Morphological, Molecular and MALDI-TOF MS Identification of Bedbugs and Associated Wolbachia Species in Rural Senegal.

Bed bugs are known to carry several microorganisms. The purpose of this study was to assess the prevalence of bed bug infestation in two rural areas of Senegal and determine the species present in the population. A screening was conducted to detect some arthropod associated pathogenic bacteria in bed bugs and to evaluate the prevalence of endosymbiont carriage. One survey took place in 17 villages in Niakhar and two surveys in Dielmo and Ndiop and surroundings area in the same 20 villages. Bed bugs collected were identified morphologically and by MALDI-TOF MS tools. Microorganisms screening was performed by qPCR and confirmed by sequencing. During the survey in the Niakhar region, only one household 1/255 (0.4%) in the village of Ngayokhem was found infested by bed bugs. In a monitoring survey of the surroundings of Dielmo and Ndiop area, high prevalence was found during the two rounds of surveys in 65/314 (21%) in 16/20 villages (January-March) and 93/351 (26%) in 19/20 villages (December). All bed bugs were morphologically identified as the species Cimex hemipterus, of which 285/1,637 (17%) were randomly selected for MALDI-TOF MS analysis and bacteria screening. Among the Bacteria tested only Wolbachia (Alphaproteobacteria, Rickettsiales, Rickettsiaceae) DNA was found in 248/276 (90%) of the bedbugs. We briefly describe a high level of non-generalized bed bug infestation in rural Senegal and the diversity of Wolbachia strains carried by C. hemipterus. This study opens perspectives for raising household awareness of bed bug infestations and possibilities for appropriate control.

Morphological and Molecular Identification of Tropical Bed Bugs From Two Cities of the Pearl River Delta in China.

From the 1960s to the 1980s, with the implementation of nationwide 'Four-Pest Elimination' campaigns (bed bugs, Cimex lectularius L. and Cimex hemipterus (F.) (Hemiptera: Cimicidae), were listed as one of the targeted pests), bed bug infestations were gradually eliminated in most provinces in China. However, during the last two decades, reports of bed bug infestations in the Pearl River Delta of China have shown an upward trend. Up to now, the bed bug species occurring in this area was much less frequently reported. In this study, we used both morphological and molecular methods to accurately identify the species of bed bugs collected from the cities of Guangzhou and Foshan, China. Results indicated that no significant difference was observed in the mean pronotum width-to-length ratio of Guangzhou (2.6) and Foshan (2.4) specimens; however, both were significantly lower than that of a laboratory strain C. lectularius (3.1). The genetic distances of our specimens with C. hemipterus and C. lectularius were 0-0.2% and 22.2-22.6%, respectively. On the basis of the morphological characteristics and mitochondrial DNA sequence data, it can be affirmed that bed bugs collected from Guangzhou and Foshan were C. hemipterus.

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.

Phylogenetic analyses of bat-associated bugs (Hemiptera: Cimicidae: Cimicinae and Cacodminae) indicate two new species close to Cimex lectularius.

BACKGROUND: Bats are regarded as the primary (ancestral) hosts of bugs of the family Cimicidae. The historically and economically most important species in the family is the common bedbug (Cimex lectularius), because of its worldwide occurrence and association with humans. This molecular-phylogenetic study was initiated in order to expand the knowledge on the phylogeny of cimicid bugs of bats, by investigating samples from Hungary, Romania (representing central-eastern Europe) and two further countries (South Africa and Vietnam). RESULTS: Altogether 216 cimicid bugs were collected (73 Ci. lectularius, 133 Ci. pipistrelli, nine Cacodmus ignotus and one Ca. sparsilis). Members of the Cimex lectularius species group were found both in the environment of bats (only Myotis emarginatus, which is a cave/attic-dwelling species) and on three crevice-dwelling bat species (two pipistrelloid bats and M. bechsteinii). On the other hand, Ci. pipistrelli always occurred off-host (near M. myotis/blythii, which are cave/attic-dwelling species). In addition, two Cacodmus spp. were collected from Pipistrellus hesperidus. The morphological characters of these specimens are illustrated with high resolution pictures. Analysis of cytochrome c oxidase subunit 1 (cox1) sequences generated from 38 samples indicated relative genetic homogeneity of Ci. pipistrelli, while the Ci. lectularius group had two haplotypes (collected from pipistrelloid bats in Hungary and Vietnam) highly divergent from other members of this species group. These results were confirmed with molecular and phylogenetic analyses based on the internal transcribed spacer 2 (ITS2). Bat-associated bugs morphologically identified as Ca. ignotus and Ca. sparsilis were different in their cox1, but identical in their ITS2 sequences. CONCLUSIONS: Molecular evidence is provided here on the existence of two new genotypes, most likely new species, within the Ci. lectularius species group. The relevant specimens (unlike the others) were collected from pipistrelloid bats, therefore the association of Ci. lectularius with different bat host species (pipistrelloid vs myotine bats) should be evaluated further as a possible background factor of this genetic divergence. In addition, Ca. ignotus is reported for the first time in South Africa.

