The first new species of European Ascocotyle Looss, 1899 (Digenea: Heterophyidae) described in more than half a century

Ascocotyle (Phagicola) trentinii n. sp. is described based on adults from experimentally infected ducklings (Anas platyrhynchos domesticus) fed with metacercariae from the visceral serosa of the Mediterranean banded killifish, Aphanius fasciatus (Cyprinodontiformes: Aphaniidae), from coastal lagoons in northeastern Italy (Emilia-Romagna Region). The new species is placed into the subgenus Phagicola because of the presence of a single row of circumoral spines, vitelline follicles being confined between the ventral sucker and testes, and uterine loops not reaching anterior to the ventral sucker. Ascocotyle (P.) trentinii n. sp. differs from other members of the subgenus Phagicola, as well as other species of Ascocotyle, by the number (27–33) of circumoral spines which are 13.5–17 μm long and 3.5–5 μm wide, and by the morphology of a gonotyl which is composed of about 8 large refractile pockets. The occurrence of metacercariae in A. fasciatus indicates that the life cycle of the new species is completed in brackish water lagoons. It is the fourth species of Ascocotyle described in Europe and may be endemic to the Mediterranean region because its second (fish) intermediate host is endemic to this region.

The genus Ascocotyle includes species characterized by an oral sucker armed with circumoral spines arranged usually in one or two rows (spines are absent in a very few taxa), and bearing a conical muscular prolongation (posterior appendage) of the oral sucker (Pearson, 2008). Spination patterns, i.e., the number of rows of circumoral spines, their number and size, together with some characteristics of internal organs, such as the posterior extent and shape of the intestinal caeca, morphology of the gonotyl, position and extent of vitelline follicles, etc. serve as a basis for species identification (Burton, 1958;Scholz et al., 1997a, b). Several subgenera of Ascocotyle have been proposed, but their validity, circumscription and phylogenetic relationships remain unclear, partly because molecular data are available for only a few species (Hernández-Orts et al., 2019;Santos and Borges, 2020).

Specimen collection
visceral organs (peritoneum). Previous analyses on different batches of A. fasciatus sampled from the same area showed high prevalence and high intensity (always more than 100 metacercariae/fish) belonging to the same species on the basis of the number of circumoral spines (see Table 1). Metacercariae were rinsed in saline, observed live under dissecting microscope and then fixed in 70% ethanol for further studies.
To obtain adults, four 3 days old ducklings (Anas platyrhynchos domesticus var. American pekin) were fed with A. fasciatus infected with metacercariae of the new species. Fish infection was verified at dissection microscope before feeding the ducklings. Ten fish were chopped together and fed to four ducklings. The fish were previously checked under dissection microscope to verify the presence of metacercariae, always over 100 in number. Ducklings were kept in a ground stand in a facility of the State Veterinary Institute of Lombardia and Emilia-Romagna in Forlì. They were subjected daily to coprological examination until the finding of digenean eggs, i.e., 5 days p.i. Ducklings were then euthanised by cervical dislocation and their intestine was examined to collect the parasites. The adults (several dozens) were then isolated from the gut of experimentally infected ducklings 5 days post-infection, washed in sterile saline and then fixed in 70% ethanol.

Morphological data
Adults were clarified with Amman lactophenol or stained with Mayer's carmine, dehydrated, clarified with eugenol, and mounted in Canada balsam as permanent preparations. Specimens were observed under light microscope Olympus BX 51. Line drawings were made using a drawing tube and measurements were taken using imaging software NIS-Elements (Nikon, Campi Bisenzio, FI, Italy) and QuickPhoto (Olympus, Tokyo, Japan). Measurements are expressed in micrometres (μm) and are presented as the range, with the mean followed by standard deviation (SD); n = number of measurements, usually also corresponding to the number of specimens measured, with few exceptions, such as size of circumoral spines. For scanning electron microscopy (SEM), adults were dehydrated through a graded alcohol series, dried by hexamethyldisilazane, coated with gold and then examined using a Phenom XL G2 Desktop SEM (Thermo Fisher Scientific) operating at 5 kV.

