Introduction

The whiteleg shrimp, Litopenaeus vannamei, is among the commercially valuable penaeid shrimps cultured in several countries worldwide (FAO 2020; Islam et al. 2020). Nevertheless, owing to intensive cultivation and climate shifts (Byers 2021; Kibenge 2019), L. vannamei farming has been affected by several pathogens, which have devastated the business of the shrimp aquaculture industry (Khoa et al. 2004; Kibenge 2019; Zhang et al. 2014; Zou et al. 2020). Black spot disease (BSD) is among the diseases affecting shrimp production as it claimed numerous lives, particularly in the late rearing stages (Pazir et al. 2022). This disease was first reported in the kuruma prawn, Marsupenaeus japonicus, by Ishikawa (1968) and was caused by a fungal infection. Hatai and Egusa (1978) identified the causal agent of BSD as Fusarium solani, which was then regarded as one of the most severe infections of M. japonicus in Japan (Khoa et al. 2005; Yao et al. 2022). Fusarium species are ubiquitous fungal agents affecting plants, soil, freshwater, and brackish water (Lightner 1996; Palmero et al. 2009). These fungi may spread to various plants globally, causing agricultural losses (Figueroa et al. 2018; Moretti et al. 2017; Summerell 2019).

Probiotics are solo or mixed cultures of microbial populations that, when supplied in sufficient quantities, may promote the development and health of the host (El-Saadony et al. 2021; Lara-Flores et al. 2010; Salminen et al. 1999; Yilmaz et al. 2022). Due to their unique advantages and health benefits, probiotics have been marketed and sold as an immediate water supplement and feed additive (Jahangiri and Esteban 2018; LaPatra et al. 2014). Applying probiotics directly to water is an effective method for improving water quality, but applications as feed supplements have also given positive outcomes (Jahangiri and Esteban 2018). Probiotics positively influenced the water quality parameters such as dissolved oxygen, hardness, pH, temperature, and osmotic pressure (Cha et al. 2013; Das et al. 2008).

Bacillus species are commonly used as effective probiotic supplements in aquaculture (Abdel-Tawwab et al. 2022; Liu et al. 2012). Bacillus can produce several extracellular enzymes and withstand extreme temperatures and dehydration (Yu et al. 2009). These probiotics have been extensively explored recently and may contribute greatly to aquatic organisms’ physiological, morphological, hematological, and immunological conditions when administered at optimal levels in the rearing water (Rahman et al. 2021).

The primary target of this research was to examine the influence of applying three water supplementation levels of Bacillus species probiotic (SANOLIFE®PRO-W) on the water quality, growth performance, innate immunity, antioxidant activities, and disease resistance of whiteleg shrimp to F. solani infection.

Materials and methods

Animals and experimental design

Apparent healthy 240 shrimps with an average initial body weight of 2.0 ± 0.07 g/shrimp were acquired from a special farm in Ismailia Governorate, Egypt. Shrimps were acclimatized for 14 days in an outdoor 2000-L fiberglass tank and were fed on a commercial diet of Skretting Company, Belbeis, Sharkia, Egypt. Nutritional ingredients of this diet composed of fishmeal (72% crude protein), soybean meal (48% crude protein), corn gluten (63% crude protein), wheat bran, yellow corn, soybean oil, starch, vitamin, and mineral premix. The proximate chemical composition for the diet was 89.59% dry matter, 40% crude protein, 8.5% ether extract, 3.34% fiber, 6.52% ash, 41.56% NFE, and 18.94 MJ/kg growth energy. Afterward, shrimps were randomly stocked at twelve 1-m3 concrete ponds (20 individuals per each one) in a special farm in Ismailia Governorate, Egypt, to represent four triplicate experimental treatments. Bacillus species probiotics, which are commercially sold as SANOLIFE®PRO-W (a mixture of Bacillus subtilis and B. licheniformis at 5 × 105 CFU/g; INVE Aquaculture, Belgium), were added to the rearing water at levels of 0, 0.01, 0.02, and 0.03 g/m3, representing T0 (control), T1, T2, and T3 respectively. Five liters of clean water was mixed with those different levels of the probiotic, and water (5 L) was sprinkled over the pond’s surface biweekly for 56 days. Water was changed daily at a rate of 5% for each pond. Shrimps were fed on the commercial diet at 7% in the first 28 days and 6% in the next 28 days of the shrimp biomass. The given feed was divided equally into three portions and offered to animals three times a day (8.00, 12.00, and 16.00 h). The total bacterial count in SANOLIFE®PRO-W was determined using the standard pour plate method (APHA 1998).

