Journals of Gerontology: Biological Sciences

cite as: J Gerontol A Biol Sci Med Sci, 2017, Vol. 72, No. 4, 473–480
doi:10.1093/gerona/glw248
Advance Access publication February 2, 2017

Original Article

Assessing Health Span in Caenorhabditis elegans: Lessons

From Short-Lived Mutants
Jarod A.  Rollins,1 Amber C.  Howard,2 Sarah K.  Dobbins,3 Elsie H.  Washburn,4 and
Aric N. Rogers1
1
Davis Center for Regenerative Biology and Medicine, Mount Desert Island Biological Laboratory, Bar Harbor, Maine. 2College of Arts
and Sciences, University of Maine at Augusta. 3Department of Engineering, Stanford University, California. 4College of Math and Science,
California Polytechnic University, San Luis Obispo.

Address correspondence to Aric Rogers, PhD, Davis Center for Regenerative Biology and Medicine, Mount Desert Island Biological Laboratory,
159 Old Bar Harbor Road, Bar Harbor, ME 04609. E-mail: arogers@mdibl.org

Received August 16, 2016; Editorial Decision Date November 16, 2016

Decision Editor: Rafael de Cabo, PhD

Abstract
Genetic changes resulting in increased life span are often positively associated with enhanced stress resistance and somatic maintenance.
A  recent study found that certain long-lived Caenorhabditis elegans mutants spent a decreased proportion of total life in a healthy state
compared with controls, raising concerns about how the relationship between health and longevity is assessed. We evaluated seven markers of
health and two health-span models for their suitability in assessing age-associated health in invertebrates using C elegans strains not expected
to outperform wild-type animals. Additionally, we used an empirical method to determine the transition point into failing health based on the
greatest rate of change with age for each marker. As expected, animals with mutations causing sickness or accelerated aging had reduced health
span when compared chronologically to wild-type animals. Physiological health span, the proportion of total life spent healthy, was reduced
for locomotion markers in chronically ill mutants, but, surprisingly, was extended for thermotolerance. In contrast, all short-lived mutants
had reduced “quality-of-life” in another model recently employed for assessing invertebrate health. Results suggest that the interpretation of
physiological health span is not straightforward, possibly because it factors out time and thus does not account for the added cost of extrinsic
forces on longer-lived strains.

Keywords: Health—Life-span measurement—Phenotype—Sarcopenia—Invertebrate

An assumption underlying the desire for increased life span is that
it will also improve quality of life by helping ameliorate the impact
of diseases and diminution of health associated with aging. This
possibility is generally supported by evidence from model organ-
isms showing that enhanced somatic maintenance, frequently meas-
ured by increased survival under various forms of stress, coincides
with increased longevity (1–3). Other measurements of health have
shown that prolongevity interventions maintain physiology in a
youthful state, including preservation of locomotion, feeding behav-
ior, and accumulation of autofluorescent pigments in cells (4–6).
Furthermore, when genetic models of longevity are combined with
age-associated models of disease, results often indicate amelioration
or delay in the onset of pathology (7,8). Together, the evidence indi-
cates that health span can be extended through evolutionarily con-
served genes and pathways.
Although many of the studies linking enhanced longevity with
increased stress resistance have come from investigations utilizing
Caenorhabditis elegans model system, a recent study provided evi-
dence suggesting that the actual percent of life spent in a frail state,
referred to as physiological gerospan, was increased among long-
lived C elegans mutants compared with wild-type animals (9). In their
investigation, they examined several markers of health that changed
with age in well-characterized long-lived mutants and compared
them with wild-type animals. While generally confirming enhanced
chronological health span, their results suggested that the proportion
of total life spent healthy in long-lived mutants, termed physiological
health span, was shorter than that of wild-type animals. A later study
demonstrated that the reduced physiological health span of long-
lived daf-2 mutants was a result of altered foraging behavior caused
by tracking unstimulated locomotion in the presence of food (10).

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474 Journals of Gerontology: BIOLOGICAL SCIENCES, 2017, Vol. 72, No. 4
However, the reduced physiological health span of other long-lived
mutants remains unexplained. Additionally, assessment of health
span in mouse using markers for body composition, energetics, and
movement showed little association with mortality suggesting an
uncoupling of health span and life span in more complex systems
(11). These provocative studies have raised concerns about the way
in which we examine health in animal systems and the applicabil-
ity of such results to human health. The importance of considering
health span in the context of life span was made clear in the recent
convening of the Geoscience Network, which deemed extension of
life span alone as insufficient evidence of delayed aging (12).
Obstacles to the characterization of invertebrate health span
include the choice of which strain and markers to use. For the former,
we used short-lived C elegans mutants displaying signs of accelerated
aging and/or chronic illness, allowing assessment of candidate health
markers and recently reported models that assess age-associated health.
