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Received: 6 December 2024
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Accepted: 9 January 2025
Published: 17 January 2025
Citation: Qiu, M.; Bi, X.; Liu, Y.; Li, H.;
Li, D.; Chen, G. Toxicology Effects of
Cadmium in Pomacea canaliculate:
Accumulation, Oxidative Stress,
Microbial Community, and
Transcriptome Analysis. Int. J. Mol.
Sci. 2025,26, 751. https://doi.org/
10.3390/ijms26020751
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Article
Toxicology Effects of Cadmium in Pomacea canaliculate:
Accumulation, Oxidative Stress, Microbial Community,
and Transcriptome Analysis
Mingxin Qiu 1, Xiaoyang Bi 1, Yuanyang Liu 1, Huashou Li 1, Dongqin Li 2,* and Guikui Chen 1,*
1Guangdong Laboratory for Lingnan Modern Agriculture, Guangdong Provincial Key Laboratory of
Agricultural & Rural Pollution Abatement and Environmental Safety, College of Natural Resources and
Environment, South China Agricultural University, Guangzhou 510642, China;
20222005009@stu.scau.edu.cn (M.Q.); 20221005001@stu.scau.edu.cn (X.B.); 1260710657@stu.scau.edu.cn (Y.L.);
lihuashou@scau.edu.cn (H.L.)
2Institute of Quality Standard and Monitoring Technology for Agro-Products, Guangdong Academy of
Agricultural Sciences, Guangzhou 501640, China
*Correspondence: lidongqin@gdaas.cn (D.L.); guikuichen@scau.edu.cn (G.C.)
Abstract: Cadmium (Cd) pollution poses an important problem, but limited information is
available about the toxicology effects of Cd on freshwater invertebrates. We investigated
the accumulation, oxidative stress, microbial community changes, and transcriptomic
alterations in apple snails (Pomacea canaliculata) under Cd stress. The snails were exposed to
the 10
µ
g/L Cd solution for 16 days, followed by a 16-day elimination period. Our results
showed that the liver accumulated the highest Cd concentration (17.41
µ
g/g), followed by
the kidneys (8.00
µ
g/g) and intestine-stomach (6.68
µ
g/g), highlighting these tissues as
primary targets for Cd accumulation. During the elimination period, Cd concentrations
decreased in all tissues, with the head-foot and shell exhibiting over 30% elimination rates.
Cd stress also resulted in reduced activities of superoxide dismutase (SOD), catalase (CAT),
and glutathione transferase (GST) compared to the control group. Notably, even after
16 days of depuration, the enzyme activities did not return to normal levels, indicating
persistent toxicological effects. Cd exposure significantly reduced the diversity of gut
microbiota in P. canaliculata. Moreover, transcriptome analysis identified differentially
expressed genes (DEGs) primarily associated with lysosome function, motor proteins,
protein processing in the endoplasmic reticulum, drug metabolism via cytochrome P450
(CYP450), arachidonic acid metabolism, and ECM–receptor interactions. These findings
suggest that Cd stress predominantly disrupts cellular transport and metabolic processes.
Overall, our study provides comprehensive insights into the toxicological impact of Cd on P.
canaliculata and emphasizes the importance of understanding the mechanisms underlying
Cd toxicity in aquatic organisms.
Keywords: Pomacea canaliculate; cadmium; oxidative stress; gut microbiota; transcriptome
1. Introduction
Cadmium (Cd) is a highly carcinogenic heavy metal, with concentrations of Cd in
uncontaminated water typically below 1 ug/L [
1
] However, both natural phenomena
such as volcanic eruptions and anthropogenic activities, including mining, smelting, and
fertilization, can dramatically elevate Cd levels by thousands or even tens of thousands of
times [
2
]. As a non-essential toxic element, Cd primarily exists in the aquatic environment
Int. J. Mol. Sci. 2025,26, 751 https://doi.org/10.3390/ijms26020751
Int. J. Mol. Sci. 2025,26, 751 2 of 16
in ionic form, characterized by high solubility and mobility, and it accumulates in aquatic
organisms through food chain amplification [3–5].
Extensive research has been conducted on the effects of waterborne Cd on aquatic
organisms. Cadmium can be bioaccumulated in aquatic organisms, leading to adverse
physiological and biochemical effects. Research by Yang demonstrated that prolonged
exposure to low doses of Cd over four months adversely affects various organs in tilapia,
including the gills, muscles, brain, and intestine [
6
]. This exposure not only increases the
prevalence of harmful intestinal microbiota but also impairs the immune function of tilapia
and diminishes the liver’s detoxification capacity. Similarly, Wang found that a 30-day
exposure to waterborne Cd negatively impacts Carassius auratus gibelio by suppressing
appetite, which results in reduced growth and survival rates [
7
]. This exposure also alters
the structure and composition of the gut microbiota and disrupts the functional gut barrier.
Furthermore, Cd has been shown to compromise the structural integrity of the kidneys,
particularly affecting the glomeruli and tubules, as evidenced by Cui [
8
]. The observed
reduction in ATPase activity and abnormal levels of superoxide dismutase (SOD) and
catalase (CAT) suggest that Cd interferes with mitochondrial energy metabolism. Despite
these known effects, research on the impact of Cd on freshwater invertebrates remains
limited. In particular, few studies have thoroughly examined the changes in gut microbiota
and transcriptomic responses in these organisms. This lack of comprehensive analysis
highlights a significant gap in our understanding of Cd’s impact on invertebrate health.
Consequently, there is an urgent need for detailed studies on Cd toxicology in freshwater
invertebrates to address this gap.
