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Rearing followed procedures categorical in Weeks et al.23,24. In brief, C. lectularius were acquired from a antecedents at the University of Sheffield (UK) and again reared at Rothamsted Research (UK). Bed bugs were reared in antecedents pots in a controlled ambiance at 25 °C ± 1.5 °C and 80% RH ± 5%. The antecedents was maintained in 60 × 40 mm artificial pots. A 20 mm bore aperture was fabricated in the centre of the artificial spiral top lid to admittance air exchange. A allotment of accomplished cobweb was captivated in abode amid the pot and the lid with a allotment of elastic, this prevented bed bugs from artifice and acceptable abatement of the lids to facilitate feeding. The ablaze administration was set to 14L:10D. Already per anniversary bed bugs were accustomed admission to heparinised sheep claret (TCS Biosciences, Botolph Claydon, UK) appliance bottle feeders60. These feeders were bankrupt afterwards use with balmy baptize with bactericide (1% Teepol 12–20 unperfumed detergent, Hertfordshire supplies, Welwyn Garden City, UK) followed by an acetone bathe afore actuality placed at 150 °C for at atomic 12 h.
Experimental insects, unless contrarily stated, were adults of both es that had been blood-fed 7 to 14 canicule previously. This date of bed bugs was easier to handle afterwards accident and were added alive in the amphitheatre than afresh fed bed bugs. Twelve hours afore use, beginning insects were transferred into a pot and confused into the behaviour allowance to acclimatise. Bed bugs were called about from antecedents pots, articular as adults and ed beneath a analytic microscope. Both males and females of C. lectularius were acclimated for behavioural assays testing airy extracts. However, as there was no cogent aberration in acknowledgment to the volatiles from bed bug-exposed affidavit amid the es in ahead appear experiments24, alone males of C. lectularius were acclimated to affirm the behavioural action of the constructed blends. All abstracts were conducted amid the years of 2007 to 2011.
Bed bug-exposed affidavit acclimated for airy abreast were clarify affidavit that had ahead been apparent to bed bugs. One hundred bed bugs of alloyed and date were put into artificial antecedents pots complete clarify affidavit (accordion folded, 70 × 40 mm, Whatman 125 mm, Whatman PLC, Maidstone, UK). Bed bugs acclimated the cardboard as a refuge; aggregating, defaecating and ovipositing in the folds. Afterwards one month, the bed bugs, eggs and exuviae were removed from the cardboard and the cardboard was activated for behavioural action appliance the still-air olfactometer declared by Weeks et al.24. Gloves were beat whilst administration the bed bug-exposed paper.
Volatiles from bed bug-exposed affidavit were abandoned by air entrainment. Ten bed bug-exposed clarify affidavit were independent in a bottle barge (250 mL). Ten unexposed clarify affidavit (accordion folded, 70 × 40 mm, Whatman 125 mm) were placed in a added barge as a clarify cardboard control. As a bare control, an entrainment additionally was completed of an abandoned bottle vessel. Air was pulled through a charcoal clarify (activated charcoal, 10–14 mesh, 50 g, BDH Actinic Ltd, Poole, UK) for purification, into the bottle accommodation and beyond the clarify cardboard at a breeze bulk of 600 mL/min. Air from anniversary barge was again pulled out into a Pyrex tube complete Porapak Q (50 mg, cobweb admeasurement 50/80, Supelco, Bellefonte, PA, USA), a absorptive polymer that accessories airy compounds, captivated in abode by two plugs of silanised bottle complete (Supelco, Bellefonte, PA, USA). Entrainment argosy were purged for 10 min above-mentioned to admittance of the Porapak tubes and again the entrainment was run for 7 days. Back the Porapak tubes were removed, they were eluted with 750 μL of afresh re-distilled diethyl ether into 1.1 mL acicular vials (Chromacol, Welwyn Garden City, UK). The vials were stored at −20 °C above-mentioned to analysis. The entrainment was again eight times. As anniversary entrainment independent ten papers, anniversary abstract independent the agnate of 1680 bed bug apparent cardboard hours or BBEXPH. As the extracts were 500 uL in aggregate they independent 3.36 BBEXPH per uL. Charcoal filters were bankrupt afore entrainment by adapter to a breeze of antiseptic nitrogen whilst actuality acrimonious in an oven (150 °C) for two hours. Porapak tubes were bankrupt by abrasion with dichloromethane (1 mL) followed by re-distilled diethyl ether (4 mL) and conditioned by adapter to a breeze of antiseptic nitrogen whilst in heating blocks (135 °C) for two hours. All glassware was done in balmy baptize with bactericide (1% Teepol 12–20 unperfumed detergent), again acetone and placed in an oven (150 °C) for two hours afore actuality used.
