Ultrastructural and molecular study of the microsporidian Toguebayea baccigeri n. gen., n. sp., a hyperparasite of the digenean trematode Bacciger israelensis (Faustulidae), a parasite of Boops boops (Teleostei, Sparidae)

A new microsporidian Toguebayea baccigeri n. gen., n. sp., hyperparasite of Bacciger israelensis (Digenea, Faustulidae), parasite of Boops boops (Teleostei, Sparidae) is described by means of transmission electron microscopy. The phylogenetic analysis, based on the SSU rDNA gene, places the new species in the clade containing mainly crustacean-infecting microsporidia of the genus Cucumispora, within superclade V (Marinosporidia) sensu Vossbrinck et al., 2014. Mature spores of T. baccigeri are ovoid, uninucleated and measure 2.5 × 1.4 μm. The number of coils of the polar tube is 8–10. The polaroplast is composed of an external lamellar part and an internal vesicular or granular part. The main differences that distinguish the new genus and new species from the closely related microsporidia include hyperparasitism in a digenean host infecting a marine fish, the geographic distribution (coast of Tunisia), presence of one nucleus at all developmental stages, disporoblastic sporogony, and the absence of sporophorous vacuoles.

In the present study, we describe a new genus and a new species by means of transmission electron microscopy, Toguebayea baccigeri n. gen., n. sp., hyperparasite of Bacciger israelensis Fischthal, 1980 (Digenea, Faustulidae), a parasite of the bogue Boops boops (Linnaeus, 1758) (Teleostei, Sparidae) from the coast of Tunisia. We provide an SSU rDNA-based phylogenetic analysis of relationships of T. baccigeri n. gen., n. sp. with related species/genera.

Transmission electron microscopy
Adult live flukes were rinsed with a 0.9% NaCl solution and fixed in cold (4°C) 2.5% glutaraldehyde in a 0.1 M sodium cacodylate buffer at pH 7.4 for 2 h, rinsed in 0.1 M sodium cacodylate buffer at pH 7.4, post-fixed in cold (4°C) 1% osmium tetroxide with 0.9% potassium ferricyanide in the same buffer for 1 h, rinsed in Milli-Q water (Millipore Gradient A10), dehydrated in graded ethanol series and propylene oxide, embedded in Spurr's resin and polymerized at 60°C for 72 h. Semithin sections (500 nm thick) were obtained using a Reichert-Jung Ultracut E ultramicrotome and stained with 1% methylene-blue with 1% borax. Ultrathin sections (60-90 nm thick) containing the excretory vesicle were prepared with the same ultramicrotome. Sections were placed on copper grids (200 lm mesh size) and double-stained with uranyl acetate and lead citrate according to the Reynolds procedure [12]. Stained ultrathin sections were examined in a JEOL 1010 transmission electron microscope operated at an accelerating voltage of 80 kV, equipped with a CCD camera (Gatan Orius model SC1000A1) in the "Centres Científics i Tecnològics" of the University of Barcelona (CCiTUB). All microsporidian measurements were obtained using the Gatan Microscopy Suite Digital Micrograph version 2.11.1404.0 coupled to the CCD camera.

