The fibre-associated cellulolytic bacterial community inthe hindgut of wood-feeding higher termites(Nasutitermes spp.)

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

Gaku Tokuda2 and Andreas Brune1*1Department of Biogeochemistry, Max Planck Institutefor Terrestrial Microbiology, Marburg, Germany.2Tropical Biosphere Research Center, COMB, Universityof the Ryukyus, Nishihara, Okinawa, Japan.

Summary

Termites digest lignocellulose with the help of theirsymbiotic gut microbiota. In the hindgut of evolution-ary lower termites, a dense community of cellulolyticflagellates sequesters wood particles from thehindgut content into their digestive vacuoles. Inhigher termites (family Termitidae), which possess anentirely prokaryotic microbiota, the wood particlesare available for bacterial colonization. Substantialparticle-associated cellulase activities have beendetected in the hindgut of Nasutitermes species, butthe microorganisms responsible for these activitiesand their potential association with the wood fibresremain to be studied. Here, we used density-gradientcentrifugation to separate wood fibres and adherentbacterial cells from cells freely suspended in thehindgut fluid. In Nasutitermes corniger, the fibre frac-tion contained 28% of the DNA and 45% of thecellulase activity in the luminal contents (P3 region).Community fingerprinting (terminal restriction frag-ment length polymorphism) and pyrotag sequencinganalysis of the bacterial 16S rRNA genes demon-strated that the wood fibres in the hindgut of bothN. corniger and N. takasagoensis are specificallycolonized by members of Fibrobacteres, the TG3phylum, and certain lineages of Spirochaetes charac-teristic of the gut microbiota of wood-feeding highertermites. We propose that the loss of flagellates inhigher termites provided a new niche for fibre-associated cellulolytic bacteria.

Introduction

Termites digest lignocellulose with the help of their sym-biotic gut microbiota (Brune, 2014). The breakdown ofingested wood particles is initiated by endogenouscellulases secreted by the salivary glands or the midguttissue, and completed in the voluminous hindgut, whichcarries the bulk of the symbionts (Watanabe and Tokuda,2010; Brune and Ohkuma, 2011; Ni and Tokuda, 2013). Inthe evolutionary basal lineages, also referred to as ‘lowertermites’, the microbial component of this dual cellulolyticsystem consists of a dense community of cellulolytic flag-ellates (Brugerolle and Radek, 2008; Ohkuma and Brune,2011). The flagellates sequester the wood particles intotheir digestive vacuoles and digest them with a suite ofglycosyl hydrolases, with little or no contribution byprokaryotes. However, cellulolytic flagellates are entirelyabsent in the evolutionary-derived lineage of ‘higher ter-mites’ (family Termitidae).

The loss of flagellates was accompanied by substantialchanges in the prokaryotic gut microbiota (Hongoh, 2011;Ohkuma and Brune, 2011), but the processes responsiblefor lignocellulose digestion in the hindgut of higher ter-mites are only poorly understood (reviewed by Hongoh,2011; Ni and Tokuda, 2013; Brune, 2014). While membersof the subfamily Macrotermitinae engaged in a uniquesymbiosis with a basidiomycete fungus, which breaksdown lignocellulosic substrates in fungal gardens beforethey are consumed by the termites, the members of othertermitid subfamilies must digest lignified plant fibreentirely within their digestive tract.

Although there are several reports of cellulase activitiesin hindgut homogenates of higher termites (Potts andHewitt, 1973; Rouland et al., 1986; Chararas and Noirot,1988), it was proposed that Nasutitermes species relymostly on endogenous cellulases acting in the foregut andmidgut, and that bacterial symbionts in the hindgut con-tributed at best marginally to cellulose digestion (Hoganet al., 1988; Slaytor, 1992). However, perception ofthe situation changed fundamentally with the discoverythat the cellulase activity in hindgut homogenates ofNasutitermes takasagoensis was not located in the particle-free supernatant but mostly in the post-centrifugation pellet,which had not been investigated in the earlier studies

Received 6 December, 2013; revised 21 January, 2014; accepted21 January, 2014. *For correspondence. E-mail brune@mpi-marburg.mpg.de; Tel. (+49) 6421 178 701; Fax (+49) 6421 178 999.

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Environmental Microbiology (2014) doi:10.1111/1462-2920.12425

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(Tokuda et al., 2005). Further examination of hindguthomogenates of N. takasagoensis and N. walkeri sug-gested that the particle-associated activity is that of cell-bound or fibre-associated microbial enzymes (Tokuda andWatanabe, 2007).

