Chemoreception to aggregation pheromones in the common bed bug, Cimex lectularius

Accepted Manuscript Chemoreception to aggregation pheromones in the common bed bug, Cimex lectularius Feng Liu, Caixing Xiong, Nannan Liu PII:

S0965-1748(17)30020-6

DOI:

10.1016/j.ibmb.2017.01.012

Reference:

IB 2924

To appear in:

Insect Biochemistry and Molecular Biology

Received Date: 7 October 2016 Revised Date:

30 January 2017

Accepted Date: 31 January 2017

Please cite this article as: Liu, F., Xiong, C., Liu, N., Chemoreception to aggregation pheromones in the common bed bug, Cimex lectularius, Insect Biochemistry and Molecular Biology (2017), doi: 10.1016/ j.ibmb.2017.01.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Prepared for Publication in

Address Correspondence to:

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Insect Biochemistry and Molecular Biology

Dr. Nannan Liu Department of Entomology & Plant Pathology

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301 Funchess Hall, Auburn University

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Auburn, AL 36849-5413, USA

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Phone: [001] (334) 844-2661;

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Fax: [001] (334) 844-5005

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E-mail: [email protected]

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Chemoreception to Aggregation Pheromones in the Common Bed Bug, Cimex lectularius

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Abbreviated title: Chemoreception to Aggregation Pheromones in the Common Bed Bug

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Feng Liu1, Caixing Xiong1, Nannan Liu*

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Department of Entomology and Plant Pathology, Auburn University, Auburn, AL 36849, USA

Corresponding author, FL and CX share the first author for this article

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Abstract

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The common bed bug, Cimex lectularius, is an obligate blood-feeding insect that is resurgent

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worldwide, posing a threat to human beings through its biting nuisance and disease transmission.

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Bed bug aggregation pheromone is considered a very promising attractant for use in the

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monitoring and management of bed bugs, but as yet little is known regarding the sensory

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physiology of bed bugs related to this pheromone. This study examined how the individual

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components of aggregation pheromone are perceived by the olfactory receptor neurons (ORNs)

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housed in different types of olfactory sensilla in bed bugs and the molecular basis for the ORNs’

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responses to the aggregation pheromone. We found that the ORNs in the D olfactory sensilla

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played a predominant role in detecting all the components of aggregation pheromone except for

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histamine, which was only recognized by the C sensilla. Bed bugs’ E sensilla, which include four

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functionally distinct groups, showed only a very weak but variant sensitivity (both excitatory and

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inhibitory) to the components of aggregation pheromone. Functional tests of 15 odorant

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receptors (ORs) in response to the components of aggregation pheromone revealed that most of

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these components were encoded by multiple ORs with various tuning properties. This study

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provides a comprehensive understanding of how bed bug aggregation pheromone is perceived

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and recognized in the peripheral olfactory system and will contribute useful information to

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support the development of synthetic attractants for bed bug monitoring and control.

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Keywords: bed bug, olfaction, olfactory sensillum, olfactory receptor neuron, odorant receptor

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1. Introduction

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As one of the most notorious pests affecting public health, infestations of the common bed bug

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(Cimex lectularius) are responsible for a number of adverse effects, including severe dermal

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reactions, emotional distress, paranoia and disease transmission (Sansom et al., 1992; Alexander,

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1994; Anderson and Leffler 2008; Salazar et al., 2014). Although chemical insecticide is still the

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most efficient method for the management of bed bugs, the development of insecticide resistance

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in these insects is beginning to be widely reported for many of the major insecticides commonly

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applied in the field such as pyrethroids (Moore and Miller, 2006; Romero et al., 2007),

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weakening the insect control effort considerably. The rise in second-hand furniture transactions,

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the increasing popularity of international travel, changes in pest-control tactics and the lack of an

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effective monitoring tool have also contributed to the resurgence of bed bugs right across the

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developed world (Doggett et al., 2004; Haynes and Potter, 2013). In particular, the lack of a

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suitable monitoring tool makes the task of dealing with infestations both harder and more

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complicated.

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In an effort to develop more effective strategies for monitoring bed bugs, bed bug

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aggregation pheromone has been the focus of many investigations for over 60 years, even though

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its exact components remain unclear. Initial research on the bed bug aggregation pheromone

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found that bed bugs aggregate within refugia and return to these harborages guided by a specific

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‘nest odor’ (Marx, 1955). This ‘nest odor’, which has been collected from experimental bed bug

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harborages, was identified as bed bug airborne aggregation pheromone based on a gas

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chromatography-mass spectrometry (GC-MS) analysis and dual-choice, still-air olfactometer

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bioassay (Siljander et al., 2008). Ten odors with >100 pg abundance in this airborne aggregation

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pheromone, namely nonanal, decanal, (E)-2-hexenal, (E)-2-octenal, (2E,4E)-octadienal,

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benzaldehyde, (+)- and (−)-limonene, sulcatone and benzyl alcohol, have proven to be essential

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in attracting adult male, virgin adult female, and juvenile bed bugs (Siljander et al., 2008).

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However, a more recent study has reported bed bug aggregation pheromone isolated from bed

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bug exuviae and feces to consist primarily of five volatile components (dimethyl disulfide,

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dimethyl trisulfide, (E)-2-hexenal, (E)-2-octenal, 2-hexanone) and one less-volatile component

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(histamine) (Gries et al., 2015); these five volatile components were found to be responsible for

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attracting bed bugs to safe shelters, while the less-volatile component caused bed bug arrestment

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upon contact. This dramatic variation in the components of bed bug aggregation pheromone may

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be partially due to the different sources (bed bug harborage air versus bed bug exuviae and feces),

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from which the pheromone was extracted. Nevertheless, two compounds, namely (E)-2-hexenal

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and (E)-2-octenal, were identified in both studies (Siljander et al., 2008; Gries et al., 2015),

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suggesting their important roles in eliciting aggregation behavior.

