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. 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] J. H. Kim , Inna Khozin-Goldberg and Dina Zilberg Microalgae for the remedy of parasitic and bacterial infections in fish: a substitute to conventional chemical treatments 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. 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. Materials & Methods 2. Antibacterial activity test (disc diffusion assay) 1 week growth 2 weeks growth Breakage, freeze-drying and extraction with ethanol S. Iniae on TSA agar V. harveyi on MH agar Disks with extracts Measure diameter of growth inhibition Incubation at 26.5° for 24 h 1. Antiparasitic activity test Gyrodactylus turnbulii Recording death and detachment over time Breakage by bead beating Recording no. of parasites on the tail fin before and after treatment Results 1. Antiparasitic effect of microalgal extracts Time (mins) 0 60 120 180 240 Mortality (%) 0 20 40 60 80 100 S-K1-EtOH K1-EtOH S-EtOH EtOH Control (S-K1-EtOH) c bc b b a In vitro (tail fin clips) Treatments Number parasites per fish 0 2 4 6 8 10 12 14 16 0 hour 24 hours median mean 100% (1.86) 86% (2.17) 100% (1.93) 93% (2.92) 100% (3.79) 29% (4) 100% (3.71) 0% (0) b ab a a * * In vivo Contol (FW) Contol (S-K1-ethanol) S-K1-EtOH 1.25 S-K1-EtOH 2.5 3. Attempting to identify the active compound in the algal extract: Time (mins) 0 60 120 180 240 Mortality (%) 0 20 40 60 80 100 Cont. EtOH SCC F34 F34-1a F34-1b F34-2 F34-3 F34-4 F34-5 F34-6+7 F34-8 F34-9 F34-10 * * Time (mins) 0 60 120 180 240 Mortality (%) 0 20 40 60 80 100 Cont. EtOH F1 F2 F3 F4 F5 F6 † * TLC (thin layer chromatography) Fractionation by SCC (Silica cartridge chromatography) Minutes 9 10 11 12 13 14 15 16 -0.10 -0.05 0.00 0.05 0.10 0.15 0.20 0.25 16:0 16:1 17:0 16:3 18:0 18:1w9 18:1w7 EPA 2. Antibacterial effect of microalgal extracts V. harveyi (MH agar ) Treatments Cont. DDW Cont. DMSO 1 week 2 weeks Inhibitory diameter (mm) 6 11 16 21 26 31 Streptococcus iniae Vibrio harveyi * * † S. Iniae (TSA agar ) GC analysis of active fraction 1b: