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Proteins, Transcripts, and Genetic Architecture of Seminal Fluid and Sperm in the Mosquito Aedes aegypti*

  • Author Footnotes
    ** Authors contributed equally to this work.
    Ethan C. Degner
    Footnotes
    ** Authors contributed equally to this work.
    Affiliations
    From the ‡Department of Entomology, Cornell University, Ithaca, New York;
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  • Author Footnotes
    ** Authors contributed equally to this work.
    Yasir H. Ahmed-Braimah
    Footnotes
    ** Authors contributed equally to this work.
    Affiliations
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York;
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  • Kirill Borziak
    Affiliations
    Center for Reproductive Evolution, Syracuse University, Syracuse, New York
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  • Mariana F. Wolfner
    Correspondence
    To whom correspondence may be addressed.
    Affiliations
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York;
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  • Laura C. Harrington
    Correspondence
    To whom correspondence may be addressed.
    Affiliations
    From the ‡Department of Entomology, Cornell University, Ithaca, New York;
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  • Steve Dorus
    Correspondence
    To whom correspondence may be addressed.
    Affiliations
    Center for Reproductive Evolution, Syracuse University, Syracuse, New York
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  • Author Footnotes
    * This study was supported by NIH/NIAID grant R01AI095491 to MFW and LCH, NIH/NICHD grant R21HD088910 to S.D. and MFW, a Cornell Graduate School fellowship to ECD, and a Cornell Entomology Department Griswold grant to ECD and LCH. YHAB was supported by NIH/NICHD grant R01HD059060 to MFW and Andrew G. Clark. RNA-seq data and mass spectrometry data were made possible by NIH grants 1S10OD010693-01 and 1S10OD017992-01, respectively. Proteomics computing was supported by NSF grant OAC-1541396/ACI-1541396 to Eric Sedore of the Syracuse University Information Technology Services. Mosquito images in Graphical Abstract used with permission, © 2016 David Felix Duneau.
    This article contains supplemental material.
    ** Authors contributed equally to this work.
Open AccessPublished:December 14, 2018DOI:https://doi.org/10.1074/mcp.RA118.001067
      The yellow fever mosquito, Aedes aegypti, transmits several viruses causative of serious diseases, including dengue, Zika, and chikungunya. Some proposed efforts to control this vector involve manipulating reproduction to suppress wild populations or to replace them with disease-resistant mosquitoes. The design of such strategies requires an intimate knowledge of reproductive processes, yet our basic understanding of reproductive genetics in this vector remains largely incomplete. To accelerate future investigations, we have comprehensively catalogued sperm and seminal fluid proteins (SFPs) transferred to females in the ejaculate using tandem mass spectrometry. By excluding female-derived proteins using an isotopic labeling approach, we identified 870 sperm proteins and 280 SFPs. Functional composition analysis revealed parallels with known aspects of sperm biology and SFP function in other insects. To corroborate our proteome characterization, we also generated transcriptomes for testes and the male accessory glands—the primary contributors to Ae. aegypti, sperm and seminal fluid, respectively. Differential gene expression of accessory glands from virgin and mated males suggests that transcripts encoding proteins involved in protein translation are upregulated post-mating. Several SFP transcripts were also modulated after mating, but >90% remained unchanged. Finally, a significant enrichment of SFPs was observed on chromosome 1, which harbors the male sex determining locus in this species. Our study provides a comprehensive proteomic and transcriptomic characterization of ejaculate production and composition and thus provides a foundation for future investigations of Ae. aegypti, reproductive biology, from functional analysis of individual proteins to broader examination of reproductive processes.

      Graphical Abstract

      The mosquito, Aedes aegypti, is the most important vector of arboviruses globally, transmitting viruses that cause dengue (
      • Bhatt S.
      • Gething P.W.
      • Brady O.J.
      • Messina J.P.
      • Farlow A.W.
      • Moyes C.L.
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      • Brownstein J.S.
      • Hoen A.G.
      • Sankoh O.
      • Myers M.F.
      • George D.B.
      • Jaenisch T.
      • Wint G.R.W.
      • Simmons C.P.
      • Scott T.W.
      • Farrar J.J.
      • Hay S.I.
      The global distribution and burden of dengue.
      ), Zika (
      • Chouin-Carneiro T.
      • Vega-Rua A.
      • Vazeille M.
      • Yebakima A.
      • Girod R.
      • Goindin D.
      • Dupont-Rouzeyrol M.
      • Lourenco-de-Oliveira R.
      • Failloux A.B.
      Differential susceptibilities of Aedes aegypti Aedes albopictus, from the Americas to Zika virus.
      ), chikungunya (
      • Pialoux G.
      • Gauzere B.A.
      • Jaureguiberry S.
      • Strobel M.
      Chikungunya, an epidemic arbovirosis.
      ), and yellow fever (
      • Monath T.P.
      • Vasconcelos P.F.
      Yellow fever.
      ). Consequently, Ae. aegypti, places a severe strain on public health infrastructure around the world (
      • Fredericks A.C.
      • Fernandez-Sesma A.
      The burden of dengue and chikungunya worldwide: implications for the southern United States and California.
      ). Despite decades of effort to control mosquito populations, Ae. aegypti, continues to contribute to human disease epidemics. New and improved control strategies are needed to prevent future outbreaks and mitigate disease burden.
      Some promising control strategies under development target reproduction to suppress mosquito populations. For example, sterilized males can be released to suppress populations by impairing reproduction by their wild mates (
      • Klassen W.
      • Curtis C.F.
      History of the Sterile Insect Technique.
      ,
      • Carvalho D.O.
      • McKemey A.R.
      • Garziera L.
      • Lacroix R.
      • Donnelly C.A.
      • Alphey L.
      • Malavasi A.
      • Capurro M.L.
      Suppression of a field population of Aedes aegypti, in Brazil by sustained release of transgenic male mosquitoes.
      ,
      • Macias V.M.
      • Ohm J.R.
      • Rasgon J.L.
      Gene drive for mosquito control: where did it come from and where are we headed?.
      ). Manipulating reproductive phenotypes may also provide a means of driving disease-refractory traits into a population (reviewed in 8). One such strategy employs the intracellular bacterium Wolbachia, which, when introduced into Ae. aegypti, induces cytoplasmic incompatibility that allows the bacterium to spread in a population, potentially to fixation (
      • Hoffmann A.A.
      • Montgomery B.L.
      • Popovici J.
      • Iturbe-Ormaetxe I.
      • Johnson P.H.
      • Muzzi F.
      • Greenfield M.
      • Durkan M.
      • Leong Y.S.
      • Dong Y.
      • Cook H.
      • Axford J.
      • Callahan A.G.
      • Kenny N.
      • Omodei C.
      • McGraw E.A.
      • Ryan P.A.
      • Ritchie S.A.
      • Turelli M.
      • O'Neill S.L.
      Successful establishment of Wolbachia Aedes, populations to suppress dengue transmission.
      ). Cytoplasmic incompatibility causes sperm of males with Wolbachia, to be incompatible with uninfected females' eggs, whereas Wolbachia-,positive females can reproduce with any male, regardless of infection status (reviewed in
      • LePage D.
      • Bordenstein S.R.
      Wolbachia,: Can we save lives with a great pandemic?.
      ), giving Wolbachia,-positive individuals a fitness advantage over their uninfected counterparts. This bacterium also blocks or reduces transmission of several viruses, including dengue (
      • Walker T.
      • Johnson P.H.
      • Moreira L.A.
      • Iturbe-Ormaetxe I.
      • Frentiu F.D.
      • McMeniman C.J.
      • Leong Y.S.
      • Dong Y.
      • Axford J.
      • Kriesner P.
      • Lloyd A.L.
      • Ritchie S.A.
      • O'Neill S.L.
      • Hoffmann A.A.
      The wMel Wolbachia, strain blocks dengue and invades caged Aedes aegypti, populations.
      ) and Zika (
      • Dutra H.L.
      • Rocha M.N.
      • Dias F.B.
      • Mansur S.B.
      • Caragata E.P.
      • Moreira L.A.
      Wolbachia, blocks currently circulating Zika virus isolates in Brazilian Aedes aegypti, mosquitoes.
      ). Consequently, introduction of novel Wolbachia, infections into vector populations is being explored as a transmission reducing strategy.
      Designing mosquito control strategies that target reproduction requires an intimate knowledge of the underlying cellular and molecular mechanisms. Yet, only a few functions of proteins involved in mosquito reproduction have been described to date. For example, in Ae. aegypti, seminal fluid proteins (SFPs)
      The abbreviations used are: SFP, seminal fluid protein; MS/MS, tandem mass spectrometry; APEX, absolute protein expression; FDR, false discovery rate; PSM, peptide spectral match; MAG, male accessory gland; dpe, days post eclosion; TPM, transcripts per million; GO, gene ontology; S-LAP, Sperm leucyl-aminopeptidase.
      1The abbreviations used are: SFP, seminal fluid protein; MS/MS, tandem mass spectrometry; APEX, absolute protein expression; FDR, false discovery rate; PSM, peptide spectral match; MAG, male accessory gland; dpe, days post eclosion; TPM, transcripts per million; GO, gene ontology; S-LAP, Sperm leucyl-aminopeptidase.
      induce several physiological and behavioral changes in females, including refractoriness to future mating (
      • Gwadz R.W.
      • Craig Jr, G.B.
      • Hickey W.A.
      Female sexual behavior as the mechanism rendering Aedes aegypti, refractory to insemination.
      ,
      • Helinski M.E.
      • Deewatthanawong P.
      • Sirot L.K.
      • Wolfner M.F.
      • Harrington L.C.
      Duration and dose-dependency of female sexual receptivity responses to seminal fluid proteins in Aedes albopictus Ae. aegypti, mosquitoes.
      ,
      • Duvall L.B.
      • Basrur N.S.
      • Molina H.
      • McMeniman C.J.
      • Vosshall L.B.
      A peptide signaling system that rapidly enforces paternity in the Aedes aegypti, mosquito.
      ,
      • Fuchs M.S.
      • Craig G.B.
      • Despommier D.D.
      The protein nature of the substance inducing female monogamy in Aedes aegypti.
      ,
      • Fuchs M.S.
      • Craig Jr, G.B.
      • Hiss E.A.
      The biochemical basis of female monogamy in mosquitoes. I. Extraction of the active principle from Aedes aegypti.
      ,
      • Villarreal S.M.
      • Pitcher S.
      • Helinski M.E.H.
      • Johnson L.
      • Wolfner M.F.
      • Harrington L.C.
      Male contributions during mating increase female survival in the disease vector mosquito Aedes aegypti.
      ), stimulation of oogenesis (
      • Klowden M.J.
      • Chambers G.M.
      Male accessory gland substances activate egg development in nutritionally stressed Aedes aegypti, mosquitoes.
      ), enhanced survival (
      • Villarreal S.M.
      • Pitcher S.
      • Helinski M.E.H.
      • Johnson L.
      • Wolfner M.F.
      • Harrington L.C.
      Male contributions during mating increase female survival in the disease vector mosquito Aedes aegypti.
      ), and the ability to fertilize eggs (
      • Adlakha V.
      • Pillai M.K.
      Involvement of male accessory gland substance in the fertility of mosquitoes.
      ). However, the molecular identity of active SFP components for this species remains elusive. Seminal fluid initiates sperm motility via the action of proteases in many insects (silkworm (
      • Nagaoka S.
      • Kato K.
      • Takata Y.
      • Kamei K.
      Identification of the sperm-activating factor initiatorin, a prostatic endopeptidase of the silkworm, Bombyx mori.
      ); water strider (
      • Miyata H.
      • Thaler C.D.
      • Haimo L.T.
      • Cardullo R.A.
      Protease activation and the signal transduction pathway regulating motility in sperm from the water strider Aquarius remigis.
      ); Culex, mosquito (
      • Thaler C.D.
      • Miyata H.
      • Haimo L.T.
      • Cardullo R.A.
      Waveform generation is controlled by phosphorylation and swimming direction is controlled by Ca2+ in sperm from the mosquito Culex quinquefasciatus.
      )), but the precise sperm proteins on which seminal fluid acts in Ae. aegypti, have not been identified. Similarly, sperm-associated odorant receptors may control motility in Ae. aegypti, although the exact function and ligands of these receptors are unknown (
      • Pitts R.J.
      • Liu C.
      • Zhou X.
      • Malpartida J.C.
      • Zwiebel L.J.
      Odorant receptor-mediated sperm activation in disease vector mosquitoes.
      ). Finally, the mechanism by which Wolbachia, induces cytoplasmic incompatibility has not been described in Ae. aegypti, but Wolbachia, proteins contained in sperm are hypothesized to be involved (
      • Beckmann J.F.
      • Fallon A.M.
      Detection of the Wolbachia protein WPIP0282 in mosquito spermathecae: implications for cytoplasmic incompatibility.
      ,
      • Beckmann J.F.
      • Ronau J.A.
      • Hochstrasser M.
      A Wolbachia, deubiquitylating enzyme induces cytoplasmic incompatibility.
      ).
      Identification of sperm proteins and SFPs that are transferred to females during copulation is an important objective to enable future investigations into specific reproductive processes. Components of the transferred ejaculate include sperm and seminal fluid—both of which play vital roles in mosquito reproduction. An Ae. aegypti, seminal fluid proteome was first reported by Sirot et al., (
      • Sirot L.K.
      • Hardstone M.C.
      • Helinski M.E.H.
      • Ribeiro J.M.C.
      • Kimura M.
      • Deewatthanawong P.
      • Wolfner M.F.
      • Harrington L.C.
      Towards a semen proteome of the dengue vector mosquito: protein identification and potential functions.
      ) based on mass spectrometry analyses. That study described 93 putative SFPs transferred during mating. Although not a primary focus of their work, they also identified 101 putative sperm proteins. Later work identified more than twice as many SFPs from Ae. albopictus, using similar methodology (
      • Boes K.E.
      • Ribeiro J.M.C.
      • Wong A.
      • Harrington L.C.
      • Wolfner M.F.
      • Sirot L.K.
      Identification and characterization of seminal fluid proteins in the Asian tiger mosquito, Aedes albopictus.
      ). Proteome complexity of other insects' seminal fluid (reviewed in
      • Avila F.W.
      • Sirot L.K.
      • LaFlamme B.A.
      • Rubinstein C.D.
      • Wolfner M.F.
      Insect seminal fluid proteins: identification and function.
      ,
      • Baer B.
      • Zareie R.
      • Paynter E.
      • Poland V.
      • Millar A.H.
      Seminal fluid proteins differ in abundance between genetic lineages of honeybees.
      ,
      • Sepil I.
      • Hopkins B.R.
      • Dean R.
      • Thezenas M.L.
      • Charles P.D.
      • Konietzny R.
      • Fischer R.
      • Kessler B.M.
      • Wigby S.
      ) and sperm (
      • Wasbrough E.R.
      • Dorus S.
      • Hester S.
      • Howard-Murkin J.
      • Lilley K.
      • Wilkin E.
      • Polpitiya A.
      • Petritis K.
      • Karr T.L.
      The Drosophila melanogaster, sperm proteome-II (DmSP-II).
      ,
      • Zareie R.
      • Eubel H.
      • Millar A.H.
      • Baer B.
      Long-term survival of high quality sperm: insights into the sperm proteome of the honeybee Apis mellifera.
      ,
      • Whittington E.
      • Zhao Q.
      • Borziak K.
      • Walters J.R.
      • Dorus S.
      Characterisation of the Manduca sexta, sperm proteome: Genetic novelty underlying sperm composition in Lepidoptera.
      ) suggests that more proteins remain to be identified in the Ae. aegypti, ejaculate. Here, we used tandem mass spectrometry (MS/MS) with greater sensitivity and the recently revised and expanded genome to build on the foundational work of Sirot et al., by identifying constituent proteins of both Ae. aegypti, sperm and seminal fluid. Importantly, significantly increased coverage of the sperm proteome allowed more accurate differentiation between seminal fluid and sperm proteins in the mixed ejaculate sample. We also profiled the transcriptomes of the male accessory glands (MAG; before and after mating) and testes, the major source tissues for SFPs and sperm proteins, respectively. Our proteomic characterization represents a nearly 4-fold expansion of putative SFPs and a more than 8-fold expansion in the Ae. aegypti, sperm proteome. Our results yield insights into the molecular function, genome organization, regulation, and evolution of sperm proteins and SFPs in this important disease vector. Ultimately, these proteomes provide a basis for future studies of Ae. aegypti, reproduction and, potentially, a catalogue of molecular targets for the development of novel mosquito control methods.