Support for this hypothesis came from metagenomicstudies, which identified numerous glycosyl hydrolasegenes encoding putative cellulases in metagenomiclibraries derived from the luminal contents of the enlargedP3 compartment of Nasutitermes spp. and Amitermeswheeleri (Warnecke et al., 2007; He et al., 2013). InNasutitermes spp., the majority of these genes areassigned to Fibrobacteres and Spirochaetes (Warneckeet al., 2007), two bacterial phyla abundantly representedin the hindgut of wood-feeding higher termites (Hongohet al., 2006; Köhler et al., 2012). In the dung-feedingA. wheeleri, the community is instead dominated byFirmicutes (He et al., 2013). It is noteworthy that in bothcases, the metagenomes showed an overrepresentationof gene functions indicating the presence of substrate-binding, cell-associated cellulase complexes; while thehindgut metagenome of A. wheeleri encodes for an abun-dance of putative cohesins and dockerins typical ofclostridial cellulosomes, those of Nasutitermes spp. com-prised numerous homologues presumably encodingextracytoplasmic proteins with cohesin-like function inFibrobacter succinogenes (He et al., 2013).

In all ruminants, the bacteria contributing most impor-tantly to fibre digestion are from the Fibrobacteres andFirmicutes (order Clostridiales). Their firm attachmentto food particles by extracellular cellulosomes (inRuminococcus flavefaciens) or their functional analogues(in F. succinogenes) greatly increases the efficiency ofdegradation and provides substrates for the entiremethanogenic feeding chain (Flint et al., 2008). The foodparticles in the rumen are relatively large and easily sepa-rated from the rumen fluid by simple filtration (Koike et al.,2003; Brulc et al., 2011), which has allowed extensivestudies of the structure and activities of ruminal fibre-associated communities (Brulc et al., 2009; Kim et al.,2011).

In the case of termites, however, the grinding actionof mandibles and gizzard break down the wood toparticle sizes that overlap those of the larger hindgutbacteria (Tokuda et al., 2012). Apart from preliminaryultrastructural evidence of microbial colonization (Tokudaet al., 2005), nothing is known about the existence orcomposition of a fibre-associated community in thehindgut of higher termites and its cellulolytic activities. Inthis study, we developed a new method that allows differ-entiating between fibre-associated and only cell-associated activities. By separating wood fibres andattached bacterial cells from cells freely suspended in theluminal hindgut fluid, we determined the contributions

of the different fractions to cellulose hydrolysis inN. corniger. In addition, we characterized the fibre-associated bacterial community of N. corniger and N.takasagoensis by terminal restriction fragment length poly-morphism (T-RFLP) analysis and 454-pyrosequencing ofbacterial 16S rRNA genes in the respective fractions.

Results

Distribution of cellulase activity in the hindgut

Homogenization of N. corniger hindguts with micro-pestles released only about 10% of the total cellulaseactivity into the supernatant (Table 1). The activityreleased by sonication was considerably higher, but morethan half of the total activity still remained in the pellet andwas liberated only by detergent extraction (CelLytic B).While the activity released by homogenization may rep-resent (at least in part) soluble cellulases in the hindgutfluid, the additional activity released by sonication andthe residual activity in the pellet were considered to beassociated with the particulate fraction. High particle-associated activities have been reported already in a pre-vious study of N. takasagoensis (Tokuda and Watanabe,2007). We obtained essentially the same results witha different batch of N. takasagoensis, except that theabsolute activities were lower. Comparison of theresults obtained for total hindguts and P3 compartmentindicated that in both termite species, most of thecellulase activity is confined to P3, the enlarged hindgutpaunch (Table 1).

Density-dependent enrichment of wood fibresfrom the P3 lumen

Scanning electron microscopy revealed that most woodparticles are densely colonized by various types of fila-mentous or spiral-shaped bacterial cells (Fig. 1). The sizeof most wood particles in the hindgut paunch ofN. corniger ranged between 10 and 50 μm; only a fewwere > 100 μm in length (Fig. 2). The average particlesize of wood fibres in the hindgut paunch (25 ± 18 μm)was significantly smaller than that in the crop (124 ±69 μm) and the midgut (128 ± 66 μm).

Fractionation of the luminal content by buoyant densityusing Percoll-gradient centrifugation yielded two well-separated bands, with the bulk of the wood particles con-centrated near the bottom of the tube (fibre fraction) andthe unattached cells close to the meniscus (fibre-freefraction; Fig. 3A). The fibre fraction contained 83% of thelignin and 28% of the DNA in the sample, whereas thefibre-free fraction contained only 17% of the lignin but68% of the DNA in the P3 lumen, which indicated that asubstantial part of the gut microbiota in the P3 contents is

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associated with wood particles (Fig. 3B). Inspection ofToluidine-Blue-stained preparations by bright-field andphase-contrast microscopy confirmed that the fibre frac-tion consisted mostly of wood particles and only fewunassociated cells, whereas the fibre-free fraction con-tained many suspended bacteria and only few smallerwood particles. The size distribution of wood particlesin the fibre fraction was virtually identical to that in theP3 fluid (Fig. 2), which indicated that the separation

procedure is not biased against wood particles of a par-ticular size.

