LuxS modulates motility and secretion of extracellular protease in fish pathogen Vibrio harveyi.

Vibrio harveyi can cause infections and diseases in a variety of marine vertebrates and invertebrates, which are harmful to the aquaculture industry. The LuxS quorum-sensing system regulates the expression of virulence factors in a wide variety of pathogenic bacteria. In this study, an in-frame deletion of the luxS gene was constructed to reveal the role of LuxS in the physiology and virulence of V. harveyi. Statistical analysis showed no significant differences in the growth ability, biofilm formation, antibiotic susceptibility, virulence by intraperitoneal injection, and ability of V. harveyi to colonize the spleen and liver of the pearl gentian grouper between the wild-type (WT) and luxS mutant. However, deletion of luxS decreased the secretion of extracellular protease, while increasing swimming and swarming abilities. Simultaneously, a luxS-deleted mutant showed overproduction of lateral flagella, and an intact luxS complemented this defect. Since motility is flagella dependent, 16 V. harveyi flagella biogenesis related genes were selected for further analysis. Based on quantitative real-time reverse transcription-PCR (qRT-PCR), the expression levels of these genes, including the polar flagella genes flaB, flhA, flhF, flhB, flhF, fliS, and flrA and the lateral flagella genes flgA, flgB, fliE, fliF, lafA, lafK, and motY, were significantly upregulated in the ΔluxS: pMMB207 (ΔluxS+) strain as compared with the V. harveyi 345: pMMB207 (WT+) and C-ΔluxS strains during the early, mid-exponential, and stationary growth phases. Our results indicate that LuxS plays an important role in controlling motility, flagella biogenesis, and extracellular protease secretion in V. harveyi.

Effect of nitrite exposure on oxidative stress, DNA damage and apoptosis in mud crab (Scylla paramamosain).

Nitrite is one of major environmental pollutants that can impact immunological parameters in aquatic organisms. In the present study, we investigated the effects of nitrite exposure on oxidative stress, DNA damage and apoptosis in mud crab (Scylla paramamosain). Mud crab were exposed to 0, 5, 10 and 15 mg L-1 nitrite for 72 h. These data showed that acid phosphatase (ACP) and alkaline phosphatase (ALP) activity significantly decreased in treatments with various concentrations of nitrite (5, 10 and 15 mg L-1) after 24 and 48 h, while the levels of nitric oxide (NO) significantly increased in these treatments. Nitrite exposure could suppress superoxide dismutase (SOD) and catalase (CAT) activity, and increase the formation of malondialdehyde (MDA) after 48 and 72 h of exposure. In addition, nitrite exposure decreased total haemocyte counts after 48 and 72 h of exposure. Cytological damage, DNA damage and apoptosis was observed obviously at 72 h after nitrite exposure. Moreover, nitrite exposure significantly induced the mRNA levels of phosphorylated Jun N-terminal kinases (JNK), and eventually activated p53 signaling and caspase-3. These results indicated that nitrite exposure could induce oxidative stress, which further caused DNA damage and apoptosis in mud crab. Our results will be helpful to understand the mechanism of nitrite toxicity on crustaceans.

Molecular characterization, expression and antimicrobial activities of a c-type lysozyme from the mud crab, Scylla paramamosain.

Lysozyme is an important immune protein involved in the first line of defense for crustaceans. In the present study, a c-type lysozyme gene (SpLyzC) was cloned and characterized from the mud crab, Scylla paramamosain. The full-length cDNA was 849 bp with an open reading frame of 669 bp, and encoded a polypeptide of 223 amino acids with a calculated molecular mass of 23.7 kDa and an isoelectric point of 8.90. SpLyzC shared conserved active sites with c-type lysozymes from other species, detected in all tested tissues and had higher expression levels in hepatopancreas and gill tissues. The expression of SpLyzC was up-regulated in hepatopancreas and gill after infection with Vibrio parahaemolyticus and Staphylococcus aureus. The density of bacteria in the hemolymph and the mortality of crabs increased following infection with V. parahaemolyticus after SpLyzC expression was silenced by injecting double-strand RNA of SpLyzC. The recombinant protein of the S. paramamosain c-type lysozyme (rSpLyzC) exhibited antibacterial activities against Micrococcus lysodeikticus, S. aureus, Vibrio harveyi and V. parahaemolyticus. These results indicate that SpLyzC could help eliminate bacteria in S. paramamosain and may play an important role in resistance to bacterial infection.

