The fine structure of the midgut epithelium in a centipede, Scolopendra cingulata (Chilopoda, Scolopendridae), with the special emphasis on epithelial regeneration

Arthropod Structure & Development 43 (2014) 27e42

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The fine structure of the midgut epithelium in a centipede, Scolopendra cingulata (Chilopoda, Scolopendridae), with the special emphasis on epithelial regeneration qukasz Chajec, Lidia Sonakowska, Magdalena M. Rost-Roszkowska* Department of Animal Histology and Embryology, University of Silesia, Bankowa 9, Katowice 40-007, Poland

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 March 2013 Accepted 19 June 2013

Scolopendra cingulata has a tube-shaped digestive system that is divided into three distinct regions: fore-, mid- and hindgut. The midgut is lined with a pseudostratified columnar epithelium which is composed of digestive, secretory and regenerative cells. Hemocytes also appear between the digestive cells of the midgut epithelium. The ultrastructure of three types of epithelial cells and hemocytes of the midgut has been described with the special emphasis on the role of regenerative cells in the protection of midgut epithelium. The process of midgut epithelium regeneration proceeds due to the ability of regenerative cells to proliferate and differentiate according to a circadian rhythm. The regenerative cells serve as unipotent stem cells that divide in an asymmetric manner. Additionally, two types of hemocytes have been distinguished among midgut epithelial cells. They enter the midgut epithelium from the body cavity. Because of the fact that numerous microorganisms occur in the cytoplasm of midgut epithelial cells, we discuss the role of hemocytes in elimination of pathogens from the midgut epithelium. The studies were conducted with the use of transmission electron microscope and immunofluorescent methods. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Midgut epithelium Regeneration Regenerative cells Hemocytes Ultrastructure Centipede

1. Introduction Myriapoda is a group of terrestrial invertebrates that live in most forests, grasslands and even dry deserts, where they fulfill a very important role in breaking down decaying plant and animal material and in soil decomposition. Therefore, they are treated as bioindicators of the natural environment (Triebskorn et al., 1991; Grgi c and Kos, 2005; de Godoy and Fontanetti, 2010). Studies connected with many organs of myriapods (Köhler, 2002; Nogarol and Fontanetti, 2011; Perez and Fontanetti, 2011; Souza et al., 2011) have shown that the midgut epithelium might be treated as the model organ which plays an important role in forming a barrier against external stress factors (Malagoli et al., 2010). Processes that are activated as a response to such factors are: cell death, regeneration ¼ the self-renewal of tissues and organs, the accumulation of spherites, synthesis of metallothioneins or antioxidants, etc. (Köhler et al., 1995; Descamps et al., 1996;

* Corresponding author. Tel.: þ48 32 3591 376; fax: þ48 32 2596 229. E-mail addresses: [email protected], [email protected] (M.M. Rost-Roszkowska). 1467-8039/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.asd.2013.06.002

Parthasarathy and Palli, 2007; Park and Takeda, 2008; Hakim et al., 2010; Rost-Roszkowska et al., 2010a,b, 2012; Chajec et al., 2012a; Franzetti et al., 2012). Cell and tissue regeneration enables cells which are damaged or disrupted by cell death, environmental stress factors or pathogens to be replaced. Additionally, regeneration is involved in embryogenesis, metamorphosis and the growth of tissues and organs (Hakim et al., 2010; Nardi et al., 2010). While many studies connected with Hexapoda have been performed, precise knowledge about the organs and tissues of myriapods is still poor (Rosenberg and Müller, 2009). The general morphology of the digestive system has been described (Balbiani, 1890; Kaufman, 1960, 1961, 1962; Shukla and Shukla, 1980; Lewis, 1981; Fantazzini et al., 1998; Miyoshi et al., 2005; Koch et al., 2009) with special emphasis on the ultrastructure of the ectodermal fore- and hindgut (Elzinga, 1998; Hilken and Rosenberg, 2009). Despite the fact that myriapods are treated as bio-indicators, many myriapods are nocturnal and their life is connected with the day/night cycle (Minelli, 1993). These kinds of organisms might be used in analysis of processes involved in homeostatic maintenance and in addressing the involvement of circadian rhythms in regulation of regenerative mechanisms.

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Therefore we have chosen the nocturnal centipede Scolopendra cingulata (Chilopoda, Scolopendridae) as the object of our studies, but we have also compared the processes of cell regeneration of S. cingulata with the well-known and widely distributed stone centipede Lithobius forficatus (Chajec et al., 2012a,b,c).

