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Protein-Level Interactions as Mediators of Sexual Conflict in Ants*

  • Author Footnotes
    §§ These authors contributed equally.
    Ryan Dosselli
    Footnotes
    §§ These authors contributed equally.
    Affiliations
    From the ‡ARC Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, Bayliss Building (M316),

    Centre for Evolutionary Biology, School of Biological Sciences (M092),

    Honey Bee Health Research Group, School of Molecular Sciences (M316), The University of Western Australia, Crawley WA 6009, Australia;
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  • Author Footnotes
    §§ These authors contributed equally.
    Julia Grassl
    Footnotes
    §§ These authors contributed equally.
    Affiliations
    From the ‡ARC Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, Bayliss Building (M316),

    Honey Bee Health Research Group, School of Molecular Sciences (M316), The University of Western Australia, Crawley WA 6009, Australia;
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  • Susanne P.A. den Boer
    Affiliations
    From the ‡ARC Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, Bayliss Building (M316),

    Centre for Social Evolution, Department of Biology, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen, Denmark;
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  • Madlen Kratz
    Affiliations
    From the ‡ARC Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, Bayliss Building (M316),

    Honey Bee Health Research Group, School of Molecular Sciences (M316), The University of Western Australia, Crawley WA 6009, Australia;
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  • Jessica M. Moran
    Affiliations
    From the ‡ARC Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, Bayliss Building (M316),

    Centre for Evolutionary Biology, School of Biological Sciences (M092),

    Honey Bee Health Research Group, School of Molecular Sciences (M316), The University of Western Australia, Crawley WA 6009, Australia;
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  • Jacobus J. Boomsma
    Correspondence
    To whom correspondence may be addressed:Centre for Social Evolution, Department of Biology, University of Copenhagen, 2100 Copenhagen, Denmark. Tel.:+45 35321340;
    Affiliations
    Centre for Social Evolution, Department of Biology, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen, Denmark;
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  • Boris Baer
    Correspondence
    To whom correspondence may be addressed:Center for Integrative Bee Research (CIBER), Department of Entomology, University of California, Riverside, CA 92506. Tel.:+1 951 827 5833, Fax:+1 951 827 3086;
    Affiliations
    Center for Integrative Bee Research (CIBER), Department of Entomology, The University of California, Riverside CA 92506
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  • Author Footnotes
    This article contains supplemental material Tables S1-S3, Figs. S1-S4, and Movie S1.
    §§ These authors contributed equally.
Open AccessPublished:December 31, 2018DOI:https://doi.org/10.1074/mcp.RA118.000941
      All social insects with obligate reproductive division of labor evolved from strictly monogamous ancestors, but multiple queen-mating (polyandry) arose de novo, in several evolutionarily derived lineages. Polyandrous ant queens are inseminated soon after hatching and store sperm mixtures for a potential reproductive life of decades. However, they cannot re-mate later in life and are thus expected to control the loss of viable sperm because their lifetime reproductive success is ultimately sperm limited. In the leaf-cutting ant Atta colombica, the survival of newly inseminated sperm is known to be compromised by seminal fluid of rival males and to be protected by secretions of the queen sperm storage organ (spermatheca). Here we investigate the main protein-level interactions that appear to mediate sperm competition dynamics and sperm preservation. We conducted an artificial insemination experiment and DIGE-based proteomics to identify proteomic changes when seminal fluid is exposed to spermathecal fluid followed by a mass spectrometry analysis of both secretions that allowed us to identify the sex-specific origins of the proteins that had changed in abundance. We found that spermathecal fluid targets only seven (2%) of the identified seminal fluid proteins for degradation, including two proteolytic serine proteases, a SERPIN inhibitor, and a semen-liquefying acid phosphatase. In vitro, and in vivo, experiments provided further confirmation that these proteins are key molecules mediating sexual conflict over sperm competition and viability preservation during sperm storage. In vitro, exposure to spermathecal fluid reduced the capacity of seminal fluid to compromise survival of rival sperm in a matter of hours and biochemical inhibition of these seminal fluid proteins largely eliminated that adverse effect. Our findings indicate that A. colombica, queens are in control of sperm competition and sperm storage, a capacity that has not been documented in other animals but is predicted to have independently evolved in other polyandrous social insects.

