Mating-induced patterns of spermathecal fluid protein expression in two eusocial insect species, Lasius niger and Apis mellifera

Mating-induced patterns of spermathecal fluid protein expression in two eusocial insect species, Lasius niger and Apis mellifera | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Mating-induced patterns of spermathecal fluid protein expression in two eusocial insect species, Lasius niger and Apis mellifera Alison McAfee, Félicien Delgueldre, Shelley E. Hoover, Serge Aron, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6831102/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Eusocial insect queens exhibit some of the most extreme durations of sperm storage in the animal kingdom. This extended lifespan of sperm within the queen’s storage organ (the spermatheca) after mating is largely sustained by the spermathecal fluid matrix—a rich and proteinaceous secretion that fills the void volume within the spermatheca. We conducted a comparative proteomics study on mating-induced changes in spermathecal fluid of two long-lived hymenopteran species, Lasius niger and Apis mellifera , capable of different durations of sperm storage (> 20 years for L. niger and up to 5 years for A. mellifera ). We found some similarities between species; for example, enolase and other enzymes responsible for carbohydrate metabolism were among the top differentially expressed proteins in both A. mellifera and L. niger. Additionally, both species exhibited post-mating upregulation of catalase, glutathione peroxidase, and Mn-conjugated superoxide dismutase (SOD), all of which are important antioxidant enzymes. However, we also identified notable differences, with Cu/Zn-conjugated SODs being consistently downregulated after mating in L. niger but upregulated in A. mellifera . Likewise, canonical immune effectors (phenoloxidase and lysozyme) showed similar patterns of expression in both species, (with phenoloxidase remaining unchanged and lysozyme increasing after mating), but ferritins, which are multifunctional antioxidant proteins that are also induced by immune challenges, differed, increasing in L. niger but decreasing in A. mellifera. Herein, we discuss expression patterns of these proteins and additional immune proteins, hexamerins, odorant binding proteins, and a key carbohydrate metabolism enzyme (glyceraldehyde-3-phosphate dehydrogenase) in the context of the life histories of these two social insect species. Biological sciences/Biochemistry/Proteins/Proteome Biological sciences/Physiology/Reproductive biology Biological sciences/Zoology/Animal physiology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Reproductive division of labour between queen and worker castes is one of the hallmarks of eusocial Hymenoptera (ants, eusocial bees, and eusocial wasps). Queens are specialized for reproduction, including mating and laying eggs. In contrast, workers usually do not reproduce; they are in many cases sterile or incapable of mating. Instead, they take on a variety of non-reproductive tasks that are essential for the colony's growth and survival. Queens are markedly longer lived than their non-reproductive female nestmates, with lifespans varying greatly among species 1 , 2 . Queen bumble bees ( Bombus ) for instance, live for one year ― substantially longer than their unmated daughters, who live for several weeks 3 . Queen honey bees ( Apis mellifera ) live comparatively longer, frequently reaching two years of age, with the longest recorded lifespan of eight years 4 . Queen ants, however, hold the record for being the longest-lived hymenopterans, with Atta queens living > 10 years, and L. niger and Pogonomyrmex owyheei in particular reaching > 20 years old 1 . Incredibly, queens of all these species continue laying fertilized eggs for their entire lifetime using sperm they acquired during just one mating period early in life. Given that queens may only remain productive as long as they maintain a supply of viable sperm, and that they can not re-mate later in life, their fecundity and lifespan are sperm-limited and effective sperm storage (reviewed in Degueldre & Aron 5 ) is therefore essential for their longevity. To support this function, each queen possesses a spermatheca ― a specialized sperm storage organ ― where their lifetime supply of sperm cells are densely maintained 6 in a bath of spermathecal fluid that is secreted into the reservoir 7 . This fluid, produced by the adjoining spermathecal glands, is rich in antioxidant enzymes 8 – 10 while also being depleted in oxygen 11 , 12 , which appears to offer a layered mechanism by which damage to the sperm from reactive oxygen species (ROS) can be limited long-term. When stored, sperm may also enter quiescence (lower metabolic activity), which further minimizes sperm senescence by reducing damage by ROS 13 , 14 , although this has not yet been demonstrated in eusocial Hymenoptera. Studying the protein profiles of spermathecal fluid among species with different sperm storage requirements, such as A. mellifera and L. niger (which differ in lifespan by approximately an order of magnitude), before and after mating may help determine the molecular systems that support varying degrees of long-term sperm maintenance and may eventually reveal an evolutionary origin of these critical systems. To date, there have been many molecular analyses comparing the profiles of spermathecal fluid of mated to virgin queens in A. mellifera and several ant species 8–10,15−24 . However, few investigations remove the sperm cells prior to conducting molecular analysis 8 , 10 , 23 , and elsewhere it is not clear if sperm removal has been achieved or not 9 . This may bias the interpretation of the data, as it is not clear if the molecular source is the spermathecal fluid, the sperm, or other cells associated with the spermathecal tissue ( e.g. , the epithelial cells that make up the spermathecal wall 25 ). The spermathecae of mated A. mellifera and L. niger queens contain several million sperm cells 26 – 29 ; therefore, if included in the sample, their molecular profiles would be expected to contribute greatly to any differences observed between mated and unmated queens. Among studies that investigated spermathecal fluid devoid of sperm, in A. mellifera , one early proteomic investigation identified 122 proteins in spermathecal fluid, confirming the presence of antioxidant enzymes, heat-shock proteins, and enzymes linked to carbohydrate metabolism, among other minor contributors like major royal jelly proteins (MRJPs) and odorant binding proteins (OBPs) 8 . More recently, upregulation of antioxidant enzyme expression and activity was ascertained in spermathecal fluid, as well as abundance of other antioxidant proteins (transferrin and MRJPs, which are multifunctional proteins that can reduce oxidative stress 30 ). A key carbohydrate (glyceraldehyde-3-phosphate, or GAP) and catabolic enzyme (GAP dehydrogenase, or GAPDH) have also been identified as critical to support sperm viability by “safely” (non-oxidatively) and efficiently generating ATP 11 . In analyses of Lasius spp. ( L. japonicus and L. hayashi ), transcriptomic and proteomic studies generally agree with those in A. mellifera , with evidence supporting enrichment of antioxidant capabilities, protein chaperones, and carbohydrate metabolism in the spermatheca, but these studies did not specifically deplete sperm cells from the samples 9 , 20 . As a queen ages, some sperm will inevitably die despite the substantial investment in limiting oxidative stress 31 – 33 , but the fate of non-viable (defined here as membrane-permeable 34 ) sperm cells is not always clear. In A. mellifera , dead sperm accumulate in the spermatheca over time 31 , 32 , suggesting that non-viable cells are not recycled (or at least not completely) and are expelled along with live cells during each fertilization event. This appears to be an inefficient use of space and cellular components, but given that honey bee queens release a fractional volume of spermathecal contents to fertilize each egg (and not a specific number of spermatozoa) 29 , with that volume containing anywhere from 2-100 cells, depending on the queen’s mating success 29 , 35 , 36 , the presence of some (< 50%) dead sperm in the spermatheca does not have a meaningful impact on fertilization success in the queen’s first season 37 , 38 . Ant queens ( Solenopsis invicta and A. colombica ) use sperm comparatively judiciously, at ~ 3 spermatozoa per egg for established queens, 39 , 40 and, contrary to the honey bee, paradoxically more sperm appear to be released as the queen grows older 39 . Interestingly, in the case of L. niger , sperm viability also increases as a queen ages 26 , implying that dead sperm must be broken down and possibly recycled or otherwise removed by yet unknown mechanisms, and that long-lived ant queens are under selective pressure to maintain successful fertilizations using a minimal number of sperm. The same study also found that immune activation trades off with sperm viability in established L. niger queens, suggesting that older queens may invest less in constitutive self-preservation processes and more in sperm maintenance 26 . We conducted a comparative proteomics analysis of A. mellifera and L. niger spermathecal fluid pre- and post-mating to determine if mating-induced changes in protein abundance reflect the species’ differences in sperm maintenance strategies. As queens in both species are relatively long lived, we expected to see many similarities in the types of proteins that were differentially abundant in spermathecae after mating, but we also anticipated some differences. Specifically, we hypothesized that since proteases are abundant in Lasius spermathecae 9 and evidence suggests that dead sperm may be eliminated in this species, L. niger spermathecal fluid may be enriched for proteolytic enzymes in mated queens, but the same pattern is not expected in A. mellifera . Secondly, we hypothesized that L. niger queens would invest more heavily in antioxidant systems post-mating than A. mellifera , since the latter species can afford to maintain lower sperm viability without immediate consequences for fertilization 38 , 41 . For the same reason, we thirdly hypothesized that L. niger queens may exhibit reduced investment in innate immune defences after mating, whereas A. mellifera queens may still afford relatively high constitutive immune effector expression. Finally, since GAP can be efficiently metabolised anaerobically to produce ATP, which is advantageous to both species, we hypothesized that A. mellifera and L. niger would both exhibit elevated GAPDH levels post-mating. Methods Queens Generation of the queens used in this study has been previously described 42 . Because L. niger exhibits fully claustral nest founding, where the queen is solely responsible for rearing the first workers, whereas A. mellifera colonies reproduce by fission (and new queens are supplied with an existing workforce), three reproductive time points were sampled for L. niger (virgin, incipient, and established) and two were sampled for A. mellifera (virgin and incipient). Briefly, virgin L. niger queens were captured as they exited their nest to commence their nuptial flight, incipient (newly mated) L. niger queens were collected seven days after mating (indicated by dealation), while established L. niger queens were sampled seven days after their first worker progeny emerged (9–10 weeks after mating). Virgin A. mellifera were collected 1–2 days after emerging from their pupal cell and incipient A. mellifera were collected 10–12 days after emerging (with successful mating unequivocally indicated by active laying). Sample preparation L. niger queens (n = 20 virgins, n = 20 incipient, and n = 70 established queens) were killed by decapitation and spermathecae were dissected in 200 µl of semen diluent (188.3 mM sodium chloride, 5.6 mM glucose, 574.1 nM arginine, 684.0 nM lysine, and 50 mM tris[hydroxymethyl]aminomethane, pH 8.7) 43 under a Leica EZ4 stereomicroscope. Each sperm sample was transferred in an empty 1.5 ml microtube, which was gently inverted until homogenisation. The samples were then stored at -80°C until shipping to the University of British Columbia (UBC) on dry ice. Although freeze-thaw cycles are sometimes used to lyse cells (which would be undesirable in our case), we routinely freeze sperm samples and conduct cell counting assays at a later date, at which time the sperm cells show no visible morphological differences. At UBC, the samples were thawed and centrifuged for 10 min (10,000 g , 4°C), then the supernatant was removed and again centrifuged for 15 min (18,000 g , 4°C). At both centrifugation steps, ~ 10% of the supernatant was left behind in the tube to minimize possible sperm cell carryover (Figure S1 ). Because of the low protein content (due to small spermathecal size) of L. niger samples, 3–4 samples were pooled to yield final sample