reproductive Biology of Invertebrates. Vol. IX, part B: Progress in Male Gamete Ultrastructure and Phylogeny, K.G. and R.G Adiyodi (eds), pp. 311–388. Oxford & IBH Publishing, New Delhi. Alberti, G. Reproductive systems of gamasid mites (Acari, Anactinotrichida) reconsidered. Proceedings of the IVth Sympsoium of EURAAC, Siena 2000 (in press). Alberti, G. and Coons, L.B. 1999. Acari – Mites. In: Microscopic Anatomy of Invertebrates, F.W. Harrison (ed.), Vol. 8C, pp. 515–1265. Wiley-Liss, New York. Alberti, G. and Hänel, H. 1986. Fine structure of the genital system in the bee parasite Varroa jacobsoni (Gamasida: Dermanyssina) with remarks on spermiogenesis, spermatozoa and capacitation. Exp. Appl. Acarol. 2: 63–104. Alberti, G. and Di Palma, A. Fine structure of the phytoseiid-type sperm access system (Acari, Gamasida, Phytoseiidae). Proceedings of the IVth Sympsoium of EURAAC, Siena 2000 (in press). Alberti, G., Gegner, A. and Witaliñski, W. 1999. Fine structure of the genital system in the females of Pergamasus mites (Acari: Gamasida: Pergamasidae). J. Morphol. 240: 195– 223. Alberti, G., Gegner, A. and Witaliñski, W. 2000. Fine structure of the spermatophore and spermatozoa in inseminated females of Pergamasus mites (Acari: Gamasida: Pergamasidae) J. Morphol. 245: 1–18. Alberti, G. and Storch, V. 1976. Ultrastruktur-Untersuchungen am männlichen Genitaltrakt und an Spermien von Tetranychus urticae (Tetranychidae, Acari). Zoomorphologie 83: 283–296. Alberti, G. and Storch, V. 1989. Zur Feinstruktur des weiblichen Genitaltraktes von Tubiluchus philippinensis (Tubiluchidae, Priapulida). Zool. Anz. 222: 12–26. Alberti, G. and Zeck-Kapp, G. 1986. The nurimentary egg development of the mite Varroa jacobsoni (Acari, Arachnida), an ectoparasite of honey bees. Acta Zoologica 67: 11–25. Amano, H. and Chant, D.A. 1978. Mating behaviour and reproductive mechanisms of two species of predaceous mite, Phytoseiulus persimilis Athias-Henriot and Amblyseius andersoni (Chant) (Acarina: Phytoseiidae). Acarologia 28: 201–220. Anderson, D.L. and Trueman, J.W.H. 2000. Varroa jacobsoni (Acari: Varroidae) is more than one species. Exp. Appl. Acarol. 24: 165–189. Athias-Henriot, C. 1971. Nouvelles notes sur les Amblyseiini (Gamasides Podospermiques, Phytoseiidae). 1. La dépilation des genuaux et tibias des pattes. Acarologia 13: 4–15. Boeck, P. 1984. Der Semidünnschnitt, 172pp. J.S. Bergmann-Verlag, München. Brinton, L.P. and Oliver Jr., J.H. 1971. Fine structrue of oogonial and oocyte development in Dermacentor andersoni Stiles (Acari: Ixodididae). J. Parasitol. 57: 720–747. Chant, D.A. 1985. Internal anatomy. In: Spider Mites. Their Biology, Natural Enemies and Control, W. Helle and M.W. Sabelis (eds), Vol. 1B, pp. 11–16. Elsevier, Amsterdam. Coons, L.B. and Alberti, G. 1999. Acari – Ticks. In: Microscopic Anatomy of Invertebrates, F.W. Harrison (ed.), Vol. 8B, pp. 267–514. Wiley-Liss, New York. Dosse, G. 1958. Die Spermathecae ein zusätzliches Bestimmungsmerkmal bei Raubmilben. Pflanzenschutzber 20: 1–11. Dosse, G. 1959. Über den Kopulationsvorgang bei Raubmilben aus der Gattung Typhlodromus (Acari, Phytoseiidae). Pflanzenschutzber 22: 125–133. Evans, G.O. 1992. Principles of Acarology, 563pp. C.A.B International, Wallingford. Evans, G.O. and Till, W.M. 1979. Mesostigmatic mites of Britain and Ireland (Chelicerata: Acari: Parasitiformes). An introduction to their external morphology and classification. Trans. Zool. Soc. Lond. 35: 139–270.

590 Fahrenbach, W.H. 1999. Merostomata. In: Microscopic Anatomy of Invertebrates, F.W. Harrison (ed.), Vol. 8A, pp. 21–115, Wiley-Liss, New York. Farley, R.D. 1999. Scorpiones. In: Microscopic Anatomy of Invertebrates, F.W. Harrison (ed.), Vol. 8A, pp. 117–222. Wiley-Liss, New York. Felgenhauer, B.C. 1999. Araneae. In: Microscopic Anatomy of Invertebrates, F.W. Harrison (ed.), Vol. 8A, pp. 223–266. Wiley-Liss, New York. Fain, A. 1963. La spermathéque et ses canaux adducteurs chez les acariens mésostigmatiques parasites des voies respiratoires. Acarologia 5: 463–479. Fain, A., Hyland, K.E. and Aitken, T.H.G. 1977. Flower mites of the family Ascidae phoretic in nasal cavities of birds (Acarina: Mesostigmata). Acta Zool. Pathol. Antverp. 69: 99– 154. Feldman-Muhsam, B. and Filshie, B.K. 1979. The ultrastructure of the prospermium of Ornithodoros ticks and its relation to sperm maturation and capacitation. In: The spermatozoon, D.W. Fawcett and J.M. Bedford (eds), pp. 355–369. Urban & Schwarzenberg, Baltimore-Munich. Helle, W., Bolland, H.R., Van Arendonk, R., de Boer, R., Schulten, G.G.M. and Russell, V.M. 1978. Genetic evidence for biparental males in haplo-diploid predator mites (Acarina: Phytoseiidae). Genetica 49: 165–171. Helle, W. and Sabelis, M.W. (eds) 1985. Spider Mites. Their Biology, Natural Enemies and Control. World Crop Pests, Vol. 1B, 458pp. Elsevier, Amsterdam. Jagers op Akkerhuis, G., Sabelis, M.W. and Tjailingii, W.F. 1985. Ultrastructure of chemoreceptors on the pedipalps and first tarsi of Phytoseiulus persimilis. Exp. Appl. Acarol. 1: 235–251. Karg, W. 1982. Diagnostik und Systematik der Raubmilben aus der Familie Phytoseiidae Berlese in Obstanlagen. Zool. Jb. Abt. Systematik, Ökologie u. Geogr. Tiere 109: 188– 210. Karnovsky, M.J. 1965. A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. Cell Biol. 27: 137–138. Krantz, G.W. 1978. A Manual of Acarology, 2nd edn., 509pp. Oregon State University Book Stores, Corvallis. Krantz, G.W. and Wernz, J.G. 1979. Sperm transfer in Glyptholaspis americana. In: Recent Advances in Acarology, J.G. Rodriguez (ed.), Vol. 2, pp. 441–446. Academic Press, New York. Lindquist, E.E. 1995. Remarkable convergence between two taxa of ascid mites (Acari: Mesostigmata) adapted to living in pore tubes of bracket fungi in North America, with description of Mycolaelaps new genus. Can. J. Zool. 73: 104–128. Michael, A.D. 1892. On the variations in the internal anatomy of the Gamasinae, especially in that of the genital organs and their mode of coition. Trans. Linn. Soc. Lond. 5 (ser. 2): 281–324. Norton, R.A., Kethley, J.B., Johnston, D.E. and O’Connor, B.M. 1993. Phylogenetic perspectives on genetic systems and reproductive modes of mites. In: Evolution and Diversity of Sex Ratio in Insects and Mites, D.L. Wrensch and M.A. Ebbert (eds), pp. 8–99. Chapman & Hall, New York. Nuzzaci, G., Di Palma, A. and Aldini, P. Functional morphology of the female genital system in Typhlodromus spp. (Acari: Phytoseiidae). In: Proc. 10th Intern. Congr. Acarology, R.B. Halliday, D.E Walter, H.C. Proctor, R.A. Norton and M.J. Colloff (eds), pp. 196–202. CSIRO Publ., Melbourne.

