Sex Differences in the Response to Genetic and Chemical Pro-Longevity Interventions in 

Caenorhabditis elegans 

by  

Rose Al-Saadi 

  

A dissertation accepted and approved in partial fulfillment of the 

requirements for the degree of 

Doctor of Philosophy 

in Biology 

  

Dissertation Committee:  

Cristopher Niell, Chair  

Patrick Phillips, Advisor  

Matthew Barber, Core Member  

Peter Ralph, Core Member  

Michael Wehr, Institutional Representative  

 
 

University of Oregon  
 

Summer 2025 
 
 
 
 
 



 

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© 2025 Rose Al-Saadi  
  

This work is openly licensed via CC BY 4.0. 

 
  

https://creativecommons.org/licenses/by/4.0/


 

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DISSERTATION ABSTRACT 
 
Rose Al-Saadi 
 
Doctor of Philosophy in Biology 
 
Title: Sex Differences in the Response to Genetic and Chemical Pro-Longevity Interventions in 

Caenorhabditis elegans 
 
 

Women often live longer than men yet suffer worse health outcomes in old age. This 

sexual dimorphism in lifespan and aging trajectories is robust and has been frequently observed 

and comprehensively documented. Sex differences extend beyond natural aging and are 

additionally seen in the response to pro-longevity interventions. Despite that, our understanding 

of the mechanisms underlying such differences remains limited. In this dissertation, I utilize the 

model organism Caenorhabditis elegans to explore sex differences in aging. I leverage a 

healthspan measure that is unique to male C. elegans: mating. Males display a complex mating 

behavior that strongly declines with age due to physiological deterioration, making it an ideal 

screening phenotype to investigate the efficacy of pro-longevity interventions. Using male 

reproductive success as a complex healthspan measure, I investigate the effects of DAF-2/ IGF-1 

insulin receptor degradation on male longevity and whether the effects of this intervention are 

sexually dimorphic. Additionally, I examine the effects of several pro-longevity chemical 

compounds on male lifespan and contrast them to previously published hermaphrodite data to 

determine whether their effects on longevity are sexually dimorphic or sex shared. Finally, I 

characterize the transcriptomic signatures of two chemical pro-longevity interventions, 

metformin and Thioflavin T, on aging C. elegans and determine whether the effects at the 

transcriptional level are sexually dimorphic or sex shared. This work provides a new 

experimental paradigm for the study of sex differences in aging using the model organism C. 

elegans and offers new mechanistic targets for future research. Understanding the mechanistic 

basis of sex differences in aging will aid in the development of interventions that can benefit 

individuals of any sex.  

This dissertation includes previously unpublished coauthored material. 

 
 

 



 

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ACKNOWLEDGMENTS 

I wish to express my deep and sincere gratitude to my advisor, Dr. Patrick Phillips. He has 

demonstrated to me that being a kind mentor and a rigorous scientist are not mutually exclusive. 

He has advocated for me even before I arrived at UO and has supported me every step of the 

way. He has always encouraged me to trust my scientific instincts and allowed me the freedom 

to explore my interests in his lab, which has been a truly invaluable experience. I would also like 

to thank my first academic advisor, Dr. Sarah Hall, who introduced me to C. elegans research 

and encouraged me to pursue a PhD and a career in science; without her guidance and 

mentorship, I would not be where I am today. I would like to also thank my current and previous 

committee members, Cris Niell, Matt Barber, Peter Ralph, Mike Wehr, and Nadia Singh for their 

support, mentorship, and insightful feedback on my projects throughout my PhD. 

I am deeply grateful to the Phillips lab members, both current and past, who have fostered a 

positive and supportive work environment that made being in the lab both intellectually 

stimulating and incredibly fun. I want to especially thank Zach Stevenson and Christine Sedore 

for being the best coffee run crew, and for being amazing friends inside and outside the lab. 

Their enthusiasm for science is both unparalleled and infectious and they both continue to inspire 

me to be a better scientist. I want to thank Kristin Robinson and Aubrey Mayer for being great 

office mates and for allowing me to vent about failed experiments and worms that refuse to die, 

Megan Moerdyk-Schauwecker for ensuring I always had friends to share a Thanksgiving meal 

with, Hannah Lewack for being the best undergraduate mentee I could have asked for, and Anna 

Coleman-Hulbert and Erik Johnson for adding quirky and fun vibes to the lab and making our 

Tea Times the best.  

My time as a PhD student has been enriched by a number of student groups including the 

Graduate Evolutionary Biology and Ecology Students (GrEBES), the Community for Minorities 

in STEM (CMiS), and Women in Graduate Science (WGS). These groups have offered me a 

sense of belonging, which was especially needed during the early days of the pandemic, and 

allowed me to integrate myself into a scientific community of my peers. I am also grateful for the 

financial support that I have received from all three groups that contributed to attending 

conferences and recognized my contributions in leadership roles. 



 

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I want to thank my funding sources who have made this work possible. This work was funded in 

part by the Genetics Training Program at the University of Oregon, which is funded by the 

National Institutes of Health (T32GM149387). Additional funding was provided by National 

Institutes of Health awards to Patrick C. Phillips (U01AG045829, U24AG056052, 

R01AG088629). 

I would like to thank all the friends who have made my time in Eugene a happy and exciting 

time. I want to thank my cohort mates LyAndra Lujan and Masha Korchagina for their friendship 

and support, Elizabeth Vargas, Jordan Munroe, Kona Shaw, Ethan Shaw, Emily Dennis, and 

Max Horrocks for teaching me how to work hard and play hard, Leanne Kelley and Alex 

Nichitean, my worm sisters, and Mayara Ribeiro and Niko Wagner, for their support from afar; 

reminiscing about Café Disco and Chipotle Wednesdays will always bring me joy. 

I want to thank my family for all the love and support that they have provided through the years. 

I am forever grateful for their courage to immigrate to the United States from Iraq and the 

sacrifices they made that have allowed me to pursue many opportunities I couldn’t have 

otherwise. 

I want to acknowledge the important contributions of my cats Sesame, Peppercorn, and Nimbus 

to this work through their moral support and unwavering love and purrs. 

I feel incredibly lucky to have shared my grad school journey with my best friend and partner, 

Zac Bush, who has enriched my life with wonderful people, delicious food, running, and great 

knowledge of meiotic recombination and obscure internet memes. Thank you for being my main 

support system over the last three years of my PhD, for sharing and amplifying my enthusiasm 

for science, for inspiring me to become a better and more compassionate person, and for all the 

love and joy you bring into my life. 

  



 

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DEDICATION 

 

This dissertation is dedicated to my mother Wajida. Her passion for knowledge and her drive to 

pursue education despite all obstacles has inspired me throughout my life and planted in me a love 

for science and education. 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 



 

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TABLE OF CONTENTS 

Chapter Page 
 
 
CHAPTER 1: INTRODUCTION ................................................................................ 12 

 Aging Theories and Hallmarks .............................................................................. 12 

 Sex Differences in Aging and Age-Associated Diseases ...................................... 13 

 C. elegans as an Aging Model ............................................................................... 15 

 Pro-longevity Interventions in C. elegans ............................................................. 16 

 Challenges and Benefits of C. elegans Males as an Aging Model ........................ 18 

 Dissertation Outline ............................................................................................... 19 

 Bridge to Chapter 2 ................................................................................................ 20 

CHAPTER 2: KNOCKING DOWN THE DAF-2 INSULIN-LIKE RECEPTOR  

IN MALE C. ELEGANS DRAMATICALLY INCREASES REPRODUCTIVE  

HEALTH AND LONGEVITY IN A SEX AND TISSUE-SPECIFIC MANNER ..... 21 

 Abstract .................................................................................................................. 21 

 Main ....................................................................................................................... 22 

 Discussion .............................................................................................................. 28 

 Methods.................................................................................................................. 31 

 Supplemental Figures ............................................................................................. 34 

 Supplemental Tables .............................................................................................. 37 

 Bridge to Chapter 3 ................................................................................................ 45 



 

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CHAPTER 3: PRO-LONGEVITY COMPOUNDS EXTEND  

CAENORHABDITIS ELEGANS MALE LIFESPAN AND REPRODUCTIVE  

HEALTHSPAN ........................................................................................................... 46 

 Abstract .................................................................................................................. 46 

 Introduction ............................................................................................................ 46 

 Materials and Methods ........................................................................................... 49 

 Results .................................................................................................................... 51 

 Discussion .............................................................................................................. 55 

 Supplemental Tables .............................................................................................. 61 

 Bridge to Chapter 4 ................................................................................................ 70 

CHAPTER 4: PRO-LONGEVITY INTERVENTIONS GENERATE  

SEXUALLY DIMORPHIC TRANSCRIPTOMIC SIGNATURES IN AGING 

CAENORHABDITIS ELEGANS .................................................................................. 71 

 Introduction ............................................................................................................ 71 

 Materials and Methods ........................................................................................... 73 

 Results .................................................................................................................... 75 

 Discussion .............................................................................................................. 87 

 Supplemental Figures ............................................................................................. 91 

 Supplemental Tables .............................................................................................. 93 

CHAPTER 5: CONCLUSION .................................................................................... 94 

REFERENCES CITED ................................................................................................ 96 



 

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LIST OF FIGURES 
 
Figure Page 
 
 
Fig. 2.1 Ubiquitous and tissue-specific DAF-2 degradation extends male lifespan .... 25 

Fig. 2.2 Ubiquitous DAF-2 degradation preserves late-life male reproductive  

success .......................................................................................................................... 27 

Fig. S2.1 Neuron- and Hypodermis-specific DAF-2 degradation does not extend  

male lifespan ................................................................................................................ 34 

Fig. S2.2 Tissue-specific DAF-2 degradation does not preserve late-life male  

reproductive success .................................................................................................... 35 

Fig. S2.3 Turning success and LOV efficiency are not improved upon DAF-2  

ubiquitous degradation ................................................................................................. 36 

Fig. 3.1 Pro-longevity compounds extend male lifespan ............................................. 53 

Fig. 3.2 Sex differences in the percent change in median lifespan for different  

compounds compared to the control ............................................................................ 54 

Fig. 3.3 Sulforaphane and metformin preserve late-life male reproductive success ... 56 

Fig. 4.1 FUdR interacts with pro-longevity compounds in a sex-dependent  

manner .......................................................................................................................... 77 

Fig. 4.2 Hermaphrodites and males have distinct transcriptomic profiles  

associated with aging ................................................................................................... 78 

Fig. 4.3 Metformin elicits sexually dimorphic transcriptional changes ....................... 80 

Fig. 4.4 ThioT elicits sexually dimorphic and sex-shared transcriptional changes ..... 82 

Fig. 4.5 Metformin and ThioT interact with age to modulate gene expression  

changes ......................................................................................................................... 84 



 

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Fig. 4.6 Sex interacts with compound treatments to modulate age-associated gene  

expression changes ....................................................................................................... 86 

Fig. S4.1 Principal component analysis of RNA-seq replicates .................................. 91 

Supplemental Fig. S4.2 Top genes whose expression change by compound  

treatment is sex biased ................................................................................................. 92 

 

 



 

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LIST OF TABLES 
 
Table Page 
 
 
Table S2.1 Lifespan assay summary statistics ............................................................. 37 

Table S2.2 Lifespan assay CPH model output ............................................................. 38 

Table S2.3 Mating assay summary statistics ............................................................... 39 

Table S2.4 Mating assay GLM model output .............................................................. 41 

Table S2.5 Behavior assay summary statistics ............................................................ 43 

Table S2.6 Behavior assay GLM output ...................................................................... 44 

Table S2.7 Strains used in this study ........................................................................... 45 

Table S3.1 Lifespan assay summary statistics ............................................................. 61 

Table S3.2 Lifespan assay CPH model output ............................................................. 62  

Table S3.3 Change in median lifespan by sex summary statistics .............................. 63 

Table S3.4 Median lifespan by sex CPH model output ............................................... 64  

Table S3.5 Mating assay summary statistics ............................................................... 66 

Table S3.6 Mating assay GLMM model output .......................................................... 68  

Table S4.1 Compound by FUdR by sex CPH model output ....................................... 93 

  



 

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CHAPTER1: INTRODUCTION 

Aging theories and hallmarks 

Aging is defined as the progressive deterioration of physiological and cellular functions over 

time. The study of aging as a biological process is complicated by its multifactorial nature, and 

by the variable contribution of these factors depending on the genetic background and the 

environment of an individual (Gems 2022). Several theories and mechanisms for aging have 

been proposed, with some rooted in evolutionary concepts while others focused on mechanistic 

causes. One of the most prominent evolutionary explanations of aging is George Williams’ 

antagonistic pleiotropy, which states that some genes have contrasting or “antagonistic” effects 

on fitness, beneficial in early life and detrimental in late life (Williams 1957). Given that 

selection is weaker following reproductive senescence, the detrimental effects of such alleles in 

late life are “shadowed”, leading to an overall positive selection on loci promoting beneficial 

“wild type” processes in early life and contribute to aging in late life (Williams 1957). 

