Name: surveying low-cost methods to measure lifespan and healthspan in caenorhabditis elegans
Uploaded: 2022-05-18T14:00:00
Duration: 10 min 8 s
Description: Watch this Scientific Journal Video about Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans at JoVE.com

G.G. is supported by T32AG052374 and R.H.S. is supported by R00AG065200 from the National Institute on Aging. We thank the CGC (funded by NIH Office of Research Infrastructure Program P40 OD010440) for the strains.

The authors declare no competing financial interests.

Lifespan, most simply defined as the duration of life, is a clear binary phenomenon in most organisms-either an organism is living or is not. However, longevity does not always correlate with an organism&#39;s health. For example, mitochondrial hormesis models where exposure to mitochondrial stress dramatically increases lifespan are generally some of the longest-lived animals, yet exhibit stunted growth and decreased metabolic function37,54. Similarly, animals with hyperactive endoplasmic reticulum stress responses also exhibit certain behaviors and phenotypes that can be correlated with decreased health, despite having dramatically improved protein homeostasis and lifespan36,49. Finally, many longevity paradigms in model organisms including increased HSF-1 function55, increased XBP-1 function56, and altered FoxO signaling57 are all correlated with increased cancer risk, and it is inarguable that extended lifespan is not beneficial if an organism is in a constant struggle with cancer and other health maladies. Therefore, longevity cannot be a standalone measurement in aging biology.
Thus, the concept of healthspan has been a growing field in aging biology. Healthspan, loosely defined as the period of life that one is healthy, is more difficult to ascertain than longevity. However, unlike longevity, the concept of &#34;health&#34; is complicated, as there are many different readouts to organismal health: on the organismal level, there are muscle function/strength, neuronal/cognitive function, reproductive health, etc.; on the cellular level there are protein homeostasis, lipid homeostasis, glucose homeostasis, metabolism, etc. In 2014, aging biologists have definitively characterized biological hallmarks of aging with the structured definition that it must be something that naturally breaks down during aging and can experimentally be altered such that experimental exacerbation should accelerate aging and experimental intervention should slow down aging. These nine hallmarks of aging include genomic instability, telomere attrition, epigenetic alterations, loss of protein homeostasis (proteostasis), stem cell exhaustion, altered intercellular signaling, mitochondrial dysfunction, deregulated nutrient sensing, and cellular senescence58. Since then, numerous studies argue other factors should be included, including extracellular proteins and systemic physiology such as immunity and inflammation59. Ultimately, the complex definition of healthspan mandates that organismal health be measured using multiple different methods.
Therefore, in this manuscript, multiple methods are presented to measure various aspects of healthspan using the nematode model, C. elegans. We assay locomotory behavior using thrashing assays, reproductive health using egg count and brood size, and sensitivity to stress. Indeed, locomotory behavior is a gold-standard method for measuring healthspan, as organisms exhibit significant loss of motility and movement during aging51. Loss of locomotory behavior can be ascribed to multiple hallmarks of aging, as muscle function in C. elegans is dependent on proper proteostasis60, mitochondrial dysfunction61, and neuron-muscle signaling62. While this manuscript focuses on one measurement of locomotory behavior, it is important to note that many other methods exist, including motility of animals on a solid agar plate, response to touch51, and chemotaxis assays63. However, these methods generally require more sophisticated recording devices, usage of worm-tracking software, or usage of expensive, dangerous, or volatile chemicals, all of which may be prohibitive in some research settings.
In addition, assays for egg count and brood size are presented as a method of measuring reproductive health and as the simplest method to measure cell division in adult worms, since adult worms are post-mitotic and only germ cells and embryos undergo cell division within an adult worm64. As a measure of cell division, reproductive health can be relevant for the aging hallmarks of cellular senescence and stem cell exhaustion. Reproductive health can be affected by many factors, including pathogenic infection65 or exposure to stress49, though there is no direct correlation between reproductive health and longevity. In fact, some long-lived animals exhibit a significant decrease in brood size49, and it is even possible that there exists an inverse correlation between longevity and brood size50. This is not a phenomenon specific to C. elegans, as detrimental effects of reproduction on longevity have long been observed in humans66, companion dogs67, and mice68. Still, we provide egg count and brood size as a reliable and low-cost method for measuring reproductive health with the caveat that reproductive health may not correlate with longevity or healthspan.
Finally, survival assays are offered as an indirect measure of organismal health. Importantly, cellular stress responses, including response to thermal stress69 and ER stress35 rapidly decline during the aging process and have direct relevance to the aging hallmark of proteostasis70,71. In contrast, hyperactivating stress responses can significantly increase lifespan by promoting resilience to stress35,37,38. While this study focuses on the simplest and lowest cost methods, a large number of alternative methods for stress resilience assays exist for thermotolerance41 and oxidative stress66, each requiring a different set of equipment and consumables. Beyond simple exposure studies to stressors, other physiological methods can be performed depending on access to equipment. For example, an extracellular flux analyzer can monitor mitochondrial function and cellular respiration73; fluorescent dissection microscopes will allow measurements of transcriptional reporters for stress response activation20; and high-resolution compound or confocal microscopes can be used to measure organelle morphology with fluorescent probes for mitochondria74, the endoplasmic reticulum75,76, and actin cytoskeleton77.
As a final cautionary tale for measurements of longevity, while chemical and genetic methods for sterilizing worms are offered to significantly decrease cost, it is important to note that both can directly impact lifespan. For example, exposure to FUDR has been previously reported to impact both lifespan and thermotolerance45. And while the glp-4(bn2) mutant itself does not have any direct effects on lifespan, growth at 25 &#176;C is a mild heat-stress33,34 and thus can impact lifespan2. There exists other methods for sterilizing C. elegans, including auxin-mediated sterility78 or alternative temperature-sensitive sperm-deficient mutants79. However, all methods have some caveats, and care should be taken to utilize the least detrimental assay for each laboratory&#39;s scientific needs. One final limitation of longevity studies is potential variability that can occur due to low sample sizes or simply by an objective error by the investigator. This can be circumvented as new technologies are born in automated lifespan technologies80, but again these systems are costly and require some engineering and computational equipment and skills. Ultimately, the collection of methods provided here is a reliable set of tools that can be quickly adapted and learned in almost any institution and provide a solid foundation for aging biology.

Since the time of the publication of &#39;The genetics of Caenorhabditis elegans&#39;, one of the most influential articles by Sydney Brenner in 1974, this microscopic worm has been considered an outstanding model system to study biological mysteries1. In 1977, Michael R. Klass published the method for measuring the lifespan of C. elegans and established this model system to study aging2. The investigation to understand the relationship between stress and longevity has started with the identification of a single mutation in the age-1 gene, which resulted in a lifespan extension in C. elegans3. Furthermore, contemporary studies have identified other lifespan-increasing mutations, which revealed long-lived mutant worms that exhibit increased resistance to stress4,5,6. With its many advantages including a short lifespan, easy maintenance, completely sequenced genome containing homology to about two-thirds of all human disease-causing genes, availability and ease of using RNA interference (RNAi) libraries, and physiological similarity with humans7,8,9, C. elegans has quickly been adopted as an outstanding model for stress and aging biology.
Perhaps the greatest utilities of C. elegans are its extremely low cost of maintenance, ease of experimentation, and the variety of genetic tools available for studies. C. elegans are typically grown on a solid agar medium with an E. coli food source. Two commonly used E. coli strains are standard OP50, a B strain that is perhaps the most commonly used10, and HT115, a K-12 strain that is used primarily for RNAi experiments11,12. The HT115 K-12 strain carries a deletion in RNAIII RNase, a mutation that is essential for RNAi methods, where plasmids expressing dsRNA corresponding to individual C. elegans genes are used. The dsRNA feeding vectors allow for robust knockdown of C. elegans genes without the need for complex crosses or genome editing, as bacteria carrying these plasmids can be directly fed to nematodes. Thousands of these bacterial RNAi vectors exist in the HT115 background, including the most popular Vidal RNAi library with &#62;19,000 individual RNAi constructs13 and the Ahringer RNAi library with 16,757 RNAi constructs14. However, the OP50 and HT115 bacterial diets have major differences in metabolic profile, including differences in Vitamin B1215,16. Therefore, it is recommended to perform all experiments on a single bacteria source, if possible, to avoid gene-diet interactions that may introduce multiple confounding factors as previously described17,18,19. Due to its ease, animals are maintained on OP50 for all the experimental conditions described here, but all experiments are performed on HT115 as previously described20. Briefly, animals are maintained at OP50 and transferred to HT115 post-synchronization (after bleaching) for consistency between RNAi vs. non-RNAi experiments. Alternatively, an RNAi-competent OP50 strain carrying a similar deletion of RNAIII RNase found in the E. coli K12 HT115 strain can also be used21.
