Syst Parasitol (2014) 88:153–166 DOI 10.1007/s11230-014-9495-2
Phenotypic plasticity in Caryophyllaeus brachycollis Janiszewska, 1953 (Cestoda: Caryophyllidea): does fish host play a role? Daniel Barcˇa´k • Mikula´sˇ Oros • Vladimı´ra Hanzelova´ • Toma´sˇ Scholz
Received: 13 March 2014 / Accepted: 16 April 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Recent molecular phylogenetic studies on fish tapeworms of the genus Caryophyllaeus Gmelin, 1790 (Cestoda: Caryophyllidea), parasites of cyprinid fishes in the Palaearctic Region, have revealed unexpected phenotypic plasticity that seems to be related to definitive hosts. In the present paper, Caryophyllaeus brachycollis Janiszewska, 1953 is redescribed and its two morphotypes are circumscribed on the basis of newly-collected specimens. Morphotype 1 from barbels [Barbus spp. including the type-host Barbus barbus (L.); Barbinae] and chubs (Squalius spp.; Leuciscinae) is characterised by a more robust body with spatulate scolex, which is only slightly wider than a very short neck region, and the anterior position of the testes and vitelline follicles, which begin immediately posterior to the scolex. Specimens of Morphotype 2 from breams (Abramis spp., Ballerus spp. and Blicca spp.; Abraminae), which have been previously misidentifed as Caryophyllaeus laticeps (Pallas, 1781), possess a more slender body with a flabellate scolex, which is much wider than a long neck, and the first testes begin at a considerable distance posterior to D. Barcˇa´k M. Oros (&) V. Hanzelova´ Institute of Parasitology, Slovak Academy of Sciences, Hlinkova 3, 04001 Kosˇice, Slovakia e-mail: [email protected] T. Scholz Institute of Parasitology, Biology Centre of the Academy of Science of the Czech Republic, Branisˇovska´ 31, 370 05 Cˇeske´ Budeˇjovice, Czech Republic
the first vitelline follicles. Despite conspicuous differences in the scolex morphology and the anterior extent of the testes and vitelline follicles, both morphotypes are identical in the morphology of the posterior end of the body, in particular that of the cirrus-sac, which is large, thick-walled, elongate-pyriform, and contains a long cirrus, and in the distribution of the vitelline follicles, which surround medially vas deferens near the cirrus-sac. A specimen of Morphotype 1 from B. barbus from the Argens River, France, is designated as neotype of C. brachycollis. The presence of phenotypic plasticity in morphological characteristics previously used for differentiation of species of Caryophyllaeus may confound species identification, which is crucial for biodiversity, ecological and evolutionary studies. To avoid these potential problems, combination of morphological and molecular data is strongly recommended.
Introduction Phenotypic plasticity in parasites may represent a serious obstacle to their reliable identification based on morphological characteristics only. A recent study by Bazsalovicsova´ et al. (2014) has detected an unexpectedly high extent of morphological polymorphism of monozoic (i.e. without external and internal segmentation) tapeworms of the genus Caryophyllaeus Gmelin, 1790 (Cestoda: Caryophyllidea), parasites of cyprinid fishes in the Palaearctic Region (Schmidt, 1986;
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Protasova et al., 1990; Mackiewicz, 1994). Several morphotypes from different fish hosts, such as the white-eyed bream Ballerus sapa (Pallas), the Macedonian vimba Vimba melanops (Heckel) and the common nase Chondrostoma nasus (L.), were at first considered to represent new species by the present authors (unpubl. data). However, they seem to represent distinct, hostspecific morphotypes of the polymorphic Caryophyllaeus laticeps (Pallas, 1781) parasitising a wide spectrum of cyprinid fishes as indicated by Bazsalovicsova´ et al. (2014) using genetic markers [partial sequences of the mitochondrial cytochrome c oxidase subunit I (cox1) and partial large subunit of the ribosomal DNA (lsrDNA)]. Molecular data have also revealed that Caryophyllaeus tapeworms from breams, namely freshwater bream Abramis brama (L.), zope Ballerus ballerus (L.), white-eyed bream Ballerus sapa and white bream Blicca bjoerkna (L.), with a large, thick-walled cirrussac and a flabellate scolex, previously identified as C. laticeps, represent in fact one of the two morphotypes of Caryophyllaeus brachycollis Janiszewska, 1953. This tapeworm was described from four species of cyprinid fishes of the subfamily Leuciscinae from Poland by Janiszewska (1953). So far, it has been reported from cyprinids of the subfamilies Alburninae [Alburnus alburnus (L.)], Barbinae [Barbus barbus (L.), B. lacerta Heckel, B. meridionalis Risso, B. petenyi Heckel, B. tauricus Kessler and B. tyberinus Bonaparte], Cyprininae (Cyprinus carpio L.) and Leuciscinae [Leuciscus idus (L.), Rutilus pigus (Lace´pe`de), R. rutilus (L.), R. virgo (Heckel), Squalius cephalus (L.), Vimba vimba (L.) and V. melanops] in Europe (Austria, Bulgaria, Czech Republic, France, Hungary, Italy, Serbia, Slovakia, and the Ukraine) and Palaearctic Asia (China, Iraq and Russia) (Janiszewska 1953, 1954; Chen, 1973; Scholz, 1989; Protasova et al., 1990; Moravec et al., 1997; Cakic et al., 1998; Kirin, 2001; Moravec, 2001; Pojman´ska & Cielecka, 2001; De Liberato et al., 2002; Rahemo & Mohammad, 2004; Oros & Hanzelova´, 2009). This cestode has been typified by the possession of a large, thick-walled cirrus-sac and a spatulate scolex followed by a very short neck, and the anterior position of both the testes and vitelline follicles (see key to the species of Caryophyllaeus in Dubinina, 1987; Scholz, 1989; Protasova et al., 1990). Since new materials of both morphotypes of C. brachycollis were collected, the present paper provides redescription of
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this polymorphic species and characterises morphologically the two morphotypes, which were circumscribed genetically by Bazsalovicsova´ et al. (2014).
Materials and methods The present study was based on morphological evaluation of the specimens from museum collections and newly-collected materials partly used for a molecular phylogenetic study by Bazsalovicsova´ et al. (2014); detailed list of materials studied is provided in the taxonomic summary below. Specimen voucher from which the molecular sample was derived in the recent Bazsalovicsova´ et al.’s (2014) companion paper is specified as hologenophore, and conspecific specimen voucher collected together with the molecular specimen is specified as paragenophore (Astrin et al., 2013). Freshly collected tapeworms were processed using standardised methodology as described in detail by Oros et al. (2010). Briefly, live tapeworms from fresh hosts were washed and immediately fixed with hot 4% formaldehyde solution for morphological studies, stained with acetocarmine or Mayer’s carmine, dehydrated in an ethanol series, cleared in eugenol, and mounted in Canada balsam as permanent preparations (whole mounts). Illustrations were made using a drawing attachment of Leica DM 5000B light microscope and photomicrographs were digitally captured with a Leica DFC 450C camera mounted on a Leica DM 5000B light microscope with Differential Interference Contrast (DIC). Measurements were taken using the LAS V3.8Ink (Leica) program. Histological sections (cross and longitudinal sections) were made from formalin-fixed specimens using standard protocols (thickness of sections 10–12 lm), stained with Weigert’s hematoxylin and eosin, and mounted in Canada balsam. Two specimens from Abramis brama from the Latorica River, Slovakia (Morphotype 2) and two specimens from Barbus meridionalis from the Myslavsky´ stream in Kosˇice, Slovakia (Morphotype 1), were used for scanning electron microscopy (SEM) observations; they were processed as described by Oros et al. (2010). Measurements are in micrometres (lm) unless otherwise indicated; terminology of microtriches follows Chervy (2009). Acronyms of helminthological collections are as follows: Institute of Parasitology, BC ASCR, Cˇeske´
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Budeˇjovice, Czech Republic (acronym IPCAS), Natural History Museum, Geneva, Switzerland (MHNGPLAT), Natural History Museum, London, UK (BMNH), Institute of Parasitology, SAS, Kosˇice, Slovakia (PISAS), and Zoological Institute of the Ukrainian Academy of Sciences, Kiev, Ukraine (ZIK). The scientific and common names of fish hosts follow FishBase (Froese & Pauly, 2014).
