Bed bugs (Cimex lectularius L.) are globally important human parasites. Integrated pest management (IPM) approaches, which include the use of essential oil-based insecticidal compounds, have been proposed for their control. This study aimed to define insecticidal activity and neurophysiological impacts of plant essential oil constituents. The topical and fumigant toxicity of 15 compounds was evaluated against adult male bed bugs. Neurological effects of the 6 most toxicologically active compounds were also determined. In both topical and fumigant bioassays, carvacrol and thymol were the most active compounds. The potency of bifenthrin (a pyrethroid insecticide) in topical bioassays was 72,000 times higher than carvacrol, while vapors of dichlorvos (an organophosphate insecticide) were 445 times more potent than thymol. Spontaneous electrical activity measurements of the bed bug nervous system demonstrated neuroinhibitory effects of carvacrol, thymol and eugenol, whereas linalool produced an excitatory effect. Although citronellic acid and (±)-camphor increased baseline activity of the nervous system their effects were not statistically significant. Bifenthrin also caused neuroexcitation, which is consistent with its known mode of action. These comparative toxicity and neurological impact findings provide new information for formulating effective essential oil-based insecticides for bed bug IPM and conducting mode-of-action studies on individual essential oil components.
Bed bugs (Cimex lectularius L.) are economically and medically important global human parasites. They feed on human blood and their bites can worsen psychological disorders, cause sleep deprivation and other health issues such as rashes, itching, allergies, and etc.1. The U.S. Center for Disease Control and Prevention (CDC) and the U.S. Environmental Protection Agency (EPA) consider bed bugs as a pest of significant public health importance2. A resurgence of bed bugs has occurred over the last 18 years and they continue to spread. One of the primary factors for their resurgence is due to the overuse of synthetic insecticides with similar modes of action, which has led to insecticide resistance development3-6. The application of synthetic insecticides within buildings or in indoor environments is also a public health concern due to the toxic effects that can result from prolonged exposure7-9.
Integrated pest management (IPM) approaches have been proposed for the effective management of bed bugs. This strategy includes the use of multiple control tactics: resident education, bed bug monitoring using active and passive traps, non-chemical control (removal of infested furniture, heat treatments, use of mattress encasements etc.), along with the use synthetic and essential-oil based insecticides10-12. There is also an increased demand from the public for use of efficacious "green" products for urban pest management. Botanical insecticides, including essential oils are considered safe because of their low toxicity to humans and animals13,14. Plant-derived essential oils have emerged as a potential alternative option for the management of insect pests15,16. Because they pose a minimum risk, essential oil compounds are exempt from full EPA registration (Federal Insecticides, Fungicides, and Rodenticides Act-FIFRA, 40 CFR 152.25)17. Some of the drawbacks associated with the use of essential oils for pest control are: (i) short residual life that necessitates frequent applications (ii) high volatility can lead to odor problems, which are sometimes unacceptable to residents, and (iii) field efficacy of these products is generally less documented for different insect pest species15,16.
Essential oils are secondary metabolites derived from aromatic plants that are composed of complex mixtures of chemical constituents or components with different functional groups (e.g., phenols, aldehydes, acids, hydrocarbons, etc.)18. Recent studies have shown that plant-derived essential oils exhibit contact and fumigant toxicity against field populations of bed bugs14,19,20. However, these studies have not characterized the insecticidal activity of major constituents of essential oils against bed bugs. More than a dozen essential oil-based products are available commercially for indoor use, but only two products have been found effective for bed bug control21. Therefore, there is a need for conducting comparative baseline toxicity studies with bed bugs using major components or constituents of different plant essential oils (Table S1) that have been shown to be efficacious against urban and agricultural insect pests22-31.
There is also a significant knowledge gap regarding the effects of major or active components of essential oils on the insect nervous system32,33. The possible target sites for the essential oil components thymol, eugenol, and carvacrol are gamma-amino butyric acid (GABA), octopamine/tyramine and nicotinic acetylcholine (nACh) receptors, respectively34-37. Very few studies have documented electrophysiological responses induced by application of essential oil components to the nervous system of insects. Price and Berry38 reported that the essential oil components eugenol, geraniol and citral are neurologically active against Periplaneta americana and Blaberus discoidalis. Similarly, Hertel et al.39 found the plant essential oil components quassin and cinnamaldehyde to be neurologically active against P. americana. Recent in silico molecular docking studies with major chemical constituents of marigold essential oil (α-terpinolene, piperitone and piperitenone) suggested the neurotransmitter hydrolyzing enzyme acetylcholinesterase as the potential target site in bed bugs14.
