Jingwei Jiang

Biodegradation of Benzene, Toluene, Ethylbenzene, and o-, m-, and p-Xylenes by the Newly Isolated Bacterium Comamonas sp. JB

A bacterium designated strain JB, able to degrade six benzene, toluene, ethylbenzene, and o-, m-, and p-xylene (BTEX) compounds, was isolated from petroleum-contaminated soil. Taxonomic analyses showed that the isolate belonged to Comamonas, and until now, the genus Comamonas has not included any known BTEX degraders. The BTEX biodegradation rate was slightly low on the mineral salt medium (MSM), but adding a small amount of yeast extract greatly enhanced the biodegradation. The relationship between specific degradation rate and individual BTEX was described well by Michaelis-Menten kinetics. The treatment of petrochemical wastewater containing BTEX mixture and phenol was shown to be highly efficient by BTEX-grown JB. In addition, toxicity assessment indicated the treatment of the petrochemical wastewater by BTEX-grown JB led to less toxicity than untreated wastewater.

Expression Analysis of Immune Related Genes Identified from the Coelomocytes of Sea Cucumber (Apostichopus japonicus) in Response to LPS Challenge

The sea cucumber (Apostichopus japonicus) occupies a basal position during the evolution of deuterostomes and is also an important aquaculture species. In order to identify more immune effectors, transcriptome sequencing of A. japonicus coelomocytes in response to lipopolysaccharide (LPS) challenge was performed using the Illumina HiSeq™ 2000 platform. One hundred and seven differentially expressed genes were selected and divided into four functional categories including pathogen recognition (25 genes), reorganization of cytoskeleton (27 genes), inflammation (41 genes) and apoptosis (14 genes). They were analyzed to elucidate the mechanisms of host-pathogen interactions and downstream signaling transduction. Quantitative real-time polymerase chain reactions (qRT-PCRs) of 10 representative genes validated the accuracy and reliability of RNA sequencing results with the correlation coefficients from 0.88 to 0.98 and p-value <0.05. Expression analysis of immune-related genes after LPS challenge will be useful in understanding the immune response mechanisms of A. japonicus against pathogen invasion and developing strategies for resistant markers selection.

Characterization of phenoloxidase from the sea cucumber Apostichopus japonicus.

Phenoloxidase (PO) is a crucial immune-related enzyme in invertebrates. In this study, three POs of the sea cucumber Apostichopus japonicus were detected in coelomic fluid using linear-gradient native-PAGE combined with catechol staining and then partially purified by gel excising. The results showed that the three POs had a color of mahogany (AjPO1), yellow (AjPO2) and purple (AjPO3) respectively with molecular weights smaller than 21kDa in native-PAGE after staining with catechol. Enzymatic activities analysis revealed that AjPO1, AjPO2 and AjPO3 had optimal temperature of 45, 95 and 85°C and pH of 5.0, 8.0 and 8.0, respectively. Kinetic analysis showed that the Km values of AjPO1 for catechol, l-DOPA, dopamine and hydroquinone were 3.23, 0.86, 3.98 and 1.20mmol/l, respectively, those of AjPO2 were 0.31, 0.38, 2.05 and 1.30mmol/l, respectively, and those of AjPO3 were 5.95, 1.28, 5.81 and 0.62mmol/l, respectively. These results suggest that the three POs are laccase-type phenoloxidase. The activities of all three A. japonicus POs were significantly promoted by Ca(2+), Mg(2+) and Mn(2+), and strongly inhibited by ethylenediamine tetraacetic acid disodium (EDTA), sodium diethyldithiocarbamate (DETC) and some common antioxidants. The inhibitions by EDTA and DETC suggest that the three A. japonicus POs are copper-containing metalloenzymes. Immune-responsive analysis showed that the total PO activities in coelomocytes (TPAC) increased greatly after lipopolysaccharide (LPS) challenge and declined significantly after polyinosinic-polycytidylic acid (PolyI:C) challenge, implying that A. japonicus PO immune system, which is composed of several isoenzymes with different characteristics, is closely involved in the defense against the infection of Gram-negative bacteria and double-stranded RNA viruses.

