Acute toxicity assessment of fungal secondary metabolites with Paramecium caudatum

Barbara Pernfuss Bipesco-Team Institute of Microbiology Leopold-Franzens-University Innsbruck Why Paramecium caudatum?

„ Paramecia are easy and „ There is experimental exper- cheap to rear (for nearly ience with the rearing of everybody). Paramecium caudatum and the sensitivity of the cells against fungal „toxins“ in „ Paramecia are animals living in aqueous systems (non bioassays at our institute. targets). „ We were/are looking for „lower animals“, which can „ You can directly observe the reaction of Paramecia (and possibly displace „higher their organelles) to a given animals“ (fish, mammals) in substance. bioassays one day.

„ There are already literature data of bioassays to Why Paramecium caudatum?

„ Hussain M. M., Rao L. S. P., Khan M. A. (1985). Bioassay of Dimethoa insecticide to Paramecium caudatum a ciliated protozoan. Journal of Science Research, 7(3): 131-133. „ Juchelka C. M., Snell T. W. (1995). Rapid toxicity assessment using ingestion rate of Cladocerans and Ciliates. Archives of environmental contamination and toxicology 28: 508-512.

„ Komala Z. (1975). The effect of some pesticides on Paramecium aurelia. Folia Biologica 23(3): 231-243.

„ Komala Z. (1982). Paramecium bioaassay test in studies on Cartap. Bulletin of Environmental Contamination and Toxicology 28: 660- 663.

„ Komala Z. (1984). Paramecium bioassay test in studies on the insecticide Kartox 50. Folia Biologica 32(4): 281-293.

„ Komala Z. (1985). The effect of Decis 2.5 EC, a pyrethroid insecticide, on Paramecium primaurelia. Folia Biologica 33(1-2): 9-14.

„ Komala Z. (1986). The toxicity of Enolophos for Paramecium primaurelia. Folia Biologica 34(3): 263-268.

„ Komala Z. (1987). The effect of Cymbush 25 EC, a pyrethroid insecticide, to Paramecium primaurelia. Folia Biologica 35(3-4): 165-168.

„ Komala Z. (1989). The effect of Ambush 25 EC, a pyrethroid insecticide, on Paramecium primaurelia. Folia Biologica 37(1-2): 21-24.

„ Komala Z. (1992). The effect of Isathrine 10 EC, a pyrethroid insecticide, on Paramecium primaurelia. Preliminary Report. Folia Biologica 40(1-2): 53-55.

„ Le Du A., Dive D., Guerbert M., Jouany J. M. (1993). The protozoan biotest Colpidium campylum, a tool for toxicity detection and interaction modelling. The Science of the total Environment, Suppl. 1993: 809-815.

„ Lejczak B. (1977). Effect of insecticides: Chlorphenvinphos, Carbaryl and Ropoyur on aquatic organisms. Polskie Archiewum Hydrobiologii 24(4): 583-591.

„ Miyoshi N., Kawano T., Tanaka M., Kadono T., Kosaka T., Kunimoto M.,Takahashi K., Hosoya H. (2003). Use of Paramecium species in bioassays for environmental risk management: determination of IC50 values for water pollutants. Journal of Health Science 49(6): 429- 435.

„ Pöder R. (1982). Über Nachweis, Isolierung und Charakterisierung eines auf Protozoen toxisch wirkenden Stoffes aus Hebeloma edurum (Agaricales). Dissertation an der Leopold-Franzens-Universität Innsbruck.

„ Rajini P. S., Krishnakumari M. K., Majumder S. K. (1989). Cytotoxicity of certain organic solvents and organophosphorus insecticides to the ciliated protozoan Paramecium caudatum. Microbios 59: 157-163.

