J. Parasitol., 97(3), 2011, pp. 493–502 F American Society of Parasitologists 2011

THELOHANELLUS TOYAMAI (SYN. TOYAMAI ) INFECTING THE GILLS OF KOI CYPRINUS CARPIO IN THE EASTERN UNITED STATES

Matt J. Griffin and Andrew E. Goodwin* Thad Cochran National Warmwater Aquaculture Center, Mississippi State University, PO Box 197, 127 Experiment Station Road, Stoneville, Mississippi 38776. e-mail: [email protected]

ABSTRACT: A myxozoan resembling of Thelohanellus was isolated from the gills of koi (Cyprinus carpio) cultured in North Carolina. Plasmodia measuring ,200 mm in diameter contained tear-shaped myxospores containing a single pyriform polar capsule. The spore body was concave on one side, measuring 16.2 (14.7–16.8) mm long and 5.6 (4.5–6) mm wide. The polar capsule was 6.4 (5.8– 7.2) mm long and 4.2 (3.4–4.6) mm wide, containing a polar filament coiled perpendicular to the longitudinal axis of the spore body making 8 turns. Occasionally, an oblong, irregularly shaped mass of protoplasm was observed between the polar capsule and spore capsule. Analysis of 18S small-subunit ribosomal DNA sequence demonstrated this isolate as a 99.9% match with Myxobolus toyamai from gills of C. carpio. However, the case isolate lacked the characteristic second polar capsuleofMyxobolus, morphologically placing it within the Thelohanellus. Here we supplement genetic sequence data with histopathology, an amended morphological description of the agent, and a review of the original classification. For future reference, we suggest this organism be referred to as Thelohanellus toyamai Kudo, 1933, in accordance with the original classification and the nomial M. toyamai be avoided because it is at best outdated and, at worst, incorrect.

As a consequence of its widespread introduction to temperate information and vice versa. We also attempt to clarify the waters as a food and ornamental , Cyprinus carpio (common ambiguity surrounding the of T. toyamai and carp and koi) has become one of the most widely distributed Myxobolus toyamai, as well as provide further evidence to freshwater fish species in North America. As such, an in-depth support the monophyletic origins of Myxobolus, Henneguya, knowledge of its pathogens is of significant importance for and Thelohanellus within the . fisheries biologists and fish health professionals (Dykova et al., 2003). Several Thelohanellus spp. have been described from C. MATERIALS AND METHODS carpio, most from European and Asian waters (Molna´r, 1982; Molna´r and Kova´cs-Gayer, 1982; Rhee et al., 1993; Moshu and Origin of parasite Molna´r, 1997; Yokoyama, 1997). Thelohanellus toyamai has been Koi (20–25 cm total length) from a commercial fish farm in North reported from the gills of common carp in Japan, northern Carolina were submitted for diagnosis because of unusual gill lesions that Vietnam, Asia, and Europe (Hoffman, 1999) but has not been were grossly apparent. The producer reported that some fish were gasping at the surface, even though dissolved oxygen concentrations were high. Initial reported from common carp or koi in the United States. examination revealed the fish to be infected by several different species of Alternatively, Myxobolus toyamai, suggested to be synonymous myxosporeans present in such high densities that normal gill architecturewas with T. toyamai (Shul’man, 1966; Hoffman, 1999), has been severely disturbed. Samples of gill tissue were preserved in 10% neutral reported from North American waters (Eiras et al., 2005), buffered formalin for histology and in 70% ethanol for the preservation of DNA to aid in the identification and characterization of the parasite. indicating that Thelohanellus spp. may have been previously isolated from North American but reported as Myxobolus Gross and histological examination spp. Given the morphological similarities between closely related myxozoans and the poor communication that can exist between Individual pseudocysts of the tentatively identified Thelohanellus sp. (n 5 3) were excised from ethanol-preserved tissue by sharp dissection, placedon fish health professionals from different countries, many species a microscope slide with a drop of physiological saline (0.85% [w/v] of NaCl), may have been inadequately described and identified, thus adding and mechanically ruptured. The myxospores were further diluted with saline, ambiguity to host and geographic ranges for many myxozoan cover slipped, and examined using an Olympus BX-50 microscope (Olympus parasites. In the absence of supplemental molecular data, many Corporation, Tokyo, Japan). Representative images (n 5 15) from the pooled myxospores were captured using a Spot Insight QE digital camera synonyms possibly exist (Zhang et al., 2010). Consequently, it has and analyzed using Spot Basic 3.1 Image analysis software (Diagnostic been suggested on several occasions that molecular sequence data Instruments, Sterling Heights, Michigan). A separate preparation of be included when new species are described or original myxospores was prepared and stained with Lugol’s iodine (ENG Scientific, descriptions are revised. This would eliminate the inconsistencies Clifton, New Jersey) for visualization of the iodinophilous vacuole. Tissue from formalin-preserved gills was dehydrated, embedded in that can arise from identifications based on morphology alone paraffin, and sectioned at 2 um. Sections were stained with hematoxylin (Kent et al., 2001; Lom and Dykova, 2006; Whipps and Diggles, and eosin stain and examined by light microscopy. 2006; Griffin, Khoo, et al., 2009; Griffin, Wise, et al., 2009). In the present study, we describe a myxozoan from farmed koi. Molecular analysis Morphometrics, histopathology, and 18S small-subunit (SSU) Individual pseudocysts of the tentatively identified Thelohanellus sp. ribosomal DNA (rDNA) sequence data demonstrate the impor- (n 5 3) were identified microscopically from archived tissue sections fixed tance of supplementing morphological data with DNA sequence in 70% ethanol, excised by sharp dissection, placed individually into a 1.5-ml microcentrifuge tube containing 100 ml of nuclease-free water, and the pseudocycts ruptured by mechanical agitation. The sample tube was Received 11 October 2010; revised 16 December 2010; accepted 31 confirmed to contain only the target organism by placing a drop of water December 2010. from the tube on a glass microscope slide and examining the contents *Aquaculture/Fisheries Center, University of Arkansas at Pine Bluff, carefully. Once the sample prep was determined to contain only the Mail Slot 4912, 1200 North University Drive, Pine Bluff, Arkansas Thelohanellus sp., 600 ml of PuregeneH Cell Lysis Solution (Gentra 71601. Systems, Minneapolis, Minnesota) was added to the sample tube. DOI: 10.1645/GE-2674.1 Following an initial incubation of 10 min at 95 C, 3 ml of proteinase K

493 494 THE JOURNAL OF PARASITOLOGY, VOL. 97, NO. 3, JUNE 2011

TABLE I. Primer sequences used in this study.

