- Email: [email protected]
The fine structure of the midgut in the mite Anystis baccarum (L.) (Acari, Actinedida: Anystidae)
Arthropod Structure & Development 37 (2008) 299e309 www.elsevier.com/locate/asd
The fine structure of the midgut in the mite Anystis baccarum (L.) (Acari, Actinedida: Anystidae) Svetlana A. Filimonova* Zoological Institute, Russian Academy of Sciences, 199034 St. Petersburg, Russia Received 27 June 2007; received in revised form 26 November 2007; accepted 26 November 2007
Abstract The ventriculus and the midgut caeca of the fed females of Anystis baccarum (L.) were investigated by using light and electron microscopy. In addition to the main type of polyfunctional digestive cells, special secretory cells were detected in the anterior region of the ventriculus. The shape and the ultrastructure of the digestive cells vary depending on their physiological state. Intracellular digestion, absorption or excretion processes prevail at different stages of the cell cycle. The secretory cells are characterized by the presence of extensive rough endoplasmic reticulum, filling whole space of the cell. These cells do not contain the apical network of pinocytotic canals, which are typical for the digestive cells. Three types of secretory granules were found in the cytoplasm of the secretory cells that probably correspond to three sequential stages of granulogenesis. The primary secretory granules are formed by the fusion of Golgi vesicles. The primary granules fuse to form complex vesicles with heterogeneous contents. These secondary granules aggregate to form very large inclusions of high electron density (tertiary secretory granules), which probably represent the storage of the secretory product. All types of secretory granules were observed close to the apical plasmalemma. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: Actinedida; Midgut ultrastructure; Secretory cells
1. Introduction The midgut of arachnids is generally divided into the anterior and posterior parts commonly named the anterior and posterior midgut, respectively (Alberti, 1973; Alberti and Storch, 1983; Becker and Peters, 1985; Ludwig and Alberti, 1990, 1992; Alberti and Coons, 1999). The anterior midgut, including ventriculus and midgut caeca, participates in active intracellular digestion, while the posterior midgut appears to be involved in excretion and, to some extent, in regulation of water balance. In most derived actinedid mites (such as Parasitengona) the midgut ends blindly, lacking a typical posterior midgut. Instead of the latter, a dorsomedian excretory organ exists, which opens to the outside through an uropor (Mitchell, 1964, 1970; Vistorin-Theis, 1978; Shatrov, 1989, 2003; Alberti and Coons, 1999). In the other cases the midgut * Tel.: þ7 0812 714 0151; fax: þ7 0812 714 0444. E-mail address: [email protected] 1467-8039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.asd.2007.11.005
is considered to be open, and the connection between anterior and posterior midgut is clearly demonstrated (Blauvelt, 1945; Alberti, 1973; Ehrnsberger, 1984; Vistorin, 1980; Mothes and Seitz, 1981; Alberti and Crooker, 1985; Akimov and Gorgol, 1990; Filimonova, 2001). In some species with open midgut the postventricular region is represented by a uniform tube (Mothes and Seitz, 1981; Alberti and Crooker, 1985; Akimov and Gorgol, 1990; Filimonova, 2001) in the others it is divided into colon and postcolon, which differ histologically (Alberti, 1973; Vistorin, 1980; Ehrnsberger, 1984). For most actinedid families the anatomical organization of the digestive tract is still unknown. In most examined representatives of Acari, the epithelium of the anterior midgut is composed of a single cell type, which develops from digestive to excretion cell and then undergoes degeneration as separated cell fragments in the gut lumen. Thus, most of the authors suppose the digestive cells to be polyfunctional, being involved subsequently in intracellular digestion, absorption, and excretion of waste products with
300
S.A. Filimonova / Arthropod Structure & Development 37 (2008) 299e309
each process prevailing at different stages of the cell cycle (Mothes and Seitz, 1981; Alberti and Crooker, 1985; Shatrov, 1989; Nuzzaci and Alberti, 1996; Filimonova, 2001). For most taxonomic groups of Chelicerata, such as Scorpiones (Alberti and Storch, 1983; Goyffon and Martoja, 1983), Phalangida (Becker and Peters, 1985), Araneae (Ludwig and Alberti, 1988, 1990), Uropygi, Pseudoscorpiones (Ludwig and Alberti, 1990), Palpigradi (Ludwig and Alberti, 1992), a special kind of secretory cells was detected in the midgut epithelium. However, this cell differentiation is not typical for the acariform mites (order Acariformes) studied so far. The absence of secretory cells was definitely shown for most families of actinedid mites (suborder Actinedida), such as Tetranychidae (Mothes and Seitz, 1981; Alberti and Crooker, 1985), Demodicidae (Desch, 1988), Eriophyoidea (Nuzzaci and Alberti, 1996), Calyptostomatidae (VistorinTheis, 1978), Trombiculidae (Shatrov, 1989), Myobiidae (Filimonova, 2001), Microtrombidiidae (Shatrov, 2003), as well as for some oribatid and acaridid mites (suborders Oribatida and Acaridida) (Alberti and Coons, 1999). In actinedid mites, secretory cells in the midgut epithelium were described at light microscopical level in the mites belonging to the Labidostommatidae (Vistorin, 1980), and ultrastructurally in the Bdellidae (Alberti, 1973; Alberti and Storch, 1983). In both cases these cells were provided by basophilic secretory granules, which in Bdellidae were shown to be accompanied by an extremely developed rough endoplasmic reticulum (RER). It is interesting to note, that the secretory granules of Bdellidae are extremely large (up to 9 mm), being very similar to those in other arachnids examined so far (Alberti, 1973; Ludwig and Alberti, 1990, 1992). The present paper describes the fine structure and functioning of the midgut epithelium in a predatory mite Anystis baccarum (L.). The family Anystidae is of special interest, because of the great number of primitive features common for actinedid mites (Kethley in: Norton et al., 1993). Wright and Newell (1964) investigated the midgut of Anystis in one of the first EM studies. They showed the presence of several cell categories which corresponded, according to these authors, to the gradual changing from absorptive cells to excretory ones. No specialized secretory cells were indicated in their study. In contrast to that study, the present investigation shows the existence of special secretory cells in the midgut of Anystis, with their definite position in the midgut and the fine structure clearly distinct from the other epithelial cells.
in Epon 812. The semithin sections for light microscopy were stained with a mixture of methylene blue and Azur II (rO 6.8). Ultrathin sections were stained with uranyl acetate and lead citrate. The sections were studied using a Zeiss LEO 900 electron microscope. 3. Results 3.1. General remarks The midgut is divided into a central ventriculus, two pairs of diverticula (anterior and posterior caeca) and the postventricular region, composed of a large postcolon (Figs. 1a,b and 2a). The postcolon begins posteriorly from the ventriculus, and runs backwards and ventrally to a short cuticle-lined rectum leading to the anus. The walls of the ventriculus and caeca consist of a single epithelial layer underlined by a basal lamina and muscle cells. The epithelial cells of the ventriculus and caeca are variable in shape and to some extent in size, having various nutritional inclusions in the cytoplasm, stained brightly in semithin sections. In the light microscope, the postcolon is characterized by the uniform columnar cells with no inclusions; the cells are more basophilic than those in the ventricular region. The epithelial lining of the postcolon is either folded composed of the high columnar cells, or flattened in the case when a great number of crystalline inclusions fills
2. Materials and methods Five adult females of Anystis baccarum (L.) were collected from the soil surface in the sand places in the suburbs of Kiev (Ukraine). The mites were fed on collembolans in laboratory condition under the visual control of this process and fixed 4 (two females) and 24 h (three females) after the feeding. The mite specimens were put in 2.5% glutaraldehyde solution in 0.05 N cacodylate buffer (pH 7.4) containing sucrose and CaCl2 for 2 h. Then they were transferred into 2% osmium tetroxide prepared in the same buffer. Material was embedded
Fig. 1. Schematic drawings of the midgut in Anystis baccarum, showing the location of the secretory cells (hatched area). (a) Dorsal view, (b) lateral view. Es e esophagus; MgC e midgut caeca; PC e postcolon; Ph e pharynx; R e rectum; V e ventriculus.
