The midgut epithelia of the millipedes Polyxenus lagurus, Archispirostreptus gigas and Julus scandinavius were analyzed under light and transmission electron microscopies. In order to detect the proliferation of regenerative cells, labeling with BrdU and antibodies against phosphohistone H3 were employed. A tube-shaped midgut of three millipedes examined spreads along the entire length of the middle region of the body. The epithelium is composed of digestive, secretory and regenerative cells. The digestive cells are responsible for the accumulation of metals and the reserve material as well as the synthesis of substances, which are then secreted into the midgut lumen. The secretions are of three types - merocrine, apocrine and microapocrine. The oval or pear-like shaped secretory cells do not come into contact with the midgut lumen and represent the closed type of secretory cells. They possess many electron-dense granules (J. scandinavius) or electron-dense granules and electron-lucent vesicles (A. gigas, P. lagurus), which are accompanied by cisterns of the rough endoplasmic reticulum. The regenerative cells are distributed individually among the basal regions of the digestive cells. The proliferation and differentiation of regenerative cells into the digestive cells occurred in J. scandinavius and A. gigas, while these processes were not observed in P. lagurus. As a result of the mitotic division of regenerative cells, one of the newly formed cells fulfills the role of a regenerative cell, while the second one differentiates into a digestive cell. We concluded that regenerative cells play the role of unipotent midgut stem cells.

• Klíčová slova
• Digestive cells, Midgut epithelium, Millipedes, Regenerative cells, Secretory cells, Ultrastructure,
• MeSH
• členovci fyziologie   ultrastruktura   MeSH
• epitel metabolismus   ultrastruktura   MeSH
• kmenové buňky fyziologie   ultrastruktura   MeSH
• trávicí systém metabolismus   ultrastruktura   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH

Methanogens represent the final decomposition step in anaerobic degradation of organic matter, occurring in the digestive tracts of various invertebrates. However, factors determining their community structure and activity in distinct gut sections are still debated. In this study, we focused on the tropical millipede species Archispirostreptus gigas (Diplopoda, Spirostreptidae) and Epibolus pulchripes (Diplopoda, Pachybolidae), which release considerable amounts of methane. We aimed to characterize relationships between physicochemical parameters, methane production rates, and methanogen community structure in the two major gut sections, midgut and hindgut. Microsensor measurements revealed that both sections were strictly anoxic, with reducing conditions prevailing in both millipedes. Hydrogen concentration peaked in the anterior hindgut of E. pulchripes. In both species, the intestinal pH was significantly higher in the hindgut than in the midgut. An accumulation of acetate and formate in the gut indicated bacterial fermentation activities in the digestive tracts of both species. Phylogenetic analysis of 16S rRNA genes showed a prevalence of Methanobrevibacter spp. (Methanobacteriales), accompanied by a small fraction of so-far-unclassified "Methanomethylophilaceae" (Methanomassiliicoccales), in both species, which suggests that methanogenesis is mostly hydrogenotrophic. We conclude that anoxic conditions, negative redox potential, and bacterial production of hydrogen and formate promote gut colonization by methanogens. The higher activities of methanogens in the hindgut are explained by the higher pH of this compartment and their association with ciliates, which are restricted to this compartment and present an additional source of methanogenic substrates. IMPORTANCE Methane (CH4) is the second most important atmospheric greenhouse gas after CO2 and is believed to account for 17% of global warming. Methanogens are a diverse group of archaea and can be found in various anoxic habitats, including digestive tracts of plant-feeding animals. Termites, cockroaches, the larvae of scarab beetles, and millipedes are the only arthropods known to host methanogens and emit large amounts of methane. Millipedes are ranked as the third most important detritivores after termites and earthworms, and they are considered keystone species in many terrestrial ecosystems. Both methane-producing and non-methane-emitting species of millipedes have been observed, but what limits their methanogenic potential is not known. In the present study, we show that physicochemical gut conditions and the distribution of symbiotic ciliates are important factors determining CH4 emission in millipedes. We also found close similarities to other methane-emitting arthropods, which might be associated with their similar plant-feeding habits.

