, Genesee Scientific). D. melanogaster GAL4/UAS-RNAi experiments have been performed at 25 .Cell Rep. Author manuscript; available in PMC 2021 November 10.Pu et al.PageMETHOD DETAILSAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptGeneration of GFP reporter constructs and transgenic flies–All GFP reporter constructs were generated by PCR amplification from the genomic fragments from distinctive Drosophila species and cloned in to the GFP reporter vector pS3aG by means of the AscI and SbfI internet site (All primers listed in Table S3). The initial screen to find the D. melanogaster EB enhancer focused on 4 different regions based on bond-PB μ Opioid Receptor/MOR review transcript: i) 5 of the gene (3R: 22547847..22550679), intron 1 (3R: 22550873..22554636), intron 3 (3R: 22555014..22555385) along with the three non-coding region (3R: 22556495..22558341). All constructs have been injected into the D. melanogaster Xout line and integrated into the genome applying the PhiC31 integrase system. In situ hybridization–Ejaculatory bulbs (EBs) from three-day old male adult flies had been dissected in Phosphate-Buffered Saline (PBS). In situ hybridization was performed with RNA probes as described previously (Chung et al., 2009). Probes for bond in situ hybridization had been synthesized from cDNA applying species-specific primers (Table S3). The D. melanogaster probe was utilized for in situ hybridization to D. melanogaster, D. simulans, D. yakuba, and D. erecta. D. ananassae probe was made use of for D. ananassae. D. pseudoobscura probe was applied for D. pseudoobscura and D. subobscura. D. willistoni probe was utilized for D. willistoni, D. sturtevanti, and D. nebulosa. S. lebanonensis probe was utilised to S. lebanonensis and S. rufifrons. C. procnemis probe was utilised for C. procnemis and S. latifasiaeformis. M. PDE11 list domestica probe was used for M. domestica. Phylogenetic analyses–A multilocus dataset of 17 genes from 21 species was applied for phylogenetic reconstruction. The genes included the 15 nuclear markers Amyrel (Amyrel), Distal-less (Dll), Dopa decarboxylase (Ddc), ebony (e), engrailed (en), even-skipped (eve), hedgehog (hh), Notum (Notum), patched (ptc), wingless (wg), 28S ribosomal RNA (28S), Alcohol dehydrogenase (Adh), Glycerol-3-phosphate dehydrogenase (Gpdh), Superoxide dismutase (Sod), Xanthine dehydrogenase (Xdh), plus the two mitochondrial markers cytochrome oxidase subunit 1 (COI) and cytochrome oxidase subunit two (COII). Nucleotide sequences in the 20 drosophilid species have been retrieved from the DrosoPhyla project (Finet et al., 2021), and nucleotide sequences of M. domestica were collected from the NCBI database. Alignments for every person gene were generated working with MUSCLE (Edgar, 2004) with default parameters. Unreliably aligned positions had been excluded making use of trimAl with parameters -gt 0.five and -st 0.001 (Capella-Guti rez et al., 2009). In-house Python scripts have been utilized to concatenate the aligned sequences (Finet et al., 2021). Maximum likelihood searches have been performed making use of PhyML three.0 (Guindon et al., 2010) under the GTR+4+I model, and one hundred bootstrap replicates had been performed for support estimation. RNA sequencing and analysis–RNA in the EBs of around 200 eight-day old Canton-S D. melanogaster males was extracted applying TRIzol Reagent in line with manufacturer’s instructions. Indexed RNA-Seq libraries had been prepared from 1 g of total RNA making use of the TruSeq RNA Library Prep Kit v2 (Illumina) in accordance with manufacturer’s protocol. RNA high-quality and concentration were measured on an Agilent 2100 Bio-analyz
