The chemical communication, as well as the mating behaviour, of bed bugs (Cimex lectularius) has received increased attention in recent years as bed bug infestations have increased worldwide. The occurrence of alarm pheromones has been known since the 1970 s [1] but, in the light of traumatic insemination (the only mode of copulation in the Cimicidae), new discoveries concerning the usage of alarm pheromones by bed bugs are changing the common view that alarm pheromones are used as a predator defence system only [2].

All developmental stages and both sexes of C. lectularius are obligate blood-feeders and they form large aggregations within close proximity of their human host. Bed bugs remain hidden inside their refuge where they mate, moult and oviposit until their next nocturnal blood meal on the resting host [3, 4]. Mating is exclusively traumatic, with the male piercing a secondary genital opening in the female with its lanceolate paramere and ejaculating directly into the abdominal cavity [3, 4]. Mating occurs throughout the adults' life and hence polygamous mating is common [3–5]. Multiple mating injuries have been shown to be costly for females, probably due to increased immunological threat [6], and lead to a reduced life span [7].

Mating is closely associated with blood feeding and male attention is directed to any large, newly blood-fed bed bugs [8, 9]. Hence, males display characteristic mounting behaviour when presented to an engorged individual regardless of sex [5, 10]. If the mounted mate is a male or a large nymph, the males respond by a quick dismount and mating does not occur [5]. This suggests a lack of long-distance sex identification signals and that identification occurs after mounting [10].

It is well established that stressed bed bugs of all stages and sexes release an alarm pheromone, causing the dispersal of conspecifics [4, 11]. However, the alarm pheromone has a dual function in bed bugs, where a mounted male responds by releasing alarm pheromone to prevent homosexual mating [10], in addition to adopting specific behaviours [5]. In contrast, females do not release alarm pheromone when mounted by a male [10, 11]. The alarm pheromone is produced by glands located between the first and second pairs of legs on the methatorax of adults and dorsally on the nymph abdomen [4]. The alarm pheromone is composed of (E)-2-hexenal and (E)-2-octenal [12, 13], both of which are detected by olfactory receptor neurons (ORNs) housed in smooth peg sensilla on the adult antenna [14]. Female and male bed bugs produce different ratios of the two aldehydes, with both sexes releasing a larger amount of (E)-2-hexenal than nymphs [12]. In addition to the aldehydes, last nymphal stages also produce two oxygenated aldehydes, 4-oxo-(E)-2-hexenal and 4-oxo-(E)-2-octenal [[15], Liedtke et al., unpublished data]. The behavioural role of these nymph-specific compounds, or the significance of the reversed ratio of the two aldehydes, has not yet been established.

We hypothesize that the nymph-emitted compounds are used by nymphs as an identification signal, which deters mate-seeking males. After establishing that male bed bugs are able to detect these compounds, using single sensillum recordings, we predict that by removing the possibility for nymphs to emit their pheromone, nymphs would experience a higher rate of mating than non-manipulated nymphs. In addition, the addition of the major nymph-specific compound 4-oxo-(E)-2-hexenal, as well as a reversed ratio of the two aldehydes would result in mating disruption of adult bed bugs.

This study demonstrates that the alarm pheromone produced by the dorsal abdominal glands of C. lectularius nymphs is detected by males and acts as a protection against traumatic extragenital insemination.

Male bed bugs mount any moving object of the size of a fed female bed bug [8] without discrimination of sex [5, 10], developmental stage (this study) or even species [16] prior to physical contact [3, 4]. These observations are corroborated by our study in which males mounted blood-fed females and nymphs with the same frequency (Figure 1a). However, when in physical contact, the male usually responds by a quick dismount when the mounted individual is a male [10] or a nymph (Figure 1b). By mating with a mistaken individual, the male wastes energy and sperm, and thereby decreases his reproductive success. The possible cost for the mounted individual may be mechanical damage such as gut perforation and the increased risk of infection by pathogens [6, 17]. Females have physiological and anatomical adaptations, such as the spermalege, which reduces these fitness costs imposed during mating [4, 17]. However, even for females, traumatic insemination carries a measurable fitness cost [7], which may be higher for males [10] and nymphs who lack such adaptations. It is therefore crucial for the nymphs to state their identity to males in order to avoid traumatic extragenital insemination. This statement is strengthened by our observation of silenced nymphs, which were unable to emit their alarm pheromone, and hence were mated (Figure 1b). To confirm the hypothesis that nymphs state their identity via their alarm pheromone, we tried to disrupt the mating of silenced females by adding alarm pheromone signals that are associated with nymph emission. We found that both (E)-2-hexenal:(E)-2-octenal in a nymph-specific ratio [2:5] and 4-oxo-(E)-2-hexenal, which all elicit ORN responses at biologically relevant concentrations, are sufficient to deter males from copulating with the manipulated female (Figure 3b). In contrast, the female and male specific ratio of the two aldehydes did not elicit dismounting (Figure 3b). This non-repulsive effect of the alarm pheromone emitted by adults may be linked, as mentioned by [2], to the dual interpretation of this blend in relation to the amount emitted. Indeed, males, in addition to releasing alarm pheromone while mounted [10], adopt specific behaviours of non-acceptance by bending their abdomen and shaking their body in order to escape piercing (personal observations).

The unusual mating behaviour of the bed bug, with traumatic insemination, gives new insights to their chemical communication systems. Like males avoiding homosexual mistakes [10], nymphs also emit an alarm pheromone to avoid being chosen as partner. By further analysing the alarm pheromone ratios and content, we find that a specific ratio of aldehyde compounds, characteristic of nymphs, are sufficient to avoid mating, but we also conclude that 4-oxo-(E)-2-hexenal produced specifically by the nymphs is detected by specific sensilla on the male antennae and may contribute on its own to a repellent effect on males. This double effect may give a stronger protection to the nymphs against mating and piercing mistakes. The chemical communication system of the bed bugs is only just unfolding, and further analyses on longevity costs to nymphs as well as males who have been pierced is a high priority to fully understand the picture of traumatic insemination.