Asain Lady Beetle

April 2, 2025

Sample information
Picture
Photos by: Charlotte W
Location
Collection date 02/17/2025
Captive / Cultivated? Wild-caught
Group Berkshire Community College
Observations

Laby beetle has very slow movement and fading colors with roughly 4 spots on each side of its back. It has two large spots at its head.
Putative identification Arthropoda Hexapoda Insecta Coleoptera Coccinellidae Harmonia Harmonia axyridis

Methods
Extraction kit Monarch DNA extraction (NEB)
DNA extraction location Partial abdomen
Single or Duplex PCR Single Reaction
Gel electrophoresis system Edvotek Gel Electrophoresis
Buffer TAE
DNA stain SYBR Safe
Gel images
Protocol notes

Single PCR Reaction – CO1 Primers 3/12

Taq polymerase used: New England Biolabs OneTaq Hot Start Quick-Load 2x MM w/Standard Buffer

DNA Ladder:New England Biolabs 1kb Plus DNA Ladder for Safe Stains

Performed 1 cycle at 94 C, 30 cycles at 94 C, 49 C, and 68 C, and 1 cycles at 68 C. 55C annealing temperature.

Single PCR Reaction – Wolbachia_F and Wolbachia_R Primers 3/25

Taq polymerase used: New England Biolabs OneTaq Hot Start Quick-Load 2x MM w/Standard Buffer

DNA Ladder: New England Biolabs 1kb Plus DNA Ladder for Safe Stains

55C annealing temperature, same cycles as previously stated.

Results
Wolbachia presence No
Confidence level Medium
Explanation of confidence level
  1. Gel Electrophoresis provided clear distinct bands. In our first PCR and gel all controls appeared as expected. However, in the second PCR and gel for Wolbachia only 3 controls appeared as expected. We failed to produce a Wolbachia indicator for an arthropod known to have Wolbachia. This leads to medium confidence, with potential for false negative.
Wolbachia 16S sequence
Arthropod COI sequence
Summary The Harmonia axyridis was found to be negative for Wolbachia.
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