Caryophyllaeus brachycollis Janiszewska, 1953 Type-host: Barbus barbus (L.) (Cyprinidae, Barbinae) (see Remarks). Other hosts: Hosts verified by the present study are marked with asterisks (Morphotype 1, one asterisk; Morphotype 2, two asterisks): Alburnus alburnus (L.) (Alburninae); *Barbus barbus (L.), *B. cyclolepis Heckel, B. lacerta Heckel, *B. meridionalis Risso, B. petenyi Heckel, B. tauricus Kessler, B. tyberinus Bonaparte (Barbinae), Cyprinus carpio (L.) (Cyprininae), **Abramis brama (L.), **Ballerus ballerus (L.), **B. sapa (Pallas), **Blicca bjoerkna (L.), **Leuciscus aspius (L.), **L. idus (L.), Rutilus pigus (Lace´pe`de), R. rutilus (L.), R. virgo (Heckel), */**Squalius cephalus (L.), *S. orpheus Kottelat & Economidis (new host record), Vimba vimba (L.), V. melanops (Heckel) (Leuciscinae) (all Cypriniformes: Cyprinidae). Type-locality: Argens River near Marseille, France. Type-material: Neotype (hologenophore – TS-07/85) from Barbus barbus (host coll. number BB29), Argens River near Marseille, France, June 2007, collected by M. Da´vidova´ and A. Sˇimkova´ (IPCAS C-51/5). Site of infection: Anterior intestine. Intermediate host: Limnodrilus hoffmeisteri Clapare`de (Oligochaeta: Tubificidae) (Janiszewska, 1954). Distribution: Records verified by the present authors are marked with asterisks (Morphotype 1, one asterisk; Morphotype 2, two asterisks; see Fig. 5): Europe (**Austria, *Bulgaria, */**Czech Republic, **Finland, *France, Hungary, *Italy, *Portugal, **Russia, Serbia, */**Slovakia, and */**Ukraine) and Palaearctic Asia (Iraq, Russia and China). Sequence data: KF051092–KF051100, KF051102– KF051115, KF051117, KF051118, KF051121– KF051124, KF051126–KF051160, KF051162, KF051163, KF051166–KF051170, KF051173–
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KF051181 (for more details, see table 2 in Bazsalovicsova´ et al., 2014). Material studied: Morphotype 1 (see also tables 1, 2 in Bazsalovicsova´ et al., 2014): (i) Specimens from museum collections: 5 specimens from B. barbus, Danube and Dniester Rivers, Ukraine, 1951 & 1965 (ZIK; not numbered; host Nos. 89 & 139) and Brenta River, Italy, 1999 (IPCAS C-3/5); 3 specimens from B. cyclolepis, Arda River, Bulgaria, 2010 (PISAS Nos. BG 47/10 & 83/10); 8 specimens from B. meridionalis, Porto, Portugal (IPCAS C-51/1); 1 specimen from B. petenyi, Bulgaria, 1953 (ZIK; not numbered; host No. 103-20); 37 specimens from S. cephalus, Brenta and Orta Rivers, Italy, 1991 & 2001 (BMNH 20003.9.20.11; IPCAS C-51/3; MHNGPLAT 88064), Becˇva and Rokytna´ Rivers, Czech Republic, 1962 & 1986 (IPCAS C-51/3) and Dniester River, Ukraine, 1958 (ZIK; not numbered). (ii) Newly-collected specimens: 11 specimens from B. barbus, Argens, Cassole and Saˆone Rivers, France, 2004 & 2007 (IPCAS C-51/5; MHNG-PLAT 88065); 12 specimens from B. meridionalis, Myslavsky´ stream, Kosˇice, Slovakia, 2005 (hologenophore PISAS SR 2A); 2 specimens from S. orpheus, Arda River, Bulgaria, 2010 (hologenophore PISAS BG 33/10). Morphotype 2 (see also tables 1, 2 in Bazsalovicsova´ et al., 2014): (i) Specimens from museum collections: 5 specimens from A. brama, Saravesi Lake, Finland, 1986 (BMNH 1986.6.26.8–14), Vltava River, Czech Republic, 1986–1988 (IPCAS C-2/1), Dniester River, Ukraine, 1958 (ZIK; not numbered); 1 specimen from L. aspius, Northern Donets, Ukraine (ZIK; not numbered; host No. 159-3); 4 specimens from S. cephalus, Rokytna´ River, Czech Republic, 1986 and Latorica River, Slovakia, 2007 & 2008 (IPCAS C-51/3). (ii) Newly-collected specimens: 71 specimens from A. brama, Latorica and Tisa Rivers, Slovakia, 2008, 2009, and Cheboksary Reservoir, Volga River, Russia, 2008 & 2009 (paragenophores PISAS SR 33/08, 37/08, 59/08, 60/08, 15/09, 17/09, 19/09, 31/09, 33/09; MHNGPLAT 88066); 10 specimens from B. ballerus, Latorica River, Slovakia, 2009 (paragenophores PISAS SR 7/09, 15/09) and Rybinsk Reservoir, Volga River, Russia, 2009 (paragenophore PISAS RU 7/09); 2 specimens from B. sapa, Latorica and Tisa Rivers, Slovakia, 2004 & 2008 (paragenophores PISAS SR 192/04, 193/04, 33/08,
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59/08); 9 specimens from B. bjoerkna, Cheboksary Reservoir, Volga River, Russia, 2009 (hologenophores PISAS RU 63/09, 119–120/09; paragenophores 165–170/09; MHNG-PLAT 88067); 2 specimens from L. idus, Latorica River, Slovakia, 2007 (PISAS 47/07-3); 2 specimens from S. cephalus, Latorica River, Slovakia, 2008 (PISAS 119/08). Description of Morphotype 1 (Figs. 1–4) [Based on 6 specimens ex B. barbus and B. meridionalis from France and Slovakia; measurements from the neotype in parentheses; see also Table 1.] Caryophyllidea, Caryophyllaeidae. Body more robust, with maximum width 0.8–1.3 mm (1.1 mm) at cirrus-sac region, narrowing towards posterior end. Scolex afossate, 0.9–1.6 mm (1.0 mm) long, spatulate with tapered anterior edge, 1.2–2.1 mm (1.9 mm) wide, only slightly wider than neck, 0.8–0.9 mm (0.9 mm) wide (Figs. 1B–D, 4A). Width of neck represents 43–69% (47%) of scolex width. Anterior edge of scolex with numerous slight indentations and superficial grooves (wrinkles) on its surface. Surface covered uniformly with acicular filitriches (Fig. 4D). Inner longitudinal musculature well developed, formed by small bundles of muscle fibres (Fig. 3A–C). Testes medullary, about 170–190 in number (in some specimens, precise number difficult to count because of extensive vitelline follicles covering testes), oval to almost spherical, 88–203 9 80–196 (n = 100) (118–135 9 111–122), intermingled with vitelline follicles. Distance of anteriormost testes from anterior extremity short, 0.8–2.0 mm (1.0 mm) (Fig. 1B–D). First testes begin at same level, in some cases even anterior (Fig. 1B) or slightly posterior to first vitelline follicles (Fig. 1C, D). Posteriorly testes reach to anterior margin of cirrus-sac (Fig. 2A, B). Testicular field 7.3–20.2 mm long. Vas deferens well developed, in distal (posterior) part surrounded medially by vitelline follicles. Cirrussac large, elongate to ovoid, thick-walled, 1,075–1,227 9 518–641 (1,089 9 570), containing strongly muscular, long cirrus (Figs. 2A, B, 3A, B); width of cirrus-sac represents 54–74% (54%) of body width at cirrus-sac region. External and internal seminal vesicle absent. Ovary follicular, H-shaped, 721–903 (884) wide, arms 1,010–1,403 (1,158–1,224) long and 194–537 (235–251) wide (Fig. 2A, B). Vagina tubular, situated ventral to uterus, proximal (posterior) part sinuous, distal (anterior) part almost straight, joining uterus to
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form short common uterovaginal duct. Receptaculum seminis situated anterodorsal to ovarian isthmus, almost spherical to oval, 300 9 196 (294 9 197) (Fig. 2B). Preovarian vitelline follicles numerous, lateral and median, surrounding testes and vas deferens, including its distal (posterior) part; follicles irregularly-shaped and variable in size, 72–140 9 59–135 (n = 100) (111–120 9 77–85). Anteriormost follicles begin at short distance (0.8–1.5 mm) (0.8 mm) posterior to anterior edge of scolex (Fig. 1B–D). Vitelline follicles reach posteriorly up to level of anterior or middle part of ovarian arms, absent alongside ovary. Postovarian follicles present (Fig. 2A, B). Uterus forms several loops between ovary and level of middle or posterior part of cirrus-sac (Fig. 2A, B); preovarian part surrounded by numerous glands. Uterus occupies region 1.7–2.5 mm long (2.3 mm), 9–12% of testicular field. Eggs oval, operculate, unembryonated, smooth, 66–75 9 41–44 (66–72 9 41–43) (n = 15, intrauterine eggs on whole-mounts). Genital pores separated, close to each other, female pore opens posterior to male pore (Figs. 3B, 4C). Description of Morphotype 2 (Figs. 1–4) [Based on 44 specimens ex A. brama, B. ballerus and B. bjoerkna from Russia and Slovakia; measurements in Table 1.] Caryophyllidea, Caryophyllaeidae. Body more slender, elongate, 12–32 mm, with maximum width 0.5–1.0 mm at cirrus-sac region, narrowing towards posterior end. Scolex afossate, 1.1–1.9 mm long, flabellate, with variously curved anterior margin, 1.3–2.8 mm wide, significantly wider than neck, 0.4–0.9 mm wide (Figs. 1F–H, 4B). Width of neck represents 24–49% of scolex width. Anterior edge of scolex with numerous slight indentations and superficial grooves (wrinkles) on its surface. Surface covered uniformly with acicular filitriches. Inner longitudinal musculature well developed, formed by small bundles of muscle fibres. Testes medullary, about 140–450 in number (in some specimens, precise number difficult to count because of extensive vitelline follicles covering testes), oval to almost spherical, 75–201 9 68–183 wide (n = 100), intermingled with vitelline follicles. Distance of anteriormost testes from anterior extremity moderate, 2.6–8.3 mm (Fig. 1F–H). First testes begin at much more posterior (up to 4.3 mm) to first vitelline follicles (Fig. 1F, H). Posteriorly testes reach to anterior margin of cirrus-sac (Fig. 2C). Testicular field 7.4–20.2 mm
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Table 1 Measurements of individual morphotypes of Caryophyllaeus brachycollis Janiszewska, 1953 from different hosts and geographical regions (main morphological differences between the two morphotypes in bold) Character / Morphotype Host Locality
Body length (mm) (A)
Morphotype 1
Morphotype 1
Morphotype 2
Morphotype 2
Morphotype 2
Morphotype 2
Barbus barbus France (n = 5)
Barbus meridionalis Slovakia (n = 1)
Abramis brama Slovakia (n = 9)
Abramis brama Russia (n = 14)
Ballerus ballerus Russia (n = 1)
Blicca bjoerkna Russia (n = 2)
11–26
–
12–27
15–26
32
22–23
form
robust
robust
slender
slender
slender
slender
width
853–1,255
997–1,246
534–1,031
514–1,177
1,083
spatulate
spatulate
flabellate
flabellate
flabellate
flabellate
length
908–1,561
1,003
1,072–1,661
1,194–1,847
1,740
1,309–1,526
width
1,198–2,072
1,402
1,449–2,789
1,287–2,366
2,223
1,652–1,773
537–896
406–673
937
597–866
Scolex type
Neck width ratio to scolex width
885
823–892
770
43–69%
55%
28–42%
24–49%
42%
36–49%
88–203 9 80–196
117–140 9 95–140
75–178 9 68–147
92–201 9 78–183
156–173 9 134–155
153–166 9 113–140
number
170–190
–
230–420
140–450
290
215–240
distance from first vitelline follicle (mm)
0.14–0.56
0.06
0.4–1.9
0.8–3.0
4.3
1.1–1.3
distance from anterior extremity (mm)
0.97–2.02
0.8
2.6–3.8
3.1–5.5
8.3
3.4–3.9
reach up to
ant. to post. part of CS
ant. to post. part of CS
ant. to middle part of CS
ant. part of CS
ant. part of CS
ant. part of CS
length of testicular field (mm)
7.3–20.2
–
7.4–20.2
10.5–18.8
19.0
15.2–16.4
1,075–1,227 9 518–641
1117 9 571
537–1,098 9 311–651
567–982 9 318–567
1,093 9 646
635 9 399
64%
54–64%
43–62%
60%
40%
791
821–1,091
Testes size
Cirrus-sac size extent in relation to body width Ovary width