Theories that have since emerged to explain antagonistic pleiotropy from a mechanistic 

standpoint can be divided into senescent theories and the programmatic aging theory (Gems 

2022). The disposable soma theory is a senescent theory that was first proposed by T. B. L. 

Kirkwood (1977), which holds that given limited resources, natural selection will prioritize 

resource investment in the germline and reproduction, depleting resources from the soma and 

leading to an accumulation of damage without enough resources for maintenance mechanisms to 

reverse it. The programmatic theory, which was proposed independently by Mikhail 

Blagosklonny (2006) and João Pedro de Magalhães and Church (2005), states that although 

aging itself is not a programmed process, early programs that evolved to regulate growth and 

development can have run-on functions (hyperfunction) in late life that lead to aging and age-

associated decline. Since their proposal, these theories have received varying degrees of 

empirical support (Mc Auley 2025). Interestingly, these evolutionary theories of aging do not 

necessarily predict that there will be conserved genetic pathways that directly influence the 

process of aging itself, yet the discovery of such pathways has completely revolutionized the 

field over the last 30 years. 



 

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In addition to proposing evolutionary and mechanistic theories, the field has benefitted from 

describing a set of aging hallmarks that are defined by (1) their age-associated onset or 

progression, and (2) the ability to accelerate or decelerate aging by modifying them (López-Otín 

et al. 2013; López-Otín et al. 2023). These hallmarks are divided into primary (genomic 

instability, telomere attrition, epigenetic alterations, loss of proteostasis, disabled 

macroautophagy), antagonistic (deregulated nutrient-sensing, mitochondrial dysfunction, cellular 

senescence), and integrative (stem cell exhaustion, altered intercellular communication, chronic 

inflammation, dysbiosis) (López-Otín et al. 2023). Moreover, the trans-NIH Geroscience Interest 

Group described aging pillars (adaptation to stress, epigenetics, inflammation, macromolecular 

damage, metabolism, stem cells and regeneration, and proteostasis) and emphasized their 

interconnectedness and the importance of understanding how these interactions regulate aging 

and age-associated disease (Kennedy et al. 2014). A key goal of the aging field is to uncover 

interventions that can modulate these hallmarks and thus modulate aging progression and 

trajectories. 

Sex differences in aging and age-associated diseases 

A remarkable phenomenon in the aging field and one of its most robust features is that women 

live longer than men (Austad 2011). This phenomenon is observed across the world and 

throughout history (Rochelle et al. 2015). In fact, sex-based differences in life expectancy have 

been documented in datasets dating back to 1751 from Sweden. These differences persisted 

despite the global average life expectancy increasing by more than two-fold, from 28.5 years in 

1751 to 73.2 in 2020 (Dattani et al. 2023). Additionally, sex-based discrepancies are present even 

in extreme mortality conditions such as famine, slavery, and pandemics (Rochelle et al. 2015; 

Zarulli et al. 2018; Yan et al. 2024). While these differences may be in part due to the complex 

social structures and environmental factors in human populations, similar sex-based differences 

in longevity are observed in social groups where men and women share similar lifestyles (Luy 

2003; Lindahl-Jacobsen et al. 2013). Similarly, sex differences in longevity are seen across many 

other wild mammalian species and commonly used model organisms (Austad and Fischer 2016; 

Lemaître et al. 2020). 



 

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Aging is often measured by lifespan (the chronological time between birth and death) and 

healthspan (the portion of lifespan that is disease-free). Both measurements show sex 

differences, albeit paradoxically; while women live longer than men, their healthspan is shorter 

(Oksuzyan et al. 2008; Gordon et al. 2017). Women are more likely than men to experience 

Alzheimer’s disease and other dementias (Rajan et al. 2021), chronic conditions (Bello-Lujan et 

al. 2022), bone mineral density loss (Daly et al. 2013), inflammation (Milan-Mattos et al. 2019), 

and higher rates and levels of frailty as they age (Gordon et al. 2017). On the other hand, men 

have a higher incidence of ischemic heart disease, trachea, bronchus, and lung cancers, and colon 

and rectum cancers (Mauvais-Jarvis et al. 2020; Hägg and Jylhävä 2021). 

Why do sex differences in aging exist? Sex-chromosomal and sex-hormonal mechanisms have 

been proposed as a proximate explanation for the longevity-gap between sexes (Hägg and 

Jylhävä 2021). Some of the sex-chromosomal explanations are focused on the X chromosome, 

largely related to X-chromosome inactivation (Marais et al. 2018). In humans, the homogametic 

sex has two X chromosomes with one undergoing random inactivation, and the heterogametic 

sex has one X and one Y chromosome. In the presence of X-linked deleterious mutations, the 

heterogametic sex has an “unguarded” X chromosome and is more vulnerable to expressing such 

mutations. Although this is supported by the higher incidence of X-linked diseases such as 

Duchenne muscular dystrophy and hemophilia in individuals with XY chromosomes, the effect 

size of this mechanism is not enough to explain the magnitude of lifespan differences (Connallon 

et al. 2022). Additionally, while X-chromosome inactivation is random, a skewed inactivation is 

associated with cardiovascular disease risk and cancer incidence, and a balanced inactivation is 

associated with healthy aging and longevity (Gentilini et al. 2012; Roberts et al. 2022).  

Other sex-chromosomal explanations are centered around the Y chromosome including Y 

chromosome toxicity or loss (Marais et al. 2018). The toxic Y hypothesis postulates that the high 

density of transposable elements on the Y chromosome are activated during aging due to the age-

related loss of heterochromatin, contributing to the shorter lifespan of males (Muyle et al. 2021). 

Although some studies in Drosophila provided support for this hypothesis by detecting a 

correlation between the number of Y chromosomes and longevity, a recent study refuted it, 

showing instead that lifespan is correlated with phenotypic sex rather than the number of Y 

chromosomes (Brown et al. 2020; Delanoue et al. 2023). The mosaic loss of Y chromosome, 



 

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which is considered the most common somatic mutation in individuals with XY chromosomes, is 

correlated with cancer and several neurodegenerative and cardiovascular diseases (Bruhn-

Olszewska et al. 2025). 

Sex-hormonal mechanisms lend themselves as an additional explanation for the large disparity in 

lifespan. While both males and females experience age-related decline in sex-hormone levels, 

only females experience a sharp decline in hormones during menopause. Natural menopause has 

been associated with a greater rate of neuromuscular and cognitive decline (Weber et al. 2012; 

Piasecki et al. 2024). Similarly, surgical menopause, or oophorectomy, has been associated with 

decline in cognitive function and an increase in the risk of Alzheimer’s (Rocca et al. 2007; Bove 

et al. 2014; Gurvich et al. 2018). Longer durations of estrogen exposure are associated with 

longevity while shorter periods are associated with increased risk of cardiovascular diseases 

(Shadyab et al. 2017; Hägg and Jylhävä 2021; Mishra et al. 2021). All together, these differences 

have been hypothesized to be a major contributor to the mortality-morbidity paradox. 

C. elegans as an aging model 

A French librarian with a personal interest in biology, Émile Maupas, was the first to describe 

Caenorhabditis elegans (originally named Rhabditis elegans) in 1899, naming it for its rod-like 

shape (rhabditis) and elegant sinusoidal movement (elegans) (Maupas 1899). Although early 

work on C. elegans by Nigon and Dougherty described its development and reproductive 

systems (Nigon 1949; Dougherty et al. 1959), the establishment of C. elegans as a tractable 

genetic model system is credited to Sydney Brenner who started working on this nematode in 

1967 (Brenner 2009). Brenner recognized the power of C. elegans as a model system: they are 

small and easily maintained, their generation time is short (about 3 days), and their brood size is 

large (300-350 progeny) which creates a large nearly isogenic population from a single 

hermaphrodite that is ideal for addressing questions about genetics, development, and behavior 

(Brenner 1974). Eight years later, Brenner published the first paper outlining C. elegans genetic 

methods and identified 300 mutations in about 100 genes that mostly affect behavior and 

morphology (Brenner 1974). The establishment of this model system catalyzed many 

groundbreaking discoveries and accomplishments including describing the complete cell lineage 

of an animal and the discovery of apoptosis (Kimble and Hirsh 1979; Hedgecock et al. 1983; 



 

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Sulston et al. 1983; Ellis and Horvitz 1986), the discovery of RNAi, microRNAs and their role in 

gene regulation (Lee et al. 1993; Wightman et al. 1993; Fire et al. 1998), and the establishment 

of green fluorescent proteins as gene expression markers (Chalfie et al. 1994). C. elegans was 

also the first animal to have its genome sequenced (The C. elegans Sequencing Consortium 

1998). This small nematode facilitated one of the greatest discoveries in the aging field; that 

lifespan can be regulated genetically. 

In 1983, Michael Klass proposed a method to screen for longevity in C. elegans and conducted 

the first screen for long-lived mutants in C. elegans, identifying eight such mutants (Klass 1983). 

Several of those mutants, however, displayed reduced food intake and therefore their long 

lifespan was attributed to caloric restriction. Tom Johnson’s work later uncoupled the reduced 

food intake and the prolonged lifespan phenotypes in one of those mutants, age-1 (Friedman and 

Johnson 1988). Although age-1 mutants experienced reduced fertility which was hypothesized to 

underlie the increased longevity (in line with the disposable soma theory), they also had a 

reduced mortality rate (the probability of death at a given time); this provided the first evidence 

that mortality rate is genetically regulated (Johnson 1990). This work fascinated Cynthia Kenyon 

and fueled her curiosity about the genetic basis of aging, inspiring her to plan her own screen for 

long-lived mutants. However, before even conducting such screen, a lucky decision to use daf-2 

as the screening strain due to its dauer-constitutive phenotype (causing larval arrest) revealed 

that daf-2 itself was a long-lived mutant, becoming the longest-lived mutant at the time (Kenyon 

et al. 1993; Kenyon 2011). The gene daf-2 encodes for an insulin/IGF-1 receptor homolog that is 

conserved in mammals, a finding that revolutionized the aging field because it demonstrated that 

aging can be regulated genetically via evolutionarily conserved pathways (Kimura et al. 1997; 

Kenyon 2011). 

Pro-longevity interventions in C. elegans 

Since the discovery of daf-2 as a long-lived mutant, C. elegans became a popular aging model, 

with studies leveraging its short lifespan and amenability to genetic manipulations to investigate 

a variety of interventions including genetic, pharmacological, and lifestyle interventions. The 

three major pathways that have been implicated in C. elegans lifespan regulation are the 

insulin/IGF-1 signaling (IIS), the AMP-activated kinase (AMPK), and the mechanistic target of 



 

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rapamycin (mTOR) pathways (Kishimoto et al. 2018). In addition to daf-2, two other genes in 

the IIS pathway play a significant role in its aging regulation aging via nutrient sensing: the PI3K 

homolog age-1 and the FoxO transcription factor homolog daf-16 (Morris et al. 1996; Lin et al. 

