Environmental DNA collection method, environmental DNA collection kit, and container for environmental DNA collection

Abstract

To provide a method for collecting environmental DNA, a kit for collecting environmental DNA and a container for collecting environmental DNA by which the environmental DNA of a biological species moving on the waterside can simply be collected while suppressing the decomposition of the environmental DNA contained in a collected water sample.SOLUTION: The environmental DNA collection method includes an installation step of installing a container 1 for storing purified water W and collecting environmental DNA on a waterside, and a collection step of collecting the purified water W in the container 1 as a water sample for environmental DNA analysis after leaving the container 1 on the waterside for a predetermined period of time. The environmental DNA collection kit includes a container 1 for storing a water sample for collecting environmental DNA, and purified water W stored in the container 1. A container 1 for collecting environmental DNA has a tray-like shape opened upward, has a bottom part 4 and a side wall 5 rising from the bottom part 4, and includes a storage part 6 capable of storing purified water W.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for collecting environmental DNA, an environmental DNA collection kit, and a container for collecting environmental DNA. [Background technology]

[0002] Environmentally friendly construction work requires conservation measures, such as the development of alternative habitats for rare species. Visual identification of organisms requires the involvement of specialized investigators, but this poses challenges due to limited timeframes and the difficulty of securing and coordinating investigators. While it is possible to identify species based on environmental sound recordings or imagery, accurate identification is difficult because it requires specialized knowledge and advanced technology. For this reason, environmental DNA analysis, which obtains biological information from environmental water samples, has recently gained attention as a method for accurately identifying species. Environmental DNA is DNA (deoxyribonucleic acid) derived from organisms released into the environment (water) and is thought to originate from fragments, excrement, and other sources. Environmental DNA analysis is expected to be useful for estimating the types and abundance of organisms inhabiting aquatic areas. Conventionally, a technique for analyzing environmental DNA has been proposed, for example, in Patent Document 1, which discloses a method for pretreating environmental water samples. Patent Document 1 is characterized in that the environmental water sample is brought into contact with a disinfectant containing a quaternary ammonium salt as an active ingredient before being subjected to nucleic acid analysis. The environmental water sample pretreatment method disclosed in Patent Document 1 can suppress the decomposition of nucleic acids contained in the environmental water sample when estimating biota or the amount of specific organisms by environmental DNA analysis. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Laid-Open No. 2015-160175 Summary of the Invention [Problem to be solved by the invention]

[0004] However, the environmental water sample pretreatment method of Patent Document 1 involves the analysis of environmental DNA based on environmental water samples, which has the problem of making it difficult to collect environmental DNA from species that move around water or on land.Furthermore, the environmental water sample pretreatment method of Patent Document 1 requires pretreatment of the environmental water sample, which has the problem of making preparation for analysis time-consuming and cumbersome. The present invention aims to solve the above-mentioned problems and propose an environmental DNA collection method, an environmental DNA collection kit, and a container for environmental DNA collection that can easily collect environmental DNA from biological species that move around water while suppressing the degradation of the environmental DNA contained in collected water samples. [Means for solving the problem]

[0005] In order to solve the above problem, the environmental DNA collection method of the present invention is characterized by comprising an installation step of storing purified water and installing a container for collecting environmental DNA near water, and a collection step of leaving the container near water for a predetermined period of time and then collecting the purified water in the container as a water sample for environmental DNA analysis. In the environmental DNA collection method of the present invention, a container holding purified water is placed near a body of water, creating a portable simulated water area. By bringing organisms living in the water area into contact with the purified water, the environmental DNA of biological species dissolved or suspended in the purified water can be collected while suppressing its decomposition rate. This allows for the efficient collection of environmental DNA of biological species moving near the water.

[0006] In the setting step, it is preferable that purified water is stored in the container before or after placing the container near water. With this configuration, the timing for storing purified water in the container can be selected and the container can be placed near water, making it possible to place the container in a location that corresponds to the location where environmental DNA is to be collected.

[0007] In order to solve the above problem, the environmental DNA collection kit of the present invention is characterized by comprising a container for storing a water sample for collecting environmental DNA, and purified water stored in the container. This configuration makes it possible to conveniently collect environmental DNA from biological species that move around waterside areas without placing a burden on the collector.