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Considering the biological importance of aggregation pheromone to bed bugs and the

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high potency of aggregation pheromone for attracting these insects, it would be meaningful to

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elucidate the sensory process of aggregation pheromone in the bed bug olfactory system,

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especially the responses from olfactory receptor neurons (ORNs) and odorant receptors (ORs).

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Therefore, in an effort to better understand the reception process of bed bugs for the aggregation

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pheromone, we conducted a systematic investigation of the neuronal responses of both males and

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female bed bugs to an odorant panel composed of all the components of bed bug aggregation

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pheromones identified in previous studies. We then went on to test the sensitivity of 15

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previously characterized bed bug ORs (OR1, 5, 9b, 11, 12, 15, 17, 19, 20, 21, 36, 37, 42, 46, and

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47) to all these components, which enhanced our understanding of the molecular basis by which

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these pheromone components are perceived by the bed bug peripheral olfactory system.

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2. Materials and Methods

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2.1 Insects and chemicals

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The C. lectularius colony originated from Ft. Dix, New Jersey, USA (Bartley and Harlan, 1974)

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and is susceptible to pyrethroid insecticides (Romero et al., 2007). All the bed bugs were reared

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at 25±2°C under a photoperiod of 12:12 (L: D). Rabbit blood (Hemostat Inc, CA) was used to

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feed bed bugs in the lab. All components of bed bug aggregation pheromones were commercially

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purchased with high purity (Table 1). In addition, three long-chain human odorants, 1-

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tetradecene, lauryl chloride, and N-pentadecanoic acid, were used for functional classification of

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E sensilla in this study (Liu and Liu, 2015). 1-tetradecene was purchased from Aldrich with 97%

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of purity. Lauryl chloride was purchased from Acros Organics with 99% of purity and N-

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pentadecanoic acid was from Sigma with 99% of purity. Each of the compounds was diluted in

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dimethyl sulfoxide (DMSO) to create stock solutions at a dose of 10 fold dilution (v/v for liquid

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or w/v for solid, Harraca et al., 2010) and stored at 4ºC. Subsequent decadic dilutions (10-fold

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dilutions) were made from these stock solutions.

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2.2 Single sensillum recording

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Adult bed bugs were randomly selected at least five days after blood feeding. The bed bugs

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(male or female) were anaesthetized (2-3 min on ice) and mounted on a microscope slide (76×26

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mm) between 2 pieces of double-sided tape. Using double-sided tape, the antennae were fixed to

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a cover slip resting on a small ball of dental wax to facilitate manipulation. The cover slip was

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placed at an appropriate angle to the bed bug’s head. Once mounted, the bed bug was placed

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under a LEICA Z6 APO microscope in such a way as to ensure that the antennae were visible at

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high magnification (×720). Two tungsten microelectrodes were sharpened in 10% KNO2 at 5V to

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a ~1 µm tip diameter. The reference electrode, which was connected to ground, was inserted into

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the abdomen of the bed bug and the other electrode, which was connected to a preamplifier (10×,

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Syntech, Kirchzarten, Germany), was inserted into the shaft of the sensillum to complete the

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electrical circuit and extracellularly record the olfactory receptor neuron potentials (Den Otter et

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al. 1980). Controlled manipulation of the electrodes was performed using two micromanipulators

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(Leica, Germany). The preamplifier was connected to an analog to digital signal converter

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(IDAC, Syntech, Germany), which in turn was connected to a computer for signal recording and

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visualization. Signals were recorded for ten seconds starting one second before stimulation. As

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there are a high number of ORNs co-located in each sensillum type, we did not attempt to

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calculate the firing rate for each ORN within the same sensillum. Instead, the total numbers of

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action potentials were counted off-line for a 500 ms period before and after stimulation for the

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whole sensillum. The number of action potentials after stimulation was subtracted from the

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number of action potentials before stimulation and multiplied by two in order to quantify the

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change in the firing rate in the sensillum in spikes per second.

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2.3 Stimulation and stimuli

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Ten microliters of the diluted solutions of the individual chemical components of the aggregation

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pheromone were dispensed onto a filter paper (40×3 mm) that was then inserted into a Pasteur

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pipette to create the stimulus cartridges for each component. Cartridges containing only the

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solvent (DMSO) were used as the controls. A constant airflow across the antennae was

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maintained at 20 ml/s throughout the experiment. Humidified air was delivered to the preparation

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through a glass tube (10-mm inner diameter). The glass tube was perforated by a small hole,

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slightly larger than the tip of the Pasteur pipette, 10 cm from the end of the tube. Stimulation was

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achieved by inserting the tip of the stimulus cartridge into the hole in the glass tube. A stimulus

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controller (Syntech, Germany) diverted a portion of the air stream (0.5 l/min) to flow through the

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stimulus cartridge for 0.5 sec, thus delivering the stimulus to the sensilla. The distance between

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the end of the glass tube and the antennae was ≤1 cm. At least six replicates for each of the

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different stimuli were conducted on different individuals. The values of the spikes were obtained

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by averaging all the recordings for the response of each sensillum to each chemical. The dose-

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response data were analyzed using GraphPad Prism 5.0 (GraphPad Software Inc, CA).

