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Arthropod Structure & Development 43 (2014) 27e42

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Arthropod Structure & Development

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The fine structure of the midgut epithelium in a centipede,Scolopendra cingulata (Chilopoda, Scolopendridae), with the specialemphasis 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

Article history:Received 5 March 2013Accepted 19 June 2013

Keywords:Midgut epitheliumRegenerationRegenerative cellsHemocytesUltrastructureCentipede

* Corresponding author. Tel.: þ48 32 3591 376; faxE-mail addresses: magdalena.rost-roszkowska@

(M.M. Rost-Roszkowska).

1467-8039/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.asd.2013.06.002

a b s t r a c t

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 composedof digestive, secretory and regenerative cells. Hemocytes also appear between the digestive cells of themidgut epithelium. The ultrastructure of three types of epithelial cells and hemocytes of the midgut hasbeen described with the special emphasis on the role of regenerative cells in the protection of midgutepithelium. The process of midgut epithelium regeneration proceeds due to the ability of regenerativecells to proliferate and differentiate according to a circadian rhythm. The regenerative cells serve asunipotent stem cells that divide in an asymmetric manner.

Additionally, two types of hemocytes have been distinguished among midgut epithelial cells. Theyenter the midgut epithelium from the body cavity. Because of the fact that numerous microorganismsoccur in the cytoplasm of midgut epithelial cells, we discuss the role of hemocytes in elimination ofpathogens from the midgut epithelium. The studies were conducted with the use of transmissionelectron microscope and immunofluorescent methods.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Myriapoda is a group of terrestrial invertebrates that live inmost forests, grasslands and even dry deserts, where they fulfill avery important role in breaking down decaying plant and animalmaterial and in soil decomposition. Therefore, they are treated asbioindicators of the natural environment (Triebskorn et al., 1991;Grgi�c and Kos, 2005; de Godoy and Fontanetti, 2010). Studiesconnected with many organs of myriapods (Köhler, 2002; Nogaroland Fontanetti, 2011; Perez and Fontanetti, 2011; Souza et al.,2011) have shown that the midgut epithelium might be treatedas the model organ which plays an important role in forming abarrier against external stress factors (Malagoli et al., 2010). Pro-cesses that are activated as a response to such factors are: celldeath, regeneration ¼ the self-renewal of tissues and organs, theaccumulation of spherites, synthesis of metallothioneins or anti-oxidants, etc. (Köhler et al., 1995; Descamps et al., 1996;

: þ48 32 2596 229.us.edu.pl, mrost@us.edu.pl

All rights reserved.

Parthasarathy and Palli, 2007; Park and Takeda, 2008; Hakimet al., 2010; Rost-Roszkowska et al., 2010a,b, 2012; Chajec et al.,2012a; Franzetti et al., 2012). Cell and tissue regeneration enablescells which are damaged or disrupted by cell death, environmentalstress factors or pathogens to be replaced. Additionally, regener-ation is involved in embryogenesis, metamorphosis and thegrowth of tissues and organs (Hakim et al., 2010; Nardi et al.,2010).

While many studies connected with Hexapoda have beenperformed, precise knowledge about the organs and tissues ofmyriapods is still poor (Rosenberg and Müller, 2009). The gen-eral 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 ultrastructureof the ectodermal fore- and hindgut (Elzinga, 1998; Hilken andRosenberg, 2009). Despite the fact that myriapods are treatedas bio-indicators, many myriapods are nocturnal and their life isconnected with the day/night cycle (Minelli, 1993). These kindsof organisms might be used in analysis of processes involved inhomeostatic maintenance and in addressing the involvement ofcircadian rhythms in regulation of regenerative mechanisms.

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Therefore we have chosen the nocturnal centipede Scolopendracingulata (Chilopoda, Scolopendridae) as the object of ourstudies, but we have also compared the processes of cellregeneration of S. cingulata with the well-known and widelydistributed stone centipede Lithobius forficatus (Chajec et al.,2012a,b,c).

