Sample information

The arthropod was found on top of a mostly still pool of water at the edge of a brook.  It was skidding across the water with others of the same species.  It was collected just after dark on a day that had light rain throughout the afternoon.  The temperature at the time of collection was about 17.8 degrees C (64 degrees F).

*Note that the arthropod was identified as Chironomidae sp. by NCBI BLAST using its DNA sequence (from the CO1 gene), but that identification was not considered accurate based on the observations of the collected arthropod.  Body structure and behaviors before collection did not line up with those expected from Chironomidae sp..  Based on observations the arthropod is deemed to be a water strider of an undetermined species (Gerris sp.).*

Methods

• We used a DNA extraction protocol based on the insect adaptation of New England Biolabs’ Monarch Spin gDNA Extraction kit (Product # T3010).
• The specimen was incubated for 30 minutes in a hot water bath at 56 degrees C.

• Our first PCR reaction was set up on 10/14/25 and was a duplex reaction because we used both the Arthropod CO1 and Wolbachia 16S primers together.
• We used an annealing temperature of 49 degrees Celsius.
• We used this Taq polymerase: New England Biolabs One Taq Hot Start Quick-Load 2X Master Mix with Standard Buffer (#M0488S)
• Differed from the original protocol in that: We used 88uL of Taq Master Mix in our PCR Cocktail rather than 87.5uL.

• Our first Gel image was taken on 10/21/25 and was run at 125 volts for 13 minutes and used this DNA Ladder: New England Biolabs 1 kb Plus DNA Ladder for Safe Stains (product # N0559S)

• Our second PCR was set up on 10/28/25, used the same Taq polymerase as the first PCR reaction, and was a single reaction that used Wolbachia 16S rRNA primers.
• We used an annealing temperature of 55 degrees Celsius.
• It differed from the original protocell in that: we used 46uL of water and 88uL of Taq Master Mix in our PCR cocktail rather than 45.5uL and 87.5uL (respectively).

• Our third PCR was set up on 10/28/25, used the same Taq polymerase as the first PCR reaction, and was a single reaction that used Arthropod CO1 primers.
• We used an annealing temperature of 49 degrees Celsius.
• It differed from the original protocell in that:
• 1. We used only one collected arthropod sample (1-KM), and only the +DNA and water for controls.
• 2. We mixed a PCR cocktail using 8uL Arthropod_F Primer, 8uL Arthropod_R Primer, 26uL water, and 50uL Taq Master Mix 2x (enough for 4 total reactions).

• Our second Gel image was taken on 11/4/25 and was run at 125 volts for 25 minutes.  We used this DNA Ladder: New England Biolabs 1 kb Plus DNA Ladder for Safe Stains (product # N0559S)

• Our third Gel image was taken on 11/4/25 and was run at 125 volts for 25 minutes.  We used this DNA Ladder: New England Biolabs 1 kb Plus DNA Ladder for Safe Stains (product # N0559S)

Results

I am moderately confident in the presence of Wolbachia in my arthropod because both the first and second PCR showed a band for the Wolbachia 16S gene and both reactions had expected outcomes from the controls.

Although it was extremely faint, it looked as though my first gel electrophoresis had picked up a DNA strand of about 440 base pairs (I compared my arthropod’s lane (2) with the lane containing the -Arthropod Control (5) to identify the presence of a Wolbachia band).  Since the band for the DNA of my arthropod was also faint, it can be assumed that the DNA was fully extracted out of my arthropod, making Wolbachia DNA much harder to identify.

With a single PCR using Wolbachia 16S primers only, a more conclusive result was determined.  Because it was run at an annealing temperature of 55 degrees Celsius it had a much higher sensitivity for the Wolbachia 16S gene.  Our second gel electrophoresis showed a band at about 440 base pairs in my arthropod sample, signifying the presence of Wolbachia in my arthropod.

Even through my results are clear, it is very possible that they show a false positive.  Water striders are predatory arthropods and with the misidentification from NCBI BLAST it is likely that DNA from another arthropod that the water strider consumed was extracted.  If this is the case, it cannot be determined whether the water strider was infected or if it consumed a Wolbachia infected arthropod before collection.