Synopsis of the species of Ortholinea Shulman, 1962 (Cnidaria: Myxosporea: Ortholineidae)

A synopsis of Ortholinea Shulman, 1962 (Cnidaria: Myxosporea: Ortholineidae) is presented and identifies 26 nominal species presently allocated within this genus. Species morphological and morphometric features, tissue tropism, type-host, and type-locality are provided from original descriptions. Data from subsequent redescriptions and reports is also given. Accession numbers to sequences deposited in GenBank are indicated when available, and the myxospores were redrawn based on original descriptions. The information gathered shows that Ortholinea infect a wide taxonomic variety of freshwater and marine fish. Nonetheless, the broad host specificity reported for several species is not fully supported by morphological descriptions and requires molecular corroboration. The members of this genus are coelozoic and mainly parasitize the urinary system, with few species occurring in the gallbladder. Ortholinea visakhapatnamensis is the only exception, being histozoic in the visceral peritoneum. Molecular data of the small subunit ribosomal RNA gene (SSU rDNA) is available for about one third of Ortholinea species, with genetic interspecific variation ranging between 1.65% and 29.1%. Phylogenetic analyses reveal Ortholinea to be polyphyletic, with available SSU rDNA sequences clustering within the subclades of the highly heterogenous freshwater urinary clade of the oligochaete-infecting lineage. The life cycles of two Ortholinea species have been clarified based on molecular inferences and identify triactinomyxon actinospores as counterparts, and marine oligochaetes of the family Naididae as permissive hosts to this genus.


Introduction
The class Myxozoa Grassé, 1970 comprises microscopic obligate cnidarian parasites.There are more than 2,200 known myxozoan species, presently distributed among 66 genera and 20 families (Fiala et al., 2015;Freeman & Kristmundsson, 2015;Freeman et al., 2020).The subclass Myxosporea Bütschli, 1881 is the most diversified, encompassing species characterized by a complex life cycle that require Abstract A synopsis of Ortholinea Shulman, 1962 (Cnidaria: Myxosporea: Ortholineidae) is presented and identifies 26 nominal species presently allocated within this genus.Species morphological and morphometric features, tissue tropism, type-host, and type-locality are provided from original descriptions.Data from subsequent redescriptions and reports is also given.Accession numbers to sequences deposited in GenBank are indicated when available, and the myxospores were redrawn based on original descriptions.The information gathered shows that Ortholinea infect a wide taxonomic variety of freshwater and marine fish.Nonetheless, the broad host specificity reported for several species is not fully annelids (oligochaetes, polychaetes, and sipunculids) as definitive hosts, and vertebrates (usually fish, but also birds, reptiles, and mammals) as intermediate hosts (Lom & Dyková, 2006).The family Ortholineidae Lom & Noble, 1984 is particularly heterogenous, comprising three coelozoic genera that parasitize marine fish -Ortholinea Shulman, 1962, Neomyxobolus Chen & Hsieh, 1960and Kentmoseria Lom & Dyková, 1995 -but also, two histozoic genera parasitizing freshwater fish -Cardimyxobolus Ma, Dong &Wang, 1982, andTriangula Chen &Hsieh, 1984.
