Expansion of the fatty acyl reductase gene family shaped pheromone communication in Hymenoptera
- Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Czech Republic
- Charles University, Czech Republic
- Okinawa Institute of Science and Technology Graduate University, Japan
- Max Planck Institute for Chemical Ecology, Germany
Figures
Figure 1 with 5 supplements
Unrooted hymenopteran FAR gene tree.
Tree tips are colored according to taxonomy: red, bumble bee FARs (B. terrestris, B. lucorum, B. lapidarius, B. impatiens, B. rupestris); green, stingless bee FARs (Tetragonula carbonaria, Melipona quadrifasciata); blue, FARs from other Apidae species (i.e. A. mellifera, Euglossa dilemma, Ceratina calcarata, and Epeolus variegatus); and black, FARs from other hymenopteran species. The FAR-A ortholog group is highlighted yellow; other ortholog groups in shades of grey. Functionally characterized bumble bee FARs from this study are indicated by filled triangles and numbered. 1: BlapFAR-A1, 2: BlucFAR-A1, 3: BterFAR-A1, 4: BlapFAR-A4, 5: BlucFAR-A2, 6: BterFAR-A2, 7: BlapFAR-A5, 8: BterFAR-J, and 9: BlapFAR-J. The functionally characterized A. mellifera FAR is indicated by an empty triangle. Internal nodes highlighted with black boxes indicate bootstrap support >80%. Violet squares at the tree tips indicate FARs for which CDD search yielded all three FAR conserved features—active site, NAD(P)+ binding site and putative substrate binding site (see Figure 1—source data 1 for complete CDD search results).
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Figure 1—source data 1
Predicted protein sequence lengths and conserved domains detected in predicted FAR coding regions via Conserved Domain Database search.
The presence of a domain or conserved feature is marked with ‘+’.
- https://doi.org/10.7554/eLife.39231.009
Figure 1—figure supplement 1
Expression of FARs in male labial gland and male fat body of Bombus terrestris (A) B. lucorum (B) and B. lapidarius (C).
The expression values shown are log2-transformed normalized counts of reads (RPKM values) mapping to the FAR coding regions. FARs functionally characterized in this study are highlighted in orange.
Figure 1—figure supplement 2
FAR gene tree including non-hymenopteran (Drosophila melanogaster, Bombyx mori, Tribolium castaneum) and hymenopteran FARs.
Tree tips are colored according to taxonomy: red, bumble bee FARs (B. terrestris, B. lucorum, B. lapidarius, B. impatiens, B. rupestris); green, stingless bee FARs (Tetragonula carbonaria, Melipona quadrifasciata); blue, FARs from other Apidae species (i.e. A. mellifera, Euglossa dilemma, Ceratina calcarata, and Epeolus variegatus); black, FARs from other hymenopteran species; purple, non-hymenopteran species. The FAR-A ortholog group is highlighted in yellow; other ortholog groups are in shades of grey. The functionally characterized Drosophila melanogaster Waterproof FAR and Bombyx mori pheromone-biosynthetic FAR (Bmor-pgFAR) are indicated by filled triangles. Internal nodes highlighted with black boxes indicate bootstrap support >80%.
Figure 1—figure supplement 3
Clusters of FAR-A genes on B. terrestris genomic scaffolds Un679 (A) and Un989 (B).
The horizontal axis shows genomic coordinates of genes within the scaffold. FAR genes are labeled.
Figure 1—figure supplement 4
Protein sequence identities of bumble bee male marking pheromone (MMP)-biosynthetic FAR candidates.
The colors indicate % identity (red: highest, white: medium, blue: lowest).
Figure 1—figure supplement 5
Protein sequence alignment of bumble bee FAR candidates.
Identical amino acid residues are highlighted in dark blue, similar residues in light blue. The conserved domains FAR-N_SDR_e (accession number cd05236) and Sterile/FAR_C (accession numbers pfam03015 and cd09071) from Conserved Domain Database are underlined. The residues forming putative active site are marked with an asterisk. The alignment was generatedusing Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) tool using similarity groups: G, AVLI, FYW, CM, ST, KRH, DENQ.
Figure 2
Number of predicted FAR genes (transcripts) across hymenopteran lineages and across FAR groups.