Given the knowledge gaps associated with the unavailability of comparative toxicity data for individual essential oil constituents against bed bugs, and their impacts on the nervous system, the objectives of this research were (i) to determine topical and fumigant toxicity of fifteen essential oil components against bed bugs and (ii) identify neurological effects caused by the six most effective constituents by performing electrophysiology experiments.
Acetone-diluted essential oil components were applied to the ventral metathorax of adult male bed bugs to determine their topical toxicity. Of the fifteen different components tested, carvacrol and thymol were relatively more toxic with LD
Adult male bed bugs were exposed to vapors of essential oil components in sealed mason jars (volume of 473 ml) to determine their fumigant toxicity. Thymol was the most toxic compound with a LC
Acetone (solvent carrier) applied to control filter papers evaporated completely (100%) during the 30 sec to 5 min drying time described in the methods section. Data on evaporation of different essential oil components for the 24 h bioassay duration are presented in Table 2. Percent evaporation was highest for eucalyptol (100%), whereas it varied from ~90% for thymol to <1% for trans-cinnamaldehyde. When regression analysis was performed between compounds for which LC
Spontaneous nerve activity recordings from the fused thoracic ganglion of adult male bed bugs demonstrated no neuroexcitatory or neuroinhibitory effects of solvent controls containing either 0.1% DMSO + 0.01% Tween 20 (P = 0.790) or 0.1% absolute ethanol + 0.01% Tween 20 (P = 0.826) in comparison to the HEPES-buffered physiological saline (PS) treatment (Fig. 1a). At the Bonferroni adjusted statistical significance level of P < 0.0125 (i.e., 0.05 ÷ number of comparisons in two-sample t-tests) the concentration of 4 mM for both carvacrol (P = 0.005) and thymol (P = 0.001) caused significant neuroinhibition (Fig. 1b,c). Eugenol exhibited significant neuroinhibitory effects at the 2 mM concentration (P = 0.001; Fig. 1d).Neurophysiological effects of essential oil components, bifenthrin and solvent controls on the bed bug nervous system. Bars represent average departure ratios calculated by dividing the nervous activity spikes surpassing the threshold in post-treatment recordings (either with essential oil constituents or bifenthrin or solvent controls) with spike counts from physiological saline (PS) pre-treatment. Asterisks (*) in different graphs indicate significant differences from solvent control recordings (two-sample t-tests with Bonferroni corrected P-value i.e. 0.05 ÷ number of comparisons for each compound). (a) Solvent control treatments, PS + 0.1% dimethyl sulfoxide (DMSO) + 0.01% Tween-20 (SC-D) or PS + 0.1% absolute ethanol + 0.01% Tween 20 (SC-E) had no effect on nervous system activity (P > 0.025). (b) Carvacrol (4 mM), (c) thymol (4 mM), and (d) eugenol (2 mM) exhibited a neuroinhibitory effect as indicated by departure ratios significantly below 1 (P < 0.0125). (e) With departure ratios above 1, linalool (4 mM) led to significant neuroexcitation (P < 0.0125), but (f) citronellic acid (P > 0.01) and (g) (±)-camphor (P > 0.0125) did not cause significant neurological impacts. (h) The positive control treatment with bifenthrin (10 µM) caused significant neuroexcitation (P < 0.0125).
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For linalool, the concentration of 4 mM (P = 0.011) produced significant neuroexcitatory effects (P < 0.0125) (Fig. 1e). Citronellic acid (Fig. 1f) and (±)-Camphor (Fig. 1g) resulted in departure ratios that were >1 and were indicative of neuroexcitatory effects, however, none of the concentrations tested for these compounds caused a significant increase in nervous activity at the Bonferroni corrected significance levels of P < 0.01 and P < 0.0125, respectively. As expected, the positive control treatment with bifenthrin (a synthetic pyrethroid insecticide), caused significant neuroexcitation at the 10 µM concentration (P = 0.0001; Fig. 1h).
Linear regression analysis showed that carvacrol and thymol caused a concentration-dependent decrease in spontaneous electrical activity of the nervous system (P < 0.05, Fig. S2). In contrast, citronellic acid, linalool and bifenthrin induced concentration-dependent increase in nervous activity (P < 0.05, Fig. S2). Eugenol and (±)-camphor did not show concentration-dependent changes in neurological activity (P > 0.05, Fig. S2), likely because their effects were bi-phasic (i.e., pronounced effects at intermediate concentrations in comparison to lower or higher concentrations; Fig. 1d,g).
