Anna Faltýnková Simona Georgieva Aneta Kostadinova Isabel Blasco-Costa

Transcription

1 Syst Parasitol (2014) 89: DOI /s Diplostomum von Nordmann, 1832 (Digenea: Diplostomidae) in the sub-arctic: descriptions of the larval stages of six species discovered recently in Iceland Anna Faltýnková Simona Georgieva Aneta Kostadinova Isabel Blasco-Costa Tomáš Scholz Karl Skírnisson Received: 1 July 2014 / Accepted: 6 August 2014 Ó Springer Science+Business Media Dordrecht 2014 Abstract Frequent infections with Diplostomum spp. (Digenea: Diplostomidae) were found in the freshwater snail Radix peregra (Müller) and three fish species, the salmonids Salmo trutta fario L., Salvelinus alpinus (L.) and the gasterosteid Gasterosteus aculeatus L., collected in four lakes in south-western Iceland in Detailed analysis of the isolates integrating molecular, morphological and ecological data revealed that these belong to Diplostomum spathaceum (Rudolphi, 1819) and five putative new species (three infecting both snails and fish). This paper provides detailed descriptions of the metacercariae of the six species-level lineages of Diplostomum spp. and of the A. Faltýnková (&) S. Georgieva A. Kostadinova I. Blasco-Costa T. Scholz Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, Branišovská 31, České Budějovice, Czech Republic faltyn@paru.cas.cz S. Georgieva Faculty of Science, University of South Bohemia, Branišovská 31, České Budějovice, Czech Republic I. Blasco-Costa Department of Zoology, University of Otago, PO Box 56, Dunedin, New Zealand K. Skírnisson Laboratory of Parasitology, Institute for Experimental Pathology, University of Iceland, Keldur, 112 Reykjavík, Iceland cercariae of three of the lineages discovered in Iceland with comments on the application of ITS1 rdna for species distinction within Diplostomum von Nordmann, 1832 in the light of the novel data. We strongly suggest the use of molecular evidence based on cox1 gene sequences (in addition to ITS1-5.8S-ITS2 sequences) in association with detailed assessment of the morphology of the larval stages in future studies of Diplostomum spp. in fish and snails. Introduction In a study on digenean parasites of the freshwater snail Radix peregra (Müller) and three fish species, the salmonids Salmo trutta fario L. and Salvelinus alpinus (L.) and the gasterosteid Gasterosteus aculeatus L., in four lakes in south-western Iceland, we found frequent infections with Diplostomum spp. Detailed analysis of the isolates integrating molecular, morphological and ecological data resulted in the discovery of one described [Diplostomum spathaceum (Rudolphi, 1819)] and five putative new species that complete their life-cycles within a narrow geographic area (Blasco-Costa et al., 2014). Formal naming of the new species awaits discovery of the adults via matching of larval and adult life-cycle stages with the aid of molecular markers. However, this may be severely delayed or deemed impossible due to ethical reasons and other obstacles in examination of the bird definitive hosts.

2 196 Syst Parasitol (2014) 89: We believe that although not formally named, the distinctive genetic and morphological characteristics of the larval stages discovered in Iceland may help the advancement of the identification of Diplostomum spp. in the freshwater environments of Europe, especially in the sub-arctic. The recently built molecular framework (databases for mitochondrial cox1 and nuclear ITS1-5.8S-ITS2 rdna sequences) for Diplostomum spp. in Europe and Canada (Locke et al., 2010a, b; Behrmann-Godel, 2013; Georgieva et al., 2013; Blasco-Costa et al., 2014) appears essential for shifting the focus on the morphological and ecological characterisation in the course of identification of the larval stages of Diplostomum spp. in snails and fish. This paper provides detailed descriptions of the metacercariae of the six lineages of Diplostomum spp. and of the cercariae of three of the lineages discovered in Iceland, with comments on the application of ITS1 rdna for species distinction within the genus in the light of the novel data. Materials and methods A total of 434 Radix peregra (Müller) (Gastropoda: Lymnaeidae) was collected in May and August 2012 in Lakes Raudavatn ( N, W), Family Park ( N, W) and Nordic House ( N, W) in south-western Iceland. Snails were screened for infections with Diplostomum spp. and shipped live to the Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic. Upon arrival, snails were labelled and placed into beakers with small amount of tap water under a light source to stimulate cercarial emission. Snails were identified using Glöer (2002). Although R. peregra have recently been treated as junior synonyms of Radix balthica (L.) the name R. peregra is used herein following the molecular studies of Bargues et al. (2001) and Huňová et al. (2012) which provide sequences for snails sampled in Iceland. Totals of 59 Gasterosteus aculeatus from Lakes Hafravatn ( N, W), Family Park and Nordic House, 19 S. trutta fario and four Salvelinus alpinus from Lake Hafravatn were examined for infections with strigeid metacercariae. Cercariae and metacercariae of Diplostomum spp. were studied on live and fixed material by means of light and scanning electron microscopy (SEM; cercariae only). Series of photomicrographs were made for each isolate (live and fixed larvae) with a digital camera of an Olympus BX51 microscope prior to sequencing and measurements were taken from the digital images with the aid of Quick Photo Camera 2.3 image analysis software. Drawings of the cercariae are based on digital images of live cercariae; upon examination in vivo the cercariae were fixed in cold 4% formaldehyde solution. The structure of the secondary excretory system of the metacercariae was reconstructed from serial microphotographs and the number of excretory concretions was counted in representative isolates subjected to sequencing. Upon examination in vivo, the metacercariae were fixed in ethanol (molecular biology grade) for DNA isolation and photographed again. Samples of cercariae fixed in 4% formaldehyde solution (isolates of Lineage 4 and 6 of Blasco-Costa et al., 2014 only) were postfixed in 2% osmium tetroxide for 2 h, washed in 0.1 M phosphate buffer, dehydrated through an acetone series, critical point-dried and sputtercoated with gold. Samples were examined using a JEOL JSM 7401-F scanning electron microscope at an accelerating voltage of 4 kv. All measurements are in micrometres and are presented as the range followed by the mean in parentheses. Measurements in the descriptions are based on live specimens; measurements from fixed material are provided after each description. The following abbreviations were used in the text and tables: AOW, anterior organ width; BL, body length; FL, furca length; TSL, tail stem length; VSW, ventral sucker width. For illustrative purposes a Neighbour-Joining (NJ) tree was constructed using the Kimura 2-parameter method with MEGA v.6 (Tamura et al., 2013). Nodal support was based on 1,000 replicates (no outgroup was used). Naming scheme of larval stages of Diplostomum spp. would likely become complicated in view of the recent increased sampling effort resulting in the generation of sequences for novel genetically distinct lineages. Locke et al. (2010a, b) used Arabic numerals for the species-level lineages discovered in Canada (up to Diplostomum sp. 9). Using a numbering scheme would suffer lack of coordination e.g. current BOLD holdings reach up to Diplostomum sp. 21 (