Perhaps one major limitation to RNAi experiments in C. elegans is the concern of knockdown efficiency. While knockdown efficiency can be validated via qPCR or western blotting, these require expensive equipment and reagents and are limited to bulk analysis. This is even more of a concern looking at specific cells, such as neurons, which are refractory (less sensitive) to RNAi. While RNAi efficiency in specific cells can be enhanced via overexpression of SID-1, the transmembrane protein essential for dsRNA uptake22, this is still limited to the cell type-specific expression patterns of the promoters used for these constructs, and thus gene knockouts and mutations are the most foolproof means of depleting gene functions. Beyond RNAi-mediated knockdown, C. elegans are also highly amenable to genome editing with CRISPR-based strategies23,24,25&#160;and transgenic construct overexpression through microinjections, with the option to integrate transgenic constructs through irradiation or transposon-based integration26,27,28,29. However, these methods require expensive microinjection equipment, and the high cost of guide RNAs or Cas9 enzyme may prohibit these methods in institutions with limited funding. Instead, thousands of transgenic lines and mutants are readily available for a few dollars both at the Caenorhabditis Genetics Center (CGC) and National Bioresource Project (NBRP). The NBRP offers isolated mutants for a large number of C. elegans genes, including published and therefore verified mutant strains, mutants derived from pilot projects, and mutants that have yet to be characterized. In contrast, CGC is a depository of mostly published and established C. elegans lines from the research community. Both of them ship strains worldwide at very reasonable rates and offer a wide variety of options for those with limited capacity to synthesize strains in-house.
Here, a curated methods collection is offered, which are likely to be the lowest cost methods for assaying lifespan and healthspan in C. elegans. All the methods presented here require low-cost equipment and supplies, and only utilize strains readily available from the CGC. Perhaps most prohibitive for longevity and survival assays in C. elegans is the cost of Nematode Growth Media (NGM) plates. Since C. elegans are hermaphrodites and self-fertilize, standard survival assays require that adult animals be continuously moved away from their progeny to avoid contamination from offspring. Not only is this process time-consuming, it can become expensive due to the necessity of approximately 100 plates per condition to run a single lifespan assay. Here, two alternatives are provided: utilization of the temperature-sensitive germline-less mutant, glp-4(bn2), or chemical sterilization using 5-fluoro-2&#39;-deoxyuridine (FUDR). glp-4 encodes a valyl aminoacyl tRNA synthetase, and the temperature-sensitive glp-4(bn2) are reproductively deficient at restrictive temperatures due to decreased protein translation30,31. FUDR is a robust method to chemically sterilize C. elegans by preventing DNA replication, thus inhibiting reproduction32. Although FUDR can be prohibitively expensive for some labs, only a small amount is required to chemically sterilize worms, and its stability in powder form may make it feasible for most groups. Utilizing the temperature-sensitive glp-4(bn2) mutant is certainly the cheapest option, as the only requirement is an incubator to shift the animals to the restrictive 25 &#176;C; however, it should be noted that growth at 25 &#176;C may cause mild heat-stress33,34. Regardless of the method, using sterile animals can significantly decrease the costs of consumables required for age-related assays.
