Jing-Zhe Jiang

Identification of a cobia (Rachycentron canadum) CC chemokine gene and its involvement in the inflammatory response.

The chemokines regulate immune cell migration under inflammatory and physiological conditions. We investigated a CC chemokine gene (RcCC1) from cobia (Rachycentron canadum). The full-length RcCC1 cDNA is comprised 673 nucleotides and encodes a four-cysteine arrangement 99-amino-acid protein typical of known CC chemokines. The genomic DNA of RcCC1 consists of three exons and two introns. Phylogenetic analysis showed that RcCC1 was closest to the MIP group of CC chemokines. Quantitative real-time RT-PCR (qRT-PCR) analysis revealed RcCC1 was constitutively expressed in all tissues examined, with relative strong expression in gill, blood, kidney, spleen, and head kidney. The RcCC1 transcripts in the head kidney, spleen, and liver were quickly up-regulated after stimulation with formalin-inactivated Vibrio carchariae (bacterial vaccine) or polyriboinosinic polyribocytidylic acid (poly I:C). These results indicate RcCC1 not only plays a role in homeostasis, but also may be involved in inflammatory responses to bacterial and viral infection.

A new species of Glugea Thélohan, 1891 in the red sea bream Pagrus major (Temminck & Schlegel) (Teleostei: Sparidae) from China

A new microsporidian species is described from farmed red sea bream Pagrus major (Temminck & Schlegel) (Teleostei: Sparidae). Large numbers of spherical whitish xenomas were observed throughout the visceral organs of the host. Histological examination showed that the microsporidia caused several xenomas that were embedded in the intestinal muscularis externa or submucosa. Light and transmission electron microscopy examination of the spores also revealed morphological features typical of species of Glugea Thélohan, 1891. This microsporidian parasite has two different types of mature spores: microspores and macrospores. The spores are elongate-ovoid, with a large posterior vacuole. The polaroplast is bi-partite, with anterior and posterior parts comprising densely packed lamellae and loose membranes, respectively, and occupies approximately the anterior half of the spore. The polar filament is anisofilar, with 12–13 coils in a single layer almost touching the posterior spore wall. Comparison of the small subunit rDNA sequences revealed 92.7–98.1% identity with the sequences available from other Glugea spp. from piscine hosts. Phylogenetic analysis demonstrated that the microsporidian species studied clustered within the Glugea clade with strong support. Based on the differences in the morphological characteristics and molecular data, the microsporidian infecting P. major is considered to represent a species new to science, Glugea pagri n. sp.

Functional annotation of an expressed sequence tag library from Haliotis diversicolor and analysis of its plant-like sequences.

The small abalone, Haliotis diversicolor, is a widely distributed and cultured species in the subtropical coastal area of China. To identify and classify functional genes of this important species, a normalized expressed sequence tag (EST) library, including 7069 high quality ESTs from the total body of H. diversicolor, was analyzed. A total of 4781 unigenes were assembled and 2991 novel abalone genes were identified. The GC content, codon and amino acid usage of the transcriptome were analyzed. For the accurate annotation of the abalone library, different influencing factors were evaluated. The gene ontology (GO) database provided a higher annotation rate (69.6%), and sequences longer than 800bp were easily subjected to a BLAST search. The taxonomy of the BLAST results showed that lancelet and invertebrates are most closely related to abalone. Sixty-seven identified plant-like genes were further examined by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing, only seven of these were real transcripts in abalone. Phylogenic trees were also constructed to illustrate the positions of two Cystatin sequences and one Calmodulin protein sequence identified in abalone. To perform functional classification, three different databases (GO, KEGG and COG) were used and 60 immune or disease-related unigenes were determined. This work has greatly enlarged the known gene pool of H. diversicolor and will have important implications for future molecular and genetic analyses in this organism.

Quantitative PCR detection for abalone shriveling syndrome-associated virus.

