Modern bed bugs are resistant to multiple insecticide classes, particularly the pyrethroids. The efficacy of pyrethroid-impregnated mattress liners marketed for bed bug management has been variable. This study evaluated the efficacy of a permethrin-impregnated mattress liner, ActiveGuard, against 24 bed bug strains, consisting of both Cimex hemipterus (F.) and Cimex lectularius L. A 'mat assay', employing an allethrin-impregnated mat, was used to establish the pyrethroid resistance profile of all strains. Three experiments were conducted to evaluate the effect of ActiveGuard exposure on bed bug knockdown: 1) exposing the bed bugs continuously on the liner for up to 24 d, 2) holding the bed bugs on the liner for either 4 or 6 h, and 3) placing a noninsecticide treated fabric above the liner with the bed bugs held continuously on top. Our results indicated that all modern strains (collected within the last 15 years during the current resurgence) were pyrethroid-resistant, although the magnitude of resistance was highly variable between strains. In the continuous exposure study, an incomplete knockdown was recorded for most modern bed bug strains, with some having no knockdown even up to 7 d of constant exposure. In the 4 or 6 h exposure study, the level of knockdown was reduced even further, and very few bed bugs were knocked down in the double fabric study. The results of this study indicate that pyrethroid-impregnated mattress liners are not likely to be effective in the management of most modern bed bug infestations involving either C. hemipterus or C. lectularius.

Not all encounters with pathogens are stochastic and insects can adjust their immune management in relation to cues associated with the likelihood of infection within a life cycle as well as across generations. In this study we show that female insects (bed bugs) up-regulate immune function in their copulatory organ in anticipation of mating by using feeding cues. Male bed bugs only mate with recently fed females and do so by traumatic insemination (TI). Consequently, there is a tight temporal correlation between female feeding and the likelihood of her being infected via TI. Females that received predictable access to food (and therefore predictable insemination and infection cycles) up-regulated induced immunity (generic antibacterial activity) in anticipation of feeding and mating. Females that received unpredictable (but the same mean periodicity) access to food did not. Females that anticipated mating-associated immune insult received measurable fitness benefits (survival and lifetime reproductive success) despite laying eggs at the same rate as females that were not able to predict these cycles. Given that mating is a time of increased likelihood of infection in many organisms, and is often associated with temporal cues such as courtship and/or feeding, we propose that anticipation of mating-associated infection in females may be more widespread than is currently evidenced.

All 100+ bedbug species (Cimicidae) are obligate blood-sucking parasites [1, 2]. In general, blood sucking (hematophagy) is thought to have evolved in generalist feeders adventitiously taking blood meals [3, 4], but those cimicid taxa currently considered ancestral are putative host specialists [1, 5]. Bats are believed to be the ancestral hosts of cimicids [1], but a cimicid fossil [6] predates the oldest known bat fossil [7] by >30 million years (Ma). The bedbugs that parasitize humans [1, 8] are host generalists, so their evolution from specialist ancestors is incompatible with the "resource efficiency" hypothesis and only partially consistent with the "oscillation" hypothesis [9-16]. Because quantifying host shift fre- quencies of hematophagous specialists and generalists may help to predict host associations when vertebrate ranges expand by climate change [17], livestock, and pet trade in general and because of the previously proposed role of human pre-history in parasite speciation [18-20], we constructed a fossil-dated, molecular phylogeny of the Cimicidae. This phylogeny places ancestral Cimicidae to 115 mya as hematophagous specialists with lineages that later frequently populated bat and bird lineages. We also found that the clades, including the two major current urban pests, Cimex lectularius and C. hemipterus, separated 47 mya, rejecting the notion that the evolutionary trajectories of Homo caused their divergence [18-21].

