Bacterial Communities in the Midgut of Ponerine Ants ( Hymenoptera : Formicidae : Ponerinae )

Insects act as hosts for microorganisms, with which they share a wide range of interactions. The orders Blattaria, Hymenoptera, Coleoptera and Hemiptera are typically involved in symbiotic relationships with microorganisms (Boursaux-Eude & Gross, 2000). Among the symbionts of these species, bacteria have received the most attention because they determine the ecological success of the insect host, influencing insect development and the immune response (Shoemaker et al., 2000), reproduction (Giorgini et al., 2010), behavior (Dillon et al., 2002) and particularly Abstract Symbiotic microorganisms are directly related to the ecological success of host insects, influencing many aspects of their biology. The present study is the first to investigate the microbiota associated with ants of the subfamily Ponerinae and aims to identify the bacterial midgut communities of Dinoponera lucida, Neoponera curvinodis, Pachycondyla striata, Odontomachus brunneus and Odontomachus bauri relied on culture-dependent technique, particularly 16S rRNA sequencing. The greatest species richness was observed in O. bauri, with 15 OTUs, followed by D. lucida with five OTUs, O. brunneus, with four OTUs, and N. curvinodis and P. striata, both with three OTUs. There were representatives of the phyla Actinobacteria, Proteobacteria, Tenericutes and Firmicutes, including the genera Bartonella, Mesoplasma, Mesorhizobium, Spiroplasma, Wolbachia and Serratia in the guts of the studied Ponerine ants. Spiroplasma and Mesoplasma were found to be prevalent in the studied ants and they were the only genera of bacteria found in more than one of the analyzed ant species suggesting they might be beneficial symbionts. The low microbial diversity observed given the predatory trophic habits of the species studied suggests that there is selection for these microorganisms, predominantly preserving symbionts with functional roles that are able to colonize this environment. It is also valid to infer that the identified bacteria are predominant in the gut and exhibit mutualistic functions that are important mainly for immunity, but also to reproduction and nutrition; moreover, a subset may be parasites that could have considerable impacts on the studied ants. Sociobiology An international journal on social insects


Introduction
Insects act as hosts for microorganisms, with which they share a wide range of interactions.The orders Blattaria, Hymenoptera, Coleoptera and Hemiptera are typically involved in symbiotic relationships with microorganisms (Boursaux-Eude & Gross, 2000).Among the symbionts of these species, bacteria have received the most attention because they determine the ecological success of the insect host, influencing insect development and the immune response (Shoemaker et al., 2000), reproduction (Giorgini et al., 2010), behavior (Dillon et al., 2002) and particularly