Fig. 1. A. Schematic illustration of the digestive system of Scolopendra cingulata: crop (cr), ca(mg), Malpighian tubules (Mt), pharynx (ph), pyloric valve (pv), salivary glands (sg). B. Schem(hc), regenerative cells (rc), secretory cells (sc), muscles (mc), basal lamina (bl), midgut lumeby digestive cells (dc) and secretory cells (sc). Basal lamina (bl), visceral muscles (mc), hemepithelium. Digestive cells (dc), regenerative cells (arrow), midgut lumen (l). Light microsc

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

rdiac valve (cv), esophagus (eso), foregut (fg), proventriculus (pv), hindgut (hg), midgutatic illustration of the midgut epithelium of S. cingulata. Digestive cells (dc), hemocytesn (l). C. Transverse section of the pseudostratified columnar midgut epithelium formedocytes (arrows), Light microscope, bar ¼ 6.25 mm. D. Transverse section of the midgutope, bar ¼ 7.5 mm.

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 involvedin the regulation of regenerative processes, (f) to state what the roleof regenerative cells is in the protection of the midgut epitheliumand (g) to determine whether hemocytes help to protect themidgut epithelium.

2. Materials and methods

S. cingulata, as one of the smallest species among Scolopen-dridae, is widely distributed around the Mediterranean Sea insouthern Europe and North Africa. It lives under stones, leaf litterand rocks and prefers a dark and damp environment. Animals usedfor our studies were reared in plastic boxes (20 � 15 � 6 cm) at atemperature of 22 �C and humidity of about 70%. They were fedwith the larvae of Tenebrio molitor, Acheta domesticus and adultspecimens of Porcellio scaber. In order to determine whethermidgut regeneration is cyclic or continuous, we examined themidguts of specimens that were fixed either during the day orduring the night.

2.1. Light and transmission electron microscopy

Adult specimens were decapitated (15 specimens at noon and15 at midnight) and fixed with 2.5% glutaraldehyde in a 0.1 Msodium phosphate buffer (pH 7.4, 4 �C, 2 h). Then the material waspostfixed in 2% osmium tetroxide in a 0.1 M phosphate buffer(4 �C, 1.5 h), dehydrated in a graded concentration series ofethanol (50, 70, 90, 95 and 100%, each for 15 min) and acetone(15 min) and eventually embedded in epoxy resin (EpoxyEmbedding Medium Kit; Sigma). Semi- and ultra-thin sectionswere cut on a Leica Ultracut UCT25 ultramicrotome. Semi-thinsections (0.8 mm thick) were stained with 1% methylene blue in0.5% borax and analyzed using an Olympus BX60 light microscope.Ultra-thin sections (70 nm) were stained with uranyl acetate andlead citrate and examined with a Hitachi H500 transmissionelectron 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) andstained with Schiff’s reagent (24 h, 37 �C). Slides were analyzedusing an Olympus BX60 light microscope.

2.2.2. Detection of proteinsSemi-thin sections were treated with a 1% solution of periodic

acid to remove the osmium (10 min at room temperature) andstained with bromophenol blue (BPB) (24 h, 37 �C). Slides wereanalyzed using an Olympus BX60 light microscope.

2.2.3. Labeling of nuclei and actin filamentsIsolated 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

Fig. 3. A. The apical region of digestive cells (dc). The apical membrane forms microvilli (mmitochondria (m) and cisterns of RER (RER). Endosymbionts (es), multivesicular bodies (mvActin filaments which form the actin cortex stained red with rhodamine-phalloidin (arrowmidgut lumen (l), visceral muscles (mc). Fluorescence microscope, bar ¼ 23.8 mm. C. Multivenumerous small vesicles (arrows). Mitochondria (m), endosymbionts (es), electron-dense spsome of digestive cells (dc). Mitochondria (m), cisterns of SER (SER). TEM, bar ¼ 1.2 mm. E. SMicrovilli (mv). TEM, bar ¼ 0.33 mm.

slides and washed in Tris-buffered saline (TBS; 5 min) and in 0.1%Triton X-100 in TBS (5min). Eventually, the slides were stainedwithrhodamine-phalloidin (40 min, room temperature), washed in TBS(five times, 10 min each) and labeled with DAPI (40,6-diamidino-2-phenylindole) (30 min). Slides were analyzed using an OlympusBX60 fluorescence microscope.