For the latter, we wanted to make a distinction between health markers
strongly dependent on biological aging and those which only have a
temporal relationship or are weakly dependent on the aging process.
Given that our chosen short-lived strains displayed signs of chronic
illness, we adopted definitions from Brody and Schneider (13) used for
chronic human diseases to make this distinction for health markers. In
their manuscript, only disorders with mortality rates that increase with
age were defined as age dependent. Applying a similar logic to the pre-
sent study, markers which failed sooner chronologically in short-lived
mutants were defined to be aging dependent, as the earlier failing of
both health and longevity is presumably regulated by similar processes.
Conversely, markers were considered time dependent if they changed
with age but not in a manner consistent with life span.
For short-lived strains, we used animals with mutations in daf-
16 (abnormal dauer formation), mev-1 (abnormal methyl viologen
sensitivity), hsf-1 (heat shock factor), and sir-2.1 (silent informa-
tion regulator). daf-16 encodes a FOXO transcription factor that
acts in the insulin/insulin-like growth factor signaling (IIS) pathway
and is required for increased life span and enhanced resistance to
stress when the IIS pathway is attenuated (14). mev-1 encodes the C
elegans ortholog for human mitochondrial succinate dehydrogenase
cytochrome b560 subunit, the mutation of which is associated with
significantly decreased life span, increased reactive oxygen species
(15), and severely impaired reproduction (16). hsf-1 encodes the
heat shock transcription factor important for mediating the response
to unfolded proteins in the cytoplasm and signals a series of pro-
longevity genes (7,17). Its expression is required for life span and
stress-resistance phenotypes associated with attenuated IIS (17) and
loss-of-function leads to defects in development and reproduction
(18,19). sir-2.1 encodes a NAD-dependent deacetylase important for
metabolic homeostasis, with lost function resulting in shortened life
span and increased sensitivity to stress (20).
Using mutants for these genes, we determined which previ-
ously used markers of health span were aging dependent and thus
informative candidates for health-span determination linked to life
span. Additionally, we found maximum bending amplitude, which is
an indicator of muscle integrity (21), to be a new aging-dependent
health-span marker with possible associations with longevity. To
determine when the healthy period of life ends, we posited an empiri-
cal method based on the rate of change for a given marker. Although
chronological health span was reduced in the chosen short-lived
mutants, physiological (time normalized) health span was actually
greater for thermotolerance in short-lived strains in this model.
Although the apparent simplicity of the physiological health-span
model suggests a straightforward interpretation, these unexpected
results indicate additional consideration is required, such as the cost
of extrinsic forces on aging, which is not taken into consideration
when time is factored out.
Materials and Methods
Nematode Culture and Strains
C elegans strains were cultured at 20°C and maintained on nema-
tode growth media (NGM) plates seeded with the Escherichia coli
strain OP50. N2 wild-type, CF1038 daf-16(mu86) I, XA8203 sir-
2.1(ok434) IV, PS3551 hsf-1(sy441) I, and TK22 mev-1(kn1) III
were obtained from the Caenorhabditis Genetics Center (CGC). All
strains were permitted to grow at least three generations under nor-
mal, unstressed conditions prior to use in experiments. Except for
thermotolerance assays, all experiments were carried out at 20°C.
Life Spans
Survival was scored starting from the first day of adulthood using 100
or more worms per strain divided among three seeded NGM plates
containing 200 µM fluorodeoxyuridine (FUDR) to prevent contami-
nation by progeny. Survival status was determined by touch provo-
cation. Worms with vulval protrusion were censored from the assay.
Differences in survival curves were calculated using the log-rank test
in the Graphpad Prism 6.0 software. Maximum life span was calcu-
lated by taking the average life span of the top 10 longest-lived worms.
Development and Fecundity
Several dozen gravid adults were allowed to lay eggs on spotted
NGM plates for 1 hour and removed. Time to egg-laying adulthood
was started from this point. Eggs were allowed to hatch and 20–22
animals were randomly picked and separated onto individual plates.
Plates bearing single young adults were checked every hour for the
presence of eggs. Animals from the development assay were trans-
ferred to fresh plates every 24 hours following initiation of egg lay-
ing. Hatched progeny and unfertilized oocytes were counted for each
plate 48 hours after removal of the egg-laying adult.
