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Environmental Microbiology (2014)

doi:10.1111/1462-2920.12425

The fibre-associated cellulolytic bacterial community in

the hindgut of wood-feeding higher termites
(Nasutitermes spp.)

Aram Mikaelyan,1 Jrgen F. H. Strassert,1

Gaku Tokuda2 and Andreas Brune1*
1
Department of Biogeochemistry, Max Planck Institute
for Terrestrial Microbiology, Marburg, Germany.
2
Tropical Biosphere Research Center, COMB, University
of the Ryukyus, Nishihara, Okinawa, Japan.
Summary
Termites digest lignocellulose with the help of their
symbiotic gut microbiota. In the hindgut of evolutionary lower termites, a dense community of cellulolytic
flagellates sequesters wood particles from the
hindgut content into their digestive vacuoles. In
higher termites (family Termitidae), which possess an
entirely prokaryotic microbiota, the wood particles
are available for bacterial colonization. Substantial
particle-associated cellulase activities have been
detected in the hindgut of Nasutitermes species, but
the microorganisms responsible for these activities
and their potential association with the wood fibres
remain to be studied. Here, we used density-gradient
centrifugation to separate wood fibres and adherent
bacterial cells from cells freely suspended in the
hindgut fluid. In Nasutitermes corniger, the fibre fraction contained 28% of the DNA and 45% of the
cellulase activity in the luminal contents (P3 region).
Community fingerprinting (terminal restriction fragment length polymorphism) and pyrotag sequencing
analysis of the bacterial 16S rRNA genes demonstrated that the wood fibres in the hindgut of both
N. corniger and N. takasagoensis are specifically
colonized by members of Fibrobacteres, the TG3
phylum, and certain lineages of Spirochaetes characteristic of the gut microbiota of wood-feeding higher
termites. We propose that the loss of flagellates in
higher termites provided a new niche for fibreassociated cellulolytic bacteria.

Received 6 December, 2013; revised 21 January, 2014; accepted

21 January, 2014. *For correspondence. E-mail brune@mpimarburg.mpg.de; Tel. (+49) 6421 178 701; Fax (+49) 6421 178 999.

2014 Society for Applied Microbiology and John Wiley & Sons Ltd

Introduction
Termites digest lignocellulose with the help of their symbiotic gut microbiota (Brune, 2014). The breakdown of
ingested wood particles is initiated by endogenous
cellulases secreted by the salivary glands or the midgut
tissue, and completed in the voluminous hindgut, which
carries the bulk of the symbionts (Watanabe and Tokuda,
2010; Brune and Ohkuma, 2011; Ni and Tokuda, 2013). In
the evolutionary basal lineages, also referred to as lower
termites, the microbial component of this dual cellulolytic
system consists of a dense community of cellulolytic flagellates (Brugerolle and Radek, 2008; Ohkuma and Brune,
2011). The flagellates sequester the wood particles into
their digestive vacuoles and digest them with a suite of
glycosyl hydrolases, with little or no contribution by
prokaryotes. However, cellulolytic flagellates are entirely
absent in the evolutionary-derived lineage of higher termites (family Termitidae).
The loss of flagellates was accompanied by substantial
changes in the prokaryotic gut microbiota (Hongoh, 2011;
Ohkuma and Brune, 2011), but the processes responsible
for lignocellulose digestion in the hindgut of higher termites are only poorly understood (reviewed by Hongoh,
2011; Ni and Tokuda, 2013; Brune, 2014). While members
of the subfamily Macrotermitinae engaged in a unique
symbiosis with a basidiomycete fungus, which breaks
down lignocellulosic substrates in fungal gardens before
they are consumed by the termites, the members of other
termitid subfamilies must digest lignified plant fibre
entirely within their digestive tract.
Although there are several reports of cellulase activities
in hindgut homogenates of higher termites (Potts and
Hewitt, 1973; Rouland et al., 1986; Chararas and Noirot,
1988), it was proposed that Nasutitermes species rely
mostly on endogenous cellulases acting in the foregut and
midgut, and that bacterial symbionts in the hindgut contributed at best marginally to cellulose digestion (Hogan
et al., 1988; Slaytor, 1992). However, perception of
the situation changed fundamentally with the discovery
that the cellulase activity in hindgut homogenates of
Nasutitermes takasagoensis was not located in the particlefree supernatant but mostly in the post-centrifugation pellet,
which had not been investigated in the earlier studies

