Gyrodactylus laevisoides n. sp. (Monogenea: Gyrodactylidae) infecting northern redbelly dace Phoxinus eos Cope (Cyprinidae) from Nova Scotia, Canada

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Gyrodactylus laevisoides n. sp. (Monogenea: Gyrodactylidae) infecting northern redbelly dace Phoxinus eos Cope (Cyprinidae) from Nova Scotia, Canada Stanley D. King, David K. Cone, Michael P. Mackley & Paul Bentzen

Systematic Parasitology An International Journal ISSN 0165-5752 Volume 86 Number 3 Syst Parasitol (2013) 86:285-291 DOI 10.1007/s11230-013-9450-7

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Author's personal copy Syst Parasitol (2013) 86:285–291 DOI 10.1007/s11230-013-9450-7

Gyrodactylus laevisoides n. sp. (Monogenea: Gyrodactylidae) infecting northern redbelly dace Phoxinus eos Cope (Cyprinidae) from Nova Scotia, Canada Stanley D. King • David K. Cone • Michael P. Mackley • Paul Bentzen

Received: 25 July 2013 / Accepted: 17 September 2013 Ó Springer Science+Business Media Dordrecht 2013

Abstract Gyrodactylus laevisoides n. sp. is described from the gill rakers of red belly dace, Phoxinus eos Cope (Cyprinidae), from Nova Scotia, Canada. Gyrodactylus laevisoides n. sp. is the second species of Gyrodactylus Nordmann, 1832 described from this host and is characterised by weakly curving hamuli, a small ventral bar lacking anterolateral processes, stout dorsal bar, small marginal hooks with sickles larger proximally than distally and having a small circular process on the heel, a MCO with spines arranged in two arched rows, and lack of obvious excretory bladders. The new species most closely resembles Gyrodactylus laevis Malmberg, 1957, a Eurasian species whose principle host is Phoxinus phoxinus (L.). The two species are separated by Gyrodactylus laevisoides n. sp. having less divergent and longer hamulus root and marginal hook sickle toe with a steeper continuous angle and heel that is less prominent. The morphological description is supplemented with sequences of the 18S gene (449 bp, including the V4 region) and of the ITS region (821 bp). Gyrodactylus sedelnikowi Gvosdev, 1950 infecting

S. D. King (&)  M. P. Mackley  P. Bentzen Department of Biology, Dalhousie University, Halifax, NS B3H 4J1, Canada e-mail: [email protected] D. K. Cone Department of Biology, Saint Mary’s University, Halifax, NS B3H 3C3, Canada

Barbatula barbatula (L.) and Gyrodactylus neili Leblanc, Hansen, Burt & Cone, 2006 infecting Esox niger Lesueur are the most genetically similar species on GenBank for the 18S rRNA gene and ITS regions respectively (c.96% and c.92%). Gyrodactylus laevisoides n. sp. belongs to Malmberg’s subgenus Gyrodactylus (Gyrodactylus) and phylogenetic analysis of the ITS region groups this species with other members of the subgenus. The phylogeny has two main clades, one comprised of Eurasian species and the other of North American species, specifically Gyrodactylus laevisoides n. sp. and Gyrodactylus neili. It is suspected that this lineage, which is seemingly underrepresented in North America, likely colonised the new world with an ancestral species of Phoxinus via the Bering land connection around the time of the Pliocene.

Introduction The red belly dace, Phoxinus eos Cope is widely distributed across North America, from British Columbia to Nova Scotia, north to the Northwest Territories, and south to Colorado (Stasiak, 2006). It has only been reported to host one species of Gyrodactylus Nordmann, 1832, Gyrodactylus eos Mayes, 1977, although its Eurasian congener, Phoxinus phoxinus (L.), reportedly hosts 17 species (Harris et al., 2008; Shinn et al., 2010). During a recent parasite survey two species of Gyrodactylus were encountered infecting P. eos,


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Gyrodactylus eos and a previously undescribed species. The present study describes the latter as Gyrodactylus laevisoides n. sp. with the aid of light and scanning electron microscopy and rDNA sequences.

