© Institute of Parasitology, Biology Centre CAS #$;1-'<<2-1+ doi: '-'55''=1-'<-1+ http://folia.paru.cas.cz Research Article The good, the bad and the ugly: Emys trinacris, Placobdella costata and Haemogregarina stepanowi in Sicily (Testudines, Annelida and Apicomplexa) Vincenzo Arizza', Francesco Sacco', Debora Russo', Rita Scardino', Marco Arculeo', Melita Vamberger1 and Federico Marrone' ' Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche, Sezione di Biologia Animale ed Antropologia ;;9$%$%!6 1 Museum für Tierkunde, Senckenberg Dresden, Dresden, Germany Abstract: Endemic Sicilian pond turtles Emys trinacris Fritz, Fattizzo, Guicking, Tripepi, Pennisi, Lenk, Joger et Wink were exam7 ined for the presence of haemogregarine parasites. The presence of haemogregarines, occurring mainly in the microgametocyte stage ?'21/-'1)%;<5/-K1)%NQNRU%+V%E. trinacris. Based on the R%;X9;9'*YN[Haemogregarina stepanowi Dani7 NZ'**K[%99RNH. stepanowi9ƽ; in erythrocyte shape depending on the infective stage. ƽ99Z%NH. stepanowi N;[ƽ%N%]N;R%N%;R9 by H. stepanowi in the closely related European pond turtle Emys orbicularis (Linneaus), monitoring of the health status of the infected Sicilian populations of E. trinacris is desirable. The restricted distribution of populations of Emys infected with haemogregarines in X99;R9%7%9R\9 Keywords: %R%!7 ]%;; ?"Q % R9[99; widely distributed blood parasites in turtles worldwide of the genus Placobdella ?Q?TZ ? 1-'1Q %U N 1-'KQ involve merogony and the formation of gametocytes in Emys trinacris Fritz, Fattizzo, Guicking, Tripepi, Pen7 a vertebrate host, and gamogony and sporogony in the nisi, Lenk, Joger et Wink is a Sicilian endemic pond turtle. ;9R?1--*Q9; Although it is morphologically similar to E. orbicularis sporozoites of these parasites live in invertebrate hosts, 9?Q?#1--<Q%9U%7 the merozoites are thought to be transmitted to turtles by ic studies have unambiguously revealed the presence of ?$'+*+1--'Q_; ;[ƽRNNN turtles are better targets for leech attachment because of U?#1--K$1-''%R; ;9?1--*Q]%;7 1-'KQ!R;;97 garine species are common and widely distributed in fresh7 ary importance of this chelonian species and the reduction N9?;1--<`%$1--* of its populations caused by habitat destruction, pollution TZ 1-'5Q NZ ?'**KQ and pathogens, E. trinacris N !"Y presence of the parasite in the European pond turtle Emys _Q[R?~Z1--+Q9;;; orbicularis (Linnaeus) and described it under the name the need for further studies aimed at its characterising and Haemogregarina stepanowiNZ'**K monitoring of its health status. [%;;% ![;; vertebrate hosts is quite low and the same taxon might, leeches parasitising the Sicilian pond turtle (Marrone et al. ƽ9? 1-'<QR9%U%7 1--'TZ 1-'5Q"R9 parasites in the blood cells of wild populations of E. trina- of haemogregarines seems to be determined by the distri7 cris was investigated. !"#$%['*+-'12$% !