Natural History Sciences. Atti Soc. it. Sci. nat. Museo civ. Stor. nat. Milano, 9 (1): 11-16, 2022 DOI: 10.4081/nhs.2022.544 New distributional data on Haemogregarina stepanowi (Apicomplexa) and Placobdella costata (Hirudinea) parasitising the Sicilian pond turtle Emys trinacris (Testudines) Rita Scardino, Marco Arculeo, Vincenzo Arizza, Giuseppe Bazan, Mario Lo Valvo, Federico Marrone, Luca Vecchioni* Dipartimento di Scienze e Tecnologie biologiche, chimiche e farmaceutiche, Università degli Studi di Palermo, Via Archirafi 18, 90123 Palermo (PA), Italia. E-mail: Rita Scardino: rituccia1989@gmail.com Marco Arculeo: marco.arculeo@unipa.it Vincenzo Arizza: vincenzo.arizza @unipa.it Giuseppe Bazan: giuseppe.bazan @unipa.it Mario Lo Valvo: mario.lovalvo @unipa.it Federico Marrone: fedrico.marrone @unipa.it * Corresponding Author: luca.vecchioni @unipa.it © 2021 Rita Scardino, Marco Arculeo, Vincenzo Arizza, Giuseppe Bazan, Mario Lo Valvo, Federico Marrone, Luca Vecchioni Received for publication: 28 May 2021 Accepted for publication: 2 November 2021 Online publication: 18 March 2022 Abstract - The host-parasite system “Emys trinacris - Placob- della costata - Haemogregarina stepanowi” is known for Sicily, but scarce information is available to date about the distribution of the two parasites P. costata and H. stepanowi on the island. Therefore, an extensive sampling effort through visual census and collection and analysis of blood smears of the endemic Sicilian pond turtle E. trinac- ris was carried out in 46 water bodies scattered throughout mainland Sicily. Our findings revealed that the distribution of both parasites is lim- ited to the Nebrodi area, where the infection of H. stepanowi has shown a high incidence on the local turtle populations. Our data suggest no correlation between the current distribution of the two parasite species and environmental features. The current distribution of H. stepanowi and P. costata seems not to be relictual, but rather the outcome of a recent colonisation process. Considering the possible negative impact of both H. stepanowi and P. costata on their turtle host, their long-term effect on E. trinacris should be investigated. Key words: blood parasites, conservation, Emydidae, Glossipho- niidae, Haemogregarinidae. Riassunto - Nuovi dati sulla distribuzione di Haemogregarina stepanowi (Apicomplexa) e Placobdella costata (Hirudinea) parassiti della testuggine palustre siciliana Emys trinacris (Testudines). In Sicilia è noto il sistema ospite-parassita “Emys trinacris - Pla- cobdella costata - Haemogregarina stepanowi”, ma attualmente sono disponibili scarse informazioni sulla distribuzione in Sicilia dei paras- siti P. costata e H. stepanowi. Pertanto, è stato condotto un ampio sforzo di campionamento attraverso il censimento visivo e la raccolta e l’analisi di strisci di sangue della testuggine palustre siciliana E. trinacris in 46 corpi idrici distribuiti in tutta la Sicilia. I nostri risultati hanno rivelato che la distribuzione di entrambi i parassiti è limitata alla zona dei Nebrodi, dove l’infezione da H. ste- panowi ha mostrato un’elevata incidenza sulle popolazioni di testug- gini locali. I dati raccolti non hanno suggerito alcuna correlazione fra l’attuale distribuzione delle due specie di parassiti e le caratteristiche ambientali. L’attuale distribuzione di H. stepanowi e di P. costata non sembra essere relittuale, ma piuttosto il risultato di un recente processo di colonizzazione. Considerando il possibile impatto negativo sia di H. stepanowi che di P. costata sul loro ospite, l’effetto a lungo termine su E. trinacris dovrebbe esserne studiato. Parole chiave: conservazione, Emydidae, Glossiphoniidae, Hae- mogregarinidae, parassiti del sangue. INTRODUCTION Sicily hosts the endemic pond turtle Emys