Gonads and gametogenesis in astigmatic mites (Acariformes: Astigmata) Arthropod

Astigmatans are a large group of mites living in nearly every environment and exhibiting very diverse reproductive strategies. In spite of an uniform anatomical organization of their reproductive systems, gametogenesis in each sex is highly variable, leading to gamete formation showing many peculiar fea- tures and emphasizing the distinct position of Astigmata. This review summarizes the contemporary knowledge on the structure of ovaries and testes in astigmatic mites, the peculiarities of oogenesis and spermatogenesis, as well as provides new data on several species not studied previously. New questions are discussed and approaches for future studies are proposed. is an the BY (http://creativecommons.org/licenses/by/3.0/).

and water-filled tree holes, phytotelmata and caves (Hughes, 1976;Evans, 1992;Fashing, 1994Fashing, , 1998. The other species are successful in destroying stored food such as cereals, flour, cheese, dried fruits and meat, etc., leading to considerable crop damage (Hughes, 1976). An abundance of Astigmata is associated with other animals, mainly insects and amniotic vertebrates (e.g. all Psoroptidia with the exception of Pyroglyphidae which are free-living), frequently as serious dermicolous parasites (Fain and Lukoschus, 1986;Proctor, 2003;OConnor, 2009). They can also inhabit bird and mammalian nests or such distinct habitat as feathers. Despite a wide range of habitats and a successful adaptive radiation, these mites share many distinct features in reproductive anatomy and reproductive behavior.
Semithin cross sections were stained with an Azur II and methylene blue (1:1) mixture, whereas thin sections were collected on formvar coated grids, contrasted with uranyl acetate and lead citrate according to standard protocols (Venable and Coggeshall, 1965), and examined under a transmission electron microscope JEOL JEM 100SX (JEOL Ltd., Tokyo, Japan) at 80 kV in the Department of Cell Biology and Imaging, Institute of Zoology, Jagiellonian University.
For fluorescent staining with DAPI and Pyronin Y, the females of Rhizoglyphus echinopus were processed as follows: the posterior parts of mite bodies were fixed for 2 h at 4 C in fixative containing 4% paraformaldehyde and 2.5% sucrose in 0.01 M PBS. After fixation, the material was washed and dehydrated in a graded ethanol series then embedded in LR-White (Fluka) resin. Semithin sections were stained 30 min with DAPI, washed several seconds in PBS, and stained 20 min with Pyronin Y (SigmaeAldrich)(20 mg/ml). After brief washing in PBS sections were mounted and examined under a fluorescence microscope Olympus BX51 (Olympus Corporation, Tokyo, Japan) fitted with appropriate filters.

Reproduction of Astigmata
Both sexual and parthenogenetic species occur in Astigmata, the latter represented by arrhenotokous, thelytokous, and rare deuterotokous organisms. In species possessing both females and males (sexual and arrhenotokous species) the proportion of sexes may be only slightly biased towards females. The males use their intromittent organ, the aedeagus (¼penis) to inseminate females with sperm during copulation. Insemination never occurs through the oviporus, but via a supplementary inseminatory system. Sexual species (with diploid females and diploid males) occur for instance in Acaroidea and Glycyphagoidea, whereas arrhenotokous diplohaploid species (with diploid females and haploid males) are known in Histiostomatoidea (Histiostomatidae: Histiostoma), Hemisarcoptoidea (Winterschmidtiidae: Kennethiella, Ensliniella, Kurosaia) (Hughes and Jackson, 1958;Heinemann and Hughes, 1969;Cowan, 1984;Klompen et al., 1987;Okabe and Makino, 2003) and Sarcoptoidea. Thelytokous species occur in Histiostomatoidea (Histiostomatidae), Acaroidea (e.g. Acaridae: Schwiebea) (Okabe and OConnor, 2001;Okabe et al., 2008). Thelytokous populations are composed of females but extremely rare males can also be occasionally found; such males are non-reproducing as is believed, but their reproductive systems and/or spermatozoa have never been studied. In Knemidocoptes mutans (Analgoidea: Epidermoptidae) the frequency of males in populations is 2e4% (Dubinin, 1953); such strong bias towards males in practically sedentary mites suggests thelytoky rather than the effect of local mate competition, a phenomenon which can also lead to sex ratio distortion (Hamilton, 1967). Deuterotoky, in which both males and females are produced from unfertilized eggs, has been reported in Histiostomatidae (Heinemann and Hughes, 1969).
In most cases, the type of reproduction is only suspected and is based mainly on population structure since detailed studies are usually missing. Moreover, some phenomena concerning reproduction can be misinterpreted. For instance, a well-known cosmopolitan species, H. feroniarum (Histiostomatidae), has been for years believed to be comprised of arrhenotokous and thelytokous populations (Hughes and Jackson, 1958). Recent unpublished molecular studies (in co-operation with Dr. Miros1awa Dabert, Adam Mickiewicz University, Pozna n, Poland) revealed that there are two separate but morphology practically indistinct species, an arrhenotokous species (H. feroniarum) and a thelytokous form (Histiostoma sp.).
Finally, it should be emphasized that the most basal astigmatan group, Schizoglyphoidea, has not been studied since only several immature specimens of one species are known (OConnor, 2009), whereas living adult males and females are required for a comprehensive description of reproductive anatomy. Therefore, in this mini-review on anatomical and cytological aspects of reproduction, the superfamily Histiostomatoidea is treated as the most basal in Astigmata.
