ABSTRACT
Salinity is an important abiotic stress that affects metabolic and
physiological activities, breeding, development, and growth of
mollusks. In this study, we investigated the effects of a range of
water salinity on the apple snail Pomacea canaliculata , a highly
invasive species and an important pest of rice. To examine the
molecular response of P. canaliculata to salinity, we recorded young
snails grown in a saline water environment for 4 months and compared
their physiological and biochemical parameters with those of freshwater
snails. We used RNA‐seq analysis to identify genes and biological
processes involved in response to salinity. The results showed that
saline water stress reduced the survival rate of the snail population,
increased their feeding rate and snail weight, and led to an increase
in shell strength and thickness, as well as a significant widening of
the overall shell morphology. In female snails, the activities of CAT,
SOD, and T‐AOC were significantly enhanced, while GSH activity, MDA
content, and NOS activity showed significant decreases. In male snails,
only MDA content exhibited a significant decrease, while ACHE activity
showed a significant increase. Based on transcriptome analysis
conducted for the liver and gills of the snails, a total of
1,569,678,584 raw reads were obtained from the nine libraries on the
Illumina Novaseq 6000 platform. After preprocessing and the removal of
low‐quality sequences, 1,560,932,792 clean reads were generated. The
number of upregulated and downregulated differentially expressed genes
(DEGs) in male snails after the saline stress was higher than that in
female snails. The DEGs mainly involved oxidative stress, cellular
regulation, and response. Saline concentration inhibited the hatching
of eggs to a certain extent. Different levels of saline stress
significantly affected the contents of free water, bound water, and
enzyme activity of their eggs at different hatching stages. These
findings provide theoretical support for understanding the saline
tolerance of snails.
Keywords: Pomacea canaliculata, RNA‐seq, salinity acclimation,
transcriptome
__________________________________________________________________
In this study, we investigated the effects of a range of salinity on
the apple snail Pomacea canaliculata , a highly invasive species and an
important pest of rice. To examine the molecular response of P.
canaliculata to salinity, we recorded young snails grown in a saline
water environment for 4 months and compared their physiological and
biochemical parameters with those of freshwater snails. These findings
provide theoretical support for understanding the salt tolerance of
snails.
graphic file with name ECE3-15-e71581-g001.jpg
1. Introduction
Pomacea canaliculata is an extremely harmful aquatic invasive species
that originated in the Amazon River basin of South America and has now
spread to many countries and regions worldwide. It is considered to be
one of the most malignant waterborne organisms (Yang et al. [37]2023;
Zhou et al. [38]2016; Musri Musman et al. [39]2013). Due to its strong
acclimation, broad diet, large food intake, high egg production, and
fast reproduction rate, this invasive snail can quickly spread in
environments such as rivers, lakes, and fields. It feeds on a variety
of crops and aquatic plants, thus disrupting the food chain and posing
a threat to the diversity of freshwater organisms and the functioning
of the ecosystem in the invaded area (Ma et al. [40]2023; Corbin
et al. [41]2022). In 2000, the apple snail was included in the list of
the world's 100 worst invasive alien species (Manara et al. [42]2022).
As an invasive species, P. canaliculata exhibits a remarkable ability
to adapt to various environmental conditions. Investigating its
environmental adaptability is crucial for developing targeted
ecological management strategies to mitigate its detrimental impacts on
native species and the overall ecosystem.
Salinity is one of the most important environmental factors in aquatic
environment, which greatly affects the survival, reproduciton,
development, growth, metabolism, immune function, and physiological
functions of mollusks (Martyniuk et al. [43]2022; Koudenoukpo
et al. [44]2021; Gaiser et al. [45]2006). P. canaliculata is classified
as an osmoconforming mollusk, and its population's ability to acclimate
to saline stress indicates a potential for invading estuarine habitats
(Koudenoukpo et al. [46]2021). P. canaliculata can potentially survive
in environments with salinity levels of ≤ 5.0 ppt, with survival rates
varying by age class based on shell height, following the order:
older > mature > juvenile snails (Liu, Liu, et al. [47]2022). Qin
et al. found that P. canaliculata can survive for at least 5 days at a
salinity of 12.0 ppt, over 30 days in environments with salinity
ranging from 0 to 6 ppt, and can maintain normal physiological
activities under salinity conditions of 0 to 4 ppt. As environmental
salinity gradually increases, P. canaliculata exhibits enhanced saline
tolerance; however, higher salinity levels lead to significant
decreases in average daily weight gain, specific growth rate, and egg
hatching rate (Qin et al. [48]2022, [49]2020). Experimental results
confirmed that in low‐salinity environments, P. canaliculata can absorb
more abundant inorganic ions, thereby promoting its growth, and it is
speculated that this may further improve its salinity tolerance (Yang
et al. [50]2016). However, the tolerance and long‐term acclimation
mechanisms of freshwater and saline water environmental stress on P.
canaliculata have not been studied at the transcriptome level to
identify the genes responsible for salinity regulation, which affects
the understanding of the fundamental mechanism underlying acclimation
to fluctuations in salinity. When the environmental salinity changes
abnormally, it causes a variety of physiological stress reactions in P.
canaliculata, and the production of reactive oxygen species (ROS)
increases accordingly (Polyak et al. [51]2022; Batista
et al. [52]2023). Owing to variation in the salinity of the aquatic
environment, P. canaliculata have evolved various physiological
strategies for salinity acclimation. In the coastal regions of southern
China, the apple snails have spread to the coastal brackish water
wetland ecosystem, and have been found in habitats such as fish ponds,
tidal flats, and farmland where both fresh and saline water are
present, including the mangrove wetlands in the coastal areas of South
China, with the presence of P. canaliculata and their egg masses (Liu,
Liu, et al. [53]2022).
Enzymes and transporters play a crucial role in maintaining internal
osmotic and ionic homeostasis in response to fluctuating water
salinity, thus actively participating in salinity acclimation and
osmoregulation (Andreeva et al. [54]2020). The identification of
candidate genes associated with salinity changes is of paramount
importance in elucidating the molecular foundation underlying this
essential physiological process (Ren et al. [55]2020). The
transcriptome represents a collection of genes that exhibit dynamic
expression patterns, which are contingent upon the physiological state
of organisms and are responsive to external environmental factors
(Andreeva et al. [56]2020; Price et al. [57]2007). These investigations
have allowed the elucidation of several differentially expressed genes
(DEGs) and pathways associated with changes in water salinity.
With the rapid development of molecular technologies, it is possible to
study the ecological and physiological mechanisms of P. canaliculata
using gene expression analysis. Prior studies on P. canaliculata have
primarily focused on examining the effects of salinity on various
aspects such as survival, larval and juvenile development, oxygen
consumption, ammonia excretion, growth, and energy budget (Qin
et al. [58]2022; Yang et al. [59]2018). Although basic data such as
morphological traits and survival rates may seem sufficient to address
the issue, they only describe the phenotypic outcomes resulting from
environmental stress and fail to uncover the molecular mechanisms
driving physiological acclimation. P. canaliculata’s survival under
saline water conditions is primarily regulated by gene
expression‐mediated phenotypic changes rather than immediate
morphological alterations. Analyzing DEGs allows precise identification
of stress‐related pathways, including osmotic regulation, ion
transport, energy metabolism, and antioxidant defense, which are vital
for long‐term acclimation processes not directly observable through
morphology or survival data alone.
