Transcriptomic analysis of embryo development in the invasive snail Pomacea canaliculata

Transcriptomic analysis of embryo development in the invasive snail Pomacea canaliculata Abstract A scarcity of genomic data hinders progress in understanding gene expression of Pomacea canaliculata embryogenesis. This study used RNA-Seq (Illumina) to characterize gene expression across four stages of embryo development of P. canaliculata from early stage II (endoderm formation) to later stage V (appearance of shell pigmentation). A total of 35,100 unigenes were annotated and gene expression patterns were considered in relation to organogenesis of albumen gland, nervous system, circadian rhythm, immune system and shell formation. This is the first use of next-generation sequencing to catalogue gene expression in the developing embryo of P. canaliculata and provides primary data for future molecular ecological studies of this invasive snail. INTRODUCTION The apple snail Pomacea canaliculata is the only freshwater snail listed among the 100 worst invasive species worldwide (Dreon et al., 2014). The species originates from South America, but is now found in North America, Asia and Europe (Accorsi et al., 2017). Large size and great appetite for aquatic plants make P. canaliculata a threat with potential ecological and economical risks because of damage to wetlands and crops (Youens & Burks, 2008; Hayes et al., 2012). Additionally, P. canaliculata is the intermediate host of the nematode Angiostrongylus cantonensis, which is responsible for potentially lethal encephalitis (Lv et al., 2009; Song et al., 2016). At present there are no accepted and efficient strategies for controlling the spread of P. canaliculata. The understanding of molecular processes underlying embryonic development may contribute to the generation of efficient and reliable molecular tools to control this invasive snail. Successful establishment in invaded areas may relate to features of P. canaliculata including highly effective mechanisms to deal with adverse environments and natural predators. Notably, the reproduction of P. canaliculata involves deposition of hundreds of bright pink or reddish coloured egg masses above water. The calcareous egg shell and a neurotoxin-like protein in the perivitelline fluid are successful mechanisms to deal with environmental stresses such as UV radiation and predators (Frassa et al., 2010; Sun et al., 2012). The process of embryonic development in P. canaliculata is not well understood. Previous studies have emphasized the nutritional and protective functions of perivitelline fluid protein, but little is known about the gene-expression patterns during embryonic development. Similar to most other gastropods, the embryonic development of P. canaliculata includes the formation of a shell. The process of shell formation involves changes in body shape and deposition of minerals and pigments in a matrix of proteins (Liu et al., 2007). The dramatic changes during embryonic development require expression and action of many genes, which coordinate and modulate various developmental events (Jackson, Worheide & Degnan, 2007). Previous investigations of embryonic development in marine bivalves, including scallop and oyster species, have identified genes, involved in organogenesis, shell formation and utilization of nutrients (Cannuel & Beninger, 2006; Naimi et al., 2009a, b). The sequencing of several molluscan genomes has been accomplished, including those of Lottia gigantea, Crassostrea gigas and Aplysia californica. The availability of these resources provides opportunities to perform comparative and genome-wide analyses of development on a transcriptional level. Application of transcriptomic technologies provides insight into the spatial and temporal dynamics of gene expression during consecutive stages of embryonic development, revealing details of gene regulation. This approach has been applied to study the embryos of several species of molluscs, such as the clam Meretrix meretrix (Huan, Wang & Liu, 2012) and the gastropods A. californica (Heyland et al., 2011) and Ilyanassa obsoleta (Lambert et al., 2010). This study uses the Illumina HiSeq 4000 platform to construct a database representing an annotated transcriptome assembly for developing P. canaliculata embryos. These transcriptome data should provide a resource for future research on P. canaliculata embryonic development. MATERIAL AND METHODS Egg incubation and embryo collection Eggs clutches laid by Pomacea canaliculata were collected from a laboratory aquarium (South China Agricultural University, Guangzhou, People’s Republic of China) and incubated at 25 °C. Each day, shells of some eggs from individual clutches were broken using forceps to obtain embryos for microscopic identification of the stage of development (based on previous studies of Koch, Winik & Castro-Vazquez, 2009; Sun et al., 2010; Fig. 1). We analysed embryonic development at stage II (endoderm formation), stage III (internal and external organs formed), stage IV (shell formation) and stage V (appearance of shell pigmentation). Developing embryos that had reached the desired stage were placed in tubes and snap-frozen in liquid nitrogen. Samples were stored at −80 °C before the experiments described below. Figure 1. View largeDownload slide Embryonic development of Pomacea. canaliculata, showing embryos removed from eggs consecutively at stages II to V. Scale bars: 500 μm (stages II and III); 1000 μm (stages IV and V). Figure 1. View largeDownload slide Embryonic development of Pomacea. canaliculata, showing embryos removed from eggs consecutively at stages II to V. Scale bars: 500 μm (stages II and III); 1000 μm (stages IV and V). Transcriptome sequencing and de novo assembly Total RNA was extracted from each sample separately, using TRIzol Reagent (ThermoFisher), according to the manufacturer’s instructions. After treating total RNA samples with DNase I (TaKaRa), oligo(dT)-absorption (ThermoFisher) was used to isolate mRNA. The mRNA was fragmented by mixing with the fragmentation buffer and cDNA libraries (NEBNext® Ultra™ RNA Library Prep Kit) were synthesized from the mRNA fragments. Quality control and quantification of the sample libraries employed an Agilent 2100 Bioanalyzer and the ABI StepOnePlus Real-Time PCR System. The resulting libraries were sequenced (paired ends and select cDNA fragments preferentially of 200–250 bp in length) using an Illumina HiSeq 4000 platform. Low-quality raw reads (containing adaptors or with high content of unknown base calls) were identified using cutadapt software (Grabherr et al., 2011). High quality reads of each sample were assembled using Trinity software (Haas et al., 2013) and Tgicl (Pertea et al., 2003) to cluster transcripts to unigenes. The clustered unigenes were compared to SILVA (Quast et al., 2013) ribosomal RNA databases to exclude those matching rRNA sequences (score > 60). Functional annotation of unigenes The unigenes of the transcriptome datasets were annotated using Blast (Altschul et al., 1990) to nr GenBank database, eggNOG, KEGG and Swissprot. Blast2GO (Conesa et al., 2005) and Interproscan5 (Quevillon et al., 2005) were used for GO annotation and Interpro annotation. For transcriptome analysis, clean reads were mapped to unigenes using Bowtie2 (Langmead & Salzberg, 2012) and gene expression levels were calculated according to a previous study using the RSEM method that quantifies total expressed transcripts with the TPM (transcripts per million) value (Li & Dewey, 2011). Then PCA analysis was performed using all samples with ‘princomp’, a function of R software. Differentially expressed genes (DEG) were detected with PossionDis as described by Audic & Claverie (1997). RESULTS AND DISCUSSION We used Illumina next-generation sequencing technology to record gene expression patterns during the embryonic development of Pomacea canaliculata. All of the clean reads have been deposited in the NCBI SRA database under accession number SRX2538617. Transcriptome assembly yielded 241,904 high-confidence unigenes with average length 792 bp. Removal of short sequences (<500 bp) yielded 35,100 unigenes. Using six different approaches (NCBI-nr, Swissprot, eggNOG, Interpro, GO and KEGG) provided computational annotation for 31,475 (71.68%) unigenes (Supplementary Material). The taxonomic distribution of best matches indicated that greater than 81% of annotated sequences best matched with molluscan species. Gene ontology (GO) analysis assigned annotated unigenes to the first-level GO categories of biological process, cellular component and molecular function, respectively (Fig. 2). The most abundant second-level terms are cellular process (38.62% of annotated unigenes) and metabolic process (34.23%) for the biological process category, membrane (25.14%) and membrane part (14.58%) for the cellular component category, and binding (53.93%) and catalytic activity (35.60%) for the molecular function category. Figure 2. View largeDownload slide GO classification of unigenes. X axis represents percent of unigenes. Y axis represents GO term. Because one unigene can be attributed to more than one term, the sum of all terms is larger than 100%. Figure 2. View largeDownload slide GO classification of unigenes. X axis represents percent of unigenes. Y axis represents GO term. Because one unigene can be attributed to more than one term, the sum of all terms is larger than 100%. At present, the genetic foundation, factors and molecular mechanisms that drive the progression of stages in embryonic development of P. canaliculata are mostly unknown. Comparative analysis of gene-expression levels will extend our knowledge and understanding of growth and development-related unigenes. Annotation of DEGs provided the five most enriched GO terms for each stage of development (Table 1). Between stage II and stage III, when the embryo transitions from endoderm formation to the onset of organogenesis, the most abundant DEGs are associated with receptor activity of molecular function. At stage IV (when most external and internal organs are fully formed), the most highly expressed DEGs relate to metabolic processes. Table 1. The five most enriched GO term of DEG among embryonic stages of Pomacea canaliculata.   