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Anaerobic fungal communities differ along the horse digestive tract

December 2018
Fungal Biology 123(3)

DOI:10.1016/j.funbio.2018.12.004

Authors:

Joan Elizabeth Edwards

Wageningen University & Research

Sandra Kittelmann

Wilmar International Ltd.

Kerstin Kaerger

Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute

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Anaerobic fungi are potent fibre degrading microbes in the equine hindgut, yet our understanding of their diversity and community structure is limited to date. In this preliminary work, using a clone library approach we studied the diversity of anaerobic fungi along six segments of the horse hindgut: caecum, right ventral colon (RVC), left ventral colon (LVC), left dorsal colon (LDC), right dorsal colon (RDC) and rectum. Of the 647 ITS1 clones, 61.7 % were assigned to genus level groups that are so far without any cultured representatives, and 38.0 % were assigned to the cultivated genera Neocallimastix (35.1 %), Orpinomyces (2.3 %), and Anaeromyces (0.6 %). AL1 dominated the group of uncultured anaerobic fungi, particularly in the RVC (88 %) and LDC (97 %). Sequences from the LSU clone library analysis of the LDC, however, split into two distinct phylogenetic clusters with low sequence identity to Caecomyces sp. (94–96 %) and Liebetanzomyces sp. (92 %) respectively. Sequences belonging to cultured Neocallimastix spp. dominated in LVC (81 %) and rectum (75.5 %). Quantification of anaerobic fungi showed significantly higher concentrations in RVC and RDC compared to other segments, which influenced the interpretation of the changes in anaerobic fungal diversity along the horse hindgut. These preliminary findings require further investigation.

Phylogenetic relationships of ITS1 sequences of anaerobic fungi generated from the LDC inferred using the UPGMA method with bootstrap values from 1000 replications. The evolutionary distances were computed using the Jukes-Cantor method. The analysis involved a total of 142 nucleotide sequences (127 sequences generated from the LDC in this study and 14 of the most closely related sequences plus 1 outgroup sequence).

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Anaerobic fungal communities differ along the horse digestive tract

Erica Mura

, Joan Edwards

, Sandra Kittelmann

, Kerstin Kaerger

, Kerstin Voigt

Jakub Mr

azek

, Giuseppe Moniello

, Katerina Fliegerova

Department of Veterinary Medicine, University of Sassari, Via Vienna 2, 07100 Sassari, Italy

Laboratory of Microbiology, Wageningen University &Research, Wageningen, 6708 WE, the Netherlands

Wilmar International Ltd., Wil@NUS Corporate Lab, National University of Singapore, Singapore 117599, Singapore

Institute of Microbiology, University of Jena, Neugasse 25, 07743 Jena, Germany

Leibniz Institute for Natural Product Research and Infection Biology, Jena Microbial Resource Collection, Adolf-Reichwein-Str. 23, 07745 Jena, Germany

Institute of Animal Physiology and Genetics, CAS, Víde

nsk

a 1083, Prague 14220, Czech Republic

article info

Article history:

Received 23 July 2018

Received in revised form

23 November 2018

Accepted 19 December 2018

Available online 27 December 2018

Corresponding Editor: Pieter van West

Keywords:

Anaerobic fungi

Diversity

Equine hindgut

ITS1

Uncultured

abstract

Anaerobic fungi are potent ﬁbre degrading microbes in the equine hindgut, yet our understanding of

their diversity and community structure is limited to date. In this preliminary work, using a clone library

approach we studied the diversity of anaerobic fungi along six segments of the horse hindgut: caecum,

right ventral colon (RVC), left ventral colon (LVC), left dorsal colon (LDC), right dorsal colon (RDC) and

rectum. Of the 647 ITS1 clones, 61.7 % were assigned to genus level groups that are so far without any

cultured representatives, and 38.0 % were assigned to the cultivated genera Neocallimastix (35.1 %),

Orpinomyces (2.3 %), and Anaeromyces (0.6 %). AL1 dominated the group of uncultured anaerobic fungi,

particularly in the RVC (88 %) and LDC (97 %). Sequences from the LSU clone library analysis of the LDC,

however, split into two distinct phylogenetic clusters with low sequence identity to Caecomyces sp. (94

e96 %) and Liebetanzomyces sp. (92 %) respectively. Sequences belonging to cultured Neocallimastix spp.

dominated in LVC (81 %) and rectum (75.5 %). Quantiﬁcation of anaerobic fungi showed signiﬁcantly

higher concentrations in RVC and RDC compared to other segments, which inﬂuenced the interpretation

of the changes in anaerobic fungal diversity along the horse hindgut. These preliminary ﬁndings require

further investigation.

1. Introduction

Horses evolved as free-ranging herbivores of grassland envi-

ronments, with an enlarged hindgut adaptation enabling them to

obtain energy and nutrients from plant structural polysaccharides

through microbial fermentation. The hindgut is comprised of two

main fermentative chambers, the caecum and colon, which

together constitute two-thirds of the volume of the digestive tract.

The hindgut has a combined capacity of over 200 L (Frape, 2010)

and accounts for 75 % of the mean transit time (23e48h) of dietary

particles (van Weyenberg et al., 2006). The hindgut is home to

bacteria, anaerobic fungi, methanogenic archaea and protozoa. Of

these, the anaerobic fungi (Neocallimastigomycetes) are the most

potent in terms of degrading plant ﬁbres due to their complete and

very efﬁcient set of plant cell-wall degradation enzymes (Gruninger

et al., 2014; Haitjema et al., 2014). Despite their presence in the

horse hindgut within a few weeks of birth (Julliand et al., 1996),

almost all the current knowledge of anaerobic fungi is derived from

ruminant based studies.