Water quality measurements

Water quality indices were determined daily at 9:00 h below 30 cm of the ponds’ surface. The water temperature (T; °C) and dissolved oxygen (DO) were determined by an oxygen meter (970 portable DO meter, Jenway, London, UK). A pH meter determined the pH (Digital Mini-pH Meter, model 55, Fisher Scientific, Denver, CO, USA). The salinity was assessed by a refractometer (Erma, Japan). At the same time, the unionized ammonia (NH3) was calculated utilizing the HACH comparison device according to Boyd (1982), and total ammonia nitrogen (TAN) of the pond water was determined by an ammonia test kit (Advance Pharma, Thailand).

Growth efficiency of shrimp

The growth performance was evaluated biweekly via sampling and weighing ten animals from each pond. The body weight gain (WG), daily weight gain (DWG), specific growth rate (SGR), and survival % were calculated using the following equations:

$$\begin{array}{l}\mathrm{Body}\;\mathrm{weight}\;\mathrm{gain}\left(\mathrm{WG}\right)=\mathrm{Final}\;\mathrm{weight}\left(\mathrm Wf\left(\mathrm g\right)\right)-\mathrm{Initial}\;\mathrm{weigh}\left(\mathrm{Wi}\left(\mathrm g\right)\right).\\\mathrm{Body}\;\mathrm{weight}\;\mathrm{gain}\;\%\;\left(\mathrm{WG}\;\%\right)=\left[\mathrm{WG}/\mathrm{Initial}\;\mathrm{weight}\;\right]\times100.\\\mathrm{Daily}\;\mathrm{weight}\;\mathrm{gain}\;\left(\mathrm g/\mathrm{fish}/\mathrm{day}\right):\;\mathrm{DWG}=\mathrm{WG}/\mathrm{duration}\;\mathrm{period}\;\left(\mathrm T\right)\;\left(56\;\mathrm{days}\right).\\\mathrm{Feed}\;\mathrm{Intake}\;\left(\mathrm g/\mathrm{fish}\right):\;\mathrm{The}\;\mathrm{quantity}\;\mathrm{of}\;\mathrm{feed}\;\mathrm{given}\;\mathrm{or}\;\mathrm{offered}\;\mathrm{during}\;\mathrm{the}\;\mathrm{trial}\;\mathrm{duration}/\mathrm{shrimp}\;\left(\mathrm g\right).\\\mathrm{Feed}\;\mathrm{conversion}\;\mathrm{ratio}\;\left(\mathrm{FCR}\right)=\mathrm{Feed}\;\mathrm{intake}\;\left(\mathrm g\right)/\mathrm{Weight}\;\mathrm{gain}\;\left(\mathrm g\right).\\\mathrm{Specific}\;\mathrm{growth}\;\mathrm{rate}\;\left(\mathrm{SGR}\right)=100\times\;\left(\ln\;\mathrm Wf-\;\ln\;\mathrm{Wi}\right)/\mathrm T\\\mathrm{Shrimp}\;\mathrm{biomass}\;\left(\mathrm g\right)=\mathrm Wf\times\;\mathrm{Final}\;\mathrm{number}\;\mathrm{of}\;\mathrm{shrimps}\\\mathrm{Survival}\;\mathrm{rate}\;\left(\mathrm{SR}\right)=\left(\mathrm{Survival}\;\mathrm{shrimp}\;\mathrm{number}/\mathrm{Initial}\;\mathrm{shrimp}\;\mathrm{number}\right)\times100.\end{array}$$

Proximate chemical analysis of body

When the experiment was ended, five shrimps from each pond were weighed and dried in a furnace at 105 °C for 180 min, and then crushed and tested according to AOAC (1995) for moisture, crude protein, crude fat, and ash.

