Vladimír Šustr

Application of ARDRA and PLFA analysis in characterizing the bacterial communities of the food, gut and excrement of saprophagous larvae ofPenthetria holosericea (Diptera: Bibionidae): a pilot study

Amplified ribosomal DNA restriction analysis (ARDRA) was used to compare the bacterial communities of the food, the gut sections (ceca, anterior and posterior midgut, hindgut) and the excrement of the litter feeding bibionid larvae ofPenthetria holosericea. For universal eubacterial primers ARDRA patterns were complex with only minor differences among samples. Taxon specific primers were also applied to characterize the samples. Fragment composition was transformed to presence/absence binary data and further analyzed. Cluster analysis revealed that bacterial communities of gut highly resembled each other with the exception of the ceca. ARDRA patterns of consumed leaves clustered together with the intact leaves but differed from those of the excrement. ARDRA results were compared with microbial community structure based on phospholipid fatty acid (PLFA) fingerprints. The cluster analysis of PLFA (presence/absence binary) data resulted in a pattern similar to the ARDRA data. The PCA analysis of PLFA relative content separated microbial communities into five groups: (1) anterior and posterior midgut, (2) hindgut, (3) ceca, (4) consumed and intact litter, (5) excrement. Both methods indicated that conditions in the larval gut result in formation of a specific microbial community which differs from both the food and excrement ones. Particularly ceca — (blind appendages, harbor very specific microbial community) are divided from the rest of the gut by perithropic membrane.

Changes in amount of bacteria during gut passage of leaf litter and during coprophagy in three species of Bibionidae (Diptera) larvae

To elucidate the interaction between bacteria and saprophagousDiptera larvae, the amounts of bacteria in leaf litter, individual gut compartments, and feces of three species ofBibionidae (Bibio pomonae, Bibio marci, andPenthetria holosericea), feeding either directly on leaf litter or on fecal pellets produced from leaf litter by larvae of the same species, were assessed by determining total direct counts and viable counts on solid media at different pH. InP. holosericea, the effect of various cultivation temperatures on direct counts of bacteria in individual compartments was also demonstrated. In all species, the amount of bacteria in the anterior mesenteron was lower than in the consumed food, regardless of whether the larvae were feeding on leaf litter or feces, and increased again in the posterior part of the gut. The amount of bacteria in these compartments was generally higher in larvae feeding on feces than in those feeding on leaf litter, whereas the amount of bacteria found in the ceca varied. InB. marci, the amount of bacteria in the mesenteron sections able to grow on alkaline medium (pH 9) was higher than that of bacteria able to grow on slightly acidic medium (pH 5.5) during both the first and the second gut passage. InB. pomonae andP. holosericea, this increase was observed only during the second gut passage. The effect of gut passage inP. holosericea on changes in direct counts of bacteria was more pronounced when the larvae were fed at 5 °C as compared to 20 °C. Radiolabeled bacteria were digested in the gut and utilized as a source of energy and nutrients by the larvae; digested bacteria represented up to 10 % of the material assimilated by the larvae. Lysozyme activity in whole-gut extracts ofP. holosericea had a pH optimum of at pH 7, indicating a lowin situ activity in the alkaline mesenteron. Proteinase activity, however, had an optimum at pH >12, suggesting that the digestion of bacteria in the bibionid gut is caused by a combination of digestive proteinases and alkaline pH in the anterior mesenteron.

Food chain effects of sublethal ultraviolet radiation on subarctic Daphnia pulex - : a field and laboratory study

In shallow subarctic ponds Daphnia and their algal food are often exposed to solar ultraviolet radiation (UVR). It was previously demonstrated that direct exposure of Daphnia to UVR may result in sublethal damage to Daphnias intestinal system. This led to the hypothesis that, as consequences of such exposures, Daphnia may be experiencing a situation similar to starvation. We examined, in controlled experiments, the indirect effect of UVR-treated food algae on Daphnia pulex which, themselves, were also exposed to UVR. We specifically tested whether exposure of D. pulex to solar UVR affects food transport and enzymatic digestion. As expected, the UVR-exposed food and UVR-treated Daphnia combination produced the strongest effects on intestinal damage, and mortality. Some of these effects, as well as the grazer-related effects, were similar to those observed during starvation. The total activities of digestive enzymes (amylase and cellulase) were somewhat reduced in UVR treated D. pulex, but the function of enzymes is not seriously damaged as is clear from the increasing trend of protein-specific activities. We conclude that, in some ways, Daphnia undergoing sublethal UVR exposures may be experiencing a condition similar to what they would experience under very low food or even fasting conditions.