• Klíčová slova
• Methanobrevibacter, Methanomassiliicoccales, digestive tract, methane, methanogenesis, methanogenic community, physicochemical parameters, tropical millipedes,
• MeSH
• Bacteria genetika   metabolismus   MeSH
• členovci mikrobiologie   MeSH
• formiáty metabolismus   MeSH
• fylogeneze MeSH
• gastrointestinální trakt metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• kyslík analýza   MeSH
• methan metabolismus   MeSH
• oxidace-redukce MeSH
• RNA ribozomální 16S genetika   MeSH
• střevní mikroflóra * genetika   MeSH
• vodík metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• formiáty MeSH
• formic acid MeSH Prohlížeč
• kyslík MeSH
• methan MeSH
• RNA ribozomální 16S MeSH
• vodík MeSH

Methane production by intestinal methanogenic Archaea and their community structure were compared among phylogenetic lineages of millipedes. Tropical and temperate millipedes of 35 species and 17 families were investigated. Species that emitted methane were mostly in the juliform orders Julida, Spirobolida, and Spirostreptida. The irregular phylogenetic distribution of methane production correlated with the presence of the methanogen-specific mcrA gene. The study brings the first detailed survey of methanogens' diversity in the digestive tract of millipedes. Sequences related to Methanosarcinales, Methanobacteriales, Methanomicrobiales and some unclassified Archaea were detected using molecular profiling (DGGE). The differences in substrate preferences of the main lineages of methanogenic Archaea found in different millipede orders indicate that the composition of methanogen communities may reflect the differences in available substrates for methanogenesis or the presence of symbiotic protozoa in the digestive tract. We conclude that differences in methane production in the millipede gut reflect differences in the activity and proliferation of intestinal methanogens rather than an absolute inability of some millipede taxa to host methanogens. This inference was supported by the general presence of methanogenic activity in millipede faecal pellets and the presence of the 16S rRNA gene of methanogens in all tested taxa in the two main groups of millipedes, the Helminthophora and the Pentazonia.

• MeSH
• biodiverzita * MeSH
• členovci mikrobiologie   MeSH
• denaturační gradientová gelová elektroforéza MeSH
• Euryarchaeota genetika   metabolismus   fyziologie   MeSH
• feces chemie   MeSH
• fylogeneze MeSH
• gastrointestinální trakt mikrobiologie   MeSH
• methan biosyntéza   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• RNA ribozomální 16S genetika   MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH
• Rumunsko MeSH
• Slovenská republika MeSH
• Názvy látek
• methan MeSH
• RNA ribozomální 16S MeSH

The process of autophagy has been detected in the midgut epithelium of four millipede species: Julus scandinavius, Polyxenus lagurus, Archispirostreptus gigas, and Telodeinopus aoutii. It has been examined using transmission electron microscopy (TEM), which enabled differentiation of cells in the midgut epithelium, and some histochemical methods (light microscope and fluorescence microscope). While autophagy appeared in the cytoplasm of digestive, secretory, and regenerative cells in J. scandinavius and A. gigas, in the two other species, T. aoutii and P. lagurus, it was only detected in the digestive cells. Both types of macroautophagy, the selective and nonselective processes, are described using TEM. Phagophore formation appeared as the first step of autophagy. After its blind ends fusion, the autophagosomes were formed. The autophagosomes fused with lysosomes and were transformed into autolysosomes. As the final step of autophagy, the residual bodies were detected. Autophagic structures can be removed from the midgut epithelium via, e.g., atypical exocytosis. Additionally, in P. lagurus and J. scandinavius, it was observed as the neutralization of pathogens such as Rickettsia-like microorganisms. Autophagy and apoptosis ca be analyzed using TEM, while specific histochemical methods may confirm it.

• Klíčová slova
• Diplopoda, cell death, digestive system, histochemistry, midgut, ultrastructure,
• MeSH
• apoptóza * MeSH
• autofagie * MeSH
• členovci * MeSH
• fagozomy ultrastruktura   MeSH
• fluorescenční mikroskopie MeSH
• lyzozomy ultrastruktura   MeSH
• mikroskopie MeSH
• Rickettsia imunologie   MeSH
• střevní sliznice imunologie   patologie   MeSH
• transmisní elektronová mikroskopie MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Pitfall trapping is one of the standard methods used for the capture of ground-active arthropod groups. Despite being frequently used, the standardization of this method is problematic due to the large range of combinations of the individual parameters of pitfall traps with varying efficacy under different environmental conditions. We evaluated the effects of the trap diameter, the fixing fluid, and their combination on the capture efficacy for harvestmen (Opiliones) and millipedes (Diplopoda). We used pitfall traps with three different diameters: 3 cm, 5 cm, and 12 cm, filled with three types of fixing fluids (saturated fluid of NaCl, 10:1 mixture of 70% ethanol and glycerol and 4% formaldehyde). Altogether, 90 traps representing nine combinations of trap diameters and fixing fluid were placed on a mown meadow in spring and autumn intervals for a total of 45 days. We sampled 1,488 individuals representing 11 harvestmen species and 881 individuals representing 11 millipede species. Large (d = 12 cm) and medium (5 cm) traps captured significantly more millipede species and individuals than the small-sized traps (3 cm). The same effect was observed for harvestmen species richness, whereas the medium traps (d = 5 cm) captured the highest mean activity of harvestmen. By analyzing the differences in the body sizes of the studied arthropods in relation to the trap diameter and fluid, we found that larger traps, as well as traps filled with NaCl solution, captured larger harvestmen more frequently than the other trap types. Our results revealed that the combination of larger traps (d = 5 and 12 cm) and formaldehyde was most effective in the capture of both studied groups. However, the disadvantage of formaldehyde is its toxicity.