ID  Description  Class  DEGs  Stage II vs stage III  GO:0060089  Molecular transducer activity  Molecular function  192  GO:0004872  Receptor activity  Molecular function  192  GO:0004888  Tansmembrane signalling receptor activity  Molecular function  135  GO:0038023  Signalling receptor activity  Molecular function  145  GO:0004871  Signal transducer activity  Molecular function  149  Stage III vs stage IV  GO:0016798  Hydrolase activity, acting on glycosyl bonds  Molecular function  72  GO:0004553  Hydrolase activity, hydrolysing O-glycosyl compounds  Molecular function  64  GO:0005576  Extracellular region  Cellular component  54  GO:0005975  Carbohydrate metabolic process  Biological process  75  GO:0006022  Aminoglycan metabolic process  Biological process  36  Stage IV vs stage V  GO:0016798  Hydrolase activity, acting on glycosyl bonds  Molecular function  67  GO:0004553  Hydrolase activity, hydrolysing O-glycosyl compounds  Molecular function  60  GO:0005576  Extracellular region  Cellular component  51  GO:0006030  Chitin metabolic process  Biological process  32  GO:1901071  Glucosamine-containing compound metabolic process  Biological process  32    ID  Description  Class  DEGs  Stage II vs stage III  GO:0060089  Molecular transducer activity  Molecular function  192  GO:0004872  Receptor activity  Molecular function  192  GO:0004888  Tansmembrane signalling receptor activity  Molecular function  135  GO:0038023  Signalling receptor activity  Molecular function  145  GO:0004871  Signal transducer activity  Molecular function  149  Stage III vs stage IV  GO:0016798  Hydrolase activity, acting on glycosyl bonds  Molecular function  72  GO:0004553  Hydrolase activity, hydrolysing O-glycosyl compounds  Molecular function  64  GO:0005576  Extracellular region  Cellular component  54  GO:0005975  Carbohydrate metabolic process  Biological process  75  GO:0006022  Aminoglycan metabolic process  Biological process  36  Stage IV vs stage V  GO:0016798  Hydrolase activity, acting on glycosyl bonds  Molecular function  67  GO:0004553  Hydrolase activity, hydrolysing O-glycosyl compounds  Molecular function  60  GO:0005576  Extracellular region  Cellular component  51  GO:0006030  Chitin metabolic process  Biological process  32  GO:1901071  Glucosamine-containing compound metabolic process  Biological process  32  Table 1. The five most enriched GO term of DEG among embryonic stages of Pomacea canaliculata.   ID  Description  Class  DEGs  Stage II vs stage III  GO:0060089  Molecular transducer activity  Molecular function  192  GO:0004872  Receptor activity  Molecular function  192  GO:0004888  Tansmembrane signalling receptor activity  Molecular function  135  GO:0038023  Signalling receptor activity  Molecular function  145  GO:0004871  Signal transducer activity  Molecular function  149  Stage III vs stage IV  GO:0016798  Hydrolase activity, acting on glycosyl bonds  Molecular function  72  GO:0004553  Hydrolase activity, hydrolysing O-glycosyl compounds  Molecular function  64  GO:0005576  Extracellular region  Cellular component  54  GO:0005975  Carbohydrate metabolic process  Biological process  75  GO:0006022  Aminoglycan metabolic process  Biological process  36  Stage IV vs stage V  GO:0016798  Hydrolase activity, acting on glycosyl bonds  Molecular function  67  GO:0004553  Hydrolase activity, hydrolysing O-glycosyl compounds  Molecular function  60  GO:0005576  Extracellular region  Cellular component  51  GO:0006030  Chitin metabolic process  Biological process  32  GO:1901071  Glucosamine-containing compound metabolic process  Biological process  32    ID  Description  Class  DEGs  Stage II vs stage III  GO:0060089  Molecular transducer activity  Molecular function  192  GO:0004872  Receptor activity  Molecular function  192  GO:0004888  Tansmembrane signalling receptor activity  Molecular function  135  GO:0038023  Signalling receptor activity  Molecular function  145  GO:0004871  Signal transducer activity  Molecular function  149  Stage III vs stage IV  GO:0016798  Hydrolase activity, acting on glycosyl bonds  Molecular function  72  GO:0004553  Hydrolase activity, hydrolysing O-glycosyl compounds  Molecular function  64  GO:0005576  Extracellular region  Cellular component  54  GO:0005975  Carbohydrate metabolic process  Biological process  75  GO:0006022  Aminoglycan metabolic process  Biological process  36  Stage IV vs stage V  GO:0016798  Hydrolase activity, acting on glycosyl bonds  Molecular function  67  GO:0004553  Hydrolase activity, hydrolysing O-glycosyl compounds  Molecular function  60  GO:0005576  Extracellular region  Cellular component  51  GO:0006030  Chitin metabolic process  Biological process  32  GO:1901071  Glucosamine-containing compound metabolic process  Biological process  32  Based on differential expression patterns among developmental stages, unigenes that are of interest for the study of embryonic development of P. canaliculata were identified (Table 2). The albumen gland of P. canaliculata secretes perivitelline proteins, including perivitelline ovorubin proteins (Sun et al., 2012), that surround the developing embryo. These proteins not only provide all nutrients and energy for embryonic development, but also protect the eggs from environmental stressors and predators (Dreon et al., 2004; Heras et al., 2008; Dreon, Ituarte & Heras, 2010). Three ovorubin-encoding genes (perivitellin ovorubin-1, perivitellin ovorubin-2, perivitellin ovorubin-3) were highly expressed in stage II, with expression levels decreased significantly in the three later stages. The underlying mechanism is unclear, considering that in animals the maternal factors that contributed to the egg cytoplasm initially control development, while the zygotic nuclear genome is quiescent. Subsequently, the genome is activated, embryonic gene products are mobilized and maternal factors are cleared (Lee, Bonneau & Giraldez, 2014). The high expression of perivitellin ovorubin protein-encoding genes during early embrogenesis suggests that the embryos may either receive mRNA transcripts from the parent to maintain a certain level of defence, or that the embryos transcribe and translate genes to contribute to their own defence against predation, in addition to that from parentally-provided perivitellin ovorubin proteins. Table 2. Select differentially expressed genes among the four embryonic stages of Pomacea canaliculata. Unigenes  Stage II/Stage I  Stage III/Stage II  Stage IV/Stage III  Protein name    Fold change    Albumen gland          TRINITY_DN119492_c0_g1_i1  −2.83  0  −2.65  Perivitellin ovorubin-1  TRINITY_DN144541_c3_g7_i1  −2.64  0  −3.71  Perivitellin ovorubin-2  TRINITY_DN124633_c0_g1_i1  −2.80  0  −3.88  Perivitellin ovorubin-3  G protein-coupled receptor          TRINITY_DN101258_c0_g1_i1  0  0  3.78  Opsin  TRINITY_DN116917_c0_g1_i2  3.42  0  0  5HT4 receptor  TRINITY_DN144870_c2_g1_i2  2.21  0  0  5HT2 receptor  TRINITY_DN122825_c0_g2_i3  2.78  0  0  NPFF2 receptors  TRINITY_DN122960_c0_g1_i1  4.34  0  0  Adenosine A2B receptor  TRINITY_DN132125_c0_g1_i1  3.47  0  0  Cholecystokinin receptor type A  TRINITY_DN134539_c0_g1_i4  8.28  0  0  Somatostatin receptor 2  TRINITY_DN136817_c0_g2_i12  7.05  0  0  Dopamine D3 receptor  TRINITY_DN137372_c0_g2_i1  2.82  0  0  Neurotensin type 1 receptor  TRINITY_DN137907_c1_g2_i1  4.28  0  0  CC chemokine receptor 6  Cytochrome P450(CYP)          TRINITY_DN126194_c0_g1_i1  0  0  7.56  CYP 3A43  TRINITY_DN127763_c0_g4_i5  0  0  2.44  CYP 3A24  TRINITY_DN128942_c0_g4_i1  7.77  0  −6.89  CYP 4F22  TRINITY_DN130575_c0_g1_i1  −3.71  0  0  CYP 3A7  TRINITY_DN144157_c3_g1_i3  7.55  0  0  CYP 2B4  TRINITY_DN143132_c0_g3_i2  0  0  −6.99  CYP 2C30  Cellulase          TRINITY_DN129366_c0_g1_i1  0  7.92  2.78  Endoglucanase 20  TRINITY_DN138509_c1_g3_i1  0  2.57  0  Endo-beta-1,4-glucanase  TRINITY_DN142135_c0_g2_i2  0  7.82  4.33  Family 10 cellulase  TRINITY_DN145404_c1_g1_i3  0  0  4.20  Cellulase EGX3  Nervous system          TRINITY_DN145021_c0_g6_i1  2.49  0  0  5-HT  TRINITY_DN136773_c0_g1_i4  4.30  0  0  FMRF1  TRINITY_DN133591_c0_g1_i3  −7.80  8.57  0  Moa  TRINITY_DN140497_c1_g5_i1  4.52  0  0  Ninjurin 1  Circadian rhythm          TRINITY_DN136298_c0_g1_i4  2.73  0  0  Clk  Handedness          TRINITY_DN135564_c1_g2_i8  7.06  0  0  Nodal  TRINITY_DN131526_c0_g1_i  −7.51  6.83  0  Pitx  TRINITY_DN138997_c3_g2_i2  0  6.82  0  Piwi  TRINITY_DN139424_c1_g2_i4  −2.37  0  2.73  Nck1  Shell formation          TRINITY_DN136576_c1_g1_i8  −8.98  0  6.94  Calponin  TRINITY_DN129910_c0_g17_i1  2.92  0  0  Calmodulin  TRINITY_DN129097_c0_g2_i3  0  0  7.12  Perlucin 1  Immune system          TRINITY_DN113417_c0_g1_i1  0  0  6.85  C-type lectin  TRINITY_DN143157_c0_g1_i2  0  0  5.11  I-type lectin-like protein 4  TRINITY_DN139530_c0_g1_i3  0  0  2.26  Ricin_B_lectin  TRINITY_DN122928_c1_g2_i1  0  0  7.56  C1q domain protein  TRINITY_DN144320_c0_g1_i1  9.50  0  0  Toll-like receptor 4  TRINITY_DN128784_c2_g1_i1  0  0  7.12  Concanavalin A-like lectin  TRINITY_DN134165_c0_g1_i1  0  0  4.59  Fibrinogen  TRINITY_DN132910_c0_g4_i3  7.22  0  0  F-type lectins  TRINITY_DN118405_c0_g1_i2  0  0  7.21  Gal_Lectin  TRINITY_DN129873_c0_g1_i2  −3.15  0  3.20  H-types lectins  Muscular contraction          TRINITY_DN136954_c0_g2_i9  3.21  0  0  Titin  Unigenes  Stage II/Stage I  Stage III/Stage II  Stage IV/Stage III  Protein name    Fold change    Albumen gland          TRINITY_DN119492_c0_g1_i1  −2.83  0  −2.65  Perivitellin ovorubin-1  TRINITY_DN144541_c3_g7_i1  −2.64  0  −3.71  Perivitellin ovorubin-2  TRINITY_DN124633_c0_g1_i1  −2.80  0  −3.88  Perivitellin ovorubin-3  G protein-coupled receptor          TRINITY_DN101258_c0_g1_i1  0  0  3.78  Opsin  TRINITY_DN116917_c0_g1_i2  3.42  0  0  5HT4 receptor  TRINITY_DN144870_c2_g1_i2  2.21  0  0  5HT2 receptor  TRINITY_DN122825_c0_g2_i3  2.78  0  0  NPFF2 receptors  TRINITY_DN122960_c0_g1_i1  4.34  0  0  Adenosine A2B receptor  TRINITY_DN132125_c0_g1_i1  3.47  0  0  Cholecystokinin receptor type A  TRINITY_DN134539_c0_g1_i4  8.28  0  0  Somatostatin receptor 2  TRINITY_DN136817_c0_g2_i12  7.05  0  0  Dopamine D3 receptor  TRINITY_DN137372_c0_g2_i1  2.82  0  0  Neurotensin type 1 receptor  TRINITY_DN137907_c1_g2_i1  4.28  0  0  CC chemokine receptor 6  Cytochrome P450(CYP)          TRINITY_DN126194_c0_g1_i1  0  0  7.56  CYP 3A43  TRINITY_DN127763_c0_g4_i5  0  0  2.44  CYP 3A24  TRINITY_DN128942_c0_g4_i1  7.77  0  −6.89  CYP 4F22  TRINITY_DN130575_c0_g1_i1  −3.71  0  0  CYP 3A7  TRINITY_DN144157_c3_g1_i3  7.55  0  0  CYP 2B4  TRINITY_DN143132_c0_g3_i2  0  0  −6.99  CYP 2C30  Cellulase          TRINITY_DN129366_c0_g1_i1  0  7.92  2.78  Endoglucanase 20  TRINITY_DN138509_c1_g3_i1  0  2.57  0  Endo-beta-1,4-glucanase  TRINITY_DN142135_c0_g2_i2  0  7.82  4.33  Family 10 cellulase  TRINITY_DN145404_c1_g1_i3  0  0  4.20  Cellulase EGX3  Nervous system          TRINITY_DN145021_c0_g6_i1  2.49  0  0  5-HT  TRINITY_DN136773_c0_g1_i4  4.30  0  0  FMRF1  TRINITY_DN133591_c0_g1_i3  −7.80  8.57  0  Moa  TRINITY_DN140497_c1_g5_i1  4.52  0  0  Ninjurin 1  Circadian rhythm          TRINITY_DN136298_c0_g1_i4  2.73  0  0  Clk  Handedness          TRINITY_DN135564_c1_g2_i8  7.06  0  0  Nodal  TRINITY_DN131526_c0_g1_i  −7.51  6.83  0  Pitx  TRINITY_DN138997_c3_g2_i2  0  6.82  0  Piwi  TRINITY_DN139424_c1_g2_i4  −2.37  0  2.73  Nck1  Shell formation          TRINITY_DN136576_c1_g1_i8  −8.98  0  6.94  Calponin  TRINITY_DN129910_c0_g17_i1  2.92  0  0  Calmodulin  TRINITY_DN129097_c0_g2_i3  0  0  7.12  Perlucin 1  Immune system          TRINITY_DN113417_c0_g1_i1  0  0  6.85  C-type lectin  TRINITY_DN143157_c0_g1_i2  0  0  5.11  I-type lectin-like protein 4  TRINITY_DN139530_c0_g1_i3  0  0  2.26  Ricin_B_lectin  TRINITY_DN122928_c1_g2_i1  0  0  7.56  C1q domain protein  TRINITY_DN144320_c0_g1_i1  9.50  0  0  Toll-like receptor 4  TRINITY_DN128784_c2_g1_i1  0  0  7.12  Concanavalin A-like lectin  TRINITY_DN134165_c0_g1_i1  0  0  4.59  Fibrinogen  TRINITY_DN132910_c0_g4_i3  7.22  0  0  F-type lectins  TRINITY_DN118405_c0_g1_i2  0  0  7.21  Gal_Lectin  TRINITY_DN129873_c0_g1_i2  −3.15  0  3.20  H-types lectins  Muscular contraction          TRINITY_DN136954_c0_g2_i9  3.21  0  0  Titin  Table 2. Select differentially expressed genes among the