Dietary lacto-sacc stimulates the immune response of gravid mud crab (Scylla olivacea)

Highlights

• •1.5 % lacto-sacc enhanced the immunostimulation of gravid mud crabs.
• •Dietary lacto-sacc improved total hemocytes count.
• •Dietary lacto-sacc reduced the mud crab mortality that were negatively correlated with phenoloxidase and prophenoloxidase.
• •Clotting time of hemolymph decreased with increasing of total hemocytes count.

Abstract

Mud crabs have a functioning innate immune system. Probiotics have a considerable effect in improving immune responses in gravid mud crab. The current study investigated the influence of three lacto-sacc concentrations (T1=0 %, T2=1 %, and T3=1.5 %) in a 45 % protein and 12 % lipid meal on the immune response of gravid mud crabs. Three groups of wild gravid mud crabs (average weight: 122.33±1.53 g) were raised individually in bamboo spawning boxes at mangrove pens for 12 weeks. Wild gravid mud crabs (T4) were also caught from the river during the breeding season and tested to offer a more exact assessment of the effect of lacto-sacc. After a 12-week feeding study, T3 therapy resulted in the highest total hemocyte count levels (P < 0.05), followed by T2 and T1. The T3 treatment results in somewhat faster hemolymph clotting, although there is no significant difference among the treatments (P < 0.05). T3 treatments significantly improved immunological parameters (PO, proPO, and SOD enzyme) and survival (61.67±2.89 %) when challenged with Vibrio parahaemolyticus. In the challenge test, the immunological markers THC, PO, and proPO correlated positively, but hemolymph clotting time and mortality linked negatively. Furthermore, T1 was compared to an untreated wild gravid female mud crab (T4), and the results were almost identical. Thus, 1.5 % lacto-sacc is recommended for gravid mud crabs.

Introduction

Mud crab commonly known as mangrove crabs belong to the family Portunidae under genus Scylla with four major species i.e., Scylla olivacea, S. serrata, paramamosain. and S. tranquebarica [1]. Among four the mud crab S. olivacea also known as orange mud crab is commercially important species and most abundant species in Sundarbans Mangrove region [2]. Disease infestation caused by infectious pathogens (virus, bacteria, fungus, etc.) is a serious restriction in crustacean aquaculture, accounting for significant economic loss in the aquaculture industry worldwide [3]. High rates of larval mortality in mud crab hatcheries are typically associated with bacterial infections, despite the fact that antibiotics are extensively used to enhance survival rates [4]. Probiotics have practical significance for crab farming sustainability since they improve immunostimulation, growth promotion, sickness control, and reduce the usage of antibiotics. Probiotic bacteria are used in aquaculture as a microbiological supplement in place of antibiotics [5]. Probiotics are live bacteria that, when consumed in enough amounts, improve the host's health [6]. Probiotics such as microalgae, yeasts, and gram-positive and gram-negative bacteria have been utilized to improve the growth, survival, health, and disease prevention of aquatic animals. Probiotics can produce inhibitory compounds including antibiotic substances, bacteriocins, and siderophores, which compete with pathogens for chemicals and energy while enhancing immunity and restoring microbial balance [7]. Probiotics are used as feed supplements in aquaculture to boost humoral immunity by increasing lysozyme activity, phagocytosis, and respiratory burst [8]. Probiotics as feed additives also enhance the gut health benefits and immune response of the crustaceans and boost up disease resistance capacity [9]. The innate immune response is a conserved feature in crustaceans that aids in pathogen defense via detection, signaling pathways, and inhibition [[10], [11]]. Lacto-sacc, a dietary feed supplement used in mud crab feed and a trademark of Alltech, Inc, USA is a light brown free-flowing powder containing three probiotics mixed-Saccharomyces cerevisiae, Lactobacillus acidophilus, and Enterococcus faecium. Clotting of mud crab hemolymph is a key immune response that protects against systemic infections caused by invading microorganisms [12]. Crustacean hemolymph contains components that aid in hemolymph clotting and serve as a wound healing defensive mechanism by reducing hemolymph loss through wound sealing, maintaining internal osmotic equilibrium, and trapping or rejecting pathogens [[13], [14], [15]]. In crustaceans, hemolymph clotting is a complex biochemical process mediated by proteins identified in mud crab hemocytes [[16], [17]].

Transglutaminases are enzymes found in crustacean hemocytes that, when released into the bloodstream, start a cascade that causes plasma coagulogen cross-linking and hemolymph clotting [[17], [18]]. The amount of time it takes for hemolymph to clot depends on the number of hemocytes in circulation. Hemocyte cells in crustaceans perform a unique role in immunity [18], since they participate in cellular immune responses such as phagocytosis, encapsulation, and cell-to-cell communication, as well as humoral immune responses such as proteolytic cascades [19].

The presence or absence of cytoplasmic granules distinguishes hemocyte cells in crustaceans, and their typical types of hemocyte cells are identified as hyaline cell (without evident granule), semi-granular cell (with some small granules), and granular cell (with high numbers of large granule) [[21], [22]], which are involved in phagocytosis and melanin production by the prophenoloxidase (proPO) system in which proPO is an important component of crustaceans' immune defense mechanism [22], and circulating hemocytes play a vital role in the release of proPO enzyme [23].

