Electrophysiologically and behaviourally active semiochemicals identified from bed bug refuge substrate

Abstract

Bed bugs are pests of public health importance due to their relentless biting habits that can lead to allergies, secondary infections and mental health issues. When not feeding on human blood bed bugs aggregate in refuges close to human hosts. This aggregation behaviour could be exploited to lure bed bugs into traps for surveillance, treatment efficacy monitoring and mass trapping efforts, if the responsible cues are identified. The aim of this study was to identify and quantify the bed bug aggregation pheromone. Volatile chemicals were collected from bed bug-exposed papers, which are known to induce aggregation behaviour, by air entrainment. This extract was tested for behavioural and electrophysiological activity using a still-air olfactometer and electroantennography, respectively. Coupled gas chromatography-electroantennography (GC-EAG) was used to screen the extract and the GC-EAG-active chemicals, benzaldehyde, hexanal, (E)-2-octenal, octanal, nonanal, decanal, heptanal, (R,S)-1-octen-3-ol, 3-carene, β-phellandrene, (3E,5E)-octadien-2-one, (E)-2-nonenal, 2-decanone, dodecane, nonanoic acid, 2-(2-butoxyethoxy)ethyl acetate, (E)-2-undecanal and (S)-germacrene D, were identified by GC-mass spectrometry and quantified by GC. Synthetic blends, comprising 6, 16, and 18 compounds, at natural ratios, were then tested in the still-air olfactometer to determine behavioural activity. These aggregation chemicals can be manufactured into a lure that could be used to improve bed bug management.

Figure 1

The effect of increasing concentration of the volatile extract from bed bug-exposed paper on the behaviour of Cimex lectularius in a still-air olfactometer (inset). Extract concentrations (E) tested were 0.0675, 0.675, and 6.75 BBEXPH (hours of bed bug-exposed paper air entrainment extract; or 0.02, 0.20 and 2.00% of the extract, respectively). The response due to a positive control of bed bug-exposed paper (BBEXP) and a negative control of solvent (re-distilled diethyl ether) are shown for comparison. Bars represent mean differences in (a) visits to pots (SED 1.193 visits), and (b) time spent in zones (s, SED 109.2 s) between odour and no-odour pots and zones, respectively. Means ± standard error; bars labelled ‘a or b’ are significantly different (at the 5% level using restricted maximum likelihood analysis) from the negative or positive control, respectively. Bars labelled ‘ns’ were not significantly different from either control.

Figure 2

Coupled gas chromatography-electroantennography (GC-EAG) of Cimex lectularius (female, inset photo) responses to a bed bug-exposed paper volatile extract. Blue line corresponds to the flame ionisation detector (FID) on the GC. Black line corresponds to the antennal response of the insect preparation. Labelled peaks were GC-EAG-active in five or more of the 20 insects tested (peaks 1–21).

Figure 3

The effect of concentration of volatile extract (E) and a 16-component synthetic blend (B) on the behaviour of Cimex lectularius in a still-air olfactometer. Concentrations tested were 6.75, 13.50, and 27.00 BBEXPH (hours of bed bug-exposed paper air entrainment extract; or 2, 4 and 8% of the extract, respectively) for both extracts and blends. The response due to a positive control of bed bug-exposed paper (BBEXP) and a negative control of solvent (re-distilled diethyl ether, CON) are shown for comparison. Bars represent mean differences in (a) visits to pots (SED 1.428 visits), and (b) time spent in zones (s, SED 206.5 s) between odour and no-odour pots and zones, respectively. Means ± standard error; bars labelled ‘a or b’ are significantly different (at the 5% level using restricted maximum likelihood analysis) from the negative or positive control, respectively.

Figure 4

The effect of 16-, 18-, and 6-component synthetic blends (SB-16, SB-18, and SB-6, respectively) on the behaviour of Cimex lectularius in a still-air olfactometer. Synthetic blends (27 BBEXPH, i.e. hours of bed bug-exposed paper air entrainment extract; or 8% of the extract) tested and compared to the response due to two positive controls, bed bug-exposed paper (BBEXP) and a volatile extract (EXT) from bed bug-exposed paper (27 BBEXPH), and a negative control of solvent (re-distilled diethyl ether). Bars represent mean differences in (a) visits to pots (SED 1.586 visits) and (b) time spent in zones (s, SED 158.8 s) between odour and no-odour pots and zones, respectively. Means ± standard error; bars labelled ‘a,b or c’ are significantly different (at the 5% level using restricted maximum likelihood analysis) from the negative control, positive control or volatile extract, respectively.