Importantly, transcriptomic data capture early molecular responses to
stress before visible phenotypic changes occur, providing crucial
insights for predicting long‐term survival and invasion potential. Many
invasive species rely on gene‐regulated phenotypic plasticity to
colonize new environments. Insufficient knowledge is available
concerning the molecular pathways associated with acclimatory
mechanisms in response to salinity changes in P. canaliculata.
Consequently, there is a compelling need to investigate the salinity
acclimation of P. canaliculata at the transcriptional level to unravel
the underlying fundamental mechanisms involved in water salinity
acclimation and it remains unclear whether this invasive species can
establish stable populations in coastal wetlands.
In this study, we aimed to investigate the survival mechanisms of P.
canaliculata under saline stress. To this end, we conducted a 4‐month
saline stress experiment. The specific objectives were to: (1) evaluate
the effects of saline stress on survival rate, food intake, body
weight, morphological characteristics, and egg mass production; and (2)
compare gene expression profiles of two major osmoregulatory organs,
the gills and liver, under control (0 ppt) and saline stress (2 ppt)
conditions using RNA sequencing (RNA‐seq).
2. Materials and Methods
2.1. Experimental Snails and Salinity Stress Treatment
Pomacea canaliculata snails were reared in open‐air cement ponds
located at the Ecological Teaching and Research Farm (113°1′E, 23°9′N)
of South China Agricultural University (SCAU) in Guangzhou, China. P.
canaliculata individuals were collected from the wild field when their
shell height reached approximately 10 mm (±2 mm) and were subsequently
brought into the laboratory for cultivation. The snails were cultivated
indoors at a temperature of 26°C ± 1°C for 10 days and placed in
plastic aquaria (45 cm ×35 cm ×35 cm) at a depth of about 20 cm. Each
day, excess food was provided, and water was timely changed. The
lettuce from the previous day was removed and replaced with fresh
lettuce to maintain a state of excess food.
Snails with normal appearance and strong vitality were selected for the
experiment. The aquariums were covered with mesh to prevent the snails
from escaping. The snails were reared under natural lighting
conditions. Every 3 days, two‐thirds of the water was pumped out, and
aerated tap water containing saturated calcium carbonate was added to a
depth of 15 cm. Observations were made during the first 10 days of the
experiment to determine a feeding amount of 100 g per aquarium (with
surplus food provided daily).
A total of 80 snails were divided into each aquarium containing aerated
water. The experiment consisted of two treatments: one had saline
concentration adjusted to 2 ppt (our previous salinity measurements in
coastal wetlands inhabited by P. canaliculata indicated an average
salinity of 2 ppt) using commercially available marine salt mixtures
(contain 98% NaCl along with trace minerals including MgSO[4], CaSO[4]
and K[2]SO[4], was produced under the product standard number
Q/320482YDWL003) and the other without saline treatment, each treatment
replicated four times. The survival mechanism of P. canaliculata was
determined by assessing its response when the operculum was pried open.
The survival rate in each aquarium was recorded every 5 days, and the
daily food intake and snail weight were recorded every 10 days. Any
dead snails were promptly removed. The duration of continuous exposure
for adult snails was 80 days. The hatched P. canaliculata snails were
not further exposed to saline conditions; they were placed in tap water
for observation and data collection.
2.2. Shell Strength and Shell Thickness Measurement
In both the saline water‐treated group and the control group, 5 male
and 5 female snails with similar body sizes were randomly selected from
the aquariums. Their shell thickness and shell compressive strength
(shell strength) were measured. The shells and flesh were carefully
separated without damaging the snail shells using dissecting scissors
and a surgical knife. The shell compressive strength was measured using
an Edgewood spring pressure tester (model HP‐200) equipped with a
digital force gauge (accuracy: 0.1 N). The snail was placed on the
loading platform with the shell opening facing downward, and the force
gauge was used to compress the shell until it was crushed. The maximum
force recorded during the shell‐crushing process represented the shell
compressive strength. The shell thickness was measured at three
randomly selected locations around the damaged area using a spiral
micrometer (model DL321025B) with an accuracy of 0.001 mm. The average
of these measurements provided an estimation of the shell thickness.
2.3. Morphological Characteristics Measurement
Morphological measurements of the snail shells were conducted using
intact individuals of the selected snail species. The specimens were
fixed and two‐dimensional images were captured using a camera. The
software Image‐Pro Plus 6.0 was employed to measure the morphological
parameters of the snail shells. The following measurements were
selected: spire length (SL), spire width (SW) (Swinehart
et al. [60]1998), height from all layers (HL), inner lip length (IL),
basal lip height (BL), aperture length (AL), aperture basal width from
the lowermost part of the aperture to the periphery (ABW), aperture
inner length (AL’), and aperture width (AW). The measurement sites and
parameters are illustrated in the Figure [61]1. To account for the
potential influence of individual size differences on the analysis of
morphological features, data correction was performed using shell
height (HL), aperture length (AL), and aperture width (AW) as
covariates for all measured parameters.
FIGURE 1.
FIGURE 1
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Morphological indicators of P. canaliculata.
2.4. Saline Stress Treatment of Egg Masses
Egg masses of P. canaliculata were collected from a natural population
on September 22, during the peak oviposition season in summer. Intact
egg masses with visible surface mucus and bright red coloration, which
indicated they were freshly laid on the same day, and randomly selected
using a tool to assist with detachment. The egg mass collection was
conducted separately from the juvenile snail experiments, which took
place at a different time. Each egg mass was placed into its respective
culture dish, with gauze netting stretched across the top to support
the egg mass. Upon hatching, the juveniles passed through the mesh into
the dish, where they were incubated and subsequently counted. Solutions
of 0, 2, and 5 ppt salinity were prepared using commercially available
sea saline crystals (same contents for the adult survival treatments)
and added into the corresponding spray bottles. The CK treatment
involved no liquid application, simulating the natural hatching
conditions of P. canaliculata eggs in the environment. The egg masses
were wetted twice daily, at 10:00 AM and 10:00 PM respectively,
ensuring the surface was just covered with water each time. The hatched
snails were transferred promptly to prevent their escape.
Using the CK group as a reference, an inhibition rate (IR) greater than
0 indicates that the treatment exerts an inhibitory effect on egg mass
hatching, whereas an IR less than 0 suggests that the treatment
promotes egg mass hatching. The hatching rate (HR, %) and inhibition
rate (IR, %) of P. canaliculata egg masses were calculated using the
following formulas:
[MATH: HR=THSTNE×100% :MATH]
[MATH: IR=HRCK−HRTREATMENTHRCK×100% :MATH]
In the formulas, THS represents the total number of hatched snails, TNE
denotes the total number of eggs, HR(CK) refers to the hatching rate of
the control group, and HR(TREATMENT) indicates the hatching rate of the
treatment group. The treatment period for egg masses was 30 days.
