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Cold Tolerance and Physiological Response of Natural Overwintering Pomacea canaliculata in South China

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Pomacea canaliculata (Lamarck 1822), a freshwater gastropod indigenous to lower Del Plata Basin of Argentina, has become the most destructive and invasive rice pests in south China since its introduction in the 1980s. In Guangdong, the main production areas for double rice, most of P. canaliculata overwinter in paddy field ditches after late‐rice harvesting in mid‐November and diapause to temporarily to avoid the damaging effects of extreme low temperatures. This pest aroused from diapause and migrated to the paddy field after early‐rice reviving in next late March. Overwintering and cold tolerance of natural P. canaliculata have a non‐negligible impact on population dynamics and distribution in the following year. We tested the supercooling capability, levels of cryoprotectant synthesis, activity of antioxidant defense system (antioxidant enzymes and reduced glutathione), and degree of oxidative damage (concentration of malondialdehyde as an index of lipid peroxidation) monthly, using natural P. canaliculata samples with a size‐gender structure (i.e., juveniles, female, and male adults) from experimental ponds during the period of mid‐November to the following April. P. canaliculata survived the winter with a monthly death rate of 7%–16.5% in coldest January. The supercooling point (SCP) of overwintering P. canaliculata decreased initially before increasing subsequently with monthly changes in water temperature. P. canaliculata accumulated a high glycogen content before December, which depleted towards the end of January, while lipid content reached peak in January and depleted since February. Activity of antioxidant defense system of P. canaliculata exhibited significant monthly differences and showed relatively higher size heterogeneity than monthly variations. The results contribute to the knowledge of adaptability in overwintering P. canaliculata and help to understand the mechanism of the invasive success of this species.
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Journal of Experimental Zoology Part A: Ecological and
Integrative Physiology
RESEARCH ARTICLE
Cold Tolerance and Physiological Response of Natural
Overwintering Pomacea canaliculata in South China
Zhong Qin
1,2,3
| Zeheng Xiao
1,2,3
| Chuang Li
1,2,3
| Jimin Liu
1,2,3
| Fucheng Yao
1,2,3
| Xiaoting Lin
1,2,3
| Jiaen Zhang
1,2,3
|
Yiman Liu
4
1
College of Natural Resources and Environment, South China Agricultural University, Guangzhou, Guangdong, China |
2
Guangdong Engineering Technology
Research Centre of Modern EcoAgriculture and Circular Agriculture, Guangzhou, Guangdong, China |
3
Key Laboratory of AgroEnvironment in the Tropics,
Ministry of Agriculture and Rural Affairs, Guangzhou, Guangdong, China |
4
Wuhan Academy of Agricultural Sciences, Wuhan, China
Correspondence: Jiaen Zhang (jeanzh@scau.edu.cn)
Received: 7 July 2024 | Revised: 31 August 2024 | Accepted: 9 September 2024
Funding: This work was supported by the National Natural Science Foundation of China (41871034, 31870525, and U1131006), the Open Project Program of
Guang dong Provincial Key Laboratory of Agricultural Artificial Intelligence (GDKLAAI2023003), Guangdong Modern Agricultural Technology Innovation
Team Construction Project (2023KJ134, 2021KJ259, and 2022KJ105).
Keywords: cold tolerance | golden apple snail | overwintering phases | physiological | water temperature
ABSTRACT
Pomacea canaliculata (Lamarck 1822), a freshwater gastropod indigenous to lower Del Plata Basin of Argentina, has become the
most destructive and invasive rice pests in south China since its introduction in the 1980s. In Guangdong, the main production
areas for double rice, most of P. canaliculata overwinter in paddy field ditches after laterice harvesting in midNovember and
diapause to temporarily to avoid the damaging effects of extreme low temperatures. This pest aroused from diapause and
migrated to the paddy field after earlyrice reviving in next late March. Overwintering and cold tolerance of natural
P. canaliculata have a nonnegligible impact on population dynamics and distribution in the following year. We tested the
supercooling capability, levels of cryoprotectant synthesis, activity of antioxidant defense system (antioxidant enzymes and
reduced glutathione), and degree of oxidative damage (concentration of malondialdehyde as an index of lipid peroxidation)
monthly, using natural P. canaliculata samples with a sizegender structure (i.e., juveniles, female, and male adults) from
experimental ponds during the period of midNovember to the following April. P. canaliculata survived the winter with a
monthly death rate of 7%16.5% in coldest January. The supercooling point (SCP) of overwintering P. canaliculata decreased
initially before increasing subsequently with monthly changes in water temperature. P. canaliculata accumulated a high
glycogen content before December, which depleted towards the end of January, while lipid content reached peak in January and
depleted since February. Activity of antioxidant defense system of P. canaliculata exhibited significant monthly differences and
showed relatively higher size heterogeneity than monthly variations. The results contribute to the knowledge of adaptability in
overwintering P. canaliculata and help to understand the mechanism of the invasive success of this species.
1 | Introduction
Pomacea canaliculata (Lamarck 1822) (Gastropoda: Ampullar-
iidae), a freshwater snail native to tropical and temperate South
America, has become a severe invasive agricultural pest in
many Asian countries including the Philippines, China,
Vietnam, Thailand, Japan, and Korea (Hayes et al. 2008;de
Brito and Joshi 2016). This snail was listed as one of 100 of the
world's worst invasive alien species due to its voracious appetite
for a wide variety of aquatic plants (Halwart 1994; Boland
et al. 2008), detrimental effects on biodiversity and the func-
tioning of natural wetlands (Carlsson and Lacoursiere 2005;
© 2024 Wiley Periodicals LLC.