trinacris Fritz, Fattizzo, Guicking, Tripepi, Pennisi, Lenk, Joger & Wink 2005, which is the only autochthonous freshwa- ter turtle occurring on the island. This turtle species has a scattered distribution (Vamberger et al., 2015; Mar- rone et al., 2016a, 2016b; Vecchioni et al., 2020b), and has suffered severe habitat destruction and fragmenta- tion (Fritz et al., 2005), which together with the species’ limited dispersal ability (Iannella et al., 2018) put it at risk. Furthermore, E. trinacris suffers the pressure of in- vasive alien species (Marrone & Naselli-Flores, 2015; Naselli-Flores & Marrone, 2019; Liuzzo et al., 2020), occasional hybridisation with the congeneric E. orbicu- laris (Linnaeus, 1758) (Vamberger et al., 2015; Scardino et al., 2020), possible parasite spill-over (Arizza et al., 2016), and potential threats due to angling (Vecchioni et al., 2020a). The only Palaearctic representative of the glossiphoni- id leech genus Placobdella Blanchard 1893 is P. costata (Müller 1846) which has a strict, but not exclusive, tro- phic relationship with aquatic turtles of the genus Emys Duméril 1806 (Bielecki et al., 2012; Romero et al., 2014; Kvist et al., 2022). These two taxa constitute a host-pa- rasite system in which a further species is involved i.e. Haemogregarina stepanowi (Danilewsky 1885) a wide- spread apicomplexan haemoparasite of aquatic turtles (Dvořáková et al., 2014; Arizza et al., 2016). This last species has a complex life cycle that requires an inter- mediate host (e.g. E. trinacris) and a definitive host (i.e. 12 P. costata), in which the haemogregarine’s gametogene- sis occurs in both, with the subsequent formation of the zygote (Siddall & Desser, 1990; Mihalca et al., 2008; Dvořáková et al., 2014). Marrone et al. (2016a) and Vecchioni et al. (2021) in-(2021) in- vestigated the system “E. trinacris + P. costata” in an at- tempt to assess the possible existence of a cophylogenetic pattern between them. However, no evidence supporting this hypothesis was retrieved. We here describe the distribution of the “E. trinacris - P. costata - H. stepanowi” system in Sicily, in light of the possible impact of the two parasites on the populations of the endemic Sicilian pond turtle. MATERIALS AND METHODS Samples were collected in 46 sites located on main- land Sicily from 2014 to 2017 (no record of E. trinacris is available for the small circum-Sicilian islands) as shown in Fig. 1 and Tab. 1, including both low-altitude water bo- dies and the highest localities where E. trinacris is known to occur (Marrone et al., 2016b; Ottonello et al., 2021), including permanent lakes and ponds, brackish lakes and watercourses. Pond turtles were caught by hand or with baited hoop traps as described in Marrone et al. (2016a). After the identification of the species in situ, a blood sam- ple was obtained from the dorsal coccygeal vessel of each turtle. Each pond turtle was also carefully inspected for the presence of leeches; when these were present, they were collected and fixed in situ in 90% ethanol and identi- fied according to Minelli (1977). Afterwards, the animals were immediately released in nature. Blood smears were prepared in situ according to Arizza et al. (2016). Leech samples and blood smears are stored at the Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF) of the University of Palermo under the curatorial responsibility of one of the authors (LV). The distribution of the sampled population of E. tri- nacris in Sicily, the occurrence of its parasites, and the presence of parasitized individuals were mapped in ESRI ArcGIS 10.2. The distribution of the captured turtles and parasitized individuals has been analysed with respect to bioclimatic data (Bazan et al., 2015), lithological features (Basilone, 2018) and habitats