Effects of medical herbs in Tian- Dong- Tang- Gan powder on non- specific immune responses and resistance to acute ammonia stress in Litopenaeus vannamei

Tian- Dong- Tang- Gan powder (TDTGP) is a newly developed immune enhancer in Litopenaeus vannamei . In vitro, TDTGP was non- toxic to haemolymph at 400 µg ml −1 and increased the activities of phenol oxidase (PO), acid phosphatase (ACP) and su peroxide dismutase (SOD) after incubation with haemolymph for 8– 48 h. In vivo, the activities of PO, SOD, ACP and alkaline phosphatase (AKP) in the shrimp haemolymph could be increased during the 28- day feeding trials. An acute ammonia stress test was performed after the feeding trials were terminated. We studied the immunological parameters and histology diagnosis. In the drug group, the immune index burst following acute ammonia exposure was higher than that in the negative control and control groups. Histological detection showed that the integrity of the hepatopancreas cells in the TDTGP groups was higher than that in the negative control group at 72 h after ammonia exposure. The results showed that feeding shrimp TDTGP for 28 days could improve the immunity and resistance to ammonia stress by Litopenaeus vannamei and protect the integrity of the hepatopancreas during ammonia stress. These findings showed that TDTGP can be developed and used as an immunoenhancer in Litopenaeus vannamei .


| INTRODUC TI ON
The Pacific white shrimp Litopenaeus vannamei is one of the pillar species in China's aquaculture industry. However, with the rapid development of the shrimp breeding industry in recent years, the continuous expansion of the shrimp breeding area and scale has led to increased breeding intensity and the deterioration of the breeding environment. During the cultivation process, due to the high degree of intensification, high stocking density and high feeding amount, with the extension of the cultivation time, the contents of ammonia and harmful microorganisms in the aquaculture water gradually increase (Duan et al., 2018;Liu, 2004). Excess ammonia can reportedly reduce the immune ability of Litopenaeus vannamei, and the residual bait will lead to the accumulation of harmful microorganisms (Cui et al., 2017;Si et al., 2019). Litopenaeus vannamei can readily develop various diseases due to excess ammonia such as acute hepatopancreatic necrosis disease, white spot syndrome and those caused by the white spot syndrome virus, Taura syndrome virus, Vibrio harveyi and Vibrio damsela (Ananda Raja et al., 2017;Leu et al., 2013;Pang et al., 2019) in culture, resulting in large economic losses. At present, the prevention and treatment of these diseases primarily depend on antibiotics and chemicals, but the use of antibiotics and chemicals is increasingly restricted due to the shortcomings associated with drug residues, drug resistance, pollution and other concerns (Johansson et al., 2014;Valitalo et al., 2017).
Traditional Chinese medicine has been used for thousands of years as a medicine to prevent and treat diseases in China. Modern research shows that many traditional Chinese medicines and their extracts can improve immunity and can be used as alternatives to antibiotics to prevent and treat diseases of Litopenaeus vannamei, for example polysaccharides from Angelica sinensis , San-Huang-San (Zhai & Li, 2019) and rose myrtle Rhodomyrtus tomentosa seed extract (Dang et al., 2019). In traditional Chinese medicine, Asparagus cochinchinensis is used to nourish yin and moisten dryness. Modern research shows that Asparagus extract pregnane glycosides and polysaccharides have anti-oxidant and anti-neuroinflammatory properties (Cho & Yang, 2018;Jian et al., 2013). Panax notoginseng is a type of precious Chinese medicine, and it is primarily composed of polysaccharides and saponins.
Research shows that the total saponins in Panax notoginseng are primarily composed of Panax notoginseng R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, ginsenoside Rd, and others, and the total saponins in Panax notoginseng can be used to treat inflammation and bleeding caused by internal and external injuries, among others, and it can improve cardiovascular activity function (Wang et al., 2016;Yang et al., 2014).
In this study, according to early-stage exploration in the laboratory, the total polysaccharides in Asparagus cochinchinensis were extracted by an alcohol alkali extraction method, and the total saponins in Panax notoginseng were extracted by an alcohol extraction method. After they were mixed in a specific proportion, an appropriate amount of water-soluble silica was added as the auxiliary material, and then, the extracts were evenly granulated, dried and comminuted into a powder named Tian-Dong-Tang-Gan powder (TDTGP). The total polysaccharide and total saponin contents of TDTGP were determined, followed by an in vitro immune test, a 28-day feeding test and an acute ammonia stress test to study the effects of TDTGP on the non-specific immunity of Litopenaeus vannamei.

