1. Effects of agricultural pesticides and the chytrid fungus
Batrachochytrium dendrobatidis on the health of post-
metamorphic northern leopard frogs (Lithobates pipiens)
M.Sc. thesis defense
Linda Joan Paetow
2. Objectives
• does exposure to pesticides increase the susceptibility of frogs to
Batrachochytrium dendrobatidis (Bd)?
• what are the combined effects of exposure to pesticides and Bd on frog
health ?
Joyce Longcore
3. Introduction (pathogen)
Batrachochytrium dendrobatidis Longcore, Pessier and Nichols 1999
- (Bd) (Phylum Chytridiomycota, Class Chytridiomycetes, Order Chytridiales)
- fungal pathogen that causes the disease chytridiomycosis
Vance Vredenburg
Global distribution of Bd in 2008
Jesse Brunner
Sporangium producing and
releasing flagellated
zoospores
6. Introduction (atrazine)
• “no adverse effect on aquatic communities at 20 μg/L”
(Solomon et al. 1996)
but:
• endocrine disruptor at 0.1 μg/L (Hayes et al. 2006)
• disrupts functions of white blood cells in leopard
frogs at 0.1 μg/L (Brodkin et al. 2007)
• increases susceptibility to trematode and viral infections
at ≤ 16 µg / L (Kiesecker 2002; Forson and Storfer 2006)
target concentration 2.1 μg/L
7. Introduction (glyphosate)
Glyphosate
• half-life: 2 – 14 days
• “no adverse effects on amphibians chronically exposed
to < 740 μg a.e./L” (Giesy et al. 2000)
Polyoxyethylene tallowamine (POEA)
• half-life: 21 – 42 days
• ≈ 2000X more toxic to frog embryos than glyphosate
(Perkins et al. 2000)
target concentration: 100 μg a.e./L
9. Frogs captured in August, 2007:
Animal collection & general husbandry
Montreal
•Boucherville
Laboratory conditions:
• individually isolated
• 16h / 8h light-dark cycle
• 21 ± 1.3°C
• daily water changes
• could enter / exit water
• crickets every 2 days
• vitamin and mineral powder
once weekly
10. Day 1
• frogs weighed & measured
(found no difference in baseline mass or SVL)
• transferred to 1L Mason jars
(sterilized, acid-washed)
• frogs could enter / exit the water
11. Euthanized at Day 21
Bd control
Exposed to Bd
Pesticide control Exposed to atrazine Exposed to glyphosate
Experimental design
12. Bd exposures (day 22 – 55)
• Bd culture JEL423
• maintained in 1% tryptone broth, at 4°C
• passaged every 3 months
• grown on 1% tryptone Petri plates to harvest zoospores
• 50 μL of inoculum added directly into exposure jars
Bd growing on Petri plates
Sprorangia stained with 0.4%
trypan blue
Exposure Exposure concentrations
(zoospores / mL)
1st 79 600
2nd 86 400
3rd 130 000
4th 67 200
Frequency of skin shedding over 33 days
13. • survivorship
• growth
• organosomatic indices
• melanomacrophage aggregates
• hepatic granulomas
• differential white blood cell counts
• ratio of neutrophils to lymphocytes
• ratio of white to red blood cells
Biomarkers of animal health and immune function
14. Biomarkers of animal health and immune function
day 1 – 21:
pesticide exposure
day 22 – 94:
Bd exposure
and subsequent
maintenance
Survival
Growth
• change in mass
• change in SVL
Survival
Growth
• change in mass
• change in SVL
Survival
Growth
• change in mass
• change in SVL
15. Biomarkers of animal health and immune function
day 1 – 21:
pesticide exposure
day 22 – 94:
Bd exposure
and subsequent
maintenance
Hepatosomatic index (HSI)
= liver weight / total body weight
Splenosomatic index (SSI)
= spleen weight / total body weight
16. Biomarkers of animal health and immune function
HSI
• frequently increases in polluted conditions
• an increase may signal:
- induction of biotransformation enzymes
- inflammation (tissue damage or pathogenic infection)
SSI
• a crude measure of immune function
• a decrease may signal immunosuppression
• an increase may signal immune response to a pathogen
