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Experimental Parasitology 122 (2009) 165–168
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Research Brief
Eimeria trichosuri: Phylogenetic position of a marsupial coccidium, based on 18S rDNA sequences M.L. Power a,*, C. Richter a, S. Emery a, J. Hufschmid b, M.R. Gillings a a b
Department of Biological Sciences, Macquarie University, North Ryde, NSW 2109, Sydney, Australia Department of Veterinary Sciences, University of Melbourne, Australia
a r t i c l e
i n f o
Article history: Received 5 December 2008 Received in revised form 12 February 2009 Accepted 13 February 2009 Available online 25 February 2009 Keywords: Eimeria Marsupial 18S rDNA Phylogeny
a b s t r a c t Phylogenetic analysis of the genus Eimeria suggests that parasite and host have coevolved over broad evolutionary timescales. Here we extend this analysis by determining the 18S rDNA gene sequence of the marsupial coccidium, Eimeria trichosuri, and assessing its phylogenetic position relative to Eimeria from birds, reptiles and placental mammals. This analysis placed E. trichosuri clones in a clade that diverged before the major clade comprising species from placental mammals. The position of E. trichosuri is consistent with host phylogeny where marsupials represent an ancient evolutionary line that predates the placental mammal line. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction Parasitic protists of the genus Eimeria have been described in a diverse range of vertebrate host groups. Several species cause significant mortality or morbidity in some hosts and are of particular concern in animal production industries (Barta et al., 1997; Cere et al., 1995; Yabsley and Gibbs, 2006). Species identification of Eimeria has primarily been based on oocyst characteristics, host specificity, pathology and geographic distribution data (Duszynski and Wilber, 1997). However, some species of Eimeria are morphologically similar and occur in several hosts. These factors may compromise species identification (Zhao and Duszynski, 2001). Consequently, the application of molecular tools for Eimeria identification and differentiation have become important. Molecular analyses for identification of Eimeria have enabled a better understanding of the evolutionary relationships within this genus. More than 1100 Eimeria species have been described in vertebrate hosts, but our understanding on the relationships between these species is limited. Phylogenetic studies of Eimeria are limited to domestic fowl (Barta et al., 1997; Lew et al., 2003), other bird species (Matsubayashi et al., 2005; Yabsley and Gibbs, 2006), rodents (Hnida and Duszynski, 1999; Reduker et al., 1987; Slapeta et al., 2001), bats (Zhao et al., 2001) and rabbits (Cere et al., 1995; Kvicerova et al., 2008). A proposal for an improved classification system for parasitic protists indicates the need to include molecular data (Tenter et al., 2002). This will enable phylogenetic * Corresponding author. Fax: +612 9850 8245. E-mail address:
[email protected] (M.L. Power). 0014-4894/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2009.02.008
inferences to be made and result in a more stable taxonomy. The inclusion of Eimeria species from host groups of ancient evolutionary lineages will help expand our knowledge of the diversity and evolution of this group. Marsupials are an ancient evolutionary lineage. More than forty Eimeria species have been described from marsupial hosts in Australasia and the Americas. These hosts include kangaroos and wallabies (Macropodidae), wombats (Vombatidae), possums (Phalangeridae), bandicoots (Peramelidae) and opossums (Didelphidae) (Barker et al., 1988a, 1988b, 1989a, 1988c; Bennett et al., 2006; Heckscher et al., 1999; O’Callaghan et al., 1998, 2001). To date there has been no attempt to determine the phylogenetic relationships of Eimeria of marsupials or to catalogue the genetic diversity of Eimeria species from this host group. In this study, we determined the phylogenetic position of the marsupial coccidium Eimeria trichosuri, a species found in brushtail-possums of the genus Trichosurus. 2. Material and methods 2.1. Parasite sources, screening and purification Faecal samples were collected from live-trapped mountain brushtail possums (Trichosurus cunninghami) at Boho South, north-east Victoria (36°48’S, 145°450 E). Animals were retained in individual enclosures with concrete floors for 10 weeks. The concrete floors were covered with newspaper which was replaced every day, and all faecal material was collected from the floor at the same time. Faecal samples were stored in 2% (w/v) potassium
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dichromate at room temperature until examination. Samples were screened for the presence of Eimeria oocysts using flotation over saturated zinc sulphate and microscopy (Daugschies et al., 1999). Sporulated oocysts were purified from faeces using a modified sucrose flotation method (Truong and Ferrari, 2006) and stored in sterile water at 4 °C. 2.2. Morphologic and morphometric analyses Oocysts were examined at 1000 magnification using an Olympus BH2 epifluorescent microscope (Ziess) with differential interference contrast. Images were captured using a Nikon DXM digital camera and measurements were performed using ImageJ (http://rsbweb.nih.gov/ij/).