2.5. Enzyme Activity Measurement
The specific tissues were dissected and promptly frozen in liquid
nitrogen for preservation at −80°C in a freezer. During the
measurements, a mass‐volume ratio of 1:9 (tissue to pre‐chilled
physiological saline) was prepared. The tissue was homogenized in an
ice‐water bath using an electric high‐speed tissue homogenizer to
obtain a 10% tissue homogenate. Subsequently, the homogenate was
centrifuged at 3000 rpm for 10 min at 4°C, and the resulting
supernatant was collected for the determination of activities of CAT,
GSH, ACHE, NOS, SOD, T‐AOC and MDA content (Catalan et al. [63]2006)
2.6. Statistical Analyses
Statistical analysis was conducted using one‐way analysis of variance
(ANOVA) followed by least significant difference (LSD) or Games–Howell
post hoc tests for multiple comparisons on survival rate, food intake,
weight, shell strength and shell thickness, morphological changes, and
enzyme activity of snails. IBM SPSS Statistics 26 software was used for
the analysis, and graphs were created using Origin 8.0. Significance
was set at p < 0.05, or p < 0.01 (Table [64]1).
TABLE 1.
Statistical methods used for data analysis.
Variable Statistical test Post hoc test Significance level
Survival rate One‐way ANOVA Games–Howell p < 0.05, or p < 0.01
Food intake One‐way ANOVA Games–Howell p < 0.05, or p < 0.01
Weight One‐way ANOVA Games–Howell p < 0.05, or p < 0.01
Shell strength One‐way ANOVA LSD p < 0.05, or p < 0.01
Shell thickness One‐way ANOVA LSD p < 0.05, or p < 0.01
Morphological changes One‐way ANOVA LSD p < 0.05, or p < 0.01
Enzyme activity One‐way ANOVA LSD p < 0.05, or P < 0.01
Hatchability and inhibition rate One‐way ANOVA LSD p < 0.05
Physiological and biochemical substance content One‐way ANOVA LSD
p < 0.05
One‐way ANOVA LSD p < 0.05
Antioxidative System Indices One‐way ANOVA LSD p < 0.05
[65]Open in a new tab
2.7. Total RNA Extraction, Library Construction and Illumina Sequencing
The integrity, concentration, and purity of the RNA samples were
assessed using agarose gel electrophoresis and Nanodrop analysis. To
construct a strand‐specific library, a method for removing ribosomal
RNA (rRNA) was employed. Firstly, total RNA was subjected to rRNA
depletion. Subsequently, the RNA was fragmented into short fragments of
250–300 bp. The fragmented RNA served as a template for the synthesis
of the first cDNA strand using random oligonucleotides as primers.
Then, the RNA template was degraded using ribonuclease H, and the
second cDNA strand was synthesized using DNA polymerase I and four
deoxyribonucleotide triphosphates. The purified double‐stranded cDNA
was subjected to end repair, A‐tailing, and ligation with sequencing
acclimator. The cDNA library was size‐selected using AMPure XP beads to
obtain fragments of approximately 350–400 bp. The second strand of
cDNA, which contained uracil, was selectively degraded using
uracil‐specific excision reagents. Finally, PCR amplification was
performed to generate the library. If the library exhibited an insert
fragment length distribution of approximately 250–300 bp and an
effective concentration greater than 2 nM, it was subjected to
paired‐end 150 bp sequencing using the Illumina platform.
2.8. Analysis of Differentially Expressed Protein Codes
The raw data were processed using Fastp to remove acclimator sequences
and low‐quality sequences, resulting in clean data with a minimum read
length of 75 bp. The genome sequence and annotation file of snail were
obtained from the NCBI database
([66]http://www.ncbi.nlm.nih.gov/protein/). The clean data was aligned
to the reference genome using Hisat2 with default parameters. Stringtie
software was then used to assemble and identify novel transcripts based
on the genome‐aligned results.
Using the merged transcripts generated by Stringtie as the reference,
gene‐level quantification was performed, and a read count matrix was
generated. The read count matrix was imported into R 3.6.3 software,
and the R package edgeR was used for differential expression analysis
of protein‐coding genes. Genes with a fold change greater than 1
(|log2(Fold change)| > 1) and a false discovery rate (FDR) less than
0.05 were considered as differentially expressed genes between groups.
Additionally, Tbtools was used for gene ontology (GO) and Kyoto
Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of
the differentially expressed genes (Table [67]2).
TABLE 2.
Water salinity and control treatments for the snails and their eggs.
Information on the salinity treatments Experimental treatments for
snails Experimental egg masses
CK 0 ppt Untreated
0 ppt N/A 0 ppt
2 ppt 2 ppt 2 ppt
5 ppt N/A 5 ppt
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Note: ‘N/A’ (not applicable) is used when a specific treatment is not
applicable. “Untreated” refers to experimental subjects that have not
undergone any treatment. All artificial water used in this study was
aerated tap water.
3. Results
3.1. Survival
Compared with the control group (0 ppt), the survival rate of P.
canaliculata declined in a 2 ppt water salinity environment on July 8th
(Figure [69]2). The survival rates of P. canaliculata in the control
and 2 ppt salinity treatment were 99% and 91%, respectively, showing a
significant difference at a highly significant level (p < 0.01). After
July 8th, the survival rates of the 2 ppt salinity treatment group were
significantly lower than the control until August 17th. Subsequently,
with increasing treatment time, the difference in survival rates
between the 2 ppt treatment group and the control group gradually
diminished. At the end of the experiment, the survival rates in the
control and 2ppt treatment groups were 75% and 73%, respectively, with
no significant difference (p > 0.05). The results of repeated measures
analysis of variance indicated a highly significant effect of treatment
time on the survival rate of P. canaliculata (F [2,16] = 102.992;
p < 0.001), and a significant interaction between the water salinity
stress and the treatment time (F [2,16] = 4.481; p < 0.05).
FIGURE 2.
FIGURE 2
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Survival, food intake, and weight of P. canaliculata in different
saline treatments. CK represents the treatment that snails were kept in
a freshwater environment without salinity. The error bars represent the
standard error (SE). The feeding amount and snail weight values in the
figure represent the average feeding amount and body weight per
individual. A single asterisk (*) indicates p < 0.05, while double
asterisks (**) indicate p < 0.01.
3.2. Food Consumption
In both treatments, P. canaliculata exhibited normal eating activity at
the beginning of the treatments. While from July 8th, the snails in the
saline environment showed significantly higher food intake compared
with the control (p < 0.05), with daily average eating amounts of 1.30
and 0.98 g for the saline water and control treatments, respectively.
Throughout the experiment, the snails in the saline treatment
consistently demonstrated significantly higher food intake than the
control (p < 0.05), with the greatest difference observed on July 28th,
where the food intake in the saline environment was 1.50 times higher
than that of the control group. The results of the repeated measures
analysis of variance indicated a highly significant effect of the
treatment time on food intake (F [2,9] = 5.197; p < 0.05), and a
significant interaction between the water salinity stress and the
treatment time (F [2,9] = 3.233; p < 0.05).
3.3. Weight
Overall, the population weight of the snails in both treatments showed
a gradual increase, although there were some periods during which the
average weight gain slightly reduced due to the occurrence of mortality
among larger individuals. After June 8th, the snail weight in the
saline water treatment was significantly higher than the CK, with
average weights of 3.72 and 3.28 g, respectively. In the later stage of
the experiment, the snail population in the saline treatment
consistently exhibited significantly higher weight than the CK, with a
highly significant difference observed between September 6th and
September 16th. The body weight growth rate exhibited a pattern of
initial increase followed by a subsequent decline. In the CK group, the
highest growth rate (19%) was observed on August 17th, whereas in the
2 ppt treatment group, the peak (15%) occurred on July 15th, which was
approximately the midpoint of the experiment. By the end of the
experiment, the average body weight in the CK had increased from 3.015
to 4.51 g per individual, while in the 2 ppt group, it increased from
3.66 to 5.235 g per individual. The snail weight in the saline
treatment was 1.17 and 1.16 times higher than the corresponding CK. The
results of the repeated measures analysis of variance indicated a
highly significant effect of the treatment time on weight (F
[2,9] = 60.084; p < 0.001), while the interaction between the salinity
stress and the treatment time was not significant (F [2,9] = 2.153;
p > 0.05) (Figure [71]2).
