Hao Song

De novo transcriptome sequencing and analysis of Rapana venosa from six different developmental stages using Hi-seq 2500

The carnivorous whelk Rapana venosa is regarded as a biological invader with strong ecological fitness in the United States, Argentina, France and other countries. R. venosa may seriously damage bivalve resources. Nonetheless, in China, R. venosa is an important commercial species. Larval development, especially metamorphosis, influences the natural population and industrial breeding. However, there are few studies on the early development of R. venosa, and our understanding is further limited by a lack of genomic information. In this study, de novo sequencing was performed to obtain a comprehensive transcriptome profile during early development. A Hi-seq 2500 sequencing run produced 148,737,902 raw reads that were assembled into 1,137,556 unigenes (average length of 619 nucleotides, of which 49,673 could be annotated). The unigenes were assigned to biological processes and functions after annotation in Gene Ontology, eukaryotic Ortholog Groups and Kyoto Encyclopedia of Genes and Genomes. We also identified 93,196 simple sequence repeats among the unigenes. Six unique sequences associated with neuroendocrine function were analyzed by quantitative real-time PCR. Our data represent the first comprehensive transcriptomic resource for R. venosa. Functional annotation of the unigenes involved in various biological processes could stimulate research on the mechanisms of early development in this species. Understanding the mechanism of early development and metamorphosis would benefit antifouling research and aquaculture of R. venosa.

Comprehensive and Quantitative Proteomic Analysis of Metamorphosis-Related Proteins in the Veined Rapa Whelk, Rapana venosa

Larval metamorphosis of the veined rapa whelk (Rapana venosa) is a pelagic to benthic transition that involves considerable structural and physiological changes. Because metamorphosis plays a pivotal role in R. venosa commercial breeding and natural populations, the endogenous proteins that drive this transition attract considerable interest. This study is the first to perform a comprehensive and quantitative proteomic analysis related to metamorphosis in a marine gastropod. We analyzed the proteomes of competent R. venosa larvae and post-larvae, resulting in the identification of 5312 proteins, including 470 that were downregulated and 668 that were upregulated after metamorphosis. The differentially expressed proteins reflected multiple processes involved in metamorphosis, including cytoskeleton and cell adhesion, ingestion and digestion, stress response and immunity, as well as specific tissue development. Our data improve understanding of the physiological traits controlling R. venosa metamorphosis and provide a solid basis for further study.

Transcriptomic Analysis of Differentially Expressed Genes During Larval Development of Rapana venosa by Digital Gene Expression Profiling

During the life cycle of shellfish, larval development, especially metamorphosis, has a vital influence on the dynamics, distribution, and recruitment of natural populations, as well as seed breeding. Rapana venosa, a carnivorous gastropod, is an important commercial shellfish in China, and is an ecological invader in the United States, Argentina, and France. However, information about the mechanism of its early development is still limited, because research in this area has long suffered from a lack of genomic resources. In this study, 15 digital gene expression (DGE) libraries from five developmental stages of R. venosa were constructed and sequenced on the IIIumina Hi-Sequation 2500 platform. Bioinformaticsanalysis identified numerous differentially and specifically expressed genes, which revealed that genes associated with growth, nervous system, digestive system, immune system, and apoptosis participate in important developmental processes. The functional analysis of differentially expressed genes was further implemented by gene ontology, and Kyoto encyclopedia of genes and genomes enrichment. DGE profiling provided a general picture of the transcriptomic activities during the early development of R. venosa, which may provide interesting hints for further study. Our data represent the first comparative transcriptomic information available for the early development of R. venosa, which is a prerequisite for a better understanding of the physiological traits controlling development.

Selection of housekeeping genes as internal controls for quantitative RT-PCR analysis of the veined rapa whelk (Rapana venosa)

