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Increased ROS Production: A Component of the
Longevity Equation in the Male Mygalomorph,
Brachypelma albopilosa
Francois Criscuolo
1
*, Candide Font-Sala
2
, Frederic Bouillaud
2
, Nicolas Poulin
1
, Marie Trabalon
1
1Institut Pluridisciplinaire Hubert Curien, De
´partement Ecologie, Physiologie et Ethologie, CNRS-UDS, UMR 7178, Strasbourg, France, 2BIOTRAM, Universite
´Paris
Descartes, CNRS UPR9078, Faculte
´de Me
´decine Necker-Enfants Malades, Paris, France
Abstract
Background:
The diversity of longevities encountered in wildlife is one of the most intriguing problems in biology.
Evolutionary biologists have proposed different theories to explain how longevity variability may be driven by bad genes
expression in late life or by gene pleiotropic effects. This reflexion has stimulated, in the last ten years, an active research on
the proximal mechanisms that can shape lifespan. Reactive oxygen species (ROS), i.e., the by-products of oxidative
metabolism, have emerged as the main proximate cause of ageing. Because ROS are mainly produced by the mitochondria,
their production is linked to metabolic rate, and this may explain the differences in longevity between large and small
species. However, their implication in the sex difference in longevity within a species has never been tested, despite the fact
that these differences are widespread in the animal kingdom.
Methodology/Principal Findings:
Mitochondrial superoxide production of hemolymph immune cells and antioxidant and
oxidative damages plasma levels were measured in adult male and female B. albopilosa at different ages. We found that
female spiders are producing less mitochondrial superoxide, are better protected against oxidative attack and are then
suffering less oxidative damages than males at adulthood.
Conclusions/Significance:
In tarantulas, once reaching sexual maturity, males have a life expectancy reduced to 1 to 2 years,
while females can still live for 20 years, in spite of the fact that females continue to grow and moult. This study evidences an
increased exposure of males to oxidative stress due to an increase in mitochondrial superoxide production and a decrease
in hemolymph antioxidant defences. Such a phenomenon is likely to be part of the explanation for the sharp reduction of
longevity accompanying male tarantula maturity. This opens several fundamental research roads in the future to better
understand how reproduction and longevity are linked in an original ageing model.
Citation: Criscuolo F, Font-Sala C, Bouillaud F, Poulin N, Trabalon M (2010) Increased ROS Production: A Component of the Longevity Equation in the Male
Mygalomorph, Brachypelma albopilosa. PLoS ONE 5(10): e13104. doi:10.1371/journal.pone.0013104
Editor: Samuli Helle, University of Turku, Finland
Received March 4, 2010; Accepted September 9, 2010; Published October 1, 2010
Copyright: ß2010 Criscuolo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by finding from Centre National de la Recherche Scientifique (CNRS). The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: francois.criscuolo@c-strasbourg.fr
Introduction
The origin of the chelicerata phylum finds its roots at the
precambrian ages and this long history has allowed extensive
diversification of the chelicerate life history traits. Mygalomorphae
are large, predatory spiders of the family Theraphosidae and adult
sizes range from 4 to 10 inches across the outspread legs, spiders
being able to catch prey size up to mice and birds [1]. Theraphosidae
are renowned for their longevity. For example in the spiders of the
subfamily Theraphosinae, the males can reach maturity in 4–8 years,
living then only one – two year after the last molt (imaginal molt).
In the same species, females reach maturity in 5–10 years and then
live 20 more years while continuing to grow and resuming
numerous reproductive cycles [2].
Sex differences in longevity are widespread in the animal
kingdom and are indeed in general biased towards females [3,4].
In a recent study, genetic modified mice having only female genes
are living longer than control mice [5]. However, the mechanisms
of sex differences in longevity remain to be determined and are
likely to be species specific. For example, telomere length which is
linked to longevity is longer in females than males in humans [6,7].
Additionally, telomere loss after an infection is greater in males
than in females [8]. Because telomere loss is mainly triggered by
oxidative stress [9], differences in oxidative stress may be an
important factor in determining female and male life expectancy
[10,11]. Therefore intra-specific differences in longevity would
result from a more rapid senescence in males due to a ‘‘live fast,
die young’’ trade-off, which can be traduced, for example, by
higher chronic oxidative stress [12,13].
