A new Myxobolus (Cnidaria: Myxosporea) infecting the ornamental catfish Corydoras schwartzi from the Purus River in Brazil

A new microscopic cnidarian is described, infecting five of thirty (16.6%) specimens of Corydoras schwartzi caught in the Purus River, State of Amazonas, Brazil. Histological analysis showed that cyst development occurred in the serosa layer of the intestine. Mature myxospores are ovoid in body shape in frontal view, 22.4 ± 0.3 μm in total length and 16.3 ± 0.1 μm in width. Internally, two aubergine-shaped, elongate symmetrical polar capsules occupy more than half the length of the spore, 14.3 ± 0.2 μm in length and 6.5 ± 0.1 μm in width. Ultrastructural analysis provided evidence of five polar filament coils inside the polar capsule and binucleated sporoplasm containing a moderate number of sporoplasmosomes. The valvogenic cells abutting each other form a sutural ridge and frequently a thin layer of homogeneous material separates the cells. The outer surface of the myxospore valves is smooth, with no evidence of formation of ridges in the valves. Immature myxospores in various stages of development were observed. This study is the first report of a myxosporean parasitizing C. schwartzi and the first report of a myxosporean infection in the intestine of an ornamental fish from South America.


Introduction
Within Phylum Cnidaria, myxozoans are microscopic endoparasites of worldwide distribution and with approximately 2400 species they represent around 20% of cnidarian species diversity (Zhan et al. 2013;Atkinson et al. 2018). Myxozoans have a complex life cycle that typically involves invertebrates and vertebrates as defi nitive and intermediate hosts, respectively (Okamura et al. 2015).
In the last years, a large number of myxosporean species has been described infecting diff erent organs and tissues of wild and farmed South American freshwater fi sh (Mathews et al. 2015;Velasco et al. 2016;Vidal et al. 2017;Abrunhosa et al. 2017;Capodifoglio et al. 2019), with some species causing considerable pathological issues such as reduction of epithelial area of the gill, myocarditis of the heart, deformation, displacement, retraction and compression of capillaries of the gill lamellae, large skin nodules, thickening of the tunica externa of the swim bladder with granulomatous reaction, perivascular edema in the interlamellar area, and stretching of the corneal epithelium and urinary bladder (Barassa et al. 2003;Adriano et al. 2009;Naldoni et al. 2009;Matos et al. 2014).
Although there are several myxosporean infection surveys of South American teleosts, information on ornamental freshwater fi sh is still scarce, particularly from the Amazon region (Mathews et al. 2018) given the richness in species living in the greatest interconnected freshwater fl uvial system in the world (Junk et al. 2007;Moreau & Coomes 2007). Within the Amazonian ornamental teleosts, smaller catfi shes, particularly species belonging to the genus Corydoras Lacépède, 1803, have great diversity with approximately 162 described species and are of considerable economic importance due to wide commercialization in the international aquarium pet industry (Reis 1998;Prang 2007). Corydoras schwartzi Rössel, 1963 is a member of Siluriformes belonging to the family Callichthyidae Bonaparte, 1838, endemic of the Purus River basin in Brazil. It can reach up to 5 cm in total length and inhabits blackwater-fl ooded forests and the littoral zone of slow fl ow rivers (Froese & Pauly 2018).
To our knowledge, nothing is known about myxosporeans infecting specimens of C. schwartzi. The present study describes a new species of Myxobolus Bütschli, 1882 infecting the intestine of C. schwartzi from Lábrea Municipality in the Amazon region of Brazil, a key supply region of this species to international aquarium markets.

Material and methods
Between October and November 2018, thirty specimens of C. schwartzi (ranging from 3.9 to 4.2 cm in total length and 2.3 to 2.7 g in weight) (Fig. 1A) were caught in the Purus River, near the Municipality of Lábrea (7°15′32″ S, 64°47′52″ W), State of Amazonas, Brazil. The fi sh sample access was authorized by the Brazilian Ministry of the Environment (SisGen Process No. A5BD085). Fish were transported alive to the fi eld laboratory and euthanized by a benzocaine overdose (400 mg l −1 ), in accordance with Brazilian law for scientifi c use of animals (Federal Law No. 11794, dated 8 October 2008) and all organs were examined for myxosporean infection using a light microscope. Mature myxospores fi xed in 10% formalin were transported to the Department of Biophysics, Federal University of São Paulo, and morphological and morphometric analyses were performed based on the criteria outlined by Lom & Arthur (1989). Measurements and photographs were taken of 30 myxospores using a Leica DM1000 LED compound microscope equipped with Leica Application Suite version 1.6.0 image capture software. Smears containing free myxospores were air-dried, fi xed with methanol and stained with Giemsa solution to mount on permanent slides that were deposited in the collections of the Museum of Zoology of the University of São Paulo -USP, São Paulo, Brazil (MZUSP). in 2% osmium tetroxide (OsO 4 ) for 4 to 5 h. After dehydration in an ascending concentration of ethanol series, the samples were embedded in EMbed 812 resin (Electron Microscopy Sciences, Hatfi eld, PA, USA). Ultrathin sections, double stained with uranyl acetate and lead citrate, were examined in a LEO 906 electron microscope operating at 60 kV.
For the histological analysis, fresh fragments of infected tissues containing cysts were fi xed in a 10% buff ered formalin solution, dehydrated in an increasing concentration series of ethanol, diaphanized, embedded in paraffi n, cut into serial sections 5 μm thick using a Leica RM2255 automated microtome and stained with hematoxylin-eosin. Images were captured using a Leica DM 1000 microscope coupled to a computer and using Leica Application Suite software ver. 1.6.0 for image capture.

