Fatal Chytridiomycosis in the Tyrrhenian Painted Frog
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- Bielby, J., Bovero, S., Sotgiu, G. et al. EcoHealth (2009) 6: 27. doi:10.1007/s10393-009-0232-2
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Batrachochytrium dendrobatidis (Bd), the causative agent of the amphibian disease chytridiomycosis, is an important factor in the global decline of amphibians. Within Europe, animals that exhibit clinical signs of the disease have only been reported in Spain despite the pathogen’s wide, but patchy, distribution on the continent. Recently, another occurrence of chytridiomycosis was reported in Euproctus platycephalus, the Sardinian brook newt, on the Mediterranean island of Sardinia, but without any evidence of fatal disease. We report further evidence of the emergence of Bd on Sardinia and the first evidence of lethal chytridiomycosis outside of Spain. Unusual mortalities of the Tyrrhenian painted frog (Discoglossus sardus) were found at three sites in the Limbara mountains of northern Sardinia. Molecular and histological screens of corpses, frogs, and tadpoles from these sites revealed infection with Bd. Infection and mortality occurred at locations that are unusual in terms of the published habitat requirements of the pathogen. Given the endemicity, the IUCN Red List status of the amphibian species on Sardinia, and the occurrence of infection and mortality caused by chytridiomycosis, there is serious reason for concern for the impact that disease emergence may have on the conservation of the amphibians of the island.
The emergence of Batrachochytrium dendrobatidis (Bd), the etiological agent of chytridiomycosis, is responsible for regional mass mortality events of amphibian hosts, as well as host population declines and extinctions (Lips et al., 2008; Schloegel et al., 2006; La Marca et al., 2005; Laurance et al., 1996). These regional effects are detectable at broad geographic scales: the pathogen occurs on six continents, infects hundreds of amphibian species, and is widely recognized as a major contributor to the global decline of amphibians (Bielby et al., 2008; Lips et al., 2008). Within Europe, the Bd knowledge base has grown slowly during the past decade: Bd has been detected in wild amphibians from many European countries, including Italy (Garner et al., 2005; Simoncelli et al., 2005; Stagni et al., 2002). However, records of mass mortalities and local extirpation of host species exist only from Spain (Bosch and Rincón, 2008; Bosch and Martínez-Solano, 2006; Bosch et al., 2001). Recently chytridiomycosis has been detected on the island of Sardinia, affecting the endangered Sardinian brook newt, Euproctus platycephalus (Bovero et al., 2008). Signs of disease in E. platycephalus included ulcerative damage, degradation of digits, and skin abnormalities. The greater consequences of Bd emergence for Sardinian amphibians remain unclear because unusual mortalities that may be the result of disease have not been observed in any of the island’s amphibians. This may be due to a lack of surveillance rather than any lack of disease-related mortality, because declines of Sardinian amphibians have been reported since the early 1980s (Colomo and Ticca, 1984; Puddu et al., 1988), which coincides roughly with published dates for Bd-driven declines in other parts of the world (Lips et al., 2008; Laurance et al., 1996). Here we outline some results of a 5-year survey of amphibian populations of the three main mountain ranges of Sardinia and report lethal infection in the Tyrrhenian painted frog, Discoglossus sardus, and evidence of substantial mortality due to disease in this host species at three breeding sites.
Field surveys were initiated in 2004. Surveys were initially conducted as part of a study of the distribution and life history of E. platycephalus, but a specific Bd sampling component was incorporated from 2007 onwards. Surveys were opportunistic, in that they focussed on locations that appeared to have habitat suitable for amphibians on Sardinia and were conducted in mountainous regions. We surveyed pools, lakes, and mountain streams for both metamorphosed caudate and anuran amphibians, as well as their larvae. We found the first evidence of unusual mortality in June 2004 at a site located in the Limbara Mountains. We discovered six dead adult Discoglossus sardus in a stream feeding a freshwater lake, Laghetto dei Pompieri located in northern Sardinia (N 40°50′20.0″, E 9°8′35.0″). At least two of the corpses had extensive damage to the tips of the digits of both fore- and hind-limbs. Corpses showed no other signs of decomposition or predator damage, although in some cases invasion of the corpse with hyphal fungus was advanced. No corpses were collected at this time. We returned to this site in August 2007 and found the stream to be completely dried. We surveyed Laghetto dei Pompieri itself and captured 17 apparently healthy and recently metamorphosed D. sardus. All animals were handled using disposable gloves and kept individually in sealed plastic bags to prevent any cross-contamination amongst frogs. We swab-sampled (Hyatt et al., 2007) each frog by firmly running a swab repeatedly over the lower abdomen, limbs, and feet. We also collected ten dead larval Sardinian tree frogs (Hyla sarda) found near the confluence of the stream bed and the lake and preserved them in 70% ethanol: thousands of recently metamorphosed tree frogs were in evidence at the lake. We visited this site again in early May 2008. We found hundreds of breeding tree frogs in and around the lake, but no sign of D. sardus at the lake site. We also surveyed the stream, this time running with water, and could find no evidence of D. sardus breeding activity, eggs, or tadpoles. However, in the same sampling period we collected and preserved 19 D. sardus tadpoles from a shallow, ephemeral pool approximately 1 meter in diameter located in the same drainage as Laghetto dei Pompieri (N 40°50′54.8″, E 9°09′06.5″). The pool was located less than 2 km from the lake but was less than 20 m from the far upstream reaches of a small tributary that eventually led into the stream feeding into Laghetto dei Pompieri. Surveys of this upstream reach revealed no evidence of D. sardus larvae or any sign of breeding activity.
