A998 4

The methanol (MeOH) used to extract the samples was of analytical grade obtained from R & M chemicals (United Kingdom). Other solvents used for the mobile phase selection were all HPLC - grade solvents with percentage purities greater than 99.9%. They include acetonitrile (ACN) [A998-4, Fisher Scientific, United State of America (USA)], acetone (A949-1, Fisher Scientific, USA), MeOH (106007, Merck KGaA, Darmstadt, Germany), dichloromethane (DCM) (106044, Merck KGaA, Darmstadt, Germany), acetic acid (02002123, Fisher Scientific, USA), chloroform (102445, Merck KGaA, Darmstadt, Germany), and toluene (108327, Merck KGaA, Darmstadt, Germany). The mixed aflatoxin STD (Pribolab, STD#1089) at a certified concentration of 20 μg/ml for each of the four aflatoxins (B1, B2, G1, and G2) in 5 ml ACN solution was supplied by Pribolab, Singapore.

Live queens were briefly restrained in plastic marking tubes (Betterbee, Greenwich, CT, USA). Using forceps, heads were swiftly removed to expose the open neck cavity. Using a graduated glass capillary tube, a measured quantity of hemolymph (5–20 ul per bee) was collected from the cavity and placed into a mix of 50 ul acetonitrile (Fisher Scientific A998–4, Waltham, MA, USA) and 50 ul 0.9% sodium chloride solution (Fisher Scientific S271–500) contained within a 9-mm autosampler insert (Fisher Scientific C4010-630) inside an autosampler vial (Fisher Scientific C5000-1W) with a vial cap (Fisher Scientific C5000-54B). This method prevents hemolymph melanization and preserves JH in suspension, following Kai et al. (2018). Samples were vortexed and JH was twice extracted into 100-μl volumes of hexanes (Fisher Scientific H306–1) containing 10 ng of citronellol (Sigma-Aldrich W230915, St. Louis, MO, USA) as an internal standard. After each extraction, the JH–hexane–citronellol phase (upper layer) was transferred into a new autosampler vial (with insert and cap). JH extracts were stored at −80°C until they were run on a gas chromatography–mass spectrometry machine according to methods in Kai et al. (2018) (see Supporting Information for details). Bees were stored at −80°C until they were processed for dissection.

Five ml of a certified mix of four standard aflatoxin solutions containing an equal ratio of the four aflatoxins [Aflatoxin B1 (AFB1), Aflatoxin B2 (AFB2), Aflatoxin G1 (AFG1), and Aflatoxin G2 (AFG2)] was supplied by Pribolab, China (STD#1089). Two sets of dilutions of the mixed aflatoxin standard solution were prepared in (75% MeOH) (first set) and 100% HPLC-grade acetonitrile (ACN) (A998-4, Fisher Scientific Malaysia) solvent (second set). Each set contains 16 concentration levels (0, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, and 90 ng/ml) of the mixed aflatoxin standard solution. Each concentration was prepared in 7 different vials, making a total of 112 vials for each set (first set and second set).