Introduction

The giant freshwater prawn, Macrobrachium rosenbergii, is a commercially important freshwater crustacean species (Lalrinsanga et al. 2014; Nguyen et al. 2019). However, the rapid development of prawn industry has promoted the incidence of infectious diseases, mainly caused by viruses and bacteria (Chen et al. 2001a; Hsu et al. 2005; Suanyuk and Dangwetngam 2014). Lactococcus garvieae has proven to be one of the significant coccus pathogens for prawns, in which L. garvieae infection in M. rosenbergii causes cumulative mortality of around 30 to 75% (Chen et al. 2001b; Chen and Wang 2001). Several strategies have been implemented to deal with bacterial infections (Serrano 2005). However, antibiotics have adverse biosafety and biosecurity implications (Guo et al. 2011; Lakshmi et al. 2013; Sørum et al. 2006). Therefore, a solution is needed to solve the problem of antibiotics, such as plant-based medicines recently used as therapeutic alternatives (Disch et al. 2017; Valladão et al. 2015).

In aquaculture, medicinal herbs have been reported to promote growth, improve immunity, and possess antimicrobial properties (Direkbusarakom et al. 1998; Jian and Wu 2004; Sivaram et al. 2004; Yahaya et al. 2015). Radix Scutellariae is the dried root of the medicinal herb Scutellariae baicalensis Georgi. In addition, it shows various therapeutic effects (e.g., anti-inflammation, anti-cancer, and anti-virus-related diseases), and it has been proved that flavonoids are the most abundant constituents and stimulate these therapeutic effects. The major bioactive flavones in Radix Scutellariae existing are aglycones (baicalein, wogonin, oroxylin A) and glycosides (baicalin, wogonoside, oroxylin A-7-glucuronide) (Li et al. 2011). Meanwhile, the main bioactive component in the water extract of Radix Scutellaria (WERS) is baicalin (Yan et al. 2022). Noticing the promising potential of Radix Scutellariae, several studies have been conducted to evaluate the effect of Radix Scutellariae supplementation on aquatic animals, such as olive flounder (Cho et al. 2013) and tilapia (Yin et al. 2006). However, to the best of our knowledge, no studies have considered the effects of Radix Scutellariae on prawns.

Injection, immersion, and oral administrations are the most common delivery routes of plant extracts in aquaculture (Reverter et al. 2014). However, as previously reviewed, each method has advantages and disadvantages (Assefa and Abunna 2018; Galindo-Villegas and Hosokawa 2004). In the present study, through the prawn’s anus, WERS was directly introduced to the region of the anterior midgut connecting with the hepatopancreas for stimulating immune responses. This method is called reverse gavage feeding (RGF) (Aranguren and Lightner 2009; Tran et al. 2013; Xie et al. 2017). Delivering WERS to the midgut directly at least partially avoids the first-pass metabolism effect and the possibility of WERS-food interactions when applied WERS incorporation in the feed. In addition, the RGF method is less traumatic than oral gavage and makes it possible to deliver a dose of WERS into the midgut precisely. Therefore, the immunomodulatory effects of WERS associated with each dose on prawn immunity can be accurately compared. For this reason, it is necessary to investigate the immunomodulatory effects of WERS when applied to prawns by RGF.

Materials and methods

Prawn acclimatization

Selected M. rosenbergii (20.0 ± 2.0 g) were purchased from a local commercial shrimp farm (Pingtung, Taiwan). Two weeks before treatment, prawns were maintained at room temperature in a 400 l tank (25 prawns/tank) fitted with a freshwater circulator and fed with commercial feed twice daily, equal to 5% of prawn body weight.

Plant extraction

Selected Radix Scutellariae was purchased from a local market (Pingtung, Taiwan). Following a previous method to prepare WERS (Zhao et al. 2016), 10 g of Radix Scutellariae powder was boiled in 150 ml of double-distilled water at 95 °C for 15 min. Then, the supernatant was obtained by centrifugation at 1000 × g at 4 °C for 10 min before evaporating using a freeze dryer (EYELA FDU-1100). Lyophilized powder of WERS was then stored at 4 °C until used.