The schematic phylogenetic tree of Hymenoptera was adapted from Peters et al. (2017). The rightmost column indicates whether FARs were predicted from genome or transcriptome assemblies. The red triangle points to the presumed onset of FAR-A expansion in the common ancestor of bumble bees (Bombini) and stingless bees (Meliponini).
Figure 3
Average TE densities in 10 kb windows around groups of B. terrestris (A, B) and B. impatiens (C, D) genes.
(A and C) Distributions of overall TE densities around FAR genes (N, non-FAR-A genes, in green; A, FAR-A genes, in red) with respect to the genome-wide distribution of TE densities around RefSeq genes (in grey) are shown for the B. terrestris (A) and B. impatiens (C) genomes. (B and D) Densities of individual TE classes and families for FAR-A (in red) and non-FAR-A (in green) genes are depicted for B. terrestris (B) and B. impatiens (D). The average TE densities for the whole gene group are compared to the genome-wide average. Asterisks indicate statistical significance obtained by permutation test.
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Figure 3—source data 1
List of TE densities for FAR-A and non-FAR-A genes.
- https://doi.org/10.7554/eLife.39231.012
Figure 4 with 2 supplements
Expression pattern of FAR candidates across bumble bee tissues.
The FAR transcripts were assayed using quantitative PCR on cDNA from tissues of 3-day-old B. lapidarius, B. lucorum and B. terrestris males and queens (N = 3; queen LG: one biological replicate represents tissues from two queens).
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Figure 4—source data 1
List of Cp differences for FAR transcripts.
The file contains a list of Cp difference values for all assayed FAR transcripts. See Figure 4—figure supplement 1—source data 1 for Cp values.
- https://doi.org/10.7554/eLife.39231.016
Figure 4—figure supplement 1
Relative expression of FAR candidates in the tissues of B. lapidarius, B. lucorum and B. terrestris males and queens.
The FAR transcripts were assayed using RT-qPCR on cDNA from tissues of 3-day-old B. lapidarius, B. lucorum and B. terrestris males and queens (N = 3; queen LG: one biological replicate represents tissues from two queens). Significant differences (p < 0.05, one-way ANOVA followed by post-hoc Tukey’s HSD test) are marked with different letters; the statistical testing was performed on Cp difference values.
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Figure 4—figure supplement 1—source data 1
List of relative transcript levels and Cp values for FAR candidates.
The file contains a list of relative transcript level and individual Cp values for all assayed FAR and reference transcripts and results of statistical testing.
- https://doi.org/10.7554/eLife.39231.018
Figure 4—figure supplement 2
Relative expression of FAR-A1 and FAR-A1-short in male and queen LGs of B. lapidarius.
The transcripts were determined in cDNA from tissues of 3-day-old B. lapidarius males and queens (N = 3; queen LG: one biological replicate represents tissues from two queens). Significant differences (p < 0.05, two-tailed t-test) are indicated with an asterisk.
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Figure 4—figure supplement 2—source data 2
List of relative transcript level, Cp difference and Cp values for FAR-A1 and FAR-A1-short.
The file contains a list of relative transcript level, Cp difference and individual Cp values for FAR-A1, FAR-A1-short and reference transcripts and the results of statistical testing.
- https://doi.org/10.7554/eLife.39231.017
Figure 5 with 1 supplement
Fatty alcohols and fatty acyls of bumble bee male LGs together with the proposed participation of FARs in biosynthesis.
The fatty alcohols and fatty acyls (determined as methyl esters) were extracted from LGs of 3-day-old males of B. lucorum, B. terrestris and B. lapidarius (N = 3) and quantified by GC. The area (size) of fatty alcohol and fatty acyl circle represents the mean quantity per a single male LG (Figure 5—source data 1). The FARs which could be involved in the fatty alcohol biosynthesis based on their specificity are appended to the left side of the corresponding fatty alcohol circle.
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Figure 5—source data 1
List of fatty alcohol and fatty acyl quantities in LGs and FBs of bumble bee males.
The file contains a list of quantified fatty alcohols and fatty acyl methyl esters (transesterifiable fatty acyls) in male LGs and FBs of 3-day-old B. terrestris, B. lucorum and B. lapidarius. In the case of 22:1 alcohols in FB of B. terrestris, the assignment of individual isomers was not possible due to their low amount, so they are reported as a sum of isomers.
