Antioxidant Responses Induced by Short-Term Activity–Estivation–Arousal Cycle in Pomacea canaliculata

Abstract

Long-term estivation (45 days) in the apple snail Pomacea canaliculata induces an increase of non-enzymatic antioxidants, such as uric acid and reduced glutathione (GSH), which constitutes an alternative to the adaptive physiological strategy of preparation for oxidative stress (POS). Here, we studied markers of oxidative stress damage, uric acid levels, and non-enzymatic antioxidant capacity, enzymatic antioxidant defenses, such as superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST), and transcription factors expression [forkhead box protein O (FOXO), hypoxia-inducible factor-1 alpha (HIF1α), and nuclear factor erythroid 2-related factor 2 (Nrf2)] in control active animals, 7-day estivating and aroused snails, in digestive gland, gill, and lung tissue samples. In the digestive gland, SOD and CAT activities significantly increased after estivation and decreased during arousal. Meanwhile, GST activity decreased significantly during the activity–estivation–arousal cycle. Gill CAT activity increased significantly at 7 days of estivation, and it decreased during arousal. In the lung, the CAT activity level increased significantly during the cycle. FOXO upregulation was observed in the studied tissues, decreasing its expression only in the gill of aroused animals during the cycle. HIF1α and Nrf2 transcription factors decreased their expression during estivation in the gill, while in the lung and the digestive gland, both transcription factors did not show significant changes. Our results showed that the short-term estivation induced oxidative stress in different tissues of P. canaliculata thereby increasing overall antioxidant enzymes activity and highlighting the role of FOXO regulation as a possible underlying mechanism of the POS strategy.

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Frontiers in Physiology | www.frontiersin.org 1 February 2022 | Volume 13 | Article 805168
BRIEF RESEARCH REPORT
published: 02 February 2022
doi: 10.3389/fphys.2022.805168
Edited by:
Youji Wang,
Shanghai Ocean University, China
Reviewed by:
Liu Lan Zhao,
Sichuan Agricultural University, China
Marie-Agnes Coutellec,
Institut National de recherche pour
l’agriculture, l’alimentation et
l’environnement (INRAE), France
*Correspondence:
Maximiliano Giraud-Billoud
mgiraudbilloud@gmail.com
Specialty section:
This article was submitted to
Invertebrate Physiology,
a section of the journal
Frontiers in Physiology
Received: 16 November 2021
Accepted: 05 January 2022
Published: 02 February 2022
Citation:
Giraud-Billoud M, Campoy-Diaz AD,
Dellagnola FA, Rodriguez C and
Vega IA (2022) Antioxidant
Responses Induced by Short-Term
Activity–Estivation–Arousal Cycle in
Pomacea canaliculata.
Front. Physiol. 13:805168.
doi: 10.3389/fphys.2022.805168
Antioxidant Responses Induced by
Short-Term Activity–Estivation–Arousal
Cycle in Pomacea canaliculata
MaximilianoGiraud-Billoud
1,2,3*, AlejandraD.Campoy-Diaz
1,2,3, FedericoA.Dellagnola
1,2,4,
CristianRodriguez
1,2,4 and IsraelA.Vega
1,2,4
1 IHEM, CONICET, Universidad Nacional de Cuyo, Mendoza, Argentina, 2 Facultad de Ciencias Médicas, Instituto de
Fisiología, Universidad Nacional de Cuyo, Mendoza, Argentina, 3 Departamento de Ciencias Básicas, Escuela de Ciencias de
la Salud-Medicina, Universidad Nacional de Villa Mercedes, San Luis, Argentina, 4 Departamento de Biología, Facultad de
Ciencias Exactas y Naturales, Universidad Nacional de Cuyo, Mendoza, Argentina
Long-term estivation (45 days) in the apple snail Pomacea canaliculata induces an increase
of non-enzymatic antioxidants, such as uric acid and reduced glutathione (GSH), which
constitutes an alternative to the adaptive physiological strategy of preparation for oxidative
stress (POS). Here, westudied markers of oxidative stress damage, uric acid levels, and
non-enzymatic antioxidant capacity, enzymatic antioxidant defenses, such as superoxide
dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST), and transcription
factors expression [forkhead box protein O (FOXO), hypoxia-inducible factor-1 alpha (HIF1α),
and nuclear factor erythroid 2-related factor 2 (Nrf2)] in control active animals, 7-day estivating
and aroused snails, in digestive gland, gill, and lung tissue samples. In the digestive gland,
SOD and CAT activities signicantly increased after estivation and decreased during arousal.
Meanwhile, GST activity decreased signicantly during the activity–estivation–arousal cycle.
Gill CAT activity increased signicantly at 7 days of estivation, and it decreased during arousal.
In the lung, the CAT activity level increased signicantly during the cycle. FOXO upregulation
was observed in the studied tissues, decreasing its expression only in the gill of aroused
animals during the cycle. HIF1α and Nrf2 transcription factors decreased their expression
during estivation in the gill, while in the lung and the digestive gland, both transcription
factors did not show signicant changes. Our results showed that the short-term estivation
induced oxidative stress in different tissues of P. canaliculata thereby increasing overall
antioxidant enzymes activity and highlighting the role of FOXO regulation as a possible
underlying mechanism of the POS strategy.
Keywords: hypometabolism, oxidative stress, preparation for oxidative stress, redox-sensitive transcription
factors, apple snails (Pomacea spp.)
INTRODUCTION
Several organisms live under harsh environmental conditions and therefore have evolved dierent
strategies to cope with them. Particularly during hypometabolic situations where the imbalance
of oxidative stress can damage self-cells or tissues, many animals show physiological protective
strategies known collectively as “preparation for oxidative stress (POS)” (Hermes-Lima et al.,

Giraud-Billoud et al. Snail’s POS Strategy in Short-Term Estivation
Frontiers in Physiology | www.frontiersin.org 2 February 2022 | Volume 13 | Article 805168
1998, 2015; Giraud-Billoud etal., 2019). Although, the evidence
has conrmed the POS strategy in more than 80 animal species
from eight dierent phyla: Cnidaria, Nematoda, Annelida,
Tardigrada, Arthropoda, Mollusca, Echinodermata, and Chordata
(Moreira etal., 2016), the underlying mechanisms are still not
fully understood.
Molecular mechanisms putatively involved in the POS strategy
include (a) DNA methylation and histone modications, (b)
regulation of transcription factors, (c) control of mRNA translation
by microRNAs, and (d) post-translational modications of
antioxidant enzymes (Giraud-Billoud et al., 2019). In particular,
antioxidant response elements (ARE) are cytoprotective genes,
which are upregulated under situations of high levels of reactive
oxygen species (ROS) and electrophilic compounds, in order
to reduce the damage to intracellular macromolecules that could
lead to cell death (Pamplona and Costantini, 2011). Under
hypoxia, the upregulation of genes like forkhead box protein
O (FOXO), nuclear factor erythroid 2-related factor 2 (Nrf2),
and hypoxia-inducible factor-1 alpha (HIF1α), among others,
has been described (Kobayashi and Yamamoto, 2006; Webb
et al., 2009; Malik and Storey, 2011). ese REDOX-sensitive
transcription factors cause an increase of endogenous antioxidants
to cope with ROS overproduction (Ensminger et al., 2021).
FOXO regulates expression of target genes controlling dierent
cell responses like stress tolerance, and inducing, for example,
an increase of mRNA and concentration of superoxide dismutase
(SOD), and it regulates catalase (CAT) expression during
hypometabolic situations (Kops et al., 2002; Malik and Storey,
2011; Ponugoti et al., 2013). HIF1α is expressed in response to
hypoxia and activates antioxidants such as SOD, CAT, glutathione
S-transferase (GST), and glutathione peroxidase (Song et al.,
2015; Lacher et al., 2018). Nrf2 is a transcription factor that is
negatively regulated by Kelch-like ECH-associated protein 1
(Keap1). Electrophile molecules and ROS may modify cysteine
residues in Keap1, which triggers transcriptional regulation of
antioxidant proteins throughout Nrf2 stimulation. ese include
SOD, CAT, GST, and other proteins involved in scavenging ROS
and reduced glutathione (GSH) biosynthesis (Zhu et al., 2005;
Hayes and McMahon, 2009; Kovac et al., 2015; Suzuki and
Yamamoto, 2015). Changes in the expression of these genes
have not been studied until now in the context of estivation
in mollusks. In order to evaluate this role and to shed light
on the molecular physiology of this phenomenon, we have
established a short-term experimental model of activity–estivation–
arousal cycle in the apple snail Pomacea canaliculata
(Caenogastropoda, Ampullariidae).
Apple snails are a conspicuous clade of amphibious snails
that show broad adaptive capacities to survive extreme
environmental conditions such as desiccation, cold, or high
salinity (Giraud-Billoud et al., 2013, 2018; Hayes et al., 2015;
Yang et al., 2018). In particular, P. canaliculata develops a
peculiar form of POS when it is exposed to prolonged periods
(45 days) of environmental stress (estivation or hibernation;
Giraud-Billoud et al., 2013, 2018). Aer long-term estivation,
non-enzymatic antioxidant defense mechanisms (e.g., increased
circulating levels of GSH and uric acid) allow arousing safely
from the hypometabolic state, restoring the physiological
conditions of active animals within 24 h of re-immersion in
water (Giraud-Billoud et al., 2011, 2013). Besides, proteomic
studies in this species have shown that CAT expression increases
aer a 30-day-period of estivation (Sun et al., 2013). ese
ndings suggest that, in the initial stages of the activity–estivation–
arousal cycle, P. canaliculata prepares for oxidative stress through
activating enzymatic antioxidant defense mechanisms, as is
shown for other animal species (Hermes-Lima et al., 2015).
us, a plausible hypothesis to test is that, once antioxidant
enzymatic defenses are diminished by molecular or cellular
alterations induced by stress, non-enzymatic defense mechanisms
would play a leading role in tissue protection.
Attempting to answer this question, we evaluated the
physiological response at the tissue level, including the expression
of REDOX-sensitive transcription factors, in a model of a
short-term (7 days) activity–estivation–arousal cycle in P.
canaliculata. us, we aimed (a) to determine the production
of ROS and the consequent damage to macromolecules from
oxidative stress, and (b) to evaluate the participation of
non-enzymatic and enzymatic antioxidant defense mechanisms
during the cycle. In addition, we (c) assessed for changes in
the expression of REDOX-sensitive transcription factors (FOXO,
HIF1α, and Nrf2) as potential molecular mechanisms that lead
to the development of the POS strategy during the activity–
estivation–arousal cycle.
MATERIALS AND METHODS
Animals
e adult animals (both sexes) used in all experiments came
from our laboratory strain (stock origin and culturing conditions
have been previously reported, Giraud-Billoud et al., 2013).
Procedures for snail culture, sacrice, and tissue sampling were
approved by the Institutional Committee for the Care and Use
of Laboratory Animals (CICUAL, Facultad de Ciencias Médicas,
Universidad Nacional de Cuyo), Approval Protocol No 55/2015.
Short-Term Activity–Estivation–Arousal
Cycle Induction and Tissue Sampling
Experimental groups comprised by six adult animals were
allotted to the following categories: (1) active control snails
(Ctrl); (2) estivated snails for 7 days (Est); and (3) aroused
snails (Ar), 20 min aer the operculum was detached from
the shell aperture following water exposure (Giraud-Billoud
et al., 2011).
Tissue samples from the gill, lung, and digestive gland
(midgut gland or hepatopancreas) were dissected, immediately
frozen in liquid nitrogen, and stored at −80°C until use.
ROS Production
Reactive oxygen species production was evaluated according
to Wang and Joseph (1999). Tissue samples were homogenized
(1:5 w/v) in 100 mM Tris-HCl buer with 5 mM MgCl2 and
2 mM EDTA. Homogenates were centrifuged at 10,000g (4°C)
for 20 min, and the supernatants were incubated with 40 mM

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2′,7′-dichlorouorescein diacetate (DCFH-DA) in buered
solution (30 mM HEPES, 200 mM KCl, and 1 mM MgCl2) for
20 min (37°C). e released DCFH-DA was oxidized by ROS,
forming a uorescent compound, DCF, which was excited at
485 nm and detected at 538 nm. Results were expressed as
arbitrary units of orescence (AUF) per milligram of wet tissue
per minute (AUF/mg/min).
Hypoxic Damage Markers
Protein oxidative damage (concentration of carbonyl groups,
CG) was determined as described by Levine et al. (1990),
based on the addition of carbonyl group to
2,4-dinitrophenylhydrazine (DNPH). Tissues were homogenized
in 20 mM potassium phosphate (pH 7.4) and then centrifuged
at 10,000g for 30 min. e supernatants containing proteins
were incubated for 60 min in a solution of 2 M HCl, 10 mM
DNPH, this solution was then precipitated with 20% (w/v)
trichloroacetic acid. Aer that, the precipitates were washed
with ethanol/ethyl acetate (1:1) and dissolved in a solution of
20 mM potassium phosphate (pH 2.3) containing 6M guanidine
hydrochloride. CG content was determined by measuring the
absorbance at 360 nm (Reznick and Packer, 1994). Results were
expressed as nanomole of carbonyl groups per milligram of
protein (nmol/mg). Protein concentration was measured
according to the method of Bradford (1976).
iobarbituric acid reactive substances (TBARS) were
quantied as an index of lipid peroxidation. Tissue samples
(~100 mg) were homogenized (Ultraturrax® homogenizer) in
900 μl of 0.1 M sodium phosphate buer (pH 7.0) and centrifuged
(10,500g, 5 min), and the supernatants were kept frozen until
TBARS quantication with the method described by Wasowicz
etal. (1993) and modied by Lapenna etal. (2001). e aliquots
were mixed with 1 ml of working solution (15% w/v trichloroacetic
acid, 0.25 M hydrochloric acid, 0.67% w/v thiobarbituric acid,
2.25 mM butylated hydroxytoluene solution, and 0.1 ml of 8.1%
SDS) and kept at 4°C during the process. Aer that, the samples
were heated at 95°C for 30 min and 3 ml of butanol were
added. Finally, tubes were stirred for 5 min and centrifuged
at 1,500g for 10 min. Organic layers were collected and placed
in glass cuvettes and TBARS were spectrophotometrically
determined at 540 nm, with an extinction coecient of 156 mM−1.
e concentration was expressed as nanomole of TBARS per
gram of wet tissue (nmol/g). Lactate concentration was used
as an anaerobic glycolysis marker and was measured using a
commercial kit (Wiener Lab), following the manufacturer’s
instructions. Concentrations of lactic acid were expressed as
micromole per milligram of wet tissue (umol/mg).
Antioxidant Prole Characterization
Free radical scavenging capacity (oxidation of 2,2′-azino-bis-
3-ethylbenzothiazoline-6-sulfonic acid radical, ABTS+),
concentration of soluble uric acid as non-enzymatic antioxidant,
antioxidant enzymes activity (SOD, CAT, and GST), and proteins
were measured as antioxidant prole characterization. Frozen
tissue samples (approximately 100 mg) were processed using
an UltraTurrax® homogenizer in a buered solution (20 mM
Tris-HCl, 1 mM EDTA, 0.15 mM KCl, 1 mM dithioerythritol,
0.5 M sucrose, 0.1 mM phenylmethylsulfonyl uoride, and pH
7.6), supplemented with Halt™ Protease Inhibitor Cocktail
(ermo Fisher) and then centrifuged 30 min at 4°C (10,500g).
