Anti-lipopolysaccharide factor and crustin-III, the anti-white spot virus peptides in Penaeus monodon: Control of viral infection by up-regulation

White Spot Syndrome Virus (WSSV) is the most devastating disease affecting shrimp culture around the world. Though, considerable progress has been made in the detection and molecular characterization of WSSV in recent years, information pertaining to immune gene expression in shrimps with respect to WSSV infection remains limited. In this context, the present study was undertaken to understand the differential expression of antimicrobial peptide (AMP) genes in the haemocytes of Penaeus monodon in response to WSSV infection on a time-course basis employing semi-quantitative RT-PCR. The present work analyzes the expression profile of six AMP genes (ALF, crustin-1, crustin-2, crustin-3, penaeidin-3 and penaeidin-5), eight WSSV genes (DNA polymerase, endonuclease, immediate early gene, latency related gene, protein kinase, ribonucleotide reductase, thymidine kinase and VP28) and three control genes (18S rRNA, β-actin and ELF) in P. monodon in response to WSSV challenge. Penaeidins were found to be up-regulated during early hours of infection and crustin-3 during late period of infection. However, ALF was found to be up-regulated early to late period of WSSV infection. The present study suggests that AMPs viz. ALF and crustin-3 play an important role in antiviral defense in shrimps. WSSV gene transcripts were detected post-challenge day 1 itself and increased considerably day 5 onwards. Evaluation of the control genes confirmed ELF as the most reliable control gene followed by 18S rRNA and β-actin for gene expression studies in shrimps. This study indicated the role of AMPs in the protection of shrimps against viral infection and their possible control through the up-regulation of AMPs

A new isoform of anti-lipopolysaccharide factor identified from the blue swimmer crab, Portunus pelagicus: Molecular characteristics and phylogeny

Anti-lipopolysaccharide factors are small proteins that bind and neutralize lipopolysaccharide and exhibit potent antimicrobial activities. This study presents the molecular characterization and phylogenetic analysis of the first ALF isoform (Pp-ALF1; JQ745295) identified from the hemocytes of Portunus pelagicus. The full length cDNA of Pp-ALF1 consisted of 880 base pairs encoding 293 amino acids with an ORF of 123 amino acids and contains a putative signal peptide of 24 amino acids. Pp-ALF1 possessed a predicted molecular weight (MW) of 13.86 kDa and theoretical isoelectric point (pI) of 8.49. Two highly conserved cysteine residues and putative LPS binding domain were observed in Pp-ALF1. Peptide model of Pp-ALF1 consisted of two α-helices crowded against a four-strand β-sheet. Comparison of amino acid sequences and neighbor joining tree showed that Pp-ALF1 has a maximum similarity (46%) to ALF present in Portunus trituberculatus followed by 39% similarity to ALF of Eriocheir sinensis and 38% similarity to ALFs of Scylla paramamosain and Scylla serrata. Pp-ALF1 is found to be a new isoform of ALF family and its characteristic similarity with other known ALFs signifies its role in protection against invading pathogens.

Two isoforms of anti-lipopolysaccharide factors identified and characterized from the hemocytes of portunid crabs, Portunus pelagicus and Scylla tranquebarica

Anti-lipopolysaccharide factors (ALFs), a type of cationic antimicrobial peptides (AMPs), and their derivatives are becoming predominant candidates for potential drugs in viral and bacterial diseases. This study reports the first ALF from the mud crab Scylla tranquebarica (StALF, JQ899453) and the second ALF isoform from the blue swimmer crab Portunus pelagicus (PpALF2, JQ899452). Both sequences encoded for precursor molecules, starting with a signal peptide containing 26 amino acid residues, followed by a highly cationic mature peptide, containing two conserved cysteine residues flanking a putative lipopolysaccharide (LPS)-binding domain. BLAST analysis revealed that both PpALF2 and StALF exhibited significant similarity with crustacean ALF sequences. The predicted molecular mass of the mature ALFs was 11.2 kDa with an estimated pI of 10.0. PpALF2 and StALF also showed the typical pattern of alternating hydrophobic and hydrophilic residues in their putative disulphide loop, suggesting that they comprise the same functional domain. Phylogenetic analysis showed that PpALF2 and StALF have similar evolutionary status and they were phylogenetically ancient immune effector molecules which may play an essential role in the host defense mechanism. The spatial structures of PpALF2 and StALF possessed four beta-strands and two alpha-helices. The results indicated that there were more than one ALF involved in crab immunity against various pathogens. ALFs would provide candidate promising therapeutic or prophylactic agents in health management and diseases control in crustacean aquaculture

Development of a cell culture system from the ovarian tissue of African catfish Clarias gariepinus

A growth medium with Leibovitz-15 L-15.as the base, supplemented with foetal bovine serum 10% vrv., fish muscle extract 10% vrv., prawn muscle extract 10% vrv., lectin concanavalin A. 0.02 mg mly1., lipopolysaccharide 0.02 mg mly1., glucose D 0.2 mg mly1., ovary extract 0.5% vrv.and prawn haemolymph 0.5%. has been formulated with 354"10 mOsm for the development and maintenance of a cell culture system from the ovarian tissue of African catfish, Clarias gariepinus. For its subculturing, a cell dissociationrextracting solution, composed of equal portions of trypsin phosphate versene glucose TPVG. containing 0.0125% wrv.trypsin and 25% vrv.non-enzymatic cell dissociation solution 1 and 2, has also been developed with which the cell culture can be passaged 15 times after which they cease to multiply and consequently perish. The cell cultures can be maintained for 12–15 days without fluid change between the passages. This is the first report of a cell culture system from the ovarian tissues of African catfish

Molecular and Functional Characterization of Antimicrobial Peptides in Crustaceans

Most living organisms are constantly exposed to potentially harmful pathogens. It is the immune system of the organism that enables it to survive in an environment loaded with dangerous pathogenic microorganisms. The innate immunity provides organisms with a rapid and non-specific first line of defense against pathogens. It includes physical barriers such as skin and mucous membranes and chemical barriers including the high acidity of gastric juice, and specialized soluble molecules that possess antimicrobial activity. One of the well-known innate immune defense mechanisms is the production of antimicrobial substances by specific cells or tissues of the organisms. Antimicrobial peptides (AMPs) are such natural substances that