[0008] To solve the above problems, the container for collecting environmental DNA of the present invention is a tray-shaped container that opens upward and is placed near water. The container for collecting environmental DNA has a bottom and side walls that rise from the bottom, and is characterized by including a reservoir that can store purified water and a roof that covers at least a portion of the reservoir from above. This configuration allows organisms living near water to easily come into contact with the purified water stored in the storage section, making it possible to effectively collect environmental DNA of biological species. Furthermore, the roof section effectively prevents raindrops, fallen leaves, and other contaminants from entering the storage section, allowing for more accurate collection of environmental DNA of biological species. Furthermore, the roof section creates a dark area in the storage section, providing a hiding place for organisms, making it easier for organisms to enter the storage section.

[0009] Preferably, an inclined surface extending toward the installation surface is formed continuously at the upper edge of the side wall. In this configuration, organisms can be effectively guided into the reservoir via the inclined surface, which is expected to enable the collection of environmental DNA from a greater number of biological species. [Effects of the Invention]

[0010] The environmental DNA collection method, environmental DNA collection kit, and container for collecting environmental DNA of the present invention can effectively collect environmental DNA from biological species that move around waterside areas while suppressing the degradation of environmental DNA contained in collected water samples. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a perspective view showing a state in which a container for collecting environmental DNA according to an embodiment is installed near water. [Figure 2] FIG. 2 is a longitudinal cross-sectional view taken along line II-II of FIG. [Figure 3] FIG. 3 is a longitudinal sectional view taken along line III-III in FIG. [Figure 4]FIG. 10 is a perspective view showing a first modified example of a container for collecting environmental DNA. [Figure 5] FIG. 5 is a vertical cross-sectional view taken along line VV of FIG. 4. [Figure 6] FIG. 10 is a perspective view showing the roof portion of a second modified example of a container for collecting environmental DNA. [Figure 7] FIG. 10 is a perspective view showing a third modified example of a container for collecting environmental DNA. [Figure 8] 10 is a table showing the results of environmental DNA analysis using a container and environmental water according to a comparative example. [Figure 9] 1 is a table showing the results of an analysis of environmental DNA using the container of this embodiment and purified water in an example. DETAILED DESCRIPTION OF THE INVENTION

[0012] The environmental DNA collection method, environmental DNA collection kit, and container for environmental DNA collection of the present invention will be described in detail below with reference to the accompanying drawings. In the embodiments, common parts are designated by common reference numerals, and redundant explanations will be omitted. In the following description, the terms "front / back," "left / right," and "up / down" refer to the directions shown in FIG. 1.

[0013] (Environmental DNA collection container) Fig. 1 is a perspective view showing a state in which a container for collecting environmental DNA according to an embodiment is installed near water, and Fig. 2 is a longitudinal sectional view taken along line II-II in Fig. 1. As shown in Figures 1 to 3, the container 1 for collecting environmental DNA stores a water sample for collecting environmental DNA. The container 1 has a tray-like body 2 that opens upward, and a roof 3. The body 2 and roof 3 are made of a lightweight plastic material that is easy to transport. Plastic materials generally have low thermal conductivity and do not easily store heat, making them suitable for the container 1 for collecting environmental DNA. Examples of plastic materials that can be used include polypropylene, polyethylene, and polyethylene. Note that at least one or a portion of the body 2 and roof 3 may be made of stainless steel. Furthermore, since environmental DNA analysis requires cleaning using hypochlorite or the like, it is preferable that the container 1 be made of a material that can withstand this cleaning.

[0014] The main body 2 has a rectangular bottom 4, side walls 5 rising from the front, rear, left, and right edges of the bottom 4, and a storage section 6 defined by the bottom 4 and each side wall 5. Purified water W is stored in the storage section 6. The storage section 6 (main body 2) may have any shape as long as it is structured to store purified water W, and may be of various shapes as long as it allows living organisms to come into contact with the purified water W. The size of the main body 2 is not particularly limited, but it is preferable that the size be such that it is easy to transport and install. The height of each side wall 5 is preferably set to about 5 cm.

[0015] A continuous inclined surface 7 is formed on the upper edge of each of the front, left, and right side walls 5. Each inclined surface 7 extends toward the installation surface S on the water's edge in front, left, and right of the main body 2. Each inclined surface 7 functions as a guideway to guide living organisms into the reservoir 6.