54–74%
445–924
433–1,093
829
length of ovarian arms
721–903 1,150–1,403
1,010–1,047
834–1,766
815–1,747
1,620–1,651
1,113–1,176
width of ovarian arms
204–537
194–241
113–304
117–252
235–271
178–277
distance from posterior end (B)
2,265–2,951
2,601
1,264–2,389
1,450–2,120
2,101
1,920–2,019
Proportion (B) : (A)
8–14%
–
7–9%
8–9%
7%
8–9%
Size of vitelline follicles
72–125 9 59–100
104–140 9 83–135
47–137 9 43–110
77–200 9 57–140
113–149 9 87–120
99–154 9 82–123
1.4–2.7
1.7–3.2
4.0
2.5–2.6
ant. to middle part of ovary
ant. to middle part of ovary
anterior part of ovary
anterior part of ovary
distance from anterior extremity (mm)
0.83–1.49
posterior extent of preovarian follicles
anterior part of ovary
Uterus extent (mm)
0.82 anterior part of ovary
1.3–2.9
1.6–2.9
3.5
2.1–2.3
9–12%
–
13–18%
14–19%
19%
14%
Size of intrauterine egg
66–75 9 41–44
68–72 9 41–43
59–65 9 40–45
62–65 9 42–44
59–61 9 43–45
59–65 9 40–42
Receptaculum seminis
294–300 9 196–197
–
–
303–489 9 229–370
–
–
extent in relation to length of testicular area
1.7–2.5
1.9
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Fig. 1 Line drawings of Caryophyllaeus brachycollis Janiszewska, 1953 (A–D, Morphotype 1, B, hologenophore; E–H, Morphotype 2, G, paragenophore). A, E, Outlines of adults, note the indication of the first testes and vitelline follicles; B–D, F–H, Anterior part of the body with the first vitelline follicles and testes. Abbreviations: cs, cirrus-sac; fte, first testes; fvf, first vitelline follicles; ov, ovary. Scalebars: A, E, 1 mm; B–D, F–H, 500 lm
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long. Vas deferens well developed, in distal (posterior) part surrounded medially by vitelline follicles. Cirrus-sac large, elongate to ovoid, thick-walled, 537–1,098 9 311–651, containing strongly muscular, long cirrus (Fig. 2C); width of cirrus-sac represents 40–64% of body width at cirrus-sac region. External and internal seminal vesicle absent. Ovary follicular, H-shaped, 433–1,093 wide, with arms 815–1,747 long and 113–304 wide (Fig. 2C). Vagina tubular, situated ventral to uterus, proximal (posterior) part sinuous, distal (anterior) part almost straight, joining uterus to form short common uterovaginal duct. Receptaculum seminis situated anterodorsal to ovarian isthmus, almost spherical to oval, 303–489 9 229–370 (n = 5). Preovarian vitelline follicles numerous, lateral and median, surrounding testes and vas deferens, including its distal (posterior) part; follicles irregularly-shaped and variable in size, 47–200 9 43–140 (n = 100). Anteriormost follicles begin far, 1.4–4.0 mm, from anterior extremity (Fig. 1G, H). Vitelline follicles reach posteriorly up to level of anterior or middle part of ovarian arms, absent alongside ovary. Postovarian follicles present (Fig. 2C). Uterus forms several loops between ovary and level of middle or posterior part of cirrus-sac (Fig. 2C); preovarian part surrounded by numerous glands. Uterus occupies region 1.3–3.5 mm long, 13–19% of testicular field. Eggs oval, operculate, unembryonated, smooth, 59–65 9 40–45 (n = 30, intrauterine eggs on wholemounts). Genital pores separated, close to each other, female pores opens posterior to male pore (Fig. 2C). Remarks Janiszewska (1953) described C. brachycollis from four species of cyprinids of two subfamilies, B. barbus and B. petenyi (Barbinae), and S. cephalus and L. idus (Leuciscinae), from several rivers in Poland (Odra and Vistula with its tributaries Barycz, Raba and Ropa). However, she did not designate a type-host. Common barbel, B. barbus, was most heavily infected (prevalence 27%; n = 48; maximum intensity 7 tapeworms). Moreover, type-specimens were not designated nor their deposition was indicated in the original description. Subsequent search for the types of C. brachycollis was unsuccessful and all our enquiries indicate that the type-material is not extant. Therefore, we designate one of the specimens from B. barbus (this
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fish host was listed first in the original description) from the Argens River near Marseille, France, deposited at the Institute of Parasitology, BC ASCR, Cˇeske´ Budeˇjovice, Czech Republic (IPCAS C-51/5), as the neotype of C. brachycollis. Since its original description in 1953, the species has been reported from several European countries including the European part of Russia, whereas records from Asia are rare and require verification (vouchers of tapeworms reported are not available). This concerns the record of C. brachycollis from Barbus lacerta in Iraq by Rahemo & Mohammad (2004), because Khawia armeniaca (Cholodkovsky, 1915), which possesses a similar scolex, occurs frequently in barbels in Iraq (Scholz et al., 2011), and the record from common carp Cyprinus carpio in Huangsha Lake, Hubei Province in China by Chen (1973). Janiszewska (1954) reported a procercoid (= plerocercoid according to Chervy, 2002) of C. brachycollis from Limnodrilus hoffmeisteri Clapare`de from the Odra River in Poland. Caryophyllaeus brachycollis was easily differentiated from congeneric species by scolex shape and the anterior extent of the first testes that begin at the same level or just slightly posterior to the first vitelline follicles (see Dubinina, 1987; Scholz, 1989; Protasova et al., 1990). However, molecular data provided recently by Bazsalovicsova´ et al. (2014) have shown that the species is polymorphic and individuals from leuciscine fishes, especially breams (Abramis, Ballerus and Blicca), which were previously identified as Caryophyllaeus laticeps, represent a separate morphotype of C. brachycollis. This morphotype, called herein C. brachycollis Morphotype 2, differs from the ‘nominotypical’ morphotype (called C. brachycollis Morphotype 1 herein) mainly in the following characteristics: (i) the shape of the scolex, which is wider and flabellate (vs spatulate in Morphotype 1); (ii) the length of the neck, which is much longer in Morphotype 2 (vs very short in Morphotype 1 as expressed in the species name); (iii) a much more posterior extent of the testes and vitelline follicles from the scolex (the testes in Morphotype 1 begin at the same level as the first vitelline follicles or only slightly more posterior; both begin just posterior to the scolex); (iv) the anterior edge of the scolex with numerous shallow superficial grooves or wrinkles (vs almost smooth or with only a few wrinkles in Morphotype 1); (v) a more slender,
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Fig. 2 Line drawings of Caryophyllaeus brachycollis Janiszewska, 1953, posterior part of the body. (A, B, Morphotype 1, ventral view; C, Morphotype 2, dorsal view). Note large, elongate, thick-walled cirrus-sac. Abbreviations: cs, cirrus-sac; mgp, male gonopore; ov, ovary; pvf, postovarian vitelline follicles; rs, receptaculum seminis; te, testes; ug, uterine glands; ut, uterus; uvp, uterovaginal pore; va, vagina; vd, vas deferens; vf, vitelline follicles. Scale-bars: 500 lm
longer body (vs body more robust in relation to its total length) (see Figs. 1–4; Table 1). However, both morphotypes of C. brachycollis share the identical morphology of the posterior part of the body, in particular the possession of a large, elongate ovoid, thick-walled cirrus-sac (its width represents 40–74% of body width) containing a long, strongly muscular cirrus of lamellar structure
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(Fig. 3A, B), and the median position of the vitelline follicles at the level of the distal (posterior) part of the external sperm duct (vas deferens) (Fig. 2). The morphology and structure of the cirrus-sac were uniform in all C. brachycollis specimens and both morphotypes that occur in a number of fish hosts within a large distribution area (Fig. 5). Therefore, the cirrus-sac is considered to represent a taxonomically
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important structure suitable for circumscription of this species. Caryophyllaeus laticeps, which may occur quite commonly with C. brachycollis in co-infections, differs from the latter species in the morphology of the posterior end of the body, especially in the possession of a small, subspherical, thin-walled cirrus-sac containing a well-developed internal seminal vesicle, and in the absence of vitelline follicles medially at the level of posterior loops of vas deferens (external sperm duct). This absence of vitelline follicles alongside the median line of the body makes the region with vas deferens to appear conspicuous (see figures 2J and 2M in Scholz, 1989). Mixed infections with both C. laticeps and Morphotype 2 of C. brachycollis were detected in four species of leuciscine cyprinids from the Latorica and Tisa Rivers in eastern Slovakia and the Volga River in the European part of Russia, namely A. brama (percentage of C. laticeps vs C. brachycollis specimens 79%/21%, n = 333 specimens counted), B. ballerus (79%/21%, n = 33), B. sapa (90%/10%, n = 21) and B. bjoerkna (44%/ 56%, n = 16). In total, C. laticeps tapeworms represented 78% of the specimens counted, whereas those of C. brachycollis Morphotype 2 represented only 22%. This proportion differed slightly between individual localities (Latorica, 74%/26%, n = 105; Tisa, 85%/15%, n = 122; Volga, 77%/23%, n = 176). In the possession of a large, thick-walled cirrus-sac, C. brachycollis is almost indistinguishable from C. fimbriceps Annenkova-Khlopina, 1919 (see figures 2L and 2M in Scholz, 1989). This species was described from common carp C. carpio in Russia and then reported as a carp parasite of veterinary importance in the former USSR and eastern Europe, causing mortalities of heavily infected fish (Bauer et al., 1973). Both species differ allegedly from each other by the morphology of the scolex, which is cuneicrispitate in C. fimbriceps (flabellate with the fimbriate anterior margin; see figure 2D, E in Scholz, 1989 and figure 1I in Oros et al., 2010). However, some C. brachycollis tapeworms of Morphotype 2 possess scolex resembling that of C. fimbriceps, which has also been reported from breams (Leuciscinae) (Protasova et al., 1990). It is obvious that the independent status of the latter species has to be confirmed, preferably by molecular data. Unfortunately, no ethanol-fixed material of C. fimbriceps has been available and the species seems to have disappeared globally, possibly as a result of the
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introduction of another caryophyllidean cestode specific to carp, Khawia sinensis Hsu¨, 1935, to the European part of the former USSR and Eastern Europe (Oros et al., 2009). Morphological and molecular evaluation of new material of C. fimbriceps, which is currently absent, may reveal its conspecificity with C. brachycollis.
