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Applied and Environmental Microbiology, July 2009, p. 4324-4332, Vol. 75, No. 13
0099-2240/09/$08.00+0     doi:10.1128/AEM.00455-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Bacterial Associates of Arboreal Ants and Their Putative Functions in an Obligate Ant-Plant Mutualism ^,

Sascha Eilmus¹ and Martin Heil^1,2*

Department of General Botany-Plant Ecology, University of Duisburg-Essen, Universitätsstraße 5, D-45117 Essen, Germany,¹ Departamento de Ingeniería Genética, CINVESTAV—Irapuato, Km. 9.6 Libramiento Norte, Carretera Irapuato-León, 36821 Irapuato, Guanajuato, Mexico²

Received 24 February 2009/ Accepted 5 May 2009

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Bacterial communities are highly diverse and have great ecological importance. In the present study, we used an in silico analysis of terminal restriction fragments (tRF) to characterize the bacterial community of the plant ant Pseudomyrmex ferrugineus. This species is an obligate inhabitant of Acacia myrmecophytes and feeds exclusively on plant-derived food sources. Ants are the dominant insect group in tropical rain forests. Associations of ants with microbes, which contribute particularly to the ants’ nitrogen nutrition, could allow these insects to live on mostly or entirely plant-based diets and could thus contribute to the explanation of the high abundances that are reached by tropical ants. We found tRF patterns representing at least 30 prokaryotic taxa, of which the Acidobacteria, Actinobacteria, Bacteroidetes, Firmicutes, Planctomycetes, Proteobacteria, and Spirochaetes comprised 93%. Because most bacterial taxa were found in all ant-derived samples studied and because the bacteria detected on the ants’ host plant revealed little overlap with this community, we regard our results as reliably representing the bacterial community that is associated with P. ferrugineus. Genera with a likely function as ant symbionts were Burkholderia, Pantoea, Weissella, and several members of the Enterobacteriaceae. The presence of these and various other groups was confirmed via independent PCR and cultivation approaches. Many of the bacteria that we detected belong to purportedly N-fixing taxa. Bacteria may represent important further partners in ant-plant mutualisms, and their influences on ant nutrition can contribute to the extraordinary abundance and evolutionary success of tropical arboreal ants.

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Bacteria are ubiquitous and play important roles in almost every ecological community, including various functions as further partners in the interactions among higher organisms (5, 16, 19, 24, 36, 59). Unfortunately, we lack suitable cultivation protocols for many environmental bacteria, and our knowledge of their distribution, diversity, and ecological function thus remains restricted. Similarly, bacteria such as "Candidatus Blochmannia," Buchnera, Rickettsia, and Wolbachia are extremely common intracellular endosymbionts of insects (10, 22, 40, 46, 54), but many further symbionts of insects likely remain to be discovered. Therefore, we need methods for the unbiased description of the entire microbial community of insects in order to gain a more general overview of their effects on the ecology and evolution of their hosts.

Almost the same problems apply to ants. Ants are the dominant insect group and can comprise more than 80% of the arthropods in tropical rain forests (9, 23, 47), but it is not understood how ants can reach this abundance. Strikingly, ants reach much higher biomass values than their supposed prey organisms, a phenomenon termed "Tobin's ant biomass paradoxon" (8, 9, 47). It has been suggested that dominant ant taxa might be "more herbivorous" than originally assumed (8). However, plant-derived food sources appear too low in nitrogen to allow the construction of the ant's bodies, and the question thus remained open how these "vegetarian" ants can obtain balanced alimentation.

One possible explanation would be the existence of ant-microbe mutualisms (8, 59), because many insects depend on symbiotic bacteria for their nutrition (see, e.g., references 5, 36, and 57). Camponotus ants harbor intracellular "Ca. Blochmannia" symbionts (4), which play an important role in their hosts’ nitrogen metabolism (16, 57, 59). Similarly, arboreal Tetraponera ants carry gut bacteria that might be able to recycle waste nitrogen or fix atmospheric nitrogen (46, 48). While many symbionts appear to be engaged in a mutualism with their insect host, Wolbachia has been reported to parasitize many insects through changing their sex ratios (25). The impact of Wolbachia infection in ants, however, remains enigmatic (52). In summary, no generalizations on the interactions between bacteria and ants are possible thus far, because our knowledge of bacteria that are associated with the most successful eusocial insects on earth remains scattered.