2 A. Mikaelyan et al.
(Tokuda et al., 2005). Further examination of hindgut
homogenates of N. takasagoensis and N. walkeri suggested that the particle-associated activity is that of cellbound or fibre-associated microbial enzymes (Tokuda and
Watanabe, 2007).
Support for this hypothesis came from metagenomic
studies, which identified numerous glycosyl hydrolase
genes encoding putative cellulases in metagenomic
libraries derived from the luminal contents of the enlarged
P3 compartment of Nasutitermes spp. and Amitermes
wheeleri (Warnecke et al., 2007; He et al., 2013). In
Nasutitermes spp., the majority of these genes are
assigned to Fibrobacteres and Spirochaetes (Warnecke
et al., 2007), two bacterial phyla abundantly represented
in the hindgut of wood-feeding higher termites (Hongoh
et al., 2006; Khler et al., 2012). In the dung-feeding
A. wheeleri, the community is instead dominated by
Firmicutes (He et al., 2013). It is noteworthy that in both
cases, the metagenomes showed an overrepresentation
of gene functions indicating the presence of substratebinding, cell-associated cellulase complexes; while the
hindgut metagenome of A. wheeleri encodes for an abundance of putative cohesins and dockerins typical of
clostridial cellulosomes, those of Nasutitermes spp. comprised numerous homologues presumably encoding
extracytoplasmic proteins with cohesin-like function in
Fibrobacter succinogenes (He et al., 2013).
In all ruminants, the bacteria contributing most importantly to fibre digestion are from the Fibrobacteres and
Firmicutes (order Clostridiales). Their firm attachment
to food particles by extracellular cellulosomes (in
Ruminococcus flavefaciens) or their functional analogues
(in F. succinogenes) greatly increases the efficiency of
degradation and provides substrates for the entire
methanogenic feeding chain (Flint et al., 2008). The food
particles in the rumen are relatively large and easily separated from the rumen fluid by simple filtration (Koike et al.,
2003; Brulc et al., 2011), which has allowed extensive
studies of the structure and activities of ruminal fibreassociated communities (Brulc et al., 2009; Kim et al.,
2011).
In the case of termites, however, the grinding action
of mandibles and gizzard break down the wood to
particle sizes that overlap those of the larger hindgut
bacteria (Tokuda et al., 2012). Apart from preliminary
ultrastructural evidence of microbial colonization (Tokuda
et al., 2005), nothing is known about the existence or
composition of a fibre-associated community in the
hindgut of higher termites and its cellulolytic activities. In
this study, we developed a new method that allows differentiating between fibre-associated and only cellassociated activities. By separating wood fibres and
attached bacterial cells from cells freely suspended in the
luminal hindgut fluid, we determined the contributions

of the different fractions to cellulose hydrolysis in

N. corniger. In addition, we characterized the fibreassociated bacterial community of N. corniger and N.
takasagoensis by terminal restriction fragment length polymorphism (T-RFLP) analysis and 454-pyrosequencing of
bacterial 16S rRNA genes in the respective fractions.

Results
Distribution of cellulase activity in the hindgut
Homogenization of N. corniger hindguts with micropestles released only about 10% of the total cellulase
activity into the supernatant (Table 1). The activity
released by sonication was considerably higher, but more
than half of the total activity still remained in the pellet and
was liberated only by detergent extraction (CelLytic B).
While the activity released by homogenization may represent (at least in part) soluble cellulases in the hindgut
fluid, the additional activity released by sonication and
the residual activity in the pellet were considered to be
associated with the particulate fraction. High particleassociated activities have been reported already in a previous study of N. takasagoensis (Tokuda and Watanabe,
2007). We obtained essentially the same results with
a different batch of N. takasagoensis, except that the
absolute activities were lower. Comparison of the
results obtained for total hindguts and P3 compartment
indicated that in both termite species, most of the
cellulase activity is confined to P3, the enlarged hindgut
paunch (Table 1).

Density-dependent enrichment of wood fibres

from the P3 lumen
Scanning electron microscopy revealed that most wood
particles are densely colonized by various types of filamentous or spiral-shaped bacterial cells (Fig. 1). The size
of most wood particles in the hindgut paunch of
N. corniger ranged between 10 and 50 m; only a few
were > 100 m in length (Fig. 2). The average particle
size of wood fibres in the hindgut paunch (25 18 m)
was significantly smaller than that in the crop (124
69 m) and the midgut (128 66 m).
Fractionation of the luminal content by buoyant density
using Percoll-gradient centrifugation yielded two wellseparated bands, with the bulk of the wood particles concentrated near the bottom of the tube (fibre fraction) and
the unattached cells close to the meniscus (fibre-free
fraction; Fig. 3A). The fibre fraction contained 83% of the
lignin and 28% of the DNA in the sample, whereas the
fibre-free fraction contained only 17% of the lignin but
68% of the DNA in the P3 lumen, which indicated that a
substantial part of the gut microbiota in the P3 contents is

2014 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology

The fibre-associated community of higher termites

Table 1. Cellulase activities present in the supernatant after homogenization or sonication of entire hindguts or P3 compartments of Nasutitermes
species and after detergent extraction of the sonicated pellet.
Cellulase activitya in supernatant (units g1)
Species

Compartment

Homogenizationb

Sonicationc

Detergent treatmentd

Totale

Particle-associated
activityf

N. corniger

Hindgut
P3
Hindgut
Hindgut
P3

0.023 0.009
0.017 0.008
0.014 0.053g
n.d.i
n.d.

