CZS_Slideshow
- 1. Combined effect of a Gyrodactylus sp. and the fungal pathogen Batrachochytrium dendrobatidis on bullfrog tadpole (Rana catesbeiana) survival Linda J Paetow1*, BD Pauli2 , JD McLaughlin3, DJ Marcogliese1 1 Saint-Lawrence Centre, Environment Canada, 105 McGill, 7th floor, Montreal, Qc. H2Y 2E7 2 NWRC Environment Canada, 1125 Colonel By Drive, Ottawa, Ont. K1A 0H3 3 Department of Biology, Concordia University, 7141 Sherbrooke St W, Montreal, Qc.H4B 1R6 Linda Paetow
- 2. Introduction • 48% of amphibian species are in decline (Stuart et al., 2004; Science 306(5702): 1092-1100) • Important emerging infectious diseases caused by: - iridoviruses - Batrachochytrium dendrobatidis (parasitic fungus) Linda Paetow
- 3. Batrachochytrium dendrobatidis • Two-stage life cycle (free-swimming zoospores; intracellular sporocysts) • Lethal to adults of many spp. (infects keratinized skin cells; causes osmotic imbalance) • Non-lethal to larvae of many spp. (infects keratinized mouthparts) • Bullfrogs are asymptomatic carriers (both larvae and adults) Bd sporocysts (stained with tyrpan blue) Linda Paetow
- 4. Anthropogenic causes of declines • Include: - climate change, increased UV irradiation, habitat destruction, introduced species, pollution • Pollution includes chemical contamination of: - air, soil, water - e.g., by agricultural pesticides • Pesticides affect amphibian: - growth, development, behavior, reproductive success, immune response Linda Paetow
- 5. Objective • Do pesticides increase vulnerability to Bd? • Pesticides used: - Atrazine: - challenges the immune system - causes intersex - Glyphosate: - ‘harmless’ active-ingredient - toxic surfactants Linda Paetow
- 6. Experimental design Treatments: 1) No pesticide + No Bd (control) 2) No pesticide + Bd (singly challenged) 3) Glyphosate + Bd (doubly challenged) 4) Atrazine + Bd (doubly challenged) 4 replicates / treatment (5 for controls) 6 tadpoles / replicate Bullfrog tadpole (Rana catesbeiana)
- 7. Exposures & timeline Pesticide exposure Bd inoculation Monitoring 14 days 29 days Total time: 45 days 2x
- 8. Culture of Bd • Virulent Bd strain • Maintained in 1% tryptone broth (4ºC) • Plated on agar and incubated (10 days at 22ºC) • Zoospores / plate counted • 2 plates / aquarium: - Day 14 (24 hrs) - Day 16 (24 hrs) • Total exposure : = 41, 010 zoospores / ml * 48 hrs Linda Paetow B. dendrobatidis colonies on 1% tryptone agar following incubation Linda Paetow
- 9. General husbandry • 33 L aquariums containing 8 L water • 6 L changed daily • 16h/8h day/night cycle • 21ºC (+/- 1.3ºC). • Fed boiled lettuce + Spirulina tablets (proteins & vitamins) • Reduced water level with mortality Linda Paetow Linda Paetow Linda Paetow
- 10. Endpoints Recorded daily: • Survival • General body condition (symptom development) - Scoliosis - Lethargy - Emaciation - Depimentation Linda Paetow Linda Paetow Scoliosis Emaciation Depigmentation Linda Paetow
- 11. Confounding factor: gyrodactylids! Linda Paetow Additional endpoint: Degree of skin erosion (0, +, ++, +++) (measure of pathology of infection) Linda Paetow Skin erosion on side and belly exposing inner dermal tissues Linda Paetow Heavy infections (eye & tail) Gyro pathology Linda Paetow Linda Paetow
- 12. End of experiment - Euthanization (0.8% MS-222) - Examination - Survival curves by treatment Linda Paetow J-F Lafond
- 13. Kaplan-Meyer Survival Analysis Control No Pesticide + Bd Glyphosate + Bd Atrazine + Bd Survival with gyros Logrank p = 0.0080
- 14. Proportional Hazard Regression Analysis The TPHREG Procedure Contrast Test Results Contrast DF Wald Chi-Square Pr > ChiSq 1-2 1 8.9876 0.0027 1-3 1 6.3263 0.0119 1-4 1 5.7895 0.0161 2-3 1 0.4007 0.5267 2-4 1 0.6477 0.4209 3-4 1 0.0264 0.8710 Treatment 1) Control 2) No Pesticide + Bd 3) Glyphosate + Bd 4) Atrazine + Bd Survival with Gyros
- 15. Mortality with gyros Mortality at end (45 Days) Treatment % Mortality 1) Control 10 2) No pesticide + Bd 54.2 3) Glyphosate + Bd 44.2 4) Atrazine + Bd 41.7 Linda Paetow
- 16. Survival without and with gyro pathology considered Log-rank test with Skin Erosion as a covariate Test Chi-Square DF Pr > Chi-Square Log-Rank 15.1671 3 0.0017 Log-rank test for homogeneity over treatments Test Chi-Square DF Pr > Chi-Square Log-Rank 11.8343 3 0.0080
- 17. Role of pesticides • Were pesticides improving survival? - Did pesticides adversely affect gyros? Bd + pesticides Bd only
- 18. + Bd + Bd = = + Bd = + Bd = Conclusion Other studies: Our study:
- 19. Implication for conservation Bd + Bd+ A NEW CONCERN X X X X
- 20. Acknowledgements Stéphanie Arsenaeault Dr. Grant Brown Ken Doe Andrée Gendron Dr, James Grant Paula Jackman Jean-François Lafond Sean Locke Dr. Joyce Longcore Linda Paetow Pesticide Fund