| Herb sources and extraction
Asparagus cochinchinensis and Panax notoginseng were purchased from Nanning Traditional Medicine Market in Guangxi, China.
Asparagus cochinchinensis was extracted twice in a water bath at 80°C for 2.5 h with 10% ethanol at pH = 9 in a 1:10 ratio after soaking for 1.5 h, combining the two 300 mesh and concentrate filtrates in boiling water and concentrating it all to a density of 1.2 g ml −1 , adding 5 times the volume of 95% ethanol for 24 h and discarding the supernatant, and precipitating and weighing it for later analysis. Panax notoginseng was extracted twice and concentrated to 1.15 g ml −1 in a water bath at 90°C with 70% ethanol in a 1:10 ratio for 2 h. It was purified with D101 macroporous adsorption resin (Solarbio, M0041), concentrated in a water bath at 100°C and freeze-dried (Christ ALPHA 1-2 LD plus) to obtain the refined total saponins.
The total polysaccharides from asparagus, total saponins of notoginseng and the silica were mixed in a specific proportion. Last, after drying at 60°C, the 100 meshes were crushed and stored at room temperature for further study.

| Content determination
The total polysaccharide content was measured by anthrone sulphuric acid method. The standard curve for the polysaccharide content absorbance value was established with dextran as the standard, and the absorbance value of the sample was placed within the standard curve to calculate the polysaccharide content.
The total saponin content of the TDTGP was determined by high-performance liquid chromatography (HPLC, Waters e2695).

| Cell counting kit-8 (CCK-8) assay
The viability of the haemolymph cells was detected using a CCK-8 assay (Beyotime Biotechnology) in this research (Li et al., 2018). A

| Immune response in vitro
According to the cells of haemolymph viability results, the control group, APS group (100 μg ml −1 ) and TDTGP I to IV groups (25, 50, 100 and 200 μg ml −1 ) were established for the experiment, and each group had four repetitions. One ml of 2.2 × 10 6 ml −1 cells of haemolymph suspension was added to each hole in the 24-well plates and placed in an incubator at 28°C under 5% CO 2 for 2 h. The culture solution in each hole was replaced with one containing each group of drugs. The supernatant and cells were collected after 8, 12 and 24 h of co-culture. The supernatant was used to determine the PO, SOD, AKP and ACP activities.

| Experimental diets
Various drug-containing diets and a control diet for the experimental shrimp were prepared according to the feed formulation shown in Table 2. In brief, the basal diet was formulated to contain approximately 417.33 g kg −1 crude protein and 72 g kg −1 crude fat, which are sufficient to support the optimal growth of Litopenaeus vannamei.  Tian-Dong-Tang-Gan powder was supplemented separately in the basal diet at the expense of cellulose to obtain the final drug dose for the different experimental groups as follows: control (0 mg kg −1 ), negative control (0 mg kg −1 ), APS (2 g kg −1 ), TDTGP-1

TA B L E 2 Formulation and chemical proximate composition of the experimental diets
(2 g kg −1 ), TDTGP-2 (4 g kg −1 ) and TDTGP-3 (8 g kg −1 ). The ingredients were grounded and sieved through a 300μm mesh to obtain a fine powder. The powder was then mixed thoroughly with fish oil and then tap water was gradually added until a stiff dough was obtained.
Later, the dough was extruded through a mincer, ripened at 70°C for 5 h, air-dried in the dark and then sieved into pellets. The feed pellets were stored in plastic bags at −20°C until use. were maintained throughout the experiment at a constant level of salinity (10 ± 1.0‰), temperature (27 ± 1.0°C), pH (8.0 ± 0.2) and dissolved oxygen (above 5 ppm). Water exchange (30%) was performed daily until the end of the feeding experiment. Each treatment group was fed with the aforementioned diet four times daily for 28 days. The unfed food, remains and waste were removed before feeding.

| Acute ammonia exposure
At the end of the 28-day feeding experiment, we adjusted the water level for each group to 60 L, added the appropriate concentration of NH 4 Cl solution to the other groups except the control group, adjusted the concentration of NH 4 Cl to 100 mg L −1 (the actual ammonia molecular concentration was 2.10 mg L −1 ), and measured the ammonia nitrogen concentration by indophenol blue method every 24 h and adjusted the ammonia nitrogen concentration of the experimental water in time, and we performed the ammonia stress experiment for 72 continuous hours.

| Histomorphology
After the acute ammonia exposure experiment, four shrimp from each tank were randomly collected for histomorphology analysis and fixed in a 4% formalin solution for 48 h following fixation, and the histomorphology samples were processed and stained with haematoxylin and eosin (H&E) using standard histological techniques (Howard et al., 2004) and examined for cell morphology under a light microscope (Olympus, DP72).

