Fluorescent Probe Combined with Photoelectric Analysis Technology for Detection of Escherichia coli.

Food safety is facing great challenges in preventing foodborne diseases caused by pathogenic pollution, especially in resource-limited areas. The rapid detection technique of microorganisms, such as immunological methods and molecular biological methods, plays a crucial key in timely bioanalysis and disease treatment strategies. However, it is difficult for these methods to simultaneously meet the criteria of simple operation, high specificity, and sensitivity, as well as low cost. Coconut water is known as the "water of life" in Hainan. It is a refreshing and nutritious beverage which is widely consumed due to its beneficial properties to health. Coconut water processing is an important pillar industry in Hainan. The detection of pathogenic microorganisms, such as Escherichia coli, in coconut water has become an important factor which has restricted the upgrading and development of this industry. Based on the needs of industrial development, we developed a microbial photoelectric detection system which was composed of a fluorescent probe detection reagent and a photoelectric sensor detection device. This system combined microbial enzyme targets, selective fluorescent substrate metabolism characteristics, and a photoelectric sensor signal transduction mechanism, which produce a strong signal with a high signal-to-noise ratio. The microbial detection system developed here has a simple structure, simple and convenient operation, short detecting time (≥2 h), and high sensitivity (1 CFU/mL). This system may also enable early warning and monitoring programs for other pathogenic microorganisms in order to promote the overall competitiveness of the Hainan coconut water industry.

Gold nanoparticles-mediated fluorescent chemical nose sensor for pathogenic diagnosis and phenotype.

Pathogens are one of the important factors affecting national economic construction. An ideal detection system for pathogen control with excellent sensitivity, high specificity, and time-saving is needed. Here, we reported a method for bacterial detection using gold nanoparticles-mediated fluorescent "chemical nose" sensors (GFCEs). The technique consists of gold nanoparticles-coated magnetic particle using benzaldehyde, octyl aldehyde, and pyrimidine-4-formaldehyde modified, respectively. And these positively charged nanocompound interacting with three different fluorescent proteins (FPs) to form three kinds of GFCEs, respectively, named GFCE1, GFCE2, and GFCE3. Upon binding with pathogenic cells, functionalized gold nanoparticles could identify patches on hydrophobic/functional surfaces of microorganisms, and self-assemble with living bacteria by complementary electrostatic interactions. The binding ability between GFCEs and bacteria determines the change of fluorescence response of three FPs from GFCEs. These feature fluorescent level are pathogen-specific, highly repeatable, and can be analyzed by Linear Discriminant Analysis (LDA). The combination of GFCE1 and GFCE2 has the best performance when detecting pathogens with concentrations of 106 cfu mL-1 . The first discriminant within 15 minutes is 93.8%, which could be used for subsequent identification of unknown samples. The commonly applicable system provides a simple way for the rapid bacterial detection without preprocessing procedures.

Multi-particle interaction in AC electric field driven by dielectrophoresis force.

When the dielectrophoresis technology is used to manipulate micron-sized particles, the interaction between particles should not be ignored because of the particle-particle interaction. Especially, when multiple particles (number of particles is above 2) are simultaneously manipulated, the interaction between neighboring particles will affect the results of the manipulation. This research investigates the interaction of particles caused dielectrophoresis effect by the Arbitrary Lagrangian-Eulerian (ALE) method based on the hypothesis of the thin layer of the electric double layer at the microscale. The mathematics model can be solved simultaneously by the finite element method for the AC electric field, the flow field around the suspended particles and the particle mechanics at the micrometer scale. In this study, the particle conductivity and the direction of the electric field are investigated, we find that particle conductivity and electric field direction pose an impact on particle movement, and the research reveal the law of microparticle dielectrophoresis movement, which could offer theoretical and technology support to profoundly understand the precise manipulation of particles in microfluidic chips by the dielectrophoresis effect.