The gut microbiota is essential for aquatic organism health, as it maintains intestinal
barrier integrity, promotes metabolism, and enhances immunity [
9
]. Waterborne Cd can
detrimentally impact the gut of aquatic organisms, leading to alterations in gut length
and weight as well as changes in microbial community structure and taxonomic composi-
tions [
10
]. While the majority of research has concentrated on the toxic effects of Cd on the
intestinal tracts of fish, studies on the gut microbiota response of freshwater invertebrates
under Cd stress are relatively scarce [
6
,
11
–
13
]. Understanding the effects of Cd on these
organisms is crucial for assessing overall ecosystem health.
Transcriptomics is the study of gene expression profiles. In recent years, transcrip-
tomics has been widely used to study the effects of heavy metals on aquatic organisms [
14
].
This approach has helped to deepen our understanding of the potential effects of Cd on the
environment and aquatic organisms [
15
]. For instance, Nair demonstrated that Cd stress
induces changes of oxidative enzyme and CYP450, which are related to cellular antioxidant
activity and detoxification [
16
]. Myosin was significantly decreased under Cd exposure,
which plays an important role in cell movement and intracellular material transport [
17
].
Likewise, this technique allowed us to perform transcriptome analysis of apple snails to
further reveal how Cd affects apple snails at the molecular level.
In the present study, the apple snail (Pomacea canaliculata) was chosen as the indicator
species due to its remarkable adaptability and extensive distribution across North America,
Africa, and Asia despite its native origin in South America [
18
]. This species is capable
of thriving in a wide temperature range from 10
◦
C to 35
◦
C, which contributes to its
widespread presence. Notably, the apple snail’s digestive glands have a significant ability
to accumulate heavy metals, making it an ideal subject for environmental monitoring.
Histopathological examinations have identified substantial changes in the digestive glands
under Cd stress, such as increased cupping, degeneration of columnar cells, and narrowing
of the lumen [19,20].
This study aimed to investigate the effects of waterborne Cd on apple snails, focusing
on enzyme activity, gut microbiota abundance, and transcriptome changes. To achieve
Int. J. Mol. Sci. 2025,26, 751 3 of 16
this, apple snails were exposed to Cd for 16 days, followed by a depuration period of 16
days. Samples were collected from various tissues, including the heart, intestine-stomach,
kidneys, liver, shell, head-foot, and gonads, to provide a comprehensive understanding
of Cd accumulation across different parts of the organism. By analyzing changes in Cd
accumulation, enzyme activity, gut microbiota, and transcriptome, the study sought to
elucidate the mechanisms underlying Cd toxicity in apple snails.
2. Results
2.1. Cd Accumulation and Elimination in the Tissues of P. canaliculata
Figure 1shows the accumulation and elimination of Cd in the heart, intestine-stomach,
kidneys, liver, shell, head-foot, and gonads of P. canaliculata. The results revealed that the
accumulation of Cd in various tissues of P. canaliculata increased significantly during the
exposure period (p< 0.05).
Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 3 of 17
this, apple snails were exposed to Cd for 16 days, followed by a depuration period of 16
days. Samples were collected from various tissues, including the heart, intestine-stomach,
kidneys, liver, shell, head-foot, and gonads, to provide a comprehensive understanding
of Cd accumulation across dierent parts of the organism. By analyzing changes in Cd
accumulation, enzyme activity, gut microbiota, and transcriptome, the study sought to
elucidate the mechanisms underlying Cd toxicity in apple snails.
2. Results
2.1. Cd Accumulation and Elimination in the Tissues of P. canaliculata
Figure 1 shows the accumulation and elimination of Cd in the heart, intestine-stom-
ach, kidneys, liver, shell, head-foot, and gonads of P. canaliculata. The results revealed that
the accumulation of Cd in various tissues of P. canaliculata increased signicantly during
the exposure period (p < 0.05).
Figure 1. Cadmium accumulation and elimination in the dierent tissues of Pomacea canaliculata on
day -16 and day -32. (a) Cd concentration in shell; (b) Cd concentration in head-foot; (c) Cd concen-
tration in heart; (d) Cd concentration in gonads; (e) Cd concentration in intestine-stomach; (f) Cd
concentration in liver; (g) Cd concentration in kidneys. * indicates a signicant dierence compared
with the control (* p < 0.05, ** p < 0.01, and *** p < 0.01). # p < 0.05 and ## p < 0.01 are relative to day
32.
Cadmium accumulation was highest in the liver (17.41 μg/g), followed by the kid-
neys (7.99 μg/g), intestine-stomach (6.68 μg/g), head-foot (0.52 μg/g), heart (0.51 μg/g),
shell (0.15 μg/g), and gonads (0.14 μg/g). These levels were approximately 1–3 times
higher than those observed in the control group. At the end of the elimination period,
signicant reductions in Cd concentrations were observed in the intestine-stomach, kid-
neys, liver, shell, and head-foot in the Cd exposure group compared with those on day 16
(p < 0.01). The elimination eciency of various tissues was as follows: head-foot (40.98%),
shell (32.97%), gonads (26.86%), intestine-stomach (17.58%), liver (14.02%), kidneys
(12.46%), and heart (8.62%) (Table S1).
Figure 1. Cadmium accumulation and elimination in the different tissues of Pomacea canaliculata
on day-16 and day-32. (a) Cd concentration in shell; (b) Cd concentration in head-foot; (c) Cd
concentration in heart; (d) Cd concentration in gonads; (e) Cd concentration in intestine-stomach;
(f) Cd concentration in liver; (g) Cd concentration in kidneys. * indicates a significant difference
compared with the control (* p< 0.05, ** p< 0.01, and *** p< 0.01). # p< 0.05 and ## p< 0.01 are
relative to day 32.