Materials and methods Sample collection Red belly dace were collected by seine net from Meadow Pond near Windsor, Nova Scotia (44°58.0700 N, 64°05.2970 W) on 22–24 June 2012. Fish were transported to Dalhousie University and either examined live using a stereomicroscope or stored in 100% ethanol for future study. Gyrodactylids were removed from the fish and were either mounted on slides, fixed in 5% formalin heated to 65°C or preserved in 100% ethanol. Morphological studies Ethanol-preserved specimens were individually mounted on glass slides holding a single drop of water. Of these, the haptoral hard parts of 12 individuals were released from the soft tissue using a digestion buffer (Harris et al., 1999). Specimens, both whole and digested, were then mounted in a 50% glycerol solution and examined using bright field, phase and differential interference contrast optics. Additional parasites, fixed in 5% formalin, were also mounted whole on glass slides in a 50% glycerol solution. Of these, the holotype and five paratypes were prepared by removing the cover-slip of the flattened specimens and staining with Gomori’s trichrome for approximately 1 min (Kritsky et al., 1978). These type-specimens were then dehydrated using absolute ethanol, cleared in xylene and mounted in Canada balsam. Diagnostic measurements follow Malmberg (1970) with the exception of the dorsal and ventral bar width, referred to here as length, and vice versa. In addition, hamuli and marginal hook aperture distances were taken in accordance with Shinn et al. (2004). All measurements were obtained using Zeiss Axioplan 2 digital software (Zeiss, Thornwood, New York) and are reported in micrometres (mean ± SD followed by range in parentheses) rounded to the nearest 0.5 lm. Scanning electron microscopy Ethanol-preserved worms were placed in a drop of distilled water on a 12 mm glass cover-slip which in


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turn was superficially affixed to a glass slide using a small amount of distilled water. Soft tissue was removed using a digestion buffer (Harris et al., 1999) followed by incubation at 55°C for 10 min. The digestion buffer was then removed using paper towel and the released hard parts washed 3–4 times with distilled water to remove excess debris and airdried. The cover-slip was then attached to an aluminium stub using double-sided carbon tape and sputtercoated with gold prior to examination with a LEO 1450VP scanning electron microscope (Oberkochen, Germany). Molecular characterisation DNA was extracted from two preserved parasites individually using a DNeasy blood and tissue kit (Qiagen, Valencia, California) following manufacturer’s instructions. For both individuals the ITS region was amplified using the primer pairs P3b (50 TAG GTG AAC CTG CAG AAG GAT CAT-30 ) and P4 (50 -GTC CGG ATC CTC CGC TTA TTG ATA TGC-30 ) (Cable et al., 1999). In addition 449 bases of the 18S rRNA gene, including the V4 region, were amplified using the primer pair PBS18SF (50 -CGC GCA ACT TAC CCA CTC TC-30 ) and PBS18SR (50 ATT CCA TGC AAG ACT TTT CAG GC-30 ) (Cone et al., 2010). Each 12.5 ll PCR consisted of 1 ll DNA template, 19 Titanium Taq buffer (Clontech), 0.2 mM of dNTP, 0.1 lm of each primer and 0.59 Titanium Taq DNA polymerase (Clontech). Amplification was performed in an Eppendorf thermal cycler using the following protocol: 95°C for 3 min, 5 touchdown cycles of 95°C for 30 sec, 65°C for 30 sec (decreasing by 3°C for each of the 5 touchdown cycles), 72°C for 60 sec, then 30 amplification cycles of 95°C for 30 sec, 50°C for 30 sec and 72°C for 60 sec. This was followed by a 300 sec final hold at 72°C. Products were visualised on a 1% agarose gel stained with GelGreen (Biotium). The PCR products were purified using Exonuclease I and Antarctic Phosphatase (New England BioLabs, Beverly, Massachusetts) and sequenced (ABI dideoxy) in both directions by Genewiz Inc. (South Plainfield, New Jersey) using the same primers that generated the PCR products. The sequence data were edited using CodonCode V. 3.5 (CodonCode Co., Dedham, Massachusetts) and using the consensus sequence GenBank databases were searched for similar sequences using BLAST (Basic Local Alignment Search Tool). Using MEGA 5 (Tamura

