Dirk Schmitt-Wagner, John A. Breznak and Andreas Brune
In preparation
Abstract
The molecular diversity and community structure of spirochetes from the gut of the soil-feeding higher termite Cubitermes ugandensis was analyzed using amplified rDNA restriction analysis (ARDRA), and terminal restriction fragment length polymorphism (T-RFLP) analysis, and by cloning and sequencing amplified 16S rRNA genes with spirochete-specific primers. Of 67 clones analyzed with ARDRA, 34 different ribotypes were identified, which reflected the high diversity of spirochetes in the different hindgut sections of C. ugandensis. Nearly all clones were affiliated with the "termite cluster" in the genus Treponema, which exclusively consists of termite clone sequences, but grouped in higher-termite or soil-feeding-termite-specific clusters, distinct from clone sequences obtained in studies with lower termites. T-RFLP analysis revealed a dramatic shift in diversity and relative abundance between the highly alkaline hindgut section P1 and the following hindgut sections (P3 and P4). A decrease in diversity and abundance also for the slightly acidic hindgut section P5 was detected. The hindgut sections P3 and P4 showed the highest abundance and diversity of termite-gut-specific spirochetes. The results of this study revealed the great reservoir of bacterial diversity in the gut of soil-feeding higher termites and pointed to a coevolution of spirochetes within the termite host.
Introduction
Termites are terrestrial, social insects that comprise the order Isoptera, consisting of six families of lower termites and one family of higher termites (Krishna, 1970; Wood and Johnson 1986; Noirot 1992; Kambhampati and Eggleton, 2000). The great majority of termites live in tropical and subtropical regions, but they extend also into the temperate zone to about 48° N and about 45° S – between these latitudes lie about two-thirds of the Earth's land surface (Lee and Wood, 1971). In tropical and subtropical regions their number exceeds 6000 individuals m–2 and their biomass density ranges between 5 and 50 g m–2, thereby often surpassing biomass densities of mammalian herbivores (0.01–17.5 g m–2) (Lee and Wood, 1971; Collins and Wood 1984).
In recent publications, it has been shown that termite guts contain a large diversity of previously unrecognized bacteria (Ohkuma, 1996; Friedrich et al., 2001) and that the greater than 2000 described species of termites (Wood and Johnson 1986) are still an abundant reservoir of unrecognized microbial diversity. This has especially been shown for the diversity of spirochetes in the termite gut (Paster et al., 1996; Lilburn et al., 1999; Ohkuma et al., 1999). For the termite hindgut it was proposed that spirochetes are one of the most abundant, consistently present bacterial groups (Breznak, 1984), accounting for as much as 50% of all prokaryotes (Paster et al., 1996). However, in the higher soil-feeding termite Cubitermes ugandensis spirochetes do not exceed 11% of all bacteria in a single gut segment evaluated by direct DAPI cell counts (Schmitt-Wagner et al., 2003a). But due to the versatile metabolic possibilities observed with recently isolated spirochete strains from the termite gut (Leadbetter et al 1999; Lilburn et al., 2001), with homoacetogenic acetate production from H2 and CO2 and nitrogen-fixation activity, suggest that spirochetes also play an important role in the gut of soil-feeding termites. Especially the production of acetate from H2 and CO2 could assign a role to the spirochetes, in addition to the methanogenic archaea, as a hydrogen sink in the termite gut. The accumulation of hydrogen to remarkable partial pressures in the mixed segment and the P3 gut segment has been shown in a microsensor study (Schmitt-Wagner, 1999).