Abstract
Symbiotic microorganisms are directly related to the ecological success of host insects, influencing many aspects of their biology.The present study is the first to investigate the microbiota associated with ants of the subfamily Ponerinae and aims to identify the bacterial midgut communities of Dinoponera lucida, Neoponera curvinodis, Pachycondyla striata, Odontomachus brunneus and Odontomachus bauri relied on culture-dependent technique, particularly 16S rRNA sequencing.The greatest species richness was observed in O. bauri, with 15 OTUs, followed by D. lucida with five OTUs, O. brunneus, with four OTUs, and N. curvinodis and P. striata, both with three OTUs.There were representatives of the phyla Actinobacteria, Proteobacteria, Tenericutes and Firmicutes, including the genera Bartonella, Mesoplasma, Mesorhizobium, Spiroplasma, Wolbachia and Serratia in the guts of the studied Ponerine ants.Spiroplasma and Mesoplasma were found to be prevalent in the studied ants and they were the only genera of bacteria found in more than one of the analyzed ant species suggesting they might be beneficial symbionts.The low microbial diversity observed given the predatory trophic habits of the species studied suggests that there is selection for these microorganisms, predominantly preserving symbionts with functional roles that are able to colonize this environment.It is also valid to infer that the identified bacteria are predominant in the gut and exhibit mutualistic functions that are important mainly for immunity, but also to reproduction and nutrition; moreover, a subset may be parasites that could have considerable impacts on the studied ants.coevolution.However, little is currently known about these interactions.In the tribe Camponotini, the omnivorous ants of the genus Camponotus have established an association with the intracellular bacteria Blochmannia and it has been a fundamental partner by improving colony growth and the host immune system (Souza et al., 2009).The congruence in the topology of bacteria and ant phylogeny suggests it has initiated in a common ancestral followed by the coevolution between the partners (Sameshima et al., 1999;Sauer et al., 2000).Moreover, genomic analysis of Blochmannia from host divergent lineages has showed differential loss of genes that affect cellular functions and metabolic pathways and this variation have been linked to distinct host-associated pressures (Williams and Wernegreen, 2015).
It was also observed the symbiosis between Tetraponera ants and nitrogen-fixing root-nodule bacteria which might be related to recycling nitrogen-rich metabolic waste (Borm et al., 2002).Nitrogen-fixing bacteria were also observed in a great diversity of ants and it is has been linked with the evolution of herbivory in ants (Russel et al., 2009).In this sense, many researches have attempted to characterize microbial communities associated to ants and their putative role to improve host fitness (Eilmus & Heil, 2009;Ishak et al., 2011;Anderson et al., 2012;He et al., 2014).
In the subfamily Ponerinae, although the existence of bacteria in the midgut of Odontomachus bauri Emery 1892 has been reported using an ultrastructural approach (Caetano et al., 2008;Caetano et al., 2010), the identity of these microorganisms remains to be determined.Although these ants exhibit plesiomorphic behavioral and morphological characteristics, such as very small colonies, reduced fertility rates and a poor capacity for dispersal and colonizing new areas, they present a wide geographical distribution (Bolton, 2003;Martins et al., 2007;Werren et al., 2008), which could be related to the presence of symbiotic microorganisms.
As a first step toward understanding the mutual benefits of symbiotic microorganisms in these ants, the purpose of the present study was to describe the bacterial communities in the midguts of the ants Dinoponera lucida (currently included in the red list of endangered species in Brazil (Ministério do Meio Ambiente, 2003), Neoponera curvinodis, Pachycondyla striata, Odontomachus brunneus, Odontomachus bauri and to discuss the putative functional roles of bacteria-host interactions.

Biological material and collection
Individuals of the Ponerine species D. lucida, N. curvinodis, P. striata, O. brunneus and O. bauri.For each species, approximately 15 individuals were collected from a single nest.The individuals collected were processed immediately to avoid the possible alteration of their intestinal microbiota.The collected specimens were identified and deposited in the collection of the Myrmecology Laboratory of the Cocoa Research Center (Centro de Pesquisas do Cacau -CEPEC) under accession number # 5676.

DNA extraction and amplification of 16S rRNA
A total of 8 to 13 individuals of each species were used to increase the amount of DNA extracted.The ants were anesthetized by freezing at -20°C for 2 min and then immersed in 70% ethanol, followed by washing in 0.9% NaCl for external disinfection.The dissection was conducted under a dissecting microscope, with the materials placed on a glass slide containing a drop of 0.9% NaCl.Then, the midgut was washed three times in 0.9% NaCl, and DNA extraction was performed using standard proteinase K digestion in TNES buffer (250 mM Tris, 2 M NaCl, 100 mM EDTA and 2.5% SDS, pH 7.5) and incubated in 1.5 ml tubes for 3 h at 55°C with 5 µL of proteinase K (20 mg/mL); then incubated at 37°C for 30 min after addition of 3 µL of RNAse A (4 mg/mL).Proteins were precipitated with 200 µL of 5-molar NaCl and centrifugation; DNA was precipitated from the supernatant using 600 µL of isopropanol and washing with 70% ethanol.Samples were re-suspended with 30 µL of TE (10 mM Tris, pH 8; 1 mM EDTA pH 8) and extraction was confirmed in 1% agarose gel.
For N. curvinodis, the amplification of the bacterial 16S rRNA region was conducted via PCR, which was performed using the PuReTaq Ready-To-Go PCR Beads kit (GE Healthcare).Each reaction contained 1.0 µL of DNA (~ 30 ng) and 5 pmol of each of the universal bacterial primers 27F (5´-AGAGTTTGATCA/CTGGCTCAG) and 1492R (5´-TACGGT/CTACCTTGTTACGACTT) (Lane, 1991) in a final reaction volume of 25 mL.For all of the other species, the reactions consisted of 3 µL of 1X buffer ((NH-4 ) 2 SO 4 ), 2.4 µL of MgCl 2 (2 mM), 1.5 µL of dNTPs (0.2 mM), 0.3 µL of each primer (10 pmol), 1 µL of DNA (~ 30 ng) and 0.2 µL of Taq polymerase (1 U) (Fermentas) in a final reaction volume of 30 µL.The PCR program consisted of an initial denaturation at 95°C for 5 min, followed by 35 cycles at 95°C for 1 min, 50°C for 1 min and 72°C for 3 min for N. curvinodis; there was an additional final extension at 72°C for 10 min for the other species.The primers amplified a region of approximately 1.5 kb.The PCR products obtained from N. curvinodis were purified using the GFX PCR Gel Band and DNA Purification Kit (GE Healthcare).For the other species, the purification was performed using a Nucleic Acid and Protein Purification kit (Macherey-Nagel).

