Rotifer resting eggs
Nadav Y. Denekamp
National Institute of Oceanography
Israel Oceanographic & Limnological Research
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Outline
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Participation in workpackages
Previous meeting report summary
Current report in detail
Future directions
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Workpackages
 The NIO research group participates in
workpackages: WP1, WP2, WP4, WP5 and WP6.
 WP1 deliverables:
D4: Optimal conditions for the indcution of asexually
and sexually reproducing rotifers and their resting
eggs (M9)
D5: cDNA libraries, EST sequencing and database
construction (M16)
D6-D8: Scheduled for M30-M36
 WP2, WP4, WP5 and WP6 are scheduled to M30M36
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Last meeting report
 Setting up asexually reproducing rotifer cultures
(high salinity media)
 Setting up sexually reproducing rotifer cultures
(low salinity media)
 Hatching experiments
 Development of an RNA extraction method for
rotifers.
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The current report
 Collection of samples for cDNA libraries from
rotifer cultures
 Collection of sample for cDNA libraries from
resting eggs and various stages of hatching
 Future experiments
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Reproduction of rotifers
High salinity: ~100% of
sea water (40 ppt)
Low salinity: ~50% of
sea water (20 ppt)
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Sample collection for RNA extraction
 Mictic an amictic
females
 Females bearing resting
eggs
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Picking females bearing resting eggs one by one…
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Samples collection From RE toward
hatching
Dormant
stage
10 hr
20 hr
30 hr
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RNA amounts obtained from rotifers
and RE
 12-32 mg RNA were extracted from 1000-2000
rotifers (300-800 ng/ml at 40 ml)
 4-12 mg RNA were extracted from 2000-3000 RE
(100-300 ng/ml at 40 ml)
 The amount of mRNA in RE is not known.
 Large amounts of RE are needed for the
extraction of RNA from different stages toward
hatching
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Production of RE
 Non clonal rotifer cultures were set up in low
salinity media (~10 ppt)
 Cultures were fed with Nannochloropsis
 Females bearing RE appeared after 5 days
 RE were collected after 11-12 days.
 30,000 RE were collected from 6 liter cultures.
 RE were divided in to batches of 2000-3000 RE
and were stored for 84 days at 25oC in the dark.
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RNA extraction from rotifers
“Classic” total eukaryotic RNA looks like:
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http://www.ambion.com/techlib/tn/83/8313.html
Degradation of 26S rRNA is expected due to its
thermal instability (Kaneko et al., 2002, Fisheries
Scieneces, Collier JR, 1983, Biological bulletin)
9 kb
7 kb
26S
18S
5 kb
3 kb
2 kb
1 kb
0.5 kb
5S
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PCR experiments
 In order to evaluate the mRNA quality PCR
experiments were performed for expression of
actin and eft1a.
 The sequence of actin for B. plicatilis is known
(AB111352)
 The sequence of eft1a for B. plicatilis had to be
found
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Degenerated primers were designed by MSA of
eft1a genes from: C. elegans, Nereis, S. cerevisiae
and Human.
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500 bp
PCR with degenerated primers for eft1a
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Temperature gradient PCR with primers
of eft1a 12-342 and eft1a 101-317
cDNA source: mictic and amictic rotifers
53.2
55.5 58.1 60.8
63.5
55.5 58.1 60.8
63.5
500bp
200bp
Temperatures in Celsius
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PCR with primers for actin
cDNA source: mictic amictic amictic rotifers
1mM
Mg2+
500 bp
The correct sequence for actin
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PCR experiments with cDNA synthesized
from mRNA of RE
PCR with primers for actin
Random
primers
Oligo(dT)
500 bp
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PCR with primers for eft1a
Random
primers Oligo(dT)
200 bp
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Proposed cDNA libraries to be
constructed from the RNA samples
(A) Normalized library of sexual and asexual
reproducing rotifers (Clonal and no clonal cultures)
(B) Normalized library of RE in dormant stage
(C) Non-normalized library of RE in dormant stage
(D) Normalized library of RE 20 and 30 hours after
hatching initiation.
(E) Subtractive library of female bearing RE against
sexual+asexual reproducing rotifers, both from the
same clone.
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Future experiments
 Gene expression analysis toward production of
resting eggs
– HSP70: May stabilize proteins structure during
dormancy. Its sequence in B. plicatilis is known.
– Mn SOD: Antioxidant enzyme. Oxidative stress may
cause damage during hatching. Sequence in B.
plicatilis is known
– TPS1: Trehalose phosphate synthase. Sequence in B.
plicatilis is not known
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Role of trehalose in RE survival
 Trehalose enhances survival during anhydrobiosis in
other organisms such as yeasts and artemia.
 Cloning of trehalose producing genes resulted in
desiccation tolerance in mammalian cells (Crowe and
Crowe, 2000, Nature).
 Trehalose was not found in desiccated Bdelloid rotifers
(Tunnacliffe and Lapinski, 2002, R. SOC., Caprioli et al.,
2004, CBP).
 Very little ammount of trehalose was found in B.
plicatilis RE (Caprioli et al., 2004,CBP)
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Attempts to find the sequence for tps1
in B. plicatilis
 No significant similarity was found between nucleotide
sequences of Drosophila, Arabidopsis, S. cerevisiae and
C. elegans.
 Degenerated primers were designed after MSA of
protein sequences
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 The sequence of the PCR products did not
match any known tps1 genes.
 Further trials will be done using the AUAP
primer and one of the degenerated primers.
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Experiment design
Population
density
Sexually
reproducing
culture
Asexually
reproducing
culture
Time
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