Accepted on 1 October 2010

 2011 Blackwell Verlag GmbH J Zool Syst Evol Res doi: 10.1111/j.1439-0469.2010.00601.x

1
Department of Zoology, Universität Stuttgart, Biological Institute, Stuttgart, Germany; 2Kristianstad University, School of
Teacher Education, Aquatic Biology & Chemistry Group, Kristianstad, Sweden

Food of tardigrades: a case study to understand food choice, intake and digestion
Ralph O. Schill1, K. Ingemar Jönsson2, Martin Pfannkuchen1 and Franz Brümmer1

Abstract
Mosses are an excellent habitat for tardigrades because of their ability to ensure a high humidity and to provide a rich food supply for both
carnivorous and herbivorous species. Food choice can be correlated with the morphology of the buccal apparatus, and consequentially, their
distribution is sometimes linked to food availability (nematodes, rotifers, plant cells, algae, yeast and bacteria). In many species, material
containing chlorophyll is often observed in the midgut. However, little information has been available until now on the actual food preference of
tardigrades. Since trophic interactions within soil food webs are difficult to study, here we use a polymerase chain reaction–based approach as a
highly sensitive detection method. The study was carried out to investigate the presence of chlorophyll matter in the gut of active specimens, based
on sequence analyses of the chloroplast ribulose 1,5-bisphosphate carboxylase ⁄ oxygenase large subunit (rbcL) gene from mosses and algae. The
sequences found in the gut of Macrobiotus sapiens were derived from the moss families Pottiaceae and Erpodiaceae, in Macrobiotus persimilis and
Echiniscus granulatus from the moss family Grimmiaceae, and in Richtersius coronifer from the green algae genus Trebouxia. Furthermore, we
show the emission of green autofluorescence from the chloroplasts in the algae within the gut of tardigrades and followed the progress of digestion
over a 48-h period. The autofluorescent emission level declined significantly, and after 2 days, the signal level was similar to the level of the starved
control.

Key words: Algae – diagnostic PCR – gut-content analysis – invertebrate – moss – predation

Introduction and Traunspurger 2005). Several other tardigrade species,

Tardigrades are small, motile metazoa that inhabit terrestrial,
freshwater, brackish water and marine environments. They
colonized nearly all habitats from the deep ocean trenches to
the highest mountains around the world (Marcus 1928a;
Nelson 2002). Some tardigrades have a cosmopolitan distri-
bution, while others are endemic (Nelson 2002). Despite their
overall abundance, with more than 1070 described species
(Degma et al. 2010), they have received little attention over the
last decades and there are few ecological studies relating to
their life history and food preference. Although a variety of
food sources have been reported for tardigrades, including
plant cell fluids, bacteria, algae, protozoa and other small
invertebrates (Marcus 1928a; Greven 1980; Kinchin 1994),
more detailed studies on food preference are scarce. The
carnivorous species Milnesium tardigradum Doyère, 1840
(Milnesiidae) is known to feed on nematodes and rotifers.
Such knowledge has enabled this species to be cultured in the
laboratory (Suzuki 2003; Schill et al. 2004; Schill and Fritz
2008). Several bdelloid rotifer species and mouthparts and
even entire individuals of another tardigrade species (Diphas-
con sp.) have been identified in the gut contents of mounted
Milnesium cfr. tardigradum specimens from the field (McInnes
et al. 2001). Hohberg and Traunspurger (2005) investigated
the consumption rate of Paramacrobiotus richtersi (Murray,
1911) (Macrobiotidae) with nematode prey Pelodera teres
(Schneider, 1860) (Rhabditidae) and Acrobeloides nanus (de
Man, 1880) (Cephalobidae). Pelodera teres was consumed at a
lower rate than the larger Acrobeloides nanus, which indicates
that the type of prey affected the consumption rate (Hohberg
Corresponding author: Ralph O. Schill (ralph.schill@bio.
uni-stuttgart.de)
Contributing authors: K. Ingemar Jönsson (ingemar.jonsson@hkr.se),
Martin Pfannkuchen (pfannkuchen@cim.irb.hr), Franz Brümmer
(franz.bruemmer@bio.uni-stuttgart.de)
which can be described as carnivores or omnivores, can also be
cultured (Guidetti et al. 2009; Hengherr et al. 2009).
