Patterns and determinants of the global herbivorous mycobiome.

Despite their role in host nutrition, the anaerobic gut fungal (AGF) component of the herbivorous gut microbiome remains poorly characterized. Here, to examine global patterns and determinants of AGF diversity, we generate and analyze an amplicon dataset from 661 fecal samples from 34 mammalian species, 9 families, and 6 continents. We identify 56 novel genera, greatly expanding AGF diversity beyond current estimates (31 genera and candidate genera). Community structure analysis indicates that host phylogenetic affiliation, not domestication status and biogeography, shapes the community rather than. Fungal-host associations are stronger and more specific in hindgut fermenters than in foregut fermenters. Transcriptomics-enabled phylogenomic and molecular clock analyses of 52 strains from 14 genera indicate that most genera with preferences for hindgut hosts evolved earlier (44-58 Mya) than those with preferences for foregut hosts (22-32 Mya). Our results greatly expand the documented scope of AGF diversity and provide an ecologically and evolutionary-grounded model to explain the observed patterns of AGF diversity in extant animal hosts.

The ultrastructure of the apical organ of the Müller's larva of the tiger flatworm Prostheceraeus crozieri.

The tiger flatworm Prostheceraeus crozieri (Polycladida) develops via an eight-lobed, and three-eyed planktonic Müller's larva. This larva has an apical organ, ultrastructural details of which remain elusive due to a scarcity of studies. The evolution and possible homology of the polyclad larva with other spiralian larvae is still controversial. Here, we provide ultrastructural data and three-dimensional reconstructions of the apical organ of P. crozieri. The apical organ consists of an apical tuft complex and a dorso-apical tuft complex. The apical tuft complex features a central tuft of five long cilia, which emerge from four or five individual cells that are themselves encircled by two anchor cells. The necks of six multibranched gland cells are sandwiched between ciliated tuft cell bodies and anchor cells. The proximal parts of the ciliated cell bodies are in contact with the lateral brain neuropil via gap junctions. Located dorsally of the apical tuft complex, the dorso-apical tuft complex is characterized by several long cilia of sensory neurons, these emerge from an epidermal lumen and are closely associated with several gland cells that form a crescent apically around the dorsal anchor cell, and laterally touch the brain neuropil. Such ciliated sensory neurons emerging from a ciliated lumen are reminiscent of ampullary cells of mollusc and annelid larvae; a similar cell type can be found in the hoplonemertean decidula larva. We hypothesize that the ampullary-like cells and the tuft-forming sensory cells in the apical organs of these spiralian larvae could be homologous.

No time to die: Comparative study on preservation protocols for anaerobic fungi.

Anaerobic fungi (AF, phylum Neocallimastigomycota) are best known for their ability to anaerobically degrade recalcitrant lignocellulosic biomass through mechanic and enzymatic means. While their biotechnological potential is well-recognized, applied research on AF is still hampered by the time-consuming and cost-intensive laboratory routines required to isolate, maintain, and preserve AF cultures. Reliable long-term preservation of specific AF strains would aid basic as well as applied research, but commonly used laboratory protocols for AF preservation can show erratic survival rates and usually exhibit only moderate resuscitation success for up to one or two years after preservation. To address both, the variability, and the preservation issues, we have set up a cross-laboratory, year-long study. We tested five different protocols for the preservation of AF. The experiments were performed at three different laboratories (Austria, Germany, Switzerland) with the same three morphologically distinct AF isolates (Anaeromyces mucronatus, Caeocmyces sp., and Neocallimastix cameroonii) living in stable co-culture with their naturally occurring, syntrophic methanogens. We could show that handling greatly contributes to the variability of results, especially in Anaeromyces mucronatus. Cryopreservation of (mature) biomass in liquid nitrogen had the highest overall survival rates (85-100%, depending on the strain and laboratory). Additionally, preservation on agar at 39°C had surprisingly high survival rates for up to 9 months, if pieces of agar containing mature AF thalli were resuscitated. This low-cost, low-effort method could replace consecutive batch cultivation for periods of up to 6 months, while long-term preservation is best done by cryopreservation in liquid nitrogen. Regardless of the method, however, preserving several replicates (>three) of the same strain is highly advisable.

Why eDNA fractions need consideration in biomonitoring.