Spherical or subspherical ovaries are located symmetrically in the rear part of the idiosoma, usually on both sides of the last section of the alimentary tract, i.e. the postcolon, anal atrium and anus. They occupy a considerable part of the idiosoma, but in C. sellnicki are very small, spherical and located directly above the ventral cuticle (unpublished). In Psoroptidia, ovaries are located dorsally as in Sarcoptes scabiei and N. cati e Sarcoptidae (Witali nski and Walzl, 1995), Psoroptes spp. e Psoroptidae (Lekimme et al., 2005) (Fig. 2BeD), and F. rostratus e Falculiferidae, but in the latter species they are additionally shifted anteriorly (Fig. 4D). Oviducts emerge at the ventral, anteroventral or lateroventral surface of the ovaries and run forward either straight to the oviporal region of the mite body as in the sarcoptid mites: S. scabiei and N. cati (Witali nski and Walzl, 1995), or are S-shaped bending twice to reach the oviporal region as in Acarus siro, Tyrophagus perniciosus, or Sancassania berlesei (Acaridae) (Walzl et al., 2004) Lekimme et al. (2005) (CeF). Abbreviations: asterisk e location of the ovarian nutritive cell (ONC), a e anus, ae e aedeagus, AG e male accessory gland, chg e chorional gland, chg1, 2 e two parts of chorional gland in Sarcoptes, covd e common oviduct, de e ejaculatory duct, ic e inseminatory canal, io e inseminatory opening, mg e midgut, oc e oocyte, op e oviporus, ov e ovary, ovd e oviduct, pch e preoviporal chamber, rs e spermatheca, scc e spermconveying cord, sz e spermatozoa in spermatheca, t e testis, vd e deferent duct.
Scale bars: 100 mm in (A, B, E, F); 200 mm in (C, D). showing eccentric ONC with ramified nucleus and large nucleoli (nu). Oocytes in various developmental stages (oc) are connected with the ONC via intercellular bridges with diaphragm-crossed lumina (arrows). Germinal line cells are embedded in somatic ovarian stroma cells (osc). Thin ovarian wall cells (ow) lying on basal lamina (bm) encompass the ovary. (B) In the ovary of Acaridae and Carpoglyphidae the ONC is located subcentrally and oocytes (oc) are connected with the ONC via funnel-type intercellular bridges filled by electron-dense material (arrows). Other abbreviations as in (A). (C) The ONC in the ovary of Glycyphagus is in a dorsal position whereas the ventral part of the gonad contains oocytes (oc) in different developmental stages. Other abbreviations as in (A). (D) The LR-White semithin section through the ovary in Rhizoglyphus echinopus stained sequentially with DAPI and pyronine Y. DAPI (blue) reveals DNA whereas pyronine Y counterstains RNA. Note very strong signal for DNA from the ONC nucleus as compared to oocyte (oc) nuclei. oc e oocytes, ONC e the ovarian nutritive cell, ow e ovarian wall cells. Scale bars: 10 mm.  Fig. 2A). Ovaries in F. rostratus are located both dorsally and more anteriorly, while oviducts emerge to run posteriorly and then turn ventrally toward the oviporal region of the idiosoma. Consequently, in F. rostratus two distinct parts of the oviduct are present: a proximal upper section and a distal lower section. In all species, the oviducts close to the oviporus coalesce to form an unpaired common oviduct (¼uterus or chorion gland in sarcoptid and psoroptid mites e Fig. 4D and F). The common oviduct passes forward into a preoviporal chamber (sometimes incorrectly termed vagina), lined with a thin, usually plicated cuticle. This part can form a short-tube ovipositor by eversion which allows precise egg manipulation during oviposition. The genital aperture, the oviporus, is a longitudinal, transversal, or inverted V-shaped slit located in the mid region of the mite venter. Its structure is complex because the cuticular walls are folded and two pairs of finger-like genital papillae are located between folds (for details see: Witali nski et al., 1990;Evans, 1992;Walzl, 1992;Alberti and Coons, 1999). The pregenital sclerite (epigynum) rarely occurs in free-living astigmatans (many Glycyphagidae) but is common in parasitic Astigmata (Ehrnsberger et al., 2001).
Female accessory glands occurring as paired, separate structures connected with the oviducts are absent. Paired accessory glands have been reported in some species (Kuo and Nesbitt, 1970;Baker and Krantz, 1985;Witali nski et al., 1990), but subsequent studies with serial semithin sections have shown that the interpretation of the distal third portion of the oviduct as separate accessory glands was erroneous (Witali nski, 1993;Witali nski and Walzl, 1995). However, in non-psoroptidian species the distal third portion of the oviducts has a thickened glandular wall, whereas in psoroptidians the common oviduct forms dorsally a large, more or less distinct glandular pocket, the chorion gland. In S. scabiei and N. cati the chorion gland comprises only one type of secretory cells, but in F. rostratus it contains two types of cells located in two distinct regions ( Fig. 4D and F) (Witali nski, 1993).
Sperm-conveying cords are solid cellular structures, conical in A. siro, but thin and cord-like in most other species in which the spermatheca is placed far from the ovaries, as, for instance, in F. rostratus and P. fuchsi where their diameter is 4.0e4.5 mm and ca. 3.0 mm, respectively. Spermatozoa migrate between conveying cord cells ( Fig. 6A) from the basal part of the spermatheca towards the ovaries (A. siro e Witali nski et al., 1990; F. rostratus e unpublished). Syngamy occurs within the ovary with previtellogenic oocytes before they are covered by a vitelline envelope; consequently, in sperm cells of Astigmata the acrosome is absent (Liana and Witali nski, 2005).
