DOI: 10.5061/dryad.gb5mkkx2j
title: Sperm length and seminal fluid proteins promote male reproductive success in D. melanogaster

description: Spermatozoal morphology is highly variable both within and among species,
 often corresponding to variation in the shape of the female sperm storage
 organs in ways that can significantly impact fertilization success. In an
 effort to understand genetic mechanisms of sperm length variation, we
 compared gene expression patterns in the testes of Drosophila melanogaster
 males that produce either long or short sperm. We found that genes
 upregulated in long sperm testes are enriched for long noncoding RNAs
 (lncRNAs) and seminal fluid proteins (Sfps). Transferred in seminal fluid
 to the female during mating, Sfps are secreted by the male accessory
 glands and affect female remating rate, physiology, and behavior with
 concomitant advantages for male reproductive success. While sperm and Sfps
 are both critical for male reproductive success, they are largely
 considered to be functionally, genetically, and developmentally
 independent and despite being upregulated in long sperm testes, Sfps have
 no known function in testis tissue. We found that knockouts of two Sfps
 upregulated in long sperm males, Sex Peptide (SP) and ovulin (Acp26Aa)
 resulted in shorter sperm, which altogether suggests that Sfps may play a
 role in the development of sperm length during spermatogenesis. Consistent
 with this, knockout of accessory gland function did not affect sperm
 length, suggesting that accessory gland expression had no influence on
 spermatogenic processes. We also found that long sperm males were better
 able to delay female remating. These results might suggest that long sperm
 males have a double advantage in sperm competition by both delaying female
 remating, likely through transfer of more Sfps, and by resisting sperm
 displacement. However, we found that the delay in female remating does not
 necessarily translate to more progeny or higher paternity success. Thus,
 we found that multiple components of the ejaculate promote male
 reproductive success at different stages of reproduction, but the realized
 fitness advantages in sperm competition are uncertain.
description: # Sperm length and seminal fluid proteins promote male reproductive
 success in *D. melanogaster* Dataset DOI:
 [10.5061/dryad.gb5mkkx2j](10.5061/dryad.gb5mkkx2j) ## Description of the
 data and file structure # README for Datasets Associated with Galvin J, EL
 Larson, S Yedigarian, M Rahman, K Borziak, M DeNieu, and MK Manier
 **Title:** Genetic coordination of sperm morphology and seminal fluid
 proteins promotes male reproductive success in *Drosophila melanogaster*
 ## Experiment 1: RNAseq of Testes Producing Long or Short Sperm ###
 Datasets: * **Supp1_rpkm_all.csv** * **Supp2_DEgene.summary.csv** *
 **rpkm.sfps.csv** ### **Supp1\_rpkm\_all.csv** | Column | Description | |
 :--------------------------------------------- |
 :--------------------------------------------------------------- | | ID |
 FBgn gene identifier | | chr | Chromosome on which each gene is located |
 | H08\_A, H08\_B, H08\_C, H20\_A, H20\_B, H20\_C | Reads Per Kilobase
 Million (RPKM) for each gene in these samples | | L08\_A, L08\_B, L17\_B,
 L17\_C | Reads Per Kilobase Million (RPKM) for each gene in these samples
 | ### **Supp2\_DEgene.summary.csv** | Column | Description | |
 :--------------------------------- |
