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RESEARCH PAPER
Exploring Lifespan Modulation in C. elegans Through RNAi-Mediated DAF-2 Knockdown
Across Variable Temperature Conditions

Vashnavi Vashist
Department of STEM, Capilano University
SCI 400- Research Project
Mark Vaughan, Eunice Chin, Mahta Khosravi
December 9th, 2024

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ABSTRACT
One important factor influencing lifespan and aging in Caenorhabditis elegans (C. elegans)
is the insulin/IGF-1 signaling (IIS) pathway, which is controlled by the daf-2 gene. In C. elegans,
environmental factors like temperature are known to significantly alter lifespan and stress
responses, while RNA interference (RNAi) provides a reliable technique for examining gene
function. With an emphasis on comprehending the interplay between genetic and environmental
factors in aging, this study investigates the effects of RNAi-mediated knockdown of daf-2 on
lifespan across four different temperature conditions (4°C, 10°C, 20°C, and 30°C). Synchronized
populations of C. elegans were exposed to daf-2 RNAi and maintained under controlled
temperature conditions to assess their lifespan. Although the study is ongoing and results have
not yet been collected, it is hypothesized that daf-2 knockdown will result in reduced lifespan
relative to controls, with the most pronounced effects occurring at temperature extremes (4°C
and 30°C). This research seeks to elucidate the complex interplay between IIS signaling and
environmental stressors in lifespan regulation, contributing to the broader understanding of
genetic and environmental influences on aging and resilience mechanisms.

1. INTRODUCTION
1.1 Environmental Influence on Aging
Aging, a universal biological process, is marked by a gradual decline in physiological and
cellular functions, leading to increased disease susceptibility and mortality (López-Otín et al.,
2013; Kirkwood & Austad, 2000). Among environmental factors influencing aging, temperature
plays a crucial role, particularly in ectothermic organisms such as Caenorhabditis elegans (C.
elegans). Lower temperatures have been shown to enhance lifespan by suppressing metabolic

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rates and activating stress resistance pathways, including proteostasis and autophagy (Honda &
Honda, 1999; Lee et al., 2014). Conversely, higher temperatures accelerate aging by increasing
metabolic demands, oxidative damage, and proteostatic challenges (Rikke & Johnson, 2004;
Toth et al., 2008). As shown in Figure 1, lower temperatures promote extended lifespans,
optimal temperatures support moderate lifespans, and higher temperatures significantly reduce
lifespan (Honda & Honda, 1999; Rikke & Johnson, 2004; Toth et al., 2008). This intricate
relationship highlights the importance of environmental stressors in shaping aging dynamics and
highlights the potential to uncover fundamental mechanisms of longevity through the study of C.
elegans (López-Otín et al., 2013; Kenyon, 2010).

Figure 1: Relationship between temperature and lifespan in C. elegans. Cold temperatures
extend lifespan by reducing metabolic activity and oxidative stress, optimal temperatures
maintain a moderate lifespan, and high temperatures shorten lifespan due to increased metabolic

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demands and oxidative damage. This schematic illustrates the role of temperature as a key
environmental factor influencing aging.
1.2 C. elegans as a Model Organism in Aging Studies
C. elegans is an exceptional model organism for aging research due to its wellcharacterized lifecycle, genetic simplicity, and conservation of critical aging-related pathways
such as the insulin/IGF-1 signaling (IIS) pathway (Kenyon, 2010; López-Otín et al., 2013). The
lifecycle of C. elegans, as shown in Figure 2, progresses through distinct stages: embryogenesis,
four larval stages (L1–L4), and adulthood. At 20°C, the lifecycle from egg to reproductive adult
spans approximately 3 days, and the entire lifespan is about 3 weeks (Brenner, 1974).
Embryogenesis occurs within 14 hours, followed by hatching into the L1 larval stage. Worms
pass through L2, L3, and L4 stages, undergoing molting at each transition (Brenner, 1974). The
adult stage is marked by reproduction and eventual aging, with visible changes such as decreased
motility, accumulation of lipofuscin, and increased susceptibility to environmental stressors
(Gruber et al., 2007; Herndon et al., 2002). Synchronization techniques, such as bleaching,
ensure uniform developmental stages, reducing variability in experiments and enhancing the
reliability of lifespan studies (Lewis & Fleming, 1995).

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Figure 2: Lifecycle of Caenorhabditis elegans (C. elegans) at 20°C. The lifecycle progresses
through embryogenesis, four larval stages (L1–L4), and adulthood, with the entire development
occurring over approximately three weeks under optimal laboratory conditions. Under
environmental stressors such as crowding, starvation, or high temperature, the L1 larvae may
enter an alternative dauer stage, which is a dormant and stress-resistant state. Dauer refers to a
specialized larval form adapted for survival during unfavorable conditions. The adaptability of C.
elegans, coupled with its rapid lifecycle, makes it an ideal model organism for studying
development, genetics, and aging. Patrycja Vasilyev Missiuro. (2010, August 26). Predicting
Genetic Interactions in Caenorhabditis elegans using Machine Learning.
https://www.researchgate.net/publication/266342950_Predicting_Genetic_Interactions_in_Caeno
rhabditis_elegans_using_Machine_Learning

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When compared to other model organisms such as Drosophila melanogaster (fruit flies) and
mammals, C. elegans stands out for its simplicity and experimental flexibility (Kenyon, 2010;
Mitchell et al., 2015). Mammalian models (Figure 3a) provide a closer physiological and
genomic resemblance to humans and are invaluable for translational research, particularly in the
study of complex tissue interactions and aging-related diseases. However, the long lifespan of
mammals (~2–3 years in mice) and ethical considerations often make them less practical for
high-throughput or exploratory research (Mitchell et al., 2015).

