By Heather PaulHeather Paul By Leein HisroomLeein HisroomBy Renato GaigaRenato Gaiga By Brian GratwickeBrian Gratwicke By Brian GratwickBrian Gratwick By Andreas KayAndreas Kay

The frog-killing fungus in Nanaimo, BC An undergraduate research project by Patrick Reid

Part 1 WORLDWIDE Part 2 The local AREA Part 3 Implications The frog-killing fungus

PART 1 WORLDWIDE By Geoff GalliceGeoff Gallice

AMPHIBIAN STATUS  Recent worldwide declines  1/3 of all identified species are at risk of extinction (Stuart et al. 2004)

Amphibians are the most threatened vertebrate group. SAD FROG By Elvir BElvir B

Amphibian Declines Habitat Loss Over Exploitation Invasive Species Pollution Wildlife Diseases Climate Change

Declines in pristine mountain areas. How is this possible?

Chytrid Fungus (Batrachochytrium dendrobatidis)  Potentially lethal skin infection on amphibians (Berger et al. 1998)  Caused decline or extinction of 200 amphibian species worldwide (Skerrat et al. 2007) Many declines in prestine mountain areas Most effeted mountasin species Associated with amphibian mortality events on every continent (except Antarctica) (Lips et al., 2005; Berger et al., 1998; Bosch et al., 2001; Muths et al., 2003)

Amphibia.web The emergence of Bd may represent the greatest pathogen-associated biodiversity loss in recorded history (Skerrat et al. 2007)

Why the recent increase in Bd- associated mortalities?

Batrcachochytriumdendrobatidis Batrachochytrium dendrobatidis Strain level (variants) Species level KIll (no/yes ) Multiple variants: some kill some don't (Berger et al. 2005)

Spread of Bd Past Present Global Spread of pathogenic variants of Bd (Example: not actual locations) Strain A Strain B Strain C

 Pathogenic strains may have been spread through the amphibian trade  American bullfrogs are resistant to Bd and me be acting as global carriers (Daszak et al. 2004; Schloegel et al. 2012) Bullfrogs as disease carriers By Goly ShevGoly Shev By Jonathan E. KolbyJonathan E. Kolby Amphibians for worldwide shipmentBullfrogs being sold in Shanghai

Part 2 The local AREA By Ken WalkerKen Walker

Local species infected with BD American bullfrog (Lithobates catesbeianus ) Western Toad (Anaxyrus boreas) Photo courtesy of Bill Pennell Photo courtesy of Elke Wind Detected on Vancouver Island (Garner et al., 2006) Detected in Nanaimo (Baxter, 2012 unpublished data)

Western toads  Blue-listed in BC (at risk of becoming threatened)  Bd-caused declines of Western Toads in the United States raises concern (Muths et al. 2003) Photo courtesy of Elke Wind

American bullfrogs  Invasive species on Vancouver Island  May have brought foreign strains to local area  May spread infections among local toads

research objective Look for Bd on Western Toads and American bullfrogs in Nanaimo Sequence Bd strains found on toads and bullfrogs to determine the possibility of between-species transfer. Compare the DNA sequence to the global Bd database to determine the strain’s origin and any previously recorded pathogenicity und on toads and bullfrogs to determine the possibility of between-species transfer Evaluate presence of Bd on toads and bullfrogs in Nanaimo Use strain data to asses bullfrogs role in the local spread and arrival of Bd Find out what strain (variant) is present on local toads and bullfrogs

Swabbing for presence of Bd

Analysis of Skin Swabs Bd DNA from the nested PCR assay was put into a plasmid and cloned into bacteria in preparation for sequencing.  Subsequently purified product was sent to the NAPS unit at the University of British Columbia for sequencing and then compared to the GenBank database for strain identification. Subsequently purified product was sent to the NAPS unit at the University of British Columbia for sequencing and then compared to the GenBank database for strain identification. Extra ct Detect Identify Strain

Part 3 Implications

The strain detected (CW 34) is likely pathogenic to amphibians, heightening the risk for toad mortalities locally.

 How did it get to Nanaimo?

bullfrogs Role in the spread of Bd Possibili ty Evidenc e resul t Arrived to Nanaimo on bullfrogs Transferred between bullfrogs and toads CW 34 on bullfrogs in the amphibian trade and locally CW 34 present on both bullfrogs and toads in Nanaimo Does not refute Support

Take home messages  Amphibians are declining, with Bd acting as a strong contributor  Toads and bullfrogs in the local area are infected with the Bd  The presence of a pathogenic strain of Bd on local toads heightens the importance of monitoring toad populations

By Heather PaulHeather Paul By Leein HisroomLeein HisroomBy Renato GaigaRenato Gaiga By Brian GratwickeBrian Gratwicke By Brian GratwickBrian Gratwick By Andreas KayAndreas KayBy Heather PaulHeather Paul By Leein HisroomLeein HisroomBy Renato GaigaRenato Gaiga By Brian GratwickeBrian Gratwicke

Acknowledgment I would like to thank Hitomi Kimura for helping me with the research and my supervisors Dr. Eric Demers for guiding me through the project and Dr. Catherine Thomson for her molecular knowledge. I am grateful for the help of Wendy Simms and Dr. Timothy Goater in collecting bullfrog samples. samples.

References Berger, L., G. Marantelli, L.F. Skerratt and R. Speare. 2005b. Virulence of the amphibian chytrid fungus Batrachochytrium dendrobatidis varies with the strain. Disease of Aquatic Organisms 68: Berger, L., R. Speare, P. Daszak, D.E. Green, A.A. Cunningham, C.L. Goggin, R. Slocombeh, M.A. Ragani, A.D. Hyatt, K.R. MCDonald, H.B. Hinesk, K.R. Lips, G. Marantellim and H. Paresb Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America. Proceedings of the National Academy of Sciences USA 95: Daszak, P., A. Strieby, A.A. Cunningham, J.E. Longcore, C.C. Brown and D. Porter Experimental evidence that the bullfrog (Rana catesbeiana) is a potential carrier of chytridiomycosis, an emerging fungal disease of amphibians. Herpetol. J. 14: Muths, E, P.S. Corn, A.P. Pessier, and D.E Green Evidence for disease-related amphibian decline in Colorado. Biological Conservation 110: Schloegel, L.M., L.F. Toledo, J.E. Longcore, S.E. Greenspan, C.A. Vieira, M. Lee, S. Zhao, C. Wangen, C.M. Ferreira, M.R. Hipolito, A.J. Davies, C.A. Cuomo, P. Daszak and T.Y. James Novel, panzootic and hybrid genotypes of amphibian chytridiomycosis associated with the bullfrog trade. Molecular Ecology 21:5162–5177. Skerratt, L.F., L. Berger, R. Speare, S. Cashins, K.R. McDonald, A.D. Phillott, H.B. Hines and N. Kenyon Spread of chytridiomycosis has caused the rapid global decline and extinction of frogs. EcoHealth 4 : Stuart, S.N., M. Hoffmann, J.S. Chanson, N.A. Cox, R.J. Berridge, P. Ramani and B.E. Young Threatened Amphibians of the World. Lynx Edicions, Barcelona, Spain.