A novel bacterial phylum that participates in carbon and osmolyte cycling in the Challenger Deep sediments.

Large amounts of detrital organic matter and osmolytes accumulate in the sediments of hadal trenches (>6000 m depth) due to the funnelling effect. It is still unknown whether there are novel active microbes that depend on specific carbon sources in extreme and isolated environments. In this study, we present a novel active bacterial phylum, Candidatus Tianyabacteria in the FCB superphylum, which was enriched in sediments collected from the Challenger Deep. Genome binning resulted in high-quality Ca. Tianyabacteria genomes representing two Ca. Tianyabacteria lineages (L1 and L2) in sediments 0-21 cm below the surface (cmbsf); L1 tends to be abundant in the upper layers (0-9 cmbsf), and L2 seems to be more prevalent in the deeper layers (12-21 cmbsf). Gene annotation and transcriptomics results indicate that the two lineages might import and catalyse amino acids and myo-inositol into central carbon metabolism for a heterotrophic lifestyle. Probably due to differences in environmental oxygen levels, the L2 genomes harbour gene clusters responsible for denitrification and fermentation, while the L1 genomes encode octahaem cytochrome c and multicopper oxidase using unknown substrates. The Ca. Tianyabacteria are thus novel heterotrophic organisms that participate in processes of carbon, nitrogen and organic osmolyte cycling in hadal sediments.

Fluorescent and Opt-Electric Recording Bacterial Identification Device for Ultrasensitive and Specific Detection of Microbials.

Since microbial detection is an important aspect for the prevention and control of foodborne diseases, an ideal detection system with high sensitivity, strong specificity, and timeliness is needed. Here, we proposed a fluorescent and opt-electric recording bacterial identification device (FORBID) for fully automatic real-time photoelectric sensing analysis of microbials by integrating the metabolic characteristics of microbial and selective substrate catalysis. It simplifies the testing process (one-step) and decreases the need of professional technicians. Besides, the system exhibits ultrasensitive (1 CFU/mL) and specific detection (99%) in both microbials, Escherichia coli and Pseudomonas aeruginosa. More importantly, the timeliness of this system is even better than that of the traditional culture methods. It is believed that this system can be extended to the detection of other microorganisms and provide a potential alternative for the detection of pathogens.

Phylogenomics of expanding uncultured environmental Tenericutes provides insights into their pathogenicity and evolutionary relationship with Bacilli.

BACKGROUND: The metabolic capacity, stress response and evolution of uncultured environmental Tenericutes have remained elusive, since previous studies have been largely focused on pathogenic species. In this study, we expanded analyses on Tenericutes lineages that inhabit various environments using a collection of 840 genomes. RESULTS: Several environmental lineages were discovered inhabiting the human gut, ground water, bioreactors and hypersaline lake and spanning the Haloplasmatales and Mycoplasmatales orders. A phylogenomics analysis of Bacilli and Tenericutes genomes revealed that some uncultured Tenericutes are affiliated with novel clades in Bacilli, such as RF39, RFN20 and ML615. Erysipelotrichales and two major gut lineages, RF39 and RFN20, were found to be neighboring clades of Mycoplasmatales. We detected habitat-specific functional patterns between the pathogenic, gut and the environmental Tenericutes, where genes involved in carbohydrate storage, carbon fixation, mutation repair, environmental response and amino acid cleavage are overrepresented in the genomes of environmental lineages, perhaps as a result of environmental adaptation. We hypothesize that the two major gut lineages, namely RF39 and RFN20, are probably acetate and hydrogen producers. Furthermore, deteriorating capacity of bactoprenol synthesis for cell wall peptidoglycan precursors secretion is a potential adaptive strategy employed by these lineages in response to the gut environment. CONCLUSIONS: This study uncovers the characteristic functions of environmental Tenericutes and their relationships with Bacilli, which sheds new light onto the pathogenicity and evolutionary processes of Mycoplasmatales.

