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Pathogens vs. Pesticides: The Threat to Eastern Hellbenders
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Pathogens vs. Pesticides: The Threat to Eastern Hellbenders Center for Wildlife Health UT Institute of Agriculture Matthew J. Gray
Transcript of Pathogens vs. Pesticides: The Threat to Eastern Hellbenders
Pathogens vs. Pesticides: The Threat to Eastern Hellbenders
Center for Wildlife HealthUT Institute of Agriculture
Matthew J. Gray
Collaborators and Funding:
Dr. Debra MillerBill Reeves
Dale McGinnitySherri ReinschDr. Bill Sutton
Dr. Louise Rollins-SmithDr. David BemisDr. Becky Wilkes
Dr. Rachel HillDr. Yi GengJenny Asper
Jenny HowardAllison Graham
Davis CarterMorgan GaynorJessica NelsonCarson Lillard
Ben WilsonReilly JacksonJordan Chaney
Center of Excellence in Livestock Diseases and
Human Health
Limited Information on Salamanders (hellbenders)
Pesticides and Amphibians
Glyphosate and Atrazine: Linked to amphibian mortality and pathology
Pathogens and AmphibiansBatrachochytrium dendrobatidis Ranavirus
Photo by M. Neimiller
• Decline of >200 species• Mostly anurans• Tropics in high
elevation streams
• Infection & disease >175 poikilothermic species
• Anurans and salamanders• Temperate and Tropics
Ranavirus and Cryptobranchidae
Oryx: 2015
Concerning??r = 85%: Infection & Disease
Brunner et al. (2015)
Hiwassee River = 24% Little River = 60%
Rv in 2009:
JWD: 2012
Shaanxi: ~2500 farms, 8500 sal/farm, Rv causing 80-100% mortality, $21 Million USD ($2,000/sal)
Objectives1) Test the susceptibility of eastern hellbender
larvae to ranavirus• Temperatures: 15 and 22 C
• Normal water temperature range in eastern TN
2) Test the toxicity of glyphosate herbicides to eastern hellbender larvae
• Formulations: Cornerstone® and Roundup®
50% glyphosate
+ proprietary surfactant
41% glyphosate
+ proprietary surfactant
“Generic Roundup”
Study AnimalsField Collection of Eggs Raised in Captivity
• October 2013: Captive Bred• October 2014:
• (Polk Co, TN)
• Nashville Zoo• Flow-through tanks:
recirculating (15-18 C)
TWRA Scientific Collection Permit #1525
Ranavirus Challenges15oC: Pilot Study 22oC: Expanded Studyntotal= 7 larvae (4 mo) ntotal= 18 larvae
• 12 hr Exposure• 24 hr Exposure• 48 hr Exposure• 72 hr Exposure
n = 1 / trt; 3 controls
Bath (Static) Exposure: 103 PFU/mL (FV3-like)
• 48 hr Exposure
n = 6 / trt; 6 controls
Bath: 103 PFU/mL
IC Injection:103 PFU
Flow-through System
Static Exposure
UT IACUC #2140
Ranavirus Challenges15oC = No Mortality22oC = Mortality Starting 15-18 days PE
Injection = 5 of 6 Died Water Bath = 4 of 6 Died
Ranavirus Challenges
Injection = One survivor was infectedWater = Two survivors were not infected
Ranavirus: Classic Gross Signs
Epidermal Hemorrhage
Edema
Swollen and hemorrhagic liver
Ranavirus: Classic Pathology
Liver: NecrosisSkin: Hemorrhage
Ranavirus: Novel PathologySkin Lesions
(Polyps)
Skin polyp-Epithelial proliferation (thickened outer layer), necrosis (cellular debris) and mixed inflammatory cells
Inflammatory cells extend deep in tissue
Many inflammatory cells within the epidermis
Pesticide Exposures
Trial 1: Cornerstone®
ntotal= 18 larvae (6-7 mo)
• 0.03 ppm• 0.3 ppm• 3.0 ppm
24 hr “Pulse” Exposure
n = 5 / trt; 3 controls; duration = 14 days per trial
Trial 2: Cornerstone®
• 3 ppm for 48 hrs• 3 ppm for 96 hrs• 6 ppm for 96 hrs
“Chronic” Exposure
No Mortality
No Mortality
Trial 3: Roundup®
No Mortality
• 3 ppm for 48 hrs• 3 ppm for 96 hrs• 6 ppm for 96 hrs
“Chronic” Exposure
Relyea and Jones (2009)
LC5096-h = 4 ppm for 4 salamander species
with Roundup®
UT IACUC #2321
• 12 ppm for 96 hrs• 24 ppm for 96 hrs
“Severe” ExposureTrial 4: Roundup®
Herbicide: Trial 4 Results
100% Mortality in 24 ppm Roundup®
Cornerstone 12 ppm
Degeneration (vacuolation), Necrosis (fragmented cells), and ultimate loss of epidermis
Toe of control hellbender
Toe of Hellbender exposed to Roundup®
Cartilage
Cartilage
Skin
Skin
Experimental DesignTreatment Number of
animalsExposure
Bd only 5 48 hours Bd
Ranavirus only 5 48 hours Rv
Bd+Ranavirus 5 48 hours Bd, then 48 hours Rv
Bd+Herbicide 5 96 hours Roundup, then 48 hours Bd
Ranavirus+Herbicide 5 96 hours Roundup, then 48 hours Rv
Control 3 NONE
Treatments: 5Bd: 105 zoosporesRanavirus: 103 PFU/mlHerbicide: Roundup, 6 ppm (sublethal)
Replicates: 5Controls: 3
Methods: Zoospore Enumeration
SETUP DECANTING ZOOSPORE WATER USING THE HEMACYTOMETER
ENUMERATION INOCULATION
Methods: Inoculation
Survival
Infection + Mortality
Gross Observations• Gross signs
shedding skinskin polypshemorrhage (mouth, skin, kidneys)
• Behavioral changesanimals not eatinganimals not hidinganimals floating / buoyancy issueslethargy
BdRv2
BdRv3
RvH3
Rv2
hemorrhage
shedding
polyp
Behavior Changes
Summary and DiscussionRanavirus
Chytrid Fungus (Bd)
• Ranavirus was pathogenic at warmer water temperatures• FV3-like ranaviruses replicate faster (Ariel et al. 2009)• Impaired watershed: no buffers or reduced flow
• No mortality of juvenile hellbenders after 28 days• Lack of keratin in skin?
Histology
Hellbender Mucosome
• Did not inactivate Bd• Inactivated ranavirus
Summary and DiscussionHerbicide
• Larval hellbenders were able to survive up to 4X the concentration of Roundup® compared to other salamander species (Ambystomatidae, Salamandridae)
• Roundup may be more toxic than Cornerstone
Herbicide + Pathogens• Herbicide + Bd = 20% mortality (0% Bd only)• Herbicide + Rv = 100% mortality (80% Rv only)• Synergistic effect
Sublethal Effects• Pathogen and Herbicide Exposure Altered Behavior!