Digestive enzyme activities

Hepatopancreases from shrimps were weighed, and 1.0 g was blended in 1.5 mL of distilled water. Before being examined, these samples were stored in 1-mL Eppendorf tubes at − 20 °C. Supernatants were obtained after centrifugation at 13,000 × g for 5 min at 4 °C. Triplicate tests were performed after diluting the homogenate with the appropriate buffers. Enzyme activity units were measured in milligrams of soluble protein (U mg−1).

Trypsin and chymotrypsin activities were measured kinetically (Geiger 1984; Tseng et al. 1982) using N-α-benzoyl-dl-arginine p-nitroanilide (BAPNA, Sigma, B4875) and N-succinyl-ala-ala-pro-phe (SAPPNA, Sigma, S7388) as substrates. A spectrophotometer recorded both reactions at 407 and 405 nm every 0.9 s for 3 min. Reaction and incubation temperatures were 25 °C. Trypsin and chymotrypsin activities produced 1 μmol of 4-nitroaniline per minute per milligram of protein. Calculations used an ε405 = 10.2 L mmol−1 cm−1 extinction coefficient (Geiger 1984; Geiger and Fritz 1981).

Protease enzyme activities were determined via azocasein (Sigma A2765, Sigma Chemical, St. Louis, USA) as a substrate, as Svåsand et al. (2007) described. Lipase enzyme activity was determined following the method of Versaw et al. (1989) using β-naphthyl caprylate (Sigma, N8875). A 0.001 U/min rise in absorbance at 440 nm and 540 nm was utilized to quantify protease and lipase activity.

Using the method described by Rick and Stegbauer (1974), alpha-amylase activity was measured. Maltose generated a calibration curve (Kanto Chemical, Tokyo, Japan). At 550 nm, a spectrophotometer measured the concentration of the samples and the standards used to create the calibration curve (Jenway, Essex, UK). Amylase value was determined as mmol of maltose emitted per minute per µg of protein.

The innate immunity induces

Hemolymph was collected from 10 animals from each treatment. Hemolymph was extracted from the cardio-coelom near the hindmost side of the carapace by a 1-mL medically disinfected needle and syringe. Anticoagulant (0.34 M NaCl, 0.01 M KCl, 0.01 M EDTA-Na2, and 0.01 M HEPES, pH 7.45 and 780 mOsm. kg−1) was included in the syringe (Vargas-Albores et al. 1993). Hemolymph was collected using a 1:1 anticoagulant ratio. After pipetting 1 mL of hemolymph into an Eppendorf tube and centrifugation at 3000 rpm for 15 min at 4 °C, the blue supernatant was packed into fresh tubes as a plasma sample at a temperature of 80 °C.

A total hemocyte count (THC) was determined by a hemocytometer and examined under a stereomicroscope (Supamattaya et al. 2000). The plasma lysozyme content was evaluated by lysis of the Gram-positive bacteria Micrococcus luteus solution (M3770-25G Lot 117K8707, Sigma-Aldrich), following Zanuzzo et al. (2017).

The respiratory burst (RB) activity was determined in a portion of the whole hemolymph with pre-cooling anticoagulant by the method described by Anderson and Siwicki (1995). Prophenoloxidase (PO) activity was assessed spectrophotometrically by measuring l-DOPA-derived dopachrome production (Liu et al. 2004). Phagocytosis (PP) and the phagocytic index were measured using the procedures outlined by Rengpipat et al. (2000).

Antioxidant activity and lipid peroxidation

Enzymatic antioxidant activities, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) enzyme activities, were evaluated in the hepatopancreatic samples. CAT activity was measured using the auto-oxidation of pyrogallol described by Marklund and Marklund (1974). The CAT activity was detected after the H2O2 reduction at 240 nm, as Claiborne (1985) described. The GPx activity was measured by tracking the rates of NADPH oxidation by the two-step reaction with glutathione reductase at 340 nm utilizing an extinction coefficient of 6.22 mM−1 cm−1, following Gunzler and Flohe (1985). The units of enzyme activity were reported as specific activities (IU/mg protein). We employed the technique mentioned by Draper and Hadley (1990) for generating TBARS through an acid-heating reaction to measure lipid peroxidation using malondialdehyde. MDA equivalents are shown as nmol/mg protein.