To study aging, standard lifespan assays are conventional as paradigms that alter longevity have direct impacts on aging. However, measurements of healthspan and stress tolerance present additional information on the health of the organism. Here, several methods are offered to measure healthspan: 1) fecundity as a measure of reproductive health; 2) brood size as a measure of developmental health and viability of laid offspring; and 3) locomotory behavior as a measure of muscle function and motility, both of which are directly correlated with aging. Additionally, assays of stress tolerance are offered: survival to ER stress, mitochondrial/oxidative stress, and thermal stress survival. Indeed, animals with increased resistance to ER stress35,36, mitochondrial stress37, and thermal stress38 exhibit increased lifespan. ER stress is applied by exposing C. elegans to tunicamycin, which blocks N-linked glycosylation and causes the accumulation of misfolded proteins39. Mitochondrial/oxidative stress is induced by exposure to paraquat, which induces superoxide formation specifically in the mitochondria40. Heat stress is applied through the incubation of animals at 34-37 &#176;C33,41. All the assays described here can be performed with minimal equipment and funds, and offer a variety of tools to study aging in diverse groups.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>APEX IPTG</td>
			<td>Genesee</td>
			<td>18-242</td>
			<td>for RNAi</td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>VWR</td>
			<td>90000-764</td>
			<td>for NGM plates</td>
		</tr>
		<tr>
			<td>Bacto Peptone</td>
			<td>VWR</td>
			<td>97064-330</td>
			<td>for NGM plates</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>VWR</td>
			<td>97061-904</td>
			<td>for NGM plates</td>
		</tr>
		<tr>
			<td>Carbenicillin</td>
			<td>VWR</td>
			<td>76345-522</td>
			<td>for RNAi</td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>VWR</td>
			<td>80057-932</td>
			<td>for NGM plates</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>VWR</td>
			<td>BDH1115-1LP</td>
			<td>solvent for drugs</td>
		</tr>
		<tr>
			<td>LB Broth</td>
			<td>VWR</td>
			<td>95020-778</td>
			<td>for LB</td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate</td>
			<td>VWR</td>
			<td>97062-132</td>
			<td>for NGM plates, M9</td>
		</tr>
		<tr>
			<td>Paraquat</td>
			<td>Sigma-Aldrich</td>
			<td>36541</td>
			<td>for oxidative/mitochondrial stress</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>VWR</td>
			<td>97061-566</td>
			<td>for bleach soluton</td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>VWR</td>
			<td>EM-PX1570-2</td>
			<td>for NGM plates</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>VWR</td>
			<td>EM-PX1565-5</td>
			<td>for M9</td>
		</tr>
		<tr>
			<td>S7E Dissecting Scope</td>
			<td>Leica</td>
			<td>10450840</td>
			<td>Standard dissecting microscope</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>VWR</td>
			<td>EM-SX0420-5</td>
			<td>for NGM plates, M9</td>
		</tr>
		<tr>
			<td>Sodium hypochlorite</td>
			<td>VWR</td>
			<td>RC7495.7-32</td>
			<td>for bleach solution</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>VWR</td>
			<td>71003-472</td>
			<td>for M9</td>
		</tr>
		<tr>
			<td>Tetracycline hydrochloride</td>
			<td>VWR</td>
			<td>97061-638</td>
			<td>for RNAi</td>
		</tr>
		<tr>
			<td>Tunicamycin</td>
			<td>Sigma-Aldrich</td>
			<td>T7765-50MG</td>
			<td>for ER stress</td>
		</tr>
	</tbody>
</table>

surveying low-cost methods to measure lifespan and healthspan in caenorhabditis elegans

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many historical and contemporary studies have identified thousands of lifespan-altering paradigms, including genetic mutations, transgenic gene expression, and hormesis, a beneficial, low-grade exposure to stress. With its many advantages, including a short lifespan, easy and low-cost maintenance, and fully sequenced genome with homology to almost two-thirds of all human genes, C. elegans has quickly been adopted as an outstanding model for stress and aging biology. Here, several standardized methods are surveyed for measuring lifespan and healthspan that can be easily adapted into almost any research environment, especially those with limited equipment and funds. The incredible utility of C. elegans is featured, highlighting the capacity to perform powerful genetic analyses in aging biology without the necessity of extensive infrastructure. Finally, the limitations of each analysis and alternative approaches are discussed for consideration.

