Males induce premature demise of the opposite sex by multifaceted strategies

Sexual interactions can influence organismal health independently of reproduction across nematodes, flies, and mammals. In Caenorhabditis, males shorten the lifespan of females or hermaphrodites via sperm, seminal fluid, pheromones, and other secreted compounds. Despite identification of some mediators (for example, transcription factors such as DAF-16/FOXO and HLH-30/TFEB, chromatin regulators like UTX-1, insulin-like peptides, and roles for self-sperm), a systematic understanding of the pathways that drive male-induced lifespan shortening and their overlap with canonical longevity mechanisms has been lacking. The study aims to define transcriptional responses to sexual interactions, identify functionally causal genes, determine contributions of distinct male components (sperm, seminal fluid, pheromones), and test whether combined interventions can protect against male-induced demise, thereby clarifying how sexual environment modulates longevity.

Prior work shows males can reduce lifespan and alter physiology of the opposite sex across species: in C. elegans, mating and male pheromones shorten lifespan; in Drosophila, mating and sex peptides incur costs to females; in mice, male presence increases female body mass and stress responses independent of fertilization. In C. elegans, male-induced demise involves male sperm, seminal fluid, and secreted pheromones, with hermaphrodite-side mediators including insulin signaling (INS-11, INS-7), transcription factors (DAF-16/FOXO, HLH-30/TFEB), chromatin regulators (UTX-1/KDM6A), and protective effects of self-sperm. However, comprehensive transcriptomic and functional mapping of male-induced pathways, their tissue specificity, and their interactions with canonical longevity mutants remained to be established.

• Organisms and strains: C. elegans wild type (N2) and multiple mutants/ transgenics including glp-1(e2144/e2141), daf-2(e1370), nuo-6(qm200), isp-1(qm150), jmjd-1.2 overexpression, delm-1(ok1226), delm-2(ok1822), acd-1(bz90), tissue-specific RNAi strains, various fluorescent reporters (hsp-6p::GFP; fat-5p::fat-5::GFP; fat-7p::fat-7::GFP). Maintenance on NGM with OP50-1 at 20°C; temperature-sensitive strains at 15°C (maintenance) and 25°C (assays).
• RNA sequencing (RNA-seq): Sterile glp-1(e2144) hermaphrodites were exposed to males either briefly (1 day) or longer (5 consecutive days), starting at adulthood, and sampled at day 3 and day 7 of life. Males were included at defined ratios; exposure initiated at adult day 1–2 for long exposure and day 2 or 6 for brief exposure. 75 hermaphrodites per condition per replicate (3–8 biological replicates). Male-enriched transcripts were quantified from males sampled in parallel to filter male-derived reads. Library prep used SMART-Seq v4 and Nextera XT; paired-end 75 bp sequencing on Illumina NextSeq. Analysis: STAR alignment to WBcel235, DESeq2 for differential expression, variance-stabilizing transformation for PCA, filtering of male-enriched genes, gene set enrichment via WormCat.
• Targeted RNAi screen and lifespan assays: In wild-type fertile hermaphrodites, RNAi knockdown of male-induced genes identified from RNA-seq (and controls) was performed via HT115 feeding. Lifespans were measured in the absence and presence of males (various exposure paradigms: continuous, 1-day exposure, and male-conditioned plates (MCP)). Kaplan–Meier and Cox proportional hazard models were used to assess lifespan effects and gene x male presence interactions.
• Dissecting male components: Microarrays were used to distinguish gene sets induced by (i) male sperm vs. seminal fluid using mating with WT males vs. fer-6(hc6) sperm-less males for 24 h (2:1 male:hermaphrodite), and (ii) male pheromones via exposure to MCP (plates conditioned by fog-2(q71) males). SAM identified differentially expressed genes; WormCat provided enrichment analysis.
• Combinatorial interventions: Double RNAi (delm-2 + acbp-3) compared to single knockdowns to test additivity against male-induced demise.
• Tissue specificity: Tissue-specific RNAi strains (neuron-specific TU3401; intestine-specific VP303 and IG1839) used to knock down delm-2 (and paralogs) and acbp-3 in specific tissues; single-cell RNA-seq (L2 data reanalysis) to map expression of delm-2/acd-1 and acbp-3 across cell types (UMAP with Seurat/Harmony).
• Lipid metabolism assays: Oil Red O staining quantified neutral lipids over time in hermaphrodites under control vs delm-2 RNAi, with and without males; fat-5 and fat-7 translational reporters measured MUFA-related enzyme expression.
• Additional assays: DAF-16::GFP nuclear localization after heat shock with vs without males; mitochondrial UPR reporter hsp-6p::GFP in nuo-6 and isp-1 backgrounds with vs without males. CRISPR-Cas9 used to create acd-1 deletion in delm-2 background and triple mutants with delm-1; mating efficiency assays ensured RNAi did not impair mating rates.
• Statistics: Log-rank tests for survival curves; Cox proportional hazards modeling for interaction (gene x male presence), with protective (HR<1, P<0.05) and detrimental (HR>1, P<0.05) classifications; Mann–Whitney tests and two-way ANOVA for imaging quantifications; multiple hypothesis corrections for transcriptomic analyses.

