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.

Biological control of Cimex lectularius with Beauveria bassiana: Effects of substrate, dosage, application strategy, and bed bug physiology.

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.

Effect of Bed Bug (Hemiptera: Cimicidae) Aldehydes on Efficacy of Fungal Biopesticides.

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.

Characterization of New Defensin Antimicrobial Peptides and Their Expression in Bed Bugs in Response to Bacterial Ingestion and Injection.

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.

A Comparative Study of Body Lice and Bed Bugs Reveals Factors Potentially Involved in Differential Vector Competence for the Relapsing Fever Spirochete Borrelia recurrentis.

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.

A Francisella tularensis-Like Bacterium in Tropical Bed Bugs from Madagascar.

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.

Molecular and MALDI-TOF MS identification of swallow bugs Cimex hirundinis (Heteroptera: Cimicidae) and endosymbionts in France.

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.

Time- and tissue-specific antimicrobial activity of the common bed bug in response to blood feeding and immune activation by bacterial injection.

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 (MIC1) bioassays and the Kirby-Bauer protocol to evaluate antimicrobial peptide (AMP2) activity in tissue extracts from the midguts or 'rest of body' (RoB3) 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.

Characterization of bacterial communities associated with blood-fed and starved tropical bed bugs, Cimex hemipterus (F.) (Hemiptera): a high throughput metabarcoding analysis.

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.

Molecular analysis of the blood meals and bacterial communities of bed bugs (Cimex lectularius L.) to assess interactions with alternative hosts.

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.

Blood deprivation and heat stress increase mortality in bed bugs (Cimex lectularius) exposed to insect pathogenic fungi or desiccant dust.

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.

An Earliest Endosymbiont, Wolbachia massiliensis sp. nov., Strain PL13 from the Bed Bug (Cimex hemipterus), Type Strain of a New Supergroup T.

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 Cimexlectularius. 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 Cimexhemipterus, designated as wChem strain PL13, and proposed Wolbachiamassiliensis 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.

Chitinase production by Trichoderma koningiopsis UFSMQ40 using solid state fermentation.

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.

Ex vivo characterization of the circulating hemocytes of bed bugs and their responses to bacterial exposure.

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.

Mating changes the genital microbiome in both sexes of the common bedbug Cimex lectularius across populations.

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.

Case not Closed: Arguments for New Studies of the Interactions between Bed Bugs and Human Pathogens.

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.

Female immunity in response to sexually transmitted opportunistic bacteria in the common bedbug Cimex lectularius.

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.

Insecticidal Activity of Doxycycline against the Common Bedbug.

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.

Lack of influence by endosymbiont Wolbachia on virus titer in the common bed bug, Cimex lectularius.

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.

Female bed bugs (Cimex lectularius L) anticipate the immunological consequences of traumatic insemination via feeding cues.

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.