Supernatants were collected, aliquoted, and frozen until use.
Oxidation of ABTS+ was measured by the method of Miller
and Rice-Evans (1997). Briey, in presence of persulphate
anions, a colorless salt generates the greenish-blue cationic
radical ABTS+, which decolorizes when reacts with antioxidants
in the sample, hence extinguishing spectrophotometric reading
at 734 nm. An ascorbic acid standard curve was used (Giraud-
Billoud et al., 2018), and results were expressed as percent
ABTS+ oxidation.
Uric acid concentration was measured in 100 μl aliquots
sample, measuring the amount of hydrogen peroxide formed
aer treatment with urate oxidase. A colored quinoneimine
product generated was quantied at 510 nm, according to
Trinder (1969). Uric acid concentration was expressed as
millimole of compound per milligram of protein (mmol/mg).
Superoxide dismutase activity was determined by the method
described by McCord and Fridovich (1969), where the compound
formazan red is formed from mixing xanthine and the enzyme
xanthine oxidase, as generators of O2− and 2-(4-iodophenyl)-
3-(4-nitrophenol)-5-phenyltetrazolium (INT) chloride, which
reacts with O2−. e activity was quantied as the percentage
of inhibition compared to a calibration curve performed with
puried SOD. CAT activity was quantied by the method of
Aebi (1984), which from decomposition of 10 mM H2O2 in
50 mM phosphate buer (pH 7.0) and 20 μl of the tissue extract,
the enzyme activity was estimated at 240 nm. GST activity
was measured according to Habig etal. (1974). GST determination
was carried out at constant temperature (25°C) in homogenization
buer, 50 mM 1-chloro-2,4-dinitrobenzene, and 100 mM reduced
glutathione. e increase in absorbance (wavelength 340 nm)
was measured every 30 s for 120 s. Results of enzyme activity
were expressed as Units of SOD and CAT or milliUnits of
GST per milligram of protein (U/mg or mU/mg).
Protein concentration was estimated according to the method
of Lowry et al. (1951), using 50 μl of sample, and the colored
complex was measured at 690 nm.
All the results were expressed as mean ± SEM (N = 6 per group).
REDOX-Sensitive Transcription Factors
Expression
Total RNA was extracted from tissue homogenates of four
animals for each experimental group, using a NucleoSpin RNA
Set for NucleoZOL (Macherey-Nagel), following the supplier’s
recommendations. RNA was quantied (NanoDrop ND-100
spectrophotometer) and stored at −20°C. Expression levels of
FOXO, HIF1α, and NRf2 genes were assessed by quantitative
RT-qPCR. Around 500 ng of total RNA was used for reverse
transcription (M-MLV Reverse transcriptase, Invitrogen
Cat.#28025-021). Quantitative PCR was performed in a nal
volume of 10 μl containing 50 ng of cDNA, iTaq Universal
SYBR Green Supermix reaction mix (BIORAD) and 0.5 μM
of each specic primer (Supplementary Table 1) using a

Giraud-Billoud et al. Snail’s POS Strategy in Short-Term Estivation
Frontiers in Physiology | www.frontiersin.org 4 February 2022 | Volume 13 | Article 805168
CFX-96 thermocycler (BIORAD Cat.#1725122). Each specic
pair of primers was designed using the genomic information
of P. canaliculata (Sun etal., 2019). To ensure that amplicons
were from mRNA and not from genomic DNA amplication,
controls without reverse transcription were included. Validation
was performed based on amplicon size and melting point.
e relative expression levels of FOXO, HIF1α, and Nrf2 genes
in all samples were normalized to β-actin and relative
quantication was performed using the 2−ΔΔCT method
(Schmittgen and Livak, 2008).
All the results were expressed as relative expression units
(REU), each value showed in Tab l e  1 represents mean ± SEM
(N = 4 per g roup).
Statistical Analysis
For multigroup comparisons, variable distribution was evaluated
by Shapiro–Wilk normality test, and equal variance Bartlett’s
test was used to evaluate homoscedasticity for each set of
experimental variables. Dierences among experimental groups
(control, estivation, and arousal) and also between tissues
(digestive gland, gill, and lung) of each condition of the
activity–estivation–arousal cycle were evaluated by one-way
ANOVA followed, when signicant, with a Newman–Keuls post
hoc test for multiple comparisons. An ANOVA table with F
(Dfn, Dfd) and p-values obtained is available in Supplementary
Table 2 .
RESULTS
ROS Production and Oxidative Damage
Both estivation-induced ischemia and arousal-induced
reperfusion cause an increase in ROS levels. Comparing the
studied tissues of each experimental set, the ROS levels in
the digestive gland were 7–8-fold higher than the gill and the
lung (Figure 1A). On the other hand, the comparison of ROS
production between active, estivating, and aroused animals for
a same organ, showed that in the digestive gland, ROS levels
signicantly increased aer estivation and arousal, compared
to control group (Ctrl = 542.4 ± 8.5; Est = 834.6 ± 74.8; and
Ar = 781.0 ± 57.9 AUF/mg/min; Figure1A). Besides, in the gill,
ROS levels almost doubled during 7-day estivation and 20 min
of arousal (Ctrl = 89.4 ± 6.1; Est = 159.3 ± 6.5; and Ar = 143.1 ± 13.2
AUF/mg/min; Figure 1A). Also, lung showed an increase of
ROS production during estivation (Ctrl = 63.9 ± 14.6;
Est = 154.6 ± 47.1; and Ar = 155.4 ± 48.9 AUF/mg/min; Figure1A).
If the studied tissues had not eective antioxidant protection
mechanisms to counteract the ROS overproduction during activity–
estivation–arousal cycle, the damage to molecules such as proteins
and lipids would have become evident. In the control groups,
the levels of CG were 3-fold higher in the gill compared to the
digestive gland and 2-fold higher than in the lung (Figure 1B).
Protein damage, measured as CG, raised 2-fold in the gill compared
to the digestive gland and the lung from estivated and aroused
animals (Figure 1B). In the activity–estivation–arousal cycle, the
protein damage in the digestive gland increased signicantly aer
estivation (Ctrl = 4.1 ± 0.5; Est = 13.3 ± 2.5; and Ar = 8.1 ± 1.2 nmol/
mg; Figure 1B), meanwhile, in the gill a signicant increase
was observed aer estivation and then decreased signicantly
aer the arousal (Ctrl = 11.3 ± 0.6; Est = 26.8 ± 1.7; and
Ar = 18.4 ± 2.2 nmol/mg; Figure 1B). Similar signicant changes
were observed in the lung (Ctrl = 9.0 ± 0.3; Est = 14.4 ± 0.2; and
Ar = 9.7 ± 0.7 nmol/mg; Figure 1B). Lipid peroxidation, evidenced
by an increase in TBARS levels, was slightly higher in the digestive
gland than in the other tissues studied for the control group.
However, TBARS levels became more than double in the digestive
gland than in the gills and lungs in the estivation and arousal
groups (Figure 1C). Furthermore, TBARS concentration of
estivating and arousal snails was signicantly higher than in
control snails, either in the gill (Ctrl = 0.94 ± 0.04; Est = 1.18 ± 0.05;
and Ar = 1.12 ± 0.05 nmol/mg; Figure 1C) or the digestive gland
(Ctrl = 1.42 ± 0.03; Est = 2.02 ± 0.14; and Ar = 1.87 ± 0.09 nmol/mg;
Figure1C). e lung did not show signicant changes in TBARS
levels (Figure 1C).
Finally, anaerobic cellular activity induced by hypoxia during
estivation was evaluated, because it may induce an increase
in lactic acid levels. Lactate concentrations were 2-fold higher
in the digestive gland than in the gill and lung of estivating
snails, while in the active and aroused groups, although, the
values in the digestive gland were signicantly higher, the
dierences between organs were lower. Nonetheless, no signicant
changes in the lactate concentrations were observed during
the activity–estivation–arousal cycle of each tissue (Figure1D).
Antioxidant Defenses
e non-enzymatic antioxidant capacity of each tissue was
evaluated by the percent ABTS+ oxidation and uric acid
concentrations. Two-fold higher ABTS+ oxidation was observed
in the digestive gland, compared to the gill for each experimental
set, while ABTS+ oxidation in the lung from control animals
was 3-fold higher than the gill. Comparing the levels across
activity, estivation and arousal groups for each tissue, the digestive
TABLE1 | REDOX-sensitive transcription factors expression in tissues of
P. canaliculata exposed to short term activity–estivation–arousal cycle.
Control Estivation Arousal
FOXO gene
Gill 0.32 ± 0.12 2.12 ± 0.35*0.28 ± 0.04**
Lung 0.72 ± 0.23 61.3 ± 12.5*64.8 ± 14.3*
Digestive gland 0.98 ± 0.41 4.90 ± 3.57 1.38 ± 0.37
HIF1α gene
Gill 1.81 ± 0.56 0.58 ± 0.16*0.20 ± 0.14*
Lung 1.09 ± 0.27 4.26 ± 1.581 2.29 ± 0.68
Digestive gland 0.82 ± 0.35 2.40 ± 1.21 1.31 ± 0.15
Nrf2 gene
Gill 3.50 ± 1.15 0.11 ± 0.03*0.49 ± 0.16*
Lung 1.62 ± 0.31 1.91 ± 0.44 1.12 ± 0.03
Digestive gland ND ND ND
*Gene expression as relative expression unit (REU). Each value represents Mean ± SEM.
N = 4 per group. ND = Not detected. *Indicates signicant differences vs. control group.
**Indicates signicant differences between estivation and arousal groups (One-way
ANOVA, Newman–Keuls’s post-test).

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gland was the only tissue that showed a signicant increase
in the percent ABTS+ oxidation during estivation and arousal,
compared to control group (Ctrl = 73.6 ± 2.8; Est = 96.9 ± 5.2; and
Ar = 92.2 ± 5.1%; Figure1E). Otherwise, due to the intracellular
uric acid deposits found in the digestive gland and the lung,
the concentrations in these tissues were approximately 10-fold
higher than those observed in the gill (Figure 1F). In the
studied conditions, uric acid concentration only increased
signicantly in the gill of arousal group (Ctrl = 73.4 ± 7.2;
Est = 93.7 ± 19.0; and Ar = 159.3 ± 26.9 mM/mg; Figure 1F).
According to our hypothesis, the response to short-term
estivation and the rapid reactivation induced by early arousal
induces an enzyme-mediated antioxidant response. SOD activity
was always higher in the digestive gland and gill compared
to the lung, but the levels went from 4-fold to 8-fold higher
activity between tissues in the control active animals and the
SOD activity of estivation and arousal animals, respectively.
Furthermore, the digestive gland showed a signicant increase
in SOD activity during estivation, and a signicant decrease
in the arousal (Ctrl = 9.5 ± 1.9; Est = 17.5 ± 1.4; and
Ar = 11.5 ± 1.8 U/mg; Figure 1G), meanwhile, in the gill and
lung no signicant changes were observed during the activity–
estivation–arousal cycle. CAT activity was around 8-fold higher
in the digestive gland than in the gill and lung. During the
activity–estivation–arousal cycle, the CAT enzyme activity
increased during estivation and arousal in the digestive gland
(Ctrl = 78.5 ± 6.3; Est = 164.5 ± 22.8; and Ar = 133.8 ± 16.5 U/mg;
Figure 1H). Also, in the gill, CAT increased its activity aer
estivation and decreased signicantly in the arousal
(Ctrl = 14.8 ± 1.8; Est = 35.9 ± 3.2; and Ar = 17.0 ± 0.9 U/mg;
ABC
DEF
GHI
FIGURE1 | Physiological responses of Pomacea canaliculata exposed to short term activity–estivation–arousal cycle. (A) Reactive oxygen species (ROS);
(B) protein oxidative damage (carbonyl groups, CG); (C) thiobarbituric acid reactive substances (TBARS); (D) lactate; (E) percent of ABTS+ oxidation; (F)
non-enzymatic antioxidant (uric acid); (G–I) enzymatic antioxidant defenses [superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST)],
in the digestive gland (green), gill (yellow), and lung (blue). Mean ± SEM. Signicant differences between activity, estivation, and arousal groups in the same
organ (p < 0.05, one-way ANOVA, Newman–Keulsʼs test) are indicated as follows: aactivity vs. estivation, bactivity vs. arousal, cestivation vs. arousal. Signicant
differences between organs at the same time of the activity–estivation–arousal cycle (p < 0.05, one-way ANOVA, Newman–Keulsʼs test) are indicated as
follows: * midgut gland vs. gill, ** midgut gland vs. lung, and *** gill vs. lung.

Giraud-Billoud et al. Snail’s POS Strategy in Short-Term Estivation
Frontiers in Physiology | www.frontiersin.org 6 February 2022 | Volume 13 | Article 805168
Figure1H). Finally, in the lung, the animals showed a signicant
increase in CAT activity during estivation and arousal
(Ctrl = 5.6 ± 0.7; Est = 15.1 ± 1.3; and Ar = 14.9 ± 3.1 U/mg;
Figure 1H). On the other hand, GST enzyme activity in the
digestive gland was 3-fold higher than in the gill and the
lung. e GST activity showed a signicant decrease in the
digestive gland from estivation and arousal groups, compared
to control (Ctrl = 573.6 ± 96.8; Est = 305.4 ± 23.6; and
Ar = 250.2 ± 43.1 mU/mg; Figure1I). Besides, GST activity showed
in lung an increase during estivation and decreased signicantly
in the arousal (Ctrl = 83.9 ± 15.2; Est = 115.6 ± 9.2; and
Ar = 80.8 ± 5.7 mU/mg; Figure 1I). In the gill no signicant
changes were observed.
REDOX-Sensitive Transcription Factors
(HIF1α, FOXO, and Nrf2) Expression
To evaluate changes in the expression of the REDOX-sensitive
transcription factors, specic primers were designed from the
genome of P. canaliculata (Sun etal., 2019). HIF1α and Nrf2
transcription factor sequences were identied, but in the cases
of FOXO, only one sequence “FOXO-like” was found. For this
reason, an in silico phylogenetic analysis of FOXO was previously
made (methodological information is available as a
Supplementary Material). e unrooted ML tree showed
sequences of FoxO1-like from chordates in a basal position,
locating FoxO3-like sequences of mollusks (P. canaliculata’s
FOXO and FOXO3 from the bivalve Sinonovacula constricta)
outside chordate FoxO3-like sequences; FoxO4-like and FoxO6
sequences were placed as derivative loci. is result showed
that the FOXO locus from invertebrates is in a basal position
compared to vertebrate FOXO 3, 4, and 6 domains loci
(Supplementary Figure 1).
Table 1 showed an increase in FOXO expression in the
studied tissues. In the gill and digestive gland from estivating
snails, FOXO increased respectively its expression around 6/5-fold
than control snails, and then decreased in aroused snails. In
the lung of estivated snails, the increase was signicantly higher
than in control group (around 85 times) and remained high
in aroused snails.
HIF1α and Nrf2 expression decreased signicantly in the
gill of P. canaliculata aer estivation, compared to the control
group. Likewise, the expression of both genes in the lung did
not show signicant changes during the activity–estivation–
arousal cycle. Also, the expression of HIF1α did not show
signicant changes in the experimental groups of the digestive
gland during the activity–estivation–arousal cycle, while the
expression levels of Nrf2 were not detectable.
DISCUSSION
Distantly-related animal species have evolved adaptive strategies
to tolerate environmental heat or lack of water (Storey, 2002).
