Insects

The susceptible Harold Harlan strain of bed bug was used for all experiments. This strain was maintained at 25 °C, 50 ± 15% relative humidity, and a photoperiod of 12:12 (L: D) h. Bed bugs were fed weekly on defibrinated rabbit blood (Hemostat Laboratories, Dixon, CA) using the membrane feeding method60. Each week, 5th instar nymphs were separated from the main colony and reared in different jars. Newly emerged adult males were separated and used in all experiments. For toxicity evaluation, 8–10 d old adult males were used (average weight = ~2 mg per insect) that were fed 4–5 d before bioassays. However, for electrophysiology studies 10–15 d old adult males that were fed 7–8 d before evaluation were used. This starvation period allowed for clean dissections due to the absence of undigested blood in the foregut and midgut (Fig. 2b).

Figure 2 Electrophysiology recording set-up (suction electrode technique), dissected bed bug and its ganglion. (a) Recording electrode (RE) was placed in gentle contact with the fused thoracic ganglion, whereas the reference electrode (RefE) was placed in contact with the carcass. The ground electrode (GE) was placed in the Petri dish, but in contact with the external cuticle of the bed bug body in the presence of saline. (b,c) Fused thoracic and abdominal ganglion of the bed bug can be seen in the metathoracic region. Segmental nerves extend from the fused ganglion (see reference number 61 for a description of the bed bug ventral nerve cord). Full size image

Chemicals

High purity essential oil components carvacrol, geraniol, eugenol, methyl eugenol, trans-cinnamaldehyde, citronellic acid, (±)-citronellal, α-pinene, linalool, R (+)-limonene, eucalyptol, (−)-terpinen-4-ol, and menthone were obtained from Sigma-Aldrich (St. Louis, MO), whereas thymol and (±)-camphor were obtained from Alfa Aesar (Hill, MA) (Table S1). These active constituents are found in various aromatic plants (Table S1). All fifteen essential oil components (Table S1) were selected based upon the previous toxicity literature on different urban and agricultural pests22,23,24,25,26,27,28,29,30,31. The positive controls dichlorvos (≤100% purity) and bifenthrin (98% purity) were obtained from Sigma Aldrich and Chem Service Inc. (West Chester, PA), respectively. Analytical grade solvents such as acetone, ethanol and dimethyl sulfoxide (DMSO) were purchased from Fisher Scientific (Pittsburgh, PA). Buffer salts and other reagents used for preparation of HEPES (4-(2-hydroxyethyl)−1-piperazineethanesulfonic acid)-buffered physiological saline were purchased from Sigma-Aldrich, Fisher Scientific and Avantor Performance Materials, LLC (Center Valley, PA).

Topical application

Initially, each essential oil component was diluted in acetone on a volume-to-volume basis to prepare stock solutions based on the density of each component (Table S1). The only exceptions were thymol and (±)-camphor, which were prepared on a weight per volume basis due to their crystalline nature or form. The stock solutions were then serially diluted to prepare a range of dilutions (at least 5 for each component). Topical applications of different concentrations (volume range 0.5–1 µL) were made on the ventral metathorax using a 25 µL micro-syringe (Hamilton, Reno, NV) attached to a PB-600-1 repeating dispenser (Hamilton, Reno, NV). Insects were immobilized by attaching them dorsally to colored labelling tape (Fisher Scientific, Pittsburg, PA). Control groups were treated with acetone only. Technical grade bifenthrin dissolved in acetone (weight to volume basis) was used as a positive control. After treatment, insects (in groups of 10) were transferred into 35 × 10 mm Petri dishes with vents (Item number: 627161, Greiner Bio-One, Frickenhausen, Germany) lined with a single layer of Whatman # 1 filter paper (GE Healthcare UK Limited, Amersham Place, UK). Petri dishes were then placed in an environmental chamber with temperature, humidity and lighting conditions similar to those used for rearing. Initial bioassay experiments suggested that mortality caused by essential oil treatments did not significantly change between observation intervals of 24 and 48 h. Therefore, mortality scoring of all treatments was performed at 24 h post-treatment. Insects that were lying on their backs and/or were unable to move upon prodding were scored as dead. In total, three replicates were performed for each concentration (n = 30). The average weight of a single adult male bed bug used for bioassays was 2 mg. Hence, the topical lethal dose values are reported as µg/mg body weight.