$32++'12*+'*-567%89 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. '-'55''=1-'<-1+ ]%;;Emys trinacris Table 1. Geographical coordinates of the sampled sites and synopsis of the studied blood samples of Emys trinacris Fritz, Fattizzo, Guicking, Tripepi, Pennisi, Lenk, Joger et Wink. Sites Coordinates Infected/examined Lago Urio Quattrocchi, Mistretta (ME) 2K5-5Y6'5125< 2=5 Lago di Spartà, S. Agata di Militello (ME) 2*-'K-Y6'52*K1 -=1 Laghetto Frattina, Corleone (PA) 2K'KY6'2'*'5 -='2 Laghetto Coda di Riccio, Godrano (PA) 2K112Y6'212KK -='' Laghetto presso Valle Maria, Godrano (PA) 2K1K<Y6'2155- 0/4 Lago presso C/da Costa del Renoso, Monreale (PA) 2K2-'Y6'2-12K -= Laghetto presso C/da Volta di Falce, Monreale (PA) 2K1-'Y6'1K51 -='* Lago Gorgo, Montallegro (AG) 2152'Y6'2'+1 -=2- Canale di Pantano Longarini, Ispica (RG) 2<511<Y6'K--21 0/4 Laghetti di Contrada Pantana, Caronia (ME) 2K<KY6'522-K <=< Chiusa Arcera, Monreale (PA) 2K5K*Y6'2121* -=1 M;;6[M[6$M$%6M;9 MATERIALS AND METHODS ber or sizes) were summarised as the mean, standard deviation Sampling. One hundred one sicilian pond turtles were sam7 (SD), standard error of the mean (SE), and range. %?R'Q Molecular evaluation.YUN9N account for the possible ecological preferenda of all the compo7 blood samples from infected E. trinacris specimens collected in 7%%;N9 Laghetti di Contrada Pantana and Lago Urio Quattrocchi with an % ƽ 9 ƽ % 9$$Y%Z?Z%QN; 9;RN79NR; %99RX99'* N7ZN9Emys trinacris (see Mar7 Y;N%[9;[%U%7 1-'<RQSpecimens of E. trinacris were captured by ?#K7""""72K77 hands or by hoop net traps left overnight in concealed sites (Ream """"""72Q;R6?1--*Q %'+<<QU%;;7 ;;%9ƾR%7 spection and detailed macroscopic inspection of the skin, plastron R;[%U? and carapace, blood samples were taken for cytological, haema7 1-'1TZ 1-'51-'K;1-'KQ tological and molecular analyses. Blood samples were obtained $"%U1)!Y'-%%9% from the dorsal coccygeal vessel of each collected turtle. After R9ƽN1K%[[;"1-5)Y$'-%[') blood samples had been obtained, the animals were immediately '-)[%-1)!Y#7XY$%7 released into their natural environments. For each individual, two 1)Y%'11)9R7N blood smears and one blood sample in ethanol were collected. 9%1-) Blood smears were prepared in situ according to Arizza et %[9 ?1-'5QRN%;7 +K"K%62K+1"5KK*"5K %N[U%7 1"+-6[U1"'-% ; $% % ?[7N 3 %77 $" 9 N Z 'V ; ; 9[ %NZ;9''-R9ƽN]Q 9;U$7!%9?9%R] 1-%N9;N1%9 Staufen, Germany) and sequenced in both directions on an ABI blood smears were examined under a Leica DMRE light micro7 22-U?%#"" 9;'--&%;[%%X9 Q9;;%2'"X9; N;%?_"#51-"_%7 (Applied Biosystems). Chromatograms were imported and edited Q?#;'Q!%;;N?_9_Q N"%_1-'?9%$_9R was used to examine the slides and perform calculations. Australia). The sequences obtained from Lago Urio Quattrocchi N%R9;'-2 red and Laghetti presso Contrada Pantana were deposited in GenBank blood cells (RBC) for each animal. For study of the erythrocyte 99%R<+'5'<+'5'* <-N%9?'K97 '*X9R;;ƽHaemogregari- 'KN'KN%;%7 naNN%Z?#;17 'KN%;%Q#9% ZY9%RQ69%X9R;; were considered: lengths (L) and widths (W) and nuclear lengths Hemolivia mauritanica ?;;'+-5Q was included ?Y_Q9N?YQ?Q in the analysis to be used as an outgroup. 9?YQN9 ?