Nine genera of anaerobic fungi validly described based on

cultivated representatives, including those with ﬁlamentous mon-

ocentric (Neocallimastix,Piromyces,Oontomyces and Buwchfa-

wromyces), ﬁlamentous polycentric (Orpinomyces,Anaeromyces and

Pecoramyces) and bulbous (Caecomyces and Cyllamyces) mycelium

(Gruninger et al., 2014; Edwards et al., 2017), have been recently

extended by new monocentric genera Feramyces (Hanafy et al.,

2018) and Liebetanzomyces (Joshi et al., 2018). These genera, how-

ever, represent only part of anaerobic fungal diversity as indicated

by the large and growing numbers of internal transcribed spacer

region 1 (ITS1) sequences in public databases that belong to

*Corresponding author. Institute of Animal Physiology and Genetics, Czech

Academy of Sciences, Víde

nsk

a 1083, Prague 14220, Czech Republic.

Fax: þ420267090500.

E-mail address: ﬂiegerova@iapg.cas.cz (K. Fliegerova).

All the authors contributed equally to this work.

Contents lists available at ScienceDirect

Fungal Biology

journal homepage: www.elsevier.com/locate/funbio

https://doi.org/10.1016/j.funbio.2018.12.004

Fungal Biology 123 (2019) 240e246

potentially novel, as yet uncultured clades within the Neo-

callimastigomycetes. Based on the re-evaluation of publicly avail-

able ITS1 sequences, which took into account both primary

sequence and secondary structure information, Kittelmann et al.

(2012) and later Koetschan et al. (2014) proposed a revised phy-

logeny and pragmatic taxonomy of anaerobic fungi, which resulted

in 37 reproducible species or genus-level clades. Eighteen of these

clades have not been cultured, containing only environmental

derived sequences, and the number of not yet cultivated clades was

recently increased to twenty-ﬁve (Paul et al., 2018).

A survey of anaerobic fungi in faeces of 30 different ruminant

and non-ruminant herbivore species, found that members of the

family Equidae clustered apart from the other non-Equidae herbi-

vores studied (Liggenstoffer et al., 2010). The different domesti-

cated and non-domesticated equines sampled in the Liggenstoffer

et al. (2010) study shared a similar anaerobic fungal community,

which was mainly composed of two novel genus level groups that

are now termed AL1 &AL3 (Koetschan et al., 2014). Cultivated

anaerobic fungal genera were found in equine samples only in

limited relative abundance (4e12 % of Caecomyces,2%ofNeo-

callimastix, 0.3 % of Piromyces,0.1e0.3 % of Anaeromyces). These

results suggest that the digestive tract of horses is largely occupied

by novel, as yet uncultured anaerobic fungi, which differ from those

previously described from foregut herbivores.

Whilst molecular based analysis of equine anaerobic fungal di-

versity has been performed on faecal samples (Liggenstoffer et al.,

2010), it is known that bacterial, protozoal and archaeal commu-

nity composition differs with hindgut segment (Julliand and

Grimm, 2016; Fliegerova et al., 2016). Therefore, there is a clear

need to assess if the anaerobic fungal community composition also

differs along the hindgut, particularly as niche differentiation

within the anaerobic fungi has previously been proposed (Grifﬁth

et al., 2009). In this preliminary study, the anaerobic fungal com-

munity composition along the hindgut of a mature horse was

determined using ITS1 based clone libraries.

2. Materials and methods

2.1. Collection of digesta samples from the horse hindgut

Gut content samples were taken from the six segments of the

hindgut of an Anglo-Arabian gelded male (24 y old) euthanised for

non-research purposes in a local abattoir. The horse was main-

tained on a mixed diet of grass, meadow hay and complementary

feed as described by Fliegerova et al. (2016), and was healthy with

no history of any intestinal disorders. The segments sampled were:

caecum, right ventral colon (RVC), left ventral colon (LVC), left

dorsal colon (LDC), right dorsal colon (RDC) and rectum (repre-

senting faeces). Gut segments were tied off to prevent mixing be-

tween neighbouring segments, and the whole content from a

section mixed thoroughly before a sample (approx 500 g) was

taken. Samples were placed in labelled plastic containers and

transported on wet ice back to the laboratory where they were

frozen (at 20



C) and then freeze-dried. Freeze-dried material was

stored at 20



C until DNA extraction.

2.2. DNA extraction

Freeze-dried gut content (5 g) was homogenized using a mortar

and pestle with liquid nitrogen. Genomic DNA was then extracted

from 400 mg of the resulting powder using the cetyl-

trimethylammonium bromide extraction protocol of Gardes and

Bruns (1993) after modiﬁcation with the respect to the amount of

sample material used. The concentration and purity of extracted

nucleic acids was checked using a NanoDrop 2000c UV-Vis

spectrophotometer (Thermo Scientiﬁc, U.S.A), and DNA extracts

were stored at 20



C until use.

2.3. PCR ampliﬁcation

Ampliﬁcation of the anaerobic fungal ITS1 region from each

sample was carried out with the combination of the fungal uni-

versal ITS1 forward primer (Gardes and Bruns, 1993) and the

Neocallimastigomycetes speciﬁc 5.8S rRNA gene reverse primer

(Edwards et al., 2008). This resulted in an amplicon of approxi-

mately 350 bp in length, as described previously by Fliegerova et al.

