Astaxanthin combined with formic acid helps increase resistance to Vibrio parahaemolyticus bacteria in whiteleg shrimp
A 90-day feeding trial was conducted to evaluate the effects of formic acid (FA) and astaxanthin (AX) on growth, survival, immune parameters and resistance to Vibrio infection parahaemolyticus in Pacific white shrimp. The study was divided into two experiments. In experiment 1, 12-day-old postlarvae were randomly assigned into six groups and then fed four times daily with six test diets containing 0.3 % FA, 0.6 % FA, 50 ppm AX, 0 .3 % FA + 50 ppm AX, .6 % FA + 50 ppm AX, and control sample (none of these supplements). After the 60-day feeding trial, the body weights of all treatment groups were not significantly different from the control group, although shrimp fed formic acid had significantly lower body weights than shrimp fed formic acid. fed 50 ppm AX. However, the AX 0.6% FA + 50 ppm group had a significantly higher survival rate (82.33 ± 8.32%) than the control group (64.33 ± 10.12%).
In experiment 2, Vibrio parahaemolyticus was added to each tank to achieve a final concentration of 10^4 CFU/mL. Each treatment group received the above-mentioned diet for an additional 30 days. At the end of this experiment, there was no difference in weight gain between all experimental groups. However, the survival rate of shrimp fed diets including formic acid, astaxanthin and their combination (range 45.83–67.50%) was significantly higher than that of the control group (20.00 ± 17.32%). Shrimp fed with FA also had significantly lower total intestinal bacteria and Vibrio spp. while the immune parameters [total blood cell count (THC), phagocytic activity, phenoloxidase (PO) activity and superoxide dismutase (SOD) activity] of AX-fed groups were significantly improved compared to other groups. In summary, Formic acid, astaxanthin and their combination are useful in stimulating the shrimp immune system to resist Vibrio parahaemolyticus bacteria.
METHOD
Experiment 1 Effect of formic acid and astaxanthin on the growth and survival of Pacific white shrimp postlarvae.
Shrimp and experimental procedures
The experiments were conducted at the Aquaculture Business Research Center Laboratory, Faculty of Fisheries, Kasetsart University, Thailand. Postlarvae-9 (PL-9) of Pacific white shrimp was obtained from a hatchery in Chachoengsao province, Thailand. After 3 days of acclimation, shrimp (PL-12) were randomly assigned to 24 × 500-L fiberglass tanks (four replicate tanks per treatment). Each tank is stocked with 75 shrimp. Each treatment group was fed one of six diets four times daily to satiety for 60 days. Salinity throughout the experiment was maintained at 25 ppt, dissolved oxygen above 4 ppm, and water temperature at 29 ± 1 °C. Food scraps and feces are vacuumed daily, with 10% water changed every 3 days. The average body weight and survival rate of shrimp were recorded after a 60-day experimental period. Ten shrimp from each tank were randomly selected and weighed individually using a two-decimal-digit scale.
Experiment 2 Effects of formic acid and astaxanthin on growth, survival, gut bacteria, and immune response of Pacific white shrimp infected with Vibrio parahaemolyticus .
Shrimp and experimental procedures
Shrimp from each tank in experiment 1 were randomly assigned to new 24 × 500-L fiberglass tanks (four replicate tanks per treatment). Stocking density is 30 fish/tank. At the beginning of this experiment (0 days), Vibrio parahaemolyticus was added to each tank to achieve a final concentration of 10 4 colony forming units (CFU)/mL, which is the normal concentration of Vibrio in water Shrimp culture as described by Sung et al. ( 2001 ) and Lavilla-Pitogo et al. ( 1998 ). V. parahaemolyticus used for the immersion challenge test in this study was collected from the EMS farm in Thailand using the method described by Joshi et al. ( 2014 ). Each treatment group received the same diet as in experiment 1 four times daily for an additional 30 days. Salinity, dissolved oxygen and water temperature were maintained as in experiment 1. Food scraps and feces were vacuumed every 2 days.
Growth and survival research
The weight of shrimp from each treatment was measured and their survival recorded at day 30 after challenge with V. parahaemolyticus at 10^4 CFU /mL.
Research on intestinal bacteria
Five shrimp from each group were randomly selected and their intestines were collected on days 10, 20, and 30. The intestines of each shrimp were homogenized and spread on TCBS (selective medium for the culture of Vibrio spp.) or NA (common medium for most bacterial cultures) using the spread plate technique, followed by incubation at 37°C for 24 hours. Finally, all bacterial colonies were counted and calculated in CFU/g units.