Mygalomorphes are an ideal model to tackle sex differences in
life history strategies, and to look at the intimate physiological
mechanisms that uphold the different trade-offs. Once reaching
sexual maturity, both sexes largely differ in their living modes.
Males adopt a very active life notably related to the active search
for reproductive partners [14] which induces higher energetic
demands and leads to a higher resting metabolic rate (RMR)
PLoS ONE | www.plosone.org 1 October 2010 | Volume 5 | Issue 10 | e13104
compared to females [15]. Based on the ‘‘free radical theory of
ageing’’, higher metabolic rate should induce higher ROS
production and without the appropriate antioxidant protective
answer, high RMR will be associated with increased senescence
rates [16]. However, counter-intuitive results have been found at
the individual level in mice, where high RMR are associated with
longer lifespan because of specific mitochondrial adaptations
[17,18]. It remains that males generally show at the end of the
reproduction period the resulting consequences of a higher rate of
ageing, by losing their abdomen hairs, losing body mass and an
overall deterioration of body condition and of locomotor abilities
[19].
In the present paper, we tested in captive tarantula Therapho-
sidae (Brachypelma albopilosa) whether sexual maturity is accompa-
nied by an increase in oxidative stress in males. To assess oxidative
stress, we measured superoxide mitochondrial production in
haemocyte cells and the ratio antioxidant defences/oxidative
damages in the hemolymph. Based on the free-radical hypothesis
of ageing, we predict that sexually mature males should have an
enhanced production of mitochondrial ROS associated with a less
efficient antioxidant barrier, increasing their chronic level of
oxidative damages.
Results
Description of life trajectories of captive tarantulas is given in
Table 1, confirming that in controlled conditions, females lived
longer than males (data presented in Table 1 were obtained from a
different set of individuals). Additionally, there was a significant
difference in body mass between sexes, males B. albopilosa being
nearly 3 fold lighter than females (10.8460.78 vs. 28.8860.80 g,
F
1, 28
= 260.7, P,0.001).
Antioxidant capacity and level of oxidative damage
Fig. 1 shows the antioxidant capacities and the hydroperoxide
levels found in male and female tarantula hemolymph. Antioxi-
dant barrier was significantly higher in females (382.3618.7 vs.
169.3620.6 mM of HClO neutralised, F
1, 19
= 58.55, P,0.001).
Conversely, the concentration of hydroperoxides was higher in
males (6.0961.00 vs. 2.9260.85 mM H
2
O
2
neutralised, F
1, 11
=
5.841, P= 0.036), thereby showing that, on average, females have
a more efficient antioxidant capacity and accumulated less
oxidative damages.
Cell superoxide measurement
MitoSOX fluorescence was not found in the nucleus (a
noticeable drawback), thereby confirming the specificity of the
probe as a superoxide indicator (Fig. 2). The rate of oxidation of
MitoSOX
TM
Red probe in haemolymph cell determined in B.
albopilosa was significantly different between sexes (Linear Mixed
Model, F
1, 26.71
= 19.14, P,0.001). Males exhibited a higher mean
rate of superoxide accumulation compared to females (1.4760.18
vs. 0.5060.18 slope value, Fig. 3) and this was independent of any
session effect (estimated covariance parameter: 0.0860.13).
Two different factors may account for this difference in
superoxide production: body mass, and the time elapsed from
the imaginal moult because of the sex differences in adult lifespan
and development. We checked for within sex-effect (Table 2) and
we found that: (i) superoxide production was not affected either by
the time or by the body mass residuals in females and (ii)
superoxide production increased with time when male tarantulas
became adult, independently of body mass variability with time
(Fig. 4).
Discussion
Male tarantulas are exposed to higher risk of oxidative stress.
Rate of superoxide production measured on haemolymph cells
revealed a significantly higher production in males. Moreover, a
lower antioxidant capacity of the haemolymph was found in
males. Therefore, reaching adulthood is synonymous of an
unbalanced ROS production which can be one of the proximal
mechanisms explaining the rapid ageing process observed in male
tarantulas. Interestingly, adult females do not suffer from an
increased oxidative stress with age, thereby suggesting that the
roots of accelerated ageing in males are associated with, or
originate from, the metabolic changes that accompanied male
sexual maturity.
