Transcriptome analysis to explore the molecular mechanisms involved in the dormancy-arousal process in Pomacea canaliculata

doi:10.21203/rs.3.rs-4941305/v1

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Transcriptome analysis to explore the molecular mechanisms involved in the dormancy-arousal process in Pomacea canaliculata

https://doi.org/10.21203/rs.3.rs-4941305/v1

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The apple snail (Pomacea canaliculata), a freshwater snail listed as a pernicious invasive alien species by the World Conservation Union (IUCN), has caused serious agricultural and ecological harm worldwide. The species enters dormancy under extreme environmental stress and does not lift the dormant state until the environment is suitable, which is an important survival strategy. In order to investigate its survival mechanism under environmental stress conditions, the present study was carried out to investigate the response of apple snails to air exposure stress through air exposure stress treatment and transcriptome analysis, using apple snails living normally under water conditions as control (CK), and to excavate the relevant mechanisms regulating their drought tolerance, dormancy and arousal. The results showed that the 5-, 15- and 30-day air-exposure stress treatment groups (DRY05, DRY15 and DRY30) showed a general down-regulation of metabolism-related pathways, including starch and sucrose metabolism, linoleic acid metabolism, glutathione metabolism and glycosaminoglycan degradation, compared with the CK group. Moreover, Apoptosis, intercellular adhesion, insulin resistance, and immune status of apple snails were also significantly changed during dormancy. These changes help the apple snail to reduce energy expenditure and maintain vital activities. In addition, this study found that pathways related to cell cycle, immune signalling and intercellular adhesion were significantly affected when apple snails enter dormancy (DRY01) and arousal (RCY01). This study provides a reference for understanding the tolerance of apple snails to extreme environments, and provides a basic theory for apple snail biocontrol research.

Biological sciences/Computational biology and bioinformatics

Biological sciences/Molecular biology

Biological sciences/Physiology

transcriptomics

air-exposure

dormancy

awakening

apple snail

The apple snail (Pomacea canaliculata) is a large freshwater snail that typically inhabits rivers, ditches, and rice fields in the tropics and subtropics ¹. Due to its rapid reproduction ability, strong environmental adaptability and the habit of migrating with the water flow, coupled with the trend of global warming, the distribution range of the apple snail has spread from the tropical regions to temperate regions ². The apple snail has a unique "gill-lung" respiratory system, which can not only breathe with gills in the water, but also exchange gas in the air through the lung ³. This snail has a wide range of food habits and a large food intake, and often eats aquatic crops such as lotus root, rice and wild rice, which not only poses a threat to agricultural production activities, but also seriously persecutes the ecological niche of local snails, causing great damage to the local ecosystem and species diversity ⁴. In addition, apple snails have a large number of internal parasites, and people can suffer from serious health problems such as eosinophilic meningoencephalitis when they accidentally ingest apple snails parasitised by zoonotic parasites ⁵. In view of its harmful effects on the ecosystem and human health, the International Union for Conservation of Nature (IUCN) recognised the apple snail as one of the world's 100 pernicious invasive alien species in 2000 ⁶ .

When faced with extreme environments, the apple snail makes a series of stress strategies to adapt to the environmental stresses by making adjustments at multiple levels: behavioural, physiological and biochemical. Air exposure stress refers to exposing aquatic animals to air away from the water body, which will cause water imbalance in the aquatic organism ⁷. The apple snail, when sensing the absence of water in the environment, rapidly secretes mucus to close the shell opening, stops feeding and and all activities, and enters a dormant state ⁸. This state can pose new challenges to the survival of the apple snail, including oxidative stress, accumulation of toxic metabolites, muscle atrophy due to prolonged inactivity, and degradation of internal organs due to prolonged starvation ⁹. This survival strategy has been found in several animals, including amphibians, reptiles, mollusks, and fish, which survive long periods of dormancy by suppressing metabolic rate and respiration to reduce water loss and energy expenditure ⁸. For example, the African clawed frog (Xenopus laevis) enters a state of dormancy by burying itself in the soil and remaining still in the face of seasonal droughts in lake habitats. During this period, African clawed frogs face severe dehydration, losing 25 to 30% of their body water, resulting in increased blood concentration and hypoxic stress due to impeded oxygen transport, and then shift from aerobic to anaerobic metabolism to meet the energy requirements of dormant hypoxic conditions. Although prolonged resting as well as starvation during dormancy can lead to atrophy of muscles, liver and intestine. However, the liver and muscle tissues remain intact and are able to resume normal activity at the end of dormancy ^10,11. The pulmonate snail (Chondrina avenacea) lives on the surface of rock walls and is exposed to environmental drought and waterlessness during the hot summer months when they go directly into dormancy. The coat membrane tightly closes the shell opening to remain highly impermeable to water during dormancy, and the mouth cap formed by mucopolysaccharides secreted by the coat membrane tightly seals the shell opening to further reduce water evaporation, and energy is conserved by intermittent opening of the respiratory pores for gas exchange and by reducing the metabolic rate to 30% of the normal state ^12,13.