Fumigant exposure and quantification of evaporation for essential oil components

Filter papers (9 cm diameter, Whatman #1) (GE Healthcare UK Limited) were treated with essential oil component solution (volume range: 9.46–1892 µL) prepared in acetone as described under “Topical application” bioassays. Treated papers were placed in glass containers (473 mL Mason jars; Anchor Glass Container Corporation, Tampa, FL) after complete evaporation of acetone. Evaporation time varied from ~30 sec to 5 mins based upon insecticide volume that was applied to the filter paper. In case of dichlorvos, only 30–45 sec of evaporation time was required because the treatment volume of ~10–15 µL was much lower in comparison to that of essential oil components. Ten adult bed bugs held in a mesh-covered glass scintillation vial (20 mL; W.W. Grainger, Inc., Lake Forest, IL) were then placed in mason jars along with treated filter papers. The mason jar was then sealed completely and transferred to an environmental chamber. Control insects were exposed to acetone treated filter papers. Acetone application volume for controls corresponded to the volume used for highest insecticide concentration or application volume of each tested compound. Three replicates (n = 30) were performed for each concentration. Mortality did not significantly change after the initial 24 h observation interval, as such all observations were recorded 24 h post exposure. Mortality was scored by following the same protocol described for topical bioassays. Fumigant lethal concentration values are expressed as amount of insecticide per liter air volume (mg/L).

To determine essential oil constituent or DDVP evaporation levels during the 24 h bioassay period, we first measured the weight (in grams) of untreated filter papers (W0) on a Mettler AE 100 weighing scale (Mettler-Toledo, Inc., Columbus, OH). After that, acetone-diluted essential oil constituents or insecticides were applied to the filter paper and the weights of these treated filter papers were recorded after the acetone (solvent carrier) evaporation period (30 sec to 5 mins) described in the previous paragraph had elapsed (W1). Control filter papers were treated with acetone only. Filter papers were then placed individually in sealed mason jars for 24 h. At 24 h, filter papers were weighed again (W2). Three concentrations (low, medium and high) were used for determining evaporation percentage for each compound. They were representative of the entire range of concentrations tested in fumigant bioassays for each compound. Three independent replicates were performed for each concentration. The following formula was used for calculating percent evaporation:

$$ \% \,{\rm{Evaporation}}=\frac{{\rm{Amount}}\,{\rm{evaporated}}\,(\mathrm{W1}-\mathrm{W2})}{{\rm{Amount}}\,{\rm{applied}}\,(\mathrm{W1}-\mathrm{W0})}\times 100$$

Electrophysiology equipment

The electrophysiology equipment used in this study was previously described by Gondhalekar and Scharf 61 and Feston62. The setup consists of three electrodes; recording, reference and ground (Fig. 2a). Recording and reference electrodes were mounted on suction electrode holders (Cat. No. 64–1035 Warner Instruments, Hamden, CT). Both electrodes were fabricated from ~4 cm lengths of 0.5 mm diameter gold wire (World Precision Instruments, Sarasota, FL) and fitted within 1.0 mm borosilicate glass capillaries (Harvard Apparatus, Holliston, MA) that were pulled to a fine point with a Micropipette puller (Narishige Co., LTD, Tokyo, Japan). Capillaries were used only for single recordings. The ground electrode consisted of #2 steel pin (Catalog #1208B2 Bio Quip Products, Rancho Dominguez, CA) which was held by a Pin Vise (#162 A The L.S. Starrett Company Athol, MA). All electrodes were connected to a model 4001 capacitance compensation head stage (Dagan Inc., Minneapolis, MN), which was connected to a Hum Bug 50/60 Hz Noise Eliminator (Quest Scientific Instruments Inc., North Vancouver, BC, Canada) and then a model EX-1 differential amplifier (Dagan Inc., Minneapolis, MN). The amplifier was interfaced with computerized digitizing hardware (PowerLab/ 4SP, ADInstruments, Milford, MA) and software that functioned as an eight-channel chart recorder (Chart version 3.5.7, ADInstruments, Milford, MA).

Dissections and neurophysiology recordings

Dissections were performed in 35 × 15 mm Petri dishes (Fisher Scientific, Hampton, NH) filled 2/3 of their volume with wax (Frey Scientific and CPO Science, Nashua, NH) (Fig. 2a) under a Leica S6D Greenough stereo microscope (Leica Microsystems Inc. Buffalo Grove, IL). Bed bugs were immobilized by four 0.15 mm stainless minutien pins (Carolina Biological Supply Company, Burlington, NC) during dissection (Fig. 2b). New Petri dishes and minutien pins were used for each recording. The general procedure described by Feston62 was used for performing dissections. Each experimental bed bug was dissected via one longitudinal incision from the dorsal abdomen up to the thorax followed by two latitudinal incisions across the wing pads to expose the fused ganglion (Fig. 2b)63. One microliter of HEPES-buffered saline, pH 7.1 was pipetted into the insect hemocoel immediately after dissection. Fat bodies, gut and other thoracic and abdominal body tissues were removed for better visualization of the ganglion (Fig. 2b,c).

