DOI: 10.5061/dryad.5qfttdzj6
title: Data from: A novel sperm-derived seminal fluid protein in Caenorhabditis nematodes

description: Nematode sperm contain subcellular vesicles known as membranous organelles
 (MOs) that fuse with the sperm cell membrane upon sperm activation to
 release their soluble contents into the extracellular space. The second
 most abundant proteins in the MOs belong to the conserved
 Nematode-Specific Peptide family, group F (NSPF) gene family. We
 hypothesize that these proteins contribute to seminal fluid and are part
 of post-insemination reproductive tract dynamics. We characterized the
 anatomical region where the NSPF proteins likely function during
 fertilization using dissected testes and whole-worm immunostaining of a
 His-tagged nspf-1 transgene. We confirmed that NSPF proteins are
 transferred to females during mating. NSPF proteins localize to the uterus
 lumen when transferred to mated females and in unmated adult
 hermaphrodites. These results suggest that the uterine localization of the
 NSPF proteins is likely a functional property of both male-derived sperm
 and self-sperm and not incidental to the point of transfer during mating.
 In males, we confirm that NSPF proteins are indeed sperm derived. We then
 used experimental evolution to compete the wildtype allele against a
 deletion allele in 10 replicate obligate-outcrossing populations. We
 calculated a mean selective disadvantage of 0.1% for the deletion allele,
 which indicated that the NSPF genes are beneficial to male fitness. This
 conclusion was reinforced by qualitative trends from lower-powered
 single-generation fertility assays. Together we demonstrate that nematodes
 use a novel approach for contributing proteins to seminal fluid and show
 that the highly abundant NSPF proteins likely have a beneficial impact on
 fitness.
description: # A novel sperm-derived seminal fluid protein in Caenorhabditis nematodes
 Dataset DOI: [10.5061/dryad.5qfttdzj6](10.5061/dryad.5qfttdzj6) ##
 Description of the data and file structure The oligonucleotides used in
 this study are available as Table S1. The immunostaining data are
 available in File S2 and the complete protocol is available in File S1.
  The fertility data are available in File S3. The raw amplicon data are
 available at NCBI SRA under accession number PRJNA1009866. The processed
 amplicon reads are available in File S4. The scripts used to analyze the
 amplicon data and immunostaining data are provided here as well as via the
 Cutter Lab GitHub repository NSPF
 ([https://github.com/Cutterlab/NSPF](https://github.com/Cutterlab/NSPF)).
 ### Files and variables #### File: analyze\_fecundity\_staining.R
 **Description:** This R script analyzes the fecundity data in File S3 and
 the immunostaining data in File S2. #### File: amplicon\_processing.sh
 **Description:** This script processes the raw amplicon sequence data and
 BLASTs each read to the wild type and deletion alleles. #### File:
 Analyze\_Amplicon\_BestBlast.R **Description:** This R script selects the
 best BLAST allele for each read (from the processed data) and analyzes the
 amplicon sequence data. #### File: FileS1\_SupplementaryMethods.docx
 **Description:** Complete immunostaining protocols and buffer recipes.
 #### File: FileS2\_IHSdata.txt **Description:** Immunostaining data across
 developmental stages and sexes. Each line represents an individual worm
 scored for fluorescence (1 = present; 0 = absent). ##### Variables * Worm:
 Given as strain*-*stage-sex * Head: pharynx region of the worms; present
 in all stages * Upper gonad: distal gonad; meitoic region of the gonad;
 present in all stages but most prominent in L4 and older * Mid
 gonad: proximal gonad; in hermaphrodites/females this is after the turn to
 the spermathecae; in males this is after the turn to the seminal vesicle;
 present in L4 and older * lower gonad: uterus in hermaphrodites/females;
 vas deferens in males; present in L4 and older * Vulva/cloaca:  mating
 "opening"; present in all stages * Tail: in males this includes
 the fan and rays * None: no staining seen * Eggs: presence or absence of
 eggs #### File: FileS3\_FecundityData.txt **Description:** Late life
 fecundity data ##### Variables * male: strain for the male used in the
 mating * D5: fecundity on day 5 of adulthood * D6: fecundity on day 6 of
 adulthood * D7: fecundity on day 7 of adulthood * D8: fecundity on day 8
 of adulthood * D9: fecundity on day 9 of adulthood * D10: fecundity on day
 10 of adulthood * D11: fecundity on day 11 of adulthood * D12: fecundity
 on day 12 of adulthood #### File: FileS4\_BestBlast.txt.zip
 **Description:** Processed amplicon sequencing reads. ##### Variables *
 ID: sequence ID * Amplicon: Best BLAST allele (WT or DEL) * Identity:
 Percent identity to called allele * Alignment.length: Number of aligned
 base pairs * Mismatches: Number of base pair mismatches * gap.opens *
 q.start: Query start * q.end: Query end * s.start: sequence start * s.end:
 sequence end * evalue * bit.score * replicate:
 generation.expevolrep.seqrep * replicate2: generation.expevol.rep ####
 File: TableS1\_Primers.csv **Description:**  Primers used in this study.
 ##### Variables * primer name * sequence 5' to 3' * purpose ####
 File: TableS2\_Strains.csv **Description:** Strains used in this study.
 ##### Variables * Strain Name: strain * Primary Strain Background: genetic
 background for transgenics * Genotype: full genotype *
 Backcrossing/Inbreeding: number of generations backcrossed or inbred *
 Method: method by which the strain was generated * Source: strain source 
 #### File: TableS3\_ModelComparison.csv **Description:** Linear model
 comparisons for amplicon sequencing data. All six models fit are given
 along with their summaries (from R). #### File: nspf001-deconv.nd2
 **Description:** Deconvoluted image of stained, dissected gonads. To be
 analyzed along with file nspf001.nd2 to create Figure 1. #### File:
 nspf001.nd2 **Description:** Confocal image of stained, dissected gonads.
 To be analyzed along with file nspf001-deconv.nd2 to create Figure 1. ####
 File: nspf005-deconv.nd2 **Description:** Deconvoluted image of stained,
 dissected gonads. To be analyzed along with file nspf005.nd2 to create
 Figure 1. #### File: nspf005.nd2 **Description:** Confocal image of
 stained, dissected gonads. To be analyzed along with
 file nspf005-deconv.nd2 to create Figure 1. #### File: nspf006-deconv.nd2
 **Description:** Deconvoluted image of stained, dissected gonads. To be
 analyzed along with file nspf006.nd2 to create Figure 1. #### File:
 nspf006.nd2 **Description:** Confocal image of stained, dissected gonads.
 To be analyzed along with file nspf006-deconv.nd2 to create Figure 1. ##
 Code/software A bash script for processing the raw amplicon sequencing
 data is provided along with R scripts to analyze innumostaining,
 fecundity, and sequencing data. Open source software needed are R and
 ImageJ. ## Access information Other publicly accessible locations of the
 data: *
 [https://github.com/Cutterlab/NSPF](https://github.com/Cutterlab/NSPF)
 Data was derived from the following sources: * Kasimatis, K.R.,
 Moerdyk-Schauwecker, M.J., Timmermeyer, N. & Phillips, P.C. 2018.
 Proteomic and evolutionary analyses of sperm activation identify
 uncharacterized genes in *Caenorhabditis* nematodes. *BMC Genomics*
 **19**: 593.