Vol.:(0123456789)1 3 Curr Microbiol (2017) 74:589–597 DOI 10.1007/s00284-017-1201-6 Intracellular Symbiotic Bacteria of Camponotus textor, Forel (Hymenoptera, Formicidae) Manuela O. Ramalho1  · Cintia Martins2 · Larissa M. R. Silva1 · Vanderlei G. Martins1 · Odair C. Bueno1  Received: 17 November 2016 / Accepted: 19 January 2017 / Published online: 6 March 2017 © Springer Science+Business Media New York 2017 Keywords Endosymbiont · Camponotini · Weaver ant · Wolbachia · Blochmannia Introduction Symbiotic relationships between insects and bacteria occur in virtually all orders, including Hymenoptera, and may be divided into primary (obligate) and secondary (facultative) interactions, both types have been identified in Formicidae [1, 2]. Obligate endosymbionts are the result of an ancient association with the host; they usually live inside special- ized cells called bacteriocytes and contribute to ant nutri- tion. As a result of this association, the bacterial genome, which is vertically transmitted, may shrink [3]. Bloch- mannia is an example of an obligate symbiont that is com- monly found in Camponotus species in the Northern Hemi- sphere [4]. In contrast, facultative symbionts are characterized by a more recent association and may be transmitted vertically or horizontally [5]. Wolbachia, for example, stands out for interfering in the reproduction of its hosts, but its role in the worker caste of ants is unknown. According to recent esti- mates, between 20 and 35% of arthropods are infected with Wolbachia [6]. Wolbachia has a large diversity of strains, which are divided into supergroups (A–F); the strains found in insects belong exclusively to supergroups A and B. Traditionally, Wolbachia infections were detected with the wsp gene [7]; however, due to its high rate of recom- bination [8] and strong selection for diversification [9] a multilocus sequence typing (MLST) approach has gained popularity [10]. In studies of intracellular bacteria, the term cospecia- tion is often used, and these associations between insects (host) and bacteria are well documented in the literature. Abstract This study focuses on the weaver ant, Cam- ponotus textor, Forel which occurs in some areas of the Brazilian Cerrado and Atlantic Forest, and its symbionts: Blochmannia, an obligate symbiont of Camponotus, and Wolbachia, known for causing reproductive alterations in their hosts. The main goal of this study was to investigate the presence, frequency of occurrence, and diversity of Wolbachia and Blochmannia strains in C. textor colonies. We found high infection rates (100%) and the occurrence of at least two distinct strains of Blochmannia (H_1 or H_7) in the same species. The observed haplotype variation within a single species may result from the high mutation rate of the symbiont. Similarly, the Wolbachia was found in all colonies with different rates of infections and a new strain (supergroup A) was deposited in the MLST database. The diversity found in the present study shows that there is still much to explore to understand about these symbiotic interactions. * Manuela O. Ramalho manuramalho2010@gmail.com Cintia Martins martins.c@ufpi.edu.br Larissa M. R. Silva larissamedski@yahoo.com.br Vanderlei G. Martins martv@uol.com.br Odair C. Bueno odaircb@rc.unesp.br 1 Universidade Estadual Paulista “Júlio de Mesquita Filho” UNESP – Campus Rio Claro, Biologia, CEIS, Av. 24A, 1515, Bela Vista, Rio Claro, SP 13506-900, Brazil 2 Universidade Federal do Piauí - Campus Ministro Reis Velloso, Av. São Sebastião, 2819, 64.202-020, Parnaíba, Piauí, Brazil http://orcid.org/0000-0002-8144-6172 http://crossmark.crossref.org/dialog/?doi=10.1007/s00284-017-1201-6&domain=pdf 590 M. O. Ramalho et al. 1 3 Examples include aphids, the tsetse fly, cockroaches, and their respective symbionts [11–13]. In ants, this type of interaction also results in a high degree of congruence between host and symbiont phylogenetic trees, indicating the occurrence of parallel diversification and of maternal transmission of the infection. However, the geographic dis- tribution may only partially reflect the congruence between host and symbiont phylogenetic trees [14]. One of the best known genera of ants for having symbi- otic relationships with bacteria is Camponotus Mayr, 1861 [15, 16] and in a recent