Isolation and Characterization of Vibrio kanaloae as a Major Pathogen Associated with Mass Mortalities of Ark Clam, Scapharca broughtonii, in Cold Season

High temperature is a risk factor for vibriosis outbreaks. Most vibrios are opportunistic pathogens that cause the mortality of aquatic animals at the vibrio optimal growth temperature (~25 °C), whereas a dominant Vibrio kanaloae strain SbA1-1 is isolated from natural diseased ark clams (Scapharca broughtonii) during cold seasons in this study. Consistent symptoms and histopathological features reappeared under an immersion infection with SbA1-1 performed at 15 °C. The pathogenicity difference of SbA1-1 was assessed under different temperatures (15 °C and 25 °C). The cumulative mortality rates of ark clams were significantly higher at the low temperature (15 °C) than at the high temperature (25 °C); up to 98% on 16th day post SbA1-1 infection. While the growth ratio of SbA1-1 was retarded at the low temperature, the hemolytic activity and siderophores productivity of SbA1-1 were increased. This study constitutes the first isolation of V. kanaloae from the natural diseased ark clams (S. broughtonii) in cold seasons and the exposition of the dissimilar pathogenicity of SbA1-1 at a different temperature. All the above indicates that V. kanaloae constitutes a threat to ark clam culture, especially in cold seasons.

Purification and Characterization of a New CRISP-Related Protein from Scapharca broughtonii and Its Immunomodulatory Activity

More and more attention has been paid to bioactive compounds isolated from marine organisms or microorganisms in recent years. At the present study, a new protein coded as HPCG2, was purified from Scapharca broughtonii by stepwise chromatography methods. The molecular weight of HPCG2 was determined to be 30.71 kDa by MALDI-TOF-MS. The complete amino acid sequence of HPCG2 was obtained by tandem mass spectrometry combined with transcriptome database analysis, and its secondary structure was analyzed using circular dichroism. HPCG2 comprised 251 amino acids and contained 28.4% α-helix, 26% β-sheet, 18.6% β-turn, and 29.9% random coil. HPCG2 was predicted to be a cysteine-rich secretory protein-related (CRISP-related) protein by domain prediction. Moreover, HPCG2 was proved to possess the immunomodulatory effect on the murine immune cells. MTT assay showed that HPCG2 promoted the proliferation of splenic lymphocytes and the cytotoxicity of NK cells against YAC-1 cells. Flow cytometry test revealed that HPCG2 enhanced the phagocytic function of macrophages and polarized them into M1 type in RAW264.7 cells. In particular, Western blot analysis indicated that the immunomodulatory mechanism of HPCG2 was associated with the regulation on TLR4/JNK/ERK and STAT3 signaling pathways in RAW 264.7 cells. These results suggested that HPCG2 might be developed as a potential immunomodulatory agent or new functional product from marine organisms.

Effects of Acute Low-Salinity Stress on the Activities of Catalase (CAT), Superoxide Dismutase (SOD) and Glutathiones-Transferase (GST) in Scapharca broughtonii

Salinity is an important environmental factor of osmotic pressure which is closely related to growth and development of aquatic organisms. Some physiological studies regarding to challenge of salinity have been reported in marine shellfish. However, information about the effects of acute low-salinity stress on the enzyme activities in ark shell, Scapharca broughtonii, is still limited now. In this study, the enzyme activities of catalase (CAT), superoxide dismutase (SOD) and glutathiones-transferase (GST) in five tested tissues of S. broughtonii under different salinity (10, 15, 20, 25 and 30‰) for different challenged time (24, 48, 72 and 96h) were investigated. The dynamic change of the three enzyme activities after challenged were further analyzed according to the detected data. Results revealed that low-salinity stress generated great change of CAT, SOD and GST activities in organism, but the change peculiarity in different tissues was different in some degree. Especially, two of the five tissues, hepatopancreas and foot, showed special characteristics responding to the low-salinity stress. It also showed that the variations of the three enzymes activities have tissue specificity and time sequentiality in S. broughtonii.