DOI: 10.5061/dryad.nzs7h4529
title: Data from: Bed bug preferences for host odor or aggregation odor are differentially modulated by physiological state in various odorscapes

description: Bed bugs live in an ecologically and spatially restricted indoor habitat
 comprised of overlapping aggregation and host odors, and they traverse
 relatively short distances between blood-hosts and aggregation sites.
 Although many studies demonstrated aggregation or host odor preference
 respectively, the modulation of their preferences between these divergent
 odors is poorly understood. Given the recurrent transitions of bed bugs
 between replete and hungry states, we evaluated the effects of six
 odorscapes containing aggregation and host skin odors on bed bug
 preferences. Hunger state modulated odor preference for aggregation and
 foraging in all tested odorscapes. Aggregation odor attracted both fed and
 unfed bed bugs. Host skin odor attracted unfed bed bugs but repelled
 recently fed bed bugs and the addition of carbon dioxide to host odor
 enhanced the behavioral responses. These findings suggest that orientation
 to aggregation sites in fed bed bugs is driven by two distinct odor
 processing mechanisms for attractant and aversive odors. Unfed bed bugs
 discriminate two attractive odors – aggregation and host odors – but host
 odor predominates over aggregation odor in driving their orientation
 behavior. Understanding the dynamic switching of odor preferences during
 the blood digestion cycle will guide the implementation of chemical lures
 in IPM. Host odors alone and their co-emission with aggregation pheromone
 repelled fed bed bugs from traps. Conversely, unfed bed bugs had a strong
 preference for host odor emitted either alone or with aggregation odors.
 Therefore, the independent use of either host or aggregation odor lures
 and their co-emission from the same trap should be carefully considered.
description: <strong>2          MATERIALS AND
 METHODS</strong> <strong>2.1      
 Insects</strong> The tested laboratory strain
 (Harold Harlan strain) was collected in 1973 in Fort Dix NJ, and
 maintained on a human host until December 2008. Then, in our laboratory,
 it was fed on defibrinated rabbit blood until July 2021 and on human blood
 thereafter. It was maintained at 35–45% relative humidity, 25 °C, on a
 12:12 (L:D) h cycle and fed weekly on heparinized human blood (supplied by
 the American Red Cross under IRB #00000288 and protocol #2018-026). We
 used an artificial feeding system, which has been previously described.
 The feeding system was housed in a North Carolina State
 University-approved BSL-2 facility (Biological Use Authorization
 #2020-09-836). Between feeding sessions, the glass feeders were sanitized
 with 7.5% sodium hypochlorite and 95% ethanol, and air-dried. Since we
 conducted 24 hr-observations in an open arena, only adult males (age
 unknown) were used in this study in case of accidental escape. Within 24
 hrs post feeding, fully engorged males were separated from colony jars
 into groups of 100. Each group was kept in a new rearing jar (5 cm
 diameter x 4.5 cm height) with two clean filter paper shelters (4 x 9 cm)
 using #1 Whatman filter papers (Whatman, Madistone, United Kingdom) for
 2-, 4-, 6-, 8-, 10-, 12- or 14-days. Tested males were discarded after a
 single bioassay. <strong>2.2       Open arena
 bioassay design</strong> Arena bioassays
 evaluated the preferences of bed bugs for human skin odor and aggregation
 odor. The bottom of a plastic arena (50L x 40W x 15H cm, without a lid,
 #2565519, Project Source, Baton Rouge, LA) was lined with disposable
 absorbent liner (AL2050, Jaece Industries, North Tonawanda, NY), secured
 with masking tape (2020-48TP6, 3M Scotch, Maplewood, MN), and replaced
 after each trial. A pitfall shelter was placed on each side of the arena,
 35 cm apart (Fig. 1). Pitfall shelters were made as follows: Commercial
 disposable bathroom paper cups (88.7 mL, 5 cm diameter x 5.6 cm height)
 were placed below the arena and attached to the disposable absorbent liner
 so there was no gap between the cup and the liner (Fig. 1). A filter paper
 with a test odor (see below) was placed in each cup and the cup was
 lightly covered with clean filter papers during the assay to create a dark
 shelter within the cup. Under the dark conditions in the scotophase, the
 shelter did not appear to serve as a visual landmark, but it could
 potentially be visually detected by bed bugs during the photophase.
 Importantly, the shelters did not serve as traps and therefore we refer to
 them as pitfall shelters rather than pitfall traps; the vertical test
 filter paper touched the horizontal cover filter paper, so the tested bed
 bugs could move freely between the two shelters during each bioassay.
