Zoosyst. Evol. 97 (2) 2021, 307-314 | DOI 10.3897/zse.97.64769 yee BERLIN Molecular diagnostic based on 18S rDNA and supplemental taxonomic data of the cnidarian coelozoic Ceratomyxa (Cnidaria, Myxosporea) and comments on the intraspecific morphological variation Fabricio B. Sousa’, Tiago Milanin*, André C. Morandini*:’, Luis L. Espinoza’, Anai Flores-Gonzales®, Ana L.S. Gomes®, Daniele A. Matoso®, Patrick D. Mathews? Program of Genetics, Conservation and Evolutionary Biology, National Institute of Amazonian Research, 69060-001 Manaus, Brazil Department of Basic Sciences, Faculty of Animal Science and Food Technology, University of Sdo Paulo, 13635-900 Pirassununga, Brazil Department of Zoology, Institute of Biosciences, University of Sdo Paulo, 05508-090 Sao Paulo, Brazil Laboratory of Biology and Molecular Genetics, Faculty of Veterinary Medicine, National University of San Marcos, 2800, San Borja, Lima, Peru Research Institute of Peruvian Amazon (IIAP-AQUAREC), Puerto Maldonado, Madre de Dios 17000, Peru Institute of Biological Sciences, Federal University of Amazonas, 60077-000 Manaus, Brazil Marine Biology Center CEBIMar, University of Sado Paulo, 11612-109 Sao Sebastido, Brazil N MO OO BR WYN http://zoobank. org/39565869-65A C-44C 7-98 FB-108969C8F IBC Corresponding author: Patrick D. Mathews (patrickmathews83@gmail.com) Academic editor: Pavel Stoev # Received 22 February 2021 # Accepted 2 June 2021 Published 10 June 2021 Abstract Ceratomyxa amazonensis is a cnidarian myxosporean originally described with strongly arcuate crescent-shaped myxospores, ab- sence of vegetative stages and infecting Symphysodon discus, an important Amazonian ornamental fish in the aquarium industry. As part of a long-term investigation concerning myxosporeans that infect discus fish Symphysodon spp. from different rivers of the Am- azon Basin, thirty specimens of S. discus collected from Unini River were examined. Plasmodial vegetative stages therefrom were found freely floating in the bile of gall bladders from eighteen fish. Mature myxospores were slightly crescent-shaped, measuring 4.72+0.1 (4.52-4.81) um in length, 24.2 + 0.4 (23.9-25.3) um in thickness with polar capsules 2.31 + 0.1 (2.29-2.33) um in length and 2.15 + 0.1 (2.13—2.17) um in width. Strong morphological differences were observed between the newly isolated myxospores ob- tained and the previously described C. amazonensis, however, molecular assessment, based on 18S rDNA, revealed a high similarity (99.91%), with only a single nucleotide base change. This study provides new data, expanding the original description of the species with a discussion on differences in myxospore-morphology in the context of intraspecific morphological plasticity. Key Words Brazil, ceratomyxid, morphological plasticity, ornamental fish, parasitic cnidarian Introduction The global aquarium market moves millions of orna- mental fish worldwide and is the primary mode for in- ternational transport of cnidarian myxosporean parasites (Hallett et al. 2015). As such, there is a fundamental need for constant monitoring to enable diagnosis and timely control of infections by this parasite group in aquarium fish (Mathews et al. 2018). Although the Amazon Basin is amongst the most important sources of wild-caught ornamental fishes 1n the international aquarium industry (Moreau and Coomes 2007), there are few surveys con- cerning cnidarian myxosporean infections in Amazonian ornamental fish (Mathews et al. 2015, 2017, 2020a, b). The three recognised species of the discus genus Sym- physodon Heckel, 1840, in the family Cichlidae, are popular, expensive and widely exploited ornamental fish (Bleher et al. 2007). These neotropical freshwater Copyright Sousa FB et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 308 cichlids are endemic to the Amazon Basin and restrict- ed to areas where seasonal flooding occurs (Bleher et al. 2007). The red discus Symphysodon discus Heckel, 1840 inhabits lentic aquatic environments, such as floodplains and flooded forests in the lower Rio Negro, upper Ua- tuma, Unini, Nhamunda, Trombetas and Abacaxis