Boxelder Bug 2

December 4, 2025

Sample information
Picture
Photos by: Ayush P.
Location
Collection date 11/13/2025
Captive / Cultivated? Wild-caught
Group NCSSM
Observations

After my teacher mentioned that her husband had been seeing unfamiliar insects in the window of his office, I checked the area and quickly noticed several boxelder bugs clustered near the frame. The temperature was around 55°F, which likely encouraged them to seek warmth indoors. I collected some of the bugs for closer observation.
Putative identification Arthropoda Insecta Hemiptera Rhopalidae Boisea Boisea rubrolineata

Methods
Extraction kit DNeasy (Qiagen) blood and tissue kit
DNA extraction location Whole arthropod
Single or Duplex PCR Single Reaction
Gel electrophoresis system MiniPCR
Buffer TBE
DNA stain
Gel images
Protocol notes

DNA Extraction: Because our boxelder bugs were smaller than a grain of rice, we did not dissect them. Instead, we used a standard whole-body tissue extraction method by placing a small portion of the bug into the extraction buffer and mechanically breaking down the tissue to release genomic DNA.

Gel Electrophoresis: We amplified the extracted DNA using both arthropod and Wolbachia primers. The arthropod primer served as a positive control to confirm that the PCR reaction worked and that our DNA extraction was successful. The Wolbachia-specific primer allowed us to test for the presence of Wolbachia DNA within the boxelder bug samples. After PCR, we ran the products through gel electrophoresis to visualize amplification results and determine whether Wolbachia was present.

Results
Wolbachia presence Yes
Confidence level High
Explanation of confidence level
The presence of a clear band from the arthropod primer suggests that our DNA extraction and PCR were successful, which increases our confidence that the procedure worked as intended. However, without a confirmed Wolbachia-positive sample to compare against, we cannot be fully certain that the absence or presence of a Wolbachia band is accurate.
Wolbachia 16S sequence Download AB1
ACCCTCATCCTTAGTTACCATCAGGTAATGCTGGGGACTTTAAGGAAACTGCCAGTGATAAACTGGAGGA AGGTGGGGATGATGTCAAGTCATCATGGCCCTTATGGAGTGGGCTACACACGTGCTACAATGGTGGCTAC AATGGGCTGCAAAGTCGCGAGGCTAAGC
BLAST at The Wolbachia Project   BLAST at NCBI
Arthropod COI sequence Download AB1
GATCTGGCATAGTAGGCTCATCTTTAAGATGAATTATTCGTGTAGAATTAGGGCAACCTGGTAGATTTAT TGGGGATGATCAAACATATAATGTAATTGTAACAGCACATGCTTTTATTATAATTTTCTTTATAGTTATG CCAATTATGATTGGTGGATTTGGGAATTGATTAGTGCCTTTAATAATTGGGGCCCCAGATATAGCATTTC CTCGAATAAATAATATAAGATTTTGACTTTTACCTCCTTCCCTTACTTTATTACTTGCAAGTAGTATAGT TGAAAGAGGGGCGGGAACTGGATGAACAGTTTATCCCCCTCTATCAAGAAATTTATCCCATAGAGGTGCC TCTGTAGATTTAGCAATTTTTTCATTACATTTAGCAGGTGTATCATCAATTTTAGGGGCTATTAATTTTA TTTCAACTATTATTAATATACGGCCAGCAGGAATAAGACCTGAACGAATACCATTATTTGTATGATCAGT AGGTATTACAGCTCTTCTATTATTGTTGTCTTTACCTGTTTTAGCGGGGGCCATTACAATACTTTTAACT GACCGAAACTTTAATACATCTTTCTTTGATCCTACCGGGGGAGGAG
BLAST at The Wolbachia Project   BLAST at NCBI
Summary The Boisea rubrolineata was found to be postive for Wolbachia.
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