• Klíčová slova
• body size, capture rate, fixing fluid, method selection, trap diameter,
• Publikační typ
• časopisecké články MeSH

Some species of centipedes and millipedes inhabit upper soil layers exclusively and are not recorded by pitfall trapping. Because of their sensitivity to soil conditions, they can be sampled quantitatively for evaluation of soil conditions. Soil samples are heavy to transport and their processing is time consuming, and such sampling leads to disturbance of the soil surface which land-owners do not like. We evaluated the use of hay-bait traps to sample soil dwelling millipedes and centipedes. The effectiveness of this method was found to be similar to the effectiveness of soil sampling. Hay-bait traps installed for 8-10 weeks can substitute for direct soil sampling in ecological and inventory studies.

• Klíčová slova
• Chilopoda, Diplopoda, agroecosystem, soil fauna, soil sampling,
• Publikační typ
• časopisecké články MeSH

Millipedes represent a model for the study of organic matter transformation, animal-microbial interactions, and compartmentalisation of digestion. The activity of saccharidases (amylase, laminarinase, cellulase, xylanase, chitinase, maltase, cellobiase, and trehalase) and protease were measured in the midgut and hindgut contents and walls of the millipedes Archispirostreptus gigas and Epibolus pulchripes. Assays done at pH 4 and 7 confirmed activities of all enzymes except xylanase. Hydrolysing of starch and laminarin prevailed. The hindgut of E. pulchripes was shorter, less differentiated. Micro-apocrine secretion was observed only in the midgut of A. gigas. Merocrine secretion was present in midgut and hindgut of E. pulchripes, and in the pyloric valve and anterior hindgut of A. gigas. Alpha-polysaccharidases were mostly active in the midgut content and walls, with higher activity at pH 4. The low activity of amylase (A. gigas) and laminarinase (E. pulchripes) in midgut tissue may indicate their synthesis in salivary glands. Cellulases were found in midgut. Chitinases, found in midgut content and tissue (E. pulchripes) or concentrated in the midgut wall (A. gigas), were more active at an acidic pH. Polysaccharidases were low in hindguts. Protease shows midgut origin and alkaline activity extending to the hindgut in E. pulchripes, whereas in A. gigas it is of salivary gland origin and acid activity restricted to the midgut. Some disaccharidases, with more alkaline activity, showed less apparent midgut-hindgut differences. It may indicate an axial separating of the primary and secondary digestion along the intestinal pH gradient or the presence of enzymes of hindgut parasites.

• Klíčová slova
• Cellulase, Digestive enzymes, Gut compartments, Histology, Myriapoda,
• MeSH
• celulasa metabolismus   MeSH
• chitinasy metabolismus   fyziologie   MeSH
• členovci klasifikace   enzymologie   MeSH
• gastrointestinální trakt enzymologie   MeSH
• koncentrace vodíkových iontů MeSH
• polysacharidy metabolismus   MeSH
• proteasy metabolismus   MeSH
• substrátová specifita MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• celulasa MeSH
• chitinasy MeSH
• polysacharidy MeSH
• proteasy MeSH