Discussion Classification, systematics and species identification of helminths, including fish cestodes, have been largely based on morphological characters (Wardle & McLeod, 1952; Schmidt, 1986; Khalil et al., 1994). Since the late 1970’s, cestode systematics has benefited from the application of phylogenetic systematics (cladistics), first applied in cestodes by Brooks (1978) and later also used for the assessment of phylogenetic relationships of major groups of tapeworms, including those parasitising teleost fishes (Hoberg et al., 1997, 1999; Rego et al., 1998; Oros et al., 2008). Although these studies represented a valuable contribution to our understanding of cestode relationships, they suffered from a high degree of homoplasy of morphological characters used; this decreased reliability of the conclusions inferred from morphology-based phylogenies. The application of DNA-based genetic markers in phylogenetic systematics, which started in cestodology in the late 1990’s (Mariaux, 1998; Olson & Caira, 1999; Zehnder & Mariaux, 1999; Kodedova´ et al., 2000) represented even a more important step forward to our better understanding of the evolutionary history of cestodes and their phylogenetic relationships (Olson et al., 2001; Olson & Tkach, 2005; Brabec et al., 2006; Waeschenbach et al., 2007, 2012; Littlewood, 2008). Genetic markers also made it possible to assess more reliably intraspecific variability, which resulted in detecting cryptic diversity in some groups of parasitic flatworms (Doanh et al., 2009), but also in revealing unexpected phenotypic plasticity in others (Blasco-Costa et al., 2010; Miller & Cribb, 2013; Bazsalovicsova´ et al., 2014). Phenotypic plasticity is defined as the property of a given genotype to produce different phenotypes in response to distinct environmental conditions (Pigliucci, 2001). In parasites, this ability of a genotype to present distinct phenotypic features can be influenced by physiological,
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Fig. 3 Histological sections of Caryophyllaeus brachycollis Janiszewska, 1953 from Barbus barbus, Morphotype 1. A, B, Longitudinal sections of posterior part of body. Note large, elongate, thick-walled cirrus-sac (A) and separate genital pores (B); C, Cross-section. Abbreviations: cs, cirrus-sac; ilm, inner longitudinal musculature; mgp, male gonopore; oc, osmoregulatory canal; te, testes; ut, uterus; uvp, uterovaginal pore; va, vagina; vf, vitelline follicles. Scale-bars: A, B, 500 lm; C, 250 lm
biochemical, ecological and other differences between individual hosts. In fact, a high intraspecific morphological variation caused by phenotypic plasticity may represent a serious obstacle in parasite identification and can confound species differentiation based on morphological characters only. Bazsalovicsova´ et al. (2014) have demonstrated such a case of phenotypic plasticity in two polymorphic species of fish tapeworms, C. brachycollis and C.
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laticeps. Unexpectedly, some tapeworms from breams, previously identified as C. laticeps, represented in fact a morphogically distinct, but genetically closely related lineage of C. brachycollis. This species was considered to be morphologically quite uniform and restricted to barbels (Barbinae) and chub (Squalius cephalus) (Janiszewska, 1953; Scholz, 1989; Protasova et al., 1990). In congeneric C. laticeps, type-species of the genus, as many as five
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Fig. 4 Scanning electron micrographs of Caryophyllaeus brachycollis Janiszewska, 1953. A, Anterior part of body with scolex, Morphotype 1; B, Morphotype 2; C, Separate genital pores; D, Acicular filitriches (filiform microtriches). Abbreviations: mgp, male gonopore; uvp, uterovaginal pore. Scale-bars: A, B, 200 lm; C, 100 lm; D, 2 lm
morphotypes, most of them host-specific and originally supposed to represent new species by the present authors, were distinguished; molecular data confirmed that they all belong in fact to only one genetic lineage (Bazsalovicsova´ et al., 2014). In the present study, barbels (Barbinae) harboured only Morphotype 1 of C. brachycollis, whereas breams (Leuciscinae) were infected only with tapeworms of Morphotype 2. In chub (S. cephalus, Leuciscinae), both
morphotypes of C. brachycollis were found, which indicates that other factors than those related exclusively to host physiology and microanatomy of the intestinal tract may be involved in the phenotypic plasticity of C. brachycollis. Mechanisms of possible host-induced phenotypic plasticity in cestodes are not well known and further studies are necessary to provide their explanation. Experimental cross-infections of different fish with plerocercoids of both morphotypes
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Fig. 5 Distribution map of Caryophyllaeus brachycollis Janiszewska, 1953. Triangles, Morphotype 1; circles, Morphotype 2
of C. brachycollis from oligochaetes would help confirm the existence of this phenomenon. Intraspecific plasticity in the morphology of the anterior part of the body of species of Caryophyllaeus demonstrated by Bazsalovicsova´ et al. (2014) and by the present study indicates that taxonomic importance of morphological characteristics related to the scolex and the anterior extent of the testes and vitelline follicles in other groups of caryophyllidean cestodes should be critically assessed, preferably on the basis of combined morphological and molecular studies of adequately processed materials (see Oros et al., 2010). However, the present data do not necessarily question reliability of scolex morphology in the systematics and species diagnostics of caryophyllidean cestodes in general (see Oros et al., 2010 for comprehensive data on scolex morphology of Palaearctic taxa), just call for more attention to be paid when these morphological characteristics are used in individual groups of caryophyllidean cestodes. Results of Bazsalovicsova´ et al. (2014) and present study have shown that morphology of the cirrus-sac represents a key diagnostic character in the species of Caryophyllaeus. The cirrus-sac of caryophyllideans varies greatly in size (its width represents from onefifth to three-fourths of the body width) and is strongly muscular in species of most genera (Mackiewicz, 1972, 1994). Appearance of the cirrus-sac was uniform in all specimens of both morphotypes of C. brachycollis (168 specimens examined) from several fish hosts in a large distribution area and thus
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its morphology can be considered to be stable and species-specific. The present study, which represents a follow-up of that by Bazsalovicsova´ et al. (2014), clearly shows how it is important to combine morphological and molecular data to avoid misidentifications and false conclusions based on morphological characters only. Suitability of such a combined approach has also been proven in the taxonomic revision of the species of Khawia Hsu¨, 1935, especially in the assessment of the validity of two genetically closely related, but morphologically markedly different species from different fish hosts in China (Xi et al., 2009; Scholz et al., 2011; Kra´ˇlova´-Hromadova´ et al., 2012; Orosova´ & Oros, 2012). Khawia saurogobii Xi, Oros, Wang, Wu, Gao & Nie, 2009, a parasite of gudgeons (Saurogobio spp.; Gobiinae) in China, which is morphological markedly different from, but genetically closely related to, K. sinensis Hsu¨, 1935, might be an example of morphological divergence as a result of ongoing sympatric speciation by host switching (very narrow taxonomic range of hosts). In contrast, Morphotype 2 of C. brachycollis from breams shows a large distribution area in the Palaearctic (Fig. 5) and appears to represent only a distinct morphotype of euryxenous parasite with a wide spectrum of definitive hosts. Acknowledgements The authors thank two anonymous reviewers for helpful suggestions, to Diana Kirin, Agricultural University of Plovdiv, Bulgaria, Martina Da´vidova´ and Andrea Sˇimkova´, Masaryk University, Brno, Czech Republic, Larisa G. Poddubnaya, Institute of Inland Waters, Borok, Russia, Elga Tieri, Italy, and Bahram Dezfuli, University of Ferrara, Italy, for providing specimens of C. brachycollis from Bulgaria, France, Russia and Italy, respectively. This study was supported by the Slovak Research and Development Agency (projects nos. APVV-0653-11 and LPP 0171-09), Grant Agency VEGA (No. 2/0129/12), Institute of Parasitology (RVO: 60077344) and Czech Science Foundation (project No. P505/12/G112). The work was undertaken within the framework of the Research & Development Operational Programme funded by the ERDF (code ITMS: 26220120022) (0.5).
