Comparative Microbiome Analysis of Aphelia virginiensis and Latrodectus manctans
Gwyneth Glidden, Zoe Wazocha, and Dr. Barbara Murdoch
Eastern Connecticut State University, Willimantic CT USA
1. Introduction
Kentucky Flat Millipede Aphelia virginiensis
• Sampled from Elizabeth Town, Kentucky, US
• The compound it secretes, as a form of defense, contains cyanide Southern Black Widow Latrodectus manctans
• Sampled from Baja Peninsula Sur, Mexico
• Possesses wide variety in color variation for immature females and all males
Hypothesis
The microbiomes of the two invertebrate species will exhibit distinct bacteria with minimal similarities due to their diverse evolutionary relationship.
2. Species
Methodology
DNA Extraction and Quantitation
• Calculated samples using standard curve, checked with fluorometer
Library Preparation (PCR & barcoding)
• Amplified 16S rRNA gene in L. virginiensis and L. manctans with PCR; Amplicons checked with gel electrophoresis on 1% agarose gel
• Purification of DNA samples with AMPure beads
Nanopore Sequencing
• High accuracy base calling for 16S rRNA sequencing using Flongle device (Figure 2)
3. Microbial Composition: Phyla
Figure 3. Nanopore sequencing of Southern Black Widow phyla. 134 out of 136 of the phylum sequenced were unknown. Bacteria in these phyla are very common and diverse.
Figure 4. Nanopore sequencing of Kentucky Flat Millipede phyla. 547 out of 582 of the phylum sequenced were unknown. Acidobacteriota, Elusimicrobiota, & Verrucomicrobiota are commonly found in terrestrial soils; they can be very abundant.
4. Microbial Composition: Genera
Endomicrobium
Microvirga
Variovorax
Ereboglobus
Figure 6 Nanopore sequencing of Kentucky Flat Millipede genera. 547 out of 582 of the genera sequenced were unknown. Ereboglobus: In the family of Opitutaceae, found after being isolated from cockroach hindgut (Tegtmeier et al., 2018).
5. Conclusion & Discussion
Figure 7. Results from gel electrophoresis. This technique was used to separate the charged 16S rRNA gene to evaluate sizes of each fragment relative to one another. Bands from all samples resided around 1400 bp, which was verified by DNA ladders.
The nanopore sequencing technique is an innovative method for exploring the microbiome of different organisms Invertebrate have vast microbiomes and contain many kinds of bacteria
Improvement: use other applications after using EPI2ME to find more about the “unknown”
6. Acknowledgments
Contributions of Materials
Specimen samples were provided by Dr. Matthew R. Graham Flongle flow cells were purchased from Oxford Nanopore Technologies
ECSU Research Institute Faculty
Director of Success Scholar Program: Dr. Barbara Murdoch
Peer Mentor: Zachary Kelly
Figure 1. Millipede & Black Widow. 16S rRNA from A) A. virginiensis and B) L. manctans were collected from the specimens. A & B are from Walker, 2008 & Hollenbeck, 2022.
Figure 2. Nanopore sequence workflow. (Adapted from Oxford Nanopore website).