sizes of n = 6 virgin, n = 8 incipient, and n = 22 established queen samples. A. mellifera queens (n = 7 virgins and n = 10 mated) were dissected by removing the final abdominal tergites and retrieving the spermatheca using fine forceps. The spermatheca was placed on a clean tissue paper and gently rolled to remove the tracheal net. The spermatheca was then placed in a 1.5 mL tube containing 200 µl Tris (100 mM, pH 8.0) and gently ruptured by pressing with a plastic pestle. After suspending the sperm, the samples were centrifuged following the same methods as for L. niger . For both sample sets, protein in the supernatant was precipitated by adding ice-cold acetone to a final concentration of 80%, then incubated overnight at -20°C. The protein pellet was washed twice with 250 µl ice-cold 80% acetone, discarding the supernatant. Hereon, sample preparation steps (resuspension, reduction, alkylation, digestion, and desalting) were conducted exactly as previously described for hemolymph samples 42 , except that for L. niger , given the low protein yields (in most cases precipitated pellets were not visible), there was insufficient protein to enable quantification for each sample; therefore, each sample was assumed to be 5 µg for the purposes of reduction, alkylation, and digestion. Briefly, we suspended the precipitated protein in urea buffer (8 M urea, 2 M thiourea, 100 mM Tris, pH 8.0), reduced disulfide bonds using dithiothreitol (1 µg per 50 µg protein), alkylated with iodoacetamide (1 µg per 10 µg protein) and digested with LysC/Trypsin mix (1 µg per 25 µg protein). After four hours of initial digestion, the samples were diluted in 50 mM ammonium bicarbonate and allowed to continue digesting overnight at room temperature. Digested peptides were desalted using in-house made C18 stop and go gel extraction (STAGE) tips 44 and peptides were eluted using 150 µl of 40% acetonitrile, 0.5% formic acid. After evaporating to dryness, peptides were suspended in 11 µl of injection solvent (0.5% acetonitrile, 0.1% formic acid) and 1 µl was used to quantify peptide concentrations based on A205 nm. A. mellifera samples were diluted to 10 ng/µl, whereas L. niger samples were diluted to 1 ng/µl. Liquid chromatography and mass spectrometry Because L. niger peptide concentrations were low, they were analyzed on a high-sensitivity mass spectrometry system (timsTOF SCP; Bruker Daltonics, Germany) designed for single-cell proteomics, whereas A. mellifera samples were analyzed on a timsTOF Pro2 (Bruker Daltonics, Germany). L. niger samples (10 ng each) were randomly injected and analyzed using a NanoElute UHPLC system (Bruker Daltonics) with Aurora Series Gen2 (CSI) analytical column (25cm x 75µm 1.6µm FSC C18, with Gen2 nanoZero and CSI fitting; Ion Opticks, Parkville, Victoria, Australia) heated to 50°C (by Column toaster M, Bruker Daltonics) and coupled to timsTOF SCP operated in data-independent acquisition parallel accumulation serial fragmentation (DIA-PASEF) mode. The gradient ramped from 2–12% buffer B over 15 min, then to 33% buffer B from 15 to 30 min, then to 95% buffer B over 0.5 min, and held for 7.72 min. Before each run, the analytical column was conditioned with 4 column volumes of buffer A. Buffer A consisted of 0.1% aqueous formic acid and 0.5% acetonitrile in water, and buffer B consisted of 0.1% formic acid in 99.4% acetonitrile. The NanoElute thermostat temperature was set at 7°C. The analysis was performed at 0.3 µL/min flow rate. The timsTOF SCP was set to PASEF scan mode for DIA acquisition scanning from 100–1700 m/z. The capillary voltage was set to 1800 V, drying gas to 3 L/min, and drying temperature to 200°C. The MS1 scan was followed by 8 consecutive PASEF ramps containing 24 non-overlapping 25 m/z isolation windows, covering 400–1000 m/z. As for the TIMS setting, ion mobility range (1/k 0 ) was set to 0.64–1.4 V·s/cm 2 with a 100 ms ramp time and accumulation time (100% duty cycle), and ramp rate of 9.34 Hz. This resulted in 0.96 s of total cycle time. The collision energy was ramped linearly as a function of mobility from 20 eV at 1/k 0 = 0.6 V·s/cm 2 to 59 eV at 1/k 0 = 1.6 V·s/cm 2 . Error of mass measurement is typically within 3 ppm and is not allowed to exceed 7 ppm. For calibration of ion mobility dimension, the ions of Agilent ESI-Low Tuning Mix ions were selected (m/z [Th], 1/k 0 [Th]: 622.0290, 0.9915; 922.0098, 1.1986; 1221.9906, 1.3934). A. mellifera samples (50 ng each) were analyzed in randomized injection order using the same liquid chromatography system and gradient but coupled to a timsTOF Pro2 mass spectrometer (Bruker Daltonics, Germany). As previously described 42 , the Captive Spray ionisation source was operated at 1700 V capillary voltage and 200°C drying temperature. The MS spectra were collected in positive mode from 100–1700 m/z. The TIMS was operated with equal ramp and accumulation time of 85 ms (100% duty cycle). For each TIMS cycle, seven DIA-PASEF scans were used, each with three to four steps, with a total of 25 DIA-PASEF windows spanning from 299.5–1200.5 m/z and from ion mobility range (1/k 0 ) 0.7 V·s/cm 2 to 1.3 V·s/cm 2 . Variable isolation width from 36–61 m/z was used with an overlap of 1 m/z between two neighbouring windows. The collision energy was ramped linearly as a function of mobility value from 20 eV at 1/k 0 = 0.6 V·s/cm 2 to 65 eV at 1/k 0 = 1.6 V·s/cm 2 . Data processing Raw mass spectrometry data were searched using DIA-NN 45 (1.8.1). Default parameters were used, except that ‘FASTA digest for library-free search’, ‘Deep learning-based spectra, RTs and IMs prediction’, and ‘MBR’ were selected, ‘Protein inference’ was set to protein names from FASTA, two missed cleavages were allowed and ‘Neural network classifier’ was set to double-pass mode. The FASTA database for L. niger was downloaded from Uniprot on December 5, 2022, and the A. mellifera database was downloaded on February 2, 2023. A list of 381 potential protein contaminants were appended to each database 46 . The A. mellifera database also contained all viral, bacterial, and microsporidian honey bee pathogen sequences available on Uniprot. Statistical analysis Label-free quantitation data was analyzed using the limma package 47 within R (4.3.0) 48 . First, contaminant sequences were removed, data were log2 transformed, then complete data histograms were inspected for approximate normalcy. Proteins with fewer than 25% valid values were removed. Means models with empirical Bayes variance estimation were fit to each dataset to determine significant differences between all pairwise contrasts (3 for L. niger and 1 for A. mellifera ). False discovery rates (FDRs) were controlled to 5% using the Benjamini-Hochberg method. GO term enrichments were assessed using the gene score resampling (GSR) method within ErmineJ 49 (3.1.2; default parameters) for up- and down-regulated proteins separately. The GSR method does not test for enrichment in a hit-list vs. background; rather, it tests for enrichment along the p value continuum and is not reliant on user-defined thresholds (see Lee et al. 49 for more details). Enrichment FDRs were controlled to 5% (Benjamini-Hochberg method) in all instances. Data availability The raw mass spectrometry data, search results, FASTA databases, and sample metadata are all publicly available on the MassIVE proteomics data repository under the accession MSV000092460 (https://doi.org/doi: 10.25345/C58G8FT6W ) for L. niger and MSV000096180 (https://doi.org/doi: 10.25345/C51V5BR67 ) for A. mellifera. All label-free protein quantification data, sample metadata, and statistical outputs are additionally available in Data S1. Results Overview and GO enrichment We identified 2,516 unique proteins across L. niger samples, of which 1,447 were considered quantified (present in > 25% of samples) and half (720; 49.8%) of which were differentially expressed (5% FDR, Benjamini-Hochberg method) in at least one pairwise contrast ( i.e. , virgin vs. incipient, virgin vs. established, and incipient vs. established; Fig. 1 A). Notably, more proteins were upregulated than downregulated in incipient and established queens relative to virgins, many of which were putative sperm proteins (structural components of flagella), despite the centrifugation steps taken to remove sperm cells from samples prior to processing (Fig. 1 B & C). Interestingly, while both incipient and established queen spermathecae contain sperm, putative sperm proteins were still more abundant in established queens relative to incipient queens (Fig. 1 D), though at a smaller magnitude. GO term enrichment analysis on all up- and down-regulated protein lists for each pairwise contrast detected enriched terms only among up-regulated proteins in the virgin-to-incipient comparison (Fig. 1 E). Transmembrane transporter activity (GO:0022857) and protein catabolic process (GO:0030163) were the top two most significantly enriched GO terms (adjusted p = 0.032 and 0.036, respectively). In A. mellifera , we quantified substantially more proteins (4,223 out of 4,789), 2,796 (58%) of which were differentially expressed (Fig. 2 A). Again, putative sperm proteins were among those upregulated in incipient queens (Fig. 2 B). Among upregulated proteins, many GO terms were significantly enriched, most of which are related to carbohydrate metabolism, nucleotide metabolism, and transmembrane transporter activity (Fig. 2 C; 5% FDR, Benjamini-Hochberg correction). Among downregulated proteins, most of the significantly enriched GO terms were linked to translation, vesicle coat proteins, and protein folding (Fig. 2 D). Top differentially expressed proteins While many proteins were differentially expressed and yielded enriched GO terms, we also investigated specific groups of proteins, namely, those with the highest magnitude of differential expression as well as key enzymes implicated in successful sperm storage, immune proteins, and a curious pattern of odorant binding protein expression. In L. niger , the top five most significant differentially expressed proteins were pyruvate dehydrogenase, vesicular glutamate transporter, outer dense fiber protein (a putative sperm protein), enolase, and receptor-expression enhancing protein, all of which were elevated after mating (Fig. 3 A), with the former three also upregulated after mating in A. mellifera (Data S1). The top five differentially expressed proteins in A. mellifera were cytochrome c (testis specific), disintegrin and metalloproteinase with thrombospondin motifs 12, enolase, restin homolog, and an uncharacterized protein, all of which were upregulated after mating (Fig. 3 B). Of these, orthologs of the specific cytochrome c (cytochrome c-2) and enolase were also upregulated after mating in L. niger , while the others were either not identified or were not differentially abundant. Antioxidant enzymes Most major antioxidant enzymes, including catalase, superoxide dismutase (SODs), glutathione peroxidase (GPx), peroxidase (Px), and thioredoxin-dependent peroxidase (Trx) were upregulated after mating in both species, but there were notable differences between species for SOD metalloenzymes that conjugate different metals. While Mn-conjugated SOD was elevated after establishment in L. niger , Cu/Zn-conjugated SODs were downregulated (Fig. 4 A). In A. mellifera , however, both Mn- and Cu/Zn-conjugated SODs were upregulated after mating (Fig. 4 B). The various peroxidases were generally upregulated in both species after nest establishment and mating, respectively, except for one GPx and one Trx, which did not significantly change in A. mellifera and L. niger , respectively. Immune-associated proteins In L. niger , prophenoloxidase (PO) and lysozyme (Lys) were the only canonical immune effectors quantified, with PO being unaffected by mating and Lys significantly increasing (Fig. 5 A). The same pattern was observed in A. mellifera , with immune responsive protein (IRP)30 additionally increasing and hymenoptaecin remaining unchanged (Fig. 5 B). Ferritin, a multifunctional protein involved in both mitigating oxidative stress and immunity 50 , showed differing patterns of expression in L. niger versus A. mellifera : While all four quantified ferritin isoforms increased after mating in L. niger , all isoforms decreased in A. mellifera. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) GAPDH has been previously implicated as a key enzyme in sperm storage for A. mellifera 11 . We quantified two isoforms of the enzyme in L. niger (GAPDH and GAPDH-like), with one decreasing and the other increasing after by the time of nest establishment (Fig. 6 A). In A. mellifera , GAPDH significantly increased after mating (Fig. 6 B), consistent with previous data 11 . Odorant binding proteins and hexamerins The canonical function of odorant binding proteins (OBPs) is to transport odorant molecules in the antennal hemolymph, but their expression in non-chemoreceptive tissues suggests they may have other functions, such as transporting hormones or other small molecules 51 – 54 . Likewise, hexamerins are complex, multifunctional proteins, with strong caste- and tissue-specific patterns of expression, that have also been implicated as hormone carriers 55 , 56 . We investigated members of these protein families, and found that while only one OBP was quantified in L. niger (and was not differentially expressed), OBPs in A. mellifera were abundant and displayed more complex patterns (Fig. 7 A & B). In A. mellifera , 8 OBPs were quantified, with five significantly increasing, two significantly decreasing, and one remaining unchanged after mating. All hexamerin genes were quantified in the spermathecal fluid of both species (two in L. niger and four in A. mellifera ),and again marked species differences were apparent (Fig. 7 C & D). In L. niger , one hexamerin was upregulated after mating and the other remained unchanged, while in A. mellifera , all four hexamerins were strongly downregulated after mating. Discussion Our comparisons of L. niger and A. mellifera adult queen reproductive stages offers some of the richest proteomics data yet on spermathecal fluid samples that have been depleted of sperm. Although the reproductive stages we analyzed are not precisely congruent (with virgin, incipient, and established queens for L. niger and virgin and incipient queens only for A. mellifera ), we are able to draw several key findings: 1) Our data tentatively support the notion that L. niger may have mechanisms to remove dead sperm components from the spermatheca, which could involve protein degradation machinery ( e.g. the ubiquitin-proteasome system and other proteases); 2) Despite A. mellifera having reduced (relative to L. niger ) selective pressure on efficient sperm maintenance, incipient honey bee queens appear to invest in elevating expression of a wide range of canonical antioxidant enzymes (catalase, SODs, GPx, Trx, and Px), whereas L. niger exhibits more narrow investment; 3) Canonical immune effector expression was similar between species, but L. niger consistently increased ferritin production (proteins involved in both immunity and mitigating oxidative stress) after nest establishment, possibly pointing to investment in alternative antioxidant proteins that moonlight as immune effectors; and 4) Our data confirm previous findings that A. mellifera upregulates GAPDH in the spermatheca after mating 11 but in L. niger , expression patterns of two versions of the enzyme (GAPDH and GAPDH-like) diverge, making it unclear if the enzyme has similar importance in this species. In both L. niger and A. mellifera , a large fraction of the spermathecal fluid proteome changed upon mating, with a subset of those appearing to have been derived from sperm ( i.e. , major components of flagella). This effect was more apparent in L. niger , with 21/172 (12.2%) putative sperm flagellar proteins among those intensely significant (adjusted p 4) in the virgin-to-incipient transition, 35/325 (10.8%) in the virgin-to-established transition, and 29/143 (20.3%) in the incipient-to-established transition. By contrast, in A. mellifera , only 12/301 (4.0%) were putative sperm proteins among those intensely significant in the virgin-to-incipient transition. Combined with 1) the significant enrichment of protein degradation machinery among L. niger proteins upregulated after mating (Fig. 1 E), but not in A. mellifera (Fig. 2 C), 2) the prior suggestion of a sperm degradation or removal system in L. niger queens 26 , and 3) the persistence of higher abundances of putative sperm proteins in established relative to incipient mated queens (when spermathecae from both of which contain abundant sperm) (Fig. 1 D), we suggest that this is a biologically relevant result and not simply a failure to remove sperm contaminants via centrifugation. Together, these data support our first hypothesis, that L. niger may possess a mechanism for removing dead sperm, but the reason for this process being present in L. niger but absent in A. mellifera is unclear. Differences in mating frequency cannot explain the observed pattern. Sperm from males of highly polyandrous species compete to incapacitate each other 57 , which may necessitate a recycling system of incapacitated sperm. However, L. niger queens are only facultatively polyandrous (normally mating with only one male, in which case the opportunity for sperm competition would be absent) while honey bees are highly polyandrous. Mating frequency and sperm competition are therefore not likely to be driving the need for a sperm recycling system. While the underlying reason is elusive, the possibility of eliminating dead sperm from spermathecae is not a novel idea. Da Cruz-Landim found that, in stingless bees ( Melipona bicolor ), sperm appeared to be endocytosed by cells in the spermathecal epithelium 58 . However, this phenomenon has been investigated intensively in L. niger and found not to occur 59 . The present data suggest that the cells could instead be degraded enzymatically prior to transport or recycling of their molecular residues, but without more targeted experimental data, this remains a speculation. No such sperm removal or recycling mechanism has been suggested in A. mellifera ― instead, dead sperm tend to accumulate in the spermatheca as a queen ages ― and indeed we see > 3-fold lower proportional representation of putative sperm proteins in the mated honey bee samples relative to L. niger , along with no enrichment for protein degradation machinery among differentially expressed proteins. Our investigation of antioxidant enzymes did not match our prediction that L. niger would invest more heavily in ROS protection via antioxidant enzyme expression compared to A. mellifera . On the contrary, every antioxidant enzyme quantified in A. mellifera (catalase, SODs ―both Mn- and Cu/Zn-conjugated versions ― Px, GPx, Trx) was upregulated in incipient queens shortly after mating (Fig. 4 B). In L. niger , for which we had an additional group of established queens sampled 9–10 weeks after mating, some enzymes were upregulated in this group (catalase, Mn-conjugated SOD, and GPx), while some were downregulated (Cu/Zn-conjugated SODs), and none significantly differed between incipient mated queens and virgins (Fig. 4 A). The marked difference in regulation of the different SOD metalloenzymes is intriguing but the functional relevance of this remains elusive, since all SODs catalyze the same reaction (converting superoxide radicals to hydrogen peroxide and molecular oxygen). Interestingly, previous data shows that Cu and Zn ion concentrations are elevated in the spermathecal fluid of older L. niger queens relative to virgins 59 . Since the SODs using these cofactors were conversely downregulated after nest establishment, this implies that the metal ions are serving a different purpose, which is yet to be determined. Regardless, the fact that only one SOD was elevated in established L. niger implies that these queens may have a reduced need for removal of superoxide radicals. Ant and honey bee spermathecae are reportedly similarly anoxic 11 , 12 , but some data show that ROS and H 2 O 2 are both elevated in honey bee spermathecal fluid after mating 10 , which may point to an increased need for antioxidant capabilities in this species. Whole-body Cu/Zn-conjugated SOD is expressed at lower levels in L. niger queens relative to drones and, to a lesser degree, workers 60 , despite drones having lifespans that are more than two orders of magnitude shorter than the queens. While absolute quantitation or analysis of enzyme activity levels may be more enlightening than relative quantitation between reproductive stages, together, these data suggest that Cu/Zn-conjugated SODs may be less essential when it comes to both sperm and individual longevity. Perhaps the queens have other mechanisms of limiting ROS generation at the source, rendering extensive antioxidant systems unnecessary. We expected that L. niger may exhibit reduced investment in innate immune defenses after mating, owing to the greater pressure on efficient sperm maintenance in this species, but this is generally not what we found. Among canonical immune effectors ( i.e. antimicrobial peptides, lysozymes, and phenoloxidase), only phenoloxidase and lysozyme were quantified in L. niger , which displayed consistent patterns of expression with the same proteins in A. mellifera (Fig. 5 ). However, ferritins were consistently elevated after mating in L. niger but reduced after mating in A. mellifera . This is noteworthy because ferritins appear to be non-canonical immune proteins 50 , 61 – 63 , as well as antioxidant proteins (due to iron sequestration, preventing the formation of hydroxyl radicals via the Fenton reaction) 64 . We speculate that L. niger could be achieving efficient antioxidant protection (and immunity) by investing in multifunctional proteins such as these instead of the typical antioxidant enzymes, like A. mellifera . Previous work in A. mellifera has shown that metabolism of GAP is an efficient way to produce ATP anaerobically, and generating energy in this way supports sperm viability 11 . Since this system would theoretically be advantageous in both species, we therefore expected GAPDH to become elevated in both L. niger and A. mellifera after mating. Our data confirm that GAPDH was strongly elevated after mating in A. mellifera , but the results are less clear for L. niger (Fig. 6 ). Because L. niger has two isoforms of this enzyme, and they show opposite patterns of expression (with GAPDH-like increasing upon nest establishment and GAPDH decreasing), further experimentation is necessary to determine how these enzymes’ properties differ and whether GAPDH-like expression is sufficient to support the similar ATP generation efficiency as seen in A. mellifera . Enolase (also known as phosphopyruvate hydratase) however, was among the top 5 most strongly differentially expressed proteins in both species and, like GAPDH, is a key enzyme in anaerobic glycolysis. Pyruvate dehydrogenase (which is normally inhibited under anaerobic conditions) was additionally among the same group of top proteins upregulated after mating in L. niger ; a puzzling finding, given the anoxic environment of the spermatheca. In addition to the above-discussed a priori hypotheses, here we highlight some additional and surprising patterns of expression of OBPs and hexamerins (Fig. 7 ). OBPs are typically thought to function as soluble odorant transporters, carrying odorants from the antennal sensilla, through the sensillum lymph fluid, to odorant receptors on the odorant receptor neurons 51 – 53 , 65 . However, they are widely expressed among non-olfactory tissues 54 and therefore likely carry out multiple functions, as suggested by Pelosi et al 52 . We have previously quantified a plethora of OBPs in A. mellifera ejaculates 66 and found that OBP14 was significantly linked to sperm viability in spermathecal fluid (among many other OBPs identified) 67 , in addition to quantifying eight distinct OBPs in A. mellifera here. The fact that seven of these OBPs were differentially expressed after mating suggests that they are serving an important function in this tissue, possibly as carriers of other small molecules, such as hormones or other hydrophobic signalling ligands. Only one OBP was quantified in L. niger , and it was not differentially expressed, but proteome coverage was generally low in this species and a deeper proteome will be necessary to further explore this topic. Hexamerins, while typically being described as a source of amino acid residues during metamorphosis, have also been implicated as hormone transporters (analogous to albumins in vertebrates) 56 . Here, hexamerins also showed striking patterns of expression in A. mellifera , with all four proteins becoming dramatically downregulated after mating, whereas in L. niger , one of two hexamerins changed, modestly increasing by the time of nest establishment. These data point to divergent roles in these two species’ spermathecae, but those functions remain to be clarified. Conclusion Although sperm maintenance is a common feature among all eusocial hymenopterans, different species likely have evolved different strategies for the task, given their disparate lifespans, fertilization efficiencies, and possible sperm recycling mechanisms. We indeed found similarities but also marked differences in the spermathecal fluid proteomes of L. niger and A. mellifera before and after mating. Most notably, we identified preliminary support for the notion that L. niger may have the capacity to break down sperm internally with the help of proteolytic enzymes, but further experimentation is necessary to fully ascertain this idea. Despite L. niger having intensified maintenance requirements (in terms of supporting high-efficiency fertilization and an extreme duration of storage), this species appears to narrowly invest in canonical antioxidant enzymes compared to A. mellifera , with all three Cu/Zn-conjugated SODs counterintuitively declining with nest establishment. However, this deficiency could be partly compensated by elevated production of antioxidant ferritins, which may double as immune proteins, and diversify the types of radicals that can be suppressed or neutralized. Future work should focus on measuring absolute values of enzyme activity and ROS concentrations to enable direct species comparisons, which would offer clearer data on whether long-lived hymenopterans are under more intense selection for sperm preservation, as we expect. Declarations Funding This work was supported by grants from the Natural Sciences and Engineering Research Council (RGPIN-2022-03022) to L.J.F. and the Belgian National Fund for Scientific Research (FRS-FNRS –CDR J.0004.20F) to S.A. Mass spectrometry infrastructure was supported by grants from PacifiCan (grant number 22637), the Canada Foundation for Innovation, and the BC Knowledge Development Fund (grant number 43403 for both), and the University of British Columbia Life Sciences Institute (no applicable grant number). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author Contribution AM and SA conceptualized the experiment. AM conducted the proteomics analysis, generated figures, performed statistical tests, and wrote the first draft of the manuscript. FD and SEH supplied biological material. SA and LJF provided supervision and resources. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6831102","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":483214263,"identity":"3b255a5d-de49-4e98-a0c9-aad05e2fc530","order_by":0,"name":"Alison McAfee","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABB0lEQVRIiWNgGAWjYDACdsYGBKeCgUEORB94gE8LM7KWMwwMxmAtCXi1IHOAWhLBJuDTwt/M3Pzh4w6GPAaxww8/HKiwSZ8fdvgh0BY7Od0G7FokDjO2Sc48w1DMIJ1mLHHgTFruxttpBkAtycZmB3BYA9TCzNsGdI90DoP0x7bDuRtnJ4C0HEjchkOL/GHG5s9QLcw/Dv77n244O/0DXi0GhxkbpKFa2CQONhxIkJfOwW+LIdgvbRKJbdJpZhYHjiUbbpDOKTiQYIDbL3LH2x9/+Nhmk9gvnfz4xoEaO3n52embP3yosJPD6X0IkGBggzsVrNIAr3I0IN9AiupRMApGwSgYCQAAiQpg8nPL3dEAAAAASUVORK5CYII=","orcid":"","institution":"University of British Columbia","correspondingAuthor":true,"prefix":"","firstName":"Alison","middleName":"","lastName":"McAfee","suffix":""},{"id":483214269,"identity":"c7e25b64-6b40-4d99-843b-39e0cfcb87aa","order_by":1,"name":"Félicien Delgueldre","email":"","orcid":"","institution":"Université Libre de Bruxelles","correspondingAuthor":false,"prefix":"","firstName":"Félicien","middleName":"","lastName":"Delgueldre","suffix":""},{"id":483214270,"identity":"3340a596-62ca-4382-a770-6551862239cd","order_by":2,"name":"Shelley E. 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Black tiles indicate missing values. 274 (B), 487 (C), and 485 (D) proteins were differentially expressed in the virgin-to-incipient transition, the virgin-to-established transition, and the incipient-to-established transition, respectively. Select protein functions of interest are color-coded. FC = fold-change; INDY = protein “i m not dead yet”. Structural components of sperm flagella are categorized as “putative sperm proteins.” The horizontal dotted lines indicate the 5% FDR threshold, and the vertical dotted lines indicate log\u003csub\u003e2\u003c/sub\u003e(FC) = 2. Proteins with adjusted p \u0026lt; 0.05 and log(FC) \u0026gt; 2 are considered\u003cem\u003e \u003c/em\u003eintensely significant. E) Significantly enriched GO terms (shown; 5% FDR, Benjamini-Hochberg correction) were only identified among up-regulated proteins in one contrast, the virgin-to-incipient transition. Higher multifunctionality scores indicate that enrichments are more likely to occur by chance due to component proteins being tied to many functions.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6831102/v1/760ed20be9ecfda95fa1a674.png"},{"id":86440028,"identity":"143969ae-b465-43eb-9469-694da2787a9a","added_by":"auto","created_at":"2025-07-10 16:19:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":433170,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eA. mellifera\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e differential and functional enrichment of spermathecal proteins.\u003c/strong\u003e A) 2,796 (shown) of 4,138 proteins were differentially expressed among virgin and incipient queens. B) Protein functions of interest are color-coded. The horizontal dotted line indicates the 5% FDR threshold, and vertical dotted lines indicate log\u003csub\u003e2\u003c/sub\u003e(FC) = 2. Proteins with adjusted p \u0026lt; 0.05 and log(FC) \u0026gt; 2 are considered\u003cem\u003e \u003c/em\u003eintensely significant. C \u0026amp; D) Significantly enriched GO terms among upregulated and downregulated proteins, respectively (Benjamini-Hochberg correction, 5% FDR). Higher multifunctionality scores indicate that enrichments are more likely to occur by chance due to component proteins being tied to many functions.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6831102/v1/a8219983b1361ab0bf8256f3.png"},{"id":86440026,"identity":"04eb4eac-6a14-4751-9fdb-c529a0ed5b24","added_by":"auto","created_at":"2025-07-10 16:19:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":144467,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTop five differentially expressed proteins in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eL. niger \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. mellifera\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e Pyruvate DH = pyruvate dehydrogenase. Boxes represent the interquartile range (IQR) and whiskers span 1.5 IQR. Median bars were removed if they obscured box size and colour. A) \u003cem\u003eL. niger \u003c/em\u003eproteins. B) \u003cem\u003eA. mellifera \u003c/em\u003eproteins.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6831102/v1/d1e3df469b7c3d000f4ebc9d.png"},{"id":86441079,"identity":"60b2292d-f133-4eae-9cc6-503beeba7e0d","added_by":"auto","created_at":"2025-07-10 16:27:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":143286,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntioxidant enzyme expression. \u003c/strong\u003eSOD = superoxide dismutase; GPx = glutathione peroxidase; Px = peroxidase; Trx = thioredoxin-dependent peroxidase. Boxes represent the interquartile range (IQR) and whiskers span 1.5 IQR. Asterisks indicate that the protein was differentially expressed at 5% FDR (Benjamini-Hochberg correction). Median bars were removed if they obscured box size and colour. A) \u003cem\u003eL. niger\u003c/em\u003eantioxidant enzymes. B) \u003cem\u003eA. mellifera \u003c/em\u003eantioxidant enzymes.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6831102/v1/2f84a8ea000a6824ed5fcb74.png"},{"id":86441076,"identity":"2c5f7563-570b-408b-a503-81aa2bfff829","added_by":"auto","created_at":"2025-07-10 16:27:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":168889,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmune-associated proteins. \u003c/strong\u003ePO = prophenoloxidase; PO-AF = prophenoloxidase activating factor; Lys = lysozyme; Fer = ferritin; Hym = hymenoptaecin; IRP30 = immune-responsive protein 30. Boxes represent the interquartile range (IQR) and whiskers span 1.5 IQR. Asterisks indicate that the protein was differentially expressed at 5% FDR (Benjamini-Hochberg correction). A) \u003cem\u003eL. niger \u003c/em\u003eimmune-associated proteins. B) \u003cem\u003eA. mellifera \u003c/em\u003eimmune-associated proteins.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6831102/v1/79f4264719eb740a46d2d49e.png"},{"id":86440032,"identity":"aed45477-c637-4e37-868e-3e4b91bd0753","added_by":"auto","created_at":"2025-07-10 16:19:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":73980,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGlyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. \u003c/strong\u003eBoxes represent the interquartile range (IQR) and whiskers span 1.5 IQR. Asterisks indicate that the protein was differentially expressed at 5% FDR (Benjamini-Hochberg correction). A) Enzyme isoforms in \u003cem\u003eL. niger. \u003c/em\u003eB) \u003cem\u003eA. mellifera.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6831102/v1/7f876d2c9f847f0d67f86543.png"},{"id":86441916,"identity":"fc5f9d04-049b-4dfe-8523-6b334bf80138","added_by":"auto","created_at":"2025-07-10 16:43:01","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":200239,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOdorant binding protein and hexamerin expression. \u003c/strong\u003eOBP = odorant binding protein; Hex = hexamerin. Boxes represent the interquartile range (IQR) and whiskers span 1.5 IQR. Asterisks indicate that the protein was differentially expressed at 5% FDR (Benjamini-Hochberg correction). A) \u003cem\u003eL. niger \u003c/em\u003eOBP (only one was quantified in the dataset). B) \u003cem\u003eA. mellifera \u003c/em\u003eOBPs. C) \u003cem\u003eL. niger \u003c/em\u003ehexamerins.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-6831102/v1/066012639b68fc4583f1cf97.png"},{"id":95564039,"identity":"d01a3c13-1748-4a8f-bc08-741e4bc77e24","added_by":"auto","created_at":"2025-11-10 16:06:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2510659,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6831102/v1/ce33aa24-bd85-47f1-b580-41d5b1de331f.pdf"},{"id":86441075,"identity":"f05ee992-b67a-43c9-ac57-e226cef0b8e4","added_by":"auto","created_at":"2025-07-10 16:27:01","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":127622,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFileFigureS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6831102/v1/6af665b05bc8782b8c21d413.docx"},{"id":86440034,"identity":"d168fc4c-ef60-41b5-bf3b-6b0abacf6a54","added_by":"auto","created_at":"2025-07-10 16:19:01","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2361221,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFileDataS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6831102/v1/8f8ca3d12f5967d4b35ec652.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mating-induced patterns of spermathecal fluid protein expression in two eusocial insect species, Lasius niger and Apis mellifera","fulltext":[{"header":"Introduction","content":"\u003cp\u003eReproductive division of labour between queen and worker castes is one of the hallmarks of eusocial Hymenoptera (ants, eusocial bees, and eusocial wasps). Queens are specialized for reproduction, including mating and laying eggs. In contrast, workers usually do not reproduce; they are in many cases sterile or incapable of mating. Instead, they take on a variety of non-reproductive tasks that are essential for the colony's growth and survival. Queens are markedly longer lived than their non-reproductive female nestmates, with lifespans varying greatly among species\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Queen bumble bees (\u003cem\u003eBombus\u003c/em\u003e) for instance, live for one year ― substantially longer than their unmated daughters, who live for several weeks\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Queen honey bees (\u003cem\u003eApis mellifera\u003c/em\u003e) live comparatively longer, frequently reaching two years of age, with the longest recorded lifespan of eight years\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Queen ants, however, hold the record for being the longest-lived hymenopterans, with \u003cem\u003eAtta\u003c/em\u003e queens living\u0026thinsp;\u0026gt;\u0026thinsp;10 years, and \u003cem\u003eL. niger\u003c/em\u003e and \u003cem\u003ePogonomyrmex owyheei\u003c/em\u003e in particular reaching\u0026thinsp;\u0026gt;\u0026thinsp;20 years old\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Incredibly, queens of all these species continue laying fertilized eggs for their entire lifetime using sperm they acquired during just one mating period early in life.\u003c/p\u003e\u003cp\u003eGiven that queens may only remain productive as long as they maintain a supply of viable sperm, and that they can not re-mate later in life, their fecundity and lifespan are sperm-limited and effective sperm storage (reviewed in Degueldre \u0026amp; Aron\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e) is therefore essential for their longevity. To support this function, each queen possesses a spermatheca ― a specialized sperm storage organ ― where their lifetime supply of sperm cells are densely maintained\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e in a bath of spermathecal fluid that is secreted into the reservoir\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. This fluid, produced by the adjoining spermathecal glands, is rich in antioxidant enzymes\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e while also being depleted in oxygen\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, which appears to offer a layered mechanism by which damage to the sperm from reactive oxygen species (ROS) can be limited long-term. When stored, sperm may also enter quiescence (lower metabolic activity), which further minimizes sperm senescence by reducing damage by ROS\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, although this has not yet been demonstrated in eusocial Hymenoptera.