Identification of a novel species of Eimeria Schneider, 1875 from the woylie, Bettongia penicillata Gray (Diprotodontia: Potoroidae) and the genetic characterisation of three Eimeria spp. from other potoroid marsupials

Faecal samples (n = 1,093) collected from the woylie Bettongia penicillata Gray, in south-western Australia were examined for the presence of coccidian parasites. Eimeria sp. oöcysts were detected in 15.2% of samples. Faecal samples obtained from the eastern bettong Bettongia gaimardi (Desmarest) (n = 4) and long-nosed potoroo Potorous tridactylus (Kerr) (n = 12) in Tasmania, were also screened for the presence of Eimeria spp. (prevalence 50% and 41.7%, respectively). Morphological and genetic comparison with other known species of Eimeria indicates that the material identified in woylies is novel. This study aimed to (i) morphologically describe and genetically characterise Eimeria woyliei n. sp. found in woylies; and (ii) genetically characterise Eimeria gaimardi Barker, O’Callaghan & Beveridge, 1988, Eimeria potoroi Barker, O’Callaghan & Beveridge, 1988, and Eimeria mundayi Barker, O’Callaghan & Beveridge, 1988, from other potoroid marsupials. Molecular phylogenetic analyses conducted at the 18S rDNA and mitochondrial cytochrome c oxidase subunit 1 (cox1) loci revealed that E. woyliei n. sp. was most closely related to Eimeria setonicis Barker, O’Callaghan & Beveridge, 1988, at the 18S rDNA locus, and Eimeria trichosuri O’Callaghan & O’Donoghue, 2001, at the cox1 locus. Eimeria woyliei n. sp. is the sixth species of Eimeria to be formally described from potoroid marsupials.


Introduction
Coccidian parasites are known to infect potoroid marsupials, including the critically endangered woylie This article was registered in the Official Register of Zoological Nomenclature (ZooBank) as 01B2910A-6B10-40B0-ABB3-40725DDA9FD4. This article was published as an Online First article on the online publication date shown on this page. The article should be cited by using the doi number. This is the Version of Record. or brush-tailed bettong Bettongia penicillata Gray. Although morbidity and mortality associated with coccidial infection in free-ranging macropods is uncommon (Vogelnest & Portas, 2008), disease has been documented in macropods under stress (e.g. the eastern grey kangaroo Macropus giganteus Shaw; Barker et al., 1972). Given the reliance of many threatened species on interventional management practices such as translocation, a process which has been identified as a significant stressor (Hing et al., 2017), it is imperative that we gain a greater understanding of the parasite species infecting wildlife, particularly those with the potential to cause disease in their host (e.g. coccidian parasites).
During this study we aimed to (i) morphologically describe and genetically characterise E. woyliei n. sp. from woylies; and (ii) genetically characterise E. gaimardi from the eastern bettong, and E. potoroi and E. mundayi from the long-nosed potoroo Potorous tridactylus (Kerr).

Sample collection
Between 2014 and 2018, woylie faecal samples (n = 1,093) were collected from various sites within southwestern Australia (Table 1) as part of a collaborative project with the Department of Biodiversity, Conservation and Attractions (DBCA), Kensington, Australia. Newspaper was placed beneath each trap to collect faeces, which were stored in 70% ethanol, 10% buffered formalin and/or 2% potassium dichromate until processing.
In 2018, faecal samples collected from the eastern bettong (n = 4) were obtained from a captive population at Bonorong Wildlife Sanctuary in Brighton,Tasmania (42.71°S,147.27°E). Samples from the long-nosed potoroo (n = 12) were acquired from wild-caught animals within the Peter Murrell reserves, south of Hobart, Tasmania (43.00°S, 147.18°E). Faecal samples from the eastern bettong and long-nosed potoroo were collected directly from traps and samples were stored in 2% potassium dichromate prior to analysis.

Identification of coccidian oöcysts in faecal samples
For woylies, the majority of faecal samples (n = 1,073) were examined for the presence of coccidian oöcysts using simple faecal flotation with sodium nitrate (NaNO 3 ) as described by Northover et al. (2017). These samples were formalin-fixed. To describe the morphology of E. woyliei n. sp., an additional 20 samples were sporulated (using potassium dichromate) and underwent faecal flotation using zinc sulphate (ZnS0 4 ) in distilled water (SG 1.20). Briefly, faeces were placed into a 10 ml centrifuge tube (up to the 1.5-2.0 ml mark), emulsified in distilled water (tube filled to the 10-ml mark) and centrifuged (2,000 rpm for 2 min). The supernatant was removed before filling the tube with zinc sulphate solution and reemulsifying, before final centrifugation (2,000 rpm for 2 min). A sterile wire loop was used to transfer 2-3 loops from the surface of the tube to a glass slide, and a 22 9 22 mm coverslip was placed on top. Each sample was examined at 1009 magnification using an Olympus BX50 microscope. All other potoroid samples were examined using zinc sulphate. To calculate the prevalence of infection, each faecal sample was scored as positive or negative for the presence of coccidian oöcysts.