four embryonic stages of Pomacea canaliculata. Unigenes  Stage II/Stage I  Stage III/Stage II  Stage IV/Stage III  Protein name    Fold change    Albumen gland          TRINITY_DN119492_c0_g1_i1  −2.83  0  −2.65  Perivitellin ovorubin-1  TRINITY_DN144541_c3_g7_i1  −2.64  0  −3.71  Perivitellin ovorubin-2  TRINITY_DN124633_c0_g1_i1  −2.80  0  −3.88  Perivitellin ovorubin-3  G protein-coupled receptor          TRINITY_DN101258_c0_g1_i1  0  0  3.78  Opsin  TRINITY_DN116917_c0_g1_i2  3.42  0  0  5HT4 receptor  TRINITY_DN144870_c2_g1_i2  2.21  0  0  5HT2 receptor  TRINITY_DN122825_c0_g2_i3  2.78  0  0  NPFF2 receptors  TRINITY_DN122960_c0_g1_i1  4.34  0  0  Adenosine A2B receptor  TRINITY_DN132125_c0_g1_i1  3.47  0  0  Cholecystokinin receptor type A  TRINITY_DN134539_c0_g1_i4  8.28  0  0  Somatostatin receptor 2  TRINITY_DN136817_c0_g2_i12  7.05  0  0  Dopamine D3 receptor  TRINITY_DN137372_c0_g2_i1  2.82  0  0  Neurotensin type 1 receptor  TRINITY_DN137907_c1_g2_i1  4.28  0  0  CC chemokine receptor 6  Cytochrome P450(CYP)          TRINITY_DN126194_c0_g1_i1  0  0  7.56  CYP 3A43  TRINITY_DN127763_c0_g4_i5  0  0  2.44  CYP 3A24  TRINITY_DN128942_c0_g4_i1  7.77  0  −6.89  CYP 4F22  TRINITY_DN130575_c0_g1_i1  −3.71  0  0  CYP 3A7  TRINITY_DN144157_c3_g1_i3  7.55  0  0  CYP 2B4  TRINITY_DN143132_c0_g3_i2  0  0  −6.99  CYP 2C30  Cellulase          TRINITY_DN129366_c0_g1_i1  0  7.92  2.78  Endoglucanase 20  TRINITY_DN138509_c1_g3_i1  0  2.57  0  Endo-beta-1,4-glucanase  TRINITY_DN142135_c0_g2_i2  0  7.82  4.33  Family 10 cellulase  TRINITY_DN145404_c1_g1_i3  0  0  4.20  Cellulase EGX3  Nervous system          TRINITY_DN145021_c0_g6_i1  2.49  0  0  5-HT  TRINITY_DN136773_c0_g1_i4  4.30  0  0  FMRF1  TRINITY_DN133591_c0_g1_i3  −7.80  8.57  0  Moa  TRINITY_DN140497_c1_g5_i1  4.52  0  0  Ninjurin 1  Circadian rhythm          TRINITY_DN136298_c0_g1_i4  2.73  0  0  Clk  Handedness          TRINITY_DN135564_c1_g2_i8  7.06  0  0  Nodal  TRINITY_DN131526_c0_g1_i  −7.51  6.83  0  Pitx  TRINITY_DN138997_c3_g2_i2  0  6.82  0  Piwi  TRINITY_DN139424_c1_g2_i4  −2.37  0  2.73  Nck1  Shell formation          TRINITY_DN136576_c1_g1_i8  −8.98  0  6.94  Calponin  TRINITY_DN129910_c0_g17_i1  2.92  0  0  Calmodulin  TRINITY_DN129097_c0_g2_i3  0  0  7.12  Perlucin 1  Immune system          TRINITY_DN113417_c0_g1_i1  0  0  6.85  C-type lectin  TRINITY_DN143157_c0_g1_i2  0  0  5.11  I-type lectin-like protein 4  TRINITY_DN139530_c0_g1_i3  0  0  2.26  Ricin_B_lectin  TRINITY_DN122928_c1_g2_i1  0  0  7.56  C1q domain protein  TRINITY_DN144320_c0_g1_i1  9.50  0  0  Toll-like receptor 4  TRINITY_DN128784_c2_g1_i1  0  0  7.12  Concanavalin A-like lectin  TRINITY_DN134165_c0_g1_i1  0  0  4.59  Fibrinogen  TRINITY_DN132910_c0_g4_i3  7.22  0  0  F-type lectins  TRINITY_DN118405_c0_g1_i2  0  0  7.21  Gal_Lectin  TRINITY_DN129873_c0_g1_i2  −3.15  0  3.20  H-types lectins  Muscular contraction          TRINITY_DN136954_c0_g2_i9  3.21  0  0  Titin  Unigenes  Stage II/Stage I  Stage III/Stage II  Stage IV/Stage III  Protein name    Fold change    Albumen gland          TRINITY_DN119492_c0_g1_i1  −2.83  0  −2.65  Perivitellin ovorubin-1  TRINITY_DN144541_c3_g7_i1  −2.64  0  −3.71  Perivitellin ovorubin-2  TRINITY_DN124633_c0_g1_i1  −2.80  0  −3.88  Perivitellin ovorubin-3  G protein-coupled receptor          TRINITY_DN101258_c0_g1_i1  0  0  3.78  Opsin  TRINITY_DN116917_c0_g1_i2  3.42  0  0  5HT4 receptor  TRINITY_DN144870_c2_g1_i2  2.21  0  0  5HT2 receptor  TRINITY_DN122825_c0_g2_i3  2.78  0  0  NPFF2 receptors  TRINITY_DN122960_c0_g1_i1  4.34  0  0  Adenosine A2B receptor  TRINITY_DN132125_c0_g1_i1  3.47  0  0  Cholecystokinin receptor type A  TRINITY_DN134539_c0_g1_i4  8.28  0  0  Somatostatin receptor 2  TRINITY_DN136817_c0_g2_i12  7.05  0  0  Dopamine D3 receptor  TRINITY_DN137372_c0_g2_i1  2.82  0  0  Neurotensin type 1 receptor  TRINITY_DN137907_c1_g2_i1  4.28  0  0  CC chemokine receptor 6  Cytochrome P450(CYP)          TRINITY_DN126194_c0_g1_i1  0  0  7.56  CYP 3A43  TRINITY_DN127763_c0_g4_i5  0  0  2.44  CYP 3A24  TRINITY_DN128942_c0_g4_i1  7.77  0  −6.89  CYP 4F22  TRINITY_DN130575_c0_g1_i1  −3.71  0  0  CYP 3A7  TRINITY_DN144157_c3_g1_i3  7.55  0  0  CYP 2B4  TRINITY_DN143132_c0_g3_i2  0  0  −6.99  CYP 2C30  Cellulase          TRINITY_DN129366_c0_g1_i1  0  7.92  2.78  Endoglucanase 20  TRINITY_DN138509_c1_g3_i1  0  2.57  0  Endo-beta-1,4-glucanase  TRINITY_DN142135_c0_g2_i2  0  7.82  4.33  Family 10 cellulase  TRINITY_DN145404_c1_g1_i3  0  0  4.20  Cellulase EGX3  Nervous system          TRINITY_DN145021_c0_g6_i1  2.49  0  0  5-HT  TRINITY_DN136773_c0_g1_i4  4.30  0  0  FMRF1  TRINITY_DN133591_c0_g1_i3  −7.80  8.57  0  Moa  TRINITY_DN140497_c1_g5_i1  4.52  0  0  Ninjurin 1  Circadian rhythm          TRINITY_DN136298_c0_g1_i4  2.73  0  0  Clk  Handedness          TRINITY_DN135564_c1_g2_i8  7.06  0  0  Nodal  TRINITY_DN131526_c0_g1_i  −7.51  6.83  0  Pitx  TRINITY_DN138997_c3_g2_i2  0  6.82  0  Piwi  TRINITY_DN139424_c1_g2_i4  −2.37  0  2.73  Nck1  Shell formation          TRINITY_DN136576_c1_g1_i8  −8.98  0  6.94  Calponin  TRINITY_DN129910_c0_g17_i1  2.92  0  0  Calmodulin  TRINITY_DN129097_c0_g2_i3  0  0  7.12  Perlucin 1  Immune system          TRINITY_DN113417_c0_g1_i1  0  0  6.85  C-type lectin  TRINITY_DN143157_c0_g1_i2  0  0  5.11  I-type lectin-like protein 4  TRINITY_DN139530_c0_g1_i3  0  0  2.26  Ricin_B_lectin  TRINITY_DN122928_c1_g2_i1  0  0  7.56  C1q domain protein  TRINITY_DN144320_c0_g1_i1  9.50  0  0  Toll-like receptor 4  TRINITY_DN128784_c2_g1_i1  0  0  7.12  Concanavalin A-like lectin  TRINITY_DN134165_c0_g1_i1  0  0  4.59  Fibrinogen  TRINITY_DN132910_c0_g4_i3  7.22  0  0  F-type lectins  TRINITY_DN118405_c0_g1_i2  0  0  7.21  Gal_Lectin  TRINITY_DN129873_c0_g1_i2  −3.15  0  3.20  H-types lectins  Muscular contraction          TRINITY_DN136954_c0_g2_i9  3.21  0  0  Titin  G protein-coupled receptors (GPCRs) constitute a vast protein family that encompasses a wide range of functions, including various autocrine, paracrine and endocrine processes. Based on considerable diversity at the sequence level, GPCRs can be separated into distinct groups (Vassilatis et al., 2003). Many rhodopsin-like GPCRs (GPCRA) genes were differentially expressed during embrogenesis. GPCRA represent a widespread protein family that includes hormone, neurotransmitter and light receptors, all of which transduce extracellular signals through interaction with guanine nucleotide-binding proteins. Visual pigments (opsins) are the light-absorbing molecules that mediate vision. In the central nervous system, 5-hydroxytryptamine (5-HT) or serotonin receptors can influence various neurological processes, such as aggression, anxiety and appetite and, as a result, these are the target of a variety of pharmaceutical drugs, including many antidepressants, antipsychotics and anorectics (Nichols & Nichols, 2008). Neuropeptide FF receptors (Parker et al., 2000) belong to a family of neuropeptides containing an RF-amide motif at their C terminus, which have a high affinity for the pain modulatory peptide neuropeptide NPFF (Elshourbagy et al., 2000). GPCRs are major drug targets and may also be a target for control of P. canaliculata development. Cytochrome P450 enzymes are a superfamily of haem-containing mono-oxygenases that are found in all kingdoms of life and show extraordinary diversity in their reaction chemistry. Cytochrome P450 enzymes are important for the detoxification and clearance of various xenobiotics compounds. We identified about 60 unigenes encoding cytochrome P450 enzymes and were able to identify six that were differentially expressed during embryo developing. These findings indicate the potential for rational design of selective molluscicides—for example, by inhibiting unique cytochrome P450s or by activation of the molluscicide by cytochrome P450s only expressed in P. canaliculata juveniles (Adema et al., 2017). The nervous system appears at early developmental stages in bivalves (Dyachuk, 2016). Ninjurin (nerve injury-induced protein) is involved in nerve regeneration (Araki & Milbrandt, 1996) and we found ninjurin genes up-regulated in stage III embryos. Antibodies raised against serotonin(5-HT) and FMRFamide have proved to be especially useful as markers for the ganglia and major pathways of developing nervous systems, and imunohistochemical studies using antibodies against various neurotransmitters have also detected the development of nervous systems in early embryonic stages of gastropods (Barlow & Truman, 1992; Dickinson, Nason & Croll, 1999; Dickinson & Croll, 2003). Shell formation is important for molluscan development and begins at an early stage, e.g. the shell of Ostrea edulis first begins to form in the gastrula (Medaković et al., 1997). Several gene products related to shell formation and growth have been characterized, like calponin, Perlucin 1, calmodulin and ferritin (Fang et al., 2008; Wang, Liu & Xiang, 2009). Analysis of nervous system and shell formation genes present possible targets for control of invasive molluscan species like P. canaliculata. Invertebrates rely on innate immunity for their internal defence against pathogens. Lectins play an important role in gastropods for recognition and clearance of invaders (Bulat et al., 2016). Our analyses revealed a diversity of lectins that are differentially expressed during embyonic developing, including C-type lectins, I-type lectins, F-type lectins, Ricin_B_lectin and Gal_Lectin. A voracious appetite for aquatic plants renders the invasive P. canaliculata an ecological and economic threat in wetlands and crop fields (Youens & Burks, 2008; Hayes et al., 2012). The degradation of cellulose and xylans by the digestive system of P. canaliculata requires several types of enzymes (Gilkes et al., 1991). Expression of several cellulases was up-regulated in the later stages of embryonic development, such as endoglucanase 20, endo-beta-1,4-glucanase and cellulase EGX3. In addition, the titin gene is a critical contributor to muscular contraction (Heyland et al., 2011). Clk is a clock gene and modification of expression of clock genes may interrupt the circadian rhythm of P. canaliculata and affect feeding and egg-laying (Adema et al., 2017). In summary, we conducted a large-scale RNAseq analysis of the developing embryo of P. canaliculata. Analysis of the sequences recorded identified several DEGs with functions that likely relate to albumen gland, nervous system, circadian rhythm, immune system and shell formation. These transcriptome data will provide fundamental information for molecular ecological studies of the invasive freshwater snail P. canaliculata. SUPPLEMENTARY MATERIAL Supplementary material is available at Journal of Molluscan Studies online. ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (grant no. U1131006). The authors themselves are responsible for the content and writing of the paper. We acknowledge the help from reviewers and comments from Associate Editor C. Adema. REFERENCES Accorsi, A., Benatti, S., Ross, E., Nasi, M. & Malagoli, D. 2017. A prokineticin-like protein responds to immune challenges in the gastropod pest Pomacea canaliculata. Developmental and Comparative Immunology , 72: 37– 43. Google Scholar CrossRef Search ADS   Adema, C.M., Hillier, L.W., Jones, C.S., Loker, E.S. et al.   2017. Whole genome analysis of a schistosomiasis-transmitting freshwater snail. Nature Communications , 8: 15451. Google Scholar CrossRef Search ADS   Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. 1990. Basic local alignment search tool. Journal Molecular Biology , 215: 403– 410. Google Scholar CrossRef Search ADS   Araki, T. & Milbrandt, J. 1996. Ninjurin, a novel adhesion molecule, is induced by nerve injury and promotes axonal growth. Neuron , 17: 353– 361. Google Scholar CrossRef Search ADS   Audic, S. & Claverie, J.M. 1997. The significance of digital gene expression profiles. Genome Research , 7: 986– 995. Google Scholar CrossRef Search ADS   Barlow, L.A. & Truman, J.W. 1992. Patterns of serotonin and SCP immunoreactivity during metamorphosis of the nervous system of the red abalone, Haliotis rufescens. Journal of Neurobiology , 23: 829– 844. Google Scholar CrossRef Search ADS   Bulat, T., Smidak, R., Sialana, F.J., Jung, G., Rattei, T., Bilban, M., Sattmann, H., Lubec, G. & Aradska, J. 2016. Transcriptomic and proteomic analysis of Arion vulgaris—proteins for probably successful survival strategies? PLoS One , 11: e150614. Google Scholar CrossRef Search ADS   Cannuel, R. & Beninger, P.G. 2006. Gill development, functional and evolutionary implications in the Pacific oyster Crassostrea gigas (Bivalvia: Ostreidae). Marine Biology , 149: 547– 563. Google Scholar CrossRef Search ADS   Conesa, A., Gotz, S., Garcia-Gomez, J.M., Terol, J., Talon, M. & Robles, M. 2005. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics , 21: 3674– 3676. Google Scholar CrossRef Search ADS   Dickinson, A. & Croll, R.P. 2003. Development of the larval nervous system of the gastropod Ilyanassa obsoleta. Journal of Comparative Neurology , 466: 197– 218. Google Scholar CrossRef Search ADS   Dickinson, A.J.G., Nason, J. & Croll, R.P. 1999. Histochemical localization of FMRFamide, serotonin and catecholamines in embryonic Crepidula fornicata (Gastropoda, Prosobranchia). Zoomorphology , 119: 49– 62. Google Scholar CrossRef Search ADS   Dreon, M.S., Fernández, P.E., Gimeno, E.J. & Heras, H. 2014. Insights into embryo defenses of the invasive apple snail Pomacea canaliculata: egg mass ingestion affects rat intestine morphology and growth. PLoS Neglected Tropical Diseases , 8: e2961. Google Scholar CrossRef Search ADS   Dreon, M.S., Ituarte, S. & Heras, H. 2010. The role of the proteinase inhibitor ovorubin in apple snail eggs resembles plant embryo defense against predation. PLoS One , 5: e15059. Google Scholar CrossRef Search ADS   Dreon, M.S., Schinella, G., Heras, H. & Pollero, R.J. 2004. Antioxidant defense system in the apple snail eggs, the role of ovorubin. Archives Biochemistry and Biophysics , 422: 1– 8. Google Scholar CrossRef Search ADS   Dyachuk, V.A. 2016. Hematopoiesis in bivalvia larvae: cellular origin, differentiation of hemocytes, and neoplasia. Developmental and Comparative Immunology , 65: 253– 257. Google Scholar CrossRef Search ADS   Elshourbagy, N.A., Ames, R.S., Fitzgerald, L.R., Foley, J.J., Chambers, J.K. et al.   2000. Receptor for the pain modulatory neuropeptides FF and AF is an orphan G protein-coupled receptor. Journal of Biological Chemistry , 275: 25965– 25971. Google Scholar CrossRef Search ADS   Fang, Z., Yan, Z., Li, S., Wang, Q., Cao, W., Xu, G., Xiong, X., Xie, L. & Zhang, R. 2008. Localization of calmodulin and calmodulin-like protein and their functions in biomineralization in P. fucata. Progress in Natural Science , 18: 405– 412. Google Scholar CrossRef Search ADS   Frassa, M.V., Ceolín, M., Dreon, M.S. & Heras, H. 2010. Structure and stability of the neurotoxin PV2 from the eggs of the apple snail Pomacea canaliculata. Biochimica et Biophysica Acta , 1804: 1492– 1499. Google Scholar CrossRef Search ADS   Gilkes, N.R., Henrissat, B., Kiburn, D.G., Miller, R.C. & Warren, R.A. 1991. Domains in microbial beta-1, 4-glycanases: sequence conservation, function, and enzyme families. Microbiology Review , 55: 303– 315. Grabherr, M.G., Haas, B.J., Yassour, M., Levin, J.Z., Thompson, D.A., Amit, I. & Regev, A. 2011. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nature Biotechnology , 29: 644– 652. Google Scholar CrossRef Search ADS   Haas, B.J., Papanicolaou, A., Yassour, M., Grabherr, M., Blood, P.D., Bowden, J., Couger, M.B., Eccles, D., LI, B., Lieber, M., Macmanes, M.D., Ott, M., Orvis, J., Pochet, N., Strozzi, F., Weeks, N., Westerman, R., William, T., Dewey, C.N., Henschel, R., Leduc, R.D., Friedman, N. & Regev, A. 2013. De novo transcript sequence reconstruction from RNA-seq using the trinity platform for reference generation and analysis. Nature Protocols , 8: 1494– 1512. Google Scholar CrossRef Search ADS   Hayes, K.A., Cowie, R.H., Thiengo, S.C. & Strong, E.E. 2012. Comparing apples with apples: clarifying the identities of two highly invasive neotropical Ampullariidae (Caenogastropoda). Zoological Journal of the Linnean Society , 166: 723– 753. Google Scholar CrossRef Search ADS   Heras, H., Frassa, M.V., Fernández, P.E., Galosi, C.M., Gimeno, E.J. & Dreon, M.S. 2008. First egg protein with a neurotoxic effect on mice. Toxicon , 52: 481– 488. Google Scholar CrossRef Search ADS   Heyland, A., Vue, Z., Voolstra, C.R., Medina, M. & Moroz, L.L. 2011. Developmental transcriptome of Aplysia californica. Journal of Experimental Zoology Part B—Molecular and Developmental Evolution , 31: 113– 134. Google Scholar CrossRef Search ADS   Huan, P., Wang, H. & Liu, B. 2012. Transcriptomic analysis of the clam Meretrix meretrix on different larval stages. Marine Biotechnology , 14: 69– 78. Google Scholar CrossRef Search ADS   Jackson, D.J., Worheide, G. & Degnan, B.M. 2007. Dynamic expression of ancient and novel molluscan shell genes during ecological transitions. BMC Evolutionary Biology , 7: 160. Google Scholar CrossRef Search ADS   Koch, E., Winik, B.C. & Castro-Vazquez, A. 2009. Development beyond the gastrula stage and digestive organogenesis in the apple snail Pomacea canaliculata (Architaenioglossa, Ampullariidae). Biocell , 33: 49– 65. Lambert, J.D., Chan, X.Y., Spiecker, B. & Sweet, H.C. 2010. Characterizing the embryonic transcriptome of the snail Ilyanassa. Integrative and Comparative Biology , 50: 768– 777. Google Scholar CrossRef Search ADS   Langmead, B. & Salzberg, S.L. 2012. Fast gapped-read alignment with Bowtie 2. Nature Methods , 9: 357– 359. Google Scholar CrossRef Search ADS   Lee, M.T., Bonneau, A.R. & Giraldez, A.J. 2014. Zygotic genome activation during the maternal-to-zygotic transition. Annual Review of Cell and Developmental Biology , 30: 581– 613. Google Scholar CrossRef Search ADS   Li, B. & Dewey, C.N. 2011. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics , 12: 323. Google Scholar CrossRef Search ADS   Liu, H., Liu, S., Ge, Y., Liu, J., Wang, X., Xie, L., Zhang, R. & Wang, Z. 2007. Identification and characterization of a biomineralization related gene PFMG1 highly expressed in the mantle of Pinctada fucata. Biochemistry , 46: 844– 851. Google Scholar CrossRef Search ADS   Lv, S., Zhang, Y., Liu, H., Zhang, C., Steinmann, P., Zhou, X. & Utzinger, J. 2009. Angiostrongylus cantonensis: morphological and behavioral investigation within the freshwater snail Pomacea canaliculata. Parasitology Research , 104: 1351– 1359. Google Scholar CrossRef Search ADS   Medaković, D., Popović, S., Gržeta, B., Plazonić, M. & Hrsbrenko, M. 1997. X-ray diffraction study of calcification processes in embryos and larvae of the brooding oyster Ostrea edulis. Marine Biology , 129: 615– 623. Google Scholar CrossRef Search ADS   Naimi, A., Martinez, A.S., Specq, M.L., Diss, B., Mathieu, M. & Sourdaine, P. 2009a. Molecular cloning and gene expression of Cg-Foxl2 during the development and the adult gametogenetic cycle in the oyster Crassostrea gigas. Comparative Biochemistry Physiology Part B: Biochemistry and Molecular Biology , 154: 134– 142. Google Scholar CrossRef Search ADS   Naimi, A., Martinez, A.S., Specq, M.L., Mrac, A., Diss, B., Mathieu, M. & Sourdaine, P. 2009b. Identification and expression of a factor of the DM family in the oyster Crassostrea gigas. Comparative Biochemistry Physiology Part A: Molecular and Integrative. Physiology , 152: 189– 196. Nichols, D.E. & Nichols, C.D. 2008. Serotonin receptors. Chemical Reviews , 108: 1614– 1641. Google Scholar CrossRef Search ADS   Parker, R.M., Copeland, N.G., Eyre, H.J., Liu, M., Gilbert, D.J., Crawford, J., Couzens, M., Sutherland, G.R., Jenkins, N.A. & Herzog, H. 2000. Molecular cloning and characterisation of GPR74 a novel G-protein coupled receptor closest related to the Y-receptor family. Molecular Brain Research , 77: 199– 208. Google Scholar CrossRef Search ADS   Pertea, G., Huang, X., Liang, F., Antonescu, V., Sultana, R., Karamycheva, S., Lee, Y., White, J., Cheung, F., Parvizi, B., Tsai, J. & Quackenbush, J. 2003. TIGR gene indices clustering tools (TGICL): a software system for fast clustering of large EST datasets. Bioinformatics , 19: 651– 652. Google Scholar CrossRef Search ADS   Quast, C., Pruesse, E., Yilmaz, P., Gerken, J., Schweer, T., Yarza, P., Peplies, J. & Glockner, F.O. 2013. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Research , 41: D590– D596. Google Scholar CrossRef Search ADS   Quevillon, E., Silventoinen, V., Pillai, S., Harte, N., Mulder, N., Apweiler, R. & Lopez, R. 2005. InterProScan: protein domains identifier. Nucleic Acids Research , 33: W116– W120. Google Scholar CrossRef Search ADS   Song, L., Wang, X., Yang, Z., Lv, Z. & Wu, Z. 2016. Angiostrongylus cantonensis in the vector snails Pomacea canaliculata and Achatina fulica in China: a meta-analysis. Parasitology Research , 115: 913– 923. Google Scholar CrossRef Search ADS   Sun, J., Zhang, Y., Thiyagarajan, V., Qian, P. & Qiu, J. 2010. Protein expression during the embryonic development of a gastropod. Proteomics , 10: 2701– 2711. Google Scholar CrossRef Search ADS   Sun, J., Zhang, H., Wang, H., Heras, H., Dreon, M.S., Ituarte, S., Ravasi, T., Qian, P. & Qiu, J. 2012. First proteome of the egg perivitelline fluid of a freshwater gastropod with aerial oviposition. Journal of Proteome Research , 11: 4240– 4248. Google Scholar CrossRef Search ADS   Vassilatis, D.K., Hohmann, J.G., Zeng, H., Li, F., Ranchalis, J.E., Mortrud, M.T., Brown, A., Rodriguez, S.S., Weller, J.R., Wright, A.C., Bergmann, J.E. & Gaitanaris, G.A. 2003. The G protein-coupled receptor repertoires of human and mouse. Proceedings of the National Academy of Sciences of the USA , 100: 4903– 4908. Google Scholar CrossRef Search ADS   Wang, X., Liu, B. & Xiang, J. 2009. Cloning, characterization and expression of ferritin subunit from clam Meretrix meretrix in different larval stages. Comparative Biochemistry and Physiology Part B: Biochemisry and Molecular Biology , 154: 12– 16. Google Scholar CrossRef Search ADS   Youens, A.K. & Burks, R.L. 2008. Comparing apple snails with oranges: the need to standardize measuring techniques when studying Pomacea. Aquatic Ecology , 42: 679– 684. Google Scholar CrossRef Search ADS   © The Author(s) 2018. Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Molluscan Studies Oxford University Press

Transcriptomic analysis of embryo development in the invasive snail Pomacea canaliculata

Loading next page...
 
/lp/ou_press/transcriptomic-analysis-of-embryo-development-in-the-invasive-snail-DsBOKcakXw
Publisher
Oxford University Press
Copyright
© The Author(s) 2018. Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved. For Permissions, please email: journals.permissions@oup.com
ISSN
0260-1230
eISSN
1464-3766
D.O.I.
10.1093/mollus/eyy024
Publisher site
See Article on Publisher Site

Abstract

Abstract A scarcity of genomic data hinders progress in understanding gene expression of Pomacea canaliculata embryogenesis. This study used RNA-Seq (Illumina) to characterize gene expression across four stages of embryo development of P. canaliculata from early stage II (endoderm formation) to later stage V (appearance of shell pigmentation). A total of 35,100 unigenes were annotated and gene expression patterns were considered in relation to organogenesis of albumen gland, nervous system, circadian rhythm, immune system and shell formation. This is the first use of next-generation sequencing to catalogue gene expression in the developing embryo of P. canaliculata and provides primary data for future molecular ecological studies of this invasive snail. INTRODUCTION The apple snail Pomacea canaliculata is the only freshwater snail listed among the 100 worst invasive species worldwide (Dreon et al., 2014). The species originates from South America, but is now found in North America, Asia and Europe (Accorsi et al., 2017). Large size and great appetite for aquatic plants make P. canaliculata a threat with potential ecological and economical risks because of damage to wetlands and crops (Youens & Burks, 2008; Hayes et al., 2012). Additionally, P. canaliculata is the intermediate host of the nematode Angiostrongylus cantonensis, which is responsible for potentially lethal encephalitis (Lv et al., 2009; Song et al., 2016). At present there are no accepted and efficient strategies for controlling the spread of P. canaliculata. The understanding of molecular processes underlying embryonic development may contribute to the generation of efficient and reliable molecular tools to control this invasive snail. Successful establishment in invaded areas may relate to features of P. canaliculata including highly effective mechanisms to deal with adverse environments and natural predators. Notably, the reproduction of P. canaliculata involves deposition of hundreds of bright pink or reddish coloured egg masses above water. The calcareous egg shell and a neurotoxin-like protein in the perivitelline fluid are successful mechanisms to deal with environmental stresses such as UV radiation and predators (Frassa et al., 2010; Sun et al., 2012). The process of embryonic development in P. canaliculata is not well understood. Previous studies have emphasized the nutritional and protective functions of perivitelline fluid protein, but little is known about the gene-expression patterns during embryonic development. Similar to most other gastropods, the embryonic development of P. canaliculata includes the formation of a shell. The process of shell formation involves changes in body shape and deposition of minerals and pigments in a matrix of proteins (Liu et al., 2007). The dramatic changes during embryonic development require expression and action of many genes, which coordinate and modulate various developmental events (Jackson, Worheide & Degnan, 2007). Previous investigations of embryonic development in marine bivalves, including scallop and oyster species, have identified genes, involved in organogenesis, shell formation and utilization of nutrients (Cannuel & Beninger, 2006; Naimi et al., 2009a, b). The sequencing of several molluscan genomes has been accomplished, including those of Lottia gigantea, Crassostrea gigas and Aplysia californica. The availability of these resources provides opportunities to perform comparative and genome-wide analyses of development on a transcriptional level. Application of transcriptomic technologies provides insight into the spatial and temporal dynamics of gene expression during consecutive stages of embryonic development, revealing details of gene regulation. This approach has been applied to study the embryos of several species of molluscs, such as the clam Meretrix meretrix (Huan, Wang & Liu, 2012) and the gastropods A. californica (Heyland et al., 2011) and Ilyanassa obsoleta (Lambert et al., 2010). This study uses the Illumina HiSeq 4000 platform to construct a database representing an annotated transcriptome assembly for developing P. canaliculata embryos. These transcriptome data should provide a resource for future research on P. canaliculata embryonic development. MATERIAL AND METHODS Egg incubation and embryo collection Eggs clutches laid by Pomacea canaliculata were collected from a laboratory aquarium (South China Agricultural University, Guangzhou, People’s Republic of China) and incubated at 25 °C. Each day, shells of some eggs from individual clutches were broken using forceps to obtain embryos for microscopic identification of the stage of development (based on previous studies of Koch, Winik & Castro-Vazquez, 2009; Sun et al., 2010; Fig. 1). We analysed embryonic development at stage II (endoderm formation), stage III (internal and external organs formed), stage IV (shell formation) and stage V (appearance of shell pigmentation). Developing embryos that had reached the desired stage were placed in tubes and snap-frozen in liquid nitrogen. Samples were stored at −80 °C before the experiments described below. Figure 1. View largeDownload slide Embryonic development of Pomacea. canaliculata, showing embryos removed from eggs consecutively at stages II to V. Scale bars: 500 μm (stages II and III); 1000 μm (stages IV and V). Figure 1. View largeDownload slide Embryonic development of Pomacea. canaliculata, showing embryos removed from eggs consecutively at stages II to V. Scale bars: 500 μm (stages II and III); 1000 μm (stages IV and V). Transcriptome sequencing and de novo assembly Total RNA was extracted from each sample separately, using TRIzol Reagent (ThermoFisher), according to the manufacturer’s instructions. After treating total RNA samples with DNase I (TaKaRa), oligo(dT)-absorption (ThermoFisher) was used to isolate mRNA. The mRNA was fragmented by mixing with the fragmentation buffer and cDNA libraries (NEBNext® Ultra™ RNA Library Prep Kit) were synthesized from the mRNA fragments. Quality control and quantification of the sample libraries employed an Agilent 2100 Bioanalyzer and the ABI StepOnePlus Real-Time PCR System. The resulting libraries were sequenced (paired ends and select cDNA fragments preferentially of 200–250 bp in length) using an Illumina HiSeq 4000 platform. Low-quality raw reads (containing adaptors or with high content of unknown base calls) were identified using cutadapt software (Grabherr et al., 2011). High quality reads of each sample were assembled using Trinity software (Haas et al., 2013) and Tgicl (Pertea et al., 2003) to cluster transcripts to unigenes. The clustered unigenes were compared to SILVA (Quast et al., 2013) ribosomal RNA databases to exclude those matching rRNA sequences (score > 60). Functional annotation of unigenes The unigenes of the transcriptome datasets were annotated using Blast (Altschul et al., 1990) to nr GenBank database, eggNOG, KEGG and Swissprot. Blast2GO (Conesa et al., 2005) and Interproscan5 (Quevillon et al., 2005) were used for GO annotation and Interpro annotation. For transcriptome analysis, clean reads were mapped to unigenes using Bowtie2 (Langmead & Salzberg, 2012) and gene expression levels were calculated according to a previous study using the RSEM method that quantifies total expressed transcripts with the TPM (transcripts per million) value (Li & Dewey, 2011). Then PCA analysis was performed using all samples with ‘princomp’, a function of R software. Differentially expressed genes (DEG) were detected with PossionDis as described by Audic & Claverie (1997). RESULTS AND DISCUSSION We used Illumina next-generation sequencing technology to record gene expression patterns during the embryonic development of Pomacea canaliculata. All of the clean reads have been deposited in the NCBI SRA database under accession number SRX2538617. Transcriptome assembly yielded 241,904 high-confidence unigenes with average length 792 bp. Removal of short sequences (<500 bp) yielded 35,100 unigenes. Using six different approaches (NCBI-nr, Swissprot, eggNOG, Interpro, GO and KEGG) provided computational annotation for 31,475 (71.68%) unigenes (Supplementary Material). The taxonomic distribution of best matches indicated that greater than 81% of annotated sequences best matched with molluscan species. Gene ontology (GO) analysis assigned annotated unigenes to the first-level GO categories of biological process, cellular component and molecular function, respectively (Fig. 2). The most abundant second-level terms are cellular process (38.62% of annotated unigenes) and metabolic process (34.23%) for the biological process category, membrane (25.14%) and membrane part (14.58%) for the cellular component category, and binding (53.93%) and catalytic activity (35.60%) for the molecular function category. Figure 2. View largeDownload slide GO classification of unigenes. X axis represents percent of unigenes. Y axis represents GO term. Because one unigene can be attributed to more than one term, the sum of all terms is larger than 100%. Figure 2. View largeDownload slide GO classification of unigenes. X axis represents percent of unigenes. Y axis represents GO term. Because one unigene can be attributed to more than one term, the sum of all terms is larger than 100%. At present, the genetic foundation, factors and molecular mechanisms that drive the progression of stages in embryonic development of P. canaliculata are mostly unknown. Comparative analysis of gene-expression levels will extend our knowledge and understanding of growth and development-related unigenes. Annotation of DEGs provided the five most enriched GO terms for each stage of development (Table 1). Between stage II and stage III, when the embryo transitions from endoderm formation to the onset of organogenesis, the most abundant DEGs are associated with receptor activity of molecular function. At stage IV (when most external and internal organs are fully formed), the most highly expressed DEGs relate to metabolic processes. Table 1. The five most enriched GO term of DEG among embryonic stages of Pomacea canaliculata.   ID  Description  Class  DEGs  Stage II vs stage III  GO:0060089  Molecular transducer activity  Molecular function  192  GO:0004872  Receptor activity  Molecular function  192  GO:0004888  Tansmembrane signalling receptor activity  Molecular function  135  GO:0038023  Signalling receptor activity  Molecular function  145  GO:0004871  Signal transducer activity  Molecular function  149  Stage III vs stage IV  GO:0016798  Hydrolase activity, acting on glycosyl bonds  Molecular function  72  GO:0004553  Hydrolase activity, hydrolysing O-glycosyl compounds  Molecular function  64  GO:0005576  Extracellular region  Cellular component  54  GO:0005975  Carbohydrate metabolic process  Biological process  75  GO:0006022  Aminoglycan metabolic process  Biological process  36  Stage IV vs stage V  GO:0016798  Hydrolase activity, acting on glycosyl bonds  Molecular function  67  GO:0004553  Hydrolase activity, hydrolysing O-glycosyl compounds  Molecular function  60  GO:0005576  Extracellular region  Cellular component  51  GO:0006030  Chitin metabolic process  Biological process  32  GO:1901071  Glucosamine-containing compound metabolic process  Biological process  32    ID  Description  Class  DEGs  Stage II vs stage III  GO:0060089  Molecular transducer activity  Molecular function  192  GO:0004872  Receptor activity  Molecular function  192  GO:0004888  Tansmembrane signalling receptor activity  Molecular function  135  GO:0038023  Signalling receptor activity  Molecular function  145  GO:0004871  Signal transducer activity  Molecular function  149  Stage III vs stage IV  GO:0016798  Hydrolase activity, acting on glycosyl bonds  Molecular function  72  GO:0004553  Hydrolase activity, hydrolysing O-glycosyl compounds  Molecular function  64  GO:0005576  Extracellular region  Cellular component  54  GO:0005975  Carbohydrate metabolic process  Biological process  75  GO:0006022  Aminoglycan metabolic process  Biological process  36  Stage IV vs stage V  GO:0016798  Hydrolase activity, acting on glycosyl bonds  Molecular function  67  GO:0004553  Hydrolase activity, hydrolysing O-glycosyl compounds  Molecular function  60  GO:0005576  Extracellular region  Cellular component  51  GO:0006030  Chitin metabolic process  Biological process  32  GO:1901071  Glucosamine-containing compound metabolic process  Biological process  32  Table 1. The five most enriched GO term of DEG among embryonic stages of Pomacea canaliculata.   ID  Description  Class  DEGs  Stage II vs stage III  GO:0060089  Molecular transducer activity  Molecular function  192  GO:0004872  Receptor activity  Molecular function  192  GO:0004888  Tansmembrane signalling receptor activity  Molecular function  135  GO:0038023  Signalling receptor activity  Molecular function  145  GO:0004871  Signal transducer activity  Molecular function  149  Stage III vs stage IV  GO:0016798  Hydrolase activity, acting on glycosyl bonds  Molecular function  72  GO:0004553  Hydrolase activity, hydrolysing O-glycosyl compounds  Molecular function  64  GO:0005576  Extracellular region  Cellular component  54  GO:0005975  Carbohydrate metabolic process  Biological process  75  GO:0006022  Aminoglycan metabolic process  Biological process  36  Stage IV vs stage V  GO:0016798  Hydrolase activity, acting on glycosyl bonds  Molecular function  67  GO:0004553  Hydrolase activity, hydrolysing O-glycosyl compounds  Molecular function  60  GO:0005576  Extracellular region  Cellular component  51  GO:0006030  Chitin metabolic process  Biological process  32  GO:1901071  Glucosamine-containing compound metabolic process  Biological process  32    ID  Description  Class  DEGs  Stage II vs stage III  GO:0060089  Molecular transducer activity  Molecular function  192  GO:0004872  Receptor activity  Molecular function  192  GO:0004888  Tansmembrane signalling receptor activity  Molecular function  135  GO:0038023  Signalling receptor activity  Molecular function  145  GO:0004871  Signal transducer activity  Molecular function  149  Stage III vs stage IV  GO:0016798  Hydrolase activity, acting on glycosyl bonds  Molecular function  72  GO:0004553  Hydrolase activity, hydrolysing O-glycosyl compounds  Molecular function  64  GO:0005576  Extracellular region  Cellular component  54  GO:0005975  Carbohydrate metabolic process  Biological process  75  GO:0006022  Aminoglycan metabolic process  Biological process  36  Stage IV vs stage V  GO:0016798  Hydrolase activity, acting on glycosyl bonds  Molecular function  67  GO:0004553  Hydrolase activity, hydrolysing O-glycosyl compounds  Molecular function  60  GO:0005576  Extracellular region  Cellular component  51  GO:0006030  Chitin metabolic process  Biological process  32  GO:1901071  Glucosamine-containing compound metabolic process  Biological process  32  Based on differential expression patterns among developmental stages, unigenes that are of interest for the study of embryonic development of P. canaliculata were identified (Table 2). The albumen gland of P. canaliculata secretes perivitelline proteins, including perivitelline ovorubin proteins (Sun et al., 2012), that surround the developing embryo. These proteins not only provide all nutrients and energy for embryonic development, but also protect the eggs from environmental stressors and predators (Dreon et al., 2004; Heras et al., 2008; Dreon, Ituarte & Heras, 2010). Three ovorubin-encoding genes (perivitellin ovorubin-1, perivitellin ovorubin-2, perivitellin ovorubin-3) were highly expressed in stage II, with expression levels decreased significantly in the three later stages. The underlying mechanism is unclear, considering that in animals the maternal factors that contributed to the egg cytoplasm initially control development, while the zygotic nuclear genome is quiescent. Subsequently, the genome is activated, embryonic gene products are mobilized and maternal factors are cleared (Lee, Bonneau & Giraldez, 2014). The high expression of perivitellin ovorubin protein-encoding genes during early embrogenesis suggests that the embryos may either receive mRNA transcripts from the parent to maintain a certain level of defence, or that the embryos transcribe and translate genes to contribute to their own defence against predation, in addition to that from parentally-provided perivitellin ovorubin proteins. Table 2. Select differentially expressed genes among the four embryonic stages of Pomacea canaliculata. Unigenes  Stage II/Stage I  Stage III/Stage II  Stage IV/Stage III  Protein name    Fold change    Albumen gland          TRINITY_DN119492_c0_g1_i1  −2.83  0  −2.65  Perivitellin ovorubin-1  TRINITY_DN144541_c3_g7_i1  −2.64  0  −3.71  Perivitellin ovorubin-2  TRINITY_DN124633_c0_g1_i1  −2.80  0  −3.88  Perivitellin ovorubin-3  G protein-coupled receptor          TRINITY_DN101258_c0_g1_i1  0  0  3.78  Opsin  TRINITY_DN116917_c0_g1_i2  3.42  0  0  5HT4 receptor  TRINITY_DN144870_c2_g1_i2  2.21  0  0  5HT2 receptor  TRINITY_DN122825_c0_g2_i3  2.78  0  0  NPFF2 receptors  TRINITY_DN122960_c0_g1_i1  4.34  0  0  Adenosine A2B receptor  TRINITY_DN132125_c0_g1_i1  3.47  0  0  Cholecystokinin receptor type A  TRINITY_DN134539_c0_g1_i4  8.28  0  0  Somatostatin receptor 2  TRINITY_DN136817_c0_g2_i12  7.05  0  0  Dopamine D3 receptor  TRINITY_DN137372_c0_g2_i1  2.82  0  0  Neurotensin type 1 receptor  TRINITY_DN137907_c1_g2_i1  4.28  0  0  CC chemokine receptor 6  Cytochrome P450(CYP)          TRINITY_DN126194_c0_g1_i1  0  0  7.56  CYP 3A43  TRINITY_DN127763_c0_g4_i5  0  0  2.44  CYP 3A24  TRINITY_DN128942_c0_g4_i1  7.77  0  −6.89  CYP 4F22  TRINITY_DN130575_c0_g1_i1  −3.71  0  0  CYP 3A7  TRINITY_DN144157_c3_g1_i3  7.55  0  0  CYP 2B4  TRINITY_DN143132_c0_g3_i2  0  0  −6.99  CYP 2C30  Cellulase          TRINITY_DN129366_c0_g1_i1  0  7.92  2.78  Endoglucanase 20  TRINITY_DN138509_c1_g3_i1  0  2.57  0  Endo-beta-1,4-glucanase  TRINITY_DN142135_c0_g2_i2  0  7.82  4.33  Family 10 cellulase  TRINITY_DN145404_c1_g1_i3  0  0  4.20  Cellulase EGX3  Nervous system          TRINITY_DN145021_c0_g6_i1  2.49  0  0  5-HT  TRINITY_DN136773_c0_g1_i4  4.30  0  0  FMRF1  TRINITY_DN133591_c0_g1_i3  −7.80  8.57  0  Moa  TRINITY_DN140497_c1_g5_i1  4.52  0  0  Ninjurin 1  Circadian rhythm          TRINITY_DN136298_c0_g1_i4  2.73  0  0  Clk  Handedness          TRINITY_DN135564_c1_g2_i8  7.06  0  0  Nodal  TRINITY_DN131526_c0_g1_i  −7.51  6.83  0  Pitx  TRINITY_DN138997_c3_g2_i2  0  6.82  0  Piwi  TRINITY_DN139424_c1_g2_i4  −2.37  0  2.73  Nck1  Shell formation          TRINITY_DN136576_c1_g1_i8  −8.98  0  6.94  Calponin  TRINITY_DN129910_c0_g17_i1  2.92  0  0  Calmodulin  TRINITY_DN129097_c0_g2_i3  0  0  7.12  Perlucin 1  Immune system          TRINITY_DN113417_c0_g1_i1  0  0  6.85  C-type lectin  TRINITY_DN143157_c0_g1_i2  0  0  5.11  I-type lectin-like protein 4  TRINITY_DN139530_c0_g1_i3  0  0  2.26  Ricin_B_lectin  TRINITY_DN122928_c1_g2_i1  0  0  7.56  C1q domain protein  TRINITY_DN144320_c0_g1_i1  9.50  0  0  Toll-like receptor 4  TRINITY_DN128784_c2_g1_i1  0  0  7.12  Concanavalin A-like lectin  TRINITY_DN134165_c0_g1_i1  0  0  4.59  Fibrinogen  TRINITY_DN132910_c0_g4_i3  7.22  0  0  F-type lectins  TRINITY_DN118405_c0_g1_i2  0  0  7.21  Gal_Lectin  TRINITY_DN129873_c0_g1_i2  −3.15  0  3.20  H-types lectins  Muscular contraction          TRINITY_DN136954_c0_g2_i9  3.21  0  0  Titin  Unigenes  Stage II/Stage I  Stage III/Stage II  Stage IV/Stage III  Protein name    Fold change    Albumen gland          TRINITY_DN119492_c0_g1_i1  −2.83  0  −2.65  Perivitellin ovorubin-1  TRINITY_DN144541_c3_g7_i1  −2.64  0  −3.71  Perivitellin ovorubin-2  TRINITY_DN124633_c0_g1_i1  −2.80  0  −3.88  Perivitellin ovorubin-3  G protein-coupled receptor          TRINITY_DN101258_c0_g1_i1  0  0  3.78  Opsin  TRINITY_DN116917_c0_g1_i2  3.42  0  0  5HT4 receptor  TRINITY_DN144870_c2_g1_i2  2.21  0  0  5HT2 receptor  TRINITY_DN122825_c0_g2_i3  2.78  0  0  NPFF2 receptors  TRINITY_DN122960_c0_g1_i1  4.34  0  0  Adenosine A2B receptor  TRINITY_DN132125_c0_g1_i1  3.47  0  0  Cholecystokinin receptor type A  TRINITY_DN134539_c0_g1_i4  8.28  0  0  Somatostatin receptor 2  TRINITY_DN136817_c0_g2_i12  7.05  0  0  Dopamine D3 receptor  TRINITY_DN137372_c0_g2_i1  2.82  0  0  Neurotensin type 1 receptor  TRINITY_DN137907_c1_g2_i1  4.28  0  0  CC chemokine receptor 6  Cytochrome P450(CYP)          TRINITY_DN126194_c0_g1_i1  0  0  7.56  CYP 3A43  TRINITY_DN127763_c0_g4_i5  0  0  2.44  CYP 3A24  TRINITY_DN128942_c0_g4_i1  7.77  0  −6.89  CYP 4F22  TRINITY_DN130575_c0_g1_i1  −3.71  0  0  CYP 3A7  TRINITY_DN144157_c3_g1_i3  7.55  0  0  CYP 2B4  TRINITY_DN143132_c0_g3_i2  0  0  −6.99  CYP 2C30  Cellulase          TRINITY_DN129366_c0_g1_i1  0  7.92  2.78  Endoglucanase 20  TRINITY_DN138509_c1_g3_i1  0  2.57  0  Endo-beta-1,4-glucanase  TRINITY_DN142135_c0_g2_i2  0  7.82  4.33  Family 10 cellulase  TRINITY_DN145404_c1_g1_i3  0  0  4.20  Cellulase EGX3  Nervous system          TRINITY_DN145021_c0_g6_i1  2.49  0  0  5-HT  TRINITY_DN136773_c0_g1_i4  4.30  0  0  FMRF1  TRINITY_DN133591_c0_g1_i3  −7.80  8.57  0  Moa  TRINITY_DN140497_c1_g5_i1  4.52  0  0  Ninjurin 1  Circadian rhythm          TRINITY_DN136298_c0_g1_i4  2.73  0  0  Clk  Handedness          TRINITY_DN135564_c1_g2_i8  7.06  0  0  Nodal  TRINITY_DN131526_c0_g1_i  −7.51  6.83  0  Pitx  TRINITY_DN138997_c3_g2_i2  0  6.82  0  Piwi  TRINITY_DN139424_c1_g2_i4  −2.37  0  2.73  Nck1  Shell formation          TRINITY_DN136576_c1_g1_i8  −8.98  0  6.94  Calponin  TRINITY_DN129910_c0_g17_i1  2.92  0  0  Calmodulin  TRINITY_DN129097_c0_g2_i3  0  0  7.12  Perlucin 1  Immune system          TRINITY_DN113417_c0_g1_i1  0  0  6.85  C-type lectin  TRINITY_DN143157_c0_g1_i2  0  0  5.11  I-type lectin-like protein 4  TRINITY_DN139530_c0_g1_i3  0  0  2.26  Ricin_B_lectin  TRINITY_DN122928_c1_g2_i1  0  0  7.56  C1q domain protein  TRINITY_DN144320_c0_g1_i1  9.50  0  0  Toll-like receptor 4  TRINITY_DN128784_c2_g1_i1  0  0  7.12  Concanavalin A-like lectin  TRINITY_DN134165_c0_g1_i1  0  0  4.59  Fibrinogen  TRINITY_DN132910_c0_g4_i3  7.22  0  0  F-type lectins  TRINITY_DN118405_c0_g1_i2  0  0  7.21  Gal_Lectin  TRINITY_DN129873_c0_g1_i2  −3.15  0  3.20  H-types lectins  Muscular contraction          TRINITY_DN136954_c0_g2_i9  3.21  0  0  Titin  Table 2. Select differentially expressed genes among the four embryonic stages of Pomacea canaliculata. Unigenes  Stage II/Stage I  Stage III/Stage II  Stage IV/Stage III  Protein name    Fold change    Albumen gland          TRINITY_DN119492_c0_g1_i1  −2.83  0  −2.65  Perivitellin ovorubin-1  TRINITY_DN144541_c3_g7_i1  −2.64  0  −3.71  Perivitellin ovorubin-2  TRINITY_DN124633_c0_g1_i1  −2.80  0  −3.88  Perivitellin ovorubin-3  G protein-coupled receptor          TRINITY_DN101258_c0_g1_i1  0  0  3.78  Opsin  TRINITY_DN116917_c0_g1_i2  3.42  0  0  5HT4 receptor  TRINITY_DN144870_c2_g1_i2  2.21  0  0  5HT2 receptor  TRINITY_DN122825_c0_g2_i3  2.78  0  0  NPFF2 receptors  TRINITY_DN122960_c0_g1_i1  4.34  0  0  Adenosine A2B receptor  TRINITY_DN132125_c0_g1_i1  3.47  0  0  Cholecystokinin receptor type A  TRINITY_DN134539_c0_g1_i4  8.28  0  0  Somatostatin receptor 2  TRINITY_DN136817_c0_g2_i12  7.05  0  0  Dopamine D3 receptor  TRINITY_DN137372_c0_g2_i1  2.82  0  0  Neurotensin type 1 receptor  TRINITY_DN137907_c1_g2_i1  4.28  0  0  CC chemokine receptor 6  Cytochrome P450(CYP)          TRINITY_DN126194_c0_g1_i1  0  0  7.56  CYP 3A43  TRINITY_DN127763_c0_g4_i5  0  0  2.44  CYP 3A24  TRINITY_DN128942_c0_g4_i1  7.77  0  −6.89  CYP 4F22  TRINITY_DN130575_c0_g1_i1  −3.71  0  0  CYP 3A7  TRINITY_DN144157_c3_g1_i3  7.55  0  0  CYP 2B4  TRINITY_DN143132_c0_g3_i2  0  0  −6.99  CYP 2C30  Cellulase          TRINITY_DN129366_c0_g1_i1  0  7.92  2.78  Endoglucanase 20  TRINITY_DN138509_c1_g3_i1  0  2.57  0  Endo-beta-1,4-glucanase  TRINITY_DN142135_c0_g2_i2  0  7.82  4.33  Family 10 cellulase  TRINITY_DN145404_c1_g1_i3  0  0  4.20  Cellulase EGX3  Nervous system          TRINITY_DN145021_c0_g6_i1  2.49  0  0  5-HT  TRINITY_DN136773_c0_g1_i4  4.30  0  0  FMRF1  TRINITY_DN133591_c0_g1_i3  −7.80  8.57  0  Moa  TRINITY_DN140497_c1_g5_i1  4.52  0  0  Ninjurin 1  Circadian rhythm          TRINITY_DN136298_c0_g1_i4  2.73  0  0  Clk  Handedness          TRINITY_DN135564_c1_g2_i8  7.06  0  0  Nodal  TRINITY_DN131526_c0_g1_i  −7.51  6.83  0  Pitx  TRINITY_DN138997_c3_g2_i2  0  6.82  0  Piwi  TRINITY_DN139424_c1_g2_i4  −2.37  0  2.73  Nck1  Shell formation          TRINITY_DN136576_c1_g1_i8  −8.98  0  6.94  Calponin  TRINITY_DN129910_c0_g17_i1  2.92  0  0  Calmodulin  TRINITY_DN129097_c0_g2_i3  0  0  7.12  Perlucin 1  Immune system          TRINITY_DN113417_c0_g1_i1  0  0  6.85  C-type lectin  TRINITY_DN143157_c0_g1_i2  0  0  5.11  I-type lectin-like protein 4  TRINITY_DN139530_c0_g1_i3  0  0  2.26  Ricin_B_lectin  TRINITY_DN122928_c1_g2_i1  0  0  7.56  C1q domain protein  TRINITY_DN144320_c0_g1_i1  9.50  0  0  Toll-like receptor 4  TRINITY_DN128784_c2_g1_i1  0  0  7.12  Concanavalin A-like lectin  TRINITY_DN134165_c0_g1_i1  0  0  4.59  Fibrinogen  TRINITY_DN132910_c0_g4_i3  7.22  0  0  F-type lectins  TRINITY_DN118405_c0_g1_i2  0  0  7.21  Gal_Lectin  TRINITY_DN129873_c0_g1_i2  −3.15  0  3.20  H-types lectins  Muscular contraction          TRINITY_DN136954_c0_g2_i9  3.21  0  0  Titin  Unigenes  Stage II/Stage I  Stage III/Stage II  Stage IV/Stage III  Protein name    Fold change    Albumen gland          TRINITY_DN119492_c0_g1_i1  −2.83  0  −2.65  Perivitellin ovorubin-1  TRINITY_DN144541_c3_g7_i1  −2.64  0  −3.71  Perivitellin ovorubin-2  TRINITY_DN124633_c0_g1_i1  −2.80  0  −3.88  Perivitellin ovorubin-3  G protein-coupled receptor          TRINITY_DN101258_c0_g1_i1  0  0  3.78  Opsin  TRINITY_DN116917_c0_g1_i2  3.42  0  0  5HT4 receptor  TRINITY_DN144870_c2_g1_i2  2.21  0  0  5HT2 receptor  TRINITY_DN122825_c0_g2_i3  2.78  0  0  NPFF2 receptors  TRINITY_DN122960_c0_g1_i1  4.34  0  0  Adenosine A2B receptor  TRINITY_DN132125_c0_g1_i1  3.47  0  0  Cholecystokinin receptor type A  TRINITY_DN134539_c0_g1_i4  8.28  0  0  Somatostatin receptor 2  TRINITY_DN136817_c0_g2_i12  7.05  0  0  Dopamine D3 receptor  TRINITY_DN137372_c0_g2_i1  2.82  0  0  Neurotensin type 1 receptor  TRINITY_DN137907_c1_g2_i1  4.28  0  0  CC chemokine receptor 6  Cytochrome P450(CYP)          TRINITY_DN126194_c0_g1_i1  0  0  7.56  CYP 3A43  TRINITY_DN127763_c0_g4_i5  0  0  2.44  CYP 3A24  TRINITY_DN128942_c0_g4_i1  7.77  0  −6.89  CYP 4F22  TRINITY_DN130575_c0_g1_i1  −3.71  0  0  CYP 3A7  TRINITY_DN144157_c3_g1_i3  7.55  0  0  CYP 2B4  TRINITY_DN143132_c0_g3_i2  0  0  −6.99  CYP 2C30  Cellulase          TRINITY_DN129366_c0_g1_i1  0  7.92  2.78  Endoglucanase 20  TRINITY_DN138509_c1_g3_i1  0  2.57  0  Endo-beta-1,4-glucanase  TRINITY_DN142135_c0_g2_i2  0  7.82  4.33  Family 10 cellulase  TRINITY_DN145404_c1_g1_i3  0  0  4.20  Cellulase EGX3  Nervous system          TRINITY_DN145021_c0_g6_i1  2.49  0  0  5-HT  TRINITY_DN136773_c0_g1_i4  4.30  0  0  FMRF1  TRINITY_DN133591_c0_g1_i3  −7.80  8.57  0  Moa  TRINITY_DN140497_c1_g5_i1  4.52  0  0  Ninjurin 1  Circadian rhythm          TRINITY_DN136298_c0_g1_i4  2.73  0  0  Clk  Handedness          TRINITY_DN135564_c1_g2_i8  7.06  0  0  Nodal  TRINITY_DN131526_c0_g1_i  −7.51  6.83  0  Pitx  TRINITY_DN138997_c3_g2_i2  0  6.82  0  Piwi  TRINITY_DN139424_c1_g2_i4  −2.37  0  2.73  Nck1  Shell formation          TRINITY_DN136576_c1_g1_i8  −8.98  0  6.94  Calponin  TRINITY_DN129910_c0_g17_i1  2.92  0  0  Calmodulin  TRINITY_DN129097_c0_g2_i3  0  0  7.12  Perlucin 1  Immune system          TRINITY_DN113417_c0_g1_i1  0  0  6.85  C-type lectin  TRINITY_DN143157_c0_g1_i2  0  0  5.11  I-type lectin-like protein 4  TRINITY_DN139530_c0_g1_i3  0  0  2.26  Ricin_B_lectin  TRINITY_DN122928_c1_g2_i1  0  0  7.56  C1q domain protein  TRINITY_DN144320_c0_g1_i1  9.50  0  0  Toll-like receptor 4  TRINITY_DN128784_c2_g1_i1  0  0  7.12  Concanavalin A-like lectin  TRINITY_DN134165_c0_g1_i1  0  0  4.59  Fibrinogen  TRINITY_DN132910_c0_g4_i3  7.22  0  0  F-type lectins  TRINITY_DN118405_c0_g1_i2  0  0  7.21  Gal_Lectin  TRINITY_DN129873_c0_g1_i2  −3.15  0  3.20  H-types lectins  Muscular contraction          TRINITY_DN136954_c0_g2_i9  3.21  0  0  Titin  G protein-coupled receptors (GPCRs) constitute a vast protein family that encompasses a wide range of functions, including various autocrine, paracrine and endocrine processes. Based on considerable diversity at the sequence level, GPCRs can be separated into distinct groups (Vassilatis et al., 2003). Many rhodopsin-like GPCRs (GPCRA) genes were differentially expressed during embrogenesis. GPCRA represent a widespread protein family that includes hormone, neurotransmitter and light receptors, all of which transduce extracellular signals through interaction with guanine nucleotide-binding proteins. Visual pigments (opsins) are the light-absorbing molecules that mediate vision. In the central nervous system, 5-hydroxytryptamine (5-HT) or serotonin receptors can influence various neurological processes, such as aggression, anxiety and appetite and, as a result, these are the target of a variety of pharmaceutical drugs, including many antidepressants, antipsychotics and anorectics (Nichols & Nichols, 2008). Neuropeptide FF receptors (Parker et al., 2000) belong to a family of neuropeptides containing an RF-amide motif at their C terminus, which have a high affinity for the pain modulatory peptide neuropeptide NPFF (Elshourbagy et al., 2000). GPCRs are major drug targets and may also be a target for control of P. canaliculata development. Cytochrome P450 enzymes are a superfamily of haem-containing mono-oxygenases that are found in all kingdoms of life and show extraordinary diversity in their reaction chemistry. Cytochrome P450 enzymes are important for the detoxification and clearance of various xenobiotics compounds. We identified about 60 unigenes encoding cytochrome P450 enzymes and were able to identify six that were differentially expressed during embryo developing. These findings indicate the potential for rational design of selective molluscicides—for example, by inhibiting unique cytochrome P450s or by activation of the molluscicide by cytochrome P450s only expressed in P. canaliculata juveniles (Adema et al., 2017). The nervous system appears at early developmental stages in bivalves (Dyachuk, 2016). Ninjurin (nerve injury-induced protein) is involved in nerve regeneration (Araki & Milbrandt, 1996) and we found ninjurin genes up-regulated in stage III embryos. Antibodies raised against serotonin(5-HT) and FMRFamide have proved to be especially useful as markers for the ganglia and major pathways of developing nervous systems, and imunohistochemical studies using antibodies against various neurotransmitters have also detected the development of nervous systems in early embryonic stages of gastropods (Barlow & Truman, 1992; Dickinson, Nason & Croll, 1999; Dickinson & Croll, 2003). Shell formation is important for molluscan development and begins at an early stage, e.g. the shell of Ostrea edulis first begins to form in the gastrula (Medaković et al., 1997). Several gene products related to shell formation and growth have been characterized, like calponin, Perlucin 1, calmodulin and ferritin (Fang et al., 2008; Wang, Liu & Xiang, 2009). Analysis of nervous system and shell formation genes present possible targets for control of invasive molluscan species like P. canaliculata. Invertebrates rely on innate immunity for their internal defence against pathogens. Lectins play an important role in gastropods for recognition and clearance of invaders (Bulat et al., 2016). Our analyses revealed a diversity of lectins that are differentially expressed during embyonic developing, including C-type lectins, I-type lectins, F-type lectins, Ricin_B_lectin and Gal_Lectin. A voracious appetite for aquatic plants renders the invasive P. canaliculata an ecological and economic threat in wetlands and crop fields (Youens & Burks, 2008; Hayes et al., 2012). The degradation of cellulose and xylans by the digestive system of P. canaliculata requires several types of enzymes (Gilkes et al., 1991). Expression of several cellulases was up-regulated in the later stages of embryonic development, such as endoglucanase 20, endo-beta-1,4-glucanase and cellulase EGX3. In addition, the titin gene is a critical contributor to muscular contraction (Heyland et al., 2011). Clk is a clock gene and modification of expression of clock genes may interrupt the circadian rhythm of P. canaliculata and affect feeding and egg-laying (Adema et al., 2017). In summary, we conducted a large-scale RNAseq analysis of the developing embryo of P. canaliculata. Analysis of the sequences recorded identified several DEGs with functions that likely relate to albumen gland, nervous system, circadian rhythm, immune system and shell formation. These transcriptome data will provide fundamental information for molecular ecological studies of the invasive freshwater snail P. canaliculata. SUPPLEMENTARY MATERIAL Supplementary material is available at Journal of Molluscan Studies online. ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (grant no. U1131006). The authors themselves are responsible for the content and writing of the paper. We acknowledge the help from reviewers and comments from Associate Editor C. Adema. REFERENCES Accorsi, A., Benatti, S., Ross, E., Nasi, M. & Malagoli, D. 2017. A prokineticin-like protein responds to immune challenges in the gastropod pest Pomacea canaliculata. Developmental and Comparative Immunology , 72: 37– 43. Google Scholar CrossRef Search ADS   Adema, C.M., Hillier, L.W., Jones, C.S., Loker, E.S. et al.   2017. Whole genome analysis of a schistosomiasis-transmitting freshwater snail. Nature Communications , 8: 15451. Google Scholar CrossRef Search ADS   Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. 