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

Summary

Procedures are described for studying the probing and ingestion behaviors of Culex tarsalis mosquitoes on human hands using AC-DC electropenetrography.

Abstract

Mosquitoes transmit pathogens that negatively affect human and animal health. A greater understanding of their blood-feeding biology and interactions with hosts and pathogens could be exploited to develop new targets for controlling mosquito-borne diseases. Unfortunately, probing (i.e., biting) behaviors of mosquitoes are poorly understood because they occur inside host tissues. Here, a non-invasive procedure is described for using AC-DC electropenetrography (EPG) to indirectly visualize and quantify mosquito feeding behaviors by recording changes in electrical signals generated when probing and ingesting on human hands. Thin gold wires are attached to the mosquitoes using conductive silver glue and connected to the EPG instrument. The human host holds a substrate voltage probe in their hand. Probing of the host by the wired mosquito completes the electrical circuit, and electrical signals are recorded on a computer as "waveforms" that can then be measured and enumerated for analysis. This methodology has been used to characterize the probing and ingestion behaviors of Aedes aegypti and Culex tarsalis mosquitoes and can be applied to other mosquito species. EPG can be used to study the effects of pathogens, insecticides, and other factors on mosquito feeding behaviors.

Introduction

Mosquitoes cause suffering and economic loss worldwide due to their roles as disease vectors and nuisance pests¹. Anopheles mosquitoes transmit malaria, which annually causes an estimated 219 million cases globally, more than 400,000 deaths², and 12 billion US dollars in direct costs³. Aedes mosquitoes are the most notable vectors of public health importance because they have a nearly global presence and transmit agents of Chikungunya, dengue fever, lymphatic filariasis, Rift Valley Fever, yellow fever, and Zika, which claim lives, overwhelm health systems, and result in chronic suffering, life-long morbidity, and disability^4,5. In addition, Culex mosquitoes transmit agents of equine encephalitis, Japanese encephalitis, lymphatic filariasis, Saint Louis encephalitis, and West Nile fever, which impact both human and animal health^3,4.

Vector control with insecticide-based approaches is the leading method for controlling vector-borne diseases globally^6,7. However, the number of insecticides available for mosquito control is limited. In addition, insecticide resistance is evolving in many species, especially in Aedes mosquitoes, impacting our ability to combat vector-borne diseases and driving the need for new control strategies^6,8. A better understanding of mosquito biology, mosquito-host-pathogen interactions, and the processes that regulate pathogen transmission can facilitate the development of new tools for vector control⁶.

Probing and ingestion behaviors in host tissues can affect pathogen transmission and disease pathology^9,10,11,12. For instance, more microfilariae were ingested when Aedes aegypti fed directly from capillaries rather than pooled blood outside ruptured vessels¹³. Thus, fundamental and applied research on this topic can aid in developing, refining, and evaluating mosquito control strategies and other countermeasures for pathogen transmission. However, significant knowledge gaps exist in the details of cryptic blood-feeding behaviors that occur below the surface of the host's skin because these behaviors often require invasive procedures on live animal models or employ transparent artificial substrates or liquid diets^9,11,14,15. Non-invasive electropenetrography (EPG; also called electrical penetration graph) techniques have been used to study feeding behaviors of plant-feeding insects related to plant pathogen transmission, host plant resistance, and insecticide modes of action because they allow researchers to indirectly observe, record, and quantify mouthpart movements and behaviors that occur inside host tissues^10,12.

Since the 1950s, electronic penetration monitoring techniques have been applied to blood-feeding arthropods, including mosquitoes^{16,17,18,19,20}. However, EPG was never widely adopted for blood-feeding arthropods because biological activities corresponding to the waveforms were never verified²¹. In addition, the designs and electronics of early instruments resulted in over-emphasis of some electrical signals, high noise-to-signal ratios, low resolution and distortion of waveforms, and irritation to vertebrate hosts^12,22. A more recent instrument, the AC-DC EPG monitor²³, has variable settings and improved signal processing, making it better suited to record blood-feeding arthropods and decipher the biological origins of the waveforms in experimental correlation studies^10,12.

For EPG, an electrical signal is applied to the host through a substrate voltage probe (i.e., reference electrode). The arthropod is attached to a recording electrode and placed on the host. When the arthropod inserts its mouthparts (i.e., stylets) into the host, the electrical signal is conveyed to the AC-DC EPG monitor for signal processing. The electrical signals are then displayed on a computer as "waveforms" corresponding to specific behaviors such as mouthpart movements, ingestion, salivation, and pathogen acquisition/inoculation^10,12. Sequential and nonsequential response variables related to the duration and count of the waveforms^24,25 at different hierarchical levels (families, types, and subtypes) can then be calculated based on the frequency and duration of the waveforms to gain insight into the feeding behaviors that occur within and between species, and in response to biotic and abiotic factors, such as hosts, pathogens, pesticides, and environmental conditions^10,12.

So far, the AC-DC EPG monitor has been used to characterize the probing and ingestion behaviors of Ae. aegypti²⁶ and Culex tarsalis²⁷ mosquitoes feeding on human hands, and Dermacentor variabilis and Amblyomma americanum ticks feeding on bovine calves²⁸. In addition, a murine model for EPG²⁹ was recently developed to facilitate pathogen studies using this instrument. Human hands are ideal for the initial mosquito waveform characterization because human hosts can report when they feel the mosquito probing as well as other behaviors, making the correlation of waveforms with behaviors easier (Table 1). In addition, a better understanding of the plasticity in feeding behaviors among mosquitos in different human populations could contribute to the development of more specialized management interventions for mosquitoes and the pathogens they transmit.

Table 1: Description of waveform families used to classify electrical signals recorded from Culex tarsalis on human hands using AC-DC EPG. The probing phase and biological activities corresponding to each waveform family. See Cooper et al.²⁷ for more information. Abbreviation: AC = alternating current, DC = direct current, EPG = electropenetrograph. Please click here to download this Table.

To facilitate the use of EPG to study mosquito biology and host-vector interactions, methodology for quantifying the feeding behaviors of Cx. tarsalis mosquitoes on human hands using the AC-DC EPG monitor are presented here. These protocols build upon the fundamental AC-DC EPG procedures described in online resources³⁰ and assume that users have a basic working knowledge of AC-DC EPG and associated software³¹. The procedures outlined can be modified for other mosquito and host species and provide a foundation to facilitate hypothesis-driven research of mosquito feeding behaviors.

