No abstract

The objective of the study was to find out a natural way to fight white spot syndrome virus (WSSV) in cultured shrimps, as the present scenario necessitated an organic remedy for the devastating pathogen in crustaceans. Under this research programme seven mangrove plants were collected, identified and aqueous extracts screened for their protective effect on the giant tiger shrimp Penaeus monodon against WSSV. The experimental design consisted two modes of application, such as exposure of the virus to the extract and injection challenge, and oral administration of the extract coated feed followed by oral challenge. All experimental animals were monitored through a nested diagnostic PCR analysis. Of the seven mangrove extracts screened aqueous extract from Ceriops tagal imparted total protection to shrimp from WSSV when challenged by both methods. Shrimps administered with the aqueous extract from C. tagal were devoid of virions. The HPLC fingerprint of the aqueous extracts from C. tagal showed more than 25 peaks and 7 of them were larger and well separated. Preliminary phytochemical analysis revealed the presence of alkaloids, flavonoids, polyphenolics, cardiac glycosides, saponins and sterols. The study indicated suitability of the aqueous extract of C. tagal as a possible prophylaxis for WSSV infection in shrimp. This is the first report on the anti WSSV property of the mangrove plant C. tagal.

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.

a b s t r a c tSeveral oral vaccination studies have been undertaken to evoke a better protection against white spot syndrome virus (WSSV), a major shrimp pathogen. Formalin-inactivated virus and WSSV envelope protein VP28 were suggested as candidate vaccine components, but their uptake mechanism upon oral delivery was not elucidated. In this study the fate of these components and of live WSSV, orally intubated to black tiger shrimp (Penaeus monodon) was investigated by immunohistochemistry, employing antibodies specific for VP28 and haemocytes. The midgut has been identified as the most prominent site of WSSV uptake and processing. The truncated recombinant VP28 (rec-VP28), formalin-inactivated virus (IVP) and live WSSV follow an identical uptake route suggested as receptor-mediated endocytosis that starts with adherence of luminal antigens at the apical layers of gut epithelium. Processing of internalized antigens is performed in endo-lysosomal compartments leading to formation of supra-nuclear vacuoles. However, the majority of WSSV-antigens escape these compartments and are transported to the inter-cellular space via transcytosis. Accumulation of the transcytosed antigens in the connective tissue initiates aggregation and degranulation of haemocytes. Finally the antigens exiting the midgut seem to reach the haemolymph. The nearly identical uptake pattern of the different WSSV-antigens suggests that receptors on the apical membrane of shrimp enterocytes recognize rec-VP28 efficiently. Hence the truncated VP28 can be considered suitable for oral vaccination, when the digestion in the foregut can be bypassed.

White spot syndrome virus (WSSV) is a pathogen that causes considerable mortality of the farmed shrimp, Penaeus monodon. Candidate 'vaccines', WSSV envelope protein VP28 and formalin-inactivated WSSV, can provide short-lived protection against the virus. In this study, P. monodon was orally intubated with the aforementioned vaccine candidates, and protein expression in the gut of immunised shrimps was profiled. The alterations in protein profiles in shrimps infected orally with live-WSSV were also examined. Seventeen of the identified proteins in the vaccine and WSSV-intubated shrimps varied significantly compared to those in the control shrimps. These proteins, classified under exoskeletal, cytoskeletal, immune-related, intracellular organelle part, intracellular calcium-binding or energy metabolism, are thought to directly or indirectly affect shrimp's immunity. The changes in the expression levels of crustacyanin, serine proteases, myosin light chain, and ER protein 57 observed in orally vaccinated shrimp may probably be linked to immunoprotective responses. On the other hand, altered expression of proteins linked to exoskeleton, calcium regulation and energy metabolism in WSSV-intubated shrimps is likely to symbolise disturbances in calcium homeostasis and energy metabolism.