Sex-Biased Survival, Behavior Response, and Recovery Performance of Pomacea canaliculata Snails to Drought Stress and Rewatering Condition

Simple Summary In this study, we investigated the survival rate of P. canaliculata and explored the differential responses and mechanisms of female and male P. canaliculata under drought stress through a simulated drought experiment. Our findings revealed that female P. canaliculata produced a large number of eggs before burrowing into the soil when faced with drought condition. Furthermore, we observed that the feeding and activity of female P. canaliculata were more active than those of the males after rewatering following drought stress. Additionally, there were some gender differences in the antioxidant system of P. canaliculata after rewatering following drought stress. We expect that this research will be useful for a better understanding of how P. canaliculata tolerates and recovers from drought stress, which may aid in explaining the success of their invasions and predicting mollusk responses to climate change. Abstract As the frequency of droughts increases with climate change, the tolerance of aquatic organisms to abiotic stressors will become critical determinants of survival. Pomacea canaliculata has become a widely distributed agricultural and environmental pest in southern China. To evaluate their tolerance and adaptation under the drought condition, the survival, feeding, behavior, and antioxidant system changes in female and male P. canaliculata were investigated during drought stress and rewatering process through an indoor simulation experiment. The results showed that female snails laid eggs before burrowing into the soil to ensure offspring reproduction. Female P. canaliculata had higher survival rates than males under drought stress, and their recovery ability of activity after rewatering was also superior to those of males. The antioxidant system of P. canaliculata showed obvious activation with gender differences after rewatering. Overall, the survival rate of female P. canaliculata was higher after drought stress, and the resilience ability of female snails after rewatering was stronger, including in their behavior, feeding, and antioxidant system recovery. The P. canaliculata tolerance to drought and the ability to recover quickly after drought may contribute to their long-term survival and facilitate continuous invasion.


Introduction
The apple snail, Pomacea canaliculata (Lamarck, 1822), originally from the Amazon River basin in South America, was introduced in the 1980s from Argentina into Asia for human food and the aquarium trade due to its rich protein content and high nutritional

Experiment Materials
All of the tested P. canaliculata individuals were collected from irrigation ditches in a farm located at South China Agricultural University in Guangzhou (23 • 14 N, 113 • 38 E), Guangdong Province, China. A batch of eggs identified as P. canaliculata hatched in the irrigation ditches in advance, and a gauze net was installed at the inlet and outlet of the irrigation ditches to prevent apple snails from entering and escaping. Apple snails with a shell height of less than 25 mm and immature sex were selected during the collection to ensure that the selected snails had not experienced long-term drought before drought stress. Before the snails were used for the experiment, they were reared in an aquarium (70 cm long, 52 cm wide, 37 cm high) in a window-lit room with natural sunlight under temperatures of 24-30 • C, and they were fed with lettuce (Lactuca sativa) and duckweed (Lemna minor). Female and male apple snails with a shell height of more than 30 mm (mature individuals) were selected for the experiment, based on exhibiting normal behavior and appearance. We first identified their sex based on operculum morphology, because the opercula at a particular site of males become convex when they tend to mature, which is different from the females whose opercula remain concave after sexual maturity [28]. Additionally, we further examined a visible albumin gland through the shell (to identify mature females) or testes (to identify mature males).