The goal of the present study was to obtain an impression of the entire bacterial community that is associated with the ant Pseudomyrmex ferrugineus. This species is an obligate plant ant, which defends its host plants against herbivores and encroaching vegetation and feeds exclusively on host-derived food rewards, that is, cellular food bodies and extrafloral nectar (6, 26, 27). Having a very specialized life history and depending on a well-defined type of strictly plant-derived alimentation, this species appeared particularly suitable for discovery of bacteria that could play a role in the success of "vegetarian" arboreal ants. Because larvae feed on food bodies while adult ants appear to feed only on extrafloral nectar, both developmental stages were considered. We used the assignment tool TReFID (42), which employs in silico analysis of terminal restriction fragments (tRF) for the gross characterization of bacterial life in a sample, to answer the following questions: (i) Which bacterial taxa can be found to be associated with this ant species? (ii) Which of the TReFID-based identifications can be confirmed by classical cultivation and sequencing methods? (iii) Does TReFID reveal reproducible results? We finally conducted an extensive literature and database search in order to discuss likely functions of these bacteria in ant ecology and in the obligate mutualism which these ants have established with their specific host plants.

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Study site and organisms.
The study was conducted at a site ca. 15 km W of Puerto Escondido in the coastal area of Oaxaca, México (15°55.596'N and 097°09.118'W; elevation, 15 m). Pseudomyrmex ferrugineus F. Smith obligatorily inhabits Acacia myrmecophytes (27) and feeds exclusively on the plant-derived food rewards (6) to the composition of which it is physiologically adapted (21, 29). The species has never been found nesting outside a myrmecophytic Acacia plant (27, 50; personal observations by P. S. Ward and M. Heil). Workers and larvae from three colonies collected directly from Acacia hindsii host plants were used for the analysis. Additionally, larvae of a fourth colony were used for seven independent analyses to proof for reproducibility of the TReFID approach.

TReFID software.
The software TReFID (tRF identifying program [www.trefid.net]) allows scanning of an environmental sample for the presence of prokaryotic taxa that are present in its source database (15, 42). In short, TReFID applies in silico analysis of tRF obtained from digestions of fluorochrome-labeled PCR products of the 16S rRNA gene with multiple restriction enzymes (see the supplemental material). The current database comprises 22,239 entries from the NCBI database (www.ncbi.nlm.nih.gov). Since information on the taxonomy of many entries of the NCBI database needs revision, the results of the tRF length polymorphism (tRFLP) analyses were counterchecked with the Ribosomal Database Project II (http://rdp.cme.msu.edu/classifier/classifier.jsp) (49) and by a BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi). For validation purposes, this technique was accompanied by conventional cloning and cultivation technologies.

Sample processing and amplification of procaryotic DNA.
Since microbes living on the surfaces of ants can be mutualistic partners of interest (7), we did not sterilize the surfaces of the ants. For every colony, each six larvae and six workers were pooled, and prokaryotic DNA was extracted using the UltraClean Soil DNA kit according to the manufacturer's protocol (MoBio, Solana Beach, CA). Total DNA was used as a template for amplification of the 16S rRNA gene with the universal bacterial primer pairs 27F + 1495R (51) and 63F + 1387R (34) (Table 1). The PCR primer 63f was 5' labeled with the fluorescent dye 6-carboxyfluorescein (Sigma-Aldrich, Germany).

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TABLE 1. Primers used in this worka

Hot start PCR utilizing the Peqlab kit (a special kit free of Escherichia coli DNA) was carried out in a Peqlab cycler (Peqlab, Erlangen, Germany). The total reaction volume was 25 µl (2.5 µl PCR buffer, 5 µl enhancer solution, 1.5 µl 25 mM MgCl₂, 2 µl 25 mM deoxynucleoside triphosphate solution, 1 µl of each primer, 1 µl template DNA, and 11 µl H₂O); the PCR program consisted of 40 cycles (denaturation, 30 s at 94°C; annealing, 30 s at temperatures decreasing stepwise from 66 to 56°C; elongation, 45 s at 72°C). Only those PCR approaches were used for further steps whose corresponding zero sample showed no amplification. All procedures were performed as described previously (42).