Past studies on the tolerance of the apple snail to air exposure have focused on physiology, biochemistry, and behavior ¹⁴. Studies have been conducted to induce dormancy in apple snails by air exposure stress, observed behavioral changes and examined the peroxide and antioxidant contents ^8,15. The results of the study revealed that uric acid acts as a non-enzymatic antioxidant during dormancy in apple snails. In addition, superoxide dismutase and catalase, two key enzymes for defense against oxidative stress, continued to function throughout the dormancy period. By treating apple snails with air exposure, Guo et al. found that air exposure had an inhibitory effect on the growth of apple snails and that the activities of digestive enzymes and antioxidant enzymes were enhanced in apple snails ¹⁶.

Transcriptome sequencing, a technique for sequencing and analyzing mRNAs in tissues or cells using high-throughput sequencing technology, can help to gain a deeper understanding of the gene transcription of the apple snail under air-exposure treatment conditions ^17,18. In this study, we analyzed the gene transcripts of apple snails at different periods under air exposure to explore the tolerance-related genes and preliminarily analyzed the regulatory mechanism of their air exposure tolerance. Meanwhile, we also studied gene transcripts before and after exposure to air and before and after return to water as a means of uncovering the genes and signalling pathways involved in making the apple snail dormant as well as awakening it. This provides a theoretical basis for the study of apple snail biological defense and control, reduces the reproduction rate of Fusiliers, mitigates the harm to the ecological environment, and is of great practical significance for the scientific prevention and control of apple snail invasion. It also provides a reference for understanding the tolerance of other mollusks to extreme environments.

2.1. Gene Function Annotation

The results of gene function annotation of the apple snail gene sequences (ASM307304v1) in the Interpor, GO, eggNOG, KOG, Swissprot, Nr, Pfam, and KEGG databases are shown in Table S1. A total of 17,423 sequences were annotated, accounting for 95.40% of the total gene count. Among them, 17,350 sequences were annotated by Nr database, with an annotation rate of 95.00%, followed by InterPro, eggNOG, Pfam, GO, Swissprot, KOG, and KEGG databases in the order of the number of sequences getting annotated were 14,664 (80.29%), 13,858 (75.88%), 12,890 (70.58%), 12,774 (69.94%), 11,806 (64.64%), 9,440 (51.69%), 8,112 (44.42%).

2.2. Mechanisms of drought tolerance in the apple snail

2.2.1. Assessment of correlation between samples

The degree of correlation between the samples was assessed using principal component analysis and heatmap (Figure S1). The results showed good intra-group correlations with reliable biological replicates, poor inter-group correlations and significant differences between groups.

2.2.2. Differentially expressed genes analysis

The CK group was compared with the other groups and named DRY01 vs CK, DRY05 vs CK, DRY15 vs CK, DRY30 vs CK, RCY01 vs CK (Fig. 1A). To investigate the drought tolerance mechanism of apple snails under air exposure stress, we analyzed the differentially expressed genes (DEGs) between the CK group and three different air exposure duration groups (DRY05, DRY15, DRY30), respectively. A total of 5446 DEGs were screened in DRY05 vs CK; a total of 3278 DEGs were screened in DRY15 vs CK; a total of 9527 DEGs were screened in DRY30 vs CK; the number of DEGs shared by DRY05 vs CK, DRY15 vs CK, and DRY30 vs CK, was 1521 (Figure S2).

2.2.3. Functional enrichment analysis

KEGG enrichment results showed that 1521 shared DEGs were mainly enriched in Biosynthesis of secondary metabolites, Metabolism of xenobiotics by cytochrome P450, Drug metabolism – cytochrome P450, Adherens junction, Starch and sucrose metabolism, Linoleic acid metabolism, Glutathione metabolism, Adipocytokine signaling pathway, Glycosaminoglycan degradation, alpha-Linolenic acid metabolism, Caffeine metabolism, Pentose phosphate pathway, and Phenylpropanoid biosynthesis (Fig. 1B).

GSEA results showed that Adherens junction, Drug metabolism – cytochrome P450, Drug metabolism – other enzymes, Linolenic acid metabolism Metabolism of xenobiotics by cytochrome P450, alpha-Linolenic acid metabolism, Caffeine metabolism, Phenylpropanoid biosynthesis, and Starch and sucrose metabolism in the above three groups compared to CK group, were significantly down-regulated (Fig. 1C). The genes enriched in these 10 pathways in relation to the samples are shown in Fig. 1D and Table S2.

2.2.4. Weighted correlation network analysis

The soft threshold value of 16 is chosen for subsequent scale-free network construction (Figure S3). The gene cluster dendrogram is shown in Fig. 2A. All genes were classified into 49 modules, and the modules with an absolute value of module-sample correlation greater than 0.6 were filtered as key modules, which were MElightgreen, MEplum1, MEmagenta, and MEskyblue3 (Fig. 2B-C).

The results of the functional enrichment analysis of the genes in the key modules are shown in Fig. 3A-B. The results showed that the genes in the key modules were mainly enriched in pathways related to the immune system and immune signalling, including Toll-like receptor signaling pathway, NF-kappa B signaling pathway, TNF signaling pathway, IL − 17 signaling pathway, B cell receptor signaling pathway, and T cell differentiation etc. The toll-like receptor signaling pathway and Adherens junction were significantly down-regulated in all groups compared to the CK group (Fig. 3C). The heatmap of the correlation between genes (in the Toll-like receptor signaling pathway) and samples is shown in Fig. 3D. Genes in the key sub-network obtained using MCODE analysis included CASP2, CASP10 and TRAF3, etc (Fig. 3E).

2.3. Mechanisms of dormancy and awakening in apple snails

2.3.1. DEGs analysis

To investigate the mechanisms by which the apple snail enters dormancy under air-exposure stress and awakens in water, we analyzed DEGs between the DRY01 and CK groups (DRY01 vs CK) and between the RCY01 and DRY30 groups (RCY01 vs DRY30), respectively (Fig. 4A). A total of 443 DEGs were screened in DRY01 vs CK; a total of 4297 DEGs were screened in RCY01 vs DRY30 (Figure S4).

2.3.2. GO enrichment analysis

GO enrichment analysis showed that DEGs in DRY01 vs CK mainly acted in cilium organization, lipid catabolic process, cilium assembly, plasma membrane bounded cell projection assembly, cell projection assembly, and microtubule cytoskeleton organization, and most of the DEGs in DRY01 group were up-regulated compared to the CK group. The DEGs in RCY01 vs DRY30 mainly acted in cilium assembly, cilium organization, microtubule bundle formation, axoneme assembly, plasma membrane bounded cell projection assembly, intraciliary transport, and cell projection assembly, and most of the DEGs in RCY01 group were down-regulated compared to the DRY30 group (Fig. 4B).

2.3.3. Gene Set Enrichment Analysis

Gene set enrichment analysis (GSEA) obtained a total of 11 KEGG pathways which were significantly down-regulated in DRY01 vs CK and up-regulated in RCY01 vs DRY30, including Toll-like receptor signaling pathway, Regulation of actin cytoskeleton, Proteoglycans in cancer, NOD-like receptor signaling pathway, NF-kappa B signaling pathway, Necroptosis, Influenza A, Hepatitis C, Focal adhesion, Cell adhesion molecules, and Adherens junction. In addition, seven KEGG pathways significantly up-regulated in DRY01 vs CK and down-regulated in RCY01 vs DRY30 were obtained for Vitamin digestion and absorption, Protein processing in endoplasmic reticulum, Proteasome, Meiosis - yeast, Cell cycle - yeast, Cell cycle and ABC transporters (Fig. 4C).

GSEA analysis based on GO showed that in the DRY01 vs CK, cell cycle checkpoint signaling, regulation of cell cycle phase transition, G1/S transition of mitotic cell cycle, G2/M transition of mitotic cell cycle, and regulation of G2/M transition of mitotic cell cycle were up-regulated; activation of innate immune response, I-kappaB phosphorylation, regulation of T cell activation, regulation of immune effector process, and regulation of cytokine production were down-regulated; cell adhesion, cell-cell adhesion, positive regulation of cell adhesion, positive regulation of cell-cell adhesion, and regulation of cell-cell adhesion were also down-regulated. The result in RCY01 VS DRY30 is the opposite (Fig. 4D).

2.4. Validation of transcriptome data using qPCR

The real-time quantitative PCR (qPCR) validation results are shown in Fig. 5. The results showed that the qPCR results of the 8 DEGs matched with the RNA-seq results, so the results of the sequencing analysis had high confidence.

Air exposure is a phenomenon in which aquatic organisms are removed from the water environment and exposed to air. To survive the stress of air exposure, some aquatic organisms rapidly enter a state of dormancy ^19–21. Dormancy is a common physiological state in amphibians, reptiles, invertebrates, mollusks, echinoderms, and fish in response to extreme environmental stresses such as high or low temperatures, drought, or food shortages. The existence of dormancy in some organisms as early as 200 million years ago can be demonstrated by fossil evidence from Pleistocene earthworm burrows, Devonian to Cretaceous lungfish burrows, Permian earthworm burrows, and Permian to Triassic bivalve burrows ²². Apple snails tend to protect themselves from air exposure stress by entering dormancy. In this study, we induced dormancy in apple snails by air exposure and comparatively analyzed the differences in gene transcript levels in the liver tissue of apple snails in the CK group and at 5 (DRY05), 15 (DRY15), and 30 days (DRY30) of dormancy to explore the mechanisms of drought tolerance during dormancy in apple snails. KEGG enrichment analysis of shared DEGs revealed that these genes were significantly enriched in metabolism-related pathways, such as Starch and sucrose metabolism, Linoleic acid metabolism, Glutathione metabolism, Glycosaminoglycan degradation, etc. While in GSEA similarly several metabolism-related pathways were found to be significantly up-regulated in the control group compared to the experimental group. This can be interpreted as an indication that the metabolic functions of the apple snail are suppressed during dormancy, and the organism reduces energy consumption by lowering the metabolic rate to ensure that it can maintain prolonged life activities without external water and food sources ^23–25. Linoleic acid is one of the basic components of cell membranes ²⁶. Down-regulation of linoleic acid metabolism may contribute to the maintenance of normal cell morphology during dormancy in apple snails. However, the down-regulation of linoleic acid metabolism, as a precursor of arachidonic acid, also implies that neural development and growth will be affected ²⁷. Abnormalities in Glycosaminoglycan degradation and Glutathione metabolic pathways during dormancy in apple snails are of interest. Glycosaminoglycans have a variety of biological functions. For example, they are an important component of the extracellular matrix and are hydrophilic, which provides structural support for cells and retains water in cells ^28,29. When animals are dormant, metabolic activities are inhibited and the synthesis of biomolecules such as glycosaminoglycans is slowed down accordingly. Therefore, down-regulation of the glycosaminoglycan degradation pathway in the apple snail under the stress of air exposure contributes to the maintenance of intracellular glycosaminoglycan content, which can help the apple snail to maintain the stability of the cytoplasmic matrix and the storage of intracellular water. In addition, glycosaminoglycans have antioxidant properties that scavenge excess free radicals from the body ³⁰. Glutathione is also an antioxidant and plays an important role in the proper functioning of the immune system by resisting oxidative damage and integrating detoxification ³¹.

WGCNA results showed that air exposure stress significantly affected apoptosis, cell-cell adherens junction, insulin resistance and immune status in apple snails. Caspases are the main enzymes that execute apoptosis ³². In this study, CASP2, CASP10 and TRAF3 associated with apoptosis were screened as hub genes. Apple snails are starved for a long time during dormancy and need to consume their own stored energy substances to maintain their life activities, a process that may be accompanied by damage to body tissues and further lead to organ degeneration. Previous studies have shown that organs such as the gastrointestinal tract, liver and pancreas of the sea cucumber (Apostichopus japonicus) deteriorate during dormancy due to prolonged periods of non-feeding, which is closely related to apoptosis ^33,34. The onset of apoptosis is influenced by many interrelated processes. The immune system and immune signalling are involved in the regulation of apoptosis ³⁵. In the present study, significant differences in immune status were found between the control group and all other groups, and Toll-like receptor signaling pathway was screened to be significantly down-regulated in all experimental groups compared to the control group in the GSEA results. Toll-like receptor activation can further activate downstream nuclear factor kappa-B or interferon regulatory factors through a myeloid differentiation primary response protein 88-dependent pathway, which in turn induces the secretion of cytokines such as tumor necrosis factor, interleukin, and interferon, and thus exerts immune functions ³⁶. This process is significantly down-regulated during dormancy in apple snails and may be related to the level of immune cells in apple snails. The vital activities of immune cells require a lot of energy, and the reduction of autoimmune levels during dormancy may help the apple snail to achieve conservation of energy consumption ³⁷. Changes in insulin resistance are of equal interest. Many dormant animals such as the African lungfish (Protopterus annectens) increase their fat reserves prior to dormancy and rely on these reserves to provide them with metabolic fuel during dormancy, possibly through the mechanism of insulin resistance, which regulates the uptake and oxidation of glucose, promotes the process of gluconeogenesis in the liver and inhibits anabolism throughout the body to conserve energy ^38–40. In this study, the expression of PEPCK (Phosphoenolpyruvate carboxykinase; P._canaliculata11272) was significantly lower in the control group than in the groups during apple snail dormancy. PEPCK is the rate-limiting enzyme of hepatic and renal gluconeogenesis and a key regulator of the tricarboxylic acid cycle ^41,42. Elevated PEPCK expression during dormancy implies that more non-glycans may be converted to glucose in the organism via the gluconeogenesis. In addition, another gene in the insulin resistance pathway, CPT1A (carnitine O-palmitoyltransferase 1; P._canaliculata05988), was found to be significantly more expressed in the control group than in the groups during dormancy of apple snails in this study. CPT1A generates acylcarnitines by catalysing the binding of long-chain acyl coenzyme A to carnitine. This step is the first step in driving fatty acids across the mitochondrial membrane from the cytoplasmic matrix into the mitochondria for β-oxidation ⁴³. The low expression of CPT1A during dormancy corresponds to the low metabolic state of the apple snail.

In order to explore the molecular mechanisms underlying the entry into dormancy and the awakening of apple snails after rehydration, the present study comparatively analyzed the differences in gene transcript levels between the CK group and the DRY01 group shortly after entry into dormancy, as well as between the DRY30 group, which had been dormant for 30 days, and the RCY01 group, which had awakened after dormancy. The results show that functions related to cell cycle, immune status and intercellular adhesion are significantly affected when apple snails enter dormancy or awakening. Specifically, upon entering dormancy, the cell cycle-related functions of the apple snail were up-regulated, and the immune state and intercellular adhesion-related functions were down-regulated, whereas upon awakening the cell cycle-related energy was down-regulated and the immune state and intercellular adhesion-related functions were up-regulated. The relationship between the cell cycle and dormancy is complex and subtle. The cell cycle is a process of cell growth and division that includes multiple phases, whereas dormancy is a cellular state in which cells temporarily stop proliferating but remain alive ^44,45. It has been shown that the dormant state of tumour cells is associated with specific phases of the cell cycle ⁴⁶. Changes in the cell cycle during dormancy-awakening in apple snails may be due to specific cellular responses to stress, which needs to be explored in further studies. In addition, the present study suggests that the apple snail rapidly enters a hypometabolic state by significantly lowering its immune level shortly after entering dormancy, and that the immune level rapidly rebounds upon awakening in the water. While previous studies have generally concluded that normal sleep induces inflammatory activity, which strengthens the body's resistance to pathogens, dormancy due to air exposure in the apple snail does not appear to be the same as normal sleep, leading to the results of this study ^47–49. In the present study, the changes in function related to intercellular adhesion when the apple snail enters dormancy and when it rehydrates and awakens may be due to the fact that the dormant state requires a reduction in energy expenditure, and that reducing intercellular adhesion reduces the amount of energy required by the cell to maintain these adhesions. At the same time, the reduction of intercellular adhesion also helps the cells to change their state quickly when needed in order to reactivate quickly in response to appropriate signals ⁵⁰. In some cases, the reduction of intercellular adhesions due to dormancy also causes the cell to lose polarity, and the loss of cell polarity helps the cell to enter a more quiescent state ⁵¹.

In summary, this study revealed the mechanism of air exposure tolerance in apple snails by inducing dormancy through air exposure and analyzing gene expression differences at different time points. It was found that the metabolic functions of apple snails were suppressed during dormancy, especially the metabolic pathways related to starch and sucrose metabolism, linoleic acid metabolism, glutathione metabolism, and glycosaminoglycan degradation. In addition, dormancy is accompanied by significant changes in apoptosis, intercellular adhesion, insulin resistance and immune status. These changes help apple snails to reduce energy expenditure and sustain life activities in resource-poor environments. By comparing transcript levels before and after dormancy and before and after awakening, it was found that pathways related to cell cycle, immune status, and intercellular adhesion play key regulatory roles. These changes may be a specific cellular response to air exposure stress. The dormancy-awakening process of the apple snail involves complex molecular regulatory mechanisms that contribute to its survival in adversity.

5.1. Sample collection

The channeled apple snails were collected from the suburbs of Hangzhou (Zhejiang, China), and kept in tanks with water changed every two days and fed with fresh greens. After a period of feeding, 40 apple snails of similar weight and size were selected and placed in a dry tank for air exposure. After a day, the apple snails stood still and went into dormancy. The air exposure phase lasted for 30 days, after which the apple snails were resubmerged in water.

Samples were taken on four days (days 1, 5, 15, and 30) of the air exposure phase and one day after resubmerged in water. Three apple snails were randomly selected for each sampling, their livers removed and grouped in chronological order of sampling as DRY01, DRY05, DRY15, DRY30, and RCY01. In addition, livers from three non-air exposed apple snails were taken as control samples (CK). All livers were rapidly frozen using liquid nitrogen at the time of sampling and later stored in a refrigerator at a temperature set to − 80 ℃. .

5.2. Total RNA extraction

Total RNA was extracted from 18 liver tissue samples of apple snails using TRIzol (Invitrogen, USA) reagent in the following steps: 1) Take the liver tissue in a centrifuge tube, add 1 ml Trizol reagent, and homogenise thoroughly, leave for 5 min at room temperature; 2) Centrifuge at 10000 g at 4 ℃ for 10 min, take the supernatant; 3) Add 0.2 ml of chloroform, shake vigorously, and incubate for 3 min at room temperature; 4) Centrifuge the mixed sample at 12000 g for 10 min at 4°C and aspirate the supernatant; 5) Add 0.5 ml isopropanol to the supernatant, mix the liquid in the tube gently, and leave it at room temperature for 10 min; 6) Centrifuge at 12000 g for 10 min at 4°C. Remove the supernatant and retain the precipitate; 7) Add 1 ml of 75% pre-cooled ethanol to the precipitate and shake; 8) Centrifuge at 12000 g for 5 min at 4°C, remove the supernatant and retain the precipitate; 9) Add 20 µl of DEPC-treated double-distilled deionized water to dissolve RNA and incubate at 60 ℃ for 5 min; 10) RNA integrity was detected using an Agilent 2100 (Agilent, USA) instrument; sample RNA integrity was analyzed by an agarose gel electrophoresis test, and RNA concentration and purity were detected using a NanoDrop (NanoDrop, USA) instrument.

5.3. Transcriptome analysis

5.3.1. Analysis of sequencing data

Total RNA samples were sent to the Frasergen Information Company (Wuhan, China) for sequencing. Raw reads obtained from sequencing were weeded out of spliced and low-quality reads by the Trimomatic v0.39 tool to obtain high-quality clean reads. Functional prediction was performed by downloading the reference genome of the apple snail (ASM307304v1) at the National Center for Biotechnology Information (NCBI), and all protein sequences of the apple snail reference genome were aligned with Nr, InterPor, GO, eggNOG, KOG, Swissprot, Pfam, KEGG and other databases to obtain gene function and pathway annotations. HISAT2 v2.0.4 software was used to compare the clean reads to the reference genome of the apple snail, and Samtools v1.20 tool was used to organize the alignment hits in the order of the reference sequences. Based on the alignment results, the gene expression was estimated with the help of StringTie v1.3.4 software, and the number of read segments located in the exons of the genes after alignment and the FPKM values were extracted from the results using Stringtie and Ballgown.

5.3.2. Differential expression analysis

Differential expression analysis using Bioconductor's R package DESeq2: the Read counts matrix was entered, the grouping information was constructed as a condition vector, and the DESeqDataSet object was constructed, including the normalisation of expression values, and the DESeq function was used to calculate the fold change and significance p-values of genes. Input Read counts matrix, construct the condition vector from the grouping information, construct the DESeqDataSet object, including the normalization of the expression values, and use the DESeq function to calculate the gene's differential fold change and significance P value. Genes where |log₂FoldChange| > 1 and P adjust < 0.05 can be defined as DEGs.

5.3.3. Functional enrichment analysis

GO enrichment analysis and KEGG enrichment analysis of DEGs using the DAVID database (https://david.ncifcrf.gov/). GSEA used GSEA software and the MSigDB database. P < 0.05 and FDR < 0.25 indicate statistical significance.

5.3.4. Weighted correlation network analysis

WGCNA is mainly carried out using the WGCNA package in R programming language. Calculate the soft threshold using the pick Soft Threshold () function; choose a appropriate soft threshold based on the analysis of network topology for various soft thresholding powers; use the blockwiseModules() function for network construction and module identification, setting minModuleSize = 30, deepSplit = 3, mergeCutHeight = 0.1; modules were sorted and gene cluster dendrogram was drawn. The key modules were filtered by calculating the correlation between the modules and the sample-info, and then the key modules were analyzed for functional enrichment.

5.3.5. Protein-protein interaction network construction

Protein-protein interaction network was analyzed by applying the interactions in the STRING protein interaction database (http://string-db.org). The results were visualised using cytoscape software. Key subnetworks and genes are screened by the MCODE plugin.

5.4. Real-time quantitative PCR

To confirm the reliability of the transcriptome analysis results, we analyzed the relative expression of the 8 DEGs in all groups using qPCR. The methods of qPCR experiments and calculation of relative gene expression are the same as those of Pang et al ⁵². The log₂(2^−ΔΔCT/2^−ΔΔCT) was calculated to compare with the log₂FoldChang value in the transcriptome results. Information on the primers is given in Table S3.

Ethics statement

The animal study protocol was approved by the Institutional Review Board of Yancheng Teachers University.

Data availability

The datasets generated and/or analysed during the current study are available in the SRA database, [PRJNA1153304].

Acknowledgements

We would like to thank Yancheng Teachers University for providing the experimental instruments and technical support for the Jiangsu Provincial Key Laboratory of Coastal Wetland Biological Resources and Environmental Protection.

Author contributions

Gang Wang designed and took part in the whole process of the experiment; Gang Wang and Rongchen Liu wrote the draft of this manuscript; Chijie Yin and Yu Chen co-conceived the experiment; Aobo Pang and Qiuting Ji revised the draft critically for important intellectual content; Mengjun Wei, Hao Gao, Yutong Shen, Fang Wang, Shouquan Hou, Huabin Zhang and Senhao Jiang participated in the experiments; Boping Tang, Lianfu Chen and Daizhen Zhang revised the first draft. All authors have read and approved the final manuscript.

Funding

This research was financially supported by the National Natural Science Foundation of China [32270487, 32070526].

Competing interests

The authors declare no competing interests.

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Transcriptome analysis to explore the molecular mechanisms involved in the dormancy-arousal process in Pomacea canaliculata

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Abstract

Figures

1. Introduction

2. Results

2.1. Gene Function Annotation

2.2. Mechanisms of drought tolerance in the apple snail

2.2.1. Assessment of correlation between samples

2.2.2. Differentially expressed genes analysis

2.2.3. Functional enrichment analysis

2.2.4. Weighted correlation network analysis

2.3. Mechanisms of dormancy and awakening in apple snails

2.3.1. DEGs analysis

2.3.2. GO enrichment analysis

2.3.3. Gene Set Enrichment Analysis

2.4. Validation of transcriptome data using qPCR

3. Discussion

4. Conclusions

5. Materials and methods

5.1. Sample collection

5.2. Total RNA extraction

5.3. Transcriptome analysis

5.3.1. Analysis of sequencing data

5.3.2. Differential expression analysis

5.3.3. Functional enrichment analysis

5.3.4. Weighted correlation network analysis

5.3.5. Protein-protein interaction network construction

5.4. Real-time quantitative PCR

Declarations

References

Additional Declarations

Supplementary Files

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