3. Results Screening of faecal samples from 15 mountain brushtail possums by zinc floatation identified Eimeria oocysts in 2 samples. Oocysts were identified by their oval shape, thick cell wall and presence of four sporocysts (Fig 1). For one sample >100 oocysts were observed and 3 oocysts were observed in the second sample. The two samples were concentrated using sucrose gradient concentration but oocysts were only detected in the sample with >100 oocysts, this sample was used for subsequent analyses. Morphometric data of oocysts are provided in Table 1. Oocysts were ellipsoidal; with a double-layered wall, outer wall smooth, clear to yellow in colour; inner wall colourless; oocyst residuum present
2.3. DNA extraction Genomic DNA was extracted using PrepGem following the manufacturer’s instructions with modifications for oocyst wall lysis (Zygem Corporation, New Zealand). Oocysts suspended in sterile water (10 lL) were crushed with a mini-pestle (Astral Scientific) and the volume made up to 99 lL with PrepGem buffer 3. Samples were vortexted and the PrepGem enzyme (1 lL) was added. Samples were then incubated in a thermocycler (Eppendorf, Australia) for 15 min at each of 37 °C, 75 °C and 95 °C. After incubation, samples were centrifuged for 3 min at 8000 RPM followed by the addition of 1 lL TE (0.02 mM) and stored at 4 °C. 2.4. Gene amplification, cloning and sequencing Amplifications of the 18S rDNA were performed using GoTaq green 2 mastermix (Promega) and contained 20 pM each the forward primer 50 -GCTTGTCTCAAAGATTAAGCC and reverse primer 50 -AGCGACGGGCGGTGTGTACAA (Zhao and Duszynski, 2001). Reaction conditions comprised an initial denaturation at 94 °C for 3 min followed by 35 cycles of 94 °C, 40 s, 60 °C, 45 s and 72 °C for 1 min 30 s, with a final extension at 72 °C for 5 min. PCR products were visualised using 2% agarose gel electrophoresis and ethidium bromide staining (1 lg/ml). PCR products were purified using the Qiagen spin column PCR purification kit (Qiagen, Hilden, Germany) and amplicons cloned using the TA TOPO vector cloning system (Invitrogen, California, USA). Positive transformants were cultured overnight and plasmids recovered using the Qiagen miniprep kit (Qiagen, Hilden, Germany). Sequencing was performed using primers supplied with the cloning kit and gene insert specific primers C18SF2 50 -AAGGAMGGCAGCAGGCG and C18S R7 50 CGCCTGCTGCCKTCCTT to generate four overlapping fragments. The ABI 3130xl Genetic analyzer with the BigDyeTM terminator kit (Applied Biosystems, Foster City, California) was used for sequencing. 2.5. Phylogenetic analyses Sequences were searched against the GenBank database using BlastN (www.angis.org.au). 18S rDNA sequences of Eimeria sp from GenBank were aligned to E. trichosuri using ClustalW (Thompson et al., 1994). Phylogenies were inferred using parsimony, Neighbor-joining using the kimura 2 parameter distance analysis and maximum likelihood. Tree reliability was determined using bootstrap analyses of 1000 replicates. Alignments and phylogenies were conducted using MEGA 4 (Molecular Evolutionary Genetics Analysis software, Arizona State University, Tempe Arizona, USA). The nucleotide sequences generated in this study were submitted to GenBank under the Accession Nos. FJ829320–FJ829323.
