Autophagy and Apoptosis in the Midgut Epithelium of Millipedes

Microscopy and Microanalysis (2019), 25, 1004–1016 doi:10.1017/S143192761900059X

Micrographia

M.M. Rost-Roszkowska1*, J. Vilimová2, K. Tajovský3, A. Chachulska-Żymełka1, A. Sosinka1, M. Kszuk-Jendrysik1, A. Ostróżka1 and F. Kaszuba1 1Department of Animal Histology and Embryology, University of Silesia in Katowice, Bankowa 9, 40-007 Katowice, Poland; 2Department of Zoology, Charles University, Faculty of Science, Viničná 7, 128 44 Prague 2, Czech Republic and 3Institute of Soil Biology, Biology Centre CAS, Na Sádkách 7, 370 05 České Budějovice, Czech Republic

Abstract The process of autophagy has been detected in the midgut epithelium of four millipede species: Julus scandinavius, Polyxenus lagurus, Archispirostreptus gigas, and Telodeinopus aoutii. It has been examined using transmission electron microscopy (TEM), which enabled dif- ferentiation of cells in the midgut epithelium, and some histochemical methods (light microscope and fluorescence microscope). While autophagy appeared in the cytoplasm of digestive, secretory, and regenerative cells in J. scandinavius and A. gigas, in the two other species, T. aoutii and P. lagurus, it was only detected in the digestive cells. Both types of macroautophagy, the selective and nonselective processes, are described using TEM. Phagophore formation appeared as the first step of autophagy. After its blind ends fusion, the autophagosomes were formed. The autophagosomes fused with lysosomes and were transformed into autolysosomes. As the final step of autophagy, the residual bodies were detected. Autophagic structures can be removed from the midgut epithelium via, e.g., atypical exocytosis. Additionally, in P. lagurus and J. scandinavius, it was observed as the neutralization of pathogens such as Rickettsia-like microorganisms. Autophagy and apoptosis ca be analyzed using TEM, while specific histochemical methods may confirm it. Key words: cell death, digestive system, Diplopoda, histochemistry, midgut, ultrastructure (Received 17 September 2018; revised 4 January 2019; accepted 15 April 2019)

Introduction of stress, autophagy can activate a diversity of pathways such as apoptosis and/or necrosis (Semenza, 2008; Cebollero et al., Autophagy is a process which can act as a pro-survival process or 2012; Franzetti et al., 2012; Rost-Roszkowska et al., 2015; it can represent a type of cell death. In response to some stress fac- Sonakowska et al., 2016). tors, e.g., starvation, this process, as a pro-survival factor, protects Autophagy has been described as a common process that is a cell against its death (e.g., apoptosis) or against the activation of responsible for the proper functioning of the middle region in inflammation (Malagoli et al., 2010; Tettamanti et al., 2011; the digestive system (the midgut) of arthropods throughout Cebollero et al., 2012; Rost-Roszkowska et al., 2012; Lipovšek & their life cycle and embryogenesis (Malagoli et al., 2010; Novak, 2015; Lipovšek et al., 2015; Larsson & Masucci, 2016; Rost-Roszkowska et al., 2010, 2012; Mpakou et al., 2011; Sonakowska et al., 2016). However, it can also initiate the death Tettamanti et al., 2011; Franzetti et al., 2012; Khoa & Takeda, of an entire cell when too many organelles undergo internaliza- 2012; Fernandes et al., 2014; Karpeta-Kaczmarek et al., 2016; tion inside the double-membraned vacuoles (autophagosomes). Sonakowska et al., 2016; Lipovšek et al., 2018). The midgut, After the fusion of autophagosomes with lysosomes, digestion which participates in the secretion, absorption, excretion, and starts inside the just formed autolysosomes and, in some accumulation of many substances, plays an important role in instances, the death of the entire cell is activated (Tsujimoto & the maintenance of homeostasis. This organ, which is lined Shimizu, 2005; Park et al., 2013; Karpeta-Kaczmarek et al., with a simple epithelium that is composed of different types of 2016). Autophagy has been distinguished as a nonselective pro- cells (e.g., digestive cells, secretory cells, regenerative cells, etc.) cess when different organelles, structures, or substances gather appears to be a good model for the analysis of the role of autoph- inside autophagosomes. It can also be a selective process in agy (Malagoli et al., 2010; Teixeira et al., 2013). This process can which only specific organelles are digested (e.g., mitophagy, retic- be activated in the digestive system by various external factors that ulophagy, lipophagy, nucleophagy, etc.). Both of these can be enter an organism along with nutrients (Rost-Roszkowska et al., signs of a cell’s reaction to stressors. Depending on the degree 2010; Lipovšek & Novak, 2015; Karpeta-Kaczmarek et al., 2016; Sonakowska et al., 2016) or even internal mechanisms, e.g., *Author for correspondence: M.M. Rost-Roszkowska, growth factor deprivation, or ER stress (He & Klionsky, 2009). E-mail: [email protected] Autophagy and apoptosis have been described as commonly Cite this article: Rost-Roszkowska MM, Vilimová J, Tajovský K, Chachulska- occurring in the midgut epithelium of centipedes (Myriapoda, Żymełka A, Sosinka A, Kszuk-Jendrysik M, Ostróżka A, Kaszuba F (2019) Chilopoda) (Rost-Roszkowska et al., 2015, 2016), where they Autophagy and Apoptosis in the Midgut Epithelium of Millipedes. Microsc Microanal 25, 1004–1016. doi:10.1017/S143192761900059X have been described in digestive, secretory, and regenerative cells.

