Identification of a Novel Inhibitor of Cimex lectularius Acetylcholinesterase.

Acetylcholinesterase (AChE) stands as a primary target of commercial insecticides, notably organophosphates and carbamates. Despite their widespread use in agricultural and indoor pest control, concerns over their high toxicity and the emergence of resistance have restricted their efficacy. In this study, we conducted high-throughput virtual screening against both wild-type (WT) and resistant Cimex lectularius AChE utilizing a library encompassing 1 270 000 compounds. From this screening, we identified 100 candidate compounds and subsequently assessed their inhibitory effects on purified AChE enzymes. Among these candidates, AE027 emerged as a potent inhibitor against both WT and resistant AChE, exhibiting IC50 values of 10 and 43 µM, respectively. Moreover, the binding of AE027 significantly stabilized AChE, elevating its melting temperature by approximately 7 °C. Through molecular docking and molecular dynamics simulation, we delineated the binding mode of AE027, revealing its interaction with a site adjacent to the catalytic center, which is distinct from known inhibitors, with differing poses observed between WT and resistant AChE. Notably, the resistance mutation F348Y, positioned at a site directly interfacing with AE027, impedes ligand binding through steric hindrance. Furthermore, we evaluated the toxicity and pharmacokinetic properties of AE027 utilizing bioinformatics tools. These findings lay a crucial foundation for the development of a novel generation of insecticides that can combat both WT and resistant pest populations effectively and safely.

Cimex lectularius Linnaeus, 1758 (Hemiptera: Cimicidae) in Costa Rica: First Case Report Confirmed by Molecular Methods in Central America.

Cimex lectularius and Cimex hemipterus are the most common species of bedbugs that infest homes. Although case reports decreased substantially by the end of the 20th century, bed bugs, and especially C. lectularius, are currently suffering a resurgence mostly attributed to insecticide resistance, inadequate pest control, and increased travel. Here, we report, to the best of our knowledge, the first molecular confirmation of C. lectularius in Central America. Specimens were obtained from an apartment located in Heredia, Costa Rica. These specimens were identified morphologically as C. lectularius. The species identification was confirmed by amplifying and sequencing fragments of the cytochrome oxidase subunit I (COI) and the 16S rRNA (16S) genes. The phylogenetic analysis showed that the sequences obtained were more closely related to a C. lectularius mitochondrial complete genome sequence from China, with similarities of 98.84% (686/694) for COI and 98.97% (387/391) for 16S. The finding of C. lectularius in Costa Rica will require further investigation in order to determine the extent of current infestations and the susceptibility to insecticides, especially due to the impact that this species can have in human health, as well as the tourism industry in the region.

Insecticide resistance mechanisms with novel 'kdr' type gene mutations in the tropical bed bug Cimex hemipterus.

BACKGROUND: The tropical bed bug, Cimex hemipterus, is a serious indoor public health pest in tropical regions causing intense physical discomfort and mental distress to humans. At present, the application of insecticides is the major control strategy. The present study was designed to evaluate the development of resistance and resistance mechanisms in Cimex hemipterus from Kandy district, Sri Lanka. METHODS: The resistance status of the collected bed bugs was determined against the discriminative dosages of DDT, malathion, propoxur, deltamethrin and permethrin by conducting bioassays according to World Health Organization guidelines. Activities of insecticide metabolizing enzymes, i.e. esterases, glutathione S-transferases (GST) and monooxygenases, and the insensitivity of organophosphate/carbamate target site acetylcholinesterase (AChE), were evaluated by biochemical assays. Regions of the gene of the pyrethroid/DDT target site, the voltage-gated sodium channel regulatory protein (VGSC), were sequenced for possible kdr mutations. RESULTS: Survival percentages of bed bug population were 71, 68 and 51% for DDT, malathion and propoxur respectively. KT50 and KT90 values, calculated using log-probit mortality curves for deltamethrin were 62.55 and 123.96 h, respectively. These values were much higher for permethrin where KT50 was 201.10 h and the KT90 was beyond the detectable range. Results were compared with previous values reported for the same population in 2002. Resistance to propoxur has increased significantly from 11 to 51% with about a 20-fold increase in the number of individuals with elevated esterase mechanism. No significant change has occurred in malathion and DDT resistance, in GST and monooxygenase activities, and in AChE sensitivity for the past 14 years. Six kdr associated mutations (Y/L995H, V1010L, I1011F, L1014F, V1016E, L1017F/S) and a non-kdr associated mutation (A1007S mutation) were found from the α-region of the VGSC gene. Out of the kdr type mutations, only L1014F has been reported previously form C. hemipterus while the others have been reported from other insects. CONCLUSIONS: The bed bug population has developed high resistance to propoxur with increased esterase activities. KT50 for deltamethrin and permethrin has increased 125- and 20-fold, respectively, over the period 2002 to 2016. To the authors' knowledge, this is the first time that the possible involvement of a kdr type mutation in developing pyrethroid resistance in C. hemipterus has been shown in Sri Lanka.

