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.
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.
SUMMARY Concentrations of testosterone in the peripheral plasma of normal, cryptorchid and castrated male horses have been measured by a rapid radioimmuno‐assay. Basal concentrations in horses with one or two testicles range from 65 pg to 1600 pg/ml. Intravenous injection of 12000 i.u. of Human Chorionic Gonadotrophin (HCG) into horses possessing testicular tissue stimulated a rise in testosterone concentration which could be detected within 25–35 minutes of the injection. Geldings consistently showed low testosterone concentrations in the plasma (15.3±4.9 pg/ml; 15 animals), and injection of HCG did not stimulate a significant rise in testosterone concentration. Of 29 horses presented as rigs all those which were later found to have been already castrated had plasma testosterone concentrations indistinguishable from those found in geldings (nine animals) and failed to respond to HCG injection (five animals). Evidence is presented that the epididymis and spermatic cord of the horse are not capable of producing significant quantities of testosterone. The possibility is discussed of using paired blood samples, one taken prior to and the other taken after HCG injection, as a test of whether or not a horse has been castrated. RÉSUMÉ Les concentrations de testosterone dans le plasma périphérique des chevaux males normaux, cryptorchides et castrés ont été mesurés par des tests radio actifs. La concentration de base chez les chevaux avec un ou deux testicules varie de 65 pg à 1600 pg/ml. L'injection intraveineuse de 12000 U.I. de gonadotrophine chorionique humaine chez des chevaux possédant un tissu testiculaire entraina. L'augmentation du taux de testostérone détectable entre 25 et 35 minutes après l'injection. Les chevaux hongres montrèrent de basses concentrations plasmatiques de testostérone (15.3±4.9 pg—15 sujets) et l'administration de gonadotrophine ne provoqua aucune élévation significative du taux de testostérone chez eux. Des 29 chevaux réputés cryptorchides tous ceux qui furent identifiés plus tard comme ayant été castrés montrèrent des concentrations plasmatiques de testostérone de même ordre que les hongres, et ne présentérent aucune réponse à l'injection de gonadotrophine. Il semble que ni le lépididyme ni le cordon spermatique du cheval ne soient capables de secrèter des quantités significatives de testostérone. On envisage la possibilité, en comparant deux échantillons de sang, l'un prélevé avant, l'autre prélevé aprés l'injection de gonadotrophine chorionique, d'établir si un cheval a été ou n'a pas été castré. ZUSAMMENFASSUNG Die Testosteronkonzentrationen im peripheren Plasma normaler, kryptorchider und kastrierter männlicher Pferde wurden gemessen mit Hilfe eines schnellen radio‐immuno‐assay. Die Konzentrationen schwanken zwischen 65 bis 1600 pg/ml bei Pferden mit einem oder zwei Hoden. Die intravenöse Injektion von 12'000 IU menschlichen Choriongonadotropins stimulierte einen Anstieg der Testosteronkonzentration bei Pferden mit Hodengewebe nach 25–35 Minuten. Wallachen zeigten immer tiefe Testostero...
BACKGROUND: Pyrethroids are the most widely used insecticides for the control of malaria transmitting Anopheles gambiae mosquitoes and rapid increase in resistance to this insecticide class is of major concern. Pyrethroids target the Voltage Gated Sodium Channels (VGSCs), that have a key role in the normal function of the mosquitoes' nervous system. VGSC mutations L995F and L995S have long been associated with pyrethroid resistance and screening for their presence is routine in insecticide resistance management programs. Recently, a VGSC haplotype containing two amino acid substitutions associated with resistance in other species, V402L and I1527T, was identified. These two VGSC mutations are found in tight linkage and are mutually exclusive to the classical L995F/S mutations.RESULTS: We identify the presence of the V402L-I1527T haplotype in resistant An. coluzzii colonized strains and in field populations from Burkina Faso, at frequencies higher than previously reported; in some cases almost reaching fixation. Functional validation of V402L in insecticide resistance using a CRISPR/Cas9 genome modified line showed that it confers reduced mortality after exposure to all tested pyrethroids and DDT, but at lower levels compared to L995F. In contrast to L995F however, no fitness costs were identified for mosquitoes carrying V402L under laboratory conditions. CONCLUSION: The V402L substitution confers pyrethroid resistance in An. gambiae in the absence of any other VGSC substitution and/or alternative resistance mechanisms. The lower fitness cost associated with this kdr mutation may provide a selective advantage over the classical kdr in some settings and genotyping at this locus should be added in the list of resistant alleles for routine screening.
We investigated alternatives to whole blood for blood feeding of mosquitoes with a focus on improved stability and compatibility with mass rearing programs. In contrast to whole blood, an artificial blood diet of ATP-supplemented plasma was effective in maintaining mosquito populations and was compatible with storage for extended periods refrigerated, frozen, and as a lyophilized powder. The plasma ATP diet supported rearing of both Anopheles and Aedes mosquitoes. It was also effective in rearing Wolbachia-infected Aedes mosquitoes, suggesting compatibility with vector control efforts.
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