study by Bronw and Wernergreen [17] found that 95–98% of bacteria found in Camponotus chromaiodes were Blochmannia and Wolbachia, but most studies of the microbiota and host are restricted to the Northern Hemisphere. Camponotus textor, Forel as well as 14 other species of ants distributed around the world, is known as a weaver ant because it uses silk to construct its nests [18]. Although the existing literature suggests that this species is common in the forests of Central and South America, its precise distri- bution is not fully known [19]. In general, published works describe only behavioral traits, mostly related to nest con- struction using the silk produced by their larvae [20]. The taxonomy of the species is complicated. C. textor is often mistaken for C. senex (Smith) due to their mor- phological similarities. Recently, however, Ramalho et  al. [21] distinguished the two species based on molecular data and ecological traits of specimens of both species collected in the Neotropics. Their results corroborate with Longino [22], who considers them as two separate species. Although C. textor has elaborate behaviors, little is known about its feeding habits, biology, ecology, and interactions with other organisms [20, 23, 24]. Other few studies involving ants and endosymbionts from the Neotropical region have already shown how diverse these associations are [25, 26]. Since the descrip- tions of primary and secondary symbionts in Campono- tus are based on species from the Northern Hemisphere, the goal of this study was to investigate the presence, fre- quency of occurrence, and strain diversity of Wolbachia and Blochmannia in C. textor colonies, which are ants exclusively Neotropical, and evaluate the diversity of these endosymbionts. Materials and Methods Collection, Identification, and Total DNA Extraction Camponotus textor workers were collected from eight localities in different regions of Brazil with either typi- cal Cerrado or Atlantic Forest vegetation: Rio Claro, SP (22°23′42″ S, 47°32′33″ W), Araraquara, SP (21°43′29″ S, 48°1′7″ W), Ribeirão Preto, SP (21°12′42″ S, 47°48′24″ W), Santa Rita do Passa Quatro, SP (21°42′4″ S, 47°29′23″ W), São João da Boa Vista, SP (21°58′10″ S, 46°47′56″ W), Uberlândia, MG (two colonies: 18°53′10″ S, 48°15′39″ W, and 18°53′1″ S, 48°15′34″ W), and Ilhéus, BA (14°18′45″ S, 39°53′13″ W). The collected material was preserved in 80% ethanol and kept at −20 °C until DNA extraction. Specimens were identified by Dr. Jacques Dela- bie and deposited in the collection of CEPLAC, Ilhéus, BA (accession number 5692). Total DNA was extracted from eight individual workers of each colony and preserved in 80% ethanol [19, 27]. We used primers Bloch16S-462F and Bloch16S-1299R [16] to screen for Blochmannia. We used primers wsp81f and wsp691r for the initial detection of Wolbachia [7, 28] and EF1α-532f and EF1α-610r [29] as positive controls. The amplification was performed using Taq DNA Polymerase, Recombinant (Invitrogen), following the protocol of the manufacturer. We used the thermal cycler parameters rec- ommended by Baldo et al. [30] and Wernegreen et al. [16] to identify Wolbachia and Blochmannia, respectively. Purification of the PCR product was performed using the GFX PCR DNA and Gel Band Purification kit (GE Healthcare). Samples were quantified in the Thermo Scien- tific NanoDrop 2000 (Uniscience) and sequenced using the BigDye Terminator v3.1 reagent kit (Applied Biosystems). Sequence reading was carried out in a 3130 Genetic Ana- lyzer automated sequencer (Applied Biosystems). Analyses: Blochmannia After the sequences were edited, multiple Blochmannia infections were detected. As a result, cloning with the pGEM-T Vector System I (Promega) was required to isolate each sequence; we followed the protocol provided by the manufacturer. Miniprep followed Zhou et al. [31], and the sequencing reactions were prepared as described before. Samples that have succeeded in sequencing were depos- ited in GenBank (accession codes KX212263–KX212309, Ilhéus, Rio Claro, São João da Boa Vista and Santa Rita do Passa Quatro colonies). A haplotype network was con- structed using sequences with highest similarity found in Genbank (E-values of 0.0 and 98% similarity with “Candi- datus Blochmannia ulcerosus” AY334375.1, “Candidatus