Hemocytes have been proved to be the site of production and storage of AMPs in invertebrates such as horseshoe crabs, mussels and decapod crustaceans [9], [15], [16], Gemcitabine cost [17] and [18]. The brown mud crab, Scylla serrata, is a decapod crustacean of the brachyuran family, Portunidae. They are tolerant to wide range of environmental parameters and there has been a huge interest in the aquaculture of this species due to its high demand/price, high flesh content and rapid growth rates in captivity. Mud crabs are a highly delicious seafood commodity and are therefore an important candidate species for aquaculture.

S. serrata is a well known commercial species in India, Philippines and Vietnam. Like many other decapod crustaceans [8], [19], [20] and [21], the mud crab also possesses broad-spectrum antibacterial activity in its hemolymph that constitutes part of its nonspecific defenses. However, there are hardly any published works on major AMP families (ALF and crustins) present

in this species. The present study aims at the molecular characterization and phylogenetic analysis of two major families of AMPs, viz. anti-lipopolysaccharide factor and crustin from S. serrata. Healthy adults of S. serrata (∼300 g body weight) were collected from the backwater stream along Vypeen, Kochi, India. Hemolymph was collected from the base of abdominal appendages using specially designed much capillary tubes (RNase-free) rinsed using pre-cooled anticoagulant solution (RNase-free 10% sodium citrate, pH 7.0). Total RNA was extracted from the hemocytes using TRI reagent LGK-974 molecular weight (Sigma) following manufacture’s protocol. RNA was quantified by spectrophotometry at 260 and 280 nm. Only RNAs with absorbance ratios (A260:A280) greater than 1.8 were used for the present work.

First strand cDNA was generated in a 20 μl reaction volume containing 5 μg total RNA, 1x RT buffer, 2 mM dNTP, 2 μM oligo d(T20), 20 U of RNase inhibitor and 100 U of M-MLV reverse transcriptase (Ferementas, Inc.). The reaction was conducted at 42 °C for 1 h followed by an inactivation step at 85 °C for 15 min. PCR amplification of 1 μl of cDNA was performed in a 25 μl reaction volume containing 1x standard Taq buffer (10 mM Tris–HCl, 50 mM KCl, pH 8.3), 3.5 mM MgCl2, 200 μM dNTPs, 0.4 μM each primer and 1 U Taq DNA polymerase (Fermentas Inc.). PCR primers were designed using GeneTool software based on consensus sequence. Amplifications were performed using the primers (1) Sc-ALF-F (5′-ggacagaagaaacattgaggacgacgca-3′), Sc-ALF-R (5′-ggaaatcaaaaacatccattacaggtca-3′) and (2) Sc-Crus-F (5′-gagagcagaattagacactgt-3′), Sc-Crus-R (5′-atatagtataacataaccatacttc-3′). The thermal profile used was 94 °C for 2 min followed by 35 cycles of 94 °C for 15 s, 60 °C for 30 s and 68 °C for 30 s and a final extension at 68 °C for 10 min.