[0016] The roof 3 covers the rear of the reservoir 6 (main body 2) from four directions: above, from the left and right sides, and from the rear. The roof 3 has left and right walls 3a, a rear wall 3b, and a top wall 3c. The top wall 3c is formed continuously with the upper edges of the left and right walls 3a and the upper edge of the rear wall 3b. Although the roof portion 3 is shown as covering the rear portion of the storage portion 6 (main body portion 2), it is not limited to this and may cover the entire storage portion 6 (main body portion 2), the front portion of the storage portion 6 (main body portion 2), or the central portion of the storage portion 6 (main body portion 2) in the front-to-back direction.

[0017] The roof 3 may be fixed to the installation location using fixing members such as pegs (not shown), or may be weighted. By installing the roof 3 in this manner, it is possible to prevent the roof 3 from shifting position or tipping over due to wind, etc., and it can be left stationary at the installation location for a certain period of time. The container 1 may also be fixed to the installation location using fixing members (not shown), or may be weighted. Furthermore, the height of the roof portion 3 is preferably set to, for example, about 15 cm or lower, taking into consideration the sunshade effect of the roof portion 3 and the effect of the roof portion 3 being recognized as a hiding place by living creatures. Furthermore, the roof portion 3 may be given the same color and pattern as the rocks, trees, etc. at the survey site (installation location).

[0018] Purified water W is highly pure water from which impurities have been removed, and is water that does not contain impurities such as chemicals (such as chlorine) that degrade environmental DNA or microorganisms. Examples of purified water W include distilled water, reverse osmosis water, and ion-exchanged water. As an example of water quality analysis of purified water W, water of A4 level according to JIS K0557 classification, which has high purity and is suitable for precise analysis, is preferred. On the other hand, microorganisms (bacteria) that break down environmental DNA (producing the decomposing enzyme deoxyribonuclease) are ubiquitous in environmental waters near water bodies. Environmental waters that contain a lot of organic matter and turbidity generally have a large number of microorganisms, which makes it easier for decomposing enzymes to be released into the environment, accelerating the decomposition of environmental DNA.

[0019] In contrast, purified water W is highly pure water from which impurities have been removed as described above, and contains almost no enzymes that degrade organic substances or environmental DNA, making it chemically more stable than environmental water. Due to these factors, the rate at which environmental DNA decomposes is slower in purified water W than in environmental water, and even small amounts of environmental DNA resulting from brief contact are likely to leave traces. This makes it possible to collect environmental DNA that is difficult to decompose when organisms come into contact with purified water W. In this embodiment, the depth of the purified water W in the reservoir 6 is set to about 3 to 5 cm from the bottom 4, taking into consideration the ease of entry and exit of amphibians.

[0020] (Environmental DNA collection method) The method for collecting environmental DNA includes an installation step and a collection step. The installation step is a step of installing the container 1 near water. The collection step is a step of leaving the container 1 near water for a predetermined period of time, and then collecting environmental DNA from the purified water W in the container 1 as a water sample for environmental DNA analysis. In the installation process, the container 1 is washed and sterilized in advance using hypochlorite or the like. In the installation process, purified water W is stored in the container 1 before or after placing the container 1 near water. It is preferable that the purified water W be placed in a water container (not shown), such as a sealable bottle, before transporting it to the survey site, and then pouring a predetermined amount into the container 1 at the installation site. It is preferable that the sealable bottle have a wide mouth to make it easier to pour water, etc.

[0021] Here, "waterside" refers to the surrounding area of ​​a place where water meets land, such as a pond, marsh, wetland, river, lake, irrigation channel, reservoir, waterway, etc. Possible locations for installing the container 1 include an adult moving on land, or any location desired for observation (for example, a location away from the place where water meets land, etc.).

[0022] In the collection process, a predetermined amount of purified water W from the reservoir 6 is collected into a collection container, such as a sealable bottle. During collection, care must be taken to prevent contaminants from entering the collection container. It is preferable that the sealable bottle have a wide opening for ease of collection. The collected purified water W can be transported to a laboratory in the collection container and filtered, or it can be filtered at the survey site. When transporting the collection container to a laboratory, it is preferable that the container be stored in an environment with minimal temperature change, such as a refrigerated or insulated box. In the collection process, DNA is extracted from the residue collected on the filtered filter paper and the biological species is identified, similar to known environmental DNA analysis. In the above collection process, an example is shown in which the purified water W in the storage section 6 is collected into a collection container, but this is not limited to this, and the installed container 1 may be sealed as a storage container and transported to a laboratory.