Table 2: Definitions of common AC-DC EPG terminology. Definitions are based on those given in online resources^30,31 and scientific literature³². Abbreviation: AC = alternating current, DC = direct current, EPG = electropenetrograph. Please click here to download this Table.

Protocol

This protocol (IRB-10729) was approved by the Institutional Review Board (IRB) for Human Research at Kansas State University²⁷ and is intended for use with pathogen-free mosquitoes only. Human volunteers gave their informed consent to participate in the study and publish their data. Before initiating any work with animals, humans, or biohazards, ensure all procedures have been reviewed and approved by the appropriate institutional regulatory committees or oversight individuals. See the Table of Materials for details about all equipment and materials used in this protocol.

1. Initial preparation and setup

1. Set up the AC-DC EPG monitor (Figure 1A), Faraday cage (Figure 1B,C), and software as previously described^30,31 using the modifications described below (steps 1.1.1-1.1.5). Refer to online resources³⁰ for step-by-step tutorials on setting up and using the equipment and software. Save the desired setup as default in the hardware management and waveform reviewing software. Refer to Table 2 for a list of common AC-DC EPG terminology and their definitions.
   1. Connect a head stage amplifier (head amp) and a substrate voltage probe to Channel 1 of the control box.
      NOTE: Recording from a single channel is presented even though the AC-DC monitor can simultaneously accommodate four insects and hosts.
   2. Ensure that Channel 1 on the control box is switched on and the corresponding Pre-1 and Post-1 channels on the switch interface box are switched on.
      NOTE: Channels not being used must remain switched off.
   3. Use the hardware management software to enable Channels 1 and 3 on the DI-710 A/D Board (but not others) to record the pre and post rectified signals.
   4. Format the hardware management software to view/overlap the pre- and post-rectified waveforms (e.g., 1 = 1 for the pre-signal and 2 = 3 for the post-signal).
   5. Set the sampling rate on the hardware management software to 100 samples per second per channel (i.e., S/s/CHAN: 100).
2. Construct insect stubs^26,27,29,30:
   1. Preheat the soldering iron to the recommended temperature for the specific rosin core solder.
   2. Use a wire cutter-stripper tool to strip the plastic coating ~6 cm from the end of a 20 AWG copper wire roll, but avoid severing or marking the copper wire bundle inside (Figure 2A). Then, cut off the exposed copper wire bundle and unravel into individual strands (Figure 2B).
   3. Use needle-nosed pliers to wrap one strand of the copper wire around the shaft of a suitable escutcheon pin underneath the head (i.e., flat top) so that most of the copper wire extends past the head of the pin. Fold the wire's tail over the pin's head to secure the wire in place (Figure 2C).
      NOTE: Fit test escutcheon pins to identify ones that fit snuggly into the port of the head amp (when it is turned off, unplugged, and set to 10⁶ Ohms (Ω)).
   4. Clasp the shaft of the escutcheon pin in the helping hands soldering aid so the copper wire points upward. Then, apply a small dab (~0.2 cm ball) of water-soluble flux to the junction of the escutcheon pin and copper wire (Figure 2D).
      CAUTION: Flux is toxic and must be handled with care.
   5. Uncoil ~10 cm of solder and straighten it out. Hold the hot soldering iron against the head of the pin for a few seconds until the flux melts and the metal is hot. Then, press the tip of the solder to the head of the pin next to the soldering iron until the solder melts and completely coats the junction, fusing the pin and wire into an insect stub.
   6. While the insect stub cools, clean the tip of the soldering iron with steel wool and a damp sponge.
   7. Once cool, remove the insect stub from the helping hands. Rinse the insect stub in 70% ethanol and then water to remove residual flux. Trim the copper wire to the desired length (~3 cm, Figure 2E).
   8. Repeat steps 1.2.3-1.2.7 until 20-30 insect stubs are constructed.
3. Assemble an insect aspirator^26,27:
   1. Pass the tip of a glass Pasteur pipette through a flame, rotating the pipette slightly. Repeat until the end of the pipette is the appropriate size to fit snuggly around the insect's abdomen.
   2. Cover the large end of the modified Pasteur pipette with mosquito netting, then insert it directly into the silicone tubing connected to a laboratory aspirator and secure the junction with electrical tape to form an insect aspirator (Figure 2H).
      NOTE: The Pasteur pipette and netting can be inserted into a short piece of plastic tubing fitted with a quick disconnector for easy switching between different sizes (Figure 2H).

Figure 1: The AC-DC EPG monitor equipment setup for recording on human hands. (A) Components of the EPG, including the (1) control box, (2) switch interface box, (3) power supply, (4) DI-710 A/D Board, (5) computer, (6) power regulator, and (B) Faraday cage containing (C) a digital microscope (7) on a  gooseneck stand, (8) bubble wrap and air pillows, (9) substrate voltage probe, and (10) head stage amplifier on a laboratory stand. Abbreviations: AC = alternating current, DC = direct current, EPG = electropenetrograph. Please click here to view a larger version of this figure.

Figure 2: Recording electrodes for wiring mosquitoes and an insect aspirator for manipulating mosquitoes during wiring. (A) 20 AWG copper wire with 6 cm of plastic coating removed to expose the copper wire bundle inside. (B) Individual strands of the copper wire bundle. (C) One strand of copper wire wrapped around the head of a brass escutcheon pin with the wire's tail folded over the pin's head. (D) A dab of flux on the junction of the brass escutcheon pin and copper wire held by a helping hands soldering aid in preparation for soldering. (E) A completed insect stub constructed from a brass nail soldered to a copper wire. (F) A recording electrode constructed from a gold wire wrapped around the tip of the copper wire of an insect stub and secured in place with silver glue. (G) A close-up of the gold loop at the tip of the recording electrode. (H) An assembled (top) and disassembled (bottom) insect aspirator made from modified Pasteur pipettes, mosquito netting, plastic tubing, electrical tape, and male quick disconnectors, next to the laboratory aspirator tube fitted with the female quick disconnector (top right). Scale bars = 1 cm (A-F), 200 µm (G). Please click here to view a larger version of this figure.

