A SURVEILLANCE STUDY LINKS THE AFRICAN CLAWED FROG WITH THE INTRODUCTION OF

RANAVIRUS TO CHILE

Alexandra Peñafiel-Ricaurte, Stephen J. Price, Mario Alvarado-Rybak, Andrew A. Cunningham, and Claudio Soto-Azat

IPBES report 2019

• Plastics ↑10 times since 1980.

• Population x2 since 1970.

• ↓82% biomass wild mammals.

50%

González del Pliego et al. 2019. Curr. Biol.

INTRODUCED SPECIES MAY PLAY AN IMPORTANT ROLE IN RANAVIRUS EPIDEMIOLOGY

• There is evidence that X. laevis and different species of introduced fish may act as Ranavirus reservoirs orvectors for native amphibian species (Gantress et al. 2003, Robert et al. 2007, Bang Jensen et al. 2009, Gobbo et al. 2010, Bang Jensen et al.2011, Brenes et al. 2014, Chinchar et al. 2014).

Mosquito fish X. laevis

• In Chile:

• 26 introduced fish spp. have established feral populations in rivers and other water bodies in Chile (Iriarte et al. 2005).

Global distribution of Xenopus laevis

Mora et al. 2019. Manag. Biol. Inv.

03-06-19 06(47Página 1 | El Diario de Atacama

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• Captured individuals = average of 218.4 individuals/hour.

• Population size: 1,162 individuals.• Divided by area (86 m2): density of 13.5

individuals/m2.

Non-linear extrapolation model

X(t)=N(1)(1-exp(-Kt))

RANAVIRUS HAS BEEN REPORTED IN SEVERAL COUNTRIES FROM SOUTH AMERICA

FIRST EVIDENCE OF RANAVIRUS IN CHILEAN AMPHIBIANS WAS REPORTED IN 2016

Soto-Azat et al. 2016

• 2011 – 2013.

Calyptocephalella gayi9 individuals

Pleurodema thaul2 individuals

Telmatubofo bullocki1 individual

Xenopus laevis175 individuals

TOTAL: 187 individuals

RANAVIRUS WAS DETECTED IN ONE NATIVE AND 7 INVASIVE AMPHIBIANS FROM TWO SITES IN SANTIAGO

Site 1: Rinconada (4 X. laevis).

Site 3: Talagante (3 X. laevis, 1 Calyptocephalellagayi).

4.3%

FV3

RANAVIRUS SURVEILLANCE ALONG CHILE

• Rhinella spinulosa. (98 tadpoles, II region)

• Pleurodema thaul.(335 tadpoles, IV, V, VI, VII, XIV region).

• Tadpoles from native non-endangered species.

• Calyptocephalella gayi.(5 dead tadpoles, IV, VII región)(58 preserved carcasses, RM).

• Xenopus laevis.(1011 post-metamorphs, IV, V, VI, VII región).

• Adult amphibians from native species. (245 individuals from 11 species II, IV, VIII, X región).

TOTAL: 1752 Amphibians741 Native + 1011 X. laevis

FISH SURVEILLANCE

TOTAL: 526 fish395 Invasive + 101 native

Invasive freshwater fish

• Gambusia holbrooki.(321 adults, II, VI, V, VI, XIV).

• Oncorhynchus mykiss.(71 juveniles, II, X region).

• Cyprinus carpio.(3 adults, VIII region).

Native freshwater fish

• Orestias chungarensis. (30 adults, XV region).

• Cheirodon galusdae.(31 adults, VIII region).

• Galaxia maculatus.(70 adults, VIII region).

SAMPLING

Native tadpoles, X. laevis and fishAdult amphibians from native

species

• Euthanasia (MS-222) (Landsberg et al. 2013; Bayley et al. 2013).

• qPCR assay specific for amphibian-associated ranaviruses, targeting a 97bp fragmentof the MCP gene (Leung et al. 2017).

• 84bp fragment of the EBF3 gene (common from vertebrates) forviral load estimation.

• Also used as an IPC to discard PCR inhibition.

• Liver, kidney and spleen, preserved at -80°C.

• Swab tips preserved in 95% ethanol.

RESULTS: AMPHIBIAN TISSUE SAMPLES SUMMARY

• Overall apparent prevalence in X. laevis: 0.9% (9/ 1011)

0%0%0%

0%0%

Region Site Lat Long Species n Rv +True

Prevalence Lower Upper

VX Putre -18.195503 -69.568573 Rhinella spinulosa 54 0 0 0 6.641359II Alcolcha -21.027084 -68.450103 Rhinella spinulosa 32 0 0 0 10.71792

Val le de Jere -23.187166 -67.991194 Rhinella spinulosa 5 0 0 0 43.448246Calama -22.748641 -68.071030 Rhinella spinulosa 7 0 0 0 35.433044

IV Rio Elqui -29.897250 -71.244583 Pleurodema thaul 81 0 0 0 4.527809

Oval le -30.644806 -71.568204 Xenopus laevis 48 1 2.0833333 0.10686103 10.899218

Il lapel -31.619583 -71.141833 Pleurodema thaul 23 0 0 0 14.311662

Calyptocephalella gayi 2 0 0 0 65.761977V Jardin Botanico -32.039694 -71.498111 Xenopus laevis 39 0 0 0 8.966685

Vi l la Alemana -33.036251 -71.370742 Xenopus laevis 285 1 0.3508772 0.01799765 1.96041

