Age, Caste, and Social Context Shape Ovarian Morphology and Transcriptomic Profiles in Red Harvester Ants (Pogonomyrmex barbatus)

Age, Caste, and Social Context Shape Ovarian Morphology and Transcriptomic Profiles in Red Harvester Ants (Pogonomyrmex barbatus) | 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 Age, Caste, and Social Context Shape Ovarian Morphology and Transcriptomic Profiles in Red Harvester Ants (Pogonomyrmex barbatus) María Fernanda Vergara-Martínez, Dennet Guerra-Sandoval, Berenice Otero-Díaz, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6933990/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Nov, 2025 Read the published version in npj Aging → Version 1 posted 11 You are reading this latest preprint version Abstract Reproductive division of labor defines eusocial insects like ants, where queens reproduce and workers remain mostly sterile. Yet, some workers retain rudimentary ovaries, raising questions about their reproductive potential. We examined morphological and transcriptomic differences in ovaries of Pogonomyrmex barbatus queens and workers of varying ages and social contexts. Queens had large, yolk-rich oocytes, while worker ovaries showed signs of degeneration. Callow workers had more developed ovaries than mature ones, suggesting reproductive decline with age. Queenless workers showed more ovarian regression compared to queenright ones. Transcriptomic analyses revealed over 2,000 differentially expressed genes between castes, including those involved in metabolism, hormonal signaling, and epigenetic regulation. Notably, queenless workers upregulated a fertility-linked gene and downregulated lipid metabolism genes. Our results show that both age and social environment constrain worker reproductive potential, highlighting the queen’s role in maintaining worker sterility and offering insights into reproductive senescence in eusocial systems. Biological sciences/Cell biology/Senescence Biological sciences/Physiology/Ageing ovary ants morphology division of labor reproduction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Eusocial insects, including ants, exhibit a striking reproductive division of labor. While the queen specializes in reproduction, the workers mainly perform cooperative tasks such as nest building, food collection, brood care, and colony defense 1 – 3 . This social complexity, along with their global abundance 4 , 5 , makes ants important species in the functioning and maintenance of many ecosystems 5 – 7 . Notably, despite having very similar genomes, the reproductive and lifespan differences between castes are remarkable, with queens of some species capable of living up to 40 years, continuously producing offspring, while the sterile workers typically live only a few months 8 . This difference is unusual, as in most organisms, there is a trade-off between fecundity and longevity: high reproductive investment usually comes at the cost of reduced lifespan. In ants, however, this trade-off is absent, as queens are both the most fertile and longest-lived individuals in a colony 9 , 10 . This phenomenon is particularly prominent in species with fixed caste systems, the most common form of social organization among ants, where reproductive roles are determined early and remain stable throughout life. In these species, the lifespan gap between castes is especially dramatic. This notable difference in lifespan and reproductive capability underscores the specialized roles each caste plays in the survival and success of the colony. It also raises a critical question: What physiological mechanisms enable queens to sustain both high reproductive output and extended longevity, while workers remain largely sterile with shorter lifespans? A major anatomical distinction between queens and workers lies in their ovaries. The number of ovarioles in an individual ant differs by species, but within most species, the queen has the largest number of ovarioles, with several simultaneously developing oocytes at various stages, a clear reflection of their reproductive specialization within the colony. This specialization is also reflected in the presence of spermathecae, specialized sperm storage organs that release sperm to fertilize the eggs. In contrast, workers generally have fewer, often smaller, ovarioles with less developed follicles. In many species, workers have lost the ability to lay eggs completely or produce only embryo-less eggs, known as trophic eggs 11 . Structurally, the reproductive system of female ants consists of two ovaries, each with several polytrophic meroistic ovarioles 12 , 13 . In general, the anterior end of the ovarioles contains the germarium, which contains terminal filament cells and germline stem cells. Terminal filament cells represent the immediate progeny of the germline stem cells where the development of each oocyte begins. These cells differentiate into cystoblast cells in the germarium, which subsequently form multicellular cysts. Within these cysts, one cell becomes the oocyte while the others become nurse cells, forming the follicle. This structure is surrounded by follicular cells that facilitate the transfer of proteins from the hemolymph to the oocyte, such as vitellogenin 14 . The nurse cells nourish the adjacent developing oocyte by providing RNA and proteins. The follicle moves along the length of the ovariole as it grows and matures. For the production of workers or gynes, the eggs are then fertilized by sperm released from the spermatheca. Males come from unfertilized haploid eggs 15 . Additionally, in some species, both queens and workers can lay trophic eggs 11 . Despite this foundational knowledge, very little is known about how ovarian morphology differs between queens and workers across species, or how these structural differences might influence physiology and aging trajectories in each caste. While most ants exhibit fixed caste systems, some display remarkable reproductive plasticity, allowing individuals to shift between reproductive and non-reproductive states depending on environmental or social cues. Examples include queenless species such as Ooceraea biroi and Pristomyrmex pungens , which reproduce via thelytokous parthenogenesis, and facultatively thelytokous species like Platythyrea punctata and Cataglyphis cursor , where egg-laying can occur in the absence of a queen 16 , 17 . Even more strikingly, in Harpegnathos saltator , certain workers (gamergates) can take over reproduction and dramatically extend their lifespan following queen loss 18 . Despite these fascinating exceptions, most ants exhibit rigid caste systems, where only queens reproduce, and workers remain sterile. It is in these fixed systems that we observe the most extreme contrasts in ovarian structure and lifespan, yet the molecular and physiological mechanisms underlying this specialization remain poorly understood. The red harvester ant, Pogonomyrmex barbatus , provides an ideal model for addressing this gap. Native to North American arid and semi-arid regions 19 , this species has a strict reproductive division of labor: queens are the sole reproductive individuals, while workers, despite retaining ovaries, show no evidence of egg-laying 20 . Importantly, the lifespan difference between queens and workers is extreme, queens can live up to 30 years, while workers survive only about a year. This contrast far exceeds that of plastic species, where lifespan differences are often limited to just a few months or years. Despite their ecological and evolutionary significance, the morphological and physiological differences between queen and worker ovaries in this species remain largely unexplored. To address this gap, we compared ovarian structure across castes, ages, and social conditions. Additionally, we conducted transcriptomic analyses to identify molecular signatures associated with reproductive division of labor. Our findings provide new insights into the mechanisms underlying reproductive aging and caste-specific longevity in ants. Materials and methods Husbandry To compare ovaries between queens and workers of P. barbatus , we used workers from three colonies collected during 2021 and 2022 in Texcoco de Mora and Cuautitlán Izcalli, State of Mexico, Mexico. The colonies were maintained in the laboratory in artificial nests at 27°C on a 12-hour light/dark cycle, and were provided with canary seeds and water ad libitum . Workers used in the experiment were collected at the foraging arena from three different colonies. By the time of the experiment each of these colonies had between 100–200 workers in total. Cuticle color was used as an age marker in the workers, with light-colored workers ( 20 days old) considered mature (old). Queens were obtained from recently founded colonies (~ 5 months old) with 3–5 workers each. To compare queenright and queenless workers of P. barbatus , we used workers from recently founded colonies. The colonies were collected in May 2023 in Texcoco de Mora, State of Mexico, Mexico, and placed in test tubes at 27°C on a 12-hour light/dark cycle, and were provided with canary seeds and water ad libitum . The colonies contained between 6–8 ants at the start of the experiment. For queenless colonies, the queen was removed, and the workers remained in contact with the eggs, larvae, and pupae for five weeks until dissection. Dissection of ant ovarioles Female ants were anesthetized on ice for one minute and immersed for another minute in 70% ethanol before being placed in a small amount of 1X phosphate buffered saline (PBS). Ovarioles were dissected by pulling on the third tergite using a pair of fine tweezers and then removing fat and unrelated tissues. Ovaries were placed immediately into a fixative solution of 4% paraformaldehyde (PFA) in 1X PBS. Staining Ovaries were fixed overnight at 4°C in 4% PFA in 1X PBS. Following fixation, ovaries were washed 3 times for 20 minutes each with Triton (0.5% Triton in 1X PBS) at room temperature on a rotator. The samples were then washed 2 times for 10 minutes in 0.01% Tween in 1X PBS, ovaries were incubated with DAPI (1:1000, Sigma, #D9542) and Phalloidin (1:400, Invitrogen, #A22287) for 2h, and mounted in Vectashield® mounting media (Vector Laboratories, #H1000)(Fig. 2 , 5 ). Confocal microscopy and analysis Confocal images were captured using a Nikon A1R + confocal laser scanning microscope. Images were analyzed in ImageJ software for quantitative and qualitative characterizations while statistical analysis and generation of figures were performed using GraphPad Prism software. Statistical significance was assessed with a permutation test for constrained correspondence analysis ( n = 10,000 random permutations) using Python 3.7.16 within a Jupyter notebook, employing the pandas, statsmodels, numpy, and scipy libraries. Histological sections and light microscopy Ant ovaries were collected in 1X PBS and fixed overnight at 4°C in 4% PFA in 1X PBS. Following fixation, the tissues were briefly washed four times with 1X PBS and then post-fixed in a 10% formalin buffer overnight at room temperature. The samples were washed four times for 15 minutes each with distilled water at room temperature. Dehydration was carried out with a series of ethanol baths of 60%, 70%, 80%, 90%, 96%, 100% and 100% for one hour each using a Leica TP 1020 automatic tissue processor. The samples were then washed in 50% alcohol-50% xylol for one hour, 100% xylol twice for one hour each, and two washes of paraffin (Paraplast Plus®), one for 1 hour and the other for 30 minutes. The samples were then embedded in paraffin blocks at 58°C-60°C, and five-micron sections were cut using a Leica RM 2155 rotary microtome and mounted on slides. For staining, we began by deparaffinizing the samples. We placed them in an oven at 60°C for 30 minutes, then placed the slides in a coplin jar and washed them with xylol twice, first for 10 minutes, then 5 minutes. The samples were washed with 50% alcohol-50% xylol for 5 minutes. Next, we treated them with 100%, 96%, 96%, and 70% ethanol for 5 minutes each. Finally, we rinsed the samples twice in distilled water for 5 minutes each. The samples were stained with Gill’s hematoxylin 0.20% for 7 minutes, then rinsed in tap water for 3 minutes, followed by two rinses in distilled water, each for 3 minutes. They were then stained with eosin 0.25% for 10 minutes. Following staining, the samples were subjected to a series of ethanol washes (70%, 80%, 90%, 96%, 100%) for 4 minutes each. We then treated the samples with 50% alcohol-50% xylol twice for 4 minutes each. Afterward, the samples were immersed in xylene for 4 minutes, the xylene was replaced and the samples were maintained in xylene until mounting. Finally, we mounted the samples on slides with a coverslip and one or two drops of rapid mounting medium ENTELLAN®. Images were captured using a Carl Zeiss™ Axio Vert.A1 inverted microscope under bright field illumination. Transmission electron microscopy (TEM) Ovaries were dissected in PBS and fixed by immersion in 2.5% glutaraldehyde and 4% PFA in sodium phosphate buffer (0.1M, pH 7.4), post-fixed in 1% osmium tetroxide, dehydrated in a graded series of ethanol and embedded in EPON (epoxy resin). Semithin sections (1 µm) were cut using an ultramicrotome (Leica EM UC6), stained with toluidine blue to select areas in light microscopy examination (Axioskop 2 Plus). Ultrathin sections of 60–90 nm were cut and collected on slot grids previously covered with formvar membrane. Sections were stained with uranyl acetate and lead citrate. The structure for each sample was recorded using a Jeol JEM-1011 transmission electron microscope housed in the National Institute of Pediatrics in Mexico. Images were taken with PhotoImpact 10. RNA extraction and sequencing From four ant colonies, approximately 50 foragers were selected based on cuticle color (dark) and task they were doing and labeled on their gaster with Uni-paint markers. 