Efficacy of plant-based products and nonconventional pesticides for the management of tropical bed bug.

Insecticide resistance is widespread in global bed bug populations. Both common bed bugs and tropical bed bugs are pyrethroid-resistant among most field populations. Plant-based products and nonconventional pesticides offer minimal-risk strategies for managing bed bug resistance, but this strategy has yet to be formally evaluated in Cimex hemipterus (F.) (Hemiptera: Cimicidae). Here, several commercial plant-based formulations (Cedarcide, EcoRaider (also known as EcoVenger), EcoSMART, and Bio-D), a novel product, Provecta, and a pyrethroid insecticide, Pesguard FG161 were tested against pyrethroid-susceptible and resistant strains of C. hemipterus using direct spray, residual exposure, and egg dipping assays. Direct spray treatments outperform residual applications against all tested bed bug strains. Cedarcide exhibits the highest consistency in eliminating bed bugs, followed by EcoRaider, EcoSMART, and Provecta that outperform Bio-D and Pesguard FG161. In comparison to Pesguard FG161, all plant-based insecticide products and Provecta showed higher efficacy against pyrethroid-resistant strains. Although effective, product efficacy varies in terms of speed. Cedarcide kills all bed bugs within 1 min after spraying; however, other products can take up to 9 days to achieve 100% mortality. The efficacy of all products was reduced when evaluated on fabric surface (42%-65% mortality). Cedarcide and EcoRaider reduced egg hatchability by 37%-73% and 47%-70%, respectively. This study suggests that certain plant-based insecticides and an unconventional insecticide can serve as alternative direct spray treatments for managing tropical bed bugs, though their residual effects are limited.

Perception, attitudes, and knowledge on infestation and management of bed bugs in major cities of Indonesia: A cross-sectional online survey.

The resurgence of bed bugs is a global phenomenon, but until now reports on bed bug infestations in Indonesia are rare. The success of bed bug control is affected by people's knowledge and awareness. Stigma against bed bugs in Indonesia discourages the public from reporting infestations and therefore knowledge of their impact on public health is scarce. Herein, this study investigates the public's perception, attitudes, and knowledge on bed bug-related issues in several major cities in Indonesia through an online survey. Despite low case reports, three in five respondents (n = 600) have encountered bed bugs at least once; mostly in their homes (74.1%). Approximately half of the respondents correctly identified bed bugs, whereas mites were often misidentified for bed bugs (26.3%). Bite marks were not a useful indicator for detecting bed bugs. We found age, gender, and level of education affects the public's perception toward various bed bug-related issues. Regarding bed bug treatment, above 50% respondents are unaware of the availability of bed bug-specific insecticidal products and are unwilling to pay pest management professionals to control infestation. This study provides the first overview of the public's awareness and perception of bed bug infestations in some major cities of Indonesia, which can be useful for designing public health policies for bed bug management. The reported data represents the perspectives of online users, most likely from metropolitan regions. A bigger monitoring program encompassing pest professionals and hospitality businesses would give a more thorough overview of the bed bug impact in Indonesia.

The Efficacy of a Pyrethroid-impregnated Mattress Liner on Multiple International Strains of Cimex lectularius (Hemiptera: Cimicidae) and Cimex hemipterus (Hemiptera: Cimicidae).

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.

Bacterial diversity and community structure shapes pederin polymorphism but lacks association with host genotype specificity in the rove beetle, Paederus fuscipes.

Paederus beetles are notorious for their irritant haemolymph toxin pederin, which is synthesised by an unculturable bacterial symbiont, causing dermatitis when in contact with human skin. Pederin polymorphism is observed when (+) females carrying the toxin pederin and (-) females lacking this co-occur in natural populations. Despite reports detecting pederin polymorphism in several Paederina beetles, symbiont infection frequencies in their natural populations and the bacterial diversity differences underlying the polymorphism between these female types are understudied. Herein we report a high prevalence (>80%) of female Paederus fuscipes carrying bacterial ped genes in all six study populations. This finding suggests that selection pressure favouring pederin-producing females is crucial for survival in the natural environment. 16S rRNA metabarcoding analysis revealed significant dominance of the unculturable pederin-producing Pseudomonas-like bacterium (Gammaproteobacteria) in (+) females, consistent with previous studies. The microbial diversity of the (-) females revealed a significant abundance of Apibacter (Bacteroidia), previously undocumented, suggesting its importance in the functionality of (-) females. Predicted functions related to metabolisms are enriched in (-) females, suggesting fitness advantage possibilities in sustaining the population in the absence of predation. Further investigations on the possible genetic basis of the host genotype revealed no association of host mtDNA and pederin polymorphism in Pa. fuscipes.

