Molecular Ecology Resources (2011) 11, 271–278 doi: 10.1111/j.1755-0998.2010.02946.x DNA BARCODING Genetic barcoding of marine leeches (Ozobranchus spp.) from Florida sea turtles and their divergence in host specificity AUDREY E. McGOWIN,* TRIET M. TRUONG,* ADRIAN M. CORBETT,† DEAN A. BAGLEY,‡§ LLEWELLYN M. EHRHART,‡§ MICHAEL J. BRESETTE,§ STEVEN T. WEEGE§ and DAVE CLARK§ *Department of Chemistry, Wright State University, Dayton, OH 45435, USA, †Department of Neuroscience, Cell Biology and Physiology, Wright State University, Dayton, OH 45435, USA, ‡Department of Biology, University of Central Florida, Orlando, FL 32816, USA, §Inwater Research Group, Inc., 4160 NE Hyline Dr., Jensen Beach, FL 34957, USA Abstract Ozobranchus margoi and Ozobranchus branchiatus are the only two species of marine turtle leeches (Ozobranchus spp.) known to inhabit the Atlantic coast of the United States and theGulfofMexico.Inearlyreports of fibropapillomatosis (FP) in green turtles (Chelonia mydas), O. branchiatus was implicated as a vector in the transmission of Fibropapilloma- associated turtle herpesvirus (FPTHV). It is imperative that the leech species be identified to elucidate the role Ozobranchus spp. may play in disease transmission. In this study, Ozobranchus branchiatus has been identified for the first time on a log- gerhead (Caretta caretta) turtle, and the molecular data for this species is now available for the first time in GenBank. Both species of leeches were also found infecting a single C. mydas. Using morphological taxonomy combined with distance- and character-based genetic sequence analyses, this study has established a DNA barcode for both species of Ozobranchus spp. leech and has shown it can be applied successfully to the identification of leeches at earlier stages of development when morphological taxonomy cannot be employed. The results suggest a different haplotype may exist for O. branchiatus leeches found on C. caretta versus C. mydas. Leech cocoon residue collected from a C. mydas was identified using the new method. Keywords: cocoon, DNA barcoding, Fibropapillomatosis-associated turtle herpesvirus, new host record, Ozobranchus branchiatus, Ozobranchus margoi, sea turtle leech, two species infection Received 1 September 2010; revision received 11 October 2010; accepted 21 October 2010 1975). A distinguishing morphological characteristic of Introduction the Ozobranchidae family is finger-like terminally There are only two known species of marine turtle branching lateral protuberances (gills or branchidae) that leeches (Ozobranchus spp.) known to inhabit the Atlantic decrease in complexity and size from the anterior to the coast of the United States and the Gulf of Mexico, Ozo- posterior and begin on segment XIII of the abdomen branchus margoi and Ozobranchus branchiatus. The primary (Sawyer 1986). The primary difference in these two spe- host for O. margoi is the loggerhead turtle (Caretta caretta) cies of leeches is in the number of gills with one pair per but O. margoi has been reported on green turtles (Chelonia segment; O. margoi has five pairs (Davies 1978) and mydas), hawksbill turtles (Eretmochelys imbricata) and O. branchiatus sports seven pairs (Raj & Penner 1962). Kemp’s Ridley turtles (Lepidochelys kempi) as well (Bunk- Both species have a short, segmented body with two eye ley-Williams et al. 2008). Only rarely is a host other than spots, a proboscis and a large posterior sucker. These C. mydas reported for O. branchiatus, although its occur- leeches attach to the mouth, neck, cloaca and under the rence on black turtles (Chelonia agassizi a.k.a. Chelonia flippers of turtles and deposit eggs in cocoons on the car- mydas of the Eastern Pacific) (McDonald & Dutton 1990), apace. Almost nothing is known about the life cycle of hawksbill turtles (Bunkley-Williams et al. 2008), and olive sea turtle leeches, including whether they can exist in the ridley turtles (Lepidochelys olivacea) in Mexico has been absence of a turtle host or if they have alternate hosts. documented (Hernandez-Vazquez & Valadez-Gonzalez Ozobranchus spp. can be very small (millimetres in 1998). These warm water marine leeches belong to the length) with various life stages so differentiation amongst family Rhynchobdellida, Ozobranchidae (Sawyer et al. species is difficult. Only a few descriptions of Ozobran- Correspondence: Audrey E. McGowin, Fax: +1 937 775 2717; chus spp. exist in the scientific literature (Sawyer et al. E-mail: [email protected] 1975) primarily because of the challenges of studying Ó 2010 Blackwell Publishing Ltd 272 DNA BARCODING their hosts, a formidable task because of the limited aligned and unique substitutions at specific positions can knowledge of sea turtle life history patterns (Bolten 2003). be identified as