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2.4 Expression of bed bug odorant receptors (ORs) in the Xenopus oocyte system and two-

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electrode, voltage-clamp electrophysiological recordings

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The entire coding region for each of the bed bug odorant receptors (OR1, 5, 9b, 11, 12, 15, 17,

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19, 20, 21, 36, 37, 42, 46, and 47) and the co-receptor (ORCO) were amplified using primers

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with a cutting site added. The purified PCR products were cut with NotI-HF/NheI-HF (New

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England Biolabs, MA) and then cloned into pT7Ts vector (a gift from Dr. Wang at the Institute

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of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China), with a Kozak

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sequence added behind the cutting site in the forward primer. The constructed vectors were

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linearized with specific restriction enzyme and the cRNAs synthesized from linearized vectors

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with mMESSAGE mMACHINE T7 (Ambion, Carlsbad, CA). Mature healthy oocytes (stage V–

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VII) (Nasco, Salida, CA) were harvested from the African Clawed Frog (Xenopus laevis) and

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treated with collagenase I (GIBCO, Carlsbad, CA) in washing buffer (96 mM NaCl, 2 mM KCl,

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5 mM MgCl2, and 5 mM HEPES [pH = 7.6]) for about 1 h at room temperature. After being

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cultured overnight at 18°C, oocytes were microinjected with 10 ng cRNAs of both the ORs and

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ORCO. After injection, the oocytes were incubated for 4-7 days at 18°C in 1X Ringer’s solution

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(96 mM NaCl, 2 mM KCl, 5 mM MgCl2, 0.8 mM CaCl2, and 5 mM HEPES [pH = 7.6])

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supplemented with 5% dialyzed horse serum, 50 mg/ml tetracycline, 100 mg/ml streptomycin

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and 550 mg/ml sodium pyruvate. Whole-cell currents were recorded from the injected Xenopus

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oocytes with a two-electrode voltage clamp. Odorant-induced currents were recorded with an

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OC-725C oocyte clamp (Warner Instruments, Hamden, CT) at a holding potential of −80 mV.

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Odorants (components of bed bug aggregation pheromone) were dissolved in DMSO at a 1:10

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ratio to make stock solutions that were later diluted in 1× Ringer’s solution to the indicated

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concentrations. Data acquisition and analysis were carried out with Digidata 1440A and

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pCLAMP 10.2 software (Axon Instruments Inc., CA). All these ORs were challenged with

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individual components of the bed bug aggregation pheromone at a concentration of 1:104 v/v (or

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w/v for histamine). For those ORs showing a strong response to certain components at high

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concentration (104-fold dilution), their responses to a serial dilution were further investigated and

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the EC50 values (effective concentration for reaching 50% of the maximum response) calculated.

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2.5 Statistical analysis

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An unpaired t test was applied to compare the responses from male and female bed bugs; P <

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0.05 was considered a significant difference. Hierarchical cluster analysis for classification of the

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E sensilla using Ward’s method based on the Euclidian distance was performed using PASW

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Statistic 18 (IBM, NY).

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3. Results

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3.1 Neuronal responses of olfactory sensilla to individual aggregation pheromone

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components

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Previous studies have shown that odorants in different chemical classes tend to evoke variant

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responses from olfactory sensilla of diverse morphological types and that D sensilla are more

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broadly tuned than either C or E sensilla (Liu and Liu, 2015). To investigate the role of each of

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these types of olfactory sensilla in perceiving aggregation pheromone, we tested the 14 potential

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components of aggregation pheromone identified in the previous studies (Siljander et al., 2008;

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Gries et al., 2015; Table 1) on 35 sensilla, 80% of the total olfactory sensilla, from both male and

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female bed bugs.

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Generally, we found that different components presented quite different activities in activating the D olfactory sensilla (Dα, Dβ, Dγ) (Fig. 1). The saturated aldehydes (nonanal and

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decanal) elicited particularly strong responses on all three types of D sensilla, with firing

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frequencies of ≥100 spikes/s. For the unsaturated aldehydes, (2E, 4E)-octadienal evoked much

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stronger responses from Dα and Dβ than Dγ sensilla and (E)-2-octenal was more effective in

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activating Dβ and Dγ than Dα sensilla, while (E)-2-hexenal triggered responses much more

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efficiently from Dα than Dβ and Dγ sensilla. As to the ketones, Dβ and Dγ sensilla were much

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more sensitive to 2-hexanone than Dα sensilla, while Dγ sensilla showed much stronger

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responses to sulcatone than either Dα or Dβ sensilla. For the aromatics (benzal and benzyl

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alcohol), only a very weak response was elicited from the D sensilla, with benzal evoking

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slightly larger responses than benzyl alcohol in Dβ and Dγ than in Dα sensilla. Both the terpenes

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(R-(+)-limonene and S-(-)-limonene) showed high efficiency in activating Dγ but not the Dα and

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Dβ sensilla. For the only amine (histamine) component in the pheromone, almost no response

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was generated from any of the D sensilla types. However, of the two C sensilla tested, the C1

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sensilla were sensitized to several of these components, including histamine, (2E, 4E)-octadienal,

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(E)-2-hexenal and benzaldehyde, while the C2 sensilla were exclusively sensitive to histamine

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(Fig. 1). The E sensilla tested generally showed only very weak responses (<50 spikes/s) to the

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components, with some displaying inhibitory activity to components such as sulcatone, dimethyl

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trisulfide and dimethyl disulfide.

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3.2 No sexual dimorphism between the responses to individual aggregation pheromone

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components

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To determine whether there are any differences between the male and female bed bugs’ olfactory

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responses to individual components of the bed bug aggregation pheromone, we compared the

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neuronal responses of their olfactory sensilla. The C and D sensilla were chosen for this

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comparison since both types of olfactory sensilla showed high efficiency in perceiving certain

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components in the aggregation pheromone, specifically the components listed in Table 2. Those

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component-sensillum combinations that presented a strong response (≥50 spikes/s) in either sex

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were used for the statistical analysis (Table 2). The results revealed no significant differences in

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the olfactory responses of male and female bed bugs for any of the potential components of bed

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bug aggregation pheromone tested, which supports a previous study that found no sexual

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dimorphism between male and female bed bugs’ responses to a panel of 29 compounds (Harraca

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et al., 2010). Therefore, unless stated otherwise, for the remainder of this study only males were

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used in the experiments with adult bed bugs.

