First report of a histozoic Henneguya (Cnidaria, Endocnidozoa) infecting a synbranchid potamodromous fish from South America: Morphostructural and biological data

In this study, a Henneguya myxosporean species is described to infect an ecological, biological, and evolutionary important fish from Amazon biome. The myxosporean was found in the skin of only one specimen of marbled swamp eel, Synbranchus marmoratus caught in a small stream from Peruvian Amazon floodplain. Mature myxospores have ovoid shape from the valvular view, measuring 32.2 ± 0.6 μm (31.6–32.8) in total length, 21.5 ± 0.3 μm (21.2–21.8) in spore body length, 11.7 ± 0.5 μm (11.2–12.2) in width and 10.6 ± 0.9 μm (9.7–11.5) in thickness. Non-bifurcate caudal appendage, measuring 10.7 ± 0.4 μm (10.3–11.1) in length. Two polar capsules elongated aubergine in shape, equal in size and measuring 4.9 ± 0.2 μm (4.7–5.1) in length and 3.1 ± 0.5 μm (2.6–3.6) in width. Polar tubules coiled in 7–8 turns. This is the first report of a Henneguya species parasitizing a fish of the order Synbranchiformes from Amazon basin and the first to describe this parasite infecting a potamodromous fish from South America.


Introduction
Myxosporean are a biologically diverse group of microscopic cnidarians of wide distribution around the world (Atkinson et al. 2018).They mostly innocuous parasites with complex life cycles that involve invertebrate and vertebrate hosts (Okamura et al. 2015).Although, most myxosporean species have fish hosts, they have radiated sporadically into other groups of vertebrates, including amphibians, reptiles, waterfowl and small mammals (Okamura et al. 2015).Within myxosporean, Henneguya Thélohan, 1892 is one of the most species rich genera with more than 250 species described taxonomically (Eiras 2002;Rangel et al. 2023).Although the Amazon basin is one of the main biodiversity hotspots, the myxosporean fauna is poorly known.To date only 19 Henneguya species have been reported in fish from this geographic region with almost all reported data so far coming from the Brazilian part of the Amazon basin (Eiras and Adriano 2012;Mathews et al. 2016;Naldoni et al. 2018).In the Peruvian Amazon, despite a recording of over 650 fish species, there is a gap in the knowledge of myxosporean diversity.Indeed, only three Henneguya species have been described (Mathews et al. 2017(Mathews et al. , 2018(Mathews et al. , 2020)).
The marbled swamp eel Synbranchus marmoratus Bloch, 1795 is considered a potential predator and it can be found throughout flooded forests, small streams and associated swamps subject to water level changes, between the rainy season and the dry period (Heisler 1982;Favorito et al. 2005;Junges et al. 2010).This species of fish is a protogynous diandric, meaning that females will change their sex and become males (Allsop and West 2003).It is a potamodromous species which is capable of switching from exclusive water breathing to exclusive air breathing (Heisler 1982).Despite, the ecological, biological, and evolutive importance of the marbled swamp eel, little is known about its parasitic fauna, particularly the ones concerning myxosporean parasites.
This study aims to contribute to the increase of knowledge of diversity cnidarian myxosporean and their interaction with fishes from Amazon biome.Thus, spore morphology features using light, scanning and transmission electron microscopy as well as other important biological characters such as tissue tropism and host-specificity are provided.