Experiment Procedure
One hundred and eighty tested female snails and the same-quantity tested male snails were assigned to each of the three treatments: control (aquatic condition) and two drought treatments, with drought durations of 60 and 120 d, respectively. Six replicates were performed for each of three treatments. Three treatments were placed in an outdoor balcony to ensure that they were as close as possible to the environmental field temperatures. Ten snails were released into each bucket (24 cm diameter and 21 cm high) at the beginning of the experiment. Each bucket was filled with an 8 cm layer of soil at the bottom and a 10 cm layer of water on top of the soil, and the buckets were covered with mesh to prevent the snails from escaping.
After 1 d adaptation, a siphon tube was used to slowly remove water at high water level, while a pipette and an ear syringe were used to meticulously extract water at low water level. This controlled the water level to decrease to the soil surface without any noticeable moisture in the two drought treatments. The water was slowly removed without losing soil, and the soil was allowed to dry naturally. When the remaining water level was lower than half of the apple snail's shell height, the apple snails began to drill into the soil naturally, and the experiment began to record the drilling of female and male apple snails every hour. Subsequent experiments were conducted on the laboratory balcony, simulating natural soil drying without additional water input throughout the process.
Many eggs were found within the first day of lowering the water level, and all egg masses were carefully removed to a Petri dish, respectively. Eight milliliter pure aerated water was placed on the bottom cover of the petri dish, and then the top was covered with egg mass to ensure internal humidity and air circulation. Next, the dish was placed in an artificial climatic chamber with a 14/10 h (light/dark) photoperiod at 25 ± 2 • C. Hatchings were checked daily, and the incubation periods of these egg masses were calculated from the day when they were laid to the day of the first hatching [29]. In order to determine hatching success, the number of hatchlings in each egg mass was counted, and the rest of the non-incubated eggs were counted after they were dispersed in 2% sodium hydroxide solution for 1.5 h [30].
After arriving at the experimental processing time (drought duration of 60 and 120 days), 5 L aerated tap water was added to the buckets in the dry condition to bring the water level of 8 cm above the top of the soil. Survival status was confirmed when the snails were able to resume normal behavior after re-entering an aquatic condition. The behaviour of the snails was observed at 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, and 6 h after rewatering. The individual behaviour of the snails was classified into six states of activity: feeding, crawling, foot deployment, first tentacle extension, aperture agape, and closed operculum. At the same time, the surviving snails were fed with pre-weighed lettuce, and their intake was assessed by measuring the wet weight of the surplus lettuce after 24 h.
Liver tissue (0.2 g) was sampled from eight randomly chosen snails (four of each female and male) per time at control (aquatic condition), under 120 d drought, and then 24 h after rewatering treatment (recovery aquatic condition was 24 h after 120 d of drought treatment). The liver was carefully removed with a sterile scalpel and washed with 0.9% NaCl before being dissected, and then it was drained on filter paper and stored at 80 • C in an ultra-low temperature refrigerator for enzymatic activity detection.
The activity of superoxide dismutase (SOD), catalase (CAT), total antioxidant capacity (T-AOC), glutathione S-transferase (GST), reduced glutathione (GSH), and malonaldehyde (MDA) were tested according to the operation manual of SOD (Product Code A001), CAT (Product Code A007), T-AOC (Product Code A015), GST (Product Code A004), GSH (Product Code A006), and MDA (Product Code A003) from Nanjing Jiancheng Company and the methods are described in the manufacturer's operation manual ( Figure 1). were able to resume normal behavior after re-entering an aquatic condition. The behaviour of the snails was observed at 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, and 6 h after rewatering. The individual behaviour of the snails was classified into six states of activity: feeding, crawling, foot deployment, first tentacle extension, aperture agape, and closed operculum. At the same time, the surviving snails were fed with pre-weighed lettuce, and their intake was assessed by measuring the wet weight of the surplus lettuce after 24 h. Liver tissue (0.2 g) was sampled from eight randomly chosen snails (four of each female and male) per time at control (aquatic condition), under 120 d drought, and then 24 h after rewatering treatment (recovery aquatic condition was 24 h after 120 d of drought treatment). The liver was carefully removed with a sterile scalpel and washed with 0.9% NaCl before being dissected, and then it was drained on filter paper and stored at 80 °C in an ultra-low temperature refrigerator for enzymatic activity detection.

Data Analysis
Independent sample t-tests were used to analyze the differences between males and females in self-burial, reproduction, survival, and food intake. Two-way analysis of variance was used to analyze the effects of sex and drought treatments on the antioxidant system of P. canaliculata. All analyses were performed using SPSS 22.0 (IBM Corp., Armonk, NY, USA). All graphs were produced using Origin 9.1 (Origin Lab, Northampton, MA, USA).

Results
Both female and male P. canaliculata started to self-bury when the water level was less than half of the shell. There was no significant difference in the non-self-burial proportion between female and male snails within the first 4 h, but the non-self-burial proportion in female snails was significantly higher than that in male snails at the 6, 8, 9, 11, and 13 h (p < 0.05), and the difference reached the maximum at the 11th h: the proportions of non-self-buried snails in female and male snails were 64.5% and 40%, respectively (Figure 2, Table 1). Figure 1. Experimental procedures to estimate survival, behavior response, and recovery performance of P. canaliculata snails to the drought stress and rewatering condition.

