Northeastern Naturalist Vol. 22, No. 1 D.J. Harris 2015 209 2015 NORTHEASTERN NATURALIST 22(1):209–212 Survey of Hepatozoon (Apicomplexa, Hepatozoidae) Blood Parasites in Small Mammals and Snakes from the Huyck Preserve, New York D. James Harris* Abstract - Currently there is limited information on blood-parasite distribution and diversity in wildlife in North America, particularly for some groups such as reptiles, despite their potential impact on their hosts. Snakes and small mammals were surveyed in the Huyck Preserve, NY, during September 2013. No snakes were infected, but a Peromyscus leucopus (White-footed Mouse) was positive. Similarity of the 18S rRNA gene with data from Gen- Bank indicates that Hepatozoon spp. from snakes and small mammals are related, further highlighting a possible role for trophic transmission. Introduction The important role that parasites play in shaping ecosystems is now widely accepted (Poulin 1999). Despite this, knowledge on distribution and diversity of many groups of parasites is limited. Hepatozoon spp. are obligate heteroxenous parasites of many vertebrate (intermediate) and invertebrate (definitive) hosts. More than 300 species have been described, the majority from snakes and around 50 from mammals. Currently there is “a dearth of molecular information regarding Hepatozoon species cycling in North American wildlife” (Allen et al. 2011). Furthermore, recent studies have indicated probable predator–prey transmission for groups including snakes and their prey (Tomé et al. 2013) and carnivores and rodents (Allen et al. 2011), making surveys of different groups of vertebrates from the same locality particularly useful. The aim of this study was to survey Hepatozoon presence in the Huyck Preserve and Biological Research Station in New York (42°31'N, 74°9'W), in both snakes and small mammals. This information may shed light on parasite transmission between predators and prey. Methods I conducted the fieldwork during September 2013. Snakes were collected by hand, and identified using field guides (Conant and Collins 1998), while mammals were caught at night using small traps baited with peanut butter and seeds. Since some mammals can be difficult to identify to the species level in the field, I used a DNA barcoding approach to confirm species identify of key specimens. For all specimens, I took a small tissue sample for DNA analysis by cutting the tail. When this bled naturally, I smeared a drop of blood across a glass slide for *CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Campus Agrário de Vairão, 4485-661 Vairão, Portugal; james@cibio.up.pt. Manuscript Editor: Chris Ritzi Northeastern Naturalist 210 D.J. Harris 2015 Vol. 22, No. 1 examination under the microscope and afterwards released the animals at the point of capture. Blood smears were air-dried, fixed with methanol, and stained with Giemsa (Telford 2009), and examined using an Olympus CX41 microscope. I examined each slide for 15 minutes and recorded it as negative if no parasites were noted in that time. I performed DNA extraction using the standard high-salt method (Sambrook et al. 1989). Screening for Hepatozoon parasites was performed though polymerase chain reaction (PCR) using the HEMO primers (Perkins and Keller 2001). Briefly, the PCR protocol consisted of 94 ºC for 30 sec, 60 ºC for 30 sec, and 72 ºC for 1 min, repeated for 35 cycles (see Harris et al. 2011 for more details). Positive and negative controls were run with each reaction. I used PCR with universal 16S rRNA primers in the barcoding of the mammal hosts (Palumbi et al. 1991). I sent all positive products to a commercial company (Macrogen, Netherlands) for sequencing. Results In total , I collected 36 snakes belonging to 3 species: Thamnophis sirtalis (L.) (Common Garter Snake; n = 24), Storeria dekayi (Holbrook) (Brown Snake; n = 10), and Diadophis punctatus (L.) (Ring-necked Snake; n = 2). None of the snake blood smears examined under the microscope were positive for Hepatozoon. Twenty-two samples of small mammals were also examined: 5 Tamias striatus (L.) (Eastern Chipmunk), 