Molecular analyses and phylogenetic tree
Total DNA was isolated from 17 digeneans (fixed in absolute ethanol) obtained from eight fishes. For DNA isolation, a commercial FastDNA Ò Spin Kit for Soil (MPBiomedicals, Solon, OH, USA) was used following the manufacturer's instructions, with the homogenizer FastPrep-24™ 5G (MP Biomedicals) as cellular disruptor.
A fragment of the SSU rDNA of the microsporidia was amplified by PCR targeting a 1200 bp region following Sokolova et al. [14] with the primer V1f and primer 1492r [23]. The reaction mixture contained 1U Taq DNA polymerase, 1 lL of each primer (10 lM), 2.5 lL of dNTPs mix (200 lM), 2.5 lL MgCl 2 (25 mM), 2.5 lL 10Â buffer (15 mM Mg 2+ ), 5 lL of DNA template and water to a total volume of 25 lL. The cycling conditions were initial denaturation of 95°C for 5 min followed by 35 cycles of 95°C for 30 s, 45°C for 60 s at suitable temperature, and 120 s at 72°C, followed by a final extension step at 72°C for 10 min. The PCR reactions were performed in an XP Cycler (Bioer Technology, Hangzhou, PR China) thermocycler. PCR products were resolved on 2% agarose gels. The desired size band was cut from the gel and purified with an EZNA Gel Extraction Kit (Omega Bio-Tek, Norcross, GA, USA), following the manufacturer's recommendations. The purified PCR products were sequenced at Macrogen Europe (Madrid, Spain) with primers V1f, 530r, 530f, 1061f, and 1492r [23,26].
The obtained nucleotide sequences were edited with the MEGA X program [8] and subsequently aligned with other microsporidian sequences using the ClustalW program included in MEGA X. Minor corrections, to increase the aligned sequence similarity and improve the inferences on any positional homology, were then made by hand. A BLAST search was carried out in order to elucidate any homologies or similarities with the sequences previously published in the GenBank database (Supplementary material Table S1).
The molecular identification was achieved by phylogenetic analysis through the Neighbour-Joining distance method with the Kimura 2-parameter estimate [9] with at least 1000 bootstrap replications (Supplementary material Figure S1); and the Maximum Likelihood method with the Tamura-Nei model [20] in MEGA X, using the sequence of Trichonosema pectinatellae Canning, Refardt, Vossbrinck, Okamura & Curry, 2002 (AF484695.1) as the outgroup (Fig. 1). Distance estimation was carried out in MEGA X using the p-distance model distance matrix for transitions and transversions (Kimura 2-parameter), with partial deletion of position with gaps or missing data (Supplementary material Table S2). The nucleotide sequence obtained in this work was submitted to the GenBank database under accession no. MZ413057.
Toguebayea baccigeri n. gen., n. sp. urn:lsid:zoobank.org:act:B182EACC-4E16-43FC-8576-59F89824592E Description: As for the genus. Diagnosis: Presence of a microsporidian hyperparasite with descriptive features of the genus in the cytoplasm of epithelial cells of the excretory vesicle of B. israelensis infecting the pyloric caeca of B. boops. Diagnosis of morphological features by TEM as described herein. Spores ovoid and uninucleated, approximately 2.5 Â 1.4 lm; exospore around 40 nm wide; endospore around 50 nm wide and about 5 nm wide at the level of the apical part near the anchoring disk; polar sac 30-40 nm wide; isofilar polar tube with 8-10 coils arranged in one or two rows, with a diameter of 95-115 nm, composed of alternate concentric electron-dense and electron lucent layers with a central tubular element 8 nm wide; manubrium around 115 nm wide and 850 nm long; well-developed polaroplast lamellar in the external part and granular in the internal part; nucleus around        Table 1) Microsporidian infection was found in adults of Bacciger israelensis, a digenean parasite of the bogue Boops boops caught off the coast of Tunisia. The pathogen was present along the entire length of the hosts excretory vesicle epithelium (Figs. 2 and 3). There was no evidence of spore groups enclosed in sporophorous vacuoles. Each developmental stage occurred in direct contact with the cytoplasm of the host cell and had only one nucleus (Figs. 3 and 5-8).

Meronts (Figs. 3 and 4)
Meronts were uninucleated, oval to elongated cells that measure 2-3 lm and were surrounded by a thin plasmalemma (Fig. 4 inset). Their cytoplasm contained a great number of free ribosomes, a few saccules of endoplasmic reticulum and rare vacuoles (Fig. 4A, inset). The nucleus appeared oval and measured approximately 1 lm in its larger diameter. Division of merogonic stages was observed (Figs. 3 and 4): initially, the meront elongated and its nucleus divided by transverse constriction, briefly presenting a binucleated appearance. Afterwards, the cytoplasm constricted, giving rise to two cells identical to the initial meront. Division of the nucleus was sometimes faster than that of the cytoplasm. As a result, on some occasions, it was possible to observe three meronts in a chain (Fig. 4B). Sporonts (Fig. 5) After a few divisions, some meronts underwent morphological transformations to become sporonts. These were ovoid cells, easily recognized by the presence of an electron-dense wall, about 15 nm thick, surrounding the plasma membrane ( Fig. 5A, inset). Their smaller diameter (in cross-sections) was about 1 lm, while their larger diameter (in longitudinal sections) was about 2 lm. Initially, endoplasmic reticulum saccules increased in number and dilated, and a few vacuoles appeared (Fig. 5B). These sporonts had a single large central nucleus measuring about 1 lm in diameter. During the nucleus' division, the sporont became binucleated for a certain time. These dividing sporonts had an elongated shape (measuring up to 5 lm long by 1.5 lm wide). They then constricted in the middle creating two uninucleated sporoblasts (Fig. 6A).
Sporoblasts and sporogenesis (Fig. 6) Sporoblasts were uninucleated (Fig. 6A). They were, at first, irregular in shape and then they became ovoid. They were surrounded by a continuous wall about 15-20 nm thick, and were also in direct contact with the cytoplasm of the host cell. Their cytoplasm contained numerous free ribosomes (and polyribosomes) and a few flattened saccules of endoplasmic reticulum (Figs. 6A, 6C, 6D). The appearance of the polar tube was the first obvious manifestation of sporogenesis. At the anterior end of the polar tubule, there was a polar sac containing an electron-opaque material forming an anchoring disc (Fig. 6D). The limiting membrane of the polar sac was continuous with that of the polar tube. Then, the polaroplast appeared to originate from an initially amorphous, electron-dense material associated with the anterior extremity of the polar tube (Figs. 6B, 6C). Within this amorphous mass there were numerous membranes forming parallel saccules that surrounded the manubrium, which is the anterior rectilinear part of the polar tube (Figs. 6C, 6D). In older sporoblasts, the anterior saccules were very flat, while the posterior saccules were dilated, presenting an irregular contour. These saccules originated from a polaroplast made up, in the mature spore, of two distinct parts: anterior lamellar part and posterior granular part. Sporogenesis ended when the initial wall separated from the plasma membrane of the young spore, giving rise to a clear endospore. The initial electron-dense wall became the exospore.