S.A. Filimonova / Arthropod Structure & Development 37 (2008) 299e309
the lumen of the organ (Fig. 2a). The latter evidently represent waste products. As visible in semithin sections (Figs. 2aec), the epithelial lining of the midgut consists of the digestive and the secretory cells. The digestive cells are much more numerous and variable. Their size and shape greatly depend on their physiological state: from the low flat cells to the large goblet-shaped cells with round basophilic inclusions in the cytoplasm. Some of these cells form large bulb-shaped protrusions extending to the gut lumen and filled with various inclusions. The same cell fragments can be observed in the gut lumen without any connection to the epithelium (Figs. 2b,c). The secretory cells occupy exclusively the anterior and ventral sides of the ventriculus and little medial areas of the anterior caeca next to the ventricular wall (Fig. 1a). Not more than twenty secretory cells are visible per semi-thin section. The secretory cells are large with basophilic cytoplasm packed with basophilic granules of similar size. The granules are much more numerous in mites, fixed 24 h after the meal as compared to those fixed 4 h after the meal (Figs. 2b,c). In both cases the granules of the secretory cells look greenishblue whereas the inclusions in the digestive cells vary from blue to violet in semi-thin sections. Both cell types contain a single nucleus with a large central nucleolus. 3.2. The ultrastructure The digestive cells exhibit a different ultrastructure according to their functional state. Two kinds of these cells predominate. The first one is characterized by a brush border on the surface facing the gut lumen and by the accumulation of long mitochondria in the apical region of the cell (Fig. 3a). The brush border is not more than 0.8 mm high with a bundle of filaments visible inside the microvilli. The nucleus is provided with a large nucleolus (Fig. 3b). This kind of cells is commonly full of lipid droplets and conspicuous RER, both spread all over the cytoplasm (Figs. 3aec). Small Golgi bodies are frequently observed in the central part of the cell. Each of them consists of a pile of narrow cisterns surrounded by a group of small vesicles. The latter ones include more common electron-light particles as well as dense granules of the same size (0.15e0.20 mm in diameter) (Fig. 3c). Single dense granules ranging up to 0.35 mm can be visible in the cytoplasm of the cells as well as in close contact with the plasmalemma in its apical and lateral regions (Fig. 3d). Similar membranebound inclusions may be found occasionally in close contact to the basal lamina (Fig. 3e). Another kind of digestive cells is represented by the lighter cells having abare apical surface or with few microvilli of irregular shape (Fig. 4a). The nucleus looks active with a large nucleolus as in the previous kind of cells. Golgi bodies form drop-shaped granules of high electron density (Fig. 4b). The most striking feature of these cells is the presence of numerous pinocytotic canals, pinocytotic vesicles and phagosomes which occupy a wide apical layer of cytoplasm free of other organelles (Figs. 4a,c). Pinocytotic vesicles are frequently seen to merge, giving rise to larger endosomes of low electron
301
density (Fig. 4a, arrow). The connection of pino- and phagocytotic vesicles with lysosome-like structures is frequently observed as well. In the central part of the cell, a great number of secondary lysosomes with fine fibrillar or dense contents may be visible. Mitochondria concentrate mainly in the central and basal regions of the cell. Cells of the third kind are also lacking an apical brush border and are characterized by light cytoplasm, which is partly free from any components. RER and lipid droplets are not so frequent, as in the previous kinds of cells. Electron-lucent vacuoles and different concretions (or spherites) predominate in the cytoplasm. These cells are sometimes observed with their apical parts expanded into the gut lumen as if they are ready to separate from the epithelia. Such apical regions are filled with huge clear vacuoles, spherites and dense bodies, which form shapeless conglomerates. Digestive cells with a fine structure intermediate between the three cell kinds described above were also observed in the midgut lining. Electron microscopy reveals poor infoldings of the cell surface in the basal region of all the cells. Mitochondria, visible among the infoldings, are not numerous enough to form the typical basal labyrinth. They look round with an electron-lucent matrix and narrow cristae. Some of the digestive cells possess electron-dense granules lying adjacent to the basal plasma membrane (Fig. 5a). The granules have a narrow electron-lucent halo and a size about 0.15e0.20 mm. Groups of the same granules were observed inside the cell processes, occasionally discernable in the basal region of the midgut epithelial lining next to the basal lamina (Fig. 5b). In mites fixed 4 h after the experimental feeding, all kinds of digestive cells possess RER mainly in the form of short swollen cisterns or spherical particles full of material of moderate electron density. In mites fixed 24 h after the feeding, long cisterns of RER are more frequent, being sometimes arranged in whorls. The secretory cells are characterized by an apical surface either bare or with rare microvilli of irregular shape (Figs. 6aec). Numerous short fragments of microvilli may be visible in the gut lumen just above the apical plasmalemma 24 h after the meal (Fig. 6a). The apical surface of the cells is predominantly bare 4 h after the meal (Figs. 6b,c). A basal labyrinth is not present. The nuclei look active with large nucleoli mainly composed of fibrillar components. RER is the most extensive cell component. It fills all the cytoplasm, around the mitochondria and large secretory granules (Figs. 6a and 7a,b). Numerous small Golgi bodies are commonly visible in the central and basal regions of the cell (Fig. 7b). Each of them represents a heap of flat sacks, enlarged to the sides with internal dense material. Occasionally, tightly packed small vesicles of moderate electron density are seen between RER and Golgi bodies being closely associated with both organelles (Fig. 7b). Golgi sacs are often visible connected with the larger granules, or probably transform entirely into a group of granules. These primary granules are round or, more frequently, drop-shaped, about 0.2e0.3 mm in diameter with their contents varying from medium to high electron
302
S.A. Filimonova / Arthropod Structure & Development 37 (2008) 299e309
Fig. 2. The composition of the midgut of Anystis baccarum visible in semithin sections. (a) Frontal section through the mite body dorsally to the synganglion. Scale bar ¼ 50 mm. (b) Longitudinal section of the ventriculus 4 h after the meal. The digestive cells are overloaded with secondary lysosomes; the secretory cells possess much less secretory granules (arrow), than 24 h after the meal. Scale bar ¼ 33 mm. (c) Longitudinal section of the anterior part of the ventriculus 24 h after feeding. Scale bar ¼ 30 mm. FB e fat body cells; MgC e midgut caeca; N e nucleus of a digestive cell; Oo e oocytes; PC e postcolon; SC e secretory cells; SG 1, 2 e salivary glands; SLy e secondary lysosomes; VL e ventricular lumen.
S.A. Filimonova / Arthropod Structure & Development 37 (2008) 299e309
303
Fig. 3. A kind of digestive cell, which probably is active in absorption. TEM (a) The apical region of the cell. Note regular microvilli and mitochondria, concentrated in the subapical cytoplasm. Scale bar ¼ 1 mm. (b) The central region of the same cell is also rich in lipid droplets. Scale bar ¼ 2 mm. (c) Golgi bodies, producing secretory granules in the central cytoplasm. Scale bar ¼ 0.5 mm. (d) Electron-dense granules in the apical region of the digestive cell. (e) A granule in a very close position to the basal lamina of the epithelium. Scale bar ¼ 0.5 mm. BL e basal lamina; G e Golgi bodies; L e lipid droplets; M e mitochondria; ML e muscle layer; MV e microvilli; N e nucleus; Nu e nucleolus; RB e residual body; RER e rough endoplasmic reticulum; S e spherites; SG e secretory granules.