In vitro antibacterial analysis of phenoloxidase reaction products from the sea cucumber Apostichopus japonicus

Three phenoloxidases (POs) of Apostichopus japonicus, AjPOs (AjPO1, AjPO2 and AjPO3), were partially purified from the coelomocytes with an electrophoretic method, and then employed for the in vitro antibacterial analysis. Using L-3,4-dihydroxyphenylalanine (L-DOPA) as a substrate, AjPO1 and AjPO2-derived compounds inhibited the growth of Vibrio splendidus and Staphylococcus aureus, while AjPO3-derived compounds only inhibited the growth of V. splendidus. When dopamine was used as a substrate, AjPO1 and AjPO3-derived compounds inhibited the growth of V. splendidus and Vibrio harveyi, while AjPO2-derived compounds only inhibited the growth of V. splendidus. Moreover, AjPO1-derived compounds showed stronger inhibition in V. harveyi than AjPO3-derived compounds did. However, all of the three AjPO reaction products showed no inhibitions on the growth of Pseudoalteromonas nigrifaciens, Shewanella baltica, Micrococcus lysodeikticus, Streptococcus dysgalactiae and Nocardiopsis sp. with L-DOPA or dopamine as a substrate. Scanning electron microscope (SEM) observation of V. harveyi treated by AjPOs and dopamine showed that AjPO1-derived compounds resulted in massive bacteriolysis, AjPO2-derived compounds caused no obvious alteration on bacterial morphology, and AjPO3-derived compounds increased the ratio of spheroidal bacteria. All these results suggested that AjPO reaction products derived by L-DOPA and dopamine had different but limited antibacterial spectrum, and the different antibacterial effects observed among three AjPOs resulted from the different reaction products generated by AjPOs with the same substrate.

High-Density Genetic Mapping with Interspecific Hybrids of Two Sea Urchins, Strongylocentrotus nudus and S. intermedius, by RAD Sequencing.

Sea urchins have long been used as research model organisms for developmental biology and evolutionary studies. Some of them are also important aquaculture species in East Asia. In this work, we report the construction of RAD-tag based high-density genetic maps by genotyping F1 interspecific hybrids derived from a crossing between a female sea urchin Strongylocentrotus nudus and a male Strongylocentrotus intermedius. With polymorphisms present in these two wild individuals, we constructed a female meiotic map containing 3,080 markers for S. nudus, and a male meiotic map for S. intermedius which contains 1,577 markers. Using the linkage maps, we were able to anchor a total of 1,591 scaffolds (495.9 Mb) accounting for 60.8% of the genome assembly of Strongylocentrotus purpuratus. A genome-wide scan resulted in the identification of one putative QTL for body size which spanned from 25.3 cM to 30.3 cM. This study showed the efficiency of RAD-Seq based high-density genetic map construction using F1 progenies for species with no prior genomic information. The genetic maps are essential for QTL mapping and are useful as framework to order and orientate contiguous scaffolds from sea urchin genome assembly. The integration of the genetic map with genome assembly would provide an unprecedented opportunity to conduct QTL analysis, comparative genomics, and population genetics studies.

Identification and characterization of proteins with phenoloxidase-like activities in the sea urchin Strongylocentrotus nudus.

Three proteins with PO-like activities in the coelomocytes of sea urchin Strongylocentrotus nudus were identified using electrophoretic method and named as SnPO1, SnPO2 and SnPO3 according to their molecular mass from high to low. The SnPOs were characterized for substrate specificity and the effects of temperature, pH, divalent metal ions and inhibitors on PO activities. They showed oxidative activities to L-3,4-dihydroxyphenylalanine. (l-DOPA), dopamine and hydroquinone, but failed to oxidize tyrosine, which illustrated the three proteins had laccase-like PO activities. The optimum temperature for the activities of SnPO1, SnPO2 and SnPO3 was 75 °C, 70 °C, 40 °C, and the optimum pH was 7.0, 9.0, 8.0, respectively. The SnPOs were notably activated after being incubated in boiled water for 60 min, suggesting that the three proteins are thermophilic. The activity of SnPO1 was greatly enhanced by Cu(2+), Mn(2+) and Fe(2+) and inhibited by Pb(2+), Cd(2+), EDTA, DETC, sodium sulfite and ascorbic acid, but SnPO2 and SnPO3 were not obviously affected by Pb(2+) and Cd(2+), suggesting the three proteins are copper-containing, and the catalytic properties of SnPO1 might be different from those of SnPO2 and SnPO3. Taken together, SnPO1, SnPO2 and SnPO3 might play different roles in the immune and physiological processes of S. nudus.

