DOI: 10.5061/dryad.08kprr58q
title: Data from: Molecular characterisation of faecal bacterial assemblages among four species of syntopic odonates

description: Factors such as host species, phylogeny, diet, timing, and location of
 sampling are thought to influence the composition
 of gut-associated bacteria in insects. In this study, we compared
 the faecal-associated bacterial taxa for three Coenagrion and one
 Enallagma damselfly species. We expected high overlap in representation of
 bacterial taxa due to the shared ecology and diet of these species. Using
 metabarcoding based on the 16S rRNA gene, we identified 1513 sequence
 variants, representing distinct bacterial ‘taxa’. Intriguingly, the
 damselfly species showed somewhat different magnitudes of richness of
 ZOTUs, ranging from 480 to 914 ZOTUs. In total, 921 (or 60.8% of the 1513)
 distinct ZOTUs were non-shared, each found only in one species, and then
 most often in only a single individual. There was a surfeit of these
 non-shared incidental ZOTUs in the Enallagma species accounting for it
 showing the highest bacterial richness and accounting for a sample-wide
 pattern of more single-species ZOTUs than expected, based on comparisons
 to the null model. Future studies should address the extent to which
 faecal bacteria represent non-incidental gut bacteria and whether abundant
 and shared taxa are true gut symbionts.
description: To assess the faecal bacterial assemblages of damselflies, we
 targeted four predatory odonate species at a freshwater pond of
 approximately 600 m × 200 m (12 ha), located in Southern Finland
 (ETRS-TM35FIN N: 67118; E: 2460). On 1–2 June 2016, we collected 185
 individuals (20–26 males and females from each species) for faecal DNA
 analysis. All our focal damselfly species belong to the family
 Coenagrionidae: <em>Coenagrion lunulatum</em> (Charpentier,
 1840), <em>Coenagrion hastulatum</em> (Charpentier, 1825),
 <em>Coenagrion pulchellum</em> (Vander Linden, 1825), and
 <em>Enallagma cyathigerum</em> (Charpentier, 1840). Species
 identification of damselflies was based on current literature, e.g. [27].
 These four target species were selected as they were the most common
 predatory species at the study site, based on pilot surveys (K. Kaunisto,
 pers. obs.). Only sexually mature individuals with adult colours and
 hardened wings were included in the study. According to a previous study
 [28], all four focal species feed mainly on dipteran prey by open foraging
 flights and by gleaning insects from vegetation.<br>Each damselfly
 was placed into a sterile 10-ml collection tube housing a piece of
 dampened paper towel to reduce desiccation risk. To allow for defecation,
 damselflies were kept in the tubes for the next 24 h (sufficient time for
 defecation to occur, according to [18]). After the live individuals had
 defecated into the tube, we froze the entire sample without removing the
 faeces or the damselfly. All faecal material was collected from the tubes
 with sterile forceps, after which the faeces were frozen in 15-ml Falcon
 tubes at −64 °C until further processing and analysis.
