1.1 Wet method

The whole animal is removed and cut into small pieces and ground in a pestle-mortar until the mixer becomes as homogenous mass. The whole content was soaked in 1:5 ratio (w/v) of methanol: chloroform (9:1) and ethyl acetate: chloroform (9:1) for 72 hours (Sunil et al., 2016). After soaking the samples were concentrated to 100 mg/ml in methanol and ethyl acetate by rota-evaporator.

1.2 Dry method

The known amount of meat was kept in hot air oven at 55°C for 72 hours. The dried samples were powdered and dissolved in 1:5 ratio (w/v) of methanol: chloroform (9:1) and ethyl acetate: chloroform (9:1) for 72 hours (Sunil et al., 2016). After soaking the samples they were concentrated to 100 mg/ml in methanol and ethyl acetate.

1.3 Preparation of microbial cultures

Three pathogens namely, Escherichia coli, Staphylococcus aureus, Vibrio harveyi were selected to know the antagonistic activity. 2.5 gms of nutrient broth (HIMEDIA) was dissolved in 100 ml of distilled water and autoclaved at 121°C, 15 lbs. for 15 min. The broth was cooled down to room temperature and bacterial cultures were inoculated in sterilized nutrient broth and were incubated at 37°C for 24 hrs.

1.4 Antagonistic activity

The extracts were tested for their anti-bacterial activity by agar well diffusion method (Perez et al., 1990). The 18 hrs. young culture broths were taken and the pathogenic strains were spread evenly over sterile Mueller Hinton Agar plates. The 6 mm diameter wells were prepared on agar plates by using sterile cork borer. The wells were filled with 100 µL extract and controls were arranged. The plates were incubated at 37°C for 24 hours. Antibacterial activities were evaluated by measuring the zone of inhibition showed in millimeters (mm).

2 Results

The two different extracts of Cellana radiata were screened for their antibacterial activity against three different pathogenic bacteria. The methanolic dry extracts did not show any inhibitory zones but the wet extracts have shown antibacterial activity against Vibrio harveyi (Figure 2; Figure 3; Table 1). The ethyl acetate extracts (from both wet and dry methods) have shown inhibitory zones against Vibrio harveyi (Figure 2; Figure 3; Table 1).

Figure 2 Agar well diffusion method against V. harveyi, S. aureus and E.coli

3 Discussion

From ancient times the molluscs are useful organisms which support the coastal communities as food and also economically. These are used in medical applications and as bio-indicators in recent trends (Asta, 2011). The molluscs, particularly gastropods were showing antibacterial properties. In the present study three different pathogenic bacteria viz. Escherichia coli (human), Staphylococcus aureus (Fish) and Vibrio harveyi (Shrimp) were tested to know the antibacterial activity against them to methanolic and ethyl acetate crude extracts. The wet extract of methanol has shown antibacterial activity (12 mm) against V. harveyi. The ethyl acetate extracts by both dry and wet methods have shown antibacterial activity against V. harveyi, the dry extract 13 mm and wet extract 18 mm.

V. harveyi showing severe effect on commercially important resources like fish, shrimp and also pearl oysters and causes mass mortality. The pearl oyster Pinctada maxima died and damaged heavily by V. harveyi infection (Lester, 1989). Some gastropods have shown antibacterial activity against V. harveyi and in near future these metabolites may be helpful to aquaculture industries. The Donax faba shell extract has shown inhibitory zone of 3 mm to V. harveyi from ethanol extract (Giftson and Patterson, 2014). The extracts of crude peptides shown zone of inhibition to V. harveyi from pelecypods, Crassostrea madrasensis, Saccostrea cucullata, Meretrix meretrix, Perna viridis, Polymesodaerosa and from gastropods, Ficus gracilis, Hemifusus tuba, Bursa rana, Xenophora mekranensis and from cephalopods, Sepeilla inermis, Octopus dolfusii, Vampyroteuthis infernalis, Loligosp, Ancirtroclieinix leriieii (Naveen et al., 2014). The ethanolic extract of Chicerous ramous shown inhibitory zone of 6 mm to V. harveyi (Giftson et al., 2015).

4 Conclusion

The present study has shown that Cellana radiata is having antimicrobial properties against shrimp pathogen i.e., Vibrio harveyi.

Authors’ contributions

This work was carried out in all collaboration between authors. All authors read and approved the final manuscript.

Acknowledgments

Authors are grateful to the Department of Science and technology (DST), New Delhi, Govt. of India for the financial support in the form of INSPIRE Fellowship to carry out this work.

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