The labeled PCR products were purified (QiaQuick spin columns; Qiagen, Hilden, Germany) and partitioned, and aliquots were subjected to restriction digests using up to 13 different enzymes in parallel. The restriction enzymes employed were AluI, Bme1390I, Bsh1236I, BsuRI (HaeIII), Cfr13I, Hin6I, HinfI, MboI, MspI, RsaI, TaiI, TaqI, and TasI (all from Fermentas, St. Leon-Roth, Germany). Digestion with AluI (AG/CT), Bme1390I (CC/NGG), Bsh1236I (CG/CG), Cfr13I (G/GNCC), HaeIII (GG/CC), Hin6I (C/GCG), HinfI (G/ANTC), MboI (/GATC), MspI (C/CGG), or RsaI (GT/AC) was performed at 37°C, and those with TaiI (ACGT/), TacI (T/CGA), and TasI (/AATT) were performed at 65°C. Digests were performed overnight in a total volume of 100 µl (25 µl purified labeled DNA solution, 10 µl restriction enzyme buffer, 1 µl enzyme, and 65 µl H₂O) at the temperatures indicated above. The restriction fragments were precipitated with ethanol and dissolved in 10 µl of deionized water.

Data evaluation with TReFID.
Fragment size analysis was performed with an ABI 3730 instrument (Applied Biosystems) by a sequencing company (GENterprice Genomics, Mainz, Germany) using the size standard GeneScan (GS)-500 ROX (Applied Biosystems). Fragment sizes were determined by using the local Southern method, and raw data were handled in the Peak scanner software program, v1.0 (Applied Biosystems). Only fragments of a length between 25 and 500 bp were considered for TReFID analysis, as described earlier (42).

In short, all detected patterns of restriction fragments were compared to all entries of the TReFID database. Closely related bacteria may yield identical tRF patterns and then cannot be distinguished based only on TReFID results. Usually, this resolution limit occurs at the level of congeneric species, seldom at the level of genera that share very similar ribotypes. As a consequence, the score list often contains more entries than there were different tRF patterns (see the supplemental material). Our reports on the diversity of the different bacterial groups detected are therefore only at the individual taxonomic levels that could be reliably defined.

An important difference from other tRFLP approaches is that TReFID detects the presence or absence of tRF patterns, which are specific for a bacterial taxon, but does not quantify peak areas of certain fragment peaks; quantitative conclusions therefore cannot be drawn. However, TReFID allows a relative determination of the bacterial diversity in a sample depending on the number of different taxon-specific tRF patterns.

Culture conditions.
To isolate bacteria for cultivation purposes, complete workers and larvae were homogenized in 1 ml sterile phosphate-buffered solution (isotonic, pH 7) and centrifuged at 200 x g for 5 min. The same procedure was applied to each three samples of leaves and extrafloral nectaries of Acacia hindsii plants, which were included to check for bacteria present in the ants’ immediate environment. The supernatant then was plated on plates of agar or Gelrite (Roth, Karlsruhe, Germany) with YEM⁺ medium (containing mannitol [10 g/liter] or sacharose [10 g/liter] or glucose [5 g/liter] and fructose [5 g/liter], Bacto-Trypton [0.4 g/liter] [Roth, Karlsruhe], yeast extract [0.4 g/liter] [AppliChem, Darmstadt, Germany], K₂HPO₄ [0.5 g/liter], MgSO₄·7H₂O [0.4 g/liter], NaCl [0.1 g/liter], and 1 ml/liter trace element solution SL8 [1]). This medium was chosen because of its transparency and durability, which allowed long-time cultivation of isolates. After differentiation, the isolated colonies could be selected based on their colony shape and/or color in order to obtain many purportedly different isolates for further analysis. All media were supplemented with cycloheximide (1 mg/ml medium) to prevent growth of fungi or protozoa. Plates were incubated at 30°C until colonies were clearly visible. Single colonies were then suspended in distilled water and used directly for PCR amplifications of the 16S rRNA gene using the primers described above.

Test for reproducibility.
Total DNA of P. ferrugineus larvae of a fourth colony was extracted and a PCR performed as described above. The PCR amplificates were then used as a template for a PCR with fluorochrome-labeled primers in seven independent samples, and the products of each approach were divided into 12 portions for digestion with 12 different restriction enzymes. The tRF data that were obtained from these approaches were then independently analyzed using TReFID.