      DISCUSSION

      A rapidly expanding body of evidence supports the critical roles of seminal fluid proteins (SFPs) in a wide array of reproductive phenotypes (reviewed in 29). Although this has been most extensively investigated in Drosophila, seminal peptides and proteins are also associated with post-mating behavioral and physiological responses in mosquitoes such as Ae. aegypti, (
      • Duvall L.B.
      • Basrur N.S.
      • Molina H.
      • McMeniman C.J.
      • Vosshall L.B.
      A peptide signaling system that rapidly enforces paternity in the Aedes aegypti, mosquito.
      ,
      • Fuchs M.S.
      • Craig G.B.
      • Despommier D.D.
      The protein nature of the substance inducing female monogamy in Aedes aegypti.
      ,
      • Villarreal S.M.
      • Pitcher S.
      • Helinski M.E.H.
      • Johnson L.
      • Wolfner M.F.
      • Harrington L.C.
      Male contributions during mating increase female survival in the disease vector mosquito Aedes aegypti.
      ). The primary goals of this study were to comprehensively catalogue male proteins transferred to Ae. aegypti, females during insemination and establish a reliable methodology for delineating between sperm proteins and SFPs. To accomplish this, we (1) conducted an in-depth proteomic characterization of sperm, (2) used a whole-female labeling approach to identify unlabeled male proteins transferred by the male during insemination and (3) characterized the transcriptomes of the testis and male accessory gland (MAG). Importantly, we note that the whole-female labeling approach has been employed previously in Ae. aegypti, but the assignment of proteins as SFPs was limited by the lack of information regarding proteins found in sperm. Thus, distinctions between sperm proteins and SFPs were previously difficult to achieve. In the present study, we have re-assigned 39 proteins identified by Sirot et al.,: 23 previously identified SFPs and 4 sperm proteins as putative components of both sperm and seminal fluid, 10 previously identified SFPs as putative sperm proteins, and 2 previously identified sperm proteins reassigned as high-confidence SFPs. We acknowledge that our MS/MS-based approach still includes some inherent uncertainty, and that this should be kept in mind when interpreting our classifications. It is also noteworthy that advances in MS/MS sensitivity and accuracy have resulted in far greater power of detection in our study, and our analysis has also benefited tremendously from the recent resequencing and reannotation of the Ae. aegypti, genome (
      • Matthews B.J.
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      • Kingan S.
      • Koren S.
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      • Korlach J.
      • Neafsey D.E.
      • Phillippy M.
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      ). Our proteomic characterization resulted in a nearly 4-fold expansion of the current Ae. aegypti, seminal fluid proteome, and an 8-fold expansion of identified proteins in sperm. Together, this Ae. aegypti, “ejaculatome” provides a foundation for future molecular studies of mosquito reproduction and associated applications to control mosquito populations (see below).

      Proteome Characteristics Independently Validate Identification

      Our work differs from previous SFP characterization studies (
      • Sirot L.K.
      • Hardstone M.C.
      • Helinski M.E.H.
      • Ribeiro J.M.C.
      • Kimura M.
      • Deewatthanawong P.
      • Wolfner M.F.
      • Harrington L.C.
      Towards a semen proteome of the dengue vector mosquito: protein identification and potential functions.
      ,
      • Boes K.E.
      • Ribeiro J.M.C.
      • Wong A.
      • Harrington L.C.
      • Wolfner M.F.
      • Sirot L.K.
      Identification and characterization of seminal fluid proteins in the Asian tiger mosquito, Aedes albopictus.
      ,
      • Findlay G.D.
      • Yi X.
      • Maccoss M.J.
      • Swanson W.J.
      Proteomics reveals novel Drosophila, seminal fluid proteins transferred at mating.
      ) in that our classification was supported by a detailed knowledge of sperm proteome composition. Nonetheless, several independent validation approaches were helpful in assessing the quality of our proteomic characterization. For example, we quantified the proportion of proteins with predicted secretion signals and analyzed transcriptome profiles in testes and MAGs. As would be predicted, SFPs identified in this study possessed a significantly higher proportion of predicted secretion signals than sperm proteins. Although only 33% possessed predicted secretion signals in our high-confidence SFPs, this proportion is consistent with what has been reported in seminal fluid of the grasshopper Melanoplus sanguinipes, (
      • Bonilla M.L.
      • Todd C.
      • Erlandson M.
      • Andres J.
      Combining RNA-seq and proteomic profiling to identify seminal fluid proteins in the migratory grasshopper Melanoplus sanguinipes, (F).
      ). The large proportion of proteins that lacked this signal may be because much seminal fluid secretion in Ae. aegypti, has been reported to occur through both apocrine and holocrine mechanisms (
      • Ramalingam S.
      Secretion in the male accessory glands of Aedes aegypti, (L.) (Diptera: Culicidae).
      ,
      • Dapples C.C.
      • Foster W.A.
      • Lea A.O.
      Ultrastructure of the accessory gland of the male mosquito, Aedes aegypti (L.) (Diptera: Culicidae).
      ). The genes encoding high-confidence SFPs were also, on average, highly specific or biased toward expression in the MAG. RNAs encoding over 99% of the identified proteins were also represented in their target tissues' transcriptomes, adding further validation to their identification as seminal fluid and sperm proteins. Those 11 proteins with no expression in their target tissue may represent proteins that were produced outside of the testes or accessory glands (e.g., in adjoining tissues such as the vas deferentia or trafficked into these organs) or transcripts that were not expressed at the time of tissue dissection.
      Analysis of the functional composition of our proteomes revealed that they were closely aligned with the results of previous sperm (
      • Wasbrough E.R.
      • Dorus S.
      • Hester S.
      • Howard-Murkin J.
      • Lilley K.
      • Wilkin E.
      • Polpitiya A.
      • Petritis K.
      • Karr T.L.
      The Drosophila melanogaster, sperm proteome-II (DmSP-II).
      ,
      • Zareie R.
      • Eubel H.
      • Millar A.H.
      • Baer B.
      Long-term survival of high quality sperm: insights into the sperm proteome of the honeybee Apis mellifera.
      ,
      • Whittington E.
      • Zhao Q.
      • Borziak K.
      • Walters J.R.
      • Dorus S.
      Characterisation of the Manduca sexta, sperm proteome: Genetic novelty underlying sperm composition in Lepidoptera.
      ,
      • Dorus S.
      • Busby S.A.
      • Gerike U.
      • Shabanowitz J.
      • Hunt D.F.
      • Karr T.L.
      Genomic and functional evolution of the Drosophila melanogaster, sperm proteome.
      ) and SFP studies in insects (
      • Baer B.
      • Zareie R.
      • Paynter E.
      • Poland V.
      • Millar A.H.
      Seminal fluid proteins differ in abundance between genetic lineages of honeybees.
      ,
      • Findlay G.D.
      • Yi X.
      • Maccoss M.J.
      • Swanson W.J.
      Proteomics reveals novel Drosophila, seminal fluid proteins transferred at mating.
      ). For example, our expanded sperm proteome was highly enriched for proteins related to flagellar structure, including microtubules, dynein complexes, and ciliar components, and proteins likely associated with the mitochondrial derivatives, which are a predominant structure in mosquito sperm (
      • Bao S.N.
      • de Souza W.
      Ultrastructural and cytochemical studies of the spermatid and spermatozoon of Culex quinquefasciatus, (Culicidae).
      ,
      • Ndiaye M.
      • Mattei X.
      • Thiaw O.T.
      Maturation of mosquito spermatozoa during their transit throughout the male and female reproductive systems.
      ) and that of other insects. Consistent with what was described by Sirot et al., (
      • Sirot L.K.
      • Hardstone M.C.
      • Helinski M.E.H.
      • Ribeiro J.M.C.
      • Kimura M.
      • Deewatthanawong P.
      • Wolfner M.F.
      • Harrington L.C.
      Towards a semen proteome of the dengue vector mosquito: protein identification and potential functions.
      ), as well as in other insects (reviewed in
      • Avila F.W.
      • Sirot L.K.
      • LaFlamme B.A.
      • Rubinstein C.D.
      • Wolfner M.F.
      Insect seminal fluid proteins: identification and function.
      ,
      • Baer B.
      • Zareie R.
      • Paynter E.
      • Poland V.
      • Millar A.H.
      Seminal fluid proteins differ in abundance between genetic lineages of honeybees.
      ,
      • Findlay G.D.
      • Yi X.
      • Maccoss M.J.
      • Swanson W.J.
      Proteomics reveals novel Drosophila, seminal fluid proteins transferred at mating.
      ) and humans (
      • Pilch B.
      • Mann M.
      Large-scale and high-confidence proteomic analysis of human seminal plasma.
      ), proteases were highly enriched among our high-confidence SFPs, supporting the likely accuracy of our expanded characterization (reviewed in
      • Laflamme B.A.
      • Wolfner M.F.
      Identification and function of proteolysis regulators in seminal fluid.
      ). The observed enrichment of vesicle-mediated transport proteins is also consistent with the fact that Ae. aegypti, seminal fluid is in part produced by apocrine secretion (
      • Ramalingam S.
      Secretion in the male accessory glands of Aedes aegypti, (L.) (Diptera: Culicidae).
      ). Additionally, exosomes and other vesicles are believed to play a role in a variety of post-insemination cellular interactions. For example, vesicles transferred in Drosophila, seminal fluid have been reported to fuse with sperm and interact with the female reproductive tract (
      • Corrigan L.
      • Redhai S.
      • Leiblich A.
      • Fan S.J.
      • Perera S.M.
      • Patel R.
      • Gandy C.
      • Wainwright S.M.
      • Morris J.F.
      • Hamdy F.
      • Goberdhan D.C.
      • Wilson C.
      BMP-regulated exosomes from Drosophila, male reproductive glands reprogram female behavior.
      ), exosomes of the mouse epididymis have recently been implicated in the control of sperm RNA stores (
      • Sharma U.