Primer Sequence (59–39) Direction Sequence location (bp)* Reference

H9 .TTACCTGGTCCGGACATCAA .Forward 1331–1350 .Hanson et al., 2001 H2 .CGACTTTTACTTCCTCGAAATTGC .Reverse 2021–2024 .Hanson et al., 2001 MyxospecF .TTCTGCCCTATCAACTWGTTG .Forward 301–322 .Fiala, 2006 MyxospecR .GGTTTCNCDGRGGGMCCAAC .Reverse 1214–1195 .Fiala, 2006 Genmyxo3 .TGATTAAGAGGAGCGGTTGG .Forward 982–1001 .Griffin et al., 2008 Genmyxo4 .GGATGTTGGTTCCGTATTGG .Forward 957–976 .Griffin et al., 2008 Genmyxo5 .TAAGCGCAGCAACTTTGAGA .Reverse 617–598 .Griffin et al., 2008 ERIB1 .ACCTGGTTGATCCTGCCAG .Forward 2–20 .Barta et al., 1997 ERIB10 .CTTCCGCAGGTTCACCTACGG .Reverse 2079–2059 .Barta et al., 1997

* Sequence location based on the 18S SSU rDNA sequence of Henneguya exilis (AF021881).

(20 mg/ml) was added to the lysate, and the tube was mixed by inversion the most similar sequences (excluding duplicates) identified by the BLAST and incubated at 56 C overnight. The remainder of the isolation was search. From this initial dataset, the number of sequences was reduced to carried out according to the manufacturer’s suggested protocol. The include all Thelohanellus spp. sequenced to date, sequences from the 25 purified genomic DNA was then suspended in 30 ml of Puregene DNA most closely related myxobolids, as well as sequences from representative hydration solution (10 mM Tris, 1 mM EDTA, pH 7.0–8.0). members of well-defined groups within the Myxobolidae (Andree et al., 1999; Salim and Desser, 2000; Kent et al., 2001; Molna´r et al., 2002; 18S SSU rDNA gene amplification Eszterbauer, 2004; Fiala, 2006; Ferguson et al., 2008; Iwanowicz et al., 2008; Griffin, Khoo, et al., 2009; Sze´kely et al., 2009; Camus and Griffin, The 18S SSU rDNA gene was first amplified with the universal 2010). eukaryotic primers ERIB1 and ERIB10 (Barta et al., 1997; Fiala, 2006) Myxozoan sequences from representative groups within the Myxobo- (Table I). Nested PCR reactions were carried out in duplicate using the lidae were downloaded from the NCBI nucleotide database and aligned generic myxozoan primer sets H2/H9 described by Hanson et al. (2001), using the Clustal W application of the Molecular Evolutionary Genetics MyxospecF-MyxospecR from Fiala (2006), and Genmyxo3, Genmyxo4, Analysis, v. 4.0 (MEGA4) software package (Tamura et al., 2007). The and Genmyxo5 from Griffin et al. (2008) (Table I). The initial 25-ml PCR sequences used in phylogenetic analysis are listed in Table II. reaction mixture contained 2.5 ml of TaKaRa TaqH Hot Start Version 103 shasta (GenBank accession number AF001579) was chosen as an PCR Buffer (TaKaRa Bio, Otsu, Shiga, Japan), 5 mM of each outgroup for phylogenetic analysis. deoxyribonucleotide triphosphate, 10 pmol of each primer, 0.625 U of Phylogenetic and molecular evolutionary analyses were conducted on TaKaRa Taq Hot Start Taq polymerase, template, and nuclease-free the obtained 18S SSU rDNA sequence using the MEGA4 software water to volume. The reaction mixture was cycled on a PTC-100 thermal package (Tamura et al., 2007). The data were analyzed by maximum cycler with an initial denaturation step of 95 C for 10 min, followed by 30 parsimony analysis (MP) using a close-neighbor-interchange (CNI) search cycles of 95 C for 1 min, 48 C for 1 min, 72 C for 2 min, and a final level 7, in which the initial trees were obtained with the random addition extension step of 72 C for 10 min. Two microliters of PCR product from of sequences (1,000 replicates) with all alignment gaps treated as missing the initial reaction were used in nested PCR with the following primer data (Eck and Dayhoff, 1966; Nei and Kumar, 2000). Minimum evolution combinations: Erib1/Genmyxo5, H2/H9, MyxospecF/MyxospecR, Gen- (ME) distance analysis also was performed using a CNI search level 7, myxo3/H2, and Genmyxo4/H2. All reaction components remained the with evolutionary distances determined by maximum composite likelihood same except that 14.75 ml nuclease-free water was used to bring the (Rzetsky and Nei, 1992; Nei and Kumar, 2000; Tamura et al., 2004). The reaction volume to 25 ml, and the annealing temperature was 52 C instead initial tree for ME analysis was generated using the neighbor-joining of 48 C. The PCR products were detected by electrophoresis through a algorithm with all positions containing alignment gaps and missing data 1.2% agarose gel and visualized under ultraviolet light after ethidium eliminated only in pairwise sequence comparisons (pairwise deletion bromide staining. option) (Saitou and Nei, 1987; Tamura et al., 2004). Clade support for both analyses was assessed by bootstrapping (1,000 replicates for ME; DNA sequencing 1,000 replicates for MP) (Felsenstein, 1985). The PCR products were purified using the QIAquickH PCR Purification Kit (Qiagen, Valencia, California), resuspended in 20-ml Puregene DNA REDESCRIPTION hydration solution and quantified using a NanodropH spectrophotometer and the accompanying software (Nanodrop Technologies, Wilmington, Thelohanellus toyamai Kudo, 1933 Delaware). Each product was sequenced directly 3 times in each direction Synonym: Myxobolus toyamai Kudo, 1915 by dideoxy chain termination sequencing (Sanger et al., 1977) with an ABI Spore morphology: Plasmodia containing Thelohanellus myxospores Prism Dye Termination Cycle Sequencing Kit (Applied Biosystems, Foster consisted of ovate pseudocysts, approximately 200 mm in diameter, situated City, California) using approximately 40 ng of template per reaction. The within central sinus of gill filaments. Removal and subsequent rupture of products were purified using Centri-Sep Spin columns (Princeton pseudocysts during wet-mount preparation revealed many myxospores Separations, Adelphia, New Jersey) and analyzed at the USDA Mid- lacking bilateral symmetry, with single polar capsules situated toward South Area Genomics Laboratory in Stoneville, Mississippi. The obtained anterior end of spore body; concave on one side (Figs. 1, 2). Excised sequence fragments were assembled using the SeqMan utility of the myxospores elongate and pyriform, with rounded posterior, 16.2 (14.7– Lasergene software package (DNASTAR, Madison, Wisconsin), and 16.8) mm long and 5.6 (4.6–6) mm wide in valvular view. Single pyriform sequence ambiguities were clarified using corresponding ABI chromato- polar capsule measured 6.4 (5.8–7.2) mm long and 4.2 (3.4–4.6) mm wide with grams. single polar filament coiled perpendicular to long axis of spore body making 8 turns. Occasionally an oblong, irregularly shaped mass of protoplasm Phylogenetic analysis observed between polar capsule and spore capsule. Intercapsular appendix, iodinophilous vacuole, and mucous coat not observed. The obtained sequence was compared with similar myxozoan sequences identified using a BLAST (megablast; http://blast.ncbi.nlm.nih.gov/Blast. Taxonomic summary cgi) search of the NCBI nonredundant nucleotide database for highly similar sequences. Preliminary analysis (data not shown) included 75 of Host: Common carp Cyprinus carpio (Cyprinidae). GRIFFIN AND GOODWIN—T. TOYAMAI FROM KOI IN THE EASTERN UNITED STATES 495