Background Bed bugs (Heteroptera: Cimicidae) are a group of blood-feeding ectoparasites. They mainly specialize on bats and birds, but a few species are important human pests. They exhibit several unique adaptations for their parasitic lifestyle. Among those, bed bug aggregations represent a striking example of a sub-social structure. However, their benefits for the bed bugs as well as their potential for bed bug control are largely unexplored. Young nymphs are known to disperse from the aggregations much less than older ones or adults. We therefore found possible that the aggregation age structure is connected with success in finding host and tested the effect of presence of adults on nymphal feeding success. Results We tested the effect of presence of adults on feeding success of first-instar nymphs using an artificial feeding system. We found that presence of fed adults causes larger proportion of nymphs to feed. Conclusions Based on our data, fed bed bugs seem to trigger the young nymphs to actively forage. Since the first instar is much less viable than later stages, our finding points to an adaptive behavior that economizes on foraging energy cost. In the context of bed bug control, knowledge on such behavior emphasizes the prevention of fed bed bugs from returning to harborages. Bed bug traps may thus be used not just as means of bed bug monitoring, but also as means of control.

The rapid evolution of insecticide resistance remains one of the biggest challenges in the control of medically and economically important pests. Insects have evolved a diverse range of mechanisms to reduce the efficacy of the commonly used classes of insecticides, and finding the genetic basis of resistance is a major aid to management. In a previously unstudied population, we performed an F2 resistance mapping cross for the common bed bug, Cimex lectularius, for which insecticide resistance is increasingly widespread. Using 334 SNP markers obtained through RAD-sequencing, we constructed the first linkage map for the species, consisting of 14 putative linkage groups (LG), with a length of 407 cM and an average marker spacing of 1.3 cM. The linkage map was used to reassemble the recently published reference genome, facilitating refinement and validation of the current genome assembly. We detected a major QTL on LG12 associated with insecticide resistance, occurring in close proximity (1.2 Mb) to a carboxylesterase encoding candidate gene for pyrethroid resistance. This provides another example of this candidate gene playing a major role in determining survival in a bed bug population following pesticide resistance evolution. The recent availability of the bed bug genome, complete with a full list of potential candidate genes related to insecticide resistance, in addition to the linkage map generated here, provides an excellent resource for future research on the development and spread of insecticide resistance in this resurging pest species.

BACKGROUND:Bed bugs [both Cimex hemipterus (F.) and Cimex lectularius L.] are highly resistant to pyrethroids worldwide. An important resistance mechanism known as 'knockdown resistance' (kdr) is caused by genetic point mutations on the voltage-gated sodium channel (VGSC) gene. Previous studies have identified two point mutations (V419L and L925I) on the VGSC gene in C. lectularius that are responsible for kdr-type resistance. However, the kdr mutations in C. hemipterus have not been investigated.RESULTS:Four novel mutations, L899V (leucine to valine), M918I (methionine to isoleucine), D953G (aspartic acid to glycine) and L1014F (leucine to phenylalanine), were identified in the domain II region of the C. hemipterus VGSC gene. This region has been widely investigated for the study of kdr-type resistance to pyrethroids in other insect pests. The V419L and L925I kdr mutations as previously identified in C. lectularius were not detected in C. hemipterus.CONCLUSION:M918I and L1014F are considered to be probable kdr mutations and may play essential roles in kdr-type resistance to pyrethroids in C. hemipterus. Further studies are under way in the authors' laboratory to determine the non-kdr-type resistance mechanisms in C. hemipterus.

This paper reviews basic biological aspects of bedbugs and discusses current control strategies (i.e., environmental, biological, chemical) for addressing bedbug infestations.

Sexual selection, differences in reproductive success between individuals, continues beyond acquiring a mating partner and affects ejaculate size and composition (sperm competition). Sperm and seminal fluid have very different roles in sperm competition but both components encompass production costs for the male. Theoretical models predict that males should spend ejaculate components prudently and differently for sperm and seminal fluid but empirical evidence for independent variation of sperm number and seminal fluid volume is scarce. It is also largely unknown how sperm and seminal fluid variation affect future mating rate. In bedbugs we developed a protocol to examine the role of seminal fluids in ejaculate allocation and its effect on future male mating rate. Using age-related changes in sperm and seminal fluid volume we estimated the lowest capacity at which mating activity started. We then showed that sexually active males allocate 12% of their sperm and 19% of their seminal fluid volume per mating and predicted that males would be depleted of seminal fluid but not of sperm. We tested (and confirmed) this prediction empirically. Finally, the slightly faster replenishment of seminal fluid compared to sperm did not outweigh the faster decrease during mating. Our results suggest that male mating rate can be constrained by the availability of seminal fluids. Our protocol might be applicable to a range of other organisms. We discuss the idea that economic considerations in sexual conflict research might benefit from distinguishing between costs and benefits that are ejaculate dose-dependent and those that are frequency-dependent on the mating rate per se.