Health-Span Assays
Speed, bending angle, and worm size were assayed for each time
point by sampling from a population of age-synchronized animals
on seeded NGM plates. Worms were tracked in real time after gently
tapping plates to stimulate movement and distinguish the ability to
move from food-seeking behavior (10). Forward and backward speed
were recorded with a Stingray F504B ASG digital (Allied Technologies
GmbH) camera equipped with an AF Micro-Nikkor 60mm f/2.8D lens
(Nikon). 30-second intervals of continuous (unbroken) tracking were
analyzed using Wormlab ver 3.0 (MBF Bioscience). For pharyngeal
pumping, worms were observed individually with a Leica M165FC
stereo microscope and digitally recorded (VLC, ver. 2.1.3) on seeded
NGM plates. Pumps were counted manually by playing 30 seconds of
video at a reduced speed. For autofluorescence measurements, worms
were transferred to 1% agar pads containing 20-mM sodium azide
spotted on glass slides. Autofluorescence was measured on a Leica
M165FC stereo microscope in both the green fluorescent protein
(GFP) (GFP narrow band filter set, excitation ET470/40 nm, emission
ET510/10  nm) and the 4′,6-diamidino-2-phenylindole (DAPI) (ET
DAPI filter set, excitation AT350/50x nm, emission ET460/50m) chan-
nels using a 3-second exposure. Quantification of autofluorescence
was performed using ImageJ for mean pixel intensity and corrected
for background fluorescence. Thermotolerance assays were conducted
with 100 worms (aged as indicated) and distributed among two plates
at 35°C. Survival was measured every hour by touch provocation.
Journals of Gerontology: BIOLOGICAL SCIENCES, 2017, Vol. 72, No. 4
Resistance to oxidative stress was tested using worms distributed to
individual wells (12 worms/well, 4 wells/strain) of a 24-well culture
plate containing 7.5-mM hydrogen peroxide in S basal buffer. Survival
was measured every other hour by touch provocation. All health-span
assays were independently repeated three times.
Statistical Analysis
All statistics other than log-rank tests were performed in the soft-
ware package R (ver 3.1.3). For each health marker, analysis of
variance was used to estimate effects of genotype, time, and the
interaction of the two. Replicate experiment was used as a block-
ing factor. Post hoc Tukey’s honest significant difference was used
to compare means between genotypes and time points. The rate of
change in each aging-dependent health marker was modeled using
linear regression. The linear regression estimated the effect of time
with replicate experiment as a blocking factor. As maximum speed
and maximum amplitude did not decrease until Day 5 for most gen-
otypes, Day 1 was omitted from the linear regression analysis for
these parameters. For similar reasons, heat stress survival on Day 2
was omitted from the regression analysis. To determine the onset of
rapid health decline in wild type, linear regression was used as above
but with a sliding window of 3 consecutive time points. The time by
which each genotype entered gerospan was imputed using the regres-
sion function from the linear models. Because measurements were
not taken on Day 19 for thermotolerance, the missing values were
extrapolated for each genotype using the linear regression from the
other time points. Cumulative quality-adjusted survival was deter-
mined for each genotype as described by Hahm and colleagues (10)
by first calculating the normalized health metric score and then the
quality-adjusted survival for each aging-dependent trait.
Results
Reduced Whole-Life Health and Accelerated Aging
in Short-Lived Mutants
Testing was carried out to confirm the short-lived and impaired
stress response status of daf-16(mu86), sir-2.1(ok434), hsf-1(sy441),
and mev-1(kn1) mutants. Life-span survival curves are shown in
Figure  1A and median life span ranged from 26% to 38% below
that of N2 wild type (17.0  days for sir-2.1(ok434), 16.5  days for
mev-1(kn1), 15.5  days for daf-16(mu86), and 14.3  days for hsf-
1(sy441)). Information pertaining to statistics and replicates are
reported in Supplementary Table 1. Survival under heat or oxidative
stress was also reduced in these strains (Figure 1B and C), indicating
defects in somatic maintenance.
In long-lived strains, increased life span and resistance to
stress often coincide with tradeoffs in growth and reproduction.