Cloning and sequencing
Purified PCR products from P. curvinodis were ligated into pJET vectors using the Clone JET PCR Cloning Kit (Fermentas).Competent E. coli DH10β cells were transformed via heat shock.Subsequently, 100 µL of cells was inoculated into Petri dishes containing LB agar and ampicillin (20 µg/mL), then incubated overnight at 37°C.For all of the other species, the purified PCR products were ligated into pGEM vectors using the pGEM-T Easy Vector Systems kit (Promega).Competent E. coli JM109 cells were transformed via thermal shock.Subsequently, 100 µL of cells was inoculated into Petri dishes containing LB agar, ampicillin (20 µg/mL) and X-Gal (45 µL/plate) and incubated overnight at 37°C.The obtained recombinant colonies were transferred to 96-well plates containing LB agar (1 mL) and ampicillin (20 µg/mL) and grown at 37°C for approximately 22 h.The extraction of the plasmid DNA was performed following the protocol described by Vettore et al. (2001).The results of the extraction were visualized on 2% agarose gels, and only those clones that contained an insert of the expected size were used in the subsequent stages.The sequencing reactions were performed on microplates using the ABI BigDye Terminator Cycle Sequencing kit (version 3.1), 0.5 to 1 µg of DNA and 5 pmol of the pJET1.2_forwardprimer (5'-CGACTCACTATAGGGAGAGCGGC) for N. curvinodis and the M13_forward primer (5'-GTTTTCCCAGTCACGAC) for the other species.After purification, the samples were run in an ABI 3500 automated sequencer (Applied Biosystems).

Analysis of 16S rRNA sequences
The obtained 16S rRNA sequences were pre-processed using the EGene automated pipeline generation system (Durham et al., 2005).At this stage, the sequences were screened for primer and vector sequences (pJET1.2and M13), which were then removed.The base quality was also checked, with 90% considered to be the threshold for good bases within a window (Phred value > 20), using a window size of 200 bp.Subsequently, the sequences were filtered based on a minimum size of 200 bp and aligned using the ClustalW tool (Thompson et al., 1994), followed by manual refinement.Using the distance matrix generated in DNAdist, the sequences were assigned to operational taxonomic units (OTUs) using MOTHUR (version 1.8.0) (Sanchez-Contretas & Vlisidou, 2008).The obtained frequency data were used to construct rarefaction curves and to calculate the Chao1 richness estimator and the Shannon and Simpson diversity indices.The sequences were assigned to phylogenetic classes using the Classifier tool of the Ribosomal Database Project (RDP) (Xie et al., 2010) and compared with related sequences in the GenBank database using the BLASTN tool (http://www.ncbi.nlm.nih.gov/genbank/index.html).