Marcus (1928a) observed the marine genus Batillipes sp.
searching for diatoms and algae while living in among their
food. Other marine species like the Echiniscoides sigismundi
(Schultze, 1865) graze their food using their strong claws to
climb among the small filaments of Enteromorpha sp. along the
seashore.
Many freshwater and terrestrial tardigrades live on moss
stems. The cells of the mosses Polytrichum sp., Dicranium sp.,
Leucobryum sp. and Racomitrium sp. are known to be difficult
to tap with the comparatively small stylets of the tardigrades.
However, the moss species Hypnum cupressiforme, Hypnum
parietinum and Hypnum uncinatum, as well as Grimmia and
Tortula species are well known as microhabitats for tardi-
grades (Marcus 1928a) and may contain up to 20 000
individuals per 1 g of air-dried moss (Marcus 1928b). Irregular
feeding marks have been observed on leaves of mosses
(Marcus 1928a), but in general, it is largely unknown what
these moss-living tardigrades feed on and whether they are
specialized in their food choice.
In this study, we show the rate at which herbivorous
tardigrades digest their algae food by following the degradation
of chlorophyll. Furthermore, we identify the gut content of
three different eutardigrade species and one heterotardigrade
species in relation to the moss in which they live. To study food
specificity, we carried out feeding experiments offering four
species of green algae and one species of blue-green algae.
Methods
In situ food detection
To detect the natural food of the tardigrade species Macrobiotus
sapiens Binda & Pilato, 1984, Macrobiotus persimilis Binda & Pilato,
1972, Richtersius coronifer (Richters, 1903), and Echiniscus granulatus

J Zool Syst Evol Res (2011) 49 (Suppl. 1), 66–70

Food of tardigrades
(Doyère, 1840), we rehydrated three moss samples containing living
tardigrades with Volvic water for 1 h (Danone Waters Deutschland
GmbH, Frankfurt, Germany). The moss sample with the tardigrade
species Macrobiotus persimilis and Echiniscus granulatus was from
Tübingen, Germany. Richtersius coronifer was extracted from moss
from Öland, Sweden, and the moss with Macrobiotus sapiens had been
collected in Rovinj, Croatia. From each tardigrade species, 20 animals
were collected by hand using a Stereomicroscope SZX7 (Olympus
Europa GmbH, Hamburg, Germany). Subsequently, the animals were
identified and frozen in liquid nitrogen. Genomic DNA was extracted
according to (Schill and Steinbrück 2007) with the NucleoSpin Tissue
(Macherey-Nagel, Düren, Germany) method. As it was not possible to
dissect out the gut from tardigrades, prelysis and lysis was achieved by
incubation of whole specimens in a proteinase K ⁄ SDS solution at 56C
for 3 h and at 70C for 10 min, respectively. The animals were then
ground with a pestle to release the DNA and subsequently the DNA
bound to a silica membrane in a NucleoSpin Tissue column. After
two further steps of washing, the DNA was eluted with 25 ll of pure
water. Polymerase chain reaction (PCR) amplification was performed
in a final volume of 25 ll, consisting of extracted DNA, 0.5 unit Taq
DNA Polymerase (Genaxxon, Biberach, Germany) with 10· reaction
buffer added to a final concentration of 1.5 mM MgCl2, 0.25 mM
dNTP, 1 lM oligonucleotide of each primer. The primers we used to
characterize the algae or moss food in this study were designed by
hand (rbcL-for: 5¢-ATGCGTTGGAGAGAYCGTTTC-3; rbcL-rev:
5¢-AGTTACTTSACGTTCWCCTTC-3¢), based on ribulose-1,5-bis-
phosphate carboxylase ⁄ oxygenase large subunit gene (rbcL) sequences
from five Bryophyta and four Chlorophyta, obtained from the
National Centre for Biotechnology Information (NCBI) GenBank
(Table S1). Extracted DNA was amplified with an initial denaturation
step of 6 min at 95C, followed by 35 cycles of 30 s at 94C, 30 s at
55C and 45 s at 72C, respectively, with a final period of 5 min at
72C. Aliquots of the PCR products were run with SYBR Gold
1 : 1000 (Molecular Probes, Eugene, OR, USA) on a 3% agarose gel
for control of the fragment size. For sequencing, the PCR products
were cloned with the TOPO TA Cloning kit (Invitrogen, Carlsbad,
NM, USA). Five randomly collected clones from each cloned PCR
product were sequenced and identified using blast (Basic Local
Alignment Search Tool) (Altschul et al. 1997).