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Among millipedes (Myriapoda, Diplopoda), the autophagy has 2% osmium tetroxide in a 0.1 M PBS (4°C, 1.5 h), dehydrated in a been described in the cytoplasm of hepatic cells (De Godoy & graded concentration series of ethanol (50, 70, 90, 95, and four Fontanetti, 2010; Nogarol & Fontanetti, 2011; Rost-Roszkowska times 100%, each for 15 min) and acetone (15 min). Finally, the et al., 2018a) or fat body cells (Fontanetti et al., 2006). The midgut midguts were embedded in epoxy resin (Epoxy Embedding epithelium of millipedes is lined with a simple epithelium that is Medium Kit; Sigma). Semi and ultrathin sections were prepared composed of three types of cells—digestive, secretory, and regener- using a Leica Ultracut UCT25 ultramicrotome (The University ative (Hopkin & Read, 1992; Fontanetti & Camargo-Mathias, 1997; of Silesia in Katowice, Poalnd). The semi-thin sections (0.8 µm Fantazzini et al., 2002; Camargo-Mathias et al., 2004; De Godoy & thick) after staining with 1% methylene blue in 0.5% borax and Fontanetti, 2010; Souza & Fontanetti, 2011; Sosinka et al., 2014; examined using an Olympus BX60 light microscope. The ultra- Fontanetti et al., 2015; Rost-Roszkowska et al., 2018b). During thin sections (70 nm) were stained with uranyl acetate and lead our previous studies, we analyzed the structure and ultrastructure citrate and analyzed using a Hitachi H500 TEM. of the midgut epithelium in several millipede species— Archispirostreptus gigas, Julus scandinavius, Polyxenus lagurus Quantitative Analysis (Sosinka et al., 2014), and Telodeinopus aoutii (Rost-Roszkowska et al., 2018b). Here we present the results of studies that are con- The ultrathin sections (70 nm) were used in order to count the nected with autophagy and apoptosis observed commonly in the number of midgut epithelial cells with autophagosomes/autolyso- mentioned above millipedes. The aims of this study were (a) to somes/residual bodies in relation to the total number of cells analyze and describe an autophagy using transmission electron (TEM enables to distinguish digestive, secretory, and regenerative microscopy (TEM) in the midgut of some millipedes, (b) to deter- cells). The percentage of cells with autophagosomes was deter- mine the role of autophagy in the midgut epithelium in millipedes mined randomly by counting the cells (digestive, secretory, and that inhabit different environments and that consume different regenerative cells separately) in the midgut of the millipedes spe- diets, and (c) to check if the relationship between autophagy and cies examined here. apoptosis exists. The ultrathin sections were also used in order to count the number of apoptotic cells among the midgut epithelium. The percentage of apoptotic digestive, secretory, and regenerative cells was also Material and Methods determined randomly by counting the cells in the midgut. One Material specimen of each species was used for the quantitative analysis (both the autophagy and apoptosis). The sections were selected The following species, which represent three millipede orders randomly throughout the entire length of the midgut, so cells were selected: J. scandinavius (order Julida), P. lagurus (order from different regions of the organ were always used for the study. Polyxenida), and A. gigas and T. aoutii (the latter two species represent the order Spirostreptida). The specimens of J. scandinavius originated from the Podyjí Cryosections National Park, Southern Moravia (Czech Republic). P. lagurus Isolated midguts from adult specimens of millipedes (ten speci- was collected in the Ruda National Nature Monument near mens of J. scandinavius, ten specimens of P. lagurus, three spec- Veselí nad Lužnicí, Southern Bohemia and from the Czech imens of A. gigas, and five specimens of T. aoutii) were Karst Protected Landscape Area, Central Bohemia (Czech fragmented and embedded in a tissue-freezing medium (Jung) Republic). The millipede species originated from natural and at −22°C without fixation. Cryostat sections were cut (5 µm unpolluted environments. The spirostreptid millipedes, A. gigas thick) and mounted on 1% gelatin-coated slides (SuperFrost and T. aoutii, were purchased from boutiques that sell pets. The Plus slides, Menzel-Gläser). specimens were in good condition, actively moving, feeding, Some fragments from each isolated midgut were transferred to and burrowing in the organic substrate in the breeding containers. Karnovsky’s fixative and prepared for acid phosphatase staining A. gigas and T. aoutii were reared separately in 60 × 30 × 40 cm for TEM (see below). glass plastic boxes, and J. scandinavius and P. lagurus in smaller ones (20 × 15 × 6 cm) (relative humidity of about 70%, tempera- ture of 22°C). Wet decaying bark of trees that was covered with Acid Phosphatase Staining—Detection of Lysosomes and algae and lichens and leaf litter were put into all of the boxes as Autolysosomes (Light Microscopy) the food sources. A. gigas and T. aoutii were additionally fed Cryosections were initially washed in Tris-buffered saline (TBS) with fresh fruits (apple, pear, banana) and vegetables (tomato, (5 min, RT) and a 0.1 N sodium acetate-acetic acid buffer (pH champignon, cucumber). Pieces of cuttlebone (Sepia officinalis) 5.0–5.2, RT). Then they were incubated in a 0.1 N sodium acetate- were added to the laboratory cultures as a source of calcium. acetic acid buffer (pH 5.0–5.2, 1.5 h, 37°C) containing 0.01% naphthol phosphate AS-BI, 2% N-N-dimethylformamide, 0.06% Methods Fast Red Violet LB, and 0.5 mM MnCl2. Negative controls were performed by eliminating the naphthol phosphate AS-BI sub- Light and Transmission Electron Microscopy strate. The material was analyzed using an Olympus BX60 light After adaptation to the laboratory conditions, the adult specimens microscope. of millipedes, both males and females (ten specimens of J. scandinavius, ten specimens of P. lagurus, eight specimens of A. gigas, Acid Phosphatase Staining—Detection of Lysosomes and and ten specimens of T. aoutii), were anesthetized with chloro- Autolysosomes (TEM) form, decapitated, and their midguts were isolated. The material was fixed with 2.5% glutaraldehyde in a 0.1 M sodium phosphate The midguts from two specimens of each species were fixed in buffer (PBS) (pH 7.4, 4°C, 2 h), washed in 0.1 M PBS, postfixed in Karnovsky’s fixative (2 h, 4°C), washed in a cacodylate buffer

Figure 1. a: Membranous structures surrounding organelles (arrows). b, c: Double-membraned phagophore (arrows). Cisterns of endoplasmic reticulum (ER), mitochondria (m). a: A. gigas. Bar = 0.25 µm. b: J. scandinavius. Bar = 0.25 µm. c: A. gigas. Bar = 0.2 µm.

