Community Analysis of Hindgut Methanogens in Worker versus Soldier Cast Members of the

Lower Termite Species, Reticulitermes flavipes

Reticulitermes flavipes, soldier and worker cast individuals

By Susanne Juhler, Student at the MBL summer course in

Microbial Diversity, 2006

1

Abstract Despite much attention being paid to the methane production occurring in the termite hindgut it has never fully been elucidated exactly how many species of methanogens are existing in this gut system. Neither has it been investigated to which extent the different feeding behaviors of distinct termite cast members within a population might affect the methanogen community. In this study, the methanogen community of the lower termite, Reticulitermes flavipes, was investigated and compared among soldier and worker termites through a cultivation independent approach, applying PCR amplification of the 16S rRNA gene using Archaea specific primers. Amplified gene segments were cloned, sequenced and subjected to phylogenetic analysis in ARB. Furthermore, F420 fluorescence microscopy was applied to investigate differences in methanogen morphotypes attached to the gut wall, and in situ methane production rates were measured in bottle incubated termites by Gas Chromatograph (GC) analysis. Clone libraries from both soldier and worker termites showed a fairly high abundance of Thermoplasmata related sequences (47.9% and 26.4% of total sequences, respectively). No characterized isolates are found related to this group, and the physiology of these organisms therefore remains unknown, though possibly they play a significant role in the microbial gut community. The remaining clone sequences all grouped within the genus of Methanobrevibacter, and on the basis of a 97% similarity cut off, these sequences were divided into five distinct OTUs. Besides including the only three well characterized methanogen species isolated from the hindgut of R. flavipes; Mbb. cuticularis (RFM-1), Mbb. curvatus (RFM-2), and Mbb. filiformis (RFM-3), the clone libraries thus showed the additional presence of two new OTUs so far not recognized in the gut of this termite. One OTU (RFM-4) was most closely related to the isolate, Mbb. smithii, obtained from human and animal feces, while the second OTU (RFM-5) was related to the uncharacterized Mbb. sp. str. Mc30 from the gut of a higher termite. According to results obtained from Libshuff analysis, no statistically significant differences were observed among the worker and the soldier clone libraries. However, the combined results of F420 fluorescence microscopy and clone sequence distribution within each Methanobrevibacter OTU indicated some tendencies: The worker termites seemed to comprise a higher abundance of Mbb. filiformis than the soldiers. Furthermore, the Mbb. sp. str. Mc30 related OTU was only observed in soldier termites, though numbers were too low to show any significant difference. GC analysis showed rates of methanogenesis in the workers to be more than twice as high as in the soldiers (0.72 versus 0.30 � mol/g decapitated ww/h). Furthermore, hydrogen saturation significantly increased methane production in both cast members (by 163% and 201%, respectively), suggesting hydrogen limitation of the methanogens in the gut system.