1990. Basic local alignment search tool. Journal Molecular Biology , 215: 403– 410. Google Scholar CrossRef Search ADS   Araki, T. & Milbrandt, J. 1996. Ninjurin, a novel adhesion molecule, is induced by nerve injury and promotes axonal growth. Neuron , 17: 353– 361. Google Scholar CrossRef Search ADS   Audic, S. & Claverie, J.M. 1997. The significance of digital gene expression profiles. Genome Research , 7: 986– 995. Google Scholar CrossRef Search ADS   Barlow, L.A. & Truman, J.W. 1992. Patterns of serotonin and SCP immunoreactivity during metamorphosis of the nervous system of the red abalone, Haliotis rufescens. Journal of Neurobiology , 23: 829– 844. Google Scholar CrossRef Search ADS   Bulat, T., Smidak, R., Sialana, F.J., Jung, G., Rattei, T., Bilban, M., Sattmann, H., Lubec, G. & Aradska, J. 2016. Transcriptomic and proteomic analysis of Arion vulgaris—proteins for probably successful survival strategies? PLoS One , 11: e150614. Google Scholar CrossRef Search ADS   Cannuel, R. & Beninger, P.G. 2006. Gill development, functional and evolutionary implications in the Pacific oyster Crassostrea gigas (Bivalvia: Ostreidae). Marine Biology , 149: 547– 563. Google Scholar CrossRef Search ADS   Conesa, A., Gotz, S., Garcia-Gomez, J.M., Terol, J., Talon, M. & Robles, M. 2005. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics , 21: 3674– 3676. Google Scholar CrossRef Search ADS   Dickinson, A. & Croll, R.P. 2003. Development of the larval nervous system of the gastropod Ilyanassa obsoleta. Journal of Comparative Neurology , 466: 197– 218. Google Scholar CrossRef Search ADS   Dickinson, A.J.G., Nason, J. & Croll, R.P. 1999. Histochemical localization of FMRFamide, serotonin and catecholamines in embryonic Crepidula fornicata (Gastropoda, Prosobranchia). Zoomorphology , 119: 49– 62. Google Scholar CrossRef Search ADS   Dreon, M.S., Fernández, P.E., Gimeno, E.J. & Heras, H. 2014. Insights into embryo defenses of the invasive apple snail Pomacea canaliculata: egg mass ingestion affects rat intestine morphology and growth. PLoS Neglected Tropical Diseases , 8: e2961. Google Scholar CrossRef Search ADS   Dreon, M.S., Ituarte, S. & Heras, H. 2010. The role of the proteinase inhibitor ovorubin in apple snail eggs resembles plant embryo defense against predation. PLoS One , 5: e15059. Google Scholar CrossRef Search ADS   Dreon, M.S., Schinella, G., Heras, H. & Pollero, R.J. 2004. Antioxidant defense system in the apple snail eggs, the role of ovorubin. Archives Biochemistry and Biophysics , 422: 1– 8. Google Scholar CrossRef Search ADS   Dyachuk, V.A. 2016. Hematopoiesis in bivalvia larvae: cellular origin, differentiation of hemocytes, and neoplasia. Developmental and Comparative Immunology , 65: 253– 257. Google Scholar CrossRef Search ADS   Elshourbagy, N.A., Ames, R.S., Fitzgerald, L.R., Foley, J.J., Chambers, J.K. et al.   2000. Receptor for the pain modulatory neuropeptides FF and AF is an orphan G protein-coupled receptor. Journal of Biological Chemistry , 275: 25965– 25971. Google Scholar CrossRef Search ADS   Fang, Z., Yan, Z., Li, S., Wang, Q., Cao, W., Xu, G., Xiong, X., Xie, L. & Zhang, R. 2008. Localization of calmodulin and calmodulin-like protein and their functions in biomineralization in P. fucata. Progress in Natural Science , 18: 405– 412. Google Scholar CrossRef Search ADS   Frassa, M.V., Ceolín, M., Dreon, M.S. & Heras, H. 2010. Structure and stability of the neurotoxin PV2 from the eggs of the apple snail Pomacea canaliculata. Biochimica et Biophysica Acta , 1804: 1492– 1499. Google Scholar CrossRef Search ADS   Gilkes, N.R., Henrissat, B., Kiburn, D.G., Miller, R.C. & Warren, R.A. 1991. Domains in microbial beta-1, 4-glycanases: sequence conservation, function, and enzyme families. Microbiology Review , 55: 303– 315. Grabherr, M.G., Haas, B.J., Yassour, M., Levin, J.Z., Thompson, D.A., Amit, I. & Regev, A. 2011. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nature Biotechnology , 29: 644– 652. Google Scholar CrossRef Search ADS   Haas, B.J., Papanicolaou, A., Yassour, M., Grabherr, M., Blood, P.D., Bowden, J., Couger, M.B., Eccles, D., LI, B., Lieber, M., Macmanes, M.D., Ott, M., Orvis, J., Pochet, N., Strozzi, F., Weeks, N., Westerman, R., William, T., Dewey, C.N., Henschel, R., Leduc, R.D., Friedman, N. & Regev, A. 2013. De novo transcript sequence reconstruction from RNA-seq using the trinity platform for reference generation and analysis. Nature Protocols , 8: 1494– 1512. Google Scholar CrossRef Search ADS   Hayes, K.A., Cowie, R.H., Thiengo, S.C. & Strong, E.E. 2012. Comparing apples with apples: clarifying the identities of two highly invasive neotropical Ampullariidae (Caenogastropoda). Zoological Journal of the Linnean Society , 166: 723– 753. Google Scholar CrossRef Search ADS   Heras, H., Frassa, M.V., Fernández, P.E., Galosi, C.M., Gimeno, E.J. & Dreon, M.S. 2008. First egg protein with a neurotoxic effect on mice. Toxicon , 52: 481– 488. Google Scholar CrossRef Search ADS   Heyland, A., Vue, Z., Voolstra, C.R., Medina, M. & Moroz, L.L. 2011. Developmental transcriptome of Aplysia californica. Journal of Experimental Zoology Part B—Molecular and Developmental Evolution , 31: 113– 134. Google Scholar CrossRef Search ADS   Huan, P., Wang, H. & Liu, B. 2012. Transcriptomic analysis of the clam Meretrix meretrix on different larval stages. Marine Biotechnology , 14: 69– 78. Google Scholar CrossRef Search ADS   Jackson, D.J., Worheide, G. & Degnan, B.M. 2007. Dynamic expression of ancient and novel molluscan shell genes during ecological transitions. BMC Evolutionary Biology , 7: 160. Google Scholar CrossRef Search ADS   Koch, E., Winik, B.C. & Castro-Vazquez, A. 2009. Development beyond the gastrula stage and digestive organogenesis in the apple snail Pomacea canaliculata (Architaenioglossa, Ampullariidae). Biocell , 33: 49– 65. Lambert, J.D., Chan, X.Y., Spiecker, B. & Sweet, H.C. 2010. Characterizing the embryonic transcriptome of the snail Ilyanassa. Integrative and Comparative Biology , 50: 768– 777. Google Scholar CrossRef Search ADS   Langmead, B. & Salzberg, S.L. 2012. Fast gapped-read alignment with Bowtie 2. Nature Methods , 9: 357– 359. Google Scholar CrossRef Search ADS   Lee, M.T., Bonneau, A.R. & Giraldez, A.J. 2014. Zygotic genome activation during the maternal-to-zygotic transition. Annual Review of Cell and Developmental Biology , 30: 581– 613. Google Scholar CrossRef Search ADS   Li, B. & Dewey, C.N. 2011. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics , 12: 323. Google Scholar CrossRef Search ADS   Liu, H., Liu, S., Ge, Y., Liu, J., Wang, X., Xie, L., Zhang, R. & Wang, Z. 2007. Identification and characterization of a biomineralization related gene PFMG1 highly expressed in the mantle of Pinctada fucata. Biochemistry , 46: 844– 851. Google Scholar CrossRef Search ADS   Lv, S., Zhang, Y., Liu, H., Zhang, C., Steinmann, P., Zhou, X. & Utzinger, J. 2009. Angiostrongylus cantonensis: morphological and behavioral investigation within the freshwater snail Pomacea canaliculata. Parasitology Research , 104: 1351– 1359. Google Scholar CrossRef Search ADS   Medaković, D., Popović, S., Gržeta, B., Plazonić, M. & Hrsbrenko, M. 1997. X-ray diffraction study of calcification processes in embryos and larvae of the brooding oyster Ostrea edulis. Marine Biology , 129: 615– 623. Google Scholar CrossRef Search ADS   Naimi, A., Martinez, A.S., Specq, M.L., Diss, B., Mathieu, M. & Sourdaine, P. 2009a. Molecular cloning and gene expression of Cg-Foxl2 during the development and the adult gametogenetic cycle in the oyster Crassostrea gigas. Comparative Biochemistry Physiology Part B: Biochemistry and Molecular Biology , 154: 134– 142. Google Scholar CrossRef Search ADS   Naimi, A., Martinez, A.S., Specq, M.L., Mrac, A., Diss, B., Mathieu, M. & Sourdaine, P. 2009b. Identification and expression of a factor of the DM family in the oyster Crassostrea gigas. Comparative Biochemistry Physiology Part A: Molecular and Integrative. Physiology , 152: 189– 196. Nichols, D.E. & Nichols, C.D. 2008. Serotonin receptors. Chemical Reviews , 108: 1614– 1641. Google Scholar CrossRef Search ADS   Parker, R.M., Copeland, N.G., Eyre, H.J., Liu, M., Gilbert, D.J., Crawford, J., Couzens, M., Sutherland, G.R., Jenkins, N.A. & Herzog, H. 2000. Molecular cloning and characterisation of GPR74 a novel G-protein coupled receptor closest related to the Y-receptor family. Molecular Brain Research , 77: 199– 208. Google Scholar CrossRef Search ADS   Pertea, G., Huang, X., Liang, F., Antonescu, V., Sultana, R., Karamycheva, S., Lee, Y., White, J., Cheung, F., Parvizi, B., Tsai, J. & Quackenbush, J. 2003. TIGR gene indices clustering tools (TGICL): a software system for fast clustering of large EST datasets. Bioinformatics , 19: 651– 652. Google Scholar CrossRef Search ADS   Quast, C., Pruesse, E., Yilmaz, P., Gerken, J., Schweer, T., Yarza, P., Peplies, J. & Glockner, F.O. 2013. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Research , 41: D590– D596. Google Scholar CrossRef Search ADS   Quevillon, E., Silventoinen, V., Pillai, S., Harte, N., Mulder, N., Apweiler, R. & Lopez, R. 2005. InterProScan: protein domains identifier. Nucleic Acids Research , 33: W116– W120. Google Scholar CrossRef Search ADS   Song, L., Wang, X., Yang, Z., Lv, Z. & Wu, Z. 2016. Angiostrongylus cantonensis in the vector snails Pomacea canaliculata and Achatina fulica in China: a meta-analysis. Parasitology Research , 115: 913– 923. Google Scholar CrossRef Search ADS   Sun, J., Zhang, Y., Thiyagarajan, V., Qian, P. & Qiu, J. 2010. Protein expression during the embryonic development of a gastropod. Proteomics , 10: 2701– 2711. Google Scholar CrossRef Search ADS   Sun, J., Zhang, H., Wang, H., Heras, H., Dreon, M.S., Ituarte, S., Ravasi, T., Qian, P. & Qiu, J. 2012. First proteome of the egg perivitelline fluid of a freshwater gastropod with aerial oviposition. Journal of Proteome Research , 11: 4240– 4248. Google Scholar CrossRef Search ADS   Vassilatis, D.K., Hohmann, J.G., Zeng, H., Li, F., Ranchalis, J.E., Mortrud, M.T., Brown, A., Rodriguez, S.S., Weller, J.R., Wright, A.C., Bergmann, J.E. & Gaitanaris, G.A. 2003. The G protein-coupled receptor repertoires of human and mouse. Proceedings of the National Academy of Sciences of the USA , 100: 4903– 4908. Google Scholar CrossRef Search ADS   Wang, X., Liu, B. & Xiang, J. 2009. Cloning, characterization and expression of ferritin subunit from clam Meretrix meretrix in different larval stages. Comparative Biochemistry and Physiology Part B: Biochemisry and Molecular Biology , 154: 12– 16. Google Scholar CrossRef Search ADS   Youens, A.K. & Burks, R.L. 2008. Comparing apple snails with oranges: the need to standardize measuring techniques when studying Pomacea. Aquatic Ecology , 42: 679– 684. Google Scholar CrossRef Search ADS   © The Author(s) 2018. Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

Journal of Molluscan StudiesOxford University Press

Published: Jun 2, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off