0.1 M (15 min, RT), 0.2 M Tris-maleate (10 min, RT), and finally RT), and stained with a TUNEL reaction mixture (In Situ Cell incubated in the incubation medium composed of: 0.2 M Death Detection Kit, TMR red, Roche) (60 min at 37°C in the Tris-Maleate buffer and 0.1 M sodium β-glycerophosphate and dark). Finally, the material was labeled with Hoechst 33342 0.02 M lead citrate (2 h, 37°C) (Lewis & Knight, 1992). After (30 min in darkness, RT). Slides were analyzed using an washing the material with Tris-Maleate (2 × 15 min, RT), it was Olympus BX60 fluorescence microscope. Negative controls with- postfixed in 1% osmium tetroxide, dehydrated in a graded series out terminal deoxynucleotidyl transferase (TdT) were prepared of ethanol (50, 70, 90, 96, 100%, 15 min each) and prepared according to the labeling protocol (In Situ Cell Death Detection according to the standard method for TEM. The material was Kit, TMR red, Roche). then analyzed using a Hitachi H500 TEM.

Results TUNEL Assay Autophagy appeared in the cytoplasm of the digestive, secretory, Cryosections were incubated in a permeabilization solution (0.1% and regenerative cells in J. scandinavius and A. gigas, while in T. sodium citrate) (2 min on ice in 4°C), washed in TBS (3 × 5 min, aoutii and P. lagurus, but it was only detected in the apical and

Figure 2. a–e: Autophagosomes (au) in the cytoplasm of digestive cells (dc) in the midgut epithelium of millipedes. Cisterns of endoplasmic reticulum (ER), mitochondria (m), nucleus (n), lamellar bodies (lb), reserve material (rm). a, b: Nonspecific autophagy. c, d: Specific autophagy. a: T. aouti. Bar = 1 µm. b: T. aouti. Bar = 0.6 µm. c: T. aoutii. Bar = 0.6 µm. d: J. scandinavius. Bar = 0.2 µm.

perinuclear cytoplasm of digestive cells. The process occurred organelles were lamellar bodies (Fig. 2c), cisterns of the endoplas- along the entire length of the midgut. Differences between the mic reticulum (reticulophagy, Fig. 2d), spherites (Fig. 3a), and males and females were not observed. spheres with the reserve material (the material made of proteins, TEM revealed that at the beginning of autophagy, the double- lipids, and polysaccharides was described in our previous paper— membrane structure (the phagophore) appeared in the vicinity of Sosinka et al., 2014)(Fig. 3b, 3c). In about 30% of the specimens some organelles (Fig. 1a). The phagophore had two blind ends of P. lagurus and J. scandinavius that were analyzed, (Figs. 1b, 1c) that enlarged to form the autophagosome (Figs. 2a–2e). Rickettsia-like microorganisms were observed (Fig. 3d, 3e). They Nonspecific autophagy has been detected, when numerous were completely enclosed inside the autophagosomes (Fig. 3e). organelles, e.g., cisterns of the endoplasmic reticulum, mitochon- Autolysosomes with organelles and an electron-dense content dria, lamellar bodies, spheres of the reserve material, and spherites (Fig. 4a) and residual bodies with homogenous and digested accumulated together inside autophagosomes (Figs. 2a, 2b). electron-dense content were also detected (Fig. 4b). They mainly However, in some of the autophagosomes, an agglomeration of accumulated in the perinuclear (Fig. 4c) and apical cytoplasm mainly one type of organelles was observed (similar to the specific (Fig. 4b) of the digestive cells in the midgut of all of the millipedes autophagy). In this case, the majority of the degenerated that were examined. Only single autolysosomes/residual bodies

Figure 3. a–e: Specific autophagy in the midgut epithelium of millipedes. Autophagosomes (au), cisterns of endoplasmic reticulum (ER), mitochondria (m), reserve material (rm), Rickettsia-like microorganisms (asterisk) in the cytoplasm of the digestive cells, spherites (sp). a: P. lagurus. Bar = 0.3 µm. b: P. lagurus. Bar = 0.2 µm. c: P. lagurus. Bar = 0.5 µm. d: P. lagurus. Bar = 0.5 µm. e: J. scandinavius. Bar = 0.15 µm.

appeared in the cytoplasm near the nuclei in the secretory and Together with autophagy, the process of apoptosis was regenerative cells of J. scandinavius and A. gigas. detected. It occurred mainly in the digestive cells with the cyto- In A. gigas, J. scandinavius, and P. lagurus, autophagosomes/ plasm rich in autophagosomes/autolysosomes/residual bodies. autolysosomes/residual bodies were observed reaching the apical Numerous cells were shrunken and distinct extracellular spaces cell membranes (Fig. 5a). The fragments of the apical cell mem- between them and neighboring digestive cells could be observed brane devoid of microvilli formed an evagination into the midgut (Fig. 6a). These cells were observed as gradually separated from lumen (Fig. 5b). The autophagic structures, which became the basal lamina, so they contacted the basal lamina with thin enlarged, were detected as moving into the evagination strands of the cytoplasm (Fig. 7b). The cells showed signs of apo- (Fig. 5c). Also, fragments of the cell cytoplasm with the cell mem- ptosis: their cytoplasm was electron dense (Figs. 6a–6c) and their brane and autophagic structures inside were seen as expelled into nuclei achieved the lobular shape (Figs. 6c, 7a). The chromatin the midgut lumen. Therefore, this process resembled atypical exo- formed electron-dense patches (Figs. 6c, 7b), mitochondria were cytosis. The process of autophagy in the midgut epithelium was devoid of their crista, and their matrix became electron lucent confirmed using acid phosphatase labeling (Fig. 5d). A quantita- (Fig. 6a). The apoptotic cells were observed as cells which lost tive analysis revealed the autophagy in about 17% of digestive cells contact with the basal lamina gradually discharged into the mid- in J. scandinavius and A. gigas, about 26% of digestive cells in P. gut lumen (Fig. 7c). Apoptosis concerned mainly the digestive lagurus and T. aoutii, while in secretory cells and regenerative cells with numerous autophagic structures, while it was not cells, this process was scarce (J. scandinavius, A. gigas) or not observed in regenerative or secretory cells in all of the species observed (P. lagurus, T. aoutii)(Table 1). analyzed here.

Figure 4. a–c: Autophagy in the midgut epithelium of millipedes. Autophagosomes (au), autolysosomes (al), residual bodies (rb), mitochondria (m), nucleus (n), microvilli (mv), cisterns of endoplasmic reticulum (ER), Golgi complexes (G), spherites (sp), reserve material (rm). a: J. scandinavius. Bar = 0.6 µm. b: P. lagurus. Bar = 0.3 µm. c: T. aoutii. Bar = 0.8 µm.