2

Introduction Methanogenic Archaea existing in the termite hindgut are interesting for more than one reason. Not only have they previously been blamed for being the main contributors of global methane emission, but also are their coexistence with acetogenic bacteria in this particular environment very curious. Despite much focus on the termite hindgut methanogens, only three isolates have been obtained from this system; Mbb. cuticularis (RFM-1), Mbb. curvatus (RFM-2), and Mbb. filiformis (RFM-3), isolated from the termite Reticulitermes flavipes (Leadbetter and Breznak, 1996, Leadbetter et al., 1998). All three of these isolates cluster within in the Methanobrevibacter genus, and all uncultured methanogen 16S rRNA-gene sequences obtained from lower termites show close relatedness to these isolates. However, it has not thoroughly been investigated how high a diversity of methanogen species are existing in the termite gut. Neither has it been elucidated to which extent the different feeding behaviors of distinct termite cast members might affect the methanogen community. Individuals of the termite population are arranged in two distinct termite casts: worker termites which build up the nest and feed on wood, and soldier termites which defense the nest and due to their large mandibles are not able to feed on their own. Soldiers are fed by predigested material already processed by the workers and thus ingest material which is most possibly deprived of any easily degradable organic carbon and nitrogen. Investigating differences in gut biota among soldier and worker termites is a so far unexploited opportunity to evaluate the effect of feeding behavior on the hindgut microbiota excluding differences caused by variations among different species or even different termite populations within a species. In this study, the methanogen community composition of the wood-eating lower termite species, Reticulitermes flavipes, was investigated through PCR amplification, cloning, sequencing and phylogenetic analysis of the archaean 16S rRNA-gene. Furthermore, morphotypes of methanogens in the termite gut were observed through F420 fluorescence microscopy, and in situ methane production rates in living termites were measured through GC analysis. Results were compared among soldier and worker termites from the same termite population. Materials and methods The termites: The termites analyzed were of the wood-feeding lower termite species, Reticulitermes flavipes, all collected from the same termite population beneath a wood chunk in a backyard in Woods Hole, MA. Moist pieces of corrugated cardboard were laid out underneath the wood chunk and allowed to be inhabited by termites for 1-2 days before being brought to the laboratory in a zip-lock plastic bag. DNA extraction: Termite hindgut DNA extraction for 16S rRNA gene sequencing was performed within hours after collecting the termites. Four separate DNA extractions were performed containing 2 x 25 worker guts, and 25 and 11 soldier guts, respectively (named Worker 1, Worker 2, Soldier 1, and Soldier 2). Termites were dissected on clean parafilm using ethanol sterilized tweezers and needles. Hindguts were immediately transferred to a bead beating tube containing S1 solution on ice. DNA was extracted directly from the termite guts applying soil Mo Bio DNA Extraction Kit and DNA extracts were evaluated on a 0.1 % agarose gel. PCR amplification of the 16S rRNA gene: From the extracted DNA a 575bp 16S rRNA gene segment was amplified by PCR, applying Archaea specific primers (340f: 5’-CCTACGGGGCGCA(C/G)CAGG(C/G)GC-3’, Tm 70.5 ºC, and 915r: 5’-

3

GTGCTCCCCCGCCAATTCCT-3’, Tm 62.9 ºC (Löffler et al., 1997)). The primers were tested in ARB to ensure perfect match to the Methanobrevibacter genus as well as other methanogen genera and to test for unspecific binding in non-archaean domains. Positive control DNA was from an anaerobic enrichment of methanogens and the negative control contained RNAase free water in stead of DNA template. PCR was performed on three dilutions of each DNA extract (1:1, 1:10, and 1:100) to test for PCR inhibiting compounds. Each PCR reaction contained 25 �l Promega master mix, 5 �l of each primer (10 mM), 1 �l DMSO, 10 �l RNAase free water and 4 �l DNA template. PCR conditions were as follows: denaturation, 94°C (5 min); 30 cycles of 94°C (30 s), 61°C (45 s), 72°C (2 min 10 s); final elongation, 72°C (6 min). 45 �l of each PCR product was loaded on a 1.2% agarose gel, running at 50 V for 2.5 h. Positive bands of PCR products were cut out under UV light and purified using MinElute Gel Extraction Kit. 1 �l of the final extract was evaluated on a 1.2 % agarose gel to ensure the PCR product purity. Cloning and sequencing: PCR products were cloned into competent E. coli cells using TOPO TA Cloning® Kit (Invitrogen). Cloning reactions contained following: 3 �l purified PCR product, 1 �l salt solution, 1 �l H2O, and 1 �l TOPO Cloning Vector. 2 �l of reaction was added to One Shot® E.coli cells. Following cloning, host cells were plated on LB plates containing X-gal and Kanamycin to select for positive clones (cells growing due to uptake of vector-plasmid containing Kanamycin resistance gene and PCR product insert, that disrupts plasmid galactosidase gene so cells do not convert X-gal and therefore remain white). After 24h incubation at 37 ºC 50 clones were picked for sequencing from each library (Worker 1, Worker 2, Soldier 1, and Soldier 2). PCR products where sequenced in one direction using primer 915r. Backup cultures for each of the picked clones were made in micro titer plates, incubated at 37 ºC for 24h, added 25% glycerol and stored at -80 ºC. 16S rRNA gene phylogenetic analysis: Sequences were edited in 4-Peaks (quality control cut-off value 60) and imported in ARB. Each sequence where automatically aligned to the 10 closest relatives in the database (greengenes) and alignments were subsequently edited manually in ARB and added to the tree. An overall tree of methanogen clone sequences and sequences of characterized methanogen isolates were constructed using five different treeing methods, including Neighbor-Joining, Maximum Parsimony, and Maximum Likelihood, and a consensus tree was made. Neighbor Joining distance matrixes were constructed from each cluster of clone sequences and the closest related species, and based on a 97 % similarity cut-off value clone sequences were divided into distinct OTUs. The four clone libraries were analyzed in Dotur to obtain rarefaction curves and Chao values testing for library diversity coverage. Furthermore, sequences were exported to Phylip where a distance matrix were constructed and analyzed in Libshuff to test for significant differences among the separate clone libraries. F420 fluorescence microscopy of termite hindguts: Termites of both the soldier and the worker cast were dissected using clean forceps and needles. Hindguts were separated from the rest of the termite, cut open in a horizontal cut and washed 3 times in 10 �l drops of salt solution (to remove gut fluid while avoiding cell lysis). The guts were mounted in salt solution on a slide with the internal epithelia exposed, covered in a cover slip and visualized in a Zeiss fluorescence microscope. Due to autofluorescence of the F420 cofacter in the methanogenesis pathway, methanogens attached to the gut epithelia could be visualized and distinguished from other cells. Methanogen morphotypes and relative abundance was investigated and compared among soldier and worker termite hindguts. A total of 9 soldier termites and 9 worker termites were analyzed by microscopy. Furthermore, drops of salt solution containing the gut fluid wash-out were examined.