- https://doi.org/10.7554/eLife.39231.021
Figure 5—figure supplement 1
Apparent specificity of FARs in bumble bee LGs and FBs.
The fatty alcohol ratios represent the apparent specificity of all putative FARs which are active in LGs and FBs of 3-day-old B. lucorum, B. terrestris and B. lapidarius (N = 3). Ratios >99%, suggesting high conversion rates of fatty acyl to fatty alcohol, are marked with an asterisk. In the case of 22:1 alcohols in FB of B. terrestris, the assignment of individual isomers was not possible due to their low amount, so they are counted as a sum of isomers. See Equation 1 for a description of fatty alcohol ratio calculation.
Figure 6 with 8 supplements
Apparent specificity of bumble bee FARs expressed in yeasts.
The fatty alcohol ratios represent the apparent specificity of individual MMP-biosynthetic FAR candidates when expressed in yeast hosts (N = 3). The fatty alcohol production was quantified by GC analysis of yeast total lipid extracts with the recombinant FARs either acting on yeast native lipids (A) or acting on non-native substrates after supplementation of yeasts with either Z9,Z12-18:, Z9,Z12,Z15-18: or Z15-20: acyl-CoA precursors (B). The data in (B) panel of BlucFAR-A1 and BlucFAR-A2 are taken from yeasts expressing the proteins from yeast codon-optimized nucleotide sequences. Note the different y-axis scale for BterFAR-A2 and BlucFAR-A2. Significant differences (p < 0.01, one-way ANOVA followed by post-hoc Tukey’s HSD test) are marked with different letters. See Equation 1 for a description of fatty alcohol ratio calculation.
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Figure 6—source data 1
List of fatty alcohol and fatty acyl quantities in FAR-expressing yeasts and fatty alcohol ratios of FARs.
The file contains a list of quantified fatty alcohols and fatty acyl methyl esters (transesterifiable fatty acyls) in yeasts which express bumble bee FARs and of fatty alcohol ratio values for individual FARs and the results of statistical testing.
- https://doi.org/10.7554/eLife.39231.031
Figure 6—figure supplement 1
Protein expression and lipid profile in yeast expressing BlapFAR-A1-short.
(A) Protein expression pattern determined in yeast cell lysate using western blot followed by detection with anti-6×His-tag antibody. Lanes: S, protein standard (VI, AppliChem); 1, yeast carrying plasmid with the sequence of BlapFAR-A1-short. (B) Chromatogram traces of lipid extracts from the yeast strains expressing BlapFAR-A1-short and BlapFAR-A1. (C) Protein sequence alignment of BlapFAR-A1-short and BlapFAR-A1. The 22-residue sequence lacking in BlapFAR-A1-short is indicated by a black dashed line above the sequence. The conserved domains FAR-N_SDR_e (accession number cd05236) and FAR_C (accession number cd09071) from Conserved Domain Database are underlined. The amino acid residues forming putative active site are marked with an asterisk.
Figure 6—figure supplement 2
Fatty alcohol production in yeasts expressing bumble bee FARs specific for long chain fatty acyls.
The chromatogram traces show fatty alcohol and fatty acyl methyl ester profiles from lipid extracts of yeast expressing bumble bee FARs which are specific for long chain fatty acyls (C14–C18), that is BlapFAR-A1, BlucFAR-A2, BterFAR-A2, BlapFAR-A4, BlapFAR-A5.
Figure 6—figure supplement 3
Fatty alcohol production in yeasts expressing bumble bee FARs specific for very long chain fatty acyls.
The chromatogram traces show fatty alcohol and fatty acyl methyl ester profiles from lipid extracts of yeast expressing bumble bee FARs which are specific for very long chain fatty acyls (C20–C26), that is BlucFAR-A1, BterFAR-A1, BlapFAR-J, BterFAR-J.
Figure 6—figure supplement 4
Protein expression in yeasts expressing bumble bee FARs.
