bioRxiv preprint doi: https://doi.org/10.1101/560755; this version posted February 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 1 Title: Taxonomic survey of Anadenobolus monilicornis gut microbiota via shotgun 2 nanopore sequencing 3 Short Title: Nanopore sequencing of Anadenobolus monilicornis gut microbiota 4 Orlando J. Geli-Cruz1, Matias J. Cafaro1, Carlos J. Santos-Flores1, Alex J. Ropelewski2, 5 Alex R. Van Dam1* 6 University of Puerto Rico Mayagüez, Call Box 9000 Mayagüez, PR 00681-90001 7 Pittsburgh Supercomputing Center, 300 S. Craig Street, Pittsburgh, PA 152132 8 Author Contributions: AVD conceived the project; AVD, MJC, AR and CJSF contributed 9 resources; AVD and OJGC analyzed the data; OJGC, AVD, MJC, and CJSF wrote the paper. 10 Abstract 11 Millipedes constitute one of many soil-inhabiting organisms that act as important 12 components of litter decomposition and nutrient recycling in terrestrial ecosystems. This is thanks 13 in part to the microbial diversity that they contain in their gut compartments. However, millipedes 14 and their gut microbiota are understudied, compared to other arthropods. For this reason, we 15 partook in a metagenomic analysis of the gut of Anadenobolus monilicornis. We collected 16 specimens of A. monilicornis, which were starved for a varying amount of time, from different 17 municipalities of Puerto Rico. Once the DNA from their guts was extracted and sequenced using 18 the MinION nanopore sequencer, we proceeded to analyze and compile the data obtained from the 19 sequencer using programs such as Phylosift and MEGAN6 and the web-based MG-RAST. From 20 our two best samples, we obtained a total of 87,110 and 99,749 reads, respectively. After 21 comparing the data analyses and gene annotation done for both samples, we found that the bacterial bioRxiv preprint doi: https://doi.org/10.1101/560755; this version posted February 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 2 22 phyla Proteobacteria, Bacteroidetes and Firmicutes were consistently well represented; one of our 23 samples had much more Chlamydiae representation than the other, however. Sampled eukaryote 24 phyla include Arthropoda, Chordata and Streptophyta. We would need a greater sample size to 25 better determine differences in microbial diversity between millipede populations across the 26 island; considering our small sample size, however, we were able to broadly reveal the diversity 27 within the microenvironment of A. monilicornis’s gut. 28 29 Introduction 30 Millipedes are a group of arthropods belonging to the class Diplopoda and the subphylum 31 Myriapoda. With around 12,000 described species worldwide, they are a diverse group found in a 32 variety of habitats from humid rainforests to xeric deserts [1–3]. Millipedes are one of many soil- 33 inhabiting decomposers, alongside other macroinvertebrates such as earthworms and isopods [4– 34 6]. They are considered in some ecosystems as one of the more important components of terrestrial 35 litter decomposition and nutrient cycling [4–6]. However, they are an understudied arthropod 36 group and have received little attention to elucidate their interactions with their gut microbial 37 community, despite their significance in nutrient cycling at the soil leaf litter interface [7,8]. 38 In comparison, the microbial composition of the gut of leaf litter macroinvertebrates has 39 received some attention from the scientific community. Termites, for example, are known to 40 harbor diverse clades of bacteria from the phyla Bacteroidetes, Firmicutes and Spirochaetes in 41 their guts, depending on the diet and gut compartments studied [9]. The guts of terrestrial isopods, 42 which also have a similar ecological role to millipedes, are well represented by Proteobacteria 43 [10]. In a similar fashion, millipedes have been shown to host certain microorganisms in their guts bioRxiv preprint doi: https://doi.org/10.1101/560755; this version posted February 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 3 44 which aid in digestive processes [11]. Dilution plating techniques have shown that some millipede 45 species harbor an abundant diversity of proteobacteria and actinobacteria [12]. 46 There have been a small number of microbial community surveys of the gut of millipedes. 