African Journal of Microbiology Research Vol. 6(11), pp. 2639-2648, 23 March, 2012 Available online at http://www.academicjournals.org/AJMR DOI: 10.5897/AJMR11.975 ISSN 1996-0808 ©2012 Academic Journals

Full Length Research Paper

Luminal hindgut bacterial diversities of the grass and sugarcane feeding Trinervitermes trinervoides

Tendai Walter Sanyika1,3*, Konanani Justice Rashamuse2#, Fritha Hennessy2 and Dean Brady1,2

1Department of Biotechnology and Food Technology, Tshwane University of Technology, Private Bag X680, Nelson Mandela Drive, Pretoria, 0001, South Africa. 2Protein Technologies, CSIR Biosciences, Meiring Naude Road, Brummeria 0091, Pretoria, South Africa. 3Current address; Department of Biochemistry, University of Zimbabwe, P.O. Box MP167, Mount Pleasant, Harare, Zimbabwe.

Accepted 19 December, 2011

Termites depend on intestinal fauna for digestion of lignocellulose, but the variations in gut bacterial diversity between species is poorly characterised. Bacterial diversities from the third proctodeal hindgut segments (P3, the paunch) of the grass and sugarcane feeding Trinervitermes trinervoides (: ) termites were compared using 16S rRNA gene pyrosequence analysis. A total of 2274 operational taxonomic units (OTUs) were identified from the sugarcane feeding termites at 99% sequence identity, and 2943 OTUs were identified from the grass feeding termites. The bacterial communities in both termites were dominated by Spirochaetes of the genus Treponema. The Bacteroidetes, Proteobacteria and Planctomycetes were more represented in the hindgut of the sugarcane feeding termite, whereas Acidobacteria and Firmicutes were more represented in the grass feeding termite. The Fibrobacteres had a slight correlatation with a woody diet. Planctomycetes and Acidobacteria correlated with sugarcane and grass, respectively, and were the key distinguishing bacterial functional groups between these termites of the same species. Termite hindgut bacterial diversity did not cluster according to diet but termite phylogeny; this suggests diet was not the main determinant of microbial diversity.

Key words: Termite hindgut, 16S rRNA gene, pyrosequencing, bacterial diversity, substrate preference.

INTRODUCTION

Termites depend on symbionts living in the gut for 2001; Donovan et al., 2001). However, within this family, efficient degradation of lignocellulose. All higher termites the members of the genus Trinervitermes (subfamily lack the eukaryotic cellulolytic symbionts, such as Nasutitermitinae) are predominantly efficient grass protozoa, that are associated with the lower termites. feeders that are widely distributed in savannas of West Instead they have evolved complex hind gut communities Africa, Southern Africa and Indomalaya (Adam et al., entirely composed of bacteria or in some cases fungi as 2008). Using behavioural studies, the feeding in the subfamily Macrotermitinae (Eggleton and Tayasu, preferences of Trinervitermes trinervoides were shown to 2001; Donovan et al., 2001). Members of the Termitidae be determined by physiochemical properties of the family are soil, wood, litter and grass feeding termites, substrate (Adam et al., 2005). These studies did not, typically found in tropical forests (Eggleton and Tayasu, however, specifically associate the termite hindgut bacterial diversity with diet. Brauman et al. (2001) focused their study on molecular level detection and profiling of prokaryotic groups using oligonucleotide *Corresponding author. E-mail: [email protected]. Tel: probe hybridization in termites with different feeding +263 778 195 570. habits. The study targeted group level diversity, mainly at the family level but did not include sequence based # Author contributed equally to the work. identification. 2640 Afr. J. Microbiol. Res.