Data Analysis
Independent sample t-tests were used to analyze the differences between males and females in self-burial, reproduction, survival, and food intake. Two-way analysis of variance was used to analyze the effects of sex and drought treatments on the antioxidant system of P. canaliculata. All analyses were performed using SPSS 22.0 (IBM Corp., Armonk, NY, USA). All graphs were produced using Origin 9.1 (Origin Lab, Northampton, MA, USA).

Results
Both female and male P. canaliculata started to self-bury when the water level was less than half of the shell. There was no significant difference in the non-self-burial proportion between female and male snails within the first 4 h, but the non-self-burial proportion in female snails was significantly higher than that in male snails at the 6, 8, 9, 11, and 13 h (p < 0.05), and the difference reached the maximum at the 11th h: the proportions of non-selfburied snails in female and male snails were 64.5% and 40%, respectively ( Figure 2, Table 1).
Drought significantly increased the number of egg masses per female P. canaliculata (control: 0.28, drought: 0.7, p < 0.001) and the number of total eggs (control: 418.8, drought: 1056.6, p < 0.001), but significantly decreased the hatching rate (control: 75.39%, drought: 45.79%, p < 0.001) of P. canaliculata. The incubation period (control: 14 d, drought: 13.83 d, p > 0.05) did not change significantly under the drought treatment ( Figure 3).   Figure 3).   The average survival rates of female and male P. canaliculata after 60 d of drought were 90% and 72.5%, respectively, with a significant difference between female and male snails (p < 0.05, Figure 4). After 120 d of drought, the survival rates of both female and male P. canaliculata decreased compared with 60 d of drought, but the difference between female The average survival rates of female and male P. canaliculata after 60 d of drought were 90% and 72.5%, respectively, with a significant difference between female and male snails (p < 0.05, Figure 4). After 120 d of drought, the survival rates of both female and male P. canaliculata decreased compared with 60 d of drought, but the difference between female and male snails remained significant (female: 80%, male: 53.33%,* p < 0.05; Figure 4). The average survival rates of female and male P. canaliculata after 60 d of drought were 90% and 72.5%, respectively, with a significant difference between female and male snails (p < 0.05, Figure 4). After 120 d of drought, the survival rates of both female and male P. canaliculata decreased compared with 60 d of drought, but the difference between female and male snails remained significant (female: 80%, male: 53.33%,* p < 0.05; Figure 4). When rewatered after 60 d drought, the feeding ability of P. canaliculata recovered ( Figure 5). The results showed that the average 24 h food intake of female and male P. canaliculata was 1.44 g and 0.96 g, respectively, with a significant difference between female and male snails (p < 0.05, Figure 5). When rewatered after 120 d drought, the 24 h food intake of both female and male P. canaliculata was lower than that of the 60 d treatment, but there was still a significant difference between the female and male snails (female: 1.09 g, male: 0.79 g, p < 0.05; Figure 5). When rewatered after 60 d drought, the feeding ability of P. canaliculata recovered ( Figure 5). The results showed that the average 24 h food intake of female and male P. canaliculata was 1.44 g and 0.96 g, respectively, with a significant difference between female and male snails (p < 0.05, Figure 5). When rewatered after 120 d drought, the 24 h food intake of both female and male P. canaliculata was lower than that of the 60 d treatment, but there was still a significant difference between the female and male snails (female: 1.09 g, male: 0.79 g, p < 0.05; Figure 5).     Specifically, the drought and then 24 h rewatering treatment significantly decreased SOD activity of both female and male snails, and female snails have higher SOD activity at 24 h after rewatering compared with male snails (Figure 7a). On the contrary, the drought and then 24 h rewatering treatment significantly increased MDA content for both female and male snails (Figure 6b). The CAT activity of both female and male snails significantly decreased after the drought stress and increased significantly after 24 h rewatering treatment, but it was still significantly lower than the control (Figure 7c). The T-AOC activity of both female and male snails significantly decreased under the drought stress, but it could reach the control level after 24 h rewatering treatment (Figure 7d).  Figure 6b corresponds to the activity patterns of P. canaliculata after being rewatered following 120 d drought. After 30 min, 63.89% of females and all males had a sealed operculum. After 45 min, 20.37% of females first extended their tentacles, and 35.19% of females and 24.44% males opened their operculum. Meanwhile, 44.44% of females and 75.56% of males retained a closed operculum. After 2 h, all living snails showed signs of activity. The first instance of feeding was observed in females (19.91%) after 5 h, while males started feeding at least 6 h later. After 6 h, 31.94% of females and 18.89% of males kept feeding.
Through two-way analysis of variance, we found that drought stress significantly affected the activities of SOD (p < 0.001), MDA (p < 0.001), CAT (p < 0.001), T-AOC (p < 0.001), GST (p < 0.001), and GSH (p < 0.001) of P. canaliculata, while the snail sex significantly influenced the activities of SOD (p < 0.01), CAT, GST (p < 0.05), and GSH (p < 0.05) of P. canaliculata, and the interaction between the drought treatment and the snail sex significantly affected the GST activity (p < 0.05, Table 2). Specifically, the drought and then 24 h rewatering treatment significantly decreased SOD activity of both female and male snails, and female snails have higher SOD activity at 24 h after rewatering compared with male snails (Figure 7a). On the contrary, the drought and then 24 h rewatering treatment significantly increased MDA content for both female and male snails (Figure 6b). The CAT activity of both female and male snails significantly decreased after the drought stress and increased significantly after 24 h rewatering treatment, but it was still significantly lower than the control (Figure 7c). The T-AOC activity of both female and male snails significantly decreased under the drought stress, but it could reach the control level after 24 h rewatering treatment (Figure 7d). Drought significantly decreased the GST content of both female and male snails, and, at 24 h after rewatering, it did not significantly increase the GST content of female snails, but it significantly increased the GST content of male snails (Figure 7e). The GSH content of both female and male snails significantly decreased after drought but recovered to control level after 24 h rewatering (Figure 7f).

