Abstract It is widely considered that most organisms cannot survive prolonged exposure to temperatures below 0°C, primarily because of the damage caused by the water in cells as it freezes. However, some organisms are capable of surviving extreme variations in environmental conditions. In the case of temperature, the ability to survive subzero temperatures is referred to as cryobiosis. We show that the ozobranchid leech, Ozobranchus jantseanus, a parasite of freshwater turtles, has a surprisingly high tolerance to freezing and thawing. This finding is particularly interesting because the leach can survive these temperatures without any acclimation period or pretreatment. Specifically, the leech survived exposure to super-low temperatures by storage in liquid nitrogen (−196°C) for 24 hours, as well as long-term storage at temperatures as low as −90°C for up to 32 months. The leech was also capable of enduring repeated freeze-thaw cycles in the temperature range 20°C to −100°C and then back to 20°C. The results demonstrated that the novel cryotolerance mechanisms employed by O. jantseanus enable the leech to withstand a wider range of temperatures than those reported previously for cryobiotic organisms. We anticipate that the mechanism for the observed tolerance to freezing and thawing in O. jantseanus will prove useful for future studies of cryopreservation.

Citation: Suzuki D, Miyamoto T, Kikawada T, Watanabe M, Suzuki T (2014) A Leech Capable of Surviving Exposure to Extremely Low Temperatures. PLoS ONE 9(1): e86807. https://doi.org/10.1371/journal.pone.0086807 Editor: Matsuo Uemura, Iwate University, Japan Received: May 28, 2012; Accepted: December 19, 2013; Published: January 22, 2014 Copyright: © 2014 Suzuki et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was partially supported by the Global COE program for Biodiversity and Evolutionary Research (A06) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study. Competing interests: The authors have declared that no competing interests exist.

Introduction In most ectothermic organisms, prolonged exposure to temperatures below 0°C can cause the water in their tissues to freeze, resulting in permanent physiological damage and sometimes cell death. However, organisms that live in extreme environments, such as polar regions or mountain highlands, frequently endure subzero temperatures [1]–[3]. Organisms in temperate zones typically survive such temperatures by hibernating [4], [5]. Cryobiosis, which refers to the adaptation or tolerance to freezing temperatures, is a form of cryptobiosis [6]. Among ectotherms, freeze tolerance should only be used to describe an ecologically relevant hibernation strategy that includes the ability to survive long-term freezing while maintaining constant and maximal intracellular ice contents at the subzero temperatures that are naturally encountered in the hibernaculum [7]. The leech genus Ozobranchus (Annelida: Hirudinida: Ozobranchidae) contains seven species that parasitize turtles [8]. Ozobranchus jantseanus is an external parasite on the freshwater turtles Mauremys japonica and M. reevesii, and relies on its turtle hosts for all of its life stages, i.e. from the egg to the adult stage [9] (Fig. 1). The leech is distributed throughout East Asia, Japan and China [9]. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. Stereoscopic micrograph of Ozobranchus jantseanus (dorsal view). Scale bar = 1 mm. https://doi.org/10.1371/journal.pone.0086807.g001 Several turtle species experience subzero temperatures (−2 to −4°C) for 1 to 11 days in their hibernacula [10], and M. japonica and M. reevesii, the hosts of O. jantseanus, typically hibernate in environments where they are likely to periodically encounter temperatures as low as 0°C. However, it would be unusual for both the leeches and their turtle hosts to exist under such cold conditions for extended periods of time. In the present study, we examined cryoresistance in O. jantseanus. The results showed that the leech was capable of surviving exposure to extremely low temperatures (−196°C) as well as prolonged storage at −90°C. All of the individuals had been active before initiating the experiments, indicating that this cryoresistance ability is always present in O. jantseanus.