Such defects in short-lived strains would indicate illness at all life
stages. Growth was assessed by analysis of nematode size quanti-
fied on Day 5 of adulthood, by which time all strains were com-
pletely grown. Only mev-1 mutants were significantly smaller
than wild-type mutants (106.2  mm2 vs 154.7  mm2, p  =  2.87E-13,
Tukey’s test; Figure  1D). Development was assessed by measur-
ing the time required for each strain to develop from an egg to an
egg-laying adult (Figure  1E). Development was delayed in hsf-1
(92.8 h compared with 89.2 h in wild type, p = 6.85E-06, Tukey’s
test) and severely delayed in mev-1 (112.5 h, p = 1.0E-16, Tukey’s
test). Reproductive health was assessed by counting hatched progeny
(Figure  1F), and unfertilized oocytes (Supplementary Figure  1), as
well as by measuring the distribution and duration of reproduction
(Supplementary Figure 2). Significantly reduced number of progeny
475
Figure 1.  Short-lived Caenorhabditis elegans mutants demonstrate deficiencies
in health during development and adulthood. (A) Representative survival
curves of N2 wild-type and short-lived mutants from four biological repeats
(Supplementary Table  1). Survival curves were compared using Mantel-Cox
log-rank tests and all short-lived mutants had significantly (p < .05) shorter
median life spans than wild-type N2 (mean survival difference for daf-16(mu86)
was −33%, mev-1(kn1) was −29%, hsf-1(sy441) was −37%, and sir-2.1(ok434)
was −26%). (B) Mean survival at 35°C on Day 6 of adulthood was reduced
in daf-16, mev-1 and hsf-1, but not in sir-2.1 (p = 8.0E-8, 2.4E-11, 2.4E-11, and
0.79, respectively, calculated using Tukey’s test). Data were pooled from three
independent experiments, n  =  300/strain. (C) Mean survival was reduced in
all short-lived strains in response to 7.5-µM hydrogen peroxide at Day 5 of
adulthood (p < .0001, Tukey’s test). Data were pooled from three independent
experiments, n = 138/strain. (D) The size of selected strains based on imaged
area on Day 5 of adulthood showed that only mev-1 were smaller compared
with wild type (p = 2.8E-13, Tukey’s test). Data were pooled from 3 independent
experiments, n = 45/strain. Data were pooled from 3 independent experiments.
E) Time to development for each strain as measured from time to egg laying
to first day of adulthood. Compared with wild type, mev-1 and hsf-1 had a
longer development time while daf-16 and sir-2.1 were similar (p  =  1E-16,
p = 1.0E-16, p = .088 and p =.049, respectively Tukey’s test). Data were pooled
from 4 independent experiments, n = 70/strain. F) Fecundity of each selected
strain as measured by the number of eggs hatched. The mutants mev-1 and
sir-2.1 had reduced hatched progeny compared with wild type (p  =  1.0E-
12 and 1.4E-12, respectively in Tukey’s test). Data were pooled from four
independent experiments, n = 64/strain. Error bars indicate SEM. ***p < .001.
were produced in mev-1 (129 compared with 259 in wild type,
p = 1.02E-12, Tukey’s test) and sir-2.1 (199, p = 1.44E-12) mutants
(Figure 1F). Interestingly, a significant number of unfertilized oocytes
were laid in the daf-16 (78.5, p = 1.03E-12, Tukey’s test) and hsf-1
(41.7, p = 1.07E-12) backgrounds compared with wild-type N2 (0.7;
Supplementary Figure  1) near the end of the reproductive period,
indicative of dysregulated ovulation. However, there was not a sig-
nificant impediment to the production of viable progeny in daf-16
(254, p  =  .95) and hsf-1 (246, p  =  .34) compared with wild type
(259) in the conditions tested (Figure  1F). Finally, the bulk distri-
bution of reproduction in sir-2.1 animals was shifted to after the
second day of egg laying, unlike in the wild-type background where
more than 50% of viable progeny were laid within the first 48 hours
of the onset of egg laying (Supplementary Figure 2).
Together, the data for longevity, stress resistance, development,
and reproduction suggested that the hsf-1, mev-1, and sir-2.1 mutants
used here model sickness due to their deficits in early- and late-life
metrics of health. While the high number of unfertilized oocytes in
daf-16 animals suggests anomalous regulation of ovulation, total
fecundity was not significantly reduced. Conversely, daf-2 mutants
produce fewer unfertilized oocytes compared with wild type (22),
476 Journals of Gerontology: BIOLOGICAL SCIENCES, 2017, Vol. 72, No. 4
presumably due to increased daf-16 activity. Thus, with the absence
of major developmental or reproductive deficiencies, daf-16 mutants
may be considered a model of accelerated aging, although lack of
functional DAF-16 means these animals have chronically reduced
stress resistance. With these characterizations in mind, we analyzed
the lifelong performance of health markers concerning motility, feed-
ing, autofluorescence, and stress resistance.
Locomotion and Thermotolerance Are
Aging-Dependent Markers of Health
Locomotion is dependent on the integrity of muscle mass, connective
tissue, and neuronal signaling (21,23–26). We investigated changes
in movement with age using digital video recording and nematode
tracking software (Wormlab 3.1, see Methods). Mean and maxi-
mum speed declined over time in all strains tested (Figure 2A and B).
Short-lived mutants were typically slower than wild type for both
mean and maximum speed. By Day 5 of adulthood, the maximum
speeds of daf-16, sir-2.1, mev-1, and hsf-1 were slower than wild
type by 30%, 32%, 48%, and 51%, respectively. Differences in
maximum speed increased by Day 15 (slower by 60%, 70%, 59%,
and 58%, respectively). The decline in mean speed began after Day
1 for the short-lived mutants and after Day 5 for wild type, with
similar results for maximum speed except that the decline began
after Day 5 for daf-16 and hsf-1. As mean and maximum speed both
declined with age sooner in all short-lived mutants compared with
wild-type animals, we categorized these traits as aging dependent.