17. Biomarkers of animal health and immune function
day 1 – 21:
pesticide exposure
day 22 – 94:
Bd exposure
and subsequent
maintenance
Melanomacrophage aggregates in the
- liver (LMA)
- spleen (SMA)
Granulomas in the liver (LGA)
• measured numbers and sizes
• used grid on slide and ruler in ocular lens
• 10 frogs per 6 treatment groups
19. Biomarkers of animal health and immune function
day 1 – 21:
pesticide exposure
day 22 – 94:
Bd exposure
and subsequent
maintenance
• Differential WBC counts
• N/L ratio
• WBC/RBC ratio
- 10 or 11 frogs per 6 treatment groups
20. Biomarkers of animal health and immune function
Differential white blood cell (WBC) counts:
• number of eosinophils, neutrophils, basophils, lymphocytes, monocytes / 100 WBCs
• pesticides may decrease lymphocytes and eosinophils
• pathogenic infections may alter leucocyte profiles
• injuries to tissues may increase neutrophils
Ratio of neutrophils to lymphocytes (N/L):
• may increase with stress
Ratio of WBCs / RBCs:
• pesticides may decrease WBCs
21. Indicators of Bd infection and chytridiomycosis
day 1 – 21:
pesticide exposure
day 22 – 94:
Bd exposure
and subsequent
maintenance
Tissue samples
• histological analysis of toe skin:
- 63 Bd-exposed frogs
• molecular analysis (rt-PCR):
- 30 Bd-exposed frogs
(i.e. 10 per Bd-exposed subgroup)
- 15 Bd-unexposed frogs
(i.e. 5 per Bd-unexposed subgroup)
23. Statistical analysis
day 1 – 21:
pesticide exposure
day 22 – 94:
Bd exposure
and subsequent
maintenance
Survival:
• Kaplan-Meyer analysis
• log-rank statistic
Survival:
• Kaplan-Meyer analysis
• log-rank statistic
Survival:
• Kaplan-Meyer analysis
• log-rank statistic
• 3 treatment groups
• 6 treatment groups
• 6 treatment groups
24. Statistical analysis
day 1 – 21:
pesticide exposure
day 22 – 94:
Bd exposure
and subsequent
maintenance
Growth:
Growth:
Growth:
• 3 treatment groups
• one-way ANOVA
- pesticide treatment
• 6 treatment groups
• two-way ANOVA
- pesticide treatment
- Bd exposure
- pesticide X Bd
• 6 treatment groups
• two-way ANOVA
- pesticide treatment
- Bd exposure
- pesticide X Bd
25. Statistical analysis
day 1 – 21:
pesticide exposure
day 22 – 94:
Bd exposure
and subsequent
maintenance
Organosomatic indices
Hepatosomatic index (HSI)
Splenosomatic index (SSI)
• 3 treatment groups
• one-way ANOVA
- pesticide treatment
• 6 treatment groups
• two-way ANOVA
- pesticide treatment
- Bd exposure
- pesticide X Bd
26. Statistical analysis
day 1 – 21:
pesticide exposure
day 22 – 94:
Bd exposure
and subsequent
maintenance
LMAs, SMAs, sizes of LGAs:
• two-way ANOVA
- pesticide treatment
- Bd exposure
- pesticide X Bd
Numbers of LGAs:
• the GENMOD procedure
- negative binomial distribution
- log link function
- deviance/df = 1.20 (1 implies a good fit)
- number of grid squares = offset term
- model terms:
- pesticide treatment
- Bd exposure
- pesticide X Bd
27. Statistical analysis
day 1 – 21:
pesticide exposure
day 22 – 94:
Bd exposure
and subsequent
maintenance
• Differential WBC counts
• N/L ratio
• WBC/RBC ratio
Analyzed using the GENMOD procedure of SAS
- binomial distribution
- log link function
- deviance/df much > 1
- used a scaling option to handle overdispersion
- model terms:
- pesticide treatment
- Bd exposure
- pesticide X Bd
28. Statistical analysis
day 1 – 21:
pesticide exposure
day 22 – 94:
Bd exposure
and subsequent
maintenance
Analyzed using the GENMOD procedure of SAS
- binomial distribution
- log link function
- deviance/df = 1.06, implying good fit
- model terms:
- pesticide treatment
- Bd exposure
- pesticide X Bd
Frequency of skin shedding:
47. Other blood cell ratios
Normal reference range : < 0.01 – 0.67 (Davis 2009)
Pesticide
Bd
Pesticide * Bd
Pesticide
Bd
Pesticide * Bd