Reverse gavage feeding and sample collection

The selected prawns were divided into 4 treatment groups, and each treatment consisted of 3 replications (25 prawns/tank). Before WERS administration, prawns were fasted for 24 h to ensure their intestines were free of feces. Subsequently, a 10 µl mixed solution of PBS and red dye (30: 1 v/v) containing different doses of WERS (0, 1, 5, and 10 µg) was slowly introduced to the anterior midgut via anal route by RGF. The use of red dye to facilitate observation and ensure that the solution sent into the intestine through the anus can reach the anterior midgut.

Based on a previous study (Xie et al. 2017), sampling was carried out 24 h after RGF, consisting of four time intervals (3, 6, 12, and 24 h after RGF). Hemolymph (100 µl) was transferred to a 1.5-ml Eppendorf tube containing 900 μl cooled anticoagulant (0.34 g EDTA, 0.8 g sodium citrate, and 10 μl Tween 80 in 100 ml of distilled H2O, at pH 7.45 with the osmolality adjusted to 490 mOsm kg−1 with NaCl). In addition, digestive organs (hepatopancreas and midgut) were taken out from the prawns’ bodies and stored at − 20 °C in RNAlater™ solution until use.

Measurement of the immune responses

Total hemocyte count (THC) was determined by counting the number of hemocytes in the anticoagulant and hemolymph mixture on the hemocytometer fields under a microscope. Hemocytes found on the fields of the hemocytometer were calculated in three replications.

The respiratory burst (RB) activity was determined using the reduction of nitroblue tetrazolium (NBT) to formazan as a measure of superoxide anion (O2−) production (Song and Hsieh 1994) with some modification. RB activity was expressed as the amount of NBT reduction in 100 µl of hemolymph.

The hydroxamate-based colorimetric method was used to determine the TG activity in M. rosenbergii hemolymph (Gorman and Folk 1980). Initially, 50 µL of hemolymph was incubated with 150 µl of Tris–acetate buffer, 12.5 µl of hydroxylamine hydrochloride 2 M, and 37.5 µl of Z-Glu-Gly at 37 °C for 10 min. Then, 250 µl of coloring agent (contained 15 g trichloroacetic acid and 5 g iron (III) chloride hexahydrate in 100 ml of ddH2O) was added and centrifuged at 10,000 rpm for 5 min. Finally, the supernatant was measured colorimetrically at 525 nm with a microplate reader.

Lysozyme activity was determined by a turbidimetric assay which used a suspension of Micrococcus lysodeikticus as a substrate in a reaction mixture (Shugar 1952). Hemolymph (10 µl) was mixed with 200 µl of M. lysodeikticus in PBS (0.02% (w/v)) at 25 °C. Then, the absorbance at 450 nm was immediately recorded with a microplate reader every minute. Lysozyme from white egg (Amresco, USA) was used as control. Lysozyme activity was quantified in absorbance reduction per minute.

RNA extraction, cDNA synthesis, and qRT-PCR analysis of immune related genes

Total RNA was extracted from the digestive organs (hepatopancreas and midgut) using Trizol® reagent (Ambion®, USA). The RNA samples were suspended in 30 μl DECP-treated water and kept at − 80 °C until use. Total RNA concentration was qualified and quantified using BioSpectrometer ((Eppendorf BioSpectrometer® fitted with Hellma TrayCell Cap, Germany). According to the manufacturer’s instructions, first-strand cDNA was synthesized from 2 µg of total RNA using M-MuLV Reverse Transcriptase (Lucigen®, USA). The cDNA was subsequently stored at − 20 °C.

