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MTBLS362:  Molecular characterization of firefly nuptial gifts: a multi-omics approach sheds light on postcopulatory sexual selection

 Authors: Timothy Fallon

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  Submitted: 13-Jul-2016 , Release date: 22-Dec-2016 , Update date: 11-Oct-2017

 Submitted by:  Timothy R Fallon  |   Study status: Public

Study Description

Postcopulatory sexual selection is recognized as a key driver of reproductive trait evolution, including the machinery required to produce endogenous nuptial gifts. Despite the importance of such gifts, the molecular composition of the non-gametic components of male ejaculates and their interactions with female reproductive tracts remain poorly understood. During mating, male Photinus fireflies transfer to females a spermatophore gift manufactured by multiple reproductive glands. Here we combined transcriptomics of both male and female reproductive glands with proteomics and metabolomics to better understand the synthesis, composition and fate of the spermatophore in the common Eastern firefly, Photinus pyralis. Our transcriptome of male glands revealed up-regulation of proteases that may enhance male fertilization success and activate female immune response. Using bottom-up proteomics we identified 208 functionally annotated proteins that males transfer to the female in their spermatophore. Targeted metabolomic analysis also provided the first evidence that Photinus nuptial gifts contain lucibufagin, a firefly defensive toxin. The reproductive tracts of female fireflies showed increased gene expression for several proteases that may be involved in egg production. This study offers new insights into the molecular composition of male spermatophores, and extends our understanding of how nuptial gifts may mediate postcopulatory interactions between the sexes.

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  Organism(s)

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Photinus pyralis

  Study Design Description

targeted metabolites

CHMO:liquid chromatography-mass spectrometry

Protocol Description
Sample collection Photinus pyralis fireflies used in this study were collected at Mercer Meadows Pole Farm, Lawrenceville, NJ (40°18’23.4” N, 74°44’53.9”W) on 27 June and 11-12 July 2015, and identified based on male genitalia [1] and flash patterns. Both sexes were kept individually in plastic containers with sliced apple and damp paper towel. Mating status of field-collected individuals was unknown. Fireflies were kept in the lab for less than one week prior to experimentation.

Ref:
[1] Green, J. W. Revision of the Nearctic species of Photinus (Lampyridae: Coleoptera). Proc Calif Acad Sci 28, 561–613 (1956).
Extraction A single spermatophore dissected from a male Photinus pyralis was placed in 100 µl of 50% methanol. A single adult male Photinus pyralis was flash frozen in liquid nitrogen, and the posterior 2 abdominal segments (containing lantern & genetalia) were removed with a razor blade at 4 °C. The remaining anterior portion of the firefly “body” was placed in 150 µl 50% MeOH. Both tissues were macerated in the solvent, and intermittently sonicated in a water bath sonicator for 30 min, not letting the temperature rise above 40 °C. Post sonication, the extract was centrifuged in a benchtop centrifuge at 14,000 g @ 4°C for 10 min to pellet tissue debris and other particulates. The clarified extract was filtered through a 0.2 µm PFTE filter (Filter Vial, P/No. 15530-100, Thomson Instrument Company).
Chromatography 20 µl of the filtered extracts were separated on a UltiMate 3000 (Dionex) liquid chromatography system by reversed-phase chromatography on a 150 mm C18 Column (Kinetex 2.6 µm silica core shell C18 100 Å pore, P/No. 00F-4462-Y0, Phenomenex) by a gradient of Solvent A (0.1% formic acid in H2O) and Solvent B (0.1% formic acid in acetonitrile); 5% B for 2 min, 5-80% B over 40 min, 95% B for 4 min, and 5% B for 5 min; flow rate 0.8 ml/min. The flow from this chromatography was coupled to a Q-Exactive (Thermo-Scientific) mass spectrometer.
Mass spectrometry The Q-Exactive mass spectrometer was configured to perform 1 MS1 scan from m/z 120-1250 followed by 1-3 data-dependent MS2 scans using HCD fragmentation with a stepped collision energy of 10, 15, 25 normalized collision energy (NCE). Data was collected as profile data. The instrument was always used within 7 days of the last mass accuracy calibration. The ion source parameters were as follows: spray voltage (+) at 3000 V, spray voltage (-) at 2000 V, capillary temperature at 275 °C, sheath gas at 40 arb units, aux gas at 15 arb units, spare gas at 1 arb unit, max spray current at 100 (µA), probe heater temp at 350 °C, ion source: HESI-II.
Data transformation The raw profile data in Thermo format was converted to mzML format using ProteoWizard MSConvert [1] with 64-bit binary encoding precision, index writing, gzip compression of the whole file, zlib compression of peaklist data , and numpress linear compression of peaklist data. Data analysis was performed with MZmine2 2.19 [2] and Xcalibur 2.2 SP1.48 (Thermo Scientific).

