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The ultrastructural investigation of the midgut in the quill mite Syringophilopsis fringilla (Acari, Trombidiformes: Syringophilidae)
Arthropod Structure & Development 38 (2009) 303–313
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The ultrastructural investigation of the midgut in the quill mite Syringophilopsis fringilla (Acari, Trombidiformes: Syringophilidae) S.A. Filimonova* Zoological Institute, Parasitology, Russian Academy of Sciences, Universitetskaya Embankment 1, 199034 St. Petersburg, Russia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 October 2008 Accepted 27 January 2009
The midgut of the females of Syringophilopsis fringilla (Fritsch) composed of anterior midgut and excretory organ (¼posterior midgut) was investigated by means of light and transmission electron microscopy. The anterior midgut includes the ventriculus and two pairs of midgut caeca. These organs are lined by a similar epithelium except for the region adjacent to the coxal glands. Four cell subtypes were distinguished in the epithelium of the anterior midgut. All of them evidently represent physiological states of a single cell type. The digestive cells are most abundant. These cells are rich in rough endoplasmic reticulum and participate both in secretion and intracellular digestion. They form macropinocytotic vesicles in the apical region and a lot of secondary lysosomes in the central cytoplasm. After accumulating various residual bodies and spherites, the digestive cells transform into the excretory cells. The latter can be either extruded into the gut lumen or bud off their apical region and enter a new digestive cycle. The secretory cells were not found in all specimens examined. They are characterized by the presence of dense membrane-bounded granules, 2–4 mm in diameter, as well as by an extensive rough endoplasmic reticulum and Golgi bodies. The ventricular wall adjacent to the coxal glands demonstrates features of transporting epithelia. The cells are characterized by irregularly branched apical processes and a high concentration of mitochondria. The main function of the excretory organ (posterior midgut) is the elimination of nitrogenous waste. Formation of guanine-containing granules in the cytoplasm of the epithelial cells was shown to be associated with Golgi activity. The excretory granules are released into the gut lumen by means of eccrine or apocrine secretion. Evacuation of the fecal masses occurs periodically. Mitotic figures have been observed occasionally in the epithelial cells of the anterior midgut. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Mites Ultrastructure Midgut
1. Introduction Acariform mites (order Acariformes ¼ Actinotrichida) represent a great variety of different small arthropods, including parasites of man and many domestic animals. This attracts special attention to the morphological and physiological aspects of their feeding behavior and digestion. It is well known that in the majority of Chelicerata the secretory product of salivary glands serves for the preoral digestion of the meal. Inside the digestive tract the most important events occur in the midgut, where intracellular digestion predominates. The midgut usually consists of the anterior and posterior portions. In most acariform mites special secretory cells are absent and the anterior midgut (ventriculus and midgut caeca) is composed of polyfunctional epithelial cells (Alberti and Coons, 1999). The exceptions are several families of predatory mites, all of
* Corresponding author. Tel.: þ7 0812 714 0151; fax: þ7 0812 714 0444. E-mail address: fi[email protected] 1467-8039/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.asd.2009.01.002
which belong to the suborder Trombidiformes: Bdellidae (Alberti, 1973; Alberti and Storch, 1983), Labidostommatidae (Vistorin, 1980), and Anystidae (Filimonova, 2008a). In the suborder Astigmata secretory cells have never been detected ultrastructurally (Alberti and Coons, 1999). In Acarus siro the cells producing the peritrophic matrix were also detected as a special cell type (Sˇobotnik et al., 2008). The posterior midgut forms the excretory pellets and evacuates them through the hindgut and anal opening, if these organs are present (Alberti and Coons, 1999). In the astigmatic mites the posterior midgut is divided into the colon and postcolon. Malpighian tubules enter the gut at the border of these organs. In trombidiform mites, Malpighian tubules are absent, the colon tends to be reduced, and the postventricular midgut is represented by the dorsomedian excretory organ (Ehrnsberger, 1984; Alberti and Coons, 1999). In several trombidiform taxa, for instance in the cohort Parasitengona, the excretory organ has no connection with the ventriculus (Mitchell, 1970; Vistorin-Theis, 1978; Shatrov, 1989, 2003). The excretory organ of these mites was considered to be
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a functional analog to Malpighian tubules (Mitchell, 1970). Though the connection between the two parts of the midgut was later shown in most trombidiform taxa (Blauvelt, 1945; Alberti, 1973; Vistorin, 1980; Mothes and Seitz, 1981; Ehrnsberger, 1984; Alberti and Crooker, 1985; Mothes-Wagner, 1985; Akimov and Gorgol, 1990; Filimonova, 2001, 2008a,b), the origin and functioning of the excretory organ is still unclear (Mothes-Wagner, 1985). A serious problem is the little information available about the cell replacement in the midgut epithelium of acariform mites. Special regenerative cells, as well as mitotic divisions, have recently been found in Acarus siro (Sˇobotnik et al., 2008). It seems to be an exception or a characteristic of astigmatic mites only, as in trombidiform species no mitotic divisions have ever been observed, though many authors described the extrusion of epithelial cells or part of them into the gut lumen, and their subsequent degeneration (Blauvelt, 1945; Alberti, 1973; Ehrnsberger, 1984; Alberti and Crooker, 1985; Akimov and Gorgol, 1990; Alberti and Coons, 1999). Taking into account the problems mentioned above and the great diversity of internal organization of acariform mites, the investigation of possibly many groups of these animals seems to be necessary. The aim of the present paper is to describe peculiarities of the morphology and physiology of the midgut in quill mites (Trombidiformes: Syringophilidae), which live inside the shaft of bird flight feathers and feed on tissue fluids by piercing the wall of the quill with their chelicerae. The internal anatomy of these mites has been recently studied for the first time (Filimonova, 2008b). 2. Materials and methods Females of Syringophilopsis fringilla (Fritsch) were collected from the chaffinch, Fringilla coelebs Linnaeus (Passeriformes: Fringillidae), captured for ringing in the bird-banding station ‘‘Rybachy’’ (Russia, Kaliningrad Province) in autumn of 2006. Having been taken out of the host, quill mites were put into 2.5% glutaraldehyde solution in 0.01 M phosphate buffer (pH 7.4). For better penetration of the fixative solution the cuticle of the body was pierced in some places. After that the specimens were transferred into fresh fixative solution for some days and then postfixed in 2% osmium tetroxide prepared in the same buffer. Material was embedded in Epon 812. The semithin sections for light microscopy were stained with a mixture of methylene blue and Azur II (pH ¼ 6.8). Ultrathin sections stained with uranyl acetate and lead citrate were studied using Zeiss LEO 900 and TESLA BS 500 electron microscopes. In whole 10 females were studied. 3. Results The midgut of Syringophilopsis fringilla consists of the two portions, the anterior and posterior midgut. The anterior midgut includes the unpaired ventriculus and two pairs of diverticuli: anterior and posterior midgut caeca. The posterior midgut is represented by a long tubular excretory organ, connected to the hindgut at the end of the mite body (Figs. 1a,b). 3.1. Anterior midgut The anterior midgut occupies all the free space of the body cavity. The central unpaired ventriculus is situated just behind the synganglion. It sends forwards and backwards two pairs of caeca (Figs. 1a,b). Apart from the ventral wall of the ventriculus adjacent to the coxal glands, all the remaining anterior midgut is lined by the same single-layered epithelium (Fig. 1b). The epithelium is underlain by a very thin basal lamina. Dorsally, large skeletal muscles are apposed to the ventriculus; in the other parts of the midgut, single muscle bands can be visible in some places. Both, the midgut
Fig. 1. Schematic drawings of the digestive tract in Syringophilopsis fringilla, showing the location of the modified midgut epithelium (hatched area). (a) Dorsal view, (b) lateral view. A – anus; AC – anterior caeca; Es – esophagus; EO – excretory organ; HG – hindgut; PC – posterior caeca; Ph – pharynx; Sy – synganglion; V – ventriculus.
epithelium and the surrounding muscle cells form thin processes directed to the body cavity. No signs of connective tissue were found around the midgut. In most specimens studied, the midgut lumen was completely filled with the extensions and fragments of the epithelial cells loaded by numerous basophilic inclusions and vacuoles (Figs. 2a,b). The epithelial lining varies in thickness from place to place, due to the different height of the epithelial cells (Fig. 2a). The nuclei of most epithelial cells are elongate, with the large diameter about 5–6 mm. They are typically electron-lucent and possess two or three extensively developed nucleoli, each up to 2.5 mm in size. In several cases, the nuclei contained condensed chromatin in a form resembling chromosomes. These nuclei seem to be in prophase, as nuclear envelope partly exists (Fig. 2c). Metaphase pictures were also observed at the light microscope level, while no evident anaphases were found. Dumbbell-shaped nuclei were also regularly observed in all the mites examined (Fig. 2d). They are measuring about 8–9 mm in diameter. Both kinds of unusual nuclei occur in cells lacking any special features and having RER cisterns, lysosome-like inclusions, lipid droplets, as well as glycogen rosettes (Figs. 2c,d). The ultrastructural investigation shows that the apical surface of the midgut epithelium lacks a true brush border. Locally, small apical processes can be found, but they do not have internal filaments typical of microvilli (Figs. 2b,e). Glycogen particles are common inside these processes, as well as in the cytoplasm of the cells. The lamellae delaminating from the apical surface of the cells are often observed free in the gut lumen (Fig. 2e). A basal labyrinth is absent. Mitochondria do not concentrate in any part of the cell.