The aims of this study were (a) to describe the fine structure of all of the types of cells which form the midgut epithelium in the nocturnal predator S. cingulata, (b) to analyze the process of epithelium regeneration, (c) to determine whether the regenerative cells of the midgut fulfill the role of stem cells, (d) to determine whether the process of midgut regeneration occurs in a cyclic or

Fig. 1. A. Schematic illustration of the digestive system of Scolopendra cingulata: crop (cr), cardiac valve (cv), esophagus (eso), foregut (fg), proventriculus (pv), hindgut (hg), midgut (mg), Malpighian tubules (Mt), pharynx (ph), pyloric valve (pv), salivary glands (sg). B. Schematic illustration of the midgut epithelium of S. cingulata. Digestive cells (dc), hemocytes (hc), regenerative cells (rc), secretory cells (sc), muscles (mc), basal lamina (bl), midgut lumen (l). C. Transverse section of the pseudostratified columnar midgut epithelium formed by digestive cells (dc) and secretory cells (sc). Basal lamina (bl), visceral muscles (mc), hemocytes (arrows), Light microscope, bar ¼ 6.25 mm. D. Transverse section of the midgut epithelium. Digestive cells (dc), regenerative cells (arrow), midgut lumen (l). Light microscope, bar ¼ 7.5 mm.

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Fig. 2. A. Folds of the basal cell membrane (arrows). Mitochondria (m), cisterns of RER (RER), basal lamina (bl), hemocytes (hc), nucleus (n), secretory cells (sc). TEM, bar ¼ 1.7 mm. B. Gap junction (arrows), lipid droplets (ld). TEM, bar ¼ 0.55 mm. C. Perinuclear regions of digestive cells (dc) with elongated nuclei (n), glycogen granules (g), mitochondria (m), nucleoli (nu), cytoskeleton (arrows). TEM, bar ¼ 2 mm. D. The cytoplasm of the perinuclear region rich in mitochondria (m), cisterns of RER (RER) and SER (SER), Golgi complexes (d). Lipid droplets (ld), electron-dense spheres with proteins (p). TEM, bar ¼ 0.65 mm.

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continuous manner, (e) to state if day/night cycle might be involved in the regulation of regenerative processes, (f) to state what the role of regenerative cells is in the protection of the midgut epithelium and (g) to determine whether hemocytes help to protect the midgut epithelium.

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slides and washed in Tris-buffered saline (TBS; 5 min) and in 0.1% Triton X-100 in TBS (5 min). Eventually, the slides were stained with rhodamine-phalloidin (40 min, room temperature), washed in TBS (five times, 10 min each) and labeled with DAPI (40 ,6-diamidino-2phenylindole) (30 min). Slides were analyzed using an Olympus BX60 fluorescence microscope.

2. Materials and methods S. cingulata, as one of the smallest species among Scolopendridae, is widely distributed around the Mediterranean Sea in southern Europe and North Africa. It lives under stones, leaf litter and rocks and prefers a dark and damp environment. Animals used for our studies were reared in plastic boxes (20  15  6 cm) at a temperature of 22  C and humidity of about 70%. They were fed with the larvae of Tenebrio molitor, Acheta domesticus and adult specimens of Porcellio scaber. In order to determine whether midgut regeneration is cyclic or continuous, we examined the midguts of specimens that were fixed either during the day or during the night. 2.1. Light and transmission electron microscopy Adult specimens were decapitated (15 specimens at noon and 15 at midnight) and fixed with 2.5% glutaraldehyde in a 0.1 M sodium phosphate buffer (pH 7.4, 4  C, 2 h). Then the material was postfixed in 2% osmium tetroxide in a 0.1 M phosphate buffer (4  C, 1.5 h), dehydrated in a graded concentration series of ethanol (50, 70, 90, 95 and 100%, each for 15 min) and acetone (15 min) and eventually embedded in epoxy resin (Epoxy Embedding Medium Kit; Sigma). Semi- and ultra-thin sections were cut on a Leica Ultracut UCT25 ultramicrotome. Semi-thin sections (0.8 mm thick) were stained with 1% methylene blue in 0.5% borax and analyzed using an Olympus BX60 light microscope. Ultra-thin sections (70 nm) were stained with uranyl acetate and lead citrate and examined with a Hitachi H500 transmission electron microscope. 2.2. Histochemical methods 2.2.1. Detection of glycogen and polysaccharides (PAS method) Semi-thin sections were treated with a 2% solution of periodic acid to remove the osmium (10 min at room temperature) and stained with Schiff’s reagent (24 h, 37  C). Slides were analyzed using an Olympus BX60 light microscope.

