Development and worldwide use of non-lethal, and minimal population-level impact, protocols for the isolation of amphibian chytrid fungi

Matthew C Fisher; Pria Ghosh; Jennifer M G Shelton; Kieran Bates; Lola Brookes; Claudia Wierzbicki; Gonçalo M Rosa; Rhys A Farrer; David M Aanensen; Mario Alvarado-Rybak; Arnaud Bataille; Lee Berger; Susanne Böll; Jaime Bosch; Frances C Clare; Elodie A Courtois; Angelica Crottini; Andrew A Cunningham; Thomas M Doherty-Bone; Fikirte Gebresenbet; David J Gower; Jacob Höglund; Timothy Y James; Thomas S Jenkinson; Tiffany A Kosch; Carolina Lambertini; Anssi Laurila; Chun-Fu Lin; Adeline Loyau; An Martel; Sara Meurling; Claude Miaud; Pete Minting; Serge Ndriantsoa; Simon J O'Hanlon; Frank Pasmans; Tsanta Rakotonanahary; Falitiana C E Rabemananjara; Luisa P Ribeiro; Dirk S Schmeller; Benedikt R Schmidt; Lee Skerratt; Freya Smith; Claudio Soto-Azat; Giulia Tessa; Luís Felipe Toledo; Andrés Valenzuela-Sánchez; Ruhan Verster; Judit Vörös; Bruce Waldman; Rebecca J Webb; Che Weldon; Emma Wombwell; Kelly R Zamudio; Joyce E Longcore; Trenton W J Garner

doi:10.1038/s41598-018-24472-2

Development and worldwide use of non-lethal, and minimal population-level impact, protocols for the isolation of amphibian chytrid fungi

Matthew C Fisher (Corresponding Author), Pria Ghosh, Jennifer M G Shelton, Kieran Bates, Lola Brookes, Claudia Wierzbicki, Gonçalo M Rosa, Rhys A Farrer, David M Aanensen, Mario Alvarado-Rybak, Arnaud Bataille, Lee Berger, Susanne Böll, Jaime Bosch, Frances C Clare, Elodie A Courtois, Angelica Crottini, Andrew A Cunningham, Thomas M Doherty-Bone, Fikirte GebresenbetDavid J Gower, Jacob Höglund, Timothy Y James, Thomas S Jenkinson, Tiffany A Kosch, Carolina Lambertini, Anssi Laurila, Chun-Fu Lin, Adeline Loyau, An Martel, Sara Meurling, Claude Miaud, Pete Minting, Serge Ndriantsoa, Simon J O'Hanlon, Frank Pasmans, Tsanta Rakotonanahary, Falitiana C E Rabemananjara, Luisa P Ribeiro, Dirk S Schmeller, Benedikt R Schmidt, Lee Skerratt, Freya Smith, Claudio Soto-Azat, Giulia Tessa, Luís Felipe Toledo, Andrés Valenzuela-Sánchez, Ruhan Verster, Judit Vörös, Bruce Waldman, Rebecca J Webb, Che Weldon, Emma Wombwell, Kelly R Zamudio, Joyce E Longcore, Trenton W J Garner

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Abstract

Parasitic chytrid fungi have emerged as a significant threat to amphibian species worldwide, necessitating the development of techniques to isolate these pathogens into culture for research purposes. However, early methods of isolating chytrids from their hosts relied on killing amphibians. We modified a pre-existing protocol for isolating chytrids from infected animals to use toe clips and biopsies from toe webbing rather than euthanizing hosts, and distributed the protocol to researchers as part of the BiodivERsA project RACE; here called the RML protocol. In tandem, we developed a lethal procedure for isolating chytrids from tadpole mouthparts. Reviewing a database of use a decade after their inception, we find that these methods have been applied across 5 continents, 23 countries and in 62 amphibian species. Isolation of chytrids by the non-lethal RML protocol occured in 18% of attempts with 207 fungal isolates and three species of chytrid being recovered. Isolation of chytrids from tadpoles occured in 43% of attempts with 334 fungal isolates of one species (Batrachochytrium dendrobatidis) being recovered. Together, these methods have resulted in a significant reduction and refinement of our use of threatened amphibian species and have improved our ability to work with this group of emerging pathogens.