2. Wiring of mosquitoes

1. Prepare mosquitoes and silver glue (the afternoon/evening before EPG):
   1. Rear and maintain Cx. tarsalis KNWR strain mosquitoes as previously described²⁷.
   2. Fast adult female mosquitoes, 5-17 days post-eclosion, for 8-12 h to enhance probing rates but allow access to water to avoid mortality.
   3. Prepare fresh silver glue^{26,27,28,29,30}:
      1. Add 510 mg of 4-8 µm silver flake to a 20 mL beaker. Tare the balance and add 1,100 mg of non-toxic school glue to the beaker on top of the silver flake.
      2. Tare the balance, add 510 mg of 8-10 µm silver flake to the beaker on top of the glue, and then add 1,000 µL of water using a pipette.
      3. Gently add a stir bar to the beaker and place it on the stir plate. Begin stirring slowly, gradually turn up to the 'fast' setting, and stir for 4 min. Ensure all contents are incorporated.
      4. Use a transfer pipette (with the tip cut off) to transfer the silver glue to 1.5 mL microcentrifuge tubes.
      5. Wrap the tops of the silver glue tubes with Parafilm and store on the benchtop until use.
         NOTE: Silver glue has the correct consistency for up to 2 days, but making fresh glue the evening before use is recommended³⁰.
2. Prepare recording electrodes^26,27,29,30:
   1. Vigorously vortex the silver glue before use.
   2. Wrap a foam block with fresh plastic wrap to hold the prepared insect stubs and wired mosquitoes.
      NOTE: The foam must be large enough to allow the wired mosquitoes to stand on the surface without touching each other but small enough to fit inside a storage box. CAUTION: Excretory droplets released after blood feeding may contain pathogens. Discard the plastic wrap/foam block appropriately after use.
   3. Remove tarnish (and old silver glue if reusing insect stubs) from the tip of the copper wire by scraping the top 1 cm of the copper wire with a razor blade until shiny on all sides. Prepare one insect stub for each mosquito, plus a few extras.
   4. Cut 0.025 mm diameter gold wire into 4-5 cm long pieces. Always hold the wire in self-closing forceps masked with tape to avoid denting the wire.
      NOTE: Gold wire is much easier to see against a black background.
   5. Place the end of the gold wire along the clean tip of the copper wire of the insect stub and gently wrap the gold wire 3x around the copper wire.
   6. Use a dissection probe to apply silver glue to hold the gold wire in place. Ensure there is no tail or loose end. Place the insect stub in the foam until the glue dries.
   7. Gently stroke the gold wire attached to the insect stub to smooth and shape it into a long, gentle arc (Figure 2F). Trim the wire to the desired length (~2.5-3 cm).
   8. Working under a dissection microscope, use fine-point forceps to make a gold loop at the tip of the gold wire, and bend the loop at a 90° angle as described below (steps 2.2.8.1-2.2.8.4; Figure 2E,F). Ensure the gold loop is fully closed. Return the insect stub to the foam block.
      1. Using the non-dominant hand, hold the insect stub by the brass nail, with the gold wire tip touching the microscope stage. Using the dominant hand, grasp the very end of the gold wire with fine-point forceps.
      2. Using the fine-point forceps, cross the tip of the gold wire over the copper wire of the insect stub near where the gold wire is glued on. Touch the end of the fine-point forceps and gold wire down against the microscope stage.
      3. Gently pull on the insect stub until the gold wire forms a small gold loop around the tip of the fine point forceps (do not pull too tight). Release the gold wire and carefully remove the forceps.
      4. While still holding the insect stub in the non-dominant hand, grasp the gold loop with the fine point forceps so that the forceps are at a perpendicular angle to the neck of the wire and the tip of the forceps span the middle of the gold loop, thus grasping both sides of the loop. Gently pull on the insect stub while lightly rotating the forceps away from the insect stub until the loop is bent at a 90° angle to the neck of the wire (Figure 2G).
3. Attach recording electrodes to mosquitoes^26,27,29,30 (the morning of EPG):
   1. Anesthetize fasted mosquitoes with CO₂ gas until all movement stops. Position the mosquitoes on their backs on the anesthesia pad, with their abdomens facing the researcher.
   2. Place a ~2 cm piece of laboratory tape under a dissection microscope, sticky side down. Place a 0.5 cm drop (~ 10 µL) of silver glue on the tape.
   3. Aspirate the abdomen of an anesthetized mosquito into the modified Pasteur pipette using the suction only long enough to seat the abdomen in place. Hold the mosquito under the dissecting scope above the silver glue using the aspirator.
      NOTE: The wings can be inside or outside the tip of the modified Pasteur pipette, but having them inside will help prevent the mosquito from falling out. Avoid bending or damaging the wings.
   4. Working quickly, dip a minutien pin in the silver glue and apply a thin layer to the mosquito's pronotum (i.e., the dorsal surface of the thorax). Immediately proceed to the next step if possible (or re-anesthetize the mosquito if needed).
      NOTE: It is recommended to remove scales on the pronotum by rubbing the pin back and forth to help ensure glue penetration of pores in the cuticle. Avoid getting glue on any other part of the mosquito.
   5. Quickly dip the gold loop at the tip of the recording electrode in the silver glue and place it on the silver glue spot on the mosquito's pronotum. Rock the loop back and forth to ensure that it sits flush against the pronotum. Hold the wire until the glue dries (~20-25 s).
   6. Replace the insect stub in the foam at an angle that allows the wired mosquito to stand comfortably when it wakes up. Proceed with wiring the next mosquito.
      NOTE: It is advisable to take notes about the quality of each wiring job.
   7. Once the desired number of mosquitoes is wired, place the foam block in a storage box. Maintain the mosquitoes under normal rearing conditions until use.