2.2.4. BrdU labeling of proliferating cells Regenerative cell proliferation was identified using labeling with 5-bromo-20 -deoxyuridine-50 -monophosphate e BrdU (Roche). Fourteen animals (7 at noon and 7 at midnight) were injected with 50 mg BrdU/kg body weight. After 1 h the specimens were sacrificed and their midguts were isolated. The material was then fixed in Karnovsky’s fixative (4% paraformaldehyde and 2.5% glutaraldehyde) (30 min, room temperature), washed in TBS with 0.1% Triton X-100 (5 min, room temperature) and embedded in a tissue-freezing medium (Jung). Cryostat sections (5 mm thick) were placed on 1% gelatin-coated slides. After washing in TBS (1  3 min) and incubation in 2N HCl (60 min, 37  C), the material was incubated with 50 mg/ml anti-BrdU antibodies conjugated to fluorescein (Roche), diluted in TBS with 0.1% BSA (1 h, room temperature), washed in TBS (2  5 min) and analyzed using an Olympus BX60 fluorescence microscope. 2.2.5. Immunolabeling with anti-phosphohistone H3 The midguts isolated from 10 specimens (5 decapitated at noon, 5 at midnight) were embedded in a tissue-freezing medium (Jung) without fixation. Cryostat sections (5 mm thick) were mounted on 1% gelatin-coated slides, washed in TBS (5 min, room temperature) then 0.1% Triton X-100 in TBS (5 min, room temperature) and incubated in 1% BSA in TBS (30 min, room temperature). The material was then stained overnight in a 1:100 dilution of antiphosphohistone H3 antibodies (Millipore) in 1% BSA in TBS (room temperature). After washing with TBS (2  5 min, room temperature), it was incubated in a 1:200 dilution of goat anti-rabbit IgG Alexa-Fluor 488 (Sigma) conjugated secondary antibodies in 1% BSA in TBS (1 h, room temperature), washed in TBS (2  5 min, room temperature) and mounted in 50% glycerol in TBS. The sections were analyzed using an Olympus BX60 fluorescence microscope.

3. Results 3.1. Structure of the digestive system of Scolopendra cingulata

2.2.2. Detection of proteins Semi-thin sections were treated with a 1% solution of periodic acid to remove the osmium (10 min at room temperature) and stained with bromophenol blue (BPB) (24 h, 37  C). Slides were analyzed using an Olympus BX60 light microscope. 2.2.3. Labeling of nuclei and actin filaments Isolated midguts from five specimens were embedded without fixation in a tissue-freezing medium (Jung) and frozen rapidly. Cryostat sections (5 mm thick) were placed on 1% gelatin-coated

The digestive system of S. cingulata has a tube-like shape without any digestive diverticulae or caeca. Three distinct regions can be distinguished: the foregut (pharynx, esophagus, crop and proventriculus ¼ gizzard), midgut and hindgut. The cardiac valve is located between the foregut and midgut, while the pyloric valve is between the midgut and hindgut (Fig. 1A). The foregut, which is the longest region of the digestive system, occupies about 1/2 of its length, while the hindgut is the shortest (about 1/10 of its length).

Fig. 3. A. The apical region of digestive cells (dc). The apical membrane forms microvilli (mv) and the cytoplasm with numerous vesicles with electron-lucent content (arrows), mitochondria (m) and cisterns of RER (RER). Endosymbionts (es), multivesicular bodies (mvb), midgut lumen (l), electron-dense spheres with proteins (p). TEM, bar ¼ 0.83 mm. B. Actin filaments which form the actin cortex stained red with rhodamine-phalloidin (arrows) in the apical cytoplasm. Nuclei are labeled with DAPI (blue). Midgut epithelium (e), midgut lumen (l), visceral muscles (mc). Fluorescence microscope, bar ¼ 23.8 mm. C. Multivesicular bodies (mvb) accumulated in the neighborhood of the apical cell membrane and numerous small vesicles (arrows). Mitochondria (m), endosymbionts (es), electron-dense spheres with proteins (p). TEM, bar ¼ 0.52 mm. D. Spherites (sp) in the apical cytoplasm of some of digestive cells (dc). Mitochondria (m), cisterns of SER (SER). TEM, bar ¼ 1.2 mm. E. Smooth septate junction (arrow) between apical regions of adjacent digestive cells (dc). Microvilli (mv). TEM, bar ¼ 0.33 mm.