DNA fragmentation, which occurs during apoptosis, was con- observed autophagy in the cytoplasm of the digestive, secretory, firmed with a TUNEL assay (Figs. 8a–8f): apoptotic signaling cas- and regenerative cells in J. scandinavius and A. gigas, while in cade caused the fragmentation of DNA (red signals showed nuclei T. aoutii and P. lagurus, it was only detected in the digestive with DNA fragmented). A midgut epithelium that had been incu- cells. The species J. scandinavius and A. gigas are both herbivo- bated without the TdT enzyme solution showed no signals (not rous and saprophagous (Šustr et al., 2013, authors’ own observa- shown). A quantitative analysis revealed 13.4/7.4/11/10.4% of tion). The other two species are typical herbivorous millipedes apoptotic digestive cells in the midgut epithelium of J. scandina- that primarily feed on fruit and vegetables (T. aoutii; Sigling, vius, P. lagurus, A. gigas, T. aoutii, respectively (Table 2). 2010) or preferably consume algae cells (P. lagurus; Hopkin & Read, 1992). In centipedes (Chilopoda), this process has been described as being common in all of the cells of the midgut epi- Discussion thelium, in animals that are obligatory predators (e.g., Scolopendra The midgut epithelium of millipedes is composed of three types cingulata), that are exposed to starvation when there is no avail- of cells: digestive (principal, absorptive), secretory, and regenera- able prey in the environment. Although they are carnivorous, in tive cells (midgut stem cells) (Hopkin & Read, 1992; Fontanetti & the event of a lack of animal food, other centipedes (e.g., Camargo-Mathias, 1997; Fantazzini et al., 2002; Camargo- Lithobius forficatus) can feed on litter as well as on the remains Mathias et al., 2004; De Godoy & Fontanetti, 2010; Souza & of small animals (Chajec et al., 2012, 2014). Thus, the natural Fontanetti, 2011; Sosinka et al., 2014; Fontanetti et al., 2015; period of starvation is shortened. Karasov et al. (2011) stated Rost-Roszkowska et al., 2018b). During our previous studies, we that the quality of food has an effect on the duration of digestion analyzed the precise ultrastructure of all cells which form the and midgut transit. The period needed for digestion is elongated midgut epithelium, and we managed to examine the processes when the animal feeds on low-quality food, e.g., arthropods with of autophagy and apoptosis in the animals which were not epidermis covered with cuticle (Mitra & Flynn, 2007; Karasov exposed to any stressors: they originated from the nonpolluted et al., 2011). Therefore, we can conclude that, similar to the cen- environment, were in a good condition, and fed ad libitum.We tipedes that were analyzed (Chajec et al., 2014), in millipedes the

Figure 5. a–e: Autophagosomes (au), autolysosomes (al), and residual bodies (rb) discharging. Mitochondria (m), microvilli (mv), nucleus (n), evagination of the apical cell membrane (arrows). a: P. lagurus. Bar = 0.3 µm. b: P. lagurus. Bar 0.6 µm. c: P. lagurus. Bar = 0.45 µm. d: J. scandinavius. Histochemical staining of acid phosphatase activity. Pink/red spots of acid phosphatase locality (arrowheads). Bar = 9 µm. e: T. aoutii. Cytochemical staining of acid phosphatase activity. Electron-dense spots of acid phosphatase locality (arrowheads). Bar = 0.7 µm.

type of food can prolong the duration of digestion thus activating et al., 2001, 2009; Cruz et al., 2011; Chajec et al., 2012, 2014; autophagy in the cytoplasm. Eventually, the energy during longer Sosinka et al., 2014). Secretory cells are small, long-living, and periods of digestion can be supplied. rarely found in the midgut epithelium of myriapods (Chajec Regenerative cells of the midgut epithelium of arthropods are et al., 2012, 2014; Sosinka et al., 2014; Rost-Roszkowska et al., treated as the midgut stem cells which are able to stay in a quies- 2018b) as in the other arthropods (Santos et al., 2016). The secre- cence state or divide mitotically. During the process of differentia- tory and regenerative cells, being located among the basal regions tion, they take on the features of the distinct epithelial cells (Hakim of the digestive cells, have no contact with the midgut lumen

Figure 6. a–c: Apoptosis in the midgut epithelium of millipedes. Autophagosomes (au), mitochondria (m), cisterns of endoplasmic reticulum (ER), digestive cell (dc), apoptotic cell (ac), nucleus (n), nucleoli (nu), spherites (sp). a: T. aoutii. Bar = 0.6 µm. b: J. scanidavius. Bar = 0.8 µm. c: J. scanidavius. Bar = 0.5 µm.

Table 1. Mean (x) ± Standard Deviation (SD) of Cells with Signs of Autophagy in the laboratory conditions. Therefore, starvation which could the Midgut Epithelium in Millipedes (n =5;n—Number of Sections in Which 100 enhance autophagic processes should not be activated (Eitzinger Cells were Counted; One Specimen of Each Species). et al., 2013). We can state that the autophagy that was observed Digestive Secretory Regenerative in the millipedes is a common process that participates in the Cells Cells Cells proper functioning of the midgut epithelium. It has also been con- sidered to be commonly present in the hepatic cells of millipedes J. scandinavius 17.34 ± 1.15 4.0 ± 0.0 6.0 ± 0.0 (De Godoy & Fontanetti, 2010; Nogarol & Fontanetti, 2011; P. lagurus 26 ± 3.46 ––Rost-Roszkowska et al., 2018a) where it participates in glycogen digestion. Autophagy has also been suggested as the survival A. gigas 17.2 ± 2.68 5.33 ± 1.15 7.34 ± 1.15 factor that supplies the energy derived from the digestion of poly- T. aoutii 25.8 ± 2.28 ––saccharides. This process has also been described in the fat body of millipedes as a type of degradation of the endoplasmic reticulum and the digestion of proteins (Fontanetti et al., 2006). This process takes place in the midgut epithelium in millipedes examined (a feature of the pseudostratified epithelium) (Fantazzini et al., here with the similar frequency: about 17% of digestive cells 2002; Camargo-Mathias et al., 2004; Fontanetti & de Godoy, (J. scandinavius, A. gigas) or 26% of digestive cells (T. aoutii, 2007; Sosinka et al., 2014; Moreira-de-Sousa et al., 2017; P. lagurus) showed signs of autophagy. As has been mentioned Rost-Roszkowska et al., 2018b). So, there is the possibility that above, the animals were not exposed to any stressors, their the autophagy has not been detected because the regenerative conditions were similar to the natural environment, and they and secretory cells of T. aoutii and P. lagurus having no contact were fed ad libitum. with the nourishments were not exposed to any stressor or were Degenerated or disrupted organelles, the reserved material in their quiescence state. The millipedes examined here were fed (e.g., lipids, proteins, saccharides), toxic substances (e.g., heavy ad libitum and were only decapitated after they had adapted to metals), and/or pathogens may be neutralized and digested due

