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

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

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.

Long-Term Mosquito culture with SkitoSnack, an artificial blood meal replacement.

The reliance on blood is a limiting factor for mass rearing of mosquitoes for Sterile-Insect-Technique (SIT) and other mosquito-based control strategies. To solve this problem, we have developed SkitoSnack, a formulated diet for Aedes aegypti (L) mosquitoes, as an alternative for vertebrate blood. Here we addressed the question if long-term yellow fever mosquito culture with SkitoSnack resulted in changed life history traits and fitness of the offspring compared to blood-raised mosquitoes. We also explored if SkitoSnack is suitable to raise Asian tiger mosquitos, Aedes albopictus (L.), and the human bed bug, Cimex lectularius (L). We measured life history traits for 30th generation SkitoSnack-raised Ae. aegypti and 11th generation SkitoSnack-raised Ae. albopictus, and compared them with control mosquitoes raised on blood only. We compared meal preference, flight performance, and reproductive fitness in Ae. aegypti raised on SkitoSnack or blood. We also offered SkitoSnack to bed bug nymphs. We found that long-term culture with SkitoSnack resulted in mosquitoes with similar life history traits compared to bovine blood-raised mosquitoes in both species we studied. Also, Ae. aegypti mosquitoes raised on SkitoSnack had similar flight performance compared to blood raised mosquitoes, were still strongly attracted by human smell and had equal mating success. Minimal feeding occurred in bed bugs. Our results suggest that long-term culture with the blood-meal replacement SkitoSnack results in healthy, fit mosquitoes. Therefore, artificial diets like SkitoSnack can be considered as a viable alternative for vertebrate blood in laboratory mosquito culture as well as for mosquito mass production for Sterile-Insect-Technique mosquito control interventions. SkitoSnack was not suitable to induce engorgement of bed bugs.

Surfactantless Emulsions Containing Eugenol for Imidacloprid Solubilization: Physicochemical Characterization and Toxicity against Insecticide-Resistant Cimex lectularius.

Synthetic insecticides have been used for a long time as one of the most effective tools for insect pest control. However, the re-emergence of insect pests and their fast development of resistance, as has occurred for pyrethroid-resistant bed bugs Cimex lectularius L., make it necessary to develop new and safe strategies for effective pest control. This has fostered the research on new eco-sustainable formulations based on essential oils, which allows reducing the impact associated with the intensive use of synthetic insecticides on the environment and their effects on human health. This research explores the stability of water/eugenol/ethanol surfactantless emulsions loaded with imidacloprid (0.003 wt%), and their toxicity against a resistant bed bug strain. The results have shown that these emulsions enable the solubilization of a poorly water-soluble drug, such as the imidacloprid, without any significant modification of their stability. Furthermore, the application of the obtained formulations against the pyrethroid-resistant bed bug results in mortality in the 50-85% range upon topical and spray applications, with the increase of the eugenol content enhancing the effectiveness of the formulations. It may be expected that the ternary water/eugenol/ethanol mixtures could be further developed in the preparation of ready to use formulations, enabling the dispersion of insecticides for pest control.

Susceptibility Status of Bedbugs (Hemiptera: Cimicidae) Against Pyrethroid and Organophosphate Insecticides in Dar es Salaam, Tanzania.

Cimex hemipterus resistance to residual insecticides is a growing public health concern worldwide. Herein, we report the susceptibility status of C. hemipterus against pyrethroid (permethrin) and organophosphate (dichlorvos) insecticides in Dar es Salaam. The knockdown efficacy of dichlorvos (99%) was greater than that of permethrin (43%), and mortality at the end of 24 h was also greater for dichlorvos than for permethrin. Mortality in recommended concentration of permethrin was lower than manufacturer's expectations. In contrast, similar concentrations in dichlorvos resulted into mortality of 100% at the end of 24 h. LC50 and LC95 values for permethrin were 3.36 (2.356-4.364) and 2,887.39 (2,886.37-2,888.39), respectively, whereas those of dichlorvos were 7.91 (3.85-11.97) and 44.39 (40.33-48.45). Dar es Salaam city has high coverage and usage of long-lasting insecticidal nets (LLINs) for prevent malaria. The resistance in bedbugs to permethrin may be related to the widespread use of the insecticide in LLINs. We recommend of the change from pyrethroids dichlorvos for the control of bedbugs.

Evidence for a sexually selected function of the attachment system in bedbugs Cimex lectularius (Heteroptera, Cimicidae).