Two previous studies investigated physiological differences
between male and female tarantulas and tried to established
relationships between life history sexual dimorphism (males being
active while females stayed sedentary in their burrows) and
physiological changes [20,15]. Because for some insects, repro-
ductive success largely depends on male searching ability [21], it
must have driven the selection of physiological traits that allow
males to sustain intensive locomotor activity, such as a higher
RMR. High RMR allows greater workload at peak times [22] and
it seems logical that basal metabolic rate has been found higher in
male than in female spiders [15]. However, the same authors
Table 1. Longevity of Brachypelma albopilosa in laboratory
(n = 30 for males and n = 10 for females).
Males Females
Duration of development Days Days
Emergence from egg sac to adult moult
(maturity)
1792622 2034629
Adult period: adult - death 4376105 57906712
Total longevity 22296127 78246741
These data were measured from different individuals than the ones used for
superoxide production. The duration of periods were compared between males
and females using a t-test. All inter-sex comparisons are significant, P,0.001.
doi:10.1371/journal.pone.0013104.t001
Figure 1. Antioxidant hemolymph barrier measured on male
(white cross hatched bars, n = 9) and female (grey cross
hatched bars, n = 11) tarantulas, as well as reactive oxygen
metabolite levels (5 males (white bars) and 7 females (grey
bars)). Female tarantulas showed a two fold higher antioxidant
capacity than males (P,0.001), and a two fold reduced quantity of
damaged bio-molecules in their haemolymph (P= 0.036).
doi:10.1371/journal.pone.0013104.g001
ROS in Tarantulas
PLoS ONE | www.plosone.org 2 October 2010 | Volume 5 | Issue 10 | e13104
attested that peak metabolism is not submitted to sexual
dimorphism, females being able to sustain the same maximal
locomotor effort than males [20]. Because high RMR increases the
overall energy demands of the body, one possibility is that males
are not able to meet the overall energy requirement during
reproduction, and are then facing an impossible trade-off, ending
with the sacrifice of body maintenance. Data describing free-living
male energy budget during reproduction should help to test this
possibility. However, our growing conditions did not allow intense
locomotor activity (cages being too small) and spiders were fed ad
libitum. Males apparently continued to feed until reaching an
advanced age (Trabalon, unpublished data), and despite this
apparent balanced energy state, they were still presenting
progressive body deterioration. This illustrates that male senes-
cence rate at adulthood is not the mere consequence of a male
lifestyle. Oxidative stress is the main mechanism put forward to
explain the metabolic rate theory [16,23], which stipulates that
lifespan is inversely correlated to metabolic rate. Three important
predictions derive from this metabolic rate theory of aging: (i)
death probability increases with age, (ii) metabolic rate and
lifespan should be inversely correlated to body size (large animals
will have lower metabolism and longer life expectancy) and (iii)
lifespan should be inversely correlated to ROS production and
positively with antioxidant power. Our data give some supports to
the two last predictions. However, the metabolic rate of living
theory is faced with recent contradictions [24,25,26] and there is a
need to further characterize how metabolic rate and oxidative
stress can be or could be modified throughout the life stages in a
longitudinal study of male and female tarantulas.
The metabolic shift from passive to active life may have co-
evolved with a complete redistribution of reserves towards
reproductive investment at the expense of body maintenance (the
so-called cost of reproduction, [27]). However, the initial idea of
functions competing for a limited amount of energy is still under
debate [28] and energy expenditure associated to gamete
production is supposed to be low in spider [20]. One possibility is
that males are not trading-off a limited quantity of nutrients, but key
metabolites among different functions. For example, male adult
spiders use an amount of energy and metabolites in the spermatic
web construction necessary for the transfer of sperm to copulatory
organs [29]. Variability in internal allocation of metabolites has
been previously shown in insects [30] and concerns cell fuels (lipids,
glycogen) which may change the way mitochondria are functioning
and finally drive different rates of ROS production [31]. Another
Figure 2. Representative images of tarantula haemolymph cells after incubation with MitoSOX Red probe. Fluorescence was mainly
localized in the cytoplasm (a) and there was no apparent unspecific labelling of the nucleus (b).