0.069 0.024
0.099 0.004
0.039 0.02h
0.028 0.005
0.029 0.004

0.141 0.025
0.122 0.022
0.058 0.012h
0.026 0.005
0.020 0.008

0.210 0.049
0.221 0.026
0.097 0.032h
0.054 0.010
0.049 0.012

89%
92%
86%
82%
80%

N. takasagoensis

a. One unit of enzyme activity is defined as the amount of enzyme required to release 1 mol of reducing sugar equivalents per minute from
microcrystalline cellulose.
b. Cellulase activity present in the supernatant after pestle homogenization; considered to represent particle-free activity.
c. Cellulase activity present in the supernatant after sonication of the sample; includes also any particle-free activity.
d. Cellulase activity released upon detergent extraction of the post-sonication pellet with CelLytic B.
e. Sum of the activities released by sonication and detergent treatment.
f. Cellulase activity remaining after subtraction of the particle-free activity from the total activity.
g. Results from Tokuda and colleagues (2005).
h. Results from Tokuda and Watanabe, 2007.
i. Not determined.
All activities are based on the fresh weight of the termites used in the preparation and are means of three homogenates (five termites
each) standard error.

associated with wood particles (Fig. 3B). Inspection of

Toluidine-Blue-stained preparations by bright-field and
phase-contrast microscopy confirmed that the fibre fraction consisted mostly of wood particles and only few
unassociated cells, whereas the fibre-free fraction contained many suspended bacteria and only few smaller
wood particles. The size distribution of wood particles
in the fibre fraction was virtually identical to that in the
P3 fluid (Fig. 2), which indicated that the separation

procedure is not biased against wood particles of a particular size.

When we collected wood particles and microbial cells in
the different fractions by centrifugation, we recovered
45% of the cellulase activity in the P3 luminal fluid from
the fibre fraction and 40% of the activity from the fibre-free
fraction (the sum of the activities released by sonication
and subsequent detergent treatment; Fig. 3C). The proportion of the activity released only after detergent treat-

Fig. 1. Scanning electron micrograph of bacterial cells adhering to wood fibres in the hindgut of Nasutitermes corniger (A) and
N. takasagoensis (B).

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A. Mikaelyan et al.

Frequency

0.5

P3 lumen
Fibre-fraction

0.5

50

100

150

200

Length of wood fibres (m)

Fig. 2. Histogram comparison of the length distribution of wood
fibres in the P3 fluid (top) and fibre fraction (bottom) obtained from
Nasutitermes corniger.

ment was significantly higher in the fibre fraction

(P < 0.05, KruskalWallis test).
T-RFLP analysis of the samples revealed that the bacterial community in the luminal contents of N. corniger was
unevenly distributed between the two fractions. In all rep-

licates, two of the major peaks in the luminal fluid (144 and
400 bp) were recovered almost exclusively from the fibre
fraction, whereas the dominant terminal restriction fragment (T-RF) (618 bp) was present in both fractions (Fig. 4).
Using the predicted T-RFs of the sequences from clone
libraries (Hongoh et al., 2006), we tentatively assigned
them to TG3 phylum, Fibrobacteres and Spirochaetes.
When we fractionated the P3 luminal fluid of
N. takasagoensis in the same manner, the resulting
T-RFLP profiles were similar to those of N. corniger,
except that the peak representing Fibrobacteres subphylum 2 was quite small already in the luminal fluid (Fig. 4).
Again, the dominant T-RF of 618 bp was recovered from
both fractions, representing a large number of unresolved
phylotypes in the Treponema Cluster I.
Pyrotag analysis
To increase the taxonomic resolution of the community
analysis, we amplified the V3V4 region (about 450 bp) of
the 16S rRNA genes in luminal fluid, fibre fraction and
fibre-free fraction of the two termite species and analysed
the products by 454-pyrosequencing. The sequences
(500010 000 reads per sample) were classified against a
comprehensive database that includes all homologues
previously obtained from insect guts and allows identification of groups not yet resolved in public databases,
such as the termite-specific lineages in the TG3 phylum
and the Fibrobacteres (Khler et al., 2012). We further

C
Cellulase activity (%)

Recovery (%)
0

Fibre-free
fraction

50

100

DNA
Lignin

50

100

Sonicated
Detergent

Fibre
fraction

Fig. 3. Fractionation of the particles in the luminal fluid of the P3 compartment of Nasutitermes corniger by density-gradient centrifugation (A).
Distribution of lignin and DNA between the two fractions (B). Cellulase activity released by sonication and by detergent treatment of the
sonication pellet, relative to the total activity in the luminal fluid (C).