Nearly all spirochete clone sequences investigated to date, are grouped in two Treponema clusters (Lilburn et al., 1999), later termed the "termite Treponema clusters I and II" (Ohkuma et al., 1999). The spirochete clones within these clusters are only distantly related to each other, again revealing the high diversity of bacteria in the termite gut. Therefore, it was not surprising that just the spirochete isolates from the termite gut extended the known metabolic abilities of spirochetes. Most information on spirochete diversity is based on studies of various lower termites, only one higher soil-feeding termite (Pericapritermes nitobei) from Japan, has been investigated in this respect (Ohkuma et al., 1999). In the study presented here, the axial distribution and the diversity of spirochetes in the different gut segments of an African soil-feeding higher termite (C. ugandensis) was investigated using amplified rDNA restriction analysis (ARDRA) and terminal restriction fragment length polymorphism (T-RFLP) and by cloning and sequencing spirochete 16S rRNA genes.
Materials and Methods Termites
Cubitermes ugandensis Fuller was collected in the Kakamega forest (Kenya). The termites were brought to the laboratory in polypropylene containers together with nest fragments and soil of the collection site. Within a week after collection, worker caste termites were dissected with sterile, fine-tipped forceps, and guts were separated into six sections (Fig. 1).
Fig. 1 Gut morphology of a Cubitermes sp. worker termite. Gut sections were separated at the indicated positions. The gut is drawn in its unraveled state to illustrate the sequence of the individual segments: C, crop; M, midgut; ms, mixed segment, and P1, P3, P4, and P5, the proctodeal hindgut segments (Noirot, 2001). Hydrogen partial pressure in the individual gut segments was determined with hydrogen-sensitive microsensors for Cubitermes orthognathus (Schmitt-Wagner, 1999). The average luminal pH of the major gut segments was determined with glass pH microelectrodes for Cubitermes speciosus (Brune and Kühl, 1996).
DNA extraction
Fifty gut sections each were pooled, and DNA was extracted and purified using a previously described procedure that involves bead-beating and the use of polyvinylpolypyrrolidone spin columns to remove humic substances (Friedrich et al., 2001).
PCR amplification
16S rRNA genes for cloning were specifically amplified from DNA extracts of gut, soil, and nest material using the spirochete-specific primers 63F and 1400R as previously described by Lilburn et al. (1999).
Clone libraries
Clone libraries of 16S rRNA genes were created from the PCR products of each hindgut section (Fig. 1) using the TA Cloning kit (Invitrogen). Clones were checked for correct insert size by vector-targeted PCR.
Screening and sorting clones
DNA from all clones containing full-length inserts was amplified for ARDRA using primers 63F and 1400R and digested with HpaII as previously described (Lilburn et al., 1999).
Phylogenetic analysis
Sequence data were analyzed using the ARB software package (Ludwig et al., 2003) [version 2.5b; O. Strunk and W. Ludwig, Technische Universität München (http://www.arb-home.de)] as described in Schmitt-Wagner et al. (2003a). Sequences were checked for chimerae using the Check_Chimera program (Maidak et al., 2001).
In addition, the 400 terminal sequence positions at the 5'- and 3'- ends of the sequences were subjected separately to treeing analysis (“fractional treeing”; (Ludwig et al., 1997)); significant differences in phylogenetic placement of these terminal sequence fragments were regarded as indicative of chimera formation. All chimeric sequences were excluded from further analyses.
T-RFLP analysis
T-RFLP analysis was carried out as described by Chin et al. (1999) with minor modifications. Bacterial 16S rRNA gene sequences were amplified utilizing a pentamethine-carbocyanine (IRD700)-labeled spirochete-specific primer 63F (5'-GTYTTAAGCATGCAAGT-3'; Escherichia coli position 46–62) modified of the spirochete-specific probe from Paster et al. (1996) and primer 907R (5'-CCG TCA ATT CCT TTR AGT TT-3'; E. coli position 907–926) (Lane et al., 1985). PCR was carried out as described previously (Friedrich et al., 2001) using a Touch-down-PCR program consisting of an initial denaturing step of 3 min at 94°C, followed by 20 cycles of 30 s at 94°C, 30 s at 60°C by reducing the temperature in succession 0.5°C per cycle and 1 min at 72°C, followed by 10 cycles of 30 s at 94°C, 30 s at 56°C, and 1 min at 72°C, and concluding with a 7-min extension at 72°C. Purified PCR products were quantified spectrophotometrically. PCR amplicons (40 ng DNA) were digested with the restriction endonucleases MspI and HaeIII, 2.5 U each, (MBI Fermentas), 2 µl 10× Y+/Tango buffer in a total volume of 20 µl in 0.2-ml reaction tubes for 3 h at 37°C. The endonucleases were inactivated by heating for 20 min at 80°C. Samples were analyzed on an COR sequencer 4200 (COR) using LI-COR 50–700 size standard and GelScan 5.01 Software (BioSciTec). The Gelscan software allows the automated area integration of each T-RF peak to be manually corrected.