      Graphical Abstract

      When females mate with multiple males, i.e., are polyandrous, conflicts over paternity continue after mating if sperm from different males compete for storage and egg fertilization, a process referred to as sperm competition. Due to conflicting reproductive interests between the sexes, female reproductive tracts have evolved discrimination mechanisms to secure appropriate mate choice and reproductive success despite sperm competition constraints (
      • Birkhead T.R.
      • Hosken D.J.
      • Pitnick S.
      ,
      • Peretti A.V.
      • Aisenberg A.
      ). The phenotypic manifestations of these sexual conflicts have been well studied (
      • Peretti A.V.
      • Aisenberg A.
      ,
      • Shuker D.M.
      • Simmons L.W.
      ,
      • Firman R.C.
      • Gasparini C.
      • Manier M.K.
      • Pizzari T.
      Postmating female control: 20 years of cryptic female choice.
      ), but the underlying molecular processes mediating them remain largely unknown. Resolving these mechanisms is becoming increasingly feasible (
      • Harrison P.W.
      • Wright A.E.
      • Zimmer F.
      • Dean R.
      • Montgomery S.H.
      • Pointer M.A.
      • Mank J.E.
      Sexual selection drives evolution and rapid turnover of male gene expression.
      ,
      • Wilkinson G.S.
      • Breden F.
      • Mank J.E.
      • Ritchie M.G.
      • Higginson A.D.
      • Radwan J.
      • Jaquiery J.
      • Salzburger W.
      • Arriero E.
      • Barribeau S.M.
      • Phillips P.C.
      • Renn S.C.
      • Rowe L.
      The locus of sexual selection: Moving sexual selection studies into the post-genomics era.
      ) and has the potential to provide key insights into the homology and possible convergence of genes and proteins mediating sexual conflict (
      • Sirot L.K.
      • Wong A.
      • Chapman T.
      • Wolfner M.F.
      Sexual conflict and seminal fluid proteins: A dynamic landscape of sexual interactions.
      ,
      • Lüpold S.
      • Pitnick S.
      • Berben K.S.
      • Blengini C.S.
      • Belote J.M.
      • Manier M.K.
      Female mediation of competitive fertilization success in Drosophila melanogaster.
      ,
      • Pitnick S.
      • Pfennig D.W.
      Brotherly love benefits females.
      ).
      Seminal fluid (SF) is a glandular secretion that males provide to sperm. It contains proteins that are important determinants of male reproductive success (
      • Poiani A.
      Complexity of seminal fluid: A review.
      ), as is particularly well documented in Drosophila, where the sex peptide has a range of effects on female reproductive physiology and behavior (
      • Liu H.
      • Kubli E.
      Sex-peptide is the molecular basis of the sperm effect in Drosophila melanogaster.
      ). These interactions may serve joint reproductive interests, but they tend to primarily favor male interests while reducing future reproductive success of females (
      • Avila F.W.
      • Sirot L.K.
      • LaFlamme B.A.
      • Rubinstein C.D.
      • Wolfner M.F.
      Insect seminal fluid proteins: Identification and function.
      ). Such sexual conflicts are often influenced by dynamic coevolutionary arms races, where molecular mechanisms are difficult to disentangle particularly when rates of re-mating are unpredictable. Females affect the outcome of sexual conflicts via defensive sexual strategies, but they are unlikely to gain ultimate control when re-mating continues to expose them to new male ejaculates (
      • Avila F.W.
      • Sirot L.K.
      • LaFlamme B.A.
      • Rubinstein C.D.
      • Wolfner M.F.
      Insect seminal fluid proteins: Identification and function.
      ,
      • Rice W.R.
      Sexually antagonistic male adaptation triggered by experimental arrest of female evolution.
      ).
      The mating systems of Atta, leaf-cutting ants offer unique opportunities to study male-female protein interactions during insemination and sperm storage. Queens and males have large body sizes and ejaculates are transferred directly to the spermatheca, the queen's sperm storage organ, where they encounter both rival ejaculates and queen-derived secretions (
      • Liberti J.
      • Baer B.
      • Boomsma J.J.
      Queen reproductive tract secretions enhance sperm motility in the ants.
      ). These characteristics imply that artificial insemination is a feasible experimental approach for emulating natural insemination (
      • den Boer S.P.A.
      • Boomsma J.J.
      • Baer B.
      A technique to artificially inseminate leafcutter ants.
      ). Because queens never re-mate later in life (
      • Boomsma J.J.
      • Baer B.
      • Heinze J.
      The evolution of male traits in social insects.
      ), selection can produce both female enhancement mechanisms of sperm preservation after insemination (
      • Liberti J.
      • Baer B.
      • Boomsma J.J.
      Queen reproductive tract secretions enhance sperm motility in the ants.
      ) and mechanisms to terminate sperm competition if it compromises future female reproductive success (
      • den Boer S.P.A.
      • Baer B.
      • Dreier S.
      • Aron S.
      • Nash D.R.
      • Boomsma J.J.
      Prudent sperm use by leaf-cutter ant queens.
      ). Directly after mating, Atta, queens must individually dig a burrow to establish a new colony. Thus, Atta, queens face a series of trade-offs when allocating metabolic resources to control sexual conflict and maximize survival during solitary colony-founding under significant constraints of resource availability and disease pressure (
      • den Boer S.P.A.
      • Baer B.
      • Dreier S.
      • Aron S.
      • Nash D.R.
      • Boomsma J.J.
      Prudent sperm use by leaf-cutter ant queens.
      ,
      • Baer B.
      • Armitage S.A.
      • Boomsma J.J.
      Sperm storage induces an immunity cost in ants.
      ).
      We previously found that SF of A. colombica, males reduces survival of rival sperm in vitro, a process known as sperm incapacitation, and that the queen's spermathecal secretions terminate this process of competitive elimination (
      • den Boer S.P.
      • Baer B.
      • Boomsma J.J.
      Seminal fluid mediates ejaculate competition in social insects.
      ). Here we conducted a series of experiments to unravel the molecular basis of these male-male and male-female interactions. We first show that the in vitro, incapacitation effects of SF on sperm of other males are due to proteins (
      • den Boer S.P.
      • Stürup M.
      • Boomsma J.J.
      • Baer B.
      The ejaculatory biology of leafcutter ants.
      ) and that the interactions between SF and spermathecal fluid (SpF) are also protein based. This implies that proteins play crucial roles in the phenotypic expression of sperm competition and its termination. In a second step, we used a quantitative proteomics approach by mixing SF with SpF to identify the proteins that are targeted for degradation using differential in gel electrophoresis (DIGE). In a third step, to assess whether focal proteins originated from male or female secretions, we conducted a full proteome analyses of both SF and SpF, which allowed us to identify the SF proteins that are degraded by SpF. Finally, we performed an in vivo, artificial insemination experiment and in vitro follow-up trials with general and specific protease inhibitors to obtain phenotypic confirmation for these SF proteins being molecular agents and counteragents of sperm competition. The experimental procedures and results are presented in this order in the sections below.