(A) Protein expression patterns determined in yeast cell lysates using western blot followed by detection with anti-6 ×His tag antibody. (B) Detail of the FAR-J full-length protein region with increased photograph contrast. Lanes: S, protein standard (VI, AppliChem); NC, negative control (yeast carrying empty vector); 1–11, yeast strains carrying plasmids with BlucFAR-A1 (1), BterFAR-A1 (2), BlapFAR-A1 (3), BlucFAR-A2 (4), BterFAR-A2 (5), BterFAR-J (6), BlapFAR-J (7), BlapFAR-A4 (8), BlapFAR-A5 (9), BlucFAR-A1-opt (10) and BlucFAR-A2-opt (11).
Figure 6—figure supplement 5
Apparent specificity of FARs expressed from wild-type and yeast codon-optimized sequences.
Plasmids carrying wild-type and yeast codon-optimized (‘-opt’) nucleotide sequences for two FARs from B. lucorum, BluFAR-A1 and BluFAR-A2, were transformed into yeast host and expressed (N = 3). Fatty alcohol production was analyzed by GC of yeast total lipid extracts. The fatty alcohol ratios represent the apparent specificity of expressed FARs. Significant differences (p < 0.05, two-tailed t-test) are marked with an asterisk. See Equation 1 for a description of fatty alcohol ratio calculation.
Figure 6—figure supplement 6
Fatty alcohol production in FAR-expressing yeasts supplemented with non-native fatty acyl substrates.
Following GC×GC-MS chromatogram traces show the production of fatty alcohols by BlapFAR-A1, BlapFAR-A4, BlucFAR-A1 and BterFAR-A1 determined in extracts from yeast strains supplemented with Z9,Z12-18: (A), Z9,Z12,Z15-18: (B) and Z15-20: (C) acyl precursors, that is the acyls which are non-native to the yeast. NC, negative control (yeast carrying empty vector); 1a, Z9,Z12,Z15-18:OH; 1 m, Z9,Z12,Z15-18:Me; 2a, Z9,Z12-18:OH; 2 m, Z9,Z12-18:Me; 3a, Z9-18:OH; 3 m, Z9-18:Me; 4a, 18:OH; 5a, Z15-20:OH; 5 m, Z15-20:Me; 6a, 20:OH; 6 m, 20:Me. The m/z values selected for display: (A) 55 + 67, (B) 55 + 79, (C) 55 + 74, standards 55 + 67 + 74 + 79.
Figure 6—figure supplement 7
Relative amounts of fatty alcohols produced in bumble bee FAR-expressing yeasts.
The graphs show relative weight percentages of individual fatty alcohols in the total fatty alcohol mixtures produced by FAR-expressing yeast cells.
Figure 6—figure supplement 8
The quantitative aspects of fatty alcohol production in yeasts expressing bumble bee FARs.
(A) Quantities of individual fatty alcohols produced in yeast relative to the lyophilized cell biomass. Note the differences in respective y-axis scales. (B) Quantities of total fatty alcohols produced by FAR-expressing strains relative to the culture volume.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Acromyrmex echinatior) | Acromyrmex echinatior genome | https://www.ncbi .nlm.nih.gov/bioproject | PRJNA271903 | |
| Gene (Andrena vaga) | Andrena vaga transcriptome | https://www.ncbi.nlm.nih.gov/bioproject | PRJNA252325 | |
| Gene (Apis mellifera) | Apis mellifera genome | https://www.ncbi.nlm.nih.gov/bioproject | PRJNA13343 | |
| Gene (Bombus impatiens) | Bombus impatiens genome | https://www.ncbi.nlm.nih.gov/bioproject | PRJNA70395 | |
| Gene (Bombus rupestris) | Bombus rupestris transcriptome | https://www.ncbi.nlm.nih.gov/bioproject | PRJNA252240 | |
| Gene (Bombus terrestris) | Bombus terrestris genome | https://www.ncbi.nlm.nih.gov/bioproject | PRJNA68545 | |
| Gene (Bombus terrestris) | B. terrestris LG and FB transcriptomes | https://www.ncbi.nlm.nih.gov/bioproject | PRJEB9937 | |