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et al. 2011) the consensus ITS sequence was aligned with the following sequences from GenBank based on genetic similarity: Gyrodactylus elegans Nordmann, 1832 (AY278034), Gyrodactylus prostae Ergens, 1963 (AY278038), Gyrodactylus alburnensis Prost, 1972 (AY278032), Gyrodactylus laevis Malmberg, 1957 (AY278033), Gyrodactylus perccotti Ergens & Yukhimenko, 1973 (JN603638), Gyrodactylus carassii Malmberg, 1957 (AY278033), Gyrodactylus magnificus Malmberg, 1957 (AY278035), Gyrodactylus phoxini Malmberg, 1957 (AY278037), Gyrodactylus neili LeBlanc, Hansen, Burt & Cone, 2006 (AY881175), Gyrodactylus jennyae Paetow, Cone, Huyse, McLaughlin & Marcogliese, 2009 (EU678357). The sequence of Gyrodactylus stephanus Mueller, 1937 (FJ845515), which was alignable but less similar, was included as an outgroup. Phylogenetic analyses incorporated the Minimum Evolution (ME), Neighbour-Joining (NJ) and Maximum likelihood (ML) algorithms using Kimura’s 2-parameter method (Kimura, 1980) and all phylogenies were tested with 1,000 bootstrap repeats. The final dataset included 905 positions.

Gyrodactylus laevisoides n. sp. Type-host: Red belly dace, Phoxinus eos Cope (Cyprinidae) Type-locality: Meadow Pond, Windsor, Nova Scotia (44°58.0700 N, 64°05.2970 W). Site: Gill rakers. Material studied: 12 specimens obtained on June 22–24 2012. Prevalence and mean intensity: 9 of 92 fish sampled (9.8%); 2.4 ± 1.3. Type-material: Harold Manter Laboratory of Parasitology (Accession nos. holotype HWML-49868, paratype HWML-49869); New Brunswick Museum (2 paratypes, NBM-010303 and NBM-010304), Royal BC Museum (2 paratypes, 013-00135-001 and 013-00135-002). Molecular sequence data: Partial 18S rDNA (449 bp) sequence is deposited in GenBank (accession no. KF263526) as is the ITS-1 (partial, 314 bp), 5.8S (complete, 157 bp) and ITS-2 (complete, 350 bp) fragment (accession no. KF263527). Etymology: The species is named after its morphological resemblance to Gyrodactylus laevis infecting the Eurasian minnow, P. phoxinus.


