Parasitology Research (2019) 118:1719–1730 https://doi.org/10.1007/s00436-019-06324-8 FISH PARASITOLOGY - ORIGINAL PAPER Description of a new myxozoan Kudoa eugerres n. sp. and reclassification of two Sphaerospora sensu lato species Graça Casal1,2 & Emerson C. Soares3 & Sónia Rocha2,4 & Themis J. Silva3 & Elton L. Santos3 & Renato Nascimento3 & Elsa Oliveira4 & Carlos Azevedo2,4 Received: 20 September 2018 /Accepted: 12 April 2019 /Published online: 3 May 2019 # Springer-Verlag GmbH Germany, part of Springer Nature 2019 Abstract Ultrastructural and phylogenetic description of a fish-infecting myxosporean found infecting the gallbladder wall of the teleos- tean Eugerres brasilianus Cuvier, 1830, collected from the Atlantic coast near the city of Maceió (Alagoas State), Brazil. Groups of mature pseudo-conical myxospores, agglutinated forming pseudocyst structures, occurring in the mucosa of gallbladder were 5.2 ± 0.8 μm(4.5–6.0) (n = 30) long, 4.3 ± 0.6 μm(3.8–4.7) (n = 25) thick, and 2.9 ± 0.2 μm(2.7–3.2) (n = 25) wide. The two ellipsoidal polar capsules, 1.8 ± 0.4 × 1.2 ± 0.4 μm(n = 25), opened close to the sutural line, each containing an isofilar polar tubule. The latter consisted of a single coil with five to six turns, arranged obliquely to the axis of the polar capsule. This myxosporean parasite, while being morphologically similar to Sphaerospora spp., displays tissue tropism and phylogenetic relationships distinct from the latter. Bayesian inference (BI) and maximum likelihood (ML) analyses showed the parasite and two other related species clustering within the marine clade, more specifically within a subclade of the larger Kudoa (Multivalvulida) clade. Consequently, this atypical new myxozoan species was classified as Kudoa eugerres n. sp. and two other histozoic Sphaerospora spp. sensu lato were transferred to the genus Kudoa. Keywords Electron microscopy . Phylogenetic analysis . 18S rDNA gene . Myxosporean . Teleostean Introduction invertebrate definitive host (Polychaeta or Oligochaeta), and a poikilothermic vertebrate as intermediate host (usually fresh- Myxosporeans are metazoan parasites belonging to the phy- water, brackish, or marine fishes, but also amphibians or rep- lum Cnidaria Hatschek, 1888, characterized by a complex life tiles) (Lom and Dyková 2006). Presently, it is widely recog- cycle, in which there is an alternation of stages between an nized that the main evolutionary trends are the definitive and intermediate hosts and the tissue tropism (Carriero et al. 2013; Holzer et al. 2018). Section Editor: Astrid Holzer Numerous myxosporeans have been reported from fresh- water and marine habitats in different geographic regions. The * Carlos Azevedo [email protected]; [email protected] genus Sphaerospora Thélohan, 1892 accounts for at least 102 species described from Eurasia and North America, being 1 University Institute of Health Sciences & Institute of Research and most of them coelozoic parasites of the urinary system (Lom Advanced Training in Health Sciences and Technologies, CESPU, and Dyková 2006;Jirků et al. 2007; Patra et al. 2018)and, 4585-116 Gandra, Portugal more rarely, of the gallbladder (Gb) (Su and White 1994; Zhao 2 Laboratory of Animal Pathology, Interdisciplinary Centre of Marine et al. 2015). Some occurrences in other organs which involve and Environmental Research (CIIMAR/UP), University of Porto, histozoic development have also been reported (Lom and 4450-208 Matosinhos, Portugal 3 Dyková 1992). Sphaerospora molnari infects the gill fila- Laboratory of Aquaculture, Center for Agricultural Sciences ments and skin of the common carp Cyprinus carpio (Lom (CECA), Federal University of Alagoas (UFAL), Rio Largo, Alagoas 57100-000, Brazil et al. 1983; Eszterbauer et al. 2013), S. testicularis infects testicular tissue and intestine of wild and cultured of 4 Laboratory of Cell Biology, Institute of Biomedical Sciences (ICBAS/UP), University of Porto, Rua Jorge Viterbo Ferreira No. Dicentrarchus labrax (Sitjà-Bobadilla and Alvarez-Pellitero 228, 4050-313 Porto, Portugal 1990;Fioravantietal.2004), S. ovophila was found in 1720 Parasitol Res (2019) 118:1719–1730 Canada in the ovary of the teleostean Lepomis gibbosus (Xiao 6 days in an aquarium with aerated seawater, following the and Desser 1997), and S. dicentrarchi is a systemic parasite of abiotic parameters calculated at the site of fish collection: sa- European seabass, D. labrax (Sitjà-Bobadilla and Alvarez- linity 3.0–3.5‰,pH6.0–6.5, temperature ~ 25 °C. All speci- Pellitero 1992;Fioravantietal.2004) having been described mens were periodically observed and later anesthetized with from the tissues of several organs, predominantly the Gb and MS 222 before being dissected. intestine, with a prevalence of 100% and 70.5% in wild and cultured fishes, respectively (Sitjà-Bobadilla and Alvarez- Light microscopy and transmission electron Pellitero 1993). The latter species was also reported from microscopy spotted seabass, D. punctatus, caught in the estuary of the Alvor River (southern Portugal) (Xavier et al. 2013). For LM, small fragments of tissues