Protocol for single sensillum recording from labial olfactory sensory fields in spotted lanternfly
Olfactory receptor neurons (ORNs) and their receptors detect odorants that mediate host plant finding, aggregation, and mating. Identifying these odorants for spotted lanternfly (SLF) is crucial for developing effective management strategies to limit its agricultural and ecological damage. SLF is a polyphagous planthopper that has recently spread across the United States and other countries. The spread of this insect is mediated, in part, via ORNs. , , , , , , , , ORNs in SLF are housed in structures mainly located on the antennae and labial sensory fields. , , However, the odorant receptive range of these ORNs remains largely undetermined. The following protocol provides step-by-step procedures for measuring and analyzing odorant response profiles of labial ORNs in SLF, aiming to foster research in this area across planthoppers, a group of over 9000 described species in the infraorder Fulgoromorpha, suborder Auchenorrhyncha. This protocol has also been adapted for antennal ORNs and is applicable to adults and nymphal instars of most planthopper species. Institutional permissions Insects were transferred from the field to the insect containment facility located in the Dweck Chemical Ecology Laboratory at the Connecticut Agricultural Experiment Station, Main Campus, New Haven, Connecticut, in accordance with U.S. Department of Agriculture (USDA) permit 526-24-114-68354. Preparation of odorant cartridges Timing: 2 min for each odorant cartridge 1. Prior to beginning, prepare odorant cartridges . a. Using clean forceps, place a 1.3 cm diameter disc of filter paper into the large end of a disposable borosilicate glass Pasteur pipette (2 mL volume) to prepare an odorant cartridge. b. Vortex a diluted odorant solution for a few seconds to ensure it is well-mixed and homogeneous. c. Pipette 10 μL on the filter paper disc. d. Cap the end of the large end Pasteur pipette with a 1000 μL pipette tip (without filter) to reduce odorant evaporation and to connect to the pulse flow output of the odorant delivery system. e. Prepare a cartridge with the diluent of each odorant as a control. Note: Depending on the volatility of the odorants, preparing new cartridges every three hours or at any specific interval is recommended. CRITICAL: Use a pair of clean gloves while preparing odorant cartridges and use pipette tips with filters for odorant/solvent pipetting. Mounting the labium Timing: 5–10 min 2. Using micro-scissors, carefully clip the legs and wings of the spotted lanternfly ( A and 1B). Note: This step is to prevent the insect from moving during subsequent steps. CRITICAL: Handle the insect gently to avoid damaging the labium or covering it with the oozing hemolymph. 3. Insert the spotted lanternfly into a 1000 μL plastic pipette tip with its head directed toward the narrower end ( C). Note: Ensure the insect is positioned with its body aligned laterally (sideways) relative to the length of the pipette tip. CRITICAL: This positioning is crucial for easily recording from ORNs housed in olfactory sensilla located on the apical and subapical labial sensory fields. , , 4. Place a small amount of clay on a glass microscope slide ( D). 5. Attach the pipette tip to the slide using the clay ( D). Note: Ensure that the pipette tip is securely fastened and that the body remains oriented laterally. 6. Cut a small piece of double-sided tape and attach it to a cover slip ( D). 7. Use clay to secure the cover slip to the center of the microscope slide ( D). 8. Carefully position the labium laterally against the tape under a stereomicroscope ( D). Note: Verify that the apical and subapical labial olfactory sensilla are accessible and properly oriented. CRITICAL: The labium should be fixed securely to minimize any movement during recording. Setting up equipment Timing: 1–2 h 9. Set up the electrophysiology rig . a. Choose a room that has stable and suitable temperature and relative humidity to ensure consistent recording conditions and avoid damaging the equipment. Note: Isolation from electrical and mechanical noise sources, such as refrigerators, centrifuges, or other potentially interfering equipment, is crucial to avoid any electrical or mechanical interference during recording. b. Place the microscope at the center of an anti-vibration table ( A and 2B). This minimizes vibrations that could affect the precision of the recording. c. Secure a manual manipulator to the anti-vibration table ( B). d. Attach a stainless-steel shaft to the manipulator. This shaft will hold the tungsten reference electrode ( B). e. Connect two motorized micromanipulators to the anti-vibration table using stands: i. First Micromanipulator: Equip it with a holder for the recording electrode probe ( B). ii. Second Micromanipulator: Hold a glass tube (20 cm long, ∼4 mm inner diameter) that supplies continuous humidified air to the preparation at an airflow speed of 40 cm/s ( B). iii. Place the end of the glass tube at ∼1–2 cm from the preparation ( C). The end of the tube should have a hole to introduce odorized pulsed air into the continuous humidified air during odorant application. iv. Use a stimulus air controller to generate pulsed and continuous airflow ( A). v. Use an anemometer to accurately measure the air flow speed. f. Connect the recording electrode probe to an Intelligent Data Acquisition Controller (IDAC) system or another suitable amplifier/digitizer system. This system will capture and process the electrical signals from the ORNs. g. Link the IDAC system to the computer at the workstation. This connection allows for the transfer and analysis of data from the recording electrode. h. Ground the manual and motorized manipulators to the same location within the rig. Proper grounding is crucial to prevent electrical noise and interference in your recording. i. Install the appropriate acquisition software for the IDAC system on the computer. Check compatibility of the digital acquisition drivers with the operating system to ensure smooth operation of the IDAC system.   