 However, we saw no evidence of bed bugs leaving a shelter after they
 entered it. <strong>2.3       Preparation of odor
 sources</strong> Bed bug-conditioned filter
 papers, human skin swabs and clean filter papers were used as the
 aggregation odor, host skin odor and no-odor sources, respectively. We did
 not carry out chemical analyses because we tested natural blends of skin
 and aggregation odors. For preparing the aggregation odor, two filter
 papers, each 4 x 9 cm made from #1 Whatman filter papers, were placed in a
 jar with 100 freshly fed males for 14 days. The filter papers served as
 substrate and shelter on which the bed bugs defecated. Because we used
 three arenas for each treatment, the two conditioned filter papers were
 cut into pieces, and an equal amount of filter paper was placed in each
 pitfall shelter. Thus, a single pitfall shelter contained 24
 cm<sup>2</sup> of bed bug-conditioned filter paper,
 representing approximately 33-male-equivalents of aggregation
 odor. For preparing the host odor, human skin was
 swabbed with two filter papers (#1 Whatman) following a previously
 validated protocol with IRB approval for recruitment, informed consent,
 and odor sample collections granted from North Carolina State University,
 Raleigh, NC (IRB Approval #14173). A single adult male participant
 (&gt;21 yr old) provided informed consent. Prior to collecting skin
 swabs, he was instructed to: 1) not eat ‘spicy’ food at least 24 h before
 collecting a skin swab; 2) take a morning shower; 3) not use a deodorant
 or cosmetics/lotions on the sampled surfaces; 4) not exercise or perform
 any strenuous physical activity; and 5) take the skin swabs 4–8 h after
 showering. The participant was then provided with #1 Whatman filter papers
 and glass vials (20 ml) and asked to collect skin swabs as follows: 1)
 rinse hands with water and dry before use; 2) use a single filter paper
 and swab the left arm from hand to armpit for 12 s using both sides of the
 filter paper; 3) rub the left leg from the lower thigh to ankle for 12 s
 using both sides of the filter paper; 4) rub the left armpit for 6 s using
 both sides of the filter paper; 5) place the filter paper into a glass
 vial and label the vial; and 6) repeat with a new filter paper swabbing
 the right side of the body. The papers were kept at –30
 <sup>o</sup>C and tested within 24 hrs after collection. Three
 skin swab papers were cut into pieces, which were equally distributed
 among three pitfall shelters in three replicates. Thus, a single pitfall
 shelter contained the equivalent of a single filter paper swabbed on a
 human subject for 30 sec. In <em>Experiments</em>
 <em>4</em> and <em>5</em> (see below), one pitfall
 shelter contained both aggregation and host odors represented by separate
 filter papers for aggregation odor and host odor.
 <strong>2.4       Bioassay
 observations</strong> The bioassay room was kept
 at 25 °C, 30–40% RH, and a photoperiod of 12 hrs dark (08:00 to 20:00) and
 12 hrs light (20:00 to 08:00). These environmental conditions were the
 same as in the rearing environment. Bioassays were conducted for 24 hrs
 starting at 09:00. Twenty-five males (ages unknown) were placed under an
 inverted cup in the center of the arena (release point, Fig. 1) and
 allowed to acclimate for 3 min. The cup was removed, and the positions of
 all bed bugs (the two pitfall shelters and arena floor) were recorded at
 0, 1, 3, 6, 12 and 24 hrs (09:00, 10:00, 12:00, 15:00, 21:00 and 09:00).
 Throughout, the human observer wore a face mask and gloves to minimize bed
 bug exposure to human breath and skin odors, including
 CO<sub>2</sub>. Red light was used for observation during the
 scotophase. To avoid odor contamination among different treatments in the
 bioassay room, only one type of treatment (experiment) was conducted using
 three bioassay arenas on the same day.
 <strong>2.5       Experimental
 design</strong> We generated six types of
 odorscapes for six experiments, with three arenas used as three replicates
 in each experiment (Table 1). <em>Experiment
 1.</em> Both pitfall shelters had the same odor sources (either
 aggregation odor or host (skin) odor) to evaluate the symmetry of the
 bioassay environment. Two-days post feeding (Fed) and 14-days post feeding
 (Unfed) adult males were bioassayed.
 <em>Experiment 2.</em> A narrow odorscape in which
 bed bugs were given a choice between a single odor source and no odor:
 either aggregation or host odor vs. clean filter paper. An odor source was
 placed in one pitfall shelter and clean filter paper in the other shelter.
 Two-days post feeding (Fed) and 14-days post feeding (Unfed) adult males
 were bioassayed. <em>Experiment 3.</em> A
 broader odorscape in which bed bugs were given a choice between two
 distinct odor sources: aggregation odor vs. host odor. To evaluate the
 effect of hunger state on odor preference, separate cohorts of bed bugs
 were assayed 2-, 4-, 6-, 8-, 10-, 12- and 14-days post feeding.
 <em>Experiment 4.</em> A complex odorscape in which
 bed bugs could choose between two admixed odor sources and a single odor
 source: a combination of aggregation and host odors in one pitfall shelter
 vs. host odor in the other shelter. Two-days post feeding (Fed) and
 14-days post feeding (Unfed) adult males were bioassayed.
