Varroa destructor reprodactive mites
Three types of adult female mites were collected for the different experiments: “Free moving mites”, “phoretic mites” and “reproductive mites”. Collection of phoretic mites was performed by taking a brood frame out of the hive and brushing off the adult bees. Emerging bees were allowed to naturally emerge at 26-30 °C, and phoretic adult female mites were collected from emerging bees, using fine tweezers and fine paint brush, and used up to 1 hour from emerging. Reproductive mites were collected from purple eye pupa, and used up to 1 hour from collection. Total RNA of Varroa foreleg and body devoid of forelegs was extracted using GeneAll Kit (Seoul, South Korea) as described before (Singh et al. 2016). In short, forelegs were excised from the mite using a fine surgical blade (size no. 11) under a stereo microscope (Olympus SZX12, Shinjuku-ku, Tokyo, Japan). For each replicate, 50 forelegs were homogenized using a pestle after dipping the tip of the tube in liquid nitrogen. RNA extraction was carried out according to manufacturer’s instructions. The quality and quantity of the RNA were measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). RNA samples of two replicates for each stage were processed by Technion Genome Center (Israel) according to the Ilumina TruSeq RNA Library Preparation Kit v2. The constructed libraries were sequenced in one lane on program 100 PE using SBS V reagent. The complete paired-end sequences were obtained as individual FASTQ files.