Previous studies have reported that penaeidin was found to be highly upregulated following a challenge with Vibrio harveyi in giant tiger shrimp [9] and [20] indicating that penaeidins have immune modulatory functions against both pathogens via http://www.selleckchem.com/products/azd9291.html an unknown mechanism [16]. Penaeidins were constitutively expressed in
their mature and active form in granular haemocytes of naive shrimps and were stored within cytoplasmic granules of granular hemocyte populations [21] and [22]. Recently penaeidin like antimicrobial peptide from F. indicus (Fi-Penaeidin) was reported by Antony et al. [44] and the sequence similarity were different from the present study; hence we named the present penaeidin sequence as Fein-Penaeidin. Accordingly, in the present study, (i) the cDNA of the Fein-Penaeidin gene from F. indicus was cloned and sequenced. (ii) The sequence and homology modeling of Fein-Penaeidin from F. indicus was analyzed using bioinformatics tools. (iii) The tissue distribution and variation of expression profiles of Fein-Penaeidin in peptidoglycan and V. parahaemolyticus challenged shrimp were quantified by real-time ZD6474 mw PCR. Indian white shrimp F. indicus of approximately
15–20 g were obtained from the coastal area of Nagapattinam to Chennai, Tamil Nadu, India. They were stocked under natural photoperiod in a recirculating tank system with water salinity and temperature maintained at 30‰ salinity at 30 °C, respectively. The shrimp were fed with a commercial shrimp pellet feed. A
marine pathogenic bacterium from F. indicus was cultured in Thio sulphate citrate bile salt agar (TCBS) for 2 days at 37 °C. Identification of the strain was confirmed by 16s rRNA sequence and the sequence (V. parahemolyticus DAHV1) were submitted to the Gene Bank with accession no: HQ693275. For experiments, fresh culture was prepared by inoculation of an overnight culture (1:100) into fresh medium for further culturing until it reach a density of 0.5–0.6 at OD 600 nm. Hemolymph was collected from the Indian white shrimp F. indicus from the ventral 3-mercaptopyruvate sulfurtransferase sinus cavity of the first abdominal segment using 1 ml syringe containing 0.5ml anticoagulant solution (0.45 M NaCl, 0.1 M glucose, 30 mM sodium citrate, 26 mM citric acid, 10 mM EDTA, pH 7.5, 780 mOsm kg−1) and immediately centrifuged at 500 g at 4 °C for 20 min to separate the haemocytes from the plasma. The resulting hemocyte pellet was used for total RNA isolation. After hemolymph collection, shrimp tissues were harvested by dissection and were immediately frozen in liquid nitrogen and stored at −80 °C. Haemocytes and tissues were further treated according to the various experimental procedures. Total RNA was extracted from the haemocytes of F. indicus using TRIzol reagent and quantified by Bio photometric plus (Eppendorf, Germany) at 260 and 280 nm. Only RNAs with absorbance ratios (A260:A280) greater than 1.8 were used for cDNA synthesis.