Materials and methods
Six specimens of S. marmoratus (ranging from 18.1 to 21.6 cm in length) that died during transport were donated by local fishers for ornamental fishes in March 2018.According to the fishers, these fish were caught in a small stream near of the Village Oran (3°21'0"S, 72°31'0"W), Omagua Region, Department of Loreto, Peru.
Morphometric analysis was performed following the criteria outlined by Lom and Arthur (1989).Measurements and photographs were taken from 30 randomly selected formalin-fixed mature myxospores, using a computer equipped with Axiovision 4.1 image capture software coupled to an Axioplan 2 Zeiss microscope (Carl Zeiss AG, Oberkochen, Germany).Spore length, thickness, polar capsule length, width, and caudal appendage length were measured and given in micrometers (μm) and expressed as a mean ± standard deviation, followed by the range in parentheses where appropriate.Permanent slides containing mature myxospores stained with Giemsa were mounted and deposited in the cnidarian collection of the Zoology Museum at the University of São São Paulo,Brazil (Hapantotype MZUSP 8733).
Histological analysis was performed on fresh tissue fragments containing plasmodium.Infected tissue was fixed in 10% buffered formalin solution, then dehydrated with increasing series of ethanol, diaphanized, embedded in paraffin, cut into serial sections 5 μm thick using an HM 340E electron microtome (Thermo ScientificTM, Massachusetts, USA), and stained with haematoxylin/eosin.A light microscope DM1000 (Leica, Washington, USA) coupled to a computer and using the Leica Application Suite software version 1.6.0 was used for image capture.
Surface ultrastructure observation was performed in leaked myxospores from ruptured plasmodium using a glass slide previously treated with poly-L-lysine.Samples were processed as described in Mathews et al. (2022a).Samples were visualized with a DSM 940 scanning electron microscope (Carl Zeiss, Hamburg, Germany) operating at 15 kV.For internal structural analyses, a whole intact plasmodium was fixed in 2.5% glutaraldehyde with 0.1 M buffered cacodylate (pH 7.4) for 24 h and processed routinely according to standard transmission electron microscope methods.Samples were examined under a JEOL 1200 EX II transmission electron microscope at 60 kV and micrographs were captured with a GATAN 791 camera.
For molecular diagnostic, extraction of genomic DNA (gDNA) was performed in a single plasmodium dissected from the skin and fixed in absolute ethanol.The gDNA was extracted using a DNeasy Blood & Tissue Kit (Qiagen Inc., California, USA), in accordance with the manufacturer's instructions for animal tissue protocol.Polymerase chain reactions (PCRs) were conducted in a final volume reaction of 25 μL, which comprised 10-50 ng of extracted DNA, 0.2 pmol for each primer, 12.5 μL of Dream Taq Green PCR Master Mix (Thermo Scientific) and nuclease-free water.Partial 18S rDNA sequence was amplified using routinely chosen primers paired as follows ERIB1 with ACT1r and Myxgen4F with ERIB10 (Barta et al. 1997, Kent et al. 2000, Hallett and Diamant 2001).PCRs were performed in an AG22331 Hamburg Thermocycler (Eppendorf, Hamburg, Germany) and amplification thermal cycling consisted of 95 °C for 5 min, followed by 35 cycles at 95 °C for 1 min, 58 °C for 1 min, 72 °C for 2 min, and then final elongation at 72 °C for 5 min.Amplification PCR products were electrophoresed in 2.0% agarose gel in a Tris-Acetate EDTA buffer, stained with Sybr Safe DNA gel stain (Invitrogen by Life Technologies, Carlsbad, USA), and analyzed under a Stratagene 2020E trans illuminator (Stratagene California, San Diego, USA).Band sizes of the amplicons was estimated by comparison with the concurrently run molecular weight marker 1 Kb Plus DNA Ladder (Invitrogen by Life Technologies).PCR products were purified using USB ExoSap-IT (Thermo Fisher Scientific, Waltham, USA) in accordance to the manufacturer's instructions.Purified PCR amplicons were sequenced using the same PCR primers and performed with a BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems Inc., California, USA) in an ABI 3730 DNA sequencing analyzer.

Results
Of six wild specimens of S. marmoratus, a single wild specimens of S. marmoratus examined, was infected in the skin by an unknown cnidarian myxosporean species.Based on the phenotypic characters of the mature myxospores, this species was assigned to the genus Henneguya.The fish presented five plasmodia distributed in the body skin.The same were not found in any other organ.Type host.Symbranchus marmoratus (Teleostei: Symbranchidae).
Site of infection.Stratus corneum of epidermis layer of the skin.
Surface topography analyses of mature myxospores in valvular view revealed smooth valve cell with presence of mucous in a small area (Fig. 2a).In sutural view myxospore evidence a conspicuous sutural line (Fig. 2c).The density of caudal appendage is likely be identical to that of its valve (Fig. 2b).Internal ultrastructural observations showed binucleated sporoplasm contained sev-   eral sporoplasmosomes and valve-forming materials in young sporoblast developmental myxospore stage (Fig. 3a).Polar capsule with polar tubule internalized and contained seven to eight coils at a more advanced sporoblast developmental stage (Fig. 3b).Caudal appendage and conspicuous sutural line in mature spores.(Fig. 3c).
Histologic evidenced tissue tropism of the myxosporean under study, occurring in the stratus corneum of epidermis layer of the skin (Figure 4).The parasites induced no apparent tissue destruction, ulcerations, necrosis or inflammatory response.For molecular procedures, partial 18S rDNA gene was successfully amplified by PCR (Fig. 5), however, sequencing failed.