1997; Ogg et al. 1997; Paradis and Ruvkun 1998; Uno and Nishida 2016). Under favorable 

conditions and food abundance, insulin-like peptides bind the DAF-2 receptor which modulates 

other proteins downstream that ultimately inhibit DAF-16 from translocating into the nucleus. 

Under unfavorable conditions, however, such as caloric restriction or DAF-2 defects via 

mutations or knockdowns, DAF-16 is phosphorylated and translocates into the nucleus, 

activating longevity and stress response pathways that promote longer lifespan (Kishimoto et al. 

2018). Similarly, mTOR inhibition by low nutrient conditions of mutations that decrease its 

activity promote longevity in an AMPK- and DAF-16-dependent manner (Vellai et al. 2003; Jia 

et al. 2004; Uno and Nishida 2016). The effects of mTOR are mediated by the FoxA 

transcription homolog PHA-4 via its role in regulating autophagy (Sheaffer et al. 2008). 

Although there is some level of crosstalk between these signaling pathways, the effect of mTOR 

on lifespan is independent of DAF-16, and combinatorial attenuation of both pathways increases 

lifespan in an additive manner (Chen et al. 2013). 

While genetic interventions offer invaluable mechanistic insights into longevity, they do not 

satisfy the need for interventions that are more viable for use in human health and longevity. 

Therefore, an additional important avenue of aging research is the development and investigation 

of chemical and pharmacological interventions. C. elegans represent an excellent screening 

platform to identify and characterize such interventions due to the properties mentioned 

previously and in chapter 3. This provided the basis for the formation of the Caenorhabditis 

Intervention Testing Program, a National Institute of Aging-funded program to screen for 

compound interventions in a reproducible and rigorous manner (Driscoll et al. 2025). CITP has 

screened over 75 compounds and identified 12 compounds that reliably extend hermaphrodite 

lifespan (Lucanic et al. 2017; Onken et al. 2022; Banse et al. 2024; Banse et al. 2024; Sedore et 

al. 2025). Due to this wealth of knowledge and established protocols and tools for C. elegans 

longevity, it represents an ideal model organism for the investigation of aging questions and 

mechanisms. 



 

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Challenges and benefits of C. elegans males as an aging model 

C. elegans is an androdiecious species with two sexes: hermaphrodites which are genetically 

females that evolved the ability to produce sperm and self-fertilize, and males which mate with 

hermaphrodites to produce cross progeny. The two sexes show behavioral, neuronal, and 

physiological differences in addition to differences in aging and lifespan (Barr et al. 2018; Hotzi 

et al. 2018; Reilly et al. 2021; Weng and Murphy 2024; Purice et al. 2025). However, the studies 

that have investigated hermaphrodite development and aging far outnumber those that have 

investigated males. One of the reasons for that is males are rare in the common lab strain N2 

Bristol, composing only 0.01% of the population. Moreover, males have a high drive to search 

for mates, leading them to escape the agar media they are grown on and desiccating on the 

plastic walls of petri dishes. Another feature of males is their toxic effect on other 

hermaphrodites and males (more in chapter 3 discussion). 

Despite the challenges that males present, they offer a unique behavior that can be utilized as a 

comprehensive health metric: mating. Male mating behavior is considered one of the most 

complex behaviors in C. elegans nematodes that involves multiple steps, integrates a variety of 

inputs including chemical and mechanosensory, and requires proper function of neurons, muscle, 

and the germline (Barr and Garcia 2006). Most importantly, male mating declines with age due 

to an increase in turning defects that is correlated with neuronal deficits (Guo et al. 2012; 

Chatterjee et al. 2013). 

Another unique benefit of using males is the lack of requirement of 5-Fluoro-2'-deoxyuridine 

(FUdR) supplementation. FUdR is commonly used in hermaphrodite aging studies, despite 

evidence showing its effect on hermaphrodite lifespan both directly and indirectly (more in 

chapter 3 introduction, (Mitchell et al. 1979; Gandhi et al. 1980; Anderson et al. 2016; Wang et 

al. 2019; McIntyre et al. 2021). Because males cannot produce self-progeny, the need for FUdR 

use is eliminated, allowing for the effects of interventions on lifespan to be interpreted directly 

without the confounding effect of FUdR. 



 

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Dissertation outline 

Sexual dimorphism in aging trajectories has long been noted, yet the mechanisms underlying 

such differences remain elusive. In the face of an aging population, it is becoming increasingly 

important to expand our knowledge of the basis of such difference which would aid in the 

development of pro-longevity interventions that can benefit both men and women alike. This 

dissertation aims to explore sex differences in aging using C. elegans as the model organism due 

to its short lifespan, amenability to both genetic and chemical interventions, and the abundance 

of molecular and genetic resources available in it. 

In chapter 2, I explore sex differences in the response to genetic pro-longevity interventions. In 

addition to myself, Hannah B. Lewack and Patrick C. Phillips were key contributors to this work, 

with Hannah B. Lewack as a co-lead author. We explore how IIS attenuation using an auxin-

inducible degron system affect survival and reproductive healthspan in male C. elegans. We 

uncover an extraordinary longevity phenotype and investigate the specific tissue types that 

contribute to it. We additionally develop male mating success as a novel and complex healthspan 

measure and describe a novel phenotype that inhibits mating in older males and is repressed by 

IIS disruption. 

In chapter 3, I explore sex differences in the response to chemical pro-longevity interventions at 

the physiological level. In addition to myself, Patrick Phillips was a key contributor to this work, 

which is currently under review. I describe a new compound-screening paradigm using male C. 

elegans survival and reproductive success as a complex healthspan metric. I screened several 

compounds that have been shown to promote hermaphrodite lifespan and found that their effect 

size on lifespan is sex specific. The chapter provides further support to idea that lifespan and 

healthspan are two separate aging metrics that can be uncoupled. 

In chapter 4, I explore sex differences in the response to chemical pro-longevity interventions at 

the transcriptional level. In addition to myself, Christine A. Sedore, Erik Johnson, Erik Segerdell, 

and Patrick Phillips were key contributors to this work. We characterize the effects of two 

compounds, metformin and Thioflavin T, on the transcriptomic landscape of C. elegans 

hermaphrodites and males. Additionally, we explore how biological sex modulates these effects. 



 

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We uncover some sex-biased and sex-shared mechanisms by which these compounds extend 

lifespan. 

Taken together, these chapters describe sex differences at the physiological and transcriptional 

level of a classic aging model in the response to pro-longevity interventions and propose a new 

experimental paradigm that leverages the unique benefits of C. elegans males as a screening tool 

for chemical and pharmacological interventions. 

Bridge to chapter 2 

In chapter 1, I provide an introduction into the aging field, reviewing historical evolutionary 

theories and current knowledge of the molecular pathways that contribute to aging. I highlight 

the prevalence and complexity of sex differences in aging trajectories and the growing demand 

for understanding the mechanisms that underlie differences in the response to pro-longevity 

interventions. In chapter 2, I explore sex differences in the response to genetic interventions. 

Specifically, I explore how targeting the IIS pathway in C. elegans males in a ubiquitous and 

tissue-specific manner affect lifespan and reproductive healthspan. 

  



 

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CHAPTER 2: KNOCKING DOWN THE DAF-2 INSULIN-LIKE RECEPTOR IN 

MALE C. ELEGANS DRAMATICALLY INCREASES REPRODUCTIVE HEALTH AND 

LONGEVITY IN A SEX AND TISSUE-SPECIFIC MANNER 

Rose S. Al-Saadi, Hannah B. Lewack, Patrick C. Phillips 

Author Contributions 

RSA and PCP conceptualized the project. RSA conducted the lifespan assays, the mating assays 

for ages 12–21 days, conducted the statistical analysis and visualization, and co-wrote the 

original manuscript. HBL conducted the mating success and mating behavior assays and co-

wrote the original manuscript. PCP supervised the project, reviewed and edited the manuscript, 

and acquired funding. 

Abstract 

Aging is a universal phenomenon experienced by nearly all multicellular organisms, with several 

factors including sex impacting its manifestation (Austad and Fischer 2016). In the nematode 

model Caenorhabditis elegans, most aging studies have been conducted with hermaphrodites, 

and little is known about male-specific responses to pro-longevity mutations. The auxin-

inducible degron system has been used to degrade DAF-2/IGF-1 in hermaphrodites and it was 

found that both ubiquitous and tissue-specific degradation extended lifespan (Venz et al. 2021; 

Roy et al. 2022; Zhang et al. 2022). However, the role of sex in modulating this lifespan 

extension remains unknown. Here we show that ubiquitous degradation of DAF-2 in male C. 

elegans increases median lifespan by 446%, the longest lifespan extension by a single 

intervention to date. Degrading DAF-2 in the male germline decreased lifespan, which is the 

opposite of its effect on hermaphrodites (Zhang et al. 2022). We identified a novel age-

associated male tail paralysis phenotype that prohibits aged males from mating. A significant 

reduction in this phenotype is observed with ubiquitous DAF-2 degradation and is associated 

with an increase in reproductive healthspan at old age by 39.6%. This work highlights the 

importance of studying sex differences in aging and builds upon a paradigm for examining pro-

longevity genetic interventions and their effects on both lifespan and healthspan. 



 

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Main 

Sex underlies incredibly robust differences in aging and longevity between males and females 

(Austad 2011). Such differences are seen in human populations and many other wild mammalian 

species (Rochelle et al. 2015; Lemaître et al. 2020). While complex social structures and other 

environmental factors undoubtedly contribute to this phenomenon, sex-based differences in 

longevity are nevertheless observed in social groups where both sexes share similar lifestyles 

(Luy 2003; Lindahl-Jacobsen et al. 2013). Precisely how sex regulates aging and longevity, 

especially at a molecular level, is still largely understudied. This is in part due to the complex 

nature of aging, and in part due to limited emphasis in research programs on how sex modulates 

different biological processes. Developing better tools and interventions that are effective at 

enhancing late life health and longevity for both males and females depends critically on 

increased understanding of the biological basis of sex-specific differences in aging responses. 

While the influence of sex on the natural aging trajectories is clear, over the last two decades it 

has become evident that the sexes respond differently to pro-longevity interventions including 

genetic and pharmacological interventions (Knufinke et al. 2023; Bartke et al. 2024). While we 

know these differences in response to longevity interventions exist, the molecular mechanisms 

underlying these differences remain to be fully elucidated. Here, we use Caenorhabditis elegans 

as a genetic model to investigate how tissue-specific degradation of the classic DAF-2 insulin-

like receptor enhances health and lifespan in a sex-dependent manner. 

The insulin/ insulin-like growth factor 1 (IGF-1) signaling pathway (IIS) is one of the best 

described genetic pathways that modulate aging. Knocking out components of this pathway 

confers significant longevity extensions in worms, flies, mice, and other animals (Kenyon 2010). 

Sex differences in the IIS pathway have also been well documented in humans and other species 

(Tramunt et al. 2020). For example, women develop lower sensitivity to insulin than men as they 

age and generally have lower incidences of metabolic diseases (Ciarambino et al. 2023). 

Reduced IIS pathway activity in mice leads to a larger lifespan extension and better health 

outcomes in females than in males (Garratt 2020). Despite these differences, the molecular basis 

of sex-specific responses to insulin signaling remains relatively understudied. Investigating these 



 

23 
 

 

differences ensures that sex differences are included in the design and implementation of 

therapeutics aimed at enhancing health late in life. 