2014 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology

The fibre-associated community of higher termites

TG3

Treponema I

N. corniger

P3

P3

N. takasagoensis

Relative abundance

Ff

Ff

F
200

400

600

Fragment size (bp)

Fig. 4. T-RFLP profiles (TaqI digestion) of the bacterial
communities associated with the P3 lumen, the fibre-free fraction
(Ff) and fibre fraction (F) from Nasutitermes corniger (top) and
N. takasagoensis (bottom).

improved the classification of the diverse phylotypes in

the Treponema cluster I that were not resolved by the
T-RFLP analysis by adding numerous unpublished nearfull-length sequences from both lower and higher termites
and sequences available in public databases to our reference database (A. Mikaelyan, T. Khler and A. Brune, in
preparation). Final classification yielded around 300
genus-level taxa in total, and between 33 and 154 for
each sample (see Supporting Information Table S1 for
details).
Ordination analysis showed that the bacterial communities associated with the fibre fraction and fibre-free fraction of N. corniger differed strongly from each other and
from that of the luminal content (Fig. 5). Also in the case of
N. takasagoensis, the fibre fraction and fibre-free fraction
clustered separately, but the fibre fraction was not significantly separated from the luminal content. Communities
in replicate preparations of each fraction were highly
similar for both termites.
Closer inspection of the taxonomic composition of the
respective communities confirmed the distinct differences
between fibre and fibre-free fractions of both termites
(Fig. 6). In N. corniger, two termite-specific clusters of the
Fibrobacteres were strongly enriched (40.1% and 4.2%
mean relative abundance in fibre and fibre-free fractions,

respectively; P < 0.05 in a KruskalWallis test) as were

clusters of the TG3 phylum (12.1% and 2.7%, respectively; P < 0.05), which indicated an association with wood
particles. This is in agreement with the results of T-RFLP
analysis of the same samples.
In N. takasagoensis, the number of reads in the luminal
fluid assigned to the TG3 phylum was higher than that of
Fibrobacteres. Again, the relative abundance of the TG3
phylum was higher in the fibre fraction than in the fibrefree fraction (13.4% and 4.5%, respectively; P < 0.05),
corroborating the results of the T-RFLP analysis, which in
this case was even based on a different batch of termites.
Interestingly, the relative abundance of Fibrobacteres in
the pyrotag analysis was higher than in the T-RFLP analysis of N. takasagoensis, adding to the notion that the
fraction of Fibrobacteres in N. takasagoensis may vary
between batches (Khler et al., 2012). However, in contrast with N. corniger, the difference in abundance of
Fibrobacteres between fibre and fibre-free fractions was
not significant.
The distribution of Spirochaetes between fibre and
fibre-free fractions was similar in both termite species.
Of the several well-supported genus-level clusters in
Treponema cluster I, the fibre-free fractions from both
termite species had a higher percentage of sequences
binned to Treponema subclusters Ia and If (13.6% and
28.9%, respectively, in N. corniger, and 5.8% and 20.6%,
respectively, in N. takasagoensis). However, Sequences
binned to Treponema subcluster Ic were abundant also in
the fibre fraction of N. corniger and formed the most abundant group in the fibre fraction of N. takasagoensis
(40.1%).

NMDS axis 2

Fibrobacteres
subphylum 2

F Ff P3
N. corniger
N. takasagoensis

Stress = 0.07
NMDS axis 1

Fig. 5. Ordination analysis of community dissimilarity between P3

luminal contents, fibre-free fraction (Ff) and fibre fraction (F) (three
replicates each) from Nasutitermes corniger and N. takasagoensis.

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A. Mikaelyan et al.

0.001

0.1

10

N. corniger

N. takasagoensis

Relative abundance (%)

P3

Phylum

Family

Genus

Acidobacteria

Acidobacteriaceae
Holophagaceae
Sanguibacteraceae
Porphyromonadaceae
Rikenellaceae
Termite Cluster

Uncultured 23
Uncultured 3
Sanguibacter
Paludibacter
Alistipes 2
Termite Cluster I
Termite Cluster II
Gut Cluster 2
Uncultured 3
Uncultured 24
Turicibacter
Termite Cluster
Bradyrhizobium 12
Sphingomonas 3
Desulfovibrio 3
Higher termite cluster
Trinervitermes cluster a
Uncultured 4
Treponema Ia
Treponema Ic
Treponema Ie
Treponema If
Termite Cluster I
Nasutitermes Cluster

Actinobacteria
Bacteroidetes
Fibrobacteres
Firmicutes

Planctomycetes
Proteobacteria

Spirochaetes

TG3

Peptostreptococcaceae
Ruminococcaceae
Erysipelotrichaceae
Cluster IV
Bradyrhizobiaceae
Sphingomonadaceae
Desulfovibrionaceae
Cluster 1
Spirochaetaceae

Termite Cluster
Termite-Cockroach Cluster

Ff

P3

Ff

Fig. 6. Comparison of the relative abundance of genus-level bacterial groups in the pyrotag libraries of P3 lumen, fibre-free fraction (Ff) and
fibre fraction (F) from Nasutitermes corniger and N. takasagoensis. An interactive spreadsheet with the detailed classification results at all
taxonomic levels is provided in the supplementary material (Supporting Information Table S2).

Discussion
This study establishes the existence of a distinct fibreassociated bacterial community in the hindgut of woodfeeding higher termites. Using a newly developed method
that separates wood fibres and associated microorganisms from unattached cells in the luminal fluid, we showed
that the wood fibres in the hindgut of two Nasutitermes
species are specifically colonized by members of
Fibrobacteres, Spirochaetes and the TG3 phylum. The
fibre fraction of N. corniger contained almost half of the
cellulase activity in the luminal fluid, which indicated that
the fibre-associated community contributes substantially
to the cellulolytic activity in the hindgut.
The first indication of association of cellulolytic bacteria
to wood fibres was provided by Tokuda and colleagues
(2005), who found that most of the cellulolytic activity in
hindgut homogenates of N. takasagoensis was insoluble
and trapped in the pellet after centrifugation; it was
released into the supernatant only by sonication or detergent treatment (Tokuda and Watanabe, 2007). We
obtained similar results for N. corniger, where the particle-

associated activity in the P3 compartment amounted also

to almost 90% of the total cellulase activity in the hindgut
(Table 1). After density-gradient centrifugation, almost the
entire cellulase activity in the luminal fluid was present
either in the fibre fraction (45%) or in the fibre-free bacterial fraction (40%), indicating that (i) the wood particles
contain more than half of the cellulolytic activity in the
hindgut and (ii) the majority of the remaining activity is
associated with microbial cells. It is not clear whether the
latter activity belongs to genuinely unattached bacteria or
to cells that were separated from the wood fibres during
centrifugation. Likewise, it remains open whether the
activity in the fibre fraction originated from fibreassociated cells or from cellulases that are exclusively
bound to the fibre.
The fact that the proportion of the activity released after
detergent treatment is considerably larger in the fibre
fraction than in the fibre-free fraction clearly indicates that
insoluble cellulases are not only more abundant but also
more tightly bound in the bacteria adhering to the wood
particles. The absolute numbers may have to be regarded
with the appropriate caution because we cannot exclude

2014 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology

The fibre-associated community of higher termites

that the detergent also affects the activity of the extracted
cellulases, either by improving enzyme-substrate interactions or (less likely) by increasing the solubility of the
microcrystalline substrate (Eriksson et al., 2002).
The consistent presence of several bacterial groups in
the fibre fractions from both termites clearly indicates that
the wood particles are colonized by a specialized fibreassociated community. This is most obvious in the case of
the Fibrobacteres and the TG3 phylum, which were
almost exclusively encountered in the fibre fraction. In the
case of the spirochetes consistently colonizing the wood
particles, however, the majority of the respective populations were present in the fibre-free fraction.
The uncultured Fibrobacteres colonizing the guts of
Nasutitermes species and other termites fall into a wellsupported cluster (subphylum 2; Hongoh et al., 2006).
They are only distantly related to Fibrobacteres from
mammalian guts and other environments (subphylum 1),
which comprise the two cultured representatives of the
phylum. Fibrobacter succinogenes and F. intestinalis
are important cellulose degraders in herbivore guts
(Ransom-Jones et al., 2012). Fibrobacter succinogenes
attaches to plant fibre in the rumen and produces a variety
of glycosyl hydrolases that efficiently degrade even crystalline cellulose (Miron et al., 2001). Two homologues
encoding endoglucanases of glycosyl hydrolase family 9,
which includes the most prevalent cellulases in the
genome of F. succinogenes, are abundantly represented
in the metagenomes of Nasutitermes spp. (Warnecke
et al., 2007; He et al., 2013). The cellulolytic system
of F. succinogenes is not fully understood, but the lack
of genes encoding typical structural components of
cellulosomes, such as scaffoldins and dockerins, suggests that it differs fundamentally from that of cellulolytic
clostridia (Wilson, 2009). Nevertheless, colonization of
the cellulose fibres by F. succinogenes is a prerequisite
for efficient digestion (Kudo et al., 1987; Miron and
Forsberg, 1998; Jun et al., 2007). Interestingly, the
metagenomes of Nasutitermes spp. have been shown to
contain many genes with an extracytoplasmic domain
(IPR011871) that is commonly found in F. succinogenes
proteins and has been hypothesized to serve as a
functional analogue of cohesins (He et al., 2013). It
seems likely that the distant phylogenetic relatives of
F. succinogenes in termite guts are responsible for at least
part of the cellulase activity in the fibre fraction.
Bacteria from the TG3 phylum occur in high abundance
in wood-feeding higher termites of the genera Microcerotermes (Hongoh et al., 2006) and Nasutitermes
(Hongoh et al., 2006; Warnecke et al., 2007; Khler et al.,
2012). They are a sister group of Fibrobacteres (Hongoh
et al., 2006; Sorokin et al., 2013); some authors even
consider them members of the same phylum (Warnecke
et al., 2007; He et al., 2013). There are no representatives