Estivation is a hypometabolic process that involves lowering
body mass (oen by dehydration), a low (or null) metabolic
rate, and low oxygen availability (Navas and Carvalho, 2010;
Storey and Storey, 2012). Furthermore, tissue reoxygenation
during arousal from estivation induces an acute oxidative stress
by ROS increase, without sucient antioxidant defenses to
neutralize them (Storey, 2002; Storey and Storey, 2010; Staples,
2016). In this scenario, some animals activate a stress-responsive
physiological adaptation (the POS strategy) to cope with oxidative
damage (Hermes-Lima and Storey, 1995; Giraud-Billoud etal.,
2019); if this does not happen, many macromolecules can get
damaged by ROS, which, in turn, can lead to cell death (Schieber
and Chandel, 2014; Sies, 2015; Sies et al., 2017).
Mollusks have been proposed as model organisms to study
the POS strategy aer estivation; however, freshwater gastropods
have not received attention comparable to terrestrial gastropods
(Hermes-Lima and Storey, 1995; Hermes-Lima et al., 1998;
Ramos-Vasconcelos and Hermes-Lima, 2003; Nowakowska etal.,
2009; Giraud-Billoud et al., 2011, 2013; Nowakowska et al.,
2011). e freshwater P. canaliculata is an obligate air-breather
that ventilates mainly or solely with lung when dwelling in
poorly oxygenated waters or burying in the mud, closing tightly
its operculum at estivation (Cowie, 2002; Hayes et al., 2015).
During long-term estivation (45 days), snails increase
concomitantly non-enzymatic antioxidant defenses (particularly,
uric acid) with TBARS levels, but without changes in the
antioxidant enzymatic (CAT and SOD) defense system (Giraud-
Billoud et al., 2011, 2013). A recent proteomic study focused
on hypoxia tolerance showed that P. canaliculata is more tolerant
to acute hypoxia than Pomacea diffusa, and it is related to a
metabolic suppression and conservation of cellular fuels for
extending the animal survival time under hypoxia (Mu etal.,
2018). ese results are consistent with the non-signicant
changes observed in tissue lactate concentrations of P. canaliculata
exposed to a short-term estivation (Figure 1) and with the
presence of this species in water bodies that dry seasonally
or that have low oxygen concentration (Kwong et al., 2008;
Hayes etal., 2015). On the other hand, a short-term estivation
(7 days) induced a concomitant increase of TBARS and SOD
and CAT enzyme activities (SOD only for digestive gland)
without signicant changes in the uric acid concentration
(Figure 1).
e gill and the lung of P. canaliculata are physiologically
related organs that share vasculature and innervation allowing
the alternation between breathing in the water and the air
(Rodriguez etal., 2019, 2021). Oxidative damage of gill’s proteins
and lipids by ROS (Figure 1) was evident in estivating and
aroused snails, being the oxidative burst partially compensated
by an increase in the CAT activity. On the other hand, there
was a concomitant increase in protein damage and CAT activity
in the lung of estivating and aroused animals. e greater
diculty of the gill, compared to the lung, to protect itself
from oxidative burst aer estivation may be related to the
following facts: (a) the gill has a mitochondria rich epithelium
and suers a total collapse and dehydration during estivation
(out of water), and (b) the lung may maintain some activity
mobilizing variable volumes of air with each insuation of
the cavity and also has a highly-developed urate tissue (Giraud-
Billoud et al., 2008). In fact, urate concentration in the lung
were an order of magnitude higher than that in the gill
(Figures 1, 2), suggesting that the former may access directly

Giraud-Billoud et al. Snail’s POS Strategy in Short-Term Estivation
Frontiers in Physiology | www.frontiersin.org 7 February 2022 | Volume 13 | Article 805168
to this antioxidant molecule (Becker, 1993), while the latter
could access only soluble uric acid from hemolymph through
the slow (null) microcirculation.
e digestive gland of P. canaliculata is a key organ that
participates in multiple and diverse physiological processes
(Cowie, 2002; Hayes et al., 2015), and contains a bacterial
symbiont in the digestive epithelial cells with detoxication
and digestive functions (Castro-Vazquez etal., 2002; Vega etal.,
2005, 2006, 2007; Godoy et al., 2013; Campoy-Diaz et al.,
2018, 2020; Escobar-Correas et al., 2019). Compared to the
respiratory organs, the digestive gland showed higher levels
of ROS, equivalent levels of protein and lipid damage, and
high SOD and CAT activities in estivating and aroused snails.
Also, the digestive gland and lung showed high uric acid
concentration possibly associated with the storing of urate
crystalloids in the perivascular tissue (Giraud-Billoud et al.,
2008). ese ndings indicate that the digestive gland is able
to tolerate the oxidative burst induced by the activity–estivation–
arousal cycle through the action of a robust defense system
based on a combination of enzymatic and non-enzymatic
antioxidant defenses.
Furthermore, wefound a tissue and experimental condition
dependent correlation between antioxidant enzymes activities
and REDOX-sensitive transcription factors expression (FOXO,
Nrf2, and HIF1α), in the activity–estivation–arousal cycle of
P. canaliculata. FOXO expression and CAT activity increased
in all the studied tissues of estivating snails. SOD activity
changed dierentially in each tissue from control and estivated
snails, with an increase in SOD activity only in the digestive
gland of estivated snails. Nrf2 and HIF1α expression were
downregulated in the gill of estivating and aroused snails, but
HIF1α showed a tendency to increase its levels during estivation
in the lung and digestive gland. In this scenery, it is possible
that the Nrf2 and HIFα expression in the gill is associated
with their histological and functional peculiarities, i.e., the loss
of gaseous exchange surface and dehydration aer estivation.
Future studies must clarify the inverse relationship between
Nrf2 expression and antioxidant enzyme activities during the
estivation of P. canaliculata.
In this study, wehave described for the rst time in a mollusk
the relationship between the modication in tissue expression of
transcription factors and activity of antioxidant enzymes that
protect tissues exposed to a short period of hypoxia, triggered
by estivation. In this sense, P. canaliculata is a highly resistant
species to harsh environmental conditions, which makes it one
of the 100 worst invasive species in the world (Lowe etal., 2000).
Figure2 shows the responses that wehypothesize for this animal
model. During a short-term estivation, cells are exposed to an
FIGURE2 | Schematic representation of Pomacea canaliculata responses to different periods of estivation (7 and 45 days). Unknown mechanisms have been
represented with a question mark.

Giraud-Billoud et al. Snail’s POS Strategy in Short-Term Estivation
Frontiers in Physiology | www.frontiersin.org 8 February 2022 | Volume 13 | Article 805168
increase in ROS, which generates an imbalance between oxidant
molecules and antioxidant defenses, potentially generating damage
to macromolecules. is induces, among other potential physiological
adaptive adjustments, an increase in the expression of FOXO3,
which leads to an increase in the synthesis of antioxidant enzymes
such as SOD and CAT that protect against oxidative stress (Figure2,
le pathway). When estivation is prolonged, as can occur in
periods of drought that aect the bodies of water it inhabits,
this species can also protect itself against ROS generated by
non-enzymatic antioxidants such as uric acid and glutathione
(Figure 2, right pathway), which allow it to extend its survival
in adverse conditions, waiting the return of water.
e mechanisms that allow cells and tissues to adapt to
dierent oxidative stress situations are a continuously growing
research eld. From a comparative physiology perspective, these
ndings may represent signicant biomedical advances in the
future (Hawkins and Storey, 2020). e POS strategy involves
increasing antioxidant defenses before they become necessary
to counteract damage from oxidative stress (Hermes-Lima etal.,
1998). is anticipatory response requires nely regulated cellular
and molecular mechanisms (Giraud-Billoud et al., 2019).