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Syst Parasitol (2014) 88:153–166 Schmidt, G. D. (1986). Handbook of tapeworm identification. Boca Raton, Florida: CRC Press. 675 pp. Scholz, T. (1989). Amphilinida and Cestoda, parasites of fish in Czechoslovakia. Praha: Academia, 56 pp. Scholz, T., Brabec, J., Kra´ˇlova´-Hromadova´, I., Oros, M., Bazsalovicsova´, E., Ermolenko, A., & Hanzelova´. (2011). Revision of Khawia spp. (Cestoda: Caryophyllidea), parasites of cyprinid fish, including a key to their identification and molecular phylogeny. Folia Parasitologica, 58, 197–223. Waeschenbach, A., Webster, B. L., Bray, R. A., & Littlewood, D. T. J. (2007). Added resolution among ordinal level relationships of tapeworms (Platyhelminthes: Cestoda) with complete small and large subunit nuclear ribosomal RNA genes. Molecular Phylogenetics and Evolution, 45, 311–325. Waeschenbach, A., Webster, B. L., & Littlewood, D. T. J. (2012). Adding resolution to ordinal level relationships of tapeworms (Platyhelminthes: Cestoda) with large fragments of mtDNA. Molecular Phylogenetics and Evolution, 63, 834–847. Wardle, R. A., & McLeod, J. A. (1952). The zoology of tapeworms. Minneapolis: University of Minnesota Press, 780 pp. Xi, B. W., Oros, M., Wang, G. T., Wu, S. G., Gao, D., & Nie, P. (2009). Khawia saurogobii n. sp. (Cestoda: Caryophyllidea) from freshwater fish Saurogobio spp. (Cyprinidae) in China. Journal of Parasitology, 95, 1516–1519. Zehnder, M. P., & Mariaux, J. (1999). Molecular systematic analysis of the order Proteocephalidea (Eucestoda) based on mitochondrial and nuclear rDNA sequences. International Journal for Parasitology, 29, 1841–1852.
Phenotypic plasticity in Caryophyllaeus brachycollis Janiszewska, 1953 (Cestoda: Caryophyllidea): does fish host play a role? Daniel Barcˇa´k • Mikula´sˇ Oros • Vladimı´ra Hanzelova´ • Toma´sˇ Scholz
Received: 13 March 2014 / Accepted: 16 April 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Recent molecular phylogenetic studies on fish tapeworms of the genus Caryophyllaeus Gmelin, 1790 (Cestoda: Caryophyllidea), parasites of cyprinid fishes in the Palaearctic Region, have revealed unexpected phenotypic plasticity that seems to be related to definitive hosts. In the present paper, Caryophyllaeus brachycollis Janiszewska, 1953 is redescribed and its two morphotypes are circumscribed on the basis of newly-collected specimens. Morphotype 1 from barbels [Barbus spp. including the type-host Barbus barbus (L.); Barbinae] and chubs (Squalius spp.; Leuciscinae) is characterised by a more robust body with spatulate scolex, which is only slightly wider than a very short neck region, and the anterior position of the testes and vitelline follicles, which begin immediately posterior to the scolex. Specimens of Morphotype 2 from breams (Abramis spp., Ballerus spp. and Blicca spp.; Abraminae), which have been previously misidentifed as Caryophyllaeus laticeps (Pallas, 1781), possess a more slender body with a flabellate scolex, which is much wider than a long neck, and the first testes begin at a considerable distance posterior to D. Barcˇa´k M. Oros (&) V. Hanzelova´ Institute of Parasitology, Slovak Academy of Sciences, Hlinkova 3, 04001 Kosˇice, Slovakia e-mail: [email protected] T. Scholz Institute of Parasitology, Biology Centre of the Academy of Science of the Czech Republic, Branisˇovska´ 31, 370 05 Cˇeske´ Budeˇjovice, Czech Republic
the first vitelline follicles. Despite conspicuous differences in the scolex morphology and the anterior extent of the testes and vitelline follicles, both morphotypes are identical in the morphology of the posterior end of the body, in particular that of the cirrus-sac, which is large, thick-walled, elongate-pyriform, and contains a long cirrus, and in the distribution of the vitelline follicles, which surround medially vas deferens near the cirrus-sac. A specimen of Morphotype 1 from B. barbus from the Argens River, France, is designated as neotype of C. brachycollis. The presence of phenotypic plasticity in morphological characteristics previously used for differentiation of species of Caryophyllaeus may confound species identification, which is crucial for biodiversity, ecological and evolutionary studies. To avoid these potential problems, combination of morphological and molecular data is strongly recommended.
Introduction Phenotypic plasticity in parasites may represent a serious obstacle to their reliable identification based on morphological characteristics only. A recent study by Bazsalovicsova´ et al. (2014) has detected an unexpectedly high extent of morphological polymorphism of monozoic (i.e. without external and internal segmentation) tapeworms of the genus Caryophyllaeus Gmelin, 1790 (Cestoda: Caryophyllidea), parasites of cyprinid fishes in the Palaearctic Region (Schmidt, 1986;
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Protasova et al., 1990; Mackiewicz, 1994). Several morphotypes from different fish hosts, such as the white-eyed bream Ballerus sapa (Pallas), the Macedonian vimba Vimba melanops (Heckel) and the common nase Chondrostoma nasus (L.), were at first considered to represent new species by the present authors (unpubl. data). However, they seem to represent distinct, hostspecific morphotypes of the polymorphic Caryophyllaeus laticeps (Pallas, 1781) parasitising a wide spectrum of cyprinid fishes as indicated by Bazsalovicsova´ et al. (2014) using genetic markers [partial sequences of the mitochondrial cytochrome c oxidase subunit I (cox1) and partial large subunit of the ribosomal DNA (lsrDNA)]. Molecular data have also revealed that Caryophyllaeus tapeworms from breams, namely freshwater bream Abramis brama (L.), zope Ballerus ballerus (L.), white-eyed bream Ballerus sapa and white bream Blicca bjoerkna (L.), with a large, thick-walled cirrussac and a flabellate scolex, previously identified as C. laticeps, represent in fact one of the two morphotypes of Caryophyllaeus brachycollis Janiszewska, 1953. This tapeworm was described from four species of cyprinid fishes of the subfamily Leuciscinae from Poland by Janiszewska (1953). So far, it has been reported from cyprinids of the subfamilies Alburninae [Alburnus alburnus (L.)], Barbinae [Barbus barbus (L.), B. lacerta Heckel, B. meridionalis Risso, B. petenyi Heckel, B. tauricus Kessler and B. tyberinus Bonaparte], Cyprininae (Cyprinus carpio L.) and Leuciscinae [Leuciscus idus (L.), Rutilus pigus (Lace´pe`de), R. rutilus (L.), R. virgo (Heckel), Squalius cephalus (L.), Vimba vimba (L.) and V. melanops] in Europe (Austria, Bulgaria, Czech Republic, France, Hungary, Italy, Serbia, Slovakia, and the Ukraine) and Palaearctic Asia (China, Iraq and Russia) (Janiszewska 1953, 1954; Chen, 1973; Scholz, 1989; Protasova et al., 1990; Moravec et al., 1997; Cakic et al., 1998; Kirin, 2001; Moravec, 2001; Pojman´ska & Cielecka, 2001; De Liberato et al., 2002; Rahemo & Mohammad, 2004; Oros & Hanzelova´, 2009). This cestode has been typified by the possession of a large, thick-walled cirrus-sac and a spatulate scolex followed by a very short neck, and the anterior position of both the testes and vitelline follicles (see key to the species of Caryophyllaeus in Dubinina, 1987; Scholz, 1989; Protasova et al., 1990). Since new materials of both morphotypes of C. brachycollis were collected, the present paper provides redescription of
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this polymorphic species and characterises morphologically the two morphotypes, which were circumscribed genetically by Bazsalovicsova´ et al. (2014).
Materials and methods The present study was based on morphological evaluation of the specimens from museum collections and newly-collected materials partly used for a molecular phylogenetic study by Bazsalovicsova´ et al. (2014); detailed list of materials studied is provided in the taxonomic summary below. Specimen voucher from which the molecular sample was derived in the recent Bazsalovicsova´ et al.’s (2014) companion paper is specified as hologenophore, and conspecific specimen voucher collected together with the molecular specimen is specified as paragenophore (Astrin et al., 2013). Freshly collected tapeworms were processed using standardised methodology as described in detail by Oros et al. (2010). Briefly, live tapeworms from fresh hosts were washed and immediately fixed with hot 4% formaldehyde solution for morphological studies, stained with acetocarmine or Mayer’s carmine, dehydrated in an ethanol series, cleared in eugenol, and mounted in Canada balsam as permanent preparations (whole mounts). Illustrations were made using a drawing attachment of Leica DM 5000B light microscope and photomicrographs were digitally captured with a Leica DFC 450C camera mounted on a Leica DM 5000B light microscope with Differential Interference Contrast (DIC). Measurements were taken using the LAS V3.8Ink (Leica) program. Histological sections (cross and longitudinal sections) were made from formalin-fixed specimens using standard protocols (thickness of sections 10–12 lm), stained with Weigert’s hematoxylin and eosin, and mounted in Canada balsam. Two specimens from Abramis brama from the Latorica River, Slovakia (Morphotype 2) and two specimens from Barbus meridionalis from the Myslavsky´ stream in Kosˇice, Slovakia (Morphotype 1), were used for scanning electron microscopy (SEM) observations; they were processed as described by Oros et al. (2010). Measurements are in micrometres (lm) unless otherwise indicated; terminology of microtriches follows Chervy (2009). Acronyms of helminthological collections are as follows: Institute of Parasitology, BC ASCR, Cˇeske´
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Budeˇjovice, Czech Republic (acronym IPCAS), Natural History Museum, Geneva, Switzerland (MHNGPLAT), Natural History Museum, London, UK (BMNH), Institute of Parasitology, SAS, Kosˇice, Slovakia (PISAS), and Zoological Institute of the Ukrainian Academy of Sciences, Kiev, Ukraine (ZIK). The scientific and common names of fish hosts follow FishBase (Froese & Pauly, 2014).