2.2.4. BrdU labeling of proliferating cellsRegenerative 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) wereinjected with 50 mg BrdU/kg body weight. After 1 h the specimenswere sacrificed and their midguts were isolated. The material wasthen fixed in Karnovsky’s fixative (4% paraformaldehyde and 2.5%glutaraldehyde) (30 min, room temperature), washed in TBS with0.1% Triton X-100 (5 min, room temperature) and embedded in atissue-freezing medium (Jung). Cryostat sections (5 mm thick) wereplaced on 1% gelatin-coated slides. After washing in TBS (1�3min)and incubation in 2N HCl (60 min, 37 �C), the material was incu-batedwith 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 BX60fluorescence microscope.

2.2.5. Immunolabeling with anti-phosphohistone H3The 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 on1% gelatin-coated slides, washed in TBS (5 min, room temperature)then 0.1% Triton X-100 in TBS (5 min, room temperature) andincubated in 1% BSA in TBS (30 min, room temperature). The ma-terial was then stained overnight in a 1:100 dilution of anti-phosphohistone H3 antibodies (Millipore) in 1% BSA in TBS (roomtemperature). After washing with TBS (2 � 5 min, room tempera-ture), it was incubated in a 1:200 dilution of goat anti-rabbit IgGAlexa-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 sec-tions were analyzed using an Olympus BX60 fluorescencemicroscope.

3. Results

3.1. Structure of the digestive system of Scolopendra cingulata

The digestive system of S. cingulata has a tube-like shapewithout any digestive diverticulae or caeca. Three distinct regionscan be distinguished: the foregut (pharynx, esophagus, crop andproventriculus ¼ gizzard), midgut and hindgut. The cardiac valveis located between the foregut and midgut, while the pyloric valveis between the midgut and hindgut (Fig. 1A). The foregut, which isthe longest region of the digestive system, occupies about 1/2 ofits length, while the hindgut is the shortest (about 1/10 of itslength).

v) and the cytoplasm with numerous vesicles with electron-lucent content (arrows),b), midgut lumen (l), electron-dense spheres with proteins (p). TEM, bar ¼ 0.83 mm. B.s) in the apical cytoplasm. Nuclei are labeled with DAPI (blue). Midgut epithelium (e),sicular bodies (mvb) accumulated in the neighborhood of the apical cell membrane andheres with proteins (p). TEM, bar ¼ 0.52 mm. D. Spherites (sp) in the apical cytoplasm ofmooth septate junction (arrow) between apical regions of adjacent digestive cells (dc).

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3.2. Structure of the midgut epithelium of Scolopendra cingulata

The midgut is lined with a pseudostratified columnar epithe-lium in which all cells have contact with the thick and stronglyfolded basal lamina; however, not all cells reach the midgut lumen(Fig. 1B). The midgut epithelium is surrounded by visceral mus-cles: circular muscles (the inner layer) and longitudinal muscles(the external layer) (Fig. 1B); it is covered on its luminal surface bya peritrophic membrane (not shown). Three types of cells weredistinguished in the midgut epithelium: digestive cells (Fig. 1BeD), 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 themidgut lumen and basal lamina (Fig. 1B,C). Their cytoplasmshows regionalization in the distribution of organelles that formthree distinct regions: the basal, perinuclear and apical. The basalmembrane forms numerous folds and invaginations; numerousmitochondria, free ribosomes and cisterns of the rough endo-plasmic reticulum (RER) accumulate in their neighborhood(Fig. 2A). Gap junctions occur between the basal and perinuclearregions of adjacent digestive cells (Fig. 2B). Lateral membranesare strongly folded (Fig. 2C). The perinuclear cytoplasm has anelongated nucleus with distinct patches of heterochromatin nearthe nuclear envelope and a large homogenous nucleolus (Fig. 2C).Numerous mitochondria, cisterns of RER and SER, Golgi com-plexes (Fig. 2D) and cytoskeleton (Fig. 2C) are also gathered nearthe nucleus. The apical cytoplasm is the most extensive and oc-cupies approximately half of each cell’s height. The apicalmembrane forms long microvilli (Fig. 3A), which possess theirown cytoskeleton. Actin filaments entering the apical cytoplasmform the actin cortex (Fig. 3B). The apical cytoplasm is rich inelongated 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 bodiesmainly gather in the neighborhood of the apical cell membranewhere 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 foundbetween the apical regions of adjacent digestive cells (Fig. 3E).

