Analysis of Stone Centipede and Anuroctonus phaiodactylus Microbiomes
Catherine Gaspar, Gage Mallard, Dr. Barbara Murdoch
Eastern Connecticut State University, Willimantic, CT, USA
Introduction
•Nanopore sequencing can sequence the human genome for under $1000 as compared to the 2.7 billion it cost in 2003.
•Nearly all bacteria and archaea contain the 16s rRNA gene, though most genera have a unique variation.
•Using this technology, we can determine if there is a microbiome present in a sample, and, if so, what microbes may be present.
•Many organisms have not had their microbiomes analyzed, leaving a new frontier open to be explored.
Hypothesis:
Arthropods collected in separate regions will have distinct and unique microbiomes.
Organism Samples
Lithobius forcifactus (Stone centipede)
- Sample from Elizabeth Town, KY
- Native to Europe, also found in U.S.
- Are habitat generalists; will live in any damp environment
- Eats insects and other invertebrates
- Sample from Baja California, Mexico - Native to the western parts of North America
- Relies on touch to find prey, even though the centipede has eyes Anuroctonus phaiodactylus
- Prefers dry areas with deep, well-packed soil where it can burrow - A. phaiodactylus is known for its burrowing, where it lives and hunts prey by ambushing unsuspecting targets
Methodology
Qubit Quantification
• A Qubit fluorometer was used to quantify the initial concentration of double-stranded DNA(dsDNA) in each sample.
DNA Amplification
• Polymerase Chain Reaction (PCR) was used to amplify the 16S rRNAgene and to add individual Nanopore sequencing barcodes to each sample.
Gel Electrophoresis
• PCR products (amplicons) for each sample were checked using gel electrophoresis. Purification
• AMPure bead cleanup was used to purify DNA of primers, enzymes, and other substances left over from PCR.
• Qubit quantification was used to determine the amount of DNAremaining after AMPure bead purification.
Nanopore Sequencing
• The DNA samples were pooled into one and placed into the nanopore device, which was run overnight.
Data Analysis
• EPI2ME software was used to analyze the genetic code against a known database so that a tentative identity can be assigned to each unique read.
• Microsoft Excel was used for calculations and graph production.
Results
Figure 2. Phyla identified from Lithobius forcifactus and Anuroctonus phaiodactylus samples. A 100% stacked bar graph was made to identify the phyla present and display their respective representations in each of the two samples analyzed.
A.
and the stone centipede.
Conclusions
B.
Future Directions
• Anuroctonus phaiodactylus and the stone centipede contained unique and distinct microbiomes.
• It appears our hypothesis was supported in that the microbiomes of each sample were highly dissimilar from each other, suggesting that arthropods from different regions may have different microbial compositions.
• The only shared community was the genera Serratia from the phylum Pseudomonadota, suggesting that it may be important to these organisms.
• It appears that the microbiome of Anuroctonus phaiodactylus contains a higher number of unique genera compared to the stone centipede.
References and Acknowledgements
• This technology could be used to identify bacterial pathogens or locate genetic mutations to accelerate disease treatment.
• This technology can be used to identify the microbes present in soil samples which, in turn, could be used to fix infertile soil and maximize crop yield.
• Nanopore sequencing could be used to look for antibiotic production from new, unexplored sources.
• It can be used to evaluate microbial diversity in one's body, exploring potential links between microbiome composition and health.
• It could be used to check water quality, looking for and identifying invisible contaminants.
Murdoch, B. (2025). Lecture notes. Summer Institute 2025 ChatGPT and Gemini were used to identify samples. Zachary Kelly assisted as Peer Mentor. North American Insects & Spiders. (2023, August 8). Brown Centipede - Lithobius forficatus - North American Insects & spiders. https://www.cirrusimage.com/centipedes_stone/ Dr. Matthew R. Graham provided the image of Anuroctonus phaiodactylus, as well as collected and provided the sample organisms.
ahmed et al. clinical epigenetics (2022) 14:107 https://doi.org/10.1186/s13148-022-01323- (Source of nanopore representation)
2. Genera Comparison Between Samples
1. Phyla Comparison Between Samples 3. Rarefaction Curves for Each Sample
Figure 4. Rarefaction curves of the microbiomes of Anuroctonus phaiodactylus
Figure 3. Genera found in L. forcifactuse and A. phaiodactylus Panel A is L. forcifactuse, and Panel B is A. phaiodactylus. Spiroplasma was the most prevalent in Panel A at 69%. Stenotrophomonas was the most prevalent in Panel B at 28%.
Figure 1. The Nanopore sequencing process on the molecular level. This image is a representation of active nanopore sequencing within the Flongle
(North American Insects & Spiders, 2023)
Anuroctonus phaiodactylus
Anuroctonus phaiodactylus