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.

Double cuticle barrier in two global pests, the whitefly Trialeurodes vaporariorum and the bedbug Cimex lectularius.

The integument protects the organism against penetration of xenobiotics and water that would potentially interfere with homeostasis. In insects that play key roles in a variety of agricultural and ecological habitats, this inward barrier has barely been investigated. In order to advance knowledge in this field, we studied integumental barrier (cuticle) permeability in the two global pests Trialeurodes vaporariorum (greenhouse whitefly) and Cimex lectularius (bedbug), applying a simple dye-penetration assay. In agreement with our recent findings in Drosophila melanogaster, we show that the surface of these insects is regionalised. We also show that, in contrast to the single barrier in D. melanogaster, two barriers with distinct temperature-sensitive and lipid-based physico-chemical material properties act in parallel to protect these insects against penetration of hydrophilic molecules. These findings imply the existence of unexplored mechanisms by which the cuticle acts as a protective coat against the penetration of water and xenobiotics, including pollutants and insecticides.

Investigation of the midgut structure and ultrastructure in Cimex lectularius and Cimex pipistrelli (Hemiptera: Cimicidae).

Cimicidae are temporary ectoparasites, which means that they cannot obtain food continuously. Both Cimex species examined here, Cimex lectularius (Linnaeus 1758) and Cimex pipistrelli (Jenyns 1839), can feed on a non-natal host, C. lectularius from humans on bats, C. pipistrelli on humans, but never naturally. The midgut of C. lectularius and C. pipistrelli is composed of three distinct regions-the anterior midgut (AMG), which has a sack-like shape, the long tube-shaped middle midgut (MMG), and the posterior midgut (PMG). The different ultrastructures of the AMG, MMG, and PMG in both of the species examined suggest that these regions must fulfill different functions in the digestive system. Ultrastructural analysis showed that the AMG fulfills the role of storing food and synthesizing and secreting enzymes, while the MMG is the main organ for the synthesis of enzymes, secretion, and the storage of the reserve material. Additionally, both regions, the AMG and MMG, are involved in water absorption in the digestive system of both Cimex species. The PMG is the part of the midgut in which spherites accumulate. The results of our studies confirm the suggestion of former authors that the structure of the digestive tract of insects is not attributed solely to diet but to the basic adaptation of an ancestor.

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.

[NEW FINDINGS OF BAT ECTOPARASITES (CHIROPTERA: VESPERTILIONIDAE) IN SOUTHERN SIBERIA].

The data on new findings of ectoparasites (mites and insects) of bats (Chiroptera: Vespertilionidae) in the Western Sayan and Tuva are represented. The bat fly Basilia mongolensis mongolensis Theodor, 1966 was discovered in the territory of Russia for the first time. Gamasid mite Spinturnix bregetovae Stanyukovich, 1995 is new for the region. New hosts were described for some ectoparasites.

Duplicated female receptacle organs for traumatic insemination in the tropical bed bug Cimex hemipterus: adaptive variation or malformation?

During mating, male bed bugs (Cimicidae) pierce the female abdomen to inject sperm using their needle-like genitalia. Females evolved specialized paragenital organs (the spermalege and associated structures) to receive traumatically injected ejaculates. In Leptocimex duplicatus, the spermalege is duplicated, but the evolutionary significance of this is unclear. In Cimex hemipterus and C. lectularius, in which females normally develop a single spermalege on the right side of the abdomen, similar duplication sometimes occurs. Using these aberrant morphs (D-females) of C. hemipterus, we tested the hypothesis that both of the duplicated spermaleges are functionally competent. Scars on female abdominal exoskeletons indicated frequent misdirected piercing by male genitalia. However, the piercing sites showed a highly biased distribution towards the right side of the female body. A mating experiment showed that when the normal insemination site (the right-side spermalege) was artificially covered, females remained unfertilized. This was true even when females also had a spermalege on the left side (D-females). This result was attributed to handedness in male mating behavior. Irrespective of the observed disuse of the left-side spermalege by males for insemination, histological examination failed to detect any differences between the right-side and left-side spermaleges. Moreover, an artificial insemination experiment confirmed that spermatozoa injected into the left-side spermalege show apparently normal migration behavior to the female reproductive organs, indicating an evolutionary potential for functionally-competent duplicated spermaleges. We discuss possible mechanisms for the evolutionary maintenance of D-females and propose a plausible route to the functionally-competent duplicated spermaleges observed in L. duplicatus.

Bed bugs.

The term bed bug is applied to 2 species of genus Cimex: lectularius describes the common or temperate bed bug, and hemipterus its tropical cousin. Cimex lectularius is aptly named; its genus and species derive from the Latin words for bug and bed, respectively. Though the tiny pest is receiving increased public attention and scrutiny, the bed bug is hardly a new problem.

