RNA-Seq analysis and WGCNA reveal dynamic molecular responses to air exposure in the hard clam Mercenaria mercenaria.

Intertidal bivalves are constantly exposed to air due to daily and seasonal tidal cycles. The hard clam Mercenaria mercenaria is an economically important bivalve species and often subjected to air exposure for more than 10 days during long-distance transportation. Hard clam exhibits remarkable tolerance to air exposure. In this study, we performed RNA sequencing on hemocytes of M. mercenaria exposed to air for 0, 1, 5, 10, 20 and 30 days. The overall and dynamic molecular responses of hard clams to air exposure were revealed by different transcriptomic analysis strategies. As a result, most cytochrome P450 1A and 3A, and monocarboxylate transporter family members were up-regulated during air exposure. Additionally, the dominant molecular process in response to 5-d, 10-d, 20-d and 30-d air exposure was refolding of misfolded proteins in endoplasmic reticulum, lysosome-mediated degradation of phospholipids, protein metabolism and reorganization of cytoskeleton, and activation of anti-apoptotic process, respectively. Our results facilitated comprehensive understanding of the tolerance mechanisms of intertidal bivalves to air exposure.

De novo assembly transcriptome analysis reveals the preliminary molecular mechanism of pigmentation in juveniles of the hard clam Mercenaria mercenaria.

Color plays a vital function in camouflage, sexual selection, immunity, and evolution. Mollusca possess vivid shell colors and pigmentation starts at the juvenile stage. The hard clam Mercenaria mercenaria is a widely cultivated bivalve of high economic value. To explore the molecular mechanism of pigmentation in juvenile clams, here, we performed RNA-Seq analysis on non-pigmented, white, and red M. mercenaria specimens. Clean reads were assembled into 358,285 transcripts and 149,234 unigenes, whose N50 lengths were 2107 bp and 1567 bp, respectively. Differentially expressed genes were identified and analyzed for KEGG enrichment. "Melanoma/Melanogenesis", "ABC transporters", and "Porphyrin and chlorophyll metabolism" pathways appeared to be associated with pigmentation. Pathways related to carotenoid metabolism seemed to also play a vital role in pigmentation in juveniles. Our results provide new insights into the formation of shell color in juvenile hard clams.

Integrated mRNA and miRNA transcriptomic analysis reveals the response of Rapana venosa to the metamorphic inducer (juvenile oysters).

Metamorphosis, as a critical developmental event, controls the population dynamics of most marine invertebrates, especially some carnivorous gastropods that feed on bivalves, whose population dynamics not only affect the maintenance of the ecological balance but also impact the protection of bivalve resources; therefore, the metamorphosis of carnivorous gastropods deserve attention. Here, we investigated the mechanism underlying the response of the carnivorous gastropod Rapana venosa to its metamorphic inducer juvenile oysters through integrated analysis of miRNA and mRNA profiles. According to the results, we speculated that the AMPK signaling pathway may be the critical regulator in the response to juvenile oysters in R. venosa competent larvae. The NF-kB and JAK-STAT signaling pathways that regulated apoptosis were also activated by the metamorphic inducer, which may result in the degeneration of the velum. Additionally, the significant changes in the expression of the SARP-19 precursor gene and protein cibby homolog 1-like gene may indicate that these signaling pathways also regulate growth and development during metamorphosis. This study provides further evidence that juvenile oysters can induce metamorphosis of R. venosa at the transcriptional level, which expands our understanding of the metamorphosis mechanism in carnivorous gastropods.

Symbiotic microbiome and metabolism profiles reveal the effects of induction by oysters on the metamorphosis of the carnivorous gastropod Rapana venosa.

Most marine mollusks have a pelagic larval phase, and they need to undergo metamorphosis to develop into adults. Metamorphosis is affected by many factors, including abiotic factors such as temperature, salinity and illumination as well as biological factors such as food and microorganisms. In our previous study, we found that the metamorphosis of Rapana venosa requires induction by juvenile oysters, which are the food source of R. venosa. However, the regulatory mechanism of this induction is largely unknown. In the present study, we evaluated the impacts of induction by juvenile oysters on competent larvae of R. venosa. Competent larvae were experimentally divided into two pools, and scallop shells without juvenile oysters and scallop shells with juvenile oysters were added for 2 h and 12 h to monitor alterations in critical gene expression, symbiotic microbiota and metabolomic responses. The carboxypeptidase gene was increased while the cellulase gene was decreased, which may mean that the food habit transition was induced by juvenile oysters. Meanwhile, critical genes in the neuroendocrine system were also significantly altered in juvenile oysters. Furthermore, dramatic changes in the symbiotic microbiota and metabolism profiles were observed, with many of them associated with the digestive system and neuroendocrine system. In conclusion, juveniles as food resources may induce metamorphosis in R. venosa by regulating the neuroendocrine system and promoting the development of the digestive system and changes in digestive enzymes. This study may provide evidence that induction by juvenile oysters can promote food habit transition and metamorphosis in R. venosa by regulating the metabolome and microbiome and further altering the digestive and neuroendocrine systems of R. venosa, which expands our understanding of the regulatory mechanism of metamorphosis in R. venosa. However, further studies are needed to explore the specific substance inducing metamorphosis released by juvenile oysters.