Of the markers tested in the present study, maximum speed had the
highest correlation with survival rate (Table 1).
Locomotion is also characterized by maximum bending ampli-
tude, which depends on thick filament organizing centers that trans-
mit the force of muscle contraction into locomotion (21). Maximum
bending amplitude decreased after Day 5 in all strains (Figure 2C). By
Day 12 and beyond, values for all short-lived strains were significantly
Figure  2.  Locomotion and thermotolerance were reduced sooner
chronologically in short-lived mutants. Mean and maximum speed,
maximum bending amplitude, and thermotolerance throughout life are
shown in A–D. Error bars indicate SEM. Data were pooled from three
independent experiments. n = 45/strain for locomotion parameters. n = 300/
strain for thermotolerance. *p < .05, ***p < .001, as compared with N2 wild
type on the same day by analysis of variance with post hoc Tukey’s test.
reduced compared with wild type, in accordance with an aging-
dependent marker. To our knowledge, this is the first characterization
of maximum bending amplitude with age in C elegans on solid media.
Thus, locomotion markers in Figure  2A–C are all aging dependent,
making them strong candidates for health-related diagnostics of aging.
Heat stress can disrupt cellular homeostasis though protein mis-
folding. Thermotolerance decreased with age in all strains tested,
and short-lived mutants had significantly reduced thermotolerance
compared with wild-type worms. The strain-specific reduction
was greatest for hsf-1, which was 34% (p  =  3.0E-09), followed
by 29% for mev-1 (p = 1.3E-06), 28% for sir-2.1 (p = 4.1E-06),
and 24% for daf-16 (p = 2.6E-04) on Day 13 (Figure 2D; Tukey’s
test). Results indicate that thermotolerance is an aging-dependent
marker.
Autofluorescence and Pharyngeal Pumping Are
Time-Dependent Markers of Health
Both DAPI and GFP channel autofluorescence have been used as
aging metrics in C elegans (27,28). To better understand the relation-
ship between autofluorescence and age, we measured DAPI chan-
nel (350/50ex,450/50em) and GFP channel (480/20ex, 510/20em)
autofluorescence throughout adulthood. DAPI channel fluorescence
was seen to increase with age in N2 wild-type, mev-1 and hsf-1
strains (Figure  3A). Surprisingly, daf-16 DAPI autofluorescence
stopped increasing after Day 12, whereas sir-2.1 remained steady
throughout life. Although GFP channel autofluorescence increased
with time for all strains, values in daf-16 and sir-2.1 mutants were
never greater than wild-type and were significantly lower by Day 19
(p = .039 and .002 respectively, Tukey’s test, Figure 3B). Conversely,
mev-1 mutants displayed significantly higher GFP autofluorescence
by Day 19 (p = .0002). hsf-1 mutants had significantly higher GFP
autofluorescence on Day 12 of adulthood (p = .006) but not on Day
15 (p = .99). Based on results for the strains used, DAPI and GFP
Figure 3.  Autofluorescence and pharyngeal pumping were not altered sooner
in short-lived mutants. (A) DAPI channel autofluorescence, (B) GFP channel
autofluorescence, and (C) pharyngeal pumping of wild-type and short-lived
mutants throughout life. Insufficient numbers of hsf-1 mutants survived to
Day 19 to take complete measurements for that time point. Pumping was
not significantly different at any time point for any strain compared with wild
type. Error bars indicate SEM. Data were pooled from three independent
experiments. n = 45/strain for autofluorescence measurements, n = 200/strain
for pharyngeal pumping. *p < .05, **p < .01, ***p < .001, as compared with
wild type on the same day by analysis of variance with post hoc Tukey’s test.
Journals of Gerontology: BIOLOGICAL SCIENCES, 2017, Vol. 72, No. 4
channel autofluorescence are supported as time-dependent markers
of health.
The ability of the worms to feed is based on the pumping rate of
their pharynx, which they modulate according to availability of food
(29) and which has been used as a marker for health in C elegans
(4,5,27). Pumping rates were measured on Days 1, 5, 8, 12, and 15,
after which no appreciable pumping was detected. Although pump-
ing declined over time (Figure 3C), rates were similar or higher in
short-lived mutants compared with wild type, indicating that pump-
ing is time dependent. This time dependency was also observed
for long-lived mutants (9) where pumping was not detected after
15 days.
