Formyltetrahydrofolate Synthetase Gene Diversity in the Guts of Higher Termites with Different Diets and Lifestyles

Abstract

In this study, we examine gene diversity for formyl-tetrahydrofolate synthetase (FTHFS), a key enzyme in homoacetogenesis, recovered from the gut microbiota of six species of higher termites. The “higher” termites (family Termitidae), which represent the majority of extant termite species and genera, engage in a broader diversity of feeding and nesting styles than the “lower” termites. Previous studies of termite gut homoacetogenesis have focused on wood-feeding lower termites, from which the preponderance of FTHFS sequences recovered were related to those from acetogenic treponemes. While sequences belonging to this group were present in the guts of all six higher termites examined, treponeme-like FTHFS sequences represented the majority of recovered sequences in only two species (a wood-feeding Nasutitermes sp. and a palm-feeding Microcerotermes sp.). The remaining four termite species analyzed (a Gnathamitermes sp. and two Amitermes spp. that were recovered from subterranean nests with indeterminate feeding strategies and a litter-feeding Rhynchotermes sp.) yielded novel FTHFS clades not observed in lower termites. These termites yielded two distinct clusters of probable purinolytic Firmicutes and a large group of potential homoacetogens related to sequences previously recovered from the guts of omnivorous cockroaches. These findings suggest that the gut environments of different higher termite species may select for different groups of homoacetogens, with some species hosting treponeme-dominated homoacetogen populations similar to those of wood-feeding, lower termites while others host Firmicutes-dominated communities more similar to those of omnivorous cockroaches.

INTRODUCTION

The insect order Isoptera is divided into seven major families. Six of these families are comprised of “lower termites,” which are exclusively wood and/or grass feeders. The “higher termites” are a single family (Termitidae), which nonetheless contains about 85% of known genera (10). Higher termites are able to utilize a much broader range of substrates than lower termites; in addition to wood and grass feeding, higher termite species have evolved fungus-cultivating, litter- and soil-feeding lifestyles. Higher termites with different feeding habits have been found to have vastly different complements of symbiotic bacteria (21, 32, 33, 35).

The symbiosis between termites and their gut microbes is a complex, obligate mutualism. The hindgut community acts as a highly efficient bioreactor, converting complex substrates to acetate, which constitutes the principal source of energy for the termite (22). In wood-feeding termites, H₂ is the central free intermediate in the degradation of lignocellulose, representing 18 to 26% of the termite's respiratory activity (27). The majority (83 to 100%) of this hydrogen is converted to acetate through homoacetogenesis (27). In contrast, soil-feeding and fungus-cultivating termites have been shown to have relatively lower rates of gut homoacetogenesis and higher rates of methanogenesis from H₂ and CO₂ (3), suggesting that diet can have a large impact on the homoacetogenic community.

While most termite gut homoacetogens are as yet uncultured, the diversity of organisms that may be capable of carrying out this activity can be assessed using primers that target the gene for formyl-tetrahydrofolate synthetase (FTHFS), a key enzyme in the Wood-Ljungdahl pathway of homoacetogenesis (12, 14). In lower termites, the dominant FTHFS types group phylogenetically with FTHFS genes from homoacetogenic spirochetes of the genus Treponema (26, 31). The recent metagenome of microbes inhabiting the gut of the wood-feeding higher termite Nasutitermes revealed the presence of termite treponeme-like FTHFS genes (35). However, the fragmentary nature of that data set precludes detailed phylogenetic analysis of those sequences, and a survey of FTHFS genes in other species of higher termites has not yet been presented. Here we explore the diversity of homoacetogenic organisms present in 6 species of higher termites having diverse feeding regimes.

MATERIALS AND METHODS

Insect collection.

Nasutitermes sp. Cost003 and Rhynchotermes sp. Cost004 were collected in the INBio forest preserve in Guápiles, Costa Rica. Nasutitermes sp. Cost003 was collected at a height of 1.2 m in a Psidium guajaba tree and appeared to be feeding on deadwood. Rhynchotermes sp. Cost004 was collected in the same area from a nest located under an unidentified bromeliad, with feeding trails leading to a large pile of wet, decaying plant leaves, consistent with a litter-feeding lifestyle. Microcerotermes sp. Cost008 was collected from the base of a palm tree about 100 m from the beach at Cahuita National Park in Costa Rica and appeared to be feeding on dead portions of the same plant. Amitermes sp. Cost010 was collected from the roots of dead sugar cane plants at a Costa Rican plantation. Amitermes sp. JT2 and Gnathamitermes sp. JT5 were collected from subterranean nests at Joshua Tree National Park.

