Effects of hypo-osmotic stress on osmoregulation, antioxidant response, and energy metabolism in sea cucumber Holothuria moebii under desalination environment.

With global climate changing, hypo-salinity events are increasing in frequency and duration because of continuous rainfall and freshwater inflow, which causes reduced cytosolic osmolarity and cellular stress responses in aquatic animals. Sea cucumbers are considered stenohaline because they lack osmoregulatory organs and are vulnerable to salinity fluctuations. In this study, we performed multiple biochemical assays, de novo transcriptomics, and widely targeted metabolomics to comprehensively explore the osmoregulatory mechanisms and physiological responses of sea cucumber Holothuria moebii to hypo-osmotic stress, which is a representative specie that is frequently exposed to hypo-saline intertidal zones. Our results found that H. moebii contracted their ambulacral feet and oral tentacles, and the coelomic fluid ion concentrations were reduced to be consistent with the environment. The microvilli of intestines and respiratory trees underwent degeneration, and the cytoplasm exhibited swelling and vacuolation. Moreover, the Na+, K+, and Cl- concentrations and Na+/K+-ATPase activity were significantly reduced under hypo-osmotic stress. The decrease in protein kinase A activity and increase in 5'-AMP level indicated a significant inhibition of the cAMP signaling pathway to regulate ion concentrations. And small intracellular organic molecules (amino acids, nucleotides and their derivatives) also play crucial roles in osmoregulation through oxidative deamination of glutamate, nucleotide catabolism, and nucleic acid synthesis. Moreover, lysosomes and peroxisomes removed oxidative damage, whereas antioxidant metabolites, such as N-acetyl amino acids and glutathione, were increased to resist oxidative stress. With prolonged hypo-osmotic stress, glycerophospholipid metabolism was enhanced to maintain membrane stability. Furthermore, acyl-CoA-binding protein activity was significantly inhibited, and only a small amount of acylcarnitine was significantly accumulated, which indicated a disruption in energy metabolism. PPAR signaling pathway and choline content were up-regulated to promote fatty acid metabolism under hypo-osmotic stress. Overall, our results provide new insights into the osmoregulatory mechanisms and physiological responses of sea cucumbers to hypo-osmotic stress.

Diversity and distribution of culturable fouling bacteria in typical mariculture zones in Daya Bay, South China.

The diversity and distribution of culturable fouling bacteria in shellfish, fish and non-mariculture zones in Daya Bay were investigated by using a traditional culture-dependent approach combined with an analysis of bacterial 16S rRNA gene sequences. A total of 129 isolates of fouling bacteria belonging to 37 species in 25 genera were collected and identified, which indicated that the three different mariculture zones harbored abundant and diverse fouling bacterial community. At the genus level, Pseudomonas, Arcobacter and Curtobacterium dominated the fouling bacterial community. Moreover, approximately 46% of the 37 representative isolates could form biofilms. After comparing the diversity and distribution of the biofilm-forming bacteria in three different mariculture zones, it was concluded that the ratios of biofilm-forming bacteria in shellfish (68.4%) and fish (63.4%) in mariculture zones were much greater than those in non-mariculture (42.0%) zone. These results provide important information, for the first time, regarding the fouling bacterial community in typical mariculture zones in South China, which will establish a foundation to develop strategies for biofilm control and disease defense.

Effects of Diet on Larval Survival, Growth, and Development of the Sea Cucumber Holothuria leucospilota.

