Introduction

Invasive species constitute some of the most important causes of biodiversity loss (^{WILCOVE et al., 1998}; ^{WILCOVE & MASTER, 2005}). In this context, introduced (non-native) free-living species usually arrive to a new territory with lower parasite richness when compared to what is observed in their original locality (^{TORCHIN et al., 2003}). This can be due to the fact that some parasites are not represented in the population taken from the native geographic range; other parasites are taken with the hosts, but they do not arrive to the new territory, and those parasites that do arrive to the new territory may not survive and reproduce therein (^{MACLEOD et al., 2010}). This suggests that there is an advantage for those invasive species that release from their parasites. However, some parasites can be transported to a new territory and naturalize in such a way that the introduced host may act as source of introduced parasites for native free-living species (^{SMITH & CARPENTER, 2006}; ^{LYMBERY et al., 2014}), or they can also catch native parasites (^{KELLY et al., 2009a}; ^{JOHNSON & THIELTGES, 2010}; ^{MASTITSKY & VERES, 2010}), affecting the dynamic of both native hosts and parasites (^{KELLY et al., 2009b}; ^{LYMBERY et al., 2014}). Thus, the study of parasites of invasive species fosters an understanding of the underlying processes that positively or negatively affect these species.

The clawed frog, Xenopus laevis Daudin, 1802, is native to Africa and has been introduced to Europe, Asia, North America, and South America, for both scientific use and pet trade (^{MEASEY et al., 2012}). In Chile, this frog was introduced into the wild in 1973, when an unknown number of individuals was dumped into the Caren Lagoon, which is close to Santiago’s international airport (^{JAKSIC, 1998}). The first naturalized population (stage III, after ^{COLAUTTI & MACISAAC, 2004}) was recorded at the beginning of the 1980s (^{VELOSO & NAVARRO, 1988}). Xenopus laevis spread from this lagoon on its own to other lagoons, ponds, dams, and watercourses around Santiago, although it has also translocated from Caren Lagoon to other bodies of water (^{LOBOS & JAKSIC, 2005}). The spread rate of this frog in Chile was estimated to be between 3.1 and 5.4 km/year, reaching an invaded area of about 21,200 km² in the last decade; this area accounts for four of the fifteen administrative regions (^{LOBOS & JAKSIC, 2005}) and the African clawed frog is expected to invade further north and south in Chile (^{LOBOS et al., 2013}).

At present, the only important pathogen reported in X. laevis in Chile is Batrachochytrium dendrobatidis Longcore, Pessier, Nichols, 1999, which is the etiological agent underlying chytridiomycosis – a disease that has resulted in the population decline and extinction of several anuran species worldwide (^{SOLÍS et al., 2010}) and is considered a notifiable disease by ^{OIE (2010)}. Studies on the helminth parasites of X. laevis have not been performed in Chile; however, many helminth species have been described in feral populations in North America (^{KUPERMAN et al., 2004}) and in wild populations in Africa (^{PRITCHARD, 1964}; ^{MACNAE et al., 1973}; ^{WADE, 1982}; ^{TINSLEY, 1996}). Thus, in order to describe the antecedents involved in the process X. laevis invasion in Chile, in this study, we aimed to analyze the gastrointestinal and external parasite community of this species of frog in this territory.

Materials and Methods

From 1997-2014, 179 adult X. laevis were caught from 10 localities in central Chile: El Tabo (Córdova stream; coordinates: 33°26'0.05”S, 71°38'44.61”W; n = 9 individuals), El Yali National Reserve (Los Molles dam: 33°48'3.60”S, 71°41'49.21”W; n = 2), Tejas Verdes (Maipo river: 33°37'42.25”S, 71°36'30.80”W; n = 3), Batuco (Batuco lagoon: 33°12'0.01”S, 70°50'0.01”W; n = 43), Ibacache (Ibacache stream: 33°27'5.18”S, 71°19'59.41”W; n = 24), La Pintana (Universidad de Chile: 33°34'23.88”S, 70°37'57.64”W; n = 5), Las Chilcas (Chilcas stream: 32°52'17.24”S, 70°50'43.19”W; n = 8), Alhue (Carén stream: 34° 3'57.71”S, 71°15'5.94”W; n = 3), Palmilla (Tinguiririca river: 34°35'49.14”S, 71°21'20.29”W; n = 3), and La Patagua (private dam: 34°42'59.26”S, 71°23'5.94”W; n = 79).