3 Syst Parasitol (2014) 89: boldsystems.org/index.php/taxbrowser_taxonpage? taxid=70375). Therefore, we used the lineage number of Blasco-Costa et al. (2014) plus the country of provenance, Iceland (abbreviated as Lin. 2I, Lin. 3I etc. in the text), to indicate the different Diplostomum spp. in the descriptions below. Family Diplostomidae Poirier, 1886 Genus Diplostomum von Nordmann, 1832 Diplostomum spathaceum (Rudolphi, 1819) Measurements of fixed metacercariae: Body ( ); BW/BL = (66%); oral sucker ( ); pharynx ( ); pseudosuckers (37) long; ventral sucker ( ); sucker width ratio 1: (1:0.89); holdfast organ ( ); hindbody (41). Diplostomum sp. Lin. 2I Second intermediate hosts: Gasterosteus aculeatus L. (Teleostei: Gasterosteidae); Salvelinus alpinus (L.) (Teleostei: Salmonidae). Site in host: Eye lens. Localities: Lakes Family Park and Nordic House (ex G. aculeatus); Lake Hafravatn (ex S. alpinus). Representative sequences: Lineage 1 in Blasco-Costa et al. (2014): GenBank KJ KJ (cox1); KJ KJ (ITS1-5.8S-ITS2). Description (Fig. 1.1) Metacercaria Body medium-sized, ( ), elongate-oval [width (59%) of body length], with maximum width just anterior to ventral sucker. Oral sucker subspherical, ( ). Pseudosuckers medium-sized, ( ). Oral opening terminal; prepharynx absent; pharynx elongate-oval, ( ); oesophagus very short, bifurcates close posterior to pharynx; caeca wide, usually filled with dark, fine granular content, encroach holdfast organ and terminate blindly at midlevel of excretory vesicle. Ventral sucker subspherical, ( ), smaller or slightly larger than oral sucker (sucker width ratio 1: (1:0.84), at mid-body length or slightly posterior. Holdfast organ relatively small, transversely oval, bipartite, contiguous with ventral sucker, ( ). Excretory vesicle small, V-shaped; reserve excretory system of diplostomid type; excretory concretions predominantly large, \ 300 in number, grouped into 2 lateral extracaecal and 1 median fields; concretions in median field dorsal to ventral sucker largest. Hindbody (53) long. First intermediate host: Radix peregra (Müller) (Gastropoda: Lymnaeidae). Second intermediate hosts: Gasterosteus aculeatus L. (Teleostei: Gasterosteidae); Salmo trutta fario L. (Teleostei: Salmonidae). Site in host: Hepatopancreas (ex R. peregra); eye lens (ex G. aculeatus, S. trutta fario). Localities: Lake Raudavatn (ex R. peregra); Lakes Hafravatn and Nordic House (ex G. aculeatus); Lake Hafravatn (ex S. trutta fario). Prevalence: 0.7% (ex R. peregra); for prevalences in the fish hosts see Blasco-Costa et al. (2014). Representative sequences: Lineage 2 of Blasco-Costa et al. (2014): GenBank KJ KJ (cox1); KJ KJ (ITS1-5.8S-ITS2). Description (Figs. 1.2, 2A, 3A) Cercaria Body elongate-oval, lacking yellow pigment, ( ), shorter than tail stem [TSL/BL = (1.28)]. Anterior organ elongate-oval, large, posterior margin reaches to last third of forebody length, ( ). Ventral sucker spherical, somewhat post-equatorial, ( ); width exceeds width of anterior organ [AOW/VSW = (0.73)]. Mouth opening ventro-subterminal; prepharynx very long, narrower in anterior organ; pharynx round, small, ( ); oesophagus short, bifurcates just anterior to ventral sucker; intestinal caeca conspicuous, terminate at some distance from excretory vesicle. Penetration gland-cells 2 pairs, relatively small, posterior to ventral sucker, intracaecal, posterior pair not reaching extremities of caeca;

4 198 Syst Parasitol (2014) 89: Fig. 1 Metacercariae of the six lineages of Diplostomum spp. ex Salmo trutta, Salvelinus alpinus and Gasterosteus aculeatus discovered in Iceland. Numbers correspond to lineage numbers in Blasco-Costa et al. (2014). 1, Diplostomum spathaceum (Lineage 1 of Blasco-Costa et al., 2014); 2, Diplostomum sp. Lin. 2I ; 3, Diplostomum sp. Lin. 3I ; 4, Diplostomum sp. Lin. 4I ; 5, Diplostomum sp. Lin. 5I ; 6, Diplostomum sp. Lin. 6I. Scale-bar: 200 lm ducts open antero-laterally to mouth. Anlagen of reproductive organs a compact round mass of small cells just anterior to excretory vesicle. Tail stem (258) long, (37) wide at base, shorter than furcae [TSL/FL = (0.88)], margins with somewhat rugged appearance, contains 8 9 pairs of caudal bodies with weakly-incised contours clustered along excretory duct. Furcae without fin-folds, (293) long, (22) wide at base. Flamecell formula 2[(1?1?1)?(1?1?1?[2])]=16. Excretory vesicle V-shaped, with round stem; caudal excretory duct passes through tail stem; Cort s isle present at level of anterior extremity of tail stem; excretory pores at midlength of furcae. Body armature: Pre-oral spines arranged in three groups: a median group of 15 larger spines, and 2 lateral groups with 7 smaller spines. Post-oral spines denser than spines on body, in alternate rows encircling first third of anterior organ, followed by short spineless area. Transverse rows of spines