Attachment to surfaces is a major aspect of an animal's interaction with the environment. Consequently, shaping of the attachment system in relation to weight load and substrate is considered to have occurred mainly by natural selection. However, sexual selection may also be important because many animals attach to their partner during mating. The two hypotheses generate opposing predictions in species where males are smaller than females. Natural selection predicts that attachment ability will scale positively with load, and hence body size, and so will be larger in females than males. Sexual selection predicts attachment forces in males will be larger than those in females, despite the males' smaller size because males benefit from uninterrupted copulation by stronger attachment to the female. We tested these predictions in the common bedbug Cimex lectularius, a species in which both sexes, as well as nymphs, regularly carry large loads: blood meals of up to 3 times their body weight. By measuring attachment forces to smooth surfaces and analysing in situ fixed copulating pairs and the morphology of attachment devices, we show that: (i) males generate twice the attachment force of females, despite weighing 15% less; (ii) males adhere to females during copulation using hairy tibial adhesive pads; (iii) there are more setae, and more setae per unit area, in the pads of males than in those of females but there is no difference in the shape of the tarsal setae; and (iv) there is an absence of hairy tibial attachment pads and a low attachment force in nymphs. These results are consistent with a sexually selected function of attachment in bedbugs. Controlling sperm transfer and mate guarding by attaching to females during copulation may also shape the evolution of male attachment structures in other species. More generally, we hypothesise the existence of an arms race in terms of male attachment structures and female counterparts to impede attachment, which may result in a similar evolutionary diversification to male genitalia.

Binucleation of male accessory gland cells in the common bed bug Cimex lectularius.

The insect male accessory gland (MAG) is an internal reproductive organ responsible for the synthesis and secretion of seminal fluid components, which play a pivotal role in the male reproductive strategy. In many species of insects, the effective ejaculation of the MAG products is essential for male reproduction. For this purpose, the fruit fly Drosophila has evolved binucleation in the MAG cells, which causes high plasticity of the glandular epithelium, leading to an increase in the volume of seminal fluid that is ejaculated. However, such a binucleation strategy has only been sporadically observed in Dipteran insects, including fruit flies. Here, we report the discovery of binucleation in the MAG of the common bed bug, Cimex lectularius, which belongs to hemimetabolous Hemiptera phylogenetically distant from holometabolous Diptera. In Cimex, the cell morphology and timing of synchrony during binucleation are quite different from those of Drosophila. Additionally, in Drosophila, the position of the two nuclei in the adult stage changes as a result of the mating history or the nutrient conditions; however, it remains stable in Cimex. These differences suggest that binucleation in the Cimex MAG plays a unique role in the male reproductive system that is distinct from that of Drosophila.

Survival and Transstadial Persistence of Trypanosoma cruzi in the bed bug (Hemiptera: Cimicidae).

Bed bug populations are increasing around the world at an alarming rate and have become a major public health concern. The appearance of bed bug populations in areas where Chagas disease is endemic raises questions about the role of these insects in the transmission of Trypanosoma cruzi, the etiological agent of the disease. In a series of laboratory evaluations, bed bug adults and nymphs were experimentally fed with T. cruzi-infected blood to assess the ability of T. cruzi to survive inside the bed bug and throughout the insect's molting process. Live T. cruzi were observed in gut contents of experimentally infected bed bug adults via light microscopy and the identity of the parasite was confirmed via polymerase chain reaction analysis. T. cruzi persisted at least 97-d postinfection in adult bed bugs. Nymphal stage bed bugs that were infected with T. cruzi maintained the parasite after molting, indicating that transstadial passage of T. cruzi in bed bugs took place. This report provides further evidence of acquisition, maintenance, and for the first time, transstadial persistence of T. cruzi in bed bugs.

Analysis of the life stages of Cimex lectularius captured within a medical centre suggests that the true numbers of bed bug introductions are under-reported.

Little is known about the epidemiology of bed bugs within the healthcare system, but nymphal stages predominate in natural infestations. This study determined the life stages of bed bugs captured within a medical centre, and found that older bed bugs were more likely to be captured than younger insects. The numbers of first instars, third-fifth instars and adult females captured were significantly different compared with the numbers of each life stage found in a natural infestation (P<0.01). A significant number of early-instar bed bugs introduced into the medical centre may go unnoticed by hospital staff.

Functional characterization of Aquaporin-like genes in the human bed bug Cimex lectularius.

