The seminal odorant binding protein Obp56g is required for mating plug formation and male fertility in Drosophila melanogaster

  1. Nora C Brown
  2. Benjamin Gordon
  3. Caitlin E McDonough-Goldstein
  4. Snigdha Misra
  5. Geoffrey D Findlay
  6. Andrew G Clark  Is a corresponding author
  7. Mariana Federica Wolfner  Is a corresponding author
  1. Department of Molecular Biology and Genetics, Cornell University, United States
  2. Department of Evolutionary Biology, University of Vienna, Austria
  3. Department of Biology, College of the Holy Cross, United States
6 figures, 1 table and 6 additional files

Figures

Figure 1 with 3 supplements
Seminal Obp gene expression and fecundity/remating defects in females mated to Obp56g1 null males.

(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 …

Figure 1—figure supplement 1
Whole body knockdown of Obp56g using Tubulin-GAL4 results in loss of post-mating response phenotypes in females.

(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). …

Figure 1—figure supplement 2
Male reproductive tract knockdown of Obp56g with CrebA-GAL4 is required for the post-mating response and mating plug formation.

(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 …

Figure 1—figure supplement 3
Spermatogenesis appears normal in Obp56g1;ProtB-eGFP males relative to Obp56g1/CyO;ProtB-eGFP control males.

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 …

Figure 2 with 5 supplements
CRISPR/Cas9-generated mutants of Obp22a, Obp51a, Obp56e, Obp56f, Obp56i, and Obp8a have no or marginal effects on female fecundity and remating rates.

(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. …

Figure 2—figure supplement 1
Crossing scheme to generate CRISPR mutants in autosomal (Obp22a, Obp51a, Obp56e, Obp56f, Obp56i) and X-linked (Obp8a) Obp genes used in this study, with text boxes representing chromosomes X/Y, 2, and 3 (dot chromosome not shown).

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 …

Figure 2—figure supplement 2
No effect of heterozygosity in PMR phenotypes relative to homozygous WT or homozygous CRISPR mutant males.

(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 …

Figure 2—figure supplement 3
Box plots of hatchability estimates from CS females mated to Obp56g or CRISPR mutant males.

(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 …

Figure 2—figure supplement 4
Mating latency and duration measurements from CRISPR-generated Obp mutants with CS females.

(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 …

Figure 2—figure supplement 5
Mating duration (A) and latency (B) measurements from homozygous wildtype (+/+), heterozygous mutant (+/-) and homozygous CRISPR mutant (-/-) males mated to CS females.

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 …

Figure 3 with 2 supplements
Obp56g is expressed in the Drosophila male ejaculatory bulb of the reproductive tract.

(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, …

Figure 3—figure supplement 1
Expression of Obp56g-GAL4 in the gustatory bristles of the labellum.

(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 …

Figure 3—figure supplement 2
Obp56g is the most highly expressed seminal Obp in the ejaculatory bulb.

(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., …

Figure 4 with 2 supplements
Females mated to Obp56g1 null males have defects in mating plug formation and sperm storage after mating.

(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 …

Figure 4—figure supplement 1
Obp56g1 mutant males do not have gross issues with sperm transfer during mating at the 12 min ASM time point.

(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 …

Figure 4—figure supplement 2
Western blot of major SFPs from CS females mated to Obp56g null and control males at 35 minutes ASM.

(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 …

Figure 4—figure supplement 2—source data 1

Raw film images and uncropped, labeled western blots for data shown in Figure 4—figure supplement 2.

https://cdn.elifesciences.org/articles/86409/elife-86409-fig4-figsupp2-data1-v2.zip
Figure 5 with 9 supplements
Dynamic changes in copy number, presence/absence, and evolutionary divergence rates of seminal Obp genes across the Drosophila genus.

(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 …

Figure 5—figure supplement 1
Synteny plot for Obp51a, phylogeny on the left from McGeary and Findlay, 2020.

Surrounding gene names represent gene names in D. melanogaster.

Figure 5—figure supplement 2
Synteny plot for Obp8a, phylogeny on the left from McGeary and Findlay, 2020.

Surrounding gene names represent gene names in D. melanogaster.

Figure 5—figure supplement 3
Synteny plot for Obp22a, phylogeny on the left from McGeary and Findlay, 2020.

Surrounding gene names represent gene names in D. melanogaster.

Figure 5—figure supplement 4
Synteny plot for Obp56e and Obp56f, phylogeny on the left from McGeary and Findlay, 2020.

