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Establishing of Genetic Analyses Methods of Feces from the Water Shrew, Chimarrogale Platycephalus (Erinaceidae, Eulipotyphla)

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Establishing of Genetic Analyses Methods of Feces from the Water Shrew, Chimarrogale Platycephalus (Erinaceidae, Eulipotyphla) Central JSM Biology Bringing Excellence in Open Access Research Article *Corresponding author Koji Tojo, Department of Biology, Faculty of Science, Shinshu University, Asahi 3-1-1, Matsumoto, Nagano 390- Establishing of Genetic 8621, Japan, Tel: 81-263-37-3341; Email: Submitted: 11 April 2017 Analyses Methods of Feces Accepted: 28 April 2017 Published: 30 April 2017 from the Water Shrew, ISSN: 2475-9392 Copyright Chimarrogale platycephalus © 2017 Tojo et al. OPEN ACCESS (Erinaceidae, Eulipotyphla) Keywords • River ecosystem 1 2 1 Tomohiro Sekiya , Hidetaka Ichiyanagi , and Koji Tojo * • Top predator 1Department of Biology, Faculty of Science, Shinshu University, Japan • Non-damaged sampling 2Faculty of Advanced Science and Technology, Kumamoto University, Japan • Genetic structure • Analysis method development Abstract The Japanese endemic water shrew, Chimarrogale platycephalus is a small mammal adapted to mountain streams, and is the apex predator within its hierarchy preying on fish and aquatic benthos. Therefore, it is considered to be an extremely important species in the conservation of mountain stream ecosystems. Currently, a reduction in the number of habitats available and/or a decrease in populations, indicates that it is being threatened in various areas of Japan. They have been listed as being in critical status on the red list. With respect to the conservation of such endangered species, the accumulation of basic knowledge such as population structure and an understanding of genetic structure for each population are a very important. However, the accumulated ecological knowledge and knowledge of population genetics gathered to date is still at a poor level because this water shrew is relatively difficult to capture alive. It has been reported that severe damage occurs due to the stress of the capture method. We have tried to understand their population and genetic structures without capture by using their feces. We established genetic analyses methods using their feces. INTRODUCTION Currently, with a reduction in the number of habitats available and/or a decrease in populations, this water shrew is The Japanese water shrew, Chimarrogale platycephalus being threatened in various areas of Japan. Although this water (Soricidae, Soricomorpha), is a small mammal adapted to shrew has been recorded in 41 prefectures of Japan [3], in 37 of underwater life in mountain streams (Figure 1A). This species is those prefectures, they have been listed as being in critical status endemic to Japan, inhabiting only Honshu and Kyushu. This water on the local “Red List”. With respect to the conservation of such endangered species, the accumulation of basic knowledge such as and aquatic benthos (Figure 1B) [1]. They are basically nocturnal, population structures and an understanding of genetic structures doingshrew theiris the hunting apex predator at night. within Recently, its hierarchy, it has been preying reported on thatfish this water shrew also preys on salamanders [2]. As such, this water shrew is considered to be an extremely important species befor describedeach population later), is the a significantly accumulated important. ecological However, knowledge for andthis in the conservation of mountain stream ecosystems. Because knowledgewater shrew, of whichpopulation is relatively genetics difficult gathered to capture to date aliveare still (as atwill a they depend on the stability of their habitats for their staple poor level. Therefore, it is desirable to undertake further such diet prey in their sole environment of mountain streams, they research in the future [4]. In conventional biological research, some trap capture theare extremelymountain vulnerablestream environment to any modifications (including toconstruction their habitat of methods have been utilized. However, it has been reported that erosionor environment control dams,[3]. In bank recent protection years, artificial construction modifications work, and to severe damage occurs due to stress of the capture method. This deterioration of water quality) are considered to have resulted in shrew, when captured in traps, often dies within about two deterioration in their habitats. hours [5]. When carrying out a trapping survey, it is essential to Cite this article: Sekiya T, Ichiyanagi H, Tojo K (2017) Establishing of Genetic Analyses Methods of Feces from the Water Shrew, Chimarrogale platycephalus (Erinaceidae, Eulipotyphla). JSM Biol 2(1): 1010. Tojo et al. (2017) Email: Central Bringing Excellence in Open Access 1A 1C 1E 1F 1D 1G 1B Figure 1 A typical mountain stream habitat and places for predation and excretion, which the water shrew Chimarrogale platycephalus inhabits. 