Taxonomy of Myxobolus ridouti N. Sp. and M. ridgwayi N. Sp. (Myxozoa) from Pimephales notatus and Semotilus atromaculatus (Cypriniformes) in Ontario.

Abstract

Myxobolus ridouti n. sp. and M. ridgwayi n. sp. are described, respectively, from Pimephales notatus (Rafinesque) and Semotilus atromaculatus (Mitchill) in Algonquin Park, Ontario, Canada. Both are intracellular in striated muscles of the body flank. Spores of M. ridouti n. sp. are subcircular (9.5-10.5 μm long by 9.4-10.9 μm wide); those of M. ridgwayi n. sp. are oval (10.0-12.1 μm long by 9.5-10.5 μm). Both species have spores with a small, distinct swelling (1 μm) on the spore valve near the posterior sutural ridge, similar to that described for Myxobolus insignis Eiras, Malta, Varella, and Pavanelli, 2005. It looks to be non-nuclear in origin, apparently part of the internal face of a spore valve, and could be taxonomically useful. Partial sequence data of the 18S rDNA from both species are unique among those species in the genus that have been sequenced. The sequences have the greatest overall identity with various species of Myxobolus from cyprinids in North America, Europe, and Asia, with a constructed molecular phylogeny having the 2 new species forming a separate clade.

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TAXONOMY OF MYXOBOLUS RIDOUTI N. SP. AND M. RIDGWAYI N. SP. (MYXOZOA) FROM
PIMEPHALES NOTATUS AND SEMOTILUS ATROMACULATUS (CYPRINIFORMES)
IN ONTARIO
Russell Easy and David Cone*
Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H6. e-mail: reasy@dal.ca
ABSTRACT:
Semotilus atromaculatus (Mitchill) in Algonquin Park, Ontario, Canada. Both are intracellular in striated muscles of the body flank.
Spores of M. ridouti n. sp. are subcircular (9.5–10.5 mm long by 9.4–10.9 mm wide); those of M. ridgwayi n. sp. are oval (10.0–12.1 mm
long by 9.5–10.5 mm). Both species have spores with a small, distinct swelling (1 mm) on the spore valve near the posterior sutural ridge,
similar to that described for Myxobolus insignis Eiras, Malta, Varella, and Pavanelli, 2005. It looks to be non-nuclear in origin,
apparently part of the internal face of a spore valve, and could be taxonomically useful. Partial sequence data of the 18S rDNA from
both species are unique among those species in the genus that have been sequenced. The sequences have the greatest overall identity
with various species of Myxobolus from cyprinids in North America, Europe, and Asia, with a constructed molecular phylogeny having
the 2 new species forming a separate clade.
Myxobolus ridouti n. sp. and M. ridgwayi n. sp. are described, respectively, from Pimephales notatus (Rafinesque) and
There are over 800 nominal species of Myxobolus Bu ¨tschli,
1882 (Myxozoa) described from host fishes worldwide (Eiras,
Molnar et al., 2005; Lom and Dykova ´, 2006; Ferguson et al.,
2008). These parasites are typically histozoic and tissue specific,
developing, as a group, in all host organ systems (Lom and
Dykova ´, 1992). Most species are host specific to freshwater bony
fishes, with limited radiation having occurred within coastal
marine fishes (Bahri et al., 2004). Available evidence suggests that
species of Myxobolus are among the most diverse parasite lineages
in freshwater fishes, with richness reaching as high as 5 species per
individual host (Cone et al., 2004).
The taxonomy of Myxobolus is difficult because the spores of
so many species resemble each other (see Chen and Ma, 1998).
Contemporary species descriptions address this issue by providing
as much detailed information as possible on spore and plasmodial
structure (Eiras and D’Souza, 2004), ultrastructure (Ali et al.,
2003; Tajdari et al., 2005), novel spore morphology (Eiras, Malta
et al., 2005), pathology and nature of the infections (Longshaw et
al., 2003; Levsen et al., 2004), sequence data of the 18S rDNA
(Easy et al., 2005; Molna ´r et al., 2007, 2008, 2009; Ferguson et al.,
2008), and ecological information on tissue and host specificity
(Fomena et al., 2004; Molna ´r et al., 2007). Most authors now try
to use as many of these other features with sequence data, forming
an integrated taxonomic assessment (Lom and Dykova ´, 2006;
Sze ´kely et al., 2009a, 2009b).