The recent advent of high throughput sequencing manufacturers. The DNA was eluted in 10 mM Tris-buffered water technologies has revolutionalised metagenomic DNA (pH 8.0). A total of 12-15 μg high molecular weight metagenomic analysis (Armougom and Raoult, 2009; Droege and Hill, DNA was extracted from each of the two termites samples (Gilliespie et al., 2005). 2008; Kunin et al., 2008). The advantage of utilising high thoughput sequencing is that, it provides broader depth and coverage of bacterial diversity compared to clone PCR amplification libraries (Kröber et al., 2009; Kunin et al., 2008). There has been increasing interest in exploiting the termite gut The universal full length 16S rRNA gene primer sets selected, targeted for general bacterial diversity were combinations of E9F (5’ microorganisms and their genes, for commercial - GAGTTTGATCCTGGCTCAG) with U1510R (5’ - purposes, especially for enzymes that degrade plant GGTTACCTTGTTACGCATT) and of 27F (5’ - material (Li et al., 2009; Warnecke et al., 2007). It is AGAGTTTGATCMTGGCTCAG) with 1492R (5’ - therefore important to understand the nature of these TACGGHTACCTTGTTACGACTT), respectively. The first round full bacterial communities and to confirm if specific groups length PCR products from each sample were pooled for nested are associated with preferences for specific substrates in pyrosequencing of a common region. A combination of primer sets, EukA (5’–AACCTGGTTGATCCTGCCAGT) and EukB (5’– order to be able to exploit their functional capacity fully. TGATCCTTCTGCAGGTTCACCTAC), and of ITS1 (5’ – In this study, we reported the pyrosequencing analysis TCCGTAGGTGAACCTGCGG) and ITS4 (5’ – of the bacterial 16S rRNA gene diversity from the hindgut TCCTCCGCTTATTGATATGC) respectively, were used for the bacterial communities of grass-feeding and sugarcane- detection of eukaryotic 18S rRNA gene. All the amplifications were feeding Trinervitermes trinervoides termites. These carried out using polymerase chain reactions (PCR) involving one results were also compared to the bacterial populations initial denaturation cycle at 94°C, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 50 or 55°C for 30 s and from Nasutitermes and Microtermes. extension at 72°C for 1.5 min, followed by a final extension at 72°C for 10 min. A high fidelity DNA polymerase (Phusion, Finnzymes, Finland), was used with 100 ng of template DNA. MATERIALS AND METHODS

Termite collection Pyrosequencing

Termite mounds were collected from separate grass in Pretoria (S The full length 16S rRNA gene PCR products were resolved on 25° 75’ 26”, E 28° 26’ 81”) and sugarcane fields, in Komatipoort (S 0.8% low melting agarose and were extracted using a GeneJETTM 25° 23' 7.8", E 31° 52' 53.1"), South Africa in October 2009. The Gel extraction Kit (Fermentas, Lithuania). The products were mounds were of an easily excavatable size and were wrapped in pyrosequenced (Inqaba Biotech, South Africa) using the Titanium black plastic bags and kept in cooler boxes for up to 24 h in the 454 GS FLX/Titanium Sequencing Platform (Hoffmann-La Roche, laboratory prior to dissection. Germany). A nested PCR was performed using Eubacterial specific primers RW01 (5’ – AACTGGAGGAAGGTGGGGAT) and 1492R targeting the V8 to V9 hyper-variable region, which covers the Termite hindgut dissection region from 1170 to 1492 bp of the E. coli 16S rRNA gene. The adaptors, 5'-ACGAGTGCGT-3' and 5'-ACGCTCGACA-3', attached Luminal contents were sampled separately from the grass and to the pyrosequencing primers, were used as identifiers to different- sugarcane feeding termites, respectively, originating from tiate between the grass feeding and sugarcane feeding termite geographically distinct mounds. The largest hindgut compartment, hindgut 16S rRNA gene sequences respectively, allowing for the third proctodeal segment (P3 hindgut or paunch), was separate processing of results (Ronaghi et al., 1996, 1998). aseptically dissected and the luminal contents extracted from approximately 300 worker termites on ice. The luminal contents were pooled and re-suspended in a final volume of approximately 1 Sequence analysis and classification ml in TE buffer (pH 8.0). The samples were kept on ice prior to immediate DNA extraction. A total of 18 525 and 27 188 pyrosequences were generated from hindgut samples of the grass feeding and sugarcane feeding termites, respectively. The Greengenes DNA extraction (http://greengenes.lbl.gov/cgi-bin/nph-index.cgi) Nearest Alignment Space Tool (NAST) was used to identify chimeric sequences, Metagenomic DNA was extracted using the direct lysis method perform sequence alignments and assign closely related (Gilliespie et al., 2005), that is, using the ZR Genomic DNA II Kit™ sequences from 16S rRNA gene databases. Kit (Zymo Research, USA). Glass micro beads and three volumes A total of 12 413 and 8 687 good quality, full length pyro- of the lysis buffer were added to the gut content suspension. The sequences, averaging 300 bp, were selected from the grass and cells were mechanically lysed by bead beating for 60 s using the sugarcane feeding termites, respectively, using the RDP Disruptor Genie® (Scientific Industries, USA) followed by chemical pyrosequencing pipeline of the Ribosomal Database Project (RDP) lysis at room temperature for 30 min and centrifugation at 17,000 x (http://rdp.cme.msu.edu/) Release 10 (Cole et al., 2009). Classifi- g for 5 min. The supernatant was passed through a silica column cation of the newly generated sequences was based on their (provided in the kit) and eluted by centrifugation for 1 min, followed closest known cultured relatives in the RDP, according to the by washing using ethanol buffer as recommended by the nomenclatural taxonomy and Bergey's Manual, using the trained