The reserve material accumulates in the cytoplasm of thedigestive cells: numerous spheres with homogenous or hetero-genous and electron-dense content occur in the apical and peri-nuclear regions (Figs. 2D and 3A). They contain proteins (Fig. 4A)or proteins and saccharides (Fig. 4B). Glycogen granules alsoappear in the neighborhood of the nucleus (Fig. 2C), while lipiddroplets 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 apicalmembrane, fuse with it and release their contents into themidgut lumen (Fig. 4C). During apocrine secretion, the apicalmembrane loses the microvilli and forms a large evagination into

Fig. 4. A. BPB-positive granules (arrows) in the midgut epithelium (e) of S. cingulata. Basal laLight microscope, bar ¼ 5.8 mm. B. PAS-positive granules (arrows) in the midgut epithelium (microscope, bar ¼ 5.8 mm. C. Merocrine secretion. Electron-lucent vesicles (arrows) fuse wibar ¼ 0.46 mm. D. Apocrine secretion: the portion of the apical membrane of digestive cellMitochondria (m). TEM, bar ¼ 1.28 mm. E. Apocrine secretion. Apical vesicle (star) is expell

the midgut lumen (Fig. 4D). Eventually, the evagination is sepa-rated from the entire cell and is discharged into the midgutlumen (Fig. 4E).

3.4. Secretory cells

Secretory cells, which are sparsely distributed among digestivecells (Fig. 1B,C), do not contact the midgut lumen and assume apear-like shape. They extend a narrow cytoplasmic process to thebasal lamina (Figs. 2A and 5A); and their basal membranes aredevoid of any folds. The cytoplasm does not show a regionaliza-tion in the distribution of organelles. An oval nucleus with smallpatches of heterochromatin and homogenous nucleolus occupiesthe widest region of the cell (Figs. 2A and 5A,B). The entirecytoplasm is rich in numerous small granules of different electrondensities (Fig. 5A,B), which accumulate mainly proteins or sac-charides (Fig. 4A,B). Many mitochondria and cisterns of RERappear in the neighborhood of the nucleus (Fig. 5B). Intercellularjunctions between the secretory and digestive cells were notobserved.

3.5. Regenerative cells

Regenerative cells are individually distributed among thedigestive cells along the entire length of the midgut. They possess apear-like shape with a very long and thin strand of the cytoplasmthat has contact with the basal lamina (Chajec et al., 2012c). Thelargest region of the cell is shifted toward the perinuclear or eventhe apical region of the digestive cell, but it does not have contactwith the midgut lumen (Fig. 6A). During the interphase, the ovalnucleus possesses small patches of heterochromatin located nearthe 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, cisternsof RER and free ribosomes, lipid droplets, electron-dense granules,spherites, multivesicular bodies, autophagosomes and small vacu-oles appear (Fig. 6A,C). Intercellular junctions between the regen-erative cell and adjacent digestive or secretory cells are absent.

Themitotic divisions of regenerative cells (Fig. 6DeF) depend onthe 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 to-ward the midgut lumen and all organelles segregate in the neigh-borhood of the two poles of the mitotic spindle (Fig. 6DeF).However, the mitotic cell establishes contact with the midgutlumen before the end of the mitosis (Fig. 6E). The nuclear envelopeis formed just after the mitotic division (Figs. 6F and 7D) and thencytokinesis begins (Fig. 7C). One of the two daughter cells differ-entiates into the digestive cell, while the second one fulfills the roleof a regenerative cell with its characteristic ultrastructure asdescribed above (Fig. 7D).