20 workers were returned to their respective colonies, whereas the remaining workers were kept five weeks isolated from their queen. Three different experimental groups were considered in this study: queens, workers that were kept in their colonies (queenright), and workers isolated for five weeks (queenless). Ants were anesthetized on ice and the ovaries were dissected in an ice-cold 1X PBS buffer using fine forceps and scissors. Fat bodies attached to the tissues were carefully removed. To increase the final RNA concentration, we pooled tissue for each RNA sample. Each pool contained five queens or ten workers from either group (queenright and queenless). We used four pooled biological replicates for each condition. Dissected and pooled ovaries were immediately stored in QIAzol Lysis Reagent (QIAGEN), homogenized in a FastPrep-24™ Classic Instrument (MP Biomedicals) and finally frozen at -80°C. RNA was extracted using a modified QIAZol/phenol chloroform protocol, followed by RNeasy (QIAGEN) purification with DNase I (Thermo Fisher Scientific) on-column digestion. Isolated RNA sample quality was assessed with a High Sensitivity RNA Tapestation (Agilent Technologies) and quantified by Qubit 2.0 RNA HS assay (Thermo Fisher Scientific). RNA extractions were sent to Admera Health BioPharma Services (New Jersey, USA) for Poly(A) selection library preparation and RNA-Seq. Paramagnetic beads coupled with oligo d(T)25 were combined with total RNA to isolate poly(A) + transcripts based on the NEBNext® Poly(A) mRNA Magnetic Isolation Module manual (New England BioLabs). Prior to first strand synthesis, samples were randomly primed (5´ d(N6) 3´ [N = A,C,G,T]) and fragmented based on manufacturer’s recommendations. The first strand was synthesized with the Protoscript II Reverse Transcriptase with a longer extension period, approximately 30 minutes at 42⁰C. All remaining steps for library construction were done according to the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England BioLabs). Final RNA quantity of the libraries was assessed by Qubit 2.0 (Thermo Fisher Scientific) and quality was assessed by TapeStation HSD1000 ScreenTape (Agilent Technologies). Final fragment size was about 500bp with an insert size of about 350bp. Illumina® 8-nt dual-indices were used. Equimolar pooling of libraries was performed based on QC values and sequenced on an Illumina® NovaSeq X Plus 10B (Illumina) with a read length configuration of 150 PE for 40 M PE reads per sample (20M in each direction). Reads for all sequences generated in this study are available at the National Center for Biotechnology Sequence Read Archive (NCBI - SRA; BioProject ID: PRJNA1279095). Sequence processing and differential expression analyses Quality reports of the raw sequences were generated using FastQC ( http://www.bioinformatics.babraham.ac.uk/projects/fastqc ; Andrews, 2010). Adapters and low quality nucleotide bases were trimmed using Cutadapt v4.6 21 and reexamined in FastQC. Mapping of reads onto the genome of P. barbatus 22 previously downloaded from NCBI (GenBank accession: GCF_000187915.1) was done in STAR v2.7.11a 23 and count matrices were generated estimating gene expression levels with the featureCounts tool available in the Subread package v2.0.6 24 . Differential gene expression (DGE) analyses were performed with DESeq2 v3.18 25 . To identify candidate differentially expressed genes within the two analyses (queen vs worker and queenright vs queenless worker) we kept all genes with a significantly adjusted p-value below 0.05 and a fold change greater than 2 (FC > 2). Functional annotation of the filtered candidate genes was performed using DAVID 26 , 27 paying particular attention to gene ontology (GO) terms and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. This database provides a comprehensive set of different functional annotation tools when dealing with large lists of genes. Parameters for the functional annotation analysis included: 1) Gene Ontology: GOTERM Biological Process (BP) Direct, GOTERM Cellular Component (CC) Direct, GOTERM Molecular Function (MF) Direct; and 2) Pathways: KEGG Pathway. All data visualizations were produced with R (version 4.4.1) using RStudio and the Tidyverse package suite. Results Differences in ovary structure among castes of Pogonomyrmex barbatus In addition to their overall morphological differentiation by caste (Fig. 1 a-c), queens and workers also differ markedly in their ovarian structures. Queen ovaries were fully developed and contained a high number of tracheae, whereas workers had immature, poorly developed ovaries and contained few tracheae (Fig. 1 e-f). In queens, the large mature vitellogenic oocytes were slightly elongated and surrounded by a thick layer of follicular cells, suggesting the presence of reproductive eggs (Fig. 2 a). Young workers exhibited well-developed ovarioles with a high number of oocyte follicles at many developmental stages (Fig. 2 a’), and older workers showed atrophied ovarioles, with the ovariole being partly empty and lacking early stage oocytes (Fig. 2 a’’). On average, queens had significantly more ovarioles per ovary (56.20 ± 9.78) than workers (6.70 ± 2.40)(Unpaired t test, df = 24, p < 0.001)(Fig. 2 b). There was no significant difference in ovariole number between young workers (8.300 ± 1.767) and old workers (5.100 ± 1.853)(ANOVA, df = 24, p = 0.37 after Bonferroni correction)(Fig. 2 b). Ovariole length did not differ significantly between queens (1,873 ± 262µm) and workers (1,896 ± 357.4µm)(Unpaired t test, df = 24, p = 0.89). Within the worker caste, old workers had significantly longer ovarioles (2,080 ± 352.4 µm) than young workers (1,713 ± 265.5µm)(ANOVA, df = 24, p < 0.05 after Bonferroni correction)(Fig. 2 c). However, young workers had significantly more follicles per ovariole (6.62 ± 0.84) than old workers (3.67 ± 1.43)(ANOVA, df = 24, p < 0.001 after Bonferroni correction)(Fig. 2 d). Unexpectedly, the number of follicles per ovariole in queens (1.16 ± 0.08) was significantly lower than both young workers (6.62 ± 0.84) (ANOVA, df = 24, p < 0.001 after Bonferroni correction) and old workers (3.67 ± 1.43)(ANOVA, df = 24, p < 0.001 after Bonferroni correction)(Fig. 2 a’-a’’, 2d). Queens also had significantly fewer developmental stages with vitellogenic oocytes per ovariole (1.16 ± 0.08) than workers (5.145 ± 1.89)(Mann-Whitney test, df = 24, p < 0.001). When comparing the size of the largest oocytes (Fig. 2 e), at which point the egg is fully developed and ready for fertilization, we found that the area of mature oocytes was significantly larger in queens (319,579 ± 49,133 µm²) than both young (106,435 ± 39,479 µm²)(ANOVA, df = 24, p < 0.001 after Bonferroni correction) and old (169,703 ± 92,169 µm²)(ANOVA, df = 24, p = 0.001 after Bonferroni correction) workers (Fig. 2 e). Content and structural differences in the oocytes of Pogonomyrmex barbatus queens and workers We analyzed the composition and structure of developing oocytes of queens and workers in terms of size. We found notable differences between queen and worker oocyte yolk content and follicular cell distribution through hematoxylin and eosin staining (Fig. 3 ). The larger oocytes in queens, known as vitellogenic oocytes (VO), and the previtellogenic oocytes (PVO) in workers are precisely delineated by follicular cells (FCs). These cells determine the size and shape of the oocyte, and allow the transport of nutrients to the oocyte cytoplasm 28 – 30 . However, the oocytes in workers tend to be smaller and to show a less organized distribution of the follicular epithelium, regardless of age, indicating a less advanced stage of maturation that could correspond to their infertility (Fig. 3 d-g). In queens (Fig. 3 b-c), the separation of the follicular cells surrounding the vitellogenic oocyte (VO) is clearly observed, forming intercellular spaces (black arrows) that may allow the passage of vitellogenin into the cytoplasm through patency (Raikhel & Dhadialla, 1992; Wu et al., 2021; Wyatt & Davey, 1996)(Fig. 3 a-b). There are also clear differences between young and old workers. Younger workers have a more distinct organization of follicular cells (FCs), whereas the delineation between these cells becomes less perceptible in older workers (Fig. 3 d-g). This suggests a progressive alteration in ovarian structure with age, potentially linked to a decline in reproductive potential or cellular maintenance over time. Furthermore, unlike in workers, dense acidophilic structures are also observed in the ooplasm of queens, which are yolk granules (YG) composed of vitellin, the stored form of vitellogenin in the viable oocyte (Fig. 3 b-c) 29 . None of the worker oocytes, not even the largest ones, exhibit these yolk granules (YG). This is also seen in non-mature queen oocytes where they lack these yolk granules in their cytoplasm (Fig. S1 ). However, unlike in workers, these oocytes in queens will eventually become viable. Differences in ooplasm content and oocyte ultrastructure between queen and worker ovaries were evidenced through ultrastructural analysis using TEM To explore potential differences in oocyte structure associated with caste and age, we conducted transmission electron microscopy (TEM) on ultrathin sections of the largest oocytes from each group: queens, callow workers, and mature workers. We found clear ultrastructural differences between groups. In queens, we observed numerous round and clear structures within the cytoplasm (Fig. 4 a). Ultrastructural analysis confirmed that these correspond to vacuole-like structures (Fig. 4 b). Protein plaques in the ooplasm appeared dark, indicating higher protein density, and were large, irregular or ovoid in shape (Fig. 4 c). The microvilli zone was compact, dense, and continuous, suggesting an increased surface area for nutrient exchange into the mature oocyte. On the oocyte side of this interface, small and abundant protein plaques were evident (Fig. 4 d). Follicular cells surrounding the oocyte displayed nuclei with prominent nucleoli, numerous mitochondria, and a well-developed rough endoplasmic reticulum (Fig. 4 e). In callow workers, the oocyte cytoplasm also contained vacuoles and protein plaques (Fig. 4 f), which were frequently arranged around the vacuoles (Fig. 4 g) and accumulated beneath the microvilli zone, which appeared discontinuous (Fig. 4 h-i). Within the follicular cells, mitochondria and abundant peroxisomes were observed (Fig. 4 j). In mature workers, large vacuoles were observed in the oocyte cytoplasm (Fig. 4 k). Similar to callow workers, these were surrounded by protein plaques (Fig. 4 l) and located predominantly beneath the microvilli zone, which showed marked discontinuity (Fig. 4 m-n). Follicular cells contained mitochondria, peroxisomes, and rough endoplasmic reticulum; however, signs of cellular damage were also evident, including disorganization and discontinuities in the nuclear envelope and mitochondrial membranes, suggesting a possible loss of functional integrity in these older individuals (Fig. 4 o). Effects of queen removal on ovary structure in Pogonomyrmex barbatus workers Due to the fact that the absence of a queen induces various physiological changes in worker ants 31 – 33 , we decided to study the structure of the ovarioles 5 weeks after the queen was removed. We compared ovaries from queenright workers and queenless workers in recently founded colonies. In this context, we observed larger and better-developed oocytes in queenright workers (Fig. 5 a), while the queenless workers exhibited an atrophied reproductive system, with empty ovarioles and fewer oocytes (Fig. 5 a’). Moreover, the atrophy was observed in both young and old workers. There was no significant difference in the number of ovarioles between queenright workers (6.600 ± 1.89) and queenless workers (5.16 ± 2.28)(unpaired t test, df = 26, p = 0.10)(Fig. 5 b). However, queenless workers had shorter ovarioles (queenright, 1,689 ± 350.2 µm vs. queenless, 1,316 ± 221.1 µm)(unpaired t test, df = 26, p < 0.01)(Fig. 5 c), with fewer follicles (queenright, 8.577 ± 2.84 vs. queenless, 5.31 ± 1.69)(unpaired t test, df = 26, p < 0.01) and smaller oocytes (queenright, 126,993 ± 83,403µm² vs. queenless, 69,180 ± 33,746 µm²)(unpaired t test, df = 26, p < 0.05)(Fig. 5 d). Differential gene expression in ovaries of Pogonomyrmex barbatus queens and workers To investigate caste-specific molecular differences in the ovaries, we analyzed the ovarian transcriptomes of Pogonomyrmex barbatus queens and workers using RNA-seq. A total of 2,052 genes were identified as differentially expressed (FDR < 0.05), with 909 genes upregulated in queens and 1,143 in workers (Supplementary Data 1). A heatmap of the DEGs showed consistent expression patterns across biological replicates (Fig. 6 a). Principal Component Analysis (PCA) revealed a clear separation between the two castes, indicating distinct transcriptomic profiles in ovarian tissue (Fig. 6 b). Gene Ontology (GO) and KEGG pathway enrichment analyses were performed to gain insight into the biological functions of the differentially expressed genes (Supplementary Data 3). GO enrichment analysis revealed that queen-upregulated genes were associated with biological processes such as proteolysis, peptide catabolic process, isoprenoid biosynthetic process, mevalonate pathway, cholesterol biosynthetic process, and cell division, among others (Fig. 6 c). KEGG pathway analyses, in turn, showed enrichment in metabolic pathways, peroxisome related function, terpenoid backbone biosynthesis, glutathione metabolism, motor proteins, and biosynthesis of cofactors, among others (Fig. 6 e). In contrast, queen-downregulated genes were enriched in GO terms related to carbohydrate metabolism, lipid metabolism, fatty acid biosynthesis, signal transduction, transcriptional regulation, among others (Fig. 6 d). KEGG analysis revealed enrichment in metabolic pathways, as well as in neuroactive ligand-receptor interaction, phagosome related function, biosynthesis of cofactors, lysosome related function, oxidative phosphorylation, and other processes (Fig. 6 f). Effects of queen removal on gene expression in Pogonomyrmex barbatus worker ovaries We also performed RNA-seq on ovarian tissue from P. barbatus queenright and queenless workers. A total of 26 genes were identified as differentially expressed (FDR < 0.05), with just one gene upregulated (Supplementary Data 2). Principal component analysis (PCA) and a heatmap of the differentially expressed genes are shown (Fig. 7 a, b). Most of the downregulated genes are associated with lipid and fatty acid metabolism. In contrast, the single upregulated gene in queenless workers corresponded to a microtubule-associated protein-like gene (Fig. 7 a), which has been implicated in egg development in echinoderms and fertility in mice 34 , 35 . Discussion In order to characterize active and inactive ovaries in ants, we analyzed the structure and morphology of the ovaries of Pogonomyrmex barbatus . Our morphological and structural analysis revealed significant differences across castes and worker ages, including ovariole number, oocyte development, tracheal supply, and the presence of yolk granules. These traits reflect caste-specific reproductive specialization and age-related functional decline in workers. Queens of P. barbatus exhibit a greater number of ovarioles compared to workers, and their oocytes are significantly larger, which enables them to produce multiple eggs simultaneously, a trait that could be essential for colony survival as has been documented for other ant species 13 , 36 . Although each ovariole in queens contains fewer follicles, these follicles and their developing oocytes are larger and more advanced in maturation, suggesting a reproductive strategy that prioritizes the production of fewer but higher quality oocytes. This is consistent with their role as the primary reproductive individuals in the colony. The abundant presence of yolk granules, indicative of active vitellogenesis, and the well-defined structure of queen oocytes highlight their specialization for reproduction. In contrast, workers have fewer ovarioles and smaller oocytes, indicating lower reproductive capacity. Interestingly, young (callow) workers possess a higher number of follicles per ovariole than older workers despite having longer ovarioles. How the number of follicles per ovariole relates to reproductive capacity within the worker caste is unclear, but it may suggest that they retain some reproductive potential early in life that diminishes with age 13 , 37 , 38 . Similar age-related declines in egg number and quality have been described in other social insects such as bees and wasps 39 – 41 . These changes likely reflect a transition in colony roles due to age polyethism, where older workers take on tasks such as foraging and defense activities that do not require reproductive capacity 42 – 44 . Such a shift has also been observed in Pogonomyrmex californicus , where nurses involved in brood care have more oocytes than foragers, even though brood care does not necessarily require oviposition 42 , 45 . The ultrastructural differences observed between castes and age groups not only reflect functional states of the ovary but also reveal fundamental aspects of reproductive physiology in social insects. The high density of mitochondria and the extensive development of rough endoplasmic reticulum (RER) in follicular cells surrounding queen oocytes suggest high metabolic activity, characterized by abundant mitochondria, which is consistent with the biosynthetic demands of sustained reproduction. In contrast, follicular cells in mature workers (older individuals) exhibit signs of cellular damage, such as disrupted mitochondrial integrity and nuclear membrane abnormalities, which may reflect an age-related decline in cellular function. However, young (callow) workers do not exhibit such damage, potentially reflecting the evolutionary trade-off between reproduction and longevity typical of social insects 9 , 10 . An important morphological distinction in the ovaries between castes was the separation of follicular cells, creating intercellular spaces, likely facilitating the passage of vitellogenin into the oocyte via patency 28 . This structural adaptation, essential for the accumulation of yolk precursors and nutrient reserves during vitellogenesis, was absent in worker ovaries, suggesting a structural suppression of vitellogenesis in this caste. Supporting this pattern, notable differences were also observed in the organization of the microvillar zone. In queens, the microvilli formed a dense, continuous interface at the oocyte surface, supporting enhanced exchange of nutrients and signaling molecules. In contrast, the microvilli in workers were fragmented and discontinuous, reflecting a reduced capacity for uptake at the oocyte interface. Together, the presence of intercellular spaces between follicular cells and the structure of the microvillar region support the notion that nutrient transport and vitellogenin uptake are regulated according to reproductive status and restrict the reproductive potential in workers. To better understand the molecular basis underlying reproductive divergence between castes, we performed a transcriptomic analysis in queen and worker ovaries. Genes upregulated in queens were associated with processes critical for oogenesis and sustained reproductive activity, including isoprenoid and terpenoid biosynthesis, peptide catabolism, proteolysis, cell division, and glutathione metabolism. These enriched functions reflect key biosynthetic and antioxidant demands of active ovaries and are consistent with the cellular observations in queen follicular cells indicative of high metabolic activity. In contrast, genes downregulated in queen ovaries, or up in workers, are linked mainly to nutrient transport, neurophysiology, and carbohydrate metabolism. The reduced structural integrity of follicular cells in workers, along with the absence of transcriptional signatures associated with antioxidant pathways and cellular renewal as in queens, may reflect the limited evolutionary investment in reproductive maintenance in this caste. Notably, major royal jelly protein 1 ( mrjp1 ) and a related mrjp1-like gene also showed high expression in queens. These genes, originally characterized in eusocial bees 46 , 47 , have been linked to caste differentiation and, more recently, to antioxidant and longevity-promoting functions 48 . Although only one complete copy of mrjp has been confirmed in P. barbatus , its elevated expression suggests it may play an important but yet undescribed role in ant reproduction or queen physiology. Interestingly, in bumblebees there is only one mrjp gene, which has an analogous role in reproduction and caste differentiation in bees 49 , 50 , suggesting that a single copy may be enough to fulfill these functions in ant queens of P. barbatus . This is further supported by evidence that royal jelly and related proteins extend lifespan in model organisms such as C. elegans 51 , 52 . In addition, genes involved in the mevalonate pathway, which is involved in juvenile hormone (JH) biosynthesis, a key regulator of vitellogenesis 53 , were also upregulated in queens 54 . Likewise, the vitellogenin-2-like gene was upregulated, which may explain the high levels of yolk granules found in the mature ovaries of queens. Interestingly, some components of this juvenile hormone pathway were upregulated in queens, while others in the same pathway were downregulated, suggesting a complex, possibly caste-specific modulation of hormonal signals. Similarly, within the insulin/IGF signaling (IIS) pathway many insulin-degrading enzyme-like genes, as well as the insulin-like growth factor II-B ( IGF-2 ) gene, were overexpressed in queens, while a gene encoding an insulin-like receptor was downregulated, potentially reflecting alternative signaling mechanisms that decouple reproduction from aging, as observed in other social insects 55 . Consistent with the reproduction specialized expression profile observed in P. barbatus queens, Aurora kinase B like ( AURKB ) and Aurora kinase C-like ( AURKC ) genes were also found to be upregulated. Aurora kinases are well established regulators of fecundity and oocyte development in mammals and have been demonstrated to regulate meiotic spindle assembly in oocyte maturation in the tunicate Oikopleura dioica 56 . AURKB and AURKC play essential roles in key cell cycle processes, such as regulating chromosome alignment and accurate meiotic division, which are critical for the production of functional oocytes 57 . This molecular signature is consistent with the need of ant queens to sustain continuous oogenesis throughout their reproductive lifespan. Thus, the upregulation of these genes in queens likely reflects their essential role in supporting efficient oocyte maturation in reproductively active and highly fecund individuals. KEGG pathways associated with glutathione metabolism and peroxisome activity also point to reinforced antioxidant defenses, likely supporting sustained oogenesis while mitigating oxidative stress. Notably, the gene encoding superoxide dismutase 1 ( SOD1 ) was downregulated in queens compared to workers. While SOD1 overexpression has been linked to increased lifespan in model organisms like Drosophila 58 , 59 , studies in long-lived ant queens, such as Lasius niger , suggest that extreme longevity can occur despite lower SOD1 levels 60 , 61 . This pattern, also observed in P. barbatus , supports the idea that reproductive castes may rely on non-canonical antioxidant mechanisms, possibly involving reduced mitochondrial reactive oxygen species (ROS) production rather than elevated expression of detoxifying enzymes. Such alternative strategies could represent a key adaptation for sustaining long-term reproductive function without the need for constitutively high antioxidant activity. We also observed that queen removal did not lead to ovary activation in P. barbatus workers. In many ant species, the absence of the queen triggers egg-laying by workers, either of viable or trophic eggs, due to the loss of inhibitory chemical signals such as pheromones or cuticular hydrocarbons (CHCs) emitted by the queen 62 – 65 . These cues typically maintain reproductive suppression among workers, but once removed, some individuals can activate their ovaries and initiate oogenesis. However, in P. barbatus , morphological evidence indicated the opposite trend. Workers from queenless colonies displayed clear signs of ovarian regression, including shorter ovarioles, smaller oocytes, and fewer developing oocytes. This contrasts with what is typically observed in bees and many ants, where the absence of queen pheromones lifts reproductive inhibition and leads to ovary development 16 , 31 , 66 , 67 . These findings suggest that in P. barbatus , reproductive suppression is not solely dependent on queen-derived chemical cues. At the molecular level, we found only one upregulated gene in 5-week queenless compared to queenright workers; echinoderm microtubule-associated protein-like ( EML ). Orthologs of this gene have been implicated in the control of oocyte meiotic progression in mammals and in ribosome-microtubule attachment in sea urchin eggs 34 , 35 . However, its specific role in ant ovaries remains unknown. Notably, this gene did not show differential expression between queens and queenright workers, suggesting its upregulation may not be directly linked to reproductive activation. In contrast, 25 genes were downregulated in queenless workers, 20 of which were also downregulated in queens. Among these were genes involved in CHC biosynthesis, raising the possibility that queenless workers may experience a partial chemical profile shift, perhaps mimicking queen-like traits, despite remaining non-reproductive. Further supporting the morphological and molecular evidence, five weeks of behavioral monitoring revealed no oviposition or presence of trophic or viable eggs in queenless colonies. Still, workers retain ovarian tissue, which may represent an evolutionary safeguard that allows reproductive flexibility under extreme ecological conditions, as seen in other ant species 13 , 68 – 70 . The persistent suppression of ovarian development in workers likely reflects a colony-level strategy that favors non-reproductive roles such as brood care and foraging. This is particularly relevant in P. barbatus , where workers are not known to lay eggs, even in the absence of the queen. Interestingly, this contrasts with what is seen in the closely related species Pogonomyrmex rugosus , where workers are known to produce trophic eggs when the queen is absent 11 . These interspecific differences underscore the diversity of reproductive regulation strategies among closely related ants. While no egg-laying was observed during our five-week observation period, we cannot exclude the possibility that longer timeframes or specific environmental cues might eventually allow ovary activation in P. barbatu