      RESULTS

      Our results confirm that the key interaction effects between male SF and queen SpF are driven by the protein containing (HMW) fractions of these secretions (Fig. 3A,), and that the proteomic interactions between these secretions are essential for understanding the molecular mechanisms of sexual conflict in A. colombica,. This result prompted our comparative DIGE experiment to identify changes in the sex-specific proteomes when SF interacts with SpF. The results of the DIGE experiment then triggered our efforts to obtain the SF and SpF proteomes, so we could determine the sex-specific origin of the proteins with altered abundances after the male and female secretions interact (Fig. 4) and to do validation experiments to show that serine proteases have key roles in the expression and regulation of sexual conflict (Figs. 3B,-3D,).
      Figure thumbnail gr3
      Fig. 3.Changes in sperm survival after experimentally manipulating the sperm-incapacitation efficiency of rival males' SF, by exposure to either SpF or synthetic protease inhibitors. (A,) Sperm survival after 30 min exposure to: a male's own SF (♂own; control), rival males' SF (♂rival), rival males' SF mixed with female SpF (♂rival + ♀all), rival males' SF mixed with the high molecular weight fraction of female SpF (♂rival + ♀HMW), and rival males' SF with the low molecular weight fraction of SpF (♂rival + ♀LMW). The first three bars have previously been published by Den Boer et al., 2010 (
      • Baer B.
      • Armitage S.A.
      • Boomsma J.J.
      Sperm storage induces an immunity cost in ants.
      ). (B,) Sperm survival after exposure to rival SF (pooled from 350 males), after being artificially inseminated into a queen's spermatheca and recovered at time intervals between 5 min (0 h) and 12 h. (C,) Sperm survival after 2-h exposure to Hayes saline, saline with a mixture of synthetic inhibitors of proteases (Saline + inhibitors), rival males' SF, and rival males' SF with synthetic inhibitors of proteases (SF + inhibitors); we used residuals rather than direct measurements to adjust the response variable for unexplained variation in overall sperm viability across two subsequent field seasons (see methods and for details). (D,) Sperm survival after 2-h exposure to Hayes saline, saline with synthetic inhibitors of serine proteases (Saline + AEBSF), saline with synthetic inhibitors of carboxypeptidases (Saline + EDTA), rival males' SF, rival males' SF with synthetic inhibitors of serine proteases (SF + AEBSF), and rival males' SF with synthetic inhibitors of carboxypeptidases (SF + EDTA). See for procedural details. Bars marked with different letters are significantly different at p, < 0.05 (contrasts test).
      Figure thumbnail gr4
      Fig. 4.Comparison of the SF and SpF proteomes of A. colombica,. The Venn diagram shows the total number of proteins identified from male SF and queen SpF and the number of proteins identified in both proteomes using shotgun proteomics (large font). The pie charts represent functional classes of proteins quantified in the DIGE experiment, showing the class fractions (colors) and numbers of proteins (small font) that increased (+) or decreased (−) in abundance after exposure to SpF (see ).
      The SF and SpF proteomes included 311 and 396 identified proteins, respectively, and showed that less than half of these proteins (135) were shared (Fig. 4, supplemental Fig. S2, supplemental Table S1). Gene Ontology further showed that a large number of proteins were present either in SF or SpF but not in both secretions. In particular, proteins involved in DNA replication, amino acid biosynthesis/metabolism, ATP-binding, and metal ion binding were identified in greater number in the SpF proteome than in the SF proteome (Fisher's exact probability test, two tailed, p, < 0.05), whereas proteins involved in glycan processing, signaling, and carbohydrate turnover were identified in greater number in the SF proteome (supplemental Table S2). These results also showed that the A. colombica, SF proteome is of comparable size to that of honeybee drones (
      • Grassl J.
      • Peng Y.
      • Baer-Imhoof B.
      • Welch M.
      • Millar A.H.
      • Baer B.
      Infections with the sexually transmitted pathogen Nosema apis, trigger an immune response in the seminal fluid of honey bees (Apis mellifera,).
      ) but considerably larger than that of fruit flies (
      • Ravi Ram K.
      • Wolfner M.F.
      Seminal influences: Drosophila, Acps and the molecular interplay between males and females during reproduction.
      ). The SpF proteome that we obtained extends the resolution depth by an order of magnitude compared with a previous study that identified proteins in the spermatheca of another species of Atta, leaf-cutting ants (
      • Malta J.
      • Martins G.F.
      • Marques A.E.
      • Games P.D.
      • Zanuncio J.C.
      • Baracat-Pereira M.C.
      • Salomao T.M.F.
      Insights into the proteome of the spermatheca of the leaf-cutting ant Atta sexdens rubropilosa, (Hymenoptera: Formicidae).
      ).
      The DIGE experiment (Fig. 2) enabled us to identify a total of 471 protein spots, 418 of which did not show any significant change in abundance. Of the 53 spots that changed in abundance, 39 significantly increased and 14 significantly decreased after SF and SpF interacted. These abundance changes accumulated over time after insemination, with 26 spots having changed in intensity after 30 min and 49 after 12 h. However, only four spots had changed after 30 min and not after 12 h (supplemental Table S3). Because we found no evidence for any overall changes in protein abundance in 89% (418 out of 471) gel spots, we concluded that no overall degradation of proteins had occurred during the experiment. This inference was reinforced by our finding that the average amount of protein retrieved per inseminated queen did not differ across experimental groups (32.2 ± 10.6 μg in controls, 24.1 ± 6.0 μg in the 30-min treatment, and 29.2 ± 13.4 μg in the 12-h treatment; analysis of variance, F = 0.4597, df, = 2, p, = 0.645).
      Using MS, we identified 44 protein spots where abundances were significantly changed on the DIGE gel. No significant protein identifications were found in nine spots, but we identified a total of 22 unique proteins in the remaining 35 spots (Table I, supplemental Table S3). Some of the SF proteins that changed in abundance after contact with SpF were identified in multiple spots across the gels (supplemental Table S3 and supplemental Figs. S3 and S4), suggesting these spots only contained protein fragments, also because MW and isoelectric point were substantially different from the intact protein predictions in Uniprot. Such indications of protein fragmentation were found for a total of 24 spots. Three of these proteins (serine protease inhibitor B10 (SERPIN B10), acid phosphatase, and the uncharacterized protein Acol_04113) showed a decreased abundance in the spots representing the expected molecular size and PI for the intact proteins and increased abundances in spots representing lower MWs consistent with these being fragments (Table I and supplemental Fig. S3). For all spots that were identified as representing multiple proteins, we compared the emPAI scores and report all significant identifications (supplemental Table S3).
      Table IProteins identified in DIGE spots with significant abundance changes after exposure of seminal fluid to spermathecal fluid
      ProteinFunctionSFSpFNumberSizeChange
      Serine protease Easter (Acol_08100)ProteolysisYesYes365FragmentDecreased
      368FragmentDecreased
      432FragmentDecreased
      436FragmentDecreased
      Serine protease Snake (Acol_02829)ProteolysisYesNo421IntactDecreased
      436IntactDecreased