DNA extraction.

Guts were extracted from termites within 48 h of collection. Whole guts were collected from 20 workers of each species. Extracted whole guts were suspended in 500 μl 1× TE (10 mM Tris, 1 mM EDTA, pH 7.4) and stored at −20°C until DNA purification. DNA was purified from gut samples using a bead-beating lysis followed by DNeasy purification (Qiagen), as described by Matson et al. (17).

FTHFS amplification, cloning, and RFLP analysis.

FTHFS genes were amplified from insect guts as described by Leaphart and Lovell (12). Primers with 5′ phosphate groups were purchased from Integrated DNA Technologies. Amplification reactions for cloning contained 1 μM (each) primer, 1× Failsafe premix D (Epicentre), and 0.0525 U/μl Expand high-fidelity Taq polymerase (Roche). FTHFS was amplified from Cost003 in reaction mixtures containing 1 ng/μl template and following the recommended step-down protocol (12), followed by 25 cycles at an annealing temperature of 55°C. All other termite samples contained low levels of PCR-inhibiting compounds and required further dilution; these reaction mixtures contained 0.1 ng/μl template and were amplified for an additional 5 cycles at 55°C to generate a similar final concentration of product. PCRs were purified using QIAquick PCR purification kits (Qiagen) and cloned using a GC cloning and amplification kit with the LC-Kan vector (Lucigen).

Cloned PCR products were screened by restriction fragment length polymorphism (RFLP) analysis. Isolated colonies were picked and placed in 10 μl 1× TE and then incubated at 95°C for 5 min. This lysate was used for amplification reactions to generate a template for RFLP analyses and sequencing. Inserts were amplified using the vector primers SL1 and SR2 (Lucigen), FailSafe premix A (Epicentre), and 0.05 U/μl Taq polymerase (NEB). The thermocycling protocol was as follows: 3 min as 95°C, 30 cycles (95°C for 30 s, 55°C for 30 s, and 72°C for 1 min 30 s), and then 10 min at 72°C. RFLP typing used the enzyme HinP1I (NEB): 6 μl of the PCR product was added to 0.4 μl 10× NEB buffer 2, 0.3 μl HinP1I (NEB), and 3.3 μl H₂O and then digested at 37°C for 4 h. A single representative clone of each RFLP type was amplified for sequencing using the protocol above and substituting Expand high-fidelity polymerase (Roche) for Taq DNA polymerase.

COII identification of termites.

For each species of Joshua tree termite, COII gene fragments were amplified using the supernatant of a mixture containing an individual termite head crushed in 1× TE as a template. Termite COII was amplified using the primers CI-J-1773 and B-tLys and cycling conditions described by Miura et al. (20). Reactions included FailSafe premix D (Epicentre) and Expand high-fidelity Taq polymerase (Roche).

The identification of the Nasutitermes, Microcerotermes, and Amitermes termites was confirmed to the genus level with molecular phylogeny (see Fig. S1 in the supplemental material). Nasutitermes sp. Cost003 was collected within 30 feet of the Nasutitermes sp. FK-2007 nest previously collected for metagenomic sequencing (35) and had an identical COII gene sequence. No COII genes were available in public databases for Rhynchotermes or Gnathamitermes, so identification of Cost004 and JT5 relied on termite morphology (6, 36). The COII gene from the Gnathamitermes genus groups closely with sequences from Amitermes termites. The genus Rhynchotermes is typically classified as a member of the Nasutiterminae subfamily. However, this family is paraphyletic (2, 9), and Rhynchotermes sp. Cost004 grouped phylogenetically with termites from the proposed subfamily Syntermitinae (5).

Sequence analysis.

Sequence reads were assembled and edited using the Lasergene software package (version 7.2.1; Dnastar, Inc.). FTHFS protein sequences were aligned and phylogenetic analyses were carried out using the ARB software package (16). Libraries were screened for chimeric sequences using the Bellerophon software program (8); a single putative chimeric sequence each was eliminated from the Nasutitermes sp. Cost003 and Rhynchotermes sp. Cost004 libraries.

Nucleotide sequence accession numbers.

Sequences generated in this study have been deposited in GenBank under accession numbers JF431107 to JF431248.

