Cloning, expression prolife, and immune characterization of a novel stat family member (stat5bl) in Chinese tongue sole (Cynoglossus semilaevis).

STAT plays important roles in innate immunity during JAK/STAT signaling pathway, and STAT5 is particularly focused due to the existence of duplicated forms in fish and mammal. In Chinese tongue sole, stat5bl was suggested to be a candidate related to Vibrio harveyi resistance based on previous QTL screening. In this study, the full length of stat5bl cDNA was cloned and its expression patterns were analyzed. stat5bl was predominantly expressed in immune tissues, where the highest level was observed in liver, followed by skin and gill. Time course expression patterns were examined in six tissues (liver, skin, gill, kidney, intestine, spleen) after V. harveyi infection. stat5bl could be up-regulated by V. harveyi infection in all tissues except liver, despite the timepoints of peak were different. In contrast, stat5bl was significantly downregulated in liver. To elucidate the role of stat5bl in liver, in vitro RNAi were performed using primary liver cell culture. Knockdown of stat5bl could regulate the expression of genes closely related to JAK/STAT pathway. This study would enlarge our understanding of stat5bl in fish immunity.

Cloning, characterization and functional analysis of dctn5 in immune response of Chinese tongue sole (Cynoglossus semilaevis).

In mammals, microtubule-dependent trafficking could participate the immune response, where the motor proteins are suggested to play an important role in this process, while the related study in fish was rare. In this study, dctn5, a subunit of dyactin complex for docking motor protein, was obtained by previous immune QTL screening. The full-length cDNAs of two dctn5 transcript variants were cloned and identified (named dctn5_tv1 and dctn5_tv2, respectively). Tissue distribution showed that dctn5_tv1 was widely distributed and high transcription was observed in immune tissue (skin), while dctn5_tv2 was predominantly detected in gonad and very low in other tissues. Time-course expression analysis revealed that dctn5_tv1 could be up-regulated in gill, intestine, skin, spleen, and kidney after Vibrio harveyi challenge. Moreover, recombinant Dctn5_tv1 exhibited high antimicrobial activity against Escherichia coli and Streptococcus agalactiae due to binding to bacteria cells. Taken together, these data suggest Dctn5_tv1 is involved in immune response of bacterial invasion in Chinese tongue sole.

Molecular characterization and expression analysis of a novel r-spondin member (rspo2l) in Chinese tongue sole (Cynoglossus semilaevis).

Numerous studies suggest R-spondins (Rspos) plays a role in mammalian sex development and differentiation by activating WNT signaling pathways. However, Rspos are frequently less reported in teleosts. In this study, a molecular characterization and expression analysis was conducted with a new rspondin member in the Chinese tongue sole, rspondin2-like (rspo2l). The length of rspo2l cDNA is 1251 bp with 732 bp of coding sequence. A qRT-PCR analysis revealed that the transcription of rspo2l was distributed in various tissues, with high transcription levels in the liver, skin, and gills which might indicate a possible role in immunity. We next examined a time-course of transcription levels in four immune tissues (gill, liver, spleen, and kidney) after Vibrio harveyi challenge. It was found that rspo2l was up-regulated in the gills, spleen, and kidney and down-regulated in the liver, and the greatest responses occurred at 24 and 48 h after bacterial challenge. An assessment of ß-catenin, the key regulator of the canonical WNT signaling pathway, at different time points in four immune organs revealed that its transcription profile was similar to that of rspo2l after bacterial challenge. The results suggest that tongue sole rspo2l might play a role in immune responses after bacterial challenge, while the potential link with the WNT signaling pathway still requires further investigation. This is the first report about the involvement of rspondins in fish immune responses.

[Effects of salinity on spawning and larval development of Exopalaemon carinicauda].

Female Exopalaemon carinicauda at ovarian maturation stage II rearing by artificial propagation in the laboratory were chosen as test material. The shrimps were gradually acclimated to the experimental salinity levels of 2, 5, 10, 15, 20, 25 and 30 to determine the effects of salinity on spawning, embryonic development and larval growth of E. carinicauda. The results showed that the ovaries of female E. carinicauda could mature at all test salinity levels. However, it failed to spawn at salinity 2. Salinity levels from 10 to 20 were the most favorable for female E. carinicauda spawning. Although larval development was most successful in eggs incubated at salinity levels of 5 to 30, embryonic development were significantly affected by salinity, and the egg incubation period was shorter at salinities of 15, 20 and 25 than at the other salinities. There were no significant effects of salinity on the rates of larval metamorphosis and survival, but the dry mass of individuals was significantly affected by salinity. The dry mass of shrimps reared at salinities of 15 and 20 were significantly higher than at the other salinities. The growth of 20-day old shrimps was significantly affected by salinity. The specific growth rate increased with the increasing salinity level from 5 to 20, and then decreased at the salinity above 20. The mRNA level of gill Na(+)-K(+)-ATPase was high at high and low salinities, and the lowest at the theoretical salinity 17.5, which might be equal to the hemolymph osmotic pressure of E. carinicauda. It was implied that female parent E. carinicauda could reproduce in a wide range of salinities, while 20-day old shrimps presented higher growth rates at salinities near its theoretical isosmotic point.