Fig. 1. Eimeria oocyst isolated from faeces of the mountain-brushtail possum. Based on morphological characteristics the species is E. trichosuri.
Table 1 Morphometric comparison of Eimeria oocysts from mountain brushtail possum and E. trichosuri from common brushtail possums. E. trichosuri This study
E. trichosuri O’Callaghan and O’Donoghue (2001)
Oocyst length
Mean SD Range
40.3 3.37 31.5–45.9
41.4 3.20 34.4–49.2
Oocyst width
Mean SD Range
20.8 1.22 17.7–23.6
22.7 2.67 18.4–27.8
Length:width
Mean Range
1.94 1.48–2.49
1.8 1.3–2.6
Sporocyst length
Mean SD Range
13.9 1.73 9.6–18.8
15.6 1.02 13.9–18.0
Sporocyst width
Mean SD Range
9.64 0.88 7.26–12.0
9.9 0.69 8.2–12.0
Length:width
Mean Range
1.44 1.04–1.82
1.6 1.1–2.0
Micropyle width
Mean
3.0–4.42
3.2–4.0
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Fig. 2. Inferred phylogenies using parsimony, neighbour joining and maximum likelihood of E. trichosuri based on 1420 bp of the 18S rDNA gene. Support is indicated at nodes and represents percentage of 1000 boostrap replicates. Values of less than 50% are not shown.
as loose granules; micropyle present; 1-2 refractile bodies; 4 ellipsoidal sporocysts; Stieda body present; sub-Stieda body present; sporocyst residuum an aggregation of globules; 2 sporozoites with 1 -2 refractile globules. Amplification of genomic DNA generated a clear 18S rDNA product of about 1600 bp when analysed using agarose electrophoresis (data not shown). Cloning and sequencing of four positive transformants generated a contiguous sequence of 1620 bp from four overlapping fragments. Nucleotide alignment identified two sequences with 99.9% similarity and that both sequences were represented by two clones. The sequences differed by a transversion at bp 682 (guanine and cytosine) and a transition at bp 707 (thymine and cytosine). Parsimony, neighbour joining and maximum likelihood analysis of partial 18S rDNA sequences (1421 bp) of 36 Eimeria species from rodents, birds, bats, cattle and a snake placed the marsupial derived E. trichosuri clones into a group that branched before the large monophyletic clade that comprised Eimeria from eutherians and chickens (Fig 2). Species from single host groups formed monophyletic branches except for the two species from bats, which were placed within the rodent clade. The nodes for monophyletic clades representing single host groups were supported by high bootstrap values (Fig 2). 4. Discussion Phylogenetic inferences of the genus Eimeria show a high relatedness between species from closely related hosts, and in most cases, Eimeria from single host groups are placed in monophyletic clades. However, host groups of Eimeria are under-represented with phylogenetic inferences based only on Eimeria from rodents, bats, rabbits and birds (Hnida and Duszynski, 1999; Kvicerova et al., 2008; Reduker et al., 1987; Slapeta et al., 2001; Yabsley and Gibbs, 2006; Zhao and Duszynski, 2001). Inclusion of Eimeria from a diversity of host groups, particularly from hosts represent-
ing ancient evolutionary lineages, would provide more informative and reliable inferences. Here we provide the first DNA sequence data for an Eimeria species from a marsupial host. Marsupials evolved approximately 100 my before the placental mammals and extant species are classified into 19 families that are often grouped according to distribution in Australia or the Americas (Merrick et al., 2006). In Australia marsupials underwent a major adaptive radiation, in isolation, which has given rise to more than 150 extant species. In marsupials native to Australia, approximately forty Eimeria species have been described from a limited number of host species representing six families (O’Donoghue and Adlard, 2000). Three Eimeria species have also been described from South American marsupials (Heckscher et al., 1999). E. trichosuri is the only species recorded in brushtail possums. It has uniform oocyst characteristics in isolates from different individuals and from different localities (O’Callaghan and O’Donoghue, 2001). In this study Eimeria oocysts from T. cunninghami, the mountain brushtail possum, had characteristics similar to those described for E. trichosuri from the common brushtail possum T. vulpecula (O’Callaghan and O’Donoghue, 2001). Based