(2010). The PCR reaction (50

l) was performed with a PPP Master

Mix kit (Top-Bio, Czech Republic) and 0.3

M of each primer using

the following thermal cycling conditions: 33 cycles of denaturation

at 94



C for 1 min, annealing at 58



C for 30 s and extension at 72



for 45 s with an initial cycle of 94



C for 4 min and ﬁnal cycle of



C for 2 min. Each sample was PCR ampliﬁed in quadruplicate

and then pooled after successful ampliﬁcation and veriﬁed by

agarose gel electrophoresis.

For one sample, the LDC, the ribosomal large subunit 28S rRNA

(LSU) was also ampliﬁed in quadruplicate with universal primers

NL1 and NL4 as described previously by Fliegerova et al. (2006). The

rationale for this analysis is explained in the results section.

Pooled PCR amplicons of the correct length were excised from

an agarose gel with a sterile scalpel blade, puriﬁed and concen-

trated using a QIAquick Gel Extraction Kit (Qiagen, Germany).

2.4. Construction of clone libraries and sequencing

The TOPO

TA Cloning

Kit for Sequencing (Life Technologies,

USA) was used for the preparation of the ITS1 clone libraries for

each segment of equine hindgut, and the LSU clone library of the

LDC segment. Ligation of the PCR amplicons into pCR4-TOPO vector

was performed at room temperature for 30 min followed by

transformation into competent One Shot TOP10 Escherichia coli

cells (30 min on ice). Randomly selected clones of E.coli grown

overnight in lysogeny broth plates with ampicillin (50

g/ml) were

checked by PCR with M13 primers for the presence of the ITS1 and

LSU fragments of expected length. Plasmid DNA was then isolated

from 881 ITS1 clones and 20 LSU clones using a GenElute™HP

Plasmid Miniprep Kit (SigmaeAldrich, USA). Puriﬁed plasmid DNAs

were quantiﬁed using a NanoDrop ND-1000 UV-Vis Sectropho-

tometer (NanoDrop Technologies, USA) and Sanger sequenced

(SEQme, Czech Republic). Generated sequences were checked for

errors and any vector contamination removed. Of the clones

sequenced, 647 of the ITS1 clones and 17 of the LSU clones yielded

clear and unambiguous sequence data that were used for further

analysis.

2.5. Taxonomic and phylogenetic analysis

Taxonomic assignment of ITS1 sequences (97 % identity

threshold) was done in QIIME version 1.8 (Caporaso et al., 2010)by

BLAST against the anaerobic fungal ITS1 reference database

(Koetschan et al., 2014). The 647 cloned sequences with taxonomic

assignment from the six libraries were concatenated into a single

fasta ﬁle, and a sequence map (seqs_otus.txt) was manually built

for input into the script “make_otu_table.py”. The resulting relative

abundance table was summarized at the clade level. A similar

process was followed with the LSU sequence data, except the

similarity search was carried out using BLAST against GenBank.

Evolutionary analyses of ITS1 sequences and the LSU sequences

from the LDC sample were separately performed using the software

MEGA 6 (Tamura et al., 2013) to construct phylogenetic trees, with

1000 replicates for bootstrap analysis, using the UPGMA method.

E. Mura et al. / Fungal Biology 123 (2019) 240e246 241

LSU genes of described genera Caecomyces (JQ782555, KM878679),

Cyllamyces (DQ273829, KY386297), Piromyces (JF974096, JF974119,

JN939159), Buwchfawromyces (KP205570, NG058679), Feramyces

(MG584197, MG584228, MG605676), Neocallimastix (JF974094,

JN939158, KT274174, KR920745), Pecoramyces (JN939127,

KX961618), Orpinomyces (HQ703476, JN939163, KM878680),

Anaeromyces (JN939157, JN939170, JN939172), Liebetanzomyces

(MH468763) and Oontomyces (JX017314, JX017315) were used as

reference sequences.

The ITS1 nucleotide sequences generated from the horse hind-

gut have been deposited in the GenBank database under the

following accession numbers: MH038102 eMH038269 (caecum),

MH038399 eMH038497 (RVC), MH038498 eMH038612 (LVC),

MH038272 eMH038398 (LDC), MH038613 eMH038694 (RDC)

and MH038695 eMH038784 (rectum). The LSU sequences of the

LDC have been deposited in the GenBank database under the

accession numbers MH125212 eMH125228.

2.6. qPCR analysis

The MX 3005P QPCR System (Stratagene) and the Kapa SYBR

Fast qPCR Master mix (Kapa Biosystems) was used for the qPCR

determination of ITS1 gene copy numbers in each sample using the

ITS1 primer pair and cycling conditions as described above (see PCR

ampliﬁcation). The standard curve was created (in triplicate) using

a 10-fold dilution series of a pCR4-TOPO plasmid containing the

ITS1 region of a Piromyces isolate (NCBI Accession No. KY368107).

ITS1 copy numbers were determined (in triplicate) from 1

l of ten-

fold diluted DNA, and data expressed per g of dry matter of digesta.

Three replicates of a negative control sample without DNA template

were also included in the qPCR assay. The qPCR assayefﬁciency was

101.5 %, and quantitation was linear over eight orders of magnitude

(10

to 10

gene copy

1

). The unpaired t-test (Microsoft Excel,

2010) was used to identify signiﬁcant differences in the counts of

anaerobic fungi among all the samples. Signiﬁcant differences were

declared when P <0.05. A quantitative proﬁle of the anaerobic

fungi was then calculated, using the ratio between the total ITS1

gene copy number and relative abundance of each clade for each

hindgut segment.