Because most tropical sea cucumbers have been overexploited around the world, the sea cucumber Holothuria leucospilota has become increasingly commercially important in recent years. Restocking and aquaculture of H. leucospilota using hatchery-produced seeds could both enhance declining wild populations and provide sufficient beche-de-mer product to satisfy increasing market demand. Identifying an appropriate diet is important for successful hatchery culture of the H. leucospilota. In this study, we trialed different ratios of microalgae Chaetoceros muelleri (2.00-2.50 × 106 cells/mL) and yeast (Saccharomyces cerevisiae, ~2.00 × 106 cells/mL) in diets for H. leucospilota larvae (6 d after fertilization, referred to as "day 0") at proportions 4 : 0, 3 : 1, 2 : 2, 1 : 3, and 0 : 4 by volume, in 5 treatments (A, B, C, D, and E, respectively). Larval survival rates in these treatments decreased over time, with the survival highest in treatment B (59.24 ± 2.49%) on day 15 (double that of the lowest rate in treatment E (28.47 ± 4.23%)). For any sampling event, larval body length in treatment A was always the lowest after day 3, and that for treatment B was always the highest, except on day 15. The maximum percentage of doliolaria larvae occurred in treatment B (23.33%) on day 15, followed by treatments C, D, and E (20.00%, 10.00%, and 6.67%, respectively). No doliolaria larvae occurred in treatment A, and pentactula larvae occurred only in treatment B (3.33%). On day 15 in all treatments, late auricularia larvae had hyaline spheres, but these were not prominent in treatment A. Densities of juveniles attaching to settlement plates varied with treatments, and the values were very low for the larvae only fed microalgae (A, 2.39 ± 1.95 ind per plate) or yeast (E, 2.13 ± 1.05 ind per plate)-only ~5% of the maximum number settling in treatment B (45.56 ± 7.24 ind per plate). Increased larval growth, survival and development, and juvenile attachment indicates that diets combining microalgae and yeast are more nutritionally balanced than single diets for hatchery of H. leucospilota. A combined diet of C. muelleri and S. cerevisiae at a 3 : 1 ratio is optimum for the larvae. Based on our results, we propose a larval rearing protocol to facilitate mass production of H. leucospilota.

Environmental filtering dominates bacterioplankton community assembly in a highly urbanized estuarine ecosystem.

Estuaries are important in terms of biodiversity, biogeochemical function, and ecological balance due to their intense land-sea interactions. The sustainability of estuarine ecosystem function relies on a good understanding of the ecological processes related to microbial communities. However, microbial community assembly in such ecosystems is still not well understood. Here, based on 16S rRNA sequencing, we investigated the bacterioplankton community structure in the Pearl River Estuarine system during the wet and dry seasons. Results showed that there were significant seasonal and spatial variations in the bacterioplankton communities of the estuary, with seasonal variations being more remarkable. Multiple bacterioplankton with different abundances in the wet and dry seasons were observed, e.g., the class Actinobacteria and Oxyphotobacteria were enriched in the wet season, whereas Alphaproteobacteria and Saccharimonadia were more abundant in the dry season. Both variation partitioning and null model analysis revealed that environmental filtering dominated the bacterioplankton community assembly in the Pearl River Estuary. Water physical properties (e.g., salinity and temperature), nutrient content (e.g., nitrate), and upstream land use (e.g., urban land cover) together determined the distribution of the bacterioplankton composition in this highly urbanized estuarine ecosystem. These findings would help improve our understanding of the bacterioplankton communities in estuarine ecosystems and provide a theoretical foundation for estuarine ecological health management.

Transcriptomic analysis of sea cucumber (Holothuria leucospilota) coelomocytes revealed the echinoderm cytokine response during immune challenge.