\u003c/p\u003e\u003cp\u003eStudying the protein profiles of spermathecal fluid among species with different sperm storage requirements, such as \u003cem\u003eA. mellifera\u003c/em\u003e and \u003cem\u003eL. niger\u003c/em\u003e (which differ in lifespan by approximately an order of magnitude), before and after mating may help determine the molecular systems that support varying degrees of long-term sperm maintenance and may eventually reveal an evolutionary origin of these critical systems. To date, there have been many molecular analyses comparing the profiles of spermathecal fluid of mated to virgin queens in \u003cem\u003eA. mellifera\u003c/em\u003e and several ant species\u003csup\u003e8\u0026ndash;10,15\u0026minus;24\u003c/sup\u003e. However, few investigations remove the sperm cells prior to conducting molecular analysis\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, and elsewhere it is not clear if sperm removal has been achieved or not\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. This may bias the interpretation of the data, as it is not clear if the molecular source is the spermathecal fluid, the sperm, or other cells associated with the spermathecal tissue (\u003cem\u003ee.g.\u003c/em\u003e, the epithelial cells that make up the spermathecal wall\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e). The spermathecae of mated \u003cem\u003eA. mellifera\u003c/em\u003e and \u003cem\u003eL. niger\u003c/em\u003e queens contain several million sperm cells\u003csup\u003e\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e; therefore, if included in the sample, their molecular profiles would be expected to contribute greatly to any differences observed between mated and unmated queens.\u003c/p\u003e\u003cp\u003eAmong studies that investigated spermathecal fluid devoid of sperm, in \u003cem\u003eA. mellifera\u003c/em\u003e, one early proteomic investigation identified 122 proteins in spermathecal fluid, confirming the presence of antioxidant enzymes, heat-shock proteins, and enzymes linked to carbohydrate metabolism, among other minor contributors like major royal jelly proteins (MRJPs) and odorant binding proteins (OBPs)\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. More recently, upregulation of antioxidant enzyme expression and activity was ascertained in spermathecal fluid, as well as abundance of other antioxidant proteins (transferrin and MRJPs, which are multifunctional proteins that can reduce oxidative stress\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e). A key carbohydrate (glyceraldehyde-3-phosphate, or GAP) and catabolic enzyme (GAP dehydrogenase, or GAPDH) have also been identified as critical to support sperm viability by \u0026ldquo;safely\u0026rdquo; (non-oxidatively) and efficiently generating ATP\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In analyses of \u003cem\u003eLasius\u003c/em\u003e spp. (\u003cem\u003eL. japonicus\u003c/em\u003e and \u003cem\u003eL. hayashi\u003c/em\u003e), transcriptomic and proteomic studies generally agree with those in \u003cem\u003eA. mellifera\u003c/em\u003e, with evidence supporting enrichment of antioxidant capabilities, protein chaperones, and carbohydrate metabolism in the spermatheca, but these studies did not specifically deplete sperm cells from the samples\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAs a queen ages, some sperm will inevitably die despite the substantial investment in limiting oxidative stress\u003csup\u003e\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, but the fate of non-viable (defined here as membrane-permeable\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e) sperm cells is not always clear. In \u003cem\u003eA. mellifera\u003c/em\u003e, dead sperm accumulate in the spermatheca over time\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, suggesting that non-viable cells are not recycled (or at least not completely) and are expelled along with live cells during each fertilization event. This appears to be an inefficient use of space and cellular components, but given that honey bee queens release a fractional volume of spermathecal contents to fertilize each egg (and not a specific number of spermatozoa)\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, with that volume containing anywhere from 2-100 cells, depending on the queen\u0026rsquo;s mating success\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, the presence of some (\u0026lt;\u0026thinsp;50%) dead sperm in the spermatheca does not have a meaningful impact on fertilization success in the queen\u0026rsquo;s first season\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Ant queens (\u003cem\u003eSolenopsis invicta\u003c/em\u003e and \u003cem\u003eA. colombica\u003c/em\u003e) use sperm comparatively judiciously, at ~\u0026thinsp;3 spermatozoa per egg for established queens,\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e and, contrary to the honey bee, paradoxically more sperm appear to be released as the queen grows older\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Interestingly, in the case of \u003cem\u003eL. niger\u003c/em\u003e, sperm viability also increases as a queen ages\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, implying that dead sperm must be broken down and possibly recycled or otherwise removed by yet unknown mechanisms, and that long-lived ant queens are under selective pressure to maintain successful fertilizations using a minimal number of sperm. The same study also found that immune activation trades off with sperm viability in established \u003cem\u003eL. niger\u003c/em\u003e queens, suggesting that older queens may invest less in constitutive self-preservation processes and more in sperm maintenance\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWe conducted a comparative proteomics analysis of \u003cem\u003eA. mellifera\u003c/em\u003e and \u003cem\u003eL. niger\u003c/em\u003e spermathecal fluid pre- and post-mating to determine if mating-induced changes in protein abundance reflect the species\u0026rsquo; differences in sperm maintenance strategies. As queens in both species are relatively long lived, we expected to see many similarities in the types of proteins that were differentially abundant in spermathecae after mating, but we also anticipated some differences. Specifically, we hypothesized that since proteases are abundant in \u003cem\u003eLasius\u003c/em\u003e spermathecae\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e and evidence suggests that dead sperm may be eliminated in this species, \u003cem\u003eL. niger\u003c/em\u003e spermathecal fluid may be enriched for proteolytic enzymes in mated queens, but the same pattern is not expected in \u003cem\u003eA. mellifera\u003c/em\u003e. Secondly, we hypothesized that \u003cem\u003eL. niger\u003c/em\u003e queens would invest more heavily in antioxidant systems post-mating than \u003cem\u003eA. mellifera\u003c/em\u003e, since the latter species can afford to maintain lower sperm viability without immediate consequences for fertilization\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. For the same reason, we thirdly hypothesized that \u003cem\u003eL. niger\u003c/em\u003e queens may exhibit reduced investment in innate immune defences after mating, whereas \u003cem\u003eA. mellifera\u003c/em\u003e queens may still afford relatively high constitutive immune effector expression. Finally, since GAP can be efficiently metabolised anaerobically to produce ATP, which is advantageous to both species, we hypothesized that \u003cem\u003eA. mellifera\u003c/em\u003e and \u003cem\u003eL. niger\u003c/em\u003e would both exhibit elevated GAPDH levels post-mating.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eQueens\u003c/h2\u003e\u003cp\u003eGeneration of the queens used in this study has been previously described\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Because \u003cem\u003eL. niger\u003c/em\u003e exhibits fully claustral nest founding, where the queen is solely responsible for rearing the first workers, whereas \u003cem\u003eA. mellifera\u003c/em\u003e colonies reproduce by fission (and new queens are supplied with an existing workforce), three reproductive time points were sampled for \u003cem\u003eL. niger\u003c/em\u003e (virgin, incipient, and established) and two were sampled for \u003cem\u003eA. mellifera\u003c/em\u003e (virgin and incipient). Briefly, virgin \u003cem\u003eL. niger\u003c/em\u003e queens were captured as they exited their nest to commence their nuptial flight, incipient (newly mated) \u003cem\u003eL. niger\u003c/em\u003e queens were collected seven days after mating (indicated by dealation), while established \u003cem\u003eL. niger\u003c/em\u003e queens were sampled seven days after their first worker progeny emerged (9\u0026ndash;10 weeks after mating). Virgin \u003cem\u003eA. mellifera\u003c/em\u003e were collected 1\u0026ndash;2 days after emerging from their pupal cell and incipient \u003cem\u003eA. mellifera\u003c/em\u003e were collected 10\u0026ndash;12 days after emerging (with successful mating unequivocally indicated by active laying).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSample preparation\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eL. niger\u003c/em\u003e queens (n\u0026thinsp;=\u0026thinsp;20 virgins, n\u0026thinsp;=\u0026thinsp;20 incipient, and n\u0026thinsp;=\u0026thinsp;70 established queens) were killed by decapitation and spermathecae were dissected in 200 \u0026micro;l of semen diluent (188.3 mM sodium chloride, 5.6 mM glucose, 574.1 nM arginine, 684.0 nM lysine, and 50 mM tris[hydroxymethyl]aminomethane, pH 8.7)\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e under a Leica EZ4 stereomicroscope. Each sperm sample was transferred in an empty 1.5 ml microtube, which was gently inverted until homogenisation. The samples were then stored at -80\u0026deg;C until shipping to the University of British Columbia (UBC) on dry ice. Although freeze-thaw cycles are sometimes used to lyse cells (which would be undesirable in our case), we routinely freeze sperm samples and conduct cell counting assays at a later date, at which time the sperm cells show no visible morphological differences. At UBC, the samples were thawed and centrifuged for 10 min (10,000 \u003cem\u003eg\u003c/em\u003e, 4\u0026deg;C), then the supernatant was removed and again centrifuged for 15 min (18,000 \u003cem\u003eg\u003c/em\u003e, 4\u0026deg;C). At both centrifugation steps, ~\u0026thinsp;10% of the supernatant was left behind in the tube to minimize possible sperm cell carryover (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Because of the low protein content (due to small spermathecal size) of \u003cem\u003eL. niger\u003c/em\u003e samples, 3\u0026ndash;4 samples were pooled to yield final sample sizes of n\u0026thinsp;=\u0026thinsp;6 virgin, n\u0026thinsp;=\u0026thinsp;8 incipient, and n\u0026thinsp;=\u0026thinsp;22 established queen samples.\u003c/p\u003e\u003cp\u003e\u003cem\u003eA. mellifera\u003c/em\u003e queens (n\u0026thinsp;=\u0026thinsp;7 virgins and n\u0026thinsp;=\u0026thinsp;10 mated) were dissected by removing the final abdominal tergites and retrieving the spermatheca using fine forceps. The spermatheca was placed on a clean tissue paper and gently rolled to remove the tracheal net. The spermatheca was then placed in a 1.5 mL tube containing 200 \u0026micro;l Tris (100 mM, pH 8.0) and gently ruptured by pressing with a plastic pestle. After suspending the sperm, the samples were centrifuged following the same methods as for \u003cem\u003eL. niger\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eFor both sample sets, protein in the supernatant was precipitated by adding ice-cold acetone to a final concentration of 80%, then incubated overnight at -20\u0026deg;C. The protein pellet was washed twice with 250 \u0026micro;l ice-cold 80% acetone, discarding the supernatant. Hereon, sample preparation steps (resuspension, reduction, alkylation, digestion, and desalting) were conducted exactly as previously described for hemolymph samples\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, except that for \u003cem\u003eL. niger\u003c/em\u003e, given the low protein yields (in most cases precipitated pellets were not visible), there was insufficient protein to enable quantification for each sample; therefore, each sample was assumed to be 5 \u0026micro;g for the purposes of reduction, alkylation, and digestion. Briefly, we suspended the precipitated protein in urea buffer (8 M urea, 2 M thiourea, 100 mM Tris, pH 8.0), reduced disulfide bonds using dithiothreitol (1 \u0026micro;g per 50 \u0026micro;g protein), alkylated with iodoacetamide (1 \u0026micro;g per 10 \u0026micro;g protein) and digested with LysC/Trypsin mix (1 \u0026micro;g per 25 \u0026micro;g protein). After four hours of initial digestion, the samples were diluted in 50 mM ammonium bicarbonate and allowed to continue digesting overnight at room temperature. Digested peptides were desalted using in-house made C18 stop and go gel extraction (STAGE) tips\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e and peptides were eluted using 150 \u0026micro;l of 40% acetonitrile, 0.5% formic acid. After evaporating to dryness, peptides were suspended in 11 \u0026micro;l of injection solvent (0.5% acetonitrile, 0.1% formic acid) and 1 \u0026micro;l was used to quantify peptide concentrations based on A205 nm. \u003cem\u003eA. mellifera\u003c/em\u003e samples were diluted to 10 ng/\u0026micro;l, whereas \u003cem\u003eL. niger\u003c/em\u003e samples were diluted to 1 ng/\u0026micro;l.