Syst Parasitol (2009) 74:29–39 DOI 10.1007/s11230-009-9186-6 The translocated Liza haematocheila (Teleostei: Mugilidae) as a new host of four species of Saturnius Manter, 1969 (Digenea: Hemiuridae) within its invasive range in the Black Sea Plamen Pankov Æ David I. Gibson Æ Aneta Kostadinova Received: 19 November 2008 / Accepted: 16 February 2009 Ó Springer Science+Business Media B.V. 2009 Abstract This paper presents the first documented record of four species of Saturnius Manter, 1969 in Liza haematocheila (Temminck & Schlegel) in its invasive Pontic range. S. papernai Overstreet, 1977, S. dimitrovi Blasco-Costa, Pankov, Gibson, Balbuena, Raga, Sarabeev & Kostadinova, 2006, S. minutus Blasco-Costa, Pankov, Gibson, Balbuena, Raga, Sarabeev & Kostadinova, 2006 and Saturnius sp. are described and compared with existing data by means of multivariate morphometric analysis. The morphological and morphometric similarities between the specimens of Saturnius sp. from L. haematocheila and a small form of S. papernai from L. aurata indicate the possible existence of another, cryptic species. Although having acquired at least three species of Saturnius since its establishment in the Black Sea region, there is no evidence that a Pacific congener has P. Pankov
A. Kostadinova (&) Central Laboratory of General Ecology, Bulgarian Academy of Sciences, 2 Gagarin Street, 1113 Sofia, Bulgaria e-mail: Aneta.Kostadinova@uv.es D. I. Gibson Department of Zoology, Natural History Museum, Cromwell Rd, London SW7 5BD, UK A. Kostadinova Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, Branišovská 31, 370 05 České Budějovice, Czech Republic been introduced with this fish. This is the first report of S. minutus from the Black Sea. Introduction Liza haematocheila (Temminck & Schlegel) (syn. Mugil soiuy Basilewsky; see e.g. Parin, 2003; Fricke et al., 2007; Eschmeyer, 2008) is a mugilid fish native to the Western North Pacific. Following numerous deliberate introduction attempts to support commercial fisheries, this species established a successful breeding population in the Sea of Azov during the early 1980s (Occhipinti-Ambrogi & Savini, 2003) and is now the most abundant mugilid species present (Kottelat & Freyhof, 2007). L. haematocheila has already become established in the north-eastern Black Sea, where it has been subjected to commercial fishing by Russia and the Ukraine since 1995. A small stock has also been exploited along the Turkish coasts since the 1990s. The environmental conditions of its Black Sea range appear to favour this exotic species, whose growth rate exceeds that of the native mullet species (Okumus & Bascinar, 1997). Furthermore, its expansion in the Black Sea coincides with a decline in the native mullet species, which it apparently replaces (Kottelat & Freyhof, 2007). More recently, L. haematocheila has been recorded from the Aegean Sea (Kaya et al., 1998), and Starushenko & Kazanski (1996) have predicted its ongoing invasion towards the Western Mediterranean. 123 30 Syst Parasitol (2009) 74:29–39 Until the recent description of Saturnius overstreeti Blasco-Costa, Montero, Gibson, Balbuena, Raga, Shvetsova & Kostadinova, 2008 from the Russian coast of the Sea of Japan (Blasco-Costa et al., 2008), no bunocotyline hemiurids have been reported from L. haematocheila in its native range. Domnich & Sarabeev (1999) described Bunocotyle constrictus Domnich & Sarabeev, 1999 from this host collected in the Sea of Azov, which they later assumed to be a mis-identification of Saturnius papernai Overstreet, 1977 (see Domnich & Sarabeev, 2000a, b, c, d; Sarabeev, 2000; Sarabeev & Domnich, 2000). However, Blasco-Costa et al. (2008), based on the re-examination of type- and voucher material, considered B. constrictus a species inquirenda and rejected its identification as S. papernai. Material from subsequent records of S. papernai in L. haematocheila from the Black and/or Azov Seas (Dmitrieva & Gaevskaya, 2001; Gaevskaya & Korniychuk, 2003) was not described, and these records are, therefore, questionable. In a study of the parasites of mullets (Mugilidae) from two localities off the Bulgarian Black Sea coast, we collected four