[0023] (Environmental DNA collection kit) The environmental DNA collection kit includes a container 1 for collecting environmental DNA and purified water W stored in the container 1. In other words, because the environmental DNA collection kit includes the container 1 and purified water W, by using this kit, it is possible to set up the container 1 and collect the DNA in accordance with the collection method, ensuring consistency and reproducibility of the results and increasing the reliability of the collection. Furthermore, the environmental DNA collection kit eliminates the need to prepare the container 1 and purified water W individually, simplifies the setup process, and also saves time and costs.

[0024] According to the environmental DNA collection method, environmental DNA collection kit, and environmental DNA collection container 1 of this embodiment described above, a portable simulated water area can be created near water by placing the container 1 containing purified water W near water. By contacting organisms living near water with the purified water W, the environmental DNA of biological species dissolved or suspended in the purified water W can be collected while suppressing the rate of decomposition. In other words, biological species can be identified from genetic information derived from biological tissue fragments (such as the organism's skin or excrement) with minimal labor at the survey site. It is also possible to collect environmental DNA from juveniles and nocturnal species, which are difficult to identify visually.

[0025] Furthermore, in the installation process of the environmental DNA collection method, the timing for storing purified water W in container 1 can be selected and container 1 can be installed near water, making it possible to install container 1 in accordance with the location where environmental DNA is to be collected.

[0026] Furthermore, since the environmental DNA collection kit includes the container 1 and purified water W, environmental DNA from biological species that move around waterside areas can be suitably collected without placing a burden on the collector.

[0027] Furthermore, the container 1 for collecting environmental DNA has a roof 3, which can effectively prevent raindrops, fallen leaves, etc. from entering the storage section 6, allowing for more accurate collection of environmental DNA of biological species. The roof 3 also creates a dark area in the storage section 6, forming a hiding place where organisms can hide, making it easier for organisms to enter the storage section 6. Furthermore, the container 1 has inclined surfaces 7 on the front side wall 5, left side wall 5, and right side wall 5, so that even organisms that cannot jump or are small can easily climb over the side walls 5, and the organisms can be suitably guided into the storage section 6 via the various inclined surfaces 7. This makes it possible to expect the collection of environmental DNA from a greater number of biological species.

[0028] (First modified example of container for collecting environmental DNA) Fig. 4 is a perspective view showing a first modified example of a container for collecting environmental DNA, and Fig. 5 is a longitudinal cross-sectional view of the main body 2. The container 1A of the first modified example is based on the container 1 of the first embodiment, with the addition of a lure 8a suspended by a support rod 8. As shown in Figure 4, the support rod 8 is fixed to the rear wall 3b of the roof 3 and has a generally L-shape. The artificial lure 8a is hung from the tip of the support rod 8 so that it is immersed in the purified water W in the reservoir 6. The artificial lure 8a is preferably made of tape, yarn, or the like, and is preferably brightly colored, such as pink or orange, so that it stands out to frogs and other insects. It may also be made to reflect light, or painted with fluorescent paint to make it stand out at night.

[0029] 5, the front side wall 5 of the main body 2 has an inner surface 5a that is an inclined surface that extends rearward toward the bottom 4. The inner surface 5a is continuous with the inclined surface 7 of the front side wall 5 and functions as a guide path that guides living organisms to the reservoir 6.

[0030] The environmental DNA collection container 1A of the first modified example also provides the same effects as the above-described container 1 (see FIG. 1, etc.). In addition, since it is equipped with artificial bait 8a suspended from support rod 8, it can induce predatory behavior of organisms living near water, and efficiently attract the organisms to storage section 6. Furthermore, since the inner surface 5a of the front side wall 5 is an inclined surface, the living creatures can be attracted to the reservoir 6 without feeling uncomfortable.