Ref:
[1] Chambers, M.C., Maclean, B., Burke, R., Amodei, D., Ruderman, D.L., Neumann, S., Gatto, L., Fischer, B., Pratt, B., Egertson, J., et al. (2012). A cross-platform toolkit for mass spectrometry and proteomics. Nat Biotech 30, 918–920. doi: 10.1038/nbt.2377. PMID:23051804
[2] Pluskal, T., Castillo, S., Villar-Briones, A., and Oresic, M. (2010). MZmine 2: modular framework for processing, visualizing, and analyzing mass spectrometry-based molecular profile data. BMC Bioinformatics 11, 395. doi:10.1186/1471-2105-11-395. PMID:20650010
Metabolite identification 1. Thermo .raw data was converted to .mzML by ProteoWizard MSConvert with the parameters specified above.
2. Raw data was imported from .mzML format files.
3. MS1 and MS2 profile mass spectra were detected/centroided with the “Mass Detector” module. The following parameters were used:
- Mass Detector: Exact mass
- Noise level: 1E4
4. Continuous MS1 ions were assembled into mass traces with the “Chromatogram builder” module. The following parameters were used:
- Minimum time span (min): 0.1 the
- Minimum height: 1.0E4, and the
- m/z tolerance: 0.002 or 5ppm (whichever is greater)
5. Chromatograms were then deconvolved into individual peaks, using the “Chromatogram deconvolution” module, with the algorithm set to “Local minimum search” and following parameters:
- Chromatographic threshold: 1.0%
- Search minimum in RT range (min): 0.1
- Minimum relative height: 0.5%
- Minimum absolute height: 1.0E3
- Min ratio peak top/edge: 4
- Peak duration range (min): 0.0 – 5.0
6. The MS1 mass spectra were then deisotoped with the “Isotopic peak grouper” module. The following parameters were used:
- m/z tolerance: 0.01025 Da or 20ppm (whichever is greater)
- Retention time tolerance: 0.2 (absolute (min))
- Monotonic shape: unchecked
- Maximum charge: 2
- Representative isotope: Most intense
7. Mass features were aligned with a correction for retention time deviation between the Photinus pyralis body and spermatophore extract, using the “RANSAC aligner” module with the following parameters:
- m/z tolerance: 8.0E-4 or 4ppm (whichever is greater)
- RT tolerance: 2.0%
- RT tolerance after correction: 0.25 ( absolute (min) )
- RANSAC iterations: 1000000
- Minimum number of points: 30.0%
- Threshold value: 0.08 ( absolute (min) )
- Linear model: unchecked
- Require same charge state: unchecked
8. Putative in-source fragments were identified by the “Fragment search” module with the following parameters:
- Retention time tolerance: 0.3 (absolute (min) )
- m/z tolerance of MS2 data: 0.001 or 5ppm (whichever is greater)
- Max fragment peak height: 50%
- Min MS2 peak height: 1.0E5 9. Putative adduct ions were identified by the “Adduct search” module with the following parameters:
- Retention time tolerance: 0.3 (absolute (min) )
- m/z tolerance of MS2 data: 0.0005 or 2ppm
- Max relative adduct peak height: 20%
- The following default MZmine2 adducts were selected:
o [M+Na-H]
o [M+K-H]
o [M+NH3]
10. Putative complexed ions were detected by the “Complex search” module with the following parameters:
- Retention time tolerance: 0.3 (absolute (min) )