Preliminary data about compartmentalization of the gut of the saprophagous dipteran larvae Penthetria holosericea (Bibionidae)

Abstract To describe the compartmentalization of the gut and the microbial activity in the digestive tract, the histology of the gut wall, enzymatic activity, pH of the gut content, abundance and composition of the microbial community (direct counts, plate counts on various media, and phospholipid fatty acid analyses—PLFA) were studied in the Penthetria holosericea larvae. The highest secretion activity was observed in the caeca and in the part of the anterior midgut before caeca openings. A strong increase in pH (10.5) was observed in the anterior part of the midgut. Some enzymes produced in the caeca might have their maximum activity in a different part of the gut as a result of their pH optimum; e.g. amylase was most active in the strongly alkaline anterior part of the mesenteron. Maltase, hydrolyzing products of starch degradation, was most active in the posterior part of the gut and in the ectoperithrophic space. The microbial community was reduced in the anterior part of the midgut. In the posterior part of the gut, bacterial numbers increased and peaked in excrements. Caeca had an abundant microbial community.

Methane Production and Methanogenic Archaea in the Digestive Tracts of Millipedes (Diplopoda)

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.

Temperature Preference and Respiration of Acaridid Mites

ABSTRACT The thermal preferences in a grain mass and respiration at various temperatures in mites (Acari: Acarididae) of medical and economical importance [Acarus siro (L. 1758), Dermatophagoides farinae Hughes 1961, Lepidoglyphus destructor (Schrank 1871), and Tyrophagus putrescentiae (Schrank 1781)] were studied under laboratory conditions. Based on the distribution of mites in wheat, Triticum aestivum L., grain along a thermal gradient from 10 to 40°C, L. destructor, D. farinae, and A. siro were classified as eurythermic and T. putrescentiae as stenothermic. The lowest preferred temperature was found for D. farinae (28°C), followed by A. siro (28.5°C), L. destructor (29.5°C), and T. putrescentiae (31.5°C). The relationship between the respiration rate and the temperature was similar for all four mite species. The highest respiration was found in the range from 31 to 33°C This is ≈2°C higher than the preferred temperature of these species. The lower temperature threshold of respiration ranged from 1 to 5°C and the upper threshold ranged from 45 to 48°C Acclimatization of A. siro to temperature regimes of 5, 15, and 35°C resulted in thermal preferences between 9 and 12°C, 9 and 20°C, and 28 and 35°C, respectively. The respiration rate of acclimatized specimens increased with the temperature, reaching a maximum at 29.0°C for mites acclimatized at 5 and 15°C and a maximum at 33.7°C for those acclimatized at 30°C.

Temperature dependence and acclimatory response of amylase in the High Arctic springtail Onychiurus arcticus (Tullberg) compared with the temperate species Protaphorura armata (Tullberg).

Amylolytic activity was measured in whole body homogenates of High Arctic (Onychiurus arcticus) and temperate (Protaphorura armata) springtails (Collembola: Onychiuridae) in the temperature range 5-55 degrees C. A pH of ca. 8 was optimum for amylolytic activity in both species. A higher weight-specific amylolytic activity was observed in P. armata. In O. arcticus, amylolytic activity depended on thermal acclimation, which increased during 2 and 9 weeks of cold acclimation (5 degrees C) and decreased over 7 weeks of warming (15 degrees C) of animals that were previously acclimated to cold for 2 weeks. In cold-acclimated O. arcticus, a slower decrease of amylolytic activity occurred with lowering of temperature in the range 5-20 degrees C in comparison with warm-acclimated specimens and P. armata, which resulted in higher activity at 5 degrees C. The activation energy calculated from an Arrhenius plot for P. armata was 68.7 kJ.mol(-1). In O. arcticus it was between 30.2 and 61.5 kJ.mol(-1), being lower in cold-acclimated samples. The temperature optimum for amylolytic activity was higher in the temperate species (40 degrees C), whilst in O. arcticus it depended on the acclimation regime: it rose to 35 degrees C after warm acclimation and decreased to 20 degrees C after cold adaptation. The total soluble protein content of body tissues of O. arcticus also increased during cold acclimation. These differences between the two species suggest that amylolytic activity is an indicator of cold adaptation in the High Arctic O. arcticus.