Phagocytosis is the process of removing pathogenic microorganisms and foreign particles from the host, whereas proPO eliminates foreign particles and makes them immune to any foreign particles or pathogenic microorganisms that may enter their body by promoting cell-to-cell communication. Melanization is another essential innate defense response in mud crabs against microbial infections [24], which is regulated by the proPO activating system [25]. Few studies on crab proPO and its role in innate immunity have been reported in crustaceans [[26], [27]], but data on proPO in mud crab are still rare, demanding more investigation into the roles of proPO in the immune response [28].

Non-self-recognition proteins activate the proPO cascade, releasing proPO [29], which is found in granular and semi-granular hemocytes [30]. ProPO is the zymogen for the enzyme phenoloxidase (PO). PO is a key enzyme involved in crustacean innate immunity, cuticular sclerotization, and defensive mechanisms against many diseases and parasites. The oxidoreductase PO converts phenols to quinones, which non-enzymatically polymerize to form the pigment melanin [31]. Melanin and its metabolites have been shown to be harmful to pathogenic microorganisms.

The pathogen in the hemolymph is captured in this cascade of melanin, resulting in a darkened, paralyzed blob; this is known as the melanization reaction [33]. Melanization produces reactive oxygen and nitrogen intermediates, which have antibacterial, antifungal, and antiviral effects. Melanization can also increase encapsulation, phagocytosis, and the development of nodules [[32], [33]].

Furthermore, superoxide dismutase (SOD) is an important antioxidant enzyme in crustaceans that removes excess physiologically reactive oxygen intermediates to prevent harmful circumstances [34]. Crustaceans produce a sequence of reactive oxygen intermediates (ROIs) in reaction to environmental changes or microbial incursions in order to endure physiological alterations. However, an excess increase in ROIs has deleterious consequences on the host [35]. The antioxidant enzyme SOD function as defense mechanisms eliminate ROIs most effectively and quickly, and they also serve as a good therapeutic agent against reactive oxygen species-mediated illnesses [36]. The immune parameter assays, which include hemocytes in hemolymph, PO, proPO, and SOD activity are used to assess crustacean immune parameters. Thus, the current study examined the immune responses in female gravid mud crabs to measure the immune response (Hemocytes, PO, proPO, and SOD) following oral administration of dietary lacto-sacc. The results were matched to treated and wild samples to corroborate the results in mud crabs.

Materials & methods

Experimental design and immunostimulants diet preparation

The gravid female mud crabs (S. olivacea) were obtained from a local river in Bangladesh's Sundarbans mangrove forest and randomly assigned to one of three treatments: T1 (0 % lacto-sacc as control), T2 (1 % lacto-sacc), and T3 (1.5 % lacto-sacc) [48,74]. Each treatment includes 20 gravid females that were raised individually in a bamboo-made mud crab breeding box (L 2ft×W 1.5ft×H 1.5 ft). The wild mud crabs were collected and acclimatized in earthen mangrove pens using specially designed mud crab breeding boxes. The crabs were acclimatized in experimental ponds and habituated in formulated feeding (without lacto-sacc) for the first 7 days. Arter that, lacto-sacc based feeding were provided for the experiment. Female mud crabs were chosen because only mated and gravis mud crabs were gathered from the wild and raised on a lacto-sacc based diet for brood mud crab growth. Mud crabs in bamboo-made breeding box were reared in earthen mangrove broodstock pens (L 50 ft x W 24 ft x D 3 ft). The experimental mangrove pens were designed followed by the natural mangrove habitat at Sundarbans region. The bottom of the pens was muddy and full of salt tolerance grass plantation. The dyke of the pens was modified by planting two mangrove plants, Avicennia officinalis (locally known as "Baim") and Bruguiera gymnorrhiza (locally known as "Kankra") in a zigzag pattern to produce a natural mangrove habitat. All the pens were facilitated with inlet and outlet system and full of aeration support.

Immunostimulant diets for mud crabs were prepared using feedstuffs viz., fish meal, rice polish, maize, palm oil, wheat flour, vitamin mix, mineral mix, mycotoxin binder, pellet binder and lacto-sacc (Table 1), which contained two bacteria, L. acidophilus (1.2 × 10⁸ cfu/g) and Enterococcus faecium (7.3 × 10⁷ cfu/g), and one live yeast, Saccharomyces cerevisiae (2.7 × 10⁹ cells/g) that were fully blended homogenously through a mixer homogenizer before adding distilled water. All feeds were manually processed into 2 mm pellets. The pellets were dried overnight under aseptic conditions and kept at −20 °C until use. The pellet samples were sent to the laboratory to be tested for protein, fat, and ash content. Crude protein was evaluated using Kjeldahl after block digestion with Copper Catalyst and steam distillation into Boric Acid (AOAC Official Method 990.20). [75]. Crude fat was determined using diethyl ether, classic Soxhlet extraction method (AOAC Official Method 920.39) [76], and crude fiber was determined using the Fibertec fibercap system (AOAC 962.09). [77].

Table. 1.

