Afoxolaner is an isoxazoline compound characterized by a good safety profile and extended effectiveness against fleas and ticks on dogs following a single oral administration. In vitro membrane feeding assay data and in vivo pharmacokinetic studies in dogs established an afoxolaner blood concentration of 0.1-0.2 μg/ml to be effective against both fleas (Ctenocephalides felis) and ticks (Dermacentor variabilis). Pharmacokinetic profiles in dogs following a 2.5mg/kg oral dosage demonstrated uniform and predictable afoxolaner plasma concentrations above threshold levels required for efficacy for more than one month. Dose ranging and a 5-month multi-dose experimental study in dogs, established that the 2.5mg/kg oral dosage was highly effective against fleas and ticks, and produced predictable and reproducible pharmacokinetics following repeated dosing. Mode of action studies showed that afoxolaner blocked native and expressed insect GABA-gated chloride channels with nanomolar potency. Afoxolaner has comparable potency between wild type channels and channels possessing the A302S (resistance-to-dieldrin) mutation. Lack of cyclodiene cross-resistance for afoxolaner was confirmed in comparative Drosophila toxicity studies, and it is concluded that afoxolaner blocked GABA-gated chloride channels via a site distinct from the cyclodienes.

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Canker caused by the Ascomycete Valsa mali is the most destructive disease of apple in Eastern Asia, resulting in yield losses of up to 100%. This necrotrophic fungus induces severe necrosis on apple, eventually leading to the death of the whole tree. Identification of necrosis inducing factors may help to unravel the molecular bases for colonization of apple trees by V. mali. As a first step toward this goal, we identified and characterized the V. mali repertoire of candidate effector proteins (CEPs). In total, 193 secreted proteins with no known function were predicted from genomic data, of which 101 were V. mali-specific. Compared to non-CEPs predicted for the V. mali secretome, CEPs have shorter sequence length and a higher content of cysteine residues. Based on transient over-expression in Nicotiana benthamiana performed for 70 randomly selected CEPs, seven V. mali Effector Proteins (VmEPs) were shown to significantly suppress BAX-induced PCD. Furthermore, targeted deletion of VmEP1 resulted in a significant reduction of virulence. These results suggest that V. mali expresses secreted proteins that can suppress PCD usually associated with effector-triggered immunity (ETI). ETI in turn may play an important role in the V. mali–apple interaction. The ability of V. mali to suppress plant ETI sheds a new light onto the interaction of a necrotrophic fungus with its host plant.

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