Figure 3: Comparative insulin/IGF-1 signaling (IIS) pathways in mammals, Drosophila
melanogaster (fruit fly), and Caenorhabditis elegans (C. elegans). (A) In mammals, the IIS
pathway begins with insulin binding to the insulin receptor (hINR), leading to the activation of
phosphatidylinositol-3-kinase (PI3K) and AKT kinase. PTEN (phosphatase and tensin homolog)
acts as a negative regulator, while FOXO transcription factors control stress resistance and
longevity-related genes. (B) In Drosophila, the pathway is similarly regulated by the insulin-like

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receptor (dInR), with DP110 acting as the PI3K homolog, dPTEN as the negative regulator, and
dFOXO as the transcription factor. (C) In C. elegans, the IIS pathway is centered around DAF-2,
an insulin-like receptor, which regulates downstream components, including AGE-1 (a PI3K
homolog), DAF-18 (a PTEN homolog), and AKT kinases. DAF-16, the C. elegans FOXO
homolog, is a critical transcription factor promoting stress resistance, metabolism, and longevity
when translocated to the nucleus. Abbreviations: PI3K, phosphatidylinositol-3-kinase; PTEN,
phosphatase and tensin homolog; AKT, protein kinase B; FOXO, forkhead box O; dInR,
Drosophila insulin receptor; DP110, PI3K homolog in Drosophila; DAF-2, insulin-like receptor
in C. elegans; AGE-1, PI3K homolog in C. elegans; DAF-18, PTEN homolog in C. elegans.
Biglou, S. G., Bendena, W. G., & Chin-Sang, I. (2021). An overview of the insulin signaling
pathway in model organisms Drosophila melanogaster and Caenorhabditis
elegans. Peptides, 145, 170640. https://doi.org/10.1016/j.peptides.2021.170640

Drosophila melanogaster (Figure 3b), another widely used model organism, shares many
advantages with C. elegans, such as genetic tractability and conservation of pathways like IIS.
However, Drosophila has a longer lifespan (~2 months) and a higher level of physiological
complexity, including a structured brain and more intricate metabolic networks. This makes
Drosophila particularly useful for studying tissue-specific aging and neurodegeneration but less
efficient for rapid genetic and environmental studies (Tower, 2000).
C. elegans (Figure 3c) provides a unique blend of simplicity and relevance. The IIS pathway in
C. elegans—involving DAF-2 (insulin-like receptor) and DAF-16 (FOXO transcription factor)—
is highly conserved across species. This pathway integrates environmental signals such as

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temperature, nutrient availability, and oxidative stress to modulate stress resistance, metabolism,
and longevity (Kenyon, 2010; Uno & Nishida, 2016).
1.3 The IIS Pathway: Central to Aging in C. elegans.
The insulin/IGF-1 signaling (IIS) pathway is a highly conserved regulator of aging,
metabolism, and stress resistance across species, including C. elegans and mammals (Kenyon,
2010; Uno & Nishida, 2016). In C. elegans, the pathway is centered around the daf-2 gene,
which encodes an insulin-like receptor homologous to the mammalian IGF-1 receptor. (Kenyon,
2010; Uno & Nishida, 2016) Activation of DAF-2 initiates a signaling cascade that begins with
AGE-1, a phosphatidylinositol-3-kinase, which produces phosphatidylinositol-3,4,5-triphosphate
(PIP3). This molecule activates AKT-1 and AKT-2, serine/threonine kinases that phosphorylate
and inactivate the FOXO transcription factor DAF-16, effectively sequestering it in the
cytoplasm (Figure 4a) (Biglou et al., 2021; Dillin et al., 2002). In this active IIS state, stress
resistance and longevity-related genes are repressed, and cellular energy is directed toward
growth and reproduction. (Kenyon, 2010; Dillin et al., 2002; Uno & Nishida, 2016)

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FIGURE 4: The insulin/IGF-1 signaling (IIS) pathway and its downstream elements in C.
elegans. In the active state(4a), DAF-2, an insulin-like receptor, binds to insulin-like peptides,
triggering a signaling cascade through AGE-1, a phosphoinositide 3-kinase, and AKT-1/2,
protein kinases that phosphorylate DAF-16, a Forkhead box O (FOXO) transcription factor.
Phosphorylation prevents DAF-16 from entering the nucleus, thereby inhibiting the expression of
genes linked to stress resistance and longevity. Conversely, in the inactive state (4b) of DAF-2
(knockdown), the absence of signaling allows DAF-16 to translocate into the nucleus, where it
activates genes promoting stress resilience, metabolic adaptation, and increased longevity. This
figure highlights the intricate relationship between the IIS pathway components, including DAF2, AGE-1, AKT-1/2, and DAF-16, in regulating lifespan through environmental and genetic
inputs.
When IIS is reduced, for instance, through daf-2 knockdown, the signaling cascade is disrupted
(Kenyon, 2010; Uno & Nishida, 2016). AGE-1 fails to produce sufficient PIP3, resulting in the

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inactivation of AKT-1 and AKT-2 (Biglou et al., 2021; Dillin et al., 2002). This
dephosphorylation enables DAF-16 to translocate to the nucleus (Figure 4b), where it promotes
the expression of genes associated with stress resilience, detoxification, and longevity (Kenyon,
2010; Uno & Nishida, 2016). These include sod-3 (superoxide dismutase), ctl-1 and ctl-2
(catalases), and hsp-16.2 (heat shock protein), which are vital for managing oxidative stress and
protein misfolding (Kenyon, 2010; Dillin et al., 2002; Uno & Nishida, 2016). Furthermore, lipl-4
(involved in lipid metabolism) and lys-7 (antimicrobial defense) are also upregulated, enhancing
cellular resilience and metabolic stability (Biglou et al., 2021; Uno & Nishida, 2016).
This pathway integrates external environmental signals, such as temperature, with intrinsic
genetic processes to regulate aging and stress responses (Kenyon, 2010; Uno & Nishida, 2016).
At lower IIS levels, as induced by daf-2 knockdown, the focus of cellular resources shifts from
reproduction and growth to maintenance and stress adaptation, thereby extending lifespan
(Biglou et al., 2021; Rikke & Johnson, 2004). This regulatory balance allows C. elegans to adapt
to environmental changes, exemplifying the interplay between genetic and environmental inputs
in lifespan determination (Gems & Partridge, 2013; Uno & Nishida, 2016). The IIS pathway's
evolutionary conservation underscores its relevance across species, providing a robust model to
investigate aging mechanisms and stress resilience (Kenyon, 2010; Gems & Partridge, 2013).
Figures 4a and 4b illustrate the molecular changes in IIS signaling, highlighting the contrast
between DAF-2 active and inactive states (Biglou et al., 2021).
1.4 RNA Interference (RNAi) and IPTG Mechanism
RNA interference (RNAi) is a powerful technique that allows precise silencing of specific
genes, such as daf-2, in C. elegans (Fire et al., 1998; Grishok et al., 2001). This study utilized

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RNAi to target daf-2, a critical gene in the insulin/IGF-1 signaling (IIS) pathway (Kenyon, 2010;
Uno & Nishida, 2016). The RNAi process begins with the ingestion of genetically engineered
Escherichia coli strains producing double-stranded RNA (dsRNA) complementary to the target
gene (Timmons & Fire, 1998). Once ingested, the dsRNA is processed by the enzyme Dicer,
which cleaves it into small interfering RNAs (siRNAs) (Grishok et al., 2001). These siRNAs are
incorporated into the RNA-induced silencing complex (RISC), which guides the degradation of
complementary mRNA, effectively silencing the target gene (Fire et al., 1998; Grishok, 2005).
This pathway, illustrated in Figure 5, highlights the sequential steps from dsRNA processing to
mRNA degradation, enabling effective gene silencing.