Chronological Health Span Is Reduced in
Short-Lived Strains
To further investigate the relationship between health span and life
span, we calculated the amount of time spent healthy in wild-type
animals for each marker. Because candidate aging markers do not
always decrease (eg, autofluorescence; Figure 3) nor change by more
than 50% (maximum bending angle; Figure 2), we implemented a
method that uses the most dynamic period of change taking place
in a wild-type animal to determine when health falters for a given
marker. Thus, this method is customized to the natural transition for
the health marker being used. This was determined by measuring
rates of change over a sliding window of three time points, with the
last time point in the window that demonstrates the steepest slope
indicating the first time point of the geriatric state (see Methods).
Based on this method, which we refer to as Sliding Window Analysis
of Vigor, the onset of gerospan for aging-dependent markers in
N2 wild-type animals was Day 12 for speed and maximum speed,
Day 13 for thermotolerance, and Day 19 for maximum bending
amplitude (Table 2; Supplementary Figure 3). With these transition
points, the percent changes from peak values at the time of gero-
span onset were near middle- to late-middle age for average speed,
maximum speed, and thermotolerance (36.3%, 48.3%, and 43.6%,
respectively; Table 2). However, the onset of gerospan for maximum
bending amplitude was 72.8%, indicating that it is the only aging-
dependent marker with a transition point that occurs in late life,
closer to the time humans are typically characterized as entering
a geriatric state. In order to determine analogous transitions into
the geriatric state in the short-lived strains, interpolation using lin-
ear regression models (Supplementary Figure 4 and Supplementary
Table 2) was carried out based on wild-type transition values.
The transition to chronological gerospan using aging-depend-
ent markers, based on interpolation of linear regression data,
occurred earlier in short-lived mutants and is presented graphically
in Figure  4A–D. The transition for daf-16, mev-1, hsf-1, and sir-
2.1 for maximum speed was 8.1  ±  0.3, 5.6  ±  0.5, 4.6  ±  0.5, and
8.7 ± 0.2 days, respectively, which were all reduced compared with
wild type (13.4 ± 0.3 days; Figure 4B). Qualitatively similar results
Table 1.  Survival Rate Is Highly Correlated With Maximum Speed and Well Correlated With Other Health Markers. Spearman Correlations
Between Survival Rate and Each Health-Span Parameter
Speed Max Speed Max Amp
Coefficient 0.88 0.95 0.69
p Value 9.7E-11 1.3E-15 2.5E-05
Note: DAPI = DAPI channel autofluorescence; GFP = GFP channel autofluorescence; Max Amp = maximum bending amplitude; Max Speed = maximum move-
ment speed; Pumping = pharyngeal pumping; Speed = mean movement speed.
477
were obtained for mean speed (Figure  4A). The onset of gerospan
for maximum bending amplitude was 9.6  ±  0.5  days for daf-16,
4.5 ± 0.9 days for mev-1, 7.6 ± 0.5 days for hsf-1, and 9.4 ± 0.4 days
for sir-2.1, earlier than wild type (18.6 ± 2.0 days) (Figure 4C). The
onset of gero span for thermotolerance (Figure  4D) was also ear-
lier than wild type (15.3  ±  0.2  days) in daf-16 (13.3  ±  0.1  days),
mev-1 (13.5  ±  0.1  days), hsf-1 (12.1  ±  0.3  days), and sir-2.1
(14.0 ± 0.2 days). Results were in line with expectations for intrinsi-
cally health-deficient animals.
Physiological Comparisons Reveal That Health Span
Is Nonproportional With Life Span
We calculated the physiological health span for each aging-
dependent marker in Figure  4E–H. Health span for maximum
speed was reduced in daf-16 (40.9 ± 1.6%), sir-2.1 (25.8. ± 2.2%),
mev-1 (23.6. ± 2.5%), and hsf-1 (41.1  ±  1.2%) compared with
wild type (49.7 ± 1.3%; Figure 4F), with similar results for mean
speed (Figure  4E). Physiological health span for maximum bend-
ing amplitude was also reduced in daf-16 (48.5  ±  2.4%), hsf-1
(20.8 ± 4.0%), and sir-2.1 (44.8 ± 1.8%) as compared with wild
type (35.2  ±  1.4%; Figure  4G). Surprisingly, physiological health
span for thermotolerance was extended in all of the short-lived
mutants (daf-16: 60.8 ± 0.5%, mev-1: 55.0 ± 0.4%, hsf-1: 55.6% ±
0.5%, and sir-2.1: 58.3  ±  0.4%) compared with wild type
(47.0 ± 0.4%; Figure 4H). As the observed maximum life span of a
strain may be dependent on the size of the population, we addition-
ally calculated physiological health span for each strain normalized
by their median life span (Supplementary Figure 5). However, no
qualitative differences in the physiological health span were evi-
dent between the two methods. These results make interpreting
previously reported decreases in physiological health span markers
in long-lived mutants (9) challenging and suggest that the apparent
simplicity of health-span metrics normalized to remove time may
be misleading.