591 Petrova, V.I. 1970. Structure and development of the male genital system of the predaceous mite Phytoseiulus persimilis Athias-Henriot. Izr. Akad. Nauk. Latv. SSR 5: 24–27 (in Russian). Pound, J.M. and Oliver Jr., J.H. 1976. Reproductive morphology and spermatogenesis in Dermanyssus gallinae (De Geer) (Acari, Dermanyssidae). J. Morphol. 150: 825–842. Richardson, K.C., Jarrett, L.J. and Finke, E.H. 1960. Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Technol. 35: 313–323. Ritter, W. 1981. Varroa disease of the honeybee Apis mellifera. Bee World 62: 141–153. Sabelis, M.W. and Janssen, A. 1994. Evolution and life-history pattern in the Phytoseiidae. In: Mites: Ecological and Evolutionary Analyses of Life-History Patterns, M.A. Houck (ed.), pp. 70–98. Chapman & Hall, New York, London. Sabelis, M.W. and Harmsen, R. (eds) 1992. Population dynamics of plant-inhabiting mites. Exp. Appl. Acarol. 14 (special issue): 179–418. Schulten, G.G.M. 1985. Mating. In: Spider Mites. Their Biology, Natural Enemies and Control. World Crop Pests, W. Helle and M.W. Sabelis (eds), Vol. IB, pp. 55–65. Elsevier, Amsterdam. Schuster, R.O. and Smith, L.M. 1960. The spermatheca as taxonomic features in Phytoseiid mites of western North America. Proc. Entomol. Soc. Wash. 62: 181–188. Steiner, J., Diehl, P.A. and Vlimant, M. 1995. Vitellogenesis in Varroa jacobsoni, a parasite of honey bees. Exp. Appl. Acarol. 19: 411–422. Thomas, R.H. and Zeh, D.W. 1984. Sperm transfer and utilization strategies in arachnids: ecological and morphological constraints. In: Sperm Competition and the Evolution of Animal Mating Systems, R.L. Smith (ed.), pp. 179–221. Academic Press, New York. Walter, D.E. and Lindquist, E.E. 1989. Life history and behavior of mites in the genus Lasioseius (Acari: Mesostigmata: Ascidae) from grassland soils in Colorado, with taxonomic notes and description of a new species. Can. J. Zool. 67: 2797–2813. Walter, D. and Lindquist, E.E. 1997. Australian species of Lasioseius (Acari: Aesostigmata: Ascidae): the porulosus group and other species from rain forest canopies. Invert. Tax. 11: 525–547. Wainstein, B.A. 1973. On the structure of some organs of Phytoseiidae (Parasitiformes) important for taxonomy. Zool. Zh. 52: 1871–1872 (in Russian). Witaliñski, W. 1986. Egg-shells in mites. I. A comparative ultrastructural study of vitelline envelope formation. Cell Tissue Res. 244: 209–214. Witaliñski, W. 1987a. Egg-shell in mites: cytological aspect of vitelline envelope and chorion formation in Pergamasus barbarus Berlese (Gamasida, Pergamasidae). Intern. J. Acarol. 13(3): 189–196. Witaliñski, W. 1987b. Topographical relations between oocytes and other ovarian cells in three mite species (Acari). Acarologia 28: 297–306. Witaliñski, W. 1988. Egg shells in mites. Vitelline envelope and chorion in a water mite, Limnochares aquatica L. (Acari, Limnocharidae). J. Zool. Lond. 214: 285–294. Witaliñski, W. 1993. Egg shells in mites. Vitelline envelope and chorion in Acaridida (Acari). Exp. Appl. Acarol. 17: 321–344. Witaliñski, W., Szlendak, E. and Boczek, J. 1990. Anatomy and ultrastructure of the reproductive system of Acarus siro (Acari: Acaridae). Exp. Appl. Acarol. 10: 1–31.