Blochmannia laevigatus” AY334370.1, and “Candidatus Blochmannia herculeanus” AJ250715.1) with the median- joining method in Network 4.5.1.0 [32]. To test whether there is geographic correlation of the colony with the several strains of Blochmannia, we use the Mantel test available in “vegan” package [33] of R software [34]. The geographical coordinates of the colonies were transformed to metric UTM using the “rgdal” package [35], and the genetic distance of each Blochmannia sequence 591Intracellular Symbiotic Bacteria of Camponotus textor, Forel (Hymenoptera, Formicidae) 1 3 was calculated using the Kimura 2-parameter model [36] in PAUP 4.0 [37]. Analyses: Wolbachia MLST The sequences generated were edited and aligned using BioEdit sequence alignment editor [38] and ClustalW [39]. The sequences obtained with the wsp primers allowed us to determine if the Wolbachia infections were single or mul- tiple. If a single infection was confirmed, the Wolbachia MLST approach was initiated following the single infection protocol available at the MLST website (http://pubmlst. org/wolbachia/). All the analyses were run in triplicate (three different workers per colony). Double-infected colo- nies were excluded from the analyses. The alleles of each gene were compared one at a time with those deposited on the MLST database, and the sequence types (ST) were later confirmed through the concatenated analysis. The sequences from the wsp primers were compared with others in the same database [30]. The dataset was partitioned into the different genes, and an appropriate model of sequence evolution was chosen using the Akaike Information Criterion in ModelTest v3.06 [40]. The models selected were GTR_I_G for gatB and wsp, and GTR_G for coxA, hcpA, ftsZ, and fbpA. Phyloge- netic reconstruction based on Bayesian analysis was carried out in MrBayes 3.1.2 [41] by running a 1,000,000-genera- tion Markov chain and sampling every 1000 generations. The first 25% of the trees were discarded as burnin, and the probability values were calculated using the remaining trees. All the Formicidae STs in the MLST database were used for the comparative analysis. Since there is only one Formicidae B strain, other insects were added to compare and confirm the supergroups. The D strain was used as the outgroup. Results Blochmannia All Camponotus specimens analyzed were positive for Blochmannia, with a total of 47 clones distributed in 22 different haplotypes, with 508-bp of the 16S rRNA region (Fig.  1). The network analysis revealed that Blochmannia haplotypes found in C. textor are distant (46 different nucleotides) from other Blochmannia sequences in Cam- ponotus previously deposited in the database. As these Blochmannia from other Camponotus were selected to pos- sess the highest similarity with the present study, it empha- sizes how different the Blochmannia found in C. textor is. In addition, there was more than one Blochmannia strain per worker, and these strains were not exclusive to a given geographic location. For example, haplotype 1 (H_1) was found in all locations and haplotype 7 (H_7) was only absent from São João da Boa Vista. There was no corre- lation between the Blochmannia genetic distances and the geographic locations of the colonies using the Mantel test (r = 0.023, P = 0.3). In addition, haplotype 1 (H_1) was the most frequent, followed by haplotype 7 (H_7), the two differed by a single nucleotide. The H_1 haplotype differs by only one nucleotide of the following haplotypes: H_3, H_22, H_5, H_4, H_21, H_14, H_13, by two nucleotides: H_2, H_18, H_17, H_15, H_19, H_20; by three nucleo- tides: H_12, H_16; and finally H_6 differ by five nucleo- tides. The H_7 haplotype differs by only one nucleotide of the following haplotypes: H_8, H_11 and H_9, and by two nucleotides H_10. All these mutations happened in differ- ent loci. The haplotype network suggests that there are two dis- tinct Blochmannia lineages (H_1 and H_7) in the ant popu- lation, and that the other haplotypes derive from H_1 and H_7 (Fig. 1). This could explain the co-occurrence of H_7 with H_8, H_9, and H_10, and the co-occurrence of H_1 with H_2 and H_3. Except the haplotype H_22 being co- occurring with H_7, all other haplotypes follow the pattern of the co-occurrence happens between haplotypes