1. 1 Rotifer resting eggs Nadav Y. Denekamp National Institute of Oceanography Israel Oceanographic & Limnological Research

2. 2 Outline Participation in workpackages Previous meeting report summary Current report in detail Future directions

3. 3 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

4. 4 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.

5. 5 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

6. 6 Reproduction of rotifers High salinity: ~100% of sea water (40 ppt) Low salinity: ~50% of sea water (20 ppt)

7. 7 Sample collection for RNA extraction Mictic an amictic females Females bearing resting eggs

8. 8 Picking females bearing resting eggs one by one…

9. 9 Samples collection From RE toward hatching Dormant stage 10 hr 20 hr 30 hr

10. 10 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

11. 11 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 25 o C in the dark.

12. 12 RNA extraction from rotifers Classic total eukaryotic RNA looks like: http://www.ambion.com/techlib/tn/83/8313.html

13. 13 0.5 kb 1 kb 2 kb 3 kb 5 kb 7 kb 9 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

14. 14 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)AB111352 The sequence of eft1a for B. plicatilis had to be found

15. 15 Degenerated primers were designed by MSA of eft1 genes from: C. elegans, Nereis, S. cerevisiae and Human.

16. 16 PCR with degenerated primers for eft1 500 bp

17. 17 Temperature gradient PCR with primers of eft1 12-342 and eft1 101-317 53.2 55.5 58.163.560.855.5 58.163.560.8 Temperatures in Celsius 200bp 500bp cDNA source: mictic and amictic rotifers

18. 18 PCR with primers for actin 500 bp 1mM Mg 2+ The correct sequence for actin cDNA source: mictic amictic amictic rotifers

19. 19 500 bp Random primers Oligo(dT) PCR experiments with cDNA synthesized from mRNA of RE PCR with primers for actin

20. 20 200 bp Random primers Oligo(dT) PCR with primers for eft1

21. 21 Proposed cDNA libraries to be constructed from the RNA samples Normalized library of sexual and asexual reproducing rotifers (Clonal and no clonal cultures) Normalized library of RE in dormant stage Non-normalized library of RE in dormant stage Normalized library of RE 20 and 30 hours after hatching initiation. Subtractive library of female bearing RE against sexual+asexual reproducing rotifers, both from the same clone.

22. 22 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

23. 23 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)

24. 24 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

25. 25 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.

26. 26 Experiment design Time Population density Asexually reproducing culture Sexually reproducing culture