Feeding experiment
In the feeding experiment, we offered the tardigrade species Macro-
biotus sapiens (n = 24), Macrobiotus persimilis (n = 24), Richtersius
coronifer (n = 24) and Echiniscus granulatus (n = 24) five different
species of green algae (Haematococcus pluvialis Flotow, Micrasterias
rotata Ralfs, Chlorella vulgaris Beijerinck, Spirogyra sp. Link,
Chlorogonium elongatum (P.A. Dangeard) Francé) and one species of
blue-green algae (Glaucozystis nostochinearum Itzigsohn in Raben-
horst) as food. All algae species were courtesy of the Zoological
Institute, Eberhard-Karls-University of Tübingen, Germany. We
starved the animals for 2 days and offered them the algae in separated
wells as food. We checked the gut visually every 30 min over a period
of 6 h using a Stereomicroscope SZX7 (Olympus Europa GmbH). The
amount of algae consumed was classified into four categories (0 = no
feeding on algae, 1 = weak feeding on algae, 2 = strong feeding on
algae and 3 = very strong feeding on algae).
Digestion experiment
To study the process of digestion, we used the tardigrade species
Macrobiotus sapiens, which was collected in Rovinj, Croatia, and
cultured in the laboratory on agar plates. This tardigrade species was
reared in plastic culture dishes on a 4-mm layer of 3% agar with a
3-mm layer of Volvic water over its surface. As food, we provided
the green algae Chlorogonium elongatum twice a week. Before the
digestion experiment, we starved the animals (n = 25) for 4 days.
After this period of time, the animals were almost transparent and no
residues were visible in the gut using a Stereomicroscope SZX7.
Subsequently, we offered an excess of fresh extracted green algae
Chlorogonium elongatum and allowed the tardigrades to feed on it for
1 h. Tardigrades were then transferred to a new agar plate with no
67
food. Previous experiments showed that longer feeding times are not
necessary because the tardigrades were satiated with algae after around
45 min. We could detect autofluorescent emission of green light from
the chlorophyll in the green algae of single specimens that were
transferred after a time series (1, 12, 24, 36, 48 h) to an object slide and
observed under epifluorescence with an Axiovert 200 M (Zeiss,
Oberkochen, Germany), using the filter set 10 (Zeiss) BP450-
490|BS510|BP515-565 and blue light illumination. The decreasing
autofluorescent emission through digestion of the chlorophyll in the
algae during the time series was quantified (mean ± SD) by a
densitometric image analysis system (Herolab E.A.S.Y., Weisloch,
Germany). The highest signal level was arbitrarily set to 100%.
Results
Moss species identification
The identification of moss depends on several characters (e.g.
fruiting bodies), which are not always available. Identification
is often further complicated by different moss species cluster-
ing together. However, the following list of moss shown in
Table S2 represents the primary species in which we found
tardigrades although we cannot exclude cryptic moss species.
The moss in which the species Macrobiotus sapiens was found
consisted of three moss families: Pottiaceae (Didymodon
sinuosus Delogne, Tortula muralis Hedwig), Grimmiaceae
(Schistidium sp.) and Orthotrichaceae (Orthotrichum diapha-
num Schrader ex Bridel). The species Macrobiotus persimilis
was found only in the moss family Grimmiaceae (Schistidium
cf. crassipilum), Richtersius coronifer in the family Orthotrich-
aceae (Orthotrichum cupulatum G. F. Hoffmann ex Bridel) and
Echiniscus granulatus in the family Grimmiaceae (Schistidium
cf. crassipilum H. Blom).
In situ food detection
With the designed primer pair for the rbcL sequence, we
obtained from each extracted DNA a 392-bp PCR product,
which was cloned to capture sequences of different algae or
moss species (Table S3). Five replicate sequences of each
group were analysed to obtain information on food uptake.
The sequences found in the gut of Macrobiotus sapiens were
derived from the moss families Pottiaceae and Erpodiaceae.
