Scutellaria polysaccharide mediates the immunity and antioxidant 1 capacity of the giant freshwater prawn ( Macrobrachium rosenbergii )

24 Background: The giant freshwater prawn ( Macrobrachium rosenbergii ) is a 25 commercially valuable freshwater crustacean species that is cultivated throughout the 26 world. M. rosenbergii is frequently affected by a variety of diseases. Therefore, feed 27 additive research is necessary to improve the immunity and survival rate of M. 28 rosenbergii . Scutellaria polysaccharide (SPS) extracted from Scutellaria baicalensis (a 29 Chinese medicinal herb) can enhance the antioxidant ability of organisms. 30 Methods: In this study, M. rosenbergii were fed with 50 mg/kg, 100 mg/kg, and 150 31 mg/kg of SPS. Following a four-week feeding trial, SPS had no positive effect on the 32 growth of M. rosenbergii compared with the control group. Then, the immunity and 33 antioxidant capacity of M. rosenbergii were tested by qRT-PCR and enzyme activities. 34 Results: The results showed that the expressions of prophenoloxidase (proPO) and toll 35 receptor (Toll-R) mRNA showed no changes in hemocytes of M. rosenbergii . However, 36 the expressions of heat shock protein 70 (HSP70) and nuclear factor κB (NF-κB) 37 mRNA were up-regulated during the first two weeks of culture and were down- 38 regulated in weeks 3 and 4. The mRNA expressions of HSP70, NF-κB, and Toll-R 39 (participating in the immune response) in heart, muscle, and hepatopancreas were 40 decreased after four weeks of SPS feeding. This indicated that long-term feeding of 41 SPS could regulate the immune responses of M. rosenbergii. The activity levels of 42 antioxidant biomarkers, alkaline phosphatase (ACP), acid phosphatase (AKP), 43 polyphenol oxidase (PPO), catalase (CAT), and superoxide dismutase (SOD), in hemocytes, heart, muscle, and hepatopancreas increased during the feeding time, 45 indicating that the antioxidant capacity of M. rosenbergii was improved by SPS 46 supplementation in the feed. 47 Conclusions: In summary, SPS was conducive to enhancing the antioxidant capacity 48 of M. rosenbergii . These results provide a theoretical basis in supporting of SPS 49 addition to the feed of M. rosenbergii.


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The giant freshwater prawn (Macrobrachium rosenbergii) is a commercially 66 valuable freshwater crustacean species, which is cultured all over the world. Numerous   Scutellaria polysaccharide (SPS) is extracted from the root of Scutellaria 85 baicalensis, which is a traditional Chinese herb and widely prescribed to treat bacterial 86 infections in humans [14]. A recent study showed that the stem and leaves (aerial parts) 87 of S. baicalensis had extensive antibacterial effects on Aeromonas hydrophila, 88 Edwardsiella tarda, V. alginolyticus, and V. harveyi [6]. The     curve analysis was performed to determine the target specificity. The relative 138 expression ratio of the target genes versus the β-actin gene was calculated using the 2 -

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∆∆CT method, and all data were given in terms of relative mRNA expression.  During the breeding period, the HSP70 level followed a decreasing trend (Fig. 3A).

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After one week of culturing prawns the expression of HSP70 was slightly decreased in 179 the 50 mg/kg and 100 mg/kg groups. Furthermore, decreases of HSP70 were observed 180 in the 100 mg/kg and 150 mg/kg groups after culturing for two weeks, especially in the 181 muscle tissue and the hepatopancreas.   After one week of culture, the activity of AKP increased in both heart and muscle 205 tissues (Fig. 5B). After two weeks of culture, the activity of the AKP enzyme increased 206 significantly in muscle tissue. After four weeks of culture, enzyme activity decreased 207 in heart and muscle tissue and increased in the hepatopancreas. These results suggest 208 that muscle AKP might be sensitive to the feeding duration of SPS.

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After two weeks of culture, the heart and muscle PPO levels decreased, and the 210 hepatopancreas PPO level increased (Fig. 5C). Over three weeks of culture, PPO levels 211 increased in the heart, muscle, and hepatopancreas. After four weeks of culture, PPO 212 decreased, which indicates that polysaccharides can increase the activity of PPO at the 213 appropriate time.

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The SOD levels increased in the heart, muscle, and hepatopancreas over the four 215 weeks of cultivation (Fig. 5D). However, a significant increase in the SOD level was 216 found in the hepatopancreas after two weeks of culture, indicating that the 217 hepatopancreas is the most sensitive organ to SPS.

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As shown in Fig. 5E, the CAT levels increased significantly in heart, muscle, and 219 hepatopancreas tissues after three weeks of culture. In the fourth week, the CAT activity 220 level was higher than levels in the third week of culture. The results showed that long-221 term feeding of SPS enhanced CAT activity and overall antioxidant capacity.

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The antioxidant capacity can also reflect the immune capacity and health status.

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ACP, AKP, and PPO play important roles in the immune system of shrimp or prawn.

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Phosphatase can catalyze the hydrolysis of various phosphorous compounds.

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According to their optimal pH characteristics, phosphorous compounds can be divided 288 into AKP and ACP. In this study, after feeding SPS at different concentrations, ACP and 289 AKP enzyme activities increased in hemocytes over the whole feeding time. They also 290 increased in heart and muscle tissues after the first feeding week, but decreased in the 291 fourth feeding week. These results indicate that long-term feeding of SPS did not 292 significantly enhance the activity of ACP and AKP. It has been demonstrated that long-       Table 1 522 Primers used in qRT-PCR on mRNA of immune parameters.