RESULTS AND DISCUSSION

FTHFS libraries were constructed from 4 species of higher termite from Costa Rica and 2 desert-adapted species from California (Table 1). Phylogenetic analysis of recovered FTHFS shows a broad diversity (Fig. 1). With the exception of a sequence that grouped with sulfate-reducing Proteobacteria and another that could not be placed reliably within the FTHFS phylogeny (Gnathamitermes clones 2F and 2E, respectively; not shown), all of the recovered sequences grouped with either Firmicutes- or Treponema-like FTHFS sequences. While the Treponema-like FTHFS types formed a monophyletic clade with other insect gut-derived clones, the Firmicutes-like FTHFS types represented a number of distinct clades, some of which most likely represent nonacetogenic organisms. Major clades, discussed in detail below, include the Clostridium sp. M62/1 group and two groups of probable purinolytic organisms, the clone E/Streptococcus group and the Clostridium acidurici group.

Table 1.

FTHFS libraries constructed in this study

Species	No. of clonesa	No. of RFLP typesa	No. of OTU (98% aa)b
Nasutitermes sp. Cost003	52	19	14
Rhynchotermes sp. Cost004	61	41	29
Microcerotermes sp. Cost008	27	16	12
Amitermes sp. Cost010	26	18	17
Amitermes sp. JT2	89	23	14
Gnathamitermes sp. JT5	60	24	22

^aExcludes non-full-length clones and RFLP types and clones determined to be chimeric.

^bOperational taxonomic unit (OTU) classification was based on at least 98% amino acid identity.

Fig. 1.

Phylogenetic analysis of higher termite FTHFS clones and selected relatives. The tree was constructed using 317 unambiguous, aligned amino acids and the PhyML maximum-likelihood algorithm. Homoacetogen similarity (HS) scores are shown for each isolate or environmental clone. Open circles indicate nodes also supported by either the Fitch distance or Phylip parsimony method. Closed circles indicate nodes supported by all three algorithms. The scale bar indicates 0.1 changes per alignment position. Inset: phylogenetic analysis of termite and roach mitochondrial COII genes. The tree was calculated using 394 unambiguous, aligned DNA bases. The scale bar indicates 0.1 changes per alignment position. A more detailed phylogenetic analysis of COII genes is presented in Fig. S1 in the supplemental material.

To complement phylogenic analysis, a homoacetogen similarity (HS) score was calculated according to the method of Henderson et al. (7). This measure represents the percentage of residues in 40 alignment positions shared with known homoacetogens. Clades identified as belonging to probable purinolytic Firmicutes and other nonacetogens had low HS scores. Many of the termite treponemes and affiliated sequences had high HS scores (≥95), as did a number of sequences in the M62/1 clade, suggesting that at least some of the sequences in these groups represent novel homoacetogens.

Higher termite treponemes.

All higher termites examined yielded at least one FTHFS sequence that affiliated with the “Termite treponemes and affiliates” clade. Most of the sequences included in this group share a hexapeptide insert absent from other homoacetogens (31). The basal Microcerotermes and Cryptocercus punctulatus clusters lack this indel but were included based on the strength of their phylogenetic association with treponeme-like sequences (robustly grouping with this clade by all three treeing methods). The majority of the treponeme-affiliated sequences amplified from higher termites formed a single cluster (detailed in Fig. 2) with a single sequence from the lower termite Reticulitermes santonensis. This cluster represents a large diversity of sequences, including an extensive radiation of sequences from the guts of Rhynchotermes termites and a highly microdiverse cluster of sequences from Nasutitermes. The only treponeme-affiliated sequences that fell outside this cluster were a group of sequences found in Microcerotermes and a single sequence identified in Amitermes sp. Cost010.

Fig. 2.

Phylogenetic analysis of the higher termite treponeme clade. Tree were constructed using 344 unambiguous, aligned amino acids and the PhyML maximum-likelihood algorithm. The HS score is shown next to each clone name. Open circles indicate nodes also supported by either the Fitch distance or Phylip parsimony method. Closed circles indicate nodes supported by all three algorithms. The scale bar indicates 0.1 changes per alignment position. The outgroup consisted of the 13 acetogenic isolates in Fig. 1 plus Treponema spp. ZAS-8 and ZAS-9.

Sequences affiliated with Clostridium sp. M62/1.

The majority of sequences recovered from two Amitermes termites affiliated with a large group of Firmicutes-like FTHFS sequences (the Clostridium sp. M62/1 clade in Fig. 1; phylogenetic detail is in Fig. 3), as do a large fraction of FTHFS sequences recovered in a similar study of the gut microbiota of the omnivorous cockroach Periplaneta americana (24). Clostridium sp. M62/1 is a butyrate-producing firmicute (13); to our knowledge, that isolate has not been examined for homoacetogenic growth. This group is highly diverse, with relatively large evolutionary distances between recovered clones. Although a large diversity of genes belonging to this clade have been identified in the guts of several termites and cockroaches, very few animal gut sequences fall within this group. The exceptions include Clostridium sp. M62/1 and single clones from FTHFS libraries of human feces (23), the bovine rumen (18), and the rumen of silage-fed deer (7).