      Serpin B10 (Acol_08024)Serine protease inhibitorYesYes257IntactDecreased
      267IntactDecreased
      388IntactDecreased
      389IntactDecreased
      399IntactDecreased
      470FragmentIncreased
      Carboxypeptidase B (Acol_07302)Metallo-carboxyproteaseYesYes262IntactDecreased
      Acid phosphatase (Acol_04502)DephosphorylationYesNo257IntactDecreased
      267IntactDecreased
      395FragmentIncreased
      HOT (Acol_07180 HOT)Energy metabolismYesNo265IntactDecreased
      3245IntactDecreased
      Uncharacterized protein Acol_04113UnknownYesYes87IntactDecreased
      192FragmentIncreased
      198FragmentIncreased
      551FragmentIncreased
      Chitinase-like protein Idgf4 (Acol_07567)MorphogenesisYesYes374FragmentIncreased
      386FragmentIncreased
      369FragmentIncreased
      371FragmentIncreased
      380FragmentIncreased
      397FragmentIncreased
      511FragmentIncreased
      517FragmentIncreased
      GAPDH (Acol_07180 GapDH)GlycolysisNoYes329IntactIncreased
      346IntactIncreased
      Beta-glucuronidase (Acol_05025)Carbohydrates metabolismNoYes156IntactIncreased
      Transketolase (Acol_07599)Pentose phosphate shuntNoYes154IntactIncreased
      213IntactIncreased
      Catalase (Acol_06379)Response to oxidative stressNoNo210FragmentIncreased
      213FragmentIncreased
      216FragmentIncreased
      SOD [Cu-Zn] (Acol_09923)Response to oxidative stressYesYes519IntactIncreased
      GST (Acol_15065)Response to oxidative stressYesYes464FragmentIncreased
      Hexamerin (Acol_04072)Oxygen transportNoYes114IntactIncreased
      120IntactIncreased
      2031IntactIncreased
      PEBP (Acol_01467)Lipid bindingYesYes464FragmentIncreased
      467FragmentIncreased
      Transgelin (Acol_06601)Actin bindingNoYes531IntactIncreased
      PpiC-type (Acol_08173)Protein foldingNoNo552IntactIncreased
      NDP (Acol_07573)GTP/UTP biosynthesisNoYes562IntactIncreased
      Arginine kinase (Acol_12832)PhosphorylationYesYes320IntactIncreased
      Transferrin (Acol_11379)Metal ion bindingNoYes198FragmentIncreased
      551FragmentIncreased
      Actin (Acol_04097)CytoskeletonYesYes213IntactIncreased
      We identified 22 unique proteins from the 2D-DIGE gels, of which seven showed a decrease and 15 an increase in abundance (Table I, Fig. 4). Of the seven SF proteins with reduced abundance, four (serine protease: snake, acid phosphatase, the uncharacterized protein Acol_04113, and hydroxyacid-oxoacid transhydrogenase) were found in the SF proteome only, and three (serine protease: easter, SERPIN B10, and carboxypeptidase B) were present in both the SF and SpF proteomes (Table I, supplemental Tables S1 and S3, and supplemental Figs. S3 and S4).
      Of the 15 proteins that increased in abundance after transfer of SF to the spermatheca, seven were found in both proteomes (chitinase-like protein imaginal disk growth factor 4, superoxide dismutase, glutathione S-transferase, phosphatidylethanolamine-binding protein, arginine kinase, actin, and transferrin (Table I)). The remaining eight proteins were identified only in the SpF proteome, including glyceraldehyde-3-phosphate dehydrogenase, beta-glucoronidase, transketolase, catalase, hexamerin, transgelin, peptidyl-prolyl cis-trans isomerase, and nucleoside diphosphate kinase (Table I). Because no intact proteins that were exclusively found in the SF increased in abundance after contact with SpF, we inferred that all observed increases in abundances were the result of proteins that were introduced by the SpF (Table I).
      SF proteins that decreased in abundance after contact with SpF predominantly belonged to the proteolysis functional category (serine proteases: easter and snake, SERPIN B10, and carboxypeptidase B). It is noteworthy that three of the proteins that changed in abundance after contact with SpF showed a decreased abundance in spots with a MW of the intact protein, while some smaller fragments increased in abundance (SERPIN B10, acid phosphatase, and the uncharacterized protein Acol_04113). This suggests that the intact protein was fragmented, consistent with these proteins being specific targets of SpF proteins directly after insemination (Table I).
      Most of the proteins that increased in abundance are involved in energy metabolism (glyceraldehyde-3-phosphate dehydrogenase, beta-glucoronidase, transketolase, and arginine kinase) and oxidative stress responses, including redox and immunity (catalase, superoxide dismutase, and glutathione S-transferase) (Table I, Fig. 4; see supplemental Table S3 for further details on all proteins). Of the eight SpF-specific proteins that we did not identify in the SF, five appeared only on the gels where SF was exposed to SpF for 30 min and 12 h. The remaining three SpF-specific proteins plus all seven proteins shared between SF and SpF were all found in higher abundances after the SF was exposed to SpF (Table I), suggesting that increased abundances of SF proteins resulted from the mixing with SpF proteins.
      As already mentioned, the seven SF proteins that decreased in abundance after exposure to SpF (Table I) included two serine proteases (easter and snake), SERPIN B10, and a carboxypeptidases B. Snake appeared to be SF-specific, whereas easter and SERPIN B10 were found in both SpF and SF. Carboxypeptidases have a rather broad array of biological functions (
      • Gatehouse J.A.
      Insect Carboxypeptidases.
      ), but SERPINs can target serine proteases involved in sperm-egg recognition (see Discussion). We detected a total of five different SERPIN proteins in the SpF proteome, three of which were also present in the SF proteome. Our results thus provide several lines of evidence that serine proteases and SERPINs are targeted by SpF to terminate mutual sperm incapacitation in A. colombica,. We also detected a fragment of SERPIN B10 in SF after in vivo, exposure to SpF (Table I and supplemental Fig. S3). Interestingly, although we identified a total of three different SERPINs in the SF proteome, only SERPIN B10 was found to be targeted by SpF for degradation, suggesting that SpF targets individual proteins with high specificity.
      Of the remaining three SF-specific proteins that were reduced in abundance, uncharacterized protein Acol_04113 was degraded into at least three fragments that all increased in abundance after male and female secretions came into contact (Table I, supplemental Fig. S3), consistent with this protein also being targeted for destruction by SpF. A similar fragmentation effect could be observed for acid phosphatase (supplemental Fig. S3) where we detected one fragment spot that increased 21-fold after 12 h of SpF exposure. Acid phosphatase has fertility-reducing effects (see Discussion for further details). The final SF-specific protein identified as being targeted by SpF was hydroxyacid-oxoacid transhydrogenase, an enzyme that catalyzes oxidation-reduction reactions (Table I). We did not detect any fragments for this protein, but the intact protein decreased in abundance after exposure to SpF (supplemental Fig. S3).
      Five of the 15 proteins that increased in abundance (chitinase-like protein imaginal disk growth factor 4, catalase, phosphatidylethanolamine-binding protein, glutathione S-transferase, and transferrin) were identified as fragments, but it was not possible to ascertain whether fragmentation occurred before or after SF was put in contact with SpF (Table I and supplemental Fig. S3). Imaginal disk growth factor 4 appeared to be already split into several fragments in the male body because it was identified in six spots of the pure SF (369, 371, 380, 386, 397, and 511; Table I, supplemental Fig. S3), but two new degradation products (spots 374 and 517) appeared when exposed to SpF, suggesting some further fragmentation had occurred.

