First instar and adult male bed bugs, Cimex lectularius (Hemiptera: Cimicidae), increase feeding activity in the presence of adult females.

BACKGROUND: Bed bugs, Cimex lectularius, form day-time aggregations from which they depart at night to feed on human blood. Obtaining an initial blood meal is a critical step in the development of first instars. Previous research had shown that first instars had greater success in obtaining this essential meal when in the presence of adults than when they were alone. METHODS: Feeding by bed bugs was tested in upright vertical cylindrical chambers fitted with a paper ramp to aid in climbing toward a blood feeder suspended across the upper end of the cylinder. Feeding success by the first instars was tested when they were alone in the chamber or when they were in the presence of adult females, males, or both together.  RESULTS: The mean proportions of the first instars that fed were significantly higher when they were confined with adults of both sexes or adult females than when they were confined alone or with males. Feeding by adult males was also enhanced by confinement with females. When first instars and adult females were confined together, the mean duration before first instars began feeding was longer than for females. There was no difference in feeding success by first instars confined with their mothers or nonmothers. CONCLUSIONS: Elevated feeding by first instars and adult males in the presence of females may be adaptive traits that enhance fitness. First instars must feed to avoid dehydration and starvation and to obtain resources needed for development. Adult males would benefit not only by increased feeding success but also by greater likelihood of finding a recently engorged female with which to mate. The lack of any difference in feeding success of first instars in the presence of their mothers or nonmothers argues against parental care in this species.

Sexual conflict and sexual networks in bed bugs: the fitness cost of traumatic insemination, female avoidance and male mate choice.

Sexual conflict is prevalent among animals and is primarily caused by the fact that the optimal mating rates are often higher in males than in females. While there is a growing appreciation that females can also gain from multiple matings, we still know relatively little about which sex controls the observed mating rates and how close it is to the optimal female mating rates. To address this issue, we tracked female bed bugs (Cimex lectularius) inseminated daily versus weekly and found that weekly inseminated females lived longer and produced over 50% more offspring. In a follow-up experiment employing a social network framework, we placed 24 bed bugs into a semi-naturalistic arena and recorded all sexual interactions. While recently inseminated females did not avoid males more often, they were more frequently rejected by males. Finally, we tracked avoidance behaviour in a single cohort of female bed bugs as they received six successive daily inseminations. Avoidance rates increased and insemination durations decreased with increasing number of prior inseminations. Overall, our results indicate high costs of polyandry. Although females possess some plastic avoidance strategies, the observed rates of insemination fall closer to the male rather than female optimum.

Species identification and pyrethroid resistance genotyping of recently resurgent Cimex lectularius and Cimex hemipterus in Korea.

The global resurgence of bed bug infestations, exacerbated by increasing international travel, trade, and insecticide resistance, has significantly impacted Korea. This study identified the bed bug species and performed pyrethroid resistance genotyping of recently resurgent bed bugs in Korea. Thirty-one regional bed bug samples were collected from 5 administrative regions: Gyeonggi-do (n=14), Seoul (n=13), Busan (n=2), Jeonllanam-do (n=1), and Chungcheongbuk-do (n=1). The samples underwent morphological and molecular identification. Twenty-four regional samples (77.4%) were identified as the tropical bed bug, Cimex hemipterus, and the remaining 7 regional samples (22.6%) were identified as the common bed bug, Cimex lectularius. The C. hemipterus regional samples carried at least three mutations associated with knockdown resistance (kdr), including 2 super-kdr mutations. The 7 C. lectularius regional samples possessed at least one of the 3 kdr-related mutations associated with pyrethroid resistance. This study confirms that the prevalent bed bug species recently in Korea is C. hemipterus, replacing the previously endemic C. lectularius. Additionally, the rise in bed bug populations with pyrethroid resistance underscores the necessity of introducing alternative insecticides.

A novel prolixicin identified in common bed bugs with activity against both bacteria and parasites.

