Myxobolus rasmusseni, Tilley & Barry & Hanington & Goater, 2024

Tilley, Molly F., Barry, Danielle, Hanington, Patrick C. & Goater, Cameron P., 2024, Description, life cycle, and development of the myxozoan Myxobolus rasmusseni n. sp. in fathead minnows, Pimephales promelas: A possible emerging pathogen in southern Alberta, Canada, International Journal for Parasitology: Parasites and Wildlife 24, pp. 100944-100944 : 100944-

Genus Myxobolus Bütschli, 1882

Myxobolus rasmusseni n. sp.

Type host: Fathead minnow Pimephales promelas Rafinesque, 1820 ( Cypriniformes : Cyprinidae )

Type locality: University Pond , University of Lethbridge, Lethbridge, Alberta, Canada (49.680631, – 112.870276)

Site of infection: Disfiguring lesions containing myxospore-filled plasmodia located primarily on surface of circumorbital sinus and oral cavities; secondarily on epithelial tissues associated with mandibular and opercular regions of head and pectoral girdle.

Type specimens: 1 syntype hematoxylin-stained histological slide and one formalin-preserved, fathead minnow with lesions deposited in the University of Alberta freshwater invertebrate collection, Edmonton , Canada under accession numbers #IN5001 (histological slide) and #IN5002 (whole fish). GenBank voucher PP375284, GenBank triactinospore voucher PP591956 .

Prevalence: Prevalence: 92.3% of 196 1-yr old fathead minnows sampled from 5 reservoirs near Lethbridge, AB, Canada in July 2019 contained plasmodium-filled lesions.

Etymology: Species named after Dr. Joseph Rasmussen, Department Biological Sciences, University of Lethbridge, AB, Canada in recognition of his theoretical and empirical contributions to fisheries biology and aquatic ecosystem ecology.

3.1.1. Morphological description

Myxospores pyriform in valvular view, contained within various sized and shaped, membrane bound plasmodia ( Figs. 3–5 ). Myxospores present within plasmodia as singles or couplets ( Fig. 4A ). Myxospore dimensions - 14.7–15.5 μm long (15.1 ± 0.4; n = 50) X 7.2–8.0 μm (7.6 ± 0.4; n = 50) wide. Myxospore comprised of two smooth symmetrical valves containing straight sutural ridge running lengthwise along midline ( Fig. 4A ). Sutural ridge with two projections most visible at posterior end of side-on myxospore ( Fig. 4A ). Projections run lengthwise proximal to surface of frontal-view myxospore ( Fig. 4A ). Mature myxospore with 2 pyriform, equal sized and symmetrical polar capsules 7.0–8.2 μm long (7.6 ± 0.6; n = 50), 2.3–2.9 μm wide (2.6 ± 0.3; n = 50); polar filament with 8–9 turns in coil ( Figs. 3 and 4 ). Basal sporoplasm located within posterior third of myxospore, containing one or two nuclei and large iodinophilous vacuole ( Figs. 3 and 4A ). Mucus coat enveloping posterior two-thirds of myxospore, obvious in stained wet mounts ( Fig. 3A ) and TEM preparations ( Fig. 4A ). Morphology of pre-sporogonic stages in fathead minnows or Tubifex were not assessed.

3.1.2. Molecular and phylogenetic characterization

BLASTn search of the 18S rDNA M. rasmusseni n. sp gene sequence in GenBank showed that it was dissimilar to all other submitted myxozoan sequences. When entered into GenBank, the M. rasmusseni n. sp. sequence was most similar to the newly-described M. carlhubbsi from the gills of southern striped shiners from Arkansas, USA (McAllister et al., 2023, 95.2%), and M. bilobus from golden shiner in Ontario, Canada ( Cone et al., 2005; DQ008579, 92.2%). The M. rasmusseni n. sp. 18S rDNA sequence aligned up to 97% with other Myxobolus spp. most of which are associated with infection of the gill lamellae and filaments of cyprinid fishes ( Fig. 5 ; Supplementary Table 1). This group included M. bilobus ( Cone et al., 2005) , M. pseudokoi (Li and Desser, 1985) , and M. carlhubbsi (McAllister et al., 2023) , forming a monophyletic clade of cyprinid-infecting species from North America ( Fig. 5 ). This clade was sister to a clade of cyprinid-infecting species mostly from Europe with the exception of Myxobolus sp. EzoUgui (Yustinasari et al. unpublished; LC544125, 93.1%), M. obesus (Gurley, 1893) , M. Hungary-EE-2003, ( Eszterbauer et al., 2015), M. eirasianus (Cech et al., 2012) , M. intimus

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(Cech et al., 2012) and M. dujardini (Mitchell et al., 1985) .

3.1.3. Remarks

In addition to RNA gene sequence dissimilarities, the new species is distinguished from its closest relatives M. pseudokoi (Li and Desser, 1985) , M. bilobus ( Cone et al., 2005) , and M. carlhubbsi (McAllister et al., 2023) in host tissue site selection within the head cavities and in myxospore morphology. Individual plasmodia of each of the other clade members infect the gills. Furthermore, M. rasmusseni n. sp. is the only species in the clade whose pattern of development leads to large, conspicuous host lesions that are filled with multiple myxospore-filled plasmodia.