      • Sun F.
      • Conine C.C.
      • Reichholf B.
      • Kukreja S.
      • Herzog V.A.
      • Ameres S.L.
      • Rando O.J.
      Small RNAs are trafficked from the epididymis to developing mammalian sperm.
      ), and the abundance of exosome markers in avian SFPs has led to speculation about vesicle-mediated mechanisms in post-testicular sperm maturation (
      • Borziak K.
      • Alvarez-Fernandez A.
      • T L.K.
      • Pizzari T.
      • Dorus S.
      The seminal fluid proteome of the polyandrous red junglefowl offers insights into the molecular basis of fertility, reproductive ageing and domestication.
      ). Therefore, the accuracy of our expanded proteomic characterization of sperm and SFP proteomes is corroborated by several independent lines of evidence.
      It is important to note that, despite the application of stringent proteomic thresholds, some proteins could not be definitively assigned as either sperm protein or SFP. Previous studies in Drosophila, and Lepidoptera have consistently identified known SFPs (such as Drosophila, Acp36DE) at appreciable abundance levels in sperm that have yet to be combined with MAG secretions (
      • Wasbrough E.R.
      • Dorus S.
      • Hester S.
      • Howard-Murkin J.
      • Lilley K.
      • Wilkin E.
      • Polpitiya A.
      • Petritis K.
      • Karr T.L.
      The Drosophila melanogaster, sperm proteome-II (DmSP-II).
      ,
      • Whittington E.
      • Forsythe D.
      • Borziak K.
      • Karr T.L.
      • Walters J.R.
      • Dorus S.
      Contrasting patterns of evolutionary constraint and novelty revealed by comparative sperm proteomic analysis in Lepidoptera.
      ,
      • Dorus S.
      • Busby S.A.
      • Gerike U.
      • Shabanowitz J.
      • Hunt D.F.
      • Karr T.L.
      Genomic and functional evolution of the Drosophila melanogaster, sperm proteome.
      ). Our identification of a relatively large protein set that is highly MAG-biased in expression but also present in sperm further suggests that the incorporation of “SFPs” during testicular sperm maturation occurs and is worthy of additional functional investigation. Although Drosophila, expression profiles in the testis and accessory gland are quite distinct, many SFPs exhibit low levels of co-expression in the testis (Dorus, unpublished data). Our transcriptomic analyses here further support such patterns of co-expression. As such, dichotomous distinctions between sperm proteins and SFPs may be an oversimplification of a more nuanced relationship between these reproductive systems. We acknowledge this ambiguity in our classification of MAG-biased proteins that were also identified in our sperm proteome. We also note that our sperm purification method could have allowed the inclusion of seminal fluid proteins (that is, nonsperm ejaculate proteins) that are produced in the seminal vesicle, vas deferentia, or testes. Similarly, the possibility exists that some male accessory gland proteins may migrate into the seminal vesicle. Although the contribution of such proteins to the sperm proteome, if any, is likely small, we cannot rule out this uncertainty.
      We also note that despite our expanded proteomic coverage, several proteins that we anticipated to be identified were absent. The most notable case was Head Peptide-1, a seminal fluid peptide which has been shown to be transferred in the ejaculate (
      • Naccarati C.
      • Audsley N.
      • Keen J.N.
      • Kim J.H.
      • Howell G.J.
      • Kim Y.J.
      • Isaac R.E.
      The host-seeking inhibitory peptide, Aea-HP-1, is made in the male accessory gland and transferred to the female during copulation.
      ,
      • Chen P.S.
      • Stumm-Zollinger E.
      • Aigaki T.
      • Balmer J.
      • Bienz M.
      • Bohlen P.
      A male accessory gland peptide that regulates reproductive behavior of female D. melanogaster.
      ) and has been reported to induce short term monogamy in the female after mating (
      • Duvall L.B.
      • Basrur N.S.
      • Molina H.
      • McMeniman C.J.
      • Vosshall L.B.
      A peptide signaling system that rapidly enforces paternity in the Aedes aegypti, mosquito.
      ). Head Peptide-1, like many SFPs, undergoes extensive post-translational modification and may therefore be challenging to identify bioinformatically without a priori, knowledge of the biochemical composition of the proteolytic products (such as in the case of the well-studied Drosophila, Sex Peptide; 82). Another example was adipokinetic hormone (AAEL011996), which did not meet our two unique peptide inclusion threshold, although we did identify five copies of one peptide from its precursor protein that was also identified in Ae. albopictus, seminal fluid (
      • Boes K.E.
      • Ribeiro J.M.C.
      • Wong A.
      • Harrington L.C.
      • Wolfner M.F.
      • Sirot L.K.
      Identification and characterization of seminal fluid proteins in the Asian tiger mosquito, Aedes albopictus.
      ). This protein has been postulated to contribute to sperm protection from oxidative stress (
      • Bednarova A.
      • Kodrik D.
      • Krishnan N.
      Adipokinetic hormone exerts its anti-oxidative effects using a conserved signal-transduction mechanism involving both PKC and cAMP by mobilizing extra- and intracellular Ca2+ stores.
      ) and the regulation of feeding behavior (
      • Konuma T.
      • Morooka N.
      • Nagasawa H.
      • Nagata S.
      Knockdown of the adipokinetic hormone receptor increases feeding frequency in the two-spotted cricket Gryllus bimaculatus.
      ) in other insects. We suggest that the complexity of proteolytic pathways, governed both by male and female interacting proteins, is a major barrier in the use of shotgun proteomics to study SFP identity and function in the female reproductive tract. Conducting similar analyses with an alternative digestive enzyme or de novo, peptide sequencing may allow the detection of additional proteins whose tryptic products could not be identified using standard database searches. Searching for proteins in the supernatant of our sperm samples may also identify soluble proteins that associate with the surface of sperm, as has been shown with sex peptide in D. melanogaster, (
      • Peng J.
      • Chen S.
      • Busser S.
      • Liu H.
      • Honegger T.
      • Kubli E.
      Gradual release of sperm bound sex-peptide controls female postmating behavior in Drosophila.
      ). Finally, future investigations would also likely benefit from the inclusion of a targeted proteomic approach (reviewed in
      • Borras E.
      • Sabido E.
      What is targeted proteomics? A concise revision of targeted acquisition and targeted data analysis in mass spectrometry.
      ). Such approaches require an a priori, list of candidate peptides; in Ae. aegypti, the neuropeptides and protein hormones catalogued by Predel et al., (
      • Predel R.
      • Neupert S.
      • Garczynski S.F.
      • Crim J.W.
      • Brown M.R.
      • Russell W.K.
      • Kahnt J.
      • Russell D.H.
      • Nachman R.J.
      Neuropeptidomics of the mosquito Aedes aegypti.
      ,
      • Swanson W.J.
      • Vacquier V.D.
      The rapid evolution of reproductive proteins.
      ) represent a useful pool of potentially important molecules.

