Ultrastructure of spherites in the midgut diverticula and Malpighian tubules of the harvestman Amilenus aurantiacus during the winter diapause.

Amilenus aurantiacus overwinter in diapause, a natural starvation period, in hypogean habitats. The structure of spherites in the midgut diverticula (MD) and Malpighian tubules (MT) has been studied comparatively by light microscopy and TEM to detect eventual differences in mineral consumption in the beginning and at the end of the starvation period in these organs (MD and MT) associated with digestive processes. The chemical composition of spherites was examined by combining energy-dispersive X-ray spectroscopy (EDXS), electron energy-loss spectroscopy (EELS) and energy-filtered TEM (EFTEM). The structure of the spherites changed during overwintering in both organs. At the beginning of overwintering, the spherites were composed of densely packed concentric layers of electron-dense and electron-lucent material. In the middle and at the end of overwintering, the electron-lucent layers between the layers of material indicated the loss of some material. The chemical composition of the spherites changed only in the MD; at the beginning of overwintering, these contained Si, O, C and Fe, while later there was no more Fe. In contrast, spherites in the MT were composed of Si, O, C and Ca throughout overwintering. A less intensive exploitation of the MD spherites was probably due to complete cessation of digestive and other cell activity in this organ during the winter diapause; activity of the MT slowed, but continued removing the cell metabolites.

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.

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.

Malpighian tubules in harvestmen.

In arachnids, the Malpighian tubules (MTs), coxal glands and stercoral pockets are capable of collecting and removing excreta from the body. The presence of the MTs among Opiliones was evidenced for the first time in Amilenus aurantiacus in 2015. Individuals undergo a winter diapause subterranean habitats. Here, we provided the morphological and cytological description of the MTs and asked whether their structure and ultrastructure change during the winter diapause. We studied the changes using light and transmission electron microscopy. The MTs consisted of the ureter and a pair of long, lateral blind-ended tubules, forming a long loop in the opisthosoma, and a coiled, terminal ball in the prosoma. The MTs were uniform, composed of a single-cell type, a monolayer of cuboidal epithelial cells, and the basal lamina. The cell ultrastructure was quite comparable to those in other arthropods, except for very long infoldings of the basal membrane protruding close to the nucleus. Except for spherite exploitation, no changes were observed in the ultrastructure of the MT epithelial cells during overwintering. We suggest that the analogous MTs in A. aurantiacus, and the nephron anatomies, along with a single-cell-type MT epithelium, might be of advantage in modelled studies of the nephron.

Notes on coexistence of closely related Leiobunum rupestre and L. subalpinum (Opiliones, Eupnoi, Sclerosomatidae).

Two closely related, syntopic species, Leiobunum rupestre and L. subalpinum, spend the day at apparently randomly distributed resting sites. In this preliminary research, we studied differences in their microclimatic preferences at a locality in the Pohorje Mts., Slovenia. We measured air temperature and relative humidity at individual resting sites, and found that L. subalpinum occupied a slightly wider air temperature-relative humidity ecospace as compared to L. rupestre. Individuals of L. rupestre rested at more humid sites than L. subalpinum. Regression analyses revealed that the presence of L. rupestre was most likely at resting sites with air temperature in the range of 1217°C and with relative air humidity of 98.1%. In contrast, the presence of L. subalpinum was most likely at resting sites with air temperature 23.1°C and relative air humidity in the range of 6585%. Nevertheless, microclimatic niches of both species overlapped almost completely, indicating that niche filtering may allow the coexistence of the two species.

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.

Methyl-ketones in the scent glands of Opiliones: a chemical trait of cyphophthalmi retrieved in the dyspnoan Nemastoma triste.

The homologous and phylogenetically old scent glands of harvestmen-also called defensive or repugnatorial glands-represent an ideal system for a model reconstruction of the evolutionary history of exocrine secretion chemistry ("phylogenetic chemosystematics"). While the secretions of Laniatores (mainly phenols, benzoquinones), Cyphophthalmi (naphthoquinones, chloro-naphthoquinones, methyl-ketones) and some Eupnoi (naphthoquinones, ethyl-ketones) are fairly well studied, one open question refers to the still largely enigmatic scent gland chemistry of representatives of the suborder Dyspnoi and the relation of dyspnoan chemistry to the remaining suborders. We here report on the secretion of a nemastomatid Dyspnoi, Nemastoma triste, which is composed of straight-chain methyl-ketones (heptan-2-one, nonan-2-one, 6-tridecen-2-one, 8-tridecen-2-one), methyl-branched methyl-ketones (5-methyl-heptan-2-one, 6-methyl-nonan-2-one), naphthoquinones (1,4-naphthoquinone, 6-methyl-1,4-naphthoquinone) and chloro-naphthoquinones (4-chloro-1,2-naphthoquinone, 4-chloro-6-methyl-1,2-naphthoquinone). Chemically, the secretions of N. triste are remarkably reminiscent of those found in Cyphophthalmi. While naphthoquinones are widely distributed across the scent gland secretions of harvestmen (all suborders except Laniatores), methyl-ketones and chloro-naphthoquinones arise as linking elements between cyphophthalmid and dyspnoan scent gland chemistry.