Fig. 4. A kind of digestive cell, probably involved in intracellular digestion. (a) The apical region of the cell with numerous pinocytotic vesicles and lysosomes. Bare surface is highly typical. Note the junction of pinocytotic vesicles and a large endosome (arrow). Scale bar ¼ 0.5 mm. (b) Golgi body with lysosomes in the vicinity. Scale bar ¼ 0.5 mm. (c) The formation of a macropinocytotic vesicle on the apical surface of the cell. Smaller vesicles are formed at the end of the thin pinocytotic canals. Scale bar ¼ 0.25 mm. G e Golgi body; Ly e primary lysosomes; MgL e midgut lumen; PC e pinocytotic canals; PV e pinocytotic vesicles; S e spherites; SLy e secondary lysosomes.
S.A. Filimonova / Arthropod Structure & Development 37 (2008) 299e309
305
between the primary and secondary granules can be observed in the same cells (Figs. 7a,b). Both the primary and the secondary granules are frequently noticed to merge into large tertiary granules, which predominate in the cytoplasm of the secretory cells (Fig. 7a). The tertiary granules are spherical membrane-bound inclusions up to 8 mm in size. Their electron-dense contents appear to change from homogenous to heterogeneous with several particles of another density inside the granule. The homogeneous granules prevail 24 h after the feeding (Figs. 6a and 7a), while 4 h after the feeding all the granules contain heterogeneous contents (Figs. 6b,c). All kinds of secretory granules, mentioned before, are visible just underneath the apical plasma membrane. No pinocytotic canals are seen in this region of the secretory cells (Figs. 6aec). Mites fixed 4 h after the experimental feeding, demonstrate numerous polymorphic vesicles in close contact to the tertiary secretory granules (Fig. 6b, arrow). The same vesicles were also seen in close position to the apical plasmalemma as they were ready to be released into the gut lumen (Fig. 6c). Apart from the secretory granules, some lipid droplets, spherites and lysosomes are also observed in this cell type, but they are always much fewer than those in the digestive cells. 4. Discussion
Fig. 5. Endocrine-like elements in the midgut epithelium. (a) Secretory granules in the cytoplasm of the digestive cell in the vicinity of the basal lamina. Scale bar ¼ 0.25 mm. (b) Process with endocrine-like granules (arrow) at the base of midgut epithelium. Scale bar ¼ 1 mm. BL e basal lamina; FB e fat body cell; L e lipid droplets; M e mitochondria; N e nucleus; RER e rough endoplasmic reticulum; SG e secretory granules.
density (Figs. 7a,b). The primary granules are commonly tightly packed close to more complicated secondary inclusions with their diameter ranging from 1.5 to 2.0 mm. The latter typically contain a mixture of tiny tubes, dense particles and microvesicles (0.03 mm in diameter), as well as a few lamellate components (Figs. 7a,b). All transitional forms
The presence of a wide opening between the ventriculus and postcolon in A. baccarum confirms the alimentary tract of this mite to be open, as it was previously suggested by Alberti (1973). This fact might be of special interest, since the family Anystidae is considered to be close to the cohort Parasitengona (Norton et al., 1993), which is characterized by a blind gut (Mitchell, 1964, 1970; Vistorin-Theis, 1978; Shatrov, 1989, 2003). On the other hand, the division of the postventricular region into colon and postcolon, typical to some other groups of actinedid mites with open midgut (Alberti, 1973; Vistorin, 1980; Ehrnsberger, 1984), was not observed in A. baccarum. A large postcolon, commonly packed with waste products directly connects ventriculus and rectum. There is a point of view, that reduction of the colon is a progressive tendency among actinedid mites (Alberti and Coons, 1999). The data obtained in the present paper show that the epithelium of the postcolon, lacking any nutritive inclusions, is distinct from that of the anterior midgut. The postcolon of Anystis, probably takes part in the formation of the excretes by means of their gradual concentration in the lumen, which is provided by the intense dilation of the organ. As for the anterior midgut there is no ultrastructural difference between the ventriculus and caeca in A. baccarum, which is similar to what was observed in other actinedid mites (Alberti and Coons, 1999). It is interesting to note, that no special contacts were shown between midgut and underlined fat body in the females studied. For some other representatives of acariform mites it was shown that the cells of the surrounding connective tissue (or other tissues lying close to the midgut) send finger-like processes penetrating the basal lamina
306
S.A. Filimonova / Arthropod Structure & Development 37 (2008) 299e309
Fig. 6. Ultrastructure of the apical region of the secretory cells of Anystis baccarum. (a) 24 h after the meal, all kind of secretory granules can be found underneath the apical plasmalemma, being ready to be released into the gut lumen. Scale bar ¼ 0.3 mm. (b,c) 4 h after the meal numerous secretory vesicles emerge from the tertiary secretory granules in the apical cytoplasm (arrow). Scale bar ¼ 0.5 mm. Lu e midgut lumen; M e mitochondria; MV e microvilli; PSG e primary secretory granules; RER e rough endoplasmic reticulum; SSG e secondary secretory granules; SV e secretory vesicles; TSG e tertiary secretory granules.
S.A. Filimonova / Arthropod Structure & Development 37 (2008) 299e309
307
Fig. 7. Formation of the secretory product in the secretory cells. (a) The common view of the central cytoplasm. Scale bar ¼ 0.5 mm. (b) Details of the granulogenesis. Scale bar ¼ 0.5 mm. G e Golgi bodies; M e mitochondria; PSG e primary secretory granules; RER e rough endoplasmic reticulum; SSG e secondary secretory granules; TSG e tertiary secretory granules.
308
S.A. Filimonova / Arthropod Structure & Development 37 (2008) 299e309
of the midgut epithelium (Alberti and Storch, 1983; Alberti and Coons, 1999; Filimonova, 2001). The digestive cells are the most abundant in the anterior midgut of A. baccarum. They look very similar to those described in other actinedid mites (Alberti, 1973; Mothes and Seitz, 1981; Alberti and Storch, 1983; Shatrov, 1989, 2003; Alberti and Coons, 1999; Filimonova, 2001), as well as some other arachnid taxa (Goyffon and Martoja, 1983; Becker and Peters, 1985; Ludwig and Alberti, 1990, 1992). The digestive cells of Anystis demonstrate relatively low transport activity as they do not form a typical basal labyrinth. At the same time they are characterized by a high level of intracellular digestion, which takes place even 24 h after the meal. Pino- and phagocytosis can be observed simultaneously in a single digestive cell, as well as it was shown in the representatives of Tetranychidae (Mothes and Seitz, 1981), Trombiculidae (Shatrov, 1989), Myobiidae (Filimonova, 2001). The digestive cells can greatly change their size and shape during the digestion, as it has also been shown for Anystis sp. (Wright and Newell, 1964). In the fed mites examined in the present study, three kinds of digestive cells are most typical. 1. Cells bearing a well developed brush border on their apical surface and commonly demonstrating a high concentration of lipid droplets in their cytoplasm and relatively few lysosome-like structures. This kind of cells is practically lacking pinocytotic canals and vesicles, being evidently specialized in absorption of simple compounds from the gut lumen. 2. Cells with poor microvilli or a smooth luminal surface and provided with an extensive apical network of pinocytotic tubules, numerous endosomes and lysosomes in the central region. These cells may be predominantly engaged in the intracellular digestion. 3. Cells rich in dense bodies and spherites, commonly free from either regular microvilli or pinocytotic tubules, containing sparse lysosomes and lipid granules. The main function of these cells is probably excretion. Although distinguishable, these three populations of digestive cells exhibit a lot of similarities and transitional states. This leads to the conclusion that they represent different physiological states of the same cell type. The same has been also suggested for the midgut cells of Anystis sp. (Wright and Newell, 1964). Perhaps, because of using less suitable fixation techniques in those days, these authors could not see as many details, as it is possible nowadays, but they considered the variety seen in the cell structure as the reflection of their continuous development through some transitional stages and subsequent degeneration, when they finally are extruded into the gut lumen. In the course of the present investigation we did not observe the detachment of whole cells into the gut lumen, but their fragments were frequently detected. However, the absence of unfed animals in our study might partly explain this difference in observations. Contrary to the previous work (Wright and Newell, 1964), a special type of secretory cells was identified in the gut
