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 coli cells (NEB) following the manufacturer’s specifications. Seventy-five microlitres of undiluted transformed 10-beta cells was poured into an ampicillin 20 mg/ml (+Amp) lysogeny broth (LB) plate and incubated O/N at 37 °C; (E) ten clones were recovered from the LB plate and grown in 3 ml of liquid LB +Amp O/N at 37 °C and 250 rpm. To obtain the plasmids from clones, the ZR Plasmid Miniprep Kit (Zymo) was used following the manufacturer’s specifications and evaluated with the BpmI, XhoI, and NotI restriction enzymes following the same protocols as described above for restriction digestion and electrophoresis; (F) three positive plasmids for the selected restriction cutting sites were sequenced using Oxford Nanopore and aligned to the in silico construct to confirm the sequence identity. kb kilobase, MW molecular weight; Fig. S3: containers used for fly rearing and crosses. (A) plastic bottle used for fly rearing; (B) sandglass-shaped arrangement made using two 5-oz clear plastic cups joined from removed bottoms used for fly crosses after embryo injections. These containers also included: a top holed cap and a bottom cap with an opening of ~ 1 inch in diameter; a rounded piece of white paper towel; a small glass vial filled with tap water and a small protein cookie made with yeast, milk and egg powder and cane sugar; Fig. S4: fly crosses completed after embryos microinjections. G generation, wt wild-type; Note S2: primers used for LcupOrco insert detection of landing site. Three types of pair of primer associated with three different strategies were used to determine whether the landing site of the LcupOrco insert was withing the LcupOrco locus in the L. cuprina genome. The first was associated with the left side (LS) of the insert including the upstream genome region, the hsp83 promoter, and part of the green marker; the second was designed to amplify the right side (RS) of the insert including the part of tub 3′-UTR and the downstream genome region; and the third was to amplify the whole LcupOrco insert (All), including from upstream to downstream L. cuprina genome regions flanking the LcupOrco locus ; Table S1: RNA-Seq data overview. (A) each sample represents an RNA-Seq library. Sample names, description, and number of raw, trimmed, and mapped reads per library are provided; (B) total sequences identified for the RNA-Seq experiment, divided by RNA type; Fig. S5: principal component analysis (PCA) completed using transcripts with TPM expression ≥ 5 for all libraries (samples) associated with EXP-1 (larval stages). Samples WL1_1 to 3, WL2_1 to 3, and WL3_1 to 6 were collected from the LA07 colony. Samples WL1_4 and 5 and WL2_4 and 5 were collected from heterozygous individuals obtained from the crossing between the LA07 and the SLAM5X colonies. The PCA analysis did not show differences between samples of the same larval stage, collected from different colonies. L1 first larval state, L2 second larval stage, L3 third larval stage, WL whole larva sample, TPM transcript per million; Fig. S6: ABC transporters preliminary neighbor-Joining phylogenetic analysis. Dmel Drosophila melanogaster, Lcup Lucilia cuprina, Lser Lucilia sericata; Fig. S7: guide RNA (gRNA) cutting efficiency tested in vitro using the EnGen Spy Cas9. The amplicon size of LcupOrco gene fragment without cutting was 439 bp and after Cas9 in vitro cutting generated two overlapped amplicons of 224 and 215 bp, respectively. C control, uncut genomic DNA, MW molecular weight, T treated, gDNA + Cas9, Fig. S8: Lucilia cuprina eggs, larva stage 3 (L3) and adult flies expressing the ZsGreen marker before the genotyping analysis to determine LcupOrco insert landing site. (A) L3 showing transient expression of the marker 7 days after wt eggs injection; (B) eggs obtained from crossing a LcupOrco mosaic male with a wt female; (C) wt L3 on top versus a heterozygous ZsGreen L3 at the bottom; (D) heterozygous ZsGreen L3 on top versus homozygous for the same marker at the bottom; (E) dorsal view of a wt adult male (left) versus G1 heterozygous ZsGreen male (right) under bright field; (F) same males showed in E under a green filter; ventral view of the same males using bright field (G) and a green filter (H); Fig. S9: PCR amplification of gDNA of samples collected from LcupOrco larval diet preference test and female olfaction assay: (A) samples of larval olfaction assay; (B) samples of adult female olfaction assay. Primers, amplicon sequence, and cycling parameters are detailed in Note S1. MW molecular weight; Fig. S10: Sanger sequencing results for samples collected from LcupOrco larval diet preference test and adult female olfaction assay. (A) genotyped samples from nos. 1 to 48 of larval diet preference test; and (B) genotyped samples from nos. 1 to 72 of adult female olfaction assay. Synthego software was used to analyze sample’s chromatograms, and indels % = 0 corresponds to wt samples, from 1% to 89% to heterozygous samples, and ≥ 90% to homozygous samples. Results were compiled along with single larva preferences in Table S2A, B; Table S2: compiled data of samples associated with LcupOrco behavioral assays. (A) larval diet preference test; (B) adult female olfaction assay. The numbers 1–8 after the letters in B, refer to the main chambers of the olfactometer, and numbers after dots, to single females from the same chamber. Fr fresh beef (room temperature), FrCo fresh cold beef, FrHo fresh hot beef, HET heterozygous, HOM homozygous, NC non-choice, NG nongenotyped, Ro rotten beef (room temperature), RoCo rotten cold beef (25 ± 1 °C); RoHo rotten hot beef (33 ± 1 °C), wt wild-type. Hyphen symbol means nontested by Sanger sequencing; Fig. S11: eggs obtained from LcupOrco mutated females: (A) eggs obtained from 8-day-old LcupOrco+/− females 3 days after crossing them to LcupNPF−/− males expressing the ZsGreen marker. The same marker was removed from LcupOrco females after confirming that the LcupOrco insert landing site was not located withing the LcupOrco gene locus in the L. cuprina genome. The insert was removed by selecting nonfluorescent larvae and crossing males obtained from them for two generations versus wt females. In addition, males from each generation were genotyped using protocols described in Note S1 to confirm the presence of indels and point mutations within the LcupOrco coding region. The fluorescence in eggs confirmed the mating between the LcupOrco+/− females and LcupNPF−/− males and egg fertilization. LcupOrco−/− females did not lay eggs at this time; (B) part of LcupOrco−/− females laid eggs 10 days after mixing them with LcupNPF−/− males, but eggs were not fertilized as shown under green filter (C), and no larvae emerged from them; part of the LcupOrco−/− females laid fertilized eggs 17 days after mixing with LcupNPF−/− males, as shown under bright field (D–F) and green filter (E-G); these eggs produced healthy progeny; Fig. S12: ovaries from 10-day-old L. cuprina wt and LcupOrco mutated females. Three females per condition were dissected (columns). All females were provided with tap water and sugar, but the presence of protein in the diet and mating condition changed between groups; wt virgin females not fed (A–C) and fed with protein (D–F); wt mated females not fed (G–I) and fed with protein (J–L); LcupOrco−/− mutated females fed with protein (M–O).