When we collected wood particles and microbial cells inthe different fractions by centrifugation, we recovered45% of the cellulase activity in the P3 luminal fluid fromthe fibre fraction and 40% of the activity from the fibre-freefraction (the sum of the activities released by sonicationand subsequent detergent treatment; Fig. 3C). The pro-portion of the activity released only after detergent treat-

Table 1. Cellulase activities present in the supernatant after homogenization or sonication of entire hindguts or P3 compartments of Nasutitermesspecies and after detergent extraction of the sonicated pellet.

Species Compartment

Cellulase activitya in supernatant (units g−1)Particle-associatedactivityfHomogenizationb Sonicationc Detergent treatmentd Totale

N. corniger Hindgut 0.023 ± 0.009 0.069 ± 0.024 0.141 ± 0.025 0.210 ± 0.049 89%P3 0.017 ± 0.008 0.099 ± 0.004 0.122 ± 0.022 0.221 ± 0.026 92%

N. takasagoensis Hindgut 0.014 ± 0.053g 0.039 ± 0.02h 0.058 ± 0.012h 0.097 ± 0.032h 86%Hindgut n.d.i 0.028 ± 0.005 0.026 ± 0.005 0.054 ± 0.010 82%P3 n.d. 0.029 ± 0.004 0.020 ± 0.008 0.049 ± 0.012 80%

a. One unit of enzyme activity is defined as the amount of enzyme required to release 1 μmol of reducing sugar equivalents per minute frommicrocrystalline 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 termiteseach) ± standard error.

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

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

ment was significantly higher in the fibre fraction(P < 0.05, Kruskal–Wallis test).

T-RFLP analysis of the samples revealed that the bac-terial community in the luminal contents of N. corniger wasunevenly distributed between the two fractions. In all rep-

licates, two of the major peaks in the luminal fluid (144 and400 bp) were recovered almost exclusively from the fibrefraction, whereas the dominant terminal restriction frag-ment (T-RF) (618 bp) was present in both fractions (Fig. 4).Using the predicted T-RFs of the sequences from clonelibraries (Hongoh et al., 2006), we tentatively assignedthem to TG3 phylum, Fibrobacteres and Spirochaetes.

When we fractionated the P3 luminal fluid ofN. takasagoensis in the same manner, the resultingT-RFLP profiles were similar to those of N. corniger,except that the peak representing Fibrobacteres subphy-lum 2 was quite small already in the luminal fluid (Fig. 4).Again, the dominant T-RF of 618 bp was recovered fromboth fractions, representing a large number of unresolvedphylotypes in the Treponema Cluster I.

Pyrotag analysis

To increase the taxonomic resolution of the communityanalysis, we amplified the V3–V4 region (about 450 bp) ofthe 16S rRNA genes in luminal fluid, fibre fraction andfibre-free fraction of the two termite species and analysedthe products by 454-pyrosequencing. The sequences(5000–10 000 reads per sample) were classified against acomprehensive database that includes all homologuespreviously obtained from insect guts and allows identifi-cation of groups not yet resolved in public databases,such as the termite-specific lineages in the TG3 phylumand the Fibrobacteres (Köhler et al., 2012). We further

0 50 100 150 200

0

0.5

0.5 P3 lumenFibre-fraction

Length of wood fibres (μm)

Freq

uenc

y

Fig. 2. Histogram comparison of the length distribution of woodfibres in the P3 fluid (top) and fibre fraction (bottom) obtained fromNasutitermes corniger.

Fibre-freefraction

Fibrefraction

0 50 100

A C

0 50 100

Recovery (%)

LigninDNA

B

DetergentSonicated

Cellulase activity (%)

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 thesonication pellet, relative to the total activity in the luminal fluid (C).

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improved the classification of the diverse phylotypes inthe Treponema cluster I that were not resolved by theT-RFLP analysis by adding numerous unpublished near-full-length sequences from both lower and higher termitesand sequences available in public databases to our ref-erence database (A. Mikaelyan, T. Köhler and A. Brune, inpreparation). Final classification yielded around 300genus-level taxa in total, and between 33 and 154 foreach sample (see Supporting Information Table S1 fordetails).