Within these families, we obtained hits in the NCBI GenBank
for the species Tortula obtusissima (C. Müll.), Aulacopilum
hodgkinsoniae Brotherus, and Venturiella sinensis C. Müller,
1897 with a maximum identity of 97%. The sequences
obtained from the gut of Macrobiotus persimilis all resulted
in the moss family Grimmiaceae (Grimmia elongata (maximum
identity of 99%), Coscinodon cribrosus Spruce (maximum
identity of 99%), Schistidium strictum (maximum identity of
98%)). Echiniscus granulatus contained sequences of the family
Grimmiaceae and the species Schistidium strictum Loeske
ex Mart. (maximum identity of 98%), Grimmia elongata
G. Kaulfuss in Sturm (maximum identity of 98%) and
Coscinodon cribrosus (maximum identity of 98%). In contrast
to the other three tardigrade species, guts of Richtersius
coronifer contained sequences of the green algae genus
Trebouxia Puymaly (maximum identity of 97%).
Feeding experiment
We offered the tardigrade species Macrobiotus sapiens,
Macrobiotus persimilis, Richtersius coronifer and Echiniscus
granulatus six different species of algae as food (Table S4).
J Zool Syst Evol Res (2011), 49 (Suppl. 1), 66–70
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68
(a)
Fig. 1. (a) Overview screen from Marcobiotus sapiens after feeding on green algae Chlorogonium elongatum. (b) Emission of green autofluo-
rescence from the chloroplasts in the algae after excitation with blue light (Zeiss filter set 10 BP450-490|BS510|BP515-565). (c) The level of
autofluorescent emission from the gut was calculated by densitometric analysis
Richtersius coronifer and Echiniscus granulatus showed no
uptake of algae during the whole time of the experiment. In
contrast to these two species, Macrobiotus sapiens fed strongly
on the algae Haematococcus pluvialis, very strongly on
Chlorogonium elongatum and weakly on Glaucozystis nosto-
chinearum. Macrobiotus persimilis fed in the same manner as
Macrobiotus sapiens. Micrasteria rotata, Chlorella vulgaris and
Spirogyra sp. were not eaten.
Digestion experiment
The autofluorescent emission of green light from the chlo-
roplasts was arbitrarily set to 100% after the tardigrade
species Macrobiotus sapiens had been feeding for 45 min on
the green algae Chlorogonium elongatum (Fig. 1a,b,c). During
the following 48 h of digestion, the autofluorescent emission
level declined significantly (Fig. 2). After the first 12 h, the
level dropped from 100% (±10.61%) to 77.52% (±17.03%).
A further 12 h later, the level had decreased to 42.83%
(±11.39%). After 36 h of digestion, autofluorescent emission
decreased approximately fivefold (22.40 ± 11.12%) com-
pared to the well-fed animals, and after 2 days, the signal
level of 2.73% (±1.53%) was similar to the level of the
starved control (1.00 ± 1.37%).
Autofluorescence emission of
100.00
green algae (%)
75.00
50.00
25.00
0.00
Control 0 12
Time of digestion (h)
Fig. 2. Levels (mean ± SD) of the autofluorescent emission of green
algae in the gut of Marcobiotus sapiens starved as control, after feeding
(0 h) and a time series of digestion (12, 24, 36 and 48 h; n = 5)
J Zool Syst Evol Res (2011), 49 (Suppl. 1), 66–70
 2011 Blackwell Verlag GmbH
Schill, Jönsson, Pfannkuchen and Brümmer
(b) (c)
200 µm
Discussion
The availability and choice of food is an important factor for
life history parameters and for understanding dynamics of
populations. Knowledge on food choice is also of utmost
importance for culturing a species under laboratory condi-
tions. Our study represents one of the first attempts to study
the choice and preference of food in tardigrades. The moss
identification, after extraction of tardigrades, showed that the
tardigrade species Macrobiotus sapiens originated from a
cluster of several mosses, belonging to the families Pottiaceae,
Grimmiaceae and Orthotrichaceae. Macrobiotus persimilis and
Echiniscus granulatus were found in the family Grimmiaceae,
and Richtersius coronifer in the family Orthotrichaceae.
By the amplification of DNA from the gut of Macrobiotus
sapiens, we were able to identify rbcL sequences from three
different moss species. As we found no hits with 100%
sequence similarity (identity), at species level, in the GenBank,
we limited our results to the genus and family of the moss.
