Philippine Journal of Science 150 (3): 753-763, June 2021 ISSN 0031 - 7683 Date Received: 30 Sep 2020

Diversity of Bacteria in (: Formicidae) from Canopy and Understory of Selected Trees at Mount Makiling Forest Reserve, Laguna, Philippines

Michael P. Gatpatan1, Mia Beatriz C. Amoranto1, Alfredo Jose C. Ballesteros3, Noel G. Sabino1, Jocelyn T. Zarate2, Ma. Anita M. Bautista3, and Lucille C. Villegas1*

1Microbiology Division, Institute of Biological Sciences 2National Institute of Molecular Biology and Biotechnology (BIOTECH) University of the Philippines Los Baños, College, Laguna 4031 Philippines 3National Institute of Molecular Biology and Biotechnology (NIMBB) University of the Philippines Diliman, Quezon City 1101 Philippines

The Mount Makiling Forest Reserve (MMFR) is a hotspot and listed as one of the 170 conservation priority areas established by the Philippine government. Its flora and fauna diversity has been reported, but knowledge gap has been identified concerning the bacterial communities associated with the flora and fauna. This study focused on ants (Hymenoptera: Formicidae), which are dominant in forest canopy and play essential roles in the ecosystem functionality. A metagenomic sequencing approach based on amplified V3–V4 regions of the 16S rRNA was employed to investigate the bacterial communities associated with five arboreal collected from MMFR. The collected ants were identified as thoracicus, sp., leonardi, mindanaensis, and Polyrhachis semiinermis. The sequence analyses revealed that Proteobacteria, Spirochaetes, Firmicutes, Bacteroides, and Actinobacteria were the most abundant phyla. Individual analysis of the bacterial genera associated with the five ant species showed that unclassified members of Rhizobiaceae, Orbaceae, and Burkholderiaceae were dominant in D. thoracicus. Unclassified members of Rhizobiaceae, Spirochaetaceae, and Ruminococcaceae were dominant in Myrmicaria sp. On the other hand, Candidatus Blochmannia, and Wolbachia were abundant in ants C. leonardi, P. mindanaensis, and P. semiinermis. Bray-Curtis distance and UPGMA cluster analyses showed that the microbiomes of the Camponotini group clustered together, while D. thoracicus and Myrmicaria sp. exhibited unique bacterial profiles. Predictive gene profile analysis showed that the most functional categories were those associated with metabolism and biosynthesis of amino acids, pathways for metabolism of nucleotide, amino sugars and nitrogen, and utilization of different carbon sources.

Keywords: bacterial diversity, canopy and understory, Formicidae, metagenomics, Mount Makiling Forest Reserve

*Corresponding Author: [email protected]

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INTRODUCTION excess of carbon from carbohydrate-rich diets of canopy foraging ants facilitate their ecological dominance. One of the many significant roles played by bacteria is being symbiotic partners of various living organisms The MMFR is a biodiversity hotspot and is listed as including such as ants (Chandler et al. 2008; one of the 170 conservation priority areas established Neuvonen et al. 2016). Bacterial associations with by the Philippine government. To date, there has been ants help in compensating for the low nutritional no report on the bacterial diversity associated with ants diet of the host through the synthesis of essential (Hymenoptera: Formicidae) in MMFR. Therefore, this amino acids (Feldhaar et al. 2007). Previous studies study was aimed at generating baseline information on the showed that the gammaproteobacteria Candidatus bacterial diversity associated with the arboreal ants from Blochmannia, Buchnera, Rickettsia, and Wolbachia are selected trees in MMFR using the 16S rDNA metagenomic extremely common intracellular bacterial endosymbionts. sequencing. Candidatus Blochmannia is commonly found in carpenter ants ( Camponotus) and is known to be host- specific (Eilmus and Heil 2009). Bacterial associations are also linked to the feeding habit like, for instance, the MATERIALS AND METHOD association of Rhizobiales to herbivory (Russell et al. 2009). Also, some bacteria colonize the tegument Sample Collection and are involved in the defense mechanism against Arboreal ant samples were collected from March until entomopathogens (Mattoso et al. 2012). December 2017 within the 2-ha Molawin-Dampalit long Insects are foremost in the canopy ecosystems (Basset term ecological plot of MMFR. MMFR is located in the et al. 2015). Among the rain forest invertebrates, ants southern part of Metro Manila, Luzon Island, Philippines dominate the rain forest canopies, which represent 50% at 14°08'14” N and 121°11'33” E (Castillo et al. 2018; of the forest biomass and 90% of the individual Gonzalez et al. 2020). Three large-diameter trees – . Their abundance is attributed to their diet – namely, narra (Pterocarpus indicus Willd.) located in Area which includes plant materials, extrafloral nectar, pollen, 46 at Quadrant 1, “bagtikan” [Parashorea malaanonan fungal spores and mycelia, and sap (Dejean et al. 2007). (Blco) Merr.] located in Area 32 at Quadrant 2, and McGlyn and Parra (2016) reported that stoichiometric another bagtikan in Area 12 at Quadrant 3 – were chosen

