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Comparative evaluation of three herbal extracts on growth performance, immune response, and resistance against Vibrio parahaemolyticus in Litopenaeus vannamei

BMC Veterinary Research volume 21, Article number: 166 (2025) Cite this article

Abstract

This study evaluated the effects of dietary supplementation with herbal extracts from Artemisia herba-alba, Lonicera japonica, and Lilium candidum on growth performance, survival, feed utilization, antioxidant capacity, and immune response in Litopenaeus vannamei. The efficacy of these herbal-supplemented diets was assessed in enhancing resistance against Vibrio parahaemolyticus-induced Acute Hepatopancreatic Necrosis Disease (Vp AHPND). A total of 2,400 shrimp post-larvae (initial weight 0.74 ± 0.02 g) were randomly assigned to four triplicate groups. Shrimp were fed isonitrogenous and isocaloric diets: T1 (control, basal diet), T2 (basal diet + 250 mg/kg A. herba-alba), T3 (basal diet + 250 mg/kg L. japonica), and T4 (basal diet + 250 mg/kg L. candidum). Herbal-supplemented groups showed significantly improved (P ≤ 0.05) growth performance, feed utilization, and survival rates compared to the control, with T4 exhibiting the highest values. Significant enhancements of immune assays were observed in total hemocyte count, phagocytosis activity, total protein, glutathione peroxidase, and lysozyme activity in herbal-supplemented groups. Antioxidant indicators (catalase, superoxide dismutase, and phenoloxidase) were boosted while malondialdehyde levels decreased in herbal-treated shrimp. Following V. parahaemolyticus challenge, herbal diets effectively reduced cumulative mortality in L. vannamei. Histopathological examination revealed milder AHPND-associated alterations in A. herba-alba and L. candidum-treated groups, contrasting with atrophy, necrosis, and epithelial cell sloughing observed in the positive control. These findings demonstrate the immunostimulatory potential of A. herba-alba, L. japonica, and L. candidum as dietary supplements to enhance growth performance, immune function, and disease resistance in L. vannamei aquaculture, offering a promising strategy for sustainable shrimp farming.

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Introduction

Aquaculture has emerged as one of the most promising food-producing sectors, demonstrating the greatest capacity to meet rising global food demand [1, 2]. Among the cultivated species, Litopenaeus vannamei stands out as an omnivorous and vigorous shrimp species, widely distributed and exhibiting robust environmental adaptability and rapid growth [3, 4]. Over the past two decades, L. vannamei cultivation has experienced exponential growth worldwide, becoming the most significant commercial aquaculture industry [5,6,7]. However, the expansion of shrimp culture practices has exacerbated various threats, including environmental degradation, disease emergence, and devastating impacts on the industry [8, 9].

Vibriosis, a serious bacterial disease in shrimp aquaculture, continues to impede the progress of shrimp farming, leading to substantial economic losses [6, 10, 11]. Several pathogenic Vibrio species have been identified as causative agents of Vibriosis in shrimp, including V. harveyi, V. anguillarum, V. splendidus, V. parahaemolyticus, and V. alginolyticus [11, 12]. Of particular concern is Acute Hepatopancreatic Necrosis Disease (AHPND), an emerging bacterial disease caused by V. parahaemolyticus Vp AHPND. AHPND is considered one of the most dangerous diseases affecting farmed shrimp, with mortality rates reaching 70–100% [13,14,15]. Infected shrimp with AHPND display symptoms such as lethargy, poor growth, empty stomachs, pale hepatopancreas, reddish carapace, and redness of the swimming and walking legs [15, 16]. In Egypt, Vibriosis outbreaks have been documented, causing mortalities in L. vannamei cultured in earthen ponds [17]. Moreover, V. parahaemolyticus infections pose additional threats due to their zoonotic potential and increasing concerns regarding multi-resistant bacteria [18].

Traditionally, chemotherapeutics, including chemicals, antibiotics, and disinfectants, have been used to prevent and treat AHPND [13, 19]. However, the overuse of antimicrobials has resulted in resistant bacteria and antibiotic residues in food, posing dangers to both the aquatic environment and human health [20]. In response to these challenges, eco-friendly aquaculture models have been proposed to enhance immune responses, control disease outbreaks, and improve aquaculture production. These models incorporate prebiotics, probiotics, synbiotics, and algal and herbal phytobiotics [1, 21,22,23].

Herbal extracts have gained attention as potential alternatives to chemotherapeutics due to their safety, ease of use, high effectiveness, and eco-friendly nature for both farmed animals and humans [24]. These extracts offer numerous benefits to aquatic animals, including enhanced immune system function, stimulated growth and maturation, inhibition of pathogenic bacteria, viruses, fungi, and parasites, and reduced stress [25, 26]. The bioactive compounds in herbal extracts can promote physiological metabolism, and protein synthesis, improve growth performance and feed utilization, and even enhance the quality of aquatic products [27]. In shrimp aquaculture, certain herbal plants, such as myrtle leaf extract and Eclipta alba, have demonstrated protective effects against various pathogenic Vibrio species and AHPND-causing V. parahaemolyticus Vp AHPND, thereby reducing the incidence of AHPND [28,29,30].

This study evaluates three herbal extracts as potential immunostimulants, growth promoters, and antibacterial agents in shrimp culture: Artemisia herba-alba, Lonicera japonica Thunb, and Lilium candidum L. A. herba-alba, whose main active ingredient is Artemisinin (ART), has shown promising pharmacological effects, including antibacterial [31], antiviral, and anti-inflammatory properties, which could be beneficial in addressing shrimp diseases [31]. Artemisinin (ART) possesses antioxidant capacity through the activation of CAT, SOD, and GSH-Px creating a reduction of lipid peroxidation [29]. In addition, ART-supplemented diets enhanced the non-specific immunity markers including the phenol oxidase, lysozyme, and alkaline phosphatase [32]. Lonicera japonica Thunb, a species of honeysuckle, has been used in traditional medicine for centuries and has recently gained attention for its potential antimicrobial and immunomodulatory properties [33]. Lonicera has been proven their immunostimulant, antimicrobial, antiviral, anti-inflammatory, antioxidant, and hepatoprotective activities associated with major active compounds including flavonoids, glycosides, iridoids, triterpenoids, organic acids, and lignans, volatile oil providing [34,35,36]. L. candidum L., commonly known as the white lily, has been used in folk medicine and has shown potential for antimicrobial activity, which may apply to aquaculture settings [37]. L. candidum contains phytochemicals including kaempferol, linalool, citronellal, and humulene which have immunostimulatory potential for the secretion of pro-inflammatory cytokines IL-6 and IL-8 [37, 38]. Despite the potential of herbal extracts in controlling shrimp bacterial diseases, comprehensive studies on their efficacy in aquaculture remain limited [39, 40]. Furthermore, scientific evidence supporting the antibacterial activity of herbal extracts against pathogenic bacteria in shrimp farming is insufficient to encourage widespread adoption by aquaculturists. This study investigated the effects of three eco-friendly herbal extracts (A. herba-alba, L. japonica, and L. candidum) on intensively cultured L. vannamei. We assessed their impact on growth performance, feed utilization efficiency, and immune function parameters. Additionally, we evaluated the disease resistance of herbal-treated L. vannamei against V. parahaemolyticus.

Materials and methods

Ethical approval

This study adhered to animal welfare guidelines as outlined by the American Fisheries Society (AFS, 2014). The experimental procedures, including fish handling, examination, and rearing, were conducted following the recommendations and approval of the Committee for Ethical Care and Use of Animals and Aquatic Animals at the National Institute of Oceanography and Fisheries (NIOF, Egypt) under the protocol number NIOFAQ2F23R029.

Herbal extract preparation

Fresh samples of A. herba-alba, L. japonica, and L. candidum (1 kg each) were obtained from the National Agriculture Research Center (NARC), Egypt. The plant materials underwent cleaning, air-drying at 40 °C for 72 h, and subsequent grinding into a powder form. For each herb, 100 g of powder was mixed with 70% ethanol (1:10 w/v) and left at ambient temperature for 72 h. The mixtures were then stirred magnetically at 150 rpm for 24 h and filtered twice using Whatman No. 1 papers (42 μm pore size). A rotary evaporator (BÜCHI Rotavapor R-124) with a water bath (B-480) was employed to remove the alcohol. The resulting concentrated extracts were stored in dark, sterile glass containers at −20 °C until further use [24, 41].

Phytochemical analysis of crude extracts

Total phenolic compound determination

Total phenolics in the extracts were quantified using a UV spectrophotometer (Jenway-6405-UV/VIS) and the Folin-Ciocalteu reagent, based on a colorimetric oxidation/reduction reaction. The procedure involved mixing 0.5 mL of diluted extract with 2.5 mL of diluted Folin-Ciocalteu reagent and 2 mL of Na2CO3 solution (75 g/L) [42]. After incubation at 50 °C for 5 min and subsequent cooling, absorbance was measured at 763 nm. Distilled water was used as a control. Total phenolic content was expressed as gallic acid equivalents (GAE), calculated using the equation: y = 0.0187x + 0.0786 (R2 = 0.998), where (y) represents absorbance, (x) denotes concentration (mg GAE g⁻1 extract) and (R2) is the correlation coefficient (Table 1).

Table 1 Analysis of total flavonoids, and phenolic content of the selected plant
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Determination of total flavonoids

Total flavonoid content was assessed following a modified method from Ordon et al. (2006). The process involved adding 3 mL of 10 g L⁻1 aluminum chloride (AlCl3) ethanolic solution to 0.5 mL of extract. After 1 h of dark incubation, absorbance was measured at 420 nm at room temperature. Extract samples were evaluated at a final concentration of 1 mg/mL. Total flavonoid content was expressed as quercetin equivalent (QE), calculated using the equation: y = 0.0148x—0.0118 (R2 = 0.9996), where x represents absorbance and y denotes concentration (μg QE) and R2 is the correlation coefficient (Table 1 ).

Formulation of basal and functional diets

The basal and herbal-incorporated diets were formulated to meet the optimal nutritional requirements for L. vannamei (Guimaraes et al. 2008; Niu et al. 2011). All diets were designed to be isoproteic and isoenergetic. The composition included dry matter (90.39%), crude protein (40.69%), crude fat (7.36%), crude fiber (3.68%), ash (10.02%), nitrogen-free extract (NFE) (28.64%), and gross energy (18.15 MJ kg1) (Table 2 ). For diet preparation, dry ingredients were initially weighed, combined, and homogenized in a Hobart-type mixer. Oil was then incorporated and mixed thoroughly for 5 min. Subsequently, herbal extracts were added at a concentration of 250 mg kg1 and mixed with deionized hot water (40% of the dry ingredient mixture) for an additional 5 min. The resulting wet mixture was extruded through a 1.2 mm die using an electric kitchen meat grinder (Moulinex ME605131—HV8—1600 W, Paris, France). The extruded pellets were initially air-dried at room temperature (25 °C) for 3 h using an air conditioner and an electric fan in a well-ventilated laboratory. Final drying was conducted at 45 °C for 24 h. The dried diets were then stored at −20 °C until use. Four experimental diets were prepared for the study. The control diet (T1) consisted of the basal diet without any herbal supplements (Table 2 ). The A. herba-alba-treated diet (T2) contained the basal diet supplemented with 250 mg kg1 A. herba-alba. The L. japonica-treated diet (T3) comprised the basal diet with 250 mg kg1 L. japonica added. The L. candidum-treated diet (T4) was formulated by incorporating 250 mg kg1 L. candidum into the basal diet.

Table 2  Feed formulation and proximate chemical composition of the shrimp basal diet (% on dry matter (DM) basis)
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Influence of dietary herbal supplements on growth and immune responses

Shrimp post-larvae were purchased from the National Company for Fishery & Aquaculture. Before initiating the official trial, these larvae underwent a two-week acclimation period of physical, clinical, and bacteriological examinations to ensure disease-free status and to establish uniform, robust, and vigorous health conditions. This study utilized 2400 shrimp post-larvae (PL) with an initial mean weight of 0.74 ± 0.02 g. These PLs were randomly allocated to four treatment groups (T1, T2, T3, and T4), each with three replicates. Each replicate consisted of 200 PLs housed in a 2 m3 capacity fiberglass tank. The control group (T1) received the basal diet without supplements. The experimental groups T2, T3, and T4 were fed the basal diet supplemented with A. herba-alba, L. japonica, and L. candidum, respectively. All diets were administered at a feeding rate of 5–7% of body weight. The experiment was conducted over four months. Throughout this duration, the feeding rate was regularly adjusted based on the shrimps' increasing body weight to maintain optimal nutrition.

Rearing and clinical observation

Feeding was scheduled four times daily at 07:00, 11:00, 17:00, and 21:00, with a photoperiod (12L: 12D). Shrimp vitality was observed one hour after feeding with. Water exchange rates ranged from 1/3–1/2 of the tank volume every other day initially, progressing to daily exchanges two weeks before the experiment's conclusion. Tanks were equipped with aeration and filtration systems and cleaned daily by siphoning. Water parameters were measured using testing kits and electronic probes [43]. Water quality was continuously maintained including temperature at 27.7 ± 0.98°C, pH at 7.5 ± 0.42, dissolved oxygen at 6.49 ± 0.86 mg/L, and salinity at 32 ± 1.75 ppt. Shrimp were regularly inspected for the appearance of behavioral or clinical signs, including anorexia, lethargy, empty gut, expanded chromatophores, and pale hepatopancreas.

Growth parameters

The growth rate in weight and length of the shrimp was recorded throughout the experiment and at its conclusion. Growth performance was evaluated using several parameters. Weight gain (WG, %): WG = 100 × (final weight – initial weight) / initial weight. The specific growth rate (SGR) was determined in percent per day, computed as 100 × (ln final weight – ln initial weight) / days of feeding experiment. Feed conversion ratio (FCR): FCR = feed consumed / weight gain. Survival rate (SR) was calculated as a percentage, using the equation: 100 × (final number of shrimp / initial number of shrimp). The protein efficiency ratio (PER) was determined by dividing the shrimp weight gain by the protein intake.

Analysis of immune parameters

Hemolymph Sampling

Before hemolymph collection, shrimp were subjected to a 24-h fasting period. Five shrimp specimens were randomly selected from each replicate and anesthetized using MS-222 (0.1 g L⁻1 tricaine methanesulfonate; Sigma-Aldrich). Hemolymph was extracted from the ventral sinus at the base of the first abdominal segment using 1-cm3 syringes. The collected hemolymph was divided into two portions. The first portion was placed in microtubes containing an anticoagulant solution (30 mM trisodium citrate, 0.34 M NaCl, and 10 mM EDTA; pH 7.55), with its osmolality adjusted to 780 mOsm kg⁻1 using glucose. This hemolymph and anticoagulant mixture was prepared at a ratio of 1:9 in a sterile tube and placed on ice [44]. The second portion was placed in a tube without anticoagulant and temporarily stored at 4°C for 12 h. It was then centrifuged at 4000 rpm for 10 min, and the resulting serum was stored at −80°C for subsequent use in biochemical and immunological assays.

Immune assays

Several immune parameters were assessed using the collected hemolymph samples. Total hemocyte count (THC) was determined using a hemocytometer under an inverted phase-contrast microscope [45]. Phenoloxidase (PO) activity was measured based on the formation of dopachrome from L-dihydroxyphenylalanine (L-DOPA) substrate using a spectrophotometer [46, 47]. Superoxide dismutase (SOD) activity was assessed using a modified method from [48] and further verified following the method of McCord and Fridovich [49]. Lysozyme (LYZ) activity was analyzed using the turbidimetric method as described by [46]. The phagocytic activity of hemocytes was determined in a 24-well plate and expressed as a percentage of phagocytic hemocytes to total observed hemocytes [50]. Protein concentration was measured using an ELISA microplate format (Bradford 1976). Glutathione peroxidase (GPx) activity was measured following the methodology of Flohe and Gunzler (1984). Lipid peroxidation levels were determined by measuring malondialdehyde (MDA) levels during fatty acid oxidation [51]. All spectrophotometric measurements were performed using a UV–visible spectrophotometer, and all assays were conducted in triplicate to ensure the reliability of the results.

Evaluation of the resistance of the herbal-treated shrimp to V. parahaemolyticus Vp AHPND

The pathogenic V. parahaemolyticus SHA used in this study was initially isolated from diseased Litopenaeus vannamei shrimp during an infection outbreak, fulfilling Koch's postulates. The diseased shrimp exhibited symptoms including anorexia, lethargy, empty gut, pale hepatopancreas, and expanded chromatophores. This V. parahaemolyticus SHA was identified by 16S rRNA sequencing and submitted to GenBank under accession number OP659038. The bacterial strain was purchased from the Laboratory of Marine Microbiology, National Institute of Oceanography and Fisheries (NIOF), Alexandria, Egypt.

Following the 4-month feeding period with herbal-supplemented diets, the disease resistance efficacy of treated L. vannamei was evaluated through a challenge against the pathogenic V. parahaemolyticus SHA. Before the challenge test, an LD50 assay was conducted to determine the infective dosage.

LD50 assay

To determine the median lethal dose (LD50), a total of 210 shrimp with an average weight of 12.54 ± 0.029 g were equally distributed into seven triplicated groups (T1, T2, T3, T4, T5, T6, and T7), with 30 shrimp per group. The V. parahaemolyticus SHA was cultured in tryptic soy broth (TSB) supplemented with 3% NaCl and incubated at 27°C for 24 h. The bacterial cells were then harvested by centrifugation at 10,000 rpm for 1 min at 4°C. The resulting bacterial pellet was resuspended in 0.9% NaCl solution and adjusted to a concentration of 3 × 108 CFU/mL (equivalent to McFarland standard #1) to serve as the stock bacterial suspension for injection [52]. This standardized suspension was then serially diluted with PBS to obtain the required concentrations ranging from 3 × 103 to 3 × 108 CFU/mL.

The negative control group (T1) received an intramuscular injection of 50 µL sterile 0.9% saline solution in the third abdominal segment. The remaining groups (T2 to T7) were injected intramuscularly with 50 µL of the corresponding five serial dilutions of V. parahaemolyticus SHA, ranging from 3.0 × 103 to 3.0 × 108 CFU/mL [53]. The cumulative mortality was recorded over 72 h post-injection, and based on these results, the LD50 was determined to be 3 × 104 CFU/mL.

Bacterial challenge

The bacterial challenge was conducted following modified procedures from [53]. A total of 150 shrimp were used in this experiment, randomly chosen from the original experimental groups, with 30 shrimp taken from each of the five groups that had received different dietary treatments during the feeding trial. The selected shrimp were then redistributed into five new triplicated groups of 10 shrimp each. These new groups were housed in 500-L capacity fiberglass tanks and designated as a negative control group (T1), a positive control group (T2), and three herbal-treated groups (T3-T5). The V. parahaemolyticus SHA suspension used for injection was prepared as previously described, with the concentration adjusted to 3 × 104 CFU/mL based on the LD50 determination. Each shrimp in the positive control T2, and herbal-treated groups (T3, T4, and T5) received an intramuscular injection of 50 μL of this bacterial suspension in the third abdominal segment, while the negative control group received an equivalent volume of sterile 0.9% saline solution.

Throughout the one-week challenge period, water quality parameters were maintained within optimal ranges through proper aeration, filtration, regular water renewal, and adherence to hygienic practices. The experimental shrimp were fed daily with a diet containing 40% protein at a rate of 5% of their biomass. Shrimp were monitored daily for clinical signs, morbidity, and mortality. Bacteriological samples from dead and moribund shrimp were aseptically collected and cultured on thiosulfate-citrate-bile salts-sucrose (TCBS) agar to confirm V. parahaemolyticus as the causative agent of mortality. Mortalities were attributed to V. parahaemolyticus only when a pure culture of the bacterium was isolated from the challenged shrimp, thereby fulfilling Koch's postulates. This rigorous approach ensured the accurate identification of V. parahaemolyticus as the primary cause of observed mortalities.

Histopathological Examination

Hepatopancreas samples from challenged shrimp were processed for histopathological examination following standard protocols [54, 55]. Tissues were fixed in Davidson's AFA fixative, dehydrated through a graded ethanol series, cleared in xylene, and embedded in paraffin wax. Sections of 5–6 μm thickness were prepared using a rotary microtome (KD-2258), mounted on glass slides, and stained with hematoxylin and eosin (H&E). Tissue sections were examined using a light microscope (BEL photonic), and photographs were captured using a digital camera (TOUPCAM TM-UCMOS-3.1MP1/2, Model No.3.2).

Statistical analysis

Data were analyzed using one-way ANOVA and Duncan's multiple range test (SPSS for Windows, version 22, USA). The significance level was set at P ≤ 0.05. Standard errors were estimated. Principal Correspondence Analysis (PCA) was performed using the Brodgar statistical program (Brodgar version 2.7.5, 2017, Highland Statistics Ltd., www.highstat.com).

Results

Influence of dietary herbal supplements on growth performance and immune response

Clinical Signs and Health Indicators

The L. vannamei treated with herbal supplements exhibited improved health indicators compared to the control group. These shrimps displayed more active swimming patterns, enhanced appetite, and improved growth. Their body parts generally appeared clearer and more brightly colored, with a harder shell texture. Additionally, the treated shrimp showed full guts and good overall coloration without signs of melanization, all indicative of better health status.

Growth performance

Significant differences (P ≤ 0.05) were observed in various growth parameters among the herbal-treated and non-herbal-treated groups. The L. candidum-treated group demonstrated significantly higher FW, WG, SGR, RGR, and survival ratio (P ≤ 0.05) compared to both the A. herba-alba-treated and L. japonica-treated groups. Overall, shrimp receiving herbal supplementation showed significantly improved growth performance compared to the untreated control group, as detailed in (Table 3).

Table 3 Growth and survival performance of L. vannamei fed herbal-supplemented diet under an intensive culture system
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Feed utilization and productivity parameters

L. vannamei fed herbal-treated diets exhibited significantly (P ≤ 0.05) enhanced feed utilization and productivity parameters compared to the non-herbal-treated group. The L. candidum-treated group demonstrated significantly lower feed intake, improved FCR, and a higher protein efficiency ratio than those in the A. herba-alba and L. japonica-treated groups. Consequently, the highest productivity was achieved in the L. candidum-treated group, followed by the L. japonica-treated group, and then the A. herba-alba-treated group (Table 4).

Table 4 Feed utilization and productivity parameters of L. vannamei fed herbal-supplemented diet under an intensive culture system
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Immune response of herbal-treated L. vannamei

Herbal-treated L. vannamei exhibited significantly enhanced (P ≤ 0.05) immune parameters compared to non-treated shrimp. The A. herba-alba, L. japonica, and L. candidum-treated groups showed increased (P ≤ 0.05) values of total hemocyte count, total protein concentration, and phagocytosis activity relative to the control. Glutathione peroxidase (GPx) and phenoloxidase (PO) activities were significantly higher in the A. herba-alba-treated group (T2) compared to other treated groups. The L. japonica-treated group (T3) demonstrated maximum lysozyme (LYS) activity. Higher antioxidant activity, as measured by catalase (CAT) and superoxide dismutase (SOD), was observed in the L. candidum-treated group (T4). Conversely, malondialdehyde (MDA) levels were significantly lower in all herbal-treated groups compared to the control group. These results, presented in Table 5 and Fig. 1, indicate that herbal extracts efficaciously enhanced the immune response of cultured whiteleg shrimp compared to the control (Fig. 1).

Table 5 Immune, and antioxidant parameters of L. vannamei fed herbal-supplemented diets under an intensive culture system
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Fig. 1
figure 1

Serum antioxidative status of white leg shrimp, L. vannamei fed herbal-supplemented diets under an intensive culture system for 120 days. Catalase, (CAT), Superoxide Dismutase (SOD), phenol oxidase (PO), and Malondialdehyde (MDA). The bars (mean ± SE) with different letters are significantly different (P ≤ 0.05). Treatments: T1: shrimp fed a basal diet (control) without any herbal supplements; T2: shrimp fed a basal diet supplemented with 250 mg kg−1 A. herba-alba; T3: shrimp fed a basal diet supplemented with 250 mg kg−1 L. japonica; T4: shrimp fed a basal diet supplemented with 250 mg kg−1 L. candidum

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Bacterial challenge with V. parahaemolyticus

Mortality and survival rates

The challenge data, including cumulative mortality and relative survival percentage of L. vannamei challenged with V. parahaemolyticus (Table 6). No mortality was recorded in the negative control group (T1), which was inoculated with physiological saline, during the seven days post-challenge. Herbal supplementation significantly increased (P ≤ 0.05) the survival rate of challenged L. vannamei compared to the positive control. Lower mortality rates of 23.33%, 20%, and 13.33% were significantly (P ≤ 0.05) recorded in the challenged herbal-treated groups T3, T4, and T5, respectively, compared to 66.67% in the positive control (T2).