BACKGROUND: The sea cucumber Holothuria leucospilota belongs to echinoderm, which is evolutionally the most primitive group of deuterostomes. Sea cucumber has a cavity between its digestive tract and the body wall that is filled with fluid and suspended coelomic cells similar to blood cells. The humoral immune response of the sea cucumber is based on the secretion of various immune factors from coelomocytes into the coelomic cavity. The aim of this study is to lay out a foundation for the immune mechanisms in echinoderms and their origins in chordates by using RNA-seq. RESULTS: Sea cucumber primary coelomocytes were isolated from healthy H. leucospilota and incubated with lipopolysaccharide (LPS, 10 µg/ml), polyinosinic-polycytidylic acid [Poly (I:C), 10 µg/ml] and heat-inactived Vibrio harveyi (107 cell/ml) for 24 h, respectively. After high-throughput mRNA sequencing on an Illumina HiSeq2500, a de novo transcriptome was assembled and the Unigenes were annotated. Thirteen differentially expressed genes (DEGs) were selected randomly from our data and subsequently verified by using RT-qPCR. The results of RT-qPCR were consistent with those of the RNA-seq (R2 = 0.61). The top 10 significantly enriched signaling pathways and immune-related pathways of the common and unique DEGs were screened from the transcriptome data. Twenty-one cytokine candidate DEGs were identified, which belong to 4 cytokine families, namely, BCL/CLL, EPRF1, IL-17 and TSP/TPO. Gene expression in response to LPS dose-increased treatment (0, 10, 20 and 50 µg/ml) showed that IL-17 family cytokines were significantly upregulated after 10 µg/ml LPS challenge for 24 h. CONCLUSION: A de novo transcriptome was sequenced and assembled to generate the gene expression profiling across the sea cucumber coelomocytes treated with LPS, Poly (I:C) and V. harveyi. The cytokine genes identified in DEGs could be classified into 4 cytokine families, in which the expression of IL-17 family cytokines was most significantly induced after 10 µg/ml LPS challenge for 24 h. Our findings have laid the foundation not only for the research of molecular mechanisms related to the immune response in echinoderms but also for their origins in chordates, particularly in higher vertebrates.

Sea cucumber physiological response to abiotic stress: Emergent contaminants and climate change.

The ocean is facing a multitude of abiotic stresses due to factors such as climate change and pollution. Understanding how organisms in the ocean respond to these global changes is vital to better predicting consequences. Sea cucumbers are popular echinoderms with multiple ecological, nutritional, and pharmaceutical benefits. Here, we reviewed the effects of environmental change on an ecologically important echinoderm of the ocean, aiming to understand their response better, which could facilitate healthy culture programs under environmental changes and draw attention to knowledge gaps. After screening articles from the databases, 142 studies were included on the influence of emergent contaminants and climate variation on the early developmental stages and adults of sea cucumbers. We outlined the potential mechanism underlying the physiological response of sea cucumbers to emerging contaminants and climate change. It can be concluded that the physiological response of sea cucumbers to emergent contaminants differs from their response to climate change. Sea cucumbers could accumulate pollutants in their organs but are aestivated when exposed to extreme climate change. Research showed that the physiological response of sea cucumbers to pollutants indicates that these pollutants impair critical physiological processes, particularly during the more susceptible early phases of development compared to adults, and the accumulation of these pollutants in adults is often observed. For climate change, sea cucumbers showed gradual adaptation to the slight variation. However, sea cucumbers undergo aestivation under extreme conditions. Based on this review, critical suggestions for future research are presented, and we call for more efforts focusing on the co-occurrence of different stressors to extend the knowledge regarding the effects of environmental changes on these economically and ecologically important species.

Taxonomic Diversity, Predicted Metabolic Pathway, and Interaction Pattern of Bacterial Community in Sea Urchin Anthocidaris crassispina.

Bacterial assemblages associated with sea urchin are critical to their physiology and ecology within marine ecosystems. In this study, we characterized the bacterial communities in wild sea urchin Anthocidaris crassispina captured in Daya Bay, South China Sea. A total of 363 amplicon sequence variants belonging to nine phyla and 141 genera were classified from intestine, body surface, and surrounding seawater samples. Proteobacteria, Firmicutes, and Bacteroidetes were the dominant bacteria phyla found in this study. A network analysis of bacterial interspecies interactions revealed varying complexity, stability, connectivity, and relationship patterns across the samples, with the most intricate network observed in the surrounding seawater. Metagenomic predictions highlighted the distinct bacterial metabolic pathways, with significant differences between intestine and seawater samples. Notably, pathways associated with polysaccharide degradation, including chitin derivatives, starch, and CoM biosynthesis, were markedly abundant, underscoring the gut microbiota's key role in digesting algae. In addition, other metabolic pathways in intestine samples were linked to immune response regulation of sea urchins. Overall, this study provides a comprehensive overview of the bacterial community structure and potential functional roles in A. crassispina.

The potential for large-scale kelp aquaculture to counteract marine eutrophication by nutrient removal.

Large-scale kelp aquaculture offers several opportunities to counteract eutrophication. Here, six sites from Ailian Bay to its offshore waters in Rongcheng, northern China, were selected to investigate the spatiotemporal variations of kelp (Saccharina japonica) and seawater concentrations of C, N, and P in 2017 and 2019 in order to assess nutrient removal by kelp farming. Variations in biological parameters and elemental nutrient concentrations of whole thalli were also quantified in 2015 and 2016. We found that kelp farming in Ailian Bay can remove 1309 tons of C, 104 tons of N, and 12 tons of P. The increase of total suspended matter and nutrient concentrations from the coast to offshore waters supports the hypothesis of nutrient removal by farmed kelp. This study provides evidence of the bioremediation potential of farmed kelp.

Transcriptome and metabolome analyses reveal gender-specific expression genes in sea cucumber (Holothuria leucospilota).

The sea cucumber Holothuria leucospilota, a nutritive and commercial marine species, has a high protein and low lipid content. To date, the mechanisms underlying gender determination and differentiation in sea cucumbers remain unclear. Identifying gender-specific molecular markers is an effective method of revealing the genetic basis of gender determination and differentiation. The inability to distinguish between male and female individuals causes reproductive efficiency to decline in aquaculture. In this study, we used the gonads of the sea cucumber H. leucospilota as samples to conduct the experiment. The differentially abundant metabolites (DAMs) detected by liquid chromatography-mass spectrometry were enriched in pathways associated with prolactin metabolism, insulin metabolism, hypoxia-inducible factor-1 signaling, and calcium signaling. At the transcriptome level, Illumina sequencing was performed on H. leucospilota, demonstrating that gender-specific expression genes were enriched in the retinoic acid-inducible gene I-like receptor signaling pathway, C-type lectin receptor signaling pathway, alpha-linolenic acid metabolism, and ether lipid metabolism by the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. By analyzing the common pathways between DAMs and differentially expressed genes, we found that gender-related genes of H. leucospilota were mostly enriched in the necroptosis pathway and the cysteine and methionine metabolism pathways. According to the common pathways, uch-sc1 and uch-sc2 are male-specific expression genes, and uch-sc3 and bhmt are female-specific expression genes at the mRNA level. These results provide information on gender differences in H. leucospilota.

Cancer Cell Inhibiting Sea Cucumber (Holothuria leucospilota) Protein as a Novel Anti-Cancer Drug.

Cancer remains the primary cause of death worldwide. To develop less toxic anti-cancer drugs to relieve the suffering and improve the survival of cancer patients is the major focus in the anti-cancer field. To this end, marine creatures are being extensively studied for their anti-cancer effects, since extracts from at least 10% of the marine organisms have been shown to possess anti-tumor activities. As a classic Chinese traditional medicine, sea cucumbers and compounds extracted from the sea cucumbers, such as polysaccharides and saponins, have recently been shown to exhibit anti-cancer, anti-inflammatory, and anti-oxidant effects. Holothuria leucospilota (H. leucospilota) is a tropical edible sea cucumber species that has been successfully cultivated and farmed in large scales, providing a readily available source of raw materials to support the development of novel marine anti-cancer drugs. However, very few studies have so far been performed on the biological activities of H. leucospilota. In this study, we first investigated the anti-cancer effect of H. leucospilota protein on three cancer cell lines (i.e., HepG2, A549, Panc02) and three normal cell lines (NIH-3T3, HaCaT, 16HBE). Our data showed that H. leucospilota protein decreased the cell viabilities of HepG2, A549, HaCaT, 16HBE in a concentration-dependent manner, while Panc02 and NIH-3T3 in a time- and concentration-dependent manner. We also found that the inhibitory effect of H. leucospilota protein (≥10 µg/mL) on cell viability is near or even superior to EPI, a clinical chemotherapeutic agent. In addition, our data also demonstrated that H. leucospilota protein significantly affected the cell cycle and induced apoptosis in the three cancer cell lines investigated; in comparison, it showed no effects on the normal cell lines (i.e., NIH-3T3, HaCaT and 16HBE). Finally, our results also showed that H. leucospilota protein exhibited the excellent performance in inhibiting cell immigrations. In conclusion, H. leucospilota protein targeted the cancer cell cycles and induced cancer cell apoptosis; its superiority to inhibit cancer cell migration compared with EPI, shows the potential as a promising anti-cancer drug.

Molecular cloning, expression and functional analysis of NF-kB1 p105 from sea cucumber Holothuria leucospilota.

The nuclear factor-κB (NF-κB) family is evolutionary conserved and plays key roles in the regulation of numerous basic cellular processes. In this study, a sea cucumber Holothuria leucospilota NF-κB1 p105 named HLp105 was first obtained. The full-length cDNA of HLp105 is 6564 bp long, with a 219 bp 5' untranslated region (UTR), a 2979 bp 3' UTR, and a 3366 bp open reading frame (ORF) encoding for 1121 amino acids with a deduced molecular weight of 123.92 kDa and an estimated pI of 5.31. HLp105 protein contains the conserved domain RHD, IPT, ANK and DEATH. HLp105 mRNA can be detected in all tissues examined, with the highest level in the intestine, followed by the transverse vessel, rete mirabile, coelomocytes, respiratory tree, bolishiti, cuvierian tubules, body wall, oesophagus and muscle. Challenged by LPS or poly (I:C), the transcription level of HLp105 was apparently up-regulated in the tissues examined. Besides, Over-expression of HLp105 in HEK293T cells, the apoptosis was inhibited, and the cytokines IL-1ß and TNF-α were activated. The results are important for better understanding the function of NF-κB1 p105 in sea cucumber and reveal its involvement in immunoreaction.

First echinoderm alpha-amylase from a tropical sea cucumber (Holothuria leucospilota): Molecular cloning, tissue distribution, cellular localization and functional production in a heterogenous E.coli system with codon optimization.

Holothuria leucospilota (Echinodermata: Holothuroidea) is a widespread tropical sea cucumber with strong value for the ecological restoration of coral reefs. Therefore, some studies regarding the artificial breeding and cultivation of H. leucospilota have been undertaken recently. However, the biological functions of the digestive system of this species have not been elucidated. In this study, a cDNA coding for α-amylase, an indicator of digestive maturity in animals, was identified from H. leucospilota and designated Hl-Amy. The full-length cDNA of the Hl-Amy gene, which is 1734 bp in length with an open reading frame (ORF) of 1578 bp, encodes a 525 amino acid (a.a.) protein with a deduced molecular weight of 59.34 kDa. According to the CaZy database annotation, Hl-Amy belongs to the class of GH-H with the official nomenclature of α-amylase (EC 3.2.1.1) or 4-α-D-glucan glucanohydrolase. The Hl-Amy protein contains a signal peptide at the N-terminal followed by a functional amylase domain, which includes the catalytic activity site. The mRNA expression of Hl-Amy was abundantly exhibited in the intestine, followed by the transverse vessel with a low level, but was hardly detected in other selected tissues. During embryonic and larval development, Hl-Amy was constitutively expressed in all stages, and the highest expression level was observed in the blastula. By in situ hybridization (ISH), positive Hl-Amy signals were observed in different parts of the three different intestinal segments (foregut, midgut and hindgut). The Hl-Amy recombinant protein was generated in an E. coli system with codon optimization, which is necessary for Hl-Amy successfully expressed in this heterogenous system. The Hl-Amy recombinant protein was purified by immobilized metal ion affinity chromatography (IMAC), and its activity of starch hydrolysis was further detected. The optimal temperatures and pH for Hl-Amy recombinant protein were 55°C and 6.0, respectively, with an activity of 62.2 U/mg. In summary, this current study has filled a knowledge gap on the biological function and expression profiles of an essential digestive enzyme in sea cucumber, which may encourage future investigation toward rationalized diets for H. leucospilota in artificial cultivation, and optimized heterogenous prokaryotic systems for producing recombinant enzymes of marine origins.

The complete mitochondrial genome of a tropical sea cucumber, Holothuria leucospilota.

The mitochondrial genome of Holothuria leucospilota was 15,906 bp in length, containing 13 protein-coding genes (PCGs), 22 tRNA genes, and two rRNA genes. There were four initiation codons (ATG, ATT, ATC, and ATA) for the PCGs, and the termination codon of most PCGs was TAA, except for nad4 (TAG) and nad6 (TAG). Only one PCG (nad6) and five tRNA genes (tRNASer(UCN) , tRNAGln , tRNAAla , RNAVal , and tRNAAsp ) were encoded on the light chain; the other genes were encoded on the heavy chain. H. leucospilota was most closely related to Holothuria scabra in a phylogenetic tree.

Nutrient release from fish cage aquaculture and mitigation strategies in Daya Bay, southern China.

Finfish cage culture is the most predominant form of mariculture. The rapid expansion of fish cage culture systems has raised concerns about their environmental impact, such as nutrient release. In this study, for the first time, we estimated the release of nitrogen (N) and phosphorus (P) from fish cage culture in Daya Bay, southern China, by constructing N and P budget models based on a mass balance principle. In addition, the contribution and importance of nutrients from fish culture and other nutrient sources, including submarine groundwater discharge, benthic sediments, local rivers, and atmospheric deposition were assessed. The annual amount of N and P released from fish cage culture was 205.6 metric tons (hereafter tons) of N and 39.2 tons of P, including 142.7 tons of dissolved inorganic nitrogen (DIN) and 15.1 tons of dissolved inorganic phosphorus (DIP). Among the analyzed nutrient sources, the contributions of DIN and DIP from fish culture were 7.0% and 2.7%, respectively. For cages consuming conventional trash fish, 142 kg of N and 26 kg of P were released into the environment per ton of fish products, much higher than the values (72 kg N and 17.3 kg P) for cages using formulated feed. In fish culture, the dissolved nutrients were more N rich, but the particulate nutrients were more P rich. The ratio of cage-derived N and P was 21.1, higher than the ratio of coastal seawater (27.1), indicating that cage culture may also impact the local nutrient forms around farming regions. Oyster cultivation and harvest removed 126.3 tons of N and 35.1 tons of P from of the bay. Replacing trash fish with formulated feed and co-culturing of nutrient extractive species (e.g., bivalves, macroalgae) and deposit-feeding species (e.g., sea cucumber) in fish culture zones can be efficient nutrient mitigation strategies.

First echinoderm trehalase from a tropical sea cucumber (Holothuria leucospilota): Molecular cloning and mRNA expression in different tissues, embryonic and larval stages, and under a starvation challenge.

