Research ArticleEVOLUTIONARY BIOLOGY

Increased proteomic complexity in Drosophila hybrids during development

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Science Advances  07 Feb 2018:
Vol. 4, no. 2, eaao3424
DOI: 10.1126/sciadv.aao3424
  • Fig. 1 The proteome in developing hybrid embryos is more complex than the proteome of both species combined.

    Peptides were grouped according to their presence in either the hybrid or internal standard sample. (A) The proteome of developing hybrid embryos (yellow) from a cross of D. mel. ♀ with D. sim. ♂ was analyzed with bottom-up proteomics and compared to an internal standard that is a 1:1 mixture of the respective D. melanogaster (blue) and D. simulans (green) embryos. The relative percentage and standard deviation (SD) of peptides (experimental triplicates) are indicated below the nonproportional Venn diagrams, and the larger, sample-specific fraction of the proteome in either sample is highlighted (bold). Handles within each Venn diagram indicate pairs of two species-specific orthologs that were detected in both or hybrids or internal standard. The relative contribution (percentage) to all pairs of orthologs identified in a proteome that comprises all biological replicates is depicted next to the handle. The largest contribution is highlighted in bold. (B) In addition, the head-specific proteome of adult hybrid female flies of the same cross (D. mel. ♀ × D. sim. ♂) was compared to the corresponding internal standard. (C) Hybrid embryos of the reverse cross (D. sim. ♀ × D. mel. ♂) are not viable (white). The proteome of these nondeveloping hybrid embryos was compared to the same internal standard as in (A).

  • Fig. 2 Protein levels in hybrids differ between the proteomes that are specific to the genome of the father and mother.

    (A) Peptide nodes were classified according to species specificity in D. melanogaster (Dm) or D. simulans (Ds) or indistinguishable (DmDs) and the distribution of peptide node ratios [isobaric isotopolog ratio (Ri)] of developing embryos (D. mel. ♀ × D. sim. ♂ cross) versus the internal standard plotted. Individual points in the graph (left) indicate the relative frequency of peptide nodes per ratio bin, which is the fold change in peptide abundance in hybrids in comparison to the internal standard. The best fit of a Gaussian (line) for each subset of peptide nodes is included. The distribution of all measurements is shown in a box-and-whisker graph (middle). The statistical significance between D. melanogaster– and D. simulans–specific peptide nodes was determined with unpaired t test with Welch’s correction for unequal variances, assuming that groups are independent (****P < 0.0001). A scatterplot (right) of ratio versus spectrum weight highlights peptide nodes (large dots) that were significantly regulated according to an FDR ≤ 0.05. The spectrum weight (y axis) is inversely proportional to the variance of measurement of each peptide node. All three plots are repeated for the proteome in heads of adult hybrid flies of the D. mel. ♀ × D. sim. ♂ cross (B) and in nondeveloping embryos of the D. sim. ♀ × D. mel. ♂ cross (C).

  • Fig. 3 HP1a in hybrid embryos and adult flies shows distinct, species-specific protein levels.

    All peptide nodes (rectangles) that are part of specific HP1a proteoforms and orthologous proteins (ovals) are connected by edges that show the relationship between peptide nodes and protein nodes in a bipartite protein-peptide network. Different HP1a proteoforms and orthologs are indicated by their respective UniProt identifiers in developing embryos of the D. mel. ♀ × D. sim. ♂ cross (A), nondeveloping embryos of the D. sim. ♀ × D. mel. ♂ cross (B), and in adult fly heads of the D. mel. ♀ × D. sim. ♂ cross (C). Colors indicate either species specificity (light blue and green) or relative abundance wherein a peptide node is present in both samples (hybrids and internal standard) and measured with a relative ratio (white) or present in either hybrids or internal standard (red and dark blue, respectively) or was identified but not quantified (gray). The log2-transformed value of the fold change (Rc value) per peptide node or its presence in hybrids only (Infinity; red rectangles) or the internal standard (−Infinity; dark blue rectangles) is indicated. Peptides subsumed in peptide nodes with the value “N/A” were identified, but the relative abundance of the peptide node was not quantified because the number of ratio measurements was too low (<3 independent measurements). The asterisk indicates a ±Infinity value (Rc-based data analysis). An outline of a peptide node in red as well as an edge in red indicates that the peptide node (>4-fold or highest difference value) deviates from the two additional peptide nodes in a pair of two proteins with unique peptide nodes. The pair of peptide nodes is indicated by a classification number attributed to the edge, and the classification number “3” indicates that unique peptide nodes are different (>4-fold or highest difference value) from the remaining peptide nodes in the protein pair.

  • Fig. 4 D. simulans–specific orthologs of the proteostasis network are increased in developing hybrid embryos.

    The graph displays presence and relative expression levels of orthologs and groups these according to their contributions to a protein’s life cycle in specific cell biological pathways. Pathways that are part of the proteostasis network are printed in bold. Species specificity is indicated by color (blue, D. melanogaster; green, D. simulans; black, no species specificity), and relative abundance is indicated by the color fill of the circle (Rc-values, light circle, >2-fold down; bold circle, 2-fold down ≤ R ≤ 2-fold up; filled circle, >2-fold up).

  • Fig. 5 The hybrid proteome is increased in complexity during early embryonic development and decreased in adult hybrid flies.

