Tenebenal: a meta-diamide with potential for use as a novel mode of action insecticide for public health.

BACKGROUND: There is an urgent need for insecticides with novel modes of action against mosquito vectors. Broflanilide is a meta-diamide, discovered and named Tenebenal™ by Mitsui Chemicals Agro, Inc., which has been identified as a candidate insecticide for use in public health products. METHODS: To evaluate its potential for use in public health, Tenebenal™ was screened using an array of methodologies against Anopheles and Aedes strains. Initially it was assessed for intrinsic efficacy by topical application. Tarsal contact bioassays were then conducted to further investigate its efficacy, as well as its potency and speed of action. The potential of the compound for use in indoor residual spray (IRS) applications was investigated by testing the residual efficacy of a prototype IRS formulation on a range of typical house building substrates, and its potential for use in long-lasting insecticidal nets (LLIN) was tested using dipped net samples. Finally, bioassays using well-characterized insecticide-resistant mosquito strains and an in silico screen for mutations in the insecticide's target site were performed to assess the risk of cross-resistance to Tenebenal™. RESULTS: Tenebenal™ was effective as a tarsal contact insecticide against both Aedes and Anopheles mosquitoes, with no apparent cross-resistance caused by mechanisms that have evolved to insecticides currently used in vector control. Topical application showed potent intrinsic activity against a Kisumu reference strain and an insecticide-resistant strain of Anopheles gambiae. Applied to filter paper in a WHO tube bioassay, Tenebenal™ was effective in killing 100% of susceptible and resistant strains of An. gambiae and Aedes aegypti at a concentration of 0.01%. The discriminating concentration of 11.91 µg/bottle shows it to be very potent relative to chemistries previously identified as having potential for vector control. Mortality occurs within 24 h of exposure, 80% of this mortality occurring within the first 10 h, a speed of kill somewhat slower than seen with pyrethroids due to the mode of action. The potential of Tenebenal™ for development in LLIN and IRS products was demonstrated. At least 12 months residual efficacy of a prototype IRS formulation applied at concentrations up to 200 mg of AI/sq m was demonstrated on a range of representative wall substrates, and up to 18 months on more inert substrates. A dipped net with an application rate of around 2 g/sq m Tenebenal™ killed 100% of exposed mosquitoes within a 3-min exposure in a WHO cone test. CONCLUSIONS: Tenebenal™ is a potent insecticide against adult Aedes and Anopheles mosquitoes, including strains resistant to classes of insecticide currently used in vector control. The compound has shown great potential in laboratory assessment and warrants further investigation into development for the control of pyrethroid-resistant mosquitoes.

Characterization of a novel glycosylated glutathione transferase of Onchocerca ochengi, closest relative of the human river blindness parasite.

Filarial nematodes possess glutathione transferases (GSTs), ubiquitous enzymes with the potential to detoxify xenobiotic and endogenous substrates, and modulate the host immune system, which may aid worm infection establishment, maintenance and survival in the host. Here we have identified and characterized a σ class glycosylated GST (OoGST1), from the cattle-infective filarial nematode Onchocerca ochengi, which is homologous (99% amino acid identity) with an immunodominant GST and potential vaccine candidate from the human parasite, O. volvulus, (OvGST1b). Onchocerca ochengi native GSTs were purified using a two-step affinity chromatography approach, resolved by 2D and 1D SDS-PAGE and subjected to enzymic deglycosylation revealing the existence of at least four glycoforms. A combination of lectin-blotting and mass spectrometry (MS) analyses of the released N-glycans indicated that OoGST1 contained mainly oligomannose Man5GlcNAc2 structure, but also hybrid- and larger oligommanose-type glycans in a lower proportion. Furthermore, purified OoGST1 showed prostaglandin synthase activity as confirmed by Liquid Chromatography (LC)/MS following a coupled-enzyme assay. This is only the second reported and characterized glycosylated GST and our study highlights its potential role in host-parasite interactions and use in the study of human onchocerciasis.