The hematophagous common bed bug, Cimex lectularius, is not known to transmit human pathogens outside laboratory settings, having evolved various immune defense mechanisms including the expression of antimicrobial peptides (AMPs). We unveil three novel prolixicin AMPs in bed bugs, exhibiting strong homology to the prolixicin of kissing bugs, Rhodnius prolixus, and to diptericin/attacin AMPs. We demonstrate for the first time sex-specific and immune mode-specific upregulation of these prolixicins in immune organs, the midgut and rest of body, following injection and ingestion of Gr+ (Bacillus subtilis) and Gr- (Escherichia coli) bacteria. Synthetic CL-prolixicin2 significantly inhibited growth of E. coli strains and killed or impeded Trypanosoma cruzi, the Chagas disease agent. Our findings suggest that prolixicins are regulated by both IMD and Toll immune pathways, supporting cross-talk and blurred functional differentiation between major immune pathways. The efficacy of CL-prolixicin2 against T. cruzi underscores the potential of AMPs in Chagas disease management.

Repellency of N,N-diethyl-3-methylbenzamide (DEET) during host-seeking behavior of bed bugs (Hemiptera: Cimicidae) in binary choice olfactometer assays.

The bed bug (Cimex lectularius L.) is one of the most prolific and burdensome indoor pests, and suppression of bed bug populations is a global priority. Understanding bed bug behavior is important to the development of new tactics for their control. Major gaps exist in our understanding of how host cues, insecticide resistance, and exposure modality impact the repellency of formulated products to bed bugs. Here, we validate the use of a binary choice olfactometer for assessing bed bug repellency behaviors using N,N-diethyl-3-methylbenzamide (DEET) in a dose-dependent manner, while considering the role of host-associated stimuli (with vs. without CO2), exposure modality (olfactory vs. olfactory and contact), and resistance status (susceptible vs. resistant) on repellency. We observed that host-seeking insecticide-susceptible bed bugs were repelled only when olfactorily exposed to high concentrations of DEET. However, exposure to DEET by contact repelled insecticide-susceptible bed bugs at 100-fold lower dose of DEET. Further, we demonstrate for the first time that insecticide-resistant bed bugs were significantly more responsive to DEET than susceptible bed bugs. We conclude that the 2-choice olfactometer is an effective tool for assessing the behavioral responses of bed bugs to spatial and contact repellents.

Activation of immune pathways in common bed bugs, Cimex lectularius, in response to bacterial immune challenges - a transcriptomics analysis.

The common bed bug, Cimex lectularius, is an urban pest of global health significance, severely affecting the physical and mental health of humans. In contrast to most other blood-feeding arthropods, bed bugs are not major vectors of pathogens, but the underlying mechanisms for this phenomenon are largely unexplored. Here, we present the first transcriptomics study of bed bugs in response to immune challenges. To study transcriptional variations in bed bugs following ingestion of bacteria, we extracted and processed mRNA from body tissues of adult male bed bugs after ingestion of sterile blood or blood containing the Gram-positive (Gr+) bacterium Bacillus subtilis or the Gram-negative (Gr-) bacterium Escherichia coli. We analyzed mRNA from the bed bugs' midgut (the primary tissue involved in blood ingestion) and from the rest of their bodies (RoB; body minus head and midgut tissues). We show that the midgut exhibits a stronger immune response to ingestion of bacteria than the RoB, as indicated by the expression of genes encoding antimicrobial peptides (AMPs). Both the Toll and Imd signaling pathways, associated with immune responses, were highly activated by the ingestion of bacteria. Bacterial infection in bed bugs further provides evidence for metabolic reconfiguration and resource allocation in the bed bugs' midgut and RoB to promote production of AMPs. Our data suggest that infection with particular pathogens in bed bugs may be associated with altered metabolic pathways within the midgut and RoB that favors immune responses. We further show that multiple established cellular immune responses are preserved and are activated by the presence of specific pathogens. Our study provides a greater understanding of nuances in the immune responses of bed bugs towards pathogens that ultimately might contribute to novel bed bug control tactics.

The puncture mechanics: an example from the bed bug Cimex lectularius showing traumatic insemination using the paramere.

