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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 ?'2 1 / - '1 )% ; < 5 / - K1 )% N Q N R U % +V % E. trinacris. Based on the R % ; X9 ; 9 '* Y N [ Haemogregarina stepanowi Dani7 N Z '**K [ % 9 9 R N H. stepanowi 9 ƽ ; in erythrocyte shape depending on the infective stage. ƽ 9 9Z % N H. stepanowi N ; [ ƽ % N % ] N ; R % N % ; R 9 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 X9 9 ; R 9% 7% 9 R \ 9 Keywords: % R % ! 7
] % ; ; ?" Q % R9 [ 9 9 ; 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. ;9 R ? 1--*Q 9; Although it is morphologically similar to E. orbicularis sporozoites of these parasites live in invertebrate hosts, 9 ? Q ? # 1-- !" # $ % [ '* +-'12 $ % ! $ 32+ +' 12* +'*-56 7% 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) 2 K5-5Y6 '5 125< 2=5 Lago di Spartà, S. Agata di Militello (ME) 2* -'K-Y6 '5 2*K1 -=1 Laghetto Frattina, Corleone (PA) 2 K'KY6 '2 '*'5 -='2 Laghetto Coda di Riccio, Godrano (PA) 2 K112Y6 '2 12KK -='' Laghetto presso Valle Maria, Godrano (PA) 2 K1K<Y6 '2 155- 0/4 Lago presso C/da Costa del Renoso, Monreale (PA) 2 K2-'Y6 '2 -12K -= Laghetto presso C/da Volta di Falce, Monreale (PA) 2 K1-'Y6 '1 K51 -='* Lago Gorgo, Montallegro (AG) 2 152'Y6 '2 '+1 -=2- Canale di Pantano Longarini, Ispica (RG) 2< 511<Y6 'K --21 0/4 Laghetti di Contrada Pantana, Caronia (ME) 2 K<KY6 '5 22-K <=< Chiusa Arcera, Monreale (PA) 2 K5K*Y6 '2 121* -=1 M ; ; 6 [ M [ 6 $ M $ % 6 M ;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. Y U N 9 N account for the possible ecological preferenda of all the compo7 blood samples from infected E. trinacris specimens collected in 7 % % ; N 9 Laghetti di Contrada Pantana and Lago Urio Quattrocchi with an % ƽ 9 ƽ % 9$ $ Y % Z ? Z % Q N ; 9 ; R N7 9 N R ; % 9 9 R X9 9 '* N 7Z N 9 Emys trinacris (see Mar7 Y ; N % [ 9; [ % U %7 1-' # $ ; 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 stepanowi N Z '**K % Emys trinacris Fritz, Fattizzo, Guicking, Tripepi, Pennisi, Lenk, Joger et Wink N [ 7 N % 7 % N Z % . A–D M ; % ƽ ; maturity, A, B M % ; % N ; 9 9 9 ? N M 9 Q6 C, D – microgametocyte with a dense, homogeneous, 9 9 N N % ; % 6 E, 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%7 Z 7 Y R % 9 ; [ lihood (ML) analyses were then performed as implemented in % ; ; 7 ; ; X9 9 [ 2 1 < ? X9 1-'1Q $ [_ 2 ?9 % ! [ % ! N 7 1-'-Q N % 9 ; ; 9 KM'--V 9 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 ' + ? R 1-'1Q Y 9 N R +1 9 were evaluated by their posterior probabilities in the BI tree and the other nine sampling sites, all of them located outside N ' --- R [_ Y R % 9 ; ; R The BI analysis was performed with two independent runs overall percentage of mean prevalence in Sicily stood at 1 --- --- ; 9 [ Z 9 ; 9 U % +V ; heating values. Trees and parameter values were sampled every '-- ; 9 ; 1- --- Morphology of haemogregarine parasites and infected K --- ?1KVQ N 7 erythrocytes QR9 7 R ! ! In the examined infected specimens, we most frequent7 X9 9 N - -'' 9 ly observed three developmental stages located inside the 9 N ' --- % red cells of the peripheral blood as reported for haemogre7 indicating convergence of the runs. ; 9 R ?