Changes in the midgut cells in the European cave spider, Meta menardi, during starvation in spring and autumn

  • Saška Lipovšek
  • Gerd Leitinger
  • Tone Novak
  • Franc Janžekovič
  • Szymon Gorgoń
  • Karolina Kamińska
  • Magdalena Rost-Roszkowska
Original Paper
  • 46 Downloads

Abstract

During the growth period, in surface habitats, spiders catch enough prey to feed normally. In contrast, in the cave entrance zone, prey may be relatively scarce. Meta menardi inhabits this cave section, resulting in temporary starvation. We studied structural changes in the midgut epithelial cells of M. menardi during a short-term and a medium-term controlled starvation to mimic the occasional starvation in caves, during spring and autumn. Digestive cells, secretory cells and adipocytes were examined before the experimental starvation, in the middle and at the end of starvation. We used light microscopy, transmission electron microscopy and specific histochemical methods for the detection of lipids, polysaccharides and proteins. Detection of lysosomes, autolysosomes and apoptosis was also carried out. The general structures of the cells did not change during the experimental starvation in either season, while some specific differences in the ultrastructure were observed. In both sexes, in both seasons, the amounts of lipids, glycogen and proteins decreased during starvation. Larger amounts of lipids were found in autumn, while there were no significant differences in the amounts of glycogen and proteins. In both sexes, in both seasons, autophagy and apoptosis intensified with starvation in progress, but more intensively in females. Thus, autumn individuals, in contrast to spring ones, compile energy-supplying stores to confront the subsequent winter deficiency of prey in caves, while the cellular ultrastructures undergo the same starvation-dependant changes at any time during the growth period.

Keywords

Autophagy Apoptosis Energy compound supply Arachnids Starvation 

Introduction

The European cave spider, Meta menardi (Latreille, 1804) (Araneae, Tetragnathidae), is an opportunistic predator inhabiting the twilight zone of most hypogean habitats across Europe. Measuring up to 17 mm (Fritzén and Koponen 2011; Hörweg et al. 2011; Nentwig et al. 2017), it is among the most prominent invertebrates in the cave entrance zone (Leruth 1939; Tercafs 1972; Dresco-Derouet 1960; Bourne 1976, 1977; Růžička 1990; Marusik and Koponen 1992; Smithers 1996, 2005a, b; Novak et al. 2010, 2012; Deltshev 2011; Fritzén and Koponen 2011; Hörweg et al. 2011; Isaia et al. 2011; Mammola and Isaia 2014, 2016; Helsdingen 2015; Manenti et al. 2015).

According to the original ecological classification of subterranean animals (Schiner 1854; Racoviță 1907; Boutin 2004), trogloxenes are not adapted to the subterranean habitat, troglobionts are well adapted, and troglophiles are in between. Troglophiles either alternate between the epigean and hypogean habitats or live permanently in the subterranean, but show only moderate adaptation to the subterranean habitat, e.g., partly reduced eyes and adaptations to compensate for the lack of visual orientation, and moderate tolerance for below-zero temperatures (Kirchner 1987; Novak et al. 2014).

Meta menardi ranks among the troglophiles, but do not complete their life cycle underground. They are active throughout the year and live about 2 years. The life cycle consists of two ecophases: a hypogean and an epigean phase (Szymczakowski 1953; Smithers 2005a; Novak et al. 2010; Hörweg et al. 2011; Mammola and Isaia 2014). Adults mate in hypogean habitats in spring. In summer, females produce egg sacs (cocoons) (Lepore et al. 2012; Chiavazzo et al. 2015). Juveniles hatch from the egg sacs in late autumn or in winter, and the second-instar spiderlings leave the subterranean habitats and spread outside by ballooning. Until becoming fourth-instars, they live outside, and then return to the hypogean habitat, where they live until their death (Hörweg et al. 2011; Mammola and Isaia 2014).

Spiders tolerate starvation well and can go without food for months. Abiotic features and prey availability are major determinants of habitat suitability for cave spiders (Manenti et al. 2015). In central Europe, during the growth season, when most epigean invertebrates are active, two main groups of potential prey for M. menardi appear in caves with respect to their abundance and mobility (Novak et al. 2010). About 50, mostly sparse prey species are present on the ceiling and walls throughout the year, and a massive immigration of about a dozen estivating species appears during the driest and warmest months (Novak et al. 2010). However, once in place, estivating individuals do not change their place, or do so rarely, and gradually some of them move into fissures, stone interspaces and similar microhabitats, and thus are no longer available to M. menardi. Estivating individuals leave the caves after at least a few days of cold, wet weather. Such prey dynamics provides only seasonally limited access to migratory prey for the spiders within caves. This could be the reason for the evolution of a special preying strategy in M. menardi that combine catching flying prey in webs and crawling prey on cave walls (Tercafs 1972; Bourne and Robert 1978; Eckert and Moritz 1992; Smithers 2005a; Novak et al. 2010; Mammola and Isaia 2014). The relatively small orb web of M. menardi is rudimentary; its widely spaced spirals cannot ensnare small flying insects (Eckert and Moritz 1992; Novak et al. 2010; Fritzén and Koponen 2011; Mammola and Isaia 2014). In caves, M. menardi prey on a range of prey, like slugs, non-flying arthropods and flying insects (Tercafs 1960; Pötzsch 1966; Smithers 1996, 2005b; Nyffeler and Symondson 2001; Novak et al. 2010; Mammola and Isaia 2014).

In spiders, digestion begins extra-corporally, and the liquefied food is pumped into the midgut, where it is finally digested and the nutrients are absorbed. The epithelium of the midgut is composed of four cell types: basal, secretory and digestive cells and guanocytes (Foelix 1996; Felgenhauer 1999). Basal cells are non-differentiated cells, which transform into secretory and digestive cells (Foelix 1996; Felgenhauer 1999). Secretory cells are characterized by an abundant rough endoplasmic reticulum, and many electron-dense granules containing digestive enzymes (Foelix 1996; Felgenhauer 1999). The digestive cells are most numerous in the midgut epithelium. They are characterized by digestive vacuoles (Foelix 1996). Guanocytes are specialized absorptive cells, which metabolize and store products like purine, guanine and uric acid (Foelix 1996; Felgenhauer 1999).

Autophagy is an adaptation process of cells to stress conditions (Mizushima et al. 2008). In cells sufficiently supplied with nutrients to maintain their normal functioning, autophagy is suppressed. Otherwise, it is induced by hormone stimulation (Tettamanti et al. 2011; Franzetti et al. 2012), microsporidian infection (Rost-Roszkowska et al. 2011), chemical substances (Wilczek et al. 2014) and starvation (Munafo and Colombo 2001; Romanelli et al. 2014; Wilczek et al. 2014; Lipovšek et al. 2014). In arthropods, overwintering in hypogean habitats, autophagy is an important pro-survival process (Lipovšek et al. 2014, 2015, 2017; Lipovšek and Novak 2016).