4

Measuring in situ methane production: In situ methane production rates in whole individuals of worker and soldier termites were measured through Gas Chromatograph (GC) analysis. A total of 6 60 ml serum bottles were prepared: 2 bottles containing 15 worker termites each, 2 containing 15 soldier termites, and two bottles with no termites as a control. Bottles were closed with rubber stoppers and crimped. In one bottle of each pair, 6 ml air was removed with a sterile syringe and replaced with pure H2 (obtaining a 10% H2 atmosphere, saturating methanogenesis). Methane production was measured with a 1 hour interval by taking out three replicate air samples from each bottle (250 �l in a gas tight syringe) and injecting in a Gas Chromatograph. The Gas chromatograph was calibrated using methane standards of 0, 0.888, 2.22, 11.1, 22.2, 44.4, and 111 nmol CH4 injections in order to make detection limit sufficiently low. Termites were weighted before and after decapitation, and methane production was calculated as emission rates pr. g. decapitated body weight (fresh wet weight). Results Phylogenetic analysis of clone libraries: A total of 145 clone sequences were obtained and analyzed. The sequences showed to be distributed in two phylogenetic groups within the Euryarchaeota; the Thermoplasmata and the Methanobrevibacter. The distribution is shown in table 1. Library Thermoplasmata Methanobrevibacter Total Worker 1 9 (22.0) 31 (78.0) 40 Worker 2 10 (31.3) 22 (68.7) 32 Soldier 1 13 (38.2) 21 (61.8) 34 Soldier 2 22 (56.4) 17 (43.6) 39 Workers pooled 19 (26.4) 53 (73.6) 72 Soldiers pooled 35 (47.9) 38 (52.1) 73

The Thermoplamata contained 26.6% and 47.9% of the worker termite clones and soldier termite clones, respectively. A phylogenetic tree of these clones and the most closely related sequences is shown in figure 1. The Neighbor Joining distance matrix showed clone 16S rRNA gene sequences to be more than 99% similar to each other. The two closest relatives were a partial sequence obtained from the gut of a higher termite, and sequences obtained from cockroach guts, showing 96-98% and 93-99.1% similarity to the clone sequences, respectively. No characterized isolates showed any close relatedness to this group of sequences.

Tabel 1: Number of clone sequences distributed within Thermoplasmata and Methanobrevibacter, and total number of sequences obtained from each clone library. Numbers in parentheses show the distribution in percentage of total sequences within the given library.

5

The rest of the sequences, 73.6% and 52.1% from the worker and soldier libraries, respectively, grouped within the Methanobacteriales all in the genus Methanobrevibacter. The clone sequences clustered in very closely related units, and based on the Neighbor Joining distance matrix the sequences were divided in to 5 distinct OTUs, each only including clone sequences more than 97% similar to each other. All sequences within each OTU showed less than 97% similarity with any of the other defined OTUs. A consensus tree was made from trees obtained by 5 different treeing methods, including Neighbor-Joining, Maximum Likelihood, and Maximum parsimony. The trees were constructed from one representative clone from each of the five OTUs, their closest related species and several other species within the Methanobrevibacter genus (fig. 2). The distinct OTUs are marked, given the names RFM-1 to RFM-5 (RFM, reticulitermes flavipes methanogens).