Correlation between the activity of digestive enzymes and nonself recognition in the gut of Eisenia andrei earthworms

Earthworms Eisenia andrei, similarly to other invertebrates, rely on innate defense mechanisms based on the capability to recognize and respond to nonself. Here, we show a correlation between the expression of CCF, a crucial pattern-recognition receptor, and lysozyme, with enzyme activities in the gut of E. andrei earthworms following a microbial challenge. These data suggest that enzyme activities important for the release and recognition of molecular patterns by pattern-recognition molecules, as well as enzymes involved in effector pathways, are modulated during the microbial challenge. In particular, protease, laminarinase, and glucosaminidase activities were increased in parallel to up-regulated CCF and lysozyme expression.

Community composition and cold tolerance of soil Collembola in a collapse karst doline with strong microclimate inversion

Abstract The study compared communities of soil Collembola along the inversed microclimatic gradient of the collapse doline of the Silicka ľadnica Ice Cave (Slovakia) in spring and autumn of 2005. Kruskal-Wallis ANOVA and the Mann- Whitney test revealed significant differences in abundance between sites and both seasons. Significantly higher abundance means and species richness were observed at most sites during the spring compared with the autumn. NMS ordination documented a clear delimitation of communities with remarkably different soil microclimates. The community pattern of the coldest section of the gradient, with low species richness and high mean abundance, was analogous to communities living in the harsh alpine and polar soils. The collapse doline with inversed microclimate hosted a high number of species (72) and a broad variety of montane forms (13), thus documenting that these karst landforms enhance local diversity of edaphic Collembola and serve as local refugia of specialized cold-tolerant species. The cold tolerance of the four abundant species at the doline cold sites, namely Ceratophysella sigillata, Tetrodontophora bielanensis, Protaphorura armata and Desoria tigrina, was tested in the laboratory using one-hour exposition survival tests. Within a temperature range from -2.4 to -7.8◦C, T. bielanensis was the most cold-sensitive species, with a lethal dose LD50 of -4.4◦C, while D. tigrina was the most cold-resistant, showing LD50 of -5.8◦C.

Microprofiles of oxygen, redox potential, and pH, and microbial fermentation products in the highly alkaline gut of the saprophagous larva of Penthetria holosericea (Diptera : Bibionidae)

The saprophagous larvae of bibionid flies harbor bacteria in their alkaline intestinal tracts, but little is known about the contribution of the gut microbiota to the digestion of their recalcitrant diet. In this study, we measured oxygen and hydrogen partial pressure, redox potential and pH in the midgut, gastric caeca and hindgut of larvae of the bibionid fly Penthetria holosericea with Clark-type O2 and H2 microsensors, platinum redox microelectrodes, and LIX-type pH microelectrodes. The center of the midgut lumen was anoxic, whereas gastric caeca and hindgut were hypoxic. However, redox potential profiles indicated oxidizing conditions throughout the gut, with lowest values in the midgut (+20 to +60mV). Hydrogen production was not detected. The midgut was extremely alkaline (pH around 11), whereas hindgut and gastric caeca were neutral to slightly alkaline. While HPLC analysis showed high concentrations of glucose in the midgut (15mM) and gastric caeca (27mM), the concentrations of microbial fermentation products such as lactate (2-4mM), acetate (<1mM) and succinate (<0.5mM) were low in all gut regions, suggesting that the contribution of microorganisms to the digestive process, particularly in the alkaline midgut, is only of minor importance. We conclude that the digestive strategy of the saprophytic larva of P. holosericea, which feeds selectively on decomposed leaves and its own microbe-rich faeces, differs fundamentally from those of detritivorous and humivorous insects, which host a highly active, fermentative microbiota in their alkaline midgut or hindgut compartments.