Resistance to F. solani infection.

Preparation of the fungal strain

F. solani was obtained from the Microbiological Unit, Fish Diseases Department, Animal Health Institute, Agricultural Research Center, Dokki, Giza, Egypt. F. solani was cultured on potato dextrose agar (PDA) for 7 days at 25 °C. Then, 20 mL of sterilized distilled water was inserted into each culture plate to collect the conidial material in 30-mL tubes. After that, the samples were filtered to confirm fungal conidia. Finally, enumeration was performed to evaluate the count of the conidia and then optimized to 5 × 104/mL in sterilized distilled water.

Experimental infection procedures

Treated shrimps were obtained and grouped at a rate of 10 individuals per 50-L tank; three replicates represented each treatment. Shrimps were artificially infected in the mid-lateral part of the carapace or the abdominal tergal and pleural plates, carapace, and gills, as described by Hose et al. (1984). Shrimp wounding was performed by removal of the trailing edge of the carapace or the abdominal tergal and pleural plates, carapace, and gills. A cotton swab dipped in a thick solution of F. solani conidiospores (5 × 104 conidia/mL; obtained from a 7-day-old Sabouraud dextrose agar culture) was used to swab wounds quickly. Before returning the shrimp to their aquariums, the leaking hemolymph was permitted to clot and capture the spores. In addition, a cotton swab infected with F. solani was spun (under mild pressure) against the gills. The control treatment was not infected but injured like the other groups. Over 14 days, animals were given the same corresponding diets used from starting the experiment. Abnormal behaviors and shrimp mortalities were detected. Salinity, dissolved oxygen, and water temperature were preserved throughout the challenge tests under the same conditions as the feeding experiment. Every 2 days, leftover feed and waste were removed.

Statistical analysis

Shapiro–Wilk and Bartlett’s tests verified data normality and variance homogeneity before statistical analysis. Each parameter’s mean and SEM were determined. After a one-way analysis of variance (ANOVA) by IPM SPSS (Version 26), Duncan’s test was applied to compare means at P < 0.05 (Dytham 2011).

Results

Water quality parameters

All water quality indices were suitable for shrimp culture (Table 1). Total ammonia nitrogen (TAN) and unionized ammonia were affected by Bacillus species probiotics as their values decreased by increasing the probiotic levels. The control treatment had a high level of TAN and ammonia, while its lowest levels were observed at T2 and T3, which received 0.02 and 0.03 g/m3 of Bacillus species probiotics. On the other hand, the DO levels were significantly increased with probiotic addition, while pH was reduced. In contrast, the probiotic addition to water did not significantly affect the water temperature and salinity levels (P > 0.05).

Table 1 Changes in water quality parameters of ponds treated with different levels of Bacillus species probiotic and stocked with whiteleg shrimp for 56 days
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Digestive enzyme activities

The hepatopancreatic digestive enzymes of shrimp were increased significantly by applying probiotics in the rearing water (P < 0.05; Table 2). The chymotrypsin, trypsin, proteases, lipase, and alpha-amylase enzyme activities were improved significantly with increasing probiotics levels than the control group. There was no substantial variance among T2 and T3 for all digestive enzymes except for chymotrypsin.

Table 2 Digestive enzymes of whiteleg shrimp stocked at ponds treated with different levels of Bacillus species probiotic in water for 56 days
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Growth performance and feed utilization

When compared with the control group, the result showed that water application of the probiotics significantly enhanced (P < 0.05) the growth performance and the weight of shrimp (Table 3). Final weight, weight gain, weight gain %, ADG, SGR, and feed intake significantly increased with water application, without differences between T2 and T3 (P > 0.05). Conversely, FCR and survival rate were significantly enhanced in probiotic groups (P < 0.05) than the control, without any difference between T2 and T3.

Table 3 Growth performance of whiteleg shrimp stocked at ponds treated with different levels of Bacillus species probiotic in water for 56 days
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Whole-body chemical composition

Compared with the control group, the total protein and ash contents of the probiotic-treated shrimp were improved. However, the lipid content was significantly reduced (Table 4). There was no substantial variance in moisture content among the different treatments (P > 0.05).

Table 4 Proximate chemical analysis of whiteleg shrimp body composition stocked at ponds treated with different levels of Bacillus species probiotic in water for 56 days
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Antioxidant status and lipid peroxidation

The activities of SOD, CAT, and GPx enzymes were significantly improved by adding probiotic Bacillus to water than the control treatment (P < 0.05; Table 5). Conversely, the MDA levels were decreased with increasing probiotic Bacillus levels in pond water than the control treatment (P < 0.05).

Table 5 Enzymatic antioxidant activities and lipid peroxidation (MDA concentrations) of whiteleg shrimp stocked at ponds treated with different levels of Bacillus species probiotic in water for 56 days
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Innate immunity status

All immunological parameters significantly improved by increasing the probiotic Bacillus than the control treatment (P < 0.05; Table 6). THC, PO activity, RB activity, lysozyme activity, and phagocytic index were significantly increased in shrimps reared in ponds with probiotic Bacillus than in the control group, particularly at T3 and T4.

Table 6 Immunological response of whiteleg shrimp stocked at ponds treated with different levels of Bacillus species probiotic in water for 56 days
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Resistance to F. solani infection

Shrimps reared in water containing Bacillus species probiotics were more resistant to F. solani infection than the control group, which displayed the highest mortality rates. When the probiotic doses increased in the rearing water, fatality rates dropped for each treatment (Fig. 1). The survival rate of shrimp in the T3 group was the highest (75%); however, it was 0% for the control group without probiotic addition (Fig. 2).

Fig. 1
figure 1

Cumulative mortality of whiteleg shrimp reared in ponds containing different levels of Bacillus species probiotics in the rearing water for 56 days and post-challenged by F. solani infection for 14 days

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Fig. 2
figure 2

The survival rate of whiteleg shrimp reared in ponds containing different levels of Bacillus species probiotics in the rearing water for 56 days and post-challenged by F. solani infection for 14 days. Bars having different letters are significantly different at P < 0.05

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Discussion

In the present study, the water quality parameters were within the levels approved for shrimp culture (Boyd and Tucker 1998). The water application of Bacillus species probiotics to pond water lowered TAN and NH3 levels. This is owing to the presence of the Bacillus mixture, which plays essential functions in the nitrogen cycle via ammonification (Hui et al. 2019), nitrification (Rout et al. 2017), and denitrification (Verbaendert et al. 2011) as well as nitrogen fixation (Yousuf et al. 2017). Hence, Bacillus sp. can eliminate the various kinds of nitrogen from aquaculture wastewater. B. amyloliquefaciens DT, for instance, transformed organic nitrogen to ammonium (Hui et al. 2019), while B. cereus PB8 eliminated \({\mathrm{NO}}_{-2}-\mathrm N\) from wastewater (Barman et al. 2018).

The most crucial water quality indicator for aquaculture activities is dissolved oxygen (DO) since the aquatic environment is rich with ammonia, phosphorus, organic waste, copper, and other elements that could reduce the DO levels (dos Santos Simoes et al. 2008). In the current study, DO increased markedly with the addition of Bacillus species probiotics in a dose-dependent manner compared to the control, and this is because Bacillus could optimize the decomposition of organic matter load (Hai 2015), thereby recycling nutrients in the water column and reducing sludge accumulation (Soltani et al. 2019). Furthermore, the decomposition of organic matter increases DO (Boyd and Gross 1998; Cha et al. 2013). Our study agrees with previous results which found that Bacillus sp. improved DO levels in rearing water, such as in the case of B. megaterium (Hura et al. 2018), a combination of Bacillus (Hainfellner et al. 2018), Bacillus species mixture (composed of B. subtilis, B. licheniformis, B. megaterium, and B. laterosporus) (Gomes et al. 2008; Zink et al. 2011).

In the present study, it was noticed that the Bacillus mixture decreased pH values compared with the control group, based on the water application dose. These findings might be explained by the fact that Bacillus efficiently transforms organic matter to CO2, which is then used as a carbon source by β- and γ-proteobacteria (Koops and Pommerening-Röser 2001), which convert most of the organic matter into slime or bacterial biomass (Mohapatra et al. 2013; Zorriehzahra et al. 2016). Moreover, CO2 is known to lower pH levels. The basic character of rearing waters is preferable to acidic waters, as Hura et al. (2018) found positive properties in carp culture due to the conservation of alkalinity by B. megaterium. In Bacillus-treated tilapia ponds, a rise in pH was also detected in the study performed by Elsabagh et al. (2018). In acidic circumstances, Bacillus may raise the pH of the water, making it ideal for fish production. Contrary to what was reported for pH, some reports showed that probiotic Bacillus lowered pH toward neutral (Gomes et al. 2008; Nimrat et al. 2012; Wu et al. 2016).

Herein, the Bacillus mixture did not affect the temperature, confirming prior studies that found no substantial temperature regulation by Bacillus sp. (Banerjee et al. 2010; Ghosh et al. 2008; Nimrat et al. 2012). As Velmurugan and Rajagopal (2009) noted, as a conservative measure, the temperature is unaffected by biological activities.

In the present investigation, the Bacillus sp. combination did not affect the water salinity levels. Aftabuddin et al. (2013) concluded that combining B. megaterium and Streptomyces fradiae did not noticeably impact water salinities. Furthermore, Velmurugan and Rajagopal (2009) found that biological processes do not readily influence salinity since it is a conservative water quality indicator. These authors further demonstrated that Bacillus is incapable of modulating salinity.

There is a strong relationship between the kinds and amounts of nutrients in feed and the number and activity of digestive enzymes in aquatic animals. Herein, it was observed that water application of Bacillus enhanced digestive enzyme activities compared to the control group. Likewise, it was reported that B. licheniformis could boost the nutritional digestibility of aquatic animals by increasing enzymatic production, such as amylase, protease, and cellulase. Ziaei-Nejad et al. (2006) found that probiotic Bacillus strains increased such enzymes in Indian shrimp. Here, the increase of protease, amylase, and lipase enzymes may be the primary factor that led to improved growth performance. It seems that B. licheniformis could enhance the absorption, digestion, and availability of certain nutrients (Yaqub et al. 2022).

Enhancing the functioning of digestive enzymes may improve digestion and absorption of food, hence enhancing growth performance and feed efficiency (Xie et al. 2019). The current research findings indicate that adding a probiotic Bacillus mixture to rearing water increased the growth rate of shrimp. This may be due to their positive growth-promoting role (Yanbo and Zirong 2006). Chen et al. (2020) found that B. licheniformis considerably enhanced the weight gain and specific growth rates of prawns. Moreover, it was also found that Bacillus improved the growth performance of L. vannamei (Cao et al. 2022) and Indian shrimp (Ziaei-Nejad et al. 2006). On the other hand, Kewcharoen and Srisapoome (2019) found that the probiotic B. subtilis AQAHBS001 could enhance the histomorphology of the midgut of white shrimp, increasing intestinal villi height/width, which increased nutrient uptake and shrimp development. Verschuere et al. (2000) and Zokaeifar et al. (2012) demonstrated that the Bacillus secretes a vast array of enzymes that help in the nutritional improvement of the host, hence promoting growth. The improved feed digestion may be linked to higher digestive enzyme activity (Zokaeifar et al. 2012).

The present research showed that probiotic Bacillus–added water increases crude protein and ash of shrimp compared to the control. These findings were consistent with those reported by Cao et al. (2022), who found that applying a particular concentration of probiotics may increase the crude muscle protein content of whiteleg shrimp compared to the control group. Seenivasan et al. (2014) declared that oral administration of a combination of Lactobacillus sporogenes, B. subtilis, and Saccharomyces cerevisiae enhanced the crude protein and ash contents of chicken carcasses. In contrast to our findings, Yu et al. (2009) and Heizhao et al. (2008) confirmed that dietary probiotics did not affect the body composition of whiteleg shrimp.

The oxidative stress–related enzymes (SOD, CAT, and GPx) and lipid peroxidation (represented by MDA) are bioindicators of oxidative cell injury, which are implicated in pathological processes and the etiology of numerous fish diseases (Abdel-Tawwab and Wafeek 2017; Hermes-Lima 2004; Kehrer 1993; Storey 1996). In the current investigation, probiotic application enhanced the antioxidative properties, as evidenced by increased SOD, CAT, and GPx and decreased MDA levels in shrimp hepatopancreas. Such findings might be associated with the elevated Bacillus sp. counts in the water of the rearing ponds. Shen et al. (2010) concluded that probiotics added to water improved the antioxidant enzyme activity of L. vannamei (Shen et al. 2010). In comparison with the control group, Amoah et al. (2019) observed a substantial increase in SOD (serum) and GPx (serum and liver) activities in whiteleg shrimp treated with B. coagulans ATCC 7050. Such an antioxidant impact has been found in investigations on different commercial probiotics (Abdel-Tawwab et al. 2020; Župan et al. 2015).

In shrimp, lysozyme and polyphenol oxidase (PO) activities are essential immunological enzymes contributing to innate immune responses (Magnadóttir 2006; Whyte 2007). In the present investigation, lysozyme and PO activities were increased in shrimps reared in ponds treated with probiotics than in the control group. These results may be attributable to the B. subtilis probiotic in water, which helps to enhance the innate immunity of L. vannamei (Amoah et al. 2019; Wongsasak et al. 2015; Zokaeifar et al. 2012). Moreover, Kewcharoen and Srisapoome (2019) observed that probiotic B. subtilis AQAHBS001 induced lysozyme activity in L. vannamei.

In the current research, the probiotic administered to shrimp ponds increased phagocytosis, THC, and respiratory burst activities. Phagocytes are responsible for attacking foreign pathogens, activating T cells (Parham 2014), and producing antibody signals linked to innate immune system activation. Shrimps have three kinds of hemocyte cells: hemocytes, hyalinocytes, granulocytes, and semi-granulocytes (Martin and Graves 1985). Hemocytes are protective cells in the hemolymph, shared defense systems versus foreign particles. Hemocyte cells function in phagocytosis, encapsulation, nodule formation, wound healing, and coagulation (Aguirre-Guzman et al. 2015; Martínez 2007). These hemocytes are concerned with phagocytosis and engulfing pathogens and foreign substances. During phagocytosis, a molecular mechanism recognized as respiratory leukocyte burst increases oxygen uptake, producing oxygen reduction and superoxide anion (Biller-Takahashi and Urbinati 2014). The present investigation showed a substantial improvement in immunological markers compared to the control group, suggesting that a probiotic mixture may positively affect immunity in L. vannamei hemolymph.

Probiotics are crucial in developing innate immunity in aquatic species, enabling them to combat pathogenic microorganisms and environmental stressors (Abdel-Latif et al. 2022; Abdel-Tawwab et al. 2022; El-Saadony et al. 2021; Rahman et al. 2021; Yilmaz et al. 2022). In the current investigation, F. solani infection resulted in significant mortality rates in the control group, but animals raised in ponds supplemented with probiotics had decreased mortality rates. B subtilis and B. licheniformis regulated the immune response when added to the water as probiotics, resulting in enhanced resistance to fungal infection. Hence, probiotic bacteria may compete with other invading microorganisms, reducing susceptibility and mortality rates (Yaqub et al. 2022). Similar findings were previously reported by Balcázar et al. (2006), who demonstrated the efficacy of dietary B. subtilis UTM 126 in preventing vibriosis in white shrimp. Furthermore, Zokaeifar et al. (2012) demonstrated that B. subtilis supplementation decreased mortalities following experimental challenge with Vibrio harveyi.

Conclusion

In summary, the current research indicated that water application of Bacillus species probiotic composed of B. subtilis and B. licheniformis had a positive influence on growth performance, chemical composition, digestive enzymes, antioxidant status, immunological indices, and disease resistance in whiteleg shrimp, with the greatest results achieved in T2 and T3, which received 0.02 and 0.03 g/m3 respectively.