Research on immune parameters
Immune parameters were measured at the end of the feeding trial. Ten shrimp per treatment were used for immunoassay. A blood sample of 250 µL from each shrimp was taken from the base of the 3rd leg using a syringe containing 750 µL of pre-cooled (4°C) anticoagulant (0.114 M trisodium citrate, 450 mM NaCl, 10 mM KCl, 10 mM HEPES at pH 7.4) (Nonwachai et al. 2010 ). The anticoagulant-hemolytic mixture was used to measure total blood cell count (THC), phagocytic activity, phenoloxidase (PO) activity, superoxide dismutase (SOD) activity, and bactericidal activity.
1. Total blood cell count
After blood collection, blood cells were counted using a hemocytometer and calculated as THC (cells/mL) = count × 104 × dilution factor.
2. Phagocytic activity
Phagocytic activity was determined according to Itami et al. ( 1994 ). The collected shrimp erythrocytes were washed with shrimp brine (NaCl solution 28.4 g, MgCl 2·6H 2O 1.0 g, MgSO4·7H 2O 2.0 g, CaCl 2·2H 2O 2.25 g, KCl 0.7 g, glucose 1.0 g, and HEPES 2.38 g/L) and the live cell count was adjusted to 1 × 10^6 cells/mL. The cell suspension (200 µL) was seeded onto a glass slide. After 20 minutes, remove the cell suspension and rinse with shrimp brine three times. Heat-reducing yeast preparation (2 mL) was added and incubated for 2 hours. Next, the thermocidal yeast preparation was removed and the cell suspension was washed with shrimp brine 5 times to reach a concentration of 5 × 10 8 cells/mL and fixed with 100 % methanol. Slides were then stained with Giemsa stain and mounted with Permount slide mounting fluid. Two hundred blood cells were counted for each sample. Phagocytic activity, defined as the rate of phagocytosis expressed as:
Phagocytosis ratio = (phagocytic blood cells/total blood cells) × 100
3. Phenoloxidase activity
Phenoloxidase activity was measured spectrophotometrically by recording the formation of dopachrome generated from l-dihydroxyphenylalanine, after modification of a published procedure (Supamattaya et al. 2000 ). The hemolytic-anticoagulant mixture was washed three times with shrimp brine and centrifuged at 1000 rpm and 4°C for 10 min. Blood cell lysate was prepared from blood cells in cacodylate buffer (pH 7.4; 0.01 M sodium cacodylate, 0.45 M sodium chloride, 0.01 M calcium chloride, and 0.26 M magnesium chloride ; pH 7.0) using an ultrasound at amplitude 30 for 5 s and the suspension was then centrifuged at 10,000 rpm at 4°C for 20 min and the supernatant was collected. Then, 200 µL of 0.25% trypsin in cacodylate buffer was mixed into 200 µL of blood cell lysate, followed by 200 µL of l-dihydroxyphenylalanine at 4 mg/mL as substrate. Enzyme activity was measured by absorbance of dopachrome at 490 nm. The protein content of the blood cell lysate was measured according to a published procedure (Lowry et al. 1951 ). Phenoloxidase activity was calculated as the optimal density increase per minute per milligram of protein.
4. Superoxide dismutase activity
SOD activity was measured by its ability to inhibit superoxide radical-dependent reactions using the Ransod Kit (Randox, Crumlin, UK). The method is based on the formation of red formazan in the reaction of 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride (INT) and superoxide radical, analyzed in a spectrophotometer at wavelength 505 nm. The reaction mixture (1.7 mL) contained 0.05 mM xanthine and 0.025 mM INT dissolved in 50 mM CAPS (pH 10.2) and 0.94 mM EDTA. In the presence of xanthine oxidase, superoxide and uric acid are generated from xanthine. The superoxide radicals then react with INT to produce the red formazan dye. The hemolytic-anticoagulant mixture was centrifuged at 3000 rpm and 4°C for 10 min. The plasma was removed and the residue was resuspended with 3 mL of 0.9% NaCl and centrifuged again. The supernatant was discarded and the residue was resuspended with 2 mL of triple distilled water at 4°C. An aliquot of 50 µL of resuspended blood cells was placed into each well of a 96-well plate containing 200 µL of reaction mixture. Fifty microliters of xanthine oxidase solution was added to each well and the absorbance was measured at 505 nm and 37°C. The reaction rate was estimated from absorbance readings at 0.5 and 3 min after addition of xanthine oxidase. A SOD reference standard has been provided with the Ransod Kit. One unit of SOD is defined as the amount required to inhibit the rate of xanthine reduction by 50%. Specific activity is expressed in units of SOD/mL.