Sanyika et al. 2641

RDP Naïve Bayesian rRNA Classifier (Wang et al., 2007), (Version 1492R, respectively. Eukaryotic rRNA genes were not 2.2, March 2010) of the RDP. The RDP was also used to compare detected using PCR (results not shown), indicating that the diversities between the libraries, alignment, cluster analysis and eukaryotic symbionts (fungi and protozoa) were not determination of OTUs. Removal of sequencing adaptors and all manual manipulations and correction of alignments were done present in the hindgut of these termites, nor was the using BioEdit software (Hall, 1999). material contaminated by termite DNA. Archaeal popu- lations were not investigated. In order to minimize PCR errors such as chimeras and DNA polymerase related Statistical analysis errors, a low number of PCR amplification cycles were

OTUs were determined at 97, 98, 99 and 100% similarity using the used according to Acinas et al. (2005). A high fidelity RDP based hierarchical clustering. The OTU statistics were used to DNA polymerase was used and precautions were taken calculate the Chao (Chao, 1987) and the Shannon Wiener diversity to remove low quality sequences from the analysis. indices (Shannon and Weaver, 1949) at 95% confidence interval, and for rarefaction analysis. The Multivariate Statistical Package v3.12a (Kovach Computing Services, United Kingdom) was used Estimation of OTUs and diversity richness for cluster analysis and correspondence analysis (CA). For cluster and correspondence analysis, the phylum level percentage diversities of termite hindgut bacteria published in literature A total of 2 274 OTUs were identified from 8 687 (Hongoh et al., 2005; Warnecke et al., 2007) were used. All the 16S sugarcane feeding termite sequences at 99% similarity rRNA gene sequences of the Nasutitermes species hindgut were (Table 1). The Chao non-parametric estimator (Chao, downloaded through the database project repository 1987), based on the singletons projected the number of (http://www.ncbi.nlm.nih.gov/genomeprj/19107). The sequences of expected OTUs at an average of 3 806. A total of 2 943 the representative OTUs quantified in the study were classified using the RDP classifier at 80% bootstraps, accounting for their OTUs were observed from 12 413 grass feeding termite abundance. For the Microcerotermes species, all the sequences hindgut sequences but were estimated at an average of (accession numbers AB191790 to AB192133) were downloaded 3 330 OTUs using the Chao estimator. In both cases, and classified in the same manner. The Nasutitermes and good coverage was obtained, but those bacterial groups Microcerotermes species data were used as comparative data for present as minor contributors to the population may not our results. The data was arranged as a matrix of percentage phylum have been detected (Figure 1). The rarefaction curves diversity for termites of different feeding substrates. The diversity tended to approach maximum diversity (rarefaction was then clustered using the Unweighted Pair Group Method with asymptote) at 97 and 98% sequence identity thresholds Arithmetic Mean (UPGMA) using the Bray Curtis coefficient with less OTUs observed. The diversity measures of dissimilarity matrix. For CA, the Kaiser’s rule (Legendre and evenness and richness between the two termites (Table Legendre, 1983) was used to identify the number of axes to extract 1) were comparable at all levels of similarity. based on a square root transformed data matrix, with outlier species removed and rare species down-weighted. Eigen analysis was based on the Hill reciprocal averaging algorithm and the eigenvalue scores were used for the conjoint ordination bi-plots. Phylogenetic classification of sequences

