Chemically Mediated Arrestment of the Bed Bug, Cimex lectularius, by Volatiles Associated with Exuviae of Conspecifics

Abstract

Extracts of the exuviae (cast skins) of nymphal bed bugs (Cimex lectularius) were analyzed for volatile compounds that might contribute to arrestment of adult bed bugs. Four volatile aldehydes, (E)-2-hexenal, 4-oxo-(E)-2-hexenal, (E)-2-octenal, and 4-oxo-(E)-2-octenal were consistently detected in the headspace of freshly shed exuviae regardless of the developmental stages from which the exuviae were obtained. Quantification of the aldehydes in the solvent extracts of homogenized fresh, 45- or 99-d aged 5th instar exuviae indicated that the aldehydes are present in the exuviae and dissipate over time, through evaporation or degradation. Microscopic observation of the fifth instar exuviae indicated that the dorsal abdominal glands on the exuviae maintained their pocket-like structures with gland reservoirs, within which the aldehydes might be retained. Two-choice olfactometer studies with the volatiles from exuviae or a synthetic blend mimicking the volatiles indicated that adult bed bugs tend to settle close to sources of the aldehydes. Our results imply that the presence and accumulation of bed bug exuviae and the aldehydes volatilizing from the exuviae might mediate bed bugs' interaction with their microhabitats.

Fig 1. Tubing olfactometer.

A piece of fine fabric screen (A) was placed before inserting vials (B) into the 15-cm PVC tubing (C) to prevent the test insect from physically contacting the contents of the vials. A small slit (D) at the center of the tubing was used to introduce a single adult bed bug into the olfactometer. The bed bug (E) was typically found on the fabric screen of the chosen vial after 18 h.

Fig 2. Regression lines of four aldehydes detected from exuviae of 5th instar nymphs.

(A) (E)-2-hexenal, (B) 4-oxo-(E)-2-hexenal, (C) (E)-2-octenal, (D) 4-oxo-(E)-2-octenal. Units for the chemical quantity per exuvia is μg. Dotted lines indicate 95% confidence intervals.

Fig 3. Dorsal abdominal gland morphology study with 5th instar exuviae.

(A) Dorsal abdominal glands on an exuvia observed with optical dissection microscope. (B) Scanning electron microscope image showing the longitudinal cut section of dorsal abdominal glands. (C) Optical microscope image showing the cut section of dorsal area of an exuvia embedded in resin (D) Two exuviae with dorsal abdominal glands that were visualized by taking up a colored solvent into the gland reservoirs. For (A), (B), and (C), the arrow indicates the anterior direction of the exuvia. DAGs 1–3 indicate dorsal abdominal glands on the 3rd, 4th, and 5th abdominal tergites, respectively.

Fig 4. Olfactometer study with two different sources of aldehyde pheromones.

For assays with the exuviae, the treatment vial contained 70–76 exuviae from 3rd, 4th, and 5th instar nymphs. For assays with the synthetic aldehydes, the treatment vial contained a small piece of cotton that was treated with 40 μl of paraffin oil containing the synthetic aldehyde blend. Control vials were either left empty (exuviae study) or contained a small piece of cotton treated with clean paraffin oil (synthetic blend study). Numbers in the bar show the number of individuals found on either treatment or control vials at the end of 18-h assay. See text for statistical analyses.