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Bed bugs (Hemiptera: Cimicidae) are widely distributed, obligately blood-feeding insects, but they have never been linked to pathogen transmission in humans. Most other hematophagous insects that frequently bite humans transmit pathogens, and it is unclear why bed bugs do not. One hypothesis is that bed bugs have evolved a highly robust immune system because their mating system, traumatic insemination, exposes females to consistent wounding and bacterial infections. Although this has been proposed, very little is known about the bed bug immune system and how bed bugs respond to microbial challenges introduced by wounding. Similarly, there is little known about how the bed bug immune system responds to human pathogens. Understanding the bed bug immune system could give insight to why bed bugs appear not to transmit disease and under what circumstances they could, while also facilitating biological control efforts involving microbes. To investigate the transcriptomic response of bed bugs to immune challenges, we exposed female bed bugs to three bacterial challenges. 1.) Pseudomonas fluorescens, an entomopathogen known to have harmful effects to bed bugs, 2.) bacteria cultured from a bed bug enclosure (99.9 % Bacillus spp.), likely encountered during traumatic insemination, and 3.) Borrelia duttoni, a human vector-borne pathogen that causes relapsing fever. We compared the transcriptomes of infected bed bugs with uninfected matched controls in a pairwise fashion, focusing on immune-related genes. We found many known antimicrobial effector genes upregulated in response to P. fluorescens and traumatic insemination-associated bacteria, but interestingly, not in response to B. duttoni. In the differentially expressed genes that were shared between experiments, we found significant overlap in the P. fluorescens treatment and the traumatic insemination bacteria treatment, and between the P. fluorescens and B. duttoni treatments, but not between the traumatic insemination bacteria treatment and the B. duttoni treatment. Finally, we identify previously overlooked candidates for future studies of immune function in bed bugs, including a peroxidase-like gene, many putative cuticle-associated genes, a laccase-like gene, and a mucin-like gene. By taking a comprehensive transcriptomic approach, our study is an important step in understanding how bed bugs respond to diverse immune challenges., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jose E. Pietri reports financial support was provided by US Army Contracting Command. Jonas G. King reports financial support was provided by Foundation for Food and Agriculture Research. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Background: Bartonella quintana is a body louse-borne bacterium causing bacteremia and infective endocarditis. We aimed to describe B. quintana detection among arthropods and their hosts., Methods: We searched databases in PubMed Central/MEDLINE, Scopus, Embase, and Web of Science from January 1, 1915 (the year of B. quintana discovery) to January 1, 2024, to identify publications containing specific search terms relating to B. quintana detection among arthropods. Descriptive statistics and meta-analysis of pooled prevalence using random-effects models were performed for all arthropods and body and head lice., Results: Of 1265 records, 62 articles were included, describing 8839 body lice, 4962 head lice, and 1692 other arthropods, such as different species of fleas, bedbugs, mites, and ticks. Arthropods were collected from 37 countries, of which 28 had arthropods with B. quintana DNA. Among articles that reported B. quintana detection among individual arthropods, 1445 of 14,088 (0.1026, 95% CI [0.0976; 0.1077]) arthropods tested positive for B. quintana DNA, generating a random-effects model global prevalence of 0.0666 (95% CI [0.0426; 0.1026]). Fifty-six studies tested 8839 body lice, of which 1679 had B. quintana DNA (0.1899, 95% CI [0.1818; 0.1983]), generating a random-effects model pooled prevalence of 0.2312 (95% CI [0.1784; 0.2843]). Forty-two studies tested 4962 head lice, of which 390 head lice from 20 studies originating from 11 different countries had B. quintana DNA (0.0786, 95% CI [0.0713; 0.0864]). Eight studies detected B. quintana DNA exclusively on head lice. Five studies reported greater B. quintana detection on head lice than body lice; all originated from low-resource environments., Conclusions: Bartonella quintana is a vector-borne bacterium with a global distribution, disproportionately affecting marginalized populations. Bartonella quintana DNA has been detected in many different arthropod species, though not all of these arthropods meet criteria to be considered vectors for B. quintana transmission. Body lice have long been known to transmit B. quintana. A limited number of studies suggest that head lice may also act as possible vectors for B. quintana in specific low-resource contexts., (© 2024. The Author(s).)

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., Competing Interests: Declaration of Competing Interest The authors declared that they have no potential conflicts of interest with respect to the research, authorship and publication of this article., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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., (© 2024. The Author(s).)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Background: Cimex lectularius L. (bed bug) (Hemiptera: Cimicidae) is a serious indoor pest worldwide, and this nuisance needs to be controlled using different methods in integrated pest management (IPM). Beauveria bassiana (Bals.-Criv.) Vuill. (Hypocreales: Cordycipitaceae) kills bed bugs, and insect pathogenic fungi may be utilized to control bed bugs in IPM. To increase knowledge of this methodology, forced exposure experiments were conducted with different formulations, doses, and substrates, using bed bugs in variable physiological states., Results: Both oil- and water-formulated fungal products showed significant improvement when conidial concentrations were raised in five steps from 0.02 to 2.0%. At low concentrations (0.02% in water) effects from substrate and application strategy were observed. Application on soft substrates (cotton and polyester) yielded significantly higher bed bug mortality rates than on harder substrates (paper, wood, and linoleum) with a final mortality of 35-63% against 8-10%. Multiple applications over time also improved B. bassiana's ability to kill bed bugs, and at low concentrations only a triple application on cotton showed 100% final mortality. Bed bug age and reproductive status significantly affected survival. Older and reproducing individuals showed higher mortality compared to newly emerged adults. Differences in feeding status also yielded differences in mortality timing, but only minor differences in final mortality rates. Egg production and hatching success were significantly reduced by some treatments., Conclusion: B. bassiana appears to be an asset in the fight against bed bugs. Substrate, dosage, application strategy, and bed bug physiology are important factors to consider for optimal efficacy and safe indoor control with insect pathogenic fungi. © 2023 Society of Chemical Industry., (© 2023 Society of Chemical Industry.)