Description (Figs. 1–4) [Based on 12 specimens.] Body 258 ± 42.6 (207–343) long; 75 ± 18.1 (56.5–105) wide at mid-body. Haptor ovate, 62.5 ± 3.9 (57.5–68) long, 86.5 ± 15.9 (74.5–109.5) wide. Cephalic lobes distinct, with a small protuberance in the tegument at the apex. Head organs large, divided into 2 unequal size masses. Cephalic glands posterolateral to pharynx. Pharynx 21.5 ± 2.5 (18.5–25.5) long, with 8 short processes. Excretory bladders not evident. Male copulatory organ (MCO) spherical, 11.5 ± 1.2 (10–14), immediately posterior to pharynx, with 1 principal spine and 9–11 small terminal spines forming 2 arched rows. Intestinal crura ending blindly near posterior edge of uterus. Testes immediately anterior to ovary. Hamuli 35.5 ± 1.2 (34–38) long, surrounded in musculature ending near curvature of point; root 14 ± 1.4 (12–17); shaft 26.5 ± 0.9 (25–27.5); point 15 ± 0.6 (14–16); aperture 17.5 ± 0.9 (16.5–19). Ventral bar 14 ± 1.1 (12–15) long, 13.5 ± 1.3 (11–14) wide, median length of 3.5 ± 0.1 (3–3.5). Anterolateral processes of ventral bar absent. Ventral bar membrane rectangular, 9 ± 0.5 (8–9.5) long, difficult to observe without staining. Dorsal bar robust, 9.5 ± 0.7 (8.5–10) long, 2 ± 0.2 (1.5–2) wide. Marginal hooks small, 17.5 ± 0.7 (17–19) long; handle 12 ± 0.6 (11.5–13.5) long, tapering slightly at each end; sickle 5.5 ± 0.1 long with prominent process or ‘button’ on heel and a small anterior protrusion from proximal side of base; 2 ± 0.1 (1.5–2) wide distally; 3.5 ± 0.2 (3–4) wide proximally; aperture 5 ± 0.2 (5–5.5); filament loop 9 ± 1 (8.5–9.5) long. Remarks Gyrodactylus laevisoides n. sp. most closely resembles Gyrodactylus laevis Malmberg, 1957 infecting the Eurasian minnow, P. phoxinus. The two species can be differentiated based on the shape of the marginal hook sickle and the hamuli roots, with Gyrodactylus laevisoides n. sp. having a marginal hook toe with a steeper angle and a heel that is less prominent and a hamulus root which is longer and less divergent. Gyrodactylus laevisoides n. sp. occurred in mixed infections with G. eos. The two species are easily separated by the morphology of the ventral bar, G. eos having large anterolateral processes and a linguiform


Author's personal copy 288 Figs. 1–3 Line drawings of Gyrodactylus laevisoides n. sp. c infecting Phoxinus eos. 1, whole mount; 2, opisthaptoral central hook complex; 3, male copulatory organ. Scale-bars: 1, 100 lm; 2, 10 lm; 3, 5 lm

membrane, the MCO, G. eos having spines arranged in one arched row, and the shape of the marginal hook. These two species were also separated by microhabitat, G. eos infecting the fins and body and Gyrodactylus laevisoides n. sp. infecting the gill rakers. These species also occurred at differing prevalence and mean intensity; G. eos was common (56%, 3.7 ± 4.0) whereas infections of Gyrodactylus laevisoides n. sp. were less frequent (9.8%, 2.4 ± 1.3).

Molecular taxonomy The ITS region fragment generated was 821 bp long which included a partial ITS-1 (314 bp), complete 5.8S (157 bp) and complete ITS2 (350 bp). A BLAST query of the 5.8S fragment returned eight identical matches, all of which were members of the subgenus G. (Gyrodactylus): G. alburnensis, G. carassii, G. elegans, G. magnificus, G. neili, G. percotti, G. phoxini and G. prostae. A search of the whole ITS fragment returned Gyrodactylus neili as having the greatest molecular similarity (92%; AY881175). When queried, the partial 18S fragment (449 bp) returned Gyrodactylus sedelnikowi Gvosdev, 1950 as most genetically similar (96%; AY566387). Phylogenetic analysis of the ITS region consistently grouped Gyrodactylus laevisoides n. sp. with G. neili (Fig. 5) amongst a larger group of Eurasian members of the G. (Gyrodactylus), with identical tree topology and high bootstrap support for all algorithms used (ME, NJ, ML).

