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The social behaviour of anuran amphibians. Anim. tion in a free-ranging wildlife species? EcoHealth 3:35–40. Behav. 25:666–693. Herpetological Review, 2012, 43(3), 425–427. © 2012 by Society for the Study of Amphibians and Reptiles Ranavirus Infection in Native Amphibians at La Selva Biological Station, Costa Rica: First Report of Ranavirus in Central America Ranaviruses are a globally widespread group of iridoviruses with a very well-known herpetofauna (Guyer and Donnelly 2005; capable of causing mass mortality events in amphibian popula- Whitfield et al. 2007). We collected toe clip tissue samples from tions (Gray et al. 2009; Green et al. 2002), and are acknowledged amphibians between July 2006 and May 2008 in association with to be a significant threat to amphibian populations in many parts a mark-recapture study using 19 study plots within primary for- of the world. Central America hosts a diverse, unique, and highly est. Our sampling for this study was focused on forest-floor am- threatened amphibian fauna (Whitfield et al., in press), yet there phibians, the majority of which are direct-developing species have been only modest efforts to detect and characterize rana- with no free-living tadpole stage. Toe clips were stored in 70% viruses in the region. No information exists on impacts of rana- ethanol until we performed laboratory analyses. viruses to amphibian hosts or populations in the Neotropics—a We haphazardly selected 104 of these toe clip samples for mo- significant gap in knowledge that may inhibit effective conserva- lecular detection of ranavirus using quantitative real-time PCR tion planning. (qPCR). We extracted nucleic acids from toe clips with Qiagen Speare (1991) reported an iridovirus, likely a ranavirus, from DNeasy spin columns, and used the extract to perform qPCR fol- Rhinella marina in Costa Rica. Picco and Collins (2007) tested lowing the standard ranavirus protocol (Kerby and Storfer 2009). 41 amphibians from Monteverde, Costa Rica and 43 amphibians We used 13µL reactions containing 2µL of extracted DNA tem- from Las Cruces, Costa Rica but did not detect ranavirus at ei- plate, 900-nmol forward primer, 900-nmol reverse primer, 250 ther site. Lips et al. (2006) assayed 38 amphibians during a mass mortality event at El Copé, Panama and detected no ranavirus. STEVEN M. WHITFIELD* To our knowledge, there are only two reports of infection by University of South Dakota, Biology Department, 414 E. Clark St, ranaviruses anywhere in the American tropics: Galli et al.(2006) Vermillion, South Dakota 57069, USA; e-mail: [email protected] reported ranavirus infections in cultivated, non-native American MAUREEN A. DONNELLY Bullfrogs, Lithobates catesbeianus, in Brazil; and Zupanovic et al. Florida International University, Department of Biological Sciences, (1998) reported infection by “Guatopo Virus”—a novel strain of UP Campus OE167, Miami, Florida 33199, USA ranavirus similar to frog virus 3 (FV3)—from wild frogs in Vene- ERICA GEERDES zuela. Here, we provide the first robust evidence for the presence JACOB KERBY of ranavirus in amphibians in Central America. University of South Dakota, Biology Department, 414 E. Clark St, V We conducted this study at La Selva Biological Station, a 16 ermillion, South Dakota 57069, USA 2 km private biological reserve in the northeastern lowlands of * Author for correspondence Sarapiquí, Heredia Province, Costa Rica (10.433°N, 83.983°W) Herpetological Review 43(3), 2012 426 AMPHIBIAN DISEASES these infected individuals. The sequence produced in this study taBLe 1. Results of qPCR assays for ranavirus at La Selva Biological was identical to basepairs 205 to 611 of seven FV3 strains from Station in Costa Rica. Genbank. Taxon Number positive / number tested This study provides strong evidence of infection by ranavirus in wild native amphibians in the lowlands of Costa Rica. Preva- Bufonidae lence of infection among all species at La Selva was relatively low Rhaebo haematiticus 0 / 2 (3.8%; 95% CI 1.1–9.5%), although prevalence in the one species Hylidae in which we detected infections, Craugastor bransfordii, was Scinax eleaochroa 0 / 3 considerably higher (14.2%, 95% CI: 4.0–32.6%). We noted no ob- Eleutherodactylidae vious symptoms of ranaviral disease in these frogs, but have no data available to determine whether infections have any adverse Diasporus diastema 0 / 9 effect on individuals or populations. Our genetic characteriza- Strabomantidae tion of this FV3 strain is currently insufficient to assess whether Pristimantis cerasinus 0 / 1 this represents a strain native to Central America or a strain re- Prisimantis ridens 0 / 3 cently introduced from other areas. Craugastoridae This study represents the first report of ranavirus infection Craugastor bransfordii 4 / 28 in amphibians from Central America, and one of very few re- Craugastor crassidigitus 0 / 1 ports from the hyper-diverse Neotropical region. Other studies Craugastor fitzingeri 0 / 7 from Central America have failed to detect ranaviruses (Lips et Craugastor megacephalus 0 / 8 al. 2006; Picco and Collins 2007; but see Speare et al. 1991), but Craugastor mimus 0 / 34 were based on small sample sizes (each <100 samples) and used Craugastor noblei 0 / 1 traditional PCR for pathogen detection, which is less sensitive than the qPCR used in this study. We suspect that the absence Craugastor talamancae 0 / 5 of reports of ranaviruses from tropical regions is likely a result Total amphibians sampled 4 / 104 of limited sampling rather than a true absence of these patho- gens. We expect that further studies of ranavirus in tropical re- gions are very likely to increase the known distribution of these nmol probe, and 2X Taqman Fast Universal Master Mix (Applied pathogens. It will be important to determine how the ranavirus Biosystems, Foster City, California). We ran plates for one cycle we detected is related to other ranaviruses, whether this strain of 95°C (20 sec) and 50 cycles of 95°C (3 sec) and 60°C (20 sec) on is endemic or introduced, and whether it presents a risk to the a StepOnePlus qPCR machine (Applied Biosystems, Foster City, neotropical amphibian assemblages already decimated by chy- California) in a molecular core facility at the University of South tridiomycosis-associated declines. Dakota. Each plate included a negative control and a standard curve of 1 x 103 – 1 x 107 copies of the complete major capsid pro- Literature Cited tein sequence of FV3 (Mao et al. 1997), 3900bp in length, derived from a bacterial plasmid (pCRII) obtained from V. G. Chinchar. Brunner, J. L., and J. P. CoLLins. 2009. Testing assumptions of the trade- All samples were run in triplicate and samples with two or more off theory of the evolution of parasite virulence. Evol. Ecol. Res. positive wells were considered positive. Samples with only one 11:1169–1188.