Cimicidae are well-known for traumatic insemination, and males pierce females with their parameres and transfer sperm through them. The shape of parameres is relatively stable in the family, but in some genera, the paramere is elongated, appearing less resistant against lateral deflection. To understand the mechanical limitations of the paramere, we studied its penetration mechanics of the common bed bug, Cimex lectularius. We examined the post-abdominal morphology, paramere geometry and material properties and conducted breaking stress experiments on the paramere under wet and dry conditions. Mechanical property gradients are present with the paramere tip as the stiffest region and the base as the most flexible one. These mechanical properties relate to the presence of Ca, Zn and Si. The basal wing-shaped structure is flexible, enabling it to interlock with the anal region during mating. The paramere is slightly twisted; the tip region is circular in cross-section, and the geometry of the rest is rather complex. In the mechanical tests, wet parameres mainly buckled, while dried parameres broke off. The level of structural failures depended on directions from which the compression forces were applied. Structural, material and mechanical strengthening mechanisms preventing the paramere from mechanical failure are discussed.

First detection of Rickettsia felis and Ehrlichia canis in the common bed bug Cimex lectularius.

Bed bugs, common blood-feeding pests, have received limited attention regarding their potential involvement in emerging pathogen transmission. This study aimed to investigate the main vector-borne bacteria within bed bugs collected from Tunisian governorates and to genetically characterize the identified species. Molecular screening was conducted on field-collected bed bug samples, targeting zoonotic vector-borne bacteria from the Anaplasmataceae family, as well as the genera Rickettsia, Ehrlichia, Bartonella, and Borrelia. A total of 119 Cimex lectularius specimens were collected and grouped into 14 pools based on sampling Tunisian sites. Using genus-specific PCR assays, DNA of Rickettsia and Ehrlichia spp. was detected in a single pool. Sequencing and BLAST analysis of the obtained partial ompB and dsb sequences from positive samples revealed 100% similarity with those of Ehrlichia canis and Rickettsia felis available in GenBank. Obtained partial sequences showed phylogenetic similarity to R. felis and E. canis isolates found in dogs and ticks from American and European countries. To the best of our knowledge, this study is the first to investigate bed bugs in Tunisia and to report the worldwide identification of R. felis and E. canis DNA in the common bed bug, C. lectularius. These findings highlight the need for further research to explore the potential role of bed bugs in the epidemiology of these vector-borne bacteria.

Identification of a Novel Inhibitor of Cimex lectularius Acetylcholinesterase.

Acetylcholinesterase (AChE) stands as a primary target of commercial insecticides, notably organophosphates and carbamates. Despite their widespread use in agricultural and indoor pest control, concerns over their high toxicity and the emergence of resistance have restricted their efficacy. In this study, we conducted high-throughput virtual screening against both wild-type (WT) and resistant Cimex lectularius AChE utilizing a library encompassing 1 270 000 compounds. From this screening, we identified 100 candidate compounds and subsequently assessed their inhibitory effects on purified AChE enzymes. Among these candidates, AE027 emerged as a potent inhibitor against both WT and resistant AChE, exhibiting IC50 values of 10 and 43 µM, respectively. Moreover, the binding of AE027 significantly stabilized AChE, elevating its melting temperature by approximately 7 °C. Through molecular docking and molecular dynamics simulation, we delineated the binding mode of AE027, revealing its interaction with a site adjacent to the catalytic center, which is distinct from known inhibitors, with differing poses observed between WT and resistant AChE. Notably, the resistance mutation F348Y, positioned at a site directly interfacing with AE027, impedes ligand binding through steric hindrance. Furthermore, we evaluated the toxicity and pharmacokinetic properties of AE027 utilizing bioinformatics tools. These findings lay a crucial foundation for the development of a novel generation of insecticides that can combat both WT and resistant pest populations effectively and safely.

Optimization and establishment of laboratory rearing conditions for Cimex lectularius L. against variable temperature and relative humidity.