TABLE II. Myxozoan sequences (GenBank accession number) used in phylogenetic analysis.

Species GenBank accession no.

Thelohanellus spp. T. hovorkai .DQ231155 T. kitauei .GQ396677 T. nikolskii .DQ231156 T. sinensis .DQ452013 T. wuhanensis .AY165181 T. zahrahae .EU643622 Myxobolus spp. M. ampullicapsulatus .DQ339482 M. basilamellaris .AF507971 M. bilobus .DQ008579 M. bizerti .AY129318 M. carassii .DQ452012 M. cerebralis .U96492 M. csabai .EU643628 M. cultus .AB121146 M. cycloides .DQ439810 M. dogieli .EU003978 M. ellipsoides .DQ439813 M. episquamalis .AY129312 M. gayerae .DQ439809 M. hungaricus .AF448444 M. koi .FJ710800; FJ841887 M. lentisuturalis .AY278563 M. leptobarbi .EU643623 M. longisporus .AY364637 M. macrocapsularis .FJ716095 M. margitae .EU598803 M. musseliusae .FJ710801 FIGURE 1. Wet-mount preparations of the myxospore stage of M. neurobius .AF085180 Thelohanellus toyamai demonstrating the coiled polar filaments within a M. neurophilus .FJ468489 single polar capsule. Spores resting in both valvular (V) and sutural (S) M. obesus .AY325286 view; Nomarski interference; scale bars 5 25 mm. M. pellicides .AF378339 M. pendula .AF378340 Locality: Fish cultured in the North Carolina. The producer has a M. pseudokoi .AF186839 history of importing brood fish from Asia. M. rotundus .FJ851449 Other localities: None. M. tasikkenyirensis .EU643626 Site of tissue development: Within connective tissue of the gill filament. M. toyamai .FJ710802 Prevalence of infection: Unknown. M. wulii .EF690300 Gross and histological examination Henneguya spp. H. adiposa .EU492929 Microscopic examination of wet mounts revealed that all gill filaments H. akule .EU016076 were filled with a series of nodules of various sizes distorting the normal architecture of the gill (Fig. 3). When the cysts were mechanically H. cutanea .AY676460 ruptured, the smaller cysts ruptured easily and were difficult to isolate H. daoudi .EU643625 because of their small size and proximity to each other. However, these H. doneci .EU344899 smaller cysts contained large numbers of myxospores morphologically H. doori .U37549 consistent with descriptions of Myxobolus longisporus and Myxobolus koi H. gurlei .DQ673465 (Yokoyama et al., 1997; Dykova et al., 2003; Eiras et al., 2005). H. ictaluri .AF195510 Alternatively, the larger cysts (,200 mm in diameter), which were easily H. lateolabracis .AB183747 isolated due to their size and structural integrity, consistently contained a H. lesteri .AF306794 myriad of pyriform, tear-shaped spores with a single ellipsoid polar H. rhinogobii .AB447993 capsule, consistent with Thelohanellus spp. (Hoffman, 1999). H. salminicola .AF031411 Histologically, pseudocysts up to 200 mm in diameter located in the H. shaharini .EU643630 central sinus of the gill filaments immediately adjacent to the gill filament cartilage. Distention of the gill filament was pronounced, and lamellae H. weishanensis .AY165182 adjacent to the cyst were no longer present. There was very little evidence H. zschokkei .AF378344 of inflammation associated with the pseudocyst surface (Fig. 4).

