Wolbachia #2: Pillbug

March 30, 2023

Sample information
Picture
Photos by: Kamilia B.
Location
Collection date 09/14/2022
Captive / Cultivated? Wild-caught
Group Woburn Memorial High School
Observations

– pillbug was very active and was still alive while being shoved in the ethanol tube

– had a hard shell and many legs

– gray color

– size of a pea
Putative identification Arthropoda Crustacea Malacostraca Isopoda Armadillidae Armadillidium Armadillidium vulgare

Methods
Extraction kit Edwards Buffer
DNA extraction location Abdomen
Single or Duplex PCR Duplex Reaction
Gel electrophoresis system MiniOne
Buffer TBE
DNA stain GelGreen
Gel images
Protocol notes

– cut off abdomen of mosquito in order to use the sex organs to find Wolbachia

– Cell lysis the DNA of the insect and then precipitate the DNA

– Then drain the DNA pellets in absorb the supernatant for PCR testing

– after running PCR use the DNA to run a 2% agarose gel electrophoresis

– Well 1: Ladder (100 bp)
– Well 2: arthropod #1 mosquito
– Well 3: arthropod #2 pill bug
– Well 4: (+) Arthropod Control
– Well 5: (-) Arthropod Control
– Well 6: (+) DNA Control
– Well 7: (-) DNA Control (Water)
– Well 8: nothing

– compare results with control DNA in order to identify wolbachia in the insect

Results
Wolbachia presence Unknown
Confidence level Medium
Explanation of confidence level

– As there is no arthropod band or Wolbachia band in well 3, there may have been in an error in the PCR process. Since there was identifiable cross contamination, it cannot be confidently confirmed that the arthropod has Wolbachia
Wolbachia 16S sequence
Arthropod COI sequence
Summary
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