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Fig. 2. General morphology of the midgut epithelium visible on the LM (a) and EM (b–e) levels. (a) Cross semithin section of the ventriculus with the coxal glands (CG) invading into the ventricular epithelium (E) ventrally. (b) Midgut lumen (Lu) occluded by the remnants of the epithelial cells. (c) Nucleus (N) with condensing chromatin (Ch) belonging to differentiated epithelial cell. (d) Dumbbell-shaped nucleus (N) with several nucleoli (Nu). (e) Formation of macropinocytotic vesicles (PV) on the apical surface of the digestive cells. Scale bars: a ¼ 15 mm, b ¼ 1.5 mm, c, d ¼ 2 mm, e ¼ 1.0 mm. AP – apical processes; Cu – cuticle; Ep – epidermis; F – cell fragments floating in the gut lumen; Gly – glycogeneous granules; Hly – heterolysosomes; L – lipids; Lm – surface lamellae; RER – rough endoplasmic reticulum; SII – spherites of the second type.
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Most of the lateral surface of the neighboring epithelial cells shows deep interdigitations. In the apical region the epithelial cells are connected by a zonula adherens followed by septate desmosomes below. In the middle region some isolated septate desmosomes were seen. Ultrastructural analysis shows four kinds of epithelial cells in the anterior midgut (Fig. 3). The most common are the digestive cells, which may be referred to as the light (L) and dark (D) cells based on the density of their cytoplasm (Figs. 2b, 3 and 4a). Both of these cells may be either flat or columnar and are characteristically
rich in rough endoplasmic reticulum (RER). The L cell is characterized by swollen RER cisternae with electron-lucent content (Figs. 3 and 4b), while in the D cell, flat RER patterns are packed more tightly in the cytoplasm, and contain material of moderate electron density (Figs. 3 and 4a). Both cell types possess numerous small Golgi bodies commonly forming an indistinct Golgi field, which is connected to the RER. Secretory granules originating from the Golgi body lie nearby (Fig. 4a). The mature secretory granules represent spherical inclusions 0.5 mm in size with homogeneous contents of moderate or high electron density (Fig. 4b). They are formed by the
Fig. 3. Scheme, showing the main kinds of epithelial cells in the anterior midgut of Syringophilopsis fringilla and their possible relationships (arrows). E1, E2 – sequential stages of the excretory cell development; F – cell fragment, freely floating in the gut lumen. D1, D2 – sequential stages of differentiation of the dark-stained digestive cell, L –the light-stained digestive cell; S – the secretory cell. AP – apical processes; BL – basal lamina; Gly – glycogeneous granules; Hly – heterolysosomes; L – lipids; M – mitochondria; N – nucleus; Nu – nucleolus; PV – pinocytotic vesicles; R – ribosomes; RB – residual body; RER – rough endoplasmic reticulum; SI, SII – spherites of the first and second types; SG – secretory granules.
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Fig. 4. Fine structure of the digestive and excretory cells of Syringophilopsis fringilla. (a) The border between a dark (D) (to the left) and a light (L) (to the right) digestive cell (arrows). (b) General view of L cell. (c) D2 cell with the basal region rich in RER and the apical part loaded with heterolysosomes (Hly) and spherites (SII). (d) Crystalline inclusions (CI) in the lumen of the midgut caeca (Lu). The apical region of an E1 cell is also visible. (e) The apical region of an E2 cell (E), and a cell fragment (F) floating in the lumen of the ventriculus (Lu). G – Golgi bodies; N – nucleus; Nu – nucleolus; RER – rough endoplasmic reticulum; SI, SII – spherites; SG – secretory granule. Scale bars: a ¼ 0.5 mm, b, c ¼ 2 mm, d, e ¼ 3 mm.
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fusion of the primary granules and commonly lie not far from the Golgi zone (Figs. 4a,b). In both L and D cells large lipid droplets are always present, as well as a great number of glycogen particles (a-form). The latter can be seen either inside the large electronlucent vacuoles or lying freely in the cytoplasm. The D cell population is not uniform, composed of the most common flat D1 cells and columnar D2 cells, which contain more or less electron-lucent vacuoles in the apical region (Fig. 3). These vacuoles, ranging from 0.5 to 1.2 mm in diameter, originate from the apical invaginations or by the fusion of the tops of the apical processes and may be considered as macropinocytotic vesicles (Fig. 2e). In the cytoplasm, the vacuoles join and form larger vesicles with variable heterogeneous material inside, which probably represents a kind of heterolysosomes (Figs. 3 and 4c). Intermediate states between L and D1 cells are often observed being characterized by the relatively dark cytoplasm with swollen RER cisternae, not so abundant as in D1 cells. The third kind of the epithelial cells is the excretory cell (E) characterized by the apical region extended into the gut lumen (Figs. 3 and 4c–e). RER and Golgi bodies are considerably less numerous in this region, which is filled by various residual bodies, heterolysosomes and spherites. The high concentration of spherites is the most striking feature of this cell (Figs. 4c,d). They seem to be formed inside certain vacuoles (or may be heterolysosomes) by gradual concentration of dense material from the center to the periphery. Two kinds of such inclusions are clearly distinguishable: an electron-lucent inclusion with concentric layers of different electron density (SI type), and that with a highly dense granule inside, which tends to be partly destroyed during preparation (S II type) (Figs. 4c,d). Both kinds of spherites are of the same size, ranging from 2 to 3 mm, and are clearly identified at LM level owing to their birefringence. Both of them were shown to be released into the gut lumen by eccrine secretion. The E cell population can be divided into two subtypes, E1 and E2 cells. The E1 cell has the basal region similar to that in the typical D1 cell, including conspicuous RER and Golgi bodies (Figs. 3 and 4c), whereas the E2 cell is completely full of large vacuoles, heterolysosomes and spherites (Fig. 3), practically lacking RER cisternae and Golgi bodies. The most striking feature of E2 cells is the presence of a huge apical vacuole, apparently formed by the junction of the smaller vacuoles typical of E1 cells (Fig. 4e). Cell fragments with the same vacuoles surrounded by numerous spherites were commonly visible in the midgut lumen of all specimens (Figs. 2a, 3 and 4e). Occasionally, pycnotic nuclei were found inside them. Crystalline particles, typically observed in the gut lumen, are believed to be derivates of the spherites II (Fig. 4d). The secretory cells (S cells) were found only in two specimens (from 10 females examined in the present study) (Fig. 3). These cells contain an extremely large amount of RER in the form of long narrow cisternae tightly packed all over the cytoplasm, and numerous dense membrane-bounded granules (Figs. 3 and 5a–d). The nucleus is larger than in other cell types, measuring 9–10 mm in its large dimension. A single nucleolus is up to 7 mm in diameter (Fig. 5a). The apical surface of the S cell may be either bare (Fig. 5b) or with long thin microvilli-like processes lacking internal filaments (Fig. 5d). Golgi bodies are represented by a large heap of small vesicles originating from RER cisternae. The primary secretory granules look similar to those in the digestive cells (L and D1) (Fig. 5c). They fuse with each other to form the mature secretory granules which are typically about 2 mm in size (Fig. 5a). Cells containing larger inclusions (4 mm in size) were also seen (Fig. 5b). Releasing of the secretory product was not observed. Lipid and glycogen inclusions are fewer than in other cell types, as well as vacuoles and spherites.