47 In some species, the most dominant bacteria were found to belong to the Enterobacteriaceae 48 family; in addition, ascomycetes were the most common yeast strains found [13]. A desert- 49 dwelling millipede species possesses gut bacteria that can degrade cellulose, contributing to 50 nutrient cycling in deserts [1]. As well, certain species from the millipede orders Julida, 51 Spirobolida, and Spirostreptida harbor an association between methanogenic archaea and ciliate 52 protozoa in their hindguts, contributing to methane production [14]. The diversity of bacteria and 53 other microorganisms that occur in millipede guts might be of interest to field ecologists and 54 microbiologists alike, as the interactions between these organisms affects both soil nutrient 55 recycling and organic matter decomposition [15,16]. A full genetic or metagenomic approach to 56 these problems, however, has yet to take off. 57 For this study, we will be focusing on the microbiota that inhabits Anadenobolus 58 monilicornis’s digestive tract. A. monilicornis is a species native to the Caribbean which has also 59 been introduced to Florida (U.S.A.), where it is treated as a pest [17,18]. It is considered the most 60 common millipede in the karst zones of Puerto Rico [3]. Previous microbiota studies have been 61 morphohology based for A. monilicornis; specifically, Contreras & Cafaro [19] conducted a 62 morphometric study of the protozoa Enterobryus luteovirgatus, which forms a commensalistic 63 relationship with the millipede. Beyond this study, very little is known about this species. 64 To identify the microbial diversity inside A. monilicornis’s gut, we will utilize a shotgun 65 metagenomic analysis. Shotgun sequencing of environmental samples, most notably from bioRxiv preprint doi: https://doi.org/10.1101/560755; this version posted February 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 4 66 microbial communities [20], can lead to the discovery of microorganisms that are otherwise 67 difficult or impossible to culture in a laboratory setting [21]. Several metagenomic sequencing 68 studies have successfully revealed complex host-symbiont relationships, such as those that occur 69 in deep-sea tube worms [22], termites [23] and Daphnia species [21]. Particularly, metagenomic 70 studies involving arthropods have focused mostly on insects [24–27], successfully revealing their 71 microbial diversity. However, there are few comprehensive metagenomics sequencing surveys of 72 the microbiota of other non-insect arthropods, millipedes included [28,29]. 73 To analyze the DNA of A. monilicornis, Oxford Nanopore Sequencing Technology (ONT) 74 will be used. This relatively new technique works by identifying the order of nucleotides in a DNA 75 sequence as it passes through individual nanopore channels. ONT has already proven useful for 76 producing relatively long DNA sequences, real-time analysis, and detection of structural sequence 77 variants [30]. Specifically, we will be using the MinION, a portable nanopore sequencer developed 78 by Oxford Nanopore Technologies. Our objective is to begin to develop protocols for shotgun 79 sequencing of millipede guts via ONT, and to develop an initial fingerprint of the microbial 80 diversity found within A. monilicornis using ONT as a tool to elucidate this complex system. 81 82 Materials and Methods 83 Millipede Sampling 84 Anadenobolus monilicornis millipedes were collected from two locations collected in July 85 2017 from western Puerto Rico: The University of Puerto Rico, Mayagüez Campus, and the 86 municipality of Rincón. These were kept alive and starved for 24 hours (Mayagüez samples), and 87 ten days (Rincón samples). The millipedes were collected and kept in small glass containers with bioRxiv preprint doi: https://doi.org/10.1101/560755; this version posted February 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 5 88 moist filter paper. The Rincón sample was kept until it began to eat the filter paper and the frass 89 showed little signs of plant material. 90 Gut and DNA extraction 91 The gut extraction and DNA extraction work were done in the Symbiosis laboratory at the 92 University of Puerto Rico, Mayagüez Campus. Following workstation and lab material 93 sterilization with 10% bleach, the head and the last two or three segments of the abdomen of the 94 specimens were cut and removed with a scalpel; the abdomen was cut to facilitate gut extraction. 95 The guts were removed and placed in 2mL tissue disruption tubes, where they were liquified by 96 manually shaking the tubes. 97 We followed the Qiagen Fast DNA Tissue Kit (cat. No. 51404) protocol to purify the DNA 98 samples from the specimens. The buffer for the Qiagen Fast DNA Tissue Kit was prepared before 99 use as the protocol specified: 40 mL of ethanol were added to the AW1 and AW2 Buffer 100 concentrates, and 25mL of isopropanol were added to the Buffer MVL concentrate.