However, there is growing evidence of interaction between
dierent REDOX-sensitive transcription factors (Klotz et al.,
2015; Klotz and Steinbrenner, 2017; Lacher et al., 2018; Gille
et al., 2019), such as those studied in the experimental model
proposed in this work, which could balance responses according
to the cellular antioxidant demand during variable hypometabolic
periods. FOXO3 appears to bethe FOXO subclass present in
P. canaliculata (Supplementary Figure 1) and their expression
changes along the studied activity–estivation–arousal cycle may
represent a response to enhancement of ROS during hypoxia,
but also to other biological processes as the protein turnover,
and cell survival and death regulation (Tzivion et al., 2011;
Davy etal., 2018). Further studies could explore other associated
responses of FOXO3 in P. canaliculata and their regulation by
acetylation, ubiquitination, methylation, phosphorylation, and
miRNA binding (Brown and Webb, 2018). Also, other
mechanisms have been related to adjust cellular responses in
animal models of estivation like epigenetic changes, such as
DNA methylation that regulates the metabolism of Apostichopus
japonicus during estivation (Zhao et al., 2015), or upregulation
of miRNA like it has been described in the foot muscle of
Otala lactea aer 10-day estivation (Hoyeck et al., 2019).
e characterization of the adaptive physiological defense
responses, in the face of adverse environmental conditions in
animals that use the POS strategy, implies studying phenomena
that are unknown until now, and therefore it is of interest in
the eld of comparative animal physiology.
DATA AVAILABILITY STATEMENT
e raw data supporting the conclusions of this article will
be made available by the authors, without undue reservation.
ETHICS STATEMENT
Procedures for snail culture, sacrice, and tissue sampling were
approved by the Institutional Committee for the Care and Use
of Laboratory Animals (CICUAL, Facultad de Ciencias Médicas,
Universidad Nacional de Cuyo), Approval Protocol No. 55/2015.
AUTHOR CONTRIBUTIONS
All authors listed have made a substantial, direct, and intellectual
contribution to the work, and approved it for publication.
FUNDING
is work was supported by grants from Ministerio de Ciencia,
Tecnología e Innovación, Agencia Nacional de Promoción de
la Investigación, el Desarrollo Tecnológico y la Innovación,
Argentina (PICT-2018-03966-BID) and Secretaría de
Investigación, Internacionales y Posgrado, Universidad Nacional
de Cuyo, Argentina (Proyecto Tipo I, 06/J511).
ACKNOWLEDGMENTS
e authors would like to thank Sergio Carminati for
technical assistance.
SUPPLEMENTARY MATERIAL
e Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fphys.2022.
805168/full#supplementary-material
REFERENCES
Aebi, H. (1984). Catalase in vitro. Methods Enzymol. 105, 121–126. doi: 10.1016/
s0076-6879(84)05016-3
Becker, B. (1993). Towards the physiological function of uric acid. Free Radi c.
Biol. Med. 14, 615–631. doi: 10.1016/0891-5849(93)90143-I
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem. 72, 248–254. doi: 10.1016/0003-2697(76)90527-3
Brown, A. K., and Webb, A. E. (2018). Regulation of FOXO factors in mammalian
cells. Curr. Top. De v. Biol . 127, 165–192. doi: 10.1016/bs.ctdb.2017.10.006
Campoy-Diaz, A. D., Arribére, M. A., Guevara, S. R., and Vega, I. A. (2018).
Bioindication of mercury, arsenic and uranium in the apple snail Pomacea
canaliculata (Caenogastropoda, Ampullariidae): bioconcentration and
depuration in tissues and symbiotic corpuscles. Chemosphere 196, 196–205.
doi: 10.1016/j.chemosphere.2017.12.145
Campoy-Diaz, A. D., Escobar-Correas, S., Canizo, B. V., Wuilloud, R. G., and
Vega, I. A. (2020). A freshwater symbiosis as sensitive bioindicator of
cadmium. Environ. S ci. Pollut. Res. Int . 27, 2580–2587. doi: 10.1007/
s11356-019-07082-x
Castro-Vazquez, A., Albrecht, E., Vega, I., Koch, E., and Gamarra-Luques, C.
(2002). Pigmented corpuscles in the midgut gland of Pomacea canaliculata

Giraud-Billoud et al. Snail’s POS Strategy in Short-Term Estivation
Frontiers in Physiology | www.frontiersin.org 9 February 2022 | Volume 13 | Article 805168
and other neotropical apple-snails (Prosobranchia, Ampullariidae): a possible
symbiotic association. Biocell 26, 101–109.
Cowie, R. (2002). “Apple snails (Ampullariidae) as agricultural pests: their
biology, impacts and management,” in Molluscs as Crop Pests. ed. G. Baker
(Wallingford: CABI Publishing), 145–192.
Davy, P. M., Allsopp, R. C., Donlon, T. A., Morris, B. J., Willcox, D. C., and
Willcox, B. J. (2018). FOXO3 and exceptional longevity: insights from hydra
to humans. Curr. Top. Dev. Biol. 127, 193–212. doi: 10.1016/bs.ctdb.2017.10.001
Ensminger, D. C., Salvador-Pascual, A., Arango, B. G., Allen, K. N., and
Vázquez-Medina, J. P. (2021). Fasting ameliorates oxidative stress: a review
of physiological strategies across life history events in wild vertebrates. Comp.
Biochem. Physiol. A Mol. Integr. Physiol. 256:110929. doi: 10.1016/j.
cbpa.2021.110929
Escobar-Correas, S., Mendoza-Porras, O., Dellagnola, F. A., Colgrave, M. L.,
and Vega, I. A. (2019). Integrative proteomic analysis of digestive tract
glycosidases from the invasive golden apple snail, Pomacea canaliculata. J.
Proteome Res. 18, 3342–3352. doi: 10.1021/acs.jproteome.9b00282
Gille, A., Turkistani, A., Tsitsipatis, D., Hou, X., Tauber, S., Hamann, I., et al.
(2019). Nuclear trapping of inactive FOXO1 by the Nrf2 activator diethyl
maleate. Redox Biol. 20, 19–27. doi: 10.1016/j.redox.2018.09.010
Giraud-Billoud, M., Abud, M. A., Cueto, J. A., Vega, I. A., and Castro-Vazquez, A.
(2011). Uric acid deposits and estivation in the invasive apple-snail, Pomacea
canaliculata. Comp. Biochem. Physi ol. A Mol. Integr. Physi ol. 158, 506–512.
doi: 10.1016/j.cbpa.2010.12.012
Giraud-Billoud, M., Castro-Vazquez, A., Campoy-Diaz, A. D., Giurida, P. M.,
and Vega, I. A. (2018). Tolerance to hypometabolism and arousal induced
by hibernation in the apple snail Pomacea canaliculata (Caenogastropoda,
Ampullariidae). Comp. Biochem. Physiol. B Biochem. Mol. Biol. 224, 129–137.
doi: 10.1016/j.cbpb.2017.12.015
Giraud-Billoud, M., Koch, E., Vega, I., Gamarra-Luques, C., and Castro-Vazquez, A.
(2008). Urate cells and tissues in the south American apple snail Pomacea
canaliculata. J. Molluscan Stud. 74, 259–266. doi: 10.1093/mollus/eyn017
Giraud-Billoud, M., Rivera-Ingraham, G. A., Moreira, D. C., Burmester, T.,
Castro-Vazquez, A., Carvajalino-Fernández, J. M., et al. (2019). Twenty years
of the ‘preparation for oxidative stress’ (POS) theory: ecophysiological
advantages and molecular strategies. Comp. Biochem. Phy siol. A Mol. Inte gr.