Caryophyllaeus brachycollis Janiszewska, 1953 Type-host: Barbus barbus (L.) (Cyprinidae, Barbinae) (see Remarks). Other hosts: Hosts verified by the present study are marked with asterisks (Morphotype 1, one asterisk; Morphotype 2, two asterisks): Alburnus alburnus (L.) (Alburninae); *Barbus barbus (L.), *B. cyclolepis Heckel, B. lacerta Heckel, *B. meridionalis Risso, B. petenyi Heckel, B. tauricus Kessler, B. tyberinus Bonaparte (Barbinae), Cyprinus carpio (L.) (Cyprininae), **Abramis brama (L.), **Ballerus ballerus (L.), **B. sapa (Pallas), **Blicca bjoerkna (L.), **Leuciscus aspius (L.), **L. idus (L.), Rutilus pigus (Lace´pe`de), R. rutilus (L.), R. virgo (Heckel), */**Squalius cephalus (L.), *S. orpheus Kottelat & Economidis (new host record), Vimba vimba (L.), V. melanops (Heckel) (Leuciscinae) (all Cypriniformes: Cyprinidae). Type-locality: Argens River near Marseille, France. Type-material: Neotype (hologenophore – TS-07/85) from Barbus barbus (host coll. number BB29), Argens River near Marseille, France, June 2007, collected by M. Da´vidova´ and A. Sˇimkova´ (IPCAS C-51/5). Site of infection: Anterior intestine. Intermediate host: Limnodrilus hoffmeisteri Clapare`de (Oligochaeta: Tubificidae) (Janiszewska, 1954). Distribution: Records verified by the present authors are marked with asterisks (Morphotype 1, one asterisk; Morphotype 2, two asterisks; see Fig. 5): Europe (**Austria, *Bulgaria, */**Czech Republic, **Finland, *France, Hungary, *Italy, *Portugal, **Russia, Serbia, */**Slovakia, and */**Ukraine) and Palaearctic Asia (Iraq, Russia and China). Sequence data: KF051092–KF051100, KF051102– KF051115, KF051117, KF051118, KF051121– KF051124, KF051126–KF051160, KF051162, KF051163, KF051166–KF051170, KF051173–
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KF051181 (for more details, see table 2 in Bazsalovicsova´ et al., 2014). Material studied: Morphotype 1 (see also tables 1, 2 in Bazsalovicsova´ et al., 2014): (i) Specimens from museum collections: 5 specimens from B. barbus, Danube and Dniester Rivers, Ukraine, 1951 & 1965 (ZIK; not numbered; host Nos. 89 & 139) and Brenta River, Italy, 1999 (IPCAS C-3/5); 3 specimens from B. cyclolepis, Arda River, Bulgaria, 2010 (PISAS Nos. BG 47/10 & 83/10); 8 specimens from B. meridionalis, Porto, Portugal (IPCAS C-51/1); 1 specimen from B. petenyi, Bulgaria, 1953 (ZIK; not numbered; host No. 103-20); 37 specimens from S. cephalus, Brenta and Orta Rivers, Italy, 1991 & 2001 (BMNH 20003.9.20.11; IPCAS C-51/3; MHNGPLAT 88064), Becˇva and Rokytna´ Rivers, Czech Republic, 1962 & 1986 (IPCAS C-51/3) and Dniester River, Ukraine, 1958 (ZIK; not numbered). (ii) Newly-collected specimens: 11 specimens from B. barbus, Argens, Cassole and Saˆone Rivers, France, 2004 & 2007 (IPCAS C-51/5; MHNG-PLAT 88065); 12 specimens from B. meridionalis, Myslavsky´ stream, Kosˇice, Slovakia, 2005 (hologenophore PISAS SR 2A); 2 specimens from S. orpheus, Arda River, Bulgaria, 2010 (hologenophore PISAS BG 33/10). Morphotype 2 (see also tables 1, 2 in Bazsalovicsova´ et al., 2014): (i) Specimens from museum collections: 5 specimens from A. brama, Saravesi Lake, Finland, 1986 (BMNH 1986.6.26.8–14), Vltava River, Czech Republic, 1986–1988 (IPCAS C-2/1), Dniester River, Ukraine, 1958 (ZIK; not numbered); 1 specimen from L. aspius, Northern Donets, Ukraine (ZIK; not numbered; host No. 159-3); 4 specimens from S. cephalus, Rokytna´ River, Czech Republic, 1986 and Latorica River, Slovakia, 2007 & 2008 (IPCAS C-51/3). (ii) Newly-collected specimens: 71 specimens from A. brama, Latorica and Tisa Rivers, Slovakia, 2008, 2009, and Cheboksary Reservoir, Volga River, Russia, 2008 & 2009 (paragenophores PISAS SR 33/08, 37/08, 59/08, 60/08, 15/09, 17/09, 19/09, 31/09, 33/09; MHNGPLAT 88066); 10 specimens from B. ballerus, Latorica River, Slovakia, 2009 (paragenophores PISAS SR 7/09, 15/09) and Rybinsk Reservoir, Volga River, Russia, 2009 (paragenophore PISAS RU 7/09); 2 specimens from B. sapa, Latorica and Tisa Rivers, Slovakia, 2004 & 2008 (paragenophores PISAS SR 192/04, 193/04, 33/08,
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59/08); 9 specimens from B. bjoerkna, Cheboksary Reservoir, Volga River, Russia, 2009 (hologenophores PISAS RU 63/09, 119–120/09; paragenophores 165–170/09; MHNG-PLAT 88067); 2 specimens from L. idus, Latorica River, Slovakia, 2007 (PISAS 47/07-3); 2 specimens from S. cephalus, Latorica River, Slovakia, 2008 (PISAS 119/08). Description of Morphotype 1 (Figs. 1–4) [Based on 6 specimens ex B. barbus and B. meridionalis from France and Slovakia; measurements from the neotype in parentheses; see also Table 1.] Caryophyllidea, Caryophyllaeidae. Body more robust, with maximum width 0.8–1.3 mm (1.1 mm) at cirrus-sac region, narrowing towards posterior end. Scolex afossate, 0.9–1.6 mm (1.0 mm) long, spatulate with tapered anterior edge, 1.2–2.1 mm (1.9 mm) wide, only slightly wider than neck, 0.8–0.9 mm (0.9 mm) wide (Figs. 1B–D, 4A). Width of neck represents 43–69% (47%) of scolex width. Anterior edge of scolex with numerous slight indentations and superficial grooves (wrinkles) on its surface. Surface covered uniformly with acicular filitriches (Fig. 4D). Inner longitudinal musculature well developed, formed by small bundles of muscle fibres (Fig. 3A–C). Testes medullary, about 170–190 in number (in some specimens, precise number difficult to count because of extensive vitelline follicles covering testes), oval to almost spherical, 88–203 9 80–196 (n = 100) (118–135 9 111–122), intermingled with vitelline follicles. Distance of anteriormost testes from anterior extremity short, 0.8–2.0 mm (1.0 mm) (Fig. 1B–D). First testes begin at same level, in some cases even anterior (Fig. 1B) or slightly posterior to first vitelline follicles (Fig. 1C, D). Posteriorly testes reach to anterior margin of cirrus-sac (Fig. 2A, B). Testicular field 7.3–20.2 mm long. Vas deferens well developed, in distal (posterior) part surrounded medially by vitelline follicles. Cirrussac large, elongate to ovoid, thick-walled, 1,075–1,227 9 518–641 (1,089 9 570), containing strongly muscular, long cirrus (Figs. 2A, B, 3A, B); width of cirrus-sac represents 54–74% (54%) of body width at cirrus-sac region. External and internal seminal vesicle absent. Ovary follicular, H-shaped, 721–903 (884) wide, arms 1,010–1,403 (1,158–1,224) long and 194–537 (235–251) wide (Fig. 2A, B). Vagina tubular, situated ventral to uterus, proximal (posterior) part sinuous, distal (anterior) part almost straight, joining uterus to
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form short common uterovaginal duct. Receptaculum seminis situated anterodorsal to ovarian isthmus, almost spherical to oval, 300 9 196 (294 9 197) (Fig. 2B). Preovarian vitelline follicles numerous, lateral and median, surrounding testes and vas deferens, including its distal (posterior) part; follicles irregularly-shaped and variable in size, 72–140 9 59–135 (n = 100) (111–120 9 77–85). Anteriormost follicles begin at short distance (0.8–1.5 mm) (0.8 mm) posterior to anterior edge of scolex (Fig. 1B–D). Vitelline follicles reach posteriorly up to level of anterior or middle part of ovarian arms, absent alongside ovary. Postovarian follicles present (Fig. 2A, B). Uterus forms several loops between ovary and level of middle or posterior part of cirrus-sac (Fig. 2A, B); preovarian part surrounded by numerous glands. Uterus occupies region 1.7–2.5 mm long (2.3 mm), 9–12% of testicular field. Eggs oval, operculate, unembryonated, smooth, 66–75 9 41–44 (66–72 9 41–43) (n = 15, intrauterine eggs on whole-mounts). Genital pores separated, close to each other, female pore opens posterior to male pore (Figs. 3B, 4C). Description of Morphotype 2 (Figs. 1–4) [Based on 44 specimens ex A. brama, B. ballerus and B. bjoerkna from Russia and Slovakia; measurements in Table 1.] Caryophyllidea, Caryophyllaeidae. Body more slender, elongate, 12–32 mm, with maximum width 0.5–1.0 mm at cirrus-sac region, narrowing towards posterior end. Scolex afossate, 1.1–1.9 mm long, flabellate, with variously curved anterior margin, 1.3–2.8 mm wide, significantly wider than neck, 0.4–0.9 mm wide (Figs. 1F–H, 4B). Width of neck represents 24–49% of scolex width. Anterior edge of scolex with numerous slight indentations and superficial grooves (wrinkles) on its surface. Surface covered uniformly with acicular filitriches. Inner longitudinal musculature well developed, formed by small bundles of muscle fibres. Testes medullary, about 140–450 in number (in some specimens, precise number difficult to count because of extensive vitelline follicles covering testes), oval to almost spherical, 75–201 9 68–183 wide (n = 100), intermingled with vitelline follicles. Distance of anteriormost testes from anterior extremity moderate, 2.6–8.3 mm (Fig. 1F–H). First testes begin at much more posterior (up to 4.3 mm) to first vitelline follicles (Fig. 1F, H). Posteriorly testes reach to anterior margin of cirrus-sac (Fig. 2C). Testicular field 7.4–20.2 mm
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Table 1 Measurements of individual morphotypes of Caryophyllaeus brachycollis Janiszewska, 1953 from different hosts and geographical regions (main morphological differences between the two morphotypes in bold) Character / Morphotype Host Locality
Body length (mm) (A)
Morphotype 1
Morphotype 1
Morphotype 2
Morphotype 2
Morphotype 2
Morphotype 2
Barbus barbus France (n = 5)
Barbus meridionalis Slovakia (n = 1)
Abramis brama Slovakia (n = 9)
Abramis brama Russia (n = 14)
Ballerus ballerus Russia (n = 1)
Blicca bjoerkna Russia (n = 2)
11–26
–
12–27
15–26
32
22–23
form
robust
robust
slender
slender
slender
slender
width
853–1,255
997–1,246
534–1,031
514–1,177
1,083
spatulate
spatulate
flabellate
flabellate
flabellate
flabellate
length
908–1,561
1,003
1,072–1,661
1,194–1,847
1,740
1,309–1,526
width
1,198–2,072
1,402
1,449–2,789
1,287–2,366
2,223
1,652–1,773
537–896
406–673
937
597–866
Scolex type
Neck width ratio to scolex width
885
823–892
770
43–69%
55%
28–42%
24–49%
42%
36–49%
88–203 9 80–196
117–140 9 95–140
75–178 9 68–147
92–201 9 78–183
156–173 9 134–155
153–166 9 113–140
number
170–190
–
230–420
140–450
290
215–240
distance from first vitelline follicle (mm)
0.14–0.56
0.06
0.4–1.9
0.8–3.0
4.3
1.1–1.3
distance from anterior extremity (mm)
0.97–2.02
0.8
2.6–3.8
3.1–5.5
8.3
3.4–3.9
reach up to
ant. to post. part of CS
ant. to post. part of CS
ant. to middle part of CS
ant. part of CS
ant. part of CS
ant. part of CS
length of testicular field (mm)
7.3–20.2
–
7.4–20.2
10.5–18.8
19.0
15.2–16.4
1,075–1,227 9 518–641
1117 9 571
537–1,098 9 311–651
567–982 9 318–567
1,093 9 646
635 9 399
64%
54–64%
43–62%
60%
40%
791
821–1,091
Testes size
Cirrus-sac size extent in relation to body width Ovary width
