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Effects of dietary supplementation with Bacillus velezensis on the growth performance, body composition, antioxidant, immune-related gene expression, and histology of Pacific white shrimp, Litopenaeus vannamei
BMC Veterinary Research volume 20, Article number: 368 (2024)
Abstract
In recent decades, probiotics have become an acceptable aquaculture strategy for shrimp growth promotion and immune modulation. This study aimed to evaluate the effect of Bacillus velezensis on Litopenaeus vannamei following a 60-day trial. L. vannamei (3 ± 0.4 g) were distributed into four groups with three replicates per group and fed an isonitrogenous diet supplemented with B. velezensis at 0, 1 × 107, 1 × 108, and 1 × 109 CFU/g, which were defined as the control, G1, G2, and G3 groups, respectively. B. velezensis significantly improved the growth, survival rate, and proximate body composition of L. vannamei (P < 0.05). All groups fed the B. velezensis diet showed significant increases in digestive enzymes (lipase, amylase, and protease), superoxide dismutase (SOD; G3), catalase (CAT; G3, G2, and G1), lysozyme activity (G3 and G2), immunoglobulin M (IgM), bactericidal activity BA%, alkaline phosphatase (AKP), and acid phosphatase (ACP) compared with the control group (P < 0.05). Malondialdehyde (MDA), triglycerides, cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels were significantly decreased in all groups fed B. velezensis diet compared with the control group (P < 0.05). The expression levels of SOD (G3), LZM, and serine proteinase genes were significantly higher in L. vannamei fed diets containing B. velezensis than in the control group (P < 0.05). This is the first study to address the effects of B. velezensis on the expression of the LZM and serine proteinase genes in L. vannamei. L. vannamei fed diet containing B. velezensis had more B and R cells in its hepatopancreas than did the control group. In conclusion, B. velezensis is a promising probiotic that can be safely added to the diet of L. vannamei with 1 × 109 CFU/g. Its application had a positive influence on the health status, survival rate, nutritional value, and immunity of L. vannamei.
Introduction
Shrimp farming, particularly of Litopenaeus vannamei, is increasing significantly in many countries throughout the world [1,2,3], including Egypt, which has recently taken on the challenge of achieving self-sufficiency in fish and fish products through megaprojects like Ghalioun and Sharq Al-Tafria.
Litopenaeus vannamei, also known as Vannamei shrimp, Penaeus vannamei, Pacific white shrimp, and whiteleg shrimp, is a shrimp species that efficiently utilises the natural productivity of its ponds, even under intensive culture conditions. Furthermore, it has lower feed costs due to its lower protein requirement (18–35%) compared to more carnivorous shrimp species (36–42%) [4] and is considered a major commercial food commodity due to its high price, high resistance to disease, excellent flavour, great nutritional value, well-understood aquaculture technologies, and vast consumption [5,6,7].
Farming intensification practices and climatic changes during L. vannamei aquaculture increase its susceptibility to different types of diseases, leading to great economic losses, and the demand for environmentally friendly alternatives to antibiotics is critical. Probiotics (non-pathogenic bacteria) are a strategy used to improve shrimp aquaculture by promoting digestion and disease resistance. They can also be employed as antibiotic alternatives, thereby preventing drug resistance to routinely used antimicrobials and antibiotic residues [5, 8].
Probiotics play a major role in fish and shellfish aquaculture productivity. They positively impact survival and growth rates, preserve water quality in ponds, and aid in the digestion and absorption of nutrients. The above reasons led to higher growth performance and a higher feed conversion ratio [9,10,11]. They improve the innate and adaptive immune responses of shrimp, resulting in increased resistance to infectious diseases through increased hemocyte counts, phenoloxidase activity, respiratory bursts, and the upregulation of antimicrobial gene expression. They also improve antioxidant enzymes, such as SOD&CAT, and decrease oxidative stress marker e.g. MDA which in turn makes the incorporation of probiotics a key benefit in shrimp aquaculture [12,13,14].
Bacillus spp., Lactobacillus spp., Pediococcus spp., and Enterococcus spp. are among the bacterial species employed as probiotics in aquaculture. Bacillus spp. are excellent examples of probiotics with superior properties, such as the production of non-pathogenic and non-toxic antipathogenic materials and the ability to produce spores, which extend the shelf life and make them more resistant to adverse environmental conditions [15,16,17,18,19,20,21].
Despite the use of Bacillus spp. in aquaculture of different aquatic species e.g. B. subtilis in L. vannamei, B. velezensis in grass carp, and B. amyloliquefaciens in Oreochromis niloticus [22,23,24], studies on B. velezensis in shrimp diets are scarce [14, 25]. Therefore, this study aimed to assess the impact of this probiotic on L. vannamei growth performance, survival rate, feed utilisation, body composition, biochemical parameters, antioxidants, and immune-related gene expression, in addition to histopathological alterations.
Material and methods
The current study followed a standard working methodology approved by the Animal Use and Care Committee of the Faculty of Fish and Fisheries Technology at Aswan University, Egypt (Protocol No. 5/2022).
The probiotic used and its safety
B. velezensis used in this study was a locally isolated Egyptian strain obtained from a healthy fish pond (National Research Center Project No. 12050419–2019, Egypt). It was genetically identified as B. velezensis (unpublished data).
In fibreglass tanks, 120 white shrimp with an average initial body weight of 3 ± 0.4 g/shrimp were randomly divided into four groups in triplicate (10 shrimp per tank) to test the safety of used B. velezensis for this shrimp species. Diets supplemented with four different levels of B. velezensis (CG: 0 CFU/g as a control group; G1: 1 × 107 CFU/g; G2: 1 × 108 CFU/g; and G3: 1 × 109 CFU/g) were fed to L. vannamei ad libitum according to García-Bernal et al. [26]. Activity, behavior, and survival rates of L. vannamei were checked daily for 3 days.
Diets preparation
A commercial isonitrogenous basal diet specifically formulated for shrimp from a factory in Kafer Elsheikh, Egypt was used in this study. The ingredients and chemical analysis of the basal diet is shown in Table 1. According to Cunniff and AOAC [27], moisture (7.45%), crude protein (40.28%), crude fat (6.85%), and crude ash (9.84%) were present. Moisture content was measured by drying the diet sample in a hot-air oven at 135 °C then gets the difference between the sample weight before and after drying. Protein was measured using the Kjeldahl method, in which sulfuric acid was used for digestion, copper sulfate was used as a catalyst, and ammonia was finally titrated. The Soxhlet apparatus was used to determine the crude lipid content with chloroform–methanol extraction. Gross energy was measured using a bomb calorimeter. B. velezensis was added to the basic diet according to Toften and Jobling [28] and stored at 4°C until use. In summary, B. velezensis was mixed with sterile saltwater to form suspensions. Diets were prepared by sprinkling the feed with water suspensions containing B. velezensis at different concentrations according to Chen et al. [25] (1 × 107 CFU/g, 1 × 108 CFU/g, and G3: 1 × 109 CFU/g), mixing well, and allowing the feed to dry in a cool dry place. An equivalent quantity of sterile saltwater was added to the diet of the control group. The plate count method using tryptic soyagar plates was used to verify the bacterial load in the diet. To ensure high probiotic levels in the supplemental diet, it was made on a weekly basis.