In May 2006, the bodies of approximately 20 D. sardus adults were discovered at Monte Olia forest workers’ camp (N 40°44′45″, E 9°21′40″), in a Limbara mountain drainage system well separated from that of Laghetto dei Pompieri. We again observed tissue loss on the digits, some evidence of decomposition, but none of predator damage on other parts of the body. One specimen from the 2006 Monte Olia mass mortality event was collected and preserved in 70% ethanol. No other amphibian species were in evidence at this location at this time. We returned to this site in August 2007 and found the stream to be completely dried. However, in May 2008 when the stream was again running, we found two D. sardus corpses: a severely decomposed juvenile and an adult male with some evidence of hyphal fungal invasion. Both animals were preserved in 70% ethanol. Additionally, we captured and swab sampled 4 adult or juvenile D. sardus and collected and euthanized 27 larvae, storing them in 70% ethanol. No other amphibian species were observed at this location.
Again in May 2008, we surveyed a first-order tributary (Affluente Pisciaroni; N 40°51′40.2″, E 9°8′46.6″) of the Pisciaroni stream (N 40°51′42.1″, E 9°08′16.4″), a large, cascading, mountainous stream located in the Limbara Mountains in a drainage adjacent to, but separate from, the Laghetto dei Pompieri drainage. We surveyed the majority of the length of this tributary, swabbed one adult D. sardus near the start of the tributary and discovered one moribund, juvenile D. sardus in a deep pool not far downstream, located below a small fall. E. platycephalous also were observed at this location. The juvenile died soon after collection and was subsequently preserved in 70% ethanol. Further downstream we encountered a broadening of the tributary near a large cascade unshielded by the stream-side canopy. We found D. sardus tadpoles in several small, sun-exposed pools and two dead adult D. sardus, exhibiting severe decomposition and hyphal invasion, in two separate pools. We collected and preserved the two corpses and from one of those pools we collected 18 tadpoles that were observed to be feeding on the corpse. Tadpoles of D. sardus were rarely observed below this location and a survey of the Pisciaroni itself revealed no evidence of amphibian activity, even though we had encountered E. platycephalus adults and larvae and D. sardus larvae in low numbers along this river the previous year.
All corpses, tadpoles, and swabs were taken to the Institute of Zoology in London and assessed for the molecular signal of infection with Bd. We sampled skin tissue from a hind limb digit and the drink-patch from corpses, and we sampled keratinized mouthparts of tadpoles. Skin samples, tadpole mouthparts, and swabs were extracted following the protocol of Boyle et al. (2004). Extractions were diluted 1/10 with distilled water and then subjected to real-time quantitative polymerase chain reaction (qPCR) developed specifically for the detection of Bd (Boyle et al., 2004). All samples were screened in duplicate and compared to four standard concentrations of Bd and a negative control. We also made standard histology preps from digits of the moribund, juvenile D. sardus collected along the Affluente Pisciaroni in May 2008 (Hyatt et al., 2007). Toes were sectioned and sections stained with hematoxylin and eosin and examined using 40X magnification.