deriv- ing from. For many others haplotypes found, we could not exclude the possibility of sequencing artifacts. Therefore, we decided to focus our discussions in these haplotypes that were repeatedly detected in different ant specimens (H_1 and H_7). Wolbachia Wolbachia was detected in all colonies analyzed with the wsp gene, but its rates of infection varied: 75% in Rio Claro, 37.5% in Santa Rita do Passa Quatro, 87.5 and 80% in Uberlândia I and II, and 100% in Araraquara, Ribeirão Preto, Ilhéus, and São João da Boa Vista. The electrophero- gram revealed both single and double infections. The only colonies with single infections were in Santa Rita do Passa Quatro (SP) and São João da Boa Vista (SP). The triplicate MLST sequences from the single-infected colonies were compared with the sequences deposited in the Wolbachia MLST database. There was no variation within the species; in other words, all alleles for all individ- uals were identical. However, we found a new allele, coxA allele 185, and a novel ST, ST347, was deposited in the MLST database as a consequence. The sequences gener- ated by wsp were included in an additional analysis, which revealed hypervariable regions (HVR1: allele 37, HVR2: allele 38, HVR3: allele 41, and HVR4: allele 37) and 100% similarity with wsp allele 58. The Bayesian inference analysis (using Bayesian poste- rior probabilities, BPP) of the 42 concatenated sequences http://pubmlst.org/wolbachia/ http://pubmlst.org/wolbachia/ 592 M. O. Ramalho et al. 1 3 in different supergroups revealed that ST347 was more closely related to supergroup A STs, thus characterizing this new strain (Fig. 2). However, the strain found in the present study is separated from the strains from North America and the Old World. Combined with ST45, this new strain forms a strongly supported clade (BPP = 90) that is separated from other supergroup A strains. Super- group A and supergroup B were separated by 100 bp. Discussion Blochmannia Corroborating the findings of Sameshima et  al. [15] and Wernegreen et  al. [16], all analyzed individuals had Blochmannia suggesting that the bacteria are fixed within populations of species in the ant genus Camponotus, as Fig. 1 Blochmannia haplo- types in Camponotus textor. a Haplotype network showing the higher frequency of H_1, followed by H_7. b Distribu- tion of the different haplotypes from each analyzed individual. The haplotype size represents the frequency found, and the point in red was added by the software as hypothetical haplo- type. *C. textor from this study. (Color figure online) 593Intracellular Symbiotic Bacteria of Camponotus textor, Forel (Hymenoptera, Formicidae) 1 3 observed in Formica truncorum and its obligate endosym- biont Wolbachia [42]. Additionally, Blochmannia strains found in the present work were very different to “Candi- datus Blochmannia ulcerosus” (AY334375.1, USA), “Can- didatus Blochmannia laevigatus” (AY334370.1, USA), and “Candidatus Blochmannia herculeanus” (AJ250715.1, USA), which confirms the high interspecific diversity. We believe that this huge difference could be because (I) there are few studies in South America that analyzed the diversity of this endosymbiont, and (II) this bacterium has a high rate of mutation [43]. Besides that, there was no correlation between the geographical location and strain similarity based on the intraspecific haplotype network of the Blochmannia sequences found in C. textor, i.e., shared strains occurred in different geographical regions (Fig. 3). The intracellular endosymbionts that live in bacte- riocytes are vertically transmitted [44]. The fact that the bacteria are located inside a specialized organ associated with female reproductive tissues suggests that the specia- tion processes of the host and its endosymbiont are inter- connected [45, 46]. Phylogenetic congruence suggests the absence of horizontal transfer [43], contrasted to the recur- rent recombination among strains of free-living bacteria [47]. The present study analyzed different colonies from different locations and showed the same pattern of diver- sity of Blochmannia (H_1 and H_7, in different locations). Therefore, there is no evidence of horizontal transmission of Blochmannia among these populations of C. textor, sug- gesting that the long-distance migration