Remarks Lom and Dykova (2006) described 2 distinct morphotypes of Thelohanellus, one containing a pyriform, tear-shaped polar capsule with 496 THE JOURNAL OF PARASITOLOGY, VOL. 97, NO. 3, JUNE 2011

FIGURE 2. Wet-mount preparations and schematic of the myxospore stage of Thelohanellus toyamai demonstrating the coiled polar filaments within the single polar capsule and the oblong protoplasmic mass sometimes mistaken as a second, stunted polar capsule (arrows). Scale bars 5 10 mm.

a single coil of polar filament similar to the case isolate. The second morphotype possessed a subspherical polar capsule with a second, inner coil. Comparisons of key morphological characteristics of several Thelohanellus spp. similar to the case isolate are presented in Table III. Sequence analysis for a 1964 bp region of the 18S SSU rDNA gene revealed this isolate to be 99.9% similar (1962 of 1964 bp) to M. toyamai reported from C. carpio in Asia (FJ10802). Phylogenetically, this isolate clusters (98% bootstrap confidence for NJ; 100% for ME) with other myxobolids isolated from the gills of common carp (Figs. 5, 6), demonstrating tissue tropism within the clade that has been well documented by previous investigators (Martyn et al., 2002; Zhao et al., 2008; Zhang et al., 2010). A multiple alignment of sequences from the most closely related myxozoan species obtained from GenBank revealed 3 highly variable regions within the 18S SSU rDNA locus, similar to those described previously by Iwanowicz et al. (2008). Pairwise comparisons revealed multiple bases differing between the case isolate and sequences of several other Myxobolus spp. However, there was high degree of sequence similarity between the case isolate and M. toyamai (Fig. 7), suggesting they are the same organism, even though the case isolate lacked the characteristic second polar capsule of Myxobolus spp.

FIGURE 4. Hematoxylin and eosin-stained histological sections of koi gill demonstrating two irregular coalescing plasmodia disrupting the normal lamellar architecture (A), and at higher magnification (B), the asynchronous development of the myxospores, with the mature myxo- FIGURE 3. A gill arch from a koi (Cyprinus carpio) infected with several spores (black arrows) and the amorphous immature spores (white arrows) myxozoan parasites. Arrows depict pseudocysts containing T. toyamai. located at the periphery of the plasmodia. TABLE III. Key morphological characteristics of Thelohanellus spp. including several formerly classified as Myxobolus (Kudo, 1933). LPC, length of polar capsule; LSB, length of spore body; PCS, polar capsule shape; SI, site of infection; WPC, width of polar capsule; WSB, width of spore body. All measurements are provided in micrometers (mm). Values in parentheses denote size ranges when reported in conjunction with mean values. Dash 5 data not reported.

Species LSB WSB LPC WPC PCS SI Host References

Thelohanellus toyamai 16.2 (14.7–16.8) 5.6 (4.5–6.0) 6.4 (5.8–7.2) 4.2 (3.4–4.6) Pyriform Gills Cyprinuscarpio This paper (case isolate) Thelohanellus toyamai 15 7–8 7–8 3–4 Pyriform Gills Cyprinus carpio Kudo, 1919, 1933 syn. Myxobolus toyamai Hoffman, 1999 Thelohanellus fuhrmanni 18–20 8 9–10 — Pyriform Many organs Several fishes Kudo, 1919, 1933 syn. Myxobolus fuhrmanni Hoffman, 1999 GOODWIN— AND GRIFFIN Thelohanellus kitauei 26.3 (23–29) 9.2 (8–11) 16.8 (14–18) 7.4 (6–9) Pyriform Intestinal wall Cyprinus carpio Egusa and Nakajima, 1981 Hoffman, 1999 Thelohanellus misgurni 14.–15.5 6–7.3 6.3 2–3 Pyriform Gall bladder Misgurnus Kudo, 1919, 1933 syn. Myxobolus misgurni anguillicaudatus Thelohanellus notatus 17–18 7.5–8.0 7.0 4.0 Pyriform Subdermal Several fishes Kudo, 1919, 1933 syn. Myxobolus notatus Hoffman, 1999 Thelohanellus oculi-leucisci 9–10 4.5–5.5 5 2 Pyriform Vitreous humor Several fishes Kudo, 1919, 1933 syn. Myxobolus oculi-leucisci of eyes Hoffman, 1999 Thelohanellus pyriformis 16–18 7–8 7.5 3.5 Pyriform Many organs Several fishes Kudo, 1919, 1933 TOYAMAI T. syn. Myxobolus pyriformis Hoffman, 1999 Thelohanellus rohitae 30–32 7–8 22–23 — Pyriform Gills Labeo rohita Kudo, 1919, 1933 syn. Myxobolus rohitae Thelohanellus seni 13.2–13.6 10.1–10.3 4 — Pyriform Fins Labeo rohita Kudo, 1919, 1933 497 STATES UNITED EASTERN THE IN KOI FROM syn. Myxobolus seni Thelohanellus unicapsulatus 12–13 7–8 6 3 Pyriform Skin Labeo niloticus Kudo, 1919, 1933 syn. Myxobolus unicapsulatus Thelohanellus zahrahae 23.8 (21.7–26.3) 9.0 (8.5–9.4) 9.9 (7.9–10.8) 6.3 (5.3–6.6) Pyriform Gills Barbonymus Sze´kely et al., 2009 gonioinotus 498 THE JOURNAL OF PARASITOLOGY, VOL. 97, NO. 3, JUNE 2011