48. Indicators of infection and disease
• histology of toe skin from 63 Bd-exposed frogs:
negative for infection and disease
No Bd
• frequency of skin shedding significantly affected by
exposure to Bd
• rt-PCR of toes from 30 Bd-exposed frogs:
1 positive result for infection (1.6 DNA copies)
Pesticides
Bd
Pesticides X Bd
0.3664
< 0.0001
0.6493
51. Atrazine & growth
• atrazine reduced gain in mass over 94 days, but not gain in SVL
Other studies:
• atrazine disrupted normal endocrine functions in fish and amphibians (Rohr
and McCoy 2010)
• atrazine elevated the metobolic rate of larval African clawed frogs
(Langerveld et al. 2009)
• Atrazine caused dehydration in juvenile streamside salamanders (Rohr and
Palmer 2005)
52. Glyphosate & growth
• the glyphosate formulation reduced the gain in SVL during the 21-day
exposure; gain in mass unaffected
• previously observed in larval amphibians exposed to POEA-containing
formulations (Howe et al. 2004; Cauble and Wagner 2005; Relyea 2009)
• POEA may be an endocrine disruptor
• POEA has longer aquatic half-life (21 - 42 days) than glyphosate (2 - 14 days)
(Giesy et al. 2000)
53. Pesticides & the liver
• Toxins in the liver can cause:
- hepatocellular degeneration
- nodular lesions (e.g. granulomas)
- increased number/size of macrophage aggregates
- inflammation
• HSI, LMAs, LGAs unaffected by the pesticides
• suggests the pesticides were not hepatotoxic
54. Pesticides & immune function
• SSI, blood cell ratios unaffected by the pesticides
• does not rule out potential immunosuppressive effects of the
pesticides
• uncontrolled factors also affected these measurements
55. Bd & survivorship
• survivorship unaffected by Bd and the combined exposures
• frogs exposed to > 3 million zoospores during each exposure event
In contrast:
• Western toads exposed to 104 zoospores experienced 100% mortality in 42
days (Carey et al. 2006)
• Australian frogs exposed to 5,000 zoospores for 15 hrs experienced 65-95%
mortality (Woodhams et al. 2007)
56. Bd & the other biomarkers
• exposure to Bd did not affect growth, HSI, LMAs, SMAs, LGAs
• exposure to Bd did not cause stress, based on N/L ratio (Davis 2009)
• exposure to Bd did not affect biomarkers of immune function (SSI, blood cell ratios)
In contrast:
• foothill yellow-legged frogs exposed to Bd were 50% smaller (Davidson et al. 2007)
• pathology reports suggest that HSI may be elevated (Densmore and Green 2007)
• other studies observed neutrophila, neutropenia, eosinophelia, basophilia
(Woodhams et al. 2007; Davis et al. 2010)
57. Indicators of infection and disease
• no positive results for infection or disease by histological methods
• one positive result for infection by molecular methods
• frogs may be resistant to Bd as previously observed in northern leopard
frogs (Ouellet et al. 2005; Longcore et al. 2007; Woodhams et al. 2008;
Voordouw et al. 2010)
• infections may have been controlled by excessive shedding of the skin
• antimicrobial peptides secreted by skin glands may have helped to
combat infection (monitoring attempted but failed)
58. Summary
Pesticides:
• each reduced growth at low environmental levels
• could contribute to population declines in leopard frogs
Bd:
• increased the frequency of skin shedding
• had no other observed effect on animal health or immune function
Combined exposures:
• had no observed effect on animal health or immune function
Frogs:
• appeared resistant to Bd
59. Conclusion
• pre-exposure to the pesticides did not
increase the susceptibility of the leopard
frogs to Bd
• should be verified using species of
amphibians that are more susceptible to Bd
60. References
• Allran and Karasov 2001. Environmental Toxicology and Chemistry 20: 769–775.