of the ants hap- pened in the past and that the common ancestor of this ants has been carrying these strains ever since [15, 16, 44]. The high intraspecific diversity of Blochmannia hap- lotypes in this species should also be noted. Based on the frequencies observed, we cannot exclude the possibility of Fig. 2 Bayesian inference analysis of the sequences in the Wolbachia with the host/location available in MLST database. The Wolbachia strain found in Camponotus textor and identified through this work is highlighted (ST347), and belongs to the supergroup A clade. The symbol “-” means that the information was unavailable 594 M. O. Ramalho et al. 1 3 at least two distinct haplotypes coexisting in the same host species. Degnan et  al. [43] observed a high mutation rate in Blochmannia, which might explain the occurrence of haplotype variation within a single species. Nevertheless, we should consider three hypotheses: (1) this diversity is a sequencing artifact; (2) there are multiple copies of 16S in the Blochmannia genome; and (3) there are multiple strains of Blochmannia in C. textor. Given the high fre- quency of haplotypes identified in this study, the possibility of a sequencing artifact may be rejected, at least for H_1 and H_7, which were frequently found in different colo- nies. The second hypothesis merits further investigation. Because we did not analyze the whole genome of Bloch- mannia, we do not know if there are multiple copies of the 16S genome. However, we checked whether there is prec- edence in the publicly available genome sequence of the Blochmannia endosymbiont from C. obliquus strain 757, GenBank accession # NZ_CP010049, and it was found in only one copy of the 16S gene [48]. Therefore, we also believe that this hypothesis does not apply to C. textor. The third hypothesis is the most plausible; although we sampled different colonies from different locations, we observed the same two haplotypes in each one of them (either H_1 or H_7). Due to the high mutation rate of Blochmannia, it is possible that these diversified strains of Blochmannia had already been present in the ancestor of C. textor, and that they spread along with their host species as the geographic range of C. textor expanded. In our study, a pattern emerged where every worker analyzed harbored either haplotype H_1 or H_7 (Fig. 1b). Interestingly, however, these two dominant haplotypes never co-occurred, that is, with our sampling protocol we were unable to find haplotypes H_1 and H_7 in the same individual. More studies are needed to address the possi- bility of incompatibility or competition between these hap- lotypes. It is important to note that little is known of the general biology of this host ant, and in particular if the colonies are polygynous or monogynous. If this species is monogynous, the single queen may harbor both haplotypes, excluding the possibility of incompatibility or competition. However, an additional question may be raised: why would these multiple strains not be quickly lost due to genetic drift? Genetic drift does not prevent the co-occurrence of multiple strains of Wolbachia within the same individual [25, 26, 49, 50], and we believe the same could be the case for Blochmannia, explaining its diversity. If functional Fig. 3 Distribution map of Blochmannia haplotypes found in Camponotus textor. Haplotype 1 (H_1) was the most common and it is highlighted in pink. Haplotype 7 (H_7) is highlighted in blue. Note that there are colonies with the pres- ence of both haplotypes. Loca- tions where we confirmed the presence of Blochmannia, but we cannot define the haplotype by the technique of cloning, are highlighted in gray. (Color figure online) 595Intracellular Symbiotic Bacteria of Camponotus textor, Forel (Hymenoptera, Formicidae) 1 3 divergence has occurred, akin to the recent diversification of symbionts in cicadas reported by Campbell et  al. [51], more studies will be needed to understand the diversity of Blochmannia in Camponotus sp. Wolbachia Camponotus textor had a high rate of Wolbachia infection compared to Solenopsis spp. from the same region [25], suggesting that the bacteria may be at or near fixation, as suggested by Wenseleers et al. [42] in F. truncorum, Fab- ricius. However, the ST and the wsp gene did not vary among different