different forms of Saturnius Manter, 1969 from L. haematocheila. Three were identified as S. papernai Overstreet, 1977, S. dimitrovi BlascoCosta, Pankov, Gibson, Balbuena, Raga, Sarabeev & Kostadinova, 2006 and S. minutus Blasco-Costa, Pankov, Gibson, Balbuena, Raga, Sarabeev & Kostadinova, 2006, whereas the fourth, Saturnius sp., exhibited differences from existing species of Saturnius (recently revised by Blasco-Costa et al., 2008). This paper presents the first documented record of four species Saturnius in L. haematocheila in its invasive Pontic range and includes morphological descriptions and multivariate morphometric analysis supporting the identifications. examined as permanent mounts in Canada balsam. Worms from frozen fish were stored in 70% ethanol and processed as above. Measurements are taken from illustrations, which were made using a drawing apparatus attached to an Olympus-BX51 microscope at 9400 magnification. Fully-developed, non-collapsed eggs were measured using an Olympus ColorView III digital camera and Olympus Cellp software (ver. 2.4). All measurements are in micrometres; mean values in the text are given within parentheses. The data for 28 metrical variables (see e.g. Table 1; the length of the oesophagus and egg-size were excluded) were ln-transformed and a principal components analysis (PCA) run to examine the multivariate relationship between the specimens of Saturnius ex L. haematocheila [22 specimens identified as S. minutus (n = 9), Saturnius sp. (n = 11), S. papernai (n = 1) and S. dimitrovi (n = 1)] and those of the dataset of Blasco-Costa et al. (2008) [i.e. S. minutus (n = 12); S. dimitrovi (n = 26); S. papernai ex M. cephalus (n = 31) and S. papernai ex L. aurata (n = 36)]. The following abbreviations for ratios are used in the text and tables: FO/BL, forebody as a percentage of body length; LSL/BL, posteriormost pseudosegment length as a percentage of body length; LSW/ LSL, posteriormost pseudosegment width as a percentage of its length; MFL/VSL, muscular flange length as a percentage of ventral sucker length; LSW/BW, posteriormost pseudosegment width as a percentage of body width; VSW/BWVS, ventral sucker width as a percentage of body width at the level of the anterior margin of the ventral sucker; MFW/BWVS, muscular flange width as a percentage of body width at the anterior border of ventral sucker. Materials and methods Saturnius dimitrovi Blasco-Costa, Pankov, Gibson, Balbuena, Raga, Sarabeev & Kostadinova, 2006 A total of 96 Liza haematocheila was collected and examined during 2004–2005 from off the Bulgarian Black Sea coast. Trematodes were recovered from fresh and frozen fish. Live worms were fixed by being pipetted into nearly-boiling saline solution (c.80°C) and immediately transferred to 70% ethanol, stained with iron acetocarmine, dehydrated through a series of alcohols, cleared in dimethyl phthalate and 123 Host: Liza haematocheila (Temminck & Schlegel). Locality: River Kamchia estuary (Bulgarian Black Sea coast). Site: Embedded in the cardiac stomach lining. Prevalence: 4.2%. Intensity: 1–20 (mean 7.5). Voucher material: BMNH 2009.1.27.5. Syst Parasitol (2009) 74:29–39 31 Table 1 Morphometric data for Saturnius minutus and Saturnius sp. Species S. minutus S. minutus Saturnius sp. Host Mugil cephalus Liza haematocheila L. haematocheila Locality Western Mediterranean Black Sea (off Sozopol) Black Sea (River Kamchia) Source Blasco-Costa et al. (2006) Present study (n = 9) Present study (n = 11) Range Mean Range Mean Range Mean Body length Body width (at ventral sucker flange) 364–542 73–115 431 92 405–523 87–112 461 99 400–540 66–108 474 87 Body width (at anterior margin of ventral sucker) – 74 61–80 72 62–101 79 Forebody length 133–184 155 139–186 164 143–187 164 Oral sucker 31–54 9 39–70 41 9 56 40–53 9 50–67 45 9 57 36–58 9 32–64 50 9 53 Measurements Ventral sucker 56–74 9 56–74 67 9 66 58–75 9 57–72 67 9 65 46–56 9 47–58 51 9 52 Pharynx 20–32 9 19–31 25 9 25 18–33 9 26–31 29 9 29 26–35 9 21–32 30 9 28 Oesophagus length Sinus-sac 7–29 23–41 9 15–26 19 32 9 21 17–29 24–37 9 19–31 23 29 9 24 17–29 23–40 9 18–34 22 32 9 26 Pars prostatica 18–38 9 12–24 27 9 17 22–41 