The spermatheca in Astigmata (Figs. 2A, D and 4C, D) (Witali nski et al., 1990;Desch, 2001;Lekimme et al., 2005) is a complex saccular organ which consists of cellular and cuticular elements. The cuticular lining of the inseminatory canal amalgamates with the solid cuticular basal part of the spermatheca, the main part of the spermatheca visible in mites on microscopical slides examined under a light microscope. The cuticle of the basal part is supported by a thick layer of cells which continue anteriorly to form the saccular part of the spermatheca. The wall of the saccular part is thin and its cells form many long, internally projecting microvilli. The margin of the basal part of the spermatheca protrudes to form two very thin, more or less continuous, fine cuticular lamellae which encompass the internal space of the spermatheca. Thus, the lumen of the spermatheca is divided into two compartments, (1) an external one, penetrated with microvilli of spermatheca cells, and (2) an internal one, delimited by double lamellae, to which sperm and other male-derived substances are introduced. It is believed that sperm cells leave the spermatheca basis to enter conveying cords and then ovaries via a pair of openings in the basal part of the spermatheca located near two minute V-shaped cuticular appendages visible in light microscopical images. Klien and Walzl (2010) demonstrated that in S. berlesei, sperm cells aggregate in the spermatheca close to the entrance into such V-shaped appendages. These appendages, named the ducti conjunctivi, consist of a sclerotized part, the funnel, which is ca. 40 mm long. Its diameter varies from 1 mm at the spermatheca to 20 mm at the distal end. These interesting structures, however, are still waiting for precise descriptions at the ultrastructural level.

The ovary
The ovaries in Astigmata (Fig. 3) are of nutrimental type and, as in other animals, are composed of two elements: germinal and somatic cells. Oogonia and previtellogenic oocytes belong to germinal cells, as well as one very large and spherical cell of nutritive nature, the so-called ovarian central cell. All these germline cells are embedded in only several somatic cells with peripherally positioned nuclei. The ovary is surrounded by a layer of thin epithelium which is difficult to discern under a light microscope and, e.g., is not mentioned in the sarcoptid mite N. cati (Witali nski, 1988). Vitellogenic oocytes occur in the transient zone between the ovary and oviduct and fill the oviductal lumen, in which vitellogenesis is completed and egg envelopes begin to form. Prasse (1968) was the first to observe the ovarian central cell and later observations confirmed its presence in all studied species (Witali nski et al., 1990;Walzl, 1992;Witali nski and Walzl, 1995;Desch, 2001;Lekimme et al., 2005;Schwaha et al., 2008;Witali nski et al., 2014). Indeed, the central cell in many species is located centrally or subcentrally (A. siro, R. echinopus, C. sellnicki) within the ovary ( Fig. 3B and D), but in some cases is shifted adaxially, as in H. feroniarum (Figs. 3A and 4B), Histiostoma sp., abaxially as in C. lactis, abaxially and anteriorly as in Psoroptes spp. (Lekimme et al., 2005), antero-dorsally as in S. scabiei (Desch, 2001), dorsally as in N. cati, or is located either dorsally or subcentrally as in G. domesticus (Figs. 3C and 4C). Since it can actually take central or quite eccentric positions, I propose to use the term ovarian nutritive cell (ONC), as a more proper name instead of central cell.  The ONC (Figs 3 and 4AeC, E) is a very large cell (diameter from ca. 20 mm in Falculifer and Pseudolichus, 25e30 mm in Sarcoptes, Scutulanyssus and Grallolichus, 30e35 mm in Canestrinia, Glycyphagus, Notoedres, 40 mm in Pterolichus, up to 50 mm in Histiostoma, Psoroptes and Dermatophagoides) (Desch, 2001;Liana, 2004;Lekimme et al., 2005) containing an elaborated nucleus with a number of prominent nucleoli. In most species the ONC has been described as multinucleate (Pyroglyphidae: D. farinae, Dermatophagoides pteronyssinus e Walzl, 1992) or mononucleate with a multilobular nucleus (Acaridae: A. siro e Witali nski et al., 1990), but recent studies with serial sectioning following 3-D reconstruction performed on representatives of three families (Schwaha et al., 2008): S. berlesei (Acaridae), G. domesticus (Glycyphagidae), Chorioptes bovis and Otodectes cynotis (Psoroptidae) led to the conclusion that in all Astigmata in ONCs only one extensively branched nucleus is present. DNA-specific fluorescence after DAPI staining suggests polyploidy (Fig. 3D). The nuclear envelope forms many concavities rich in nuclear pore complexes with frequently adhering fine granular or flocculent nuage material. The rest of the cytoplasm is packed with free ribosomes; it also contains mitochondria and scarce ER and profiles of Golgi bodies. Lysosome-like bodies with non-homogenous content (H. feroniarum: Witali nski et al., 2014) as well as crystalline inclusions (S. scabiei: Desch, 2001) can also be found.
Eccentrically located ONCs are partly covered by oogonia and previtellogenic oocytes distributed usually in one to several layers; in this area the plasmalemma of the ONC can form many short protrusions described, e.g. in S. scabiei (Desch, 2001), as microvilli. In G. domesticus and C. osmiae the ONC also forms irregular, sometimes terminally expanded protrusions, penetrating the ovarian stroma cells at some distance (Figs. 5C and 6B). Oogonia and oocytes are connected with the ONC through conspicuous intercellular bridges (Figs. 4A, C, 5A, B and 6B). In general, intercellular bridges in all Astigmata except Histiostomatidae are funnel-shaped and filled with an electron-dense granular material ( Fig. 6B) (Witali nski et al., 1990;Lekimme et al., 2005;Schwaha et al., 2008;Florek and Witali nski, 2010b;Klien and Walzl, 2010). The bridge opening at the ONC side is ca. 6 mm wide and is smaller than at the oocyte side. Oogonia and small, young oocytes have elongated and much thinner bridges, but their diameter increases with the growth of the cells. Recent preliminary studies (Florek and Witali nski, 2010b) indicated that in histiostomatid mites (H. feroniarum) a second, unusual type of intercellular bridge is present (Figs. 3A and 4B). This bridge is practically twodimensional and very large in advanced previtellogenic oocytes (10 mm or more) but its lumen is crossed with a thin diaphragm of electron dense material, hence it has been termed a diaphragmcrossed bridge. At higher magnification, the dense material is arranged in a rectangular lattice. The same was observed in another undescribed Histiostoma species (Witali nski et al., 2014). In another histiostomatid mite, Bonomoia opuntiae, the bridge has somewhat intermediate structure since the diaphragm dense material is thickened in the center of the bridge lumen (preliminary observations).