• Brodkin et al. 2007. Environmental Toxicology and Chemistry 26: 80–84.
• Carey et al. 2006. EcoHealth 3: 5-21.
• Cauble and Wagner 2005. Bulletin of Environmental Contamination and Toxicology 75: 429–435.
• Christin et al. 2004. Aquatic Toxicology 67: 33-43.
• Davidson et al. 2007. Environmental Science and Technology 41: 1771-1776.
• Davis 2009. http://wildlifehematology.uga.edu (accessed June 22, 2010).
• Davis et al. 2010. Comparative Clinical Pathology 19: 49–55.
• Densmore and Green 2007. ILAR Journal 48: 235-254.
• Feng et al. 2006. Journal of Agriculture and Food Chemistry 38: 1110-1118.
• Forson and Storfer 2006. Ecological Applications 16: 2325-2332.
• Giesy et al. 2000. Reviews of Environmental Contamination and Toxicology 167: 35-120.
• Hayes et al. 2006. Environmental Health Perspectives 114: 40-50.
• Howe et al. 2004. Environmetal Toxicology and Chemistry 23: 1928-1938.
• Kiesecker 2002. Procedures of the National Academy of Sciences (USA) 99: 9900-9904.
• Langerveld et al. 2009. Environmental Research 109: 379–389.
• Longcore, Pessier and Nichols 1999. Mycologia 91: 219-227.
• Longcore et al. 2007. The Journal of Wildlife Management 71: 435-444.
• Ouellet et al. 2005. Conservation Biology 19: 1431–1440.
• Perkins et al. 2000. Environmental Toxicology and Chemistry 19: 940–945.
• Relyea 2009 Oecologia 159: 363–376.
• Rohr and McCoy 2010. Environmental Health Perspectives 118: 20-32.
61. References
• Rohr and Palmer 2005. Environmental Toxicology and Chemistry 24: 1253–1258.
• Solomon et al. 1996. Environmental Toxicology and Chemistry 15: 31-76.
• Voordouw et al. 2010. BMC Ecology 10:6 doi: 10.1186/1472-6785-10-6
• Woodhams et al. 2007. Animal Conservation 10: 409–417.
• Woodhams et al. 2008. Herpetological Review 39: 66–68.
62. Dr. David Marcogliese
Dr. Daniel McLaughlin
Bruce Pauli
Andrée Gendron
Claude Lessard
Malorie Gélinas
Hubert Désilets
Sophie Trépanier
Kimberly Buhl
Simon Despaties
Jean-François Lafond
Stéphanie Arseneault
Violaine Peltier
Coralie Beaudry
Ariane Laurence
Emilie Lessard
Mixime Guérard
Dr. Roger Cue
Dr. Todd Smith
Dr. Colin Rousseaux
Dr. Sylvia Ruby
Dr. Joyce Longcore
Dr. Robert Lumsden, DMV
Dr. Paul Widden
Dr. Ian Fergusson
Dr. Grant Brown
Dr. Marc Champagne
Dr. Chris Blanar
Janet Rokas
Michel Harvey
Kelly Pingel
Elaine Muise
Sean Locke
Rachel Krause
Angela Rose-Lapierre
Simon Daoust
Dominic Dodge
Christophe Achard-Dodge
Adam Dodge
Gyrodactylus jennyae and
Jenny Cooke
Pesticide Fund
Acknowledgements
64. Kaplan-Meyer Survival Analysis
Test of Equality over Strata
Test Chi-Square DF Pr > Chi-Square
Log-Rank 2.6608 5 0.7521
Wilcoxon 2.6644 5 0.7516
-2Log(LR)* 3.9537 5 0.5561
65. Introduction
• 48% of amphibian species are in decline
• Important diseases are caused by:
- Batrachochytrium dendrobatidis (Bd)
- iridoviruses
• Pesticides increase susceptibility to
parasitic infection and disease
Christin et al. 2004. Aquat. Toxicol. 67: 33-43.