      Exposure of SF to SpF Reduces Rival Sperm Incapacitation Quickly

      To confirm the efficiency of SpF in eliminating sperm incapacitation over time, we replicated the experiment of Fig. 1 and transferred pooled SF mixtures from six unrelated rival males to the spermathecae of virgin queens, which we then recovered by dissection after time intervals between 5 min and 12 h. These recovered SF samples were then exposed to newly collected sperm of a different male, which showed that sperm survival was higher when the SF had been exposed to SpF for longer periods of time (χ2 = 25.308, df, = 4, p, < 0.001) (Fig. 3B,).

      Protease Inhibitors Confirm the Identity of Antagonistic Proteins in SF

      We finally tested whether SF proteases are indeed the prime agents of sperm incapacitation by incubating SF mixtures of competing males with or without adding three commercially available cocktails of protease inhibitors. We found that sperm survival increased significantly when general protease inhibitors were present (χ2 = 10.419, df, = 3, p, = 0.015, Fig. 3C,). We subsequently tested protease inhibitors that are more specifically effective against the two main categories of proteases, carboxypeptidases, and serine proteases, as identified in the DIGE experiment. This confirmed that only the serine protease inhibitor AEBSF reduced the incapacitation ability of SF toward rival sperm (χ2 = 62.271, df, = 5, p, < 0.001, Fig. 3D,). These combined results provide further support that spermathecal secretions have a general and indiscriminate preservation function for the viability of newly stored sperm mixtures (see (
      • den Boer S.P.
      • Baer B.
      • Boomsma J.J.
      Seminal fluid mediates ejaculate competition in social insects.
      ) for phenotypic evidence) by neutralizing those SF proteases that mediate sperm incapacitation between ejaculates.