Molecular and biochemical characterization of the bed bug salivary gland cholinesterase as an acetylcholine-sequestering enzyme.

The common bed bug, Cimex lectularius, possesses a cholinesterase expressed exclusively in the salivary gland (ClSChE). In this study, we investigated the molecular forms, tissue distribution patterns and biochemical properties of ClSChE and showed that ClSChE exists as a soluble monomeric form or a soluble dimeric form connected by a disulfide bridge. Immunohistochemical analysis confirmed that ClSChE was expressed in the epithelial cells of both the salivary gland and the duct. In addition, the secretion of monomeric ClSChE through the proboscis during feeding was confirmed by western blotting using a ClSChE-specific antibody. To predict the role of ClSChE injected into the tissue of an animal host, we analyzed the extent of hydrolysis of acetylcholine (ACh) by ClSChE by ultra-performance liquid chromatography-tandem mass spectrometry. ClSChE binding to ACh was not clearly resolved in the binding assay format used in this study, probably due to the weak but detectable ACh-hydrolytic activity of ClSChE. Nevertheless, kinetic analysis revealed that ClSChE possesses extremely low Km (high affinity to ACh) and Vmax values. These findings suggest that ClSChE functions virtually as an ACh-sequestering protein by having a very strong affinity to ACh but an extremely long turnover time. Given that ACh regulates a wide variety of host physiologies, we discuss the tentative roles of ClSChE in blood vessel constriction and itch/pain regulation in the host.

Transcriptome profiling reveals differential gene expression of detoxification enzymes in a hemimetabolous tobacco pest after feeding on jasmonate-silenced Nicotiana attenuata plants.

BACKGROUND: The evolutionary arms race between plants and insects has driven the co-evolution of sophisticated defense mechanisms used by plants to deter herbivores and equally sophisticated strategies that enable phytophagous insects to rapidly detoxify the plant's defense metabolites. In this study, we identify the genetic determinants that enable the mirid, Tupiocoris notatus, to feed on its well-defended host plant, Nicotiana attenuata, an outstanding model for plant-insect interaction studies. RESULTS: We used an RNAseq approach to evaluate the global gene expression of T. notatus after feeding on a transgenic N. attenuata line which does not accumulate jasmonic acid (JA) after herbivory, and consequently accumulates very low levels of defense metabolites. Using Illumina sequencing, we generated a de novo assembled transcriptome which resulted in 63,062 contigs (putative transcript isoforms) contained in 42,610 isotigs (putative identified genes). Differential expression analysis based on RSEM-estimated transcript abundances identified 82 differentially expressed (DE) transcripts between T. notatus fed on wild-type and the defenseless plants. The same analysis conducted with Corset-estimated transcript abundances identified 59 DE clusters containing 85 transcripts. In both analyses, a larger number of DE transcripts were found down-regulated in mirids feeding on JA-silenced plants (around 70%). Among these down-regulated transcripts we identified seven transcripts possibly involved in the detoxification of N. attenuata defense metabolite, specifically, one glutathione-S-transferase (GST), one UDP-glucosyltransferase (UGT), five cytochrome P450 (P450s), and six serine proteases. Real-time quantitative PCR confirmed the down-regulation for six transcripts (encoding GST, UGT and four P450s) and revealed that their expression was only slightly decreased in mirids feeding on another N. attenuata transgenic line specifically silenced in the accumulation of diterpene glycosides, one of the many classes of JA-mediated defenses in N. attenuata. CONCLUSIONS: The results provide a transcriptional overview of the changes in a specialist hemimetabolous insect associated with feeding on host plants depleted in chemical defenses. Overall, the analysis reveals that T. notatus responses to host plant defenses are narrow and engages P450 detoxification pathways. It further identifies candidate genes which can be tested in future experiments to understand their role in shaping the T. notatus-N. attenuata interaction.