The short pyrosequence reads generated in this study RESULTS are unsuitable for phylogenetic detailed inference based on distances (Liu et al., 2007) but can be confidently PCR amplification of the termite hindgut 16S rRNA assigned to phylogenetic groups using the naïve gene diversity Bayesian rRNA classifier (Wang et al., 2007) (Figure 2). A total of ten phyla, Acidobacteria, Actinobacteria, In this study, the strategy involves amplifying full length Bacteroidetes, Deferribacteres, Firmicutes, 16S rRNA genes from a cell free metagenome library Planctomycetes, Proteobacteria, Spirochaetes, using two sets of universal primers, followed by a nested Synergistetes and Verrucomicrobia were identified in the PCR of a common region using one set of pyro- hindguts of the two termites (Figure 2). Unclassified sequencing primers. More than one set of universal bacterial sequences accounted for 4.9% in the hindgut of primers was initially used in order to increase the the sugarcane termite and 7.2% in the grass feeding coverage of bacterial diversity, since universal primers termite. Amongst the sequences identified from the usually fail to cover the diversity of members from all sugarcane feeding termite hindgut, approximately 83% known bacterial phyla (Forney et al., 2004). The aim of could be classified into twenty four genera. By contrast, obtaining full length regions was to ensure adequate for the grass feeding termite, 84% of the sequences coverage of the genetic material present. Approximately, could be classified into twenty seven genera (data not 1500 and 1465 bp 16S rRNA gene PCR products were shown). obtained using the primer pairs E9F-U1510R and 27F- Spirochaetes of the genus Treponema were predominant

2642 Afr. J. Microbiol. Res.

Table 1. The Chao and Shannon Wiener diversity estimates of bacterial diversity from the grass feeding and sugarcane feeding termites at difference sequence identity thresholds.

100% 99% 98% 97% Sg Gs Sg Gs Sg Gs Sg Gs N 8686 12413 8686 12413 8686 12413 8686 12413 OTUs 3723 4683 2274 2943 1223 1651 911 1168 chao 6967 5419 3806 3330 1839 1888 1353 1320 LCI95 6636 5334 3596 3270 1721 1840 1254 1283 UCI95 7334 5515 4051 3401 1985 1949 1481 1370 H' 6.99 7.32 6.15 6.50 4.99 5.44 4.57 4.81 varH 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 E 0.85 0.87 0.80 0.81 0.70 0.73 0.67 0.68

Sg = sugarcane feeding termite, Gs = grass feeding termite, N = number of sequences analyzed, *Lower limit (LCI95) and upper limit (ULCI95) Chao estimates at 95% confidence interval, H' = The Shannon Wiener diversity index (Shannon and Weaver, 1949), var H' = variance of H', E = The Shannon Wiener equitability index.

4000

3500 Unique 3000 99% 98% 2500 97%

2000

1500

No. of of observedOTUsNo. 1000

500

0 0 2000 4000 6000 8000 10000 (a) No. of sequences sampled

5000 4500 4000 3500 3000 2500 2000

1500 No. of of observedOTUsNo. 1000 500 0 0 2000 4000 6000 8000 10000 12000 14000 (b) No. of sequences sampled

Figure 1. Rarefaction curves depicting the number of OTUs, defined at different sequence identitity thresholds, observed with sampling effort between the hindguts of the (a) sugarcane and (b) grass feeding termites. Sanyika et al. 2643

Sugarcane (%)

Grass (%)

7.0 6.0 5.0 4.0

% Composition% 3.0 2.0 1.0 0.0

(a)

0.90 0.80 0.70 0.60 0.50 0.40 0.30

% Composition% 0.20 0.10 0.00

(b)

Figure 2. Distribution of non-Spirochaete bacterial sequences identified in the hindgut of a grass feeding and a sugarcane feeding Trinervitermes trinervoides termite using the RDP Classifier (Wang et al., 2007) Version 2.2 at 50% confidence limit (a) Phylum level classification and (b) the proportion classified to genus level. In both termites, Spirochaetes of the genus Treponema accounted for approximately 82%.

