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