Hepatic coccidiosis in Serrasalmus rhombeus Linnaeus, 1766 from the Amazon basin: morphological and histopathological aspects

michele.velasco.mv@gmail.com Abstract Calyptospora species are coccids that commonly cause liver infections in fish all around the world. This paper describes the morphology and histopathological characteristics of liver infection caused by Calyptospora sp. in black piranha Serrasalmus rhombeus , from the Capim River, in the municipality of Ipixuna do Pará, state of Pará (Brazil). Specimens were collected, analyzed and necropsied and tissue fragments containing parasites were prepared for histology and scanning electron microscopy. Parasitism was detected in 33.0% of the analyzed specimens, which had spherical oocysts in the liver, with four pyriform sporocysts presenting sporozoites internally. A histological examination revealed oocysts positioned close to blood vessels, causing necrosis and degeneration of hepatic parenchyma, while the presence of mononuclear cell infiltrate and melanomacrophages indicated the onset of an inflammatory process. This is the first record of the genus Calyptospora in fish from the Capim This describes the morphology of Calyptospora sp. and histopathological aspects of hepatic coccidiosis in , captured


Introduction
Coccids, a subclass of the phylum Apicomplexa, are intracellular parasites that have a wide variety of host fish, from Chondrichthyes (elasmobranches) to Osteicthyes, which are represented by freshwater and saltwater teleosts that occur worldwide (Dyková & Lom, 1981;Davies & Ball, 1993;Molnár, 2006). In fact, there is phylogenetic evidence that coccids of terrestrial vertebrate emerged from fish coccidian strains (Rosenthal et al., 2016).
The family Calyptosporidae is represented by species with a heteroxenous life cycle, where by the oocysts are transmitted from the intermediate host, an invertebrate, to the definitive host, the fish (Overstreet et al., 1984;Whipps et al., 2012). The genus Calyptospora Overstreet, Hawkins and Fournie, 1984 is known to cause mainly liver pathologies, but it can also be found in the pancreas, gallbladder, mesentery, gonads and other tissues (Overstreet & Hawkins, 2017;Ramos et al., 2018;Negrão et al., 2019).
Serrasalmus rhombeus Linnaeus, 1766 stands out among the ichthyofauna of the Amazon basin. Commonly known as black or red-eyed piranha, its carnivorous habits render it notoriously aggressive. This species is part of the family Serrasalmidae, endemic to South America, usually found in northeastern Brazil and in rivers of the Amazon basin, which local communities sell as ornamental fish (Nakayama et al., 2001;Acácio et al., 2012;Sá-Oliveira et al., 2017;Anjos et al., 2018).
This paper describes the morphology of Calyptospora sp. and histopathological aspects of hepatic coccidiosis in S. rhombeus, captured in the Capim River, in the northeast of the state of Pará, eastern Amazonia. The fish were anesthetized using the MS-222 protocol (50 mg/L tricaine methanesulfonate) and then necropsied, as authorized by the institution's Ethics Committee on Animal Use, under Protocol No. 011/2014). A parasitology examination was performed using a stereomicroscope to search for lesions/cysts on the body surface and in the coelomic cavity, and the findings were confirmed under an ordinary light microscope (LM), using fresh slides.

Histology
Were small fragments of parasitized liver tissue were fixed in Davidson's fluid for a period of 24 hours, processed using paraffin techniques, 5 um thick cut and stained with Hematoxylin-Eosin (HE) and Zielh-Neelsen (ZN) (Luna, 1968).

Oocyst morphometry
Mature oocysts (n=30) were measured from images captured by a Zeiss Axiocam ICc 1 camera installed in a Zeiss Axioskop 40 microscope. Means and standard deviations were measured in micrometers (µm). A morphometric comparison was made with other species described in the literature, using principal component analysis (PCA), and analyzing the following parameters: oocyst diameter (OD), sporocyst length and width (SL) and the length-to-width ratio of oocyst to sporocyst (L:W), as described by Da Silva et al. (2019) for Calyptospora. The hierarchical clustering method UPGMA was also adopted. Both analyses were performed using PAST 3.0 software (Hammer et al., 2001).

Scanning Electron Microscopy (SEM)
Fragments of parasitized liver tissue were fixed in 5% glutaraldehyde buffered with sodium cacolydate at pH 7.2 for 12h at 4ºC. They were then washed in buffer solution, fixed in 2% OsO 4 and buffered for three hours without varying the temperature. After this, the fragments were dehydrated in increasing ethanol and propylene oxide series, coated with a thin layer of gold and photographed in a TESCAN MIRA3 FEG-SEM field emission scanning electron microscope at the Emílio Goeldi Museum in Belém, state of Pará (Brazil).