During the differentiation, the regenerative cell changes itsshape into a columnar one. The apical membrane of the differen-tiating cell gradually forms microvilli which protrude into themidgut lumen (Fig. 6A). The nucleus of the differentiating cellmoves into the perinuclear region of the epithelium. The cellular

mina (bl), digestive cells (dc), hemocytes (hc), secretory cell (sc), visceral muscles (mc).e). Basal lamina (bl), digestive cells (dc), secretory cells (sc), visceral muscles (mc). Lightth the apical cell membrane of digestive cells (dc). Midgut lumen (l), vacuole (v). TEM,s (dc) devoid of microvilli forms large protrusions (arrows) into the midgut lumen (l).ed 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, bar¼ 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, bar¼ 1.2mm.D. Mitotic division of the regenerative cell (rc). Chromosomes (ch), digestive cells (dc),midgut lumen (l), mitochondria(m), electron-dense granules (arrows). TEM, bar¼ 1.1 mm. E. The organelles form two groups near themitotic spindle poles. The regenerative cell (rc) contacts (arrow) themidgut lumen (l)before theendofmitosis. Chromosomes (ch), digestive cell (dc), endosymbionts (es), lipiddroplets (ld),mitochondria (m). TEM,bar¼1mm.F. After themitoticdivisionof the regenerative cell(rc), the nuclear envelopes of two daughter nuclei (n) are formed. Digestive cells (dc), endosymbionts (es), microvilli (mv). TEM, bar¼ 1.4 mm.

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). Crosssection 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 digestivecell, 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 thisarticle.)

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content of reserved material gradually increases (Fig. 7D). Region-alization in the distribution of organelles, which have accumulatedin cytoplasm, and intercellular junctions appear between newlyformed and digestive cells (Fig. 6A). Regenerative cells differentiateinto only digestive ones; they do not form secretory cells.

3.6. Hemocytes

Hemocytes were found in all of the specimens analyzed, bothmales and females, between visceral muscles and just beneath thebasal 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 todifferences in the ultrastructure of hemocytes, two types of thesecells can be distinguished (Fig. 8B). The majority of hemocytes(about 85%) belong to I type, while the II type is rather rare (about15%).

Large hemocytes of the I type possess an ameboid shape. Anirregular nucleus is located in the central cytoplasm which hasnumerous small and irregular electron-dense granules (Fig. 8BeD).Many vacuoles with parallel strands of a crystalline or tubularmaterial occur (Fig. 8D) together with numerous mitochondria,cisterns of SER and RER, Golgi complexes, vesicles with an electron-lucent content, multivesicular and lamellar bodies and autopha-gosomes (Fig. 8C). All organelles accumulate in the neighborhood ofthe nucleus making the cortical cytoplasm poor in organelles(Fig. 8C).

Smaller hemocytes of the II type are much more regular inshape and possess a large but lobular nucleus with a smallnucleolus. The characteristic feature of these hemocytes is thepresence 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 anelectron-lucent content accumulate near the nucleus, hence thecortical cytoplasm is poor in organelles (Fig. 8E,F). Intercellularjunctions between hemocytes and midgut epithelium cells areabsent.

3.7. Microorganisms

Numerous bacillus-like microorganisms which are probablyendosymbionts were observed in the cytoplasm of digestive cells inall of the specimens analyzed, both males and females. They mainlyaccumulate in the apical cytoplasm near the apical cell membrane(Figs. 3A,C and 9A). In addition, some endosymbionts occur in thecytoplasm of regenerative cells (Fig. 6AeF) and in the midgutlumen. Gregarines are a second type of microorganisms observed inabout 10% of specimens. They accumulate in both the cytoplasm ofdigestive cells and in the midgut lumen near the apical cell mem-branes (Fig. 9B,C).