3. EPG setup and preparation (the morning before EPG)

1. Turn on the AC-DC EPG monitor (by flipping the switch on the power supply) at least 30 min prior to recording to allow for instrument equilibration and any troubleshooting.
   NOTE: Channels in use on the control box and switch interface box can be left switched on.
2. Turn on the computer and open the hardware management software.
   NOTE: If the signals do not appear as flat thin lines, troubleshooting is required (see discussion section).
3. Perform the touch test^27,30 to verify equipment functionality:
   1. While grounded (i.e., touching the Faraday cage with bare skin), turn the dial on the head amp to 10⁶ Ω and insert the shaft of an insect stub into the port.
      ​NOTE: Conducting the touch test at a higher input resistance (Ri) level may damage the head amp.
   2. While grounded, switch the head amp on and firmly grasp the copper wire of the insect stub between the index finger and thumb three times. If the EPG is working correctly, three large peaks will appear in both the pre- and post-signals on the screen. If this is not the case, troubleshooting is required (see discussion section).
4. Adjust the instrument settings to 150 mV alternating current (AC) with an Ri of 10⁷ Ω, a hardware gain of 20-40x, a negative offset, a native baseline, and a software gain of 8-16x^27,30, as described below (steps 3.4.1-3.4.3).
   1. Set the voltage, current type, hardware gain (range, gain, and multiplier), offset, and baseline (zero) using the switches and dials on Channel 1 of the control box.
      NOTE: The appropriate hardware gain setting depends on the quality of the wiring job and silver glue, so variation in optimal gain settings from mosquito to mosquito and day-to-day is expected. The hardware gain may need to be adjusted mid-recording if the signal peaks out (defined in Table 2 and shown in Figure 3A,C) or is too small to visualize. Any hardware gain changes will permanently affect the recording (shown in Figure 3A,C).
   2. Set the Ri level using the dial on the head amp.
   3. Set and adjust the software gain using the hardware management software (e.g., select the pre or post signal and then left-click to raise the software gain or right-click to lower the software gain).
      NOTE: Any gain changes made in the software will NOT permanently affect the recording.
5. Set up a new recording in the hardware management software, but do not start recording until step 4.3 in the next section.

4. EPG on human hands^26,27 :

1. After informed consent is obtained (as specified by the home institution's IRB), have the host sit beside the Faraday cage with one arm inside while grasping the substrate voltage probe and holding it below the head amp.
   NOTE: The arm must rest comfortably on bubble wrap or a similar non-conductive substrate. The hand and substate voltage probe must not contact the Faraday cage or head amp while recording. To avoid damage, it is imperative that the volunteer does not touch the head amp once it is turned on. If the head amp is touched accidentally, perform the touch test described above to assess its functionality.
2. Once the host is in position, turn the head amp to the side. Remove a wired mosquito from the storage box and connect it to the instrument by inserting the brass pin of the insect stub into the port on the head amp. Leave the mosquito dangling.
   NOTE: Keep a spray bottle of 70% ethanol nearby to control any mosquitoes that detach from the wire.
3. Start the recording using the hardware management software. Position the head amp above the hand so that the mosquito can comfortably stand and probe.
4. Switch the head amp on while grounded. Start a 10 min timer. Adjust the host hand position as needed to assist in probing success.
   NOTE: When using mosquitoes inclined to probe immediately, turn the head amp on before positioning the mosquito on the hand. A thin wooden dowl or nonconductive paintbrush handle can be used to bend the copper wire of the insect stub to further adjust the position of the mosquito. A magnifying glass or digital microscope on a gooseneck stand can aid in observing the mosquito during EPG, especially for behavioral correlation studies.
5. Use the hardware management software to make timestamped observations (e.g., event markers) during the recording regarding host responses, mosquito behaviors, gain changes, etc.). Adjust the position of the signals on the screen and the software gain as needed to keep both signals clearly visible on the screen. Adjust the hardware gain if the signal(s) peaks out (Figure 3A,C and Table 2) or if the waves appear too small when the software gain is maxed out. Adjust the offset if rectifier foldover is observed (Figure 3C,C' and Table 2). Refer to the software manual³¹ and online resources³⁰ for detailed instructions on how to make these adjustments.
   1. To adjust the position of the signals on the screen, select the pre or post signal (e.g., by clicking on 
      1 = 1 or 2 = 3) and then drag the signal to the desired position on the screen (ideally near the middle of the screen but not overlapping).
   2. To adjust the software gain, use the hardware management software described in step 3.4.3.
   3. To adjust the hardware gain, start by turning the Gain Dial for Channel 1 to the right or left to raise or lower the gain, respectively. If that adjustment is not sufficient to eliminate peaking out (gain too high; Figure 3A,C) or allow the details of the signal to be clearly visualized (gain too low), flip the Channel 1 Range and Multiplier Switches to adjust the gain further.
      NOTE: Using event markers to record when the hardware gain was adjusted is recommended to aid waveform data interpretation.
   4. To adjust the offset, flip the Channel 1 Offset Switch on the control box to neg, then turn the Channel 1 Offset Dial until the peaks in the pre and post signals occur in the same direction on the computer screen. If the Offset Dial is turned too far, the post-signal will flip again.
6. If the mosquito does not probe within 10 min (or other defined time point), switch the head amp off. If the mosquito is mid-probe at the time mark, allow it to finish probing prior to turning the head amp off. Have the host carefully remove their hand, then remove the mosquito with the attached insect stub. Replace the mosquito in the storage box.
7. Stop/close the recording using the hardware management software. Proceed with EPG on the next mosquito (Step 4.2).
   NOTE: Remember to change the file name after each mosquito.
8. Switch off the head amp and power source at the session end and close the software.
9. Review the Participant Debriefing Statement or similar with the volunteer as specified in the approved IRB.
10. Dispose of mosquitoes per protocol requirements. Remove the plastic wrap from the foam block and discard it appropriately.
11. After all data collection is complete, review and measure the waveforms and analyze the data as previously described^{24,25,26,27,29,30}. Refer to online resources³⁰ for step-by-step tutorials for using the waveform reviewing software, making figures depicting the waveforms, measuring the waveforms, exporting the data for analysis, conducting the statistical analysis, and a detailed explanation of common waveform response variables.

