Tadpole

In order to obtain metacercariae experimentally, we attempted to infect laboratory-reared snails [Biomphalaria glabrata (Say, 1818)] and fish (Poecilia reticulata Peters, 1859). These species were used as experimental hosts due to their availability in the laboratory and previous knowledge on the involvement of snails and fish as second intermediate hosts of echinostomes. The behavior of cercariae in the presence of these potential hosts was observed under a stereomicroscope. After 24hs of exposure to cercariae, the snails and fish were necropsied. We also searched for metacercariae in samples of insects, fishes, snails, and tadpoles collected in the same water bodies where snails were found infected.
Metacercariae found in tadpoles collected in the stream where snails were found infected were used for an experimental infection study. We suspected they could be of the same species based on the number of excretory corpuscles. Aiming to obtain adult parasites for taxonomic identification, a sub-sample of 50 metacercariae was orally administered to one specimen of a dexamethasone-immunosuppressed (50 mg/kg) male Swiss mouse. The infected mouse was maintained on a 12/12h light–dark cycle and allowed access to food and water ad libitum. Coproparasitological examinations by the sedimentation technique were conducted daily, starting from seven days post-infection. The mouse was euthanized via barbituric overdose (sodium pentobarbital, injected intraperitoneally) and necropsied for the search of adult parasites 14 days post-infection.

We collected samples for ranavirus detection during September–December in Northern Chile, and during the Austral summer (December–March) in Central and Southern Chile, coinciding with the warmer months and the breeding season at all sites. All individuals were collected during day sessions, and each site was visited only once. We established 57 as the minimum number of individuals to be collected at each site in order to detect at least one positive individual if the virus was present. We considered a 95% confidence interval, and assumed 100% sensitivity of the ranavirus detection molecular techniques and a previous ranavirus prevalence of 4.3% for amphibians in central Chile (Soto-Azat et al., 2016 (link)). For most species, sampling stopped either when the minimum required number of individuals from each species present at a site was achieved, or at the end of the day. In the case of Xenopus laevis, because of Chilean regulations, all individuals captured during the sampling session were euthanized and analyzed for ranavirus presence.
We sampled tadpoles of two abundant, widely distributed native amphibian species: the Andean spiny toad (Rhinella spinulosa) and the four-eyed frog (Pleurodema thaul), as well as adults of the invasive Xenopus laevis for ranavirus detection. In addition, we collected and sampled five Calyptocephalella gayi tadpoles found dead in the wild, and ethanol preserved carcasses of 58 recently metamorphosed C. gayi from a mortality event that occurred in a ranaculture facility in Santiago in 2015 were provided to us for ranavirus detection (Table 1). We also sampled two native fish species: galaxia (Galaxias maculatus) and pochas (Cheirodon galusdae), as well as the invasive Gambusia holbrooki, Oncorhynchus mykiss and Cyprinus carpio (Table S1). All the above were considered as the target species of this study, and were euthanized for tissue sampling as decribed below. In addition, when found, we captured adults of R. spinulosa and P. thaul that were present at sampling sites, plus individuals from other native amphibian species found in sympatry with the target species (Table S2). These individuals were non-invasively sampled and released as described below.
For amphibians, we used nets to collect tadpoles of R. spinulosa, P. thaul, and dead C. gayi tadpoles. We collected adults of X. laevis either by hand nets or using chicken liver baited funnel traps. When traps were used, we set them late in the afternoon and checked early the next morning. We collected adults from native species by hand, and these specimens were released at the same site where captured immediately after sampling. For fish, we used hand nets to collect adult Galaxia maculatus, Cheirodon galusdae, Gambusia holbrooki and Cyprinus carpio. We collected juvenile Oncorhynchus mykiss using hand nets or fishing rods (Table S2). Each captured individual was handled with a new pair of vinyl gloves. To minimize any contamination of samples or the spread of pathogens within and between sites, a strict field sampling and disinfection protocol was followed, with reference to Phillott et al. (2010) (link).
According to Gray, Miller & Hoverman (2012) (link) and Goodman, Miller & Ararso (2013) (link), analysis of tissue samples increases the probability of detecting ranaviruses compared to the analysis of non-invasively acquired samples. Therefore, to minimize the impact on native amphibian populations, we only collected tadpoles for tissue sampling. In the case of X. laevis, only adults were collected, as we did not find tadpoles at the study sites where this species was present.
Collected amphibian tadpoles, adult X. laevis and fish were euthanized at their capture sites using an overdose of the anaesthetic tricaine methane sulfonate (Dolical 80%, Centrovet), buffered (pH 7) with sodium bicarbonate (Bayley, Hill & Feist, 2013 (link)). Calyptocephalella gayi carcasses from the ranaculture facility were rinsed with distilled water before necropsy. Gross examination of amphibian and fish viscera were conducted by a veterinarian following Miller, Gray & Storfer (2011) (link). Most histopathological changes associated with ranavirus occur in liver, kidney and spleen (Miller et al., 2015 ). Thus, for ranavirus detection, we obtained samples of these three organs and placed them in individual vials containing 95% ethanol. In addition, we obtained non-invasive oral swab samples from all adult native amphibians captured (see Table S2), by rotating a sterile rayon tipped swab (Medical Wire) for 3–5 s against the buccal mucosa (Gray, Miller & Hoverman, 2012 (link); Goodman, Miller & Ararso, 2013 (link)); swab tips were stored in 1.5 ml sterile vials containing 95% ethanol prior to nucleic acid extraction.