Experimental design
Obviously healthy 500 L. vannamei, nearly of the same size, were obtained from a private farm in Port Said Governorate, Egypt, (Elshamy farm), where the research was conducted. Informed consent was obtained from the farm’s owner before conducting the trial on the shrimp. Shrimp were acclimatized for 2 weeks in outdoor 1000-L fibreglass tanks and fed a basal diet without probiotics. After adaptation, 300 shrimp with an average initial body weight of 3 ± 0.4 g/shrimp were randomly allocated into four groups in triplicate in 1 m3 fibreglass tanks (25 shrimp per tank). L. vannamei fed diets supplemented with four different levels of B. velezensis (CG: 0 CFU/g as a control group; G1: 1 × 107 CFU/g; G2: 1 × 108 CFU/g; and G3: 1 × 109 CFU/g) for 60 successive days as a feeding trial. Shrimp were fed a diet at 6% of its weight their weight within the first 30 days and 4.5% within the next 30 days. Feeding rates were altered every two weeks as shrimp body weights changed. The provided feed was divided into four equal portions and given to the shrimp four times per day (06:00, 12:00, 17:00, and 22:00). Every other day, over 20% of the tank water was exchanged. The cumulative mortality rate of shrimp was recorded daily. Water quality metrics were measured using HANNA instruments (model HI 9829 – Multiparameter, USA) and maintained throughout the study period at: temperature, 27.5–31.1 °C; pH, 8–8.4; dissolved oxygen, 5.1–6.2 ppm; and salinity, 39.9–41.7 ppt. HANNA instrument 733 Ammonia High Range was used to measure ammonia to be 0–0.01 ppm during the study period. Four times a day, the tanks were cleared of unfed diets and shrimp poop.
Extraction of hemolymph, serum and plasma from L. vannamei
Hemolymphs were collected from 5 L. Vannamei in each group. Individual shrimp hemolymph (100 μL) was extracted from the pleopod base of the first abdominal segment using a sterile 1-mL syringe (25 G × 13 mm needle) without anticoagulant. Next, the hemolymph was refrigerated at 4°C for 2 h before centrifugation at 10,000 rpm for 15 min to obtain serum. The serum was kept at -20 °C for further examination [29].
Another sample of hemolymph from 5 L. vannamei / group was withdrawn in a sterile 1-mL syringe loaded with a precooled (4 °C) solution (SIC-EDTA, Na2) (450 mM NaCl, 10 mM KCl, 10 mM hepes, and 10 mM EDTA, Na2 at pH 7.3) as an anticoagulant [30]. Individual eppendorf tubes were used to retain the hemolymph, which was kept on ice to separate the plasma. Samples of hemolymph were immediately centrifuged at 800 rpm for 10 min at 4 °C, and the plasma was frozen at − 80 °C.
Growth performance and survival rate
After the feeding trial, the remaining L. vannamei were counted, and all growth parameters, such as initial body weight (g), final body weight (FBW), weight gain rate (WG%), specific growth rate (SGR), feed conversion rate (FCR), and survival rate (SR), were calculated as follows, according to Tekinay and Davies [31]:
Determination of proximate body composition of L. vannamei
Following a 60-day feeding study, five randomly selected L. vannamei, virtually identical in size, were held at − 20℃ to assess their contents of moisture, dry matter DM, protein, lipid, growth energy GE (Kcal/g), and ash, as per Cunniff and AOAC [27].
Digestive enzyme activity
The Gastrointestinal tissues were homogenized in cold PBS. GIT homogenate of L. vannamei was centrifuged at 18,894 rpm at 4 °C for 5 min, and the supernatant was carefully separated to analyze different digestive enzymes. Lipase was determined using kits from Spectrum Company for Biotechnology, Egypt, slightly modifying the procedure outlined by Moss and Henderson [32]. To put it briefly, lipase splits a synthetic substrate (DGMRE) to produce the colorful end product, methylresorufin. The increasing absorbance of red methylresorufin was measured colorimetrically at a wavelength of 580 nm [32]. Amylase was determined using starch as substrate; in which 2 flasks (test and control) were used. In each flask, 5 ml of starch was added and placed in a water bath at 37 °C for 5 min. In the test flask, 0.1 ml of enzyme extract was added and mixed well, whereas no addition was made in the control flask; the mixture was mixed well and left for 7.5 min. The 2 flasks removed from water bath and added 5 ml of working iodine solution to each flask, diluted to 50 ml with water, and mixed well. At a wavelength of 660 nm, the result was measured colorimetrically [33]. Proteases were determined using a protease activity assay kit (Fluorometric—Green) (ab112152) (Abcam Co, UK), in which casein conjugate served as a generic substrate. A green fluorescent dye was used to label the casein, which significantly quenched the fluorescence. Fluorescence intensity increased in a direct proportion with protease activity. With the FITC filter set, the signal was easily observed at Ex/Em = 490/525 nm using a fluorescent microplate reader [34].
Antioxidant, immune response and biochemical parameters
A bead homogenizer was used to homogenize several samples, each comprising one part of the hepatopancreas and nine parts of 0.9% saline, in an ice-filled container for 10 min. After centrifugation for 10 min at 4 °C and 3,500 rpm, the supernatant was collected and stored at -80 °C to be used for the detection of superoxide dismutase (SOD), catalase CAT, and lipid peroxide [11]. According to Nishikimi et al. [35], Satoh [36], and Aebi [37], superoxide dismutase (SOD), lipid peroxide (Malondialdehyde) (MDA), and catalase were assessed colorimetrically using kits from Biodiagnostic Co., Egypt at wave lengths of 560, 510, and 534 nm, respectively.
Following the manufacturer’s instructions, a fish lysozyme ELISA kit (Sunlong Biotech Co., China) was used to measure serum lysozyme activity using the ELISA micro-well technique at a wavelength of 450 nm using a microplate ELISA reader. Using a commercial kit (Sunlong Biotech Co., China) and the manufacturer’s instructions, immunoglobulin M (IgM) was quantified by ELISA.
Iida et al. [38] provided a methodology for determining the bactericidal activity BA%, which involved diluting serum samples three, four, and five times in Tris buffer (pH 7.5). The diluted solutions were treated with a bacterial solution (0.001 g/mL, Aeromonas hydrophila) for 24 h at 25 °C. Reaction solutions in 50 µL were incubated on TSA for a whole day at 25 °C. The colony-forming unit (CFU) was computed using the plate counting method. A survival index (SI) was created using the data [39] according to the following formula:
Department of Fish Health and Management, Central Laboratory for Aquaculture Research, Egypt kindly provided the Aeromonas hydrophila strain (1.7 × 106 CFU) for the serum bactericidal activity test.
From the collected L. vannamei serum, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were measured using kits (Biodiagnostic Co., Egypt) according to Reitman and Frankel [40]. Triglycerides, cholesterol, HDL-cholesterol, and LDL-cholesterol were also tested using kits from Biodiagnostic Co., Egypt, according to Fossati and Prencipe [41], Richmond [42], Burstein et al. [43], and Wieland and Seidel [44], respectively.
Using kits from Biodiagnostic Co., Egypt, alkaline phosphatase (AKP) and acid phosphatase (ACP) (Biodiagnostic Co., Egypt) were quantified colorimetrically at a wavelength of 510 nm in accordance with Belfield and Goldberg [45] and Kind and King [46], respectively.
Total RNA extraction, cDNA synthesis, and real-time quantitative PCR analysis of antioxidant and immune-related genes
Total RNA was extracted from 50 mg of L. vannamei hepatopancreatic tissues using trizol (iNtRON Biotechnology) according to the manufacturer’s instructions. The integrity of RNA was confirmed using 2% agarose gel electrophoresis with ethidium bromide. The concentration and purity of RNA were determined using a Nanodrop BioDrop spectrophotometer (Biochrom Ltd, Cambridge CB23 6DW, UK) based on the A260/A280 nm ratio. Two μg of RNA sample were reverse transcribed using the ABT 2X RT Mix cDNA synthesis kit according to manufacturer’s Protocol. Gene expression profiling was performed in Rotor Gene-Q (Qiagen-Germany) using gene-specific primer sequences for the amplification of the antioxidation-related gene (SOD) and immune genes serine proteinase and lysozyme (LZM) genes (Table 2). The amplification reaction was performed using an ABT 2X qPCR Mix (SYBR) kit. The reaction volumes was 20 μl consisting of 10 µL SYBR Green, 0.6 µL of forward and reverse specific primers, 1 µL of cDNA template, and nuclease-free water, to make a final volume 20 µL. The PCR program was performed with the following conditions: activation at 95 °C for 15 min, followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at the primer-specific temperature for 15 s, and extension at 72 °C for 25 s. This was followed by a melt curve analysis to assess the specificity of amplification at 72 °C to 95 °C. All genes were tested in triplicate. CT values for each sample were determined and incorporated into “fold change” (2 − ΔΔCT), calculation based on Livak and Schmittgen [47], and mRNA expressions for each sample were normalized against beta actin as a housekeeping gene.
Histological examination
L. vannamei hepatopancreas (3 samples) were collected from each group, fixed for 24 h in Davidson’s fixative, and then transferred to 70% ethanol. Subsequently, they were passed through ascending grades of ethyl alcohol for dehydration and then cleared in xylene. Cleared tissues were embedded in paraffin wax. The paraffin blocks were sectioned into 5µm-thick sections and stained with H&E according to Bell and Lightner [54]. The numbers of E, B, and R cells were counted using ImageJ software with a cell counter plugin in 20 randomly chosen tubules from each treatment according to Abd El-Naby et al. [34].
Statistical analysis
The data were subjected to one-way ANOVA using IBM SPSS Statistics (Version 22), and the results were displayed as mean ± standard error (SE). Significant differences between the examined groups were determined using a multiple-range test [55]. Differences between groups were considered statistically significant at P < 0.05. For each parameter, values in the same row with distinct superscript letters (a, b, c, and d) indicate significantly different mean values.
Results
Bacillus velezensis safety
The application of different concentrations of B. velezensis in L. vannamei diets did not cause any abnormal activity, behavior, or mortality in this shrimp species.
Growth performance, proximate body composition and digestive enzymes
The growth performance and survival rate of test diets fed to L. vannamei and supplemented with varying amounts of B. velezensis for 60 days are presented in Table 3. Although only the G3 and G2 groups showed a significant increase in the specific growth rate compared with the control, all B. velezensis-supplemented groups had significantly higher final body weight, weight gain rate, and survival rate. Compared with the control group fed a diet free of B. velezensis, the L. vannamei feed conversion ratio was significantly improved after B. velezensis supplementation. Among the supplemented groups, L. vannamei fed a diet containing 1 × 109 CFU/g of B. velezensis exhibited the best overall growth performance (final body weight, weight gain rate, specific growth rate, and feed conversion ratio).
The proximate body composition of L. vannamei-fed test diets supplemented with varying amounts of B. velezensis for 60 days was significantly improved compared to the control. All groups fed the studied diets, especially G3, showed significant increases (P < 0.05) in DM, protein, and GE compared with the control group. The B. velezensis-supplemented diet groups had considerably lower moisture, fat content, and ash content than the control group (P < 0.05) (Table 4).
All groups fed B. velezensis diets showed a significant (P < 0.05) improvement in the digestive enzymes of L. vannamei GIT (lipase, amylase, and protease) compared with the control group; G3 was the highest in all groups (Table 5).
Antioxidant enzymes and immune variables
The antioxidant enzyme SOD was significantly enhanced (P < 0.05) in G3 only compared with the other groups; while CAT showed a significant increase (P < 0.05) in all groups fed the BV diet compared with the control group. MDA showed a significant decrease (P < 0.05) in all groups fed the B. velezensis diet compared with the control group, especially G3, G2, and G1 in succession.
The lysozyme activities of G3 and G2 were significantly higher (P < 0.05) than those of G1 and the control group as immune response biomarkers. Compared with the control group, all groups fed B. velezensis diets had a substantial increase (P < 0.05) in IgM, with G2 exhibiting the highest value. In all groups fed B. velezensis diets, BA% increased significantly (P < 0.05) compared with the control group (Table 6).
Biochemical parameters
The addition of dietary B. velezensis at varying concentrations to the diet of L. vannamei resulted in a significant decrease in the activity of enzymes linked to hepatopancreatic function, including AST and ALT. AKP and ACP levels were significantly increased in all groups fed the test diets compared with the control group (Table 7).
Lipids profile
Regarding the lipid profile presented in Table 8, all groups of L. vannamei fed B. velezensis diets had a significant (P < 0.05) decreases in triglycerides, cholesterol, high-density lipoprotein, and low-density lipoprotein compared with the control group. A dose-dependent decrease in cholesterol and triglyceride levels was observed.
Hepatopancreatic antioxidant
Hepatopancreatic antioxidant gene expression (Fig. 1) showed no significant difference in SOD mRNA levels between the control, G1, and G2 groups. However, G3 cells exhibited significantly higher (P < 0.05) SOD expression compared to these groups. On the other hand, in the hepatopancreatic immune response for LZM and serine proteinase genes, G3 had the highest expression level, followed by G2, whereas G1 lacked a statistically significant difference in gene expression compared with the control group. Interestingly, in this study, the hepatopancreatic SOD and serum lysozyme activities using the diagnostic kits, as well as the transcript levels of these genes in the hepatopancreas, were identical.
Histological examination
The hepatopancreas of L. vannamei fed B. velezensis-supplemented diets did not exhibit any histopathologic changes. However, L. vannamei fed the control diet showed a significant increase in the number of strongly basophilic cuboidal embryonic cells (E) with large centrally located nuclei compared with the B. velezensis-supplemented groups (P < 0.05). In contrast, the B. velezensis-supplemented groups displayed a significant increase (P < 0.05) in large globular-shaped vesicular secreting cells (B) with basal nuclei and large apical vacuoles, large reabsorption cells (R) with round basal nuclei and multivacuolar granular cytoplasm, as well as star-shaped and polygonal liver tubules (Fig. 2 and Table 9).
Discussion
Nowadays, natural compounds like probiotics that boost growth, antioxidant capacity, and immune responses have received much attention in L. vannamei aquaculture, particularly in Egypt. Moreover, B. velezensis has not received as much attention as other species in its family [21, 23, 53, 56]. Accordingly, this study was interested in the locally isolated probiotic B. velezensis, which has been studied as a natural feed additive for L. vannamei.
Feeding different doses of B. velezensis did not cause mortality in L. vannamei, indicating that this strain is safe for the species. Chen et al. [25] and Yang et al. [14] also showed that B. velezensis was not toxic to L. vannamei.
According to the current study, L. vannamei improved feed utilization, survival rate, proximate body composition, digestive enzyme activities, antioxidants, and immune response when fed B. velezensis-enriched diets containing 1 × 109 CFU/g for eight weeks which reflected in increased growth performance, WG rate and SGR, as well as decreased FCR compared with the control group. The improved growth performance observed in this study may be a result of Bacillus species having been shown to improve the secretion of digestive enzymes, increase the digestibility and absorption of nutrients, increase fish appetite by producing vitamins and organic acids, produce certain essential micronutrients, detoxify harmful compounds in the diet, secrete several metabolites with antimicrobial properties, and suppress pathogenic microorganisms that reduce growth [22, 57,58,59,60,61,62,63]. Chen et al. [25] and Zhang et al. [64] reported similar findings in L. vannamei and Amur minnow Rhynchocypris lagowskii, respectively. Furthermore, Ji et al. [65] reported that B. velezensis YFI-E109 contained genes involved in metabolic regulation that facilitate amino acid and carbohydrate transport and metabolism, thereby enhancing hybrid yellow catfish growth.
Several factors influence the proximate body composition of aquatic animals, including age, sex, season, shrimp feed, density, and water quality [66]. All groups fed B. velezensis diets demonstrated a significant rise in DM, protein, and GE but lower moisture, fat content, and ash content than the control group. Protein is the most essential biochemical component of shrimp bodies, and its content is influenced by diet digestibility [66]. The differences in body composition between L. vannamei-fed B. velezensis and the control group indicate improvements in the nutritional value of L. vannamei-fed B. velezensis.
Digestive enzyme activity can help understand the digestive physiology and nutritional requirements of white shrimp [67]. This study showed that digestive enzymes, including amylase, lipase, and protease, were significantly increased in L. vannamei fed B. velezensis diets compared with the control group. Increased digestive enzyme levels reflect improved growth performance of L. vannamei. Numerous studies have also demonstrated the same result; Yang et al. [14] reported enhancements in lipase and α-amylase activities in L. vannamei fed with B. velezensis at feed concentrations of 0.3 and 0.4 g/kg. Wang [68] observed similar outcomes in L. vannamei-fed probiotics, including Bacillus sp. and photosynthetic bacteria. Amoah et al. [17] reported the same results in L. vannamei-fed diets supplemented with B. coagulans. Probiotic strains, particularly Bacillus sp., are renowned for their ability to synthesize a wide array of exoenzymes that improve diet digestibility and increase the burden of beneficial bacteria linked with probiotics used in L. vannamei [21, 25]. Therefore, higher enzymatic activity improves digestibility and maximizes feed use.
Reactive oxygen species (ROS) are natural metabolites of ordinary cellular metabolism [69]. They are created and negated at equilibrium; however, any stressor can upset this equilibrium, leading to toxic and harmful effects in live cells [70]. Living cells counteract these effects and preserve their equilibrium via a variety of defensive systems, including antioxidant defense mechanisms [71, 72]. On the other hand, measuring the antioxidant enzymes SOD and CAT can indicate the oxidative stress status and antioxidant capacity of aquatic organisms [18, 73,74,75]. According to our findings, the G3 group and all other groups fed the B. velezensis diet had higher SOD and CAT activities than the control group. This study revealed that the B. velezensis diet had stronger hepatopancreatic anti-oxidative effects on L. vannamei as those reported by Yang et al. [14] and Chen et al. [25], who observed elevated SOD and CAT levels in L. vannamei fed the B. velezensis diet. SODs are very effective at reducing oxidative stress because they eliminate excess reactive oxygen species (ROS) by degrading excess superoxide radicals, producing oxygen and hydrogen peroxide, and thus halting free radical damage to cells [76,77,78]. CAT can eliminate hydrogen peroxide, which is produced by SODs, from cells producing molecular oxygen and water [79]. In the current study, B. velezensis exhibited antioxidative activity by upregulating antioxidative gene expression; SOD (in G3) in addition to two immune-related genes; Serine proteinase and LZM (in G3 and G2) in L. vannamei. Similar results were reported by Chen et al. [25], who reported a significant increase in SOD expression in L. vannamei fed B. velezensis- enriched diets at 107 CFU/g (BV2) and 109 CFU/g concentrations. Shrimp depend entirely on innate immune responses against pathogenic infections through cellular and humoral responses, such as prophenoloxidase (proPO) activation, toll pathway initiation, hemolymph coagulation, complement activation, melanization, phagocytosis, encapsulation, and antimicrobial peptide synthesis interceded by serine proteinase cascades [80,81,82]. Serine proteinase (SP) is an essential proteolytic enzyme involved in various physiological processes, including digestion, blood coagulation, embryonic development, and immune response [83]. Thus, B. velezensis has a positive impact on the expression of serine proteinase in L. vannamei, which improves digestive and immune responses. LZM is an antimicrobial peptide that is produced primarily by shrimp hemocytes and plays an important role in the innate immune response of shrimp to various microbial infections [84,85,86]. LZM is mainly expressed in the hepatopancreas [87]. To the best of our knowledge; this is the first study to address the effects of B. velezensis on the expression of the serine proteinase and LZM genes in L. vannamei.
The non-enzymatic antioxidant MDA is a lipid peroxidation product that can detect the body's lipid oxidative stress [87, 88]. The hepatopancreatic MDA level of L. vannamei-fed B. velezensis diets was much lower than that of shrimp fed the control diet, indicating that B. velezensis did not cause oxidative stress and may even reduce it. In accordance, Amoah et al. [17] found decreased levels of MDA in L. vannamei-fed B. coagulans-enriched diets.
Lysozyme is a functional antimicrobial protein and key component of shrimp innate immunity that contributes significantly to host responses to infectious agents [7, 89,90,91,92]. Lysozyme activity in L. vannamei-fed B. velezensis diets was significantly higher in G3 and G2 compared to G1 and the control group. Similarly, prior studies found that L. vannamei fed B. coagulans-enriched diets exhibited increased lysozyme activity compared with the control diet [17].
Shrimp lacks an acquired immune system and relies mostly on innate immunological responses via cellular and humoral mechanisms [93,94,95]. The primary humoral reaction involves IgM release to destroy invading microorganisms. Our study found a rise in IgM levels in L. vannamei fed B. velezensis diets, indicating an enhanced immunological state. In fish, IgM is the first antibody generated during infection and plays a crucial role in systemic and mucosal immune tissues [96]. B. velezensis enhances IgM gene expression in grass carp [28]. Similarly, B. velezensis was among the three Bacillus species that increased IgM levels in Oreochromis niloticus (Kuebutornye et al. 2020). Rhynchocypris lagowskii fed on B. velezensis had elevated IgM levels [64].
In this study, B. velezensis exhibited potent bactericidal activities compared with the control group, which modulated the immune response of L. vannamei, thereby increasing disease resistance. This could be a result of several products produced by B. velezensis with antibacterial effects, such as bacteriocin produced by B. velezensis strain BUU004 isolated from shrimp pond sediment [97, 98].
AST and ALT enzymes reflect the health status of the shrimp hepatopancreas [99]. Dietary B. velezensis decreased the activities of these two enzymes in L. vannamei, indicating improved function of the hepatopancreas. The results obtained were identical to those reported by Chen et al. [25] and Yang et al. [14].
Crustaceans’ immune systems rely primarily on phosphorylation and dephosphorylation, both of which require AKP and ACP [100]. Furthermore, AKP is a regulatory enzyme involved in metabolism and phagolysis, whereas ACP is a key component of lysozyme enzymes that destroy pathogens in invertebrates [101, 102]. In this study, all groups fed the B. velezensis diet had higher AKP and ACP levels than the control group. Chen et al. [25] reported similar results for L. vannamei fed a B. velezensis-rich diet. Amoah et al. [17] also found an increase in ACP in L. vannamei-fed diets enriched with B. coagulans.
In the current study, all groups of L. vannamei fed a B. velezensis-enriched diet showed significant decreases in triglycerides, cholesterol, high-density lipoprotein, and low-density lipoprotein compared with the control group, suggesting that B. velezensis may play a vital role in regulating lipid metabolism. Lipid metabolism is positively correlated with blood triglyceride levels [103]. B. velezensis YFI-E109 in diets enhanced lipid utilization and reduced lipid deposition in fish, resulting in decreased triglyceride levels in hybrid yellow catfish [104]. In the same regard, Amoah et al. [17] reported lower triglyceride levels in L. vannamei-fed B. coagulans-enriched diets. Lee et al. [60] reported that Bacillus strains can synthesize vitamins and thus lower cholesterol levels.
The hepatopancreas is a vital organ in shrimp that regulates several functions of the digestive system, including steroid hormone production, digestive enzyme secretion, carbohydrate and fat metabolism, and nutrient absorption, distribution, and storage. In addition, the hepatopancreas is the main detoxification organ of shrimp [34, 105, 106].
In this study, groups of L. vannamei fed B. velezensis-enriched diets showed higher numbers of B cells (secreting cells; the main producer of digestive enzymes) and R cells (absorption, lipid and glycogen storage cells), which are involved in the digestion, absorption, and storage of nutrients, which in turn indicated the improvement in growth performance in these groups compared with the control group, which had an increase in undifferentiated embryonic cells (E). The above result is consistent with those of Chen et al. [19], in addition to García-Bernal et al. [26], who reported an increase in B cells in L. vannamei fed with Streptomyces probiotics relative to the control group.
Conclusion
In summary, the current study indicated that Bacillus velezensis is a promising probiotic that can be safely added to the diet of Litopenaeus vannamei with 1 × 109 CFU/g. Its application had a positive influence on the health status, survival rate, nutritional value, and immunity of L. vannamei.
Availability of data and materials
The data sets used in the present study are accessible on reasonable request from the corresponding author.
References
Toledo A, Frizzo L, Signorini M, Bossier P, Arenal A. Impact of probiotics on growth performance and shrimp survival: a meta-analysis. Aquaculture. 2019;500:196–205.
Barreto A, Peixoto D, Fajardo C, Pinto W, Rocha RJM, Conceição LEC, et al. Health-promoting additives supplemented in inert microdiets for whiteleg shrimp (Penaeus vannamei) post-larvae: effects on growth, survival, and health status. Animals. 2023;13:726.
Rattanadilog Na Phuket T, Charoensapsri W, Amparyup P, Imjongjirak C. Antibacterial activity and immunomodulatory role of a proline-rich antimicrobial peptide SpPR-AMP1 against Vibrio campbellii infection in shrimp Litopenaeus vannamei. Fish Shellfish Immunol. 2023;132:108479.
FAO. Penaeus vannamei. Cultured Aquatic Species Information Programme. Text by Briggs, M. In: Fisheries and Aquaculture. Rome. Updated 2006–08–17. https://www.fao.org/fishery/en/culturedspecies/penaeus_vannamei/en (2024). Accessed 20 Jan 2024.
Amiin MK, Lahay AF, Putriani RB, Reza M, Putri SME, Sumon MAA, et al. The role of probiotics in Vannamei shrimp aquaculture performance – a review. Veterinary World. 2023;16:638–49.
Liu Y, Zhuang Z, Liao Z, Yao R, Chen M, Wei H. Effects of low-fish-meal diet supplemented with coenzyme q10 on growth performance, antioxidant capacity, intestinal morphology, immunity and hypoxic resistance of Litopenaeus vannamei. Antioxidants. 2023;12:2042.
Yudiati E, Widiasa IN, Sunaryo S, Ridhuwan R, Tadeus DY, Arifin Z, Mangkusasmito F, Sugiyanto S, Setyawan DA. Oral supplementation of low alginate dose in diet stimulates immune response of Litopenaeus vannamei at concrete circle pond mass culture. IOP Conference Series: Earth and Environmental Science. 2023. https://doi.org/10.1088/1755-1315/1260/1/012006.
Dawood M, Abo-Al-Ela HG, Hasan MT. Modulation of transcriptomic profile in aquatic animals: probiotics, prebiotics and synbiotics scenarios. Fish Shellfish Immunol. 2020;97:268–82.
Ahmadifar E, Moghadam MS, Dawood MAO, Hoseinifar SH. Lactobacillus fermentum and/or ferulic acid improved the immune responses, antioxidative defence and resistance against Aeromonas hydrophila in common carp (Cyprinus carpio) fingerlings. Fish Shellfish Immunol. 2019;94:916–23.
Sadeghi FR, Ahmadifar E, Shahriari Moghadam M, Ghiyasi M, Dawood MA, Yılmaz S. Lemon, Citrus aurantifolia, peel and Bacillus licheniformis protected common carp, Cyprinus carpio, from Aeromonas hydrophila infection by improving the humoral and skin mucosal immunity, and antioxidative responses. J World Aquaculture Soc. 2020;52:124–37.
Yousefi M, Ahmadifar M, Mohammadzadeh S, Kalhor N, Eslimi Esfahani D, Bagheri A, et al. Individual and combined effects of the dietary Spirulina platensis and Bacillus licheniformis supplementation on growth performance, antioxidant capacity, innate immunity, relative gene expression and resistance of goldfish, Carassius auratus to Aeromonas hydrophila. Fish Shellfish Immunol. 2022;127:1070–8.
Hoseinifar SH, Sun Y-Z, Wang A, Zhou Z. Probiotics as means of diseases control in aquaculture, a review of current knowledge and future perspectives. Front Microbiol. 2018;9:2429.
Yılmaz S, Yılmaz EŞ, Dawood MA, Ringø E, Ahmadifar E, Abdel-Latif HM. Probiotics, prebiotics, and synbiotics used to control vibriosis in fish: a review. Aquaculture. 2022;547:737514.
Yang H, Du D, Zhang Q, Teame T, Wang A, Hao Q, et al. Dietary Bacillus velezensis T23 fermented products supplementation improves growth, hepatopancreas and intestine health of Litopenaeus vannamei. Fish shellfish immunol. 2024;149:109595.
Fadl SE, Elsabagh M, El- Habashi NM, Shehab El-Din MT. Some studies on Pediococcus acidilactici as a feed additive in tilapia finger lings. Egyptian J Nutrition and Feeds. 2013;16:351–64.
Kavitha M, Raja M, Perumal P, Evaluation of probiotic potential of bacillus spp. isolated from the digestive tract of freshwater fish Labeo calbasu (Hamilton,. Aquac. Reports. 1822;2018(11):59–69.
Amoah K, Huang QC, Tan BP, Zhang S, Chi SY, Yang QH, et al. Dietary supplementation of probiotic Bacillus coagulans ATCC 7050, improves the growth performance, intestinal morphology, microflora, immune response, and disease confrontation of Pacific white shrimp. Litopenaeus vannamei Fish Shellfish Immunol. 2019;87:796–808.
Dawood MAO, Moustafa EM, Gewaily MS, Abdo SE, AbdEl-kader MF, SaadAllah MS, et al. Ameliorative effects of Lactobacillus plantarum L-137 on Nile tilapia (Oreochromis niloticus) exposed to deltamethrin toxicity in rearing water. Aquat Toxicol. 2020;219:105377.
Kuebutornye FKA, Tang J, Cai J, Yu H, Wang Z, Abarike ED, et al. In vivo assessment of the probiotic potentials of three host -associated bacillus species on growth performance, health status and disease resistance of Oreochromis niloticus against Streptococcus agalactiae. Aquaculture. 2020;527:735440.
Khalid F, Khalid A, Fu Y, Hu Q, Zheng Y, Khan S, et al. Potential of Bacillus velezensis as a probiotic in animal feed: a review. J Microbiol. 2021;59:627–33.
Monier MN, Kabary H, Elfeky A, Saadony S, Abd El-Hamed N, Eissa M, et al. The effects of Bacillus species probiotics (Bacillus subtilis and B. licheniformis) on the water quality, immune responses, and resistance of whiteleg shrimp (Litopenaeus vannamei) against Fusarium solani infection. Aquacult Int. 2023;31:3437–55.
Reda RM, Selim KM. Evaluation of Bacillus amyloliquefaciens on the growth performance, intestinal morphology, hematology and body composition of Nile tilapia. Oreochromis niloticus Aquaculture International. 2015;23:203–17.
El-Barbary Y, Gaafar A, Younes A, El-Ashram A. The influence of continuous and intermittent Bacillus subtilis AQUA-GROW® application on the white leg shrimp, Litopenaeus vannamei, immune-related genes. Egyptian Journal of Aquatic Biology & Fisheries. 2021;25:241–61.
Wu Z, Qi X, Qu Sh, Ling F, Wang G. Dietary supplementation of Bacillus velezensis B8 enhances immune response and resistance against Aeromonas veronii in grass carp. Fish Shellfish Immunol. 2021;115:14–21.
Chen L, Lv C, Li B, Zhang H, Ren L, Zhang Q, et al. Effects of Bacillus velezensis supplementation on the growth performance, immune responses, and intestine microbiota of Litopenaeus vannamei. Front Mar Sci. 2021;8:744281.
García-Bernal M, Medina-Marrero R, Rodríguez‐Jaramillo C, Marrero-Chang O, Campa-Córdova ÁI, Medina-García R, et al. Probiotic effect of Streptomyces spp. on shrimp (Litopenaeus vannamei) postlarvae challenged with Vibrio parahaemolyticus. Aquaculture Nutr. 2018;24:865–71.
Cunniff P, AOAC (Association of Official Analytical Chemists). Official methods of analysis of AOAC international. 16th ed. Washington, DC: Association of Official Analytical Chemists; 1995.
Toften H, Jobling M. Feed intake and growth of Atlantic salmon, Salmo salar L., fed diets supplemented with oxytetracycline and squid extract. Aquac Nutr. 1997;3:145–51.
Xu B, Zhang G, Wang L, Sagada G, Zhang J, Shao Q. The influence of dietary β-1,3- glucan on growth performance, feed utilization, antioxidative and immune status of Pacific white shrimp. Litopenaeus vannamei Aquaculture Nutrition. 2021;27:1590–601.
Vargas-Albores F, Guzmán-Murillo MA, Ochoa JL. An anticoagulant solution for haemolymph collection and prophenoloxidase studies of Penaeid shrimp (Penaeus californiensis). Comp Biochem Phys. 1993;A 106:299–303.
Tekinay S, Davies AA. Dietary carbohydrate level influencing feed intake, nutrient utilisation and plasma glucose concentration in the rainbow trout, Oncorhynchus mykiss. Turk J Vet Anim Sci. 2001;25:657–66.
Moss DW, Henderson AR. Digestive enzymes of pancreatic origin. In: Burtis CA, Ashwood ER, editors. Tietz Textbook of Clinical Chemistry. 3rd ed. Philadelphia: W.B Saunders Company; 1999. p. 689–708.
Caraway WT. A stable starch substrate for the determination of amylase in serum and other body fluids. Am J Clin Pathol. 1959;32:97–9.
Abd El-Naby AS, Eid AE, Gaafar AY, Sharawy Z, Khattaby AA, El-sharawy MS, et al. Overall evaluation of the replacement of fermented soybean to fish meal in juvenile white shrimp, Litopenaeus vannamei diet: growth, health status, and hepatopancreas histomorphology. Aquacult Int. 2023;32:1665–83.
Nishikimi M, Appaji N, Yagi K. The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen. Biochem Biophys Res Commun. 1972;46:849–54.
Satoh K. Serum lipid peroxide in cerebrovascular disorders determined by a new colorimetric method. Clin Chim Acta. 1978;90:37–43.
Aebi H. Catalase in vitro. Methods Enzymol. 1984;105:121–6.
Iida T, Takahashi T, Wakabayashi H. Decrease in the bactericidal activity of normal serum during the spawning period of rainbow trout. Nippon Suisan Gakkaishi. 1989;55:463–5.
Wardlaw AC, Unkles ShE. Bactericidal activity of coelomic fluid from the sea urchin Echinus Esculentus. J Invertebr Pathol. 1978;32:25–34.
Reitman S, Frankel S. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am J Clin Pathol. 1957;28:56–63.
Fossati P, Prencipe L. Serum triglycerides determined colorimetrically with an enzyme that produces hydrogen peroxide. Clin Chem. 1982;28:2077–80.
Richmond W. Preparation and properties of a cholesterol oxidase from Nocardia sp. and its application to the enzymatic assay of total cholesterol in serum. Clin Chem. 1973;19:1350–6.
Burstein M, Scholnick HR, Morfin R. Rapid method for the isolation of lipoproteins from human serum by precipitation with polyanions. J Lipid Res. 1970;11:583–95.
Wieland H, Seidel D. A simple specific method for precipitation of low density lipoproteins. J Lipid Res. 1983;24:904–9.
Belfield A, Goldberg DM. Revised assay for serum phenyl phosphatase activity using 4-amino-antipyrine. Enzyme. 1997;12:561–73.
Kind PR, King EJ. Estimation of plasma phosphatase by determination of hydrolysed phenol with amino-antipyrine. J Clin Pathol. 1954;7:322–6.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C (T)) Method. Methods. 2001;25(4):402–8.
Flores-Miranda MDC, Luna-González A, Cortés-Espinosa DV, Álvarez-Ruiz P, Cortés-Jacinto E, Valdez-González FJ, et al. Effects of diets with fermented duckweed (Lemna sp.) on growth performance and gene expression in the pacific white shrimp, Litopenaeus vannamei. Aqua Int. 2015;23:547–61.
Fierro Coronado JA, Luna González A, Caceres Martínez CJ, Álvarez Ruiz P, Escamilla-Montes R, González Ocampo HA, et al. Effect of microbial immunostimulants on WSSV infection percentage and the expression of immune-related genes in white shrimp (Litopenaeus vannamei). Revista Colombiana De Ciencias Pecuarias. 2019;32:221–31.
Wang YC, Chang PS, Chen HY. Differential time-series expression of immune-related genes of Pacific white shrimp Litopenaeus vannamei in response to dietary inclusion of β-1, 3-glucan. Fish Shellfish Immunol. 2008;24:113–21.
Han-Ching Wang K, Tseng CW, Lin HY, Chen IT, Chen YH, Chen YM, et al. RNAi knock-down of the Litopenaeus vannamei Toll gene (LvToll) significantly increases mortality and reduces bacterial clearance after challenge with Vibrio harveyi. Dev Comp Immunol. 2010;34:49–58.
Jiménez-Vega F, Vargas-Albores F, Söderhäll K. Characterisation of a serine proteinase from Penaeus vannamei haemocytes. Fish Shellfish Immunol. 2005;18:101–8.
Zokaeifar H, Balcázar JL, Saad CR, Kamarudin MS, Sijam K, Arshad A, et al. Effects of Bacillus subtilis on the growth performance, digestive enzymes, immune gene expression and disease resistance of white shrimp, Litopenaeus vannamei. Fish Shellfish Immunol. 2012;33:683–9.
Bell TA, Lightner DV. A handbook of normal penaeid shrimp histology. USA: World Aquaculture Society; 1988.
Duncan DB. Multiple Range and Multiple F Tests. Biometrics. 1955;11:1–42.
Interaminense JA, Vogeley JL, Gouveia CK, Portela RS, Oliveira JP, Silva SMBC, et al. Effects of dietary Bacillus subtilis and Shewanella algae in expression profile of immune-related genes from hemolymph of Litopenaeus vannamei challenged with Vibrio parahaemolyticus. Fish Shellfish Immunol. 2019;86:253–9.
Irianto A, Austin B. Probiotics in aquaculture. J Fish Dis. 2002;25:633–42.
Chen XH, Koumoutsi A, Scholz R, Schneider K, Vater J, Süssmuth R, et al. Genome analysis of Bacillus amyloliquefaciens FZB42 reveals its potential for biocontrol of plant pathogens. J Biotechnol. 2009;140:27–37.
Molinatto G, Puopolo G, Sonego P, Moretto M, Engelen K, Viti C. Complete genome sequence of Bacillus amyloliquefaciens subsp. plantarum S499, a rhizobacterium that triggers plant defenses and inhibits fungal phytopathogens. J Biotechnol. 2016;238:56–9.
Lee N, Kim W, Paik H. Bacillus strains as human probiotics: characterization, safety, microbiome, and probiotic carrier. Food Sci Biotechnol. 2019;28:1297–305.
Rabbee MF, Ali M, Choi J, Hwang BS, Jeong SC, Baek K. Bacillus velezensis: a valuable member of bioactive molecules within plant microbiomes. Molecules. 2019;24:1046.
Xie J, Liu Q, Liao S, Fang H, Yin P, Xie S, et al. Effects of dietary mixed probiotics on growth, non-specific immunity, intestinal morphology and microbiota of juvenile pacific white shrimp, Litopenaeus vannamei. Fish Shellfish Immunol. 2019;90:456–65.
Liu G, Deng Y, Cheng C, Ma H, Jiang J, Feng J, et al. Antibacterial characterization of Bacillus velezensis LG37 and mining of genes related to biosynthesis of antibacterial substances. The Israeli Journal of Aquaculture – Bamidgeh. 2022. https://doi.org/10.46989/001c.57533.
Zhang Y, Yu M, Lin L, Wang J, Zhang D, Wang Q, et al. Effects of dietary Bacillus velezensis LSG2-5 on growth, immunity, antioxidant capacity, and disease resistance of amur minnow (Rhynchocypris lagowskii Dybowski). Aquac Nutr. 2022. https://doi.org/10.1155/2022/7199145.
Ji P-F, Yao C-L, Wang Z-Y. Immune response and gene expression in shrimp (Litopenaeus vannamei) hemocytes and hepatopancreas against some pathogen-associated molecular patterns. Fish Shellfish Immunol. 2009;27:563–70.
Faghih S, Alizadeh A, Babadaei Samani R, Honarvar M, Dashtiannasab A. Effect of dietary supplementation with Gontscharovia popovii on growth performance, whole body composition, and hematological parameters in Litopenaeus vannamei. Iran J Fish Sci. 2023;22:790–808.
Jahan I, Dar Sh, Anand G, Singh Sh, Reddy AK, Sudhagar A, et al. Enzymatic alterations in Litopenaeus vannamei (Boone, 1931) juveniles exposed to different levels of dietary potassium and magnesium reared in inland saline water. Int J Curr Microbiol App Sci. 2017;6:773–80.
Wang Y-B. Effect of probiotics on growth performance and digestive enzyme activity of the shrimp Penaeus vannamei. Aquaculture. 2007;269:259–64.
Redza-Dutordoir M, Averill-Bates DA. Activation of apoptosis signaling pathways by reactive oxygen species. Biochimica Biophysica Acta. 2016;1863:2977–92.
Elbialy ZI, Salah AS, Elsheshtawy A, Elkatatny NM, Fouad AM, Abo-Al-Ela HG. Differential tissue regulation of nrf2/keap1 crosstalk in response to Aeromonas infection in Nile tilapia: a comparative study. Aquacult Int. 2024;32:545–62.
Apel K, Hirt H. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol. 2004;55:373–99.
Kurutas EB. The importance of antioxidants which play the role in cellular response against oxidative/nitrosative stress: current state. Nutr J. 2015;15:71.
Yang H, Yang M, Sun J-J, Guo F, Lan J, Wang X, et al. Catalase eliminates reactive oxygen species and influences the intestinal microbiota of shrimp. Fish Shellfish Immunol. 2015;47:63–73.
Yu Q, Fu Z, Huang M, Xu Ch, Wang X, Qin J, et al. Growth, physiological, biochemical, and molecular responses of Pacific white shrimp Litopenaeus vannamei fed different levels of dietary selenium. Aquaculture. 2021;535:736393.
Hamed M, Soliman HAM, Said REM, Martyniuk CJ, Osman AGM, Sayed AE. Oxidative stress, antioxidant defense responses, and histopathology: biomarkers for monitoring exposure to pyrogallol in Clarias gariepinus. J Environ Manage. 2024;351:119845.
Kohen R, Nyska A. Oxidation of biological systems: Oxidative stress phenomena, antioxidants, redox reactions, and methods for their quantification. Toxicol Pathol. 2002;30:620–50.
Umasuthan N, Bathige SDNK, Revathy KS, Lee Y, Whang I, Choi CY, et al. A manganese superoxide dismutase (MnSOD) from Ruditapes philippinarum: comparative structural- and expressional-analysis with copper/zinc superoxide dismutase (Cu/ZnSOD) and biochemical analysis of its antioxidant activities. Fish Shellfish Immunol. 2012;33:753–65.
Li J, Xu Y, Jin L, Li X. Effects of a probiotic mixture (Bacillus subtilis YB-1 and Bacillus cereus YB-2) on disease resistance and non-specific immunity of sea cucumber, Apostichopus japonicus (Selenka). Aquacult Res. 2015;46:3008–19.
Suryono ChA, Yudiati E, Azhar N. Immune Profile of Litopenaeus vannamei in monoculture and IMTA ponds system. Jurnal Kelautan Tropis. 2023;26:255–62.
Cerenius L, Söderhäll K. The prophenoloxidase-activating system in invertebrates. Immunol Rev. 2004;198:116–26.
Jiravanichpaisal P, Lee BL, Söderhäll K. Cell-mediated immunity in arthropods: hematopoiesis, coagulation, melanization and opsonization. Immunobiology. 2006;211:213–36.
Liu Y, Hou F, He Sh, Qian Z, Wang X, Mao A, et al. Identification, characterization and functional analysis of a serine protease inhibitor (Lvserpin) from the Pacific white shrimp. Litopenaeus vannamei Developmental and Comparative Immunology. 2014;43:35–46.
Siyu Y, Yuxin H, Jiao Y, Yichen Z, Yichen L, Xuyun G. Molecular cloning and characterization of a novel serine protease homologues (Lv-SPH) in immune response from Litopenaeus vannamei. Journal of Fisheries of China. 2022;2014(46):815–24.
Hong XP, Xu D, Zhuo Y, Liu HQ, Lu LQ. Identification and pathogenicity of Vibrio parahaemolyticus isolates and immune responses of Penaeus (Litopenaeus) vannamei (Boone). J Fish Dis. 2016;39:1085–97.
Tang T, Liu J, Li S, Li H, Liu F. Recombinant expression of an oriental river prawn anti-lipopolysaccharide factor gene in Pichia pastoris and its characteristic analysis. Fish Shellfish Immunol. 2020;98:414–9.
Matos GM, Rosa RD. On the silver jubilee of crustacean antimicrobial peptides. Rev Aquacult. 2022;11:594–612.
Trenzado C, Hidalgo MC, García-Gallego M, Morales AE, Furné M, Domezain A, et al. Antioxidant enzymes and lipid peroxidation in sturgeon Acipenser naccarii and trout Oncorhynchus mykiss: a comparative study. Aquaculture. 2006;254:758–67.
Fan J, Zhang Y, Zhou H, Liu Y, Cao Y, Dou X, et al. Dietary malondialdehyde damage to the growth performance and digestive function of hybrid grouper (Epinephelus fuscoguttatus♀ × E. lanceolatu♂). Animals (Basel). 2023;13:3145.
Lin Y, Chen J, Chen Y, Yeh S, Chen L, Huang Ch, et al. Crowding of white shrimp Litopenaeus vananmei depresses their immunity to and resistance against Vibrio alginolyticus and white spot syndrome virus. Fish Shellfish Immunol. 2015;45:104–11.
Hu F, Wang Y, Hu J, Bao Z, Wang M. A novel c-type lysozyme from Litopenaeus vannamei exhibits potent antimicrobial activity. Fish Shellfish Immunol. 2022;131:729–35.
Wu J, Tian Sh, Luo K, Zhang Y, Pan H, Zhang W, Mai K. Dietary recombinant human lysozyme improves the growth, intestinal health, immunity and disease resistance of Pacific white shrimp Litopenaeus vannamei. Fish Shellfish Immunol. 2022;121:39–52.
Shakweer MS, Elshopakey GE, Abdelwarith AA, Younis EM, Davies SJ, Elbahnaswy S. Comparison of immune response of Litopenaeus vannamei shrimp naturally infected with Vibrio species, and after being fed with florfenicol. Fishes. 2023;8:148.
Hauton C. The scope of the crustacean immune system for disease control. J Invertebr Pathol. 2012;110:251–60.
Okamura Y, Mekata T, Elshopakey GE, Itami T. Molecular characterization and gene expression analysis of hypoxia-inducible factor and its inhibitory factors in kuruma shrimp Marsupenaeus japonicus. Fish Shellfish Immunol. 2018;79:168–74.
Kulkarni A, Krishnan S, Anand D, Kokkattunivarthil Uthaman S, Otta SK, Karunasagar I, et al. Immune responses and immunoprotection in crustaceans with special reference to shrimp. Rev Aquac. 2021;13:431–59.
Xia H, Lu J, Yang P, Chen F, Zhang Y, Liu L, et al. Research Progress in Molecular Biology of Fish Immunoglobulin M (IgM). The Israeli Journal of Aquaculture – Bamidgeh• IJA. 2023. https://doi.org/10.46989/001c.73925.
Vanichkul K, Areechon N, Kongkathip N, Srisapoome P, Chuchird N. Immunological and bactericidal effects of turmeric (Curcuma longa Linn.) extract in pacific white shrimps (Litopenaeus vannamei Boone). Kasetsart J (Nat Sci). 2010;44:850–8.
Soodsawaeng P, Rattanamangkalanon N, Boonthai T, Vuthiphandchai V, Nimrat S. Bacteriocin from Bacillus velezensis BUU004 as a seafood preservative: Antibacterial potential, and physical and chemical qualities of dried, seasoned, and crushed squids. Suan Sunandha Sci & Tech [Internet]. 2023;10:105–19.
Chen S, Zhuang Z, Yin P, Chen X, Zhang Y, Tian L, et al. Changes in growth performance, haematological parameters, hepatopancreas histopathology and antioxidant status of pacific white shrimp (Litopenaeus vannamei) fed oxidized fish oil: regulation by dietary myoinositol. Fish Shellfish Immunol. 2019;88:53–64.
Deng B, Wang Z, Tao W, Li W, Wang C, Wang M, et al. Effects of polysaccharides from mycelia of Cordyceps sinensis on growth performance, immunity and antioxidant indicators of the white shrimp Litopenaeus vannamei. Aquac Nutr. 2015;21:173–9.
Yang C, Kong J, Wang Q, Liu Q, Tian y, Luo K. Heterosis of haemolymph analytes of two geographic populations in Chinese shrimp Fenneropenaeus chinensis. Fish Shellfish Immunol. 2007;23:62–70.
Yin X-L, Li Z-J, Yang K, Lin H-Z, Guo Z-X. Effect of guava leaves on growth and the non-specific immune response of Penaeus monodon. Fish Shellfish Immunol. 2014;40:190–6.
Austin MA, Hokanson JE, Edwards KL. Hypertriglyceridemia as a cardiovascular risk factor. Am J Cardiol. 1998;81(4A):7B–12B.
Ji Z, Lu X, Xue M, Fan Y, Tian J, Dong L, et al. The probiotic effects of host-associated Bacillus velezensis in diets for hybrid yellow catfish (Pelteobagrus fulvidraco ♀ × Pelteobagrus vachelli ♂). Anim Nutr. 2023;15:114–25.
Ruiz Th, Rossetto Vidal M, Ribeiro K, Vicentini C, Vicentini I. Histology of the hepatopancreas and anterior intestine in the freshwater prawn Macrobrachium carcinus (Crustacea, Decapoda). Nauplius. 2020. https://doi.org/10.1590/2358-2936e2020023.
Goh JX, Tan LT, Law JW, Khaw KY, Zengin G, Chan K, et al. Probiotics: Comprehensive Exploration of the Growth Promotion Mechanisms in Shrimps. Progress in Microbes & Molecular Biology. 2023. https://doi.org/10.36877/pmmb.a0000324.
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Abdelsamad, A.E.M., Said, R.E.M., Assas, M. et al. Effects of dietary supplementation with Bacillus velezensis on the growth performance, body composition, antioxidant, immune-related gene expression, and histology of Pacific white shrimp, Litopenaeus vannamei. BMC Vet Res 20, 368 (2024). https://doi.org/10.1186/s12917-024-04207-4
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DOI: https://doi.org/10.1186/s12917-024-04207-4