 <strong>Sample Processing and Molecular
 Analysis</strong> Total DNA was extracted as
 described in a previous study using NucleoSpin Tissue XS Kit (product nr
 740901, Macherey-Nagel, Düren, Germany) [28]. To characterize the
 bacterial assemblages of the focal species, we used established
 metabarcoding protocols for dragonflies building on earlier optimization
 [1828]. To amplify bacterial 16S rRNA gene (hypervariable region v4), we
 used primers 515F-Parada (also known as 515FB: 5′-GTG YCA GCM GCC GCG GTA
 A-3′; Parada et al. 2016) and 806R-Apprill (also known as 806RB: 5′-GGA
 CTA CNV GGG TWT CTA AT-3′; [29]). Each DNA sample was amplified in two
 separate reactions that were individually tagged and sequenced. The
 locus-specific PCR setup followed Kankaanpaa <em>et al.</em>
 [30] and included 5 μl of 2× MyTaq HS Red Mix (Bioline, UK), 2.4 μl of
 H2O, 150 nM of each primer (two forward and two reverse primer versions;
 total primer mix concentration 600 nM), and 2 μl of DNA extract per each
 sample in 10 μl volume. CycAQ6ling conditions were 3 min at 95 °C, then 35
 cycles of 45 s at 95 °C, 1 min at 50 °C, and 1 min 30 s at 72 °C, ending
 with 10 min at 72 °C. In the second PCR stage, the first PCR products were
 modified by attaching Illumina-specific adapters and sample-specific
 indices. For a reaction volume of 10 μl in the indexing PCR, we mixed 5 μl
 of MyTaq HS RedMix, 500 nM of each tagged and indexed primer (i7 and i5),
 and 3 μl of locus-specific PCR product from the first PCR phase. For this
 second PCR, we used the following protocol: initial denaturation for 3 min
 at 98 °C, then 15 cycles of 20 s at 95 °C, 15 s at 60 °C, and 30 s at 72
 °C, followed by 3 min at 72 °C. All the indexed reactions were then pooled
 and purified using magnetic beads [3132].<br>Sequencing was done on
 an Illumina MiSeq v3 PE 2×300 (Illumina Inc., San Diego, CA, USA) run,
 including the PhiX control library by the Turku Centre for Biotechnology,
 Turku, Finland. After sequencing, the reads were demultiplexed into each
 original sample and uploaded onto CSC servers (IT Center for Science,
 https://www.csc.fi/ ) for bioinformatic analysis. Paired-end reads
 (13,027,754) were merged and trimmed for quality using 64-bit vsearch
 version 2.14.2 [33] command ‘fastq_mergepairs’ with the default options
 and ‘fastq_allowmergestagger’. Primers were removed from the merged reads
 (11,179,018) using software cutadapt version 1.14 (Martin 2011) with 20%
 mismatch rate, minimum length of 240 bp and truncate length of 270 bp (the
 excess nucleotides were trimmed from 3′ end). Trimmed reads (11,050,385)
 reads were then collapsed into unique sequences (singletons removed) with
 command ‘fastx_uniques’ and option ‘minuniquesize’ set to 10 (49,832
 uniques retrieved). Finally, reads were corrected for point errors to
 obtain an accurate set of amplicon sequences (=denoised) and filtered of
 chimeric amplicons (=chimeras were removed) resulting in 3803 ZOTUs
 (‘ZOTU’, ‘zero-radius OTU’) through command ‘unoise3’ using USEARCH
 version 11.0.667 with settings minsize = 8 and unoise_alpha = 2. The
 median and mean length of ZOTUs was 253 bp (SD ± 2.50 bp) Then ZOTUs were
 mapped back to the original trimmed reads with command ‘usearch_global’ to
 establish the total number of reads in each sample using vsearch. We were
 able to map 10,627,197 of 11,050,385 (96.17%) to our original samples. The
 ZOTUs (sequence variants) were assigned to taxa using 16 RDP database with
 SINTAX (Edgar, 2010) probabilistic algorithm implemented in vsearch. The
 database ‘16S RDP training set v18’ (21k seqs) was downloaded from the
 usearch website (https://drive5.com/usearch/manual/sintax_downloads.html;
  accessed 19th April 2023). For the chosen database, the genus level is
 the lowest taxonomic level. For any taxonomic level, we only accepted
 assignations with 100% probability. The data was further filtered to
 remove artefacts, spurious reads, and non-targets based on information on
 the numerous control samples, technical replicates, and taxonomy. First,
 we removed those ZOTUs from any sample that had fewer reads than
 extraction or PCR controls (9,833,618 reads retained). Then, we collapsed
 reads based on the taxonomy per each sample, that is, all the reads that
 were assigned to the same taxa per sample were summarized. Out of the 3803
 ZOTUs, we identified 983 to genus, 1570 to family, 2002 to order, 3063 to
 class, 3319 to phylum, and 3482 to domain level. From the total ~10M
 reads, we identified 4.0M to genus, 4.4M to family, 8.5M to order, and
 9.5M to the higher levels. Then, we removed taxa that were present in a
 sample by only one of the two replicates and finally summed the reads in
 both replicates (9,678,663 reads left). Then, to remove potentially leaked
 ‘tag-jumped’ reads from the data, we removed all taxa from the samples
 with less than 0.05% proportion of the total reads in one sample
 (9,636,233 reads saved). We removed all the taxa outside domains Bacteria
 or Archaea, as well as Class Chloroplast (9,006,117 reads passed the
 filtering). The non-targets included mainly plants (~6200 reads) and Fungi
 (~250 reads). Altogether 284,351 reads could not be assigned with the
 strict 100% probability threshold. Finally, very rare occurrences
 (sequence count &lt; 20) were removed (9,004,996 final
 reads). 
