Insects
Bed bugs were reared in plastic jars (30 mm id) covered by nylon netting (0.5 mm) and placed in an incubator (KB8400 FL, Termaks, Bergen, Norway) under a 12 h:12 h light:dark cycle at 25°C and 70% humidity, as previously described [14]. Bed bugs were fed every 1-2 weeks on defibrinated chicken blood using an in vitro feeding system as described by [18]. After the blood meal, last instar nymphs were isolated individually until they moulted as adults, to ensure virginity of the experimental individuals.
For single sensillum recordings, one-to-three weeks old unmated and unfed adults were used in order to pinpoint possible sexual dimorphism in ORN responses. For behavioural experiments, large nymphs and unmated females were fed on chicken blood for 10 min prior to each observation, whereas males had been unfed for 1-2 weeks.
Single sensillum recordings
For single sensillum recordings, a bed bug was dorsally restrained under a Nikon Eclipse (E600-FN) microscope, which allowed us to identify individual sensilla at high magnification (×750), in order to test all the different functional types previously characterized by [14]. Using a piezoelectric micromanipulator (DC-3K, Märzhauser Wetzlar, Berlin, Germany), an electrolytically sharpened tungsten microelectrode was introduced into the shaft or base of a sensillum and the reference tungsten electrode was inserted into the head capsule through the neck region. The recording electrode was connected to a preamplifier (×10, Syntech, Kirchzarten, Germany) and the electrical signals were fed through an analogue-digital signal converter (IDAC-4, Syntech) and then visualized and recorded on a computer using the Autospike (v3.3, Syntech, Germany) software.
The insect antenna was placed in a humidified charcoal-filtered air stream delivered at 1 m/s via a glass tube (6 mm id), and a stimulus controller (CS-02, Syntech) permitted us to divert a 2 mL/s airflow through a Pasteur pipette, containing a stimulus, for 500 ms. The interval between each stimulation lasted until the neuronal activity reached the prestimulus frequency level.
The headspace collection of four large nymphs (Leidtke et al. unpublished data) was puffed over the antenna in order to measure the detection of the nymph-emitted volatiles. As the nymph head space collections were prepared in hexane, this solvent was used as a control during these experiments. Synthetic chemicals, (E)-2-hexenal (98%, Aldrich), (E)-2-octenal (>94%, Sigma), 4-oxo-(E)-2-hexenal and 4-oxo-(E)-2-octenal (96% and 98%, respectively, synthesized and supplied by E Hedenström, Mid Sweden University), were dissolved in paraffin oil (Merck, Darmstadt, Germany). During dose-response experiments, all compounds were tested in a series from the lower to the higher dilution and paraffin oil was used as a control.
Behavioural experiments
Male mounting of nymphs
In order to test differences in male attraction to females and nymphs, a recently fed female or late instar nymph was carefully placed into the centre of an arena (glass Petri dish, 50 mm diameter), with filter paper (renewed after each experiment) covering the base to facilitate the bed bugs' movement. A randomly selected unfed virgin male was then introduced and the behaviour of the couple was monitored for 5 min. The male mounting and dismounting behaviours were observed and timed to one tenth of a second, using a customized data logger application written in Delphi 6.0. All mounted individuals were inspected at the end of the experiment for the presence of ejaculate in the abdomen as a proof of a mating resulting from a successful mounting. All the behavioural experiments were carried out at room temperature in dimmed light conditions.
Removal of nymph pheromone
When testing the effect of nymph odour on male mounting behaviour, large nymphs were randomly divided into three treatment groups: silenced nymphs, sham nymphs and control. The silenced nymphs' dorsal abdomen was carefully coated with nail polish in order to cover their dorsal abdominal glands, as described in [10], whereas sham nymphs had the sides of their abdomen painted. The last group of nymphs, as well as a group of females, were not manipulated. Observations were conducted as described above and individuals whose natural behaviour was altered by the painting were discarded.
Addition of alarm pheromone
Prior to the observation, the methathoracic glands of an unfed female were covered by nail polish to prevent emission of alarm pheromones. In order to mimic a male/nymph interaction, odours were mechanically puffed onto a male/female pair as soon as the male mounted the silenced female. The couple was exposed to a single puff of stimulus delivered via a Pasteur pipette equipped with a 5 mL rubber bulb as a manual release system. The Pasteur pipette contained a filter paper strip (~5 × 15 mm) impregnated with 10 μL of a stimulus solution. The chemicals used in the behavioural experiments were diluted singly in hexane (99%), down to a 100 ng/μL level (10-4 dilution step). The (E)-2-hexenal: (E)-2-octenal blend was then formulated to obtain three different ratios: a female ratio of 5:4, a male ratio of 1:1 and a nymph ratio of 2:5 [Liedtke et al., unpublished data]. The three ratios of aldehydes, the oxo-aldehyde and a hexane control were tested on four different groups of male/female pairs, and observations were conducted in the same manner as described above.
Analysis
Single sensillum recordings
The antennal olfactory system of bed bugs is composed of nine grooved peg sensilla, six smooth peg sensilla and 29 trichoid sensilla [19]. As bed bug sensilla contain a high number of ORNs, we were unable to differentiate single ORNs using the shape and amplitude of their spikes (action potentials) (Figure 2c) [14]. For this reason, the total number of all spikes was manually counted for each recording, allowing us to assess the overall ORN activity elicited by stimulation within a specific sensillum. The net number of spikes per second (number of spikes 500 ms before stimulus onset subtracted from the number of spikes 500 ms after stimulus onset and then multiplied by two) in response to the blank were subtracted from the net number of spikes in response to stimuli. In addition, the mean temporal pattern of response was assessed by counting the total number of spikes within 250 ms duration bins. For this, the mean spike frequency of the eight first bins before stimulation (2 s) was calculated and was subtracted from each bin in order to obtain the net firing rate change per bin.
For response analysis, the Mann Whitney U-test was used to compare the response to test stimuli and solvent (Figure 2a and 2b). The same test was used for temporal response analysis, in which firing rate changes to the test stimulus were compared bin by bin (250 ms interval) to firing rate changes to the control stimulus (Figure 2c). All statistical analysis was performed with SPSS software (release 17.0, SPSS, USA).
Behavioural experiments
The number of trials with mountings were added and divided by the total number of trials to give the mounting frequency. The percentage of successful mountings (matings) was calculated by dividing the number of mated partners by the number of mounted partners.
The Kruskal Wallis test was applied to compare general differences in sample groups larger than two. It was followed by the Mann Whitney U-test in order to statistically compare the proportions between two samples. The time of mounting was log transformed in order to reach normality (Kolmogorov Smirnov test) and as the variance was statistically similar between the two groups (Levene test), one way analysis of variance was then applied. All the statistical analyses were performed with SPSS software (release 17.0, SPSS, USA).