doi:10.1371/journal.pone.0013104.g002
Figure 3. Mean superoxide production measured using
MitoSOX Red fluorescence intensity levels in haemolymph
tarantula cells. Data were collected during three independent
sessions. Rate of superoxide production were standardized among
the three sessions of measurement and examined relatively to each
mean values. Males (n = 15) presented a relative superoxide production
significantly higher than females (n = 14, Table 1).
doi:10.1371/journal.pone.0013104.g003
Table 2. General linear model testing the effect of two
factors, the time elapsed from the last moult and the residuals
between body mass and the time elapsed from the last moult
on mitochondrial ROS production in males or females
tarantula.
df F
P
CELL SUPEROXIDE PRODUCTION
Males
Time from last moult 1,14 11.20 0.005
Residuals Body mass/Time from last moult 1,14 2.96 0.111
Females
Time from last moult 1,13 0.22 0.645
Residuals Body mass/Time from last moult 1,13 1.50 0.244
Linear regression values for males were: ROS = 0.006 x Time from last moult
+0.88, r = 0.67. Confidence interval at 95% for the slope value: 0.002–0.009; for
the constant: 0.341–1.417.
doi:10.1371/journal.pone.0013104.t002
ROS in Tarantulas
PLoS ONE | www.plosone.org 3 October 2010 | Volume 5 | Issue 10 | e13104
possibility is that sexual maturation alters the nature of cell
components (lipid membrane or proteins) in a way that their
intrinsic resistance to oxidative stress is reduced in males [32].
Additionally, the active life style of adult male tarantulas exposed
them to increased risks of mortality, either due to predation, sexual
cannibalism, or to heat stress and desiccation since they abandon
their burrows [14,33]. Therefore, selection could have driven a
lower investment in somatic maintenance in adult males and a
greater allocation of metabolic resources to reproduction, leading to
a reduced lifespan as predicted by the soma disposable theory [34].
Energy demanding sexual-related activities can alter immunity
in vertebrates [35] as well as in invertebrates [36]. Most of the
time, this cost is reflected as a diminution of immune efficiency,
increasing the risk of infection [37,38,39] or of co-lateral
autoimmune damages due to non-specific ROS production mainly
by innate immune cells [40,41]. In insects, haemocytes are
circulating cells that are responsible for the cellular response by
encapsulation and phagocytosis [42] during which ROS produc-
tion will serve for pathogen destruction [43]. The available
information on the immune system of spiders is scarce. However,
ROS production during infection also occurs in arachnid
haemocytes [44]. Therefore, because we mainly measured
superoxide production in haemocytes, one question arises from
our results: is it possible that male tarantulas suffer from
autoimmune processes that may be involved in their fast ageing
rate? It will be worthwhile to look at the importance of
autoimmune disorders in old male vs. female tarantulas.
To conclude, our study suggests that oxidative stress is part of
the proximal mechanisms explaining the great difference in
longevity between male and female spider’s B. albopilosa. Male
reproductive strategy demands a sharp metabolic shift that is likely
to be supported by changes in metabolism, which is apparently
achieved at a cost for longevity. For example, juvenile hormone
and insulin-like growth factors are known to affect longevity in
insects [45,46,47], hypothetically by altering the immune system.
An experimental approach manipulating this pathway or directly
inhibiting the immune activation would help to better understand
why male tarantulas present a semelparous reproductive strategy
while females are iteroparous. As suggested in vertebrates [48], we
confirmed that ROS production may be different between sexes at
the intra-specific level, and that oxidative stress is likely to play a
great role in shaping life history parameters.