| Post-analysis after administration
Haemolymph was collected once every 7 days for the feeding experiment and once every 24 h during the ammonia stress experiment from each experiment using insulin syringes preloaded with 0.2 ml of anticoagulant (5% sodium citrate at pH 7) to avoid haemolymph clotting, and the samples were stored at −80°C for further analysis.

| Immunological parameters
The PO activity of the haemolymph samples was determined spectrophotometrically by recording the formation of dopachrome using l-dihydroxyphenylalanine (l-dopa) as a substrate.
We used l-dopa as the substrate and modified it according to Söderhäll's and Mason's method (Mason, 1956;Söderhäll, 1983).

| Data analysis
One-way ANOVA was performed using SPSS software (SPSS version 22.0 for Windows). The means were compared at the 0.05 levels and with the subsequent post hoc multiple comparison with the Duncan test, and the results are presented in a histogram prepared in GraphPad Prism 6.0.

| Viability of haemolymph
The viability of the haemolymph can be improved by co-culture with the drugs and shrimp haemolymph in each dose group for 4, 12 and 24 h for which the effect was the best at 12 and 24 h (Figure 4).
There was no effect at or below TDTGP 400 μg ml −1 , and the activity of each group gradually decreased; the decrease of TDTGP 800 μg ml −1 was the most obvious (p < 0.05) when co-incubation was performed for 48 h (Figure 4). The results showed that TDTGP

| Cell immune response in vitro
TDTGP can promote the activity of immune factors in cells of haemolymph ( Figure 5). At 8 h after co-incubation, the activity of SOD in the supernatant of each drug group was significantly higher than that of the control (p < 0.05); the activity of PO in the supernatant of the TDTGP 200 µg ml −1 group was significantly higher than that of the control (p < 0.05), the activity of AKP in the supernatant of TDTGP in the 50 µg ml −1 group was significantly higher than that of the control (p < 0.05), and at 12 h after co-incubation, the activity of AKP in the supernatant of each dose group of TDTGP was significantly higher than that of the control (p < 0.05). The ACP activity was significantly higher in the control (p < 0.05).

| Feeding experiment
The PO, ACP and AKP activities first increased and then decreased at different time points after providing the shrimp with asparagine powder during the 28-day feeding experiment (Figure 6). On the 7th day, the PO activity in the haemolymph of the TDTGP-1 and TDTGP-2 groups was significantly higher than that of the control (p < 0.05), and the activity of ACP and AKP in the TDTGP-2 group was significantly higher than that of the blank control group (p < 0.05). On the 14th day of the feeding test, the SOD activities of APS, TDTGP-1 and TDTGP-3 were significantly higher than that of the control (p < 0.05). On the 28th day, the SOD activity in the TDTGP-1, TDTGP-2 and TDTGP-3 groups was significantly higher than that of the control (p < 0.05).

| Acute ammonia stress experiment
The changes in the immune parameters in the shrimp haemolymph under acute ammonia stress are shown in Figure 7. Over the 24 h of acute ammonia stress, the PO activity in the haemolymph of each ammonia stress model group was significantly higher than that of the control group (p < 0.05), and the PO activity in the haemolymph of the APS and TDTGP-2 groups was significantly higher than that of the negative control group (p < 0.05). The ACT activity in the TDTGP-3 group was significantly higher than that in the other groups (p < 0.05) at 24 h after ammonia stress (Figure 6), and there was no significant difference between the other groups and the control group (p > 0.05). At 48 h, the ACP activity of the negative group and each treatment group was significantly higher than that of the control group (p < 0.05). The activity of AKP in the TGTGP-2 group was significantly higher (p < 0.05) than that in the negative group at 24 h of ammonia stress (Figure 6), while the activity of AKP in the APS group was significantly higher than that in the other groups at 48 h (p < 0.05).

| Effects of TDTGP on the histomorphology of the acute ammonia stress test
The shrimp exhibited marked histological alterations in the hepato-

| DISCUSS ION
Innate immunity was considered as the primary defence mechanism in crustaceans, and it includes both humoral components and cellular activities that are coordinated to eliminate foreign organisms that could be potentially hazardous to the host. In the crustacean, cellular defence was directly performed by the haemolymph, by  Söderhäll, 1983) that are involved the reactions of phagocytosis, encapsulation and nodule formation, etc (Holmblad & Söderhäll, 1999;Mapanao et al., 2018). In these cells, hyaline cells are the main phagocytic cells, while granular cells contain many secretory granules containing that components of the proPO system. Semi-granular cells appear to be the most sensitive ones and react first during an immune response, by degranulating. These two cells main to take part in coagulation, release of agglutinins and/or synthesis of melanin. (Holmblad & Söderhäll, 1999;Mapanao et al., 2018;Ratcliffe & Rowley, 1979).
Humoral components include the activation and release of molecules stored within haemocytes, such as agglutinins, phenol oxidase (PO), antimicrobial peptides, acid phosphatase (ACP) and superoxide dismutase (SOD) (Holmblad & Söderhäll, 1999). SOD plays a crucial role in the phagocytosis process or antioxidant stress, and following hyaline cells phagocytosis, the infection pathogen can induce a reactive oxygen reaction to synthesise reactive oxygen intermediates (ROIs). However, the excessive accumulation of ROIs may induce oxidative stress in the host. In a normal physiological state, harmful effects of ROIs are effectively neutralized by the antioxidant defence system of organisms, which in general comprises enzymes like SOD, and SOD degrades excess O 2 − , thereby inhibiting ROIs generation to prevent harm to the host (Chang et al., 2013;Holmblad & Söderhäll, 1999;Wu et al., 2016). Many studies have found that activated PO catalyses the stepwise oxidation of phenols to quinones, which polymerize non-enzymatically and form insoluble melanin deposits, then encapsulation pathogens. Therefore, the activity of PO has a positive correlation with shrimp disease resistance (Nappi et al., 1995). ACP is an important component of phagocytic lysosomes, and in the phagocytosis and encapsulation of haemocytes, phagocytic lysosomes have bactericidal action with the release of ACP. AKP is an important component of lysosomal enzymes, and it plays a role in invertebrate immune responses (Cheng, 1978(Cheng, , 1989Kuo et al., 2019). Therefore, the activities of PO, SOD, ACP and AKP were used which is consistent with the report by Yu-Sheng Wu. Wu (Wu et al., 2016) found that the Litopenaeus vannamei oral different concentrations beta 1,3/1,6-glucan effectively enhanced O 2 − production and phenoloxidase and superoxide dismutase activity, and oral administration with 0.2 g/kg of vitamin C presented beneficial nonspecific immune responses and enzyme activity. This shows that TDTGP can improve the nonspecific immunity of Litopenaeus vannamei like beta 1,3/1,6-glucan and vitamin C.
During the high-density culture of Litopenaeus vannamei, ammonia is the final product of organic matter and protein decomposition.
Ammonia-N (>5 mg/L) and NH 3 -N (>0.357 mg/L) will affect the expression of coagulation and immune genes (Chang et al., 2015;Frías-Espericueta et al., 2000). Chen (Chen et al., 1988) found that during the culture of Penaeus penicillatus, the highest ammonia content can reach 46 mg/L. Zhang  found that long-term ammonia stress (46 mg/L) can lead to decreases in the total haemolymph, phagocytic activity and antibacterial activity, and Xiao et al. anti-oedema, anti-thromboembolism, anti-coagulation, antihyperlipidaemia and anti-hyperglycaemia effects (Yang et al., 2014).
In this experiment, histopathological studies found that after 72 h of ammonia stress in the negative and APS groups, the number of cells in the hepatopancreas decreased, and even part of the structure disappeared. However, the hepatopancreas cells in each group fed with TDTGP could maintain their integrity, which may be due to the anti-inflammatory and anti-oxidation effects of Asparagus extract and the total saponins of notoginseng. In future studies, the mechanism of TDTGP to improve the anti-ammonia stress effect of Litopenaeus vannamei will be further studied.

| CON CLUS ION
In this study, we addressed the immunoregulatory effect of asparagine powder on Litopenaeus vannamei from three perspectives: in vitro, in vivo and under acute ammonia stress. We found that asparagine powder can improve the activities of PO, ACP, AKP, SOD and other immune factors in Litopenaeus vannamei and can help cells resist acute ammonia stress by maintaining the integrity of the hepatopancreas cells. The results showed that the Tian-Dong-Tang-Gan powder could improve the immune ability of Litopenaeus vannamei and its ability to resist ammonia stress. This powder can be used as a new immune enhancer, but its exact mechanism requires further study. In addition, this study found that Astragalus polysaccharides had no protective effect on the hepatopancreas cells of Litopenaeus vannamei under ammonia stress, but it could enhance its immunity.

ACK N OWLED G EM ENTS
This work was financially supported by the Innovation Driven

CO N FLI C T O F I NTE R E S T
There are no conflict of interest on any front.

AUTH O R CO NTR I B UTI O N S
Xiao-Dong Xie and Mei-lan Mo designed and performed research, analysed data and wrote the paper; Shu-Mian Zhou and Jing Cheng, and Mei-Ling Yu performed research and analysed data; Ying-Yi Wei revised and proved the paper; Ting-Jun Hu designed research analysed data and revised the paper.

DATA AVA I L A B I L I T Y S TAT E M E N T
Some or all data, models, or code generated or used during the study are available from the corresponding author by request.