Metabolic response of Mercenaria mercenaria under heat and hypoxia stress by widely targeted metabolomic approach.

In the context of global climatic changes, marine organisms have been exposed to environmental stressors including heat and hypoxia. This calls for the design of multi-stressors to uncover the impact of oceanic factors on aquatic organisms. So far, little is known about the metabolic response of marine organisms, especially bivalves, to the combined effects of heat and hypoxia. In this study, we employed widely targeted metabolomic analysis to study the metabolic response of gills in hard clam, a heat- and hypoxia-tolerant bivalve. A total of 810 metabolites were identified. Results showed that the heat group (HT) and heat plus hypoxia group (HL) had a higher number of differential metabolites than the hypoxia group (LO). Glycolysis was affected by the heat and heat plus hypoxia stress. Moreover, anaerobic metabolic biomarkers were accumulated marking the onset of anaerobic metabolism. Environmental stresses may affect Tricarboxylic acid (TCA) cycle. Accumulation of carnitine and glycerophospholipid may promote fatty acid ß oxidation and maintain cell membrane stability, respectively. The high content of oxidized lipids (i.e., Leukotriene) in HL and HT groups implied that the organisms were under ROS stress. The significantly differential metabolites of organic osmolytes and vitamins might relieve ROS stress. Moreover, accumulation of thermoprotective osmolytes (monosaccharide, Trimethylamine N-oxide (TMAO)) accumulation was helpful to maintain protein homeostasis. This investigation provided new insights into the adaptation mechanisms of hard clam to heat, hypoxia and combined stress at the metabolite level and highlighted the roles of molecules and protectants.

Genome-wide analysis of the hard clam mitogen-activated protein kinase kinase gene family and their transcriptional profiles under abiotic stress.

Mitogen-activated protein kinase kinase (MAPKK) was the hub component of the Mitogen-activated protein kinase (MAPK) signaling pathway and played an important role in the cellular response to environmental stress. In this study, we identified five MmMAPKK genes in hard clam Mercenaria mercenaria and found that all MmMAPKK genes contain a conserved protein kinase domain. The MmMAPKK genes derived from dispersed duplication were unevenly distributed in three chromosomes. Although the genome size was highly variable among different bivalve mollusks, the number of MAPKK genes was relatively stable. Phylogenetic analysis showed that bivalve MAPKK was divided into five clades, and amino acid sequences of MAPKK from the same clade consisted of similar conserved motifs. The syntenic analysis demonstrated that MmMAPKKs had the highest number of homologous gene pairs with Cyclina sinensis. MmMAPKKs were ubiquitously expressed in all examined tissues, and all MmMAPKK genes were highly expressed in the ovary. MmMAPKK genes showed stress-specific expression under envirionmental stress. MmMAPKK7 showed an upregulated in heat and heat plus hypoxia stress while MmMAPKK1 showed an upregulated in hypoxic stress groups. Dynamic changes of MmMAPKK7, MmMAPKK6 and MmMAPKK1 in hemocytes were observed in response to air exposure. MmMAPKK4 significantly downregulated after air exposure for five days. MmMAPKK7 and MmMAPKK6 might participate in adaptation to low salinity stress. Our results provided useful information about MAPKK and laid a foundation for further studies on MAPKK evolution in the bivalve.

Identification and characterization of olfactory receptor genes and olfactory perception in rapa whelk Rapana venosa (Valenciennes, 1846) during larval settlement and metamorphosis.

The rapa whelk Rapana venosa, an economically important marine fishery resource in China but a major invader all over the world, changes from a phytophagous to a carnivorous form following settlement and metamorphosis. However, the low settlement and metamorphosis rates (<1%) of larvae limit the abundance of R. venosa. This critical step (settlement and metamorphosis) remains poorly characterized but may be related to how larvae perceive the presence of shellfish, their new source of food. Here, we report that larvae may use olfactory perception to sense shellfish. Olfactory receptor (OR) genes are involved in odor sensing in animals. We identified a total of 463 OR genes, which could be grouped into nine clades based on phylogenetic analysis. When assessing the attraction of larvae at different developmental stages to oyster odor, R. venosa showed active settlement and metamorphosis behavior only at the J4 stage (competent larva, 1000-1500 µm shell length) and in the presence of shellfish odor at the same time. Expression of OR gene family members differed between stage 2 (four-spiral whorl stage) and stage 1 (single- to three-spiral whorl stage), indicating significant changes in the olfactory system during larval development. These findings broaden our understanding of olfactory perception, settlement, and metamorphosis in gastropods and can be used to improve R. venosa harvesting, as well as the sustainable development and utilization of this resource.

