2.2. Bioassays to test the effect of pyrethroids on Psylliodes chrysocephala

P. chrysocephala samples were tested for resistance to the pyrethroid lambda-cyhalothrin using a glass vial bioassay based on IRAC (Insecticide Resistance Action Committee) Method 031 (www.irac-online.org/methods/weevils-and-flee-beetles/2014). Glass vials (14 ml: 7 cm tall/2 cm diameter) (S Murray and Co, Surrey, UK) were prepared by coating the inner surface with different concentrations of the insecticide. Initial stock solutions were prepared by diluting the technical grade insecticide in technical grade acetone. Three doses, equivalent to 4%, 20% and 100% of the recommended field application rate of lambda-cyhalothrin (7.5 g a.i./ha) were used. The controls were glass vials treated with acetone only. To coat vials, 500 μl of solution was pipetted into the vials which were then placed horizontally without lids on a roller in a fume hood. Vials were rotated at room temperature for at least 2 h until all the acetone had evaporated. Vials were then left vertically at 4 °C overnight before attaching the screw tops the following day.

The adult beetles (see 2.1) were used within a few days of collection and only those capable of walking or jumping when released onto a tray inside a three-sided Perspex cage were collected, using a hand-held battery-powered pooter. A minimum of ten beetles were transferred from the inverted pooter through a small funnel into each vial. The vials were then resealed and left at 18 ± 1 °C under a 16:8 h light:dark photoperiod. After 24 h, the beetles were transferred to untreated glass vials without lids under upturned 200 ml plastic disposable cups (VWR International Ltd, Dublin, Ireland), to allow for a potential recovery which can occur in insects with metabolic resistance. After a further 24 h, the beetles were released onto a tray and individuals scored using a fine paint brush according to three categories: ‘mobile’ (capable of jumping or walking in a coordinated way), ‘affected’ (incapable of jumping or coordinated movement) or ‘dead’ (no movement). Scoring of the beetles from each vial was done for 10 min to avoid adults that were simulating death, a behaviour shown by this species that has probably evolved through predation pressure. Results were expressed as percentage mortalities. Following scoring, beetles in each category were transferred to Eppendorf tubes and snap frozen using liquid nitrogen before being stored in a freezer at −80 °C.