Insights into the strategy of micro-environmental adaptation: Transcriptomic analysis of two alvinocaridid shrimps at a hydrothermal vent.

Diffusing fluid at a deep-sea hydrothermal vent creates rapid, acute physico-chemical gradients that correlate strongly with the distribution of the vent fauna. Two alvinocaridid shrimps, Alvinocaris longirostris and Shinkaicaris leurokolos occupy distinct microhabitats around these vents and exhibit different thermal preferences. S. leurokolos inhabits the central area closer to the active chimney, while A. longirostris inhabits the peripheral area. In this study, we screened candidate genes that might be involved in niche separation and microhabitat adaptation through comparative transcriptomics. The results showed that among the top 20% of overexpressed genes, gene families related to protein synthesis and structural components were much more abundant in S. leurokolos compared to A. longirostris. Moreover, 15 out of 25 genes involved in cellular carbohydrate metabolism were related to trehalose biosynthesis, versus 1 out of 5 in A. longirostris. Trehalose, a non-reducing disaccharide, is a multifunctional molecule and has been proven to act as a protectant responsible for thermotolerance in Saccharomyces cerevisiae. Putative positively selected genes involved in chitin metabolism and the immune system (lectin, serine protease and antimicrobial peptide) were enriched in S. leurokolos. In particular, one collagen and two serine proteases were found to have experienced strong positive selection. In addition, sulfotransferase-related genes were both overexpressed and positively selected in S. leurokolos. Finally, genes related to structural proteins, immune proteins and protectants were overexpressed or positively selected. These characteristics could represent adaptations of S. leurokolos to its microhabitat, which need to be confirmed by more evidence, such as data from large samples and different development stages of these alvinocaridid shrimps.

Genomic differences within the phylum Marinimicrobia: From waters to sediments in the Mariana Trench.

Marinimicrobia are widespread from the marine surface to the hadal zone. Major clades of Marinimicrobia have evolved to different ecotypes along with energy gradients, but their genomes in deeper waters and sediments have rarely been studied. Here we obtained 11 Marinimicrobia draft genomes from the water column in the full-ocean depth and the hadal sediments in the Mariana Trench. All the predicted genomic capabilities of the metagenome-assembled genomes (MAGs) are indicative of heterotrophic lifestyle. The MAGs from the hadal depths are distinct from those from the mesopelagic and bathypelagic depths by enrichment of the genes involved in amino acids metabolism and mismatch repair. Compared with the MAGs from waters, those from the sediments were dramatically expanded by acquiring the genes responsible for chemotaxis, mobility and the two-component systems. Marinimicrobia were apparently differentiated in the environments with different depths, organic matters and electronic acceptors. Our results also posit a potential evolutionary relationship between the species inhabiting the waters and sediments, indicating the occurrence of allopatric speciation in Marinimicrobia.

In situ meta-omic insights into the community compositions and ecological roles of hadal microbes in the Mariana Trench.

The low temperature and elevated hydrostatic pressure in hadal trenches at water depths below 6000 m render sample collection difficult. Here, in situ hadal water microbial samples were collected from the Mariana Trench and analysed. The hadal microbial communities at different depths were revealed to be consistent and were dominated by heterotrophic Marinimicrobia. Thirty high-quality metagenome-assembled genomes (MAGs) were retrieved to represent the major hadal microbes affiliated with 12 prokaryotic phyla. Most of the MAGs were newly reported and probably derived from novel hadal inhabitants as exemplified by a potentially new candidate archaeal phylum in the DPANN superphylum. Metabolic reconstruction indicated that a great number of the MAGs participated in nitrogen and sulfur cycling, in which the nitrification process was driven sequentially by Thaumarchaeota and Nitrospirae and sulfur oxidization by Rhodospirillales in the Alphaproteobacteria class. Moreover, several groups of hadal microbes were revealed to be potential carbon monoxide oxidizers. Metatranscriptomic result highlighted the contribution of Chloroflexi in degrading recalcitrant dissolved organic matter and Marinimicrobia in extracellular protein decomposition. The present work provides an in-depth view on the hadal microbial communities regarding their endemism and element cycles.

New Microbial Lineages Capable of Carbon Fixation and Nutrient Cycling in Deep-Sea Sediments of the Northern South China Sea.

Metagenomics of marine sediments has uncovered a broad diversity of new uncultured taxa and provided insights into their metabolic capabilities. Here, we detected microbial lineages from a sediment core near the Jiulong methane reef of the northern South China Sea (at 1,100-m depth). Assembly and binning of the metagenomes resulted in 11 genomes (>85% complete) that represented nine distinct phyla, including candidate phyla TA06 and LCP-89, Lokiarchaeota, Heimdallarchaeota, and a newly described globally distributed phylum (B38). The genome of LCP-89 has pathways for nitrate, selenate, and sulfate reduction, suggesting that they may be involved in mediating these important processes. B38 are able to participate in the cycling of hydrogen and selenocompounds. Many of these uncultured microbes may also be capable of autotrophic CO2 fixation, as exemplified by identification of the Wood-Ljungdahl (W-L) pathway. Genes encoding carbohydrate degradation, W-L pathway, Rnf-dependent energy conservation, and Ni/Fe hydrogenases were detected in the transcriptomes of these novel members. Characterization of these new lineages provides insight to the undescribed branches in the tree of life.IMPORTANCE Sedimentary microorganisms in the South China Sea (SCS) remain largely unknown due to the complexity of sediment communities impacted by continent rifting and extension. Distinct geochemical environments may breed special microbial communities including microbes that are still enigmatic. Functional inference of their metabolisms and transcriptional activity provides insight in the ecological roles and substrate-based interactivity of these uncultured Archaea and Bacteria These microorganisms play different roles in utilizing inorganic carbon and scavenging diverse organic compounds involved in the deep-sea carbon cycle. The genomes recovered here contributed undescribed species to the tree of life and laid the foundation for future study on these novel phyla persisting in marginal sediments of the SCS.

Genomic Characterization of a Novel Gut Symbiont From the Hadal Snailfish.

Hadal trenches are characterized by not only high hydrostatic pressure but also scarcity of nutrients and high diversity of viruses. Snailfishes, as the dominant vertebrates, play an important role in hadal ecology. Although studies have suggested possible reasons for the tolerance of hadal snailfish to high hydrostatic pressure, little is known about the strategies employed by hadal snailfish to cope with low-nutrient and virus-rich conditions. In this study, the gut microbiota of hadal snailfish was investigated. A novel bacterium named "Candidatus Mycoplasma liparidae" was dominant in the guts of three snailfish individuals from both the Mariana and Yap trenches. A draft genome of "Ca. Mycoplasma liparidae" was successfully assembled with 97.8% completeness by hybrid sequencing. A set of genes encoding riboflavin biosynthesis proteins and a clustered regularly interspaced short palindromic repeats (CRISPR) system was present in the genome of "Ca. Mycoplasma liparidae," which was unusual for Mycoplasma. The functional repertoire of the "Ca. Mycoplasma liparidae" genome is likely set to help the host in riboflavin supplementation and to provide protection against viruses via a super CRISPR system. Remarkably, genes encoding common virulence factors usually exist in Tenericutes pathogens but were lacking in the genome of "Ca. Mycoplasma liparidae." All of these characteristics supported an essential role of "Ca. Mycoplasma liparidae" in snailfish living in the hadal zone. Our findings provide further insights into symbiotic associations in the hadal biosphere.

Periodic and Spatial Spreading of Alkanes and Alcanivorax Bacteria in Deep Waters of the Mariana Trench.