Figure 3: Examples of suboptimal recordings due to peaking out, control box gain changes, unclear voltage changes from inferior wiring, rectifier foldover, and noise. Waveforms from Culex tarsalis mosquitoes feeding to repletion on human hands, distorted by (A) peaking out (blue horizontal arrow) and subsequent control box gain changes from 100x to 80x to 40x to 60x to 40x to 20x (orange vertical arrows), (B) unclear voltage changes, and (C) rectifier fold over in the post- (black) signal compared to the pre- (red) signal (see waveform J, closeup shown in panel C'), line noise (11 Hz sine wave visible in pre-rectification (red) signal only, closeup shown in panel C'), peaking out (blue horizontal arrow), and a control box gain change from 500x to 200x (orange vertical arrow). Waveform names are shown along the top of each panel. The purple arrows in the lower right corner of panel C indicate when the head amp was turned off after the probe was complete and provide another example of rectifier foldover. Red horizontal lines associated with the pre-rectification signal in panel C indicate when Event Markers were used to record time-stamped notes. Ri levels, applied signals, time scales, and software gains are (A) 10⁷ Ω, 150 mV DC, 13.2 s/div, 8x; (B) 10⁷ Ω, 150 mV DC, 4.4 s/div, 128x; and (C) 10⁷ Ω, 150 mV AC, 7.4 s/div, 2x; (C') 0.2 s/div, 8x. Abbreviations: AC = alternating current, DC = direct current, Ri = input resistance, mV = millivolt, Ω = Ohms, s/div = seconds per division. This figure was prepared with data from Cooper et al.²⁷. Please click here to view a larger version of this figure.

Results

AC-DC EPG recordings of Cx. tarsalis mosquitoes' blood feeding on human hands (Figure 4) generated interpretable waveforms suitable for analysis and publication²⁷. Interpretable waveforms contained distinct, distinguishable repeating patterns that could be classified into waveform families (Table 1 and Figure 4), types, and subtypes when the compression (defined in Table 2) and software gain were adjusted in waveform reviewing software^27,31. All waveform families (J, K, L, M, N) and types (L1, L2, M1, M2) previously described in Ae. aegypti feeding on human hands²⁶ were identified in the waveforms generated by Cx. tarsalis feeding on human hands, verifying the validity of the methods described here. In addition, three new waveform types (M3, N1, N2) and numerous subtypes of L1, L2, M1, and M2 were identified²⁷. In addition, the voltage drop back to baseline when the mouthparts are withdrawn from the host was named waveform family W²⁷.

Systematically varying the instrument settings (current type and Ri levels) while following the procedures outlined here allowed for the determination of the optimal settings for recording Cx. tarsalis feeding on human hands, the electrical origins of each waveform, the average duration in seconds of complete and incomplete probes per mosquito, and for statistical comparison of the waveform duration in seconds per insect (WDI), the number of waveform events per insect (NWEI), and the waveform duration in seconds per event per insect (WDEI) across the settings²⁷. Mosquitoes were also interrupted during different waveforms and dissected to determine which waveform was correlated with blood ingestion²⁷. A similar investigation was performed for Ae. aegypti using comparable methodology²⁶.

Due to EPG's sensitive and intricate nature, not all recordings were as pristine as those shown in Figure 4. Peaking out (Figure 3A,C), hardware gain changes (Figure 3A,C), inferior wiring jobs (Figure 3B), rectifier foldover in waveform J (Figure 3C,C'), and electrical noise (Figure 3C,C') occasionally distorted and masked the waveforms, rendering them ill-suited for use in publication quality figures and making scoring and analysis difficult. Thus, front-end effort is recommended to perfect wiring skills, optimize gain and voltage settings, adjust the offset, and control electrical noise. Interestingly, rectifier foldover was only observed in waveform family J for Cx. tarsalis (Figure 3C,C') and did not recur once the offset was initially adjusted, even when other species of mosquitoes (Ae. aegypti and Culex quinquefasciatus) were used (data not shown). In addition, troubleshooting revealed that the uncontrolled electrical noise shown in Figure 3 (panels C and C') resulted from a faulty Faraday cage. The recordings made in the faulty Faraday cage were still usable, but noise issues were resolved when an improved Faraday cage was built (Figure 1B).

Even when all precautions and front-end optimizations were taken, slight variations in the quality of the electrode attachment to the mosquito and other unknown factors that distorted the signal and altered the signal-to-noise ratio periodically made it necessary to adjust the hardware gain mid-recording to ensure that the signal was in the visible range and the resolution of the peaks in each waveform were sufficient for waveform identification and characterization. Unfortunately, adjusting the hardware gain mid-recording can result in abrupt voltage shifts (Figure 3A, C), rendering the waveforms ill-suited for publication but still usable (in whole or part) for measurement and analysis.

Figure 4: Representative waveforms from a Culex tarsalis probe on a human hand recorded with an Ri of 10⁷ Ω using an applied signal of 250 mV AC. Family-level (NP, J, K, L, M, W) waveform names are shown along the top of each panel. Lower panels show enlargements of select regions in panel A. Inserted panels show enlargements of indicated regions. Time scales and software gains are (A) 7.0 s/div,16x; (B) 1.0 s/div, 64x; (B') 0.2 s/div, 64x; (C) 1.0 s/div, 64x; (C') 0.2 s/div, 32x; (D) 1.0 s/div, 64x; (D') 0.2 s/div, 64x. Abbreviations: AC = alternating current, Ri = input resistance, mV = millivolt, Ω = Ohms, s/div = seconds per division. This figure was prepared with data from Cooper et al.²⁷. Please click here to view a larger version of this figure.

Discussion

When using the AC-DC EPG, it is critical to have a basic understanding of hardware and software operation³⁰ and carefully follow the protocols described herein to avoid damaging the equipment. Most notably, avoid touching the head amp when it is turned on unless grounded on the Faraday cage. Recording as far back in the Faraday cage as possible is recommended³⁰. The cage doors do not necessarily need to be closed to obtain interpretable waveforms unless recording in an environment with excessive ambient electrical noise. Nothing motorized can be in the Faraday cage. It is important to note that the Faraday cage itself must be electrically grounded solely through the head amp for it to work properly. This is accomplished by connecting the head amp to a metal laboratory stand with the paint sanded off one foot of the stand to allow a conductive pathway for the electricity captured by the Faraday cage to flow to the laboratory stand, into the metal casing of the head amp, down the cord to the control box, through the power supply into a properly grounded wall outlet, and into the earth through the building ground³⁰. A multimeter can be used to check the quality of the connection among the components inside the cage by measuring the resistivity (which is the inverse of conductivity) between various points. A wireless AM radio can also be placed inside the Faraday cage to verify that the sound is significantly reduced (or ideally eliminated) when the doors are closed, confirming that the Faraday cage is adequately capturing airborne electrical signals (E. Backus per. Comm.).

