T. castaneum Control and Pesticide-selected Colony Maintenance
We used six T. castaneum populations (Adairville, Coffee, Dorris, RR, Snavely, WF Ware) collected from stored grain facilities in the southeast USA . In these environments with other stored product pests,T. castaneum are exposed to a variety of parasites, both within and between species in the community, and may be incidentally or intentionally exposed to a range of different pesticides. Beetle colonies had been maintained under laboratory control conditions (standard diet of whole wheat flour + 5% yeast; 30°C; 70% humidity; in the dark) for at least 20 generations prior to the start of the experiment.
For each of the six ancestral T. castaneum populations, we initially exposed approximately 200 larvae per population to control conditions (no pesticides) or to pesticides (organophosphate [OP] or pyrethroid [Pyr]) (Suppl. Fig. 1; 18 populations total). Initial exposure doses were calculated as one tenth the manufacturer’s recommended dose (OP [Malathion 50% EC, Southern Ag]- 0.103 mg/ml; Pyr [Demon WP, 40% cypermethrin, Syngenta]- 0.0251 mg/ml). We approximated LC50 pesticide concentrations for ancestral populations using dose response curves , and adjusted these after the second generation (LC50: 5.14 mg/ml OP and 0.188 mg/ml Pyr). To create pesticide exposure diets, we combined 0.15 g/ml standard diet with 10 ml of pesticide solutions diluted in DI water . This diet slurry was added to 1 L plastic colony containers and allowed to dry overnight at 55°C. For each pesticide-selected population, we allowed approximately 200 pupae and larvae to develop on the pesticide diets for one week before supplementing colonies with 100ml of fresh standard diet. We transferred individuals to new pesticide exposure containers every four weeks. After six generations of selection, we tested for increased pesticide resistance in F2 larvae (reared for two generations without selection to avoid parental effects) from the pesticide-regime (here, used interchangeably with evolution-regime) populations and found these populations had significantly increased survival against pesticides compared to control-regime populations . Meanwhile, we continued selection in all populations for another two generations (to generation 8).