FIGURE 6. Phylogenetic tree generated by minimum evolution analysis of the 18S rDNA sequences of selected myxosporeans, rooted at Ceratomyxa shasta. Members of the Thelohanellus are highlighted in gray. Evolutionary distances were computed using the maximum composite likelihood method and are in the units of the number of base substitutions per site. Numbers at FIGURE 5. Phylogenetic tree generated by maximum parsimony analysis of the 18S rDNA sequences of selected myxosporeans, rooted at Ceratomyxa nodes indicate bootstrap confidence values (1,000 replications). M., shasta. Members of the Thelohanellus are highlighted in gray. Numbers Myxobolus;H.,Henneguya;T.,Thelohanellus;C.,Ceratomyxa. at nodes indicate bootstrap confidence values (1,000 replications). M., Myxobolus;H.,Henneguya;T.,Thelohanellus;C.,Ceratomyxa. known about the group. According to a review by Lom and DISCUSSION Dykova (2006), there are at least 75 known species, of which only T. hovorkai, Thelohanellus kitauei, and T. nikolskii are thought to There have been several reports of Thelohanellus spp. from be pathogenic. Recently Sze´kely and others (2009) added to the common carp; however, outside of Thelohanellus hovorkai and list of potentially pathogenic species when they identified Thelohanellus nikolskii (Sze´kely et al., 1998), relatively little is Thelohanellus zahrahae from the java barb (Barbonymus goniono- GRIFFIN AND GOODWIN—T. TOYAMAI FROM KOI IN THE EASTERN UNITED STATES 499

FIGURE 7. Alignment of 18S SSU rDNA sequences emphasizing 3 highly variable regions within the gene locus. Periods designate conserved regions; dashes designate gaps or missing data. Nucleotide positions are based on the 18S SSU rDNA gene sequence of Myxobolus longisporus (AY364637). tus), suggesting the parasite may have pathological significance in identifying 6 from the common carp, including T. toyamai. Malaysian fish culture during cases of heavy infections. However, this number may be higher as Thelohanellus dogieli was Several Thelohannelus spp. have been the subject of studies in listed as a synonym of T. hovorkae and T. nikolskii, both of which Europe and Asia, focusing on everything from the life cycle, to have since been molecularly confirmed as distinct species pathology associated with infection, to the development of (Eszterbauer et al., 2006). Of the 13 species reported by Hoffman chemotherapeutic treatments of myxozoan infections in the host (1999) that could potentially infect North American fishes, only 4 (Rhee et al., 1993; Yokoyama, 1997; Liyanage et al., 1998; Sze`kely described species have actually been collected from North et al., 1998; Yokoyama et al., 1999; Molna´r, 2002; Yokoyama et American waters, none of which was T. toyamai. al., 2006). Hoffman (1999) cited 13 different Thelohanellus spp. With the advent of molecular techniques and the increasing that could potentially infect fishes of North American waters, availability of sequencing technology, it is important to encourage 500 THE JOURNAL OF PARASITOLOGY, VOL. 97, NO. 3, JUNE 2011 the simultaneous submission of supplemental sequence data with al., 2008; Iwanowicz et al., 2008; Griffin, Khoo, et al., 2009; all species descriptions, new or otherwise. However, the original Griffin, Wise, et al., 2009). As such, phylogenetic classifications description of T. toyamai was published well before the advent of should not be made based on 18S SSU rDNA sequences alone, molecular techniques (Kudo, 1919, 1933). Additionally, the optics because isolates identified morphologically as Myxobolus or available at the time were significantly inferior to the digital Thelohanellus may group molecularly with Henneguya spp. and imagery and high-resolution optics available today. As such, vice versa. Thus, it is not surprising to find this isolate to fall discrepancies between morphological descriptions made today genetically within a well-recognized clade of Myxobolus, although and those made nearly a century ago are inevitable. morphologically it has been classified as a Thelohanellus. Similar to Thelohanellus pyriformis, Thelohanellus notatus, and Current taxonomic classifications are based largely on spore several other Thelohanellus spp., T. toyamai was originally morphology, discriminating between species and genera based on classified as a Myxobolus containing a single polar capsule the number and configuration of shell valves, polar capsules, and (Kudo, 1919, 1933). The original description of M. toyamai presence or absence of caudal processes. Unfortunately this (Kudo, 1919) depicts a pyriform myxospore, with attenuated classification system has limitations, especially at the family level, anterior and rounded posterior ends, no bilateral symmetry, since differences in key morphological characteristics between curved lateral sides, a single pyriform polar capsule containing a closely related genera can be ambiguous (Lom and Dykova, distinct coiled polar filament at the anterior end with a small, 2006). Recently several studies using 18S rDNA sequences to oblong mass of protoplasm between the polar capsule and the determine taxonomic relationships have shown that molecular shell. In 1933, to accommodate the several species of Myxobolus phylogenies for many myxozoans demonstrate interrelatedness containing