The consequences of stress involve changes in polypeptide synthesis, denaturation of proteins, DNA damage, disorders in intracellular respiration, and disintegration of cellular structures (Mizushima et al. 2008; Klionsky et al. 2016). When a cell can no longer stand the extent of the damage, it carries out apoptotic and/or necrotic changes and dies. Apoptotic mechanisms contribute to achieving a proper balance between the proliferation intensity and the rate of elimination of destroyed cells (Zakeri and Lockshin 2002). Necrosis or necrotic cell death is a passive process, initiated by physical, chemical or/and biotic factors that initiate a number of morphological changes, loss of osmotic pressure and swelling of cells (McCall 2010).

In this research study, we considered that in natural habitats, M. menardi feed whenever they catch prey. However, spiders may not catch any prey for shorter or longer periods, e.g., 2 months, and consequently undergo natural starvation. We asked which characteristic changes appear in the midgut epithelial cells in starved M. menardi at the beginning (spring) and at the end (autumn) of the growth period. For this purpose, we arranged a short-term and a medium-term starvation experiment with M. menardi in captivity under control. Since autophagy, apoptosis and necrosis are efficient indicators of the effects of nutrient deficiency, we focused on changes in these processes. We hypothesized that in spring, adults are fully vital but poorly fed, while in autumn they are aging, well fed and, consequently, showing both starvation-induced and gerontological changes. In the experiment, we expected more intensive apoptotic activity, greater amounts of stored energy-supplying compounds and less intensive autophagic activity in the autumn individuals, as compared to the spring ones.

Materials and Methods

Experimental design

We collected nine males and nine females from three caves in northern Slovenia (locality centroid 46°24 × 55″N, 15°10 × 31″E; altitudes 600–740 m) in May (“spring spiders”), and an equal number of specimens in October (“autumn spiders”). All the specimens were adults, so they had undergone their natural period of winter starvation. During spring, adults recover from the overwintering malnutrition and prepare for mating at the beginning of summer. During autumn, M. menardi accumulate reserve materials as a source of energy during overwintering (own observation). The specimens were held individually in glasses with wet paper, in a refrigerator at 8 °C. We assumed that after about 2 weeks of starvation, a conspicuous number of autophagic and apoptotic structures would appear, while there would not yet be any necrotic structures. After about 7 weeks of starvation, we expected to find all these structures. In accordance with data from the literature (Park et al. 2009) and with the species biology, spiders of one group were starved for 18 days, and those of the other group for 45 days. The spiders collected from natural habitats were randomly divided into three experimental groups of 6 individuals each. Cells from all the individuals, from different diverticules and different diverticule segments (proximal, middle, distal) were equivalently taken for the analysis.

Group 1 (beginning of the starvation experiment; control): three males and three females were analysed just after collection from their natural habitat.

Group 2 (middle of the starvation experiment): three males and three females were analysed after 18 days of starvation.

Group 3 (end of the starvation experiment): three males and three females were analysed after 45 days of starvation.

Light and transmission electron microscopy (TEM)

For structural and ultrastructural studies, small pieces of the midgut diverticula were quickly extracted and fixed. The tissue was fixed in 2.45% glutaraldehyde and 2.45% paraformaldehyde in a 0.1 M sodium cacodylate buffer (pH 7.4) at room temperature for 2 h and at 4 °C for 12 h, washed in a 0.1 M sodium cacodylate buffer (pH 7.4) at room temperature for 3 h and postfixed with 2% OsO4 at room temperature for 2 h. The samples were dehydrated in a graded series of ethanol (50, 70, 90, 96, 100%, each for 30 min at room temperature) and embedded in TAAB epoxy resin (Agar Scientific Ltd., Essex, England). For light microscopy, semi-thin sections (5 μm) were used, stained with 0.5% toluidine blue in aqueous solution and analysed using a light microscope Nikon Eclipse E800. We used a Nikon DN100 camera. For TEM, ultra-thin sections (70–75 nm) of the midgut were transferred onto copper grids, stained with uranyl acetate and lead citrate and analyzed with a Zeiss EM 902 transmission electron microscope. The percentage of cells with autophagic structures was determined by random counting in 100 midgut epithelium cells. Cells, observed at the 3000× magnification, containing autophagic structures were considered autophagic cells.

Histochemical methods for light microscopy

Semi-thin sections of the samples embedded in TAAB were used in order to quantify changes in the amounts of lipids, glycogen and other polysaccharides and proteins.

Detection of lipids (Sudan black B staining)

Semi-thin sections were stained with Sudan black B (15 min, room temperature) (Litwin 1985). Thereafter, the semi-thin sections were washed in 50% ethanol and afterwards in distilled water, and analyzed using a Nikon Eclipse E800 light microscope equipped with a Nikon DN100 digital camera.

Detection of polysaccharides (PAS method)

Semi-thin sections were treated with a 2% solution of periodic acid (10 min, room temperature), washed in 70% ethanol and stained with Schiff’s reagent (24 h, 37 °C) (Litwin 1985). After washing in water, the slides were examined using a Nikon Eclipse E800 light microscope equipped with a Nikon DN100 digital camera.

Detection of proteins (Bonhag method)

Semi-thin sections were treated with a 1% solution of periodic acid (10 min, room temperature), washed in water and stained with bromophenol blue (BPB) (24 h, 37 °C) (Litwin 1985). Thereafter, the slides were washed in water and examined using a Nikon Eclipse E800 light microscope equipped with a Nikon DN100 digital camera.

Detection of lysosomes and autolysosomes (Acid phosphatase reaction)

The midgut diverticula were cut into small pieces and embedded in a tissue-freezing medium (Tissue-Tek, O.C.T Compound, Sakura Finetek, USA) without fixation. The material was cut into semi-thin cryosections (5 μm) using a Tissue-Tek II cryostat (Tissue-Tek, Naperville, IL, USA) and mounted on slides. After washing the slides in TBS for 5 min, these were washed in a 0.1 M sodium acetate–acetic acid buffer (25 °C, pH 5.0–5.2). Thereafter, cryosections were incubated in a 0.1 M sodium acetate–acetic acid buffer containing 2% N-N-dimethylformamide (Sigma-Aldrich), 0.06% Fast Red Violet LB (Sigma-Aldrich), 0.5 mM MnCl2 and 0.01% naphthol phosphate AS–BI (Sigma-Aldrich) (1.5 h, 37 °C). For negative control, cryosections were incubated in the mixture as given above, but without naphthol phosphate AS–BI. Cryosections were examined using an Olympus BX60 microscope equipped with an Olympus XC50 digital camera.

Detection of apoptosis (TUNEL assay)

The TUNEL assay was used for labeling of apoptotic cells. The midgut diverticula were cut into small pieces and embedded in a tissue-freezing medium (Tissue-Tek, O.C.T Compound, Sakura Finetek, USA) without fixation. The material was cut into semi-thin cryosections (5 μm) using a Tissue-Tek II cryostat (Tissue-Tek, Naperville, IL, USA) and mounted on slides. Cryosections were washed in TBS (3 × 5 min), and then stained with the TUNEL reaction mixture (In Situ Cell Death Detection Kit, TMR red, Roche) for 60 min (37 °C). After washing in TBS, the cryosections were stained with DAPI (1 μg/mL, Sigma). Cryosections were examined under an Olympus BX60 microscope equipped with an Olympus XC50 digital camera and appropriate filters. The percentage of apoptotic cells was determined by randomly counting 100 cells in the midgut epithelium—7 tissue samples from each sex in each season were analyzed.