Cadmium accumulation was highest in the liver (17.41
µ
g/g), followed by the kidneys
(7.99
µ
g/g), intestine-stomach (6.68
µ
g/g), head-foot (0.52
µ
g/g), heart (0.51
µ
g/g), shell
(0.15
µ
g/g), and gonads (0.14
µ
g/g). These levels were approximately 1–3 times higher
than those observed in the control group. At the end of the elimination period, significant
reductions in Cd concentrations were observed in the intestine-stomach, kidneys, liver,
shell, and head-foot in the Cd exposure group compared with those on day 16 (p< 0.01).
The elimination efficiency of various tissues was as follows: head-foot (40.98%), shell
(32.97%), gonads (26.86%), intestine-stomach (17.58%), liver (14.02%), kidneys (12.46%),
and heart (8.62%) (Table S1).
2.2. Enzyme Activity Response of Snails Under Cd Stress
On day 16, the activities of superoxide dismutase (SOD), catalase (CAT), and glu-
tathione transferase (GST) in the Cd-exposed group were measured at 286.45, 2.08, and
24.60 U/mg protein, respectively. In contrast, the control group exhibited higher enzyme
activities, with values of 292.45, 2.81, and 28.51 U/mg protein, as illustrated in Figure 2.
Int. J. Mol. Sci. 2025,26, 751 4 of 16
Notably, the activities of CAT and GST in the Cd-exposed group were lower than those in
the control group (p< 0.05). On day 32, the enzyme activities in the Cd exposure group
exhibited significant differences compared with the control group (p< 0.05). After 16 days
of purification, each enzyme activity had a different reaction pattern. SOD presented no
differences on day 16 but had a significant increase in activity on day 32. For CAT, there
were significant decreases in activity, compared to the control, for both sampling points, but
on day 32, the significance level was different. For GST, significant differences were found
for both time periods, but for day 16, the activity of GST was decreased after Cd exposure,
while for day 32, there was significant increase in the activity for the snails previously
exposed to Cd, as significant differences were still observed between the Cd-treated group
and control group.
Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 4 of 17
2.2. Enzyme Activity Response of Snails Under Cd Stress
On day 16, the activities of superoxide dismutase (SOD), catalase (CAT), and gluta-
thione transferase (GST) in the Cd-exposed group were measured at 286.45, 2.08, and 24.60
U/mg protein, respectively. In contrast, the control group exhibited higher enzyme activ-
ities, with values of 292.45, 2.81, and 28.51 U/mg protein, as illustrated in Figure 2. Nota-
bly, the activities of CAT and GST in the Cd-exposed group were lower than those in the
control group (p < 0.05). On day 32, the enzyme activities in the Cd exposure group exhib-
ited signicant dierences compared with the control group (p < 0.05). After 16 days of
purication, each enzyme activity had a dierent reaction paern. SOD presented no dif-
ferences on day 16 but had a signicant increase in activity on day 32. For CAT, there were
signicant decreases in activity, compared to the control, for both sampling points, but on
day 32, the signicance level was dierent. For GST, signicant dierences were found
for both time periods, but for day 16, the activity of GST was decreased after Cd exposure,
while for day 32, there was signicant increase in the activity for the snails previously
exposed to Cd, as signicant dierences were still observed between the Cd-treated group
and control group.
Figure 2. (a) Superoxide dismutase (SOD), (b) catalase (CAT), and (c) glutathione transferase (GST)
changes in Pomacea canaliculata on day 16 and day 32, respectively. * indicates a signicant dierence
compared with the control (* p < 0.05, ** p < 0.01, and *** p < 0.01). ### p < 0.001 is relative to day 32.
2.3. Characterization of the Gut Microbiota
The abundance and diversity of the gut microbiota of P. canaliculata were assessed by
Chao index, observed features, and community diversity indices (Shannon) and Simpson
diversity index (Simpson). As shown in Figure 3, Cd stress signicantly reduced the Chao
index, observed features, and Shannon index (p < 0.01), indicating that Cd exposure neg-
atively impacted the diversity of the gut microbiota in P. canaliculata.
Figure 2. (a) Superoxide dismutase (SOD), (b) catalase (CAT), and (c) glutathione transferase (GST)
changes in Pomacea canaliculata on day 16 and day 32, respectively. * indicates a significant difference
compared with the control (* p< 0.05, ** p< 0.01, and *** p< 0.01). ### p< 0.001 is relative to day 32.
2.3. Characterization of the Gut Microbiota
The abundance and diversity of the gut microbiota of P. canaliculata were assessed by
Chao index, observed features, and community diversity indices (Shannon) and Simpson
diversity index (Simpson). As shown in Figure 3, Cd stress significantly reduced the
Chao index, observed features, and Shannon index (p< 0.01), indicating that Cd exposure
negatively impacted the diversity of the gut microbiota in P. canaliculata.
Taxonomic analyses revealed variations in the composition of the gut microbiota of
P. canaliculata. Proteobacteria, Firmicutes, Bacteroidota, Fusobacteriota, and Actinobacteri-
ota made up the dominant bacterial phyla and accounted for 40.14%, 38.40%, 8.16%, 6.91%,
and 3.77% of the relative abundance in Cd-exposed group, respectively. They accounted
for 47.77%, 25.75%, 14.35%, 4.6%, and 4.44%, respectively, of the relative abundance in the
control group (Figure 4a).
