Su-Kyoung Kim

Selectively enhanced expression of prophenoloxidase activating enzyme 1 (PPAE1) at a bacteria clearance site in the white shrimp, Litopenaeus vannamei

BackgroundThe prophenoloxidase-activating (PO activating) system plays an important role in the crustacean innate immunity, particularly in wound healing and pathogen defense. A key member of this system is prophenoloxidase-activating enzyme (PPAE), which is the direct activator of prophenoloxidase (proPO). Despite their importance in crustacean PO activating system, the studies on them remain limited.ResultsHere we report on a PPAE of white shrimp, Litopenaeus vannamei (lvPPAE1), which showed 94% similarity to PPAE1 of Penaeus monodon. We found that lvPPAE1 in fluid hemocytes was down regulated after challenge by Vibrio harveyi but was enhanced when shrimps were exposed to a bacteria-rich environment for long-term. In vivo gene silence of lvPPAE1 by RNAi can significantly reduce the phenoloxidase activity (PO) and increase the susceptibility of shrimps to V. harveyi. Although lvPPAE1 was down-regulated in fluid hemocytes by Vibrio challenge, its expression increased significantly in gill after bacteria injection, which is the primary bacteria-clearance tissue.ConclusionSuppressed expression in fluid hemocytes and enhanced expression in gill indicates selectively enhanced expression at the bacterial clearance site. This is a novel feature for PPAE expression. The results will contribute to our understanding of the PO activating system in crustaceans.

Effect of multiple infections with white spot syndrome virus and Vibrio anguillarum on Pacific white shrimp Litopenaeus vannamei (L.): mortality and viral replication

Multiple infections are commonly found in practical shrimp culture and may cause more serious consequences than infections by one pathogen only. Therefore, this study was conducted to evaluate the effect of multiple infections with white spot syndrome virus (WSSV) and Vibrio anguillarum on Pacific white shrimp Litopenaeus vannamei (L.), mortality, WSSV replication in vivo and host immune response. In the WSSV single-infection group (WSSV load, 2 × 10(2) copies μL(-1)), mean cumulative mortality was 29.2%. In the V. anguillarum single-infection group, cumulative mortality was 12.5% when shrimp were challenged by 10(5) CFU mL(-1) of bacteria. In the co- and super-infection groups, 37.5% and 50% cumulative mortalities, respectively, were observed at a lower bacterial concentration of 10(3) CFU mL(-1), suggesting that shrimp with multiple infections died earlier and more frequently than singly infected shrimp. WSSV load after injection was tracked over time by TaqMan quantitative PCR. WSSV load increased more rapidly in the multiple-infection groups than in the single-infection group. Additionally, mRNA expression of the genes encoding prophenoloxidase 1 and 2, which are closely involved in innate immunity in shrimp, was down-regulated more extensively in multiple-infection groups than in single-infection groups, as indicated by quantitative reverse-transcription PCR.

Comparison of immune response of Pacific white shrimp, Litopenaeus vannamei, after multiple and single infections with WSSV and Vibrio anguillarum.

Our previous study demonstrated that Pacific white shrimp (Litopenaeus vannamei) infected by multiple pathogens showed higher mortality and death occurred more quickly than those infected by a single pathogen (Jang et al., 2014). For better understanding the defense mechanism against white spot syndrome virus (WSSV) and Vibrio anguillarum, immune responses of shrimp were evaluated in this study. The mRNA expression levels of five immune-related genes were analyzed by quantitative reverse real-time PCR, which included proPO-activating enzyme 1 (PPAE1), PPAE2, proPO activating factor (PPAF), masquerade-like serine proteinase (Mas) and ras-related nuclear gene (Ran). Results demonstrated that the transcription was suppressed more intensively in the multiple infection group than those in single infection groups. The transcriptional suppression was directly related to the higher mortality. The hypoimmunity could benefit pathogen invasion, replication and release of toxin in vivo. Results in this study will help to understand immune defense mechanism after shrimp were infected by multiple pathogens in aquaculture.