The analysis of environmental DNA (eDNA) is revolutionizing the monitoring of biodiversity as it allows to assess organismic diversity at large scale and unprecedented taxonomic detail. However, eDNA consists of an extracellular and intracellular fraction, each characterized by particular properties that determine the retrievable information on when and where organisms live or have been living. Here, we review the fractions of eDNA, describe how to obtain them from environmental samples and present a four-scenario concept that aims at enhancing spatial and temporal resolution of eDNA-based monitoring. Importantly, we highlight how the appropriate choice of eDNA fractions precludes misinterpretation of eDNA-based biodiversity data. Finally, future avenues of research towards eDNA fraction-specific analyses are outlined to unravel the full potential of eDNA-based studies targeting micro- and macro-organisms.

Evaluation of cellulose based films comprising tea tree oil against dermatophytes and yeasts.

BACKGROUND: Onychomycosis is defined as infection caused by nondermatophytic molds and yeasts: tinea unguium is caused by dermatophytes. PURPOSE: Within this study, hydroxyethyl cellulose (HEC) as an important non-ionic, water-soluble cellulose derivative was chosen to develop formulations containing tea tree oil as active antifungal agent were developed and evaluated for their potential in the treatment of onychomycosis. METHODS: Two polymeric films based on HEC (HEC-B-04 and HEC-E-10) were obtained by solvent evaporation method and characterized in terms of appearance, disintegration, stickiness, elongation, rheological behavior and adhesiveness. Moreover, different strains of dermatophytes such as Trichophyton rubrum and yeasts as Candida albicans were treated with polymeric films containing tea tree oil (0.5 - 2 % v/v) in order to determine their antifungal potential by the inhibition zone assay. RESULTS: HEC-B-04 and HEC-E-10 were investigated by SEM measurements resulting in confluent surface morphology. HEC-B-04 and HEC-E-10 showed disintegration after 32.7 min and 34.0 min, respectively. Furthermore, HEC-E-10 revealed a moisture index of 1.74 and underpinned adhesive properties in terms of required detachment force with 4.86 N. HEC-E-10 pointed to the most antifungal one among the others against Trichophyton rubrum and Candida albicans. CONCLUSION: Taking these findings in consideration, promising adhesive onychial formulations were developed as forthcoming approach in treatment of nail infections.

The masking effect of extracellular DNA and robustness of intracellular DNA in anaerobic digester NGS studies: A discriminatory study of the total DNA pool.

Most commonly, next generation sequencing-based microbiome studies are performed on the total DNA (totDNA) pool; however, this consists of extracellular- (exDNA) and intracellular (iDNA) DNA fractions. By investigating the microbiomes of different anaerobic digesters over time, we found that totDNA suggested lower species richness considering all and/or only common species and yielded fewer unique reads as compared to iDNA. Additionally, exDNA-derived sequences were more similar to those from totDNA than from iDNA and, finally, iDNA showed the best performance in tracking temporal changes in microbial communities. We postulate that abundant sequences present within the exDNA fraction mask the overall results of totDNA and provide evidence that exDNA has the potential to qualitatively bias microbiome studies at least in the anaerobic digester environment as it contains information about cells that were lysed hours or days ago. iDNA, however, was found to be more appropriate in providing reliable genetic information about potentially alive as well as rare microbes within the target habitat.

Quantities of Intra- and Extracellular DNA Reveal Information About Activity and Physiological State of Methanogenic Archaea.

Although being a common aim of many microbial ecology studies, measuring individual physiological conditions of a microbial group or species within a complex consortium is still a challenge. Here, we propose a novel approach that is based on the quantification of sequentially extracted extracellular (exDNA) and intracellular DNA (iDNA) and reveals information about cell lysis and activity of methanogenic archaea within a biogas-producing microbial community. We monitored the methane production rates of differently treated batch anaerobic cultures and compared the concentrations of the alpha subunit of the methyl coenzyme M reductase gene of methanogenic archaea in extracellular and intracellular DNA fractions and in the classically extracted total DNA pool. Our results showed that this fine-tuned DNA approach coupled with the interpretation of the ratio between free exDNA and iDNA considerably improved microbial activity tracking compared to the classical extraction/quantification of total DNA. Additionally, it allowed to identify and quantify methanogenic populations that are inactive and those that are strongly influenced by cell lysis. We argue that despite the need of further studies, this method represents a novel approach to gain specific physiological information from a complex environmental sample and holds the potential to be applied to other microbes of interest.

Simple yet effective: Microbial and biotechnological benefits of rumen liquid addition to lignocellulose-degrading biogas plants.