At genus level, the dominant microbiota of the P. canaliculata gut microbiota in the
Cd-exposed group were Lactococcus,Rhodoblastus,Cetobacterium,Bacteroides, and Pleomor-
phomonas, accounting for 37.84%, 8.70%, 6.91%, 3.66%, and 4.15% of the relative abundances,
respectively. These proportions were 24.78%, 13.34%, 4.60%, 5.21%, and 4.39%, respec-
tively, in the control group. Compared to the control group, the diversity of Lactococcus
and Cetobacterium increased in the Cd-exposed group, while the diversity of Rhodoblastus,
Bacteroides, and Pleomorphomonas decreased (Figure 4b).
Int. J. Mol. Sci. 2025,26, 751 5 of 16
Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 5 of 17
Figure 3. Changes in alpha diversity of Pomacea canaliculata. (a) Chao index of species richness; (b)
observed features in the microbiota; (c) Shannon index of species diversity; and (d) Simpson index
of species diversity. ** p < 0.01.
Taxonomic analyses revealed variations in the composition of the gut microbiota of
P. canaliculata. Proteobacteria, Firmicutes, Bacteroidota, Fusobacteriota, and Actinobacte-
riota made up the dominant bacterial phyla and accounted for 40.14%, 38.40%, 8.16%,
6.91%, and 3.77% of the relative abundance in Cd-exposed group, respectively. They ac-
counted for 47.77%, 25.75%, 14.35%, 4.6%, and 4.44%, respectively, of the relative abun-
dance in the control group (Figure 4a).
Figure 4. The taxonomic composition of Pomacea canaliculata gut microbiota at the phylum (a) and
genus levels (b).
At genus level, the dominant microbiota of the P. canaliculata gut microbiota in the
Cd-exposed group were Lactococcus, Rhodoblastus, Cetobacterium, Bacteroides, and Pleomor-
phomonas, accounting for 37.84%, 8.70%, 6.91%, 3.66%, and 4.15% of the relative abun-
dances, respectively. These proportions were 24.78%, 13.34%, 4.60%, 5.21%, and 4.39%,
Figure 3. Changes in alpha diversity of Pomacea canaliculata. (a) Chao index of species richness;
(b) observed features in the microbiota; (c) Shannon index of species diversity; and (d) Simpson index
of species diversity. ** p< 0.01.
Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 5 of 17
Figure 3. Changes in alpha diversity of Pomacea canaliculata. (a) Chao index of species richness; (b)
observed features in the microbiota; (c) Shannon index of species diversity; and (d) Simpson index
of species diversity. ** p < 0.01.
Taxonomic analyses revealed variations in the composition of the gut microbiota of
P. canaliculata. Proteobacteria, Firmicutes, Bacteroidota, Fusobacteriota, and Actinobacte-
riota made up the dominant bacterial phyla and accounted for 40.14%, 38.40%, 8.16%,
6.91%, and 3.77% of the relative abundance in Cd-exposed group, respectively. They ac-
counted for 47.77%, 25.75%, 14.35%, 4.6%, and 4.44%, respectively, of the relative abun-
dance in the control group (Figure 4a).
Figure 4. The taxonomic composition of Pomacea canaliculata gut microbiota at the phylum (a) and
genus levels (b).
At genus level, the dominant microbiota of the P. canaliculata gut microbiota in the
Cd-exposed group were Lactococcus, Rhodoblastus, Cetobacterium, Bacteroides, and Pleomor-
phomonas, accounting for 37.84%, 8.70%, 6.91%, 3.66%, and 4.15% of the relative abun-
dances, respectively. These proportions were 24.78%, 13.34%, 4.60%, 5.21%, and 4.39%,
Figure 4. The taxonomic composition of Pomacea canaliculata gut microbiota at the phylum (a) and
genus levels (b).
2.4. Transcriptomic Analysis
We also performed transcriptomic analysis of P. canaliculata among the control group
and Cd-exposed group through comparative analysis of differentially expressed genes
(DEGs) between treated groups. There were a total of 1086 DEGs, in which 478 and
608 unigenes were up-regulated and down-regulated.
To elucidate the functions of these differentially expressed genes (DEGs), they were
mapped to the Gene Ontology (GO) database and categorized into three functional areas:
biological process (BP), molecular function (MF), and cellular component (CC), compris-
ing 166, 181, and 25 subclasses, respectively, as shown in Figure 5. In the comparison
Int. J. Mol. Sci. 2025,26, 751 6 of 16
between the 10
µ
g/L Cd treatment and the control, six GO terms in the BP category were
significantly enriched: oxidation-reduction process (GO:0055114), macromolecule modifica-
tion (GO:0043412), transmembrane transport (GO:0055085), cellular protein modification
process (GO:0006464), phosphorus metabolic process (GO:0006793), and carbohydrate
derivative metabolic process (GO:1901135), accounting for 14.84%, 13.28%, 11.72%, 10.16%,
and 7.81%. Within the MF category, enzyme regulator activity (GO:0030234) and en-
dopeptidase inhibitor activity (GO:0004866) emerged as the most prominent GO terms,
representing 3.92% and 3.14%. Additionally, two DEGs were notably enriched in sev-
eral CC categories, including the extracellular region (GO:0044421) and cytoskeletal part
(GO:0044430), accounting for 8.45% and 4.23%, as depicted in Figure 6d.
Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 7 of 17
Figure 5. GO annotations of single genes of the transcriptome, where each annotated sequence was
assigned at least one of the following GO terms: biological process (BP), cellular component (CC),
and molecular function (MF).
