The impact of the Amphibian Chytrid Fungus Batrachochytrium dendrobatidis on a Green and Golden Bell Frog Litoria aurea reintroduction program at the Hunter Wetlands Centre Australia in the Hunter Region of NSW M.P. Stockwell, S. Clulow, J. Clulow and M. Mahony Amphibian Research Group, School of Environmental and Life Sciences The University of Newcastle, Callaghan Drive, Callaghan NSW 2308 Email: [email protected]. au Reintroduction programs are used widely in conservation to reduce a species’ risk of extinction and amphibians are considered suitable candidates for such programs because of their behavioural T simplicity and high reproductive output. The Green and Golden Bell Frog Litoria aurea is an endangered C species that has been reintroduced into several areas within its natural range, but the outcome of A these programs remain unknown. This paper presents the results from the first release of the bell frog in the Hunter Region of New South Wales. This reintroduction released 850 tadpoles into a closed R system of three ponds and rehabilitated habitat. Tadpole survival was high but following metamorphosis T a decline in numbers began that continued for 13 months and resulted in the disappearance of all S released bell frogs. The cause of this decline was investigated and eventually attributed to infection by the Amphibian Chytrid Fungus Batrachochytrium dendrobatidis. These results emphasize the importance B of including regular chytrid testing in the monitoring of both natural populations and reintroduction A programs, particularly as few sick and dead animals were found to indicate its presence. Key words: Litoria aurea, green and golden bell frog, reintroduction, translocation, amphibian chytrid fungus, Batrachochytrium dendrobatidis, chytridiomycosis. Introduction Reintroduction programs are used in conservation to reduce allowing them to establish large populations within several a species’ risk of extinction by establishing new populations generations (Bloxam and Tonge 1995). The Green and in areas that they have declined or disappeared from Golden Bell Frog Litoria aurea is an endangered species (Jungius 1985; IUCN 1987; Dodd and Seigel 1991; Serena that has been declining for the last 30 years. The and Williams 1995; Seddon et al 2007). They also raise cause of this decline has not been determined although the perceived value of land, increase public awareness and habitat modification, predation by the introduced fish participation in conservation efforts and provide information Gambusia holbrooki and the Amphibian Chytrid Fungus on the population dynamics, ecology and management Batrachochytrium dendrobatidis have been implicated (Pyke of the study species (Dodd and Seigel 1991; Serena and and White 2001; Pyke et al 2002; DEC NSW 2005). As Williams 1995). Reintroduction programs require extensive such, the introduction of the bell frog into rehabilitated planning, research and years of monitoring and should result or supplementary habitat has been widely used in its in a self sustaining population that interacts freely with its conservation with programs currently occurring in Botany, environment and requires minimal management (Dodd Marrickville, Collaroy, Arncliffe and Pambula (DEC and Seigel 1991; Serena and Williams 1995). However, NSW 2005; Daly et al 2008; Pyke et al 2008) . reintroduction programs are rarely successful in achieving Despite the number of bell frog reintroduction programs these outcomes and their value in conservation is constantly underway, their design, methodology and progress remain questioned (Jungius 1985; Conant 1988; Wirth 1990; unknown because records have rarely been published in Lindburg 1992). Despite this, reintroduction programs peer-reviewed journals. This paper describes the outcome are widely implemented (Jungius 1985; Griffith et al 1989; of the first release of the green and golden bell frog in a Dodd and Seigel 1991; Seddon et al 2007) particularly for reintroduction program occurring in the Hunter Region amphibians (Bloxam and Tonge 1995). of New South Wales (NSW). The aim of this release was Amphibians are suitable candidates for reintroduction to establish the first generation of bell frogs on site in the programs as they are behaviorally simple, requiring no absence of the threatening processes considered to be pre-release training and have high reproductive outputs, responsible for its decline. Australian October 2008 Zoologist volume 34 (3) 379 Stockwell et al. Methods a. Study Site Hunter Wetlands Centre Australia owns and manages the Shortland Wetlands in the Hunter Region of NSW (32°52’19”S 151°42’90”E) including 45 hectares of rehabilitated wetlands and a visitor centre. It is currently listed as a Ramsar wetland of international importance and functions as a community based environmental, educational and ecotourism