9 15–26 30 9 20 23–42 9 13–29 31 9 19 Seminal vesicle 12–47 9 11–37 33 9 22 28–52 9 18–40 38 9 29 21–46 9 19–41 32 9 28 Anterior testis 22–47 9 22–47 33 9 33 28–35 9 24–40 32 9 30 23–35 9 22–44 31 9 32 Posterior testis 22–45 9 20–57 34 9 35 18–38 9 21–44 30 9 33 26–43 9 22–43 34 9 29 Ovary 22–37 9 21–46 30 9 33 23–55 9 31–47 35 9 40 20–49 9 40–63 38 9 46 Vitellarium 29–64 9 22–51 42 9 37 27–53 9 32–53 43 9 42 37–63 9 32–46 50 9 40 Last pseudosegment 65–150 86 9 63 97–130 9 71–85 108 9 77 114–158 9 84–116 137 9 97 Ventral sucker flange cone 34–59 46 9 23 36–50 9 16–24 46 9 21 27–39 9 9–16 34 9 13 Eggs 19–24 9 9–12 21 9 10 –a –a 22–27 9 11–14 24 9 13 Sucker length ratio 1:1.21–2.25 1:1.66 1:1.23–1.76 1:1.55 1:0.91–1.21 1:1.02 Sucker width ratio 1:0.90–1.82 1:1.19 1:1.07–1.37 1:1.17 1:0.78–1.56 1:1.01 BW/BL (%) 19–25 21 18–25 22 15–22 18 VSW/BWVS (%) – 89 80–103 91 52–81 66 FO/BL (%) MFL/VSL (%) 28–39 – 36 69 31–39 62–81 36 69 32–38 54–78 35 66 MFW/BWVS (%) – 31 22–33 29 9–22 16 Ratios LSL/BL (%) 17–29 20 21–26 24 26–35 29 LSW/BW (%) – 68 71–86 78 100–135 112 LSW/LSL (%) – 73 65–82 71 61–85 71 a Eggs collapsed Description (Fig. 1B) [Based on 30 specimens. Measurements from single well-fixed adult specimen.] Body elongate, cylindrical, 678 long, with maximum width of 121 at level of ventral sucker flange; width at mid-level of ventral sucker 86. Tegument unarmed, with slight longitudinal and transverse striations. Three circular muscular flanges present around body: oral sucker flange located at posterior level of oral sucker; strongly developed ventral sucker flange (MFL/VSL = 85%; MFW/BWVS = 27%) in third pseudosegment, with 123 32 Fig. 1 Saturnius spp. ex Liza haematocheila from the Bulgarian Black Sea coast. A. S. papernai, ventral view; B. S. dimitrovi, dorsal view; C. S. minutus, dorsal view; D. Saturnius sp., dorsal view. Scale-bar: 200 lm 123 Syst Parasitol (2009) 74:29–39 Syst Parasitol (2009) 74:29–39 2 lateral nipple-shaped conical protuberances [cone length (anterior-posterior) 68; width (lateral) 23]; well-developed third flange located in posterior third of last pseudosegment. Body with 7 pseudosegments separated by 6 transverse fibrous septa. First, second and third pseudosegments similar in length; fourth smallest; last largest, 194 9 89 (LSL/BL = 29%; LSW/BW = 100%). Oral sucker thick-walled, with terminal opening and flared anterior border, 61 9 74. Ventral sucker strongly muscular (VSW/BWVS = 93%), spherical, larger than oral sucker (sucker length ratio 1:1.31; sucker width ratio 1:1.08), in middle third of body, 80 9 80. Forebody 195 (29% of body length). Prepharynx absent; pharynx muscular, subglobular, 40 9 37. Oesophagus thick-walled, shorter than pharynx, 27. Intestinal bifurcation just anterior to genital pore. Caeca relatively wide, not observed posterior to ventral sucker (masked by uterus filled with eggs). Testes 2, subtriangular, smooth, oblique, slightly separated; anterior testis in fifth pseudosegment, 55 9 33; posterior testis in sixth pseudosegment, 50 9 34. Seminal vesicle thick-walled, elongateoval, antero-dorsal to ventral sucker, larger than sinus-sac, 74 9 55. Pars prostatica external, oval, vesicular, with lining of anuclear blebs, slightly smaller than sinus-sac, 31 9 18, surrounded by large prostatic cells. Sinus-sac subglobular, 40 9 35, contains hermaphroditic duct lined by small cells. Genital pore a wide transverse slit, submedian, at level of first septum. Ovary elongate-oval, in anterior part of last pseudosegment, contiguous with posterior testis, overlaps anterior margin of vitellarium, 69 9 38. Uterine seminal receptacle present; Laurer’s canal not observed. Vitellarium compact, elongate-oval, large, 82 9 53. Uterus thin-walled, forms numerous coils in hindbody. Eggs numerous (c.162), 22–25 (24) 9 11–14 (12) (n = 8). Excretory system not visible; pore wide, terminal. Remarks These specimens key down to S. dimitrovi using the key to species of Saturnius in Blasco-Costa et al. (2008) due to the strong development of the second muscular flange and the two anterior transverse fibrous septa. Other characteristic features of S. dimitrovi and these Black Sea specimens