The bed bug Cimex lectularius is a blood-feeding re-emerging annoyance pest insect that has the ability to transmit Trypanosoma cruzi under experimental laboratory conditions. Aquaporins (AQPs) are water channel proteins that are essential in biological organisms. C. lectularius are constantly exposed to water-related stress, suggesting that AQPs may offer novel control avenues. We identified and cloned four AQPs from C. lectularius, assessed tissue and lifestage-specific expression, and characterized biochemical functions in vitro and in vivo. We identified an efficient water-specific AQP (ClAQP1), two aquaglyceroporins (ClGlp1 and ClGlp2) and a homolog of Drosophila melanogaster big brain (ClBib). ClGlp1 was only functional when co-expressed with the water-specific AQP. Simultaneous RNAi gene silencing of ClAQP1 and ClGlp1 significantly reduced water and urea excretion post blood feeding. The Bib homologue was enriched in embryos, exclusively expressed in ovaries, and when silenced, dramatically increased bug fecundity. Our data demonstrate that AQPs have critical roles in excretion, water homeostasis and reproduction in C. lectularius, and could be potential targets for control in this notorious pest.

Effect of Feeding History and Time Elapsed From Field Collection on the Movement Behavior and Response to Stimulation in Cimex lectularius (Hemiptera: Cimicidae).

The common bed bug (Cimex lectularius L.) (Hemiptera: Cimicidae) is an obligate blood-sucking insect that has been resurging in many countries. Researching this pest's behavior will help design more effective control methods. In this study, we evaluated the effect of feeding history and time elapsed from field collection on bed bug movement behavior and response to chemical lure or carbon dioxide stimulation in the laboratory. After CO2 was released, bed bugs unfed for 3 d began to return to harborages; in contrast, the ones unfed for 2 and 4 wk spent significantly more time outside their harborages during the first 1 h after than the 1 h before CO2 release. After CO2 release, there was an increase in activity (time spent moving outside harborage) in all bed bugs with different feeding history or time elapsed from field collection. During the 8-h observation period when CO2 was present, bed bug males unfed for 4 wk spent significantly more time exploring outside harborages than the ones unfed for 3 d, 1 wk, and 2 wk. Nymphs collected 1-2 wk and 1 yr ago spent significantly more time exploring outside harborages than the ones collected 43 yr ago. Bed bug's exploratory activity (the total percentage of bed bugs trapped in both baited and unbaited interceptors) was significantly affected by their time elapsed from field collection and their exploratory activity level was 1-2 wk > 6 mo > 5 and 43 yr. Both feeding history and time elapsed from field collection significantly affected bed bug movement, whereas bed bug's response to chemical lure or CO2 (the percentage of bed bugs trapped in the baited interceptor, summarized as the number of bed bugs trapped in the baited interceptor divided by the total number of trapped bed bugs in both baited and unbaited interceptors) was unaffected by the time elapsed from field collection.

Impact of sublethal exposure to a pyrethroid-neonicotinoid insecticide on mating, fecundity and development in the bed bug Cimex lectularius L. (Hemiptera: Cimicidae).

Sublethal exposure to an insecticide may alter insect feeding, mating, oviposition, fecundity, development, and many other life history parameters. Such effects may have population-level consequences that are not apparent in traditional dose-mortality evaluations. Earlier, we found that a routinely used combination insecticide that includes a pyrethroid and a neonicotinoid (Temprid® SC) had deleterious effects on multiple bed bug (Cimex lectularius, L.) behaviors. Here, we demonstrate that sublethal exposure impacts physiology and reproduction as well. We report that sublethal exposure to Temprid SC has variable aberrant effects on bed bugs depending on the strain, including: a reduction in male mating success and delayed oviposition by females. However, after sublethal exposure, egg hatch rate consistently declined in every strain tested, anywhere from 34%-73%. Conversely, impact on fifth instar eclosion time was not significant. While the strains that we tested varied in their respective magnitude of sublethal effects, taken together, these effects could reduce bed bug population growth. These changes in bed bug behavior and fecundity could lead to improved efficacy of Temprid SC in the field, but recovery of impacted bugs must be considered in future studies. Sublethal effects should not be overlooked when evaluating insecticide efficacy, as it is likely that other products may also have indirect effects on population dynamics that could either aid or inhibit successful management of pest populations.

Bed Bug (Hemiptera: Cimicidae) Detection in Low-Income, High-Rise Apartments Using Four or Fewer Passive Monitors.