Discussion
Our results provide experimental evidence that both female and male P. canaliculata were able to survive extended periods of drying conditions that stimulated their natural burrowing behavior and allowed them to aestivate. After 120 d drought stress, 80% of female snails and 53.34% of males remained viable, which supports the snails' strong drought tolerance. Darby et al. recorded P. paludosa had a 70% survival rate after 84 d in a comparable simulated outdoor dry down event [31]. In another study [19], more females (44.7%) survived than males (32.1%) in the drying conditions created by full burial after 47 d. Moreover, they could survive in dry conditions for up to 13 months, and they were buried in wet mud for up to 29 months. Differences in exposure method, snail size, and

Discussion
Our results provide experimental evidence that both female and male P. canaliculata were able to survive extended periods of drying conditions that stimulated their natural burrowing behavior and allowed them to aestivate. After 120 d drought stress, 80% of female snails and 53.34% of males remained viable, which supports the snails' strong drought tolerance. Darby et al. recorded P. paludosa had a 70% survival rate after 84 d in a comparable simulated outdoor dry down event [31]. In another study [19], more females (44.7%) survived than males (32.1%) in the drying conditions created by full burial after 47 d. Moreover, they could survive in dry conditions for up to 13 months, and they were buried in wet mud for up to 29 months. Differences in exposure method, snail size, and species may contribute to the significant difference in survival rates between previous studies and our study.
In addition, the survival rate of females was significantly higher than that of males under drought treatments, indicating that P. canaliculata females exhibited greater viability than males in terms of drought tolerance. Sex-biased drought tolerance has also been found in other animals. Male elephants (Loxodonta africana) are more sensitive to drought than female elephants [32]. Previous research has shown that Pomacea sp. females exhibited greater viability than males in terms of desiccation tolerance [33]. Other aspects of P. canaliculata have also shown sexual differences. In addition to a longer lifespan [34], females also exhibit faster growth rates than males [26]. When confronted with predation risks, females have been shown to respond more strongly to alert signals from conspecifics, resulting in a higher survival rate than males in interactions with natural predators [35]. In terms of susceptibility to high temperatures and fasting, males seem to be more vulnerable than females [36,37]. These dissimilarities between female and male Pomacea sp. might be attributed to a female survival strategy that prioritizes preserving reserves and nutritional condition, while males tend to deplete their energy reserves to increase their reproductive success [37].
The high survival rate of P. canaliculata that we observed after 60 and 120 d of dry conditions is best explained by their diverse suite of behavioral and physiological adaptations for tolerating abiotic stressors. P. canaliculata with a dual respiratory system consisting of a gill and a lung, has a variety of traits, such as having a flexible operculum, burrowing behavior, and living both in water and on dryland, which all probably contribute to the survival [27,38]. In this study, it was observed that the recovery was not immediate, and the transition out of dry conditions progressed gradually. Surviving P. canaliculata also survived and resumed normal activities of their return to water condition. From opening the aperture to first extending a tentacle and beginning movement, P. canaliculata became active, then deployed their foot, crawled, and resumed feeding. Giraud-Billoud et al.
recorded similar behavioral recovery of P. canaliculata individuals [39]. Glasheen et al. [40] further observed that snails of the Pomacea species resume mating and can reproduce successfully after extended exposure to drying conditions. The recovery process of activity after rewatering following 60 and 120 d of drought was found to be better in female P. canaliculata than in males. After 60 d drought, females began feeding at 3 h after rewatering, while males needed 4 h to recover feeding. Females and males resumed feeding at 5 and 6 h after 120 d drought, respectively. Additionally, after 24 h, the feeding amount of female snails was significantly higher than that of males. This suggests that females have better resilience to recover from drought than males, and the tolerance to drought and high population resilience after rewatering may partially account for the success of P. canaliculata as expanding global invaders [41,42].