Results
In the present study, cysts of an unknown species of Myxobolus were found in the intestine of fi ve specimens of C. schwartzi. Cysts were ellipsoidal elongated in shape, measuring up to 213.6 μm in length and 38.8 μm in width (

Etymology
The specifi c name, adrianoi, is in homage to Dr. Edson Adriano, Professor at Federal University of São Paulo, Brazil, who has been contributing to improving our knowledge on the diversity of South American Myxosporea.

Site of infection
Serosa layer of intestine.

Description
Mature myxospores ovoid in body shape in frontal view, showing two aubergine-shaped, elongate symmetrical polar capsules occupying more than half length of spore. Total myxospore length 22.4 ± 0.3 μm and width 16.3 ± 0.1 μm. Two polar capsules, 14.3 ± 0.2 μm in length and 6.5 ± 0.1 μm in width (  Ultrastructural analysis showed myxospores in various stages of development. In young developmental myxospore stage, the polar fi lament could still be observed out of the polar capsules and valvogenic cells were readily recognized by valve-forming materials (Fig. 4A). In myxospores at a more advanced developmental stage, a polar fi lament inside the polar capsule and binucleated sporoplasms containing a moderate number of sporoplasmosomes were observed ( Fig. 4B-C). Transverse sections of a polar capsule provided evidence of a polar fi lament with fi ve coils ( Fig. 4B-C). Sections of immature myxospores show the valvogenic cells abutting each other to form a sutural ridge, and frequently the two cells were separated by a thin layer of homogenous material (Fig. 4D). Almost mature myxospores showed sutural lines, valve-forming material, valves, two nuclei and sporoplasmosomes at sporoplasms (Fig. 4E-F). The outer surface of the myxospore valves was smooth, with no evidence of formation of ridges in the valves (Fig. 4E-F).