Results
After pre-processing, 603 high-quality sequences were obtained; 129 were from D. lucida, 76 from P. curvinodis, 159 from P. striata, 125 from O. brunneus and 116 from O. bauri.These sequences were classified into known bacterial phyla using RDPClassifier from the Ribosomal Database Project public database.Using the 97% similarity criterion, the sequences were grouped into OTUs, and a representative sequence for each OTU was compared with sequences deposited in GenBank.
The following phyla were detected: Proteobacteria (in D. lucida, N. curvinodis, P. striata and O. bauri), Tenericutes (in D. lucida, N. curvinodis, P. striata and O. brunneus ), Actinobacteria (in N. curvinodis and O. bauri) and Firmicutes (in O. bauri) (Tables 1, 2 and 3 In D. lucida were detected five OTUs, in which were detected two species of the genera Spiroplasma (Table 1).In N. curvinodis, three OTUs were found, with a predominance of the genera Wolbachia and Mesoplasma.Three OTUs were also detected in P. striata, in which two Spiroplasma species were predominant (Table 2).In these three species of ants, OTUs were found that showed less than 97% similarity with homologous GenBank sequences, indicating that they potentially represented new species of the genera Spiroplasma and Mesoplasma.
Four OTUs were detected in O. brunneus and 15 in O. bauri (Table 3).Bacteria of the Mesoplasma and Spiroplasma genera were found in O. brunneus, whereas in O. bauri, there was a predominance of species from the genus Serratia as well as a large number of OTUs represented by single or only a few sequences from other genera.
Rarefaction analysis showed no significant differences in the richness of the bacterial communities present in D. lucida, N. curvinodis, O. brunneus and P. striata whereas O. bauri showed a significantly greater richness compared with the other species (Figure 1).The curves reached plateaus at 3% difference, except for O. bauri, between sequences (95% confidence), indicating that the number of clones was sufficient to cover the diversity of bacteria in the midgut of these ants.In addition, according to the Chao1 values, the highest species richness is expected in the bacterial community in O. bauri, followed by D. lucida and O. brunneus, and there was no difference in the bacterial species richness between the two Pachycondyla and Neoponera (Table 4).
In terms of diversity, according to the Shannon index, the Odontomachus and Dinoponera samples differed significantly from the Pachycondyla samples.However, using the Simpson index, differences were detected both between genera and between the Odontomachus species studied, but there was no significant difference when compared with Dinoponera.
and are known to be present in a number of ant species (Funaro et al., 2011;Ishak et al., 2011;Sapountzis et al., 2015).However, previous studies (Funaro et al., 2011) have found that these bacteria are not always present in different subcastes within a single ant colony or different colonies from the same population, indicating that these microorganisms are not essential for the development of the host species.Nevertheless, in certain cases, there is specificity between associated bacteria and ant species.Sapountzis et al. (2015) suggest that their function might be related to the processing of chitin, the main component of the cuticles of insect prey.
In the present study, Spiroplasma and Mesoplasma were found to be prevalent in the studied ants and they were the only genera of bacteria found in more than one of the ant species analyzed, and more than one species from the same genus was present in several cases, indicating that ants of this tribe is a natural reservoir of this group of microorganisms.It was observed that in the ant species in which both of these genera occurred (N.curvinodis, P. striata and O. brunneus), the diversity of other phyla was reduced.In the presence of these bacteria, there is likely a mechanism for the inhibition of other prokaryotes.
Spiroplasma has been reported to be responsible for causing the death of males originating from infected females (Anbutsu & Fukatsu, 2003;Hurst & Jiggins, 2000).This effect distorts the sex ratio, increasing the number of females in the population (Wang et al., 2007).On the other hand, Spiroplasma was reported as a mutualistic symbiont in Drosophila associated with an increased tolerance against infection by nematodes and parasitic attack.Moreover, no other symbiont bacteria was detected, and possibly this is the only symbiont responsible for defense against parasites (Jaenike et al, 2010;Xie et al, 2010) what could be essential for these ants considering their predatory habits which can bring several entomopathogenic microorganisms.
Another relatively common bacterium found in O. bauri in the present study was the enteropathogenic species Serratia marcescens.This bacterium has been used as a model in studies addressing the biological control of insects due to its low to moderate pathogenicity and its ability to infect a wide variety of insects (Connick et al., 2001;Dillon et al., 2005).
Bacteria from the genus Wolbachia were found in N. curvinodis.This genus includes intracellular bacteria that are widely distributed in arthropods.It is estimated that more than 20% of insect species are infected by a strain of Wolbachia (Russel et al., 2009).This occurrence is even more widespread in ants and may be related to their mode of colony formation, as Wolbachia are more common in species in which the queen depends on workers to found the colony (Watts et al., 2009).
Wolbachia belongs to the order Rickettsiales (phylum Proteobacteria, class Alphaproteobacteria), and its members are considered to be reproductive parasites because they induce reproductive changes in their hosts, such as the death of males, the feminization of males, thelytokous parthenogenesis  The values that are followed by different letters are significantly different (95% confidence interval).