Ammonia toxicity in the mud crab (Scylla paramamosain): The mechanistic insight from physiology to transcriptome analysis.

Ammonia is a major aquatic environmental pollutants. However, the underlying molecular mechanism of ammonia-induced toxicity is not fully understood. In this study, we investigated the physiological response and molecular mechanism in mud crab (Scylla paramamosain) exposed to the acute total ammonia (30 mg L-1) for 48 h. The results shown that ammonia exposure induced oxidative stress, and subsequently led to cytological damage and DNA damage. Transcriptome analysis was applied to investigate the key genes and pathways involved in the responses to ammonia exposure. A total of 722 differentially expressed genes (DEGs) (526 up-regulated and 196 down-regulated) were identified. DEGs mainly involved in pathways including metabolism, cellular processes, signal transduction and immune functions. Additionally, transcriptome analysis revealed that ATM/p53-Caspase3 pathway involved in apoptosis induced by ammonia stress. These results provided a new insight into the mechanism of the potential toxic effects of ammonia on crustaceans.

Virulence regulation of cel-EIIB protein mediated PTS system in Streptococcus agalactiae in Nile tilapia.

Streptococcus agalactiae is a major pathogen of tilapia causing significant economic losses for the global aquatic industry yearly. To elucidate the role of cel-EIIB protein-mediated phosphotransferase systems (PTS) in the virulence regulation of S. agalactiae, cel-EIIB gene deletion in a virulent strain THN0901 was achieved by homologous recombination. The cellobiose utilization of △cel-EIIB strain was significantly decreased relative to S.a.THN0901 strain incubating in LB with 10 mg/ml cellobiose (p < 0.05). The biofilm formation ability of △cel-EIIB strain was also significantly decreased when cultured in BHI medium (p < 0.05). Under a lower infection dose, the accumulative mortality of tilapia caused by △cel-EIIB strain was dramatically decreased (20%), of which S.a.THN0901 strain and △cel-EIIB::i strain were 53.33% and 50%, respectively. The competition experience using tilapia model indicated the invasion and colonization ability of △cel-EIIB strain was significantly weaker than that of S.a.THN0901 strain (p < 0.05). Compared to △cel-EIIB::i strain, the mRNA expression of csrS, csrR, rgfA, rgfC, bgrR and bgrS was significantly downregulated in △cel-EIIB strain (p < 0.05). In conclusion, cel-EIIB protein-mediated cel-PTS not only contributes to biofilm formation and virulence regulation, but also plays an important role in the invasion and colonization of S. agalactiae.

Major surfome and secretome profile of Streptococcus agalactiae from Nile tilapia (Oreochromis niloticus): Insight into vaccine development.

Streptococcus agalactiae is a major piscine pathogen that is responsible for huge economic losses to the aquaculture industry. Safe recombinant vaccines, based on a small number of antigenic proteins, are emerging as the most attractive, cost-effective solution against S. agalactiae. The proteins of S. agalactiae exposed to the environment, including surface proteins and secretory proteins, are important targets for the immune system and they are likely to be good vaccine candidates. To obtain a precise profile of its surface proteins, S. agalactiae strain THN0901, which was isolated from tilapia (Oreochromis niloticus), was treated with proteinase K to cleave surface-exposed proteins, which were identified by liquid chromatography-tandem spectrometry (LC-MS/MS). Forty surface-associated proteins were identified, including ten proteins containing cell wall-anchoring motifs, eight lipoproteins, eleven membrane proteins, seven secretory proteins, three cytoplasmic proteins, and one unknown protein. In addition, culture supernatant proteins of S. agalactiae were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and all of the Coomassie-stained bands were subsequently identified by LC-MS/MS. A total of twenty-six extracellular proteins were identified, including eleven secretory proteins, seven cell wall proteins, three membrane proteins, two cytoplasmic proteins and three unknown proteins. Of these, six highly expressed surface-associated and secretory proteins are putative to be vaccine candidate of piscine S. agalactiae. Moreover, immunogenic secreted protein, a highly expressed protein screened from the secretome in the present study, was demonstrated to induce high antibody titer in tilapia, and it conferred protection against S. agalactiae, as evidenced by the relative percent survival (RPS) 48.61± 8.45%. The data reported here narrow the scope of screening protective antigens, and provide guidance in the development of a novel vaccine against piscine S. agalactiae.

Comparative proteome analysis of two Streptococcus agalactiae strains from cultured tilapia with different virulence.

Streptococcus agalactiae is a major piscine pathogen, which causes significant morbidity and mortality among numerous fish species, and results in huge economic losses to aquaculture. Many S. agalactiae strains showing different virulence characteristics have been isolated from infected tilapia in different geographical regions throughout South China in the recent years, including natural attenuated S. agalactiae strain TFJ0901 and virulent S. agalactiae strain THN0901. In the present study, survival of tilapia challenged with S. agalactiae strain TFJ0901 and THN0901 (10(7)CFU/fish) were 93.3% and 13.3%, respectively. Moreover, there are severe lesions of the examined tissues in tilapia infected with strain THN0901, but no significant histopathological changes were observed in tilapia infected with the strain TFJ0901. In order to elucidate the factors responsible for the invasive potential of S. agalactiae between two strains TFJ0901 and THN0901, a comparative proteome analysis was applied to identify the different protein expression profiles between the two strains. 506 and 508 cellular protein spots of S. agalactiae TFJ0901 and THN0901 were separated by two dimensional electrophoresis, respectively. And 34 strain-specific spots, corresponding to 27 proteins, were identified successfully by MALDI-TOF mass spectrometry. Among them, 23 proteins presented exclusively in S. agalactiae TFJ0901 or THN0901, and the other 4 proteins presented in different isomeric forms between TFJ0901 and THN0901. Most of the strain-specific proteins were just involved in metabolic pathways, while 7 of them were presumed to be responsible for the virulence differences of S. agalactiae strain TFJ0901 and THN0901, including molecular chaperone DnaJ, dihydrolipoamide dehydrogenase, thioredoxin, manganese-dependent inorganic pyrophosphatase, elongation factor Tu, bleomycin resistance protein and cell division protein DivIVA. These virulence-associated proteins may contribute to identify new diagnostic markers and help to understand the pathogenesis of S. agalactiae.

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).

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.

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.

[Effects of abrupt salinity stress on osmoregulation of juvenile Rachycentron canadum].

Rachycentron canadum is a thriving mariculture species for offshore cage in southern Mainland and Taiwan of China, due to its rapid growth rate and high quality flesh. In this paper, the gill Na(+)-K+ ATPase (NKA) activity and iono- and osmoregulation of juvenile R. canadum were investigated in a 12 h stress of ambient salinities (0-45), and the results showed that after an abrupt transfer to the salinities of 0, 5, 15, 25, 37 (control) and 45, the death of juvenile R. canadum only occurred in salinity 0, with a mortality of 100% by the end of the experiment. In all treatments, the gill NKA activity and serum osmolality fluctuated in first 3 h, and then changed smoothly. The NKA activity varied with salinity grade in U shape, being significantly (P < 0.05) higher in salinity 5 and the lowest in salinity 15 in 12 h, while the serum osmolality (ranged 293-399 mOsmol x kg(-1)) presented a positive correlation with salinity. Serum [Na+] and [Cl-] concentration slightly increased with salinity within the period of 3-12 h, while serum [K+] displayed a reverse pattern. The isosmotic point was estimated as 328.2 mOsm x kg(-1) and corresponded to salinity 11.48. The isoionic points for serum [Na+], [K+] and [Cl-] were estimated as 155.2, 6.16, and 137.1 mmol x L(-1), which corresponded to the salinities of 10.68, 20.44 and 8.41, respectively. It was concluded that R. canadum could be characterized physiologically as a "higher-NKA-in-hyposmotic media" marine euryhaline teleost with the capability of rapid and effective hyper/hypo iono- and osmoregulation.