The present study describes a new species from striated muscle
of Pimephales notatus (Rafinesque) (Cyprinidae) and a second
from Semotilus atromaculatus (Mitchill) (Cyprinidae), both in
Ontario. We use spore morphology and 18S rDNA sequence data
as the basis for the descriptions.
MATERIALS AND METHODS
Bluntnose minnow (P. notatus) and creek chub (S. atromaculatus) were
caught on 17 May 2004 in baited minnow traps set in Brewer Lake,
Algonquin Park, Ontario, Canada. Live fish were anaesthetized, and
organs were examined microscopically for myxozoans, by teasing of
tissues with forceps, using incident lighting that helps create contrast
between infected and non-infected cells. Plasmodia were examined first in
wet mounts using coverslips and identified by spore morphology. These
plasmodia were then scraped from the slide and placed in 5% formalin (for
later morphological study, during which spores were photographed in
bright field optics of a Zeiss AxioPlan microscope and studied with the
Zeiss Image Analysis System [Zeiss, AxioVision 4.5 software, 2006, Carl
Zeiss Canada Ltd., Toronto, Canada]) or in 95% ethanol for molecular
taxonomy. Tissues with plasmodia were sectioned histologically as
described by Cone et al. (2004). Measurements in the description are
means in mm ± SD, followed by ranges in parentheses.
DNA was extracted from myxozoans using the methods from Easy et al.
(2005). Briefly, tissues containing parasitic spores were lysed in 10 mM
Tris-Cl pH 8.0, 1 mM EDTA (TE), 1% SDS with proteinase K (200 mg/
ml) for 2 hr in a 37 C water bath. Lysates were extracted twice with
phenol:chloroform:isoamyl alcohol (25:24:1), and the DNA was precip-
itated with cold 100% ethanol and 3 M sodium acetate (pH 7.0), followed
by centrifugation at 9,300 g for 10 min. The pellet was washed once with
70% ethanol and air-dried at room temperature. Genomic DNA was re-
suspended in 50 ml TE and stored at 4 C. Quantification of DNA was
completed using a Beckman Spectrophotometer (Beckman Coulter
Canada, Mississauga, Ontario, Canada). The 18S rDNA was amplified
using primers 18r (59-CTACGGAAACCTTGTTACG-39) (Whipps et al.,
2003) and 18e (59-TGGTTGATCCTGCCAGT-39) (Hillis and Dixon,
1991). Primers were synthesised by IDT (Stender Way, Santa Clara,
California) and re-suspended in ddH2O to a stock concentration of
10 mM. PCR was performed in 50-ml reaction volumes containing 1.5 mM
MgCl2, 0.2 mM dNTP, 1.25 units Taq polymerase, 1 mM of each primer,
and 300 ng DNA. Amplifications were performed on a Perkin-Elmer
Gene Amp 9700 Thermocycler (Perkin Elmer, Life and Analytical
Services, Waltham, Massachusetts). Cycling parameters were: initial
denaturation at 95 C for 5 min, followed by 35 cycles of 94 C for
1 min, 55 C for 1 min, 72 C for 1.5 min, and a final extension at 72 C for
10 min. The PCR products were excised from an agarose gel and purified
using UltraClean from ABgene (ABgene, Mississauga, Ontario). Sequenc-
ing reactions were performed using ET terminator chemistry (Amersham
Biosciences, Piscataway, New Jersey) and sequenced on a MegaBACE
1000 capillary sequencer (Amersham Biosciences). The data were edited
using Sequencher (Gene Codes, Ann Arbor, Michigan) and submitted for
database searching using BLASTX (Altschul et al., 1997). Sequences were
aligned using ClustalX software (Thompson et al., 1997). Phylogenetic
analyses were conducted using PAUP version 4.0b10 (PAUP, Sinauer
Associates Inc., Sunderland, Massachusetts) and included character-based
analysis (maximum parsimony) and distance methods (UPGMA, mini-
mum evolution and neighbor joining), with bootstrap analysis of 1,000
replicates using Ceratomyxa shasta as the outgroup.