Extracts were analysed appliance a Hewlett Packard (HP-6890) GC with a non-polar (HP-1) and a arctic (DB-WAX) cavalcade (J & W Scientific, Folsom, CA, USA). HP-1 (50 m × 0.32 mm; blur thickness, 0.52 μm) and DB-WAX (30 m × 0.32 mm; blur thickness, 0.5 µm) columns were adapted with a cool-on-column (COC) injector, hydrogen carrier gas and a blaze ionisation detector (FID). The oven temperature was maintained at 30 °C for 0.5 min and again programmed at 5 °C/min to 150 °C, captivated for 0.1 min, again 10 °C/min to 230 °C and again captivated for 35 min.
Compounds aural the samples were quantified about by allegory with an bang (1 μL) of 100 ng/μL band-aid of n-alkanes (C7-C25) in hexane, and again concentrated to 50 µL so that the better aiguille was at about 100 ng/μL, appliance a affable breeze of antiseptic nitrogen afore actuality re-analysed. As anniversary abstract was concentrated 10-fold, from 500 uL to 50 uL, the extracts now independent 33.6 BBEXP per uL. Assimilation indices (RI) were affected afterward Bartle61. An aliquot (30 μL) of anniversary of the eight airy extracts were accumulated for added analysis.
The electrodes acclimated to accomplish recordings were Ag/AgCl affairs (diameter 0.37 mm; Harvard Apparatus, Edenbridge, UK) amid into bottle pipettes, fabricated from borosilicate bottle capillaries (outer bore 2.00 mm; close bore 1.16 mm; Harvard Apparatus, Edenbridge, UK). The bottle pipettes were abounding with Ringer’s band-aid (7.55 g NaCl, 0.64 g KCl, 0.22 g CaCl2, 1.73 g MgCl2, 0.86 g NaHCO3 and 0.61 g Na3PO4 per L water). Afore abutting the insect, the tips of the bottle pipettes/electrodes were put into acquaintance to affirm electrical connectivity. The bed bug was again algid on ice for one minute afore dissection. The arch was removed by an cavity through the pronotum acceptance the bust and belly to be discarded. The bill and one antenna were amputated to abate noise. One of the bottle pipettes was again amid into the aback of the head, through the pronotum, and the tip of the actual antenna was amid into the recording electrode that was already in position (Fig. 2, inset). Affairs were captivated in a continuous, humidified, charcoal filtered air breeze (1 L) advancing from a bottle tube, which was positioned 5 mm from the antennal preparation. Already the antennal alertness and EAG accessories were in position the microscope ablaze was switched off to abate noise.
The appraisal actuality in band-aid (1 µL), was activated to a clarify cardboard band (4 × 60 mm) appliance a disposable micropipette (Microcaps, Drummond Scientific Co., USA) at 100% or 33.6 BBEXPH. Afterwards 30 s, to acquiesce the bread-and-butter to evaporate, the clarify cardboard band was amid into a disposable bottle Pasteur pipette (150 mm, SLS Select Education, Nottingham, UK). Back closed this created a distinct use “odour cartridge”. A bang commitment accessory (Syntech Bang Air Controller CS-O5), which was controlled by a bottom pedal, generated a “puff” of air as the tip of the odour armament was amid into the air flow. For 2 s, air was anesthetized through the odour armament into the capital air flow, which again went over the antennal preparation. The bang commitment accessory ensured a connected air breeze by compensating for the air advancing through the pipette. The appliance of a 1 mV beating was performed at the alpha of anniversary agreement to acquiesce for arrangement of EAG responses. Amplification of the signals was set at ×10,000. Signals were analysed appliance the computer-based software package, EAG 2000 (Syntech, The Netherlands62). Controls consisted of a complete ascendancy of ammonia, activated as 35% ammonium hydroxide (FSA Laboratory Supplies, Loughborough, UK), and its agnate abrogating control, distilled water. An added abrogating ascendancy of re-distilled diethyl ether was all-important for allegory with extracts. Controls were completed at the alpha and end of anniversary agreement and at approved intervals throughout. The complete ascendancy (ammonia) and its abrogating ascendancy (water) were activated immediately, i.e. afterwards the 30 s dehydration period.
EAG responses of males and females of C. lectularius to the airy abstract from an air entrainment of bed bug-exposed affidavit were recorded to actuate EAG-activity of the extracts. Three males and three females were able as aloft and activated with a 1 μL of anniversary ascendancy (ammonia, distilled water, and re-distilled diethyl ether) and the airy extract. The antennae were angry already every two account for best antennal accretion and constancy (data not shown).
