Comparative physiological, biochemical and transcriptomic analyses to reveal potential regulatory mechanisms in response to starvation stress in Cipangopaludina chinensis.

Cipangopaludina chinensis, as a financially significant species in China, represents a gastropod in nature which frequently encounters starvation stress owing to its limited prey options. However, the underlying response mechanisms to combat starvation have not been investigated in depth. We collected C. chinensis under several times of starvation stress (0, 7, 30, and 60 days) for nutrient, biochemical characteristics and transcriptome analyses. The results showed that prolonged starvation stress (> 30 days) caused obvious fluctuations in the nutrient composition of snails, with dramatic reductions in body weight, survival and digestive enzyme activity (amylase, protease, and lipase), and markedly enhanced the antioxidant enzyme activities of the snails. Comparative transcriptome analyses revealed 3538 differentially expressed genes (DEGs), which were significantly associated with specific starvation stress-responsive pathways, including oxidative phosphorylation and alanine, aspartate, and glutamate metabolism. Then, we identified 40 candidate genes (e.g., HACD2, Cp1, CYP1A2, and GPX1) response to starvation stress through STEM and WGCNA analyses. RT-qPCR verified the accuracy and reliability of the high-throughput sequencing results. This study provides insights into snail overwintering survival and the potential regulatory mechanisms of snail adaptation to starvation stress.

Performance and characterization of snail adhesive mucus as a bioflocculant against toxic Microcystis.

Toxic Microcystis blooms are widespread in aquatic bodies, posing major threats to aquatic and human life. Recently, bioflocculants have attracted considerable attention as a promising biomaterial for Microcystis management. In search of a novel organism that can produce an efficient bioflocculant for controlling harmful algae sustainably, the native gastropod Cipangopaludina chinensis was co-cultured continuously with toxic Microcystis under different initial algal cell densities. The bioflocculation effect of snail mucus on toxic Microcystis, microcystin removal, and toxin accumulation in snails was investigated. In addition, the properties of the adhesive mucus were characterized using microscopic, X-ray diffraction, infrared spectroscopy, and polysaccharide and proteome analyses. Microcystis cells were captured and flocculated by the snail mucus; removal efficiencies of up to 89.9% and 84.8% were achieved for microalgae and microcystin-leucine arginine (MC-LR), respectively, when co-cultured with C. chinensis for only one day. After nine-day exposure, less than 5.49 µg/kg DW microcystins accumulated in the snails, indicating safety for human consumption. The snail mucus contained 104.3 µg/mg protein and 72.7 µg/mg carbohydrate, which provide several functional groups beneficial for Microcystis bioflocculation. The main monosaccharide subunits of polysaccharides are galactose, galactosamine, glucosamine, fucose, glucose, and mannose. Most of them are key components of polysaccharides in many bioflocculants. Gene Ontology analysis indicated the protein enrichment in binding processes and catalytic activity, which may account for Microcystis bioflocculation via protein binding or enzymatic reactions. The findings indicate that native C. chinensis secretes adhesive mucus that can act as bioflocculant for toxic Microcystis from ambient water and can be an effective and eco-friendly tool for Microcystis suppression.

Characterization of the shell proteins in two freshwater snails Pomacea canaliculata and Cipangopaludina chinensis.

Uncovering the molecular mechanism of shell formation not only reveals the evolution of molluscs but also lay a foundation for shell-inspired biomaterial synthesis. Shell proteins are the key macromolecules of the organic matrices that guide the calcium carbonate deposition during shell mineralization and have thus been intensively studied. However, previous studies on shell biomineralization have mainly focused on marine species. In this study, we compared the microstructure and shell proteins in the apple snail Pomacea canaliculata which is an alien species that has invaded Asia, and a freshwater snail Cipangopaludina chinensis which is native to China. The results showed that although the shell microstructures were similar in these two snails, the shell matrix in C. chinensis contained more polysaccharides. Moreover, the compositions of shell proteins were quite different. While the shared 12 shell proteins (including PcSP6/CcSP9, Calmodulin-A, and proline-rich protein) were supposed to play key roles in shell formation, the differential proteins were mainly immune components. The presence of chitin in both shell matrices and the chitin-binding domains containing PcSP6/CcSP9 underpinned the relevance of chitin as a major fraction in gastropods. Interestingly, carbonic anhydrase was absent in both snail shells, suggesting that freshwater gastropods might have unique pathways to regulate the calcification process. Our study suggested that shell mineralization might be very different in freshwater and marine molluscs, and therefore, the field should pay more attention to the freshwater species to achieve a more comprehensive insight into biomineralization.