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3.2. Structure of the midgut epithelium of Scolopendra cingulata The midgut is lined with a pseudostratified columnar epithelium in which all cells have contact with the thick and strongly folded basal lamina; however, not all cells reach the midgut lumen (Fig. 1B). The midgut epithelium is surrounded by visceral muscles: circular muscles (the inner layer) and longitudinal muscles (the external layer) (Fig. 1B); it is covered on its luminal surface by a peritrophic membrane (not shown). Three types of cells were distinguished in the midgut epithelium: digestive cells (Fig. 1Be D), secretory cells (Fig. 1B,C) and regenerative cells (Fig. 1B,D). Hemocytes also occurred between the midgut digestive cells (Fig. 1B,C). 3.3. Digestive cells and the types of secretion The digestive cells have columnar shapes and contact the midgut lumen and basal lamina (Fig. 1B,C). Their cytoplasm shows regionalization in the distribution of organelles that form three distinct regions: the basal, perinuclear and apical. The basal membrane forms numerous folds and invaginations; numerous mitochondria, free ribosomes and cisterns of the rough endoplasmic reticulum (RER) accumulate in their neighborhood (Fig. 2A). Gap junctions occur between the basal and perinuclear regions of adjacent digestive cells (Fig. 2B). Lateral membranes are strongly folded (Fig. 2C). The perinuclear cytoplasm has an elongated nucleus with distinct patches of heterochromatin near the nuclear envelope and a large homogenous nucleolus (Fig. 2C). Numerous mitochondria, cisterns of RER and SER, Golgi complexes (Fig. 2D) and cytoskeleton (Fig. 2C) are also gathered near the nucleus. The apical cytoplasm is the most extensive and occupies approximately half of each cell’s height. The apical membrane forms long microvilli (Fig. 3A), which possess their own cytoskeleton. Actin filaments entering the apical cytoplasm form the actin cortex (Fig. 3B). The apical cytoplasm is rich in elongated mitochondria, cisterns of RER and SER, free ribosomes, and rare vacuoles and vesicles with an electron-lucent content (Fig. 3A). Near the apical membrane, multivesicular bodies mainly gather in the neighborhood of the apical cell membrane where they disintegrate releasing small vesicles (Fig. 3A,C). Spherites occur in both the perinuclear and apical cytoplasm (Fig. 3D). Smooth septate junctions (zonula continua) are found between the apical regions of adjacent digestive cells (Fig. 3E). The reserve material accumulates in the cytoplasm of the digestive cells: numerous spheres with homogenous or heterogenous and electron-dense content occur in the apical and perinuclear regions (Figs. 2D and 3A). They contain proteins (Fig. 4A) or proteins and saccharides (Fig. 4B). Glycogen granules also appear in the neighborhood of the nucleus (Fig. 2C), while lipid droplets are observed in the entire cytoplasm of the digestive cell (Fig. 2D). Two types of secretion occur in the digestive cells: merocrine (Fig. 4C) and apocrine (Fig. 4D,E). During merocrine secretion, small and electron-lucent vesicles move toward the apical membrane, fuse with it and release their contents into the midgut lumen (Fig. 4C). During apocrine secretion, the apical membrane loses the microvilli and forms a large evagination into

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the midgut lumen (Fig. 4D). Eventually, the evagination is separated from the entire cell and is discharged into the midgut lumen (Fig. 4E). 3.4. Secretory cells Secretory cells, which are sparsely distributed among digestive cells (Fig. 1B,C), do not contact the midgut lumen and assume a pear-like shape. They extend a narrow cytoplasmic process to the basal lamina (Figs. 2A and 5A); and their basal membranes are devoid of any folds. The cytoplasm does not show a regionalization in the distribution of organelles. An oval nucleus with small patches of heterochromatin and homogenous nucleolus occupies the widest region of the cell (Figs. 2A and 5A,B). The entire cytoplasm is rich in numerous small granules of different electron densities (Fig. 5A,B), which accumulate mainly proteins or saccharides (Fig. 4A,B). Many mitochondria and cisterns of RER appear in the neighborhood of the nucleus (Fig. 5B). Intercellular junctions between the secretory and digestive cells were not observed. 3.5. Regenerative cells Regenerative cells are individually distributed among the digestive cells along the entire length of the midgut. They possess a pear-like shape with a very long and thin strand of the cytoplasm that has contact with the basal lamina (Chajec et al., 2012c). The largest region of the cell is shifted toward the perinuclear or even the apical region of the digestive cell, but it does not have contact with the midgut lumen (Fig. 6A). During the interphase, the oval nucleus possesses small patches of heterochromatin located near the nuclear envelope and an oval and homogenous nucleolus (Fig. 6B). The electron-lucent cytoplasm of the regenerative cells (Fig. 6A) is poor in organelles: only sporadic mitochondria, cisterns of RER and free ribosomes, lipid droplets, electron-dense granules, spherites, multivesicular bodies, autophagosomes and small vacuoles appear (Fig. 6A,C). Intercellular junctions between the regenerative cell and adjacent digestive or secretory cells are absent. The mitotic divisions of regenerative cells (Fig. 6DeF) depend on the day/night cycle: during the day the cell is in the interphase, while during the night it begins to divide to form two daughter cells (Fig. 7A,B). During the division, the regenerative cell elongates slightly toward the midgut lumen and all organelles segregate in the neighborhood of the two poles of the mitotic spindle (Fig. 6DeF). However, the mitotic cell establishes contact with the midgut lumen before the end of the mitosis (Fig. 6E). The nuclear envelope is formed just after the mitotic division (Figs. 6F and 7D) and then cytokinesis begins (Fig. 7C). One of the two daughter cells differentiates into the digestive cell, while the second one fulfills the role of a regenerative cell with its characteristic ultrastructure as described above (Fig. 7D). During the differentiation, the regenerative cell changes its shape into a columnar one. The apical membrane of the differentiating cell gradually forms microvilli which protrude into the midgut lumen (Fig. 6A). The nucleus of the differentiating cell moves into the perinuclear region of the epithelium. The cellular