Frogs of La Patagua were actively caught from a dam with use of an ad hoc mesh; in this dam, the frogs were apparent in high densities. In the other localities, the frogs were captured using simple funnel traps (buckets with tight-fitting lids, modified by the lateral insertion of open cones) and baited with liver. After they were euthanized in benzocaine immersion (see ^{CHARBONNEAU et al., 2010}), the frogs were preserved in 70% ethanol and examined in the laboratory under stereomicroscope. They were assessed for helminths through the gastrointestinal tube, cavities, lungs, liver, and skin. Nematodes were preserved in 70% ethanol and cleared with lactophenol for light microscope examination. For the prevalence of infection, refer to ^{Margolis et al. (1982)}. The Comité de Ética of the Facultad de Ciencias Veterinarias – Universidad de Concepción approved and certified the study (certificate number CBE 23‐12) Specimens of nematode parasites were deposited into the Helminthological Collection of Centro de Ecología Aplicada del Litoral (CECOAL 16110301; there were a total of 7 specimens).

Results and Discussion

Only nine specimens of nematodes were found in six frogs (prevalence: 3.4%) from La Patagua. They were found encapsulated in the intestinal serosa and were subsequently identified as larvae of the genus Contracaecum Railliet and Henry, 1912. Genus identification was based on morphological attributes (^{HARTWICH, 2009}): the presence of a posterior ventricular appendix, an anterior intestinal caecum, the excretory pore at the base of the ventral labia, and a rounded tail (Figure 1). Measurements (µm; mean ± standard deviation) of eight larvae (unless otherwise stated) were: total length 2,100 ± 380, width: 116.66 ± 12.74, cecum length: 173.75 ± 69.7, esophagus length: 269 ± 54, distance excretory pore – anterior end: 15 ± 42 (two individuals), distance nerve ring - anterior end: 47 (one individual), tail length: 76.25 ± 11.08 (four individuals). Given the small number of specimens and the fact that they were larvae, it was not possible to identify the species. No external parasites were found.

Figure 1 Contracaecum sp. larvae from the intestinal serosa of Xenopus laevis from La Patagua, Chile: (A) Anterior end; (B) Lateral view of the cephalic portion; (C) Dorsal view of the cephalic portion; (D) Lateral view of the posterior end. Scale bars: A, D: 100 μm; B, C: 50 μm. 

Given the various processes that make the arrival and establishment of parasites to new territories difficult (^{MACLEOD et al., 2010}), the reduced parasite richness of an invasive host species in the colonized territory, when compared to its original geographic range, is expected. Thus, our results were in line with our expectations, particularly as a greater richness of gastrointestinal and cavity helminths have been recorded in X. laevis in South Africa. For instance, some of the helminth species reported in African clawed frogs include Cephalochalamys namaquensis Cohn, 1906 (cestode); Protopolystoma xenopodis Price, 1943; Gyrdicotylus gallieni Vercammen-Grandjean, 1960 (monogenean); Oligolecithus jonkershoekensis Pritchard, 1964; and Progonimodiscus doyeri Ortlepp, 1926 (digenean) (^{PRITCHARD, 1964}; ^{THEUNISSEN et al., 2014}). For a further review, see ^{Tinsley (1996)}. In addition, in this study, the prevalence of Contracaecum sp. was low, and the parasites were present in only one geographical location, La Patagua. This lack of helminths in most clawed frogs means that there is a lack of enemies (parasitic helminths), enhancing the survival, which may favor the process of invasion by X. laevis. More studies are necessary to confirm this hypothesis. The parasitic richness found in our study is also lower than what was previously found in other territories invaded by this frog, including California, where at least seven species of helminths have been reported in the same anatomical parts of the frogs investigated in this study. These helminths included the following: C. namaquensis, G. gallieni, P. xenopodis, Clinostomum sp., Contracaecum sp., Eustrongylides sp., and Acanthocephalus sp. (^{LAFFERTY & PAGE, 1997}; ^{KUPERMAN et al., 2004}). Further studies are necessary to test the possible reasons underlying this difference.