While the moss family Erpodiaceae appeared in the gut
sequences and not in the morphologically identified samples of
moss, we conclude that the Erpodiaceae moss was one of the
unidentified moss species, mentioned before, or the tardigrades
moved from one cushion to another moss cushion. The family
Grimmiaceae was identified from both the gut and the habitat
with a further GenBank hit for another species within the
Grimmiaceae. However, we found no rbcL sequence corre-
sponding to the families Pottiaceae or Orthotrichaceae.
Macrobiotus sapiens will only eat these two moss families
when Grimmiaceae is not available. Alternatively, the Potti-
aceae and Orthotrichaceae may have already been digested,
and the DNA fragments destroyed before samples were taken.
We identified the Grimmiaceae (Schistidium sp.) in the gut of
Macrobiotus persimilis which was also identified in the habitat.
Additionally, we found rbcL sequences from two other genera
from the family Grimmiaceae. Results for Echiniscus granula-
tus were similar. Considering also the results for Macrobiotus
24 36 48 sapiens, we can conclude that Grimmiaceae mosses are
apparently a good food source for tardigrades and suitable
to tap into and suck.
In comparison, Richtersius coronifer had a dark green gut
that was attributable to the presence of several species of green
Food of tardigrades
algae as no rbcL DNA was identified from mosses. The fact that
these tardigrades did not contain DNA of the moss in which
they live does not of course prove that they never feed on it.
However, algae would seem to be a more likely food source for
this tardigrade. In fact, algae may generally be a more common
food source for tardigrades than was previously thought.
To study the choice of algae as food, we offered the same
tardigrade species six different species of algae. Surprisingly,
Richtersius coronifer, which contained the green algae in the
gut, was not at all interested in the offered algae. The
morphology of the algae appears to be quite important for
tardigrades because many algae have a gelatinous cover which
could prove difficult to negotiate. However, Macrobiotus
sapiens and Macrobiotus persimilis preferred Haematococcus
pluvialis and Chlorogonium elongatum, with a diameter of
5–25 lm (Fan et al. 1994) and 10–25 lm (Hoops and Witman
1985), respectively. It is possible that the morphology of the
algae Micrasteria rotata, Chlorella vulgaris and Spirogyra sp. is
responsible for them not being eaten. Micrasteria rotata has a
polar lobe, lateral wings and is approximately 200 lm in size
(Lacalli 1975). This alga seems to be too large for the mouth of
the tardigrades examined and the cell wall too strong for the
stylets. In contrast, Chlorella vulgaris, the smallest algae with a
size of around 6 lm, may be too small for the tardigrades.
Spirogyra sp. is a filamentous alga that contains cells of
different sizes and with cell walls of different thicknesses. The
preferred diameter of algae as food seems to be >10 lm such as
the blue-green algae Glaucozystis nostochinearum (40–50 lm).
Of course, these observations only consider the morphology.
Various unknown factors, e.g. metabolites, may also play a
role.
There is little information about digestion rates of inverte-
brates in general. Also, most experiments have been conducted
with carnivorous species and enzyme-linked immunosorbent
assays. Fichter and Stephen (1981) observed the time-related
decay in prey antigens ingested by the predator Podisus
maculiventris (Say) (Hemiptera, Pentatomidae). They showed a
linear degradation of moth larva [Orgyia pseudotsugata
(McDunnough, 1921)] antigens at 24 C in the predator over
7 days, suggesting a constant rate of digestion. Other arthro-
pods like the pirate bugs [Orius insidiosus (Say)] showed,
regardless of the number of prey eaten (pink bollworm eggs),
recognizable quantities of prey in their gut after 12 h, but not
24 h after feeding. The decline in the proportion of positive
responses was relatively linear over a 24-h period (Hagler and
Naranjo 1997). The immunological determination of digestive
rates in the hepatopancreas of syntopic scorpions Urodacus
armatus Pocock, 1888 and Urodacus novaehollandiae Peters,
1861 feeding on cockroaches showed that detectable prey
antigens had disappeared within 3 days of ingestion (Quinlan
et al. 1993).
Marcus (1928a) described the changing pH values in
tardigrades from the foregut, with an acidic environment
(owing to digestions enzymes), to the midgut, with a basic
environment. The nutriments will be absorbed in the individ-
ual storage cells, freely floating in the body fluid. The size of
the storage cells is correlated with the nutritional condition of
the animals (Marcus 1928a; Szymanska 1994). Our digestion
experiment showed a strong decrease in the green autofluo-
rescent emission from the chloroplasts in the green algae. This
indicated a continuous digestion with a reduction in the
available intact chloroplasts (chlorophyll molecules) over 12 h.
