The ability to isolate and generate a DNA profile from human DNA recovered from tropical bed bugs (Cimex hemipterus) for identifying individuals can be useful for public health, forensic, and medical entomology. In this study, genomic DNA was recovered from both male and female bed bugs at every time interval tested (0, 1, 3, 5, 7, 14, 30, and 45 days post blood meal). The total DNA concentrations recovered from male bed bugs ranged from 12.93 to 65.97 ng/µL, while the total DNA concentrations from female bed bugs ranged from 8.93 to 44.53 ng/µL. However, based on the results from the BLAST search and PCR products, human DNA could be detected from female bed bugs at 0, 3, 5, 14, and 30 days post blood meal using the D18S51 marker. Concentrations of PCR products of the D18S51 locus from male bed bugs ranged from 4.20 to 35.50 ng/µL, whereas, for female bed bugs, concentrations ranged from 4.31 to 22.47 ng/µL. These were generally higher compared to the PCR products of the first hypervariable part (HVR1) marker. The results indicate the HVR1 locus was less sensitive than the D18S51 locus.
The tropical bed bug is scientifically recognized as a significant public health problem. While there is an increased awareness about their resurgence by medical and life science committees, efficient bed bug management still remains unresolved. The solution may soon arise, as information about bed bugs' infestation dynamics and systematics are becoming more distinguishable. Recent developments in studies about bed bugs are based on molecular intervention by determining their genetic variation and phylogeography. The aim of this study is to assess the phylogenetic relationships and genetic diversity among the populations of tropical bed bugs inhabiting Malaysia. A molecular genotyping study was conducted with 22 tropical bed bug populations composed of three individuals per population. The mitochondrial (COI) gene was used as a marker. The data obtained were analyzed using the T-Coffee, ClustalX, MEGA 6.0, and PAUP software. The results showed one main monophyletic clade that consisted of two groups: Ch01 and Ch02. Ch02 consists of samples from the Bandar Hilir population, differing from the other populations studied by one singleton base. However, as there were no changes in the amino acid, this singleton genetic variation was considered to have no effect on genetic differentiation. Ch01 shows similarity with some sequence of Cimex hemipterus (F.) from Thailand, suggesting an international diversity connection. The disparity index apparently suggests that all isolates are homogeneous populations and are supported by the low value of the mean pairwise distance between isolates. This study will increase the knowledge about phylogeographic diversity of tropical bed bug in Malaysia.
The surge in tropical bed bug Cimex hemipterus (Fabricius) (Hemiptera: Cimicidae) infestations has led to an increase in genomic studies. In this study, the population genetics and breeding patterns of 22 Malaysian populations were analyzed, including genetic differentiation and genetic distance. For seven microsatellite loci, the number of alleles varied from 6 to 14. The allelels per loci contrasted sharply between the overall population and within the populations. The average observed and expected heterozygosity was 0.280 and 0.828 for the overall population and 0.281 and 0.657 among the populations, respectively. Based on polymorphic information criteria, the markers with a value >0.5 were highly polymorphic. In the Hardy-Weinberg equilibrium, the loci of Ch 09ttn, Ch 01dn, and Ch 13dn of the overall population showed signs of a null allele. The stutter peaks caused no scoring errors; large allele dropouts were not detected for any loci; and a correlation imbalance was not indicated. The genetic differentiation among populations was moderate, with a coefficient of genetic differentiation (FST) of 0.144. The bed bug populations showed strong inbreeding, with highly positive coefficients of inbreeding (FIS). The molecular variation attributed to inbreeding was 83% within the populations, compared with 17% among the populations. The admixture individuals in STRUCTURE and neighbor-joining phylogenetic trees also indicated weak genetic structure in the geographical populations, suggesting moderate gene flows between populations. Thus, moderately active dispersion and human-mediated transport shaped the genetic structure of C. hemipterus populations in Malaysia.
The metagenomic datasets of the microbial DNA from tropical bed bugs (Cimex hemipterus) after feeding on human blood were presented. Next-generation sequencing of the community DNA was carried out on an Illumina Miseq platform and the raw fastq files were analyzed using QIIME (version 1.9.1). The metagenome of three samples comprised of 108,198 sequences representing 44,646,263 bps with a mean length of 412.63 bps. The sequence data is accessible at the NCBI SRA under the bioproject number PRJNA600667. Community analysis showed Proteobacteria was the most abundance (more than 99%) microbial community that present in the guts of fully fed tropical bed bugs.