In subduction zones, serpentinization and biological processes may release alkanes to the deep waters, which would probably result in the rapid spread of Alcanivorax However, the timing and area of the alkane distribution and associated enrichment of alkane-degrading microbes in the dark world of the deep ocean have not been explored. In this study, we report the richness (up to 17.8%) of alkane-degrading bacteria, represented by Alcanivorax jadensis, in deep water samples obtained at 3,000 to 6,000 m in the Mariana Trench in two cruises. The relative abundance of A. jadensis correlated with copy numbers of functional almA and alkB genes, which are involved in alkane degradation. In these water samples, we detected a high flux of alkanes, which probably resulted in the prevalence of A. jadensis in the deep waters. Contigs of A. jadensis were binned from the metagenomes for examination of alkane degradation pathways and deep sea-specific pathways, which revealed a lack of nitrate and nitrite dissimilatory reduction in our A. jadensis strains. Comparing the results for the two cruises conducted close to each other, we suggest periodic release of alkanes that may spread widely but periodically in the trench. Distribution of alkane-degrading bacteria in the world's oceans suggests the periodic and remarkable contributions of Alcanivorax to the deep sea organic carbon and nitrogen sources.IMPORTANCE In the oligotrophic environment of the Mariana Trench, alkanes as carbohydrates are important for the ecosystem, but their spatial and periodic spreading in deep waters has never been reported. Alkane-degrading bacteria such as Alcanivorax spp. are biological signals of the alkane distribution. In the present study, Alcanivorax was abundant in some waters, at depths of up to 6,000 m, in the Mariana Trench. Genomic, transcriptomic, and chemical analyses provide evidence for the presence and activities of Alcanivorax jadensis in deep sea zones. The periodic spreading of alkanes, probably from the subductive plates, might have fundamentally modified the local microbial communities, as well as perhaps the deep sea microenvironment.

Genomics insights into ecotype formation of ammonia-oxidizing archaea in the deep ocean.

Various lineages of ammonia-oxidizing archaea (AOA) are present in deep waters, but the mechanisms that determine ecotype formation are obscure. We studied 18 high-quality genomes of the marine group I AOA lineages (alpha, gamma and delta) from the Mariana and Ogasawara trenches. The genomes of alpha AOA resembled each other, while those of gamma and delta lineages were more divergent and had even undergone insertion of some phage genes. The instability of the gamma and delta AOA genomes could be partially due to the loss of DNA polymerase B (polB) and methyladenine DNA glycosylase (tag) genes responsible for the repair of point mutations. The alpha AOA genomes harbour genes encoding a thrombospondin-like outer membrane structure that probably serves as a barrier to gene flow. Moreover, the gamma and alpha AOA lineages rely on vitamin B12 -independent MetE and B12 -dependent MetH, respectively, for methionine synthesis. The delta AOA genome contains genes involved in uptake of sugar and peptide perhaps for heterotrophic lifestyle. Our study provides insights into co-occurrence of cladogenesis and anagenesis in the formation of AOA ecotypes that perform differently in nitrogen and carbon cycling in dark oceans.

The Enigmatic Genome of an Obligate Ancient Spiroplasma Symbiont in a Hadal Holothurian.