Insects were transferred from the field to the insect containment facility located in the Dweck Chemical Ecology Laboratory at the Connecticut Agricultural Experiment Station, Main Campus, New Haven, Connecticut, in accordance with U.S. Department of Agriculture (USDA) permit 526-24-114-68354.   Timing: 2 min for each odorant cartridge 1. Prior to beginning, prepare odorant cartridges . a. Using clean forceps, place a 1.3 cm diameter disc of filter paper into the large end of a disposable borosilicate glass Pasteur pipette (2 mL volume) to prepare an odorant cartridge. b. Vortex a diluted odorant solution for a few seconds to ensure it is well-mixed and homogeneous. c. Pipette 10 μL on the filter paper disc. d. Cap the end of the large end Pasteur pipette with a 1000 μL pipette tip (without filter) to reduce odorant evaporation and to connect to the pulse flow output of the odorant delivery system. e. Prepare a cartridge with the diluent of each odorant as a control. Note: Depending on the volatility of the odorants, preparing new cartridges every three hours or at any specific interval is recommended. CRITICAL: Use a pair of clean gloves while preparing odorant cartridges and use pipette tips with filters for odorant/solvent pipetting.   Timing: 5–10 min 2. Using micro-scissors, carefully clip the legs and wings of the spotted lanternfly ( A and 1B). Note: This step is to prevent the insect from moving during subsequent steps. CRITICAL: Handle the insect gently to avoid damaging the labium or covering it with the oozing hemolymph. 3. Insert the spotted lanternfly into a 1000 μL plastic pipette tip with its head directed toward the narrower end ( C). Note: Ensure the insect is positioned with its body aligned laterally (sideways) relative to the length of the pipette tip. CRITICAL: This positioning is crucial for easily recording from ORNs housed in olfactory sensilla located on the apical and subapical labial sensory fields. , , 4. Place a small amount of clay on a glass microscope slide ( D). 5. Attach the pipette tip to the slide using the clay ( D). Note: Ensure that the pipette tip is securely fastened and that the body remains oriented laterally. 6. Cut a small piece of double-sided tape and attach it to a cover slip ( D). 7. Use clay to secure the cover slip to the center of the microscope slide ( D). 8. Carefully position the labium laterally against the tape under a stereomicroscope ( D). Note: Verify that the apical and subapical labial olfactory sensilla are accessible and properly oriented. CRITICAL: The labium should be fixed securely to minimize any movement during recording.   Timing: 1–2 h 9. Set up the electrophysiology rig . a. Choose a room that has stable and suitable temperature and relative humidity to ensure consistent recording conditions and avoid damaging the equipment. Note: Isolation from electrical and mechanical noise sources, such as refrigerators, centrifuges, or other potentially interfering equipment, is crucial to avoid any electrical or mechanical interference during recording. b. Place the microscope at the center of an anti-vibration table ( A and 2B). This minimizes vibrations that could affect the precision of the recording. c. Secure a manual manipulator to the anti-vibration table ( B). d. Attach a stainless-steel shaft to the manipulator. This shaft will hold the tungsten reference electrode ( B). e. Connect two motorized micromanipulators to the anti-vibration table using stands: i. First Micromanipulator: Equip it with a holder for the recording electrode probe ( B). ii. Second Micromanipulator: Hold a glass tube (20 cm long, ∼4 mm inner diameter) that supplies continuous humidified air to the preparation at an airflow speed of 40 cm/s ( B). iii. Place the end of the glass tube at ∼1–2 cm from the preparation ( C). The end of the tube should have a hole to introduce odorized pulsed air into the continuous humidified air during odorant application. iv. Use a stimulus air controller to generate pulsed and continuous airflow ( A). v. Use an anemometer to accurately measure the air flow speed. f. Connect the recording electrode probe to an Intelligent Data Acquisition Controller (IDAC) system or another suitable amplifier/digitizer system. This system will capture and process the electrical signals from the ORNs. g. Link the IDAC system to the computer at the workstation. This connection allows for the transfer and analysis of data from the recording electrode. h. Ground the manual and motorized manipulators to the same location within the rig. Proper grounding is crucial to prevent electrical noise and interference in your recording. i. Install the appropriate acquisition software for the IDAC system on the computer. Check compatibility of the digital acquisition drivers with the operating system to ensure smooth operation of the IDAC system.      