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Adults of Clinostomum spp. are digenetic trematodes found in fish-eating birds, reptiles and occasionally mammals, including humans. Freshwater snails serve as first intermediate hosts and many fish species and amphibians as second intermediate hosts. To date, amphibian hosts of Clinostomum metacercariae include members of urodele and anuran families in North America, but no data are available on infections of European amphibians, including newts. In this study, we characterize infections of Clinostomum complanatum metacercariae in four smooth (Lissotriton vulgaris) and 18 Italian crested newts (Triturus carnifex) from an artificial pond located in a protected area in Tuscany, Italy. Parasites were surgically removed from the infected newts and identified both morphologically and using sequences of a mitochondrial gene, cytochrome c oxidase I, and the ribosomal markers, internal transcribed spacers. This is the first record of C. complanatum in European newts and, more generally, in amphibians in Europe.

AIM. Clinostomum is a Digenetic Trematode living at the adult stage in the oral cavity, pharynx or oesophagus of fish-eating birds, reptiles and occasionally mammals, including man and recognize as second intermediate host many fish species. Metacercariae of the genus Clinostomum have been described from a variety of American anurans and urodeles. We report the first record of Clinostomum complanatum in European Amphibians, with the finding of metacercariae in Italian crested newt (Triturus carnifex) and smooth newt (Lissotriton vulgaris). MATERIALS AND METHODS. From October 2010 to March 2012, eighteen Italian crested newts (4 metamorphic males, 4 metamorphic females, 2 paedomorphic females and 8 larvae) and four smooth newts (1 paedomorphic male, 3 paedomorphic females) with evident yellow grub cysts were observed in an artificial pond in the protected area A.N.P.I.L. “Podere la Querciola” (Sesto Fiorentino, Tuscany, 43.824703N, 11.173299E). The infected newts were anesthetized with tricaine methanesulphonate (MS222). The cysts were incised with a scalpel and the metacercariae removed and fixed in 90% ethanol. The newts were rinsed with freshwater until recovery and then a small amount of ciprofloxacin ophthalmic ointment was placed on the wounds. The metacercariae were subjected to morphological and molecular identification amplifying the ITS rDNA region and the COI mtDNA genes (Caffara M et al., 2011, J Parasitol, 97: 884-891) RESULTS. A total of 35 cysts were collected, 30 from the Italian crested newts and 5 from smooth newts. In L. vulgaris all the cyst (5) were in the head, while in T. carnifex the cysts were recovered from different part of the body as head (14), snout (6), throat (4), mouth (5) and tail (1). The morphological observations carried out on 11 metacercariae allowed to refer them to Clinostomum complanatum (Digenea: Clinostomidae). The analysis of the sequences of both genes ITS rDNA and COI mtDNA showed 99.9-100% identity with this species. CONCLUSIONS. The taxonomy of the genus Clinostomum is still confused and should be addressed, in order to avoid misidentification, by combining the morphological and the molecular approach. The presence of Clinostomum spp. in Amphibia has been recorded in North America and Mexico since many years (McAllister CT et al, 1990, J Helminth Soc Wash, 57: 69-71; Miller DL et al, 2004, J Helmith, 78: 373-376; McAllister CT et al, 2007, Texas J Sci, 59: 321-326; McAllister CT et al, 2010, Comp Parasitol, 77: 25-30; Cabrera-Guzman E et al, 2010, J Parasitol, 96: 736-739) where the species described so far are C. marginatum, C. attenuatum and C. complanatum, even if most of the works do not include detailed morphological and/or molecular descriptions. Concerning our specimens the morphological characters clearly allowed to refer the parasites to the species C. complanatum, data supported by the molecular analysis. This is the first record of C. complanatum in European newts and, more in general, in amphibians from Europe. Further analyses are required in order to establish the impact of this parasite on wild populations of European newts.