Sensory and chemical basis of off-host aggregation behavior by bed bugs, Cimex lectularius

Abstract

After feeding on hosts, bed bugs, Cimex lectularius L., aggregate in cracks and crevices near their hosts. Off-host aggregation is mediated by sensory organs on the bed bug antennae and chemical stimuli associated with bug feces. This dissertation examined the sensory bases of bed bug off-host aggregation behavior, and results are presented in four chapters. Chapter one provides a basic overview of existing literature on the sensory structures located on adult antennae and the chemical stimuli that influence bed bug behavior. The chapter concludes with a discussion of practical applications for bed bug control. In chapter two, behavioral assays and microscopy were used to study sensilla on the bed bug antenna. A multi-choice behavioral assay using fecal stained filter papers determined which antennal segments mediate off-host aggregation. Both scanning and transmission electron microscopy techniques were used to determine the type and function of sensilla on the pedicel of adults and nymphs. In addition to an abundance of serrated hairs, several smooth hairs with gustatory function were sparsely distributed throughout the segment and a distal patch of sensilla with olfactory function was also described. The identification of sensilla with olfactory and gustatory function on the pedicel suggests off-host aggregation by bed bugs may be mediated by a volatile or semi-volatile compound or compounds. In chapter three, the chemical stimulus associated with bed bug feces was analyzed, including stimulus volatility, extraction, isolation, and separation of component molecules. Solid phase microextration (SPME) techniques were used to assess the presence of known bed bug pheromones, (E)-2-hexenal (E2H) and (E)-2-octenal (E2O) on fecal stained papers that were heat treated for several days. In addition, multi-choice behavioral assays were used to assess aggregation response to fecal stained papers that were heated, to papers washed in various solvents, and to concentrated methanol extracts and extracts separated by solid phase extraction (SPE) techniques. Results demonstrated that E2H and E2O decrease significantly with heat exposure; however, aggregation response to fecal stained disks remained relatively constant, suggesting that the chemical stimulus is less volatile compared to previous reports. The chemical stimulus was soluble in methanol and water, with bed bug response greatest to concentrations of fecal extracts above 30 mg/ml methanol. Separation of the active components was possible using a C18 SPE cartridge and gas chromatography techniques, which prepared the chemical stimulus for further identification. In chapter four, crude extracts from bed bug feces were analyzed by a gas chromatograph coupled with an electro-antennogram detector (GC-EAD) and mass spectrometer (GC-MS) to identify essential components of the off-host aggregation pheromone. Adult antennae responded to compounds associated with three elution regions of the crude extract. Several chemical compounds were identified in each of the active regions, and selected groups of compounds were evaluated in multiple choice assays to assess aggregation response. A combination of two compounds, dimethyl trisulfide (DMTS) and methyldiethanolamine (MDEA) resulted in an aggregation response that was equivalent to original extract. This final chapter concludes with a discussion of potential applications of a synthetic aggregation pheromone for surveillance and bed bug control.