The common bedbug, Cimex lectularius L. (Hemiptera: Cimicidae), is a globally re-emerging pest that is playing an increasing role in legal disputes and compensation claims as a result of its unpleasant feeding activity. However, there is little information about the feeding frequency of bedbugs outside controlled laboratory cultures. Here, we present a simple method of estimating the average time since the last bloodmeal of individual female bedbugs in a single sampling event, applicable to a single bedbug harbourage or an entire room. Using the temperature-dependent rate of decrease in the abdomen size of the bedbug after a bloodmeal, we found that, in a highly infested room kept at a constant temperature of 26 degrees C, females fed every 2.5 days on average. Our method corrects for variations in body size across different populations and determines the shrinkage that occurs when individuals are preserved in ethanol. This method should, therefore, be widely applicable. It is cheap, rapid and, if coupled with information on the total number of bedbugs present in a room, allows for the estimation of the minimum number of times persons lodging in a room have been bitten by bedbugs. This method can also be used to calculate the feeding rate of other blood-sucking insects on their hosts. The sex ratio in the infestation was female-biased. Finally, our case study suggests that individual female bedbugs within a harbourage do not seem to feed at a regular rate, but tend to synchronize feeding patterns.

The common bed bug, Cimex lectularius (L.) (Hemiptera: Cimicidae) is known to become associated, from time to time, with clothing or linen. In such locations, it may escape insecticide treatment, and may be carried to new locations. We test the suggestion that laundering is sufficient to kill all life stages and thus help prevent reinfestation and dispersion. We establish minimum temperatures for washing and minimum temperatures and times for tumble-drying, as well as testing cold soaking, dry cleaning, and freezing as alternative strategies for delicate items. Data loggers were used to confirm temperature settings and monitor temperature changes during the treatments. Adult bed bugs, nymphs, and eggs were sealed into small, permeable cotton pouches, which were then placed into garments of clothing. Washing at 60 degrees C was found to be effective against all life stages, as was tumble drying on a hot cycle (>40 degrees C) for at least 30 min, dry cleaning with perchloroethylene, and freezing at -17 degrees C for at least 2 h. Using data loggers it was also shown that 2.5 kg of loosely packed, dry laundry takes approximately 8 h to reach -17 degrees C. Soaking for 24 h in detergent-free water was found to be effective against active stages but had no effect on eggs.

Increased female reproductive rates usually result in accelerated senescence. This correlation provides a link between the evolutionary conflict of the sexes and aging when ejaculate components elevate female reproductive rates at the cost of future reproduction. It is not clear whether this female cost is manifest as shorter lifespan or an earlier onset or a steeper rate of reproductive senescence. It also is unclear whether beneficial ejaculates release females from reproductive trade-offs and, if so, which senescence parameters are affected. We examined these issues in the bedbug, Cimex lectularius, a long-lived insect that shows reduced female lifespan as well as female reproductive senescence at the male-determined mating frequency. We demonstrate experimentally that, independently of the mating frequency, females receiving more ejaculate show increased reproductive rates and enter reproductive senescence later than females receiving less ejaculate. The rate of reproductive senescence did not differ between treatments, and reproductive rates did not predict mortality. The ejaculate effects were consistent in inter-and intra-population crosses, suggesting they have not evolved recently and are not caused by inbreeding. Our results suggest that ejaculate components compensate for the costs of elevated female reproductive rates in bedbugs by delaying the onset of reproductive senescence. Ejaculate components that are beneficial to polyandrous females could have arisen because male traits that protect the ejaculate have positive pleiotropic effects and/or because female counter-adaptations to antagonistic male traits exceed the neutralization of those traits. That males influence female reproductive senescence has important consequences for trade-offs between reproduction and longevity and for studies of somatic senescence.