Figure 7. a–c: Apoptosis in the midgut epithelium of millipedes. Autophagosomes (au), basal lamina (bl), mitochondria (m), microvilli (mv), cisterns of endoplasmic reticulum (ER), digestive cell (dc), apoptotic cell (ac), Golgi complexes (G), reserve material (rm), residual bodies (rb), spherites (sp), nucleus (n), nucleoli (nu), patches of the heterochromatin (white arrowheads), extracellular spaces between cells (asterisks), thin strands of the cytoplasm (white arrows). a: P. lagurus. Bar = 1.1 µm. b: A. gigas. Bar = 1 µm. c: J. scanidavius. Bar = 0.5 µm.

to autophagy. TEM shows two types of autophagy—a nonselective reticulum or spheres with the reserve material (mainly lipids) process (different organelles and structures are closed inside the accumulated and were targeted with the autophagosomes. autophagosomes) and a selective process (specific organelles are Reticulophagy results in the disposal of damaged/unfolded pro- neutralized). Depending on the organelles that have been enclosed teins or even eliminates damaged membranes, thereby protecting in the autophagosomes, we can differentiate mitophagy, reticu- a cell against their accumulation (Bernales et al., 2006; Cebollero lophagy, or lipophagy (Bernales et al., 2007; Narendra et al., et al., 2012). Spheres of the reserve material (mainly lipids or gly- 2008; Demine et al., 2012; Mijaljica et al., 2012;Włodarczyk colipids) can be utilized inside autophagosomes/autolysosomes in et al., 2017). In the millipedes that were examined here, we order to supply the energy which originates from the products of observed both types of autophagy––the nonselective and selective digestion. The pathway of lipid metabolism through the lysosomal processes. Among selective autophagy, reticulophagy and lipoph- degradative pathway of autophagy has been described as lipophagy were observed when many cisterns of the endoplasmic agy (Liu & Czaja, 2013). In the reserve material described in

Figure 8. a–f: Fluorescence micrograph of red TUNEL-positive signals (white circles) and Hoechst 33342 staining in midgut epithelium (e)ofT. aoutii (a–c) and P. lagurus (d–f). Midgut lumen (l), nuclei (blue signals). a–c: Bar = 32 µm. d–f: Bar = 45 µm.

the cytoplasm of digestive cells in millipedes, we detected lipids, which are damaged or have many autophagic structures, xenobi- polysaccharides, proteins, or glycolipids (Sosinka et al., 2014; otics, or pathogens, from the digestive epithelium. In the midgut Rost-Roszkowska et al., 2018b). The lamellar bodies degraded lumen, they are eventually digested and the inflammation is not due to autophagy in millipedes are concentric membrane layers activated (Franzetti et al., 2012; Rost-Roszkowska et al., 2012). which function in secretion and lipid storage (Schmitz & Apoptotic and autophagic morphologies may coexist in the Müller, 1991) or the degradation of glycolipids and glycoproteins same cells, so the cell death may be a mixed type even at the (Allegranza et al., 1989; Hariri et al., 2000). This confirms the fact molecular level. In addition, these two processes may depend that autophagy is used in millipede species to digest the reserve on each other in some tissues/organs (Gozuacik & Kimchi, material as a source of energy. We conclude that different organ- 2007). The same upstream signaling pathways may interfere on elles (mitochondria, cisterns of endoplasmic reticulum—RER, both apoptotic and autophagic directions. The knockdown of etc.) that are damaged by any of the xenobiotics that originate key autophagy genes can activate nutrient deprivation-induced from food may be utilized without any harmful effect on the apoptotic cell death (Boya et al., 2005; Gozuacik & Kimchi, entire cell. In this case, the nonspecific autophagy is activated. 2007). In addition, autophagy and apoptosis can also be activated Autophagy is also a survival factor against infection by pathogens simultaneously (Lee et al., 2003). Therefore, a distinct crosstalk in millipedes. They were enclosed inside the autophagosomes and between autophagy and apoptosis and necrosis has been removed from the midgut epithelium into the midgut lumen. The described (Orrenius, 2004; Franzetti et al., 2012; Lipovšek et al., similar mechanism has been described in the midgut epithelium 2018; Rost-Roszkowska et al., 2018c). In the digestive epithelia of the other invertebrates. However, when too many autophagic of many invertebrates, apoptosis is a natural process which, due structures occur in the cytoplasm, the cell must degenerate to the neutralization of epithelial cells, prevents the tissue against (Levine & Klionsky, 2004; Giusti et al., 2007; Rost-Roszkowska inflammation (Vaidyanathan & Scott, 2006; Maghsoudi et al., et al., 2010, 2015; Franzetti et al., 2012). The process of cell degen- 2012; Lipovšek et al., 2018). However, the autophagy does not eration (e.g., apoptosis, necrosis) enables the elimination of cells activate apoptosis and/or necrosis in the hepatic cells of

Table 2. Mean (x) ± Standard Deviation (SD) of Apoptotic Cells in the Midgut Intensive autophagy can activate apoptosis, but only in the diges- Epithelium in Millipedes (n =5; n – Number of Sections in Which 100 Cells tive cells of the midgut epithelium. were Counted; One Specimen of Each Species). Acknowledgments. We would like to express our gratitude to Dr. Danuta Digestive Secretory Regenerative Urbańska-Jasik and Dr. Łukasz Chajec (University of Silesia in Katowice) Cells Cells Cells for their technical assistance. J. scandinavius 13.4 ± 2.19 –– Conflict of Interest. The authors declare that they have no conflict of P. lagurus 7.4 ± 1.67 ––interests. –– A. gigas 11.0 ± 2.58 Research Involving Human Participants and/or Animals. To collect the T. aoutii 10.4 ± 2.3 ––material, no specific permissions were required for locations/activities. No human material was analyzed. The studies did not involve endangered or protected species.