- m/z tolerance of MS2 data: 0.001 or 5ppm (whichever is greater)
- Max complex peak height: 80.0%
11. The resulting peaklist was exported as “spermatophore_vs_body_final_unfiltered.mzTab”
12. The peaklist was filtered to remove putative complexes, adducts, and fragments, using the “peaklist rows filter” module, with the custom parameters:
- Text in identity: “Adduct” or “Complex” or “Fragment”
- Keep or remove rows: “Remove rows that match all criteria”
This step used a custom fork of MZmine2 2.1.9 (https://github.com/photocyte/mzmine2/commit/609bb3b1811b3df4ff632100591a2e449564eaf2). The change has been contributed to the main MZmine2 sourcecode repository.
13. The resulting peaklist without annotated adducts, complexes, and in-source fragment ions was exported as “spermatophore_vs_body_final_filtered.mzTab”
14. Resulting compounds were manually annotated using a combination of the “Online database search” module & “Formula prediction” module. When possible, MS2 spectra were compared with the Metlin metabolite database (metlin.scripps.edu)
Source Name Organism Organism part Protocol REF Sample Name
spermatophore Photinus pyralis spermatophore/nuptial gift Sample collection Ppyr_Spermatophore_1
body Photinus pyralis Body - 2 posterior lantern segments excised Sample collection Ppyr_UM_TRF0001_body
pre_spermatophore_blank_injection blank blank Sample collection pre_spermatophore_blank
pre_body_blank_injection blank blank Sample collection pre_body_blank
Validations marked with (*) are specially approved by the MetaboLights Curators.
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Condition Status Description Requirement Group Message
PASSES Study Title MANDATORY STUDY OK
PASSES Study Description MANDATORY STUDY OK
PASSES Study text successfully parsed OPTIONAL STUDY OK
PASSES Study Contact(s) have listed email MANDATORY CONTACT OK
PASSES Sample(s) MANDATORY SAMPLES OK
PASSES Sample Name consistency check MANDATORY ASSAYS OK
PASSES Publication(s) associated with this Study MANDATORY PUBLICATION OK
PASSES Minimal Experimental protocol MANDATORY PROTOCOLS OK
PASSES Comprehensive Experimental protocol OPTIONAL PROTOCOLS OK
PASSES Sample Collection protocol MANDATORY PROTOCOLS OK
PASSES Protocols text successfully parsed OPTIONAL PROTOCOLS OK
PASSES Organism name MANDATORY ORGANISM OK
PASSES Organism part MANDATORY ORGANISM OK
* PASSES Study Factors MANDATORY FACTORS No Study Factor information is provided
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PASSES Assay has raw files referenced MANDATORY FILES OK
PASSES Assay referenced raw files detection in filesystem MANDATORY FILES OK
PASSES Raw files in the Assay(s) have the correct format MANDATORY FILES OK
PASSES Assay(s) MANDATORY ASSAYS OK
PASSES All Assays have Metabolite Assignment File (MAF) referenced OPTIONAL FILES OK
PASSES Metabolite Assignment File (MAF) is present in Study folder MANDATORY FILES OK
PASSES Metabolite Assignment File (MAF) has correct format MANDATORY FILES OK
PASSES Metabolite Identification File (MAF) content MANDATORY FILES OK
PASSES ISA-Tab investigation file check MANDATORY ISATAB OK