Surrounding gene names represent gene names in D. melanogaster.

Figure 5—figure supplement 5
Synteny plot for Obp56g, phylogeny on the left from McGeary and Findlay, 2020.

Surrounding gene names represent gene names in D. melanogaster.

Figure 5—figure supplement 6
Synteny plot for Obp56i, phylogeny on the left from McGeary and Findlay, 2020.

Surrounding gene names represent gene names in D. melanogaster.

Figure 5—figure supplement 7
RAXML-NG maximum likelihood inferred trees for genes in the (A) Obp56 cluster across melanogaster group species, or (B) Obp51a cluster, where genes are colored as in Figure 5—figure supplement 1.

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’ …

Figure 5—figure supplement 8
Gene trees for Obp22a and Obp51a.

(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 …

Figure 5—figure supplement 9
Positively selected sites in Obp22a cluster on the outward-facing region of the protein.

(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 …

Figure 6 with 1 supplement
Seminal Obp genes show changes in expression pattern across species from bulk RNAseq data published in Yang et al., 2018.

(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 …

Figure 6—figure supplement 1
Semi-quantitative RT-PCR data from dissected tissues (head, accessory gland +ejaculatory duct, ejaculatory bulb, and carcass) from D. melanogaster (Dmel), D. ananassae (Dana), D. pseudoobscura (Dpse), D. virilis (Dvir), and D. mojavensis (Dmoj) males after 35 cycles of PCR. NTC = no template control.
Figure 6—figure supplement 1—source data 1

Raw and uncropped, labeled gel images for data shown in Figure 6—figure supplement 1.

https://cdn.elifesciences.org/articles/86409/elife-86409-fig6-figsupp1-data1-v2.zip

