Hai-Yan Wang

Population Genetic Structure and Demographic History of Atrina pectinata Based on Mitochondrial DNA and Microsatellite Markers

The pen shell, Atrina pectinata, is one of the commercial bivalves in East Asia and thought to be recently affected by anthropogenic pressure (habitat destruction and/or fishing pressure). Information on its population genetic structure is crucial for the conservation of A. pectinata. Considering its long pelagic larval duration and iteroparity with high fecundity, the genetic structure for A. pectinata could be expected to be weak at a fine scale. However, the unusual oceanography in the coasts of China and Korea suggests potential for restricted dispersal of pelagic larvae and geographical differentiation. In addition, environmental changes associated with Pleistocene sea level fluctuations on the East China Sea continental shelf may also have strongly influenced historical population demography and genetic diversity of marine organisms. Here, partial sequences of the mitochondrial Cytochrome c oxidase subunit I (COI) gene and seven microsatellite loci were used to estimate population genetic structure and demographic history of seven samples from Northern China coast and one sample from North Korea coast. Despite high levels of genetic diversity within samples, there was no genetic differentiation among samples from Northern China coast and low but significant genetic differentiation between some of the Chinese samples and the North Korean sample. A late Pleistocene population expansion, probably after the Last Glacial Maximum, was also demonstrated for A. pectinata samples. No recent genetic bottleneck was detected in any of the eight samples. We concluded that both historical recolonization (through population range expansion and demographic expansion in the late Pleistocene) and current gene flow (through larval dispersal) were responsible for the weak level of genetic structure detected in A. pectinata.

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.

Detecting One-Hundred-Year Environmental Changes in Western China Using Seven-Year Repeat Photography

Due to its diverse, wondrous plants and unique topography, Western China has drawn great attention from explorers and naturalists from the Western World. Among them, Ernest Henry Wilson (1876 –1930), known as ‘Chinese’ Wilson, travelled to Western China five times from 1899 to 1918. He took more than 1,000 photos during his travels. These valuable photos illustrated the natural and social environment of Western China a century ago. Since 1997, we had collected E.H. Wilsons old pictures, and then since 2004, along the expedition route of E.H. Wilson, we took 7 years to repeat photographing 250 of these old pictures. Comparing Wilsons photos with ours, we found an obvious warming trend over the 100 years, not only in specific areas but throughout the entire Western China. Such warming trend manifested in phenology changes, community shifts and melting snow in alpine mountains. In this study, we also noted remarkable vegetation changes. Out of 62 picture pairs were related to vegetation change, 39 indicated vegetation has changed to the better condition, 17 for degraded vegetation and six for no obvious change. Also in these photos at a century interval, we found not only rapid urbanization in Western China, but also the disappearance of traditional cultures. Through such comparisons, we should not only be amazed about the significant environmental changes through time in Western China, but also consider its implications for protecting environment while meeting the economic development beyond such changes.

Morphological and Genetic Identification of the Validity of the Species Atrina chinensis (Bivalvia: Pinnidae)

ABSTRACT Identification of pinnid species is based largely on morphological characteristics that are highly plastic; thus, classification of pinnids remains controversial. We identified a species of Atrina, found along the southern China coast, as Atrina chinensis Deshayes, 1841, but other authors have treated it as a synonym of Atrina pectinata Linnaeus, 1767. The objective of this study was to clarify the taxonomic status of this species by comparing both morphological and genetic data with data from other Atrina species. Of the 4 shell parameters analyzed, only 1 (size of the posterior adductor) differed significantly between A. pectinata and A. chinensis. However, these species did not form a clade on the phylogenetic trees constructed based on nuclear 28S rRNA or the mitochondrial cytochrome oxidase I (mtCOI) and 16S rRNA genes. Moreover, A. chinensis is, genetically, is a sister taxon to Atrina vexillum instead of A. pectinata. We suggest that A. chinensis is a valid taxon and not a synonym of A. pectinata.

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.

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