Figure 5: RNA Interference (RNAi) Mechanism. This diagram illustrates the RNAi process,
where double-stranded RNA (dsRNA) is processed by Dicer into small interfering RNA (siRNA)
molecules. These siRNAs are incorporated into the RNA-induced silencing complex (RISC),
guiding it to complementary messenger RNA (mRNA) for targeted degradation. The process

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effectively silences gene expression by preventing translation of the mRNA into protein. This
mechanism is widely used in gene knockdown experiments, such as silencing the daf-2 gene in
Caenorhabditis elegans. Waseem, Q. ul A. (2022, February 28). RNA Interference or RNAi.
Microbiology Notes. https://microbiologynotes.org/rna-interference-or-rnai/

A critical component of the RNAi protocol is the induction of dsRNA production in the bacterial
host using IPTG (isopropyl-β-D-thiogalactopyranoside) (Sambrook et al., 1989; Grishok, 2005).
IPTG is a synthetic analog of allolactose, a natural inducer of the lac operon in bacteria (Jacob &
Monod, 1961). Structurally, IPTG resembles lactose and its derivative allolactose but possesses
unique properties that make it especially effective for experimental applications (Malakar et al.,
2007). As shown in Figure 6, lactose is a disaccharide of galactose and glucose, which upon
hydrolysis or cellular metabolism, forms allolactose (Beckwith, 1987). Allolactose serves as the
natural inducer of the lac operon by binding to and inactivating the lac repressor protein, thereby
facilitating transcription of downstream genes. IPTG mimics this function, binding to the lac
repressor and releasing it from the operator region of the lac operon (Sambrook et al., 1989).

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Figure 6 Structural Comparison of Lactose, Allolactose, and IPTG: This figure depicts the
chemical structures of lactose, allolactose, and isopropyl-β-D-thiogalactopyranoside (IPTG),
highlighting their roles in gene regulation. Lactose is a natural disaccharide consisting of
galactose and glucose linked by a β-1,4 glycosidic bond. It serves as both a substrate for
metabolism and a precursor to allolactose, which is a β-1,6 isomer of lactose. Allolactose acts as
the natural inducer of the lac operon by binding to and deactivating the lac repressor protein,
thereby facilitating gene transcription. IPTG is a synthetic analog of allolactose designed to
mimic its function. Due to its structural similarity, IPTG effectively binds to the lac repressor but
is resistant to metabolic degradation, providing a stable and consistent method for operon

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induction in controlled experimental settings, such as RNA interference (RNAi) studies. Lactose
Operon - an overview | ScienceDirect Topics. (n.d.). Www.sciencedirect.com.
https://www.sciencedirect.com/topics/medicine-and-dentistry/lactose-operon
However, unlike lactose or allolactose, IPTG is non-metabolizable, which means it is not
degraded or consumed during bacterial growth (Grabski et al., 2019). This ensures sustained
induction of the operon and continuous transcription of the dsRNA-producing gene (Sambrook
& Russell, 2001). This property makes IPTG an ideal inducer for RNAi experiments, providing
consistent and long-term gene expression (Timmons & Fire, 1998). The molecular interaction
between IPTG and the lac operon, depicted in Figure 7, demonstrates how IPTG inactivates the
repressor, enabling RNA polymerase to initiate transcription of the desired gene (Chen et al.,
2015).

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Figure 7: Mechanism of Lac Operon Regulation. This figure illustrates the regulation of the
lac operon in bacteria, highlighting the role of the lac repressor and inducer molecules. In the
absence of an inducer (top), the lac repressor binds to the operator sequence, physically blocking
RNA polymerase from transcribing the downstream genes (lacZ, lacY, and lacA) required for
lactose metabolism. When an inducer such as allolactose or its analog IPTG (isopropyl-β-Dthiogalactopyranoside) is present (bottom), it binds to the repressor, causing a conformational
change that releases the repressor from the operator. This allows RNA polymerase to bind to the
promoter region and initiate transcription of the lac operon, facilitating gene expression. IPTG is
particularly useful in experimental setups due to its stability and resistance to degradation,

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ensuring consistent induction of target genes. Khan Academy. (2021). The lac operon . Khan
Academy. https://www.khanacademy.org/science/ap-biology/gene-expression-andregulation/regulation-of-gene-expression-and-cell-specialization/a/the-lac-operon

The molecular interaction between IPTG and the lac operon, depicted in Figure 7, demonstrates
how IPTG inactivates the repressor, enabling RNA polymerase to initiate transcription of the
desired gene (Chen et al., 2015). RNAi-induced gene silencing using IPTG-inducible bacterial
strains allows for precise genetic modulation, creating a controlled environment to study the
effects of daf-2 knockdown on lifespan (Timmons & Fire, 1998). However, without sterilization,
the presence of progeny in experimental populations could introduce significant variability,
confounding the analysis of lifespan and stress response (Grishok et al., 2000).
1.5 Experimental Consistency with FUdR
To ensure experimental consistency in aging studies, 5-fluoro-2’-deoxyuridine (FUdR)
plays a crucial role by sterilizing adult C. elegans and preventing progeny from complicating
lifespan observations (Mitchell et al., 1979). FUdR, a thymidine analog, incorporates into DNA
and inhibits replication during cell division, effectively halting reproduction (Gandhi et al.,
1980). This ensures that population synchronization is maintained, enabling accurate assessment
of aging phenotypes (Hosono et al., 1982). Importantly, FUdR is applied after worms reach the
L4 larval stage to avoid interference with developmental processes (Anderson et al., 2009). This
approach facilitates the study of genetic and environmental interactions, such as the role of
RNAi-induced daf-2 knockdown and temperature variations, in regulating lifespan without
confounding effects from progeny (Aitlhadj & Stürzenbaum, 2010). By leveraging FUdR, the

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study achieves a controlled experimental environment, critical for elucidating the molecular
mechanisms underlying aging (Figure 8).

