Project PXD004005

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Summary

Title

Molecular composition of male nuptial gifts in fireflies: shedding light on postcopulatory sexual selection

Description

Postcopulatory sexual selection is now widely recognized as a key driver of reproductive trait evolution, including the machinery required to produce endogenous nuptial gifts. Despite the importance of such gifts, molecular composition of the non-gametic components of male ejaculates and their interactions with female reproductive tracts remain poorly understood. .  Here we combined transcriptomic studies of both male and female reproductive glands with proteomic and metabolomic approaches to better understand the synthesis, composition and fate of this spermatophore gift in the common Eastern firefly, Photinus pyralis.

Sample Processing Protocol

One hour after the initiation of stage II copulation (Lewis & Wang, 1991), a mating pair of Photinus pyralis fireflies was separated, and the spermatophore was carefully dissected out from the female’s reproductive tract. Upon removal from storage, the spermatophore was transferred into 50 µL of 2x Laemmli Sample Buffer (Bio-Rad) with 2% β-mercaptoethanol, and heated to 95˚C for 5 min. Sample (25 µL) was loaded onto a 12% percent discontinuous Laemelli SDS-PAGE gel. BLUEstain™ Protein ladder (Gold Biotechnology) was loaded in a neighboring well for inferring protein size. Eight sections containing proteins ranging from >180 kDa to ~6 kDa were cut from the gel, and provided to the Whitehead Institute Proteomics Core Facility (Cambridge, MA). Thereafter the samples were digested with trypsin, and run individually on a Dionex Ultimate 3000 RSLCnano nanoflow LC coupled to a ThermoFisher Scientific Orbitrap Elite mass spectrometer.

Data Processing Protocol

In silico translated ORFs from the Trinity de novo transcriptome were used as a search database to identify tryptic peptides from the samples. Mascot (Matrix Science, London, UK; version 2.5.1) was used as the proteomic search engine. Verification of peptide and protein identification and general analysis was performed in Scaffold (Supplementary methods 1; Scaffold version 4.4.8, Proteome Software Inc., Portland, OR).

Contact

Tim Fallon, MIT
Jing-Ke Weng, Whitehead Institute for Biomedical Research ( lab head )

Submission Date

18/07/2016

Publication Date

06/12/2016

Cell Type

spermatozoon

Disease

disease free

Modification

Oxidation
Carbamidomethyl

Quantification

Spectrum counting

Experiment Type

Gel-based experiment

Assay count

8

Publication

Publication pending

Assay

Showing 1 - 8 of 8 results
# Accession Title Proteins Peptides Unique Peptides Spectra Identified Spectra View in Reactome
1 66000 no assay title provided (mzIdentML) 362 4071 1190 3210 3210
2 65994 no assay title provided (mzIdentML) 480 3873 1227 2835 2835
3 65993 no assay title provided (mzIdentML) 447 4337 1635 3421 3421
4 65996 no assay title provided (mzIdentML) 410 3510 1256 2778 2778
5 65995 no assay title provided (mzIdentML) 330 3375 1187 2717 2717
6 65998 no assay title provided (mzIdentML) 531 3506 1486 2776 2776
7 65997 no assay title provided (mzIdentML) 604 3863 1512 2844 2844
8 65999 no assay title provided (mzIdentML) 463 2874 1104 2105 2105