Human profiling from STR and SNP analysis of tropical bed bug, Cimex hemipterus, for forensic science.

Tropical bed bugs, Cimex hemipterus, which commonly feeds on human blood, may be useful in forensic applications. However, unlike the common bed bug, Cimex lectularius, there is no information regarding tropical bed bug, C. hemipterus, being studied for its applications in forensics. Thus, in this study, lab-reared post-feeding tropical bed bugs were subjected to Short Tandem Repeat (STR) and Single Nucleotide Polymorphism (SNP) analyses to establish the usage of tropical bed bugs in forensics. Several post-feeding times (0, 5, 14, 30, and 45 days) were tested to determine when a complete human DNA profile could still be obtained after the bugs had taken the blood meal. The results showed that complete STR and SNP profiles could only be obtained from the D0 sample. The profile completeness decreased over time, and partial STR and SNP profiles could be obtained up to 45 days post-blood meal. The generated SNP profiles, complete or partial, were also viable for HIrisPlex-S phenotype prediction. In addition, field-collected bed bugs were also used to examine the viability of the tested STR markers, and the STR markers detected mixed profiles. The findings of this study established that the post-blood meal of tropical bed bugs is a suitable source of human DNA for forensic STR and SNP profiling. Human DNA recovered from bed bugs can be used to identify spatial and temporal relations of events.

MALDI-TOF MS identification of Cimex lectularius and Cimex hemipterus bedbugs.

Bedbugs (Cimex lectularius and C. hemipterus) have reemerged as a major public health problem around the world. Their bites cause various skin lesions as well as discomfort and anxiety. Their role as potential vectors of various infectious agents is discussed. Accordingly, all suspected cases of bedbug infestations need to be documented thoroughly, with an unequivocal identification of the arthropods involved, if any are present. Although morphological identification is easily and quickly performed by entomologists or professionals, it can be challenging otherwise. Also, distinguishing Cimex lectularius and C. hemipterus requires entomological expertise. MALDI-TOF mass spectrometry has been recently presented as an additional tool for arthropod identification. In this study, we assess the use of MALDI-TOF MS for the identification of laboratory and wild strains of C. lectularius and C. hemipterus. Several body parts of laboratory reared C. lectularius specimens were used to develop a MALDI-TOF MS protocol for bedbug identification, which was later validated using five other laboratory and wild populations of C. hemipterus and C. lectularius. A total of 167C. lectularius and C. hemipterus bedbug specimens (98 laboratory specimens and 69 wild specimens) were submitted to MALDI-TOF MS analysis. 143/167 (85.63%) provided high quality MS spectra. The in-lab database was then upgraded with a total of 20 reference spectra from all bedbug populations and the rest of the MS spectra (123 bedbugs) were blind tested. All specimens were properly identified to the species level using MALDI-TOF MS and 86,25% (69/80) were aptly identified according to their origin with LSVs ranging from 1.867 to 2.861. MALDI-TOF MS appears as a reliable additional tool for the identification of these two anthropophilic species.

Electrophysiologically and behaviourally active semiochemicals identified from bed bug refuge substrate.

Bed bugs are pests of public health importance due to their relentless biting habits that can lead to allergies, secondary infections and mental health issues. When not feeding on human blood bed bugs aggregate in refuges close to human hosts. This aggregation behaviour could be exploited to lure bed bugs into traps for surveillance, treatment efficacy monitoring and mass trapping efforts, if the responsible cues are identified. The aim of this study was to identify and quantify the bed bug aggregation pheromone. Volatile chemicals were collected from bed bug-exposed papers, which are known to induce aggregation behaviour, by air entrainment. This extract was tested for behavioural and electrophysiological activity using a still-air olfactometer and electroantennography, respectively. Coupled gas chromatography-electroantennography (GC-EAG) was used to screen the extract and the GC-EAG-active chemicals, benzaldehyde, hexanal, (E)-2-octenal, octanal, nonanal, decanal, heptanal, (R,S)-1-octen-3-ol, 3-carene, ß-phellandrene, (3E,5E)-octadien-2-one, (E)-2-nonenal, 2-decanone, dodecane, nonanoic acid, 2-(2-butoxyethoxy)ethyl acetate, (E)-2-undecanal and (S)-germacrene D, were identified by GC-mass spectrometry and quantified by GC. Synthetic blends, comprising 6, 16, and 18 compounds, at natural ratios, were then tested in the still-air olfactometer to determine behavioural activity. These aggregation chemicals can be manufactured into a lure that could be used to improve bed bug management.