Rivers in Brazil (Amado et al. 2011). Myxosporeans are endoparasitic microscopic cnidari- ans with worldwide distributions (Atkinson et al. 2018). With over 2,400 species recorded from aquatic and ter- restrial hosts, there 1s evidence of extensive diversifica- tion in and dispersion of this group of parasitic cnidar- ians (Atkinson et al. 2018). Annelids are the definitive hosts, releasing infective actinospores into the aquatic environment (Fiala et al. 2015). Although virtually all vertebrate groups can be infected, fish comprise the larg- est number of known secondary hosts (Fiala et al. 2015). Amongst the myxosporeans, species of the genus Cera- tomyxa Thélohan, 1892 are mostly highly host-specific coelozoic parasites with approximately 300 species that mainly parasitise the gall bladders of a wide range of fish species (Eiras et al. 2018), with some species reportedly generating pathologies in their hosts (Alama-Bermejo et al. 2011, Barreiro et al. 2017). Despite the enormous di- versity of fish species in the Amazon Basin, only seven Ceratomyxa species have been reported (Eiras et al. 2018, Da Silva et al. 2020). Ceratomyxa amazonensis Mathews, Naldoni, Maia & Adriano, 2016 was described as para- sitising S. discus from the Rio Negro River, Amazonas State, Brazil, with the first published nucleotide sequence of a Ceratomyxa species from a strictly freshwater envi- ronment (Mathews et al. 2016). As part of a long-term investigation concerning myxo- sporeans that infect discus fish Symphysodon spp. from dif- ferent rivers of the Amazon Basin, specimens of S. discus collected from the Unini River were examined. This study supplements the original description of the cnidarian myx- osporean C. amazonensis, providing new data on the stag- es of and morphological variation in myxospores, thereby extending the original description of the species. Further- more, differences in myxospore morphology are discussed in the context of intraspecific morphological plasticity. Materials and methods In August 2019, thirty specimens of S. discus (ranging from 10.3 + 1.2 cm in total length and 23.4 + 4.2 g in weight) were collected from the Unini River, near Bar- celos Municipality (0°58'30"S, 62°55'26"W), Amazonas State, Brazil. The fishes were sampled under a collection licence issued by the Brazilian Ministry of the Environ- ment (SISBIO Process No. 73241-2). The euthanasia procedure was approved by the Federal University of Amazonas Ethics Committee for Scientific Use of Ani- mals (CEUA-UFAM No. 025/2019). After necropsy, gall bladders were carefully removed and placed in small Pe- tri dishes for further examination under stereo and opti- zse.pensoft.net Sousa, FB. et al.: Supplementary data of Ceratomyxa amazonensis from Amazon Basin cal microscopes. Samples of the bile were collected by puncturing the gall bladder using a pointed glass pipette; a drop of bile was then pipetted on to a microscope slide, covered with a cover slip and observed under an Olympus BX53 light microscope at 400 magnification. Morphological and morphometric analyses were per- formed on 30 randomly selected mature myxospores us- ing a computer, equipped with Axivision 4.1 image cap- ture software coupled to an Axioplan 2 Zeiss microscope. Following the criteria outlined by Lom and Arthur (1989) and Heiniger et al. (2008), measurements taken for each myxospore included spore length (SL), spore thickness (ST), polar capsule length (PCL) and polar capsule width (PCW) in micrometres (um) and posterior angle (PA) in degrees (°). The myxospore dimensions were expressed as mean and standard deviation, followed by the range in parentheses. Smears containing free myxospores were air-dried, fixed with methanol and stained with Giemsa to mount on permanent slides. Slides with stained myx- ospores and vials containing formalin-fixed plasmodia were deposited in the cnidarian collection of the Zoology Museum at the University of SAo Paulo — USP, Sado Paulo, Brazil (MZUSP). For transmission electron microscopy, infected gall bladders were fixed for two days in 2.5% glutaralde- hyde, diluted in 0.1 M