C. elegans are an excellent model organism for aging research due to a large majority of aging mechanisms being conserved with humans. Importantly, they have a very low cost in maintenance and experimentation with minimal requirements for equipment and consumables, making them a coveted model system for institutions with limited funds. Moreover, a plethora of simple assays with shallow learning curves makes them an excellent system for even the youngest investigator with little to no experience. All these factors combined with the powerful genetics of C. elegans including the ease of genome editing, thousands of available mutants and transgenic animals at nominal costs, and available RNAi libraries for genetic knockdown of virtually every gene make them an ideal system for undergraduate institutions. Here, some of the lowest cost methods to study aging in C. elegans are surveyed, focusing primarily on assays with minimal equipment and consumable cost, as well as shallow learning curves. In fact, the entirety of the protocols and data collection were written/performed by junior investigators with &#60;5 months of research experience, mostly undergraduate students.
Longevity studies in C. elegans are very simple due to the short lifespan of animals, ranging from 14-20 days. Importantly, lifespan assays are highly standardized and only require an incubator, a standard dissection microscope, a standard worm pick, and consumables for preparing NGM-agar plates. Perhaps the most cost-prohibitive aspect of lifespan measurements in C. elegans is consumables required. This is because C. elegans are hermaphrodites that self-fertilize; therefore, adults being tracked for longevity assays need to be moved away from progeny daily. However, animals can be sterilized by exposing them to FUDR or using mutants, such as the temperature-sensitive germline-less glp-4(bn2) mutant grown at the prohibitive 25 &#176;C to reduce the amount of consumables required30,31,32. Here, lifespan assays were performed with FUDR or with the glp-4(bn2) germline-less mutants, which show similar results to standard lifespans performed on non-sterile animals. While the wild-type lifespans are not identical due to the effects of FUDR45 or growth at 25 &#176;C on lifespan2, the short-lived hsf-1 knockdown animal reliably shows a significant decrease in lifespan for all conditions (Figure 1). hsf-1 encodes the heat-shock factor-1 transcription factor, which is involved in the regulation of the thermal stress response, and its knockdown results in a significant decrease in lifespan38,46.
While longevity is an important factor to consider in aging biology, often, longevity does not correlate with increased health, even in C. elegans47. Thus, as a complementary approach, we offer several methods of measuring organism health, including reproductive health, locomotory behavior, and stress resilience. Reproductive health can be measured in one of two ways. First, measurements of egg count will give a direct measurement of how many eggs are laid by a single self-fertilizing hermaphrodite. However, since animals produce more oocytes than sperm, some unfertilized eggs that would never produce viable progeny are also laid48. Therefore, to get a better understanding of the true reproductive capacity of an animal, measurements of the brood size provide a measure of how many viable offspring are produced. Oftentimes, increased stress resilience can actually decrease reproductive capacity, potentially due to the inherent effect of perceived stress on reproduction49. Similarly, a significant decrease in both the number of eggs laid and brood size is found in hsf-1 overexpression animals compared to wild-type controls (Figure 2A,B). In fact, some hsf-1 overexpression animals exhibit full sterility, providing evidence that reproductive health can be inversely correlated with longevity.
While reproductive health is important to understanding germline health, functional meiosis, and reproductive capacity, in general, there is no direct correlation between longevity and brood size50. Thus, as a complementary approach, locomotory behavior is offered as a gold-standard method for assaying C. elegans healthspan during aging51. There are many methods to measure locomotory behavior, but most methods require sophisticated cameras, tracking software, or expensive chemicals. In contrast, thrashing assays require virtually no equipment beyond what a standard C. elegans lab is equipped with: dissecting microscope, worm pick, pipette, and consumables for making NGM-agar plates. Thrashing rates provide a reliable method for measuring healthspan during aging, as measured by a significant decrease in thrashing in old animals compared to young animals (Figure 2C).