      DISCUSSION

      Sexual conflicts are known to continue after insemination when the reproductive interests of females and males are not fully aligned, which is essentially always the case when females mate with multiple males. In the leaf-cutting ant A. colombica, male SF proteins incapacitate rival sperm within a brief time window, and queens are known to neutralize hostility among competing ejaculates upon storing sperm, an adaptation that seems logical because they cannot re-mate later in life (
      • den Boer S.P.A.
      • Baer B.
      • Dreier S.
      • Aron S.
      • Nash D.R.
      • Boomsma J.J.
      Prudent sperm use by leaf-cutter ant queens.
      ,
      • den Boer S.P.
      • Baer B.
      • Boomsma J.J.
      Seminal fluid mediates ejaculate competition in social insects.
      ). We here combined proteomic methods with the experimental approaches that are typically used in evolutionary and behavioral ecology to obtain first insights in the expression and regulation of sexual conflicts in social insects. The SF and SpF proteomes that we obtained are of sufficient size and depth to make meaningful comparisons and we are confident that they enabled us to identify potential proteins of interest that are essential for the expression of sexual conflict in leaf-cutting ants.
      We found that proteins secreted by the sperm storage organ, the spermatheca, target a very small subset of male SF proteases and we show that their degradation or biochemical inhibition increases the survival of sperm that would otherwise have died due to exposure to SF of other males. Our results are also consistent with many components of SF having an overall positive effect on sperm survival, similar to what we have reported previously in both ants and bees (
      • den Boer S.P.A.
      • Baer B.
      • Dreier S.
      • Aron S.
      • Nash D.R.
      • Boomsma J.J.
      Prudent sperm use by leaf-cutter ant queens.
      ,
      • den Boer S.P.
      • Baer B.
      • Boomsma J.J.
      Seminal fluid mediates ejaculate competition in social insects.
      ), and that this support is independent of whether SF originates from the same or a different male (
      • den Boer S.P.
      • Baer B.
      • Boomsma J.J.
      Seminal fluid mediates ejaculate competition in social insects.
      ). This is why sperm survival is typically higher in SF from rival males compared with Hayes saline. Phenotypic studies of differential sperm survival in scenarios of sperm competition therefore always require both own-SF and Hayes saline controls.
      Our study shows, to our knowledge for the first time, that it is the proteins in the SpF that neutralize the antagonistic effects of rival SF on sperm survival. Our finding that SpF contains a larger number of proteins involved in oxidation-reduction supports the idea that the queen′s spermatheca evolved a highly specific biochemical machinery to keep sperm viable for long periods of time, consistent with Atta, queens potentially surviving for 20 years and producing in the order of 100 million fertilized eggs (
      • den Boer S.P.
      • Baer B.
      • Boomsma J.J.
      Seminal fluid mediates ejaculate competition in social insects.
      ,
      • den Boer S.P.A.
      • Boomsma J.J.
      • Baer B.
      Seminal fluid enhances sperm viability in the leafcutter ant Atta colombica.
      ,
      • Paynter E.
      • Millar A.H.
      • Welch M.
      • Baer-Imhoof B.
      • Cao D.
      • Baer B.
      Insights into the molecular basis of long-term storage and survival of sperm in the honeybee (Apis mellifera,).
      ). Both proteomes appeared to contain a large number of proteins involved in protein degradation and proteolysis, which we were able to functionally link to both the initiation and termination of mutual sperm incapacitation (supplemental Table S2 and supplemental Fig. S2).
      When we inspected the 22 proteins that had changed in abundance after SF had been exposed to SpF (DIGE experiment), we found four that increased in abundance to have known functions in energy metabolism and that three are involved in response to oxidative stress and immunity (Table I, Fig. 4, and supplemental Table S3). Both these functions seem logical as adaptations to maximize queen survival and fertility. However, the SF proteins that were significantly reduced in abundance provided direct insights into the expression and control of sexual conflicts in leaf-cutting ants. Using a serine-specific protease inhibitor, we were able to reproduce the SpF neutralizing effect in vitro, which confirmed that serine proteases are directly involved in sperm incapacitation (Fig. 3C,) and that they are eliminated by SpF proteins. Indeed, four proteins reduced in abundance are involved in proteolysis, including the serine proteases easter and snake.
      In Drosophila, snake (specific to SF in Atta,) and easter (present in both SpF and SF proteomes of Atta,) are part of a proteolytic cascade that ultimately activates the Toll receptor during embryo development (
      • Misra S.
      • Hecht P.
      • Maeda R.
      • Anderson K.V.
      Positive and negative regulation of Easter, a member of the serine protease family that controls dorsal-ventral patterning in the Drosophila, embryo.
      ,
      • Smith C.L.
      • Giordano H.
      • Schwartz M.
      • DeLotto R.
      Spatial regulation of Drosophila, Snake protease activity in the generation of dorsal-ventral polarity.
      ), which initiates the innate immune system (
      • Valanne S.
      • Wang J.-H.
      • Rämet M.
      The Drosophila, Toll signaling pathway.
      ) by responding to the protein spätzle (
      • Jang I.H.
      • Chosa N.
      • Kim S.H.
      • Nam H.J.
      • Lemaitre B.
      • Ochiai M.
      • Kambris Z.
      • Brun S.
      • Hashimoto C.
      • Ashida M.
      • Brey P.T.
      • Lee W.J.
      A spatzle-processing enzyme required for toll signaling activation in Drosophila, innate immunity.
      ). This suggests that there is a connection between the incapacitation of rival sperm and cellular immunity functions in SF, consistent with other insect homologs of easter and snake being involved in the pro-phenoloxidase cascade (
      • Kim M.S.
      • Baek M.J.
      • Lee M.H.
      • Park J.W.
      • Lee S.Y.
      • Soderhall K.
      • Lee B.L.
      A new easter-type serine protease cleaves a masquerade-like protein during prophenoloxidase activation in Holotrichia diomphalia, larvae.
      ,
      • Wang Y.
      • Jiang H.
      Reconstitution of a branch of the Manduca sexta, prophenoloxidase activation cascade in vitro: Snake-like hemolymph proteinase 21 (HP21) cleaved by HP14 activates prophenol oxidase- activating proteinase-2 precursor.
      ), which determines innate immune defenses mediated by self-non-self-recognition. Sperm incapacitation by rival SF could therefore have evolved via modifications of immune proteins that originally functioned to recognize pathogens and minimize disease transmission during mating in the strictly monogamous ancestors of the leaf-cutting ants. Proteins of the innate immune system are also very abundant in the SF of polyandrous honeybees (
      • Grassl J.
      • Peng Y.
      • Baer-Imhoof B.
      • Welch M.
