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.

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.

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.

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.

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.

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.

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.

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.

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.

Repellent effect of two anthranilates against the tropical bed bug Cimex hemipterus (Hemiptera: Cimicidae) demonstrated in laboratory assays.

BACKGROUND: The bites and blood sucking of bed bugs (Cimex spp.) (Hemiptera: Cimicidae) pose a serious threat to human physical and mental health. Application of an effective repellent can prevent or reduce bed bug bites. Previous studies on repellent screening mainly focused on Cimex lectularius L. In this study, we investigated the repellent effect of two safe food additives, ethyl anthranilate (EA) and butyl anthranilate (BA), against Cimex hemipterus (F.), and also explored the role of antennae and mouthparts on C. hemipterus perception of repellents. RESULTS: Both EA and BA had a strong repellent effect against tropical bed bugs and their repellency was similar or lower than that of N,N-diethyl-3-methyl benzoyl amide, depending on whether or not a CO2 source was present. EA had higher repellency than BA and exhibited repellency to C. hemipterus for 3 days when a CO2 source was present. C. hemipterus avoided resting on 20% EA- and BA-treated harborages. Applying 20% EA and BA on rabbit skin significantly reduced the blood intake of C. hemipterus within 2 h. C. hemipterus could perceive EA and BA after their antennae or mouthparts or both antennae and mouthparts were removed. CONCLUSION: Both EA and BA had strong repellency against C. hemipterus, with EA being more repellent. Ablation of antennae and mouthparts did not affect the perception of EA and BA by C. hemipterus. © 2023 Society of Chemical Industry.

Changes in body size and fertility due to artificial and natural feeding of laboratory common bed bugs (Heteroptera: Cimicidae).

Rearing common bed bugs (Cimex lectularius L.) and other hematophagous insects is essential for basic, medical, and pest-control research. Logistically, acquiring fresh blood can be a challenge, while biologically, the eventual effects of different rearing and blood preparation protocols on bed bug genotype and phenotype pose a risk of biased research results. Using bed bug populations that are either bat- (BL) or human-related (HL), we tested the short- and long-term effects of rearing bugs on live bats or human volunteers, or artificially on CPDA (citrate phosphate dextrose, adenine)-treated blood, measuring meal size, body size, and fertility. We found that artificial feeding did not affect meal size compared with feeding on natural hosts. Long-term rearing across many generations of HL on CPDA-preserved blood led to reduced body size and fertility compared with populations reared on human volunteers. Blood preservatives increased the proportion of sterile eggs even after a single feed. Finally, our results indicated that laboratory reared bed bugs were smaller, regardless of the blood source, than wild bugs. Similar effects of artificial feeding or laboratory rearing alone should be considered in future studies using bed bug cultures to choose an appropriate rearing protocol. With regard to switching between bat and human hosts, HL took smaller meals and BL had lower fertility when fed on bats than when fed on humans. We attribute these results to methodological constrains, specifically the inconsistency of bat feeding, rather than to host specialization. Nevertheless, BL can be easily reared using human blood and artificial feeding systems.

The evolution of female-biased genital diversity in bedbugs (Cimicidae).

Rapid genitalia evolution is believed to be mainly driven by sexual selection. Recently, noncopulatory genital functions have been suggested to exert stronger selection pressure on female genitalia than copulatory functions. In bedbugs (Cimicidae), the impact of the copulatory function can be isolated from the noncopulatory impact. Unlike in other taxa, female copulatory organs have no function in egg-laying or waste-product expulsion. Males perform traumatic mating by piercing the female integument, thereby imposing antagonistic selection on females and suspending selection to morphologically match female genitalia. We found the location of the copulatory organ evolved rapidly, changing twice between dorsal and ventral sides, and several times along the anteroposterior and the left-right axes. Male genital length and shape varied much less, did not appear to follow the positional changes seen in females, and showed no evidence for coevolution. Female genitalia position evolved 1.5 times faster than male genital length and shape and showed little neutral or geographic signals. Instead, we propose that nonmorphological male traits, such as mating behavior, may drive female genitalia morphology in this taxon. Models of genitalia evolution may benefit from considering morphological genital responses to nonmorphological stimuli, such as male mating behavior or copulatory position.

Morphological, molecular, and MALDI-TOF MS identification of bed bugs and associated Wolbachia species from Cameroon.

After vanishing from the public eye for more than 50 years, bed bugs have resurged to become one of the most widely discussed and heavily researched insect pests in the world. This study presents the basic information of infestations of tropical bed bugs, Cimex hemipterus (Hemiptera: Cimicidae), in Cameroon. A total of 248 immature stage and adult bed bug specimens were collected from households and a travel agency in Yaoundé and Douala, Cameroon. The ability of MALDI-TOF MS to identify bed bugs was tested using heads for adults and cephalothoraxes for immature stages. Microorganism screening was performed by qPCR and confirmed by regular PCR and sequencing. Based on morphometrical criteria, four stages of immature bed bugs are represented. Of the 248 bed bug specimens morphologically identified as Cimex hemipterus, 246 (77 males, 65 females and 104 immature specimens) were submitted to MALDI-TOF MS analysis. Of the 222 adults and immature specimens tested, 122 (59.9 %) produced good quality MALDI-TOF MS spectra (35 adults and 87 immature specimens). Blind testing allowed species level identification of 98.21 % of adult and immature C. hemipterus. Among the bacteria tested, only Wolbachia DNA was found in 12/246 (4.8 %) bed bugs. More surveys in the country are warranted to assess the true level of bed bug infestations, in order to take appropriate action for their control.