epithelium of A. baccarum. The secretory cells, typical for midgut epithelium in most arachnid taxa, are not common in acariform mites studied before (Alberti and Coons, 1999). However some points made us consider the granular cells of Anystis midgut as a special type of secretory cells. The secretory cells occupy exclusively the anterior ventricular wall. They are clearly distinguished from the digestive cells both at the light and electron microscopical levels by the extensive RER and large protein-like granules. The granules differ from the various nutritional vacuoles by their homogeneous content (at least 24 h after feeding) and very large size. The secretory cells demonstrate no pinocytotic tubules or any nutritional vacuoles as well as secondary lysosomes, indicating a low activity in intracellular digestion. The apical brush border of the secretory cells is either poor or absent, indicating low absorptive activity as well. Large dense granules have been shown in all arachnids where secretory cells have been found (Alberti and Storch, 1983; Goyffon and Martoja, 1983; Becker and Peters, 1985; Ludwig and Alberti, 1990, 1992; Alberti and Coons, 1999). Having a proteinaceous content, these granules remain unchanged through starvation of the animals. This suggests the secretory product more likely to be exoenzymes than stored material (Goyffon and Martoja, 1983; Becker and Peters, 1985; Ludwig and Alberti, 1992). Among actinedid mites, similar secretory granules were demonstrated at EM level only in Bdellidae (Alberti, 1973; Alberti and Storch, 1983), in which they have a size very close to that in A. baccarum (up to 9 mm). In all cases the presence of the granules associates with a high-developed RER, which confirms the secretory nature of their content. The maturation of the secretory product, as observed in the secretory cells of A. baccarum, is shown to be particular and far from the classical scheme of granulogenesis. The complicated Golgi body appears to produce several kinds of vesicles at a time, as the primary granules associated to its structures are of various shape and size. Some of the inclusions soon become heterogeneous. Then they pass several transitional stages, merging and forming the secondary inclusions, which are characterized by a great variety of size and internal contents. At this stage the granules resemble secondary lysosomes more than a secretory product, but a careful analysis of intermediate stages of their formation and their ability to merge into huge mature granules leads us to the conclusion that they all represent stages of granulogenesis. Not only the large tertiary granules but also the primary and the secondary ones often lie in an extremely apical position indicating their imminent extrusion. Thus, the secretory product might be released into the gut lumen at different stages of its maturation. If this is true, the large mature granules should be accounted as the temporary storage of the secretory material. Microapocrine secretion is a possible way of releasing the secretory material, as numerous fragments of microvilli have been noticed in the gut lumen close to the apical surface of the secretory cells. The study did not reveal endocrine cells in the midgut epithelium of A. baccarum. As it was shown (Fig. 5a), dense
S.A. Filimonova / Arthropod Structure & Development 37 (2008) 299e309
granules with light halo are regularly observed in the basal region of the digestive cells closely associated to the outer cell membrane. The same granules were shown inside the cell processes, which can be found in the basal region of the epithelium (Fig. 5b). It seems likely that the digestive cells have additional endocrine-like function. Neither replacement cells nor mitotic figures were observed in the midgut epithelium of A. baccarum. This situation is typical for most acariform mites examined up to now (Alberti and Coons, 1999). The process of cell replacement is still unclear in these arachnids. Acknowledgements The author is deeply grateful to Dr. I. Badanin from Zoological Institute (Ukrainian Academy of Sciences) for the material supplied and to the engineers A.E. Tenisson and P.I. Henkin (Zoological Institute of Russian Academy of Sciences) for their technical assistance. The work was supported by the Russian Foundation for Basic Research; grant No. 06-04-48538-a. References Akimov, L.A., Gorgol, V.T., 1990. Predatory and Parasitic Mites e Cheyletidae. Naukova Dumka, Kiev, 120 p (In Russian). Alberti, G., 1973. Erna¨hrungsbiologie und Spinnvermo¨gen der Schnabelmilben (Bdellidae, Trombidiformes). Zeitschrift f}ur Morphologie der Tiere 76, 285e338. Alberti, G., Coons, L.B., 1999. Acari: mites. In: Harrison, F.W., Foelix, R.F. (Eds.), Microscopic Anatomy of Invertebrates, vol. 8C. Wiley-Liss, New York, pp. 515e1217. Alberti, G., Crooker, A.R., 1985. Internal anatomy. In: Helle, W., Sabelis, M.V. (Eds.), Spider Mites. Their Biology, Natural Enemies and Control, vol. 1A. Elsevier, Amsterdam, pp. 29e62. Alberti, G., Storch, V., 1983. Zur Ultrastruktur der Mitteldarmdru¨ssen von Spinnentieren (Scorpiones, Araneae, Acari) unter verschiedenen Erna¨hrungsbedingungen. Zoologischer Anzeiger 3/4, 145e160. Blauvelt, W.E., 1945. The internal anatomy of the common red spider mite (Tetranychus telarius L.). Cornell University Experiment Station Memoirs 270, 1e46. Becker, A., Peters, W., 1985. Fine structure of the midgut gland of Phalangium opilio (Chelicerata, Phalangida). Zoomorphology 105, 317e325. Desch Jr., C.E., 1988. The digestive system of Demodex folliculorum (Acari: Demodicidae) of man: a light and electron microscope study. In:
309
Channabasavanna, G.P., Viraktamath, C.A. (Eds.), Progress in Acarology, vol. 1. Oxford and IBH Publishing, New Delhi, pp. 187e195. Ehrnsberger, R., 1984. Anatomie des Verdauungstraktes der Rhagidiidae (Acari, Trombidiformes). Osnabru¨cker Naturwissenschaftliche Mitteilungen 11, 67e90. Filimonova, S.A., 2001. The fine structure of the midgut in the mite Myobia murismusculi. Cytology 43, 425e431 (In Russian). Goyffon, M., Martoja, R., 1983. Cytophysiological aspects of digestion and storage in the liver of scorpion, Androctonus australis (Arachnida). Cell and Tissue Research 228, 661e675. 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, 709e718. Ludwig, M., Alberti, G., 1990. Peculiarities of arachnid midgut glands. Acta Zoologica Fennica 190, 255e259. Ludwig, M., Alberti, G., 1992. Fine structure of the midgut of the Prokoenenia wheeleri (Arachnida: Palpigradi). Zoologische Beitrage 34, 127e134. Mitchell, R., 1964. The anatomy of an adult chigger mite Blankaartia acuscutellaris (Walch). Journal of Morphology 114, 373e391. Mitchell, R., 1970. The evolution of a blind gut in trombiculid mites. Journal of Natural History 4, 221e229. Mothes, U., Seitz, K., 1981. Functional microscopic anatomy of the digestive system of Tetranychus urticae (Acari, Tetranychidae). Acarologia 22, 257e270. Norton, R.A., Kethley, J.B., Johnston, D.E., O’Connor, B.M., 1993. Phylogenetic perspectives on genetic systems and reproductive modes of mites. In: Wrensch, D.L., Ebbert, M.A. (Eds.), Evolution and Diversity of Sex Ratio in Insects and Mites. Chapman & Hall, New York, London, England, pp. 8e99. Nuzzaci, G., Alberti, G., 1996. Internal anatomy and physiology. In: Lindquist, E.E., Sabelis, M.W., Bruin, J. (Eds.), Eriophyoid Mites e Their biology, Natural Enemies and Control. World Crop Pests, vol. 6. Elsevier, Amsterdam, pp. 101e150. 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, 28e37 (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, 227e239. Vistorin, H.E., 1980. Erna¨hrungsbiologie und Anatomie des Verdauungstraktes der Nicoletiellidae (Acari, Trombidiformes). Acarologia 21, 204e215. Vistorin-Theis, G., 1978. Anatomische Untersuchungen an Caliptostomiden (Acari, Trombidiformes). Acarologia 19, 242e257. Wright, K.A., Newell, J.M., 1964. Some observations on the fine structure of the midgut of the mite Anystis sp. Annals of the Entomological Society of America 57, 684e693.