Discussion Gyrodactylus laevisoides n. sp. is a member of Malmberg’s subgenus G. (Gyrodactylus), having an MCO with spines arranged in two rows, no obvious excretory bladders, ventral bar lacking anterolateral processes and a marginal hook sickle of distinct shape. This group is commonly reported in Eurasia,


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Fig. 4 Scanning electron micrographs of Gyrodactylus laevisoides n. sp. infecting Phoxinus eos from Nova Scotia, Canada. A, hamuli; B, marginal hook sickle; C, marginal hook. Scale-bars: A, 10 lm; B, 1 lm; C, 3 lm

especially from cyprinid hosts, but is seemingly not as abundant in the New World. Seven species of this subgenus have been reported from North America: G. aquilinus Threlfall, 1974; G. albeoli Rogers, 1968; G. baeacanthus Wellborn & Rogers, 1967; G. chologastris Mizell, Whittaker & McDougal, 1969; G. lingulatus Rogers 1968, G. fryi Cone & Dechtiar 1984; and G. neili. Interestingly, both G. fryi and G. neili infect esocid hosts (Esox niger and E. lucius L., respectively) which are thought to have been

colonised via host switch from cyprinids to an ancestor of contemporary Esox (see Cone & Dechtiar, 1984; Leblanc et al., 2006), the latter being a predator of cyprinids. The phylogeny presented here seems to support this hypothesis as the two North American members included (Gyrodactylus laevisoides n. sp. and G. neili) group together with high bootstrap support. The Eurasian members form a separate clade that is subdivided into two groups, which correspond to Malmberg’s elegans (including G. elegans,


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Fig. 5 Dendogram depicting the relationship between Gyrodactylus laevisoides n. sp. and related species of Gyrodactylus, based on ITS sequence data. Phylogeny was tested with 1,000 bootstrap repeats using Minimum Evolution, Neighbour-Joining and Maximum Likelihood methods with values reported respectively

G. prostae, G. alburnensis and G. laevis) and phoxini (including G. carassii, G. perccotti, G. magnificus and G. phoxini) species groups. The phylogenetic separation of this lineage into clades infecting Eurasian hosts, consisting mostly of cyprinids, and North American hosts that include cyprinids and esocids, coupled with the absence of members of G. (Gyrodactylus) reported from Eurasian species of Esox, suggests that this particular lineage colonised the New World with an ancestral species of Phoxinus via the Bering land connection around the time of the Pliocene (Lindsay & McPhail, 1986; Chen, 1994) before host switching to Esox. However, other members of G. (Gyrodactylus) may have colonised North America prior to this event. The SEM micrographs revealed three features of the marginal hook sickle which were not evident under light microscopy: a small anterior protrusion from the base of the marginal hook sickle adjacent to the connection point of the handle, a deposit of hardened tissue on the distal side of the marginal hook toe, adjacent to the base of the shaft, and a distinct process or ‘button’ on the heel of marginal hook sickle. Recent studies employing SEM on related taxa have found similar processes on the marginal hook sickle of Ieredactylus rivuli Schelkle, Paladini, Shinn, King, Johnson, Oosterhout, Mohammed & Cable, 2011 (see Schelkle et al., 2011),


Paragyrodactylus sp. (personal observation), and to a lesser degree in Gyrodactylus salinae Paladini, Huyse & Shinn, 2011 (see Paladini et al., 2011). This feature, which is a suspected point of muscle attachment (Schelkle et al., 2011), is seemingly a plesiomorphic trait possessed by basal members of the Gyrodactylidae and one that is revealed best through SEM. Acknowledgements The authors thank Xiang Zhang for technical assistance as well as the New Brunswick Museum (Canada) and the Royal BC Museum (Canada) for funding taxonomic work. We acknowledge the support of the Canadian Healthy Oceans Network. This work was funded in part by a Natural Sciences and Engineering Research Council (NSERC) CGS-D grant awarded to SDK and a NSERC Discovery grant awarded to PB.