5 Syst Parasitol (2014) 89: Fig. 2 Cercariae of three lineages of Diplostomum spp. ex Radix peregra discovered in Iceland. A, Diplostomum sp. Lin. 2I ; B, Diplostomum sp. Lin. 4I ; C, Diplostomum sp. Lin. 6I. Scale-bars: 100 lm lacking, entire body covered with dense, conspicuous, evenly distributed spines; spineless area around ventral sucker lacking. Ventral sucker armed with at least 240 spines of similar in size to body spines, arranged in 4 5 rows. Tail stem and furcae with conspicuous, pointed spines similar in size to body spines; spines arranged in 2 ventral and 2 dorsal bands along entire tail stem, each consisting of 5 rows of spines and single bands along furcal margins consisting of c.4 rows of spines. Resting position: Tail stem bent at 90. Measurements of fixed cercariae: Body ( ); anterior organ ( ); ventral sucker ( ); AOW/VSW = (1.24). Metacercaria Body small, ( ), robust [width 50 85% (60%) of body length], with maximum width just anterior to ventral sucker. Oral sucker subspherical, ( ). Pseudosuckers large, strongly muscular, ( ), typically invaginated, forming a pouch-like structure on each side of oral sucker. Oral opening terminal; prepharynx absent; pharynx slightly elongate-oval, ( ); oesophagus very short, bifurcates close posterior to pharynx; caeca wide, usually filled with dark, fine granular content, encroach holdfast organ and terminate blindly at midlevel of excretory vesicle. Ventral sucker subspherical, ( ), similar to or slightly larger than oral sucker (sucker width ratio 1: (1:1.19), at mid-body length or slightly posterior. Holdfast organ small, transversely oval, bi-partite, contiguous with and partially overlapping ventral sucker dorsally, ( ). Excretory vesicle small, V-shaped; reserve excretory system of diplostomid type but excretory concretions very small, \300 in number, scattered but generally grouped into two lateral extracaecal and one median field; concretions in median field dorsal to ventral sucker largest. Hindbody not developed. Measurements of fixed metacercariae: Body ( ); BW/BL = (66%); oral sucker ( ); pharynx

6 200 Syst Parasitol (2014) 89: Fig. 3 Body armature of cercariae of the three lineages of Diplostomum spp. ex Radix peregra discovered in Iceland. A, Diplostomum sp. Lin. 2I ; B, Diplostomum sp. Lin. 4I ; C, Diplostomum sp. Lin. 6I. Scale-bars: bodies, 100 lm; tails, 200 lm ( ); pseudosuckers ( ); ventral sucker ( ); sucker width ratio 1: (1:1.21); holdfast organ ( ). Remarks The most characteristic feature of the cercariae of Diplostomum sp. Lin. 2I is the absence of transverse rows on body, typical for the known cercariae of Diplostomum spp. as well as for the two other cercariae from Iceland described below. Instead, the surface of the entire body, tail stem and furcae is covered by robust tegumental spines. This characteristic tegumental spination of the material from Iceland shows similarities only with the description of Diplostomum commutatum (Diesing, 1850) by Niewiadomska & Kiseliene (1994). However, the present cercaria differs from the cercaria of D. commutatum in the number and arrangement of the tegumental spines as follows: (i) the number of the pre-oral spines in the median group (15 vs 11 12); (ii) the number of pre-oral spines in each lateral group (7 vs 6); (iii) the number of post-oral rows of spines (10 11 vs 12 14); (iv) the number of spine rows on ventral sucker (4 5 vs 3); and (v) the total number of spines on ventral sucker (at least 240 vs 180). Furthermore, the tail stem of the cercaria of Diplostomum sp. Lin. 2I is shorter than the furcae and contains a smaller number of caudal bodies (8 9 vs 10 pairs), and the penetration gland-cells although located similarly to those in D. commutatum, are small (see also Table 1). The metacercaria of Diplostomum sp. Lin. 2I possesses a shorter but wider body than the metacercaria of D. commutatum (mean vs lm), somewhat wider oral (mean vs lm) and ventral sucker (mean vs lm) and smaller holdfast organ (mean vs lm) (see Niewiadomska, 2003). These data support the distinctness of the form described from Iceland.

7 Syst Parasitol (2014) 89: Table 1 Comparative data for the cercariae of Diplostomum sp. Lineage 2 Iceland, Diplostomum sp. Lineage 4 Iceland and similar species Species/Feature Diplostomum sp. Lineage 2 Iceland D. commutatum (Diesing, 1850) Source Present study Niewiadomska & Kiseliene (1994) Diplostomum sp. Lineage 4 Iceland Present study Niewiadomska & Kiseliene (1994) D. baeri Dubois, 1937 D. gasterostei Williams, 1966 Williams (1966); Blair (1977) Relation BL-TSL-FL BL\TSL\FL BL\TSL[FL BL\TSL=FL BL\TS\FL BL\TSL[FL Relation VSW/AOW VSW\AOW VSW[AOW VSW\AOW VSW=AOW VSW\AOW No. of pre-oral spines in the (mode 9) median group No. of pre-oral spines in each 7 6 absent absent absent lateral group No. of post-oral rows of spines Transverse rows of spines on absent absent (9 a ) body No. of spine rows on ventral sucker complete? 1 incomplete No. of spines on ventral sucker at least 240 c per row (c.120 in total) per row Penetration gland-cells small, do not cover ends of caeca large, do not cover ends of caeca large, cover ends of caeca large, cover ends of caeca cover ends of caeca Spines on tail stem present present present absent present Spines on furcae present present present absent present Resting position tail stem bent at 90 tail stem bent at 90 tail stem straight tail stem straight tail stem straight a Data from the published drawing by Williams (1966) Abbreviations: AOW, anterior organ width; BL, body length; FL, furca length; TSL, tail stem length; VSW, ventral sucker width