The fine structure of the midgut in the mite Anystis baccarum (L.) (Acari, Actinedida: Anystidae) Svetlana A. Filimonova* Zoological Institute, Russian Academy of Sciences, 199034 St. Petersburg, Russia Received 27 June 2007; received in revised form 26 November 2007; accepted 26 November 2007
Abstract The ventriculus and the midgut caeca of the fed females of Anystis baccarum (L.) were investigated by using light and electron microscopy. In addition to the main type of polyfunctional digestive cells, special secretory cells were detected in the anterior region of the ventriculus. The shape and the ultrastructure of the digestive cells vary depending on their physiological state. Intracellular digestion, absorption or excretion processes prevail at different stages of the cell cycle. The secretory cells are characterized by the presence of extensive rough endoplasmic reticulum, filling whole space of the cell. These cells do not contain the apical network of pinocytotic canals, which are typical for the digestive cells. Three types of secretory granules were found in the cytoplasm of the secretory cells that probably correspond to three sequential stages of granulogenesis. The primary secretory granules are formed by the fusion of Golgi vesicles. The primary granules fuse to form complex vesicles with heterogeneous contents. These secondary granules aggregate to form very large inclusions of high electron density (tertiary secretory granules), which probably represent the storage of the secretory product. All types of secretory granules were observed close to the apical plasmalemma. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: Actinedida; Midgut ultrastructure; Secretory cells
1. Introduction The midgut of arachnids is generally divided into the anterior and posterior parts commonly named the anterior and posterior midgut, respectively (Alberti, 1973; Alberti and Storch, 1983; Becker and Peters, 1985; Ludwig and Alberti, 1990, 1992; Alberti and Coons, 1999). The anterior midgut, including ventriculus and midgut caeca, participates in active intracellular digestion, while the posterior midgut appears to be involved in excretion and, to some extent, in regulation of water balance. In most derived actinedid mites (such as Parasitengona) the midgut ends blindly, lacking a typical posterior midgut. Instead of the latter, a dorsomedian excretory organ exists, which opens to the outside through an uropor (Mitchell, 1964, 1970; Vistorin-Theis, 1978; Shatrov, 1989, 2003; Alberti and Coons, 1999). In the other cases the midgut * Tel.: þ7 0812 714 0151; fax: þ7 0812 714 0444. E-mail address: [email protected] 1467-8039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.asd.2007.11.005
is considered to be open, and the connection between anterior and posterior midgut is clearly demonstrated (Blauvelt, 1945; Alberti, 1973; Ehrnsberger, 1984; Vistorin, 1980; Mothes and Seitz, 1981; Alberti and Crooker, 1985; Akimov and Gorgol, 1990; Filimonova, 2001). In some species with open midgut the postventricular region is represented by a uniform tube (Mothes and Seitz, 1981; Alberti and Crooker, 1985; Akimov and Gorgol, 1990; Filimonova, 2001) in the others it is divided into colon and postcolon, which differ histologically (Alberti, 1973; Vistorin, 1980; Ehrnsberger, 1984). For most actinedid families the anatomical organization of the digestive tract is still unknown. In most examined representatives of Acari, the epithelium of the anterior midgut is composed of a single cell type, which develops from digestive to excretion cell and then undergoes degeneration as separated cell fragments in the gut lumen. Thus, most of the authors suppose the digestive cells to be polyfunctional, being involved subsequently in intracellular digestion, absorption, and excretion of waste products with
300
S.A. Filimonova / Arthropod Structure & Development 37 (2008) 299e309
each process prevailing at different stages of the cell cycle (Mothes and Seitz, 1981; Alberti and Crooker, 1985; Shatrov, 1989; Nuzzaci and Alberti, 1996; Filimonova, 2001). For most taxonomic groups of Chelicerata, such as Scorpiones (Alberti and Storch, 1983; Goyffon and Martoja, 1983), Phalangida (Becker and Peters, 1985), Araneae (Ludwig and Alberti, 1988, 1990), Uropygi, Pseudoscorpiones (Ludwig and Alberti, 1990), Palpigradi (Ludwig and Alberti, 1992), a special kind of secretory cells was detected in the midgut epithelium. However, this cell differentiation is not typical for the acariform mites (order Acariformes) studied so far. The absence of secretory cells was definitely shown for most families of actinedid mites (suborder Actinedida), such as Tetranychidae (Mothes and Seitz, 1981; Alberti and Crooker, 1985), Demodicidae (Desch, 1988), Eriophyoidea (Nuzzaci and Alberti, 1996), Calyptostomatidae (VistorinTheis, 1978), Trombiculidae (Shatrov, 1989), Myobiidae (Filimonova, 2001), Microtrombidiidae (Shatrov, 2003), as well as for some oribatid and acaridid mites (suborders Oribatida and Acaridida) (Alberti and Coons, 1999). In actinedid mites, secretory cells in the midgut epithelium were described at light microscopical level in the mites belonging to the Labidostommatidae (Vistorin, 1980), and ultrastructurally in the Bdellidae (Alberti, 1973; Alberti and Storch, 1983). In both cases these cells were provided by basophilic secretory granules, which in Bdellidae were shown to be accompanied by an extremely developed rough endoplasmic reticulum (RER). It is interesting to note, that the secretory granules of Bdellidae are extremely large (up to 9 mm), being very similar to those in other arachnids examined so far (Alberti, 1973; Ludwig and Alberti, 1990, 1992). The present paper describes the fine structure and functioning of the midgut epithelium in a predatory mite Anystis baccarum (L.). The family Anystidae is of special interest, because of the great number of primitive features common for actinedid mites (Kethley in: Norton et al., 1993). Wright and Newell (1964) investigated the midgut of Anystis in one of the first EM studies. They showed the presence of several cell categories which corresponded, according to these authors, to the gradual changing from absorptive cells to excretory ones. No specialized secretory cells were indicated in their study. In contrast to that study, the present investigation shows the existence of special secretory cells in the midgut of Anystis, with their definite position in the midgut and the fine structure clearly distinct from the other epithelial cells.
in Epon 812. The semithin sections for light microscopy were stained with a mixture of methylene blue and Azur II (rO 6.8). Ultrathin sections were stained with uranyl acetate and lead citrate. The sections were studied using a Zeiss LEO 900 electron microscope. 3. Results 3.1. General remarks The midgut is divided into a central ventriculus, two pairs of diverticula (anterior and posterior caeca) and the postventricular region, composed of a large postcolon (Figs. 1a,b and 2a). The postcolon begins posteriorly from the ventriculus, and runs backwards and ventrally to a short cuticle-lined rectum leading to the anus. The walls of the ventriculus and caeca consist of a single epithelial layer underlined by a basal lamina and muscle cells. The epithelial cells of the ventriculus and caeca are variable in shape and to some extent in size, having various nutritional inclusions in the cytoplasm, stained brightly in semithin sections. In the light microscope, the postcolon is characterized by the uniform columnar cells with no inclusions; the cells are more basophilic than those in the ventricular region. The epithelial lining of the postcolon is either folded composed of the high columnar cells, or flattened in the case when a great number of crystalline inclusions fills
2. Materials and methods Five adult females of Anystis baccarum (L.) were collected from the soil surface in the sand places in the suburbs of Kiev (Ukraine). The mites were fed on collembolans in laboratory condition under the visual control of this process and fixed 4 (two females) and 24 h (three females) after the feeding. The mite specimens were put in 2.5% glutaraldehyde solution in 0.05 N cacodylate buffer (pH 7.4) containing sucrose and CaCl2 for 2 h. Then they were transferred into 2% osmium tetroxide prepared in the same buffer. Material was embedded
Fig. 1. Schematic drawings of the midgut in Anystis baccarum, showing the location of the secretory cells (hatched area). (a) Dorsal view, (b) lateral view. Es e esophagus; MgC e midgut caeca; PC e postcolon; Ph e pharynx; R e rectum; V e ventriculus.