Discussion
Despite the growing description of myxosporean infecting South American fishes (Sousa et al. 2021;Adriano and Oliveira 2022), the diversity of these ancient metain this neotropical realm remains largely unknown (Okamura et al. 2018;Mathews et al. 2022b).In this context, our study describes a histozoic myxosporean species of Henneguya, infecting skin of the Amazonian potamodromous fish S. marmoratus.In the Amazon biome, Henneguya encompasses 22 recognized species (Table 1), reported infecting Characiform, Perciform, Cichliform, Gymnotiform and Siluriform fishes (Mathews et al. 2017(Mathews et al. , 2018;;Adriano and Oliveira 2022;Rangel et al. 2023).However, to the best of our knowledge, this is the first report of a Henneguya species parasitizing a fish of the order Synbranchiformes from Amazon basin and the first to describe this parasite infecting a potamodromous fish from South America.Thus, our results contribute to freshwater myxobolids taxonomy and to increasing our knowledge of cnidarian myxosporean diversity.
The morphological data of the mature myxospore isolated were first compared considering Henneguya species previously described from Peruvian Amazon freshwater fishes.Nevertheless, these differ from the new isolated in myxospore body length (18.7 ± 0.9 μm in length for H. multiradiatus, 14.3 ± 0.1 μm for H. loretoensis, 13.4 ± 0.9 μm for H. peruviensis and 21.5 ± 0.3 μm to the new isolated), polar capsule length (9.1 ± 0.1 μm in H. multiradiatus, 5.1 ± 0.2 μm in H. loretoensis, 3.3 ± 0.2 μm in H. peruviensis and 4.9 ± 0.2 μm in the new isolated), number of coils of the polar tubule (10-11 in H. multiradiatus, five in H. loretoensis, four to five in H. peruviensis and seven to eight in the new isolated) and in the length of the caudal appendage (25.8 ± 0.6 μm in H. multiradiatus, 21.9 ± 0.1μm in H. loretoensis, 10.7 ± 0.1 in H. peruviensis and 10.7 ± 1.2 μm in the new isolated).Compared with the all other freshwater Henneguya species reported to infect Amazonian fishes, the new isolated differed in at least one characteristic (shape of spore, size of spore or polar capsule, presence or absence or number of valve striations, size of caudal appendage and number of polar tubules turns), tissue and host preference as showed in the Table 1.
Regarding molecular data, from South America of the around hundred recognized species, large number of species lack molecular data (Milanin et al. 2017).Indeed, for much of the myxosporean described myxospore morphology was used by ichthyopathologist researchers for species discrimination, because myxo-spore is a unique structure possessing many characters important for classification (Lom and Dyková 1992).In our study, the partial 18S rDNA gene was successfully amplified by PCR using general eukaryotic and specific primers to myxosporean parasites (Fig. 5).However, after the amplification process, sequencing failed so it was not possible to carry out phylogenetic analysis.In addition, to the few samples with only five plasmodia to perform morphological, ultrastructural, histological and molecular analysis and limitations in accessing new samples of the same region.Although, we were not able to provide the phylogenetic data, the new isolate was strongly characterized based on spore morphometrically features as well as biological traits such as tissue preference and host specificity both important taxonomic keys for classification of freshwater histozoic myxobolids.Future molecular phylogenetic studies are highly recommended, since this would permit stronger taxonomic comparison.

Figure 1 .
Figure 1.Henneguya sp.parasite from the skin of Synbranchus marmoratus.a: formalin-fixed myxospores in valvular view showing appendage caudal (large arrows) and two polar capsules in the anterior pole of spore occupied only the anterior third of the myxospore body (small blue arrows).b: mature myxospores stained with Giemsa with noticeable binucleate sporoplasm (double arrow) and polar capsules with aubergine shape (large arrow).c: schematic illustration of mature myxospore with polar tubule inside of polar capsule.Scale bars: 5 µm.

Figure 2 .
Figure 2. Surface topography by SEM of Henneguya sp.infecting skin of Synbranchus marmoratus.a: mature myxospore in valvular view showing smooth valve cell with presence of mucous (white star) in a small area and caudal appendage.Scale bar. 1 µm.b: amplified area of the caudal appendage evidencing density of caudal appendage likely to be identical to that of its valve.Scale bar.200 nm c: myxospore evidence a conspicuous sutural line in sutural view.Scale bar: 100 nm.

Figure 4 .
Figure 4. Histological sections of the host-tissue infected of Synbranchus marmoratus with Henneguya sp.a: Intact plasmodium located in the stratus corneum of epidermis layer of the skin.Scale bar.50 µm.b: mature myxospore in sutural view with noticeable caudal appendage.Scale bar. 10 µm.