s workers. This work represents the first detailed morphological, structural, and molecular characterization of ovaries exhibiting extreme differences in reproductive activity and longevity within a fixed caste species. By examining reproductive constraints in a system where genetically similar individuals show vast disparities in lifespan and reproductive output, this study provides valuable insights into the mechanisms underlying reproductive aging. These findings contribute to a deeper understanding of how reproductive division of labor shapes aging trajectories in social insects and offer a solid foundation for future research into the regulation of longevity and fertility within complex societies. Declarations Author Contribution Investigation and data analysis: M.F.V-M., D.G-S., B.O-D., E.S-S. and I.F-P. Writing: M.F.V-M., D.G-S., B.O-D. and I.F-P. Methodology: M.F.V-M., D.G-S., B.O-D., E.S-S, P.M-G., R.R-R., R.M.V-V., A.G-M. and I.F-P. Resources: A.G-M., R.M.V-V. and I.F-P. Funding acquisition and supervision: I.F-P. All authors reviewed the manuscript. Acknowledgement We thank César Maximiliano Vázquez Franco for taking the Pogonomyrmex barbatus ant photographs shown in Figure 1. We are also grateful to Miguel Tapia Rodríguez from the Microscopy Unit at IIBO for technical assistance. We thank Andrea Luna Méndez and Mayra Franco Sariñana for their dedicated care of the ant colonies. We are also thankful to Ian A. E. Butler for his valuable suggestions on the manuscript and assistance with editing. We thank Natália Faraj Murad for advice on transcriptomic analysis. Finally, we are grateful to everyone at the AntLab UNAM for their helpful suggestions and support. The electron microscopy work was supported by Program E022 from the Instituto Nacional de Pediatría in Mexico City. This work was supported by the Global Consortium for Reproductive Longevity and Equality at the Buck Institute for Research on Aging, made possible by the Bia-Echo Foundation (GCRLE-0620 Junior Scholar Award) to IF-P. This work was also supported by UNAM-PAPIIT IN21072 funding to IF-P, and Secretaría de Ciencia, Humanidades, Tecnología e Innovación (SECIHTI) scholarship CVU 1249398 to MFV-M and is part of the requirements for obtaining the doctoral degree in experimental biology at the Posgrado en Ciencias Biológicas UNAM of MFV-M. Data Availability Reads for all sequences generated in this study were deposited into the National Center for Biotechnology Sequence Read Archive (NCBI - SRA) BioProject ID: PRJNA1279095 and are available at the following URL: https://dataview.ncbi.nlm.nih.gov/object/PRJNA1279095?reviewer=upe3k3keq6j9n34p7o6g8hju6r References Gordon, D. M. 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(a) Queen. (b) Mature worker (\u0026gt;20 days old), with darker cuticle coloration. (c) Callow worker (\u0026lt;20 days old) with lighter cuticle coloration. (d) Schematic drawing of the ovariole structure of \u003cem\u003eP. barbatus.\u003c/em\u003e(e) Queen ovaries. (f) worker ovaries. Samples were cleaned to get rid of tracheas as much as possible, but queen ovarioles possess a large number of them making their complete removal difficult (arrows). Scale bar represents 1000 µm.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6933990/v1/84b1447c1c2171dbcdcf7920.png"},{"id":85558444,"identity":"ce3953a0-deb4-460c-b5ec-4b52b1954e26","added_by":"auto","created_at":"2025-06-27 11:58:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2341427,"visible":true,"origin":"","legend":"\u003cp\u003eOvary morphology in \u003cem\u003ePogonomyrmex barbatus\u003c/em\u003e varies with age and caste. (a-a’’) Confocal images of \u003cem\u003eP. barbatus\u003c/em\u003e ovarioles for queen, callow and mature workers. (b) Quantification of the number of ovarioles per individual. (c) Quantification of the length of ovarioles per individual. (d) Quantification of number of follicles per ovariole. (e) Quantification of the largest oocyte area per individual. Green, yellow and orange points represent different colonies. Scale bar represents 500 µm. * significant via one-way anova, p=0.05.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6933990/v1/71681e981ce3721f2d3d155b.png"},{"id":85557880,"identity":"3d6d3c6a-468d-43a3-9e10-1a9ac0be319e","added_by":"auto","created_at":"2025-06-27 11:50:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":364367,"visible":true,"origin":"","legend":"\u003cp\u003eLight micrographs of \u003cem\u003ePogonomyrmex barbatus\u003c/em\u003e queen and worker ovaries reveal caste-specific differences in oocyte maturation and vitellogenesis. (a) Comparative diagram of oocytes during vitellogenesis showing the patency (left) and non-patency (right) states. (b, c) Queen oocytes exhibit yolk granules all over the oocyte surface. Gaps between follicular cells are well distinguished surrounding the bigger queen oocyte indicating active patency. (d, e) Callow worker oocyte. (f, g) Mature worker oocyte. No yolk granules were found in worker oocytes. (VO) Vitellogenic oocyte, (PVO) Previtellogenic oocyte, (FC) Follicular cells, (YG) Yolk granules, (black arrows) Intercellular spaces. Scale bars represent 100 µm.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6933990/v1/a9380d0a66324175bd4bf1d4.png"},{"id":85559090,"identity":"6064b7c2-a64d-435a-921a-2edfff6ba70c","added_by":"auto","created_at":"2025-06-27 12:14:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":211391,"visible":true,"origin":"","legend":"\u003cp\u003eLight and electron microscopy images of \u003cem\u003ePogonomyrmex barbatus\u003c/em\u003e oocytes, highlighting caste- and age-related ultrastructural differences between queens and workers. The vitellogenic (VO) and previtellogenic (PVO) oocytes are shown. All oocytes are surrounded by follicular cells (fc). (a-e) The ooplasm of the queen is dominated by vacuole-like structures (*) and protein plates (p). Lipidic structures (l) and lysosomes (ly) are also found. The band of the microvilli (empty arrowheads) is wide and electrodense. Follicular cells (fc) have a prominent nucleus (nc). (e) The cytoplasm of a follicular cell shows mitochondria (mt) and rough endoplasmic reticulum (ER). (f-j) In the ooplasm of the callow worker ant there are large vacuoles (*) surrounded by protein plates (p). Lipidic structures (l) and lysosomes (ly) are also found. The follicular cells have a large nucleus (n). The microvilli zone is discontinuous (empty arrows). The cytoplasm of follicle cells (fc) contains mitochondria (mt) and peroxisomes (black arrowheads). (k-o) In the ooplasm of mature ants, the presence of large vacuoles (*) surrounded by protein plates (p), lipidic structures (l) and lysosomes (ly) are shown. There is also discontinuity in the microvilli zone (empty arrows). There is damage to the double membrane of the cell nucleus (nc) and mitochondria (mt) (arrows). Amplification: a, f, k = 200x (Toluidine blue stain); b, g, l = 5000x; c, h, m = 8,330X; d, i, n = 25,000x; e, j, o = 83,300x.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6933990/v1/2a6c18ed830448243f6306db.png"},{"id":85558449,"identity":"d73678b0-3832-464a-869e-97dcaecc0d81","added_by":"auto","created_at":"2025-06-27 11:58:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2818836,"visible":true,"origin":"","legend":"\u003cp\u003eOvaries of\u003cem\u003e Pogonomyrmex barbatus \u003c/em\u003eworkers change structure with the removal of the queen. (a-a’) Confocal images of queenright and queenless worker ovarioles. (b) Quantification of the number of ovarioles. (c) Quantification of the length of ovarioles. (d) Quantification of number of follicles. (e) Quantification of the area of the largest oocyte. Green triangles represent old workers while blue squares represent young workers. Scale bar: 500 µm.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6933990/v1/537a3d40ba8d37838a9f0ad1.png"},{"id":85558510,"identity":"97883168-575e-43d8-b675-d9af8bea2891","added_by":"auto","created_at":"2025-06-27 12:06:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":414403,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptomic profiling of ovarian tissues reveals caste-specific gene expression in \u003cem\u003ePogonomyrmex barbatus \u003c/em\u003eants. (a) Heatmap of 2052 differentially expressed genes in ovaries of\u003cem\u003e P. barbatus\u003c/em\u003e queens and workers. Expression values are shown as Z-scores of normalized counts. (b) Principal component analysis (PCA) of gene expression profiles in queen and worker ovaries of\u003cem\u003e P. barbatus\u003c/em\u003e. Each dot represents a biological replicate. (c–d) Gene Ontology (GO) enrichment analysis for biological processes associated with genes upregulated (c) and downregulated (d) in queen ovaries relative to worker ovaries. (e–f) KEGG pathway enrichment analysis for genes upregulated (e) and downregulated (f) in queen ovaries relative to worker ovaries.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6933990/v1/8e679953639e0f9e8a323042.png"},{"id":85557904,"identity":"d8666ebe-8a30-4097-9a3a-7617259e4e05","added_by":"auto","created_at":"2025-06-27 11:50:08","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":530310,"visible":true,"origin":"","legend":"\u003cp\u003eOvarian transcriptomic changes in \u003cem\u003ePogonomyrmex barbatus\u003c/em\u003e workers after 5 weeks of queen absence. (a) Heatmap of differentially expressed genes (DEGs) between ovaries of workers from queenless colonies (5 weeks) and queenright colonies. Expression values are shown as Z-scores of normalized counts. (b) Principal component analysis (PCA) of ovarian transcriptomes between queenless and queenright worker samples. Each point represents one biological replicate.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-6933990/v1/f5ca69a87bff559bea9522f9.png"},{"id":96105008,"identity":"4f612a82-dc03-475b-892c-df7d8b8481a8","added_by":"auto","created_at":"2025-11-17 16:06:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10178856,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6933990/v1/f04ab3a3-f4c9-440b-8710-a779afb8b151.pdf"},{"id":85557877,"identity":"fc6d46a0-b0eb-4adf-8a63-94ea26dd1db0","added_by":"auto","created_at":"2025-06-27 11:50:07","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1737798,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6933990/v1/2783b77381a91b02b2311647.docx"},{"id":85558445,"identity":"5360cc27-8ce3-40b7-bc2e-1c9ee2a715a0","added_by":"auto","created_at":"2025-06-27 11:58:07","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":404087,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryData1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6933990/v1/575514ccf0fdae5b110fd328.xlsx"},{"id":85557884,"identity":"a3ed3199-dfb2-46cb-afbc-316bb182e1f1","added_by":"auto","created_at":"2025-06-27 11:50:07","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":54456,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryData2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6933990/v1/9b7188db4613ee5dcc37f2aa.xlsx"},{"id":85557914,"identity":"e4417f2b-8148-4a04-91e1-3447d8f51791","added_by":"auto","created_at":"2025-06-27 11:50:08","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":331494,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryData3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6933990/v1/6192001fcc1ee0af07493a0e.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Age, Caste, and Social Context Shape Ovarian Morphology and Transcriptomic Profiles in Red Harvester Ants (Pogonomyrmex barbatus)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEusocial insects, including ants, exhibit a striking reproductive division of labor. While the queen specializes in reproduction, the workers mainly perform cooperative tasks such as nest building, food collection, brood care, and colony defense\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. This social complexity, along with their global abundance\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, makes ants important species in the functioning and maintenance of many ecosystems\u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Notably, despite having very similar genomes, the reproductive and lifespan differences between castes are remarkable, with queens of some species capable of living up to 40 years, continuously producing offspring, while the sterile workers typically live only a few months\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. This difference is unusual, as in most organisms, there is a trade-off between fecundity and longevity: high reproductive investment usually comes at the cost of reduced lifespan. In ants, however, this trade-off is absent, as queens are both the most fertile and longest-lived individuals in a colony\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThis phenomenon is particularly prominent in species with fixed caste systems, the most common form of social organization among ants, where reproductive roles are determined early and remain stable throughout life. In these species, the lifespan gap between castes is especially dramatic. This notable difference in lifespan and reproductive capability underscores the specialized roles each caste plays in the survival and success of the colony. It also raises a critical question: What physiological mechanisms enable queens to sustain both high reproductive output and extended longevity, while workers remain largely sterile with shorter lifespans?