Sexual interactions significantly impact organismal health, independent of reproduction, across various species, including nematodes, flies, and mammals. In mice, for instance, males induce weight gain and reduce lifespan in females, even without fertilization. However, this phenomenon is poorly understood due to the prevalence of single-sex experimental environments. *C. elegans*, with its short lifespan, presents an ideal model for systematically investigating the effects of sexual interactions on longevity. Previous research has demonstrated that male-hermaphrodite interactions shorten the hermaphrodite lifespan, involving contributions from both sexes. Males utilize sperm, seminal fluid, pheromones, and secreted compounds to induce premature death, especially in the presence of numerous males. Several molecular pathways have been implicated in this process, including those involving transcription factors (FOXO/DAF-16, TFEB/HLH-30), chromatin regulators (KDM6A/UTX-1), insulin ligands (INS-11, INS-7), and even self-sperm. However, a comprehensive study exploring the underlying pathways and their interplay with established longevity pathways is lacking. This study aims to address this gap by systematically identifying and characterizing the genes and pathways involved in male-induced demise in *C. elegans*, and understanding how they interact with known longevity pathways.

The literature review section summarizes existing knowledge on the impact of sexual interactions on lifespan across various species. Studies in nematodes, *Drosophila*, and mice demonstrate that male presence can negatively affect female healthspan. In *C. elegans*, previous work pinpointed specific male components (sperm, seminal fluid, pheromones) as contributors to hermaphrodite demise. Some molecular mechanisms, such as the roles of specific transcription factors and signaling pathways, were identified but a holistic understanding of the regulatory network was missing. This research builds on this foundation, using a systematic approach to broaden our understanding of the complex interplay between sex, genes, and aging.

This study employed RNA sequencing (RNA-seq) and RNA interference (RNAi) to systematically investigate the effects of sexual interactions on *C. elegans* lifespan. RNA-seq was conducted on young (day 3) and middle-aged (day 7) sterile *glp-1* hermaphrodites exposed to males for brief (1 day) and long (5 days) periods. Exposure commenced at adulthood to eliminate developmental effects. Transcriptomic changes were analyzed using principal component analysis (PCA) and differential gene expression analysis. The study identified genes upregulated in hermaphrodites in response to male presence, focusing on those affected by both short and long exposures. A targeted RNAi screen was performed using wild-type hermaphrodites to assess the functional role of the male-induced genes. Hermaphrodite lifespan was measured in the presence and absence of males, using Cox proportional hazard models to determine the interaction between gene knockdown and male presence. The study classified genes into categories based on their interaction with males: specifically protective, generally protective, no effect, and detrimental. Classical long-lived mutants were also assessed to investigate the interaction between longevity manipulations and male presence. Additional experiments analyzed the responses of hermaphrodites to specific male components (sperm, seminal fluid, pheromones) using microarrays. Lifespan assays assessed the effect of combined loss-of-function of multiple genes. Tissue-specific RNAi was performed to determine the location of gene action. Oil Red O (ORO) staining was used to measure neutral lipids to examine the role of *delm-2* in lipid metabolism. Single-cell RNA sequencing data was reanalyzed to determine the cellular expression of key genes. CRISPR-Cas9 was used to generate a triple mutant to better understand *delm-2* and its paralogs.

The study revealed that male presence induces significant transcriptional changes in *C. elegans* hermaphrodites, primarily affecting lipid metabolism, collagen production, and stress response. A targeted RNAi screen identified genes whose knockdown specifically protected against male-induced death (*delm-2*, *acbp-3*, *acd-1*). Other genes (*sri-40*, *wrt-10*, *sre-28*) exhibited broader lifespan extension regardless of male presence. Classical longevity mutants, unexpectedly, exhibited increased susceptibility to male-induced demise, suggesting males may repress downstream components of longevity pathways, like FOXO/DAF-16. Male-induced genes responded differently to sperm, seminal fluid, and pheromones, indicating distinct mechanisms of action. Combinatorial manipulation of these genes, particularly *delm-2* and *acbp-3*, offered enhanced protection against male-induced death. Knockdown of *ceh-60*, a transcription factor, also provided strong protection. *delm-2* and *acbp-3* were found to act in multiple tissues (nervous system and intestine) to mediate male-induced demise. *delm-2* regulates lipid metabolism, preventing fat loss induced by males. This suggests that inter-tissue communication is vital for this phenomenon.

This research significantly advances our understanding of male-induced demise in *C. elegans*. It identifies specific genes and pathways involved in this process, demonstrating that males employ multiple strategies to shorten hermaphrodite lifespan. The finding that classical longevity mutants exhibit increased susceptibility to male-induced death highlights the importance of considering the sexual environment when evaluating longevity interventions. The identification of combinatorial strategies to counteract male-induced death opens new avenues for research. The conserved nature of many identified genes suggests potential relevance to human aging and age-related diseases.

This study provides a comprehensive analysis of male-induced demise in *C. elegans*, identifying key genes and pathways involved in this process. It reveals the importance of considering the sexual environment in longevity studies and demonstrates the effectiveness of combinatorial strategies to counteract the negative impact of males on lifespan. The involvement of *delm-2* in lipid metabolism highlights a potential mechanism for the observed effects. Future research could focus on understanding the inter-tissue communication involved in male-induced demise and explore the translational potential of these findings to human health.

The study primarily focuses on *C. elegans*, a model organism. While many genes identified are conserved in mammals, the precise translation of findings to higher organisms requires further investigation. RNAi knockdown does not always represent complete gene knockout, so some effects may be due to incomplete gene silencing. Also, the study uses a laboratory setting and its relevance to natural populations requires further exploration.