Materials and Methods
Animal study
Brachypelma albopilosa [49] is a native of Costa Rica and
Honduras terricolous spider. Listed on appendix II accordingly
to the Convention on International Trade in Endangered Species
(CITES), all spiders B. albopilosa used in the different tests came
from the IPHC laboratory stock (permits Nu540048 – Pre´fecture
de Meurthe et Moselle, France). The spiders studied here emerged
from five different egg sacs built by spiders in the laboratory. The
spiderlings were reared and maintained until death. Twenty days
after their emergence from the egg sac, the spiderlings were
housed individually in 1 litter plastic containers (1668cm68 cm)
during the larval period (1 year and four moult) and, from the four
juvenile moults were maintained in 8 litters glass box (27618 cm
616 cm), before finally being transferred to 14 litters plastic
containers (32622 cm 620 cm) after the pre-adult moult. All
containers were lined with 2 cm of compost, which provided a
substratum for locomotion, web fixation, refuge, attachment and
ecdysis. The spiders were maintained under ambient conditions of
temperature, humidity and luminosity. The air temperature and
relative humidity were monitored daily using a hydro -
thermograph. Breeding was induced at 2562uC, under a
12:12 h photoperiod and 60610% of humidity. Spiders were
fed ad libitum with a standardized diet of Tenebrionidae larvae
Figure 4. Standardized superoxide production in 15 males and 14 females tarantula, measured in haemolymph cells, in relation to
the time elapsed from the imaginal moult. Each point represents the rate of accumulation of superoxide during two hours measured using
MitoSOX fluorescence in one individual (see text for statistics).
doi:10.1371/journal.pone.0013104.g004
ROS in Tarantulas
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(Zophobas morio) and of larvae or adults Blattidae (Blabera fusca,
Pleriplaneta americana). These preys were selected according to the
suitability of their body size for the experimental animals at
different developmental stages.
Prior to sampling, spiders were anaesthetized by chilling at 4uCfor
30 min and heamolymph was then sampled (0.5–1 ml) from
punctured dorsal aorta at the edge between the cephalothorax and
the opisthosoma into a capillary and flushed out into an Eppendorf
tube kept on ice. Immediately after collection, haemolymph was
diluted (1:1) in anti-coagulant solution (NaCl 119 mM, NaHCO
3
14.9 mM, KCl 4.7 mM, KH
2
PO
4
1.18 mM, MgSO
4
1.17 mM,
CaCL
2
1.6 mM, EDTA 0.026 mM, glucose 5.5 mM). Assessment of
superoxide production was conducted immediately after haemo-
lymph collection.
Superoxide measurement was conducted on 15 adult virgin males
(1558.9638.7 days) and 14 adult virgin females (3695.7640.1 days),
females being older than males (F
1, 28
= 1471.8, P,0.001). Males
and females tarantula have different moulting frequency. When the
males reach sexual maturity and resume their imaginal moult, they
stop growing and do not moult anymore [25]. On the contrary, once
their imaginal moult is accomplished, females continue to grow and
enter regular moulting periods through their adult life. In
consequence, there was a great difference in the laps of time since
the nympho-imaginal moult between sexes: males entered their
reproductive life for 105.1646.3 days while females did so for
431.6647.9 days (F
1, 28
=24.06, P,0.001). In particular, if females
have all reached sexual maturity since a long time, only four males
were ‘‘old’’ mature spider (279.5623.7 days since the imaginal
moult) while eleven reached sexual maturity only for 41.6615.2
days. Consequently, the post-moult period (days) was taken into
account as a fixed factor in the data analysis (see Statistics).
Antioxidant capacity and level of oxidative damage
Oxidative balance was assessed on frozen haemolymph from the
same animals, depending on the volume of haemolymph
remaining after the ROS production measurement. Consequently,
the antioxidant barrier (in 11 females and 9 males B. albopilosa) and
the concentration of reactive oxygen metabolites (primilary
hydroperoxides, in 7 females and 5 males) were measured using
OXY-Adsorbent and d-ROMs tests (Diacron, Italy). Detailed
description of these measurements has been previously published
[50,51]. In Oxy test, the sample is subjected to massive oxidation
through hypochlorous acid (HOCl), and the efficiency of its
antioxidant capacity (including enzymatic and non-enzymatic
compounds) is assessed by quantification of the unreacted (excess)
of acid by a spectrophotometric method (l= 492 nm). In D-ROM
test, the concentration of cell hydroperoxides generated by
oxidative attack on various organic substrates like proteins, lipids
or DNA is measured, in presence of iron (produced by the R2
reagent), after generation of alkoxyl and peroxyl radicals which
will in turn oxidize an alkyl-substituted aromatic amine (R1
reagent), thus transforming them into a pink derivate. Color
development was quantified by spectrophotometry (l= 492 nm),
in proportion to the initial hydroperoxide concentration. Taran-
tula heamolymph was diluted 1/200 for linear determination using
a linear standard curve method. Intra-specific (between male and
female tarantulas, dilution in distilled water 1:200) differences in
the antioxidant capacity was determined. All measurements were
run in duplicates. Intra-individual coefficient of variation was
4.9% for the Oxy test and 8.3% for the D-ROM test.