Treatment of bed bugs with the solvent carrier (acetone) did not induce any poisoning symptoms such as hyperactivity, paralysis or leg tremors at 2 and 4 h after treatment (Table 3). However, hyperactivity, defined as uncoordinated movement and wandering behavior, was observed in bed bugs treated with five of the six most toxic essential oil components (carvacrol, thymol, eugenol, linalool and (±)-camphor) at the 2 h interval (Table 3). Citronellic acid treated insects did not show hyperactivity symptoms. Bed bugs treated with all six toxic plant essential oil components were paralyzed, i.e., they were unable to walk or right themselves upon prodding at the 4 h observation interval (Table 3). Paralysis was also observed in thymol and (±)-camphor treated insects 2 h after treatment. Leg tremors (involuntary leg spasms, twitching and quivering) were observed in knocked-down insects treated with thymol, linalool and (±)-camphor (Table 3). Death of treated insects was first observed ~6 h after treatment with some of the compounds and hence observations on non-quantitative poisoning symptoms were not recorded after the 4 h observation interval.
Initially we characterized the inherent toxicity of fifteen different plant essential oil components against bed bugs. Carvacrol and thymol were the most active compounds in topical application bioassays. Both compounds exhibited similar levels of contact toxicity and were 13-15 times more potent than the least toxic constituent, methyl eugenol in topical bioassays. Carvacrol and thymol were previously reported as being effective, with contact and fumigant toxicity against several insect pests including cockroaches, kissing bugs and house flies22-26. As found in other insects, increased toxicity of carvacrol and thymol towards bed bugs might be due to two major properties: (i) they are saturated compounds (contain carbon-carbon single bonds outside the benzene ring) and (ii) the presence of functional hydroxyl groups on the benzene ring22,25. These structural properties may also have allowed thymol and carvacrol to penetrate rapidly through the cuticle, undergo slow detoxification and interact effectively with their target sites22,25,40. The lipophilicity of essential oil compounds is another important property that plays a role in penetration through the insect cuticle22. The LogP or octanol-water partition coefficients (higher values indicate greater lipophilicity)25 for carvacrol were higher than thymol (Table S1). Similarly, the LogP coefficient for the third most toxic compound in topical assays (citronellic acid) was higher than the LogP coefficient for eugenol (Table S1). In previous studies, citronellic acid and eugenol have been shown to possess contact toxicity against Muscadomestica and Tetranychus urticae22,28.
When considering the fumigant toxicity of essential oil constituents, thymol was most potent, followed by carvacrol, linalool, and (±)-camphor (Table 2). As stated in the previous paragraph, thymol and carvacrol have contact and fumigant toxicity against several insect species22-26. Fumigant effects of linalool have been demonstrated against Thrips palmi, Plutella xylostella and Blattellagermanica27,29,30. Whereas, (±)-camphor was reported as having contact and fumigant action against the P. xylostella30, but not against stored product pests31.
Determination of 24 h evaporation levels for essential oil constituents revealed large variations among compounds. The amount of initially applied chemical that evaporated during the 24 h bioassay period ranged from <0.5% for trans-cinnamaldehyde to 100% for eucalyptol (Table 2). A series of regression analyses conducted between LC
Several essential oil-based products have already been commercialized, especially for bed bug control. However, of the nine different natural compound products, only EcoRaider
Electrophysiology recordings were performed using the suction electrode technique to investigate the effects of essential oil components on the bed bug nervous system. Four of the six most active components identified collectively from topical and fumigant bioassays impacted baseline electrical activity of the bed bug nervous system. The neurophysiology data for carvacrol, thymol, eugenol and linalool provides a basis for understanding their toxicity against bed bugs. Bifenthrin (a positive control insecticide used in this study) and other synthetic pyrethroids modify the gating characteristics of voltage-sensitive sodium channels that lead to a delay in their closure, and thereby cause a neuroexcitatory effect on the insect nervous system41. In this study, bifenthrin caused significant neuroexcitation of baseline nervous system activity. Effects of bifenthrin at the 10 µM concentration on the bed bug nervous system were similar with a study that employed the suction electrode electrophysiology technique against the mole crickets42. Both neuroexcitatory (linalool) and neuroinhibitory (carvacrol, thymol and eugenol) essential oil constituents were neurologically active at millimolar (mM) concentrations. The structural and chemical property differences between essential oil components and bifenthrin may have led to significant differences in toxicity at the nervous system level40. In this regard, higher lipophilicity of bifenthrin (LogP value of 6, Table S1) in comparison to that of essential oil constituents may allow bifenthrin to effectively penetrate and interact with the membrane bound target site(s) within the nervous system at micromolar concentrations. Overall, low potency of neurological effects caused by essential oil compounds is consistent with their relatively lower topical and fumigant toxicity to different insect pest species and bed bugs. The effective concentration range or quantity of essential oil components (2 to 4 mM or 1.5 × 10
Neurological impacts of essential oil components against bed bugs were concentration-dependent for most test compounds (P < 0.05; Fig. S2). Similarly, Price and Berry38 found concentration-dependent neurological effects of essential oil components on the ventral nerve cord of P. americana and B. discoidalis. The effective concentration ranges for essential oil constituents tested in this study were similar to those of Price and Berry for citral, eugenol and geraniol38. The neurological impacts of eugenol and (±)-camphor were not concentration-dependent and showed a biphasic effect in our study (Figs 1 and S2). A previous study also revealed biphasic effects of geraniol on cockroach nervous system activity38.