54–74%
445–924
433–1,093
829
length of ovarian arms
721–903 1,150–1,403
1,010–1,047
834–1,766
815–1,747
1,620–1,651
1,113–1,176
width of ovarian arms
204–537
194–241
113–304
117–252
235–271
178–277
distance from posterior end (B)
2,265–2,951
2,601
1,264–2,389
1,450–2,120
2,101
1,920–2,019
Proportion (B) : (A)
8–14%
–
7–9%
8–9%
7%
8–9%
Size of vitelline follicles
72–125 9 59–100
104–140 9 83–135
47–137 9 43–110
77–200 9 57–140
113–149 9 87–120
99–154 9 82–123
1.4–2.7
1.7–3.2
4.0
2.5–2.6
ant. to middle part of ovary
ant. to middle part of ovary
anterior part of ovary
anterior part of ovary
distance from anterior extremity (mm)
0.83–1.49
posterior extent of preovarian follicles
anterior part of ovary
Uterus extent (mm)
0.82 anterior part of ovary
1.3–2.9
1.6–2.9
3.5
2.1–2.3
9–12%
–
13–18%
14–19%
19%
14%
Size of intrauterine egg
66–75 9 41–44
68–72 9 41–43
59–65 9 40–45
62–65 9 42–44
59–61 9 43–45
59–65 9 40–42
Receptaculum seminis
294–300 9 196–197
–
–
303–489 9 229–370
–
–
extent in relation to length of testicular area
1.7–2.5
1.9
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Fig. 1 Line drawings of Caryophyllaeus brachycollis Janiszewska, 1953 (A–D, Morphotype 1, B, hologenophore; E–H, Morphotype 2, G, paragenophore). A, E, Outlines of adults, note the indication of the first testes and vitelline follicles; B–D, F–H, Anterior part of the body with the first vitelline follicles and testes. Abbreviations: cs, cirrus-sac; fte, first testes; fvf, first vitelline follicles; ov, ovary. Scalebars: A, E, 1 mm; B–D, F–H, 500 lm
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long. Vas deferens well developed, in distal (posterior) part surrounded medially by vitelline follicles. Cirrus-sac large, elongate to ovoid, thick-walled, 537–1,098 9 311–651, containing strongly muscular, long cirrus (Fig. 2C); width of cirrus-sac represents 40–64% of body width at cirrus-sac region. External and internal seminal vesicle absent. Ovary follicular, H-shaped, 433–1,093 wide, with arms 815–1,747 long and 113–304 wide (Fig. 2C). Vagina tubular, situated ventral to uterus, proximal (posterior) part sinuous, distal (anterior) part almost straight, joining uterus to form short common uterovaginal duct. Receptaculum seminis situated anterodorsal to ovarian isthmus, almost spherical to oval, 303–489 9 229–370 (n = 5). Preovarian vitelline follicles numerous, lateral and median, surrounding testes and vas deferens, including its distal (posterior) part; follicles irregularly-shaped and variable in size, 47–200 9 43–140 (n = 100). Anteriormost follicles begin far, 1.4–4.0 mm, from anterior extremity (Fig. 1G, H). Vitelline follicles reach posteriorly up to level of anterior or middle part of ovarian arms, absent alongside ovary. Postovarian follicles present (Fig. 2C). Uterus forms several loops between ovary and level of middle or posterior part of cirrus-sac (Fig. 2C); preovarian part surrounded by numerous glands. Uterus occupies region 1.3–3.5 mm long, 13–19% of testicular field. Eggs oval, operculate, unembryonated, smooth, 59–65 9 40–45 (n = 30, intrauterine eggs on wholemounts). Genital pores separated, close to each other, female pores opens posterior to male pore (Fig. 2C). Remarks Janiszewska (1953) described C. brachycollis from four species of cyprinids of two subfamilies, B. barbus and B. petenyi (Barbinae), and S. cephalus and L. idus (Leuciscinae), from several rivers in Poland (Odra and Vistula with its tributaries Barycz, Raba and Ropa). However, she did not designate a type-host. Common barbel, B. barbus, was most heavily infected (prevalence 27%; n = 48; maximum intensity 7 tapeworms). Moreover, type-specimens were not designated nor their deposition was indicated in the original description. Subsequent search for the types of C. brachycollis was unsuccessful and all our enquiries indicate that the type-material is not extant. Therefore, we designate one of the specimens from B. barbus (this
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fish host was listed first in the original description) from the Argens River near Marseille, France, deposited at the Institute of Parasitology, BC ASCR, Cˇeske´ Budeˇjovice, Czech Republic (IPCAS C-51/5), as the neotype of C. brachycollis. Since its original description in 1953, the species has been reported from several European countries including the European part of Russia, whereas records from Asia are rare and require verification (vouchers of tapeworms reported are not available). This concerns the record of C. brachycollis from Barbus lacerta in Iraq by Rahemo & Mohammad (2004), because Khawia armeniaca (Cholodkovsky, 1915), which possesses a similar scolex, occurs frequently in barbels in Iraq (Scholz et al., 2011), and the record from common carp Cyprinus carpio in Huangsha Lake, Hubei Province in China by Chen (1973). Janiszewska (1954) reported a procercoid (= plerocercoid according to Chervy, 2002) of C. brachycollis from Limnodrilus hoffmeisteri Clapare`de from the Odra River in Poland. Caryophyllaeus brachycollis was easily differentiated from congeneric species by scolex shape and the anterior extent of the first testes that begin at the same level or just slightly posterior to the first vitelline follicles (see Dubinina, 1987; Scholz, 1989; Protasova et al., 1990). However, molecular data provided recently by Bazsalovicsova´ et al. (2014) have shown that the species is polymorphic and individuals from leuciscine fishes, especially breams (Abramis, Ballerus and Blicca), which were previously identified as Caryophyllaeus laticeps, represent a separate morphotype of C. brachycollis. This morphotype, called herein C. brachycollis Morphotype 2, differs from the ‘nominotypical’ morphotype (called C. brachycollis Morphotype 1 herein) mainly in the following characteristics: (i) the shape of the scolex, which is wider and flabellate (vs spatulate in Morphotype 1); (ii) the length of the neck, which is much longer in Morphotype 2 (vs very short in Morphotype 1 as expressed in the species name); (iii) a much more posterior extent of the testes and vitelline follicles from the scolex (the testes in Morphotype 1 begin at the same level as the first vitelline follicles or only slightly more posterior; both begin just posterior to the scolex); (iv) the anterior edge of the scolex with numerous shallow superficial grooves or wrinkles (vs almost smooth or with only a few wrinkles in Morphotype 1); (v) a more slender,
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Fig. 2 Line drawings of Caryophyllaeus brachycollis Janiszewska, 1953, posterior part of the body. (A, B, Morphotype 1, ventral view; C, Morphotype 2, dorsal view). Note large, elongate, thick-walled cirrus-sac. Abbreviations: cs, cirrus-sac; mgp, male gonopore; ov, ovary; pvf, postovarian vitelline follicles; rs, receptaculum seminis; te, testes; ug, uterine glands; ut, uterus; uvp, uterovaginal pore; va, vagina; vd, vas deferens; vf, vitelline follicles. Scale-bars: 500 lm
longer body (vs body more robust in relation to its total length) (see Figs. 1–4; Table 1). However, both morphotypes of C. brachycollis share the identical morphology of the posterior part of the body, in particular the possession of a large, elongate ovoid, thick-walled cirrus-sac (its width represents 40–74% of body width) containing a long, strongly muscular cirrus of lamellar structure
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(Fig. 3A, B), and the median position of the vitelline follicles at the level of the distal (posterior) part of the external sperm duct (vas deferens) (Fig. 2). The morphology and structure of the cirrus-sac were uniform in all C. brachycollis specimens and both morphotypes that occur in a number of fish hosts within a large distribution area (Fig. 5). Therefore, the cirrus-sac is considered to represent a taxonomically
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important structure suitable for circumscription of this species. Caryophyllaeus laticeps, which may occur quite commonly with C. brachycollis in co-infections, differs from the latter species in the morphology of the posterior end of the body, especially in the possession of a small, subspherical, thin-walled cirrus-sac containing a well-developed internal seminal vesicle, and in the absence of vitelline follicles medially at the level of posterior loops of vas deferens (external sperm duct). This absence of vitelline follicles alongside the median line of the body makes the region with vas deferens to appear conspicuous (see figures 2J and 2M in Scholz, 1989). Mixed infections with both C. laticeps and Morphotype 2 of C. brachycollis were detected in four species of leuciscine cyprinids from the Latorica and Tisa Rivers in eastern Slovakia and the Volga River in the European part of Russia, namely A. brama (percentage of C. laticeps vs C. brachycollis specimens 79%/21%, n = 333 specimens counted), B. ballerus (79%/21%, n = 33), B. sapa (90%/10%, n = 21) and B. bjoerkna (44%/ 56%, n = 16). In total, C. laticeps tapeworms represented 78% of the specimens counted, whereas those of C. brachycollis Morphotype 2 represented only 22%. This proportion differed slightly between individual localities (Latorica, 74%/26%, n = 105; Tisa, 85%/15%, n = 122; Volga, 77%/23%, n = 176). In the possession of a large, thick-walled cirrus-sac, C. brachycollis is almost indistinguishable from C. fimbriceps Annenkova-Khlopina, 1919 (see figures 2L and 2M in Scholz, 1989). This species was described from common carp C. carpio in Russia and then reported as a carp parasite of veterinary importance in the former USSR and eastern Europe, causing mortalities of heavily infected fish (Bauer et al., 1973). Both species differ allegedly from each other by the morphology of the scolex, which is cuneicrispitate in C. fimbriceps (flabellate with the fimbriate anterior margin; see figure 2D, E in Scholz, 1989 and figure 1I in Oros et al., 2010). However, some C. brachycollis tapeworms of Morphotype 2 possess scolex resembling that of C. fimbriceps, which has also been reported from breams (Leuciscinae) (Protasova et al., 1990). It is obvious that the independent status of the latter species has to be confirmed, preferably by molecular data. Unfortunately, no ethanol-fixed material of C. fimbriceps has been available and the species seems to have disappeared globally, possibly as a result of the
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introduction of another caryophyllidean cestode specific to carp, Khawia sinensis Hsu¨, 1935, to the European part of the former USSR and Eastern Europe (Oros et al., 2009). Morphological and molecular evaluation of new material of C. fimbriceps, which is currently absent, may reveal its conspecificity with C. brachycollis.