\u003c/p\u003e\n\u003ch3\u003eLiquid chromatography and mass spectrometry\u003c/h3\u003e\n\u003cp\u003eBecause \u003cem\u003eL. niger\u003c/em\u003e peptide concentrations were low, they were analyzed on a high-sensitivity mass spectrometry system (timsTOF SCP; Bruker Daltonics, Germany) designed for single-cell proteomics, whereas \u003cem\u003eA. mellifera\u003c/em\u003e samples were analyzed on a timsTOF Pro2 (Bruker Daltonics, Germany). \u003cem\u003eL. niger\u003c/em\u003e samples (10 ng each) were randomly injected and analyzed using a NanoElute UHPLC system (Bruker Daltonics) with Aurora Series Gen2 (CSI) analytical column (25cm x 75\u0026micro;m 1.6\u0026micro;m FSC C18, with Gen2 nanoZero and CSI fitting; Ion Opticks, Parkville, Victoria, Australia) heated to 50\u0026deg;C (by Column toaster M, Bruker Daltonics) and coupled to timsTOF SCP operated in data-independent acquisition parallel accumulation serial fragmentation (DIA-PASEF) mode. The gradient ramped from 2\u0026ndash;12% buffer B over 15 min, then to 33% buffer B from 15 to 30 min, then to 95% buffer B over 0.5 min, and held for 7.72 min. Before each run, the analytical column was conditioned with 4 column volumes of buffer A. Buffer A consisted of 0.1% aqueous formic acid and 0.5% acetonitrile in water, and buffer B consisted of 0.1% formic acid in 99.4% acetonitrile. The NanoElute thermostat temperature was set at 7\u0026deg;C. The analysis was performed at 0.3 \u0026micro;L/min flow rate.\u003c/p\u003e\u003cp\u003eThe timsTOF SCP was set to PASEF scan mode for DIA acquisition scanning from 100\u0026ndash;1700 m/z. The capillary voltage was set to 1800 V, drying gas to 3 L/min, and drying temperature to 200\u0026deg;C. The MS1 scan was followed by 8 consecutive PASEF ramps containing 24 non-overlapping 25 m/z isolation windows, covering 400\u0026ndash;1000 m/z. As for the TIMS setting, ion mobility range (1/k\u003csub\u003e0\u003c/sub\u003e) was set to 0.64\u0026ndash;1.4 V\u0026middot;s/cm\u003csup\u003e2\u003c/sup\u003e with a 100 ms ramp time and accumulation time (100% duty cycle), and ramp rate of 9.34 Hz. This resulted in 0.96 s of total cycle time. The collision energy was ramped linearly as a function of mobility from 20 eV at 1/k\u003csub\u003e0\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.6 V\u0026middot;s/cm\u003csup\u003e2\u003c/sup\u003e to 59 eV at 1/k\u003csub\u003e0\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.6 V\u0026middot;s/cm\u003csup\u003e2\u003c/sup\u003e. Error of mass measurement is typically within 3 ppm and is not allowed to exceed 7 ppm. For calibration of ion mobility dimension, the ions of Agilent ESI-Low Tuning Mix ions were selected (m/z [Th], 1/k\u003csub\u003e0\u003c/sub\u003e [Th]: 622.0290, 0.9915; 922.0098, 1.1986; 1221.9906, 1.3934).\u003c/p\u003e\u003cp\u003e\u003cem\u003eA. mellifera\u003c/em\u003e samples (50 ng each) were analyzed in randomized injection order using the same liquid chromatography system and gradient but coupled to a timsTOF Pro2 mass spectrometer (Bruker Daltonics, Germany). As previously described\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, the Captive Spray ionisation source was operated at 1700 V capillary voltage and 200\u0026deg;C drying temperature. The MS spectra were collected in positive mode from 100\u0026ndash;1700 m/z. The TIMS was operated with equal ramp and accumulation time of 85 ms (100% duty cycle). For each TIMS cycle, seven DIA-PASEF scans were used, each with three to four steps, with a total of 25 DIA-PASEF windows spanning from 299.5\u0026ndash;1200.5 m/z and from ion mobility range (1/k\u003csub\u003e0\u003c/sub\u003e) 0.7 V\u0026middot;s/cm\u003csup\u003e2\u003c/sup\u003e to 1.3 V\u0026middot;s/cm\u003csup\u003e2\u003c/sup\u003e. Variable isolation width from 36\u0026ndash;61 m/z was used with an overlap of 1 m/z between two neighbouring windows. The collision energy was ramped linearly as a function of mobility value from 20 eV at 1/k\u003csub\u003e0\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.6 V\u0026middot;s/cm\u003csup\u003e2\u003c/sup\u003e to 65 eV at 1/k\u003csub\u003e0\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.6 V\u0026middot;s/cm\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eData processing\u003c/h3\u003e\n\u003cp\u003eRaw mass spectrometry data were searched using DIA-NN\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e (1.8.1). Default parameters were used, except that \u0026lsquo;FASTA digest for library-free search\u0026rsquo;, \u0026lsquo;Deep learning-based spectra, RTs and IMs prediction\u0026rsquo;, and \u0026lsquo;MBR\u0026rsquo; were selected, \u0026lsquo;Protein inference\u0026rsquo; was set to protein names from FASTA, two missed cleavages were allowed and \u0026lsquo;Neural network classifier\u0026rsquo; was set to double-pass mode. The FASTA database for \u003cem\u003eL. niger\u003c/em\u003e was downloaded from Uniprot on December 5, 2022, and the \u003cem\u003eA. mellifera\u003c/em\u003e database was downloaded on February 2, 2023. A list of 381 potential protein contaminants were appended to each database\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. The \u003cem\u003eA. mellifera\u003c/em\u003e database also contained all viral, bacterial, and microsporidian honey bee pathogen sequences available on Uniprot.\u003c/p\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eLabel-free quantitation data was analyzed using the limma package\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e within R (4.3.0)\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. First, contaminant sequences were removed, data were log2 transformed, then complete data histograms were inspected for approximate normalcy. Proteins with fewer than 25% valid values were removed. Means models with empirical Bayes variance estimation were fit to each dataset to determine significant differences between all pairwise contrasts (3 for \u003cem\u003eL. niger\u003c/em\u003e and 1 for \u003cem\u003eA. mellifera\u003c/em\u003e). False discovery rates (FDRs) were controlled to 5% using the Benjamini-Hochberg method. GO term enrichments were assessed using the gene score resampling (GSR) method within ErmineJ\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e (3.1.2; default parameters) for up- and down-regulated proteins separately. The GSR method does not test for enrichment in a hit-list vs. background; rather, it tests for enrichment along the p value continuum and is not reliant on user-defined thresholds (see Lee et al.\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e for more details). Enrichment FDRs were controlled to 5% (Benjamini-Hochberg method) in all instances.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eThe raw mass spectrometry data, search results, FASTA databases, and sample metadata are all publicly available on the MassIVE proteomics data repository under the accession MSV000092460 (https://doi.org/doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.25345/C58G8FT6W\u003c/span\u003e\u003cspan address=\"10.25345/C58G8FT6W\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for \u003cem\u003eL. niger\u003c/em\u003e and MSV000096180 (https://doi.org/doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.25345/C51V5BR67\u003c/span\u003e\u003cspan address=\"10.25345/C51V5BR67\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for \u003cem\u003eA. mellifera.\u003c/em\u003e All label-free protein quantification data, sample metadata, and statistical outputs are additionally available in Data S1.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eOverview and GO enrichment\u003c/h2\u003e\u003cp\u003eWe identified 2,516 unique proteins across \u003cem\u003eL. niger\u003c/em\u003e samples, of which 1,447 were considered quantified (present in \u0026gt;\u0026thinsp;25% of samples) and half (720; 49.8%) of which were differentially expressed (5% FDR, Benjamini-Hochberg method) in at least one pairwise contrast (\u003cem\u003ei.e.\u003c/em\u003e, virgin vs. incipient, virgin vs. established, and incipient vs. established; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Notably, more proteins were upregulated than downregulated in incipient and established queens relative to virgins, many of which were putative sperm proteins (structural components of flagella), despite the centrifugation steps taken to remove sperm cells from samples prior to processing (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB \u0026amp; C). Interestingly, while both incipient and established queen spermathecae contain sperm, putative sperm proteins were still more abundant in established queens relative to incipient queens (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), though at a smaller magnitude. GO term enrichment analysis on all up- and down-regulated protein lists for each pairwise contrast detected enriched terms only among up-regulated proteins in the virgin-to-incipient comparison (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Transmembrane transporter activity (GO:0022857) and protein catabolic process (GO:0030163) were the top two most significantly enriched GO terms (adjusted p\u0026thinsp;=\u0026thinsp;0.032 and 0.036, respectively).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn \u003cem\u003eA. mellifera\u003c/em\u003e, we quantified substantially more proteins (4,223 out of 4,789), 2,796 (58%) of which were differentially expressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Again, putative sperm proteins were among those upregulated in incipient queens (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Among upregulated proteins, many GO terms were significantly enriched, most of which are related to carbohydrate metabolism, nucleotide metabolism, and transmembrane transporter activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC; 5% FDR, Benjamini-Hochberg correction). Among downregulated proteins, most of the significantly enriched GO terms were linked to translation, vesicle coat proteins, and protein folding (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eTop differentially expressed proteins\u003c/h2\u003e\u003cp\u003eWhile many proteins were differentially expressed and yielded enriched GO terms, we also investigated specific groups of proteins, namely, those with the highest magnitude of differential expression as well as key enzymes implicated in successful sperm storage, immune proteins, and a curious pattern of odorant binding protein expression. In \u003cem\u003eL. niger\u003c/em\u003e, the top five most significant differentially expressed proteins were pyruvate dehydrogenase, vesicular glutamate transporter, outer dense fiber protein (a putative sperm protein), enolase, and receptor-expression enhancing protein, all of which were elevated after mating (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), with the former three also upregulated after mating in \u003cem\u003eA. mellifera\u003c/em\u003e (Data S1). The top five differentially expressed proteins in \u003cem\u003eA. mellifera\u003c/em\u003e were cytochrome c (testis specific), disintegrin and metalloproteinase with thrombospondin motifs 12, enolase, restin homolog, and an uncharacterized protein, all of which were upregulated after mating (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Of these, orthologs of the specific cytochrome c (cytochrome c-2) and enolase were also upregulated after mating in \u003cem\u003eL. niger\u003c/em\u003e, while the others were either not identified or were not differentially abundant.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eAntioxidant enzymes\u003c/h2\u003e\u003cp\u003eMost major antioxidant enzymes, including catalase, superoxide dismutase (SODs), glutathione peroxidase (GPx), peroxidase (Px), and thioredoxin-dependent peroxidase (Trx) were upregulated after mating in both species, but there were notable differences between species for SOD metalloenzymes that conjugate different metals. While Mn-conjugated SOD was elevated after establishment in \u003cem\u003eL. niger\u003c/em\u003e, Cu/Zn-conjugated SODs were downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In \u003cem\u003eA. mellifera\u003c/em\u003e, however, both Mn- and Cu/Zn-conjugated SODs were upregulated after mating (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The various peroxidases were generally upregulated in both species after nest establishment and mating, respectively, except for one GPx and one Trx, which did not significantly change in \u003cem\u003eA. mellifera\u003c/em\u003e and \u003cem\u003eL. niger\u003c/em\u003e, respectively.