Finally, survival to stress assays is an additional physiological measurement of resilience. The capacity to activate stress responses generally declines during the aging process, making animals less resilient and more sensitive to stress. Thus, stress resilience can be used as a reliable proxy for organismal health. Here, methods are offered for surveying sensitivity to 1) ER stress in response to tunicamycin exposure, a chemical agent that blocks N-linked glycosylation and results in accumulation of misfolded proteins in the ER; 2) mitochondrial/oxidative stress through exposure to paraquat, a chemical agent that induces superoxide formation in mitochondria; and 3) thermal stress through exposure to elevated temperatures. For tunicamycin and paraquat assays, the drug is incorporated into the NGM-agar plate during plate production. For high concentrations of tunicamycin, progeny generally does not develop, and thus sterilization techniques do not need to be used. The protocol presented here recommends 25 ng/&#181;L as a final concentration of tunicamycin, but for those with limited funds, 10 ng/&#181;L also shows a significant reduction in survival (Figure 3A). Both concentrations limit progeny development, and thus no sterilization methods are needed, although the DMSO control will require a sterilization technique or movement of animals onto new plates. This is because tunicamycin toxicity prevents the development of progeny, but DMSO is virtually nontoxic, which allows progeny to develop fully when grown on tunicamycin.
For paraquat assays, either a sterilization technique or movement of animals is required as paraquat treatment does not prevent progeny from developing to adulthood. High levels of paraquat (4 mM) significantly shorten lifespan, while low levels of paraquat (0.25 mM) increase lifespan due to a hormetic effect (Figure 3B), consistent with previously published results52. Finally, thermotolerance assays only require an incubator that can reach 30-37 &#176;C, and no additional reagents are required. Overexpression of hsf-1 increases thermotolerance at 37 &#176;C (Figure 3C) as previously published53. However, as others have shown previously and from the present data, the major problem with thermotolerance assays is their variability. Many factors can contribute to variability within thermotolerance assays, including differences between incubators and the time animals spend outside of the incubator while scoring thermotolerance each hour. For a thorough guideline of thermotolerance, refer to the reference 41.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64091/64091fig01.jpg" /> 
Figure 1: Comparison of lifespan measurements with and without sterilization. (A) Lifespans of wild-type N2 nematodes grown on NGM-agar plates seeded with empty vector (ev) or hsf-1 RNAi bacteria at 20 &#176;C. Animals were moved away from progeny on days 1, 3, 5, and 7 of adulthood. (B) Lifespans of wild-type N2 nematodes grown on NGM-agar-FUDR plates seeded with empty vector (ev) or hsf-1 RNAi bacteria at 20 &#176;C. Animals were grown to adulthood on standard ev or hsf-1 RNAi plates, and then moved to FUDR plates on day 1 of adulthood. (C) Lifespans of glp-4(bn2) mutant animals grown on NGM-agar plates seeded with empty vector (ev) or hsf-1 RNAi at 25 &#176;C. For all conditions, animals were scored for death every 2 days until all animals were recorded as dead or censored. Animals with bagging, protrusion, or explosion of the vulva, or those that crawled up the sides of the plates and desiccated were censored. All statistics were performed using Log-Rank Mantel-Cox testing and can be found in Table 2. <a href="https://www.jove.com/files/ftp_upload/64091/64091fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64091/64091fig02.jpg" /> 