Descrição das Infestações e estudo morfométrico de percevejos de cama (Hemiptera: Cimicidae) do Estado de São Paulo / Description of infestations and morphometric study Bed Bug (Hemiptera: Cimicidae) in the State of Sao Paulo

Os cimicídeos, conhecidos popularmente como percevejos-de-cama, são insetos hematófagos durante os estágios de ninfas e adulto. Algumas espécies apresentam marcante antropofilia e apesar de não transmitirem patógenos, podem causar algumas complicações a saúde humana, como alergias graves e anemia. O número de infestações nos últimos anos em muitos países tem aumentado significativamente e a relação com lugares de baixo IDH não é mais observada, inclusive na cidade de São Paulo. A precariedade nos estudos dificulta o conhecimento sobre as infestações. No presente estudo, teve-se o objetivo descrever infestações no estado de São Paulo e seus aspectos, e estudar a morfologia dos percevejos de cama encontrados, através de análise morfométrica. Foram coletadas e descritas cinco infestações, quatro na cidade de São Paulo e uma na cidade de Mirandópolis. Os resultados sugerem haver falta de conhecimento da população sobre esta praga urbana e comfirmam Cimex lectularius como principal espécie presente no estado de São Paulo, assim como sugere uma proximidade populacional das infestações dentro da cidade de São Paulo em relação a infestações de outras localidades. Porém os estudos devem ser estimulados no intuito de enriquecer o conhecimento sobre estes insetos, haja vista o grande fluxo de pessoas e a possibilidade de agravamento das infestações, uma vez que grandes eventos internacionais ocorrerão em capitais brasileiras.

The Cimicidae, commonly known as bedbugs are bloodsucking insects throughout all their life cycle. Some species show striking anthropophily and despite they dont transmit diseases they can cause some complications for human health, such as severe allergies and anemia. The number of infestations over the last few years in many countries has increased significantly compared to places with low HDI that is no longer observed, including São Paulo city. The lack of studies hinders knowledge about infestations. The present study aims to describe infestations in the state of São Paulo and its aspects, and to study the morphology of bedbugs found, through morphometric analysis. Five infestations in the city of São Paulo and one in the city of Mirandópolis were collected and described. The results suggest lack of knowledge of the population about this urban blight and confirm Cimex lectularius as the main species present in the state of São Paulo. Also suggests proximity of infestations within the city of São Paulo in relation to infestations of other localities. However, the studies should be encouraged in order to enrich the knowledge about these insects, considering the large flow of people and the possibility of aggravating the infestation, since large international events will occur in the largest Brazilian cities.

Mitochondrial DNA and morphology show independent evolutionary histories of bedbug Cimex lectularius (Heteroptera: Cimicidae) on bats and humans.

The bedbug, Cimex lectularius, is a well-known human ectoparasite that is reemerging after a long absence of several decades in developed countries of North America and Western Europe. Bedbugs' original hosts were likely bats, and the bedbugs are still common in their roosts. Using morphometry and sequences of mitochondrial cytochrome oxidase subunit I and 16S genes, we showed that the populations on bats and humans are largely isolated and differ in morphology. The character of the morphological difference suggests it to be due to adaptation to different hosts, namely adaptations to different sensory, feeding, and dispersal needs. Using the molecular data, we estimated the time of splitting into bat- and human-parasitizing groups using the isolation-with-migration model. The estimate is surprisingly long ago and seems to predate the expansion of modern human from Africa. The gene flow between bat- and human-parasitizing bedbugs is limited and asymmetric with prevailing direction from human-parasitizing populations to bat-parasitizing populations. The differentiation of the populations fits the concept of host races and supports the idea of sympatric speciation. Furthermore, our findings contradict recently formulated hypotheses suggesting bat roosts as a source of bedbug's resurgence as a human pest. Also, we extend the known host range of the bedbug by two bat species.

Bedbugs (Cimicidae infestation): the worldwide renaissance of an old partner of human kind.

Bedbugs have been known as a human parasite for thousands of years, but scientific studies about this insect are recent and limited. Cimex lectularius, the common bedbug, was a well-known parasite in human dwellings until the end of the Second World War. Nowadays, bedbugs are considered uncommon in the industrialized world. Anecdotal reports suggest that bedbugs are getting more common in the United States, Canada, and United Kingdom. In Brazil, there are few reports about bedbug infestations in the literature. The aim of this article was to alert physicians, especially in Brazil, about this ectoparasitosis, including aspects of the bedbug biology, their parasitism in human host, treatment and prophylaxis.

Bedbugs (Cimicidae infestation): the worldwide renaissance of an old partner of human kind

Bedbugs have been known as a human parasite for thousands of years, but scientific studies about this insect are recent and limited. Cimex lectularius, the common bedbug, was a well-known parasite in human dwellings until the end of the Second World War. Nowadays, bedbugs are considered uncommon in the industrialized world. Anecdotal reports suggest that bedbugs are getting more common in the United States, Canada, and United Kingdom. In Brazil, there are few reports about bedbug infestations in the literature. The aim of this article was to alert physicians, especially in Brazil, about this ectoparasitosis, including aspects of the bedbug biology, their parasitism in human host, treatment and prophylaxis.