SMRT Sequencing Reveals Candidate Genes and Pathways With Medicinal Value in Cipangopaludina chinensis.

Cipangopaludina chinensis is an economically important aquatic snail with high medicinal value. However, molecular biology research on C. chinensis is limited by the lack of a reference genome, so the analysis of its transcripts is an important step to study the regulatory genes of various substances in C. chinensis. Herein, we conducted the first full-length transcriptome analysis of C. chinensis using PacBio single-molecule real-time (SMRT) sequencing technology. We identified a total of 26,312 unigenes with an average length of 2,572 bp, of which the largest number of zf-c2h2 transcription factor families (120,18.24%) were found, and also observed that the majority of the 8,058 SSRs contained 4-7 repeat units, which provided data for subsequent work on snail genetics Subsequently, 91.86% (24,169) of the genes were successfully annotated to the four major databases, while the highest homology was observed with Pomacea canaliculata. Functional annotation revealed that the majority of transcripts were enriched in metabolism, signal transduction and Immune-related pathways, and several candidate genes involved in drug metabolism and immune response were identified (e.g., CYP1A1, CYP2J, CYP2U1, GST, ,PIK3, PDE3A, PRKAG). This study lays a foundation for future molecular biology research and provides a reference for studying genes associated with the medicinal value of C. chinensis.

Comparison of gut microbiome in the Chinese mud snail (Cipangopaludina chinensis) and the invasive golden apple snail (Pomacea canaliculata).

Background: Gut microbiota play a critical role in nutrition absorption and environmental adaptation and can affect the biological characteristics of host animals. The invasive golden apple snail (Pomacea canaliculata) and native Chinese mud snail (Cipangopaludina chinensis) are two sympatric freshwater snails with similar ecological niche in southern China. However, gut microbiota comparison of interspecies remains unclear. Comparing the difference of gut microbiota between the invasive snail P. canaliculata and native snail C. chinensis could provide new insight into the invasion mechanism of P.canaliculata at the microbial level. Methods: Gut samples from 20 golden apple snails and 20 Chinese mud snails from wild freshwater habitats were collected and isolated. The 16S rRNA gene V3-V4 region of the gut microbiota was analyzed using high throughput Illumina sequencing. Results: The gut microbiota dominantly composed of Proteobacteria, Bacteroidetes, Firmicutes and Epsilonbacteraeota at phylum level in golden apple snail. Only Proteobacteria was the dominant phylum in Chinese mud snail. Alpha diversity analysis (Shannon and Simpson indices) showed there were no significant differences in gut microbial diversity, but relative abundances of the two groups differed significantly (P < 0.05). Beta diversity analysis (Bray Curtis and weighted UniFrac distance) showed marked differences in the gut microbiota structure (P < 0.05). Unique or high abundance microbial taxa were more abundant in the invasive snail compared to the native form. Functional prediction analysis indicated that the relative abundances of functions differed significantly regarding cofactor prosthetic group electron carrier and vitamin biosynthesis, amino acid biosynthesis, and nucleoside and nucleotide biosynthesis (P < 0.05). These results suggest an enhanced potential to adapt to new habitats in the invasive snail.

Comparison of the composition and function of gut microbes between adult and juvenile Cipangopaludina chinensis in the rice snail system.