Bacterial symbionts influence host susceptibility to fenitrothion and imidacloprid in the obligate hematophagous bed bug, Cimex hemipterus.

The use of insecticides remains important in managing pest insects. Over the years, many insects manifested physiological and behavioral modifications resulting in reduced efficacy of insecticides targeted against them. Emerging evidence suggests that bacterial symbionts could modulate susceptibility of host insects against insecticides. Here, we explore the influence of host microbiota in affecting the susceptibility of insect host against different insecticides in the blood-sucking bed bug, Cimex hemipterus. Rifampicin antibiotic treatment resulted in increased susceptibility to fenitrothion and imidacloprid, but not against deltamethrin. Meanwhile, the host fitness parameters measured in the present study were not significantly affected by rifampicin treatment, suggesting the role of bacterial symbionts influencing susceptibility against the insecticides. 16S metagenomics sequencing revealed a drastic shift in the composition of several bacterial taxa following rifampicin treatment. The highly abundant Alphaproteobacteria (Wolbachia > 90%) and Gammaproteobacteria (Yersinia > 6%) in control bed bugs were significantly suppressed and replaced by Actinobacteria, Bacilli, and Betaproteobacteria in the rifampicin treated F1 bed bugs, suggesting possibilities of Wolbachia mediating insecticide susceptibility in C. hemipterus. However, no significant changes in the total esterase, GST, and P450 activities were observed following rifampicin treatment, indicating yet unknown bacterial mechanisms explaining the observed phenomena. Re-inoculation of microbial content from control individuals regained the tolerance of rifampicin treated bed bugs to imidacloprid and fenitrothion. This study provides a foundation for a symbiont-mediated mechanism in influencing insecticide susceptibility that was previously unknown to bed bugs.

Multiple Mechanisms Conferring Broad-Spectrum Insecticide Resistance in the Tropical Bed Bug (Hemiptera: Cimicidae).

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.

Cuticle thickening associated with fenitrothion and imidacloprid resistance and influence of voltage-gated sodium channel mutations on pyrethroid resistance in the tropical bed bug, Cimex hemipterus.

BACKGROUND: The common bed bug, Cimex lectularius L., and the tropical bed bug, Cimex hemipterus (F.), are now widely regarded as important public health pests following their rapid global resurgence, largely due to insecticide resistance and an increased rate of global travel. The insecticide resistance mechanisms are well documented in C. lectularius, however, only one mechanism is validated in C. hemipterus thus far. This demands further understanding on the resistance mechanisms involved in C. hemipterus. RESULTS: Here, we identified differences in resistance to fenitrothion (organophosphate) and imidacloprid (neonicotinoid) related cuticle thickness in C. hemipterus. There is evidence of a possible association between cuticle thickness and resistance, but the association can be tenuous, likely because resistance is multifactorial in C. hemipterus. We also discovered a novel T1011 residue in domain IIS6 of the voltage-gated sodium channel that likely enhanced susceptibility to deltamethrin (pyrethroid) despite the presence of a L1014F mutation known to confer pyrethroid resistance in C. hemipterus. Our findings also confirmed that the M918I mutation enhanced resistance to pyrethroid when present with the L1014F mutation, which was consistent with a super-kdr phenotype, as reported previously. Multiple resistance mechanisms can be found within a single C. hemipterus population, and the presence of both M918I + L1014F mutations likely masked the influence of cuticle thickness in conferring resistance against deltamethrin. The elevated metabolic enzyme activities in some strains were not necessarily associated with increased insecticide resistance. CONCLUSION: This study has enhanced our understanding on the penetration resistance mechanism and target site insensitivity of sodium channels in C. hemipterus.

Multifaceted interactions between the pseudomonads and insects: mechanisms and prospects.

Insects and bacteria are the most widespread groups of organisms found in nearly all habitats on earth, establishing diverse interactions that encompass the entire range of possible symbiotic associations from strict parasitism to obligate mutualism. The complexity of their interactions is instrumental in shaping the roles of insects in the environment, meanwhile ensuring the survival and persistence of the associated bacteria. This review aims to provide detailed insight on the multifaceted symbiosis between one of the most versatile bacterial genera, Pseudomonas (Gammaproteobacteria: Pseudomonadaceae) and a diverse group of insect species. The Pseudomonas engages with varied interactions with insects, being either a pathogen or beneficial endosymbiont, as well as using insects as vectors. In addition, this review also provides updates on existing and potential applications of Pseudomonas and their numerous insecticidal metabolites as biocontrol agents against pest insects for the improvement of integrated pest management strategies. Here, we have summarized several known modes of action and the virulence factors of entomopathogenic Pseudomonas strains essential for their pathogenicity against insects. Meanwhile, the beneficial interactions between pseudomonads and insects are currently limited to a few known insect taxa, despite numerous studies reporting identification of pseudomonads in the guts and haemocoel of various insect species. The vector-symbiont association between pseudomonads and insects can be diverse from strict phoresy to a role switch from commensalism to parasitism following a dose-dependent response. Overall, the pseudomonads appeared to have evolved independently to be either exclusively pathogenic or beneficial towards insects.

