Biodiversity at gene level; DNA sampling for water quality monitoring in Kanagawa Prefecture, 1

 

Since last year, I listed myself as a citizen monitor for the water quality of Kanagawa‘s rivers. They supply tap water to us living in Kanagawa (; my 5 consecutive posts from October 21 to November18, and one for 30 December). Our main approach for the task is good-ol‘ nature observation, sometimes with lab study with stereo microscopes. The method has its history starting 2007. It has accumulated data for biodiversity of the river environment and its surrounding forests and towns. Of course, it has limitation. Aside from familiar man-power or the like problems, there is inevitable difficulty to check wild habitat by humans. Not every point of earth is easy for homo sapience to go near, even if it‘s with simple landing nets and vats.

What‘s there?

Say, you walk in Tanzawa mountains and try to collect specimens of aquatic creatures for water monitoring. You encounter an almost vertical fall of stream. It has large and extremly slippery stone wall. The wall looks suspiciously rich for water lifes. They could cling to moss-eaten rock 5m high above from our narrow and precarious trecking route. CAUTION: don‘t try to collect specimen from such dangerous part of water flow. Or, below your feet on a simple mountain bridge, some 20m below, there is a waterfall basin. With binoculars we recognize fishes down there. But fishing from narrow and unstable mountain crossing cannot guarantee you a catch … CAUTION: don’t try to collect specimen from such dangerous part of water flow. You got the point, don’t you? Don’t worry. There is a possible solution for studying ecosystem of this kind of environment, thanks to the 21st century technology. It’s DNA sampling.

DNA sampling demonstration

Originally, the method of using DNA for cataloguing water creatures was started to study ocean. Well, no one expects humans to study comprehensively the entire sea of this planet. On the other hand, all the living things shed and spread the remnants of gene within the environment. Majority of us wash away our dirt peeled from the body in regular shower, or using toilet, don‘t we? It means our body accumulates and discards old body parts made by gene everytime. (er ... it sounds ... yack. But let‘s face the reality!) Same is happening for the water dwellers. Living or dead, they continuously leave the traces of their gene, aka DNA, in water they live. Scientists noticed this, and develped the way to collect DNA from a bucket of sea and the other natural waters. They feed the DNA data from water to powerful computer and analyze the result. Starting this fiscal year, Kanagawa Prefecture introduced this way of monitoring water quality by citizen monitors ... For more detail of the project scheme, I tell you later. This post and next are for my adventure with actual DNA collecting in Yadoriki Water Source Forest やどりき水源林.

Pacific Ocean!

Kanagawa‘s way to collect DNA from rivers has two approaches. One is using syringes. Another is literly collecting a bucket of water. For both approaches once the sample is taken, they must be at least cilled or frozen. Monitors must rush to a courier’s office to send it to the Prefecture’s lab by express. I realized, yeah DNA is ubiquitous in natural water but they are very fragile. DNA is broken apart quickly unless treated carefully. That’s the reason why there is no “DNA pollution” of river, lake, pond, sea, … Come to think of it, it would be why the ecosystem is happy to accept “natural” garbage and circulate everything in a sustainable way ... Anyway, let me show you what kind of thing we’ve done this fiscal year for DNA sampling. Let’s start with the syringe method. Here is a kit I received from the lab of the Prefecture.

The first thing we monitors must do is to wear gloves before collecting water. In the same token, we’re to stand downstream, when we collect water in a stream. It’s to avoid contamination. Researchers don’t need human DNA in the sample, you know.

Wearing rubber gloves

Or, standing downstream.

Next, a usage of bucket. Unlike oceanic study, we don’t need a large bucket to collect water. So, a plastic cup in a size of coffee mag is enough. I attached a rope to it in order to collect water from a point where we cannot have direct access, like a waterfall basin. Throwing the “mag” and pulling the string toward us would do the job. Also, we have to sanitize it before usage with dishwashing liquid, dry it under sunshine completely, then rinse the cup just before collecting sample with the same natural water from which we collect water.

A washed cup with a rope attached.

With it, we can collect water from a point like this, or

From a waterfall basin like this.

For syringe method, 50ml of collected water is sucked up into a syringe from a cup and discard the remaining water downstream. At the tip of the tool, we attach a Sterivex that is a filter to catch DNA. We push water from a syringe through the filter. Once 50ml is filtered, we repeat the same process until we draw1000m through the filter. But it is often the case we cannot filtrate 1000ml as the pump of syringe reaches to the point where it cannot go any further. Inorganic material like dust? Some other things? Very large DNA? We stop the process at this point, and record how much water we filtered for the Sterivex.

A syringe with Sterivex.
The amount of water is for one cycle.

Filtering ...

Once the filtering reaches to the max, we pump it for the last with air and squeez any remaining water inside the Sterivex. Mr. Hasebe of Prefecture told us, “This is VERY IMPORTANT process to preserve DNA!“ Then, remove the Sterivex from the syringe and seal the tip with a rubber cap securely.

We then pour 2mL of RNAlater in the Sterivex with a sterilized dropper. It‘s to suppress the break-down of DNA. Once all the RNAlater is added, close the end of Sterivex tightly, like this.

The next step must be taken quickly. We enclose the DNA sample filter in a cooler bag with frozen ice packs, and send it in prompt as a “frozen matter” from the nearest courier office. If the preservation of DNA goes OK with RNAlater and frozen packs, the DNA can reach the freezer of the Prefecture office in no time and the sample can be kept for a long period as long as the freezer works OK. Some people said the process is very tedious, but I found it very interesting. Actually the most difficult part of the task was to reach to the courier office smoothly (because traffic jam, etc.) and to send the sample as “water filter,” not a biological matter … sending biological or medical thing as courier is EXTREMELY difficult in Japan, of course ... I think I don’t tell a lie at the courier … It’s filtered water, to be sure. Phew! Next week, I tell you the “bucket method,” and more about the adventure of dipping up water in Yadoriki Water Source Forest for DNA. Please stay tuned!

If you find environmental issues in waters of Kanagawa Prefecture, please make a contact with Kanagawa Environmental Research Center 神奈川県環境科学センター

1-3-39 Shinomiya, Hiratsuka City, 254-0014
〒254-0014平塚市四之宮1-3-39
Phone: 0463-24-3311
FAX: 0463-24-3300

k-center@k-erc.pref.kanagawa.jp

https://www.pref.kanagawa.jp/docs/b4f/index.html