Unpacking WHO and CDC Bottle Bioassay Methods: A Comprehensive Literature Review and Protocol Analysis Revealing Key Outcome Predictors.

Background: Resistance monitoring is a key element in controlling vector-borne diseases. The World Health Organization (WHO) and Centres for Disease Control and Prevention (CDC) have each developed bottle bioassay methods for determining insecticide susceptibility in mosquito vectors which are used globally. Methods: This study aimed to identify variations in bottle bioassay methodologies and assess the potential impact on the data that is generated. Our approach involved a systematic examination of existing literature and protocols from WHO and CDC, with a focus on the specifics of reported methodologies, variation between versions, and reported outcomes. Building on this, we experimentally evaluated the impact of several variables on bioassay results. Results: Our literature review exposed a significant inconsistency in the how bioassay methods are reported, hindering reliable interpretation of data and the ability to compare results between studies. The experimental research provided further insight by specifically identifying two key factors that influence the outcomes of bioassays: mosquito dry weight and relative humidity (RH). This finding not only advances our comprehension of these assays but also underscores the importance of establishing precisely defined methodologies for resistance monitoring. The study also demonstrates the importance of controlling bioassay variables, noting the significant influence of wing length, as an indicator of mosquito size, on mortality rates in standardized bioassays. Conclusions: Generating data with improved protocol consistency and precision will not only deepen our understanding of resistance patterns but also better inform vector control measures. We call for continued research and collaboration to refine and build consensus on bioassay techniques, to help bolster the global effort against vector-borne diseases like malaria.

A new WHO bottle bioassay method to assess the susceptibility of mosquito vectors to public health insecticides: results from a WHO-coordinated multi-centre study.

BACKGROUND: The continued spread of insecticide resistance in mosquito vectors of malaria and arboviral diseases may lead to operational failure of insecticide-based interventions if resistance is not monitored and managed efficiently. This study aimed to develop and validate a new WHO glass bottle bioassay method as an alternative to the WHO standard insecticide tube test to monitor mosquito susceptibility to new public health insecticides with particular modes of action, physical properties or both. METHODS: A multi-centre study involving 21 laboratories worldwide generated data on the susceptibility of seven mosquito species (Aedes aegypti, Aedes albopictus, Anopheles gambiae sensu stricto [An. gambiae s.s.], Anopheles funestus, Anopheles stephensi, Anopheles minimus and Anopheles albimanus) to seven public health insecticides in five classes, including pyrethroids (metofluthrin, prallethrin and transfluthrin), neonicotinoids (clothianidin), pyrroles (chlorfenapyr), juvenile hormone mimics (pyriproxyfen) and butenolides (flupyradifurone), in glass bottle assays. The data were analysed using a Bayesian binomial model to determine the concentration-response curves for each insecticide-species combination and to assess the within-bioassay variability in the susceptibility endpoints, namely the concentration that kills 50% and 99% of the test population (LC50 and LC99, respectively) and the concentration that inhibits oviposition of the test population by 50% and 99% (OI50 and OI99), to measure mortality and the sterilizing effect, respectively. RESULTS: Overall, about 200,000 mosquitoes were tested with the new bottle bioassay, and LC50/LC99 or OI50/OI99 values were determined for all insecticides. Variation was seen between laboratories in estimates for some mosquito species-insecticide combinations, while other test results were consistent. The variation was generally greater with transfluthrin and flupyradifurone than with the other compounds tested, especially against Anopheles species. Overall, the mean within-bioassay variability in mortality and oviposition inhibition were < 10% for most mosquito species-insecticide combinations. CONCLUSION: Our findings, based on the largest susceptibility dataset ever produced on mosquitoes, showed that the new WHO bottle bioassay is adequate for evaluating mosquito susceptibility to new and promising public health insecticides currently deployed for vector control. The datasets presented in this study have been used recently by the WHO to establish 17 new insecticide discriminating concentrations (DCs) for either Aedes spp. or Anopheles spp. The bottle bioassay and DCs can now be widely used to monitor baseline insecticide susceptibility of wild populations of vectors of malaria and Aedes-borne diseases worldwide.

