AC-DC Electropenetrography for the Study of Probing and Ingestion Behaviors of Culex tarsalis Mosquitoes on Human Hands

Our group is using EPG to study how arboviruses and other factors affect probing and ingestion behaviors that occur during blood feeding, as many pathogens will manipulate vector behavior to enhance transmission. Basically, we're studying what happens during a mosquito bite and how those behaviors influence pathogen transmission and disease pathology. Historically, EPG was predominantly used to study plant feeding insects.

The recent development of AC-DC EPG procedures for blood-feeding arthropods allows us to study the probing and ingestion behaviors that occur during blood feeding in unprecedented detail without the need for invasive procedures, possibly allowing for the development of novel management strategies. The biggest challenge of using EPG on blood-feeding arthropods is correlating the waveforms with biological activities like salivation and pathogen transmission. We have strong hypotheses based on the electrical origins of the waveforms and the literature, but we still need empirical evidence.

To begin, pass the tip of a glass pasture pipette through a flame, rotating the pipette slightly. Inspect the diameter and repeat the process to bring the pipette to the desired size. To make an insect aspirator, cover the large end of the modified pasture pipette with mosquito netting and inserted into the silicone tubing connected to a laboratory aspirator securing the junction with electrical tape.

For the recording electrodes wrap a foam block with fresh plastic wrap for holding the prepared insect stubs and wired mosquitoes. Scrape the top one centimeter of the copper wire with a razor blade until shiny on all sides. Then cut 0.025 millimeter diameter gold wire into four to five centimeter long pieces.

Hold the wire in self-closing forceps masked with tape to avoid denting the wire, gently wrap the gold wire three times around the copper wire of the insect stub starting along the clean tip of the copper wire. Use a dissection probe to apply silver glue to hold the gold wire in place. After ensuring there is no loose end, place the insect stub in the foam until the glue dries.

Then gently stroke the gold wire attached to the insect stub to shape it into a long, gentle arc, and trim the wire to approximately 2.5 to 3 centimeters in length. Working under a dissection microscope, hold the insect stub by the brass nail with the non-dominant hand while the gold wire touches the dissection microscope stage and grasp the very end of the gold wire with fine-point forceps using the dominant hand, use the fine-point forceps to cross the tip of the gold wire over the copper wire of the insect stub near the glued area. Then gently pull on the insect stub until the gold wire forms a small gold loop around the fine-point forceps.

Release the gold wire and carefully remove the forceps. While holding the insect stub in the non-dominant hand, grasp the gold loop with fine point forceps. Pull on the insect stub while lightly rotating the forceps away from the stub until the loop bends at a 90 degree angle to the neck of the wire.

Ensure the gold loop is fully closed before returning the insect stub to the foam block. To begin, prepare the insect aspirator and the recording electrodes for the wiring of mosquitoes. Position the anesthetized mosquitoes on their backs on the anesthesia pad with their abdomens facing the researcher.

After placing a laboratory tape, apply a 0.5 centimeter drop of freshly mixed silver glue on the tape. Then aspirate the abdomen of an anesthetized mosquito into the aspirator. Using the suction only long enough to seat the abdomen in place and hold the mosquito under the dissecting scope above the silver glue using the aspirator.

After dipping a minuchin pin in the silver glue, apply a thin layer to the pronotum of the mosquito. Quickly dip the gold loop at the tip of the recording electrode in the silver glue. Place it on the silver glue spot on the mosquito's pronotum and rock the loop back and forth to ensure it sits flush.

Next, replace the insect stub in the foam at an angle that allows the wired mosquito to stand comfortably when it wakes up. Once the desired number of mosquitoes are wired, place the foam block in a storage box and maintain it. To begin, attach the recording electrodes to the mosquitoes on the electropenetography or EPG experiment day.

Once the host is in position, turn the head amplifier to the side. After removing a wired mosquito, insert the brass pin of the insect stub into the port on the head amplifier. Start the recording Using the hardware management software.

Position the head amplifier above the hand so that the mosquito can comfortably stand and probe. Switch the head amplifier on while grounded. Adjust the host hand position as needed to assist in probing success.

Use the hardware management software to make timestamped observations regarding host responses, mosquito behaviors, gain changes, and other relevant events during the recording. Adjust the position of the signals on the screen and the software gain using the hardware management software to keep both signals clearly visible. To adjust the hardware gain, flip the channel one range and multiplier switches.

If that adjustment is not sufficient, turn the gain dial for channel one to the right or left to raise or lower the gain respectively to avoid peaking out or unclear peaks. For tuning the offset, flip the channel one offset switch on the control box to neg. Then turn the channel one offset dial until the peaks in the pre and post signals occur in the same direction on the computer screen.

If the mosquito is in the middle of a probe, allow it to finish. If it does not probe within 10 minutes, switch the head amplifier off. Have the host carefully remove their hand, then remove the mosquito with the attached insect stub.

Finally, replace the mosquito in the storage box. AC-DC EPG recordings of Culex tarsalis mosquitoes feeding on human hands generated interpretable waveforms suitable for classification into families, types, and subtypes.