0.01

G CloneG11 WorkerTermite2 SJ, 609 G CloneE02 SoldierCommunity1 SJ, 615 G CloneE02 WorkerTermite1 SJ, 622 G CloneG09 WorkerTermite2 SJ, 611 G CloneC01 WorkerTermite1 SJ, 602

G CloneD09 SoldierTermite2 SJ, 620 G CloneA10 SoldierTermite2 SJ, 611 G CloneG02 SoldierCommunity1 SJ, 620 G CloneB01 SoldierCommunity1 SJ, 502

G CloneG10 WorkerTermite2 SJ, 611 G CloneF12 SoldierTermite2 SJ, 608 G CloneE03 SoldierCommunity1 SJ, 615 G CloneD04 WorkerTermite1 SJ, 664 G CloneC05 WorkerTermite1 SJ, 589

G CloneF02 WorkerTermite1 SJ, 615 G CloneH09 WorkerTermite2 SJ, 600 G CloneB03 SoldierCommunity1 SJ, 649 G CloneC03 SoldierCommunity1 SJ, 665 G CloneD05 SoldierCommunity1 SJ, 588 G CloneG03 SoldierCommunity1 SJ, 601 G CloneG05 SoldierCommunity1 SJ, 569 G CloneB10 SoldierTermite2 SJ, 588

G CloneC10 SoldierTermite2 SJ, 569 G CloneA09 SoldierTermite2 SJ, 619 G CloneE12 SoldierTermite2 SJ, 611 G CloneD11 WorkerTermite2 SJ, 613 G CloneC12 SoldierTermite2 SJ, 609 G CloneF09 SoldierTermite2 SJ, 618 G CloneE09 SoldierTermite2 SJ, 644 G CloneE10 SoldierTermite2 SJ, 520

G CloneB12 WorkerTermite2 SJ, 601 G CloneA11 SoldierTermite2 SJ, 620

G CloneH10 SoldierTermite2 SJ, 599 G CloneC09 SoldierTermite2 SJ, 603 G CloneF09 WorkerTermite2 SJ, 662 G CloneD02 WorkerTermite1 SJ, 648

G CloneC11 WorkerTermite2 SJ, 476 G CloneE08 WorkerTermite2 SJ, 659

G CloneH02 SoldierCommunity1 SJ, 464 G CloneF08 SoldierTermite2 SJ, 624

G CloneC11 SoldierTermite2 SJ, 462 G CloneA06 SoldierTermite2 SJ, 377

G CloneB06 SoldierTermite2 SJ, 530 G CloneC06 SoldierTermite2 SJ, 421 G CloneE10 WorkerTermite2 SJ, 507

G CloneD11 SoldierTermite2 SJ, 644 G CloneH04 WorkerTermite1 SJ, 611 G CloneF02 SoldierCommunity1 SJ, 600

G CloneE01 WorkerTermite1 SJ, 327 G CloneG05 WorkerTermite1 SJ, 889

G CloneH01 SoldierCommunity1 SJ, 404 G CloneF01 SoldierCommunity1 SJ, 404

G CloneH05 SoldierTermite2 SJ, 172 isolate xylophagous cockroach clone PA203, 1313

G CloneD08 SoldierTermite2 SJ, 110 AF293578termite.P3

Ar 9, 789 isolate xylophagous cockroach clone PA201, 1312

isolate xylophagous cockroach clone PA204, 1314 isolate xylophagous cockroach clone PA202, 1314

0.01

G CloneG11 WorkerTermite2 SJ, 609 G CloneE02 SoldierCommunity1 SJ, 615 G CloneE02 WorkerTermite1 SJ, 622 G CloneG09 WorkerTermite2 SJ, 611 G CloneC01 WorkerTermite1 SJ, 602

G CloneD09 SoldierTermite2 SJ, 620 G CloneA10 SoldierTermite2 SJ, 611 G CloneG02 SoldierCommunity1 SJ, 620 G CloneB01 SoldierCommunity1 SJ, 502

G CloneG10 WorkerTermite2 SJ, 611 G CloneF12 SoldierTermite2 SJ, 608 G CloneE03 SoldierCommunity1 SJ, 615 G CloneD04 WorkerTermite1 SJ, 664 G CloneC05 WorkerTermite1 SJ, 589