The still-air olfactometer declared by Weeks et al.24 was acclimated for the behavioural assays. Clean affection gloves were beat back administration olfactometers and added equipment. New pots and mesh, which were done afore use, were acclimated for anniversary replicate. All accessories was done with balmy baptize and bactericide (1% Teepol 12–20 unperfumed detergent), rinsed with 50% ethanol, followed by baptize and dried. New unexposed clarify affidavit were acclimated for anniversary replicate.
Data were recorded appliance the video recording equipment, accoutrement and software as declared in Weeks et al.24. EthoVision Adaptation 3.1 software (Noldus Information Technology, Wageningen, Netherlands) was acclimated to abduction video images to clue bed bugs during behavioural assays. Afterwards ambience up the equipment, an alone bed bug was alien into the centre of the amphitheatre appliance a accomplished acrylic brush. The continuance of the bioassay was 20 min.
The complete ascendancy consisted of an odour pot complete a distinct bed bug-exposed paper, which was able as declared above, compared to a no-odour pot complete an unexposed clarify cardboard (accordion folded, 70 × 40 mm, Whatman 125 mm). The abrogating ascendancy consisted of two no-odour pots, but the clarify cardboard was advised with re-distilled diethyl ether (10 µL). The airy extracts and constructed blends were removed from the freezer one hour above-mentioned to the aboriginal bioassay and were maintained at allowance temperature throughout the day. The airy abstract or constructed alloy (10 µL) was activated assimilate the unexposed clarify cardboard in the appointed odour pot and the agnate no-odour pot was advised with re-distilled diethyl ether (10 µL).
The position of the odour and no-odour pots was randomised amid replicates. For the complete control, the accessories was set up 20 min afore the alpha of the bioassay. Back testing the acknowledgment of bed bugs to solvent, airy extracts, and constructed blends, the pots were larboard for 30 s to acquiesce the bread-and-butter to evaporate, afore the olfactometer was assembled.
As the airy agreement appear by a bed bug-exposed cardboard during a behavioural appraisal has been begin to be adorable to bed bugs previously23,24, the bed bug-exposed paper hours (BBEXPH), which refers to the time of air entrainment, is reported. The 100% extract, which is agnate to 33.6 BBEXPH per uL, was adulterated in re-distilled diethyl ether to appraisal the acknowledgment of bed bugs to assorted concentrations. The appliance of 10 uL was taken into consideration. In behavioural appraisal 1, three concentrations were tested, 0.0675 (0.02%), 0.675 (0.2%), and 6.75 (2%) BBEXPH. In behavioural appraisal 2, two added concentrated solutions were activated alongside the 6.75 BBEXPH, 13.50 BBEXP (4%) and 27.00 BBEXP (8%) alongside the constructed blends. The solutions were stored in 1.1 mL acicular vials (Chromacol, Welwyn Garden City, UK) at −20 °C above-mentioned to use in bioassays.
The acknowledgment of bed bugs to altered concentrations of the airy abstract from bed bug-exposed affidavit was advised by behavioural assay. Assay (i.e. abrogating ascendancy (solvent), complete ascendancy (bed bug-exposed paper) and three airy abstract concentrations), , and odour pot position were advised as factors and randomised. In anniversary block (comprising one day) males and females of C. lectularius were activated with anniversary of the bristles treatments. Therefore, one block comprised 10 bioassays (n = 20). Acknowledgment variables were: cardinal of visits to pots and time spent in zones (see inset in Fig. 1). The bioassays were completed in the aboriginal scotophase, amid 10:00 and 15:00.
The GC acclimated was an Agilent-6890N, complete a HP-1 cavalcade (30 m × 0.32 mm; blur thickness, 0.52 μm; J & W Scientific, Folsom, CA, USA) with a COC injector, hydrogen carrier gas and an FID. The oven temperature was maintained at 40 °C for 2 min and again programmed at 5 °C/min to 100 °C and again at 10 °C/min to 250 °C. The end of the cavalcade was amid into the centre of the capital air breeze so that the eluent went over the antennal preparation. A acrimonious alteration band ensured that the compounds did not air-conditioned and abbreviate central the cavalcade already they larboard the GC oven. Amplification of the signals was set at ×10,000. Signals were analysed appliance the computer-based software package, EAD 2000 (Syntech, The Netherlands62). The airy abstract (1 μL, at 100% or 33.6 BBEXPH) from an air entrainment of bed bug-exposed affidavit was injected assimilate the column.
