Additional file 1 of Genetic and behavioral analyses suggest that larval and adult stages of Lucilia cuprina employ different sensory systems to detect rotten beef

Additional file 1: Fig. S1: larval stages and tissues used for the RNA-Seq experiment. (A) whole larva stage 1 (L1); (B) whole larva stage 2 (L2); (C) lateral view of a whole late larva stage 3 (L3) and (D) ventral view; (E) whole early larval stage 3 (WL); (F) first segment designated as “head” (H) from an early L3, detailed within a dashed white circle; and (G) gut from an early L3, where crop have been separated from the cardia originally connected by the foregut (not present in the picture); Note S1: LcupOrco gene, guide RNA (gRNA) sequence, and primers used to determine potential indels and point mutations at the gRNA cutting site (genotyping); Fig. S2: LcupOrco insert construction. (A) LcupOrco gene fragment including two sequences corresponding to the left (LHA) and right (RHA) homology arms of 1000 bp in length each, necessary for the CRISPR homology directed repair (HDR) protocol cloned using plasmid pUCIDT-AMP GoldenGate (ordered from IDT). In addition, a DNA section including the restriction cutting sites (RCS) for enzymes XhoI (NEB) and NotI (NEB) was added in between of both sequences in the same plasmid; (B) donor plasmid including the ZsGreen Marker followed by the hsp83 promoter located between positions 1891 and 7441 [2]; (C) ZsGreen Marker obtained from the donor plasmid. The plasmid was linearized using restriction enzymes BpmI (NEB), XhoI and NotI at 37 °C for 60 min, and the reaction was finished at 65 °C for 20 min. After restriction digestion, the mix was electrophoresed on a 1.5% agarose gel at 60 V for 120 min. The band with the size corresponding to the marker (~5 kb) was recovered from the gel and purified using the Zymoclean Gel DNA recovery kit (Zymo) following the manufacturer’s specifications; (D) to obtain the final construct, 50 ng of linearized pUCIDT-AMP GoldenGate plasmid + 175.5 ng of the marker fragment (1:3 ratio) was ligated overnight (O/N) at 16 °C using the T4 DNA ligase (NEB). After ligation, 1 μl of the reaction mix was used to transform 10-beta competent Escherichia ...