a single polar capsule, Thelohanellus was recognized as based more on tissue tropism, host species, and geographic a distinct genus, differentiated from the Myxobolus by the absence distribution rather than spore morphology (Eszterbauer, 2002; of a second polar capsule. This resulted in the immediate Molna´r et al., 2002; Eszterbauer, 2004; Easy et al., 2005; Molnar reclassification of 11 species of Myxobolus, including M. toyamai et al., 2008). Still, for other groups, such as the ictalurid-infecting (Kudo, 1933). Several of these species are listed along with their clade of Henneguya spp., taxonomic organization based on spore key morphological characteristics in Table III. The data present- morphology and host predilections are in agreement with ed here support the original description identifying the absence of molecular phylogenies (Iwanowicz et al., 2008; Griffin, Khoo, et a second polar capsule. According to the defined characteristics of al., 2009; Griffin, Wise, et al., 2009). Although useful, there are the Myxobolus and Thelohanellus genera (Kudo, 1933; Shul’man, limitations with determining taxonomic relationships based on 1966; Hoffman, 1999), the pyriform, tear-shaped spore with a molecular systematics alone. Most molecular phylogenies for the single pyriform, tear-shaped polar capsule places this isolate Myxozoa, because of convenience and sequence availability, are within the Thelohanellus. based on a single gene (18S SSU rDNA), with a majority of the Lom and Dykova (2006) cited the difficulties in discriminating sequences obtained from GenBank. As a consequence, these between Thelohanellus spp. with only 1 polar capsule and studies are dependent upon accurate species identifications and Myxobolus spp. with 2 polar capsules, 1 of which is extremely descriptions for the deposited sequences. Accordingly, ambiguous stunted. This is also evident within the literature, as many descriptions have significant impacts on taxonomic classifications. synonyms between the 2 genera exist (Kudo, 1933; Shul’man, The rapidity at which new species of Myxozoa are being identified 1966; Hoffman, 1999). This ambiguity is suggestive of the close and previously described species are being genetically character- interrelatedness of Myxobolus and Thelohanellus, which has been ized stresses the importance of supplementing genetic sequence supported by molecular phylogenetics. The data presented in this data with accurate morphological descriptions, as well as study also demonstrate the similarity of the 2 genera, placing this sufficient data regarding host type, site of infection, and isolate more closely to Myxobolus spp. isolated from common geographic locale in order to maintain constancy within the carp in Asia than to Thelohanellus spp. (100% bootstrap support group (Lom and Dykova, 2006). The findings presented here for both MP and ME analysis) isolated from different hosts. further demonstrate the limitations of using a single gene (18S Similarly, based on 18S rDNA sequences, there is significant SSU rDNA) for taxonomic classifications within the Myxozoa, as genetic divergence between different members of the Thelohanel- they do not always correlate directly with morphological lus, as some species are more closely related to Myxobolus spp. classifications. Instead, molecular data are most useful when and Henneguya spp. than other Thelohanellus spp. This suggests a used to supplement morphological classification, providing a monophyletic origin for Thelohanellus, Myxobolus, and Henne- second level of distinction to assist in confirmation of species guya, which supports previous claims regarding the monophyletic identification or to aid in differentiating between morphologically origins of Myxobolus and Henneguya within the Myxobolidae similar isotypes from different hosts and geographic regions. (Kent et al., 2001; Fiala, 2006; Lom and Dykova, 2006). The The original description for M. toyamai, prior to the acceptance significant genetic separation between species of Thelohanellus, of Thelohanellus as a distinct genus, reported the absence of a coupled with the considerable geographic separation and vari- second polar capsule (Kudo, 1919). Shu’lman (1966) later ability in preferred habitats of these different host families, mentions a second stunted polar capsule significantly smaller suggests that paired polar capsules have developed (or resolved) than the first, mentioning that it is often not recognized as a polar during separate evolutionary events within the Myxobolidae, capsule. This is likely referring to the oblong irregular mass of although the selective pressures that have led to these develop- protoplasm from the original description (Kudo, 1919), which ments remain unclear. In a similar fashion, Henneguya spp. was occasionally observed in this study. Both Shul’man (1966) isolated from ictalurids in the southeastern United States indicate and Hoffman (1999) list M. toyamai and T. toyamai as closer relatedness to some Myxobolus spp. than Henneguya spp. synonymous; however, by definition the absence of a second from other host families in different geographic locales (Griffin et polar capsule classifies this organism within the Thelohanellus GRIFFIN AND GOODWIN—T. TOYAMAI FROM KOI IN THE EASTERN UNITED STATES 501