      • Millar A.H.
      • Baer B.
      Infections with the sexually transmitted pathogen Nosema apis, trigger an immune response in the seminal fluid of honey bees (Apis mellifera,).
      ) and have been shown to kill sexually transmitted diseases with high efficiency (
      • Peng Y.
      • Grassl J.
      • Millar A.H.
      • Baer B.
      Seminal fluid of honeybees contains multiple mechanisms to combat infections of the sexually transmitted pathogen Nosema apis.
      ).
      We detected that fragments of SERPIN B10 and of several serine proteases increased in abundance, indicating that the intact proteins were cleaved when SF came into contact with SpF (Table I). SERPINs are a class of protease inhibitors that irreversibly target serine proteases, which can mediate sperm-egg recognition (reviewed (
      • Laflamme B.A.
      • Wolfner M.F.
      Identification and function of proteolysis regulators in seminal fluid.
      ,
      • Huntington J.A.
      Serpin structure, function and dysfunction.
      )). Serine proteases and SERPINs are further known to affect proteolytic cascades responsible for incapacitating and preserving sperm in mammals and solitary insects by protecting sperm against damage from female immune reactions (
      • Laflamme B.A.
      • Wolfner M.F.
      Identification and function of proteolysis regulators in seminal fluid.
      ). This is once more consistent with the conjecture that immune proteins might have been precursors for the evolution of sperm competition proteins (
      • Laflamme B.A.
      • Wolfner M.F.
      Identification and function of proteolysis regulators in seminal fluid.
      ). We detected a total of five different SERPIN proteins in the total SpF proteome, two of which were also present in the SF proteome, including SERPIN B10, the protein we found to be specifically targeted by female secretions. This suggests that female spermathecal secretions specifically eliminate easter and snake (especially the latter, which is SF specific), possibly with the three SpF-specific SERPINs.
      As to the remaining proteins targeted by the SpF (Table I), carboxypeptidase B, acid phosphatase, and hydroxyacid-oxoacid transhydrogenase have many biological functions, including protein digestion and regulating the insect molting process (
      • Gatehouse J.A.
      Insect Carboxypeptidases.
      ,
      • Ote M.
      • Mita K.
      • Kawasaki H.
      • Daimon T.
      • Kobayashi M.
      • Shimada T.
      Identification of molting fluid carboxypeptidase A (MF-CPA) in Bombyx mori.
      ). The uncharacterized protein Acol_04113, the most abundant protein in A. colombica, SF but not detected in SpF, shows strong homology with a family of uncharacterized ant proteins (
      • Nygaard S.
      • Hu H.
      • Li C.
      • Schiøtt M.
      • Chen Z.
      • Yang Z.
      • Xie Q.
      • Ma C.
      • Deng Y.
      • Dikow R.B.
      • Rabeling C.
      • Nash D.R.
      • Wcislo W.T.
      • Brady S.G.
      • Schultz T.R.
      • Zhang G.
      • Boomsma J.J.
      Reciprocal genomic evolution in the ant-fungus agricultural symbiosis.
      ) that all share an Armadillo-like domain normally used in protein binding. Uncharacterized protein Acol_04113 also has some degree of homology with mucins, which are glycoproteins belonging to the extracellular matrix, and with the BRO-1 domain involved in protein targeting to the lysozyme. Acol_04113 is degraded into at least three fragments when SF and SpF come into contact (supplemental Fig. S3), highlighting its importance as target for elimination by SpF.
      The acid phosphatase, which has also been found in moth testes (
      • Gilbert L.I.
      • Huddleston C.J.
      Testicular acid phosphatase in giant silkmoths.
      ), cleaves phosphate groups from other proteins and has been shown to liquefy human ejaculates, where it has consistently negative effects on male fertility in conjunction with the serine protease kallikrein (
      • Brillard-Bourdet M.
      • Réhault S.
      • Juliano L.
      • Ferrer M.
      • Moreau T.
      • Gauthier F.
      Amidolytic activity of prostatic acid phosphatase on human semenogelins and semenogelin-derived synthetic substrates.
      ). Finally, Atta, SpF reduced the abundance of hydroxyacid-oxoacid transhydrogenase in SF, an enzyme that catalyzes several oxidation-reduction reactions, including the interconversion of 2-hydroxyglutarate and 2-oxoglutarate, which are key intermediates in the TCA cycle and amino acid metabolism (
      • Kardon T.
      • Noël G.
      • Vertommen D.
      • Schaftingen E.V.
      Identification of the gene encoding hydroxyacid-oxoacid transhydrogenase, an enzyme that metabolizes 4-hydroxybutyrate.
      ). Further research will be required to resolve the complex molecular functioning of these less-well-studied proteins in more detail, but our results suggest that sexual conflict over sperm storage is mediated by very few proteins and that the known characteristics of these proteins are consistent with them being instruments of antagonism between male and female reproductive interests (Table I).
      Our follow-up experiments generated several lines of independent evidence that the proteins we identified are indeed instrumental in the dynamics of sperm competition and sperm preservation, even though further details remain to be studied in the future. Increased exposure time of SF to SpF in vivo, increased the survival of rival sperm, and a mixture of general protease inhibitors and a specific serine protease inhibitor both terminated the harmful effect of SF on rival sperm. These in vitro results corroborate the idea that spermathecal secretions have a general and indiscriminate preservation function for the viability of newly stored sperm mixtures (see (
      • den Boer S.P.
      • Baer B.
      • Boomsma J.J.
      Seminal fluid mediates ejaculate competition in social insects.
      ) for phenotypic evidence) by neutralizing hostile SF proteins. In particular, serine proteases from SF seem to have an important role and will likely be gratifying targets in follow-up studies.
      A mechanistic understanding of sexual conflict does not only require knowledge of the interactions between male and female proteomes but also depends on the morphological traits that determine the efficiency by which the sexes can impose and control their respective interests. Ants and many other evolutionary-derived hymenopteran social bees are peculiar in that males are unable to physically enhance their individual interests because they either die shortly after copulation or continue to seek copulations with other queens (
      • Boomsma J.J.
      • Baer B.
      • Heinze J.
      The evolution of male traits in social insects.
      ). This provides newly inseminated queens with considerable opportunities to control the sperm storage process in ways that maximise female reproductive success. Consistent with this logic, dissection of artificially and naturally inseminated A. colombica, queens revealed that spermathecae are highly muscular and able to contract continuously between the two spermathecal lobes (supplemental Movie S1). These contractions are likely instrumental for queens to mix newly stored sperm with spermathecal gland secretions, and their existence is consistent with queens rapidly gaining control over hostile interactions between rival ejaculates.