Distinct regulation patterns of the two prophenoloxidase activating enzymes corresponding to bacteria challenge and their compensatory over expression feature in white shrimp (Litopenaeus vannamei)

Prophenoloxidase activating enzyme 2 (PPAE2), which belongs to the second PPAE family of prawns, was isolated from white shrimp Litopenaeus vannamei. The currently identified lvPPAE2 and lvPPAE1 from our former report were taken as model candidates to analyze the relationship of the two shrimp PPAE families as well as the regulation mechanism of shrimp PPAEs. The tissue expression of lvPPAE2 was more ubiquitous than lvPPAE1. The mRNA abundance of lvPPAE2 was about 10 percent of lvPPAE1 in co-existed tissues. When challenged with Vibrio harveyi. LvPPAE2 showed a distinct transcriptional regulation pattern compared to lvPPAE1. Silence of lvPPAE2 significantly increased shrimps susceptibility to V. harveyi, suggesting the lvPPAE2 plays essential role in shrimp host defense. A novel PPAE specific compensatory over expression feature was found in the two lvPPAEs. Single gene specific silence of lvPPAE1 and lvPPAE2 resulted in a significant difference in reduction of hemolymph PO activity. Double silence of the two lvPPAEs failed to cause a further reduction on PO activity or shrimp mortality to bacteria, despite that double silence sufficiently suppressed both of the two lvPPAEs. Our findings suggest both lvPPAEs contribute to shrimp melanization cascade and host defense against bacteria. Distinct regulation pattern corresponding to the same pathogen invasion suggests the two lvPPAEs are actually under different regulation ways. A novel PPAE specific compensatory over expression mechanism found in our study offered us a clue in understanding the robustness of shrimp innate immunity and network of crustacean proPO activating system.

Effect of Biofloc on the Survival and Growth of the Postlarvae of three Penaeids (Litopenaeus Vannamei, Fenneropenaeus Chinensis, and Marsupenaeus Japonicus) and their Biofloc Feeding Efficiencies, as Related to the Morphological Structure of the Third Maxilliped

Bioflocs contribute to the nutrition and growth of some penaeid shrimp, such as L. vannamei, by providing an additional food source in intensive, limited exchange production systems. However, it remains unclear whether other penaeids could use bioflocs as food sources. Furthermore, little is known concerning the feeding mechanism of shrimp for harvesting and consuming bioflocs. This study determines whether three penaeid species could use bioflocs as food sources and suggests mechanical functions of the third maxilliped to collect bioflocs in a water column. The postlarvae of L. vannamei, F. chinensis, and M. japonicus were assessed in 14-day culture trials with four treatments: biofloc water with feeding (BF-f), natural seawater with feeding (SW-f), biofloc water without feeding (BF-nf), and natural seawater without feeding (SW-nf). Litopenaeus vannamei exhibited a significantly higher specific growth rate (SGR) in the BF-f treatment than in the SW-f treatment, whereas the other two species did not demonstrate any differences in their SGR and survival rate between BF-f and SW-f treatments, suggesting that bioflocs might contribute to the growth of L. vannamei postlarvae but do not affect the survival and growth of F. chinensis or M. japonicus. The results obtained from the non-feeding experiments implied a filter feeding habit of L. vannamei and a cannibalistic habit of F. chinensis. Differences in the third maxillipeds are relevant in this regard. The feeding mechanism of M. japonicus with intermediate features of setae requires further study. The present study may contribute to understanding the different feeding abilities of bioflocs as food source in currently farmed penaeid shrimp in the Far East Asian countries.

Analysis of Population Distribution on Stocking Area of Sandy Beach Clam, Gomphina melanaegis

The clam, Gomphina melanaegis, which is commonly called the sandy beach clam because of its habitat, is a valuable organism in the sandy coast of East Sea, Korea. It is frequently observed in large populations between 0.5 and 5 meters. We have released 50,000 seedling, ranging from the diameter of approximately 3 mm, 2007, and 1,000,000 seedling, ranging from the diameter of approximately 0.2 mm, plus 100,000 adult individuals, ranging from the shell length of approximately 20~40 mm, 2008 at Jinha beach. The spawning period of G. melanaegis, was from June to August, and the main spawning occurred in July. This investigation was carried out to elucidate population distribution on the stocking area of sandy beach clam, G. melanaegis. The relationship between shell length and ring radius in each ring was investigated as a regression line. The relationship between the shell length and shell height of released young clam was SH=1.0105SL-4.7764 (). The relationship between the shell length and total weight of released young clam was (). It draws a deduction that the ring of this clam was produced once a year during the duration between June and August

Expression of c-Type Lysozyme from the Fleshy Shrimp Fenneropenaeus chinensis Is Upregulated Following Vibrio anguillarum and Lipopolysaccharide Injection

Chicken-type lysozyme (c-lysozyme) is present in shrimp and is active against some bacteria. To further understand the regulation of c-lysozyme in the fleshy shrimp Fenneropenaeus chinensis, we determined the tissue-specific gene expression of c-lysozyme and the time-course of mRNA expression in response to Vibrio anguillarum and lipopolysaccharide (LPS) injection by quantitative reverse real-time polymerase chain reaction. The results showed that c-lysozyme was expressed in all tissues tested, including gill, eyestalk, eye, hemocytes, hepatopancreas, intestine, heart, and pleopod. It was most highly expressed in the intestine followed by the eyestalk, gill, hemocytes and hepatopancreas. The mRNA expression level began to decline in a short time after V. anguillarum challenge and was then upregulated by two fold or more at 24 h post injection (hpi) compared to that at 0 h. Expression was suppressed shortly after LPS injection and began to increase with higher levels of 5.8-, 5.2- and 8.4-fold at 24, 48, and 72 hpi, respectively. Higher expression was sustained and showed a gradual increasing trend until the end of the experiment (72 hpi). These results increase our understanding of the regulation of defense mechanisms and facilitate an evaluation of the effects of probiotics or immunostimulants in shrimp culture.

Acute Toxicity of Cadmium on Gene Expression Profiling of Fleshy Shrimp, Fenneropenaeus Chinensis Postlarvae Using a cDNA Microarray

Abstract Microarray technology provides a unique tool for the determination of gene expression at the level of messenger RNA (mRNA). This study, the mRNA expression profiles provide insight into the mechanism of action of cadmium in Fleshy shrimp (Fenneropenaeus chinensis). The ability of genomic technologies was contributed decisively to development of new molecular biomarkers and to the determination of new possible gene targets. Also, it can be approach for monitoring of trace metal using oligo-chip microarray-based in potential model marine user level organisms. 15K oligo-chip for F. chinensis that include mostly unique sets of genes from cDNA sequences was developed. A total of 13,971 spots (1,181 mRNAs up- regulated and 996 down regulated) were identified to be significantly expressed on microarray by hierarchical clustering of genes after exposure to cadmium for different conditions (Cd24-5000 and Cd48-1000). Most of the changes of mRNA expression were observed at the long time and low concentration exposure of Cd48-1000. But, gene ontology analysis (GO annotation) were no significant different between experiments groups. It was observed that mRNA expression of main genes involved in metabolism, cell component, molecular binding and catalytic function. It was suggested that cadmium inhibited metabolism and growth of F. chinensis .Key words:Microarray, Fenneropenaeus chinensis, Cadmium, Toxicity1)

Seasonal Change of RNA/DNA Ratio and Blood Characteristics of Black Sea Bream Acanthopagrus schlegeli Habituated in Geojae Costal Area, Kyungnam Province, Korea

The monthly variations of blood characteristics and RNA/DNA of black sea bream, Acanthopagrus schlegeli, habituated in Geojae costal area were analysed to determine health condition of natural stocks in terms of gonad maturation and spawning season from March 2010 to February 2011. Spawning season determinated by gonadosomatic index is from June to August. RNA/DNA ratio of black sea bream muscle was strongly correlated with spawning season. During the gonad maturation RNA/DNA ratio in dorsal muscle tissue was decreased contrast to rapid increase during spawning season. Blood composition factors increased in terms of gonad maturation are aspartate aminotransferase, cholesterol, triglyceride, total protein, glucose, globulin, alkaline phosphatase and inorganic phosphate. Other blood factors increased during spawning season are alanine aminotransferase, blood urea nitrogen, uric acid and lactate dehydrogenase.

Changes in the Growth and Body Composition of Juvenile White Shrimp Litopenaeus vannamei Fed diets Containing Fish Meal and Soybean Meal as Protein Sources

A 4-week feeding experiment was conducted with juvenile white shrimp Litopenaeus vannamei to compare the growth of shrimp fed different protein sources: fish meal (FM) and soybean meal (SM). By the end of the experiment, the shrimp grew to g (FM) and g (SM). The food and protein conversion rates were significant (P and , respectively. The proximate compositions of the entire body and edible part of the shrimp were similar between FM and SM, except for crude protein. The total amino acid levels were slightly lower in SM than FM, but the difference was not significant. During the experiment, the total ammonia, nitrate, and nitrite concentrations increased continuously in both groups and were slightly higher in SM than FM. In conclusion, soybean meal as a substitute for fish meal results in satisfactory growth and survival of white shrimp.