G CloneF02 WorkerTermite1 SJ, 615 G CloneH09 WorkerTermite2 SJ, 600 G CloneB03 SoldierCommunity1 SJ, 649 G CloneC03 SoldierCommunity1 SJ, 665 G CloneD05 SoldierCommunity1 SJ, 588 G CloneG03 SoldierCommunity1 SJ, 601 G CloneG05 SoldierCommunity1 SJ, 569 G CloneB10 SoldierTermite2 SJ, 588

G CloneC10 SoldierTermite2 SJ, 569 G CloneA09 SoldierTermite2 SJ, 619 G CloneE12 SoldierTermite2 SJ, 611 G CloneD11 WorkerTermite2 SJ, 613 G CloneC12 SoldierTermite2 SJ, 609 G CloneF09 SoldierTermite2 SJ, 618 G CloneE09 SoldierTermite2 SJ, 644 G CloneE10 SoldierTermite2 SJ, 520

G CloneB12 WorkerTermite2 SJ, 601 G CloneA11 SoldierTermite2 SJ, 620

G CloneH10 SoldierTermite2 SJ, 599 G CloneC09 SoldierTermite2 SJ, 603 G CloneF09 WorkerTermite2 SJ, 662 G CloneD02 WorkerTermite1 SJ, 648

G CloneC11 WorkerTermite2 SJ, 476 G CloneE08 WorkerTermite2 SJ, 659

G CloneH02 SoldierCommunity1 SJ, 464 G CloneF08 SoldierTermite2 SJ, 624

G CloneC11 SoldierTermite2 SJ, 462 G CloneA06 SoldierTermite2 SJ, 377

G CloneB06 SoldierTermite2 SJ, 530 G CloneC06 SoldierTermite2 SJ, 421 G CloneE10 WorkerTermite2 SJ, 507

G CloneD11 SoldierTermite2 SJ, 644 G CloneH04 WorkerTermite1 SJ, 611 G CloneF02 SoldierCommunity1 SJ, 600

G CloneE01 WorkerTermite1 SJ, 327 G CloneG05 WorkerTermite1 SJ, 889

G CloneH01 SoldierCommunity1 SJ, 404 G CloneF01 SoldierCommunity1 SJ, 404

G CloneH05 SoldierTermite2 SJ, 172 isolate xylophagous cockroach clone PA203, 1313

G CloneD08 SoldierTermite2 SJ, 110 AF293578termite.P3

Ar 9, 789 isolate xylophagous cockroach clone PA201, 1312

isolate xylophagous cockroach clone PA204, 1314 isolate xylophagous cockroach clone PA202, 1314

Figure 1: Phylogenetic tree obtained by Neighbor Joining, showing distribution of clone sequences and their closest relatives within the Thermoplasmata.

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Analysis of each of the four clone libraries in Dotur resulted in Rarefaction curves and Chao values showing saturation for all libraries (fig. 3a-3d).

0.1

G CloneB05 SoldierCommunity1 SJ, 666

Methanobrevibacter smithii , 1343 94

G CloneD03 WorkerTermite1 SJ, 666

Methanobrevibacter curvatus , 1358 91

G CloneG08 WorkerTermite2 SJ, 668

Methanobrevibacter filiformis , 1361 100

Methanobrevibacter ruminantium str. M1, 1260

66

Methanobrevibacter acididurans str. ATM, 1358

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G CloneC04 WorkerTermite1 SJ, 624

Methanobrevibacter cuticularis , 1312 84

Methanobrevibacter arboriphilus str. SA, 1441

77

G CloneG11 SoldierTermite2 SJ, 609

Methanobrevibacter sp. str . Mc30, 1310

82

74

90

Arcaheal methanogenic sludges UASB granular sludge, 1327

Methanobacterium thermoautotrophicum , 1330 78

Methanobacterium bryantii str. MOH, 1260

82

Methanosphaera stadtmaniae, 1419

RFM- 4

RFM- 2

RFM- 3

RFM- 1

RFM- 5

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G CloneB05 SoldierCommunity1 SJ, 666

