Paleoparasitological analysis of a coprolite assigned to a carnivoran mammal from the Upper Pleistocene Touro Passo Formation, Rio Grande do Sul, Brazil

A paleoparasitological analysis was carried out on a large coprolite assigned to a carnivoran mammal, recovered from the Municipality of Uruguaiana, in the western region of the State of Rio Grande do Sul, Brazil, where the Upper Pleistocene Touro Passo Formation crops out. For this, an individual sample was extracted from the specimen using an electric drill, dissociated with 10% hydrochloric acid solution, washed with distilled water, and sifted through a 500 mesh Tyler sieve. After laboratory processing, the sediment retained on the sieve was mixed with glycerin and examined by optical microscopy, which revealed the presence of 14 protozoan oocysts and three nematode eggs. The morphological characteristics of the oocysts (i.e., spherical shape, thickwalled, internal zygote apparently at the beginning of sporulation, as well as their size) and of the eggs (i.e., ovoidal shape, rounded ends, smooth surface, thin-shelled, embryo in their interior, along with their morphometry) suggest that these specimens belong respectively to the orders Eucoccidiorida and Strongylida (Family Ancylostomatidae) represented by several parasitic species of the alimentary tract of modern carnivore. This is the fi rst record of paleoparasites discovered in a vertebrate host from the Touro

The term coprolite, denotes petrified feces, droppings or excrement and is used in paleontological and archaeological studies (Ferreira et al. 2008, Hunt et al. 2012. Coprolites provide interesting paleoecological evidences, such as the diet and physiology of the producer, the presence of organic inclusions, intestinal microbiota and endoparasitic fauna, which can not be accessed through body remains (De Baets & Littlewood 2015, Bajdek et al. 2016, Vajda et al. 2016, Dentzien-Dias et al. 2018, De Baets et al. 2020. Additionally depending on the type of paleoparasite found in a coprolite, it is also possible to infer about their probable hosts and pathogenic effects on them, as well as food chains, paleoenvironmental and paleoclimatic conditions of the ecosystems in the geological moment which these organisms were fossilized, using the necessary requirements for survival and maintenance of the biological cycles of the species of similar modern parasites as proxies. Despite advances in paleoparasitological analysis of human coprolites, including the current possibility of DNA extraction from parasites, especially by some South American parasitologists since the 20 th century (Ferreira et al. 2011), there are few contributions on this topic regarding animal coprolites worldwide. Nevertheless these studies have increased in recent years and have already been able to reveal the presence of parasitic protozoans and helminths in coprolites of sharks (Dentzien-Dias et al. 2013), dinosaurs (Poinar & Boucot 2006), crocodyliforms (Cardia et al. 2018, 2019a, b, Dentzien-Dias et al. 2018, dinornithiform birds (Wood et al. 2013), non-mammalian therapsids (Hugot et al. 2014, Silva et al. 2014, Bajdek et al. 2016, Francischini et al. 2018, and different mammals, such as rodents (Sardella & Fugassa 2009a, b, 2011, Sardella et al. 2010, Beltrame et al. 2012, Mowlavi et al. 2014), ruminants (Fugassa et al. 2008, Taglioretti et al. 2015, Beltrame et al. 2017a, b, Nunes et al. 2017 and carnivorans (Fugassa et al. 2006, Beltrame et al. 2010, 2016, Sianto et al. 2014, Mowlavi et al. 2015, Fugassa & Petrigh 2017, Perri et al. 2017, Petrigh et al. 2019, Tietze et al. 2019. The oldest confidently identified paleoparasitological record in a coprolite derives from the Permian and is dated in up to 259.8 million years (Dentzien-Dias et al. 2013), although possible even older Carboniferous remains have been reported (Zangerl & Case 1976), demonstrating an universe of possibilities still to be explored in the Veterinary Paleoparasitology and Paleoichnology areas, mainly in geological units with rich and abundant paleofaunas, such as the Touro Passo Formation.
Therefore the present study describes the first record of paleoparasites in a unique coprolite found in the Touro Passo Formation, assigned to a carnivoran mammal.