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--11-2K 1K='-=1-'' $Y[71-'57---***5. us ?# ; ' Q % 9 ; '1 + / - < )% ; < - / - < )% N The microgametes RESULTS had a R ; 9 9 ?# ; '" Q N '2 1 / - ' )% ; < 5 / - K )% Blood smear analysis N 9 9 N * < / ' - )% ; Based on the microscopic study of blood smear sam7 2 / - < )% N The mature trophozoites were % 9 '-' 9 U % 7 N % 9 X9 9 %7 N % ; ; ? R 'Q U 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 11 1 / ' 25 '1 < / - ' < / - -K 1'5 1 / '5 K < K* / ' 5+ K 2 / - 11 ' 1 / - 11 1 + / KK - '1 / - -2 M 12 < / - +2 '2 + / - +' ' / - '* 1K* K / 1' 5+ < 55 / ' -K K < / - +2 ' ' / - '< 1+ 1 / * 55 - '' / - -2 T 1K K / - 2 'K / - K- ' <1 / - K 2' ' / '+ 1 '- 2+ / - '< 5 / - -+ 1 ' / - -' 2* 5 / 2 5* - '1 / - -' 3% / 6 _ M ; 6 M N 6 _= M 6 M 6 Y_ M 9 9 ; 6 Y M 9 9 N 6 Y M 9 9 6 Y N 9 N %9 ? U Q Haemogregarina sp. (KF257925) Haemogregarina sp. (KF257923) 100 / * Haemogregarina sp. (KF257924) 87 / 63 H. sacaliae (KM887507) 87 / 60 H. pellegrini (KM887509) H. balli (HQ224959) 83 / 64 H. stepanowi SYRIA (KF257927) H. stepanowi SERBIA (KT749877) 79 / 64 H. stepanowi ALGERIA (KF257929) BULGARIA (KF257928) 54 / 73 H. stepanowi H. stepanowi IRAN (KF257926) Haemogregarina sp. ITALY - Laghetti di Contrada Pantana Haemogregarina sp. ITALY - Lago Urio Quattrocchi Hemolivia mauritanica (KF992710) Fig. 2. ; % R ;% 9 '* Y Y 9 R R = [_ R U ; 6 9 R N K- N Z 9%R X9 from GenBank are shown in parentheses. R ?# ; ' #Q % 9 DISCUSSION 1' < / ' K )% ; + / - + )% N [ % % ; 7 The predominant stages in our samples were macrog7 garines in the erythrocytes of the endemic Sicilian pond % ?+VQ N % ; % tro7 turtle Emys trinacris. Based on the morphology of the in7 phozoites were rarely observed (1V 'V Q 7 ; ; [ ?1--1Q Infected erythrocytes had a normal or elongated shape we were able to identify the observed haemogregarine spe7 % ? R 1 cies as Haemogregarina stepanowi6 [ N measurements). The nucleus was displaced to a marginal [ % R X9 ; ;% 97 ?# ; ' #Q ?# ; ' "Q ! '* Y ; H. stepanowi is a widespread species some cases, a vacuole was observed near the trophozoite in the Palaearctic region, ranging from Croatia to Iran and ?# ; ' Q N R 7 [ ?[ '+* N N % ; % N U ?# ; ' Q '+'- [ 1--* TZ 1-'5 7 or with rarely occurring double infections. R Z [ 1-'5 Q N able to infect freshwater turtles belonging to the families of Emydidae and Geoemydidae. Until now H. stepanowi has X9 ; '* Y ;% [ % not reported from Italy, nor, to the best of our knowledge, presence of Haemogregarina sp. in the blood of the Sicil7 is any record of the presence of this apicomplexan parasite ian pond turtles. The topologies of the phylogenetic trees R 9 9 N '2 ! 