Microscopy and Microanalysis (2019), 25, 1004–1016 doi:10.1017/S143192761900059X Micrographia Autophagy and Apoptosis in the Midgut Epithelium of Millipedes 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. © Microscopy Society of America 2019 Downloaded from https://www.cambridge.org/core. University of Athens, on 06 Oct 2021 at 22:40:11, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S143192761900059X Microscopy and Microanalysis 1005 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 num- ber of apoptotic cells among the midgut epithelium. The percent- age 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 rep- resent 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