5. Antibacterial activity
Bactericidal activity was measured as described by Supamattaya et al. (2000). Serum was separated from blood cells of each shrimp sample before diluting in 2.6% NaCl at ratios of 1:2, 1:4, 1:8, 1:16 and 1:32. Then, 0.5 mL of each serum dilution was used for testing. For the negative control, 0.1 mL NaCl was used in the assay. One-tenth of a milliliter of Vibrio harveyi suspension (8.2 × 10^6 CFU/mL) was added to each serum and control dilution. Treatments were incubated at room temperature for 3 h before bacterial enumeration. Results were recorded from dilutions that could reduce V. harveyi by 50% compared to the control.
RESULT
Experiment 1 Effect of formic acid and astaxanthin on growth and survival rate of Pacific white shrimp postlarvae
After 60 days of feeding, shrimp fed AX 50 ppm had the highest average body weight (4.45 ± 0.45 g), followed by shrimp fed 0.3 % FA + 50 ppm AX ( 4.38 ± 0.37 g), 0.6 % FA + 50. ppm AX (4.05 ± 0.21 g) and control group (4.18 ± 0.05 g). However, the body weight of all shrimp fed formic acid and astaxanthin was not significantly different from the control group. The average survival rate of shrimp fed with 0.6 % FA + 50 ppm AX was 82.33 ± 8.32 %, the highest among all other groups and significantly higher than the control group (64 ,33 ± 10.12 %) (Supplementary material 1 : Table S1).
Experiment 2 Effects of formic acid and astaxanthin on growth, survival, gut bacteria, and immune response of Pacific white shrimp infected with Vibrio parahaemolyticus.
At the end of the feeding trial, the average weight gain of 0.3 % FA + 50 ppm shrimp fed with AX was the highest, 2.97 ± 0.83 g. However, no significant differences between the six experimental groups were observed. The average survival rate of all shrimp fed FA and AX was significantly higher than that of the control group (20.00 ± 17.32%), and the best results were obtained in the group fed 0.6% FA + 50 ppm AX (67.50 ± 3.33%) ( Additional file 1 : Table S2).
For gut bacteria research, both Vibrio spp. number and total bacteria of all four groups fed FA (namely 0.3% FA, 0.6% FA, 50 ppm AX + 0.3% FA and 50 ppm AX + 0.6% FA ) was significantly lower than the control group and 50 ppm. Groups were fed with AX throughout the feeding trial. The lowest numbers of intestinal bacteria were observed in shrimp fed diets containing high doses of FA (i.e., 0.6% FA). On the 30th day of the experiment, two Vibrio spp. amounts observed at 50 ppm AX + 0.6 % FA and 0.6 % FA (1.30 ± 0.58 and 1.60 ± 0.70 × 10^6 CFU/g, respectively), in where the highest number was in the control group (47.20 ± 25.40 × 10 6 CFU/g). Similarly, the two lowest total bacterial counts were in the 0.6 % FA and 50 ppm AX + 0.6 % FA groups (2.80 ± 1.30 and 3.10 ± 0.70 × 10 6, respectively). CFU/g), while the highest number was in the control group. (45.00 ± 27.40 × 10^6 CFU/g) (Figs. 1,,22).
Figure 1 Total Vibrio spp. (10^6 CFU/g) in the intestines of Pacific white shrimp (n = 5) after challenge with V. parahaemolyticus at 10^4 CFU/ml. Data are presented as mean ± standard deviation. Different letters above the bars indicate whether the means are significantly different from each other (p < 0.05)
Figure 2 Total bacterial count (10^6 CFU/g) in the gut of Pacific white shrimp (n = 5) after infection with Vibrio parahaemolyticus at a concentration of 10^4 CFU/mL. Data are presented as mean ± standard deviation. Different letters above the bars indicate whether the means are significantly different from each other (p < 0.05)
Shrimp immune parameters were significantly affected by AX in shrimp feed. Shrimp fed diets containing AX (specifically, 50 ppm AX + 0.3% FA, 50 ppm AX + 0.6% FA, and 50 ppm AX) had higher total hemocyte counts (THC) (Figure 2). 3), phagocytic activity (Figure 2). 4) and phenoloxidase (PO) activity (Figure 2). 5) significantly higher than the control group and FA group. Superoxide dismutase (SOD) activity (Figure 2). 6) of shrimp fed 50 ppm AX but not in the 50 ppm AX + 0.3 % FA and 50 ppm AX + 0.6 % FA groups showed a significant increase compared to shrimp not fed AX. However, the bactericidal activity of shrimp serum in all groups was at the same serum dilution, which was 1:4 (Additional file 1 : Table S3).
Figure 3 Total hemocytes count (105 cells/ml) of Pacific white shrimp (n = 10) after challenge with Vibrio parahaemolyticus at 104 CFU /mL. Data are presented as mean ± standard deviation. Different letters above the bars indicate whether the means are significantly different from each other (p < 0.05)
Figure 4 Phagocytic activity (%) of Pacific white shrimp (n = 10) after infection with Vibrio parahaemolyticus at a concentration of 10^4 CFU/mL. Data are presented as mean ± standard deviation. Different letters above the bars indicate whether the means are significantly different from each other (p < 0.05)
Figure 5 Phenoloxidase activity (units/min/mg protein) of Pacific white shrimp (n = 10) after challenge with Vibrio parahaemolyticus at 10^4 CFU /ml. Data are presented as mean ± standard deviation. Different letters above the bars indicate whether the means are significantly different from each other (p < 0.05)
Figure 6 Superoxide dismutase activity (units of SOD/mL) of Pacific white shrimp (n = 10) after challenge with Vibrio parahaemolyticus at 10^4 CFU /ml. Data are presented as mean ± standard deviation. Different letters above the bars indicate whether the means are significantly different from each other (p < 0.05)
Discussions
Organic acids are widely used as animal food additives and preservatives to prevent food spoilage. As a group, these compounds mainly consist of saturated linear monocarboxylic acids and their derivatives (Ricke 2003). Many of them are available as sodium, potassium, or calcium salts because they are typically odorless, easier to handle, less corrosive, and more soluble than free acids (Papatsiros and Billinis 2012 ). Organic acids have antibacterial activity against certain pathogenic bacteria such as Escherichia coli, Salmonella spp., and Vibrio spp. (Ricke 2003 ; Papatsiros and Billinis 2012 ; da Silva et al. 2013 ). Undissociated forms of organic acids can easily penetrate the bacterial cell membrane and dissociate into anions and H + in the cytoplasm (Ricke 2003 ; Beales 2004 ; Lückstädt and Mellor 2011 ). Once inside bacterial cells, they reduce intracellular pH and disrupt the cytoplasmic membrane, protein synthesis system, genetic material and metabolic enzymes. Additionally, because bacterial cells use ATP to pump excess H+ out of the cell, organic acids also deplete ATP and affect the cell’s ability to maintain pH homeostasis (Ricke 2003 ; Beales 2004 ; Lückstädt and Mellor 2011 ). However, not all organic acids are effective against bacteria. In fact, the organic acids involved in specific antibacterial activity are short-chain acids (C1–C7) and are simple monocarboxylic acids such as formic, acetic, propionic and butyric acids, or are carboxylic acids bearing the hydroxyls such as lactic, malic, tartaric, and citric acids (Dibner and Buttin 2002 ; Papatsiros and Billinis 2012 ).
Organic acids are mainly used as feed additives to improve the growth performance of pigs and poultry (Dibner and Buttin 2002 ; Franco et al. 2005 ; Lückstädt and Mellor 2011 ; Papatsiros and Billinis 2012 ); There are also reports on the benefits of organic acids for aquatic animals, including red hybrid tilapia (Ng et al. 2009 ; Koh et al. 2014 ), yellowtail fish (Sarker et al. 2012 ). , sturgeon (Khajepour and Hosseini 2012 ), rohu (Baruah et al. 2007 ), black tiger shrimp (Ng et al. 2015 ), and Pacific white shrimp (Walla et al. 2012 ; da Silva et al. 2013 ; Su et al. 2014 ; Romano et al. 2015 ). However, results from experiment 1 showed that shrimp fed FA had significantly lower body weight than shrimp fed 50 ppm AX and slightly worse growth than the control group. This indicates that formic acid does not promote shrimp growth and may have some negative effects on shrimp growth. Other short-chain fatty acids can promote growth, for example, 2% organic acid mixture (including a mixture of formic, lactic, malic and citric acids) (Romano et al. 2015 ) or 2 g/kg citric acid (Su et al. 2014 ).
Although there was no obvious improvement in the growth rate and survival rate of uninfected shrimp postlarvae in our study, the use of formic acid increased the survival rate of infected shrimp postlarvae. significantly infected with V. parahaemolyticus compared to the control group. This result is consistent with research on gut bacteria, i.e. shrimp fed formic acid had significantly lower Vibrio spp. and total bacterial count compared to animals not fed formic acid. Similarities between Vibrio spp. The number and total number of bacteria showed that Vibrio spp. is an important component of the shrimp intestinal microflora (Moss et al. 2000 ; Oxley et al. 2002 ; Liu et al. 2011 ). Antibacterial effect of formic acid on Vibrio spp. also reported in vitro (Mine and Boopathy 2011 ; Adams and Boopathy 2013 ; da Silva et al. 2013 ). Considering all these aspects, the antibacterial properties of formic acid may reduce Vibrio infection of Pacific white shrimp (Papatsiros and Billinis 2012 ; Adams and Boopathy 2013 ; da Silva et al. 2013 ). by entering the bacterial cell wall in an undissociated form, which then releases H + and destabilizes the intracellular pH of the bacterial cytoplasm, leading to death (da Silva et al. 2013 ).
Astaxanthin is a pigment of the xanthophyll class (oxidized derivatives of carotenoids) and is widely used in salmon and crustacean aquaculture to produce the desired red-orange color. Astaxanthin possesses strong antioxidant properties and plays an important role in larval development and reproductive success of crustaceans. It occurs naturally in the green microalga Haematococcus pluvialis and the red yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma). However, because farmed crustaceans often do not have access to natural sources of astaxanthin and they cannot synthesize carotenoids de novo, total astaxanthin must be obtained from their diet (Higuera-Ciapara et al. 2006 ; Seabra and Pedrosa 2010 ).
The growth of shrimp fed with astaxanthin in experiment 1 was significantly better than that of shrimp fed with FA, but not significantly different from the control group. The survival rate of shrimp fed with astaxanthin was not significantly different from the control group. However, the survival rate of shrimp infected with V. parahaemolyticus in experiment 2 was significantly improved. However, unlike formic acid, astaxanthin did not inhibit intestinal bacterial populations, suggesting that other mechanisms may be responsible for the increased survival rate. In fact, many immune parameters of shrimp fed astaxanthin were improved, including total blood cell count (THC), phagocytic activity, phenoloxidase (PO) activity, and superoxide dismutase (SOD) activity. ). These results suggest that astaxanthin has immunostimulatory properties that prevent V. parahaemolyticus infection in Pacific white shrimp.
The antioxidant activity of carotenoids may be related to immunomodulatory effects; By quenching singlet oxygen and free radicals, carotenoids can protect white blood cells from oxidative damage (Bendich 1989 ). Superoxide dismutase (SOD) is an antioxidant enzyme that protects cells against oxidative stress by removing superoxide anion (O 2 − ) and it is used as an indicator of immune response (Campa-Cordova et al. al. 2002a , 2002b ). Given that astaxanthin also has antioxidant properties, this suggests that such a mechanism must be involved in immunoregulation. Furthermore, the effects of carotenoids in enhancing cell-mediated and humoral immune responses of vertebrates have also been documented (Bendich 1989 ; Chew and Park 2004 ). Several studies have reported that dietary carotenoids can increase immune parameters, enhance survival rates, or act as a pathogen preventative for many aquatic animals such as carp. common (Anbazahan et al. 2014 ; Sowmya and Sachindra 2015 ), rainbow trout (Amar). et al. 2001 ), Pacific white shrimp (Flores et al. 2007 ; Niu et al. 2009 ), black tiger shrimp (Supamattaya et al. 2005 ), kuruma shrimp (Chien and Shiau 2005 ) and giant freshwater shrimp (Angeles et al. 2009 ). Our results are somewhat similar to these studies.
Even though formic acid and astaxanthin have different modes of action on shrimp, both have a positive impact on their ability to resist bacterial challenge. Meanwhile, our results showed that the combination of formic acid and astaxanthin was not better than either alone or in combination. The only exception was that uninfected shrimp fed 0.6% FA + 50 ppm AX had a significantly higher survival rate than the control group. Overall, formic acid (FA 0.3 and 0.6%) and astaxanthin (50 ppm AX) were equally effective in preventing V. parahaemolyticus infection in Pacific white shrimp. Due to the fact that formic acid is cheaper than astaxanthin, the use of formic acid as a feed additive in shrimp farming can be considered more economically valuable.
Conclusion
Astaxanthin (50 ppm AX) can be used as a growth promoter in uninfected Pacific white shrimp, while formic acid (0.3 and 0.6% FA) and AX can enhance high survival rate of shrimp infected with Vibrio parahaemolyticus under laboratory conditions. Additionally, shrimp fed with FA had the bacteria Vibrio spp. and total bacterial counts, while shrimp fed with AX showed improvements in many immune parameters. The results of our study show that using FA, AX and their combination as feed additives can prevent Vibrio parahaemolyticus infection in shrimp.
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