Biochemical and toxicological properties of two acetylcholinesterases from the common bed bug, Cimex lectularius.

We examined the molecular and enzymatic properties of two acetylcholinesterases (AChEs; ClAChE1 and ClAChE2) from the common bed bug, Cimex lectularius. Native polyacrylamide gel electrophoresis followed by activity staining and Western blotting revealed that ClAChE1 is the main catalytic enzyme and is abundantly expressed in various tissues. Both ClAChEs existed in dimeric form connected by a disulfide bridge and were attached to the membrane via a glycophosphatidylinositol anchor. To determine their kinetic and inhibitory properties, both ClAChE1 and ClAChE2 were in vitro expressed in Sf9 cells using a baculovirus expression system. ClAChE1 showed higher catalytic efficiency toward acetylcholine, supporting the hypothesis that ClAChE1 plays a major role in postsynaptic transmission. An inhibition assay revealed that ClAChE1 is generally more sensitive to organophosphates and carbamates examined although ClAChE2 was >4000-fold more sensitive to malaoxon than ClAChE1. The relatively higher correlation between the in vitro ClAChE1 inhibition and the in vivo toxicity suggested that ClAChE1 is the more relevant toxicological target for organophosphates and carbamates. Although the physiological function of ClAChE2 remains to be elucidated, ClAChE2 also appears to have neuronal functions, as judged by its tissue distribution and molecular and kinetic properties. Our findings help expand our knowledge on insect AChEs and their toxicological properties.

RNA interference of NADPH-cytochrome P450 reductase results in reduced insecticide resistance in the bed bug, Cimex lectularius.

BACKGROUND: NADPH-cytochrome P450 reductase (CPR) plays a central role in cytochrome P450 action. The genes coding for P450s are not yet fully identified in the bed bug, Cimex lectularius. Hence, we decided to clone cDNA and knockdown the expression of the gene coding for CPR which is suggested to be required for the function of all P450s to determine whether or not P450s are involved in resistance of bed bugs to insecticides. METHODOLOGY/PRINCIPAL FINDINGS: The full length Cimex lectularius CPR (ClCPR) cDNA was isolated from a deltamethrin resistant bed bug population (CIN-1) using a combined PCR strategy. Bioinformatics and in silico modeling were employed to identify three conserved binding domains (FMN, FAD, NADP), a FAD binding motif, and the catalytic residues. The critical amino acids involved in FMN, FAD, NADP binding and their putative functions were also analyzed. No signal peptide but a membrane anchor domain with 21 amino acids which facilitates the localization of ClCPR on the endoplasmic reticulum was identified in ClCPR protein. Phylogenetic analysis showed that ClCPR is closer to the CPR from the body louse, Pediculus humanus corporis than to the CPRs from the other insect species studied. The ClCPR gene was ubiquitously expressed in all tissues tested but showed an increase in expression as immature stages develop into adults. We exploited the traumatic insemination mechanism of bed bugs to inject dsRNA and successfully knockdown the expression of the gene coding for ClCPR. Suppression of the ClCPR expression increased susceptibility to deltamethrin in resistant populations but not in the susceptible population of bed bugs. CONCLUSIONS/SIGNIFICANCE: These data suggest that P450-mediated metabolic detoxification may serve as one of the resistance mechanisms in bed bugs.

Deep sequencing of pyrethroid-resistant bed bugs reveals multiple mechanisms of resistance within a single population.