Christin et al., 2003. Env. Toxicol. Chem.22: 1127-1133.
Gendron et al. 2004. Oecologia. 135: 469-476.
66. Immatures
• Immatures:
- 1 in Atrazine, No Bd, Male
- 1 in Glyphosate, No Bd, Male
- 1 in Pesticide control, Bd, Female
- 1 in Glyphosate, Bd, Male
68. Introduction
• Pesticides increase susceptibility to parasitic
infection and disease:
- malathion linked with heavier bacterial infections
- atrazine linked with heavier trematode infections
- cocktails linked with heavier lungworm infections
Taylor et al. 1999. J. Wildl. Dis. 536-541.
Rohr et al. 2008. Nature 455: 1235-1239.
Christin et al. 2004. Aquat. Toxicol. 67: 33-43.
Christin et al., 2003. Env. Toxicol. Chem.22: 1127-1133.
Gendron et al. 2004. Oecologia. 135: 469-476.
69. pesticide solutions
Field results:
Glyphosate: 0.91 μg a.e./L Aminomethylphosphonic acid: 1.2 μg/L
NOEC for chronic exposures: 740 μg/L
Experimental concentrations:
Nominal Test solution Sample from jars
Atrazine: 2.1 µg/L 4.28 ± 0.04 μg/L 1.70 ± 0.26 μg/L
Glyphosate: 100 µg a.e./L 3.83 ± 0.95 μg ae/L 8.13 ± 1.27 μg ae/L
Watershed in BC: glyphosate dissipated from 162 to < 1 μg/L in 24h (Feng et al. 2006)
70. Introduction (Bd)
Mehgan Murphy Linda Paetow
Species that
display resistance
to Bd.
American bullfrog
Northern leopard frog
Patrizia Corsaro
African clawed frog
Resistance may be tied to:
- antimicrobial peptides secreted by host skin
- frequency with which hosts shed their skin
- cutaneous bacteria with anti-fungal properties
- host behaviour
- environmental conditions
71. Biomarkers of animal health and immune function
Growth
pesticides may disrupt:
- feeding rates
- efficiency of food assimilation
- endocrine functions
energy may be diverted from growth toward:
- detoxification
- tissue repair following chemical exposure
- immune response
- tissue repair following exposure to a pathogen
72. Pesticide concentrations:
Target Sample 1 (day 20) Sample 2 (day 21)
Atrazine: 2.1 µg/L 4.28 ± 0.04 μg/ L 1.70 ± 0.26 μg/L
Glyphosate: 100 µg a.e./L 3.83 ± 0.95 μg ae/L 8.13 ± 1.27 μg ae/L
In a watershed in B.C., glyphosate dissipated from 162 to < 1 μg/L in 24h
(Feng et al. 2006)
73. Pesticides & survivorship
Atrazine
• survival of adult northern leopard frogs not directly threatened by atrazine;
• agrees with other studies (Allran and Karasov 2001; Christin et al. 2004)
Glyphosate
• No adverse effects concentration for amphibians = 740 µg a.e./L
• POEA increases toxicity of glyphosate formulations
• effects of POEA-containing formulations difficult to predict
• the concentration of POEA should also be presented