Informed Consent. On behalf of my co-authors, I declare that the manu- millipedes (Rost-Roszkowska et al., 2018a). It would probably be script has not been published anywhere and has not been submitted to any the mechanism that enables the digestion of the reserve material: other Journal. the cytoplasm of hepatic cells is rich in glycogen granules digested due to autophagy (De Godoy & Fontanetti, 2010; Nogarol & Fontanetti, 2011; Rost-Roszkowska et al., 2018a). In some short- References living arthropods, in which regenerative cells were not detected, Allegranza A, Tredici G, Marmirolli P, di Donato S, Franceschetti S & the autophagy enables to fulfill the digestive cells their functions Mariani C (1989). Sialidosis type I: Pathological study in an adult. Clin as long as possible (Rost-Roszkowska et al., 2012). Therefore, Neuropathol 8, 266–271. we can conclude that, if autophagy appears in the cytoplasm of Baton LA & Ranford-Cartwright LC (2007). Morphological evidence for pro- digestive, secretory, and/or regenerative cells, it plays the role of liferative regeneration of the Anopheles stephensi midgut epithelium following a survival factor. However, when the stressor is too strong, it Plasmodium falciparum ookinete invasion. J Invertebr Pathol 96, 244–254. accelerates cell death. In some invertebrates’ digestive systems, a Bernales S, McDonald KL & Walter P (2006). Autophagy counterbalances distinct crosstalk between autophagy and necrosis has been endoplasmic reticulum expansion during the unfolded protein response. PLoS Biol 4(12), e423. described: intensive autophagy activates necrosis, while the apo- Bernales S, Schuck S & Walter P (2007). Selective autophagy of the endoplas- ptosis was not detected even using immunochemical methods mic reticulum. Autophagy 3, 285–287. (Rost-Roszkowska et al., 2018c). Here we suggest a crosstalk Boya P, Gonzales-Polo RA, Casares N, Perfettini JL, Dessen P, between autophagy and apoptosis in the midgut epithelium of Larochette N, Métivier D, Meley D, Souquere S, Yoshimori T, millipedes. Apoptosis was observed in the digestive cells of centi- Pierron G, Codogno P & Kroemer G (2005). Inhibition of macroautoph- pedes, but they never occurred in the midgut secretory or regen- agy triggers apoptosis. Mol Cell Biol 25, 1025–1040. erative cells (Rost-Roszkowska et al., 2015). As has been Camargo-Mathias MI, Fantazzini ER & Fontanetti CS (2004). mentioned above, the secretory and regenerative cells are closed Ultrastructural features of the midgut of Rhinocricus padbergi – cell types and their cell membranes do not contact the midgut (Diplopoda: Spirobolida). Braz J Morphol Sci 21,65 71. lumen and food masses (Chajec et al., 2012, 2014; Sosinka Cebollero E, Reggiori F & Kraft C (2012). Reticulophagy and ribophagy: Regulated degradation of protein production factories. Int J Cell Biol. et al., 2014; Rost-Roszkowska et al., 2018a, 2018b, 2018c). The 2012: 182834 (it is published abstract) digestive cells are the only cells which contact the nourishments Chajec Ł, Rost-Roszkowska MM, Vilimova J & Sosinka A (2012). so they are exposed to numerous stressors which can enter the Ultrastructure and regeneration of midgut epithelial cells in Lithobius forfi- animals’ body from the midgut lumen (Vaidyanathan & Scott, catus (Chilopoda, Lithobiidae). Inv Biol 131, 119–132. 2006; Baton & Ranford-Cartwright, 2007; Franzetti et al., 2012; Chajec Ł, Sonakowska L & Rost-Roszkowska MM (2014). The fine structure Wilczek et al., 2014; Lipovšek et al., 2018). This process undergoes of the midgut epithelium in a centipede, Scolopendra cingulata (Chilopoda, in the midgut epithelium with a similar frequency in the milli- Scolopendridae) with the special emphasis on epithelial regeneration. pedes examined here: 13.4, 7.4, 11, and 10.4% suggesting that Arthropod Struct Dev 43,27–42. they were not exposed to any stressors. Cruz LC, Araújo VA, Dolder H, Araújo APA, Serrão JE & Neves CA (2011). Autophagy is also a survival factor against infection by patho- Morphometry of the midgut of Melipona quadrifasciata anthidioides (Lepeletier) (Hymenoptera: Apidae) during metamorphosis. Neotrop gens in millipedes. Rickettsia-like microorganisms were observed Entomol 40(6), 677–681. in about 30% of the P. lagurus and J. scandinavius specimens. De Godoy JAP & Fontanetti CS (2010). Diplopods as bioindicators of soils: We can suspect that these microorganisms are probably patho- Analysis of midgut of individuals maintained in substract containing sewage gens which can get to the midgut epithelium due to endocytosis sludge. Water Air Soil Pollut 210, 389–398. (Sokolova et al., 2006). In addition, they were enclosed inside the Demine S, Michel S, Vannuvel K, Wanet A, Renard P & Arnould T (2012). autophagosomes and discharged into the midgut lumen. A simi- Macroautophagy and cell responses related to mitochondrial dysfunction, lar neutralization of pathogens was reported for centipede species lipid metabolism and unconventional secretion of proteins. Cells 1(2), by Chajec et al. (2012, 2014) or Hexapoda (Rost-Roszkowska 168–203. et al., 2010; Boya et al., 2005). In summary, we can conclude Eitzinger B, Micic A, Körner M, Traugott M & Scheu S (2013). Unveiling that autophagy is a survival process that is commonly observed soil food web links: New PCR assays for detection of prey DNA in the gut of soil arthropod predators. Soil Biol Biochem 57, 943–945. in the digestive cells of midgut epithelium in millipedes. Fantazzini ER, Fontanetti CS & Camargo-Mathias MI (2002). Midgut of the Furthermore, it may occur in all epithelial cells: digestive, secre- millipede, Rhinocricus padbergi (Verhoeff, 1938) (Diplopoda: Spirobolida): tory, and regenerative cells. However, the apoptosis was activated Histology and histochemistry. Arthropoda Sel 11, 135–142. only in the digestive cells of the midgut epithelium, while we did Fernandes KM, Neves CA, Serrão JE & Martins GF (2014). Aedes aegypti not manage to detect it in the secretory and regenerative cells. midgut remodeling during metamorphosis. Parasitol Int 63(3), 506–512.