After 24 h, there were almost no chloroplasts remaining and
69
therefore no food in the gut. We have also shown that it is
possible to identify the food with rbcL sequences from DNA
extracted from the gut for up to 2 days after feeding. Further
studies will give a more detailed overview of what tardigrades
eat. Trophic interactions within food webs are difficult to study
with conventional methods. Therefore, a PCR-based approach
as used in this study is a highly sensitive detection method for
the qualitative characterization of trophic interactions as has
already proved to be the case for other invertebrates (Juen and
Traugott 2005, 2006, 2007; Admassu et al. 2006).
Acknowledgements
Many thanks to Roger Worland for critical discussion and manuscript
pre-review. We thank Inge Polle for technical assistance, Steffen
Hengherr for help with the tardigrade culture, and Martin Nebel for
the identification of moos. This study was enabled using the equip-
ments made available by the project FUNCRYPTA (0313838A),
funded by the German Federal Ministry of Education and Research,
BMBF.
Zusammenfassung
Nahrung von Tardigraden: Untersuchungen zum Verständnis der Nah-
rungsauswahl, Nahrungsaufnahme und Verdauung
Moose sind aufgrund ihrer Fähigkeiten, eine hohe Luftfeuchtigkeit zu
gewährleisten und für carnivore und herbivore Tardigradenarten
genügend Nahrung zur Verfügung zu stellen, ein hervorragender
Lebensraum für Tardigraden. Die Wahl der Nahrung korreliert mit
der Morphologie des Buccalapparates und ihre Verbreitung wird von
der Nahrungsverfügbarkeit (Nematoden, Rotatorien, Pflanzenzellen,
Algen, Hefen und Bakterien) bestimmt. Bei vielen Arten kann
chlorophyllhaltiges Material im Mitteldarm beobachtet werden. Es
gibt bis jetzt jedoch nur wenige Informationen über die Nahrungsprä-
ferenz von Tardigraden. Da trophische Interaktionen innerhalb des
Nahrungsnetzes im Boden schwierig zu untersuchen sind, haben wir
eine sehr sensitive Nachweismethode, basierend auf der Polymerasen-
Kettenreaktion, angewandt. Basierend auf der Sequenzanalyse des
Gens Ribulose-1,5-bisphosphat-Carboxylase ⁄ -Oxygenase (rbcL) in
den Chloroplasten der Moose und Algen wurde das chlorophyllhaltige
Material im Darm aktiver Tardigraden untersucht.
Die Sequenzen, die im Darm von Macrobiotus sapiens gefunden
wurden, gehören zu Moosen der Familien Pottiaceae und Erpodiaceae,
die in Macrobiotus persimilis und Echiniscus granulatus zu Moosen der
Familie Grimmiaceae und die in Richtersius coronifer zu Grünalgen
der Gattung Trebouxia.
Zusätzlich zeigen wir die Emission der grünen Autofluoreszenz der
gefressenen Chloroplasten im Darm der Tardigraden und die an-
schließende Verdauung über einen Zeitraum von 48 Stunden. Die
Autofluoreszenz nahm signifikant ab und nach zwei Tagen war das
Signal ähnlich stark wie bei der ausgehungerten Kontrolle.
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Supporting Information
Additional Supporting Information may be found in the online
version of this article:
Table S1. Ribulose-1,5-bisphosphate carboxylase ⁄ oxygen-
ase large subunit gene (rbcL) sequences from five Bryophyta
and four Chlorophyta, obtained from National Center for
Biotechnology Information (NCBI) GenBank.
Table S2. Identified species of moss in which tardigrades
have been found.
Table S3. Different sequence obtained from the gut DNA
and identified by the most similar sequences found in the
NCBI GenBank.
Table S4. Species of algae that were offered as a food source
to four different species of tardigrades. The amount of algae was
classified in four categories (0 = did not feed, 1 = fed weak on
algae, 2 = fed strong on algae, and 3 = fed very strong on
algae).
Please note: Wiley-Blackwell are not responsible for the
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by the authors. Any queries (other than missing material)
should be directed to the corresponding author for the article.