description: # Data from: Molecular characterisation of faecal bacterial assemblages
 among four species of syntopic odonates
 [https://doi.org/10.5061/dryad.08kprr58q](https://doi.org/10.5061/dryad.08kprr58q) Molecular data from faecal PCR-based 16S sRNA DNA analysis from four damselfly species. ## Description of the data and file structure DNA extraction using Macherey-Nagel NucleoSpin Tissue XS Kit (product nr 740901, Macherey-Nagel, Düren, Germany), followed by 16S gene PCR using primers 515F-Parada 5′-GTG YCA GCM GCC GCG GTA A-3′ (Parada et al. 2016) and 806R-Apprill 5′-GGA CTA CNV GGG TWT CTA AT-3′ (Apprill et al. 2015), and library preparation. Sequencing was carried on Illumina MiSeq v3 2x300bp run. Data consists of raw FASTQ files with two technical replicates per sample. Retrieved sequence variants (ZOTUs), ZOTU tables, and ZOTU assignations are also included. See the original article for more detailed information. ### File contents and Column names ##### File = rdp\_16s\_v18\_plus\_ZymoMock\_out.txt, tab-separated, no header-line Column 1 = ZOTU ID. This is the sequence variant ID based on the bioinformatics carried out up to this step. Column 2 = Taxonomic identification of each ZOTU. This column contains the whole taxonomic path from Domain to the genus level based on probabilistic assignation of the ZOTUs. The file contents are a standard output from USEARCH SINTAX algorithm. Each taxonomic level is separated with a comma and taxa abbreaviation. The probability of each ZOTU for each taxonomic level is given in parentheses. All taxonomic levels are abbreviated as follows: d = Domain p = Phylum c = Class o = Order f = Family g = Genus ##### File = zotutab\_global.txt, space-separated, includes a header-line First column = ZOTU ID. This is the sequence variant ID based on the bioinformatics carried out up to this step. Columns 2-379 = Each column contains a sample, and each samples is represented as two technical PCR replicates. The naming of the samples follows this logic: ---. Odonate species contain species information (COH = Coenagrion hastulatum; COL = Coenagrion lunulatum, COP = Coenagrion pulchellum, and ENC = Enallagma cyathigerum). Sex and individual number are as follows: sex (F = female; M = male) and a running number for each individual in the group. *For example: Bac-1-ENC-M4 is the first PCR replicate of the Enallagma cyathigerum male number 4 sample.* ##### File = zotus.fa, fasta-formatted file Each header line begins with character '&gt;', followed by ZOTU ID. The ZOTU nucleotide sequence begins always after the header line. The sequence is wrapped to 80 character rows. ##### File = ODO\_Bac\_17Feb2018.tgz, tar-ball file containing compressed (guzip) FASTQ-formatted raw sequence files Each file is named as follows, separated by dash '-' project-tag = Always Bac for this dataset. replicate number 1 or 2 = Technical PCR replicate 1 or 2. Odonate species = Odonate species information (COH = Coenagrion hastulatum; COL = Coenagrion lunulatum, COP = Coenagrion pulchellum, and ENC = Enallagma cyathigerum) Sex code and individual number = sex (F = female; M = male) and a running number for each individual in the group. ILLUMINA TECHNICAL CODE = everything after the underscore is technical Illumina sample code and relevant to the study or data. File name extensions = The extension FASTQ refers to FASTQ format sequence file and extension GZ refers to gzip-compressed file. *For example: **Bac-1-ENC-M4**_S25_L001_R1001.fastq.gz is the first PCR replicate of the Enallagma cyathigerum male number 4 sample. Everything after the underscore is technical Illumina sample code and relevant to the study or data. The extension FASTQ refers to FASTQ format sequence file, and extension GZ refers to gzip-compressed file.*