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https://doi.org/10.1002/jez.2874
O'Neil et al. 2023) and vector of some parasites (e.g., Angios-
trongylus cantonensis) relevant to human health (Kim
et al. 2014; Pandian, Najer, and Modrý 2023). One reason ac-
counting for successful range expansion and colonization of
P. canaliculata is its notable adaption to environmental changes
in temperature and food or water availability (Albrecht, Car-
reño, and CastroVazquez 1999) and shows the ability to enter
the hypometabolic states of either estivation (GiraudBilloud
et al. 2013,2022) or hibernation (Matsukura et al. 2009;
Seuffert, Burela, and Martín 2010). As a pantropical freshwater
gastropod, low winter temperatures in invaded regions have
direct effects on survival, growth, and reproduction of P. ca-
naliculata, thereby influencing its distribution and population
dynamics in the subsequent year. Invasive P. canaliculata have
developed a variety of adaptions to avoid the damaging effects
of low winter temperatures in temperate regions (Matsukura
and Wada 2007). For instance, P. canaliculata can increase their
cold tolerance before winter by slowing down metabolism and
entering dormancy in paddy fields, irrigation canals, ponds, and
other bodies of water (Wada and Matsukura 2007; Liu
et al. 2020). Their physiological responses to enhance cold
resistance include the reduction of water retention in the body,
the accumulation of lowmolecularweight compounds (e.g.,
glucose, glycerol, glutamine, and carnosine), and the upregu-
lation of the expression of macromolecular antifreeze proteins
(Matsukura et al. 2008; Xiao et al. 2022).
The dormant state induced by estivation or hibernation repre-
sents a challenging hypometabolic condition that can arouse
imbalances between oxyradical production and antioxidant
defenses, both during dormancy and after arousal (Giraud
Billoud et al. 2011). P. canaliculata developed a peculiar strategy
of preparation for oxidative stress (POS)when it was exposed
to prolonged periods (e.g., 45 days) of environmental stress
during estivation or hibernation (GiraudBilloud et al. 2018).
This species was able to tolerate the oxidative burst induced by
the activityestivationarousal cycle through the action of a
robust defense system based on a combination of enzymatic and
nonenzymatic antioxidant defenses. The regulation of enzy-
matic (superoxide dismutase, SOD and catalase, CAT) and
nonenzymatic antioxidants (uric acid and reduced glutathione)
as well as heat shock protein expression (Hsc70, Hsp70) during
hypometabolism may constitute a mechanism to minimize
oxidative stress in cycles of hibernation and awakening (Gao
et al. 2022; Rodriguez, CampoyDiaz, and GiraudBilloud 2023).
On the other hand, some studies indicated that P. canaliculata
of different sizes or genders might differ in cold tolerance ability
and respond differently to lowtemperature stress (Yusa, Wada,
and Takahashi 2006; Guo et al. 2019). These studies, most of
them done in the laboratory control environment, contributed
to the knowledge of the adaptive strategies and involved
mechanisms of P. canaliculata to tolerate adverse temperature
conditions. Comparable data on P. canaliculata acclimated in
natural environmental conditions, however, are relatively rare,
and little is known about the dynamic physiological adjust-
ments that may accompany hibernation in P. canaliculata.
P. canaliculata was intentionally introduced to Zhongshan city,
Guangdong province, China in the early 1980s. During the past
four decades, this species has extended its range from south to
as far north as 31.0° N and is widely distributed across more
than 17 provinces (Yang, Wu, and Lun 2013). This species could
develop three generations per year in southern provinces of
Hainan and Guangdong dominated by double cropping rice
system, causing serious damage to both rice production and
agroecosystem function (Zhou, Wu, and Yang 2003). The snails
were observed to bury themselves in drained paddy fields or
move to adjacent ditches or canals in early or midNovember,
until the fields are irrigated for rice planting in midMarch of
next year (Liu et al. 2014). The snails tend to stay motionless
(i.e., the state of diapause), safely overwinter with low mortality
(10%27%, unpublished data) in natural environments (Zhou,
Wu, and Yang 2003). Field observation also showed that snails
in this region usually exhibit a rather quick response to
instantaneous temperature changes, being able to reactivate as
soon as conditions are favorable (He et al. 2011). Snails col-
lected in Guangzhou displayed the similar responses to cold
stress as those originating from temperate populations and
showed enhanced cold hardiness after cold acclimation
(Qin et al. 2020). Despite this, the importance of cold stress on P.
canaliculata has not been well studied in this subtropical region,
compared with those conducted in temperate regions. Entering
diapause temporarily during the winter allows P. canaliculata to
avoid the damaging effects of extreme temperatures. Several
studies have been done on physiological aspects or biochemical
changes taking place in the diapausing individuals (Matsukura
et al. 2008; GiraudBilloud et al. 2018), little is known about the
seasonal changes in the cold hardiness capacity of overwintering
P. canaliculata populations and the role of the markers of the
energy metabolism and oxidative stress in them during prepa-
ration for diapause and after arousal.