Recording neuronal responses to odorants Timing: 3–5 min for each odorant This step aims to measure neuronal responses to each odorant. 1. Place the preparation slide on the microscope stage with a low magnification (10x) objective in position ( C). 2. Move the stage until the tip of the labium is in focus and centered in the field of view at low magnification. 3. Switch to a high magnification (50x) objective to ensure that the sensory fields at the tip of the labium are visible ( D). Note: The tip of the labium contains the apical and subapical sensory fields, which bear olfactory sensilla. 4. Start the signal-recording (AutoSpike) software and turn on the audio output to monitor neuronal firing activity. 5. Insert the reference electrode into the abdomen using the low magnification objective ( E). Note: Use tungsten rods (127 μm diameter and 76.2 mm length) for both reference and recording electrodes. Sharpen the electrodes to approximately 1 μm diameter at the tip by dipping them repeatedly for several seconds in either a 10% KNO3 (∼1 M) solution or a 10% KOH (1.8 M) solution. CRITICAL: This solution requires a current of less than 10 mA to facilitate the sharpening process. 6. Under high magnification (50x), use the motorized micromanipulator to carefully insert the recording electrode into one of the two placoid sensilla that form the subapical labial sensory field ( F). 7. Listen for neuronal firing activity through the audio output of the IDAC system ( A) as you insert the recording electrode. This indicates that you have successfully targeted a neuron. 8. Once a stable signal is established, connect the odorant cartridge to the pulse flow outlet of the stimulus controller system using a flexible tube. 9. Insert the narrow end of the odorant cartridge into the hole of the 20 cm tube that supplies continuous humidified air to the preparation. 10. For odorant application, press the pedal. Note: Pressing the pedal initiates the application of odorized pulsed air for 0.5 s and starts signal recording for 10 s or any desired period. CRITICAL: The introduction of odorized pulsed air will increase airflow speed from 40 cm/s to 50 cm/s. The duration of the pulsed air should be maintained at 0.5 s for standard recording. Data analysis Timing: 1–3 min for each odorant The aim of this step is to determine the number of neurons within a sensillum, measure neuronal spontaneous firing activities, identify modes of neuronal responses to odorants and their dynamics, and quantify these responses in spikes per second. 11. Use the AutoSpike Software to Sort Spikes. a. Open the “AutoSpike” software on the computer connected to the IDAC system. Note: This software will allow you to analyze the neuronal activity recorded from the ORNs. b. Load the recorded data from the session where you captured the neuronal firing activity from the subapical labial sensory field. c. Use the ‘Create Amplitude Histogram’ function within the AutoSpike software to analyze the recorded spikes. Note: This function will sort the spikes based on their amplitude, allowing you to differentiate between different neurons within the sensillum. The number of distinct peaks or groups of spikes in the histogram corresponds to the number of neurons within the sensillum. 12. For each neuron, calculate responses to odorants as the increase (or decrease) in the action potential frequency by subtracting the number of action potentials during the 0.5 s preceding the odorant stimulation from the number of action potentials during the 0.5 s of odorant stimulation. 13. Multiply the difference by two to convert the response into units of spikes per second (spikes/s). Note: The software includes a function to “insert signal-frequency”, which can be used to analyze the response dynamics of the recorded neuronal signals. This feature allows you to examine how the frequency of neuronal firing changes over time in response to the odorant stimulation. AutoSpike also supports the exportation of data in various formats, enabling further analysis using other tools or software.   Timing: 3–5 min for each odorant This step aims to measure neuronal responses to each odorant. 1. Place the preparation slide on the microscope stage with a low magnification (10x) objective in position ( C). 2. Move the stage until the tip of the labium is in focus and centered in the field of view at low magnification. 3. Switch to a high magnification (50x) objective to ensure that the sensory fields at the tip of the labium are visible ( D). Note: The tip of the labium contains the apical and subapical sensory fields, which bear olfactory sensilla. 4. Start the signal-recording (AutoSpike) software and turn on the audio output to monitor neuronal firing activity. 5. Insert the reference electrode into the abdomen using the low magnification objective ( E). Note: Use tungsten rods (127 μm diameter and 76.2 mm length) for both reference and recording electrodes. Sharpen the electrodes to approximately 1 μm diameter at the tip by dipping them repeatedly for several seconds in either a 10% KNO3 (∼1 M) solution or a 10% KOH (1.8 M) solution. CRITICAL: This solution requires a current of less than 10 mA to facilitate the sharpening process. 6. Under high magnification (50x), use the motorized micromanipulator to carefully insert the recording electrode into one of the two placoid sensilla that form the subapical labial sensory field ( F). 