      Evolution of Male Reproductive Proteomes

      Male reproductive proteins, including SFPs, are consistently among the fastest evolving classes of protein (reviewed in 88). Although initially a goal of our study, conducting a robust analysis of the molecular evolution of proteins identified in this study was limited by the availability of genomic resources appropriate for both inter- and intraspecific tests of positive selection. Obtaining high quality genomic data for different populations of Ae. aegypti, has proven difficult, given the genome's repetitive nature (
      • Matthews B.J.
      • Dudchenko O.
      • Kingan S.
      • Koren S.
      • Antoshechkin I.
      • Crawford J.E.
      • Glassford W.J.
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      • Redmond S.N.
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      • Wu Y.
      • Batra S.S.
      • Brito-Sierra C.A.
      • Buckingham S.D.
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      • Chan S.
      • Cox E.
      • Evans B.R.
      • Fansiri T.
      • Filipovic I.
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      • Gloria-Soria A.
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      • Kodali V.
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      • Muehling J.
      • Omer A.
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      • Peluso P.
      • Aiden A.P.
      • Ramasamy V.
      • Rasic G.
      • Roy S.
      • Saavedra-Rodriguez K.
      • Sharan S.
      • Sharma A.
      • Smith M.
      • Turner J.
      • Weakley A.M.
      • Zhao Z.
      • Akbari O.S.
      • Black W.C.
      • Cao H.
      • Darby A.C.
      • Hill C.
      • Johnston J.S.
      • Murphy T.D.
      • Raikhel A.S.
      • Sattelle D.B.
      • Sharakhov I.V.
      • White B.J.
      • Zhao L.
      • Aiden E.L.
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      • Tu Z.
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      • Korlach J.
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      • Phillippy M.
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      Improved reference genome of Aedes aegypti, informs arbovirus vector control.
      ,
      • Nene V.
      • Wortman J.R.
      • Lawson D.
      • Haas B.
      • Kodira C.
      • Tu Z.J.
      • Loftus B.
      • Xi Z.Y.
      • Megy K.
      • Grabherr M.
      • Ren Q.H.
      • Zdobnov E.M.
      • Lobo N.F.
      • Campbell K.S.
      • Brown S.E.
      • Bonaldo M.F.
      • Zhu J.S.
      • Sinkins S.P.
      • Hogenkamp D.G.
      • Amedeo P.
      • Arensburger P.
      • Atkinson P.W.
      • Bidwell S.
      • Biedler J.
      • Birney E.
      • Bruggner R.V.
      • Costas J.
      • Coy M.R.
      • Crabtree J.
      • Crawford M.
      • deBruyn B.
      • DeCaprio D.
      • Eiglmeier K.
      • Eisenstadt E.
      • El-Dorry H.
      • Gelbart W.M.
      • Gomes S.L.
      • Hammond M.
      • Hannick L.I.
      • Hogan J.R.
      • Holmes M.H.
      • Jaffe D.
      • Johnston J.S.
      • Kennedy R.C.
      • Koo H.
      • Kravitz S.
      • Kriventseva E.V.
      • Kulp D.
      • LaButti K.
      • Lee E.
      • Li S.
      • Lovin D.D.
      • Mao C.H.
      • Mauceli E.
      • Menck C.F.M.
      • Miller J.R.
      • Montgomery P.
      • Mori A.
      • Nascimento A.L.
      • Naveira H.F.
      • Nusbaum C.
      • O'Leary S.
      • Orvis J.
      • Pertea M.
      • Quesneville H.
      • Reidenbach K.R.
      • Rogers Y.H.
      • Roth C.W.
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      • Schatz M.
      • Shumway M.
      • Stanke M.
      • Stinson E.O.
      • Tubio J.M.C.
      • VanZee J.P.
      • Verjovski-Almeida S.
      • Werner D.
      • White O.
      • Wyder S.
      • Zeng Q.D.
      • Zhao Q.
      • Zhao Y.M.
      • Hill C.A.
      • Raikhel A.S.
      • Soares M.B.
      • Knudson D.L.
      • Lee N.H.
      • Galagan J.
      • Salzberg S.L.
      • Paulsen I.T.
      • Dimopoulos G.
      • Collins F.H.
      • Birren B.
      • Fraser-Liggett C.M.
      • Severson D.W.
      Genome sequence of Aedes aegypti, a major arbovirus vector.
      ). Further, this mosquito's ability to move globally as diapausing eggs has allowed for frequent mixing and a complex population structure (
      • Moore M.
      • Sylla M.
      • Goss L.
      • Burugu M.W.
      • Sang R.
      • Kamau L.W.
      • Kenya E.U.
      • Bosio C.
      • Munoz Mde L.
      • Sharakova M.
      • Black W.C.
      Dual African origins of global Aedes aegypti, s.l. populations revealed by mitochondrial DNA.
      ,
      • Shi Q.M.
      • Zhang H.D.
      • Wang G.
      • Guo X.X.
      • Xing D.
      • Dong Y.D.
      • Xiao L.
      • Gao J.
      • Liu Q.M.
      • Sun A.J.
      • Li C.X.
      • Zhao T.Y.
      The genetic diversity and population structure of domestic Aedes aegypti, (Diptera: Culicidae) in Yunnan Province, southwestern China.
      ). The development of appropriate population level genetic data for the analysis of recent selective sweeps should be a priority in Ae. aegypti, as it has been in Anopheles gambiae, (
      • Lawniczak M.K.
      • Emrich S.J.
      • Holloway A.K.
      • Regier A.P.
      • Olson M.
      • White B.
      • Redmond S.
      • Fulton L.
      • Appelbaum E.
      • Godfrey J.
      • Farmer C.
      • Chinwalla A.
      • Yang S.P.
      • Minx P.
      • Nelson J.
      • Kyung K.
      • Walenz B.P.
      • Garcia-Hernandez E.
      • Aguiar M.
      • Viswanathan L.D.
      • Rogers Y.H.
      • Strausberg R.L.
      • Saski C.A.
      • Lawson D.
      • Collins F.H.
      • Kafatos F.C.
      • Christophides G.K.
      • Clifton S.W.
      • Kirkness E.F.
      • Besansky N.J.
      Widespread divergence between incipient Anopheles gambiae, species revealed by whole genome sequences.
      ,
      • Reidenbach K.R.
      • Neafsey D.E.
      • Costantini C.
      • Sagnon N.
      • Simard F.
      • Ragland G.J.
      • Egan S.P.
      • Feder J.L.
      • Muskavitch M.A.
      • Besansky N.J.
      Patterns of genomic differentiation between ecologically differentiated M and S forms of Anopheles gambiae, in West and Central Africa.
      ). Furthermore, given the extent of molecular divergence between Ae. aegypti, and Ae. albopictus, (
      • Boes K.E.
      • Ribeiro J.M.C.
      • Wong A.
      • Harrington L.C.
      • Wolfner M.F.
      • Sirot L.K.
      Identification and characterization of seminal fluid proteins in the Asian tiger mosquito, Aedes albopictus.
      ), the development of genomic resources for a more closely related outgroup to Ae. aegypti, will assist in understanding evolutionary patterns at the gene level. Despite these limitations, our analysis of orthology did reveal that the suite of proteins contributing to seminal fluid, but not sperm, has diverged substantially from other Dipterans. Although sperm proteins and SFPs possess levels of orthology to the Drosophila, genome that are comparable to the genome as a whole, only 59 and 4% of orthology was observed when comparing the Ae. aegypti, sperm and SFP proteomes (respectively) with those of Drosophila, (
      • Wasbrough E.R.
      • Dorus S.
      • Hester S.
      • Howard-Murkin J.
      • Lilley K.
      • Wilkin E.
      • Polpitiya A.
      • Petritis K.
      • Karr T.L.
      The Drosophila melanogaster, sperm proteome-II (DmSP-II).
      ,
      • Findlay G.D.
      • Yi X.
      • Maccoss M.J.
      • Swanson W.J.
      Proteomics reveals novel Drosophila, seminal fluid proteins transferred at mating.
      ). Although some of this disparity may be attributed to differences in overall proteome size and coverage, such a stark contrast is nonetheless compelling evidence of tissue-specific evolutionary patterns. Orthology between Ae. aegypti, SFPs and Ae. albopictus, SFPs (
      • Boes K.E.
      • Ribeiro J.M.C.
      • Wong A.
      • Harrington L.C.
      • Wolfner M.F.
      • Sirot L.K.
      Identification and characterization of seminal fluid proteins in the Asian tiger mosquito, Aedes albopictus.
      ), whereas more extensive (43%), was still lower than orthology between the sperm proteomes of Ae. aegypti, and D. melanogaster,—two distantly related Dipterans. These general patterns of orthology among Ae. aegypti, Ae. albopictus, and D. melanogaster, are consistent with those described by Boes et al., (
      • Boes K.E.
      • Ribeiro J.M.C.
      • Wong A.
      • Harrington L.C.
      • Wolfner M.F.
      • Sirot L.K.
      Identification and characterization of seminal fluid proteins in the Asian tiger mosquito, Aedes albopictus.
      ), although the absolute level of orthology between studies varies considerably because of methodological differences. The patterns we observe suggest a process of “turn-over” in seminal fluid proteomes, whereby overall protein composition diverges rapidly even when there is evidence for conservation with regard to overarching molecular functions represented in seminal fluid. For example, a priori, expectations about Gene Ontology enrichment were met for both Ae. aegypti, sperm (e.g., cilium and mitochondrial proteins) and SFPs (extracellular localization and hydrolase activity), despite overall SFP divergence. SFPs are a pronounced target of selection and have been discussed as a driver of sexual conflict (reviewed in 94), and thus they are expected to rapidly diverge. By contrast, we note that strong conservation of sperm proteins exists across distant taxa, with different insect orders displaying 25% orthology between sperm proteomes (
      • Whittington E.
      • Zhao Q.
      • Borziak K.
      • Walters J.R.
      • Dorus S.
      Characterisation of the Manduca sexta, sperm proteome: Genetic novelty underlying sperm composition in Lepidoptera.
      ), and even D. melanogaster, and mammals with 20% sperm proteome orthology (
      • Wasbrough E.R.
      • Dorus S.
      • Hester S.
      • Howard-Murkin J.
      • Lilley K.
      • Wilkin E.
      • Polpitiya A.
      • Petritis K.
      • Karr T.L.
      The Drosophila melanogaster, sperm proteome-II (DmSP-II).
      ). The overall lack of conservation in seminal fluid proteomes makes comparing the roles of specific SFPs across species difficult, but conserved molecular functions among SFPs will nevertheless allow the wealth of knowledge in Drosophila, to be leveraged toward an understanding of SFP function in nonmodel insects.
      Unlike other mosquitoes with heteromorphic sex chromosomes, Culicine mosquitoes (e.g. Aedes and Culex,) harbor male-determining loci on undifferentiated, homomorphic sex chromosomes (
      • Toups M.A.
      • Hahn M.W.
      Retrogenes reveal the direction of sex-chromosome evolution in mosquitoes.
      ). Theory predicts the evolution of heteromorphic sex chromosomes following the acquisition of a sex determining locus, suppression of recombination, and expansion of the nonrecombining region. It remains unclear why homomorphic sex chromosomes appear to be retained in some taxa (
      • Charlesworth B.
      The evolution of chromosomal sex determination and dosage compensation.
      ,
      • Charlesworth B.
      • Charlesworth D.
      The degeneration of Y chromosomes.
      ). One proposed mechanism to mediate the selective effect of sexually antagonistic alleles on the promotion of recombination suppression is the establishment of efficient sex-biased expression (
      • Vicoso B.
      • Kaiser V.B.
      • Bachtrog D.
      Sex-biased gene expression at homomorphic sex chromosomes in emus and its implication for sex chromosome evolution.
      ). Although previously lacking, the significant enrichment of SFPs on chromosome 1 is the first evidence in support of this hypothesis in Ae. aegypti., This trend was restricted to SFPs and was not observed for genes solely over-expressed in the MAG or testis. It is intriguing to speculate that this distinction between SFPs and other male reproductive genes might be because of the prevalence (and selective strength) of sexually antagonistic alleles specifically among SFPs, which may favor their localization on chromosome 1. This is consistent with their putative role as drivers of sexual conflict (reviewed in
      • Sirot L.K.
      • Wong A.
      • Chapman T.
      • Wolfner M.F.
      Sexual conflict and seminal fluid proteins: a dynamic landscape of sexual interactions.
      ), including the mediation of female post-mating responses such as sexual receptivity and longevity (
      • Helinski M.E.
      • Deewatthanawong P.
      • Sirot L.K.
      • Wolfner M.F.
      • Harrington L.C.
      Duration and dose-dependency of female sexual receptivity responses to seminal fluid proteins in Aedes albopictus Ae. aegypti, mosquitoes.
      ,
      • Villarreal S.M.
      • Pitcher S.
      • Helinski M.E.H.
      • Johnson L.
      • Wolfner M.F.
      • Harrington L.C.
      Male contributions during mating increase female survival in the disease vector mosquito Aedes aegypti.
      ).