In this paper, the growth requirements, fermentation pattern, and hydrolytic enzymatic activities of anaerobic ciliates collected from the hindgut of the African tropical millipede Archispirostreptus gigas are described. Single-cell molecular analysis showed that ciliates from the millipede hindgut could be assigned to the Nyctotherus velox and a new species named N. archispirostreptae n. sp. The ciliate N. velox can grow in vitro with unspecified prokaryotic populations and various plant polysaccharides (rice starch-RS, xylan, crystalline cellulose20-CC, carboxymethylcellulose-CMC, and inulin) or without polysaccharides (NoPOS) in complex reduced medium with soluble supplements (peptone, glucose, and vitamins). Specific catalytic activity (nkat/g of protein) of α amylase of 300, xylanase of 290, carboxymethylcellulase of 190, and inulinase of 170 was present in the crude protein extract of N. velox. The highest in vitro dry matter digestibility was observed in RS and inulin after 96 h of fermentation. The highest methane concentration was observed in xylan and inulin substrates. The highest short-chain fatty acid concentration was observed in RS, inulin, and xylan. In contrast, the highest ammonia concentration was observed in NoPOS, CMC, and CC. The results indicate that starch is the preferred substrate of the N. velox. Hydrolytic enzyme activities of N. velox showed that the ciliates contribute to the fermentation of plant polysaccharides in the gut of millipedes.

• Klíčová slova
• Nyctotherus, ciliates, enzymatic activities, hindgut protozoa, in vitro growth, polysaccharide fermentation, tropical millipedes,
• Publikační typ
• časopisecké články MeSH

The total species richness in the myriapod assemblages of the lowland Altais near Charyshskoe Village, Altai Province, southwestern Siberia, Russia is estimated to be at least 19 species from ten genera, eight families, five orders, and two classes. The following species are new to SW Siberia: Lithobius (Ezembius) ostiacorum Stuxberg, 1876, L. vagabundus Stuxberg, 1876, and L. (Monotarsobius) nordenskioeldii Stuxberg, 1876, while L. (E.) proximus Sseliwanoff, 1880 and L. (M.) insolens Dányi & Tuf, 2012 are recorded for the first time from the Altai Province of Russia. A species of Strigamia which is morphologically similar to Strigamia cf. transsilvanica (Verhoeff, 1928) has been found in the study area but its true specific identity is yet to be determined. The seasonal dynamics of myriapod assemblages in terms of the species diversity, density, sex-age structure, and vertical distribution along the soil profile have been studied with regard to the different slope exposures.

• Klíčová slova
• Altai, Siberia, centipedes, distribution, ecology, lowland, millipedes, new records,
• Publikační typ
• časopisecké články MeSH

Methane emission by terrestrial invertebrates is restricted to millipedes, termites, cockroaches, and scarab beetles. The arthropod-associated archaea known to date belong to the orders Methanobacteriales, Methanomassiliicoccales, Methanomicrobiales, and Methanosarcinales, and in a few cases also to non-methanogenic Nitrososphaerales and Bathyarchaeales. However, all major host groups are severely undersampled, and the taxonomy of existing lineages is not well developed. Full-length 16S rRNA gene sequences and genomes of arthropod-associated archaea are scarce, reference databases lack resolution, and the names of many taxa are either not validly published or under-classified and require revision. Here, we investigated the diversity of archaea in a wide range of methane-emitting arthropods, combining phylogenomic analysis of isolates and metagenome-assembled genomes (MAGs) with amplicon sequencing of full-length 16S rRNA genes. Our results allowed us to describe numerous new species in hitherto undescribed taxa among the orders Methanobacteriales (Methanacia, Methanarmilla, Methanobaculum, Methanobinarius, Methanocatella, Methanoflexus, Methanorudis, and Methanovirga, all gen. nova), Methanomicrobiales (Methanofilum and Methanorbis, both gen. nova), Methanosarcinales (Methanofrustulum and Methanolapillus, both gen. nova), Methanomassiliicoccales (Methanomethylophilaceae fam. nov., Methanarcanum, Methanogranum, Methanomethylophilus, Methanomicula, Methanoplasma, Methanoprimaticola, all gen. nova), and the new family Bathycorpusculaceae (Bathycorpusculum gen. nov.). Reclassification of amplicon libraries from this and previous studies using this new taxonomic framework revealed that arthropods harbor only CO2 and methyl-reducing hydrogenotrophic methanogens. Numerous genus-level lineages appear to be present exclusively in arthropods, suggesting long evolutionary trajectories with their termite, cockroach, and millipede hosts, and a radiation into various microhabitats and ecological niches provided by their digestive tracts (e.g., hindgut compartments, gut wall, or anaerobic protists). The distribution patterns among the different host groups are often complex, indicating a mixed mode of transmission and a parallel evolution of invertebrate and vertebrate-associated lineages.

• Klíčová slova
• Bathyarchaeia, Nitrososphaerales, archaea, cockroaches, gut microbiota, methanogens, millipedes, termites,
• Publikační typ
• časopisecké články MeSH