Ten macho affairs and ten changeable affairs for a complete of 20 GC-EAG runs were completed. GC-EAG-active peaks from the accompanying GC-EAG appraisal were bent by comparing traces visually on a ablaze box to bout agnate EAG responses to peaks. Responses were akin amid males and females alone assimilate adept traces for anniversary . Breadth there were bristles or added responses in the aforementioned abode on the electroantennograms, the agnate abode on the chromatogram was apparent and the assimilation time of the aiguille was recorded. Adept traces of males and females were again overlaid to analyze breadth responses occurred in both es. A band-aid of n-alkanes (C7-C25, 1 μL of 100 ng/μL) in hexane additionally was analyzed to account the assimilation indices (RI) of any GC-EAG-active peaks for allegory with GC and accompanying GC-MS traces61.
Volatile extracts (1 μL at 100% or 33.6 BBEXPH) from the bed bug-exposed cardboard and two ascendancy air entrainments were analysed by accompanying GC-MS, appliance a Micromass Autospec Ultima alluring breadth MS, accompanying to an Agilent 6980 GC able with a HP-1 capillary cavalcade (50 m × 0.32 mm; blur thickness, 0.52 µm; J & W Scientific, Folsom, CA, USA) and adapted with a COC injector. The carrier gas was helium. The oven temperature was maintained at 30 °C for 5 min, and again programmed at 5 °C/min to 250 °C. Ionization was by electron appulse at 70 eV, antecedent temperature 220 °C. Electrophysiologically-active peaks begin to be specific to airy extracts from bed bug-exposed papers, but absent from controls, were tentatively articular by allegory with MS databases (NIST, 2002) and accepted by co-injection for aiguille accessory on the two columns with anchored phases of differing polarity63. Already confirmed, assorted point alien standards were acclimated to quantify anniversary of the GC-EAG-active compounds articular from the air entrainment abstract of the bed bug-exposed cardboard (n = 3). Most of the articular compounds were purchased commercially from a array of sources (≥95% purity). Compounds that were not accessible commercially, i.e. β-phellandrene (90% purity), (S)-germacrene D (92% purity), and (3E,5E)-octadien-2-one (83% purity), were provided via aqueous chromatography/chemical synthesis. (R)-Germacrene D (11% by GC) was antiseptic from “gum haggar”, a gum adhesive from Commiphora holtziana (Engl., 1904)64, whilst (S)-germacrene D (92% pure) was antiseptic from a commercially accessible sample (RC Treatt, UK, 40% pure) by small-scale aqueous chromatography over silica abounding with argent nitrate. The two enantiomers of germacrene D were again accumulated in band-aid in according amounts for co-injection. 3,5-Octadien-2-one (40 mg, 83% authentic by GC) was actinic in one footfall starting from (E)-2-pentenal (Sigma-Aldrich, Dorset, UK, 95% pure)65. β-Phellandrene was acquired from a bartering antecedent (no best in production) as acclimated by Barata et al.66.
For behavioural appraisal 2, the absorption acknowledgment experiment, a 16-component constructed alloy (SB-16) was activated that independent the articular GC-EAG-active compounds except for those that bare to be actinic or antiseptic afore they could be quantified and acclimated in behavioural assays (Table 1). All compounds were acquired commercially and were added to the alloy in the amounts affected by the assorted point alien accepted adjustment (Table 1). Anniversary admixture was able in a 1 or 5 mg/mL banal band-aid and again added to a concentrated admixture in the actual ratios. Gas chromatography on HP-1 and DB-WAX columns (methods declared earlier) was acclimated to assay that the ratios of the chemicals in the constructed blends were authentic afore they were adulterated to 6.75 (2%), 13.50 (4%), and 27.00 (8%) BBEXPH. The constructed blends were adulterated in afresh re-distilled diethyl ether and stored in 1.1 mL acicular vials (Chromacol) at −20 °C above-mentioned to use in bioassays.
For behavioural appraisal 3, to analyze altered constructed blends, an 18-component (SB-18) and a aesthetic 6-component (SB-6) constructed alloy additionally were able (Table 1) and all three were activated at 27 BBEXPH (8%). SB-18 independent the 16 chemicals included in SB-16 and, additionally, the two chemicals that were provided via aqueous chromatography/chemical synthesis, (S)-germacrene D and (3E,5E)-octadien-2-one. SB-6 independent the six compounds that gave statistically cogent GC-EAG responses afterward the Sloane and Sullivan adjustment for automated detection67.
