0044-5231/01/240/03–04-341 $ 15.00/0

How to Crawl and Dehydrate on Moss*

Hartmut GREVEN and Lutz SCHÜTTLER

Institut für Zoomorphologie und Zellbiologie der Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany

Abstract. Tardigrades (Macrobiotus sp., Echiniscus testudo) were videotaped when moving on single plants ofmosses with different water retaining capacities. On a fully hydrated Encalyptra contorta plant encased in a con-tinuous water column, tardigrades could hardly crawl rapidly in a specific direction. This was probably due to theirlow specific weight and the poor ability of their claws to cling to the substrate. As tardigrades were not able towalk on the undersurface of the leaves under these conditions, they were rinsed out of a fully soaked moss cushionin the course of time. Fully hydrated Polytrichum formosum plants did not show a visible water film and locomo-tion of tardigrades could not be observed on this moss. Activity of animals appeared to be confined to the dropletsin the leaf axils. During dehydration, the tardigrades stopped moving and formed a tun on the spot.

Key words. Tardigrada, locomotion, water retaining-capacity, secondary habitat.

1. INTRODUCTION

Direct observation of tardigrades in mosses and soil isdifficult to accomplish. Therefore previous tardi-gradologists gained data on locomotion, tun formation,etc., under artificial or at best seminatural conditions(for review see MARCUS 1929). More recent authorsseem to have abstained totally from such unsatisfacto-ry investigations. Recently we reviewed studies on thelocomotion pattern of tardigrades confirming andbroadening previous observations (SCHÜTTLER &GREVEN 2000/2001). In addition, we watched speci-mens of Echiniscus testudo Doyère, 1840 (Hetero-tardigrada) and Macrobiotus sp., partly by videotech-niques, when crawling and dehydrating on singleplants of the mosses Polytrichum formosum Hedw.(known as not being very suitable for tardigrades, butpopulated occasionally) and Encalyptra contortaHedw. (densely populated by the mentioned species).

2. MATERIALAND METHODS

E. testudo and Macrobiotus sp. were collected from E. contor-ta. Single plants and leaflets of this and the moss P. formosum

were isolated from cushions and soaked for at least 1 h in tapwater to become fully hydrated. At least five tardigrades ofeach species were placed singly on the leaves with a pipetteand videotaped. For videography we used a conventional digi-tal camcorder (Sony Digital Handycam DCR-VX 1000E) witha zoom (×20, f = 59 mm), a digital videorecorder (Sony DHR1000VC), and a monitor (Sony KV 25×50). For the PC-aidedevaluation of videosequences see BRENNER & GREVEN (1999)and SCHÜTTLER & GREVEN (2000/2001). Hydrated and dehy-drating individual moss plants were videotaped in an uprightposition. For P. formosum fixed on plastic material we used thezoom; for E. contorta we adapted the camcorder to a binoculartilted at 90°. For scanning electron microscopy single leaveswere fixed in 2.5% glutaraldehyde in 0.2 mol/l cacodylatebuffer, pH 7.4, postfixed in 1% osmiumtetroxide and critical-point-dried. Moss plants and leaves with tardigrades were air-dried. Both preparations were sputter-coated with gold andviewed in a Leitz AMR 1000 scanning electron microscope.

3. OBSERVATIONS AND DISCUSSION

When walking on a hydrated moss leaf, both speciessporadically showed the locomotion pattern of the firstthree pairs of legs known from the marine tardigradespecies Batillipes mirus Richters, 1909 (see MARCUS

1929). In E. testudo the fourth pair pushed the body for-ward by alternating movements independent of themovements of the first three pairs. In Macrobiotus sp.the fourth legs were drawn together under the body,

Zool. Anz. 240 (2001): 341–344© by Urban & Fischer Verlaghttp://www.urbanfischer.de/journals/zoolanz

* Contribution to the 8th International Symposium onTardigrada, Copenhagen, Denmark, 30 July–5 August 2000.

