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.

This study shows that the disease resistance and survival rate of Penaeus monodon in a larval rearing systems can be enhanced by supplementing with antagonistic or non-antagonistic probiotics. The antagonistic mode of action of Pseudomonas MCCB 102 and MCCB 103 against vibrios was demonstrated in larval mesocosm with cultures having su⁄cient concentration of antagonistic compounds in their culture supernatant. Investigations on the antagonistic properties of Bacillus MCCB 101, Pseudomonas MCCB 102 and MCCB 103 and Arthrobacter MCCB 104 against Vibrio harveyi MCCB 111under in vitro conditions revealed that Pseudomonas MCCB 102 and MCCB 103 were inhibitory to the pathogen. These inhibitory properties were further con¢rmed in the larval rearing systems of P. monodon. All these four probionts signi¢cantly improved larval survival in long-term treatments as well as when challenged with a pathogenic strain ofV. harveyi MCCB111. We could demonstrate that Pseudomonas MCCB 102 and MCCB 103 accorded disease resistance and a higher survival rate in P. monodon larval rearing systems through active antagonism of vibrios, whereas Bacillus MCCB 101 and Arthrobacter MCCB 104 functioned as probiotics through immunostimulatory and digestive enzyme-supporting modes of action.

Pyocyanin is a redox active phenazine pigment produced by Pseudomonas aeruginosa, with broad antibiotic activity having pharmacological, aquaculture, agriculture and industrial applications. In the present work cytotoxicity induced by pyocyanin is demonstrated in a human embryonic lung epithelial cell line (L-132), a rainbow trout gonad cell line (RTG-2) and a Spodoptera frugiperda pupal ovarian cell line (Sf9). For toxicity evaluation, cellular morphology, mitochondrial function (XTT), membrane leakage of lactate dehydrogenase, neutral red uptake, affinity of electrostatic binding of protein with sulforhodamine B dyes, glucose metabolism, and reactive oxygen species, were assessed. Results showed that higher pyocyanin concentration is required for eliciting cytotoxicity in L-132, RTG-2 and Sf9. The microscopic studies demonstrated that the cell lines exposed to pyocyanin at higher concentrations alone showed morphological changes such as clumping and necrosis. Among the three cell lines L-132 showed the highest response to pyocyanin than the others. In short, pyocyanin application at concentrations ranging from 5 to 10 mg l(-1) were not having any pathological effect in eukaryotic systems and can be used as drug of choice in aquaculture against vibrios in lieu of conventional antibiotics and as biocontrol agent against fungal and bacterial pathogens in agriculture. This is besides its industrial and pharmacological applications.

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