Introduction
As long-lived apex predators closely associated with the coastal zone, bottlenose dolphins (Tursiops spp.) can be environmental sentinels by providing early indication of health stressors, including parasites (Bossart Reference Bossart2011; Moore Reference Moore2008). Obtaining samples from marine mammal carcasses via necropsies allows for opportunistic insight into the health of these animals (Rowles et al. Reference Rowles, Van Dolah, Hohn, Dierauf and Gulland2001). Bottlenose dolphins have been reported to be infected with broad tapeworms Diphyllobothrium spp. around the world (e.g., Hatsushika and Shirouzu Reference Hatsushika and Shirouzu1990; Quiñones et al. Reference Quiñones, Giovannini, Raga and Fernández2013; Shirouzu et al. Reference Shirouzu, Hatsushika and Okino1999; Zam et al. Reference Zam, Caldwell and Caldwell1971) including D. stemmacephalum in the Gulf of Mexico (Sánchez et al. Reference Sánchez, Goldstein and Dronen2018; Ward and Collins Reference Ward and Collins1959). However, none has been identified to species level in the Northwestern Atlantic Ocean (Ridgway Reference Ridgway1965; Zam et al.,Reference Zam, Caldwell and Caldwell1971). Herein we report the finding and molecular identification of D. stemmacephalum from the intestine of a bottlenose dolphin Tursiops sp. that was stranded on the coast of the Northwestern Atlantic Ocean in South Carolina (SC), USA.
Material and methods
The bottlenose dolphin (Field Number SC1910) was found dead, stranded on Sullivan’s Island, SC (32°76’18”N, 79°86’25”W) on 3 March 2019 and identified as Tursiops truncatus at the time (Figure 1). However, the species T. erebennus was reinstated as a valid species residing in SC after examination of the individual occurred (Costa et al. Reference Costa, Mcfee, Wilxoc, Archer and Rosel2022; see discussion below) and no further identification was possible. Therefore, as it is unclear whether this animal belongs to T. truncatus or T. erebennus, herein we identify it as Tursiops sp.
(a), stranding location of bottlenose dolphin SC1910 on Sullivan’s Island, South Carolina, USA; (b), stranded dolphin Tursiops sp. on site; (c), gastrointestinal tract excised during necropsy; (d), strobila of cestode (later identified molecularly as Diphyllobothrium stemmacephalum) collected from one segment of the dolphin’s small intestine.
The individual was a ~1.5-year-old, 169 cm long male. Organs, including the gastrointestinal tract, were stored at −20°C for later examination (Figure 1). A tapeworm infection in the intestine was noted during a later assessment of the dolphin’s gastrointestinal tract on 18 October 2022. The frozen stomach and intestines were thawed, weighed, and examined. During dissection, the entire length of the intestine was divided into eight equal-length samples, which sectioned the parasite(s). Part of the strobilae was fixed in 10% neutral buffered formalin and part in 100% ethanol for molecular studies. However, the integrity of intestinal helminths, and cestodes in particular, is quickly compromised when collected from stranded and/or frozen hosts (Kuchta and Scholz Reference Kuchta, Scholz, Caira and Jensen2017), as in this case. Hence, identification of this worm was based solely on sequencing data.
Genomic DNA from the parasite specimen was extracted using a Qiagen DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) following the protocol of the manufacturer. Portions of the mitochondrial cytochrome c oxidase I (COI) and nuclear large subunit ribosomal RNA (28S rRNA) genes were amplified and sequenced. The COI PCR was done using primers JB3 (5´-TTTTTTGGGCATCCTGAGGTTTAT-3´; Bowles et al. Reference Bowles, Blair and McManus1995) and CO1-R trema (5´- CAACAAAATCATGATGCAAAAGG-3´; Miura et al. Reference Miura, Kuris, Torchin, Hechinger, Dunham and Chiba2005). A 25-μl total reaction contained 1X Promega GoTaq® Flexi PCR Buffer (Madison, WI, USA), 0.4X Invitrogen Rediload™ loading buffer (Thermo Fisher Scientific, Waltham, MA, USA), 2 mM MgCl2, 0.5 mM dNTPs, each primer at 0.3 μM, 1 U Promega GoTaq® DNA polymerase, and 3 μl template DNA. Cycling was as follows: 5 min at 95°C was followed by 35 cycles at 95°C for 30 s, 48°C for 30 s, and 72°C for 45 s, followed by 72°C for 5 min. The 28S PCR was done using primers digl2 (5´-AAGCATATCACTAAGCGG-3´; Tkach et al. Reference Tkach, Grabda-Kazubska, Pawlowski and Swiderski1999) and 1500R (5´-GCTATCCTGAGGGAAACTTCG-3´; Tkach et al. Reference Tkach, Littlewood, Olson, Kinsella and Swiderski2003), and reagents were the same as above except 1.5 mM MgCl2, 0.2 mM dNTPs, 0.5 μM of each primer, and 1 μl of template were used. Cycling was as follows: 3 min at 94°C was followed by 40 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min, followed by 72°C for 7 min. Products, including PCR positive and negative controls, were electrophoresed on 1% agarose gels stained with GelRed (Biotium, Freemont, CA, USA) and visualised under UV light. The product was then cleaned using ExoSAP-IT (Affymetrix, Santa Clara, CA, USA) and sent to Eurofins MWG Operon LLC (Louisville, KY, USA) for direct, bi-directional sequencing using the same primers as above in addition to internal primer 300F (5´-CAAGTACCGTGAGGGAAAGTTG-3´; Littlewood et al. Reference Littlewood, Curini-Galletti and Herniou2000) for the 28S rRNA gene sequence. Complementary sequences were assembled, compared to their chromatograms, and edited accordingly using Sequencher version 5.4.6 (Gene Codes Corp., Ann Arbor, MI, USA). The resulting sequences were compared to those in the National Center for Biotechnology Information’s GenBank database using megaBLAST (Morgulis et al. Reference Morgulis, Coulouris, Raytselis, Madden, Agarwala and Schäfffer2008).
Results
Upon necropsy this dolphin was determined to have a body condition of Code 3 (fair to moderate decomposition, organs still intact according to Geraci and Lounsbury (Reference Geraci and Lounsbury2005)). Gross pathology from necropsy indicated that this dolphin suffered from scoliosis, oral lesions, circular, depressed skin lesions, probable verminous pneumonia caused by lungworms, ruptured spleen, possible hypertrophic cardiomyopathy, and malnourishment (SC1910 necropsy report, NOAA NCCOS, Charleston, SC, USA). The dolphin’s intestine was 23.75 m long and contained 375 g of contents (wet weight), 320 g of which was cestode (wet weight). Strobilae were found in six of the eight sections of intestine with none present in the proximal and distal ends. Strobilae within each section was weighed and the total length was estimated to be approximately 63 m (Figure 1d). Hence, although no scolex was recovered and no number of cestodes could be confirmed, this dolphin was most certainly infected by several tapeworm individuals. No cestode was present in the stomach, which contained 110 g of contents (wet weight) and two cephalopod beaks.