recorded in the “Carta della Natura” (ISPRA, 2012). Because the Shapiro-Wilk test (Shapiro & Wilk, 1965) showed that data were not nor- mally distributed, non-parametric Spearman correlation test was used in order to explore the possible link between turtle distribution/presence and environmental features. Fig. 1 - Sites in Sicily where the occurrence of E. trinacris, P. costata and H. stepanowi has been observed. / Siti in Sicilia in cui è stata osservata la presenza di E. trinacris, P. costata e H. stepanowi. RITA SCARDINO, MARCO ARCULEO, VINCENzO ARIzzA, GIUSEPPE BAzAN, MARIO LO VALVO, FEDERICO MARRONE, LUCA VECCHIONI 13 Tab. 1 - List of the sampled sites, captured individuals of E. trinacris, collected P. costata individuals and synopsis of the studied blood samples of E. trinacris in which H. stepanowi occurred. Geographical coordinates are expressed as decimal degrees (MapDatum: WGS84). The type of water body is shown in brackets: A) Artificial; N) Natural. / Elenco dei siti campionati, individui catturati di E. trinacris, individui raccolti di P. costata e sinossi dei campioni di sangue studiati di E. trinacris in cui H. stepanowi era presente. Le coordinate geografiche sono espresse in gradi decimali (MapDatum: WGS84). Tra parentesi è indicato il tipo di corpo idrico: A) Artificiale; N) naturale. * Pond turtles kept in captivity. / Testuggini palustri tenute in cattività. Province Municipality Site name Latitude N Longitude E Elevation (m a.s.l.) E. trinacris (n. ind) Naturalness Evaluation Index (“NEI”) P. costata (p/a) H. stepanowi (p/a) N. parasitized individuals (by H. stepanowi) Agrigento Casteltermini Fiume Platani (N) 37.493897 13.687256 138 7 0.38 - - - Agrigento Montallegro Lago Gorgo (A) 37.408766 13.324239 65 26 0.72 - - - Agrigento Siculiana RNO “Torre Salsa” (N) 37.374883 13.329954 18 17 0.49 - - - Caltanissetta Gela Biviere di Gela (N) 37.020923 14.345073 6 - 0.77 - - - Caltanissetta Riesi C.da Pietralunga (N) 37.337828 14.126060 220 - 0.40 - - - Catania Randazzo Lago Gurrida (A) 37.857812 14.900734 855 - 0.49 - - - Enna Enna Fiume Imera (N) 37.494888 14.145615 261 11 0.51 - - - Enna Enna Pergusa (N) 37.511532 14.304555 671 14 0.89 - - - Enna Nicosia Laghetti Sambuchetti- Campanito (N) 37.832023 14.390823 1252 22 0.41 1 1 18 Enna Nicosia Laghetti Sambuchetti- Campanito 2 (N) 37.831846 14.390189 1261 1 0.39 1 - - Messina Alcara Li Fusi Alcara Li Fusi (A) 37.991153 14.719617 644 3 0.50 - 1 2 Messina Alcara Li Fusi Lago di Maulazzo (A) 37.94059 14.672646 1470 - 0.98 - - - Messina Caronia Lago Gianferraro (N) 37.951383 14.49746 1010 2 0.96 1 1 2 Messina Caronia Marina di Caronia (N) 38.027674 14.476765 65 - 0.36 - - - Messina Caronia Marina di Caronia 2 (A) 38.022166 14.477595 159 1 0.42 - 1 1 Messina Caronia San Fratello (A) 37.98346 14.566497 500 2 0.73 1 1 1 Messina Caronia San Fratello 2 (A) 37.979295 14.542903 403 8 0.57 1 1 7 Messina Caronia Stagno di Pantana (N) 37.949314 14.551364 922 44 0.83 1 n.a. n.a. Messina Caronia Torre del Lauro (A) 38.03035 14.531056 246 2 0.94 - 1 1 Messina Caronia Torre del Lauro 2 (A) 38.039912 14.547666 179 - 0.54 - - - Messina Cesarò Lago Biviere (N) 37.95211 14.715968 1279 - 0.70 - - - Messina Mistretta Lago Urio Quattrocchi (N) 37.901245 14.396302 1039 3 0.84 1 1 3 Messina Sant’Agata di Militello Lago di Spartà (A) 38.030587 14.647807 670 20 0.43 - - - Palermo Godrano Gorgo Lungo (N) 37.901286 13.408673 894 11 0.70 - - - Palermo Godrano Laghetto Coda di Riccio (A) 37.873226 13.398654 862 17 0.86 - - - Palermo Godrano Laghetto Coda di Riccio 2 (A) 37.872622 13.401166 868 5 0.81 - - - Palermo Godrano Laghetto presso Valle Maria (A) 37.882222 13.411111 651 4 0.71 - - - Palermo Monreale Chiusa Arcera (A) 37.916111 13.391111 584 2 0.94 - - - Palermo Monreale Laghetto agricolo di Tagliavia (A) 37.896613 13.299692 589 14 0.37 - - - Palermo Monreale Lago presso C/da Costa del Renoso (A) 37.883611 13.043056 216 7 0.41 - - - Palermo Monreale Ponte Calatrasi (N) 37.844453 13.119358 203 - 0.36 - - - Palermo Monreale Stagno agricolo di Gallitello (A) 37.866954 12.961537 132 23 0.41 - - - Palermo Palermo Laghetto agricolo del Frattina (A) 37.865877 13.30409 478 11 0.52 - - - NEW DISTRIBUTIONAL DATA ON HAEMOgrEgArINA StEPANOwI AND PlAcObDEllA cOStAtA PARASITISING THE SICILIAN POND TURTLE EMyS trINAcrIS 14 Province Municipality Site name Latitude N Longitude E Elevation (m a.s.l.) E. trinacris (n. ind) Naturalness Evaluation Index (“NEI”) P. costata (p/a) H. stepanowi (p/a) N. parasitized individuals (by H. stepanowi) Ragusa Ispica Canale Arezzi (A) 36.707172 14.957663 3 - 0.33 - - - Ragusa Ispica Canale presso C/da Carruba (A) 36.751374 14.980002 27 - 0.33 - - - Ragusa Ispica Pantano Gariffi (A) 36.733481 14.938490 2 - 0.33 - - - Ragusa Ragusa Fiume Irminio (N) 36.788669 14.602031 18 - 0.42 - - - Siracusa Noto Canale Longarini (A) 36.735227 14.996854 8 6 0.33 - - - Siracusa Noto Pantani di Vendicari (N) 36.807613 15.095825 2 13 0.69 - - - Siracusa Pachino Ispica (n.a.) -* -* n.a. 7 - - - - Siracusa Pachino Pantano Longarini 1 (N) 36.713826 15.01244 0 - 0.75 - - - Siracusa Pachino Pantano Longarini 2 (N) 36.708364 15.00215 0 - 0.87 - - - Siracusa Pachino Pantano Longarini 3 (N) 36.707618 15.00772 1 - 0.68 - - - Trapani Calatafimi- Segesta Laghetto presso C/da Anguillara (A) 37.856747 12.914465 204 - 0.36 - - - Trapani Custonaci Lentina (A) 38.051115 12.674649 90 3 0.37 - - - Trapani Mazara del Vallo RNI “Lago Preola e Gorghi Tondi” (N) 37.609118 12.653975 3 30 0.55 - - - To evaluate the influence of landscape features on species distribution, the “Naturalness Evaluation Index” (“NEI”, Baiamonte et al., 2015) of the area surrounding the sites (defined with a 500 m buffer) was calculated. The index was used to measure the conservation status of habitats where 0 means total artificiality and 1 maximum natural- ness. Values of NEI were correlated with the occurrence of turtles in each site and the occurrence of parasitized individuals. RESULTS Overall, 336 individuals of E. trinacris were captured in 30 different sites out of the 46 sampled water bodies (Tab. 1). In 15 cases, sites consisted of natural habitats including lakes (4), ponds (6), and watercourses (5). In 14 cases the species was present in anthropogenic habitats including ar- tificial lakes (2) and farm ponds (12). One sample includes pond turtles kept in captivity. Conversely, no E. trinacris were recorded in the other 16 surveyed sites. Sampling si- tes were located within different landscape contexts such as agricultural systems, pastures, wood plantations, and natural forests from sea level up to 1470 m a.s.l. The NEI mean value of sites with the absence and presence E. tri- nacris was respectively 0.54 and 0.61. The Spearman correlation test between occurrence of turtles and NEI of surrounding landscape was not significant (r= 0.211). Among the sites where pond turtles were found, only seven showed the occurrence of P. costata. These are both natural (5 sites) and artificial ponds (2 sites), all located within areas of high naturalness: the Nebrodi Natural Park and the Sambuchetti-Campanito Natural Reserve. The fo- renamed areas are characterized by geological substrata of Numidian Flysch formation and by three different al- titudinal vegetation belts (Quercus suber L., Q. cerris L. and Fagus sylvatica L. forests). The NEI value of the sites where P. costata has been detected ranges from 0.39 to 0.96, with a mean value of 0.67. Based on the study of Emys blood smear samples, 35 individuals from eight sites out of the 336 pond turtles examined were positive for