include: body with seven 33 pseudosegments separated by strongly muscular transverse septa; ventral sucker large in relation to body; and a large saccular seminal vesicle. The metrical data for the measured specimens from L. haematocheila fall within the range of measurements and ratios given for S. dimitrovi by Dimitrov et al. (1998) (as S. papernai) and Blasco-Costa et al. (2006) with the exception of the slightly smaller testicular width (33 and 34 vs 42–80 and 44–84 lm, respectively). To date, S. dimitrovi has been recorded from Mugil cephalus L., Liza aurata (Risso), L. ramado (Risso) and Chelon labrosus (Cuvier) in the Black Sea (the first host) and the western Mediterranean (the last three hosts) (Dimitrov et al., 1998; Blasco-Costa et al., 2008). L. haematocheila is a new host record for S. dimitrovi. Saturnius minutus Blasco-Costa, Pankov, Gibson, Balbuena, Raga, Sarabeev & Kostadinova, 2006 Host: Liza haematocheila (Temminck & Schlegel). Locality: Off Sozopol (Bulgarian Black Sea coast). Site: Embedded in the cardiac stomach lining. Prevalence: 3.1%. Intensity: 1–8 (mean 3.7). Voucher material: BMNH 2009.1.27.6. Description (Fig. 1C; Table 1) [Based on 11 worms. Measurements for 9 well-fixed, whole-mounted adult specimens.] Body minute, elongate, cylindrical, with rounded posterior extremity and maximum width at level of ventral sucker flange. Tegument unarmed, with fine longitudinal and transverse striations. Three circular muscular flanges present around body: oral sucker flange weakly developed, located posterior to mid-level of oral sucker; ventral sucker flange strongly muscular, located in posterior third of second pseudosegment, with 2 lateral nippleshaped conical protuberances; third flange relatively well developed, located in posterior third of last pseudosegment. Body with 6 pseudosegments separated by 5 very thin transverse fibrous septa, with anteriormost septum being particularly thin; no septum present at level of ventral sucker. Oral sucker simple, transverse-oval, with slightly flared anterior border in some specimens and terminal opening. Ventral sucker strongly 123 34 Syst Parasitol (2009) 74:29–39 6 5 S. dim itrovi 4 S. m inutus 3 Factor 2: 9.44% 2 1 0 Saturnius sp. -1 S. papernai ex M. cephalus -2 S. papernai ex L. aurata -3 -4 -5 -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 Factor 1: 66.31% Fig. 2 Plot of the 127 specimens of Saturnius spp. in the first plane of the PCA. Legend: S. minutus (triangles); S. dimitrovi (diamonds); S. papernai ex Mugil cephalus (circles); ‘Small form’ of S. papernai ex Liza aurata (squares; frozen specimens in grey). Black symbols indicate specimens from L. haematocheila in the Black Sea muscular, subspherical, larger than oral sucker, at about mid-body. Prepharynx absent; pharynx muscular, subspherical. Oesophagus short. Intestinal bifurcation at about mid-distance between genital pore and pharynx. Caeca form ‘Drüsenmagen’ anterior to first septum and cyclocoel dorsally to posterior third of vitellarium (observed in single specimen). Testes 2, subspherical to elongate-oval, smooth, oblique, contiguous or slightly separated, in fourth and fifth pseudosegments. Seminal vesicle thinwalled, elongate-oval, antero-dorsal to ventral sucker, similar in size to or larger than sinus-sac. Pars prostatica external, elongate-oval, similar in size to sinus-sac, lined with anuclear blebs, surrounded by small number of large prostatic cells. Sinus-sac thinwalled, subglobular, contains hermaphroditic duct lined by small intensely staining cells. Genital pore a 123 wide transverse slit, submedian, at level of first septum. Ovary oval, in anterior part of last pseudosegment, overlaps vitellarium antero-ventrally. Uterine seminal receptacle present; Laurer’s canal not observed. Vitellarium compact, elongate-oval. Uterus thinwalled, occupies free space in hindbody; metraterm not seen. Eggs not numerous, large in relation to body size. Excretory vesicle Y-shaped; arms unite dorsally at level of pharynx; stem subspherical; pore relatively wide, terminal. Remarks This material from L. haematocehila keys down to, and exhibits the characteristic features of, S. minutus, Syst