Neuropeptidomics of the Bed Bug Cimex lectularius.

The bed bug Cimex lectularius is a globally distributed human ectoparasite with fascinating biology. It has recently acquired resistance against a broad range of insecticides, causing a worldwide increase in bed bug infestations. The recent annotation of the bed bug genome revealed a full complement of neuropeptide and neuropeptide receptor genes in this species. With regard to the biology of C. lectularius, neuropeptide signaling is especially interesting because it regulates feeding, diuresis, digestion, as well as reproduction and also provides potential new targets for chemical control. To identify which neuropeptides are translated from the genome-predicted genes, we performed a comprehensive peptidomic analysis of the central nervous system of the bed bug. We identified in total 144 different peptides from 29 precursors, of which at least 67 likely present bioactive mature neuropeptides. C. lectularius corazonin and myosuppressin are unique and deviate considerably from the canonical insect consensus sequences. Several identified neuropeptides likely act as hormones, as evidenced by the occurrence of respective mass signals and immunoreactivity in neurohemal structures. Our data provide the most comprehensive peptidome of a Heteropteran species so far and in comparison suggest that a hematophageous life style does not require qualitative adaptations of the insect peptidome.

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.

Two compounds in bed bug feces are sufficient to elicit off-host aggregation by bed bugs, Cimex lectularius.

BACKGROUND: After feeding, bed bugs aggregate in cracks and crevices near a host. Aggregation and arrestment are mediated by tactile and chemical stimuli associated with the bugs' feces and exuviae. Volatiles derived from fecally stained filter papers were analyzed by solid-phase microextraction (SPME) and evaluated using a multichoice behavioral assay to determine their impact on bed bug aggregation. In addition, crude fecal extracts were collected in methanol, analyzed by gas chromatography coupled with electroantennogram detection (GC-EAD) and mass spectrometry (GC-MS) and evaluated in open-air multichoice behavioral assays. RESULTS: The SPME method was used to detect (E)-2-hexenal and (E)-2-octenal in heated bed bug feces. The presence of these two volatile components did not affect aggregation. Analysis of the crude fecal extracts revealed several semi-volatile nitrogenous compounds, a carboxylic acid and a sulfur-based compound. Adult antennae responded to compounds eluted from three regions of the crude extract using GC-EAD. A combination of two compounds, dimethyl trisulfide and methyldiethanolamine, resulted in aggregation responses equivalent to the original crude extract. CONCLUSION: Bed bug aggregation is mediated by semi-volatile compounds derived from fecal extracts, and two compounds are sufficient to elicit aggregation. The two compounds identified here could be used to enhance the effectiveness of insecticidal applications or improve monitoring techniques. © 2016 Society of Chemical Industry.

Bed bug aggregation pheromone finally identified.

Bed bugs have become a global epidemic and current detection tools are poorly suited for routine surveillance. Despite intense research on bed bug aggregation behavior and the aggregation pheromone, which could be used as a chemical lure, the complete composition of this pheromone has thus far proven elusive. Here, we report that the bed bug aggregation pheromone comprises five volatile components (dimethyl disulfide, dimethyl trisulfide, (E)-2-hexenal, (E)-2-octenal, 2-hexanone), which attract bed bugs to safe shelters, and one less-volatile component (histamine), which causes their arrestment upon contact. In infested premises, a blend of all six components is highly effective at luring bed bugs into traps. The trapping of juvenile and adult bed bugs, with or without recent blood meals, provides strong evidence that this unique pheromone bait could become an effective and inexpensive tool for bed bug detection and potentially their control.

Aggregation responses of Cimex hemipterus F. to semiochemicals identified from their excreta.

BACKGROUND & OBJECTIVES: Bedbug Cimex hemipterus lives in cracks and crevices, and shows aggregation activity in the harbourage containing its fecal matter. Limited information is available on the chemicals influencing this aggregation behaviour in C. hemipterus. Possible components responsible for aggregation, if identified and evaluated can be helpful in designing bedbug management. METHODS: Study on the isolation and identification of chemicals from the excreta extract responsible for aggregation has been carried by GC-MS. The compounds identified were evaluated at different concentrations to C. hemipterus and their attraction index was determined. RESULTS: The GC-MS analysis of the excreta extract resulted in identification of 33 different types of semiochemicals such as esters, carboxylic acids, alcohols, aldehydes, ketones and other hydrocarbons. Among these 33 compounds of various groups, the carboxylic acid (E)-2-hexenoic acid and the aldehyde (E)-2-hexenal found to elicit highest positive aggregation behavioural response in all the stages followed by hexenal. INTERPRETATION & CONCLUSION: The fecal matter influence the aggregation behaviour in C. hemipterus. (E)-2-hexenoic acid, hexanoic acid, (E)-2-hexenal and hexanal found to exhibit aggregation in various stages of bedbug life cycle. These chemical components can be further exploited for designing and development tool for management of bedbugs.