Emerging infestations of bed bugs are affecting normal human lifestyle globally. This study has been designed to optimize the rearing conditions for Cimex lectularius L. (Hemiptera), to support the scientific research on them. Bed bugs have been projected onto three different temperature (20 °C, 25 °C, and 30 °C) and relative humidity (50%, 70%, and 90%) conditions to check their overall growth and survival rate. Adult mortality, weight loss, egg laying, percentage hatching, hatching initiation and completion, nymph mortality, and molting have been evaluated to optimize the best conditions. The temperature at 25 °C with 90% RH showed minimum mortality for adults (female 13.33 ± 3.33% and male 6.67 ± 3.33%) and nymphs (13.33 ± 3.33%), while maximum egg laying (40.33 ± 1.86), with highest percentage hatching (98.23 ± 0.58%). At 30 °C with 90% RH, hatching initiation and completion (5.19 ± 0.12 days and 7.23 ± 0.16 days) as well as molting initiation and completion (3.73 ± 0.12 days and 7.00 ± 0.24 days) were found to be fastest. Thus, it can be concluded that 25 °C with 90% RH is ideal for rearing of adults and 30 °C with 90% RH is appropriate for rapid growth of nymphs.

Lethal and sublethal heat-exposure of bed bugs (Cimex lectularius L.) causes alarm pheromone emission and elicits a movement response in nearby recipients.

Many gregarious insect species use aggregation and alarm pheromones. The bed bug, Cimex lectularius L., emits an alarm pheromone (AP), a 70/30 blend of (E)-2-hexenal and (E)-2-octenal, when threatened. Bed bugs avoid temperatures above 43 °C, which are lethal to bugs and used commercially as spatial heat treatments to manage infestations. However, the interaction of bed bug AP in heat avoidance has not been investigated. The goal of this research was to: 1) determine if bed bugs emit AP as an alarm response to heat exposure, and 2) quantify the behavioral responses of conspecifics to AP emitted by heat-exposed bed bugs. Using a selected ion flow tube mass spectrometer, we found that bed bugs responded to lethal and sublethal heat exposure by emitting AP. The Harlan laboratory population emitted more pheromone than a laboratory adapted field population from Florida (McCall). Harlan females emitted the most AP, followed by Harlan males, McCall females and males. In separate behavioral experiments, we showed that conspecifics (i.e., recipients) reacted to AP released by heat exposed bed bugs (i.e., emitters) by frantically moving within 50 mm and 100 mm test arenas. The Harlan recipients reacted to AP in 100 mm areas, whereas the McCall strain did not, indicating a short area of effectiveness of the AP. Synthetic AP components tested in behavioral experiments caused identical effects as the natural AP blend released by heat-exposed bed bugs.

Dynamics of cuticle-associated transcript profiles during moulting of the bed bug Cimexlectularius.

The bed bug Cimex lectularius is a worldwide human pest. The sequenced genome allows molecular analyses of all aspects of bed bug biology. The present work was conducted to contribute to bed bug cuticle biology. As in other insect species, the C. lectularius cuticle consists of the three horizontal layers procuticle, epicuticle and envelope. To analyse the genes needed for the establishment of the stratified cuticle, we studied the expression pattern of 42 key cuticle-related genes at the transition of the penultimate nymphal stage to adult animals when a new cuticle is formed. Based on gene expression dynamics, in simplified model, we distinguish two key events during cuticle renewal in C. lectularius. First, upon blood feeding, modulation of ecdysone signalling culminates in the transcriptional activation of the transcription factor Clec-Ftz-F1 that possibly controls the expression of 32 of the 42 genes tested. Second, timed expression of Clec-Ftz-F1 seems to depend also on the insulin signalling pathway as RNA interference against transcripts of the insulin receptor delays Clec-Ftz-F1 expression and stage transition. An important observation of our transcript survey is that genes needed for the construction of the three cuticle layers are largely expressed simultaneously. Based on these data, we hypothesise a considerable synchronous mechanism of layer formation rather than a strictly sequential one. Together, this work provides a basis for functional analyses of cuticle formation in C. lectularius.