Haliotis diversicolor (small abalone) is an important seafood found along the southern coast of China. Since 1999, the yields of cultured abalone in China have been severely affected by an epidemic of continuous outbreaks of a fatal disease. A novel double-stranded DNA virus, abalone shriveling syndrome-associated virus (AbSV), was proven to be one of the main causative agent. Although the pathogenicity and genome of AbSV has been ascertained, the epidemiology of AbSV remains to be investigated. In this study, four pairs of AbSV-specific primers were designed on the basis of the AbSV genome, and were tested for their specificities and sensitivities in quantitative real-time PCRs (qPCRs) after optimization of the annealing temperature. The 3F3/3B3 primer pair was finally chosen with a good specificity and high efficiency of amplification, with a detection limit of about 10 copies of recombinant plasmid containing AbSV genes in a 20-μL reaction mixture. In the detection of AbSV in abalone samples along the southern coast of China, most of the diseased samples had more than 80 virus copies in 1ng host genome DNA. AbSV was also demonstrated in mature hybrid (LY) and juvenile (JH) abalones from assays of healthy animals collected in recent years.

Real-time isothermal detection of Abalone herpes-like virus and red-spotted grouper nervous necrosis virus using recombinase polymerase amplification

Abstract Abalone herpes-like virus (AbHV) and Red-spotted grouper nervous necrosis virus (RGNNV) are two serious viruses that infect animal populations in aquaculture. Both viruses cause diseases associated with high mortality rates, resulting in dramatic economic losses in the aquaculture industry. There are currently no effective treatments for either of these two viral diseases. Thus, early, rapid, and accurate diagnosis plays a fundamental role in disease prevention and control in aquaculture. Traditional methods of diagnosis, such as virus culture, enzyme-linked immunoassay, and polymerase chain reaction (PCR), are either time consuming or require sophisticated temperature control devices. In this study, one sets of specific primers and probes were designed for the real-time quantitative recombinase polymerase amplification (qRPA) detection of AbHV and RGNNV separately. The sensitivity and specificity of detection were evaluated by comparison with detection by conventional PCR and quantitative PCR. The optimal reaction temperature and time for virus detection is 37°C for 20min. The detection limit is 100 copies per reaction, making this approach faster and more sensitive than qPCR in this study. In a field application, the detection percentage of qRPA was higher than that of qPCR for both AbHV and NNV. Additionally, good correlation was found between qRPA and qPCR detection (R2 >0.8). The methods presented here can be used as alternatives to qPCR for quick and quantitative detection of pathogens infecting aquaculture species.

Characterization and transcriptional analysis of a new CC chemokine associated with innate immune response in cobia (Rachycentron canadum)

Chemokines are small, secreted cytokine peptides, known principally for their ability to induce migration and activation of leukocyte populations under both pathological and physiological conditions. On the basis of previously constructed express sequence tags (ESTs) of the head kidney and spleen cDNA library of the perciform marine fish Rachycentron canadum (common name cobia). We used bi-directional rapid amplification of cDNA ends (RACE) and obtained a full-length cDNA of a new CC chemokine gene (designated RcCC3). The RcCC3 putative peptide exhibits sequence similarity to the group of CCL19/21/25 CC chemokines. The reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used in transcript expression studies of RcCC3. We examined the constitutive expression of the transcripts in 12 tissues of non-stressed cobia; RcCC3 transcripts were detected in all tissues examined, with the highest expression in gill and liver, following by head kidney, kidney, spleen, skin, intestine, muscle, stomach, heart, blood and brain. Transcript expression of RcCC3 was examined in immune-related organs, including head kidney, spleen and liver, following intraperitoneal injection of phosphate-buffered saline (control), polyriboinosinic polyribocytidylic acid (poly(I:C)) and formalin-killed Vibrio carchariae (bacterial vaccine). The transcripts in these tissues were quickly up-regulated by the injection of poly(I:C) and bacterial vaccine at early time points, although with different expression profiles. These results indicate RcCC3 represents an important component of innate immunity in cobia.

Nested PCR detection of abalone shriveling syndrome-associated virus in China.

Haliotis diversicolor (small abalone) is an economic seafood found off the Southern coast of China. Since 1999, the cultured abalone yields in China have been affected severely by continual outbreaks of a fatal epidemic disease caused by abalone shriveling syndrome associated virus (AbSV), a double-stranded DNA virus. Although the pathogenicity and genome of AbSV have been ascertained, the epidemiology of AbSV infection remains to be investigated. In the present study, four pairs of AbSV-specific primers were designed on the basis of open reading frame (ORF)24 and ORF25 sequences in the AbSV genome. Two nested PCR detection methods were established by optimization of the annealing temperatures of primers. The results showed that the specificity of primers for AbSV detection could not be interfered with by the host genome and other aquaculture species or viruses. The detection limits of the two methods were about 10 copies of recombinant plasmid containing AbSV genes in 20μL reaction mixture. The results of detection of the AbSV epidemic showed that AbSV was still present in juvenile abalones in some farms along the Southern coast of China (Fujian and Guangdong).

Real-time quantitative isothermal detection of Ostreid herpesvirus-1 DNA in Scapharca subcrenata using recombinase polymerase amplification

Ostreid herpesvirus-1 (OsHV-1) is a well-known pathogen associated with high mortality rates in hatchery-reared larvae and juveniles of different bivalve species worldwide. Early, rapid and accurate diagnosis plays a fundamental role in disease prevention and control in aquaculture. Recombinase polymerase amplification (RPA) is a novel isothermal amplification method, which can amplify detectable amount of DNA at 37 °C-39 °C within 20 min. In the present study, two sets of specific primers and probes were designed for the real-time quantitative RPA (qRPA) detection of OsHV-1 DNA. The sensitivity and specificity of detection were evaluated by comparison with quantitative polymerase chain reaction (qPCR). The detection limit for qRPA assays was shown to be 5 copies DNA/reaction for the primer set ORF95, which was lower than the 100 copies required for the qPCR test. The optimal reaction temperature and time were 37 °C for 20 min, making this approach faster than qPCR. This is the first study to apply qPCR and qRPA methods to detect OsHV-1 in Scapharca subcrenata. The percentage of viral load sample detected by the two methods was 22% and the correlation of the two virus quantitative results was 0.8. Therefore, qRPA assays is sensitive, fast, and high-temperature independent relative to qPCR and is suitable for critical clinical diagnostics use and rapid field analysis in resource-limited settings.

Relationships between and formation dynamics of the microbiota of consumers, producers, and the environment in an abalone aquatic system

An ecosystem is a community comprising living and nonliving components of the environment. Microbes are ubiquitous elements in each of these components. The dynamics of microbiota formation in an ecosystem is important to elucidate, because how the different components of a system exchange microbes, and how the microbes control ecological processes remain unresolved. In this study, an abalone, Haliotis diversicolor, seed-nursing pond was used as a model system. We first examined changes in bacterial communities during the seedling cultivation of this herbivorous juvenile aquatic invertebrate animal. Denaturing gradient gel electrophoresis (DGGE) and pyrosequencing were used to analyze bacterial community dynamics and spatio-temporal interactions of different system components: consumers (abalone), producers (algae or a substrate), and the environment (water). DGGE fingerprints revealed that the developmental stages of abalone influences bacterial communities of both the abalone and substrate. Although the communities in water fluctuated daily, they could be divided into two clusters that coincided with abalone stages, reflecting the transition from larva to juvenile at around day 21. Pyrosequencing showed that the microbiota in the abalone and substrate had more operational taxonomic units in common than that of either with water. The Bray-Curtis similarity index was used to quantify the formation dynamics of microbiota among the various components of the system. The bacterial communities in producers and consumers showed similar changes. These communities were unstable at the beginning and then slowly stabilized over time. The environmental bacterial community was more stable than the bacterial communities in consumers and producers, and may have been the basis for stability in the system. Our research provides insights into the dynamics of microbiota formation in various biotic elements of a system that will contribute to predictive systems modeling.