Cipangopaludina chinensis is an important economic value snail species with high medicinal value. The gut microbes of aquatic animals plays a vital role in food digestion and nutrient absorption. Herein, we aimed at high-throughput sequencing of 16S rRNA to further investigate whether there were differences in the composition and function of gut microbes of adult and juvenile C. chinensis snails, as well as sediments. This study found that the microbial diversity of the sediment was significantly higher than that of the snails gut (P < 0.001), but there was no significant difference between the gut flora of adult and juvenile snails (P > 0.05). A total of 47 phyla and 644 genera were identified from all samples. Proteobacteria and Verrucomicrobia were the two dominant phyla in all samples, and overall relative abundances was 48.2% and 14.2%, respectively. Moreover, the relative abundances of Aeromonas and Luteolibacter in the gut of juvenile snails (30.8%, 11.8%) were higher than those of adults (27.7%, 10.6%) at the genus level (P > 0.05). Then, four indicator genera were found, namely Flavobacterium, Silanimonas, Geobacter and Zavarzinella, and they abundance in the gut of juvenile snails was significantly higher than that of adults (P < 0.05). This results imply the potential development of Silanimonas as a bait for juvenile snail openings. We observed that Aeromonas was the primary biomarker of the snail gut and sediments (P < 0.001), and it may be a cellulose-degrading bacteria. Function prediction revealed significantly better biochemical function in the snail gut than sediments (P < 0.001), but no significant differences in adult and juvenile snail (P > 0.05). In conclusion, studies show that the snail gut and sediment microbial composition differ, but the two were very similar. The microbial composition of the snail gut was relatively stable and has similar biological functions. These findings provide valuable information for in-depth understanding of the relationship between snails and environmental microorganisms.

Echinostoma macrorchis Metacercariae in Cipangopaludina chinensis malleata from Xiengkhuang Province, Lao PDR and Morphologies of Adults from Experimental Animals.

We identified the echinostome metacercariae in Chinese mystery snails, Cipangopaludina chinensis malleata, from Xiengkhuang Province, Lao PDR with morphologies of adult worms recovered. Total 20 snails were examined with artificial digestion method and then the collected metacercariae were orally infected to a mouse and a rat. Adult worms recovered from experimental animals were observed with a light microscope and a SEM. The metacercariae were round, 125×123 µm in average size, with a moderately thick cyst wall, collar spines distributed in the head collar and excretory granules in 2 canals of excretory tube. Adult flukes (3-week-old in a rat) were elongated, ventrally curved and 5.310×1.023 mm in average size. Head collar distinct, bearing 43 collar spines with 5 end group ones on each side. Oral sucker subterminal, prepharynx very short, pharynx well developed, and esophagus relatively short. Cirrus sac well developed, with a saccular seminal vesicle, and ventral sucker very large. Ovary round and on the median line of the body. Testes tandom and elongated. Eggs operculated, elliptical and 90×57 µm in average size. In the SEM observation, the head crown prominent, with 43 collar spines resembled with horns of younger stag. Scale-like tegumental spines were densely distributed on the surface between the head collar and ventral sucker, and their densities were decreased posteriorly. Conclusively, the metacercariae detected in C. chinensis malleata from Lao PDR were identified as those of Echinostoma macrorchis based on the morphological characteristics of adult worms.

Echinostoma macrorchis Metacercariae in Cipangopaludina chinensis malleata from Xiengkhuang Province, Lao PDR and Morphologies of Adults from Experimental Animals

We identified the echinostome metacercariae in Chinese mystery snails, Cipangopaludina chinensis malleata, from Xiengkhuang Province, Lao PDR with morphologies of adult worms recovered. Total 20 snails were examined with artificial digestion method and then the collected metacercariae were orally infected to a mouse and a rat. Adult worms recovered from experimental animals were observed with a light microscope and a SEM. The metacercariae were round, 125×123 μm in average size, with a moderately thick cyst wall, collar spines distributed in the head collar and excretory granules in 2 canals of excretory tube. Adult flukes (3-week-old in a rat) were elongated, ventrally curved and 5.310×1.023 mm in average size. Head collar distinct, bearing 43 collar spines with 5 end group ones on each side. Oral sucker subterminal, prepharynx very short, pharynx well developed, and esophagus relatively short. Cirrus sac well developed, with a saccular seminal vesicle, and ventral sucker very large. Ovary round and on the median line of the body. Testes tandom and elongated. Eggs operculated, elliptical and 90×57 μm in average size. In the SEM observation, the head crown prominent, with 43 collar spines resembled with horns of younger stag. Scale-like tegumental spines were densely distributed on the surface between the head collar and ventral sucker, and their densities were decreased posteriorly. Conclusively, the metacercariae detected in C. chinensis malleata from Lao PDR were identified as those of Echinostoma macrorchis based on the morphological characteristics of adult worms.

