Note applicative

Let's catch tardigrades!

Tardigrades live in all kinds of environments, and more than 1,400 species have been discovered. Tardigrades are aquatic animals, and terrestrial tardigrades commonly live in pore water in the soil or in water trapped in moss. Certain species of tardigrade are often found in drought-tolerant moss Bryum argenteum which can be found in sunny areas and exhibits recognizable white tips (Fig. 1). This particular moss is a good habitat because many rivals and predators live in other types of moss with higher moisture content. To find tardigrades, first collect the Bryum argenteum and moisten it by dripping water on it. Leave it for several tens of minutes to several hours. Aspirate water several times with a dropper and drip onto a glass slide or dish. A stereo microscope is recommended because it allows you to explore moving tardigrades in a wide field of view, regardless of the container, and allows you to observe them with a 3D perspective. If using an upright microscope, place the drop onto a glass slide and slowly cover it with a cover glass from one side. When using an inverted microscope, apply the drop onto a thin dish with a bottom glass thickness of 170 μm. In addition, if you fill an agar medium with mineral water and supply live chlorella for medaka and daphnia food dissolved in water, you can keep it for a while at room temperature (22-25℃) (the lifespan of tardigrades is 1 to several months). However, there are also carnivorous species (eating rotifers, nematodes, etc.).

Let's observe the details!

Next, the details were observed using Macrobiotus shonaicus (Tardigrada: Macrobiotidae), kindly provided by Dr. Kazuharu Arakawa (Institute for Advanced Biosciences, Keio University). This species was described in 2018 by Professor Arakawa and his collaborators in Yamagata Prefecture, Japan. The eyespots appear larger (Fig. 3, 4, 5) than the species collected in Kanagawa Prefecture, Japan (Fig. 2b, c).
It also has four distinctive pairs of limbs, each with four claws (Fig. 3a). Of the four pairs of limbs, only the last pair has claws facing backwards, while the rest have claws facing forward (Fig. 3a). The way the claws grow differs greatly between the Macrobiotidae and the Echiniscidae (Fig. 3b, c, d). The length and shape of these claws are one of the key points to distinguish tardigrade species.

Next, the mouth was observed. It looks like a bow and arrow, with a tube extending from its small, round mouth and slightly arched tubes on both sides of the tube (Fig. 4). The round pharynx is located further back (Fig. 4, bottom middle). Tardigrades extend toothed needles from their mouths, trap food, and use their pharynx to retract it up and grind it for subsequent digestion. The shape of this pharynx is also one of the characteristics used to distinguish tardigrade species.

The digestive system continues from the pharynx to the esophagus to the midgut, and food accumulated in the midgut can be seen through the transparent body (Fig. 5a). Surprisingly, it is excreted in one lump that is nearly 1/3 the size of the body (Fig. 5a).

Interestingly, this species exhibits internal fertilization prior to shedding the eggs for hatching. As the eggs grow larger, they occupy almost half of the body (black arrows in Fig. 5b).

Tardigrade’s life cycle

Since tardigrades were captured and imaged in various states, the life cycle of tardigrades was visually summarized. There are species of tardigrades that reproduce parthenogenetically and those that reproduce sexually. There are also species that lay eggs directly into the external environment and those that lay eggs within their own outer shells. Macrobiotus shonaicus observed in this note reproduces sexually and lays eggs inside its outer shell (Fig. 6). It is also possible to observe the eggs growing larger within the ovary (Fig. 6). The newborn tardigrade then repeatedly molts its skin and grows larger (Fig. 6).