Protective symbiosis has been reported in many organisms, but the molecular mechanisms of the mutualistic interactions between the symbionts and their hosts are unclear. Here, we sequenced the 424-kbp genome of "Candidatus Spiroplasma holothuricola," which dominated the hindgut microbiome of a sea cucumber, a major scavenger captured in the Mariana Trench (6,140 m depth). Phylogenetic relationships indicated that the dominant bacterium in the hindgut was derived from a basal group of Spiroplasma species. In this organism, the genes responsible for the biosynthesis of amino acids, glycolysis, and sugar transporters were lost, strongly suggesting endosymbiosis. The highly decayed genome consists of two chromosomes and harbors genes coding for proteolysis, microbial toxin, restriction-methylation systems, and clustered regularly interspaced short palindromic repeats (CRISPRs), composed of three cas genes and 76 CRISPR spacers. The holothurian host is probably protected against invading viruses from sediments by the CRISPRs/Cas and restriction systems of the endosymbiotic spiroplasma. The protective endosymbiosis indicates the important ecological role of the ancient Spiroplasma symbiont in the maintenance of hadal ecosystems.IMPORTANCE Sea cucumbers are major inhabitants in hadal trenches. They collect microbes in surface sediment and remain tolerant against potential pathogenic bacteria and viruses. This study presents the genome of endosymbiotic spiroplasmas in the gut of a sea cucumber captured in the Mariana Trench. The extreme reduction of the genome and loss of essential metabolic pathways strongly support its endosymbiotic lifestyle. Moreover, a considerable part of the genome was occupied by a CRISPR/Cas system to provide immunity against viruses and antimicrobial toxin-encoding genes for the degradation of microbes. This novel species of Spiroplasma is probably an important protective symbiont for the sea cucumbers in the hadal zone.

Comparative Transcriptomic Analysis Reveals Candidate Genes and Pathways Involved in Larval Settlement of the Barnacle Megabalanus volcano.

Megabalanus barnacle is one of the model organisms for marine biofouling research. However, further elucidation of molecular mechanisms underlying larval settlement has been hindered due to the lack of genomic information thus far. In the present study, cDNA libraries were constructed for cyprids, the key stage for larval settlement, and adults of Megabalanus volcano. After high-throughput sequencing and de novo assembly, 42,620 unigenes were obtained with a N50 value of 1532 bp. These unigenes were annotated by blasting against the NCBI non-redundant (nr), Swiss-Prot, Cluster of Orthologous Groups (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Finally, 19,522, 15,691, 14,459, and 10,914 unigenes were identified correspondingly. There were 22,158 differentially expressed genes (DEGs) identified between two stages. Compared with the cyprid stage, 8241 unigenes were down-regulated and 13,917 unigenes were up-regulated at the adult stage. The neuroactive ligand-receptor interaction pathway (ko04080) was significantly enriched by KEGG enrichment analysis of the DEGs, suggesting that it possibly involved in larval settlement. Potential functions of three conserved allatostatin neuropeptide-receptor pairs and two light-sensitive opsin proteins were further characterized, indicating that they might regulate attachment and metamorphosis at cyprid stage. These results provided a deeper insight into the molecular mechanisms underlying larval settlement of barnacles.

Lirex: A Package for Identification of Long Inverted Repeats in Genomes.

Long inverted repeats (LIRs) are evolutionarily and functionally important structures in genomes because of their involvement in RNA interference, DNA recombination, and gene duplication. Identification of LIRs is highly complicated when mismatches and indels between the repeats are permitted. Long inverted repeat explorer (Lirex) was developed and introduced in this report. Written in Java, Lirex provides a user-friendly interface and allows users to specify LIR searching criteria, such as length of the region, as well as pattern and size of the repeats. Recombinogenic LIRs can be selected on the basis of mismatch rate and internal spacer size from identified LIRs. Lirex, as a cross-platform tool to identify LIRs in a genome, may assist in designing following experiments to explore the function of LIRs. Our tool can identify more LIRs than other LIR searching tools. Lirex is publicly available at http://124.16.219.129/Lirex.

Deinococcus planocerae sp. nov., isolated from a marine flatworm.

A Gram-positive, non-motile and coccoid strain, designated XY-FW106T, was isolated from a marine flatworm identified to be Planocera sp. The 16S rRNA gene sequence of this pink organism was consistent with membership of the genus Deinococcus, with high sequence similarity to Deinococcus aetherius ST0316T (94.7%). The optimum growth temperature range of the strain XY-FW106T was found to be 25-30 °C and optimum growth occurs at pH 7.2-7.4 without NaCl. The strain XY-FW106T was found to contain unidentified glycolipids, unidentified phosphoglycolipids, unidentified phospholipids and unidentified lipids, which differed from those of closely related species. Menaquinone MK-8 was identified as the major respiratory quinone and the predominant cellular fatty acids were found to be Summed Feature 3 (C16:1 ω7c/C16:1 ω6c), C16:0, iso-C15:0, and Summed Feature 8 (C18:1 ω7c/C18:1 ω6c). The DNA G+C content was determined to be 70.2 mol%. The biochemical and chemotaxonomic data together suggest that the strain represents a new species for which the name Deinococcus planocerae sp. nov. is proposed. The type strain is XY-FW106T (=MCCC 1K01499T=KCTC 33809T).

