**2.3. Evaluation of gut microbiota**

Three prawns were collected from each experimental unit before and after the trial for gut microbiota evaluation. Each prawn was deactivated in freezer at −20°C for 10 min and sterilised using formalin (50 ppm). The guts were aseptically collected and weighed into sterile universal bottles containing peptone water (0.1%) to release the available bacteria for a period of 2 h. 1 mL was taken from each sample bottle and diluted 10-folds and subsequently serially


**2.4. Bacterial challenge**

To evaluate prawn resistance to disease infection, a challenge test using the pathogenic bacteria, *Pseudomonas aeruginosa* and *Aeromonas hydrophila* was performed following 84 days feeding trial. Prior to the challenge test, a preliminary experiment was performed to determine the LD50 (lethal dose) of the pathogenic bacteria. For the challenge test, 10 prawn were selected from each replicate tank and transferred into another tank filled with dechlorinated freshwater. *Pseudomonas aeruginosa* was grown nutrient agar broth and *Aeromonas hydrophila* on PBS broth for 24 h at 35°C in an incubator in Microbiology Laboratory. Bacterial cells were then centrifuged at 3000× g for 30 min to form pellets. The pellets were re-suspended in 1 mL of 0.1% peptone water and after which 100 mL of the suspension were diluted at 4:10 with water from experimental tank. The prawn were exposed to bacterial solution of both *Pseudomonas aeruginosa* (1 × 107 CFU/mL) and *Aeromonas hydrophila* (5 × 105 CFU/mL) for 30 min and returned into experimental set up [13, 14]. The fish were fasted for 24 before infection and feeding with experimental diets resumed 12 h later. All prawn groups were kept under observation for 14 days to record any abnormal clinical signs and the daily mortality.

Gut Microbiota and Innate Immune Response of *Macrobrachium vollenhovenii* Infected…

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Three prawns were randomly selected from each experimental unit. The haemolymph was collected from the ventral part of the haemocoel of the second abdominal segment with the aid of a sterilised syringe and a 21-gauge disposable hypodermic needle containing 1 mL of Alserver's solution and was transferred into anticoagulant bottle (EDTA). The plasma was prepared by centrifuged the haemolymph at 300× g for 10 min at 4°C. The haemocytes were

The innate immune parameters were measured using the diagnostic reagent kits (Randox® Laboratories, Crumlin, County Antrim, UK). Superoxide dismutase (SOD) activity was measured spectrophotochemically by the ferricytochrome c method using xanthine/xanthine oxidase as the source of superoxide radicals. The reaction mixture consisted of 50 mM potassium phosphate buffer (pH 7.8), 0.1 mM EDTA, 0.1 mM xanthine, 0.013 mM cytochrome C and 0.024 IU/mL xanthine oxidase. One activity unit was defined as the amount of enzyme necessary to produce a 50% inhibition of the ferricytochrome C reduction rate measured at 550 nm.

240 nm according to [15]. The reaction mixture contained 50 mm potassium phosphate buffer

The respiratory burst activity was measured using diagnostic reagent kits (Randox, London, UK) as described by [16]. Respiratory burst activity was quantified by the nitroblue tetrazolium (NBT) assay which measures the quantity of intracellular oxidative free radicals; according to [17], with some modification. Briefly, 100 mL of the haemocytes were added to each well of a 96 well microtitre plate (Nalge-Nunc, Hereford, UK). The plate was incubated at 25°C, for 2 h to allow attachment of cells. Unattached cells were washed off three times using fresh L-15 medium. L-15 medium was then supplemented with NBT (1 mg/mL) and phorbol 12-myristate 13-acetate (PMA, SigmaeAldrich; 1 mg/mL) dissolved in dimethyl sulphoxide (DMSO, Sigma), and 100 mL added to each well of the microtitre plate and incubated for 1 h at

Catalase (CAT) activity was determined by measuring the decrease of H2

freshly prepared.

cells/mL in an ice-cold.

O2

concentration at

**2.5. Determination of innate immune response parameters**

suspended and adjusted to a concentration of 5 × 106

O2

(pH 7.0) and 10.6 mM H2

Note: DCP = Dicalcium phosphate; LA = *Lactobacillus acidophilus.*\*Premixes = HI-MIX®AQUA (Fish) each 1 kg contains; vitamin A, 4000,000 International Unit (IU); vitamin D3, 8,00,000 IU; vitamin E, 40, 000 IU; vitamin K3, 1600 mg; vitamin B1, 4000 mg; vitamin B2, 3000 mg; vitamin B6, 3800 mg; vitamin B12, 3 mcg; Nicotinic acid 18,000 mg; Pantothenic acid, 8000 mg; Folic acid, 800 mg; Biotin, 100 mcg; Choline chloride 120,000 mg; Iron, 8000 mg; Copper, 800 mg; Manganese, 6000 mg; Zinc, 20,000 mg; Iodine, 400 mg; Selenium, 40 mg; Vitamin C C(coated), 60,000 mg; Inositol, 10,000 mg; Colbat, 150 mg; Lysine, 10,000 mg; Methionine, 10,000 mg; Antioxidant, 25,000 mg.

**Table 1.** Ingredients and chemical composition (%; on dry matter basis) of experimental diets containing graded levels of *Lactobacillus acidophilus*.

diluted with dilution factor of 10−4. 2 mL was taken from each diluted sample and dispensed into two Petri dishes (1 mL to each). The first dish received plate count agar (PCA, LAB M, LAB149) for total viable count (TVC), while the second Petri dish received MacConkey agar (LAB M, LAB002) for total enterobactereceae count (TEB) using the pure plate count method [11–12]. Each dilution was overlaid, respectively, with PCA and MacConkey that have been cooled to 50°C. At this temperature, agar is still in liquid form [12].

The dishes were then gently swirled to mix the bacteria with the liquid agar. The mixtures were allowed to harden. When the mixture was hardened, the individual cells were fixed in place and incubated (Newlife Laboratory Incubator NL-9052-1) for 24 h at 37°C to allow distinguished colonies to form. The colonies formed were counted using Wincom Colony Counter (16 W, 220 V ± 10%, 50 Hz). The experiments were replicated three times. The TVC and TEB were expressed in Log10CFU/g [12].