Nucleotide sequence accession numbers.
Accession numbers (GenBank and NCBI; submitted 23 June 2008) of all sequences of bacteria associated with P. ferrugineus are as follows: EU841918 to EU841962, EU842025 to EU842031, EU842055 to EU842077, EU842079 to EU842087, and EU842098 to EU842110.

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General patterns.
The TReFID analysis of P. ferrugineus ants indicated the presence of 30 microbial classes (Acidobacteria, Actinobacteria, Alphaproteobacteria, Anaerolineae, Aquificae, Bacilli, Bacteroidetes, Betaproteobacteria, Chlorobia, Chloroflexi, Clostridia, Cyanobacteria, Deferribacteres, Deinococci, Deltaproteobacteria, Epsilonproteobacteria, Flavobacteria, Fusobacteria, Gammaproteobacteria, Gemmatimonadetes, Lentisphaerae, Mollicutes, Nitrospira, Planctomycetacia, Sphingobacteria, Spirochaetes, Thermoprotei, Thermotogae, candidate division TM7, and Verrucomicrobiae). Eight divisions represented 93% of all tRF patterns that were obtained in the present study. These divisions are Acidobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, Planctomycetes, Proteobacteria, and Spirochaetes (Fig. 1).

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FIG. 1. Bacterial diversity in Pseudomyrmex ferrugineus. The relative contributions of tRF patterns (= tRF fingerprints) from different bacterial taxa are depicted separately for larvae (n = 4 colonies) and adult workers (n = 3 colonies). Statistically significant differences between the relative numbers of tRF patterns of larvae and workers are marked with an asterisk. Statistical evaluation was performed with the Statistica 6 software program (StatSoft) using the Mann-Whitney U test (P < 0.05 for Alphaproteobacteria, Sphingobacteria, Flavobacteria, and Deltaproteobacteria).

The relative proportions of tRF patterns associated with larvae and adult workers were very similar (Fig. 2) and were dominated by the Proteobacteria, which represented some 40% of all patterns, comprising 5 classes and 39 families (Fig. 1 and 2). For both developmental stages, 48% of the diversity of the Alphaproteobacteria was represented by the Rhizobiales (Rhizobium and Bartonella), followed by the Rhodospirillales, Rhodobacteriales, and Sphingomonadales (Fig. 2) (each group representing ca. 15% of the Alphaproteobacteria). tRF patterns indicating the presence of relatives of the Rickettsiales (Wolbachia) were detected in all sampled colonies. The Betaproteobacteria were dominated by the Burkholderiales (ca. 62%), while the most diverse groups within the Gammaproteobacteria were the Pseudomonadales (30%), Oceanospirillales (19%), and the Enterobacteriales (10%) (Fig. 2).

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FIG. 2. Bacteria in larvae and workers of P. ferrugineus. The major relative contributions of tRF patterns (= tRF fingerprints) from different bacterial taxa are depicted separately for larvae (n = 4 colonies) and adult workers (n = 3 colonies), and the relative compositions of Alpha-, Beta-, and Gammaproteobacteria in both host life stages are presented. -, β-, and , Alpha-, Beta-, and Gammaproteobacteria; Ac, Actinobacteria; B, Bacilli; Cy, Cyanobacteria; Sp, Spirochates; A, Acidithiobacillales; B, Thiotrichales; C, Chromatiales; D, Xanthomonadales; E, Oceanospirillales; F, Enterobacteriales; G, Alteromonadales; H, Pasteurellales, I, Pseudomonadales; J, Rickettsales; K, Caulobacterales; L, Sphingomonadales; M, Rhodobacterales; N, Rhodospirillales; O, Rhizobiales; P, Nitrosomonadales; Q, Procabacteriales; R, Hydrogenophilales; S, Methylophilales; T, Neisseriales; U, Rhodocyclales; V, Burkholderiales; X, taxa contributing less than 1% of tRF patterns.

From the leaves and nectaries of the host plants, cultivation approaches identified only Bacillus, Sphingomonas, and Methylobacterium species (Table 2). The bacterial community associated with P. ferrugineus was thus clearly different from the community of the host organs with which the ants have most intimate contact.

