A new polystomatid (Monogenea, Polystomatidae) from the mouth of the North American freshwater turtle Pseudemys nelsoni

Abstract Based on material collected from Pseudemys nelsoni (Reptilia: Chelonia: Emydidae) during a parasite survey of the herpetofauna around Gainesville, Florida, USA, Polystomoides nelsoni sp. n. is described as a new polystome species. This parasite was found in the oral and pharyngeal region of the host. In a sample of nine Pseudemys nelsoni, three specimens were found to release polystome eggs. One turtle was euthanized and dissected and found to be infected in the oral region with 19 specimens belonging to an as-yet-unknown Polystomoides. This is only the fifth Polystomoides recorded from the Nearctic realm. This species is distinguished from known species by a combination of characteristics including marginal hooklet morphology, body length and haptor dimensions.


Introduction
Although monogeneans are predominantly single host fish parasites, polystomatid flatworms (Monogenea, Polystomatidae) radiated onto the tetrapods and are known from a diverse range of hosts, including the Australian lungfish, amphibians, freshwater turtles and the hippopotamus (Raharivololoniaina et al. 2011). Of the 24 currently known polystome genera (Du Preez et al. 2014) three exclusively parasitize turtles, namely Polystomoides Ward, 1917, Polystomoidella Price, 1939and Neopolystoma Price, 1939 (see Du Preez and Moeng 2004, Du Preez et al. 2007, Du Preez et al. 2008, Morrison and Du Preez 2012, Price 1939, Wright 1879, Yamaguti 1963. Genera of the subfamily Polystomoidinae, including the three polystome genera known from turtles and the genus Nanopolystoma, known from caecilians (Du Preez et al. 2008), all have non-confluent gut caeca lacking diverticula and possess skeletal elements inside the haptoral suckers. Chelonian polystomes are further characterised by non-diverticulated gut caeca of equal length and subsequent, absence of prehaptoral or haptoral anastomoses and a compact medial testis. Polystomoides is found in the oral cavity, nasal cavity, cloaca and urinary bladder of the host and has two pairs of hamuli, with the outer pair being larger than the inner pair. Polystomoidella parasitizes the urinary bladder of turtles and has a single pair of hamuli. Neopolystoma is found in the oral cavity, nasal cavity, ocular cavity, cloaca and urinary bladder and has no hamuli.
At During a survey of freshwater turtles around Gainesville, Florida, USA, Pseudemys nelsoni (Reptilia: Chelonia: Emydidae) was found to be infected with an as-yetunknown Polystomoides. This paper provides the formal description of this previously unknown parasite.

Material and methods
During April-June 2004 baited crayfish traps were set to capture terrapins in ponds in and around Gainesville, Florida, USA. Captured turtles were individually placed in 20 L plastic buckets with dechlorinated tap water to a depth of about 50 mm. After a period of 24 hours turtles were removed and the water screened for the presence of polystome eggs. The water from the containers in which turtles were housed was poured through two plankton sieves with respective mesh sizes of 500 µm and 100 µm. The first sieve removed the coarse debris in the water while the second retained finer debris and any polystome eggs that might be present. The contents of both sieves were then washed into separate glass Petri dishes and examined under a dissecting microscope. The Petri dish with contents from the course sieve was scanned for adult parasites that may have dislodged, and the Petri dish with contents from the fine sieve was scanned for polystome eggs.
Recovered eggs were removed and incubated at room temperature in Petri dishes containing clean water. Freshly hatched oncomiracidia were collected and mounted semi-permanently using ammonium picrate as mounting medium to clear the parasites and reveal the marginal hooklets. Turtles that were found not to be infected with polystome eggs were screened a second and third period of 24 hours. A single infected turtle was euthanized by injecting 0.5 mL of sodium pentobarbitone diluted with water (0.5 mL pentobarbitone and 4.5 mL water) straight into the heart. After 15 minutes the specimen was dissected. The cloaca, urinary bladder and accessory bladders as well as the oral cavities, nasal cavities, pharyngeal cavities, eye surface and cavity under the nictitating membrane were examined for polystomes, with the aid of a stereo microscope. The remainder of the turtles were released where collected.
Polystome whole mounts were prepared as follows: individual mature polystome species collected from the host species were fixed under cover-slip pressure in 10% neutral buffered formalin (NBF). Representative sub-adult specimens were fixed in 70% molecular grade ethanol for future molecular studies.
Parasites earmarked for permanent mounts were hydrated using 30% EtOH, stained overnight in a weak aceto-carmine staining solution, gradually dehydrated to absolute EtOH, cleared in a 1:1 ratio mix of absolute ethanol-xylene and then pure xylene, and mounted in Canada balsam. Preparations were studied using a Nikon E800 compound microscope fitted with a Nikon DXM1200 digital microscope camera connected to a PC. Measurements were taken using Eclipse network software (Nikon). Marginal hooklet measurements were obtained from the oncomiracidia that hatched from incubated eggs, following the protocol developed by Du Preez and Maritz (2006).

