Parasitology International 66 (2017) 116–118 Contents lists available at ScienceDirect Parasitology International journal homepage: www.elsevier.com/locate/parint Short communication Establishment of the complete life cycle of Spirometra (Cestoda: Diphyllobothriidae) in the laboratory using a newly isolated triploid clone Tetsuya Okino a,⁎, Hiroshi Ushirogawa a, Kumiko Matoba a, Shin-ichiro Nishimatsu b,MinekiSaitoa,⁎ a Department of Microbiology, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0192, Japan b Department of Molecular Biology, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0192, Japan article info abstract Article history: Methods to maintain the life cycle of pathogenic organisms become powerful tools for studying molecular and Received 7 June 2016 cellular bases of infectious diseases. Spirometra erinaceieuropaei is a parasitic tapeworm that causes sparganosis Received in revised form 19 December 2016 in humans. Because S. erinaceieuropaei has a complex life cycle with different stages and host species require- Accepted 22 December 2016 ments, there have been no reports to establish the complete life cycle in the laboratory. In this study, using Cy- Available online 24 December 2016 clops as the first intermediate host, mouse as the experimental second intermediate host, and dog as the final Keywords: host, we succeeded in maintaining S. erinaceieuropaei in the laboratory. By repeating the established life cycle fi Triploid ve times, we obtained a clonal population of S. erinaceieuropaei from a single adult worm. A karyotype study Spirometra erinaceieuropaei showed that the chromosome of this clone is triploid (3n = 27), indicating that a genetically uniform strain is Complete life cycle established by apomictic reproduction. The strain was named Kawasaki triploid (Kt). A partial sequence of mito- Sparganosis chondrial cytochrome c oxidase subunit 1 gene of the strain Kt showed more than 98% similarity with those of S. New model organism erinaceieuropaei isolates from Australia, China, and South Korea, and the resultant phylogeny indicated that the strain Kt is a member of a distinctive clade from East Asia and Oceania. Our system will be particularly useful for studies of S. erinaceieuropaei infection and human sparganosis. © 2016 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND li- cense (http://creativecommons.org/licenses/by-nc-nd/4.0/). Spirometra erinaceieuropaei is an intestinal tapeworm of wild and taxonomic situation, we adopted the tentatively used specific name domesticated carnivores that is most commonly seen in Asian countries “erinaceieuropaei” for this study. [1]. It is well known that invasion and migration of S. erinaceieuropaei Since plerocercoids of S. erinaceieuropaei produce growth hormone- larva (plerocercoid) into subcutaneous tissue, abdominal cavity, eye, like factors and enhance the growth of its host (mouse) through an un- and central nervous system cause sparganosis, an important parasitic known mechanism [7], the establishment of reliable and reproducible zoonosis in humans. Humans become infected by eating raw and model system based on a genetically uniform parasite is needed for undercooked meats of plerocercoid-infected animals (frog, snake and studying metabolic pathways and their alterations during infection. In etc.), by placing the raw meats on open wounds, skin ulcers, and eyes, order to investigate the pathogenesis of S. erinaceieuropaei infection, an- and by drinking water contaminated with procercoid-infected cope- imal studies are essential. However, performing animal studies has been pods (Cyclops spp.) [1]. Recent molecular and morphological analyses a difficult task for S. erinaceieuropaei because of its complex life cycle, proposed that S. erinaceieuropaei in Asia is a cryptic species complex in- which passes through three distinct, well-defined larval stages. There- cluding S. erinaceieuropaei and S. dicipiens [1–6]. However, the proposal fore, maintaining the complete life cycle of S. erinaceieuropaei in the lab- has not been fully accepted because maternally inherited mitochondrial oratory was considered impossible until now. Both diploid and triploid DNA (mtDNA) was only used for the species identification without con- forms exist in populations of S. erinaceieuropaei, and the parthenogenet- siderations of introgressive hybridization between the two species. Pop- ic triploid is desirable as a model organism because of its genetic unifor- ulation genetic analyses using nuclear DNA markers are, therefore, mity [8]. Therefore, in the present study, we attempted to establish the required to clarify their species status, and particularly to identify the complete