S.A. Filimonova / Arthropod Structure & Development 37 (2008) 299e309
the lumen of the organ (Fig. 2a). The latter evidently represent waste products. As visible in semithin sections (Figs. 2aec), the epithelial lining of the midgut consists of the digestive and the secretory cells. The digestive cells are much more numerous and variable. Their size and shape greatly depend on their physiological state: from the low flat cells to the large goblet-shaped cells with round basophilic inclusions in the cytoplasm. Some of these cells form large bulb-shaped protrusions extending to the gut lumen and filled with various inclusions. The same cell fragments can be observed in the gut lumen without any connection to the epithelium (Figs. 2b,c). The secretory cells occupy exclusively the anterior and ventral sides of the ventriculus and little medial areas of the anterior caeca next to the ventricular wall (Fig. 1a). Not more than twenty secretory cells are visible per semi-thin section. The secretory cells are large with basophilic cytoplasm packed with basophilic granules of similar size. The granules are much more numerous in mites, fixed 24 h after the meal as compared to those fixed 4 h after the meal (Figs. 2b,c). In both cases the granules of the secretory cells look greenishblue whereas the inclusions in the digestive cells vary from blue to violet in semi-thin sections. Both cell types contain a single nucleus with a large central nucleolus. 3.2. The ultrastructure The digestive cells exhibit a different ultrastructure according to their functional state. Two kinds of these cells predominate. The first one is characterized by a brush border on the surface facing the gut lumen and by the accumulation of long mitochondria in the apical region of the cell (Fig. 3a). The brush border is not more than 0.8 mm high with a bundle of filaments visible inside the microvilli. The nucleus is provided with a large nucleolus (Fig. 3b). This kind of cells is commonly full of lipid droplets and conspicuous RER, both spread all over the cytoplasm (Figs. 3aec). Small Golgi bodies are frequently observed in the central part of the cell. Each of them consists of a pile of narrow cisterns surrounded by a group of small vesicles. The latter ones include more common electron-light particles as well as dense granules of the same size (0.15e0.20 mm in diameter) (Fig. 3c). Single dense granules ranging up to 0.35 mm can be visible in the cytoplasm of the cells as well as in close contact with the plasmalemma in its apical and lateral regions (Fig. 3d). Similar membranebound inclusions may be found occasionally in close contact to the basal lamina (Fig. 3e). Another kind of digestive cells is represented by the lighter cells having abare apical surface or with few microvilli of irregular shape (Fig. 4a). The nucleus looks active with a large nucleolus as in the previous kind of cells. Golgi bodies form drop-shaped granules of high electron density (Fig. 4b). The most striking feature of these cells is the presence of numerous pinocytotic canals, pinocytotic vesicles and phagosomes which occupy a wide apical layer of cytoplasm free of other organelles (Figs. 4a,c). Pinocytotic vesicles are frequently seen to merge, giving rise to larger endosomes of low electron
301
density (Fig. 4a, arrow). The connection of pino- and phagocytotic vesicles with lysosome-like structures is frequently observed as well. In the central part of the cell, a great number of secondary lysosomes with fine fibrillar or dense contents may be visible. Mitochondria concentrate mainly in the central and basal regions of the cell. Cells of the third kind are also lacking an apical brush border and are characterized by light cytoplasm, which is partly free from any components. RER and lipid droplets are not so frequent, as in the previous kinds of cells. Electron-lucent vacuoles and different concretions (or spherites) predominate in the cytoplasm. These cells are sometimes observed with their apical parts expanded into the gut lumen as if they are ready to separate from the epithelia. Such apical regions are filled with huge clear vacuoles, spherites and dense bodies, which form shapeless conglomerates. Digestive cells with a fine structure intermediate between the three cell kinds described above were also observed in the midgut lining. Electron microscopy reveals poor infoldings of the cell surface in the basal region of all the cells. Mitochondria, visible among the infoldings, are not numerous enough to form the typical basal labyrinth. They look round with an electron-lucent matrix and narrow cristae. Some of the digestive cells possess electron-dense granules lying adjacent to the basal plasma membrane (Fig. 5a). The granules have a narrow electron-lucent halo and a size about 0.15e0.20 mm. Groups of the same granules were observed inside the cell processes, occasionally discernable in the basal region of the midgut epithelial lining next to the basal lamina (Fig. 5b). In mites fixed 4 h after the experimental feeding, all kinds of digestive cells possess RER mainly in the form of short swollen cisterns or spherical particles full of material of moderate electron density. In mites fixed 24 h after the feeding, long cisterns of RER are more frequent, being sometimes arranged in whorls. The secretory cells are characterized by an apical surface either bare or with rare microvilli of irregular shape (Figs. 6aec). Numerous short fragments of microvilli may be visible in the gut lumen just above the apical plasmalemma 24 h after the meal (Fig. 6a). The apical surface of the cells is predominantly bare 4 h after the meal (Figs. 6b,c). A basal labyrinth is not present. The nuclei look active with large nucleoli mainly composed of fibrillar components. RER is the most extensive cell component. It fills all the cytoplasm, around the mitochondria and large secretory granules (Figs. 6a and 7a,b). Numerous small Golgi bodies are commonly visible in the central and basal regions of the cell (Fig. 7b). Each of them represents a heap of flat sacks, enlarged to the sides with internal dense material. Occasionally, tightly packed small vesicles of moderate electron density are seen between RER and Golgi bodies being closely associated with both organelles (Fig. 7b). Golgi sacs are often visible connected with the larger granules, or probably transform entirely into a group of granules. These primary granules are round or, more frequently, drop-shaped, about 0.2e0.3 mm in diameter with their contents varying from medium to high electron
302
S.A. Filimonova / Arthropod Structure & Development 37 (2008) 299e309
Fig. 2. The composition of the midgut of Anystis baccarum visible in semithin sections. (a) Frontal section through the mite body dorsally to the synganglion. Scale bar ¼ 50 mm. (b) Longitudinal section of the ventriculus 4 h after the meal. The digestive cells are overloaded with secondary lysosomes; the secretory cells possess much less secretory granules (arrow), than 24 h after the meal. Scale bar ¼ 33 mm. (c) Longitudinal section of the anterior part of the ventriculus 24 h after feeding. Scale bar ¼ 30 mm. FB e fat body cells; MgC e midgut caeca; N e nucleus of a digestive cell; Oo e oocytes; PC e postcolon; SC e secretory cells; SG 1, 2 e salivary glands; SLy e secondary lysosomes; VL e ventricular lumen.
S.A. Filimonova / Arthropod Structure & Development 37 (2008) 299e309
303
Fig. 3. A kind of digestive cell, which probably is active in absorption. TEM (a) The apical region of the cell. Note regular microvilli and mitochondria, concentrated in the subapical cytoplasm. Scale bar ¼ 1 mm. (b) The central region of the same cell is also rich in lipid droplets. Scale bar ¼ 2 mm. (c) Golgi bodies, producing secretory granules in the central cytoplasm. Scale bar ¼ 0.5 mm. (d) Electron-dense granules in the apical region of the digestive cell. (e) A granule in a very close position to the basal lamina of the epithelium. Scale bar ¼ 0.5 mm. BL e basal lamina; G e Golgi bodies; L e lipid droplets; M e mitochondria; ML e muscle layer; MV e microvilli; N e nucleus; Nu e nucleolus; RB e residual body; RER e rough endoplasmic reticulum; S e spherites; SG e secretory granules.
Fig. 4. A kind of digestive cell, probably involved in intracellular digestion. (a) The apical region of the cell with numerous pinocytotic vesicles and lysosomes. Bare surface is highly typical. Note the junction of pinocytotic vesicles and a large endosome (arrow). Scale bar ¼ 0.5 mm. (b) Golgi body with lysosomes in the vicinity. Scale bar ¼ 0.5 mm. (c) The formation of a macropinocytotic vesicle on the apical surface of the cell. Smaller vesicles are formed at the end of the thin pinocytotic canals. Scale bar ¼ 0.25 mm. G e Golgi body; Ly e primary lysosomes; MgL e midgut lumen; PC e pinocytotic canals; PV e pinocytotic vesicles; S e spherites; SLy e secondary lysosomes.