Ordination analysis showed that the bacterial commu-nities associated with the fibre fraction and fibre-free frac-tion of N. corniger differed strongly from each other andfrom that of the luminal content (Fig. 5). Also in the case ofN. takasagoensis, the fibre fraction and fibre-free fractionclustered separately, but the fibre fraction was not signifi-cantly separated from the luminal content. Communitiesin replicate preparations of each fraction were highlysimilar for both termites.

Closer inspection of the taxonomic composition of therespective communities confirmed the distinct differencesbetween fibre and fibre-free fractions of both termites(Fig. 6). In N. corniger, two termite-specific clusters of theFibrobacteres were strongly enriched (40.1% and 4.2%mean relative abundance in fibre and fibre-free fractions,

respectively; P < 0.05 in a Kruskal–Wallis test) as wereclusters of the TG3 phylum (12.1% and 2.7%, respec-tively; P < 0.05), which indicated an association with woodparticles. This is in agreement with the results of T-RFLPanalysis of the same samples.

In N. takasagoensis, the number of reads in the luminalfluid assigned to the TG3 phylum was higher than that ofFibrobacteres. Again, the relative abundance of the TG3phylum was higher in the fibre fraction than in the fibre-free fraction (13.4% and 4.5%, respectively; P < 0.05),corroborating the results of the T-RFLP analysis, which inthis case was even based on a different batch of termites.Interestingly, the relative abundance of Fibrobacteres inthe pyrotag analysis was higher than in the T-RFLP analy-sis of N. takasagoensis, adding to the notion that thefraction of Fibrobacteres in N. takasagoensis may varybetween batches (Köhler et al., 2012). However, in con-trast with N. corniger, the difference in abundance ofFibrobacteres between fibre and fibre-free fractions wasnot significant.

The distribution of Spirochaetes between fibre andfibre-free fractions was similar in both termite species.Of the several well-supported genus-level clusters inTreponema cluster I, the fibre-free fractions from bothtermite species had a higher percentage of sequencesbinned to Treponema subclusters Ia and If (13.6% and28.9%, respectively, in N. corniger, and 5.8% and 20.6%,respectively, in N. takasagoensis). However, Sequencesbinned to Treponema subcluster Ic were abundant also inthe fibre fraction of N. corniger and formed the most abun-dant group in the fibre fraction of N. takasagoensis(40.1%).

TG3Fibrobacteressubphylum 2 Treponema I

Rel

ativ

e ab

unda

nce

400200 600

Fragment size (bp)

P3

P3

Ff

F

Ff

F

N. c

orni

ger

N. t

akas

agoe

nsis

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

N. cornigerN. takasagoensis

P3FfF

NMDS axis 1

NM

DS

axi

s 2

Stress = 0.07

Fig. 5. Ordination analysis of community dissimilarity between P3luminal contents, fibre-free fraction (Ff) and fibre fraction (F) (threereplicates each) from Nasutitermes corniger and N. takasagoensis.

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Discussion

This study establishes the existence of a distinct fibre-associated bacterial community in the hindgut of wood-feeding higher termites. Using a newly developed methodthat separates wood fibres and associated microorgan-isms from unattached cells in the luminal fluid, we showedthat the wood fibres in the hindgut of two Nasutitermesspecies are specifically colonized by members ofFibrobacteres, Spirochaetes and the TG3 phylum. Thefibre fraction of N. corniger contained almost half of thecellulase activity in the luminal fluid, which indicated thatthe fibre-associated community contributes substantiallyto the cellulolytic activity in the hindgut.

The first indication of association of cellulolytic bacteriato wood fibres was provided by Tokuda and colleagues(2005), who found that most of the cellulolytic activity inhindgut homogenates of N. takasagoensis was insolubleand trapped in the pellet after centrifugation; it wasreleased into the supernatant only by sonication or deter-gent treatment (Tokuda and Watanabe, 2007). Weobtained similar results for N. corniger, where the particle-

associated activity in the P3 compartment amounted alsoto almost 90% of the total cellulase activity in the hindgut(Table 1). After density-gradient centrifugation, almost theentire cellulase activity in the luminal fluid was presenteither in the fibre fraction (45%) or in the fibre-free bacte-rial fraction (40%), indicating that (i) the wood particlescontain more than half of the cellulolytic activity in thehindgut and (ii) the majority of the remaining activity isassociated with microbial cells. It is not clear whether thelatter activity belongs to genuinely unattached bacteria orto cells that were separated from the wood fibres duringcentrifugation. Likewise, it remains open whether theactivity in the fibre fraction originated from fibre-associated cells or from cellulases that are exclusivelybound to the fibre.

