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

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.

Rab3a ablation related changes in morphology of secretory vesicles in major endocrine pancreatic cells, pituitary melanotroph cells and adrenal gland chromaffin cells in mice.

In this work we have compared the ultrastructural characteristics of major pancreatic endocrine cells, pituitary melanotrophs and adrenal chromaffin cells in the normal mouse strain (wild type, WT) and mice with a known secretory deficit, the Rab3a knockout strain (Rab3a KO). For this purpose, pancreata, pituitary glands and adrenal glands from the Rab3a KO and from the WT mice were analysed, using conventional transmission electron microscopy (TEM). In order to assess the significance of the presence of Rab3a proteins in the relevant cells, we focused primarily on their secretory vesicle morphology and distribution. Our results showed a comparable general morphology in Rab3a KO and WT in all assessed endocrine cell types. In all studied cell types, the distribution of secretory granules along the plasma membrane (number of docked and almost-docked vesicles) was comparable between Rab3a KO and WT mice. Specific differences were found in the diameters of their secretory vesicles, diameters of their electron-dense cores and the presence of autophagic structures in the cells of Rab3A KO mice only. Occasionally, individual electron-dense round vesicles were present inside autophagosome-like structures; these were possibly secretory vesicles or their remnants. The differences found in the diameters of the secretory vesicles confirm the key role of Rab3a proteins in controlling the balance between secretory vesicle biogenesis and degradation, and suggest that the ablation of this protein probably changes the nature of the reservoir of secretory vesicles available for regulated exocytosis.

Starvation hardiness as preadaptation for life in subterranean habitats.

Most subterranean habitats, especially caves, are considered extreme environments, mainly because of the limited and erratic food supply and constant darkness. In temperate regions, many climatic conditions, such as temperature and air humidity, are periodically less adverse or even more favourable in caves than the harsh seasonal weather on the surface. Accordingly, many animal species search for hibernacula in caves. These overwintering, non-specialized subterranean species (non-troglobionts) show various modes of dormancy and ongoing development. Since they do not feed, they all undergo periodic starvation, a preadaptation, which might evolve in permanent starvation hardiness, such as found in most specialized subterranean species (troglobionts). To this end, we performed a comparative analysis of energy-supplying compounds in eleven most common terrestrial non-troglobiont species during winter in central European caves. We found highly heterogeneous responses to starvation, which are rather consistent with the degree of energetic adaptation to the habitat than to overwintering mode. The consumption of energy-supplying compounds was strongly higher taxa-dependant; glycogen is the main energy store in gastropods, lipids in insects, and arachnids rely on both reserve compounds. We assume that permanent starvation hardiness in specialized subterranean species might evolved in many different ways as shown in this study.

Towards understanding partial adaptation to the subterranean habitat in the European cave spider, Meta menardi: An ecocytological approach.

The European cave spider, Meta menardi, is a representative of the troglophiles, i.e. non-strictly subterranean organisms. Our aim was to interpret the cytological results from an ecological perspective, and provide a synthesis of the hitherto knowledge about M. menardi into a theory of key features marking it a troglophile. We studied ultrastructural changes of the midgut epithelial cells in individuals spending winter under natural conditions in caves, using light microscopy and TEM. The midgut diverticula epithelium consisted of secretory cells, digestive cells and adipocytes. During winter, gradual vacuolization of some digestive cells appeared, and some necrotic digestive cells and necrotic adipocytes appeared. This cytological information completes previous studies on M. menardi starved under controlled conditions in the laboratory. In experimental starvation and natural winter conditions, M. menardi gradually exploit reserve compounds from spherites, protein granules and through autophagy, and energy-supplying lipids and glycogen, as do many overwintering arthropods. We found no special cellular response to living in the habitat. Features that make it partly adapted to the subterranean habitat include starvation hardiness as a possible preadaptation, an extremely opportunistic diet, a partly reduced orb, tracking and capturing prey on bare walls and partly reduced tolerance to below-zero temperatures.