on relatedness of hosts and oocyst characteristics we have concluded that the oocysts from mountain brushtail possums are E. trichosuri. As oocyst material is difficult to preserve (Duszynski and Wilber, 1997) a major limitation in undertaking DNA analyses on described species of Eimeria from marsupials is the lack of availability of holotypes. In this study, a host species with a single described Eimeria was used, making identification of the parasite species straightforward. However, sequence analyses of E. trichosuri did indicate some minor diversity in the 18S rDNA gene with two almost identical sequences (99.9% similarity) identified from cloned fragments. Although co-infections of Eimeria species have been recorded for several marsupial host species (Barker et al., 1989b; Heckscher et al., 1999; O’Callaghan et al., 1998), the oocyst characteristics in this isolate were highly uniform suggesting that only a single species of Eimeria was present in this individual, and that the
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sequence differences represent within species polymorphism in the 18S rDNA. Prior to DNA analyses of Eimeria from marsupial hosts susceptible to multiple Eimeria species, it will be necessary to use methods to separate oocysts of different morphologies to ensure that DNA sequences are correctly determined for individual Eimeria species. The phylogeny of the marsupial coccidium E. trichosuri indicates that the marsupial species diverged prior to Eimeria from placental mammals, which is consistent with host evolution. In this phylogeny, species from chickens share a common ancestor with species from placental mammals. This strongly suggests that the chicken Eimeria were derived from an ancestor resident in a placental mammal. Expansion of phylogenies to include species from more diverse hosts will assist with clarifying inferred Eimeria ancestries. This study has provided a platform for a larger study investigating the phylogeny and co-evolutionary relationships of Eimeria from all marsupial hosts where species from a diverse range of marsupial hosts will be analysed across several genes. Molecular data from Eimeria species of ancient origin will provide a greater understanding on the evolution and host-parasite relationship of this important genus. Acknowledgments The Authors thank Professor Ian Beveridge for comments and suggestions. Funding for this project was provided though the Macquarie University Research Fellowship scheme. References Barker, I.K., O’Callaghan, M.G., Beveridge, I., 1988a. Eimeria-spp. Apicomplexa Eimeriidae Parasitic in the Rat Kangaroos Hypsiprymnodon-Moschatus Potorous-Tridactylus Aepyprymnus-Rufescens and Bettongia-Gaimardi Marsupialia Potoroidae. International Journal for Parasitology 18, 947–954. Barker, I.K., O’Callaghan, M.G., Beveridge, I., 1988b. Eimeria-spp. Apicomplexa Eimeriidae Parasitic in Wallabies and Kangaroos of the Genera Setonix Thylogale Wallabia Lagorchestes and Dendrolagus marsupialia Macropodidae. International Journal for Parasitology 18, 955–962. Barker, I.K., O’Callaghan, M.G., Beveridge, I., 1989a. Host–parasite associations of Eimeria spp. (Apicomplexa: Eimeriidae) in kangaroos and wallabies of the genus Macropus (Marsupiala: Macropodidae). International Journal for Parasitology 19, 241–263. Barker, I.K., O’Callaghan, M.G., Beveridge, I., 1989b. Host–parasite associations of Eimeria-spp. picomplexa Eimeriidae in Kangaroos and Wallabies of the Genus Macropus Marsupialia Macropodidae. International Journal for Parasitology 19, 241–264. Barker, I.K., O’Callaghan, M.G., Beveridge, I., Close, R.L., 1988c. Host-Parasite Associations of Eimeria-Spp Apicomplexa Eimeriidae in Rock Wallabies Petrogale-Spp Marsupialia Macropodidae. International Journal for Parasitology 18, 353–364. Barta, J.R., Martin, D.S., Liberator, P.A., Dashkevicz, M., 1997. Phylogenetic relationships among eight Eimeria species infecting domestic fowl inferred using complete small subunit ribosomal DNA sequences. Journal of Parasitology 82, 262–271. Bennett, M.D., Woolford, L., O’Hara, A.J., Nicholls, P.K., Warren, K.S., Hobbs, R.P., 2006. A new Eimeria species parasitic in western barred bandicoots, Perameles
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