Physiol. 234, 36–49. doi: 10.1016/j.cbpa.2019.04.004
Giraud-Billoud, M., Vega, I. A., Rinaldi Tosi, M. E., Abud, M. A., Calderón, M. L.,
and Castro-Vazquez, A. (2013). Antioxidant and molecular chaperone defenses
during estivation and arousal in the south American apple-snail Pomacea
canaliculata. J. Exp. Biol. 216, 614–622. doi: 10.1242/jeb.075655
Godoy, M. S., Castro-Vasquez, A., and Vega, I. A. (2013). Endosymbiotic and
host proteases in the digestive tract of the invasive snail Pomacea canaliculata:
diversity, origin and characterization. PLoS One 8:e66689. doi: 10.1371/
journal.pone.0066689
Habig, W. H., Pabst, M. J., and Jakoby, W. B. (1974). Glutathione S-transferases.
J. Biol . Chem. 249, 7130–7139. doi: 10.1016/S0021-9258(19)42083-8
Hawkins, L. J., and Storey, K. B. (2020). Advances and applications of environmental
stress adaptation research. Comp. Biochem . Physiol. A Mol . Integr. Physiol.
240:110623. doi: 10.1016/j.cbpa.2019.110623
Hayes, K., Burks, R., Castro-Vazquez, A., Darby, P., Heras, H., Martín, P., et al.
(2015). Insights from an integrated view of the biology of apple snails
(Caenogastropoda: Ampullariidae). Malacologia 58, 245–302. doi:
10.4002/040.058.0209
Hayes, J. D., and Mcmahon, M. (2009). NRF2 and KEAP1 mutations: permanent
activation of an adaptive response in cancer. Trends Biochem. Sci. 34, 176–188.
doi: 10.1016/j.tibs.2008.12.008
Hermes-Lima, M., Moreira, D. C., Rivera-Ingraham, G. A., Giraud-Billoud, M.,
Genaro-Mattos, T. C., and Campos, É. G. (2015). Preparation for oxidative
stress under hypoxia and metabolic depression: revisiting the proposal two
decades later. Free Radic. Biol. Med. 89, 1122–1143. doi: 10.1016/j.
freeradbiomed.2015.07.156
Hermes-Lima, M., and Storey, K. B. (1995). Antioxidant defenses and metabolic
depression in a pulmonate land snail. Am. J. Phys. 268, R1386–R1393. doi:
10.1152/ajpregu.1995.268.6.R1386
Hermes-Lima, M., Storey, J. M., and Storey, K. B. (1998). Antioxidant defenses
and metabolic depression. e hypothesis of preparation for oxidative stress
in land snails. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 120, 437–448.
doi: 10.1016/S0305-0491(98)10053-6
Hoyeck, M. P., Hadj-Moussa, H., and Storey, K. B. (2019). Estivation-responsive
microRNAs in a hypometabolic terrestrial snail. Peer J. 7:e6515.
Klotz, L.-O., Sánchez-Ramos, C., Prieto-Arroyo, I., Urbánek, P., Steinbrenner, H.,
and Monsalve, M. (2015). Redox regulation of FoxO transcription factors.
Redox Biol. 6, 51–72. doi: 10.1016/j.redox.2015.06.019
Klotz, L.-O., and Steinbrenner, H. (2017). Cellular adaptation to xenobiotics:
interplay between xenosensors, reactive oxygen species and FOXO transcription
factors. Redox Biol. 13, 646–654. doi: 10.1016/j.redox.2017.07.015
Kobayashi, M., and Yamamoto, M. (2006). Nrf2–Keap1 regulation of cellular
defense mechanisms against electrophiles and reactive oxygen species. Adv.
Enzym. Regul. 46, 113–140. doi: 10.1016/j.advenzreg.2006.01.007
Kops, G. J., Dansen, T. B., Polderman, P. E., Saarloos, I., Wirtz, K. W., Coer, P. J.,
et al. (2002). Forkhead transcription factor FOXO3a protects quiescent cells
from oxidative stress. Nature 419, 316–321. doi: 10.1038/nature
01036
Kovac, S., Angelova, P. R., Holmström, K. M., Zhang, Y., Dinkova-Kostova, A. T.,
and Abramov, A. Y. (2015). Nrf2 regulates ROS production by mitochondria
and NADPH oxidase. Biochim. Biophys. Acta 1850, 794–801. doi: 10.1016/j.
bbagen.2014.11.021
Kwong, K.-L., Wong, P.-K., Lau, S. S., and Qiu, J.-W. (2008). Determinants of
the distribution of apple snails in Hong Kong two decades aer their initial
invasion. Malacologia 50, 293–302. doi: 10.4002/0076-2997-50.1.293
Lacher, S. E., Levings, D. C., Freeman, S., and Slattery, M. (2018). Identication
of a functional antioxidant response element at the HIF1A locus. Redox
Biol. 19, 401–411. doi: 10.1016/j.redox.2018.08.014
Lapenna, D., Ciofani, G., Pierdomenico, S. D., Giamberardino, M. A., and
Cuccurullo, F. (2001). Reaction conditions affecting the relationship
between thiobarbituric acid reactivity and lipid peroxides in human
plasma. Free Radic. Biol. Med. 31, 331–335. doi: 10.1016/
S0891-5849(01)00584-6
Levine, R. L., Garland, D., Oliver, C. N., Amici, A., Climent, I., Lenz, A.-G.,
et al. (1990). Determination of carbonyl content in oxidatively modied
proteins. Methods Enzymol. 186, 464–478.
Lowe, S., Browne, M., Boudjelas, S., and De Poorter, M. (2000). 100 of the
World’s Worst Invasive Alien Species a Selection From the Global Invasive
Species Database. Published by e Invasive Species Specialist Group (ISSG),
a Specialist Group of the Species Survival Commission (SSC) of the World
Conservation Union (IUCN), 12pp. First published as Special Li-Out
in Aliens.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein
measurement with the folin phenol reagent. J. Biol. Ch em. 193, 265–275.
doi: 10.1016/S0021-9258(19)52451-6
Malik, A. I., and Storey, K. B. (2011). Transcriptional regulation of antioxidant
enzymes by FoxO1 under dehydration stress. Gene 485, 114–119. doi:
10.1016/j.gene.2011.06.014
Mccord, J. M., and Fridovich, I. (1969). Superoxide dismutase: an enzymatic
function for erythrocuprein (hemocuprein). J. Biol. Chem . 244, 6049–6055.
doi: 10.1016/S0021-9258(18)63504-5
Miller, N. J., and Rice-Evans, C. A. (1997). Factors inuencing the antioxidant
activity determined by the ABTS•+ radical cation assay. Free Radic. Res. 26,
195–199. doi: 10.3109/10715769709097799
Moreira, D. C., Venancio, L. P., Sabino, M. A., and Hermes-Lima, M. (2016).
How widespread is preparation for oxidative stress in the animal kingdom?
Comp. Biochem. Physiol . A Mol . Integr. Physiol . 200, 64–78. doi: 10.1016/j.
cbpa.2016.01.023
Mu, H., Sun, J., Cheung, S. G., Fang, L., Zhou, H., Luan, T., et al. (2018).
Comparative proteomics and codon substitution analysis reveal mechanisms
of dierential resistance to hypoxia in congeneric snails. J. Proteome 172,
36–48. doi: 10.1016/j.jprot.2017.11.002
Navas, C. A., and Carvalho, J. E. (2010). Aestivation. Berlin, Germany:
Springer.
Nowakowska, A., Caputa, M., and Rogalska, J. (2011). Defence against oxidative
stress in two species of land snails (Helix pomatia and Helix aspersa) subjected
to estivation. J. Exp. Zool. A Eco l. Genet. Phy siol. 315A, 593–601. doi: 10.1002/
jez.713
Nowakowska, A., Swiderska-Kolacz, G., Rogalska, J., and Caputa, M. (2009).
Antioxidants and oxidative stress in Helix pomatia snails during estivation.
