Real-time measurement of volatile chemicals released by bed bugs during mating activities

Abstract

In recent years, bed bug (Hemiptera: Cimicidae) problems have increased dramatically in many parts of the world, leading to a renewed interest in their chemical ecology. Most studies of bed bug semiochemicals have been based on the collection of volatiles over a period of time followed by chemical analysis. Here we present for the first time, a combination of proton transfer reaction mass spectrometry and video analysis for real-time measurement of semiochemicals emitted by isolated groups of bed bugs during specific behavioural activities. The most distinct peaks in the proton transfer reaction mass spectrometry recordings were always observed close to the termination of mating attempts, corresponding to the defensive emissions that bed bugs have been suspected to exploit for prevention of unwanted copulations. The main components of these emissions were (E)-2-hexenal and (E)-2-octenal recorded in ratios between 1:3 and 3:1. In the current study, the quantity varied over 1000 fold for both of the compounds with up to 40 µg total release in a single emission. Males also emit defensive compounds due to homosexual copulation attempts by other males, and no significant differences were observed in the ratio or the amount of the two components released from males or females. In summary, this study has demonstrated that combining proton-transfer-reaction mass spectrometry with video analysis can provide detailed information about semiochemicals emitted during specific behavioural activities.

Figure 1. Example of aldehyde emissions and correlation with behavioural observation.

Results from a PTR-MS recording on four male and four female bed bugs. The thick horizontal lines indicate mating attempts of male on female (red), male on male (blue) or a second male trying to copulate an already established male-female couple (orange). A. Estimated concentrations of (E)-2-hexenal and (E)-2-octenal over the entire recording period. B. Observed (dashed black line) and calculated (green line) ion counts for the mass 57 which is produced from both (E)-2-hexenal and (E)-2-octenal.

Figure 2. Examples of aldehyde emissions and correlation with behavioural observations.

Results from two PTR-MS recordings on either eight males (A) or four males and four females (B). The thick horizontal lines indicate mating attempts of male on female (red), male on male (blue) or a second male trying to copulate an already established male-female couple (orange). The amount of (E)-2-hexenal and (E)-2-octenal emitted over the entire experimental period of A is also shown in Table 2. In B, the bed bugs were kept in the closed experimental container without access to blood during the first 14 minutes (until the first arrow), then the container was placed on the arm of a human volunteer and the bed bugs were allowed to feed for the last 12 minutes (from the second arrow).

Figure 3. Concentrations of (E)-2-hexenal and (E)-2-octenal in the PTR-MS recordings from three experiments shown in Table 2 .

In A with four females no mating attempts were observed, in B with four males and four females there was a small peak, and in C there were too many bed bugs for the video analysis. Note that the recording and collection periods were slightly different.

Figure 4. Doses of (E)-2-hexenal and (E)-2-octenal in individual emissions.

All observations are based on PTR-MS recordings combined with video recordings. The dashed line indicates equal amounts of (E)-2-hexenal and (E)-2-octenal. The ratio of (E)-2-hexenal and (E)-2-octenal released during the three types of copulation formations were not significantly different from one (♂-♀: t ₉ = 1.30; P = 0.23; ♂-♂: t ₂₈ = −1.09; P = 0.28; ♂-♀-♂: t ₂ = 0.53; P = 0.65).