Turtles screened and polystomes retrieved
Nine Florida red-bellied turtles (Pseudemys nelsoni) were collected and screened. Specimens were collected from Lake Griffin, Lake Lochloosa, Lake Orange, and ponds at the U.S. Geological Survey (USGS) research facility in Gainesville.

Levels of infection
Of the nine turtles examined three were found to be infected (prevalence 33%). Only one turtle was dissected and found to have 19 polystomes in the oral region. These specimens were identified as belonging to Polystomoides; however, they did not conform to any of the 38 known Polystomoides species.

Molecular studies
Material collected was also studied at the molecular level. Based on 18S and 28S rDNA sequences, the newly discovered polystome differs from all other known turtle polystomes for which molecular data are available and occupies a distinct position basal to other Nearctic chelonian polystomes (see Etymology. The species is named after the host.

Site. Mouth.
Description. Based on ten egg-producing adults. The average measurement is given, followed by the range given in parentheses. Measurements are given in micrometres (µm). Larval (oncomiracidia) measurements are given for the marginal hooklets.
The main feature distinguishing Polystomoides from other turtle polystomes is the presence of two unequal pairs of hamuli. The other genera that parasitize turtles either have a single pair of hamuli as in Polystomoidella or the hamuli are lacking altogether as in Neopolystoma. Polystomoides and Neopolystoma species can also occasionally be distinguished from Polystomoidella in terms of the additional sites (the cavity of the eye and nose, pharynx, cloaca, and mouth) that these species parasitize, as Polystomoidella parasites are found to infect only the urinary bladder of their host species.
Unlike most other polystomes, these parasitizing chelonians have a broad geographical distribution. Both Neopolystoma and Polystomoides have been reported from the realms around the globe known to be inhabited by freshwater turtles. On the other hand, Polystomoidella is mainly known from the Nearctic realm where it is represented by five species. However, Richardson and Brooks (1987) described Polystomoidella mayesi from the urinary bladder of a Malaysian box turtle, Cuora amboinensis. The presence of Polystomoidella in the Oriental realm raises questions of possible misidentifications or a possible parasite transfer. According to Du Preez and Lim (2000) the possibility of transfer from an introduced American turtle can only be confirmed or refuted if and when P. mayesi is found in this chelonian species. Part of the evolutionary success of chelonian polystomes is the fact that they are site-specific and occupy various sites, including the oral and nasal cavities, eye cavity and the cloaca and urinary bladder. Littlewood et al. (1997) stated that congeneric species infecting the same site in different hosts are more closely related than congeneric species infecting different sites in the same host individuals. The high degree of site specificity allows for speciation and could explain the polystome diversity found in freshwater turtles. With the huge diversity of freshwater turtles globally it is likely that a vast number of chelonian polystomes remain to be discovered.