Figure 6. RNA-Seq analysis to identify the dierentially expressed genes of P. canaliculata upon Cd
treatment. (a) Volcano plot of DEGs; (b) heatmap of DEGs; (c) KEGG pathway enrichment analysis
Figure 5. GO annotations of single genes of the transcriptome, where each annotated sequence was
assigned at least one of the following GO terms: biological process (BP), cellular component (CC),
and molecular function (MF).
To further elucidate the pathways affected by Cd exposure in P. canaliculata, all DEGs
underwent KEGG pathway analysis. As illustrated in Figure 6c, 71 pathways were anno-
tated for the 10 µg/L Cd exposure compared to the control.
Int. J. Mol. Sci. 2025,26, 751 7 of 16
Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 7 of 17
Figure 5. GO annotations of single genes of the transcriptome, where each annotated sequence was
assigned at least one of the following GO terms: biological process (BP), cellular component (CC),
and molecular function (MF).
Figure 6. RNA-Seq analysis to identify the dierentially expressed genes of P. canaliculata upon Cd
treatment. (a) Volcano plot of DEGs; (b) heatmap of DEGs; (c) KEGG pathway enrichment analysis
Figure 6. RNA-Seq analysis to identify the differentially expressed genes of P. canaliculata upon Cd
treatment. (a) Volcano plot of DEGs; (b) heatmap of DEGs; (c) KEGG pathway enrichment analysis of
DEGs; (d) GO function analysis of DEGs. Pathway enrichment bubble plot colors represent adjusted
p-values, and bubble sizes represent the number of genes enriched.
3. Discussion
3.1. Cd Accumulation and Elimination
The accumulation of Cd in mollusks is primarily influenced by biological species,
Cd concentration, and exposure duration. In this study, after Cd exposure, all tissues
of P. canaliculata exhibited significantly higher Cd levels compared to the control group
(p< 0.05). The highest accumulation was found in the liver, kidneys, and intestine-stomach.
Yu observed that in Mytilus galloprovincialis, Cd accumulates most in the gills, followed
by the digestive glands, and least in the gonads [
21
]. Similarly, Zhao reported that in
Chlamys farreri, Cd concentration was highest in the kidneys, followed by the digestive
glands, mantle, gills, gonads, and muscle [
22
]. In Crassostrea angulata, Li found that Cd
accumulation was highest in the gills, followed by the mantle. In contrast, the adductor
muscle showed the least accumulation compared to the other tissues examined [
23
]. These
results indicated that Cd accumulation patterns are species-specific. Additionally, this
finding contrasts with previous studies that identified the intestine-stomach as the main
organ for Cd accumulation. The discrepancy may be due to exposure duration. Short-
term exposure causes Cd to accumulate in the gut mucosal layer, reducing its thickness
and mucin content, which prevents transfer to the liver and kidneys [
24
,
25
]. Long-term
exposure studies have demonstrated that Cd has a high affinity for sulfhydryl groups,
enabling it to form Cd-sulfhydryl complexes that enter renal epithelial cells through protein-
binding sites. Additionally, Cd activates the protective mechanism of metallothionein (MT)
in the liver, forming Cd-MT complexes that are filtered by glomeruli and reabsorbed by
proximal tubular epithelial cells. These complexes are then transported to endosomes and
lysosomes, where MT is hydrolyzed by proteases, and Cd is taken up by cation transporter
proteins [7,26].
Int. J. Mol. Sci. 2025,26, 751 8 of 16
Cadmium has a long half-life in mollusks, and its metabolism occurs mainly through
excretion. Cadmium removal efficiency is affected by mollusk species, Cd concentration,
and tissue recovery ability under Cd stress [
27
]. In this research, the elimination rates
of Cd from various tissues were as follows: cephalopods (40.98%), shells (32.97%), gut
(17.58%), liver (14.02%), kidneys (12.46%), heart (8.62%), and gonads (3.48%), indicating
tissue-specific variations in Cd elimination. The highest elimination rates in the head-foot
and shell were hypothesized to be due to the direct contact with the water [
28
]. In contrast,
Cd elimination in the liver and kidney was only about 10%, which may be due to the
redistribution of Cd in the tissues prior to elimination, and as noted by de Conto Cinier
this slow excretion rate may be due to the strong binding of Cd to ligands in the liver and
kidney [
29
]. In the present study, we found that the elimination level of Cd in the intestine
was higher than that in other organs during the decontamination process, suggesting that
unabsorbed Cd is mainly eliminated from the body through the intestine [30].
3.2. Effect of Cd Stress on the Enzyme Activity
Cadmium exposure has been demonstrated to impair the oxidase system, resulting
in the overproduction of reactive oxygen species (ROS), which subsequently interact with
macromolecules such as lipids and proteins. This interaction leads to cellular oxidative
stress, apoptosis, and membrane lipid peroxidation [
31
]. In aquatic ecosystems, cadmium
from water sources enters fish and other aquatic organisms through water and food, where
it displaces iron ions and induces oxidative stress via the Fenton reaction, causing immune
and reproductive disorders [
32
]. To counteract excessive ROS production, aquatic animals
have developed robust antioxidant defenses, primarily involving antioxidant enzymes.
Among these enzymes, SOD, CAT, and GST play crucial roles as key antioxidant defense
mechanisms. The activities of these enzymes serve as reliable and sensitive indicators for
assessing oxidative stress in Cd-exposed aquatic animals. Furthermore, cadmium exposure
disrupts mitochondrial membrane potential, promotes the release of cytochrome c, and
activates caspase-9 and caspase-3, thereby initiating an intrinsic mitochondria-mediated
apoptotic pathway [33,34]. Additionally, a study by Wang found that high concentrations
of Cd induced caspase-8 gene expression and activated caspase-3, leading to apoptosis and
further damage to the hepatopancreas of bighead carp (Hypophthalmichthys nobilis) [35].
Mitochondria are the primary sources of ROS, with complexes I (NADH–coenzyme
Q reductase), II (succinate dehydrogenase), and III (coenzyme Q–cytochrome c reductase)
in the electron transport chain (ETS) serving as the main sites of ROS production. Under
normal conditions, electrons can leak from these complexes, reducing O
2
to superoxide
(O
2−
), which acts as a precursor to hydrogen peroxide (H
2
O
2
), hydroxyl radicals (-OH), and
peroxynitrite (ONOO-). Both hydroxyl radicals and peroxynitrite are capable of inducing
protein and DNA damage as well as lipid peroxidation [
36
,
37
]. SOD serves as the first line
of defense against oxidative stress by converting free superoxide to hydrogen peroxide
and oxygen [
31
], while CAT further detoxifies hydrogen peroxide into non-toxic water
and oxygen [
38
]. GST, a family of phase II detoxification enzymes, not only reduces the
cytotoxicity of cellular metabolites and environmental chemicals by catalyzing the coupling
of reduced glutathione (GSH) to various electrophilic compounds but also participates
in the transport of lipids, hemoglobin, and steroids [
32
,
39
]. In this study, during the Cd-
exposure period, the activities of SOD, CAT, and GST at a concentration of 10
µ
g/L were
lower than those in the control group, aligning with previous research findings [
8
,
40
]. This
reduction in enzyme activity is likely related to the ability of enzymes in P. canaliculata to
manage cellular damage caused by a low concentration of Cd (10
µ
g/L) [
41
]. After 16 days
of purification, the results showed distinct response patterns for each enzyme activity. On
day 16, there was no significant difference in SOD activity; however, a significant increase
Int. J. Mol. Sci. 2025,26, 751 9 of 16
was observed on day 32. This indicates that the snails mitigated oxidative damage caused
by Cd stress through the activation of their antioxidant defense system [
42
]. In contrast,
for GST, significant differences were found at both time points. On day 16, GST activity
was reduced following Cd exposure, whereas on day 32, GST activity, which is crucial for
glutathione (GSH) metabolism, significantly increased. This suggests that GST played a
critical role in cellular detoxification during the purification stage, thereby alleviating the
toxic effects of Cd stress on the snails [4,43].
3.3. Taxonomic Composition Changes of Gut Microbiota
In this study, cadmium stress significantly affect the gut microbiota of P. canaliculata.
Specifically, alpha diversity indices indicated that 10
µ
g/L Cd decreased the diversity of gut
microbiota in P. canaliculata. The results indicated that Cd extensively accumulates in the
gut. This accumulation causes the death of bacteria with low tolerance to Cd, significantly
reducing microbial diversity within the gut of the apple snails. Ultimately, this change
disrupts the stability of the intestinal microbiota, adversely affecting the health of the
apple snails. Consequently, it can be inferred that Cd exposure jeopardizes the gut health
of P. canaliculata by diminishing the diversity of its gut microbiota [
12
]. The dominant
phyla of gut microbiota in P. canaliculate are Proteobacteria, Firmicutes, Fusobacteriota,
Bacteroidota, and Actinobacteriota, which is consistent with earlier studies [
44
,
45
]. The
findings further indicated that Cd stress led to a decrease in the abundance of Proteobac-
teria and an increase in the abundance of Firmicutes and Fusobacteriota. Notably, most
pathogenic microbiota belonged to Proteobacteria, while most Firmicutes were beneficial
microbiota [
46
]. Firmicutes are primarily associated with immune regulation, nutrient
absorption, and metabolism, whereas Fusobacteriota produce butyric acid under Cd stress,
which provides energy and ameliorates gut inflammation [47–49].
Moreover, Cd stress stimulated an increase in probiotics such as Lactococcus and Ce-
tobacterium in the gut microbiota. These probiotics play a crucial role in preventing Cd
from entering the body circulation by (1) competitively inhibiting gut absorption of Cd,
(2) stimulating gut peristalsis to promote exocytosis of Cd, and (3) improving barrier func-
tion [
50
,
51
]. Compared with the control, the abundance of Lactococcus at the Cd-exposed
group was higher, which may be attributed to the negatively charged polysaccharides
and membrane proteins on the surface of Lactococcus that facilitate Cd adsorption [
52
].
Cetobacterium, commonly found in fish, has also shown beneficial effects. Xie reported that
consumption of Cetobacterium fermentation products decreased serum lipopolysaccharide
(LPS) and diamine oxidase (DAO) activity while promoting the expression of SOD, oc-
cludin, and ZO-1, thereby enhancing the gut mucosal barrier in carp [
53
]. Additionally,
previous studies found that Cetobacterium produces vitamin B12, a growth factor associated
with protein synthesis [10].
In summary, the increased abundance of Firmicutes, Fusobacteriota, Lactococcus, and
Cetobacterium suggests that these bacteria play a crucial role in resisting Cd stress, highlight-
ing adaptive mechanisms involving beneficial microbiota that mitigate the negative effects
of Cd stress.