Infestation Pattern and Population Dynamics of the Tropical Bed Bug, Cimex hemipterus (F.) (Hemiptera: Cimicidae) Based on Novel Microsatellites and mtDNA Markers.

The tropical bed bug, Cimex hemipterus (F.), has now emerged as an important public health pest in the tropics. Despite its alarming infestation rate, the information on its population genetics remains scarce. Here, we described the infestation structure and population dynamics of C. hemipterus in the tropics, especially Malaysia and Singapore, based on eight novel microsatellites and two mtDNA markers, including cytochrome c oxidase I (COI) and 16S rRNA genes. Across populations, microsatellite data revealed high genetic diversity with significant genetic differentiation and restricted gene flow. Analysis within populations revealed evidence of a recent bottleneck. Nonetheless, elevated genetic diversity in nearly all populations suggests that the propagule in C. hemipterus populations were much diverse, distantly related (mean r = 0.373), and not significantly inbred (mean FIS = 0.24) than that observed in Cimex lectularius from previous studies. We observed seven mtDNA haplotypes across the 18 populations studied (Hd = 0.593) and several populations displayed more than one matrilineal descent. The two markers were generally congruent in suggesting a common, genetically diverse (especially at the nuclear region) source population with possibilities of multiple introductions for the bed bug populations in the present study.

Identification and characterization of jasmonic acid- and linolenic acid-mediated transcriptional regulation of secondary laticifer differentiation in Hevea brasiliensis.

Hevea brasiliensis remains the primary crop commercially exploited to obtain latex, which is produced from the articulated secondary laticifer. Here, we described the transcriptional events related to jasmonic acid (JA)- and linolenic acid (LA)-induced secondary laticifer differentiation (SLD) in H. brasiliensis clone RRIM 600 based on RNA-seq approach. Histochemical approach proved that JA- and LA-treated samples resulted in SLD in H. brasiliensis when compared to ethephon and untreated control. RNA-seq data resulted in 86,614 unigenes, of which 2,664 genes were differentially expressed in JA and LA-induced secondary laticifer harvested from H. brasiliensis bark samples. Among these, 450 genes were unique to JA and LA as they were not differentially expressed in ethephon-treated samples compared with the untreated samples. Most transcription factors from the JA- and LA-specific dataset were classified under MYB, APETALA2/ethylene response factor (AP2/ERF), and basic-helix-loop-helix (bHLH) gene families that were involved in tissue developmental pathways, and we proposed that Bel5-GA2 oxidase 1-KNOTTED-like homeobox complex are likely involved in JA- and LA-induced SLD in H. brasiliensis. We also discovered alternative spliced transcripts, putative novel transcripts, and cis-natural antisense transcript pairs related to SLD event. This study has advanced understanding on the transcriptional regulatory network of SLD in H. brasiliensis.

Circadian rhythms in insecticide susceptibility, metabolic enzyme activity, and gene expression in Cimex lectularius (Hemiptera: Cimicidae).

Many insect species display daily variation of sensitivity to insecticides when they are exposed to the same concentration at different times during the day. To date, this has not been investigated in bed bugs. To address this, we explored circadian rhythms in insecticide susceptibility, xenobiotic metabolizing (XM) gene expressions, and metabolic detoxification in the common bed bug, Cimex lectularius. An insecticide susceptible Monheim strain of C. lectularius was most tolerant of deltamethrin during the late photophase at ZT9 (i.e. nine hours after light is present in the light-dark cycle (LD) cycle) and similarly repeated at CT9 (i.e. nine hours into the subjective day in constant darkness (DD)) suggesting endogenous circadian involvement in susceptibility to deltamethrin. No diel rhythm was observed against imidacloprid insecticide despite significant daily susceptibility in both LD and DD conditions. Rhythmic expressions of metabolic detoxification genes, GSTs1 and CYP397A1 displayed similar expression patterns with total GST and P450 enzyme activities in LD and DD conditions, respectively. The oscillation of mRNA levels of GSTs1 and CYP397A1 was found consistent with peak phases of deltamethrin susceptibility in C. lectularius. This study demonstrates that circadian patterns of metabolic detoxification gene expression occur within C. lectularius. As a consequence, insecticide efficacy can vary dramatically throughout a 24 hour period.