3.4. Shell Strength and Thickness
Under saline stress treatment, the shell thickness of male P.
canaliculata significantly increased (p < 0.01), with the saline
treated snails having a shell thickness approximately 1.6 times greater
than the control. The response of shell thickness to saline treatment
varied slightly between male and female P. canaliculata. In the
control, the shell thickness of males was lower than that of females,
while the opposite trend was observed under saline treatment. Both
snail sex and saline treatment had a noticeable impact on shell
strength in P. canaliculata, with males exhibiting greater shell
strength than females in both saline stress and freshwater treatments.
After saline treatment, both male and female snails showed a
significant enhancement in shell strength (p < 0.05), particularly in
males, where the saline treatment group had a snail shell strength 1.4
times greater than the control group (Figure [72]3).
FIGURE 3.
FIGURE 3
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Shell thickness and shell strength of P. canaliculata in saline
treatments. CK represents the treatment that snails were kept in a
freshwater environment without salinity. The error bars represent the
standard error. The data in the figure represent the average shell
thickness and shell strength per individual. A single asterisk (*)
indicates p < 0.05, while double asterisks (**) indicate p < 0.01.
3.5. Morphological Changes
For morphological parameters of P. canaliculata, the results showed no
significant difference in spire length (SL) between the two groups
(p > 0.05) after correcting for body whorl height (HL) as a covariate.
However, there was a significant difference in spire width between the
two groups, with values of 0.79 and 0.74 cm for the long‐term saline
treated snails and the control, respectively (p < 0.01). When aperture
length (AL) was used as a covariate, the inner lip length (IL) of
snails in the saline treatment was 0.48 cm, significantly longer than
0.46 cm of the control group (p < 0.05). The difference in basal lip
(BL) height between the two groups was not statistically significant
(p > 0.05). After correcting for aperture width (AW), the aperture
basal width (ABW) exhibited mean values of 0.85 cm, while only 0.80 cm
in the control group (p < 0.01). The aperture inner lengths (AL’)
between the two groups were similar and showed no significant
difference (p > 0.05) (Figure [74]4).
FIGURE 4.
FIGURE 4
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Morphological changes of P. canaliculata in different saline
treatments. CK represents the treatment that snails were kept in a
freshwater environment without salinity. The error bars represent the
standard error. A single asterisk (*) indicates p < 0.05, while double
asterisks (**) indicate p < 0.01.
3.6. Enzyme Activity
In the long‐term saline water environment, the activities of CAT, SOD,
and T‐AOC in female snails significantly enhanced, while GSH, MDA, and
NOS acitvities significantly decreased. In male snails, only MDA
content exhibited a significant decrease, and ACHE activity showed a
significant increase (p < 0.05). There existed a significant
interaction between salt treatment and sex for GSH (F [1,8] = 7.272,
p < 0.05), MDA (F [1,8] = 17.191, p < 0.01), NOS (F [1,8] = 14.657,
p < 0.01), and T‐AOC (F [1,8] = 7.963, p < 0.05) (Figure [76]5).
FIGURE 5.
FIGURE 5
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Enzyme activity of P. canaliculata in different saline treatments. CK
represents the treatment that snails were kept in a freshwater
environment without salinity. The error bars represent the standard
error. All antioxidant system indicators were normalized based on
protein content. A single asterisk (*) indicates p < 0.05, while double
asterisks (**) indicate p < 0.01. All enzyme activities were normalized
to protein content. The units for CAT, ACHE, NOS, SOD, and T‐AOC are
expressed as U/mg protein; GSH is expressed as μmol/g tissue, and MDA
as nmol/g tissue.
3.7. Transcriptome Sequencing, De Novo Assembly and Alignment
High‐throughput sequencing was used to systematically analyze gene
expressions in livers and gills with or without saline treatment. We
performed deep sequencing of RNA samples from snail gills and livers.
In total, 1,569,678,584 raw reads (150 bp) were obtained from the nine
libraries on the Illumina Novaseq 6000 platform. After preprocessing
and removal of low‐quality sequences, 1,560,932,792 clean reads were
generated. Additionally, the proportion of Q30 bases exceeded 95%,
which is essential for ensuring accurate sequencing (Table [78]3). The
clean reads were mapped to the P. canaliculata reference genome. In
all, the uniquely mapping ratios for the 79.4% and 80.4% were obtained
from saline‐treated liver tissue of female and male snails, 80.3% and
79.6% for saline‐treated gill tissue of female and male snails, 79.0%
and 78.4% control liver tissue of female and male snails, and 81.3% and
81.5% for control gills of female and male snails, respectively,
indicating high levels of gene expression in both groups.
TABLE 3.
Summary of trimming and reads results of the sequences generated from
livers and gills of the P. canaliculata with or without the saline
treatments.
Sample Raw reads Clean reads Q30 (%) GC content (%)
SFL1 4,51,56,894 4,49,25,618 95.24 49.09
SFL2 5,12,44,282 5,10,04,706 95.63 51.4
SFL3 5,07,67,440 5,04,74,208 95.66 51.75
SFL4 5,70,31,684 5,67,22,334 95.63 51.4
SML1 4,74,91,730 4,72,17,796 95.13 49.11
SML2 4,78,35,368 4,75,52,158 95.54 51.57
SML3 4,53,24,356 4,49,16,414 95.83 51.15
SML4 4,47,39,836 4,45,10,574 95.52 51.08
SFG1 4,81,80,600 4,79,24,068 95.46 45
SFG2 4,82,87,456 4,80,60,452 95.47 44.43
SFG3 4,89,89,448 4,87,56,954 95.18 43.85
SFG4 5,44,25,312 5,41,47,474 95.55 44.71
SMG1 4,24,06,540 4,21,86,766 95.09 43.38
SMG2 4,40,50,950 4,38,43,060 95.49 43.38
SMG3 4,29,52,140 4,27,21,446 95.4 44.3
SMG4 4,16,63,690 4,14,48,540 95 43.2
CFL1 4,39,73,042 4,35,98,566 95.61 50.95
CFL2 4,35,61,302 4,32,45,780 95.54 49.18
CFL3 4,72,80,110 4,69,19,564 95.57 49.63
CFL4 5,79,74,724 5,76,44,298 95.84 51.06
CML1 4,30,61,962 4,27,86,376 95.64 49.82
CML2 6,16,53,842 6,12,94,066 95.62 48.51
CML3 5,21,53,760 5,18,53,138 95.43 51.34
CML4 5,66,24,968 5,63,26,906 95.63 48.55
CFG1 5,53,52,954 5,50,82,678 95.25 44.38
CFG2 4,48,41,194 4,46,21,496 95.56 44.29
CFG3 4,85,34,750 4,83,12,988 95.42 44.48
CFG4 4,14,86,286 4,12,98,174 95.11 43.66
CMG1 5,86,09,936 5,83,27,806 95.47 45.29
CMG2 5,57,16,670 5,54,48,608 95.43 45.06
CMG3 4,83,92,476 4,81,32,426 95.5 45.45
CMG4 4,99,12,882 4,96,27,354 95.12 45.2
Total 1,56,96,78,584 1,56,09,32,792
[79]Open in a new tab
Note: SFL represents saline‐treated liver tissue of female snails; SML
represents saline‐treated liver tissue of male snails; SFG represents
saline‐treated gills tissue of female snails; SMG represents
saline‐treated gills tissue of male snails; CFL represents without
saline‐treated liver tissue of female snails; CML represents without
saline‐treated liver tissue of male snails; CFG represents without
saline‐treated gills tissue of female snails; CMG represents without
saline‐treated gills tissue of male snails. Four replicates of each
treatment were carried out in RNA‐seq analysis.