7. Listen for neuronal firing activity through the audio output of the IDAC system ( A) as you insert the recording electrode. This indicates that you have successfully targeted a neuron. 8. Once a stable signal is established, connect the odorant cartridge to the pulse flow outlet of the stimulus controller system using a flexible tube. 9. Insert the narrow end of the odorant cartridge into the hole of the 20 cm tube that supplies continuous humidified air to the preparation. 10. For odorant application, press the pedal. Note: Pressing the pedal initiates the application of odorized pulsed air for 0.5 s and starts signal recording for 10 s or any desired period. CRITICAL: The introduction of odorized pulsed air will increase airflow speed from 40 cm/s to 50 cm/s. The duration of the pulsed air should be maintained at 0.5 s for standard recording.   Timing: 1–3 min for each odorant The aim of this step is to determine the number of neurons within a sensillum, measure neuronal spontaneous firing activities, identify modes of neuronal responses to odorants and their dynamics, and quantify these responses in spikes per second. 11. Use the AutoSpike Software to Sort Spikes. a. Open the “AutoSpike” software on the computer connected to the IDAC system. Note: This software will allow you to analyze the neuronal activity recorded from the ORNs. b. Load the recorded data from the session where you captured the neuronal firing activity from the subapical labial sensory field. c. Use the ‘Create Amplitude Histogram’ function within the AutoSpike software to analyze the recorded spikes. Note: This function will sort the spikes based on their amplitude, allowing you to differentiate between different neurons within the sensillum. The number of distinct peaks or groups of spikes in the histogram corresponds to the number of neurons within the sensillum. 12. For each neuron, calculate responses to odorants as the increase (or decrease) in the action potential frequency by subtracting the number of action potentials during the 0.5 s preceding the odorant stimulation from the number of action potentials during the 0.5 s of odorant stimulation. 13. Multiply the difference by two to convert the response into units of spikes per second (spikes/s). Note: The software includes a function to “insert signal-frequency”, which can be used to analyze the response dynamics of the recorded neuronal signals. This feature allows you to examine how the frequency of neuronal firing changes over time in response to the odorant stimulation. AutoSpike also supports the exportation of data in various formats, enabling further analysis using other tools or software.   Despite detailed morphological studies on the sensilla, including the olfactory sensilla, located on the antenna and labium—the main olfactory organs in spotted lanternfly (SLF)—our understanding of their functional characteristics remains limited. , SLF is an invasive, polyphagous planthopper that poses a significant threat to the grape industry and various other sectors in the United States. This insect partly relies on its sense of smell (olfaction) to locate host plants, feeding sites, mating partners, and oviposition sites. , , , , , , , , However, key aspects of its olfactory system are not well understood, including the number of functional olfactory neurons, their odorant response profiles, sensitivity and specificity, and response dynamics. Understanding these aspects is crucial not only for gaining insights into how the olfactory system works in SLF and other planthoppers but also for the development of novel natural repellents and attractants. Such discoveries could play a significant role in mitigating the impact of this destructive insect on agriculture, residential areas, and forests. The animal preparation and single sensillum recording steps outlined in this protocol enabled us to successfully, for the first time to our knowledge, record the spontaneous firing rates of neurons within the paired placoid sensilla of the subapical labial sensory organ in SLF ( A). By monitoring neuronal activity over an extended period (approximately 100 s), we were able to distinguish between two distinct populations of spikes, corresponding to two separate neurons ( A and 3B). This distinction was initially based on the differing amplitudes of the spikes and was further confirmed using the 'Create Amplitude Histogram' function of the AutoSpike software. We call the neuron with the large amplitude the ‘A neuron’ and the neuron with the small amplitude the ‘B neuron’. In our single sensillum measurements in SLF using several odorants that were identified from SLF host plants, honeydew, or the insect itself, , , , , , , , we observed that the A-neuron of the placoid sensilla exhibits robust excitatory responses to three structurally similar aldehydes ( C) and inhibitory responses to many other odorants ( D). By contrast, the B-neuron shows robust excitatory responses to a wide range of odorants, with inhibitory responses to few or none ( D). Additionally, these measurements revealed that the A and B neurons exhibit distinct excitatory odorant response profiles, even at the highest tested dilution (10⁻ 2 ), and that the responses in each neuron to odorants are dose-dependent . Furthermore, higher concentrations of certain odorants, such as acetophenone, produce longer response durations compared to lower concentrations . Note: Using this technique, we not only gained new insights into how olfaction (i.e., the sense of smell) works in SLF but also identified several odorants that were not previously known to be physiologically or behaviorally active. These newly identified compounds could prove useful in developing strategies to mitigate the impact of this invasive insect.   