\u003c/p\u003e \u003cp\u003eA major anatomical distinction between queens and workers lies in their ovaries. The number of ovarioles in an individual ant differs by species, but within most species, the queen has the largest number of ovarioles, with several simultaneously developing oocytes at various stages, a clear reflection of their reproductive specialization within the colony. This specialization is also reflected in the presence of spermathecae, specialized sperm storage organs that release sperm to fertilize the eggs. In contrast, workers generally have fewer, often smaller, ovarioles with less developed follicles. In many species, workers have lost the ability to lay eggs completely or produce only embryo-less eggs, known as trophic eggs\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eStructurally, the reproductive system of female ants consists of two ovaries, each with several polytrophic meroistic ovarioles\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. In general, the anterior end of the ovarioles contains the germarium, which contains terminal filament cells and germline stem cells. Terminal filament cells represent the immediate progeny of the germline stem cells where the development of each oocyte begins. These cells differentiate into cystoblast cells in the germarium, which subsequently form multicellular cysts. Within these cysts, one cell becomes the oocyte while the others become nurse cells, forming the follicle. This structure is surrounded by follicular cells that facilitate the transfer of proteins from the hemolymph to the oocyte, such as vitellogenin\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The nurse cells nourish the adjacent developing oocyte by providing RNA and proteins. The follicle moves along the length of the ovariole as it grows and matures. For the production of workers or gynes, the eggs are then fertilized by sperm released from the spermatheca. Males come from unfertilized haploid eggs\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Additionally, in some species, both queens and workers can lay trophic eggs\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Despite this foundational knowledge, very little is known about how ovarian morphology differs between queens and workers across species, or how these structural differences might influence physiology and aging trajectories in each caste.\u003c/p\u003e \u003cp\u003eWhile most ants exhibit fixed caste systems, some display remarkable reproductive plasticity, allowing individuals to shift between reproductive and non-reproductive states depending on environmental or social cues. Examples include queenless species such as \u003cem\u003eOoceraea biroi\u003c/em\u003e and \u003cem\u003ePristomyrmex pungens\u003c/em\u003e, which reproduce via thelytokous parthenogenesis, and facultatively thelytokous species like \u003cem\u003ePlatythyrea punctata\u003c/em\u003e and \u003cem\u003eCataglyphis cursor\u003c/em\u003e, where egg-laying can occur in the absence of a queen\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Even more strikingly, in \u003cem\u003eHarpegnathos saltator\u003c/em\u003e, certain workers (gamergates) can take over reproduction and dramatically extend their lifespan following queen loss\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDespite these fascinating exceptions, most ants exhibit rigid caste systems, where only queens reproduce, and workers remain sterile. It is in these fixed systems that we observe the most extreme contrasts in ovarian structure and lifespan, yet the molecular and physiological mechanisms underlying this specialization remain poorly understood.\u003c/p\u003e \u003cp\u003eThe red harvester ant, \u003cem\u003ePogonomyrmex barbatus\u003c/em\u003e, provides an ideal model for addressing this gap. Native to North American arid and semi-arid regions\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, this species has a strict reproductive division of labor: queens are the sole reproductive individuals, while workers, despite retaining ovaries, show no evidence of egg-laying\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Importantly, the lifespan difference between queens and workers is extreme, queens can live up to 30 years, while workers survive only about a year. This contrast far exceeds that of plastic species, where lifespan differences are often limited to just a few months or years. Despite their ecological and evolutionary significance, the morphological and physiological differences between queen and worker ovaries in this species remain largely unexplored.\u003c/p\u003e \u003cp\u003eTo address this gap, we compared ovarian structure across castes, ages, and social conditions. Additionally, we conducted transcriptomic analyses to identify molecular signatures associated with reproductive division of labor. Our findings provide new insights into the mechanisms underlying reproductive aging and caste-specific longevity in ants.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eHusbandry\u003c/h2\u003e \u003cp\u003eTo compare ovaries between queens and workers of \u003cem\u003eP. barbatus\u003c/em\u003e, we used workers from three colonies collected during 2021 and 2022 in Texcoco de Mora and Cuautitl\u0026aacute;n Izcalli, State of Mexico, Mexico. The colonies were maintained in the laboratory in artificial nests at 27\u0026deg;C on a 12-hour light/dark cycle, and were provided with canary seeds and water \u003cem\u003ead libitum\u003c/em\u003e. Workers used in the experiment were collected at the foraging arena from three different colonies. By the time of the experiment each of these colonies had between 100\u0026ndash;200 workers in total. Cuticle color was used as an age marker in the workers, with light-colored workers (\u0026lt;\u0026thinsp;20 days old) considered callows (young) and dark-colored workers (\u0026gt;\u0026thinsp;20 days old) considered mature (old). Queens were obtained from recently founded colonies (~\u0026thinsp;5 months old) with 3\u0026ndash;5 workers each.\u003c/p\u003e \u003cp\u003eTo compare queenright and queenless workers of \u003cem\u003eP. barbatus\u003c/em\u003e, we used workers from recently founded colonies. The colonies were collected in May 2023 in Texcoco de Mora, State of Mexico, Mexico, and placed in test tubes at 27\u0026deg;C on a 12-hour light/dark cycle, and were provided with canary seeds and water \u003cem\u003ead libitum\u003c/em\u003e. The colonies contained between 6\u0026ndash;8 ants at the start of the experiment. For queenless colonies, the queen was removed, and the workers remained in contact with the eggs, larvae, and pupae for five weeks until dissection.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDissection of ant ovarioles\u003c/h3\u003e\n\u003cp\u003eFemale ants were anesthetized on ice for one minute and immersed for another minute in 70% ethanol before being placed in a small amount of 1X phosphate buffered saline (PBS). Ovarioles were dissected by pulling on the third tergite using a pair of fine tweezers and then removing fat and unrelated tissues. Ovaries were placed immediately into a fixative solution of 4% paraformaldehyde (PFA) in 1X PBS.\u003c/p\u003e\n\u003ch3\u003eStaining\u003c/h3\u003e\n\u003cp\u003eOvaries were fixed overnight at 4\u0026deg;C in 4% PFA in 1X PBS. Following fixation, ovaries were washed 3 times for 20 minutes each with Triton (0.5% Triton in 1X PBS) at room temperature on a rotator. The samples were then washed 2 times for 10 minutes in 0.01% Tween in 1X PBS, ovaries were incubated with DAPI (1:1000, Sigma, #D9542) and Phalloidin (1:400, Invitrogen, #A22287) for 2h, and mounted in Vectashield\u0026reg; mounting media (Vector Laboratories, #H1000)(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eConfocal microscopy and analysis\u003c/h3\u003e\n\u003cp\u003eConfocal images were captured using a Nikon A1R\u0026thinsp;+\u0026thinsp;confocal laser scanning microscope. Images were analyzed in ImageJ software for quantitative and qualitative characterizations while statistical analysis and generation of figures were performed using GraphPad Prism software. Statistical significance was assessed with a permutation test for constrained correspondence analysis (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10,000 random permutations) using Python 3.7.16 within a Jupyter notebook, employing the pandas, statsmodels, numpy, and scipy libraries.\u003c/p\u003e\n\u003ch3\u003eHistological sections and light microscopy\u003c/h3\u003e\n\u003cp\u003eAnt ovaries were collected in 1X PBS and fixed overnight at 4\u0026deg;C in 4% PFA in 1X PBS. Following fixation, the tissues were briefly washed four times with 1X PBS and then post-fixed in a 10% formalin buffer overnight at room temperature. The samples were washed four times for 15 minutes each with distilled water at room temperature. Dehydration was carried out with a series of ethanol baths of 60%, 70%, 80%, 90%, 96%, 100% and 100% for one hour each using a Leica TP 1020 automatic tissue processor. The samples were then washed in 50% alcohol-50% xylol for one hour, 100% xylol twice for one hour each, and two washes of paraffin (Paraplast Plus\u0026reg;), one for 1 hour and the other for 30 minutes. The samples were then embedded in paraffin blocks at 58\u0026deg;C-60\u0026deg;C, and five-micron sections were cut using a Leica RM 2155 rotary microtome and mounted on slides.\u003c/p\u003e \u003cp\u003eFor staining, we began by deparaffinizing the samples. We placed them in an oven at 60\u0026deg;C for 30 minutes, then placed the slides in a coplin jar and washed them with xylol twice, first for 10 minutes, then 5 minutes. The samples were washed with 50% alcohol-50% xylol for 5 minutes. Next, we treated them with 100%, 96%, 96%, and 70% ethanol for 5 minutes each. Finally, we rinsed the samples twice in distilled water for 5 minutes each.\u003c/p\u003e \u003cp\u003eThe samples were stained with Gill\u0026rsquo;s hematoxylin 0.20% for 7 minutes, then rinsed in tap water for 3 minutes, followed by two rinses in distilled water, each for 3 minutes. They were then stained with eosin 0.25% for 10 minutes. Following staining, the samples were subjected to a series of ethanol washes (70%, 80%, 90%, 96%, 100%) for 4 minutes each. We then treated the samples with 50% alcohol-50% xylol twice for 4 minutes each. Afterward, the samples were immersed in xylene for 4 minutes, the xylene was replaced and the samples were maintained in xylene until mounting. Finally, we mounted the samples on slides with a coverslip and one or two drops of rapid mounting medium ENTELLAN\u0026reg;. Images were captured using a Carl Zeiss\u0026trade; Axio Vert.A1 inverted microscope under bright field illumination.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTransmission electron microscopy (TEM)\u003c/h2\u003e \u003cp\u003eOvaries were dissected in PBS and fixed by immersion in 2.5% glutaraldehyde and 4% PFA in sodium phosphate buffer (0.1M, pH 7.4), post-fixed in 1% osmium tetroxide, dehydrated in a graded series of ethanol and embedded in EPON (epoxy resin). Semithin sections (1 \u0026micro;m) were cut using an ultramicrotome (Leica EM UC6), stained with toluidine blue to select areas in light microscopy examination (Axioskop 2 Plus). Ultrathin sections of 60\u0026ndash;90 nm were cut and collected on slot grids previously covered with formvar membrane. Sections were stained with uranyl acetate and lead citrate. The structure for each sample was recorded using a Jeol JEM-1011 transmission electron microscope housed in the National Institute of Pediatrics in Mexico. Images were taken with PhotoImpact 10.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRNA extraction and sequencing\u003c/h3\u003e\n\u003cp\u003eFrom four ant colonies, approximately 50 foragers were selected based on cuticle color (dark) and task they were doing and labeled on their gaster with Uni-paint markers. 20 workers were returned to their respective colonies, whereas the remaining workers were kept five weeks isolated from their queen. Three different experimental groups were considered in this study: queens, workers that were kept in their colonies (queenright), and workers isolated for five weeks (queenless).\u003c/p\u003e \u003cp\u003eAnts were anesthetized on ice and the ovaries were dissected in an ice-cold 1X PBS buffer using fine forceps and scissors. Fat bodies attached to the tissues were carefully removed. To increase the final RNA concentration, we pooled tissue for each RNA sample. Each pool contained five queens or ten workers from either group (queenright and queenless). We used four pooled biological replicates for each condition. Dissected and pooled ovaries were immediately stored in QIAzol Lysis Reagent (QIAGEN), homogenized in a FastPrep-24\u0026trade; Classic Instrument (MP Biomedicals) and finally frozen at -80\u0026deg;C. RNA was extracted using a modified QIAZol/phenol chloroform protocol, followed by RNeasy (QIAGEN) purification with DNase I (Thermo Fisher Scientific) on-column digestion. Isolated RNA sample quality was assessed with a High Sensitivity RNA Tapestation (Agilent Technologies) and quantified by Qubit 2.0 RNA HS assay (Thermo Fisher Scientific).\u003c/p\u003e \u003cp\u003eRNA extractions were sent to Admera Health BioPharma Services (New Jersey, USA) for Poly(A) selection library preparation and RNA-Seq.\u0026nbsp;Paramagnetic beads coupled with oligo d(T)25 were combined with total RNA to isolate poly(A)\u0026thinsp;+\u0026thinsp;transcripts based on the NEBNext\u0026reg; Poly(A) mRNA Magnetic Isolation Module manual (New England BioLabs). Prior to first strand synthesis, samples were randomly primed (5\u0026acute; d(N6) 3\u0026acute; [N\u0026thinsp;=\u0026thinsp;A,C,G,T]) and fragmented based on manufacturer\u0026rsquo;s recommendations. The first strand was synthesized with the Protoscript II Reverse Transcriptase with a longer extension period, approximately 30 minutes at 42⁰C.