Oocytes entering meiotic prophase are small roundish cells with a relatively large spherical nucleus in which synaptonemal complexes may be visible (Witali nski et al., 2014). After the first meiotic division, the oocytes start to grow at previtellogenic phase. In S. berlesei, meiosis is completed much later in oocytes moving along the second, backward-curved part of the oviduct, whereas the first cleavage into two blastomeres occurs at the beginning of the third, forward-curved part of the oviduct (Walzl et al., 2004).
Detailed studies on the architecture of the ovary and oocyte pathways within the ovary during their development are absent, but light-microscopical observations of semithin sections suggest that previtellogenic oogenesis occurs generally around the ONC towards the pole where the oviduct emerges. Thus, in most cases oogonia and early previtellogenic oocytes are distributed dorsally or dorso-laterally, whereas the oocytes entering vitellogenesis are distributed ventrally or ventro-laterally in the ovary, i.e. close to the oviduct entrance.

Vitellogenesis
Vitellogenesis in Astigmata occurs in oviductal rather than ovarian oocytes (Fig. 4CeG) (Witali nski, 1995;Walzl et al., 2004), but this process is known only superficially, since no investigations have dealt with the subject and data are only available from papers focused on other aims (Walzl et al., 2004;Lekimme et al., 2005;Witali nski et al., 2014). The nucleus in early vitellogenic oocytes is similar to that in previtellogenic ones but its outline is irregular (Fig. 4E); cytoplasmic organelles accumulate mainly at the oocyte periphery, whereas the rest of the cytoplasm is filled with two types of inclusions: electron-dense protein yolk spheres and electron-lucent inclusions, which are lipid droplets (Fig. 6CeF). The protein yolk spheres are usually several times larger than the lipid droplets; in Psoroptes (Lekimme et al., 2005) their size is 8 mm and 0.9 mm, respectively. Peripherally located protein yolk spheres are small, ca. 1.6 mm in His- The origin of yolk protein (vitellogenin, Vg) varies among mite groups. In mites possessing an arachnid-type ovary (i.e. a hollow, tubular ovary with oocytes protruding outside on stalks and covered with basement membrane only), as in ticks and some Parasitengonina (for further references see Evans, 1992;Alberti and Coons, 1999;Coons and Alberti, 1999), the Vg is either produced by the oocyte itself or, rather, derived from external sources. Fat bodies, midgut cells, and specialized subepidermal cells have been proposed as external sources (for further discussion and references see: Cabrera et al., 2009). Externally produced Vgs are supplied through the hemolymph and absorbed via pinocytosis into oocytes. In many mites, however, the ovary is a compact structure and more or less evidently of nutrimental type (e.g. higher Gamasina: Arctacarina e Alberti and Krantz, 2007; Parasitina e Dermanyssina e Alberti and Zeck-Kapp, 1986;Di Palma and Alberti, 2001;Nuzzaci et al., 2001;Di Palma et al., 2012; or some Parasitengonina: Erythraeidae e Witte, 1975). In such cases, the oocytes are supplied by nutritive cords from nurse cells with mitochondria, ribosomes and other cytoplasmic components (Steiner et al., 1995), as well as ribonucleoproteins involved in oocyte growth, but Vgs are synthetized and absorbed from hemolymph as above. In Oribatida, nutritive cells/tissues are absent in the ovary (for further references see Liana and Witali nski, 2012). In Astigmata, despite their close evolutionary relations to Oribatida, the ovaries are of nutrimental type, but the ONC seems to be a source of ribosome/ ribosome subunits and probably mRNA for Vg authosynthesis rather than of Vg itself, since 1) cytoplasm of previtellogenic oocytes is highly saturated with free ribosomes (e.g. Fig. 5), 2) vitellogenesis starts and progresses in oocytes which are no longer connected via bridges with the ONC, and 3) when oocytes are transported along oviducts, they do not show any signs of intensive pinocytotic uptake; moreover, they are coated with a vitelline envelope (VE) transformed later into an impermeable chorion which can effectively block uptake from the hemolymph. It should be noted, however, that newly formed VE in S. berlesei is lamellated and contains pores at regular intervals; it was suggested (Walzl et al., 2004) that yolk or yolk precursors can be transported from the oviduct wall into the egg via these pores. A similar lamellated VE also grows on oviductal oocytes in C. osmiae (Fig. 6C and D).
The molecular foundations of Vgs and their genes in Astgmata are, as in other mites, fragmentary and are known in only a few species (Blomia tropicalis, D. farinae, D. pteronyssinus, G. domesticus, S. scabiei and Suidasia medanensis) (Cabrera et al., 2009). Equally unexplored is the regulation of vitellogenesis and, in particular, the identification and physiological role of ecdysteroids and juvenile hormon (JH) in endocrine regulation of vitellogenesis. In mites, ecdysteroids such as ecdysone, 20E, 2-deoxyecdysone, and makisterone A have been identified in the gamasid mites, Dermanyssus gallinae (Chambers et al., 1996) and Varroa jacobsoni (Feldlaufer and Hartfelder, 1997), and an astigmatan, Tyrophagus putrescentiae (Sakagami et al., 1992). Because JH and its analogs have a major role in influencing oogenesis/vitellogenesis in most insects, investigations aimed at finding these substances in mites have been conducted. Only farnesol, a precursor of JH, was identified in deutonymphs of Tetranychus urticae Cone, 1975, 1976). However, farnesol is present in plants and can be sequestered from food by females which may use this substance as a sex attractant. On the other hand it was shown that exogenous farnesol increase oviposition.
