Australian sheep blowfly

Sample information

Picture
Photos by: Sarah B.
Location
Collection date 08/30/2020
Captive / Cultivated? Wild-caught
Group Bordenstein Lab
Observations

This blowfly was buzzing around my patio, landed in my glass of water, and drowned. It was collected by using forceps to pull out of the water.

Putative identification Hexapoda Insecta Diptera Calliphoridae Lucilia Lucilia cuprina

Methods

Extraction kit DNeasy (Qiagen)
DNA extraction location Abdomen
Gel electrophoresis system MiniOne
Buffer TBE
DNA stain GelGreen
Gel images
Protocol notes

The fly was stored in 70% ethanol at room temperature for about 3 weeks prior to DNA extraction.

DNA Extraction: The fly was incubated in lysis buffer at 56C for 2 hours. Because there was cell debris, I did a 30 sec spin and transferred the supernatant to a fresh tube of 200ul ethanol. This was placed in the freezer overnight and DNA was purified the following day. Eluted DNA was immediately incubated at 65C for one hour prior to PCR.

PCR: MiniOne Taq polymerase was used.

Gel electrophoresis: The arthropod gel looks whispy; however, that went away as the gel ran longer. This could have been caused by not adding loading dye to samples. The results were very clear, though. I re-colored the gel images in PowerPoint. The MiniOne ladder contains 5 bands: 100, 300, 500, 1000, and 2000 bp. This sample was labeled as “blowfly”

Results

Wolbachia presence No
Confidence level High
Explanation of confidence level

The DNA extraction was successful because the arthropod COI amplified. Both (+) and (-) controls worked. The Wolbachia 16S rRNA band was absent.

Sanger sequencing revealed, with 100% identity, that it was an Australian sheep blowfly and not a housefly, as originally predicted.

Wolbachia 16S sequence
Arthropod COI sequence Download FASTA    Download AB1
AATTGGAACTTCTTTAAGAATTCTAATTCGAGCTGAATTAGGACACCCTGGAGCTTTAATTGGAGATGATCAAATTTATAATGTAATTGTTACAGCTCATGCTTTTATTATAATTTTTTTTATAGTAATGCCAATTATAATTGGAGGATTTGGAAATTGATTAGTTCCATTAATACTAGGAGCTCCAGATATAGCATTCCCTCGAATAAATAATATAAGTTTTTGACTTTTACCTCCTGCATTAACTTTATTATTAGTTAGTAGTATAGTAGAAAACGGAGCTGGAACAGGATGAACAGTTTACCCTCCCCTATCTTCTAATATTGCTCATGGAGGAGCTTCTGTTGATTTAGCTATTTTCTCTCTTCATTTAGCAGGAATTTCTTCAATTTTAGGAGCTGTAAATTTTATTACTACAGTTATTAATATACGATCAACAGGAATTACTTTTGATCGAATACCTTTATTTGTTTGATCAGTAGTAATTACAGCTTTATTGCTTTTATTATCATTACCAGTATTAGCAGGAGCTATTACAATACTTTTAACAGACCGAAATCTTAATACATCATTCTTTGATCCTGCAGGAGGAGGAGATCCAATTTTATACCAACATTTATTTT
BLAST at The Wolbachia Project   BLAST at NCBI
Summary The Lucilia cuprina was found to be negative for Wolbachia.
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