Bed bug, Cimex lectularius L., management in low-income, high-rise housing for the elderly and disabled can be difficult. Early detection is key to slowing their spread, and reducing management cost and time needed for control. To determine the minimum number of passive monitors needed to detect low-level bed bug infestations in this environment, we evaluated three monitors placed one, two, or four per apartment in a 3 by 3 experimental design. One sticky monitor, The Bedbug Detection System, and the two pitfall monitors, ClimbUp Insect Interceptors BG and BlackOut BedBug Detectors, were evaluated. Bed bugs were trapped by the ClimbUp Insect Interceptors BG and the BlackOut BedBug Detector in 88% and 79% of apartments, respectively, but only in 39% of the apartments monitored with The Bedbug Detection System. The Bedbug Detection System required significantly longer time to detect bed bugs than either the ClimbUp Insect Interceptor BG or the BlackOut BedBug Detector. With the less effective Bedbug Detection System data removed from analyses, detection rates ranged from 80 to 90%, with no significant differences among one, two, or four monitors per apartment. Results indicate it is especially important to include a bed placement when only placing a few monitors. Future work should compare the combination of cursory visual inspections with various monitor numbers and placements per apartment to determine the most efficient, cost-effective system that will be accepted and implemented in low-income housing.

Evaluation of the Potential for Secondary Kill for Ingested Insecticides in the Common Bed Bug (Hemiptera: Cimicidae).

Baits are a preferred method of urban pest management. Baits enable more targeted insecticide applications with a fraction of the active ingredient used in residual sprays. Bait translocation by foragers, and consequent secondary kill of nonforagers, enhances bait effectiveness in social insects, and in other group-living species like German cockroaches (Blattella germanica L.). We investigated the potential for secondary kill in bed bugs (Cimex lectularius L.), another gregarious species, using a liquid bait. We first investigated whether blood-fed adults enhance nymph survivorship within aggregations by increasing the local relative humidity (RH) and providing fecal nutrients. Higher RH (50% and 95%) resulted in greater survivorship of first instars compared with 0% RH. Therefore, in subsequent experiments, we controlled RH to decouple its effect on nymph survivorship from effects of fecal nutrients. The presence of fed or unfed adults did not increase unfed first instar survivorship, suggesting that if nymphs ingested feces, its nutritional benefits were minimal. Nymph survivorship was unaffected by the presence of adult males fed fipronil or clothianidin, suggesting that unlike in cockroaches, highly effective insecticides might not be effective as secondary kill toxicants in bed bugs. To directly compare secondary kill in first-instar bed bugs and B. germanica, we exposed both to insecticide-laden adult B. germanica feces. Whereas first-instar B. germanica died in the presence of insecticide-laden feces, bed bugs did not. We, therefore, conclude that secondary kill with neuroactive insecticides will likely not be a significant factor in bed bug population suppression.

Differences in Climbing Ability of Cimex lectularius and Cimex hemipterus (Hemiptera: Cimicidae).

The climbing abilities of two bed bug species, Cimex lectularius L. and Cimex hemipterus (F.), were determined by evaluating their escape rates from smooth surface pitfall traps using four commercial bed bug monitors (Verifi Bed Bug Detector, ClimbUp Insect Interceptor, BlackOut Bed Bug Detector, and SenSci Volcano Bed Bug Detector). All detectors were used in the absence of lures or attractants. Unlike C. lectularius, adult C. hemipterus were able to escape from all traps. On the other hand, no or a low number nymphs of both species escaped, depending on the evaluated traps. Examination of the vertical friction force of adults of both species revealed a higher vertical friction force in C. hemipterus than in C. lectularius. Scanning electron microscope micrograph observation on the tibial pad of adult bed bugs of C. hemipterus showed the presence of a greater number of tenent hairs on the tibial pad than on that of adult C. lectularius. No tibial pad was found on the fourth and fifth instars of both species. Near the base of the hollow tenent hairs is a glandular epithelium that is better developed in adult C. hemipterus than in adult C. lectularius. This study highlights significant morphological differences between C. lectularius and C. hemipterus, which may have implications in the monitoring and management of bed bug infestations.

Comparison of ingestion and topical application of insecticides against the common bed bug, Cimex lectularius (Hemiptera: Cimicidae).

