Molecular characterization of a crustin-like, putative antimicrobial peptide, Fi-crustin, from the Indian white shrimp, Fenneropenaeus indicus.

Antimicrobial peptides are important innate immune defense, especially in those animals which lack adaptive immunity [1e8]. Due to their small size, amphipathic structure and cationic character they can rapidly diffuse to the point of infection [9], a mechanism that presumably makes it easier to circumvent microbial resistance against the peptides [10]. Besides providing an immediate and broad-spectrum microbicidal activity, AMPs can kill bacteria in micromolar range, are promptly synthesized at low metabolic cost, and are easily stored in large amounts and readily available shortly after an infection [11e13]. Many AMPs show a remarkable specificity for prokaryotes with low toxicity for eukaryotic cells; a phenomenon which has favored their investigation and exploitation as potential new antibiotics [14]. AMP gene expression and distribution are regulated through haemocyte reactions [15]. Transcripts of crustin-encoding genes have also been observed in gills, heart and intestine [16e18] but as these tissues are highly vascularised, it is assumed the transcripts from these organs are due primarily to the haemocytes. In penaeid shrimps, four main families of AMPs have been currently described and characterized from the haemocytes: penaeidins, crustins, anti-lipopolysaccharide factors (ALFs) and lysozymes. Penaeidins are mainly active against Gram-positive bacteria, filamentous fungi [19], viruses and protozoans [20] whereas ALFs have a broader antimicrobial spectrum including Gram-negative bacteria [21,22]. Conversely, crustins are reported to have a more-restricted activity spectrum, affecting mainly marine Gram-positive bacteria [17,23,24] Crustins, a widely distributed family of AMPs was first isolated from the shore crab, Carcinus maenas as an 11.5 kDa peptide [23]. Crustins are cationic, cysteinerich antimicrobial AMPs having molecular weight of 7e14 kDa, with an isoelectric point in the range of 7.0e8.7, and contain one whey-acidic protein (WAP) domain at the carboxy terminus [25]. Crustins have been proved to be an important antimicrobial protein in the plasma and haemocyte granules of crustaceans and described as a component of the innate immune system [8]. These AMPs are dominantly synthesized and stored in haemocytes [4,8,16,18,23,24,26e30] and their release from haemocytes is induced by bacterial infection [15,27,31]. Crustin mechanisms of action and function are still largely unknown, although they contain a whey-acidic protein (WAP) domain common to proteinase inhibitory activities as well as antimicrobial activities [8]. Many full-length cDNA and several ESTs of crustins have been described in a wide range of penaeid prawns including Litopenaeus vannamei [8,24,30,32], Litopenaeus setiferus [24,32,33], Penaeus monodon [16,17,29,30,34e37], Marsupenaeus japonicus [17,38], Litopenaeus schmitti [33], Fenneropenaeus chinensis [17,29], Farfantepenaeus brasiliensis [33], Farfantepenaeus paulensis [33] and Farfantepenaeus subtilis [33]. However, no antimicrobial peptide sequences have been reported from Fenneropenaeus indicus. In the current study a crustin cDNA has been characterized from the Indian White Shrimp, F. indicus. Healthy adult F. indicus (8e10 g body weight) were purchased from a local shrimp farm in Vypeen, Kochi. Theywere transferred to aquaria of 500 l capacity and acclimatized for one week under laboratory conditions. Prawns were fed with a standard feed (Higashimaru, India). Aeration was provided in all tanks during the experiment and bioreactorwas set in all the aquaria for the removal of ammonia and nitrate. Only shrimps in the intermoult stage were sampled during the study. Haemolymph was collected from the rostral sinus using specially designed capillary tubes (RNase-free) rinsed using precooled anticoagulant solution (RNase-free, 10% sodium citrate, pH 7.0). Total RNA was extracted from the haemocytes using TRI * Corresponding author. Tel.: þ91 484 2368120; fax: þ91 484 2381120. E-mail addresses: [email protected], [email protected] (R. Philip).