A very thin water film on a moss leaf or filter paper ofapproximately the thickness of the animal presses thetardigrade against the substrate allowing locomotion.Due to capillary forces it may be an energy consumingmode of walking. It was not possible to estimate thethickness of these thin water films in our preparations.In a thicker water film or single droplets, however,specimens had problems remaining attached to the sub-strate. This was obviously due to their low specificweight (approximately 1.04; see JENNINGS 1975) andthe ineffectiveness of the claws in holding on. (Forspeculation on holding-on capacity of different claw-types of Tardigrada, see MARCUS 1929). Therefore,tardigrades very often lost hold, particularly whencrawling on a non horizontal substrate. When crawlingbeyond the edge of a leaflet of a fully hydrated mossplant (type E. contorta, see below), tardigrades reachedthe water film bridging the space to the next leafletwhere they felt down onto this leaf, after a while ontothe next and so on (Fig. 1). The longer the moss wasfully hydrated, the more tardigrades were washed away.

342 H. GREVEN and L. SCHÜTTLER

Fig. 1. Echiniscus testudo Doyère in a drop of water under asingle moss leaf. The animal has lost contact with the under-surface, moving freely in the droplet (I). When crawlingbeyond the edge of the leaf, the animal reaches the water underthe leaflet (II), falls down onto the next leaflet and so on (III).

Fig. 2–7. 2. Fully hydrated Encalyptra contorta plant covered with a continuous prominent water film (arrow). (Edited videopicture). Bar 0.5 mm. 3. Structured upper surface of an E. contorta leaflet. Bar 25 µm. 4. Fully hydrated Polytrichum formo-sum plant. Note absence of a visible water film. Bar 1 mm. 5. Upper surface of a P. formosum leaf with longitudinally arrangedassimilation lamellae. Bar 10 µm. 6. Tun of Macrobiotus sp. in a wrinkled leaf of E. contorta. Bar 50 µm. 7. Tun of Macrobio-tus sp. on the undersurface of a rolled up leaf of P. formosum. Bar 100 µm.

pushing the animal away from the substrate, or weredragged along passively. During climbing, animals usedthe fourth legs as a clasping organ holding onto the sub-strate when the other legs tried to reach a new leaf(MARCUS 1929; SCHÜTTLER & GREVEN 2000/ 2001).

Mosses have been differentiated into three typesdepending on their ability to hold water (OVERGAARD

1948). E. contorta and P. formosum each represent aseparate type. Fully hydrated E. contorta plants arecovered by a continuous water film (Fig. 2) and have arelatively high water-retaining capacity. The surface ofits leaves is well structured (Fig. 3), the undersurface isweakly structured. Leaves were arranged around thestem tristichously. After 35 min dehydration (25% rel-ative humidity, 21 °C) some droplets still were seen inthe leaf axils. Between the 45th and 50th minute, curl-ing of the leaves against the stem was completed. Thedecreasing water film and the resulting optical distor-tion as well as curling of the leaves allowed no furtherobservations. During dehydration leaves bend alongthe midrib. Tuns of tardigrades were found often nearthe midrib in the folded leaf (Fig. 6).Fully hydrated P. formosum plants were not covered bya conspicuous water film (Fig. 4). Water added to suchplants ran off, leaving a drop in the leaf axil at best andbetween leaves at the base where they were more closetogether. A water film was not seen and tardigrades didnot appear to move along the stem or on the relativelyhard leaves under these conditions. Their activityseemed to be restricted to the droplets, i.e. in leaf axils.Leaves of P. formosum are conspicuously structured ontheir upper surface showing longitudinally arrangedassimilation lamellae whose interspaces contain capil-lary water (Fig. 4; for ecto- and endohydric structuresin mosses that conduct and store water, see LÖSCH

2001). Margins of the leaves possess small “denticles”(Fig. 7). During dehydration (31% relative humidity,21 °C) leaflets curled against the stem beginning at thetop of the plant. Axil droplets evaporated before curl-ing. Curling was finished at the base of the plant afterabout 2 h. Dehydrated leaves rolled up forming tubesthat had a small opening along their middle line. Tunsof tardigrades were found on the upper and the undersurface, but seldom in the tube (Fig. 7).Dehydration of tardigrades was observed on singleleaves. Findings, however, are not conclusive as yetand confirmed essentially previous descriptions (sum-marised by MARCUS 1929). During the dehydrationprocess, we had the impression that the animals hadalready stopped walking (not moving) in a water filmstill suitable for crawling. There was no evidence thatanimals searched selectively for moist places whendehydration started, i.e. in the leaf axils (E. contorta) orin “tubes” produced by rolled up leaflets (P. formosum).

