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

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 suspended and adjusted to a concentration of 5 × 106 cells/mL in an ice-cold.

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. Catalase (CAT) activity was determined by measuring the decrease of H2 O2 concentration at 240 nm according to [15]. The reaction mixture contained 50 mm potassium phosphate buffer (pH 7.0) and 10.6 mM H2 O2 freshly prepared.

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 room temperature. After incubation, the supernatant removed from the plate and NBT reduction fixed with 100% methanol for 10 min. The plate was then washed with 70% methanol, and left to air dry. A mixture of 120 mL of 2 M potassium hydroxide and 140 mL DMSO was added to dissolve the resulting formazan blue crystals. The NBT reduction was measured using the microplate reader (Optica, Mikura Ltd., UK) at 630 nm, and respiratory burst activity was expressed as NBT reduction.

Total haemocyte count (THC) was performed in a haemocytomter using microscope. The phenolosidase activity (PO) was evaluated by measuring the formation of dopachrome L-dihydrophenylclamine (L-DOPA) at 490 nm with the aid of spectrophotometer. While reactive oxygen intermediates (ROI) were used to measure H2 O2 by horseadish peroxidase (dependent oxidation of phenol red) while chemiluminescence was used to measure the light emission from reactive oxygen intermediates [18]. Lysozyme activity of fish sera was determined by using lysoplate technique [19]. In brief, 0.60 mg/mL *Micrococcus luteus* was cast in 1% agarose gel (Difco, USA) with 50 mM phosphate buffer (pH 6.2). Wells (6 mm) were created nutrient agar plates and were filled 25 μL of serum samples and incubated for 20 h at 25°C. Lysozyme activity was calculated from a standard curve prepared with lysozyme from chicken egg white. The respiratory burst activity was measured using diagnostic reagent kits (Randox, London, UK) as described by Chiu et al. (2007). Relative protection level (RPL) was estimated as RPL = [(1−%mortality in treatment)/% mortality in control) × 100] [20].