Here, we investigated variation patterns of synthesis and bio-
chemical substances accompanying the change in the super-
cooling capability of the overwintering P. canaliculata
populations with a sizegender structure, with the aim to (1)
explore changes of the supercooling ability of P. canaliculata in
response to water temperature under natural conditions; (2)
examine whether survival and physiological defense responses
of P. canaliculata differ among populations of different sizes
and between genders; (3) understand the role of metabolism
and antioxidant regulation in P. canaliculata during over-
wintering stages.
Summary
Overwintering and cold tolerance of natural Pomacea
canaliculata populations were investigated.
Supercooling capability, synthesis, and biochemical
substances of P. canaliculata were measured for
6 months.
P. canaliculata survived the winter with a monthly
death rate of 7%16.5% in coldest January
Fluctuations in main biochemical contents and activities
of antioxidant defense system may constitute a signifi-
cant physiological advantage and peculiarity for safe
survival of P. canaliculata in winter.
Survival and physiological defense responses of P. ca-
naliculata differed among populations of different sizes
and between genders.
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2 | Materials and Methods
2.1 | Snails and Experimental Conditions
P. canaliculata snails were collected from an irrigation canal
near rice fields at the teaching and scientific research base of
South China Agricultural University in central Guangdong
Province, China (113°21E, 23°9N) in midNovember. The
region has a humid and subtropical monsoon climate with an
average annual air temperature of 21.5°C. January is the
coldest month with an average temperature of 13.0°C while the
hottest is July at 28.5°C. Annual precipitation ranges from 1612
to 1909 mm, with more than 80% occurring between April and
September (Cheng et al. 2016).
A batch of eggs identified as P. canaliculata hatched in the canal
in advance, and a gauze net was installed at the inlet and outlet
of the canal to prevent apple snails from entering and escaping
(Zhang et al. 2023). Three sizegender groups of snails (each
group containing 96 individuals) were randomly selected and
marked with paint before their use in the experiments: juveniles
(gender was not distinguished) with a shell height of
15.025.0 mm, male and female individuals in the same size of
25.035.0 mm. Each sampled group was put into plastic bags
containing moist towels, placed in a cooler with ice, and
transported immediately to the ecological farm of South China
Agricultural University, where experiments described below
were conducted.
Twentyfour cemental ponds (each size: 1 m × 1 m × 1 m) were
set in the ecological farm of South China Agricultural Univer-
sity, each had a 20cmdeep soil layer at the bottom. The sedi-
ment originated from a paddy field and was airdried, crushed,
and mixed before use. Then, CaCO
3
saturated tap water
(renewed every 2 days) was introduced to a depth of 15 cm
above the soil surface. These ponds, each having a bottom hole,
were connected together via a water pipe to keep the same
water depth. Twelve individuals from each sizegender group
were arranged into one pond randomly. Throughout the ex-
periment, the snails were fed with preweighed fresh lettuce
(35 g for each individual snail) three or four times a week,
based on our previous study on foraging of the snail (Xu
et al. 2011). Any leftover lettuce was carefully removed the
next day. During every 2 days' monitoring, snail was confirmed
dead when an odoriferous rotting body was detected or the
operculum did not contract after touching with a probe (Wada
and Matsukura 2007). Any dead individuals were removed after
counted when monitoring. The survival of the snails was
recorded on the day when sampling for index measurement.
During the study period, water temperatures were monitored
automatically through a digital thermometer having an accu-
racy of ± 0.1°C.
2.2 | Sampling and Measurements
Late every month from November 23, 2021 until April 22,
2022, P. canaliculata snails from each sizegender group were
collected randomly and divided into three subsamples to
conduct each measurement separately. All snails were
weighed individually (BW; ± 0.01 g) and shell height and
width were measured with a Vernier caliper (0.02 mm preci-
sion) before testing.
2.2.1 | Supercooling and Freezing
The supercooling point (SCP) was measured (with six repli-
cates) using a thermocouple connected to a DT9205A Type
Digital Multimeter (Guo et al. 2014). The specimens were
attached to the thermocouple by adhesive tape and placed in-
side a refrigerated chamber that was cooled at a rate of 0.5°C
per minute. The SCPs were defined as the lowest temperature
before an exothermic reaction, as indicated by a sudden tem-
perature increase. The freezing point (FP) was reached when
the thermocouple recorded the heat released by a crystallization
event (Ansart, Vernon, and Daguzan 2001). For each size
gender group, both SCP and FP were tested on six individuals.
2.2.2 | Water Content and Cryoprotectant Synthesis
The selected snails were frozen at 40°C for 10 min and
weighted to evaluate the body mass. The fresh tissue of each
individual was weighed after removing the shell and the oper-
culum (fresh mass: FM). After that, the soft tissue of each snail
was dried at 60°C for 24 h and weighed (M). The tissue was then
placed in a drying oven at 105°C and weighed every 12 h until a
constant mass (dry mass: DM) was obtained. Total water con-
tent (TWC, %) and free water content (FWC, %) of tested snail
were calculated respectively from the following equation:
T
WC = (FM DM)/FM × 100%
,
F
WC = (FM M)/FM × 100%.
The bound water content (BWC, %) was differences in values of
TWC and FWC. Each water content measurement was replicated
six times. After the water content measurement, the specimen was
ground into a powder and used to measure the total lipid content
using solvent extraction (chloroformmethanol) method as
described by (Folch, Lees, and Stanley 1957) with modifications
(Matsukura et al. 2008). Glycerol content was measured
as described by (Guo et al. 2019), while the glycogen content was
determined using the method of (Dreiling et al. 1987). Each
cryoprotectant for specific sizegender snail group was tested on
four individuals.