A frightening resurgence of bed bug infestations has occurred over the last 10 years in the U.S. and current chemical methods have been inadequate for controlling this pest due to widespread insecticide resistance. Little is known about the mechanisms of resistance present in U.S. bed bug populations, making it extremely difficult to develop intelligent strategies for their control. We have identified bed bugs collected in Richmond, VA which exhibit both kdr-type (L925I) and metabolic resistance to pyrethroid insecticides. Using LD(50) bioassays, we determined that resistance ratios for Richmond strain bed bugs were ∼5200-fold to the insecticide deltamethrin. To identify metabolic genes potentially involved in the detoxification of pyrethroids, we performed deep-sequencing of the adult bed bug transcriptome, obtaining more than 2.5 million reads on the 454 titanium platform. Following assembly, analysis of newly identified gene transcripts in both Harlan (susceptible) and Richmond (resistant) bed bugs revealed several candidate cytochrome P450 and carboxylesterase genes which were significantly over-expressed in the resistant strain, consistent with the idea of increased metabolic resistance. These data will accelerate efforts to understand the biochemical basis for insecticide resistance in bed bugs, and provide molecular markers to assist in the surveillance of metabolic resistance.

A calcium-activated nucleotidase secreted from Ostertagia ostertagi 4th-stage larvae is a member of the novel salivary apyrases present in blood-feeding arthropods.

Apyrases (ATP-diphosphohydrolase) comprise a ubiquitous class of glycosylated nucleotidases that hydrolyse extracellular ATP and ADP to orthophosphate and AMP. One class of newly-described, Ca2+-dependent, salivary apyrases known to counteract blood-clotting, has been identified in haematophagous arthropods. Herein, we have identified a gene (Oos-apy-1) encoding a protein that structurally conforms to the Ca2+-activated apyrase from the bed bug, Cimex lectularius, by immunologically screening an Ostertagia L4 cDNA expression library. The expressed protein (rOos-APY-1) was biochemically functional in the presence of Ca2+ only, with greatest activity on ATP, ADP, UTP and UDP. Host antibodies to the fusion protein appeared as early as 14 days post-infection (p.i.) and increased through 30 days p.i. Immunohistochemical and Western blot analyses demonstrated that the native Oos-APY-1 protein is present in the glandular bulb of the oesophagus and is confined to the L4. A putative signal sequence at the N-terminus and near 100% identity with a Teladorsagia circumcincta L4 secreted protein is consistent with the native protein being secreted at the cellular level. Predicated upon substrate specificity, the native protein may be used by the parasite to control the levels of host extracellular nucleotides released by locally-damaged tissues in an effort to modulate immune intervention and inflammation.

Insect-specific irreversible inhibitors of acetylcholinesterase in pests including the bed bug, the eastern yellowjacket, German and American cockroaches, and the confused flour beetle.

Insecticides directed against acetylcholinesterase (AChE) are facing increased resistance among target species as well as increasing concerns for human toxicity. The result has been a resurgence of disease vectors, insects destructive to agriculture, and residential pests. We previously reported a free cysteine (Cys) residue at the entrance to the AChE active site in some insects but not higher vertebrates. We also reported Cys-targeting methanethiosulfonate molecules (AMTSn), which, under conditions that spared human AChE, caused total irreversible inhibition of aphid AChE, 95% inhibition of AChE from the malaria vector mosquito (Anopheles gambia), and >80% inhibition of activity from the yellow fever mosquito (Aedes aegypti) and northern house mosquito (Culex pipiens). We now find the same compounds inhibit AChE from cockroaches (Blattella germanica and Periplaneta americana), the flour beetle (Tribolium confusum), the multi-colored Asian ladybird beetle (Harmonia axyridis), the bed bug (Cimex lectularius), and a wasp (Vespula maculifrons), with IC(50) values of approximately 1-11muM. Our results support further study of Cys-targeting inhibitors as conceptually novel insecticides that may be free of resistance in a range of insect pests and disease vectors and, compared with current compounds, should demonstrate much lower toxicity to mammals, birds, and fish.

Bacteriolytic activity in the ejaculate of an insect.