Coprolite
The coprolite analyzed in this study was preliminarily described by Kerber & Oliveira (2008a) and it comes from the Milton Almeida outcrop (29°40'20.57" S; 56°51'59.20" W), in the margins of the Touro Passo stream, located in the Municipality of Uruguaiana, in the western region of the State of Rio Grande do Sul, in Southern Brazil (Fig. 1). This fossil locality belongs to the Touro Passo Formation, which is Lujanian (Late Pleistocene) in age and lithologically composed by silty-sandy rocks of fluvial origin with abundant carbonate concretions layers (Kerber & Oliveira 2008a). The coprolite was originally housed in the Setor de Paleovertebrados of the This coprolite was preserved through phosphatization and is apparently complete, with a size of 18.0 cm in length and of 4.0 cm in diameter. Additionally the coprolite had grayish white coloration and cylindrical with rounded ends, consolidated and homogeneous shape, divided into four blocks by constrictions, besides the presence of large amount of organic material, such as parts of undigested bones. This morphology resembles that proposed for big felids by Chame (2003), such as the genera Panthera and Puma, however the large size of the coprolite and the finding of bones may also suggest other extinct large Carnivora mammals, such as the genera Smilodon, Protocyon, Theriodictis and Arctotherium, which were able to hunt large prey like ground sloths or feed on their carcasses. Fossil remains of carnivorans are still unknown from the Touro Passo Formation, but other Late Pleistocene (Lujanian) records from the Rio Grande do Sul, include material assigned to canids (Dusicyon cf. D. avus, Protocyon troglodytes, and Theriodictis sp.), felids (Smilodon populator) and ursids (cf. Arctotherium and indeterminate remains) (Rodrigues et al. 2004, Ribeiro & Scherer 2009, Pereira et al. 2012, Lopes 2013 and references therein).

Laboratory processing
Two individual samples (≅ 1.0 g) were extracted from the coprolite surface and its internal portion using an electric drill, resulting in some macerated material (following the procedure made by Silva et al. 2014). The resulting product of each sample was stored individually in properly labeled two Falcon 15 mL polypropylene tubes. A 10% hydrochloric acid solution was added to the first tube, as proposed by Ferreira et al. (2011). Upon dissociation of the minerals, the reaction was stopped by adding a double volume of distilled water.
The resulting solution was washed several times with distilled water, and then sifted through a 500 mesh Tyler sieve, following Bouchet et al. (1999). The residual material from sample retained on the sieve was washed again in distilled water. A drop of this material was then placed on a microscope slide, three drops of glycerin were added, and the slide was covered with a coverslip. The material was analyzed by bright field microscopy at 100x and 400x magnifications using a Quimis ® optical microscope and images of the paleoparasites observed were recorded with a Sony ® digital camera coupled to it. Each paleoparasite was measured individually using a Bel Photonics ® ocular micrometer. Measurements of the length and width of the paleoparasitic specimens were expressed in micrometers (μm), as mean ± standard deviation and range in parentheses.
The macerated material of the second tube was rehydrated by immersion in a 0.5% solution of trisodium phosphate and submitted to spontaneous sedimentation following to Iñiguez et al. 2006. Upon 72 h, aliquots of the resulting sediment were used for the extraction and detection of the Toxoplasma gondii DNA. For this, these aliquots were grinded by Biovortex homogenizer (Biospec Products Inc, USA). DNA extraction was carried out by using the IllustraTM Tissue & Cells Genomic Prep Mini Spin kit (GE Healthcare, USA) and quantification in a spectrophotometer (Epoch-Biotek, USA). PCR reactions were performed by employing the primers described by Homan et al. (2000) to amplify a 529bp fragment. Primers TOX4 (5'-CGCTGCAGGGAGGAAGACGAAAGTTG-3') and TOX5 (5'-CGCTGCAGACACAGTGCATCTGGATT-3') were used. The reactions (25 µL final volume) were run with the following reagents: 10 mM Tris HCl (pH 8.0), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTP, 10 ρmol of each primer, 0.2 units of Taq DNA polymerase, and 10 ng of DNA template. Amplification was performed in a Veriti (Life Technologies ® , Carlsbad, USA). Initial denaturation for 7 minutes at 94°C was followed by 35 cycles of 1 minute at 94°C, 1 minute at 60°C and 1 minute at 72°C, and final extension for 10 minutes at 72°C. The sequence was analyzed by electrophoresis in 1.5% agarose added with 0.1 μL/mL of SYBR ® safe DNA gel stained (Invitrogen, USA), recorded using the transilluminator (Syngene, USA), and the image was captured by the digital documentation system.