The fact that the proportion of the activity released afterdetergent treatment is considerably larger in the fibrefraction than in the fibre-free fraction clearly indicates thatinsoluble cellulases are not only more abundant but alsomore tightly bound in the bacteria adhering to the woodparticles. The absolute numbers may have to be regardedwith the appropriate caution because we cannot exclude

Acidobacteria

ActinobacteriaBacteroidetes

Fibrobacteres

Firmicutes

PlanctomycetesProteobacteria

Spirochaetes

TG3

AcidobacteriaceaeHolophagaceaeSanguibacteraceaePorphyromonadaceaeRikenellaceaeTermite Cluster

PeptostreptococcaceaeRuminococcaceaeErysipelotrichaceaeCluster IVBradyrhizobiaceaeSphingomonadaceaeDesulfovibrionaceaeCluster 1Spirochaetaceae

Termite ClusterTermite-Cockroach Cluster

Uncultured 23Uncultured 3SanguibacterPaludibacterAlistipes 2Termite Cluster ITermite Cluster IIGut Cluster 2Uncultured 3Uncultured 24TuricibacterTermite ClusterBradyrhizobium 12Sphingomonas 3Desulfovibrio 3Higher termite clusterTrinervitermes cluster aUncultured 4Treponema IaTreponema IcTreponema IeTreponema IfTermite Cluster INasutitermes Cluster

N. takasagoensisN. corniger

FFfP3 FFfP3

0.001 0.1 10Relative abundance (%)

Phylum Family Genus

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

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that the detergent also affects the activity of the extractedcellulases, either by improving enzyme-substrate interac-tions or (less likely) by increasing the solubility of themicrocrystalline substrate (Eriksson et al., 2002).

The consistent presence of several bacterial groups inthe fibre fractions from both termites clearly indicates thatthe wood particles are colonized by a specialized fibre-associated community. This is most obvious in the case ofthe Fibrobacteres and the TG3 phylum, which werealmost exclusively encountered in the fibre fraction. In thecase of the spirochetes consistently colonizing the woodparticles, however, the majority of the respective popula-tions were present in the fibre-free fraction.

The uncultured Fibrobacteres colonizing the guts ofNasutitermes species and other termites fall into a well-supported cluster (subphylum 2; Hongoh et al., 2006).They are only distantly related to Fibrobacteres frommammalian guts and other environments (subphylum 1),which comprise the two cultured representatives of thephylum. Fibrobacter succinogenes and F. intestinalisare important cellulose degraders in herbivore guts(Ransom-Jones et al., 2012). Fibrobacter succinogenesattaches to plant fibre in the rumen and produces a varietyof glycosyl hydrolases that efficiently degrade even crys-talline cellulose (Miron et al., 2001). Two homologuesencoding endoglucanases of glycosyl hydrolase family 9,which includes the most prevalent cellulases in thegenome of F. succinogenes, are abundantly representedin the metagenomes of Nasutitermes spp. (Warneckeet al., 2007; He et al., 2013). The cellulolytic systemof F. succinogenes is not fully understood, but the lackof genes encoding typical structural components ofcellulosomes, such as scaffoldins and dockerins, sug-gests that it differs fundamentally from that of cellulolyticclostridia (Wilson, 2009). Nevertheless, colonization ofthe cellulose fibres by F. succinogenes is a prerequisitefor efficient digestion (Kudo et al., 1987; Miron andForsberg, 1998; Jun et al., 2007). Interestingly, themetagenomes of Nasutitermes spp. have been shown tocontain many genes with an extracytoplasmic domain(IPR011871) that is commonly found in F. succinogenesproteins and has been hypothesized to serve as afunctional analogue of cohesins (He et al., 2013). Itseems likely that the distant phylogenetic relatives ofF. succinogenes in termite guts are responsible for at leastpart of the cellulase activity in the fibre fraction.

Bacteria from the TG3 phylum occur in high abundancein wood-feeding higher termites of the genera Micro-cerotermes (Hongoh et al., 2006) and Nasutitermes(Hongoh et al., 2006; Warnecke et al., 2007; Köhler et al.,2012). They are a sister group of Fibrobacteres (Hongohet al., 2006; Sorokin et al., 2013); some authors evenconsider them members of the same phylum (Warneckeet 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 froma hypersaline soda lake. It can grow on chitin as the solecarbon source, and its genome codes for numerousglycosyl hydrolases from CAZy families that also com-prise cellulases (Sorokin et al., 2013). Interestingly, thechitinase activity produced by the cultures is not solublebut cell associated. It is likely that also the distantly relatedTG3 bacteria in termite guts contribute to the cellulaseactivity in the fibre fraction.