life cycle of S. erinaceieuropaei using a new triploid clone. parthenogenetic triploid populations (clones). In such an ambiguous All experimental animal protocols were approved by the Animal Re- search Committee of Kawasaki Medical School (Approval Nos.: 08-083, 09-083, 10-062, 11-087, 12-062, 13-102, 14-052, and 15-097), and the experiments were conducted in accordance with institutional guide- ⁎ Corresponding authors. E-mail addresses: [email protected] (T. Okino), lines for the care and use of animals. To establish the life cycle stages [email protected] (M. Saito). of S. erinaceieuropaei in the laboratory, we used the large copepod http://dx.doi.org/10.1016/j.parint.2016.12.011 1383-5769/© 2016 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). T. Okino et al. / Parasitology International 66 (2017) 116–118 117 modifications. Namely, the wheat extract solution was diluted 5-fold with tap water to culture tens of copepods per Petri dish. As an animal diet, Euglena gracilis were provided for the beginning and middle stages, and then brine shrimp (Artemia nauplii) [10] were provided for the later stage. The copepods were maintained in an incubator at 22 °C under a 12-h light/dark cycle. We collected Spirometra plerocercoids from Japanese striped snakes (Elaphe quadrivirgata) in Okayama Prefecture, Japan, and these plero- cercoids were tentatively identified as S. erinaceieuropaei by molecular analysis as shown in Fig. 2. We chose this location because high rates of triploidy of the parasite have been observed across Okayama Prefec- ture [11]. Collected plerocercoids were orally administered to female ICR mice, and the mice were maintained in the laboratory. A single ple- rocercoid derived from the mouse was perorally administered to a dog. At 40 days post infection, the feces of the infected dog were dissolved in tap water to create a suspension, and S. erinaceieuropaei eggs were col- lected from the sieved fecal suspension. These eggs were placed on a piece of filter paper and incubated for 10–14 days at 25 °C according to the method of Harada and Mori [12]. In order to obtain coracidia, em- Fig. 1. Mitotic metaphase chromosomes and the karyotype of Spirometra erinaceieuropaei bryonated eggs were incubated at 4 °C for 5 min, and then incubated in strain Kt. Scale bar: 10 μm. tap water at 25 °C for 2 h. The hatched active coracidia were incubated with Cyclops at room temperature for approximately 3 h. Then, 4–5- species Mesocyclops leuckarti as the first intermediate host, mouse as the week-old female ICR mice were orally infected with Cyclops containing experimental second intermediate host, and dog as the final host. The 15 or 16-day-old procercoids with a feeding needle. At one month post copepods were collected with a plankton net (a simple conical net infection, mice were euthanized via CO2 asphyxiation, and the plerocer- with 60 μm mesh) from canals in Kurashiki City. Since copepods change coids were recovered at autopsy. Furthermore, a dog was infected with the diet from plant to animal during metamorphosis, it is very difficult a single plerocercoid from the mouse. The remaining plerocercoids to farm copepods for a long period. To overcome this, we cultured the were maintained in mice. Since the plerocercoid of S. erinaceieuropaei copepods in accordance with the methodology of Kumazawa [9] with can regenerate a body from the head piece, the heads were cut from Fig. 2. A midpoint-rooted phylogenetic tree inferred from mitochondrial cox1 showing relationships among taxa of Spirometra erinaceieuropaei. The sample examined in this study is shown in bold. Bootstrap values less than 50% are not shown. Abbreviations are as follows. Se: Spirometra erinaceieuropaei, KR: Korea, CH: China, AU: Australia, JP: Japan, ID: Indonesia, IN: India, NZ: New Zealand, Rnigr: Dark-spotted frog (Rana nigromaculata), Lcaer: White's tree frog (Litoria caerulea), Equad: Japanese striped snake (Elaphe quadrivirgata), Pregi:Ball python (Python regius). Insertion figure: Gravid proglottids of Se Kawasaki (AB522603) Scale bar: 500 μm. 118 T. Okino et al. / Parasitology International 66 (2017) 116–118 the living bodies, and the 8 to 13 heads were orally administered to each Although pathogenic organisms responsible for human sparganosis in of mice. The regenerated plerocercoids could be maintained in mice for Asia can be divided into two major groups by mtDNA sequences [5,6], a long time, because plerocercoids have a long lifespan (10 or more their specific status is highly ambiguous, particularly in parthenogenetic years). The remaining bodies were used for DNA sequencing and triploid clones. Further taxonomic reconsiderations are needed to clarify