The use of the entomopathogenic fungus Beauveria bassiana (Bals. - Criv.) Vuill. (Hypocreales: Cordycipitaceae) has been recently incorporated in the management of bed bugs, Cimex lectularius L. (Hemiptera: Cimicidae). Bed bugs produce a set of aldehydes that are known to affect the growth of some fungi. Considering that bed bugs or their exuviae release these aldehydes, it was suspected that the bed bugs' aggregation sites would contain an increased level of the bed bug aldehydes. The current study examined if elevated levels of the bed bug aldehydes in the microhabitats would impact the efficacy of B. bassiana. Following a brief exposure to the residues of commercial products containing B. bassiana, the treated bed bugs were kept in a vial with or without a natural or artificial blend of bed bug aldehydes (i.e., exuviae or synthetic compounds). For a B. bassiana product that is not currently registered for bed bugs control, the presence of aldehydes significantly reduced 15-d mortality (61-62%) compared to the no aldehydes control (97.7%). However, when tested with a B. bassiana formulation designed for bed bug control, the aldehydes only caused delayed mortality for the treated bed bugs. When tested in culture, the growth rate of B. bassiana on a medium was significantly reduced when the bed bug aldehydes were provided in the headspace. Implications on practical bed bug management using fungal biopesticides are discussed., (© The Author(s) 2022. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Common bed bugs, Cimex lectularius , can carry, but do not transmit, pathogens to the vertebrate hosts on which they feed. Some components of the innate immune system of bed bugs, such as antimicrobial peptides (AMPs), eliminate the pathogens. Here, we determined the molecular characteristics, structural properties, and phylogenetic relatedness of two new defensins (CL-defensin1 (XP_024085718.1), CL-defensin2 (XP_014240919.1)), and two new defensin isoforms (CL-defensin3a (XP_014240918.1), CL-defensin3b (XP_024083729.1)). The complete amino acid sequences of CL-defensin1, CL-defensin2, CL-defensin3a, and CL-defensin3b are strongly conserved, with only minor differences in their signal and pro-peptide regions. We used a combination of comparative transcriptomics and real-time quantitative PCR to evaluate the expression of these defensins in the midguts and the rest of the body of insects that had been injected with bacteria or had ingested blood containing the Gram-positive (Gr+) bacterium Bacillus subtilis and the Gram-negative (Gr-) bacterium Escherichia coli . We demonstrate, for the first time, sex-specific and immunization mode-specific upregulation of bed bug defensins in response to injection or ingestion of Gr+ or Gr- bacteria. Understanding the components, such as these defensins, of the bed bugs' innate immune systems in response to pathogens may help unravel why bed bugs do not transmit pathogens to vertebrates.

Borrelia recurrentis is the causative agent of louse-borne relapsing fever and the only Borrelia species transmitted by an insect rather than a tick vector. While bed bugs (Cimex lectularius L.) are not established vectors of any human pathogens, a recent study reported that they may be competent vectors of B. recurrentis. However, many aspects of infection and transmission remain unclear in this possible secondary vector. Here, we carried out several quantitative laboratory studies to gain a better understanding of the host suitability of bed bugs relative to the established body louse vector as well as the factors that may affect the ability of bed bugs to transmit the pathogen. We fed bed bugs B. recurrentis and estimated the level and duration of infection in the hemolymph using live imaging. We performed quantitative PCR (qPCR) to examine whole-body spirochete levels and the occurrence of vertical transmission to progeny. We also developed an assay to compare the amounts of force required to release infectious hemolymph from recently engorged bed bugs and body lice. Finally, we analyzed humoral antibacterial activity in the hemolymph, hemolymph pH, and hemocyte activity in both insect species. Our results confirm that within 24 h of ingestion, B. recurrentis can penetrate the midgut epithelium of bed bugs and enter the hemolymph, overcoming a major host barrier, as in body lice. Once in the hemolymph, spirochetes remain visible for at least 4 days. Moreover, we show that bed bugs are more physically susceptible to crushing than body lice, suggesting that crushing is a feasible route for the natural dissemination of B. recurrentis from the hemolymph of bed bugs, as for body lice. Nonetheless, our data also indicate that bed bugs are suboptimal hosts for B. recurrentis , as the bacterium does not appear to proliferate to high levels or stably colonize the hemolymph and exhibits pleomorphism in this environment. In particular, our data suggest that hemolymph pH and unique cellular immune responses, rather than humoral effectors, may be involved in limiting spirochete survival in bed bugs. Notably, we document the formation of extracellular DNA traps by bed bug hemocytes for the first time. For these reasons, while bed bugs may be capable of limited transmission given their ecology, vector competence is probably minimal relative to body lice. Additional mechanistic studies of human pathogen infection of bed bugs may provide much-needed insight into the biological factors that restrict their ability to act as vectors and may reveal novel mechanisms of immunity.

The genus Francisella includes several highly virulent human pathogens and some tick endosymbionts. Francisella infections are acquired by humans through contact with vertebrate animal reservoirs or contaminated water or dust. The species Francisella tularensis can also be transmitted by arthropods including ticks, mosquitoes, and flies. For the first time, we describe the molecular detection of an F. tularensis- like bacterium in bed bugs from samples collected in rural Madagascar. This finding suggests a potential involvement of bed bugs in the ecology of Francisella . The role of bed bugs as possible hosts, reservoirs, or vectors of Francisella spp. should be further investigated.