„ Tandon R. S., Lal R., Narayana Rao V. V. S. (1987). Effects of Malathion and Endosulfan on the growth of Parameciun aurelia. Acta Protozoologica 26(4): 325-328. Paramecium caudatum

Taxonomic Hierarchy

„ Eukarya (Crown group: Alveolata) „ Kingdom „Protozoa“ „ Phylum Ciliophora „ Class Ciliatea „ Subclass Rhabdophorina „ Order Hymenostomatida „ Suborder Peniculina „ Family Parameciidae „ Genus Paramecium „ Species Paramecium caudatum Ehr. Ö is a single celled animal ITIS Standard Report Page … http://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=all&search_value=Paramecium+caudatum Paramecium caudatum

http://biocab.org/files/Paramecium_English.jpg Paramecium caudatum

In vivo 170 µm - 300 µm!

On average: 230 µm ± 19 µm x 68 µm ± 9 µm; n = 200 (Simpson 1902)

Macronucleus ellipsoidic, in vivo 50 µm - 60 µm

Micronucleus approx. 8 µm in diameter

Two contractile vacuoles with each seven trichocysts

Longitudinale Cilia (80 - 120)

Photo: Reinhold Poeder and Judith Stemer Foissner et al. (1994) Rearing of Paramecium caudatum

Paramecium caudatum was cultured in salad-extract medium with Enterobacter aerogenes as food source. For cultivation, 10 mL glass- tubes and newly designed “culture-boxes” with a different number of straws as interior were used providing test organisms with increased surface areas on which feed-bacteria could form biofilms. From the “culture-boxes” (100 mL glass beakers with straws providing a total inner surface area of 193 cm2 and approximatly 30 mL culture broth) a very high number of cells (5.7 x 103 mL-1) was harvested.

Paramecium caudatum Ehrenberg was friendly provided by Prof. Görtz, University Stuttgart Britta Gierner (2005) Pilzmetabolite (Destruxine) in Toxizitätstests mit Paramecium caudatum. Experimental design – Bioassays

Cavity slides

Microscope

Oosporein

P. caudatum B. brongniartii B. The chemical insecticide Agritox® containing Chlorpyrifos was used as positive control. During experiment, samples were stored in a moist chamber. Primary data (number of dead and alive cells) were statistically analysed with Probit analysis (Throne, 1995) to determine Lethal Time

(LT50) and PriProbit (Sakuma, Ver. 1.63) for analysis of Effective Dose (ED50). Substances

Destruxin A – R1 = -CH2-CH=CH2 Destruxin B – R1 = -CH2-CH(CH3)2 Destruxin E – R1 = -CH2-CH-CH2 O

Relativ amounts of the cyclic hexadepsipeptides (dtx A, B, and E) are produced by the anamorphic fungus Metharizium anisopliae. Before M. anisopliae can be widly used as biocontrol agent (BCA) to fight against a range of insect pests, the potential risk of destruxins entering the food chain must be assessed. Test organisms were exposed to destruxins for 120 – 200 minutes in small volumes (drops à 20 μL) on cavity slides. Each experiment was accompanied by a negative control (solvents only) and by a positive control [chemical insecticide (Agritox® with Chlorpyrifos)].

Liu et al. 2000 Substances

O H C OH 3 O OH HO Oosporein (C14H10O8) O HO CH3 O

Oosporein, a dihydroxybenzochinon, which is a secondary metabolite of Beauveria brongniartii (Saccardo) Petch (Ascomycota, Hypocreales, Clavicipitaceae, Cordyceps), was used as a model for potentially toxic fungal metabolites. Beauveria brongniartii forms the main part of Melocont®-Pilzgerste, which is successfully inserted (applied) for decades to fight against maybeetle larvae.

Cole and Cox 1981; Kögl and Van Wessem 1944 Results - oosporein

P. caudatum : pure oosporein, culture filtrate and crude extract of B. brongniartii Paramecium caudatum, 120 minutes:

-1 1.00 LD50 pure oosporein – 0.483 mM (148 mg L ) Pure 0.5 mM 0.75 -1 Filtrate 0.5 mM LD50 culture filtrate – 1.103 mM (338 mg L ) 0.50 Pure 0.4 mM -1 Filtrate 0.4 mM LD50 crude extract – 0.343 mM (105 mg L ) 0.25 Pure 0.3 mM -1 Filtrate 0.3 mM LD50 Chlorpyrifos – 0.016 mM (5.6 mg L ) 0.00 Extract 0.5 mM Proportion killed 0 100 200 300 400 500 600 Time [min] Paramecium caudatum, growth test (four days):