Methanobrevibacter smithii , 1343 94

G CloneD03 WorkerTermite1 SJ, 666

Methanobrevibacter curvatus , 1358 91

G CloneG08 WorkerTermite2 SJ, 668

Methanobrevibacter filiformis , 1361 100

Methanobrevibacter ruminantium str. M1, 1260

66

Methanobrevibacter acididurans str. ATM, 1358

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G CloneC04 WorkerTermite1 SJ, 624

Methanobrevibacter cuticularis , 1312 84

Methanobrevibacter arboriphilus str. SA, 1441

77

G CloneG11 SoldierTermite2 SJ, 609

Methanobrevibacter sp. str . Mc30, 1310

82

74

90

Arcaheal methanogenic sludges UASB granular sludge, 1327

Methanobacterium thermoautotrophicum , 1330 78

Methanobacterium bryantii str. MOH, 1260

82

Methanosphaera stadtmaniae, 1419

0.1

G CloneB05 SoldierCommunity1 SJ, 666

Methanobrevibacter smithii , 1343 94

G CloneD03 WorkerTermite1 SJ, 666

Methanobrevibacter curvatus , 1358 91

G CloneG08 WorkerTermite2 SJ, 668

Methanobrevibacter filiformis , 1361 100

Methanobrevibacter ruminantium str. M1, 1260

66

Methanobrevibacter acididurans str. ATM, 1358

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G CloneC04 WorkerTermite1 SJ, 624

Methanobrevibacter cuticularis , 1312 84

Methanobrevibacter arboriphilus str. SA, 1441

77

G CloneG11 SoldierTermite2 SJ, 609

Methanobrevibacter sp. str . Mc30, 1310

82

74

90

Arcaheal methanogenic sludges UASB granular sludge, 1327

Methanobacterium thermoautotrophicum , 1330 78

Methanobacterium bryantii str. MOH, 1260

82

Arcaheal methanogenic sludges UASB granular sludge, 1327

Methanobacterium thermoautotrophicum , 1330 78

Methanobacterium bryantii str. MOH, 1260

82

Methanosphaera stadtmaniae, 1419

RFM- 4

RFM- 2

RFM- 3

RFM- 1

RFM- 5

RFM- 4

RFM- 2

RFM- 3

RFM- 1

RFM- 5

Figure 2: Phylogenetic consensus tree obtained by several treeing approaches, showing representatives for each clone sequences cluster and the closest relatives within the Methanobrevibacter.

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Rarefaction curve: Worker library 2

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Figure 3: Results from clone library analysis in Dotur showing rarefaction curves and Chao values for each library.

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Table 2 shows the number of clone sequences from each library distributed within each of the five OTUs. The distribution in percentage of Methanobrevibacter clone sequences from the worker and soldier libraries, respectively (libraries beeing pooled) are shown in figure 4. OTU Worker 1 Worker 2 Soldier 1 Soldier 2 RFM-1 2 0 0 0 RFM-2 8 1 8 6 RFM-3 16 18 7 6 RFM-4 5 3 3 4 RFM-5 0 0 3 1 Total 31 22 21 17

Worker libraries

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Libshuff analysis showed no difference among the four clone libraries, neither the entire libraries nor the libraries of Methanobrevibacter only. Pooling the two libraries of workers and soldiers, respectively, did not change this result.

A

B

Figure 4: Distribution of clone sequences within the distinct OTUs of Methanobrevibacter. Distiributions are given as the percentage of total Methanobrevibacter sequences obtained from each clone library. The two clone libraries of workers and of soldiers are pooled.

Tabel 1: Number of clone sequences from each library distributed within the distinct OTUs of Methanobrevibacter

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F420 Microscopy of termite hindguts A total of 9 worker and 9 soldier hindguts were dissected and examined by microscopy. Results showed no clear differences in methanogen morphotypes existing in the two casts, neither was there any robust quantitative difference in the abundance of each morphotype. However, the Methanobrevibacter filiformis morphotype (fig. 5b) generally seemed to be present in higher abundance in the worker termites than in the soldier termites. Similarly, soldier termites seemed to contain a higher abundance of straight rod shaped methanogens associated with the surface of segmented filamentous spore forming bacteria (fig. 5c). In both termite casts a high abundance of the Mbb. curvatus was observed associated with the gut wall, while cells of the Mbb. cuticularis morphotype were hard to detect (except the rods associated with the filamentous bacteria).