Termite guts are colonized by a morphologically anddiverse assemblage of Spirochaetes (Breznak andPankratz, 1977; Hogan et al., 1988). The majority falls intoTreponema cluster I (Lilburn et al., 1999; Ohkuma et al.,1999). Members of this cluster are found exclusively intermite guts and account for as much as 70% of the 16SrRNA genes of the hindgut community in Nasutitermesspp. (Hongoh et al., 2006; Köhler et al., 2012). Theirdiversity has been studied in detail (Hongoh et al., 2005;Warnecke et al., 2007). Phylogenetic analysis of thesesequences allows the identification of several distinct lin-eages (subclusters Ia–f), which were used to refine thereference database used for classification of the pyrotagreads (A. Mikaelyan and A. Brune, unpubl. results).

Spirochetes from Treponema subcluster Ia dominate inlower termites but are much less abundant in higher ter-mites (Dietrich et al., 2014). The most abundant lineagesin the hindgut of Nasutitermes spp. are subclusters Ic andIf. In contrast with subcluster Ia, which did not show anaffinity to wood particles, subclusters Ic and If were con-sistently associated with the fibre fraction, where theyoccur in similar proportions as Fibrobacteres and/ormember of the TG3 phylum. However, their abundantpresence in the fibre-free fraction underlines that theymay not be firmly associated with the wood particles. Apotential contribution of spirochetes to fibre digestion isconsistent with the finding that the metagenomes ofNasutitermes spp. comprise numerous genes binned tothe genus Treponema that encode putative cellulasesbelonging to various CAZy families (Warnecke et al.,2007; He et al., 2013).

So far, subcluster Ia is the only lineage with culturedrepresentatives: Treponema primitia (Graber and Breznak,2004; Graber et al., 2004), T. azotonutricium (Graber andBreznak, 2004; Graber et al., 2004) and T. isoptericolens(Dröge et al., 2008). None of the three species hasbeen described as cellulolytic, but T. azotonutricium andT. isoptericolens utilize cellobiose and oligosaccharidebreakdown products of cellulose (Graber and Breznak,2004; Graber et al., 2004; Dröge et al., 2008). Notably, cor-responding enzyme activities have been found to be cell-associated (Dröge et al., 2008), which would explain thecell-associated cellulase activity observed in the fibre-freefraction.

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Based on the distinct physiological capabilities andnutritional requirements even of closely related species(Graber and Breznak, 2004; Graber et al., 2004), it islikely that members of the different subclusters differ intheir metabolic potential. While some of them may bedirectly involved in fibre digestion, others may rely on thedepolymerization products formed by cellulose-degradingpopulations. The high motility of treponemes, combinedwith an abundance of genes putatively involved inchemotaxis (Warnecke et al., 2007), may preclude theneed to firmly associate with wood fibres in order to par-ticipate in the digestion process. Nevertheless, it may benecessary or advantageous to make at least temporarycontact.

The abundance of spirochetes in the fibre-associatedcommunities is in agreement with the results of electronmicroscopy, which documents the presence of spirilloidbacteria on the wood particles in the hindgut of bothN. corniger and N. takasagoensis. However, at leastsome of the numerous morphotypes colonizing the woodparticles most likely represent the fibre-associated line-ages of Fibrobacteres and the TG3 phylum. Hongohand colleagues (2006) documented a spirilloid morphol-ogy for the bacterial cells in gut homogenates ofN. takasagoensis that hybridized with fluorescence-labelled oligonucleotide probes specific for the sequencesof Fibrobacteres and TG3. Their relative abundancematched the proportion of Fibrobacteres and TG3sequences in the corresponding clone library of the entirehindgut. Although Hongoh and colleagues (2006) explic-itly mention that the cells were not associated with woodparticles, it seems plausible that the cells becamedetached during the fixation procedure. We found thata direct observation of the bacteria colonizing the woodparticles by staining with fluorescent dyes is extremelydifficult because of the strong autofluorescence oflignin.

Digestion of wood particles in the hindgut of lower ter-mites is accomplished by cellulolytic flagellates, which fillup the bulk of the hindgut volume and engulf the woodparticles as they pass through the enteric valve (Brune,2014). The absence of free wood particles would explainwhy cellulolytic bacteria seem to be of little importancein cellulose degradation in lower termites. With the lossof the flagellates by higher termites sometime in theearly Eocene (Engel et al., 2009), the wood particlesbecame accessible for microbial colonization, which alsoopened a large niche for cellulolytic bacteria. Theobvious affinity of Fibrobacteres and TG3 phylum forwood fibres and their presumed cellulolytic activity wouldexplain the enormous difference in their abundancebetween lower termites and wood-feeding higher ter-mites (Hongoh et al., 2006; C. Dietrich, T. Köhler and A.Brune, submitted).

Experimental procedures

Termites

Nasutitermes corniger stemmed from a colony maintained inthe laboratory of R. Scheffrahn, University of Florida.Nasutitermes takasagoensis was collected on IriomoteIsland, Japan. Specimens were air-shipped to our laboratoryin Marburg and maintained for a few weeks on a diet ofbirch wood and water. Worker termites were used for allexperiments.