Due to the growing public health and tourism awareness, Cimex hemipterus Fabricius (Hemiptera: Cimicidae) has gained a great interest in increasing reported infestation cases in tropical regions of the world, including Malaysia. Since the information on the molecular ecology and population biology of this species are tremendously lacking, the isolation and development of molecular markers can be used to determine its genetic structure. In this study, novel microsatellite primers isolated from enriched genomic libraries of C. hemipterus were developed using 454 Roche shotgun sequencing. Seven validated polymorphic microsatellite primers were consistently amplified and characterized from 70 tropical bed bugs collected from seven locations throughout Malaysia. The number of alleles per locus identified ranged from 6 to 14. Comparison of loci for overall and between population were done with mean observed and expected heterozygosity were determined at 0.320 and 0.814, 0.320 and 0.727, respectively. Polymorphic information criteria (PIC) valued the markers as highly informative as PIC >0.5. Overall population, they are possibly in Hardy-Weinberg equilibrium with loci Ch_09ttn, Ch_01dn, and Ch_13dn showing signs of a null allele. There were no scoring errors caused by stutter peaks, no large allele dropout was detected for all loci and showed no evidence of linkage disequilibrium. In conclusion, all seven molecular microsatellite markers identified can be beneficially used to gain more information on the population genetic structure and breeding patterns of C. hemipterus as well as the relationship of dispersal and infestation.
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.
Pepijat telah dikumpul dari seluruh Semenanjung Malaysia untuk melihat tahap kerintangan terhadap dua kelas racun serangga, piretroid dan karbamat. Dalam kajian ini, deltamethrin dan propoxur yang tersedia secara komersial digunakan untuk menguji status kerintangan pepijat tropika, Cimex hemipterus di Malaysia. Status kerintangan mereka dinilai dengan menggunakan kertas yang diresapi racun serangga, suatu kaedah bioesei yang disyorkan oleh Pertubuhan Kesihatan Sedunia (WHO). Sepuluh pepijat dewasa dalam setiap populasi diletakkan di dalam setiap tiub dengan kertas yang diresapi 0.05% deltamethrin dan 0.1% propoxur. Pada mulanya, pepijat terdedah selama 24 jam, kemudian tempoh pendedahan dilanjutkan hingga 14 hari. Bioesei dilakukan dalam triplikat. Tahap kerintangan tertinggi dipamerkan oleh populasi AR dengan nilai masa maut (LT50) sebanyak 466.119 j di dalam tiub yang mengandungi deltametrin. Sebaliknya, propoxur sangat berkesan terhadap populasi IP (Ipoh) kerana nilai masa maut yang paling rendah, (LT50 = 153.032 j). Perbezaan yang signifikan (p < 0.005) didapati antara kelas racun serangga dan populasi pepijat. Purata kematian di kalangan populasi AR, SW, HM, PY, dan PC menunjukkan perbezaan yang signifikan apabila terdedah kepada deltametrin. Sebagai kesimpulan, propoxur menunjukkan keberkesanan yang tinggi dalam mengawal populasi pepijat. Walau bagaimanapun, keberkesanannya hanya di tempat tertentu. Sementara itu, rawatan menggunakan deltamethrin harus dielakkan oleh profesional kawalan perosak kerana keberkesanannya yang rendah.
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.
Bed bugs [both Cimex hemipterus (F.) and Cimex lectularius L.] are highly resistant to pyrethroids worldwide. An important resistance mechanism known as 'knockdown resistance' (kdr) is caused by genetic point mutations on the voltage-gated sodium channel (VGSC) gene. Previous studies have identified two point mutations (V419L and L925I) on the VGSC gene in C. lectularius that are responsible for kdr-type resistance. However, the kdr mutations in C. hemipterus have not been investigated.
Modern bed bugs are resistant to multiple insecticide classes, particularly the pyrethroids. The efficacy of pyrethroid-impregnated mattress liners marketed for bed bug management has been variable. This study evaluated the efficacy of a permethrin-impregnated mattress liner, ActiveGuard, against 24 bed bug strains, consisting of both Cimex hemipterus (F.) and Cimex lectularius L. A 'mat assay', employing an allethrin-impregnated mat, was used to establish the pyrethroid resistance profile of all strains. Three experiments were conducted to evaluate the effect of ActiveGuard exposure on bed bug knockdown: 1) exposing the bed bugs continuously on the liner for up to 24 d, 2) holding the bed bugs on the liner for either 4 or 6 h, and 3) placing a noninsecticide treated fabric above the liner with the bed bugs held continuously on top. Our results indicated that all modern strains (collected within the last 15 years during the current resurgence) were pyrethroid-resistant, although the magnitude of resistance was highly variable between strains. In the continuous exposure study, an incomplete knockdown was recorded for most modern bed bug strains, with some having no knockdown even up to 7 d of constant exposure. In the 4 or 6 h exposure study, the level of knockdown was reduced even further, and very few bed bugs were knocked down in the double fabric study. The results of this study indicate that pyrethroid-impregnated mattress liners are not likely to be effective in the management of most modern bed bug infestations involving either C. hemipterus or C. lectularius.