Parasitol (2009) 74:29–39 i.e. small size, large muscular ventral sucker, long forebody and well-developed ventral sucker with nipple-shaped muscular lateral cone (Blasco-Costa et al., 2006, 2008). Comparison with the data of Blasco-Costa et al. (2006) revealed an overlap in the ranges for all metrical features, the material from L. haematocheila exhibiting higher upper size limits (and subsequently slightly higher means) for the length of the forebody and the size of the seminal vesicle, ovary and last pseudosegment; the latter was also larger in relation to body size, which results in higher means for the ratios LSL/BL and LSW/BW (Table 1). S. minutus was originally described from M. cephalus and has subsequently been recorded from L. aurata and L. saliens in the Spanish Mediterranean (Blasco-Costa et al., 2006, 2008). Our study represents the first record of S. minutus from the Black Sea, and L. haematocheila is a new host record for this species. Saturnius papernai Overstreet, 1977 Host: Liza haematocheila (Temminck & Schlegel). Locality: River Kamchia estuary (Bulgarian Black Sea coast). Site: Embedded in the cardiac stomach lining. Prevalence: 8.3%. Intensity: 1–5 (mean 2.6). Voucher material: BMNH 2009.1.27.4. Description (Fig. 1A) [Based on 21 worms. Measurements for 2 well-fixed, whole-mounted adult specimens.] Body large, elongate, cylindrical, 722–916 long, with maximum width of 113 at level of ventral sucker flange; width at midlevel of ventral sucker 96. Tegument unarmed, with slight longitudinal and transverse striations. Three circular muscular flanges present around body: oral sucker flange located at mid-level of oral sucker; ventral sucker flange relatively poorly developed (MFL/VSL = 117–123%; MFW/BWVS = 20–24%) in third pseudosegment, with 2 lateral mound-shaped conical protuberances [cone length (anterior-posterior) 63–75; width (lateral) 19–23 (MFW/VSW = 34– 39%)]; third flange well developed, located in posterior third of last pseudosegment. Body with 7 pseudosegments separated by 6 thick transverse fibrous septa. First and second 35 pseudosegments similar in length; fourth smallest; last largest, 238–299 9 115–121 (LSL/BL = 33%; LSW/BW = 102–107%). Oral sucker thick-walled, with terminal aperture, 53–63 9 62–63. Ventral sucker relatively small (VSW/BWVS = 58–60%), spherical, similar in size to or smaller than oral sucker (sucker length ratio 1:0.91–1.00; sucker width ratio 1:0.70–0.92), in middle third of body, 53–61 9 56–58. Forebody 247–305 (33–34% of body length). Prepharynx absent; pharynx muscular, subglobular, 34–42 9 31–39. Oesophagus shorter than pharynx, 29–38. Intestinal bifurcation anterior to first septum. Caeca relatively wide, not observed posterior to testes (masked by uterine coils filled with eggs). Testes 2, subtriangular, smooth, oblique, slightly separated; anterior testis in fifth pseudosegment, 40–73 9 37–55; posterior testis in sixth pseudosegment, 47–71 9 32–59. Seminal vesicle thick-walled, elongate-oval, antero-dorsal to ventral sucker, larger than sinus-sac, 69–76 9 44–47. Pars prostatica external, large, 44-62 9 37–40, larger than sinus-sac, elongate-oval, vesicular, lined with large anuclear blebs; prostatic cells relatively large, surrounding base of and partly overlapping sinus-sac. Sinus-sac elongate-oval, 44–47 9 35, contains hermaphroditic duct lined by small cells. Genital pore median, at level of first septum. Ovary elongate-oval to subtriangular, in anterior third of last pseudosegment, overlaps anterior margin of vitellarium, 66–93 9 60–71. Uterine seminal receptacle large, postero-dorsal to vitellarium; Laurer’s canal not observed. Vitellarium compact, elongateoval, large, 50–95 9 46–93. Uterus thin-walled, strongly developed, extends to near posterior extremity. Eggs numerous (c.167), 20–28 (23) 9 12–16 (13) (n = 22). Excretory system not visible; pore wide, dorsosubterminal. Remarks These specimens from L. haematocheila key down to S. papernai using the key of Blasco-Costa et al. (2008) due to: (i) the poor development of the ventral sucker flange, with two mound-shaped protuberances visible in lateral view; (ii) the presence of six thick