8 202 Syst Parasitol (2014) 89: Diplostomum sp. Lin. 3I Second intermediate hosts: Salmo trutta fario L., Salvelinus alpinus (L.) (Teleostei: Salmonidae). Site in host: Eye vitreous humour. Locality: Lake Hafravatn. Representative sequences: Lineage 3 of Blasco-Costa et al. (2014): GenBank KJ KJ (cox1); KJ KJ (ITS1-5.8S-ITS2). Description (Fig. 1.3) Metacercaria Body large, ( ), oval [width (70%) of body length], with maximum width in first body third. Oral sucker subspherical, ( ). Pseudosuckers large, strongly muscular, ( ). Oral opening terminal; prepharynx absent; pharynx elongate-oval, ( ); oesophagus very short, bifurcates close posterior to pharynx; caeca narrow, poorly visible, encroach holdfast organ and terminate blindly at mid-level of excretory vesicle. Ventral sucker transversely oval, ( ), similar to or slightly larger than oral sucker (sucker width ratio 1: (1:1.15), at mid-body length or just posterior. Holdfast organ massive, transversely oval, bi-partite, partially overlaps ventral sucker dorsally, ( ). Excretory vesicle very small, V-shaped; reserve excretory system of diplostomid type; excretory concretions medium-sized, in number, scattered throughout body except ventrally to holdfast organ where only few present; concretions in median field and ventral to holdfast organ largest. Hindbody (44) long. Measurements of fixed metacercariae: Body ( ); BW/BL = (73%); oral sucker ( ); pharynx ( ); pseudosuckers ( ); ventral sucker ( ); sucker width ratio 1: (1:1.18); holdfast organ ( ). Diplostomum sp. Lin. 4I First intermediate host: Radix peregra (Müller) (Gastropoda: Lymnaeidae). Second intermediate host: Gasterosteus aculeatus L. (Teleostei: Gasterosteidae). Site in host: Hepatopancreas (ex R. peregra); eye retina and brain (ex G. aculeatus). Localities: Lake Nordic House (ex R. peregra); Lakes Nordic House and Hafravatn (ex G. aculeatus). Prevalence: 2.8% (ex R. peregra); for prevalences in the fish host see Blasco-Costa et al. (2014). Representative sequences: Lineage 4 of Blasco-Costa et al. (2014): GenBank KJ KJ (cox1); KJ KJ (ITS1-5.8S-ITS2). Description (Figs. 1.4, 2B, 3B, 4A C) Cercaria Body elongate-oval, lacking yellow pigment, ( ), equal to or shorter than tail stem [TSL/BL = (1.13)]. Anterior organ elongateoval, posterior margin not reaching half-length of forebody, ( ). Ventral sucker subspherical, post-equatorial, ( ); width smaller than width of anterior organ [AOW/ VSW = (1.26)]. Mouth opening ventrosubterminal; prepharynx long, narrower in anterior organ; pharynx spherical, ( ); oesophagus long, bifurcates at mid-length between pharynx and ventral sucker; intestinal caeca voluminous, enlarged distally, terminate at some distance from excretory vesicle. Penetration gland-cells 2 pairs, large, with fine granular content, posterior to ventral sucker, overlap caeca, posterior pair reaching extremities of caeca; ducts open antero-laterally to the mouth. Anlagen of reproductive organs a compact mass of small cells just anterior to excretory vesicle. Tail stem (243) long, (44) wide at base, slightly shorter than or equal to furcae [TSL/FL = (0.96)] (longer in fixed cercariae, see below), contains 6 pairs of caudal bodies with smooth contours clustered along excretory duct. Furcae without fin-folds, (241) long, (27) wide at base. Flame-cell formula of excretory system 2[(1?1?1)?(1?1?1?[2])]=16. Excretory vesicle V-shaped, with round stem; caudal excretory duct passes though tail stem; Cort s isle present at level of anterior extremity of tail stem; excretory pores at mid-length of furcae. Body armature: Pre-oral spines 8, large, arranged in a single median group of 3 rows. Post-oral spines

9 Syst Parasitol (2014) 89: Fig. 4 Scanning electron microscopy micrographs of cercariae of Diplostomum spp. ex Radix peregra discovered in Iceland. A C, Diplostomum sp. Lin. 4I ; D F, Diplostomum sp. Lin. 6I. Scale-bars: A, B, E, F, 10 lm; C, D, 5 lm larger and denser than spines on body, in 5 6 alternate rows encircling body to level of first third of anterior organ, posterior spines smaller. A wide zone of smaller, less dense, irregularly dispersed spines present posterior to post-oral spines up to level of posterior margin of anterior organ, followed by a narrow spineless area dorsally delimitating this zone from transverse rows on body. Transverse rows of spines 9, extending to mid-level of ventral sucker ventrally and to posterior margin of ventral sucker dorsally. Rows 1 5 and 7 complete (i.e. encircling body), rows 8 9 discontinuous ventrally and dorsally, row 6 discontinuous dorsally. Rows 1 2 double; additional lateral spines present in rows 1 4. Two ventro-lateral nonconfluent fields of evenly distributed smaller spines present between level of posterior margin of ventral