Single-molecule long-read (SMRT) transcriptome sequencing of Mercenaria mercenaria reveals a powerful anti-apoptotic system critical for air exposure endurance.

Mercenaria mercenaria is an economically important clam species and exhibits an outstanding resistance to multiple environmental stressors. However, our understanding of their stress adaptability is limited due to a lack of genomic information, such as transcriptome resources. In this study, single-molecule long-read (SMRT) mRNA sequencing was performed to obtain the full-length gill transcriptome reference sequences of M. mercenaria under air exposure stress. In all, 14.5 G subreads were obtained and assembled into 64,603 unigenes, among which 50,613 were successfully annotated. Additionally, 56,295 SSRs, 1457 transcription factors, and 5924 lncRNAs were identified in M. mercenaria transcriptome. Furthermore, numerous apoptosis-related transcripts were identified according to Swiss-Prot annotation and their numbers were counted. We also found that most apoptosis-related transcripts exhibited typical domains of a certain protein family through conserved domain prediction. Additionally, eight typical sequences related to apoptosis pathway were detected by RT-PCR, with the aim to show the sequential variation of gene expression levels under air exposure. These results implied that the complicated apoptosis system, especially the powerful anti-apoptotic system was critical for M. mercenaria to endure air exposure.

Changes in Symbiotic Microbiota and Immune Responses in Early Development Stages of Rapana venosa (Valenciennes, 1846) Provide Insights Into Immune System Development in Gastropods.

The symbiotic microbiota can stimulate modulation of immune system, which also can promote immune system mature in critical developmental periods. In this study, we have investigated the symbiotic microbiota in Rapana venosa at five early development stages using Illumina high-throughput sequencing, and detected immune responses in larvae. Analysis of the symbiotic microbiota sequences identified that the most abundant phylum was Proteobacteria. Beta diversity analysis indicated that the structure of the symbiotic microbiota dramatically shifted in early development stages. The abundance of immune-related KEGG Orthologs (KOs) also increased in competent larval (J4, 30-day post-hatching) and postlarval after 3 days of metamorphosis (Y5, 33-day post-hatching) stages. Acid phosphatase activity decreased significantly in the Y5 stage, and alkaline phosphatase activity also at a lower level in Y5 stage, whereas lysozyme activities exhibited no remarkable change. Also, the activities of catalase and superoxide dismutase activities decreased dramatically during early development stages of R. venosa. Dramatic changes in the symbiotic microbiota and the immune response mainly occurred in the initially hatched veliger (C1), competent larval (J4) and postlarval (Y5) stages, during which the hosts might experience substantial environmental changes or changes in physiological structure and function. These findings expand our understanding of the stage-specific symbiotic microbiota in R. venosa and the close association between immune system and symbiotic microbiota in mollusks, however, the specific relationship may need more researches are needed to investigated in the future.

Complete mitochondrial genome of the hard clam (Mercenaria mercenaria).

The hard clam (Mercenaria mercenaria) is an important economic and ecological bivalve. In this study, the mitochondrial genome was sequenced. The sequenced genome size was 18,360 bp. The nucleotide composition was asymmetric with a AT bias. Mitogenome contained 13 protein-coding genes (PCGs), 2 rRNA genes, and 22 tRNA genes. Of 13 PCGs, 3 genes (cox3, nad3, and cox2) had incomplete stop codons. Furthermore, phylogenetic analysis using 12 PCGs (except atp8) figured out that M. mercenaria was closely related to genus Dosinia. The complete mitogenome of M. mercenaria provides essential information for further phylogenetic and evolutionary analysis in Veneridae.

Transcriptome analysis of shell color-related genes in the hard clam Mercenaria mercenaria.