Materials and Methods A total of 205 O. jantseanus individuals were collected from their hosts, the freshwater turtles M. japonica or M. reevesii, which were captured at two sites in the Kyoto and Chiba prefectures of Japan. A further six leech species were collected for comparative purposes: one marine species that is a parasite on sea turtles, O. margoi (n = 10), and five nonparasitic, freshwater species, Glossiphonia complanata (n = 1), Alboglossiphonia lata (n = 2), Helobdella stagnalis (n = 5), Erpobdella octoculata (n = 5), and E. japonica (n = 4). The O. margoi specimens were collected from a loggerhead turtle, Caretta caretta, along the Muroto coast of Japan, and the freshwater species (including O. jantseanus) were collected in the Kamogawa River in Kyoto Prefecture, Japan. The Guidelines for Animal Experiments at Tokyo University of Marine Science and Technology were employed in this study, and the study was approved by Professor Nobuaki Okamoto, President of Tokyo University of Marine Science and Technology. The Guidelines for Animal Experiments at Tokyo University of Marine Science and Technology are based on the Guidelines for Animal Experiments that were promulgated by the Science Council of Japan and the Ministry of Education, Culture, Sports, Science and Technology, Japan on June 1, 2006. The ozobranchid leeches were removed from the turtles using forceps, and the other leech species were collected from stones on the riverbed. The leech species and freshwater turtles are not subject to any animal protection laws in Japan and no permits were required to handle them and/or capture them by hand or by using cage traps. The use of traps is not illegal at the sampling sites in this study, and the turtles and the leeches were not injured during sampling. Freshwater turtles were released after the leeches were collected. The sea turtle, which is a protected species, was caught accidentally as fishing by-catch and was not injured by the collection of leeches. The turtle was released back into the sea once the leeches were removed. Cold tolerance in adults (total length 1–15 mm), hatchlings (total length: <1 mm), and eggs (containing approximately 20 eggs and the size of an egg was about 0.75 mm×0.75 mm) of O. jantseanus, was examined by placing the leeches in a deep freezer (−90°C) or in liquid nitrogen (−196°C; only adult O. jantseanus) for 24 hours. For adult leech of other species (total length about 5–50 mm), the same cooling tests by a deep freezer were conducted. Prior to freezing, all individuals were maintained in either freshwater or seawater at room temperature, and water on their surface was wiped just before the freezing examination. For the deep freezer test, the samples were put into a thin plastic film bag individually or one cocoon, and the bag was put into immediately the deep freezer where was kept at −90°C. On the other hand, for the liquid nitrogen immersion test, the samples were directly immersed individually. The exact cooling rates of specimens could not be measured since leech was so small and the weight was less than 20 mg. However, the actual cooling times to attain the purpose temperature −90°C or −196°C were at least within 10 seconds. Essentially, the purpose of this examination was to know the effect of end-point temperature. After the cryoexposure tests, the frozen leeches were pic up from the bag and thawed by immersion in either distilled water or seawater at room temperature. In this thawing, also the speeds were not measured, but the thawing to room temperature was finished within a few second. After thawing, specimens that restarted moving again under their own power (such as action of the gills and shrinking of the body) within five hours were judged as being alive. In the case of eggs, we thawed the frozen cocoons by storing in a petri dish containing distilled water, and kept them in the dish for a week. As the leeches hatched out from the cocoons that experienced eh cryoexposure test, we considered the eggs to have the cryotolerance. To clarify long-term freeze tolerance in O. jantseanus, the survival rates of leeches stored at −90°C for 3 to 32 months were compared. The number of individuals and duration of freezing were 10, 3, 2, 7, 112, 36, and 11 leeches, and 3, 8, 9, 15, 20, 28, and 32 months, respectively. The freezing and thawing methods, and the judgment of survival were same to above test. Using differential scanning calorimetry (DSC; Shimadzu DSC-50, Japan), we also counted how many freeze-thaw cycles adult O. jantseanus specimens were capable of surviving. Ten adult individuals of the leech were put into aluminum DSC cell without seal, and were set on DSC. A freeze-thaw cycle test was conducted as following protocol; the sample were cooled down at −10°C/min from 20°C to −100°C, immediately without holding at −100°C, and warmed at +10°C/min to 20°C. The survival at each cycle was judged from whether they started moving or not within 20 minutes after reached to 20°C. After assessing survival in this way, the freeze-thaw process was repeated until all of the specimens were classified as dead. We also examined the physicochemical characteristics such as freezing points, glass transition by DSC. The one individual was put into aluminum DSC cell and hermetically sealed. Samples were cooled down from 20°C to −120°C and warmed to 20°C at ±10°C/min. Prior to this experiment, any water on the body surface of the leeches was wiped and removed using an air dryer to exclude the effect of water out of body which may affect DSC results by their freezing.

Acknowledgments We thank T. Imadu, A. Katayama, T. Nakano, K. Takemura, and H. Takeuchi for collecting leeches and host turtles, M. Nakagawa for providing the traps for capturing turtles, and T. Yamauchi, T. Nakano, R. Kobayashi, Y. Matsuura, and T. Hikida for valuable feedback regarding the study.

Author Contributions Conceived and designed the experiments: TS. Performed the experiments: TM DS. Analyzed the data: TM DS TK. Contributed reagents/materials/analysis tools: TS MW TK. Wrote the paper: DS. Assisted with writing the manuscript: TS TM TK MW.