3.8. Identification and Analysis of Differentially Expressed Genes
To explore the potential response of P. canaliculata to salinity
stress, we conducted transcriptomic analysis on liver and gill tissues
collected from the endpoint of the study. A Venn diagram was employed
to identify the commonly DEGs across the libraries. We focused on the
analysis of DEGs that exhibited transcriptional regulation at specific
time points. Within the livers and gills, a total of 9265 upregulated
DEGs and 15,248 downregulated DEGs were identified, representing the
shared gene expression changes in response to the experimental
conditions (Figure [80]6).
FIGURE 6.
FIGURE 6
[81]Open in a new tab
The different colored circles represent the transcripts of DEGs in a
sample based on expression screening, and the values represent the
number of DEGs to different samples. SFL represents saline‐treated
liver tissue of female snails; SML represents saline‐treated liver
tissue of male snails; SFG represents saline‐treated gills tissue of
female snails; SMG represents saline‐treated gills tissue of male
snails; CFL represents without saline‐treated liver tissue of female
snails; CML represents without saline‐treated liver tissue of male
snails; CFG represents without saline‐treated gills tissue of female
snails; CMG represents without saline‐treated gills tissue of male
snails. Four replicates of each treatment were carried out in RNA‐seq
analysis.
3.9. Functional Annotation and Gene Ontology Classification
To analyze the transcriptome profile of P. canaliculata and its gene
models, we first filtered reads from two tissues and mapped them
separately to the P. canaliculata reference genome. Functional
annotation gave information on the transcripts, and genes were aligned
with public protein databases such as GO, KEGG, EggNOG, NR, Swiss‐Prot,
and Pfam. In total, there were 22,407 (96.14%) genes and 42,203
(97.35%) transcripts successfully annotated (Table [82]4).
TABLE 4.
Functional annotation of transcriptome data in six public protein
databases.
Type Gene number (percent) Transcript number (percent)
GO 15,807 (67.82%) 29,288 (67.56%)
KEGG 11,752 (50.42%) 22,913 (52.85%)
EggNOG 15,202 (65.23%) 29,353 (67.71%)
NR 22,403 (96.12%) 42,199 (97.34%)
Swiss‐Prot 14,751 (63.29%) 28,523 (65.79%)
Pfam 16,531 (70.93%) 31,275 (72.14%)
Total annotation 22,407 (96.14%) 42,203 (97.35%)
Total 23,307 (100.00%) 43,354 (100.00%)
[83]Open in a new tab
Transcriptome genes in the livers and gills of the male and female P.
canaliculata were annotated and assigned into three categories:
biological process (BP), cellular component (CC) and molecular function
(MF). Among those assigned to the category of BP, in the following
order as livers of the female snails (SFL vs. CFL), gills of the female
snails (SFG vs. CFG), gills of the male snails (SMG vs. CMG) and livers
of the male snails (SML vs. CML), cellular process (37.1%, 37.9%,
39.0%, 37.6%), metabolic process (27.4%, 22.4%, 24.0%, 27.8%),
bioligical regulation (15.5%, 14.2%, 13.1%, 13.1%) were the highly
represented. Among those assigned to the category of CC, membrane part
(34.0%, 43.6%, 37.0%, 32.8%), cell part (25.2%, 20.6%, 23.7%, 25.4%)
and oranelle (11.1%, 10.0%, 10.7%, 11.7%) were the highly represented.
Among those assigned to the category of MF, binding (41.5%, 40.6%,
43.1%, 40.2%), catalytic activity (41.3%, 40.9%, 41.0%, 40.1%) and
transporter activity (7.7%, 7.3%, 6.0%, 6.8%) were the highly
represented (Figure [84]7).
FIGURE 7.
FIGURE 7
[85]Open in a new tab
Histogram of GO classifications of P. canaliculata consensus sequences.
Results are summarized for the three main GO categories: biological
process, cellular component, and molecular function. The left axis
indicates the number of genes in each category. SFL represents
saline‐treated liver tissue of female snails; SML represents
saline‐treated liver tissue of male snails; SFG represents
saline‐treated gills tissue of female snails; SMG represents
saline‐treated gills tissue of male snails; CFL represents without
saline‐treated liver tissue of female snails; CML represents without
saline‐treated liver tissue of male snails; CFG represents without
saline‐treated gills tissue of female snails; CMG represents without
saline‐treated gills tissue of male snails. Four replicates of each
treatment were carried out in RNA‐seq analysis.
By employing KEGG pathway analysis, molecular interaction networks
within cells can be identified, facilitating the elucidation of
potential biological functions attributed to the analyzed genes
(Figure [86]8).
FIGURE 8.
FIGURE 8
[87]Open in a new tab
Scatter plot showing KEGG pathway enrichment among the DEGs. The
vertical axis represents the pathway categories, and the horizontal
axis shows the enrichment factor. The point size shows the number of
DEGs among the pathway. The bigger the point size, the more genes in
the pathway. SFL represents saline‐treated liver tissue of female
snails; SML represents saline‐treated liver tissue of male snails; SFG
represents saline‐treated gills tissue of female snails; SMG represents
saline‐treated gills tissue of male snails; CFL represents without
saline‐treated liver tissue of female snails; CML represents without
saline‐treated liver tissue of male snails; CFG represents without
saline‐treated gills tissue of female snails; CMG represents without
salt‐treated gills tissue of male snails. Four replicates of each
treatment were carried out in RNA‐seq analysis.
In the livers of female snails (SFL vs. CFL), KEGG analysis revealed
the significant enrichment of 25 pathways. Among these, the “Fatty acid
elongation” pathway exhibited the highest rich factor, playing a
crucial role in lipid metabolism and cellular energy balance. The
“Pathways of neurodegeneration” showed the highest number of DEGs,
suggesting potential neurophysiological stress responses under salinity
conditions. In the gills of female snails (SFG vs. CFG), KEGG analysis
identified 19 highly enriched pathways. The “Sulfur metabolism”
pathway, which is critical for detoxification and oxidative stress
regulation, demonstrated the highest rich factor. The “Chemical
carcinogenesis—DNA adducts” pathway exhibited the largest number of
DEGs, indicating possible DNA damage and repair mechanisms triggered by
saline stress. In the gills of male snails (SMG vs. CMG), KEGG analysis
revealed the significant enrichment of 16 pathways, the “Sulfur
metabolism” pathway possessed the highest rich factor, reinforcing its
role in stress adaptation. The “Amyotrophic lateral sclerosis” pathway
had the most DEGs, suggesting that oxidative stress‐related
neurodegenerative mechanisms may be involved in salinity adaptation. In
the livers of male snails (SML vs. CML), KEGG analysis identified 19
highly enriched pathways, the “Biosynthesis of unsaturated fatty acids”
pathway displayed the highest rich factor, which is essential for
maintaining membrane fluidity and energy storage. The “Coronavirus
disease—COVID‐19” pathway had the most DEGs, likely due to its broad
association with immune response and cellular stress pathways in KEGG
annotations.