| Gene (Camponotus floridanus) | Camponotus floridanus genome | https://www.ncbi.nlm.nih.gov/bioproject | PRJNA50201 | |
| Gene (Camptopoeum sacrum) | Camptopoeum sacrum transcriptome | https://www.ncbi.nlm.nih.gov/bioproject | PRJNA252153 | |
| Gene (Ceratina calcarata) | Ceratina calcarata genome | https://www.ncbi.nlm.nih.gov/bioproject | PRJNA340002 | |
| Gene (Colletes cunicularius) | Colletes cunicularius transcriptome | https://www.ncbi.nlm.nih.gov/bioproject | PRJNA252324 | |
| Gene (Dufourea novaeangliae) | Dufourea novaeangliae genome | https://www.ncbi.nlm.nih.gov/bioproject | PRJNA311229 | |
| Gene (Epeolus variegatus) | Epeolus variegatus transcriptome | https://www.ncbi.nlm.nih.gov/bioproject | PRJNA252262 | |
| Gene (Euglossa dilemma) | Euglossa dilemma transcriptome | https://www.ncbi.nlm.nih.gov/bioproject | PRJNA252310 | |
| Gene (Harpegnathos saltator) | Harpegnathos saltator genome | https://www.ncbi.nlm.nih.gov/bioproject | PRJNA273397 | |
| Gene (Megachile rotundata) | Megachile rotundata genome | https://www.ncbi.nlm.nih.gov/bioproject | PRJNA87021 | |
| Gene (Melipona quadrifasciata) | Melipona quadrifasciata genome | https://www.uniprot.org/proteomes/ | UP000053105 | |
| Gene (Melitta haemorrhoidalis) | Melitta haemorrhoidalis transcriptome | https://www.ncbi.nlm.nih.gov/bioproject | PRJNA252208 | |
| Gene (Nasonia vitripenis) | Nasonia vitripenis genome | https://www.ncbi.nlm.nih.gov/bioproject | PRJNA20073 | |
| Gene (Panurgus dentipes) | Panurgus dentipes transcriptome | https://www.ncbi.nlm.nih.gov/bioproject | PRJNA252205 | |
| Gene (Polistes canadensis) | Polistes canadensis genome | https://www.ncbi.nlm.nih.gov/bioproject | PRJNA301748 | |
| Gene (Tetragonula carbonaria) | Tetragonula carbonaria transcriptome | https://www.ncbi.nlm.nih.gov/bioproject | PRJNA252285 | |
| Gene | B. lucorum and B. lapidarius transcriptomes | This paper, https://www.ncbi.nlm.nih.gov/bioproject | PRJNA436452 | |
| Gene (Bombus lapidarius) | Cloned CDS of FAR-A1, FAR-A1-short, FAR-A4, FAR-A5, FAR-J | This paper, https://www.ncbi.nlm.nih.gov/genbank/ | MG450698; MG450699; MG450702; MG450703; MG450701 | |
| Gene (Bombus lucorum) | Cloned CDS of FAR-A1, FAR-A1-opt, FAR-A2, FAR-A2-opt | This paper,https://www.ncbi.nlm.nih.gov/genbank/ | MG930980; MG450697; MG930982; MG450704 | |
| Gene (Bombus terrestris) | Cloned CDS of FAR-A1, FAR-A2, FAR-J | This paper, https://www.ncbi.nlm.nih.gov/genbank/ | MG930981; MG930983; MG450700 | |
| Strain, strain background (Saccharomyces cerevisiae) | BY4741 | Brachmann et al., 1998 (DOI: 10.1002/(SICI)1097-0061 (19980130)14:2 < 115::AID-YEA204 > 3.0.CO;2–2) | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 | |
| Strain, strain background (Saccharomyces cerevisiae, BY4741) | FAR expressing yeast strains | This paper | See Supplementary file 2 | |
| Biological sample (Bombus lapidarius) | Labial gland (LG) from males and females; faty body (FB); flight muscle; gut | This paper | Dissected by standard techniques from individuals reared in laboratory colonies | |
| Biological sample (Bombus lucorum) | Labial gland (LG) from males and females; faty body (FB); flight muscle; gut | This paper | Dissected by standard techniques from individuals reared in laboratory colonies | |
| Biological sample (Bombus terrestris) | Labial gland (LG) from males and females; faty body (FB); flight muscle; gut | This paper | Dissected by standard techniques from individuals reared in laboratory colonies | |
| Antibody | Anti-6×His tag antibody-HRP conjugate, mouse monoclonal | Merck | A7058; RRID:AB_258326 | (1:2000) |