sodium cacodylate buffer (pH 7.4), washed in a glucose-saline solution for 2 h and post-fixed in 2% osmium tetroxide (OsO,) for 4 to 5 h. After dehy- dration in an ascending concentration series of ethanol, the samples were embedded in EMbed 812 resin (Elec- tron Microscopy Sciences, Hatfield, PA, USA) (Mathews et al. 2020c). Ultra-thin sections, double stained with ura- nyl acetate and lead citrate, were examined under a LEO 906 electron microscope operating at 60 kV in the Center for Electronic Microscopy (CEME) at the Federal Uni- versity of Sao Paulo. Genomic DNA (gDNA) was extracted from infect- ed bile of a fish sample and preserved in absolute eth- anol. The sample was pelleted through centrifugation at 8,000 rpm for 12 min and the ethanol removed. The gDNA was extracted from the pellet using a DNeasy Blood & Tissue Kit (animal tissue protocol) (Qiagen Inc., California, USA), in accordance with the manufacturer’s instructions. The gDNA concentration was quantified in a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, USA) at 260 nm. Polymerase chain reaction (PCR) was performed tn accordance with Mathews et al. (2016) with a final reaction volume of 25 wl, which com- prised 1 wl of DNA (10-50 ng), 0.5 ul of each specific primer (0.2 uM), 12.5 ul of Dream Taq Green PCR Mas- ter Mix (Thermo Scientific) and 10.5 ul of nuclease-free water. Partial 18S rDNA sequences were amplified using the universal eukaryotic primer pair ERIB1 (forward: ACCTGGTTGATCCTGCCAG) and ERIB10 (reverse: CTTCCGCAGGTTCACCTACGG) (Barta et al. 1997). The amplification of the partial 18S rDNA was per- formed on a Mastercycler nexus (Eppendorf, Hamburg, Germany) and the PCR cycle consisted of an initial Zoosyst. Evol. 97 (2) 2021, 307-314 309 Figure 1. Light photomicrographs of Ceratomyxa amazonensis plasmodia. a, b. Slightly elongated plasmodia showing mature myxospores (white asterisks) and few early sporogonic stages (arrows); ¢. Spherical plasmodium with two slightly crescent-shaped mature myxospores (ms) and containing early sporogonic stages (arrows); d. Differential interference contrast microscopy snapshot of a slightly crescent-shaped mature myxospore. Scale bars: 10 um. denaturation step at 95 °C for 5 min, followed by 35 de- naturation cycles at 95 °C for 1 min, annealing at 60 °C for 1 min and extension at 72 °C for 2 min, with a termi- nal extension at 72 °C for 5 min. A control reaction was processed in order to check for possible contamination. The amplified PCR product was subjected to electropho- resis on 1.0% agarose gel (BioAmerica, California, USA) in a TAE buffer (Tris—Acetate EDTA: Tris 40 mM, acetic acid 20 mM, EDTA 1 mM), stained with Sybr Safe DNA gel stain (Invitrogen by Life Technologies, California, USA) and then analysed with a Stratagene 2020E trans-il- luminator. For sizing and approximate quantification of PCR product, 1 Kb Plus DNA Ladder (Invitrogen by Life Technologies, USA) was used. The PCR product was purified on a USB ExoSap-IT (Thermo Fisher Scientific, Massachusetts, USA) in accordance with the manufac- turer’s instructions and sequenced using the PCR primer pair, as well as the additional primer pair MCS, CCT- GAGAAACGGCTACCACATCCA and MC3, GATT- AGCCTGACAGATC ACTCCACGA (Molnar 2002). This additional primer pair was used in the sequencing to connect the overlapping fragments. Sequencing was per- formed with a BigDye Terminator v.3.1 cycle sequenc- ing kit (Applied Biosystems Inc., California, USA) on an ABI 3730 DNA sequencing analyser. The sequences ob- tained were visualised, assembled and edited in BioEdit 7.1.3.0 (Hall 1999) to produce a consensus sequence. A basic local alignment search (BLASTn) was performed to evaluate the similarity of our sequence with other myxosporean sequences available in the NCBI database (Altschul et al. 1997). The newly-acquired 18S rDNA gene sequence was aligned with all available Amazonian Ceratomyxa spp. sequences, in order to evaluate pairwise genetic distance using the p-distance model in MEGA 6.0 (Tamura et al. 2013). Results Freely-floating plasmodia were found in the gall bladder bile of 18 (60%) out of the 30 S. discus specimens col- lected in the Unini River. After rupturing the plasmodia, slightly crescent-shaped mature myxospores were ob- served with sub-spherical polar capsules. These capsules were located close to the myxospore suture line in a plane perpendicular to it, at the anterior myxospore pole, thus defining classification within the genus Ceratomyxa. No signs of infection were observed in the parasitised organs. Description Plasmodia were asymmetric and slightly elongated, with mean length 62.3 (range 58.4—64.2) um and mean width 7.8 (range 6.6—8.9) um; they contained both mature myxo- spores and early sporogonic stages (Fig. 1A, B). Some zse.pensoft.net 310 Sousa, FB. et al.: Supplementary data of Ceratomyxa amazonensis from Amazon Basin Figure 2. Transmission electron microscopy images of Ceratomyxa amazonensis isolated of Symphysodon discus from the Unini River, Amazonas State, Brazil. a. Myxospore showing two sub-spherical polar capsules and sporoplasm (sp) occupying most of the myxospore volume; b. Detail of the apical suture (black arrow) and sporoplasmosomes (arrowheads); ¢. Detail of lateral suture (black arrow); d. Polar capsule displaying still uncoiled internal polar tubule (black arrow). Scale bars: 2 um (a); 1 um (¢); 500 nm (b, d). plasmodia were spherical, mean diameter 30 (range 27— 32) um, n= 11, containing both mature myxospores and visible sporogonic stages (Fig. 1C). Mature myxospores were slightly crescent-shaped, measuring 4.72 + 0.1 (4.52-4.81) um in length and 24.2 + 0.4 (23.9-25.3) um in thickness (Fig. 1D). Shell valves were approximately equal in size with rounded extremities (Fig. 1D). Apical and lateral sutures were noticeable at the junction of the valves with fine material between them (Fig. 2B, C). The posterior angle was 154° (153°-156°). Polar capsules zse.pensoft.net were equal in size, sub-spherical, measuring 2.31 + 0.1 (2.29—2.33) um in length and 2.15 + 0.1 (2.13-2.17) um in width (Figs 1D and 2A) and displayed a still uncoiled internal polar tubule (2D). Sporoplasm occupied most of the myxospore volume (Figs 1D and 2A) with sporoplas- mosomes present (Fig. 2B). Host: Symphysodon discus Heckel, 1840 (Perci- formes: Cichlidae). Type locality: Unini River, near Barcelos Municipali- ty (0°58'30"S, 62°55'26"W), Amazonas State, Brazil. Zoosyst. Evol. 97 (2) 2021, 307-314 ial Table 1. Pairwise genetic distance of 18S rDNA sequences from Ceratomyxa species described from strictly Amazonian fish hosts. The upper triangular matrix shows the number of different nucleotide positions; the lower triangular matrix shows the percentage of differing nucleotide positions. Species (GenBank ID) 1 1. Unini River isolates (this study) (MNO64752) - 2. Ceratomyxa gracillima (KY934184.1) yh 3. Ceratomyxa vermiformis (KX278420.1) 4 4. Ceratomyxa brasiliensis (KU978813.1) 2 5. Ceratomyxa amazonensis (KX236169.1) 0.1 Sites of infection: Within gall bladder (plasmodia floating free in the bile). Material deposited: The partial 18S rDNA gene se- quence was deposited in GenBank (accession number MN064752). Slides with stained myxospores and vials containing formalin-fixed plasmodia were deposited in the cnidarian collection of the Zoology Museum at the University of Sao Paulo — USP, Sao Paulo, Brazil (MZUSP 8469). Molecular Analysis The BLAST search revealed a high similarity between the newly-obtained 18S rDNA gene sequence and a pre- viously-published sequence of C. amazonensis (query cover 100%, maximum identity 99.91%), a parasite of S. discus from Rio Negro River. The pairwise compar- ison between the new isolate from the Unini River and a previously deposited 18S rDNA gene sequence of C. amazonensis found an overall genetic divergence of 0.1% with just a single nucleotide base change between the two sequences (Table 1). Discussion As pointed out by Lom and Arthur (1989) in their guide- lines for the description of myxosporean cnidarians, it is indispensable to provide as much information as possi- ble about the plasmodial stage, such as the site of infec- tion, structure, shape and size. In contrast to the