Cell superoxide measurement
Mitochondrial accumulation of superoxide was measured with
MitoSOX
TM
Red (Invitrogen), a highly selective fluorescent
probe for the detection of ROS generated within the mitochon-
dria. MitoSOX
TM
Redwasaddedtothedilutedhemolymphat
a final concentration of 5 mM in the final media and incubated at
ambient temperature (20uC) for 2 hours (time course of the
measurement). Fluorescence intensity was recorded immediately
after the addition of the probe (t0), and then during a kinetic
measurement after 60 min (t60) and 2 hours of incubation (t120).
Fluorescent signal emitted from the oxidized MitoSOX
TM
Red
reagent was detected by flow cytometry (Epics Coulter XL4,
Beckman-Coulter instrument, System II acquisition software).
Rate of superoxide production during the kinetic measurement
was quantified by plotting the fluorescent values with time (0, 60,
120 min) and by calculating the slope of the fit curve [52]. This
slope value was used for statistical analysis. Measurements have
been conducted in three different sessions and to control for
inter-session variability in fluorescent data, all individual values
have been standardized using the corresponding mean superox-
ide production value (of all samples measured in the same
session). Values used for the statistical analysis are then expressed
in relation to the mean session value (mean value = 1).
Superoxide production in both sexes has been measured for
each session. MitoSOX superoxide measurement was repeated
the same day for 4 individuals allowing the estimation of intra-
individual coefficient of variation (9.3%). MitoSOX fluorescent
signal may be affected by non-specific labelling (i.e. independent
of the level of mitochondrial superoxide production) more
specifically in the nucleus. Therefore, we checked for nuclear
fluorescent signal by microscopy (Nikon Eclipse E600) and
digital images of cells incubated with the fluorescent probe were
taken using 60x oil immersion objective lens (digital camera
DXM1200F).
Statistics
Differences in antioxidant and oxidative damage levels were
tested using a General Linear Model (GLM), with age and sex as
fixed factors. Differences in superoxide production were tested
using a Linear Mixed Model with sex used as a fixed factor and
session number as a random factor since measures have been
performed in 3 different sessions. Age, time from the imaginal
moult and body mass were found to covary linearly, since animals
get lighter with time. To avoid multilinearity, we chose to
withdraw age from the analysis because (i) age of males and
females were not overlapping and (ii) we were interested in adult
differences in superoxide production. Consequently, time from the
imaginal moult was preferred and used as a fixed factor. To
discriminate the potential effect of body mass on superoxide
production, we used the residuals of the relationship between body
mass and time from the last moult, calculated separately for males
and females because body masses did not overlap. These residuals
gave us information on the effect of body mass on superoxide
production independently of the body mass changes with time.
Normality of the residuals was tested using Kolmogorov-Smirnov
test. Analyses were performed using SPSS 16.0, with two-tailed
tests and p values #0.05. Means are quoted 6S.E.
Acknowledgments
We would like to thank J. Couturier and J.C. Olry for animal maintenance.
Author Contributions
Conceived and designed the experiments: FC MT. Performed the
experiments: FC CFS FB. Analyzed the data: FC NP. Contributed
reagents/materials/analysis tools: FC CFS FB MT. Wrote the paper: FC.
ROS in Tarantulas
PLoS ONE | www.plosone.org 5 October 2010 | Volume 5 | Issue 10 | e13104
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ROS in Tarantulas
PLoS ONE | www.plosone.org 6 October 2010 | Volume 5 | Issue 10 | e13104
