Philippine Journal of Science 150 (3): 753-763, June 2021 ISSN 0031 - 7683 Date Received: 30 Sep 2020 Diversity of Bacteria in Ants (Hymenoptera: 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 biodiversity 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 ant species collected from MMFR. The collected ants were identified as Dolichoderus thoracicus, Myrmicaria sp., Colobopsis leonardi, Polyrhachis 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 Camponotini 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] 753 Philippine Journal of Science Gatpatan et al.: Bacterial Diversity in Ants Vol. 150 No. 3, June 2021 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 insects 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 (genus 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 insect 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 arthropod biomass and 90% of the individual Gonzalez et al. 2020). Three large-diameter trees – arthropods. 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). 754 Philippine Journal of Science Gatpatan et al.: Bacterial Diversity in Ants Vol. 150 No. 3, June 2021 (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