Discussion
Although Corydoras spp. display a broad diversity and are widely exploited as an important source in the international aquatic pet trade, there are few studies about myxosporean infections in these smaller catfi shes (Mathews et al. 2017). Among approximately thirty-two species of Myxobolus described as infecting wild and farmed fi sh from the Amazon River basin and its main tributaries (Eiras et al. 2014;Abrunhosa et al. 2017;Naldoni et al. 2018;Capodifoglio et al. 2019), only one species was reported to infect Corydoras spp., i.e., Myxobolus niger Mathews, Maia & Adriano, 2016 described from the gill arch of C. melini Lönnberg & Rendahl, 1930(Mathews et al. 2016. Our study reports, for the fi rst time, a myxosporean species infecting C. schwartzi from the Amazon region and is hence the second to report a Myxobolus infection in Corydoras fi sh from South America. The morphometric data of the myxospore sample obtained were fi rst compared with those of M. niger, the unique species of Myxobolus previously described infecting Corydoras fi sh. Indeed, the comparison showed a large number of noticeable morphological and morphometric diff erences, with mature myxospores ovoid in shape for the myxobolids obtained in our study and ellipsoidal elongate for M. niger. The newly identifi ed myxospores are substantially larger in length and width (22.4 ± 0.3 μm in length and width of 16.3 ± 0.1 μm in the present study and 11.3 ± 0.4 μm in length and 6.8 ± 0.2 μm in width in M. niger). In the same way, the two polar capsules are longer and wider (14.3 ± 0.2 × 6.5 ± 0.1 μm in the present study and 5.0 ± 0.3 × 2.0 ± 0.1 μm in M. niger). Diff erences may also be observed in the number of coils in the polar fi lament (four coils in the present study and six to seven coils in M. niger).
The new species was compared with the four species of Myxobolus previously described infecting the intestine of South American freshwater fi sh: M. cunhai Penido, 1927 Actually, our results are in agreement with these studies, given that although M. niger has previously been described in C. melini, a genetically close related species to C. schwartzi, this myxosporean species was not found in the present study, thus reinforcing the fi ne-scale of the host specifi city of species of Myxobolus. According to Morand & Guégan (2000), host-specifi city may be more pronounced in populations inhabiting restricted spaces, due the fact that areas of host endemism are also areas of parasite endemism on a regional scale for freshwater fi sh. Indeed, the majority of Corydoras spp. usually inhabit restricted areas of endemism within the Amazon Basin (Reis 1998), as is the case with C. schwartzi and C. melini, both species with a restricted distribution. Actually, the distribution of C. schwartzi is restricted to the Purus River and C. melini to the Rio Negro River, both rivers separated by a distance of 703 km from each other, which is suffi cient to allow speciation of their myxosporean species, considering that generally, geographic isolation plays an important role in population divergence (Wang et al. 2019).
On the same premise, organ and/or tissue specifi city has been considered a taxonomic key for the identifi cation of freshwater histozoic platysporines, particularly for species belonging to the genus Myxobolus (Molnár 2002;Molnár et al. 2014). Accordingly, diff erences are observed in the infected tissue in the intestine when comparing the four species of Myxobolus for which information is European Journal of Taxonomy 620: 1-14 (2020) 8 available in the literature to the new species described herein (intestinal wall in M. cunhai and M. pygocentrus, mucosa layer in M. colossomatis, muscle layer in M. marajoensis and serosa layer in the new species). Furthermore, these species of Myxobolus were found parasitizing fi sh belonging to the families Pimelodidae, Heptapteridae, and Characidae. Considering M. niger, which was described from Corydoras melini, the closest host species to C. schwartzi, the comparison showed that both species diff er in the organ/tissue they were found to have infected, with the new species in the serosa layer of the intestine and M. niger in the gill arch. In the present study, a species of Myxobolus is thus described for the fi rst time infecting the intestine of a fi sh belonging to Callichthyidae, which is endemic to South America. Moreover, this is the fi rst report of intestinal infection by myxosporeans in an ornamental fi sh from the Amazon Basin.
According to Zatti et al. (2018a), host behavior may drive both parasite endemism and the radiation of myxosporeans within the Amazon Basin. These authors observed diff erences in the number of host species infected by a given species of myxosporean in a study conducted on species of the genus Cichla Schneider, 1801, which have diff erences in their distribution within the Amazon Basin. Similarly, Zatti et al. (2018b) described the presence of Ceratomyxa gracillima Zatti, Atkinson, Maia, Bartholomew & Adriano, 2018 in populations of the Amazonian catfi sh Brachyplatystoma rousseauxii Castelnau, 1855 collected at localities of wide geographic separation and considered that this fi nding is a result of the exceptionally long-distance migration of this fi sh. In addition, Whipps & Kent (2006) suggested that host distribution and migration are important factors in maintaining parasite gene fl ow over broad geographic areas. The above-mentioned studies reinforce the conclusion that host ecological characteristics may be important in aiding the characterization of the myxobolid fauna within the Amazon Basin, particularly for those species that infect Corydoras spp., considering the endemic character and the absence of migratory reproduction in this group of small catfi shes. Thus, taking into account the ecological characteristics of the host examined in our study, which are its high endemism and nonmigratory behavior, the designation of the Myxobolus found herein as a new species is reinforced.
From a pathological point of view, several studies have reported that myxosporeans are implicated in causing histological damage and mortality in wild and cultivated ornamental fi sh (Crawshaw & Sweeting 1986;Padrós et al. 2001;Camus & Griffi n 2010;Saha & Bandyopadhyay 2017). Although Myxobolus adrianoi sp. nov. induced no apparent tissue destruction or infl ammatory response in the specimens of Corydoras schwartzi in our study, previous studies of Amazonian ornamental fi sh have reported lymphocytic meningoencephalomyelitis associated with infection by Myxobolus sp. in the central nervous system of Eigenmannia sp. (Sindeaux-Neto et al. 2016). Furthermore, Camus et al. (2017) in  an investigation of mortality in a group of cardinal tetras suggested that infection by Myxobolus axelrodi Camus, Dill, Rosser, Pote & Griffi n, 2016 in ventricles of the brain and the ocular retina predisposed this group to opportunistic bacterial infection, including bacterial dermatitis and meningitis. There are, however, few published surveys of myxosporeans in Amazonian ornamental fi sh. Thus, further studies are necessary not only to increase our knowledge of the diversity of myxosporeans, but furthermore to evaluate their pathogenicity, taking into account that the Amazon Basin is the most important source of wild-caught freshwater fi shes for the global aquarium trade and considering that increased global connectivity with shorter transportation times, favor hosts and their parasites arriving in a viable state to new environments (Hallett et al. 2015).
In our study, DNA extraction was performed, but no suffi cient DNA was obtained for standardizing PCR reactions; hence, the amplifi cation of SSU rDNA from the myxospores failed. In addition, there are limitations in accessing new samples from the same region. However, based on the morphological/ morphometric features observed under light and transmission electron microscopy, geographic isolation, host endemism and host-, tissue-and organ-specifi city, the surveys corroborate the establishment of M. adrianoi sp. nov. as a new species o Myxobolus. Furthermore, the results contribute to studies of platysporine taxonomy and in extending our knowledge about myxosporean parasites of ornamental fi sh from the Amazon Basin, which is a key supplier of wild freshwater fi shes to the multi-billion dollar global aquarium trade (Moreau & Coomes 2007).