1A: Typical habitat landscape (Upper basin of the Kuma-gawa River, Hitoyoshi, Kumamoto Prefecture), 1B: A water shrew catching a Japanese white spotted char Salvelinus leucomaenis Feces found on the rock, 1E-F: An accumulation of water shrew feces excreted under a large rock in the center of a mountain stream (E, F), and 1G: Feces found on the rock, and the color, while of the swimming rock around around shrew a mountainfeces has a stream, different 1C: appearance. Typical rock on which to find feces of the water shrew, 1D: patrol any traps set frequently throughout the night. Although platycephalus utilizing its feces, samples were taken under some important measures have been undertaken to improve this various conditions for use as source material. Basically, we used situation, such as the development of a trap which minimizes In addition, the feces of individuals bred in an aquarium were alsofeces addedcollected to thein the analysis. field from Furthermore, mountain asstreams a control, (Figure tissue 1). datastress on [6], this this water has shrew. only reduced researchers’ difficulty slightly, specimens of C. platycephalus and it is still hard to efficiently obtain a large amount of biological As a result, we have tried to understand the population methods of the feces, we tried, which three were post fixed collection and stored methods in a and genetic structures of this water shrew without capture by asfreezer, follows: were (1) also Frozen used. storage;With regard (2) Fixationto the fixation and preservation and storage using their droppings. Because animal feces usually contain at utilizing 100% ethanol in 20 ml glass vials; (3) Preservation with least some remnants of epithelial cells from the gastrointestinal “InhibitEX Buffer” contained in the QIAamp Fast DNA Stool Mini tract, it is possible to analyze the genetic information of the Kit (QIAGEN, Hilden). One piece of feces was preserved in 2.5 ml corresponding individuals. In addition, for this water shrew, of this buffer. The specimens preserved using these buffers were Chimarrogale platycephalus, the feces can be relatively easily mainly used for analysis of nuclear DNA. found on boulders in mountain streams. The water shrew has a strong tendency to defecate on top of relatively large boulders DNA extraction with a good surrounding view within mountain streams. This feature allow us to conduct genetic analyses on such water shrew With respect to the extraction of total genomic DNA from the feces and is a breakthrough which will enable us to develop a feces of Chimarrogale platycephalus, a QIAamp Fast DNA Stool deeper understanding of their populations and genetic structures Mini Kit (QIAGEN, Hilden) was used. For the control specimens, in their natural habitats via a non-lethal and non-invasive method. the total genomic DNA extraction from the frozen C. platycephalus tail tissue was obtained using a DNeasy Blood & Tissue Kit MATERIALS AND METHODS (QIAGEN, Hilden). The total genomic DNA was extracted and Collection, fixation and preservation of the feces of Chimarrogale platycephalus littlepurified, in order predominantly to improve the according results. to the manufacturer’s recommended instructions; however, we modified the process a To establish a genetic analysis technique of Chimarrogale For the DNA extraction process from frozen fecal specimens, JSM Biol 2(1): 1010 (2017) 2/10 Tojo et al. (2017) Email: Central Bringing Excellence in Open Access was decreased, taking the range of samples from the standard 200 mtDNA Cyt b (wcytb-f/H15985), BSA+, 60 annealing temperature mg down to about 50 mg. In addition, the time for the centrifugal ℃ separation of impurities from a suspension containing feces was M 1 2 3 4 5 6 7 8 9 extended as appropriate. Amplification of DNA fragments using the PCR method cytochrome b (Cyt b) region, and the nuclear DNA apolipoprotein 1000bp B (ApoThe amplificationB) and breast of DNA cancer fragments susceptibility of the mitochondrial gene I (BRCA1) DNA regions using the PCR method was attempted using the total 500bp genomic DNA of C. platycephalus as a template. As the primer set Tail Feces (bred) Feces (wild) 100bp for amplification of the mtDNACyt b region, L14734/H15985 [7], Frozen 100% EtOH and wcytb-f/wcytb-r [8] were used (Table 1). Also, as the primer used,set for respectively. amplification The of Takara the nuDNA Ex Taq BRCA1 Kit was and used Apo to provide B regions, the BRCA1f/BRCA1r [9], and our designed ApoBf2/ApoBr2 were Figure 2 A PCR result of a DNA fragment of the mitochondrial DNA Cyt polymerases for the PCR reagent and was prepared according to the manufacturer’s protocol. In addition, since the DNA extracted in which the BSA solution was added. PCR was carried out using an from feces contains substances that inhibit the effectiveness of annealingb region using temperature the primer of 60°C. set, “wcytb-f/H15985”,M: The molecular size under marker conditions of the the PCR, BSA (bovine serum albumin) was added as a stabilizer for the PCR. The temperature conditions for the PCR which led to 100-bp ladder. Results in column 1-9 respectively correspond to optimum results are shown in Table (2). General electrophoresis specimen No. 30 (column 1), No. 27-1 (col. 2), No. 28-1 (col. 3), No. tail29-2 tissue (col. 4),used No. as 29-3 a control (col. 5),experiment, No. 5 (col. Feces 6), No. (bred): 10 (col. DNA 7), samples 1 No.
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