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3.3 Functional classification of E sensilla in the adult bed bug antennae

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The hair-like E sensillum, which is the most abundant type of olfactory sensillum, possesses

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about 60% (27 out of 44) of the total number of olfactory sensilla on the last flagellum of a bed

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bug antenna. Previous researchers have distinguished these morphologically into two groups (E1

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and E2) (Levinson et al., 1974; Harraca et al., 2010), although it seems likely that this is an

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oversimplification of a complex situation. As an alternative way to categorize bed bugs’ E

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sensilla, we opted to functionally classify all 27 E sensilla based on their responses to the 14

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possible components of aggregation pheromone plus three long-chain human odorants (1-

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tetradecene, lauryl chloride, and N-pentadecanoic acid) that were used to distinguish E1 and E2

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sensilla in a previous study (Liu and Liu, 2015). Given the consistency of the sensilla locations

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on the antennae, we labeled and tested each individual E sensillum for all 17 potential

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aggregation pheromone components or human odorants.

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In our recordings for 27 E sensilla and the panel of 17 odorants, the E sensilla generally exhibited sparse spontaneous firing rates and weak neuronal responses, with the highest

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excitatory response being 32 spikes/s (Fig. 2A). Different response patterns were observed

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among individual E sensilla when they were challenged by the same panel of odorants. To

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examine the relationships and potential interactions between the E sensilla, hierarchical cluster

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analyses were carried out based on the Euclidean distance between each pair of E sensilla with

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different responses to the odorants. The resulting dendrogram is shown in Fig. 2B. Four clusters

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of sensilla, designated EI, EII, EIII and EIV, were particularly well represented. Sensilla within

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the same group had similar response patterns to the odorants tested, while sensilla falling into

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different groups demonstrated distinctly different patterns of responses. For example, the EIV

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group consisting of E5, E18 and E21, which had regular and relatively high spontaneous firing

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rates, exhibited inhibitory effects for most of the potential components of the aggregation

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pheromone tested but not to the three human odorants (Fig. 3D). Another distinct group, EIII,

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which included 7 E sensilla at different locations along the antennae, that was sensitive to human

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odorants, had previously been classified as E2 sensilla as they presented irregular spontaneous

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firing rates (Fig. 3C) (Liu and Liu, 2015). The EII group also included 7 E sensilla and typically

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showed a small excitatory response to some of the potential bed bug aggregation pheromone

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components, namely decanal, nonanal and sulcatone (1:100 v/v), suggesting that this group of

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sensilla is involved in the detection of pheromone-related odorants (Fig. 3B). The largest group

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E1, consisting of 10 sensilla, showed no significant (excitatory or inhibitory) response to any of

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the 17 odorants tested (Fig. 3A).

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We updated the sensilla distribution map proposed by Steinbrecht and Müller (1976) by labelling the position of each functional type of E sensilla on the antenna (Fig. 4). The number

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and positions of the sensilla were relatively consistent between individual male or female adult

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bed bugs, with no sexual dimorphism (Levinson et al., 1974). The different types of olfactory

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sensilla were widely dispersed and intermingled with each other, particularly the E sensilla. No

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specific spatial aggregation of functionally-assembled olfactory sensillum was observed, in

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strong contrast to the functional aggregation of olfactory sensilla on the antennae of the fruit fly

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(Drosophila melanogaster) (De Bruyne et al., 2001).

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3.4 Dose-dependent sensillum responses

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To clarify the effect of compound dosage in triggering neuronal responses from different

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olfactory sensilla, components showing high activity in evoking responses at a dose of 1:104 v/v

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were selected. In general, both excitatory and inhibitory responses followed a dose-dependent

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pattern (Fig. 5). For instance, as the histamine dose rose from 1:105 v/v to 1:10 v/v, the

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excitatory response of the C2 sensillum increased dramatically, rising from 8 ± 4 to 184 ± 8

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spikes/s. Similarly, the inhibitory effect was enhanced on the E5 sensillum when the dose of

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sulcatone was increased from 1:105 v/v to 1:10 v/v. Different components also displayed

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different efficiencies in eliciting a neuronal response from the same olfactory sensillum. For

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example, (E)-2-hexenal had a threshold that was 10-fold lower than that of (2E,4E)-octadienal

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and nonanal when evoking 50 spikes/s on the Dα sensillum; dimethyl trisulfide had a threshold

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that was 10-fold lower than that of (E)-2-octenal when triggering 50 spikes/s on the Dβ sensillum;

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and sulcatone had a threshold that was almost 100-fold lower than R-(+)-limonene in activating

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the ORNs in the Dγ sensillum.

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3.5 Temporal dynamics of the olfactory responses

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Numerous studies have indicated that the temporal dynamics of neuronal responses play a

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critical role in the odorant coding process within the insect peripheral olfactory system (De

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Bruyne et al., 2001; Liu and Liu, 2015; Ye et al., 2016). In this study, we characterized the

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temporal dynamics of the responses of D sensilla (Dα, Dβ, and Dγ) to several components of the

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bed bug’s aggregation pheromone. Different components were found to elicit neuronal responses

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with very diverse temporal dynamics (Fig. 6), with (2E, 4E)-octadienal, (E)-2-hexenal and

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nonanal generating a typical tonic neuronal response with a prolonged ending on the Dα

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sensillum, while (E)-2-octenal and dimethyl trisulfide activated a typical phasic neuronal

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response with an abrupt ending on the Dβ sensillum. However, for the Dγ sensillum, R-(+)-

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limonene and S-(-)-limonene triggered phasic responses whereas sulcatone elicited a tonic

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response.

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Temporal dynamics can also represent the kinetic structure of the binding affinity of an

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odorant to ORNs. Both (2E, 4E)-octadienal and (E)-2-hexenal evoked rapid increases in firing

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rates in Dα sensilla, which may indicate high binding affinities to one or more of the ORNs

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housed in this sensillum (Fig. 6A). On the other hand, other components like nonanal, dimethyl

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trisulfide and sulcatone evoked slowly increasing slopes in the Dα, Dβ and Dγ sensilla (Fig. 6A-

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C), respectively, suggesting relatively weak binding affinities to the ORNs. The temporal dynamics of neuronal responses not only give information on the odorants’

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qualities but also suggest the quantity of odorants in the environment (Liu and Liu, 2015). In this

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study, low doses of (E)-2-hexenal (1:105 to 1:103 v/v) were more likely to initiate phasic

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neuronal responses with a fast recovery after excitation, while high doses of (E)-2-hexenal (1:102

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to 1:10 v/v) tended to trigger tonic neuronal responses that were prolonged over several seconds

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after the stimulus application (Fig. 6D). Similarly, as the doses of histamine increased, the

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temporal dynamics became more and more tonic (Fig. 6E).