    Hybrid embryos undergo early development with an increased proteome complexity following a cross of D. melanogaster with D. simulans. Proteome complexity returns to a minimal proteome in adult hybrid flies that is closer to the proteomes of each parental species.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/2/eaao3424/DC1

    table S1. Significantly regulated peptide nodes (FDR < 0.05) and associated proteins in D. mel. ♀ × D. sim. ♂ embryos, D. sim. ♀ × D. mel. ♂ embryos, and D. mel. ♀ × D. sim. ♂ heads.

    table S2. PSEA-Quant results obtained for D. mel. ♀ × D. sim. ♂ embryos, D. sim. ♀ × D. mel. ♂ embryos, and heads of the D. mel. ♀ × D. sim. ♂ cross are presented in individual tabs as indicated in the Excel file.

    network S1. D. mel. ♀ × D. sim. ♂ embryos; >2 isobaric peptide counts per peptide node.

    network S2. D. sim. ♀ × D. mel. ♂ embryos; >2 isobaric peptide counts per peptide node.

    network S3. D. mel. ♀ × D. sim. ♂ heads; >2 isobaric peptide counts per peptide node.

    network S4. D. mel. ♀ × D. sim. ♂ embryos; FDR < 0.01 and Ri > 0.0.

    network S5. D. mel. ♀ × D. sim. ♂ embryos; FDR < 0.01 and Ri < 0.0.

    network S6. D. sim. ♀ × D. mel. ♂ embryos; FDR < 0.01 and Ri > 0.0.

    network S7. D. sim. ♀ × D. mel. ♂ embryos; FDR < 0.01 and Ri < 0.0.

    network S8. D. mel. ♀ × D. sim. ♂ heads; FDR < 0.01 and Ri > 0.0.

    network S9. D. mel. ♀ × D. sim. ♂ heads; FDR < 0.01 and Ri < 0.0.

    network S10. D. mel. ♀ × D. sim. ♂ embryos; proteasomal proteins.

    network S11. D. sim. ♀ × D. mel. ♂ embryos; proteasomal proteins.

    network S12. D. mel. ♀ × D. sim. ♂ hybrid heads; proteasomal proteins.

    Data repositories

    fig. S1. Only hybrids of D. melanogaster females crossed with D. simulans males are viable.

    fig. S2. Relative abundance of peptide nodes according to species specificity and sample origin.

    fig. S3. The Venn diagram displays the relative overlap of peptides retrieved for the D. melanogaster and D. simulans proteome databases when digested in silico with the endoprotease LysC.

    fig. S4. Venn diagram of proteomes identified in each of the replicate experiments.

    fig. S5. Sample analysis workflow and detection of species-specific peptides.

  • Supplementary Materials

    This PDF file includes:

    • Legends for tables S1 and S2
    • network S1. D. mel. ♀ × D. sim. ♂ embryos; >2 isobaric peptide counts per peptide node.
    • network S2. D. sim. ♀ × D. mel. ♂ embryos; >2 isobaric peptide counts per peptide node.
    • network S3. D. mel. ♀ × D. sim. ♂ heads; >2 isobaric peptide counts per peptide node.
    • network S4. D. mel. ♀ × D. sim. ♂ embryos; FDR < 0.01 and Ri > 0.0.
    • network S5. D. mel. ♀ × D. sim. ♂ embryos; FDR < 0.01 and Ri < 0.0.
    • network S6. D. sim. ♀ × D. mel. ♂ embryos; FDR < 0.01 and Ri > 0.0.
    • network S7. D. sim. ♀ × D. mel. ♂ embryos; FDR < 0.01 and Ri < 0.0.
    • network S8. D. mel. ♀ × D. sim. ♂ heads; FDR < 0.01 and Ri > 0.0.
    • network S9. D. mel. ♀ × D. sim. ♂ heads; FDR < 0.01 and Ri < 0.0.
    • network S10. D. mel. ♀ × D. sim. ♂ embryos; proteasomal proteins.
    • network S11. D. sim. ♀ × D. mel. ♂ embryos; proteasomal proteins.
    • network S12. D. mel. ♀ × D. sim. ♂ hybrid heads; proteasomal proteins.
    • Data repositories
    • fig. S1. Only hybrids of D. melanogaster females crossed with D. simulans males are viable.
    • fig. S2. Relative abundance of peptide nodes according to species specificity and sample origin.
    • fig. S3. The Venn diagram displays the relative overlap of peptides retrieved for the D. melanogaster and D. simulans proteome databases when digested in silico with the endoprotease LysC.
    • fig. S4. Venn diagram of proteomes identified in each of the replicate experiments.
    • fig. S5. Sample analysis workflow and detection of species-specific peptides.

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • table s1 (Microsoft Excel format). Significantly regulated peptide nodes (FDR < 0.05) and associated proteins in D. mel. ♀ × D. sim. ♂ embryos, D. sim. ♀ × D. mel. ♂ embryos, and D. mel. ♀ × D. sim. ♂ heads.
    • table S2 (Microsoft Excel format). PSEA-Quant results obtained for D. mel. ♀ × D. sim. ♂ embryos, D. sim. ♀ × D. mel. ♂ embryos, and heads of the D. mel. ♀ × D. sim. ♂ cross are presented in individual tabs as indicated in the Excel file.

    Files in this Data Supplement: