Comparative profiling of hepatopancreas transcriptomes in satiated and starving Pomacea canaliculata
© The Author(s). 2017
Received: 18 September 2016
Accepted: 11 February 2017
Published: 22 February 2017
Although Pomacea canaliculata is native to South and Central America, it has become one of the most abundant invasive species worldwide and causes extensive damage to agriculture and horticulture. Conventional physical and chemical techniques have been used to eliminate P. canaliculata, but the effects are not ideal. Therefore, it is important to devise a new method based on a greater understanding of the biology of P. canaliculata. However, the molecular mechanisms underlying digestion and absorption in P. canaliculata are not well understood due to the lack of available genomic information for this species, particularly for digestive enzyme genes.
In the present study, hepatopancreas transcriptome sequencing produced over 223 million high-quality reads, and a global de novo assembly generated a total of 87,766 unique transcripts (unigenes), of which 19,942 (22.7%) had significant similarities to proteins in the UniProt database. In addition, 296,675 annotated sequences were associated with Gene Ontology (GO) terms. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment was performed for the unique unigenes, and 262 pathways (p-value < 10−5) in P. canaliculata were found to be predominantly related to plant consumption and coarse fiber digestion and absorption. These transcripts were classified into four large categories: hydrolase, transferase, isomerase and cytochrome P450. The Reads Per Kilobase of transcript per Million mapped reads (RPKM) analysis showed that there were 523 down-regulated unigenes and 406 up-regulated unigenes in the starving apple snails compared with the satiated apple snails. Several important genes are associated with digestion and absorption in plants: endo-beta-1, 4-glucanase, xylanase, cellulase, cellulase EGX1, cellulase EGX3 and G-type lysozyme genes were identified. The qRT-PCR results confirmed that the expression patterns of these genes (except for the longipain gene) were consistent with the RNA-Seq results.
Our results provide a more comprehensive understanding of the molecular genes associated with hepatopancreas functioning. Differentially expressed genes corresponding to critical metabolic pathways were detected in the transcriptome of starving apple snails compared with satiated apple snails. In addition to the cellulase gene, other genes were identified that may be important factors in plant matter metabolism in P. canaliculata, and this information has the potential to expedite the study of digestive physiology in apple snails.
KeywordsPomacea canaliculata Hepatopancreas, Transcriptome Digestion
Pomacea canaliculata (apple snail) is a member of Gastropoda and originates from South and Central America, and it is beginning to emerge in locations worldwide, including China, representing one of the 100 most invasive species in the world [1–4]. P. canaliculata features high reproductive capacity, strong adaptability, large food intake and a lack of effective predators, which indicate that it is a considerable threat to the ecosystem balance of fields and ponds [5–7]. This species has rapidly adapted to new environments and is now found in at least 11 provinces in southern China .
Presently, medication and predation approaches are applied to eradicate P. canaliculata. However, the results are not satisfactory. Moreover, these approaches are associated with drug residues and animals such as geese and ducks, which exert a negative influence on crops. In addition, the control of P. canaliculata in wetland ecosystems has received limited attention [9–15].
The hepatopancreas is an important digestive organ in P. canaliculata , and the expression of digestive enzyme genes in P. canaliculata may be closely related to nutrient metabolism and energy balance. The relative lack of information on enzyme genes has impeded research on the digestive physiology of P. canaliculata. Because of the advancements in DNA sequencing technology, generating a large amount of sequence data from non-model organisms such as the apple snail has become increasingly affordable. Although transcriptomic analyses of the hepatopancreas have been performed in several snail species [17, 18] using RNA-Seq, limited information is available on the P. canaliculata hepatopancreas transcriptome . Moreover, the available reports were confined to topics such as invasion prevention, population control, species discrimination, etc. [20, 21]. Therefore, a dearth of research has been conducted on the digestive physiology of P. canaliculata. A transcriptome analysis can enrich the supply of genetic information on the apple snail, explore its digestion and the molecular mechanisms of its growth and development, and provide a reference for research on targeted drugs for snail control.
In this study, we divided snails into two groups, starving apple snails and satiated apple snails; collected total hepatopancreatic RNA from both groups; and constructed cDNA libraries. The cDNAs were sequenced using an Illumina/Solexa platform. Approximately eight gigabytes of high-quality sequencing data were generated. For the functional annotation of the assembled gene transcripts (contigs), we searched against the NCBI nonredundant (NR) and UniProt databases using BlastX. In addition, we compared the gene expression in the hepatopancreatic tissue between satiated apple snails and starving apple snails. This strategy can help further our understanding of the biological responses of P. canaliculata and the hepatopancreas transcriptome dynamics of plant consumption in P. canaliculata.
Sample collection and RNA extraction
P. canaliculata specimens (15–20 mm shell length; 21.11 ± 0.23 g; 20 individuals) were collected from the Hunan Academy of Agricultural Sciences experimental paddy and then sent to the laboratory. The specimens were divided into hunger and satiety feeding groups. After 7 days of rearing in the aquarium, total RNA was extracted from two sets of snail hepatopancreases. The RNA quality was verified with an absorbance microplate reader (260/280 ratio > 2.0) and by agarose gel electrophoresis. Spare samples were stored at−80 °C.
Construction of a cDNA library for sequencing
The cDNA libraries were constructed via PCR amplification using random hexamer priming (the process was conducted in strict accordance with the manual). After library construction, cluster formation and Illumina sequencing were conducted on a HiSeq 2000 platform (Illumina, SY-401-2501) by Bohao Biotechnology Corporation (Shanghai, China) using the methods previously reported by our group .
Data preprocessing and de novo assembly
For the preprocessing, all the adaptor sequences, low-quality reads, ambiguous bases and sequences containing fewer than 20 nucleotides were removed from further analysis prior to contig assembly.
The sequencing data from the two samples were merged into the pooled reads, and de novo assembly was performed using CLC Genomics Workbench (version 6.0.4, CLC Bio, Aarhus, Denmark). De novo sequence assembly was performed on all remaining reads using a contig scaffolding algorithm with a minimum contig length of 200 bp or longer and a word size of 24 characters. The sequences resulting from this phase were designated contigs. Then, we applied CAP3 EST to the spliced primary unigenes to obtain the final unigenes.
Functional annotation and classification
The final unigene sequences were compared with the NR database and the UniProt database (filter: E-value < 1e-5). The NR annotations of the resulting unigenes were searched with Blast2GO Gene Ontology (GO) functional classification algorithms, and the unigenes were functionally classified using cluster of orthologous groups (COG). The unigenes were searched in the CDD using rpstblastn, and all results with E-values < 1e-5 were assigned KOG functional classification predictions and mapped to each COG level of classification, whereas the online KAAS pathway alignment analysis tool was applied to the unigene splices for the KEGG mapping analysis.
Screening of differentially expressed unigenes
Putative molecular markers and open reading frames (ORFs)
The flanking sequences of a particular microsatellite are usually highly conserved, and the repeat unit is species specific. Using this characteristic, an SSR analysis was conducted on the unigene sequences with MISA software (http://pgrc.ipk-gatersleben.de/misa/). SSR markers can be used to develop STMS, one of the most commonly used microsatellite markers, using the principle of simple sequence repeat length polymorphisms (SSLPs). EMBOSS (6.4.0)-getorf  was used to generate ORF predictions for all unigenes, and the ORF greater than 300 bp was considered the ORF of the unigene.
Differentially expressed gene validation by real-time quantitative RT-PCR (qRT-PCR)
We screened the protein-coding genes associated with plant digestion and absorption using RT-PCR validation. The following six genes were selected: cellulase, cellulase EGX3, cellulase EGX1, endo-beta-1, 4-glucanase, xylanase and G-type lysozyme. The primer sequences (Additional file 1: Table S1) for the qRT-PCR were designed utilizing the Roche Universal Probe Library Assay Design Center (https://lifescience.roche.com/en_cn.html) and synthesized by Sangon Biotechnology Corporation (Shanghai, China). The cDNA synthesis and RT-PCR processes were conducted according to the manufacturer’s instructions. A reaction volume of 20 μL was used. The amplification program was as follows: 95 °C for 30 s, followed by 34 cycles of 95 °C for 10 s and annealing at 60 °C for 30 s.
Results and discussion
RNA sequencing and de novo assembly of the hepatopancreas transcriptome
After pre-processing the sequencing data of the two samples, we obtained 95,126,010 and 128,258,226 clean reads. The clean ratios (clean ratio = clean reads/raw reads) (%) were 94.9 and 94.6%.
Statistical summary of the SG transcriptome for assembly
Total length (bp)
Average length (bp)
Functional annotation and classification
Results of blasting against the NCBI NR and UniProt protein databases
No. sequence with hits
No. unknown sequence
Percentage of annotation (%)
KAAS was used for the KEGG map analysis of 20,885 unigenes using comparative analysis tools. These unigenes were mapped to a 262-article pathway, and most were concentrated in the following categories: metabolic: biosynthesis of secondary metabolites; microbial metabolism in diverse environments; and protein processing in the endoplasmic reticulum.
Functional enrichment analysis of DEGs
Top 30 functional classifications of DEGs determined by comparisons with the GO and KEGG databases
Counts of DEGs
Based on the GO database
Carbohydrate metabolic process
Hydrolase activity, hydrolyzing O-glycosyl czompounds
Pyrimidine nucleoside metabolic process
Pyrimidine nucleobase metabolic process
transferase activity, transferring pentosyl groups
Pyrimidine-nucleoside phosphorylase activity
Electron transport chain
NADH dehydrogenase (ubiquinone) activity
ATP synthesis coupled electron transport
Enzyme regulator activity
Serine-type endopeptidase inhibitor activity
Nucleic acid binding
Regulation of catalytic activity
Peptidoglycan catabolic process
Hydrolase activity, acting on glycosyl bonds
Glutathione transferase activity
Respiratory electron transport chain
Based on the KEGG database
Metabolism of xenobiotics by cytochrome P450
Drug metabolism - cytochrome P450
Starch and sucrose metabolism
Steroid hormone biosynthesis
Biosynthesis of secondary metabolites
Arachidonic acid metabolism
Pentose phosphate pathway
Ascorbate and aldarate metabolism
Proteoglycans in cancer
Degradation of aromatic compounds
Microbial metabolism in diverse environments
Cyanoamino acid metabolism
Glycine, serine and threonine metabolism
Carbohydrate digestion and absorption
Linoleic acid metabolism
Cardiac muscle contraction
Drug metabolism - other enzymes
A total of 148 pathways were found to be enriched with DEGs, and DEGs were primarily identified in the following GO categories: metabolic pathways (104, 63.4%); chemical carcinogenesis (18, 10.98%); metabolism of xenobiotics by cytochrome P450 (16, 9.76%); and drug metabolism - cytochrome P450 (14, 8.53%). Notably, up-regulated genes were mostly found in the metabolic pathways of starch and sucrose metabolism and glutathione metabolism, and only up-regulated genes were found in the pathways of galactose metabolism, caprolactam degradation, and carbohydrate digestion and absorption (Table 3). The full lists of DEGs is shown in Additional file 7: Table S7 Collectively, these transcriptome sequences and pathway annotations provide an essential resource for further screening and expression analyses of candidate genes related to digestion and absorption in P. Canaliculata.
SSR analysis and ORF prediction
Our analyses identified 35,582 SSRs (or microsatellites) in total using MISA (Microsatellite) software, and 3171 of these SSRs (8.91%) were hybrid nucleotide repeats, 21,203 SSRs (59.59%) were mononucleotide repeats, 10,748 SSRs (30.21%) were dinucleotide repeats, and 3139 SSRs (8.82%) were trinucleotide repeats (Additional file 8: Table S8). In addition, 775 ORFs were predicted.
Analysis of digestive enzymes and genes in hepatopancreas
Categories of transcripts potentially involved in plant feed digestion
Best match to NR database
Endoglucanase-like isoform ×3 [Biomphalaria glabrata]
Endo-beta-1,4-glucanase [Ampullaria crossean]
Endoglucanase-like isoform ×3
Family 10 cellulase
Family 10 cellulase (EGXA) [Ampullaria crossean]
Cellulase EGX1 [Pomacea canaliculata]
Cellulase EGX3 [Pomacea canaliculata]
Xylanase [Ampullaria crossean]
Probable beta-D-xylosidase 7 [Aplysia californica]
Probable beta-D-xylosidase 5
Probable beta-D-xylosidase 7-like [Aplysia californica]
Maltase-glucoamylase, intestinal-like [Aplysia californica]
Beta-1,3-glucan-binding protein-like [Biomphalaria glabrata]
Glucose-6-phosphate 1-dehydrogenase-like [Crassostrea gigas]
Thymidine phosphorylase [Chelonia mydas]
Aldose 1-epimerase-like [Aplysia californica]
Cytochrome P450 3A16-like [Aplysia californica]
Cytochrome P450 II f2-like protein II
Heat shock protein 60 (HSP60) gene [Pomacea canaliculata]
Comparative analysis between the two hepatopancreas libraries
Genes encoding secreted proteins that mediate digestive absorption
We concentrated on the most significant sequenced transcripts encoding secreted proteins. This de novo assembly of the hepatopancreas transcriptome of P. canaliculata provided us with valuable raw material for the identification and analysis of P. canaliculata hepatopancreas proteins. Similar to other herbivores, P. canaliculata produces various secreted proteins that are involved in the digestion of the meal or modulation of host defenses for survival. Among these modulatory molecules, cellulase, cellulase EGX1, cellulase EGX3, endo-beta-1, 4-glucanase, xylanase and G-type lysozyme enzymes are vital for P. canaliculata survival and feeding.
We found that only the above six digestive and absorption-related enzymes were differentially expressed in this experiment, which may be related to the characteristics of P. canaliculata digestive organs themselves, the distribution of enzymes and the role of symbiotic gastrointestinal bacteria in digestion . Our group is continuing to perform related research.
Quantitative expression and comparative analysis for genes associated with digestive absorption and host defenses
In this study, cellulase EGX1, cellulase, cellulase EGX3 and G-type lysozyme were up-regulated in the hepatopancreases of fully fed P. canaliculata, and this finding is consistent with previous studies (xylanase , cellulase EGX3 , endo-beta-1, 4-glucanase [35, 36] and G-type lysozyme ), which indicates that these enzymes play an important role in the digestion and absorption of fibrous food sources. Cellulase  and cellulase EGX1  may be involved in this process. These results showed that the hepatopancreatic secretion of proteins plays an important role in the regulation of plant digestion and utilization in P. canaliculata.
In this study, to explore the digestive function of P. canaliculata as a starting point, the de novo hepatopancreatic transcriptome of P. canaliculata was initially characterized using NGS technology and bioinformatics. The transcriptomic approach provides opportunities to identify pharmacologically active proteins and further insights into the digestive and metabolic functions of P. canaliculata. In total, 90,141 contigs and 87,766 unigenes have been discovered via high-throughput sequencing of the hepatopancreas. By comparing unigenes with the NCBI NR and UniProt databases, analyzing NR results via GO functional classification analysis and analyzing unigenes via KOG and KEGG pathways, we have shown that the hepatopancreas of P. canaliculata is an essential digestive organ that plays a significant role in the digestion of plant matter. In addition, a comparative analysis of the hepatopancreas transcriptomes of the two groups indicated six genes that may be associated with plant feed digestion, and the qRT-PCR verification, RNA-Seq analysis and qRT-PCR results are consistent. Our research has enriched the genome database of P. canaliculata hepatopancreas and provided a reference for environmentally significant research on the digestive physiology of P. canaliculata. Moreover, our results provide additional opportunities to screen and clone functional genes.
Cluster of orthologous groups
False discovery rate
- P. canaliculata :
Reads per kb per million reads
Web gene ontology annotation plot
The authors thank the Shanghai Bohao Biotechnology Corporation for the assistance with data processing and bioinformatic analysis. We thank Dr. XingLi Xu and Dr. Lei Liu for the valuable comments and revision of the manuscriptIn addition, we also thank American Journal Experts his help to improve our language.
Hunan agricultural university postdoctoral fund (NO.540390205004).
Availability of data and materials
The data supporting the results of this article are included within the article and in its supplementary files.
Conceived and designed the experiments: TYC and LY. Performed the experiments: FYZ and LY. Analyzed the data:LY. Contributed reagents/materials/analysis tools: FYZ and LY. Wrote the paper: FYZ and LY. All authors have read and approved the final manuscript.
I confirm that I have read BioMed Central's guidance on competing interests and have included a statement in the manuscript on any competing interests. The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
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