TECHNICAL NOTE New primers reveal the presence of a duplicate histone H3 in the marine turtle leech Ozobranchus branchiatus Philip Lavretsky • Triet M. Truong • Audrey E. McGowin • George H. Balazs • Jeffrey L. Peters Received: 16 November 2011 / Accepted: 18 November 2011 / Published online: 1 December 2011 Ó Springer Science+Business Media B.V. 2011 Abstract Marine leeches, specific to sea turtles, have been implicated as potential vector organisms in the spread of fibropapillomatosis (FP), a pandemic neoplastic disease with Chelonia mydas having the highest affliction rate. Polymerase chain reaction identified two independent, seemingly functional histone H3 loci for marine turtle leeches Ozobranchus branchiatus collected from C. mydas in Florida and Hawaii. Primers were developed to amplify each product separately. Among the two loci, sequence differentiation (U ST ) ranged from 0.161 to 0.182 with identical amino acid translations among the 22 samples. A maximum parsimony tree of GenBank histone H3 sequences from annelids indicated the gene duplication occurred within the Ozobranchidae family. Geographically separated populations yielded U ST values of 0.004–0.005 but were phylogenetically distinctive. These novel markers will be useful in identifying ectoparasites in FP research, evaluating other histone variants, and chromatin dynamics regulation studies. Keywords Ozobranchus branchiatus Á Fibropapillomatosis Á Histone H3 Á Primers Á Gene duplication Á Chelonia mydas Histone genes are found in all eukaryotes. The histone com- plex is composed of five major groups: H1/H5, H2A, H2B, H3, and H4 (Bhasin et al. 2006). Particular attention has been given to histone H3 genes because they encode proteins involved in the formation of the core-nucleosome, which is vital for proper chromatin organization (Chaboute ´ et al. 1993). The slow-evolving nature of histone H3 has made it a pre- ferred locus for phylogenetic analyses. Colgan et al. (1998) first designed primers (H3af and H3ar) to amplify the H3 gene in arthropods, but these primers have since been used in Annelida (Lund et al. 2010; Rousset et al. 2007), Sipuncula (Maxmen et al. 2003), and Mollusca (Okusu et al. 2003; Mikkelsen et al. 2006; Dennis and Hellberg 2010). Ozobranchus branchiatus leeches are parasites known historically to use green sea turtles (Chelonia mydas) as hosts, but in recent years, have been documented on other marine turtle species (Bunkley-Williams et al. 2008; McGowin et al. 2011). The need to study marine turtle leeches has risen due to the species’ possible connection to the sea turtle neoplastic disease, fibropapillomatosis (Greenblatt et al. 2004). Until now, histone genes have not been sequenced for leech species of the Ozobranchidae family. Initially, the Colgan et al. (1998) primers were used to sequence the histone H3 locus of O. branchiatus lee- ches, but without specificity; multi-polymorphic sequences were obtained across all samples. Two different reverse primers compatible with H3af were designed, H3R1 (5 0 -GGT CAA CAA ATC ATA AAG ATA TTG G-3 0 ) and H3R2 (5 0 -TAA ACT TCA GGG TGA CCA AAA AAT CA-3 0 ) to target each locus independently. Electronic supplementary material The online version of this article (doi:10.1007/s12686-011-9581-6) contains supplementary material, which is available to authorized users. P. Lavretsky Á J. L. Peters Department of Biological Sciences, Wright State University, Dayton, OH 45435, USA T. M. Truong Á A. E. McGowin (&) Department of Chemistry, Wright State University, Dayton, OH 45435, USA e-mail: audrey.mcgowin@wright.edu G. H. Balazs National Marine Fisheries Service, Southwest Fisheries Science Center, Honolulu Laboratory, 2570 Dole Street, Honolulu, HI 96822-2396, USA 123 Conservation Genet Resour (2012) 4:487–490 DOI 10.1007/s12686-011-9581-6