Do bed bugs transmit human viruses, or do humans spread bed bugs and their viruses? A worldwide survey of the bed bug RNA virosphere.

BED BUGS: (Hemiptera: Cimicidae) are a globally distributed hematophagous pest that routinely feed on humans. Unlike many blood-sucking arthropods, they have never been linked to pathogen transmission in a natural setting, and despite increasing interest in their role as disease vectors, little is known about the viruses that bed bugs naturally harbor. Here, we present a global-scale survey of the bed bug RNA virosphere. We sequenced the metatranscriptomes of 22 individual bed bugs (Cimex lectularius and Cimex hemipterus) from 8 locations around the world. We detected sequences from two known bed bug viruses (Shuangao bedbug virus 1 and Shuangao bedbug virus 2) which extends their geographical range. We identified three novel bed bug virus sequences from a tenui-like virus (Bunyavirales), a toti-like virus (Ghabrivirales), and a luteo-like virus (Tolivirales). Interestingly, some of the bed bug viruses branch near to insect-transmitted plant-infecting viruses, opening questions regarding the evolution of plant virus infection. When we analyzed the viral sequences by their host's collection location, we found unexpected patterns of geographical diversity that may reflect humans' role in bed bug dispersal. Additionally, we investigated the effect that Wolbachia, the primary bed bug endosymbiont, may have on viral abundance and found that Wolbachia infection neither promotes nor inhibits viral infection. Finally, our results provide no evidence that bed bugs transmit any known human pathogenic viruses.

Identification of the pan-allergen tropomyosin from the common bed bug (Cimex lectularius).

Tropomyosin is a muscle contraction protein documented across all animal life. Despite its ubiquity, its unique structure in invertebrates leads to allergic responses in humans that vertebrate tropomyosin does not. High degrees of homology can explain cross-reactivity between tropomyosin derived from distantly-related arthropod species and establishes tropomyosin as a panallergen. Given this cross-reactivity and that they are commonly found in high numbers indoors, research on the potential of the common bed bug (Cimex lectularius L.) to contribute tropomyosin to the indoor environment is needed. Therefore, we investigated tropomyosin homology between bed bugs and known tropomyosin allergens from other taxa, tropomyosin in bed bug bodies, feces, and exuviae (cast skins), tropomyosin persistence over time, and impacts of common bed bug treatment strategies on detectable tropomyosin. Tropomyosin was detected in mechanically fractured bed bug cadavers and was detectable in bed bugs cadavers aged for 18 months. Additionally, a survey of pest management professionals showed dead bed bugs are not cleaned up following treatment. As such, dead bed bugs could act as tropomyosin reservoirs following bed bug treatment and exposure to tropomyosin from bed bugs could sensitize individuals and lead to increased responses to other arthropod tropomyosin.

Population genetic structure of tropical bed bug (Hemiptera: Cimicidae) populations and their breeding pattern in Iraq.

A study was conducted to investigate the population genetic structure and breeding pattern of 140 tropical bed bugs, Cimex hemipterus (F.) (Hemiptera: Cimicidae), collected from 14 infested sites in major cities in Iraq. The samples were genotyped using a set of 7 polymorphic microsatellite markers. High genetic variety was seen among populations, with an average of 2-9 alleles per locus. The number of alleles across 7 microsatellite loci was between 6 and 18. There was a notable disparity in the alleles per loci when comparing the overall population to those within it. The overall population exhibited an average observed heterozygosity of 0.175 and an average expected heterozygosity of 0.730. Among the population, the average observed heterozygosity was 0.173, while the average expected heterozygosity was 0.673. Analysis of molecular variance (AMOVA) revealed that 93% of the genetic variability was within the populations, and 7% was among them. The genetic differentiation coefficient (FST = 0.045), indicates a low degree of genetic differentiation and a high degree of inbreeding (FIS = 0.761), as indicated by notably significant positive inbreeding coefficients. Admixed individuals were revealed using STRUCTURE and neighbor-joining phylogenetic trees, demonstrating moderate gene flow between populations and a lack of genetic structure in the regional groups. Thus, both active dispersion and human-mediated dispersion possess the potential to influence the low population genetic structure of tropical bed bug C. hemipterus populations in Iraq, which can have implications toward tropical bed bug and management strategies.