Hindgut preparation

Termites were degutted with sterile fine-tipped forceps, andthe intact hindguts or the enlarged paunch (third proctodealcompartment, P3) were separated from the rest of the gutwith a scalpel. For the cellulase activity assays, intacthindguts or P3 compartments (five per replicate) were col-lected in 100 μl protease inhibitor solution (cOmplete MiniEDTA-free, Roche Molecular Biochemicals, Mannheim,Germany). P3 luminal fluid was obtained by grazing 10freshly dissected P3 compartments with a razor blade andplacing them in 100 μl of PBS in a sterile tube to release thecontents into the buffer. The ruptured P3 compartments wererepeatedly aspirated with a pipette to release bacteria notfirmly bound to the gut wall, and the luminal content wascollected in a fresh tube.

Preparation of fibre and fibre-free fractions

Wood fibres were separated from unattached bacteria freelysuspended in the luminal fluid based on differences inbuoyant density using Percoll, an inert silica-based self-forming gradient material widely used for the isolation ofviable cells and cellular components (Pertoft, 2000). Percollconcentration and centrifugation conditions were adjusted tooptimize the separation. Wood fibres were stained by mixingeach fraction with an equal volume of Toluidine Blue O solu-tion (0.05% in 0.9 M NaCl), and all fractions were inspectedfor wood fibres and bacterial cells using bright-field andphase-contrast microscopy.

Optimal separation was achieved with the following proto-col; nine parts of the original Percoll solution (GE Life Sci-ences, Munich, Germany) were mixed with one part of10 × PBS. Dilution of 83 ml of this solution with 17 ml 1 × PBSyielded the final working solution. One hundred microlitres ofP3 luminal fluid (prepared as described earlier) was mixedwith 2 ml Percoll working solution in 2 ml microcentrifugetubes precooled to 4°C. The tubes were centrifuged at20 000 g for 30 min in an Eppendorf centrifuge at 4°C, yield-ing a turbid band of cells cushioned at the top of the gradient(fibre-free fraction), which was collected from above (200 μl).The brown band of wood fibres near the bottom of the gra-dient was collected by puncturing the side of the tube andwithdrawing 200 μl with a syringe needle (fibre fraction). Bothfractions were washed with three volumes of 1 × PBS andrecovered in another centrifugation step. Washing and cen-trifugation were repeated three more times to removeresidual Percoll. For cellulase assays, the pellets wereresuspended in 100 μl protease inhibitor solution. For DNA

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extraction, the pellets were resuspended in 100 μl of1 × PBS. Samples for lignin analysis were suspended inwater.

DNA content of the fractions was measured using a dye-binding assay specific for double-stranded DNA (Qubit,Invitrogen, Karlsruhe, Germany) and used as a proxy for thedistribution of microbial biomass. Lignin content of the frac-tions was determined by measuring the absorbance at490 nm after acid alcohol extraction and incubation withphloroglucinol (Zimmer, 1999).

Assay of cellulolytic activity

Cellulase activity was assayed as described by Tokuda andWatanabe (2007). The samples were sonicated, and the par-ticulate material was sedimented by centrifugation at20 000 g for 10 min at 4°C. The supernatants were collectedand are referred to as crude extracts. The centrifugationpellet was washed three times with 100 μl protease inhibitorsolution and finally re-suspended in 100 μl CelLytic B (Sigma-Aldrich, Hamburg, Germany). The samples were vortexed for15 s to release membrane-bound enzymes. After 10 min ofincubation on ice, the tubes were centrifuged to sediment thedebris, and the supernatant was collected and is referred toas pellet extract. Crude or pellet extracts were incubated with200 μl of 2% microcrystalline cellulose (Sigma-Aldrich) inMcIlvaine’s buffer (pH 6.5) at 37°C for 1 h with agitation(1200 revolutions per minute) on a mixer (Eppendorf,Hamburg, Germany). Reducing sugars formed during theincubation were measured as previously described (Tokudaet al., 2005).

DNA extraction

DNA was extracted from the P3 luminal contents and thePercoll fractions using the method proposed by Zhou andcolleagues (1996) with some modifications. Briefly, thesamples were re-suspended in 1 ml of 1 × PBS and mixedwith 675 μl 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 treatmentwas replaced by bead-beating with zirconium beads, followedby addition of 75 μl of 20% SDS and incubation in a heatblock at 65°C for 1 h with periodic inversion of the tubes. Theremainder of the procedure followed the original protocol(Zhou et al., 1996). DNA was precipitated with 0.6 volumes ofisopropanol.