Original language	English
Article number	7772
Journal	Scientific Reports
Volume	8
DOIs	https://doi.org/10.1038/s41598-018-24472-2
Publication status	Published - 17 May 2018

Keywords

biological techniques
ecological epidemiology
microbiology

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Development and worldwide use of non-lethal, and minimal population-level impact, protocols for the isolation of amphibian chytrid fungi
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Fisher, M. C., Ghosh, P., Shelton, J. M. G., Bates, K., Brookes, L., Wierzbicki, C., Rosa, G. M., Farrer, R. A., Aanensen, D. M., Alvarado-Rybak, M., Bataille, A., Berger, L., Böll, S., Bosch, J., Clare, F. C., A Courtois, E., Crottini, A., Cunningham, A. A., Doherty-Bone, T. M., ... Garner, T. W. J. (2018). Development and worldwide use of non-lethal, and minimal population-level impact, protocols for the isolation of amphibian chytrid fungi. Scientific Reports, 8, [7772]. https://doi.org/10.1038/s41598-018-24472-2

Fisher, MC, Ghosh, P, Shelton, JMG, Bates, K, Brookes, L, Wierzbicki, C, Rosa, GM, Farrer, RA, Aanensen, DM, Alvarado-Rybak, M, Bataille, A, Berger, L, Böll, S, Bosch, J, Clare, FC, A Courtois, E, Crottini, A, Cunningham, AA, Doherty-Bone, TM, Gebresenbet, F, Gower, DJ, Höglund, J, James, TY, Jenkinson, TS, Kosch, TA, Lambertini, C, Laurila, A, Lin, C-F, Loyau, A, Martel, A, Meurling, S, Miaud, C, Minting, P, Ndriantsoa, S, O'Hanlon, SJ, Pasmans, F, Rakotonanahary, T, Rabemananjara, FCE, Ribeiro, LP, Schmeller, DS, Schmidt, BR, Skerratt, L, Smith, F, Soto-Azat, C, Tessa, G, Toledo, LF, Valenzuela-Sánchez, A, Verster, R, Vörös, J, Waldman, B, Webb, RJ, Weldon, C, Wombwell, E, Zamudio, KR, Longcore, JE & Garner, TWJ 2018, 'Development and worldwide use of non-lethal, and minimal population-level impact, protocols for the isolation of amphibian chytrid fungi', Scientific Reports, vol. 8, 7772. https://doi.org/10.1038/s41598-018-24472-2

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title = "Development and worldwide use of non-lethal, and minimal population-level impact, protocols for the isolation of amphibian chytrid fungi",

abstract = "Parasitic chytrid fungi have emerged as a significant threat to amphibian species worldwide, necessitating the development of techniques to isolate these pathogens into culture for research purposes. However, early methods of isolating chytrids from their hosts relied on killing amphibians. We modified a pre-existing protocol for isolating chytrids from infected animals to use toe clips and biopsies from toe webbing rather than euthanizing hosts, and distributed the protocol to researchers as part of the BiodivERsA project RACE; here called the RML protocol. In tandem, we developed a lethal procedure for isolating chytrids from tadpole mouthparts. Reviewing a database of use a decade after their inception, we find that these methods have been applied across 5 continents, 23 countries and in 62 amphibian species. Isolation of chytrids by the non-lethal RML protocol occured in 18% of attempts with 207 fungal isolates and three species of chytrid being recovered. Isolation of chytrids from tadpoles occured in 43% of attempts with 334 fungal isolates of one species (Batrachochytrium dendrobatidis) being recovered. Together, these methods have resulted in a significant reduction and refinement of our use of threatened amphibian species and have improved our ability to work with this group of emerging pathogens.",

keywords = "biological techniques, ecological epidemiology, microbiology",

author = "Fisher, {Matthew C} and Pria Ghosh and Shelton, {Jennifer M G} and Kieran Bates and Lola Brookes and Claudia Wierzbicki and Rosa, {Gon{\c c}alo M} and Farrer, {Rhys A} and Aanensen, {David M} and Mario Alvarado-Rybak and Arnaud Bataille and Lee Berger and Susanne B{\"o}ll and Jaime Bosch and Clare, {Frances C} and {A Courtois}, Elodie and Angelica Crottini and Cunningham, {Andrew A} and Doherty-Bone, {Thomas M} and Fikirte Gebresenbet and Gower, {David J} and Jacob H{\"o}glund and James, {Timothy Y} and Jenkinson, {Thomas S} and Kosch, {Tiffany A} and Carolina Lambertini and Anssi Laurila and Chun-Fu Lin and Adeline Loyau and An Martel and Sara Meurling and Claude Miaud and Pete Minting and Serge Ndriantsoa and O'Hanlon, {Simon J} and Frank Pasmans and Tsanta Rakotonanahary and Rabemananjara, {Falitiana C E} and Ribeiro, {Luisa P} and Schmeller, {Dirk S} and Schmidt, {Benedikt R} and Lee Skerratt and Freya Smith and Claudio Soto-Azat and Giulia Tessa and Toledo, {Lu{\'i}s Felipe} and Andr{\'e}s Valenzuela-S{\'a}nchez and Ruhan Verster and Judit V{\"o}r{\"o}s and Bruce Waldman and Webb, {Rebecca J} and Che Weldon and Emma Wombwell and Zamudio, {Kelly R} and Longcore, {Joyce E} and Garner, {Trenton W J}",

