New species of Polystomoides (Monogenoidea: Polystomatidae) parasitizing the urinary bladder of a freshwater turtle in Brazil

Abstract Trachemys dorbigni is the most abundant freshwater turtle species in Rio Grande do Sul, southern Brazil. Chelonians are known to host a wide variety of pathogens, including viruses, bacteria, hemoparasites and helminths. Among these, nine genera of polystomatid flatworms (Monogenoidea; Polystomatidae) infect freshwater turtles: Apaloneotrema, Aussietrema, Fornixtrema, Manotrema, Pleurodirotrema, Polystomoidella, Polystomoides, Uropolystomoides and Uteropolystomoides. However, little is known about the biology of these parasites in the Neotropical Realm. Through investigative cystoscopy, specimens of Polystomatidae were located inside the urinary bladder of the host T. dorbigni. Retrieved specimens were fixed and stained whole mounts prepared for taxonomic identification. In the present paper, a new species of Polystomoides (Monogenoidea: Polystomatidae) parasitizing the urinary bladder of a freshwater turtle of the species T. dorbigni in Brazil is described. Polystomoides santamariensis n. sp. differs from the congeneric species on the length of the genital spines, which are longer. Given the enormous diversity of freshwater turtles around the world, it is likely that a large number of chelonian polystomatids are still unknown.


Introduction
With 795 of the 11,733 known reptiles species of the world from Brazil, it is the country with the third-largest diversity of reptiles (Costa & Bérnils, 2018;Uetz et al., 2023). The order Testudines comprises turtles, tortoises and terrapins (freshwater turtles) and is composed of two suborders: Pleurodira and Cryptodira. The first has distribution only in the southern hemisphere, across the Australian, Ethiopian and Neotropical Realms, while Cryptodira has the largest number of living species, with distribution on all continents, with the exception of Australia and Antarctica (Van Dijk et al., 2014;Rhodin et al., 2021;Du Preez et al., 2022). In Brazil, 36 species of Testudines have been described (Costa & Bérnils, 2018) and in the state of Rio Grande do Sul, there are 11 chelonian species, among which five are marine and six are freshwater species (Lema & Ferreira, 1990). Among the latter, Trachemys dorbigni Duméril & Bibron, 1835, is the most abundant (Bujes et al., 2011). In Brazil, it occurs naturally in Rio Grande do Sul and Santa Catarina, and it also inhabits other countries such as Argentina and Uruguay (Rhodin et al., 2021).
Species of Trachemys Agassiz, 1857 present the widest geographical distribution among New World chelonians, such that their range extends from the United States to Argentina (Seidel, 2002). This genus comprises 15 species and, in addition to Trachemys dorbigni, in South America Trachemys callirostris Gray, 1856, is found in Colombia andVenezuela andTrachemys adiutrix Vanzolini, 1995, in Brazil (Van Dijk et al., 2014).
Through investigative cystoscopy after video-assisted ovariosalpingectomy performed on an individual of the species T. dorbigni, specimens of Polystomatidae were found. In the present paper, a new species of Polystomoides (Monogenoidea: Polystomatidae) parasitizing the urinary bladder of this freshwater turtle in Brazil is described.

Material and Methods
One specimen of T. dorbigni (D'Orbigny's slider or "Tartaruga-tigre-d'agua") was subjected to investigative cystoscopy after video-assisted ovariosalpingectomy in the Veterinary Hospital of the Federal University of Santa Maria (UFSM). This animal came from the São Braz conservation breeding station ("Criadouro Conservacionista São Braz"), located in Santa Maria, Rio Grande do Sul, Brazil (29º 41′ 03" S; 53º 48′ 25" W). Elective castration was performed on this individual as a population control measure.
During the cystoscopy, using a 30° scope Karl Storz TM , Tuttlingen, Germany (1.9-2.0 mm) inside in a working sheath (9.6 Fr.) and semi-rigid forceps (Karl Storz TM , Tuttlingen, Germany), three helminth specimens that were found attached to the urinary bladder were collected. These were sent to the Veterinary Parasitology Laboratory of UFSM for identification at the species level, and subsequently to the Laboratory for Helminth Parasites of Fish, Oswaldo Cruz Institute, Oswaldo Cruz Foundation. The samples were fixed in 70% alcohol and stored. Specimens were stained with Langeron's alcoholic acid carmine, dehydrated in an ethyl alcohol series, cleared in beechwood creosote and mounted in Canada balsam as permanent slides (Eiras et al., 2006).
Measurements are presented in micrometers, unless otherwise stated, as range values followed by mean values, when more than two, and the number of structures measured, where applicable, in parentheses. Specimens were illustrated with the aid of a camera lucida coupled to a Zeiss Axioskop light microscope. Light microphotographs were made with the use of the ZEN 2 software (Blue edition) ® ; Carl Zeiss Microscopy, 2011. The authorship of the taxa followed the recommendation of Article 50.1 of the International Code of Zoological Nomenclature (ICZN), which deals with the identity of the authors. The holotype and paratypes were deposited in the Helminthological Collection of the Oswaldo Cruz Institute ("Coleção Helmintológica do Instituto Oswaldo Cruz", CHIOC), Rio de Janeiro, Brazil.
According to Du Preez et al. (2022), the inter-vaginal distance that corresponds to Polystomoides infecting cryptodires ranges from 84.2-99.4% and the intervaginal distance observed in P. santamariensis fits this range. Nonetheless, these authors stated that the number of genital spines for Polystomoides infecting cryptodires ranges from 24 to 44, which is not in agreement to the number presented in the specimens described herein.
Du Preez & Theunissen (2021) proposed four types of haptoral suckers for polystomes. Their Type III corresponds to Type 2 of Pichelin (1995) and is the type present in all polystomes infecting chelonian hosts. These suckers are spherical, symmetrical, firm, directed ventrolaterally and characterized by having embedded skeletal elements that provide a secure grip on the host tissue. The morphology of the haptoral suckers of Polystomoides santamariensis n. sp. is in agreement with type III proposed by these authors.

Discussion
Polystomoides was originally described as a sugbgenus of Polystoma Zeder, 1800 by Ward (1917), based on the presence of a short ovary with a single egg, being elevated to genus rank by Ozaki (1935) who pointed to the absence of a uterus as a diagnostic character (Chaabane et al., 2022). Species of the genus are found in the mouth, esophagus, nasal cavities and urinary bladder of turtles and had been charaterized by the presence of two pairs of hamuli in the haptor (Vieira et al., 2008;Chaabane et al., 2022). Price (1939) proposed two new genera for polystomatids of chelonians: Polystomoidella Price, 1939 parasites of the urinary bladder of their hosts and Neopolystoma Price, 1939 found in the urinary bladder and nasal cavities, differing in the absence and number of hamuli, Polystomoides spp. have 2 pair of hamuli, Polystomoidella spp. have 1 pair and Neopolystoma spp. none (Vieira et al., 2008;Du Preez & Van Rooyen, 2015;Du Preez & Verneau, 2020;Chaabane et al., 2022). Only recently, the polystomatid genera parasitizing chelonians received considerable attention, with the description of Uropolystomoides; Uteropolystomoides; Aussietrema; Fornixtrema; Apaloneotrema; Manotrema and Pleurodirotrema; and the synonymizing of Neopolystoma to a junior synonym of Polystomoides [see Tinsley (2017); Tinsley & Tinsley (2016); Du Preez & Verneau (2020); Du ]. Some species previously allocated in Polystomoides were transferred to Uropolystomoides, Uteropolystomoides, and Manotrema based in one or more characteristics Price, 1939;Tinsley, 2017;Tinsley & Tinsley, 2016).
All the South American species of Polystomoides infecting the oral region Pleurodira freshwater turtles were transferred to Manotrema: Manotrema uruguayensis (Mané-Garzón & Gil, 1961), Manotrema fuquesi (Mané-Garzón & Gil, 1962) and Manotrema brasiliensis (Vieira et al., 2008. Chaabane et al. (2022) proposed the name Polystomoides for the clade composed by Neopolystoma and Polystomoides, considering the similarity in the morphology of the vaginae, reassigning nine species, previously attributed to Neopolystoma, for Polystomoides, and redefined the genus as polystomes species infecting oral cavity and urinary bladder of cryptodires, presenting or not hamuli and peripheral vaginae. Based on its unique morphology they retained Uteropolystomoides although it shares the same clade (Chaabane et al., 2022). Considering this proposition, the new species is allocated in Polystomoides, being the first species of the genus described in Brazil.
Considering the recent studies regarding the taxonomy of the polystomes parasites of turtles, further molecular studies are needed to clarify the phylogenetic relationships of these genera, to be compared to morphological and biological findings. Polystomes are grouped mainly by the site of infection considering that they present site specificity, which can lead to the process of speciation and explain the diversity of parasite species found in freshwater turtles (Du Preez & Van Rooyen, 2015). Given the enormous diversity of freshwater turtles around the world, it is likely that a large number of chelonian polystomatids are still unknown.
The present paper highlighted the value of non-invasive and non-lethal procedures to investigate the polystomatid fauna since the specimens were collected during surgery.