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.

Monogenea , Polystomatidae , Polystomoides , freshwater turtle, Florida, USA

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, 1939 and 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 present 54 turtle polystome species are known from 55 host species. Although chelonian polystomes have a broad geographical distribution, only seven Neopolystoma, two Polystomoidella and four Polystomoides species are known from the Nearctic realm. The Polystomoides species currently known from this region include Polystomoides coronatum (Leydi, 1888) Ozaki, 1935 from Trachemys dorbigni; Polystomoides multifalx Stunkard, 1924 from Pseudemys concinna (LeConte, 1830); Polystomoides oris Paul, 1938 and Polystomoides pauli Timmers & Lewis, 1979, both from Chrysemys picta.

During a survey of freshwater turtles around Gainesville, Florida, USA, Pseudemys nelsoni (Reptilia: Chelonia: Emydidae) was found to be infected with an as-yet-unknown Polystomoides. This paper provides the formal description of this previously unknown parasite.

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).

All polystome species are host-specific, with chelonian polystomes being strictly site-specific. As a result of this strict site specificity a single host could be infected by more than one polystome species. Chelonian polystomes have been fairly well studied in the USA, with 11 polystomes known from various freshwater turtle hosts (Du Preez and Lim 2000, Morrison and Du Preez 2012).

The two Polystomoidella species known from North America are Polystomoidella oblongum Wright, 1879 and Polystomoidella whartoni Wright, 1879. The seven Neopolystoma species known from the USA are: Neopolystoma elizabethae Platt, 2000; Neopolystoma fentoni Platt, 2000; Neopolystoma grossi Morrison & Du Preez, 2012; Neopolystoma moleri Morrison & Du Preez, 2012; Neopolystoma orbiculare Stunkard, 1916; Neopolystoma rugosa MacCallum, 1918; and Neopolystoma terrapenis Harwood, 1932. The four Polystomoides species known from the USA are: P. coronatum Leidy, 1888; P. multifalx Stunkard, 1924; P. oris Paul, 1938; and P. pauli Timmers & Lewis, 1979 (Morrison and Du Preez 2012).

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.

Polystomoides nelsoni sp. n. can be distinguished from the other Polystomoides species by the number of genital spines. Polystomoides nelsoni sp. n. has 123 (108–132) genital spines compared to P. fuquesi with 2, P. brasiliensis with 8–9, P. bourgati with 26–29, P. asiaticus with 34–40, P. ludhianae with 54–64, P. godavarii with 64–66, and P. australiensis with 74–95. However, P. multifalx (120–124) and Polystomoides stunkardi (92–109) are two species that also have a large number of genital spines. Compared to Neopolystoma species, Polystomoides nelsoni sp. n. also has a larger number of genital spines. Neopolystoma chelodinae has 14 (12–16), N. elizabethae 8 and Neopolystoma euzeti 34 (33–36), while P. oblongum and P. whartoni both have 16 genital spines.

The total length of the genital spines of Polystomoides nelsoni sp. n. 101 (93–106) is longer compared to those of other Polystomoides species, such as Polystomoides siebenrockiella 58 (54–60), Polystomoides rohdei 34–52, Polystomoides platynota, 60–70, Polystomoides nabedei 42–46, Polystomoides microrchis 75–88 and Polystomoides chabaudi 27 (22–31). The genital spines for Polystomoides nelsoni sp. n. are in the same size range as those of P. australiensis 93 (78–105). Polystomoides nelsoni sp. n. also has larger genital spines compared to those of Neopolystoma species, such as N. chelodinae 23.6 (20.8–27.2), N. euzeti 57 and N. elizabethae 10, as well as compared to those of Polystomoidella species, such as P. oblongum 18–22 and P. whartoni 15–18.

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.

We are indebted to the USGS in Gainesville, Florida and in particular to Dr. Tim Gross, the host for this study.