The effect of temperature and humidity on the survival and water loss of the tropical bed bug, Cimex hemipterus (F.), was studied using two field-collected strains. Insects were exposed to temperatures ranging from 20 to 45 degrees C and relative humidities (RHs) of 33, 75, and 100%. C. hemipterus survived longest under the interaction of low temperature (20 degrees C) and high RH (75-100%). Survival and water loss were significantly affected (P < 0.01) by temperature and RH (either singly, or in interaction). Strain and sex significantly (P < 0.01) influenced bed bug survival, but not on water loss. Eggs, first instars, and adults reached their upper thermal lethal limit within 1 h at 39 degrees C, 44 degrees C, and 46 degrees C, respectively. The survival and water loss profiles showed that starved C. hemipterus started to die after losing 35-45% of their body weights.
This study examined the effects of different life stages (first, second, third, fourth, and five instars; adult females and adult males) and feeding regimes (starved and blood fed) on the active movement activity of the tropical bed bug, Cimex hemipterus (F.), under mixed-stage conditions. We used an extended arena made from Tygon tube coils and observed the movement frequency and movement distance at selected time intervals up to 120 h. The fifth instars and adult males and females showed significantly (P < 0.01) greater movement frequency compared with the other stages. The first and second instars showed limited movement (< 8 m) over the experimental period. Starved bed bugs showed greater movement frequency compared with blood-fed bed bugs, with the exception of adult females. Blood-fed adult females exhibited significantly (P < 0.01) greater movement frequency and distance compared with starved females. Blood-fed females moved up to 42.3 m over 120 h. Regression analysis between movement distance of the fifth instars and adults and the time intervals revealed a positive relationship (r2 = 0.6583; P < 0.01), suggesting that delays in bed bug control efforts will increase the risk of the greater infestation. During bed bug inspection, the presence of only late instars and adults in premises would indicate a new infestation, whereas an established infestation likely would consist of mixed stages.
Five formulated insecticides (lambda-cyhalothrin at 10 mg m⁻², bifenthrin at 50 mg m⁻², fipronil at 10 mg m⁻², fenitrothion at 50 mg m⁻², imidacloprid at 5 mg m⁻²) and one active ingredient (DDT at 500 mg m⁻²) were evaluated using a surface contact method against early and late instars and adults of two strains of the tropical bed bug, Cimex hemipterus (F.). Synergism of lambda-cyhalothrin and fipronil using piperonyl butoxide (PBO) was also assessed.
The performance of five insecticides (bendiocarb, deltamethrin, DDT, malathion, and imidacloprid) using three application methods (oil-based insecticide films on filter paper, and acetone-based insecticide deposits on two substrates: filter paper and glass) was assessed against a susceptible strain of Cimex lectularius (L.) and two resistant strains of Cimex hemipterus (F.). Substrate type significantly affected (P
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.
This study examined the presence of insecticide resistance in different developmental stages (adults, first instars, and eggs) of the tropical bed bug, Cimex hemipterus (F.) using several insecticide formulations. Adults and first instars of five strains (Queensland, Kuala Lumpur, Bukit Mertajam, Saujana, and Krystal Point) were evaluated using the surface contact method and compared with a susceptible strain (Monheim) of the common bed bug Cimex lectularius L. The insecticide formulations were used at their label rates in this study: Tandem (thiamethoxam [11.6%], lambda-cyhalothrin [3.5%]) at 183.96 mg/m2; Temprid SC (imidacloprid [21%], beta-cyfluthrin [10.5%]) at 106.13 mg/m2; Sumithion 20CS (fenitrothion [20%]) at 250 mg/m2; Pesguard FG161 (d-tetramethrin [4.4%], cyphenothrin [13.2%]) at 110 mg/m2; and Sumithrin 10SEC (d-phenothrin [10%]) at 100 mg/m2. Results showed a very high level of resistance to Pesguard FG161 (388.3 to >605.0 times) and Sumithrin (302.9 to >365.5 times) in all adults of the strains tested, whereas low to high levels of resistance were registered for Tandem (1.4-4.7 times), Temprid (7.3-16.7 times), and Sumithion (1.2-14.6 times) for adults of all bed bug strains. For first instars, resistance to the former two formulations were high to very high (31.4-118.1 times). In contrast, they showed lower resistance to Tandem, Temprid, and Sumithion (1.0-10.2 times). An immersion method used to test on bed bug eggs found high to very high resistance toward all tested formulations. Results demonstrate that the resistance level varies between bed bug developmental stages.