Sequences used in the phylogeny were: Ceratomyxa shasta Noble, 1950
(AF001579), M. pseudodispar Gorbunova, 1936 (EF466088.1), Myxobolus
sp. CMW-2004 (AY591531.1), M. stanlii nomen nudum (DQ779996.2), M.
pendula Kent, Andree, and Bartholomew, 2001 (AF378340), M. procerus
Easy,Johnson,andCone,2005(AY665926),M.intramusculiEasy,Johnson,
and Cone, 2005 (AY665297), M. musculi Keysselitz, 1908 (AF380141), M.
cyprini Doflein, 1898 (AF380140), M. bartai Salim and Desser, 2000
(AF186835), M. terengganuensis Sze ´kely, Shaharom-Harrison, Cech,
Ostoros, and Molna ´r, 2009 (EU643629.1), M. intimus Zaika, 1965
(AY325285), M. bilobus Cone, Jang, Sun, and Easy, 2005 (DQ008579.1),
Received 22 June 2009; revised 24 July 2009; accepted 24 July 2009.
*Department of Biology, Saint Mary’s University, Halifax, Nova Scotia,
Canada B3H 3C3.
DOI: 10.1645/GE-2232.1
J. Parasitol., 95(6), 2009, pp. 1446–1450
F American Society of Parasitologists 2009
1446

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M. dujardini The ´lohan, 1892 (DQ439804.1), M. hungaricus Jaczo ´, 1940
(AF448444.1), Myxobolus sp. Hungary-EE-2003 (AY325283.1), and M.
obesus Gurley, 1893 (AY325286).
DESCRIPTIONS
Myxobolus ridouti n. sp.
(Figs. 1–4, 9)
Diagnosis:Plasmodiaintracellularinstriated musclecells,fusiformandup
to 300 long; polysporous. Formalin-fixed spores mostly subcircular in
frontal view, 9.9 ± 0.3 (9.5–10.5) (n 5 15) long, 10.1 ± 0.4 (9.4–10.9) wide,
and 6.7±0.01(6.7–6.8) (n5 2) thick. Fiveor6often indistinct,suturalridge
folds. Prominent swelling (1 mm) in posterior of shell of the majority of
spores. Spore length–width 0.98 ± 0.04 (0.92–0.05). Polar capsules pyriform,
converging anteriorly, 5.2 ± 0.3 (4.6–5.6) (n 5 30) long by 3.0 ± 0.27 (2.6–
3.6) (n 5 30) wide; 3–4 filament coils arranged loosely within capsule. No
intercapsular appendix, iodinophilous vacuole, or mucous coat.
Taxonomic summary
Type host: Pimephales notatus (Rafinesque) (bluntnose minnow,
Cyprinidae).
Site of infection: Intracellular in striated muscle of body flank.
Type locality: Brewer Lake, Algonquin Park, Ontario (45u359N,
78u199W).
Prevalence and intensity: Six of 15 fish were infected with an
undetermined number of plasmodia.
Etymology: This species is named for Gary Ridout of the Ontario
Ministry of Natural Resources, who has helped parasitologists collect fish
hosts for many years.
Phototypes: A black and white photograph of a portion of the contents
(photosyntypes) from a single cyst is deposited in the United States
National Parasite Collection (USNPC) (Accession no. 102126.00),
Beltsville, Maryland.
Molecular sequence data: A 765-base pair segment of the 18S rDNA is
deposited in GenBank (GQ292745).
Remarks
Spores of M. ridouti n. sp. resemble most closely those of M. endovasus
(Davis, 1947) from gill filaments of Ictiobus bubalis (Catostomidae), M.
bartai Salim and Desser, 2000, M. nodosus Kudo, 1934 from integument of
P. notatus, and M. transversalis Fantham, Porter, and Richardson, 1939
from muscle of Notropis cornutus (Cyprinidae) in overall size and shape of
the spores. However, spores of M. endovaus and M. bartai have polar
capsules that fill much more of the spore interior and miniscule sutural
ridge folds. Spores of M. nodosus have a markedly irregular posterior edge
as opposed to being smooth in M. ridouti. Spores of M. transversalis have
a small intercapsular appendix.