A comparative study of phenoloxidase activity between Cipangopaludina chinensis and Oncomelania hupensis / 中国血吸虫病防治杂志

Objective To study the differences of the phenoloxidase (PO) relative activity among ribbed shelled Oncomelania hupensis, smooth shelled O. hupensis and Cipangopaludina chinensis. Methods The crude PO fluid was extracted from the soft tissue of O. hupensis and C. chinensis by homogenation and centrifugation. The PO activity was detected with catechol as the substrate in the reaction systems. Results The PO relative activities in the ribbed shelled O. hupensis, smooth shelled O. hupensis and C. chinensis were (25.72 ± 2.27), (14.56 ± 1.24) U / mL and (13.72 ± 1.06) U / mL. The PO relative activity in the smooth shelled O. hupensis was higher than that in the ribbed shelled O. hupensis (q = 21.46, P < 0.05) and C. chinensis (q = 12.00, P < 0.05), while the difference between the PO relative activities of the latter two was not statistically significant (q = 1.62, P > 0.05) . Conclusion There is a difference in the relative PO activity between O. hupensis and C. chinensis, which may be related to the living environment of snails.

[A comparative study of phenoloxidase activity between Cipangopaludina chinensis and Oncomelania hupensis].

OBJECTIVE: To study the differences of the phenoloxidase (PO) relative activity among ribbed shelled Oncomelania hupensis, smooth shelled O. hupensis and Cipangopaludina chinensis. METHODS: The crude PO fluid was extracted from the soft tissue of O. hupensis and C. chinensis by homogenation and centrifugation. The PO activity was detected with catechol as the substrate in the reaction systems. RESULTS: The PO relative activities in the ribbed shelled O. hupensis, smooth shelled O. hupensis and C. chinensis were (25.72 ± 2.27), (14.56 ± 1.24) U / mL and (13.72 ± 1.06) U / mL. The PO relative activity in the smooth shelled O. hupensis was higher than that in the ribbed shelled O. hupensis (q = 21.46, P < 0.05) and C. chinensis (q = 12.00, P < 0.05), while the difference between the PO relative activities of the latter two was not statistically significant (q = 1.62, P > 0.05) . CONCLUSIONS: There is a difference in the relative PO activity between O. hupensis and C. chinensis, which may be related to the living environment of snails.

A comparative study of phenoloxidase activity between Cipangopaludina chinensis and Oncomelania hupensis / 中国血吸虫病防治杂志

Objective To study the differences of the phenoloxidase (PO) relative activity among ribbed shelled Oncomelania hupensis, smooth shelled O. hupensis and Cipangopaludina chinensis. Methods The crude PO fluid was extracted from the soft tissue of O. hupensis and C. chinensis by homogenation and centrifugation. The PO activity was detected with catechol as the substrate in the reaction systems. Results The PO relative activities in the ribbed shelled O. hupensis, smooth shelled O. hupensis and C. chinensis were (25.72 ± 2.27), (14.56 ± 1.24) U / mL and (13.72 ± 1.06) U / mL. The PO relative activity in the smooth shelled O. hupensis was higher than that in the ribbed shelled O. hupensis (q = 21.46, P < 0.05) and C. chinensis (q = 12.00, P < 0.05), while the difference between the PO relative activities of the latter two was not statistically significant (q = 1.62, P > 0.05) . Conclusion There is a difference in the relative PO activity between O. hupensis and C. chinensis, which may be related to the living environment of snails.

Echinostoma macrorchis (Digenea: Echinostomatidae): Metacercariae in Cipangopaludina chinensis malleata Snails and Adults from Experimental Rats in Korea.

A survey was performed to know the infection status of echinostome metacercariae in Cipangopaludina chinensis malleata snails from Korea. Total 75 snails collected in 5 localities, i.e., Imsil-gun, Jeollabuk-do, Hwasun-gun and Shinan-gun (Aphae and Jido), Jeollanam-do, and Jinju-si, Gyeongsangnam-do, were examined for metacercariae by the artificial digestion method. Infection rates of metacercariae were 80.0%, 66.7%, 100%, 60.0%, and 73.3%, and their densities were 39, 32, 183, 19, and 30 per snail infected, respectively. The metacercariae were round, 105-118×105-118 µm in size, with a thin cyst wall, collar spines on the head collar, and excretory granules in 2 canals of excretory tube. Adult flukes were elongated, ventrally curved, and 5,167×939 µm in average size. Head collar distinct, bearing 45 collar spines with 5 end groups on each side. Oral sucker subterminal, pharynx well developed, and esophagus somewhat short. Cirrus sac well developed, with a saccular seminal vesicle, and ventral sucker very large. Ovary elliptical and on the median line of the body. Testes tandem and slightly lobed. Eggs operculated, elliptical, and 90-103×55-60 µm in size. By scanning electron microscopy, the head collar was prominent with 45 collar spines resembling horns of younger stags. Scale-like tegumental spines were densely distributed on the body surface between the head collar and ventral sucker. Conclusively, it has been first confirmed that the life cycle of E. macrorchis is indigenously maintained in Korea, and C. chinensis malleata snails are popularly infected with the metacercariae of this echinostome.