The total peak area of a T-RFLP profile was defined as the sum of the peak areas of all peaks >50 bp. The relative peak area of a given peak was determined by dividing its peak area by the total peak area of the profile. When determining the number of distinct T-RFs in a given profile, only T-RFs with a relative peak area larger than 1% of the total peak area were taken into account.
Sequence data
16S rDNA sequences of clones obtained in this study were deposited with Genbank under accession numbers AY160775 to AY160876.
Results
T-RFLP analysis together with ARDRA revealed the diversity of the spirochetes in the different gut sections of the soil-feeding termite Cubitermes ugandensis. The number of clones selected for sequencing and comparative database analysis was determined by the results of the fingerprint analyses. The highest number of different phylotypes
was found in the P4 segment, as shown by the T-RFLP profile and the number of different ARDRA patterns.
Fig. 2. Rarefaction analysis of all spirochete 16S rRNA gene clones recovered from the different hindgut sections (P1, P3, P4 and P5) of Cubitermes ugandensis. The expected number of phylotypes was calculated from the number of clones and the number of different phylotypes revealed with ARDRA for each hindgut section. P1 hindgut segment ( ), P3 hindgut section (), P4 hindgut segment ({), and P5 hindgut segment (
à
). The slope of the curves indicates whether the diversity was covered (zero or low slope) or whether further phylotypes might expected by analyzing more clones (steep slope). Rarefaction analysis was carried out as described previously (Friedrich et al., 2001).A rarefaction analysis was calculated based on the number of different ribotypes revealed by ARDRA (Fig. 2). The saturated curves for all gut sections, except for segment P4, showed that the diversity was sufficiently described by the number of detected ribotypes, whereas the unsaturated curve for the P4 segment indicated the presence of unrecognized ribotypes. The highest number of clones from the P4 clone library were therefore sequenced (Table 1). From all other clone libraries, a smaller number of clones was sequenced since almost all T-RFs obtained in the fingerprint profiles of P1, P3 and P5 the gut sections were also detected in the P4 profile (Fig. 3).
Sixteen clones of the P1 clone library were tested by ARDRA using the restriction enzyme HpaII; seven different restriction patterns were revealed. Four clones, each representing a different restriction pattern were sequenced. To all four clones T-RFs of the P1 profile (T-RFs 124 and 170 bp) could be assigned (Table 2; Fig. 3). The ARDRA restriction patterns of these four clones were also present in nine other clones of the P1 clone library (Table 1).
0 2 4 6 8 10 12 14
0 5 10 15 20
Clones sampled
Expected number of phylotypes
Table 1. Number of clones and ARDRA ribotypes compared to the T-RF profiles of the
a Percentage of the total peak area in the T-RF profile that could be assigned to clones of the respective clone library.
b Percentage of all clones in the clone libraries also present in the T-RF profiles with their ARDRA pattern.
ARDRA of the 17 clones from the clone library of gut section P3 resulted in seven different restriction patterns. Four clones with different ARDRA patterns were sequenced. In the T-RFLP profile of the gut section P3, two of the major peaks (T-RFs 124 bp and 243/246 bp) (Table 2; Fig. 3) could be assigned to two of the sequenced clones. Based on the specific ARDRA restriction patterns of these two clones, seven extra clones of the P3 clone library were represented by these two sequenced clones (Table 1).