in the rumen, but the first isolate of the TG3 phylum,

Chitinivibrio alkaliphilus, has recently been obtained from
a hypersaline soda lake. It can grow on chitin as the sole
carbon source, and its genome codes for numerous
glycosyl hydrolases from CAZy families that also comprise cellulases (Sorokin et al., 2013). Interestingly, the
chitinase activity produced by the cultures is not soluble
but cell associated. It is likely that also the distantly related
TG3 bacteria in termite guts contribute to the cellulase
activity in the fibre fraction.
Termite guts are colonized by a morphologically and
diverse assemblage of Spirochaetes (Breznak and
Pankratz, 1977; Hogan et al., 1988). The majority falls into
Treponema cluster I (Lilburn et al., 1999; Ohkuma et al.,
1999). Members of this cluster are found exclusively in
termite guts and account for as much as 70% of the 16S
rRNA genes of the hindgut community in Nasutitermes
spp. (Hongoh et al., 2006; Khler et al., 2012). Their
diversity has been studied in detail (Hongoh et al., 2005;
Warnecke et al., 2007). Phylogenetic analysis of these
sequences allows the identification of several distinct lineages (subclusters Iaf), which were used to refine the
reference database used for classification of the pyrotag
reads (A. Mikaelyan and A. Brune, unpubl. results).
Spirochetes from Treponema subcluster Ia dominate in
lower termites but are much less abundant in higher termites (Dietrich et al., 2014). The most abundant lineages
in the hindgut of Nasutitermes spp. are subclusters Ic and
If. In contrast with subcluster Ia, which did not show an
affinity to wood particles, subclusters Ic and If were consistently associated with the fibre fraction, where they
occur in similar proportions as Fibrobacteres and/or
member of the TG3 phylum. However, their abundant
presence in the fibre-free fraction underlines that they
may not be firmly associated with the wood particles. A
potential contribution of spirochetes to fibre digestion is
consistent with the finding that the metagenomes of
Nasutitermes spp. comprise numerous genes binned to
the genus Treponema that encode putative cellulases
belonging to various CAZy families (Warnecke et al.,
2007; He et al., 2013).
So far, subcluster Ia is the only lineage with cultured
representatives: Treponema primitia (Graber and Breznak,
2004; Graber et al., 2004), T. azotonutricium (Graber and
Breznak, 2004; Graber et al., 2004) and T. isoptericolens
(Drge et al., 2008). None of the three species has
been described as cellulolytic, but T. azotonutricium and
T. isoptericolens utilize cellobiose and oligosaccharide
breakdown products of cellulose (Graber and Breznak,
2004; Graber et al., 2004; Drge et al., 2008). Notably, corresponding enzyme activities have been found to be cellassociated (Drge et al., 2008), which would explain the
cell-associated cellulase activity observed in the fibre-free
fraction.

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A. Mikaelyan et al.

Based on the distinct physiological capabilities and

nutritional requirements even of closely related species
(Graber and Breznak, 2004; Graber et al., 2004), it is
likely that members of the different subclusters differ in
their metabolic potential. While some of them may be
directly involved in fibre digestion, others may rely on the
depolymerization products formed by cellulose-degrading
populations. The high motility of treponemes, combined
with an abundance of genes putatively involved in
chemotaxis (Warnecke et al., 2007), may preclude the
need to firmly associate with wood fibres in order to participate in the digestion process. Nevertheless, it may be
necessary or advantageous to make at least temporary
contact.
The abundance of spirochetes in the fibre-associated
communities is in agreement with the results of electron
microscopy, which documents the presence of spirilloid
bacteria on the wood particles in the hindgut of both
N. corniger and N. takasagoensis. However, at least
some of the numerous morphotypes colonizing the wood
particles most likely represent the fibre-associated lineages of Fibrobacteres and the TG3 phylum. Hongoh
and colleagues (2006) documented a spirilloid morphology for the bacterial cells in gut homogenates of
N. takasagoensis that hybridized with fluorescencelabelled oligonucleotide probes specific for the sequences
of Fibrobacteres and TG3. Their relative abundance
matched the proportion of Fibrobacteres and TG3
sequences in the corresponding clone library of the entire
hindgut. Although Hongoh and colleagues (2006) explicitly mention that the cells were not associated with wood
particles, it seems plausible that the cells became
detached during the fixation procedure. We found that
a direct observation of the bacteria colonizing the wood
particles by staining with fluorescent dyes is extremely
difficult because of the strong autofluorescence of
lignin.
Digestion of wood particles in the hindgut of lower termites is accomplished by cellulolytic flagellates, which fill
up the bulk of the hindgut volume and engulf the wood
particles as they pass through the enteric valve (Brune,
2014). The absence of free wood particles would explain
why cellulolytic bacteria seem to be of little importance
in cellulose degradation in lower termites. With the loss
of the flagellates by higher termites sometime in the
early Eocene (Engel et al., 2009), the wood particles
became accessible for microbial colonization, which also
opened a large niche for cellulolytic bacteria. The
obvious affinity of Fibrobacteres and TG3 phylum for
wood fibres and their presumed cellulolytic activity would
explain the enormous difference in their abundance
between lower termites and wood-feeding higher termites (Hongoh et al., 2006; C. Dietrich, T. Khler and A.
Brune, submitted).

