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
3. Microbial Composition: Phyla
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.
4 2 1
1
Pseudomonadota
Elusimicrobiota Pseudomonadota Verrucomicrobiota
27
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.
2. Species A
3
Bacillota
Acidobacteriota
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
B 3 2 1
1
1
2
Enterococcus
Paludibaculum Endomicrobium Microvirga
Klebsiella
Variovorax
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.
27
Ereboglobus
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
Figure 5. Nanopore sequencing of Southern Black Widow genera. 134 out of 136 of the genera sequenced were unknown. Enterococcus has been found in the guts of many arthropods, including Southern Black Widows (Swartzman et al., 2024).
Nanopore Sequencing • High accuracy base calling for 16S rRNA sequencing using Flongle device (Figure 2) DNA Extraction/ Quantitation
Library Preparation
Nanopore Sequences
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 • 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”
EPI2ME Taxonomic Analysis
6. Acknowledgments
Figure 2. Nanopore sequence workflow. (Adapted from Oxford Nanopore website).
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.
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