Fig. 4. A. BPB-positive granules (arrows) in the midgut epithelium (e) of S. cingulata. Basal lamina (bl), digestive cells (dc), hemocytes (hc), secretory cell (sc), visceral muscles (mc). Light microscope, bar ¼ 5.8 mm. B. PAS-positive granules (arrows) in the midgut epithelium (e). Basal lamina (bl), digestive cells (dc), secretory cells (sc), visceral muscles (mc). Light microscope, bar ¼ 5.8 mm. C. Merocrine secretion. Electron-lucent vesicles (arrows) fuse with the apical cell membrane of digestive cells (dc). Midgut lumen (l), vacuole (v). TEM, bar ¼ 0.46 mm. D. Apocrine secretion: the portion of the apical membrane of digestive cells (dc) devoid of microvilli forms large protrusions (arrows) into the midgut lumen (l). Mitochondria (m). TEM, bar ¼ 1.28 mm. E. Apocrine secretion. Apical vesicle (star) is expelled from the cell into the midgut lumen (l). Microvilli (mv). TEM, bar ¼ 1 mm.

Fig. 5. A. The ultrastructure of secretory cell (sc). Numerous electron-dense granules (arrows), digestive cells (dc), mitochondria (m), large nucleus (n), nucleolus (nu). TEM, bar ¼ 1.67 mm. B. The cytoplasm of secretory cell (sc) with mitochondria (m) and cisterns of RER (RER). Digestive cells (dc), nucleus (n), nucleolus (nu), electron-dense granules (arrows). TEM, bar ¼ 1.1 mm.

Fig. 6. A. The ultrastructure of regenerative cells in the midgut epithelium of S. cingulata. Regenerative cells (rc1) with electron-lucent cytoplasm distributed between digestive cells (dc). Regenerative cells are separated from the midgut lumen (l) by thin fragments of the apical cytoplasm of digestive cells (dc). Cisterns of RER (RER), endosymbionts (es), mitochondria (m), microvilli (mv), differentiating cell (rc2), electron-dense granules (arrows), spherite (sp), smooth septate junction (ssj). TEM, bar ¼ 0.83 mm. B. Oval nuclei (n) of regenerative cells (rc). Digestive cells (dc), nucleolus (nu). TEM, bar ¼ 1.3 mm. C. Electron-lucent cytoplasm of regenerative cells poor in organelles. Autophagosomes (au), cisterns of RER (RER), digestive cells (dc), lipid droplets (ld), mitochondria (m), spherite (sp). TEM, bar ¼ 1.2 mm. D. Mitotic division of the regenerative cell (rc). Chromosomes (ch), digestive cells (dc), midgut lumen (l), mitochondria (m), electron-dense granules (arrows). TEM, bar ¼ 1.1 mm. E. The organelles form two groups near the mitotic spindle poles. The regenerative cell (rc) contacts (arrow) the midgut lumen (l) before the end of mitosis. Chromosomes (ch), digestive cell (dc), endosymbionts (es), lipid droplets (ld), mitochondria (m). TEM, bar ¼ 1 mm. F. After the mitotic division of the regenerative cell (rc), the nuclear envelopes of two daughter nuclei (n) are formed. Digestive cells (dc), endosymbionts (es), microvilli (mv). TEM, bar ¼ 1.4 mm.

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Fig. 7. A. Fluorescence micrograph showing BrdU-labeled regenerative cells (green) (arrows). Longitudinal section of the midgut epithelium (e). Midgut lumen (l), visceral muscles (mc), basal lamina (arrowhead). Fluorescence microscope, bar ¼ 29 mm. B. Fluorescence micrograph showing anti-phosphohistone H3 (green) regenerative cell (arrow). Cross section of the midgut epithelium (e). Midgut lumen (l), muscles (mc), basal lamina (arrowhead). Fluorescence microscope, bar ¼ 8.1 mm. C. Cytokinesis (arrows). Digestive cells (dc), newly formed regenerative cells (rc), mitochondria (m), nuclei (n). TEM, bar ¼ 0.8 mm. D. After the mitosis one of newly formed daughter cells differentiates (rc2) into the digestive cell, while the second one plays a role as the midgut regenerative cell (rc1). Cisterns of SER (SER), digestive cells (dc), electron dense granules (black arrows), mitochondria (m), nucleus (n), reserved material (white arrow). TEM, bar ¼ 1.2 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

content of reserved material gradually increases (Fig. 7D). Regionalization in the distribution of organelles, which have accumulated in cytoplasm, and intercellular junctions appear between newly formed and digestive cells (Fig. 6A). Regenerative cells differentiate into only digestive ones; they do not form secretory cells.