3. Results

3.1. ITS1 based diversity analysis of anaerobic fungi in the horse

hindgut

Of the 647 successfully sequenced ITS1 clones from all six seg-

ments of the horse hindgut, the 645 clones appeared to be anaer-

obic fungal in origin and only 2 of these sequences (0.3 %) could not

be further taxonomically assigned within the class Neo-

callimastigomycetes using either the anaerobic fungal ITS1 refer-

ence database or GenBank. On average 61.7 % of all the sequences

were represented by genus-level groups that have so far only been

described in the literature based on sequence data: AL1, AL7, DT1

and KF1 (as deﬁned by Koetschan et al., 2014). The cultured genera

Neocallimastix,Orpinomyces and Anaeromyces represented the

remaining 38.0 % sequences that could be taxonomically assigned.

Phylogenetic relationships of ITS1 sequences of uncultured anaer-

obic fungi generated from the horse hindgut and cultured genera is

shown in Fig. S1.

3.2. Distribution and quantiﬁcation of anaerobic fungi along the

horse hindgut

The relative abundance of the different anaerobic fungal clades

(as deﬁned by Koetschan et al., 2014) along the six segments of the

horse hindgut are shown (Fig. 1). The AL1 clade represented 53 % of

the total sequences and was present throughout the hindgut,

however, its relative abundance differed among gut segments. AL1

represented almost all of the anaerobic fungi in the RVC (88 %) and

LDC (97 %), and was the most abundant clade in the caecum (62 %)

and RDC (53 %). In contrast, the relative abundance of AL1 in the

LVC (4 %) and the rectum (3 %) was very low. Generally, the

decreased abundance of AL1 in certain gut segments seemed to be

associated with an increase in the abundance of the Neocallimastix

1 clade. This clade was the second most abundant (35.1 % of the

total sequences) and was detected in all segments. The third most

abundant clade was AL7 (4.3 % of the total sequences), and was also

detected in all segments. In contrast, the KF1 clade (4.2 % of the

total sequences) was only detected in the LVC (7 %), RDC (3 %) and

rectum (17 %). Sequences of the other less abundant fungi (Orpi-

nomyces 1a, Orpinomyces 1b, Anaeromyces 1 and DT1) were rela-

tively minor (13.7 % of total sequences) and detected only in some

hindgut segments.

Total anaerobic fungal concentrations differed between seg-

ments, with the exception of the caecum and LDC (Fig. 2). The RVC

followed by RDC was found to contain the highest concentrations of

anaerobic fungi, and the lowest concentrations of anaerobic fungi

were detected in the LVC and rectum. It can be clearly seen that the

lower concentrations of total anaerobic fungi in these segments is

associated with a substantial decrease in the amount of the AL1

clade, rather than an increase in the Neocallimastix 1 clade (as

indicated by Fig. 1).

3.3. LSU based diversity analysis of the LDC

As 97 % of the ITS1 sequences from the LDC segment belonged to

the monophyletic AL1 clade (Fig. 3), this segment was also analysed

using an LSU based clone library in order to better elucidate the

taxonomic position of this uncultured clade. Tree topology showed

that the LSU sequences obtained (n ¼17) formed two distinct

clusters (Fig. 4). The clones representing cluster I (n ¼8) had

highest sequence identity (94e96 %) to a sequence belonging to a

cultured Caecomyces communis (KM878679), and formed a sister

group to the Caecomyces/Cyllamyces group. The clones representing

cluster II (n ¼9) had highest sequence identity (91e92 %) to a se-

quences belonging to a cultured Liebetanzomyces sp. (MH468763)

and Anaeromyces sp. (MG605690), however, the cloned sequences

clustered distantly from the reference anaerobic fungi present in

the tree.

4. Discussion

Gut microbes are essential colonizers of the mammalian

digestive tract. They are involved in food decomposition, produc-

tion of vitamins and micronutrients. Microbial fermentative end-

products (especially acetate, propionate and butyrate) supply the

host body by energy. Microbiota of digestive tract is also involved in

a variety of other metabolic and physiological functions including

the shaping of the immune system. The importance of the equine

intestinal microbial ecosystem for animal health and performance

is well established (Dicks et al., 2014), however, research has pri-

marily focussed on only bacteria. The investigation of the anaerobic

fungi in the equine hindgut to date has been limited and, to the best

of our knowledge, there is no study of the diversity of anaerobic

fungi in the different anatomo-physiological segments of the horse

hindgut.

Our work showed that an uncultured Neocallimastigales clade

named AL1 was prevalent in the equine hindgut. This ﬁnding is

in agreement with an ITS1 based next generation sequencing

study that found the NG1 (¼AL1 in Kittelmann et al., 2012) group

E. Mura et al. / Fungal Biology 123 (2019) 240e246242

could account for 56.7%e99.9 % of the total anaerobic fungi in

the faeces of certain horses and zebra (Liggenstoffer et al., 2010).

It was surprising to observe that the AL1 clade was greatly

decreased in the LVC and rectum, with the reason for these de-

creases not clear.

The AL1 clade is not speciﬁc to equines, as it was also detected

(17.8e49.5 %) in a variety of other foregut fermenters (Liggenstoffer

et al., 2010). However, ITS1 sequences of the AL1 clade retrieved in

this study were most similar or identical only with those from the

zebra and horse (deposited in the GenBank database). The effort to

elucidate the phylogenetic relationships of the ITS1 deﬁned AL1

clade using the LSU resulted in splitting of the sequences into two

non-related clusters. As this contrasted the monophyletic clade

seen with ITS1, further work is needed to conﬁrm the basis of this

ﬁnding.