transverse fibrous septa which divide the body into seven pseudosegments; (iii) the sucker ratios; and (iv) the large sizes of the pars prostatica and seminal vesicle 123 36 (in relation to the sinus-sac). All metrical data fall within the ranges given for S. papernai by Overstreet (1977) and Blasco-Costa et al. (2006, 2008). S. papernai was originally described from Mugil cephalus in Bardawil Lagoon (Sinai coast, Eastern Mediterranean, see Overstreet, 1977). This description and two recent studies (Blasco-Costa et al., 2006, 2008) represent the only documented record of this species in the Mediterranean basin, the latter authors adding four additional hosts (i.e. Liza aurata, L. ramado, L. saliens and Chelon labrosus). L. haematocheila is a new host record for S. papernai. Saturnius sp. Host: Liza haematocheila (Temminck & Schlegel). Locality: River Kamchia estuary (Bulgarian Black Sea coast). Site: Embedded in the cardiac stomach lining. Prevalence: 0.9%. Intensity: 123. Voucher material: BMNH 2009.1.27.1-3. Description (Fig. 1D; Tables 1, 2) [Based on 25 worms. Measurements for 11 dorsoventrally mounted adult specimens.] Body minute, elongate-oval, with rounded posterior extremity and maximum width at mid-level of last pseudosegment. Tegument unarmed, with longitudinal and transverse striations. Three circular muscular flanges present around body: oral sucker flange poorly developed, located at mid-level of oral sucker (when protruded; n = 2); ventral sucker flange small, located in middle of second pseudosegment, with 2 lateral moundshaped conical protuberances; third flange well developed, located just posterior to mid-length of last pseudosegment. Body with 7 pseudosegments separated by 6 thick transverse fibrous septa. Oral sucker simple, subglobular, with terminal opening. Ventral sucker muscular, spherical, similar in size to or larger than oral sucker, in middle third of body. Prepharynx absent; pharynx muscular, subspherical. Oesophagus short. Intestinal bifurcation at level of genital pore. ‘Drüsenmagen’ and cyclocoel not seen. Testes 2, subspherical, smooth, oblique, contiguous or slightly separated, in fourth and fifth 123 Syst Parasitol (2009) 74:29–39 pseudosegments. Seminal vesicle thick-walled, subspherical, antero-dorsal to ventral sucker, similar in size to sinus-sac. Pars prostatica external, elongateoval, similar in size to sinus-sac, lined with anuclear blebs, surrounded by large number of large prostatic cells. Sinus-sac thick-walled, subglobular, contains eversible hermaphroditic duct lined by relatively large intensely staining cells. Genital pore median, at level of first septum. Ovary transversely elongate-oval, in anterior third of last pseudosegment, overlaps vitellarium anteroventrally. Uterine seminal receptacle present; Laurer’s canal not observed. Vitellarium compact, elongateoval. Uterus thin-walled, occupies almost entire hindbody; metraterm not seen. Eggs numerous, large in relation to body size. Excretory system not observed; pore wide, dorsosubterminal. Remarks This material from L. haematocheila exhibits the diagnostic characteristics of Saturnius and keys down to S. papernai using the key to species of BlascoCosta et al. (2008) due to: the less well-developed ventral sucker flange with mound-shaped lateral cones; the body divided into seven pseudosegments; similar sucker ratios; and a sinus-sac similar in size to the pars prostatica and seminal vesicle. However, the specimens studied, which we denote as ‘Saturnius sp.’ here, are on average only half the size of those recently described from mullets in both the Mediterranean and Black Seas (Blasco-Costa et al., 2006), with the measurements for a number of features varying outside the lower limits for S. papernai. Examples of the latter are: the size of the body, oral sucker, testes and ventral sucker flange; the length of the forebody, pars prostatica and seminal vesicle; and the width of the ventral sucker and vitellarium. A further set of metrical characters exhibits smaller lower limits in Saturnius sp.: the size of the sinus-sac and ovary; the width of the body at the mid-level of the ventral sucker, pars prostatica and seminal