colonies, suggesting that the Wolbachia infection may have occurred a long time ago in the com- mon ancestor of the populations. According to Watanabe et  al. [52], there are three possible explanations for the presence of similar strains of Wolbachia in related species: vertical transmission by a common ancestor [53], hori- zontal transmission [54], and introgressive hybridization between the hosts [55, 56]. Introgressive hybridization may be discarded because we are reporting similar strains of Wolbachia within a spe- cies C. textor. Therefore, the observed distribution of Wol- bachia may be caused by(a) vertical transmission by a com- mon ancestor, maintained despite the geographic separation of the colonies, or (b) horizontal transmission induced by similar host–parasitoid or predator–prey interactions [52]. These two hypotheses are also supported by the results of Salunke et al. [57], who used MLST to study Wolbachia in butterflies. Phylogenetically, several strains of Wolbachia have been detected in different species of ants using the MLST meth- odology, and the great majority belongs to supergroup A and the ST347 strain. The distribution of the supergroups (A, B, and D) in the haplotype network and the recon- structed phylogeny confirm that the supergroups are com- pletely separated. Haplotype 31 and haplotype 10 (which are equivalent to ST347 and ST45, respectively) were closely related, both in the network and in the phylogenetic tree, but they are distant from the other strains in super- group A. The similarity among the variants in family For- micidae was also confirmed through MLST, indicating that there are differences between the strains found in ants and those from other insects, and between the variants in the New World and the Old World [6]. Conclusion Research into symbiotic interactions of ants of the genus Camponotus often focuses on Blochmannia, but the actual diversity of the bacterial community of this genus is still unknown. In the general context, we were able to find at least two strains of Blochmannia present in the same spe- cies of C. textor, an ant occurring only in the Neotropi- cal region. One possible explanation for the occurrence of these strains could be the high mutation rate of Bloch- mannia. In the same species, the high infection rate was also observed for Wolbachia, and a new strain was depos- ited in the MLST database. However, these new ST and wsp genes were the same for all C. textor colonies ana- lyzed, suggesting that the Wolbachia infection occurred in the past in the common ancestor of these populations, before the colonies split. New studies with C. textor using next-generation sequencing technologies are needed to obtain more data on the role of symbiotic relationships and their implication for the biology of the host. Acknowledgements We thank all our colleagues for their assis- tance: Dr Kleber Del-Claro, Dr Jacques Hubert Charles Delabie, Dr Viviane Cristina Tofolo Chaud, Dr Maria Santina Castro Morini, Dr Ana Carolina Marchiori, Dr Cynara de Melo Rodovalho, and Dr Cor- rie S. Moreau. We thank reviewers and editors who provided valuable suggestions for elaboration in this study. We thank Marilia de Oliveira Ramalho for editing images. This work supported by the CAPES Foundation, Brasilia, Brazil [Grant Number 007343/2014-00]. 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Appl Environ Microbiol 78:4458–4467. doi:10.1128/ AEM.07298-11 http://dx.doi.org/10.1111/j.1365-2958.1996.tb02557.x http://dx.doi.org/10.1128/JB.173.22.7257-7268.1991 http://dx.doi.org/10.7717/peerj.881 http://dx.doi.org/10.1186/1471-2148-8-55 http://dx.doi.org/10.1073/pnas.1421386112 http://dx.doi.org/10.1073/pnas.1421386112 http://dx.doi.org/10.1007/s00248-012-0042-x http://dx.doi.org/10.1111/j.1365-294X.2009.04448.x http://dx.doi.org/10.1007/s00284-007-0055-8 http://dx.doi.org/10.1111/j.1558-5646.2008.00533.x http://dx.doi.org/10.1128/AEM.07298-11 http://dx.doi.org/10.1128/AEM.07298-11 Intracellular Symbiotic Bacteria of Camponotus textor, Forel (Hymenoptera, Formicidae) Abstract Introduction Materials and Methods Collection, Identification, and Total DNA Extraction Analyses: Blochmannia Analyses: Wolbachia MLST Results Blochmannia Wolbachia Discussion Blochmannia Wolbachia Conclusion Acknowledgements References