Quantification of reserve lipids, glycogen and proteins by TEM

To estimate conditions with respect to these reserve compounds in the midgut epithelial cells during starvation, in each season, time frame and sex, we measured the diameters of 125 lipid droplets per sample, counted glycogen rosettes in 30 1-μm2 squares on micrographs, and measured the diameters of 30 protein granules per sample.

Statistical analysis

The data distribution of lipid droplet and protein granule diameters, and the glycogen counts were tested for normality using the Kolmogorov–Smirnov test. The test showed a moderate significance in lipid droplets and glycogen granules, we, therefore, Log10-transformed the data for testing of means. Factorial ANOVA was used in testing differences between means for sex, time frame and season.

Results

Structure of the midgut diverticula epithelial cells

In all the specimens examined, the midgut consisted of a branched system of diverticula (Fig. 1). We found no differences in the general morphology of the midgut during the experiment. The diverticula were composed of a pseudostratified epithelium consisting of three cell types: the digestive cells, the secretory cells and the adipocytes (Fig. 1). Under light microscopy, the cytoplasm of the secretory cells was conspicuously darker than the cytoplasm of the digestive cells and the adipocytes (Fig. 1). Light microscopy observations showed structural changes in the midgut diverticula epithelium in tissue sections from the starved spiders as compared to controls (Fig. 1). In both sexes, in both seasons and in the two starved groups, vacuolated cytoplasm was a prominent feature in the digestive cells and adipocytes (Fig. 1). In spring and autumn in both sexes, in each time frame, lipids, carbohydrates and proteins were visualized histochemically. Figure 2 shows the outcomes of histochemical reactions in non-starved individuals in spring. In both seasons, in all time frames the outcomes were quite comparable and were, therefore, not evaluated statistically; for this purpose, TEM images were used. For this reason, only the histochemical results for spring are presented.

Fig. 1

Semithin section of the midgut diverticula of Meta menardi in spring a before the experimental starvation (male), b starved for 18 days (male) and c starved for 45 days (female). In starved individuals, the cytoplasm of some epithelial cells is vacuolized (arrows). AC adipocyte, DC digestive cell, SC secretory cell. Scale bars: 50 µm

Fig. 2

Semithin section of the midgut diverticula of Meta menardi before the experimental starvation in spring. Detection of lipids in male (a) and female (b). Detection of glycogen and other polysaccharides in male (c) and female (d). Detection of proteins in male (e) and female (f). F female, M male. Arrows refer to positive reactions. Scale bars: 50 µm

Spring spiders

Beginning of the experiment

Here, the general characteristics of the three cell types, the secretory cells, the digestive cells and the adipocytes, are briefly described. The secretory cells contained an abundant rough endoplasmic reticulum (RER). Their apical plasma membrane was differentiated into microvilli. Electron-dense secretory granules were characteristic of the secretory cells (Fig. 3a). A round to oval nucleus was located centrally in the cell. Beside RER and electron-dense protein granules, the Golgi complexes, mitochondria, spherites and lipid droplets were observed in the cytoplasm of the secretory cells. The apical plasma membrane of the digestive cells was differentiated into microvilli (Fig. 3b). The cytoplasm contained many lipid droplets, mitochondria and spherites (Fig. 3b). In the adipocytes, there were many lipid droplets, glycogen granules and spherites (Fig. 3a). In males, in only a few adipocytes, autophagosomes containing glycogen granules were seen (Fig. 3c). In females, autophagosomes, autolysosomes and residual bodies were present in a few secretory cells, adipocytes and digestive cells. TEM observations were supported by the outcomes of the acid phosphatase staining (Fig. 4a, d). In both seasons and all three time frames, the outcomes of the acid phosphatase test and the TUNEL assay were quite comparable; therefore, only the results for spring are shown. Autophagic structures (Fig. 4a, d) and apoptotic cells (Fig. 5a, d) were rare and detected in only a few midgut epithelial cells (Table 1).

Fig. 3

Ultrathin section of the midgut diverticula of Meta menardi before the experiment in spring. a Adipocyte (AC) and secretory cell (SC); male. b Digestive cell (DC); female. c Adipocyte (AC); male. Ultrathin section of the midgut diverticula of Meta menardi starved for 18 days in spring. d, e Secretory cell (SC) and adipocyte (AC); female. f Digestive cell (DC); female. AP autophagosome, G glycogen granules, L lipid droplet, LU lumen of the midgut diverticulum, M mitochondria, MV microvilli, N nucleus, RB residual body, RER rough endoplasmic reticulum, S spherite, SG secretory granulum, *necrotic cell; arrow, vacuole. Scale bars: a, b 1 µm; c 500 nm; df 2 µm

Fig. 4

Semithin section of the midgut diverticula of Meta menardi in spring. Acid phosphatase staining. a Male before the experimental starvation. b Male starved for 18 days. c Male starved for 45 days. d Female at the beginning of the experiment. e Female starved for 18 days. f Female starved for 45 days. The acid phosphatase activity (arrows). Scale bars: a 30 µm; b, c 20 µm; df 30 µm

Fig. 5

Semithin transverse section of the midgut diverticula of Meta menardi in spring. TUNEL assay. a Male before the experimental starvation. b Male starved for 18 days. c Male starved for 45 days. d Female at the beginning of the experiment. e Female starved for 18 days. f Female starved for 45 days. TUNEL-positive nuclei of apoptotic cells (arrows). Scale bars: a 20 µm; b 30 µm; c 20 µm; d 10 µm; e 10 µm; f 30 µm

Table 1

Percentage rates of midgut epithelial cells with autophagic structures in Meta menardi, as observed by TEM, and percentage rates of apoptotic midgut epithelial cells in Meta menardi, as observed by the TUNEL assay

Time frame sex

Spring

Autumn

Beginning

Middle

End

Beginning

Middle

End

Percentage rates of cells with autophagic structures (TEM)

 ♂

14

49

67

17

52

61

 ♀

19

62

71

21

65

79

Percentage rates of apoptotic cells (TUNEL assay)

 ♂

1

10

39

2

12

38

 ♀

4

20

44

4

22

43

Spiders starved for 18 days

In all three cell types, the general structures were comparable to those at the beginning of the experiment. In males, in contrast to females, differences appeared only in the digestive cells; autophagy was more intensive with respect to the beginning and, consequently, the entire cytoplasm was rich in autophagosomes. In females, the majority of secretory cells showed characteristic ultrastructure, with abundant RER (Fig. 3d, e) and electron-dense secretory granules (Fig. 3e). A few necrotic adipocytes were seen (Fig. 3e). In some secretory and digestive cells (Fig. 3f), autophagic structures were present. Besides the changes described, vacuolization of the cytoplasm was observed in some digestive cells (e.g., Fig. 3f). No differences were found in the ultrastructure of adipocytes as compared to the beginning of starvation. Autophagic structures (Fig. 4b, e) and apoptotic cells (Fig. 5b, e; Table 1) were more abundant than at the beginning of the experiment.