\u003c/p\u003e \u003cp\u003eAll remaining steps for library construction were done according to the NEBNext\u0026reg; Ultra\u0026trade; II Directional RNA Library Prep Kit for Illumina\u0026reg; (New England BioLabs). Final RNA quantity of the libraries was assessed by Qubit 2.0 (Thermo Fisher Scientific) and quality was assessed by TapeStation HSD1000 ScreenTape (Agilent Technologies). Final fragment size was about 500bp with an insert size of about 350bp. Illumina\u0026reg; 8-nt dual-indices were used. Equimolar pooling of libraries was performed based on QC values and sequenced on an Illumina\u0026reg; NovaSeq X Plus 10B (Illumina) with a read length configuration of 150 PE for 40 M PE reads per sample (20M in each direction).\u003c/p\u003e \u003cp\u003eReads for all sequences generated in this study are available at the National Center for Biotechnology Sequence Read Archive (NCBI - SRA; BioProject ID: PRJNA1279095).\u003c/p\u003e\n\u003ch3\u003eSequence processing and differential expression analyses\u003c/h3\u003e\n\u003cp\u003eQuality reports of the raw sequences were generated using FastQC (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.bioinformatics.babraham.ac.uk/projects/fastqc\u003c/span\u003e\u003cspan address=\"http://www.bioinformatics.babraham.ac.uk/projects/fastqc\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; Andrews, 2010). Adapters and low quality nucleotide bases were trimmed using Cutadapt v4.6\u003csup\u003e21\u003c/sup\u003e and reexamined in FastQC. Mapping of reads onto the genome of \u003cem\u003eP. barbatus\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e previously downloaded from NCBI (GenBank accession: GCF_000187915.1) was done in STAR v2.7.11a\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e and count matrices were generated estimating gene expression levels with the featureCounts tool available in the Subread package v2.0.6\u003csup\u003e24\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDifferential gene expression (DGE) analyses were performed with DESeq2 v3.18\u003csup\u003e25\u003c/sup\u003e. To identify candidate differentially expressed genes within the two analyses (queen vs worker and queenright vs queenless worker) we kept all genes with a significantly adjusted p-value below 0.05 and a fold change greater than 2 (FC\u0026thinsp;\u0026gt;\u0026thinsp;2).\u003c/p\u003e \u003cp\u003eFunctional annotation of the filtered candidate genes was performed using DAVID\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e paying particular attention to gene ontology (GO) terms and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. This database provides a comprehensive set of different functional annotation tools when dealing with large lists of genes. Parameters for the functional annotation analysis included: 1) Gene Ontology: GOTERM Biological Process (BP) Direct, GOTERM Cellular Component (CC) Direct, GOTERM Molecular Function (MF) Direct; and 2) Pathways: KEGG Pathway. All data visualizations were produced with R (version 4.4.1) using RStudio and the Tidyverse package suite.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eDifferences in ovary structure among castes of\u003c/b\u003e \u003cb\u003ePogonomyrmex barbatus\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn addition to their overall morphological differentiation by caste (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-c), queens and workers also differ markedly in their ovarian structures. Queen ovaries were fully developed and contained a high number of tracheae, whereas workers had immature, poorly developed ovaries and contained few tracheae (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee-f). In queens, the large mature vitellogenic oocytes were slightly elongated and surrounded by a thick layer of follicular cells, suggesting the presence of reproductive eggs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Young workers exhibited well-developed ovarioles with a high number of oocyte follicles at many developmental stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u0026rsquo;), and older workers showed atrophied ovarioles, with the ovariole being partly empty and lacking early stage oocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u0026rsquo;\u0026rsquo;).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOn average, queens had significantly more ovarioles per ovary (56.20\u0026thinsp;\u0026plusmn;\u0026thinsp;9.78) than workers (6.70\u0026thinsp;\u0026plusmn;\u0026thinsp;2.40)(Unpaired t test, df\u0026thinsp;=\u0026thinsp;24, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001)(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). There was no significant difference in ovariole number between young workers (8.300\u0026thinsp;\u0026plusmn;\u0026thinsp;1.767) and old workers (5.100\u0026thinsp;\u0026plusmn;\u0026thinsp;1.853)(ANOVA, df\u0026thinsp;=\u0026thinsp;24, p\u0026thinsp;=\u0026thinsp;0.37 after Bonferroni correction)(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eOvariole length did not differ significantly between queens (1,873\u0026thinsp;\u0026plusmn;\u0026thinsp;262\u0026micro;m) and workers (1,896\u0026thinsp;\u0026plusmn;\u0026thinsp;357.4\u0026micro;m)(Unpaired t test, df\u0026thinsp;=\u0026thinsp;24, p\u0026thinsp;=\u0026thinsp;0.89). Within the worker caste, old workers had significantly longer ovarioles (2,080\u0026thinsp;\u0026plusmn;\u0026thinsp;352.4 \u0026micro;m) than young workers (1,713\u0026thinsp;\u0026plusmn;\u0026thinsp;265.5\u0026micro;m)(ANOVA, df\u0026thinsp;=\u0026thinsp;24, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 after Bonferroni correction)(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). However, young workers had significantly more follicles per ovariole (6.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84) than old workers (3.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.43)(ANOVA, df\u0026thinsp;=\u0026thinsp;24, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 after Bonferroni correction)(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Unexpectedly, the number of follicles per ovariole in queens (1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08) was significantly lower than both young workers (6.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84) (ANOVA, df\u0026thinsp;=\u0026thinsp;24, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 after Bonferroni correction) and old workers (3.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.43)(ANOVA, df\u0026thinsp;=\u0026thinsp;24, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 after Bonferroni correction)(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u0026rsquo;-a\u0026rsquo;\u0026rsquo;, 2d). Queens also had significantly fewer developmental stages with vitellogenic oocytes per ovariole (1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08) than workers (5.145\u0026thinsp;\u0026plusmn;\u0026thinsp;1.89)(Mann-Whitney test, df\u0026thinsp;=\u0026thinsp;24, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eWhen comparing the size of the largest oocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee), at which point the egg is fully developed and ready for fertilization, we found that the area of mature oocytes was significantly larger in queens (319,579\u0026thinsp;\u0026plusmn;\u0026thinsp;49,133 \u0026micro;m\u0026sup2;) than both young (106,435\u0026thinsp;\u0026plusmn;\u0026thinsp;39,479 \u0026micro;m\u0026sup2;)(ANOVA, df\u0026thinsp;=\u0026thinsp;24, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 after Bonferroni correction) and old (169,703\u0026thinsp;\u0026plusmn;\u0026thinsp;92,169 \u0026micro;m\u0026sup2;)(ANOVA, df\u0026thinsp;=\u0026thinsp;24, p\u0026thinsp;=\u0026thinsp;0.001 after Bonferroni correction) workers (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eContent and structural differences in the oocytes of\u003c/b\u003e \u003cb\u003ePogonomyrmex barbatus\u003c/b\u003e \u003cb\u003equeens and workers\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe analyzed the composition and structure of developing oocytes of queens and workers in terms of size. We found notable differences between queen and worker oocyte yolk content and follicular cell distribution through hematoxylin and eosin staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe larger oocytes in queens, known as vitellogenic oocytes (VO), and the previtellogenic oocytes (PVO) in workers are precisely delineated by follicular cells (FCs). These cells determine the size and shape of the oocyte, and allow the transport of nutrients to the oocyte cytoplasm\u003csup\u003e\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. However, the oocytes in workers tend to be smaller and to show a less organized distribution of the follicular epithelium, regardless of age, indicating a less advanced stage of maturation that could correspond to their infertility (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed-g).\u003c/p\u003e \u003cp\u003eIn queens (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-c), the separation of the follicular cells surrounding the vitellogenic oocyte (VO) is clearly observed, forming intercellular spaces (black arrows) that may allow the passage of vitellogenin into the cytoplasm through patency (Raikhel \u0026amp; Dhadialla, 1992; Wu et al., 2021; Wyatt \u0026amp; Davey, 1996)(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-b). There are also clear differences between young and old workers. Younger workers have a more distinct organization of follicular cells (FCs), whereas the delineation between these cells becomes less perceptible in older workers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed-g). This suggests a progressive alteration in ovarian structure with age, potentially linked to a decline in reproductive potential or cellular maintenance over time. Furthermore, unlike in workers, dense acidophilic structures are also observed in the ooplasm of queens, which are yolk granules (YG) composed of vitellin, the stored form of vitellogenin in the viable oocyte (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-c)\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. None of the worker oocytes, not even the largest ones, exhibit these yolk granules (YG). This is also seen in non-mature queen oocytes where they lack these yolk granules in their cytoplasm (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). However, unlike in workers, these oocytes in queens will eventually become viable.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDifferences in ooplasm content and oocyte ultrastructure between queen and worker ovaries were evidenced through ultrastructural analysis using TEM\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo explore potential differences in oocyte structure associated with caste and age, we conducted transmission electron microscopy (TEM) on ultrathin sections of the largest oocytes from each group: queens, callow workers, and mature workers.\u003c/p\u003e \u003cp\u003eWe found clear ultrastructural differences between groups. In queens, we observed numerous round and clear structures within the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Ultrastructural analysis confirmed that these correspond to vacuole-like structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Protein plaques in the ooplasm appeared dark, indicating higher protein density, and were large, irregular or ovoid in shape (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). The microvilli zone was compact, dense, and continuous, suggesting an increased surface area for nutrient exchange into the mature oocyte. On the oocyte side of this interface, small and abundant protein plaques were evident (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Follicular cells surrounding the oocyte displayed nuclei with prominent nucleoli, numerous mitochondria, and a well-developed rough endoplasmic reticulum (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eIn callow workers, the oocyte cytoplasm also contained vacuoles and protein plaques (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef), which were frequently arranged around the vacuoles (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg) and accumulated beneath the microvilli zone, which appeared discontinuous (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh-i). Within the follicular cells, mitochondria and abundant peroxisomes were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej).\u003c/p\u003e \u003cp\u003eIn mature workers, large vacuoles were observed in the oocyte cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ek). Similar to callow workers, these were surrounded by protein plaques (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003el) and located predominantly beneath the microvilli zone, which showed marked discontinuity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003em-n). Follicular cells contained mitochondria, peroxisomes, and rough endoplasmic reticulum; however, signs of cellular damage were also evident, including disorganization and discontinuities in the nuclear envelope and mitochondrial membranes, suggesting a possible loss of functional integrity in these older individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eo).