Figure 8: Chemical Structure of 5-Fluoro-2′-Deoxyuridine (FUdR). This molecule, a
thymidine analog, is used in Caenorhabditis elegans experiments to inhibit DNA synthesis,
thereby sterilizing the worms. By preventing reproduction, FUdR ensures synchronized
populations and eliminates the confounding effects of progeny, allowing precise investigation of
genetic and environmental influences on aging. Floxuridine. (2023, January 29). Wikipedia.
https://en.wikipedia.org/wiki/Floxuridine
1.6 Integration of Genetics and Environment in Aging Research
This study focuses on understanding the dynamic interplay between genetic and environmental
factors influencing aging, specifically through RNAi-mediated daf-2 knockdown and exposure to
variable temperatures. Aging research has extensively studied genetic pathways and
environmental stressors independently, but a significant gap remains in how these factors interact
to modulate lifespan (Rikke & Johnson, 2004; Gems & Partridge, 2013). It is anticipated that
RNAi-mediated knockdown of daf-2, combined with variable temperature exposures, will extend

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the lifespan of Caenorhabditis elegans (C. elegans) by enhancing stress resilience and activating
protective genetic pathways. Specifically, lower temperatures are expected to further augment
the longevity effects of daf-2 knockdown by reducing metabolic demands and oxidative stress,
while higher temperatures may limit these benefits due to increased metabolic strain.
This research bridges the gap in aging studies by addressing how IIS suppression interacts with
environmental temperature variations to regulate lifespan. By leveraging the advantages of C.
elegans as a model organism and combining genetic and environmental interventions, this study
aims to contribute valuable insights to the broader field of aging research with potential
translational applications for human health.
2. MATERIALS AND METHODS
2.1 Study design
The experiment was designed to investigate the interaction between daf-2 knockdown
and temperature variations on the lifespan of C. elegans. Eight experimental groups were
established (Table 1), combining two factors: RNAi treatment (knockdown vs. no knockdown)
and temperature exposure (4°C, 10°C, 20°C, and 30°C). Each group consisted of synchronized
populations of worms treated and maintained under controlled conditions as shown in Figure 8.
The independent variables were the RNAi treatment (knockdown of daf-2) and the
environmental temperatures, while the dependent variable was the lifespan of the worms,
measured in days.

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Table 1: Experimental design outlining the independent and dependent variables for the study.
The independent variables include temperature (4°C, 10°C, 20°C, and 30°C) and RNA
interference (RNAi) treatment targeting the daf-2 gene. The dependent variable is the lifespan of
Caenorhabditis elegans, measured in days. Eight experimental groups were established: (1)
RNAi treatment + 4°C, (2) RNAi treatment + 10°C, (3) RNAi treatment + 20°C, (4) RNAi
treatment + 30°C, (5) No RNAi treatment + 4°C, (6) No RNAi treatment + 10°C, (7) No RNAi
treatment + 20°C, and (8) No RNAi treatment + 30°C. RNAi refers to the gene silencing
technique used to suppress the daf-2 gene. Lifespan is recorded for all groups to assess the
interaction between genetic and environmental factors.

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Figure 9: Overview of the experimental workflow and group allocation for assessing the effects
of temperature and daf-2 RNAi knockdown on C. elegans lifespan. Synchronized worms were
cultured on Agar plates prepared with or without Isopropyl β-D-1-thiogalactopyranoside (IPTG).
Worms were fed Escherichia coli expressing daf-2 dsRNA for RNAi induction or control E. coli
(non-RNAi treatment). On Day 0, synchronized worms were plated, and Day 3 worms were
exposed to RNAi-induced daf-2 knockdown. Experimental groups (1–8) were then subjected to
four temperature conditions (4°C, 10°C, 20°C, and 30°C) with or without daf-2 RNAi treatment.
Day 5 5-fluoro-2′-deoxyuridine (FUdR) was added to inhibit reproduction and maintain
synchronized populations during lifespan monitoring. This workflow illustrates the integration of
genetic and environmental factors in the experimental design.
2.2 Establishing a C. elegans Colony and maintenance.

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Wild-type C. elegans (N2 strain) were cultured on nematode growth medium (NGM)
plates seeded with Escherichia coli OP50, using the C. elegans Culture Kit (Carolina Biological
Supply, Catalog No. 173525). (“Caenorhabditis Elegans Culture Kit,” 2023) The kit provided
essential materials, ensuring a standardized and contamination-free environment for maintaining
healthy populations. Worms were kept at a standard laboratory temperature of 20°C, ideal for
their growth and reproduction.
To prepare NGM plates, 3 g Beef Extract, 15 g agar, and 5 g peptone were dissolved in 1L
distilled water, autoclaved, and allowed to cool. (Leboffe & Pierce, 2012) The medium (30ml)
was poured into sterile Petri dishes and allowed to solidify at room temperature. Plates were
dried for 24 hours at room temperature to remove excess moisture before seeding. (Stiernagle,
2019)
For the E. coli OP50 stock culture, nutrient broth was prepared by dissolving 3 g beef extract and
5 g peptone in 1 L distilled water. (Leboffe & Pierce, 2012) The medium was poured in multiple
tubes and autoclaved to ensure sterility, whenever needed tubes were inoculated with E. coli
OP50, and incubated overnight at 37°C to establish a robust bacterial stock. The bacterial culture
was then centrifuged to concentrate cells, and the resulting suspension was used to seed the
NGM plates. A small aliquot of the bacterial culture was pipetted onto the center of each plate,
spread evenly, and incubated at room temperature for 24 hours to establish a uniform lawn.
C. elegans were transferred to fresh plates by aseptically cutting a chunk of agar containing
actively growing worms and placing it onto the new seeded plate. This method minimized
handling stress and ensured the transfer of all life stages. Worms were maintained at 20°C and
transferred to fresh plates every 3–4 days to prevent overcrowding and starvation. Contamination

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was monitored daily, and compromised plates were promptly discarded. By combining the
preparation of nutrient broth, bacterial stock, and NGM plates, this approach ensured a consistent
and contamination-free environment for maintaining C. elegans populations under experimental
conditions.
2.3 Age Synchronization Protocol
To achieve uniform developmental stages for experimental worms, synchronization was
conducted at the L4 larval stage, a critical step to ensure consistent and reliable experimental
outcomes. (Figure 2) The process began by dislodging worms from NGM plates using M9
buffer, a solution prepared according to the recipe in Table 2 and autoclaved before use (Table 3Step 1). The resulting suspension was transferred to sterile centrifuge tubes (Table 3-Step 2),
centrifuged to separate the worms from the buffer (Table3-Step 3), and the supernatant was
removed (Table3-Step 4).