Figure 2: Egg count, brood size, and thrashing as measurements of healthspan. (A) Thrashing assays were performed on glp-4(bn2) mutant animals grown on NGM-agar plates seeded with empty vector at 25 &#176;C on day 1 (blue), day 4 (red), and day 9 (green) animals. Thrashing was scored in animals placed into M9 solution on an NGM-agar plate, video recorded using a standard smartphone camera mounted onto an eyepiece of a standard dissecting scope, and thrashing scored in slow motion for accuracy. n = 15 animals per condition. (B) Egg counts were measured in wild-type N2 (blue) and sur-5p::hsf-1 (red) animals. Animals were grown at 20 &#176;C and moved onto fresh plates, and eggs were counted every 12 h. The total number of eggs laid was summed. n = 7 animals for wildtype and 9 animals for sur-5p::hsf-1. (C) Brood assays were measured on the same animals as (B) where eggs were grown at 20 &#176;C for 2 days to allow hatching, and all hatched eggs were counted. *** = p &#60; 0.001 calculated using non-parametric Mann-Whitney testing. Each dot represents a single animal, and lines represent the median and interquartile range. <a href="https://www.jove.com/files/ftp_upload/64091/64091fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64091/64091fig03.jpg" /> 
Figure 3: Stress resilience as a proxy to organismal health. (A) Survival assay of N2 animals grown on the empty vector RNAi bacteria at 20 &#176;C. Animals were moved to plates containing either 1% DMSO, 10 ng/&#181;L tunicamycin (TM), or 25 ng/&#181;L TM on day 1 of adulthood. (B) Survival assay of N2 animals grown on the empty vector RNAi bacteria at 20 &#176;C. Animals were grown from the hatch on plates containing either water, 0.25 mM paraquat (PQ), or 4 mM PQ. For A-B, animals were scored for death every 2 days until all animals were recorded as dead or censored. Animals with bagging, protrusion, or explosion of the vulva, or those that crawled up the sides of the plates and desiccated were censored. All statistics were performed using Log-Rank Mantel-Cox testing (Table 2). (C) Pooled data of all 37 &#176;C thermotolerance assays for wild-type N2 animals versus overexpression of hsf-1 (sur-5p::hsf-1). Data are represented as percent alive at time = 9 h of a thermotolerance assay, with each line representing a matched experiment performed on the same day. Animals were grown on the empty vector RNAi bacteria at 20 &#176;C and moved to 37 &#176;C on day 1 of adulthood for assay. n = 60 animals per strain per replicate. <a href="https://www.jove.com/files/ftp_upload/64091/64091fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td colspan="3">Recipe</td>
		</tr>
		<tr>
			<td>Bleach solution</td>
			<td colspan="3">1.8% (v/v) sodium hypochlorite, 0.375 M KOH</td>
		</tr>
		<tr>
			<td>Carbenicillin</td>
			<td colspan="3">100 mg/mL stock solution (1000x) in water. Store at 4 &#176;C for up to 6 months or -20 &#176;C for long-term storage</td>
		</tr>
		<tr>
			<td>FUDR</td>
			<td colspan="3">10 mg/mL solution in water. Store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td colspan="3">1 M solution in water.</td>
		</tr>
		<tr>
			<td>Lysogeny Broth (LB)</td>
			<td colspan="3">In this protocol, commercial LB was used (see Table of Materials), but all standard LB home-made recipes using Bacto-tryptone, yeast extract, and NaCl are sufficient.</td>
		</tr>
		<tr>
			<td>M9 solution</td>
			<td colspan="3">22 mM KH2PO4 monobasic, 42.3 mM Na2HPO4, 85.6 mM NaCl, 1 mM MgSO4</td>
		</tr>
		<tr>
			<td>Nematode Growth Media (NGM)</td>