facility. Bell frogs were once widespread throughout Shortland but became locally extinct in the 1970s when the wetlands were filled for development. In May 2002 construction of ponds began in a secluded area of overgrown grasslands following the specifications of Pyke and White (1996). Three ponds of varying sizes were excavated (dimensions 15.7 x 12.4 m, 14.5 x 11.6 m and 12.8 x 4.9 m) and vegetated with b. the emergent Cumbungi Typha orientalis. Garden beds containing locally endemic grass and shrub species were placed around and between each pond and six rock piles were also placed either between or extending into each pond (Fig 1, 2). Figure 2. The Hunter Wetlands Centre Australia Green and Golden Bell Frog reintroduction site showing: (a) one of the three ponds with emergent Cumbungi vegetation, a rock pile extending into the water and a mesh holding basket being placed into the pond and, (b) the 1 m tall fence surrounding the reintroduction site. Ponds were filled with fish-free groundwater, pumped from a nearby creek with hoses covered in 2 mm mesh to Figure 1. Site map of the Hunter Wetlands Centre exclude any fish. Prior to the release of bell frog tadpoles Australia Green and Golden Bell Frog reintroduction site into the reintroduction site the ponds were left to stand in the Hunter Region of NSW (source: Jamieson and for 12 months. During this time two species of tree frog, Mahony 2003). the Dwarf Green Treefrog Litoria fallax and the Emerald A 1 m tall mesh fence was placed around the Spotted Treefrog Litoria peronii, breached the fence and reintroduction site, containing the ponds and 2700m2 also became established within the enclosure. of surrounding grassland (Fig 2). This fence was buried Release and Survey 20 cm into the ground, had 20 cm overhangs at the top on both sides and was designed to keep bell frogs inside Bell frog tadpoles were initially intended to be bred from the enclosure so population numbers could be monitored wild animals from the Sandgate population, less than 3 km closely. It was also to keep other species out to prevent away (32°53’00”S 151°42’05”E) from the reintroduction interspecies competition, predation and to reduce the site. However, the source population appears to have chances of chytrid being carried into the site. However, undergone a decline and possible localised extinction (M. Mahony pers. obs.) so source animals could not be prior to the construction of this fence one species of found. Tadpoles were instead bred from 2 captive animals ground frog, the Common Eastern Froglet Crinia signifera on display at the Hunter Wetlands Centre Australia that became established within the site despite efforts to were initially from the Sandgate population. remove it. Six of these froglets were sent to the CSIRO Australian Animal Health Laboratory for chytrid testing In the summer of 2004, 20 bell frog tadpoles were bred and all came back negative. at the University of Newcastle and released into a mesh Australian 380 Zoologist volume 34 (3) October 2008 Impact of the Chytrid Fungus on a Green and Golden Bell Frog reintroduction program basket (dimensions 1 m3) submerged in one of the ponds water bodies to determine whether bell frogs were able at the Hunter Wetlands Centre Australia reintroduction to disperse out of the enclosure. Any dead animals found site (Fig 2). This release was intended to assess whether were swabbed with sterile fine-tip MW-100 swabs and the habitat was likely to be suitable for the growth stored in 80% ethanol. and survival of bell frogs. Eleven of these tadpoles On individual nights in June and December a sample metamorphosed and were collected from the basket and of bell frogs were located with spotlight searches and released freely into the pond. The remaining 8 tadpoles caught by hand, where the hand was covered in a single- disappeared from the basket and may have either died use plastic bag to prevent the spread of disease. Once and decomposed or metamorphosed and climbed out. an animal was caught the bag was inverted to contain At least 3 of these metamorphs survived for more than the animal where it was weighed on digital scales and two years within the reintroduction site. This confirmed swabbed for chytrid infection by wiping the swab over that the enclosure was likely to be suitable and the the abdomen, inner thigh, hands and feet of each animal reintroduction program continued. in a standardised manner. The DNA on these swabs was In the summer of 2005, 850 bell frog tadpoles were bred extracted at The University of Newcastle and DNA from at the University of Newcastle and grown to Gosner the chytrid fungus was amplified using real-time Taqman stages 35-38 (Gosner 1960) to prevent the loss of animals PCR assays, following the methods described by Boyle to small invertebrate predators (Pyke and White 2001). et al. (2004). This test quantifies the number of chytrid Prior to release the mouthparts of 60 randomly selected zoospore equivalents present and is then multiplied by tadpoles were checked under a stereomicroscope for 10 to account for a 1/10 dilution step in the extraction signs of pigment loss associated with chytrid infection process. This zoospore count provides a measure of (Fellers et al 2001; Marantelli et al 2004; Rachowicz and infection severity that is comparable between animals. Vredenburg 2004). No pigment loss was found in any of In order to prevent the spread of disease in and out the animals tested so the release continued. The sample of the site all equipment and footwear of investigators size of 60 animals was chosen throughout this study for and grounds keepers were washed in hot tap water the monitoring of chytrid infection because it allows the (~50 °C) and sprayed with a 2% sodium hypochlorite detection of one positive animal with a likelihood of solution prior to entry, both of which will kill chytrid 95%, if the prevalence within the population is 5% or zoosporangia within 30 seconds (Johnson et al 2003) and greater (DiGiacomo and Koepsell 1986). are recommended in hygiene protocols (NPWS 2000). Upon release 650 of the tadpoles were placed freely Wader use was restricted to this site only. into the ponds and 200 were distributed into four mesh baskets partially submerged into each of the ponds. Results The tadpoles in the baskets were easily accessible and In the 2005 release 73% of tadpoles placed into 3 of could be monitored closely to ensure that the water the mesh baskets survived and metamorphosed with an quality of the ponds was suitable for their growth and average weight (± SD) of 1.09 g (± 0.34) by the end of survival. The tadpoles in the mesh baskets were counted March 2005. The tadpoles placed into the fourth mesh daily until metamorphosis when they were weighed basket escaped when the lid floated open during a heavy and released freely into the ponds. The free-living rain event. The visual encounter survey of free-living bell metamorph numbers were then monitored over time frogs also found metamorphs emerging throughout March using time-constrained standardised visual encounter and individuals were observed to survive for up to a year surveys (Crump and Scott 1994) at a frequency of 2-4 in this enclosure although numbers decreased throughout times per month. Species-time curves were generated to the year (Figure 3). A gradual decline occurred from May determine the minimal search time in each pond that 2005 and continued until the end of August. The rate of detected the maximum number of frogs visible and it decline then appeared to slow until November when it was found that 20 minutes in the two larger ponds and increased again (Figure 3). No bell frogs have been seen 10 minutes in the smaller pond were appropriate. These in the enclosure since the end of March 2006. Six frogs surveys were standardised by using the same investigator (only bell frogs) were found dead within the enclosure and following the same paths within the ponds each between June and August 2005. Only five of these animals time. The number of bell frogs, dwarf treefrogs and were in an early enough state of decay to swab and four spotted treefrogs seen in each survey were recorded. were found to be infected with chytrid. The number of Due to the cryptic nature of the eastern froglets, their zoospores from each of these was highly variable with numbers were estimated based on the number heard an average (± SD) of 3489˚ (± 6455˚) spores. None of calling at the beginning of each survey. these frogs showed any skin lesions, excess shed skin or ventral reddening. At each survey event the ponds were also monitored for predators and water depth was measured using On one night in June, 60 bell frog juveniles were caught permanent depth markers placed at the deepest point and found to have an average weight (± SD) of 3.76 g in each pond. The pH of the water in each pond was (± 1.27). These 60 animals were swabbed for chytrid and measured using a Hanna Instruments pHep meter while analysis revealed it was present on 53% of individuals. the dissolved oxygen and salinity were measured using a The number of zoospores from each sample was highly YSI Model 85 water test kit. Opportunistic night surveys variable with an average (± SD) of 50 (± 124) spores. were also conducted during rain events around near-by The December night survey took place but was only able Australian October 2008 Zoologist volume 34 (3) 381 Stockwell et al. Figure 3. The number of Green and Golden Bell Frogs encountered in each survey over the first year following release at the Hunter Wetlands Centre. to locate 4 bell frogs, none of which could be caught heard at a lesser frequency and in lower numbers from so growth and infection data could not be collected. September 2005 to February 2006, but increased after However, in the absence of bell frogs, 60 dwarf treefrogs, this period. 