Genomic characterization of symbiotic mycoplasmas from the stomach of deep-sea isopod bathynomus sp.

Deep-sea isopod scavengers such as Bathynomus sp. are able to live in nutrient-poor environments, which is likely attributable to the presence of symbiotic microbes in their stomach. In this study we recovered two draft genomes of mycoplasmas, Bg1 and Bg2, from the metagenomes of the stomach contents and stomach sac of a Bathynomus sp. sample from the South China Sea (depth of 898 m). Phylogenetic trees revealed a considerable genetic distance to other mycoplasma species for Bg1 and Bg2. Compared with terrestrial symbiotic mycoplasmas, the Bg1 and Bg2 genomes were enriched with genes encoding phosphoenolpyruvate-dependent phosphotransferase systems (PTSs) and sodium-driven symporters responsible for the uptake of sugars, amino acids and other carbohydrates. The genome of mycoplasma Bg1 contained sialic acid lyase and transporter genes, potentially enabling the bacteria to attach to the stomach sac and obtain organic carbons from various cell walls. Both of the mycoplasma genomes contained multiple copies of genes related to proteolysis and oligosaccharide degradation, which may help the host survive in low-nutrient conditions. The discovery of the different types of mycoplasma bacteria in the stomach of this deep-sea isopod affords insights into symbiotic model of deep-sea animals and genomic plasticity of mycoplasma bacteria.

Transcriptomics Analysis of Crassostrea hongkongensis for the Discovery of Reproduction-Related Genes.

BACKGROUND: The reproductive mechanisms of mollusk species have been interesting targets in biological research because of the diverse reproductive strategies observed in this phylum. These species have also been studied for the development of fishery technologies in molluscan aquaculture. Although the molecular mechanisms underlying the reproductive process have been well studied in animal models, the relevant information from mollusks remains limited, particularly in species of great commercial interest. Crassostrea hongkongensis is the dominant oyster species that is distributed along the coast of the South China Sea and little genomic information on this species is available. Currently, high-throughput sequencing techniques have been widely used for investigating the basis of physiological processes and facilitating the establishment of adequate genetic selection programs. RESULTS: The C.hongkongensis transcriptome included a total of 1,595,855 reads, which were generated by 454 sequencing and were assembled into 41,472 contigs using de novo methods. Contigs were clustered into 33,920 isotigs and further grouped into 22,829 isogroups. Approximately 77.6% of the isogroups were successfully annotated by the Nr database. More than 1,910 genes were identified as being related to reproduction. Some key genes involved in germline development, sex determination and differentiation were identified for the first time in C.hongkongensis (nanos, piwi, ATRX, FoxL2, ß-catenin, etc.). Gene expression analysis indicated that vasa, nanos, piwi, ATRX, FoxL2, ß-catenin and SRD5A1 were highly or specifically expressed in C.hongkongensis gonads. Additionally, 94,056 single nucleotide polymorphisms (SNPs) and 1,699 simple sequence repeats (SSRs) were compiled. CONCLUSIONS: Our study significantly increased C.hongkongensis genomic information based on transcriptomics analysis. The group of reproduction-related genes identified in the present study constitutes a new tool for research on bivalve reproduction processes. The large group of molecular markers discovered in this study will be useful for population screening and marker assisted selection programs in C.hongkongensis aquaculture.