Fig. 3.

Phylogenetic analysis of the Clostridium sp. M62/1 clade. The tree was constructed using 327 unambiguous, aligned amino acids and the PhyML maximum-likelihood algorithm. The HS score is shown next to each clone. Open circles indicate nodes also supported by either the Fitch distance or Phylip parsimony method. Closed circles indicate nodes supported by all three algorithms. The scale bar indicates 0.1 changes per alignment position. The 13 acetogenic isolates in Fig. 1 were used as an outgroup.

It remains unclear whether the M62/1 clade includes CO₂-reductive acetogens. As mentioned above, M62/1 itself is not known to be capable of homoacetogenic growth, and the low HS score for its FTHFS gene and absence of a recognizable carbon monoxide dehydrogenase/acetyl coenzyme A synthetase gene in the draft genome are not suggestive of homoacetogenic capacity. However, this does not preclude homoacetogenic capacity for other members of the M62/1 clade; many members have intermediate (>90) to high (>95) HS scores. P. americana is known to exhibit low but detectable rates of acetate synthesis from CO₂ (4, 11), and organisms belonging to this clade remain the best candidates for potential acetogens of the FTHFS sequences recovered in libraries of P. americana (24). Observations of soil-feeding Cubitermes termites, which also have low measured rates of in situ CO₂ fixation to acetate (3, 34), also show a robust population of CO₂-reductive acetogens, detectable when gut homogenates are incubated with inhibitors of methanogenesis (34). While we have not yet determined the native diets or rates of gut methanogenesis of the Amitermes and Gnathamitermes termites examined in this study, it is suggestive that they yielded FTHFS populations more similar to those found in an omnivorous cockroach than to those found in their wood-feeding relatives.

Potentially purinolytic FTHFS clades.

Finally, although the primers utilized in this study were designed for specific detection of homoacetogenic FTHFS types, several intriguing probable nonacetogenic FTHFS types were identified in higher termite libraries. In these organisms, FTHFS seems likely to be playing a role in degradation of purines and/or amino acids.

One such clade, described as the Clostridium acidurici clade (Fig. 4 A), included sequences that affiliated with FTHFS sequences from the purinolytic firmicutes C. acidurici (1), Clostridium cylindrosporum (1), and Eubacterium acidaminophilum (37). In these organisms, anaerobic degradation of purines results in the transfer of a formimino group to tetrahydrofolate (THF). Formimino-THF is converted to formyl-THF, and FTHFS catalyzes the release of formate and THF and the generation of ATP via substrate-level phosphorylation. Recycling of uric acid by gut bacteria has been hypothesized to play a role in termite nitrogen conservation (28), and the presence of this FTHFS clade suggests that members of the Firmicutes may be carrying out this activity within the guts of Rhynchotermes and Gnathamitermes termites. Litter-feeding Rhynchotermes termites have been shown to have lower rates of nitrogen fixation than wood-feeding Nasutitermes (30). While this may be attributed to higher nitrogen content in their food source, uric acid recycling may also play a role.

Fig. 4.

Phylogenetic analysis of the Clostridium acidurici (A) and clone E/Streptococcus (B) clades. Trees were constructed using 344 (C. acidurici) or 343 (clone E) unambiguous, aligned amino acids and the PhyML maximum-likelihood algorithm. Open circles indicate nodes also supported by either the Fitch distance or Phylip parsimony method. Closed circles indicate nodes supported by all three algorithms. Scale bars indicate 0.1 changes per alignment position. The 13 acetogenic isolates in Fig. 1 were used as an outgroup for both trees.

Another group of FTHFS types potentially linked to purine or amino acid degradation is the clone E/Streptococcus clade (Fig. 4B). While the genomic contexts of the FTHFS genes from most of the firmicutes in this group do not provide obvious functional clues, in Enterococcus gallinarum the FTHFS is near a histidine-ammonia lyase and glutamate formiminotransferase, and the clone E-like FTHFS sequences in the three streptococci are part of a conserved histidine degradation operon. While the use of FTHFS to generate ATP from the release of formate during histidine degradation has not been formally reported for bacteria, the presence of glutamate formimidoyltransferase in certain bacterial histidine degradation operons has been observed in other bioinformatic analyses (25). This entire clade may represent FTHFS types employed in this manner, or alternatively it may represent FTHFS types adapted more generally to catalyzing formyl-THF metabolism rather than synthesis. One of the uricolytic strains isolated from Reticulitermes flavipes by Potrikus and Breznak (29) was a Streptococcus species; the termite-derived sequences may represent FTHFS genes from similar organisms. Interestingly, all of the Streptococcus species in this cluster have a second FTHFS variant that is closely related to those from other streptococci and which falls within a general Lactobacillus radiation (data not shown). It is therefore tempting to speculate that these Streptococcus species acquired the clone E-like FTHFS variant via lateral gene transfer from the Firmicutes and that it is primarily utilized in the context of histidine degradation, while the other FTHFS variant is used for general cellular folate metabolism.

