Microalgae for the remedy of parasitic and bacterial infections in fish: a substitute to conventional chemical treatments
J. H. Kim, Inna Khozin-Goldberg and Dina Zilberg The French Associates Institute for Dryland Agriculture and Biotechnology, The Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Midreshet Ben- Gurion, 8499000, Israel. Email: [email protected]; [email protected] Abstract The potential of Phaeodactylum tricornutu (a diatom) as a treatment against monogenean and bacterial infections of fish was studied. A monogenean parasite, Gyrodactylus turnbulli, and two bacterial pathogens affecting fish, Streptococcus iniae and Vibrio harveyi were used. The microalgae was cultured at different conditions and extracts were prepared using different solvents, aiming to optimize the conditions for effective therapeutic preparation. Extracts were screened in vitro against G. turnbulli followed by in vivo immersion treatment against G. turnbulli-infected guppies. In vitro exposure to 5 ppt of ethanolic P. tricornutum extract killed 97% of the parasites within 4 hours. Fish were effectively treated by immersion in 2.5 ppt over 24 hours, showing significant reduction of both infection prevalence and intensity to almost complete clearance of the parasites. Anti-bacterial effect against S. iniae and V. harveyi was analysed by the disc diffusion assay. Significant antibacterial effect was confirmed in vitro. Ethanolic extract from 2 weeks-cultured microalgae showed 28 and 14 mm of growth inhibition against S. iniae and V. harveyi, respectively. Introduction Disease outbreaks cause substantial losses in aquaculture. Traditional chemical and antibiotic treatments pose adverse effects on human health and the environment and there is therefore a growing trend and need to develop natural therapeutants for aquaculture. Phaeodactylum tricornutum is a microalga (of the diatoms) with proven anti-bacterial effect. Our aim was to test the potential of P. tricornutum as a treatment against monogenean and bacterial infections of fish. Materials & Methods
1. Antiparasitic activity test 2. Antibacterial activity test (disc diffusion assay) Disks with extracts Recording death and detachment over time Breakage, freeze-drying 1 week growth and extraction with ethanol S. Iniae on TSA agar V. harveyi on MH agar
Gyrodactylus Incubation at turnbulii Breakage by bead 26.5° for 24 h beating 2 weeks growth
Recording no. of parasites on the Measure diameter of growth tail fin before and after treatment inhibition
Results 2. Antibacterial effect of microalgal extracts * † 31 Streptococcus iniae 1. Antiparasitic effect of microalgal extracts Vibrio harveyi * 26 In vitro (tail fin clips)
21 100 S-K1-EtOH c K1-EtOH S-EtOH EtOH Control (S-K1-EtOH) 16 80 bc
60 (mm)diameter Inhibitory 11 b b 40 6
Mortality (%) Mortality S. Iniae (TSA agar ) V. harveyi (MH agar ) 1 week 2 weeks Cont. DDWCont. DMSO 20 Treatments
0 a 0 60 120 180 240 3. Attempting to identify the active compound in the algal extract: Time (mins)
TRACE GC-PTV FID AF34 NL 1b Name
0.45 Fractionation by SCC (Silica cartridge chromatography) TLC (thin layer chromatography) 0.40 0.35
In vivo 0 hour median 0.30
0.25 16 24 hours mean 16:1 GC analysis of active 0.20 * Volts 0.15 * 100 F1 100 F34-1a fraction 1b: 0.10
F34-1b 16:0
14 100% 100% F2 EPA * F34-2 0.05 F3 18:0 (3.79) (3.71) 0.00 17:0
F34-3 18:1w9
18:1w7
F4 16:3 24:1 12 80 F5 80 F34-4 -0.05 -0.10 F34-5 9 10 11 12 13 14 15 16 17 18 19 a F6 † Minutes F34-6+7 10 93% Cont. EtOH F34-8 a (2.92) ab * F34-9 60 60 F34-10 8 100% 86% 100% 29% SCC F34 Cont. EtOH (1.86) (2.17) (1.93) (4) 6 40 40
Mortality (%) Mortality
Mortality (%) Mortality 4 b * 20 Number per parasites fish 20 2 0% (0) 0 0 0 0 60 120 180 240 0 60 120 180 240 Contol (FW) Contol (S-K1-ethanol) S-K1-EtOH 1.25 S-K1-EtOH 2.5 Time (mins) Treatments Time (mins)
Conclusion Phaeodactylum tricornutum-based preparations were confirmed to be affective against a parasite and bacteria affecting fish and can be potentially used to develop a natural therapeutant for aquaculture. Identification of the active ingredient in the extract is underway. Further research will be directed to evaluate anti-bacterial treatments in vivo, using infected fish. The application of this preparation against other parasites of significance to aquaculture should be explored. Finally, a cost-effective production of the extract needs to be developed.