Within C. elegans, daf-2 encodes for an insulin-like receptor that, when mutated, can double 

individual lifespan (Kenyon et al. 1993; Kimura et al. 1997; Kenyon 2010). This lifespan-

extension phenotype is dependent on the downstream FoxO transcription factor DAF-16 

(Kenyon et al. 1993). Since the discovery of this phenotype, the molecular components and the 

signaling cascade regulated by DAF-2 have been extensively studied (Kenyon 2011). Recently, 

the auxin-inducible degron (AID) system has been used to knock down DAF-2 in a spatially and 

temporally controlled manner, demonstrating that DAF-2 regulates hermaphrodite lifespan 

primarily through the intestine, without negatively impacting development and reproduction 

(Venz et al. 2021; Roy et al. 2022; Zhang et al. 2022).  

Here, we examined the sex-specific differences in longevity and healthspan in C. elegans by 

using the AID system to contrast the tissue-specific effects of DAF-2 degradation in males and 

hermaphrodites. Male mating represents an ideal measure of reproductive healthspan because it 

is a neurologically complex behavior that uses the majority of the male’s 93 sex-specific neurons 

to achieve (Sulston et al. 1980; Barr et al. 2018; Molina-García et al. 2020). It also declines 

rapidly with age (Barr and Garcia 2006; Chatterjee et al. 2013) due to behavioral and neuronal 

deficits (Guo et al. 2012; Chatterjee et al. 2013) rather than a decrease in mating drive or sperm 

quality. We find that ubiquitous downregulation of insulin-like signaling leads to a much larger 

increase in lifespan in males compared to hermaphrodites and greatly enhances male sexual 

function late in life. In contrast, downregulating insulin-like signaling in the male germline 

decreases lifespan (Zhang et al. 2022). This study demonstrates the importance of studying sex 

differences in longevity and adds to a platform for investigating these effects in one of the most 

important model systems for the study of the biology of aging. 

DAF-2 degradation drastically extends male lifespan 

The two major components of the AID system are: 1. a short sequence called a degron tag that is 

fused to the protein of interest, and 2. the plant F-box protein Transport Inhibitor Response 1 

(TIR1) (Nishimura et al. 2009; Zhang et al. 2015). TIR1, along with other endogenous proteins, 



 

24 
 

 

forms an E3 ubiquitin ligase complex that, when auxin is added, will target the protein of interest 

for degradation. However, auxin treatment alone has been previously shown to increase 

hermaphrodite lifespan (Loose and Ghazi 2021). To account for the effects of TIR1 and auxin 

treatment on male lifespan independent of protein degradation, we measured survival using the 

CA1200 strain that contains ubiquitously expressed TIR1 but no degron tag. We found that 

although there was a significant difference in survival between the ethanol control and auxin (p = 

0.0008), there was no difference in the median lifespan for the two treatments (Supplemental 

Fig. S2.1a). 

Three different groups have previously shown that ubiquitous DAF-2 degradation increased 

hermaphrodite lifespan significantly (Venz et al. 2021; Roy et al. 2022; Zhang et al. 2022). 

However, the effects of DAF-2 degradation on male lifespan remains unexplored. Using 

previously published AID strains, we asked whether the effect of ubiquitous DAF-2 degradation 

on lifespan is sexually dimorphic. We degraded DAF-2 in both males and hermaphrodites and 

measured their lifespan. We note that the whole-body DAF-2 AID strain used here produced 

occasional spontaneous dauers, indicative of leaky DAF-2 degradation without auxin exposure. 

Therefore, we used the TIR1-only strain on auxin as our negative control as reported previously 

(Zhang et al. 2022). We placed 40 animals per plate (in triplicates), with hermaphrodites and 

males housed separately, and measured their lifespan, finding that DAF-2 degradation, sex, and 

the interaction between the two factors have a significant effect on survival. Ubiquitous DAF-2 

degradation in males led to a significant increase in survival (p < 0.0001) accompanied by a 

dramatic increase in median lifespan (+446%, Fig. 2.1a), with a maximum lifespan of 127 days 

compared to 38 days in controls. This is much greater than the increase in hermaphrodite lifespan 

reported here (+109%, Fig. 2.1a) and previously (+70–135% in Venz et al. 2021, +167% in 

Zhang et al. 2022, and +88–117% in Roy et al. 2022). Sex clearly plays a key role in regulating 

the response to IIS signaling disruption, consistent with previous findings in the canonical daf-2 

mutant (Hotzi et al. 2018).  

Male-specific pheromones in C. elegans have been shown to decrease both male and 

hermaphrodite lifespan, resulting in “male-induced demise” (Maures et al. 2014; Shi and Murphy 

2014; Shi et al. 2017). To eliminate the effect of male pheromones on lifespan, we housed males  



 

25 
 

 

Fig. 2.1 Ubiquitous and tissue-specific DAF-2 degradation extends male lifespan. (a) 
Kaplan-Meier curves showing survival of C. elegans hermaphrodites and males following 
ubiquitous DAF-2 degradation. Yellow lines denote hermaphrodites and purple lines denote 
males. Dashed lines denote the negative controls treated with ethanol and solid lines denote 
DAF-degradation with 1mM auxin treatment. Each line represents at least two biological 
replicates with total n = 183-235. (b, c) Kaplan-Meier curves showing survival of C. elegans 
males following DAF-2 degradation in the (b) intestine and (c) germline. Gray lines denote the 
negative controls treated with ethanol and purple lines denote DAF-degradation with 1mM auxin 
treatment. The intestine lifespan curve represents one biological replicate with n = 80-109 and 
the germline represents two biological replicates with n = 137 – 202. The black dashed lines 
denote the age at which 50% of the population has died. The asterisks denote p-values from a 
Cox Proportional Hazards model where ****p<.0001. For additional information and the output 
of the CPH model, see Supplemental Tables S2.1 and S2.2 

on individual plates then measured the effects of DAF-2 degradation on their lifespan. We found 

that when males are housed individually, ubiquitous DAF-2 degradation resulted in a significant 

increase in lifespan (+230%, Supplemental Fig. S2.1b). We hypothesized that individually 

housed males would display increased lifespan. Indeed, control males lived longer when housed 

individually. However, DAF-2 AID males did not display an added benefit of being housed 

individually. This indicates the DAF-2 degradation confers the maximum benefit to lifespan 

extension in males, and eliminating male pheromones provides no added benefits. This increase 

in the baseline lifespan also explains the decreased effect size of this intervention on single males 

compared grouped males. 

Next, we wanted to identify which tissues contribute to this dramatic lifespan extension. Using 

strains with tissue-specific promoters driving DAF-2 degradation, we targeted DAF-2 in tissues 

known to regulate hermaphrodite lifespan (intestine, germline, neurons, hypodermis) and 

measured male lifespan (Venz et al. 2021; Roy et al. 2022; Zhang et al. 2022). We found that 

intestinal degradation of DAF-2 in males extended median lifespan by 83% (p < 10-14, Fig. 2.1b). 

Germline degradation of DAF-2, however, decreased median lifespan by 19% (p =0.00001, Fig. 

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2.1c). This negative effect of germline-specific degradation on lifespan is the opposite of what 

was observed previously in hermaphrodites (Zhang et al. 2022). Degradation of DAF-2 in the 

neurons and hypodermis did not alter survival (Supplemental Fig. S2.1c, d). Overall, we find that 

sex plays a significant role in modulating the magnitude of response to DAF-2 degradation in C. 

elegans, with males displaying a much greater increase in lifespan in response to this 

intervention compared to hermaphrodites. 

Ubiquitous DAF-2 degradation prolongs reproductive healthspan in old males 

Lifespan and healthspan can often be decoupled, and it has become increasingly important to test 

the effects of pro-longevity interventions on both metrics (Bansal et al. 2015; Hahm et al. 2015; 

Banse et al. 2024). Therefore, we tested whether the extension in male lifespan produced by 

DAF-2 degradation was accompanied by an extension in reproductive healthspan. We used 

reproductive success as our healthspan metric because it is a complex behavior that requires 

fidelity of multiple tissues and systems (muscles, neurons, reproductive organs) (Barr and Garcia 

2006), and declines with age due to physiological changes and not due to a decrease in 

motivation (Guo et al. 2012; Chatterjee et al. 2013). We tested the mating success of young (day 

1 of adulthood), middle-aged (day 7), and old (day 9) adults. Because hermaphrodites produce 

sperm and can self-fertilize, we used fog-2 pseudo-females, which are essentially hermaphrodites 

that are unable to produce sperm (Schedl and Kimble 1988), as mating partners for the males to 

ensure that only cross progeny were counted. To assess mating success, one male and two virgin 

fog-2 pseudo-females were allowed to mate for 24 hours, then mating success was measured. 

Using a logistic regression model, we asked whether age, DAF-2 degradation, and the interaction 

between the two factors have an effect on mating success. When DAF-2 is degraded 

ubiquitously, mating success at both days 7 and 9 was maintained while the no-auxin controls 

displayed the anticipated decline in mating success. Because the males were still capable of 

mating at day 9, we additionally tested days 12, 15, 18, and 21 and found that ubiquitous DAF-2 

degradation decreased the overall rate of reproductive decline by 40% (p = 4 x 10-5) compared to 

the no-auxin control (Fig. 2.2a). Ubiquitous DAF-2 AID males mate successfully later in life, 

with successful mating events occurring until day 18 of adulthood, a substantial extension 

compared to the no-auxin controls that stop mating successfully at day 9 of adulthood (Fig. 2.2a). 

Despite extending lifespan, intestinal DAF-2 degradation was not sufficient to preserve 



 

27 
 

 

Fig. 2.2 Ubiquitous DAF-2 degradation preserves late-life male reproductive success. (a) 
Logistic regression lines showing mating success for C. elegans males following ubiquitous 
DAF-2 degradation. Gray lines denote untreated controls, and purple lines denote DAF-2 
degradation with 1mM auxin treatment. Shapes represent biological replicates, with 4–58 
technical replicates in each. The gray shading around the regression lines represents SEM. A 
generalized linear mixed model with a binomial distribution was used to assess the effect of 
treatment and age (and the interaction) on mating success. (b, c) Bar graphs showing (b) the time 
on the vulva and (c) the probability of tail incoordination in C. elegans males following 
ubiquitous DAF-2 degradation. Gray bars denote untreated controls, and purple bars denote 
DAF-2 degradation with 1 mM auxin. Each bar represents at least two biological replicates, with 
a total n = 48–52. Error bars represent SEM. A generalized linear mixed model with a binomial 
distribution for tail incoordination and a gaussian distribution for the time on the vulva was used 
to assess the effect of treatment and age (and the interaction) on behavior. The asterisks denote 
p-values the generalized linear models followed by planned comparisons where *p<.05, **p<.01, 
****p<.0001. For additional information and the output of the linear models, see Supplemental 
Tables S2.3–S2.6 

reproductive success in late life (Supplemental Fig. S2.2a). In fact, degradation of DAF-2 in any 

single tissue type did not preserve reproductive success in late life (Supplemental Fig. S2.2b-d). 

Overall, we found that ubiquitous degradation of DAF-2 is the only intervention that prolongs 

reproductive healthspan in males. 

DAF-2 degradation lowers the incidence of male tail incoordination 

For a mating event to be successful, a male must complete a series of complex behaviors. Males 

must locate a hermaphrodite, scan the body using its tail via backward movement, turn once it 

reaches the head or the tail of the hermaphrodite, locate the vulva, insert the spicule, and transfer 

sperm (Barr and Garcia 2006). This sequence of behaviors, which is considered the most 

complex in C. elegans, declines rapidly with age (Chatterjee et al. 2013). To investigate which 

changes at the behavioral level contribute to sustained mating success, we degraded DAF-2 

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allowed them to mate with immobilized hermaphrodites then quantified different mating 

behaviors: response rate (whether or not a male made contact with a hermaphrodite), turning 

ability, location of vulva (LOV) efficiency (vulva location success “1” or failure “0” divided by 

the number of passes), and the amount of time on the vulva. We note that the sample size for 

turning success, LOV, and time on the vulva at day 9 is limited due to many assayed males not 

making contact with hermaphrodites and therefore not reaching a point at which those behaviors 

are relevant. DAF-2 degradation in males did not have an effect on either turning ability or LOV 

efficiency (Supplemental Fig. S.2.3a, b). The amount of time on the vulva, however, was 

significantly lower at both one and seven days of adulthood when DAF-2 was degraded (Fig. 