In contrast, many studies explored the effects of exogenous JH and its analogs or anti-JHs on mite reproduction (see Cabrera et al., 2009: Table 2), but in the case of Astigmata there was either no effect (farnesol, JH analogs: methoprene and pyriproxyfen on T. putrescentiae) or the effect was negative (JH analogs: fenoxycarb, hydroprene and methoprene on D. farinae; ecdysone analog: halofenozide on T. putrescentiae). The only positive effect was shown by the JH analog, fenoxycarb, on female reproduction in A. siro. In conclusion, studies with JH precursors and anti-JHs were equivocal in establishing that mites have insect JH or they use it to regulate reproduction; thus a new concept for the regulation of female reproduction in mites was proposed in which ecdysteroids instead of JHs play the main role in stimulation of Vg gene expression in the fat body and midgut in ticks, or the midgut and ovaries in other mites (Cabrera et al., 2009).

Egg envelopes
Eggs of Astigmata are protected by envelopes of complex origin (Witali nski, 1993). First, an early vitellogenic oocyte entering the oviduct starts to secret a VE, which is therefore of primary origin. TEM studies on VE structure and formation indicated that at the beginning the VE material is either lamellated or coarse-fibrillar (A. siro, Tyrophagus, S. berlesei e Acaridae; Witali nski, 1993; Walzl et al., 2004), but is not penetrated by oocyte microvilli as suggested by Reger (1977) for Caloglyphus anomalus (¼Sancassania anomala) (Acaridae), a species with a VE of the same appearance in TEM as A. siro (Witali nski, 1993) and C. osmiae (Fig. 6C and D). In Psoroptes spp. (Lekimme et al., 2005), the VE is homogenous and electron-lucent, and its thickness is 0.2e0.3 mm. An electron-lucent VE also appears on oocytes in F. rostratus; the oocyte, as in other psoroptid mites, forms many deep concavities (Figs. 4D, F and 6F).
As observed in several astigmatans, the thickness of the VE changes: at the beginning of VE formation its thickness increases, but decreases later when the VE lamellae or fibrils disappear, leading to a homogenous VE (see Fig. 6D and E). Interestingly, the disappearance of VE substructure is concomitant with up to a twofold decrease in VE thickness and occurs when the eggs are passing through the distal third portion of the oviduct. Its wall is thick and possibly contains secretory cells. Secretion was not evidenced, but secretory activity was suggested to be a source of VE modifications in structure and properties (Witali nski, 1993). A modified, homogenous VE is 0.3e0.4 and 0.7 mm thick in A. siro and T. perniciosus, respectively, and in A. siro it was named the chorion since it was the only layer enveloping a deposited egg. In other species, additional, exochorional material can be deposited on the egg chorion (which therefore is termed the endochorion) prior to laying. In the distal portion of oviducts in T. perniciosus and putatively Tyrophagus longior, an exochorion of three types is secreted: dense patches, granules, and locular chambers. In Aleuroglyphus ovatus, tiny spherical patches were found instead of locular chambers. In psoroptid mites S. scabiei, N. cati, and F. rostratus, a VE of flocculent appearance also transforms into a homogeneous chorion.
Chorional glands in sarcoptid mites release exochorion material on the egg surface and form a vesicular monolayer (Witali nski, 1993). In F. rostratus, the chorional gland ( Fig. 4D and F)produces a substance which is used to glue the egg on the feather barb of its host (the pigeon). It seems likely that the function of adhesive exochorion material is mainly egg fixation to substratum (Sarcoptes, Notoedres, Falculifer, pterolichoid feather mites e Dubinin, 1953), but additional functions were also proposed, e.g. limited water loss from eggs due to locular chambers in Tyrophagus (Witali nski, 1993).

The male reproductive system
Male reproductive systems in Astigmata were studied on several occasions by light-microscopy (Michael, 1901;Rohde and Oemick, 1967;Prasse, 1968;Heinemann and Hughes, 1970;Kuo and Nesbitt, 1970;Vijayambika and John, 1975;Baker and Krantz, 1985;Witali nski and Walzl, 1995) and ultrastructurally (Witali nski et al., 1990;Walzl, 1992;Lekimme et al., 2005) and are more variable in organization compared to female reproductive systems. They comprise paired spherical or ellypsoidal testes located usually symmetrically in the rear part of the idiosoma, two deferent ducts (vasa deferentia), and one, two or no accessory glands (e.g. Fig. 2E and F). The proximal part of deferent ducts serves as a sperm reservoir, while the distal one has a glandular character (Witali nski et al., 1990). In males of analgesoids A. passerinus (Analgidae) and Trouessartia appendiculata (Trouessartiidae), deferent ducts empty into vesicular structure described by Dubinin (1951) as a seminal vesicle; however, further studies seem necessary to clarify its structure and function. Both deferent ducts merge together with duct(s) of accessory gland(s), if present, to form a short unpaired deferent duct passing into an ectodermal, cuticlelined ejaculatory duct (ductus ejaculatorius). In Pterodectes (Proctophyllodidae) the ejaculatory duct is an extremely complicated structure functioning during insemination as a sperm pump (Popp, 1967;Alberti and Coons, 1999). The ejaculatory duct enters the aedeagus to terminate at its apex.