			<td colspan="3">1 mM CaCl2, 5 &#181;g/mL cholesterol, 25 mM KPO4 pH 6.0, 1 mM MgSO4, 2% (w/v) agar, 0.25% (w/v) Bacto-Peptone, 51.3 mM NaCl</td>
		</tr>
		<tr>
			<td>NGM RNAi plates</td>
			<td colspan="3">1 mM CaCl2, 5 &#181;g/mL cholesterol, 25 mM KPO4 pH 6.0, 1 mM MgSO4, 2% (w/v) agar, 0.25% (w/v) Bacto-Peptone, 51.3 mM NaCl, 1 mM IPTG, 100 &#181;g/mL carbenicillin/ampicillin. Store at 4 &#176; C in dark for up to 3 months</td>
		</tr>
		<tr>
			<td>NGM RNAi DMSO</td>
			<td colspan="3" rowspan="2">1 mM CaCl2, 5 &#181;g/mL cholesterol, 25 mM KPO4 pH 6.0, 1 mM MgSO4, 2% (w/v) agar, 0.25% (w/v) Bacto-Peptone, 51.3 mM NaCl, 1 mM IPTG, 100 &#181;g/mL carbenicillin/ampicillin; 1% DMSO</td>
		</tr>
		<tr>
			<td>(control for tunicamycin)</td>
		</tr>
		<tr>
			<td>NGM RNAi TM</td>
			<td colspan="3">1 mM CaCl2, 5 &#181;g/mL cholesterol, 25 mM KPO4 pH 6.0, 1 mM MgSO4, 2% (w/v) agar, 0.25% (w/v) Bacto-Peptone, 51.3 mM NaCl, 1 mM IPTG, 100 &#181;g/mL carbenicillin/ampicillin; 1% DMSO, 25 ng/&#181;L tunicamycin</td>
		</tr>
		<tr>
			<td>Paraquat</td>
			<td colspan="3">400 mM solution in water &#8211; should be prepared fresh</td>
		</tr>
		<tr>
			<td>Tetracycline</td>
			<td colspan="3">10 mg/mL stock solution (500x) in 100% ethanol. Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Tunicamycin</td>
			<td colspan="3">2.5 mg/mL stock solution in 100% DMSO. Store at -80 &#176;C for long-term storage. This is a 100x solution (25 ng/&#181;L working solution)</td>
		</tr>
	</tbody>
</table>
Table 1. Recipes for reagents and media for protocols.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Corresponding Figure panel</td>
			<td colspan="2">Strain, Treatment</td>
			<td>Median Lifespan</td>
			<td># Deaths/# Total</td>
			<td>p-value 
			(Log-Rank)</td>
		</tr>
		<tr>
			<td rowspan="2">1A</td>
			<td colspan="2">N2, vector RNAi, 20 &#176;C</td>
			<td>17</td>
			<td>74/120</td>
			<td>--</td>
		</tr>
		<tr>
			<td colspan="2">N2, hsf-1 RNAi, 20 &#176;C</td>
			<td>11</td>
			<td>65/120</td>
			<td>&#60;0.001</td>
		</tr>
		<tr>
			<td rowspan="2">1B</td>
			<td colspan="2">N2, vector RNAi, FUDR, 20 &#176;C</td>
			<td>19</td>
			<td>120/120</td>
			<td>--</td>
		</tr>
		<tr>
			<td colspan="2">N2, hsf-1 RNAi, FUDR, 20 &#176;C</td>
			<td>11</td>
			<td>116/120</td>
			<td>&#60;0.001</td>
		</tr>
		<tr>
			<td rowspan="2">1C</td>
			<td colspan="2">N2, glp-4(bn2), vector RNAi, 25 &#176;C</td>
			<td>13</td>
			<td>115/121</td>
			<td>--</td>
		</tr>
		<tr>
			<td colspan="2">N2, glp-4(bn2), hsf-1 RNAi, 25 &#176;C</td>
			<td>4</td>
			<td>120/120</td>
			<td>&#60; 0.001</td>
		</tr>
		<tr>
			<td rowspan="3">2A</td>
			<td colspan="2">N2, vector RNAi, 20 &#176;C, 1% DMSO</td>
			<td>19</td>
			<td>85/120</td>
			<td>--</td>
		</tr>
		<tr>
			<td colspan="2">N2, vector RNAi, 20 &#176;C,&#160; 10 ng/&#181;L tunicamycin</td>
			<td>15</td>
			<td>109/120</td>
			<td>&#60;0.001</td>
		</tr>
		<tr>
			<td colspan="2">N2, vector RNAi, 20 &#176;C,&#160; 25 ng/&#181;L tunicamycin</td>
			<td>12</td>
			<td>117/120</td>
			<td>&#60;0.001</td>
		</tr>
		<tr>
			<td rowspan="3">2B</td>
			<td colspan="2">N2, vector RNAi, 20 &#176;C</td>
			<td>19</td>
			<td>84/120</td>
			<td>--</td>
		</tr>
		<tr>
			<td colspan="2">N2, vector RNAi, 20 &#176;C,&#160; 0.25 mM paraquat</td>
			<td>24</td>
			<td>91/120</td>
			<td>&#60;0.001</td>
		</tr>
		<tr>
			<td colspan="2">N2, vector RNAi, 20 &#176;C,&#160; 4 mM paraquat</td>
			<td>6</td>
			<td>50/120</td>
			<td>&#60;0.001</td>
		</tr>
	</tbody>
</table>
Table 2. Statistics for lifespan and stress resilience.

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