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TABLE 2. Bacteria from Acacia hindsii host plants

Method validation.
Repeated processing and analysis of subsamples revealed a high reproducibility of the tRFLP method (Fig. 3). Two thousand five hundred fifty (47%) of 5,401 tRF patterns found in this sample were identified in all 7 repetitions, and only 11 (0.2%) tRF patterns were found in 3 or fewer of the parallel samples. The diversity of tRF patterns in all seven repetitions was quantitatively dominated by the Proteobacteria, followed by the Firmicutes, Actinobacteria, Bacteroidetes, Cyanobacteria, Acidobacteria, Spirochaetes, and Planctomycetes (Fig. 3).

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FIG. 3. Reproducibility of TReFID-obtained results. The relative composition of tRF patterns (= tRF fingerprints) of the bacterial community in larvae of P. ferrugineus ants is depicted separately for seven independent analyses (1 to 7) of the same original sample. The divisions Proteobacteria (a), Firmicutes (b), Bacteroidetes (c), Actinobacteria (d), Cyanobacteria (e), Acidobacteria (f), Spirochaetes (g), and Planctomycetes (h) represent about 93% of all detected tRF patterns.

When tests with an artificial mixture of Escherichia coli, Azospirillum brasilense, and Rhizobium leguminosarum were conducted using 10 different restriction enzymes, we obtained 75 tRF patterns specific for E. coli, 5 for A. brasilense, and 47 for R. leguminosarum. Therefore, the method used successfully identified all bacteria present in the sample and revealed no false identification, but the number of specific patterns detected by TReFID for each taxon was not proportional to the abundance of bacteria.

Using standard cultivation approaches, relatives of Acinetobacter, Bacillus, Burkholderia, Cupriavidus, Mesorhizobium, Pandoraea, Pantoea, Serratia, Streptomyces, Variovorax, and Weissella were confirmed successfully (Table 3). Sequencing of the 16S rRNA gene of cultured microbes added some further genera that were identified using the naive Bayesian rRNA classifier, version 2.0 (49), as Curtobacterium, Erwinia, and Streptococcus (Table 3). In total, 16 of 115 sequences retrieved from sequencing could be assigned to the Enterobacteriaceae and 11 to the Lactobacillales. All taxa that were identified via sequencing or cultivation were comprised in the TReFID result lists (see Table 3).

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TABLE 3. Congruency among bacterial taxa identified with TReFID with those confirmed by classical approachesa

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Symbiotic bacteria exhibit many effects on their insect hosts, including manipulation of their hosts’ sex ratio and life cycle (35, 37, 55), dispersal and fecundity (32), host plant choice by herbivorous insects (24), the success of pest species on particular host plants (32), and even host protection from pathogen infection (5, 19). Both the presence and the effects of such bacteria depend, however, on environmental conditions, such as the temperature regime and host nutrition (5, 11, 43), and on the host genotype (10). Because detailed studies of certain preselected taxa of microorganisms can give only a restricted impression of the overall microbial diversity, unbiased and fast characterizations of the gross bacterial communities associated with insects are required to fully understand their ecological and ultimately evolutionary importance. We therefore searched for a rapid and cost-effective method allowing the unbiased detection and preliminary characterization of putative bacterial symbionts of ants.

The bacterial community associated with P. ferrugineus turned out to be much more diverse than could have been expected from earlier studies of other ant species (16, 33, 46, 52, 57, 59). Our study revealed tRF pattern indicating the presence of 30 prokaryotic classes, while earlier studies reported 4 (48) or 2 (46) different classes of microorganisms from related ants. Does this result point to artifacts or rather to the presence of a yet-undiscovered biodiversity? When discussing this surprisingly high diversity of tRF patterns, we must recall that little is known about the diversity of environmental bacteria (39), particularly those that are associated with insects. Microorganisms in termites, for instance, have been investigated for a long time, but still an ever-increasing number of microorganisms are newly discovered and described for termites (13, 14, 17, 30, 41, 53). Similarly, our study aimed at getting an impression of an as yet undiscovered diversity of microorganisms.