2.2.3 | Antioxidant Parameters and Protein
Tissue samples from the digestive gland of tested snails were
dissected and washed with icecold saline solution. The dis-
sected organ was individually homogenized with nine volumes
of physiological saline solution in an ice bath. The resulting
suspension was centrifuged at 3000 r/min for 10 min at 4°C.
Supernatants were then collected, aliquoted, and frozen for
determining concentrations of protein and antioxidant enzyme
activities within 24 h. Antioxidant activities of superoxide dis-
mutase (SOD), peroxidase (POD), and catalase (CAT) as well as
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concentrations of malondialdehyde (MDA) and reduced gluta-
thione (GSH) were determined with commercially available
standard assay kits (Nanjing Jiancheng Science and Technology
Company Ltd., Nanjing, China). These enzymes were measured
using a UVspectrophotometer (Beijing Purkinje General
Instrument Co., Ltd.) at 550, 470, 405, 532, and 420 nm as
described by the manufacturer's instructions, respectively. Total
protein concentration in the homogenates was determined by
the method described by (Lowry et al. 1951) using bovine serum
albumin (Sigma Chemical) as a standard. The colored complex
was measured at 690 nm. For each sizegender group of snails,
all quantifications for assays were repeated four times and ex-
pressed as means ± standard errors (SEM).
2.3 | Statistical Analysis
A splitplot analysis of variance (ANOVA) model was used to
examine differences in survival, cold tolerance, and physiolog-
ical response of P. canaliculata, followed by Tukey multiple
comparison tests (at the 0.05 level). The main factor was six
sampling months from late November 2021 until April 2022.
The subfactor was three sizegender groups of snails (i.e., ju-
veniles, female, and male adults), and the block was the cement
pond. Statistical analyses were performed using the SPSS soft-
ware (version 25.0; SPSS, Chicago, IL, USA). To assess the ef-
fects of water temperature on snail supercooling capacity during
the activitywinter diapausearousal cycle, redundancy analysis
(RDA), a canonical community ordination method was used, in
which a total of seven variables including cryoprotectant solutes
(free water content, glycogen, lipid and glycerol) and body
weight of snail were correlated with five enzymatic and non-
enzymatic antioxidants (SOD, POD, CAT, GSH and MDA)
derived from monthly sampled snail groups. Factors signifi-
cantly related to enzyme activities need to be selected by Monte
Carlo analysis before the redundancy analysis, as implemented
in Canoco for Windows version 5.0 (Microcomputer Power,
Ithaca, NY, USA).
3 | Results
3.1 | Temperature, Survival and the Supercooling
Capability
Average water temperature (T
mean
) in experimental pond was
24.7°C in November, declining to 17.2°C in December and
13.3°C in January, then increasing to 22.5°C in February. The
minimum monthly water temperature (T
min
) was below 20°C
from November to the end of April, with the lowest in January
(6°C). The maximum monthly water temperature (T
max
) ex-
ceeded 20°C during the observed months (Figure 1a). At low
temperatures under 17.5°C, over 50% of the P. canaliculata
entered into a state of diapause temporarily, and under 10°C,
snails began to withdraw into their shells.
Survival of the snail differed significantly throughout the
observed months, with a mean survival rate of 90% in the initial
overwintering period followed by a low percentage (no more
than 70%) during the overwintering maintenance. The lowest
survival rate of 42.5% was recorded for the snails at the
termination of overwintering. There were no significant differ-
ences between survival of the male and female adults, but the
survival was relatively higher in female adults. The highest cold
tolerance during the observed months was found in juvenile
snails. Survival of the juveniles decreased from November,
remained 87.5% survival in January with minimum water
temperature, and reached to 62.5% survival after overwintering,
nearly two times of adult snails (Figure 2).
During the experimental period, SCPs of the snails varied from
(7.80 ± 0.456°C) (detected in April, male adults) to
(6.15 ± 0.119°C) (detected in November, juveniles). Juvenile
snail had significantly lower supercooling points, about
0.23°C0.67°C lower than that of adults during overwintering
periods (Table 1). Significant differences were found between
the SCPs of overwintering stages. The SCPs of the snails were at
the greatest levels in the overwintering initiation (mean value of
SCPs in November was 6.42°C), reached moderate levels
(SCPs in December and late January next year were higher than
7.0°C) in the overwintering maintenance, and decreased since
the termination of overwintering (mean value of SCPs from
February to April was higher than 7.6°C). The phase features
of SCPs were relatively obvious in juvenile snail population
(Figure 3). Similar trends were observed in freezing points (FPs)
of the snail across the seasonal periods. The minimum freezing
point (4.29 ± 0.122°C) was recorded in juveniles, significantly
lower than those in adult snails (Figure 3).
3.2 | Water Content and Cryoprotectant
Synthesis
Freewater content (FWC) for the snails decreased significantly
from 88.5%91.5% (in November) to the lowest level
(81.9%89.8%) during the overwintering maintenance, then
increased after arousal. The boundwater content (BWC) for the
snails decreased to the lowest value of 6.45%7.86% during
winter diapause, followed by a significant increase at the ter-
mination of overwintering (Figure 4). Significant differences in
FIGURE 1 | (a) Variations of pond water temperature; (b) Varia-
tions of lipid content in the overwintering Pomacea canaliculata pop-
ulation with a sizegender structure (juveniles, female, and male adults)
from November 2021 to April 2022. Mean survival and standard errors
(n= 4) for each group were also shown.