The rapid evolution of ejaculate components is considered to be largely driven by sexual selection. Less attention has been paid to the fact that sperm and microorganisms frequently meet; we consequently predict selection for substances that protect a male's ejaculate. We report, for the first time, bacteriolytic activity (lysozyme-like immune activity [LLA]) in the ejaculate of an animal, the common bedbug Cimex lectularius. We also show that in almost half the males LLA in the seminal fluid exceeded LLA in the hemolymph. We detected no antimicrobial peptide activity in seminal fluid. Because lysozymes degrade only bacteria, our results suggest that sperm-microbe interactions are probably important in the evolution of ejaculate components and thereby provide a route for natural selection to account for some of the diversity of seminal components.

Evaluation of piperonyl butoxide as a deltamethrin synergist for pyrethroid-resistant bed bugs.

An understanding of the mechanisms of insecticide resistance in the bed bug, Cimex lectularius L., has the potential to lead to new approaches for the control of resistant populations. We used the cytochrome P450 monooxygenase (P450) inhibitor piperonyl butoxide (PBO) to assess the role of P450s in deltamethrin resistance in three field-collected bed bug strains, LA-1, CIN-1 and WOR-1. In addition, we exposed two highly resistant strains, CIN-1 and WOR-1 (resistance ratio [RR] >2,500-fold), to dry residues of piperonyl butoxide-synergized pyrethroid formulations to determine the utility of synergism by PBO. Piperonyl butoxide synergized deltamethrin in all three strains, but its impact was variable. The synergistic ratio varied from 40 in CIN-1 to 176 in WOR-1. Because the resistance ratio for each strain after piperonyl butoxide treatment was 174 and 39, respectively, our results suggest that P450s have some involvement in deltamethrin resistance, but other resistance mechanisms must be involved as well. No significant synergistic effect of formulated deltamethrin was observed with the addition of synergized pyrethrins or formulated piperonyl butoxide in the CIN-1 strain, but synergism occurred in the WOR-1 strain. Addition of PBO to pyrethroids is not a comprehensive solution to pyrethroid resistance because strains vary in both overall resistance level and the proportion of that resistance attributable to P450s.

Biochemical and molecular analysis of deltamethrin resistance in the common bed bug (Hemiptera: Cimicidae).

This study establishes deltamethrin resistance in a common bed bug, Cimex lectularius L., population collected from New York City (NY-BB). The NY-BB population was 264-fold more resistant to 1% deltamethrin in contact bioassay compared with an insecticide-susceptible population collected in Florida (FL-BB). General esterase, glutathione S-transferase, and 7-ethoxycoumarin O-deethylase activities of NY-BB were not statistically different from those of FL-BB. cDNA fragments that encoded the open reading frame of voltage-sensitive sodium channel alpha-subunit genes from the FL-BB and NY-BB populations, respectively, were obtained by homology probing polymerase chain reaction (PCR) and sequenced. Sequence alignment of the internal and 5' and 3' rapid amplification of cDNA ends (RACE) fragments generated a 6500-bp cDNA sequence contig, which was composed of a 6084-bp open reading frame (ORF) encoding 2027 amino acid residues and 186-bp 5' and 230-bp 3' untranslated regions (5' and 3' UTRs, respectively). Sequence comparisons of the open reading frames of the alpha-subunit genes identified two point mutations (V419L and L925I) that were presented only in the NY-BB population. L925I, located the intracellular loop between IIS4 and IIS5, has been previously found in a highly pyrethroid-resistant populations of whitefly (Bemisia tabaci). V419L, located in the IS6 transmembrane segment, is a novel mutation. A Val to Met mutation at the corresponding position of the bed bug V419, however, has been identified in the tobacco budworm as a kdr-type mutation. This evidence suggests that the two mutations are likely the major resistance-causing mutations in the deltamethrin-resistant NY-BB through a knockdown-type nerve insensitivity mechanism.

Xenopus apyrase (xapy), a secreted nucleotidase that is expressed during early development.

We have characterized a cDNA encoding a Xenopus laevis apyrase (XAPY) that is expressed during embryogenesis. XAPY is highly homologous to two recently described mammalian apyrases, human SCAN-1 and rat Ca2+-NDPase, and to a lesser extent the salivary apyrase of the blood-feeding arthropod Cimex lectularis. RT-PCR analysis shows that Xapy is expressed at all the developmental stages tested, from oocytes through to tadpoles. Xapy transcripts are widely distributed in the embryo, but from late neurulae through to late tailbud stages they are highly enriched in the cement gland, an adhesive organ in the epidermis of the head. When expressed in HEK 293 cells, XAPY is largely retained in the endoplasmic reticulum, although some is also secreted. XAPY conditioned media hydrolyses UDP and UTP, confirming that it is a functional apyrase.