Molecular data
Total DNA, from both a metacercaria and an adult, were extracted using PureLink Genomic DNA Kit (Life Technologies, Carlsbad, California) following the manufacturer's protocol. The 28S rRNA gene was amplified with the primers U178 (forward 5 ′ -GCACCCGCTGAAYT-TAAG-3 ′ ) and L1642 (reverse 5 ′ -CCAGCGCCATCCATTTTCA-3 ′ ) of Lockyer et al. (2003). The thermal cycler program (Tpersonal, Biometra) was a denaturation step at 94 • C for 2 min, 40 cycles of 30 s at 94 • C, 30 s at 52 • C and 2 min at 72 • C and followed by an extended elongation step at 72 • C for 10 min. Amplified products were resolved on 1% agarose gel stained with SYBR Safe DNA Gel Stain in 0.5X TBE (Molecular Probes, Life Technologies). For sequencing, bands were excised and purified by NucleoSpin Gel and PCR Cleanup (Mackerey-Nagel, Düren, Germany) and sequenced with an ABI 3730 DNA analyzer at StarSEQ GmbH (Mainz, Germany).
Consensus sequences were assembled with Vector NTI AdvanceTM 11 software (Thermo Fisher Scientific, Carlsbad, California). The sequences were compared with previously published data by BLAST tools (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple sequence alignments were constructed by ClustalW in BioEdit 7.2.5 (Hall, 1999). Phylogenetic tree and models of nucleotide evolution (Bayesian Information Criterion) were calculated using MEGA version X (Kumar et al., 2018). To infer the evolutionary history Maximum-Likelihood (ML) method based on GTR + G + I model with 1000 replicates were used. The newly generated sequences were aligned with the sequences reported by Hernández-Orts et al. (2019) to build the ML tree. The new sequences are published in GenBank under the following accession numbers MZ654879 (metacercaria) and MZ654880 (adult). Etymology: the species is named after Professor Massimo Trentini, our dear colleague and friend who participated in the initial study of this parasite and prematurely passed away.

Ascocotyle trentinii n. sp. Figs. 1 and 2
Representative DNA sequences: newly generated sequences of 28S rDNA (length of 1,622 bp) of an adult and a metacercaria were identical, which confirms they represent different ontogenetic stages of the same species.
SEM observation of adults revealed the presence of tegumental spines with different shape: bifid in almost whole-body surface (Fig. 2H), with some spines characterised by two main tips plus a small and less developed median tip randomly present. The last type, slightly surrounding the base of both suckers, shows a single sharp apex (Fig. 2I).
Oral sucker bearing 27-33 (30 ± 2; n = 11) circumoral spines and muscular posterior appendage (posterior prolongation). Intestinal caeca reaching posteriorly to level of ventral sucker; they do not contain spherical discs. Region between pharynx and intestinal bifurcation filled with large concentration of gland cells with darker content. Primordia of genital organs (ovary and symmetrical testes) well-developed; primordium of gonotyl visible in some specimens. Excretory vesicle Y-shaped, with short and wide stem, filled with dark excretory granules.

Differential diagnosis
The new species belongs to Ascocotyle because it possesses a single row of circumoral spines, vitelline follicles confined to the region between the ventral sucker and testes, and uterine loops not reaching anterior to the ventral sucker. It is placed to the subgenus Phagicola Faust, 1920 based on the above-mentioned characteristics, but Pearson (2008) did not recognize individual subgenera of Ascocotyle (and molecular data support this conclusion; see also Fig. 3 in the present paper).
Ascocotyle (Phagicola) trentinii n. sp. differs from the other members of this subgenus as well as other species of Ascocotyle by the number (29-33) of circumoral spines 12-18 μm long and 3-6 μm wide (see Table 1 for the number of spines in all species of Ascocotyle), and by the morphology of the gonotyl, which is composed of about 8 large refractile pockets.
The tegumental spination pattern of the new species observed by SEM differs from that described in other congeneric species that were studied using SEM. The most recently described species, A. (P.) cameliae Hernández-Orts, Georgieva, Landete et Scholz, 2019 from the Magellanic penguin, Spheniscus magellanicus (Forster), in Argentina, possesses pectinate tegumental spines, but the number of their terminal points decreases continuously from three on the anterior third of the body to a single point in the posterior part of body surface (Hernández-Orts et al., 2019). Ascocotyle (P.) pindoramensis shows digitiform tegumental spines and is devoid of circumoral spines (Simões et al., 2006). Despite these morphological differences, A. (P.) pindoramensis is the sister taxa of A. (P.) trentinii n. sp. as showed in the ML tree of the 28S rDNA. Both these species are reported in cyprinodontiform fish, Poecilia vivipara Bloch et Schneider and Aphanius fasciatus, respectively, and live in similar habitats (brackish waters), but in different continents (South America versus Europe). Culurgioni et al. (2014) reported unidentified metacercariae of Ascocotyle as Ascocotyle (Phagicola) sp. 3 from A. fasciatus sampled in Santa Gilla lagoon in Sardinia, Italy. Because of a similar number of circumoral spines (>30) and their arrangement in a single row, it is likely that the specimens were conspecific with A. (P.) trentinii n. sp.

Molecular data
The ML tree showed the new species forming a well-supported cluster within the Ascocotyle clade (including species placed in the subgenus Phagicola), with A. (P.) pindoramensis (0.036 distance, no circumoral spines) as its sister taxon and A. (P.) longa (0.080 distance, a single row of circumoral spines) basal to both species (Fig. 3).