Table 2: Recipe for M9 Solution. This table outlines the components and their respective
quantities required to prepare 1 liter of M9 solution. The solution includes sodium phosphate
dibasic heptahydrate (64 g), potassium phosphate monobasic (15 g), sodium chloride (2.5 g), and
ammonium chloride (5 g). These components are dissolved in distilled water up to a 1-liter mark.
M9 solution is an essential buffer used for washing and maintaining C. elegans in experimental

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protocols, providing an isotonic environment that supports the viability and integrity of the
nematodes. M9 Minimal Salts Preparation and Recipe | AAT Bioquest. (2024). Aatbio.com.
https://www.aatbio.com/resources/buffer-preparations-and-recipes/m9-salts

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Table 3: Age Synchronization Protocol for C. elegans
This table outlines the step-by-step procedure for synchronizing C. elegans populations at the L4
larval stage. The materials required include live C. elegans, 20M bleaching solution, M9
solution, a centrifuge, sterile centrifuge tubes, and an orbital shaker. The protocol begins with
dislodging worms from plates using M9 solution (Step 1) and progresses through centrifugation
(Step 3), the application of a bleaching solution to isolate eggs (Step 5), multiple washes to
remove residual bleach (Steps 8–10), and incubation on an orbital shaker to allow eggs to hatch
synchronously into L1 larvae (Step 12). The final step involves transferring larvae to fresh NGM
plates seeded with E. coli for further growth and experimental preparation. This standardized
approach ensures uniformity in developmental stages across experimental populations. Porta-dela-Riva, M., Fontrodona, L., Villanueva, A., & Cerón, J. (2012). Basic Caenorhabditis elegans
Methods: Synchronization and Observation. Journal of Visualized Experiments, 64.
https://doi.org/10.3791/4019
The synchronization employed a bleaching method, which used a 20M bleaching solution to
dissolve adult worm bodies while leaving behind viable eggs. This 20M bleaching solution was
prepared by combining of 20mL bleach solution with 80mL of distilled water. This does not
require autoclaving because sodium hypochlorite is a strong disinfectant that naturally eliminates
contaminants and heating it can release toxic chlorine gas and reduce its effectiveness. This step
was crucial, as the bleaching solution breaks down adult cuticles and body tissues, but the robust
eggshells remain intact (Table3-Step 5). After adding the bleaching solution, the tubes were
shaken to ensure proper exposure (Table3-Step 6). The bleaching solution was removed (Table3Step 7), and the eggs were washed multiple times with M9 buffer (Table3-Steps 8-10) to
eliminate any residual bleaching agent, ensuring the safety and viability of the eggs.

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Following the washes, a final centrifugation was performed to isolate the eggs (Table3-Step 11).
The eggs were incubated on an orbital shaker overnight (Step 12) to allow synchronous hatching
into L1 larvae. The freshly hatched larvae were then transferred to fresh NGM plates seeded with
E. coli (Table3-Step 13) to grow into the L4 stage under controlled conditions. (Figure 3)
This method ensured that all experimental worms began at the same developmental stage,
minimizing variability and enhancing the accuracy of lifespan analysis. As illustrated in Figure
3, the C. elegans lifecycle provides a clear progression from egg to adulthood, highlighting the
importance of precise synchronization. The stepwise bleaching protocol detailed in Table 2
ensured consistency and reproducibility, forming a robust foundation for experiments involving
RNAi and temperature variations.
2.4 RNA Interference by feeding.
The RNA interference (RNAi) technique was utilized to silence the daf-2 gene in
Caenorhabditis elegans to study aging-related pathways. The experimental setup relied on the
use of pre-transformed pAD48-daf-2 RNAi plasmid (Figure 10), specifically designed to express
double-stranded RNA (dsRNA) targeting the daf-2 gene. This plasmid integrates essential
components of the lac operon to facilitate IPTG-inducible dsRNA production, ensuring precise
and effective gene silencing.

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Figure 10 Map of the pAD48-daf-2 RNAi Plasmid: The pAD48-daf-2 RNAi plasmid (3128
bp) is specifically engineered for the RNAi-mediated knockdown of the daf-2 gene in
Caenorhabditis elegans. The plasmid contains the daf-2 gene insert (highlighted in red), flanked
by essential regulatory elements, including the T7 promoter and lac operator, to facilitate IPTGinducible dsRNA production. Additional features include the origin of replication (Ori),
antibiotic resistance gene (AmpR) for plasmid selection, and multiple restriction sites for genetic
modification. This vector enables precise gene silencing in RNAi experiments, aiding in the
study of IIS pathway regulation and lifespan modulation. Addgene: pAD48-daf-2 RNAi. (2024).
Addgene.org. https://www.addgene.org/34834/
2.4.1 Preparation of RNAi Plates: For the experiments, standard agar plates were prepared and
supplemented with 1 mM isopropyl-β-D-thiogalactoside (IPTG) prepared by 0.23831 g of IPTG
and filling it upto 1 L mark with distilled water. To ensure even distribution and absorption, 1

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mL of IPTG solution was pipetted onto the surface of the agar, followed by four days of drying
at room temperature. Once the plates were fully dried, bacterial cultures containing the pAD48daf-2 plasmid were seeded onto the surface and incubated at room temperature for 24 hours to
establish uniform bacterial lawns. This preparation method allowed robust induction of dsRNA
in the bacterial strains.
2.4.2 Worm Feeding Protocol: Synchronized L1-stage worms, obtained via the bleaching
method, were gently transferred onto the prepared RNAi plates. The worms were then allowed to
feed on the IPTG-induced bacterial lawns at 20°C as they progressed through their larval stages.
This ensured effective knockdown of the daf-2 gene by the ingestion of dsRNA-producing
bacteria, enabling a reliable exploration of the gene's role in aging and stress response pathways.
The RNAi feeding technique used in this study provided a robust framework for gene silencing,
leveraging IPTG pre-treatment for consistent dsRNA induction in the bacterial strains. By
combining RNAi with precisely synchronized worm populations, this approach ensured
reproducibility and reliability in assessing the functional impacts of daf-2 knockdown on aging
and related phenotypes.