Changes in the midgut diverticula epithelial cells of the European cave spider, Meta menardi, under controlled winter starvation.

The European cave spider, Meta menardi, is among the most common troglophile species inhabiting the cave entrance zone in Europe, where prey is scarce in winter. Spiders feed only if prey is available; otherwise, they are subjected to long-term winter starvation. We carried out a four-month winter starvation of M. menardi under controlled conditions to analyze ultrastructural changes in the midgut diverticula epithelial cells at the beginning, in the middle and at the end of the starvation period. We used light microscopy, TEM and quantified reserve lipids and glycogen. The midgut diverticula epithelium consisted of secretory cells, digestive cells and adipocytes. During starvation, gradual vacuolization of some digestive cells, and some necrotic digestive cells and adipocytes appeared. Autophagic structures, autophagosomes, autolysosomes and residual bodies were found in all three cell types. Spherites and the energy-reserve compounds were gradually exploited, until in some spherites only the membrane remained. Comparison between spring, autumn and winter starvation reveals that, during the growth period, M. menardi accumulate reserve compounds in spherites and protein granules, and energy-supplying lipids and glycogen, like many epigean, overwintering arthropods. In M. menardi, otherwise active all over the year, this is an adaptive response to the potential absence of prey in winter.

Ultrastructure of fat body cells and Malpighian tubule cells in overwintering Scoliopteryx libatrix (Noctuoidea).

The herald moths, Scoliopteryx libatrix, overwinter in hypogean habitats. The ultrastructure of their fat body (FB) cells and Malpighian tubule (MT) epithelial cells was studied by light microscopy and transmission electron microscopy, and essential biometric and biochemical measurements were performed. The FB was composed of adipocytes and sparse urocytes. The ultrastructure of both cells did not change considerably during this natural starvation period, except for rough endoplasmic reticulum (rER) which became more abundant in March females. In the cells, the reserve material consisted of numerous lipid droplets, glycogen rosettes, and protein granula. During overwintering, the lipid droplets diminished, and protein granula became laminated. The MTs consisted of a monolayer epithelium and individual muscle cells. The epithelial cells were attached to the basal lamina by numerous hemidesmosomes. The apical plasma membrane was differentiated into numerous microvilli, many of them containing mitochondria. Nuclei were surrounded by an abundant rER. There were numerous spherites in the perinuclear part of the cells. The basal plasma membrane formed infoldings with mitochondria in between. Nuclei were located either in the basal or in the central part of the cells. During overwintering, spherites were gradually exploited, and autophagic structures appeared: autophagosomes, autolysosomes, and residual bodies. There were no statistical differences between the sexes in any measured biometric and biochemical variables in the same time frames. The energy-supplying lipids and glycogen, and spherite stores were gradually spent during overwintering. In March, the augmented rER signified the intensification of synthetic processes prior to the epigean ecophase.

Malpighian Tubule Cells in Overwintering Cave Crickets Troglophilus cavicola (Kollar, 1833) and T. neglectus Krauss, 1879 (Rhaphidophoridae, Ensifera).

During winter, cave cricket larvae undergo dormancy in subterranean habitats; this dormancy is termed diapause in second year Troglophilus cavicola larvae because they mature during this time, and termed quiescence in T. neglectus, because they mature after dormancy. Here we used electron microscopy to analyze ultrastructural changes in the epithelial cells in the Malpighian tubules (MTs) of T. cavicola during diapause, in order to compare them with previous findings on T. neglectus. Moreover, the autophagosomes were studied with immunofluorescence microscopy in both species. Although the basic ultrastructure of the cells was similar, specific differences appeared during overwintering. During this natural starvation period, the nucleus, rER, the Golgi apparatus and mitochondria did not show structural changes, and the spherites were exploited. The abundances of autophagic structures in both species increased during overwintering. At the beginning of overwintering, in both species and sexes, the rates of cells with autophagic structures (phagophores, autophagosomes, autolysosomes and residual bodies) were low, while their rates increased gradually towards the end of overwintering. Between sexes, in T. cavicola significant differences were found in the autophagosome abundances in the middle and at the end, and in T. neglectus at the end of overwintering. Females showed higher rates of autophagic cells than males, and these were more abundant in T. cavicola. Thus, autophagic processes in the MT epithelial cells induced by starvation are mostly parallel in diapausing T. cavicola and quiescent T. neglectus, but more intensive in diapausing females.