Spiders starved for 45 days

In all the three cell types, the general structures were comparable to those at the beginning of the experiment. A few digestive cells were necrotic in appearance (Fig. 6a). The apical plasma membrane of the necrotic digestive cells was still differentiated into the microvilli; the nucleus was oval, but the cytoplasm was electron lucent, and the number of cell compartments was low; only a few mitochondria and many vacuoles were seen in the cytoplasm (Fig. 6a). Some digestive cells showed apoptotic features. The cytoplasm of apoptotic cells was electron dense, the nucleus was lobular, and the chromatin was very condensed. In the cytoplasm of an apoptotic digestive cell, a large autophagosome could be observed (Fig. 6b). In both sexes, autophagic structures were found only in the digestive cells (Fig. 6d, e) and adipocytes. In females, the secretory cells had the typical general ultrastructure, while cisterns of RER were more abundant in comparison to males (Fig. 6f). Autophagy (Fig. 4c, f) and apoptosis were both intensified during starvation (Fig. 5c, f; Table 1).

Fig. 6

Ultrathin section of the midgut diverticula of Meta menardi starved for 45 days in spring. ad male, e, f female. a Necrotic digestive cell (DC). b Apoptotic digestive cell (DC). ce The digestive cell (DC). f Secretory cell (SC). AL autolysosome, AP autophagosome, M mitochondria, MV microvilli, N nucleus, RER rough endoplasmic reticulum, S spherite. Scale bars: a 2 µm; b 1 µm; ce 2 µm; f 1 µm

Autumn spiders

Beginning of the experiment

The structures of the secretory cells, digestive cells and adipocytes were comparable to those in spring individuals. In males, some digestive cells contained residual bodies (Fig. 7a), while these structures were not detected in females. The cytoplasm contained mitochondria, glycogen granules and a few vacuoles (Fig. 7a). The adipocytes contained numerous lipid droplets with diameters of up to 10 µm in males (Fig. 7b), and up to 7 µm in females, glycogen granules and protein granules. The nucleus was compressed by lipid droplets and had an irregular shape (Fig. 7b). In some female adipocytes, the residual bodies were present, while these were missing in males. As in spring, autophagic structures and apoptotic cells were rare and detected only in a few midgut epithelial cells (Table 1).

Fig. 7

Ultrathin section of the midgut diverticula of Meta menardi at the beginning of the experiment in autumn. a, b male. a Perinuclear cytoplasm of the digestive cell (DC) with some bigger vacuoles (arrows). b The adipocyte (AC). The irregular shaped nucleus (N) compressed by lipid droplets. Ultrathin section of the midgut diverticula of Meta menardi starved for 18 days in autumn. c, d male. e, f female. c Electron-dense cytoplasm of the apoptotic digestive cell (DC) containing shrinked nucleus (N). d Perinuclear cytoplasm of the digestive cell (DC). e Adipocyte (AC) with lipid droplets (L), residual bodies (RB) and some larger vacuoles in the cytoplasm (arrows). f Rough endoplasmic reticulum (RER), Golgi apparatus (GA) and secretory granulum (SG) in the secretory cell (SC). AL autolysosome, G glycogen granules, L lipid droplet, M mitochondria, N nucleus, P protein granula, RB residual body, RER rough endoplasmic reticulum. Scale bars: a, b 2 µm; ce 2 µm; f 500 nm

Spiders starved for 18 days

In all three cell types, the general structures were comparable to those at the beginning of the experiment. Some digestive cells were apoptotic, and in some cells necrosis (Fig. 7c) appeared. Apoptotic digestive cells contained shrunken nucleus; the chromatin formed electron-dense clusters near the nuclear envelope (Fig. 7c). The cytoplasm of apoptotic digestive cells was electron dense, containing some larger vacuoles (Fig. 7c). The apoptotic digestive cells had shrunk and become detached from the neighbouring cells (Fig. 7c). Necrotic digestive cells were characterized by electron-lucent cytoplasm and damaged cellular compartments (Fig. 7c). In both sexes, some digestive cells contained autophagic structures and areas of huge vacuoles (Fig. 7c, d). The adipocytes were characterized by lipid droplets, glycogen granules, protein granules, residual bodies and some large vacuoles in the cytoplasm (Fig. 7e). Many secretory cells had well developed Golgi complexes, cisterns of the rough endoplasmic reticulum and secretory granules (Fig. 7f). As in spring, autophagic structures and apoptotic cells were more abundant with respect to the beginning of the experiment (Table 1).

Spiders starved for 45 days

In all the three cell types, the general structures were comparable to those at the beginning of the experiment. In both sexes, in all the cell types autophagic structures were present (Fig. 8a–f). The most abundant autophagic structures were residual bodies (Fig. 8a–c) and autophagosomes (Fig. 8c, inset). In both sexes, in the perinuclear and the apical cytoplasm of a few digestive cells, specific, irregularly shaped, electron-dense structures were present, containing lipid droplets (Fig. 8c). Some digestive cells and adipocytes contained areas of huge vacuoles (Fig. 8d, f). Individual adipocytes (Fig. 8e) and digestive cells (Fig. 8f) showed some features of necrosis. Some autophagosomes and large vacuoles were present in the cytoplasm (Fig. 8f). As in spring, autophagy and apoptosis were intensified during starvation (Table 1).

Fig. 8

Ultrathin section of the midgut diverticula of Meta menardi starved for 45 days in autumn. ac Male, df female. a Perinuclear cytoplasm of the adipocyte (AC). b Basal cytoplasm of the adipocytes (AC). c Apical cytoplasm of the digestive cell (DC). Inset: autophagosomes in the digestive cell. d Perinuclear cytoplasm of the digestive cell (DC). e Adipocyte (AC) and secretory cell (SC). f Digestive cell (DC). AP autophagosome, L lipid droplet, M mitochondria, MV microvilli, N nucleus, RB residual body, S spherite, SG secretory granulum. Scale bars: af 2 µm; the inset in c 500 nm

Quantification of reserve lipids, glycogen and proteins

Table 2 summarizes the measurements of lipid droplet diameters. In simple and combined parameters referring to the experiment, only those including time frames differed significantly. In both sexes, larger diameters were measured in autumn, and the diameters diminished during the experiment in both seasons (Table 2). In spring, the average lipid droplet diameters diminished by 0.01 µm/day in both sexes during the experiment. In autumn, until the middle, the average lipid droplet diameter diminished by 0.05 and 0.07 µm/day in males and females, respectively, while afterwards the diminution was 0.01 µm/day in both sexes.

Table 2

Descriptive statistics of lipid droplet diameters (µm) in the midgut epithelial cells of Meta menardi collected in spring and autumn in natural habitats, and experimentally exposed to starvation; No. of counts per sample (i.e., each season, sex and the time frame) = 125

Sex

Time frames of starvation experiment

Spring

Mean ± Std. Dev

Min–Max

Autumn

Mean ± Std. Dev

Min–Max

Beginning

1.4 ± 0.6

0.5–2.9

2.0 ± 1.1

0.7–7.5

Middle

1.2 ± 0.4

0.5–2.4

1.1 ± 0.4

0.1–1.9

End

0.9 ± 0.2

0.4–2.0

0.7 ± 2.2

0.3–1.4

Beginning

1.3 ± 0.5

0.4–2.5

2.3 ± 1.2

0.6–7.4

Middle

1.2 ± 4.4

0.5–2.4

1.0 ± 0.3

0.4–1.9

End

0.9 ± 0.3

0.3–2.0

0.7 ± 0.2

0.3–1.3

Counts of glycogen granules during the spring and autumn experiments are summarized in Table 3. In simple and combined parameters referring to the experiment, only counts in different time frames differed significantly. In both sexes, counts of spring and autumn glycogen granules were quite comparable, and diminished during the experimental starvation. There were no significant differences between the sexes.