10 eastern froglets and 8 spotted treefrogs were caught During these visual encounter and night surveys bell and swabbed. Of these animals 2 dwarf treefrogs tested frogs were often observed making use of the constructed positive for chytrid with an average (± SD) zoospore habitat, basking and foraging on emergent and surrounding count of 1.8 (± 0.3). All other animals were negative for vegetation and hiding in rock piles within the enclosure. the presence of chytrid. The range of depth and water quality values was found The number of dwarf treefrogs andspotted treefrogs to be similar within the three ponds (Table 1). Several encountered during surveys did not decline in parallel frog predators were observed within the enclosure with bell frogs (Figure 4). Both species were observed less during surveys including the White Faced Heron Egretta frequently and in lower numbers from May to August but novaehollandiae, the Australian Raven Corvus coronoides, reappeared in October. Dwarf treefrogs were observed in the Pied Butcherbird Cracticus nigrogularis and the almost three times the numbers of bell frogs and peaked Eastern Water Skink Eulamprus quoyii. The scats of a in number during December 2005 and January 2006. large rodent and the tracks of a fox Vulpes vulpes have spotted treefrog numbers remained consistently low also been found within the enclosure. Each of these throughout the study (Fig. 4). The number of calling predators was observed on fewer than 3 occasions each. eastern froglets at the start of each survey also did not The Mosquito Fish Gambusia holbrok predator was never decline in parallel with the bell frogs (Fig. 5). It was observed in the reintroduction site. Figure 4. The number of Dwarf Green Treefrogs Litoria fallax and Emerald Spotted Treefrogs Litoria peronii encountered in each survey over the first year at the Hunter Wetlands Centre bell frog reintroduction site. Australian 382 Zoologist volume 34 (3) October 2008 Impact of the Chytrid Fungus on a Green and Golden Bell Frog reintroduction program Figure 5. The number of Common Eastern Froglets Crinia signifera heard calling at the beginning of each survey over the first year at the Hunter Wetlands Centre bell frog reintroduction site. Table 1. Range of depth, pH, dissolved oxygen, and salinity like that of over-winter sheltering could also reduce the of the three ponds at the Hunter Wetlands Centre bell animal’s visibility and explain an observed decline. Bell frogs frog reintroduction site. generally become less visible during winter as they retreat Depth pH Diss. O (mg/L) Salinity (ppt) to shelter sites such as thick vegetation, mud and rock piles 2 Pond 1 69-118 6.9-8.1 9.04-11.66 0.8-1.2 (Pyke and White 2001) that have higher temperatures than surrounding areas, allowing them to sustain higher body Pond 2 86-118 6.8-8 7.61-14.05 0.1-1.1 temperatures (Duellman and Trueb 1994; Pyke and White Pond 3 69-120 6.8-7.9 7.37-10.86 0.1-0.5 2001; Hamer et al 2003). This behaviour could explain the initial winter decline in bell frog numbers in this study, Discussion but an increase in spring would be expected. This increase in numbers with increasing temperature was observed for This paper describes the outcome of the first release of dwarf treefrogs and spotted treefrogs which also shelter bell frogs at the Hunter Wetlands Centre reintroduction overwinter (Fig. 4) but not for the bell frog (Fig. 3) making site in the Hunter Region of NSW. A total of 850 bell frog it unlikely that the observed decline was due to a change in tadpoles were released into three ponds and monitored 2-4 times a month. Two months after metamorphosis the the animal’s behaviour. number of bell frogs encountered during visual surveys The observed decline may also be the result of animals began to decline (Figure 3). This decline continued dispersing from the enclosure. Many amphibian species throughout the year and within 13 months of the release, are known to disperse from their natal ponds as recently bell frogs appeared to have disappeared from the site. metamorphosed young and juveniles (Duellman and Trueb There is a possibility that a small number of animals persist 1994). Bell frogs are known to move long distances as both but have not been observed and continued monitoring juveniles (Goldingay and Newell 2005) and adults (Pyke will determine this. Despite this possibility it is unlikely and White 2001; Hamer 2002). The enclosure was fenced that this release will achieve its aim of establishing the to prevent this dispersal but the ability of dwarf treefrogs and first generation of bell frogs at this site. However, it has spotted treefrogs to climb this fence raises the