Trehalases are a group of enzymes that catalyse the conversion of trehalose to glucose, and they are observed in most organisms. In this study, the first echinoderm trehalase, designated Hl-Tre, was identified from a tropical sea cucumber, Holothuria leucospilota. The full-length cDNA of H. leucospilota trehalase (Hl-Tre) is 2461 bp in length with an open reading frame (ORF) of 1788 bp that encodes a 595-amino-acid protein with a deduced molecular weight of 67.95 KDa. The Hl-Tre protein contains a signal peptide at the N-terminal and a functional trehalase domain, which includes the signature motifs 1 and 2. The mRNA expression of Hl-Tre was ubiquitously detected in all selected tissues, with the highest level being detected in the intestine. By in situ hybridization (ISH), the positive Hl-Tre signals were observed in the brush borders of the intestinal mucosa. In embryonic and larval stages, the transcript levels of Hl-Tre decreased during embryonic development and increased after the pentactula stage. After a challenge of starvation, the intestinal Hl-Tre mRNA levels were observed to be first decreased and partially recovered thereafter. Overall, our study provided the first evidence for trehalase in echinoderms and showed that this enzyme was potentially linked to a trehalose metabolic pathway in sea cucumbers.

Effect of ocean acidification on growth, calcification, and gene expression in the pearl oyster, Pinctada fucata.

In this study, shell growth, shell microstructure, and expression levels of shell matrix protein genes (aspein, n16, and nacrein) that play a key role in the CaCO3 crystal polymorphism (calcite and aragonite) of the shell were investigated in the pearl oyster Pinctada fucata at pH 8.10, 7.70, and 7.40. We found that the shell length and total weight index did not vary significantly between oysters reared at pH 8.10 and 7.70, but was significantly lower at pH 7.40. Calcium content and shell hardness were not significantly different between pH 8.10 and 7.70, but were significantly different at pH 7.40. At pH 7.40, the shell exhibited a poorly organized nacreous microstructure, and showed an apparent loss of structural integrity in the nacreous layer. The prismatic layer appeared morphologically dissimilar from the samples at pH 8.10 and 7.70. The internal layer was corroded and had dissolved. At pH 7.40, the expression levels of nacrein, aspein, and n16 decreased on day 1, and remained low between days 2 and 42. The expression levels of these genes were significantly lower at pH 7.40 than at pH 8.10 and 7.70 during days 2-42. These results suggest that ocean acidification will have a limited impact on shell growth, calcification, and associated gene expression levels at a pH of 7.70, which is projected to be reached by the end of the century. The negative effects were found on calcification and gene expression occurred at the lowest experimental pH (7.40).

Size-dependent depletion and community disturbance of phytoplankton under intensive oyster mariculture based on HPLC pigment analysis in Daya Bay, South China Sea.

In this study, we conducted a 14-month investigation in Daya Bay, southern China to understand the effects of oyster farming on phytoplankton community and biomass by using size-fractionated phytopigments. Results proved the filtering effects of oysters on phytoplankton biomass. During the oyster culture period, the average concentration of total chlorophyll a (sum of size-fractionated Chl a) within the farming area was approximately 60% lower than that at the reference site. Phytoplankton depletion in the aquaculture zone mainly occurred in micro-sized fractions (>20 µm) of Chl a, fucoxanthin, and peridinin. The influence of oyster filtration on nano-sized (2.7-20 µm) pigments seemed less than that on micro-sized ones. The depletion of peridinin and 19'-hex-fucoxanthin in aquaculture zone was higher than those of the other pigments, which indicated that flagellated cells might be selectively filtered by oysters and could be more easily influenced by oyster aquaculture. The pico-sized Chl a (<2.7 µm) comprised 24% of total Chl a on the average in the aquaculture zone during the cultural period compared to 6% in the reference site. Picoeukaryote abundance, which was determined via flow cytometry, was significantly higher in the aquaculture zone than in the non-aquaculture areas. The abundance of picoeukaryote is significantly and positively correlated with the concentrations of pico-sized prasinoxanthin, violaxanthin, and neoxanthin, indicating that picoeukaryote is dominated by those in prasinophyte. The results suggest that oyster aquaculture might stimulate the growth of prasinophyte, although the seasonal variations are mainly controlled by the water temperature in the study area. This research highlights the successful use of size-fractionated phytopigments to estimate size-specific phytoplankton biomass and community, which can be applied as a routine method to monitor the environmental effect and food resources of bivalve aquaculture.