Background: The Cimicidae are obligatory blood-feeding ectoparasites of medical and veterinary importance. We aim in the current study to assess the ability of MALDI-TOF MS to identify Cimex hirundinis swallow bugs collected in house martin nests., Methods: Swallow bugs were picked out from abandoned nests of house martin swallows and identified morphologically to the species level. The bugs were randomly selected, dissected and then subjected to MALDI-TOF MS and molecular analyses., Results: A total of 65 adults and 50 nymphs were used in the attempt to determine whether this tool could identify the bug species and discriminate their developmental stages. Five adults and four nymphs of C. hirundinis specimens were molecularly identified to update our MS homemade arthropod database. BLAST analysis of COI gene sequences from these C. hirundinis revealed 98.66-99.12% identity with the corresponding sequences of C. hirundinis of the GenBank. The blind test against the database supplemented with MS reference spectra showed 100% (57/57) C. hirundinis adults and 100% (46/46) C. hirundinis nymphs were reliably identified and in agreement with morphological identification with logarithmic score values between 1.922 and 2.665. Ninety-nine percent of C. hirundinis specimens tested were positive for Wolbachia spp. The sequencing results revealed that they were identical to Wolbachia massiliensis, belonging to the new T-supergroup strain and previously isolated from C. hemipterus., Conclusions: We report for the first time to our knowledge a case of human infestation by swallow bugs (C. hirundinis) in France. We also show the usefulness of MALDI-TOF MS in the rapid identification of C. hirundinis specimens and nymphs with minimal sample requirements. We phylogenetically characterized the novel Wolbachia strain (W. massiliensis) infecting C. hirundinis and compared it to other recognized Wolbachia clades., (© 2021. The Author(s).)

Unlike almost all hematophagous insects, common bed bugs, Cimex lectularius, are not known to transmit pathogens to humans. To help unravel the reasons for their lack of vector competence, we studied the time- and tissue-dependent expression of innate immune factors after blood feeding or immune activation through the intrathoracic injection of bacteria. We used minimum inhibitory concentration (MIC 1 ) bioassays and the Kirby-Bauer protocol to evaluate antimicrobial peptide (AMP 2 ) activity in tissue extracts from the midguts or 'rest of body' (RoB 3 ) tissues (containing hemolymph and fat body AMPs) against Gram-positive and Gram-negative bacteria. We compared AMP activity between blood-fed female bed bugs and yellow fever mosquitoes, Aedes aegypti and determined how female and male bed bugs respond to immune challenges, and how long AMP gene expression remains elevated in bed bugs following a blood meal. Blood meal-induced AMP activity is 4-fold stronger in female bed bugs than in female mosquitoes. Male bed bugs have elevated AMP activity within 8 h of a blood meal or an intrathoracic injection with bacteria, with the strongest activity expressed in RoB tissue 24 h after the immune challenge. Female bed bugs have a stronger immune response than males within 24 h of a blood meal. The effects of blood meal-induced elevated AMP activity lasts longer against the Gram-positive bacterium, Bacillus subtilis, than against the Gram-negative bacterium Escherichia coli. Unravelling the specific immune pathways that are activated in the bed bugs' immune responses and identifying the bed bug-unique AMPs might help determine why these insects are not vectors of human parasites., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

With the development of new metagenomic techniques, the microbial community structure of common bed bugs, Cimex lectularius, is well-studied, while information regarding the constituents of the bacterial communities associated with tropical bed bugs, Cimex hemipterus, is lacking. In this study, the bacteria communities in the blood-fed and starved tropical bed bugs were analysed and characterized by amplifying the v3-v4 hypervariable region of the 16S rRNA gene region, followed by MiSeq Illumina sequencing. Across all samples, Proteobacteria made up more than 99% of the microbial community. An alpha-proteobacterium Wolbachia and gamma-proteobacterium, including Dickeya chrysanthemi and Pseudomonas, were the dominant OTUs at the genus level. Although the dominant OTUs of bacterial communities of blood-fed and starved bed bugs were the same, bacterial genera present in lower numbers were varied. The bacteria load in starved bed bugs was also higher than blood-fed bed bugs.

Common bed bugs (Cimex lectularius L.) are hematophagous pests present in urban environments across the globe. It is widely established that they have a strong host preference for humans. However, there are records of C. lectularius feeding upon a range of mammalian and avian hosts, including rodents, in the field. There is little information available about how frequently common bed bugs feed on alternative hosts in residential settings, but understanding this phenomenon has implications for both management of infestations and public health. Here, we examined cohorts of C. lectularius collected from 13 different dwellings in the state of New Jersey, USA, that were known to be simultaneously infested with house mice (Mus musculus domesticus). Host-specific quantitative polymerase chain reaction (qPCR) was used to determine if blood meals were taken from mice, while 16S rRNA gene amplicon sequencing was used to screen the bed bugs for the presence of zoonotic bacterial pathogens. We found no evidence that any of the bed bugs we collected fed on mice. Furthermore, the insects harbored depauperate bacterial communities that did not include known human pathogens. However, host-specific qPCR detected feline DNA in a pool of bed bugs from one dwelling, suggesting that interaction with domestic pets should be further investigated. Although sampling in this study was limited, the approach described herein will be useful for additional studies of the interactions between bed bugs and alternative blood meal hosts.

Bed bugs (Cimex lectularius L.) have returned as a nuisance pest in the last 20 years. Different bed bug control measures in combination have not been thoroughly studied, although induction of multiple stressors may improve extermination. The effects of heat stress only, heat stress followed by exposure to insect pathogenic fungi, and heat stress followed by exposure to desiccant dust on starved and blood-fed bed bugs were investigated. Five days at 22 °C (control), 32 °C, 34 °C, or 36 °C (heat stress) did not cause mortality in adults. However, their starved first instar nymphs produced after heat stress suffered mortalities of 33%, 56% and 100%, respectively. Exposure to insect pathogenic fungi after heat stress increased the mortality of adults and their progeny compared to exposure to fungi without heat stress. The beneficial effects of heat stress were not observed in blood-fed bed bugs. Desiccant dust killed all nymphs within 2 days and all adults within 3 days regardless of previous heat stress, but survival time was prolonged by access to blood. This study highlights the advantage of combining different methods in pest management, and points to heat stress combined with blood deprivation as possible management elements to increase the control success., (© 2020 The Authors. Medical and Veterinary Entomology published by John Wiley & Sons Ltd on behalf of The Royal Entomological Society.)