Anti-inflammatory and anti-angiogenic activities of a purified polysaccharide from flesh of Cipangopaludina chinensis.

The purpose of this paper was to investigate the anti-inflammatory and anti-angiogenic activities of a purified polysaccharide (CCPSn) from flesh of Cipangopaludina chinensis in lipopolysaccharide (LPS) activated RAW264.7 macrophages and human umbilical vein endothelial cells (HUVECs), respectively. Anti-inflammatory results showed that CCPSn not only greatly decreased the pro-/anti-inflammatory cytokine secretion ratios, including TNF-α/IL-10, IL-6/IL-10 and IL-1ß/IL-10, but also reduced release levels of nitric oxide (NO) and prostaglandin E2 (PGE2), and suppressed expressions of cyclooxygenase2 (COX2) and inducible nitric oxide synthase (iNOS). The results from anti-angiogenic activities revealed that proliferation, migration, tube formation and vascular endothelial growth factor (VEGF) of HUVECs were significantly inhibited by CCPSn treatment. Even more remarkable was the fact that anti-inflammatory and anti-angiogenic activities of CCPSn exhibited a clear dose-response manner, and these activities were irrelevant to the cytotoxicities of CCPSn to RAW264.7 macrophages and HUVECs. These results indicated that CCPSn possessed strong anti-inflammatory and anti-angiogenic activities.

Lifelong neurogenesis in the cerebral ganglion of the Chinese mud snail, Cipangopaludina chinensis.

INTRODUCTION: A small group of Gastropods possessing giant neurons have long been used to study a wide variety of fundamental neurophysiological phenomena. However, the majority of gastropods do not have large neurons but instead have large numbers of small neurons and remain largely unstudied. We explored neuron size and rate of increase in neuron numbers in the Chinese mud snail, Cipangopaludina chinensis. METHODS: Using histological sections and whole mounts of the cerebral ganglia, we collected cross-sectional data on neuron number and size across the lifespan of this animal. Neurogenesis was verified using Click-it EdU staining. RESULTS: We found that total neuron number in the cerebral ganglia increases throughout the lifespan of this species at a constant rate. New neurons arise primarily near the nerve roots. Females live longer (up to 7 years) than males (up to 5 years) and thus achieve larger numbers of neurons in the cerebral ganglion. Neuron size is consistently small (<10 µm) in the cerebral ganglia at all ages, however, cells in the posterior section of the cerebral ganglia are modestly but significantly larger than cells at the anterior. CONCLUSIONS: These features suggest that C. chinensis and similar species of Caenogastropoda are good candidates for studying gastropod neurogenesis, senescence, and sex differences in the nervous system.

Echinostoma macrorchis (Digenea: Echinostomatidae): Metacercariae in Cipangopaludina chinensis malleata Snails and Adults from Experimental Rats in Korea