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.

Autophagy in the fat body cells of the cave cricket Troglophilus neglectus Krauss, 1878 (Rhaphidophoridae, Saltatoria) during overwintering.

The cave cricket Troglophilus neglectus regularly overwinters for 4-5 months in hypogean habitats. Winter dormancy is a natural starvation period, providing the opportunity to study autophagy under natural conditions. We aimed to evaluate the autophagic activity in adipocytes and urocytes of the fat body in three time frames: directly before overwintering, in the middle of dormancy, and at its end. For this purpose, we sampled individuals in caves. The cell ultrastructure was studied by transmission electron microscopy (TEM) and the abundance of autophagosomes by immunofluorescence microscopy (IFM), applying the widely used, specific immunolabeling marker microtubule-associated protein 1 light chain 3 (LC3). Before overwintering, TEM revealed scarce autophagosomes and residual bodies in the adipocytes and none in the urocytes. Congruently, IFM showed a very limited or no reaction. In the middle and at the end of overwintering, in both cell types, phagophores, autophagosomes, autolysosomes, and residual bodies were identified by TEM, while LC3 immunolabeling for detecting autophagosomes showed a conspicuous positive reaction. Both methods revealed that there were no significant differences between the sexes in any time frame. Minimal autophagic activity was detected before the winter dormancy, and it gradually intensified till the end of overwintering, probably because reserve proteins in protein granula are not composed of all the required amino acids. We conclude that in T. neglectus, autophagy is a substantial response to starvation and supports homeostatic processes during winter dormancy by supplying cells with nutrients.

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.

Hadzinia ferrani, sp. n. (Opiliones: Nemastomatidae), a highly specialized troglobiotic harvestman from Slovenia.

A highly specialized, endemic troglobiotic harvestman, Hadzinia ferrani sp. n., recently found in the Ferranova buza cave in the karst of Mt. Ulovka in central Slovenia is described. It is characterized by its small, 1.1-1.4 mm long body and very long, thin appendages, with leg II more than 20-times as long as the body. Although very similar to Nemaspela Silhavý, 1966, the genus Hadzinia Silhavý, 1966 can be clearly differentiated from Nemaspela by its truncated glans and short stylus, well delimited from the glans. According to these characteristics, Hadzinia is assumed most closely related to Pyza Starega, 1976 and Vestiferum Martens, 2006 rather than to Nemaspela with the glans gradually tapering into a mostly long stylus. Genital morphology is the most appropriate and probably the only morphological approach for investigating relationships among the taxa under study. 

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.

Structure of the Malpighian tubule cells and annual changes in the structure and chemical composition of their spherites in the cave cricket Troglophilus neglectus Krauss, 1878 (Rhaphidophoridae, Saltatoria).

Periodical changes in the structure of spherites in the Malpighian tubule cells of the cave cricket Troglophilus neglectus were studied to elucidate their role during the cricket's life cycle in natural circumstances. Special interest was given to the dormant overwintering period when we hypothesized that the primary role of spherites is to supply minerals for basic vital processes. The investigation was carried out by light and transmission electron microscopy, energy dispersive X-ray spectroscopy, electron energy-loss spectroscopy and energy-filtering TEM. Spherites are present only in the middle Malpighian tubule segment, consisting of Type 1 cells, characterized, among other features, by a round, apically placed nucleus and numerous spherites, and a few Type 2 cells with an elongated nucleus in the centre and sparse spherites. At the beginning of dormancy in November juveniles, minerals are accumulated in spherites and then decline until March. In one-year-old May larvae, spherites are commonly rich in minerals, and from July onwards they are progressively exploited in the adults. Spherite destruction starts with apoptosis in senile October individuals. The findings suggest that the mineral supply of spherites in Malpighian tubules is crucial to supporting vital processes throughout the life cycle of T. neglectus.