The second most numerous clade of uncultivated anaerobic

fungi identiﬁed in horse faeces by Liggenstoffer et al. (2010), clade

NG3 (¼AL3 in Kittelmann et al., 2012), has not been found in any

part of the horse hindgut sampled in this study. On the other hand,

sequences of the group NG7 (¼AL7 in Kittelmann et al., 2012)

retrieved by Liggenstoffer et al. (2010) in low relative abundance

only from Somali wild ass faeces (0.6 %), represented 4 % of total

clones in the present study. Sequences of another group of anaer-

obic fungi, the KF1 clade, which represented 17 % of the sequences

in the horse rectum in this study, were ﬁrst detected in cow manure

and were described as an undistinguished Cyllamyces/Caecomyces

cluster of uncultured fungi (Fliegerova et al., 2010).

Regarding the known cultivable anaerobic fungi, only the clade

Neocallimastix 1 colonized the hindgut of the studied horse with a

dominant relative abundance in the LVC and rectum. However,

Fig. 1. Relative abundances of anaerobic fungal clades (as deﬁned by Koetschan et al., 2014) in the ITS1 clone libraries constructed from six segments of the horse hindgut. The total

number of clones in each library is indicated in parentheses on the x-axis. The arrow indicates the pelvic ﬂexure position between the LVC and LDC.

Fig. 2. Quantitative proﬁles of anaerobic fungal clades (as deﬁned by Koetschan et al., 2014) in the ITS1 clone libraries constructed from six segments of the horse hindgut. The top

of the bars indicate mean concentrations (n ¼3) of total anaerobic fungal ITS1 gene copies in six different segments of horse hindgut, and different letters indicate signiﬁcant

differences (P <0.05). Quantitative proﬁles were then generated from clade relative abundances and the ITS1 concentrations in each segment. The arrow indicates the pelvic

ﬂexure position between the LVC and LDC.

E. Mura et al. / Fungal Biology 123 (2019) 240e246 243

quantitative proﬁling showed that this was due to the absence of

clade AL1, rather than an increased amount of Neocallimastix 1in

these segments. The Neocallimastix 1 sequences were mostly

similar to those obtained from ruminants, especially from cow,

bison and yak. Neocallimastix is known by its excellent hydrolytic

properties and multi-functional cellulosomal enzymes, and is

presumed to be a very effective (hemi)cellulose degrader

(Gruninger et al., 2014; Wei et al., 2016). In horse, zebra and donkey

faeces analysed by Liggenstoffer et al. (2010) this genus was either

absent or represented at very low relative abundance (<2 %).

However, in a sample from a Somali wild ass, Neocallimastix rep-

resented 45 % of all sequences.

Whilst the cause of the considerable difference in anaerobic

fungal diversity between the RVC and LVC is not clear, the differ-

ence between the neighbouring LVC and LDC may be related to the

pelvic ﬂexure. This narrow junction is responsible for the selective

retention of coarse particles. Less digested particles are retained in

the caecum and LVC, whereas liquid and ﬁner particles move on

into the LDC. These retropulsive-propulsive movements keep the

caecum and the whole ventral colon ﬁlled, increasing the fermen-

tation time of plant biomass. These segments of the hindgut are,

therefore, the sites with the highest digesta mean retention time of

(about 9 h) and represents the main site of lignocellulose degra-

dation (Van Weyenberg et al., 2006). As well as a longer time to

degrade the structural polysaccharides, this also provides more

time for the propagation of microorganisms.

The implication of the RVC ecosystem in forage degradation has

been highlighted already by de Fombelle et al. (2003), due to the

decreased proportion of cellulose in the digesta after passing

through the RVC. This corresponds well with our ﬁnding of highest

anaerobic fungal concentrations in the RVC compared to the other

hindgut segments. On the other hand, the segments with the

lowest anaerobic fungal concentrations (LVC and rectum) were

associated with a decrease in the amount of the AL1 clade. Varia-

tions in the concentration of anaerobic fungi found among different

gut segments in this study, however, contrasts with study of Dougal

et al. (2012) where no effect of gut segment was seen based on the

caecum, RDC and rectum sites that were sampled.

The RDC is the site of another selective mechanism (known as

the colonic separation mechanism). This mechanism is responsible

for the prolonged retention of ﬂuid and smaller food particles

(Drogoul et al., 2000). How this mechanism may contribute to the

decrease of the AL1 clade in the rectum is not clear. Digesta samples

from this part of the hindgut, could in future studies be separated

into a liquid and a solid fraction to provide further insight, as

anaerobic fungal vegetative biomass (as opposed to the motile

zoospores) is tightly attached to plant particles.

Faeces samples are often used as reference samples to describe

the microbial population of the digestive tract, because the faecal

microbiota might be expected to contain representatives from all

regions of the large intestine. Our study provides evidence that

horse faecal samples can serve as qualitative reference samples, as

almost all clades of anaerobic fungi found in the hindgut were also

present in the faeces sample. The only exception was Anaeromyces,

which was only detected in some segments and at very low relative

abundances. However, the relative abundance and concentrations

of anaerobic fungi differed considerably along the horse hindgut

and the functional implications of this variation requires further

investigation.

Differences in anaerobic fungal concentrations along the rumi-

nant digestive tract is known to occur (Davies et al., 1993).

Furthermore, a cultivation based study by Grifﬁth et al. (2009)

compared the anaerobic fungi in the rumen and faeces of the

same cow, and found considerable differences in the abundance of

Fig. 3. Phylogenetic relationships of ITS1 sequences of anaerobic fungi generated from the LDC inferred using the UPGMA method with bootstrap values from 1000 replications. The

evolutionary distances were computed using the Jukes-Cantor method. The analysis involved a total of 142 nucleotide sequences (127 sequences generated from the LDC in this

study and 14 of the most closely related sequences plus 1 outgroup sequence).