GC analysis of in situ methane emission rates GC measurements of the methane production rates were associated with quite a bit of uncertainty due to incorrect positioning of the baseline in the GC software leading to incorrect readings of peak areas. However, methane production over time showed clear differences between soldier and worker incubations, as well as atmospheric versus hydrogen enriched incubations (figure 6a). The methane production pr. decapitated body weight was more than twice as high in the worker termites as compared to the soldier termites (0.72 versus 0.30 �mo l/g decapitated ww/h). In both termite casts, the production rates were highly stimulated by addition of 10% hydrogen to the bottle headspace (rates increased with 201% and 163% for soldier and worker termites, respectively).

A B C

Figure 5: Pictures obtained from F420 microscopy of termite hindguts, showing morpholgies of a) Mbb. curvatus, b) Mbb. filiformis, and c) rod shaped methanogen associated with filamentous bacteria.

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Discussion Phylogenetic analysis and microscopy of hindgut communities The first surprising outcome of the phylogenetic community analysis was the fairly high abundance of Thermoplasma related sequences within all four libraries. About one fourth of the clones from the worker hindguts and almost the half of the clones from the soldier hindguts were distributed within this phylogenetic group (table 1). Previous community analyses of the termite hindgut have shown archaean sequences mainly clustering within the Methanobacteriacaea. However, a few Thermoplasmales sequences (3) have been found in the lower termite Reticulitermes speratus (Shinzato et al., 1999) and also Thermoplasmales sequences have been shown abundant in the higher termite species, Cubitermes orthognathus (Friedrich et al., 2001). All the sequences from the worker and soldier libraries clustered quite closely within this group, showing more than 99% similarity to one another. The closest relatives were sequences obtained from cockroach guts (93-99.1% similarity) and a partial 16S rRNA gene sequence obtained from the gut of the higher termite, Cubitermes orthognathus (96-98.0% similarity). Among closely related sequences, also clones from the sheep rumen were found.

In situ Methane production

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Figure 6: Results of GC analysis showing a) Increase in methane concentration in each bottle as a function of time, b) calculated methane production rates pr. g. decapitated termite weight.

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B

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No sequences from characterized isolates were found to be related, and therefore the physiology of this phylogenetic group remains unknown. However, the group seems to comprise sequences associated with a variety of gut systems, and given their high abundance in the clone libraries from this study, it seems very likely that they would play a significant role in the microbial community of the R. flavipes hindgut. In the future work on investigating the termite hindgut, it would thus be highly interesting to obtain an isolate from this group in order to characterize its physiology and potential role in the gut community. All methanogen sequences obtained clustered within the Methanobrevibacter genus. Distance matrixes showed that, based on a 97% similarity level, the sequences could be divided into five distinct OTUs, possibly representing different species (fig. 2). In the two worker and the two soldier libraries 4, 3, 4, and 4 of these OTUs were represented, respectively. When including the extra OTU of the Thermoplasmata, these numbers did not quite correspond with the Dotur Chao estimates showing one less OTU for each library, than estimated from the distance matrix. However, under the assumption that the five Methanobrevibacter OTUs are representing different species, these results then include two additional methanogen species than was previously recognized in the gut of Reticulitermes flavipes. In addition to the characterized isolates of Mbb. cuticularis, Mbb. curvatus, and Mbb. filiformis (RFM-1, RFM-2, and RFM-3), a Mbb. smithii-like OTU (RFM-4) and an OTU including Mbb. sp. str. Mc30 (RFM-5) were represented among the clones. Mbb. smithii is an isolate from feces of humans and other animals. The sequence showed 96.7-97.2% similarity with the clone sequences and thus may be closely related but not necessarily belong to the same species. The Mbb. sp. str. Mc30 is found in the gut of a higher termite and shows 97.9-99% similarity to the clone sequences in this OTU. However it is not a characterized isolate, and the second closest relative, Mbb. arboriphilus, which is isolated from rotting wood, is more closely related to the RFM-1 than to the RFM-5. Libshuff analysis comparing the four clone libraries showed no significant difference among soldier and worker communities, neither in the entire community nor the Methanobrevibacter community. Therefore, no statistically robust conclusions can be drawn on this question. However, combined with results of F420 microscopy, the distribution of clone sequences within the different OTUs may suggest some tendencies in the worker versus soldier libraries (fig. 4). Both the microscopy of the gut wall and the distribution of clone sequences suggest a higher abundance of Mbb. filiformis in the worker hindgut as compared to the soldier hindgut. During microscopy, the morphotype of Mbb. cuticularis was hardly detectable, which corresponded well with the very low frequency of clones within this OTU (represented by only two clone sequences from the Worker 1 library). In both termite casts Mbb. cuticularis showed fairly high abundance in both clone libraries and microcopy observations. The Mbb. sp. str. Mc30 related OTU (RFM-5) was only represented in the two soldier libraries (with 3 and 1 sequence, respectively). Due to the low number of sequences from this OTU, this result might not be statistically significant, but it suggest the possibility of this OTU being either soldier specific or more abundant in soldier versus worker hindguts. One might speculate, whether these methanogens are the ones associated with filamentous sporeforming bacteria, which seemed to be more frequently observed in the soldier hindguts. To interpret these results further, it would be interesting to construct specific primers for the distinct Methanobrevibacter OTUs and hybridize them to the termite guts. This would allow a more quantitative approach in comparing the soldier and the worker communities, as well as the identification of the morphotypes belonging to each OTU and their general abundance in the