Optimal male and female mating rates rarely coincide. Males often shift the rate in their favor by either increased signaling and by overcoming female resistance to copulation. The concept of sensory exploitation posits that males produce signals that mimic naturally selected benefits and so deceitfully attract females. However, males also have to overcome female resistance to actual copulation. Males may do so by copulating during situations when the female's ability to resist is decreased because of competing naturally selected demands. Males of the common bedbug, Cimex lectularius, an obligate blood feeder, mate at a rate, and in a manner that is harmful to females. Females have to feed regularly to produce eggs, and during feeding female body volume increases by 300%. Choice trials using unfed and either fed or experimentally enlarged but unfed females showed that the increased postfeeding body volume of females attracted more male mating attempts, strongly reduced female resistance to male mating attempts and resulted in a net increase in female mating rate. Our results, therefore, suggest that males have increased mating success in a situation that females cannot avoid because it is naturally selected. Such "situation exploitation" of low resistance may be a common phenomenon.

The rapid evolution of ejaculate components is considered to be largely driven by sexual selection. Less attention has been paid to the fact that sperm and microorganisms frequently meet; we consequently predict selection for substances that protect a male's ejaculate. We report, for the first time, bacteriolytic activity (lysozyme-like immune activity [LLA]) in the ejaculate of an animal, the common bedbug Cimex lectularius. We also show that in almost half the males LLA in the seminal fluid exceeded LLA in the hemolymph. We detected no antimicrobial peptide activity in seminal fluid. Because lysozymes degrade only bacteria, our results suggest that sperm-microbe interactions are probably important in the evolution of ejaculate components and thereby provide a route for natural selection to account for some of the diversity of seminal components.

Bats and other mammals perform a wide range of avoidance and defence strategies in response to parasites or the threat of being parasitized (Marshall, 1981; Lehane, 2005; Reinhardt and Siva-Jothy, 2007). This includes behavioural, morphological and physiological traits. Hosts may shift their activity phases in response to parasitism, they may avoid infested sites (see e.g., Brown and Brown, 1986 for a bird example) or roost and forage at sites which are unfavourable for the parasites. Some animals, mainly insects, when parasitised have been shown to choose warm areas in order to rid themselves of pathogenic bacteria and fungi (‘behavioural fever’ — Adamo, 1998; Blanford et al., 1998). For endotherms, Lehane (2005) lists examples of mammals choosing sites whose wind and other microclimatic conditions deter attack or feeding by bloodsucking ectoparasites. Many ectoparasites settle close to their hosts presumably in order to reduce travel costs to the hosts. Therefore, thermoregulating endotherms may be able to roost at sites with elevated external temperatures that ectothermic parasites may not be able to tolerate. It appears unknown whether bats or other mammals choose such hightemperature sites in order to prevent a settlement of blood-sucking ectoparasites in their close proximity. However, such host behaviour may not be unlikely, given that many blood-sucking insects harbour microbes that assist in blood digestion (Buchner, 1965; Usinger, 1966) and that these microbes are very sensitive to the exposure of high temperatures (e.g., Buchner, 1965; Chang, 1974). Several ectoparasites are also sensitive to low humidity values. Whilst they can survive dry conditions they are unlikely to reproduce (e.g., Benoit et al., 2007). If host defence occurs by choosing sites that are unfavourable to parasites, temperature or humidity differences can be expected: a) between the site of host and of parasite activity and b) between thermal conditions found to be optimal and those actually observed in the field.