vesicle; and the length of the vitellarium and last pseudosegment. Finally, the ventral sucker and the last pseudosegment in Saturnius sp. are larger in relation to body size, which result in higher upper limits and means for the ratios VSW/BWVS, LSW/ LSL and LSW/BW (Table 2). Syst Parasitol (2009) 74:29–39 37 Table 2 Comparative data for S. papernai and Saturnius sp. Species S. papernai Saturnius sp. Blasco-Costa et al. (2006) S. papernai (small form, frozen material) Blasco-Costa et al. (2008) Source Range Range Range Mean Present study (n = 11) Mean Measurements Body length 687–1,183 994 454–593 400–540 474 Body width (at ventral sucker flange) 107–165 141 81–104 66–108 87 Body width (at anterior margin of ventral sucker) 95–136 119 72–93 62–101 79 Forebody length 240–412 326 160–233 143–187 164 Oral sucker 55–91 9 57–91 71 9 78 38–57 9 38–56 36–58 9 32–64 50 9 53 Ventral sucker 47–79 9 57–84 66 9 73 38–47 9 44–62 46–56 9 47–58 51 9 52 Pharynx 27–65 9 27–48 44 9 40 25–37 9 23–33 26–35 9 21–32 30 9 28 Oesophagus length – 37 – 17–29 22 Sinus-sac 37–74 9 27–45 54 9 37 29–39 9 19–31 23–40 9 18–34 32 9 26 Pars prostatica 40–77 9 23–54 62 9 38 37–49 9 16–28 23–42 9 13–29 31 9 19 Seminal vesicle 52–109 9 32–81 82 9 57 33–67 9 23–45 21–46 9 19–41 32 9 28 Anterior testis Posterior testis 34–113 9 44–94 38–105 9 42–105 72 9 67 73 9 70 23–53 9 19–52 24–59 9 24–58 23–35 9 22–44 26–43 9 22–43 31 9 32 34 9 29 Ovary 42–101 9 36–120 62 9 71 28–44 9 32–48 20–49 9 40–63 38 9 46 Vitellarium 45–124 9 50–103 87 9 76 37–61 9 26–56 37–63 9 32–46 50 9 40 Last pseudosegment 176–358 9 80–173 278 9 141 120–160 9 81–105 114–158 9 84–116 137 9 97 Ventral sucker flange cone 46–91 9 19–36 65 9 28 35–54 9 12–21 27–39 9 9–16 34 9 13 Eggs 21–26 9 9–14 24 9 12 20–26 9 10–15 22–27 9 11–14 24 9 13 Ratios Sucker length ratio 1:0.91–0.92 1:0.93 1:0.7–1.1 1:0.91–1.21 1:1.02 Sucker width ratio 1:0.70–1.20 1:0.95 1:0.9–1.3 1:0.78–1.56 1:1.01 BW/BL (%) 12–19 14 15–19 15–22 18 VSW/BWVS (%) 52–71 61 55–68 52–81 66 FO/BL (%) 30–37 33 34–42 32–38 35 MFL/VSL (%) 76–118 99 77–123 54–78 66 MFW/BWVS (%) 17–32 23 13–26 9–22 16 MFW/VSW (%) 28–49 – 21–39 17–30 24 LSL/BL (%) LSW/LSL (%) 25–35 31–72 28 51 23–30 60–78 26–35 61–85 29 71 LSW/BW (%) 51–120 101 94–114 100–135 112 However, the material described above resembles the ‘small form’ of S. papernai from L. aurata distinguished by Blasco-Costa et al. (2008) in its longer forebody, relatively wider last pesudosegment, and the size of the body and most other metrical features. A morphometrical comparison between the present specimens, originating from a single frozen fish, and the frozen material of Blasco-Costa et al. (2008) revealed slightly higher upper limits of variation in the latter for the size of the testes and muscular flange at ventral sucker level (also in relation to the body and ventral sucker width, i.e. MFW/BWVS and MFW/VSW), and the lengths of the forebody, pars prostatica and seminal vesicle (Table 2). A comparison with S. minutus is also necessary here, especially because of the small size of the worms 123 38 from L. haematocheila (body length \550 lm). Although many metrical features exhibit a range overlap, Saturnius sp. can be clearly distinguished from this species by having: a body comprised of seven pseudosegments formed by six strongly developed fibrous septa; a less developed ventral sucker flange (also narrower in relation to the body, see MFW/BWVS in Table 1); a distinctly smaller ventral sucker (also in relation to the body size, resulting in values for VSW/BWVS below the lower range of S. minutus); a much larger posterior segment; and larger eggs. Saturnius sp. resembles the third species parasitising mullets in the Mediterranean basin, S. dimitrovi, in the number of septa/pseudosegments, but differs in the remaining distinguishing features listed in the comparison with S. minutus above. Overall, the above comparisons suggest that Saturnius sp. and the ‘small form’ of S. papernai ex L. aurata described by Blasco-Costa et al. (2008) may in fact represent a new species more closely related to S. papernai than to the other Mediterranean species of Saturnius. However, the lack of