Cloning and Expression of PirA gene of Vibrio parahaemolyticus Strain K5 Causing Acute Hepatopancreatic Necrosis Disease in Whiteleg Shrimp in E. coli Host Cell

Background: Acute hepatopancreatic necrosis disease (AHPND), is a bacterial disease of whiteleg shrimp, which has a high mortality rate (100%) and incurs economic losses. Our objective was to identify the genes which lead to cell and organ damage and investigate bioproducts to prevent and treat. Methods: Litopenaeus vannamei shrimp in Thua Thien Hue province, Vietnam were collected from an infected pond and analysed at the Institute of Biotechnology, Hue University. The PirA gene of Vibrio parahaemolyticus strain K5 was isolated and analyzed for nucleotide sequence and paired with the expression vector pQE30. The expression vector was transformed into E. coli strain M15, the PirA recombinant protein was expressed in the form of 6xHis-PirA fusion protein of about 15 kDa. PirA recombinant protein was purified and determined the PirAvp binding ratio, cloning and sequencing of PirA gene from Vibrio parahaemolyticus strain K5 causing AHPND by PCR method with specific primers and molecular weights of PirAvp and the PirAvp complex. Results: PirA gene from Vibrio parahaemolyticus strain K5 was cloned into pGEM-T easy vector (Promega, USA) and screened E. coli TOP10 colonies containing pGEM T easy/PirA recombinant plasmid on LB agar/ampicillin/IPTG/X-Gal medium. PCR showing a band of about 347 bp, matching the size of PirA gene and two nucleotide sequences (BamHI and HindIII). The results showed that PirA gene has a length of 336 bp and similar to PirA gene on GenBank (Code: KU556825.1). The results of protein extracted from E. coli M15 recombinant cells and 6xHis-PirA target protein was collected in elution fractions from EF2 to EF6, showed that the concentration of 6xHis-PirA protein and EF3 elution fraction collected a highest protein concentration (1,586.54 µg/ml). Conclusions: The purified PirA recombinant protein will provide materials for development research to create biological products to prevent and treat AHPND.

Moringa oleifera Leaves’ Extract Enhances Nonspecific Immune Responses, Resistance against Vibrio alginolyticus, and Growth in Whiteleg Shrimp (Penaeus vannamei)

Moringa is widely known as a plant with high medicinal properties. Therefore, moringa has a high potential for use as an immunostimulant in shrimp. This study investigated the effect of a moringa water extract on the immune response, resistance against V. alginolyticus, and growth performance of whiteleg shrimp. To perform the in vitro assay, hemocytes were incubated with different concentrations of the moringa extract. Furthermore, the moringa extract was incorporated at 0 (control), 1.25 g (ME1.25), 2.5 g (ME2.5), and 5.0 g (ME5.0) per kg of diet for the in vivo assay. During the rearing period, immune responses, namely the total hemocyte count (THC), phenoloxidase (PO) activity, phagocytosis activity, superoxide anion production, and immune-related gene expression were examined on days 0, 1, 2, 4, 7, 14, 21, and 28. Growth performance was measured 60 days after the feeding period. Furthermore, the shrimp were challenged with V. alginolyticus after being fed for different feeding durations. The results of the in vitro assay revealed that 100–250 ppm of the moringa extract enhanced the PO activity, phagocytic rate (PR), and superoxide anion production. The findings of the in vivo assay demonstrated that the THC, PO activity, PR, and immune-related gene expression, including alpha-2-macroglobulin, prophenoloxidase II, penaeidin2, penaeidin3, anti-lipopolysaccharide factor, crustin, lysozyme, superoxide dismutase, and clotting protein, were higher in the group of ME.25 and ME5.0 than in the control and ME1.25 at several time points. Growth performance was significantly increased (p < 0.05) in the ME2.5 group compared to the control group. Furthermore, the dietary ME2.5 resulted in a higher survival rate compared to that of the control group after challenging with V. alginolyticus, especially at ME2.5 administered for 4 and 7 days. This study indicated that the incorporation of the moringa extract at 2.5 g per kg of diet enhanced the immune response, the growth performance of the whiteleg shrimp, and the resistance against V. alginolyticus infection.

Sustainable Marine Aquaponics: Effects of Shrimp to Plant Ratios and C/N Ratios

Integrated aquaponic food production systems are capable of producing more food on less land using less water than conventional food systems, and marine systems offer the potential of conserving freshwater resources. However, there have been few evaluations of species combinations or operational parameters in marine aquaponics. The goal of this experiment was evaluation of stocking density ratio of Pacific whiteleg shrimp (Litopenaeus vannamei) to three edible halophytes (Atriplex hortensis, Salsola komarovii, and Plantago coronopus) with two C/N ratios in a 3 × 2 factorial design. There were three stocking density ratios (shrimp: plant), 2:1, 3:1, and 5:1; and two C/N ratios, 12 and 15. The results indicated that stocking density ratio exerted a significant impact on shrimp growth. Shrimp reared in 2:1 and 3:1 treatments had better growth performance. In contrast, plants were affected by both stocking density ratio and C/N ratio. Halophytes grown in stocking density ratios of 3:1 and 5:1 with a C/N ratio of 15 had better growth performance and nutrient content. The concentrations of TAN and NO2– were below 0.2 mg/L throughout the experiment, including the higher stocking density ratio treatments. In conclusion, the stocking density ratio of 3:1 with a C/N ratio of 15 was suggested as the optimal condition for the operation of marine aquaponics in which whiteleg shrimp and the three halophytes are target crops.

Prospects for the whiteleg shrimp Penaeus vannamei (Boone, 1931) cultivation in different regions of Russia, taking into account the hydrochemical characteristics of watebodies and the seasonal dynamics of temperature indicators

Whiteleg shrimp Penaeus vannamei (Boone, 1931) is one of the most popular and promising objects of world aquaculture. In this article possibilities and prospects of cultivating this species in Russia, the results of studies of temperature and salinity effects on the survival rate, growth, activity and food intake of white-footed shrimp were discussed. The critical values of temperature and salinity for this species have been determined. The most progressive for whiteleg shrimp cultivation are the southern regions of Russia, which have an extended sea coastline or a lot of hyperhaline or brackish waterbodies. Technologies combining commercial pond rearing with preliminary rearing of juveniles in RAS are recommended to use to increase the efficiency of cultivation.