Proper and standardized preparation of the silver glue and attachment of the recording electrodes to insects are critical to obtaining usable recordings. Insect cuticle is an insulator and acts as a capacitor if the silver glue does not adequately penetrate the pores of the cuticle to create a pathway for electricity to travel from inside the insect to the gold wire on the surface of the pronotum³⁰. Prepare the silver glue at the same time before each EPG session, work with fresh silver glue from the tube, and replace it with vigorously vortexed silver glue frequently during the wiring procedure. Ensure the gold loop is positioned against the thorax so the entire loop is in contact with the cuticle and not tilted to one side or separated by a thick layer of silver glue and scales to help form a strong electrical connection as well as prevent detachment and escape of the mosquito from the wire.

The protocols described here must be optimized when working with different mosquito species. The optimal EPG settings (Ri level, voltage, current type, and hardware gain) for recording on a given arthropod must be experimentally determined^12,30. However, once the offset is adjusted, it will not need to be readjusted when working with similar mosquitoes. Optimal settings have been determined for AC-DC EPG with Ae. aegypti²⁶ and Cx. tarsalis²⁷. Waveform libraries (defined in Table 2) for additional species will facilitate the adoption of EPG for mosquitoes. It is also necessary to optimize the anesthesia for the mosquito of interest, as blood-feeding behavior can be differentially affected based on mosquito species and anesthesia choice. For instance, Cx. tarsalis require a 4 h recovery period after 15 min of anesthesia with CO₂, whereas for Ae. aegypti, a 15 min recovery period is sufficient after a 15 min anesthesia period with ice. Culex tarsalis, however, are difficult to knock down with cold exposure, so CO₂ is predominantly used despite the long recovery period.

While the AC-DC EPG monitor is straightforward to use after reviewing online resources^30,31 or training with an experienced user, some troubleshooting is required. Initially, the author's EPG recordings of mosquito probes had an unexpected 11 Hz sign wave in the pre-signal that had not been observed or reported by Wayadande and colleagues²⁶. Consultation with E. Backus indicated that the Faraday cage was likely the issue because of interfering ground loops. A new Faraday cage constructed from a highly conductive copper screen connected to an aluminum frame bolted to a large aluminum floor plate supported by rubber feet to isolate it from the table below (Figure 1B), solved the 11 Hz noise signal (Figure 3C,C'). Additionally, occasionally, when the EPG power supply was first turned on, the pre-and post-rectification signals would appear as thick, fuzzy lines instead of straight, smooth, thin lines. This was usually fixed by turning off the instrument and checking all the cord connections. If that failed, unplugging the instrument for 1-3 min usually helped. Very rarely, the pre-and post-signals become thick and fuzzy during operation, which was addressed by turning off the instrument and plugging it into a different outlet or using a voltage regulator and power conditioner to plug the EPG into before connecting it to the wall. Ideally, a dedicated line for the EPG monitor would eliminate line noise stemming from refrigerators and other equipment plugged into the same electrical circuit as the EPG³⁰.

Currently, there are two main limitations to using EPG to study mosquitoes. First, few correlation studies have been performed to verify which probing behaviors correspond to the waveforms. While there are strong hypotheses about what is happening during each waveform family (Table 1) and some of the waveform types (M1 and M2), few of these hypotheses have been experimentally confirmed, except that blood ingestion is correlated with waveform family M^26,27. Most importantly, waveforms corresponding to salivation events must be identified because many pathogens are transmitted in the saliva. Correlation of EPG waveforms with biological activities in mosquitoes will be required to meaningfully interpret significant differences in waveform duration and counts between treatment groups. Second, probing and ingestion rates during EPG can be much lower than expected²⁷. Individually tethering mosquitoes to short wires and placing them directly on the host could prevent long-distance host-seeking and social feeding behaviors, which may affect probing and ingestion behaviors. Correctly wired insects can walk and fly normally. Still, mosquitoes often pull against the wires and sometimes get tangled or hold themselves at unnatural angles, indicating that being wired affects their behavior to some degree. The authors tested many strategies to enhance probing rates during EPG, and fasting the mosquitoes overnight, using older individuals, exposing them to human breath immediately before placing them on the hand, and applying human saliva to the hand were the most effective, as well as using hosts that constantly made fine-scale adjustments to the position of their hand to ensure the mosquito was in the ideal position to probe²⁷.

Despite these limitations, AC-DC EPG has numerous advantages over other approaches for studying probing and ingestion behaviors. Systems like the BiteOScope¹¹, the flyPAD¹⁵, and the Automated Biomaterial Platform¹⁴ that were designed to quantify probing and ingestion behaviors and salivary secretions during blood feeding utilize transparent artificial substrates and liquid diets that lack normal host tissue systems and host cues. While these synthetic platforms are useful for many experimental purposes, a clear advantage of AC-DC EPG is the ability to study these arthropod behaviors and variables of interest within natural host systems. Further, the user-defined adaptability of the AC-DC EPG system offers an additional advantage over other EPG instruments^22,33 and EPG-like electronic monitoring devices^16,21, which do not have adjustable Ri levels, current types, and voltage levels that are needed to obtain waveforms with the maximum amount of information and minimize adverse effects on a broad range of the hosts and arthropods^22,23. Electromyography (EMG) techniques are also used to study blood feeding. However, the electrical circuit usually only includes the arthropod and only muscle potentials nearest the recording electrode are recorded, so the waveforms do not provide information on detailed activities occurring at the host-insect interface like cell ruptures, stylet locations and activities, and streaming potentials through the mouthparts^28,34. Intravital microscopy techniques can be used to visualize detailed mouthpart movements and activities in living host tissues directly, but these techniques involve invasive procedures on live animal models⁹. Only the AC-DC EPG monitor can provide highly detailed information in real-time about probing, ingestion, and salivation behaviors that occur in living opaque host tissues without the need for invasive procedures and is adaptable for use in a wide variety of arthropods^10,12.