Changes in the midgut diverticula in the harvestmen Amilenus aurantiacus (Phalangiidae, Opiliones) during winter diapause.

The harvestmen Amilenus aurantiacus overwinter in diapause in hypogean habitats. The midgut diverticula have been studied microscopically (light microscopy, TEM) and biochemically (energy-storing compounds: lipids and glycogen) to analyze changes during this programmed starvation period. Throughout the investigated period, the epithelium of the midgut diverticula is composed of secretory cells, digestive cells and adipocytes. Additionally, after the middle of overwintering, the excretory cells appear, and two assemblages of secretory cells are present: the SC1 secretory cells are characterized by electron-dense cytoplasm with numerous protein granules, and the SC2 cells by an electron-lucent cytoplasm with fewer protein granules. The autophagic activity is observed from the middle of overwintering, indicating its vital role in providing nutrients during this non-feeding period. Lipids and glycogen are present in the midgut diverticula cells, except in the excretory cells. Measurements of the lipid droplet diameters and the lipid quantities yielded quite comparable information on their consumption. Lipids are gradually spent in both sexes, more rapidly in females, owing to ripening of the ovaries. Glycogen rates decrease towards the middle, and increase just before the end of overwintering, indicating that individuals are preparing for the epigean active ecophase.

Autophagic activity in the midgut gland of the overwintering harvestmen Gyas annulatus (Phalangiidae, Opiliones).

Juvenile harvestmen Gyas annulatus overwinter in dormancy in hypogean habitats for 4-5 months. The ultrastructure of the autophagic structures in their midgut epithelium cells was studied by light microscopy, transmission electron microscopy (TEM) and immunofluorescence microscopy (IFM) during this non-feeding period. Before overwintering (November), autophagic structures were scarce. In the middle (January) and at the end of overwintering (March), phagophores, autophagosomes and autolysosomes were present in the cytoplasm of both the secretory and the digestive midgut epithelium cells, gradually increasing their abundance during overwintering. In addition, vacuolization of the cytoplasm intensified. Both processes are induced by starvation. Autophagic structures and cytoplasm vacuolization enable the reuse of the cell's own components required for the maintenance of vital processes during dormancy. While TEM is a much more convenient method for recognition of the autophagic structure types and their ultrastructure, IFM enables exact counting of these structures.

Role of the fat body in the cave crickets Troglophilus cavicola and Troglophilus neglectus (Rhaphidophoridae, Saltatoria) during overwintering.

The cave crickets Troglophilus cavicola and Troglophilus neglectus are the most widely distributed European species of the family Rhaphidophoridae. Their life cycles span two years. They overwinter twice in caves in 4-6 months lasting diapause, T. cavicola in warmer microhabitats. In caves, older T. cavicola undergo sexual maturation, while T. neglectus do not. We hypothesized that the use of energy-supplying compounds and reserve proteins in the fat body is more extensive in T. cavicola than in T. neglectus. We analyzed the contents and morphology of lipid droplets, glycogen rosettes and protein granula at the beginning, the middle and the end of overwintering applying optic, TEM and biochemical methods. In all individuals, the fat body is composed of about 40 oval ribbons consisted of gradually changing adipocytes and urocytes. T. cavicola use glycogen continuously, and stop using lipids in the middle of overwintering, while this is inverse in T. neglectus. Till the middle of overwintering, all individuals exploit proteins, afterwards they are unevenly exploited. We found that the fat body is differently engaged in metabolism of both cave crickets during overwintering, supporting a more glycogen-dependent metabolism in T. cavicola, and a more lipid-dependent one in T. neglectus.