Discussion Classification, systematics and species identification of helminths, including fish cestodes, have been largely based on morphological characters (Wardle & McLeod, 1952; Schmidt, 1986; Khalil et al., 1994). Since the late 1970’s, cestode systematics has benefited from the application of phylogenetic systematics (cladistics), first applied in cestodes by Brooks (1978) and later also used for the assessment of phylogenetic relationships of major groups of tapeworms, including those parasitising teleost fishes (Hoberg et al., 1997, 1999; Rego et al., 1998; Oros et al., 2008). Although these studies represented a valuable contribution to our understanding of cestode relationships, they suffered from a high degree of homoplasy of morphological characters used; this decreased reliability of the conclusions inferred from morphology-based phylogenies. The application of DNA-based genetic markers in phylogenetic systematics, which started in cestodology in the late 1990’s (Mariaux, 1998; Olson & Caira, 1999; Zehnder & Mariaux, 1999; Kodedova´ et al., 2000) represented even a more important step forward to our better understanding of the evolutionary history of cestodes and their phylogenetic relationships (Olson et al., 2001; Olson & Tkach, 2005; Brabec et al., 2006; Waeschenbach et al., 2007, 2012; Littlewood, 2008). Genetic markers also made it possible to assess more reliably intraspecific variability, which resulted in detecting cryptic diversity in some groups of parasitic flatworms (Doanh et al., 2009), but also in revealing unexpected phenotypic plasticity in others (Blasco-Costa et al., 2010; Miller & Cribb, 2013; Bazsalovicsova´ et al., 2014). Phenotypic plasticity is defined as the property of a given genotype to produce different phenotypes in response to distinct environmental conditions (Pigliucci, 2001). In parasites, this ability of a genotype to present distinct phenotypic features can be influenced by physiological,
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Fig. 3 Histological sections of Caryophyllaeus brachycollis Janiszewska, 1953 from Barbus barbus, Morphotype 1. A, B, Longitudinal sections of posterior part of body. Note large, elongate, thick-walled cirrus-sac (A) and separate genital pores (B); C, Cross-section. Abbreviations: cs, cirrus-sac; ilm, inner longitudinal musculature; mgp, male gonopore; oc, osmoregulatory canal; te, testes; ut, uterus; uvp, uterovaginal pore; va, vagina; vf, vitelline follicles. Scale-bars: A, B, 500 lm; C, 250 lm
biochemical, ecological and other differences between individual hosts. In fact, a high intraspecific morphological variation caused by phenotypic plasticity may represent a serious obstacle in parasite identification and can confound species differentiation based on morphological characters only. Bazsalovicsova´ et al. (2014) have demonstrated such a case of phenotypic plasticity in two polymorphic species of fish tapeworms, C. brachycollis and C.
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laticeps. Unexpectedly, some tapeworms from breams, previously identified as C. laticeps, represented in fact a morphogically distinct, but genetically closely related lineage of C. brachycollis. This species was considered to be morphologically quite uniform and restricted to barbels (Barbinae) and chub (Squalius cephalus) (Janiszewska, 1953; Scholz, 1989; Protasova et al., 1990). In congeneric C. laticeps, type-species of the genus, as many as five
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Fig. 4 Scanning electron micrographs of Caryophyllaeus brachycollis Janiszewska, 1953. A, Anterior part of body with scolex, Morphotype 1; B, Morphotype 2; C, Separate genital pores; D, Acicular filitriches (filiform microtriches). Abbreviations: mgp, male gonopore; uvp, uterovaginal pore. Scale-bars: A, B, 200 lm; C, 100 lm; D, 2 lm
morphotypes, most of them host-specific and originally supposed to represent new species by the present authors, were distinguished; molecular data confirmed that they all belong in fact to only one genetic lineage (Bazsalovicsova´ et al., 2014). In the present study, barbels (Barbinae) harboured only Morphotype 1 of C. brachycollis, whereas breams (Leuciscinae) were infected only with tapeworms of Morphotype 2. In chub (S. cephalus, Leuciscinae), both
morphotypes of C. brachycollis were found, which indicates that other factors than those related exclusively to host physiology and microanatomy of the intestinal tract may be involved in the phenotypic plasticity of C. brachycollis. Mechanisms of possible host-induced phenotypic plasticity in cestodes are not well known and further studies are necessary to provide their explanation. Experimental cross-infections of different fish with plerocercoids of both morphotypes
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Fig. 5 Distribution map of Caryophyllaeus brachycollis Janiszewska, 1953. Triangles, Morphotype 1; circles, Morphotype 2
of C. brachycollis from oligochaetes would help confirm the existence of this phenomenon. Intraspecific plasticity in the morphology of the anterior part of the body of species of Caryophyllaeus demonstrated by Bazsalovicsova´ et al. (2014) and by the present study indicates that taxonomic importance of morphological characteristics related to the scolex and the anterior extent of the testes and vitelline follicles in other groups of caryophyllidean cestodes should be critically assessed, preferably on the basis of combined morphological and molecular studies of adequately processed materials (see Oros et al., 2010). However, the present data do not necessarily question reliability of scolex morphology in the systematics and species diagnostics of caryophyllidean cestodes in general (see Oros et al., 2010 for comprehensive data on scolex morphology of Palaearctic taxa), just call for more attention to be paid when these morphological characteristics are used in individual groups of caryophyllidean cestodes. Results of Bazsalovicsova´ et al. (2014) and present study have shown that morphology of the cirrus-sac represents a key diagnostic character in the species of Caryophyllaeus. The cirrus-sac of caryophyllideans varies greatly in size (its width represents from onefifth to three-fourths of the body width) and is strongly muscular in species of most genera (Mackiewicz, 1972, 1994). Appearance of the cirrus-sac was uniform in all specimens of both morphotypes of C. brachycollis (168 specimens examined) from several fish hosts in a large distribution area and thus
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its morphology can be considered to be stable and species-specific. The present study, which represents a follow-up of that by Bazsalovicsova´ et al. (2014), clearly shows how it is important to combine morphological and molecular data to avoid misidentifications and false conclusions based on morphological characters only. Suitability of such a combined approach has also been proven in the taxonomic revision of the species of Khawia Hsu¨, 1935, especially in the assessment of the validity of two genetically closely related, but morphologically markedly different species from different fish hosts in China (Xi et al., 2009; Scholz et al., 2011; Kra´ˇlova´-Hromadova´ et al., 2012; Orosova´ & Oros, 2012). Khawia saurogobii Xi, Oros, Wang, Wu, Gao & Nie, 2009, a parasite of gudgeons (Saurogobio spp.; Gobiinae) in China, which is morphological markedly different from, but genetically closely related to, K. sinensis Hsu¨, 1935, might be an example of morphological divergence as a result of ongoing sympatric speciation by host switching (very narrow taxonomic range of hosts). In contrast, Morphotype 2 of C. brachycollis from breams shows a large distribution area in the Palaearctic (Fig. 5) and appears to represent only a distinct morphotype of euryxenous parasite with a wide spectrum of definitive hosts. Acknowledgements The authors thank two anonymous reviewers for helpful suggestions, to Diana Kirin, Agricultural University of Plovdiv, Bulgaria, Martina Da´vidova´ and Andrea Sˇimkova´, Masaryk University, Brno, Czech Republic, Larisa G. Poddubnaya, Institute of Inland Waters, Borok, Russia, Elga Tieri, Italy, and Bahram Dezfuli, University of Ferrara, Italy, for providing specimens of C. brachycollis from Bulgaria, France, Russia and Italy, respectively. This study was supported by the Slovak Research and Development Agency (projects nos. APVV-0653-11 and LPP 0171-09), Grant Agency VEGA (No. 2/0129/12), Institute of Parasitology (RVO: 60077344) and Czech Science Foundation (project No. P505/12/G112). The work was undertaken within the framework of the Research & Development Operational Programme funded by the ERDF (code ITMS: 26220120022) (0.5).
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