10 204 Syst Parasitol (2014) 89: sucker and posterior extremity of body. Ventral sucker armed with 3 rows of spines (c.38 per row, c.120 spines in total). Tail stem and furcae armed with spines smaller than tegumental spines; spines arranged in 2 ventral and 2 dorsal bands along entire tail stem each consisting of 2 rows of small scale-like spines and single bands along furcal margins consisting of 3 rows of small, pointed spines each. Resting position: Tail stem straight, body slightly bent ventrally. Measurements of fixed cercariae: Body ( ); tail stem ( ); furcae (233) long, (29) wide at base; anterior organ ( ); ventral sucker ( ); pharynx ; AOW/VSW = (1.14); TSL/BL = (1.35); TSL/FL = (1.08). Metacercaria Body small, ( ), oval [width 52 80% (61%) of body length], with maximum width at level of ventral sucker. Oral sucker spherical, ( ). Pseudosuckers relatively small, ( ). Oral opening terminal; prepharynx absent; pharynx subspherical, ( ); oesophagus very short, bifurcates close posterior to pharynx; caeca wide, usually filled with dark, fine granular content, encroach holdfast organ and terminate blindly at mid-level of excretory vesicle. Ventral sucker subspherical, ( ), smaller than to slightly larger than oral sucker (sucker width ratio 1: (1:1.07), at mid-body length. Holdfast organ massive, transversely oval, bi-partite, contiguous with ventral sucker, ( ). Excretory vesicle small, V-shaped; reserve excretory system of diplostomid type; excretory concretions relatively small, in number, scattered but generally grouped into two lateral extracaecal and one median field. Hindbody (49). Measurements of fixed metacercariae: Body ( ); BW/BL = (65%); oral sucker ( ); pharynx ( ); pseudosuckers ( ); ventral sucker ( ); sucker width ratio 1: (1:0.97); holdfast organ ( ). Remarks In the arrangement of the pre-oral spines in a single median group, the cercaria of Diplostomum sp. Lin. 4I resembles the cercariae of D. spathaceum (Rudolphi, 1819), D. mergi Dubois, 1932 and D. parviventosum Dubois, 1932 but differs in a number of distinguishing features as follows: (i) the arrangement of the spines on the ventral sucker (3 vs 2 rows); (ii) the number of transverse rows of spines on body (9 vs 9 10 in D. spathaceum, 10 in D. mergi and D. parviventosum); (iii) the spination at posterior extremity of the body (spines not confluent vs confluent); (iv) the width relation of the anterior organ and ventral sucker (VSW \ AOW vs VSW [ AOW); (v) the arrangement of penetration gland-cells (second pair covering vs not reaching extremities of the caeca); and (vi) the resting position (tail stem straight vs bent). The cercaria of Diplostomum sp. Lin. 4I further differs from those of D. spathaceum and D. mergi in having spines on the tail stem and furcae; although the cercaria of D. parviventosum was described with spined tail stem and furcae, the spines are reported as dispersed (Niewiadomska & Kiseliene, 1994). Finally, the metacercariae of the three species compared above develop in the eye lenses of the fish intermediate hosts whereas the metacercarial isolates of Diplostomum sp. Lin. 4I were recovered from the eye retina and brain of G. aculeatus. The morphology of the present cercaria resembles the description of the cercaria of Diplostomum baeri Dubois, 1937 by Niewiadomska & Kiseliene (1994)in the absence of lateral groups of pre-oral spines, the presence of large penetration gland-cells covering the extremities of the caeca, the presence of caudal bodies with smooth contours, and in the resting position with a straight tail stem. However, in Diplostomum sp. Lin. 4I there are fewer post-oral (5 6 vs 7 9 in D. baeri) and transverse rows of spines (9 vs 10); of the latter, rows 1 2 are double in Diplostomum sp. Lin. 4I (vs row 1 only), rows 6 and 8 9 are incomplete (vs rows 8 10), rows 1 4 bear additional lateral spines (vs rows 2 8). Furthermore, both the tail stem and furcae are armed with spines in Diplostomum sp. Lin. 4I and the width of the ventral sucker is smaller than the width of the anterior organ. The bodies of the fixed cercariae examined here are on average shorter than those of D. baeri of Niewiadomska & Kiseliene (1994) (mean 189

11 Syst Parasitol (2014) 89: vs 216 lm) at a similar body width (48 vs 50 lm); the tail stem and furcae are also shorter (mean 252 vs 261 and 233 vs 259 lm, respectively). A comparison with the metrical data for the metacercariae of D. baeri sensu stricto of Niewiadomska (1988) recovered from eye ball outside the eye lens of Perca fluviatilis L. and Acerina cernua (L.) revealed that the metacercariae of Diplostomum sp. Lin. 4I exhibit on average smaller body dimensions (mean vs lm) but similarly-sized oral sucker ( vs lm), ventral sucker ( vs lm) and holdfast organ ( vs lm). However, there is a considerable difference in the number of the excretory concretions, in Diplostomum sp. Lin. 4I vs in D. baeri [see Niewiadomska, 1988; recently reported by Niewiadomska (2003) as (mean 562) ]. The present cercaria strongly resembles morphologically the cercaria of Diplostomum gasterostei Williams, 1966 in the presence of only a median group of pre-oral spines, in the number of transverse rows of spines on body (9) and the similar number of rows of post-oral spines (5 6 and 6, respectively), in the relative width of the anterior organ and ventral sucker (VSW \ AOW), as well as in having three rows of spines on the ventral sucker, spined tail and furcae, large penetration gland-cells covering the caeca and a resting position with a straight tail stem. The differences include the length relation of tail stem and furcae (TSL = FL in Diplostomum sp. Lin. 4I vs TSL [ FL in D. gasterostei) and the wider range for the number of median pre-oral spines in D. gasterostei (6 11, mode 9 vs 8inDiplostomum sp. Lin. 4I ; see Blair, 1977; Table 1). Compared with the cercariae of D. gasterostei described by Williams (1966), the fixed cercariae of Diplostomum Lin. 4I exhibit lower values for the length of body (mean 189 vs 250 lm), tail stem (mean 252 vs 330 lm) and furcae (mean 233 vs 270 lm) which are also wider at the base (mean 29 vs 20 lm). Although there is an overlap with the metrical data of Williams (1966) for fixed metacercariae of D. gasterostei, the present metacercariae are longer (mean 332 vs 280 lm), with larger oral ( vs lm) and ventral ( vs lm) suckers, holdfast organ ( vs lm) and pharynx ( vs lm). McKeown & Irwin (1995) completed experimentally the life-cycle of a species they identified as D. volvens Nordmann, 1832 starting with metacercariae from the retina of P. fluviatilis. Unfortunately, these authors provided rather limited morphological data for the cercariae and metacercariae of this species. The cercaria of Diplostomum sp. Lin. 4I can be differentiated from the cercaria of D. volvens by the larger body (fixed cercariae; mean vs lm) and tail stem (mean vs lm) but possess similarly-sized furcae (mean 233 vs 231 lm) and ventral sucker (mean vs lm). Further differences include body armature: cercariae of Diplostomum sp. Lin. 4I possess fewer post-oral rows of spines (5 6 vs 10 rows in D. volvens) and nine transverse rows on body (vs 10 rows). Notably, the cercaria of D. volvens described by McKeown & Irwin (1995) is markedly smaller when compared with that of D. baeri described by Niewiadomska & Kiseliene (1994): body (mean vs lm); tail stem (mean length 226 vs 261 lm); and furcae (mean 231 vs 259 lm). The above comparisons suggest the existence in Europe of at least four morphologically similar but distinct forms that may comprise the D. baeri species complex. Diplostomum sp. Lin. 5I Second intermediate hosts: Salmo trutta fario L., Salvelinus alpinus (L.) (Teleostei: Salmonidae). Site in host: Eye retina. Locality: Hafravatn. Representative sequences: Lineage 5 of Blasco-Costa et al. (2014): GenBank KJ KJ (cox1); KJ KJ (ITS1-5.8S-ITS2). Description (Fig. 1.5) Metacercaria Body medium-sized, ( ), elongate-oval to round [width (64%) of body length], with maximum width at level of ventral sucker. Oral sucker transversely oval, ( ). Pseudosuckers small, ( ), typically protruding, forming small elevations on each side of oral sucker. Oral opening terminal; prepharynx absent; pharynx spherical, ( ); oesophagus very short, bifurcates close posterior to pharynx; caeca narrow, poorly visible, encroach holdfast organ, terminate blindly at mid-level