Morphological description of the new species
Eighty-six sporulated oöcysts from a single woylie originating from Perup Sanctuary were examined at magnifications of 400-1,0009, using an Olympus BX50 microscope with a Olympus DP71 Universal Camera with Cellsens software. Photographs of sporulated oöcysts were taken using bright field microscopy. Measurements of oöcyst and sporocyst length and width, and oöcyst wall thickness were acquired using ImageJ software (US National Institute of Health, Bethesda, Maryland). All measurements are recorded in micrometres (lm) with the range followed by the mean in parentheses. We measured a single laterally positioned sporocyst within each oöcyst; if we could not identify a sporocyst in the correct position, we did not measure sporocyst length or width.
Morphological identification of other potoroid Eimeria spp. Three distinct morphotypes of sporulated oöcysts were identified from the faeces of the eastern bettong and long-nosed potoroo. Based on their size and unique oöcyst and sporocyst characters as described by Barker et al. (1988) we identified E. gaimardi from the eastern bettong, and E. mundayi and E. potoroi from the long-nosed potoroo.
Genetic characterisation of Eimeria spp. in potoroid marsupials For the woylie, nine faecal samples (eight ethanol-and one potassium dichromate-preserved) were used to genetically characterise E. woyliei n. sp. at the 18S rDNA and mitochondrial cytochrome c oxidase subunit 1 (cox1) loci. Eimeria gaimardi, E. mundayi and E. potoroi were genetically characterised using a single faecal sample (potassium dichromate-preserved) for each Eimeria species; as outlined above, morphological identification of sporulated oöcysts confirmed the identity of each species prior to genetic characterisation.

DNA extraction and PCR amplification
Samples stored in 70% ethanol and/or 2% potassium dichromate were exposed to four freeze/thaw cycles as described by Yang et al. (2016a) in order to achieve oöcyst lysis. Following the freeze/thaw step, faecal samples stored in 2% potassium dichromate were subjected to a wash step prior to lysis in order to remove the fixative, by centrifuging at 3000 rpm for 10 min and resuspending in phosphate buffered solution. DNA was isolated from 0.25 g of faecal sample using the PowerFecal DNA Isolation Kit (MolBio, Carlsbad, California) as per the manufacturer's instructions. A negative control was included to rule out contamination.
Faecal samples from the woylie were screened at the 18S rDNA locus using a nested PCR with the external Eimeria spp. primers EiF1 (5 0 -GCT TGT CTC AAA GAT TAA GCC-3 0 ) and EiR3 (5 0 -ATG CAT ACT CAA AAG ATT ACC-3 0 ), and the internal Eimeria spp. primers EiR4 (5 0 -ACT CAA AAG ATT ACC TAG AC-3 0 ) and EiF4 (5 0 -CTA TGG CTA ATA CAT GCG CAA T-3 0 ) (Yang et al., 2016a). PCR reactions were carried out in a total volume of 25 ll containing 12.5 ll of 29 KAPA HiFi Hotstart ReadyMix (Millennium Science Pty. Ltd, Victoria, Australia), 0.75 ll primer (10 lM) and 2 ll DNA template. All PCR reactions were performed as described by Yang et al. (2016a) consisting of a pre-PCR step of 94°C for 3 min, followed by 45 cycles of 94°C for 30 s, 55°C annealing temperature for 30 s and 72°C for 2 min, and a final extension step of 72°C for 5 min.
Faeces from the eastern bettong and long-nosed potoroo were screened using the 18S external primers EiGTF1 (5 0 -TTC ACT GGT CCC TCC GAT C-3 0 ) and EiGTR1 (5 0 -AAC CAT GGT AAT TCT ATG G-3 0 ) (Yang et al., 2016b), and the internal primers EiGTF2 (5 0 -TTA CGC CTACTA GGC ATT CC-3 0 ) and EiTR2 (5 0 -TGA CCT ATC AGC TTT CGA CG-3 0 ). The PCR reaction contained 10 ll 29 GoTaq PCR master mix (Promega, Alexandria NSW, Australia), 1 ll DNA (50 ng), 10 ll of each primer (10 lM stock) and 7 ll distilled water. PCR cycling conditions for the external PCR were 1 cycle of 94°C for 3 min, followed by 35 cycles of 94°C for 30 s, 58°C for 30 s and 72°C for 2 min, and a final extension step of 72°C for 5 min. The conditions for the secondary PCR were the same except for 45 cycles instead of 35. All amplicons were visualised on a 1.5% agarose gel and bands were cut and purified using an in-house filter tip method defined by Yang et al. (2013).