2.2b). This potentially explains the lower mating output of the ubiquitous DAF-2 AID in the 

early ages (Fig. 2.2a, day 1). We also noted a novel male aging phenotype that we termed “tail 

incoordination” (Fig. 2.2c). While young males are able to perform swift sinusoidal movements, 

older males seem to have a stiff tail that remains immobile while the rest of their body continues 

in the sinusoidal motion. Males with this phenotype were rarely able to begin the mating 

sequence. We found that DAF-2 degradation significantly reduces the incidence of tail 

incoordination in older males at both days 7 (-72%, p = 0.03) and 9 of adulthood (-64%, p = 

0.003, Fig. 2.2d). Overall, our results support the hypothesis that the decrease in tail 

incoordination incidence following DAF-2 degradation likely underlies the sustained 

reproductive healthspan in males. 

Discussion 

Sex differences in aging and the response to pro-longevity interventions have been documented 

widely (Austad 2011; Austad and Bartke 2016). Yet, the molecular mechanisms that underlie 

these differences remain to be fully elucidated. Here, we show that tissue-specific and ubiquitous 

knockdown of the insulin-like signaling pathway confer sexually dimorphic changes in 

longevity. We targeted the protein DAF-2 for degradation in the whole body of C. elegans males 

and hermaphrodites and found that the lifespan extension in males significantly exceeded that in 

hermaphrodites, leading to one of the largest reported lifespan extensions in C. elegans via a 

single intervention. Three different studies have previously shown that DAF-2 degradation in the 

intestine and neurons extends median lifespan in hermaphrodites (Venz et al. 2021; Roy et al. 

2022; Zhang et al. 2022). Here, we show that DAF-2 degradation in male intestine also prolongs 



 

29 
 

 

median lifespan dramatically, while neuronal degradation had no effect on lifespan. This 

difference in the effect of neuronal degradation is likely due to the sexually dimorphic nature of 

C. elegans neurons. The two sexes have several sex-specific neurons (8 in hermaphrodites and 

93 in males), and some of the 294 shared neurons are sexually dimorphic (Kim and Kim 2022). 

Additionally, it has been shown recently that neuronal aging in C. elegans results in sexually 

dimorphic transcriptional changes (Weng and Murphy 2024). Future work could explore whether 

the differences in the neuronal effect of DAF-2 degradation is via sex-specific neurons or 

sexually dimorphic shared neurons. 

Zhang et al. (2022) also found that DAF-2 degradation in the hypodermis and germline lead to 

median lifespan extension in hermaphrodites, while Venz et al. (2021) observed no effects of 

either of these tissues and Roy et al. (2022) observed no effects of germline degradation on 

lifespan. Here, we show that hypodermis degradation had no effects on median lifespan in males 

while degradation in the germline led to a decrease in their median lifespan. Because the AID 

system produces a strong knockdown but not a fully null mutation, it is possible that the 

discrepancies between these studies is due to variable levels of DAF-2 knockdown in the 

hypodermis and germline. 

Males are known to produce pheromones that are toxic to other males (Shi et al. 2017; Ludewig 

et al. 2019), therefore, we chose to culture them in groups as well as individually. Although it 

has been shown that daf-2 and other classical long-lived mutant hermaphrodites are especially 

susceptible to male-induced demise (Booth et al. 2022), our results show that DAF-2 AID males 

are still extraordinarily long-lived. When DAF-2 is ubiquitously degraded, both single and 

grouped males live significantly longer, with comparable median lifespans of 76 and 71 days, 

respectively. On the other hand, the DAF-2 AID strain without auxin exposure and DAF-2 

degradation suffered from male-induced demise, as evident by the difference of median lifespans 

of single and grouped males (23 and 15 days, respectively). This alludes to a mechanism in 

DAF-2 AID males that is protective against this male pheromone-induced demise. Future studies 

could explore the effects of DAF-2 degradation in the context of male pheromones and whether 

this degradation is protective of male-induced demise, thus contributing to the dramatic lifespan 

extension. 



 

30 
 

 

Incorporating both lifespan and healthspan measures into aging studies is important especially in 

the context of our aging population; people are living longer but suffering more diseases and 

disabilities in old age (Olshansky 2018). Here, we provide additional evidence for the 

uncoupling of lifespan and healthspan. Despite the lifespan extension conferred by intestinal 

DAF-2 degradation in males, a similar effect on reproductive healthspan was not observed. 

However, previous work had shown that in the canonical daf-2 mutant, several health metrics 

such as pharyngeal pumping (Huang et al. 2004), mobility (Mulcahy et al. 2012; Hahm et al. 

2015), learning (Weng et al. 2024), and resistance to microbial pathogens (Murphy et al. 2003) 

are improved compared to wild type animals. The discrepancy is attributed to the complexity of 

the health metric we used in our study. In order to maintain reproductive success into old age, 

several systems and tissues need to maintain their function. Therefore, DAF-2 likely needs to be 

degraded in multiple tissues to produce a meaningful effect on reproductive success, which is 

what we observed in the ubiquitous DAF-2 AID strain.  

In Roy et al. (2022) the dauer phenotype of daf-2 mutants could not be recapitulated by 

degrading DAF-2 in single tissues. Additionally, the phenotype of reduced motility in early life 

and increased motility in late life was under combinatorial regulation of both neurons and 

muscles (Roy et al. 2022). Consistent with these results, we show here that male reproductive 

success in late life could not be promoted by single-tissue degradation but requires DAF-2 to be 

degraded in multiple tissues, accomplished here only via ubiquitous degradation. Future work 

could explore the effects of combined degradation in neurons and muscles, tissues known to play 

a role in motility and mating, on reproductive success in late life.  

It has been shown previously that older males display deficits in turning behavior that is 

abolished in daf-2 mutants, accompanied with improvements in the responsiveness to 

hermaphrodites, LOV efficiency, and turning ability (Chatterjee et al. 2013). We anticipated 

observing similar changes in the ubiquitous DAF-2 AID. However, we did not see differences in 

the response rate of LOV efficiency and saw a reduction in the time on the vulva. Instead, we 

discovered a novel tail immobility phenotype in aging males is the likely cause of sexual 

dysfunction, one that is ameliorated by DAF-2 degradation in the whole body. Future work could 

explore the basis of this phenotype and its relation to muscle health and function which had been 



 

31 
 

 

shown previously to decline with age, leading to motor deficits that are reduced in daf-2 mutants 

(Glenn et al. 2004; Roy et al. 2022)  

Our work shows that genetic interventions targeting the insulin-like signaling pathway extend 

not only the lifespan of the animal, but also the reproductive healthspan. While we focus on 

reproductive healthspan here, future studies could explore other healthspan metrics such as 

oxidative stress resistance, motility, pharyngeal pumping, and lipofuscin accumulation (Bansal et 

al. 2015). Our study expands on a framework for measuring healthspan in males as well as 

describing a novel tail paralysis phenotype. Taken together, these results suggest that males 

represent a useful tool for future aging research as they may be more susceptible to certain 

interventions and provide a complex behavior that can be used to assess the efficacy of 

interventions on reproductive healthspan.  

Methods 

C. elegans strains and maintenance 

The strains used in this study were provided by the CGC, which is funded by NIH Office of 

Research Infrastructure Programs (P40 OD010440). The list of strains used in this study is 

provided in Supplemental Table S.2.7, many of which were sourced from Zhang et al. (2022). 

Strains were maintained at 20°C on 60 mm plates filled with standard nematode growth medium 

(NGM) seeded with 10 mm of Escherichia coli OP50, unless specified otherwise. Worms were 

transferred every Monday and Friday to fresh plates. Plant auxin at a final concentration of 1mM 

for knockdown experiments was prepared by adding 400 mM auxin stock (0.350g indole-3-

acetic acid added to 5mL of 190-proof ethanol) to cooled agar in a 2.5mL to 1 L ratio and poured 

onto plates once mixed. Plates were then covered with foil/towels and stored in light-proof boxes 

at 4°C after being seeded with OP50. To decrease the likelihood of spontaneous dauer formation, 

the ubiquitous DAF-12 AID strain was kept in a separate box from other strains, and only a few 

worms during maintenance were transferred to prevent overcrowding. To maintain male stocks, 

8–10 males and 4–5 hermaphrodites were transferred to new plates 1–2 times a week. Plates 

older than 1–2 weeks were discarded frequently. 



 

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Lifespan assay 

The lifespan protocol used here is based on the previously published CITP lifespan protocol 

(Lucanic et al. 2017). A day prior to the start of the experiment, L4 males are staged visually 

using the male tail morphology and picked to 60 mm NGM plates seeded with E. coli OP50 at a 

concentration of 50 males per plate. Approximately 24 hours later, the adult males are transferred 

to experimental plates (60 mm NGM plates seeded with E. coli OP50, 40 males per plate, 3 

plates per condition). Worms were scored three times a week and transferred to new plates 

weekly until the worms were all dead. Worms were scored as either alive, dead, or lost (if 

burrowed, missing, or walled). Mobile worms were scored as alive. Immobile worms were 

assayed by touching their tail or head with a platinum worm pick. If they do not respond to 

touch, they are scored as dead. Dead worms are removed from the plate, and live worms are 

counted. 

Mating assay 

For each replicate, a single male was picked at the L4 larval stage and placed on its respective 

treatment plate: a small 35 mm NGM plate or a 35 mm auxin plate seeded with 10 mm of E. coli 

OP50. Each trial consisted of 16 replicates per treatment. We tested the effects of DAF-2 

degradation on young, middle-age, and old males, looking at days 1, 7, and 9 of adulthood, 

respectively. Once the males reached their target age, they were moved to a mating plate with 

two 1-day old adult feminized fog-2 hermaphrodites. These hermaphrodites do not produce 

sperm, therefore any progeny observed must be a product of mating with a male (Schedl and 

Kimble 1988). Worms were allowed to mate for 24 hours at 20°C before being assayed. Mating 

success was scored as 1 for successful mating (progeny present), and 0 for unsuccessful mating 

(progeny absent). Plates with contamination or missing/dead males were censored.  

Behavior assay 

For each strain, 30 males were picked at their L4 stage and put on either a 100 mm NGM plate or 

a 100 mm auxin plate. They were then aged to their respective days of adulthood (one, seven, 

and nine). The day prior to the behavior assay, 300, L4 immobilized unc-31 hermaphrodites were 

picked at their L4 stage and aged to day one of adulthood on a 150 mm NGM plate seeded with 



 

33 
 

 

E. coli OP50. Behavior assays were based on methods described in Chatterjee et al. (2013). Five 

unc-31 hermaphrodites were allowed to equilibrate on a 60 mm NGM plate seeded with 10 mm 

E. coli OP50 for 10 minutes. One aged male was placed on the plate and its behavior recorded 

for four minutes using the Dinocam software. If the worm did not respond to the hermaphrodites 

after four minutes, it was excluded from mating measures, if it began mating in the final minute, 

recording time was extended to six minutes to include the entirety of the behavior. Mating was 

evaluated using six measurements: contact with vulva rate (the amount of trials where males 

made and held contact with the vulva over total trials), Number of Passes (the amount of passes a 

worm made over the vulva with its tail before successfully mating), LOV efficiency (Successful 

vulva location (1) or unsuccessful (0) divided by the number of passes over the vulva), the 

amount of time on the vulva (the amount of time the male kept contact with the vulva), turning 

success (the percentage of worms that can successfully complete turning behaviors), and tail 

paralysis (the amount of worms that had upper bodies that were mobile but whose tails were 

immobile, over the total amount of trials).  