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FWC were detected between the three snail groups, for which
the juveniles had the highest FWC with a mean of 90.6% while
female snails had the lowest. The BWC in the juveniles was
significantly higher than adult snails (Table 1).
Glycogen, glycerol, and lipid were detected as major cryopro-
tectants in snail collected from Autumn to Spring of next year.
Significant differences in the levels of the three cryoprotectants
were detected in different observed months. For all groups of
snails, mean glycerol content increased (F
5, 15
= 16.264,
p= 0.000, Table 2) from 20.283 mg·g
1
in November and
reached the maximum level of 29.147 mg·g
1
in December.
Massive depletion of glycerol deposits was observed between
December and January, followed by a partial reaccumulation
during early spring. The seasonal pattern of glycogen was
similar for glycerol (Figure 5). Season change of lipid in the
snails exhibited an opposite tendency to water temperature
(Figure 1b). The amounts of lipid increased during autumn,
reached a peak of 79.833 mg·100 mg
1
in January and
decreased, followed by a partial reaccumulation after March.
Both lipid and glycogen contents differed significantly between
the three snail groups (Table 2). The lipid content was
the highest in male snails and was lowest in juveniles by
comparing the female snails. Maximum lipid content of
87.373 ± 1.661 mg·100 mg
1
was recorded in male adults in
January, which amounted to 1.05 and 1.27 times of female
adults and juveniles, respectively. Unlike the lipid, maximum
glycogen content of 8.805 ± 0.7382 mg·100 mg
1
was recorded
in juvenile snail in January. The order of glycerol content was
opposite from that of the lipid. No difference in glycerol content
was seen between the three snail groups (Table 2).
3.3 | Antioxidant Profile
Activities or concentrations differed significantly throughout
the observed months for all analyzed antioxidant enzymes or
FIGURE 2 | Survivorship of overwintering Pomacea canaliculata
population with a sizegender structure (juveniles, female, and male
adults) from November 2021 to April 2022. Mean survival and standard
errors (n= 4) for each group were also shown.
TABLE 1 | Splitplot ANOVA for water contents, supercooling and freezing points (each with six replicates) in overwintering Pomacea canaliculata population with a sizegender structure. Main factor:
six sampling months from late November 2021 until April 2022. Subfactor: juveniles, female, and male snail adults.
Main plot Subplot
Physiological
parameters
Month (df = 5) Plot (df =5)
Mainplot
error (df = 25) Sizegender (df =2)
Month × Size
gender (df = 10)
Subplot
error (df = 60)
MS FMS FMS MS FMS FMS
Free water 128.055 10.458* 10.302 0.841 11.418 554.761 45.304* 16.420 1.341 12.245
Bound water 30.080 63.233* 0.947 1.991 0.612 6.551 13.772* 1.665 3.500* 0.476
Supercooling point 3.297 9.628* 0.374 1.091 0.364 1.734 5.063* 1.025 2.992* 0.342
Freezing point 6.295 15.158* 0.381 0.918 0.570 5.793 13.948* 0.580 1.398 0.415
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antioxidants in tested P. canaliculata snails (Table 2), with the
highest values recorded either in initial overwintering or during
the overwintering maintenance period. Among these enzymes,
the content of GSH displayed around a 1.86fold increase in last
December, while POD displayed 1.33fold increase in January
in comparison to those in April.
The three snail groups did not have a consistent variation in
activity/content of each antioxidant (Figure 6). Except GSH and
FIGURE 3 | Variations of the supercooling point and freezing points in
overwintering Pomacea canaliculata population from November 2021 to
April 2022. (ac) Variation of SCP (violin with quartile plot) and FP (box
plot) in juveniles, female, and male snail adults, respectively. Mean survival
and standard errors (n= 6) for each group were also shown.
FIGURE 4 | Variations of freewater content (FWC) and bound
water content (BWC) in overwintering Pomacea canaliculata popula-
tion. (ac) Variations of FWC (violin with quartile plot) and BWC (box
plot) in juveniles, female, and male snail adults, respectively. Mean
survival and standard errors (n= 6) for each group were also shown.
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POD, enzyme activities of the snail were also sizegender
depended. Further analysis with Tukey post hoc test showed
interaction between observed months and snail groups. In gen-
eral, the activities of SOD and CAT were significantly higher in
adults compared with juveniles, while the MDA content of adults
TABLE 2 | Splitplot ANOVA for cryoprotectants antioxidant parameters (each with four replicates) in overwintering Pomacea canaliculata population with a sizegender structure. Main factor and sub
factor were the same as Table 1.