Mature spores (Figs. 3 and 7-10)
Spores were ovoid and uninucleated, measuring around 2.5 Â 1.4 lm. They were surrounded by an exospore with a maximum width around 40 nm and an endospore with a maximum width around 50 nm and about 5 nm wide at the level of the apical part near the anchoring disk (Figs. 7-10). The polar sac was about 30-40 nm wide. The anterior straight part of the polar tube measured about 850 nm long and 115 nm in diameter (Figs. 7B and 9A). The polar tube was isofilar with a diameter of 95-115 nm presenting 8-10 coils arranged in one or, more frequently, in two slightly overlapping rows (Fig. 7A). It was composed of alternate concentric electrondense and electron lucent layers, with a central tubular element 8 nm in diameter (Fig. 9B). The polaroplast was well-developed and made of a lamellar anterior and external part and a granular internal part (Fig. 8A). Numerous polyribosomes were observed around the polaroplast, nucleus and polar tube (Figs. 7A, 8A and 9B). The nucleus was ovoid, measuring about 650 nm long and 400 nm wide (Fig. 7). The posterior vacuole was irregularly round with a diameter around 375 nm (Fig. 7B).

Molecular analyses
One consensus sequence of 1228 bp was obtained (MicroBac 01) for T. baccigeri n. gen., n. sp. The results of the BLAST (Supplementary material Table S1 Figure S1) and the Maximum Likelihood (Fig. 1) analyses based on SSU rDNA showed similar results. In both phylogenetic analyses, the novel sequence fell in a well-supported branch together with Cucumispora Ovcharenko, Bacela, Wilkinson, Ironside, Rigaud & Wattier, 2010 and several unidentified sequences of crustacean-infecting microsporidia (Microsporidium spp.). This