Fontanetti CS & Camargo-Mathias MI (1997). Histoanatomy of the digestive Malagoli D, Abdalla FC, Cao Y, et al. (2010). Autophagy and its physiological tract in Plusioporus setiger diplopod (Brolemann, 1901) (Spirostreptida, relevance in arthropods current knowledge and perspectives. Autophagy 6, Spirostreptidae). Braz J Morphol Sci 14, 205–211. 575–588. Fontanetti CS & de Godoy JAP (2007). Ultrastructural alterations observed in Mijaljica D, Prescott M & Devenish RJ (2012). The intriguing life of autopha- the midgut of the diplopod Rhinocricus padbergi exposed to sewage mud. gosomes. Int J Mol Sci 13, 3618–3635. Acta Microsc 16, 191–192. Mitra A & Flynn KJ (2007). Importance of interactions between food quality, Fontanetti CS, Tiritan B & Camargo-Mathias MI (2006). Mineralized bodies quantity, and gut transit time on consumer feeding, growth, and trophic in the fat body of Rhinocricus padbergi (Diplopoda). Braz J Morphol Sci 23, dynamics. Am Nat 169, 632–646. 487–493. Moreira-de-Sousa C, Iamonte M & Fontanetti CS (2017). Midgut of the dip- Fontanetti CS, Moreira-de-Sousa C, Pinheiro TG, Souza RB & Francisco A lopod Urostreptus atrobrunneus: Structure, function, and redefinition of (2015). Diplopoda-digestive system. In The Myriapoda. Treatise on hepatic cells. Braz J Biol 77(1), 132–139. Zoology—Anatomy, Taxonomy, Biology, Minelli A (Ed.), pp. 109–127. the Mpakou VE, Velentzas AD, Velentzas PD, et al. (2011). Programmed cell death Netherlands, Brill, Leiden, Boston: Printforce. of the ovarian nurse cells during oogenesis of the ladybird beetle Adalia Franzetti E, Huang ZJ, Shi Y, et al. (2012). Autophagy precedes apoptosis bipunctata (Coleoptera: Coccinellidae). Dev Growth Differ 53, 804–815. during the remodeling of silkworm larval midgut. Apoptosis 17, 305–324. Narendra D, Tanaka A, Suen DF & Youle RJ (2008). Parkin is recruited Giusti F, Dallai L, Beani L, et al. (2007). The midgut ultrastructure of the selectively to impaired mitochondria and promotes their autophagy. J Cell endoparasite Xenos vesparum (Rossi) (Insecta, Strepsiptera) during post- Biol 183, 795–803. embryonic development and stable carbon isotopic analyses of the nutrient Nogarol LR & Fontanetti CS (2011). Ultrastructural alterations in the midgut uptake. Arthropod Struct Dev 36, 183–197. of diplopods after subchronic exposure to substrate containing sewage mud. Gozuacik D & Kimchi A (2007). Autophagy and cell death. Curr Top Dev Biol Water Air Soil Pollut 218, 539–547. 78, 217–245. Orrenius S (2004). Mitochondrial regulation of apoptotic cell death. Toxicol Hakim RS, Baldwin KM & Loeb M (2001). The role of stem cells in midgut Lett 149,19–23. growth and regeneration. In Vitro Cell Dev Biol Animal 37(6), 338–342. Park MS, Park P & Takeda M (2013). Roles of fat body trophocytes, myceto- Hakim RS, Caccia S & Loeb M (2009). Primary culture of insect midgut cells. cytes and urocytes in the American cockroach, Periplaneta americana In Vitro Cell Dev Biol Animal 45(3–4), 106–110. under starvation conditions: An ultrastructural study. Arthropod Struct Hariri M, Millane G, Guimond MP, Guay G, Dennis JW & Nabi IR (2000). Dev 42, 287–295. Biogenesis of multilamellar bodies via autophagy. Mol Biol Cell 11(1), 255– Rost-Roszkowska M, Machida R & Fukui M (2010). The role of cell death in 268. the midgut epithelium in Filientomon takanawanum (Protura). Tissue Cell He C & Klionsky D (2009). Regulation Mechanisms and Signaling Pathways 42,24–31. of Autophagy. Annu Rev Genet 43,67–93. Rost-Roszkowska MM, Vilimova J, Sosinka A, Skudlik J & Franzetti E (2012). Hopkin SP & Read HJ (1992). The Biology of Millipedes, pp. 1–233. The role of autophagy in the midgut epithelium of Eubranchipus grubii New York: Oxford University Press. (Crustacea, Branchiopoda, Anostraca). Arthropod Struct Dev 41, 271–279. Karasov WH, Martinez del Rio C & Caviedes-Vidal E (2011). Ecological Rost-Roszkowska MM, Chajec Ł, Vilimova J, Tajovský K & physiology of diet and digestive systems. Annu Rev Physiol 73,69–93. Kszuk-Jendrysik M (2015). Does autophagy in the midgut epithelium of Karpeta-Kaczmarek J, Augustyniak M & Rost-Roszkowska M (2016). centipedes depend on the day/night cycle? Micron 68, 130–139. Ultrastructure of the gut epithelium in Acheta domesticus after long- term Rost-Roszkowska MM, Chajec Ł, Vilimova J & Tajovsky K (2016). Apoptosis exposure to nanodiamonds supplied with food. Arthropod Struct Dev 45, and necrosis during the circadian cycle in the centipede midgut. 253–264. Protoplasma 253(4), 1051–1061. Khoa DB & Takeda M (2012). Expression of autophagy 8 (Atg8) and its role Rost-Roszkowska MM, Vilimová J, Tajovský K, et al. (2018a). The ultra- in the midgut and other organs of the greater wax moth, Galleria mellonella, structure of the hepatic cells in millipedes (Myriapoda, Diplopoda). Zool during metamorphic remodeling and under starvation. Insect Mol Biol 21, Anz 274,95–102. 473–487. Rost-Roszkowska M, Kszuk-Jendrysik M, Marchewka A & Poprawa I Larsson N-G & Masucci MG (2016). Scientific Background: Discoveries of (2018b). Fine structure of the midgut epithelium in the millipede Mechanisms for Autophagy. Nobel Media AB, 2014. The Nobel assembly Telodeinopus aoutii (Myriapoda, Diplopoda) with special emphasis on epi- at Karolinska Institutet. http://www.nobelprizemedicine.org/wpcontent/ thelial regeneration. Protoplasma 255,43–55. uploads/2016/10/Scientific-Background-2016.pdf Rost-Roszkowska M, Janelt K & Poprawa I (2018c). The role of autophagy Lee CY, Clough EA, Yellon P, Teslovich TM, Stephan DA & Baehrecke EH in the midgut epithelium of Parachela (Tardigrada). Zoomorphology 137, (2003). Genome wide analyses of steroid—and radiation triggered pro- 501–509. grammed cell death in Drosophila. Curr Biol 13(4), 350–357. Santos DE, Zanuncio JC, Gonçalves de Oliveira AA & Serrao JE (2016). Levine B & Klionsky DJ (2004). Development by self-digestion: Molecular Endocrine cells in the midgut of bees (Hymenoptera: Apoidea) with differ- mechanisms and biological functions of autophagy. Dev Cell 6, 463–477. ent levels of sociability. J Apicult Res 54(4), 394–398. Lewis PR & Knight DP (1992). Cytochemical Staining Methods for Electron Schmitz G & Müller G (1991). Structure and function of lamellar bodies, Microscopy. USA: Elsevier, University of Michigan. lipid-protein complexes involved in storage and secretion of cellular lipids. Lipovšek S & Novak T (2015). Autophagy in the fat body cells of the cave J Lipid Res 32, 1539–1570. cricket Troglophilus neglectus Krauss, 1878 (Rhaphidophoridae, Saltatoria) Semenza GL (2008). Mitochondrial autophagy: Life and breath of the cell. during overwintering. Protoplasma 253, 457–466. Autophagy 4(4), 534–536. Lipovšek S, Novak T, Janžekovič F & Leitinger G (2015). Changes in the Sigling S (2010). Professional Breeders Series: Millipedes, 205 pp. Edition midgut diverticula in the harvestmen Amilenus aurantiacus (Phalangiidae, Chimaira, Germany: Frankfurt am Main. Opiliones) during winter diapause. Arthropod Struct Dev 44, 131–141. Sokolova YY, Lange CE & Fuxa JR (2006). Development, ultrastructure, nat- Lipovšek S, Leitinger G, Novak T, Janžekovič F, Gorgoń S, Kamińska K & ural occurrence, and molecular characterization of Liebermannia patagon- Rost-Roszkowska M (2018). Changes in the midgut cells in the European ica n. g., n. sp., a microsporidian parasite of the grasshopper Tristira cave spider, Meta menardi, during starvation in spring and autumn. magellanica (Orthoptera: Tristiridae). J Inv Pathol 91, 168–182. Histochem Cell Biol 149(3), 245–260. Sonakowska L, Włodarczyk A, Wilczek G, Wilczek P, Student S & Rost- Liu K & Czaja MJ (2013). Regulation of lipid stores and metabolism by lip- Roszkowska MM (2016). Cell death in the epithelia of the intestine and ophagy. Cell Death Differ 20,3–11. hepatopancreas in Neocaridina heteropoda (Crustacea, Malacostraca). Maghsoudi N, Zakeri Z & Lockshin RA (2012). Programmed cell death and PLoS ONE, 11, e0147582. apoptosis–where it came from and where it is going: From Elie Metchnikoff Sosinka A, Rost-Roszkowska MM, Vilimova J, Tajovský K, Kszuk- to the control of caspases. Exp Oncol 34, 146–152. Jendrysik M, Chajec Ł, Sonakowska L, Kamińska K, Hyra M &