Real-time measurement of volatile chemicals released by bed bugs during mating activities.

In recent years, bed bug (Hemiptera: Cimicidae) problems have increased dramatically in many parts of the world, leading to a renewed interest in their chemical ecology. Most studies of bed bug semiochemicals have been based on the collection of volatiles over a period of time followed by chemical analysis. Here we present for the first time, a combination of proton transfer reaction mass spectrometry and video analysis for real-time measurement of semiochemicals emitted by isolated groups of bed bugs during specific behavioural activities. The most distinct peaks in the proton transfer reaction mass spectrometry recordings were always observed close to the termination of mating attempts, corresponding to the defensive emissions that bed bugs have been suspected to exploit for prevention of unwanted copulations. The main components of these emissions were (E)-2-hexenal and (E)-2-octenal recorded in ratios between 1:3 and 3:1. In the current study, the quantity varied over 1000 fold for both of the compounds with up to 40 µg total release in a single emission. Males also emit defensive compounds due to homosexual copulation attempts by other males, and no significant differences were observed in the ratio or the amount of the two components released from males or females. In summary, this study has demonstrated that combining proton-transfer-reaction mass spectrometry with video analysis can provide detailed information about semiochemicals emitted during specific behavioural activities.

Semiochemicals of the common bed bug, Cimex lectularius L. (Hemiptera: Cimicidae), and their potential for use in monitoring and control.

The recent resurgence of the common bed bug, Cimex lectularius L., has driven an increase in research into the biology and behaviour of this pest. Current control is reliant on the application of insecticides, but, owing to the development of insecticide resistance, there is a need for new tools and techniques. Semiochemicals (behaviour- and physiology-modifying chemicals) could be exploited for management of bed bugs. The aim of this review was to evaluate studies undertaken in bed bug chemical ecology to date, with particular reference to how the research could be exploited for monitoring and control. Bed bugs, like many other insects, have a complex olfactory system. Recent studies have characterised the olfactory sensilla, located on the terminal segment of the antennae, to functional classes by electrophysiological screening. Behavioural studies have revealed the presence of an alarm pheromone and potential airborne aggregation semiochemicals, but it is not yet understood if bed bugs use a sex pheromone during mating. Host location cues have been investigated, and carbon dioxide has been found to be highly attractive both in laboratory and in field studies. Recent field trials have tested blends of other potential kairomones, which have been shown to have an additive effect when used in a heated bed bug trap with carbon dioxide. The trap, which combines heat and kairomones, is the only trap currently available with proven efficacy in the field. In order for semiochemicals to be useful for bed bug management, an increased knowledge and understanding of the biology, behaviour and chemical ecology of this insect is essential.

A bioassay for studying behavioural responses of the common bed bug, Cimex lectularius (Hemiptera: Cimicidae) to bed bug-derived volatiles.

The common bed bug, Cimex lectularius (Hemiptera: Cimicidae), has recently re-emerged in increasing numbers, distribution and intensity of infestation in many countries. Current control relies on the application of residual pesticides; but, due to the development of insecticide resistance, there is a need for new tools and techniques. Semiochemicals (behaviour and physiology modifying chemicals) could be exploited for management of bed bugs. However, in order to identify semiochemicals that can be utilised in monitoring or control, a suitable olfactometer is needed that enables the study of the responses of bed bugs to volatile chemicals. Previous studies have used olfactometers that do not separate olfactory responses from responses to physical contact. In this study, a still-air olfactometer was used to measure behavioural responses to different bed bug-derived volatiles presented in an odour pot. Bed bugs were significantly more likely to visit the area above the odour pot first, and more frequently, in the presence of volatiles from bed bug-exposed paper but not in the presence of volatiles from conspecific bed bugs. Bed bug activity was found to be dependent on the presence of the volatiles from bed bug-exposed paper, the time during the scotophase and the sex of the insect being tested. The still-air olfactometer could be used to test putative semiochemicals, which would allow an understanding of their behavioural role in bed bug ecology. Ultimately, this could lead to the identification of new semiochemical tools for bed bug monitoring and control.

