Ascocotyle trentinii, Gustinelli & Caffara & Scaravelli & Fioravanti & Toma & Scholz, 2022

Gustinelli, Andrea, Caffara, Monica, Scaravelli, Dino, Fioravanti, Maria Letizia, Toma & Scholz, s, 2022, The first new species of European Ascocotyle Looss, 1899 (Digenea: Heterophyidae) described in more than half a century, International Journal for Parasitology: Parasites and Wildlife 17, pp. 327-334 : 329-332

3.1. Ascocotyle trentinii n. sp. Figs. 1 and 2

Type-host: Anas platyrhynchos domesticus (experimental host); natural host of metacercariae: Aphanius fasciatus .

Site of infection: Small intestine.

Type-locality: Cervia salt marshes, Emilia-Romagna Region, Italy (44 ◦ 15 ′ 15.17 ′′ N; 12 ◦ 20 ′ 02.57 ′′ E), the site where metacercariae, used to experimentally infect the type host, were collected from Aphanius fasciatus .

Other localities: Santa Gilla lagoon, Sardinia, Italy ( Culurgioni et al., 2014 – as Ascocotyle sp. 3 ).

Type-material: Holotype and 13 paratypes are deposited at the Helminthological Collection of the Institute of Parasitology, Biology Centre of the Czech Academy of Sciences , Cesk ˇ´e Budˇejovice, Czech Republic ( IPCAS D-846 ) and the Natural History Museum , Geneva, Switzerland (MHNG-PLAT-0138866 – paratypes; MHNG-PLAT-0138867 – vouchers).

ZooBank number: urn:lsid:zoobank.org:act:A0A8E5E9-86FC-45A1-9DFA-065AB568E533 .

Second intermediate host: Mediterranean banded killifish, Aphanius fasciatus ( Cyprinodontiformes: Aphaniidae ); infected 29 of 35 killifish, i. e., prevalence of 83%, and intensity of infection reaching up to 100 metacercariae/fish; even most heavily infected fish host were sexually mature.

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.

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.

3.2. Description of adults ( Figs. 1 , 2D–I )

Description (based on 29 mounted specimens and 2 specimens studied using SEM): Body pyriform, 440–635 (566 ± 57; n = 20) long and 290–470 (365 ± 43; n = 20) wide, with maximum width at midhindbody or slightly more anterior ( Fig. 2D, E ). Body densely covered with tegumental spines except for region posterior to circumoral spines ( Fig. 2F ), around opening of ventrogenital sac ( Fig. 2G ) and posterior extremity ( Figs. 1A and 2D, E ). Anterior (preacetabular) part of body covered with flat, long and narrow spines with two short, tooth-like projections, 3.5–5.3 (4.2 ± 0.4; n = 20) long and 1.6–2.5 wide (2.0 ± 0.2; n = 20) ( Fig. 2H ). Posterior (postacetabular) part of body with simple spines with sharp terminal point (tip), 4.4–5.9 (5.1 ± 0.4; n = 20) long and 1.3–1.5 (1.4 ± 0.1; n = 20) wide ( Fig. 2I ).

Oral sucker subterminal, 58–85 (72 ± 8; n = 27) long and 64–100 (85 ± 9; n = 27) wide, with conical, short posterior appendage, 37–45 (41 ± 6; n = 2) long, reaching anterior margin of pharynx posteriorly. Oral sucker surrounded by single row of 29–33 (30 ± 1) massive, spearhead-like circumoral spines ( Figs. 1A–E , 2F ); spines 12–18 (14.5 ± 1; n = 50) long and 3–6 (4.5 ± 1; n = 50) wide (in frontal view). Prepharynx short 6–26 (15 ± 7; n = 6) long, but distinct; pharynx widely elongate, strongly muscular, 52–85 (72 ± 8; n = 29) long and 42–80 (65 ± 11; n = 29) wide ( Figs. 1A and 2D ). Oesophagus short, 29–67 (47 ± 15; n = 6) long; intestinal caeca 114–170 (143 ± 18; n = 7) long and 18–42 (31 ± 8; n = 7) wide, short, reaching posteriorly only level of posterior margin of ventral sucker. Ventral sucker well-developed, deeply embedded, spherical to subspherical, wider than long, 54–79 (67 ± 7; n = 26) long and 60–85 (77 ± 7; n = 26) wide, nearly equatorial (slightly dextral), situated at 43–49% (45% ± 3%; n = 5) of body length from anterior extremity. Opening of ventral sucker transversely oval to

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widely oval; ratio of width of suckers (OS vs VS) 1: 0.94–1.33 (1.11 ± 0.12).