• Males robustly shorten lifespan of hermaphrodites (Kaplan–Meier; P < 0.0001 for exposure paradigms). PCA showed separation by age and male exposure; prolonged exposure induced more and larger-magnitude transcriptional changes than brief exposure.
• Differential expression (after removing male-enriched genes) increased with exposure duration: approximately 16 (day 3, +males 1 day), ~90 (day 7, +males 1 day), and 1,563 (day 7, +males 5 days) significant genes (P ≤ 0.05). Commonly induced genes across conditions included sri-40, ins-11, acbp-3, delm-2, acd-1, nhx-2, cyp-25A3, col-125, sre-28, lpr-6. Upregulated categories were enriched for lipid metabolism/transport, collagens (ECM), and stress response (detoxification); some lipid β-oxidation genes were downregulated.
• RNAi screen identified: male-specific protective knockdowns (significant protective interaction by Cox model) including delm-2 (DEG/ENaC ion channel), col-43 (collagen), acbp-3 (acyl-CoA binding protein), acox-1.3 (acyl-CoA oxidase). These extended lifespan in the presence of males, often without extending lifespan in hermaphrodite-only conditions.
• General (male-independent) protective knockdowns (extended lifespan with and without males; no significant interaction): serpentine receptors sri-40 and sre-28, Hedgehog-like wrt-10, and other targets extended lifespan in +males conditions.
• Classical longevity manipulations varied with sexual context: utx-1 RNAi and nuo-6 mutation were generally protective. However, several long-lived mutants showed detrimental interactions with male presence (more sensitive than WT): daf-2(e1370), glp-1(e2144), isp-1(qm150), and jmjd-1.2 overexpression (significant detrimental interaction by Cox model). Males blunted mitochondrial UPR activation in nuo-6 mutants (hsp-6p::GFP; P < 0.0001) and reduced heat-shock-induced DAF-16 nuclear localization (P = 0.011), suggesting suppression of pro-longevity effectors.
• Distinct male components induced distinct gene sets: • Sperm-induced genes (WT mating but not fer-6 sperm-less mating) were enriched for metabolism and included acbp-3 and ins-11. RNAi of these sperm-responsive genes partially protected lifespan. • Seminal fluid-induced genes (induced by both WT and fer-6 males) included sodium channel genes delm-2 and paralog acd-1; acd-1 RNAi specifically protected against male-induced demise. While single delm-1/delm-2/acd-1 mutants did not protect, the acd-1; delm-2; delm-1 triple mutant specifically protected (Cox HR = 0.3643, P = 1.36×10^-6). • Pheromone/secreted compounds (MCP)-induced genes included solute carrier hmit-1.1; hmit-1.1 RNAi extended lifespan on MCP with a significant protective interaction (Cox HR = 0.629, P = 0.034; Mantel–Cox P = 0.0003 on MCP).
• Combined interventions were more effective: Double RNAi of delm-2 (and paralogs) plus acbp-3 provided additive protection, bringing lifespan in the presence of males close to hermaphrodite-only lifespan (Mantel–Cox P = 0.03 vs control her-only; Cox protective interaction P = 1.43×10^-5). Knockdown of the upstream transcription factor ceh-60 (PBX3/4 ortholog) also strongly and specifically protected (Cox P = 4.39×10^-9).
• Tissue specificity: Gene sets induced by sperm, seminal fluid, and pheromones were enriched in nervous and digestive systems; sperm/seminal-fluid responses also enriched in reproductive/epithelial tissues. Tissue-specific RNAi showed delm-2 knockdown in either nervous system or intestine was sufficient to protect; acbp-3 knockdown protected only when targeted to intestine.
• Mechanism via lipid metabolism: delm-2 knockdown prevented male-induced neutral lipid depletion (Oil Red O), increased neutral lipid levels in both sexes of exposure and in tissue-specific knockdowns, and upregulated lipid desaturases FAT-5 and FAT-7 (P ≤ 0.0022), suggesting protection through MUFA synthesis and fat preservation.

This study identifies distinct, functionally important gene modules in hermaphrodites that mediate male-induced demise and demonstrates that sexual environment drastically alters the outcomes of longevity interventions. Specific mediators (for example, ACBP-3 and DELM-2/paralogs) mainly act to counter male-induced damage, while other targets (for example, SRI-40) confer general longevity. In contrast, classical long-lived mutants (for example, daf-2, isp-1, glp-1, jmjd-1.2 OE) are particularly vulnerable to the presence of males, likely because males suppress downstream pro-longevity pathways, including DAF-16/FOXO nuclear localization and mitochondrial UPR. Dissecting male signals revealed that sperm, seminal fluid, and pheromones each induce discrete gene programs, enabling rational combination therapies. Combining sperm-responsive (acbp-3) and seminal-fluid-responsive (delm-2/paralogs) interventions yields near-complete protection from male-induced lifespan shortening; upstream regulation by CEH-60 provides an alternative convergent strategy. Tissue-level analyses indicate a multi-tissue response with key roles for the nervous system and intestine. Mechanistically, DELM-2 influences lipid metabolism, preserving neutral lipids and enhancing MUFA-related enzyme expression, linking ion channel activity to metabolic resilience under sexual stress. These findings underscore sexual interactions as a potent, underappreciated modulator of aging biology.

The work systematically maps the transcriptional and functional landscape of male-induced demise in C. elegans, uncovers specific protective targets (delm-2, acbp-3, acd-1, hmit-1.1), identifies general longevity mediators (sri-40, utx-1), and reveals that many canonical longevity mutants are susceptible in mixed-sex environments. It demonstrates that distinct male components (sperm, seminal fluid, pheromones) trigger different gene programs and that combining interventions across these pathways (for example, delm-2 + acbp-3 knockdown or targeting CEH-60) can largely neutralize male-induced lifespan shortening. DELM-2 acts in nervous and intestinal tissues to modulate lipid metabolism, suggesting cross-tissue signaling in sexual interaction-induced aging. Future directions include defining the specific seminal fluid factors that induce DELM-2–dependent responses, identifying secreted molecules mediating tissue-to-tissue communication, systematically testing additional combinatorial interventions, and exploring conservation of these mechanisms in other species, including mammals.

• RNAi specificity and paralog overlap: The delm-2 RNAi construct has high sequence similarity to delm-1 and acd-1; thus, some effects likely reflect combined knockdown. Differences between RNAi knockdown and null mutations (for example, triple vs single mutants) complicate interpretation.
• Context of transcriptomic assays: ChIP-Atlas and motif analyses derive from whole-worm data across developmental stages/conditions, which may not perfectly reflect adult tissues under male-induced demise. Microarray assays to parse male components used different genotypes (for example, glp-1 sterile animals) and conditions; pheromone effects required separate assays in wild-type.
• Experimental constraints: Not all lifespan comparisons could be blinded (presence vs absence of males); sample sizes followed field standards but were not predetermined by power analysis.
• Generalizability: Findings are in C. elegans laboratory strains; extension to other species, natural environments, and varying male densities requires further validation. Male abundance and timing in nature may modulate selection on these pathways.