Assay 

Assay file name: a_photinus_pyralis_spermatophore_comparative_lc-esi-hram-ms_metabolite_profiling_mass_spectrometry.txt
Measurement: metabolite profiling
Technology: mass spectrometry
Platform: Exactive (Thermo Scientific)

Data

Sample Name Protocol REF Post Extraction Derivatization Extract Name Protocol REF Chromatography Instrument Column model Column type Labeled Extract Name Label Protocol REF Scan polarity Scan m/z range Instrument Ion source Mass analyzer MS Assay Name Protocol REF Raw Spectral Data File Normalization Name Derived Spectral Data File Protocol REF Data Transformation Name Metabolite Assignment File
pre_spermatophore_blank Extraction Positive ion mode DDMS2 run none 50% MeOH blank Chromatography Thermo Scientific Dionex UltiMate 3000 System Kinetex C18 100 Å (2.6 µm, 2.1 mm x 150 mm; Phenomenex) reverse phase Mass spectrometry positive 120-1250 Thermo Scientific Q Exactive electrospray ionization orbitrap preblank Data transformation preblank.mzML.gz Metabolite identification m_photinus_pyralis_spermatophore_comparative_lc-esi-hram-ms_metabolite_profiling_mass_spectrometry_v2_maf.tsv
pre_spermatophore_blank Extraction Negative ion mode DDMS2 run none 50% MeOH blank Chromatography Thermo Scientific Dionex UltiMate 3000 System Kinetex C18 100 Å (2.6 µm, 2.1 mm x 150 mm; Phenomenex) reverse phase Mass spectrometry negative 120-1250 Thermo Scientific Q Exactive electrospray ionization orbitrap preblank_2_neg Data transformation preblank_2_neg.mzML.gz Metabolite identification m_photinus_pyralis_spermatophore_comparative_lc-esi-hram-ms_metabolite_profiling_mass_spectrometry_v2_maf.tsv
Ppyr_Spermatophore_1 Extraction Positive ion mode DDMS2 run none Spermatophore extract Chromatography Thermo Scientific Dionex UltiMate 3000 System Kinetex C18 100 Å (2.6 µm, 2.1 mm x 150 mm; Phenomenex) reverse phase Mass spectrometry positive 120-1250 Thermo Scientific Q Exactive electrospray ionization orbitrap Ppyralis_spermatophore_pos_20uL Data transformation Ppyralis_spermatophore_pos_20uL.mzML.gz Metabolite identification m_photinus_pyralis_spermatophore_comparative_lc-esi-hram-ms_metabolite_profiling_mass_spectrometry_v2_maf.tsv
Ppyr_Spermatophore_1 Extraction Negative ion mode DDMS2 run none Spermatophore extract Chromatography Thermo Scientific Dionex UltiMate 3000 System Kinetex C18 100 Å (2.6 µm, 2.1 mm x 150 mm; Phenomenex) reverse phase Mass spectrometry negative 120-1250 Thermo Scientific Q Exactive electrospray ionization orbitrap Ppyralis_spermatophore_neg_20uL Data transformation Ppyralis_spermatophore_neg_20uL.mzML.gz Metabolite identification m_photinus_pyralis_spermatophore_comparative_lc-esi-hram-ms_metabolite_profiling_mass_spectrometry_v2_maf.tsv
pre_body_blank Extraction Positive/negative ion mode DDMS2 run none 'Body' extract Chromatography Thermo Scientific Dionex UltiMate 3000 System Kinetex C18 100 Å (2.6 µm, 2.1 mm x 150 mm; Phenomenex) reverse phase Mass spectrometry alternating 120-1250 Thermo Scientific Q Exactive electrospray ionization orbitrap 50MeOH_blank_160202004253 Data transformation 50MeOH_blank_160202004253.mzML.gz Metabolite identification m_photinus_pyralis_spermatophore_comparative_lc-esi-hram-ms_metabolite_profiling_mass_spectrometry_v2_maf.tsv
Ppyr_UM_TRF0001_body Extraction Positive/negative ion mode DDMS2 run none 50% MeOH blank Chromatography Thermo Scientific Dionex UltiMate 3000 System Kinetex C18 100 Å (2.6 µm, 2.1 mm x 150 mm; Phenomenex) reverse phase Mass spectrometry alternating 120-1250 Thermo Scientific Q Exactive electrospray ionization orbitrap Ppyr_1-body_20uL Data transformation Ppyr_1-body_20uL.mzML.gz Metabolite identification m_photinus_pyralis_spermatophore_comparative_lc-esi-hram-ms_metabolite_profiling_mass_spectrometry_v2_maf.tsv

Pathways - Assay 



MetExplore Pathways Mapping

Name DB Identifier Mapped Metabolite(s)
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Aspera Download Details:

List of study files   Subset

File
audit
a_photinus_pyralis_spermatophore_comparative_lc-esi-hram-ms_metabolite_profiling_mass_spectrometry.txt
i_Investigation.txt
s_Photinus pyralis spermatophore comparative LC-ESI-HRAM-MS.txt
preblank.mzML.gz
Ppyr_1-body_20uL.mzML.gz
Ppyralis_spermatophore_neg_20uL.mzML.gz
preblank_2_neg.mzML.gz
Ppyralis_spermatophore_pos_20uL.mzML.gz
50MeOH_blank_160202004253.mzML.gz
metexplore_mapping.json
spermatophore_vs_body_final_unfiltered.mzTab
spermatophore_vs_body_final_filtered.mzTab
m_photinus_pyralis_spermatophore_comparative_lc-esi-hram-ms_metabolite_profiling_mass_spectrometry_v2_maf.tsv
spermatophore_vs_body_final_filtered.tsv
spermatophore_vs_body_final_unfiltered.tsv

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