previous description of C. amazonensis from S. discus from the Rio Negro River, where only free myxospores were re- ported (Mathews et al. 2016), in our study, plasmodia containing mature myxospores and early stages were found floating freely in the bile within host gall bladders. Here, we provide new data on the plasmodial stage, ex- tending and thereby improving the original description of C. amazonensis. Morphological plasticity in myxospores are acknowl- edged to be one of the main factors responsible for the difficulties encountered in myxosporean taxonomy and species identification, resulting in taxonomic dilemmas (Zhai et al. 2016, Guo et al. 2018, Xi et al. 2019). It is widely recognised that Ceratomyxa spp. myxospores can display a high degree of morphological plasticity (At- kinson et al. 2015, BartoSova-Sojkova et al. 2018); thus, 2 3 4 5 77 48 32 1 o2 109 109 Bue - 82 74 6.8 6:1 - 38 9 4.7 2.4 classifications, based strictly on morphology, can result in ambiguous descriptions, especially considering that there 1s a high level of natural morphological and mor- phometric variation in myxospores both within and be- tween hosts (Atkinson et al. 2015). One reason for this variation is that myxosporean infections typically feature asynchronous myxospore development, so that changes in shape and size during maturation result in a range of myxospore morphologies (Atkinson et al. 2015). A fur- ther problem in using myxospore morphology alone to distinguish species concerns the fixative methods used, because it is known that fixatives can affect the morpho- logical dimensions of myxospores relative to fresh sam- ples (Parker and Warner 1970, Zhai et al. 2016). In our study, no changes were observed in the dimensions of formalin-fixed myxospores compared to fresh samples; this could be explained by the type of fixative used, which is reported to have little, if any, effect on myxospore di- mensions (Parker and Warner 1970). These observations are consistent with those reported for Myxobolus drjagini Akhmerov, 1954, where myxospores, fixed in 10% for- malin since the 1980s, showed little shrinkage compared with fresh myxospores (Xi et al. 2019). However, mor- phological variations in some Ceratomyxa spp. have been described as created by deformations of their presumably thin-walled shell valves (Morrison et al. 1996), thus lim- iting the use of myxospore features as a sole approach to taxonomic classification. Under this scenario, in order to accurately identify new myxosporeans species, it is high- ly recommended to use a combination of morphological and biological traits, factors related to host ecology and molecular characteristics, particularly within genera with high intraspecific variation in myxospores, with Cerato- myxa being a remarkable example (Atkinson et al. 2015). In our study, the morphological comparison between the new myxospore isolate from Unini River, S. discus and previously described C. amazonensis myxospores found in specimens from the Rio Negro River, shows some dissimilar characteristics (Table 2). Unini Riv- er myxospores were slightly crescent-shaped, shorter in length and significantly thicker compared to the Rio Negro River myxospores which were strongly arcuate shaped, comparatively longer and less thick (Mathews et al. 2016). Although we noticed some discordance in myxospore morphology between the new isolate obtained and previously described C. amazonensis from Rio Negro River, the molecular analysis revealed a high similarity (99.91%) in 18S rDNA sequence data, with only a single zse.pensoft.net S12 Sousa, FB. et al.: Supplementary data of Ceratomyxa amazonensis from Amazon Basin Table 2. Comparative morphometric data for the newly-isolated myxospores and other Ceratomyxa parasites of Amazonian fish. T: thickness; L: length; PCL: length of polar capsule; PCW: width of polar capsule; PA°: Polar angle; —: no data. All measurements expressed as mean + SD. Species T L PCL New isolate 24.2+0.4 4.7+0.1 Ar3dgOs1 C. amazonensis 15.8+0.4 FIEOIS Se te, 3 C. fonsecai 28.9 42.7 2.6.20. 1 90.3 C. mylei 24.6 +08 5.1 0:2 227 0.3 C. brasiliensis 41.2429 6:2.