In the specimens, where secretory cells were not detected, single secretory granules of similar appearance were occasionally observed in other kinds of midgut cells (except for E2 cells). In the ventral part of the ventriculus, the epithelium forms large folds surrounding the proximal tube of the coxal gland (Fig. 2a). The epithelial layer adjacent to the tubes is very low in most places (sometimes not more than 1 mm in height), closely resembling connective tissue (Figs. 6a,b). The cells possess numerous branching apical processes with deep invaginations at their base. Pinocytotic vesicles of various size and shape are formed at the apical surface of these cells; their content is electron-lucent (Figs. 6c). The cytoplasm of the cells is of low electron density, lacking most inclusions, which are typical to the rest of the midgut epithelium (heterolysosomes, secretory granules or spherites) (Fig. 6a). RER is poorly-developed and Golgi bodies are rarely visible. The main component of the cytoplasm is numerous mitochondria with dense matrix and narrow cristae (Figs. 6a,b). The basal protrusions of the midgut cells may be very long and thin; however, they do not reach the coxal gland epithelium, as they never penetrate the midgut basal lamina.
3.2. Posterior midgut The posterior midgut or excretory organ is a long tube running backwards from the short, posterior extension of the ventriculus (Figs. 1a,b and 7a). At the border to the ventriculus the excretory organ forms a valve, composed of higher epithelial cells (Fig. 7b). In other regions the epithelial lining is very thin, in some places not more than 1.5 mm and about 3–4 mm at the nuclear site (Figs. 7c,d). The basal lamina is as thin as in the anterior midgut, but muscles are more numerous and are represented by relatively thick longitudinal bundles (Fig. 7c). The volume of the lumen varies greatly, relating to the quality of excretes. The free floating fragments of the epithelial cells in the lumen of the organ are very similar to those seen in the anterior midgut. Occasionally pycnotic nuclei can be found inside them (Fig. 7e). The anterior part of the excretory organ is lined by an epithelium with apical processes, which are more regular and abundant than in the anterior midgut (Fig. 7c). In the posterior part of the organ these processes disappear, but the apical surface of the cells remains irregularly shaped with sharp edges visible in a section. The apical plasmalemma is covered by a thin superficial layer of high density (Fig. 7d). The cells are predominantly flat with their lateral sides strongly interdigitated. The elongate nuclei lie along the basal lamina and usually possess one or two prominent nucleoli. The basal plasmalemma is smooth; the number of mitochondria is higher than in the anterior midgut, though they do not concentrate in any region. Some of them are up to 2 mm long. RER is not conspicuous being represented by single short cisternae. Numerous small Golgi bodies can be observed in all parts of the cytoplasm. Each of them commonly shows an ordinary stack of flat cisternae producing very small moderately dense vesicles (Fig. 7d). Joining together, these vesicles give rise to larger irregularly shaped inclusions of the same density. The latter gradually transform into granules of a very high density with a crystalline component inside, typically being destroyed during EM preparation (Fig. 7c). Both, the whole granules and their crystalline fragments are clearly visible in the lumen of the organ. Their releasing occurs in the form of eccrine or apocrine secretion. The latter was found in the specimens, where the lumen of the organ was enlarged with a great amount of waste products. In
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Fig. 5. Fine structure of the secretory cells in the anterior midgut of Syringophilopsis fringilla. (a) A central part of S cell with large nucleus (N) and numerous secretory granules (SG). Note RER cisternae filling the cytoplasm. (b) The apical portions of two S cells facing each other. (c) A heap of Golgi vesicles marks indistinct Golgi complexes (G), where primary secretory granules (PSG) are forming. (d) Microvilli-like apical processes on the luminal surface of the S cell. Scale bars: a, b ¼ 2 mm, c ¼ 1 mm, d ¼ 1.5 mm. Lu – the midgut lumen; Nu – nucleolus.
this case, the same secretory products are also released into the intercellular spaces. Irregularly shaped vacuoles (0.2–0.3 mm in diameter), glycogen and lipid inclusions are also common in the epithelial cells, as well as spherites and myeline bodies.
4. Discussion As it was shown in the previous paper (Filimonova, 2008b), the digestive tract of S. fringilla is typical to that in the cohort Eleutherengona: the anterior midgut is in open connection with the
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Fig. 6. Fine structure of the ventricular epithelium in the region adjacent to the coxal gland. (a) A common view of the fold of the ventriculus with the large E cell on its one side and a very thin epithelial lining on the opposite side attached to the coxal gland (CG). (b) Thin ventricular epithelium at a higher magnification. (c) The branching apical processes (AP) probably associated with the formation of pinocytotic vesicles (PV). Scale bars: a ¼ 2 mm, b, c ¼ 1.5 mm. AP – apical processes; E – ventricular epithelium; Hly – heterolysosomes; Lu – the lumen of the ventriculus; M – mitochondria; N – nucleus; PV – pinocytotic vesicles; SI, SII – spherites.
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Fig. 7. The structure of the excretory organ in Syringophilopsis fringilla. (a) Frontal semithin section through the posterior part of the mite body. The lumen of the excretory organ (EO) is wide. (b) The most anterior part of the excretory organ. (c) An epithelial cell of the anterior portion of the excretory organ with mature crystalline granule inside. (d) Golgi complex (G) in an epithelial cell of the posterior portion of the organ. Note irregular secretory granules (SG) in close contact with the Golgi vesicles. (e) A cell fragment (F) in the lumen of the organ, containing a pycnotic nucleus (N). Scale bars: a ¼ 50 mm, b, e ¼ 2 mm, c ¼ 0.5 mm, d ¼ 1 mm. AP – apical processes; CI – crystalline inclusions; E – the epithelium of the organ; G – Golgi body; Lu – the lumen of the organ; ML – muscle layer; N – nucleus, Nu – nucleolus; PC – posterior caecum; SC – surface coat; SII – spherites.
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postventricular region represented by a simple dorsomedian tube known as the excretory organ (Alberti and Coons, 1999). 4.1. Anterior midgut The data obtained in the present study show that the epithelium of ventriculus and caeca is uniform except for the region adjacent to the coxal glands. The cells of the latter region seem to be not involved in the digestion as they are lacking heterolysosomes. They demonstrate most features of transporting epithelia: a complex system of apical processes accompanied by a great number of large mitochondria. The main function of this region is evidently osmoregulation. A very similar modification of the midgut epithelium has been found in tetranychids both near the coxal gland and in the region closely attaching to the excretory organ (Mothes and Seitz, 1981; Mothes-Wagner, 1985; Alberti and Crooker, 1985). In some trombidiform species, finger-like protrusions of coxal gland cells were observed passing through the basal laminae of both midgut and coxal gland and entering the basal portion of the midgut epithelium (Alberti and Coons, 1999; Filimonova, 2001). In S. fringilla, such protrusions were not seen. Other regions of the anterior midgut do not demonstrate high transport activity: the cells lack a distinct brush border, as well as a basal labyrinth. Short apical processes seem to appear or disappear on the surface of the epithelial cells depending on their physiological state. Ultrastructural analysis shows four kinds of epithelial cells in the anterior midgut (Fig. 3). In all specimens examined, digestive L and D1 cells were most common, both rich in RER cisternae and Golgi bodies producing small secretory vesicles. L cells are characterized by less numerous RER and probably represent the precursors of the flat D1 cells. Two directions of D1 cell development can be suggested according to the observations obtained in the present study (Fig. 3). The first one leads to the formation of the secretory cells (S cells) with hypertrophied RER and Golgi bodies, producing dense, membrane-bounded granules of proteinaceous nature. In the specimens where S cells were not noted single granules of the same appearance were indicated in the other cell types. This leads to the conclusion that the secretory cells temporarily exist in the syringophilid midgut, associated with a certain stage of digestion. In the horseshoe crab Limulus polyphemus, the accumulation of the secretory granules was demonstrated in the midgut gland of starved animals, but these granules were quickly released into the gut lumen just after the meal (Herman and Preus, 1972). A very similar sequence of events was shown during digestion in some spiders (Alberti and Storch, 1983; Ludwig and Alberti, 1988). Fast liberating of the secretory granules in answer to the food uptake may be the cause, why the secretory cells were not observed in most trombidiform mites (Alberti and Coons, 1999). Up to date, they have been demonstrated ultrastructurally only in the representatives of Bdellidae (Alberti, 1973; Alberti and Storch, 1983) and Anystidae (Filimonova, 2008a). In both cases secretory cells are characterized by conspicuous RER and large dense granules with homogeneous content corresponding to the S cells found in S. fringilla. It is interesting to note that in other arachnids the secretory granules are also very large, measuring from 3 to 6 mm (Herman and Preus, 1972; Goyffon and Martoja, 1983; Becker and Peters, 1985a; Ludwig and Alberti, 1988, 1990, 1992), which is comparable to those found in S. fringilla (where they are up to 4 mm in diameter). Most authors consider the secretory product to be the enzymes for extracellular digestion, which is followed by the intracellular one (Herman and Preus, 1972; Becker and Peters, 1985a; Ludwig and Alberti, 1988, 1990, 1992; Alberti and Coons, 1999).
