MOLECULAR CHARACTERIZATION OF HEPATOZOON SPECIES IN REPTILES FROM THE SEYCHELLES D. James Harris, Joa ˜o P. M. C. Maia, and Ana Perera CIBIO, Centro de Investigac ¸a ˜ o em Biodiversidade e Recursos Gene ´ ticos, Campus Agra ´ rio de Vaira ˜ o, 4485–661 Vaira ˜ o, Portugal. e-mail: james@mail.icav.up.pt ABSTRACT: Hepatozoon parasites were examined for the first time in reptiles from the Seychelles Islands. Although both prevalence and intensity were low, Hepatozoon species were detected in individuals from 2 endemic species, the lizard Mabuya wrightii and the snake Lycognathophis seychellensis. This was confirmed using visual identification and through sequencing part of the 18s rRNA gene. Phylogenetic analysis indicates that the Hepatozoon on the Seychelles form a monophyletic lineage, although more data are clearly needed to stabilize estimates of relationships based on this marker. Members of Hepatozoon (Miller, 1908) are the most common, widest distributed, and speciose intracellular protozoans found in reptiles (Telford, 2009), as well as other vertebrates, including amphibians and mammals. All have a heteroxenous life cycle, with sporogeny taking place in an invertebrate vector. Such vectors are also very diverse, and include ectoparasites, bugs, biting flies, and mosquitoes (reviewed in Telford, 2009). Since many Hepatozoon species are morphologically very similar and are capable of infecting multiple hosts and vectors, the number of species is difficult to assess using just morphological identification methods (Telford, 1984). Studies on snakes have indicated very different effects on the hosts, ranging from reports of only trivial consequences for host fitness, to severe effects on host growth rate and reproductive output (Madsen et al., 2005; Brown et al., 2006). Studies of these parasites are, therefore, necessary not only to better characterize this component of biodiversity, but also to assess if parasites may pose a risk to host populations (Pedersen et al., 2007). Such assessments are particularly important in oceanic islands where host endemicity levels are typically high, while populations are small and fragmented, leading to increased extinction risks (Frankham, 2005) and possibly higher host susceptibility (Lyles and Dobson, 1993). The Seychelles is an archipelago of more than 150 islands located across the Indian Ocean at 4–11uS and 45–56uE. Many of the islands are coralline, but they also include a central group of about 40 granitic islands that are the remnants of the Seychelles microcontinent, which was isolated following the breakup of Gondwana approximately 65 million years ago (Scotese, 2001). Many are quite mountainous, so that even when global sea levels were at their highest, most of the granitic islands remained above sea level, allowing the survival of unique flora and fauna. Native reptiles make up around 30 species, of which 70% are endemic (Gerlach, 2007). To date, no studies have been made of blood parasites from these reptiles from the granitic islands, although an assessment of 143 reptiles from Aldabra Island reported finding only a single Monocercomonas (Lowery, 1971). In the present study, we assess for the first time the existence of Hepatozoon species in reptiles from the Seychelles Islands using molecular and blood smear examination methods. Various studies have indicated that detection of parasites using polymerase chain reaction (PCR) protocols and DNA sequencing are more effective than the traditional examination of blood smears, although with differing degrees of variation (e.g., Merino et al., 2009; Vilcins et al., 2009). On the other hand, parasitemia levels can be assessed using blood smears. Here both methods were used and compared. For the molecular analyses, the 18S rRNA region has been sequenced for a number of Hepatozoon species from a variety of endemic hosts. Therefore, a phylogenetic assessment was carried out to determine if Hepatozoon species from reptiles are monophyletic, and how species from the Seychelles are related to other known Hepatozoon species. MATERIALS AND METHODS Sampling and slide examination Sampling was carried out across the Seychelles, as part of an assessment of the reptile fauna of this region (details in Rocha et al., 2009). Animals were collected by hand, identified, and digitally photographed. Seventy- one individuals from 11 species were examined (Table I). Tail tips were removed and stored in 96% ethanol for DNA samples. Blood drops were also stored on Whatman paper kept in bags with silica gel. Blood from the tail was used to make blood smears for microscopic examination. Slides were air-dried and later fixed with methanol and stained with Giemsa following Telford (2009). Parasitemia levels were estimated based on counts of 5,000 erythrocytes when possible and repeated 3 times for each positive slide, except 2 (23SP and 11CNE). Molecular techniques DNA was extracted from tissue and blood drop samples using DNeasy Blood & Tissue kit (Qiagen, Valencia, California), following the manufacturer’s instructions. Detection of the presence of Hepatozoon species was initially made using PCR reactions with the primers HEMO1 and HEMO2 targeting part of the 18S rRNA region following Perkins and Keller (2001). Samples were then also used in a further PCR reaction using the primers HepF300 and HepR900, targeting another part of the 18S rRNA region, following Ujvari et al. (2004). The PCR reactions using the HEMO primers were run in a 20 ml reaction mixture containing 0.5 U of Taq DNA polymerase (Invitrogen, Carlsbad, California), 3.75 mM MgCl 2 , 0.2 mM of each nucleotide, 13 PCR buffer (200 mM Tris-HCl, 500 mM KCl), 0.5 mM of each primer, 0.4 mg/ml of albumin (BSA; Roche Applied Science, Madison, Wisconsin), and 2 ml of DNA. The reaction mix was heated to 94 C for 3 min, and then amplification was performed through 35 cycles at 94 C for 30 sec, 48 C for 30 sec, and 72 C for 1 min, followed by a final 10 min extension at 72 C. The PCR reactions using the HEP primers were run in a 20 ml reaction mixture containing 1 U of Taq DNA polymerase (Invitrogen), 1.5 mM MgCl 2 , 0.125 mM of each nucleotide, 13 PCR buffer (200 mM Tris-HCl, 500 mM KCl), 0.6 mM of each primer, 0.4 mg/ml of albumin (BSA; Roche), and 2 ml of DNA. The reaction mix was heated to 94 C for 3 min, and then amplification was performed through 35 cycles at 94 C for 30 sec, 61 C for 30 sec, and 72 C for 1 min, followed by a final 10 min extension at 72 C. Negative and positive controls were run with each reaction. The positive PCR products were purified and sequenced by a commercial sequencing facility (Macrogen Inc., Seoul, Korea). All sequences were performed in both directions. Received 25 February 2010; revised 5 September 2010; accepted 7 September 2010. DOI: 10.1645/GE-2470.1 J. Parasitol., 97(1), 2011, pp. 106–110 F American Society of Parasitologists 2011 106