The shell color of marine bivalves shows great diversity and is considered as an economic trait. In China, the hard clam, Mercenaria mercenaria, commonly has three shell colors in the wild: red, white, and mottled. The genetic mechanisms controlling color segregation are not fully understood. In this study, RNA-seq was performed to exploit the related shell color genes and determine the genetic basis of the different shell colors. Nine sequence libraries with those three shell colors of hard clam were constructed; 406,377 transcripts and 248,251 unigenes were obtained with N50 values of 1365 and 1682 base pairs, respectively. Cluster analysis identified 363, 392, and 220 genes exclusively highly expressed in red, white, and mottled clams, respectively. We further classified differentially expressed genes (DEGs), the genes involved in lipid binding and transport, signal transduction, ATP synthesis, and other processes in the red vs white comparison were found, which may participate in red shell formation. DEGs related to signal transduction, particularly G protein-coupled receptor activity, were found in the red vs mottled comparison, suggesting that these genes might be important in mottled shell formation. In the white vs mottled comparison, DEGs involved in zinc ion binding were found. Our results provide new insights into shell color formation mechanisms in molluscs. This information could be used in selective breeding and marker-assisted breeding of this economically important clam species.

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.

The veined rapa whelk, Rapana venosa, a poikilotherm that is susceptible to temperature, is an important and valuable fishery resource in China but a major invader around the world. We studied the effects of abnormal temperature on the digestive tract microflora of R. venosa to investigate how temperature impacts its digestion and ingestion. We characterized the microflora in nine samples by sequencing the 16S rRNA gene. To assess the species diversity within the samples, effective tags were clustered at 97% similarity by default. Mycoplasma was the most abundant genus among the three groups, and the Proteobacteria phylum had the highest diversity. However, the microflora structure in the digestive tract was significantly different at different temperatures. The top five most abundant genera in the samples housed at 16 °C were Mycoplasma, Phyllobacterium, Aliivibrio, Psychromonas, and Delftia, whereas those in the samples housed under 22 °C were Mycoplasma, Phyllobacterium, Delftia, Spirochaeta_2, and Sphingomonas, and those in the samples housed at 28 °C were Mycoplasma, Phyllobacterium, Vibrio, Delftia, and Aliivibrio. The family Flavobacteriaceae was more abundant in R. venosa housed at 22 °C and 28 °C, whereas a significant decrease in Flavobacteriaceae abundance and a substantial increase in Mycoplasmataceae abundance were observed in R. venosa housed at 16 °C. The alteration in the digestive tract microflora might further affect the function of the R. venosa digestive tract. The results presented herein might provide further insight into investigations on the effects of temperature on the digestion and ingestion of gastropods.

Analysis of microbial abundance and community composition in esophagus and intestinal tract of wild veined rapa whelk (Rapana venosa) by 16S rRNA gene sequencing.

The rapa whelk Rapana venosa is a commercially important gastropod in China, but a major invader worldwide. There is little information on the bacterial community composition in the digestive system of this species, despite the microflora has important roles in digestion, nutrition, disease resistance, and immune response. The present study investigated the bacterial flora community in the esophagus and intestinal tract of wild R. venosa by 16S rRNA gene sequencing. The esophagus and intestine had 1175 and 526 unique operational taxonomic units, respectively, with 616 common to both tissues. The 10 most highly represented microbial genera in the esophagus were Mycoplasma, Bifidobacterium, Escherichia, Shewanella, Vibrio, Lactobacillus, Octadecabacter, Enterococcus, Streptococcus, and Sphingomonas, which accounted for 41.35% of microbes. In the intestine, the most abundant genera were Mycoplasma, Bifidobacterium, Escherichia, Shewanella, Vibrio, Psychrilyobacter, Peptoniphilus, Sporobacterium, Octadecabacter, and Mobiluncusin (representing 62.74% of total microbes). A linear discriminant analysis, coupled with effect size, revealed that 31 taxa were differentially represented in esophagus and intestine bacterial communities. These results demonstrate the diversity of microbiota in the esophagus and intestinal tract of R. venosa and provide a basis for investigations into the physiological and immunological roles of these microorganisms in whelk.

Performance Improvement and Characterization of Spray-Coated Organic Photodetectors.

Low dark current organic photodetectors (OPDs) with a conventional structure consisting of poly(3-hexylthiophene) and [6,6]-phenyl-C61-butyric acid methyl ester (PCBM) as active layer have been fabricated by spray-coating. Tuning the thickness of active layer and thermal annealing process for the spray-coated OPDs results in a remarkable performance with a low dark current density ( Jd) of 2.90 × 10-8 A/cm2 at reverse bias of 1 V. The impact of thermal annealing on the performance of sprayed OPDs is also investigated by the impedance analysis for mechanistic understanding. Our results demonstrate that the optimization of PCBM cluster and interfacial contact between the active layer and the metal electrode tailored by thermal annealing, respectively, could effectively reduce the Jd and increase the sensitivity of sprayed OPDs. The control of PCBM cluster is more important than the interfacial contact between the layers for improving Jd. In addition, structural characterization of the active layer studied by synchrotron small-angle X-ray scattering technique reveals why the spray-coated process can achieve the lowest dark current due to the favorable structure.

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.

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.