Fengying Zhang

Multiplex immune-related genes expression analysis response to bacterial challenge in mud crab, Scylla paramamosain

Crabs lack an acquired adaptive immune system and host defense is believed to depend entirely on innate, non-adaptive mechanisms to resist invasion by pathogens. Discovery of immune-related factors are helpful for understanding the molecular response of crabs to pathogens. The mud crab Scylla paramamosain is an important marine species for aquaculture in China because of its high nutritional value for humans. In recent years, the crab is prone to being infected by microbes with the enlargement of breeding scale. In this study, eight immune-related genes were analyzed by multiplex genes expression analysis using the GenomeLab GeXP analysis system (Beckman Coulter). The expression levels of all the detected genes rose after challenged by the live bacteria, but the levels of only four genes (C-type lectin, alpha 2-macroglobulin, HSP70 and thioredoxin 1) increased after challenge in heat-killed bacteria group. So the live bacteria were more effective in motivating expressions of immune factors than heat-killed bacteria. However, the transcript of C-type lectin firstly increased at 1 h after challenge in both heat-killed and live bacteria group. This indicated that C-type lectin was a quite susceptive immune factor responding to external pathogen. In group challenged by live bacteria, the genes of alpha 2-macroglobulin, HSP40, thioredoxin 1 and prophenoloxidase activating factor (PPAF) showed response earlier than the other genes. The rise of PPAF expression preceded prophenoloxidase (proPO), which suggested that PPAF might trigger production of proPO transcripts in the early stage of phenoloxidase reaction system. C-type lectin, proPO, thioredoxin 1, HSP40, and alpha 2-macroglobulin are very important immunity factors in response to bacterial infection. According to the result of heat-killed group, HSP70 is a sensitively inductive factor to foreign stimulus compared with the other genes. The multi-gene analysis presented an alternative approach for screening of immune-related genes, and provided a more global overview of genes transcript alteration in response to bacterial challenge.

ISOLATION AND CHARACTERIZATION OF A FERRITIN cDNA FROM THE MUD CRAB SCYLLA PARAMAMOSAIN

Abstract Ferritin is an important protein for iron storage in cells. A hepatopancreas cDNA library from the mud crab Scylla paramamosain was constructed using the SMART technique. A complete cDNA sequence that showed high identity with the conserved sequence of the ferritin gene was cloned from the cDNA library and subjected to further investigation. The full-length ferritin gene of Scylla paramamosain (SpFer) consists of 767 bp and contains a complete open reading frame of 513 bp and a 26-bp iron-respective element in the 5′-untranslated region. The gene encodes a polypeptide of 170 amino acids, constituting a predicted molecular weight of 19.44 kDa and a theoretical isoelectric point of 5.24. The deduced protein shares 84% identity with the ferritin protein of the crab Eriocheir sinensis. Quantitative real-time PCR analyses revealed that the expression of ferritin was ubiquitous in different organs of S. paramamosain, including muscle, heart, ovary, testis, and hepatopancreas. The highest expression level was found in the heart, while testis tissue showed the lowest level. Ferritin mRNA expression in continuous developmental stages in zoeal phases, including Z1, Z2, Z3, Z4, and Z5, as well as megalopa and juvenile crab I stages, were also examined by quantitative real-time PCR. The expression level of ferritin was highest in the Z1 stage and lowest in the megalopa stage. This study provides useful information regarding the structure and function of ferritin and will play an important role in immunity and resistance research in S. paramamosain.

The Gut Microbial Community of Antarctic Fish Detected by 16S rRNA Gene Sequence Analysis

Intestinal bacterial communities are highly relevant to the digestion, nutrition, growth, reproduction, and a range of fitness in fish, but little is known about the gut microbial community in Antarctic fish. In this study, the composition of intestinal microbial community in four species of Antarctic fish was detected based on 16S rRNA gene sequencing. As a result, 1 004 639 sequences were obtained from 13 samples identified into 36 phyla and 804 genera, in which Proteobacteria, Actinobacteria, Firmicutes, Thermi, and Bacteroidetes were the dominant phyla, and Rhodococcus, Thermus, Acinetobacter, Propionibacterium, Streptococcus, and Mycoplasma were the dominant genera. The number of common OTUs (operational taxonomic units) varied from 346 to 768, while unique OTUs varied from 84 to 694 in the four species of Antarctic fish. Moreover, intestinal bacterial communities in individuals of each species were not really similar, and those in the four species were not absolutely different, suggesting that bacterial communities might influence the physiological characteristics of Antarctic fish, and the common bacterial communities might contribute to the fish survival ability in extreme Antarctic environment, while the different ones were related to the living habits. All of these results could offer certain information for the future study of Antarctic fish physiological characteristics.

Transcriptome Sequencing, De Novo Assembly and Differential Gene Expression Analysis of the Early Development of Acipenser baeri.

The molecular mechanisms that drive the development of the endangered fossil fish species Acipenser baeri are difficult to study due to the lack of genomic data. Recent advances in sequencing technologies and the reducing cost of sequencing offer exclusive opportunities for exploring important molecular mechanisms underlying specific biological processes. This manuscript describes the large scale sequencing and analyses of mRNA from Acipenser baeri collected at five development time points using the Illumina Hiseq2000 platform. The sequencing reads were de novo assembled and clustered into 278167 unigenes, of which 57346 (20.62%) had 45837 known homologues proteins in Uniprot protein databases while 11509 proteins matched with at least one sequence of assembled unigenes. The remaining 79.38% of unigenes could stand for non-coding unigenes or unigenes specific to A. baeri. A number of 43062 unigenes were annotated into functional categories via Gene Ontology (GO) annotation whereas 29526 unigenes were associated with 329 pathways by mapping to KEGG database. Subsequently, 3479 differentially expressed genes were scanned within developmental stages and clustered into 50 gene expression profiles. Genes preferentially expressed at each stage were also identified. Through GO and KEGG pathway enrichment analysis, relevant physiological variations during the early development of A. baeri could be better cognized. Accordingly, the present study gives insights into the transcriptome profile of the early development of A. baeri, and the information contained in this large scale transcriptome will provide substantial references for A. baeri developmental biology and promote its aquaculture research.

Isolation of the C-type lectin like-domain cDNAs from the mud crab Scylla paramamosain Estampador, 1949, and its expression profiles in various tissues, during larval development, and under Vibrio challenge

Scylla paramamosain Estampador, 1949 is one of the most precious marine crabs farmed in China. The crab is prone to infection by microbes at various stages during its growth, leading to high mortality. C-type lectin has a characteristic carbohydrate recognition domain and plays an important role in the immunity system. A cDNA library from the mud crab S. paramamosain was constructed using the SMART technique. Two complete cDNA sequences showing high identities with the C-type lectin gene were isolated from the library. The two full-length C-type lectin cDNAs of S. paramamosain (SpLec1 and SpLec2) consist of 746 and 834 bp, respectively. Quantitative realtime PCR analyses revealed that the C-type lectins were expressed mainly in haemocytes, muscle and hepatopancreas, with the highest expression level found in hepatopancreas. The C-type lectin mRNA expression in continuous developmental stages during zoeal phases were also examined by quantitative real-time PCR. After infection with Vibrio parahaemolyticus (Sakazaki, 1963) at a concentration of 2.3 × 10 6 cfu/ml, the temporal expression of SpLec1 and SpLec2 mRNA in the megalopa stage was first increased, reached a maximum, and then dropped to the original level again. The research of C-type lectins in Scylla paramamosain could shed new light on studies on immunity and moulting in the mud crab.

The complete mitochondrial genome of Chionodraco hamatus (Notothenioidei: Channichthyidae) with phylogenetic consideration

Abstract The complete mitochondrial genome of Chionodraco hamatus was obtained, which was 17 457 bp in length. This genome consists of 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and a putative control region. Of the 37 genes, 28 were encoded by heavy strand, while 9 were encoded by light strand. Overall base composition of mitogenome is estimated to be 26.38% for A, 17.44% for G, 26.00% for T, 30.18% for C, respectively, with a slight A + T bias (52.38%). The phylogenetic analysis based on 13 concatenated protein-coding genes suggested that C. hamatus as a sister species to Chionodraco myersi was clustered in family Chionodraco. The complete mitochondrial genome sequence of C. hamatus could provide a basic data for the studies on evolution for low temperature adaptability, population structure, molecular systematic, stock evaluation and conservation genetics.

[Macrophage migration inhibitory factor in mud crab Scylla paramamosain: molecular cloning, expression profiles in various tissues and under Vibrio challenge].