This is the first detailed step-by-step procedure for wiring mosquitoes, constructing an insect aspirator, and performing EPG on human hosts. These protocols are expected to facilitate the adoption of AC-DC EPG on mosquitoes feeding on human hosts. Examples of future research efforts employing AC-DC EPG may include studies on fundamental mechanisms of blood feeding and host tissue damage¹⁰, including identification of stereotypical sequences of probing behaviors preceding blood ingestion, investigation of blood flow through mouthparts, and comparison of feeding plasticity within and between arthropod species/developmental stages²⁶. EPG can also be used to quantify the effects of insecticides, antifeedants, and repellents¹². The procedures presented here for using AC-DC EPG to study mosquitoes on human hands will accelerate these studies and enable novel insights into mosquito blood-feeder biology.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Instrumentation
AC-DC 4-channel electropenetrograph	EPG Technologies, Inc.		For EPG, includes the power supply, control box, switch interface box, four head stage amplifiers, four substrate voltage probes, and associated cords
Air pillows	Amazon.com		for the human host to rest their hand/arm on during EPG
DI-710 Data acquisition hardware (AD Board)	DATAQ Instruments Inc.		for digitizing the analogue EPG signals so they can be visualized on a computer
Dimmable Led Magnifying Lamp, Large 5X Hands Free Magnifying Glass with Light and Stand for Reading Hobbies Crafts Sewing Workbench	Amazon.com		for behavioral observation during EPG; only use unplugged during EPG or noise may result
Dino-Lite Edge Digital Microscope (250x magnification)	Dino-Lite	AF4915ZT	for behavioral observation during EPG
Dino-Lite Flex-arm Desktop Stand	Dino-Lite	MS22B	for holding and manipulating the camera
Eisco Steel Retort Base Stand and Rod Set	Fisher Scientific	S41740	for holding the head stage amplifier & grounding the Faraday cage (must be made of metal)
EPG Online Workshop	UF IFAS Extension	https://ifas-crec-entomology.catalog.instructure.com/courses/epg-workshop-2022	AKA, "online resources" for learning EPG (instrument set up, data collection and analysis, Faraday construction, silver glue preparation, insect stub construction, etc.)
Furman P-1800 AR Power Regulator/Power Conditioner	Amazon.com		for reducing line noise
Small Bubble Cushioning Wrap	Amazon.com		for use as a barrier between the host and Faraday cage
Troemner Talboys Labjaws Regular Clamp Holder	Fisher Scientific	02-217-121	for holding the head stage amplifier & grounding the Faraday cage (must be made of metal and not coated in plastic)
Windows computer	Best Buy		for EPG & statistical analysis
Software
Backus 2.0 Data Analysis SAS program	UF IFAS Extension	https://crec.ifas.ufl.edu/extension/epg/data-analysis/	for statistical analysis of waveform data
Ebert 1.0 File Manipulator and Error Checker SAS programs	UF IFAS Extension	https://crec.ifas.ufl.edu/extension/epg/data-analysis/	for preperation of files for statistical analysis of waveform data
SAS version 9.4	SAS Institute Inc.		for statistical analysis of waveform data
WinDaq Lite	DATAQ Instruments Inc.		for recording and reviewing EPG data.  Includes the WinDaq Hardware Manager software (AKA, "hardware management software") and WinDaq Waveform Browser software (AKA, "waveform reviewing software"). For use on Windows computers only.
Faraday Cage
Aluminum screen frame	Home Depot	MSCF5167	for  the walls of the Faraday cage
Aluminum Sheet, 0.125 (1/8) thick 3003-H14	Metals Depot	S318	for the base of the Faraday cage
Corner braces, 1"	Home Depot	339547	for  the walls of the Faraday cage
Flat head Phillips machine screws, #6-32 x 1"	Home Depot	459999	for Faraday cage construction
Flat washers, #6	Home Depot	638977	for Faraday cage construction
Fluke 15B+ Digital Multimeter, for Electrical Applications, Measures AC/DC Voltage and Current Measurements up to 1000V and 10A, Along with Resistance, Continuity, Diode, and Capacitance Capabilities	Amazon.com		for testing the Faraday Cage
Portable Radio AM FM, Goodes Transistor Radio with Loud Speaker, Headphone Jack, 2AA Battery Operated Radio for Long Range Reception, Pocket Radio for Indoor, Outdoor and Emergency Use	Amazon.com		for testing the Faraday Cage
Pure copper screen, 16 x 16 mesh with 0.11 wire diameter, 36" x 12'	Metro Screenworks		for  the walls of the Faraday cage
Round head combo machine screws, #6-32 x 3/4"	Home Depot	528466	for Faraday cage construction
Sand paper, 1/2" by 1", 150 Grit	Home Depot	19036-20-CC	for removing the paint from the bottom of the laboratory stand
Screen frame corners, 5/16"	Home Depot	WCORN516	for  the walls of the Faraday cage
Screen spline, 0.14"	Home Depot	3034719	for  the walls of the Faraday cage
Insect Stubs
Brass Escutcheon Pins	Home Depot	801294	for constructing insect stubs
Copper wire, 20 AWG	Amazon.com		for constructing insect stubs
Harris water soluble flu	Home Depot	355398	for helping solder coat the insect stub wire and form a strong connection
Needle-nosed pliers	Home Depot		for bending wire when constructing insect stubs
Pro's Kit 900-015 Helping Hands Soldering Aid	Amazon.com		for holding escutcheon pins during soldering
Solder (with rosin core)	Home Depot	296497	for constructing insect stubs
Wire cutter stripper tool	Home Depot		for stripping and cutting copper wire when constructing insect stubs
YIHUA 928D-III Soldering Iron, 110W High Power, fully Digital Display Soldering Iron	Amazon.com		for constructing insect stubs; comes with a stand, sponge, and steel wool for cleaning
Insect Aspirator
Accuris ASPIRE Laboratory Aspirator	Accuris instruments	V0020	for applying suction to the insect aspirator; includes the handheld vacuum controller, the fine-tip (40 mm) stainless steel adaptor, and the pipette tip adapter
Chemglass Life Sciences Pasteur pipettes, 5 3/4" long	Fisher Scientific	NC0230004	for use as an insect aspirator when wiring mosquitoes