Comp. Biochem. Physiol. C Toxicol. Pharmacol. 150, 481–486. doi: 10.1016/j.
cbpc.2009.07.005

Giraud-Billoud et al. Snail’s POS Strategy in Short-Term Estivation
Frontiers in Physiology | www.frontiersin.org 10 February 2022 | Volume 13 | Article 805168
Pamplona, R., and Costantini, D. (2011). Molecular and structural antioxidant
defenses against oxidative stress in animals. Am. J. P hys. Reg ul. Integr. Comp.
Phys. 301, R843–R863. doi: 10.1152/ajpregu.00034.2011
Ponugoti, B., Xu, F., Zhang, C., Tian, C., Pacios, S., and Graves, D. T. (2013).
FOXO1 promotes wound healing through the up-regulation of TGF-β1 and
prevention of oxidative stress. J. Cell Biol. 203, 327–343. doi: 10.1083/jcb.201305074
Ramos-Vasconcelos, G., and Hermes-Lima, M. (2003). Hypometabolism,
antioxidant defenses and free radical metabolism in the pulmonate land
snail Helix aspersa. J. Exp. Biol. 206, 675–685. doi: 10.1242/jeb.00124
Reznick, A. Z., and Packer, L. (1994). Oxidative damage to proteins:
spectrophotometric method for carbonyl assay. Methods Enzymol. 233,
357–363. doi: 10.1016/S0076-6879(94)33041-7
Rodriguez, C., Prieto, G. I., Vega, I. A., and Castro-Vazquez, A. (2019). Functional
and evolutionary perspectives on gill structures of an obligate air-breathing,
aquatic snail. PeerJ 7:e7342. doi: 10.7717/peerj.7342
Rodriguez, C., Prieto, G. I., Vega, I. A., and Castro-Vazquez, A. (2021). Morphological
grounds for the obligate aerial respiration of an aquatic snail: functional and
evolutionary perspectives. PeerJ 9:e10763. doi: 10.7717/peerj.10763
Schieber, M., and Chandel, N. S. (2014). ROS function in redox signaling and
oxidative stress. Cur r. Bio l. 24, R453–R462. doi: 10.1016/j.cub.2014.03.
034
Schmittgen, T. D., and Livak, K. J. (2008). Analyzing real-time PCR data by
the comparative CT method. Nat. Protoc. 3, 1101–1108. doi: 10.1038/
nprot.2008.73
Sies, H. (2015). Oxidative stress: a concept in redox biology and medicine.
Redox Biol. 4, 180–183. doi: 10.1016/j.redox.2015.01.002
Sies, H., Berndt, C., and Jones, D. P. (2017). Oxidative stress. Annu. Re v.
Biochem. 86, 715–748. doi: 10.1146/annurev-biochem-061516-045037
Song, J., Yoon, D., Christensen, R. D., Horvathova, M., iagarajan, P., and
Prchal, J. T. (2015). HIF-mediated increased ROS from reduced mitophagy
and decreased catalase causes neocytolysis. J. Mol. Med. 93, 857–866. doi:
10.1007/s00109-015-1294-y
Staples, J. F. (2016). Metabolic exibility: hibernation, torpor, and estivation.
Compr. Physiol. 6, 737–771. doi: 10.1002/cphy.c140064
Storey, K. (2002). Life in the slow lane: molecular mechanisms of estivation.
Comp. Biochem. Physiol . A Mol . Integ r. Phys iol . 133, 733–754. doi: 10.1016/
S1095-6433(02)00206-4
Storey, K. B., and Storey, J. M. (2010). “Metabolic regulation and gene expression
during aestivation,” in Aestivation. eds. C. A. Navas and J. E. Carvalho
(Berlin Heidelberg: Springer), 25–45.
Storey, K. B., and Storey, J. M. (2012). Aestivation: signaling and hypometabolism.
J. Exp. Biol . 215, 1425–1433. doi: 10.1242/jeb.054403
Sun, J., Mu, H., Ip, J. C. H., Li, R., Xu, T., Accorsi, A., et al. (2019). Signatures
of divergence, invasiveness, and terrestrialization revealed by four apple
snail genomes. Mol. Biol. Evol. 36, 1507–1520. doi: 10.1093/molbev/ms
z084
Sun, J., Mu, H., Zhang, H., Chandramouli, K. H., Qian, P.-Y., Wong, C. K.
C., et al. (2013). Understanding the regulation of estivation in a freshwater
snail through iTRAQ-based comparative proteomics. J. Proteome Res. 12,
5271–5280. doi: 10.1021/pr400570a
Suzuki, T., and Yamamoto, M. (2015). Molecular basis of the Keap1–Nrf2
system. Free Radic. Biol. Med. 88, 93–100. doi: 10.1016/j.
freeradbiomed.2015.06.006
Trinder, P. (1969). Determination of glucose in blood using glucose oxidase
with an alternative oxygen acceptor. Ann. Clin. Biochem. 6, 24–27. doi:
10.1177/000456326900600108
Tzivion, G., Dobson, M., and Ramakrishnan, G. (2011). FoxO transcription
factors; regulation by AKT and 14-3-3 proteins. Biochim. Biophys. Acta
1813, 1938–1945. doi: 10.1038/mp.a000945.01
Vega, I., Damborenea, M., Gamarra-Luques, C., Koch, E., Cueto, J., and
Castro-Vazquez, A. (2006). Facultative and obligate symbiotic associations
of Pomacea canaliculata (Caenogastropoda, Ampullariidae). Biocell 30,
367–375.
Vega, I., Gamarra-Luques, C., Koch, E., Bussmann, L., and Castro-Vazquez, A.
(2005). A study of corpuscular DNA and midgut gland occupancy by putative
symbiotic elements in Pomacea canaliculata (Caenogastropoda, Ampullariidae).
Symbiosis 39, 37–45.
Vega, I., Giraud-Billoud, M., Koch, E., Gamarra-Luques, C., and Castro-Vazquez, A.
(2007). Uric acid accumulation within intracellular corpuscles of the midgut
gland in Pomacea canalaculata (Caenogastropoda, Ampullariidae). Veliger
48, 276–283.
Wang, H., and Joseph, J. A. (1999). Quantifying cellular oxidative stress by
dichlorouorescein assay using microplate reader. Free Radic. Biol. Med. 27,
612–616. doi: 10.1016/S0891-5849(99)00107-0
Wasowicz, W., Neve, J., and Peretz, A. (1993). Optimized steps in uorometric
determination of thiobarbituric acid-reactive substances in serum: importance
of extraction pH and inuence of sample preservation and storage. Clin.
Chem. 39, 2522–2526. doi: 10.1093/clinchem/39.12.2522
Webb, J. D., Coleman, M. L., and Pugh, C. W. (2009). Hypoxia, hypoxia-
inducible factors (HIF), HIF hydroxylases and oxygen sensing. Cell. Mol .
Life Sci. 66, 3539–3554. doi: 10.1007/s00018-009-0147-7
Yang, S., Zhong, J.-R., Zhao, L.-L., Wu, H., Du, Z.-J., Liu, Q., et al. (2018).
e salinity tolerance of the invasive golden apple snail (Pomacea canaliculata).
Molluscan Res. 38, 90–98. doi: 10.1080/13235818.2017.1386260
Zhao, Y., Chen, M., Storey, K. B., Sun, L., and Yang, H. (2015). DNA methylation
levels analysis in four tissues of sea cucumber Apostichopus japonicus based
on uorescence-labeled methylation-sensitive amplied polymorphism
(F-MSAP) during aestivation. Comp. Biochem. Physiol. B. Biochem. Mol. Biol.
181, 26–32. doi: 10.1016/j.cbpb.2014.11.001
Zhu, H., Itoh, K., Yamamoto, M., Zweier, J. L., and Li, Y. (2005). Role of
Nrf2 signaling in regulation of antioxidants and phase 2 enzymes in
cardiac broblasts: protection against reactive oxygen and nitrogen species-
induced cell injury. FEBS Lett. 579, 3029–3036. doi: 10.1016/j.
febslet.2005.04.058
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