Myxobolus ridgwayi n. sp.
(Figs. 5–8, 10)
Diagnosis: Plasmodium intracellular in striated muscle cells, fusiform,
and up to 300 long; polysporous. Formalin-fixed spores oval in frontal
view, 11.3 ± 0.5 (10.0–12.1) (n 5 15) long, 10.4 ± 0.3 (9.5–10.5) wide, 6.5
± 0.01 (6.6, 6.7) (n 5 2). Spore length–width 1.13 ± 0.06 (1.03–1.2). Five
or 6 indistinct sutural ridge folds. Prominent swelling (1 mm) in posterior
FIGURES 1–8. (1–4) Fixed spores of Myxobolus ridouti n. sp. (5–8) Fixed spores of Myxobolus ridgwayi n. sp. Bar 5 10 mm.
FIGURES 9–10.
(9) Myxobolus ridouti n. sp. (10) Myxobolus ridgwayi n. sp. Bar 5 5 mm.
Diagrammatic drawings of fixed spores in frontal view.
EASY AND CONE—NEW SPECIES OF MYXOBOLUS1447

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of shell of most spores. Polar capsules pyriform, converging anteriorly, 6.5
± 0.3 (5.8–7.0) (n 5 13) long, 3.2 ± 0.3 (2.0–3.8) wide; 4–5 polar filament
coils, arranged loosely in capsule and oblique to capsule length. No
intercapsular appendix, iodinophilous vacuole, or mucous coat.
Taxonomic summary
Type host: Semotilus atromaculatus (Mitchill) (Creek Chub, Cyprinidae).
Site of infection: Intracellular in striated muscle of body flank.
Type locality: Brewer Lake, Algonquin Park, Ontario (45u359N,
78u199W).
Prevalence and intensity: Two of 11 fish infected with undetermined
number of intracellular plasmodia.
Etymology: This species is named for Mark Ridgway, the director of the
Harkness Fisheries Research Laboratory, Algonquin Park, Ontario, for
his long-time support of fish parasitology research.
Phototypes: A black and white photograph of spores (photosyntypes)
from a single cyst is deposited in the United States National Parasite
Collection (USNPC) (Accession no. 102127.00), Beltsville, Maryland.
Molecular sequence data: An 839-base pair segment of the 18S rDNA is
deposited in GenBank (GQ292746).
Remarks
Overall spore size and shape of M. ridgwayi n. sp. resembles most closely
M. enoblei Lom and Cone, 1996 from gills of Ictiobus bubalis (Catostomi-
dae). However, spores of this latter species have polar capsules that are more
globose in shape and larger relative to the size of the spore. Myxobolus
ridgwayi also resembles M. wellerae Li et Desser, 1985 from muscle of
Notropis cornutus (Cyprinidae) and M. heterolepis Li and Desser, 1985 from
nervous tissue and eye of N. heterolepis (Cyprinidae), but both these species
have spores with a well-developed intercapsular appendix. Also, M. wellerae
has spores with a prominent posterior mucous coat. The new species differs
significantly from M. pendula Gurley, 1964 (syn. M. pellicides Li and Desser,
1985) in base pair makeup of the 18S rDNA.
A posterior swelling, similar to that observed near the sutural ridge in
spores of M. ridouti n. sp. and M. ridgwayi n. sp., has been reported (Eiras,
Malta et al., 2005) in Myxobolus insignis Eiras, Malta, Varella, and
Pavanelli, 2005. We initially thought it was a degenerating nucleus.
However, in Giemsa-stained histological sections valvular, capsular, and
sporoplasmic nuclei stained brilliantly blue–purple while the swelling
remained unstained and hyaline. This suggests it is not nuclear in origin.
The fact that the structure was not seen when spores were viewed from the
side suggests that it is positioned on the internal face of a spore valve (this
also was the impression one had from histological sections of spores) and
may be a character of taxonomic significance.
Molecular phylogeny
Analysis of a 765-base pair region of the 18S rDNA from M. ridouti n.
sp. and M. ridgwayi n. sp. showed .10% sequence divergence between the
2 species. However, both species formed a distinct separate clade in a
phylogeny (Fig. 11) prepared of those myxobolids, with the greatest
sequence similarity from the BLAST search. Distance methods revealed
similar topology to character-based analyses (maximum parsimony).