[0031] (Second modified example of the container for collecting environmental DNA) 6 is a perspective view showing a roof portion of a container for collecting environmental DNA according to a second modified example. Roof portion 3A according to the second modified example is provided with an eave portion 9 extending forward. The eaves portion 9 is formed of a transparent resin material such as an acrylic plate. The eaves portion 9 has left and right side portions 9a, 9a extending forward from the left and right wall portions 3a, 3a, and an upper portion 9c extending forward from the top wall portion 3c. The left and right side portions 9a, 9a and the upper portion 9c are integrally connected. Note that the eaves portion 9 is not limited to being transparent and may be colored.

[0032] A plurality of ventilation holes 3e are formed in the left and right wall portions 3a, 3a of the roof portion 3A. The number of ventilation holes 3e is not particularly limited. Note that a plurality of ventilation holes 3e of different sizes may be formed. Providing a plurality of ventilation holes 3e enables effective ventilation within the roof portion 3A, and allows for suitable temperature and humidity control within the roof portion 3A, adapting to the surrounding environment.

[0033] The environmental DNA collection container 1A of the second modified example also provides the same effects as the container 1 described above (see FIG. 1, etc.). In addition, the roof 3A has a transparent eaves 9, which effectively prevents raindrops, fallen leaves, etc. from entering the storage compartment while allowing natural light to effectively enter. Also, organisms that have entered the storage compartment 6 can be visually confirmed through the eaves 9.

[0034] (Third variant of the container for collecting environmental DNA) 7 is a perspective view showing a third modified example of a container for collecting environmental DNA. The roof 3B of the container 1B according to the third modified example has a mesh structure in which a net 3g is stretched over a roof-shaped frame 3f. The frame is made of lightweight and durable plastic or metal materials. The net 3g forms the left and right walls 3a, 3a, rear wall 3b, and top wall 3c of the roof 3B. The net 3g is made of a durable material such as polyester. The mesh size of the net 3g is not limited to a specific size, but it is preferable that it does not obstruct air flow and is relatively strong enough to withstand repeated use. It is also preferable that the net is properly tensioned.

[0035] In the environmental DNA collection container 1B of the third modification, the left and right walls 3a, 3a, rear wall 3b, and top wall 3c of the roof 3B are formed by a net 3g, which provides the advantages of ensuring natural light and ventilation, being lightweight, and being easy to transport, while effectively preventing fallen leaves and the like from entering the storage section 6. Another advantage is that organisms that have entered the storage section 6 can be easily visually confirmed. [Example]

[0036] Next, we will explain examples and comparative examples of the present invention that verify the effects of the environmental DNA collection method, environmental DNA collection kit, and container for environmental DNA collection of this embodiment. Figure 8 is a table showing the results of environmental DNA analysis using a container and environmental water according to a comparative example. Figure 9 is a table showing the results of environmental DNA analysis using the container of this embodiment and purified water according to an example. In the comparative example, water collected from a water body around one point in the desired research area was prepared as environmental water. Meanwhile, in this example, distilled water produced by a distilled water production device (manufactured by Wakenyaku Co., Ltd., model FRD343NC) was prepared as purified water W. This distilled water has A4 level water quality in accordance with JIS K0557.

[0037] (Comparative Example) A container (not shown) containing 2 liters of environmental water was placed at one point in the desired survey area. This container was tray-shaped, with a rectangular bottom and side walls rising from each edge of the bottom. Note that each side wall did not have the inclined surface 7 (see Figure 1, etc.) described in this embodiment, but was formed in a plate shape. The containers were set up at the study site for three days. Visual observation of organisms around the study site was conducted between 1:00 PM and 2:00 PM on the third day. After setting up, environmental water was collected twice a day at different sampling times (see (a) to (f) in Figure 8), and a predetermined amount of environmental water was collected from the containers into sealable bottles for transport. After collection, the sealable bottles for transport were refrigerated and transported to the laboratory. The day after transport to the laboratory, the collected purified water W was passed through filter paper, and the environmental DNA was concentrated on the filter paper and stored frozen. The filter paper was prepared so that it could capture even small DNA fragments. During analysis, environmental DNA was extracted from the filtration residue collected on the filter paper, and the extracted environmental DNA was confirmed using a next-generation sequencer or other device.