Formulation of immunostimulants diet

Ingredients	Feed-1 (control)	Feed-2 (1 % lacto-sacc)	Feed-3 (1.5 % lacto-sacc)
Fish meal (g)	75.90	75.90	75.90
Rice polish (g)	4.00	4.00	4.00
Maize (g)	4.00	4.00	4.00
Palm oil (g)	4.00	4.00	4.00
Wheat flour (g)	3.00	2.00	1.50
Vitamin mix (g)	2.00	2.00	2.00
Mineral mix (g)	2.00	2.00	2.00
Pellet binder (g)	5.00	5.00	5.00
Mycotoxin binder (g)	0.10	0.10	0.10
Lacto-sacc (g)	0.00	1.00	1.50
Total (g)	100.00	100.00	100.00

Note: The vitamin mix/kg (Rovithai, Thailand) contained vitamin A 50 million IU, vitamin D3 10 million IU, vitamin E 130 g, vitamin K3 10 g, vitamin B1 10 g, vitamin B2 25 g, vitamin B6 16 g, vitamin B12 100 mg, vitamin B3 200 g, vitamin B5 56 g, vitamin B7 500 mg, folic acid 8 g, antioxidant 0.200 g and anticake 20 g and mineral mix/kg contained calcium phosphate 397.5 g, calcium lactate 327 g, ferrous sulphate 25 g, magnesium sulphate 137 g, potassium chloride 50 g, sodium chloride 60 g; potassium iodide 150 mg, copper sulphate 780 mg, manganese oxide 800 mg, cobalt carbonate 100 mg, zinc oxide 1.5 g and sodium selenite 20 mg.

The immunostimulant diet was made every three days and fed to gravid female mud crabs twice a day, in the early morning and late evening. Wild gravid mud crab (T4) was also instantly caught from the river during breeding season (Similar size, weight and 100 % light check gravid not significantly different with 0 % lacto-sacc control sample) and evaluated for the comparison of the effect of lacto-sacc. The experiment remained 12 weeks, with mortalities and health status recorded at 12-hour intervals while feeding. Following a 12-week feeding study, hemolymph and tissue samples were randomly obtained from each treatment and transported to the research laboratory. The average length, width and weight of the gravid female mud crab were 6.61±0.21 cm, 9.39±0.46 cm, and 122.33±1.53 g respectively. During sample collection, water temperature ranged from 26.6 to 28.4 °C, pH was 7.4–8.2, dissolved oxygen concentration was 4.8–5.3 mg/L, and salinity was 26.13–26.63 ppt observed among the treatments. Each treatment contains 20 individuals with three replications in which 9 samples from each treatment were used for immunological assessment.

Total and differential hemocytes count

The total hemocyte count (THC) was used to calculate the total number of hemocytes per milliliter of mud crab (S. olivacea) hemolymph. Hemolymph samples were collected from the crabs' walking legs using a sterile 1 mL syringe equipped with a 26-gauge hypodermic needle syringe containing anticoagulant (0.03 M tri sodium citrate, 0.45 M sodium chloride, 0.01 M EDTA, 0.1 M glucose, and 0.026 M citric acid, pH 7.5) in a 1:1 ratio and immediately placed on ice to prevent clotting [37]. A 20 µL aliquot of each sample was placed in a tube with 5 µL of 1.2 % Rose Bengal Dye Solution (Sigma-Aldrich, USA) in 50 % ethanol and incubated at room temperature (25 ºC) for 20 min [21]. A drop (about 15 µL) of anticoagulant-hemolymph combination was placed in a hemacytometer to determine the number of hemocytes microscopically using an inverted phase-contrast Euromex microscope (iScope IS-1153PLi/SLC, Netherlands). The THC was determined using the following formula proposed by Song and Hsieh [37]: THC (per cubic millimeter) is calculated as (count of total hemolymph x dilution factor × 2 × 10⁴) divided by the number of squares counted.

The differential hemocyte count (DHC) was used to determine the relative quantity of each hemocyte cell type per milliliter of mud crab hemolymph. Mud crab hemolymph-anticoagulant combination was wet mounted on a clean glass slide and cover slip to count DHC [18]. Each slide contained approximately 150–200 hemocyte cells, depending on the thickness of the smear. The hemocytes were categorized as hyalinocytes (HC), granulocytes (GC), and semigranulocytes (SGC) using the criteria established by Jussila et al. [38].

Counting of hemolymph clotting time

The hemolymph clotting time was assessed immediately after collection of hemolymph samples from live mud crabs following protocol of Jussila et al. [39]. The 25 µL of neat hemolymph sample taken inside a precooled (in ice) plain soda lime glass capillary tube. The inner diameter of glass capillary tube was 1.1–1.2 mm and length 75 mm. The hemolymph sample was then inserted to glass capillary tube and the tube was turned to a straight vertical position with the sample in the upper end. The tube was maintained vertically until the gravity forced hemolymph column reached the lower end of the tube, after which the tube was turned 180 and repeated the process until the hemolymph fully clotted.

Analysis of superoxide dismutase activity assay

The SOD activity of gravid mud crabs was tested using the Ghosh et al. [42] method and the Creative BioMart, Inc., USA procedure (EC 1.15.1.1) [42]. This methodology was designed to determine the activity of superoxide dismutase in a food or enzyme sample. One g of tissue sample obtained from the chelate legs of mud crab was finely pulverized and homogeneously mixed with 9.0 mL of deionized water for 10 min. The mixture was transferred to a centrifuge tube containing 10 mL deionized water and centrifuged at 4000 g for 15 min. The supernatant was collected and stored at −20 °C for subsequent analysis. A Tris-EDTA buffer solution (solution A) and a 0.2 mM pyrogallol solution (solution B) were prepared according to the manufacturer's instructions (EC 1.15.1.1). Mix 2.35 mL of solution A with 2.00 mL of deionized water, then add 0.15 mL of solution B and vortex immediately. An aliquot was collected from the test tube and measured the absorbance at 325 nm using spectrophotometer (Peak instruments, C-7200, USA). Another aliquot was obtained after 1 min to measure the differences in absorbance between the two aliquots. Finally, the SOD activity was estimated using the procedure below.

$$SODactivity\left(U/mg\right)=\frac{\frac{\Delta A_{325blank}-\Delta A_{325sample}}{\Delta A_{325blank}}\times 100\%}{50\%}\times 4.5\times \frac{D}{V}\times \frac{V_1}{m}$$

Where, U/mg is the SOD activity unit; ΔA_{325 blank} is the auto-oxidation rate determined in the blank test; ΔA_{325 sample} is the auto-oxidation rate determined using a sample; V is the volume of sample used for testing in mL; D is the dilution factor of the sample; V₁ is the total volume of the sample solution in mL; m is the weight of the solid sample in gram and 4.5 is the total volume of the reaction mixture in mL.