Discussion
Studies on the occurrence and functional role of bacterial symbionts in ants are still very scarce, making it difficult to understand how these bacteria affect their hosts or how host diet and intestinal physiology and structure affect the bacterial community.
A predominance of species from the genera Mesoplasma, Spiroplasma, Wolbachia and Serratia was observed in the guts of the Ponerine ants studied.Mesoplasma and Spiroplasma are genera of intracellular bacteria from the order Entomoplasmatales (phylum Tenericutes; class Mollicutes) that are commonly found in the guts of insects and cytoplasmic incompatibility (Wenseleers & Billen, 2000).In ants, cytoplasmic incompatibility is likely the main mode of action of these bacteria (Wenseleers et al., 1998;Wenseleers et al., 2002;Watts et al., 2009).
In other insects, it has been observed that Wolbachia interfere with the expression of ferritin and with iron metabolism.These bacteria reduce the concentration of iron, protecting the cell from oxidative stress and apoptosis.This phenomenon has been observed both in species in which these bacteria occur as mutualists and in species in which they occur as facultative parasites (Keller et al., 2001).The occurrence of Wolbachia has also been related to supplementation with vitamin B (Hosokawa et al., 2010).Despite the high incidence of these bacteria in ants, there are no conclusive studies addressing their effects, and little is known about this topic (Wenseleers et al., 1998;Jaffe et al., 2001;Rani et al., 2009;Schloss et al., 2009).
Interestingly, the three species of ants found in this study to harbor bacteria that alter the reproduction or sex ratio, such as Wolbachia and Spiroplasma, are polygynous.This characteristic of their colonies could be evolved in the ants to reduce the effects of these bacteria by allowing uninfected queens to maintain the production of males in the nest.However, the opposite was observed to the ant Solenopsis, where Wolbachia infection was prevalent in monogynous colonies (Ishak et al., 2011).
Species belonging to the order Rhizobiales (class Alphaproteobacteria, phylum Proteobacteria) were found in the present analysis, such as Mesorhizobium and Bartonella in D. lucida, Pseudochrobactrum and Agrobacterium in O. bauri and Bartonella in P. striata.Rhizobiales includes known nitrogenfixing species.The presence of these bacteria has been reported in the guts of other ants, and it has been suggested that these microorganisms are responsible for dietary supplementation through nitrogen fixation or possibly through the recycling of nitrogen (Reuter et al., 2005).
The occurrence of Micrococcus, which was found in N. curvinodis, has also been reported in the midgut of other insects based on dependent and independent culture methods (Broderick et al., 2004;Kremer et al., 2009;Rafagopal, 2009).These bacteria are not pathogenic.Their presence is related to the immune response via the production of compounds that act against fungal antagonists (Cardoza et al., 2006;Hillesland et al., 2008).
Overall, there was a low diversity of bacteria in the midguts of N. curvinodis, P. striata and O. brunneus; the genera Spiroplasma and Mesoplasma were always present in these species but not necessarily together.In contrast, the bacterial diversity found in O. bauri was high (13 genera) and included S. marcescens, an entomopathogenic species.In O. bauri, neither of the two bacterial genera mentioned above were detected.Ishak et al. (2011) explored the microbiome of the ant Solenopsis geminata, which has a more granivorous diet, using a massive parallel sequencing, and they found a high abundance of Spiroplasma in workers and that is why they suggested it to be an important partner for this ant.Therefore, considering that these bacteria infect ants with different diet it is possible to predict that Spiroplasma and Mesoplasma are likely mutualists and have more immunologic instead of nutritional role by inhibiting the occurrence of a greater diversity of bacteria, including potential pathogens, protecting the host ants against massive infection by other prokaryotic microorganisms.
Taking into account the predatory trophic habits of the studied species, the low microbial diversity observed suggests that there is selection of these microorganisms by the host ants, which primarily maintain symbionts that play functional roles and that are able to colonize this environment.Feeding from other insects may bring entomopathogenic bacteria to the ant gut, thus it can also be concluded that the predominant identified bacterial species might exhibit mutualistic functions that are important mainly for immunity, but also to reproduction and nutrition.

Table 1 .
).The phylogenetic grouping of the 16S rRNA sequences from the midguts of the ant Dinoponera lucida.

Table 2 .
The phylogenetic grouping of the 16S rRNA sequences from the midguts of the ants of the genus Neoponera and Pachycondyla.

Table 3 .
The phylogenetic grouping of the 16S rRNA sequences from the midguts of the ants of the genus Odontomachus.

Table 4 .
The richness of OTUs (S), estimated richness of OTUs and diversity indices for the 16S rRNA clone libraries.