The modern resurgence of the common (Cimex lectularius L.) and tropical bed bugs (C. hemipterus [F.]) is thought to be primarily due to insecticide resistance. While there are many reports on insecticide resistance mechanisms in C. lectularius, such information in C. hemipterus is limited. We examined dichloro-diphenyl-trichloroethane (DDT), malathion, deltamethrin, permethrin, lambda-cyhalothrin resistance, and the underlying mechanisms in several C. hemipterus strains (Australia: Queensland [QLD-AU]; Malaysia: Kuala Lumpur [KL-MY], Tanjung Tokong [TT-MY], Christian [CH-MY], and Green Lane [GL-MY]). We used a surface contact method, synergism studies (utilizing piperonyl butoxide [PBO], S,S,S-tributyl phosphorotrithioate [DEF], and diethyl maleate [DEM]), and molecular detection of kdr mutations. Results demonstrated that all C. hemipterus strains possessed high resistance to DDT and the pyrethroids and moderate to high resistance to malathion. Synergism studies showed that deltamethrin resistance in all strains was significantly (P < 0.05) inhibited by PBO. In contrast, deltamethrin resistance was not affected in DEF or DEM. Similar findings were found with lambda-cyhalothrin resistance. Malathion resistance was significantly (P < 0.05) reduced by DEF in all strains. Resistance to DDT was not affected by DEM in all strains. Multiple kdr mutations (M918I, D953G, and L1014F) were detected by molecular analyses. TT-MY strain was found with individuals possessing three kdr mutation combinations; D953G + L1014F (homozygous susceptible: M918), M918I + D953G + L1014F (heterozygous resistant: I918), and M918I + D953G + L1014F (homozygous resistant: I918). Individuals with M918I + D953G + L1014F (homozygous resistant: I918) survived longer on deltamethrin (>12 h) than those (≤1 h) with other combinations. M918I + L1014F mutations most likely conferred super-kdr characteristic toward pyrethroids and DDT in C. hemipterus.
A total of 54 bed bug-infested sites (hotels, public accommodations, and residential premises) in Malaysia and Singapore was surveyed between July, 2005 and December, 2008. Only one species of bed bug was found, the tropical bed bug Cimex hemipterus (Fabricius). Bed bug infestations were common in hotels and public accommodations when compared to residential premises. The three most common locations of infestation within an infested premise were the bedding (31.1%), the headboard (30.3%), and cracks and crevices surrounding the baseboard, wall, or floor (23.5%). We speculate that the route of movement of bed bugs in hotels and public accommodations is more direct than in residential premises.
The residual performance of two pyrethroid-neonicotinoid mixture formulations: Temprid SC (10.5% beta-cyfluthrin and 21% imidacloprid) and Tandem (3.5% lambda-cyhalothrin and 11.6% thiamethoxam) on two substrates (glass and filter paper) against eight pyrethroid-resistant strains (BM-MY, BP-MY, CH-MY, GL-MY, KL-MY, SAJ-MY, TT-MY, and QLD-AU) of the tropical bed bug, Cimex hemipterus (F.) (Hemiptera: Cimicidae) collected from Malaysia, and Australia were evaluated. The aging effect of treatment residues on glass was also investigated. A susceptible C. lectularius L. strain (Monheim) was used for comparison. Temprid SC showed varying levels of performance against all C. hemipterus strains: TT-MY (PR50 = 6.5-fold, high performance), BM-MY, GL-MY, SAJ-MY, and QLD-AU (12.8-21.6-fold, moderate performance), BP-MY, and KL-MY (48.2-49-fold, poor performance), CH-MY (128.2-fold, very poor performance). On the other hand, Tandem displayed high performance against all C. hemipterus strains (1.8-8.3-fold). Tandem caused faster mortality than Temprid SC for all strains. Temprid SC and Tandem residues killed C. hemipterus significantly faster on glass than filter paper. Compared with fresh residues, the efficacy of Temprid SC residues significantly declined after one week of aging, while the effectiveness of Tandem residues declined after two weeks of aging. Further investigations using the topical assay method with a diagnostic dose of imidacloprid found two strains (CH-MY and GL-MY) resistant to imidacloprid. The six other strains (BM-MY, BP-MY, KL-MY, SAJ-MY, TT-MY, and QLD-AU) were susceptible.