E. Mura et al. / Fungal Biology 123 (2019) 240e246244

the different taxa. Fungi with bulbous morphotypes (Caecomyces

and Cyllamyces) were the most abundant genera in fresh faeces,

where they comprised a 5-fold greater proportion of the total

fungal population than in the rumen. Polycentric morphotypes

(Orpinomyces and Anaeromyces) were less frequently isolated from

fresh faeces as compared to rumen digesta. Conversely, a cultiva-

tion independent study showed no difference in anaerobic fungal

community composition in the rumen, duodenum and faeces of

three different cows (Jimenez et al., 2007).

We acknowledge that no general conclusion can be made

from the analysis of samples from one horse, and the ﬁndings of

this preliminary study should be used cautiously. Nevertheless,

the data presented here provide an important next step in

revealing the considerable, as yet largely uncharacterised,

anaerobic fungal diversity in the equine hindgut. It also high-

lights the need to conduct further studies to look at the activity,

concentrations and diversity of the anaerobic fungi throughout

the digestive tract, due to evidence of differences between

hindgut segments.

Acknowledgement

This work was supported by the Ministry of Education, Youth

and Sports of the Czech Republic, grant no. CZ.02.1.01/0.0/0.0/

15_003/0000460 OP RDE) and programs of the University of Sassari

Erasmusþ, Master and Back 2014/2015 and Visiting Professor 2016/

2017 Regione Autonoma Sardegna. JEE acknowledges funding from

an EU H2020 Marie Curie Fellowship (706899).

Appendix A. Supplementary data

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.funbio.2018.12.004.

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An extended incubation strategy to culture slow growing members of anaerobic fungi resulted in the isolation of a novel anaerobic fungus from the rumen of a goat after 15 days. The novel genus, represented by type strain G1SC, showed filamentous monocentric thallus development and produced uniflagellate zoospores, hence, showing morphological similarity to the genera Piromyces , Buwchfawromyces , Oontomyces and Pecoramyces . However, strain G1SC showed genetic similarity to the genus Anaeromyces , which, though produces uniflagellate zoospore, also exhibits polycentric thallus development. Moreover, unlike Anaeromyces , strain G1SC did not show hyphal constrictions, instead produced a branched, determinate and anucleate rhizoidal system. This fungus also displayed extensive sporangial variations, both exogenous and endogenous type of development, short and long sporangiophores and produced septate sporangia. G1SC utilised various complex and simple substrates, including rice straw and wheat straw and produced H 2 , CO 2 , formate, acetate, lactate, succinate and ethanol. Phylogenetic analysis, using internal transcribed spacer 1 (ITS1) and D1/D2 domain of large-subunit (LSU) rRNA locus, clearly showed a separate lineage for this strain, near Anaeromyces . The ITS1 based geographical distribution studies indicated detection of environmental sequences similar (93–96%) to this strain from cattle faeces. Based on morphological and molecular characterisation results of strain G1SC, we propose a novel anaerobic fungus Liebetanzomycespolymorphusgen. et sp. nov. , in the phylum Neocallimastigomycota .

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Anaerobic fungi (phylum Neocallimastigomycota) are common inhabitants of the digestive tract of mammalian herbivores, and in the rumen, can account for up to 20% of the microbial biomass. Anaerobic fungi play a primary role in the degradation of lignocellulosic plant material. They also have a syntrophic interaction with methanogenic archaea, which increases their fiber degradation activity. To date, nine anaerobic fungal genera have been described, with further novel taxonomic groupings known to exist based on culture-independent molecular surveys. However, the true extent of their diversity may be even more extensively underestimated as anaerobic fungi continue being discovered in yet unexplored gut and non-gut environments. Additionally many studies are now known to have used primers that provide incomplete coverage of the Neocallimastigomycota. For ecological studies the internal transcribed spacer 1 region (ITS1) has been the taxonomic marker of choice, but due to various limitations the large subunit rRNA (LSU) is now being increasingly used. How the continued expansion of our knowledge regarding anaerobic fungal diversity will impact on our understanding of their biology and ecological role remains unclear; particularly as it is becoming apparent that anaerobic fungi display niche differentiation. As a consequence, there is a need to move beyond the broad generalization of anaerobic fungi as fiber-degraders, and explore the fundamental differences that underpin their ability to exist in distinct ecological niches. Application of genomics, transcriptomics, proteomics and metabolomics to their study in pure/mixed cultures and environmental samples will be invaluable in this process. To date the genomes and transcriptomes of several characterized anaerobic fungal isolates have been successfully generated. In contrast, the application of proteomics and metabolomics to anaerobic fungal analysis is still in its infancy. A central problem for all analyses, however, is the limited functional annotation of anaerobic fungal sequence data. There is therefore an urgent need to expand information held within publicly available reference databases. Once this challenge is overcome, along with improved sample collection and extraction, the application of these techniques will be key in furthering our understanding of the ecological role and impact of anaerobic fungi in the wide range of environments they inhabit.