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3.6 Responses of bed bug odorant receptors (ORs) to the components of aggregation

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pheromone

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Insect odorant receptors are known to be responsible for their neuronal responses to odorants,

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including pheromones (Carey and Carlson, 2011; Leal, 2014). In a previous study, we

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characterized 15 bed bug ORs that presented a variety of sensitivities to odorants of different

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chemical classes (Liu et al., submitted for review). In order to reveal the molecular basis of the

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bed bug’s neuronal responses to the components of bed bug aggregation pheromone, we tested

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the electrophysiological responses of these 15 ORs to 14 chemical components of bed bug

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aggregation pheromone. In general, different ORs displayed different response profiles to the

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components of aggregation pheromone, with some showing very strong current responses while

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others produced barely any response (Fig. 7). OR1, 5, 20, 21, 36, 42 and 47 all elicited responses

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of more than 250 nA from at least one component; and OR9b, 12, 15, 19 and 37 generated

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responses of more than 100 nA from at least one component; while OR11, 17 and 46 produced

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only very weak responses to all the components of bed bug aggregation pheromones tested.

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Components from different chemical classes demonstrated very different activities in activating

318

the bed bug ORs. For instance, ORs 5, 9b and 21 were very sensitive to the two saturated

319

aldehydes (nonanal and decanal), while the unsaturated aldehydes ((2E, 4E)-octadienal, (E)-2-

320

hexenal and (E)-2-octenal) stimulated only a mild response from OR15. The two aromatics

321

(benzaldehyde and benzyl alcohol) elicited very strong responses from OR1 and OR42, along

322

with mild responses from OR37 and OR47, with benzaldehyde generally being more effective

323

than benzyl alcohol in most of the ORs. For the ketones, although 2-hexanone was much more

324

efficient in evoking responses from ORs 19, 36, 37, and 42 than sulcatone, sulcatone showed

325

greater efficiency in evoking responses from OR12 than 2-hexanone; both 2-hexanone and

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sulcatone evoked comparable strong responses from OR1 and OR20. As for the sulfides

327

(dimethyl disulfide and dimethyl trisulfide), both elicited very strong responses from ORs 1 and

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47, and mild responses from ORs 20 and 42. The terpenes (R-(+)-limonene and S-(-)-limonene)

329

elicited very strong responses from ORs 20 and 36. The only amine, histamine, which functions

330

in the bed bug’s arresting behavior (Gries et al., 2015), showed no activity on any of the tested

331

ORs. Previous studies also indicated that amines were recognized by the ionotropic receptors

332

(IRs) responsible for detecting polar molecules such as amines and acids (Spletter and Luo, 2009;

333

Abuin et al., 2011; Missbach et al., 2014; Joseph and Carlson, 2015). The bed bugs’ grooved peg

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C sensilla also shared similar cuticle and pore structure with the coeloconic sensilla of other

335

insects (Levinson et al., 1974), which have been shown to express IRs in Drosophila

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melanogaster (Spletter and Luo, 2009; Abuin et al., 2011). Therefore, given that the results

337

reported here showed amines to be exclusively detected by the C sensilla, which agrees with the

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findings of a previous study (Liu and Liu, 2015), we propose that IRs may be expressed in the

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neurons housed in the C type olfactory sensilla of bed bugs. To examine the effect of dosage on the responses of ORs to the various components of

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the aggregation pheromone, we tested the responses for doses ranging either from 1:108 to 1:104

342

or from 1:107 to 1:103 v/v. The resulting dose-response curves revealed that some ORs are

343

extremely sensitive to certain pheromone components (Fig. 8). For instance, OR19 showed a

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dose-dependent response to 2-hexanone with a EC50 value of 1.3x10-6 fold dilution; OR20 had a

345

dose-dependent response to both sulcatone and S-(-)-limonene, with EC50 values of 8.6x10-7 and

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3.159x10-6 fold dilution, respectively; and OR21 showed very strong sensitivity to decanal, with

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an EC50 value of 4.3x10-6 fold dilution.

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4. Discussion

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In this study, we characterized the olfactory responses of adult bed bugs to all the components of

351

aggregation pheromone so far identified. No sexual dimorphism was observed in the neuronal

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responses of male and female bed bugs to aggregation pheromones, which is consistent with the

353

behavioral responses of both bed bug sexes to the aggregation pheromone reported in

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olfactometer bioassays (Siljander et al., 2008; Gries et al., 2015). These unbiased responses of

355

both male and female bed bugs to aggregation pheromones may facilitate mate-finding for adults,

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which is extremely important as no sex pheromone has yet been identified in bed bugs. In

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addition, aggregated bed bugs within refugia may also benefit individuals by marking harborages

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with suitable microclimates, thus reducing the risk of desiccation for both male and females

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(Benoit et al. 2007).

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In testing the neuronal responses of the E sensilla to different components of the aggregation pheromone, dramatically different results were obtained for different groups of E

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sensilla, with EII showing only a small excitatory responses to most components while EIV

363

displayed remarkable inhibitory responses to most of the components tested, especially sulcatone,

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dimethyl disulfide and dimethyl trisulfide at high doses. The majority of these components also

365

elicited very strong responses in the D sensilla of bed bugs. This striking difference in the

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sensitivity of different olfactory sensilla to the same components suggests that bed bugs may

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apply a ‘push and pull’ strategy in responding to these components, where certain sensilla are

368

activated while others are inhibited. A similar phenomenon has also been reported in both

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Drosophila and mosquitoes, where the same odorants were found to activate certain ORs while

370

inhibiting others (Hallem and Carlson, 2006; Carey et al., 2010). For bed bugs, the inhibitory

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effect of specific components was enhanced as the doses increased, which may help reduce the

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input noise or balance the neuronal activities/excitation at different doses, as suggested in the

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studies by Zhu et al. (2013) and Hong and Wilson (2013).