| Recombinant DNA reagent | Escherichia coli DH5α | Thermo Fisher Scientific | 18265017 | |
| Recombinant DNA reagent | Escherichia coli One Shot TOP10 | Thermo Fisher Scientific | C4040 | |
| Recombinant DNA reagent | pYEXTHS-BN (plasmid) | Holz et al., 2002 (DOI: 10.1016/S1046-5928 (02)00029–3) | pUC and 2μ origin; LEU2, URA3 and AmpR selectable markers; PCUP1 inducible promoter; N-terminal 6 × His tag and C-terminal Strep II tag | |
| Recombinant DNA reagent | pYEXTHS-BN plasmids carrying FAR CDSs | This paper | See Supplementary file 2 | |
| Sequence- based reagent | Cloning primers | This paper | See Supplementary file 2 | |
| Sequence- based reagent | RT-qPCR primers | This paper; Horňáková et al., 2010 (DOI: 10.1016/j.ab.2009.09.019) | See Supplementary file 2 | |
| Commercial assay or kit | In-Fusion HD Cloning kit | Clontech (Takara) | 639649 | |
| Commercial assay or kit | LightCycler 480 SYBR Green I Master | Roche | 04707516001 | |
| Commercial assay or kit | RNeasy Mini Kit | Qiagen | 74104 | |
| Commercial assay or kit | S.c. EasyComp Transformation Kit | Thermo Fisher Scientific | K505001 | |
| Commercial assay or kit | SMART cDNA Library Construction Kit | Clontech (Takara) | 634901 | |
| Commercial assay or kit | SuperSignal West Femto Maximum Sensitivity Substrate | Thermo Fisher Scientific | 34096 | |
| Commercial assay or kit | TOPO TA Cloning kit | Thermo Fisher Scientific | 450640 | |
| Software, algorithm | Batch conserved domain search | DOI: 10.1093/nar/gku1221 | ||
| Software, algorithm | BLAST | DOI: 10.1016/S0022 -2836 (05)80360–2 | ||
| Software, algorithm | bowtie2 v2.2.6 | DOI: 10.1038/nmeth.1923 | ||
| Software, algorithm | CLC Genomics Workbench software v. 7.0.1 | http://www.clcbio.com | ||
| Software, algorithm | ggtree | DOI: 10.1111/2041-210X.12628 | ||
| Software, algorithm | ht-seq v0.9.1 | DOI: 10.1093/ bioinformatics/btu638 | ||
| Software, algorithm | IQTREE v1.5.5 | DOI: 10.1093/ molbev/msu300 | ||
| Software, algorithm | mafft v7.305 | DOI: 10.1093/nar/gkf436 | ||
| Software, algorithm | MAUVE 2.4.0 | DOI: 10.1101/gr.2289704 | ||
| Software, algorithm | Primer BLAST | DOI: 10.1186/1471-2105-13-134 | ||
| Software, algorithm | R programming language | R Core Team. R: A language and environment for statistical computing. (2016) |
Additional files
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Supplementary file 1
Genomic linkage group or scaffold and ortholog group of B. terrestris and A. mellifera FARs.
B. terrestris FARs functionally characterized in this study and A. mellifera FAR previously functionally characterized are highlighted in green; FAR-A gene orthologs are highlighted in orange; genomic scaffolds not placed into linkage groups are grey.
- https://doi.org/10.7554/eLife.39231.032
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Supplementary file 2
List of primers and synthetic genes and of generated plasmids and strains.
- https://doi.org/10.7554/eLife.39231.033
-
Supplementary file 3
Scripts for the analysis of repeat content around FAR genes in bumble bee genomes, statistical analysis and graphics generation.
- https://doi.org/10.7554/eLife.39231.034
-
Transparent reporting form
- https://doi.org/10.7554/eLife.39231.035
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Expansion of the fatty acyl reductase gene family shaped pheromone communication in Hymenoptera
eLife 8:e39231.
https://doi.org/10.7554/eLife.39231