Statistical analysis 

All the statistical analyses reported here were conducted in RStudio version 4.5.1 (R Core Team, 

2024). A mixed effects Cox-Proportional Hazards (CPH) model was used for each strain (single 

tissue degradation) to analyze survival data, where treatment (control or auxin) was used as the 

fixed effect and technical (Rep) and biological replicates (StartDate) were used as the random 

effects. To test for the effect of sex, treatment, and the interaction between the two on survival in 

the ubiquitous DAF-2 AID strain, a CPH model was fit where, sex, treatment, and their 

interaction were the fixed effects, and technical (Rep) and biological replicates (StartDate) were 

the random effects. The coxme package version 2.2-22 (Therneau 2024) was used to fit the CPH 

model and the survival package version 3.8-3 (Therneau and Grambsch 2000; Therneau 

2024) was used to construct the Kaplan-Meier survival curves. To analyze mating success, we fit 

a generalized linear model with a binomial distribution followed by paired comparisons to test 

for the effects of age, treatment, and their interaction on mating success within each strain. To 

analyze the effect of treatment behavior, we fit a generalized linear model with a binomial 

distribution followed by planned comparisons to test the effects of age, treatment, and their 

interaction on turning success, tail incoordination, and LOV efficiency and a gaussian 



 

34 
 

 

distribution for the time on the vulva. The lme package version 1.1-37 (Bates et al. 2015) was 

used to fit the linear model and the emmeans package version 1.11.2 (Lenth 2025) was used for 

planned comparisons. 

Supplemental Figures

 

Supplemental Fig. S2.1 Neuron- and Hypodermis-specific DAF-2 degradation does not 
extend male lifespan. (a) Kaplan-Meier curves showing survival of CA1200 TIR1-only C. 
elegans males. Gray lines denote the negative controls treated with ethanol and purple lines 
denote 1mM auxin treatment. Each line represents at least two biological replicates with total n = 
179–184. (b) Kaplan-Meier curves showing survival of C. elegans males aged on individual 
plates. Gray lines denote the negative controls treated with ethanol and purple lines denote 
ubiquitous DAF-degradation with 1mM auxin treatment. Each line represents three biological 
replicates with total n = 62–70. (c, d) Kaplan-Meier curves showing survival of C. elegans males 
following DAF-2 degradation in the (b) neurons and (c) hypodermis. Gray lines denote the 
negative controls treated with ethanol and purple lines denote DAF-degradation with 1mM auxin 
treatment. Each line represents at least two biological replicates with total n = 180-202. The 
black dashed lines denote the age at which 50% of the population has died. The asterisks denote 
p-values from a Cox Proportional Hazards model where ****p<.0001, ***p<.001. For additional 
information and the output of the CPH model, see Supplemental Tables S2.1 and S2.2 

0.00

0.25

0.50

0.75

1.00

0 10 20 30 40
Age at death (days)

Fr
ac

tio
n 

su
rv

iv
in

g

Control
Neuron

0.00

0.25

0.50

0.75

1.00

0 30 60 90 120
Age at death (days)

Fr
ac

tio
n 

su
rv

iv
in

g

Single male ubiquitous
Single male control

****

0.00

0.25

0.50

0.75

1.00

0 10 20 30 40
Age at death (days)

Fr
ac

tio
n 

su
rv

iv
in

g

TIR1 control
TIR1 ubiquitous***

0.00

0.25

0.50

0.75

1.00

0 10 20 30
Age at death (days)

Fr
ac

tio
n 

su
rv

iv
in

g

Control
Hypodermis

40

a
Figure 1

b

c d

a
Figure 3

b

0.00

0.25

0.50

0.75

1.00

1 7 9
Age (days of adulthood)

Tu
rn

in
g 

su
cc

es
s

Control
Ubiquitous

0.0

0.2

0.4

0.6

1 7 9
Age (days of adulthood)

LO
V

 e
ffi

ci
en

cy

Control
Ubiquitous

0.00

0.25

0.50

0.75

1.00

1 7 9
Age (days of adulthood)

M
at

in
g 

su
cc

es
s

Control
Intestine

0.00

0.25

0.50

0.75

1.00

1 7 9
Age (days of adulthood)

M
at

in
g 

su
cc

es
s

Control
Germline

0.00

0.25

0.50

0.75

1.00

1 7 9
Age (days of adulthood)

M
at

in
g 

su
cc

es
s

Control
Neuron

0.00

0.25

0.50

0.75

1.00

1 7 9
Age (days of adulthood)

M
at

in
g 

su
cc

es
s

Control
Hypodermis

a
Figure 2

b

c d



 

35 
 

 

Supplemental Fig. S2.2 Tissue-specific DAF-2 degradation does not preserve late-life male 
reproductive success. (a) Bar graphs showing mating success for C. elegans males following 
DAF-2 degradation in the (b) intestine, (c) germline, (d) neurons, and (e) hypodermis. Gray bars 
denote untreated controls, and purple bars denote DAF-2 degradation with 1 mM auxin. Each bar 
represents at least two biological replicates, with total n =49–59. Error bars represent SEM. A 
generalized linear mixed model with a binomial distribution was used to assess the effect of 
treatment and age (and the interaction) on mating success. For additional information and the 
output of the linear model, see Supplemental Tables S2.3 and S2.4 
  

0.00

0.25

0.50

0.75

1.00

0 10 20 30 40
Age at death (days)

Fr
ac

tio
n 

su
rv

iv
in

g

Control
Neuron

0.00

0.25

0.50

0.75

1.00

0 30 60 90 120
Age at death (days)

Fr
ac

tio
n 

su
rv

iv
in

g

Single male ubiquitous
Single male control

****

0.00

0.25

0.50

0.75

1.00

0 10 20 30 40
Age at death (days)

Fr
ac

tio
n 

su
rv

iv
in

g

TIR1 control
TIR1 ubiquitous***

0.00

0.25

0.50

0.75

1.00

0 10 20 30
Age at death (days)

Fr
ac

tio
n 

su
rv

iv
in

g

Control
Hypodermis

40

a
Figure 1

b

c d

a b

0.00

0.25

0.50

0.75

1.00

1 7 9
Age (days of adulthood)

Tu
rn

in
g 

su
cc

es
s

Control
Ubiquitous

0.0

0.2

0.4

0.6

1 7 9
Age (days of adulthood)

LO
V

 e
ffi

ci
en

cy

Control
Ubiquitous

0.00

0.25

0.50

0.75

1.00

1 7 9
Age (days of adulthood)

M
at

in
g 

su
cc

es
s

Control
Intestine

0.00

0.25

0.50

0.75

1.00

1 7 9
Age (days of adulthood)

M
at

in
g 

su
cc

es
s

Control
Germline

0.00

0.25

0.50

0.75

1.00

1 7 9
Age (days of adulthood)

M
at

in
g 

su
cc

es
s

Control
Neuron

0.00

0.25

0.50

0.75

1.00

1 7 9
Age (days of adulthood)

M
at

in
g 

su
cc

es
s

Control
Hypodermis

a b

c d



 

36 
 

 

 
Supplemental Fig. S2.3 Turning success and LOV efficiency are not improved upon DAF-2 
ubiquitous degradation. Bar graphs showing (a) turning success and (b) LOV efficiency for C. 
elegans males following ubiquitous DAF-2 degradation. Gray bars denote untreated controls, 
and purple bars denote DAF-2 degradation with 1 mM auxin. Each bar represents at least two 
biological replicates, with total n =44–52. Error bars represent SEM. A generalized linear mixed 
model with a binomial distribution was used to assess the effect of treatment and age (and the 
interaction) on behavior. For additional information and the output of the linear model, see 
Supplemental Tables S2.5 and S2.6 
  

0.00

0.25

0.50

0.75

1.00

0 10 20 30 40
Age at death (days)

Fr
ac

tio
n 

su
rv

iv
in

g

Control
Neuron

0.00

0.25

0.50

0.75

1.00

0 30 60 90 120
Age at death (days)

Fr
ac

tio
n 

su
rv

iv
in

g

Single male ubiquitous
Single male control

****

0.00

0.25

0.50

0.75

1.00

0 10 20 30 40
Age at death (days)

Fr
ac

tio
n 

su
rv

iv
in

g

TIR1 control
TIR1 ubiquitous***

0.00

0.25

0.50

0.75

1.00

0 10 20 30
Age at death (days)

Fr
ac

tio
n 

su
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iv
in

g

Control
Hypodermis

40

a
Figure 1

b

c d

a b

0.00

0.25

0.50

0.75

1.00

1 7 9
Age (days of adulthood)

Tu
rn

in
g 

su
cc

es
s

Control
Ubiquitous

0.0

0.2

0.4

0.6

1 7 9
Age (days of adulthood)

LO
V

 e
ffi

ci
en

cy

Control
Ubiquitous

0.00

0.25

0.50

0.75

1.00

1 7 9
Age (days of adulthood)

M
at

in
g 

su
cc

es
s

Control
Intestine

0.00

0.25

0.50

0.75

1.00

1 7 9
Age (days of adulthood)

M
at

in
g 

su
cc

es
s

Control
Germline

0.00

0.25

0.50

0.75

1.00

1 7 9
Age (days of adulthood)

M
at

in
g 

su
cc

es
s

Control
Neuron

0.00

0.25

0.50

0.75

1.00

1 7 9
Age (days of adulthood)

M
at

in
g 

su
cc

es
s

Control
Hypodermis

a b

c d



 

37 
 

 

Supplemental Tables 

Supplemental Table S2.1 Lifespan assay summary statistics 
Sex (housing) Strain Tissue 

knockdown 
Conditi
on 

Numb
er 
dead 

Number 
censore
d 

Median 
Time 

Mean Std 
Error 

Lower 
95% 

Upper 
95% 

90th 
perce
ntile 

Male (group) CA1200 NA Ethanol 184 13 13 14.20 0.32 13 13 20 

Male (group) CA1200 NA Auxin 179 20 13 12.87 0.31 13 13 15 

Male (group) MQD2356 Neuron Ethanol 202 38 15 15.98 0.39 15 15 24 

Male (group) MQD2356 Neuron Auxin 192 48 15 16.24 0.36 15 15 24 

Male (group) MQD2374 Intestine Ethanol 109 10 12 15.08 0.55 12 15 26 

Male (group) MQD2374 Intestine Auxin 80 41 22 24.03 0.91 22 26 NA 

Male (group) MQD2375 Germline Ethanol 137 103 16 17.26 0.48 14 19 26 

Male (group) MQD2375 Germline Auxin 202 38 13 14.47 0.37 13 13 22 

Male (group) MQD2378 Hypodermis Ethanol 180 60 13 13.98 0.32 13 15 20 

Male (group) MQD2378 Hypodermis Auxin 202 38 13 13.98 0.30 13 15 19 

Male (group) MQD2453 Whole body Ethanol 200 36 15 16.97 1.09 15 17 22 

Male (group) MQD2453 Whole body Auxin 183 61 71 71.40 1.94 66 78 106 
 

Hermaphrodite 
(group) 

MQD2453 Whole body Ethanol 235 41 33 35.29 0.71 32 37 51 

Hermaphrodite 
(group) 

MQD2453 Whole body Auxin 223 106 46 46.96 1.27 42 51 72 
 

Male (single) MQD2453 Whole body Ethanol 70 25 23 26.23 1.43 21 26 45 

Male (single) MQD2453 Whole body Auxin 62 31 76 74.49 2.68 72 82 100 
 

Hermaphrodite 
(single) 

MQD2453 Whole body Ethanol 55 20 40 41.62 1.67 37 46 60 

Hermaphrodite 
(single) 