Table 3

Descriptive statistics of glycogen granule abundance in the midgut epithelial cells of Meta menardi collected in spring and autumn in natural habitats, and experimentally exposed to starvation; No. of counts per sample (i.e., each season, sex and the starvation time frame) = 30

Sex

Time frames of starvation experiment

Spring

Mean ± Std. Dev

Min–Max

Autumn

Mean ± Std. Dev

Min–Max

Beginning

8 ± 2

5–12

9 ± 2

6–12

Middle

7 ± 1

5–10

7 ± 2

5–10

End

5 ± 1

3–8

5 ± 1

3–7

Beginning

9 ± 2

5–12

8 ± 2

6–12

Middle

7 ± 1

5–10

7 ± 1

5–10

End

5 ± 1

3–7

5 ± 1

3–7

Table 4 summarizes measurements of protein granule diameters. Only those simple and combined parameters including time frames differed significantly. In both sexes, the diameters diminished during the experiment in both seasons. In spring, the average protein granule diameters diminished by 0.04 µm/day in males, and 0.06 µm/day in females. In males, protein granule diameters were reduced in size by 0.06 µm/day until the middle, and after the middle by 0.03 µm/day until the end of the experimental starvation. In females, in both time frames the average protein granule diameter diminished by 0.08 µm/day. In autumn, in both sexes the diameters diminished by 0.03 µm/day until the middle, and by 0.06 µm/day from the middle until the end of the experiment.

Table 4

Descriptive statistics of protein granule diameters (µm) in the midgut epithelial cells of Meta menardi collected in spring and autumn in natural habitats, and experimentally exposed to starvation; No. of counts per sample (i.e., each season, sex and the time frame) = 30

Sex

Time frames of starvation experiment

Spring

Mean ± Std. Dev

Min–Max

Autumn

Mean ± Std. Dev

Min–Max

Beginning

4.2 ± 0.8

2.5–5.6

4.2 ± 1.0

2.3–5.7

Middle

3.2 ± 0.8

1.9–5.1

3.7 ± 0.8

1.8–5.1

End

2.5 ± 0.8

1.2–4.1

2.0 ± 0.7

0.6–3.7

Beginning

4.7 ± 0.8

3.2–6.1

4.2 ± 0.9

2.4–5.7

Middle

3.5 ± 0.9

1.9–5.2

3.6 ± 0.7

2.1–5.1

End

2.1 ± 0.7

0.9–3.5

1.9 ± 0.7

0.8–3.4

Rates of cells with autophagic structures, and rates of apoptotic cells

In both spring and autumn, at the beginning, before experimental starvation, cells with autophagic structures were present in both sexes, but they were more abundant in females (Table 1). During the experiment, rates of autophagic cells increased. In the middle of the experiment, the percentage of autophagic cells was about three times that in the beginning. At the end of the experiment, in spring males, this percentage was five times greater, and in autumn males, four times the beginning value. In spring and autumn females, there were four times as many autophagic structures as in the beginning.

In both seasons, spring and autumn, before the experimental starvation, apoptotic cells were rare. During the starvation, rates of apoptotic cells increased and were more abundant in females in each time frame (Table 1).

Discussion

Ultrastructure of the midgut diverticula epithelial cells

The midgut diverticules of M. menardi constitute a branched system, as described in other spiders (Foelix 1996; Felgenhauer 1999; Wilczek et al. 2014) and harvestmen (Becker and Peters 1985a, b; Ludwig and Alberti 1990) In M. menardi, the epithelium of the midgut diverticula consisted of digestive cells, secretory cells and adipocytes. Digestive cells and secretory cells were also recorded in the spider Coelotes terrestris (Ludwig and Alberti 1988, 1990), while adipocytes were absent. In the harvestmen Phalangium opilio (Becker and Peters 1985a), Gyas annulatus and G. titanus (Lipovšek et al. 2004), the epithelium of the midgut diverticula consists of digestive cells, secretory cells and excretory cells. Excretory cells were not found in the examined M. menardi. The ultrastructure of the digestive cells and the secretory cells in M. menardi was comparable to that in the spider Coelotes terrestris (Ludwig and Alberti 1988, 1990), as well as in those of the harvestmen Phalangium opilio (Becker and Peters 1985a, b), Gyas annulatus and G. titanus (Lipovšek et al. 2004).

We found no differences in the general morphology of the midgut epithelial cells during the experimental starvation of M. menardi in spring and autumn. In starved individuals, vacuolized cytoplasm appeared in the digestive cells and adipocytes. In previously studied species, the vacuolization of the cytoplasm was reported for some columnar cells in the midgut epithelium nidi of starved cockroaches Periplaneta americana (Park et al. 2009) and for some epithelial cells in the midgut of starved larvae of the antlion Euroleon nostras (Lipovšek et al. 2012). In P. americana, vacuoles in the cytoplasm were assumed to originate from degenerated RER (Park et al. 2009).

In both seasons, autophagy was less intensive at the beginning and gradually increased during the starvation experiment. However, prior to starvation, autophagic structures were relatively abundant, revealing that autophagy is a permanent process in the cells. Autophagy was intensified during both spring and autumn starvation experiments, which is related to the release of essential compounds supporting cell processes during starvation. In both seasons, autophagy was more intensive in females, presumably because of oogenesis.

In both seasons, apoptosis was rarely detected prior to the starvation experiment, and gradually increased during starvation. The intensity levels of apoptosis and autophagy were correlated during the experiment. Cells with numerous autophagic structures could obviously not endure this condition and underwent apoptosis. Similar conditions have been reported in starved P. americana, where apoptosis began to increase after about 2 weeks of starvation, and a significant increase was observed after 4 weeks of starvation (Park et al. 2009) and in X. nemoralis (Wilczek et al. 2014). In M. menardi, the apoptotic process began with structural changes in the nucleus, which became lobular and shrunken, and the cytoplasm became electron dense. Moreover, some apoptotic cells shrank, and consequently distinct extracellular spaces appeared between the apoptotic cell and the neighbouring epithelial cells. Formation of apoptotic bodies and phagocytosis was not observed in the examined M. menardi. Phagocytosis of apoptotic bodies was described in the midgut epithelium of the mosquito Culex pipiens (Vaidyanathan and Scott 2006). In some species, e.g., the greater wax moth Galleria mellonela (Uwo et al. 2002) and the proturans Filientomon takanawanum (Rost-Roszkowska et al. 2010), the apoptotic cells were discharged into the midgut lumen, where they were digested. In M. menardi, in each season and each time frame, the apoptotic cells were more numerous in females. This is in accordance with our expectations, since autophagic structures were more abundant in females, as well. Depending on the strength of a stressor, autophagy can either protect the cell against death and the cell recovers, or it can activate the apoptosis and/or necrosis (Rost-Roszkowska et al. 2015, 2016; Sonakowska et al. 2016a, b).

