During the experiment, the caged bees were kept in a dark room at 32.2 °C with 50% relative humility. Each cage was equipped with a water feeder and a 50% (w/v) sucrose water-based diet feeder. The feeders were made by cutting a hole 6 mm in diameter on the top of a 2 mL micro-centrifuge tube. Bees could access water or food easily through the opening. Water was provided ad libitum; the water feeder was replaced every 5 days or whenever it appeared to be nearly empty. The diet feeders were replaced daily, just after the daily survival check of the caged bees. Approximately 1.5 mL sucrose water-based diet was used to fill each feeder in every cage; this amount was more than sufficient to feed all of the bees in each cage. The assay continued until all test subjects had died.
To determine the effects of pesticides on longevity, three types of amendments were made to the base diet: 4 ppm bifenthrin (N-11203, ChemService, Inc., West Chester, PA, USA), 0.5 ppm β-cyfluthrin (N-11191, ChemService, Inc., West Chester, PA, USA) and no amendments (control). The concentrations used for the tested pesticides concentration were based on pilot bioassays [23 ]. Within each pesticide treatment, two base diets were compared: protein-rich (protein:carbohydrate = 1:12, [17 (link)]) and protein-free. Casein, an animal-derived protein product free from phytochemicals, was used here as a supplemental protein supplement, as it has been used in many insect artificial diets [24 (link)]. Within each base diet, phytochemical amendments were compared; these amendments included 0.5 mM p-coumaric acid (C9008, Sigma-Aldrich Co. LLC., St. Louis, MO, USA) (PC), 0.25 mM quercetin (Q4951, Sigma-Aldrich Co. LLC., St. Louis, MO, USA) treatment (Qc), 0.5 mM p-coumaric acid and 0.25 mM quercetin-combined (PQ), and no phytochemicals (Control, CD). The phytochemical concentrations selected for testing in this work, also used in previous studies [2 (link),25 (link)], were designed to fit within the range of concentrations ingested by worker bees over the course of their adult lives. Phytochemical concentrations vary in honey bee diets depending on plant species, tissue type, season, and geographic locality. Yet another source of variation is the age- and task-related polyethism of adult bees, whereby tasks in the hive are associated with different nutritional demands as bees age or colony conditions change. The concentrations tested are within the natural range documented in honey, pollen and beebread, the three primary sources of ingested phytochemicals. The concentration of p-coumaric acid used, 0.5 mM (82 μg/g), is within the range of concentrations in local honey and beebread [2 (link)] and our tested concentration of quercetin (0.25 mM = 75.6 mg/kg), although higher than concentrations typically encountered in honey (up to 4.86 mg/kg) [26 (link)], is lower than concentrations in pollen (up to 529.8 mg/kg) [27 (link),28 (link)] and in beebread (495.8 mg/kg) [29 ]. There were 24 different treatments in each experimental replicate, and each treatment had five replicates (Table 1 ).
The protein-rich (casein+) stock syrup was prepared by adding 25 g casein (C3400, Sigma-Aldrich Co. LLC., St. Louis, MO, USA) into 600 g 50% (w/v) sucrose water. The pesticides and phytochemicals were first dissolved in dimethyl sulfoxide (DMSO; D128, Fisher Scientific International, Inc., Pittsburgh, PA, USA) to make the 400× concentrated stock solutions. Finally, the sucrose water-based diets were prepared by adding 0.125 mL 400× phytochemical stock solutions into protein-free (casein−) or protein-rich (casein+) 50% syrups to make a total volume of 50 mL. In addition, the unamended phytochemical-free control diet was prepared by adding 0.125 mL DMSO to protein-free or protein-rich 50% syrup for a volume of 50 mL. As the result, all of the diets contained equal amounts of 0.25% DMSO.
To determine the effects of pesticides on longevity, three types of amendments were made to the base diet: 4 ppm bifenthrin (N-11203, ChemService, Inc., West Chester, PA, USA), 0.5 ppm β-cyfluthrin (N-11191, ChemService, Inc., West Chester, PA, USA) and no amendments (control). The concentrations used for the tested pesticides concentration were based on pilot bioassays [23 ]. Within each pesticide treatment, two base diets were compared: protein-rich (protein:carbohydrate = 1:12, [17 (link)]) and protein-free. Casein, an animal-derived protein product free from phytochemicals, was used here as a supplemental protein supplement, as it has been used in many insect artificial diets [24 (link)]. Within each base diet, phytochemical amendments were compared; these amendments included 0.5 mM p-coumaric acid (C9008, Sigma-Aldrich Co. LLC., St. Louis, MO, USA) (PC), 0.25 mM quercetin (Q4951, Sigma-Aldrich Co. LLC., St. Louis, MO, USA) treatment (Qc), 0.5 mM p-coumaric acid and 0.25 mM quercetin-combined (PQ), and no phytochemicals (Control, CD). The phytochemical concentrations selected for testing in this work, also used in previous studies [2 (link),25 (link)], were designed to fit within the range of concentrations ingested by worker bees over the course of their adult lives. Phytochemical concentrations vary in honey bee diets depending on plant species, tissue type, season, and geographic locality. Yet another source of variation is the age- and task-related polyethism of adult bees, whereby tasks in the hive are associated with different nutritional demands as bees age or colony conditions change. The concentrations tested are within the natural range documented in honey, pollen and beebread, the three primary sources of ingested phytochemicals. The concentration of p-coumaric acid used, 0.5 mM (82 μg/g), is within the range of concentrations in local honey and beebread [2 (link)] and our tested concentration of quercetin (0.25 mM = 75.6 mg/kg), although higher than concentrations typically encountered in honey (up to 4.86 mg/kg) [26 (link)], is lower than concentrations in pollen (up to 529.8 mg/kg) [27 (link),28 (link)] and in beebread (495.8 mg/kg) [29 ]. There were 24 different treatments in each experimental replicate, and each treatment had five replicates (
The protein-rich (casein+) stock syrup was prepared by adding 25 g casein (C3400, Sigma-Aldrich Co. LLC., St. Louis, MO, USA) into 600 g 50% (w/v) sucrose water. The pesticides and phytochemicals were first dissolved in dimethyl sulfoxide (DMSO; D128, Fisher Scientific International, Inc., Pittsburgh, PA, USA) to make the 400× concentrated stock solutions. Finally, the sucrose water-based diets were prepared by adding 0.125 mL 400× phytochemical stock solutions into protein-free (casein−) or protein-rich (casein+) 50% syrups to make a total volume of 50 mL. In addition, the unamended phytochemical-free control diet was prepared by adding 0.125 mL DMSO to protein-free or protein-rich 50% syrup for a volume of 50 mL. As the result, all of the diets contained equal amounts of 0.25% DMSO.
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