Acute toxicity assessment
of fungal secondary
metabolites
with Paramecium caudatum
Barbara Pernfuss
Bipesco-Team
Institute of Microbiology
Leopold-Franzens-University
Innsbruck
Why Paramecium caudatum?
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Paramecia are easy and
cheap to rear (for nearly
everybody).
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Paramecia are animals living
in aqueous systems (non
targets).
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You can directly observe the
reaction of Paramecia (and
their organelles) to a given
substance.
„
There are already literature
data of bioassays to
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There is experimental experience with the rearing of
Paramecium caudatum and
the sensitivity of the cells
against fungal „toxins“ in
bioassays at our institute.
„
We were/are looking for
„lower animals“, which can
possibly displace „higher
animals“ (fish, mammals) in
bioassays one day.
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: 660663.
„
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.
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Komala Z. (1986). The toxicity of Enolophos for Paramecium primaurelia. Folia Biologica 34(3): 263-268.
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Komala Z. (1987). The effect of Cymbush 25 EC, a pyrethroid insecticide, to Paramecium primaurelia. Folia Biologica 35(3-4): 165-168.
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Komala Z. (1989). The effect of Ambush 25 EC, a pyrethroid insecticide, on Paramecium primaurelia. Folia Biologica 37(1-2): 21-24.
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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): 429435.
„
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
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Eukarya (Crown group: Alveolata)
Kingdom
Phylum
Class
Subclass
Order
Suborder
Family
Genus
Species
„Protozoa“
Ciliophora
Ciliatea
Rhabdophorina
Hymenostomatida
Peniculina
Parameciidae
Paramecium
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 glasstubes 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
OH
H 3C
O
OH
HO
O
HO
Oosporein (C14H10O8)
CH 3
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 oospore in, culture filtrate and crude e xtract
of B. brongniartii
Proportion killed
1.00
Pure 0.5 mM
Filt rate 0.5 mM
0.75
Pure 0.4 mM
Filt rate 0.4 mM
0.50
Pure 0.3 mM
Filt rate 0.3 mM
0.25
Paramecium caudatum, 120 minutes:
LD50 pure oosporein
LD50 culture filtrate
LD50 crude extract
LD50 Chlorpyrifos
– 0.483 mM (148 mg L-1)
– 1.103 mM (338 mg L-1)
– 0.343 mM (105 mg L-1)
– 0.016 mM (5.6 mg L-1)
Ext ract 0.5 mM
0.00
0
100
200
300
400
500
Time [min]
600
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)
Conclusion
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
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 in culture filtrate - 1.2 mM oosporein
P. caudatum with 2 mM pure oosporein
P. caudatum in 30 µM Chlorpyrifos
Photos: Reinhold Pöder and Judith Stemer
Results - destruxins
LT50 with dtxA Lower limit
Upper limit
Time [min]
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
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Pure destruxins solved in NaHCO3 buffer were tested:
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the ciliates survived concentrations up to 25 mM (1.48 x 104 mg
L-1) destruxin E,
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approximately 6 mM (3.56 x 103 mg L-1) destruxin B,
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but were killed at 9 mM (5.19 x 103 mL -1) 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?
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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
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Paramecium caudatum is fast, easy and cheap to rear
⇒ but cannot be cryopreserved!
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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
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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
Q L K 1 -C T - 2 0 0 1 - 0 1 3 9 1
http://www.rafbca.com