 <em>Experiment 5</em>. In an inverse experiment to
 <em>Experiment 4</em>, a complex odorscape was tested in which
 bed bugs were given a choice between two admixed odor sources and a single
 odor source: a combination of aggregation and host odors in one pitfall
 shelter vs. aggregation odor in the other shelter. Two-days post feeding
 (Fed) and 14-days post feeding (Unfed) adult males were
 bioassayed. <em>Experiment 6</em>. To add
 complexity to the odorscape, <em>Experiment 5</em> was
 repeated, but with CO<sub>2</sub> added to the combination of
 aggregation odor and host odor in one pitfall shelter vs. aggregation odor
 in the other shelter. Because CO<sub>2</sub> is an effective
 host cue in bed bugs, we added CO<sub>2</sub> to host odor to
 mimic more complex and realistic host cues. As shown in Fig. 1, in this
 experiment only, both pitfall shelters received clean air (Medical quality
 air, Airgas Healthcare, Radnor, PA) that was passed through a humidifying
 jar (100 mL/min for each shelter). Carbon dioxide (2% or 20,000 ppm,
 Airgas Healthcare, Radnor, PA) was added to the pitfall shelter that
 contained the aggregation and host skin odors. To avoid the accumulation
 of CO<sub>2</sub> in the arena, observation time was limited
 to 0, 1, 3 and 6 hrs, all in the scotophase.
 <strong>2.6       Statistical
 analysis</strong> Binary choice odor preferences
 across multiple treatment groups were tested by full factorial repeated
 measures ANOVA (<em>α</em> = 0.05) with Tukey’s HSD in JMP
 (Student edition 18, Cary, NC), which enabled multiple comparisons of main
 (fixed) effects including the number of bed bugs in each of the two
 pitfall shelters (Pitfall shelter main effect) across all time points
 (Time main effect) and the interaction of these two factors (Pitfall
 shelter * Time). Notably, in all except one experiment, there was a
 significant effect of Time (<em>P</em> ≤ 0.05). Therefore, in
 the main manuscript we report the <em>P</em>-values of only
 the main effect (Pitfall shelter). The complete test results
 (<em>F</em>-values and df) for both main effects (Pitfall
 shelter, Time) and their interaction (Pitfall shelter * Time) are reported
 in the Supplementary Material. When a significant interaction effect was
 detected, we implemented Tukey’s HSD post-hoc test, and significant
 differences between the two pitfall shelters by time
 (<em>P</em> ≤ 0.05) are indicated by an asterisk (*) on the
 figures and reported in the Supplementary Material. The
 effects of different odorscapes on odor preferences were compared using
 the numbers of bed bugs recorded in pitfall shelters at the 6, 12 and 24
 hr time points and analyzed them with one-way ANOVA and Tukey’s
 HSD.
description: # Data from: Bed bug preferences for host odor or aggregation odor are
 differentially modulated by physiological state in various odorscapes
 Dataset DOI: [10.5061/dryad.nzs7h4529](10.5061/dryad.nzs7h4529) ##
 Description of the data and file structure ### Files and variables ####
 File:
 DRYAD_Bed_bug_preferences_for_host_odor_or_aggregation_odor_are_differentially_modulated_by_physiological_state_in_various_odorscapes.xlsx **Description:** Data sats of the results of two-choice bioassays ##### Variables The file contains the seven sheets: * Read me, description of dataset contains Name of Sheets, Column, Entry, Row, and Explanation. * Experiment1, Tested insect post feeding (day), Number of Replication, Recorded site, The number of insects observed in recorded site was shown in 0, 1hr, 3hr, 6hr, 12hr, and 24hr of observation time point. * Experiment2, Tested insect post feeding (day), Number of Replication, Recorded site, The number of insects observed in recorded site was shown in 0, 1hr, 3hr, 6hr, 12hr, and 24hr of observation time point. * Experiment3, Tested insect post feeding (day), Number of Replication, Recorded site, The number of insects observed in recorded site was shown in 0, 1hr, 3hr, 6hr, 12hr, and 24hr of observation time point. * Experiment4, Tested insect post feeding (day), Number of Replication, Recorded site, The number of insects observed in recorded site was shown in 0, 1hr, 3hr, 6hr, 12hr, and 24hr of observation time point. * Experiment5, Tested insect post feeding (day), Number of Replication, Recorded site, The number of insects observed in recorded site was shown in 0, 1hr, 3hr, 6hr, 12hr, and 24hr of observation time point. * Experiment6, Tested insect post feeding (day), Number of Replication, Recorded site, The number of insects observed in recorded site was shown in 0, 1hr, 3hr, and 6hr of observation time point. ## Code/software N/A