S.A. Filimonova / Arthropod Structure & Development 37 (2008) 299e309
305
between the primary and secondary granules can be observed in the same cells (Figs. 7a,b). Both the primary and the secondary granules are frequently noticed to merge into large tertiary granules, which predominate in the cytoplasm of the secretory cells (Fig. 7a). The tertiary granules are spherical membrane-bound inclusions up to 8 mm in size. Their electron-dense contents appear to change from homogenous to heterogeneous with several particles of another density inside the granule. The homogeneous granules prevail 24 h after the feeding (Figs. 6a and 7a), while 4 h after the feeding all the granules contain heterogeneous contents (Figs. 6b,c). All kinds of secretory granules, mentioned before, are visible just underneath the apical plasma membrane. No pinocytotic canals are seen in this region of the secretory cells (Figs. 6aec). Mites fixed 4 h after the experimental feeding, demonstrate numerous polymorphic vesicles in close contact to the tertiary secretory granules (Fig. 6b, arrow). The same vesicles were also seen in close position to the apical plasmalemma as they were ready to be released into the gut lumen (Fig. 6c). Apart from the secretory granules, some lipid droplets, spherites and lysosomes are also observed in this cell type, but they are always much fewer than those in the digestive cells. 4. Discussion
Fig. 5. Endocrine-like elements in the midgut epithelium. (a) Secretory granules in the cytoplasm of the digestive cell in the vicinity of the basal lamina. Scale bar ¼ 0.25 mm. (b) Process with endocrine-like granules (arrow) at the base of midgut epithelium. Scale bar ¼ 1 mm. BL e basal lamina; FB e fat body cell; L e lipid droplets; M e mitochondria; N e nucleus; RER e rough endoplasmic reticulum; SG e secretory granules.
density (Figs. 7a,b). The primary granules are commonly tightly packed close to more complicated secondary inclusions with their diameter ranging from 1.5 to 2.0 mm. The latter typically contain a mixture of tiny tubes, dense particles and microvesicles (0.03 mm in diameter), as well as a few lamellate components (Figs. 7a,b). All transitional forms
The presence of a wide opening between the ventriculus and postcolon in A. baccarum confirms the alimentary tract of this mite to be open, as it was previously suggested by Alberti (1973). This fact might be of special interest, since the family Anystidae is considered to be close to the cohort Parasitengona (Norton et al., 1993), which is characterized by a blind gut (Mitchell, 1964, 1970; Vistorin-Theis, 1978; Shatrov, 1989, 2003). On the other hand, the division of the postventricular region into colon and postcolon, typical to some other groups of actinedid mites with open midgut (Alberti, 1973; Vistorin, 1980; Ehrnsberger, 1984), was not observed in A. baccarum. A large postcolon, commonly packed with waste products directly connects ventriculus and rectum. There is a point of view, that reduction of the colon is a progressive tendency among actinedid mites (Alberti and Coons, 1999). The data obtained in the present paper show that the epithelium of the postcolon, lacking any nutritive inclusions, is distinct from that of the anterior midgut. The postcolon of Anystis, probably takes part in the formation of the excretes by means of their gradual concentration in the lumen, which is provided by the intense dilation of the organ. As for the anterior midgut there is no ultrastructural difference between the ventriculus and caeca in A. baccarum, which is similar to what was observed in other actinedid mites (Alberti and Coons, 1999). It is interesting to note, that no special contacts were shown between midgut and underlined fat body in the females studied. For some other representatives of acariform mites it was shown that the cells of the surrounding connective tissue (or other tissues lying close to the midgut) send finger-like processes penetrating the basal lamina
306
S.A. Filimonova / Arthropod Structure & Development 37 (2008) 299e309
Fig. 6. Ultrastructure of the apical region of the secretory cells of Anystis baccarum. (a) 24 h after the meal, all kind of secretory granules can be found underneath the apical plasmalemma, being ready to be released into the gut lumen. Scale bar ¼ 0.3 mm. (b,c) 4 h after the meal numerous secretory vesicles emerge from the tertiary secretory granules in the apical cytoplasm (arrow). Scale bar ¼ 0.5 mm. Lu e midgut lumen; M e mitochondria; MV e microvilli; PSG e primary secretory granules; RER e rough endoplasmic reticulum; SSG e secondary secretory granules; SV e secretory vesicles; TSG e tertiary secretory granules.
S.A. Filimonova / Arthropod Structure & Development 37 (2008) 299e309
307
Fig. 7. Formation of the secretory product in the secretory cells. (a) The common view of the central cytoplasm. Scale bar ¼ 0.5 mm. (b) Details of the granulogenesis. Scale bar ¼ 0.5 mm. G e Golgi bodies; M e mitochondria; PSG e primary secretory granules; RER e rough endoplasmic reticulum; SSG e secondary secretory granules; TSG e tertiary secretory granules.
308
S.A. Filimonova / Arthropod Structure & Development 37 (2008) 299e309
of the midgut epithelium (Alberti and Storch, 1983; Alberti and Coons, 1999; Filimonova, 2001). The digestive cells are the most abundant in the anterior midgut of A. baccarum. They look very similar to those described in other actinedid mites (Alberti, 1973; Mothes and Seitz, 1981; Alberti and Storch, 1983; Shatrov, 1989, 2003; Alberti and Coons, 1999; Filimonova, 2001), as well as some other arachnid taxa (Goyffon and Martoja, 1983; Becker and Peters, 1985; Ludwig and Alberti, 1990, 1992). The digestive cells of Anystis demonstrate relatively low transport activity as they do not form a typical basal labyrinth. At the same time they are characterized by a high level of intracellular digestion, which takes place even 24 h after the meal. Pino- and phagocytosis can be observed simultaneously in a single digestive cell, as well as it was shown in the representatives of Tetranychidae (Mothes and Seitz, 1981), Trombiculidae (Shatrov, 1989), Myobiidae (Filimonova, 2001). The digestive cells can greatly change their size and shape during the digestion, as it has also been shown for Anystis sp. (Wright and Newell, 1964). In the fed mites examined in the present study, three kinds of digestive cells are most typical. 1. Cells bearing a well developed brush border on their apical surface and commonly demonstrating a high concentration of lipid droplets in their cytoplasm and relatively few lysosome-like structures. This kind of cells is practically lacking pinocytotic canals and vesicles, being evidently specialized in absorption of simple compounds from the gut lumen. 2. Cells with poor microvilli or a smooth luminal surface and provided with an extensive apical network of pinocytotic tubules, numerous endosomes and lysosomes in the central region. These cells may be predominantly engaged in the intracellular digestion. 3. Cells rich in dense bodies and spherites, commonly free from either regular microvilli or pinocytotic tubules, containing sparse lysosomes and lipid granules. The main function of these cells is probably excretion. Although distinguishable, these three populations of digestive cells exhibit a lot of similarities and transitional states. This leads to the conclusion that they represent different physiological states of the same cell type. The same has been also suggested for the midgut cells of Anystis sp. (Wright and Newell, 1964). Perhaps, because of using less suitable fixation techniques in those days, these authors could not see as many details, as it is possible nowadays, but they considered the variety seen in the cell structure as the reflection of their continuous development through some transitional stages and subsequent degeneration, when they finally are extruded into the gut lumen. In the course of the present investigation we did not observe the detachment of whole cells into the gut lumen, but their fragments were frequently detected. However, the absence of unfed animals in our study might partly explain this difference in observations. Contrary to the previous work (Wright and Newell, 1964), a special type of secretory cells was identified in the gut