LT50 (0.5 mM Oosporein) – 114 min (Lower Limit 94.2, Upper Limit 133.7 of 95 % confidence limits) MIC ~ 100 µM oosporein in salad extract (30.6 mg L-1) NOEC ~ 0.5 -1 µM oosporein in SE (0.15 - 0.3 mg Conclusion L-1) ¾ To kill 50 % of Paramecia in a pond with 1000 m3 water (50 x 10 x 2 meters) we need 148 kg pure oosporein. ¾ 1 kg Melocont®-Pilzgerste contains ~ 7 mg oosporein.

® ¾ We would need 21 Million kg of Melocont -Pilzgerste to achieve this LD50 (recommended application rate 50 kg / ha).

Pernfuss et al. 2004 Results - oosporein

P. caudatum in 0.1 % NaHCO3 P. caudatum with 2 mM pure oosporein

P. caudatum in culture filtrate - 1.2 mM oosporein P. caudatum in 30 µM Chlorpyrifos

Photos: Reinhold Pöder and Judith Stemer Results - destruxins

LT50 with dtxA Lower limit Time [min] Upper limit

Britta Gierner 2005 Results - destruxins

ED50 Ö 9.87 mM dtxA

Log(ED50) Ö 0.99 mM dtxA

„Effective Dose“ (ED) of dtxA [mM] to Paramecium caudatum (120 min exposition)

Britta Gierner 2005 Results - destruxins

„ Pure destruxins solved in NaHCO3 buffer were tested:

„ the ciliates survived concentrations up to 25 mM (1.48 x 104 mg L-1) destruxin E,

„ approximately 6 mM (3.56 x 103 mg L-1) destruxin B,

3 -1 „ but were killed at 9 mM (5.19 x 10 mL ) destruxin A (LT50 = 160 min). Conclusion Because one litre culture extract of M. anisopliae contains on average 3.71 x 101 mg dtx A, 2.21 x 101 mg dtx B, and 2.87 x 101 mg dtx E, it is very unlikely that destruxin levels in the environment can reach concentrations tested in this study: they were about 140fold (dtx A), 300fold (dtx B), and 500fold (dtx E) higher than found in the culture extract.

Britta Gierner 2005 Preservation of Paramecium caudatum?

„ Lumsden W. H. R. (1972). Principles of viable preservation of parasitic protozoa. International Journal for Parasitology 2: 327-332.

„ Miyake Y., Karanis P., Uga S. (2004). Cryopreservation of protozoan parasites. Cryobiology 48: 1-7.

„ Nsabimana E., Kiŝidayová S., Macheboeuf D., Newbold C. J., Jouany J. P. (2003). Two-step freezing procedure for cryopreservation of rumen ciliates, an effective tool for creation of a frozen rumen protozoa bank. Applied and Environmental Microbiology 69(7): 3826-3832.

Ö Did not work! Comparison

Skrobek et al. (2005) Evaluation of different biological test systems to assess the toxicity of meatabolites from fungal biocontrol agents. Overall conclusion

„ Paramecium caudatum is fast, easy and cheap to rear ⇒ but cannot be cryopreserved!

„ Bioassays with Paramecium caudatum are very cheap and for nearly everybody easily and reliably to conduct and to interpret ⇒ but no automating until now! ⇒ not standardised or validated until now!

„ Paramecium caudatum cells were the most sensible organisms tested in the RAFBCA project ⇒ but is also very sensitive to osmotic pressure! ⇒ usable if solvent is aqueous and osmotic pressure equilibriated! Thanks to

„ COST 862 for the invitation! „ The auditorium for the attention! „ The BIPESCO-Team! „ The RAFBCA-Team! „ All my friends for a lot of pleasant hours!

Supported by the European Commission, Quality of Life and Management of Living Resources Programme (QoL) Key Action 1 on Food, Nutrition and Health (Contract n°QLK1-CT-2001-01391)".

http://bipesco.uibk.ac.at

QLK1-CT-2001-01391 http://www.rafbca.com