Pairwise comparisons were conducted to identify DEGs between the 2 ppt
salinity treatment group and the control group. In the gills and livers
of female and male snails, 22,215, 22,284, 22,314, and 21,984 genes,
respectively, met the criteria of having a fold change greater than 2
and an adjusted p < 0.05. In the comparison of the same organ, both the
upregulation and the downregulation of DEGs were greater in males than
in females after salt stress. In a comparison of different organs of
the same sex, the proportion of changes in DEGs following liver
response to stress was greater than that of gills, with the greatest
upregulation and downregulation of DEGs in male liver (Figure [88]9).
FIGURE 9.
FIGURE 9
[89]Open in a new tab
Volcano plot of DEGs in livers and gills of the male and female snails
after salinity treatment. The data for all genes were plotted as log2
fold change versus the –log10 of the adjusted p‐value. Significant
differentially expressed genes comparing different treatments were
highlighted in red dot (upregulation) and blue (downregulation), while
genes with no significant differences were drawn in gray. SFL
represents saline‐treated liver tissue of female snails; SML represents
saline‐treated liver tissue of male snails; SFG represents
saline‐treated gills tissue of female snails; SMG represents
saline‐treated gills tissue of male snails; CFL represents without
saline‐treated liver tissue of female snails; CML represents without
saline‐treated liver tissue of male snails; CFG represents without
saline‐treated gills tissue of female snails; CMG represents without
saline‐treated gills tissue of male snails. Four replicates of each
treatment were carried out in RNA‐seq analysis.
3.10. Impact of Saline Stress on Hatchability and Inhibition Rate of Eggs
In the collected experimental samples, each egg mass exhibited varying
degrees of hatching. The hatching rate decreased with increasing
salinity of the treatments, while the hatching inhibition rate
increased with the higher salinity levels. Among all the egg mass
samples, the average hatching rate of all eggs at the end of the
experiment was 59.0%. As depicted in Figure [90]10, the average
hatching rate was 83.0% in the control group (CK), with the highest
average observed in the 0 ppt group at 94.7%. The hatching rate in the
0 ppt group was significantly higher than that in the other two saline
treatment groups, with a rate of 37.3% at 2 ppt treatment and the
lowest average rate of 19.0% at 5 ppt treatment. Using the CK group as
a reference, the experiment revealed that 0 ppt had a promotive effect
on the hatching of eggs, with a promotion rate of 14.7%. Conversely, at
a salinity of 2 ppt treatment, the hatching of eggs was significantly
inhibited, with an inhibition rate of 53.5%. At the highest
concentration treatment, the inhibition rate reached 77.0%, indicating
that salinity has a certain inhibitory effect on the hatching of eggs
(Figure [91]10).
FIGURE 10.
FIGURE 10
[92]Open in a new tab
Hatchability and inhibition rate of P. canaliculata eggs under saline
stress. CK represents the condition that involved no liquid
application, simulating the natural hatching conditions of eggs in the
environment. The error bars represent the standard error. Different
capital letters indicate significant differences (p < 0.05) between
treatments.
3.11. Impact of Saline Stress on Physiological and Biochemical Substances of
Eggs
The free and bound water contents of eggs after the saline stress
treatment were determined. The results revealed that by the 7th day of
the experiment, the free water content in all four treatments
significantly decreased. In the CK group, the absence of stress
resulted in a reduction in free water levels due to the involvement of
free water in various physiological activities during egg hatching. The
decline of the 5 ppt group was the most dramatic. The bound water
content in the CK group remained unchanged throughout the experiment.
According to the trends in free and bound water content across
different treatment groups (as shown in the Figure [93]11), the ratio
of free water to bound water in the 0 ppt group increased gradually
over the course of the experiment, while the ratio in the 2 and 5 ppt
groups rose slowly from 7th day to the end of the experiment. The ratio
in the CK group remained constant throughout the study. Among the four
treatment groups, glycogen content in the 0 ppt group significantly
decreased by 13th day and was higher than in the other three groups.
Glycogen levels in the other groups also significantly decreased by 7th
day, with no significant differences among them until the end of the
experiment. Fat content increased progressively in all treatment
groups, with the 2 ppt group showing the most pronounced increase in
fat content over the course of the experiment (Figure [94]11).
FIGURE 11.
FIGURE 11
[95]Open in a new tab
Physiological and biochemical substance content of P. canaliculata eggs
under long‐term saline stress treatments. CK represents the condition
that involved no liquid application, simulating the natural hatching
conditions of P. canaliculata eggs in the environment. The error bars
represent the standard error. Capital letters indicate differences
between the treatments; lowercase letters indicate differences in
treatment duration; different letters represent significant intergroup
differences (p < 0.05).
3.12. Impact of Saline Stress on Antioxidant Enzymes of Eggs
Various oxidative stress‐related indicators of eggs at different stages
were determined. Except for the CK group, ATPase activity in all
treatment groups initially increased and then decreased throughout the
experiment. On the final day, ATPase activity was significantly lower
in the 2 and 5 ppt treatment groups compared with the CK and 0 ppt
groups. CAT activity in the CK group decreased from 13th day onward and
continued to decline until the end of the experiment, showing a
significant difference from the 5 ppt group, while other groups
exhibited minimal enzyme activity changes. In both the CK and 0 ppt
groups, which were not subjected to saline stress, MDA content showed
an overall trend of initial increase followed by a decrease, whereas
saline treatment led to a reduction in MDA content in the egg masses.
POD activity increased significantly in all treatment groups from the
start to 7th day, with CK and 0 ppt groups showing notably higher POD
activity compared with the 2 and 5 ppt groups. From 7th day to the end
of the experiment, POD activity gradually returned to baseline levels.
T‐CHO content was significantly lower than the initial value on 7th day
for all treatment groups, with CK, 0, and 2 ppt groups showing a
gradual increase in T‐CHO content, while the 5 ppt group had the lowest
T‐CHO content on 13th day. SOD activity significantly decreased early
in the experiment and then stabilized, with all groups showing stable
SOD activity after 7th day. T‐AOC activity increased throughout the
experiment in all treatment groups, with the CK group showing
significantly higher levels than the other groups from 13th day onward.
GSH activity in the CK, 0 ppt, and 2 ppt groups exhibited an initial
increase followed by a decrease during the experiment period
(Figure [96]12).
FIGURE 12.
FIGURE 12
[97]Open in a new tab
Antioxidative system indices of P. canaliculata eggs under long‐term
saline stress. CK represents the condition that involved no liquid
application, simulating the natural hatching conditions of eggs in the
environment. The error bars represent the standard error. Capital
letters indicate differences between treatments; lowercase letters
indicate differences in treatment duration; different letters represent
significant intergroup differences (p < 0.05). All biochemical
parameters were normalized to protein content. ATP is expressed as
nmol/mg protein. CAT, POD, SOD, and T‐AOC are expressed as U/mg
protein. MDA is expressed as nmol/mg protein. GSH is expressed as
μmol/mg protein. T‐CHO is expressed as μg/mg protein.