haemogregarines (Tab. 1). Within the pond turtle populations where the presence of H. stepanowi was recorded, all of which located in the Nebrodi area, 81% of the individuals of E. trinacris pro- ved to be infected with haemogregarines. H. stepanowi was detected in individuals living in quite well-preserved environmental contexts in terms of vegetation diversity (mean of NEI equal to 0.67) where the main anthropoge- nic disturbance is represented by grazing. However, the Spearman correlation test coefficient between occurrence of parasitized individuals and NEI value of surrounding landscape was 0.21. Conversely, no evidence for the presence of the api- complexan and leech parasites was observed in the 293 E. trinacris collected in the remaining 21 sites where the Sicilian pond turtle was found to occur (Fig. 1). DISCUSSION Based on the results, the system “Emys trinacris - Pla- cobdella costata - Haemogregarina stepanowi” proved to occur in the Nebrodi area only. Oddly, in three different sites (i.e. “Alcara li Fusi”, “Marina di Caronia 2” and “Torre del Lauro”: see Tab. 1) we recorded the occurrence of H. stepanowi but not the occurrence of P. costata; this is possibly related to the limited number of Sicilian pond turtles collected in the- se sites. Conversely, in two locations (i.e. “Sambuchetti- RITA SCARDINO, MARCO ARCULEO, VINCENzO ARIzzA, GIUSEPPE BAzAN, MARIO LO VALVO, FEDERICO MARRONE, LUCA VECCHIONI 15 Campanito 2” and "Stagno di Pantana", see Tab. 1), despi- te the occurrence of P. costata, no haemogregarine were detected; however, a single blood smear was examined, from the single pond turtle individual collected there. No blood smears were prepared for the turtles collected in “Stagno di Pantana”. Interestingly, no correlation was observed between the water bodies origin (i.e. natural or artificial, see Tab. 1), altitude or landscape features and the occurrence of P. costata and/or H. stepanowi. In fact, the occurrence of le- eches and apicomplexans was observed in different water bodies in a bioclimatic range from thermomediterranean dry to supramediterranean subhumid (between 159 and 1252 m a.s.l.), and occurring in various landscapes (Ba- zan et al., 2015), from cultivated to forested, highlighting the euryecy of these species. The collected evidence thus suggests that no ecological factors possibly determined the observed pattern, as already reported for other leech species (e.g., Marrone et al., 2021). This interpretation is supported by a not significant Spearman correlation between presence of E. trinacris and H. stepanowi and NEI values. Moreover, it is unlikely that the absence of Haemogre- garina spp. in other Sicilian areas where E. trinacris oc- curs is linked to constraints in species dispersal, especially in the case of Placobdella leeches, which are able to take advantage of several biological dispersal vectors (Davies et al., 1982; Vamberger & Trontelj, 2007). Conversely, a recent natural or human-mediated colonisation of P. co- stata in Sicily, with a point-introduction in the Nebrodi area along with translocated E. orbicularis specimens from mainland Italy (e.g., Vamberger et al., 2015), can be possibly advocated to explain the observed pattern. In sites where haemogregarines were found, the inci- dence of the apicomplexan infection was high, interesting 81% of the captured pond turtles (i.e., 35 infected turtles out of 43 captured individuals, see Tab. 1), thus showing how H. stepanowi has great efficiency in exploiting its definitive host (i.e., P. costata) to infect E. trinacris indi- viduals. To date, scant information is available on the health status of the Emys spp. populations infected by haemo- gregarines (Ozvegy et al., 2015; Úngari et al., 2018). 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