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termite gut. Confocal Laser Scanning Microscopy may be of usage in order to obtain a better focus when examining the hindgut wall. GC analysis of in situ methane production rates In situ methane production showed to be more than twice as high in the worker as in the soldier termites. In both casts, methanogenesis was highly increased when saturated with hydrogen. These results suggest that methanogenesis in the termite hindgut is hydrogen limited. Micro electrode measurements in the hindgut of Reticulitermes flavipes have shown that the hydrogen concentration reaches saturation in the center of the gut, while it is decreasing towards the gut epithelia where the methanogens are mainly located (Ebert & Brune, 1997). Acetogens competing with methanogens for hydrogen are located in the gut lumen, and combined with the results showing methanogens to be hydrogen limited, these differences in bacterial location may partly explain why acetogens are able to compete efficiently and coexist with methanogens in the termite gut. The lower methane production rates in the soldier hindgut might be caused by a lower hydrogen pressure in these cast members (e.g. caused by differences in fermentation rates due to distinct feeding-behavior). Using micro electrodes to test this hypothesis would be an interesting approach in the further investigation of possible differences among soldier and worker hindgut biotas. Aknowledgements A special thanks to Jared Leadbetter, David Walsh, Kou-San Ju, and Dionysios Antonopoulos for spending incredible amounts of time and energy providing help and advice for this study. Also a thanks to all other faculty, teaching assistants and fellow student in the course of Microbial Diversity, 2006 for helping out and sharing experiences. References

• Ebert, A., Brune, A. 1997. Hydrogen concentration profiles at the oxic-anoxic interface: a microsensor study of the hindgut of the wood-feeding lower termite Reticulis flavipes. Appl. Environ. Microbiol. 63(10): 4039-4046.

• Friedrich, M. W., Schmitt-Wagner, D., Lueders, T. 2001. Axial differences in community structure of Crenarchaeota and Euryarchaeota in the highly compartmentalized gut of the soil-feeding termite Cubitermes orthognathus. Appl. Environ. Microbiol. 67(10):4880-4890

• Leadbetter, J.R. and Breznak, J.A. 1996. Physiological ecology of Methanobrevibacter cuticularis sp. nov. and Methanobrevibacter curvatus sp. nov., isolated from the hindgut of the termite Reticulitermes flavipes. Appl. Environ. Microbiol. 62:3620-3631

• Leadbetter, J.R., Crosby, L.D., and Breznak, J.A. 1998. Methanobrevibacter filiformis sp. nov., a filamentous methanogen from termite hindguts. Arch. Microbiol. 169:287-292

• Löffler, F.E., Ritalahti, K.M., and Tiedje, J.M. 1997. Dechlorination of Chloroethenes Is Inhibited by 2-Bromoethanesulfonate in the Absence of Methanogens. Appl. Environ. Microbiol. 63(12):4982-4985

• Shinzato, N., Matsumoto, T., Yamaoka, I., Oshima, T., and Yamagishi, A. 1999. Phylogenetic diversity of symbiotic methanogens living in the hindgut of the lower termite Reticulitermes speratus analyzed by PCR and in situ hybridization. Appl. Environ. Microbiol. 65:837-840.

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