4. Discussion
The exposure to low‐concentration saline treatment significantly
reduced the survival rate of P. canaliculata in the early stages
(Figure [98]13). As the salinity increased and the duration of exposure
extended, the survival rate gradually declined (Figure [99]2). This
initial lack of defense mechanisms against external stress explains the
observed decrease in survival. However, in the later stages, the
survival rate approached that of the control group, indicating the
snails had an ability to acclimate to the changing salinity. This
adapation may involve the regulation of intracellular ion
concentrations and the synthesis and secretion of osmoregulatory
substances. Accumulation of these substances helps maintain osmotic
balance and enables the snails to cope with saline stress. This gradual
convergence of survival rates with the control group suggests an
acclimatory response to the stressors. In addition, saline stress may
induce changes in the energy metabolism of P. canaliculata to meet the
demands of the changing environment (Liu, Liu, et al. [100]2022;
Purwaningsih et al. [101]2015). Studies have shown that mollusks can
maintain their internal energy levels in low‐saline environments
through increased feeding and reduced metabolic rates (Liu, Liu, Zhao,
Li, et al. [102]2022). This metabolic acclimation likely contributes to
the gradual reduction of the mortality rate in P. canaliculata under
long‐term low‐concentration saline stress (Bassler‐Veit
et al. [103]2013; Carral et al. [104]2023). Consequently, in this
experiment, the snails subjected to saline stress exhibited
significantly higher food intake and body weights compared with the
control group.
FIGURE 13.
FIGURE 13
[105]Open in a new tab
Schematic diagrams showing changes in physiology of P. canaliculata
under saline stress treatments, based on the results obtained.
After saline stress treatment, the shell strength and thickness of the
snails increased, and there was a significant widening of the overall
shell morphology (Figures [106]3 and [107]4). The reason for these
changes may involve an increase in calcium deposition since the snails
face higher concentrations of calcium ions in saline environments
(Vokhshoori et al. [108]2023; Fernández et al. [109]2023). To adapt to
this environment, snails may increase the rate of calcium deposition,
making the shell tissue harder and stronger (Casado‐Coy
et al. [110]2022). This can also trigger the synthesis of shell
proteins, promoting shell formation (Li et al. [111]2023; Hu
et al. [112]2021), and allowing for greater energy reserve storage
within the shell to withstand the stress (Garcia et al. [113]2021). It
is worth mentioning that saline environments may impose higher demands
on the snail shell, such as increased strength and protective
functions, to cope with more adverse environmental conditions (Thakur
et al. [114]2021). Thickening and strengthening of the shell provide
better protection and support, helping the snail with stand external
pressures and predation threats (Alati et al. [115]2020; Sakalauskaite
et al. [116]2020).
In the present study, transcriptomic analysis revealed that the highest
content in the “Biological Process” category was attributed to the
regulation and response of various cellular processes at the cellular
level, which were induced by the saline treatment. When exposed to a
saline environment, aquatic organisms accumulate specific organic
solutes such as glycerol and proline to increase the intracellular
osmotic pressure, thereby attracting water molecules into the cells to
prevent cellular dehydration (Ma et al. [117]2019). They also regulate
the expression and activity of ion channels and transport proteins for
ions like Na^+, K^+, and Ca^2+ to control intracellular ion
concentrations (Iqbal et al. [118]2023). Additionally, the activity of
antioxidant enzymes undergoes corresponding changes to eliminate
reactive oxygen species and neutralize free radicals, while some
proteins undergo abnormal folding and degradation (Serba
et al. [119]2016). In this study, the levels of CAT increased under
saline stress, which aided in the breakdown of hydrogen peroxide.
Concurrently, the upregulation of SOD activity played a crucial role in
scavenging superoxide anions (O^2−), while the overall antioxidant
capacity was enhanced. The regulation of ACHE activity helped maintain
neural transmission and adapt to environmental changes. The observed
decrease in MDA levels was attributed to the strengthened antioxidant
system, which effectively reduced lipid peroxidation. Additionally, the
reduction in NOS activity likely minimized the production of reactive
nitrogen species, thereby mitigating cellular damage (Figure [120]5).
Under conditions of long‐term saline stress, the liver and gill tissues
of P. canaliculata need to acclimate to the cellular environmental
changes induced by saline stress by regulating various cellular
processes (Gao et al. [121]2022).
Studies have shown that mollusks can increase the activity of
antioxidant enzymes to reduce oxidative stress on cells (Cao
et al. [122]2020; Zhang et al. [123]2017). The changes in the lipid
composition of the cell membrane and the activation of cellular
regulatory mechanisms are caused by saline stress (Xiong
et al. [124]2018). This leads to an increase in the expression of genes
related to lipid synthesis to enhance membrane lipid production, the
modulation of genes related to lipid metabolism to adjust the rate of
membrane lipid synthesis and metabolism, and the regulation of protein
synthesis and modification to acclimate to changes in membrane
composition and adjust the structure and function of the cell membrane
(Liu et al. [125]2020). These adjustments aim to maintain the stability
of the intracellular and extracellular environments, regulate the
membrane potential, and ensure proper cellular excitability (Liu, Li,
et al. [126]2022). In the GO analysis of this experiment, it was found
that the cellular membrane components of liver cells and protein
expression levels were significantly elevated after saline stress
(Figure [127]7).
Saline treatment induced various cellular functional and metabolic
changes. These changes primarily involve the regulation of metabolic
enzymes to acclimate to alterations in energy metabolism (Wang
et al. [128]2022; Ma et al. [129]2018). The expression and
functionality of binding proteins, such as receptors and signal
transduction molecules, are modulated to accommodate changes in signal
transduction (Zhou et al. [130]2020). Under saline stress, freshwater
snails may initiate a series of cellular protective mechanisms to
mitigate damage to cell structure and function (Si et al. [131]2018;
Cui et al. [132]2019). The results of the volcano plot analysis
revealed that the majority of significantly differentially expressed
genes, both in the liver and gills, were associated with metabolism.
Among the highly expressed metabolism‐related pathways in these tissues
were “Metabolism of xenobiotics‐cytochrome P450”, “Drug
metabolism‐cytochrome P450”, and “Alanine, aspartate and glutamate
metabolism” (Figure [133]8). Saline stress may affect the sulfur
metabolism pathway in the gills of snails, as the gills are involved in
nitrogen metabolism processes, regulating the expression and activity
of sulfite reductases, as well as adjusting the synthesis and
degradation rates of sulfates (Sharma et al. [134]2019; Planells
et al. [135]2019; Al‐Tobasei et al. [136]2017). At the same time,
oxidative stress, apoptosis, and inflammation occurred, leading to
abnormal gene expression related to ALS in the gill tissue (Li
et al. [137]2018). When aquatic animals are exposed to carcinogenic
substances in the environment, these substances can enter their bodies
through gill tissues. The gills participate in the metabolism and
detoxification processes of carcinogenic substances through metabolic
reactions. DNA can be damaged by environmental factors and chemical
substances, including DNA damage caused by carcinogenic agents. Gill
cells possess various DNA repair mechanisms to correct and repair DNA
damage, including base repair, nucleotide repair, and DNA strand break
repair. These repair processes help protect the integrity of DNA and
prevent cell mutations and carcinogenesis caused by DNA damage induced
by carcinogens (Quigley et al. [138]2016; Buselic et al. [139]2018; Gao
et al. [140]2018). Following saline stress, the liver exhibited more
upregulated and downregulated DEGs than gills, indicating differences
in structure, function, and metabolism (Figure [141]9). This may be
attributed to the liver's important role as a metabolic organ
responsible for substance metabolism and detoxification in the snail.