3.6. Hemocytes Hemocytes were found in all of the specimens analyzed, both males and females, between visceral muscles and just beneath the basal lamina (Chajec et al., 2012b). They are able to move through

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the basal lamina (Fig. 8A) and enter the midgut epithelium, especially its basal and perinuclear regions (Fig. 8B). According to differences in the ultrastructure of hemocytes, two types of these cells can be distinguished (Fig. 8B). The majority of hemocytes (about 85%) belong to I type, while the II type is rather rare (about 15%). Large hemocytes of the I type possess an ameboid shape. An irregular nucleus is located in the central cytoplasm which has numerous small and irregular electron-dense granules (Fig. 8BeD). Many vacuoles with parallel strands of a crystalline or tubular material occur (Fig. 8D) together with numerous mitochondria, cisterns of SER and RER, Golgi complexes, vesicles with an electronlucent content, multivesicular and lamellar bodies and autophagosomes (Fig. 8C). All organelles accumulate in the neighborhood of the nucleus making the cortical cytoplasm poor in organelles (Fig. 8C). Smaller hemocytes of the II type are much more regular in shape and possess a large but lobular nucleus with a small nucleolus. The characteristic feature of these hemocytes is the presence of large, irregular and electron-dense granules (Fig. 8B,E,F). Mitochondria, single cisterns of RER, free ribosomes, vacuoles, lamellar bodies, autophagosomes and vesicles with an electron-lucent content accumulate near the nucleus, hence the cortical cytoplasm is poor in organelles (Fig. 8E,F). Intercellular junctions between hemocytes and midgut epithelium cells are absent. 3.7. Microorganisms Numerous bacillus-like microorganisms which are probably endosymbionts were observed in the cytoplasm of digestive cells in all of the specimens analyzed, both males and females. They mainly accumulate in the apical cytoplasm near the apical cell membrane (Figs. 3A,C and 9A). In addition, some endosymbionts occur in the cytoplasm of regenerative cells (Fig. 6AeF) and in the midgut lumen. Gregarines are a second type of microorganisms observed in about 10% of specimens. They accumulate in both the cytoplasm of digestive cells and in the midgut lumen near the apical cell membranes (Fig. 9B,C). 4. Discussion Myriapods live in soil and litter so they are exposed to both pathogens and endosymbionts, which may enter the body through the digestive system. Endosymbionts are responsible for synthesizing the digestive enzymes, supplying nutrients and protecting the organism against pathogens (Crawford et al., 1983; Tajovsky, 1992). However, to date only pathogens have been described in the digestive system of Chilopoda (Lewis, 1981). Bacillus-like microorganisms have been found in the apical cytoplasm of the digestive cells in both S. cingulata and L. forficatus (our observation). Endosymbionts commonly occur in the midgut, midgut muscles or gonads of arthropods, where they are present in all specimens of the culture. They can be transmitted from one generation to the _  ski, 1999; Szklarzewicz next transovarially (Zelazowska and Bilin et al., 2006; Nehme et al., 2007). Because the microbes are present in the apical cytoplasm of digestive cells and in the cytoplasm of regenerative cells in all of analyzed specimens of S. cingulata, we postulate that they must play a role of endosymbionts. Additionally they are not discharged from the cell when apocrine secretion occurs (see Fig. 4D). Numerous endopathogens appear in Chilopoda, e.g. pathogenic bacteria, pathogenic fungi, gregarines, coccidia, ciliates or even insects and leeches (Lewis, 1967, 1981). Coccidia and gregarines are characteristic for Lithobiomorpha and Scolopendromorpha

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(Singotam and Dass, 1977; Lewis, 1981). While in L. forficatus rickettsia-like microorganisms, coccidia and gregarines are present (our unpublished observation), only gregarines appear in S. cingulata. Pathogens which enter the midgut lumen may move through the midgut epithelium and its basal lamina and eventually infect the entire organism. Therefore, the midgut epithelium must play a role as a barrier against infection. 4.1. Regenerative cells and midgut regeneration Regenerative cells are responsible for the regeneration of the epithelium in the digestive system of animals, especially their middle region ¼ midgut. They are treated as multipotential midgut stem cells (Loeb and Hakim, 1996; Hakim et al., 2001, 2010; Loeb et al., 2001; Baton and Ranford-Cartwright, 2007; Parthasarathy and Palli, 2007, 2008; Casali and Batlle, 2009; Cruz et al., 2011; Mehrabadi et al., 2012; Teixeira et al., 2013). In Arthropoda, especially in Hexapoda, regenerative cells form groups: regenerative nests (Hung et al., 2000; Rost, 2006b; Rost-Roszkowska et al., 2010c; Mehrabadi et al., 2012) or regenerative crypts (Bigham, 1931; Raes et al., 1994; Wanderley-Teixeira et al., 2006; Nardi et al., 2010), or they are individually distributed among digestive cells (Rost, 2006a; Okuda et al., 2007; Nardi et al., 2009; Roelfstra et al., 2010; Rost-Roszkowska et al., 2010a). Likewise, in some species of Myriapoda, regenerative cells form regenerative crypts (Kaufman, 1961; Lewis, 1981; Minelli, 1993), or are individually distributed along the entire length of the midgut (Köhler and Alberti, 1992; Fontanetti et al., 2001; Fantazzini et al., 2002; Camargo-Mathias et al., 2004; de Godoy and Fontanetti, 2010; Nogarol and Fontanetti, 2011; Chajec et al., 2012a,c). However, their ultrastructure and the role in tissue regeneration have not been described (Balbiani, 1890; Léger and Duboscq, 1902; Kaufman, 1961). In S. cingulata as in L. forficatus (Chajec et al., 2012a,c), regenerative cells are poor in organelles, which start to accumulate in the cytoplasm just after the beginning of cell differentiation. Numerous mitochondria form two groups in the neighborhood of regenerative cell nuclei in L. forficatus, while in S. cingulata they are regularly distributed around the nucleus. Their high number suggests that a great amount of energy must be supplied for their proper proliferation and differentiation. Additionally, the reserve material gathered in the cytoplasm of regenerative cells supports such a hypothesis. The cytoplasm of the regenerative cells of S. cingulata is rich in autophagosomes and multivesicular bodies, which have not been found in L. forficatus (Chajec et al., 2012a). Autophagy promotes both cell survival and cell death. Since autophagy eliminates damaged organelles and proteins, it is considered as a survival mechanism (Das et al., 2012). The absence of autophagosomes in the cytoplasm of regenerative cells in L. forficatus, might be connected with the fact that they are located in the basal region of the midgut epithelium, and consequently they are shielded to some extent from mechanical damage and toxic substances that come from the midgut lumen (Chajec et al., 2012a). However, in S. cingulata, regenerative cells reach the apical region of the midgut epithelium and are separated from the midgut lumen by only thin processes of adjacent digestive cells. Therefore, they are exposed to factors that injure the entire epithelium, and autophagosomes and spherites accumulated in the cytoplasm take part in protecting the epithelium (Vandenbulcke et al., 1998; Pigino et al., 2005; Dwivedi and Ahnn, 2009; Rost-Roszkowska et al., 2012). Cyclic regeneration has been found to be connected with molting in many arthropods (Humbert, 1979; Rost, 2006a,b). In S. cingulata proliferation combined with the day/night cycle is

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probably caused not only by molting but also by other factors. First of all, as this species is nocturnal, it hunts and feeds during the night and it avoids the light by hiding under stones and digging itself in the soil. Another factor that causes the proliferation to be cyclic might be the structure of the digestive system. In S. cingulata the foregut is the longest part and is differentiated into the pharynx, esophagus, crop and gizzard lined with cuticle and possessing structures made of chitin that cut and chop the food (Lewis, 1981; Koch et al., 2009). Consequently, finely macerated food enters the midgut lumen without evident abrasion of midgut epithelium. Stem cells can divide in a symmetric or asymmetric way. During symmetric division, two identical daughter cells appear. In the case of asymmetric division, one stem cell and one progenitor cell are produced (Loeb and Hakim, 1996; Morrison et al., 1997; Hakim et al., 2001; Teixeira et al., 2013). In Myriapoda regenerative cells presumably differentiate into both the digestive and secretory cells of the midgut epithelium (Balbiani, 1890; Lewis, 1981). However, this process has not been described at the ultrastructural level, and therefore it has not been confirmed. In S. cingulata as in L. forficatus (Chajec et al., 2012a,c), regenerative cells divide in an asymmetric manner to form one regenerative cell ¼ midgut stem cell and one midgut progenitor cell, which starts to differentiate into a digestive cell. The midgut progenitor cell in S. cingulata as in L. forficatus has not been found to differentiate into a secretory cell. Because secretory cells in both species do not have contact with the midgut lumen, they are not exposed to any mechanical damage caused by food, toxic substances or pathogens, so they probably do not have to be renewed. Therefore, we postulate that the regenerative cells in S. cingulata play the role of unipotent stem cells. 4.2. Hemocytes and their role in the protection of the organism Hemocytes are cells that are observed in hemocoels of both millipedes (de Godoy and Fontanetti, 2010; Nogarol and Fontanetti, 2011; Perez and Fontanetti, 2011) and centipedes (Nevermann et al., 1991; Xylander, 2009a; Chajec et al., 2012a). However, they have also been observed between muscles or in the neighborhood of the fat body in the hemocoel (de Godoy and Fontanetti, 2010; Nogarol and Fontanetti, 2011; Perez and Fontanetti, 2011). As in insects, they are able to migrate and disperse throughout the body (Tepass et al., 1994; Nardi et al., 2003). During our studies on the midgut of centipedes, we found that hemocytes may enter the midgut epithelium extending through its basal lamina, where they can be observed between the digestive, secretory and regenerative cells, as has been described in L. forficatus (Chajec et al., 2012a) and S. cingulata (Chajec et al., 2012b). It is worth noting that the number of hemocytes in centipedes is much higher than in millipedes (Xylander, 1992, 2009a), a difference that might be attributed to the fact that hepatic cells are observed together with hemocytes in millipedes (Hopkin and Read, 1992; Fontanetti et al., 2001; de Godoy and Fontanetti, 2010; Perez and Fontanetti, 2011). Among all of the types of hemocytes described in Arthropoda, six of them: prohemocytes ¼ prehemocytes, plasmatocytes,