Figure 1. Map of the 2-ha Molawin-Dampalit Long Term Ecological Plot within the MMFR showing the three sampling sites. The location map is derived from Castillo et al. (2018) and Gonzalez et al. (2020).

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(Figure 1). using thermocycling conditions, which were as follows: initial denaturation at 95 °C for 3 min, followed by eight Samples were collected using sterile forceps and carefully cycles of denaturation at 95 °C for 30 s, annealing at 55 transferred to sterile bags. Worker ants were taken from °C for 30 s, and extension at 72 °C for 30 s. After PCR, a the actively foraging colonies. To ensure that the samples second PCR clean-up was performed to the final library belonged to the same colony, worker ants were collected before quantification. DNA integrity and concentrations from the foraging trails. The collected samples were were checked with the Qubit dsDNA BR assay kit using designated as C1 (n = 20), C2 (n = 15), C3 (n = 15), C4 the Qubit 3.0 fluorometer (Life Technologies, USA). The (n = 4), or C5 (n = 7). C1, C2, and C3 were collected from 16S libraries (n = 5) were sequenced at the Philippine Bagtikan, while C4 and C5 were collected from narra. Genome Center using Illumina MiSeq 300 bp Paired-End The samples for DNA extraction were preserved in 95% (301 cycles × 2). ethanol and stored at –20 °C until processing (Hammer et al. 2015). Voucher samples were separately preserved and stored for identification. Data Processing Raw reads were processed using the MiSeq Standard Operation Procedure of Mothur v1.39.5 for analyzing 16S Identification of Arboreal Ant Species rRNA amplicon sequencing data (http://www. mothur.org/ The samples were identified based on morphological wiki/MiSeq SOP, d.d. 2015-11-23). Forward and reverse characteristics – including body color, head shape, density reads were combined using the make.contigs command, of hairs on head and body, and prominence of eyes (Narain in which the sequence and quality scores were extracted et al. 2012). The samples were identified at the Museum before merging the reads into contigs. The sequences were of the Natural History, University of the Philippines screened to remove the sequences with ambiguous bases. Los Baños by myrmecologist Dr. David Emmanuel M. Improved sequences were optimized for downstream com- General. putational steps by removing duplicate sequences. Then, using align.seqs command the reads were aligned to the DNA Extraction, Amplicon Library Construction, SILVA reference database (Silva 132; Quast et al. 2012). and Sequencing Reads that failed to align to the correct location within The ant samples were washed with and preserved in 95% the 16S rRNA gene were removed using the screen.seqs ethanol prior to DNA extraction. Briefly, lysate preparation command. Aligned reads were simplified (via removing was done by maceration and homogenization of the ant noninformative columns (using the filter.seqs command), samples using a sterile micropestle. About 250 mg of the dereplicated (via the unique.seqs command), and denoised macerated sample was used for DNA extraction using with Mothur application of the Single Linkage Precluster- Invitrogen™ PureLink™ Genomic DNA Mini Kit (Life ing algorithm via the pre-cluster command and chimera. Technologies, CA, USA), following the manufacturer’s uchime command to remove chimeric sequences. Finally, protocol. The total DNA was diluted to obtain the standard sequences are clustered into OTUs using the cluster.split concentration for the 16S rRNA gene library protocol by command. Rarefaction analysis was carried out to estimate the Illumina MiSeq System. the depth of diversity of the data sets. Alpha diversity anal- ysis included Shannon index, Simpson, Chao1, Pielou’s Briefly, 16S rRNA V3–V4 regions were amplified using evenness, and Shannon’s effective genus number. For –1 the following conditions: 5 ng μL of total DNA, 1 μM of comparative study, samples were analyzed for Bray-Curtis amplicon PCR (polymerase chain reaction) forward prim- distance and UPGMA cluster analyses. After data process- er 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGA- ing with Mothur, 16S rRNA gene sequences were utilized CAGCCTACGGGNGGCWGCAG-3’ and reverse primer using an open-source R package Tax4Fun (Aßhauer et al. 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGA- 2015), and the SILVA database 119 (Quast et al. 2012) as CAGGACTACHVGGGTATCTAATCC3’ (Klindworth et a required tool. SILVA-based 16S rRNA profile was first al. 2013), and 2× KAPA HiFi HotStart ReadyMix (KAPA transformed to the taxonomic profile of KEGG organ- Biosystems, USA) for a 25-μL reaction mixture. PCR isms. OTU table was generated using SILVAngs based amplification was performed with a 3-min denaturing on an automatic software pipeline version 1.9.5 (https:// step at 95 °C; 25 cycles at 95 °C for 30 s, 55°C for 30 s, www.arb-silva.de/ngs/). OTU table and reference data and 72 °C for 30 s; and a final extension at 72 °C for 5 were loaded to R package Tax4Fun. Functional profiling min. The amplicon was purified by binding to an equal of the bacterial communities based on 16S data samples volume of AMpure beads (Beckman Coulter Inc., USA) was computed using the precomputed KEGG Ortholog eluted to 10-mM Tris-HCl, pH 8.5, followed by ligation (Kanehisa et al. 2014) reference profiles. The functional of dual indices and Illumina sequencing adapters using reference profiles were computed based on 100-bp reads. the Nextera XT Index Kit. PCR reactions were performed The taxonomic profiles were then normalized by the copy