In acarid mite Caloglyphus (¼Sancassania) berlesei, the testes are situated asymmetrically: the left testis is located dorsally whereas the right one is located ventrally as a result of the occupation of the left side of the body by a very large accessory gland (Witali nski and Walzl, 1995). In Lardoglyphus konoi (Acaroidea: Lardoglyphidae), one testis is in front of the other, and two different accessory glands (one of them named a chambered organ) are present (Vijayambika and John, 1975). In the hemisarcoptoid mite C. osmiae two testes e each with its own germarium e adhere very tightly to one another filling the right side of the idiosoma, whereas the left side is occupied by a large accessory gland which opens into the left deferent duct (Fig. 7B). In the glycyphagoid G. domesticus two testes are displaced symmetrically (Fig. 8A), but the left one is shorter as a large accessory gland is located anteriorly (Fig. 8B). In Psoroptes ovis (Psoroptidae) (Fig. 2E and F), paired testes are secondarily fused at their proximal parts, thus being actually unpaired (Lekimme et al., 2005). Similarly, in N. cati (Sarcoptidae), testes are interconnected by a narrow bridge which is filled with an electron-dense, flocculent material (Witali nski, 1988). In D. farinae and D. pteronyssinus (Pyroglyphidae), the testis is unpaired (Walzl, 1992). In the sarcoptid mites S. scabiei and N. cati, testes are situated anteriorly to the aedeagus due to its posterior shifting while short, quickly merging deferent ducts run postero-ventrally rather than anteroventrally (as in most other astigmatans), to empty into the beginning of the ejaculatory duct (Witali nski and Walzl, 1995).
The aedeagus is a sclerotized organ located midventrally, protruded by hydrostatic pressure and retracted by muscles attached to sclerites in the genital atrium (Prasse, 1970). Its tip is shaped to fit the female bursa copulatrix opening; in Proctophyllodes males, the aedeagus is extremely long extending far behind the body of the male (OConnor, 2009).
The germinal part (germarium) of the testis can be located dorsally or dorsolaterally (in S. scabiei and N. cati e Witali nski and Walzl, 1995;C. lactis e Florek and Witali nski, 2010a), but if the testis is elongated then the dorsally located germarium may be found either in the anterior or posterior part of the gonad, as in G.
The germarium is composed of a compact group of early germ cells, spermatogonia, adhering tightly to one another ( Fig. 8C and D) and to the so-called testicular central cell (TCC), if such a cell is present in the adult gonad. The TCC (Fig. 8C) has been reported in A. siro (Witali nski et al., 1990), Sancassania (¼Caloglyphus) berlesei and S. (¼Caloglyphus) michaeli (Prasse, 1968), Rhizoglyphus robini (Baker and Krantz, 1985) and H. feroniarum (Florek and Witali nski, 2010a), but is absent in many other species: D. pteronyssinus, D. farinae (Walzl, 1992), G. domesticus, S. scabiei, N. cati (Witali nski and Walzl, 1995), C. lactis and F. rostratus (Florek and Witali nski, 2010a). The TCCs are of special interest since their origin e germinal or somatic e and function were for a long time enigmatic. The very similar placement of ONCs and TCCs in gonads suggested a germinal origin and, moreover, a nutritive function. However, intercellular bridges connecting spermatogonia with the TCC were not observed, thus the question remained open. Studies performed recently on gonad development in Histiostoma provided evidence that ONCs and TCCs belong to the germinal cell line (Witali nski et al., 2014), because both are connected by bridges with surrounding gonial cells in a quite similar way. The nutritive role of TCCs is rather doubtful; instead, it was postulated (Florek and Witali nski, 2010a) that the TCC can "suppress and/or drive the proliferation of adjacent spermatogonia" (for further discussion see Florek and Witali nski, 2010a). Indeed, growing and subsequently proliferating spermatogonia lose tight contact with the TCC, as was observed in H. feroniarum (unpublished) and A. siro (Fig. 8C), or separate from a compact mass of germarial earliest spermatogonia in species in which the TCCs in testes are absent (Fig. 8D).
The deferent duct entrance is located opposite to the germarium, usually in the ventral area of the testis (Fig. 7A). The entrance of the deferent duct and its vicinity are packed with sperm cells (Figs. 8A and 9A). Spermatocytes and spermatids disperse after leaving the germarium which disrupts the sequential distribution of spermatogenesis stages (Fig. 9A). They disperse singly or in small clonal groups which do not form wall-encapsulated cysts; rather, they are hosted within large, sometimes multinucleate somatic

Spermatogenesis
As mentioned above, the earliest spermatogonia located in the germarium adhere to the TCC or, if a TCC is absent, they adhere to the other spermatogonia so tightly that their borders can be detected only by TEM (Fig. 8C and D). A relatively large spermatogonial nucleus contains a prominent central nucleolus (Fig. 8D). The nuclear envelope shows shallow concavities with adhering nuage material. Mitochondria are distributed close to the nucleus. Spermatogonia located at the periphery of the germarium are larger and make contact with the germarium one-sidedly (Fig. 8D), eventually to separate and enter the spermatocyte stage.
Early spermatocytes (Fig. 8E) are singular spherical cells with a roundish, "empty" nucleus devoid of condensed chromatin, but usually with a nucleolus located peripherally. Nuage material is still present. As the spermatocyte grows, the peripheral cytoplasm hosts many mitochondria and Golgi bodies; the latter participate in the formation of a characteristic superficial spongy layer of anastomosing membranes/cisternae covering the cell (Figs. 8E, 9B and 10A). The contribution of Golgi bodies to spongy layer formation was observed in many species (Witali nski et al., 1986(Witali nski et al., , 1990Liana, 2004;Florek and Witali nski, 2010a), nevertheless, subplasmalemmal cisternae of ER were also proposed to be a source of spongy layer membranes in Astigmata spermatocytes (Witali nski and Afzelius, 1987;Lekimme et al., 2005).