Comparative analysis of immunocompetence between females and males in the sea cucumber Apostichopus japonicus

Abstract In order to preliminarily understand the immune difference between females and males in the sea cucumber Apostichopus japonicus, the activities assay of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), phenoloxidase (PO), acid phosphatase (ACP) and alkaline phosphatase (ALP) with biochemical methods, the detection of PO isozymes with native‐PAGE and catechol staining, and the test of antibacterial activities with bacterial growth curve determination method were performed in this study using cell‐free coelomic fluid (CCF) and coelomocyte lysate supernatant (CLS) from females and males as the samples. The PO activities were not detected in the CLS and showed no significant difference between the CCF from females and males. However, totally five PO isozyme bands were detected in the CLS of females while only four were detected in the CLS of males after zymogram analysis. These results implied that the PO isozymes in the coelomocytes of viripotent A. japonicus were inactive under natural condition and may be activated by some certain treatments during native‐PAGE, and PO might play different immune and physiological roles between females and males. In addition, the activities of SOD, CAT, POD and ALP in the CCF and the activities of CAT, POD, ACP and ALP in the CLS from males were all significantly higher than those from females. The results collectively suggested that in viripotent A. japonicus, the gender had a remarkable effect on the immunity, and the immunocompetence of males might have an advantage over that of females. Furthermore, the activities of all determined enzymes except PO and the number of detected PO isozymes showed higher values in CLS than in CCF, implying that in viripotent A. japonicus, the coelomocytes might take more immune responsibility in comparison with CCF. HighlightsThe gender has remarkable effect on A. japonicus immunity.The reproductive development has important effect on A. japonicus immunity.The immunity of male A. japonicus might have an advantage over that of females.The effects of gender on immunity are species‐specific in invertebrates.

Unrevealing variation of microbial communities and correlation with environmental variables in a full culture-cycle of Undaria pinnatifida

Bacteria are the most abundant organisms in natural environment and dominant drivers of multiple geochemical functions. Drawing a global picture of microbial community structure and understanding their ecological status remain a grand challenge. As a typical artificial process, aquaculture provides a large amount of foods and creates great economic benefits for human beings. However, few studies are aimed at the microbial community in the aquaculture environment of aquatic plants. We analyzed microbial communities from 21 water samples in a coastal aquaculture area during the whole cultural process of Undaria pinnatifida by using high-throughout sequencing of 16S rRNA gene. The progression of U. pinnatifida aquaculture can be divided into three stages, named Seeding, Growth, and Maturity, respectively. Microbial community structures in water of the aquaculture area were significantly changed during the progression of U. pinnatifida aquaculture. The relative abundance of Flavobacteriia and Thaumarchaeota classes increased in Growth stage, and β-proteobacteria and Acidimirobiia classes decreased with the growth of U. pinnatifida. Meanwhile, environmental factors shaping the microbial community structures were uncovered during the U. pinnatifida aquaculture by using canonical correspondence analysis and Mantel test, in which temperature, dissolved oxygen, pH and nitrogen could be the major influencing factors. In addition, the microbial functions based on KEGG pathways were predicted from the microbial community compositions by PICRUSt. The comparison of predicted functions suggested that Environmental Information Processing and Genetic Information Processing were the functional categories with the most obvious shift in abundance among different stages of U. pinnatifida aquaculture. The findings of this study allowed us to better understand the microbial community in coastal aquaculture systems and the impact of seaweed cultivation on coastal ecosystems.

Proteomic analysis reveals the important roles of alpha-5-collagen and ATP5β during skin ulceration syndrome progression of sea cucumber Apostichopus japonicus

Apostichopus japonicus is one of the most important aquaculture species in China. Skin ulceration syndrome (SUS) of sea cucumber is a common and serious disease affected the development of A. japonicus culture industry. To better understand the response mechanisms of A. japonicus during SUS progression, the protein variations in the body wall of A. japonicus at different stages of SUS were investigated by a comparative proteomic approach based on isobaric tags for relative and absolute quantification. A total of 1449 proteins were identified from the samples at different SUS stages. Among these proteins, 145 proteins were differentially expressed in the SUS-related samples compared to those of healthy A. japonicus. These differentially expressed proteins involved a wide range of functions. Among these differentially expressed proteins, only two proteins, alpha-5-collagen and an unknown function protein, were differentially expressed during the whole progression of SUS compared with healthy A. japonicus. In addition, ATP synthase subunit beta (ATP5β) interacted with a variety of proteins with different functions during the SUS progression. These results implied that alpha-5-collagen and ATP5β could play important roles during the SUS progression of A. japonicus. Our study provided a new sight to understand the molecular responses of sea cucumber during the SUS progression and accumulated data for the prevention of SUS in sea cucumber aquaculture. BIOLOGICAL SIGNIFICANCE The current study aimed to reveal how the body wall of Apostichopus japonicus response to skin ulceration syndrome (SUS). To the best of our knowledge, this is the first proteomic study analyzing the differences in protein profile of sea cucumber during the whole SUS progression. By analyzing the expression differences of the proteome via isobaric labeling-based quantitative proteomic, we identified some proteins which may play important roles during the SUS progression. According to the enrichment analyses of these proteins based on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes, a draft view of how the sea cucumber affected by SUS has been drawn. The common and unique differentially expressed proteins by Venn analysis showed that alpha-5-collagen was down-regulated at all stages of SUS, which had the potential as a target component for the host-directed SUS therapy. In addition, ATP5β, a subunit of mitochondrial ATP synthase, interacting with a variety of proteins with different functions during the SUS progression. This result illustrated that energy production and metabolism could play an important role in the formation of skin ulceration and resistance to pathogens in sea cucumber. The results of this study will be helpful for researchers to gain insights into the complex molecular mechanism of SUS in sea cucumber.

In-depth profiling of miRNA regulation in the body wall of sea cucumber Apostichopus japonicus during skin ulceration syndrome progression

ABSTRACT MicroRNAs (miRNAs) are small non‐coding RNAs that mediate mRNA degradation or translation repression. Previous study showed that the expression of miRNAs was significantly changed in the body wall of sea cucumber Apostichopus japonicus after skin ulceration syndrome (SUS) infection, which is a dynamic process. However, the critical miRNAs from body wall that involved in different infection stages of SUS remain unknown. In this study, four cDNA libraries were constructed with the body wall from healthy and three SUS‐infected stages of A. japonicus. A total of 248 conserved miRNAs and five novel miRNAs were identified through Illumina HiSeq 2000 platform. Compared to the control, 238 miRNAs showed significant differential expression at three stages of SUS progression. Totally, 3149 miRNA‐mRNA pairs were identified by target prediction and 314 miRNA‐mRNA pairs showed negative correlation. It is noteworthy that 15 miRNAs and four mRNAs were located at the crucial positions of the network built with the anti‐correlated miRNA‐mRNA pairs. GO and KEGG enrichment analysis indicated that the predicted targets were involved in many immune‐related processes. Deep analysis of miR‐31c‐5p, miR‐29b‐3p, NF‐kB, mucin 2 and titin showed that they may play important roles in the pathogens attachment and recognition, signaling transduction and lesions repair of A. japonicus after SUS infection. These results would be useful for further investigating the potential roles of critical miRNAs and mRNAs in A. japonicus immune regulation. HIGHLIGHTS. total of 248 conserved miRNAs and 238 differentially expressed miRNAs were identified from Apostichopus japonicus.By target prediction, 3149 miRNA‐mRNA pairs including 2131 mRNAs and 229 miRNAs were obtained.A network was built with 314 miRNA‐mRNA pairs which presented negative correlation.Analyses about miR‐31c‐5p, miR‐29b‐3p, NF‐&kgr;B, mucin 2 and titin showed their immune functions in A. japonicus.