755 Philippine Journal of Science Gatpatan et al.: Bacterial Diversity in Ants Vol. 150 No. 3, June 2021 number of 16S rRNA gene obtained from NCBI genome annotations and the functional reference profiles were colonies form obvious columns, forage in the ground and computed based on the normalized taxonomic abundance in foliage, while some nest on trees (Wriedt et al. 2009; that linearly combined with precomputed profiles from the General and Alpert 2012). The Camponotini ant tribe KEGG database (Aßhauer et al. 2015). Box-plots were is composed of , Camponotus, Chimaero- generated using R. myrma, Colobopsis, , , , , Polyrhachis, and Pseudocamponotus (Ward et al. 2016). Members of this tribe have vertically inherited specific bacterial symbiont (Eilmus and Heil 2009; Ward RESULTS AND DISCUSSION et al. 2016). Camponotus and Polyrhachis can be found foraging in the cavities of wood, tree trunks on the ground, Sample Identification foliage, or in the canopy (General and Alpert 2012). The arboreal ants were found to belong to three ant subfamilies – namely, , , and Bacterial Composition and Richness (Figure 2). C1 was identified as Dolichoderus Rarefaction analysis carried out at a maximum depth of thoracicus. C2 is possibly a new species of Myrmicaria 9,838 counts per sample and 10 replicates per iteration and C3 is Colobopsis leonardi, previously known as Cam- to compare richness in genus count suggests that C1 had ponotus leonardi (Ward et al. 2016). C4 is Polyrhachis sufficient size to represent all identified taxa (Figure 3). mindanaensis and C5 is Polyrhachis semiinermis. C4 and The rest of the samples need more sequences to represent C5 are endemic in the Philippines (General and Alpert all identified taxa. Rarefaction analysis allows estimation 2012). C. leonardi (C1), P. mindanaensis (C4) and P. of sampling coverage but typically rely on a high-quality semiinermis (C5) are members of Camponotini ant tribe assembly, comprehensive reference data sets, or both – (General and Alpert 2012; Ward et al. 2016). which are often unavailable for complex or poorly char- acterized communities (Rodriguez-R and Konstantinidis In the Philippines, Dolichoderus is the largest ant genus 2014). C2 had the highest approximation of taxa classified (General and Alpert 2012). The ant colonies can be found to genus level (200–220), followed by C3 (70–85), C5 nesting in the ground, rotten logs, or tree hollows and (40–65), C1 (40–50), and C4 (20–40). branches in the canopy (General and Alpert 2012; Saripah and Azhar 2012). Some species derive their diet from other The α-diversity indices comparing the bacterial even- arthropods, while some are known herbivores (General ness and diversity revealed differences among samples and Alpert 2012; Hu et al. 2018; Saripah and Azhar 2012). (Table 1). The Shannon effective genus number showed Myrmicaria, on the other hand, is a morphologically that C2 had the highest genera counts, followed by C1. and ecologically diverse genus (Wriedt et al. 2009). The