The second direction of D1 cell differentiation is the typical digestive cell (D2) (Fig. 3) with its apical region usually containing macropinocytotic vesicles of different size and various vacuoles similar to those detected as heterolysosomes in many arachnid taxa (Alberti and Storch, 1983; Becker and Peters, 1985a,b; Ludwig and Alberti, 1988, 1990, 1992; Shatrov, 1989, 2003; Filimonova, 2001, 2008a). Transformation of these vacuoles into typical residual bodies was frequently observed during the study. The last stage of D2 cell development is represented by the excretory cell (E2 cell), which is completely filled with vacuoles, residual bodies and spherites (Fig. 7). Two types of spherites are clearly distinguishable. The type I is mineral spherites characterized by concentric layers of different electron density. The II type contains a sphere of high electron density, which is typically destroyed during preparation. Being released into the gut lumen, the second type of spherites gives rise to crystalline waste products. The same crystalline particles were typically observed in the excretory organ of S. fringilla and seem to be guanine granules, which look birefringent in light microscope. Similar guanine-containing spherites have been indicated in some scorpions, spiders and oribatid mites (Goyffon and Martoja, 1983; Lopez, 1983; Alberti et al., 2003), but they seem to be more widely dispersed among Chelicerata. Both excretory cells and their apical portions were shown to be extruded into the gut lumen contributing to the formation of free floating cell fragments with high concentration of waste products inside. They typically contain a single huge vacuole, where digestion is being continued. Similar pictures have been observed in most trombidiform mites studied up to now. The authors described free floating cell fragments either as derivatives of whole cells (Thor, 1904; Vistorin, 1980; Mothes and Seitz, 1981; Ehrnsberger, 1984; Alberti and Crooker, 1985) or of their apical region (Shatrov, 1989, 2003; Filimonova, 2001, 2008a). In the case of S. fringilla, the presence of pycnotic nuclei in several cell fragments suggests elimination of the whole cells. On the other hand, in many cells with accumulated waste products in their apical region, the rest of the cytoplasm is typical for D cells. This suggests that in most cases only the apical zone of D2 cells is extruded, and thereafter the lost region regenerates. Thus, D cells appear to go through several digestive cycles before they completely change into the E2 cells and die in the gut lumen. The same conclusion was made by Becker and Peters (1985a), who carefully studied different stages of digestion in the midgut glands of opilionids (Chelicerata: Phalangida). This way of cell regeneration appears to occur, when cell divisions are impossible by some reasons. This seems to be typical for most trombidiform mites. Contrary to astigmatic mites (Sˇobotnik et al., 2008), in most trombidiform mites, the regenerative cells are characteristically absent (Alberti, 1973; Vistorin, 1980; Mothes and Seitz, 1981; Alberti and Crooker, 1985; Filimonova, 2001, 2008a; Shatrov, 2003). Though undifferentiated cells have been described in some taxa (Shatrov, 1989; Akimov and Gorgol, 1990), no mitotic figures have been commonly demonstrated. During the present study several prophases and metaphases have been detected in the midgut epithelium of S. fringilla at the light microscope level. Midgut cells with condensed chromatin resembling chromosomes were shown ultrastructurally in the anterior midgut. These cells had the typical appearance of midgut epithelial cells with moderately developed RER and lysosomes. Such pictures suggest certain mitotic activity of differentiated epithelial cells. The excretory organ in most trombidiform mites eliminates fecal material coming from the anterior midgut as well as the end products of nitrogeneous metabolism (guanine) (Alberti and Coons, 1999). In contrast to that in tetranychid mites (Alberti and Crooker, 1985; Mothes-Wagner, 1985), the excretory organ of S. fringilla does not show a high transport activity, as the epithelial cells lack both
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a basal labyrinth and a typical brush border. The most striking feature of the epithelial cells is the presence of numerous Golgi bodies and membrane-bounded inclusions varying in shape and density. The inclusions are formed by the fusion of different Golgi vesicles and vacuoles, and gradually transform into the mature granules of very high density. The mature granules seem to contain a guanine component as they strongly resemble such inclusions described in other Acari (McEnroe, 1961). The guanine-containing granules are finally released into the gut lumen by means of eccrine extrusion. But in the specimens, where the excretory organ looked especially active with its lumen enlarged by the waste products, apocrine secretion was noted. The number of waste products in the lumen of the excretory organ varies greatly in different specimens (Figs. 7a,b) suggesting periodicity of their evacuation. Acknowledgments The author is deeply grateful to Dr. A.V. Bochkov and Dr. S.V. Mironov (Zoological Institute, Russian Academy of Sciences, Saint Petersburg, Russia) for providing with the field collected material and to the engineers A.E. Tenison and P.I. Genkin (from the same Institute) for their technical assistance. The work was supported by the Russian Foundation for Basic Research; grant No 06-04-48538-a and No 08-04-90412 Ukr_a. References Akimov, L.A., Gorgol, V.T., 1990. Predatory and Parasitic Mites – Cheyletidae. Naukova Dumka, Kiev, 120 p, In Russian. Alberti, G., 1973. Erna¨hrungsbiologie und Spinnvermo¨gen der Schnabelmilben } r Morphologie der Tiere 76, 285–338. (Bdellidae, Trombidiformes). Zeitschrift fu Alberti, G., Coons, L.B.,1999. In: Harrison, F.W., Foelix, R.F. (Eds.), Microscopic Anatomy of Invertebrates. Acari: Mites, vol. 8C. Wiley-Liss, New York, pp. 515–1217. Alberti, G., Crooker, A.R., 1985. In: Helle, W., Sabelis, M.V. (Eds.), Spider Mites. Their Biology, Natural Enemies and Control. Internal anatomy, vol. 1A. Elsevier, Amsterdam, pp. 29–62. Alberti, G., Storch, V., 1983. Zur Ultrastruktur der Mitteldarmdru¨sen von Spinnentieren (Scorpiones, Araneae, Acari) unter verschiedenen Erna¨hrungsbedingungen. Zoologischer Anzeiger 3/4, 145–160. Alberti, G., Seniczak, A., Seniczak, S., 2003. The digestive system and fat body of an early-derivative oribatid mite, Archegozetes longisetosus, Aoki (Acari: Oribatidae, Trhypochthoniidae). Acarologia 43, 149–219. Becker, A., Peters, W., 1985a. Fine structure of the midgut gland of Phalangium opilio (Chelicerata, Phalangida). Zoomorphology 105, 317–325. Becker, A., Peters, W., 1985b. The ultrastructure of the midgut and the formation of peritrophic membranes in a harvestman, Phalangium opilio (Chelicerata Phalangida). Zoomorphology 105, 326–332.
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Blauvelt, W.E., 1945. The internal anatomy of the common red spider mite (Tetranychus telarius L.). Cornell University Experiment Station Memoirs 270, 1–46. Ehrnsberger, R., 1984. Anatomie des Verdauungstraktes der Rhagidiidae (Acari, Trombidiformes). Osnabru¨cker Naturwissenschaftliche Mitteilungen 11, 67–90. Filimonova, S.A., 2001. The fine structure of the midgut in the mite Myobia murismusculi. Tsytologia 43, 425–431. In Russian, with English summary. Filimonova, S.A., 2008a. The fine structure of the midgut in the mite Anystis baccarum (L.) (Acari, Actinedida: Anystidae). Arthropod Structure and Development 37, 299–309. Filimonova, S.A., 2008b. Peculiarity of the internal anatomy in the mite family Syringophilidae by the example of Syringophilopsis fringilla a parasite of chaffinch. Parazitologia 42, 395–404 (In Russian, with English summary). Goyffon, M., Martoja, R., 1983. Cytophysiological aspects of digestion and storage in the liver of scorpion Androctonus australis (Arachnida). Cell and Tissue Research 228, 661–675. Herman, W.S., Preus, D.M., 1972. Ultrastructure of the hepatopancreas and associated tissues of the chelicerate arthropod, Limulus polyphemus. Zeitschrift fu¨r Zellforschung 134, 255–271. Lopez, A., 1983. Etat de nos connaissance sur l’Araigne`e Telema tenella Simon (Telemidae), ‘‘fossile vivant’’ du Canigou. Bulletin de la Socie´te´ d’Etude des Sciences Naturelles de Be´ziers 9, 20–28. Ludwig, M., Alberti, G., 1988. Digestion in spiders: histology and fine structure of the midgut gland of Coelotes terrestris (Agelenidae). Journal of Submicroscopical Cytology and Pathology 20, 709–718. Ludwig, M., Alberti, G., 1990. Peculiarities of arachnid midgut glands. Acta Zoologica Fennica 190, 255–259. Ludwig, M., Alberti, G., 1992. Fine structure of the midgut of the Prokoenenia wheeleri (Arachnida: Palpigradi). Zoologische Beitra¨ge 34, 127–134. McEnroe, W.D., 1961. Guanine excretion by the two-spotted spider mite Tetranychus telarius. Annals of the Entomological Society of America 54, 925–926. Mitchell, R., 1970. The evolution of a blind gut in trombiculid mites. Journal of Natural History 4, 221–229. Mothes, U., Seitz, K., 1981. Functional microscopic anatomy of the digestive system of Tetranychus urticae (Acari, Tetranychidae). Acarologia 22, 257–270. Mothes-Wagner, U., 1985. Fine structure of the ‘hindgut’ of the two-spotted spider mite, Tetranychus urticae, with special reference to origin and function. Experimental and Applied Acarology 1, 253–272. Shatrov, A.B., 1989. The principal peculiarities of the midgut structure of the active stages of the life cycle of trombiculid mites (Acariformes, Trombiculidae). Zoologichesky Zhurnal 68, 28–37. In Russian. Shatrov, A.B., 2003. Comparative midgut ultrastructure of unfed larvae and adult mites of Platytrombidium fasciatum (C.L. Koch, 1836) and Camerotrombidium pexatum (C.L. Koch, 1837) (Acariformes: Microtrombidiidae). Arthropod Structure and Development 32, 227–239. Sˇobotnik, J., Alberti, G., Weyda, F., Hubert, J., 2008. Ultrastructure of the digestive tract in Acarus siro (Acari, Acaridida). Journal of Morphology 269, 54–71. Thor, S., 1904. Recherches sur l’anatomie compare´e des Acariens prostigmatiques. Annales des Sciences Naturelles. Zoologie se´rie 8 (19), 1–190. Vistorin, H.E., 1980. Erna¨hrungsbiologie und Anatomie des Verdauungstraktes der Nicoletiellidae (Acari, Trombidiformes). Acarologia 21, 204–215. Vistorin-Theis, G., 1978. Anatomische Untersuchungen an Calyptostomiden (Acari, Trombidiformes). Acarologia 19, 242–257.
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The ultrastructural investigation of the midgut in the quill mite Syringophilopsis fringilla (Acari, Trombidiformes: Syringophilidae) S.A. Filimonova* Zoological Institute, Parasitology, Russian Academy of Sciences, Universitetskaya Embankment 1, 199034 St. Petersburg, Russia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 October 2008 Accepted 27 January 2009
The midgut of the females of Syringophilopsis fringilla (Fritsch) composed of anterior midgut and excretory organ (¼posterior midgut) was investigated by means of light and transmission electron microscopy. The anterior midgut includes the ventriculus and two pairs of midgut caeca. These organs are lined by a similar epithelium except for the region adjacent to the coxal glands. Four cell subtypes were distinguished in the epithelium of the anterior midgut. All of them evidently represent physiological states of a single cell type. The digestive cells are most abundant. These cells are rich in rough endoplasmic reticulum and participate both in secretion and intracellular digestion. They form macropinocytotic vesicles in the apical region and a lot of secondary lysosomes in the central cytoplasm. After accumulating various residual bodies and spherites, the digestive cells transform into the excretory cells. The latter can be either extruded into the gut lumen or bud off their apical region and enter a new digestive cycle. The secretory cells were not found in all specimens examined. They are characterized by the presence of dense membrane-bounded granules, 2–4 mm in diameter, as well as by an extensive rough endoplasmic reticulum and Golgi bodies. The ventricular wall adjacent to the coxal glands demonstrates features of transporting epithelia. The cells are characterized by irregularly branched apical processes and a high concentration of mitochondria. The main function of the excretory organ (posterior midgut) is the elimination of nitrogenous waste. Formation of guanine-containing granules in the cytoplasm of the epithelial cells was shown to be associated with Golgi activity. The excretory granules are released into the gut lumen by means of eccrine or apocrine secretion. Evacuation of the fecal masses occurs periodically. Mitotic figures have been observed occasionally in the epithelial cells of the anterior midgut. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Mites Ultrastructure Midgut
1. Introduction Acariform mites (order Acariformes ¼ Actinotrichida) represent a great variety of different small arthropods, including parasites of man and many domestic animals. This attracts special attention to the morphological and physiological aspects of their feeding behavior and digestion. It is well known that in the majority of Chelicerata the secretory product of salivary glands serves for the preoral digestion of the meal. Inside the digestive tract the most important events occur in the midgut, where intracellular digestion predominates. The midgut usually consists of the anterior and posterior portions. In most acariform mites special secretory cells are absent and the anterior midgut (ventriculus and midgut caeca) is composed of polyfunctional epithelial cells (Alberti and Coons, 1999). The exceptions are several families of predatory mites, all of
* Corresponding author. Tel.: þ7 0812 714 0151; fax: þ7 0812 714 0444. E-mail address: fi[email protected] 1467-8039/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.asd.2009.01.002
which belong to the suborder Trombidiformes: Bdellidae (Alberti, 1973; Alberti and Storch, 1983), Labidostommatidae (Vistorin, 1980), and Anystidae (Filimonova, 2008a). In the suborder Astigmata secretory cells have never been detected ultrastructurally (Alberti and Coons, 1999). In Acarus siro the cells producing the peritrophic matrix were also detected as a special cell type (Sˇobotnik et al., 2008). The posterior midgut forms the excretory pellets and evacuates them through the hindgut and anal opening, if these organs are present (Alberti and Coons, 1999). In the astigmatic mites the posterior midgut is divided into the colon and postcolon. Malpighian tubules enter the gut at the border of these organs. In trombidiform mites, Malpighian tubules are absent, the colon tends to be reduced, and the postventricular midgut is represented by the dorsomedian excretory organ (Ehrnsberger, 1984; Alberti and Coons, 1999). In several trombidiform taxa, for instance in the cohort Parasitengona, the excretory organ has no connection with the ventriculus (Mitchell, 1970; Vistorin-Theis, 1978; Shatrov, 1989, 2003). The excretory organ of these mites was considered to be
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a functional analog to Malpighian tubules (Mitchell, 1970). Though the connection between the two parts of the midgut was later shown in most trombidiform taxa (Blauvelt, 1945; Alberti, 1973; Vistorin, 1980; Mothes and Seitz, 1981; Ehrnsberger, 1984; Alberti and Crooker, 1985; Mothes-Wagner, 1985; Akimov and Gorgol, 1990; Filimonova, 2001, 2008a,b), the origin and functioning of the excretory organ is still unclear (Mothes-Wagner, 1985). A serious problem is the little information available about the cell replacement in the midgut epithelium of acariform mites. Special regenerative cells, as well as mitotic divisions, have recently been found in Acarus siro (Sˇobotnik et al., 2008). It seems to be an exception or a characteristic of astigmatic mites only, as in trombidiform species no mitotic divisions have ever been observed, though many authors described the extrusion of epithelial cells or part of them into the gut lumen, and their subsequent degeneration (Blauvelt, 1945; Alberti, 1973; Ehrnsberger, 1984; Alberti and Crooker, 1985; Akimov and Gorgol, 1990; Alberti and Coons, 1999). Taking into account the problems mentioned above and the great diversity of internal organization of acariform mites, the investigation of possibly many groups of these animals seems to be necessary. The aim of the present paper is to describe peculiarities of the morphology and physiology of the midgut in quill mites (Trombidiformes: Syringophilidae), which live inside the shaft of bird flight feathers and feed on tissue fluids by piercing the wall of the quill with their chelicerae. The internal anatomy of these mites has been recently studied for the first time (Filimonova, 2008b). 2. Materials and methods Females of Syringophilopsis fringilla (Fritsch) were collected from the chaffinch, Fringilla coelebs Linnaeus (Passeriformes: Fringillidae), captured for ringing in the bird-banding station ‘‘Rybachy’’ (Russia, Kaliningrad Province) in autumn of 2006. Having been taken out of the host, quill mites were put into 2.5% glutaraldehyde solution in 0.01 M phosphate buffer (pH 7.4). For better penetration of the fixative solution the cuticle of the body was pierced in some places. After that the specimens were transferred into fresh fixative solution for some days and then postfixed in 2% osmium tetroxide prepared in the same buffer. Material was embedded in Epon 812. The semithin sections for light microscopy were stained with a mixture of methylene blue and Azur II (pH ¼ 6.8). Ultrathin sections stained with uranyl acetate and lead citrate were studied using Zeiss LEO 900 and TESLA BS 500 electron microscopes. In whole 10 females were studied. 3. Results The midgut of Syringophilopsis fringilla consists of the two portions, the anterior and posterior midgut. The anterior midgut includes the unpaired ventriculus and two pairs of diverticuli: anterior and posterior midgut caeca. The posterior midgut is represented by a long tubular excretory organ, connected to the hindgut at the end of the mite body (Figs. 1a,b). 3.1. Anterior midgut The anterior midgut occupies all the free space of the body cavity. The central unpaired ventriculus is situated just behind the synganglion. It sends forwards and backwards two pairs of caeca (Figs. 1a,b). Apart from the ventral wall of the ventriculus adjacent to the coxal glands, all the remaining anterior midgut is lined by the same single-layered epithelium (Fig. 1b). The epithelium is underlain by a very thin basal lamina. Dorsally, large skeletal muscles are apposed to the ventriculus; in the other parts of the midgut, single muscle bands can be visible in some places. Both, the midgut
Fig. 1. Schematic drawings of the digestive tract in Syringophilopsis fringilla, showing the location of the modified midgut epithelium (hatched area). (a) Dorsal view, (b) lateral view. A – anus; AC – anterior caeca; Es – esophagus; EO – excretory organ; HG – hindgut; PC – posterior caeca; Ph – pharynx; Sy – synganglion; V – ventriculus.