2644 Afr. J. Microbiol. Res.

in both termites, accounting for 82.4% of the sequences, from published sources (Hongoh et al., 2005; Warnecke which is exceptionally high compared to other termite gut et al., 2007). systems. Work done on the fungus-cultivating termite Macrotermes michaelseni had a far lower proportion of Spirochaetes, with the predominant genus being Correspondence analysis of termite diet with members of the Cytophaga-Flexibacter-Bacteriodes bacterial diversity group (MacKenzie et al., 2007). Similar results were seen Spirochaetes and Fibrobacteres were previously from a soil feeding termite species from Senegal (Fall et identified as the major bacterial sources of plant material al., 2007). The representation of Spirochaetes of the degrading genes in the hindgut of the wood feeding genera Spirochaeta and Turneriella was considerably Nasutitermes species through use of metagenomic lower in both termites, accounting for a total of only sequence data (Warnecke et al., 2007; Brune, 2007). The 0.06% (7 sequences). The twenty most dominant OTUs Fibrobacteres were the most critical phylum for in both termites were also Spirochaetes of the genus lignocellulose degradation, which is further supported by Treponema. This is consistent with other studies wherein the novel genes and efficient cellulolytic activities present the termite hindgut has been found to be dominated by in Fibrobacter succinogenes (Morrison et al., 2009). Spirochaetes (Hongoh, 2010). In the present study, correspondence analysis biplots Based on sequence identity, using the BLAST defined the majority association between the diet and algorithm (Altschul et al., 1990), most of the identical bacterial diversity (71%) on the first axis (Figure 3a), and sequences retrieved from 16S rRNA gene databases only 18% of the correlations occurred on the second axis. were typically from anaerobic environments, especially The most notable correlations were between the other termite and hindgut samples. The closely Fibrobacteres (coefficient, 0.97), Synergistes (coefficient, related sequences retrieved using the Greengenes NAST 0.71) and Actinobacteria (coefficient, 0.70), which for all pyrosequences showed similar results. In addition, correlated with the wood feeding Microcerotermes termite the sequences from this study had high identities (95- species. The Trinervitermes trinervoides termites that fed 100% for the most dominant OTUs) to those in the on grass and sugarcane respectively were closer in the databases, suggesting that they were reliable PCR ordination space but could be differentiated by key amplicons. bacterial groups. The Planctomycetes (coefficient, 0.57)

correlated with the sugarcane along the second axis and

Comparison of termite hindgut bacterial diversities Acidobacteria (coefficient, 0.39) with the grass feeding Trinervitermes trinervoides. The proportions of these The sugarcane feeding termite was populated by groups were significantly different between the termites. sequences of the phyla Bacteroidetes (5.2%), The wood feeding termites were further separated in the Proteobacteria (3.6%) and Firmicutes (3%), amongst the ordination space when compared to the Trinervitermes non-Spirochaetes (Figure 2). Amongst these, trinervoides termites (Figure 3a) suggesting that most of predominant genera were Pirellula (0.25%), the functional groups in their hindguts were different. Ruminococcus (0.12%), Bacteroides (0.1%), Geothrix In order to reduce the bias that may have arisen from (0.07%) and Eubacterium (0.07%). For the grass feeding possible outlier bacterial groups or from differences in termite, the other non-Spirochaete phyla present were microbial diversity as a result of termite phylogeny, Firmicutes (5.2%), Proteobacteria (1.9%) and correspondence analysis was repeated using only the Acidobacteria (1.6%) as shown in Figure 2. The genera more representative groups. Each of the bacterial present included Holophaga (0.54%), Geothrix (0.29%), species selected represented at least 1% of the total Desulfovibrio (0.19%), Acetivibrio (0.1%), Papillibacter bacterial population in all termites under investigation (0.08%) and Eubacterium (0.07%). (Figure 3b). The Fibrobacteres and Synergistes were the In comparison, sequences of Bacteroidetes, key functional groups that correlated with both wood Proteobacteria and Planctomycetes were significantly feeding termites, showing correlation coefficients of 0.72 more represented in the hindgut of the termites that and 0.98, respectively. Thus, specific bacterial functional subsisted on sugar cane. Although, Acidobacteria and groups correlated with a woody diet amongst the different Firmicutes were significantly more represented in the feeding termites. The Trinervitermes trinervoides termites grass feeding termite, these were still minor populations did not significantly correlate with any bacterial groups in in comparison to the dominant Spirochetes. this case.

These bacterial groups could still be involved in functions related to the assimilation of diet and could be DISCUSSION key determinants of the termite diet preferences between these two different substrates. Correspondence analysis PCR amplification of the termite hindgut 16S rRNA was therefore used to identify the correlations between gene diversity bacterial diversity and dietary preferences in higher termites. This was performed using secondary data taken Most of the diversity reported in this study was biased Sanyika et al. 2645

NaS (Wd) 1.9

1.5

1.1

0.7

AcB 0.4

Axis 2 (18%) 2 Axis Fib TvM (gr) PtB SpC BcD FiM SyN -1.1 -0.7 -0.4 0.4 0.7 1.1 1.5 1.9

-0.4 AcT PlM McT (Wd)

-0.7 TvM (sg)

(a) -1.1

Axis 1 (71%)

TvM (sg) 1.5

1.2

0.9

0.6

0.3 BcD SyN PtB SpC FiM Fib McT (Wd)

Axis 2 (12%) 2 Axis NaS (Wd) -1.5 -1.2 -0.9 -0.6 -0.3 0.3 0.6 0.9 1.2 1.5 AcB -0.3

-0.6

-0.9

-1.2 (b) TvM (gr) -1.5 Axis 1 (78%)

Figure 3. Correspondence Analysis biplot measuring the correlation between diet and bacterial diversity in the third proctodeal hindgut segments (P3, the paunch) of the cases: grass feeding Trinervitermes trinervoides, TvM (gr); sugarcane feeding Trinervitermes trinervoides, TvM (sg); wood feeding Nasutitermes species, NaS (Wd) and the wood feeding Microcerotermes species, McT (Wd). Phylogenetic groups (variables): AcB, Acidobacteria; AcT, Actinobacteria; DfB, Deferribacteres; Fib, Fibrobacteres; ChB, Chlorobi; SyN, Synergistes; FiM, Firmicutes; BcD, Bacteroidetes; PtB, Proteobacteria; SpC, Spirochaetes and PlM, Planctomycetes. The circles represent the relative position of each feeding termite (diet) as measured by CCA scores. Inverted triangles represent the direction of correlation and position of each bacterial phylum relative to each feeding termite based on correlation measures. The relative distance of each phylum from the origin is a measure of the strength of the correlation coefficient towards a particular feeding termite. Two of these termites were phylogenetically closely related (belonging to the same genus) and the other two termites fed specifically on the same diet (wood feeders). Figure 3a shows analysis of most bacterial diversity except for outliers and Figure 3b, the major groups (>1%) in all termites. 2646 Afr. J. Microbiol. Res.

NaS (Wd) McT (Wd) TvM (gr) TvM (Sg)

0.48 0.4 0.32 0.24 0.16 0.08 0

Bray Curtis similarity index

Figure 4. Unweighted Pair Group Method with Arithmetic Mean (UPGMA) clustering of the Bray Curtis coefficient similarity matrix of bacterial diversity from the hindguts of higher worker termites that feed on four different substrates: grass feeding Trinervitermes trinervoides, TvM gr; sugarcane feeding Trinervitermes trinervoides, TvM sg; wood feeding Nasutitermes species, NaS (Wd) and the wood feeding Microcerotermes species, McT (Wd).