Figure 2. Representative ant samples collected from the three sampling sites identified as A) C1, ; B) C2, Myrmicaria sp.; C) C3, Colobopsis leonardi; D) C4, Polyrhachis mindanaensis; and E) C5, Polyrhachis semiinermis.

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Figure 3. Rarefaction curves of each arboreal ant species: Dolichoderus thoracicus (C1), Myrmicaria sp. (C2), Colobopsis leonardi (C3), Polyrhachis mindanaensis (C4), Polyrhachis semiinermis (C5), and vertebrate controls: [Ptilinopus occipitalis (S6) and Pycnototus urosticus (S7)].

Table 1. Richness and evenness of bacterial communities from the five arboreal ant species. Shannon effective genus number Sample Effective genus Simpson index Pielou's evenness Index number D. thoracicus (C1) 2.797961 1.028891 0.5545818 0.263007 Myrmicaria sp. (C2) 10.582893 2.359239 0.7662954 0.442408 C. leonardi (C3) 1.523725 0.421158 0.1337689 0.091453 P. mindanaensis (C4) 1.739419 0.553551 0.2612723 0.17197 P. semiinermis (C5) 1.685528 0.522079 0.2275161 0.134148

C3, C4, and C5 were dominated solely by specific genus. Comparative Analysis of Bacterial Community of Meanwhile, Pielou’s evenness showed that there was low Five Arboreal Ants evenness in most of the samples – especially in C3, C4, Bray-Curtis distance and UPGMA cluster analyses revealed and C5. Shannon-Weiner and Simpson indices revealed that the microbiomes of the Camponotini group (C. leonar- low bacterial diversity for each sample, which was consis- di, P. mindanaensis, and P. semiinermis) were more similar tent in other ant microbiome studies (Russell et al. 2009; compared to the microbiomes of Myrmicaria and Dolicho- Ramalho et al. 2017a) It may be a result of insects having derus (Figure 4). This suggests that Myrmicaria sp. and D. specific structures (e.g. bacteriocytes) that are used to thoracicus contain unique microbiome profiles compared house large numbers of specific bacterial lineages (Jones to the Camponotini ants. This finding may be attributed to et al. 2013). It may also be due to the negative interac- the host specificity of the microbial symbionts. Most of the tions between established insect symbionts and invading sequences recovered from the Colobopsis and Polyrhachis bacteria (Jones et al. 2012). species were from the genera Candidatus Blochmannia and

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Figure 4. Correspondence map of bacterial communities: Dolichoderus thoracicus (C1), Myrmicaria sp. (C2), Colobopsis leonardi (C3), Polyrhachis mindanaensis (C4), Polyrhachis semiinermis (C5), and vertebrate controls [Ptilinopus occipitalis (S6) and Pycnototus urosticus (S7)].