Bat ectoparasites: chigger mites (Trombiculidae), ticks (Ixodidae and Argasidae), and bugs (Cimicidae) in the Eastern Palaearctic.

Nine species-level taxa of bat ectoparasites, three chigger mites (Trombiculidae), three hard (Ixodidae), and one soft tick (Argasidae) species, as well as two bug (Cimicidae) species from nine bat species hosts were detected in the Eastern Palaearctic. Trombiculid larvae of Leptotrombidium schlugerae, Leptotrombidium album, and Ascoschoengastia latyshevi were first recorded on bats in the temperate zone of eastern Russia. L. schlugerae was more abundant than A. latyshevi in the same study sites in Eastern Siberia, and the main hosts of both chigger species were Plecotus ognevi and Eptesicus nilssonii. Ixodid ticks Dermacentor marginatus, Ixodes simplex, and Ixodes sp. were sampled from bats in Kazakhstan, the Far East, and Eastern Siberia, respectively. Phylogenetic analysis based on Cox1, 16S rDNA, and ITS2 sequences of I. simplex showed that the specimens from the Far East grouped into a clade distributed in the Eastern Palaearctic and India. In turn, the specimen of Ixodes sp. from Eastern Siberia was most closely related to Ixodes soricis and Ixodes angustus with p-distance of 9.8-10.7% (Cox1), suggesting that this tick probably belongs to a new species. Argas vespertilionis larvae were collected from three widespread bat species in Kazakhstan. Two bug species, Cimex pipistrelli and Cimex aff. lectularius, were recorded in the Far East and Eastern Siberia, respectively. Specimens from Transbaikalia were morphologically identified as Cimex lectularius. However, they differed from the latter by 12.5-12.9% of Cox1 sequences, indicating that C. aff. lectularius may be a new species.

Experimental Acquisition, Maintenance, and Transmission of Methicillin-Resistant Staphylococcus aureus by the Common Bed Bug, Cimex lectularius.

Here, we fed bed bugs through a membrane contaminated with methicillin-resistant Staphylococcus aureus (MRSA) at a concentration naturally present on human skin. We then determined the amount of viable MRSA present on their surface and internally over a period of 7 days. We also determined whether bed bugs that fed through the contaminated membrane could transmit MRSA to an uncontaminated membrane when taking a second blood meal 7 days later. Bed bugs acquired MRSA both externally on the cuticle surface as well as internally when feeding. MRSA was found to persist for 7 days both externally and internally in some bed bugs. Furthermore, MRSA replicated internally but not externally. Most importantly, bed bugs were able to transmit MRSA to an uncontaminated membrane feeder in 2 of 3 trials. These findings provide the first experimental support for the hypothesis that bed bugs may contribute to the transmission of MRSA in some settings.

Effectiveness of heat treatment in rapid control of bed bugs in environmental conditions resembling their natural habitats.

We evaluated lethal temperatures and times for killing bed bugs in diverse covered and uncovered conditions simulating their natural habitats. A total of 5400 adult bed bugs were collected alive from 17 infested locations in Paris. They were morphologically identified in laboratory as Cimex lectularius. They were then distributed in multiple sets of 30 specimens to examine in covered (tissue, furniture, mattress or blanket) and uncovered (direct exposure) conditions and in diverse step-function temperatures (50, 55 and 60°C) and times (15, 30, 60 and 120 minutes), replicated three times. Effective mortality was observed in 1080 specimens exposed directly to 50°C for 60 minutes. In specimens covered by tissue (1080 specimens), furniture (1080) or mattress (1080), all were dead at 60°C within 60 minutes. The specimens covered by blanket (1080) at the same temperature were dead after 120 minutes. A 60-minutes delay in reaching to lethal temperature within blanket compared to uncovered thermometer was observed.