We analyzed tRF patterns to study the bacterial community associated with P. ferrugineus, an obligate plant ant that lives on a strictly host plant-derived diet (6). Several TReFID results were confirmed by classical cultivation and sequencing approaches (Table 3). In seven independent analyses of the same original sample, 47% out of all tRF patterns were identified in all repetitions and only 0.2% of the patterns showed up in fewer than half of the samples, which indicates a high reproducibility of the method chosen (Fig. 3). Moreover, the analysis of an artificially mixed bacterial sample successfully identified all bacteria added and did not reveal false identifications. We therefore regard our results as qualitatively reliable. However, many more tRF patterns representative of E. coli and R. leguminosarum than of A. brasilense were found in the artificial samples, although the same amounts of all three species had been used. The number of specific patterns detected by TReFID for a taxon is thus not proportional to the abundance of bacteria and apparently depends in part on the composition of the database, which in fact contains more entries of different Escherichia or Rhizobium strains than of Azospirillum. Classical diversity indices, such as the Shannon index (45), therefore cannot be applied to data obtained with TReFID because the program was developed as a qualitative identification tool for diverse microbial communities (42).

The bacterial associates of P. ferrugineus were dominated by the three groups of Proteobacteria (Alpha-, Beta-, and Gammaproteobacteria), which comprise most of the known symbionts of ants ("Ca. Blochmannia," Gammaproteobacteria; Wolbachia, Alphaproteobacteria; gut symbionts of Tetraponera, Alpha-, Beta-, and Gammaproteobacteria). Several genera have already been reported as symbionts of other insects (Table 4), while others are generally regarded as typical symbionts of plants rather than animals (see Table S1 in the supplemental material) (Azorhizobium, Bradyrhizobium, Devosia, Ensifer, and Mesorhizobium [Rhizobiales], Swaminathania [Rhodospiralles], and Acidovorax [Burkholderiales]) or they are usually considered free-living terrestrial or marine bacteria (Table 4) (Fulvimarina and Hyphomicrobium [Rhizobiales] and Acetobacter [Rhodospirillales]). However, only three of these taxa could be cultivated from samples taken from the surfaces of the Acacia host plants (Table 2), although the same media allowed the cultivation of many more bacteria from the ant samples (Table 3). We therefore conclude that the majority of the bacteria that we identified exclusively in the ant-derived samples indeed are regularly associated with P. ferrugineus rather than representing environmental contaminations.

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TABLE 4. Bacteria found in Pseudomyrmex ferrugineus that have been described previously as symbionts of other antsa

We found almost no overlap between bacteria associated with the ants and those that could be cultivated from plant-derived samples. This observation opens the question of where the ants obtain the bacteria. Extrafloral nectar of Acacia myrmecophytes possesses highly active pathogenesis-related proteins, such as chitinases, glucanases, and peroxidases, which keep this nectar virtually free of microbes (18). The extrafloral nectar can therefore be excluded as a major source of bacteria. All larvae, workers, and reproductive individuals in an ant colony are continuously exchanging gut content via trophallaxis (23). Horizontal (that is, among members of the same generation) and vertical (that is, transgenerational) transfers of bacteria within an ant colony are thus highly likely. This assumption is in line with the observation that samples of larvae and adult workers revealed very similar bacterial communities (Fig. 1 and 2), although the two stages feed on different food sources. A direct and continuous ingestion of food-derived bacteria therefore cannot explain the patterns found in the present study. However, future studies will have to examine in detail whether the high bacterial diversity that we encountered in association with P. ferrugineus is indeed transmitted from one generation to the other, as has been described for (endo)symbiotic bacteria (59).

While several taxa represented typical intestinal bacteria (Enterobacteria), we detected seven taxa of potentially N-fixing organisms (Rhizobiales, Burkholderiales, Pseudomonadales, Spirochaeta, Cyanobacteria, and the two genera of the Gammaproteobacteria, Pantoea and Serratia), which comprised a large part of the overall bacterial diversity (Fig. 2). The last two genera were identified by tRFLP, sequencing, and cultivation (Table 3) and thus were beyond doubt present in or on P. ferrugineus.