Quantitative real-time PCR (qRT-PCR) was performed to investigate the expression of immune-related genes in the prawn digestive organs after WERS administration by RGF (Table 1). qRT-PCR was performed using the ABI StepOnePlus™ Real-Time PCR System (Applied Biosystems™, USA). The amplification was performed in a total volume of 10 μl, containing 1 μl of cDNA template, 5 μl of TB Green Premix Ex Taq II (Tli RNaseH Plus) (Takara Bio Inc., Japan), 0.2 μl of each primer, 0.2 μl of ROX reference dye (50x), and 3.4 μl of DEPC treated water. The real-time PCR program was 95 °C for 1 min, followed by 40 cycles of 95 °C for the 15 s and 60 °C for 1 min. Dissociation and melting curves of amplification products were performed at the end of each PCR to confirm that only one PCR product was amplified and detected. The relative expression of related genes were calculated by the 2−ΔΔCT method (Livak and Schmittgen 2001).

Table 1 RT-qPCR primers of immune-related genes
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Statistical analysis

ANOVA (one-way analysis of variance) was applied to evaluate data obtained from the experiments. Multiple comparisons (Duncan test) were conducted to investigate significant differences among treatments using SPSS 17. Results were present as mean ± SD. Differences between different groups at each time point were considered significant at p < 0.05.

Results

Effect of Radix Scutellariae water extract (RSWE) on the immune responses

As shown in Fig. 1, after administration with 1 and 5 µg of WERS by RGF, THC levels increased significantly (p < 0.05) compared with the other treatments for 24 h (Fig. 1). Moreover, RSWE administration by RGF was shown to enhance the TG levels of M. rosenbergii, which persist for up to 24 h, and administration of 5 µg RSWE showed the highest TG activity (Fig. 2). Furthermore, RSWE administration by RGF was shown to enhance the RB levels of M. rosenbergii. However, administration of high doses of RSWE (10 µg) by RGF showed excessive RB activity, in which RB activity increased significantly (p < 0.05) 3 h after RGF and persisted for up to 24 h (Fig. 3). In addition, RSWE administration by RGF was shown to enhance the lysozyme levels of M. rosenbergii but administration of high doses of RSWE (5 and 10 µg) by RGF showed an earlier increase in lysozyme activity than low doses of RSWE (Fig. 4).

Fig. 1
figure 1

Total hemocyte count in hemolymph of M. rosenbergii in response to S. radix water extract (RSWE) administration at 3, 6, 12, and 24 h post-RGF. Data are presented as mean ± SD (n = 3). Different letters indicate significant differences among treatments at each time point (p < 0.05)

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Fig. 2
figure 2

Transglutaminase activity in hemolymph of M. rosenbergii in response to S. radix water extract (RSWE) administration at 3, 6, 12, and 24 h post-RGF. Data are presented as mean ± SD (n = 3). Different letters indicate significant differences among treatments at each time point (p < 0.05)

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Fig. 3
figure 3

Respiratory burst activity in hemolymph of M. rosenbergii in response to S. radix water extract (RSWE) administration at 3, 6, 12, and 24 h post-RGF. Data are presented as mean ± SD (n = 3). Different letters indicate significant differences among treatments at each time point (p < 0.05)

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Fig. 4
figure 4

Lysozyme activity in hemolymph of M. rosenbergii in response to S. radix water extract (RSWE) administration at 3, 6, 12, and 24 h post-RGF. Data are presented as mean ± SD (n = 3). Different letters indicate significant differences among treatments at each time point (p < 0.05)

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Effect of Radix Scutellariae water extract (RSWE) on the immune-related genes

Hepatopancreas

The expressions of LGBP in M. rosenbergii HP were decreased post-RGF in all treatments for 24 h, except for an increase of LGBP (2.1-fold) at 12 h post-RGF in T3 (Fig. 5). The downregulation of PE in T2 and T3 was observed as early as 3 h (0.2-fold) post-RGF (Fig. 6). An increase in PE was only shown 12 h post-RGF in T3 (non-significant).

Fig. 5
figure 5

Expression of LGBP in M. rosenbergii hepatopancreas post-RGF in response to S. radix water (RSWE) extract administration at 3, 6, 12, and 24 h post-RGF. Data are presented as mean ± SD (n = 3). Different letters indicate significant differences among treatments at each time point (p < 0.05)

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Fig. 6
figure 6

Expression of PE in M. rosenbergii hepatopancreas post-RGF in response to S. radix water (RSWE) extract administration at 3, 6, 12, and 24 h post-RGF. Data are presented as mean ± SD (n = 3). Different letters indicate significant differences among treatments at each time point (p < 0.05)

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Midgut

The expressions of LGBP in M. rosenbergii MG were downregulated as early as 3 h post-RGF at T2 and T3, while T1 expression was not significantly different to the control group (Fig. 7). The upregulation of LGBP was observed in all treatments at 6 h. Furthermore, an increase of LGBP in MG was also seen at 12 and 24 h post-RGF in T2 and T1 (2.6- and 5.35-fold, respectively). The expression of proPO was upregulated in all treatments for 3 h and only T2 for 12 h (1.77-fold), while at 6 and 24 h showed no significance among treatments (Fig. 8). The upregulation of PE in T1 was observed as early as 3 h (10.64-fold) post-RGF (Fig. 9). An increase in PE also was shown at 6 h post-RGF in T3 (Fig. 9).

Fig. 7
figure 7

Expression of LGBP in M. rosenbergii midgut post-RGF in response to S. radix water (RSWE) extract administration at 3, 6, 12, and 24 h post-RGF. Data are presented as mean ± SD (n = 3). Different letters indicate significant differences among treatments at each time point (p < 0.05)

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Fig. 8
figure 8

Expression of proPO in M. rosenbergii midgut post-RGF in response to S. radix water (RSWE) extract administration at 3, 6, 12, and 24 h post-RGF. Data are presented as mean ± SD (n = 3). Different letters indicate significant differences among treatments at each time point (p < 0.05)

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Fig. 9
figure 9

Expression of PE in M. rosenbergii midgut post-RGF in response to S. radix water (RSWE) extract administration at 3, 6, 12, and 24 h post-RGF. Data are presented as mean ± SD (n = 3). Different letters indicate significant differences among treatments at each time point (p < 0.05)

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Discussion

Most of the immune responses occur in hemolymph, which contains three different types of hemocytes, namely semigranular, granular, and hyaline (Interaminense et al. 2019). Meanwhile, the digestive tract of prawn consists of three main parts, the foregut, midgut, and hindgut (McGaw and Curtis 2013). The midgut is the main absorptive area of the digestive tract, which represents a vital route of pathogen entry (Barreto et al. 2018; Rosa and Barracco 2010) and is closely associated with hemocytes. Another essential organ is the hepatopancreas, the prawn’s primary digestive gland similar to the liver in vertebrates. The hepatopancreas, which occupies a substantial part of the posterior cephalothorax and surrounds the posterior stomach and anterior midgut, is an essential site for the expression of immune-related genes in prawn (Du et al. 2013; Robalino et al. 2007; Zeng et al. 2013).

Effect of Radix Scutellariae water extract (RSWE) on the immune responses

Total hemocyte count (THC)

The circulating hemocytes play primary roles in crustaceans’ immune response to invaded microbial pathogens and external stimulations. Hemocytes conduct various physiological and pathological functions, such as antigen recognition, phagocytosis, encapsulation, nodule formation, and releasing of humoral factors (Chang et al. 2015). However, the number of hemocytes is related to many factors, such as environmental stresses (Perazzolo et al. 2002), infections (Feng et al. 2008), immunostimulant administration (Chen et al. 2014), and endocrine activities (Estrada et al. 2016). In the present study, we evaluated the effects of RSWE administration by RGF on the THC of M. rosenbergii. As shown in Fig. 1, after administration with 1 and 5 µg of RSWE by RGF, THC levels increased significantly compared with the other treatments for 24 h (Fig. 1). The similar results described by Wu et al. (Wu et al. 2015) showed that the water extract of Gynura bicolor could enhance THC of Litopenaeus vannamei at 7th day post-feeding treatment. Noni leaves extract was also shown to increase M. rosenbergii THC at 3rd day post-feeding treatment (Halim et al. 2017). It is supposed that RSWE administration by RGF may trigger immune cell proliferation in prawns, thereby increasing the secretion of cytokines (e.g., astakine), which can increase hemocyte synthesis in hematopoietic tissues.

Transglutaminase (TG)

Coagulation is initiated by releasing factors and enzymes from hemocytes, which effectively polymerizes the hemolymph clotting protein (Martin et al. 1991). In crustaceans, clotting is mediated through coagulates presented in the plasma and TG within circulating hemocytes (Liu et al. 2011). TG prevents hemocyte loss during the microbial invasion and inhibits their growth in the hemocoel (Fagutao et al. 2012). Besides, hemolymph clotting is responsible for binding and killing pathogens in horseshoe crabs. This process involves inflammatory responses, wound closure, and healing (Theopold et al. 2004). In the present study, RSWE administration by RGF was shown to enhance the TG levels of M. rosenbergii, which persist for up to 24 h, and administration of 5 µg WERS showed the highest TG activity (Fig. 2). A previous study using Eichhornia crassipes supplemented feed to enhance the immune system of M. rosenbergii also showed a similar result (Chang et al. 2013). Together with an increase in THC, our data support a functional involvement of TG in the acceleration of hemolymph coagulation of M. rosenbergii.

Respiratory burst (RB)

RB is the rapid release of reactive oxygen species (ROS) from phagocytes during phagocytosis. Increased RB activity is related to increased bactericidal activity by phagocytes or hemocytes within the cellular immune system (Song and Hsieh 1994). However, overproduction of ROS may induce oxidative stress that can potentially damage host cells (Pohanka 2013). In this study, RSWE administration by RGF was shown to enhance the RB levels of M. rosenbergii. However, administration of high doses of RSWE (10 µg) by RGF showed excessive RB activity, in which RB activity increased significantly 3 h after RGF and persisted for up to 24 h (Fig. 3). A previous study showed that RB activity of L. vannamei was increased at 24 h post-feeding with P. binghamiae hot water extract at the concentration of 10 µg/g (Chen et al. 2014). Furthermore, crude extract of Cardiospermum halicacubum leaves supplemented with commercial feed enhanced the RB activity of Penaeus monodon from 48 to 120 h after feeding (Rajasekar et al. 2011). These results suggest that the appropriate administration of RSWE by RGF can effectively induce RB activity of hemocytes in prawns.

Lysozyme

Lysozyme is found in hemolymph and most shrimp tissues, but lysozyme production is related to the number of hemocytes in the hemolymph (Burge et al. 2007; Mai and Hu 2009; Qiao et al. 2013). Lysozyme is an antimicrobial agent, and it is proven that the increased activity of lysozyme can protect the host by acting as an essential defense enzyme against infectious diseases (Ragland and Criss 2017; Saurabh and Sahoo 2008). In this study, RSWE administration by RGF was shown to enhance the lysozyme levels of M. rosenbergii. However, administration of high doses of RSWE (5 and 10 µg) by RGF showed an earlier increase in lysozyme activity than low doses of WERS (Fig. 4). An increase in lysozyme activity was also found in P. monodon and L. vannamei, given guava leaf extract and zingerone, respectively (Chang et al. 2012; Yin et al. 2014). In contrast, Radix Scutellariae could not enhance lysozyme activity in tilapia, Oreochromis niloticus (Yin et al. 2006). These results suggest that administering high doses RSWE by RGF can effectively induce RB activity of hemocytes in prawns.

Effect of Radix Scutellariae water extract (RSWE) on the immune related-genes

Lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP).

LGBP was mainly expressed in the hepatopancreas (Pan et al. 2005; Yeh et al. 2009) and is involved in cellular and humoral defenses using PAMP (pathogen-associated molecular patterns) and stimulates a cascade of responses, such as phagocytosis, clotting activation, antibacterial peptides production, and proPO cascades (Iwanaga 1994; Lai et al. 2011; Roux et al. 2002; Yeh et al. 2009). In our study, the significant upregulation of the LGBP gene in hepatopancreas was observed at 12 h after administration of 10 µg RSWE by RGF (Fig. 5). A previous study reported that LGBP expression was increased in the hepatopancreas of L. vannamei at 72 h and 7 days post-feeding treatment of diets containing β-1,3-glucan (Wang et al. 2008). In addition, LGBP expression in the hepatopancreas of M. rosenbergii was upregulated at 12 h after LPS or poly I:C administration (Yeh et al. 2009). Meanwhile, at 6 h after RSWE administration by RGF, the expression of LGBP in the midgut was significantly upregulated, and this trend persisted for prawn groups with 5 and 10 µg RSWE (Fig. 7). The increased expression of LGBP in the midgut is originates from the infiltration of hemocytes (Silveira et al. 2018). Hemocytes perform a migratory behavior to digestive tissues and contribute to gut immunity (Arts 2006; Silveira et al. 2018; Yeh et al. 2009). Based on the statements above, we speculated that the increased LGBP in MG was due to their content of hemocytes. These results suggest that the difference in LGBP expression in the digestive organs was modulated by types of immunostimulants, delivery method, and migratory behavior of hemocytes.

Prophenoloxidase (proPO)

The proPO-activating system is believed to be an essential innate defense in invertebrate immunity (Charoensapsri et al. 2011). Activation of proPO is shown to generate cytotoxic products, melanin production (melanization), and encapsulation of pathogen (Cerenius et al. 2008). The shrimp’s proPO expression was found in the hemolymph, gill, stomach, hepatopancreas, and intestine (Ai et al. 2008; Charoensapsri et al. 2009; Pang et al. 2014). It showed that proPO plays a central role in killing and eliminating invading pathogens (Tassanakajon et al. 2018). Besides, upregulation of proPO expression in diverse tissues was used as a hemocyte marker to determine hemocyte infiltration into various tissues (Soonthornchai et al. 2010).

In the present study, proPO was expressed in midgut M. rosenbergii. However, in the midgut, the expression of proPO showed a rapid on–off response after stimulation with RSWE by RGF (Fig. 8). This result is consistent with previous studies on Morinda citrifolia (Halim et al. 2017), Musa acuminate (Rattanavichai and Cheng 2015), and Solanum nigrum (Harikrishnan et al. 2011). In contrast, another study showed that the regulatory mechanism for proPO gene expression after stimulation with pUC57-CpG is a long-lasting response (Yi et al. 2014). Besides, the present study considered the upregulation of proPO in the midgut caused by infiltrating hemocytes in the midgut because proPO is synthesized in granular hemocytes and converted into an active PO form by a serine proteinase (Okumura 2007).

Peroxinectin (PE)

Peroxinectin is a cell adhesion protein essential to invertebrates for many physiological processes, including immune responses (Johansson 1999). In addition, active PE can bind to integrin receptors on the cell surface to encourage the degranulation process in a positive reaction loop and play a multifunctional agent during pathogen invasion (Sricharoen et al. 2005). In the present study, the expression of PE in the hepatopancreas of M. rosenbergii was significantly upregulated after administration of RSWE, particularly when stimulated with 1 and 10 µg RSWE at 3 and 12 h after RGF (Fig. 6). Meanwhile, the expression of PE in the midgut has a similar pattern to the expression of proPO in the midgut (Figs. 8 and 9). These circumstances are understandable since PE is a protein associated with the proPO system, and its cell adhesion activity is triggered when the proPO system is activated (Sritunyalucksana et al. 2001).

Overall, there was no correlation between the administered doses of RSWE by RGF with the immune responses and the expression of immune-related genes on M. rosenbergii. Moreover, there is no single dose of RSWE that can simultaneously boost all immune responses in circulating hemocytes and the expression of immune-related genes in the digestive organs of M. rosenbergii. However, stimulation with 5 μg RSWE by RGF consistently increased immune responses and immune-related genes in M. rosenbergii. In contrast, administration of 10 μg RSWE may inhibit some immune responses and immune-related genes in M. rosenbergii.

Conclusion

The results demonstrated that administration of RSWE by RGF improved immune responses (THC, TG, RB, and lysozyme activity) and induced the expression of immune-related genes (LGBP, proPO, and PE) in the hepatopancreas and midgut. The RGF method can be used to investigate the immunomodulatory effect of RSWE on shrimp, and the immune response associated with each dose can be compared precisely.