The salivary apyrase of the blood-sucking sand fly Phlebotomus papatasi belongs to the novel Cimex family of apyrases.

Apyrases are enzymes that hydrolyze nucleotide di- and triphosphates to orthophosphate and mononucleotides. At least two families of enzymes, belonging to the 5'-nucleotidase and to the actin/heat shock 70/sugar kinase superfamily, have evolved independently to serve the apyrase reaction. Both families require either Ca(2+) or Mg(2+) for their action. A novel apyrase enzyme sequence, with no homology to any other known protein sequence, was found recently in the salivary glands of the hematophagous bed bug Cimex lectularius. This enzyme functions exclusively with Ca(2+). Here, we report the finding of a cDNA similar to that of the C. lectularius salivary apyrase isolated from a salivary gland cDNA library of Phlebotomus papatasi. Transfection of insect cells with the P. papatasi salivary gland apyrase cDNA resulted in the secretion of a Ca(2+)-dependent apyrase whose activity was indistinguishable from that in salivary homogenates of P. papatasi. Homologous sequences were found in humans, in another sand fly (Lutzomyia longipalpis), in the fruit fly Drosophila melanogaster, in the nematode Caenorhabditis elegans and in the protozoan Cryptosporidium parvum, indicating that this family of enzymes is widespread among animal species.

Purification, cloning, and expression of an apyrase from the bed bug Cimex lectularius. A new type of nucleotide-binding enzyme.

An enzyme that hydrolyzes the phosphodiester bonds of nucleoside tri- and diphosphates, but not monophosphates, thus displaying apyrase (EC 3.6.1.5) activity, was purified from salivary glands of the bed bug, Cimex lectularius. The purified C. lectularius apyrase was an acidic protein with a pI of 5.1 and molecular mass of approximately 40 kDa that inhibited ADP-induced platelet aggregation and hydrolyzed platelet agonist ADP with specific activity of 379 units/mg protein. Amplification of C. lectularius cDNA corresponding to the N-terminal sequence of purified apyrase produced a probe that allowed identification of a 1.3 kilobase pair cDNA clone coding for a protein of 364 amino acid residues, the first 35 of which constituted the signal peptide. The processed form of the protein was predicted to have a molecular mass of 37.5 kDa and pI of 4.95. The identity of the product of the cDNA clone with native C. lectularius apyrase was proved by immunological testing and by expressing the gene in a heterologous host. Immune serum made against a synthetic peptide with sequence corresponding to the C-terminal region of the predicted cDNA clone recognized both C. lectularius apyrase fractions eluted from a molecular sieving high pressure liquid chromatography and the apyrase active band from chromatofocusing gels. Furthermore, transfected COS-7 cells secreted a Ca2+-dependent apyrase with a pI of 5.1 and immunoreactive material detected by the anti-apyrase serum. C. lectularius apyrase has no significant sequence similarity to any other known apyrases, but homologous sequences have been found in the genome of the nematode C. elegans and in mouse and human expressed sequence tags from fetal and tumor EST libraries.

Free amino acids in seminal fluid & haemolymph of Cimex lectularious L. & their possible role in sperm metabolism.

PIP: Previously it was shown that glucose and trehalose are present in the hemolymph of Cimex lectularious L. and that the active bed bug sperm can utilize these sugars oxidatively. A study was conducted on the occurrence of amino acids in the seminal fluid and the hemolymph of the bed bug and the enzymes of Cimex sperm concerned with amino acid metabolism. It was determined that the hemolymph contains 12 free amino acids and in the seminal fluid, there are 8. In the Cimex sperm, transaminase activity is limited, with L-alanine the only amino acid involved in transamination. Although it is understood that amino acids constitute a very limited source of energy to the bed bug sperm! an oxidative deamination of L-alanine may occur, since oxygen is available to the sperm and alanine and glutamic acid are present in the seminal fluid and hemolymph.^ieng