As one of the first found cytokines, macrophage migration inhibitory factor (MIF) plays an important role in several physiological processes in crabs. In this study, a full-length MIF cDNA (GenBank accession number: JX131610) from mud crab Scylla paramamosain (Sp) was cloned based on a sequence of S. paramamosain cDNA library. The full length of SpMIF was 734 bp consisting of a 363 bp open reading frame encoding the SpMIF, a 120 amino acid peptide chain. The molecular weight of SpMIF was 13.46 kDa with the pI of 6.82. The alignment analysis showed that SpMIF appeared to be closely related to the counterpart from Eriocheir sinensis (68%). Quantitative real-time PCR analysis revealed that SpMIF was highly expressed in hepatopancreas and hemocytes. In addition, the expression level of SpMIF was increased significantly after a 6-h challenge by Vibrio parahaemolyticus (4.00 × 106 CFU/mL), peaked at 8 h, and then declined to the common level in 48 h. This data indicated that SpMIF was cloned successfully, and suggested that it participated in the immune system of mud crabs.

Two transcripts of HMG-CoA reductase related with developmental regulation from Scylla paramamosain: Evidences from cDNA cloning and expression analysis.

3‐Hydroxy‐3‐methylglutaryl coenzyme‐A (HMG‐CoA) reductases (HMGRs), which catalyze the conversion of HMG‐CoA to mevalonate, may have an important role in the synthesis of methyl farnesoate (MF). In this study, we obtained two HMGR cDNA sequences termed Sp‐HMGR1 (membrane‐bound form) and Sp‐HMGR2 (soluble form), which encode 967 and 654 amino acids, respectively. The two cDNAs possess entirely identical sequences except that Sp‐HMGR1 is 1,382 bp, which encodes a sterol‐sensed domain (SSD; a membrane‐bound domain) and was first found in crustacean HMGR, larger than Sp‐HMGR2. Thus, it was deduced that these cDNAs might be derived from a single genomic DNA sequence. Sp‐HMGRs have the typical features of the HMGR class of proteins. However, residue 844 in Sp‐HMGR1, which is usually occupied by a Ser residue in other species, has an unusual Ala substitution. This Ser is thought to be involved in enzyme activity regulation by reversible phosphorylation. A putative “PEST” sequence that, until now, has only been found in crustacean species was also identified in the C‐terminus of both transcripts, and a sterol‐sensing domain, which was first found in crustacean species, was identified in Sp‐HMGR1; these findings suggest that Sp‐HMGR might function in some special regulatory mechanism. Furthermore, the quantitative real‐time polymerase chain reaction results showed that the two transcripts have different expression patterns; Sp‐HMGR2 was mainly expressed in the mandibular organ (MO) of adult crabs, whereas Sp‐HMGR1 was mainly expressed in other tissues and fertilized eggs up until the fourth juvenile crab stage. The fluctuating gene expression seemed to suggest a relationship between Sp‐HMGRs and the development of the crab, especially during the larval stage. Besides, the fluctuation of Sp‐HMGR1 in ovary, brain, and thoracic ganglia during the ovary development seemed to have some correlation with the nutrition accumulation of ovaries, whether the SSD domain evolved in this process deserve further investigation. Moreover, it remains unclear whether the significant variation in ovary, brain, and thoracic ganglia during ovary development suggests that other tissues in addition to the MO could synthesize MF.

Sensitive and rapid detection of two toxic microalgae Alexandrium by loop-mediated isothermal amplification

A loop-mediated isothermal amplification (LAMP) assay was designed and evaluated for rapid detection of the toxic microalgae Alexandrium catenella and A. minutum, which can produce paralytic shellfish poisoning (PSP). Two sets of four specific primers targeting these two species were derived from the sequence of internal transcribed spacer (ITS) of ribosomal DNA. The method worked well in less than an hour under isothermal conditions of 65°C. LAMP specificity was validated in closely related algae as a comparison, suggesting the strict specificity of the LAMP primers. Two visual inspection approaches were feasible to interpret the positive or negative results. The detection limits of A. catenella and A. minutum samples using the LAMP assay were found to be 5.6 and 4.5 pg DNA, respectively. The sensitivity of this LAMP assay was 10 or 100-fold higher than Polymerase Chain Reaction (PCR) method in detecting the two microalgae. These characteristics of species specificity, sensitivity, and rapidity suggest that this method has the potentiality in the monitoring of red tide caused by A. catenella and A. minutum.

Identification of a trypsin gene from Scylla paramamosain and its expression profiling during larval development

) was 881 bp, with a 780-bp open reading frame encoding a polypeptide of 259 amino acid residues. Comparison with genomic DNA revealed that it contained two introns. The deduced monomer of trypsin had a molecular weight of 28.15 KD and the isoelectric point was 4.49. Alignment analysis and structure prediction showed that