2.5 Exposure to Variable Temperatures
Worms will be exposed to variable temperature conditions after synchronization and
RNAi treatment. Plates from previous step (day 4) were transferred to incubators set at the
specified temperatures of 4°C, 10°C, 20°C, or 30°C. These temperature conditions were
maintained consistently to study their impact on the lifespan of C. elegans under controlled

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experimental setups. The exposure to variable temperatures allowed for the examination of
environmental influences on aging mechanisms.
2.6 FUdR Optimization
To inhibit reproduction in adult C. elegans while ensuring normal growth and
development, 5-fluoro-2′-deoxyuridine (FUdR) was added to the plates at the L4 larval stage
(day 5), just before the worms transitioned into adulthood. This timing was critical to avoid
interference with growth and ensured that reproduction alone was suppressed. Four
concentrations of FUdR (10 µM, 15 µM, 20 µM, and 25 µM) were prepared by dissolving FUdR
powder in sterile water. As FUdR has a molecular weight of approximately 246.2 g/mol, the
required quantities were calculated as 0.00246 g for 10 µM, 0.00369 g for 15 µM, 0.00492 g for
20 µM, and 0.00615 g for 25 µM, each dissolved in 1 liter of water. These stock solutions were
filter-sterilized and stored at 4°C until use.
For experimental setup, 1mL of the respective FUdR solution was pipetted onto the agar surface
of each plate. The plates were left at room temperature for 24 hours to allow the solution to
absorb evenly into the agar, ensuring consistent distribution and effective incorporation. Adding
FUdR at the L4 stage specifically targeted reproduction without affecting the development or
physiology of the adult worms, allowing for accurate lifespan analysis focused solely on the
parental population. The careful optimization of four different concentrations ensured that a
range of FUdR levels was tested for effectiveness. This approach maintained synchronized
populations and eliminated confounding factors such as overcrowding, resource depletion, or
progeny interference, enhancing the reliability and reproducibility of the experimental results.

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3. RESULTS
The foundational phase of this study focused on establishing essential experimental
conditions and validating initial methodologies for aging research in Caenorhabditis elegans (C.
elegans). Key achievements include the successful maintenance and growth of a healthy C.
elegans colony under controlled laboratory conditions, ensuring experimental reproducibility and
reliability. Synchronization of worm populations was achieved, with larvae successfully aligned
at the same developmental stage, eliminating variability associated with asynchronous growth.
This step is critical for ensuring consistency in lifespan analyses and environmental
interventions.
Additionally, optimization of 5-fluoro-2’-deoxyuridine (FUdR) concentrations was conducted to
determine the ideal sterilization conditions without adversely affecting survival outcomes.
Worms exposed to FUdR concentrations of 10 µM, 15 µM, and 20 µM demonstrated sustained
viability, while concentrations of 25 µM led to reduced survival. Based on these observations,
lower concentration 20 µM was selected for future experiments to maintain a sterile environment
without impacting the health or stress response of the worms.
Although the research is ongoing and lifespan results are not yet available, these initial successes
lay a strong foundation for the subsequent analysis of RNAi-induced daf-2 knockdown effects
under variable temperature conditions. The establishment of healthy synchronized populations
and optimization of sterilization protocols provide the experimental consistency required for
robust and meaningful findings in aging research.
4. DISCUSSION

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This study aims to explore the interaction between RNAi-mediated daf-2 knockdown and
temperature variations in modulating the aging process of Caenorhabditis elegans. While
experimental data is not yet available, the groundwork laid in establishing synchronized colonies
and optimizing FUdR concentration demonstrates the study's feasibility and scientific rigor.
These foundational steps ensure accurate and reproducible assessment of lifespan in response to
genetic and environmental interventions.
The expected results anticipate that RNAi-induced daf-2 knockdown will extend the lifespan of
C. elegans at all tested temperatures, albeit with variations based on the specific thermal
conditions. At lower temperatures, such as 4°C, the combination of IIS suppression and reduced
metabolic activity is hypothesized to result in the most significant lifespan extension. This is
likely due to lower rates of metabolic stress and oxidative damage, which complement the
protective effects of DAF-16 activation. Moderate temperatures (10°C and 20°C) are expected to
show lifespan benefits from RNAi treatment, though less pronounced than at 4°C due to
relatively higher metabolic demands. At 30°C, while daf-2 knockdown may provide some
resilience against heat stress, the protective effects of IIS suppression are expected to be limited
by the accelerated metabolic rates and cellular damage associated with higher temperatures.
A major accomplishment of this study was the successful synchronization of C. elegans at the L4
larval stage, a critical step to ensure uniform developmental stages across experimental groups.
This synchronization reduces variability, allowing for a more accurate evaluation of genetic and
environmental influences on aging. Additionally, determining the effective FUdR concentration
(25 µM) ensures sterility without affecting the worms' growth or behavior, providing consistent
conditions for lifespan analysis.