Discussion
In the present study, we describe the type species of a novel genus of microsporidian, namely Toguebayea baccigeri n. gen, n. sp., hyperparasite of the faustulid digenean Bacciger israelensis, a parasite of the sparid fish Boops boops in the Mediterranean Sea (coast of Tunisia). The description of this new genus and new species is based upon ultrastructural characteristics of its lifecycle, parasitized host, geographical distribution and on sequencing of a partial fragment of the SSU rDNA of this microsporidian hyperparasite.
The representatives of the genera Cucumispora, Hyperspora, Myospora Stentiford, Bateman, Small, Moss, Shields, Reece & Tuck, 2010, Paradoxium Stentiford, Ross, Kerr, Bass & Bateman, 2015 and Thelohania Henneguy, 1892 that also fell in Clade V share relatively high sequence similarity with the new species; however, they can be differentiated from the latter [10,18,19]. In T. baccigeri n. gen., n. sp., the sporogony is disporoblastic and all the developmental stages are uninucleated and always in contact with the host cell cytoplasm (epithelial cells of the excretory vesicle) and sporophorous vacuoles are absent. It clearly differs from Cucumispora dikerogammari Ovcharenko, Bacela, Wilkinson, Ironside, Rigaud & Wattier, 2010 [10], which presents diplokaryotic meronts, sporonts and mature spores (see Table 1). In Paradoxium irvingi Stentiford, Ross, Kerr, Bass & Bateman, 2015 all stages are also in close contact with the host cell cytoplasm, but this genus differs from the new genus Toguebayea in the formation of binucleated and later tetranucleated meronts during merogony [19]. In Thelohania butleri Johnston, Vernick & Sprague, 1978, the most evident difference is the presence of groups of eight spores enclosed in sporophorous vacuoles [2]. In Myospora metanephrops Stentiford, Bateman, Small, Moss, Shields, Reece & Tuck, 2010, a myosporid microsporidian closely related to T. butleri (see Fig. 1), all lifecycle stages (meronts, sporonts, sporoblasts and spores) are diplokaryotic. Moreover, both merogony and sporogony are clearly different from those of T. baccigeri n. gen., n. sp. Thus, in M. metanephrops diplokaryotic meronts divide into tetranucleated meronts (with two diplokaryotic nucleus) and later into octonucleated meronts (with four diplokaryotic nucleus), and tetranucleated sporonts (with two diplokaryotic nucleus) form two diplokaryotic sporoblasts [17]. Finally, H. aquatica shows the most similar development with T. baccigeri n. gen., n. sp.; all the developmental stages are uninucleated and in close contact with the host cell cytoplasm. The most evident morphological differences are the size of the mature spores (1.2 Â 1.0 lm in H. aquatica vs. 2.5 Â 1.4 lm T. baccigeri n. gen., n. sp.) and the number of polar tube coils (4 in H. aquatica vs. 8 to 10 in T. baccigeri n. gen., n. sp.) [18] (see Table 1). Traditional taxonomy based on morphological features and the molecular SSU rDNA-based phylogenetic analyses show great discrepancies, and this fact has been emphasized by several authors [11,17,24]. For example, Vossbrinck and Debrunner-Vossbrinck [24] highlighted the importance of ecological trends, such as the parasitised host and its habitat, in the molecular phylogenetic grouping of the Microsporidia. Clearly, the use of these ecological characteristics is more consistent with evolutionary relationships of microsporidia than traditional classification based on morphological and lifecycle features. According to these authors, the clade Marinosporidia includes microsporidia parasitising predominantly marine hosts, with a few exceptions inhabiting the freshwater environment [2,10,17,19]. All species mentioned above are parasites of marine and freshwater crustaceans (Decapoda and Amphipoda), with only one exception: Hyperspora, which is a hyperparasite of M. collichia (Paramyxida) in the marine mollusc Cerastoderma edule in Ría de Arousa [18]. Toguebayea baccigeri n. gen., n. sp. is strikingly different in this respect: it is a hyperparasite of the adult stage of a digenean trematode parasitising a marine fish.
As to the geographical distribution, only the new species T. baccigeri was found in the Mediterranean Sea, off Salakta (Tunisia). All other species of Clade V discussed above were found in freshwater environments, such as ponds, lakes, reservoirs, oases or rivers in continental areas, or in other marine areas (North Sea, coast of Galicia (Spain), Pacific coast of Canada and New Zealand) (Table 1).
Thus, ultrastructure of developmental stages, the host species, habitat, geographical distribution, and phylogenetic position within Clade V identify the discovered microsporidium as a new species of a new genus. Our study also confirms for the first time microsporidian infection in a digenean of the Faustulidae family and adds a new genus and species, Toguebayea baccigeri, to the list of hyperparasitic microsporidia.

Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgements. The authors wish to thank the staff of the "Centres Científics i Tecnològics" of the University of Barcelona (CCiTUB) for their assistance in the preparation of samples. JM is a member of the 2017-SGR-1008 research group. EBG was granted an FPI predoctoral scholarship by "Gobierno de Canarias" co-funded by "Fondo Social Europeo" (85%) and "Consejería de Economía, Industria, Comercio y Conocimiento". Funding from the University of La Laguna and the Canary Council of Economy, Knowledge and Employment (CEI program) is acknowledged.

Supplementary material
The supplementary materials of this article are available at https://www.parasite-journal.org/10.1051/parasite/2022007/olm. Figure S1. Phylogenetic analysis, based on the Neighbour-Joining method, of Microsporidia sequences based on the SSU rDNA gene. Trichonosema pectinatellae (AF484695.1) was used as the outgroup. MicroBac01 corresponds to the sequence obtained for Toguebayea baccigeri n. gen., n. sp. Table S1. Result of the BLAST analyses for the nucleotide sequence obtained in this study MicroBac 01. (QC: query cover; Idt: identity). Table S2. Comparison of small subunit gene (SSU rDNA) sequences among Microsporidia parasites. Pairwise distance (bottom diagonal) obtained by p-distance.