4. Discussion

Myriapods live in soil and litter so they are exposed to bothpathogens and endosymbionts, which may enter the body throughthe digestive system. Endosymbionts are responsible for synthe-sizing the digestive enzymes, supplying nutrients and protectingthe organism against pathogens (Crawford et al., 1983; Tajovsky,1992). However, to date only pathogens have been described inthe digestive system of Chilopoda (Lewis, 1981). Bacillus-like mi-croorganisms have been found in the apical cytoplasm of thedigestive cells in both S. cingulata and L. forficatus (our observation).Endosymbionts commonly occur in the midgut, midgut muscles orgonads of arthropods, where they are present in all specimens ofthe culture. They can be transmitted from one generation to thenext transovarially ( _Zelazowska and Bili�nski, 1999; Szklarzewiczet al., 2006; Nehme et al., 2007). Because the microbes are pre-sent in the apical cytoplasm of digestive cells and in the cytoplasmof regenerative cells in all of analyzed specimens of S. cingulata, wepostulate that theymust play a role of endosymbionts. Additionallythey are not discharged from the cell when apocrine secretion oc-curs (see Fig. 4D).

Numerous endopathogens appear in Chilopoda, e.g. pathogenicbacteria, pathogenic fungi, gregarines, coccidia, ciliates or eveninsects and leeches (Lewis, 1967, 1981). Coccidia and gregarinesare characteristic for Lithobiomorpha and Scolopendromorpha

(Singotam and Dass, 1977; Lewis, 1981). While in L. forficatusrickettsia-like microorganisms, coccidia and gregarines are pre-sent (our unpublished observation), only gregarines appear inS. cingulata. Pathogens which enter the midgut lumen may movethrough the midgut epithelium and its basal lamina and eventu-ally infect the entire organism. Therefore, the midgut epitheliummust play a role as a barrier against infection.

4.1. Regenerative cells and midgut regeneration

Regenerative cells are responsible for the regeneration of theepithelium in the digestive system of animals, especially theirmiddle region ¼midgut. They are treated as multipotential midgutstem cells (Loeb and Hakim, 1996; Hakim et al., 2001, 2010; Loebet al., 2001; Baton and Ranford-Cartwright, 2007; Parthasarathyand Palli, 2007, 2008; Casali and Batlle, 2009; Cruz et al., 2011;Mehrabadi et al., 2012; Teixeira et al., 2013). In Arthropoda, espe-cially in Hexapoda, regenerative cells form groups: regenerativenests (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; Nardiet al., 2010), or they are individually distributed among digestivecells (Rost, 2006a; Okuda et al., 2007; Nardi et al., 2009; Roelfstraet al., 2010; Rost-Roszkowska et al., 2010a). Likewise, in somespecies of Myriapoda, regenerative cells form regenerative crypts(Kaufman, 1961; Lewis, 1981; Minelli, 1993), or are individuallydistributed along the entire length of the midgut (Köhler andAlberti, 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, theirultrastructure and the role in tissue regeneration have not beendescribed (Balbiani, 1890; Léger and Duboscq, 1902; Kaufman,1961).

In S. cingulata as in L. forficatus (Chajec et al., 2012a,c), regen-erative cells are poor in organelles, which start to accumulate in thecytoplasm just after the beginning of cell differentiation. Numerousmitochondria form two groups in the neighborhood of regenerativecell nuclei in L. forficatus, while in S. cingulata they are regularlydistributed around the nucleus. Their high number suggests that agreat amount of energy must be supplied for their proper prolif-eration and differentiation. Additionally, the reserve materialgathered in the cytoplasm of regenerative cells supports such ahypothesis.

The cytoplasm of the regenerative cells of S. cingulata is rich inautophagosomes and multivesicular bodies, which have not beenfound in L. forficatus (Chajec et al., 2012a). Autophagy promotesboth cell survival and cell death. Since autophagy eliminatesdamaged organelles and proteins, it is considered as a survivalmechanism (Das et al., 2012). The absence of autophagosomes inthe cytoplasm of regenerative cells in L. forficatus, might be con-nected with the fact that they are located in the basal region ofthe midgut epithelium, and consequently they are shielded tosome extent from mechanical damage and toxic substances thatcome from the midgut lumen (Chajec et al., 2012a). However, inS. cingulata, regenerative cells reach the apical region of themidgut epithelium and are separated from the midgut lumen byonly thin processes of adjacent digestive cells. Therefore, they areexposed to factors that injure the entire epithelium, and auto-phagosomes and spherites accumulated in the cytoplasm takepart in protecting the epithelium (Vandenbulcke et al., 1998;Pigino et al., 2005; Dwivedi and Ahnn, 2009; Rost-Roszkowskaet al., 2012).