Reviewing the WHO Tube Bioassay Methodology: Accurate Method Reporting and Numbers of Mosquitoes Are Key to Producing Robust Results.

Accurately monitoring insecticide resistance in target mosquito populations is important for combating malaria and other vector-borne diseases, and robust methods are key. The "WHO susceptibility bioassay" has been available from the World Health Organization for 60+ years: mosquitoes of known physiological status are exposed to a discriminating concentration of insecticide. Several changes to the test procedures have been made historically, which may seem minor but could impact bioassay results. The published test procedures and literature for this method were reviewed for methodological details. Areas where there was room for interpretation in the test procedures or where the test procedures were not being followed were assessed experimentally for their impact on bioassay results: covering or uncovering of the tube end during exposure; the number of mosquitoes per test unit; and mosquito age. Many publications do not cite the most recent test procedures; methodological details are reported which contradict the test procedures referenced, or methodological details are not fully reported. As a result, the precise methodology is unclear. Experimental testing showed that using fewer than the recommended 15-30 mosquitoes per test unit significantly reduced mortality, covering the exposure tube had no significant effect, and using mosquitoes older than 2-5 days old increased mortality, particularly in the resistant strain. Recommendations are made for improved reporting of experimental parameters.

The Residual Efficacy of SumiShield™ 50WG and K-Othrine® WG250 IRS Formulations Applied to Different Building Materials against Anopheles and Aedes Mosquitoes.

Insecticides with novel modes of action are required to complement the pyrethroids currently relied upon for controlling malaria vectors. One example of this is the neonicotinoid clothianidin, the active ingredient in the indoor residual spray (IRS) SumiShield™ 50WG. In a preliminary experiment, the mortality of insecticide-susceptible and resistant An. gambiae adults exposed to filter papers treated with this IRS product reached 80% by 3 days post-exposure and 100% by 6 days post-exposure. Next, cement, wood, and mud tiles were treated with the clothianidin or a deltamethrin-based IRS formulation (K-Othrine WG250). Insecticide resistant and susceptible Anopheles and Aedes were exposed to these surfaces periodically for up to 18 months. Pyrethroid resistant Cx. quinquefasciatus was also exposed at 9 months. Between exposures, tiles were stored in heat and relative humidity conditions reflecting those found in the field. On these surfaces, the clothianidin IRS was effective at killing both susceptible and resistant An. gambiae for 18 months post-treatment, while mortality amongst the resistant strains when exposed to the deltamethrin IRS was not above that of the negative control. Greater efficacy of clothianidin was also demonstrated against insecticide resistant strains of An. funestus compared to deltamethrin, though the potency was lower when compared with An. gambiae. In general, higher efficacy of the clothianidin IRS was observed on cement and mud compared to wood, though it demonstrated poorer residual activity against Ae.aegypti and Cx. quinquefasciatus.

Review and Meta-Analysis of the Evidence for Choosing between Specific Pyrethroids for Programmatic Purposes.

Pyrethroid resistance is widespread in malaria vectors. However, differential mortality in discriminating dose assays to different pyrethroids is often observed in wild populations. When this occurs, it is unclear if this differential mortality should be interpreted as an indication of differential levels of susceptibility within the pyrethroid class, and if so, if countries should consider selecting one specific pyrethroid for programmatic use over another. A review of evidence from molecular studies, resistance testing with laboratory colonies and wild populations, and mosquito behavioural assays were conducted to answer these questions. Evidence suggested that in areas where pyrethroid resistance exists, different results in insecticide susceptibility assays with specific pyrethroids currently in common use (deltamethrin, permethrin, α-cypermethrin, and λ-cyhalothrin) are not necessarily indicative of an operationally relevant difference in potential performance. Consequently, it is not advisable to use rotation between these pyrethroids as an insecticide-resistance management strategy. Less commonly used pyrethroids (bifenthrin and etofenprox) may have sufficiently different modes of action, though further work is needed to examine how this may apply to insecticide resistance management.