4-oxo-aldehydes from the dorsal abdominal glands of the bed bug (Hemiptera: Cimicidae).

Analyses of the dorsal abdominal glands of fourth- and fifth-instar nymphs of the bed bud, Cimex lectularius L. (Hemiptera: Cimicidae), indicated the predominant constituents were (E)-2-hexenal and (E)-2-octenal, with lesser amounts of 4-oxo-(E)-2-hexenal and 4-oxo-(E)-2-octenal. The latter two compounds have not been reported previously as occurring in bed bugs. There were no differences in the chemical composition of the dorsal abdominal glands excised from exuviae left behind by either male or female adults, nor from glands excised from fourth-instar exuviae. Because the two oxo-aldehydes made up at least 16% of the gland contents, further study of the functional role of these chemicals seem advisable.

Detection of seminal fluid proteins in the bed bug, Cimex lectularius, using two-dimensional gel electrophoresis and mass spectrometry.

The global increase of the human parasite, the common bed bug Cimex lectularius, calls for specific pest control target sites. The bed bug is also a model species for sexual conflict theory which suggests that seminal fluids may be highly diverse. The species has a highly unusual sperm biology and seminal proteins may have unique functions. One-dimensional PAGE gels showed 40-50% band sharing between C. lectularius and another cimicid species, Afrocimex constrictus. However, adult, sexually rested C. lectularius males were found to store 5-7 microg of seminal protein and with only 60 microg of protein we obtained informative 2-D PAGE gels. These showed 79% shared protein spots between 2 laboratory populations, and more than half of the shared protein spots were detected in the mated female. Further analysis using liquid chromatography electrospray ionization tandem mass spectrometry revealed that 26.5% of the proteins had matches among arthropods in databases and 14.5% matched Drosophila proteins. These included ubiquitous proteins but also those more closely associated with reproduction such as moj 29, ubiquitin, the stress-related elongation factor EF-1 alpha, a protein disulfide isomerase and an antioxidant, Peroxiredoxin 6.

Heme-assisted S-nitrosation of a proximal thiolate in a nitric oxide transport protein.

Certain bloodsucking insects deliver nitric oxide (NO) while feeding, to induce vasodilation and inhibit blood coagulation. We have expressed, characterized, and determined the crystal structure of the Cimex lectularius (bedbug) nitrophorin, the protein responsible for NO storage and delivery, to understand how the insect successfully handles this reactive molecule. Surprisingly, NO binds not only to the ferric nitrophorin heme, but it can also be stored as an S-nitroso (SNO) conjugate of the proximal heme cysteine (Cys-60) when present at higher concentrations. EPR- and UV-visible spectroscopies, and a crystallographic structure determination to 1.75-A resolution, reveal SNO formation to proceed with reduction of the heme iron, yielding an Fe-NO complex. Stopped-flow kinetic measurements indicate that an ordered reaction mechanism takes place: initial NO binding occurs at the ferric heme and is followed by heme reduction, Cys-60 release from the heme iron, and SNO formation. Release of NO occurs through a reversal of these steps. These data provide, to our knowledge, the first view of reversible metal-assisted SNO formation in a protein and suggest a mechanism for its role in NO release from ferrous heme. This mechanism and Cimex nitrophorin structure are completely unlike those of the nitrophorins from Rhodnius prolixus, where NO protection is provided by a large conformational change that buries the heme nitrosyl complex, highlighting the remarkable evolution of proteins that assist insects in bloodfeeding.

A novel inhibitor of factor X activation from the salivary glands of the bed bug Cimex lectularius.

Cimex lectularius salivary gland homogenate delayed the recalcification time of human citrated plasma. Separation of the salivary gland homogenate by molecular sieving HPLC chromatography resulted in a single major peak of anticlotting activity with an apparent molecular mass of 17,000. The anticoagulant principle inhibited the activation of factor X to factor Xa in the tenase complex (FVIII, FIXa, FX, phospholipids, and calcium). However, it did not directly inhibit already activated factor Xa, suggesting that the anticlotting activity is not an anti-factor Xa. Additionally, this salivary gland anticoagulant further retarded the recalcification time of factor VIII- and factor IX-deficient plasmas, suggesting that the anticlotting principle is not directly inhibiting either the coagulation factor VIII or factor IXa. Altogether these data suggest that the anticlotting activity is an inhibitor of the activation of factor X to factor Xa in the tenase complex.