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In characterizing the responses of ORs to the possible components of bed bug

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aggregation pheromone, we confirmed that most of these components were indeed capable of

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activating single or multiple ORs among the 15 ORs tested, with several ORs showing extreme

377

sensitivity to certain components. In particular, sulcatone activated OR20 with an EC50 value in

378

the nanomolar range, suggesting that sulcatone may be one of the cognate ligands for OR20. We

379

also found that none of the three unsaturated aldehydes were very effective in evoking responses

380

from ORs, even though they elicited very strong responses from the ORNs. This may be due to

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the limited number (15) of ORs investigated for this study, which included only about 1/3 of the

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total number of bed bug ORs. Further research focusing on characterizing the remaining ORs’

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responses to these components could provide more nuanced and detailed pictures regarding how

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these components of aggregation pheromones are recognized in the bed bug peripheral olfactory

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system. Interestingly, structure-based activity was demonstrated in the responses of both ORNs

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and ORs to these components of the aggregation pheromone. For example, two stereoisomers of

388

limonene (R-(+)-limonene and S-(-)-limonene) that showed very similar activities in eliciting

389

responses from ORs also elicited very consistent responses from bed bug ORNs in the olfactory

390

sensilla, which supports our previous finding that bed bugs present analogous responses to

391

certain stereoisomers of terpenes or terpenoids (Liu et al., 2014). Similarly, ORNs and ORs

392

followed similar patterns in response to the two aromatic components of the aggregation

393

pheromone, benzaldehyde and benzyl alcohol, albeit with benzaldehyde showing much higher

394

efficiency in activating the ORs (e.g. OR1, OR37 and OR42) compared to benzyl alcohol,

395

probably due to their different binding affinity with the ORs. Although other factors (e.g. odorant

396

binding proteins or odorant-degrading enzymes) may also be involved in determining the

397

responses of ORNs to benzaldehyde and benzyl alcohol, it does appear that ORs alone have a

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huge impact on the responses from ORNs. Further studies that determine the crystal structures of

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odorant receptors would dramatically enhance our understanding of the interactions between

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chemical stimuli and odorant receptors.

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Acknowledgement

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The project described was supported by Award AAES Hatch/Multistate Grants ALA08-045,

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ALA015-1-10026, and ALA015-1-16009 to N.L.

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Authors' contributions

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Conceived and designed the study: NL and FL. Performed the experiments: CX, FL. Prepared

408

the materials: NL. Wrote the paper: NL, FL CX.

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References

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Abuin, L., Bargeton, B., Ulbrich, M.H., Isacoff, E.Y., Kellenberger, S., Benton, R., 2011.

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Functional architecture of olfactory ionotropic glutamate receptors. Neuron 69, 44-60.

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Alexander, J.O., 1994. Infestation by hemiptera. In: Arthropods and the human skin. London:

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Springer Verlag, 57-74.

Anderson, A.L., Leffler, K., 2008. Bed bug infestations in the news: a picture of an emerging

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public health problem in the United States. J. Environ. Health 70, 24-27. Benoit, J.B., Del Grosso, N.A., Yoder, J.A., Denlinger, D.L., 2007. Resistance to dehydration between bouts of blood feeding in the bed bug, Cimex lectularius, is enhanced by water

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conservation, aggregation, and quiescence. Am. J. Trop. Med. Hyg. 76, 987-993.

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Bartley, J., Harlan, H., 1974. Bed bug infestation: its control and management. Mil. Med. 139,

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Carey, A.F., Carlson, J.R., 2011. Insect olfaction from model system to disease control. Proc. Natl. Acad. Sci. U.S.A. 108, 12987-12995. Carey, A.F., Wang, G., Su, C.Y., Zwiebel, L.J. Carlson, J.R. 2010. Odorant reception in the malaria mosquito Anopheles gambiae. Nature 464, 66-71.

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Den Otter, C.J., Behan, M., Maes, F.W., 1980. Single cell response in female Pieris brassicae

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(Lepidoptera: Pieridae) to plant volatiles and conspecific egg odours. J. Insect Physiol.

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Doggett, S.L., Geary, M.J., Russell, R.C., 2004. The resurgence of bed bugs in Australia: with notes on their ecology and control. Environ. Health 4, 30-38.

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De Bruyne, M., Foster, K., Carlson, J.R., 2001. Odor coding in the Drosophila antenna. Neuron

Gries, R., Britton, R., Holmes, M., Zhai, H., Draper, J., Gries, G., 2015. Bed bug aggregation pheromone finally identified. Angew. Chemie. 127, 1151-1154.

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Hallem, E.A., Carlson, J.R. 2006. Coding of odors by a receptor repertoire. Cell 125, 143-160.

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Harraca, V., Ignell, R., Löfstedt, C., Ryne, C., 2010. Characterization of the antennal olfactory

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system of the bed bug (Cimex lectularius). Chem. Senses. 35, 195–204. Haynes, K.F., Potter, M.F., 2013. Recent progress in bed bug management. In Advanced

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Technologies for Managing Insect Pests, eds Ishaaya, I., Palli, S.R., Horowitz, A.R.

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(Springer, New York), pp 269-278.

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Hong, E.J., Wilson, R.I., 2013. Olfactory neuroscience: normalization is the norm. Curr. Biol. 23, R1091-R1093. Joseph, R.M., Carlson, J.R., 2015. Drosophila chemoreceptors: A molecular interface between

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the chemical world and the brain. Trends Genet. 31, 683-695.

Leal, W.S., 2014. Decipher the rosetta stone of insect chemical communication. Am. Entomol. 60, 223-230.