 :---------------------------------------------------------------------------------------------------------------------------------------------- | | ID | FBgn gene identifier | | length | Total length of exons in base pairs | | start | Coordinate on chromosome where gene begins | | end | Coordinate on chromosome where gene ends | | name | Gene name | | chr | Chromosome | | gene.type | Type of gene | | logFC | Log fold change | | PValue | P-value | | FDR | P-value corrected for false discovery rate | | mE\_mRNA\_A\_MateM\_4d\_testis | Expression in testis of 4-day old adult mated males. Blank cells indicate that a given gene was not identified in this dataset. | | mE\_mRNA\_A\_MateM\_4d\_acc\_gland | Expression in accessory gland of 4-day old adult mated males. Blank cells indicate that a given gene was not identified in this dataset. | | mE\_mRNA\_A\_MateM\_4d\_head | Expression in head of 4-day old adult mated males. Blank cells indicate that a given gene was not identified in this dataset. | | mE\_mRNA\_A\_MateM\_4d\_carcass | Expression in carcass of 4-day old adult mated males. Blank cells indicate that a given gene was not identified in this dataset. | | mE\_mRNA\_A\_4d\_dig\_sys | Expression in digestive system of 4-day old adult mated males. Blank cells indicate that a given gene was not identified in this dataset. | | mE\_mRNA\_A\_MateF\_4d\_ovary | Expression in ovary of 4 day old adult mated females. Blank cells indicate that a given gene was not identified in this dataset. | | mE\_mRNA\_A\_MateF\_4d\_head | Expression in head of 4 day old adult mated females. Blank cells indicate that a given gene was not identified in this dataset. | | mE\_mRNA\_P8\_CNS | Expression in central nervous system of stage 8 pupae. Blank cells indicate that a given gene was not identified in this dataset. | | mE\_mRNA\_L3\_CNS | Expression in central nervous system of 3rd instar wandering larvae. Blank cells indicate that a given gene was not identified in this dataset. | | mE\_mRNA\_L3\_Wand\_carcass | Expression in carcass of 3rd instar wandering larvae. Blank cells indicate that a given gene was not identified in this dataset. | | mE\_mRNA\_L3\_Wand\_dig\_sys | Expression in digestive system of 3rd instar wandering larvae. Blank cells indicate that a given gene was not identified in this dataset. | | mE\_mRNA\_L3\_Wand\_fat | Expression in fat body of 3rd instar wandering larvae. Blank cells indicate that a given gene was not identified in this dataset. | | mE\_mRNA\_L3\_Wand\_imag\_disc | Expression in imaginal disc of 3rd instar wandering larvae. Blank cells indicate that a given gene was not identified in this dataset. | | mE\_mRNA\_L3\_Wand\_saliv | Expression in salivary gland of 3rd instar wandering larvae. Blank cells indicate that a given gene was not identified in this dataset. | | tsi | Tissue specificity parameter. Blank cells indicates tsi was not calculated. | | test.exp | Expressed in testis | | L.exp | Expressed in L samples (short sperm) | | H.exp | Expressed in H samples (long sperm) | | test.induc | Induced in testis | | test.high | Highest expression in testis | | ag.induc | Induced in accessory gland | | ag.high | Highest expression in accessory gland | | lncRNA | Classified as lncRNA | | asRNA | Classified as asRNA | | sfps | Classified as Seminal fluid protein (Sfp) | ### **rpkm.sfps.csv** | Column | Description | | :--------------------------------------------- | :--------------------------------------------------------------- | | ID | Gene FBgn number | | chr | Chromosome | | H08\_A, H08\_B, H08\_C, H20\_A, H20\_B, H20\_C | Reads Per Kilobase Million (RPKM) for each gene in these samples | | L08\_A, L08\_B, L17\_B, L17\_C | Reads Per Kilobase Million (RPKM) for each gene in these samples | ## Experiment 2: Sperm Size and Testis Length ### Datasets: * **sperm.csv** * **testis.csv** ### **sperm.csv** | Column | Description | | :----- | :---------------------- | | Male | Identifier for male | | Length | Sperm length in microns | ### **testis.csv** | Column | Description | | :----- | :----------------------- | | Male | Identifier for male | | Testis | Testis length in microns | ## Experiment 3: Sfp Knockout ### Datasets: * **SP_130x325_2.csv** * **SP_325x130.csv** * **OV.csv** * **AG_Length.csv** * **AG_phenotypes.csv** * **SP_130x325.zip** (images that correspond to ImageID in SP_130x325_2.csv) * **SP_325x130.zip** (images that correspond to ImageID in SP_325x130.csv) * **OV_26Aa1xBL7500.zip** (images that correspond to ImageID in OV.csv) * **AG_KD.zip** (images that correspond to ImageID in AG_Length) ### **SP\_130x325\_2.csv** | Column | Description | | :------------ | :-------------------------------------------------------------- | | ImageID | Identifier for image file (some images measured multiple times) | | Length | Sperm length in microns | | Magnification | Objective used on microscope when image captured | | Scale | Scale used to convert pixels to microns | | Name | Name of measurer | | Date | Date image measured | ### **SP\_325x130.csv** | Column | Description | | :------------ | :----------------------------------------------- | | ImageID | Identifier for image file | | Length | Sperm length in microns | | Magnification | Objective used on microscope when image captured | | Scale | Scale used to convert pixels to microns | | Name | Name of measurer | | Date | Date image measured | ### **OV.csv** | Column | Description | | :------------ | :----------------------------------------------- | | ImageID | Identifier for image file | | Length | Sperm length in microns | | Magnification | Objective used on microscope when image captured | | Scale | Scale used to convert pixels to microns | | Name | Name of measurer | | Date | Date image measured | ### **AG\_Length.csv** | Column | Description | | :------------ | :----------------------------------------------- | | ImageID | Identifier for image file | | Length | Sperm length in microns | | Magnification | Objective used on microscope when image captured | | Scale | Scale used to convert pixels to microns | | Name | Name of measurer | | Date | Date image measured | ### **AG\_phenotypes.csv** | Column | Description | | :------- | :------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ | | Rep | Replicate vial identifier | | Male | Male identifier for a given vial replicate | | Date | Date male dissected | | Wing | Phenotype of wing, Cy (curly) or WT (wild type) | | Bristle | Phenotype of bristles, Sb (stubby) or WT (wild type) | | Eye | Phenotype of eye color, w (white), or (orange), red (red) | | Genotype | Genotype inferred from other phenotypes | | AG | Accessory gland size, scored from 0 (shrunken, empty, nonfunctional) to 4 (normal-sized, full). (Note there is no value of 1; in the course of analysis, scores for 2, 3, 4 are replaced with 1, 2, 3.) | | Notes | Experimenter notes | ## Experiment 4: Female Post-Mating Response ### Datasets: * **PMR_P2.csv** * **PMR_Prior.csv** * **PMR_Remating.csv** * **PMRLength.csv** * **M3.csv** * **PMR.zip** (images that correspond to ImageID in PMRLength.csv) ### **PMR\_P2.csv** | Column | Description | | :----- | :--------------------------------------------------------------------------------------- | | FemID | Female identifier consisting of last two digits of male isoline identifier, period, 1-60 | | Line | DSPR identification code for male isoline | | Sperm | Sperm length (long or short) | | Male2 | Number of sons sired by second male | | Male1 | Number of sons sired by first male | ### **PMR\_Prior.csv** | Column | Description | | :----------- | :--------------------------------------------------------------------------------------- | | FemID | Female identifier consisting of last two digits of male isoline identifier, period, 1-60 | | Line | DSPR identification code for male isoline | | Sperm | Sperm length (long or short) | | DaysToRemate | Number of days to remote | | PriorProg | Number of prior progeny | ### **PMR\_Remating.csv** | Column | Description | | :--------- | :---------------------------------------- | | Line | DSPR identification code for male isoline | | Sperm | Sperm length (long or short) | | Day | Day remated | | PercRemate | Cumulative percent remated | ### **PMRLength.csv** | Column | Description | | :------ | :---------------------------------------- | | ImageID | Identifier for image file | | Line | DSPR identification code for male isoline | | Sperm | Sperm length (long or short) | | Male | Identifier for male | | Length | Sperm length in microns | ### **M3.csv** | Column | Description | | :------ | :------------------------ | | ImageID | Identifier for image file | | Length | Sperm length in microns |