T-RFLP analysis

The bacterial community structure in the fibre fraction, fibre-free fraction and total P3 luminal contents of both N. cornigerand N. takasagoensis was studied using T-RFLP. The appro-priate restriction enzyme for optimal resolution of the bacte-rial community members was chosen based on in-silicoanalysis with the TRF-CUT program (Ricke et al., 2005), anadd-on for the ARB software package (Ludwig et al., 2004).

Bacterial 16S rRNA genes were amplified by polymerasechain reaction (PCR) using primers U341F (5′-CCTACGGGRSGCAGCAG-3′) (Baker, 2003) and 1390R (5′-ACGG

GCGGTGTGTACAA-3′) (Thongaram et al., 2005); theforward primer was labelled with fluorescein amidite. PCRproducts were purified and digested with the restrictionenzyme TaqI (at 65°C for 4 h). The digests were then ana-lysed on an automated sequence analyser (ABI 3130;Applied Biosystems, Carlsbad, California, USA). Details ofthe procedure were as previously described (Schauer et al.,2012).

Pyrotag sequencing of the 16S rRNA genes

Bacterial communities in P3 fluids from N. corniger andN. takasagoensis, and the respective fibre and fibre-free frac-tions were analysed using pyrotag sequencing. 16S rRNAgenes were amplified using primers 343F and 753R, whichare optimized to improve coverage of bacterial taxa com-monly found in insect guts (Köhler et al., 2012). Ampliconswere mixed in equimolar amounts and sequenced commer-cially (454 GS FLX Titanium Technology; GATC Biotech,Konstanz, Germany). Pyrotag sequence reads weredenoised using Acacia (version 1.52) to correct for homo-polymer errors (Bragg et al., 2012), further processed forquality under stringent conditions (reads > 200 bp, noambiguous bases, maximum number of homopolymers ≤ 8)(Schloss et al., 2011) and aligned using the mothur softwaresuite (Schloss et al., 2009) (version 1.29.0). The entire dataset was submitted to the NCBI Short Read Archive (acces-sion number SRP032939).

Classification of the pyrotag sequences

Sequence reads were taxonomically classified using theNaïve Bayesian Classifier implemented in mothur with a boot-strap value of 60% as cut-off. Classification success withpublic reference databases was highest with the databases ofsilva (http://www.arb-silva.de/) and the Ribosomal DatabaseProject (http://rdp.cme.msu.edu) but still limited because oflack of taxonomic resolution in the groups represented intermites and cockroaches. To improve resolution, we used acustomized reference database that is based on the silvanon-redundant database but contains a curated taxonomy toimprove genus-level classification, incorporating publishedphylogenies of relevant groups and most recent clone librariesthat document hitherto unresolved monophyletic groups. Thereference database (DictDB v. 2.2) is available from theauthors upon request.

Statistical analyses

The community similarity between different samples was esti-mated with the Morisita–Horn index and then visualized withnon-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 wereidentified using principal component analysis of the occur-rence and abundance of genus-level taxa using the R softwaresuite, followed by ordering of the taxa based on the rotatedcomponent loadings (Abdi and Williams, 2010). Differences inthe distribution of bacterial groups between the fractions wereassessed using the Kruskal–Wallis test.

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Scanning electron microscopy

The P3 contents of ten individuals of both N. cornigerand 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 sampleswere post-fixed on ice for 1 h in 1% OsO4 in buffer andwashed again three times (15 min). The samples were thenpipetted into small cups covered with plankton gauze anddehydrated in a graded series of ethanol. After drying using aBalzer CPD 030, the gut contents were coated with gold in aBalzer SCD 040. The samples were examined with a FEIQuanta 200 ESEM scanning electron microscope.

Acknowledgements

This study was supported by the Max Planck Society. AMreceived a doctoral fellowship from the International MaxPlanck Research School for Environmental, Cellular andMolecular Microbiology. The authors thank Rudolf Scheffrahnfor providing N. corniger, Katja Meuser for excellent technicalassistance and Karen A. Brune for linguistic comments on themanuscript.

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Supporting information

Additional Supporting Information may be found in the onlineversion of this article at the publisher’s web-site:

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Table S1. Characteristics of 16S rRNA gene amplicon librar-ies obtained by pyrotag sequencing of the bacterial commu-nities in different hindgut fractions of Nasutitermes cornigerand N. takasagoensis. Classification success is given forselected taxonomic levels.

Table S2. Relative read abundance in the pyrotag libraries ofthe bacterial communities in the different hindgut fractions ofNasutitermes corniger and N. takasagoensis. The interactivetable allows to display classification results for different taxo-nomic levels (1, phylum; 2, class; 3, order; 4, family; 5, genus).

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