      Functional Relevance of Abundant Sperm Proteins and SFPs

      Our sperm and SFP proteomes exhibited skewed abundance distribution with the top ten most abundant proteins comprising 25 and 17% of protein composition in sperm and SFPs, respectively. Interestingly, the most abundant sperm protein, cytosol aminopeptidase (AAEL006975), accounted for more than 7.4% of all protein and two other cytosol aminopeptidases were in the top ten most abundant proteins (AAEL000108, AAEL023987). These three proteins are orthologs of the eight sperm-leucyl aminopeptidases (S-LAPs) in Drosophila, with similar expression patterns, including ∼1000-fold higher expression in testes than in MAG, ∼50 times more transcript in whole male carcasses than gonadectomized carcasses (
      • Akbari O.S.
      • Antoshechkin I.
      • Amrhein H.
      • Williams B.
      • Diloreto R.
      • Sandler J.
      • Hay B.A.
      The developmental transcriptome of the mosquito Aedes aegypti, an invasive species and major arbovirus vector.
      ), and upregulation during later stages of spermatogenesis (
      • Sutton E.R.
      • Yu Y.
      • Shimeld S.M.
      • White-Cooper H.
      • Alphey A.L.
      Identification of genes for engineering the male germline of Aedes aegypti Ceratitis capitata.
      ). S-LAP orthologs constitute a significant proportion of the protein composition of Drosophila, (
      • Dorus S.
      • Wilkin E.C.
      • Karr T.L.
      Expansion and functional diversification of a leucyl aminopeptidase family that encodes the major protein constituents of Drosophila, sperm.
      ) and Lepidoptera sperm (
      • Whittington E.
      • Zhao Q.
      • Borziak K.
      • Walters J.R.
      • Dorus S.
      Characterisation of the Manduca sexta, sperm proteome: Genetic novelty underlying sperm composition in Lepidoptera.
      ,
      • Whittington E.
      • Forsythe D.
      • Borziak K.
      • Karr T.L.
      • Walters J.R.
      • Dorus S.
      Contrasting patterns of evolutionary constraint and novelty revealed by comparative sperm proteomic analysis in Lepidoptera.
      ). Little is known about the specific function of S-LAPs, although it has been postulated that they may serve a structural function given the inferred loss of enzymatic capacity of several S-LAPs during Drosophila, evolution (
      • Dorus S.
      • Wilkin E.C.
      • Karr T.L.
      Expansion and functional diversification of a leucyl aminopeptidase family that encodes the major protein constituents of Drosophila, sperm.
      ). Additionally, a Y-linked S-LAP in D. pseudoobscura, has been implicated in a cryptic meiotic drive system, where suppression of this locus results in aberrant spermatogenesis and a higher proportion of X-bearing sperm (
      • Ellison C.
      • Leonard C.
      • Landeen E.
      • Gibilisco L.
      • Phadnis N.
      • Bachtrog D.
      Rampant cryptic sex chromosome drive in Drosophila.
      ). It will be of great interest to establish the specific function of these proteins in Ae. aegypti, sperm, given their high abundance and expression patterns during spermiogenesis. Furthermore, the proteins and transcripts involved in spermatogenesis described in this study may assist in the identification of other genes involved in meiotic drive systems (reviewed in
      • Lindholm A.K.
      • Dyer K.A.
      • Firman R.C.
      • Fishman L.
      • Forstmeier W.
      • Holman L.
      • Johannesson H.
      • Knief U.
      • Kokko H.
      • Larracuente A.M.
      • Manser A.
      • Montchamp-Moreau C.
      • Petrosyan V.G.
      • Pomiankowski A.
      • Presgraves D.C.
      • Safronova L.D.
      • Sutter A.
      • Unckless R.L.
      • Verspoor R.L.
      • Wedell N.
      • Wilkinson G.S.
      • Price T.A.R.
      The ecology and evolutionary dynamics of meiotic drive.
      ), which have been proposed as potential genetic means to reduce wild populations through the induction of sex ratio biases (
      • Hammond A.M.
      • Galizi R.
      Gene drives to fight malaria: current state and future directions.
      ).
      Although no SFP was as abundant as cytosol aminopeptidase in sperm, the top ten most abundant SFPs ranged from 1.2–2.6% of the protein in our ejaculate sample. l-asparaginase (AAEL002796) was the most abundant SFP (61% more abundant than the next protein) and the tenth most abundant mRNA transcript in the MAG out of over 11,000 transcripts. Although the relevance of the abundance of this enzyme is currently unclear, it may relate to several other notable observations. First, transcript AAEL020035, whose protein product is comprised of ∼60% asparagine residues, is the single most abundant MAG transcript and was also, by far, the most abundant putatively transferred transcript. (We did not identify AAEL020035 in our SFP proteome but note that it results in few identifiable peptides because of its extreme amino acid composition). Second, asparagine tRNA-ligase (AAEL006577) was two times more abundant in seminal fluid than any other tRNA-ligase. Third, asparagine tRNA-ligase was upregulated in the MAG after mating and was the most abundant tRNA-ligase transcript. Together, these suggest that MAGs are well-equipped to produce ample protein with a strong asparagine amino acid bias. Finally, two other enzymes, aspartate transaminase (AAEL002399) and citrate synthase (AAEL004297), are abundantly present in seminal fluid and could convert aspartate produced by asparaginase to oxaloacetate and citric acid, respectively. Although it is premature to draw any firm conclusions based on these observations alone, it is intriguing to speculate that the SFP proteome has the capacity to conduct gluconeogenesis (of asparagine and potentially other amino acids) and that this may feed into to the citric acid cycle. This hypothesis is supported by the results of our KEGG analysis, in which carbon metabolism, gluconeogenesis, and alanine, aspartate, and glutamate metabolism were enriched in the seminal fluid proteome. The citric acid cycle is believed to be functional in mammalian sperm (reviewed in
      • Visconti P.E.
      Sperm bioenergetics in a nutshell.
      ), and our KEGG analysis reveals an enrichment of citric acid cycle enzymes in sperm but not seminal fluid. Whether metabolite precursors to the citric acid cycle are transported from seminal fluid to sperm remains to be determined.
      The most abundant seminal fluid proteins also exhibited a strong enrichment for function in protein cleavage. Proteins with protease, dipeptidase, and aminopeptidase activity represent 12 of the top 28 most abundant proteins present in the seminal fluid proteome. Proteolytic functions have been described previously in the seminal fluid of Ae. aegypti, (
      • Sirot L.K.
      • Hardstone M.C.
      • Helinski M.E.H.
      • Ribeiro J.M.C.
      • Kimura M.
      • Deewatthanawong P.
      • Wolfner M.F.
      • Harrington L.C.
      Towards a semen proteome of the dengue vector mosquito: protein identification and potential functions.
      ), Ae. albopictus, (
      • Boes K.E.
      • Ribeiro J.M.C.
      • Wong A.
      • Harrington L.C.
      • Wolfner M.F.
      • Sirot L.K.
      Identification and characterization of seminal fluid proteins in the Asian tiger mosquito, Aedes albopictus.
      ), Cx. quinquefasciatus, (
      • Stephens K.
      • Cardullo R.A.
      • Thaler C.D.
      Culex pipiens sperm motility is initiated by a trypsin-like protease from male accessory glands.
      ), and several nonmosquito taxa (
      • Nagaoka S.
      • Kato K.
      • Takata Y.
      • Kamei K.
      Identification of the sperm-activating factor initiatorin, a prostatic endopeptidase of the silkworm, Bombyx mori.
      ,
      • Miyata H.
      • Thaler C.D.
      • Haimo L.T.
      • Cardullo R.A.
      Protease activation and the signal transduction pathway regulating motility in sperm from the water strider Aquarius remigis.
      , reviewed in
      • Laflamme B.A.
      • Wolfner M.F.
      Identification and function of proteolysis regulators in seminal fluid.
      ,
      • Baer B.
      • Heazlewood J.L.
      • Taylor N.L.
      • Eubel H.
      • Millar A.H.
      The seminal fluid proteome of the honeybee Apis mellifera.
      ), and are a common function of many insects' seminal fluid. Based on studies in other insects, functions of these enzymes may include the activation of sperm motility or the cleavage of propeptides into their active forms (
      • Rhea J.M.
      • Wegener C.
      • Bender M.
      The proprotein convertase encoded by amontillado (amon,) is required in Drosophila, corpora cardiaca endocrine cells producing the glucose regulatory hormone AKH.
      ). Our seminal fluid proteome also contains abundant enzymes that catabolize smaller substrates, such as amino acids and carbohydrates. Taken together, the enzymatic mixture in seminal fluid may be well equipped to break down many of the molecules they contain. Seminal fluid proteins were also enriched for proteins involved in maintaining proton and redox homeostasis. We identified several proteins contributing to V-type proton ATPases, which use ATP to regulate pH via proton transport. Maintaining an optimal pH in seminal fluid may allow for efficient sperm motility (reviewed in
      • Werner M.
      • Simmons L.W.
      Insect sperm motility.
      ). Regulating pH may also create an ideal environment in which enzymatic reactions occur, either in organelles such as phagosomes and lysosomes (whose constituents were enriched in our KEGG analyses), in sperm, or in the extracellular environment. Seminal fluid also contained several proteins that function to neutralize free radicals, such as catalase (AAEL013407), peroxidase (AAEL013171), and several dehydrogenases. Regulating the physiochemical environment in seminal fluid is likely critical for the function and protection of sperm before their storage in the female's long term storage organs (spermathecae).