Sequencing and phylogenetic analysis
Purified amplicons were sequenced using an ABI Prism TM Dye Terminator Cycle Sequencing Kit (Applied Biosystems, California, USA) according to the manufacturer's instructions. Samples were sequenced in the forward and reverse direction and the denaturation step was extended to 10 min to allow efficient primer binding. The sequences were analysed in Geneious v.8.1 (Kearse et al., 2012) and aligned using reference libraries with the MUSCLE (Edgar, 2004) plugin for Geneious v.8.1. All novel sequences were deposited in GenBank.

Results
The prevalence of coccidial infection in woylies is summarised by site in Table 1. It is important to note that Dryandra contains both resident woylies (endemic to the region), and translocated woylies originating from the Upper Warren region (specifically Balban, Boyicup, Corbal, Dudijup, Dwalgan and Winnejup). Likewise, Walcott and Warrup East contain both resident and translocated (originating from Perup Sanctuary) woylies. In the eastern bettong, 2 out of 4 (50%) samples were positive for E. gaimardi. In the long-nosed potoroo, 3 out of 12 (25.0%) samples were positive for E. potoroi, while 4 out of 12 (33.3%) samples were positive for E. mundayi; mixed infections with both coccidian parasites were identified in 2 out of 12 (16.7%) samples. Etymology: The name woyliei reflects the host species local name 'woylie', which is the Aboriginal name given to this animal by the Noongar people of southwestern Western Australia (Abbott, 2001).

Genetic characterisation of Eimeria spp. in potoroids
The phylogeny of the four potoroid Eimeria spp. was investigated using Bayesian analyses at two gene loci (18S and cox1). An alignment was generated for the 18S rDNA locus (1,158 bp, Fig. 3) and two alignments for the cox1 locus (686 bp, Fig. 4; 211 bp, Fig. 5). All alignments contained the novel sequences belonging to the four potoroid species as well as reference sequences downloaded from GenBank including an outgroup (Toxoplasma gondii). Similar phylogenetic relationships were observed for both loci, although some species were not observed in the cox1 tree due to lower availability of genetic data. A second cox1 alignment was generated (211 bp) in order to incorporate relevant marsupial species of smaller fragment size.
Using the 18S rDNA locus, E. woyliei was grouped within the marsupial clade and was most similar to E. setonicis from the quokka. Eimeria woyliei oöcysts can be morphologically distinguished from oöcysts of E. setonicis by shape (pyriform vs ellipsoidal) (Austen et al., 2014). Despite the morphological similarity between oöcysts of E. woyliei and E. gaimardi, E. woyliei was more genetically similar to E. mundayi and E. potoroi, rather than E. gaimardi, though this difference was minimal.
Within Australia various coccidial species (Eimeria and less commonly Isospora) have been recorded in captive and free-ranging macropods (Mykytowycz, 1964;Barker et al., 1989;Yang et al., 2012;Duszynski, 2016). Traditional morphological criteria have been useful for identifying coccidial species infecting wildlife. However, the implementation of genetic characterisation combining both 18S rDNA and cox1 loci helps to discriminate between morphologically similar species and provides an accurate measure of the evolutionary relationship between coccidial species   (Power et al., 2009;Yang et al., 2012). As some Eimeria spp. are capable of infecting more than one marsupial host (Barker et al., 1989) and marsupials tend to harbour multiple Eimeria spp. (Power et al., 2009), this knowledge may be useful for predicting potential avenues of disease spread during the management of threatened populations (e.g. during fauna translocation). This study contributes toward our knowledge of Eimeria spp. infecting potoroid marsupials. Eimeria woyliei parasitising woylies is the sixth Eimeria spp. to be formally described from potoroid marsupials and we have genetically characterised four of the six known potoroid Eimeria species.
Funding This study was principally funded by the Australian Research Council (LP130101073). We have also been supported by grants from the Holsworth Wildlife Research Endowment (HOLSW2015-1-F149) and the Ecological Society of Australia, The Royal Zoological Society of New South Wales (Paddy Pallin Grant) and the Australian Wildlife Society.

Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.
Ethical approval All applicable international, national, and/ or institutional guidelines for the care and use of animals were followed. Samples from the woylie were collected under DBCA Scientific Licenses (Regulation 4: written notice of lawful authority; and 17: licence to take fauna for scientific purposes) and with approval from the Murdoch University Animal Ethics Committee (RW2659/14). Samples from the eastern bettong were collected with permission from Bonorong Wildlife Sanctuary. Samples from the long-nosed potoroo were collected under authorities and permits issued to the Department of Primary Industries, Parks, Water and Environment (DPIPWE) staff to live-trap wildlife on reserved land in Tasmania, following the Standard Operating Procedures for Live-trapping and Handling of Wild Tasmanian Mammals 2013 by the DPIPWE.
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