Main plot Subplot
Physiological
parameters
Month (df = 5) Plot (df =3)
Mainplot
error (df = 15) Sizegender (df =2)
Month × Size
gender (df = 10)
Subplot
error (df = 36)
MS FMS FMS MS FMS FMS
Cryoprotectants
Glycogen 18.325 13.666* 0.599 0.446 1.797 13.829 10.313* 7.618 5.681* 1.341
Glycerol 164.078 16.264* 13.939 1.382 7.373 3.889 0.385 24.220 2.401* 10.088
Lipid 882.176 31.751* 12.486 0.449 40.895 2901.635 104.433* 117.633 4.234* 27.785
Antioxidant parameters
SOD 12.146 4.980* 2.949 1.209 2.599 31.122 12.760* 5.192 2.129 2.439
POD 8.270 10.730* 0.744 0.965 0.613 0.968 1.256 0.333 0.432 0.771
MDA 1.995 46.488* 0.076 1.767 0.097 1.139 26.557* 0.325 7.581* 0.043*
GSH 1535.420 49.466* 2.580 0.083 38.825 101.625 3.274 10.762 0.347 31.040
CAT 283.411 10.173* 41.003 1.472 53.610 158.141 5.676* 84.035 3.016* 27.860
FIGURE 5 | Variations of glycerol and glycogen concentrations in
overwintering Pomacea canaliculata population. (ac) Variations of
glycerol and glycogen concentrations in juveniles, female, and male
snail adults, respectively. Mean survival and standard errors (n= 4) for
each group were also shown.
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was significantly lower than that of the juveniles. The male and
female snails had the same level of the enzyme activities.
Six factors, including average water temperature (T
mean
), su-
percooling points (SCPs), freezing points (FPs), freeand
boundwater content (FWC and BWC), and body weight of the
snails (BW) were used as explanatory variables, while the
activities or contents of the five enzymes were used as response
variables for redundancy analysis. Among these variables, WT
explained most variations among snail enzyme activities
(39.9%), followed by BW (17.2%) and SCPs (11.0%) (Table 3).
These three variables (WT, BW, and SCPs) explained together
68.1% of total variation and were considered important because
their effects on snail enzyme activities/contents reached a sig-
nificant level. The other factors were unselected according to
the forward selection step of RDA, because each of them ex-
plained < 10% of the variations. The characteristic values of the
snail enzyme activities/contents in the first and second axes
were 0.4839 and 0.1721, respectively, while the correlations
between the snail enzyme activities/contents and explanatory
variables were 0.9451 and 0.7529, respectively (Table 4). The
first two redundancy axes accounted for 65.60% of the varia-
bility in snail enzyme activities/contents (Axis 1 = 48.39%, Axis
2 = 17.21%) and accounted for 96.36% of the variability in snail
antioxidative functionsexplanatory variables relation (Axis
1 = 71.08%, Axis 2 = 25.28%). The amount rose to 82.73% of the
variation in antioxidative enzymes was explained by all
canonical axes (Table 4). The twodimensional ranking of snail
enzyme functions and explanatory variables revealed that the
first axis was primarily related to WT, while the second and
third axes were mainly associated with BW and SCPs, respec-
tively (Tables 3and 4).
Looking from the horizontal central axis, samples from juvenile
snails clustered to the upper of Axis 1, which was clearly sep-
arated from adults and was related to lower values of body
weight and supercooling points (SCPs). All samples from adult
snails clustered to the lower of Axis 1, in which male and
female snails did not constitute welldefined groups. Looking
from the vertical central axis, samples from adult snails could
be divided into three groups (from right to left), with one right
group consisting of four sample snails (two male and two
female) in December and January of 2022, one upperleft group
consisting of six sample snails (three samples for each gender)
in February, March, and April, and the lowerleft group con-
sisting of two samples (a male and a female) in November 2021
(Figure 7).
4 | Discussion
4.1 | Survival and Supercooling Capability
P. canaliculata groups survived the coldest January (T
mean
was
13.3°C) with a monthly death rate of 7%16.5%. The most
obvious death rate occurred at milder temperatures in February
(for juveniles, monthly death rate was 17.14%) and March (for
adults, monthly death rate was 14.82%17.90%). The findings
FIGURE 6 | Variations of antioxidant activities of superoxide dis-
mutase (SOD), peroxidase (POD), catalase (CAT) as well as concen-
trations of malondialdehyde (MDA) and reduced glutathione (GSH) in
overwintering Pomacea canaliculata population. (ac) Variations of
these antioxidant enzymes and antioxidants in juveniles, female, and
male snail adults, respectively. Mean survival and standard errors (n=
4) for each group were also shown.
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reinforced the previous evidence that the development of cold
tolerance of P. canaliculata was not a countermeasure to
freezing but served as avoidance or reduction of indirect chill-
ing injury at moderately low temperatures (Matsukura
et al. 2009).
Changes in supercooling capacity and the levels of biochemical
substances in P. canaliculata during overwintering phases were
investigated in this study. The SCP values for P. canaliculata
groups were consistent with a previous study of our workgroup
(ZHAO Benliang et al. 2012). Mean SCP values observed for the
coldest month (January) ranged from 7.06°C to 6.47°C. That
is, SCP was at least 10°C lower than the minimum ambient
temperature. The results showed that P. canaliculata had a high
supercooling capacity to avoid freezing in winter. They can
resist cold stress and adapt to low winter temperatures in the
field of Guangzhou. The greatest threat to survival of P. cana-
liculata was the cumulative effect of exposure in the cold above
the freezing temperature, rather than freezing.