It is involved in multiple metabolic pathways. Therefore, when facing
stressful conditions, the liver may be more sensitive to metabolic
regulation in order to cope with changes in energy metabolism and
metabolic balance compared with the gills (Swinehart et al. [142]1998;
Cheney et al. [143]2008). Due to its rich metabolic activity and
involvement in oxygen metabolism processes, the liver may be more
susceptible to oxidative stress. The liver is also an important site
for immune response, with abundant distribution of immune cells and
immune factors (Pan and Han [144]2023; Wang et al. [145]2023). Under
stress conditions, the snail liver may initiate an immune response to
counter potential pathogen invasion and infection (Chen
et al. [146]2022; Martemyanov et al. [147]2021). This may lead to the
regulation of immune‐related genes in the liver, making it more
sensitive to stress (Jeyavani et al. [148]2022; Anagha
et al. [149]2022; Chakraborty and Joy [150]2020). In the liver of male
snails, genes related to fatty acid synthesis were significantly
differentially expressed under saline treatment conditions. The snails
may require adjustments in the fatty acid synthesis pathway to
acclimate to changes in the stability and functional demands (Pan
et al. [151]2019). The process of fatty acid elongation involves the
participation of multiple enzymes and substrates in the synthesis of
long‐chain fatty acids within cells (Wang et al. [152]2018). This
regulatory response may be aimed at acclimation to the energy demands
and metabolic adjustments under saline stress conditions (Zhang
et al. [153]2018).
The volcano plot analysis revealed that the number of upregulated and
downregulated DEGs in male snails after saline stress was higher than
in female snails, indicating differences in gene expression patterns
between male and female individuals of the snail species
(Figure [154]9). The regulation of genes and hormones may lead to
different transcriptional responses to saline stress in male and female
individuals, as well as different physiological acclimatory capacities
to cope with saline stress (Xu et al. [155]2019). These DEGs may be
involved in pathways related to cell membrane stability, osmotic
regulation, ion balance, and cellular stress (Slattery
et al. [156]2018). Male and female snails may have sex‐specific gene
regulatory networks, which may include sex‐determining genes, sex
hormone receptors, and sex‐specific transcription factors (Yang
et al. [157]2023; Liu, Li, et al. [158]2022; Li and Zou [159]2019).
Long‐term saline treatment may impact the snail's nervous system,
leading to an increased number of DEGs in this pathway (Serba
et al. [160]2016). Saline stress could trigger cellular disturbances
such as oxidative stress and inflammation, which may have adverse
effects on the nervous system. Additionally, saline treatment may
affect the synthesis of neurotransmitters, neuronal function, and
interactions between nerve cells, resulting in an increased number of
DEGs in the “Pathways of neurodegeneration” (Sharma et al. [161]2019).
In a saline environment, P. canaliculata eggs undergo metabolic
adjustments to acclimate to the new conditions. Salinity has a negative
impact on egg hatching, as increased salinity raises osmotic pressure,
leading to water loss. As salinity levels rise, the increasing osmotic
pressure increases the dehydration of the eggs. Excessive dehydration
and deformation of the eggs disrupt their normal hatching process,
resulting in lower hatching rates (Wang et al. [162]2012). This
experiment also confirmed that higher saline concentrations in the
water environment correlated with lower hatching rates for P.
canaliculata eggs. The 0 ppt condition exhibited the highest hatching
rate and the highest free water to bound water ratio; it also had the
highest glycogen reserves. Compared with the control, the 0 ppt group
further promoted the development of egg masses, suggesting that it
helps maintain the normal shape of the eggs, prevents dehydration, and
thus facilitates the hatching process (Figure [163]10).
The saline environment negatively affects the stability of egg cell
membranes, which are critical for maintaining cellular structure and
function. High salinity can cause membrane rupture or surface
macromolecule denaturation, disrupting embryonic development.
Additionally, elevated saline concentrations increase metabolic stress
on the eggs. In response to hyperosmotic conditions, embryos may
require more energy to sustain normal physiological processes,
potentially altering their growth and developmental cycles (Dreon
et al. [164]2007; Salleh and Arbain [165]2015; Liu et al. [166]2023).
Our experiment revealed that high‐concentration saline stress reduced
the total cholesterol content in P. canaliculata eggs, with cholesterol
likely being utilized to address membrane damage caused by stress.
Cholesterol also plays a role in modulating membrane fluidity, enabling
cells to better acclimate to high‐salinity environments. Furthermore,
compared with the control group, the variations in MDA and POD levels
during the entire hatching period under high saline conditions
indicated that high salinity altered the developmental trajectory of
the eggs (Figure [167]11).
5. Conclusion
This study conducted a comprehensive analysis of the survival rates,
physiological and biochemical characteristics, and transcriptomes of P.
canaliculata across different growth stages to saline stress, as well
as the related response mechanisms to saline stress. The results
indicated that early survival rates significantly declined under saline
stress, but the snails were able to maintain energy balance by
increasing food intake and reducing metabolic rates, eventually
reaching a survival rate close to that of the control group. P.
canaliculata exhibited enhanced shell strength, thickness, and
significant shell widening through increased calcium content and shell
protein synthesis under saline stress, which improved their
self‐protective functions. Additionally, changes in antioxidant enzyme
activity displayed sexual dimorphism. Saline stress caused the
regulation of cellular processes, where the accumulation of organic
solutes and the modulation of ion channels were key mechanisms for
acclimating to osmotic changes and preventing cellular damage. The
liver and gills showed abundant expression of metabolism‐related
pathways, with the liver being more sensitive to metabolic and immune
responses than the gills. Saline stress also had negative effects on
the eggs, with hatching rates decreasing as salinity levels increased.
Moreover, saline stress negatively impacted membrane stability and
metabolism, reducing total cholesterol content in the eggs and altering
their developmental process. These findings not only provide a
theoretical basis for the invasion mechanisms of P. canaliculata but
also reveal the physiological, biochemical, and molecular acclimation
strategies which the species employs in response to saline stress.
Author Contributions
Yingtong Chen: conceptualization (equal), data curation (lead), formal
analysis (lead), investigation (lead), methodology (lead), validation
(lead), visualization (lead), writing – original draft (lead). Fucheng
Yao: inverstigation (equal), methodolgy (equal). Zhaoji Shi: methodolgy
(equal), software (equal), visualization (equal). Chunxia
Zhang: investigation (equal). Jimin Liu: investigation (equal). Jiaen
Zhang: conceptualization (lead), writing‐review and editing (lead),
project adminstration (lead), resources (lead), funding acquisition
(lead), supervision (lead). Zhong Qin: methodolgy (equal), validation
(equal).
Conflicts of Interest
The authors declare no conflicts of interest.
Acknowledgments