3.4. Effects of Cd Exposure on Gene Regulation in the Transcriptome
Cadmiumstress promotes cellular oxidative stress and generates large amounts of
ROS, necessitating detoxification as a crucial adaptive strategy for P. canaliculata to maintain
physiological homeostasis. In this study, KEGG pathway enrichment analysis identified sev-
eral major cellular transport and metabolic pathways, including ECM–receptor interaction,
arachidonic acid metabolism, glycerolipid metabolism, alpha-linolenic acid metabolism,
and nitrogen metabolism. This suggests that Cd stress triggers the activation of cellular
Int. J. Mol. Sci. 2025,26, 751 10 of 16
metabolic pathways in P. canaliculata. Previous research found that Cd exposure in mice
increased alanine transaminase (ALT) and aspartate transaminase (AST) activities, induced
high expression of cyclooxygenases (COX-2), caused abnormal lipid metabolism, and el-
evated arachidonic acid metabolism [
54
]. Additionally, the findings indicated that most
genes are enriched in lysosomes and the CYP450 system. Lysosomes are primarily involved
in abnormal cell clearance and energy metabolism. Experiments have shown that Cd
affects lysosomes in two ways: it inhibits autophagosome–lysosome binding, leading to the
accumulation of autophagosomes and excessive ROS production, which induces oxidative
stress in rats; and it promotes the expression of protein hydrolase CTSB, which is associated
with lysosome degradation, suggesting that Cd enhances lysosome degradation [55,56].
The CYP450 system, a critical family of enzymes for toxicant metabolism, predom-
inantly exists in the liver and gastrointestinal tract and serves as an essential phase I
detoxification enzyme. CYP450 is highly sensitive to heavy metals, and its cysteine residues
are highly conserved. Overexpression of CYP450 generates numerous free radical interme-
diates that can bind to DNA and proteins, inducing cellular damage [42,57,58].
Furthermore, this study found that Cd induces the expression of numerous genes in-
volved in endoplasmic reticulum (ER) protein processing and up-regulates cytoskeleton-
related genes. Under Cd stress, the number of misfolded proteins in the ER increases, activat-
ing the unfolded protein response (UPR) through three major pathways: ATF6, IRE1
α
, and
PERK-eIF2α. If ER stress (ERS) is not alleviated, the UPR induces apoptosis [59,60].
The cytoskeleton, which consists of microtubules, microfilaments, and intermediate fil-
aments, is also affected by Cd exposure. Templeton demonstrated that short-term exposure
to Cd can prevent cytoskeletal disruption and enhance cell survival [
61
]. This protective
effect is mediated through the activation of extracellular signal-regulated kinase (Erk) and
the inhibition of Ca
2+
/calmodulin-dependent protein kinase II (CaMK-II). Additionally,
the activation of the epidermal growth factor (EGF) receptor and the initiation of the PI3
kinase/Akt pathway contribute to these protective mechanisms. In contrast, prolonged
exposure to Cd leads to caspase-independent apoptosis, highlighting the dual nature of
Cd’s impact on cellular structures and survival pathways.
4. Materials and Methods
4.1. Animals and Experimental Conditions
Adult P. canaliculata with similar size (7.11
±
0.072 g) were obtained from a paddy field
at South China Agricultural University, Guangzhou (23
◦
9
′
N, 113
◦
1
′
E). After collection,
P. canaliculata were washed with tap water to remove sediment and other impurities
and incubated for 2 weeks in containers with purified water, and P. canaliculata were
cultured under following aquaculture environmental conditions: 12 h:12 h light/dark
regime, temperature 25
◦
C, pH of 7.85. During the acclimation period, tap water was
changed every two days to maintain optimal water quality, and lettuce constituting 3% of
the body weight of the apple snails was provided daily. Ethics approval for snail use and
care protocols was granted by the committee of the South China Agricultural University
Animal Care and Use (20110107-1).
4.2. Experimental Design
Snails of similar size and weight were randomly selected from the same culture
environment and divided into two treatment groups: control and Cd-exposed. The Cd-
exposed snails were kept in the water with the Cd concentration of 10
µ
g/L during the
initial 16-day exposure period, followed by a 16-day depuration period, during which
they were kept in tap water continuously. The 16-day interval was based on the previous
research to ensure that snails had a long enough time to accumulate waterborne Cd during
Int. J. Mol. Sci. 2025,26, 751 11 of 16
the Cd-exposure period [
62
]. Waterborne Cd usually depends on nearby anthropogenic
activities, with low Cd concentrations in affected coastal waters typically ranging from
0.1 to 10 ug/L [
63
–
65
]. In contrast, the snails in the control group were kept in the tap water.
Each group included three replicates, each containing 30 snails. The tested water was
changed every two days during the experimental period, and lettuce was supplied daily.
The snails were randomly sampled after treatment at day 16 and day 32 post treatment
to assess the effects of the treatment. Five snails were taken from each replicate of the
treatment and control groups, resulting in a total of 15 snails. These snails were sampled for
the following groups: heart, intestine-stomach, kidneys, liver, shell, head-foot, and gonads,
the samples of which were frozen immediately in liquid nitrogen and stored in a
−
80
◦
C
refrigerator for the next analysis.
4.3. Enzyme Activity Assay
About 0.5 g of five snails’ liver from each group were ground in liquid nitrogen using
a mortar, and 4.5 mL of cold saline was added during grinding for rapid homogenization;
each treatment consisted of three sets of replicates. The liver was selected as the target tissue
because it is the main organ for accumulation and metabolism [
66
,
67
]. The homogenate was
centrifuged at 3000 rpm for 10 min at 4
◦
C. The supernatant obtained after centrifugation
was used immediately for the determination of biochemical parameters. These parameters
included SOD, CAT, and GST.