2 Sorex sp. (shrews), 9 Microtus pennsylvanicus (Ord.) (Meadow Vole), and 6 Peromyscus spp. (mice). One sample of a Peromyscus (Sample code DB21794) was positive for PCR for Hepatozoon. Sequencing and blast comparison on GenBank confirmed this. Although the parasite could not be identified to the specific level, the haplotype was identical to one previously found in a North African snake (Tomé et al. 2013). There were 2 nucleotide differences from a Hepatozoon isolated from a Peromyscus leucopus (Rafinesque) (White-footed Mouse) specimen from Oklahoma (Allen et al. 2011). The 16S sequence from the positive host showed 5 nucleotide differences from the published sequence of P. leucopus, which strongly identifies the host as this species (99% similarity). Sequences have been submitted to GenBank with Accession Numbers KM225832 and KM225833. Discussion Hepatozoon sauritus Telford, Wozniak, and Butler has been reported from Common Garter Snake and Ring-necked Snake in Florida with around 20% and 7% prevalence, respectively—much lower than prevalence in snakes such as Coluber constrictor L. (Eastern Racer) (Telford et al. 2004). Both species can also host Hepatozoon eurytopis Telford (Telford 2010). Davis et al. (2012) did not report Hepatozoon in these species, although only two individuals of each were sampled, while prevalence was 48% in Agkristrodon contortrix. (L.) (Copperhead Snake; n = 25), all from urban woodland in Tennessee. These findings seem to indicate that prevalence is lower in species that do not typically consume lizards or mammals, as found in studies in the Mediterranean region (Tome et al. 2013). Furthermore, Northeastern Naturalist Vol. 22, No. 1 D.J. Harris 2015 211 prevalence can be patchy, with 4 of 20 Common Garter Snakes infected in Florida, compared to 0 of 24 in this study. Low prevalence is always difficult to detect, but there does seem to be differences between regions as well as between species. The cause of these differences deserves further investigation. Phylogenetic assessments of Hepatozoon repeatedly identify a group consisting of isolates from snakes, small mammals, and lizards (Allen et al. 2011, Tomé et al. 2013) regardless of geography. The sample reported in this study is identical for this marker to a Hepatozoon from a snake, Psammophis elegans Shaw (Elegant Sand Racer; Tomé et al. 2013), but differs from a sample from another White-footed Mouse by 2 nucleotide differences (Allen et al. 2011). Faster-evolving markers are needed to try to delimit better relationships of Hepatozoon from this group. On the other hand, the value of this genetic tool for furthering knowledge on diversity and distribution of this poorly known parasite genus is clear. Additional surveys for Hepatozoon should also help in determining why some hosts are more infected than others, and why there seem to be differences in prevalence between localities of the same host species. Acknowledgments Fieldwork was funded by a Huyck research grant. Thanks to Dawn O’Neal for her help associated with this grant. All samples were collected under New York State Department License Number 1895. I thank Samantha Banfield and Beatriz Tomé for their help during the microscopy and laboratory work involved in this study. This work was supported by “Genomics and Evolutionary Biology”, co-financed by the North Portugal Regional Operational Programme 2007/2013 (ON.2 – O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF). Literature Cited Allen, K.E., M.J. Yabsley, E.M. Johnson, M.V. Reichard, R.J. Panciera, S.A. Ewing, and S.E. Little. 2011. Novel Hepatozoon in vertebrates from the southern United States. Journal of Parasitology 97:648–653. Conant, R., and J.A. Collins. 1998. A Field Guide to the Reptiles and Amphibians of Eastern and Central North America. Houghton Mifflin Co., Boston, MA. Davis, J.R., S.A. Boyle, A.A. Khan, A.L.J. Gay, J.M. Grisham, and L.E. Luque. 2012. Snake parasitism in an urban old-growth forest. Urban Ecosystems 15:739–752. Harris, D.J., J.P.M.C. Maia, and A. Perera. 2011. 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