      Comparisons between our results and those obtained for honeybees (
      • Grassl J.
      • Peng Y.
      • Baer-Imhoof B.
      • Welch M.
      • Millar A.H.
      • Baer B.
      Infections with the sexually transmitted pathogen Nosema apis, trigger an immune response in the seminal fluid of honey bees (Apis mellifera,).
      ,
      • Baer B.
      • Heazlewood J.L.
      • Taylor N.L.
      • Eubel H.
      • Millar A.H.
      The seminal fluid proteome of the honeybee Apis mellifera.
      ) and fruit flies (
      • Ravi Ram K.
      • Wolfner M.F.
      Seminal influences: Drosophila, Acps and the molecular interplay between males and females during reproduction.
      ,
      • Findlay G.D.
      • Sitnik J.L.
      • Wang W.
      • Aquadro C.F.
      • Clark N.L.
      • Wolfner M.F.
      Evolutionary rate covariation identifies new members of a protein network required for Drosophila melanogaster, female post-mating responses.
      ) showed that the A. colombica, SF proteins targeted by SpF are not expressed in the SF of these other insects (supplemental Table S4), even though homologs are universally present in their genomes. This mutual exclusiveness is consistent with the independent evolution of polyandry in these insect lineages (
      • Boomsma J.J.
      Beyond promiscuity: Mate-choice commitments in social breeding.
      ) but also suggests rapid evolutionary dynamics and continuous recruitment (over evolutionary time) of novel proteins for attack and defense during insemination. In a more general comparative sense, the 24% overlap between SF and SpF proteomes in Atta, (Fig. 4) was similar to the 20% overlap reported in honeybees (
      • Baer B.
      • Heazlewood J.L.
      • Taylor N.L.
      • Eubel H.
      • Millar A.H.
      The seminal fluid proteome of the honeybee Apis mellifera.
      ). This overall proportional similarity may reflect functional evolutionary convergence across social insect lineages that independently evolved obligate polyandry from ancestral ants and bees that were monandrous (
      • Boomsma J.J.
      Beyond promiscuity: Mate-choice commitments in social breeding.
      ). These similarities across taxa are consistent with SF of polyandrous social insects being universally selected to enhance short-term sperm survival and to express proteolytic activity to reduce the survival of rival sperm as shown phenotypically in previous studies of Atta, leaf cutter ants and honeybees (
      • den Boer S.P.
      • Baer B.
      • Boomsma J.J.
      Seminal fluid mediates ejaculate competition in social insects.
      ). In contrast, female secretions are expected to have evolved molecular mechanisms to sustain long-term sperm storage and to neutralize hostile SF proteins, but there is no compelling reason to expect that ants and bees recruited the same proteins for their sexually antagonistic arms races.
      Taken together, the results of our study underline that complete lack of re-mating later in life imposes strong selective pressure on polyandrous ant queens to prevail in evolutionary arms races with their mates to secure maximal storage of both viable and genetically diverse sperm. Whether queens are capable to discriminate between sperm from different males during this process is unknown, but any such cryptic female choice would have to be based on mechanisms that are more complex than self-non-self-recognition, which appears to drive the interactions we document here. In this context, it might be of interest that the SF-specific protein Acol_04113 sharply decreases in abundance in the spermatheca after insemination. The partial homology of this protein with mucins, which are glycoproteins known to mediate complex recognition processes via differential glycosylation (
      • Hollingsworth M.A.
      • Swanson B.J.
      Mucins in cancer: Protection and control of the cell surface.
      ), suggests that Acol_04113 could mediate preferential recognition and discrimination between variants of the same protein molecules. Interactions of this kind have been shown to be important for male fertility in humans because they affect sperm binding to oocytes (
      • Saraswat M.
      • Joenväärä S.
      • Tomar A.K.
      • Singh S.
      • Yadav S.
      • Renkonen R.
      N-Glycoproteomics of human seminal plasma glycoproteins.
      ,
      • Pang P.-C.
      • Chiu P.C.
      • Lee C.-L.
      • Chang L.-Y.
      • Panico M.
      • Morris H.R.
      • Haslam S.M.
      • Khoo K.-H.
      • Clark G.F.
      • Yeung W.S.
      • Dell A.
      Human sperm binding is mediated by the sialyl-Lewis (x) oligosaccharide on the zona pellucida.
      ). Further research in this area will be required to understand the mechanisms that inseminated queens might use to discriminate between ejaculates.
      To our knowledge, we provide the first study that documents proteomic interactions at a high level of detail in any social insect. The insights that we obtained match predictions from evolutionary theory and underline the unique opportunities that ants and other social insects provide for studying the dynamics of sexual conflict. The interactions that we elucidated appear to involve a limited set of male and female proteins as molecular agents or counteragents (either alone or as key parts of molecular cascades) for sperm competition and sperm survival during insemination and sperm storage. The involvement of rather few specific proteases and protease inhibitors will facilitate future studies of the molecular dynamics that induce or avoid harm to sperm cells during the sperm storage process in Atta, and possibly also other polyandrous social insects such as the honeybee and some vespine wasps (
      • Boomsma J.J.
      Beyond promiscuity: Mate-choice commitments in social breeding.
      ).

      DATA AVAILABILITY

      The datasets supporting this article have been submitted to PRIDE (PXD011306 for the SF and SpF proteomes and PXD011320 for the DIGE experiment) and are available for download online. Eleven additional files are available as supplement of the online version of this paper, including four supplemental figures, four supplemental tables, two supplemental files, and a supplemental movie.

      Acknowledgments

      This study was funded by a Queen Elizabeth II Fellowship, a Future Fellowship and a Discovery Project from the Australian Research Council (ARC) to B.B., a start-up grant to B.B. offered by the University of California Riverside, a Centre of Excellence grant by the Danish National Research Foundation (DNRF57) and an Advanced ERC grant to J.J.B., and a Marie-Curie International Outgoing Fellowship to S.P.A.dB. We gratefully acknowledge the Smithsonian Tropical Research Institute in Panama for providing facilities and logistic support, and the Autoridad Nacional de Ambiente (ANAM) for issuing collection and export permits for collected ant samples. We thank Marlene Stürup for help with the field experiments and Clelia Gasparini, Joanito Liberti, and Harvey Millar for helpful discussions.

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