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The RNAi methodology utilized in this study highlights the utility of gene-silencing techniques
in addressing fundamental biological questions. By silencing the daf-2 gene, this study provides
a platform to investigate how IIS signaling integrates with environmental factors to modulate
aging. The ability to conduct such experiments in a controlled system underscores the potential
of C. elegans as a model for exploring complex interactions between genetic pathways and
environmental variables.
The anticipated outcomes of this research represent a significant contribution to aging biology, as
they will provide novel insights into how temperature interacts with IIS suppression to regulate
lifespan. This study addresses a critical gap in understanding the combined effects of genetic and
environmental factors on aging, offering a fresh perspective on the dynamic interplay between
these influences. The results have broader implications for translational research, particularly in
identifying strategies to enhance stress resilience and mitigate age-related conditions in higher
organisms, including humans.
Future studies can build on this work by incorporating additional environmental variables, such
as oxidative stress or dietary restrictions, to explore multifaceted interactions with IIS signaling.
Validation of RNAi efficiency through techniques such as qRT-PCR or Western Blotting would
further strengthen the study's conclusions and provide a more detailed understanding of the
molecular mechanisms underlying observed lifespan changes.
Some limitations of the study include the reliance on feeding-based RNAi, which can result in
variability in gene knockdown efficiency among individual worms. While ELISA was
considered as a validation method for RNAi efficiency, future research could benefit from
molecular approaches like qRT-PCR or Western Blotting for precise quantification of

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knockdown effects. Additionally, the use of FUdR to prevent reproduction, while effective, may
subtly impact stress response or metabolic pathways. The synchronization method involving
bleaching, although widely used, could introduce stress to early-stage larvae. Expanding sample
sizes and incorporating alternative synchronization or sterilization techniques may help mitigate
these concerns.
In summary, this study establishes a solid foundation for exploring the molecular and
environmental determinants of aging. By integrating genetic interventions with temperature
variability, it offers a unique approach to understanding aging dynamics and lays the groundwork
for future research into strategies for improving healthspan and resilience to stress in both model
organisms and humans.
CONCLUSION
This study investigated the interplay between RNAi-mediated daf-2 knockdown and
temperature variations in modulating the lifespan of Caenorhabditis elegans, focusing on the
integration of genetic and environmental factors in aging. By silencing the IIS pathway, the
research anticipates notable lifespan extension at lower temperatures due to reduced metabolic
stress and enhanced cellular resilience, while higher temperatures are expected to diminish these
benefits due to increased heat-induced cellular damage. The establishment of synchronized worm
populations and optimized experimental parameters demonstrated the robustness of this model
for aging research. These findings not only reinforce the utility of C. elegans as a model
organism but also provide a foundation for understanding how genetic pathways and
environmental factors converge to influence longevity. This research opens avenues for future

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studies to refine these findings and develop interventions aimed at mitigating age-related decline
and enhancing stress resilience in higher organisms, including humans.
ACKNOWLEDGMENTS:
I would like to express my deepest gratitude to my instructors at Capilano University's
STEM Faculty, including Eugene Chu, Mark Vaughan, Eunice Chin, Mahta Khosravi, Mahshid
Atapour, Nazar Kovalevskyy, Ann Meitz, Matt Berry, Casper McWilliam, and Christine
Kondratev, for their invaluable guidance and support throughout this research project. Their
expertise and encouragement have been instrumental in shaping this study.
I extend my heartfelt thanks to my lab partner, Maria Jose Pena Pena, for her collaboration and
dedication to our shared research goals. Working alongside her has been a rewarding experience
that greatly enriched the quality of this project.
Special thanks to my husband, Ashish Nayyar, for his unwavering support and encouragement,
which have been a constant source of strength throughout this endeavor. I am also immensely
grateful to my classmates and friends—Emily Derrick, Alice, Patrick, and Roberto—for their
camaraderie and insightful discussions, which provided both inspiration and motivation.
Lastly, I acknowledge the resources and infrastructure provided by Capilano University, without
which this research would not have been possible. Thank you all for your contributions and
support in making this project a reality.

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REFERENCES:
Addgene: pAD48-daf-2 RNAi. (2024). Addgene.org. https://www.addgene.org/34834/
Abdisa, T. (2018). Serological and immunological test. International Journal of Veterinary
Science and Research, 078–083. https://doi.org/10.17352/ijvsr.s1.111
Alhajj, M., Zubair, M., & Farhana, A. (2023, April 23). Enzyme linked immunosorbent assay.
StatPearls - NCBI Bookshelf. https://www.ncbi.nlm.nih.gov/books/NBK555922/
Ain. (2022, February 28). RNA Interference or RNAi. Microbiology Notes.
https://microbiologynotes.org/rna-interference-or-rnai/#google_vignette
Biglou, S. G., Bendena, W. G., & Chin-Sang, I. (2021). An overview of the insulin signaling
pathway in model organisms Drosophila melanogaster and Caenorhabditis
elegans. Peptides, 145, 170640. https://doi.org/10.1016/j.peptides.2021.170640
Bulger, D. A., Fukushige, T., Yun, S., Semple, R. K., Hanover, J. A., & Krause, M. W. (2017).
Caenorhabditis elegans DAF-2 as a model for human insulin receptoropathies. G3 Genes
Genomes Genetics, 7(1), 257–268. https://doi.org/10.1534/g3.116.037184
Cho, J., & Park, Y. (2024). Development of aging research in Caenorhabditis elegans: From
molecular insights to therapeutic application for healthy aging. Current Research in Food
Science, 9, 1-6. https://doi.org/10.1016/j.crfs.2024.100809
Carpenter, E. P., Beis, K., Cameron, A. D., & Iwata, S. (2008). Overcoming the challenges of
membrane protein crystallography. Current Opinion in Structural Biology, 18(5), 581–
586. https://doi.org/10.1016/j.sbi.2008.07.001
De-Souza, E. A., Camara, H., Salgueiro, W. G., Moro, R. P., Knittel, T. L., Tonon, G., Pinto, S.,
Pinca, A. P. F., Antebi, A., Pasquinelli, A. E., Massirer, K. B., & Mori, M. A. (2019).

RESEARCH PAPER

34

RNA interference may result in unexpected phenotypes in Caenorhabditis elegans.
Nucleic Acids Research, 47(8), 3957–3969. https://doi.org/10.1093/nar/gkz154
Engvall, E., & Perlmann, P. (1971). Enzyme-linked immunosorbent assay (ELISA) quantitative
assay of immunoglobulin G. Immunochemistry, 8(9), 871–874.
https://doi.org/10.1016/0019-2791(71)90454-x
Feldman, N., Kosolapov, L., & Ben-Zvi, A. (2014). Fluorodeoxyuridine Improves
Caenorhabditis elegans Proteostasis Independent of Reproduction Onset. PLoS
ONE, 9(1), e85964. https://doi.org/10.1371/journal.pone.0085964
Floxuridine. (2023, January 29). Wikipedia. https://en.wikipedia.org/wiki/Floxuridine
Grishok, A. (2005). RNAi mechanisms in Caenorhabditis elegans. FEBS Letters, 579(26), 5932–
5939. https://doi.org/10.1016/j.febslet.2005.08.001
Huang, J., Wang, K., Wang, M., Wu, Z., Xie, G., Peng, Y., Zhang, Y., Zhang, X., & Shao, Z.
(2023). One-day thermal regime extends the lifespan in Caenorhabditis elegans.
Computational and Structural Biotechnology Journal, 21, 495–505.
https://doi.org/10.1016/j.csbj.2022.12.017
Hannon, G. J. (2002). RNA interference. Nature, 418(6894), 244–251.
https://doi.org/10.1038/418244a
Hamilton, B. (2005). A systematic RNAi screen for longevity genes in C. elegans. Genes &
Development, 19(13), 1544–1555. https://doi.org/10.1101/gad.1308205
Kamath, R. S., Martinez-Campos, M., Zipperlen, P., Fraser, A. G., & Ahringer, J. (2000).
Effectiveness of specific RNA-mediated interference through ingested double-stranded
RNA in Caenorhabditis elegans. Genome Biology, 2(1). https://doi.org/10.1186/gb-20002-1-research0002