note = "T.W.J.G., M.C.F., D.S.S., A.L., E.C., F.C.C., J.B., A.A.C., C.M., F.S., B.R.S., S.O., were supported through the Biodiversa project RACE: Risk Assessment of Chytridiomycosis to European Amphibian Biodiversity (NERC standard grant NE/K014455/1 and NE/E006701/1; ANR-08-BDVA-002-03). M.C.F., J.S., C.W., P.G. were supported by the Leverhulme Trust (RPG-2014-273), M.C.F., A.C., C.W. were supported by the Morris Animal Foundation. J.V. was supported by the Bolyai J{\'a}nos Research Grant of the Hunagrian Academy of Sciences (BO/00597/14). F.G. and D.G. were supported by the Conservation Leadership Programme Future Conservationist Award. C.S.A. was supported by Fondecyt (No. 1181758). M.C.F. and A.C. were supported by. Mohamed bin Zayed Species Conservation Fund Project (152510704). GMR held a doctoral scholarship (SFRH/BD/69194/2010) from Funda{\c c}{\~a}o para a Ci{\^e}ncia e a Tecnologia. L.F.T., C.L., L.P.R. K.R.Z., T.Y.J., T.S.J. were supported by S{\~a}o Paulo Research Foundation (FAPESP #2016/25358-3), the National Counsel of Technological and Scientific Development (CNPq #300896/2016–6) and a Catalyzing New International Collaborations grant from the United States NSF (OISE-1159513). C.S.A. was supported by Fondecyt (No. 1181758). T.M.D. was supported by the Royal Geographical Society and the Royal Zoological Society of Scotland. B.W. was supported by the National Research Foundation of Korea (2015R1D1A1A01057282).",

year = "2018",

month = may,

day = "17",

doi = "10.1038/s41598-018-24472-2",

language = "English",

volume = "8",

journal = "Scientific Reports",

issn = "2045-2322",

publisher = "Nature Publishing Group",

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TY - JOUR

T1 - Development and worldwide use of non-lethal, and minimal population-level impact, protocols for the isolation of amphibian chytrid fungi

AU - Fisher, Matthew C

AU - Ghosh, Pria

AU - Shelton, Jennifer M G

AU - Bates, Kieran

AU - Brookes, Lola

AU - Wierzbicki, Claudia

AU - Rosa, Gonçalo M

AU - Farrer, Rhys A

AU - Aanensen, David M

AU - Alvarado-Rybak, Mario

AU - Bataille, Arnaud

AU - Berger, Lee

AU - Böll, Susanne

AU - Bosch, Jaime

AU - Clare, Frances C

AU - A Courtois, Elodie

AU - Crottini, Angelica

AU - Cunningham, Andrew A

AU - Doherty-Bone, Thomas M

AU - Gebresenbet, Fikirte

AU - Gower, David J

AU - Höglund, Jacob

AU - James, Timothy Y

AU - Jenkinson, Thomas S

AU - Kosch, Tiffany A

AU - Lambertini, Carolina

AU - Laurila, Anssi

AU - Lin, Chun-Fu

AU - Loyau, Adeline

AU - Martel, An

AU - Meurling, Sara

AU - Miaud, Claude

AU - Minting, Pete

AU - Ndriantsoa, Serge

AU - O'Hanlon, Simon J

AU - Pasmans, Frank

AU - Rakotonanahary, Tsanta

AU - Rabemananjara, Falitiana C E

AU - Ribeiro, Luisa P

AU - Schmeller, Dirk S

AU - Schmidt, Benedikt R

AU - Skerratt, Lee

AU - Smith, Freya

AU - Soto-Azat, Claudio

AU - Tessa, Giulia

AU - Toledo, Luís Felipe

AU - Valenzuela-Sánchez, Andrés

AU - Verster, Ruhan

AU - Vörös, Judit

AU - Waldman, Bruce

AU - Webb, Rebecca J

AU - Weldon, Che

AU - Wombwell, Emma

AU - Zamudio, Kelly R

AU - Longcore, Joyce E

AU - Garner, Trenton W J

N1 - T.W.J.G., M.C.F., D.S.S., A.L., E.C., F.C.C., J.B., A.A.C., C.M., F.S., B.R.S., S.O., were supported through the Biodiversa project RACE: Risk Assessment of Chytridiomycosis to European Amphibian Biodiversity (NERC standard grant NE/K014455/1 and NE/E006701/1; ANR-08-BDVA-002-03). M.C.F., J.S., C.W., P.G. were supported by the Leverhulme Trust (RPG-2014-273), M.C.F., A.C., C.W. were supported by the Morris Animal Foundation. J.V. was supported by the Bolyai János Research Grant of the Hunagrian Academy of Sciences (BO/00597/14). F.G. and D.G. were supported by the Conservation Leadership Programme Future Conservationist Award. C.S.A. was supported by Fondecyt (No. 1181758). M.C.F. and A.C. were supported by. Mohamed bin Zayed Species Conservation Fund Project (152510704). GMR held a doctoral scholarship (SFRH/BD/69194/2010) from Fundação para a Ciência e a Tecnologia. L.F.T., C.L., L.P.R. K.R.Z., T.Y.J., T.S.J. were supported by São Paulo Research Foundation (FAPESP #2016/25358-3), the National Counsel of Technological and Scientific Development (CNPq #300896/2016–6) and a Catalyzing New International Collaborations grant from the United States NSF (OISE-1159513). C.S.A. was supported by Fondecyt (No. 1181758). T.M.D. was supported by the Royal Geographical Society and the Royal Zoological Society of Scotland. B.W. was supported by the National Research Foundation of Korea (2015R1D1A1A01057282).

PY - 2018/5/17

Y1 - 2018/5/17

N2 - Parasitic chytrid fungi have emerged as a significant threat to amphibian species worldwide, necessitating the development of techniques to isolate these pathogens into culture for research purposes. However, early methods of isolating chytrids from their hosts relied on killing amphibians. We modified a pre-existing protocol for isolating chytrids from infected animals to use toe clips and biopsies from toe webbing rather than euthanizing hosts, and distributed the protocol to researchers as part of the BiodivERsA project RACE; here called the RML protocol. In tandem, we developed a lethal procedure for isolating chytrids from tadpole mouthparts. Reviewing a database of use a decade after their inception, we find that these methods have been applied across 5 continents, 23 countries and in 62 amphibian species. Isolation of chytrids by the non-lethal RML protocol occured in 18% of attempts with 207 fungal isolates and three species of chytrid being recovered. Isolation of chytrids from tadpoles occured in 43% of attempts with 334 fungal isolates of one species (Batrachochytrium dendrobatidis) being recovered. Together, these methods have resulted in a significant reduction and refinement of our use of threatened amphibian species and have improved our ability to work with this group of emerging pathogens.

AB - Parasitic chytrid fungi have emerged as a significant threat to amphibian species worldwide, necessitating the development of techniques to isolate these pathogens into culture for research purposes. However, early methods of isolating chytrids from their hosts relied on killing amphibians. We modified a pre-existing protocol for isolating chytrids from infected animals to use toe clips and biopsies from toe webbing rather than euthanizing hosts, and distributed the protocol to researchers as part of the BiodivERsA project RACE; here called the RML protocol. In tandem, we developed a lethal procedure for isolating chytrids from tadpole mouthparts. Reviewing a database of use a decade after their inception, we find that these methods have been applied across 5 continents, 23 countries and in 62 amphibian species. Isolation of chytrids by the non-lethal RML protocol occured in 18% of attempts with 207 fungal isolates and three species of chytrid being recovered. Isolation of chytrids from tadpoles occured in 43% of attempts with 334 fungal isolates of one species (Batrachochytrium dendrobatidis) being recovered. Together, these methods have resulted in a significant reduction and refinement of our use of threatened amphibian species and have improved our ability to work with this group of emerging pathogens.

KW - biological techniques

KW - ecological epidemiology

KW - microbiology

U2 - 10.1038/s41598-018-24472-2

DO - 10.1038/s41598-018-24472-2

M3 - Article

C2 - 29773857

VL - 8

JO - Scientific Reports

JF - Scientific Reports

SN - 2045-2322

M1 - 7772

ER -

Development and worldwide use of non-lethal, and minimal population-level impact, protocols for the isolation of amphibian chytrid fungi

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