Background The veined rapa whelk Rapana venosa is an important commercial shellfish in China and quantitative real-time PCR (qRT-PCR) has become the standard method to study gene expression in R. venosa. For accurate and reliable gene expression results, qRT-PCR assays require housekeeping genes as internal controls, which display highly uniform expression in different tissues or stages of development. However, to date no studies have validated housekeeping genes in R. venosa for use as internal controls for qRT-PCR. Methods In this study, we selected the following 13 candidate genes for suitability as internal controls: elongation factor-1α (EF-1α), α-actin (ACT), cytochrome c oxidase subunit 1 (COX1), nicotinamide adenine dinucleotide dehydrogenase (ubiquinone) 1α subcomplex subunit 7 (NDUFA7), 60S ribosomal protein L5 (RL5), 60S ribosomal protein L28 (RL28), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-tubulin (TUBB), 40S ribosomal protein S25 (RS25), 40S ribosomal protein S8 (RS8), ubiquitin-conjugating enzyme E2 (UBE2), histone H3 (HH3), and peptidyl-prolyl cis-trans isomerase A (PPIA). We measured the expression levels of these 13 candidate internal controls in eight different tissues and twelve larvae developmental stages by qRT-PCR. Further analysis of the expression stability of the tested genes was performed using GeNorm and RefFinder algorithms. Results Of the 13 candidate genes tested, we found that EF-1α was the most stable internal control gene in almost all adult tissue samples investigated with RL5 and RL28 as secondary choices. For the normalization of a single specific tissue, we suggested that EF-1α and NDUFA7 are the best combination in gonad, as well as COX1 and RL28 for intestine, EF-1α and RL5 for kidney, EF-1α and COX1 for gill, EF-1α and RL28 for Leiblein and mantle, EF-1α, RL5, and NDUFA7 for liver, GAPDH, PPIA, and RL28 for hemocyte. From a developmental perspective, we found that RL28 was the most stable gene in all developmental stages measured, and COX1 and RL5 were appropriate secondary choices. For the specific developmental stage, we recommended the following combination for normalization, PPIA, RS25, and RL28 for stage 1, RL5 and RL28 for stage 2 and 5, RL28 and NDUFA7 for stage 3, and PPIA and TUBB for stage 4. Discussion Our results are instrumental for the selection of appropriately validated housekeeping genes for use as internal controls for gene expression studies in adult tissues or larval development of R. venosa in the future.

Understanding microRNA Regulation Involved in the Metamorphosis of the Veined Rapa Whelk (Rapana venosa)

The veined rapa whelk (Rapana venosa) is widely consumed in China. Nevertheless, it preys on oceanic bivalves, thereby reducing this resource worldwide. Its larval metamorphosis comprises a transition from pelagic to benthic form, which involves considerable physiological and structural changes and has vital roles in its natural populations and commercial breeding. Thus, understanding the endogenous microRNAs (miRNAs) that drive metamorphosis is of great interest. This is the first study to use high-throughput sequencing to examine the alterations in miRNA expression that occur during metamorphosis in a marine gastropod. A total of 195 differentially expressed miRNAs were obtained. Sixty-five of these were expressed during the transition from precompetent to competent larvae. Thirty-three of these were upregulated and the others were downregulated. Another 123 miRNAs were expressed during the transition from competent to postlarvae. Ninety-six of these were upregulated and the remaining 27 were downregulated. The expression of miR-276-y, miR-100-x, miR-183-x, and miR-263-x showed a >100-fold change during development, while the miR-242-x and novel-m0052-3p expression levels changed over 3000-fold. Putative target gene coexpression, gene ontology, and pathway analyses suggest that these miRNAs play important parts in cell proliferation, migration, apoptosis, metabolic regulation, and energy absorption. Twenty miRNAs and their target genes involved in ingestion, digestion, cytoskeleton, cell adhesion, and apoptosis were identified. Nine of them were analyzed with real-time polymerase chain reaction (PCR), which showed an inverse correlation between the miRNAs and their relative expression levels. Our data elucidate the role of miRNAs in R. venosa metamorphic transition and serve as a solid basis for further investigations into regulatory mechanisms of gastropod metamorphosis.

Cannibalism by the juveniles of the gastropod Rapana venosa (Muricidae) reared under laboratory conditions

We report extreme cannibalistic behaviour in juveniles (1.5–10 mm) of the rapa whelk, Rapana venosa, when they transition from a phytophagous to a carnivorous feeding habit after metamorphosis, as appropriate food becomes scarce during this stage. Although cannibalism is beneficial for the individual, it can result in a high mortality rate of juvenile R. venosa as a whole, therefore juvenile cannibalism is an important determinant of population size. We observed both hole-drilling and enveloping methods of cannibalism. The inner borehole diameter on prey was positively correlated with shell height of the predator (P < 0.001). A higher cannibalism rate was mainly caused by small body size, high levels of hunger and high juvenile densities. The cannibalism rate was effectively controlled by offering bivalves, such as Ruditapes philippinarum and Argopecten irradians, as food. Our results broaden the understanding of cannibalistic behaviour in gastropods and can be used to develop or improve commercial breeding strategies for R. venosa.

Genome survey on invasive veined rapa whelk (Rapana venosa) and development of microsatellite loci on large scale

The veined rapa whelk (Rapana venosa) is an economically important gastropod in China, but is considered as an invasive species globally. Only a few studies have examined the R. venosa genome, a genomewide survey is necessary for improving our understanding of the genome structure and size of this organism. Microsatellite markers are powerful tools for characterizing germplasms, genetic diversity and kinship among individuals. The resultant data are applicable to breeding efforts in commercial aquaculture or for understanding invasion mechanisms. Here, we investigated the genome structure of R. venosa on an Illumina Hi-seq platform with

Effect of temperature on the microflora community composition in the digestive tract of the veined rapa whelk (Rapana venosa) revealed by 16S rRNA gene sequencing

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