This technique is undeniably powerful and versatile, offering substantial benefits for measuring neuronal spontaneous activities and their responses to various odorants in most insects. It also proves invaluable in educational settings, particularly in teaching laboratories, where it can be used to effectively demonstrate fundamental principles of sensory biology within a short time frame. However, despite its strengths, there are significant challenges that researchers may encounter, particularly when working with complex sensory structures. One of the primary difficulties lies in improving the signal-to-noise ratio during recording, especially when dealing with sensilla that contain a large number of neurons. For example, the placoid sensilla of parasitoid wasps can house up to 30 neurons in some species. , This densely packed neuronal environment can create substantial background noise, making it difficult to isolate and accurately measure the activity of individual neurons. Another challenge arises when neurons are activated using their most potent ligands at higher concentrations. In such cases, the increased neuronal activity can result in an overwhelming number of spikes, making it exceedingly difficult to accurately count and analyze these spikes.   Problem 1: There is no signal Sometimes inserting a recording electrode into a sensillum produces no signal ( A). This problem is related to steps 6 and 7. Potential solution • Verify that the grounding electrode is properly inserted and check that the animal is still alive. • If the problem continues, consider using a new animal for recording. Problem 2: The signal-to-noise ratio is weak Sometimes inserting a recording electrode into a sensillum produces a weak signal-to-noise ratio ( B). This problem is related to steps 6 and 7. Potential solution To enhance the signal-to-noise ratio. • Ensure that the recording electrode is sharp enough to penetrate the sensillum cuticular wall. • If the electrode is not penetrating properly, try inserting it at a different location. • If the problem continues, sharpen the recording electrode. • If the issue persists, consider using a new animal for recording. Problem 3: Losing contact while recording If the labium moves during the recording, this can lead to loss of contact ( C). This problem is related to steps 8 to 10. Potential solution To overcome this problem. • Secure the labium firmly on the cover slip using strong double-sided tape. • Ensure the anti-vibration table is functioning properly. An unbalanced table can cause movement of the recording electrode during recording.   Sometimes inserting a recording electrode into a sensillum produces no signal ( A). This problem is related to steps 6 and 7.   • Verify that the grounding electrode is properly inserted and check that the animal is still alive. • If the problem continues, consider using a new animal for recording.   Sometimes inserting a recording electrode into a sensillum produces a weak signal-to-noise ratio ( B). This problem is related to steps 6 and 7.   To enhance the signal-to-noise ratio. • Ensure that the recording electrode is sharp enough to penetrate the sensillum cuticular wall. • If the electrode is not penetrating properly, try inserting it at a different location. • If the problem continues, sharpen the recording electrode. • If the issue persists, consider using a new animal for recording.   If the labium moves during the recording, this can lead to loss of contact ( C). This problem is related to steps 8 to 10.   To overcome this problem. • Secure the labium firmly on the cover slip using strong double-sided tape. • Ensure the anti-vibration table is functioning properly. An unbalanced table can cause movement of the recording electrode during recording.   Lead contact Further information and requests for resources should be directed to and will be fulfilled by the lead contact, Hany Dweck ( hany.dweck@ct.gov ). Technical contact Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Hany Dweck ( hany.dweck@ct.gov ). Materials availability Materials associated with protocol are available through the . Data and code availability • Original data for figures in the paper is available in Dweck and Claire. • Any additional information on the data is available from the upon request. • This study did not generate code.   Further information and requests for resources should be directed to and will be fulfilled by the lead contact, Hany Dweck ( hany.dweck@ct.gov ).   Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Hany Dweck ( hany.dweck@ct.gov ).   Materials associated with protocol are available through the .   • Original data for figures in the paper is available in Dweck and Claire. • Any additional information on the data is available from the upon request. • This study did not generate code.   We thank Noelle Khalil for commenting on the manuscript. This work was supported by the 2024 CAES Board of Control Research Award and NIH grant K01 DC020145 (to H.K.M.D.).   Conceptualization, methodology, investigation, writing, and funding acquisition, H.K.M.D.   The author declares no competing interests.   