(A) Egg counts from CS females mated to Df(2 R)/+, CyO/+Obp56 g1/CyO, or Obp56g1/Df(2 R) males from 1 to 4 days after mating. Significance indicated from pairwise comparisons of male genotypes …
Remating counts and percentages for data shown in Figure 1B.
(A) Counts of eggs from mated CS females over 4 days. Females mated to Tubulin-GAL4 >Obp56gRNAi males lay significantly fewer eggs than females mated to control males (p<0.001, n=20–24). …
Remating counts and percentages for data shown in Figure 1—figure supplement 1B.
(A) CS females mated to CrebA-GAL4 >Obp56gRNAi males are significantly more likely to remate 4 days post-mating relative to control males (p=0.001, n=55–60). (B) A significantly reduced proportion …
Counts and percentages for data shown in Figure 1—figure supplement 2A and B.
Left: testes, accessory glands, and ejaculatory ducts dissected from 3- to 5-day-old males. Tissue morphology and major stages of sperm development are present in both genotypes (white arrows point …
(A) Egg counts from CS females mated to homozygous null or heterozygous control males (except for Obp8a, the control of which is from an unedited sibling line) from 1 to 4 days after mating. …
Remating counts and percentages for data shown in Figure 2B.
The kinked line represents the Y chromosome. Obp and ebony CRISPR editing takes place in the germline of individuals in the P1 generation. ebony editing can happen on either the gRNA or Cas9 …
(A) Egg counts from CS females mated to +/+, +/-, -/- CRISPR mutant males from 1 to 4 days after mating. Significance indicated from pairwise comparisons of male genotypes within days using emmeans …
Counts and percentages for data shown in Figure 2—figure supplement 2B.
(A) Proportion of eggs hatched over 4 days from females mated to Df(2 R)/+, CyO/+Obp56 g1/CyO, or Obp56g1/Df(2 R) males. Significance indicated from pairwise comparisons of male genotypes across …
(A & B) Obp8aΔ390 mutant flies mate for longer duration than Obp8aWT control flies (p<0.001)though no other statistically significant differences were observed between mating duration of mutant or …
Significance indicated from pairwise comparisons between male genotype using emmeans in a linear mixed effects model (p-value adjusted for multiple comparisons). n for A and B=37–42. Significance …
(A) Brightfield and (B) GFP fluorescent microscopy image of a reproductive tract dissected from a Obp56g-GAL4>UAS-CD4-tdGFP male, where the following tissues are labeled: AG, accessory gland. TS, …
(A) GFP expression from Obp56g-GAL>UAS-CD4-tdGFP males in the head. This sample is not placed under a coverslip. (B) GFP expression in the same genotype, with the proboscis dissected off and gently …
(A) Seurat tSNE dimensionality reduction plot of single nucleus RNAseq expression data from the male reproductive tract (without testes) and their major cell type annotations according to Li et al., …
(A) Fluorescent GFP microscopy image of the bursa of a CS female mated to a Obp56g1/CyO;ProtB-eGFP control male, with the mating plug surrounded by a dotted white line. Females were frozen in liquid …
Counts and proportions for data shown in Figure 4C.
(A) Reproductive tract of a representative CS female mated to Obp56g1/CyO;ProtB-eGFP control males, showing DAPI (mating plug), GFP (sperm heads), and merge +transillumination microscopy images and …
(A) Females mated to Obp56g1 null males have reduced amounts of Acp36DE in their bursas at 35 min ASM. Representative Western blots for SFPs in (lane 1) unmated female reproductive tracts (FV=female …
Raw film images and uncropped, labeled western blots for data shown in Figure 4—figure supplement 2.
(A) Inferred copy number of seminal Obp genes across Drosophila. Species without a dot represent an inferred loss based on syntenic analysis. Increased size of the dot represents increased gene copy …
Surrounding gene names represent gene names in D. melanogaster.
Surrounding gene names represent gene names in D. melanogaster.
Surrounding gene names represent gene names in D. melanogaster.
Surrounding gene names represent gene names in D. melanogaster.
Surrounding gene names represent gene names in D. melanogaster.
Surrounding gene names represent gene names in D. melanogaster.
