Henneguya sacacaensis n. sp. (Myxozoa: Myxosporea) parasitizing gills of the acará bicudo Satanoperca jurupari (Osteichthyes: Cichlidae) in eastern Amazon

marcela.videira@ueap.edu.br Abstract This study describes Henneguya sacacaensis n. sp. in specimens of the Osteichthyes Satanoperca jurupari (Heckel, 1840), collected in the Rio Curiaú Environmental Protection Area in the city of Macapá, state of Amapá Brazil. Using optical microscopy and molecular analysis, these cyst-shaped parasites were analyzed. The gills of 57.14% of the analyzed S. jurupari contained hundreds of spores. The cysts found on the gill lamellae were oval-shaped and whitish. The Henneguya spores had an average length of 46.5 (41.3-56.92) µm. The fusiform body of the Henneguya measured 16.5 (13.16-20.01) µm long and 5.1 (3.91-6.12) µm in width, the two polar capsules had a taper of 3.83 (3.4-4.32) µm and a width of 1.68 (1.4-1.99) µm, and the tail measured 30 (22.47-41.67) µm in length, containing a polar filament coiled seven to nine times. Morphogical and phylogenetic analysis allowed the preposition of a new species, Henneguya sacacaensis n. sp, that belongs to the family Myxobolidae and the genus Henneguya


Introduction
Myxozoa are endoparasites that can infect various organs and present high specificity (László et al., 2002). The genus Henneguya Thélohan, 1892 (Cnidaria: Myxobolidae) is one of the most diverse for the described species (Lom & Dyková, 1992) within the Myxosporea class and is the second largest genus, with about 190 described species worldwide (Eiras & Adriano, 2012).
Knowledge of parasitic biodiversity in South America is limited, especially in the Amazon region, due to the high ichthyofauna diversity and remoteness (Reis et al., 2016;Zatti et al., 2018). Recently Zatti et al. (2018) claim that 55 species of Henneguya spp. infect fish in Brazil. Of these, about 20 were found infecting fish species in the Amazon region (Velasco et al., 2016;Naldoni et al., 2018;Abrunhosa et al., 2018;Zatti et al., 2018).
The morphology of this genus is characterized by the number and shape of the spore valves, the position, number, and shape of the polar capsules (round, piriform, or ellipsoid), the relative position of the suture line, the presence of grooves and superficial appendages, and the number of coiled polar filaments (Feist & Longshaw, 2006;Lom & Dyková, 2006;Kaur & Attri, 2015).
This study describes the morphological characteristics and molecular aspects of a new species of Myxozoa, Henneguya sacacaensis n. sp., found in the gills of S. jurupari. To date, no Myxobolidae has been recorded to infect this host.

Material and Methods
The 63 specimens used for this study were collected from the Curiaú River, Macapá, Amapá, Brazil (0°8'43.6"N, 51°2'30.3"W) ( Figure 1). They were captured between September 2018 and September 2019 by the team at the Laboratory of Morphophysiology and Animal Health (LABMORSA) of the State University of Amapá (UEAP), with the help of local fishermen using a 30 mm cast net. The experiment was approved by the Animal Use Committee of the Brazilian Agricultural Research Company -Amapá (012-2018) and registered in the Biodiversity Authorization and Information System, IBAMA (SISBIO/ICMBIO License number 50376-1). The captured animals were conditioned and transported alive to LABMORSA/UEAP in refrigerated boxes with portable pumps. They were acclimatized and stored in glass aquariums with electric pumps for aeration. Subsequently, parasitological analyses were performed.
In the aquariums, the animals were anesthetized with tricaine methanesulfonate (MS222 SIGMA) at a concentration of 50 mg L -1 . Their body surface was examined with binocular stereoscopic microscopy to detect lesions (cysts) or loss of coating (scales and epidermis). Infectious foci of parasites in their developing and spore phases were found in the gall bladder. Small fragments were removed and observed with light microscopy (LM) to confirm parasitosis. Gall bladder fragments were fixed in Davidson's solution (70% alcohol, formaldehyde, acetic acid, and distilled water) for 24 hours before paraffin embedding for histopathology. They were then stained with hematoxylin and eosin (H&E) and Ziehl-Neelsen (ZN) and photographed at Carlos Azevedo Research Laboratory at the Federal Rural University of the Amazon (LPCA/UFRA). The methodology proposed by Bush et al. (1997) was used to calculate prevalence.
Cysts of eukaryotic microparasites were collected and stored in 80% ethyl alcohol at 4 °C. The total DNA of each sample was extracted using the Wizard  Genomic DNA. DNA samples were quantified by spectrophotometry (Biodrop DUO).
All molecular analyses were based on the 18S rDNA sequences, which were amplified using MC5/MC3 primers (Molnár, 2002). The final volume of the polymerase chain reaction (PCR) was of 25 μL using taq DNA polymerase Master Mix (PROMEGA, Madison, USA), with 0.5 μM of each primer and 2 μL of the DNA sample. With the initial amplification being initiated by denaturation of 10 s at 95 °C, followed by 40 cycles of denaturation at 95 °C for 1 min, hybridisation at 48 °C for 2 min, and polymerisation for 4 min 30s at 72 °C, with final polymerisation of 10 min at 92 °C.
The amplicons were subjected to electrophoretic analyses through 1.5% agarose gel purified using GFX™ PCR DNA and Gel Band Purification Kit reagents were used to purify the material after PCR, as described by the manufacturer. These steps were performed at the Laboratory of Applied Genetics at the Federal Rural University of the Amazon (LGA/UFRA).
The amplification product was sequenced on an ABI 3730 automatic DNA analyzer using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). To confirm the observed mutations, the sample was sequenced with both forward and reverse primers. The nucleotide sequences obtained were edited and aligned using the BioEdit program (Hall, 1999). The phylogenetic relationship was obtained through maximum parsimony and Bayesian analyses, using the PAUP 4.0 b10 program (Swofford, 2003) for the former and the MrBayes 3.1.2 program (Ronquist & Huelsenbeck, 2003) for the latter. Maximum parsimony analysis was performed with a heuristic search algorithm, in which equal weight was given for transitions and transversions, while insertions and deletions (indels) were treated as lost data. The confidence level of the most parsimonious tree nodes was evaluated by 1,000 bootstrap replicates (Felsenstein, 2004).
In the Bayesian analysis (BI), two parallel runs of four simultaneous searches were performed using the Markov chain Monte Carlo method (MCMC) for 5,000,000 generations each, sampling one tree every 1,000 generations and discarding the results of the first 1,250 trees (25% of the sample). The remaining 3,750 trees were used to estimate the confidence level (subsequent probability) of each node in the phylogenetic reconstruction. In the cladogram analysis, the DNA sequences of the organisms used were obtained directly from GenBank.

Vegetative phase
Henneguya parasites can form cysts at various sites in the gill filaments, and infection can be characterized as lamellar (Molnár, 2002). Stereomicroscope analysis under incident light showed whitish cysts and ellipsoids in a thin layer of connective tissue (Figure 2A).

Spores
Fresh spores were measured (n=31) ( Figure 2B, C, D). Average total lengths were 46.5±5.47 µm; spores bodies averaged 16.5±2.64 µm in length and 5.1±0.94 µm in width. The two polar capsules have a taper 3.83±0.31 µm in length and 1.68±0.20 µm in width, with the tail measuring 30±6.87 µm long. Each capsule contained a polar filament coiled seven to nine times ( Table 1) Etymology: The epithet of this genus is in honor of Raimundo dos Santos Souza, popularly known as "Sacaca", due to his contribution to Amapá popular culture.
The partial sequence of the SSU rDNA obtained from the species under study contains 1335 bp (GenBank accession number MT137652). For phylogenetic analysis, were used 38 South American sequences of Myxobolus spp. and Henneguya spp. available on Genbank. In the Bayesian inference analysis, Henneguya spp. formed a monophyletic group distributed in two clades, clade A, which includes Henneguya species that parasitize several fish species, mainly from the families Cichlidae and Pimelodidae, and clade B, which includes Myxobolus spp. parasites that infect organisms from the Amazon region (Figure 4). In comparisons between Henneguya spp. species that parasitize cichlids in the Amazon, there was a genetic p-distance ranging from 2.911 to 8.597% (Table 2).  Braz J Vet Parasitol 2020; 29(2): e000620

Discussion
The characteristics of this myxosporidian are typical of the myxospore morphology of histozoic infection and development of cysts in host gills described by Lom & Dyková (2006). The morphology of Henneguya sacacaensis n. sp. in this study was compared with other species of Henneguya described in the Amazon region.
In South America, about 100 species of the myxosporidian have been recorded in freshwater, being most of these species belonging to the Myxobolidae family, and the genera Myxobolus and Henneguya the most well-studied (Adriano & Oliveira, 2018;Naldoni et al., 2018). In the Amazon region, the most species for this genus have been recorded in the state of Pará with 75%, followed by Amazonas with 15%, and Amapá with 10%.
In the state of Amapá, only one species of myxosporidian has been described, Henneguya jariensis, parasitizing the fin of Cichla monoculus (Agassiz, 1831) (Zatti et al., 2018). Therefore, Henneguya sacacaensis n. sp. is the second myxosporidian species described in Amapá.   The Henneguya spp. have been described to infect various sites in Amazonian fish including gills, kidneys, gallbladder, medulla, testicles, and brain. Velasco et al. (2016) described that the genus Henneguya has a strong tendency to form clades, taking into consideration aspects such as environment, order, and family of host fish. Other researchers describe this trend in their studies as well (Fiala, 2006;Ferguson et al., 2008;Adriano et al., 2012;Carriero et al., 2013;Moreira et al., 2014;Abrunhosa et al., 2018). The Henneguya sacacaensis n. sp. behaves similarly and group together with others that parasitize the Cichlidae family.
Among the Henneguya spp. that infect fish from the Amazon, the Henneguya amazonica stands out (Rocha et al., 1992), with the longest total length (59.3 µm) and tail length (45.4 µm). Being that Henneguya sacacaensis n. sp. has a total length of 46.5 µm and tail length of 30 µm, only Henneguya astyanax (Vita et al., 2003) approaches it in relation to the total length (47.8 µm) and Henneguya paraensis (Velasco et al., 2016) is similar in tail length (29.5 µm).
Regarding polar capsule length, it is clear that Henneguya malabarica (Azevedo & Matos, 1996) is similar to Henneguya sacacanesis n. sp., with lengths of 3.7 µm and 3.8 µm, respectively, but the capsule width most closely resembled other species of the genus Henneguya. Henneguya sacacaensis n. sp. capsule width measure about 1.6 µm, which is more closely related to that of H. melini (1.70 µm) and H. quelen (1.68 µm).
Although the morphometric aspects are very similar to H. astyanax, the morphology of Henneguya sacacaensis n. sp. differs in the position of the binucleated cell. The binucleated cell of H. astyanax is just below the polar capsules, while in Henneguya sacacaensis n. sp., it is located at opposite ends in the sporoplasma. The sporoplasma also differs in its shape. Furthermore, the tail reinforces the difference in its arrangement, although there are no H. astyanax SSU rDNA data.

Conclusion
Thorough analysis of the morphological, morphometric, and molecular aspects allow us to describe a new species, Henneguya sacacaensis n. sp., with characteristics that differ from all other species previously described.