towards the primer 1492R, which was used for both the of the related sequences have also been identified in initial PCR and nested PCR during pyrosequencing. higher, fungus growing worker termites such as Both amplicon size and primer pairs can affect overall Macrotermes gilvus (Hongoh et al., 2006). bias of results (Engelbrektson et al., 2010). Nested PCR In comparison to the higher termite hindguts, the may increase primer bias, but Fan et al. (2009) showed human gut (Claesson et al., 2009; Qin et al., 2010), that the bias does not significantly affect the detection of gorilla (Gorilla beringei) feces (Frey et al., 2006), bovine diversity, richness and abundance within the bacterial rumen (Brulc et al., 2009), yak rumen (An et al., 2005; communities (Fan et al., 2009). Nested PCR also allows Yang et al., 2010) and pig gastrointestinal tract (Leser et for increased sensitivity of the detection of target al., 2002) bacterial diversities were found to be domi- sequences (Fan et al., 2009). Although, shorter gene nated by Bacteroidetes, Firmicutes, and in some cases amplicons could be better at detecting the most poorly Proteobacteria or Verrucomicrobia. In all these cases, the represented species (Engelbrektson et al., 2010), these Spirochaetes were far less ubiquitous in mammalian guts were unnecessary in this study, as they were not as compared to termite hindguts. expected to reveal significant differences between the termite populations. Substrate preference amongst higher termites

Comparison of diversity A comparison was made between the bacterial diversity from hindguts of the two Trinervitermes trinervoides The majority of the bacterial phylotypes from this study termites and the wood feeding Nasutitermes species and were related to sequences identified in samples from the the Microcerotermes species described in literature. hindgut of a Nasutitermes termite species from Costa Although, it has been observed that termite hindgut Rica (Warnecke et al., 2007). Other gene sequences bacterial diversity is also influenced by the environmental have been discovered that are related to those identified factors, including the diet (Yang et al., 2005), in our from the hindguts and gut walls of other termites such as findings, diet was not a determinant of bacterial diversity the Microcerotermes species 1 from since the wood feeding termites did not cluster together Thailand (Hongoh et al., 2005), Reticulitermes speratus (Figure 4). The bacterial diversities were primarily (Hongoh et al., 2003), Reticulitermes santonensis (Yang dependent on the termite host species with the diversity et al., 2005), Cubitermes species (TD43) from Kenya, from the Trinervitermes trinervoides diversity distinctly Reticulitermes flavipes (Lilburn et al., 1999) and clustering together and the wood feeding termites Zootermopsis nevadensis (Ottesen et al., 2006). separated. This is attributed to the inheritance pattern in Reticulitermes speratus (Ohkuma and Kudo, 1996) is a which termite microbial gut population is acquired by lower termite that harbours protozoan flagellates. Some physical transmission from parent termites and therefore, Sanyika et al. 2647

depends on the relatedness of the termite species PCR-induced sequence artifacts and bias: Insights from comparison (Hongoh et al., 2005). of two 16S rRNA clone libraries constructed from the same sample. Appl. Environ. Microbiol., 71: 8966–8969. The Spirochaetes identified in all termites Adam RA, Mitchell JD, van der Westhuizen MC (2005). Food predominantly belonged to the genus Treponema. They preferences in laboratory colonies of the harvester termite were more abundant in the hindguts of the grass and Trinervitermes trinervoides (Sjöstedt) (Termitidae: Nasutitemitinae). sugarcane feeding Trinervitermes trinervoides Afr. Entomol., 13: 193-200. Adam RA, Mitchell JD, van der Westhuizen MC (2008). Aspects of (approximately 82%) and less represented in the foraging in the harvester termite, Trinervitermes trinervoides Microcerotermes (54%) and the Nasutitermes (32%) (Sjöstedt) (Termitidae: Nasutitemitinae). Afr. Entomol., 16: 153-161. wood feeding termites. The Spirochaetes, especially of Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990). Basic the genus Treponema, specialize in carbon fixation local alignment search tool. J. Mol. Biol., 215:403-10. An D, Dong X, Dong Z (2005). Prokaryote diversity in the rumen of yak through reductive acetogenesis to produce acetate, the (Bos grunniens) and Jinnan cattle (Bos taurus) estimated by 16S major source of energy for the termites (Brauman et al., rDNA homology analyses. Anaerobe, 11: 207–215. 1992). They are also major contributors to nitrogen Armougom F, Raoult D (2009). Exploring microbial diversity using 16S fixation and maintenance of the carbon and nitrogen rRNA high-throughput methods. Comput. Sci. Syst. Biol., 2: 74-92. Brauman A, Kane MD, Labat M, Breznak JA (1992). Genesis of balances in the termite systems (Breznak, 2002). The acetatae and methane by gut bacteria of nutritionally diverse lower abundance of Spirochaetes in wood feeding termites. 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