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Rotifer resting eggs

Nadav Y. DenekampNational Institute of Oceanography

Israel Oceanographic & Limnological Research

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Outline

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 M30-M36

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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 g RNA were extracted from 1000-2000 rotifers (300-800 ng/l at 40 l)

4-12 g RNA were extracted from 2000-3000 RE (100-300 ng/l at 40 l)

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:

http://www.ambion.com/techlib/tn/83/8313.html

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0.5 kb

1 kb

2 kb3 kb

5 kb

7 kb9 kb

18S

26S

Degradation of 26S rRNA is expected due to its thermal instability (Kaneko et al., 2002, Fisheries Scieneces, Collier JR, 1983, Biological bulletin)

5S

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PCR experiments

In order to evaluate the mRNA quality PCR experiments were performed for expression of actin and eft1.

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 eft1 genes from: C. elegans, Nereis, S. cerevisiae and Human.

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PCR with degenerated primers for eft1

500 bp

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Temperature gradient PCR with primers of eft1 12-342 and

eft1 101-317

53.2 55.558.1 63.560.8 55.558.1 63.560.8

Temperatures in Celsius

200bp

500bp

cDNA source: mictic and amictic rotifers

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PCR with primers for actin

500 bp

1mM Mg2+

The correct sequence for actin

cDNA source: mictic amictic amictic rotifers

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500 bp

Randomprimers

Oligo(dT)

PCR experiments with cDNA synthesized from mRNA of RE

PCR with primers for actin

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200 bp

Random primersOligo(dT)

PCR with primers for eft1

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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

Time

Populationdensity

Asexually reproducing culture

Sexually reproducing culture