Experimental procedures
Termites
Nasutitermes corniger stemmed from a colony maintained in
the laboratory of R. Scheffrahn, University of Florida.
Nasutitermes takasagoensis was collected on Iriomote
Island, Japan. Specimens were air-shipped to our laboratory
in Marburg and maintained for a few weeks on a diet of
birch wood and water. Worker termites were used for all
experiments.

Hindgut preparation
Termites were degutted with sterile fine-tipped forceps, and
the intact hindguts or the enlarged paunch (third proctodeal
compartment, P3) were separated from the rest of the gut
with a scalpel. For the cellulase activity assays, intact
hindguts or P3 compartments (five per replicate) were collected in 100 l protease inhibitor solution (cOmplete Mini
EDTA-free, Roche Molecular Biochemicals, Mannheim,
Germany). P3 luminal fluid was obtained by grazing 10
freshly dissected P3 compartments with a razor blade and
placing them in 100 l of PBS in a sterile tube to release the
contents into the buffer. The ruptured P3 compartments were
repeatedly aspirated with a pipette to release bacteria not
firmly bound to the gut wall, and the luminal content was
collected in a fresh tube.

Preparation of fibre and fibre-free fractions

Wood fibres were separated from unattached bacteria freely
suspended in the luminal fluid based on differences in
buoyant density using Percoll, an inert silica-based selfforming gradient material widely used for the isolation of
viable cells and cellular components (Pertoft, 2000). Percoll
concentration and centrifugation conditions were adjusted to
optimize the separation. Wood fibres were stained by mixing
each fraction with an equal volume of Toluidine Blue O solution (0.05% in 0.9 M NaCl), and all fractions were inspected
for wood fibres and bacterial cells using bright-field and
phase-contrast microscopy.
Optimal separation was achieved with the following protocol; nine parts of the original Percoll solution (GE Life Sciences, Munich, Germany) were mixed with one part of
10 PBS. Dilution of 83 ml of this solution with 17 ml 1 PBS
yielded the final working solution. One hundred microlitres of
P3 luminal fluid (prepared as described earlier) was mixed
with 2 ml Percoll working solution in 2 ml microcentrifuge
tubes precooled to 4C. The tubes were centrifuged at
20 000 g for 30 min in an Eppendorf centrifuge at 4C, yielding a turbid band of cells cushioned at the top of the gradient
(fibre-free fraction), which was collected from above (200 l).
The brown band of wood fibres near the bottom of the gradient was collected by puncturing the side of the tube and
withdrawing 200 l with a syringe needle (fibre fraction). Both
fractions were washed with three volumes of 1 PBS and
recovered in another centrifugation step. Washing and centrifugation were repeated three more times to remove
residual Percoll. For cellulase assays, the pellets were
resuspended in 100 l protease inhibitor solution. For DNA

2014 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology

The fibre-associated community of higher termites

extraction, the pellets were resuspended in 100 l of
1 PBS. Samples for lignin analysis were suspended in
water.
DNA content of the fractions was measured using a dyebinding assay specific for double-stranded DNA (Qubit,
Invitrogen, Karlsruhe, Germany) and used as a proxy for the
distribution of microbial biomass. Lignin content of the fractions was determined by measuring the absorbance at
490 nm after acid alcohol extraction and incubation with
phloroglucinol (Zimmer, 1999).

GCGGTGTGTACAA-3) (Thongaram et al., 2005); the

forward primer was labelled with fluorescein amidite. PCR
products were purified and digested with the restriction
enzyme TaqI (at 65C for 4 h). The digests were then analysed on an automated sequence analyser (ABI 3130;
Applied Biosystems, Carlsbad, California, USA). Details of
the procedure were as previously described (Schauer et al.,
2012).

Pyrotag sequencing of the 16S rRNA genes

Assay of cellulolytic activity
Cellulase activity was assayed as described by Tokuda and
Watanabe (2007). The samples were sonicated, and the particulate material was sedimented by centrifugation at
20 000 g for 10 min at 4C. The supernatants were collected
and are referred to as crude extracts. The centrifugation
pellet was washed three times with 100 l protease inhibitor
solution and finally re-suspended in 100 l CelLytic B (SigmaAldrich, Hamburg, Germany). The samples were vortexed for
15 s to release membrane-bound enzymes. After 10 min of
incubation on ice, the tubes were centrifuged to sediment the
debris, and the supernatant was collected and is referred to
as pellet extract. Crude or pellet extracts were incubated with
200 l of 2% microcrystalline cellulose (Sigma-Aldrich) in
McIlvaines buffer (pH 6.5) at 37C for 1 h with agitation
(1200 revolutions per minute) on a mixer (Eppendorf,
Hamburg, Germany). Reducing sugars formed during the
incubation were measured as previously described (Tokuda
et al., 2005).