Testes double, unlobed, widely oval to subspherical, symmetrical, situated close to posterior margin of body, lateral to excretory bladder, 43–107 (75 ± 20; n = 7) long and 37–74 (57 ± 14; n = 7) wide (dextral testis) and 58–78 (75 ± 13; n = 7) long and 48–78 (60 ± 9; n = 7) wide (sinistral testis) ( Fig. 1A ). Seminal vesicle voluminous 59–92 (75 ± 12; n = 8) long and 42–72 (58 ± 9; n = 8) wide, difficult to observe because of numerous overlaying eggs, posterior to ventral sucker. Ventrogenital sac formed, containing obliquely situated pad-like gonotyl 12–22 (17 ± 4; n = 7) long and 43–53 (48 ± 4; n = 7) wide, composed of about 7–9, usually 8 refractile bodies. Opening of ventrogenital sac narrow, transverse, slit-like, slightly sinistral to longitudinal body axis.

Ovary spherical 34–55 (41 ± 6; n = 9) long and 32–69 (49 ± 15; n = 9) wide, median, at level of testes. Seminal receptacle 43–74 (59 ± 11; n = 12) long and 39–104 (79 ± 11; n = 12) wide, voluminous, transversely oval, median, posterior to ovary. Laurer’ s canal not observed. Vitellarium formed by irregularly-shaped follicles grouped in two posterolateral groups between posterior margin of ventral sucker to posterior half of testes. Common vitelline ducts slightly sinuous, ventrally passing over anterior part of seminal receptacle, medially joined and widened to form vitelline reservoir situated posterolateral to ovary.

Uterus tubular, forming numerous loops between anterior margin of ventral sucker and posterior margin of testes, reaching slightly posterior to testes. Metraterm opening sinistrally into ventrogenital sinus. Eggs thick-walled, operculate, 17–21 (19 ± 1; n = 20) long and 11–12 (11.5 ± 0.5; n = 20) wide. Excretory vesicle V-shaped, with short and wide stem.

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 ).

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3.3. Description of metacercariae ( Fig. 2 A–C )

Description (based on 20 live metacercariae from the visceral serosa of Aphanius fasciatus ). Encysted metacercariae oval, 173–250 long (213 ± 26) and 140–160 (154 ± 8). Cyst wall double, with hyaline, elastic outer wall 3.5 μm thick and transparent membraneous inner layer. Excysted metacercariae pyriform, covered with tiny tegumental spines. 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,

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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 Hern´andez-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 (Hern´andez-Orts et al., 2019). Ascocotyle (P.) pindoramensis shows digitiform tegumental spines and is devoid of circumoral spines ( Sim˜oes 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).

3.5. Remarks

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.

Figures

Fig. 1. Ascocotyle (Phagicola) trentinii n. sp. from experimentally infected duckling (Anas platyrhynchos); holotype (IPCAS D-846). A – total view, ventrally; B–E – oral sucker with one row of circumoral spines and posterior prolongation; F – ventral sucker with ventrogenital sac, ventrally; note gonotyl with 8 refractile pockets.

Fig. 2. Ascocotyle (Phagicola) trentinii n. sp. metacercariae from Aphanius fasciatus: (A) encysted metacercaria; (B) circumoral spines of metacercaria; (C) excysted metacercaria with emptied cyst. Ascocotyle (Phagicola) trentinii n. sp. adults from experimentally infected duckling (Anas platyrhynchos): (D) total view, ventrally; light microscopy and (E) Scanning Electron Microscopy (SEM); (F) SEM details of oral sucker with circumoral spines and (G) of ventral sucker; SEM details of (H) bifid tegumental spines and (I) single pointed spines at the bases of both suckers.