The oldest species presently included in the genus Ortholinea were originally described as belonging to the genus Sphaerospora Thélohan, 1892, family Myxidiidae Thélohan, 1892 (Thélohan, 1892).Davis (1917) considered the latter to be extremely heterogenous and, therefore, instituted the family Sphaerosporidae to include the genera Sphaerospora and Myxoproteus Doflein, 1898.This author also created the genus Sinuolinea to better encompass Sinuolinea dimorpha (Davis, 1916), originally described as belonging to the genus Sphaerospora.Later, Shulman (1959) instituted the family Sinuolineidae to include the genus Sinuolinea, comprising species with myxospores having a sinuous suture line, and the genus Davisia Laird, 1953, comprising species with myxospores having lateral processes.Shulman (1962) expanded the family Sinuolineidae with the creation of the genus Ortholinea for encompassing species with myxospores having a straight suture line, i.e., O. divergens Thélohan, 1895, O. orientalis Shulman &Shulman-Albova, 1953 andO. polymorpha Davis, 1917 (originally included in the genus Sphaerospora).Ortholinea divergens was established as type species.Lom and Noble (1984) created the family Ortholineidae within the also newly established suborder Variisporina, which united the members of the former suborders Bipolarina Tripathi, 1948emend. Shulman, 1959and Eurysporea Kudo, 1919emend. Shulman, 1962.The family Ortholineidae was created to accommodate the genera Ortholinea and Neomyxobolus, which are coelozoic and develop myxospores with polar capsules that are in the same plane as the suture line, unlike the remaining genera of the family Sinuolineidae, in which the polar capsules are located perpendicular to the suture line (Lom & Noble, 1984).The genera Cardimyxobolus, Triangula and Kentmoseria were later added to the family Ortholineidae, despite the first two differing from all other genera included in this family based on their histozoic development (Lom & Dyková, 2006).Currently, the taxonomic classification of the genus Ortholinea is: Phylum Cnidaria Hatschek, 1888Class Myxozoa Grassé, 1970Subclass Myxosporea Bütschli, 1881Order Bivalvulida Shulman, 1959Suborder Variisporina Lom & Noble, 1984Family Ortholineidae Lom & Noble, 1984Genus Ortholinea Shulman, 1962 Type species Ortholinea divergens (Thélohan, 1895) Shulman, 1962 Following the creation of the family Ortholineidae, other Sphaerospora species were ultimately transferred to the genus Ortholinea, namely Sphaerospora undulans Meglitsch, 1970and Sphaerospora sphaerocapsularae Wierzbicka 1986(Arthur & Lom, 1985;Sitjà-Bobadilla & Álvarez-Pellitero, 1994).In turn, three species have been transferred from Ortholinea to other genera.Parvicapsula irregularis (Kabata, 1962) was originally described as Sphaerospora irregularis, and later redescribed by Gaevskaya and Kovaleva (1984) as Myxoproteus irregularis.Unknowing of this taxonomic alteration, Arthur and Lom (1985) transferred S. irregularis to the genus Ortholinea.The validity of this species, however, was questioned by Køie et al. (2007), who suggested it to be more related with Parvicapsula Shulman, 1953; an assumption that was ultimately confirmed by Kodádková et al. (2014) through molecular analyses.The species Triangula perccotti (Dogiel & Akhmerov, 1960in Akhmerov, 1960) was also originally described as a member of the genus Sphaerospora (Akhmerov, 1960), and soon after transferred to the genus Myxosoma by Shulman (1962), who renamed it M. percotti, thus dropping one 'c' from the specific name.Later, Lom and Noble (1984) proposed the demise of the genus Myxosoma and synonymy of its species with Myxobolus Bütschli, 1882.However, unknowing of the taxonomic alteration proposed by Shulman (1962), Arthur and Lom (1985) transferred S. perccotti to the genus Ortholinea.Curiously, Landsberg and Lom (1991) later transferred Myxosoma percotti to the genus Triangula, and Sokolov (2013) redescribed the myxospores and recovered the 'c' of the specific name to rename it Triangula perccotti.Lastly, the species Kentmoseria alata (Kent & Moser, 1990) was originally described as Ortholinea alata, despite its myxospores possessing Page 3 of 16 37 Vol.: (0123456789) valvular projections atypical for the genus.Lom and Dyková (1995) considered this feature as being sufficient for creating the genus Kentmoseria, in which this species is presently allocated.