Commercial Electric Colored Vinyl Electrical Tape	Home Depot		for attaching the modified Pasteur pipette to the vacuum line
Mosquito netting utility fabric, 10 x 10 cm square	Joann Fabrics	16651622	for preventing mosquitoes from being aspirated into the vacuum pump
Saint Gobain Performance Plastics Tube Tygon 1/4 x 7/16	Fisher Scientific	50-206-8914	for quick and easy switching between different modified Pasteur pipettes
Thermo Scientific Nalgene HDPE Quick Disconnects (3/8 to 7/16 in. I.D.)	Fisher Scientific	15-315B	for quick and easy switching between different modified Pasteur pipettes
Silver Glue
Elmer's Glue-All Multi Purpose White Glue	Amazon.com		for silver glue preparation
Silver Flake 99.9% pure,  4-8 µm	ThermoFisher	786.09	for silver glue preparation
Silver Flake 99.95% pure, 8-10 µm	Inframat Advanced Materials	47MR-12F	for silver glue preparation
Insect Anesthesia
CO2 bubbler kit, 1000 mL, #9 Rubber Stopper	FlyStuff	59-181	for preventing insect desiccation while using the flypad
Flypad frame, standard size	FlyStuff	59-118	for prevent mosquitoes from falling off the flypad
Flystation Brush	FlyStuff	59-205	for reducing static when working on a fly pad
Flystuff Flypad standard size	FlyStuff	59-114	AKA "anesthesia pad" for anesthetizing mosquitoes
Funnel (4 inch diameter)	Amazon.com		for gassing down cups of mosquitoes before transferring to the flypad
Plexi glass or a flat plastic lid (cut to size)	Home Depot	MC-27	for covering the flypad if the mosquitoes start to wake up
Shut Off Plastic Tubing Hose Clamps Single Position and Adjustable Open (variety 1/8" to 1/2")	Amazon.com		for diverting gas between the flypad and funnel
Single Stage CO2 regulator	FlyStuff	59-142	for regulating the flow of gas from the CO2 tank
“T” Fitting for 1/8in ID Tubing	FlyStuff	59-123	for connecting both the flypad and funnel to the CO2 tank
Tubing, clear	FlyStuff	59-124C	for connecting the funnel and flypad to the regulator on the CO2 tank
Insect Wiring
Black cardstock, (65Lb, 176 g/m2) 8.5" x 11"	Michael's Stores Inc.	327826	to make gold wire easier to visualize
Black construction paper square 3.5" x 4.0"	Staples	956221	to make gold wire easier to visualize
Cotton balls	Amazon.com		for fasting mosquitoes
Craft foam #12 block (3.1 cm x 17.2 cm x 30.2 cm)	Joann Fabric and Craft Store	1941079	for holding wired mosquitoes
Deflecto Ribbon Dispenser, Clear	Amazon.com		for storing gold wire
Featherweight entomology forceps	Fisher Scientific	S72110	for insect manipulation
Fine forceps (FST by Dumont: #5 91150-20, Inox-Electronic)	Fine Science Tools	91150-20	for making a gold loop when wiring mosquitoes
Fine point paint brush	Amazon.com		for manipulating mosquitoes during wiring and EPG; must have a plastic or wooden handle if used for EPG
Fine-tip standard transfer pipets, narrow, 5.8 mL	Thomas Scientific	7761D21	for silver glue preparation
Gold wire, 99.99% pure, 0.025 mm thickness	Goodfellow	AU005121	for wiring mosquitoes
Minutien pins, tip 0.2 mm, 1 cm	Fisher Scientific	NC9681411	for applying glue to the mosquitoes
Paper cups	Rigid Paper Tube		for fasting mosquitoes
Pin vice	Fisher Scientific	50-996-811	for holding the minutien pin during insect wiring
Plastic storage box with lid (7 inch x 9 inch x 14 inch)	Amazon.com		for transporting wired mosquitoes
Plexi glass or a flat plastic lid (cut to size)	Home Depot	MC-27	for covering the fly pad if the mosquitoes start to wake up
Self-closing forceps	Fisher Scientific	08-906	for holding and manipulating gold wire
Compressed Gas
CO2 gas tank	Matheson Gas		for anesthetizing mosquitoes with a flypad
Common Lab Items
Biohazard disposable autoclave bags, 8 1/2" x 11"	Fisher Scientific	03-410-591	for disposal of blood-fed mosquitoes
Bunsen burner or propane torch	Amazon.com		for modification of Pasteur pipettes
Colored laboratory tape	Daigger Supplies	EF9763R	for labeling, preparing silver glue, and masking forceps
Disposable pipette tips, 1000 µL	Fisher Scientific		for aliquoting water when making silver glue
Dissection microscope	Leica Microsystems		for wiring mosquitoes
Dissection probe	Fisher Scientific	NC9109673	for stirring  and applying silver glue
Erlenmeyer flask, 1000 mL	Fisher Scientific		for preventing insect desiccation while using the flypad
Ethanol, 70%	Fisher Scientific	04-355-122	for recovering escaped mosquitoes
Glass petri dishes	Fisher Scientific	08-747B	for anesthetizing mosquitoes with ice
Gloves	Fisher Scientific		for personal protection
Kimwipes KimTech Science brand delicate task wipers	Fisher Scientific	06-666	for crushing mosquitoes
Lab coats	Fisher Scientific		for personal protection
LiBa Spray Bottles, Refillable Empty Spray Bottles (rubbing alcohol safe)	Amazon.com		for recovering escaped mosquitoes
Magnetic stir bar, 2 cm long	Fisher Scientific		for mixing up silver glue
Mechanical Single-Channel pipette, 1000 µL	Fisher Scientific	13-690-032	for aliquoting water when making silver glue
Microcentrifuge tubes, 1.5 mL	Fisher Scientific	780420	for storing silver glue
Parafilm M	Fisher Scientific	50-998-944	for sealing tubes of silver glue
Plastic wrap	Amazon.com		for covering the foam block
Pyrex 20 mL beaker	Fisher Scientific	07-250-051	for mixing up silver glue
Semi-Micro Balance	Fisher Scientific	10-919-370	for weighing glue and silver flake
Sharpie pen, fine point	Amazon.com		for labeling
Single-edge razor blade	Electron Microscopy Sciences	71980	for cleaning insect stubs
Standard Disposable Transfer Pipettes	Fisher Scientific	133-711-7mL	for transferring silver glue to a storage tube
Stir plate	Fisher Scientific		for mixing silver glue
Timer/stop watch	Fisher Scientific		for timing how long the host is exposed to each insect
Ultrapure (18 MΩ) water	Fisher Scientific		for silver glue preparation
Utility scissors	Fisher Scientific	08-945	for cutting gold wire
Vortex mixer	Fisher Scientific		for mixing silver glue
Study Organisms
Culex tarsalis mosquitoes	USDA ARS Center for Grain and Animal Health Research in Manhattan KS		for use as study subjects