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of queen removal on ovary structure in\u003c/b\u003e \u003cb\u003ePogonomyrmex barbatus\u003c/b\u003e \u003cb\u003eworkers\u003c/b\u003e\u003c/p\u003e \u003cp\u003eDue to the fact that the absence of a queen induces various physiological changes in worker ants\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, we decided to study the structure of the ovarioles 5 weeks after the queen was removed. We compared ovaries from queenright workers and queenless workers in recently founded colonies. In this context, we observed larger and better-developed oocytes in queenright workers (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), while the queenless workers exhibited an atrophied reproductive system, with empty ovarioles and fewer oocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea\u0026rsquo;). Moreover, the atrophy was observed in both young and old workers.\u003c/p\u003e \u003cp\u003eThere was no significant difference in the number of ovarioles between queenright workers (6.600\u0026thinsp;\u0026plusmn;\u0026thinsp;1.89) and queenless workers (5.16\u0026thinsp;\u0026plusmn;\u0026thinsp;2.28)(unpaired t test, df\u0026thinsp;=\u0026thinsp;26, p\u0026thinsp;=\u0026thinsp;0.10)(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). However, queenless workers had shorter ovarioles (queenright, 1,689\u0026thinsp;\u0026plusmn;\u0026thinsp;350.2 \u0026micro;m vs. queenless, 1,316\u0026thinsp;\u0026plusmn;\u0026thinsp;221.1 \u0026micro;m)(unpaired t test, df\u0026thinsp;=\u0026thinsp;26, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01)(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec), with fewer follicles (queenright, 8.577\u0026thinsp;\u0026plusmn;\u0026thinsp;2.84 vs. queenless, 5.31\u0026thinsp;\u0026plusmn;\u0026thinsp;1.69)(unpaired t test, df\u0026thinsp;=\u0026thinsp;26, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and smaller oocytes (queenright, 126,993\u0026thinsp;\u0026plusmn;\u0026thinsp;83,403\u0026micro;m\u0026sup2; vs. queenless, 69,180\u0026thinsp;\u0026plusmn;\u0026thinsp;33,746 \u0026micro;m\u0026sup2;)(unpaired t test, df\u0026thinsp;=\u0026thinsp;26, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05)(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDifferential gene expression in ovaries of\u003c/b\u003e \u003cb\u003ePogonomyrmex barbatus\u003c/b\u003e \u003cb\u003equeens and workers\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate caste-specific molecular differences in the ovaries, we analyzed the ovarian transcriptomes of \u003cem\u003ePogonomyrmex barbatus\u003c/em\u003e queens and workers using RNA-seq.\u0026nbsp;A total of 2,052 genes were identified as differentially expressed (FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with 909 genes upregulated in queens and 1,143 in workers (Supplementary Data 1). A heatmap of the DEGs showed consistent expression patterns across biological replicates (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Principal Component Analysis (PCA) revealed a clear separation between the two castes, indicating distinct transcriptomic profiles in ovarian tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eGene Ontology (GO) and KEGG pathway enrichment analyses were performed to gain insight into the biological functions of the differentially expressed genes (Supplementary Data 3). GO enrichment analysis revealed that queen-upregulated genes were associated with biological processes such as proteolysis, peptide catabolic process, isoprenoid biosynthetic process, mevalonate pathway, cholesterol biosynthetic process, and cell division, among others (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). KEGG pathway analyses, in turn, showed enrichment in metabolic pathways, peroxisome related function, terpenoid backbone biosynthesis, glutathione metabolism, motor proteins, and biosynthesis of cofactors, among others (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eIn contrast, queen-downregulated genes were enriched in GO terms related to carbohydrate metabolism, lipid metabolism, fatty acid biosynthesis, signal transduction, transcriptional regulation, among others (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). KEGG analysis revealed enrichment in metabolic pathways, as well as in neuroactive ligand-receptor interaction, phagosome related function, biosynthesis of cofactors, lysosome related function, oxidative phosphorylation, and other processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of queen removal on gene expression in\u003c/b\u003e \u003cb\u003ePogonomyrmex barbatus\u003c/b\u003e \u003cb\u003eworker ovaries\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe also performed RNA-seq on ovarian tissue from \u003cem\u003eP. barbatus\u003c/em\u003e queenright and queenless workers. A total of 26 genes were identified as differentially expressed (FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with just one gene upregulated (Supplementary Data 2). Principal component analysis (PCA) and a heatmap of the differentially expressed genes are shown (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, b). Most of the downregulated genes are associated with lipid and fatty acid metabolism. In contrast, the single upregulated gene in queenless workers corresponded to a microtubule-associated protein-like gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea), which has been implicated in egg development in echinoderms and fertility in mice\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn order to characterize active and inactive ovaries in ants, we analyzed the structure and morphology of the ovaries of \u003cem\u003ePogonomyrmex barbatus\u003c/em\u003e. Our morphological and structural analysis revealed significant differences across castes and worker ages, including ovariole number, oocyte development, tracheal supply, and the presence of yolk granules. These traits reflect caste-specific reproductive specialization and age-related functional decline in workers.\u003c/p\u003e \u003cp\u003eQueens of \u003cem\u003eP. barbatus\u003c/em\u003e exhibit a greater number of ovarioles compared to workers, and their oocytes are significantly larger, which enables them to produce multiple eggs simultaneously, a trait that could be essential for colony survival as has been documented for other ant species\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Although each ovariole in queens contains fewer follicles, these follicles and their developing oocytes are larger and more advanced in maturation, suggesting a reproductive strategy that prioritizes the production of fewer but higher quality oocytes. This is consistent with their role as the primary reproductive individuals in the colony. The abundant presence of yolk granules, indicative of active vitellogenesis, and the well-defined structure of queen oocytes highlight their specialization for reproduction.\u003c/p\u003e \u003cp\u003eIn contrast, workers have fewer ovarioles and smaller oocytes, indicating lower reproductive capacity. Interestingly, young (callow) workers possess a higher number of follicles per ovariole than older workers despite having longer ovarioles. How the number of follicles per ovariole relates to reproductive capacity within the worker caste is unclear, but it may suggest that they retain some reproductive potential early in life that diminishes with age\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Similar age-related declines in egg number and quality have been described in other social insects such as bees and wasps\u003csup\u003e\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. These changes likely reflect a transition in colony roles due to age polyethism, where older workers take on tasks such as foraging and defense activities that do not require reproductive capacity\u003csup\u003e\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Such a shift has also been observed in \u003cem\u003ePogonomyrmex californicus\u003c/em\u003e, where nurses involved in brood care have more oocytes than foragers, even though brood care does not necessarily require oviposition\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe ultrastructural differences observed between castes and age groups not only reflect functional states of the ovary but also reveal fundamental aspects of reproductive physiology in social insects. The high density of mitochondria and the extensive development of rough endoplasmic reticulum (RER) in follicular cells surrounding queen oocytes suggest high metabolic activity, characterized by abundant mitochondria, which is consistent with the biosynthetic demands of sustained reproduction. In contrast, follicular cells in mature workers (older individuals) exhibit signs of cellular damage, such as disrupted mitochondrial integrity and nuclear membrane abnormalities, which may reflect an age-related decline in cellular function. However, young (callow) workers do not exhibit such damage, potentially reflecting the evolutionary trade-off between reproduction and longevity typical of social insects\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAn important morphological distinction in the ovaries between castes was the separation of follicular cells, creating intercellular spaces, likely facilitating the passage of vitellogenin into the oocyte via patency \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. This structural adaptation, essential for the accumulation of yolk precursors and nutrient reserves during vitellogenesis, was absent in worker ovaries, suggesting a structural suppression of vitellogenesis in this caste. Supporting this pattern, notable differences were also observed in the organization of the microvillar zone. In queens, the microvilli formed a dense, continuous interface at the oocyte surface, supporting enhanced exchange of nutrients and signaling molecules. In contrast, the microvilli in workers were fragmented and discontinuous, reflecting a reduced capacity for uptake at the oocyte interface. Together, the presence of intercellular spaces between follicular cells and the structure of the microvillar region support the notion that nutrient transport and vitellogenin uptake are regulated according to reproductive status and restrict the reproductive potential in workers.\u003c/p\u003e \u003cp\u003eTo better understand the molecular basis underlying reproductive divergence between castes, we performed a transcriptomic analysis in queen and worker ovaries. Genes upregulated in queens were associated with processes critical for oogenesis and sustained reproductive activity, including isoprenoid and terpenoid biosynthesis, peptide catabolism, proteolysis, cell division, and glutathione metabolism. These enriched functions reflect key biosynthetic and antioxidant demands of active ovaries and are consistent with the cellular observations in queen follicular cells indicative of high metabolic activity. In contrast, genes downregulated in queen ovaries, or up in workers, are linked mainly to nutrient transport, neurophysiology, and carbohydrate metabolism. The reduced structural integrity of follicular cells in workers, along with the absence of transcriptional signatures associated with antioxidant pathways and cellular renewal as in queens, may reflect the limited evolutionary investment in reproductive maintenance in this caste.\u003c/p\u003e \u003cp\u003eNotably, major royal jelly protein 1 (\u003cem\u003emrjp1\u003c/em\u003e) and a related \u003cem\u003emrjp1-like\u003c/em\u003e gene also showed high expression in queens. These genes, originally characterized in eusocial bees\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, have been linked to caste differentiation and, more recently, to antioxidant and longevity-promoting functions\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Although only one complete copy of \u003cem\u003emrjp\u003c/em\u003e has been confirmed in \u003cem\u003eP. barbatus\u003c/em\u003e, its elevated expression suggests it may play an important but yet undescribed role in ant reproduction or queen physiology. Interestingly, in bumblebees there is only one \u003cem\u003emrjp\u003c/em\u003e gene, which has an analogous role in reproduction and caste differentiation in bees\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, suggesting that a single copy may be enough to fulfill these functions in ant queens of \u003cem\u003eP. barbatus\u003c/em\u003e. This is further supported by evidence that royal jelly and related proteins extend lifespan in model organisms such as \u003cem\u003eC. elegans\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn addition, genes involved in the mevalonate pathway, which is involved in juvenile hormone (JH) biosynthesis, a key regulator of vitellogenesis\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e, were also upregulated in queens\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Likewise, the \u003cem\u003evitellogenin-2-like\u003c/em\u003e gene was upregulated, which may explain the high levels of yolk granules found in the mature ovaries of queens. Interestingly, some components of this juvenile hormone pathway were upregulated in queens, while others in the same pathway were downregulated, suggesting a complex, possibly caste-specific modulation of hormonal signals. Similarly, within the insulin/IGF signaling (IIS) pathway many \u003cem\u003einsulin-degrading enzyme-like\u003c/em\u003e genes, as well as the \u003cem\u003einsulin-like growth factor II-B\u003c/em\u003e (\u003cem\u003eIGF-2\u003c/em\u003e) gene, were overexpressed in queens, while a gene encoding an insulin-like receptor was downregulated, potentially reflecting alternative signaling mechanisms that decouple reproduction from aging, as observed in other social insects\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eConsistent with the reproduction specialized expression profile observed in \u003cem\u003eP. barbatus\u003c/em\u003e queens, \u003cem\u003eAurora kinase B like\u003c/em\u003e (\u003cem\u003eAURKB\u003c/em\u003e) and \u003cem\u003eAurora kinase C-like\u003c/em\u003e (\u003cem\u003eAURKC\u003c/em\u003e) genes were also found to be upregulated. Aurora kinases are well established regulators of fecundity and oocyte development in mammals and have been demonstrated to regulate meiotic spindle assembly in oocyte maturation in the tunicate \u003cem\u003eOikopleura dioica\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eAURKB\u003c/em\u003e and \u003cem\u003eAURKC\u003c/em\u003e play essential roles in key cell cycle processes, such as regulating chromosome alignment and accurate meiotic division, which are critical for the production of functional oocytes\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. This molecular signature is consistent with the need of ant queens to sustain continuous oogenesis throughout their reproductive lifespan. Thus, the upregulation of these genes in queens likely reflects their essential role in supporting efficient oocyte maturation in reproductively active and highly fecund individuals.