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Jun 2016
J ANIM SCI

In the early 1990s, the equine hindgut microbial ecosystem looked like a "black box." Its vital role in hydrolyzing and fermenting fiber, thus providing energy to the host, was recognized. Although there was a critical lack of information on the hindgut microbes, their role in preventing intestinal diseases was suggested. Traditionally, the microbes of the horse hindgut were studied using culture-dependent techniques. More recently, culture-independent methods have been used and provided further insight. This review presents the history and updated knowledge regarding the microbes that live inside the different intestinal ecosystems and which collective genomes compose the hindgut microbiome. In the first section, the quantification and diversity are described for each microbial community as well as the implication of plant fiber degradation and their crucial role for an herbivore host. The microbial communities are presented in chronological order of discovery: due to their large size, protozoa were brought to light as early as 1843 in the horse cecum; in 1897, bacteria were described in the horse intestine; as early as 1910, monoflagellated eukaryotic organisms resembling protozoa were observed in the horse cecum; since then, they have been identified to be zoospores of anaerobic fungi; in 1970, bacteriophage-like particles were recognized in the cecum and colon of pony and horse; and finally, in 1996, archaea were identified in the horse cecum. The second section discusses the variations that can occur between digestive segments or between individuals. The representativeness of the fecal microbiota to the hindgut one is debated, especially as the majority of recent studies conducted on the horse hindgut are in fact focused on the feces, rather than the cecum or colon. Also, the representation of microbiota between individuals is questioned. It has long been suggested in the literature that some ponies or horses that were more susceptible to intestinal diseases may harbor a specific intestinal microbiota. Alternatively, some new studies aim at identifying a core microbiome between all individual equine. A deeper knowledge of the microbiome and its core may allow improvement of nutrition and health, understanding of the onset of digestive diseases, and the development tools for health monitoring and disease prevention.

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May 1996
PFERDEHEILKUNDE

The faecal microflora of 18 normal, two month-old foals was composed of aerobes and facultative anaerobes, mainly Enterococci (107 CFU/g of faeces) and Lactobacilli (108 CFU/g) and strict anaerobes, cellulolytic bacteria (105 CFU/g) and Clostridia (104 CFU/g). At this early age different bacterial strains colonized the gut according to a specific sequence which could be modified by the oral administration of a commercial colostrum replacer. The faecal microflora of 5 foals fed this colostrum at birth and once every two weeks during the first three months appeared to contain less cellulolytic bacteria and no Lactobacilli. The small number of foals treated limited our interpretation.

Anaerobic Fungi (Phylum Neocallimastigomycota): Advances in understanding of their taxonomy, life cycle, ecology, role, and biotechnological potential

Article

Full-text available

Jul 2014
FEMS MICROBIOL ECOL

Anaerobic fungi (phylum Neocallimastigomycota) inhabit the gastrointestinal tract of mammalian herbivores, where they play an important role in the degradation of plant material. The Neocallimastigomycota represent the earliest diverging lineage of the zoosporic fungi; however, understanding of the relationships of the different taxa (both genera and species) within this phylum is in need of revision. Issues exist with the current approaches used for their identification and classification, and recent evidence suggests the presence of several novel taxa (potential candidate genera) that remain to be characterised. The life cycle and role of anaerobic fungi has been well characterised in the rumen, but not elsewhere in the ruminant alimentary tract. Greater understanding of the ‘resistant’ phase(s) of their life cycle is needed, as is study of their role and significance in other herbivores. Biotechnological application of anaerobic fungi, and their highly active cellulolytic and hemi-cellulolytic enzymes, has been a rapidly increasing area of research and development in the last decade. The move towards understanding of anaerobic fungi using –omics based (genomic, transcriptomic and proteomic) approaches is starting to yield valuable insights into the unique cellular processes, evolutionary history, metabolic capabilities and adaptations that exist within the Neocallimastigomycota. An overview of the lifecycle, taxonomy, ecology, metabolic capabilities, and genomics of a poorly studied group of fungi: The anaerobic gut fungi of the phylum Neocallimastigomycota. An overview of the lifecycle, taxonomy, ecology, metabolic capabilities, and genomics of a poorly studied group of fungi: The anaerobic gut fungi of the phylum Neocallimastigomycota.

Feramyces austinii , gen. nov, sp. nov., an anaerobic gut fungus from rumen and fecal samples of wild Barbary sheep and fallow deer

Article

Apr 2018

Anaerobic gut fungi are common inhabitants of the alimentary tracts of herbivorous animals. Nine different Neocallimastigomycota genera have been described so far. However, culture-independent diversity surveys suggest the presence of numerous, yet-uncultured Neocallimastigomycota genera. Here, we report on the isolation and characterization of the first cultured representatives of Neocallimastigomycota clade AL6, originally identified in culture-independent surveys of fecal samples from captive wild animals. Six strains were isolated from rumen and fecal samples of a wild Barbary sheep (Ammotragus lervia) and a wild fallow deer (Dama dama) in Texas, USA. The isolates displayed medium-sized (3-7 mm) circular beige colonies with filamentous edges and a dark center on agar roll tubes. Microscopic analysis revealed monocentric thalli with both endogenous and exogenous sporangial development patterns. Zoospores were spherical, with a diameter of 9.6 ±1.9 μm, and polyflagellated, with 7-16 flagella. Phylogenetic analysis based on nuc rDNA ITS1 region and D1-D2 domains of nuc 28S rDNA revealed that the isolated strains formed a single monophyletic and bootstrap-supported clade distinct from all currently described Neocallimastigomycota genera. Substrate utilization experiments using the type strain (F3a) demonstrated robust and fast growth on sugars and plant biomass, as well as the capability to metabolize a wide range of mono-, oligo-, and polysaccharides including galactose, arabinose, alginate, and pectin. On the basis of the morphological, physiological, and phylogenetic analyses, we propose to accommodate these isolates in a new genus Feramyces (derived from the Latin word for “wild” to reflect their isolation and apparent distribution in undomesticated herbivores), and a new species, F. austinii. The type species is Feramyces austinii strain F3a.