Currently, there are 26 species of Ortholinea, as listed in Table 1.However, this species list may be expected to change based on the increasing molecular data.For instance, the broad host specificity  6.9 ± 0.7 (5.9-8.2) 6.5 ± 0.4 (6.1-6.9)Ortholinea species have been described from both freshwater and marine fish worldwide.Based on original descriptions, Ortholinea have been recorded from 31 distinct fish species belonging to 25 families and 17 orders.Eight species have been reported from Europe, another 8 from Asia, 4 from Africa, 3 from Australia, 2 from South America, and a single species has been reported from North America.Reports show that this genus is present in the Atlantic, Indian, and Pacific oceans, as well as in the Mediterranean Sea and Black Sea.
The genus typically groups marine species.The few exceptions are O. fluviatilis Lom & Dyková, 1995, O. africanus Abdel-Ghaffar et al., 2008, O. lauquen Alama-Bermejo et al., 2019, and Ortholinea sphaerocapsularae (Wierzbicka, 1986) Sitjà-Bobadilla & Álvarez-Pellitero, 1994.The last was originally described from specimens of the European eel Anguilla anguilla (Linnaeus) caught from freshwater in Poland.However, the European eel is a catadromous fish that spawns in the Sargasso Sea, with its larvae migrating to European waters carried by ocean currents of the North Atlantic Drift (Wright et al., 2022).Therefore, it is possible that infection by O. sphaerocapsularae is acquired in marine or estuarine environments.
Most Ortholinea species have been reported from a single fish host.Exceptions are O. divergens, reported from a wide array of taxonomically distinct fish hosts; O. orientalis, reported from both Clupeiformes and Gadiformes; O. clupeidae and O. undulans, reported from two distinct families of Clupeiformes and Pleuronectiformes, respectively; O. australis Lom, Rohde & Dyková, 1992, reported from two different sparid genera; and O. polymorpha, reported from two different batrachoidid species belonging to the genus Opsanus Rafinesque, 1818.The molecular data available for O. orientalis confirm that this species is not host specific, given that infections could be verified in two different genera of Clupeidae (Karlsbakk & Køie, 2011).This suggests that Ortholinea species might be capable of infecting closely related fish hosts, as in the cases of O. australis and O. polymorpha.The broader host specificity reported for other species is not fully supported by morphological descriptions and requires molecular corroboration.For instance, Karlsbakk & Køie (2011) noticed differences in the dimensions of O. orientalis myxospores between Clupeiformes and Gadiformes that are consistent with the existence of a cryptic species.Reports of O. divergens from different fish hosts and geographic localities, sometimes atypically from the gall bladder, are accompanied by significant morphological variations that suggests O. divergens constitutes a species complex.Considering that the latter is the type species of the genus Ortholinea, it is imperative that future studies seek to resolve this complex, by providing a comprehensive morphological and molecular redescription of O. divergens from its type host.
Ortholinea are coelozoic, with 23 species (88.5%) reported from the organs of the urinary system.The urinary bladder is the organ most frequently infected (69.2%), followed by the kidney (42.3%), the ureters (19.2%), and the urethra (3.8%); with several species being able to develop in more than one of these organs.Three species of Ortholinea parasitize the gallbladder (O.asymmetrica Kovaleva, Velev & Vladev, 1993, O. australis and O. orientalis), with O. orientalis also occurring in the urinary bladder.Ortholinea undulans parasitizes both the urinary system and the oviducts, and O. visakhapatnamensis is the only exception to the coelozoic nature of the genus, forming cysts in the visceral peritoneum.Cysts measure up to 1 mm and form disporic pansporoblasts.All other Ortholinea species develop mono-, di-to polysporic plasmodia.Polysporic plasmodia are the most frequently observed, but several species also develop mono-or disporic plasmodia simultaneously.Plasmodia have variable shape and size, and display both ectoplasm (hyalin and transparent) and endoplasm (finely granular).The presence of pseudopods, lobes, peripheral projections, or other villi-like projections is also characteristic of the plasmodial membrane of this genus.
Despite the diversity of terms used to describe Ortholinea myxospores (spherical, subspherical, rounded, egg-shaped, triangular, subcircular, etc.), these stages are, in fact, restricted to three general shapes in valvular plane: ellipsoid to spherical, suboval to oval, and triangular.In sutural plane, myxospores are ellipsoidal to ovoid, and generally less flattened than in the valvular plane.The two polar capsules are symmetric, either being subspherical to spherical or pyriform, and typically open to opposite sides.The reported number of turns of the polar tubule varies between a minimum of 3 turns and a maximum of 7 turns, with 3-5 turns being the most common.The suture line is generally straight and conspicuous, except for O. fluviatilis and O. undulans, which have more sinuous or undulating suture lines.The valves usually display surface ridges, except for O. gadusiae Sarkar, 1999a, 1999b, O. indica Sarkar, 1999a, 1999band O. saudii Abdel-Baki et al., 2015, which have smooth valves.
There is few information regarding the pathogenicity of Ortholinea species.These parasites appear to be harmless to their vertebrate hosts, given that macroscopic alterations to infected organs, and external signs of disease and/or mortality have never been reported.Thus far, only microscopic damages have been described in reports of O. australis, O. lauquen and O. saudii.Ortholinea australis was reported to cause enlargement of the gallbladder and thickening of its wall, leading to the stagnation of bile outflow, and hepatic disorder (Lom et al., 1992).Ortholinea lauquen was associated with both physical and pathological changes to the host kidneys, causing cellular necrosis, disintegration of the tubular epithelium, and occlusion of the tubule lumina due to plasmodia adhesion (Alama-Bermejo et al., 2019).The developmental stages of O. saudii were also reported to cause obstruction of the kidney tubules (Abdel-Baki et al., 2015).
Values of prevalence of infection by Ortholinea vary considerably, with about one third of the known species displaying values that range between 50 and 100%, and the remaining species displaying values lower than 50%.Nonetheless, molecular-based studies suggest that prevalence of infection may be highly underestimated.For instance, a prevalence of infection of only 7% was reported for O. lauquen in Galaxias maculatus (Jenyns) based on microscopic observations, but molecular analysis revealed this value to be significantly higher (49%) (Alama-Bermejo et al., 2019).Similarly, prevalence of infection of O. concentrica in Acanthistius patachonicus (Jenyns) was determined to be 30% and 60%, based on microscopic and molecular analyses, respectively (Alama-Bermejo & Hernández-Orts, 2018).
About one third of Ortholinea species (9/26) have the small subunit ribosomal RNA gene (SSU rDNA) sequences available in GenBank, most of which were obtained from infections in their type hosts and provided in the original descriptions.The only exception is O. orientalis, of which SSU rDNA sequences were provided from infections in fish other than the original host (Karlsbakk & Køie, 2011).Another 6 Ortholinea SSU rDNA sequences are available, but are unpublished records (DQ333433, KP637274, MH197371, MK937851, MZ474836, KU301052).A single large subunit ribosomal RNA gene (LSU rDNA) sequence is also available for Ortholinea nupchi Shin et al., 2023.Genetic interspecific variation between Ortholinea SSU rDNA sequences ranges between 1.65% and 29.1%.Phylogenetic analyses reveal the polyphyly of this genus, whose members cluster within the freshwater urinary clade of the oligochaete-infecting lineage, alongside the sequences of Hoferellus spp., Myxobilatus gasterostei (Parisi, 1912) et al. (2014).This heterogeneous clade is composed by several distinct genera/myxospore morphologies, but also species infecting fish or amphibian hosts, either in freshwater or marine environments.The only features that unite the members of the freshwater urinary clade is their tropism to the urinary system, and the fact that they most likely use oligochaetes as invertebrate hosts (Holzer et al., 2018).This assumption is supported by the life cycles of O. auratae Rangel et al., 2014 andO. labracis Rangel et al., 2017, which involve oligochaetes as invertebrate hosts (Rangel et al., 2015(Rangel et al., , 2017)).