In biogas plants, lignocellulose-rich biomass (LCB) is particularly slowly degraded, causing high hydraulic retention times. This fact lowers the interests for such substrates. To enhance LCB-degradation, cattle rumen fluid, a highly active microbial resource accruing in the growing meat industry, might be used as a potential source for bioaugmentation. This study compares 0%, 20% and 40% rumen liquid in a batch anaerobic digestion approach. Moreover, it determines the biogas- and methane-potentials as well as degradation-speeds of corn straw, co-digested with cattle manure. It inspects microbial communities via marker-gene sequencing, qPCR and RNA-DGGE and draws attention on possible beneficial effects of rumen addition on the biogas-producing community. Bioaugmentation with 20% and 40% v/v rumen liquid accelerated methane yields by 5 and 6 days, respectively (i.e. reaching 90% of total methane production). It also enhanced LCB- as well as (hemi)cellulose- and volatile fatty acid degradation. These results are supported by increased abundances of bacteria, methanogens and anaerobic fungi in treatments with rumen liquid amendment, and point towards the persistence of specific rumen-borne microorganisms especially during the first phase of the experiment. The results suggest that rumen liquid addition is a promising strategy for enhanced and accelerated exploitation of LCB for biomethanisation.

The interplay between total mercury, methylmercury and dissolved organic matter in fluvial systems: A latitudinal study across Europe.

Large-scale studies are needed to identify the drivers of total mercury (THg) and monomethyl-mercury (MeHg) concentrations in aquatic ecosystems. Studies attempting to link dissolved organic matter (DOM) to levels of THg or MeHg are few and geographically constrained. Additionally, stream and river systems have been understudied as compared to lakes. Hence, the aim of this study was to examine the influence of DOM concentration and composition, morphological descriptors, land uses and water chemistry on THg and MeHg concentrations and the percentage of THg as MeHg (%MeHg) in 29 streams across Europe spanning from 41°N to 64 °N. THg concentrations (0.06-2.78 ng L-1) were highest in streams characterized by DOM with a high terrestrial soil signature and low nutrient content. MeHg concentrations (7.8-159 pg L-1) varied non-systematically across systems. Relationships between DOM bulk characteristics and THg and MeHg suggest that while soil derived DOM inputs control THg concentrations, autochthonous DOM (aquatically produced) and the availability of electron acceptors for Hg methylating microorganisms (e.g. sulfate) drive %MeHg and potentially MeHg concentration. Overall, these results highlight the large spatial variability in THg and MeHg concentrations at the European scale, and underscore the importance of DOM composition on mercury cycling in fluvial systems.

Extracellular DNA in natural environments: features, relevance and applications.

Extracellular DNA (exDNA) is abundant in many habitats, including soil, sediments, oceans and freshwater as well as the intercellular milieu of metazoa. For a long time, its origin has been assumed to be mainly lysed cells. Nowadays, research is collecting evidence that exDNA is often secreted actively and is used to perform a number of tasks, thereby offering an attractive target or tool for biotechnological, medical, environmental and general microbiological applications. The present review gives an overview on the main research areas dealing with exDNA, depicts its inherent origins and functions and deduces the potential of existing and emerging exDNA-based applications. Furthermore, it provides an overview on existing extraction methods and indicates common pitfalls that should be avoided whilst working with exDNA.

The use of extracellular DNA as a proxy for specific microbial activity.

The ubiquity and relevance of extracellular DNA (exDNA) are well-known and increasingly gaining importance in many fields of application such as medicine and environmental microbiology. Although sources and types of exDNA are manifold, ratios of specific DNA-molecules inside and outside of living cells can give reliable information about the activity of entire systems and of specific microbial groups or species. Here, we introduce a method to discriminate between internal (iDNA), as well as bound and free exDNA, and evaluate various DNA fractions and related ratios (ex:iDNA) regarding their applicability to be used as a fast, convenient, and reliable alternative to more tedious RNA-based activity measurements. In order to deal with microbial consortia that can be regulated regarding their activity, we tested and evaluated the proposed method in comparison to sophisticated dehydrogenase- and RNA-based activity measurements with two anaerobic microbial consortia (anaerobic fungi and syntrophic archaea and a microbial rumen consortium) and three levels of resolution (overall activity, total bacteria, methanogenic archaea). Furthermore, we introduce a 28S rRNA gene-specific primer set and qPCR protocol, targeting anaerobic fungi (Neocallimastigomycota). Our findings show that the amount of actively released free exDNA (fDNA) strongly correlates with different activity measurements and is thus suggested to serve as a proxy for microbial activity.