Discovery, structural characterization and functional analysis of alpha-2-macroglobulin, a novel immune-related molecule from Holothuria atra.

The non-specific protease inhibitor alpha-2-macroglobulin (A2M) is a key macromolecular glycoprotein that involved in host immune defense against pathogens in vertebrates and invertebrates. However, no research regarding A2M has been developed in echinoderms to date. In this study, the full-length cDNA of A2M was cloned from the sea cucumber (Holothuria atra), which is a tropical species widely distributed along the coasts of the South China Sea and designated HaA2M. HaA2M possesses all three conserved functional domains of known A2M proteins, including the bait region domain, thioester domain and receptor-binding domain. Compared to fish and shrimp A2Ms, the histidine residue from the catalytical regions is well conserved in HaA2M. HaA2M mRNA was predominantly expressed in coelomocytes and, to a lesser extent, in the body wall, intestine and respiratory tree. A2M activity was detected in the coelomic fluids of H. atra. The mRNA expression and activity levels were investigated in the major immune tissues and coelomic fluids of H. atra after challenge with inactivated Vibrio alginolyticus or polyriboinosinic polyribocytidylic acid [Poly (I: C)]. RNA interference (RNAi)-mediated knockdown of HaA2M resulted in a significant reduction of HaA2M gene transcript level (86%). RNAi-mediated silencing of HaA2M gene significantly decreased the A2M activity (38%) and increased the number of viable bacteria (2.8-fold) in the coelomic fluids of H. atra infected by V. alginolyticus. Our study, as a whole, supplied the evidences for HaA2M as an immune-relevant molecule and it might have multiple functions in the innate immune system of H. atra.

Complete mitochondrial genome of the sandfish Holothuria scabra (Holothuroidea, Holothuriidae).

The complete mitochondrial genome (mitogenome) sequence of Holothuria scabra, an economically and ecologically important tropical sea cucumber, was first sequenced and annotated. The mitochondrial DNA is 15,779 bp in length and contains 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes, and a 456 bp putative control region, of which gene order is identical to the echinoderm ground pattern. Comparative analyses between H. scabra and other holothurians revealed three new findings: (1) the mitogenome of H. scabra is highly compact having five regions with overlapping genes and least intergenic nucleotides among the sequenced holothurians, only accounting for 3.58% of its mitogenome; (2) the genus Holothuria mitogenomes show a pattern of high interspecies divergence at the 13 PCGs, and the genetic p-distance reaches 25.68% between H. scabra and H. forskali; (3) the incomplete stop codon T of cox2 shared with H. forskali may be a common feature in the genus Holothuria.

Bioremediation and fodder potentials of two Sargassum spp. in coastal waters of Shenzhen, South China.

In this study, the bioremediation potentials of two seaweeds (Sargassum hemiphyllum and S. henslowianum) against pollution in a coastal mariculture area of Shenzhen, South China, were investigated by comparing the growth, nutrient bioaccumulation capacity of plants from the seaweed bed (control site) with plants from the fish farm. Results indicated that both species are potential candidates for bioremediation in the fish farm areas in terms of their high growth rates and high bioaccumulation capacities on inorganic nutrients. Both Sargassum spp. contain high levels of crude protein (11.7-14.0%) and crude fat (2.2-2.7%), suggesting high nutritional values. The S. hemiphyllum may serve as a good aquaculture fodder with high nutritional compositions and low heavy metal contents. However, heavy metals (Cr, Pb and Cd) of S. henslowianum exceed the maximum allowable concentrations as aquatic feed, which restricts its fodder application. In general, the results of this study may contribute to the marine pollution bioremediation in the coastal areas of South China, especially in mariculture zones.