12 206 Syst Parasitol (2014) 89: of excretory vesicle. Ventral sucker subspherical, ( ), similar to or slightly larger than oral sucker (sucker width ratio 1: (1:1.17), at mid-body length or slightly posterior. Holdfast organ massive, transversely oval, bi-partite, partially overlaps ventral sucker dorsally, ( ). Excretory vesicle small, V-shaped; reserve excretory system of diplostomid type but excretory concretions large, in number, grouped into two lateral extracaecal and one median field; concretions in median field dorsal to ventral sucker largest. Hindbody not developed. Measurements of fixed metacercariae: Body ( ); BW/BL = (79%); oral sucker ( ); pharynx ( ); pseudosuckers ( ); ventral sucker ( ); sucker width ratio 1: (1:0.96); holdfast organ ( ). Diplostomum sp. Lin. 6I First intermediate host: Radix peregra (Müller) (Gastropoda: Lymnaeidae). Second intermediate host: Gasterosteus aculeatus L. (Teleostei: Gasterosteidae). Site in host: Hepatopancreas (R. peregra); eye retina (G. aculeatus). Localities: Lake Nordic House (ex R. peregra); Lakes Nordic House and Family Park (ex G. aculeatus). Prevalence: 4.7% (ex R. peregra); for prevalences in the fish hosts see Blasco-Costa et al. (2014). Representative sequences: Lineage 6 of Blasco-Costa et al. (2014): GenBank KJ KJ (cox1); KJ KJ (ITS1-5.8S-ITS2). Description (Figs. 1.6, 2C, 3C, 4D F) Cercaria Body elongate-oval, ( ), shorter than tail stem [TSL/BL = (1.60)], with aggregations of yellow pigment in the parenchyma anteriorly. Anterior organ elongate-oval, posterior margin reaches to nearly mid-length of forebody, ( ). Ventral sucker subspherical, ( ), somewhat pre-equatorial; width equal to width of anterior organ [AOW/VSW = (1.10)]. Mouth opening ventro-subterminal; prepharynx very long, narrower in anterior organ; pharynx spherical, ( ); oesophagus relatively long, bifurcates at some distance from ventral sucker; intestinal caeca conspicuous, enlarged distally, terminate fairly close to excretory vesicle. Penetration gland-cells 2 pairs, relatively large, with fine granular content, posterior to ventral sucker, overlap caeca partially, posterior pair not reaching extremities of caeca; ducts open anterolateral to mouth. Anlagen of reproductive organs a compact round mass of small cells just anterior to excretory vesicle. Tail stem (268) long, (44) wide at base, as long as furcae [TSL/FL = (1.01)], contains 6 pairs of caudal bodies with smooth contours clustered along excretory duct. Furcae without fin-folds, (277) long, (28) wide at base. Flame-cell formula 2[(1?1?1)? (1?1?1?[2])]=16. Excretory vesicle small, V-shaped, with round stem; caudal excretory duct passes through tail stem; Cort s isle present at level of anterior extremity of tail stem; excretory pores at midlength of furcae. Body armature: Pre-oral spines arranged in 3 groups: a median group of 11 large spines and two lateral groups of 5 smaller spines each. Post-oral spines more robust than spines on body, in 7 9 alternate rows, encircling body to nearly half-length of anterior organ, posterior spines smaller. A zone of smaller, less dense, irregularly dispersed spines present posterior to post-oral spines up to level of posterior margin of anterior organ, followed by 11 transverse rows of spines extending to mid-level of ventral sucker. Rows 1 6 complete (i.e. encircling body), rows 7 9 discontinuous dorsally, row 10 discontinuous ventrally and dorsally, row 11 discontinuous ventrally. Rows 1 3 double, row 11 double laterodorsally. Two ventro-lateral fields of smaller spines present posterior to ventral sucker; spines less dense and confluent ventrally just posterior to ventral sucker and ventrally and dorsally near posterior extremity of body. Ventral sucker armed with 3 rows of spines (c.25 per row; in total), a fourth row may be partly formed. Tail stem and furcae with scale-like spines, smaller than tegumental spines; spines arranged in 2 ventral and 2 dorsal bands along tail stem except for its anterior extremity, each consisting of 3 4 rows of scale-like spines and single bands along