The acknowledgment of bed bugs to altered concentrations of SB-16 was advised by behavioural assay. The factors were assay (i.e. abrogating ascendancy (solvent), complete ascendancy (bed bug-exposed paper), three concentrations of the airy abstract and the aforementioned three concentrations of SB-16 (6.75, 13.50, 27.00 BBEXPH)) and odour pot position. The adjustment of testing and the odour pot position were randomised aural anniversary block. In anniversary block (comprising one day) males of C. lectularius were activated with anniversary of the eight treatments. Therefore, one block comprised eight bioassays (n = 20). Acknowledgment variables were: cardinal of visits to pots and time spent in zones. The bioassays were completed in the aboriginal scotophase, amid 10:00 and 15:00 (see inset in Fig. 1).
In accession to SB-16, two added constructed blends were tested, SB-18 and SB-6. The factors were assay (i.e. abrogating ascendancy (solvent), complete ascendancy (volatile abstract at 8% or 27.00 BBEXPH), and the three constructed blends, SB-18, SB-16, and SB-6, all at 8% or 27.00 BBEXPH), and odour pot position. The adjustment of testing and the odour pot position were randomised aural anniversary block. Two blocks were completed anniversary day, and, in anniversary block, males of C. lectularius were activated with anniversary of the bristles treatments. Above-mentioned to the alpha of behavioural testing, an alien complete ascendancy was completed with the bed bug-exposed cardboard to ensure that any abridgement of allure in the treatments was not due to alien conditions. Therefore, one block comprised bristles bioassays and anniversary day 11 bioassays were completed, i.e. two blocks additional the alien complete ascendancy (n = 20). Acknowledgment variables were: cardinal of visits to pots and time spent in zones (see inset in Fig. 1). The bioassays were completed in the aboriginal scotophase, amid 10:00 and 15:00.
All EAG responses were connected by adding the complete amplitude by the 1 mV arrangement recording. For normalization, the abrogating controls, afore and afterwards anniversary appraisal stimulation, were averaged and the aberration amid this bulk and the adapted EAG acknowledgment (mV) to the admixture or abstract was calculated. Electrophysiological action of the air entrainment abstract was bent by comparing the beggarly aberration amid the EAG acknowledgment (mV) to the airy abstract and the abrogating ascendancy by appliance a commutual t-test.
Chemicals were quantified by acute the absorption (ng/µL) adjoin the aiguille breadth and assuming a logistic corruption appraisal on the bulk of accepted actinic injected and the agnate breadth of the aiguille to accord a arrangement curve. Appliance the blueprint from the acknowledgment ambit the bulk of anniversary actinic aural 1 µL (33.6 BBEXPH) of the airy abstract was again calculated.
For appraisal of the behavioural assays, advance breadth the bed bug bootless to move any ambit greater than from the centre of the amphitheatre to the bend (non-responders), or back the accessories bootless and the bed bug was inaccurately tracked (failure of software), were afar from the analysis. Non-responders were according to beneath than 4% of insects. The abortion bulk of the EthoVision software was beneath than 5%. Furthermore, all advance aural a block were afar if there was no acknowledgment to the complete ascendancy of bed bug-exposed paper. In this case, no acknowledgment was authentic by no visits to either pot or added visits to the no-odour pot.
EthoVision was acclimated to analyse the x, y coordinates to account variables afterward Weeks et al.24. Again the abstracts were exported and the aberration amid the odour and no-odour abstracts (O-NO) was calculated. A complete boilerplate value, therefore, adumbrated a greater beggarly for the odour than the no-odour area or pot. The aberration in the time spent in anniversary area (in s) and the aberration in the cardinal of visits to anniversary pot was calculated. Atomic cogent differences (LSDs) at the 5% akin were acclimated to analyze agency amid assay factors to actuate significance. Assay agency were compared to the abrogating ascendancy (labelled ‘a’ if decidedly different), bed bug-exposed cardboard (labelled ‘b’) and airy abstract from bed bug-exposed cardboard (labelled ‘c’).
The abstracts were commonly broadcast as adumbrated by the balance plots for anniversary acknowledgment variable. However, the dataset independent several missing ethics due to casual tracking issues, which resulted in an asymmetric design. Therefore, a belted best likelihood (REML) adjustment was acclimated to appraisal effects. The absent hypotheses were that there was no cogent aberration in the behaviour of bed bugs in the attendance of cardboard that had been ahead apparent to conspecifics (bed bug-exposed paper), airy extracts of this paper, or constructed blends apery the airy abstract compared with the controls.
All statistical analyses of all abstracts calm during this abstraction was completed appliance GenStat adaptation 11.068.
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