+. 0,6 226. OS C. gracillima 28.0+3.4 44+1.1 1.9+0.4 C. microlepis Sor6 a OrS 5.2+0.4 Zig EAS C. vermiformis 8440.4 nucleotide base change. Previous studies in several ge- ographic regions have reported different values for 18S rDNA intraspecific divergence in Ceratomyxa (Sanil et al. 2017, Barto8ova-Sojkova et al. 2018) and there is no universal criterion regarding what constitutes a sufficient level of sequence variation to represent distinct species within the genus Ceratomyxa (BartoSova-Sojkova et al. 2018). However, the low 18S rDNA genetic difference (0.1%) observed between the isolate obtained in the pres- ent study and the previously available C. amazonensis sequence is not sufficient to designate them as different species, taking into account that 18S rDNA intraspecific sequence variation of < 1% is common (Zhao et al. 2013, Atkinson et al. 2015, Wang et al. 2019). The low genet- ic divergence observed between isolates from these two widely-separated localities (511 km apart), indicates that geography has had limited impact in terms of genetic dif- ferentiation. Our finding is consistent with previous stud- ies where samples of the same myxosporeans species, collected in distant geographic areas, had zero or very low genetic divergence in their 18S rDNA sequences (Urawa et al. 2011, Adriano et al. 2012, Wang et al. 2019). Ac- cording to Whipps and Kent (2006), host distribution and migration can be equally important factors in maintain- ing parasite gene flow over broad geographic areas. The high genetic similarity between the Unini River isolate and the Rio Negro River C. amazonensis is likely a result of adaptation of the host, S. discus, to floodwater habitats of the Amazon Region, ensuring that populations share a continuous habitat across a large geographical area, de- spite dry periods between the floods. This adaptation to a specific ecological niche may lead to high parasite gene flow over broad geographic areas. For instance, Zatti et al. (2018) observed an absence of genetic variation in the typically more variable ITS-1 region in widely separated Ceratomyxa gracillima samples infecting the gall bladder of the Amazonian catfish Brachyplatystoma rousseauxii Castelnau, 1855. They suggest high gene flow as a result of panmixia in the parasite populations due to migratory behaviour of the fish host. Based on the discussion above, we infer that the myxo- spores, newly isolated from Unini River S. discus, should be regarded as belonging to the previously described spe- cies C. amazonensis. Furthermore, the observations made during this study highlight that classifications, based zse.pensoft.net Ae ject Ol PCW PA° Source bx Ay sigent Sl 154 This study 2.6. 40.2 103.7 Mathews et al. (2016) lik: 0.2 164.8 Silva et al. (2020) - - Azevedo et al. (2011) 2.5+0.4 147 Zatti et al. (2017) 1.9+0.4 36.6 Zatti et al. (2018) Ze ae O53 Azevedo et al. (2013) PAP de ode alk 30.2 Adriano and Okamura (2017) strictly on morphology, can result in ambiguous descrip- tions and reinforce the importance of molecular methods (DNA sequencing) for identifying and distinguishing be- tween Ceratomyxa species. Acknowledgements The authors thank the fishermen of the Municipality of Barcelos, Amazonas State, for the provision of the fishes, Dr. Berger for reviewing the English idiom and Prof. R. Sinigaglia-Coimbra from the Electron Microscopy Cen- ter (CEME) at UNIFESP for supporting the TEM analy- sis. PD. Mathews thanks the Sao Paulo Research Foun- dation, FAPESP, for a Post-Doc fellowship (grant No. 2018/20482-3). A.C. Morandini was funded by CNPq (304961/2016-7) and FAPESP (2015/21007-9). T. Mi- lanin thanks FAPESP for a Post-Doc fellowship (grant No. 2015/18807-3). This work was supported by the CNPq (465540/2014-7) and FAPEAM (062.1187/2017). References Adriano EA, Carriero MM, Maia AA, Silva MR, Naldoni J, Ceccarel- li PS, Arana S (2012) Phylogenetic and host parasite relationship analysis of Henneguya multiplasmodialis n. sp. infecting Pseudo- platystoma spp. in Brazilian Pantanal wetland. 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