-34.185358 -70.799575 Pleurodema thaul 58 0 0 0 6.211786MR Criadero rana chi lena -33.385197 -71.645147 Calyptocephalella gayi 58 0 0 0 6.211786

Talagante -33.686389 -70.908333 Xenopus laevis 211 1 0.4739336 0.02430962 2.635253

Rinconada de Maipu -33.496111 -70.829722 Xenopus laevis 297 6 2.020202 0.92908604 4.336631VI Rancagua -34.185358 -70.799575 Pleurodema thaul 59 0 0 0 6.112937

-34.168433 -70.854356 Xenopus laevis 60 0 0 0 6.017185VII Hualañé -34.974352 -71.850175 Xenopus laevis 71 0 0 0 5.132795

Calyptocephalella gayi 3 0 0 0 56.149703Río Mataquito -35.045768 -71.737533 Pleurodema thaul 57 0 0 0 6.313883

XIV Valdivia -39.872720 -73.160637 Pleurodema thaul 57 0 0 0 6.313883

TOTAL 1507 9

LOW VIRAL LOADS WERE DETECTED IN ALL POSITIVE SAMPLES

Region Study Site Sample ID # Viral copiesIV - Coquimbo Ovalle MUR12/17 3.01V - Valparaiso Villa Alemana VA20/16 8.11Metropolitan Talagante ETA29/17 3.52Metropolitan Rinconada ERI36/17 2.71Metropolitan Rinconada ERI49/17 4.06Metropolitan Rinconada MRI04/17 2.61Metropolitan Rinconada MRI13/17 8.7Metropolitan Rinconada MRI15/17 6.57Metropolitan Rinconada MRI27/17 4.61

Field signs and lesions absence Carrier - Reservoir

Risk for native amphibian species

RESULTS: ADULT NATIVE AMPHIBIANS SAMPLES SUMMARY

Region Site Lat Long Species n Rv +Apparent

Prevalence

I I Rio Loa -22.748641 -68.071030 Telmatobius dankoi 50 0 0Calama -22.748641 -68.071030 Rhinella spinulosa 18 0 0

Telmatobius vilamensis 10 0 0Val le de Jere -23.187166 -67.991194 Rhinella spinulosa 30 0 0

IV Rio Elqui -29.897250 -71.244583 Pleurodema thaul 51 0 0Calyptocephalella gayi 5 0 0

VIII Nahuelbuta -37.882251 -73.371236 Batrachyla taeniata 4 0 0 Calyptocephalella gayi 11 0 0 Rhinoderma darwinii 8 0 0 Eusophus contulmoensis 11 0 0 Pleurodema thaul 14 0 0X Puyehue -40.723249 -72.433281 Rhinoderma darwinii 7 0 0 Eusophus calcaratus 23 0 0 Eusophus vertebralis 2 0 0 Eusophus emiliopigini 1 0 0

TOTAL 245 0

NATIVE AMPHIBIANS NON-INVASIVE SAMPLES SUMMARY

0%

RESULTS: FRESHWATER FISH SAMPLES SUMMARY

Region Site Lat Long Species n Rv +Apparent

Prevalence XV Lago Chungará -18.235944 -69.181638 Orestias chungarensis 30 0 0

II Calama -22.501987 -68.960164 Gambusia holbrooki 70 0 0

Oncorhynchus mykiss 11 0 0

IV Rio Elqui -29.897250 -71.244583 Gambusia holbrooki 82 0 0

V Jardin Botanico -32.039694 -71.498111 Gambusia holbrooki 40 0 0

Vi l la Alemana -33.036251 -71.370742 Gambusia holbrooki 5 0 0

VI Rancagua -34.185358 -70.799575 Gambusia holbrooki 64 0 0

VIII Nahuelbuta -37.882251 -73.371236 Cyprinus carpio 3 0 0

Cheirodon galusdae 31 0 0Galaxia maculatus 70 0 0

X Puyehue -40.723249 -72.433281 Oncorhynchus mykiss 60 0 0XIV Valdivia -39.872720 -73.160637 Gambusia holbrooki 60 0 0

TOTAL 526 0

FRESHWATER FISH SAMPLES SUMMARY (TISSUE)

0%

PARTIAL SEQUENCES

531bp CONTIGQUALITY

ASSESSMENT AND TRIMMING

RAW SEQUENCES ALIGNMENT

(Bowtie 2)

ALIGNMENTAGAINST 22

PUBLISHED RV SEQUENCES

(Bowtie 2)

Next generationsequencing using Illumina

MiSeq System

POSITIVE SAMPLES WERE INCLUDED IN LIBRARIES FOR WGS

PHYLOGENETIC ANALYSIS CONFIRMED THE VIRUS TO BE FV3

100% SIMILARITY

SUMMARY

1 positive X. laevis(IV Region)

1 positive X. laevis(V Region)

7 positive X. laevis (Metropolitan Region)

FV3

CONCLUSIONS

• Ranavirus prevalence in Chilean amphibians and fish is lower than 5% (95% CI).

• Ranavirus DNA in 9 X. laevis from 3 different regions. No virus in any of the native amphibians.

• FV3 appears to be restricted to central Chile, which coincides with the Africanclawed-frog distribution.

• Results suggest FV3 is not endemic to Chile, and that X. laevis may act as Ranavirusreservoir for native amphibians (Mora et al. 2019).

• No Ranavirus DNA in freshwater fish, but their role as reservoirs depend on the Ranavirus type and the host fish species (Jancovich et al. 2011).

• Monitoring programs at X. laevis invaded ponds, and efforts should be made toprevent the spread of this species to new water bodies.