For comparison, we also evaluated strains using a cumula-
tive quality-adjusted survival metric (10,30) (Figure 4I–L). In this
method, survival rate and health performance at each time point
are first normalized to the maximum value from wild type and
then multiplied together to yield a survival curve adjusted by the
quality of life experienced for each strain. The area under the qual-
ity-adjusted survival curve between each consecutive time point is
then calculated and cumulatively summed to provide a diagnostic
of both total quality and duration of life. Based on this analysis,
wild-type worms have the highest quality of life for all aging-
dependent markers. A similar reduced total quality of life for daf-
16 was reported for maximum speed, as compared with wild type
(10). Unlike physiological health span, the quality-adjusted metric
does not factor out time, which may be important if extrinsic fac-
tors that influence aging play a significant role in health among
differently lived strains.
Pumping DAPI GFP Thermotolerance
0.89 −0.74 −0.37 0.89
7.3E-11 3.7E-06 0.045 7.9E-11
478 Journals of Gerontology: BIOLOGICAL SCIENCES, 2017, Vol. 72, No. 4

Table  2. Empirical Determination of the Geriatric State for from movement speed, as C elegans mutants deficient in components
Individual Health Markers of force-transmitting sarcomeres exhibited differences in bending
amplitude but not locomotion (21). Out of the aging-dependent
Marker Day Mean Max % Max
markers, bending amplitude was found to fail latest in life. A previ-
Speed (µm/s) 12 75.2 207.3 36.3 ous characterization of the locomotion of C elegans while swimming
Max speed (µm/s) 12 188.7 390.2 48.3 reported an increase in the maximum difference in curvature with
Max amplitude (µm) 19 150.6 206.9 72.8 age. These two opposing results suggest that bending amplitude on
Thermotolerance (hours) 13 2.3 5.3 43.6 solid media and curvature while swimming measure different aspects
of health and behavior. Supporting this notion, the kinematics of
Note: % Max  =  the percentage of the maximal value when geriatric; crawling has been shown to be distinct from that of swimming and
Day = day of adulthood geriatric onset for wild-type N2 worms; Max = max- is characterized by a greater amplitude and smaller frequency (32).
imum value each marker observed in wild-type over life span; Max
Finally, although the pharyngeal muscle may show variable deterio-
Amp = maximum bending amplitude (µm); Max Speed = maximum movement
ration among strains (not tested), cessation of pumping was largely
speed (µm/s); Mean = mean value for each marker when N2 becomes geriatric;
invariant among wild-type and short-lived strains, similar to findings
Speed = mean movement speed (µm/s).
in long-lived mutants (9), suggesting that this marker is related to
longevity mainly by virtue of being dependent on the passage of time.
Cellular autofluorescence, a second category with the poten-
tial to be a diagnostic of aging, can occur due to accumulation of
endogenous fluorescent particles such as lipofuscin, advanced glyca-
tion end products, flavins, dihydronicotinamide-adenine dinucleo-
tide phosphate, elastin, and collagen (6,33), each with distinct but
sometimes overlapping spectral excitation and emission properties.
Of these particles, the accumulation of lipofuscin, the oxidized bi-
products of lysosomal degradation, has been a focus in mammals
as a marker of aging (34). In worms, lipofuscin accumulation has
been proposed as a marker of aging by measuring changes in auto-
fluorescence using a DAPI channel filter (27). However, Coburn and
colleagues (28) demonstrated that the presence of DAPI channel
fluorescence did not increase with induced oxidative stress nor with
age in C elegans. Instead, they showed that this autofluorescence
occurs as a burst upon death and concluded that the signal was due
to anthranilate acid esters originating from gut granules. Although
that study and the present study were able to detect significant
increases in GFP channel autofluorescence with age, they were not
Figure  4.  Physiological health span is decreased for locomotion markers and
consistently greater in short-lived strains and so failed the criteria to
increased for thermotolerance in short-lived mutants. Chronological health be considered an aging-dependent marker. However, it is important
span and gerospan for movement speed, maximum speed, maximum bending to note that there are some differences in the way in which channel-
amplitude, and thermotolerance markers were calculated in A–D, respectively. specific fluorescence is measured (eg, see DAPI filter settings in (6,35)
(E,H) Same as in A–D, but for physiological health span and gerospan. Health compared with (9,28), and ones used in the present study). Thus,
span and gerospan are indicated by lighter and darker hues, respectively. Error
autofluorescence cannot be ruled out as a possible aging-dependent
bars indicate SEM for the prediction of the onset of gero span. The cumulative
marker, but will require standardization in approach and further
quality-adjusted survival, at metric sensitive to both the total quality and duration
of life was calculated for speed, maximum speed, maximum amplitude, and characterization of what is actually measured at specific wavelengths
thermotolerance in I–L. in C elegans.