MQD2453 Whole body Auxin 44 31 39 46.89 3.28 34 60 79 

 
 
  



 

38 
 

 

Supplemental Table S2.2 Lifespan assay CPH model output 
Male (group) 

 CA1200 Cox Proportional Hazards 

Compound Effect Stderr z-value p-value 

Auxin 0.36 0.11 3.37 0.0008 
 

 MQD2356_neurons Cox Proportional Hazards 

Compound Effect Stderr z-value p-value 

Auxin -0.09 0.10 -0.87 0.38 

 

MQD2374_intestine Cox Proportional Hazards 

Compound Effect Stderr z-value p-value 

Auxin -1.20 0.16 -7.75 8.88E-15 

 

MQD2375_germline Cox Proportional Hazards 

Compound Effect Stderr z-value p-value 

Auxin 0.50 0.11 4.39 0.00001 

 

MQD2378_hypodermis Cox Proportional Hazards 

Compound Effect Stderr z-value p-value 

Auxin 0.02 0.10 0.18 0.86 
 

 MQD2453_wholebody Cox Proportional Hazards 

Compound Effect Stderr z-value p-value 

Auxin -4.00 0.29 -0.14 <2E-16 

Hermaphrodite (group) 

 MQD2453_wholebody Cox Proportional Hazards 

Compound Effect Stderr z-value p-value 

Auxin -0.91 0.11 -8.67 <2E-16 

Males and Hermaphrodites (single) interaction model 

 MQD2453_wholebody Cox Proportional Hazards 
 

Effect Stderr z-value p-value 

Auxin -0.58 0.22 -2.63 0.008 

Sex 1.02 0.19 5.49 4.07E-08 

Interaction -2.30 0.29 -8.03 9.38E-16 

 



 

39 
 

 

Supplemental Table S2.3 Mating assay summary statistics 
Strain Tissue 

Knockdown 
Age Compound Mean Mating 

Success 
SD Count SEM 

CA1200 NA 1 No auxin 0.75 0.44 51 0.06 

CA1200 NA 1 Auxin 0.71 0.46 52 0.06 

MQD2356 neuron 1 No auxin 0.76 0.43 58 0.06 

MQD2356 neuron 1 Auxin 0.74 0.44 57 0.06 

MQD2374 intestine 1 No auxin 0.89 0.32 54 0.04 

MQD2374 intestine 1 Auxin 0.89 0.31 55 0.04 

MQD2375 germline 1 No auxin 0.81 0.40 52 0.06 

MQD2375 germline 1 Auxin 0.87 0.34 52 0.05 

MQD2378 hypodermis 1 No auxin 0.84 0.37 51 0.05 

MQD2378 hypodermis 1 Auxin 0.92 0.27 53 0.04 

MQD2453 whole body 1 No auxin 0.76 0.43 51 0.06 

MQD2453 whole body 1 Auxin 0.58 0.50 59 0.06 

CA1200 NA 7 No auxin 0.04 0.19 52 0.03 

CA1200 NA 7 Auxin 0.10 0.30 51 0.04 

MQD2356 neuron 7 No auxin 0.06 0.24 49 0.03 

MQD2356 neuron 7 Auxin 0.10 0.30 51 0.04 

MQD2374 intestine 7 No auxin 0.06 0.24 52 0.03 

MQD2374 intestine 7 Auxin 0.19 0.40 52 0.06 

MQD2375 germline 7 No auxin 0.15 0.36 52 0.05 

MQD2375 germline 7 Auxin 0.25 0.43 53 0.06 

MQD2378 hypodermis 7 No auxin 0.14 0.35 51 0.05 

MQD2378 hypodermis 7 Auxin 0.10 0.30 51 0.04 

MQD2453 whole body 7 No auxin 0.29 0.46 52 0.06 

MQD2453 whole body 7 Auxin 0.35 0.48 52 0.07 

CA1200 NA 9 No auxin 0.04 0.19 56 0.03 

CA1200 NA 9 Auxin 0.06 0.24 52 0.03 

MQD2356 neuron 9 No auxin 0.06 0.24 52 0.03 

MQD2356 neuron 9 Auxin 0.08 0.27 52 0.04 

MQD2374 intestine 9 No auxin 0.00 0.00 51 0.00 

MQD2374 intestine 9 Auxin 0.06 0.24 51 0.03 

MQD2375 germline 9 No auxin 0.12 0.33 51 0.05 

MQD2375 germline 9 Auxin 0.06 0.24 51 0.03 

MQD2378 hypodermis 9 No auxin 0.08 0.27 52 0.04 

MQD2378 hypodermis 9 Auxin 0.00 0.00 49 0.00 

MQD2453 whole body 9 No auxin 0.04 0.21 46 0.03 

MQD2453 whole body 9 Auxin 0.30 0.46 56 0.06 



 

40 
 

 

Strain Tissue 
Knockdown 

Age Compound Mean Mating 
Success 

SD Count SEM 

MQD2453 whole body 12 No auxin 0.00 0.00 17 0.00 

MQD2453 whole body 12 Auxin 0.35 0.49 20 0.11 

MQD2453 whole body 15 No auxin 0.00 0.00 23 0.00 

MQD2453 whole body 15 Auxin 0.12 0.33 58 0.04 

MQD2453 whole body 18 No auxin 0.00 0.00 4 0.00 

MQD2453 whole body 18 Auxin 0.03 0.17 33 0.03 

MQD2453 whole body 21 No auxin 0.00 0.00 11 0.00 

MQD2453 whole body 21 Auxin 0.00 0.00 34 0.00 

Supplemental Table S2.3, continued 
  



 

41 
 

 

Supplemental Table S.2.4 Mating assay GLM model output 
 CA1200 Generalized Linear Model 

Treatment Effect Stderr z-value p-value 

Intercept 1.07 0.32 3.34 0.0008 

Auxin -0.17 0.44 -0.38 0.70 

Age 7 -4.29 0.79 -5.44 5.45E-08 

Age 9 -4.37 0.79 -5.54 3.02E-08 

Auxin: Age 7 1.17 0.97 1.21 0.23 

Auxin: Age 9 0.67 1.03 0.65 0.52 
 

 Germline Generalized Linear Model 

Treatment Effect Stderr z-value p-value 

Intercept 1.44 0.35 4.08 0.00005 

Auxin 0.43 0.54 0.79 0.43 

Age 7 -3.14 0.52 -6.03 1.69E-09 

Age 9 -3.45 0.56 -6.17 6.85E-10 

Auxin: Age 7 0.16 0.73 0.21 0.83 

Auxin: Age 9 -1.18 0.91 -1.30 0.19 
 

 Hypodermis Generalized Linear Model 

Treatment Effect Stderr z-value p-value 

Intercept 1.68 0.39 4.37 0.00001 

Auxin 0.82 0.65 1.27 0.20 

Age 7 -3.52 0.56 -6.28 3.31E-10 

Age 9 -4.17 0.65 -6.44 1.22E-10 

Auxin: Age 7 -1.20 0.90 -1.34 0.18 

Auxin: Age 9 -16.90 931.81 -0.02 0.99 
 

 Intestine Generalized Linear Model 

Treatment Effect Stderr z-value p-value 

Intercept 2.08 0.43 4.80 0.000001 

Auxin 0.02 0.61 0.03 0.97 

Age 7 -4.87 0.74 -6.62 3.51E-11 

Age 9 -20.65 913.35 -0.02 0.98 

Auxin: Age 7 1.34 0.92 1.45 0.15 

Auxin: Age 9 15.77 913.35 0.02 0.99 
 



 

42 
 

 

 Neuron Generalized Linear Model 

Treatment Effect Stderr z-value p-value 

Intercept 1.15 0.31 3.73 0.0001 

Auxin -0.12 0.43 -0.27 0.79 

Age 7 -3.88 0.67 -5.78 7.39E-09 

Age 9 -3.94 0.67 -5.89 3.99E-09 

Auxin: Age 7 0.63 0.87 0.72 0.47 

Auxin: Age 9 0.42 0.90 0.47 0.64 

 

Whole body Generalized Linear Model 

Treatment Effect Stderr z-value p-value 

Intercept 1.18 0.33 3.57 0.0004 

Auxin -0.87 0.42 -2.063 0.04 

Age 7 -2.08 0.45 -4.624 0.000004 

Age 9 -4.27 0.79 -5.372 7.78E-08 

Age 12 -19.74 1581.97 -0.012 0.99 

Age 15 -19.74 1360.06 -0.015 0.99 

Age 18 -19.74 3261.32 -0.006 0.99 

Age 21 -19.74 1966.65 -0.01 0.99 

Auxin: Age 7 1.14 0.60 1.905 0.057 

Auxin: Age 9 3.13 0.89 3.534 0.0004 

Auxin: Age 12 18.82 1581.97 0.012 0.99 

Auxin: Age 15 17.45 1360.06 0.013 0.99 

Auxin: Age 18 15.97 3261.32 0.005 1.00 

Auxin: Age 21 0.87 2262.53 0 1.00 

Supplemental Table S.2.4, continued 
 
  



 

43 
 

 

Supplemental Table S.2.5 Behavior assay summary statistics 

Variable Strain Tissue 
Knockdown 

Age Compound Mean SD Count SEM 

LOV MQD2453 Whole body 1 No auxin 27.92 16.37 44 2.44 

LOV MQD2453 Whole body 1 Auxin 41.33 34.43 47 4.77 

LOV MQD2453 Whole body 7 No auxin 39.67 26.79 51 3.72 

LOV MQD2453 Whole body 7 Auxin 0.80 0.41 45 0.06 

LOV MQD2453 Whole body 9 No auxin 0.68 0.36 52 0.05 

LOV MQD2453 Whole body 9 Auxin 0.64 0.41 52 0.06 

Tail incoordination MQD2453 Whole body 1 No auxin 0.27 0.39 44 0.06 

Tail incoordination MQD2453 Whole body 1 Auxin 0.25 0.40 47 0.06 

Tail incoordination MQD2453 Whole body 7 No auxin 0.29 0.38 51 0.05 

Tail incoordination MQD2453 Whole body 7 Auxin 0.27 0.39 45 0.05 

Tail incoordination MQD2453 Whole body 9 No auxin 0.00 0.00 52 0.00 

Tail incoordination MQD2453 Whole body 9 Auxin 0.00 0.00 52 0.00 

Time on vulva MQD2453 Whole body 1 No auxin 0.29 0.46 44 0.07 

Time on vulva MQD2453 Whole body 1 Auxin 0.29 0.46 47 0.06 

Time on vulva MQD2453 Whole body 7 No auxin 0.50 0.50 51 0.07 

Time on vulva MQD2453 Whole body 7 Auxin 0.44 0.50 45 0.07 

Time on vulva MQD2453 Whole body 9 No auxin 64.61 73.06 52 10.13 

Time on vulva MQD2453 Whole body 9 Auxin 59.81 56.06 52 7.77 

Turning success MQD2453 Whole body 1 No auxin 0.02 0.15 44 0.02 

Turning success MQD2453 Whole body 1 Auxin 60.00 93.10 47 13.44 

Turning success MQD2453 Whole body 7 No auxin 34.53 29.68 51 4.33 

Turning success MQD2453 Whole body 7 Auxin 35.17 33.62 45 4.66 

Turning success MQD2453 Whole body 9 No auxin 73.07 69.88 52 9.79 

Turning success MQD2453 Whole body 9 Auxin 17.00 4.47 52 0.62 

  



 

44 
 

 

Supplemental Table S.2.6 Behavior assay GLM output 

 Turning difficulty Generalized Linear Model 

Treatment Effect Stderr z-value p-value 

Intercept -0.98 0.48 -2.05 0.04 

Auxin -0.41 0.74 -0.55 0.58 

Age 7 0.11 0.72 0.15 0.88 

Age 9 -15.59 1199.77 -0.01 0.99 

Auxin: Age 7 1.15 1.05 1.10 0.27 

Auxin: Age 9 16.75 1199.77 0.01 0.99 

 