A metatranscriptomic evaluation of viruses in field-collected bed bugs.

Cimex lectularius, known as the common bed bug, is a widespread hematophagous human ectoparasite and urban pest that is not known to be a vector of any human infectious disease agents. However, few studies in the era of molecular biology have profiled the microorganisms harbored by field populations of bed bugs. The objective of this study was to examine the viruses present in a large sampling of common bed bugs and related bat bugs (Cimex pipistrelle). RNA sequencing was undertaken on an international sampling of > 500 field-collected bugs, and multiple workflows were used to assemble contigs and query these against reference nucleotide databases to identify viral genomes. Shuangao bed bug virus 2, an uncharacterized rhabdovirus previously discovered in Cimex hemipterus from China, was found in several bed bug pools from the USA and Europe, as well as in C. pipistrelle, suggesting that this virus is common among bed bug populations. In addition, Shuangao bed bug virus 1 was detected in a bed bug pool from China, and sequences matching Enterobacteria phage P7 were found in all bed bug pools, indicating the ubiquitous presence of phage-derived elements in the genome of the bed bug or its enterobacterial symbiont. However, viral diversity was low in bed bugs in our study, as no other viral genomes were detected with significant coverage. These results provide evidence against frequent virus infection in bed bugs. Nonetheless, our investigation had several important limitations, and additional studies should be conducted to better understand the prevalence and composition of viruses in bed bugs. Most notably, our study largely focused on insects from urban areas in industrialized nations, thus likely missing infrequent virus infections and those that could occur in rural or tropical environments or developing nations.

The common bed bug Cimex lectularius synthesizes hemozoin as an essential defense against the toxic effects of heme.

The common bed bug Cimex lectularius (Linnaeus 1758) is an ectoparasite that feeds preferably on human blood, being considered an important public health issue. Blood-feeding is a challenging process for hematophagous organisms, and one of the inherent risks with this kind of diet is the liberation of high doses of free heme after the digestion of hemoglobin. In order to deal with this potent cytotoxic agent, such organisms have acquired different defense mechanisms. Here, we use UV-visible spectrophotometry and infrared spectroscopy to show that C. lectularius crystalizes free heme to form the much less dangerous compound, hemozoin. According to our results, the peak of formation of hemozoin in the intestinal contents occurred 4-5 days after the blood meal, primarily in the posterior midgut. The quantification of the rate of conversion of heme to hemozoin revealed that at the end of digestion all the heme was in the form of hemozoin. Inhibition of the synthesis of hemozoin using the anti-malarial drug quinine led to an increase in both catalase activity in the intestinal epithelium and the mortality of the bed bugs, indicating that the insects were unable to cope with the oxidative stress generated by the overload of free heme. The data presented here show for the first time how C. lectularius deals with free heme, and how the process of formation of hemozoin is essential for the survival of these insects. Since resistance to insecticides is a common feature among field populations of bed bugs, there is an urgent need to develop alternative control methods. Thus, targeting the synthesis of hemozoin emerges as a possible novel strategy to fight bed bugs.

Molecular characterization and genetic diversity of Wolbachia endosymbionts in bed bugs (Hemiptera; Cimicidae) collected in Paris.

PURPOSE: This study aimed to investigate the genetic diversity of Wolbachia in field-caught bed bug species in Paris areas. METHODS: The bed bug specimens were captured from various infested localities in Paris and surrounding cities. They belonged to diverse life stages, including egg, nymph, and adult. They were then identified using morphological and molecular approaches. Furthermore, Wolbachia was detected, and its genetic diversity was investigated by conventional PCR of 16S-rRNA and Wolbachia surface protein (wsp) genes. RESULTS: A total of 256 bed bug specimens belonging to various life stages [adult (183 specimens), nymph (48), and egg (25)] were captured from seven private apartments, five social apartments, three houses, two immigrant residences, and one retirement home situated in 10 districts of Paris and 8 surrounding cities. They were identified as Cimex lectularius (237 specimens) and C. hemipterus (19) using morphological and molecular approaches. The presence and diversity of Wolbachia were ascertained by targeting 16S-rRNA and wsp genes. Based on molecular analysis, 182 and 148 out of 256 processed specimens were positive by amplifying 16S-rRNA and wsp fragments, respectively. The inferred phylogenetic analysis with 16S-rRNA and wsp sequences displayed monophyletic Wolbachia strains clustering each one in three populations. The median-joining network, including the Wolbachia 16S-rRNA and wsp sequences of C. lectularius and C. hemipterous specimens, indicated a significant genetic differentiation among these populations in Paris areas which was consent with Neighbor-Joining analyses. A phylogenetic analysis of our heterogenic Wolbachia sequences with those reported from other arthropod species confirmed their belonging to supergroup F. Moreover, no difference between Wolbachia sequences from eggs, nymphs, and adults belonging to the same clade and between Wolbachia sequences of C. lectularius and C. hemipterus were observed after sequence alignment. Furthermore, no significant correlation was found between multiple geographical locations (or accomodation type) where bed bugs were collected and the genetic diversity of Wolbachia. CONCLUSIONS: We highlight a significant heterogeneity within Wolbachia symbionts detected in C. lectularius and C. hemipterus. No correlation between Wolbachia species and bed bug species (C. lectularius versus C. hemipterus), physiological stages (egg, nymph, and adult), and sampling location was recorded in this study.