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eImmune-associated proteins\u003c/h2\u003e\u003cp\u003eIn \u003cem\u003eL. niger\u003c/em\u003e, prophenoloxidase (PO) and lysozyme (Lys) were the only canonical immune effectors quantified, with PO being unaffected by mating and Lys significantly increasing (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The same pattern was observed in \u003cem\u003eA. mellifera\u003c/em\u003e, with immune responsive protein (IRP)30 additionally increasing and hymenoptaecin remaining unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Ferritin, a multifunctional protein involved in both mitigating oxidative stress and immunity\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, showed differing patterns of expression in \u003cem\u003eL. niger\u003c/em\u003e versus \u003cem\u003eA. mellifera\u003c/em\u003e: While all four quantified ferritin isoforms increased after mating in \u003cem\u003eL. niger\u003c/em\u003e, all isoforms decreased in \u003cem\u003eA. mellifera.\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eGlyceraldehyde-3-phosphate dehydrogenase (GAPDH)\u003c/h2\u003e\u003cp\u003eGAPDH has been previously implicated as a key enzyme in sperm storage for \u003cem\u003eA. mellifera\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. We quantified two isoforms of the enzyme in \u003cem\u003eL. niger\u003c/em\u003e (GAPDH and GAPDH-like), with one decreasing and the other increasing after by the time of nest establishment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). In \u003cem\u003eA. mellifera\u003c/em\u003e, GAPDH significantly increased after mating (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), consistent with previous data\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eOdorant binding proteins and hexamerins\u003c/h2\u003e\u003cp\u003eThe canonical function of odorant binding proteins (OBPs) is to transport odorant molecules in the antennal hemolymph, but their expression in non-chemoreceptive tissues suggests they may have other functions, such as transporting hormones or other small molecules\u003csup\u003e\u003cspan additionalcitationids=\"CR52 CR53\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Likewise, hexamerins are complex, multifunctional proteins, with strong caste- and tissue-specific patterns of expression, that have also been implicated as hormone carriers\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. We investigated members of these protein families, and found that while only one OBP was quantified in \u003cem\u003eL. niger\u003c/em\u003e (and was not differentially expressed), OBPs in \u003cem\u003eA. mellifera\u003c/em\u003e were abundant and displayed more complex patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA \u0026amp; B). In \u003cem\u003eA. mellifera\u003c/em\u003e, 8 OBPs were quantified, with five significantly increasing, two significantly decreasing, and one remaining unchanged after mating. All hexamerin genes were quantified in the spermathecal fluid of both species (two in \u003cem\u003eL. niger\u003c/em\u003e and four in \u003cem\u003eA. mellifera\u003c/em\u003e),and again marked species differences were apparent (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC \u0026amp; D). In \u003cem\u003eL. niger\u003c/em\u003e, one hexamerin was upregulated after mating and the other remained unchanged, while in \u003cem\u003eA. mellifera\u003c/em\u003e, all four hexamerins were strongly downregulated after mating.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur comparisons of \u003cem\u003eL. niger\u003c/em\u003e and \u003cem\u003eA. mellifera\u003c/em\u003e adult queen reproductive stages offers some of the richest proteomics data yet on spermathecal fluid samples that have been depleted of sperm. Although the reproductive stages we analyzed are not precisely congruent (with virgin, incipient, and established queens for \u003cem\u003eL. niger\u003c/em\u003e and virgin and incipient queens only for \u003cem\u003eA. mellifera\u003c/em\u003e), we are able to draw several key findings: 1) Our data tentatively support the notion that \u003cem\u003eL. niger\u003c/em\u003e may have mechanisms to remove dead sperm components from the spermatheca, which could involve protein degradation machinery (\u003cem\u003ee.g.\u003c/em\u003e the ubiquitin-proteasome system and other proteases); 2) Despite \u003cem\u003eA. mellifera\u003c/em\u003e having reduced (relative to \u003cem\u003eL. niger\u003c/em\u003e) selective pressure on efficient sperm maintenance, incipient honey bee queens appear to invest in elevating expression of a wide range of canonical antioxidant enzymes (catalase, SODs, GPx, Trx, and Px), whereas \u003cem\u003eL. niger\u003c/em\u003e exhibits more narrow investment; 3) Canonical immune effector expression was similar between species, but \u003cem\u003eL. niger\u003c/em\u003e consistently increased ferritin production (proteins involved in both immunity and mitigating oxidative stress) after nest establishment, possibly pointing to investment in alternative antioxidant proteins that moonlight as immune effectors; and 4) Our data confirm previous findings that \u003cem\u003eA. mellifera\u003c/em\u003e upregulates GAPDH in the spermatheca after mating\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e but in \u003cem\u003eL. niger\u003c/em\u003e, expression patterns of two versions of the enzyme (GAPDH and GAPDH-like) diverge, making it unclear if the enzyme has similar importance in this species.\u003c/p\u003e\u003cp\u003eIn both \u003cem\u003eL. niger\u003c/em\u003e and \u003cem\u003eA. mellifera\u003c/em\u003e, a large fraction of the spermathecal fluid proteome changed upon mating, with a subset of those appearing to have been derived from sperm (\u003cem\u003ei.e.\u003c/em\u003e, major components of flagella). This effect was more apparent in \u003cem\u003eL. niger\u003c/em\u003e, with 21/172 (12.2%) putative sperm flagellar proteins among those intensely significant (adjusted p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and fold-change\u0026thinsp;\u0026gt;\u0026thinsp;4) in the virgin-to-incipient transition, 35/325 (10.8%) in the virgin-to-established transition, and 29/143 (20.3%) in the incipient-to-established transition. By contrast, in \u003cem\u003eA. mellifera\u003c/em\u003e, only 12/301 (4.0%) were putative sperm proteins among those intensely significant in the virgin-to-incipient transition. Combined with 1) the significant enrichment of protein degradation machinery among \u003cem\u003eL. niger\u003c/em\u003e proteins upregulated after mating (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), but not in \u003cem\u003eA. mellifera\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), 2) the prior suggestion of a sperm degradation or removal system in \u003cem\u003eL. niger\u003c/em\u003e queens\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, and 3) the persistence of higher abundances of putative sperm proteins in established relative to incipient mated queens (when spermathecae from both of which contain abundant sperm) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), we suggest that this is a biologically relevant result and not simply a failure to remove sperm contaminants via centrifugation.\u003c/p\u003e\u003cp\u003eTogether, these data support our first hypothesis, that \u003cem\u003eL. niger\u003c/em\u003e may possess a mechanism for removing dead sperm, but the reason for this process being present in \u003cem\u003eL. niger\u003c/em\u003e but absent in \u003cem\u003eA. mellifera\u003c/em\u003e is unclear. Differences in mating frequency cannot explain the observed pattern. Sperm from males of highly polyandrous species compete to incapacitate each other\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e, which may necessitate a recycling system of incapacitated sperm. However, \u003cem\u003eL. niger\u003c/em\u003e queens are only facultatively polyandrous (normally mating with only one male, in which case the opportunity for sperm competition would be absent) while honey bees are highly polyandrous. Mating frequency and sperm competition are therefore not likely to be driving the need for a sperm recycling system.\u003c/p\u003e\u003cp\u003eWhile the underlying reason is elusive, the possibility of eliminating dead sperm from spermathecae is not a novel idea. Da Cruz-Landim found that, in stingless bees (\u003cem\u003eMelipona bicolor\u003c/em\u003e), sperm appeared to be endocytosed by cells in the spermathecal epithelium\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. However, this phenomenon has been investigated intensively in \u003cem\u003eL. niger\u003c/em\u003e and found not to occur\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. The present data suggest that the cells could instead be degraded enzymatically prior to transport or recycling of their molecular residues, but without more targeted experimental data, this remains a speculation. No such sperm removal or recycling mechanism has been suggested in \u003cem\u003eA. mellifera\u003c/em\u003e ― instead, dead sperm tend to accumulate in the spermatheca as a queen ages ― and indeed we see\u0026thinsp;\u0026gt;\u0026thinsp;3-fold lower proportional representation of putative sperm proteins in the mated honey bee samples relative to \u003cem\u003eL. niger\u003c/em\u003e, along with no enrichment for protein degradation machinery among differentially expressed proteins.\u003c/p\u003e\u003cp\u003eOur investigation of antioxidant enzymes did not match our prediction that \u003cem\u003eL. niger\u003c/em\u003e would invest more heavily in ROS protection via antioxidant enzyme expression compared to \u003cem\u003eA. mellifera\u003c/em\u003e. On the contrary, every antioxidant enzyme quantified in \u003cem\u003eA. mellifera\u003c/em\u003e (catalase, SODs ―both Mn- and Cu/Zn-conjugated versions ― Px, GPx, Trx) was upregulated in incipient queens shortly after mating (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In \u003cem\u003eL. niger\u003c/em\u003e, for which we had an additional group of established queens sampled 9\u0026ndash;10 weeks after mating, some enzymes were upregulated in this group (catalase, Mn-conjugated SOD, and GPx), while some were downregulated (Cu/Zn-conjugated SODs), and none significantly differed between incipient mated queens and virgins (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eThe marked difference in regulation of the different SOD metalloenzymes is intriguing but the functional relevance of this remains elusive, since all SODs catalyze the same reaction (converting superoxide radicals to hydrogen peroxide and molecular oxygen). Interestingly, previous data shows that Cu and Zn ion concentrations are elevated in the spermathecal fluid of older \u003cem\u003eL. niger\u003c/em\u003e queens relative to virgins\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Since the SODs using these cofactors were conversely downregulated after nest establishment, this implies that the metal ions are serving a different purpose, which is yet to be determined. Regardless, the fact that only one SOD was elevated in established \u003cem\u003eL. niger\u003c/em\u003e implies that these queens may have a reduced need for removal of superoxide radicals. Ant and honey bee spermathecae are reportedly similarly anoxic\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, but some data show that ROS and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e are both elevated in honey bee spermathecal fluid after mating\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, which may point to an increased need for antioxidant capabilities in this species. Whole-body Cu/Zn-conjugated SOD is expressed at lower levels in \u003cem\u003eL. niger\u003c/em\u003e queens relative to drones and, to a lesser degree, workers\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e, despite drones having lifespans that are more than two orders of magnitude shorter than the queens. While absolute quantitation or analysis of enzyme activity levels may be more enlightening than relative quantitation between reproductive stages, together, these data suggest that Cu/Zn-conjugated SODs may be less essential when it comes to both sperm and individual longevity. Perhaps the queens have other mechanisms of limiting ROS generation at the source, rendering extensive antioxidant systems unnecessary.