Herpetology Notes, volume 8: 461-463 (published online on 14 August 2015)

Molecular screening of Hepatozoon (Apicomplexa: Adeleorina) infections in Python sebae from West Africa using 18S rRNA gene sequences

Daniela Rosado1, José Carlos Brito1,2 and David James Harris1,2,*

Members of the genus Hepatozoon Miller 1908 are (Haklová et al., 2014). It is clear therefore that data the most common intracellular protozoan parasites from different geographical regions and snake hosts are reported in snakes (Wozniak et al., 1994). Although needed, both to improve knowledge on the distribution the morphology of gamonts in reptile blood cells is of these parasites, but also to evaluate diversity within generally very conserved (Telford, 1984), many species the 18S rRNA gene, and in turn highlight its utility as have been described, historically sometimes according a marker for describing new Hepatozoon species. In to the reptile species infected. The recent development this study, we aim to detect Hepatozoon parasites from of PCR-based methods for amplifying the 18S rRNA African rock pythons, Python sebae (Gmelin 1789), of parasites directly from host blood and tissue samples from a region of their range where few studies on has shed light on various aspects of the evolutionary snake parasites have been conducted, namely southern history of Hepatozoon (Barta et al., 2012), and also Mauritania, northern Senegal and western Mali. The diet on their distribution across vertebrate hosts in which of these snakes (Luiselli et al., 2001) is different from Hepatozoon had not previously been reported, such most snakes so far assessed for Hepatozoon prevalence as bats (Pinto et al., 2013) and caecilians (Harris et al., such as Natrix, Coronella and Malpolon (Tomé et 2014). Furthermore, finding of similar genetic lineages al. 2014). Thus, data from West Africa may provide in predators and prey, both in mammal (Allen et al., additional information about trophic transmission of 2011; Maia et al., 2014) and squamate (Tomé et al., Hepatozoon. Finally, we then compare identified 18S 2013; 2014) systems indicated the potential for trophic rRNA haplotypes with published data to assess diversity transmission. Identification of distinct haplotypes of 18S within and between various snake hosts. rRNA has also been used to support the description of Twelve P. sebae were collected by hand during various new species of Hepatozoon from snakes (O’Dwyer et al., periods of fieldwork from 2008-2014 (Figure 1). In each 2013; Han et al., 2015). On the other hand, other studies case a small tail-tissue sample was taken and stored in of Hepatozoon from snakes have indicated limited 96% ethanol, and animals were then released at the geographic patterns or congruency in host association collection site. DNA was extracted using standard high salt methods (Sambrook et al., 1989). Hemogregarine- specific primers HepF300 and HepR900 (Ujvari et al., 2004) were used to target part of the 18S rRNA of potential parasites. PCR conditions consisted of 94°C for 30secs, 60°C for 30 secs and 72°C for 1 minute, with 1 CIBIO/InBIO Research Centre in Biodiversity and Genetic 35 cycles. Positive and negative controls were included, Resources, University of Porto, Campus Agrário de Vairão, and positive PCR products were purified and sequenced Rua Padre Armando Quintas, Nº 7, 4485-661 Vairão, Vila do by a commercial facility (Beckman-Coultier, UK). Conde, Portugal 2 Departamento de Biologia da Faculdade de Ciências da New sequences were deposited in GenBank; accession Universidade do Porto, Rua Campo Alegre, 4169-007 Porto, numbers KR653312 and KR653313. Portugal Two of the twelve specimens gave positive PCR * Corresponding author e-mail: [email protected] reactions, and in both cases obtained sequences were 462 Daniela Rosado et al.

Figure 1. Geographic location of the samples of P. sebae screened for Hepatozoon infection in this study.

compared against published data from GenBank using mammal hosts (Maia et al., 2014). The new sequences the BLAST algorithm. Both sequences were 537 bp obtained in this study from P. sebae are identical to long, and were identical to each other. They were also samples from C. cerastes, and P. schokari, and are identical to Hepatozoon sequences on GenBank, from a therefore another member of this lineage, along with Saharan horned viper Cerastes cerastes Linnaeus 1758 the newly described H. chinensis from king ratsnakes (EF125058, unpublished), four isolates of Hepatozoon from China (Han et al., 2015). Python sebae can prey chinensis Han et al. 2015 from king ratsnakes, Elaphe on larger mammals such as dogs and goats (Luiselli carinata (Gunther 1864), from China (isolates 1,5,6 and et al., 2001), but in general prey on small mammals, 8, Han et al., 2015), from the sand racer Psammophis so the recovery of the Hepatozoon as members of schokari (Forskal 1775) (KC696569, Tomé et al., this lineage is not unexpected. However, the finding 2013), and from the mangrove snake, Boiga denrophila of an identical haplotype in very different groups of (Boie 1827) from Thailand (KF524356, unpublished). snakes, and from as far apart as China, Thailand and A sequence from Hepatozoon domergui Landau et West Africa does raise questions about the use of this al. 1970, isolated from Madagascarophis colubrinus marker in supporting new species diagnoses. A larger Schlegel 1837, from Madagascar, differed by a single segment of the 18S rRNA gene can be obtained by nucleotide, as did other isolates of H. chinensis from using multiple primers, but the section amplified by the king ratsnakes E. carinata. Hep primers is generally more variable, and estimates Various phylogenetic assessments of Hepatozoon of phylogeny based on the longer region tend to be very species from snakes have indicated that those from Africa similar or identical to those estimated from the shorter and Madagascar and the Mediterranean region form a fragment (Maia et al., 2012). Furthermore, Haklová et lineage, along with Hepatozoon recovered from small al. (2014), using sequences from another region of the Molecular screening of Hepatozoon infections in Python sebae from West Africa 463

18S rRNA gene, also reported identical haplotypes of Maia, J.P.M.C., Perera, A., Harris, D.J. (2012): Molecular survey Hepatozoon recovered from different snake species from and microscopic examination of Hepatozoon Miller, 1908 geographically diverse regions and Sumrandee et al. (Apicomplexa: Adeleorina) in lacertid lizards from the western Mediterranean. Folia Parasitologica 59: 241–248. (2015) report this for snakes from Thailand as well, with Maia, J.P., Álvares, F., Boratynski, Z., Brito, J.C., Leite, J.V., identical Hepatozoon in different snake hosts, however Harris, D.J. (2014): Molecular assessment of Hepatozoon with some genetic differences between the Hepatozoon (Apicomplexa: Adeleorina) infections in wild canids and rodents recovered from two different tick species infecting from North Africa, with implications for transmission dynamics them. It may be that the 18S rRNA marker used in across taxonomic groups. 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