These biochemical parameters were analyzed using assay kits purchased from Nanjing
Jiancheng Bioengineering Institute, Nanjing, Jiangsu Province, China. Detailed procedures
for the measurement of biochemical parameters can be found in the Supplementary Mate-
rial Text S1.
4.4. Cd Concentration Determination
About 0.05–0.1 g of the powder was weighed, and 10 mL of HNO
3
was added and
digested using a microwave digestor (Mars 6; CEM Corporation, Matthews, NC, USA).
A blank method was used during the digestion of each batch to eliminate the interference
of reagents and solvents. For quality control, standard shrimp material (GBW10050a;
Institute of Geophysical and Geochemical Exploration, Chinese Academy of Geological
Sciences) was digested under the same conditions to ensure that the recoveries of tissues
were controlled at 95–105%. Cadmium concentrations were determined for each tissue
using ICP-MS, and Cd concentrations were expressed as µg-g−1dry weight (w/w).
4.5. Analysis of Gut Microbiota
To investigate the effects of Cd exposure on the composition of the snail microbiota,
we performed 16S rDNA gene sequencing analysis. Genomic DNA was extracted from
the samples using sodium dodecyl sulfate (SDS), and its purity and concentration were
assessed by gel electrophoresis. After quantification with Qubit, the amplified V3–V4
regions were sequenced on the Illumina HiSeq platform. The library was constructed using
the NEB Next Ultra DNA Library Preparation Kit. Microbial diversity was analyzed using
the Chao index, observed features, Shannon, and Simpson indices calculated with QIIME 2
software (version 2024.10).
4.6. RNA Extraction and Transcriptome Analysis
Total RNA was isolated from different groups of tissues using TRIzol kits (Invitrogen,
Carlsbad, CA, USA). The RNA was initially quantified using a Qubit 2.0 Fluorometer
(Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), and its integrity and total
amount were assessed with an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto,
CA, USA). The raw data obtained were then filtered using fastp software (Version 0.24.0).
Int. J. Mol. Sci. 2025,26, 751 12 of 16
Subsequently, the bipartite sequences were aligned with the reference genome using Hisat2
(v2.0.5). Differential expression analysis of the genes was conducted using DESeq2 (1.20.0)
software, with the criteria set as |log2(FoldChange)|
≥
1 and padj < 0.05. Furthermore,
Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment
pathway analyses were performed using cluster Profiler software (3.8.1).
4.7. Statistical Analysis
To ensure the accuracy and reliability of the experiment, three replications were
conducted, and the data were analyzed using IBM SPSS Statistics27.0. A normality test was
performed through Shapiro–Wilk test. The data used in the experiment were confirmed
to meet the assumptions of normality. Differences between treatments were assessed
using Student’s t-test. Equality of variances were determined using the Levene test. Data
were averaged from two independent experiments and are shown as means
±
standard
deviations (S.D.). * represents differences compared to the control group (p< 0.05).
The elimination rate (%) is the percentage decrease in the initial value (the 16 days of
exposure), and the following definition was used:
Elimination rate = (Celimination −Cexposure)/Cexposure ×100%
C
elimination
is the concentration of Cd in each tissue after a 16-day elimination period;
Cexposure is the concentration before elimination [28].
5. Conclusions
This study investigated the effects of Cd exposure on P. canaliculata, focusing on Cd
accumulation, enzyme activity, gut microbiota, and transcriptome analysis. Our findings
confirmed that Cd accumulates in various tissues, including the liver, kidneys, intestine-
stomach, head-foot, heart, shell, and gonads, with the highest concentrations observed in
the liver, followed by the kidneys and intestine-stomach. In terms of elimination efficiency,
the head-foot exhibited the greatest capacity, followed by the shell and intestine-stomach.
Under Cd stress, P. canaliculata showed reduced activities of SOD, CAT, and GST compared
to the control group, and these enzyme activities did not return to normal levels during
the depuration period. Furthermore, Cd stress led to a reduction in the diversity of gut
microbiota. Specifically, exposure to low concentrations of Cd resulted in an increased
abundance of Firmicutes and Fusobacteriota while reducing the abundance of Proteobacte-
ria, Bacteroidota, and Actinobacteriota. Transcriptome analysis further revealed that Cd
exposure affects cellular transport and metabolism. These findings enhance our under-
standing of the chronic toxicological effects of Cd on P. canaliculata and provide valuable
insights into the accumulation and metabolic processes of Cd in aquatic invertebrates.
Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/ijms26020751/s1.
Author Contributions: M.Q., data curation, formal analysis, and writing—original draft; X.B., formal
analysis; Y.L., data curation; H.L., writing—review and editing; D.L., investigation, conceptualiza-
tion, and writing—review and editing; G.C., conceptualization, funding acquisition, investigation,
supervision, and writing—review and editing. All authors have read and agreed to the published
version of the manuscript.
Funding: This work was co-funded by the National Key Research and Development Program of
China (2023YFC3707600) and the Guangzhou basic research planning for basic and applied basic
research project (2024A04J3256).
Int. J. Mol. Sci. 2025,26, 751 13 of 16
Institutional Review Board Statement: Ethics approval for snail use and care protocols was granted
by the committee of the South China Agricultural University Animal Care and Use (20110107-1).
Informed Consent Statement: Not applicable.
Data Availability Statement: Guikui Chen should be contacted to request data from this study.
Acknowledgments: The authors would like to thank Novogene for the analysis of the gut microbiota
and transcriptome of Pomacea canaliculate.
Conflicts of Interest: The authors declare no conflicts of interest.
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