Baseline electrical or nerve activity recordings were performed in HEPES-buffered physiological saline (volume: 1.5–2 µL; 185 mM sodium chloride, 10 mM potassium chloride, 5 mM HEPES sodium salt, 5 mM calcium chloride, 5 mM magnesium chloride and 20 mM glucose; pH 7.1)61,62,64. The recording electrode, fitted with a pulled glass capillary and filled with HEPES-buffered saline, was placed in gentle contact with the fused ganglia (Fig. 2a–c) with the help of a micromanipulator (model MNJR, World Precision Instruments). The reference electrode was prepared identically and placed in contact with the carcass (Fig. 2a). A ground electrode was placed in the dissection dish outside the bed bug carcass, but in contact with physiological saline (Fig. 2a). The total electrical activity recording for each insect was done for 10 minutes (Fig. 3). For the first 5 mins, spontaneous pretreatment electrical activity (i.e., baseline) was recorded by setting a threshold for the “counter” function on the Chart software (Fig. 3). The baseline electrical activity recording in physiological saline was briefly paused after the first 5 mins to enable application of 1 µL of essential oil component solution gently onto the ganglion. Multiple concentrations of essential oil constituents ranging from 0.5 to 5 mM were tested (approx. 0.5 to 5 mM or 3.75 × 10−12 to 4.25 × 10−10 µg of constituent per insect preparation). This solution was prepared by diluting essential oil components initially in DMSO (used for thymol, carvacrol, eugenol, citronellic acid and linalool dilution) or ethanol (used for (±)-camphor dilution) and then further dilutions were made in physiological saline containing 0.01% Tween 20. Recordings were resumed approximately 10–15 sec after the application of essential oil-containing solution. The waiting period of 10–15 sec was included to allow the ganglion to recover from the physical disturbance (if any) caused by application of 1 µL essential oil constituent solution. The threshold for the “counter” function remained constant for the 5 min pre-treatment and 5 min post-treatment nerve activity recordings (Fig. 3). For control recordings, a solution containing physiological saline + 0.1% DMSO or 0.1% ethanol + 0.01% Tween 20, but no essential oil component was used. To compare or see the effect of solvent controls on nervous activity, recordings were performed using physiological saline for the 5 min pre-treatment and 5 mins post-treatment recordings.

Figure 3 An example of 10-minute electrophysiological nerve activity trace from the Chart Software for 2 mM eugenol. Determination of spontaneous electrical activity bursts or spikes in pre-treatment or baseline recordings in physiological saline (for 5 mins) and post-treatment recordings in 2 mM Eugenol (5 mins) were enabled by setting the threshold using the “counter” function in the Chart software. The threshold was maintained constant between the pre-and post-treatment recordings. Data for the total number of spikes surpassing the threshold before and after treatment were used to calculate ratios representing a departure from baseline activity. Full size image

Departure ratios that represent deviation from the baseline electrical activity were calculated by dividing the total number of spike counts surpassing the threshold in post-treatment 5 min recordings (with essential oil constituents) with the total number of spike counts above threshold in 5 min of pre-treatment recordings (with physiological saline). Departure ratios that were significantly greater than “1.0” indicated neuroexcitatory action and ratios that were significantly less than “1.0” were indicative of neuroinhibition61. Similar procedures were followed to calculate departure ratios for solvent control preparations.

For the positive control treatment using bifenthrin (a pyrethroid insecticide), the same procedures were followed, however, the treatment volume was higher (2 µL). The use of a higher volume was necessary for bifenthrin based on preliminary experiments. In a preliminary study, 1 µL volume of 1.25–10 µM bifenthrin did not significantly excite the bed bug ventral nerve cord. Each bed bug or dissection represented one replicate and ten replications were performed for each essential oil component or positive control (bifenthrin) concentration, solvent controls and physiological saline controls. The recordings in which bed bugs were dead during or after 10 minutes were discarded and a new recording was performed with a new insect preparation to account for the loss.

Topical bioassays to observe poisoning symptoms

To observe poisoning symptoms at the whole organism level caused by the six most toxic essential oil components, topical application bioassays were performed at the LD 50 for each compound. Acetone-diluted compounds were applied to the metathoracic region using identical procedures outlined for “Topical application” bioassays. Control insects were treated with acetone. Poisoning symptoms exhibited by adult male bed bugs were observed at 2 and 4 h post treatment either directly in the bioassay Petri dish or under a microscope. Short videos (~30 secs) of bed bugs from various treatments were also recorded at the 2 and 4 h intervals and were used to confirm or cross-check the presence or absence poisoning symptoms. In total 30 insects were observed for each compound. Specifically, the presence or absence of three symptoms was observed: (1) hyperactivity (uncoordinated movement and wandering behavior), (2) paralysis (inability to walk or right themselves up on prodding) and (3) tremors (insects lying on their back and exhibiting involuntary leg spasms, twitching and quivering).

Statistical analysis