The third category of health used to investigate aspects of aging
in C elegans involves proteostasis and the ability to maintain somatic
Discussion
function, measured by survival, in the presence of acute stress.
Evaluation of Health-Span Markers Mechanisms contributing to proteostasis are necessary for survival
Three major categories of health are frequently used to investigate under heat stress (36) and decline with age (37). On the first day of
aspects of aging in C elegans. They include muscle function/integ- adulthood, proteostatic maintenance undergoes a dramatic transi-
rity, cellular accumulation of autofluorescent pigments, and resist- tion in C elegans that can be measured in hours (38,39). As one of
ance to acute perturbation of homeostasis, particularly proteostasis. the most robust aging-dependent markers, we find a second transi-
In the clinic, muscle function and integrity with age are judged by tion that occurs in midlife for wild type that is delayed in short-lived
muscle wasting, or sarcopenia, which is diagnosed according to gait strains relative to their life span. Although the result is surprising, the
speed, grip strength, and muscle mass (31). In C elegans, movement ability to maintain proteostasis may be more influenced by extrinsic
speed is akin to human gait speed and maximum movement speed aging factors than other markers.
has been suggested to be the one of the most informative metrics of
health in C elegans due to its high correlation with longevity and Considering a Role for Extrinsic Factors in
ability to predict remaining life span (10). In agreement with this, Health Span
maximum speed was the highest correlated with survival out of the Mortality is determined by a combination of intrinsic and extrin-
markers tested in the present study. We also considered maximum sic factors. Intrinsic aging is thought to be dependent on somatic
bending amplitude as a marker akin to grip strength that is distinct maintenance and senescence, whereas extrinsic aging is driven by
Journals of Gerontology: BIOLOGICAL SCIENCES, 2017, Vol. 72, No. 4
environmental conditions (40). Although intrinsic and extrinsic fac-
tors can affect longevity and health, the relative contribution on each
is still largely unknown. Our assessment of aging-dependent health
parameters included the binary separation of health span and gero-
span based on the physiological age of each strain. When comparing
health span physiologically, time is factored out, but this discounts
the impact of extrinsic factors in longer-lived animals, which in the
current study is the N2 wild-type strain. For example, analysis of
thermotolerance indicated physiologically extended health span in
all short-lived mutants compared with wild type (Figure 4H). This
disproportionality between health span and life span could be due to
a requirement of the passage of time to allow extrinsic factors, like
background radiation (41), to damage proteins to the point where
thermotolerance is impacted. Thus, although physiological compari-
sons have limited use in accessing the overall quality of life a particu-
lar intervention may provide, they may be beneficial in estimating
the susceptibility of a particular health marker to extrinsic forces.
The Relative Importance of Life Span and
Health Span
The question remains, is the disproportionality between life span
and health span cause for concern in the translation of aging
research? On one hand, the extended period of frailty observed
in C elegans long-lived mutants could have “disastrous economic
and social consequences if applied to humans” (9). On the other
hand, we could apply reduction ad absurdum to this notion with
our results from short-lived mutants. If longer life span without a
proportional increase in health span is disastrous, then conversely,
shorter life span with a disproportionately longer health span, like
we observed for thermotolerance, would be advantageous. Although
it may be true that there are economic benefits of having a shorter-
lived, healthier population, such a proposition brings to mind images
of a dystopian world akin to that portrayed in Logan’s Run, where
the aged are culled on their 30th birthday before they can burden
society. Additionally, this view of longevity research is missing one
important consideration, that time itself is a precious commod-
ity which scientists and economists alike have strived to appraise
(42–44). Therefore, while chronological versus physiological com-
parisons are useful to evaluate the effect environment plays in the
deterioration of health, evaluating the utility of pathways that affect
aging for therapeutic potential is best considered using metrics that
account for both health and time.
Supplementary Material
Supplementary data are available at The Journals of Gerontology,
Series A: Biological Sciences and Medical Sciences online.
Funding
This work was supported by grants from the National Institute on Aging of
the National Institutes of Health (R00AG037621) and by the Ellison Medical
Foundation (AG-NS-1087-13), both to A.R. Additionally, this project was
supported by Institutional Development Awards (IDeA) from the National
Institute of General Medical Sciences of the National Institutes of Health
(grant numbers P20GM0103423 and P20GM104318, respectively).
Acknowledgments
The authors thank George Sutphin and Santina Snow for discussions and
critical comments. Some strains were gratefully provided by the CGC, which
479
is funded by the NIH Office of Research Infrastructure Programs (P40
OD010440).
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