Table 6 Total cumulative mortality, and Relative percent survival RPS of challenged L. vannamei with V. parahaemolyticus
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Clinical signs

Post-challenge, the severity of signs associated with V. parahaemolyticus infection varied between herbal-treated groups and controls. In herbal-treated groups, most challenged shrimp displayed lethargy and anorexia on the second-day post-challenge. At 72 h post-challenge, some moribund shrimp exhibited expanded chromatophores, melanization, and pale hepatopancreas, with progressive mortality. In contrast, the positive control group experienced earlier signs at 12 h post-challenge, including anorexia, weakness, lethargy, and abnormal up-and-down swimming. These signs progressed to loss of feeding, weakness, immobility at the tank bottom, expanded chromatophores, melanization, and pale hepatopancreas, accompanied by continued morbidity and mortality between 24–48 h post-challenge. In all groups, morbid and dead shrimp displayed flaccid and mushy texture, discolored faded shells, whitish discoloration of the abdominal muscle, and reddened, necrotic tail fans (Fig. 2). Moribund shrimp also revealed empty guts and pale, atrophied hepatopancreas.

Fig. 2
figure 2

A Healthy L. vannamei with normal bright color, firm body texture, and active responsiveness before being challenged with V. parahaemolyticus. B Side view of diseased L. vannamei post-challenged with V. parahaemolyticus showing expanded chromatophores, reddened and necrotic legs, and tail fan. The scale bar = 1 cm

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Histopathological examination

Histopathological examination revealed differences in hepatopancreas tissue between challenged shrimp in non-herbal-treated and herbal-treated groups. The hepatopancreas of the negative control showed normal histological structures with firmly arranged blind tubules, narrow lumens, and intact epithelial membranes. These tubules typically exhibited a stellate structure containing four functional cell types: E-cells (embryonalzellen), R-cells (Restzellen), F-cells (Fibrillenyellen), and B-cells (Blasenzellen) (Fig. 3A). In contrast, the positive control shrimp displayed notable characteristics of V. parahaemolyticus infection, including atrophy, sloughing into the tubular lumen, necrosis, massive and progressive degeneration of epithelial cells in hepatopancreatic tubules, and hemocyte infiltration between the tubules (Fig. 3B).

Fig. 3
figure 3

Photomicrographs of hepatopancreas in L. vannamei. A T.s in the non-challenged negative-control treatment showing normal hepatopancreatic tubules, with different cells (E, R, F& B-cells) (10 × magnification). B T.s in the challenged positive control treatment with degeneration in epithelial membrane and appearance of hemocytes infiltration (10 × magnification). C, D T.s in the challenged A. herba-alba-treated group showed mild atrophy of hepatopancreatic B-cells and R-cells with tetra-shaped star tubular lumens (10&40 × magnification). E, F T.s in the challenged L. candidum-treated group with mild regularly arranged hepatopancreas tissue, well developed B-cells, and F-cells detected in contact epithelial membrane around a Penta or hexa-shaped star lumenar tubules (10&40 × magnification) Epi.M; epithelium membrane, HI; hemocytes infiltration, L; lumen, V;vacuoles. H&E stain

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The A. herba-alba-treated group showed mild atrophy and deformation of B-cells and R-cells in hepatopancreatic tubules. The lumen appeared as a tetra-shaped star occupying most of the tubule space, with irregularly arranged tubules and scattered vacuoles (Fig. 3C & D). The L. candidum-treated group displayed a more regularly arranged hepatopancreas tissue structure. The epithelial membrane appeared intact around the tubules, and the lumen occupied regular space, forming penta- or hexa-shaped star tubules with well-developed B-cells and F-cells (Fig. 3E & F). Notably, V. parahaemolyticus was not detected in the herbal-treated groups compared to the positive control. These histological changes suggest the ameliorating and recovery potential of herbal supplements in enhancing L. vannamei resistance against V. parahaemolyticus infection.

Discussion

The cultured white-leg shrimp L. vannamei is highly susceptible to Vibrio infections which were demonstrated as potential hotspots for aquaculture sustainability and zoonotic concerns [56]. In particular, V. parahaemolyticus is the causative of AHPND, a destructive emerging bacterial disease affecting the shrimp aquaculture industry, causing substantial economic losses [13].

Herbal phytobiotics have emerged as a promising approach for controlling various microbial infections in fish, and shellfish [57, 58]. Herbal extracts contain high levels of bioactive substances including biomass acids, alkaloids, glycosides, polysaccharides, and flavonoids, which possess effective medicinal properties and numerous healthcare functions in animal, and human medicine [21, 59, 60]. Certain herbal extracts have already been included commercially and supplemented for aquafeeds as growth promoters, antimicrobial compounds, and sources of vital nutrients [25, 61]. However, the therapeutic effectiveness of numerous extracts in the aquaculture field has not been sufficiently investigated in terms of their usage, antimicrobial potential, immune response, growth performance, and disease resistance activities [62, 63].

Herein, the used herbal extracts; A. herba-alba, L. japonica, and L. candidum contain various active components that contribute to their immune-stimulating and antibacterial properties. A. herba-alba is rich in artemisinin, flavonoids, and phenolic compounds, which have been shown to possess potent antioxidant and antimicrobial activities [64]. Artemisinin, in particular, has demonstrated the ability to enhance immune function and disease resistance in aquatic species [65]. Recent studies have shown that artemisinin can effectively counteract bacterial-induced inflammation and metabolic changes in various aquatic species, highlighting its potential as a natural alternative to traditional antibiotics in aquaculture [65]. L. japonica contains chlorogenic acid, luteolin, and quercetin, which exhibit strong anti-inflammatory and antioxidant effects [33]. These compounds have been found to modulate immune responses and improve gut health in various animal models [66]. L. candidum is known for its content of steroidal saponins, alkaloids, and flavonoids such as kaempferol [37]. These compounds have shown anti-inflammatory properties and the ability to modulate immune responses [67].

Following a 4-month herbal-supplementation experiment, we observed significant improvements in growth performance, survival, feed utilization, and increased shrimp productivity in herbal-supplemented diets compared to the control, with the greatest enhancement mainly in the Lilium-treated diet. This evident improvement in growth performance may be partly related to the better utilization of the offered feeds in the herbal-treated groups, which is consistent with many studies [68, 69]. It has been demonstrated that herbs can improve feed palatability by stimulating the taste buds of farmed fish, resulting in increased feed consumption [60]. As well, the nucleotides, free amino acids, and alkaloids in herbal extracts are responsible for fish attractiveness [70]. Moreover, herbal products have a particular odor and flavor that play an important role in increasing feed intake, and they can accelerate the release of digestive enzymes, promote intestinal motility, and subsequently increase feed intake and feed utilization [60]. Abdel-Rahim et al. [68] have proven that total flavonoids and phenolics are the major growth promoters for L. vannamei fed sargassum aquatic plants.

Enhancement of immune response was significantly displayed in the herbal-treated L. vannamei compared to non-herbal-treated shrimp. Higher counts of total hemocyte, phagocytosis activity, and total protein concentration were recorded in the herbal-treated groups. In particular, the highest activities of GPx and PO were significantly observed mainly in the Artemisia-treated group, maximum activities of LYS in the Lonicera-treated group, and higher antioxidant activity (CAT and SOD) in the Lilium-treated group. Regardless of the herbal supplements, these significantly higher levels of SOD, CAT, GSH-Px, and PO proved a good job of the herbal extracts for removing harmful superoxide anion radicals [29, 71, 72]). In related studies on Artemisinin, the elevated antioxidant capacity could be one of the protective effects of Artemisinin achieved by superoxide anion radicals and reducing lipid peroxidation [64, 73]. Our results regarding A. herba-alba were in line with some recently demonstrated studies. Elevation of immune parameters was significantly recorded in A. herba-alba-treated L. vannamei [59]. The latter species fed cottonseed protein concentrate meal diets, artemisinin supplementation positively influences immune response, antioxidant capacity, growth, gut health, and disease resistance against V. parahaemolyticus [74]. Similarly, in common carp, Cyprinus carpio, A. absinthium was found to potentially enhance growth performance, oxidative capacity, and immune responses [32].