possibility that provided an opportunity to investigate why the bell frog bell frogs may also have done so. The surveys of surrounding was unable to persist and to improve the success of future water bodies outside of the enclosure found no sign of bell reintroductions. frogs making dispersal unlikely to be the cause of decline. There are a number of reasons why observations of However, this does not rule out the possibility that animals individuals in a population may decline. These include dispersed beyond these ponds. decreased visibility, dispersal and death. An observed decline The final possibility is that the reintroduced bell frog may occur during visual encounter surveys if a change in population was observed to decline because of mortalities. the environment, such as increased vegetation growth or increased habitat complexity reduced the animal’s visibility. The suitability of habitat to meet all of a species’ requirements Should such an increase occur gradually over time it could is essential to the success of reintroduction programs (Dodd give the appearance of a decline. However, no environmental and Seigel 1991; Kleiman and Beck 1994; IUCN/SSC 1995). change was observed at the reintroduction site. In addition, For this reason habitat conditions were monitored closely in such change would reduce the visibility of all species and this study. The metamorphosis of 73% of bell frog basket as the pattern of fluctuations in the number of bell frogs tadpoles is much higher than the survival rate normally differed from that of dwarf treefrogs, spotted treefrogs and observed for amphibians in nature (5-10 %, Duellman and eastern froglets it is unlikely to have been the cause of Trueb 1994), indicating that the habitat was suitable for this the observed decline. A change in the animal’s behaviour species. The bell frog is tolerant of a wide range of conditions Australian October 2008 Zoologist volume 34 (3) 383 Stockwell et al. and the range of water quality measurements for each pond animals were sampled to draw conclusions with a high (Table 1) was found to remain within the range observed level of confidence. The observation that only the bell in natural habitat (Pykeet al 2002) including nearby ponds frog declined in this study suggests that it may be more on Kooragang Island (Hamer 2002). The use of habitat susceptible or more affected by chytrid infection than the for foraging and basking by bell frogs in the reintroduction other three species. These species may therefore be acting site, their high rate of growth from March to June during as reservoirs for the Chytrid Fungus, contributing to the the initial decline and the survival and reproduction of the bell frog decline. The observation of declines in the bell other species that often occur with bell frogs in nature also frog but not sympatric species in this study is echoed in indicate that the habitat was suitable. Competition and nature suggesting these species may also be contributing predation are also processes that could result in a decline to the decline of the bell frog throughout its range. in a less competitive or prey species. However, none of the The finding that 2 dwarf treefrogs had low levels of chytrid other species would be strong competitors of bell frogs at this and spotted treefrogs and eastern froglets can be infected site due to the low abundance of spotted treefrogs and the also suggests that they may have carried the fungus into much smaller size of dwarf treefrogs and eastern froglets; nor the site and swabbing all species more regularly may have would it be likely that the low number of predators observed confirmed this at an earlier stage of the reintroduction caused the decline. process. If these species did introduce chytrid into the Finally, bell frog mortalities may have been due to disease reintroduction site, more effective fencing may also have and the presence of the Chytrid Fungus within the prevented the population decline. Being carried by other reintroduction site supports this as a cause of decline. The frog species is not the only way that chytrid could have Chytrid Fungus causes the disease chytridiomycosis which entered the site. A lapse in hygiene protocols may have can result in mass mortalities in amphibian populations and resulted in chytrid being carried in on equipment or is thought to be the cause of the global amphibian decline footwear. Recent studies have found that chytrid may that has been occurring for the past 30-40 years (Berger be carried by other organisms such as on bird feathers et al 1998). It currently infects at least 93 species around (Johnson and Speare 2005) and reptile skin (Piotrowski et the world, 46 of which are Australian species (Speare and al 2004), both of which were able to move freely in and out Berger 2004, 2005). The chytrid fungus has been found