Node values are bootstrap support estimates based on 1,000 replicates. CG43101 is a gene located next to Obp51a in D. melanogaster, which has 6 cysteines in a pattern reminiscent of the Obp ‘domain’ …
(A) Obp22a and (B) Obp51a maximum likelihood inferred gene trees, where branch lengths are proportional to either estimates of dN (left) or dS (right) from PAML. Values indicated on the dS tree …
(A) Alphafold predicted protein structure (yellow) of Obp22a with the positively selected sites (Pr >0.90 BEB from model M8 of PAML) shown in maroon (Jumper et al., 2021). (B) The same structure as …
(A) log2 normalized TPM expression values (averaged across four biological replicates) of seminal Obp genes and their associated orthologs and paralogs in male reproductive tissue (including …
Raw and uncropped, labeled gel images for data shown in Figure 6—figure supplement 1.
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Gene (Drosophila melanogaster) | Obp56g | FlyBase | FLYB: FBgn0034474 | |
Gene (D. melanogaster) | Obp56i | FlyBase | FLYB: FBgn0043532 | |
Gene (D. melanogaster) | Obp56f | FlyBase | FLYB: FBgn0043533 | |
Gene (D. melanogaster) | Obp56e | FlyBase | FLYB: FBgn0034471 | |
Gene (D. melanogaster) | Obp22a | FlyBase | FLYB: FBgn0043539 | |
Gene (D. melanogaster) | Obp51a | FlyBase | FLYB: FBgn0043530 | |
Gene (D. melanogaster) | Obp8a | FlyBase | FLYB: FBgn0030103 | |
Genetic reagent (D. melanogaster) | Obp56g1 | Bloomington Drosophila Stock Center | BDSC:55079; FBst0055079; RRID:BDSC_55079 | FlyBase genotype: w*; TI{GAL4}Obp56g1 |
Genetic reagent (D. melanogaster) | UAS-CD4-tdGFP | Bloomington Drosophila Stock Center | BDSC:35836; FBst0035836; RRID:BDSC_35836 | FlyBase genotype: w1118; PBac{UAS-CD4-tdGFP}VK00033 |
Genetic reagent (D. melanogaster) | Lhm; PBac{Ubnls-eGFP, ProtB-eGFP}(3) | Gift from John Belote and Scott Pitnik | ||
Genetic reagent (D. melanogaster) | w;Df(2 R)/CyO | Bloomington Drosophila Stock Center | BDSC:25678; FBst0025678; RRID:BDSC_25678 | FlyBase genotype: w1118; Df(2 R)BSC594/CyO |
Genetic reagent (D. melanogaster) | w;CyO/Bl;TM2/TM6B | Bloomington Drosophila Stock Center | BDSC:3704 (formerly, stock no longer available) | Genotype: w[1118]/Dp(1;Y)y[+]; CyO/Bl[1]; TM2/TM6B, Tb[1] |
Genetic reagent (D. melanogaster) | nos-Cas9attP2 | BestGene/Shu Kondo & Ryu Ueda | Genotype: y1 w1118; attP2{nos-Cas9}/TM6C,Sb Tb | |
Genetic reagent (D. melanogaster) | w;vasa-Cas9 | Bloomington Drosophila Stock Center | BDSC:51324; FBst0051324; RRID:BDSC_51324 | FlyBase genotype: w1118; PBac{vas-Cas9}VK00027 |
Genetic reagent (D. melanogaster) | Obp56gRNAi | Vienna Drosophila Resource Center | VDRC:23206 (GD); FBst0454878 | FlyBase genotype: w1118; P{GD13268}v23206/TM3 |
Genetic reagent (D. melanogaster) | CrebA-GAL4 | Bloomington Drosophila Stock Center | BDSC: 49409; FBst0049409; RRID:BDSC_49409 | FlyBase genotype: w1118; P{GMR64E07-GAL4}attP2 |
Genetic reagent (D. melanogaster) | Tubulin-GAL4 | Findlay et al., 2014; 10.1371/journal.pgen.1004108 | ||
Genetic reagent (D. melanogaster) | FM7c | Gift from Susan Younger | Genotype: C(1)DX, y[1] w[1] f[1]/FM7c, Kr-GAL4[DC1], UAS-GFP[DC5], sn[+];;; | |
Genetic reagent (D. melanogaster) | Phi-C31 integrase attP9A | Bloomington Drosophila Stock Center / Rainbow Transgenics | BDSC: 35569; FBst0035569; RRID:BDSC_35569 | FlyBase genotype: y1 w* P{nanos-phiC31\int.NLS}X; PBac{y+-attP-9A}VK00027 |
Genetic reagent (Drosophila ananassae) | Wildtype (Cebu, Philippines) | National Drosophila Species Stock Center | SKU: 14024–0371.37 | |