Nodal support for trees constructed by the 4 methods revealed groupings
of lower confidence; however, the separate clade of M. ridouti and M.
ridgwayi showed $80 bootstrap support in all analyses. All of these
represent species reported from cyprinids in North America, Europe, and
Asia.
DISCUSSION
With the 2 new additions, there are now 126 nominal species of
Myxobolus described or reported from freshwater fishes in North
FIGURE 11.
accession numbers are shown in parentheses. Bootstrap values represent 1,000 replicates.
Maximum parsimony tree of 18S rDNA sequences of Myxobolus species described in this paper with other myxozoan species. GenBank
1448THE JOURNAL OF PARASITOLOGY, VOL. 95, NO. 6, DECEMBER 2009

Page 4

America. Of these, 12 are suspected to represent misidentifica-
tions, i.e., M. artus Akhmerov, 1960, M. cyprini Doflein, 1898, M.
cyprinicola Reuss, 1906, M. dujardini (The ´lohan, 1892), M.
microlatus Li and Nie, 1973, M. muelleri Butschli, 1882, M.
multiplicatus (Reuss, 1906), M. musculi Keysselitz, 1908, M.
nemachili Weiser, 1949, M. neurobius Schuberg et Schro ¨der, 1905,
M. scardini Reuss, 1906, and M. squamae Keysselitz, 1908. We
suspect these taxa are misidentified because these parasites infect
fishes on other continents with no zoogeographical connection to
the hosts examined herein (Grinham and Cone, 1990).
The remaining 114 species are distributed throughout 13 host
families (Cyprinidae 42 species, Catostomatidae 23, Centrarchidae
21, Salmonidae 8, Cyprinodontidae 6, Esocidae 3, Percidae 4,
Percopsidae 2, Clupeidae 1, Cottidae 1, Ictaluridae 1, Gasterostei-
dae 1, and Poeciliidae 1) and have been reported mostly from hosts
inNorth America. One ofthese, M. cerebralis Hofer,1903, isa well-
documented introduction from Europe. Interestingly, Myxobolus
catostomi, Farnham, Porter and Richardson, 1939 is known to
occur in Catostomus catostomus in eastern Asia (Pugachev, 1980).
The molecular phylogeny of freshwater species of Myxobolus,
based on GenBank depositions of about 120 species, most of
which are from North America, Europe, and Asia, shows
paraphyly–polyphyly and an overall poor correlation between
strict spore morphology, tissue specificity, features of the
actinospores, and the geographical distribution in freshwater
host families (Fiala, 2006). Embedded in this complex phylogeny,
however, are terminal clades showing some distinction with these
factors. For example, Molna ´r et al. (2008) describe a lineage
involving 4 myxobolids with ellipsoidal shaped spores, all with
similar 18S rDNA sequences and all parasitizing gills of the roach
(Rutilus rutilus) and bleak (Alburnus alburnus) in Europe, that
forms a terminal clade embedded among 24 other species of
Myxobolus from cyprinid fishes. Cone et al. (2005) found that
Myxobolus bilobus Cone, Yang, Sun, and Easy, 2005 occurred,
with high bootstrap support, within a clade that included 10 other
species of the genus from gills or muscle tissue of various cyprinid
fishes from North America and Eurasia. Similarly, Ferguson et al.
(2008) examined species of Myxobolus from salmonids in western
North America and Japan, most of which had pyriform-shaped
spores and most of which developed in the brain, spinal cord, or
peripheral nerves. These species formed another terminal clade
positioned among a complex phylogeny of different spore types
infecting a variety of fish families. The present results indicate that
the greatest percent similarity of the 18S rDNA of the new species
lies with those species in the genus described from other cyprinid
fishes in the Northern Hemisphere. The molecular phylogeny
further suggests that amongst these species, M. ridouti n. sp. and
M. ridgwayi n. sp. are members of a separate clade.
ACKNOWLEDGMENTS
The authors thank J. Yang for technical help and the staff of the
Harkness Fisheries Research Laboratory for field assistance and
hospitality. This work was supported by an NSERC Discovery Grant
awarded to D. K. C. and by Saint Mary’s University.
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