Significant changes in monthly SCP values in P. canaliculata
were observed, which accompanied the seasonal variation in
water temperature. The highest level of SCP was recorded at the
onset of the overwintering (for juveniles), in the phase of
overwintering maintenance (for female snails), at the termina-
tion of the overwintering (for male snails), respectively. The
mean SCP of P. canaliculata achieved the lowest during arousal
when mean water temperature was higher than 20°C (Figure 3),
indicating that the cold tolerance of P. canaliculata in the pond
was not lost at warm temperature. The findings supported the
results from a previous indoor test on P. canaliculata
(Matsukura et al. 2009). On the other hand, fluctuation of SCP
suggested that the association between the minimum water
temperature and the mean SCP was not strong. The super-
cooling point of overwintering P. canaliculata decreased ini-
tially before increasing subsequently with monthly changes in
water temperature. Similar findings were revealed from
overwintering wheat pest Eurygaster integriceps Puton (Hem.:
Scutelleridae) (Hasanvand, Izadi, and Mohammadzadeh 2020),
larvae of Kermania pistaciella (Lep.: Tineidae) (Mollaei
et al. 2016) and the linden bug Pyrrhocoris apterus (Ditrich
et al. 2018).
4.2 | Water Content and Cryoprotectant
Synthesis
Both freeand boundwater were found to decrease before and
during overwintering maintenance, then increase during
arousal in this study. Similar patterns were reported in many
studies on insects (Feng et al. 2016). Likewise, winter diapause
of P. canaliculata could also be associated with an increase in
water content to resume morphogenesis, which has been
revealed in most of the insects (Rozsypal et al. 2013). P. cana-
liculata increased metabolites to provide the necessary cryo-
protectants and energy sources for overwintering. This species
was observed to accumulate a high glycogen content before
winter diapause (December), which depleted towards the end of
the diapause period, while lipid content reached peak in Jan-
uary and depleted since February. It is likely that P. canalicu-
lata may alter their metabolic energy reserves during different
phases of overwintering, for which it mostly relied on glycogen
as the main energy reserve in the winter diapause initiation and
switched to lipid during the diapause maintenance and the
termination. The energy levels of the metabolic fuels in P. ca-
naliculata increased rapidly with winter diapause termination
to meet the energy demands for the postdiapause development
and reproduction. Such energy management strategy employed
by P. canaliculata was in accordance with previous studies on
some overwintering pests (e.g. Lin et al. 2020). However, the
findings were inconsistent with the work on P. canaliculata
from a drained paddy field by (Matsukura et al. 2008). They
recorded the increased glycerol in overwintering P. canaliculata
with decreasing glycogen concentration and the stable amount
TABLE 3 | RDA analysis of antioxidant levels/activities in overwintering Pomacea canaliculata population and the influencing variables
determined by interactive forward selection procedure with unrestricted permutation tests.
Variable Explains %
Contribution % of variation
explained PseudoFpvalue
p
value (adj)
Average water temperature
(T
mean
)
39.9 58.5 10.6 0.002 0.006
Body weight (BW) 17.2 25.3 6.0 0.002 0.003
Supercooling points (SCPs) 11.0 16.2 4.8 0.004 0.004
TABLE 4 | Eigenvalues and cumulative interpretation of RDA sequence of antioxidant levels/activities in overwintering Pomacea canaliculata
population.
Statistic
Eigen
values
Explained
variationcumulative
%
Pseudo
canonicalcorrelation
Explained fitted
variationcumulative %
Sum of all
canonicalEigen
values
Axis 1 0.4839 48.39 0.9451 71.08 0.8273
Axis 2 0.1721 65.60 0.7529 96.36
Axis 3 0.0248 68.08 0.6516 100
Axis 4 0.1465 82.73
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of lipids. Discrepancy of chemical compound changes might
attribute to water and food conditions associated with the
development of cold tolerance of P. canaliculata. Despite this
discrepancy, both studies suggested that carbohydrate meta-
bolic pathways were altered in P. canaliculata during seasonally
exposure to low temperatures (Matsukura et al. 2009). The
observed variation in depletion of energy reserves and fluctua-
tion in main biochemical contents may constitute a significant
physiological advantage and peculiarity for safe survival of P.
canaliculata in winter. We described a pattern of use of meta-
bolic fuels typical in P. canaliculata under natural environment
in this study. Further work in this regard as well as low
molecularweight carbohydrates (e.g., trehalose) and amino
acids in relation to diapause development of P. canaliculata,
would be valuable in elucidating cold stress tolerance mecha-
nisms of the snail in its natural habitat.
4.3 | Antioxidant Profile
Antioxidant defense is essential for animals to cope with
homeostasis disruption during hibernation (HermesLima,
Storey, and Storey 1998). Cold stress in winter causes oxida-
tive stress in the organism mainly by the accumulation of
reactive oxygen species (ROS), while antioxidant enzymes in
the organism can remove them or transform them into less
toxic compounds (HermesLima et al. 2015). In this experiment,
the antioxidant system of P. canaliculata showed significant
activation during winter diapause and following arousal, pos-
sibly in preparation for physiological oxidative stress due to the
overproduction of ROS that accompanies the disruption of
oxygenmetabolic homeostasis induced by lowtemperature
exposure. However, it does not seem possible to make the
general conclusion that upregulation of antioxidant enzymes
represents an adaptive response to winter diapause on a broad
based multitissue, because the current study determined the
changes in the antioxidant defenses in whole soft tissue mass
of P. canaliculata, rather than separate tissues. The adaptive
response of P. canaliculata to winter diapause and arousal
might be tissuespecific (Lian et al. 2019), further study in this
regard may help to understand the mechanisms that lead to the
development of the POS strategy during the overwintering
phases.