N-fixing gut bacteria or N-recycling intracellular bacteria can be an important general source of diet supplementation for arthropods that feed on nitrogen-poor food sources (2, 31, 36). For example, termites can feed exclusively on wood or cellulose only due to their association with microorganisms, some of which (Treponema and Pantoea) are capable of nitrogen fixation (3, 38). Interestingly, Pantoea has already been found in the gut flora of Tetraponera pilosa (46), and its presence in the P. ferrugineus samples was confirmed via tRFLP, 16S rRNA sequencing, and cultivation (Table 3). Little further knowledge exists for ants, but carpenter ants (Camponotus) harbor intracellular bacterial symbionts (4), which apparently function as an N-recycling system (58). These "Ca. Blochmannia" bacteria are capable of synthesizing essential amino acids and play a role in their host's nitrogen metabolism (16, 57, 59). Similarly, aphids are able to live exclusively on amino acid-deficient phloem sap because they are associated with N-recycling Buchnera bacteria that provide their host with essential amino acids (12), and tRF patterns very similar to those of Buchnera were detected in all of our samples. N recycling and N fixation by symbiotic bacteria have been suggested as a general explanation for the tremendous ecological success of arboreal ants, since -¹⁵N ratios of ants from Amazonia and Borneo demonstrated that many arboreal species obtain little nitrogen through predation (8). Although final experimental evidence for this hypothesis is lacking, our findings demonstrate that putatively N-fixing bacteria in ants can be much more common and taxonomically diverse than has been known so far.

We decided not to sterilize the surface of the ants used here, since important bacterial symbionts of fungus-growing ants were reported to live on the exoskeleton (7). Although typical gut bacteria comprise a large part of the microorganisms that were identified in the present study, it is not possible to decide which percentage of the bacteria that we have found to be associated with P. ferrugineus represents endosymbionts. However, all samples displayed a very similar composition of the bacterial community, demonstrating that most of these bacteria are commonly associated with these ants and that they likely represent symbionts. Nevertheless, some results, such as the presence of cyanobacteria, remain enigmatic and require further investigation of the putative role of these bacteria in the life of ants.

Which other functions might be fulfilled by the bacterial associates of P. ferrugineus? All samples tested positive for infection with Rickettsiales-like bacteria. Burkholderia (confirmed by all three attempts; Table 3) was isolated from fungus gardens of the leaf cutting ant Atta sexdens rubropilosa and found to produce antibiotics (44) and was also found in Tetraponera ants (48). Interestingly, patterns of bacterial genera found in lower termites and in our present study were strikingly similar: even some uncommon groups, such as the Bacteroidales, methanogenic Archaea, and methanotrophic bacteria, were found in termites (see Table S1 in the supplemental material) and in P. ferrugineus samples. At least some of these bacteria appear related to fermentation processes in the insect gut, since we detected members of Lactobacillales, Enterobacteriales, Bacillales, Bifidobacteriales, and Clostridiales. Members of Lactobacillales and Enterobacteriales are also well known from other hymenopterans, such as honey bees (28).

Conclusions.
We are just starting to realize the importance of associations among bacteria and ants, and much more has to be done before we can understand the roles that bacteria play in the life of ants, which represent the most successful eusocial insects, or in the mutualisms of ants with higher organisms. Unbiased investigation of the entire bacterial flora of ants—or other arthropods—appears to be an important step toward the discovery of as yet unknown bacteria that fulfill this or other ecologically important functions.

TReFID turned out to be a highly suitable approach to obtain a first insight into the high diversity of the bacterial community which was found to be regularly associated with the ant P. ferrugineus. Both the high overall diversity detected and the occurrence of various potentially N-fixing taxa point to important and manifold roles of these bacteria in the life style of P. ferrugineus as an obligate plant ant. Apparently, we must widen the current concept of ant-plant mutualisms (20) to include bacterial partners, which might alter the ants’ physiological capacities and thereby significantly affect their life-style as obligate plant ants and specialized "vegetarians."

 
We thank Heike Feldhaar (Biozentrum, Universität Würzburg) and Gabriela Olmedo (CINVESTAV, Irapuato, Mexico) for discussions and comments on earlier versions of the manuscript.

Financial support came from the DFG (Deutsche Forschungsgesellschaft: He 3169/4-2) and CONACyT (Consejo Nacional de Ciencia y Tecnología de México).

 
* Corresponding author. Mailing address: Departamento de Ingeniería Genética, CINVESTAV—Irapuato, Km. 9.6 Libramiento Norte, Carretera Irapuato-León, 36821 Irapuato, Guanajuato, Mexico. Phone: 52 462 6239657. Fax: 52 462 6239650. E-mail: mheil{at}ira.cinvestav.mx

Published ahead of print on 15 May 2009.

Supplemental material for this article may be found at http://aem.asm.org/.

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Applied and Environmental Microbiology, July 2009, p. 4324-4332, Vol. 75, No. 13
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