Wolbachia. Also, D. thoracicus and Myrmicaria sp. shared Table 2. Overall abundance of bacterial phyla from the five arboreal bacterial taxa belonging to family Rhizobiaceae at 59.21% ant species. and 46.21% relative abundances, respectively. Previous Phylum Relative abundance (%) studies reported that hosts’ diet and other environmental Proteobacteria 89.96 conditions influence the structure of bacterial communities (Chandler et al. 2008; Geib et al. 2009). Spirochetes 3.37 Firmicutes 1.80 Bacterial Communities Associated with Arboreal Ants Bacteroides 1.45 At the phylum level, Proteobacteria was the most domi- Actinobacteria 1.05 nant – representing 89.96% of the total sequences (Table Other phyla 2.37 2). Other phyla with more than 1% relative abundance were Spirochaetes, Firmicutes, Bacteroidetes, and Actino- bacteria. The dominance of Proteobacteria was previously most dominant (Figure 5A–B). Dolichoderus consumes observed in marine and terrestrial microbiomes, including a plant-based diet, and some other member species are arthropods (Jones et al. 2013; Degli-Esposti and Martinez- known herbivores (Hu et al. 2018). The plant-based Romero 2017), although their distribution remained diet of D. thoracicus may be linked to the abundance unexplained (Degli-Esposti and Martinez-Romero 2017). of Rhizobiaceae, as some members of Rhizobiales are usually associated with herbivory (Russell et al. 2009). Bacterial Communities Associated with Subfamily Previous phylogenetic analysis of Rhizobiales identified Dolichoderinae: Dolichoderus from Dolichoderus revealed a close association with the The majority of the sequences obtained from C1 belonged genus Bartonella and, more distantly, to the mutualistic to unclassified members of Rhizobiaceae, Orbaceae, and nitrogen-fixing rhizobia symbionts from leguminous Burkholderiaceae. Rhizobiaceae was found to be the plants (Russell et al. 2009). Furthermore, a Rhizobiales symbiont from , which is a leaf-cutting ant,

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Figure 5. Relative abundance of A) families and B) genera associated with the five arboreal ant species (cut off, ≥ 1.0%). was found to produce NifH protein, which is associated On the other hand, Spirochaetes are known to exist in with nitrogen fixation (Sapountzis et al. 2015). The termite gut communities and were proposed to be involved abundance of unclassified members of Rhizobiaceae in in hydrogen metabolism (Rosenthal et al. 2011). In the Dolichoderus may imply that this bacterial group plays a present study, an abundance of Treponema – particularly significant role as an ant symbiont. in the termite cluster – was documented (Figure 5B). Treponema azotonutricium sp. nov. and Treponema The bacterial taxon Orbaceae was also found to be primitia sp. nov. were the first spirochaetes isolated dominant in D. thoracicus. This bacterial group was in pure culture from the termite gut and were found to previously identified in other insect orders – including be involved in dinitrogen fixation, and H2 plus CO2 Hymenoptera, Coleoptera, Lepidoptera, and Diptera acetogenesis, respectively (Graber et al. 2004). Other (Martinson et al. 2011). Currently, the authors did not bacterial taxa associated with termites were documented find previous reports/studies about the abundance of this as well. These include species belonging to Clostridiales, bacterial taxon in ants. A reported functional analysis specifically Ruminococcaceae and Lachnospiraceae, of bee gut symbionts showed that Gilliamella apicola, which have been previously characterized for having a member of Orbaceae, derived fatty acids from plant plant cell wall-degrading glycoside hydrolase (Auer et polysaccharides and helped maintain the host’s health al. 2017). Members of the genus Dysgonomonas, which by breaking down toxins from their diet (Zheng et al. are known for their cellulolytic activity, were also found 2016). Also, the Orbaceae group may have been brought in the termite gut (Yang et al. 2014). by another insect mutualist since Dolichoderus species develop a mutualistic relationship with other hemipterans. The association of the bacterial taxa with Myrmicaria found A previous study showed that D. thoracicus derived its in this study may be traced to the foraging habits of the diet from the honeydew of its mutualist (Saripah and host. Myrmicaria foraging sites range from being ground Azhar 2012). dwellers, arboreal foragers, and some forage on nests on trees (Wriedt et al. 2009). Some species of Myrmicaria were found to attack insect prey, including termites (Kenne and Subfamily Myrmicinae: Myrmicaria Dejean 1999). subcarinata (Smith) Myrmicaria (C2) showed the greatest number of taxa for example, mostly feeds on prey as well as on identified to the genus level among all the samples. The herb particles and plant juices. It was found to possess dominant taxa were Rhizobiaceae, Spirochaetaceae, trophobioses with several bugs (Coreinae) and other Ruminococcaceae, and Fibrobacterales (Figure 5A). hemipteran species (Wriedt et al. 2009). Rhizobiaceae is the most abundant family with 46.16%, while the unclassified Rhizobiaceae and Candidatus tokpelaia were the most dominant genera (Figure 5B). Subfamily Formicinae: Colobopsis and Polyrhachis Ca. tokpelaia was recently found to be associated with The Camponotini ants from the subfamily Formicinae predatory ants and Dolichoderus (Neuvonen were represented by C. leonardi, P. mindanaensis, and et al. 2016; Bisch et al. 2018). P. semiinermis. Members of Colobopsis strictly thrive in arboreal habitats, while some members of Polyrhachis are

