The effect of a high-grain diet on the rumen microbiome of goats with a special focus on anaerobic fungi

This work investigated the changes of the rumen microbiome of goats switched from a forage to a concentrate diet with special attention to anaerobic fungi (AF). Female goats were fed an alfalfa hay (AH) diet (0% grain; n = 4) for 20 days and were then abruptly shifted to a high-grain (HG) diet (40% corn grain, 60% AH; n = 4) and treated for another 10 days. Rumen content samples were collected from the cannulated animals at the end of each diet period (day 20 and 30). The microbiome structure was studied using high-throughput sequencing for bacteria, archaea (16S rRNA gene) and fungi (ITS2), accompanied by qPCR for each group. To further elucidate unclassified AF, clone library analyses were performed on the ITS1 spacer region. Rumen pH was significantly lower in HG diet fed goats, but did not induce subacute ruminal acidosis. HG diet altered prokaryotic communities, with a significant increase of Bacteroidetes and a decrease of Firmicutes. On the genus level Prevotella 1 was significantly boosted. Methanobrevibacter and Methanosphaera were the most abundant archaea regardless of the diet and HG induced a significant augmentation of unclassified Thermoplasmatales. For anaerobic fungi, HG triggered a considerable rise in Feramyces observed with both ITS markers, while a decline of Tahromyces was detected by ITS2 and decrease of Joblinomyces by ITS1 only. The uncultured BlackRhino group revealed by ITS1 and further elucidated in one sample by LSU analysis, formed a considerable part of the AF community of goats fed both diets. Results strongly indicate that the rumen ecosystem still acts as a source for novel microorganisms and unexplored microbial interactions and that initial rumen microbiota of the host animal considerably influences the reaction pattern upon diet change.Fil: Fliegerova, Katerina O.. Czech Academy of Sciences; República ChecaFil: Podmirseg, Sabine M.. Universidad de Innsbruck; AustriaFil: Vinzelj, Julia. Universidad de Innsbruck; AustriaFil: Grilli, Diego Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza; Argentina. Universidad Nacional de Cuyo. Facultad de Cs.médicas. Departamento de Patología. Area de Microbiología; ArgentinaFil: Kvasnová, Simona. Czech Academy of Sciences; República ChecaFil: Schierová, Dagmar. Czech Academy of Sciences; República ChecaFil: Sechovcová, Hana. Czech Academy of Sciences; República ChecaFil: Mrázek, Jakub. Czech Academy of Sciences; República ChecaFil: Siddi, Giuliana. Università degli Studi di Sassari; ItaliaFil: Arenas, Graciela Nora. Universidad Nacional de Cuyo. Facultad de Cs.médicas. Departamento de Patología. Area de Microbiología; ArgentinaFil: Moniello, Giuseppe. Università degli Studi di Sassari; Itali

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

Effect of Growth Media on the Diversity of Neocallimastigomycetes from Non-Rumen Habitats

Joshi A, Young D, Huang L, et al. Effect of Growth Media on the Diversity of Neocallimastigomycetes from Non-Rumen Habitats. Microorganisms. 2022;10(10): 1972.Anaerobic fungi (AF), belonging to the phylum Neocallimastigomycota, are a pivotal component of the digestive tract microbiome of various herbivorous animals. In the last decade, the diversity of AF has rapidly expanded due to the exploration of numerous (novel) habitats. Studies aiming at understanding the role of AF require robust and reliable isolation and cultivation techniques, many of which remained unchanged for decades. Using amplicon sequencing, we compared three different media: medium with rumen fluid (RF), depleted rumen fluid (DRF), and no rumen fluid (NRF) to enrich the AF from the feces of yak, as a rumen control; and Przewalski’s horse, llama, guanaco, and elephant, as a non-rumen habitats. The results revealed the selective enrichment of Piromyces and Neocallimastix from the feces of elephant and llama, respectively, in the RF medium. Similarly, the enrichment culture in DRF medium explicitly manifested Piromyces-related sequences from elephant feces. Five new clades (MM1-5) were defined from llama, guanaco, yak, and elephant feces that could as well be enriched from llama and elephant samples using non-conventional DRF and NRF media. This study presents evidence for the selective enrichment of certain genera in medium with RF and DRF from rumen as well as from non-rumen samples. NRF medium is suggested for the isolation of AF from non-rumen environments

Presentation_1_No time to die: Comparative study on preservation protocols for anaerobic fungi.pdf

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.</p

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