Implications.

The diversity of lifestyles and feeding strategies employed by higher termites coincides with a diversity of population structures among symbiotic homoacetogens (Table 2). FTHFS sequences amplified from wood-feeding Nasutitermes termites and palm-feeding Microcerotermes termites affiliate with the homoacetogenic treponemes that dominate the guts of wood-feeding lower termites. The Rhynchotermes termites used in this study came from a colony that appeared to be feeding from a nearby compost pile with a mixture of woody and leaf detritus and had approximately equal representation of treponeme-like FTHFS and sequences affiliated with purinolytic firmicutes. The remaining termites were recovered from subterranean nests, although the degree to which they were feeding on soil or nearby plant material remains uncertain. These termites, whose diets may include some level of soil feeding (and who certainly experience greater exposure to soil), yielded a diversity of sequences that affiliate with the Firmicutes but few treponeme-like FTHFS sequences. The majority of the FTHFS types recovered from both Amitermes termites fell within the M62/1 clade, while the Gnathamitermes library was split between M62/1 and purinolytic FTHFS types. UniFrac measures of community distance (15) support these observations (see Fig. S3 and S4 in the supplemental material), grouping the Nasutitermes- and Microcerotermes-derived libraries with those from wood-feeding cockroaches and lower termites while identifying the two Amitermes-derived libraries as more similar to the omnivorous cockroach P. americana. When purinolytic FTHFS clades were included in the analysis, Rhynchotermes and Gnathamitermes termites were identified as distinct from other termite groups, but when the clone E and C. acidurici clades were excluded from the analysis, Rhynchotermes grouped with the wood-feeding termites while Gnathamitermes was grouped with the Amitermes spp. and P. americana.

Table 2.

Composition of FTHFS libraries from the hindgut microbiota of termites and relatives^a

Species	Food source	% Sequence abundance
		Termite treponemes	Clostridium sp. M62/1	Clone E/Streptococcus	Clostridium acidurici	Other
P. americana	Unknown	NF	41	NF	NF	59
C. punctulatus adult	Wood	88	2	2	NF	8
C. punctulatus nymph	Wood	50	3	41	NF	6
Z. nevadensis	Wood	77	NF	4	NF	19
C. secundus	Wood	97	NF	NF	NF	2
Incisitermes sp. Pas1	Wood	100	NF	NF	NF	NF
R. santonensis	Wood	98	NF	1	NF	1
Nasutitermes sp. Cost003	Woodb	98	NF	NF	NF	2
Microcerotermes sp. Cost008	Palmb	89	NF	NF	NF	11
Rhynchotermes sp. Cost004	Litterb	38	7	NF	46	10
Amitermes sp. Cost010	Sugarcane/soilb	12	69	NF	NF	19
Amitermes sp. JT2	Grass/soilb	1	82	NF	NF	17
Gnathamitermes sp. JT5	Grass/soilb	2	25	37	10	27

^aSequence abundance for each major FTHFS clade is given as a percentage of total clones examined; NF, not found. P. americana, C. punctulatus, and Incisitermes data are from the work of Ottesen and Leadbetter (24), Z. nevadensis data are from Salmassi and Leadbetter (31), and C. secundus and R. santonensis data are from Pester and Brune (26).

^bFood source is unknown; probable sources are based on nest location and/or feeding trails.

The Rhynchotermes, Amitermes, and Gnathamitermes termites examined here represent the first examples of termite gut communities that are not dominated by FTHFS sequences from the termite treponeme clade. Given that treponeme-associated FTHFS types are present in these termites, it seems likely that this shift in community structure is due to the presence of conditions that favor this group over homoacetogenic treponemes. In numerous studies of FTHFS genes associated with animal guts (7, 12, 18, 19, 23), an abundance of Firmicutes-like and no treponeme-like FTHFS sequences have been discovered. It has been broadly observed that wood-feeding termites (both higher and lower) have higher rates of homoacetogenesis than soil feeders; this may correlate with a uniquely favorable environment for homoacetogenic treponemes.