In general, growing spermatocytes undergo division ( Fig. 10B  and C), however, division is doubtful in some astigmatic species (see below). During division neither the nuclear envelope nor condensed chromatin with synaptonemal complexes is visible. This suggests that normal meiosis may be absent. Daughter cells remain interconnected by bridges delimited by unthickened cisterns of the spongy layer, thus typical contractile rings lined with electrondense material typical for conventional intercellular bridges or ring canals (permanent intercellular bridges in gametogenesis) are likely absent (Florek and Witali nski, 2010a). The number of interconnected spermatids is no more than four, since in most species, sectioned groups of spermatids contain two (C. lactis e Florek and Witali nski, 2010a) or three to four (A. siro, T. putrescentiae e Witali nski et al., 1986, D. columbae e Fig. 10C) cells in section, whereas in some species, e.g. in G. domesticus and C. osmiae, spermatocytes and spermatids are singular. Moreover, the size of spermatids is only somewhat smaller than that of spermatocytes, decreasing progressively. In such cases, male germ cells differentiate, but evidently do not divide.
As spermiogenesis progresses, cisterns of the spongy layer can aggregate to form a spherical spongy body (Fig. 11E, inset) (Florek and Witali nski, 2010a); cisterns of the spongy layer sometimes participate in the formation of very conspicuous structures ( Fig. 11A and C). In consequence, the spermatid is no longer covered by a spongy layer, but its plasmalemma seems to be thickened.
Chromatin appears in spermatid cytoplasm as progressively thickening threads. Electron-dense lamellae (most species) or tubules (Sarcoptes and Notoedres) derived from ER also occur. In some cases, a band of granular material (C. sellnicki) or many chains of small vesicles (Scutulanyssus obscurus) occur in the spermatids, but dense lamellae or tubules are absent (Liana, 2004).
The most striking feature of sperm cells is the lack of a nucleus; instead, chromosomal material is visible as threads embedded directly in the cytoplasm and located more or less centrally in the cell (e.g. Alberti, 1980;Lekimme et al., 2005;Liana and Witali nski, 2005;Florek and Witali nski, 2010a). Chromatin has a star-like appearance in two of three genera in the Pterolichidae family, suggesting that several threads cross at some sites. The diameter of chromatin threads varies from 40 to 60 nm in most species up to 170e190 nm in H. feroniarum.
Other peculiarities of Astigmata sperm are electron-dense lamellae derived from flat ER cisternae which are present in spermatids. In C. lactis there is only one lamella partly surrounding the chromatin threads (Florek and Witali nski, 2010a). In the Histiostoma spermatozoon (Liana and Witali nski, 2005;Witali nski et al., 2014) two lamellae run parallel to each other and this tandem is located laterally to the chromatin threads. Two to several lamellae occur in the vicinity of chromatin threads in Tyrophagus (Witali nski et al., 1986). In several other species shorter or longer profiles of lamellae, frequently in parallel arrangement, are placed mostly around the chromatin (Acarus, Glycyphagus, Dermatophagoides, Falculifer, Grallolichus, Psoroptes, Myocoptes), whereas in Pterolichus   (1) shows an electron-dense lamella (dl) which separates chromatin threads (ch) from circular profiles (arrows). Mitochondrial derivatives (m) are also present. (C) Spermatid fragment containing a conspicuous cisternal arrangement which later transforms into a spongy body of the spermatozoon. There are also electron-dense lamellae (dl) and mitochondria (m). (D) Several spermatozoa in a deferent duct (vd) containing chromatin (ch), dense lamellae (dl), and mitochondria (m). Material filling the deferent duct is highly electron-dense. (E) Spermatozoon at higher magnification showing chromatin (ch), dense lamellae (dl), and mitochondria. In the lower spermatozoon a fibrillar bundle (arrow) is present. Inset shows a spongy body. Scale bars: 1 mm in (AeC); 5 mm in (D, E); 1 mm in (E inset). they are visible in between chromatin threads; in Pseudolichus lamellae are scarce and curved showing circular profiles. Spermatozoa in Trouessartia are packed with stacks of parallel and short lamellae. In contrast, lamellae are absent in Canestrinia and Scutulanyssus sperm; in the former species sperm contains a band of granular material, whereas the Scutulanyssus sperm cell is filled with branching chains of vesicles (Liana and Witali nski, 2005). In sarcoptid mites (Notoedres and Sarcoptes), ramifying electrondense tubules distributed within the cell are visible in place of lamellae; these tubules originate during spermiogenesis from tubular profiles of ER rather than flat ER cisternae (Witali nski and Afzelius, 1987). Psoroptes sperm shows intermediate lamellar structures since spermatozoa contain many parallel lamellae organized in groups and electron-dense tubules which seem to grow out from the lamellae margins (Alberti, 1984;Liana and Witali nski, 2005).
As was mentioned earlier, mitochondria during spermiogenesis in Astigmata either persist e at least to some degree e in their normal structure, or they transform into mitochondrial derivatives of variable appearance and distribution within the cell. Nearly unmodified mitochondria have been found in sperm of P. obtusus (Liana and Witali nski, 2005), in which they form large assemblages with rod-shaped mitochondria aggregating end-to-end and sideby-side in a bundle meandering within the cell. Distinct and large assemblages of mitochondria are also present in Dermatophagoides sperm. In other studied species mitochondrial derivatives are more or less altered and their internal structures, especially cristae, are no longer discernible; such poorly visible vesicular structures occur, for instance, in Acarus and Tyrophagus sperm (Witali nski et al., 1986(Witali nski et al., , 1990. Thus, in most cases the function of mitochondrial derivatives as energy (ATP) donors for spermatozoon movement seems unlikely; their deeply disintegrated structure is in accordance with the uniparental theory of maternal mitochondrial inheritance (e.g. Giles et al., 1980;Sutovsky et al., 1999).