A testing cascade to identify repurposed insecticides for next-generation vector control tools: screening a panel of chemistries with novel modes of action against a malaria vector.

Background: With insecticide resistance in malaria vectors spreading in geographical range and intensity, there is a need for compounds with novel modes of action to maintain the successes achieved to date by long-lasting insecticidal nets and indoor residual sprays, used as part of an insecticide resistance management strategy. Screening existing registered pesticides, predominantly those developed for use in agriculture, may provide a more rapid and less logistically challenging route to identifying active ingredients of value to public health than screening and chemical synthesis programmes for novel compounds. Methods: Insecticides and acaricides from all IRAC classes, including those with unclassified modes of action, were assessed for inclusion in a laboratory bioassay testing cascade against adult female Anopheles gambiae mosquitoes. A longlist of representative candidate compounds was selected, excluding those with safety concerns, unsuitable physiochemical properties, and likely hurdles to registration for public health use.  An initial screen using topical application eliminated compounds with insufficient intrinsic activity, and a tarsal contact assay identified those with activity at an appropriate concentration. Compounds of interest were ranked by relative potency using dose response assays and discriminating dose calculations. Results: Inclusion of an adjuvant enhanced the tarsal efficacy of several compounds, facilitating the promotion of chemistries with great potential, given suitable formulation, which would not progress based on activity of compound alone. Comparison of data between stages in the testing cascade suggest that a more streamlined approach, topical application to test for intrinsic activity and determining the discriminating dose to compare relative potency of compounds, may be sufficient to identify compounds with potential value for use in long lasting insecticidal nets and indoor residual spray products. Conclusions: Identified were 11 compounds of interest as vector control agents (in descending order of potency): clothianidin, spinetoram, metaflumizone, dinotefuran, indoxacarb, abamectin, sulfoxaflor, oxazosulfyl, triflumezopyrim, fenpyroximate, and tolfenpyrad.

Characterisation of Anopheles strains used for laboratory screening of new vector control products.

BACKGROUND: Insecticides formulated into products that target Anopheles mosquitos have had an immense impact on reducing malaria cases in Africa. However, resistance to currently used insecticides is spreading rapidly and there is an urgent need for alternative public health insecticides. Potential new insecticides must be screened against a range of characterized mosquito strains to identify potential resistance liabilities. The Liverpool School of Tropical Medicine maintains three susceptible and four resistant Anopheles strains that are widely used for screening for new insecticides. The properties of these strains are described in this paper. METHODS: WHO tube susceptibility bioassays were used for colony selection and to screen for resistance to the major classes of public health insecticides. Topical and tarsal contact bioassays were used to produce dose response curves to assess resistance intensity. Bioassays with the synergist piperonyl butoxide were also performed. Taqman™ assays were used to screen for known target site resistance alleles (kdr and ace-1). RT-qPCR was used to quantify expression of genes associated with pyrethroid resistance. RESULTS: Pyrethroid selection pressure has maintained resistance to this class in all four resistant strains. Some carbamate and organophosphate resistance has been lost through lack of exposure to these insecticide classes. The Anopheles gambiae (sensu lato) strains, VK7 2014, Banfora M and Tiassalé 13 have higher levels of pyrethroid resistance than the An. funestus FUMOZ-R strain. Elevated expression of P450s is found in all four strains and the 1014F kdr mutation is present in all three An. gambiae strains at varying frequencies. Tarsal contact data and overexpression of CYP4G16 and SAP2 suggest penetration barriers and/or sequestration also confer resistance in Banfora M. CONCLUSIONS: Continual selection with deltamethrin has maintained a stable pyrethroid-resistant phenotype over many generations. In conjunction with a standardized rearing regime, this ensures quality control of strains over time allowing for robust product comparison and selection of optimal products for further development. The identification of multiple mechanisms underpinning insecticide resistance highlights the importance of screening new compounds against a range of mosquito strains.