[0038] (Examples 1 and 2) In Example 1, a container 1 (see FIG. 1) containing 2 liters of purified water W was installed at one point in a desired survey area. On the other hand, in Example 2, a container 1A (see FIG. 2) containing 2 liters of purified water W was similarly installed. In Examples 1 and 2, fixed cameras were installed near the installation positions of the containers 1 and 1A. In Examples 1 and 2, after 48 hours had passed since installation, a predetermined amount of purified water W was collected from the reservoir 6 of the container 1, 1A into a sealable bottle for transportation. In Examples 1 and 2, as in the Comparative Example, after collection, the sealable bottles for transportation were transported to a laboratory under refrigeration. Then, on the day after transport to the laboratory, the collected purified water W was passed through filter paper, and the environmental DNA was concentrated on the filter paper and stored frozen. Filter paper capable of capturing even small DNA fragments was prepared. Then, during analysis, environmental DNA was extracted from the filtration residue collected on each filter paper, and the extracted environmental DNA was confirmed using a next-generation sequencer or the like.

[0039] (Evaluation Results of Examples 1 and 2 and Comparative Example) In the comparative example, visual observation confirmed the presence of Rana rugosa, Rhacophorus arboreus, Salamander nigricans, Japanese newt, and Japanese bullfrog. In the comparative example, the biological species identified by visual observation were generally confirmed by environmental DNA analysis, but the Japanese black salamander was not detected in the environmental DNA analysis. Furthermore, even on the same day, differences in the time of collection resulted in differences in whether or not environmental DNA was detected for biological species related to the Japanese brown frog, the Tokyo Daruma frog, and the Japanese black salamander.

[0040] In contrast, in Examples 1 and 2, even though only one collection was performed, the environmental DNA obtained in the analysis results of the environmental DNA of the comparative example was detected except for the Tokyo Daruma frog. Furthermore, photographs taken with a fixed camera verified the high possibility that amphibians had been present. Furthermore, because the purified water W (distilled water) stored in containers 1 and 1A is not easily decomposed, it was verified that the environmental DNA of a wide range of biological species could be detected. Furthermore, analysis using primers for amphibians and anurans showed no difference between the presence and absence of lure 8a. On the other hand, analysis of environmental DNA targeting urodeles indicated that lure 8a may have an attractant effect on organisms.

[0041] The above describes the embodiments and examples of the present invention, but the present invention is not limited to the above embodiments and examples, and each of the above components can be modified as appropriate within the scope of the present invention. For example, in the containers 1, 1A for collecting environmental DNA, the inclined surface 7 of the main body 2 does not necessarily have to be provided. Alternatively, the inclined surface 7 may be provided only on the front side wall 5. Alternatively, the inclined surface 7 may be provided on the rear side wall 5. In this case, it is preferable to remove the rear wall 3b of the roof 3, 3A, 3B to form an opening and attract organisms through the rear side wall 5.

[0042] Furthermore, the roof sections 3, 3A may be partially formed from a net material. The roof sections 3, 3A, 3B may be configured to be separable into front and rear sections, or may be configured to be stretchable in the front-to-rear direction. The roof sections 3, 3A, 3B may also be configured so that each section is foldable so that they can be folded flat. [Explanation of symbols]

[0043] 1, 1A, 1B container 3, 3A, 3B Roof section 4 Bottom 5 side wall 6 Reservoir 7 Slope W Purified water

Claims

1. an installation step of storing purified water and installing a container for collecting environmental DNA near water; A method for collecting environmental DNA, comprising a collection step of leaving the container near water for a predetermined period of time and then collecting the purified water in the container as a water sample for environmental DNA analysis.

2. The environmental DNA collection method according to claim 1, characterized in that in the installation step, purified water is stored in the container before or after the container is placed near water.

3. An environmental DNA collection kit for collecting environmental DNA, comprising: a container for storing a water sample for collecting environmental DNA; and purified water stored in the container.

4. A container for collecting environmental DNA, which has a tray-like shape with an upward opening and is installed near water, a reservoir having a bottom and a side wall rising from the bottom and capable of storing purified water; A container for collecting environmental DNA, characterized in that it is equipped with a roof portion that covers at least a portion of the storage portion from above.

5. 5. The container for collecting environmental DNA according to claim 4, wherein an inclined surface extending toward the installation surface is continuously formed on the upper edge of the side wall.

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