4. CONCLUDING REMARKS

On the basis of our observations which have to sub-stantiated in further studies, we assume:

– Moss plants that are characterised by pliableleaflets, a continuous “water column” when fullyhydrated (see OVERGAARD 1948), and a high water-retaining capacity such as Encalyptra contorta appearnot to be an ideal substrate for unhindered locomotionof tardigrades when totally saturated. Nevertheless,such mosses are often densely populated indicatingthat tardigrades benefit in other way from these mosses(see below). A surplus of water, i.e. after significantrainfall, may enhance passively subsiding into deeperregions of a moss. This disadvantage may be mitigatedat least partly by the interweaving of single plants in acushion.– Moss plants that have rather rigid and hard leaflets,no visible water film and only water droplets in theaxils when fully hydrated (see OVERGAARD 1948), apoor water-retaining capacity and that are often notdensely interwoven such as Polytrichum formosum arein principle suitable at least for some tardigradespecies (see the discussion in MARCUS 1929;GRABOWSKI 1995). However, they may give tardi-grades only a restricted living space. – Colonisation of mosses by tardigrades depends notonly on a variety of abiotic factors (i.e. MARCUS 1929;DASTYCH 1988; WRIGHT 1991; GRABOWSKI 1995), butalso on the availability of food. Hard-leaved mosses asP. formosum may hardly be punctured by the stylets oftardigrades as suggested by MARCUS (1929) and oth-ers. However, as the content of epidermal moss cells issurely not the principal item of tardigrade food, futureattention should be directed to quantitative analyses ofthe organisms associated with tardigrades in differentmosses and their suitability as food for these animals.– Assignment of tardigrade species to certain layers ina moss cushion (A, B, C) (see HALLAS 1978) and tocertain ecological factors such as exposure to insola-tion, water-retaining capacity and desiccation (i.e.GRABOWSKI 1995) may be influenced by the abovementioned phenomenon. – An optimal thickness of water films covering mossplants is postulated to make energy sparing and effec-tive locomotion of tardigrades possible. However, itsreal thickness and duration are unknown as yet.

REFERENCES

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DASTYCH, H. (1988): The Tardigrada of Poland. MonografieFauny Polski 16: 1–255.

GRABOWSKI, B. (1995): Ökologische Untersuchungen anmoosbewohnenden Bärtierchen (Tardigrada) mit einemBericht über drei neue Arten für Deutschland. Acta Biol.Benrodis 7: 77–98.

How to Crawl and Dehydrate on Moss 343

WRIGHT, J.C. (1991): The significance of four xeric parame-ters in the ecology of terrestrial Tardigrada. J. Zool. Lond.224: 59–77.

Authors’ addresses: Hartmut GREVEN (correspondingauthor) and Lutz SCHÜTTLER, Institut für Zoomorphologieund Zellbiologie der Heinrich-Heine-Universität, Universi-tätsstr. 1, D-40225 Düsseldorf, Germany;Tel.: ++49 2118112081, Fax: ++49 2118114499,e-mail: grevenh@uni-duesseldorf.de

Received: 02. 02. 2001Reviewed: 24. 07. 2001Accepted: 20. 08. 2001Corresponding Editor: Reinhardt Møbjerg KRISTENSEN

HALLAS, T. E. (1978): Habitat preferences in terrestrial tardi-grades. Ann. Zool. Fenn. 15: 66–68.

JENNINGS, P.G. (1975): The Signy Island terrestrial referencesite. V. Oxygen uptake of Macrobiotus furciger J. Murray(Tardigrada). Br. Antarct. Surv. Bull. 41: 161–168.

LÖSCH, R. (2001): Wasserhaushalt der Pflanzen. Quelle &Meyer, Wiebelsheim.

MARCUS, E. (1929): Tardigrada. In: H.G. Bronn’s Klassenund Ordnungen des Tierreichs 5, IV, 3. Akademische Ver-lagsgesellschaft, Leipzig.

OVERGAARD, C. (1948): Studies on soil microfauna. Themoss inhabiting nematodes and rotifers. Naturvidensk.Skr. Laerde. Selsk. Skr. Arhus 1: 1–98.

SCHÜTTLER, L. & GREVEN, H. (2000/2001): Beobachtungenzur Lokomotion von Tardigraden. Acta Biol. Benrodis 11:31–50.

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