Locations of infection and infection burden of individuals at infected sites within the Limbara mountains
GE ± SD
Laghetto dei Pompieri
N 40°50′20.0″, E 9°8′35.0″
3/17 metamorphic D. sardus swabs
0.1 ± 0.03, 10.8 ± 0.6, 83.2 ± 7.3
N 40°44′45″, E 9°21′40″
Digit = 55.2 + 13.5, Drink patch = 78.3 + 15.71
Digit = 53.8 + 18.8, Drink patch = 11042.5 + 1840.31
Digit = 54.8 + 13.9, Drink patch = 16.8 + 3.4
3/4 swabs of D. sardus
10/27 larval mouthparts
N 40°51′40.2″, E 9°8′46.6″
Live D. sardus swab
1.4 ± 0.4
Digit = Bd undetected, Drink patch = 1.1 ± 0.1
11/18 larval mouthparts
Digit = 5274.3 ± 905, Drink patch = 13203.1 ± 32.8
Our results provide strong evidence for lethal chytridiomycosis in D. sardus at a minimum of three geographically distinct water courses in the Limbara mountains. We also discovered the first case of morbidity in the wild due to chytridiomycosis on Sardinia with subsequent evidence of extremely heavy infection. To the best of our knowledge, this is the first report of Bd-associated morbidity and mortality in wild amphibians in a European country other than Spain. We detected infection and mortality along a wide altitudinal gradient (Laghetto dei Pompieri, 983 m.a.s.l.; Monte Olia, 463 m.a.s.l.; upper reach of Affluente Pisciaroni, 941 m.a.s.l.; location of moribund juvenile along the Affluente Pisciaroni, 838 m.a.s.l.), often below altitudes previously considered high risk areas for lethal chytridiomycosis (above 1,000 m a.s.l., Lips et al., 2008; 1,800–2,200 m a.s.l., Bosch et al., 2006). Our data provide strong evidence that D. sardus is a primary host for Bd in the Sardinian system: results from screens of syntopic newts returned much lower prevalence (data not shown) and at Monte Olia no other amphibian species were seen where we recorded dead D. sardus, infected larvae, and infected frogs. Additionally, habitats where we recorded infection and mortality also do not fit typical profiles for Bd in Europe. Monte Olia and the stream feeding into Laghetto dei Pompieri both dry completely every summer, and infection in European amphibians is commonly associated with permanent water bodies. The pools along the Affluente Pisciaroni where we discovered corpses and infected tadpoles also were not permanent water bodies and likely experience high temperatures, because they are exposed to the direct Mediterranean sun, are less than half a meter across, and only a few centimeters deep. Sardinia in general does not exemplify the habitat characteristics reported for mass mortality sites in Spain (Bosch et al., 2001; Walker et al., 2007). Given that Bd is known to optimize life history responses to enhance growth under changing climatic conditions (Woodhams et al., 2008) as well as exhibiting strong variation in virulence among pathogen genotypes (Fisher et al., 2009), it is possible that the Sardinian strain of Bd is adapted to the local environment or phenotypically plastic. Determining whether either of these possibilities is true would be an important step toward understanding disease dynamics on the island.
The emergence of lethal chytridiomycosis in one species of Sardinian amphibian and infection in two raise serious concerns regarding the conservation of amphibians on the island. The fact that we were unable to find D. sardus at one site where mass mortalities were observed previously (Laghetto dei Pompieri) and very few frogs at another (Monte Olia) is worrying. The amphibian fauna of Sardinia is unique: of the nine native amphibian species, six are endemic, and another two have extremely restricted ranges limited to Sardinia, Corsica, and nearby islands. Both small range size and island endemicity may increase extinction risk (Cooper et al., 2008; Pimm, 1991), and many of the species driven to extinction by chytridiomycosis were known from restricted ranges (La Marca et al., 2005; Schloegel et al., 2006). Six of Sardinia’s amphibians are listed as “Near Threatened,” “Vulnerable,” or “Endangered” by the IUCN (http://www.globalamphibians.org/). Sardinia’s endemic amphibian fauna include five of only nine species of plethodontid salamander found outside of the Americas, one of only six extant members of the genus Discoglossus, and one of only two currently recognized members of the genus Euproctus. Clearly, this highly endemic, evolutionary novel, and threatened species assemblage deserves immediate and concerted conservation efforts in response to any threat to Sardinian amphibian diversity. Bd has the ability to spread tens of kilometers annually (Lips et al., 2008; Alexander and Eischeid, 2001). Given the relatively small size of Sardinia (~270 km in length at its longest point), it is essential to determine the current distribution of Bd on the island before the disease has spread too far. We also recommend the urgent development of antifungal treatments for affected Sardinian species and the establishment of captive breeding programs for Sardinia’s endemics and restricted range species.
The authors thank the People’s Trust for Endangered Species, the IUCN, and the Societas Europaea Herpetologica for funding this project. J. Bielby was supported by a NERC studentship and T. Garner was supported by an RCUK Fellowship. Essential support in the field was provided by Marco Marrosu, Federico Monticone, Cecilia Fasso of the Ente Foreste della Sardegna, Campo Uomo, Sette Fratelli Mountains, Mr. Gianpiero Serra of the Ente Foreste della Sardegna, Limbara Nord Camp, Limbara Mountain, Mr. Nuccio Tucconi of the Ente Foreste della Sardegna, Monte Olia Camp and Alessio Sussarello of the Ente Foreste della Sardegna, Servizio territoriale di Tempio.