BACKGROUND: The global prevalence of Cimex lectularius infestations has challenged current intervention efforts, as pyrethroid resistance has become ubiquitous, availability of labeled insecticides for bed bugs is limited, and non-chemical treatment options, such as heat, are often unaffordable. We evaluated representative insecticides toward the goal of developing a novel, ingestible liquid bait for hematophagous arthropods. RESULTS: LC50 values were estimated for adult males and first instar nymphs of an insecticide-susceptible strain for abamectin, clothianidin, fipronil and indoxacarb, after ingestion from an artificial feeder. LD50 values were calculated based on the ingested blood volume. Ingested abamectin, clothianidin and fipronil caused rapid mortality in both life stages. Fipronil was ∼43-fold more effective by ingestion than by topical application. Indoxacarb and its bioactive metabolite decarbomethoxylated JW062 (DCJW) were ineffective at causing bed bug mortality even at concentrations as high as 1000 ng mL-1 blood. CONCLUSIONS: Fipronil, clothianidin and abamectin have potential for being incorporated into a liquid bait for bed bug control; indoxacarb and DCJW were not effective. Bed bugs are a good candidate for an ingestible liquid bait because systemic formulations generally require less active ingredient than residual sprays, they remain contained and more effectively target hematophagous arthropods. © 2016 Society of Chemical Industry.

Mortality, fecundity and development among bed bugs (Cimex lectularius) exposed to prolonged, intermediate cold stress.

BACKGROUND: Bed bugs (Cimex lectularius L.) have returned as a nuisance pest worldwide. Their ability to withstand different types of environmental stress should be explored in order potentially to increase the efficiency of control methods. RESULTS: Immediate and long-term effects of exposure to temperatures from 0 to -10 °C for 1, 2 and 3 weeks are reported. Fifth-instar nymphs and adults were exposed to constant or fluctuating temperatures. Increased cold and extended time yielded higher mortality; nymphs were more resilient than adults at the shorter durations of exposure. At intermediate temperatures, mortality was higher at constant compared with fluctuating temperatures, whereas all individuals died after 3 weeks of exposure to -7 °C. The success among survivors after cold treatment was also affected in terms of reduced egg production, hatching success and the ability of fifth-instar nymphs to advance into the adult stage; however, nymphs produced after cold treatment developed normally. CONCLUSIONS: Detrimental effects of prolonged exposure to low temperatures were seen in bed bugs both during and after cold treatment. The results suggest that temperatures below -7 °C can be applied by laymen to control this pest in small items if available treatment time is of less concern. © 2016 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Vision in the common bed bug Cimex lectularius L. (Hemiptera: Cimicidae): eye morphology and spectral sensitivity.

Bed bugs as pests of public health importance recently experienced a resurgence in populations throughout the U.S. and other countries. Consequently, recent research efforts have focused on improving understanding of bed bug physiology and behaviour to improve management. While few studies have investigated the visual capabilities of bed bugs, the present study focused specifically on eye morphology and spectral sensitivity. A 3-D imaging technique was used to document bed bug eye morphology from the first instar through adult and revealed morphological characteristics that differentiate the common bed bug from the tropical bed bug as well as sex-specific differences. Electrophysiological measurements were used to evaluate the spectral sensitivity of adult bed bugs. Male bed bugs were more responsive than females at some wavelengths. Electrophysiological studies provided evidence for at least one photoreceptor with a spectral sensitivity curve peak in the green (λmax 520 nm) region of the spectrum. The broadened long wavelength portion of the spectral sensitivity curve may potentially indicate another photoreceptor in the yellow-green (λmax 550 nm) portion of the spectrum or screening pigments. Understanding more about bed bug visual biology is vital for designing traps, which are an important component of integrated bed bug management.

Behavioral Responses of Nymph and Adult Cimex lectularius (Hemiptera: Cimicidae) to Colored Harborages.

Behavioral bioassays were conducted to determine whether bed bug adults and nymphs prefer specific colored harborages. Two-choice and seven-choice behavioral color assays indicate that red (28.5%) and black (23.4%) harborages are optimal harborage choices for bed bugs. Yellow and green harborages appear to repel bed bugs. Harborage color preferences change according to gender, nutritional status, aggregation, and life stage. Female bed bugs prefer harborages with shorter wavelengths (lilac-14.5% and violet-11.5%) compared to males, whereas males prefer harborages with longer wavelengths (red-37.5% and black-32%) compared with females. The preference for orange and violet harborages is stronger when bed bugs are fed as opposed to when they are starved. Lone bed bugs (30%) prefer to be in black harborages while red harborages appear to be the optimum harborage color for bed bugs in more natural mixed aggregations (35.5%). Bed bug nymphs preferred different colored harborages at each stage of development, which is indicative of their developing eye structures and pigments. First instars showed no significant preference for any colored harborage soon after hatching. However, by the fifth instar, 27.5% of nymphs significantly preferred red and black harborages (which was a similar preference to adult bed bugs). The proportion of oviposited eggs was significantly greater under blue, red, and black harborages compared to other colored harborages tested. The use of visual cues such as specific colors offers great potential for improving bed bug monitoring tools by increasing trap captures.