In addition to X. laevis from California (with prevalences (P) similar to our study, varying from 0 to 4%), the genus Contracaecum was reported in Argentina in another introduced amphibian species: Lithobates catesbeianus Shaw, 1802 (P = 43.7%) (^{GONZÁLEZ et al., 2014}); this genus was also reported in the native Bufo marinus Linnaeus, 1758 (^{ESPINOZA-JIMÉNEZ et al., 2007}) from Mexico (P = 4.2%); in both cases showing higher prevalences than in our study. In Chile, this genus was found in other groups of vertebrates, including birds (P > 17.4%) and fish (P = 13.3%) (^{TORRES et al., 1982}, ¹⁹⁸³, ¹⁹⁹¹, ²⁰⁰⁵; ^{TORRES & CUBILLOS, 1987}; ^{GONZÁLEZ-ACUÑA et al., 2008}). However, as far as we know, this is the first record of an anisakid species in amphibians from Chile. Other taxa of nematodes found in amphibians native to Chile include Rhabdias sp. (P = 36%) (Rhabditida, Rhabdiasidae); Parapharyngodon sceleratus Travassos, 1923 (P = 20-100%); Spauligodon maytacapaci Vicente, Ibáñez, 1968 (P = 40-92%) (Oxyurida, Pharyngodonidae); Aplectana artigasi Puga, Torres, 1997 (P = 23-100%); Aplectana chilensis Lent, Freitas, 1948 (P = 64%); Cosmocerca chilensis Lent, Freitas, 1948 (unreported prevalence) (Ascaridida, Cosmocercidae); Skrjabinelazia sp. (P = 3.3%) (Ascaridida, Seuratidae); Physaloptera cf. lutzi Cristófaro, Guimaraes, Rodríguez, 1976 (P = 3-19%); Physaloptera sp. (P = 15%) (Spirurida, Physalopteridae); Oswaldocruzia neghmei Puga, 1981 (P = 7-100%); and Oswaldocruzia sp. (P = 7%) (Sotrongylida, Molineidae) (^{GARÍN & GONZÁLEZ-ACUÑA, 2008}).

Given that Contracaecum display an indirect cycle in which the frogs are intermediate hosts, and where laboratory-bred clawed frogs serve as the source of the invasive population (^{LOBOS et al., 2014}), the source of infection for those frogs with Contracaecum sp. was more likely the native fauna of Chile, particularly native birds (definitive hosts) than the native range of X. laevis (i. e., African infection that persisted through the laboratory breeding in Chile).

Birds are frequently mentioned as hosts of Contracaecum sp. One cormorant species, Phalacrocorax brasilianus (syn olivaceus) Gmelin, 1789 (^{TORRES, 1983}; ^{TORRES et al., 1991}, ²⁰⁰⁵), and three gull species – Larus dominicanus Lichtenstein, 1823, Larus (syn. Chroicocephalus) maculipennis Lichtenstein, 1823, and Larus serranus Tschudi, 1844 (^{TORRES et al., 1983}) – were found to be parasitized with Contracaecum in Chile, with Contracaecum rudophii Hartwich, 1964 as the one parasite species found in all cases. In addition, birds, including L. dominicanus, are also the main predators of X. laevis; there are two other bird species reported as predators of X. laevis: Athene (syn. Speotyto) cunicularia Molina, 1782 and Nycticorax nycticorax Linnaeus, 1758 (^{LOBOS & JAKSIC, 2005}). While this suggests that X. laevis facilitates the infection of this gull by serving as an intermediate host for Contracaecum sp. and as prey for L. dominicanus, the low prevalence and abundance of this parasite in X. laevis may mean that this frog is of low importance in the infection of gulls by Contracaecum nematodes.