DISCUSSION
Thick-walled subspherical sporulated oocysts and thin-shelled ovoidal embryonated eggs are respectively the typical evolutive stages of most modern species of Eucoccidiorida protozoans and Strongylida nematodes (including Ancylostomatidae) that parasitize vertebrates which can be found in the feces of their final hosts during their sexual reproduction phases (University of Illinois 1933, Anderson 2000, Berto et al. 2014. Eucoccidiorids are intracellular obligatory parasites that usually injure intestinal tissues (Monteiro 2017). Oocysts of this group of protozoans contain the zygotes and, generally, are unsporulated when liberated in the feces by their hosts. Extant genera with parasitological significance for carnivoran mammals include Besnoitia, Cryptosporidium, Cystisospora, Eimeria, Hammondia, Hepatozoon, Neospora, Sarcocystis, and Toxoplasma, being some of these acquired by the predation of other vertebrates (Taylor et al. 2015). This latter, represented by the species Toxoplasma gondii, is from special interest because of its medical and veterinary importance (Rey 2010). The oocysts described here have the size and shape expected for the genera Toxoplasma, Hammondia and Neospora, but the lack of well-defined sporocysts, sporozoites and other secondary structures within the oocysts precludes a more assertive classification of the materials. The final hosts Toxoplasma and Neospora are respectively felids and canids, while Hammondia can inhabit the small intestine of both groups. However, these three genera can parasitize other mammals as intermediate hosts, besides some species of Most species of strongylids in their adult form inhabit the alimentary tract of the major lineages of modern vertebrates, where they generally consume the blood, tissues and food ingested by their final hosts (Monteiro 2017). The Family Ancylostomatidae contains the main genera of strongylids, such as Ancyclostoma, Uncinaria and Necator, which can parasitize domestic and wild carnivoran mammals (Anderson et al. 2009, Taylor et al. 2015. These hematophagous intestinal strongylids, also known as hookworms, normally have monoxenous cycles, though some species also require, during their development, other vetebrates as paratenic hosts. The life cycle of these nematodes is usually completed from ingestion or skin penetration of infective larvae that previously developed in the environment from embryonated eggs eliminated in the feces of other final hosts. Additionally, some species can also be transmitted by via lactogenic or ingestion of paratenic hosts, especially rodents (Anderson 2000). The eggs described in this paper have a similar shape and size to the genera Ancyclostoma, Uncinaria and Necator, which also does not rule out the possibility of these specimens belonging to an extinct genus of hookworm from Pleistocene, whose life cycle would be unknown. The hypothesis that the oocysts described here belong to an extinct lineage of Eucoccidiorida can also not be dismissed. Because the eggs of these ancilostomids are practically indistinguishable, normally their differentiation is made through the morphological and morphometric analysis of their infective larvae cultivated in fresh fecal cultures or adult forms recovered from the small intestine during necropsy of the final host, however this laboratory procedures were not possible, precluding also a more assertive classification of these specimens.
Although normally the type of paleoparasite found facilitate the identification of the probable producer of a coprolite (Ferreira et al. 2008(Ferreira et al. , 2011, it was not possible by means of this device to distinguish if the examined fossil material belonged to particular group of carnivorans, since both groups of parasites observed can currently be found in these mammals  It is noteworthy that most of the Pleistocene parasitic record comes from coprolites collected in caves (Ringuelet 1957, Schmidt et al. 1992, Ferreira et al. 1993, Jouy-Avantin et al. 1999, Beltrame et al. 2012, Tietze et al. 2019 or archeological sites (Ferreira et al. 1991, Perri et al. 2017, Petrigh et al. 2019. Recording paleoparasites from buried coprolites (i.e., excavated from rock outcrops) is rare and must be related to taphonomic biases that act destroying the organic remains. These biases are still understudied, but the Upper Pleistocene Sopas and Touro Passo formations are good examples of preservation of paleoparasites in this context.
Thus the present paper reveals the first vertebrate paleoparasites from the Late Pleistocene of the Rio Grande do Sul, as well as it brings clues about the composition of the endoparasitic fauna of carnivoran mammals, including possibly extinct species of these hosts, which inhabited South America during the Lujanian. any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.