\u003c/p\u003e \u003cp\u003eKEGG pathways associated with glutathione metabolism and peroxisome activity also point to reinforced antioxidant defenses, likely supporting sustained oogenesis while mitigating oxidative stress. Notably, the gene encoding superoxide dismutase 1 (\u003cem\u003eSOD1\u003c/em\u003e) was downregulated in queens compared to workers. While \u003cem\u003eSOD1\u003c/em\u003e overexpression has been linked to increased lifespan in model organisms like \u003cem\u003eDrosophila\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e, studies in long-lived ant queens, such as \u003cem\u003eLasius niger\u003c/em\u003e, suggest that extreme longevity can occur despite lower \u003cem\u003eSOD1\u003c/em\u003e levels\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. This pattern, also observed in \u003cem\u003eP. barbatus\u003c/em\u003e, supports the idea that reproductive castes may rely on non-canonical antioxidant mechanisms, possibly involving reduced mitochondrial reactive oxygen species (ROS) production rather than elevated expression of detoxifying enzymes. Such alternative strategies could represent a key adaptation for sustaining long-term reproductive function without the need for constitutively high antioxidant activity.\u003c/p\u003e \u003cp\u003eWe also observed that queen removal did not lead to ovary activation in \u003cem\u003eP. barbatus\u003c/em\u003e workers. In many ant species, the absence of the queen triggers egg-laying by workers, either of viable or trophic eggs, due to the loss of inhibitory chemical signals such as pheromones or cuticular hydrocarbons (CHCs) emitted by the queen\u003csup\u003e\u003cspan additionalcitationids=\"CR63 CR64\" citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. These cues typically maintain reproductive suppression among workers, but once removed, some individuals can activate their ovaries and initiate oogenesis. However, in \u003cem\u003eP. barbatus\u003c/em\u003e, morphological evidence indicated the opposite trend. Workers from queenless colonies displayed clear signs of ovarian regression, including shorter ovarioles, smaller oocytes, and fewer developing oocytes. This contrasts with what is typically observed in bees and many ants, where the absence of queen pheromones lifts reproductive inhibition and leads to ovary development\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e,\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. These findings suggest that in \u003cem\u003eP. barbatus\u003c/em\u003e, reproductive suppression is not solely dependent on queen-derived chemical cues.\u003c/p\u003e \u003cp\u003eAt the molecular level, we found only one upregulated gene in 5-week queenless compared to queenright workers; \u003cem\u003eechinoderm microtubule-associated protein-like\u003c/em\u003e (\u003cem\u003eEML\u003c/em\u003e). Orthologs of this gene have been implicated in the control of oocyte meiotic progression in mammals and in ribosome-microtubule attachment in sea urchin eggs\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. However, its specific role in ant ovaries remains unknown. Notably, this gene did not show differential expression between queens and queenright workers, suggesting its upregulation may not be directly linked to reproductive activation.\u003c/p\u003e \u003cp\u003eIn contrast, 25 genes were downregulated in queenless workers, 20 of which were also downregulated in queens. Among these were genes involved in CHC biosynthesis, raising the possibility that queenless workers may experience a partial chemical profile shift, perhaps mimicking queen-like traits, despite remaining non-reproductive. Further supporting the morphological and molecular evidence, five weeks of behavioral monitoring revealed no oviposition or presence of trophic or viable eggs in queenless colonies. Still, workers retain ovarian tissue, which may represent an evolutionary safeguard that allows reproductive flexibility under extreme ecological conditions, as seen in other ant species\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan additionalcitationids=\"CR69\" citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe persistent suppression of ovarian development in workers likely reflects a colony-level strategy that favors non-reproductive roles such as brood care and foraging. This is particularly relevant in \u003cem\u003eP. barbatus\u003c/em\u003e, where workers are not known to lay eggs, even in the absence of the queen. Interestingly, this contrasts with what is seen in the closely related species \u003cem\u003ePogonomyrmex rugosus\u003c/em\u003e, where workers are known to produce trophic eggs when the queen is absent\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. These interspecific differences underscore the diversity of reproductive regulation strategies among closely related ants. While no egg-laying was observed during our five-week observation period, we cannot exclude the possibility that longer timeframes or specific environmental cues might eventually allow ovary activation in \u003cem\u003eP. barbatu\u003c/em\u003es workers.\u003c/p\u003e \u003cp\u003eThis work represents the first detailed morphological, structural, and molecular characterization of ovaries exhibiting extreme differences in reproductive activity and longevity within a fixed caste species. By examining reproductive constraints in a system where genetically similar individuals show vast disparities in lifespan and reproductive output, this study provides valuable insights into the mechanisms underlying reproductive aging. These findings contribute to a deeper understanding of how reproductive division of labor shapes aging trajectories in social insects and offer a solid foundation for future research into the regulation of longevity and fertility within complex societies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eInvestigation and data analysis: M.F.V-M., D.G-S., B.O-D., E.S-S. and I.F-P. Writing: M.F.V-M., D.G-S., B.O-D. and I.F-P. Methodology: M.F.V-M., D.G-S., B.O-D., E.S-S, P.M-G., R.R-R., R.M.V-V., A.G-M. and I.F-P. Resources: A.G-M., R.M.V-V. and I.F-P. Funding acquisition and supervision: I.F-P. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank C\u0026eacute;sar Maximiliano V\u0026aacute;zquez Franco for taking the Pogonomyrmex barbatus ant photographs shown in Figure 1. We are also grateful to Miguel Tapia Rodr\u0026iacute;guez from the Microscopy Unit at IIBO for technical assistance. We thank Andrea Luna M\u0026eacute;ndez and Mayra Franco Sari\u0026ntilde;ana for their dedicated care of the ant colonies. We are also thankful to Ian A. E. Butler for his valuable suggestions on the manuscript and assistance with editing. We thank Nat\u0026aacute;lia Faraj Murad for advice on transcriptomic analysis. Finally, we are grateful to everyone at the AntLab UNAM for their helpful suggestions and support. The electron microscopy work was supported by Program E022 from the Instituto Nacional de Pediatr\u0026iacute;a in Mexico City. This work was supported by the Global Consortium for Reproductive Longevity and Equality at the Buck Institute for Research on Aging, made possible by the Bia-Echo Foundation (GCRLE-0620 Junior Scholar Award) to IF-P. This work was also supported by UNAM-PAPIIT IN21072 funding to IF-P, and Secretar\u0026iacute;a de Ciencia, Humanidades, Tecnolog\u0026iacute;a e Innovaci\u0026oacute;n (SECIHTI) scholarship CVU 1249398 to MFV-M and is part of the requirements for obtaining the doctoral degree in experimental biology at the Posgrado en Ciencias Biol\u0026oacute;gicas UNAM of MFV-M.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eReads for all sequences generated in this study were deposited into the National Center for Biotechnology Sequence Read Archive (NCBI - SRA) BioProject ID: PRJNA1279095 and are available at the following URL: https://dataview.ncbi.nlm.nih.gov/object/PRJNA1279095?reviewer=upe3k3keq6j9n34p7o6g8hju6r\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGordon, D. M. Dynamics of task switching in harvester ants. \u003cem\u003eAnim. 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Queen dominance and worker policing control reproduction in a threatened ant. \u003cem\u003eBMC Ecol. \u003c/em\u003e\u003cstrong\u003e11\u003c/strong\u003e, 21 (2011).\u003c/li\u003e\n\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":"npj-aging","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Aging](https://www.nature.com/npjamd/)","snPcode":"41514","submissionUrl":"https://submission.springernature.com/new-submission/41514/3","title":"npj Aging","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"ovary, ants, morphology, division of labor, reproduction","lastPublishedDoi":"10.21203/rs.3.rs-6933990/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6933990/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eReproductive division of labor defines eusocial insects like ants, where queens reproduce and workers remain mostly sterile. Yet, some workers retain rudimentary ovaries, raising questions about their reproductive potential. We examined morphological and transcriptomic differences in ovaries of \u003cem\u003ePogonomyrmex barbatus\u003c/em\u003e queens and workers of varying ages and social contexts. Queens had large, yolk-rich oocytes, while worker ovaries showed signs of degeneration. Callow workers had more developed ovaries than mature ones, suggesting reproductive decline with age. Queenless workers showed more ovarian regression compared to queenright ones. Transcriptomic analyses revealed over 2,000 differentially expressed genes between castes, including those involved in metabolism, hormonal signaling, and epigenetic regulation. Notably, queenless workers upregulated a fertility-linked gene and downregulated lipid metabolism genes. Our results show that both age and social environment constrain worker reproductive potential, highlighting the queen\u0026rsquo;s role in maintaining worker sterility and offering insights into reproductive senescence in eusocial systems.\u003c/p\u003e","manuscriptTitle":"Age, Caste, and Social Context Shape Ovarian Morphology and Transcriptomic Profiles in Red Harvester Ants (Pogonomyrmex barbatus)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-27 11:50:02","doi":"10.21203/rs.3.rs-6933990/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-19T20:54:18+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-17T10:07:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-04T18:52:20+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-04T09:54:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"114672070315011527400140234185982781587","date":"2025-06-26T21:12:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"160590016692768632985804377255185567751","date":"2025-06-25T11:55:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"23331900500762165127127826190958990316","date":"2025-06-24T23:59:07+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-24T20:20:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-24T13:55:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-23T09:12:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Aging","date":"2025-06-19T23:30:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-aging","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Aging](https://www.nature.com/npjamd/)","snPcode":"41514","submissionUrl":"https://submission.springernature.com/new-submission/41514/3","title":"npj Aging","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ff247062-5840-47ce-b395-dd4e0d69763b","owner":[],"postedDate":"June 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":50662171,"name":"Biological sciences/Cell biology/Senescence"},{"id":50662172,"name":"Biological sciences/Physiology/Ageing"}],"tags":[],"updatedAt":"2025-11-17T16:00:41+00:00","versionOfRecord":{"articleIdentity":"rs-6933990","link":"https://doi.org/10.1038/s41514-025-00278-1","journal":{"identity":"npj-aging","isVorOnly":false,"title":"npj Aging"},"publishedOn":"2025-11-13 15:57:27","publishedOnDateReadable":"November 13th, 2025"},"versionCreatedAt":"2025-06-27 11:50:02","video":"","vorDoi":"10.1038/s41514-025-00278-1","vorDoiUrl":"https://doi.org/10.1038/s41514-025-00278-1","workflowStages":[]},"version":"v1","identity":"rs-6933990","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6933990","identity":"rs-6933990","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}