For the P4 clone library, ARDRA revealed 12 different restriction patterns – the highest number of ribotypes for all gut sections tested. Fourteen clones were sequenced. Six different T-RFs (124, 147, 170, 187, 196 and 221–223 bp) of the P4 profile could be assigned to 12 clones (Table 2; Fig. 3). Based on the specific ARDRA restriction patterns of these 12 clones, 2 extra clones of the P4 clone library were represented (Table 1).
Sixteen clones of the P5 clone library were studied by ARDRA, seven different ribotypes were identified. Because of the low diversity in the P5 T-RF profile, only three clones were sequenced. The only relevant T-RF peak (137 bp) of the P5 profile could be assigned to one of the sequenced clones (Table 2; Fig. 3). Due to the ARDRA restriction pattern two more clones were represented by this clone (Table 1).
The various ARDRA ribotypes were represented by clones to which different T-RFs could be assigned; therefore, the P1 and P4 T-RFLP profiles reflected about 80, the P3 T-RF profile over 50 and the P5 T-RF profile about 20% of the clones in the respective clone libraries tested by ARDRA.
Table 2. Predicted T-RF lengths after MspI and HaeIII digestion. All clones except those in italics could be assigned to T-RFs in the profiles of the respective gut sections.
Clone library (gut section) T-RF
[bp] P1 P3 P4 P5
30 CUS404
91 CUS415
124 CUS126 CUS306 CUS401
137 CUS502
147 CUS403
CUS418
149 CUS523
170 CUS109 CUS405 CUS111 CUS412 CUS118 CUS425
187 CUS413
196 CUS409
221 CUS410
CUS421
222 CUS423
223 CUS416 CUS511
239 CUS304
246 CUS302
265 CUS311
The sequenced and analyzed clones of the various clone libraries described most of the total peak area of the respective T-RF patterns (66% for P1, 79% for P4, and 77% for P5). Only the clones of the P3 clone library described <40% of the total peak area of the P3 profile.
The majority of T-RFs in the different profiles of the three alkaline gut sections P1, P3 and P4 were similar. Four peaks (124, 170, 187, and 243/246 bp) were present in all of the three hindgut segment profiles (Table 2; Fig. 3). The four T-RF peaks were also be detected in the T-RF pattern of the midgut (not shown). The T-RF with 170 bp was also detectable in the profile of the crop (not shown).
The T-RF profile of the slightly acidic gut segment P5 differed greatly from the other profiles, it showed the lowest diversity and abundance of all gut segment profiles. The two T-RF peaks (137 and 170 bp) were also present in the P4 gut segment profile (Table 2; Fig. 3).
Fig. 3. Terminal-restriction-fragment length polymorphism (T-RFLP) profiles of 16S rRNA gene PCR products, amplified from DNA extracted from different gut sections of C. ugandensis.
PCR products obtained with primers 63F (IRD700-labeled) and 907R were digested with MspI and HaeIII. T-RF length of major peaks that matched the predicted T-RFs of clones in the respective clone library are in bold. For orientation, also the size of several unassigned T-RFs is indicated.
Comparative sequence analysis revealed that nearly all clones of the four different clone libraries (P1, P3, P4 and P5) belong to the termite Treponema cluster I (Ohkuma et al., 1999). The majority of the clones obtained from C. ugandensis formed therein distinct clusters specific for higher termites ("cluster 1") or specific for soil-feeding termites ("cluster 2" and "cluster 3") (Fig. 4). Although clone sequences were grouped in the same clusters, most of them were only distantly related to each other.