Tail incoordination Generalized Linear Model 

Treatment Effect Stderr z-value p-value 

Intercept -3.76 1.01 -3.72 0.0002 

Auxin -0.07 1.43 -0.05 0.96 

Age 7 2.58 1.06 2.43 0.015 

Age 9 3.45 1.05 3.29 0.001 

Auxin: Age 7 -1.39 1.58 -0.88 0.38 

Auxin: Age 9 -1.33 1.51 -0.88 0.38 

 

LOV Generalized Linear Model 

Treatment Effect Stderr z-value p-value 

Intercept -0.66 0.29 -2.29 0.02 

Auxin -0.49 0.41 -1.21 0.23 

Age 7 0.05 0.44 0.10 0.92 

Age 9 -0.54 0.72 -0.75 0.45 

Auxin: Age 7 0.63 0.63 1.00 0.32 

Auxin: Age 9 1.09 0.93 1.18 0.24 

 

Time on vulva Generalized Linear Model 

Treatment Effect Stderr z-value p-value 

Intercept 4.26 0.03 151.60 <2.00E-16 

Auxin -0.71 0.05 -14.30 <2.00E-16 

Age 7 0.04 0.04 0.85 0.39 

Age 9 -0.53 0.09 -5.68 1.39E-08 

Auxin: Age 7 -0.25 0.08 -3.15 0.002 

Auxin: Age 9 0.67 0.12 5.54 3.08E-08 

 



 

45 
 

 

Supplemental Table S.2.7 Strains used in this study 

Strain Name Genotype Tissue knockdown 

MQD2374 ieSi61 II; daf-2(hq363[daf-2::degron::mNeonGreen]) 
unc-119(ed3) III. 

Intestine 

MQD2378 hqSi9 II; daf-2(hq363[daf-2::degron::mNeonGreen]) 
unc-119(ed3) III. 

Hypodermis 

MQD2356 hqSi8 II; daf-2(hq363[daf-2::degron::mNeonGreen]) 
unc-119(ed3) III. 

Neurons 

MQD2453 ieSi57 II; daf-2(hq363[daf-2::degron::mNeonGreen]) 
unc-119(ed3) III. 

Whole body 

MQD2375 daf-2(hq363[daf-2::degron::mNeonGreen]) unc-
119(ed3) III; ieSi38 IV. 

Germline 

JK574 fog-2(q71) V. NA 

CA1200 ieSi57 II; unc-119(ed3) III. NA 

CB169 unc-31(e169) IV. NA 

 
Bridge to chapter 3 

In chapter 2, I explored the sex differences in the response to a genetic pro-longevity 

intervention, targeted DAF-2 degradation, on lifespan. Additionally, I explored whether the 

resulting beneficial effects on lifespan in males can be extended to reproductive healthspan or 

whether the two metrics are decoupled. In chapter 3, I extend this framework to test the effects of 

a second type of intervention, chemical interventions, and whether such interventions result in a 

sex-specific benefit on lifespan and healthspan.  



 

46 
 

 

CHAPTER 3: PRO-LONGEVITY COMPOUNDS EXTEND CAENORHABDITIS 

ELEGANS MALE LIFESPAN AND REPRODUCTIVE HEALTHSPAN 

(In review at GeroScience) 

Rose S. Al-Saadi, Patrick C. Phillips 

Author contributions 

RSA and PCP conceptualized the project. RSA conducted the lifespan and mating success 

assays, conducted the statistical analysis and visualization, and wrote the original manuscript. 

PCP supervised the project, reviewed and edited the manuscript, and acquired funding. 

Abstract 

Sex differences in aging are robust and ubiquitous. Demographic differences in aging generated 

by sex have long been recognized, but the underlying biological basis for these differences and 

the potential for sex-specific interventions remain understudied. To explore sex differences in the 

response to pro-longevity interventions, we utilized the C. elegans aging model and asked 

whether male lifespan and reproductive healthspan can be extended via compounds known to 

have pro-longevity effects in hermaphrodites. We tested seven different compounds at two 

concentrations each and found that lifespan was extended under all tested conditions. However, 

reproductive healthspan measured by mating success in late life improved under only two tested 

conditions, sulforaphane and metformin. These results demonstrate that lifespan and healthspan 

can be decoupled in C. elegans males and offer a new framework for screening pro-longevity 

compounds and for studying sex differences in aging in a classical aging model. 

Introduction 

Sex differences in aging are robust and ubiquitous (Austad and Fischer 2016). These differences 

are observed in both lifespan (the chronological time between birth and death) and healthspan 

(the proportion of lifespan that is disease-free). In humans, women live longer but tend to suffer 

worse health outcomes compared to men (Oksuzyan et al. 2008; Gordon et al. 2017). Several 

age-associated conditions such as chronic conditions (Bello-Lujan et al. 2022), bone mineral 



 

47 
 

 

density loss (Daly et al. 2013), inflammation (Milan-Mattos et al. 2019), and frailty (Gordon et 

al. 2017) exhibit sex differences in incidence and severity (Hägg and Jylhävä 2021). This sexual 

dimorphism extends to modulators of aging such as pro-longevity interventions, with many 

tested regimes (genetic, chemical, or environmental) showing sex differences in efficacy (Bartke 

et al. 2024). However, our knowledge of the molecular basis of these differences is limited due 

to a historic focus on male health and disease (Holdcroft 2007). 

Caenorhabditis elegans have a short lifespan (2–3 weeks), have conserved genetic pathways that 

modulate aging (Zhang et al. 2020), have genetic diversity comparable to that of human 

populations (Andersen and Rockman 2022; Teterina et al. 2022), and have longevity that is 

amenable to both genetic and chemical interventions (Zhang et al. 2020), making them a pivotal 

model system for understanding the biology of aging. C. elegans is also sexually dimorphic, with 

self-fertile hermaphrodites and rare males (Anderson et al. 2010; Emmons 2014). While a few 

recent studies have included sex differences in C. elegans aging (McCulloch and Gems 2007; 

Liggett et al. 2015; Honjoh et al. 2017; Hotzi et al. 2018; Weng and Murphy 2024), the vast 

majority of aging research using C. elegans focuses solely upon hermaphrodites. This is largely 

due to the fact that males are rare in the commonly used lab strain N2 (0.1%), as well as being 

shown to have detrimental effects on hermaphrodite lifespan (Maures et al. 2014; Shi and 

Murphy 2014; Booth et al. 2022). Overall, our understanding of sex differences in aging 

processes in C. elegans remains extremely limited. 

The Caenorhabditis Intervention Testing Program (CITP) is a National Institutes of Aging 

supported collaborative effort across three laboratories in the USA that leverages the benefits of 

Caenorhabditis nematodes as an aging model to test the effects of pharmacological interventions 

on the lifespan and healthspan of different species in the Caenorhabditis genus in a rigorous and 

robust fashion. The program has tested dozens of compounds and has found that many do indeed 

extend hermaphrodite lifespan (Driscoll et al. 2025). However, it remains unknown whether 

these compounds are similarly efficacious in males. This seems especially pertinent because the 

CITP’s long-running sister program using mice, the ITP, has found that males tend to be more 

responsive to compound interventions than females (Strong et al. 2008; Harrison et al. 2014; 

Bartke et al. 2024). 



 

48 
 

 

Male C. elegans represent an exciting tool to test pharmacological interventions for two main 

reasons. First, males provide a complex health measure that integrates the health of multiple 

tissues and structures into one output: reproductive success. Male motivation for mating remains 

high throughout life and failure to sire progeny is caused by physical deterioration rather than a 

decrease in motivation per se (Guo et al. 2012; Chatterjee et al. 2013). This is in contrast to 

hermaphrodites, which appear to change reproductive patterns due a variety of environmental 

inputs, including the presence of males (Aprison and Ruvinsky 2016). Males need to locate a 

hermaphrodite via chemotaxis, scan their mate’s body to locate the vulva via mechanosensory 

neurons, insert the spicule, and ejaculate, thereby transferring sperm to the hermaphrodite uterus 

(Barr and Garcia 2006). If any of these processes exhibit age-associated defects, reproductive 

success will decrease. The second reason males are an excellent tool for screening 

pharmacological compounds is that males do not produce self-progeny, eliminating the need to 

abrogate offspring production, for instance using the chemotherapy agent 5-Fluoro-2'-

deoxyuridine (FUdR), which blocks cell division by inhibiting DNA synthesis (Mitchell et al. 

1979; Gandhi et al. 1980). In hermaphrodite lifespan experiments, FUdR is commonly used to 

sterilize hermaphrodites and thereby eliminate the need for daily transfers during the first 5–6 

days of adulthood (which is very labor and resource intensive). However, FUdR is known to 

affect hermaphrodites directly, as well as affecting the bacteria upon which they feed (Wang et 

al. 2019; McIntyre et al. 2021). Male lifespan experiments therefore offer the same ease as 

FUdR-supplemented hermaphrodites but without confounding effects of FUdR per se. 

Here, we tested the potential pro-longevity effects of several CITP-validated compounds on male 

lifespan and healthspan, finding that all tested compounds increase male lifespan, even at lower 

concentrations than hermaphrodites. However, only two compounds reliably improved male 

healthspan, as measured via mating success in late life. The work here describes a new 

framework for pharmacological intervention screening using male C. elegans in addition to 

hermaphrodites to expand our understanding of sex differences in aging and to enable us to 

create interventions that are effective for both sexes. 



 

49 
 

 

Materials and methods 

C. elegans strains and maintenance 

All the strains used in this study were provided by the Caenorhabditis Genetics Center (CGC), 

which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). The 

strains used were CB1489 (him-8(e1489) IV) and JK574 (fog-2(q71) V). The strains were 

maintained on 60 mm plates of standard NGM media seeded with E. coli OP50-1. Worms were 

transferred to fresh plates 2–3 times weekly. Unless otherwise specified, all the stocks and assays 

were maintained at 20ºC. 

Compound treatment 

We used the following equation to determine the working stock concentration (X), where the 

treatment volume is 450 µL for water-soluble compounds and 25 µL for DMSO-soluble 

compounds and the plate volume is 10 mL for both types: 

𝑇𝑟𝑒𝑎𝑡𝑚𝑒𝑛𝑡 𝑣𝑜𝑙𝑢𝑚𝑒  ∗  𝑋  =  𝑓𝑖𝑛𝑎𝑙 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛  ∗  𝑝𝑙𝑎𝑡𝑒 𝑣𝑜𝑙𝑢𝑚𝑒	 

For water-soluble compounds (gold sodium thiomalate, metformin, thioflavin T), 450 µL of the 

working stock was added to 60 mM NGM plates seeded with OP50-1 and allowed to dry at room 

temperature for approximately 3 days. The working stock concentrations for metformin were 

0.78 M and 1.56 M for final concentrations of 35 mM and 70 mM, respectively. The working 

stock concentrations for gold sodium thiomalate were 1.11 mM and 2.22 mM for final 

concentrations of 50 µM and 100 µM, respectively. The working stock concentrations for 

thioflavin T were 0.56 mM and 1.11 mM for final concentrations of 25 µM and 50 µM, 

respectively. For DMSO-soluble compounds (all trans retinoic acid, propyl gallate, resveratrol, 

sulforaphane), 25 µL of the working stock mixed with 425 µL of water was added to 60 mM 

NGM plates seeded with OP50-1 and allowed to dry at room temperature for approximately 3 

days. The working stock concentrations for all trans retinoic acid for were 30 mM and 60 mM 

for final concentrations of 75 µM and 150 µM, respectively. The working stock concentrations 

for propyl gallate were 40 mM and 80 mM for final concentrations of 100 µM and