Discussion
Ascocotyle (P.) trentinii n. sp. is the first new species of Ascocotyle described from Europe after more than a half century. This may be caused by little attention paid to search for these tiny trematodes, which mature in fish-eating birds; these host are usually protected in most European countries and thus difficult to examine for parasites. Another reason may be relatively rare occurrence of heterophyids in Europe and their low species diversity in temperate zones, especially compared with that in subtropical and tropical regions (Scholz et al., 1997a, b;2001).
Molecular phylogenetic analysis placed the new species within a well-supported clade composed from all but two species of Ascocotyle (Fig. 3). The only species that do not belong to Ascocotyle, thus making this genus non-monophyletic, are Phocitrema fusiforme Goto et Ozaki, 1930 from seals, sea otter and Arctic fox in northern Pacific, and Pygidiopsis macrostoma Travassos, 1928, a poorly known species originally described from a single specimen from Rattus norvegicus (Erxleben) in Brazil. In fact, both species are morphologically similar to those of Ascocotyle (including distribution of internal organs and structure of the gonotyl with pocketssee Pearson, 2008), the only difference being in the absence, rather than presence, of a posterior, tapering appendage of the oral sucker. It is obvious that validity of Phocitrema Goto et Ozaki, 1930 andPygidiopsis Looss, 1907 should be critically revised, based on a broader dataset of sequenced taxa of all genera. Hernández-Orts et al. (2019) also found P. fusiforme to be closely related to species of Ascocotyle (see Fig. 6 in that paper).
The new species completes its life cycle in brackish waters as the fish second intermediate host occurs mainly in coastal lagoons and tolerates high salinity (Leonardos et al., 1996). The recent discovery of A. trentinii n. sp. is somewhat surprising considering extraordinarily high prevalence and intensity (83%, intensity of infection up to 100 metacercariae) of its infection in the Mediterranean banded killifish in Italy. This cyprinodontiform fish is a short-lived, non-commercial species widely distributed in the Mediterranean basin (Bertoli et al., 2019). It lives in saltworks, brackish habitats and lagoons with high salinity levels (up to 65‰) and tolerating a wide range of temperature variation, between 4 and 40 • C (Leonardos and Sinis, 1998;Leonardos, 2008). Due to the high relevance of brackish environments as biodiversity hot spots and buffer areas against extreme and adverse weather events, A. fasciatus is considered as an 'umbrella species' for these environments (Simberloff, 1998;Roberge and Angelstam, 2004;Valdesalici et al., 2015;Bertoli et al., 2019) and, is listed among the "Least Concern" species in the IUCN Red List (Rondinini et al., 2013) and is reported in the Annex II of the European Habitat Directive (92/43/CEE). This fish is also tested for biological control of mosquito larvae in a brackish area in northern Italy (Salines of Cervia) and the results on controlled reproduction seem to be very promising (Mordenti et al., 2012).
Since A. fasciatus is widely distributed in the Mediterranean region, one would expect that metacercariae of the new trematode would be found earlier during surveys on fish parasites in coastal lagoons. The absence of focus of fish parasitologists on this tiny fish may be one of most plausible explications why A. trentinii n. sp. was not described earlier. Another possible reason could be restricted distribution of the new species to small areas in Italy. However, an unpublished observation of metacercariae of A. trentinii n. sp. in Aphanius fasciatus from Sardinia does not support this assumption about endemicity of the new trematode in Italy (J. Culurgioni, personal communication).
The natural definitive host of A. trentinii n. sp. is still unknown. The Cervia salt marshes, where the Mediterranean banded killifish were sampled, are characterised by a very rich fauna of fish-eating birds, such as several species of the Ardeidae, Laridae and Anatidae, which may serve as possible definitive hosts of heterophyid trematodes, including the new species. Especially birds of the families Ardeidae are common definitive hosts of trematodes linked to aquatic environments that include fish.
However, most lagoons are protected areas and it is difficult to examine fish-eating birds to obtain adult worms in natural definitive hosts, to better catalogue the biodiversity and to understand better ecological connections in this peculiar ecosystem. The Cervia salt pans host more than 30 sedentary species of birds (more than 100 species in total), among which herons (Ardea cinerea, Ardea alba, Egretta garzetta), larids (Chroicocephalus ridibundus) and anatids (Anas platyrhynchos, Spatula clypeata, Mareca penelope, Tadorna tadorna). Since several years there is also a stable population of flamingos (Phoenicopterus roseus). Considering the eating behaviour of the above mentioned species, the herons seem the most promising potential definitive hosts of A. trentinii. However, they live in a protected area, which makes their parasitological examination hardly possible.
Some non-invasive methods such as eDNA screening might help to detect foci of the parasite transmission by detecting its DNA from the eggs, miracidia or cercariae in water samples. Monitoring of the local malacofauna might also help to identify the natural first intermediate host of the new species.

Declaration of competing interest
There is no conflict of interest in the submitted manuscript entitled "A new species of Ascocotyle Looss, 1899 (Digenea: Heterophyidae): the first species newly described from Europe after more than half a century."