Saška Lipovšek

Cold tolerance in terrestrial invertebrates inhabiting subterranean habitats

*tone.novak@uni-mb.si morphological adaptation and do not complete their life cycle there. 2) Troglophiles alternate between the epigean and hypogean habitats or live permanently in subterranean habitats; they show some moderate adaptation, such as partly reduced eyes and adaptations to compensate for the lack of visual orientation. Some among these do not complete their life cycle underground, while others do. 3) Troglobionts complete their life cycle in a hypogean environment, and most of them clearly show troglomorphic characteristics, like eyelessness and depigmentation. In contrast to the frequent preference for these three ecological groups, Novak et al. (2012) found that, on the one hand, trogloxenes and troglophiles together represent a group of variously adapted species, rather than two ecologically clearly separated categories, and, on the other hand, troglobionts divide into two strictly separated subgroups. Invertebrates are ectothermal and at their critical thermal minimum they enter chill-coma, where neuromuscular transmission and movement cease INTRODUCTION

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.

Application of analytical electron microscopic methods to investigate the function of spherites in the midgut of the larval antlion Euroleon nostras (Neuroptera: Myrmeleontidae).

This study presents an application of analytical electron microscopy in biology to investigate the chemical composition of the spherites and to elucidate the importance of these methods in the life sciences. The structure of the spherites in the midgut cells of first, second, and third instar larvae Euroleon nostras was investigated by a combination of transmission electron microscopy (TEM), energy dispersive X‐ray spectroscopy (EDXS), electron energy‐loss spectroscopy (EELS), and energy filtering TEM (EFTEM). The structure and chemical composition of the spherites changed during the metamorphosis. In first larvae, the spherites are composed of amorphous, flocculent material, containing C, N, and O. In second larvae and third ones, the spherites have concentric layers of alternating electron‐dense and electron‐lucent material. In second larvae, Si, P, Ca, and Fe are accumulated in the spherite organic matrix, composed of C, N, and O. In the spherites of third larvae, additionally Al was found. Therefore, the spherites are thought to store organic compounds in all three larval stages of E. nostras and additionally inorganic compounds in second and third ones. In first larvae, spherites are present in the midgut cells; in second and third larvae, they are present in the cells of the midgut and in its lumen. It could be suggested that the spherites might be involved in the regulation of the appropriate mineral composition of the internal environment and could serve as the accumulation site of nontoxic waste materials that cannot be metabolized. Microsc. Res. Tech., 2011.

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 cells 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.

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.

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.

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.

Femoral chordotonal organ in the legs of an insect,Chrysoperla carnea(Neuroptera).

The femoral chordotonal organ (FCO) inChrysoperla carneais situated in the distal part of the femur and consists of two scoloparia, which are fused at their distal end. The distal scoloparium contains 17-20 scolopidia, and the proximal one six scolopidia. Each scolopidium consists of two sensory cells and three types of enveloping cells (glial, scolopale and attachment cell). The sensory cells of different scolopidia do not lie at the same level in the FCO. Therefore the attachment cells of different scolopidia have different lengths. In the FCO, three types of ciliary roots are found in different sensory cells. The dendrite of the sensory cell terminates in a distal process, which has the structure of a modified cilium (9x2+0). The very distal part of the cilium is surrounded by an extracellular electron dense material, the cap, and ends in a terminal dilation. The scolopale cell contains the electron dense scolopale rods, consisting of plentiful microtubules. In their middle third the scolopale rods are fused and form the scolopale. In the FCO septate junctions, desmosomes and hemidesmosomes are found.

A contribution to the functional morphology of the femoral chordotonal organ in the green lacewing Chrysoperla carnea (Neuroptera).

The femoral chordotonal organ (FCO) and the subgenual organ (SGO) of the green lacewing Chrysoperla carnea were examined by conventional light and confocal laser scanning microscopy in order to search for neuroactive substances which are used for neurotransmission in sensory cells of these organs. Antibodies against serotonin, histamine and choline acetyltransferase were tested immunohistochemically. In the FCO, antiserum against serotonin strongly labelled cell bodies and axons of about 16 sensory cells. In the proximal scoloparium all 12 sensory cells showed immunoreaction with antiserotonin. In the distal scoloparium only four of 40 sensory cells were immunoreactive. These results suggest that different neuroactive substances are employed as neurotransmitters in the FCO of the green lacewing and that the proximal scoloparium and the distal scoloparium are functionally differentiated. Contrary to the FCO in the locust, acetylcholine was not found as a neurotransmitter in the FCO of the green lacewing. Additionally, histamine showed a negative result in the sensory cells of the FCO. Other neuroactive substances seem to be used as transmitters in the SGO because none of the tested antibodies showed positive reaction.

Biological pathways involved in the development of inflammatory bowel disease

SummaryApoptosis, autophagy and necrosis are three distinct functional types of the mammalian cell death network. All of them are characterized by a number of cell’s morphological changes. The inappropriate induction of cell death is involved in the pathogenesis of a number of diseases.Pathogenesis of inflammatory bowel diseases (ulcerative colitis, Crohn’s disease) includes an abnormal immunological response to disturbed intestinal microflora. One of the most important reason in pathogenesis of chronic inflammatory disease and subsequent multiple organ pathology is a barrier function of the gut, regulating cellular viability. Recent findings have begun to explain the mechanisms by which intestinal epithelial cells are able to survive in such an environment and how loss of normal regulatory processes may lead to inflammatory bowel disease (IBD).This review focuses on the regulation of biological pathways in development and homeostasis in IBD. Better understanding of the physiological functions of biological pathways and their influence on inflammation, immunity, and barrier function will simplify our expertice of homeostasis in the gastrointestinal tract and in upgrading diagnosis and treatment.ZusammenfassungApoptose, Autophagie und Nekrose sind drei unterschiedliche Funktionsformen des Zelltodes bei Säugetieren. Alle sind durch etliche morphologische Veränderungen in den Zellen gekennzeichnet. Eine fehlerhafte Induktion des Zelltodes ist Teil der Pathogenese vieler Krankheiten.Die Pathogenese entzündlicher Darmerkrankungen (Colitis ulcerosa, Morbus Crohn) schließt auch eine anormale Immunreaktion auf die gestörte Darmmikroflora ein. Einer der wichtigsten Gründe für die Pathogenese einer chronisch-entzündlichen Krankheit und folglich einer Multiorganpathologie ist die Barrierefunktion des Darms, die für die Regelung der Zellviabilität zuständig ist. Neuere Erkenntnisse helfen zu erklären, welche Mechanismen dazu führen, dass Darmepithelzellen in einem derartigen Umfeld überleben können und wie der Verlust normaler regulativer Prozesse zu entzündlichen Darmerkrankungen führen können.Dieser Bericht beschäftigt sich mit der Regulierung biologischer Wege in der Entwicklung und Homöostase entzündlicher Darmerkrankungen. Ein besseres Verständnis der physiologischen Funktionen der biologischen Wege und deren Einfluss auf Entzündungen, Immunität und Barrierefunktion wird unser Fachwissen zum Thema Homöostase im Verdauungstrakt verbessern und sowohl Diagnose als auch Behandlung vereinfachen.