In vivo and in vitro biomineralization in the presence of the inner-shell film of pearl oyster

The inner shell surface is the biomineralization site in shell formation and an inner-shell film covers it. This surface is composed of two regions： an outer calcitic region and an inner aragonitic region. In this study, some amalgamated calcite crystals were found in the calcitic region and some aragonitic ＂imprints＂ were found in the central part of the aragonitic region. The ＂imprints＂ are probably the trace of mantle cells that adhered to the inner shell surface when the shell was produced. Furthermore, to build a novel in vitro biomineralization system, the inner-shell film was detached from the shell and introduced to the calcitic crystallization solution. Crystallization experiments showed that nacre proteins could induce aragonite crystals in the novel system but inhibited calcite growth in the absence of the inner-shell film. These data suggested that the inner-shell film may induce aragonite growth in vivo by combining nacre proteins.

Transcriptomes of Litopenaeus vannamei reveal modulation of antioxidant system induced by dietary archaeal carotenoids

Oxidative stress induced by factors such as ammonia nitrogen has become a major issue in shrimp farming.The effects of carotenoids on the growth and antioxidant capability of Litopenaeus vannamei juveniles were investigated in this study using dietary archaeal carotenoids supplementation.For four weeks,shrimp were given diets containing 0 mg/kg(Ctrl)and 55.98 mg/kg(Car)archaeal carotenoids.Dietary archaeal carotenoids significantly enhanced the astaxanthin content in shrimp muscles and carapaces,as well as the superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)activity(P<0.05).The malonaldehyde(MDA)content in Car group significantly decreased(P<0.05).The transcriptome analysis was conducted to determine the molecular processes in response to archaeal carotenoids supplementation.A total of 1536 differentially expressed genes(DEGs)were detected,including 538 upregulated DEGs and 998 downregulated DEGs.GO functional enrichment analysis between Ctrl and Car indicated that 26 GO terms including extracellular region,metabolic process,and proteolysis were enriched.The KEGG pathway enrichment analysis revealed that the amino sugar and nucleotide sugar metabolism,cysteine and methionine metabolism,glycine serine and threonine metabolism,and amino acid biosynthesis were enriched.Archaeal carotenoids influenced the expression of several important genes involved in reactive oxygen species(ROS)generation,Nrf2 signaling,and antioxidant enzymes.Seven DEGs were chosen to confirm the RNA-Seq data using qRT-PCR.The genes and pathways discovered in this work assist to elucidate the molecular processes through which archaeal carotenoid enhances L.vannamei antioxidative system.

Phenolics, fatty acids composition and biological activities of various extracts and fractions of Malaysian Aaptos aaptos

Objective: To investigate phenolics, fatty acids composition and biological activities of various extracts and fractions of Malaysian Aaptos aaptos. Methods: Fatty acid methyl ester was analyzed by gas chromatography-flame ionization detector. Antioxidant activity was determined using 2,2-diphenyl-picrylhydrazyl radical scavenging assay and total phenolics content by Folin-Ciocalteu procedure. Vero cells viability was evaluated using methyl thiazole tetrazolium and the inactivation of herpes simplex virus type 1 by neutral red uptake assay. p-Hydroxybenzamide isolated by column chromatography was characterized by utilizing nuclear magnetic resonance spectroscopy and electron impact mass spectrometry. Results: The chloroform, ethyl acetate and methanol extracts of Aaptos aaptos produced higher portions of straight-chain saturated fatty acid, while hexane extract mainly consisted of unsaturated fatty acid. The five majors of fatty acid methyl ester were identified as behenic acid, cis-10-heptadecenoic acid and cis-10-pentadecenoic acids, palmitic acid and tricosanoic acid. In addition, among all organic extracts, chloroform extract inactivated herpes simplex virus type 1 while exhibited weak cytotoxic activity against normal Vero cells and also exhibited strong cytotoxic activity on HL-60, MCF-7, K562, CEM-SS and WEHI-3 B cells. A phenolic compound, p-hydroxybenzamide was also isolated from the sponge. Conclusions: Aaptos aaptos could be a source to derive the potential antiviral and anticancer agents. However, further studies are needed to determine the mechanism involved in the process.

Exploring the Environmental Physiology of the Indo-Pacific Reef Coral <em>Seriatopora hystrix</em>with Differential Proteomics

Although reef-building corals are threatened by a number of anthropogenic impacts, certain scleractinian-dinoflagellate (genus Symbiodinium) endosymbioses have proven markedly resilient to environmental change. For instance, corals from upwelling habitats of Southern Taiwan withstand both short- and long-term increases in temperature, potentially due to their routine exposure to highly variable temperature regimes in situ. To gain a greater understanding of the proteomic basis for such acclimatization to unstable environmental conditions, specimens of the Indo-Pacific reef-building coral Seriatopora hystrix Dana 1846 were sampled during a period of stable temperature conditions from 1) a site characterized by frequent upwelling events in Southern Taiwan and 2) a nearby, non-upwelling control site in the Taiwan Strait. Two-dimensional gel electrophoresis followed by sequencing of differentially concentrated proteins with mass spectrometry unveiled significantly more proteins involved in the cellular stress response in coral hosts of the upwelling site. Although such stress protein signatures could be indicative of sub-lethal levels of cellular stress, especially given the relatively higher sediment loads characteristic of the upwelling site, these proteins may, in contrast, have been constitutively maintained at high levels in preparation for large fluctuations in temperature and other abiotic parameters (e.g., nutrient levels) brought upon by upwelling events.

Preliminary Study of Anthelmintic Potential of Terminalia catappa Fresh Leaves Following Short-Term Daily Feeding on Goats

The anthelmintic resistance was developed in many species of gastrointestinal nematodes and occurred worldwide. This phenomenon had reduced the effectiveness of anthelmintics which based on drugs. This situation has led to the scientific study on natural anthelmintic that based on traditional usage of local plants. In this study, local plant named Ketapang （Terminalia catappa） that traditionally used to treat helminth infection was chose as experimental plant. Eighteen mix Katjang goats were equally divided into three groups, where two groups were treated with mature and immature T. catappa leaves respectively, while the third group was untreated. Leaves were daily feeding in raw to the goats for four weeks. Normal goat＇s pellet was fed to the goats according to scheduled time-feediing; morning and afternoon, and water was given ad libitum. Fecal samples were collected every two days during the experimental period and subjected to modified Mc Master fecal egg count. Results for this short-term preliminary anthelmintic trial had showed significant percentage of helminth eggs reduction in goats. The reduction in goats treated with mature leaves was at 72% and 63% for the goats treated with immature leaves. Control goats did not showed significant reduction in terms of the parasite worm eggs. In conclusion, the ethnoveterinary data about this local plant was scientifically proven and can be widely promoted to the local livestock＇s owner as an alternative approach for parasitic helminths control in goats.

Temperate Stutzerimonas Phage Encoding Toxin-Antitoxin System Genes Represents a Novel Genus

Stutzerimonas have been extensively studied due to their remarkable metabolic and physiological diversity.However,research on its phages is currently limited.In this study,we isolated a novel double-stranded DNA(dsDNA)phage,vB_SstM-PG1,from the marine environment that infects Stutzerimonas stutzeri G1.Its dsDNA genome is 37204 bp long with a G/C content of 64.14%and encodes 54 open reading frames.The phage possesses a tail packaging structure that is different from known Stutzerimonas stutzeri phages and exhibits structural protein characteristics similar to those of temperate phages.In addition,two genes of toxin-antitoxin system,including YdaS_antitoxin and HEPN_SAV_6107,were found in the vB_SstM-PG1 genome and play important roles in regulating host growth and metabolism.With phylogenetic tree and comparative genomic analysis,it has been determined that vB_SstM-PG1 is not closely related to any phages previously identified in the GenBank database.Instead,it has a connection with enigmatic,uncultured viruses.Specifically,the vB_SstM-PG1 virus exhibits an average nucleotide identity of over 70%with six uncultivated viruses identified in the IMG/VR v4 database.This significant finding has resulted in the identification of a novel viral genus known as Metabovirus.

Genomic diversity and ecological distribution of marine Pseudoalteromonas phages

Pseudoalteromonas,with a ubiquitous distribution,is one of the most abundant marine bacterial genera.It is especially abundant in the deep sea and polar seas,where it has been found to have a broad metabolic capacity and unique co-existence strategies with other organisms.However,only a few Pseudoalteromonas phages have so far been isolated and investigated and their genomic diversity and distribution patterns are still unclear.Here,the genomes,taxonomic features and distribution patterns of Pseudoalteromonas phages are systematically analyzed,based on the microbial and viral genomes and metagenome datasets.A total of 143 complete or nearly complete Pseudoalteromonas-associated phage genomes(PSAPGs)were identifed,including 34 Pseudoalteromonas phage isolates,24 proviruses,and 85 Pseudoalteromonas-associated uncultured viral genomes(UViGs);these were assigned to 47 viral clusters at the genus level.Many integrated proviruses(n=24)and flamentous phages were detected(n=32),suggesting the prevalence of viral lysogenic life cycle in Pseudoalteromonas.PSAPGs encoded 66 types of 249 potential auxiliary metabolic genes(AMGs)relating to peptidases and nucleotide metabolism.They may also participate in marine biogeochemical cycles through the manipulation of the metabolism of their hosts,especially in the phosphorus and sulfur cycles.Siphoviral and flamentous PSAPGs were the predominant viral lineages found in polar areas,while some myoviral and siphoviral PSAPGs encoding transposase were more abundant in the deep sea.This study has expanded our understanding of the taxonomy,phylogenetic and ecological scope of marine Pseudoalteromonas phages and deepens our knowledge of viral impacts on Pseudoalteromonas.It will provide a baseline for the study of interactions between phages and Pseudoalteromonas in the ocean.

<i>vicK</i>Gene as Potential Identification Marker for <i>Staphylococcus aureus</i>

Staphylococcus aureus is an important human pathogen frequently detected in hospital community and has emerged as an important health concern in human medicine. Identification of S. aureus from clinical specimens by phenotypic methods may produce variable characteristics leading to ambiguity. Hence, a rapid and reliable method for identification of S. aureus is required which could expedite appropriate antibiotic therapy. This study aimed to evaluate the specificity of polymerase chain reaction (PCR) targeting a signal transduction gene, vicK, among S. aureus isolates of Hospital Sultanah Nur Zahirah, Kuala Terengganu, Malaysia. A total of 118 bacterial isolates were screened, which consisted of one hundred S. aureus isolates, ten Staphylococcus spp. and eight non-Staphylococci. Results indicated that PCR targeting vicK was able to identify 98% of S. aureus isolates with high sensitivity and specificity, while the remaining isolates of Staphylococcus spp. and non-Staphylococci did not yield any amplification of the gene. vicK thus, is highly specific within interspecies and intraspecies, which is potential to be used as a molecular identification marker for S. aureus.

Correction: Genomic diversity and ecological distribution of marine Pseudoalteromonas phages

In this article the graphics relating to Figs.2 and 3 captions had been interchanged;the fgure(s)should have appeared as shown below.The original article has been corrected.Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,which permits use,sharing.

Cloning, Characterization, and Expression Analysis of Calreticulin from Pearl Oyster Pinctada fucata

Calreticulin is a unique calcium-binding protein with multiple functions mostly located in the sarcoplasmic/endoplasmic reticulum. A large amount of calcium is absorbed from the medium and transported to mineralization sites during biomineralization in pearl oyster. This paper describes the cloning of the full-length cDNA of calreticulin from Pinctada fucata, namely PCRT. PCRT encodes a deduced 414-amino acid protein, which includes a predicted 17- amino acid signal peptide and an endoplasmic reticulum retrieval sequence HDEL. The protein shows 63%-76% sequence identity and shares some common characteristics with calreticulins from other species. Semi-quantitative RT-PCR indicates that PCRT is ubiquitously expressed in all tissues tested with the highest expression in the hemolymph and the mantle. In situ hybridization analysis of PCRT in the mantle showed strong signals in the inner fold, the inner side of middle fold, and the inner side of outer fold of the mantle epithelium, All these results suggest PCRT might be involved in Ca^2＋ transport and storage during oyster biomineralization.

Erosion of the prismatic layer by the organic matrix during the formation of the nacre-prism transition layer in the shell of Pinctada fucata (Bivalvia, Mollusca)

Three variants of the sequence of formation of the nacre-prism transition layer were observed in Pinctada fucata (Bivalvia, Mollusca) shells. In each case, the layer was formed by the organic matrix secreted by the mantle, together with the interprismatic organic envelope. The continuity of the organic phase throughout the shell was maintained as the new nacreous layer was formed on the nacre-prism transition layer. Changes in the interprismatic organic envelopes on either side of the nacre-prism transition zone indicated that the organic matrix of the nacre-prism transition layer becomes embedded into the organic phase of the prismatic layer. It is concluded that penetration and erosion of the prisms by the organic matrix generates a strong bond between the prismatic and nacreous layers.

Dynamics of phytoplankton and picoplankton over a tidal cycle in a subtropical lagoon

The influences of a tidal cycle on the distribution of autotrophic plankton were investigated in a hyper-eutrophic lagoon designated as a scenic area.Results showed that the highest concentrations of picoplankton and phytoplankton were found in the middle and inner part of the lagoon,irrespective of the tides.The MDS result also revealed that phytoplankton communities,dominated by Ceratium furca,were similar among stations in the inner bay during both flood tides and ebb tides.The time series sampling results at the inlet-outlet channel revealed that almost the same amounts of phytoplankton and picoplankton were carried through the channel during flood and ebb tides,with no trend in nutrient fluctuations except for phosphate which had a net loss from the lagoon.The results showed that tidal cycles do not effectively flush away phytoplankton and picoplankton from the lagoon,and the blooming of phytoand picoplankton is inevitable should the situation stay the same.Steps are needed to alleviate the eutrophication condition instead of depending on the natural process such as tidal cycle.

Cloning and Characterization of an mRNA Encoding F_1-ATPase Beta-Subunit Abundant in Epithelial Cells of Mantle and Gill of Pearl Oyster, Pinctada fucata

In oyster biomineralization, large amounts of calcium are absorbed from external media, transported to the mineralization site, and finally deposited via a matrix-mediated process, All these activities are very energy intensive; therefore, investigations of the energy metabolism pathways of different oyster tissues will facilitate understanding of oyster biomineralization physiology. A full-length cDNA encoding the F1- ATPase beta-subunit （the F1-β-subunit, a major calalytic subunit of F-ATPase） from the pearl oyster （Pinctada fucata） was cloned using the homology strategy with a pair of degenerated primers based on the conserved regions of other animals＇ F1-β-subunit genes. Sequencing and structural analyses showed that the obtained sequence shared high identity with other animals＇ F1-β-subunits, and had a unique phosphorylation site of PKC and CK II on the external surface of the putative protein. Results from semi-quantitative reverse transcription-polymerase chain reaction and in situ hybridization demonstrated this oyster F1-β-subunit mRNA is abundant in the gill and mantle, and distributed widely in the periostracal groove, the outer folder. and the dorsal region of the mantle and in the gill epithelial cells. These tissues were the main regions that participate in biomineralization processes such as calcium uptake, transport, and matrix secretion. The results indicate that tissues involved in biomineralization have stronger energy metabolic processes and that F1-ATPase might play an important role in oyster biomineralization by providing energy transport.

Molecular Cloning and Distribution of a Plasma Membrane Calcium ATPase Homolog from the Pearl Oyster Pinctada fucata

Plasma membrane calcium ATPase （PMCA） plays a critical role in transporting Ca^2＋ out of the cytosol across the plasma membrane which is essential both in keeping intracellular Ca^2＋ homeostasis and in biomineralization. In this paper we cloned and localized a gene encoding PMCA from the pearl oyster Pinctada fucata. This PMCA shares similarity with other published PMCAs within the functional domains. Reverse transcription-polymerase chain reaction analysis shows that it is expressed ubiquitously. Furthermore, in situ hybridization reveals that it is expressed in the inner epithelial cells of the outer fold and in the outer epithelial cells of the middle fold, as well as the edge near the shell, which suggests that PMCA may be involved in calcified layer formation. The identification and characterization of oyster PMCA can help to further understand the structural and functional properties of molluscan PMCA, as well as the mechanism of maintaining Ca^2＋ homeostasis and the mechanism of mineralization in pearl oyster.

A possible mechanism for the formation of annual growth lines in bivalve shells

We report a unique shell margin that differed from the usual shell structure of Pinctada fucata.We observed empty organic envelopes in the prismatic layer and the formation of the nacreous layer in the shell margin.All the characteristics of the growing margin indicated that the shell was growing rapidly.To explain this anomaly,we propose the concept of "jumping development".During jumping development,the center of growth in the bivalve shell jumps forward over a short time interval when the position of the mantle changes.Jumping development explains the unusual structure of the anomalous shell and the development of annual growth lines in typical shells.Annual growth lines are the result of a discontinuity in the shell microstructure induced by jumping development.

Reproductive aspects of the coastal trevally,Carangoides coeruleopinnatus in Terengganu Waters,Malaysia

This study describes for the first time the reproductive biology of the coastal trevally(Carangoides coeruleopinnatus)(Ruppel,1830)caught from Terengganu waters,Malaysia.Monthly sample collection from April 2019 to March 2020 was done at Pulau Kambing fish landing port in Terengganu,Malaysia.A total of 687 individuals comprise of 362 males(52.69%)ranged from 6.3 cm to 26.6 cm(mean±SD:13.2±3.84 cm)and 325 females(47.31%)ranged from 9.0 cm to 26.4 cm(mean±SD:13.9±3.34 cm)were observed.The sex ratio significantly deviated in favor of males(1:0.90)(χ^(2)=1.99).However,there are significant differences between sexes over months in June(χ^(2)=10.89),July(χ^(2)=11.91),August(χ^(2)=6.10)and September(χ^(2)=4.41).The monthly variation of the gonadosomatic index(IG)peaked between February and April for males and females,indicating its spawning period.The interpretation between condition factor(K)and hepatosomatic index(IH)showed that the energy mobilization from the body assists the gonad maturation.The batch fecundity of 42 mature females ranged from 15.7 cm to 21.4 cm,and mass was 142.6 g-333.0 g giving 20,438 to 121,829 eggs.The length at first maturity of males and females were 12.45 cm and 15.78 cm,respectively.The male reached earlier maturity than the female.This study increases the understanding of reproductive aspects of C.coeruleopinnatus for future formulation of rules and regulations for suitable fishery management in Terengganu waters.

Extraction and Purification of Matrix Protein from the Nacre of Pearl Oyster Pinctada fucata

A soluble matrix protein P14 with an apparent molecular mass of 14.5 kDa was isolated from fragmented nacre of pearl oysters （Pinctada fucata） treated with 10% NaOH solution to investigate the nacre matrix proteins and their effect on the CaCO3 crystal. The protein was characterized by gel exclusion chromatography and reversed-phase high performance liquid chromatography after demineralization by 10% acetic acid. The X-ray diffraction pattern of P14 crystals indicates that P14 plays an important role in nacre biomineralization. P14 can induce aragonite formation, stimulate CaCO3 crystal formation, and accelerate aragonite precipitation. Heating of the acid insoluble nacre residue, which was named conchiolin, in 10% sodium dodecyl sulfate solution supplemented with 10% β-mercaptoethanol solution for 10-20 min at about 100℃ gave two other soluble proteins having molecular masses of 19.4 kDa and 25.0 kDa. The present study suggests that these two proteins are linked to the insoluble organic matrix by disulfide bridges because the extraction yield increases when β-mercaptoethanol is added to the medium.

Cloning and Characterization of a Homologous Ca^(2+)/Calmodulin-Dependent Protein Kinase PSKH1 from Pearl Oyster Pinctada fucata

Many of the effects of Ca^2＋ signaling are mediated through the Ca^2＋/calmodulin complex and its acceptors, the Ca^2＋/calmodulin-dependent protein kinases, including PSKHI. Studies of the proteins involved in the calcium metabolism in oysters will help elucidate the pearl formation mechanism. This paper describes a full-length PSKH1 cDNA isolated from pearl oyster Pinctada fucata. Oyster PSKH1 shares 65% homology with human PSKH1 and 48% similarity with rat CaM kinase I in the amino acid sequence, and contains a calmodulin-binding domain. The results of semi-quantitative reverse transcription-polymerase chain reaction and in situ hybridization revealed that oyster PSKH1 mRNA is highly expressed in the outer epithelial cells of the mantle pallial and in the gill epithelial cells. These studies provide important information describing the complex Ca^2＋ signaling mechanism in oyster calcium metabolism.

Inhibition of Alkaline Phosphatase from Pearl Oyster Pinctada fucata by o-Phthalaldehyde: Involvement of Lysine and Histidine Residues at the Active Site

Alkaline phosphatase from Pinctada fucata was inactivated by o-phthalaldehyde （OPA）. The inactivation followed pseudo first-order kinetics with a second rate constant of 0.167 （mmol/L）^-1·min^-1 at pH 7.5 and 25℃. A Tsou＇s plot analysis showed that inactivation occurred upon formation of one isoindole group. The OPA-modified enzyme lost the ability to bind with the specific affinity column and the presence of substrates or competitive inhibitors protected the enzyme from inactivation. The results revealed that the OPA-reaction site was at the enzyme substrate binding site. Prior modification of the enzyme by lysine or histidine specific reagent abolished formation of the isoindole derivatives, suggesting that lysine and histidine residues were involved in the OPA-induced inactivation. Taken together, OPA inactivated the alkaline phosphatase from Pinctada fucata by cross-linking lysine and histidine residues at the active site and formed an isoindole group at the substrate binding site of the enzyme.

Increased susceptibility of algal symbionts to photo-inhibition resulting from the perturbation of coral gastrodermal membrane trafficking

The stability of cnidarian-dinoflagellate endosymbioses is dependent upon communication between the host gastrodermal cell and the symbionts housed within it. Although the molecular mechanisms remain to be elucidated, existing evidence suggests that the establishment of these endosymbioses may involve the sorting of membrane proteins. The present study examined the role of host gastrodermal membranes in regulating symbiont （genus Symbiodinium） photosynthesis in the stony coral Euphyllia glabrescens. In comparison with the photosynthetic behavior of Symbiodinium in culture, the Symbiodinium populations within isolated symbiotic gastrodermal cells （SGCs） exhibited a significant degree of photo-inhibition, as determined by a decrease in the photochemical efficiency of photosystem II （Fv/Fm）. This photo-inhibition coincided with increases in plasma membrane perturbation and oxidative activity in the SGCs. Membrane trafficking in SGCs was examined using the metabolism of a fluo- rescent lipid analog, N-[5-（5,7-dimethyl boron dipyrromethene difluoride）-l-pentanoyl]-D-erythro-Sphingosylpbosphoryl- choline （BODIPY-Sphingomyelin or BODIPY-SM）. Light irradiation altered both membrane distribution and trafficking of BODIPY-SM, resulting in metabolic changes. Cholesterol depletion of the SGC plasma membranes by methyl-13-cyclodextrin retarded BODIPY-SM degradation and further augmented Symbiodinium photo-inhibition. These results indicate that Symbio- dinium photo-inhibition may be related to perturbation of the host gastrodermal membrane, providing evidence for the pivotal role of host membrane trafficking in the regulation of this environmentally important coral-dinoflagellate endosymbiosis.