Levinson, H.Z., Levinson, A.R., MÜller, B., Steinbrecht, R.A., 1974. Structure of sensilla, olfactory perception, and behaviour of the bedbug, Cimex lectularius, in response to its

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alarm pheromone. J. Insect Physiol. 20, 1231-1248. Liu, F., Haynes, K.F., Appel, A.G., Liu, N., 2014. Antennal olfactory sensilla responses to insect chemical repellents in the common bed bug, Cimex lectularius. J. Chem. Ecol. 40, 522-

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Marx, R., 1955. Uber dieWirtsfindung und die Bedeutung des artspezifischen Duftstoffes bei Cimex lectularius Linné. Parasitol. Res. 17, 41-73.

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Missbach, C., Dweck, H.K., Vogel, H., Vilcinskas, A., Stensmyr, M.C., Hansson, B.S., Grosse-

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Liu, F., Liu, N., 2015. Human odorant reception in the common bed bug, Cimex lectularius. Sci.

Wilde, E., 2014. Evolution of insect olfactory receptors. eLife 3, e02115. Moore, D.J., Miller, D.M., 2006. Laboratory evaluations of insecticide product efficacy for control of Cimex lectularius. J. Econ. Entomol. 99, 2080-2086. Romero, A., Potter, M.F., Potter, D., Haynes, K.F., 2007. Insecticide resistance in the bed bug: a

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factor in the pest’s sudden resurgence? J. Med. Entomol. 44, 175–178. Salazar, R., Castillo-Neyra, R., Tustin, A.W., Borrini-Mayorí, K., Náquira, C., Levy, M.Z., 2014. Bed bugs (Cimex lectularius) as vectors of Trypanosoma cruzi. Am. J. Trop. Med. Hyg.

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Sansom, J.E., Reynolds, N.J., Peachey, R.D., 1992. Delayed reaction to bed bug bites. Arch.

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Siljander, E., Gries R., Khaskin G., Gries G., 2008. Identification of the airborne aggregation pheromone of the common bed bug, Cimex lectularius. J. Chem. Ecol. 34, 708-718.

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Spletter, M.L., Luo, L., 2009. A new family of odorant receptors in Drosophila. Cell 136, 23-25.

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Steinbrecht, R.A., Müller, B., 1976. Fine structure of the antennal receptors of the bed bug,

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Cimex lectularius L. Tissue Cell 8, 615-636. Ye, Z., Liu, F., Liu, N., 2016. Olfactory responses of southern house mosquito, Culex quinquefasciatus, to human odorants. Chem. Senses, bjv089. Zhu, P., Frank, T., Friedrich, R.W., 2013. Equalization of odor representations by a network of

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electrically coupled inhibitory interneurons. Nat. Neurosci. 16, 1678-1686.

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Figure legends

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Figure 1. Olfactory response of smooth and grooved peg (D and C) sensilla of adult bed bugs to

482

components of aggregation pheromones. Each dot represents the response (mean ± SEM, n≥6) of

483

olfactory sensilla to specific components (1:100 v/v). DMSO was used as the control.

484

Figure 2. Functional classification of E sensilla. A) Heat map of olfactory responses of ORNs

485

housed in E sensilla in response to 17 odorants (14 components from aggregation pheromone and

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3 components from human emanations) (1:100 v/v). Means of response spikes are represented

487

by gradient colors, with red representing excitatory responses and green inhibitory responses. X

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axis: 17 odorants; Y axis: 27 E sensilla. B) Dendrogram of clustered E sensilla based on their

489

responses to the odorant panels. Hierarchical cluster analysis was performed using Ward’s

490

method, based on the Euclidean distance.

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Figure 3. Representative responses of four functional types of E sensilla. Column graphs on the

492

left show mean responses to 17 odorants (1:100 v/v). Signal traces on the right are the

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representative responses of each function group of E sensilla to certain odorants, with DMSO as

494

the control. A) EI group (Mean±SEM, n=8); B) EII group (Mean±SEM, n=6); C) EIII group

495

(Mean±SEM, n=9); D) EIV group (Mean±SEM, n=6).

496

Figure 4. The distribution of different functional types of olfactory sensilla on the distal part of

497

the second flagellum of the bed bug antenna, modified from the sensilla distribution map

498

proposed by Steinbrecht and Müller (1976). Relative positions of sensilla are consistent with

499

their real positions on the antenna. Arrows with different colors represent different functional

500

types of E sensilla based on the hierarchical cluster analysis. C and D sensilla are represented by

501

circles and triangles, respectively.

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Figure 5. Dose-dependent responses of different types of olfactory sensilla to the components of

503

the aggregation pheromone. A) Dose-response curves of the Dα sensillum to (2E,4E)-octadienal,

504

(E)-2-hexenal and nonanal; B) Dose-response curves of the Dβ sensillum to dimethyl trisulfide

505

and (E)-2-octenal; C) Dose-response curves of the Dγ sensillum to sulcatone, decanal, R-(+)-

506

limonene, S-(-)-limonene and dimethyl disulfide; D) Dose-response curves of the C1 sensillum

507

to (E)-2-hexenal and histamine ; E) Dose-response curve of the C2 sensillum to histamine; F)

508

Dose-response curve of the E5 sensillum to sulcatone. X axis shows the logarithm of the dosage

509

series, exponentially increasing from 10 -5 to 10 -1 v/v. Sensilla responses are represented as

510

mean ± SEM spike/s (n ≥ 3). The dose-response curves were fitted with the Sigmoidal dose-

511

response model with variable slope using Graphpad Prism 5.