(Kudo, 1933; Shul’man, 1966, Hoffman, 1999). Close examina- carpio, with an amended morphologic description of the agent. tion of the digital images generated from the current study reveal Journal of Parasitology 96: 116–124. DYKOVA, I., I. FIALA, AND P. NIE. 2003. New data on Myxobolus no second polar capsule for this isolate; however, shadows and longisporus (Myxozoa: Myxobolidae), a gill infecting parasite of carp, other artifacts, such as the oblong protoplasmic mass, can be Cyprinus carpio haematopterus, from Chinese lakes. Folia Parasito- misleading at low magnifications and resolutions, lending logica 50: 263–268. credence to the ambiguities involved in the 2 descriptions. The EASY, R. H., S. C. JOHNSON, AND D. K. CONE. 2005. Morphological and molecular comparison of Myxobolus procerus (Kudo, 1934) and M. molecular sequence data of M. toyamai were recently submitted intramusculi n. sp. (Myxozoa) parasitizing muscles of the trout-perch ancillary to an in-depth investigation into the tissue tropism and Percopsis omiscomaycus. Systematic Parasitology 61: 115–122. redescription of a different myxozoan parasite, Myxobolus wulii. ECK, R. V., AND M. O. DAYHOFF. 1966. Atlas of protein sequence and Because M. toyamai was not the primary subject of the study, structure. National Biomedical Research Foundation, Silver Springs, Maryland, 215 p. morphological or histopathological data were not provided to EGUSA, S., AND K. NAKAJIMA. 1981. A new Myxozoa Thelohanellus kitauei, supplement the obtained DNA sequence (Zhang et al., 2010). the cause of intestinal giant cystic disease of carp. Fish Pathology 15: Nonetheless, the deposited GenBank sequence (FJ10802) is a 213–218. 99.9% match to the isolate described here, further supporting that EIRAS, J. C., K. MOLNA´ R, AND Y. S. LU. 2005. Synopsis of the species of M. toyamai and T. toyamai are one and the same. For the first Myxobolus Butschli, 1882 (Myxozoa: : Myxobolidae). Systematic Parasitology 61: 1–46. time, 18S SSU rDNA sequence data are supplemented with ESZTERBAUER, E. 2002. Molecular biology can differentiate between histological and morphological descriptions, which offer a morphologically indistinguishable myxosporean species: Myxobolus definitive identification of this isolate as T. toyamai in support elegans and M. hungaricus. Acta Veterinaria Hungarica 50: 59–62. of the original description (Kudo, 1919, 1933), eliminating ———. 2004. Genetic relationship among gill-infecting Myxobolus species (Myxosporea) of cyprinids: Molecular evidence of importance of ambiguities that can result from identifications based on tissue-specificity. Diseases of Aquatic Organisms 58: 35–40. morphology alone. For future reference, we suggest this organism ———, S. MARTON,O.Z.RACZ,M.LETENYEI, AND K. MOLNA´ R. 2006. be referred to as T. toyamai Kudo, 1933, in accordance with the Morphological and genetic differences among actinosporean stages of original reclassification and the nomial M. toyamai be avoided fish-parasitic myxosporeans (Myxozoa): Difficulties of species identification. Systematic Parasitology 65: 97–114. because it is at best outdated and, at worst, incorrect. The FELSENSTEIN, J. 1985. Confidence limits on phylogenies: An approach sequence generated here has been deposited in GenBank using the using the bootstrap. Evolution 39: 783–791. accession number HQ336729. FERGUSON, J. A., S. D. ATKINSON,C.M.WHIPPS, AND M. L. KENT. 2008. With the ubiquitous introduction of the common carp Molecular and morphological analysis of Myxobolus spp. of throughout temperate freshwaters of North America, it is salmonid fishes with the description of a new Myxobolus species. Journal of Parasitology 94: 1322–1334. important to chronicle the pathogens associated with this species FIALA, I. 2006. The phylogeny of Myxosporea (Myxozoa) based on small (Camus and Griffin, 2010). At this time, the alternate oligochaete subunit ribosomal RNA gene analysis. International Journal for host for this parasite is unknown, and it remains unclear whether Parasitology 36: 1521–1534. fish from this report became infected once they reached the GRIFFIN, M. J., L. H. KHOO,L.TORRANS,B.G.BOSWORTH,S.M.QUINIOU, P. S. GAUNT, AND L. M. POTE. 2009. New data on Henneguya pellis United States or were infected prior to importation, although (Myxozoa: Myxobolidae), a parasite of blue catfish Ictalurus furcatus. genetic analysis strongly supports the latter given the parasite’s Journal of Parasitology 95: 1455–1467. genetic similarities to Myxobolus spp. from Asia. Since the ———, D. J. WISE,A.C.CAMUS,M.J.MAUEL,T.E.GREENWAY, AND L. M. original submission, there has been no evidence of problems in POTE.2008.AnovelHenneguya sp. from channel catfish (Ictalurus punctatus) described by morphological, histological and geneologic subsequent years and no apparent impacts in the natural streams characteristics. Journal of Aquatic Health 20: 127–135. that receive water from this farm, suggesting the oligochaete host ———, ———, AND L. M. POTE. 2009. Morphology and small-subunit is absent or conditions are not conducive to completion of the life ribosomal DNA sequence of Henneguya adiposa (Myxosporea) from cycle. Additionally, the lack of a significant inflammatory Ictalurus punctatus (Siluriformes). Journal of Parasitology 95: 1076– response by the host suggests any negative impacts of infection 1085. HANSON, L. A., D. LIN,L.M.W.POTE, AND R. SHIVAJI. 2001. Small are mechanical in nature and require large numbers of plasmodia subunit rRNA gene comparisons of four actinosporean species to to result in the respiratory distress. With light infections, the establish a polymerase chain reaction test for the causative agent of parasite is likely to have little effect on the host and, therefore, proliferative gill disease in channel catfish. Journal of Aquatic Animal poses little risk in natural waters where conditions are not Health 13: 117–123. HOFFMAN, G. L. 1999. Parasites of North American freshwater fishes. 2nd conducive to the accumulation of high parasite numbers as seen in ed. Cornell University Press, Ithaca, New York, 539 p. intensive culture systems. IWANOWICZ, L. R., D. D. IWANOWICZ,L.M.POTE,V.S.BLAZER, AND W. B. SCHILL. 2008. Morphology and 18S rDNA of Henneguya gurlei LITERATURE CITED (Myxosporea) from Ameiurus nebulosus (Siluriformes) in North Carolina. Journal of Parasitology 94: 46–57. ANDREE, K. B., C. SZE´ KELY,K.MOLNA´ R,S.J.GRESOVIAC, AND R. P. KENT, M. L., K. B. ANDREE,J.L.BARTHOLOMEW,M.EL-MATBOULI,S.S. HEDRICK. 1999. Relationships among members of the genus DESSER,R.H.DEVLIN,S.W.FEIST,R.P.HEDRICK,R.W.HOFFMAN, Myxobolus (Myxozoa: ) based on small subunit ribosomal J. KHATTRA, ET AL. 2001. Recent advances in our knowledge of the DNA sequences. Journal of Parasitology 85: 68–74. Myxozoa. Journal of Eukaryotic Microbiology 48: 395–413. BARTA, J. R., D. S. MARTIN,P.A.LIBERATOR,M.DASHKEVICZ,J.W. KUDO, R. 1919. Studies on Myxosporidia: A synopsis of genera and ANDERSON,S.D.FEIGHNER,A.ELBRECHT,A.PERKINS-BARROW,M.C. species of myxosporidia. Illinois Biological Monographs 5: 239–503. JENKINS,H.D.DANFORTH, ET AL. 1997. Phylogenetic relationships ———. 1933. A taxonomic consideration of Myxosporidia. Transactions among eight Eimeria species infecting domestic fowl inferred using of the American Microscopical Society 52: 195–216. complete small subunit ribosomal DNA sequences. Journal of LIYANAGE, Y. S., H. YOKOYAMA,H.MATOYAMA,H.HOSOYA, AND H. Parasitology 83: 262–271. WAKABAYASHI. 1998. Experimentally induced hemorrhagic theloha- CAMUS, A. C., AND M. J. GRIFFIN. 2010. Molecular characterization and nellosis of carp caused by Thelohanellus hovorkai (Myxosporea: histopathology of Myxobolus koi infecting the gills of a koi Cyprinus Myxozoa). Fish Pathology 33: 489–494. 502 THE JOURNAL OF PARASITOLOGY, VOL. 97, NO. 3, JUNE 2011