13 Syst Parasitol (2014) 89: furcal margins consisting of 2 3 rows of scale-like spines. Resting position: Tail stem bent at 90. Measurements of fixed cercariae: Body ( ); tail stem ( ); furcae ( ); anterior organ ( ); ventral sucker ( ); AOW/VSW = (1.11); TSL/ BL = (1.48); TSL/FL = (1.07). Metacercaria Body large, ( ), robust [width (68%) of body length], with maximum width at first body third. Oral sucker large, subspherical, ( ). Pseudosuckers large, strongly muscular, ( ), typically protruding, forming distinct elevations on each side of oral sucker. Oral opening terminal; prepharynx absent; pharynx massive, elongate-oval, ( ); oesophagus very short, bifurcates close posterior to pharynx; caeca indistinct, encroach holdfast organ and terminate blindly at mid-level of excretory vesicle. Ventral sucker transversely oval, large, ( ), larger than oral sucker (sucker width ratio 1: (1:1.30), in third quarter of body. Holdfast organ massive, transversely oval, bi-partite, partially overlaps ventral sucker dorsally, ( ). Excretory vesicle small, V-shaped; reserve excretory system of diplostomid type but excretory concretions medium-sized, in number, scattered throughout body except dorsally to holdfast organ (few in this region in some specimens). Hindbody not developed. Measurements of fixed metacercariae: Body ( ); BW/BL = (70%); oral sucker ( ); pharynx ( ); pseudosuckers ( ); ventral sucker ( ); sucker width ratio 1: (1:1.09); holdfast organ ( ). Remarks Cercariae of two Palaearctic species of Diplostomum, D. pseudospathaceum Niewiadomska, 1984 and D. paracaudum (Iles, 1959) sensu Niewiadomska & Kiseliene (1994) appear most similar to the cercaria described above. The cercaria of Diplostomum sp. Lin. 6I resembles the cercaria of D. pseudospathaceum in: (i) the relative dimensions of body, tail and furcae (BL\TSL = FL) and anterior organ and ventral sucker (VSW = AOW); (ii) the arrangement of the preoral spines in one median and two lateral groups; (iii) the size and position of the penetration gland-cells; (iv) the presence of yellow pigment in the forebody; and (v) the resting position (Table 2). The cercariae of the two species also exhibit overlapping ranges for the numbers of the median pre-oral spines, post-oral rows of spines and rows and spines on the ventral sucker (Table 2). However, a number of differences between the two forms exist, predominantly associated with body armature. These include (i) the arrangement of the pre-oral spines in the median group (11 spines in 4 rows in Diplostomum sp. Lin. 6I vs 8 14 spines in 3 rows in D. pseudospathaceum); (ii) the number of preoral spines in each lateral group (5 vs 1 4); (iii) the number of transverse rows of spines on body (11 vs 10), with the last row (row 11 in Diplostomum sp. Lin. 6I ) being complete on dorsal side (vs incomplete row 10 in D. pseudospathaceum); (iv) the size of the spines in the anterior transverse rows (vs larger in Diplostomum sp. Lin. 6I ); (v) the presence of additional lateral spines only in row 11 in Diplostomum sp. Lin. 6I (vs in rows 3 10 in D. pseudospathaceum); (vi) the shape of the caudal bodies (rounded vs indented/ incised in D. pseudospathaceum); and (vii) the spination of the tail stem (spines along nearly entire length vs spines near distal end only). The cercaria of Diplostomum sp. Lin. 6I possesses on average shorter body (mean vs lm) than the cercaria of D. pseudospathaceum but longer tail stem (mean 259 vs 206 lm) and furcae (242 vs 201 lm) (see Niewiadomska & Kiseliene, 1994). The present cercaria resembles the cercaria of D. paracaudum in: (i) the relative dimensions of the terminal organ and ventral sucker (VSW = AOW); (ii) the presence of three groups of pre-oral spines; (iii) the size and position of penetration gland-cells; and (iv) the resting position with the tail stem bent at an angle of 90. However, the cercaria of Diplostomum sp. Lin. 6I differs from the cercaria of D. paracaudum in: (i) the presence of yellow pigment in the forebody; (ii) the number and the shape of the caudal bodies (6 pairs of rounded vs pairs of indented/incised caudal bodies); (iii) the spination of the tail stem and furcae (spined vs unspined); (iv) the number and arrangement of the pre-oral spines in the median group

14 208 Syst Parasitol (2014) 89: Table 2 Comparative data for the cercariae of Diplostomum sp. Lineage 6 Iceland and similar species Species/Feature Diplostomum sp. Lineage 6 Iceland D. pseudospathaceum Niewiadomska, 1984 Source Present study Niewiadomska & Kiseliene (1994) D. paracaudum (Iles, 1959) Niewiadomska & Kiseliene (1994) D. adamsi Lester & Huizinga, 1977 Lester & Huizinga (1977) D. scudderi (Olivier, 1941 (syn. D. baeri eucaliae Hoffmann & Hundley, 1957) Olivier (1941) a ; Hoffmann & Hundley (1957) b Relation BL-TSL-FL BL\TSL=FL BL\TSL=FL BL\TSLCFL BL\TSLCFL BL\TSL=FL a BL\TSL\FL b Relation VSW/AOW VSW=AOW VSW=AOW VSW=AOW VSW[AOW VSW=AOW No. of pre-oral spines in a the median group No. of pre-oral spines in a each lateral group No. of post-oral rows of spines Transverse rows of spines on body No. of spine rows on ventral sucker No. of spines on ventral sucker a ; 9 11 b 3 (4th row may be partly formed) c.25 per row (75 82 in total) Penetration gland-cells large, do not cover ends of caeca Spines on tail stem present, along nearly entire tail stem 2 (3rd row may be partly formed) rows many a ;3 b ? 12 in a half row (c.120 in total) large, do not cover ends of caeca large, do not cover ends of caeca large, do not cover ends of caeca present at distal end absent present b Spines on furcae present present absent present b large, do not cover ends of caeca Resting position tail stem bent at 90 tail stem bent at 90 tail stem bent at 90 tail stem straight tail stem straight a Data from Olivier (1941); b Data from Hoffmann & Hundley (1957) Abbreviations: AOW, anterior organ width; BL, body length; FL, furca length; TSL, tail stem length; VSW, ventral sucker width