References Cable, J., Harris, P. D., Tinsley, R. C., & Lazarus, C. M. (1999). Phylogenetic analysis of Gyrodactylus spp. (Platyhelminthes: Monogenea) using ribosomal DNA sequences. Canadian Journal of Zoology, 77, 1439–1449. Chen, X. Y. (1994). Morphology, phylogeny, biogeography and systematics of Phoxinus (Pisces: Cyprinidae). Ph.D. dissertation, University of Kansas, Lawrence, Kansas. Cone, D. K., Abbott, C., Gilmore, S., & Burt, M. D. (2010). A new genus and species of gyrodactylid (Monogenea) from silver hake, Merluccius bilinearis, in the Bay of Fundy, New Brunswick, Canada. Journal of Parasitology, 96, 681–684.

Author's personal copy Syst Parasitol (2013) 86:285–291 Cone, D. K., & Dechtiar, A. O. (1984). Gyrodactylus fryi n. sp. (monogenea) from Esox masquinongy Mitchill in Ontario. Canadian Journal of Zoology, 62, 1089–1090. Harris, P. D., Cable, J., Tinsley, R. C., & Lazarus, C. M. (1999). Combined ribosomal DNA and morphological analysis of individual gyrodactylid monogeneans. Journal of Parasitology, 85, 188–191. Harris, P. D., Shinn, A. P., Cable, J., Bakke, T. A., & Bron, J. E. (2008). GyroDb: Gyordactylid monogeneans on the web. Trends in Parasitology, 24, 109–111. Kimura, M. (1980). A simple method for estimating evolutionary rates base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution, 16, 111–120. Kritsky, D. C., Leiby, P. D., & Kayton, R. J. (1978). A rapid stain technique for the haptoral bars of Gyrodactylus species (monogenea). Journal of Parasitology, 64, 172–174. Leblanc, J., Hansen, H., Burt, M., & Cone, D. (2006). Gyrodactylus neili n. sp. (Monogenea: Gyrodactylidae), a parasite of chain pickerel Esox niger Lesueur (Esocidae) from freshwaters of New Brunswick, Canada. Systematic Parasitology, 65, 43–48. Lindsay, C. C., & McPhail, J. D. (1986). Zoogeography of fishes of the Yukon and Mackenzie Basins. In: Hocutt, C. H. & Wiley E. O. (Eds.), The Zoogeography of North American Freshwater fishes. New York: John Wiley and Sons, pp. 639-674. Malmberg, G. (1970). The excretory systems and the marginal hooks as a basis for the systematics of Gyrodactylus (Trematoda, Monogenea). Arkiv fo¨r Zoologi, 2, 1–235.

291 Paladini, G., Huyse, T., & Shinn, A. P. (2011). Gyrodactylus salinae n. sp. (Platyhelminthes: Monogenea) infecting the south European toothcarp Aphanius fasciatus (Valenciennes) (Teleostei, Cyprinodontidae) from a hypersaline environment in Italy. Parasites & Vectors, 4, 1–12. Schelkle, B., Paladini, G., Shinn, A. P., King, S., Johnson, M., van Oosterhout, C., Mohammed, R. S., & Cable, J. (2011). Ieredactylus rivuli gen. et sp. nov. (Monogenea, Gyrodactlyidae) from Rivulus hartii (Cyprinidontiformes, Rivulidae) in Trinidad. Acta Parasitologica, 56, 360–370. Shinn, A. P., Hansen, H., Olstad, K., Bachmann, L., & Bakke, T. A. (2004). The use of morphometric characters to discriminate specimens of laboratory-reared and wild populations of Gyrodactylus salaris and G. thymalli (Monogenea). Folia Parasitologica, 41, 239–252. Shinn, A. P., Harris, P. D., Cable, J., Bakke, T. A., Paladini, G., & Bron, J. E. (2010). GyrodDb. World Wide Web electronic publication., version (06/2010). Accessed 20 July 2013. Stasiak, R. (2006). Northern red belly dace (Phoxinus eos): a technical conservation assessment. Resource document. USDA Forest Service, Rocky Mountain Region. http://www. assessments/northernredbellydace.pdf. Accessed 20 July 2013. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., & Kumar, S. (2011). MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 28, 2731–2739.


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