LOM, J., AND I. DYKOVA. 2006. Myxozoan genera: Definition and notes on SANGER, F., S. NICKLEN, AND A. R. COULSON. 1977. DNA sequencing with taxonomy, life-cycle terminology and pathogenic species. Folia chain-terminating inhibitors. Proceedings of the National Academy Parasitologica 53: 1–36. of Sciences USA 74: 5463–5467. MARTYN, A. A., H. HONG,M.J.RINGUETTE, AND S. S. DESSER. 2002. SHUL’MAN, S. S. 1966. Myxosporidia of the USSR. Nauka Publishers, Changes in host and parasite-derived cellular and extracellular matrix Moscow-Leningrad, Russia, 631 p. components in developing cysts of Myxobolus pendula (Myxozoa). SZE´ KELY, C., A. EL-MANSY,K.MOLNA´ R, AND F. BASKI. 1998. Development Journal of Eukaryotic Microbiology 49: 175–182. of Thelohanellus hovorkai and Thelohanellus nikolskii (Myxosporea: MOLNA´ R, K. 1982. Biology and histopathology of Thelohanellus nikolskii Myxozoa) in oligochaete alternate hosts. Fish Pathology 33: 107–114. Achmerov, 1955 (myxosporea, myxozoan), a protozoan parasite of the ———, F. SHAHAROM-HARRISON,G.CECH,K.MOHAMED, AND K. common carp (Cyprinus carpio). Parasitology Research 68: 269–277. MOLNA´ R. 2009. Myxozoan pathogens of Malaysian fishes cultured ———. 2002. Differences between the European carp (Cyprinus carpio in ponds and net-cages. Diseases of Aquatic Organisms 83: 49–57. carpio) and the coloured carp (Cyprinus carpio haematopterus)in TAMURA, K., J. DUDLEY,M.NEI, AND S. KUMAR. 2007. MEGA4: susceptibility to Thelohanellus nikolskii (Myxosporea) infection. Acta Molecular evolutionary genetics analysis (MEGA) software version Veterinaria Hungarica 50: 51–57. 4.0. Molecular Biology and Evolution 24: 1596–1599. ———, M. NEI, AND S. KUMAR. 2004. Prospects for inferring very large ———, G. CECH, AND C. SZEKELY. 2008. Myxobolus species infecting the cartilaginous rays of the gill filaments in cyprinid fishes. Acta phylogenies by using the neighbor-joining method. Proceedings of the Parasitologica 53: 330–338. National Academy of Sciences USA 101: 11030–11035. WHIPPS, C. M., AND B. J. DIGGLES. 2006. Kudoa alliaria in flesh of ———, E. ESZTERBAUER,C.SZEKELY,A.DAN, AND B. HARRACH.2002. Morphological and molecular biological studies on intramuscular Argentinian hoki Macruronus magellanicus (Gadiformes; Merluccii- dae). Diseases of Aquatic Organisms 69: 259–263. Myxobolus spp. of cyprinid fish. Journal of Fish Diseases 25: 643–652. YOKOYAMA, H. 1997. Transmission of Thelohanellus hovorkai to common ———, AND E. KOVA´ CS-GAYER. 1981–1982. Occurrence of two species of carp Cyprinus carpio through the alternate oligochaete host. Systematic Thelohanellus (Myxosporea: Myxozoa) of far-eastern origin in Parasitology 36: 79–84. common carp populations of the Hungarian fish farms. Parasitolo- ———, D. INOUE,A.KUMAMARU, AND H. WAKABAYASHI. 1997. gica Hungarica 14: 51–54. Myxobolus koi (Myxozoa: Myxosporea) forms large- and small-type ´ MOSHU, A., AND K. MOLNAR. 1997. Thelohanellus (Myxozoa: Myxosporea) ‘cysts’ in the gills of common carp. Fish Pathology 32: 211–217. infection of the scales in the European wild carp Cyprinus carpio ———, J. KIM, AND S. URAWA. 2006. Differences in host selection of carpio. Diseases of Aquatic Organisms 28: 115–123. actinospores of two myxosporeans, Myxobolus arcticus and Thelo- NEI, M., AND S. KUMAR. 2000. Molecular evolution and phylogenetics. hanellus hovorkai. Journal of Parasitology 92: 725–729. Oxford University Press, New York, New York, 333 p. ———, Y. S. LIYANAGE,A.SUGAL, AND H. WAKABAYASHI. 1999. Efficacy RHEE, J. K., H. C. KIM, AND B. K. PARK. 1993. Efficacy of fumagillin of fumagillin against haemorrhagic thelohanellosis caused by against Thelohanellus kitauei infection of Israel carp, Cyprinus carpio Thelohanellus hovorkai (Myxosporea: Myxozoa) in coloured carp, nudus. Korean Journal of Parasitology 31: 57–65. Cyprinus carpio L. Journal of Fish Diseases 22: 243–245. RZHETSKY, A., AND M. NEI. 1992. A simple method for estimating and ZHANG, J. Y., H. YOKOYAMA,J.G.WANG,A.H.LI,X.N.GONG,A.RYU- testing minimum evolution trees. Molecular Biology and Evolution 9: HASEGAWA,M.IWASHITA, AND K. OGAWA. 2010. Utilization of tissue 945–967. habitats by Myxobolus wulii Landsberg and Lom, 1991 in different carp SAITOU, N., AND M. NEI. 1987. The neighbor-joining method: A new hosts and disease resistance in allogynogenetic gibel carp: Redescription method for reconstructing phylogenetic trees. Molecular Biology and of M. wulii from China and Japan. Journal of Fish Diseases 33: 57–68. Evolution 4: 406–425. ZHAO, Y., C. SUN,M.L.KENT,J.DENG, AND C. M. WHIPPS. 2008. SALIM, K., AND S. DESSER. 2000. Descriptions and phylogenetic systematics Description of a new species of Myxobolus (Myxozoa: Myxobolidae) of Myxobolus spp. from cyprinids in Algonquin Park, Ontario. based on morphological and molecular data. Journal of Parasitology Journal of Eukaryotic Microbiology 47: 309–318. 94: 737–742.