9 7 based on BI and ML analyses were largely congruent and ion, such an apparent absence is most likely to be ascribed N [ Haemogregarina species to the lack of dedicated surveys rather than to the actual ab7 occurring in the Sicilian pond turtle as H. stepanowi, which sence of this intracellular parasite in western Europe. The U R 7 U N Y Hae- intermediate (i.e. species of Mauremys [Gray] and Emys mogregarina balli ?# ; 1Q 9% U Q [ ? Placobdella # $ ; 1-'< <2 -1+ $ ; 5 < '- '55''= 1-'< -1+ ] % ; ; Emys trinacris costata [Müller]) hosts of the species are, in fact, known to % KM'--V N widely occur in western Europe. only two Sicilian sites where H. stepanowi was observed The infection with H. stepanowi 9 ƽ ? R 'Q changes in the erythrocyte shape, depending on the in7 ! ; N Z N [ Hae- fective stage. The more marked changes occur in the case mogregarina N [ Y R of infection with trophozoites because of their large size % 9 ; N [ 7 ; 6 are longer and tive host of H. stepanowi, i.e. the leech P. costata, is known ; N N ; [ ; _= to occur. Interestingly, specimens of P. costata from Sicily N 9 ? Q ; [ ; are genetically identical to those occurring in southern Ita7 % 9 ?YQ ; N ; ƽ 9 9 ; [ R9 N between their vertebrate hosts, i.e. the Sicilian E. trinacris 9 ; ; \ 9 9 N and the Italian E. orbicularis ? [ 1-'< Q ; [ ; _= Such an unexpected decoupling might be due to a recent, In erythrocytes infected with microgametocytes, the 9% 7% 9 N changes are less evident. The cells are slightly bigger, would be also supported by the evidence of the presence of N 9 9 [ ; introduced specimens of E. orbicularis from Italy in Lago % %R Y % % % 9 9 ? %R ; 1-'KQ 9 in E. trinacris N 9 R ?1-'5Q ! ;7 ; N present study, all morphometric values for the infected R R R R 7 7 erythrocytes were within the ranges reported by Arizza et R N X9 [ ? %R ; ~ 1-- ?1-'5Q U N N ; % Z 1-'1 [ 1-'< Q than in the uninfected turtles. If the introduced European pond turtles functioned ƽ 9 9Z % as vectors for the passive dispersal of P. costata and parasitised with H. stepanowi compared to data of Ariz7 H. stepanowi in Sicily, the presence of H. stepanowi in the ?1-'5Q % ; [ Sicilian pond turtle should be considered a case of ‘parasite ƽ 7 R % Z N ƽ 9 7 The consequences of a parasitosis by haemogregarines N 9 ? ; !; 1-'KQ ; 7 on the number of blood cells is not yet clear, but an in7 netic characterisation of Sicilian and Italian populations of tense parasitism by haemogregarines has been reported H. stepanowi R % % ; 7 ; % 7 to increase the number of lymphocytes and decrease the ular markers, would