However, clones within the same clone library with the same predicted T-RF site were more closely related, albeit the sequence similarity values differed over a wide range. For example, clones of the P4 clone library with a predicted T-RF of 170 bp (clones CUS405, CUS412, and CUS425; Genbank accession numbers XY–XY), which show a sequence similarity ranging from 90 to 98%, and for clones of the P1 clone library with the T-RF of 170 bp (clones CUS109, CUS111, and CUS118;
Genbank accession numbers XY–XY), which show a sequence similarity ranging from 87 to 94%. The clones of the P4 clone library with the T-RF of 170 bp show a sequence similarity to clones of the P1 clone library with the same T-RF of only 86 to 90%. Likewise, three clones CUS126 (P1 clone library), CUS306 (P3 clone library) and CUS401 (P4 clone library), which have a T-RF of 124 bp, show a sequence similarity of 90–91% (Table 2; Fig. 4)
Within the termite Treponema cluster I, five clone sequences obtained in this study (Genbank accession numbers XY–XY) grouped in Cluster 1, which consists only of clone sequences of higher termites. One clone sequence from the wood-feeding higher termite Nasutitermes lujae (Paster et al., 1996) and two clone sequences from the soil-feeding higher termite Pericapritermes nitobei (Ohkuma et al., 1999) also group in this cluster (Fig. 4). The sequence similarity of the clone sequences in this cluster differed from 86.3 to 99.5%.
Cluster 2 can be subdivided further, Cluster 2.1 contains only distantly related (78.9–
88.2% sequence similarity) clone sequences obtained only in this study (Genbank accession numbers XY–XY). Cluster 2.2, contains exclusively clone sequences obtained in this study (Genbank accession numbers XY–XY) with a sequence similarity of 91.7–95%. Cluster 2.3 contains five clone sequences from this study (Genbank accession numbers XY–XY), three clone sequences from P. nitobei (Ohkuma et al., 1999), and three clone sequences from C. orthognathus (Schmitt-Wagner et al., 2003a), with sequence similarity values of 90.4–97.8%.
Fig. 4. Phylogenetic tree of clones belonging to the spirochetes obtained in this study (shown in bold). The tree was calculated using the maximum-likelihood algorithm (Olsen et al., 1994) implemented in the ARB software package (Ludwig et al., 2003) (O. Strunk and W. Ludwig, Technische Universität München, Munich, Germany; http://www.arb-home.de) and a phylum-specific filter. For the calculation of a basic tree, only sequences with more than 1300 bp were used. Shorter sequences were added to the initial tree using the ARB parsimony tool (Ludwig et al., 1998). The scale bar represents 10% sequence difference. Clusters 1–3 are shaded. The tree was rooted using Holophaga foetida (X77215).
0.10
Clone Rs-A16 from Reticulitermes speratus, AB088892 Treponema bryantii, M57737
Clone RsDiSp3 from Reticulitermes speratus, AB032001 Clone RsDiSp1 from Reticulitermes speratus, AB031997 Spirochaeta zuelzerae, M34265 M88725
Spirochaeta caldaria, M71240
Clone MdSpiro5 from Mastotermes darwiniensis, X79548 Clone Pn37 from Pericapritermes nitobei, AB015817
Clone CUS111, ARB_FAE80BF7 Clone CUS109, ARB_D9992F2D
Clone CUS311, ARB_CD1B3995 Clone NL1 from Nasutitermes lujae, U40791 Clone CUS523, ARB_FE70860A
Clone Pn64 from Pericapritermes nitobei, AB015820 Clone CUS418, ARB_6784F627
Clone P3-9 from Cubitermes orthognathus, AY160872 Clone Pn56 from Pericapritermes nitobei, AB015818
Clone CUS118, ARB_1A65AF91
Clone Pn11 from Pericapritermes nitobei, AB015814 Clone CUS413, ARB_14938012
Clone BCf6-24 from Coptotermes formosanus, AB062834 Clone BCf11-05 from Coptotermes formosanus, AB062808
Clone RFS99 from Reticulitermes flavipes, AF068424 Isolate ZAS-9 from Zootermopsis angusticollis, AF320287 Clone NkS7 from Neotermes koshunensis, AB084955
Clone CUS416, ARB_5340774D
Clone Pn69 from Pericapritermes nitobei, AB015821 Clone P3-22 from Cubitermes orthognathus, AY160871