The feeding frequency of blood-feeding invertebrates in the wild is largely unknown but is an important predictor for the potential of disease transmission and for estimating the effects blood feeding may have on the host population. We present a method to estimate the mean feeding frequency per individual parasite from the frequency distribution of fed and unfed individuals in the wild. We used three populations of the cimicid species, Afrocimex constrictus, that parasitises the fruit bat Rousettus aegyptiacus. We found that the area occupied by a bug refugium was a good predictor of the number of bugs in that refugia. The estimated parasite population sizes ranged from ca. 25,000 to 3 million bugs. Their mean abundance was 1-15 bugs per host individual. Preventing feeding by bugs in their natural habitat showed that bugs took approximately 20 days to return to an unfed stage. A formula is presented by which the distribution of digestion stages in the samples was used to calculate that A. constrictus feeds approximately every 7-10 days. The dry weight of a full blood meal was approximated as 13.3 mg. Therefore A. constrictus is estimated to draw an average of 1-28 microL blood per host per day. We suggest that any of our methods can be adjusted to be used in other haematophagous insects to estimate host and parasite population size, mean parasite abundance and blood meal size as well as mean feeding frequency in the wild, including the bed bug species that parasitise humans.

Sexual conflict can produce several evolutionary outcomes, one of which is female-limited trait polymorphism. We examine the African bat bug Afrocimex constrictus (Cimicidae), a species where both sexes are subjected to traumatic intromission from males. We show that males possess female genital structures that in related species ameliorate the costs of traumatic insemination. Moreover, the male form of these structures differs morphologically from the standard female form. Examination of females in our isolated study population revealed a discrete polymorphism in female genitalia. Some females had the typical cimicid form, while others had genitalia that more closely resembled the distinctive male form. Males, as well as females with the distinctive male form, experienced fewer traumatic copulations than the typical female morph. We propose that some females mimic the bizarre male condition in order to reduce the frequency of costly traumatic inseminations. To our knowledge this is the first example of a distinct female-limited genital polymorphism: its nature, as well as its association with traumatic sexual interactions, strongly suggests that sexual conflict underpins this unique phenomenon.

The frequent wounding of female bedbugs (Cimex lectularius: Cimicidae) during copulation has been shown to decrease their fitness, but how females have responded to this cost in evolutionary terms is unclear. The evolution of a unique anatomical structure found in female bedbugs, the spermalege, into which the male's intromittent organ passes during traumatic insemination, is a possible counteradaptation to harmful male traits. Several functions have been proposed for this organ, and we test two hypotheses related to its role in sexual conflict. We examine the hypotheses that the spermalege functions to (i) defend against pathogens introduced during traumatic insemination; and (ii) reduce the costs of wound healing during traumatic insemination. Our results support the 'defence against pathogens' hypothesis, suggesting that the evolution of this unique cimicid organ resulted, at least partly, from selection to reduce the costs of mating-associated infection. We found no evidence that the spermalege reduces the costs of wound healing.

The hydrolysis of O-(4-nitrophenyl) 2-pyridylphosphonate possesses kinetic terms in hydroxide and hydrogen ion concentration and zwitterion and anion concentration. Comparison with suitable models where the proton is localised shows that the 'anion' term arises from hydroxide ion attack on the zwitterion rather than water attack on the monoanion; 'zwitterion' term involves attack of hydroxide ion on the cationic species. The hydrolysis of O-(4-nitrophenyl)(N-methyl-2-pyridinio)phosphonate possesses a 'neutral' term from hydroxide ion attack on the cationic species. 2-Pyridylphosphonate diesters hydrolyse via attack by water on the neutral form of the ester; there is no detectable anchimeric assistance by the pyridine nitrogen possibly due to the very low basicity of the base. The hydrolysis of O-(4-nitrophenyl) phenylphosphonate at neutral pH involves decomposition of the monoanion presumably by water attack rather than the equivalent reaction of hydroxide ion with the neutral form of the ester. O-(4-Nitrophenyl) 2-pyridyl)phosphonate is shown to complex with transition metal ions which also catalyse the hydrolysis of the ester possibly via electrophilic participation.

The enhancement in rate constant for the alkaline hydrolysis of diethyl 2-acetamidophenylphosphonate over that of the 4-isomer increases significantly with increasing dioxan content of the medium consistent with intramolecular assistance by the amido NH in the former reaction.