Genetic reagent (Drosophila pseudoobscura) | Genome line | National Drosophila Species Stock Center | SKU: 14011–0121.94 | |
Genetic reagent (Drosophila mojavensis) | Wildtype (Chocolate Mountains) | National Drosophila Species Stock Center | SKU: 15081–1352.00 | |
Genetic reagent (Drosophila virilis) | Genome line | National Drosophila Species Stock Center | SKU: 15010–1051.87 | |
Software, algorithm | CRISPR Optimal Target Finder (flyCRISPR) | Gratz et al., 2014; http://doi.org/10.1534/genetics.113.160713 | ||
Genetic reagent (D. melanogaster) | w;;gRNA(Obp8a, ebony) | This paper | Transgenic stock carrying gRNAs targeting Obp8a and ebony | |
Genetic reagent (D. melanogaster) | w;;gRNA(Obp56e, ebony) | This paper | Transgenic stock carrying gRNAs targeting Obp56e and ebony | |
Genetic reagent (D. melanogaster) | w;;gRNA(Obp56f, ebony) | This paper | Transgenic stock carrying gRNAs targeting Obp56f and ebony | |
Genetic reagent (D. melanogaster) | w;;gRNA(Obp56i, ebony) | This paper | Transgenic stock carrying gRNAs targeting Obp56i and ebony | |
Genetic reagent (D. melanogaster) | w;;gRNA(Obp22a, ebony) | This paper | Transgenic stock carrying gRNAs targeting Obp22a and ebony | |
Genetic reagent (D. melanogaster) | w;;gRNA(Obp51a, ebony) | This paper | Transgenic stock carrying gRNAs targeting Obp51a and ebony | |
Genetic reagent (D. melanogaster) | Obp8aΔ390 | This paper | CRISPR deletion of 390 bp in exon 2 of Obp8a | |
Genetic reagent (D. melanogaster) | Obp22aΔ257 | This paper | CRISPR deletion of 257 bp in exon 2 of Obp22a | |
Genetic reagent (D. melanogaster) | Obp51aΔ16 | This paper | CRISPR deletion of 16 bp in exon 1 of Obp51a | |
Genetic reagent (D. melanogaster) | Obp56eΔ239 | This paper | CRISPR deletion of 239 bp in exon 2 of Obp56e | |
Genetic reagent (D. melanogaster) | Obp56fΔ226 | This paper | CRISPR deletion of 226 bp in exon 2 of Obp56f | |
Genetic reagent (D. melanogaster) | Obp56iΔ359 | This paper | CRISPR deletion of 359 bp in exon 2 of Obp56i | |
Antibody | Anti-SP (rabbit polyclonal) | Wolfner lab | 1:1000 | |
Antibody | Anti-CG1656 (rabbit polyclonal) | Wolfner lab | 1:500 | |
Antibody | Anti-CG1652 (rabbit polyclonal) | Wolfner lab | 1:250 | |
Antibody | Anti-Acp36DE (rabbit polyclonal) | Wolfner lab | 1:12,000 | |
Antibody | Anti-Acp26Aa (rabbit polyclonal) | Wolfner lab | 1:5000 | |
Antibody | Anti-CG9997 (rabbit polyclonal) | Wolfner lab | 1:750 | |
Antibody | Anti-Antr (rabbit polyclonal) | Wolfner lab | 1:750 | |
Antibody | Anti-CG17575 (rabbit polyclonal) | Wolfner lab | 1:500 | |
Antibody | Anti-tubulin (mouse monoclonal) | Sigma-Aldrich | T5168 | 1:4000 |
Software, algorithm | RAxML-NG | Kozlov et al., 2019; http://doi.org/10.1093/bioinformatics/btz305 | ||
Software, algorithm | MEGA-11 | Tamura et al., 2021; http://doi.org/10.1093/molbev/msab120 | ||
Software, algorithm | PAML (v4.9) | Yang, 2007; https://doi.org/10.1093/molbev/msm088 | ||
Software, algorithm | R (4.2.1) | https://www.R-project.org/ |
gRNA sequences from flyCRISPR’s Optimal Target Finder tool for each Obp gene.
Primer sequences for cloning gRNAs from Supplementary file 1 into pAC-U63-tgRNA-Rev using pMGC as a PCR template (from Poe et al., 2019).
Primer sequences used in this study.
CRISPR mutant allele summary for each Obp gene.
Proportion of CS females mated to CRISPR mutant males with morphologically normal mating plugs assessed immediately after the end of mating.