characteristic attributes (CAs). A set of Interest in Ozbranchus spp. has increased in recent these CAs for any species becomes the character-based years because of its possible relationship to a panzootic ‘DNA barcode’ for species identification. The identifica- that has primarily afflicted C. mydas but has now spread tion of a new species with DNA barcoding and those not to other sea turtles (Herbst 1994; Williams et al. 1994). included in a genomic repository must be accompanied The disease, fibropapillomatosis (FP), is a condition char- by standard taxonomy (DeSalle et al. 2005). Still care acterized by the growth of multiple visceral and cutane- must be exercised when using a single gene such as COI ous fibrovascular tumours on the eyes, neck, cloaca and alone because more than one gene may be necessary to flippers of sea turtles (the same places where leeches delineate phylogenetic relationships between species attach themselves), which can interfere with the turtle’s (DeSalle et al. 2005). ability to swim, see and feed. Fibropapilloma-associated For leeches, DNA barcoding could be particularly use- turtle herpesvirus (FPTHV) has been identified as the ful, as their identification using standard taxonomic tech- causative agent of FP (Quackenbush et al. 1998), although niques can sometimes be ambiguous (Bely & Weisblat the primary vector triggering this chronic tumour-form- 2006). In addition, morphological characteristics are only ing disease is still unknown. In early reports of FP in useful when dealing with more mature specimens, not C. mydas, O. branchiatus was implicated as a vector specimens in the larval or cocoon stages, whereas DNA organism (Smith & Coates 1938; Nigrelli & Smith 1943). barcoding would be useful for all stages of development. Four separate viral variants were detected in an analysis DNA barcoding of marine leeches could facilitate marine of FPTHV in sea turtles in Florida waters (Ene et al. 2005). leech life cycle elucidation because the nucleotide The viral variant specific to Indian River Lagoon, a green sequence will be identical at every life stage. Although turtle foraging area along the Atlantic coast of Florida, the phylogeny of leeches has been studied extensively appears to be endemic to the habitat, leading to the sup- through the lens of COI sequences (Siddall & Burreson position that FP is transmitted during the juvenile phase 1998; Light & Siddall 1999; Utevsky et al. 2007) and when turtles move to nearshore feeding grounds after includes O. margoi (GenBank accession number spending several years at sea (Ene et al. 2005). Therefore, AF003268; Siddall & Burreson 1998), O. branchiatus has a vector organism could be responsible for the epizootic previously been excluded as no COI sequence had been occurring at Indian River Lagoon where as high as 72% determined. In this study, COI sequences of both species of C. mydas have been affected (Hirama & Ehrhart 2007). of leeches were obtained for specimens collected from An analysis of FPTHV concentration in five types of tur- sea turtles captured by net in the Indian River Lagoon tle ectoparasites collected in Hawaii showed only Ozo- and the St. Lucie Power Plant on Hutchinson Island in branchus spp. leeches contained sufficient viral load for Florida, resulting in the addition of O. branchiatus to the transmission of the virus between turtles (Greenblatt GenBank database. Using COI sequences of closely et al. 2004). Unfortunately, the leech species was not spec- related species already in GenBank (Siddall & Burreson ified in this publication. Knowledge of the species 1998; Light & Siddall 1999; Utevsky et al. 2007), DNA bar- removed from turtles with FP could shed light on the role codes were generated for both of these species of leeches of marine leeches as a possible vector organism in disease in the Ozobranchidae family and morphological data transmission (Williams & Bunkley-Williams 2006; Bunk- were used to corroborate DNA barcode identification. ley-Williams et al. 2008). These simple character-based attributes (sCAs) were Sequencing of particular mitochondrial genes in ani- applied to a sample of leech cocoon residue (a sample for mals, such as the cytochrome c oxidase I (COI) gene, can which taxonomic identification would be impossible) yield phylogenetic information as well as aid in the iden- taken from a C. mydas during a cold-stunning event that tification of species that have molecular data in genetic occurred in Florida in January 2010. databases such as BOLD, NCBI, GBIF, DDBJ or EMBL. The Universal Folmer primers (Folmer et al. 1994) have Materials and methods been used successfully for the amplification of COI genes in other species of leeches (Siddall & Burreson 1998; Light Collection sites & Siddall 1999; Utevsky et al. 2007).