Given that the other two herbal extracts, L. japonica and L. candidum, have never been used yet in aquaculture, our evaluation was based on the medicinally demonstrated studies in humans and animals on the immunostimulatory, antimicrobial, and growth-promotor influences of the supplemented herbal extracts. We achieved higher levels of growth, survival, feed utilization, and shrimp productivity that could be attributed to the fact that L. candidum is rich in steroidal saponins, fucoidans, and alkaloids [37]. Spirostanol and furostanol saponins were isolated from the fresh bulbs of L. candidum. Beta-sitosterol and beta-sitosterol glucoside were isolated from the butanolic extract of petals [67]. The Quillaja Saponaria-supplemented diet rich in saponins, flavonoids, and phenolics boosted both growth and feed utilization in Nile tilapia [69] via enhancing digestive enzymes (amylase, lipase), as well as growth hormone. A recent study demonstrated that L. candidum extracts kaempferol, citronellal, and humulene have anti-inflammatory activity as they significantly decreased secretion of the pro-inflammatory cytokines interleukin IL-6 and IL-8 by senescent human pulmonary fibroblasts (HPFs) and human dermal fibroblasts (HDFs) [37].

Likewise, immunostimulatory and growth performances were achieved in the Lonicera-treated L. vannamei. L. japonica was reported to have anti-inflammatory and antiviral effects that are associated with the major active components, including flavonoids and their glycosides, iridoids, triterpenoids, saponins, caffeoylquinic acids, organic acids, aromatic glycosides, lignans, volatile oils, and other compounds [75, 76]. A study in rats found that the extracts of L. japonica flower buds could be used as prebiotics to adjust intestinal flora with intestinal microflora imbalances [77] and 2012b). Further studies prove that the extracts of L. japonica flower buds could improve intestinal microbes, significantly reduce intestinal alkaline phosphatase activity, and enhance the intestinal immune barrier by regulating the content of secretory immunoglobulin SIgA and cytokines IFN-γ and IL-4 [66]. Moreover, the total flavones of L. japonica flower buds were proven to have hepatoprotective activity against liver damage induced by LPS in rats by reducing the production of free radicals, inhibiting cell membrane lipid peroxidation, and reducing the release of inflammatory mediators [78].

The disease resistance efficacy of herbal-treated L. vannamei against AHPND was proven based on the challenge potential against V. parahaemolyticus. Accordingly, the lowest significant mortality rate, 23.33, 20, and 13.33%, were respectively recorded in A. herba alba, L. japonica, and L. candidum-treated groups, compared to the highest mortality rate of 66.67% in the positive-control shrimp. Close challenge models of the V. parahaemolyticus concentration were previously demonstrated [53, 79, 80]. Post-challenge, bacteria colonize within the lymphoid, and cellular degradation can be detected about the 7th dpi (Van de Braak et al. 2002). Hence, a high bacterial load tends to overwhelm the shrimp's immune systems, and shrimp succumb to the disease (Pantoja et al. 2005). Challenged L. vannamei with V. parahaemolyticus presented clinical patterns including anorexia, lethargy, and erratic swimming, which progressed to expanded chromatophores, opaque musculature, soft shells, reddened and necrotic legs and tail, atrophied hepatopancreas, an empty gastrointestinal tract, and mortality [53],Aguilera-Rivera et al. 2019). These associated signs were a consequence of the establishment of acute hepatopancreatic necrosis disease (AHPND) in infected L. vannamei, P. monodon, P. chinensi, and Marsupenaeus japonicus [15, 81,82,83].

The challenged herbal-treated L. vannamei with V. parahaemolyticus presents moderate signs that disappear a few days later. As stated in the first experiment, different herbal supplements significantly improved growth performance, feed utilization, and survival rate. In addition, the enhancement of hematological and immunological indicators, including total hemocyte count, phagocytosis activity, total protein, glutathione peroxidase, lysozyme activity, and antioxidant indicators, was achieved. Subsequently, the lowest total cumulative mortality and highest relative percent survival were obtained in herbal-treated challenged L. vannamei with V. parahaemolyticus compared to the control treatment. The dose and duration of the herbal treatment are the main factors driving the enhancement in immune response and protection against pathogens. It was demonstrated that the proper dosage of dietary herbal supplements for fish and shrimp ranged from 0.1% to 10% and duration from 14 to 70 days [84].

Finally, evident improvement of histopathological indicators was revealed in the hepatopancreas of the herbal-treated challenged groups compared to the controls. These histological findings with the four functional cells resembled and were confirmed among different crustaceans [85]. The hepatopancreas is an important multifunction glandular organ in crustacea that plays a great role in food digestion, absorption, and storage in addition to the secretion of growth-related hormones in different crustacean animals [86]. Oppositely, the challenged control shrimp induced notable characteristics of V. parahaemolyticus infection, including atrophy, sloughing, necrosis, and massive degeneration of epithelial cells in hepatopancreatic tubule, besides lack of B, R, and F cells. Similar findings were previously reported worldwide in whiteleg L. vannamei affected by (AHPND) from farms in northwestern Mexico [87], in Korea [88], and Vietnam [89]. Milder histopathological changes were revealed in the herbal-treated groups compared to the positive control. Evident reduction in the severity of associated histological lesions of the hepatopancreatic tissue, including a tetra-shaped and penta- or hexa-shaped star tubular lumen of the A. herba-alba and L. candidum-treated groups, respectively. These histological changes revealed the ameliorating and recovery potential of herbal supplements to enhance the resistance of L. vannamei against V. parahaemolyticus infection.

In conclusion, our study demonstrates that dietary supplementation with A. herba-alba, L. japonica, and L. candidum effectively improves growth, survival, feed utilization, antioxidant capacity, and immune response in L. vannamei. The herbal additives have proven their immunoprotective and resistance-enhancing properties against AHPND-causing V. parahaemolyticus Vp AHPND. These findings highlight the potential of these herbal extracts as prophylactic and therapeutic agents against bacterial pathogens in aquaculture, offering a promising alternative to conventional antibiotics and contributing to more sustainable aquaculture practices.

Data availability

No datasets were generated or analysed during the current study.

References

  1. Abdel-Rahim MM, Elhetawy AI, Mansour AT, Mohamed RA, Lotfy AM, Sallam AE, Shahin SA. Effect of long-term dietary supplementation with lavender, Lavandula angustifolia, oil on European seabass growth performance, innate immunity, antioxidant status, and organ histomorphometry. Aquaculture Int. 2023;1–19. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s10499-023-01322-1.

  2. Caputo A, Bondad-Reantaso MG, Karunasagar I, Hao B, Gaunt P, Verner-Jeffreys D, Dorado-Garcia A. Antimicrobial resistance in aquaculture: A global analysis of literature and national action plans. Reviews in Aquaculture. 2023;15(2):568–78.

    Article  Google Scholar 

  3. Kim SK, Pang Z, Seo HC, Cho YR, Samocha T, Jang IK. Effect of bioflocs on growth and immune activity of Pacific white shrimp. L itopenaeus vannamei postlarvae Aquaculture Research. 2014;45(2):362–71.

    Article  CAS  Google Scholar 

  4. Tacon AGJ, Cody JJ, Conquest LD, Divakaran S, Forster IP, Decamp OE. Effect of culture system on the nutrition and growth performance of Pacific white shrimp Litopenaeus vannamei (Boone) fed different diets. Aquac Nutr. 2002;8(2):121–37.

    Article  Google Scholar 

  5. Aktur S, Yeumine N. Vannamei Shrimp Cultivation Including Distribution, Feed Quality and Feeding Method. Journal Siplieria Sciences. 2020;1(2):1–5.

    Article  Google Scholar 

  6. FAO. The state of world fisheries and aquaculture 2020. Sustainability in action. 2020. https://doiorg.publicaciones.saludcastillayleon.es/10.4060/ca9229en.

    Article  Google Scholar 

  7. Sun, H., Song, Y., Zhang, H., Zhang, X., Liu, Y., Wang, X., ... & Xue, C. (2020). Characterization of lipid composition in the muscle tissue of four shrimp species commonly consumed in China by UPLC− Triple TOF− MS/MS. LWT, 128, 109469.

  8. Bae J, Hamidoghli A, Djaballah MS, Maamri S, Hamdi A, Souffi I, Bai SC. Effects of three different dietary plant protein sources as fishmeal replacers in juvenile whiteleg shrimp, Litopenaeus vannamei. Fisheries and Aquatic Sciences. 2020;23:1–6.

    Article  Google Scholar 

  9. Thornber K, Verner-Jeffreys D, Hinchliffe S, Rahman MM, Bass D, Tyler CR. Evaluating antimicrobial resistance in the global shrimp industry. Rev Aquac. 2020;12(2):966–86.

    Article  PubMed  Google Scholar 

  10. Amatul-Samahah MA, Muthukrishnan S, Omar WHHW, Ikhsan NFM, Ina-Salwany MY. Vibrio sp. associated with acute hepatopancreatic necrosis disease (AHPND) found in penaeid shrimp pond from east cost of peninsular Malaysia. J Environ Biol. 2020;41:1160–70.

    Article  CAS  Google Scholar 

  11. Chatterjee SHALDAR, Haldar S. Vibrio related diseases in aquaculture and development of rapid and accurate identification methods. J Mar Sci Res Dev S. 2012;1:1–7.

    Google Scholar 

  12. Austin B, Zhang XH. Vibrio harveyi: a significant pathogen of marine vertebrates and invertebrates. Lett Appl Microbiol. 2006;43(2):119–24.

    Article  CAS  PubMed  Google Scholar 

  13. De Schryver P, Defoirdt T, Sorgeloos P. Early mortality syndrome outbreaks: a microbial management issue in shrimp farming? PLoS Pathog. 2014;10(4):e1003919. https://doiorg.publicaciones.saludcastillayleon.es/10.1371/journal.ppat.1003919.

  14. Kumar V, Roy S, Behera BK, Bossier P, Das BK. Acute hepatopancreatic necrosis disease (AHPND): virulence, pathogenesis and mitigation strategies in shrimp aquaculture. Toxins. 2021;13(8):524.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Soto-Rodriguez SA, Gomez-Gil B, Lozano-Olvera R, Betancourt-Lozano M, Morales-Covarrubias MS. Field and experimental evidence of Vibrio parahaemolyticus as the causative agent of acute hepatopancreatic necrosis disease of cultured shrimp (Litopenaeus vannamei) in Northwestern Mexico. Appl Environ Microbiol. 2015;81(5):1689–99.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Saulnier D, Haffner P, Goarant C, Levy P, Ansquer D. Experimental infection models for shrimp vibriosis studies: a review. Aquaculture. 2000;191(1–3):133–44.

    Article  Google Scholar 

  17. El Zlitne RA, Eissa AE, Elgendy MY, Abdelsalam M, Sabry NM, Sharaf MS, Abdelbaky AA. Vibriosis triggered mass kills in Pacific white leg shrimp (Litopenaeus vannamei) reared at some Egyptian earthen pond-based aquaculture facilities. Egypt J Aquat Biol Fish. 2022;26(3):261–77.

  18. Hernández-Cabanyero C, Carrascosa E, Jiménez S, Fouz B. Exploring the effect of functional diets containing phytobiotic compounds in whiteleg shrimp health: resistance to acute hepatopancreatic necrotic disease caused by Vibrio parahaemolyticus. Animals. 2023;13(8):1354.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Rowley AF, Pope EC. Vaccines and crustacean aquaculture—A mechanistic exploration. Aquaculture. 2012;334:1–11.

    Article  Google Scholar 

  20. Bondad‐Reantaso MG, MacKinnon B, Karunasagar I, Fridman S, Alday‐Sanz V, Brun E, Caputo A. Review of alternatives to antibiotic use in aquaculture. Rev Aquaculture. 2023;15(4):1421–51.

  21. Galina J, Yin G, Ardo L, Jeney Z. The use of immunostimulating herbs in fish. An overview of research. Fish Physiol Biochem. 2009;35:669–76.

    Article  CAS  PubMed  Google Scholar 

  22. Wang J, Xu C, Wong YK, Li Y, Liao F, Jiang T, Tu Y. Artemisinin, the magic drug discovered from traditional Chinese medicine. Engineering. 2019;5(1):32–9.

    Article  CAS  Google Scholar 

  23. Wu DT, Deng Y, Chen LX, Zhao J, Bzhelyansky A, Li SP. Evaluation on quality consistency of Ganoderma lucidum dietary supplements collected in the United States. Sci Rep. 2017;7(1):7792.

    Article  PubMed  PubMed Central  Google Scholar 

  24. Van Hai N. The use of medicinal plants as immunostimulants in aquaculture: A review. Aquaculture. 2015;446:88–96.

    Article  Google Scholar 

  25. Citarasu T. Herbal biomedicines: a new opportunity for aquaculture industry. Aquacult Int. 2010;18(3):403–14.

    Article  Google Scholar 

  26. Reverter M, Bontemps N, Lecchini D, Banaigs B, Sasal P. Use of plant extracts in fish aquaculture as an alternative to chemotherapy: current status and future perspectives. Aquaculture. 2014;433:50–61.

    Article  Google Scholar 

  27. Ahmadifar E, Pourmohammadi Fallah H, Yousefi M, Dawood MA, Hoseinifar SH, Adineh H, Doan HV. The gene regulatory roles of herbal extracts on the growth, immune system, and reproduction of fish. Animals. 2021;11(8):2167.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Linh NTT, Ha PTH, Van Day P, Hai LTT, Vu SH, Nghia NT, Luan NT. Efficacy of Annona glabra extract against acute hepatopancreatic necrosis disease in white-leg shrimp (Penaeus vannamei). Journal of Invertebrate Pathology. 2024;205:108142.

    Article  Google Scholar 

  29. Lu F, Haga Y, Satoh S. Effects of replacing fish meal with rendered animal protein and plant protein sources on growth response, biological indices, and amino acid availability for rainbow trout Oncorhynchus mykiss. Fish Sci. 2015;81:95–105.

    Article  CAS  Google Scholar 

  30. Tinh TH, Elayaraja S, Mabrok M, Gallantiswara PCD, Vuddhakul V, Rodkhum C. Antibacterial spectrum of synthetic herbal-based polyphenols against Vibrio parahaemolyticus isolated from diseased Pacific whiteleg shrimp (Penaeus vannamei) in Thailand. Aquaculture. 2021;533: 736070.

    Article  CAS  Google Scholar 

  31. Liu T, Lin P, Bao T, Ding Y, Lha Q, Nan P, Zhong Y. Essential oil composition and antimicrobial activity of Artemisia dracunculus L. var. qinghaiensis YR Ling (Asteraceae) from Qinghai-Tibet Plateau. Industrial Crops and Products. 2018;125:1–4.

    Article  CAS  Google Scholar 

  32. Yousefi M, Zahedi S, Reverter M, Adineh H, Hoseini SM, Van Doan H, Hoseinifar SH. Enhanced growth performance, oxidative capacity and immune responses of common carp, Cyprinus carpio fed with Artemisia absinthium extract-supplemented diet. Aquaculture. 2021;545:737167.

    Article  CAS  Google Scholar 

  33. Shang X, Pan H, Li M, Miao X, Ding H. Lonicera japonica Thunb: ethnopharmacology, phytochemistry and pharmacology of an important traditional Chinese medicine. J Ethnopharmacology. 2011;138(1):1–21.

    Article  CAS  PubMed  Google Scholar 

  34. Ge L, Xie Q, Jiang Y, Xiao L, Wan H, Zhou B, Zeng X. Genus Lonicera: New drug discovery from traditional usage to modern chemical and pharmacological research. Phytomedicine. 2022;96:153889.

    Article  CAS  PubMed  Google Scholar 

  35. Kang KB, Lee DY, Kim MS, Kim TB, Yang TJ, Sung SH. Argininosecologanin, a secoiridoid-derived guanidine alkaloid from the roots of Lonicera insularis. Nat Prod Res. 2018;32(7):788–94.

    Article  CAS  PubMed  Google Scholar 

  36. Xiao L, Liang S, Ge L, Wan H, Wu W, Fei J, Zeng X. 4, 5-di-O-caffeoylquinic acid methyl ester isolated from Lonicera japonica Thunb. targets the Keap1/Nrf2 pathway to attenuate H2O2-induced liver oxidative damage in HepG2 cells. Phytomedicine. 2020;70:153219.

  37. Zaccai M, Yarmolinsky L, Khalfin B, Budovsky A, Gorelick J, Dahan A, Ben-Shabat S. Medicinal properties of Lilium candidum L. and its phytochemicals. Plants. 2020;9(8):959.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Yarmolinsky L, Budovsky A, Ben-Shabat S, Khalfin B, Gorelick J, Bishitz Y, Yarmolinsky L. Recent updates on the phytochemistry and pharmacological properties of Phlomis viscosa Poiret. Rejuvenation Research. 2019;22(4):282–8.

    Article  CAS  PubMed  Google Scholar 

  39. Lawhavinit OA, Kongkathip N, Kongkathip B. Antimicrobial activity of curcuminoids from Curcuma longa L. on pathogenic bacteria of shrimp and chicken. Agriculture and Natural Resources. 2010;44(3):364–71.

    CAS  Google Scholar 

  40. Lawhavinit OA, Sincharoenpoka P, Sunthornandh P. Effects of ethanol tumeric (Curcuma longa Linn.) extract against shrimp pathogenic Vibrio spp. and on growth performance and immune status of white shrimp (Litopenaeus vannamei). Agriculture and Natural Resources. 2011;45(1):70–7.

    Google Scholar 

  41. Mohammadi N, Shaghaghi N. Inhibitory effect of eight secondary metabolites from conventional medicinal plants on COVID-19 virus protease by molecular docking analysis. ChemRxiv. 2020. https://doiorg.publicaciones.saludcastillayleon.es/10.26434/chemrxiv.11987475.v1.

  42. Škerget M, Kotnik P, Hadolin M, Hraš AR, Simonič M, Knez Ž. Phenols, proanthocyanidins, flavones and flavonols in some plant materials and their antioxidant activities. Food Chem. 2005;89(2):191–8.

    Article  Google Scholar 

  43. APHA. Standard methods for the examination of water and wastewater. 21st ed. Washington, DC: American Public Health Association; 2005.

    Google Scholar 

  44. Lee DC, Choi YJ, Kim JH. Toxic effects of waterborne cadmium exposure on hematological parameters, oxidative stress, neurotoxicity, and heat shock protein 70 in juvenile olive flounder, Paralichthys olivaceus. Fish Shellfish Immunol. 2022;122:476–83.

    Article  CAS  PubMed  Google Scholar 

  45. Le Moullac G, Klein B, Sellos D, Van Wormhoudt A. Adaptation of trypsin, chymotrypsin and α-amylase to casein level and protein source in Penaeus vannamei (Crustacea Decapoda). J Exp Mar Biol Ecol. 1997;208(1–2):107–25.

    Article  Google Scholar 

  46. Cheng CH, Ye CX, Guo ZX, Wang AL. Immune and physiological responses of pufferfish (Takifugu obscurus) under cold stress. Fish Shellfish Immunol. 2017;64:137–45.

    Article  CAS  PubMed  Google Scholar 

  47. Hernández-López J, Gollas-Galván T, Vargas-Albores F. Activation of the prophenoloxidase system of the brown shrimp Penaeus californiensis Holmes. Comp Biochem Physiol C: Pharmacol Toxicol Endocrinol. 1996;113(1):61–6.

    Google Scholar 

  48. Dhindsa RS, Plumb-Dhindsa PAMELA, Thorpe TA. Leaf senescence: correlated with increased levels of membrane permeability and lipid peroxidation, and decreased levels of superoxide dismutase and catalase. J Exp Bot. 1981;32(1):93–101.

    Article  CAS  Google Scholar 

  49. Aebi H. Catalase in vitro. Methods Enzymol, 105, 121–6. Bonapace G, Iuliano F, Molica S, Peta A, Strisciuglio P (2004). Cytosolic carbonic anhydrase activity in chronic myeloid disorders with different clinical phenotype. Biochim Biophys Acta. 1984;1689:179–81.

    Google Scholar 

  50. Cheng L, Liu WL, Su MP, Huang SC, Wang JR, Chen CH. Prohemocytes are the main cells infected by dengue virus in Aedes aegypti and Aedes albopictus. Parasit Vectors. 2022;15(1):1–7.

    Article  Google Scholar 

  51. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem. 1979;95(2):351–8.

    Article  CAS  PubMed  Google Scholar 

  52. Chiu CH, Guu YK, Liu CH, Pan TM, Cheng W. Immune responses and gene expression in white shrimp, Litopenaeus vannamei, induced by Lactobacillus plantarum. Fish Shellfish Immunol. 2007;23(2):364–77.

    Article  CAS  PubMed  Google Scholar 

  53. Raja RA, Sridhar R, Balachandran C, Palanisammi A, Ramesh S, Nagarajan K. Pathogenicity profile of Vibrio parahaemolyticus in farmed Pacific white shrimp, Penaeus vannamei. Fish Shellfish Immunol. 2017;67:368–81.

    Article  Google Scholar 

  54. Bell TA, Lightner DV. A handbook of normal penaeid shrimp histology. Baton Rouge: World Aquaculture Society; 1988. ISBN:0-935868-37-2.

  55. Bancroft JD, Gamble M (Eds.). Theory and practice of histological techniques. Elsevier Health Sciences. Sixth edition. 2008;33–53.

  56. Muthukrishnan S, Defoirdt T, Ina-Salwany MY, Yusoff FM, Shariff M, Ismail SI, Natrah I. Vibrio parahaemolyticus and Vibrio harveyi causing Acute Hepatopancreatic Necrosis Disease (AHPND) in Penaeus vannamei (Boone, 1931) isolated from Malaysian shrimp ponds. Aquaculture. 2019;511:734227.

    Article  Google Scholar 

  57. Butt UD, Lin N, Akhter N, Siddiqui T, Li S, Wu B. Overview of the latest developments in the role of probiotics, prebiotics and synbiotics in shrimp aquaculture. Fish Shellfish Immunol. 2021;114:263–81.

    Article  CAS  PubMed  Google Scholar 

  58. Lim SY, Loo KW, Wong WL. Synergistic antimicrobial effect of a seaweed-probiotic blend against acute hepatopancreatic necrosis disease (AHPND)-causing Vibrio parahaemolyticus. Probiotics and antimicrobial proteins. 2020;12:906–17.

    Article  CAS  PubMed  Google Scholar 

  59. Liu H, Chen G, Li L, Lin Z, Tan B, Dong X, Zhou X. Supplementing artemisinin positively influences growth, antioxidant capacity, immune response, gut health and disease resistance against Vibrio parahaemolyticus in Litopenaeus vannamei fed cottonseed protein concentrate meal diets. Fish Shellfish Immunol. 2022;131:105–18.

  60. Pu H, Li X, Du Q, Cui H, Xu Y. Research progress in the application of Chinese herbal medicines in aquaculture: a review. Engineering.

    Article  Google Scholar 

  61. AftabUddin S, Siddique MAM, Romkey SS, Shelton WL. Antibacterial function of herbal extracts on growth, survival and immunoprotection in the black tiger shrimp Penaeus monodon. Fish Shellfish Immunol. 2017;65:52–8.

    Article  PubMed  Google Scholar 

  62. Huang Y, Wu Z, Su R, Ruan G, Du F, Li G. Current application of chemometrics in traditional Chinese herbal medicine research. J Chromatogr B. 2016;1026:27–35.

    Article  CAS  Google Scholar 

  63. Pandey G, Madhuri S, Mandloi AK. Medicinal plants useful in fish diseases. Plant Archives. 2012;12(1):1–4.

    Google Scholar 

  64. Lopes-Lutz D, Alviano DS, Alviano CS, Kolodziejczyk PP. Screening of chemical composition, antimicrobial and antioxidant activities of Artemisia essential oils. Phytochemistry. 2008;69(8):1732–8.

    Article  CAS  PubMed  Google Scholar 

  65. Wang W, Zhan Y, Peng L, Gao D, Chen Y, Zhuang X. Artemisinin counteracts Edwardsiella tarda-induced liver inflammation and metabolic changes in juvenile fat greenling Hexagrammos otakii. Fish Shellfish Immunol. 2023;141: 109012.

    Article  CAS  PubMed  Google Scholar 

  66. Yang XM, Zhang FY, Xiang F, Dai ZZ, Yu CM, Li SS. Honeysuckle extract promotes host health by improving intestinal microbes and enhancing intestinal mucosal immunity. Genom Appl Biol. 2020;39:1257–63.

    Google Scholar 

  67. Mimaki Y, Satou T, Kuroda M, Sashida Y, Hatakeyama Y. Steroidal saponins from the bulbs of Lilium candidum. Phytochemistry. 1999;51:567–73.

    Article  CAS  PubMed  Google Scholar 

  68. Abdel-Rahim M, Bahattab O, Nossir F, Al-Awthan Y, Khalil RH, Mohamed R. Dietary supplementation of brown seaweed and/or nucleotides improved shrimp performance, health status and coldtolerant gene expression of juvenile whiteleg shrimp during the winter season. Mar Drugs. 2021;19(3):175.

  69. Elkaradawy A, Abdel-Rahim MM, Albalawi AE, Althobaiti NA, Abozeid AM, Mohamed RA. Synergistic effects of the soapbark tree, Quillaja saponaria and Vitamin E on water quality, growth performance, blood health, gills and intestine histomorphology of Nile tilapia. Oreochromis niloticus fingerlings Aquaculture Reports. 2021;20: 100733.

    Article  Google Scholar 

  70. Wang KHC, Tseng CW, Lin HY, Chen IT, Chen YH, Chen YM, Yang HL. RNAi knock-down of the Litopenaeus vannamei Toll gene (LvToll) significantly increases mortality and reduces bacterial clearance after challenge with Vibrio harveyi. Developmental & Comparative Immunology. 2010;34(1):49–58.

    Article  Google Scholar 

  71. Ardó L, Yin G, Xu P, Váradi L, Szigeti G, Jeney Z, Jeney G. Chinese herbs (Astragalus membranaceus and Lonicera japonica) and boron enhance the non-specific immune response of Nile tilapia (Oreochromis niloticus) and resistance against Aeromonas hydrophila. Aquaculture. 2008;275(1–4):26–33.

    Article  Google Scholar 

  72. Ji SC, Takaoka O, Jeong GS, Lee SW, Ishimaru K, Seoka M, Takii K. Dietary medicinal herbs improve growth and some non-specific immunity of red sea bream Pagrus major. Fish Sci. 2007;73:63–9.

    Article  CAS  Google Scholar 

  73. Tan RX, Zheng WF, Tang HQ. Biologically active substances from the genus Artemisia. Planta Med. 1998;64(04):295–302.

    Article  CAS  PubMed  Google Scholar 

  74. He L, Liang Y, Yu X, Peng W, He J, Fu L, Lu D. Vibrio parahaemolyticus flagellin induces cytokines expression via toll-like receptor 5 pathway in orange-spotted grouper, Epinephelus coioides. Fish & shellfish immunology. 2019;87:573–81.

    Article  CAS  Google Scholar 

  75. Ge LL, Wan HQ, Tang SM, Chen HX, Li JM, Zhang KD, Zhou BP, Fei J, Wu SP, Zeng XB. Novel caffeoylquinic acid derivatives from Lonicera japonica Thunb. flower buds exert pronounced anti-HBV activities. RSC Adv. 2018;8:35374–85. https://doiorg.publicaciones.saludcastillayleon.es/10.1039/C8RA07549B.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Guo Z, Niu X, Xiao T, Lu J, Li W, Zhao Y. Chemical profile and inhibition of α-glycosidase and protein tyrosine phosphatase 1B (PTP1B) activities by flavonoids from licorice (Glycyrrhiza uralensis Fisch). J Funct Foods. 2015;14:324–36. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.jff.2014.12.003.

    Article  CAS  Google Scholar 

  77. Yang CJ, Su DW, Wang YS, Zhang K, Ma SX, Li LQ, Cui G. The adjustment of double flower on obstructive jaundice rats’s intestinal microflora imbalance. Chin Mod Dr. 2012;50(3–4):9.

    Google Scholar 

  78. Hu CM, Jiang H, Liu HF, Li R, Li J. Protective effect of Lonicera japonica total flavone (LJTF) on immunological liver injury in mice. Pharmacol Clin Chin Mater Med. 2007;23:85–8.

    CAS  Google Scholar 

  79. Bej AK, Patterson DP, Brasher CW, Vickery MC, Jones DD, Kaysner CA. Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tl, tdh and trh. J Microbiol Methods. 1999;36(3):215–25.

    Article  CAS  PubMed  Google Scholar 

  80. Yildirim-Aksoy M, Eljack R, Peatman E, Beck BH. Immunological and biochemical changes in Pacific white shrimp, Litopenaeus vannamei, challenged with Vibrio parahaemolyticus. Microb Pathog. 2022;172: 105787.

    Article  CAS  PubMed  Google Scholar 

  81. Tran L, Nunan L, Redman RM, Mohney LL, Pantoja CR, Fitzsimmons K, Lightner DV. Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome affecting penaeid shrimp. Dis Aquat Org. 2013;105(1):45–55.

    Article  Google Scholar 

  82. CABI (Invasive Species Compendium) (2020). Acute hepatopancreatic necrosis disease. Retrieved from: https://www.cabi.org/isc/datasheet/121558#tooverview. Date of access: 10 Apr. 2020.

  83. Liu F, Li S, Yu Y, Yuan J, Yu K, Li F. Pathogenicity of a Vibrio owensii strain isolated from Fenneropenaeus chinensis carrying pirAB genes and causing AHPND. Aquaculture. 2020;735747.

  84. Harikrishnan R, Balasundaram C, Heo MS. Impact of plant products on innate and adaptive immune system of cultured finfish and shellfish. Aquaculture. 2011;317(1–4):1–15.

    CAS  Google Scholar 

  85. Gopinath R, Paul Raj R. Histological alterations in the hepatopancreas of Penaeus monodon Fabricius (1798) given aflatoxin B1-incorporated diets. Aquac Res. 2009;40(11):1235–42.

    Article  Google Scholar 

  86. Cao Z, Chen C, Wang C, Li T, Chang L, Si L, Yan D. Enterocytozoon hepatopenaei (EHP) infection alters the metabolic processes and induces oxidative stress in Penaeus vannamei. Animals. 2023;13(23):3661.

    Article  PubMed  PubMed Central  Google Scholar 

  87. Soto-Rodriguez SA, Gomez-Gil B, Lozano-Olvera R, Bolanmejia C, Aguilar-Rendon KG, Enciso-Ibarra J. Pathological, genomic and phenotypical characterization of Vibrio parahaemolyticus, causative agent of acute hepatopancreatic necrosis disease (AHPND) in Mexico. Asian Fish Sci. 2018;31:102–11.

    Google Scholar 

  88. Han JE, Choi SK, Han SH, Lee SC, Jeon HJ, Lee C, Lee KJ. Genomic and histopathological characteristics of Vibrio parahaemolyticus isolated from an acute hepatopancreatic necrosis disease outbreak in Pacific white shrimp (Penaeus vannamei) cultured in Korea. Aquaculture. 2020;524:735284.

    Article  CAS  Google Scholar 

  89. Nguyen TT, Luu TT, Nguyen TT, Pham VD, Nguyen TN, Truong QP, Hong MH. A comprehensive study in efficacy of Vietnamese herbal extracts on whiteleg shrimp (Penaeus vannamei) against Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (AHPND). Israeli J Aquaculture - Bamidgeh. 2023;75(2):1–22. https://doiorg.publicaciones.saludcastillayleon.es/10.46989/001c.81912.

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Funding

Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). This research received no external funding.

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Authors and Affiliations

  1. National Institute of Oceanography and Fisheries (NIOF), Cairo, Egypt

    Amr Fadel, Amal Khafage & Mohamed M. Abdel-Rahim

  2. Department of Aquatic Animal Medicine and Management, Faculty of Veterinary Medicine, Cairo University, P.O. 11221, Giza, Egypt

    Mohamed Abdelsalam

Authors
  1. Amr Fadel
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  2. Amal Khafage
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  3. Mohamed Abdelsalam
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  4. Mohamed M. Abdel-Rahim
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Contributions

Amr Fadel contributed to the study's supervision, conception and design, material preparation, data collection, and analysis, writing the original draft, and final manuscript. Amal Khafage investigated the histopathological examination. Mohamed M. Abdel-Rahim, and Mohamed Abdelsalam investigated the growth performance and data analysis, writing the original draft and final manuscript. All authors read and approved the final manuscript.

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Correspondence to Amr Fadel.

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Ethics approval and consent to participate

The experimental procedures, including fish handling, examination, and rearing, were conducted following the recommendations and approval of the Committee for Ethical Care and Use of Animals and Aquatic Animals at the National Institute of Oceanography and Fisheries (NIOF, Egypt) under the protocol number NIOFAQ2F23R029. It's important to note that the shrimp samples used in this research were purchased, and therefore, no specific permissions were required for their collection.

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The authors declare no competing interests.

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Fadel, A., Khafage, A., Abdelsalam, M. et al. Comparative evaluation of three herbal extracts on growth performance, immune response, and resistance against Vibrio parahaemolyticus in Litopenaeus vannamei. BMC Vet Res 21, 166 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12917-025-04588-0

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Keywords

BMC Veterinary Research

ISSN: 1746-6148

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