DNA extraction
DNA was extracted from the P3 luminal contents and the
Percoll fractions using the method proposed by Zhou and
colleagues (1996) with some modifications. Briefly, the
samples were re-suspended in 1 ml of 1 PBS and mixed
with 675 l of extraction buffer (100 mM Tris-HCl pH 8.0,
100 mM Na3PO4, 100 mM Na4EDTA, 1.5 M NaCl, 1%
cetyltrimethylammonium bromide). Proteinase K treatment
was replaced by bead-beating with zirconium beads, followed
by addition of 75 l of 20% SDS and incubation in a heat
block at 65C for 1 h with periodic inversion of the tubes. The
remainder of the procedure followed the original protocol
(Zhou et al., 1996). DNA was precipitated with 0.6 volumes of
isopropanol.

Bacterial communities in P3 fluids from N. corniger and

N. takasagoensis, and the respective fibre and fibre-free fractions were analysed using pyrotag sequencing. 16S rRNA
genes were amplified using primers 343F and 753R, which
are optimized to improve coverage of bacterial taxa commonly found in insect guts (Khler et al., 2012). Amplicons
were mixed in equimolar amounts and sequenced commercially (454 GS FLX Titanium Technology; GATC Biotech,
Konstanz, Germany). Pyrotag sequence reads were
denoised using Acacia (version 1.52) to correct for homopolymer errors (Bragg et al., 2012), further processed for
quality under stringent conditions (reads > 200 bp, no
ambiguous bases, maximum number of homopolymers 8)
(Schloss et al., 2011) and aligned using the mothur software
suite (Schloss et al., 2009) (version 1.29.0). The entire data
set was submitted to the NCBI Short Read Archive (accession number SRP032939).

Classification of the pyrotag sequences

Sequence reads were taxonomically classified using the
Nave Bayesian Classifier implemented in mothur with a bootstrap value of 60% as cut-off. Classification success with
public reference databases was highest with the databases of
silva (http://www.arb-silva.de/) and the Ribosomal Database
Project (http://rdp.cme.msu.edu) but still limited because of
lack of taxonomic resolution in the groups represented in
termites and cockroaches. To improve resolution, we used a
customized reference database that is based on the silva
non-redundant database but contains a curated taxonomy to
improve genus-level classification, incorporating published
phylogenies of relevant groups and most recent clone libraries
that document hitherto unresolved monophyletic groups. The
reference database (DictDB v. 2.2) is available from the
authors upon request.

Statistical analyses
T-RFLP analysis
The bacterial community structure in the fibre fraction, fibrefree fraction and total P3 luminal contents of both N. corniger
and N. takasagoensis was studied using T-RFLP. The appropriate restriction enzyme for optimal resolution of the bacterial community members was chosen based on in-silico
analysis with the TRF-CUT program (Ricke et al., 2005), an
add-on for the ARB software package (Ludwig et al., 2004).
Bacterial 16S rRNA genes were amplified by polymerase
chain reaction (PCR) using primers U341F (5-CCTACG
GGRSGCAGCAG-3) (Baker, 2003) and 1390R (5-ACGG

The community similarity between different samples was estimated with the MorisitaHorn index and then visualized with
non-metric multidimensional scaling using the vegan package
(Oksanen et al., 2013) in the R software suite (version 3.0.1).
Taxa contributing the most to community dissimilarities were
identified using principal component analysis of the occurrence and abundance of genus-level taxa using the R software
suite, followed by ordering of the taxa based on the rotated
component loadings (Abdi and Williams, 2010). Differences in
the distribution of bacterial groups between the fractions were
assessed using the KruskalWallis test.

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10

A. Mikaelyan et al.

Scanning electron microscopy

The P3 contents of ten individuals of both N. corniger
and N. takasagoensis were fixed for 30 min in 2.5%
glutaraldehyde in 100 mM sodium phosphate buffer (pH 7.2).
After three rinses with the same buffer (15 min), the samples
were post-fixed on ice for 1 h in 1% OsO4 in buffer and
washed again three times (15 min). The samples were then
pipetted into small cups covered with plankton gauze and
dehydrated in a graded series of ethanol. After drying using a
Balzer CPD 030, the gut contents were coated with gold in a
Balzer SCD 040. The samples were examined with a FEI
Quanta 200 ESEM scanning electron microscope.

Acknowledgements
This study was supported by the Max Planck Society. AM
received a doctoral fellowship from the International Max
Planck Research School for Environmental, Cellular and
Molecular Microbiology. The authors thank Rudolf Scheffrahn
for providing N. corniger, Katja Meuser for excellent technical
assistance and Karen A. Brune for linguistic comments on the
manuscript.

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Supporting information
Additional Supporting Information may be found in the online
version of this article at the publishers web-site:

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A. Mikaelyan et al.

Table S1. Characteristics of 16S rRNA gene amplicon libraries obtained by pyrotag sequencing of the bacterial communities in different hindgut fractions of Nasutitermes corniger
and N. takasagoensis. Classification success is given for
selected taxonomic levels.

Table S2. Relative read abundance in the pyrotag libraries of

the bacterial communities in the different hindgut fractions of
Nasutitermes corniger and N. takasagoensis. The interactive
table allows to display classification results for different taxonomic levels (1, phylum; 2, class; 3, order; 4, family; 5, genus).

2014 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology