Monday, 27 January 2014

A red algae secondary metabolite confers resistance to white spot syndrome virus and enhanced immunity in shrimp

Following an observation made by Thai shrimp farmers that shrimp cultured in ponds containing Gracilaria fisheri (a red algae) had a greater survival rate than those grown in ponds without, this team of researches isolated sulphated galactans from G. fisheri to investigate their immunostimulatory and anti-viral effects.
After extracting the sulphated galactan, its sulphate and carbohydrate content was analysed, its molecular mass determined and it was analysed by NMR and FT-IR spectroscopy. All with the aim of identifying the molecules present. These analyses revealed that the sulphated galactan of G. fisheri is a partially pyruvated and methylated agar-obiose structure with a backbone of alternating units of two sugars,  3-linked b-D-galactopyranose (G) and 4-linked 3,6-anhydro-a-L-galactopyranose.
Having identified the compound, they moved on to investigating its effects on the immune system of the shrimp Penaeus monodon. Firstly, they bioencapsulated SG in Artemia salina by storing them in two concentrations, 100µg/ml and 200µg/ml of a saline solution containing the SG. They then separated a population of shrimp into three treatments, those fed on normal A.salina, those fed on 100µg/ml SG A.salina and those fed on 200µg/ml SG A.salina. They kept the three treatments on their different diets for one week and then extracted haemolymph to compare several immune parameters: total haemocyte count, phenoloxidase activity, superoxide dismutase activity and superoxide anion production.
In SG treated shrimp, all immune parameters showed significant increase, an increase that was dose dependent. The results were as follows:
·         Total haemocyte count increased 141% with 100µg/ml and 186% with 200µg/ml
·         Phenoloxidase activity increased 360% with 100µg/ml and 1300% with 200µg/ml
·         Superoxide dismutase activity increased 555% with 100µg/ml and 635% with 200µg/ml
·         Superoxide anion production increased 204% with 100µg/ml and 341% with 100µg/ml
Having shown the immunostimulatory effect of SG, they proceeded to investigate its antiviral effects. For this they challenged each treatment of shrimp with White Spot Syndrome Virus and added a control group that underwent the same injection procedure but with an inert saline solution rather than WSSV. Over a 14 day period they measured mortality, viral load, expression of viral protein VP 28 and immune parameters. Control shrimp showed 100% mortality by day 10, whereas 100µg/ml SG treated shrimp showed 63.5% mortality at the end of the 14 days, and 200µg/ml showed just 39.5% mortality. Decreased mortality in SG treated shrimp correlated with significant increases in all immune parameters measured. At day 2, all WSSV inoculated shrimp showed VP28 gene amplification. Haemocytes collected on day 5 showed relatively low VP28 gene expression in 200mg/ml SG treated shrimp. By day 10 all inoculated controls were dead and 200mg/ml showed no VP28 gene amplification.
The mechanism of antiviral action is thought to involve an interaction between the SG’s sulphate groups and surface receptors on haemocytes. SG from G.fisheri contains two sulphate groups per disaccharide repeating unit, a relatively large amount compared to other plant or seaweed derived secondary metabolites used for antiviral functions, making it a more efficient antiviral compound. SGs from other red seaweeds have been reported to have antiviral activity against herpes simplex virus, cytomegalo virus, dengue virus and respiratory syncytial virus in humans.
Although this study only challenged SG treated shrimp with WSSV, the resulting increase in many immune parameters such as those that combat oxidative stress suggests that G.fisheri SG has the potential to be used as a non-specific immunostimulant and antiviral compound that could help protect shrimp from a range of diseases and stressors. The authors highly recommend it as a feed supplement to be used in shrimp aquaculture.

Wongprasert, K., Rudtanatip, T., & Praiboon, J. (2014). Immunostimulatory activity of sulfated galactans isolated from the red seaweed Gracilaria fisheri and development of resistance against white spot syndrome virus (WSSV) in shrimp. Fish & shellfish immunology, 36(1), 52-60.

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