Poprawa I (2014). The ultrastructure of the midgut epithelium in millipedes Tsujimoto Y & Shimizu S (2005). Another way to die: Autophagic pro- (Myriapoda, Diplopoda). Arthropod Struct Dev 43, 477–492. grammed cell death. Cell Death Differ 12, 1528–1534. Souza TS & Fontanetti CS (2011). Morphological biomarkers in the Vaidyanathan R & Scott TW (2006). Apoptosis in mosquito midgut epithelia Rhinocricus padbergi midgut exposed to contaminated soil. Ecotoxicol associated with West Nile virus infection. Apoptosis 11, 1643–1651. Environ Safety 74,10–18. Wilczek G, Rost-Roszkowska M, Wilczek P, Babczyńska A, Szulińska E, Šustr V, Tajovský K, Semanová S, Chronakova A & Simek M (2013). The giant Sonakowska L & Marek-Swędzioł M (2014). Apoptotic and necrotic African millipede, Archispirostreptus gigas (Diplopoda: Spirostreptida), a changes in the midgut glands of the wolf spiders Xerolycosa nemoralis model species for ecophysiological studies. Acta Soc Zool Bochem 77,145–158. (Lycosidae) in response to starvation and dimethoate exposure. Ecotoxic Teixeira AD, Fialho MCQ, Zanuncio JC, Ramalho FS & Serrão JE (2013). Environ Safe 101, 157–167. Degeneration and cell regeneration in the midgut of Podisus nigrispinus Włodarczyk A, Sonakowska L, Kamińska K, Marchewka A, Wilczek G, (Heteroptera: Pentatomidae) during post-embryonic development. Arthropod Wilczek P, Studnet S & Rost-Roszkowska M (2017). The effect of Struct Dev 42, 237–246. starvation and re-feeding on mitochondrial potential in the midgut Tettamanti G, Cao Y, Feng Q, Grimaldi A & de Eguileor M (2011). of Neocaridina davidi (Crustacea, Malacostraca). PLoS ONE 12(3), Autophagy in Lepidoptera: More than old wine in new bottle. ISJ 8,5–14. e0173563.