Cyclic regeneration has been found to be connected withmolting in many arthropods (Humbert, 1979; Rost, 2006a,b). InS. cingulata proliferation combined with the day/night cycle is

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Ł. Chajec et al. / Arthropod Structure & Development 43 (2014) 27e42 39

probably caused not only by molting but also by other factors.First of all, as this species is nocturnal, it hunts and feeds duringthe night and it avoids the light by hiding under stones anddigging itself in the soil. Another factor that causes the prolifer-ation to be cyclic might be the structure of the digestive system. InS. cingulata the foregut is the longest part and is differentiatedinto the pharynx, esophagus, crop and gizzard lined with cuticleand possessing structures made of chitin that cut and chop thefood (Lewis, 1981; Koch et al., 2009). Consequently, finelymacerated food enters the midgut lumen without evident abra-sion of midgut epithelium.

Stem cells can divide in a symmetric or asymmetric way.During symmetric division, two identical daughter cells appear. Inthe case of asymmetric division, one stem cell and one progenitorcell are produced (Loeb and Hakim, 1996; Morrison et al., 1997;Hakim et al., 2001; Teixeira et al., 2013). In Myriapoda regenera-tive cells presumably differentiate into both the digestive andsecretory cells of the midgut epithelium (Balbiani, 1890; Lewis,1981). However, this process has not been described at the ul-trastructural level, and therefore it has not been confirmed. InS. cingulata as in L. forficatus (Chajec et al., 2012a,c), regenerativecells divide in an asymmetric manner to form one regenerativecell ¼ midgut stem cell and one midgut progenitor cell, whichstarts to differentiate into a digestive cell. The midgut progenitorcell in S. cingulata as in L. forficatus has not been found to differ-entiate into a secretory cell. Because secretory cells in both speciesdo not have contact with the midgut lumen, they are not exposedto any mechanical damage caused by food, toxic substances orpathogens, so they probably do not have to be renewed. There-fore, we postulate that the regenerative cells in S. cingulata playthe 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 bothmillipedes (de Godoy and Fontanetti, 2010; Nogarol andFontanetti, 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 theneighborhood of the fat body in the hemocoel (de Godoy andFontanetti, 2010; Nogarol and Fontanetti, 2011; Perez andFontanetti, 2011). As in insects, they are able to migrate anddisperse throughout the body (Tepass et al., 1994; Nardi et al.,2003). During our studies on the midgut of centipedes, wefound that hemocytes may enter the midgut epithelium extendingthrough its basal lamina, where they can be observed between thedigestive, secretory and regenerative cells, as has been describedin L. forficatus (Chajec et al., 2012a) and S. cingulata (Chajec et al.,2012b). It is worth noting that the number of hemocytes in cen-tipedes is much higher than in millipedes (Xylander, 1992, 2009a),a difference that might be attributed to the fact that hepatic cellsare observed together with hemocytes in millipedes (Hopkin andRead, 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,

Fig. 8. A. Hemocytes (hc) penetrate the basal lamina (bl). Midgut epithelium (e), visceral muregions of the digestive cells (dc). Nuclei (n). TEM, bar ¼ 3.3 mm. C. The ultrastructure of hemthe central cytoplasm, while the cortical cytoplasm (star) is electron-lucent and poor in organ(d), mitochondria (m), multivesicular bodies (mvb), nucleus (n), small electron-lucent vesiclwith fibrillar material, mitochondria (m), nucleus (n). TEM, bar ¼ 0.53 mm. E. The ultrastruirregular nucleus (n) with distinct patches of heterochromatin. Cisterns of RER (RER), cortic(v), electron-lucent vesicles (arrows). TEM, bar ¼ 1.04 mm. F. Hemocytes II (hc2). Cisterns ofdense granules (g), mitochondria (m), nucleus (n), vacuoles (v). TEM, bar ¼ 0.92 mm.