_K EW K4Q=5 Y Z X9 N ; N Y ?Y_KYK4Q=5?Z"Z1-'-Q"97 ?!R "R " Q ?] '+++Q 9; N%N_=Y_=Y %%;%'152RN 99=%NY= in the sequences of Hemolivia mauritanica (GenBank accession RN[9; 9%R #++1'-Q Haemogregarina balli Paterson et Dess7 ?1-'5Q]%;R?9%7 '+<?Z9%R]115+K+QH. pel- #$;1-'<<2-1+ $;1< '-'55''=1-'<-1+ ]%;;Emys trinacris A B C 10 μm 10 μm 10 μm D E F 10 μm 10 μm 10 μm Fig. 1. Endogenous life stages of Haemogregarina stepanowiNZ'**K%Emys trinacris Fritz, Fattizzo, Guicking, Tripepi, Pennisi, Lenk, Joger et Wink N[7N%7%NZ%. A–DM;%ƽ; maturity, A, BM%;%N;999?NM9Q6C, D – microgametocyte with a dense, homogeneous, 99NN%;%6E, F – mature trophozoites are curved in a capsule with the nucleus located at the bend. legrini ?_ $ '+'-Q ?Z [**K-+Q and three specimens in Lago Urio Quattrocchi , both locat7 ?!Q ; %U%9%7Z7 YR%9;[ lihood (ML) analyses were then performed as implemented in %;;7;;X99 [21<?X91-'1Q$[_2?9 %![%!N7 1-'-QN%9;; 9KM'--V9 specimens of E. trinacris time reversible model of sequence evolution with a proportion of proved to be infected with haemogregarines. Conversely, invariable sites (GTR + I), as selected by the Akaike information no evidence for the presence of haemogregarine parasites ~[1'+?R1-'1QY9 NR+19 were evaluated by their posterior probabilities in the BI tree and the other nine sampling sites, all of them located outside N'---R[_ YR%9;;R The BI analysis was performed with two independent runs overall percentage of mean prevalence in Sicily stood at 1------;9[Z9;9 U%+V; heating values. Trees and parameter values were sampled every '--;9;1---- Morphology of haemogregarine parasites and infected K---?1KVQN7 erythrocytes QR97R!! In the examined infected specimens, we most frequent7 X99N--''9 ly observed three developmental stages located inside the 9N'---% red cells of the peripheral blood as reported for haemogre7 indicating convergence of the runs. ;9R?1--*Q%;% Ethics. Specimens of Emys trinacris were sampled and ex7 %;%?#;'Q$7 amined according to the national laws and considering all ethical cilian pond turtles were morphologically similar and con7 requirements. The investigation was carried out with permission ~9 [ R%R 9 Macrogametes, each with an oval shape and a granular del Mareof the Republic of Italy under permission code U. prot. nucleus, were usually located near the erythrocyte nucle7 $Y[71-''7--11-2K1K='-=1-''$Y[71-'57---***5. us ?#; 'Q %9 ; '1+ / -< )% ; <- / -< )% N The microgametes RESULTS had a R;99?#;'"Q N'21/-')%;<5/-K)% Blood smear analysis N 99 N *< / '- )% ; Based on the microscopic study of blood smear sam7 2/-<)%NThe mature trophozoites were %9'-'9U% 7N%9X99%7 N%;;?R'QU ing a large oval body contained in a capsule and appear7 specimens were collected in Laghetti di Contrada Pantana ing withdrawn into themselves, with the nucleus posi7 #$;1-'<<2-1+ $;2< '-'55''=1-'<-1+ ]%;;Emys trinacris Table 2. Dimensions from uninfected erythrocytes (U) of Emys trinacris Fritz, Fattizzo, Guicking, Tripepi, Pennisi, Lenk, Joger et Wink compared with erythrocytes infected with microgranulocyte (M) and trophozoite (T). Turtle EL EW L/W ES (μm1) Y_ Y Y_=Y Y?3%1) Y= U 111/'25 '1</- '</--K 1'51/'5K <K*/'5+ K2/-11 '1/-11 1+/KK -'1/--2 M 12</-+2 '2+/-+' '/-'* 1K*K/1'5+ <55/'-K K</-+2 ''/-'< 1+1/*55 -''/--2 T 1KK/-2 'K/-K- '<1/-K 2''/'+1 '-2+/-'< 5/--+ 1'/--' 2*5/25* -'1/--' 3%/6_M;6MN6_=M6M6Y_M99;6YM99 N6YM996YN9N%9?UQ Haemogregarina sp.