The chitinases have extensive biotechnological potential but have been little exploited commercially due to the low number of good chitinolytic microorganisms. The purpose of this study was to identify a chitinolytic fungal and optimize its production using solid state fermentation (SSF) and agroindustry substrate, to evaluate different chitin sources for chitinase production, to evaluate different solvents for the extraction of enzymes produced during fermentation process, and to determine the nematicide effect of enzymatic extract and biological control of Meloidogyne javanica and Meloidogyne incognita nematodes. The fungus was previously isolated from bedbugs of Tibraca limbativentris Stal (Hemiptera: Pentatomidae) and selected among 51 isolated fungal as the largest producer of chitinolytic enzymes in SSF. The isolate UFSMQ40 has been identified as Trichoderma koningiopsis by the amplification of tef1 gene fragments. The greatest chitinase production (10.76 U gds -1 ) occurred with wheat bran substrate at 55% moisture, 15% colloidal chitin, 100% of corn steep liquor, and two discs of inoculum at 30 °C for 72 h. Considering the enzymatic inducers, the best chitinase production by the isolated fungus was achieved using chitin in colloidal, powder, and flakes. The usage of 1:15 g/mL of sodium citrate-phosphate buffer was the best ratio for chitinase extraction of SSF. The Trichoderma koningiopsis UFSMQ40 showed high mortality of M. javanica and M. incognita when applied to treatments with enzymatic filtrated and the suspension of conidia.

The symbiotic Wolbachia are the most sophisticated mutualistic bacterium among all insect-associated microbiota. Wolbachia -insect relationship fluctuates from the simple facultative/parasitic to an obligate nutritional-mutualistic association as it was the case of the bedbug- Wolbachia from Cimex lectularius . Understanding this association may help in the control of associated arthropods. Genomic data have proven to be reliable tools in resolving some aspects of these symbiotic associations. Although, Wolbachia appear to be fastidious or uncultivated bacteria which strongly limited their study. Here we proposed Drosophila S2 cell line for the isolation and culture model to study Wolbachia strains. We therefore isolated and characterized a novel Wolbachia strain associated with the bedbug Cimex hemipterus , designated as wChem strain PL13, and proposed Wolbachia massiliensis sp. nov. strain wChem -PL13 a type strain of this new species from new supergroup T. Phylogenetically, T-supergroup was close to F and S-supergroups from insects and D-supergroup from filarial nematodes. We determined the 1,291,339-bp genome of wChem -PL13, which was the smallest insect-associated Wolbachia genomes. Overall, the wChem genome shared 50% of protein coding genes with the other insect-associated facultative Wolbachia strains. These findings highlight the diversity of Wolbachia genotypes as well as the Wolbachia -host relationship among Cimicinae subfamily. The wChem provides folate and riboflavin vitamins on which the host depends, while the bacteria had a limited translation mechanism suggesting its strong dependence to its hosts. However, the clear-cut distinction between mutualism and parasitism of the wChem in C . hemipterus cannot be yet ruled out.

Bed bugs ( Cimex spp.) are common ectoparasites of humans. Their ubiquity across diverse human environments combined with their blood-feeding behavior creates an ideal interface for the transmission of pathogenic microbes. Despite this potential, the current dogma is that bed bugs are not vectors of any known infectious agents. However, this conclusion is based largely on the results of studies conducted before the advent of modern molecular biology and the resurgence of bed bugs on a global scale. More importantly, a small but compelling body of modern research suggesting that bed bugs can potentially vector some human pathogens exists but is often overlooked. This article critically examines the current classification of the bed bug as an insect that does not transmit disease agents. In doing so, it highlights key knowledge gaps that still exist in understanding the potential of bed bugs as pathogen vectors and outlines several arguments for why new research on the topic is necessary.

Bed bugs (Cimex spp.) are urban pests of global importance. Knowledge of the immune system of bed bugs has implications for understanding their susceptibility to biological control agents, their potential to transmit human pathogens, and the basic comparative immunology of insects. Nonetheless, the immunological repertoire of the family Cimicidae remains poorly characterized. Here, we use microscopy, flow cytometry, and RNA sequencing to provide a basal characterization of the circulating hemocytes of the common bed bug, Cimex lectularius. We also examine the responses of these specialized cells to E. coli exposure using the same techniques. Our results show that circulating hemocytes are comprised of at least four morphologically distinct cell types that are capable of phagocytosis, undergo degranulation, and exhibit additional markers of activation following stimulation, including size shift and DNA replication. Furthermore, transcriptomic profiling reveals expression of predicted Toll/IMD signaling pathway components, antimicrobial effectors and other potentially immunoresponsive genes in these cells. Together, our data demonstrate the conservation of several canonical cellular immune responses in the common bed bug and provide a foundation for additional mechanistic immunological studies with specific pathogens of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Many bacteria live on host surfaces, in cells and in specific organ systems. In comparison with gut microbiomes, the bacterial communities of reproductive organs (genital microbiomes) have received little attention. During mating, male and female genitalia interact and copulatory wounds occur, providing an entrance for sexually transmitted microbes. Besides being potentially harmful to the host, invading microbes might interact with resident genital microbes and affect immunity. Apart from the investigation of sexually transmitted symbionts, few studies have addressed how mating changes genital microbiomes. We dissected reproductive organs from virgin and mated common bedbugs, Cimex lectularius L., and sequenced their microbiomes to investigate composition and mating-induced changes. We show that mating changes the genital microbiomes, suggesting bacteria are sexually transmitted. Also, genital microbiomes varied between populations and the sexes. This provides evidence for local and sex-specific adaptation of bacteria and hosts, suggesting bacteria might play an important role in shaping the evolution of reproductive traits. Coadaptation of genital microbiomes and reproductive traits might further lead to reproductive isolation between populations, giving reproductive ecology an important role in speciation. Future studies should investigate the transmission dynamics between the sexes and populations to uncover potential reproductive barriers.

Besides typical sexually transmitted microbes, even environmental, opportunistic microbes have been found in copulatory organs of insects and even humans. To date, only one study has experimentally investigated the sexual transmission of opportunistic microbes from male to female insects, whereas nothing is known about the transmission from females to males. Even if opportunistic microbes do not cause infection upon transmission, they might eventually become harmful if they multiply inside the female. While the immune system of females is often assumed to target sexually transmitted microbes, most studies ignore the role of mating-associated opportunistic microbes. Variation in immunity between populations has been linked to parasite or bacteria prevalence but no study has ever addressed between-population differences in immune responses to sexually transmitted opportunistic microbes. We here show that bacteria applied to the copulatory organs of common bedbugs, Cimex lectularius, are sexually transmitted to the opposite sex at a high rate, including the transmission from female to male. Bacterial growth in the female sperm-receiving organ was inhibited over the first hours after introduction, but after this initial inhibition bacterial numbers increased, suggesting a shift of investment from immune defence towards reproduction. However, 24 h after the injection of bacteria, male components, or saline as a control, the sperm-receiving organ showed lysozyme-like activity and inhibited the growth of Gram-negative and Gram-positive bacteria in vitro, potentially to mop up the remaining bacteria. Contrasting our prediction, neither bacterial growth nor immune responses differed between populations. Future studies should link transmission dynamics, immune responses and fitness effects in both sexes. Experimental manipulation of environmental bacteria could be used to investigate how transmission frequency and toxicity of sexually transmitted opportunistic microbes shapes bacteria clearance and immune responses across populations., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

There is an ongoing need for safe and effective anti-bedbug compounds. Here, we tested the toxicity of three antimicrobial agents against bedbugs when administered orally. We reveal that doxycycline has direct insecticidal activity at 250 μg/ml (0.025%) that is particularly strong against immature bedbugs and appears to be independent of antimicrobial activity. Future studies to determine the mechanisms behind this property could be useful for the development of orally active insecticides or anti-bedbug therapeutics., (Copyright © 2020 American Society for Microbiology.)

Background: The common bed bug, Cimex lectularius, is an obligatory blood-feeding ectoparasite that requires a blood meal to molt and produce eggs. Their frequent biting to obtain blood meals and intimate association with humans increase the potential for disease transmission. However, despite more than 100 years of inquiry into bed bugs as potential disease vectors, they still have not been conclusively linked to any pathogen or disease. This ecological niche is extraordinarily rare, given that nearly every other blood-feeding arthropod is associated with some type of human or zoonotic disease. Bed bugs rely on the bacteria Wolbachia as an obligate endosymbiont to biosynthesize B vitamins, since they acquire a nutritionally deficient diet, but it is unknown if Wolbachia confers additional benefits to its bed bug host. In some insects, Wolbachia induces resistance to viruses such as Dengue, Chikungunya, West Nile, Drosophila C and Zika, and primes the insect immune system in other blood-feeding insects. Wolbachia might have evolved a similar role in its mutualistic association with the bed bug. In this study, we evaluated the influence of Wolbachia on virus replication within C. lectularius., Methods: We used feline calicivirus as a model pathogen. We fed 40 bed bugs from an established line of Wolbachia-cured and a line of Wolbachia-positive C. lectularius a virus-laden blood meal, and quantified the amount of virus over five time intervals post-feeding. The antibiotic rifampicin was used to cure bed bugs of Wolbachia., Results: There was a significant effect of time post-feeding, as the amount of virus declined by ~90% over 10 days in both groups, but no significant difference in virus titer was observed between the Wolbachia-positive and Wolbachia-cured groups., Conclusions: These findings suggest that other mechanisms are involved in virus suppression within bed bugs, independent of the influence of Wolbachia, and our conclusions underscore the need for future research.

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., Competing Interests: Conflict of interest statement: R.N. owns “the Cimex store,” which conducts commercial research and activities related to bed bugs. He conducted the work in this paper as part of his PhD (prior to owning the store)., (Copyright © 2019 the Author(s). Published by PNAS.)

Bed bugs are small hematophagous insects. They are found in temperate and tropical climates around the world. Their vectorial capacity for several pathogens, including Bartonella spp., has been suspected. An experimental study of artificial infection of Cimex lectularius with Bartonella quintana and Bartonella henselae bacteria was developed to evaluate the ability of MALDI-TOF MS to simultaneously identify bed bugs and their infectious status. This experimental study confirmed the ability of MALDI-TOF MS to identify bed bugs. In addition, it was able to differentiate between control bed bugs, bed bugs infected with Bartonella quintana and bed bugs infected with Bartonella henselae, with an identification percentage above 90%., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In recent years, bed bugs have reappeared in greater numbers, more frequently, and are biting humans in many new geographic areas. Infestations by these hematophagous insects are rapidly increasing worldwide. Borrelia recurrentis , a spirochete bacterium, is the etiologic agent of louse-borne relapsing fever. The known vectors are body lice, Pediculus humanus humanus . However, previous studies have suggested that bed bugs might also be able to transmit this bacterium. Adult Cimex lectularius were artificially infected with a blood meal mixed with bacterial suspension of B. recurrentis . They were subsequently fed with pathogen-free human blood until the end of the experiment. Bed bugs and feces were collected every 5 days to evaluate the capacity of bed bugs to acquire and excrete viable B. recurrentis using molecular biology, cultures, fluorescein diacetate and immunofluorescence assays. The feces collected on the day 5 and 10 postinfection contained viable bacteria. Immunofluorescence analysis of exposed bed bugs showed the presence of B. recurrentis in the digestive tract, even in bed bugs collected on day 20 after infection. Like human body lice, bed bugs can acquire, maintain, and excrete viable B. recurrentis that might infect humans through skin lesions. This preliminary work suggests that bed bugs might be competent vectors of B. recurrentis . Because bed bugs and body lice may share the same ecological niches, the role of bed bugs in transmitting recurrent fevers deserves further study.

The common bed bug, Cimex lectularius harbors the endosymbiotic microorganism, Wolbachia (wCle), in a gonad-associated bacteriome as an obligate nutritional mutualist. The obligatory nature of this association suggests that all individuals in C. lectularius populations would be infected with wCle. However, studies spanning the past several decades have reported variation in both infection frequency and relative abundance of wCle in field-collected samples of bed bugs. Since the growth kinetics of wCle is poorly understood, the objective of this study was to quantify wCle over the life cycle of two strains of C. lectularius. Our results highlight that wCle is dynamic during bed bug development, changing relative to life stage, intermolt stage, and blood-fed status. These results suggest new hypotheses about the coordination of Wolbachia growth and regression with its host's physiology and endocrine events. The observed quantitative modulation of wCle during the bed bug life cycle and during periods of starvation may explain the disparities in wCle infections reported in field-collected C. lectularius.