granulocytes ¼ granular hemocytes, spherulocytes, coagulocytesand “discoid cells” occur in the hemocoels of Myriapods(Nevermann et al., 1991; Hilken et al., 2003; Xylander andNevermann, 2006; Xylander, 2009a). The hemocytes observed inS. cingulata resemble plasmatocytes and granulocytes. Large plas-matocytes are able tomigrate, and they possess numerous vacuoleswith lamellar or crystalline material within their cytoplasm alongwith some small electron-dense granules. The cytoplasm of smallergranulocytes accumulates numerous irregular granules with anelectron-dense content (Nevermann et al., 1991; Xylander andNevermann, 2006; Xylander, 2009a). Based on this observation,presumably all hemocytes of L. forficatus are granulocytes, whilehemocytes I of S. cingulata resemble plasmatocytes and hemocytesII are granulocytes.

Hemocytes are responsible for hemolymph coagulation afterdamage to the cuticle and epidermis, healing of injuries, opsoni-zation and phagocytosis of pathogens and the synthesis of anti-bacterial proteins (Gupta, 1986; Xylander, 1992, 2009a,b; Lavineand Strand, 2002). In L. forficatus (Chajec et al., 2012a) andS. cingulata ameboid hemocytes migrate from the hemocoelthrough the basal lamina into the midgut epithelium in order toprotect this epithelium, and eventually the entire organism, againstinfection by pathogens. In L. forficatus hemocytes accumulateamong the midgut epithelial cells only in infected specimens.Therefore, we suggest their protective role against pathogenicinfection (Chajec et al., 2012a). However, in S. cingulata those cellsoccur between the digestive cells in both infected and non-infectedspecimens. Hemocytes in S. cingulata possibly play a role not onlyagainst infection, but also against toxic substances that may enterthe organism from the midgut lumen. As is known in Diplopoda,hemocytes accumulate among cells of the fat body that surroundsthe midgut in specimens treated with toxic metals. Whennumerous degenerated cells of the fat body appeared, the numberof hemocytes in the fat body increased (de Godoy and Fontanetti,2010; Perez and Fontanetti, 2011). In S. cingulata hemocyteswhich accumulate among midgut epithelial cells possess manyautophagosomes, which take part in the elimination of organelles,proteins and pathogens (Franzetti et al., 2012; Rost-Roszkowskaet al., 2012).

5. Conclusions

(a) The midgut epithelium of S. cingulata is composed ofdigestive, secretory and regenerative cells, and hemocytes inter-spersed among cells of epithelium; (b) the process of midgutepithelium regeneration proceeds due to the ability of regenera-tive cells to proliferate and differentiate; (c) regenerative cellsfulfill the role of unipotent midgut stem cells which divide in anasymmetric manner; (d) midgut regeneration occurs in a cyclicmanner which depends on day/night cycle; (e) intensive mitoticdivisions of regenerative cells help maintain the integrity of themidgut epithelium in the event of pathogenic infection and (f)hemocytes presumably can eliminate pathogens from the midgutepithelium.

scles (mc). TEM, bar ¼ 2.5 mm. B. Hemocytes of the I (hc1) and II types (hc2) among basalocytes I (hc1). All organelles and electron-dense granules (black arrows) accumulated inelles. Autophagosomes (au), cisterns of RER (RER), digestive cells (dc), Golgi complexeses (white arrows). TEM, bar ¼ 1 mm. D. Hemocytes I (hc1). Numerous vesicles (arrows)cture of hemocytes II (hc2). Numerous large electron-dense granules (g). Lobular andal cytoplasm (star), digestive cells (dc), lamellar bodies (lb) mitochondria (m), vacuolesRER (RER), cortical cytoplasm (star), digestive cells (dc), Golgi complexes (d), electron-

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 apicalcell membrane of the digestive cells (dc). Microvilli (mv). TEM, bar ¼ 1.94 mm.

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