759 Philippine Journal of Science Gatpatan et al.: Bacterial Diversity in Ants Vol. 150 No. 3, June 2021 arboreal ants (Ramalho et al. 2017a, b). Arboreal ants mainly found in amino acid – and in nitrogen metabolism [e.g. feed on plant exudates and phytophagous insects (Ramalho glutamine synthetase, glutathione S-transferase, glutamate et al. 2017b), although exudates have low nitrogen content dehydrogenase, alanine or glycine: cation symporter, (Davidson et al. 2003). Majority of the sequences from AGCS family, cysteine desulfurase, glucosamine- C. leonardi were Enterobacteriaceae, Anaplasmataceae, fructose-6-phosphate aminotransferase (isomerizing)] Entomoplasmataceae, and Acetobacteraceae. On the other and N utilization were profiled as predicted functional hand, the two Polyrhachis species – P. mindanaensis and characteristics of bacteria associated with the samples. P. semiinermis – were dominated by Enterobacteriaceae and Anaplasmataceae at 99.16% and 98.98% relative The Camponotini ant tribe shared the same metabolic abundances, respectively (Figure 5A). Of the pathways. Thus, the dominant bacterial taxa may have Enterobacteriaceae observed, Candidatus Blochmannia greatly influenced the results of inferred functional was found to be the predominant genus (Figure 5B), profile – particularly, Rhizobiales from D. thoracicus and similar to the findings of previous studies (Ramalho et al. Myrmicaria sp. and Enterobacteriales, Ca. Blochmannia 2017b; Koto et al. 2020). Ca. Blochmannia is a well-known from the Camponotini group. intracellular symbiont of Camponotus ants (Feldhaar et al. Because arboreal ants tend to rely on plant-based food 2007; Eilmus and Heil 2009). Ca. Blochmannia helps its limited in nitrogen, the bacterial association plays an hosts in upgrading the diet through the synthesis of essential important role in host nutrition. It was hypothesized that amino acids, especially so that these ants utilize nutrient- herbivorous ant symbionts are able to recycle nitrogen deficient diets (Ramalho et al. 2017b). The symbiont waste products by degrading urea from animal wastes encodes a functional urease and glutamine synthetase and (Feldhaar et al. 2007). A study on gut bacteria of arboreal may, therefore, be involved in nitrogen recycling (Feldhaar Cephalotes ants revealed a high-efficient nitrogen et al. 2007). Furthermore, Ca. Blochmannia has a complete economy capable of recycling nitrogen to synthesize gene set for glycolysis, but it lacks essential genes for essential amino acids, and arginine metabolism to gluconeogenesis (Zientz et al. 2004). synthesize urea (Hu et al. 2018). Some ant endosymbionts The abundance of Wolbachia was also noted from the three have a complete urease gene cluster that encodes for an Camponotini ants (Figure 5B). Approximately 50–70% enzyme that hydrolyzes urea to produce CO2 and ammonia of insect species and about 30% of ant species have been (Zientz et al. 2004). Camponotus ant endosymbiont Ca. reported to be facultatively infected with Wolbachia Blochmannia recycles ammonia into the host's amino acid (Russell et al. 2012; Tolley et al. 2019). While most of the metabolism by glutamine synthetase activity (Zientz et bacterial symbionts are considered beneficial, Wolbachia al. 2004). Moreover, the complete biosynthetic pathways is detrimental and is a reproductive manipulator (Tolley et of 18 amino acids were reported in the genomes of al. 2019). It can cause manipulation of sex ratios and male Dolichoderus spp. symbionts (Bisch et al. 2018). These killing, and is a known entomopathogen (Stouthamer et symbionts have been shown to have the ability to recycle al. 1999). The high infection with Wolbachia can reduce nitrogenous waste to synthesize amino acids and vitamins the diversity of bacterial communities (Jones et al. 2013). (Russell et al. 2012; Bisch et al. 2018). Other bacterial taxa including Entomoplasmataceae and Acetobacteraceae were also predominant in C. leonardi. Entomoplasmataceae was detected as a symbiont of CONCLUSION Camponotus ants (Ramalho et al. 2017a). Likewise, The present study generated baseline information on Acetobacteraceae was reported to be a frequent and the bacterial diversity associated with five arboreal persistent gut symbiont of Camponotus ants and believed ants from the canopy to the understory of selected to be host-specific (Brown and Wernegreen 2016). trees from MMFR. Individual analysis of the bacterial taxa associated with the five species showed that Predictive Functional Gene Profile unclassified members of Rhizobiaceae, Orbaceae, and Based on predicted KEGG pathways of the five Burkholderiaceae were dominant in D. thoracicus. microbiomes, genes related to pathways involved in the Unclassified members of Rhizobiaceae, Spirochaetaceae, metabolism of carbohydrates, amino acids, lipid, and and Ruminococcaceae were dominant in Myrmicaria sp. energy were abundant in Dolichoderus, Myrmicaria, Candidatus Blochmannia and Wolbachia were abundant and Camponotini (Figure 6). Structural proteins involved in Camponotini ants C. leonardi, P. mindanaensis, and P. in the transport of sugars and amino acids (i.e. multiple semiinermis. The predicted functional profiles generated sugar transport system ATP-binding protein, ribose from the study revealed that these bacterial associations transport system ATP-binding protein, and polar amino may contribute to the host's nutrition and health. The acid transport system permease protein) – enzymes current study opens a new opportunity to further explore