512

Figure 6. Temporal dynamics of D and C sensilla in response to components of the aggregation

513

pheromone. The recording was initiated from 0s when stimulus (1: 100 v/v) was delivered for

514

0.5 s. Firing frequency (mean ± SEM spikes/0.1s, n=6) of the olfactory sensillum in 0.1s

515

intervals was consecutively calculated until t=2.2 s. A) Temporal structure of responses from Dα

516

sensilla when challenged by (2E,4E)-octadienal, (E)-2-hexenal and nonanal with a dose of 1:102

517

v/v; B) Temporal structure of responses in Dβ sensilla when challenged by 2-hexanone, (E)-2-

518

octenal, and dimethyl trisulfide with a dose of 1:102 v/v; C) Temporal structure of responses in

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Dγ sensilla when challenged by R-(+)-limonene, S-(-)-limonene and sulcatone with a dose of

520

1:102 v/v; D) Temporal structure of responses from C1 sensilla when challenged by (E)-2-

521

hexenal at a dose series from 1:105 to 1:10 v/v; E) Temporal structure of responses from C2

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sensilla when challenged by histamine at a dose series from 1:105 to 1:10 v/v.

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Figure 7. Current responses of odorant receptors to the components of the bed bug aggregation

524

pheromone. Fifteen odorant receptors were co-expressed with ORCO in the Xenopus oocyte and

525

challenged with all 14 potential components of the aggregation pheromone. Each column

526

represents the response (mean ± SEM, n≥3) of the odorant receptors to a specific component

527

(1:104 v/v) dissolved in 0.1% DMSO Ringer’s solution.

528

Figure 8. Dose-dependent responses of odorant receptors to the components of the bed bug

529

aggregation pheromone. Ten odorant receptors showing strong current response (≥100 nA) to at

530

least one component at a dose of 1:104 v/v were challenged with serial doses over the range from

531

1:109 to 1:103 v/v. Responses of the odorant receptors to these components are represented as

532

mean ± SEM spike/s (n ≥ 3). The dose-response curve was fit with the Sigmoidal dose-response

533

model with variable slope using Graphpad Prism 5.

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Table 1. Potential chemical components of bed bug aggregation pheromone tested in this study Chemicals

CAS

Purity (%)a

Company

Referencesb

Amine

Histamine

51-45-6

≥ 97

Sigma

2

Sulfide

Dimethyl disulfide

624-92-0

99

Sigma-Aldrich

2

Dimethyl trisulfide

3658-80-8

≥ 98

Sigma-Aldrich

2

(2E,4E)-octadienal

5577-44-6

95

Sigma-Aldrich

1

(E)-2-octenal

2548-87-0

≥ 94

Sigma-Aldrich

1, 2

(E)-2-hexenal

6728-26-3

99

nonanal

124-19-6

95

decanal

112-31-2

Benzaldehyde Alcohol Ketone

Terpene

SC Acros

1, 2

Aldrich

1

95

Acros

1

100-52-7

98

Alfa Aesar

1

Benzyl alcohol

100-51-6

≥ 98

Flasher Science

1

2-hexanone

591-78-6

98

Alfa Aesar

2

Sulcatone

110-93-0

≥ 98

Sigma

1

R-(+)-limonene

5989-27-5

97

Sigma

1

S-(+)-limonene

5989-54-8

96

Sigma

1

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a

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(DMSO) at a dose of 1:10 (v/v for liquid or w/v for solid) as stock dilution.

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b

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pheromone; 1) Siljander et al. (2009); 2) Gries et al. (2015).

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Purities of chemical compounds were higher than 94%; chemicals were dissolved in dimethyl sulfoxide

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Table 2. Unpaired t-test of olfactory responses between male and female bed bugs Unpaired t-test results of D sensilla Males: Means ± SEM (spikes/s)

Females: Means ± SEM (spikes/s)

(2E,4E)-octadienal

189 ± 15.8 N=6

148 ± 11.4 N=8

0.0647

(E)-2-hexenal

196 ± 17.2 N=13

250 ± 10.8 N=6

Decanal Nonanal

127 ± 14.4 N=13 190 ± 10.7 N=13

82.7 ± 14.4 N=6 194 ± 17.0 N=4

0.0555 0.0764

(2E,4E)-octadienal

114 ± 10.2 N=12

131 ± 21.7 N=6

(E)-2-octenal Decanal

136 ± 12.6 N=12 118 ± 13.4 N=12

113 ± 19.0 N=9 116 ± 18.8 N=7

Dγ

C1

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178.8 ± 11.05 N=12

171.7 ± 19.82 N=6

0.7359

116.6 ± 10.76 N=7

122.4 ± 7.837 N=7

0.6677

Dimethyl trisulfide

56.7 ± 12.0 N=9

78.5 ± 28.3 N=4

0.4123

(E)-2-octenal

85.6 ± 10.4 N=14

94.6 ± 12.1 N=10

0.5777

Sulcatone

156 ± 11.6 N=16

145 ± 23.5 N=7

Decanal Nonanal

141 ± 15.4 N=14 181 ± 13.3 N=14

128 ± 17.7 N=10 163 ± 25.3 N=10

0.633 0.5834

S-(-)-limonene

125 ± 11.1 N=13

137 ± 11.9 N=10

R-(+)-limonene Dimethyl trisulfide

120 ± 10.5 N=13 84.3 ± 9.58 N=12

144 ± 12.0 N=10 73.2 ± 8.56 N=12

(E)-2-hexenal Histamine

0.5194 0.4642 0.1363 0.3977

54.3 ± 7.05 N=6

68.0 ± 15.6 N=4

0.3932

152 ± 18.3 N=9

116 ± 28.1 N=6

0.2797

99.7 ± 10.0 N=7

96.2 ± 10.7 N=5

0.8186

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a

546

to test for sexual dimorphism.

547

b

548

considered to indicate no statistically significant difference.

chemicals eliciting strong responses (≥50 spikes/s) to certain types of sensilla in both sexes were chosen

P value of unpaired t-test. P < 0.05 was considered to show significant difference. P ≥ 0.05 was

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0.8392

Nonanal

Histamine C2

P value b

2-hexanone

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1. Diverse types of olfactory sensillum showed distinctive response profiles to the components of bed bug aggregation pheromone. 2. No differences in sensing these components were observed between male and female bed bugs. 3. Multiple functional types of olfactory sensillum were characterized based on their responses to individual pheromone components. 4. Most of the aggregation pheromone components were encoded by multiple odorant receptors (OR).