In M. menardi, a few necrotic cells appeared after the middle of the experiment. Many degenerated organelles were present in the electron-lucent cytoplasm. The necrotic cells in M. menardi are thought to be in the initial phase of necrosis, since the apical plasma membrane was partly damaged, and the microvilli could still be recognized. In the proturan Filientomon takanawanum, the degeneration of the midgut epithelium proceeds in a necrotic way as well (Rost-Roszkowska et al. 2010). We found no epithelial cells discharged into the midgut lumen, as found in F. takanawanum (Rost-Roszkowska et al. 2010). The ultrastructural changes observed in the midgut diverticula epithelial cells of M. menardi with regard to apoptosis, necrosis and autophagy were typical, as described in the midgut epithelia of other arthropods (Rost-Roszkowska et al. 2012; Teixeira et al. 2013; Wilczek et al. 2014; Lipovšek et al. 2014, 2015; Sonakowska et al. 2016a, b).

Quantification of reserve lipids, glycogen and proteins

Arthropods with life cycles spanning the winter accumulate reserve materials during the growth period in order to exploit them during the natural starvation period in winter (Lipovšek et al. 2011, 2014; Lipovšek and Novak 2016; Kamińska et al. 2016). In spring, small amounts of the reserve material were present, while in autumn, the amounts of lipid droplets, glycogen and protein granules were conspicuously greater, since M. menardi intensified the storage of energy-supplying and nutrient-supplying compounds for overwintering. In both sexes, the use of accumulated lipids was quite comparable. In spring, in both sexes the intensity of lipid exploitation was equal throughout the experiment. In contrast, in autumn the diminishing of lipid droplet diameters was 5–7 times faster until the middle of the experiment, and thereafter equal to the rate in the spring individuals. Glycogen and proteins were gradually exploited during the experiment; no significant differences between spring and autumn were found. We speculate that spring individuals, which had survived overwintering, have limited amounts of energy-supplying and nutrient-supplying compounds, and they currently used them to maintain their vital processes. In contrast, autumn individuals are assumed to be well equipped with these compounds, which were therefore intensively exploited until the middle of the experiment. Afterwards, the spiders shifted to the low-level exploitation of the reserve materials characteristic of the overwintering metabolism.

Conclusions

In M. menardi, starved in spring, structural differences in the midgut epithelial cells revealed only the starvation-dependent changes, while in spiders starved in autumn, simultaneous starvation-dependent and aging-dependent changes appeared. In spring and autumn, in both sexes, autophagy, apoptosis and necrosis were comparable with respect to cell ultrastructure as well as the intensity of the processes. The autophagy and apoptosis intensified when starvation was in progress, but more intensively in females. In both sexes, in both seasons, the amount of lipids, glycogen and proteins decreased during starvation. In both sexes, lipids were spent equally across spring starvation, but in autumn, much more intensively at the beginning of starvation, while afterwards their exploitation decreases to the spring level.

Notes

Acknowledgements

We would like to thank Elisabeth Bock and Rudi Schmied (Medical University Graz) for their excellent technical assistance. Michelle Gadpaille valuably improved the English of the manuscript.