Analysis of phenoloxidase and prophenoloxidase activity assay

The mud crab's PO activity was tested using a slightly modified Huynh et al. [40] method. The PO activity was determined spectrophotometrically by recording the production of dopachrome from l-dihydroxyphenylalanine (L-DOPA) [41]. Zymosan (0·1 %) was produced in cacodylate buffer (sodium cacodylate 10 mM, CaCl2 100 mM, pH 7.0) and centrifuged for 10 min at 2000 g. The supernatant was employed as an elicitor of the proPO system [42]. Each sampling tube contained 500 μL of diluted hemolymph, which was centrifuged at 700 g at 4 °C for 20 min. The supernatant fluid was discarded, and the pellet was washed and gently re-suspended in 500 μL cacodylate citrate buffer (10 mM sodium cacodylate, 450 mM sodium chloride, 100 mM trisodium citrate; pH 7.0) and centrifuged again at 1000 g at 4 °C for 10 min. The supernatant was discarded, and the pellet was re-suspended in 100 μL of cacodylate buffer solution (10 mM sodium cacodylate, 450 mM sodium chloride, 10 mM calcium chloride, and 260 mM magnesium chloride; pH 7.0). Cell suspensions were treated with an identical volume of zymosan for 1 hour at room temperature (25 °C) before centrifugation at 700 g for 5 min. The supernatant was transferred to an Eppendorf tube with 50 μL of l-DOPA and 800 μL of cacodylate buffer added 5 min later. To measure background PO activity, a control solution was prepared with 100 mL of cell suspension, 50 μL of cacodylate buffer, 50 μL of l-DOPA, and 800 μL of cacodylate buffer. The absorbance was measured at 490 nm using spectrophotometer (Peak instruments, C-7200, USA). The proPO activity of mud crab hemolymph was also assessed spectrophotometrically (490 nm) by recording the synthesis of dopachrome generated from l-dihydroxyphenylalanine (L-DOPA) using a modified approach of Sarathi et al. [3]. To obtain the pellet, hemolymph was centrifuged at 800 × g at 4 °C for 20 min. The pellet was then gently suspended in cacodylate buffer (0.01 M sodium cacodylate, 0.45 M sodium chloride, 0.10 M trisodium citrate, pH 7.0). The suspended pellet was centrifuged again and resuspended in 100 μL of cacodylate buffer. To activate the resuspended pellet, 50 μL of trypsin was added at 25 °C for 10 min. Next, 50 μL of DOPA was added, followed by 600 μL of cacodylate buffer 5 min later. Finally, the optical density was measured at 490 nm.

In vivo challenge test

The in-vivo challenge test was carried out using the method described by Hannan et al. [43]. Crablets were gathered from a mangrove-based natural mud crab hatchery (2.3 ± 0.56 g) and raised in a 1000 L tarpaulin tank with plastic pipe shelter. Plastic pipes were tied together and placed inside the tanks. Each treatment consists of 20 crablets, with three replications. Crablets were randomly assigned to three treatment groups based on experimental diets (T1 0 % lacto-sacc, T2 = 1 % lacto-sacc, T3 = 1.5 % lacto-sacc, and T4 = wild crablets fed with tilapia fish) with continuous aeration support. Crablets were fed twice every day based on their body weight (5 %). After 90 days of growing, crablets (14.33±2.08 g) were infected with the pathogen Vibrio sp. The bacterial inoculums were prepared by mixing 90 mL (10 mL/L water) of 24-hour TCBS broth culture with 8910 mL of sterile saline solution (2 % w/v NaCl). Before being moved to the rearing tanks, the crablets were immersed in a bacterial suspension at room temperature for 20 min at a concentration of 10 mL/L. Bacterial burdens were counted in each infection tank. The bacterial load of fresh bacterial inoculums was evaluated using the spread plate technique after 10^–3 to 10^–6 serial dilution. Bacterial samples were placed on the surface of nutrient agar plates and dispersed with a flamed, bent glass rod. After 24 h of incubation at 37 °C, the bacterial colony of 30–300 was counted using the formula cfu/mL= (number of colonies × dilution factor)/volume of culture plated in ml. The bacterial load was measured to determine the optimal pathogenic bacteria dose (cfu/mL). The Vibrio sp. pathogen was isolated from infected eggs of wild mud crabs using selective media (TCBS) and confirmed using morphological (color, size, shape, elevation, motility, growth at 4 °C, 20 °C, 30 °C and 40 °C) and biochemical tests (Growth at 0 %, 2 %, 4 %, 6 %, 8 %, and 10 % NaCl, Gram's staining, gram's test, oxidase, catalase, Voges-proskauer, methyl red, indole production, H₂S production, Sensitivity to a vibriostatic agent 0/129 (10 µg), and hemolysis test). Infected eggs were collected from the brood mud crabs (S. olivacea) that were collected from the wild. The characterization of hemolysis properties of the isolated Vibrio pathogen was tested in sheep blood agar plate containing 5 % NaCl and incubating at 37 °C for 24 h following protocol of Darmawati et al. [44]. The number of dead crablets was counted at 12-hour intervals for 14 days during the challenge test.