RESEARCH PAPER

35

Kohl, T. O., & Ascoli, C. A. (2017). Direct and indirect Cell-Based Enzyme-Linked
immunosorbent assay. Cold Spring Harbor Protocols, 2017(5), pdb.prot093732.
https://doi.org/10.1101/pdb.prot093732
Khan Academy. (2021). The lac operon . Khan Academy.
https://www.khanacademy.org/science/ap-biology/gene-expression-andregulation/regulation-of-gene-expression-and-cell-specialization/a/the-lac-operon
Life cycle of C. elegans at 20 °C. An adult hermaphrodite... (2024). ResearchGate.
https://www.researchgate.net/figure/Life-cycle-of-C-elegans-at-20-C-An-adulthermaphrodite-produces-roughly-300-eggs_fig3_265581569
López-Otín, C., Blasco, M. A., Partridge, L., Serrano, M., & Kroemer, G. (2013). The hallmarks
of aging. Cell, 153(6), 1194–1217. https://doi.org/10.1016/j.cell.2013.05.039
Lee, H. J., Alirzayeva, H., Koyuncu, S., Rueber, A., Noormohammadi, A., & Vilchez, D. (2023).
Cold temperature extends longevity and prevents disease-related protein aggregation
through PA28γ-induced proteasomes. Nature Aging, 3(5), 546–566.
https://doi.org/10.1038/s43587-023-00383-4
Li, W., Wang, C., Tao, L., Yan, Y., Zhang, M., Liu, Z., Li, Y., Zhao, H., Li, X., He, X., Xue, Y.,
& Dong, M. (2021). Insulin signaling regulates longevity through protein
phosphorylation in Caenorhabditis elegans. Nature Communications, 12(1).
https://doi.org/10.1038/s41467-021-24816-z
Lactose Operon - an overview | ScienceDirect Topics. (n.d.). Www.sciencedirect.com.
https://www.sciencedirect.com/topics/medicine-and-dentistry/lactose-operon
Leboffe, M. J., & Pierce, B. E. (2012). Microbiology: laboratory theory & application. Morton
Pub.

RESEARCH PAPER

36

M9 Minimal Salts Preparation and Recipe | AAT Bioquest. (2024). Aatbio.com.
https://www.aatbio.com/resources/buffer-preparations-and-recipes/m9-salts
Niccoli, T., & Partridge, L. (2012). Ageing as a risk factor for disease. Current Biology, 22(17),
R741–R752. https://doi.org/10.1016/j.cub.2012.07.024
Porta-de-la-Riva, M., Fontrodona, L., Villanueva, A., & Cerón, J. (2012). Basic Caenorhabditis
elegans Methods: Synchronization and Observation. Journal of Visualized
Experiments, 64. https://doi.org/10.3791/4019
Patrycja Vasilyev Missiuro. (2010, August 26). Predicting Genetic Interactions in
Caenorhabditis elegans using Machine Learning.
https://www.researchgate.net/publication/266342950_Predicting_Genetic_Interactions_in
_Caenorhabditis_elegans_using_Machine_Learning
Samuelson, A. V., Carr, C. E., & Ruvkun, G. (2007). Gene activities that mediate increased life
span of C. elegans insulin-like signaling mutants. Genes & Development, 21(22), 2976–
2994. https://doi.org/10.1101/gad.1588907
Scerbak, C., Vayndorf, E. M., Parker, J. A., Neri, C., Driscoll, M., & Taylor, B. E. (2014).
Insulin signaling in the aging of healthy and proteotoxically stressed mechanosensory
neurons. Frontiers in Genetics, 5. https://doi.org/10.3389/fgene.2014.00212
Stiernagle, T. (2019). Maintenance of C. elegans. Wormbook.org.
http://www.wormbook.org/chapters/www_strainmaintain/strainmaintain.html
Uchiyama, M., & Mihara, M. (1978). Determination of malonaldehyde precursor in tissues by
thiobarbituric acid test. Analytical Biochemistry, 86(1), 271–278.
https://doi.org/10.1016/0003-2697(78)90342-1

RESEARCH PAPER

37

Uno, M., & Nishida, E. (2016). Lifespan-regulating genes in C. elegans. Npj Aging and
Mechanisms of Disease, 2(1). https://doi.org/10.1038/npjamd.2016.10
Vakkayil, K. L., & Hoppe, T. (2022). Temperature-Dependent regulation of proteostasis and
longevity. Frontiers in Aging, 3. https://doi.org/10.3389/fragi.2022.853588
Waseem, Q. ul A. (2022, February 28). RNA Interference or RNAi. Microbiology Notes.
https://microbiologynotes.org/rna-interference-or-rnai/
Xiao, R., Zhang, B., Dong, Y., Gong, J., Xu, T., Liu, J., & Xu, X. Z. Shawn. (2013). A Genetic
Program Promotes C. elegans Longevity at Cold Temperatures via a Thermosensitive
TRP Channel. Cell, 152(4), 806–817. https://doi.org/10.1016/j.cell.2013.01.020
Zhang, B., Xiao, R., Ronan, E. A., He, Y., Hsu, A., Liu, J., & Xu, X. S. (2015). Environmental
Temperature Differentially Modulates C. elegans Longevity through a Thermosensitive
TRP Channel. Cell Reports, 11(9), 1414–1424.
https://doi.org/10.1016/j.celrep.2015.04.066