epithelium and the surrounding muscle cells form thin processes directed to the body cavity. No signs of connective tissue were found around the midgut. In most specimens studied, the midgut lumen was completely filled with the extensions and fragments of the epithelial cells loaded by numerous basophilic inclusions and vacuoles (Figs. 2a,b). The epithelial lining varies in thickness from place to place, due to the different height of the epithelial cells (Fig. 2a). The nuclei of most epithelial cells are elongate, with the large diameter about 5–6 mm. They are typically electron-lucent and possess two or three extensively developed nucleoli, each up to 2.5 mm in size. In several cases, the nuclei contained condensed chromatin in a form resembling chromosomes. These nuclei seem to be in prophase, as nuclear envelope partly exists (Fig. 2c). Metaphase pictures were also observed at the light microscope level, while no evident anaphases were found. Dumbbell-shaped nuclei were also regularly observed in all the mites examined (Fig. 2d). They are measuring about 8–9 mm in diameter. Both kinds of unusual nuclei occur in cells lacking any special features and having RER cisterns, lysosome-like inclusions, lipid droplets, as well as glycogen rosettes (Figs. 2c,d). The ultrastructural investigation shows that the apical surface of the midgut epithelium lacks a true brush border. Locally, small apical processes can be found, but they do not have internal filaments typical of microvilli (Figs. 2b,e). Glycogen particles are common inside these processes, as well as in the cytoplasm of the cells. The lamellae delaminating from the apical surface of the cells are often observed free in the gut lumen (Fig. 2e). A basal labyrinth is absent. Mitochondria do not concentrate in any part of the cell.
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Fig. 2. General morphology of the midgut epithelium visible on the LM (a) and EM (b–e) levels. (a) Cross semithin section of the ventriculus with the coxal glands (CG) invading into the ventricular epithelium (E) ventrally. (b) Midgut lumen (Lu) occluded by the remnants of the epithelial cells. (c) Nucleus (N) with condensing chromatin (Ch) belonging to differentiated epithelial cell. (d) Dumbbell-shaped nucleus (N) with several nucleoli (Nu). (e) Formation of macropinocytotic vesicles (PV) on the apical surface of the digestive cells. Scale bars: a ¼ 15 mm, b ¼ 1.5 mm, c, d ¼ 2 mm, e ¼ 1.0 mm. AP – apical processes; Cu – cuticle; Ep – epidermis; F – cell fragments floating in the gut lumen; Gly – glycogeneous granules; Hly – heterolysosomes; L – lipids; Lm – surface lamellae; RER – rough endoplasmic reticulum; SII – spherites of the second type.
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Most of the lateral surface of the neighboring epithelial cells shows deep interdigitations. In the apical region the epithelial cells are connected by a zonula adherens followed by septate desmosomes below. In the middle region some isolated septate desmosomes were seen. Ultrastructural analysis shows four kinds of epithelial cells in the anterior midgut (Fig. 3). The most common are the digestive cells, which may be referred to as the light (L) and dark (D) cells based on the density of their cytoplasm (Figs. 2b, 3 and 4a). Both of these cells may be either flat or columnar and are characteristically
rich in rough endoplasmic reticulum (RER). The L cell is characterized by swollen RER cisternae with electron-lucent content (Figs. 3 and 4b), while in the D cell, flat RER patterns are packed more tightly in the cytoplasm, and contain material of moderate electron density (Figs. 3 and 4a). Both cell types possess numerous small Golgi bodies commonly forming an indistinct Golgi field, which is connected to the RER. Secretory granules originating from the Golgi body lie nearby (Fig. 4a). The mature secretory granules represent spherical inclusions 0.5 mm in size with homogeneous contents of moderate or high electron density (Fig. 4b). They are formed by the
Fig. 3. Scheme, showing the main kinds of epithelial cells in the anterior midgut of Syringophilopsis fringilla and their possible relationships (arrows). E1, E2 – sequential stages of the excretory cell development; F – cell fragment, freely floating in the gut lumen. D1, D2 – sequential stages of differentiation of the dark-stained digestive cell, L –the light-stained digestive cell; S – the secretory cell. AP – apical processes; BL – basal lamina; Gly – glycogeneous granules; Hly – heterolysosomes; L – lipids; M – mitochondria; N – nucleus; Nu – nucleolus; PV – pinocytotic vesicles; R – ribosomes; RB – residual body; RER – rough endoplasmic reticulum; SI, SII – spherites of the first and second types; SG – secretory granules.
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Fig. 4. Fine structure of the digestive and excretory cells of Syringophilopsis fringilla. (a) The border between a dark (D) (to the left) and a light (L) (to the right) digestive cell (arrows). (b) General view of L cell. (c) D2 cell with the basal region rich in RER and the apical part loaded with heterolysosomes (Hly) and spherites (SII). (d) Crystalline inclusions (CI) in the lumen of the midgut caeca (Lu). The apical region of an E1 cell is also visible. (e) The apical region of an E2 cell (E), and a cell fragment (F) floating in the lumen of the ventriculus (Lu). G – Golgi bodies; N – nucleus; Nu – nucleolus; RER – rough endoplasmic reticulum; SI, SII – spherites; SG – secretory granule. Scale bars: a ¼ 0.5 mm, b, c ¼ 2 mm, d, e ¼ 3 mm.
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fusion of the primary granules and commonly lie not far from the Golgi zone (Figs. 4a,b). In both L and D cells large lipid droplets are always present, as well as a great number of glycogen particles (a-form). The latter can be seen either inside the large electronlucent vacuoles or lying freely in the cytoplasm. The D cell population is not uniform, composed of the most common flat D1 cells and columnar D2 cells, which contain more or less electron-lucent vacuoles in the apical region (Fig. 3). These vacuoles, ranging from 0.5 to 1.2 mm in diameter, originate from the apical invaginations or by the fusion of the tops of the apical processes and may be considered as macropinocytotic vesicles (Fig. 2e). In the cytoplasm, the vacuoles join and form larger vesicles with variable heterogeneous material inside, which probably represents a kind of heterolysosomes (Figs. 3 and 4c). Intermediate states between L and D1 cells are often observed being characterized by the relatively dark cytoplasm with swollen RER cisternae, not so abundant as in D1 cells. The third kind of the epithelial cells is the excretory cell (E) characterized by the apical region extended into the gut lumen (Figs. 3 and 4c–e). RER and Golgi bodies are considerably less numerous in this region, which is filled by various residual bodies, heterolysosomes and spherites. The high concentration of spherites is the most striking feature of this cell (Figs. 4c,d). They seem to be formed inside certain vacuoles (or may be heterolysosomes) by gradual concentration of dense material from the center to the periphery. Two kinds of such inclusions are clearly distinguishable: an electron-lucent inclusion with concentric layers of different electron density (SI type), and that with a highly dense granule inside, which tends to be partly destroyed during preparation (S II type) (Figs. 4c,d). Both kinds of spherites are of the same size, ranging from 2 to 3 mm, and are clearly identified at LM level owing to their birefringence. Both of them were shown to be released into the gut lumen by eccrine secretion. The E cell population can be divided into two subtypes, E1 and E2 cells. The E1 cell has the basal region similar to that in the typical D1 cell, including conspicuous RER and Golgi bodies (Figs. 3 and 4c), whereas the E2 cell is completely full of large vacuoles, heterolysosomes and spherites (Fig. 3), practically lacking RER cisternae and Golgi bodies. The most striking feature of E2 cells is the presence of a huge apical vacuole, apparently formed by the junction of the smaller vacuoles typical of E1 cells (Fig. 4e). Cell fragments with the same vacuoles surrounded by numerous spherites were commonly visible in the midgut lumen of all specimens (Figs. 2a, 3 and 4e). Occasionally, pycnotic nuclei were found inside them. Crystalline particles, typically observed in the gut lumen, are believed to be derivates of the spherites II (Fig. 4d). The secretory cells (S cells) were found only in two specimens (from 10 females examined in the present study) (Fig. 3). These cells contain an extremely large amount of RER in the form of long narrow cisternae tightly packed all over the cytoplasm, and numerous dense membrane-bounded granules (Figs. 3 and 5a–d). The nucleus is larger than in other cell types, measuring 9–10 mm in its large dimension. A single nucleolus is up to 7 mm in diameter (Fig. 5a). The apical surface of the S cell may be either bare (Fig. 5b) or with long thin microvilli-like processes lacking internal filaments (Fig. 5d). Golgi bodies are represented by a large heap of small vesicles originating from RER cisternae. The primary secretory granules look similar to those in the digestive cells (L and D1) (Fig. 5c). They fuse with each other to form the mature secretory granules which are typically about 2 mm in size (Fig. 5a). Cells containing larger inclusions (4 mm in size) were also seen (Fig. 5b). Releasing of the secretory product was not observed. Lipid and glycogen inclusions are fewer than in other cell types, as well as vacuoles and spherites.