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Figure 6. KEGG functional profiles of the five arboreal ant species (cut-off, ≥ 0.01). the wide ecology of bacterial communities associated REFERENCES with arboreal ants and to understand microbial interaction, ecology, and dynamics in the rainforest canopy ecosystem. AßHAUER KP, WEMHEUER B, DANIEL R, MEINICKE P. 2015. Tax4Fun: predicting functional profiles from metagenomic 16S rRNA data. Bioinformatics 31(17): 2882–2884. ACKNOWLEDGMENT AUER L, LAZUKA A, SILLAM-DUSSÈS D, MIAMBI E, O’DONOHUE M, HERNANDEZ-RAQUET The authors are grateful to the Department of Science G. 2017. Uncovering the potential of termite gut and Technology (DOST)–Philippine Council for microbiome for lignocellulose bioconversion in Agriculture, Aquatic, and Natural Resources Research anaerobic batch bioreactors. Front. Microbiol 8: 2623. and Development; DOST–Accelerated Science and Technology Human Resource Development Program; and BASSET Y, CIZEK L, CUÉNOUD P, DIDHAM RK, Makiling Center for Mountain Ecosystem. NOVOTNY V, ØDEGAARD F et al. 2015. Arthropod distribution in a tropical rainforest: tackling a four dimensional puzzle. PLoS One 10(12): e0144110.

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