Resilience of transfluthrin to oxidative attack by duplicated CYP6P9 variants known to confer pyrethroid resistance in the major malaria mosquito Anopheles funestus.

Resistance to common pyrethroids, such as deltamethrin and permethrin is widespread in the malaria mosquito Anopheles funestus and mainly conferred by upregulated cytochrome P450 monooxygenases (P450s). In the pyrethroid resistant laboratory strain An. funestus FUMOZ-R the duplicated genes CYP6P9a and CYP6P9b are highly upregulated and have been shown to metabolize various pyrethroids, including deltamethrin and permethrin. Here, we recombinantly expressed CYP6P9a and CYP6P9b from An. funestus using a baculovirus expression system and evaluated the interaction of the multifluorinated benzyl pyrethroid transfluthrin with these enzymes by different approaches. First, by Michaelis-Menten kinetics in a fluorescent probe assay with the model substrate 7-benzyloxymethoxy-4-trifluoromethylcoumarin (BOMFC), we showed the inhibition of BOMFC metabolism by increasing concentrations of transfluthrin. Second, we tested the metabolic capacity of recombinantly expressed CYP6P9 variants to degrade transfluthrin utilizing UPLC-MS/MS analysis and detected low depletion rates, explaining the virtual lack of resistance of strain FUMOZ-R to transfluthrin observed in previous studies. However, as both approaches suggested an interaction of CYP6P9 variants with transfluthrin, we analyzed the oxidative metabolic fate and failed to detect hydroxylated transfluthrin, but low amounts of an M-2 transfluthrin metabolite. Based on the detected metabolite we hypothesize oxidative attack of the gem-dimethyl substituted cyclopropyl moiety, resulting in the formation of an allyl cation upon ring opening. In conclusion, these findings support the resilience of transfluthrin to P450-mediated pyrethroid resistance, and thus, reinforces its employment as an important resistance-breaking pyrethroid in resistance management strategies to control the major malaria vector An. funestus.

Discovery of Rigidified α,ß-Unsaturated Imines as New Resistance-breaking Insecticides for Malaria Vector Control.

During our continuous search for new resistance-breaking insecticides applicable to malaria vector control, a new class of α,ß-unsaturated imines was identified by applying the principle of conformational rigidification as a powerful tool for compound optimisation. Herein we describe the successful synthesis of these compounds and their biological test results. Our lead compound 16 from this insecticidal class outperforms market standards, notably for the control of mosquito strains that exhibit either metabolic or target-site resistance to these established insecticides. In our model system for insecticide-treated mosquito nets the compound reveals long-lasting efficacy for up to several months.

Flupyradifurone (Sivanto™) and its novel butenolide pharmacophore: Structural considerations.

Flupyradifurone (4-[(2,2-difluoroethyl)amino]-2(5H)-furanone), a member of the new class of butenolide insecticides, contains a novel bioactive scaffold as pharmacophore. It is very versatile in terms of application methods to a variety of crops, exhibits excellent and fast action against a broad spectrum of sucking pest insects including selected neonicotinoid resistant pest populations such as whiteflies and aphids expressing metabolic resistance mechanisms. As a partial agonist flupyradifurone reversibly binds to insect nicotinic acetylcholine receptors (nAChRs) and lacks metabolization by CYP6CM1, a cytochrome P450 over-expressed in cotton whiteflies resistant to imidacloprid and pymetrozine. The butenolide insecticides exhibit structure-activity relationships (SAR) that are different from other nAChR agonists such as the classes of neonicotinoids and sulfoximines. The paper briefly reviews the discovery of the butenolide insecticide flupyradifurone, its SAR differentiating it from established nAChR agonists and a molecular docking approach using the binding site model of CYP6CM1vQ of Bemisia tabaci known to confer metabolic resistance to neonicotinoid insecticides.

Flupyradifurone: a brief profile of a new butenolide insecticide.

BACKGROUND: The development and commercialisation of new chemical classes of insecticides for efficient crop protection measures against destructive invertebrate pests is of utmost importance to overcome resistance issues and to secure sustainable crop yields. Flupyradifurone introduced here is the first representative of the novel butenolide class of insecticides active against various sucking pests and showing an excellent safety profile. RESULTS: The discovery of flupyradifurone was inspired by the butenolide scaffold in naturally occurring stemofoline. Flupyradifurone acts reversibly as an agonist on insect nicotinic acetylcholine receptors but is structurally different from known agonists, as shown by chemical similarity analysis. It shows a fast action on a broad range of sucking pests, as demonstrated in laboratory bioassays, and exhibits excellent field efficacy on a number of crops with different application methods, including foliar, soil, seed treatment and drip irrigation. It is readily taken up by plants and translocated in the xylem, as demonstrated by phosphor imaging analysis. Flupyradifurone is active on resistant pests, including cotton whiteflies, and is not metabolised by recombinantly expressed CYP6CM1, a cytochrome P450 conferring metabolic resistance to neonicotinoids and pymetrozine. CONCLUSION: The novel butenolide insecticide flupyradifurone shows unique properties and will become a new tool for integrated pest management around the globe, as demonstrated by its insecticidal, ecotoxicological and safety profile.

Two pathogens and one disease: detection and identification of flea-borne Rickettsiae in areas endemic for murine typhus in California.