A phylogenetic census of global diversity of gut anaerobic fungi and a new taxonomic framework

Article

Mar 2018

Obligate anaerobic fungi of the phylum Neocallimastigomycota play a key role in digesting fibrous feeds in the gut of herbivores, but little is known about their global diversity. In this study, the collective diversity of gut anaerobic fungi was examined using all curated internal transcribed spacer 1 (ITS1) sequences of anaerobic gut fungi available in GenBank. The 262,770 quality-checked fungal ITS1 sequences downloaded from GenBank were assigned to 274 operational taxonomic units (OTUs) at the approximate species level. Of these approximate species-equivalent (Sp-eq) OTUs, 119 were represented by at least five ITS1 sequences, with 38 containing known species and 81 containing no known species. Based on a rarefaction analysis, the currently available ITS1 sequences represent nearly all the major species of gut anaerobic fungi, but much more sequencing effort is needed to assess the actual richness of minor OTUs. One dataset of ITS1 reference sequences (referred to as AF-RefSeq) and one comprehensive taxonomic framework are also presented, and they are shown to be suitable for taxonomic classification of most of the ITS1 sequences in GenBank. The results of the present study may help guide future studies involving taxonomic and phylogenetic analysis of ITS1 sequences of anaerobic fungi and targeted isolation and characterization of new anaerobic fungi.

A comparison of microbial profiles of different regions of the equine hindgut

Article

May 2016
LIVEST SCI

The microbial profiles of the luminal content of five hindgut segments of one healthy horse were compared with rectal sample to elucidate the effect of anatomical region on bacterial and archaeal community structure and to evaluate the use of faeces as a representative model of large intestine. The qualitative and quantitative changes of the microbial community composition of caecum, right ventral colon, left ventral colon, left dorsal colon, right dorsal colon and faeces were monitored by denaturing gradient gel electrophoresis (DGGE) and real-time PCR using universal primers amplifying the V3 region of 16 S rDNA. DGGE fingerprints revealed extensive bacterial as well as archaeal diversity in all studied samples and reflected shifts in the community structure among the caecum, the different segments of the colon and the faeces. Archaeal DGGE pattern of the caecum differed from all the other parts of the hindgut. Microbial profile similarities were found between the left and the right dorsal colon and between the left ventral colon and the faeces. The excised DGGE bands were related to uncultured bacteria and methanogens, the dominant archaeal bands of caecum and faeces were related to Methanocorpusculum sp. Diversity indices indicated the higher diversity for bacteria than for archaea and the dominance of some methanogenic species. The real-time PCR revealed the differences in the microbial quantitative composition of each segment, showing the highest number of total bacteria and archaea in the right ventral colon. The analyses of bacterial and archaeal composition along the one equine hindgut indicate that the faecal sample is similar to that of the left ventral colon, but does not represent the microbial community of the caecum and other parts of the colon.

Equine Nutrition and Feeding: Third Edition

Book

Nov 2007

David Lawrence Frape

Equine Nutrition and Feeding has become the standard work on the subject, covering every aspect of the nutrition of breeding, growing and working horses, describing the basis upon which scientifically derived conclusions for nutrition and dietary requirements are reached. Following the last edition in 1998, the book has been extensively updated, revised and rewritten with reference to well over 400 papers that have been published since 1998 on equine science. It has been made more practical by setting out the implications of new research for feeding programmes and it includes a full account of the toxicology and metabolic and other diseases, related to diet. Their causes and control are discussed and comprehensive lists of definitions of the terms and abbreviations used are given.

Isolation, identification and fibrolytic characteristics of rumen fungi grown with indigenous methanogen from yaks ( Bos grunniens ) grazing on the Qinghai-Tibetan Plateau

Article

Mar 2016
J APPL MICROBIOL

Aim: To obtain co-cultures of anaerobic fungi and their indigenously associated methanogens from the rumen of yaks grazing on the Qinghai-Tibetan Plateau and investigate their morphology features and ability to degrade lignocellulose. Methods and results: Twenty fungus-methanogen co-cultures were obtained by Hungate roll-tube technique. The fungi were identified as Orpinomyces, Neocallimastix and Piromyces genera based on the morphological characteristics and internal transcribed spacer 1 sequences analysis. All methanogens were identified as Methanobrevibacter sp. by 16S rRNA gene sequencing. There were four types of co-cultures: Neocallimastix with Methanobrevibacter ruminantium, Orpinomyces with M. ruminantium, Orpinomyces with Methanobrevibacter millerae and Piromyces with M. ruminantium among 20 co-cultures. In vitro studies with wheat straw as substrate showed that the Neocallimastix with M. ruminantium co-cultures and Piromyces with M. ruminantium co-cultures exhibited higher xylanase, filter paper cellulase (FPase), ferulic acid esterase, acetyl esterase activities, in vitro dry matter digestibility, gas, CH4 , acetate production, ferulic acid and p-coumaric acid releases. The Neocallimastix frontalis Yak16 with M. ruminantium co-culture presented the strongest lignocellulose degradation ability among 20 co-cultures. Conclusions: Twenty fungus-methanogen co-cultures were obtained from the rumen of grazing yaks. The N. frontalis with M. ruminantium co-cultures were highly effective combination for developing a fermentative system that bioconverts lignocellulose to high activity fibre-degrading enzyme, CH4 and acetate. Significance and impact of the study: The N. frontalis with M. ruminantium co-cultures from yaks grazing on the Qinghai-Tibetan Plateau present great potential in lignocellulose biodegradation industry.