Myxospore metrics of the 22 Myxobolus spp. that infect cyprinid hosts ( Fig. 5 ) overlap with the new species ( Table 2). However, M. rasmusseni n. sp. myxospores are longer and wider than M. carlhubbsi and M. pseudokoi and smaller than M. bilobus ( Table 2). Myxospores of the new species has uniform-shaped polar capsules that are longer than the polar capsules of M. pseudokoi and M. carlhubbsi , the latter of which has irregular-shaped polar capsules. Further, M. bilobus has longer and irregular-sized polar capsules compared to the other 3 species in the clade. Myxobolus rasmusseni n. sp. has 8 to 9 coils in its polar filaments, whereas M. pseudokoi has 6–7 and M. carlhubbsi has 6 to 11 coils. The new species has a distinct sutural ridge along the midline lengthwise of the myxospore that has additional projections running adjacent on each side ( Fig. 4A ). In contrast, the sutural ridge on myxospores of M. carlhubbsi lack additional projections along the ridge, and M. bilobus has no sutural ridge. Ridge structure on M. pseudokoi myxospores is unknown. Finally, M. rasmusseni n. sp. has a distinct posterior iodinophilous vacuole located within the sporoplasm, similar to M. pseudokoi , whereas M. bilobus and M. carhubbsi lack a vacuole.

Myxobolus rasmusseni n. sp. occupies a separate branch from the sister taxa, M. pseudokoi and M. carlhubbsi . The node that distinguishes these 3 taxa has 100% bootstrap support ( Fig. 5 ). Furthermore, the branch length for M. rasmusseni n. sp. is longer than the length of the M. carlhubbsi and M. pseudokoi branch. The greater morphological outlier in this clade, M. bilobus , branches separately from the others with bootstrap support of 77% and longer branch length. The new species shares the highest percent sequence similarity with M. pseudokoi , at 95.2%, whereas other distinct species on the phylogenetic tree share greater sequence similarity, for example M. obesus and M. sp. Hungary- EE-2003 at 97.1% (Supplementary Table 1). Unfortunately, the single sequence available on GenBank for M. pseudokoi is less than half of the bp length (n = 1967) available for the M. rasmusseni n. sp. sequence.

Several Myxobolus spp. are reported from fathead minnows or hosts in the genus Pimephales but for which no sequences are available. Myxobolus hyborhynchi Fantham, Porter, and Richardson 1939 is described from bone and cartilage tissue of the head, pectoral, and caudal fins of fathead minnows (Fantham, Porter and Richardson, 1939; Cone and Frasca, 2001) but this species is not coelozoic within the head cavities and does not develop the lesions that characterize infections with Myxobolus rasmusseni n. sp. Myxospore, polar capsule, and mucus coat characteristics also differ between the two species ( Table 2). Plasmodia of Myxobolus mutabilis Kudo (1934) infects the integument of the head and fins of P. notatus . Its myxospores are ellipsoidal to spherical in shape, in stark contrast to the uniformly pyriform myxospores of

M. rasmusseni n. sp. Similarly, myxospores of M. angustus Kudo (1934) of the gill filaments and lamellae of P. promelas , P. vigilax , and P. notatus ( Table 2; McAllister et al., 2023) are slightly shorter compared to

M. rasmusseni n. sp. and have asymmetrical polar capsules ( Table 2).

Lastly, M. hendricksoni Mitchell et al. (1985) resides within brain tissue of P. promelas but has spherical/ellipsoidal myxospores, with shorter polar capsules and fewer polar filament coils ( Table 2).

Figures

Fig. 3. A. Myxospores of Myxobolus rasmusseni n. sp. prepared from a wet mount of a plasmodia-packed lesion located in the circumorbital cavity of an infected fathead minnow. A. Myxospores imaged with differential interference contrast microscope. Thin mucus coat envelopes posterior two thirds of myxospores. B. Composite line drawing of a Myxobolus rasmusseni n. sp. myxospore; PC – polar capsule; PF – polar filament; MC – mucus coat; SP – sporoplasm; IV – iodinophilous vacuole; N – nucleus.

Fig. 4. Transmission electron micrographs of plasmodia that contain M. rasmusseni n. sp. myxospores. Sections are from lesioned tissue (see inset in A) located in the circumorbital cavity of a fathead minnow. A. Side-on view of a couplet of Myxobolus rasmusseni n. sp. myxospores at 2500X magnification. Sp - Sporoplasm, Iv - Iodinophilous vacuole, Pc - Polar capsule, Pf - Polar filament; Black arrowheads indicate nuclei, orange arrowheads indicate sutural ridge along the midline of myxospore; blue arrowheads indicate posterior projections on the myxospore. B. Myxospores sectioned in various orientations with adjacent rodlet cells at 2000X magnification. Rc - Rodlet cell, Ms: myxospore.

Fig. 5. Phylogenetic tree produced by Bayesian analysis of aligned partial 18S rDNA gene sequences of M. rasmusseni n. sp. and other Myxobolus spp. infecting cyprinid fishes in Canada, Europe, and Asia. The tree is rooted with Ceratonova shasta (AF001579.1). Nodes are denoted with bootstrap probabilities generated by Bayesian analyses. Species in taxa in groups I-III are highlighted in the pairwise percent identity matrix in Supplementary Table 1.