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granulocytes ¼ granular hemocytes, spherulocytes, coagulocytes and “discoid cells” occur in the hemocoels of Myriapods (Nevermann et al., 1991; Hilken et al., 2003; Xylander and Nevermann, 2006; Xylander, 2009a). The hemocytes observed in S. cingulata resemble plasmatocytes and granulocytes. Large plasmatocytes are able to migrate, and they possess numerous vacuoles with lamellar or crystalline material within their cytoplasm along with some small electron-dense granules. The cytoplasm of smaller granulocytes accumulates numerous irregular granules with an electron-dense content (Nevermann et al., 1991; Xylander and Nevermann, 2006; Xylander, 2009a). Based on this observation, presumably all hemocytes of L. forficatus are granulocytes, while hemocytes I of S. cingulata resemble plasmatocytes and hemocytes II are granulocytes. Hemocytes are responsible for hemolymph coagulation after damage to the cuticle and epidermis, healing of injuries, opsonization and phagocytosis of pathogens and the synthesis of antibacterial proteins (Gupta, 1986; Xylander, 1992, 2009a,b; Lavine and Strand, 2002). In L. forficatus (Chajec et al., 2012a) and S. cingulata ameboid hemocytes migrate from the hemocoel through the basal lamina into the midgut epithelium in order to protect this epithelium, and eventually the entire organism, against infection by pathogens. In L. forficatus hemocytes accumulate among the midgut epithelial cells only in infected specimens. Therefore, we suggest their protective role against pathogenic infection (Chajec et al., 2012a). However, in S. cingulata those cells occur between the digestive cells in both infected and non-infected specimens. Hemocytes in S. cingulata possibly play a role not only against infection, but also against toxic substances that may enter the organism from the midgut lumen. As is known in Diplopoda, hemocytes accumulate among cells of the fat body that surrounds the midgut in specimens treated with toxic metals. When numerous degenerated cells of the fat body appeared, the number of hemocytes in the fat body increased (de Godoy and Fontanetti, 2010; Perez and Fontanetti, 2011). In S. cingulata hemocytes which accumulate among midgut epithelial cells possess many autophagosomes, which take part in the elimination of organelles, proteins and pathogens (Franzetti et al., 2012; Rost-Roszkowska et al., 2012).

5. Conclusions (a) The midgut epithelium of S. cingulata is composed of digestive, secretory and regenerative cells, and hemocytes interspersed among cells of epithelium; (b) the process of midgut epithelium regeneration proceeds due to the ability of regenerative cells to proliferate and differentiate; (c) regenerative cells fulfill the role of unipotent midgut stem cells which divide in an asymmetric manner; (d) midgut regeneration occurs in a cyclic manner which depends on day/night cycle; (e) intensive mitotic divisions of regenerative cells help maintain the integrity of the midgut epithelium in the event of pathogenic infection and (f) hemocytes presumably can eliminate pathogens from the midgut epithelium.

Fig. 8. A. Hemocytes (hc) penetrate the basal lamina (bl). Midgut epithelium (e), visceral muscles (mc). TEM, bar ¼ 2.5 mm. B. Hemocytes of the I (hc1) and II types (hc2) among basal regions of the digestive cells (dc). Nuclei (n). TEM, bar ¼ 3.3 mm. C. The ultrastructure of hemocytes I (hc1). All organelles and electron-dense granules (black arrows) accumulated in the central cytoplasm, while the cortical cytoplasm (star) is electron-lucent and poor in organelles. Autophagosomes (au), cisterns of RER (RER), digestive cells (dc), Golgi complexes (d), mitochondria (m), multivesicular bodies (mvb), nucleus (n), small electron-lucent vesicles (white arrows). TEM, bar ¼ 1 mm. D. Hemocytes I (hc1). Numerous vesicles (arrows) with fibrillar material, mitochondria (m), nucleus (n). TEM, bar ¼ 0.53 mm. E. The ultrastructure of hemocytes II (hc2). Numerous large electron-dense granules (g). Lobular and irregular nucleus (n) with distinct patches of heterochromatin. Cisterns of RER (RER), cortical cytoplasm (star), digestive cells (dc), lamellar bodies (lb) mitochondria (m), vacuoles (v), electron-lucent vesicles (arrows). TEM, bar ¼ 1.04 mm. F. Hemocytes II (hc2). Cisterns of RER (RER), cortical cytoplasm (star), digestive cells (dc), Golgi complexes (d), electrondense granules (g), mitochondria (m), nucleus (n), vacuoles (v). TEM, bar ¼ 0.92 mm.

Fig. 9. A. Endosymbionts (arrows) accumulated in the apical cytoplasm of digestive cells (dc). Mitochondria (m), multivesicular bodies (mvb). TEM, bar ¼ 0.26 mm. B. Gregarines (star) in the cytoplasm of the digestive cells (dc) in infected specimens of S. cingulata. Nucleus of gregarine (ng). TEM, bar ¼ 1.96 mm. C. Gregarines (star) in contact with the apical cell membrane of the digestive cells (dc). Microvilli (mv). TEM, bar ¼ 1.94 mm.

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