15 Syst Parasitol (2014) 89: (11 spines in 4 rows vs spines in 3 rows); (v) the number of pre-oral spines in each lateral group (5 vs 1 2); (vi) the number of spines on the ventral sucker (75 82 vs ); (vii) the number of transverse rows of spines on body (11 vs 10). Moreover, in the cercaria of Diplostomum sp. Lin. 6I the transverse rows 1 6 form complete circles (vs rows 1 5 in D. paracaudum); rows 7 9 are discontinuous dorsally and only row 10 is discontinuous ventrally and dorsally (vs rows 7 10 discontinuous both ventrally and dorsally); rows 1 3 are double (vs row 1 only). Comparisons of metrical data for fixed cercariae reveal that the cercaria of Diplostomum sp. Lin. 6I has on average smaller body than that of D. paracaudum (mean vs lm) but longer tail stem (mean 258 vs 211 lm) and furcae (mean 242 vs 208 lm) and shorter but wider anterior organ (mean vs lm) (see Niewiadomska & Kiseliene, 1994). A comparison with the known cercariae of the North American species of Diplostomum is compulsory in view of the phylogenetic relationships (Blasco- Costa et al., 2014) of the isolate described here. Unfortunately, the data available are scarce and incomplete. Comparisons with two species, D. adamsi Lester & Huizinga, 1977 and D. scudderi (Olivier, 1941) (syn. D. baeri eucaliae Hoffmann & Hundley 1957), whose metacercariae have been found in the eye retina of Perca flavescens (Mitchill) and G. aculeatus and in the brain of Culaea inconstans (Kirtland) (see Olivier, 1941; Hoffman & Hundley, 1957; Lester, 1974; Lester & Huizinga, 1977), appear most relevant for a morphological comparison. The cercariae of these species resemble the cercaria of Diplostomum sp. Lin. 6I in the similar relations of the lengths of body, tail stem and furcae, in the number of the median pre-oral spines, and in having at least three rows of spines on the ventral sucker and large penetration gland-cells that do not cover the ends of the caeca (Table 2). The cercaria of Diplostomum sp. Lin. 6I further resembles the cercaria of D. scudderi in the relative width of the anterior organ and ventral sucker (VSW = AOW), in the presence of three groups of pre-oral spines with an overlap in the number of spines in each lateral group (5 vs 4 6), in the spination of the tail stem and furcae, and in the number and shape of the caudal bodies (6 pairs with smooth contours) but differs in the resting position (tail stem bent vs straight), and in the arrangement of the transverse rows of spines on body (row 10 discontinuous dorsally vs last 1 2 transverse rows discontinuous ventrally) (Table 2). Metrical data for fixed cercariae vary within overlapping ranges for the length of the body ( vs lm in Diplostomum sp. Lin. 6I and D. scudderi, respectively), tail stem ( vs lm) and furcae ( vs lm), the size of the anterior organ ( vs lm) and the width of the ventral sucker (26 33 vs lm). A comparison with the metrical data for fixed metacercariae of D. scudderi revealed similar dimensions for the length of the body (mean 401 vs 413 lm in Diplostomum sp. Lin. 6I ) and the width of the holdfast organ (mean 103 vs 108 lm). However, the metacercaria of Diplostomum sp. Lin. 6I is characterised by on average larger oral sucker (mean width 62 vs 50 lm) and pharynx (mean vs lm) and a smaller ventral sucker (mean width 67 vs 73 lm) (see Hoffmann & Hundley, 1957). The cercaria of Diplostomum sp. Lin. 6I further resembles the cercaria of D. adamsi in the number of transverse rows (rows 1 to 6) of spines forming complete circles on body, in the number of rows of spines on the ventral sucker (3 rows, with a fourth row partly formed) but differs in: (i) the relative width of the anterior organ and ventral sucker (VSW = AOW vs VSW[ AOW); (ii) the number of transverse rows on body (11 vs 9); (iii) the number of spines on ventral sucker (75 82 vs c.108); and (iv) the resting position (tail stem bent vs straight). Both live and fixed cercariae of Diplostomum sp. Lin. 6I exhibit a shorter body ( vs lm and vs 232 lm, respectively), tail stem ( vs lm and vs 318 lm, respectively) and furcae ( vs lm and vs 292 lm, respectively) and a larger ventral sucker (width vs lm; live cercariae) (see Lester & Huizinga, 1977). No comparative data for the metacercariae of D. adamsi are available. Relationships with published isolates revealed by ITS1 rdna sequence analyses The tree inferred from NJ analysis of ITS1 sequences in Fig. 5 will be used for illustrative purposes for a discussion of the patterns and relationships inferred in Blasco-Costa et al. (2014) taking into consideration

16 210 Syst Parasitol (2014) 89: GQ Diplostomum sp. 1 L GQ D. indistinctum L GQ D. indistinctum L AY043 D. indistinctum Ga GQ Diplostomum sp. 1 L GQ Diplostomum sp. 1 L KJ SAH1 KJ GAFP1 JX D. spathaceum RAH6 Ge JX D. spathaceum LAG1 Ge JX D. spathaceum LCT1 Ge GQ Diplostomum sp. 3 L AF D. pseudospathaceum N & L JX D. pseudospathaceum LCT4 Ge JX D. pseudospathaceum LSH1 Ge JX D. pseudospathaceum LCT3 Ge GQ Diplostomum sp. 9 L 77 GQ Diplostomum sp. 8 L KJ RPNH3 KJ RPNH2 KJ RPNH5 KJ RPNH1 KJ GANH14 KJ GANH12 KJ GANH16 KJ RPNH4 KJ GANH18 99 GQ Diplostomum sp. 4 L GQ Diplostomum sp. 4 L GQ Diplostomum sp. 4 L 90 GQ D. huronense L GQ D. huronense L GQ D. huronense Ga JF D. cf. pseudospathaceum R JF D. cf. pseudospathaceum R KJ STH9 KJ STH2 KJ GAH3 KJ RPR1 JF D. cf. pseudospathaceum R JF D. cf. pseudospathaceum R 85 KJ STH3 KJ GAH6 AY042 D. baeri Ga GQ Diplostomum sp. 2 L AF D. baeri N & L JX D. baeri GGR1 Ge JX D. baeri STL1 Ge KJ GAH10 KJ GANH8 KJ SAH3 89 KJ GANH9 KJ RPNH3 KJ RPNH1 KJ SAH5 JF D. cf. baeri R JF D. cf. baeri R KJ GANH7 87 JQ D. baeri B-G KJ STH18 KJ RPNH2 KJ STH12 JF D. cf. baeri R KJ SAH6 KJ GANH6 JX D. baeri STL3 Ge JF D. cf. baeri R JF D. cf. baeri R 87 JF D. cf. baeri R KJ STH20 KJ STH19 KJ SAH10 KJ SAH11 KJ SAH13 AF D. parviventosum N & L JQ D. mergi RA97 B-G AF D. spathaceum N & L AF D. spathaceum N & L AF D. parviventosum N & L JF D. cf. parvi ventosum/spathaceum R JF D. cf. parviventosum/spathaceum R JX D. mergi 1 RAH1 Ge 100 JX D. mergi 3 GGR3 Ge JX D. mergi 3 STR10 Ge AF D. mergi N & L JX D. mergi D13 H 87 JF D. cf. mergi R JF D. cf. mergi R JF D. cf. mergi R JX D. mergi 2 RAH3 Ge JX D. mergi D14 H Lineage 1 (D. spathaceum) Lineage 6 Lineage 2 Lineage 3 + Lineage 4 Lineage 5 Clade Q D. mergi complex