The regulation of the antioxidant system in P. canaliculata as a
mechanism to minimize oxidative stress under extreme en-
vironmental temperatures has been demonstrated in some short
term controlled experiments (Matsukura et al. 2008). P. canali-
culata developed a peculiar form of POS when it was exposed to
prolonged periods (45 days) of environmental stress (e.g. estiva-
tion or hibernation) (GiraudBilloud et al. 2013,2018). Non-
enzymatic antioxidants (e.g., uric acid, reduced glutathione) were
found to be an alternative to the adaptive physiological strategy
of POS. Uric acid was not recorded in this study, however, we
observed that the nonenzymatic antioxidant GSH in the digestive
gland increased significantly before the winter diapause, which
might present a potential POS strategy.
4.4 | Responses of SizeGender Groups
In the present work, snails of different sizes and genders had
notable effects on survivals of overwintering P. canaliculata.
Juvenile snails had significant higher survival rates than adults
throughout the observed months. The findings were in general
agreement with previous reports in which snails of 1020 mm
individuals survived best over winter (Oya, Hirai, and
Miyahara 1987; Yusa, Wada, and Takahashi 2006), and also
proved by field surveys from temperate Japan (Wada and
Matsukura 2007; Wada and Matsukura 2011) and southern
China (Guo et al. 2015), where most overwintering snails were
juveniles. P. canaliculata could seasonally depress their SCPs
during overwintering stages to ensure that the freezing point
was lower than the ambient temperature and that their body
fluids remained unfrozen. Compared with those in P. canali-
culata adults, the enhanced level of juveniles supercooling
capacity may be associated with higher levels of glycogen and
glycerol contents in their body (Figure 5). P. canaliculata can
regulate their antioxidant defense to prevent or minimize
potential oxidative damage by maintaining high level of activ-
ities of the antioxidant enzymes and GSH concentration during
the winter diapause. Adult P. canaliculata snails exhibited
stronger activities of SOD, CAT, and POD as well as lower MDA
content than those in juveniles. It appeared that adult snails
were more sensitive to defense oxidative damages than
juveniles.
FIGURE 7 | Redundancy analysis (RDA) ordination triplot of the
first two axes of antioxidant levels/activities in overwintering Poma-
cea canaliculata population. The antioxidant parameters (redcolor
highlighted symbols) and the selected variables (darkbluecolor
highlighted symbols, expressed as explanatory variables) were pre-
sented as line vectors. The three variables were average water tem-
perature, T
mean
; supercooling points, SCPs; body weight of the snails,
BW. Three P. canaliculata groups, each with four replicates across the
six experimental months were also presented in the plot. For sim-
plicity, juveniles, female, and male snail adults were represented with
their capitalized first letter, respectively. The six months from
November 2021 to April 2022 were denoted sequentially by capital
letters from A to F.
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Generally, female snails had a small advantage over males in
survival during the winter diapause. Almost 76% of female
snails and 72.5% of males survived the coldest January, and the
survival rates of both snails exceeded 55% at the initial stage of
arousal, which supports the cold tolerance of P. canaliculata
during cold exposure and overwintering (Wada and
Matsukura 2007). The results were also consistent with those
obtained in a previous work showing that 95% of female snails
and 70% of males could safely survive the winter under dry
conditions in Guangzhou (Guo et al. 2019). During the over-
wintering period, supercooling point of overwintering P. cana-
liculata females was lower than that of males by 0.15°C0.23°C.
The finding was congruent with that found by (Guo et al. 2014).
Compared with P. canaliculata males, females had lower free
water contents, higher bound water contents as well as glycerol
content, which likely increased the fluid concentrations in
females (Block 2002). Relatively higher concentrations in P.
canaliculata females that could be dissolved effectively in water,
may be associated with relatively lower SCP and an improved
cold tolerance than males.
5 | Conclusions
In brief, the present study investigated monthly changes in
survival, supercooling capacity, and the levels of physiological
biochemical substances in overwintering P. canaliculata,
thereby providing new data for identifying cold hardiness of this
invasive species in natural habitats. The results indicated that
cold capacity of P. canaliculata was not only associated with the
supercooling point, but also affected by size, gender, and other
factors (e.g., genetics). More detailed information on cold tol-
erance of P. canaliculata under various spatial and temporal
effects is needed for a complete picture of stress response and
regulation strategy of this species.
Author Contributions
Jiaen Zhang conceived and designed the experiments. Zeheng Xiao
performed the experiments. Jimin Liu, Xiaoting Lin, and Chuang Li
collected the specimens; Fucheng Yao analyzed the data. Zhong Qin
wrote the manuscript. Yiman Liu provided editorial advice.
Acknowledgments
The authors thank anonymous reviewers for providing helpful com-
ments on earlier versions of this manuscript. This work was supported
by the Open Project Program of Guang dong Provincial Key Laboratory
of Agricultural Artificial Intelligence (GDKLAAI2023003), the
National Natural Science Foundation of China (41871034, 31870525,
and U1131006), Guangdong Modern Agricultural Technology Innova-
tion Team Construction Project (2023KJ134, 2021KJ259, and
2022KJ105).
Conflicts of Interest
The authors declare no conflicts of interest.
Data Availability Statement
All the experiment data sets and figures in the article can be accessed at
Dryad Digital Repository: http://doi.org/10.5061/dryad.v6wwpzh3r.
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