Sperm structure in the currently studied species also shows some special characters. In C. osmiae (Fig. 11B) the spermatozoon contains only one meandering lamella which separates chromatin threads (40e45 nm thick) and circular profiles likely derived from spermatid arcuate cisterns present around a large spongy area (Fig. 11A). The latter is putatively a remnant of the spermatocyte superficial spongy layer. Spermatozoa in G. domesticus (Fig. 11D and E) contain many electron-dense lamellae surrounding chromatin threads (70e80 nm thick), as well as many globular mitochondria with frequent incisions. In the sperm cell of P. fuchsi (Fig. 10E) centrally located, tightly packed chromatin threads (27e33 nm thick) are surrounded by radially oriented groups of lamellae arranged in parallel. Most remarkable is the sperm of D. columbae (Fig. 10D), since this is the only case of astigmatic spermatozoon without chromatin threads; instead, there are many granules of variable size (90e125 nm) and density surrounded by moderately dense, elongated or circular profiles derived from a short electrondense lamella visible in spermatids (Fig. 10C).
The acrosome is absent in sperm of Astigmata. This is the consequence of early syngamy, since sperm penetrates ovaries (Prasse, 1968;Witali nski et al., 1986;Witali nski, 1988) and encounters naked oocytes before the vitelline envelope/chorion is formed. Interestingly, spermatozoa found within the female show fine filaments regularly distributed under the cell membrane; their role in sperm motion have been suggested (Alberti, 1980;Witali nski et al., 1986).

Testicular somatic cells
Germinal cells in testis are embedded in a few somatic cells (Figs. 7A and 8C, D and 9), termed the testicular stroma cells in this review. The number of stroma cells is difficult to determine; their irregularly shaped nuclei located peripherally are scarcely visible, suggesting a low number of these cells in the testis (e.g. Sarcoptidae: N. cati e Witali nski, 1988). In some species, e.g. C. lactis, stroma cells are distinct and connected by adherent junctions, at least close to the testis-deferent duct transition (Florek and Witali nski, 2010a). However, in F. rostratus (Fig. 9) and S. scabiei, stroma cells (originally named the main somatic cells; Witali nski and Afzelius, 1987) contain many nuclei and their syncytial nature is possible. In the latter species, two additional kinds of somatic cells were found: the so-called distal somatic cells and muscle cells. The distal somatic cells are located close to the beginning of the deferent duct, thus may be considered as an element of the deferent duct wall. More interesting are muscle cells embedded in stroma cells, with contractile filaments containing appendages penetrating stroma cells and observable between germ cells in testis regions rather distant from the entrance of the deferent duct.

Conclusions and perspectives
Studies on the reproduction of Astigmata are fragmentary and focused mostly on some aspects of reproduction and reproductive behavior having implications in the evolution of reproductive strategies (Tilszer et al., 2006;Radwan, 2009) rather than gonad structure, details of gametogenesis and functioning of reproductive systems. For example, very little is known on the functioning of the spermatheca in Astigmata females (Radwan and Witali nski, 1991), as well as on events during sperm storage, migration to ovaries and details of fertilization. The same deficiency of information pertains to the role of the TCC in spermatogenesis. Recent studies on gonad development in Histiostoma (Witali nski et al., 2014) confirmed earlier suggestions (Witali nski et al., 1990) on TCC origin from the germinal line. However, the role of the TCC during the early stages of gametogenesis and reasons, why TCCs are absent in adult testes in some species whereas they remain in others, are still enigmatic. Further conclusions may stem from studies on species with adult testes devoid of TCC to evidence whether TCCs are present in developing gonads and, if so, when and how they disappear.
A very intriguing problem concerns oogenesis, in particular the structure of the unique intercellular bridges connecting previtellogenic oocytes with the ONC. In all studied Astigmata except Histiostomatidae, funnel shaped bridges filled tightly with some electron-dense material are present. The function of such structures as a gate controlling the in-and-out flow between the ONC and oocytes is only suggested; the same concerns the even more conspicuous and enigmatic diaphragm-crossed bridges found in Histiostoma species. The reasons for such profund modifications of ordinary intercellular bridges and mechanisms involved in their function should be studied in the future. Moreover, studies on ovaries in Histiostomatoidea other than Histiostoma could clarify whether the funnel-shaped intercellular bridges evolved from diaphragm-crossed bridges or vice versa, or both types appeared independently.
A different body of information should be collected and considered in the light of the evolution of gonads and reproduction in Astigmata. If we accept the idea that Astigmata evolved from within early Oribatida (Desmonomata: Trhypochthoniidae) (Norton et al., 1993) a number of problems must be considered. First, the gonads in contemporary Oribatida are quite different than in Astigmata (further literature: Alberti and Coons, 1999;Liana, 2004;Bergmann et al., 2008;Liana and Witali nski, 2012). For instance, oribatid ovaries are unpaired and composed of oocyte clusters connected via microtubule-rich protrusions with one or several enucleate centers (medullae). Such ovaries are evidently not of nutrimental type. Oribatid testes are usually paired, but consist of germinal and glandular parts. Second, sperm organization in both taxa is completely different, showing peculiarities in each group (e.g. oribatid sperm contains compact, highly condensed chromatin containing mitochondrial derivatives, whereas in astigmatic spermatozoa separate chromatin threads are freely embedded in cytoplasm). Third, differences in reproductive behavior are also striking and not easy to explain: Oribatida are inseminated through stalked spermatophores deposited on the substrate, whereas Astigmata females are inseminated during copulation via an accessory inseminatory system. Moreover, thelytokous parthenogenesis is a main reproductive strategy in Desmonomata, whereas in Astigmata thelytoky seems to be secondarily evolved in some taxa only. It is necessary to emphasize, however, that in fact we know practically nothing on the reproduction of the ancestors of Astigmata since we can only study living oribatid taxa and it is at least theoretically possible that oribatid progenitors of Astigmata were much different from contemporary Desmonomata/Trhypochthoniidae.