Results
Spherical oocysts, showing characteristics inherent to the genus Calyptospora, were found in the liver of 05 of the 15 analyzed S. rhombeus specimens, representing 33.0% of the specimens.
The oocysts had a diameter of 17.4 ± 1.0 (15.7-19) ( Figure 1A, 2B and 3A), with four pyriform sporocysts in the shape of "bowling pins" (Figure 1B, 2 A and B), 8.33 ± 1.1 (6-10) in length and 4.29 ± 0.83 (3-4.68) in width. The oocysts, which were arranged perpendicularly and covered by a thin membranous veil-like layer, and in the internal region of the sporocysts, two sporozoites were present ( Figure 1C and 3A). A suture was visible in the apical region of the external surface ( Figure 2B and D, 3B), while the posterior part contained several lateral projections, the sporopods ( Figure 2C, 3B). These morphological traits are characteristic of the genus Calyptospora (Overstreet et al., 1984).
The first two axes in the PCA, which explained 92.1% of the variance, were influenced mainly by the length-towidth ratio of oocyst to sporocyst. The Calyptospora sp. identified in this study was similar to Calyptospora sp. 4 and C. gonzaguensis, although this relationship was negative to the other species on both axes ( Figure 4). This aspect was confirmed in the similarity dendrogram, indicating that the Euclidean distance between Calyptospora sp. 4 and C. gonzaguensis was smaller, compared to Calyptospora sp. (Figure 5).
The histological results revealed oocyst clusters close to blood vessels in the hepatic parenchyma ( Figure 6 A -F), showing tissue degeneration, localized liver fibrosis and compression necrosis, with hepatic cells exhibiting pyknotic nuclei and lacey cytoplasm ( Figure 6A and 6D). Note the presence of mononuclear cell infiltrates surrounded the oocysts, indicating a mild inflammatory process ( Figure 6A and 6C), as well as melanomacrophages in both types of stains ( Figure 6B and 6F).
Calyptosporids have already been described causing liver infections in Serrasalmus niger, Serrasalmus striolatus and S. rhombeus, the host species in this study, presenting specificity for Calyptospora serrasalmi (Cheung et al., 1986;Casal et al., 2007;Whipps et al., 2012;Negrão et al., 2019). The spherical shape of oocysts, pyriform shape of sporocysts, and lateral extension of the sporopods were similar to this species. However, the morphometric comparisons showed greater proximity to Calyptospora sp. 4 found in Arapaima gigas (Bonar et al., 2006) and C. gonzaguensis described in Triportheus angulatus (Silva et al., 2020), which were particularly congruent with the diameter of the oocysts, of 19 µm and 19.6 µm, respectively ( Table 1).
The histopathological examination revealed oocyst clusters in the liver parenchyma adjacent to blood vessels. This is a typical histopathological finding of Calyptospora and has been described in several studies (Békési & Molnár, 1991;Ramos et al., 2018;Velasco et al., 2012;Santiago et al., 2012). However, minor variations occur, with some species showing tropism only in blood vessels, as observed in C. gonzaguensis, or only in the liver parenchyma, such as C. paranaidji (Silva et al., 2020;Da Silva et al., 2019).
In more intense infections, the liver changes to a visibly lighter color (Videira et al., 2013;Velasco et al., 2012), resulting from the degeneration of liver tissue. As the oocysts proliferate, they cause inflammation and formation of granulomas coated with fibrous tissue (Ramos et al., 2018;Fournie & Overstreet, 1993;Solangi & Overstreet, 1980). According to Azevedo et al. (1993), the damage caused by Calyptospora becomes more evident at the cellular level, with cells adjacent to the oocysts presenting morphological changes such as hypertrophy and destruction of cytoplasmic structures. Similar results were found in this study. Inflammatory infiltrate and the presence of mononuclear cells surrounding the oocysts were also described by Velasco et al. (2012), Ramos et al. (2018) and Santiago et al. (2012). Melanomacrophages were also found close to oocysts. These phagocytic cells belong to the host's innate immune system and are a common cell type usually found in fish liver infections. In such infections, these cells are part of the reticuloendothelial system and their function is to eliminate pathogens that use the bloodstream to reach the target organ/tissue where their life cycle is completed (Agius, 1985;Steinel & Bolnick, 2017).
Susceptibility to parasitism, as well as the degree of pathogenicity, may depend on ecological factors, as was observed in an experiment with induced C. funduli infections in Atheriniformes fish, where environmental conditions and geographical isolation acted as barriers to the prevalence of parasites in each species (Fournie et al., 2000).
The histopathological data indicated that although the prevalence of parasitism by Calyptospora sp. was not high, significant pathological changes occurred in the host's liver tissue. These changes may give rise to functional abnormalities in the liver, threatening the host's survival in nature. Brazil/ Liver (Bonar et al., 2006)