of different organs were Phylogenetic analyses show the genus Sphaerospora as checked under a microscope for a parasitological survey. one the most polyphyletic groups among myxozoans. Small fragments of infected GbW were removed and Sphaerospora (sensu stricto)(s.s.), also known as Btrue photographed using a light microscope. Sphaerosporids,^ form a basal clade in which most species For TEM, small fragments of infected GbW were fixed in are included, while Sphaerospora (sensu lato)(s.l.)appeared 4–5% glutaraldehyde in 0.2 M sodium cacodylate buffer dispersed among several subclades of marine and freshwater (pH 7.2–7.4) for 20–24 h at 4 °C and then washed overnight clades (Bartošová et al. 2013; Holzer et al. 2013). Previous in the same buffer at 4 °C, and post-fixed with 2% osmium phylogenetic studies have shown that Sphaerospora s.l. are tetroxide in the same buffer for 3 h at the same temperature, not true Sphaerosporids and, therefore, should be taxonomi- dehydrated through an ascending ethanol and propylene oxide cally reclassified (Diamant et al. 2005;Bartošová et al. 2011; series, and embedded in Epon. The semi-thin sections were Jones et al. 2011). stained with methylene blue-Azur II for LM observations. This study describes the occurrence of a Sphaerospora-like Ultrathin sections were double stained with uranyl acetate myxosporean from a South American fish of the genus and lead citrate before observation in a JEOL 100CXII TEM Eugerres (Family Gerreidae). This genus accounts for 8 spe- (JEOL Optical, Tokyo, Japan), operated at 60 kV. cies, from which no myxozoan parasitosis was ever reported. The Brazilian fish E. brasilianus is distributed along the DNA extraction, PCR amplification, and DNA Western Central Atlantic: from South Carolina (USA) to the sequencing South of Brazil, being broadly caught along the northeastern region of Brazil and in the lower São Francisco River. In the Small fragments of infected GbW were collected from three latter region, this fish represents one of the most important specimens and preserved in 80% ethanol at 4 °C before geno- marine species, much appreciated and of great commercial mic DNA extraction, which was performed using a value, namely as a potential species to be introduced in aqua- GenElute™ Mammalian Genomic DNA Miniprep Kit for culture (Soares et al. 2016). In the present study, a new spe- each sample, following the manufacturer’s instructions. The cies, Kudoa eugerres n. sp., is described from the gallbladder DNAwas stored in 50 μLofTEbufferat− 20 °C until further wall (GbW) of E. brasilianus. The morphological character- use. Amplification of the SSU rDNA gene was achieved using istics and phylogenetic positioning of marine histozoic both universal and specific myxosporean primers: the 5′-end bivalvulids are discussed, resulting in the emendation of the with the primers 18e (5′CTG GTT GAT CCT GCCAGT3′) diagnosis of the order Multivalvulida, Family Kudoidae and (Hillis and Dixon 1991)/MYX4r (5′CTGACAGATCACTC genus Kudoa in order to accommodate the parasite in the CACGAAC3 ′) (Hallett and Diamant 2001), and Kud6r (5′ study and two other known species. CGTTCATTCCGATAGTGA3′) (Whipps et al. 2003); and the 3′-end with the primers MyxospecF (5′TTCTGCCCTATCAA CTTGTTG3′)(Fiala2006), and MYX4f (5′GTTCGTGG Materials and methods AGTGATCTGTCAG3 ′)(Rochaetal.2015)/18r (5′ CTACGGAAACCTTGTTACG3′) (Whipps et al. 2003). Fish sampling PCR was carried out in 50-μL reactions using 10 pmol of each primer, 10 nmol of each dNTP, 2.5 mM MgCl2,5μL10×Taq Nineteen specimens of the teleost fish Eugerres brasilianus polymerase buffer, 1.5 units Taq DNA polymerase (Nzytech, Cuvier, 1830 (Teleostei, Gerreidae) (Brazilian common name Lisbon, Portugal), and approximately 50–100 ng of genomic Bcarapeba^ or Bmojarra^) (about 20 to 30 cm long, and about DNA. The reactions were run on a Hybaid P × E 150 to 250 g), were collected between July and December Thermocycler, with initial denaturation at 95 °C for 3 mins, 2017, from the Atlantic coast (09° 44′ S/35° 49′ W), near the followed by 35 cycles of 94 °C for 45 s, 53 °C for 45 s, and city of Maceió (Alagoas State), Brazil. Fishes were 72 °C for 90 s. The final elongation step was performed at transported alive to the laboratory and maintained for 3– 72 °C for 7 mins. Five-microliter aliquots of the PCR products Parasitol Res (2019) 118:1719–1730 1721 were electrophoresed through a 1% agarose 1 × tris–acetate– containing gaps and missing data were eliminated; all ambig- EDTA buffer (TAE) gel stained with ethidium bromide. The uous positions removed for each sequence pair. purification of PCR products was achieved using a single-step enzymatic cleanup that eliminates unincorporated primers and dNTPs by means of the ExoFast method. Purified PCR prod- Results ucts were directly sequenced using a Big Dye Terminator v.1.1 from the Applied Biosystems Kit and were run on an All internal organs were checked under a microscope for a ABI3700 DNA analyzer (Perkin-Elmer, Applied Biosystems, parasitological survey of a total of 19 specimens, of which Stabvida, Caparica, Portugal).