\u003c/p\u003e\u003cp\u003eWe expected that \u003cem\u003eL. niger\u003c/em\u003e may exhibit reduced investment in innate immune defenses after mating, owing to the greater pressure on efficient sperm maintenance in this species, but this is generally not what we found. Among canonical immune effectors (\u003cem\u003ei.e.\u003c/em\u003e antimicrobial peptides, lysozymes, and phenoloxidase), only phenoloxidase and lysozyme were quantified in \u003cem\u003eL. niger\u003c/em\u003e, which displayed consistent patterns of expression with the same proteins in \u003cem\u003eA. mellifera\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). However, ferritins were consistently elevated after mating in \u003cem\u003eL. niger\u003c/em\u003e but reduced after mating in \u003cem\u003eA. mellifera\u003c/em\u003e. This is noteworthy because ferritins appear to be non-canonical immune proteins\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan additionalcitationids=\"CR62\" citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e, as well as antioxidant proteins (due to iron sequestration, preventing the formation of hydroxyl radicals via the Fenton reaction)\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. We speculate that \u003cem\u003eL. niger\u003c/em\u003e could be achieving efficient antioxidant protection (and immunity) by investing in multifunctional proteins such as these instead of the typical antioxidant enzymes, like \u003cem\u003eA. mellifera\u003c/em\u003e.\u003c/p\u003e\u003cp\u003ePrevious work in \u003cem\u003eA. mellifera\u003c/em\u003e has shown that metabolism of GAP is an efficient way to produce ATP anaerobically, and generating energy in this way supports sperm viability\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Since this system would theoretically be advantageous in both species, we therefore expected GAPDH to become elevated in both \u003cem\u003eL. niger\u003c/em\u003e and \u003cem\u003eA. mellifera\u003c/em\u003e after mating. Our data confirm that GAPDH was strongly elevated after mating in \u003cem\u003eA. mellifera\u003c/em\u003e, but the results are less clear for \u003cem\u003eL. niger\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Because \u003cem\u003eL. niger\u003c/em\u003e has two isoforms of this enzyme, and they show opposite patterns of expression (with GAPDH-like increasing upon nest establishment and GAPDH decreasing), further experimentation is necessary to determine how these enzymes\u0026rsquo; properties differ and whether GAPDH-like expression is sufficient to support the similar ATP generation efficiency as seen in \u003cem\u003eA. mellifera\u003c/em\u003e. Enolase (also known as phosphopyruvate hydratase) however, was among the top 5 most strongly differentially expressed proteins in both species and, like GAPDH, is a key enzyme in anaerobic glycolysis. Pyruvate dehydrogenase (which is normally inhibited under anaerobic conditions) was additionally among the same group of top proteins upregulated after mating in \u003cem\u003eL. niger\u003c/em\u003e; a puzzling finding, given the anoxic environment of the spermatheca.\u003c/p\u003e\u003cp\u003eIn addition to the above-discussed \u003cem\u003ea priori\u003c/em\u003e hypotheses, here we highlight some additional and surprising patterns of expression of OBPs and hexamerins (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). OBPs are typically thought to function as soluble odorant transporters, carrying odorants from the antennal sensilla, through the sensillum lymph fluid, to odorant receptors on the odorant receptor neurons\u003csup\u003e\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. However, they are widely expressed among non-olfactory tissues\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e and therefore likely carry out multiple functions, as suggested by Pelosi \u003cem\u003eet al\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. We have previously quantified a plethora of OBPs in \u003cem\u003eA. mellifera\u003c/em\u003e ejaculates\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e and found that OBP14 was significantly linked to sperm viability in spermathecal fluid (among many other OBPs identified)\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e, in addition to quantifying eight distinct OBPs in \u003cem\u003eA. mellifera\u003c/em\u003e here. The fact that seven of these OBPs were differentially expressed after mating suggests that they are serving an important function in this tissue, possibly as carriers of other small molecules, such as hormones or other hydrophobic signalling ligands. Only one OBP was quantified in \u003cem\u003eL. niger\u003c/em\u003e, and it was not differentially expressed, but proteome coverage was generally low in this species and a deeper proteome will be necessary to further explore this topic. Hexamerins, while typically being described as a source of amino acid residues during metamorphosis, have also been implicated as hormone transporters (analogous to albumins in vertebrates)\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Here, hexamerins also showed striking patterns of expression in \u003cem\u003eA. mellifera\u003c/em\u003e, with all four proteins becoming dramatically downregulated after mating, whereas in \u003cem\u003eL. niger\u003c/em\u003e, one of two hexamerins changed, modestly increasing by the time of nest establishment. These data point to divergent roles in these two species\u0026rsquo; spermathecae, but those functions remain to be clarified.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAlthough sperm maintenance is a common feature among all eusocial hymenopterans, different species likely have evolved different strategies for the task, given their disparate lifespans, fertilization efficiencies, and possible sperm recycling mechanisms. We indeed found similarities but also marked differences in the spermathecal fluid proteomes of \u003cem\u003eL. niger\u003c/em\u003e and \u003cem\u003eA. mellifera\u003c/em\u003e before and after mating. Most notably, we identified preliminary support for the notion that \u003cem\u003eL. niger\u003c/em\u003e may have the capacity to break down sperm internally with the help of proteolytic enzymes, but further experimentation is necessary to fully ascertain this idea. Despite \u003cem\u003eL. niger\u003c/em\u003e having intensified maintenance requirements (in terms of supporting high-efficiency fertilization and an extreme duration of storage), this species appears to narrowly invest in canonical antioxidant enzymes compared to \u003cem\u003eA. mellifera\u003c/em\u003e, with all three Cu/Zn-conjugated SODs counterintuitively declining with nest establishment. However, this deficiency could be partly compensated by elevated production of antioxidant ferritins, which may double as immune proteins, and diversify the types of radicals that can be suppressed or neutralized. Future work should focus on measuring absolute values of enzyme activity and ROS concentrations to enable direct species comparisons, which would offer clearer data on whether long-lived hymenopterans are under more intense selection for sperm preservation, as we expect.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by grants from the Natural Sciences and Engineering Research Council (RGPIN-2022-03022) to L.J.F. and the Belgian National Fund for Scientific Research (FRS-FNRS \u0026ndash;CDR J.0004.20F) to S.A. Mass spectrometry infrastructure was supported by grants from PacifiCan (grant number 22637), the Canada Foundation for Innovation, and the BC Knowledge Development Fund (grant number 43403 for both), and the University of British Columbia Life Sciences Institute (no applicable grant number). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAM and SA conceptualized the experiment. AM conducted the proteomics analysis, generated figures, performed statistical tests, and wrote the first draft of the manuscript. FD and SEH supplied biological material. SA and LJF provided supervision and resources. All authors edited and approved of the final version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKeller, L. Queen lifespan and colony characteristics in ants and termites. \u003cem\u003eInsectes Soc.\u003c/em\u003e \u003cb\u003e45\u003c/b\u003e, 235\u0026ndash;246 (1998).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRemolina, S. C. \u0026amp; Hughes, K. A. 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Biology\u003c/em\u003e. \u003cb\u003e4\u003c/b\u003e, 48. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s42003-020-01586-w\u003c/span\u003e\u003cspan address=\"10.1038/s42003-020-01586-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6831102/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6831102/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEusocial insect queens exhibit some of the most extreme durations of sperm storage in the animal kingdom. This extended lifespan of sperm within the queen\u0026rsquo;s storage organ (the spermatheca) after mating is largely sustained by the spermathecal fluid matrix\u0026mdash;a rich and proteinaceous secretion that fills the void volume within the spermatheca. We conducted a comparative proteomics study on mating-induced changes in spermathecal fluid of two long-lived hymenopteran species, \u003cem\u003eLasius niger\u003c/em\u003e and \u003cem\u003eApis mellifera\u003c/em\u003e, capable of different durations of sperm storage (\u0026gt;\u0026thinsp;20 years for \u003cem\u003eL. niger\u003c/em\u003e and up to 5 years for \u003cem\u003eA. mellifera\u003c/em\u003e). We found some similarities between species; for example, enolase and other enzymes responsible for carbohydrate metabolism were among the top differentially expressed proteins in both \u003cem\u003eA. mellifera\u003c/em\u003e and \u003cem\u003eL. niger.\u003c/em\u003e Additionally, both species exhibited post-mating upregulation of catalase, glutathione peroxidase, and Mn-conjugated superoxide dismutase (SOD), all of which are important antioxidant enzymes. However, we also identified notable differences, with Cu/Zn-conjugated SODs being consistently downregulated after mating in \u003cem\u003eL. niger\u003c/em\u003e but upregulated in \u003cem\u003eA. mellifera\u003c/em\u003e. Likewise, canonical immune effectors (phenoloxidase and lysozyme) showed similar patterns of expression in both species, (with phenoloxidase remaining unchanged and lysozyme increasing after mating), but ferritins, which are multifunctional antioxidant proteins that are also induced by immune challenges, differed, increasing in \u003cem\u003eL. niger\u003c/em\u003e but decreasing in \u003cem\u003eA. mellifera.\u003c/em\u003e Herein, we discuss expression patterns of these proteins and additional immune proteins, hexamerins, odorant binding proteins, and a key carbohydrate metabolism enzyme (glyceraldehyde-3-phosphate dehydrogenase) in the context of the life histories of these two social insect species.\u003c/p\u003e","manuscriptTitle":"Mating-induced patterns of spermathecal fluid protein expression in two eusocial insect species, Lasius niger and Apis mellifera","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-10 16:18:56","doi":"10.21203/rs.3.rs-6831102/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-29T05:27:25+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-26T19:48:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-22T20:11:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"110194736262631404276998816023107346286","date":"2025-07-11T09:26:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"311682391049545959876846782298225059672","date":"2025-07-09T14:07:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-09T04:34:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-29T09:18:41+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-06-06T11:58:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-06T06:05:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-06-05T16:46:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6da88435-24f9-4055-8fac-54d8bdf4032b","owner":[],"postedDate":"July 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":51307452,"name":"Biological sciences/Biochemistry/Proteins/Proteome"},{"id":51307453,"name":"Biological sciences/Physiology/Reproductive biology"},{"id":51307454,"name":"Biological sciences/Zoology/Animal physiology"}],"tags":[],"updatedAt":"2025-11-10T16:01:26+00:00","versionOfRecord":{"articleIdentity":"rs-6831102","link":"https://doi.org/10.1038/s41598-025-22689-6","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-11-06 15:57:35","publishedOnDateReadable":"November 6th, 2025"},"versionCreatedAt":"2025-07-10 16:18:56","video":"","vorDoi":"10.1038/s41598-025-22689-6","vorDoiUrl":"https://doi.org/10.1038/s41598-025-22689-6","workflowStages":[]},"version":"v1","identity":"rs-6831102","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6831102","identity":"rs-6831102","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}