Wolbachia bacteria, vertically transmitted intracellular endosymbionts, are associated with two major host taxa in which they show strikingly different symbiotic modes. In some taxa of filarial nematodes, where Wolbachia are strictly obligately beneficial to the host, they show complete within- and among-species prevalence as well as co-phylogeny with their hosts. In arthropods, Wolbachia usually are parasitic; if beneficial effects occurs, they can be facultative or obligate, related to host reproduction. In arthropods, the prevalence of Wolbachia varies within and among taxa, and no co-speciation events are known. However, one arthropod species, the common bedbug Cimex lectularius was recently found to be dependent on the provision of biotin and riboflavin by Wolbachia, representing a unique case of Wolbachia providing nutritional and obligate benefits to an arthropod host, perhaps even in a mutualistic manner. Using the presence of presumably functional biotin gene copies, our study demonstrates that the obligate relationship is maintained at least in 10 out of 15 species of the genera Cimex and Paracimex. The remaining five species harboured Wolbachia as well, demonstrating the first known case of 100% prevalence of Wolbachia among higher arthropod taxa. Moreover, we show the predicted co-cladogenesis between Wolbachia and their bedbug hosts, also as the first described case of Wolbachia co-speciation in arthropods.

Bat bugs (Cimex adjunctus Barber) (Hemiptera: Cimicidae) collected from big brown bats (Eptesicus fuscus Palisot de Beauvoir) in Colorado, United States were assessed for the presence of Bartonella, Brucella, and Yersinia spp. using molecular techniques. No evidence of Brucella or Yersinia infection was found in the 55 specimens collected; however, 4/55 (7.3%) of the specimens were positive for Bartonella DNA. Multi-locus characterization of Bartonella DNA shows that sequences in bat bugs are phylogenetically related to other Bartonella isolates and sequences from European bats., (Published by Oxford University Press on behalf of Entomological Society of America 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Due in part to the development of insecticide resistance, the common bed bug, Cimex lectularius, has overcome human intervention efforts to make a global resurgence. The failure of chemical pesticides has created a need for novel strategies to combat bed bugs. While a number of insect pests are susceptible to the use of entomopathogenic microbes or microbial-derived toxins, biological control methods have not been thoroughly explored in bed bugs. Here, we tested the virulence of three entomopathogenic bacterial species in C. lectularius to determine their potential for bed bug control. We examined bed bug survival after inoculation with live or heat-killed Serratia marcescens, Pseudomonas fluorescens, and Bacillus thuringiensis israelensis at varying temperatures. We also analyzed the viability and growth of the same bacteria in infected bed bugs. All three bacterial species were pathogenic to bed bugs. However, the effects of S. marcescens and P. fluorescens were temperature-dependent while the lethality of B. thuringiensis israelensis was not. In addition, bacterial virulence was partly dependent on the route of infection but was not strongly associated with proliferation. Thus, our results suggest multiple possible mechanisms of microbial pathogenicity in the bed bug and indicate that entomopathogenic bacteria, or products derived from them, may have useful applications for bed bug control., (Copyright © 2017 Elsevier Inc. All rights reserved.)

In the last decade, the Chikungunya and Zika virus outbreaks have turned public attention to the possibility of the expansion of vector-borne infectious diseases worldwide. Medical entomology is focused on the study of arthropods involved in human health. We review here some of the research approaches taken by the medical entomology team of the University Hospital Institute (UHI) Méditerranée Infection of Marseille, France, with the support of recent or representative studies. We propose our approaches to technical innovations in arthropod identification and the detection of microorganisms in arthropods, the use of arthropods as epidemiological or diagnostic tools, entomological investigations around clinical cases or within specific populations, and how we have developed experimental models to decipher the interactions between arthropods, microorganisms, and humans., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: Bed bugs are a public health concern, and their incidence is increasing worldwide. Bed bug infestations are notoriously difficult to eradicate, further exacerbated by widespread resistance to pyrethroid and neonicotinoid insecticides. This study evaluated the efficacy of the newly developed fungal biopesticide Aprehend™, containing Beauveria bassiana, against insecticide-resistant bed bugs., Results: Overall mortality for the Harold Harlan (insecticide-susceptible) strain was high (98-100%) following exposure to Aprehend™ or Suspend SC (deltamethrin). The mean survival times (MSTs) for Harold Harlan bed bugs were 5.1 days for Aprehend™ and 4.8 and 3.0 days for the low and high concentrations of Suspend SC respectively. All three strains of pyrethroid-resistant bed bugs were susceptible to infection by B. bassiana, resulting in MSTs of <6 days (median = 4 days) and >94% overall mortality. Conversely, mortality of the three insecticide-resistant strains after exposure to Suspend SC was only 16-40%., Conclusion: These results demonstrate that Aprehend™ is equally effective against insecticide-susceptible and insecticide-resistant bed bugs and could provide pest control operators with a promising new tool for control of bed bugs and insecticide resistance management. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

Wolbachia symbionts are maternally inherited intracellular bacteria that have been detected in numerous insects including bed bugs. The objective of this study, the first epidemiological study in Europe, was to screen Wolbachia infection among Cimex lectularius collected in the field, using PCR targeting the surface protein gene (wsp), and to compare obtained Wolbachia strains with those reported from laboratory colonies of C. lectularius as well as other Wolbachia groups. For this purpose, 284 bed bug specimens were caught and studied from eight different regions of France including the suburbs of Paris, Bouches-du-Rhône, Lot-et-Garonne, and five localities in Alpes-Maritimes. Among the samples, 166 were adults and the remaining 118 were considered nymphs. In all, 47 out of 118 nymphs (40%) and 61 out of 166 adults (37%) were found positive on wsp screening. Among the positive cases, 10 samples were selected randomly for sequencing. The sequences had 100% homology with wsp sequences belonging to the F-supergroup strains of Wolbachia. Therefore, we confirm the similarity of Wolbachia strains detected in this epidemiological study to Wolbachia spp. reported from laboratory colonies of C. lectularius., (© M. Akhoundi et al., published by EDP Sciences, 2016.)