allow testing for the existence in Sici7 number of eosinophils in freshwater turtles (Mihalca et al. ly of autochthonous vs introduced strains of H. stepanowi, 1--1Q " Z ~ ?1--1Q [ ;7 and for the development of adequate management plans. [ ƽ ƽ R 9 On the basis of this information and considering the pos7 9ƽ7 N Z Homalopsis buccata (Linnaeus) sible anthropogenic introduction of H. stepanowi to Sicily, parasitised with Haemogregarina sp. the health status and the population dynamics of infected ] % ; ; R ; 7 populations of E. trinacris should be carefully monitored R 9 ƽ X9 9 R9 Z R % ;7 % ƽ some evidence of an impact of H. stepanowi on the occur7 haemoparasite on the conservation of the endemic Sicilian rence of shell necrosis and skin haemorrhages in Serbian pond turtle. Emys orbicularis R ? ; 1-'KQ Therefore, careful monitoring of the health status of infect7 ed populations is advisable. Acknowledgements. ! [ R %R Tutela del Territorio e del Mare is gratefully acknowledged for The nine infected Sicilian pond turtles we examined having provided the permit to allow sampling of Emys spp. in Sici7 appeared otherwise healthy, and the level of observed hae7 9 ! ?$Y[71-''7--11-2K $Y[71-'57---***5Q % ; ; N ; N 'V Two anonymous reviewers are acknowledged for their construc7 the erythrocytes contained parasites in most turtles. In con7 tive criticism, which allowed us to improve the manuscript. REFERENCES DžǍ [ NJǎǑǔǐǏ _ǂǏdž " _ǖnjdžȪ ǂǔǔ ǓNJǛǛǂ ǖǔǔǐ [ǂǓǓǐǏdž # ǂDŽDŽǐ # ǓDŽǖǍdžǐ [ ǐǘǔdžǓ ǓǐǘǏ [ ǖǓnjNJ # ǖǏǕljǐǓǏ [ ]ǂǎ7 1-'5 [ ; R ǑǍ ]džNJǔǔ ]ǐǑǑdžǏǓǂǕlj [ _ǂǓǂ Ǎdž ǂǍǍ _ _ǚǏǏ endangered Sicilian endemic pond turtle, Emys trinacris (Testu7 ] [DŽ[ǂǏǖǔ ] [NJǕDŽljdžǍǍ [ǐǛǍdžǚ7 ǕǂǏǓNJDžLjdž % Q ! ` *' 255M2K2 $ǂǓLJǓdžǚ _ $ǂǘǍǐǘǔnjNJ ǖdžDŽnjdžǓǕ ljǂDž7 ǂǓǕǂ LjdžDždžǏLjǃdž [ǂǓǕNJǏ ǎNJǕlj 1-'1 ǘNJDŽnj _ DŽljǐDŽlj " _ ǎNJǓǏǐǗ ǑNJdžLjdžǍ # 1-'1 Phylogenetic position of the adeleorinid coccidia (Myzozoa, [ 9Z 9Z [ R K+ Apicomplexa, Coccidia, Eucoccidiorida, Adeleorina) inferred 51+MK'5 9 ; '* Y X9 9Z [ R K+ ''M'*- ǓNJnjǂǏ ] "NJDŽdžnj 1-'- [ ; R NJdžǍdžDŽnjNJ "NJDŽljǐDŽnjǂ [ ǂǃǞǐȝǔnjNJ džǍdžȝ ! ǐǑdž7 % 9 9 Z 9 ] K Ǎdžǘǔnjǂ NJdžDžǖǏnjNJdžǘNJDŽǛ džǓǍdžDŽnjNJ YǐǘǂnjǐǘǔnjNJ '+M'+* $ǂnjǖǍǏNJDŽnjǂ ǛǍǂDŽljDŽNJǂnj 1-'1 " U Pla- # $ ; 1-'< <2 -1+ $ ; K < '- '55''= 1-'< -1+ ] % ; ; Emys trinacris cobdella costata ?# [ '*5 '* # R 9 1-'< '< 9 1-'< $9R '* 9;9 1-'< Cite this article as: # 9 9 [ %R ; [ [ # 1-'< ; R the ugly: Emys trinacris, Placobdella costata and Haemogregarina stepanowi in Sicily (Testudines, Annelida and Apicomplexa). # $ <2 -1+ # $ ; 1-'< <2 -1+ $ ; < <Eimeria spp. (Apicomplexa, Eimeriidae) infect7 ǕǖDŽnjǂǔ ] džǍǐ7 ǏǕǴǏ #ǂljDž ǂǍǃǐǖǔǔNJ [ ǐǖǂLj ; RR 9 ; [ R ; % 7 ǓDŽǖǍdžǐ [ [ǂǓǓǐǏdž # ǂDŽDŽǐ # ǂǎǃdžǓLjdžǓ [ ; 9 $ ; '2K 552M5K1 #ǓNJǕǛ 1-'5 9 % ; Ã ;% [ǂǓǓǐǏdž # ǂDŽDŽǐ # ǓNJǛǛǂ ǓDŽǖǍdžǐ [ 1-'