Thus far, only these two Ortholinea species have their life cycle clarified, based on molecular inferences established between myxospore and actinospore counterparts (Rangel et al., 2015(Rangel et al., , 2017)).Ortholinea auratae from the gilthead seabream Sparus aurata Linnaeus was shown to infect the oligochaete Limnodriloides agnes Hrabě (Rangel et al., 2015), while O. labracis from the European seabass Dicentrarchus labrax (Linnaeus) infects an unidentified oligochaete of the genus Tectidrilus (Rangel et al., 2017).Both oligochaete hosts belong to the family Naididae, and are distributed in estuarine and marine environments, with L. agnes typically occurring in subtidal sandy areas, and Tectidrilus in subtidal muddy sands (Erséus, 1982).The life cycles of O. auratae and O. labracis identify actinospores of the triactinomyxon collective group as counterparts for Ortholinea.This myxospore/actinospore correlation is further reinforced by the placement of the Triactinomyxon type of Rosser et al. (2014) within the freshwater urinary clade.

Ortholinea asymmetrica
Original depictions by Kovaleva et al. (1993) show the myxospores of O. asymmetrica having polar capsules positioned parallel to each other in one drawing, and convergent in another drawing.This positioning of the polar capsules does not agree with the taxonomic features of Ortholinea, best representing those of the genus Myxobolus.However, it is unclear if this representation should be considered as the result of taxonomic misidentification, given that the histozoic nature of Myxobolus does not conform with the species report performed by Kovaleva et al. (1993).It is more likely that myxospores have been poorly represented in the original drawings, as was the case of O. polymorpha, originally drawn with parallel polar capsules by Davis (1917), and later redescribed with polar capsules opening to opposite sides by Kudo (1944).Thus, solving this issue will require the morphological redescription and molecular analyses of O. asymmetrica from its original host.plasmodia.Valves with 19 or more surface ridges.Suture line straight and evident.Prevalence of infection of 51.6% (64/124) (Rangel et al., 2014).

Ortholinea mullusi
SSU rDNA sequence from the original description deposited in GenBank (MF539825).
According to Karlsbakk and Køie (2011), the original description of O. orientalis is not correct, given that the methodology (glycerin-gelatine) used by Shulman and Shulman-Albova (1953) causes myxospores to shrink, further hindering perception of important details like the presence/absence of valve ornamentations.These authors redescribed O. orientalis from the ureters of the Atlantic herring Clupea harengus Linnaeus and the European sprat Sprattus sprattus (Linnaeus) from Øresund, Denmark.Myxospores were redescribed having valves ornamented by surface ridges, one intercapsular process, and two sutural edge markings (Table 2; Fig. 31), and C. pallasi was suggested as type host, and the White Sea as type locality.The authors further considered O. antipae and O. clupeidae to be synonyms of O. orientalis, given that the main character differentiating between these species is the presence/absence of valve ornamentations.The SSU rDNA sequences of O. orientalis available in GenBank (HM770871-HM770875) were provided by Karlsbakk and Køie (2011) from infections in C. harengus and S. sprattus.

Ortholinea scatophagi
SSU rDNA sequence from the original description deposited in GenBank (MN310514).

Table 1
Myxospore morphometry of Ortholinea species.Measures are given in micrometres and, when available, include mean, standard deviation, and range in parentheses.

Table 2
Data from Ortholinea species redescriptions.Myxospore measures are given in micrometres and, when available, include mean, standard deviation, and range in parentheses.Absence of data is indicated by a hyphen.Abbreviations: F, Figure number; PCL, Polar capsule length (or diameter when width is missing); PCW, Polar capsule width; PTC, Polar tubule coils; SBL, Myxospore length; SBT, Myxospore thickness; SBW, Myxospore width; *) Measurements from fixed spores; **) Diameter.