epithelium of A. baccarum. The secretory cells, typical for midgut epithelium in most arachnid taxa, are not common in acariform mites studied before (Alberti and Coons, 1999). However some points made us consider the granular cells of Anystis midgut as a special type of secretory cells. The secretory cells occupy exclusively the anterior ventricular wall. They are clearly distinguished from the digestive cells both at the light and electron microscopical levels by the extensive RER and large protein-like granules. The granules differ from the various nutritional vacuoles by their homogeneous content (at least 24 h after feeding) and very large size. The secretory cells demonstrate no pinocytotic tubules or any nutritional vacuoles as well as secondary lysosomes, indicating a low activity in intracellular digestion. The apical brush border of the secretory cells is either poor or absent, indicating low absorptive activity as well. Large dense granules have been shown in all arachnids where secretory cells have been found (Alberti and Storch, 1983; Goyffon and Martoja, 1983; Becker and Peters, 1985; Ludwig and Alberti, 1990, 1992; Alberti and Coons, 1999). Having a proteinaceous content, these granules remain unchanged through starvation of the animals. This suggests the secretory product more likely to be exoenzymes than stored material (Goyffon and Martoja, 1983; Becker and Peters, 1985; Ludwig and Alberti, 1992). Among actinedid mites, similar secretory granules were demonstrated at EM level only in Bdellidae (Alberti, 1973; Alberti and Storch, 1983), in which they have a size very close to that in A. baccarum (up to 9 mm). In all cases the presence of the granules associates with a high-developed RER, which confirms the secretory nature of their content. The maturation of the secretory product, as observed in the secretory cells of A. baccarum, is shown to be particular and far from the classical scheme of granulogenesis. The complicated Golgi body appears to produce several kinds of vesicles at a time, as the primary granules associated to its structures are of various shape and size. Some of the inclusions soon become heterogeneous. Then they pass several transitional stages, merging and forming the secondary inclusions, which are characterized by a great variety of size and internal contents. At this stage the granules resemble secondary lysosomes more than a secretory product, but a careful analysis of intermediate stages of their formation and their ability to merge into huge mature granules leads us to the conclusion that they all represent stages of granulogenesis. Not only the large tertiary granules but also the primary and the secondary ones often lie in an extremely apical position indicating their imminent extrusion. Thus, the secretory product might be released into the gut lumen at different stages of its maturation. If this is true, the large mature granules should be accounted as the temporary storage of the secretory material. Microapocrine secretion is a possible way of releasing the secretory material, as numerous fragments of microvilli have been noticed in the gut lumen close to the apical surface of the secretory cells. The study did not reveal endocrine cells in the midgut epithelium of A. baccarum. As it was shown (Fig. 5a), dense
S.A. Filimonova / Arthropod Structure & Development 37 (2008) 299e309
granules with light halo are regularly observed in the basal region of the digestive cells closely associated to the outer cell membrane. The same granules were shown inside the cell processes, which can be found in the basal region of the epithelium (Fig. 5b). It seems likely that the digestive cells have additional endocrine-like function. Neither replacement cells nor mitotic figures were observed in the midgut epithelium of A. baccarum. This situation is typical for most acariform mites examined up to now (Alberti and Coons, 1999). The process of cell replacement is still unclear in these arachnids. Acknowledgements The author is deeply grateful to Dr. I. Badanin from Zoological Institute (Ukrainian Academy of Sciences) for the material supplied and to the engineers A.E. Tenisson and P.I. Henkin (Zoological Institute of Russian Academy of Sciences) for their technical assistance. The work was supported by the Russian Foundation for Basic Research; grant No. 06-04-48538-a. References Akimov, L.A., Gorgol, V.T., 1990. Predatory and Parasitic Mites e Cheyletidae. Naukova Dumka, Kiev, 120 p (In Russian). Alberti, G., 1973. Erna¨hrungsbiologie und Spinnvermo¨gen der Schnabelmilben (Bdellidae, Trombidiformes). Zeitschrift f}ur Morphologie der Tiere 76, 285e338. Alberti, G., Coons, L.B., 1999. Acari: mites. In: Harrison, F.W., Foelix, R.F. (Eds.), Microscopic Anatomy of Invertebrates, vol. 8C. Wiley-Liss, New York, pp. 515e1217. Alberti, G., Crooker, A.R., 1985. Internal anatomy. In: Helle, W., Sabelis, M.V. (Eds.), Spider Mites. Their Biology, Natural Enemies and Control, vol. 1A. Elsevier, Amsterdam, pp. 29e62. Alberti, G., Storch, V., 1983. Zur Ultrastruktur der Mitteldarmdru¨ssen von Spinnentieren (Scorpiones, Araneae, Acari) unter verschiedenen Erna¨hrungsbedingungen. Zoologischer Anzeiger 3/4, 145e160. Blauvelt, W.E., 1945. The internal anatomy of the common red spider mite (Tetranychus telarius L.). Cornell University Experiment Station Memoirs 270, 1e46. Becker, A., Peters, W., 1985. Fine structure of the midgut gland of Phalangium opilio (Chelicerata, Phalangida). Zoomorphology 105, 317e325. Desch Jr., C.E., 1988. The digestive system of Demodex folliculorum (Acari: Demodicidae) of man: a light and electron microscope study. In:
309
Channabasavanna, G.P., Viraktamath, C.A. (Eds.), Progress in Acarology, vol. 1. Oxford and IBH Publishing, New Delhi, pp. 187e195. Ehrnsberger, R., 1984. Anatomie des Verdauungstraktes der Rhagidiidae (Acari, Trombidiformes). Osnabru¨cker Naturwissenschaftliche Mitteilungen 11, 67e90. Filimonova, S.A., 2001. The fine structure of the midgut in the mite Myobia murismusculi. Cytology 43, 425e431 (In Russian). Goyffon, M., Martoja, R., 1983. Cytophysiological aspects of digestion and storage in the liver of scorpion, Androctonus australis (Arachnida). Cell and Tissue Research 228, 661e675. 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, 709e718. Ludwig, M., Alberti, G., 1990. Peculiarities of arachnid midgut glands. Acta Zoologica Fennica 190, 255e259. Ludwig, M., Alberti, G., 1992. Fine structure of the midgut of the Prokoenenia wheeleri (Arachnida: Palpigradi). Zoologische Beitrage 34, 127e134. Mitchell, R., 1964. The anatomy of an adult chigger mite Blankaartia acuscutellaris (Walch). Journal of Morphology 114, 373e391. Mitchell, R., 1970. The evolution of a blind gut in trombiculid mites. Journal of Natural History 4, 221e229. Mothes, U., Seitz, K., 1981. Functional microscopic anatomy of the digestive system of Tetranychus urticae (Acari, Tetranychidae). Acarologia 22, 257e270. Norton, R.A., Kethley, J.B., Johnston, D.E., O’Connor, B.M., 1993. Phylogenetic perspectives on genetic systems and reproductive modes of mites. In: Wrensch, D.L., Ebbert, M.A. (Eds.), Evolution and Diversity of Sex Ratio in Insects and Mites. Chapman & Hall, New York, London, England, pp. 8e99. Nuzzaci, G., Alberti, G., 1996. Internal anatomy and physiology. In: Lindquist, E.E., Sabelis, M.W., Bruin, J. (Eds.), Eriophyoid Mites e Their biology, Natural Enemies and Control. World Crop Pests, vol. 6. Elsevier, Amsterdam, pp. 101e150. 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, 28e37 (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, 227e239. Vistorin, H.E., 1980. Erna¨hrungsbiologie und Anatomie des Verdauungstraktes der Nicoletiellidae (Acari, Trombidiformes). Acarologia 21, 204e215. Vistorin-Theis, G., 1978. Anatomische Untersuchungen an Caliptostomiden (Acari, Trombidiformes). Acarologia 19, 242e257. Wright, K.A., Newell, J.M., 1964. Some observations on the fine structure of the midgut of the mite Anystis sp. Annals of the Entomological Society of America 57, 684e693.