Background: Bartonella quintana, the etiologic agent of trench fever and other human diseases, is transmitted by the feces of body lice. Recently, this bacterium has been detected in other arthropod families such as bed bugs, which begs the question of their involvement in B. quintana transmission. Although several infectious pathogens have been reported and are suggested to be transmitted by bed bugs, the evidence regarding their competence as vectors is unclear., Methodology/principal Findings: Bed bugs at the adult and instar developmental stages were fed three successive human blood meals inoculated with B. quintana bacterium from day one (D1) to D5; subsequently they were fed with pathogen-free human blood until the end of the experiment. Bed bugs and feces were collected in time series, to evaluate their capacities to acquire, multiply and expel viable B. quintana using molecular biology, immunohistochemistry and cultures assays. B. quintana was detected molecularly in 100% of randomly selected experimentally infected bed bug specimens (D3). The monitoring of B. quintana in bed bug feces showed that the bacterium was detectable starting on the 3rd day post-infection (pi) and persisted until day 18±1 pi. Although immunohistochemistry assays localized the bacteria to the gastrointestinal bed bug gut, the detection of B. quintana in the first and second instar larva stages suggested a vertical non-transovarial transmission of the bacterium., Conclusion: The present work demonstrated for the first time that bed bugs can acquire, maintain for more than 2 weeks and release viable B. quintana organisms following a stercorarial shedding. We also observed the vertical transmission of the bacterium to their progeny. Although the biological role of bed bugs in the transmission of B. quintana under natural conditions has yet to be confirmed, the present work highlights the need to reconsider monitoring of these arthropods for the transmission of human pathogens.

Bed bugs Cimex lectularius L. were exposed to conidia (spores) of the entomopathogenic fungus Metarhizium anisopliae by feeding, aerosol spray, or contact with a treated surface. Feeding experiments demonstrated that bed bugs were innately susceptible to this fungus. However, only at 98% humidity were mortality rates high, regardless of whether bed bugs were sprayed with a fungal solution or contacted a treated surface. Mortality in treated bed bugs at ambient humidity did not increase when these bed bugs were kept in aggregation with other bed bugs that had recently blood fed to repletion. Based on these laboratory studies, we conclude that M. anisopliae is a poor pathogen for use in control of bed bugs, particularly at humidities that would likely be encountered under field conditions., (© 2014 Entomological Society of America.)

Obligate insect-bacterium nutritional mutualism is among the most sophisticated forms of symbiosis, wherein the host and the symbiont are integrated into a coherent biological entity and unable to survive without the partnership. Originally, however, such obligate symbiotic bacteria must have been derived from free-living bacteria. How highly specialized obligate mutualisms have arisen from less specialized associations is of interest. Here we address this evolutionary issue by focusing on an exceptional insect-Wolbachia nutritional mutualism. Although Wolbachia endosymbionts are ubiquitously found in diverse insects and generally regarded as facultative/parasitic associates for their insect hosts, a Wolbachia strain associated with the bedbug Cimex lectularius, designated as wCle, was shown to be essential for host's growth and reproduction via provisioning of B vitamins. We determined the 1,250,060-bp genome of wCle, which was generally similar to the genomes of insect-associated facultative Wolbachia strains, except for the presence of an operon encoding the complete biotin synthetic pathway that was acquired via lateral gene transfer presumably from a coinfecting endosymbiont Cardinium or Rickettsia. Nutritional and physiological experiments, in which wCle-infected and wCle-cured bedbugs of the same genetic background were fed on B-vitamin-manipulated blood meals via an artificial feeding system, demonstrated that wCle certainly synthesizes biotin, and the wCle-provisioned biotin significantly contributes to the host fitness. These findings strongly suggest that acquisition of a single gene cluster consisting of biotin synthesis genes underlies the bedbug-Wolbachia nutritional mutualism, uncovering an evolutionary transition from facultative symbiosis to obligate mutualism facilitated by lateral gene transfer in an endosymbiont lineage.

Bed bugs (Cimex lectularius L.) have resurged in the United States and globally. Bed bugs are hematophagous ectoparasites of humans and other animals, including domestic pets, chickens, and bats, and their blood feeding habits contribute to their potential as disease vectors. Several species of Bartonella are re-emergent bacterial pathogens that also affect humans, domestic pets, bats and a number of other wildlife species. Because reports of both bed bugs and Bartonella have been increasing in the U.S., and because their host ranges can overlap, we investigated whether the resurgences of these medically important pathogens and their potential vector might be linked, by screening for Bartonella spp. in bed bugs collected from geographic areas where these pathogens are prevalent and from bed bugs that have been in culture in the laboratory for several years. We screened a total of 331 bed bugs: 316 bed bugs from 36 unique collections in 29 geographic locations in 13 states, 10 bed bugs from two colonies maintained in the laboratory for 3 yr, and 5 bed bugs from a colony that has been in culture since before the recent resurgence of bed bugs. Bartonella spp. DNA was screened using a polymerase chain reaction assay targeting the 16S-23S rRNA intergenic transcribed spacer region. Bartonella DNA was not amplified from any bed bug, but five bed bugs from four different apartments of an elderly housing building in North Carolina contained DNA sequences that corresponded to Burkholderia multivorans, an important pathogen in nosocomial infections that was not previously linked to an arthropod vector.

Elucidating the spatial dynamic and core constituents of the microbial communities found in association with arthropod hosts is of crucial importance for insects that may vector human or agricultural pathogens. The hematophagous Cimex lectularius (Hemiptera: Cimicidae), known as the human bed bug, has made a recent resurgence in North America, as well as worldwide, potentially owing to increased travel, climate change and resistance to insecticides. A comprehensive survey of the bed bug microbiome has not been performed to date, nor has an assessment of the spatial dynamics of its microbiome. Here we present a survey of internal and external bed bug microbial communities by amplifying the V4-V6 hypervariable region of the 16S rDNA gene region followed by 454 Titanium sequencing using 31 individuals from eight distinct collection locations obtained from residences in Cincinnati, OH. Across all samples, 97% of the microbial community is made up of two dominant OTUs, previously identified as the α-proteobacterium Wolbachia and an unnamed γ-proteobacterium from the Enterobacteriaceae. Microbial communities varied among host locations for measures of community diversity and exhibited structure according to collection location. This broad survey represents the most in-depth assessment, to date, of the microbes that associate with bed bugs.

Residual biopesticide treatments of Beauveria bassiana were tested against the bed bug Cimex lectularius. An oil formulation of conidia was applied to different substrates. Bed bugs were exposed for 1 h, transferred to an unsprayed environment and monitored for mortality. Separate bioassays evaluated the effect of bed bug strain, sex, life stage, and exposure substrate on mortality. Rapid mortality was observed in all bioassays, with bed bugs exposed to treated jersey knit cotton dying most rapidly. A further assay demonstrated efficient autodissemination of conidia from exposed bed bugs to unexposed bed bugs within artificial harborages., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Although a variety of disease agents have been reported from bed bugs, the mechanical and biological disease transmission potential of bed bugs remains unelucidated. In this study we assayed survivability of the mildly pathogenic spotted fever group rickettsia, Rickettsia parkeri, in bed bugs after feeding on R. parkeri-infected chicken blood. Two groups of 15 adult bed bugs each were fed on infected or noninfected blood, and two groups of fourth-instar bed bugs also were fed on either infected or noninfected blood. One group of 15 adult bed bugs received no bloodmeal and was included as an additional control. Two weeks postfeeding, two pools of five live bed bugs from each group were surface sterilized, macerated, and placed in Vero cell cultures in an attempt to grow live organism. The remaining five individual bed bugs from each group were dissected, their salivary glands were removed for immunofluorescence assay (IFA) staining, and the remaining body parts were processed for polymerase chain reaction (PCR) analysis. Results indicated that no immature (now molted to fifth instar) bed bugs were positive for R. parkeri by IFA or PCR, indicating that organisms did not survive the molting process. After 4 wk of cell culture, no organisms were seen in cultures from any of the treatment or control groups, nor were any cultures PCR positive. However, two of the adult bed bugs were IFA positive for rickettsia-like organisms, and these two specimens were also PCR positive using R. parkeri-specific primers. These IFA and PCR results indicate that remnants of Rickettsia parkeri (possibly whole organisms) survived in the bugs for 2 wk, but the viability of the organisms in these two specimens could not be determined.

Many insects are dependent on bacterial symbionts that provide essential nutrients (ex. aphid-Buchnera and tsetse-Wiglesworthia associations), wherein the symbionts are harbored in specific cells called bacteriocytes that constitute a symbiotic organ bacteriome. Facultative and parasitic bacterial symbionts like Wolbachia have been regarded as evolutionarily distinct from such obligate nutritional mutualists. However, we discovered that, in the bedbug Cimex lectularius, Wolbachia resides in a bacteriome and appears to be an obligate nutritional mutualist. Two bacterial symbionts, a Wolbachia strain and an unnamed gamma-proteobacterium, were identified from different strains of the bedbug. The Wolbachia symbiont was detected from all of the insects examined whereas the gamma-proteobacterium was found in a part of them. The Wolbachia symbiont was specifically localized in the bacteriomes and vertically transmitted via the somatic stem cell niche of germalia to oocytes, infecting the incipient symbiotic organ at an early stage of the embryogenesis. Elimination of the Wolbachia symbiont resulted in retarded growth and sterility of the host insect. These deficiencies were rescued by oral supplementation of B vitamins, confirming the essential nutritional role of the symbiont for the host. The estimated genome size of the Wolbachia symbiont was around 1.3 Mb, which was almost equivalent to the genome sizes of parasitic Wolbachia strains of other insects. These results indicate that bacteriocyte-associated nutritional mutualism can evolve from facultative and prevalent microbial associates like Wolbachia, highlighting a previously unknown aspect of the parasitism-mutualism evolutionary continuum.

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.

Endosymbiotic Wolbachia bacteria have been previously shown to infect laboratory colonies of the human bed bug, Cimex lectularius L. (Heteroptera: Cimicidae), but little information exists regarding the extent of infection in natural populations. We assayed C. lectularius populations from five North American regions (California, Connecticut, Florida, New York, and Toronto, Canada) and one African region (Macha, Zambia) for Wolbachia infection by the polymerase chain reaction (PCR). Wolbachia infections were prevalent in all populations assayed (83-100%). There were no significant differences in infection frequency between geographic regions, between sexes, or between life stages (adult versus nymph). The potential utility of Wolbachia for alternative bed bug control strategies is discussed.

Wolbachia symbionts are obligate intracellular bacteria that cause host reproductive alterations in many arthropods and filarial nematodes. We identified Wolbachia symbionts in the cliff swallow bug (Oeciacus vicarious Horvath) and the human bed bug (Cimex lectularius L.) (Hemiptera: Cimicidae) by polymerase chain reaction (PCR) amplification and sequencing using Wolbachia-specific 16S rDNA and FtsZ primers. Phylogenetic analyses using Bayesian, maximum likelihood, and maximum parsimony algorithms indicated, with strong support, that (1) Wolbachia infections in these two cimicid hosts form a monophyletic group, and (2) the Wolbachia strains detected belong to the F clade, previously associated with termites, weevils, and filarial nematodes.

Two different inherited bacterial symbionts from ovary tissue of the bedbug Cimex lectularius were characterized by gene amplification and sequencing analysis of their 16S rDNA gene. The first bacterium belongs to the Wolbachia subgroup of the alpha-Proteobacteria, the second is a member of the gamma-subdivision, and is closely related to the bacterial parasite of the leafhopper Euscelidius variegatus (BEV) which was shown to be capable of transovarial transmission. The high similarity (> 97%) between the C. lectularius symbiont and BEV indicates that these two microorganisms belong to the same lineage and share the ability to invade distant insect hosts to fulfill their symbiotic functions and to establish transovarial transmission to future generations.