The three compounds that produced neuroinhibition were carvacrol, thymol and eugenol. Based on in vitro studies, carvacrol is known to inhibit M. domestica nAChRs37 and its inhibitory activity was similar to dinotefuran (a neonicotinoid insecticide)43. In vertebrates, carvacrol can reversibly block the excitability of the rat sciatic nerve in a dose-dependent pattern44. However, in previous studies with insects, tyramine receptor36, transient receptor potential-like (TRPL) channels45 and GABA46 were also proposed as potential target sites for carvacrol. Thymol has been shown to bind Drosophilamelanogaster, mouse and human GABA receptors35,47,48. It was also reported as a weak inhibitor of the acetylcholinesterase enzyme49,50. Eugenol, which is a phenolic compound, was previously reported to have neuroinhibitory effects on P. americana and B. discoidalis38 and it was proposed to bind or interact with octopamine receptors in the insect nervous system34,51.
Linalool produced neuroexcitatory effects on the bed bug nervous system (Fig. 1e). Linalool was initially reported to act as a reversible competitive inhibitor of the acetylcholinesterase enzyme52. However, in subsequent studies, it was concluded that linalool does not bind to neurotransmitter enzymes53,54. It also did not produce any effect on house fly [
Bed bugs treated with the six most toxic plant essential oil components showed a range of poisoning symptoms such as hyperactivity, paralysis and leg tremors. Previously, Coats et al.57 reported hyperactivity and leg tremors as common poisoning symptoms associated with essential oil constituents. In the Madagascar cockroach (Gromphadorhina portentosa), pulegone-1,2-epoxide (an essential oil component) caused hyperactivity and muscular spasms before eventual paralysis and death58. In general, neuroinhibitory insecticides (e.g., oxadiazines and avermectins) are known to cause flaccid paralysis, wherein the muscles become limp and are unable to contract due to reduction or loss of nerve activity40,59. In contrast, rigid paralysis is caused by neuroexcitatory insecticides (e.g. organophosphates, pyrethroids, neonicotinoids)40,59. Rigid paralysis occurs because of the overstimulation of nervous system activity that causes muscles to stay in a contracted state. However, such symptoms were not visually distinguishable in bed bugs treated with neuroinhibitory (carvacrol, thymol and eugenol) or neuroexcitatory (linalool) essential oil components.
In summary, baseline toxicity of essential oil components against bed bugs as reported here provides information for development of natural product insecticides that can be used in bed bug IPM. Electrophysiology data for the most active compounds from bioassays further verifies that certain essential oil constituents affect the normal functioning of the bed bug nervous system. Collectively, these results provide insights required for identifying the target or binding sites and mode-of-action of specific essential oil constituents.
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, 5
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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 pests22-31. 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).
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.
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:%Evaporation=Amountevaporated(W1-W2)Amountapplied(W1-W0)×100
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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 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
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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.
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
Probit analysis was performed on dose-mortality and concentration-mortality data from topical application and fumigant exposure bioassays to calculate LD
We appreciate Drs Grzegorz Buczkowski and Wei Zheng fromPurdue University for their constructive feedback on this work. We thank Drs Jeff Holland (Purdue University) and Anup Amatya (New Mexico State University) for their assistance with statistical analysis of electrophysiology data. We like to thank Aaron Ashbrook, Cecilia Foley and Dr. Mahsa Fardisi and Dr. Suraj Amatya from Purdue University for technical support. We also like to thank John Lyon Obermeyer for taking photos of a dissected bed bug and its ganglion. S. G. was partially supported by the Osmun Student Innovation Funds and academic scholarships from Oser Family, BASF, Pi Chi Omega and National Conference on Urban Entomology. Research funding support was provided by the Center for Urban and Industrial Pest Management (A.D.G) and the O.W. Rollins/Orkin Endowment (M.E.S).
S.G. and A.D.G. conceived the research. S.G., A.D.G. and M.E.S. designed the experiments. S.G. performed all experiments and analyzed the data. S.G. and A.D.G. wrote the manuscript. All the authors reviewed and revised the manuscript.
The authors declare no competing interests.
Supplementary information accompanies this paper at 10.1038/s41598-019-40275-5.
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By Sudip Gaire; Michael E. Scharf and Ameya D. Gondhalekar