Phylogeography of the termite Macrotermes gilvus and insight into ancient dispersal corridors in Pleistocene Southeast Asia.

Dispersal of soil-dwelling organisms via the repeatedly exposed Sunda shelf through much of the Pleistocene in Southeast Asia has not been studied extensively, especially for invertebrates. Here we investigated the phylogeography of an endemic termite species, Macrotermes gilvus (Hagen), to elucidate the spatiotemporal dynamics of dispersal routes of terrestrial fauna in Pleistocene Southeast Asia. We sampled 213 termite colonies from 66 localities throughout the region. Independently inherited microsatellites and mtDNA markers were used to infer the phylogeographic framework of M. gilvus. Discrete phylogeographic analysis and molecular dating based on fossil calibration were used to infer the dynamics of M. gilvus dispersal in time and space across Southeast Asia. We found that the termite dispersal events were consistently dated within the Pleistocene time frame. The dispersal pattern was multidirectional, radiating eastwards and southwards out of Indochina, which was identified as the origin for dispersal events. We found no direct dispersal events between Sumatra and Borneo despite the presence of a terrestrial connection between them during the Pleistocene. Instead, central Java served as an important link allowing termite colonies to be established in Borneo and Sumatra. Our findings support the hypothesis of a north-south dispersal corridor in Southeast Asia and suggest the presence of alternative dispersal routes across Sundaland during the Pleistocene. For the first time, we also propose that a west-east dispersal through over-water rafting likely occurred across the Pleistocene South China Sea. We found at least two independent entry routes for terrestrial species to infiltrate Sumatra and Borneo at different times.

Insecticide resistance and resistance mechanisms in bed bugs, Cimex spp. (Hemiptera: Cimicidae).

The worldwide resurgence of bed bugs [both Cimex lectularius L. and Cimex hemipterus (F.)] over the past two decades is believed in large part to be due to the development of insecticide resistance. The transcriptomic and genomic studies since 2010, as well as morphological, biochemical and behavioral studies, have helped insecticide resistance research on bed bugs. Multiple resistance mechanisms, including penetration resistance through thickening or remodelling of the cuticle, metabolic resistance by increased activities of detoxification enzymes (e.g. cytochrome P450 monooxygenases and esterases), and knockdown resistance by kdr mutations, have been experimentally identified as conferring insecticide resistance in bed bugs. Other candidate resistance mechanisms, including behavioral resistance, some types of physiological resistance (e.g. increasing activities of esterases by point mutations, glutathione S-transferase, target site insensitivity including altered AChEs, GABA receptor insensitivity and altered nAChRs), symbiont-mediated resistance and other potential, yet undiscovered mechanisms may exist. This article reviews recent studies of resistance mechanisms and the genes governing insecticide resistance, potential candidate resistance mechanisms, and methods of monitoring insecticide resistance in bed bugs. This article provides an insight into the knowledge essential for the development of both insecticide resistance management (IRM) and integrated pest management (IPM) strategies for successful bed bug management.

Large-scale collection of full-length cDNA and transcriptome analysis in Hevea brasiliensis.

Natural rubber has unique physical properties that cannot be replaced by products from other latex-producing plants or petrochemically produced synthetic rubbers. Rubber from Hevea brasiliensis is the main commercial source for this natural rubber that has a cis-polyisoprene configuration. For sustainable production of enough rubber to meet demand elucidation of the molecular mechanisms involved in the production of latex is vital. To this end, we firstly constructed rubber full-length cDNA libraries of RRIM 600 cultivar and sequenced around 20,000 clones by the Sanger method and over 15,000 contigs by Illumina sequencer. With these data, we updated around 5,500 gene structures and newly annotated around 9,500 transcription start sites. Second, to elucidate the rubber biosynthetic pathways and their transcriptional regulation, we carried out tissue- and cultivar-specific RNA-Seq analysis. By using our recently published genome sequence, we confirmed the expression patterns of the rubber biosynthetic genes. Our data suggest that the cytoplasmic mevalonate (MVA) pathway is the main route for isoprenoid biosynthesis in latex production. In addition to the well-studied polymerization factors, we suggest that rubber elongation factor 8 (REF8) is a candidate factor in cis-polyisoprene biosynthesis. We have also identified 39 transcription factors that may be key regulators in latex production. Expression profile analysis using two additional cultivars, RRIM 901 and PB 350, via an RNA-Seq approach revealed possible expression differences between a high latex-yielding cultivar and a disease-resistant cultivar.