A survey was performed to know the infection status of echinostome metacercariae in Cipangopaludina chinensis malleata snails from Korea. Total 75 snails collected in 5 localities, i.e., Imsil-gun, Jeollabuk-do, Hwasun-gun and Shinan-gun (Aphae and Jido), Jeollanam-do, and Jinju-si, Gyeongsangnam-do, were examined for metacercariae by the artificial digestion method. Infection rates of metacercariae were 80.0%, 66.7%, 100%, 60.0%, and 73.3%, and their densities were 39, 32, 183, 19, and 30 per snail infected, respectively. The metacercariae were round, 105–118×105–118 μm in size, with a thin cyst wall, collar spines on the head collar, and excretory granules in 2 canals of excretory tube. Adult flukes were elongated, ventrally curved, and 5,167×939 μm in average size. Head collar distinct, bearing 45 collar spines with 5 end groups on each side. Oral sucker subterminal, pharynx well developed, and esophagus somewhat short. Cirrus sac well developed, with a saccular seminal vesicle, and ventral sucker very large. Ovary elliptical and on the median line of the body. Testes tandem and slightly lobed. Eggs operculated, elliptical, and 90–103×55–60 μm in size. By scanning electron microscopy, the head collar was prominent with 45 collar spines resembling horns of younger stags. Scale-like tegumental spines were densely distributed on the body surface between the head collar and ventral sucker. Conclusively, it has been first confirmed that the life cycle of E. macrorchis is indigenously maintained in Korea, and C. chinensis malleata snails are popularly infected with the metacercariae of this echinostome.

Characterization of a novel purified polysaccharide from the flesh of Cipangopaludina chinensis.

The purpose of this paper was to investigate the characterization of a novel polysaccharide from the flesh of Cipangopaludina chinensis, named CCPSn. The results found CCPSn was a white powder, readily soluble in hot water and slightly soluble in water. CCPSn was a homopolysaccharide composed of D-glucose (D-Glc) with molecular weight of 91.1 kDa. Based on analysis of UV-visible, FT-IR, periodic acid oxidation, Smith degradation, methylation, GM-MS and NMR, the structure of CCPSn was elucidated as follows: the backbone was composed of (1 → 3) linked α-D-Glc. The branches, consisting of a single (1 → 3) linked α-D-Glc units and terminal α-D-Glc-4-O-SO3(-), were attached to the main chain at C-4 positions. The degree of branching was calculated to be about 16.73%. The C-1 of terminal α-D-Glc-4-O-SO3(-) was linked to O-3 of (1 → 3) linked α-D-Glc in the branches. In addition, the results indicated CCPSn was a sulfated polysaccharide with the sulfate radical content of 9.12%.

Purification, characterization and immunostimulatory activity of polysaccharide from Cipangopaludina chinensis.

In this study, we investigated the purification, preliminary characterization and immunostimulatory activity in vivo of polysaccharide from Cipangopaludina chinensis (CCPS). Firstly, crude CCPS was prepared by hot water extraction. And the crude CCPS was sequentially purified by chromatography of DEAE-52 and Sephadex G-100, resulting in two purified fractions of CCPS-1 and CCPS-2. We found the two fractions were homogeneous heteropolysaccharides mainly composed of rhamnose and glucose with the average molecular weight of 226 and 235 kDa, respectively. CCPS-2 was quite different from CCPS-1. It had much higher content of uronic acid and sulfuric radical. For immunostimulatory activity in vivo, crude CCPS could significantly increase the thymus and spleen indices, enhance the macrophage function, and increase the level of serum hemolysin in cyclophosphamide-treated mice, suggesting CCPS had a potent immunostimulatory activity and could be explored as a potential natural immunomodulatory agent.

Preliminary characterization and potential hepatoprotective effect of polysaccharides from Cipangopaludina chinensis.

In the present study, we investigated the preliminary characterization, in vitro antioxidant and in vivo heptoprotective activities of polysaccharides from Cipangopaludina chinensis (CCPS). The results of chemical and gas chromatography analysis indicated that CCPS was mostly composed of glucose with high contents of uronic acid and sulfate. For antioxidant activities in vitro, CCPS showed medium lipid peroxidation inhibition effect and high Fe²âº chelating and hydroxyl radical scavenging activities. For hepatoprotective activity in vivo, the administration of CCPS significantly decreased the serum levels of alanine aminotransferase and aspartate aminotransferase, inhibited the formation of malondialdehyde in liver and tumor necrosis factor-alpha (TNF-α) in serum and restored the liver activities of superoxide dismutase, glutathione peroxidase in BCG/LPS-induced immunological liver injury mice. The results suggested that CCPS had a significant protective effect against BCG/LPS-induced immunological liver injury. The hepatoprotective effect of CSPS might be partly due to its immunoregulatory effect by inhibiting TNF-α production and antioxidant activities to protect biological systems against the oxidative stress, which were dependent on the chemical and structural properties of CCPS. Further work on the structure of CCPS is in progress.