The parasite 28S rRNA gene sequence (1,234 base pairs) was 100% similar with 100% coverage to that of the three 28S rRNA gene sequences of D. stemmacephalum Cobbold, Reference Cobbold1858 in GenBank (two from the USA found in a bottlenose dolphin (accession number KY552825) from Mississippi (Gulf of Mexico; Waescheenbach et al. 2017) and an Atlantic white-sided dolphin (Lagenorhynchus acutus) (AF286943) from Massachusetts (Olson et al. Reference Olson, Littlewood, Bray and Mariaux2001), and a third from northern Japan in a Harbour porpoise (Phocoena phocoena) (LC644720; Katahira et al. Reference Katahira, Matsuda, Maeda, Yoshida, Banzai and Matsuishi2022). The parasite COI sequence (554 bp) was 100% similar with 100% coverage to that of D. stemmacephalum (JQ268543) found in L. acutus from the USA (Massachusetts) and 99.28–99.64% similar (100% coverage) to five sequences of D. stemmacephalum in GenBank (MW034674: Monachus monachus, Adriatic Sea (unpublished), LC709257: T. truncatus, Japan (Ishisaka et al. Reference Ishisaka, Segawa, Shikawa and Itou2023), KY552885: T. truncatus, Gulf of Mexico (Waeschenbach et al. Reference Waeschenbach, Brabec, Scholz, Littlewood and Kuchta2017), LC644653: P. phocoena, Japan (Katahira et al. Reference Katahira, Matsuda, Maeda, Yoshida, Banzai and Matsuishi2022), LC042231: Homo sapiens, Japan (Yamasaki et al. Reference Yamasaki, Kumazawa, Sekikawa, Oda, Hongo, Tsuchida, Saito, Morishima and Sugiyama2016). Sequences from this study were deposited in GenBank as accession numbers OR588137 (28S) and OR558138 (COI).
Discussion
Diphyllobothrium stemmacephalum belongs to the group of broad fish tapeworms, which have an unarmed scolex and typically a long (1–30 m) strobila (Kuchta and Scholz Reference Kuchta, Scholz, Caira and Jensen2017). These tapeworms are found throughout the Northern Hemisphere, have marine mammals as definitive hosts, and are zoonotic (Yamasaki et al. Reference Yamasaki, Kumazawa, Sekikawa, Oda, Hongo, Tsuchida, Saito, Morishima and Sugiyama2016). The species is the type species of the genus; it was originally described from a Harbour porpoise (Phocoena phocoena) from the Scottish North Sea (Cobbold Reference Cobbold1858) and reported again in P. phocoena by Andersen (Reference Andersen1987) off the Netherlands and Denmark, by Katahira et al. (Reference Katahira, Matsuda, Maeda, Yoshida, Banzai and Matsuishi2022) in the western Pacific in Japan, and most recently by Striewe et al. (Reference Striewe, Wohlsein, Siebert and Lehnert2025) in the North and Baltic Seas off Germany. This species has also been identified in other marine mammals, including bottlenose dolphins from the Gulf of Mexico (Sánchez et al. Reference Sánchez, Goldstein and Dronen2018; Ward and Collins, Reference Ward and Collins1959). Additional occurrences of Diphyllobothrium infection have been reported in a variety of odontocetes, including a Beluga whale (Delphinapterus leucas) in the estuary and Gulf of Saint Lawrence (Brunel et al. Reference Brunel, Bosse and Lamarche1998), a bottlenose dolphin (T. truncatus) off Florida’s northeastern coast (Ridgway Reference Ridgway1965; Zam et al. Reference Zam, Caldwell and Caldwell1971), and pygmy sperm whales (Kogia breviceps) and short-finned pilot whales (Globicephala macrorhynchus) off the coast of Brazil (Carvalho et al. Reference Carvalho, Leal Bevilaqua, Mayo Iñiguez, Mathews-Cascon, Bezerra Ribeiro, Bezerra Pessoa, Oliveira de Meirelles, Gomes Borges, Marigo, Soares and de Lima Silva2010). However, none of these tapeworms were identified to species level. While Sánchez et al. (Reference Sánchez, Goldstein and Dronen2018) noted that the few specimens of D. stemmacephalum collected from bottlenose dolphins from the Gulf of Mexico were all significantly larger than those from other hosts and localities, to our knowledge no specimen >30 m long has ever been reported, supporting our notion that the 63 m of tapeworm collected from this dolphin was comprised of several individuals. This is the first report of D. stemmacephalum infection in a bottlenose dolphin from SC.
Bottlenose dolphins stranded in SC could be from one of several stocks that frequent coastal waters including the Charleston Estuarine System Stock, the Southern Migratory Stock, the South Carolina-Georgia Coastal Stock, the Northern Georgia-Southern South Carolina Estuarine System Stock, and the Northern South Carolina Estuarine System Stock (Hayes et al. Reference Hayes, Josephson, Maze-Foley, Rosel and Turek2020). As such, dolphins in SC may belong to T. truncatus, the Atlantic bottlenose dolphin, or to T. erebennus, the Tamanend’s bottlenose dolphin, which was previously considered to be a coastal ecotype of T. truncatus and was recently reinstated as a valid species residing within estuarine environments (Costa et al. Reference Costa, Mcfee, Wilxoc, Archer and Rosel2022). Parasitic infections of Tursiops spp. are commonly observed during necropsies, but no tapeworm infection was reported in a SC study carrying over sixteen years of data (McFee and Lipscomb Reference McFee and Lipscomb2009). Although infections by D. stemmacephalum appear to be overall infrequent (Striewe et al. Reference Striewe, Wohlsein, Siebert and Lehnert2025), their occurrence in various odontocetes around the world and in bottlenose dolphins, which are the most common marine mammals in our region, is significant as this parasite is zoonotic (Yamasaki et al. Reference Yamasaki, Kumazawa, Sekikawa, Oda, Hongo, Tsuchida, Saito, Morishima and Sugiyama2016).
The life cycle of D. stemmacephalum has yet to be unravelled. However, while this group of tapeworms was recently revised and several species of Diphyllobothrium with known life cycles were moved to genus Dibothriocephalus (see Waeschenbach et al. Reference Waeschenbach, Brabec, Scholz, Littlewood and Kuchta2017, WoRMS, 2022), it is expected that the life cycles of parasites remaining in genus Diphyllobothrium follow the same general complex pattern as other broad tapeworms (e.g., Dib. dentriticus or Dib. ditremum, see Kuchta et al. Reference Kuchta, Brabec, Kubácková and Scholz2013 and Borgstrom et al. Reference Borgstrom, Trombor, Haugen and Rosseland2017, respectively): in brief, these tapeworms use copepods and fishes as first and second intermediate hosts, respectively, and piscivorous fishes as paratenic hosts (e.g., Ikuno et al. Reference Ikuno, Akao and Yamasaki2018). To our knowledge, no fish in the Northwestern Atlantic have been reported to be infected with plerocercoids of this particular species, but it is a reasonable assumption that dolphins become infected via ingestion of infected fish. Furthermore, while no human case of infection by D. stemmacephalum is known to date from the USA, 24 human infections have been reported in Japan and Korea (Lee et al. Reference Lee, Chai, Hong, Sohn and Choi1988; Yamane et al. Reference Yamane, Kamo, Yazaki, Fukumoto and Maejima1981; in Scholz et al. Reference Scholz, Garcia, Kuchta and Wicht2009; Yamasaki et al. Reference Yamasaki, Kumazawa, Sekikawa, Oda, Hongo, Tsuchida, Saito, Morishima and Sugiyama2016). Hence, the infection of a stranded bottlenose dolphin indicates the potential for the emergence of a public health risk for raw/undercooked fish consumers in our area and warrants continuous monitoring of this sentinel species.
Acknowledgements
The authors thank the staff and volunteers of the SC Marine Mammal Stranding Network who assisted in the response and necropsy of this animal, including Francesca Battaglia, Katelyn McGlothlin, Valerie Sprinkel, and Mary Pringle. NOAA Disclaimer: The scientific results and conclusions, as well as any views or opinions expressed herein, are those of the authors and do not necessarily reflect those of NOAA or the Department of Commerce.
Financial support
Thanks to the Department of Biology (College of Charleston) and NOAA’s National Centers for Coastal Ocean Science for financial support.
Competing interests
The authors declare none.
Ethical standard
The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Declaration of Helsinki of 1975, as revised in 2008. Samples were collected under NOAA’s authority under the MMPA Section 109(h).
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