a b s t r a c tA crustin-like antimicrobial peptide from the haemocytes of giant tiger shrimp, Penaeus monodon was partially characterized at the molecular level and phylogenetic analysis was performed. The partial coding sequence of 299 bp and 91 deduced amino acid residues possessed conserved cysteine residues characteristic of the shrimp crustins. Phylogenetic tree and sequence comparison clearly confirmed divergence of this crustin-like AMP from other shrimp crustins. The differential expression of the crustin-like AMP in P. monodon in response to the administration of various immunostimulants viz., two marine yeasts (Candida haemulonii S27 and Candida sake S165) and two ␤-glucan isolates (extracted from C. haemulonii S27 and C. sake S165) were noted during the study. Responses to the application of two gram-positive probiotic bacteria (Bacillus MCCB101 and Micrococcus MCCB104) were also observed. The immune profile was recorded pre-and post-challenge white spot syndrome virus (WSSV) by semi-quantitative RT-PCR. Expressions of seven WSSV genes were also observed for studying the intensity of viral infection in the experimental animals. The crustin-like AMP was found to be constitutively expressed in the animal and a significant down-regulation could be noted post-challenge WSSV. Remarkable down-regulation of the gene was observed in the immunostimulant fed animals pre-challenge followed by a significant up-regulation post-challenge WSSV. Tissue-wise expression of crustin-like AMP on administration of C. haemulonii and Bacillus showed maximum transcripts in gill and intestine. The marine yeast, C. haemulonii and the probiotic bacteria, Bacillus were found to enhance the production of crustin-like AMP and confer significant protection to P. monodon against WSSV infection.

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AMPs are evolutional weapons, widely used by animals and plants in their innate immune system to fend off invading microbes. The present study reports characterization of a new ALF isoform (Sc-ALF; HQ638024) and the first crustin (Sc-crustin; HQ638025) from the mud crab, Scylla serrata. The full-length cDNA of Sc-ALF consisted of 477 bp with an ORF of 123 amino acids and a putative signal peptide of 26 amino acids. Sc-ALF had a predicted molecular weight (MW) of 11.17 kDa and theoretical isoelectric point (pI) of 9.95. Two highly conserved cysteine residues and putative LPS binding domain were observed in Sc-ALF. Comparison of amino acid sequences with neighbor-joining tree indicated that Sc-ALF shared maximum similarity with ALF of S. paramamosain. Peptide model of Sc-ALF created using SWISS-MODEL server was found to consist of two α-helices crowded against a four-strand β-sheet. The full-length cDNA of Sc-crustin consisted of 433 base pairs with an ORF of 111 amino acids and a putative signal peptide of 21 amino acids. Comparison of amino acid sequences with a neighbor-joining tree revealed that Sc-crustin shared high identity with other known crustins characterized from S. paramamosain, P. trituberculatus, H. araneus, C. maenas and F. chinensis. A whey-acidic-protein domain could be detected at the C-terminus with the characteristic four disulfide core. Sc-crustin had a predicted MW of 10.24 kDa and a pI of 8.76. Peptide model of Sc-crustin created using SWISS-MODEL server indicated a random coiled structure that is with two possible β-sheets but no helices.

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