In the specimens, where secretory cells were not detected, single secretory granules of similar appearance were occasionally observed in other kinds of midgut cells (except for E2 cells). In the ventral part of the ventriculus, the epithelium forms large folds surrounding the proximal tube of the coxal gland (Fig. 2a). The epithelial layer adjacent to the tubes is very low in most places (sometimes not more than 1 mm in height), closely resembling connective tissue (Figs. 6a,b). The cells possess numerous branching apical processes with deep invaginations at their base. Pinocytotic vesicles of various size and shape are formed at the apical surface of these cells; their content is electron-lucent (Figs. 6c). The cytoplasm of the cells is of low electron density, lacking most inclusions, which are typical to the rest of the midgut epithelium (heterolysosomes, secretory granules or spherites) (Fig. 6a). RER is poorly-developed and Golgi bodies are rarely visible. The main component of the cytoplasm is numerous mitochondria with dense matrix and narrow cristae (Figs. 6a,b). The basal protrusions of the midgut cells may be very long and thin; however, they do not reach the coxal gland epithelium, as they never penetrate the midgut basal lamina.
3.2. Posterior midgut The posterior midgut or excretory organ is a long tube running backwards from the short, posterior extension of the ventriculus (Figs. 1a,b and 7a). At the border to the ventriculus the excretory organ forms a valve, composed of higher epithelial cells (Fig. 7b). In other regions the epithelial lining is very thin, in some places not more than 1.5 mm and about 3–4 mm at the nuclear site (Figs. 7c,d). The basal lamina is as thin as in the anterior midgut, but muscles are more numerous and are represented by relatively thick longitudinal bundles (Fig. 7c). The volume of the lumen varies greatly, relating to the quality of excretes. The free floating fragments of the epithelial cells in the lumen of the organ are very similar to those seen in the anterior midgut. Occasionally pycnotic nuclei can be found inside them (Fig. 7e). The anterior part of the excretory organ is lined by an epithelium with apical processes, which are more regular and abundant than in the anterior midgut (Fig. 7c). In the posterior part of the organ these processes disappear, but the apical surface of the cells remains irregularly shaped with sharp edges visible in a section. The apical plasmalemma is covered by a thin superficial layer of high density (Fig. 7d). The cells are predominantly flat with their lateral sides strongly interdigitated. The elongate nuclei lie along the basal lamina and usually possess one or two prominent nucleoli. The basal plasmalemma is smooth; the number of mitochondria is higher than in the anterior midgut, though they do not concentrate in any region. Some of them are up to 2 mm long. RER is not conspicuous being represented by single short cisternae. Numerous small Golgi bodies can be observed in all parts of the cytoplasm. Each of them commonly shows an ordinary stack of flat cisternae producing very small moderately dense vesicles (Fig. 7d). Joining together, these vesicles give rise to larger irregularly shaped inclusions of the same density. The latter gradually transform into granules of a very high density with a crystalline component inside, typically being destroyed during EM preparation (Fig. 7c). Both, the whole granules and their crystalline fragments are clearly visible in the lumen of the organ. Their releasing occurs in the form of eccrine or apocrine secretion. The latter was found in the specimens, where the lumen of the organ was enlarged with a great amount of waste products. In
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Fig. 5. Fine structure of the secretory cells in the anterior midgut of Syringophilopsis fringilla. (a) A central part of S cell with large nucleus (N) and numerous secretory granules (SG). Note RER cisternae filling the cytoplasm. (b) The apical portions of two S cells facing each other. (c) A heap of Golgi vesicles marks indistinct Golgi complexes (G), where primary secretory granules (PSG) are forming. (d) Microvilli-like apical processes on the luminal surface of the S cell. Scale bars: a, b ¼ 2 mm, c ¼ 1 mm, d ¼ 1.5 mm. Lu – the midgut lumen; Nu – nucleolus.
this case, the same secretory products are also released into the intercellular spaces. Irregularly shaped vacuoles (0.2–0.3 mm in diameter), glycogen and lipid inclusions are also common in the epithelial cells, as well as spherites and myeline bodies.
4. Discussion As it was shown in the previous paper (Filimonova, 2008b), the digestive tract of S. fringilla is typical to that in the cohort Eleutherengona: the anterior midgut is in open connection with the
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Fig. 6. Fine structure of the ventricular epithelium in the region adjacent to the coxal gland. (a) A common view of the fold of the ventriculus with the large E cell on its one side and a very thin epithelial lining on the opposite side attached to the coxal gland (CG). (b) Thin ventricular epithelium at a higher magnification. (c) The branching apical processes (AP) probably associated with the formation of pinocytotic vesicles (PV). Scale bars: a ¼ 2 mm, b, c ¼ 1.5 mm. AP – apical processes; E – ventricular epithelium; Hly – heterolysosomes; Lu – the lumen of the ventriculus; M – mitochondria; N – nucleus; PV – pinocytotic vesicles; SI, SII – spherites.
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Fig. 7. The structure of the excretory organ in Syringophilopsis fringilla. (a) Frontal semithin section through the posterior part of the mite body. The lumen of the excretory organ (EO) is wide. (b) The most anterior part of the excretory organ. (c) An epithelial cell of the anterior portion of the excretory organ with mature crystalline granule inside. (d) Golgi complex (G) in an epithelial cell of the posterior portion of the organ. Note irregular secretory granules (SG) in close contact with the Golgi vesicles. (e) A cell fragment (F) in the lumen of the organ, containing a pycnotic nucleus (N). Scale bars: a ¼ 50 mm, b, e ¼ 2 mm, c ¼ 0.5 mm, d ¼ 1 mm. AP – apical processes; CI – crystalline inclusions; E – the epithelium of the organ; G – Golgi body; Lu – the lumen of the organ; ML – muscle layer; N – nucleus, Nu – nucleolus; PC – posterior caecum; SC – surface coat; SII – spherites.