ample, well-fixed material prevents its delineation at present. Statistical comparisons The first two principal components of a PCA run using the correlation matrix between 28 metrical variables of the 127 specimens of Saturnius in the combined dataset explained 75.7% of the variation in the data-set. The length of the body, forebody and last pseudosegment, as well as body width at both the anterior margin of the ventral sucker and the ventral sucker muscular flange, exhibited a greater contribution to the separation along the first axis, which explained 66.3% of the total variance, whereas the size of ventral sucker and ventral sucker flange had important contributions to the second principal component, explaining a further 9.4% of the variance. A plot of the specimens in the first plane of the PCA shows three well-separated groups that correspond to the three Mediterranean species of Saturnius; these included the specimens from L. haematocheila identified as S. dimitrovi, S. minutus and S. papernai. The specimens of Saturnius sp. grouped together with the specimens of S. papernai denoted as the ‘small form ex L. aurata’ in Blasco-Costa et al. (2008) (Fig. 2). 123 Syst Parasitol (2009) 74:29–39 As in the latter study, no clear hiatus exists in the two-dimensional plane between the samples from M. cephalus, L. aurata and L. haematocheila. Concurrent infections Although the overall prevalence of infection of L. haematocheila with Saturnius spp. was low (13.5%), the frequency of concurrent infections was quite high (38%). Thus combinations of two species (S. papernai ? S. dimitrovi and S. papernai ? S. minutus) were found in each of two fish (of 13 infected) and one fish harboured individuals of three species (5 S. papernai, 20 S. dimitrovi and 8 S. minutus). To the best of our knowledge, this is the first record of co-occurrence of Saturnius spp. in individual fish. Blasco-Costa et al. (2008) compared the prevalence patterns of the three Saturnius spp. in a large sample (676 fish) from the western Mediterranean and indicated that S. dimitrovi and S. minutus attain their highest prevalence in M. cephalus in two different ecosystems and that S. papernai reaches a maximum prevalence in L. aurata; no concurrent infections were reported in the latter study. It is, therefore, possible that our data reflect a trophic overlap between L. haematocheila and both M. cephalus and Liza spp. in the Black Sea. Concluding remarks This increased sampling effort and detailed morphological study have revealed that Liza haematocheila has acquired at least three species of Saturnius in its invasive range, and that this example of transfaunation does not appear to have introduced any Pacific species, such as S. overstreeti, into the Pontic region. However, the levels of infection of this host along the Bulgarian Black Sea coast are rather low. Further sampling and re-identification of existing material from other regions of the Black Sea may reveal the patterns of exchange of Saturnius spp. between the sympatric mullet populations and the role of L. haematocheila in the cycling of these parasites in the local communities. The morphological and morphometric similarity between the specimens of Saturnius sp. from L. haematocheila and the small form of S. papernai from L. aurata indicates the Syst Parasitol (2009) 74:29–39 possible existence of a cryptic species whose delineation might be enhanced by a molecular study. Acknowledgements This study was supported by Institute of Parasitology (Academy of Sciences of the Czech Republic) (grants Z60220518 and LC522 to AK); and the material was collected within the framework of an INTAS project (03-515998). References Blasco-Costa, I., Pankov, P., Gibson, D. I., Balbuena, J. A., Raga, J. A., Sarabeev, V. L., et al. (2006). Saturnius minutus n. sp. and S. dimitrovi n. sp. (Digenea: Hemiuridae) from Mugil cephalus L. (Teleostei: Mugilidae), with a multivariate morphological analysis of the Mediterranean species of Saturnius Manter, 1969. Systematic Parasitology, 65(7), 7–91. Blasco-Costa, I., Montero, F. E., Gibson, D. I., Balbuena, J. A., Raga, J. 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