Results of an environmental assessment conducted in a newly emergent focus of murine typhus in southern California are described. Opossums, Didelphis virginiana Kerr, infested with cat fleas, Ctenocephalides felis Buché, in the suburban area were abundant. Animal and flea specimens were tested for the DNA of two flea-borne rickettsiae, Rickettsia typhi and Rickettsia felis. R. felis was commonly detected in fleas collected throughout this area while R. typhi was found at a much lower prevalence in the vicinity of just 7 of 14 case-patient homes identified. DNA of R. felis, but not R. typhi, was detected in renal, hepatic, and pulmonary tissues of opossums. In contrast, there were no hematologic polymerase chain reaction findings of R. felis or R. typhi in opossums, rats, and cats within the endemic area studied. Our data suggest a significant probability of human exposure to R. felis in the area studied; however, disease caused by this agent is not recognized by the medical community and may be misdiagnosed as murine typhus using nondiscriminatory serologic methods.

Host (avian) biting preference of southern California Culex mosquitoes (Diptera: Culicidae).

The host preference of a vector mosquito species plays a significant role in determining human and animal risk of infection with mosquito-transmitted pathogens. Host preferences of common southern California Culex species for four bird species, American crow (Corvus brachyrhynchos), house sparrow (Passer domesticus), house finch (Carpodacus mexicanus), and mourning dove (Zenaida macroura), were examined by determining the proportion of each mosquito species that successfully engorged on each of the four bird species presented equally within a net trap to wild host-seeking mosquitoes. Bloodmeals in engorged mosquitoes captured within the net trap were identified to avian species by using a multiplex polymerase chain reaction assay targeting the cytochrome b gene sequence. There were significant differences in host selection by all three Culex species captured in numbers sufficient for analysis, with Culex erythrothorax Dyar preferentially biting American crows, Culex tarsalis Coquillett preferentially biting house sparrows, and Culex quinquefasciatus Say preferentially biting house finches. All three Culex species demonstrated more frequent engorgement on passerine birds (sparrows, finches, and crows) than the nonpasserine mourning dove. A greater preference for passerine birds might be expected to increase the transmission of pathogens, such as West Nile virus, to which passerine birds are particularly competent hosts.

Insecticidal heterolignans--tubuline polymerization inhibitors with activity against chewing pests.

Starting from natural product podophyllotoxin 1 substituted heterolignans were identified with promising insecticidal in vivo activity. The impact of substitution in each segment of the core structure was investigated in a detailed SAR study, and variation of substituents in both aromatic moieties afforded derivatives 5 and 43 with broad insecticidal activity against lepidopteran and coleopteran species. In vitro measurements supported by modeling studies indicate that heterolignans 3-134 act as tubuline polymerization inhibitors interacting with the colchicine-binding site. Insect specific structure-activity effects were observed showing that the insecticidal SAR described herein differs from reported cytotoxicity studies.

Alkaloids from stems and leaves of Stemona japonica and their insecticidal activities.

Five new alkaloids, 6beta-hydroxystemofoline (1), 16-hydroxystemofoline (2), neostemofoline (3), protostemodiol (4), and 13-demethoxy-11(S*),12(R*)-dihydroprotostemonine (5), along with 10 known alkaloids, were isolated from stems and leaves of Stemona japonica. Their structures were elucidated by 1D and 2D NMR and other spectroscopic studies. The insecticidal activity of the agonist 16-hydroxystemofoline (2) and antagonist 13-demethoxy-11(S*),12(R*)-dihydroprotostemonine (5) was demonstrated by electrophysiological in vitro tests on the insect nicotinic acetylcholine receptor and by in vivo screenings against relevant agricultural insect pests.

Incidence of a new sex-ratio-distorting endosymbiotic bacterium among arthropods.

Many intracellular micro-organisms are now known to cause reproductive abnormalities and other phenomena in their hosts. The endosymbiont Wolbachia is the best known of these reproductive manipulators owing to its extremely high incidence among arthropods and the diverse host effects it has been implicated as causing. However, recent evidence suggests that another intracellular bacterium, a Cytophaga-like organism (CLO), may also induce several reproductive effects in its hosts. Here, we present the first survey of arthropod hosts for infection by the CLO. We use a sensitive hemi-nested polymerase chain reaction method to screen 223 species from 20 arthropod orders for infection by the CLO and Wolbachia. The results indicate that, although not as prevalent as Wolbachia, the CLO infects a significant number of arthropod hosts (ca. 7.2%). In addition, double infections of the CLO and Wolbachia were found in individuals of seven arthropod species. Sequencing analysis of the 16S rDNA region of the CLO indicates evidence for horizontal transmission of the CLO strains. We discuss these results with reference to future studies on host effects induced by intracellular micro-organisms.

Variation in the NS3/NS3A gene of bluetongue viruses contained in Culicoides sonorensis collected from a single site in southern California.

To determine the variability of the NS3/NS3A gene of field strains of BTV contained in Culicoides sonorensis collected from a single site in California (CA), the NS3/NS3A gene was directly amplified and sequenced from 22 pools of C. sonorensis and compared with those of previously characterized field isolates from CA, as well as to viruses that caused recent outbreaks of bluetongue disease in ruminants in CA. Phylogenetic analysis established that the NS3/NS3A gene of strains of BTV contained in C. sonorensis collected from the site exists as a heterogeneous population. The two most divergent nucleotide sequences of the NS3/NS3A genes of these viruses differed by 2.5% (18 nucleotides). Comparison with the NS3/NS3A gene sequences from viruses that caused recent instances of bluetongue disease in ruminants in CA indicated that BTV strains from different geographic regions can exhibit a higher degree of genetic heterogeneity (up to 6.6%; 0-48 nucleotide differences) than those contained in C. sonorensis collected from a single site.