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postventricular region represented by a simple dorsomedian tube known as the excretory organ (Alberti and Coons, 1999). 4.1. Anterior midgut The data obtained in the present study show that the epithelium of ventriculus and caeca is uniform except for the region adjacent to the coxal glands. The cells of the latter region seem to be not involved in the digestion as they are lacking heterolysosomes. They demonstrate most features of transporting epithelia: a complex system of apical processes accompanied by a great number of large mitochondria. The main function of this region is evidently osmoregulation. A very similar modification of the midgut epithelium has been found in tetranychids both near the coxal gland and in the region closely attaching to the excretory organ (Mothes and Seitz, 1981; Mothes-Wagner, 1985; Alberti and Crooker, 1985). In some trombidiform species, finger-like protrusions of coxal gland cells were observed passing through the basal laminae of both midgut and coxal gland and entering the basal portion of the midgut epithelium (Alberti and Coons, 1999; Filimonova, 2001). In S. fringilla, such protrusions were not seen. Other regions of the anterior midgut do not demonstrate high transport activity: the cells lack a distinct brush border, as well as a basal labyrinth. Short apical processes seem to appear or disappear on the surface of the epithelial cells depending on their physiological state. Ultrastructural analysis shows four kinds of epithelial cells in the anterior midgut (Fig. 3). In all specimens examined, digestive L and D1 cells were most common, both rich in RER cisternae and Golgi bodies producing small secretory vesicles. L cells are characterized by less numerous RER and probably represent the precursors of the flat D1 cells. Two directions of D1 cell development can be suggested according to the observations obtained in the present study (Fig. 3). The first one leads to the formation of the secretory cells (S cells) with hypertrophied RER and Golgi bodies, producing dense, membrane-bounded granules of proteinaceous nature. In the specimens where S cells were not noted single granules of the same appearance were indicated in the other cell types. This leads to the conclusion that the secretory cells temporarily exist in the syringophilid midgut, associated with a certain stage of digestion. In the horseshoe crab Limulus polyphemus, the accumulation of the secretory granules was demonstrated in the midgut gland of starved animals, but these granules were quickly released into the gut lumen just after the meal (Herman and Preus, 1972). A very similar sequence of events was shown during digestion in some spiders (Alberti and Storch, 1983; Ludwig and Alberti, 1988). Fast liberating of the secretory granules in answer to the food uptake may be the cause, why the secretory cells were not observed in most trombidiform mites (Alberti and Coons, 1999). Up to date, they have been demonstrated ultrastructurally only in the representatives of Bdellidae (Alberti, 1973; Alberti and Storch, 1983) and Anystidae (Filimonova, 2008a). In both cases secretory cells are characterized by conspicuous RER and large dense granules with homogeneous content corresponding to the S cells found in S. fringilla. It is interesting to note that in other arachnids the secretory granules are also very large, measuring from 3 to 6 mm (Herman and Preus, 1972; Goyffon and Martoja, 1983; Becker and Peters, 1985a; Ludwig and Alberti, 1988, 1990, 1992), which is comparable to those found in S. fringilla (where they are up to 4 mm in diameter). Most authors consider the secretory product to be the enzymes for extracellular digestion, which is followed by the intracellular one (Herman and Preus, 1972; Becker and Peters, 1985a; Ludwig and Alberti, 1988, 1990, 1992; Alberti and Coons, 1999).
The second direction of D1 cell differentiation is the typical digestive cell (D2) (Fig. 3) with its apical region usually containing macropinocytotic vesicles of different size and various vacuoles similar to those detected as heterolysosomes in many arachnid taxa (Alberti and Storch, 1983; Becker and Peters, 1985a,b; Ludwig and Alberti, 1988, 1990, 1992; Shatrov, 1989, 2003; Filimonova, 2001, 2008a). Transformation of these vacuoles into typical residual bodies was frequently observed during the study. The last stage of D2 cell development is represented by the excretory cell (E2 cell), which is completely filled with vacuoles, residual bodies and spherites (Fig. 7). Two types of spherites are clearly distinguishable. The type I is mineral spherites characterized by concentric layers of different electron density. The II type contains a sphere of high electron density, which is typically destroyed during preparation. Being released into the gut lumen, the second type of spherites gives rise to crystalline waste products. The same crystalline particles were typically observed in the excretory organ of S. fringilla and seem to be guanine granules, which look birefringent in light microscope. Similar guanine-containing spherites have been indicated in some scorpions, spiders and oribatid mites (Goyffon and Martoja, 1983; Lopez, 1983; Alberti et al., 2003), but they seem to be more widely dispersed among Chelicerata. Both excretory cells and their apical portions were shown to be extruded into the gut lumen contributing to the formation of free floating cell fragments with high concentration of waste products inside. They typically contain a single huge vacuole, where digestion is being continued. Similar pictures have been observed in most trombidiform mites studied up to now. The authors described free floating cell fragments either as derivatives of whole cells (Thor, 1904; Vistorin, 1980; Mothes and Seitz, 1981; Ehrnsberger, 1984; Alberti and Crooker, 1985) or of their apical region (Shatrov, 1989, 2003; Filimonova, 2001, 2008a). In the case of S. fringilla, the presence of pycnotic nuclei in several cell fragments suggests elimination of the whole cells. On the other hand, in many cells with accumulated waste products in their apical region, the rest of the cytoplasm is typical for D cells. This suggests that in most cases only the apical zone of D2 cells is extruded, and thereafter the lost region regenerates. Thus, D cells appear to go through several digestive cycles before they completely change into the E2 cells and die in the gut lumen. The same conclusion was made by Becker and Peters (1985a), who carefully studied different stages of digestion in the midgut glands of opilionids (Chelicerata: Phalangida). This way of cell regeneration appears to occur, when cell divisions are impossible by some reasons. This seems to be typical for most trombidiform mites. Contrary to astigmatic mites (Sˇobotnik et al., 2008), in most trombidiform mites, the regenerative cells are characteristically absent (Alberti, 1973; Vistorin, 1980; Mothes and Seitz, 1981; Alberti and Crooker, 1985; Filimonova, 2001, 2008a; Shatrov, 2003). Though undifferentiated cells have been described in some taxa (Shatrov, 1989; Akimov and Gorgol, 1990), no mitotic figures have been commonly demonstrated. During the present study several prophases and metaphases have been detected in the midgut epithelium of S. fringilla at the light microscope level. Midgut cells with condensed chromatin resembling chromosomes were shown ultrastructurally in the anterior midgut. These cells had the typical appearance of midgut epithelial cells with moderately developed RER and lysosomes. Such pictures suggest certain mitotic activity of differentiated epithelial cells. The excretory organ in most trombidiform mites eliminates fecal material coming from the anterior midgut as well as the end products of nitrogeneous metabolism (guanine) (Alberti and Coons, 1999). In contrast to that in tetranychid mites (Alberti and Crooker, 1985; Mothes-Wagner, 1985), the excretory organ of S. fringilla does not show a high transport activity, as the epithelial cells lack both
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a basal labyrinth and a typical brush border. The most striking feature of the epithelial cells is the presence of numerous Golgi bodies and membrane-bounded inclusions varying in shape and density. The inclusions are formed by the fusion of different Golgi vesicles and vacuoles, and gradually transform into the mature granules of very high density. The mature granules seem to contain a guanine component as they strongly resemble such inclusions described in other Acari (McEnroe, 1961). The guanine-containing granules are finally released into the gut lumen by means of eccrine extrusion. But in the specimens, where the excretory organ looked especially active with its lumen enlarged by the waste products, apocrine secretion was noted. The number of waste products in the lumen of the excretory organ varies greatly in different specimens (Figs. 7a,b) suggesting periodicity of their evacuation. Acknowledgments The author is deeply grateful to Dr. A.V. Bochkov and Dr. S.V. Mironov (Zoological Institute, Russian Academy of Sciences, Saint Petersburg, Russia) for providing with the field collected material and to the engineers A.E. Tenison and P.I. Genkin (from the same Institute) for their technical assistance. The work was supported by the Russian Foundation for Basic Research; grant No 06-04-48538-a and No 08-04-90412 Ukr_a. References Akimov, L.A., Gorgol, V.T., 1990. Predatory and Parasitic Mites – Cheyletidae. Naukova Dumka, Kiev, 120 p, In Russian. Alberti, G., 1973. Erna¨hrungsbiologie und Spinnvermo¨gen der Schnabelmilben } r Morphologie der Tiere 76, 285–338. (Bdellidae, Trombidiformes). Zeitschrift fu Alberti, G., Coons, L.B.,1999. In: Harrison, F.W., Foelix, R.F. (Eds.), Microscopic Anatomy of Invertebrates. Acari: Mites, vol. 8C. Wiley-Liss, New York, pp. 515–1217. Alberti, G., Crooker, A.R., 1985. In: Helle, W., Sabelis, M.V. (Eds.), Spider Mites. Their Biology, Natural Enemies and Control. Internal anatomy, vol. 1A. Elsevier, Amsterdam, pp. 29–62. Alberti, G., Storch, V., 1983. Zur Ultrastruktur der Mitteldarmdru¨sen von Spinnentieren (Scorpiones, Araneae, Acari) unter verschiedenen Erna¨hrungsbedingungen. Zoologischer Anzeiger 3/4, 145–160. Alberti, G., Seniczak, A., Seniczak, S., 2003. The digestive system and fat body of an early-derivative oribatid mite, Archegozetes longisetosus, Aoki (Acari: Oribatidae, Trhypochthoniidae). Acarologia 43, 149–219. Becker, A., Peters, W., 1985a. Fine structure of the midgut gland of Phalangium opilio (Chelicerata, Phalangida). Zoomorphology 105, 317–325. Becker, A., Peters, W., 1985b. The ultrastructure of the midgut and the formation of peritrophic membranes in a harvestman, Phalangium opilio (Chelicerata Phalangida). Zoomorphology 105, 326–332.
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