      Ejaculate RNAs Transferred to Females

      There has been much conjecture about the importance of spermatozoal RNA to fertility (
      • Miller D.
      • Ostermeier G.C.
      Spermatozoal RNA: why is it there and what does it do?.
      ), and recent work has confirmed that the regulation of sperm ncRNA stores in the mammalian epididymis is necessary for proper embryogenesis (
      • Conine C.C.
      • Sun F.
      • Song L.
      • Rivera-Perez J.A.
      • Rando O.J.
      Small RNAs gained during epididymal transit of sperm are essential for embryonic development in mice.
      ). Little is known about the function of spermatozoal RNAs in insects, although they have been demonstrated to have substantial functional coherence, including an overwhelming enrichment of loci involved in translation (
      • Fischer B.E.
      • Wasbrough E.
      • Meadows L.A.
      • Randlet O.
      • Dorus S.
      • Karr T.L.
      • Russell S.
      Conserved properties of Drosophila, and human spermatozoal mRNA repertoires.
      ). New data in this study allowed us to probe for patterns in previously described transcripts that are putatively transferred to females during mating. A total of 106 transcripts were identified, including both coding and noncoding transcripts, and most of these exhibit high levels of expression in the MAG. Based on our SFP proteome, most of the protein coding transcripts are also translated at high levels. Their high expression in the MAG suggests that they may simply hitchhike into seminal fluid with other secreted molecules. Alternatively, as has been demonstrated in Drosophila, they could be transferred in intact MAG cells (
      • Leiblich A.
      • Marsden L.
      • Gandy C.
      • Corrigan L.
      • Jenkins R.
      • Hamdy F.
      • Wilson C.
      Bone morphogenetic protein- and mating-dependent secretory cell growth and migration in the Drosophila, accessory gland.
      ), or via vesicles derived from the MAG (
      • Corrigan L.
      • Redhai S.
      • Leiblich A.
      • Fan S.J.
      • Perera S.M.
      • Patel R.
      • Gandy C.
      • Wainwright S.M.
      • Morris J.F.
      • Hamdy F.
      • Goberdhan D.C.
      • Wilson C.
      BMP-regulated exosomes from Drosophila, male reproductive glands reprogram female behavior.
      ). Interestingly these vesicles, which may carry RNA cargo including miRNAs, fuse with sperm and have the capacity to interact with the female reproductive tract. Some male-derived transcripts are detectable in the female for up to 24 h post-mating (
      • Alfonso-Parra C.
      • Avila F.W.
      • Deewatthanawong P.
      • Sirot L.K.
      • Wolfner M.F.
      • Harrington L.C.
      Synthesis, depletion and cell-type expression of a protein from the male accessory glands of the dengue vector mosquito Aedes aegypti.
      ), and it has been postulated that they could be used by females in some capacity (
      • Bono J.M.
      • Matzkin L.M.
      • Kelleher E.S.
      • Markow T.A.
      Postmating transcriptional changes in reproductive tracts of con- and heterospecifically mated Drosophila mojavensis, females.
      ). In Ae. aegypti, both vesicles and RNAs are transferred in the ejaculate to the female, but their fate and function have not been investigated. Whether they impact the female or her future offspring is an intriguing, and potentially important, line of future investigation.

      Mosquito Control and Future Directions

      Understanding the molecular architecture of Ae. aegypti, reproduction holds great potential for vector control strategies. Mosquito reproduction is an ideal control target to reduce vector populations and the burden of disease transmission. The most direct application of this study will be the identification of modulators of female reproductive behavior. Mosquito SFPs induce behavioral responses that prevent female remating (
      • Helinski M.E.
      • Deewatthanawong P.
      • Sirot L.K.
      • Wolfner M.F.
      • Harrington L.C.
      Duration and dose-dependency of female sexual receptivity responses to seminal fluid proteins in Aedes albopictus Ae. aegypti, mosquitoes.
      ,
      • Fuchs M.S.
      • Craig G.B.
      • Despommier D.D.
      The protein nature of the substance inducing female monogamy in Aedes aegypti.
      ,
      • Fuchs M.S.
      • Craig Jr, G.B.
      • Hiss E.A.
      The biochemical basis of female monogamy in mosquitoes. I. Extraction of the active principle from Aedes aegypti.
      ), including short term mating refractory behavior (
      • Duvall L.B.
      • Basrur N.S.
      • Molina H.
      • McMeniman C.J.
      • Vosshall L.B.
      A peptide signaling system that rapidly enforces paternity in the Aedes aegypti, mosquito.
      ). To date, the molecule(s) responsible for long term refractoriness has yet to be identified. Given the strength and duration of responses to low SFP “doses” (
      • Helinski M.E.
      • Deewatthanawong P.
      • Sirot L.K.
      • Wolfner M.F.
      • Harrington L.C.
      Duration and dose-dependency of female sexual receptivity responses to seminal fluid proteins in Aedes albopictus Ae. aegypti, mosquitoes.
      ), identification of the responsible proteins will provide powerful tools for manipulating female reproduction in a species-specific manner. In addition, such knowledge may provide a molecular metric by which the quality of males in modified mosquito release strategies (such as those employing sterile or Wolbachia,-infected males; reviewed in
      • Lees R.S.
      • Gilles J.R.
      • Hendrichs J.
      • Vreysen M.J.
      • Bourtzis K.
      Back to the future: the sterile insect technique against mosquito disease vectors.
      ) may be monitored and optimized. Functional analysis of specific sperm proteins and SFPs may yield insights into processes such as sperm motility and activation (
      • Nagaoka S.
      • Kato K.
      • Takata Y.
      • Kamei K.
      Identification of the sperm-activating factor initiatorin, a prostatic endopeptidase of the silkworm, Bombyx mori.
      ,
      • Miyata H.
      • Thaler C.D.
      • Haimo L.T.
      • Cardullo R.A.
      Protease activation and the signal transduction pathway regulating motility in sperm from the water strider Aquarius remigis.
      ,
      • Thaler C.D.
      • Miyata H.
      • Haimo L.T.
      • Cardullo R.A.
      Waveform generation is controlled by phosphorylation and swimming direction is controlled by Ca2+ in sperm from the mosquito Culex quinquefasciatus.
      ), sperm storage (
      • Avila F.W.
      • Ravi Ram K.
      • Bloch Qazi M.C.
      • Wolfner M.F.
      Sex peptide is required for the efficient release of stored sperm in mated Drosophila, females.
      ), and sperm-egg recognition (
      • Perotti M.E.
      • Cattaneo F.
      • Pasini M.E.
      • Verni F.
      • Hackstein J.H.P.
      Male sterile mutant casanova, gives clues to mechanisms of sperm-egg interactions in Drosophila melanogaster.
      ). Very few studies have explored these processes in Ae. aegypti, (reviewed in
      • Degner E.C.
      • Harrington L.C.
      A mosquito sperm's journey from male ejaculate to egg: mechanisms, molecules, and methods for exploration.
      ). A mechanistic understanding of complex post-copulatory male-by-female interactions is critical to genetically modified mosquito release strategies that manipulate reproduction. Our detailed characterization of the male contributions to these interactions should serve as the foundation for the design and improvement of vector control strategies that limit the transmission of arboviruses that cause serious human illness and mortality.

      Data Availability

      Mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD010293 and 10.6019/PXD010293. The R analysis pipeline used for transcriptomic analysis is available as part of this project's GitHub repository (https://github.com/YazBraimah/Aegypti.ejaculatomics). Raw RNA-seq reads from this study can be obtained from the Sequence Read Archive (SRA) Accession # SRP158536. RNA-seq data from Sutton et al,. (
      • Sutton E.R.
      • Yu Y.
      • Shimeld S.M.
      • White-Cooper H.
      • Alphey A.L.
      Identification of genes for engineering the male germline of Aedes aegypti Ceratitis capitata.
      ) and Alfonso-Parra et al., (
      • Alfonso-Parra C.
      • Ahmed-Braimah Y.H.
      • Degner E.C.
      • Avila F.W.
      • Villarreal S.M.
      • Pleiss J.A.
      • Wolfner M.F.
      • Harrington L.C.
      Mating-induced transcriptome changes in the reproductive tract of female Aedes aegypti.
      ) can be accessed at SRA Accession # SRP075464 and SRA Accession # SRP068996, respectively.

      Acknowledgments

      We thank Sylvie Pitcher, Sheng Zhang, Jen Grenier, Peter Schweitzer, and the staff at the Cornell Biotechnology Resource Center for technical support, and Laura Sirot for experimental guidance and feedback. This content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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