This study reports for the natural response of female and male P. canaliculata in drying conditions found that far fewer females than males buried themselves in sediment during the first few hours of drought. Experimental observations suggest that females delayed burrowing in order to lay eggs, and most females began laying eggs at midnight, 17 h after the start of the experiment, while males continued to move or remain stationary. However, by the 24th h of the experiment, most snails of both sexes had buried themselves into the soil. Compared with controls, P. canaliculata under the dry conditions significantly increased spawning quantity at the first day of the experiment. Guo et al. found that, at the beginning of the experiment, under predation risk, P. canaliculata laid eggs faster and produced more eggs [43]. Another freshwater snail, Biomphalaria glabrata, also showed an increase in egg production shortly after exposure to a trematode parasite without parasitization [44]. Increased egg production may be a strategy for snails to maximize reproductive success when facing serious threats. The phenomenon of snails laying a large number of eggs under unfavorable conditions in the natural environment is beneficial for ensuring offspring and population reproduction. This indicates that, in addition to preserving themselves, snails will also allocate some of their energy to population reproduction when they encounter sudden changes in their environment. Invasive P. canaliculata produce large numbers of eggs with high hatching success [45,46]. Even if only a small number of P. canaliculata survive, the population would continue to grow and could cause more harm [47].
In the antioxidant system of animals, SOD, CAT, T-AOC, GST, and GSH have the function of scavenging reactive oxygen species (ROS), playing an important role in enhancing the defense and immune function of phagocytes [48,49]. However, antioxidant enzyme activities did not increase during drought, which may be because of the general inhibition of protein synthesis due to hypometabolism of the P. canaliculata during the estivation under drought [49]. Correspondingly, an increase in MDA content was observed. Lipid peroxidation is considered the result of ROS oxidizing lipids, and MDA is considered one of the final decomposition products of lipid peroxidation, reflecting the degree of oxidative damage to cell membranes indirectly [50,51]. It is worth noting that, after 24 h rewatering, the antioxidant enzyme activities of female and male P. canaliculata both increased, and their corresponding MDA content also decreased. In a previous study, an increase in TBARS (an index of oxidative stress-induced lipid peroxidation damage) was observed in the soft part of the snail during estivation, but it decreased to the level of active control after awakening [39], which is in line with the change of MDA in our study. Moreover, there were some gender differences in the antioxidant system of P. canaliculata after the rewatering under drought stress. For example, after 24 h rewatering, females exhibited stronger resilience of SOD activity while demonstrated weaker resilience of GST activity compared with males. This may indicate differences in the mobilization patterns of antioxidant enzymes in female and male P. canaliculata.
Paddy fields are the primary habitat of P. canaliculata in invaded areas of Asia, which experience alternating submerged and dry conditions during rice cultivation and fallow periods. The ability of P. canaliculata to withstand drought in this complex and changing environment facilitated its colonization. Consequently, P. canaliculata populations have been able to establish themselves in many regions and countries in Asia [6,52]. Our findings provide evidence of one of the key traits that contribute to the colonization of P. canaliculata in paddy fields.

Conclusions
The present study found that female P. canaliculata produced a large number of eggs before burrowing into the soil when facing with drought condition and had a higher survival rate than males under drought stress, and their feeding and activity after rewatering are also more active than those of males. The antioxidant system of P. canaliculata showed significant activation after 120 d drought and a gender difference. Drought tolerance and the ability to recover quickly after drought may benefit P. canaliculata invasion, which would cause more serious damage on crop production and the sustainability of aquatic ecosystems.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data for the analysis in this study are available upon request from the corresponding author.