Clone CuS421, ARB_32E81729
Clone P4b-2 from Cubitermes orthognathus, AY160869 Clone CUS405, ARB_C7DE5E0
Clone CuS502, ARB_951D91E0 Clone CUS412, ARB_467EA5BF Clone CUS425, ARB_981F7599
Clone P4b-15 from Cubitermes orthognathus, AY160870 Clone Pn58 from Pericapritermes nitobei, AB015819 Clone Pn16 from Pericapritermes nitobei, AB015815 Clone CUS415, ARB_BB5031B1
Clone Pn19 fromPericapritermes nitobei, AB015816 Clone NkS8 from Neotermes koshunensis, AB084956
Clone NkS5 fromNeotermes koshunensis, AB084954 Clone CFS6 from Coptotermes formosanus, AF068345 Clone nc5 fromNeotermes castaneus, AJ419819
Clone NkS15 from Neotermes koshunensis, AB084958 Isolate ZAS-1 from Zootermopsis angusticollis, AF093251
Isolate ZAS-2 from Zootermopsis angusticollis, AF093252 Clone HsPySp4 from Hodotermopsis sjoestedti, AB032007
Clone RFS75 from Reticulitermes flavipes, AF068421 Clone RFS3 from Reticulitermes flavipes, AF068340
Clone NkS56 from Neotermes koshunensis, AB084967 Clone CUS401, ARB_4666E7E5
Cluster 3 only consists of clones from this study (Genbank accession numbers XY–
XY). The sequence similarity of the four clone sequences in this cluster ranged from 89.8 to 93.4%. The spirochete phylogenetic tree and the formation of specific clusters (Fig. 4) clearly revealed the distinct phylogenetic positions of spirochete clones obtained from higher and lower termites.
Discussion
The results of this study clearly demonstrate that the gut of the soil-feeding termite Cubitermes ugandensis harbors a great and distinct diversity of spirochetes.
Spirochete clone sequences from higher termites, especially from soil-feeding termites, were distantly related to clone sequences obtained from lower wood-feeding termites (Lilburn et al., 1999; Ohkuma et al., 1999; Noda et al., 2002). This result is not astonishing since in lower termites, the spirochetes live mostly as ectosymbionts or endosymbionts of symbiotic flagellates in the termite gut, whereas the flagellate-free gut of soil-feeding higher termites contains only flagellate-free-living or probably gut-wall-attached spirochetes, phylogenetically distinct from the flagellate-associated spirochetes in lower termites (Fig. 4).
The high spirochete diversity is apparently an adaptation to the different physico-chemical conditions in the gut of soil-feeding termites, formed by extremely alkaline conditions and steep gradients of oxygen and hydrogen partial pressure (Brune and Kühl, 1996; Schmitt-Wagner, 1999). The highest diversity and abundance is visible in the gut sections P3 and P4 (11 and 5% of the total bacterial cell counts in the respective gut sections) (Table 3). In the extremely alkaline gut section P1 (Fig. 1), only a low diversity is present in the P1 T-RF profile. Probably owing to decreasing values of alkalinity in the P3 section (Fig. 1), the diversity increases and reaches a maximum in the nearly neutral P4 segment. In the slightly acidic P5 segment (Fig 1), the spirochetes community changed dramatically in diversity (Fig. 3) and abundance
The high spirochete diversity is apparently an adaptation to the different physico-chemical conditions in the gut of soil-feeding termites, formed by extremely alkaline conditions and steep gradients of oxygen and hydrogen partial pressure (Brune and Kühl, 1996; Schmitt-Wagner, 1999). The highest diversity and abundance is visible in the gut sections P3 and P4 (11 and 5% of the total bacterial cell counts in the respective gut sections) (Table 3). In the extremely alkaline gut section P1 (Fig. 1), only a low diversity is present in the P1 T-RF profile. Probably owing to decreasing values of alkalinity in the P3 section (Fig. 1), the diversity increases and reaches a maximum in the nearly neutral P4 segment. In the slightly acidic P5 segment (Fig 1), the spirochetes community changed dramatically in diversity (Fig. 3) and abundance