Statistical analysis

All data were analyzed using Microsoft Excel 2016 and data sets were presented as mean ± SD. The statistical analysis of this experiment was carried out using analytical software Statistix 10.0 (StatSoft, USA). A one-way ANOVA (multiple comparison) at P < 0.05 confidence level for mud crab hemolymph whereas immune parameters PO, proPO and SOD were compared at P < 0.01 confidence level. The mean values were separated by LSD posthoc statistic. Correlation of hemolymph PO, proPO, and SOD were analyzed using R software (version: R x64.4.0.3).

Results

Proximate composition of the lacto-sacc diet

The proximate content of the lacto-sacc diet was determined by three types of feed. The proximate composition of the 1.5 % lacto-sacc diet is 45 % protein, 12 % fat, 3 % ash, and 40 % carbohydrates, whereas the 1 % lacto-sacc diet has 40 % protein, 16 % fat, 3 % ash, and 41 % carbs. Furthermore, the proximate makeup of a 0 % lacto-sacc diet includes 35 % protein, 16 % fat, 3 % ash, and 46 % carbs. The proximate composition of formulated feed ingredients is provided in table 6.

Table. 6.

Proximate composition of formulated feed ingredients.

Ingredient Name	Moisture (%)	CP (%)	Dig. CP (%)	EE (%)	CF (%)	Ca (%)	T. P (%)	A. P (%)	Ash (%)	ME (Kcal/Kg)	Na (%)	Cl. (%)	K (%)
Maize	14.50	7.500	4.88	3.00	2.80	0.02	0.22	0.12	1.25	3250.00	0.05	0.04	0.32
Rice polish	9.50	13.00	8.45	19.00	11.50	0.04	1.00	0.15	9.00	2850.00	0.10	0.17	1.17
Fish meal	8.00	60.00	48.00	2.00	1.00	6.50	4.20	3.50	15.00	2800.00	0.47	0.55	0.32
Wheat flour	8.20	10.00	6.50	1.77	2.00	0.34	0.37	0.10	5.55	3300.00	0.02	0.05	0.45
Palm oil	–	38.20	30.45	1.00	3.50	0.02	650	0.370	–	3200.00	0.05	0.05	1.90
Lacto-sacc	7.5	8.200	6.50	0.02	0.50	0.10	0.10	0.10	2.20	850.00	0.20	0.20	0.20

Note: CP - Crude Protein, CF - Crude Fiber, TP -Total Phosphorous, AP - Available Phosphorous, ME - Metabolizable Energy, Ca – Calcium, EE - Ether Extract, Dig. CP - Digestible Crude Protein.

Total hemocytes count, differential hemocytes count, and clotting time

The present study implies that formulated feed containing dietary lacto-sacc increased hemocytes in mud crab hemolymph. The clotting time of mud crab hemolymph was negatively correlated (r = −0.46, p < 0.05) with total hemocytes count (Fig 1& Fig 6) but there was no significant difference in clotting time among the treatments (Fig 1). The clotting time was lower in the treatment T3 (1.5 % lacto-sacc) whereas total hemocytes count (5.75±0.23 × 10⁶ cells/mL) was higher T3. However, the clotting time was higher in T1 (0 % lacto-sacc) and total hemocytes count (1.74±0.98 × 10⁶ cells/mL) was lower in T1. The result implying that, application of 1.5 % lacto-sacc (T3) increase hemocytes 30.17 % higher than control (T1) at P < 0.05 (Fig. 1). In addition, T2 (1 % lacto-sacc) also increases the hemocytes (5.53±0.29 × 10⁶ cells/mL) that are significantly different from the control (T1) (Fig. 1). Furthermore, there was no variation in clotting time between T4 and T1 although THC has been little bit higher in wild sample T4 compared to T1 (Table 2).

Fig. 1.

Plotting of total hemocyte count (cells/ml) and clotting time.

Fig. 6.

Correlation among immune parameters (THC, PO, proPO, SOD, clotting time) and in vivo challenges test.

Table. 2.

Comparison of wild gravid female as natural control (T4) and 0 % lacto-sacc treated control (T1).

Treatment	THC (No of Cells/ml)	Clotting time (s)	PO (Unit/ml)	proPO (Unit/ml)	SOD (Unit/g)
T4 (Wild gravid as natural control)	2.11±0.15 × 106a	11.33±0.50a	0.034±0.008a	0.034±0.005a	1.99±0.76a
T1 (0 % lacto-sacc as treated control)	1.735±0.98 × 106a	11.67±0.50a	0.022±0.006a	0.031±0.032a	1.93±1.39a

Note: THC-Total hemocytes count, PO- Phenoloxidase, proPO- prophenoloxidase, SOD-Superoxide dismutase.

The differential DHC was studied in this experiment when treated with different concentration of dietary lacto-sacc. Electron microscopy of the hemolymph revealed the organelles and characteristics of the hemocytes. In according with the observed cell morphology and granularity the hemocytes were classified into five distinct types of cells viz., HC, SGC, LG, GC and SG (Fig. 2). The cells called HC were smaller, oval, rounded, or irregular in shape (raisin-like), with a dark blue or violet nucleus and little to no cytoplasm. Their nucleus lies at the center of the cell, and their cytoplasm is devoid of granules or contains only a few granules per cell. The GC were larger than HC and had a spherical, clearly visible dark or light blue central nucleus, with three possible subgroups: eosinophilic, basophilic, and neutrophilic granulocytes. The SG were either spherical or irregular in shape, with a pink or light violet nucleus and little or no cytoplasm. In addition, the SGC were spherical, fusiform, or oval shaped (Fig. 2).

Fig. 2.

Characterization of S. olivacea hemocytes HC- hyalinocytes, SGC-small granulocytes, LGC-large granulocytes, GC-granulocytes, SG-semigranulocytes, N-nucleus, C-cytoplasm.

The SGC contains small cytoplasmic granules in cytoplasm that can be seen as pink color when stained by rose Bengal dye solution. Besides, the shape of the LGC was rounded when observed under microscope. The LGC was the largest cell in hemocytes, and it contains the highest number of granules per cell. The lowest density was observed in HC, LGC, and SGC among the treatment whereas the highest density of hemocytes was observed in SG and GC cells when treated with 1.5 % lacto-sacc (T3) compared to 0 % lacto-sacc (T1) and (Table 3). The SG (%) and GC (%) were increased after 12 weeks of lacto-sacc treatments and decreased SGC (%) and LG (%) in T3 treatment whereas HC (%) was almost similar as before. On the other hand, SG (%) was also observed higher at T2 treatment but SGC (%) was lower. The other parameters were almost similar among the treatments whereas the variation of T1 and T4 was almost similar, and no significant difference was observed (Table 2).

Table. 3.

Different types of hemocytes in mud crab hemolymph.

Treatments	No. of hemocytes/ml	Different types of hemocytes
		HC (%)	SG (%)	GC (%)	SGC (%)	LG (%)
T4	2.11±0.15 × 106b	12.50±1.1b	31.53±1.8d	18.83±0.3b	18.5 ± 3.10a	18.70±0.5a
T1	1.735±0.98 × 106b	9.13±1.05b	36.63±3.6c	18.17±1.3b	18.20±1.4a	18.13±1.0a
T2	5.53±0.29 × 106a	16.63±2.1a	54.37±3.4a	12.40±2.9c	0.2 ± 0.26b	16.50±1.1b
T3	5.75±0.23 × 106a	11.07±2.4b	44.37±1.8b	33.30±2.2a	3.70±1.50b	7.53±0.42c

Note: HC-hyalinocytes, SG-semigranulocytes, GC-granulocytes, SGC-small granulocytes, LG-large granulocytes.

Analysis of phenoloxidase, prophenoloxidase and superoxide dismutase activity assay

The immune parameters of PO and ProPO activity assay (unit/mL) was measured in female gravid mud crabs (S. olivacea) hemolymph whereas the SOD activity assay (unit/g) was measured using mud crab muscle. The activity assay of PO and proPO was observed higher in T3 treatment in which the gravid mud crabs were treated with 1.5 % dietary lacto-sacc. The value of PO was 0.022±0.006, 0.076±0.077 and 0.165±0.023 whereas the value of proPO was 0.031±0.032, 0.040±0.016 and 0.121±0.014 for the treatment T1, T2 and T3 respectively which was significantly difference at P < 0.05 (Fig. 3). The application of 1 % dietary lacto-sacc also increases the value of PO and proPO, but not significantly different from the treated control samples (T1). The proPO activity assay was 3.6 times higher in T3, and 3.1 times higher in T2 compared to T1 treatment whereas the activity of PO was 4.9 times higher in T3 (1.5 % lacto-sacc), and 2.2 times higher in T2 (1 % lacto-sacc) compared to T1 (0 % lacto-sacc) treatment.

Fig. 3.

(A) Phenoloxidase (PO) and prophenoloxidase (proPO) activity asay; (B) Superoxide dismutase (SOD) activity assay.

Furthermore, the activity assay of SOD was also higher in T3 treatment but not significantly different among the treatments. The SOD value was 1.93±1.39, 3.07±1.12 and 4.89±1.61 for the treatment T1, T2 and T3 respectively (Fig 3). The SOD was increased 2.45 times higher in T3 (1.5 % lacto-sacc), and 1.59 times higher in T2 (1 % lacto-sacc) compared to T1 (0 % lacto-sacc) treatment. The linear trendline in the graph clearly indicated the increase of PO, proPO and SOD activity assay. On the other hand, the difference of PO, proPO and SOD value were almost similar between the treatment T1 and T4 (Table 2).

In vivo challenge test

The pathogen was confirmed as Vibrio parahaemolyticus through a series of biochemical tests (table 5). The pathogenicity test at sheep blood agar produced β hemolysis meaning completely breaks down the red blood cells and hemoglobin completely and leaves a clear zone around the bacterial growth (Fig. 4). The bacterial load of fresh broth culture in TCBS media was 1.2 × 10⁷±2.08×10⁶ cfu/mL when infected with the samples through immersion method. In in vivo challenge test the highest mortality (91.67±7.64 %) was observed in T1 treatment and lowest mortality (38.33±2.89 %) was observed in T3 treatment (Table 4). Whereas 60.00±13.23 % mortality was observed in T2 treatment. The mortality rate was also slightly lower in T4 samples than that of control T1 treatment (Table 4). Mortality started on day 2 after infection and it was continued at different treatments up to day 12. Mass mortality was observed in day 4 and day 5 among the treatments. Some external sign of black spot was also observed on day 12 in different parts of the body of crablets (Fig. 5).

Table. 5.

Identification of Vibrio parahaemolyticus through morphometric and biochemical test.

Type of test	List of tests	Pathogenic strains isolated from infected mud crab eggs
Colony characteristics	Color in NA media	Brownish
	Color in TCBS media	Greenish
	Size of colony	Large
	Shape of colony	Round
	Elevation of colony	Convex
Morphological characteristics	Shape	Small rod shaped
	Motility	+
	Growth at 4 °C	–
	Growth at 20 °C	+
	Growth at 30 °C	+
	Growth at 40 °C	+
Biochemical tests	Gram's staining	–
	Gram's test	–
	Growth at 0% NaCl	–
	Growth at 2% NaCl	+
	Growth at 4% NaCl	+
	Growth at 6% NaCl	+
	Growth at 8% NaCl	+
	Growth at 10% NaCl	–
	Indole production	+
	Oxidase	+
	Voges-proskauer (VP)	–
	Methyl Red (MR) test	–
	Sensitivity to a vibriostatic agent 0/129 (10 µg)	+
	Oxidase	+
	Catalase	+
	H2S production	–
	Hemolysis	β (Beta)
Identified species		Vibrio parahaemolyticus

Fig. 4.

(A) Isolation of V. parahaemolyticus in TCBS media; (B) Pathogenicity test in sheep blood agar media.

Table. 4.

In vivo challenge crablets with the pathogen Vibrio parahaemolyticus.

Treatments	Stocking density	Pathogenic sp.	CFU/mL	Mortality (%)	Survival (%)
T4	20	Vibrio parahaemolyticus	1.2 × 107±2.08×106	81.67±7.64a	18.33±7.64c
T1	20			91.67±7.64a	8.33±7.64c
T2	20			60.00±13.23b	40.00±13.23b
T3	20			38.33±2.89c	61.67±2.89a

Fig. 5.

(A). Infected crablet due to V. parahaemolyticus, black spot at different parts of the body (red circle); (B). Healthy crablet.

Correlation analysis of THC, clotting time, PO, proPO, SOD, and mortality

The correlation among the immune parameters (THC, clotting time, PO, proPO, and SOD) and mortality is plotted in Fig. 6. A positive correlation was observed among the immune parameters THC, PO and proPO but negative correlation was observed in hemolymph clotting time and mortalities in challenge test. A strong positive correlation (r = 0.71, P < 0.05) was observed between PO and proPO activity assay whereas moderate positive correlation was observed for THC-PO (r = 0.56, P < 0.05) and THC-proPO (r = 0.57, P < 0.05) correlation. Furthermore, strong negative correlation was observed between immune parameters and crablet mortalities viz., PO-mortality (−0.84, P < 0.01), proPO-mortality (−0.72, P < 0.05), THC-mortality (−0.71, P < 0.05). In the case of SOD activity assay observed moderate negative correlation (−0.58, P < 0.05) with mortality. In addition, all the immune parameters are negatively correlated with hemolymph clotting time whereas a strong negative correlation (−0.67, P < 0.05) was also observed between proPO activity assay and hemolymph clotting time.

Discussion

Since crustaceans are unable to produce immunoglobulins and must rely solely on their innate immune system to protect them from invasive pathogens (virus, bacteria, fungus, protozoa, and parasites), their innate defense systems comprise both cellular and humoral components [[45], [46],50]. The purpose of this study was to evaluate the impact of a probiotics on immune parameters such as THC, DHC, clotting time, and assays for PO, proPO, and SOD activity.

Probiotics can promote growth, reproduction, digestion, and immune response by increasing hematological parameters [[51], [52], [53], [54], [55], [56]]. The current study suggests that formulated feed including dietary lacto-sacc raised THC in mud crab (S. olivacea) hemolymph, and that increasing hemocyte count reduces hemolymph clotting time (Fig 1).

Dietary lacto-sacc is a live probiotic consisting of L. acidophilus, E. faecium, and S. cerevisiae used in this experiment because it has been reported that probiotics, which are non-digestible substances added to the diet, can specifically encourage host activity and boost immunity in crustaceans [[47], [49], [57], [58]]. The best performance was obtained when the samples were treated with 1.5 % dietary lacto-sacc, which boosted THC levels by 30.17 % with a shorter clotting time. Similarly, Battelle and Kravitz [51] discovered that clotting time in crustaceans is favorably connected with the neurohormone octopamine, which, when combined with hemocytes, reduces clotting time and prevents hemolymph losses following injuries. In crustaceans, hemolymph clotting is an important wound healing mechanism [[14], [61]], and changes in the number of circulating hemocytes in hemolymph and clotting time have been identified as stress indicators [40,39,52]. However, no significant difference in hemolymph clotting time was seen between the therapies in the current investigation. The clotting time in mud crab hemolymph is extremely short (10–12 s), maybe due to the fact that hemolymph was obtained from healthy gravid mud crabs and examined in a glass capillary tube without anticoagulant.

Furthermore, the DHC was investigated in this experiment after treatment with various concentrations of dietary lacto-sacc, revealing HC, SGC, LG, GC, and SG cells in the hemolymph whereas the changes were observed in SG (44.37±1.85 %) and GC (33.30±2.29 %) for T3 and treatment after 12 weeks. In addition, the height number of SG (54.37±3.41 %) observed in T2 treatment that was also the 1 % lacto-sacc challenge treatment.

The morphological traits were identical to those reported by Jussila et al. [38]. The most prevalent hemocytes in mud crab hemolymph were SG and GC. Semi-granular and granular cells triggered the ProPO activity assay [78,79]. At the present study the percentage of granulocyte and semigranulocytes cells was higher among the hemocytes in mud crab hemolymph when treated with 1.5 % dietary lacto-sacc (T3) as feed additives compared to other treatments. The semigranulocytes, which contain a variable number of tiny cytoplasmic granules containing PO activating enzymes, and the granulocytes, which contain a high number of cytoplasmic granules that contain proPO enzyme, the zymogen precursor to PO reported by Day et al. [53]. Granular cells' primary function is to activate ProPO, although [38] found no evidence of phagocytic activity. It has also been proposed that GC in freshwater crayfish are responsible for the creation of ProPO [20], however ProPO production in mud crabs is validated by granular cells [54]. In contrast, two crayfish species, Pacifastacus leniusculus [55] and Procambarus clarki [56], promoted ProPO synthesis by both SG and GC cells.

The functions of each hemocyte subtype are still debated [64,58]. THC and DHC serve as indicators of mud crab stress or health state, although they are lowered when infected. T3 treatments with 1.5 % lacto-sacc feed demonstrated the best results. Furthermore, hyaline cells function as phagocytic cells in the presence of yeast particles [59]. Crustacean semi-granular cells have been shown to operate as a cell encapsulation mechanism [60]. Furthermore, SG cells are recognized as the most significant hemocyte subpopulation in terms of immunological features. The SG cells are in charge of capsule formation, encapsulation, and modest phagocytic activity, as well as melanization via ProPO cascade reactions [21,52,[58], [62],63,68,69]. In addition, due to their low abundance in the bloodstream, numerous research has revealed conflicting findings on the functional activity of HC [20,63]. Hyalinocytes have been described as being released under certain conditions, particularly following immunological challenge [64,65].

The use of probiotic mixtures boosted the production of PO and SOD in gravid mud crabs. The probiotics L. acidophilus was used in the current investigation in conjunction with E. faecium and S. cerevisiae to stimulate the formation of PO and SOD activities. The results indicate that T3 treatment with 1.5 % dietary lacto-sacc resulted in greater hemocyte, PO, and SOD activity assays than T2, T1, and T4 treatments. Similarly reported by Wang and Gu [73]. According to the findings, the probiotics L. acidophilus improved mud crab immune responses by producing PO and antibacterial activity, whilst SOD activity was boosted by 8.97 % in shrimp [66]. The synthesis of SOD in hemolymph is significantly connected with innate immunity in invertebrates, which stimulates phagocytosis and protects cells from free radicals [1]. In the current study, SOD rose 2.45 times in T3 and 1.59 times in T2 as compared to T1. Campa-Córdova et al. [36] reported a 1.4-fold increase in SOD in crab muscle compared to the control group.

The PO is the final enzyme in the proPO-activating system. The production of PO in hemolymph is critical for cuticular sclerotization and the defensive mechanism against pathogenic organisms and parasites [67]. The PO enzyme converts o-diphenols and tyrosine into quinones, which are then converted into melanin through a series of non-enzymatic processes and attach to wound sites or pathogen surfaces [75,76]. Furthermore, reactive oxygen and nitrogen intermediates are formed during the melanization process, which have antibacterial, antifungal, and antiviral effects. Melanization can also increase encapsulation, phagocytosis, and the development of nodules [34,35,77].

There was a strongly negative correlation (r = −0.71) between THC and mortality because hemocytes play a vital function in pathogen removal and enhancing crustacean immunity. The study found that T3 had a higher survival (61.67±2.89 %) than T1 when challenged with V. parahaemolyticus in vivo followed by T1 treatment (8.33±7.64 %) which was similar to the research conducted by Yang et al. [70].

Crustacean hemocytes serve a vital role in eliminating foreign particles such as bacteria from mud crab hemolymph through the phagocytosis process [71] and stimulating immunity against viral infection in crustaceans such as crayfish, penaeid shrimp, and crabs [72]. Wu et al. [73] reported that the probiotic E. faecalis isolated from the intestine of mud crab (S. paramamosain) showed significant enhancement of growth and serum enzyme activities (PO, lysozyme, and SOD), as well as resistance to V. parahaemolyticus when challenged with a supplemented diet containing (10⁹ cfu/g) of probiotics for 6 weeks, with a recorded survival rate of 66.67 %. Wu et al. [73] also found that mud crab (S. paramamosain) juveniles fed a probiotic diet (10⁵ cfu/g) for 30 days showed increased expression of immune-related genes (CAT, proPO, and SOD), as well as disease resistance against V. parahaemolyticus, with a higher final survival (78.33 %).

Conclusion

The current study investigated the effect of different concentrations of dietary lacto-sacc on female gravid mud crabs. The results indicated that T3 outperformed T2 and controlled T1. The application of dietary lacto-sacc significantly increases total hemocyte count, phenoloxidase, prophenoloxidase, and superoxide dismutase, and reduced hemolymph clotting time and mortality during in vivo challenge test. The dietary lacto-sacc also increased the percentage of semigranulocytes and granulocytes cells in mud crab hemolymph. More investigations with different concentration of dietary lacto-sacc are needed to confirm the best immunological response of mud crabs during brood development.

CRediT authorship contribution statement

Md. Abdul Hannan: Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Data curation. Mohammad Bodrul Munir: Supervision, Conceptualization. Roslianah Asdari: Writing – review & editing, Supervision. Md. Shoebul Islam: Formal analysis. Rabina Akther Lima: Data curation. H.M. Rakibul Islam: Writing – review & editing. Md. Harunor Rashid: Writing – review & editing. Henry Wong Yip Hing: Resources.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.

Data availability

• Data will be made available on request.