of the reintroduction site and may have carried it in before in bell frog populations within the Hunter Region (M.P. or after the release of the bell frog. There is also some Stockwell unpubl. data) and although attempts were made evidence that chytrid may persist in the environment in the to prevent it from entering the Hunter Wetlands Centre absence of a host with laboratory studies showing chytrid Australia reintroduction site through the exclusion of other can survive in lake water for up to 7 weeks (Johnson and frog species and the adoption of strict hygiene protocols, this Speare 2003). However, chytrid has never been isolated was not successful. from the environment so it remains unknown how likely it was that chytrid was already present at the reintroduction The Chytrid Fungus was found on the skin of 53% of site prior to the release of the bell frog. bell frogs surveyed in June 2005 with individuals showing an average count of 50 zoospores. This prevalence and The results of this study illustrate the importance of zoospore count is much higher than those seen from designing and monitoring programs to allow investigation free-living and non-declining bell frog populations (M.P. into the causes of population fluctuations. For example, Stockwell unpubl. data) supporting the idea that chytrid by monitoring habitat quality, predation, dispersal and infection was the cause of decline in the reintroduced disease it could be established that chytrid induced population. Similarly, the discovery of dead bell frogs mortality was the most likely cause of decline in this infected with chytrid suggests it was the cause of death, reintroduced population, information that is essential for particularly as dead animals were found to have much the success of future bell frog reintroduction programs. higher levels of infection than live ones. However, this These results also provide the first evidence that the may be because the chytrid fungus continued to grow and amphibian chytrid fungus can cause a rapid decline in multiply after the death of the frog and as no autopsies bell frogs when a closed population containing one cohort were performed on dead frogs, other causes of mortality is exposed to it. They show that an absence of sick and cannot be ruled out. Despite this, the presence of infected dead animals or the decline of sympatric species does not animals in the reintroduction site, the knowledge that necessarily equate to an absence of the chytrid fungus, infection causes fatalities in bell frogs and is listed as a demonstrating the need for long term chytrid monitoring key threatening process of this species(DEC NSW 2005) in both reintroduced and natural populations. makes death as a result of chytrid infection the most likely Finally, this study shows the importance of publishing the cause of decline in this reintroduced population. outcome of such reintroduction programs, whether they The bell frog was not the only species to be infected are successful or unsuccessful, because publishing allows with chytrid at this site but they were the only species reintroduction and monitoring methods to be refined, observed to decline and not recover. The fungus was identifies problems, prevents mistakes from being repeated present in 3% of dwarf treefrogs sampled at the start and promotes accountability in actions ensuring that well of summer but because no bell frogs could be caught established reintroduction guidelines are followed (Jungius at that time the infection concentrations could not be 1985; Dodd and Seigel 1991; Bloxam and Tonge 1995; compared between species. There are reports that the IUCN/SSC 1995; Seddon et al 2007). It also allows us to other two species, spotted treefrog and eastern froglet, see the effect that these programs have had on decreasing can be infected by chytrid (Speare and Berger 2005) but the species risk of extinction, and therefore their value in this could not be confirmed by this study because too few its conservation. Australian 384 Zoologist volume 34 (3) October 2008 Impact of the Chytrid Fungus on a Green and Golden Bell Frog reintroduction program Acknowledgments This reintroduction program was approved by the NSW The University of Newcastle Animal Care and Ethics Department of Agriculture and NSW National Parks Committee (ACEC number 766 1002). It was conducted and Wildlife Service following peer review of the plan of as a collaborative project between the Amphibian Research management. The work was conducted under NPWS Group at The University of Newcastle, the Hunter Wetlands Scientific license (number S10382) and approved by Centre Australia and the Society of Frogs and Reptiles. References Berger, L., Speare, R., Daszak, P., Green, D.E., Cunningham, Hamer, A.J., Lane, S.J. and Mahony, M.J. 2003. 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