Compliance with ethical standards

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

References

  1. Becker A, Peters W (1985a) The ultrastructure of the midgut and the formation of peritrophic membranes in a harvestman, Phalangium opilio (Chelicerata Phalangida). Zoomorphology 105:326–332. https://doi.org/10.1007/BF00312064 CrossRefGoogle Scholar
  2. Becker A, Peters W (1985b) Fine structure of the midgut gland of Phalangium opilio (Chelicerata, Phalangida). Zoomorphology 105:317–332CrossRefGoogle Scholar
  3. Bourne JD (1976) Notes preliminaires sur la distribution spatiale du Meta menardi, Triphosa dubitata., Triphosa sabaudiata, Nelima aurantiaca et Culex pipiens au sain d’un ecosystème cavernicole (Grotte de Scierce: Mt.-Savoie). Int J Speleol 8:253–267CrossRefGoogle Scholar
  4. Bourne JD (1977) Mise en évidence de groupements temporaires de la faune pariétale dans un tunnel artificiel en fonction de l’humidité et des mouvements d’air. Rev Suisse Zool 84:527–539CrossRefGoogle Scholar
  5. Bourne JD, Robert J (1978) Remarques écologiques sur un population de l’aragnée troglophile Meta menardi Latreille. Actes du 6eme Congr suisse Spéléol. Porrentruy, pp 25–35Google Scholar
  6. Boutin C (2004) Organisms: classification. In: Gunn J (ed) Encyclopedia of Cave and Karst Science. Fitzroy Dearborn, New York, pp 548–549Google Scholar
  7. Chiavazzo E, Isaia M, Mammola S, Lepore E, Ventola L, Asinari P, Pugno NM (2015) Cave spiders choose optimal environmental factors with respect to the generated entropy when laying their cocoon. Sci Rep 5:7611CrossRefPubMedPubMedCentralGoogle Scholar
  8. Deltshev C (2011) The faunistic diversity of cave-dwelling spiders (Arachnida, Araneae) of Greece. Arachnol Mitt 40:23–32CrossRefGoogle Scholar
  9. Dresco-Derouet L (1960) Étude biologique compare de quelques espèces d’araignées lucicoles et troglophiles. Arch de Zool Exp Gén 98:271–354Google Scholar
  10. Eckert R, Moritz M (1992) Meta menardi (Latr.) and Meta merianae (Scop.): on the Biology and Habitat of the two commonest spiders in the caves of the Harz, the Kyffhauser, Thuringia and the Zittau mountains. Mitt Zool Mus Berl 68:345–350CrossRefGoogle Scholar
  11. Felgenhauer BE (1999) Araneae. In: Harrison FW, Foelix RF (eds) Microscopic anatomy of invertebrates, Vol 8a: chelicerate arthropoda. Wiley, New York, pp 223–266Google Scholar
  12. Foelix RF (1996) Biology of spiders. Oxford University Press, New YorkGoogle Scholar
  13. Franzetti E, Huang ZJ, Shi YX, Xie K, Deng XJ, Li JP, Li QR, Yang WY, Zeng WN, Casartelli M, Deng HM, Cappellozza S, Grimald A, Xia Q, Feng Q, Cao Y, Tettamanti G (2012) Autophagy precedes apoptosis during the remodeling of silkworm larval midgut. Apoptosis 17:305–324CrossRefPubMedGoogle Scholar
  14. Fritzén NR, Koponen S (2011) The cave spider Meta menardi (Araneae, Tetragnathidae)—occurrence in Finland and notes on its biology. Memoranda Soc Fauna Flora Fennica 87:80–86Google Scholar
  15. Helsdingen PJ (2015) Araneae. In: Fauna Europaea. Database European spiders and their distribution—Faunistics—Version 2015.2. http://www.european-arachnology.org/reports/fauna.shtml
  16. Hörweg C, Blick T, Zaenker S (2011) Die Große Höhlenspinne Meta menardi (LATREILLE, 1804)—Höhlentier des Jahres und Europäische Spinne des Jahres 2012. Mitt Verb dt Höhlen- u Karstforscher 57(4):108–109Google Scholar
  17. Isaia M, Paschetta M, Lana E, Pantini P, Schonhofer AL, Christian E, Badino G (2011) Aracnidi sotterranei delle Alpi Occidentali italiane / Subterranean Arachnids of the Western Italian Alps (Arachnida: Araneae, Opiliones, Palpigradi, Pseudoscorpiones). Monografie XLVII. Museo Regionale di Scienze Naturali, TorinoGoogle Scholar
  18. Kamińska K, Włodarczyk A, Sonakowska L, Ostróżka A, Marchewka A, Rost-Roszkowska MM (2016) Ultrastructure of the salivary glands in Lithobius forficatus (Myriapoda, Chilopoda, Lithobiidae) according to seasonal and circadian rhythms. Arthr Str Dev 45:536–551CrossRefGoogle Scholar
  19. Kirchner W (1987) Behavioural and physiological adaptations to cold. In: Nentwig W (ed) Ecophysiology of spiders. Springer, Berlin, pp 66–77CrossRefGoogle Scholar
  20. Klionsky DJ et al. (2016) Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edn). Autophagy 12(1):1–222CrossRefPubMedPubMedCentralGoogle Scholar
  21. Lepore E, Marchioro A, Isaia M, Buehler MJ, Pugno NM (2012) Evidence of the most stretchable egg sac silk stalk, of the European spider of the year Meta menardi. PLoS One 7(2):e30500. https://doi.org/10.1371/journal.pone.0030500 CrossRefPubMedPubMedCentralGoogle Scholar
  22. Leruth R (1939) La biologie du domaine souterrain et la faune cavernicole de la Belgique. Mém Mus r his nat Belg 87:1–506Google Scholar
  23. Lipovšek S, Novak T (2016) Autophagy in the fat body cells of the cave cricket Troglophilus neglectus Krauss, 1878 (Rhaphidophoridae, Saltatoria) during overwintering. Protoplasma 253(2):457–466CrossRefPubMedGoogle Scholar
  24. Lipovšek S, Novak T, Janžekovič F, Senčič L, Pabst MA (2004) A contribution to the functional morphology of the midgut gland in phalangiid harvestmen Gyas annulatus and Gyas titanus during their life cycle. Tissue Cell 36:275–282CrossRefPubMedGoogle Scholar
  25. Lipovšek S, Novak T, Janžekovič F, Pabst MA (2011) Role of the fat body in the cave crickets Troglophilus cavicola and Troglophilus neglectus (Rhaphidophoridae, Saltatoria) during overwintering. Arthropod Struct Dev 40(1):54–63CrossRefPubMedGoogle Scholar
  26. Lipovšek S, Letofsky-Papst I, Hofer F, Devetak D (2012) The evidence on the degradation processes in the midgut epithelial cells of the larval antlion Euroleon nostras (Geoffroy in Fourcroy, 1785) (Myrmeleontidae, Neuroptera). Micron 43(5):651–665CrossRefPubMedGoogle Scholar
  27. Lipovšek S, Janžekovič F, Novak T (2014) Autophagic activity in the midgut gland of the overwintering harvestmen Gyas annulatus (Phalangiidae, Opiliones). Arthr Str Dev 43:493–500CrossRefGoogle Scholar
  28. Lipovšek S, Novak T, Janžekovič F, Leitinger G (2015) Changes in the midgut diverticula in the harvestmen Amilenus aurantiacus (Phalangiidae, Opiliones) during winter diapause. Arthr Str Dev. https://doi.org/10.1016/j.asd.2014.12.002 Google Scholar
  29. Lipovšek S, Janžekovič F, Novak T (2017) Ultrastructure of fat body cells and Malpighian tubule cells in overwintering Scoliopteryx libatrix (Noctuoidea). Protoplasma. https://doi.org/10.1007/s00709-017-1110-3 PubMedGoogle Scholar
  30. Litwin JA (1985) Light Microscopical Histochemistry on Plastic Sections. Prog Histochem Cyto 16:1–84Google Scholar
  31. Ludwig M, Alberti G (1988) Mineral congregations, spherites in the midgut gland of Coelotes terrestris (Araneae): structure, composition and function. Protoplasma 143:43–50CrossRefGoogle Scholar
  32. Ludwig M, Alberti G (1990) Peculiarities of arachnid midgut glands. Acta Zool Fenn 190:255–259Google Scholar
  33. Mammola S, Isaia M (2014) Niche differentiation in Meta bourneti and M. menardi (Araneae, Tetragnathidae) with notes on the life history. Int J Speleol 43(3):343–353CrossRefGoogle Scholar
  34. Mammola S, Isaia M (2016) The ecological niche of a specialized subterranean spider. Invertebr Biol 135(1):20–30CrossRefGoogle Scholar
  35. Manenti R, Lunghi E, Ficetola GF (2015) The distribution of cave twilight-zone spiders depends on microclimatic features and trophic supply. Invertebr Biol 134(3):242–251CrossRefGoogle Scholar
  36. Marusik YM, Koponen S (1992) A review of Meta (Araneae, Tetragnathidae), with description of two new species. J Arachnol 20:137–143Google Scholar