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Drosophila melanogaster)Obp56gFlyBaseFLYB: FBgn0034474
Gene (D. melanogaster)Obp56iFlyBaseFLYB: FBgn0043532
Gene (D. melanogaster)Obp56fFlyBaseFLYB: FBgn0043533
Gene (D. melanogaster)Obp56eFlyBaseFLYB: FBgn0034471
Gene (D. melanogaster)Obp22aFlyBaseFLYB: FBgn0043539
Gene (D. melanogaster)Obp51aFlyBaseFLYB: FBgn0043530
Gene (D. melanogaster)Obp8aFlyBaseFLYB: FBgn0030103
Genetic reagent (D. melanogaster)Obp56g1Bloomington Drosophila Stock CenterBDSC:55079; FBst0055079; RRID:BDSC_55079FlyBase genotype: w*; TI{GAL4}Obp56g1
Genetic reagent (D. melanogaster)UAS-CD4-tdGFPBloomington Drosophila Stock CenterBDSC:35836; FBst0035836; RRID:BDSC_35836FlyBase 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)/CyOBloomington Drosophila Stock CenterBDSC:25678; FBst0025678; RRID:BDSC_25678FlyBase genotype: w1118; Df(2 R)BSC594/CyO
Genetic reagent (D. melanogaster)w;CyO/Bl;TM2/TM6BBloomington Drosophila Stock CenterBDSC: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-Cas9attP2BestGene/Shu Kondo & Ryu UedaGenotype: y1 w1118; attP2{nos-Cas9}/TM6C,Sb Tb
Genetic reagent (D. melanogaster)w;vasa-Cas9Bloomington Drosophila Stock CenterBDSC:51324; FBst0051324; RRID:BDSC_51324FlyBase genotype: w1118; PBac{vas-Cas9}VK00027
Genetic reagent (D. melanogaster)Obp56gRNAiVienna Drosophila Resource CenterVDRC:23206 (GD); FBst0454878FlyBase genotype: w1118; P{GD13268}v23206/TM3
Genetic reagent (D. melanogaster)CrebA-GAL4Bloomington Drosophila Stock CenterBDSC: 49409; FBst0049409; RRID:BDSC_49409FlyBase genotype: w1118; P{GMR64E07-GAL4}attP2
Genetic reagent (D. melanogaster)Tubulin-GAL4Findlay et al., 2014; 10.1371/journal.pgen.1004108
Genetic reagent (D. melanogaster)FM7cGift from Susan YoungerGenotype: C(1)DX, y[1] w[1] f[1]/FM7c, Kr-GAL4[DC1], UAS-GFP[DC5], sn[+];;;
Genetic reagent (D. melanogaster)Phi-C31 integrase attP9ABloomington Drosophila Stock Center / Rainbow TransgenicsBDSC: 35569; FBst0035569; RRID:BDSC_35569FlyBase 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 CenterSKU: 14024–0371.37
Genetic reagent (Drosophila pseudoobscura)Genome lineNational Drosophila Species Stock CenterSKU: 14011–0121.94
Genetic reagent (Drosophila mojavensis)Wildtype (Chocolate Mountains)National Drosophila Species Stock CenterSKU: 15081–1352.00
Genetic reagent (Drosophila virilis)Genome lineNational Drosophila Species Stock CenterSKU: 15010–1051.87
Software, algorithmCRISPR 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 paperTransgenic stock carrying gRNAs targeting Obp8a and ebony
Genetic reagent (D. melanogaster)w;;gRNA(Obp56e, ebony)This paperTransgenic stock carrying gRNAs targeting Obp56e and ebony
Genetic reagent (D. melanogaster)w;;gRNA(Obp56f, ebony)This paperTransgenic stock carrying gRNAs targeting Obp56f and ebony
Genetic reagent (D. melanogaster)w;;gRNA(Obp56i, ebony)This paperTransgenic stock carrying gRNAs targeting Obp56i and ebony
Genetic reagent (D. melanogaster)w;;gRNA(Obp22a, ebony)This paperTransgenic stock carrying gRNAs targeting Obp22a and ebony
Genetic reagent (D. melanogaster)w;;gRNA(Obp51a, ebony)This paperTransgenic stock carrying gRNAs targeting Obp51a and ebony
Genetic reagent (D. melanogaster)Obp8aΔ390This paperCRISPR deletion of 390 bp in exon 2 of Obp8a
Genetic reagent (D. melanogaster)Obp22aΔ257This paperCRISPR deletion of 257 bp in exon 2 of Obp22a
Genetic reagent (D. melanogaster)Obp51aΔ16This paperCRISPR deletion of 16 bp in exon 1 of Obp51a
Genetic reagent (D. melanogaster)Obp56eΔ239This paperCRISPR deletion of 239 bp in exon 2 of Obp56e
Genetic reagent (D. melanogaster)Obp56fΔ226This paperCRISPR deletion of 226 bp in exon 2 of Obp56f
Genetic reagent (D. melanogaster)Obp56iΔ359This paperCRISPR deletion of 359 bp in exon 2 of Obp56i
AntibodyAnti-SP (rabbit polyclonal)Wolfner lab1:1000
AntibodyAnti-CG1656 (rabbit polyclonal)Wolfner lab1:500
AntibodyAnti-CG1652 (rabbit polyclonal)Wolfner lab1:250
AntibodyAnti-Acp36DE (rabbit polyclonal)Wolfner lab1:12,000
AntibodyAnti-Acp26Aa (rabbit polyclonal)Wolfner lab1:5000
AntibodyAnti-CG9997 (rabbit polyclonal)Wolfner lab1:750
AntibodyAnti-Antr (rabbit polyclonal)Wolfner lab1:750
AntibodyAnti-CG17575 (rabbit polyclonal)Wolfner lab1:500
AntibodyAnti-tubulin (mouse monoclonal)Sigma-AldrichT51681:4000
Software, algorithmRAxML-NGKozlov et al., 2019; http://doi.org/10.1093/bioinformatics/btz305
Software, algorithmMEGA-11Tamura et al., 2021; http://doi.org/10.1093/molbev/msab120
Software, algorithmPAML (v4.9)Yang, 2007; https://doi.org/10.1093/molbev/msm088
Software, algorithmR (4.2.1)https://www.R-project.org/

Additional files

Supplementary file 1

gRNA sequences from flyCRISPR’s Optimal Target Finder tool for each Obp gene.

https://cdn.elifesciences.org/articles/86409/elife-86409-supp1-v2.docx
Supplementary file 2

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).

https://cdn.elifesciences.org/articles/86409/elife-86409-supp2-v2.docx
Supplementary file 3

Primer sequences used in this study.

https://cdn.elifesciences.org/articles/86409/elife-86409-supp3-v2.docx
Supplementary file 4

CRISPR mutant allele summary for each Obp gene.

https://cdn.elifesciences.org/articles/86409/elife-86409-supp4-v2.docx
Supplementary file 5

Proportion of CS females mated to CRISPR mutant males with morphologically normal mating plugs assessed immediately after the end of mating.

https://cdn.elifesciences.org/articles/86409/elife-86409-supp5-v2.docx
MDAR checklist
https://cdn.elifesciences.org/articles/86409/elife-86409-mdarchecklist1-v2.pdf

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