  37. McCall K (2010) Genetic control of necrosis—another type of programmed cell death. Curr Opin Cell Biol 22(6):882–888. https://doi.org/10.1016/j.ceb.2010.09.002 CrossRefPubMedPubMedCentralGoogle Scholar
  38. Mizushima N, Levine B, Cuervo AM, Klionsky DJ (2008) Autophagy fights disease through cellular self-digestion. Nature 451(7182):1069–1075CrossRefPubMedPubMedCentralGoogle Scholar
  39. Munafo DB, Colombo MI (2001) A novel assay to study autophagy: regulation of autophagosome vacuole size by amino acid deprivation. J Cell Sci 114:3619–3629PubMedGoogle Scholar
  40. Nentwig W, Blick T, Gloor D, Hanggi A, Kropf C (2017) Spiders of Europe, Version 472 02.2017. http://www.araneae.unibe.ch
  41. Novak T, Tkavc T, Kuntner M, Arnett AE, Lipovšek Delakorda S, Perc M, Janžekovič F (2010) Niche partitioning in orbweaving spiders Meta menardi and Metellina merianae (Tetragnathidae). Acta Oecol 36:522–529. https://doi.org/10.1016/j.actao.2010.07.005 CrossRefGoogle Scholar
  42. Novak T, Perc M, Lipovšek S, Janžekovič F (2012) Duality of terrestrial subterranean fauna. Int J Speleol 41(2):181–188CrossRefGoogle Scholar
  43. Novak T, Šajna N, Antolinc E, Lipovšek S, Devetak D, Janžekovič F (2014) Cold tolerance in terrestrial invertebrates inhabiting subterranean habitats. Int J Speleol 43(3):265–272. http://scholarcommons.usf.edu/ijs/vol43/iss3/3
  44. Nyffeler M, Symondson WOC (2001) Spiders and harvestmen as gastropod predators. Ecol Entomol 26:617–628. https://doi.org/10.1046/j.1365-2311.2001.00365.x CrossRefGoogle Scholar
  45. Park MS, Park P, Takeda M (2009) Starvation induces apoptosis in the midgut nidi of Periplaneta Americana: a histochemical and ultrastructural study. Cell Tissue Res 335:631 – 638CrossRefPubMedGoogle Scholar
  46. Pötzsch J (1966) Notizen zur Ernährung und Lebensweise von Meta menardi Latr. (Araneae; Araneidae). Abh Ber Naturkundemus Görlitz 41(10):1–24Google Scholar
  47. Racoviță EG (1907) Essai sur les problemes biospéologiques. Arch Zool Exp Gén (Biospéologica I), 4e serie 6:371–488Google Scholar
  48. Romanelli D, Casati B, Franzetti E, Tettamanti G (2014) A molecular view of autophagy in lepidoptera. Review article. Hindawi Publishing Corporation. Biomed Res Int. https://doi.org/10.1155/2014/902315 (Article ID 902315)Google Scholar
  49. Rost-Roszkowska MM, Machida R, Fukui M (2010) The role of cell death in the midgut epithelium in Filientomon takanawanum (Protura). Tissue Cell 42(1):24–31CrossRefPubMedGoogle Scholar
  50. Rost-Roszkowska MM, Poprawa I, Kaczmarek L (2011) Autophagy as the cell survival in response to a microsporidian infection of the midgut epithelium of Isohypsibius granulifer granulifer (Eutardigrada: Hypsibiidae). Acta Zool. https://doi.org/10.1111/j.1463-6395.2011.00552.x Google Scholar
  51. Rost-Roszkowska MM, Vilimova J, Sosinka A, Skudlik J, Franzetti E (2012) The role of autophagy in the midgut epithelium of Eubranchipus grubii (Crustacea, Branchiopoda, Anostraca). Arthr Str Dev 41:271–279CrossRefGoogle Scholar
  52. Rost-Roszkowska MM, Świątek P, Poprawa I, Rupik W, Swadźba E, Kszuk-Jendrysik M (2015) Ultrastructural analysis of apoptosis and autophagy in the midgut epithelium of Piscicola geometra (Annelida, Hirudinida) after blood feeding. Protoplasma 252(5):1387–1396CrossRefPubMedPubMedCentralGoogle Scholar
  53. Rost-Roszkowska MM, Chajec Ł, Vilimova J, Tajovsky K (2016) Apoptosis and necrosis during the circadian cycle in the centipede midgut. Protoplasma 253(4):1051–1061CrossRefPubMedGoogle Scholar
  54. Růžička V (1990) The spiders of stony debris. Acta Zool Fennica 190:333–337Google Scholar
  55. Schiner JR (1854) Fauna der Adelsberger-, Lueggerund Magdalenen-Grotte. In: Schmidl A (ed) Die Grotten und Höhlen von Adelsberg, Lueg, Planina und Laas. Braumüller, Wien, pp 231–2725Google Scholar
  56. Smithers P (1996) Observations on the prey of the cave spider Meta menardi (Latreille 1804) in South Devon. Newsl Br Arachnol Soc 77:12–14Google Scholar
  57. Smithers P (2005a) The diet of the cave spider Meta menardi (Latreille 1804) (Araneae, Tetragnathidae). J Arachnol 33:243–246CrossRefGoogle Scholar
  58. Smithers P (2005b) The early life history and dispersal of the cave spider Meta menardi (Latreille 1804), Tetragnathidae. Bull Br Arachnol Soc 13:213–216Google Scholar
  59. Sonakowska L, Włodarczyk A, Wilczek G, Wilczek P, Student S, Rost-Roszkowska MM (2016a) Cell death in the epithelia of the intestine and hepatopancreas in Neocaridina heteropoda (Crustacea, Malacostraca). PLoS One. https://doi.org/10.1371/journal.pone.0147582 PubMedPubMedCentralGoogle Scholar
  60. Sonakowska L, Włodarczyk A, Wilczek G, Wilczek P, Student S, Rost-Roszkowska MM (2016b) Cell death in the epithelia of the intestine and hepatopancreas in Neocaridina heteropoda (Crustacea, Malacostraca). PLoS One. https://doi.org/10.1371/journal.pone.0147582 PubMedPubMedCentralGoogle Scholar
  61. Szymczakowski W (1953) Preferendum temniczne jaskiniowego paja˛ka “Meta menardi” Latr. (Argiopidae). Folia Biol 1:153–168Google Scholar
  62. Teixeira A, Fialho MC, Zanuncio JC, Ramalho FS, Serrão JE (2013) Degeneration and cell regeneration in the midgut of Podisus nigrispinus (Heteroptera: Pentatomidae) during post-embryonic development. Arthr Str Dev 42:237–246CrossRefGoogle Scholar
  63. Tercafs R (1960) Notes à propos de deux araignées cavernicoles “Meta menardi Latr.” et “Nesticus cellulanus Clerck (Argiopidae)”. Ann Féd Spéléol Belg 1:14–18Google Scholar
  64. Tercafs R (1972) Biométrie spatiale dans l’écosystème souterraine: repartition du Meta menardi Latr. (Argiopidae). Int J Speleol 4:351–355CrossRefGoogle Scholar
  65. Tettamanti G, Cao Y, Feng Q, Grimaldi A, de Eguileor M (2011) Autophagy in Lepidoptera: more than old wine in new bottle. ISJ 8:5–14Google Scholar
  66. Uwo MF, Vi-Tei K, Pak P, Takeda M (2002) Replacement of midgut epithelium in the greater wax moth Galleria mellonela during larval–pupal moult. Cell Tissue Res 308:319–331CrossRefPubMedGoogle Scholar
  67. Vaidyanathan R, Scott TW (2006) Apoptosis in mosquito midgut epithelia associated with West Nile virus infection. Apoptosis 11:1643. https://doi.org/10.1007/s10495-006-8783-y CrossRefPubMedGoogle Scholar
  68. Wilczek G, Rost-Roszkowska MM, Wilczek P, Babczyńska A, Szulińska E, Sonakowska L, Marek-Swędzioł L (2014) Apoptotic and necrotic changes in the midgut glands of the wolf spider Xerolycosa nemoralis (Lycosidae) in response to starvation and dimethoate exposure. Ecotoxicol Environ Saf 101:157–167CrossRefPubMedGoogle Scholar
  69. Zakeri Z, Lockshin RA (2002) Cell death during development. J Immunol Methods 265(1–2):3–20CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2017

Authors and Affiliations

  • Saška Lipovšek
    • 1
    • 2
    • 3
  • Gerd Leitinger
    • 4
  • Tone Novak
    • 2
  • Franc Janžekovič
    • 2
  • Szymon Gorgoń
    • 5
  • Karolina Kamińska
    • 5
  • Magdalena Rost-Roszkowska
    • 5
  1. 1.Faculty of MedicineUniversity of MariborMariborSlovenia
  2. 2.Department of Biology, Faculty of Natural Sciences and MathematicsUniversity of MariborMariborSlovenia
  3. 3.Faculty of Chemistry and Chemical EngineeringUniversity of MariborMariborSlovenia
  4. 4.Institute of Cell Biology, Histology and EmbryologyMedical University of GrazGrazAustria
  5. 5.Department of Animal Histology and Embryology, Faculty of Biology and Environmental ProtectionUniversity of SilesiaKatowicePoland

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